The Biological bulletin

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

HAROLD C. BOLD, Vanderbilt University J. H. LOCHHEAD, University of Vermont

JOHN B. BUCK, National Institutes of Health E. T. MOUL, Rutgers University

T. H. BULLOCK, University of California, ARTHUR W. POLLISTER, Columbia University

Los Angeles

E. G. BUTLER, Princeton University MARY E" RAWLES> Johns *0^s Unlversitv

K. W. COOPER, University of Rochester A. R. WHITING, University of Pennsylvania

M. E. KRAHL, University of Chicago CARROLL M. WILLIAMS, Harvard University

DONALD P. COSTELLO, University of North Carolina
Managing Editor

VOLUME 113

AUGUST TO DECEMBER, 1957

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE & LEMON STS.

LANCASTER, PA.

11

THE BIOLOGICAL BULLETIN is issued six times a year at the
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn-
sylvania.

Subscriptions and similar matter should be addressed to The
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Massachusetts. Agent for Great Britain: Wheldon and Wesley,
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers $2.50. Subscription per volume (three
issues), $6.00.

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.
Donald P. Costello, P.O. Box 429, Chapel Hill, North Carolina,
during the remainder of the year.

Entered as second-class matter May 17, 1930, at the post office at Lancaster,
Pa., under the Act of August 24, 1912.

LANCASTER PRESS, INC., LANCASTER, PA.

CONTENTS

No. 1. AUGUST, 1957 PAGE

Annual Report of the Marine Biological Laboratory 1

ALLEN, M. JEAN

The breeding of polychaetous annelids near Parguera, Puerto Rico .... 49
BLASKOVICS, JOAN CORMIER, AND KENNETH B. RAPER

Encystment stages of Dictyostelium 58

VON BRAND, THEODOR, PATRICIA MCMAHON AND M. O. NOLAN

Physiological observations on starvation and desiccation of the snail

Australorbis glabratus 89

BROWN, FRANK A., JR., JOAN SHRINER AND H. MARGUERITE WEBB

Similarities between daily fluctuations in background radiation and

Oo-consumption in the living organism 103

BROWN, FRANK A., JR., H. MARGUERITE WEBB AND ERWIN J. MACEY

Lag-lead correlations of barometric pressure and biological activity. ... 112
HASTINGS, J. WOODLAND, AND DEMOREST DAVENPORT

The luminescence of the millipede, Luminodesmus sequoiae 120

HEILBRUNN, L. V., W. L. WILSON, T. R. TOSTESON, E. DAVIDSON AND R. J.

RUTMAN

The antimitotic and carcinostatic action of ovarian extracts 129

KANUNGO, M. S.

Cardiac physiology of the scorpion Palamnaeus bengalensis C. Koch. . . 135

LOWER, HARRY F.

A comparative study of the cuticular structure of three female mealy
bugs (Homoptera : Pseudococcidae) 141

LYNN, W. GARDNER, AND JAMES NORMAN DENT

Phenylthiourea treatment and binding of radioactive iodine in the
tadpole 160

PETTIBONE, MARIAN H.

Endoparasitic polychaetous annelids of the family Arabellidae with
descriptions of new species 170

RALPH, CHARLES L.

A diurnal activity rhythm in Plethodon cinereus and its modification

by an influence having a lunar frequency 188

YOST, HENRY T., JR., AND HOPE H. ROBSON

Studies on the effects of irradiation of cellular particulates. II. The
effect of gamma radiation on oxygen uptake and phosphorylation 198

No. 2. OCTOBER, 1957
BURGER, J. WENDELL

The general form of excretion in the lobster, Homarus 207

in

73264

iv CONTENTS

COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT

Body growth versus shell growth in Balanus improvisus 224

CROWELL, SEARS, AND CHARLES WYTTENBACH

Factors affecting terminal growth in the hydroid Campanularia 233

DEMEUSY, NOELLE

Respiratory metabolism of the fiddler crab Uca pugilator from two

different latitudinal populations 245

EARNER, DONALD S., AND A. C. WILSON

A quantitative examination of testicular growth in the white-crowned

sparrow 254

GROSS, WARREN J.

A behavioral mechanism for osmotic regulation in a semiterrestrial crab . 268
KANWISHER, JOHN

Freezing and drying in intertidal algae 274

MOULTON, JAMES M.

Sound production in the spiny lobster, Panulirus argus (Latreille) 286

PHILLIPS, JOHN H., JR., AND DONALD P. ABBOTT

Isolation and assay of the nematocyst toxin of Metridium senile

fimbriatum ; 296

WOOTTON, DONALD M.

Studies on the life-history of Allocreadium alloneotenicum sp. nov.

(Allocreadiidae- — Trematoda) 302

Abstracts of papers presented at the Marine Biological Laboratory:

Tuesday Evening Seminars 316

General Meetings 324

Lalor Fellowship Reports 359

No. 3. DECEMBER, 1957

BERG, WILLIAM E.

Chemical analyses of anterior and posterior blastomeres of Ciona
intestinalis 365

GREGG, JOHN R., AND MARGIT KAHLBROCK

The effects of some developmental inhibitors on the phosphorus balance

of amphibian gastrulae 376

GREGG, JOHN R., AND FRANCES L. RAY

Respiration of homogenized embryos: Rana pipiens and Rana pipiens

9 X Rana sylvatica tf 382

HEILBRUNN, L. V., AND W. L. WILSON

A rational approach to the problem of cancer chemotherapy 388

HICKOK, JOHN F., AND DEMOREST DAVENPORT

Further studies in the behavior of commensal polychaetes 397

JONES, MEREDITH L.

On the morphology of the nephridium of Nereis vexillosa Grube 407

LAZAROW, ARNOLD, S. J. COOPERSTEIN, D. K. BLOOMFIELD AND C. T. FRIZ
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 toad-
fish, using the cartesian diver microrespirometer 414

CONTENTS v

PEREZ-GONZALEZ, MARIA DOLORES

Evidence for hormone-containing granules in sinus glands of the fiddler
crab Uca pugilator ' 426

ROSENTHAL, HAROLD L.

The metabolism of strontium-90 and calcium-45 by Lebistes 442

TRAVIS, DOROTHY F.

The molting cycle of the spiny lobster, Panulirus argus Latreille.
IV. Post-ecdysial histological and histochemical- changes in the hepa-
topancreas and integumental tissues 451

RULON, OLIN

Developmental modifications in the sand dollar caused by cobaltous
chloride in combination with sodium selenite and zinc chloride 480

WOOTTON, DONALD M.

Notes on the life-cycle of Azygia acuminata Goldberger, 1911 (Azy-
giidae — Trematoda) 488

Vol. 113, No. 1 August, 1957

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE MARINE BIOLOGICAL LABORATORY
FIFTY-NINTH REPORT, FOR THE YEAR 1956 — SIXTY-NINTH YEAR

I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 10, 1956) .... 1

STANDING COMMITTEES

II. ACT OF INCORPORATION 4

III. BY-LAWS OF THE CORPORATION 4

IV. REPORT OF THE DIRECTOR 6

Statement 7

Addenda :

1. The Staff 10

2. Investigators, Lalor and Lillie Fellows, and Students 13

3. Fellowships and Scholarships 21

4. Tabular View of Attendance, 1952-1956 22

5. Cooperating and Subscribing Institutions 22

6. Evening Lectures 23

7 . Shorter Scientific Papers (Seminars) 24

8. Members of the Corporation 24

V. REPORT OF THE LIBRARIAN 41

VI. REPORT OF THE TREASURER 42

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

EMERITI

EUGENE DuBois, Cornell University Medical College

G. H. A. CLOWES, Lilly Research Laboratory

ROBERT CHAMBERS, 425 Riverside Drive, New York City

O1

-*

•'-"

2 MARINE BIOLOGICAL LABORATORY

W. C. CURTIS, University of Missouri
Ross G. HARRISON, Yale University

E. B. HARVEY, 48 Cleveland Lane, Princeton, N. J.

M. H. JACOBS, University of Pennsylvania School of Medicine

F. P. KNOWLTON, Syracuse University

A. P. MATHEWS, University of Cincinnati

W. J. V. OSTERHOUT, Rockefeller Institute

CHARLES PACKARD, Woods Hole, Mass.

LAWRASON RIGGS, 74 Trinity Place, New York 6, N. Y.

TO SERVE UNTIL 1960

H. F. BLUM, Princeton University

K. S. COLE, National Institutes of Health

L. V. HEILBRUNN, University of Pennsylvania

S. W. KUFFLER, Johns Hopkins Hospital

C. B. METZ, Florida State University

G. T. SCOTT, Oberlin College

A. H. STURTEVANT, California Institute of Technology
E. ZWILLING, University of Connecticut

TO SERVE UNTIL 1959

E. G. BUTLER, Princeton University

C. LALOR BURDICK, The Lalor Foundation, Wilmington, Delaware

D. P. COSTELLO, University of North Carolina
H. HIBBARD, Oberlin College

M. KRAHL, University of Chicago

D. MARSLAND, New York University, Washington Square College
R. RUGH, College of Physicians and Surgeons

H. B. STEINBACH, University of Minnesota

TO SERVE UNTIL 1958

W. R. AMBERSON, University of Maryland, School of Medicine

T. H. BULLOCK, University of California, Los Angeles

AURIN CHASE, Princeton University

ALBERT I. LANSING, Emory University

DANIEL MAZIA, University of California

S. MERYL ROSE, University of Illinois

MARY SEARS, Woods Hole Oceanographic Institution

ALBERT TYLER, California Institute of Technology

TO SERVE UNTIL 1957

E. G. BALL, Harvard University Medical School

F. A. BROWN, JR., Northwestern University

P. S. GALTSOFF, Woods Hole Bureau of Fisheries

E. N. HARVEY, Princeton University

L. H. KLEINHOLZ, Reed College

C. L. PROSSER, University of Illinois

A. E. SZENT-GYORGYI, Institute for Muscle Research

WM. RANDOLPH TAYLOR, University of Michigan

TRUSTEES

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES

GERARD SWOPE, JR., Chairman K. S. COLE

A. K. PARPART E. G. BUTLER

J. H. WlCKERSHAM D. MAZIA

P. B. ARMSTRONG D. P. COSTELLO

F. A. BROWN, JR. H. B. STEINBACH

THE LIBRARY COMMITTEE

MARY SEARS, Chairman E. G. BUTLER

HAROLD F. BLUM J. P. TRINKAUS

THE APPARATUS COMMITTEE

C. LLOYD CLAFF, Chairman ALBERT I. LANSING

T. H. BULLOCK

THE SUPPLY DEPARTMENT COMMITTEE

RUDOLF KEMPTON, Chairman ROBERT DAY ALLEN

C. B. METZ L. V. HEILBRUNN

THE EVENING LECTURE COMMITTEE

P. B. ARMSTRONG, Chairman L. V. HEILBRUNN

E. G. BALL MAC V. EDDS

E. G. BOETTIGER

THE INSTRUCTION COMMITTEE

S. MERYL ROSE, Chairman C. L. PROSSER

L. H. KLEINHOLZ I. M. KLOTZ

THE BUILDINGS AND GROUNDS COMMITTEE

EDGAR ZWILLING, Chairman C. B. METZ

RALPH WICHTERMAN SEARS CROWELL

THE RADIATION COMMITTEE

G. FAILLA, Chairman RAYMOND ZIRKLE

CLAUDE VILLEE ROBERTS RUGH

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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;

No^v, 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 5

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 selected 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 Com-
mittee 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 shalJ be an unincorporated
group of persons (including associations and corporations) interested in the Laboratory
and shall be organized and operated under the general supervision and authority of the
Trustees.

6 MARINE BIOLOGICAL LABORATORY

•

XI. The consent of every Trustee shall be necessary to dissolution of the Marine Bi-
ological 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 sixty-ninth session of the Marine Biological
Laboratory.

Since the close of World War II there have been certain developments at the
Laboratory which have reduced the amount of research space available to summer
investigators. It has been necessary to set aside some space formerly assigned to
investigators for special instrumentation. Seven laboratories are now used for
radiobiology, electron microscopy, dehumidified laboratories for special equipment
and the personnel responsible for these services. Eight laboratories are used by
investigators working on a year-round basis so are not available for the summer
investigators. Thus space for about thirty summer investigators has been diverted
to other uses.

Every effort has been made to accommodate as many summer investigators as
possible which has probably produced serious crowding in some areas. The estab-
lishment of the Neuromuscular Training Program will further reduce the research
space available for summer investigators. In recent years there has been a marked
increase in the number of applicants for research space with many well qualified
individuals being turned away every year. Serious consideration should be given
to the expansion of our research facilities. At least the Laboratory should pick up
the space lost to other activities. This can best be done by replacing the Rocke-
feller, Botany and Old Lecture Hall with a modern brick building. Additional
housing should be constructed at the same time to avoid over-taxing our present
housing and dining hall facilities.

1. Grants, Contracts and Contributions

The total income from these sources of support amounted to $204,034.00 in
1956. This represents 40% of the total income of the Laboratory and consists
of the following accounts :

REPORT OF THE DIRECTOR /

American Cancer Soc. — C26AS — Function of Nuclei and Nucleic Acids $ 18,705.00

American Cancer Soc. — -R-7F — Fundamental Studies in Radiobiology 6,600.00
A.E.C. — At 30-1-1343 — -Program of Research on the Physiology of

Marine Organisms Using Radioisotopes 8,450.00

N.I.H. — B643C — Encephalization in Embryonic Development 2,012.00

N.I.H. — SA43PH 423 — Investigations of the Microscopic Physiology

of Various Forms of Living Marine Life 1,350.00

N.I.H.— B799— Electrical and Mechanical Changes in Muscle 920.00

N.I.H.- — RG4359 — Biological Research on the Morphology, Ecology,

Physiology, Biochemistry and Biophysics of Marine Organisms . . . 40,000.00

N.I.H.— RG-E-45 13— Radiation-Induced Paralysis in Protozoa 14,950.00

National Science Found. — G2142 — Funds for Biological Research . . . 25,000.00
National Science Found. — G1807 — Mechano-Chemical Coupling in

Muscle 1 1,500.00

National Science Found. — G1395 — Osmoregulation of Excretion in

Tunicates 2,917.00

National Science Found. — G2655 — Structure and Function of Proteins 7,750.00

O.N.R.— 1497— Studies in Marine Biology 15,000.00

O.N.R.— 09701— Studies on Isolated Nerve Fibers 6,450.00

O.N.R. — 09702 — Investigation of Environmental Factors Influencing

Certain Marine Biological Populations in the Woods Hole area . . 5,000.00

American Philosophical Society 2,500.00

M. B. L. Associates 5,255.00

Eli Lilly Company 5,000.00

Rockefeller Foundation 20,000.00

Upjohn Company 2,000.00

Ciba Company 1,000.00

Grass Trust 1,000.00

Other . 675.00

$204,034.00

2. Ncuromiiscular Training Program

A neuromuscular training program has been set up under the supervision of
Dr. Stephen Kuffler, which will have a staff of three investigators and eight post-
doctoral Fellows. This is a multidisciplinary program which will be housed in
special quarters developed in the Crane Building. The program is being supported
by the Public Health Service.

3. Plant Improvements

During the past winter the Supply Department Building was completely recon-
structed. Extensive changes were made in the internal arrangement of the building
which adapt it much more satisfactorily to its various functions. Included in the
building will be a study museum containing specimens of the various forms avail-
able in the Woods Hole area for research. Mr. Milton B. Gray, who is very well
acquainted with the local fauna, will serve as Curator of the Museum.

MARINE BIOLOGICAL LABORATORY

The Crane Wing of the Brick Building was built in 1913. It has been in
serious need of rehabilitation for the past several years. Many of the facilities
were not adequate for much of the research currently in progress at the Laboratory.
A request for a grant of $415,000 was made to the National Science Foundation
to reconstruct and modernize this building. Early in this year, favorable action
was taken by the National Science Foundation on this grant request. This recon-
struction work will be undertaken between the 1957-1958 summer seasons. The
resulting building should be adequate for any type of research undertaken at the
Laboratory.

4. Instruction

In line with the Laboratory's policy Dr. Bostwick H. Ketchum will retire as
head of the Marine Ecology course, having served a five-year term. He will be
succeeded by Dr. Eugene P. Odum of the University of Georgia.

Dr. Stephen Kuffler has resigned as head of the Physiology course to take over
the leadership of the Neuromuscular Training Program. He will be succeeded by
Dr. W. D. McElroy of the Johns Hopkins University.

The course in Marine Ecology was originally established under Dr. Ketchum's
direction who can take real pride in the way it has developed. Dr. Kuffler main-
tained the course in Physiology at the high level of effectiveness which has charac-
terized it for many years.

5. Retirements

The Laboratory is losing the services of three of its permanent staff who have
served the Laboratory most faithfully for many years. Mr. James Mclnnis is
retiring as Manager of the Supply Department, having served in that department
for thirty-eight years.

Miss Polly Crowell has been connected with the Laboratory administration for
forty-one years and Miss Ruth Crowell with the Supply Department for thirty-
seven years. The success of the Laboratory depends in a large measure on the
effectiveness of its staff, of which they have been outstanding members.

6. Deaths

This past year the Corporation of the Marine Biological Laboratory suffered the
loss of one of its most eminent and loyal members, in the death of Dr. B. M. Duggar.
A memorial to Dr. Duggar will be presented at the Annual Meeting of 1957.

At the Annual Meeting of 1956 Dr. Mary Sears read a memorial for Mrs.
Priscilla B. Montgomery. Mrs. Montgomery was for many years the Librarian of
the Laboratory and over the years her efforts on behalf of the Library have made
it what it is today. Her name will always be remembered with affection and pride
by all who knew her.

Respectfully submitted,

PHILIP B. ARMSTRONG,

Director

REPORT OF THE DIRECTOR

MEMORIAL

PRISCILLA BRAISLIN MONTGOMERY
by

Mary Sears

28 December 1874-9 August 1956

In 1897, recent graduates of women's colleges came to Woods Hole for summer
courses just as they do today. Priscilla Braislin of Crosswicks, New Jersey, arrived
from Vassar and enrolled in the Embryology Course. The next year, Thomas Harrison
Montgomery came as an investigator and in 1900 he became an instructor in the bird
section of the nature study course. Priscilla Braislin was back to take this course after
three years of teaching school; the first at Howard Seminary in West Bridgewater and
the next two at the Pratt Institute High School in Brooklyn. Following this summer,
they became engaged and were married in Crosswicks on September 19, 1901. Within
a few years, three sons were born, Thomas Roger in Philadelphia on July 28, 1902, Hugh
in Austin, Texas, on April 17, 1904 and Raymond Braislin in Philadelphia on May 5,
1910. In 1908, the young couple purchased the house on Buzzards Bay Avenue now
owned by Norman T. Allen. No one could have foreseen then how closely the family
was to become associated with the Woods Hole scientific community !

Professor Montgomery died of pneumonia on March 19, 1912, and his widow had
to sell the Woods Hole property and go to work to support her family. At the Uni-
versity of Pennsylvania, Mrs. Montgomery worked as an assistant to the editor of the
Journal of Biological Chemistry, Professor A. N. Richards. In 1915, she had a fellow-
ship in Dr. McClung's laboratory to enable her to devote more time to work toward an
advanced degree — work she was never able to complete. She next taught at Vassar for
a year and then became an assistant professor at the University of Maine for the aca-
demic year 1918-1919. While there she taught vertebrate anatomy, histology and
embryology, as well as undertaking some original studies of her own on the embryology
of the chick.

Coming from a family of teachers, one might have supposed that Mrs. Montgomery
would have continued in the family tradition, yet she had a drive and a lack of patience
which made her temperamentally unsuited for such a career. Thus, in the fall of 1919,
she became the "resident assistant librarian in immediate charge of the library" at the
Laboratory, and in 1925, she was made Librarian.

It was due mainly to Mrs. Montgomery's respect for, and understanding of, research
that the Library grew to its present stature. When she began in 1919 the entire Library
had an annual budget of $2000 and was housed in Room 217 of the Crare Building.
However, Mrs. Montgomery felt that she was assisting the work of the laboratory in a
very real way and consequently her ambition for the Library knew no bounds. With
persistence and the continuing help and interest of Dr. Frank R. Lillie and others, she
accumulated an enviable collection, today consisting of some 75,000 volumes.

Although not a trained librarian, she knew the scientists' needs and developed an
original, yet practical, system for filing journals, and at the same time expanded the
reprint collection for circulation so that the journals themselves could always be available
in the Library. On Mrs. Montgomery's retirement at the end of 1947, she had achieved
her goal for building an outstanding library, which will long stand as a fitting tribute
to her memory.

10 MARINE BIOLOGICAL LABORATORY

The building of her home on Whitman Road in 1923 marked the end of the years
of her great personal struggle. By then her sons' education as a future business man,
a doctor and a meteorologist-oceanographer were nearly completed. From then on she
had time for sociability as well as work and she will be remembered for her genuine
hospitality by many of the summer visitors at the Laboratory from this country and
abroad.

1. THE STAFF, 1956

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

SENIOR STAFF OF INVESTIGATION
A. P. MATHEWS, Professor of Biochemistry, Emeritus, University of Cincinnati

ZOOLOGY

I. CONSULTANTS

F. A. BROWN, JR., Professor of Zoology, Northwestern University
LIBBIE H. HYMAN, American Museum of Natural History
A. C. REDFIELD, Woods Hole Oceanographic Institution

II. INSTRUCTORS

THEODORE H. BULLOCK, Associate Professor of Zoology, University of California, Los

Angeles, in charge of course

JOHN H. LOCHHEAD, Professor of Zoology, University of Vermont
NORMAN A. MEINKOTH, Associate Professor of Zoology, Swarthmore College
GROVER STEPHENS, Assistant Professor of Zoology, University of Minnesota
JOHN M. ANDERSON, Associate Professor of Zoology, Cornell University
HOWARD A. SCHNEIDERMAN, Assistant Professor of Zoology, Cornell University
MARTIN W. JOHNSON, Professor of Marine Biology, Scripps Inst. of Oceanography,

University of California, La Jolla
MORRIS ROCKSTEIN, Department of Physiology, New York University, Bellevue Medical

Center

III. LABORATORY ASSISTANTS

CHARLES H. BAXTER, University of California, Los Angeles
KENT CHRISTENSEN, Harvard University

EMBRYOLOGY

I. INSTRUCTORS

MAC V. EDDS, JR., Associate Professor of Biology, Brown University, in charge of course

PAUL B. WEISZ, Associate Professor of Biology, Brown University

NELSON T. SPRATT, JR., Professor of Zoology, University of Minnesota

J. P. TRINKAUS, Assistant Professor of Zoology, Yale University

EDGAR ZWILLING, Associate Professor of Genetics, University of Connecticut

II. LABORATORY ASSISTANT
ROBERT G. BEARD, Indiana University

REPORT OF THE DIRECTOR 11

PHYSIOLOGY

I. CONSULTANTS

MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania

OTTO LOEWI, Professor of Pharmacology, New York University, School of Medicine

ARTHUR K. PARPART, Professor of Biology, Princeton University

ALBERT SZENT-GYORGYI, Director, Institute for Muscle Research, Woods Hole

E. S. GUZMAN BARRON, Associate Professor of Biochemistry, University of Chicago

II. INSTRUCTORS

STEPHEN W. KUFFLER, Associate Professor of Ophthalmology, Wilmer Institute, Johns

Hopkins Medical School, in charge of course

FRANCIS D. CARLSON, Assistant Professor of Biophysics, Johns Hopkins University
BERNARD D. DAVIS, Professor of Pharmacology, New York University, College of

Medicine

ERIK ZEUTHEN, Lecturer, Laboratory of Zoophysiology, University of Copenhagen
RAYMOND E. ZIRKLE, Professor of Radiobiology, University of Chicago
HERMAN M. KALCKAR, National Institutes of Health

MAX A. LAUFFER, Professor and Head of Dept. of Biophysics, University of Pittsburgh
ANDREW SZENT-GYORGYI, Independent Investigator, The Institute for Muscle Research

III. LABORATORY ASSISTANT
PAUL BERNSTEIN, Department of Zoology, Columbia University

BOTANY

I. CONSULTANT

WM. RANDOLPH TAYLOR, Professor of Botany, University of Michigan

II. INSTRUCTORS

HAROLD C. BOLD, Professor of Biology, Vanderbilt University, in charge of course

ROBERT W. KRAUSS, University of Maryland

PAUL C. SILVA, Assistant Professor of Botany, University of Illinois

III. LABORATORY ASSISTANT
RAYMOND A. GALLOWAY, University of Maryland

IV. COLLECTOR
GINA ARCE, Vanderbilt University

MARINE ECOLOGY

I. CONSULTANT
ALFRED C. REDFIELD, Woods Hole Oceanographic Institution

12 MARINE BIOLOGICAL LABORATORY

II. INSTRUCTORS

BOSTWICK H. KETCHUM, Marine Microbiologist, Woods Hole Oceanographic Institu-
tion, in charge of course

EDWIN T. MOUL, Assistant Professor of Botany, Rutgers University
CHARLES JENNER, Associate Professor of Zoology, University of North Carolina

III. ASSISTANT
RUDOLF SCHELTEMA, George Washington University

THE LABORATORY STAFF, 1956
HOMER P. SMITH, General Manager

MRS. DEBORAH LAWRENCE HARLOW, ROBERT KAHLER, Superintendent,

Librarian Buildings and Grounds

CARL SCHWEIDENBACH, Manager of ROBERT B. MILLS, Manager, De-
Supply Department partment of Research Service

GENERAL OFFICE

IRVINE L. BROADBENT

POLLY L. CROWELL NANCY SHAVE

MRS. LILA MYERS GEORGIANA MARKS

LIBRARY

MARY E. CASTELLANO, Assistant Librarian
MARY A. ROHAN NAOMI BOTELHO

ALBERT NEAL

MAINTENANCE OF BUILDINGS AND GROUNDS

ROBERT ADAMS JOHN HEAD

EDMOND BOTELHO GEORGE A. KAHLER

ARTHUR CALLAHAN DONALD B. LEHY

ROBERT GUNNING ALTON J. PIERCE

JAMES S. THAYER

DEPARTMENT OF RESEARCH SERVICE

GAIL M. CAVANAUGH SEAVER HARLOW

JOHN P. HARLOW PATRICIA PHILPOTT

SUPPLY DEPARTMENT

RUTH S. CROWELL GEOFFREY LEHY

MILTON B. GRAY ROBERT O. LEHY

WALTER E. KAHLER JAMES MC!NNIS

ROBERT PERRY BRUNO TRAPASSO

PATRICIA M. CONWAY H. S. WAGSTAFF

REPORT OF THE DIRECTOR 13

2. INVESTIGATORS, LALOR AND LILLIE FELLOWS, AND STUDENTS

Independent Investigators, 1956

ABBOTT, ROBINSON S., Assistant Professor of Botany, Cornell University
ADELSON, LIONEL M., Research Associate, National Agricultural College
ALLEN, ROBERT DAY, Assistant Professor of Zoology, University of Michigan
ALSCHER, RUTH P., Associate Professor, Manhattanville College

AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland School of Medicine
ANDERSON, JOHN MAXWELL, Associate Professor of Zoology, Cornell University
ARMSTRONG, PHILIP B., Professor of Anatomy, State Univ. of New York, College of Medicine
ARNOLD, WILLIAM A., Scientific Investigator, Oak Ridge National Laboratory
BANG, FREDERICK B., Professor of Pathobiology, Johns Hopkins University School of Medicine
BENNETT, MIRIAM F., Instructor of Biology, Sweet Briar College
BENESCH, REINHOLD, Marine Biological Laboratory, Woods Hole, Massachusetts
BERGER, CHARLES A., Chairman, Department of Biology, Fordham University
BLUM, HAROLD F., Physiologist, Princeton University
BOETTIGER. EDWARD G., Associate Professor, University of Connecticut
BOLD, HAROLD C, Vanderbilt University

BRADY, ROSCOE O., National Institute of Neurological Diseases and Blindness
BRIDGMAN, ANNA J., Professor of Biology, Agnes Scott College

BROWN, FRANK A., JR., Chairman, Dept. of Biological Sciences, Northwestern University
BRYANT, S. H., Instructor of Pharmacology, University of Cincinnati, College of Medicine
BULLOCK, THEODORE H., Professor of Zoology, University of California at Los Angeles
BUTLER, ELMER G., Professor of Zoology, Princeton University
CAMPBELL, MILDRED A., Instructor of Zoology, Smith College

CARLSON, FRANCIS D., Assistant Professor of Biophysics, Johns Hopkins University
CHAET, ALFRED B., Instructor in Zoology, University of Maine
CHANG, JOSEPH J., Instructor, Department of Biology, Brown University
CHASE, AURIN M., Associate Professor of Biology, Princeton University
CHENEY, RALPH HOLT, Professor of Biology, Brooklyn College
CLAFF, C. LLOYD, Research Associate in Surgery, Harvard Medical School
CLARK, ELLIOT R., Professor Emeritus of Anatomy, University of Pennsylvania
CLEMENT, A. C., Associate Professor of Biology, Emory University
CLOWES, G. H. A., Research Director Emeritus, Lilly Research Laboratories
COHEN, MELVIN J., Instructor in Biology, Harvard University
COHEN, SEYMOUR S., Professor of Biochemistry, University of Pennsylvania
COLE, KENNETH S., Chief, Laboratory of Biophysics, National Institutes of Health
COLWIN, ARTHUR L., Associate Professor and Lecturer, Queens College
COLWIN, LAURA H., Queens College

COOPERSTEIN, SHERWIN J., Associate Professor of Anatomy, Western Reserve University Medi-
cal School
COSTELLO, DONALD P., Kenan Professor of Zoology and chairman of the Department, University

of North Carolina

COWGILL, ROBERT W., Instructor in Biochemistry, University of California, Berkeley
CROWELL, SEARS, Assistant Professor of Zoology, Indiana University
CSAPO, A., Rockefeller Institute for Medical Research
DAN, JEAN CLARK, Misaki Marine Biological Station, Japan

DAVIS, BERNARD D., Professor and Chairman of Pharmacology, New York University College
of Medicine

DODGE, FREDERICK A., JR., Graduate Fellow, Rockefeller Institute for Medical Research

DWYER, JOHN D., Director, Department of Biology, Saint Louis University

EDDS, MAC V., JR., Associate Professor of Biology, Brown University

EISEN, HERMAN N., Professor of Medicine, Washington University School of Medicine

ELLIOTT, ALFRED M., Professor of Zoology, University of Michigan

FiTzHuGH, RICHARD, Biophysicist, National Institutes of Health

FREYGANG, WALTER H., S. A. Surg. (R), U. S. Public Health Service

14 MARINE BIOLOGICAL LABORATORY

GOSSELIN, ROBERT E., Assistant Professor of Pharmacology, University of Rochester, School

of Medicine

GREEN, JAMES W., Associate Professor of Physiology, Rutgers University
GREEN, MAURICE, Instructor of Biochemistry, University of Pennsylvania
GREEN, PAUL B., Junior Fellow, Harvard University

GREIF, ROGER L., Associate Professor of Physiology, Cornell University Medical College
GROSCH, DANIEL S., Associate Professor of Genetics, North Carolina State College
GRUNDFEST, HARRY, Associate Professor of Neurology, College of Physicians and Surgeons
GUTTMAN, RITA, Assistant Professor of Biology, Brooklyn College
HAGERMAN, DWAIN D., Research Fellow, Harvard Medical School
HAGIWARA, S., Visiting Scientist, National Institutes of Health
HARVEY, ETHEL BROWNE, Research in Biology, Princeton University
HARVEY, E. NEWTON, Professor of Physiology, Princeton University
HAYASHI, TERU, Associate Professor of Zoology, Columbia University
HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College
HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania
HENLEY, CATHERINE, Research Associate, University of North Carolina
HERVEY, JOHN P., Electronic Engineer, Rockefeller Institute for Medical Research
HOLZ, GEORGE G., JR., Assistant Professor of Zoology, Syracuse University

HOLTZER, HOWARD, Assistant Professor of Anatomy, University of Pennsylvania Medical School
HOWARD, ROBERT S., Assistant Professor of Biological Sciences, University of Delaware
HUNTER, F. R., Assistant Professor, Roosevelt University

HYDE, BEAL B., Assistant Professor of Plant Sciences, University of Oklahoma
JACOBS, WILLIAM P., Associate Professor, Princeton University

JENKINS, GEORGE B., Professor Emeritus of Anatomy, George Washington University
JENNER, CHARLES E., Associate Professor of Zoology, University of North Carolina
JOHNSON, FRANK H., Associate Professor of Biology, Princeton University
JOHNSON, MARTIN W., Professor of Marine Biology, Scripps Institution of Oceanography
KALCKAR, HERMAN M., Visiting Scientist, National Institutes of Health

KARUSH, FRED, Associate Professor of Immunology, The Children's Hospital of Philadelphia
KAO, C. Y., Rockefeller Institute for Medical Research

KARLSON, PETER, Max-Planck Institut fur Biochemi, Tubingen, Western Germany
KEMP, NORMAN E., Assistant Professor of Zoology, University of Michigan
KEMPTON, RUDOLF T., Professor and Chairman, Department of Zoology, Vassar College
KEOSIAN, JOHN, Professor of Biology, Rutgers University

KIND, C. ALBERT, Assistant Professor of Biochemistry, University of Connecticut
KLEINHOLZ, L. H., Professor of Biology, Reed College

KLOTZ, IRVING M., Professor of Chemistry and Biology, Northwestern University
KOSTELLOW, ADELE B., Damon Runyon Fellow, Albert Einstein College of Medicine
KRAHL, MAURICE E., Professor of Physiology, University of Chicago
LANSING, ALBERT L, Professor of Anatomy, Emory University

LAUFFER, MAX A., Professor and Head of Dept. of Biophysics, University of Pittsburgh
LAVOIE, MARCEL E., Instructor in Zoology, University of New Hampshire
LAZAROW, ARNOLD, Professor and Head of Dept. of Anatomy, University of Minnesota
LEES, A. D., Principal Scientific Officer, Agricultural Research Council Unit of Insect Physi-
ology, England

LEHMANN, FRITZ E., Professor of Zoology, University of Berne, Switzerland
LEVINE, LAWRENCE, Instructor of Zoology, Wayne University
LEWIN, RALPH A., Grantee, National Institutes of Health
LOCHHEAD, JOHN H., Professor of Zoology, University of Vermont
LORAND, LASZLO, Assistant Professor of Chemistry, Northwestern University
LOVELACE, ROBERTA, Assistant Professor of Biology, University of South Carolina
LUBIN, MARTIN, Associate in Pharmacology, Harvard Medical School
LUMB, ETHEL SUE, Assistant Professor of Zoology, Vassar College
LYNCH, WILLIAM F., Professor of Biology, St. Ambrose College
MAAS, WERNER K., Assistant Professor of Pharmacology, New York University College of

Medicine
MARSHAK, ALFRED, Marine Biological Laboratory, Woods Hole, Massachusetts

REPORT OF THE DIRECTOR 15

MARSLAND, DOUGLAS. Professor of Biology, Washington Square College, New York University

MEINKOTH, NORMAN A., Associate Professor of Biology, Swarthmore College

MENKIN, VALY, Head of Experimental Pathology, Agnes Barr Chase Foundation, Temple

University

METZ, CHARLES B., Associate Professor of Zoology, Florida State University
MONROV, ALBERTO, Professor of Comparative Anatomy, University of Palermo, Italy
MOORE, JOHN W., Associate Chief, Laboratory of Biophysics, National Institutes of Health
MULLINS, L. J., Associate Professor of Biophysics, Purdue University
MUSACCHIA, X. J., Associate Professor of Biology, St. Louis University
NACE, PAUL FOLEY, Associate Professor of Anatomy, New York Medical College
NEEDLER, MARY E., Graduate Student and Demonstrator, University of Toronto
OSTERHOUT, W. J. V., Rockefeller Institute for Medical Research
PARKER, JOHNSON, Assistant Professor of Botany, University of Idaho

PARPART, ARTHUR K., Professor and Chairman, Department of Biology, Princeton University
PATERSON, MABEL, Instructor in Zoology, Vassar College
PERSON, PHILIP, Chief, Dental Research, V. A. Hospital
PIERCE, MADELENE E., Professor of Zoology, Vassar College
PLOUGH, HAROLD H., Professor of Biology, Amherst College
PROCTOR, NATHANIEL K., Professor of Biology, Morgan State College
PROSSER, C. LADD, Professor of Physiology, University of Illinois
RATLIFF, FLOYD, Rockefeller Institute for Medical Research
RAY, CHARLES, Assistant Professor of Biology, Emory University
REBHUN, LIONEL I., Instructor in Anatomy, University of Illinois
REED, CLARK P., School of Hygiene, Johns Hopkins University
ROCKSTEIN, MORRIS, Associate Professor of Physiology, New York University College of

Medicine

ROGERS, K. T., Assistant Professor of Zoology, Oberlin College
ROSENTHAL, THEODORE B., Associate Professor of Anatomy, Emory University
Rossi, HAROLD H., Associate Professor of Radiology, College of Physicians and Surgeons
ROTH, JAY S., Associate Professor of Biochemistry, Hahnemann Medical College
RUGH. ROBERTS, Associate Professor of Radiology, Columbia University
SAROFF, H. A., Scientist, USPHS, National Institutes of Health
SCHARRER, ERNST A., Professor and Chairman of Dept. of Anatomy, Albert Einstein College of

Medicine

SCHECHTER, VICTOR, Associate Professor of Biology, City College of New York
SCHNEIDERMAN, HOWARD A., Assistant Professor of Zoology, Cornell University
SCHUH, JOSEPH E., Assistant Professor of Biology, St. Peter's College

SCOTT, DWIGHT B. AlcNAiR, Assistant Professor of Biochemistry, University of Pennsylvania
SCOTT, SISTER FLORENCE M., Professor of Biology, Seton Hill College
SCOTT, GEORGE T., Professor of Zoology, Oberlin College
SENFT, ALFRED W., Falmouth Medical Associates
SHANES, ABRAHAM M., Physiologist, National Institutes of Health
SILVA, PAUL C., Assistant Professor of Botany, University of Illinois
SLIFER, ELEANOR H., Associate Professor of Zoology, State University of Iowa
SPEIDEL, CARL C., Professor and Chairman of Anatomy Dept., University of Virginia Medical

School

SPRATT, NELSON T., Professor of Zoology, University of Minnesota
SPYROPOULOS, COXSTANTINE, National Institutes of Health

STEARNS, RICHARD N., Instructor in Physiology, Albert Einstein College of Medicine
STEELE, RICHARD H., Visiting Investigator, Muscular Dystrophy Associations of America, Inc.
STEFANELLI, ALBERTO, Director, Universita di Rome
STEINBERG, MALCOLM S., Graduate Student, University of Minnesota
STEPHENS, GROVER C., Assistant Professor of Zoology, University of Minnesota
STUNKARD, HORACE W., Fishery Research Biologist, New York University
STURTEVANT, ALFRED H., Professor of Genetics, California Institute of Technology
SZENT-GYORGYI, ALBERT, Marine Biological Laboratory, Woods Hole, Mass.
SZENT-GYORGYI, ANDREW, Research Associate, Marine Biological Laboratory, Woods Hole, Mass.
TASAKI, I., Chief, Section on Special Senses, National Institutes of Health

16 MARINE BIOLOGICAL LABORATORY

TAYLOR, WILLIAM RANDOLPH, Professor of Botany, University of Michigan

TOKAY, ELBERT, Associate Professor of Physiology, Vassar College

TRINKAUS, J. P., Assistant Professor of Zoology, Osborn Zoological Laboratory, Yale University

TYLER, DAVID B., Professor of Pharmacology and Director of Department, University of Puerto

Rico, School of Medicine

URETZ, ROBERT B., Instructor in Biophysics, University of Chicago
DEViLLAFRANCA, GEORGE W., Assistant Professor of Zoology, Smith College
VILLEE, CLAUDE A., Assistant Professor of Biochemistry, Harvard Medical School
VINCENT, W. S., Instructor in Anatomy, Upstate Medical Center, State University of New York
WEBB, H. MARGUERITE, Assistant Professor of Physiology, Goucher College
WEISZ, PAUL B., Associate Professor of Biology, Brown University
WHITING, ANNA R., Lecturer in Zoology, University of Pennsylvania
WHITING, P. W., Professor of Zoology Emeritus, University of Pennsylvania
WICHTERMAN, RALPH, Professor of Biology, Temple University

WIERCINSKI, FLOYD J., Assistant Professor of Physiology, Hahnemann Medical College
WILBER, CHARLES G., Chief, Comparative Physiology Branch, Army Chemical Center
WILLEY, C. H., Chairman, Department of Biology, New York University, University College
WILSON, T. G., Research Associate, Princeton University
WILSON, WALTER L., Assistant Professor of Physiology and Biophysics, College of Medicine,

University of Vermont

WOOTTON, DONALD M., Woods Hole, Mass.
WITTENBERG, JONATHAN B., Assistant Professor of Biochemistry, Albert Einstein College of

Medicine

ZEUTHEN, ERIK, Lecturer, University of Copenhagen

ZIMMERMAN, ARTHUR M., Research Associate, Washington Square College, New York Uni-
versity

ZIRKLE, RAYMOND E., Professor of Radiology, University of Chicago

ZWEIFACH, B. W., Associate Professor of Pathology, New York University College of Medicine
ZWILLING, EDGAR, Associate Professor of Genetics, University of Connecticut

Beginning Investigators, 1956

AMENTA, PETER S., Pre-doctoral Student, University of Chicago

BAXTER, CHARLES H., Teaching Assistant, University of California at Los Angeles

BEARD, ROBERT G., Graduate Student, Indiana University

DAVIDSON, MARGARET E., Demonstrator-Curator in Zoology, McGill University

FRIZ, CARL T., Research Assistant, University of Minnesota

HONEGGER, CAROL M., Graduate Student, University of Pennsylvania

KANE, ROBERT EDWARD, Graduate Student, Johns Hopkins University

KURAHASHI, KIYOSHI, Research Fellow, National Institutes of Health

LARIS, PHILIP C., Graduate Student, Princeton University

LEVIN, CLINTON N., Student, New York University College of Medicine

Moos, CARL, Research Associate, Northwestern University

MORRILL, JOHN B., Graduate Student, Florida State University

NEWMAN, ANNA E., Research Fellow, Western Reserve University

PUGNO, SANDRA L., Post-doctoral Fellow, Yale University

REIMER, STANLEY M., Graduate Student, Rutgers University

STEVENSON, J. Ross, Graduate Student, Northwestern University

TAYLOR, ROBERT E., Research Physiologist, National Institutes of Health

TUCKER, MARIE, Graduate Student, University of Illinois

TUNIK, BERNARD D., Graduate Student, Columbia University

WHEAT, ROBERT W., Research Fellow, National Institutes of Health

Research Assistants, 1956

ADAMS, TERRY, 86 Chestnut Street, Boston 8, Massachusetts

ARCE, GINA, Vanderbilt University

BAIRD, SPENCER, Institute for Muscle Research

EARNER, HAZEL, Children's Hospital of Philadelphia

REPORT OF THE DIRECTOR 17

BERNSTEIN, PAUL W., New York University College of Medicine

BROWN, ROBERT A., Northwestern University

CAGLE, JULIEN, Princeton University

CRANSTON, MARGARET B., Radcliffe Graduate School

CULLERTON, JOHN M., University of Pennsylvania

DAVIS, ROGER E., University of Wisconsin

DRAKE, JOHN W., California Institute of Technology

ERDMAN, HOWARD E., North Carolina State College

FINCK, HENRY, University of Pennsylvania School of Medicine

GORMAN, DONALD J., New York University Medical School

GREENLEES, JANET, Rutgers University

HARSCH, MARY, Rutgers University

HOCHGRAF, HELEN, Smith College

KARREMAN, GEORGE, Research Associate, Institute for Muscle Research

KOGEL, JOAN, Albert Einstein College of Medicine

KRAMER, ALAN D., New York University School of Medicine

KURLAND, CHARLES G., Cornell University

LAMB, GEORGE A., State University of New York Medical School at Syracuse

LANGER, IRA J., State University of New York Medical School at Syracuse

LAURIE, JOHN S., Johns Hopkins University

LEVINE, LENORE S., New York University School of Medicine

LIGHTEN STEIN, JANET, Children's Hospital of Philadelphia

LOEB, TIMOTHY, Reed College

MCLAUGHLIN, JANE A., Institute for Muscle Research

MATHESON, GAIL E., Yale University

MIDDLEBROOK, W. R., Institute for Muscle Research

MINGIOLI, ELIZABETH S., New York University College of Medicine

PALMER, ROGER FARLEY, Florida State University

PHILPOTT, DELBERT E., Research Associate, Institute for Muscle Research

OBERLANDER, MARCIA I., State University of New York College of Medicine at Syracuse

OUTKA, DARRYLL E., University of California

PFLUEGER, OTTO H., Reed College

REICH, MELVIN, Rutgers University

ROSENTHAL, ELIZABETH, National Institutes of Health

Ross, SAMUEL M., Brooklyn College

ROWE, EDWARD C, University of Michigan

SHRINER, JOAN, Northwestern University

SIE, EDWARD, Princeton University

SMILEY, SHELDON, State University of New York

SMITH, ZOE HOLLINGSWORTH, North Carolina State College

SPERELAKIS, NICK, University of Illinois

STRAUSS, PAUL H., New York University College of Medicine

STRICKHOLM, ALFRED, University of Minnesota

SZENT-GYORGYI, EVA, Institute for Muscle Research

SZENT-GYORGYI, MARTA, Institute for Muscle Research

VOZICK, MICHAEL W., Columbia University

Library Readers, 1956

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

BENTLEY, RONALD, Assistant Professor of Biochemistry, University of Pittsburgh

BODANSKY, OSCAR, Chief, Clinical Biochemistry, Sloan-Kettering Institute

BRAITENBERG, VALENTINE, Yale Medical School

CHACE, JOHN A., Kirksville College of Osteopathy and Surgery

DEANE, HELEN W., 387 Harvard Street, Cambridge 38, Massachusetts

DIXON, F. J., JR., Professor and Chairman Department of Pathology, University of Pittsburgh

School of Medicine
DORFMAN, ALBERT, Associate Professor of Pediatrics, University of Chicago

18 MARINE BIOLOGICAL LABORATORY

FREUND, JULES, Chief, Division of Immunology, The Public Health Research Institute of New

York

FRIES, E. F. B., Associate Professor, The City College of New York
GABRIEL, MORDECAI L., Associate Professor of Biology, Brooklyn College
GINSBERG, HAROLD S., Associate Professor of Preventive Medicine, Western Reserve University,

School of Medicine

GLASS, H. BENTLEY, Professor of Biology, Johns Hopkins University
GLASSER, RICHARD L., University of Maryland

GOLDMAN, STANFORD, Professor of Electrical Engineering, Syracuse University
GOLDTHWAIT, DAVID A., Senior Clinical Instructor in Medicine, Western Reserve University
GRANT, PHILIP, Research Associate, Department of Embryology, Institute for Cancer Research
GUDERNATSCH, FREDERICK, Cornell University Medical College

GUREWICH, VLADIMIR, Associate Visiting Physician, Bellevue and Metropolitan Hospitals
HERBERT, EDWARD, Instructor in Biology, Massachusetts Institute of Technology
JACOBS, M. H., Professor Emeritus of General Physiology, University of Pennsylvania
KABAT, ELVIN A., Professor of Microbiology, Columbia University
KOHN, ROBERT R., Research Fellow, Benjamin Rose Hospital

LEIN, ALLEN, Associate Professor of Physiology, Northwestern University Medical School
LEVY, ARTHUR L., Research Biochemist, St. Vincent's Hospital
LEVINE, RACHMIEL, Chairman, Department of Medicine, Michael Reese Hospital
LING, GILBERT N., Assistant Professor of Neurophysiology, University of Illinois
LOEWI, OTTO, Research Professor of Pharmacology, New York University-Bellevue Medical

Center

LOVE, Lois H., Research Associate, National Research Council

MCDONALD, SISTER ELIZABETH, Chairman, Department of Biology, College of Mt. St. Joseph
PICK, JOSEPH, Associate Professor in Anatomy, New York Umversity-Bellevue Medical Center
RENNIE, DONALD W., Research Fellow, Harvard University School of Medicine
ROOT, WALTER S., Professor of Physiology, College of Physicians and Surgeons
ROSE, S. MERYL, Professor of Zoology, University of Illir.cis

RUBIN, SAUL H., Director, Pharmaceutical and Biochemical Research, Hoffmann-La Roche
SCOTT, ALLAN, Chairman, Department of Biology, Colby College
SCOTT, THOMAS F. M., Research Professor of Pediatrics, Children's Hospital
STOCKARD, ALFRED H., Professor of Zoology, University of Michigan
SULKIN, S. EDWARD, Professor and Chairman, Department of Microbiology, University of Texas

Southwestern Medical School

SWIFT, HEWSON, Associate Professor of Zoology, University of Chicago
TEAS, HOWARD J., Plant Physiologist, Federal Experiment Station, Mayaguez, Puerto Rico
TRURNIT, HANS J., Member of the Scientific Staff, R. I. A. S., Baltimore
TYLER, ALBERT, Professor of Embryology, California Institute of Technology
VEIS, ARTHUR, Research Chemist, Armour and Company
VISHNIAC, WOLF, Assistant Professor of Microbiology, Yale University
WAINIO, WALTER W., Associate Professor of Biochemistry, Rutgers University
WATERMAN, ALLYN J., Professor of Biology, Williams College
WTARNER, ROBERT C., Associate Professor of Biochemistry, New York University College of

Medicine

WHEELER, GEORGE E., Instructor in Biology, Brooklyn College
YNTEMA, CHESTER L., Professor of Anatomy, State University of New York College of Medicine

Lalor Fellows, 1956

S. H. BRYANT
M. COHEN
M. GREEN
P. GROSS
D. HAGERMAN
P. KARLSON
L. LEVINE
A. LEES
W. MAAS
L. REBHUN

REPORT OF THE DIRECTOR 19

Lillie Fellow, 1956

A. STEFAXELLI

Students, 1956

BOTANY

ADAIR, ELIZABETH J., Yale University
ANDERSON, ROBERT G., University of Nebraska
CORNETT, MARGARET E., Radcliffe College
DICK, STANLEY, Brooklyn College
FELITTI, VINCENT J., Dartmouth College
FORBES, PATRICIA R., Acadia University
KORN, ROBERT W., Marquette University
LAURENCOT, HENRY J., JR., Fordham University
LIND, ELIZABETH A., Wellesley College
McLEOD, GUY C, Waquoit, Massachusetts
POSEY, JANET A., West Virginia University
SMALLEY, ALFRED E., University of Georgia
SPYRIDES, GEORGE J., Dartmouth College
TALAMO, RICHARD CHARLES, Harvard University
WARD, VERNON U., Dartmouth College
WHITTIER, DEAN PAGE, University of Massachusetts
WILBOIS, ANNETTE D., Indiana University
WILLSON, DAN L., University of Oklahoma

EMBRYOLOGY

ABBOTT, JOAN, Washington University

BABCOCK, RICHARD G., University of Michigan

BRICE, MARTHA C., Oberlin College

BULL, ALICE LOUISE, Wellesley College

BURKE, JOSEPH A., S.J., Fordham University

CHAUBE, SHAKUNTALA, Osborn Biological Laboratory

COLEMAX, JOHN R., Indiana University

DECOSTA, AlARi J., Radcliffe College

FILOSA, MICHAEL F., Princeton University

FULTON, CHANDLER M., Brown University

GLASSER, JAY H., University of Connecticut

HINSCH, GERTRUDE W., Iowa State College

HOLLENBACK, JAMES G., Marquette University

HUVER, CHARLES W., University of Wisconsin

KATOH, ARTHUR K., University of Illinois

KINYOX, NANCY, Northwestern University

LAUFER, HANS, Cornell University

MCDOWELL, JAMES W., Dartmouth College

OUTTEX, LORA M., Cornell University

PAUL, SISTER CLARENCE, St. John's University

RAFF, NEIL C., Amherst College

RAY, FRAXCES L., Columbia University

ROBIXSOX, JAMES ALAX, Wesleyan University

ROTHMAX, MAXIXE, Indiana University

RUGGIERI, GEORGE D., S.J., St. Louis University

STEINMULLER, DAVID, Swarthmore College

TSAI, LIE SHA, Yale University

TWEEDELL, JOAN E. WERBER, University of Maine

PHYSIOLOGY

BADE, MARIA L., Omaha, Nebraska

BAUMAN, NORMAN, New York University College of Medicine

BRAND, EUGENE D., University of Virginia

20 MARINE BIOLOGICAL LABORATORY

CAMPBELL, JAMES W., University of Oklahoma

CHRISTIENSEN, ALBERT K., Harvard University

CLARK, ELOISE E., University of North Carolina

EVANS, DAVID R., Johns Hopkins University

GOLDSTEIN, JUDITH H., Harvard University

GONZALEZ, MARIA, University of Sao Paulo, Brazil

GORDON, MARIA F., Cox Institute, Pennsylvania University

GREENE, LEWIS J., Rockefeller Institute for Medical Research

GROSS, RUTH T., Stanford University Medical School

KIM, SOON WON, Columbia University

KOSTYO, JACK L., Cornell University

LENHERT, PAUL G., Johns Hopkins University

NEWMAN, ANNA E., Western Reserve University

PAIR, WOON Ki, Dalhousie University

PUGNO, SANDRA L., Osborn Zoological Laboratory

RUARK, MARGARET A., Yale University

SCHOOLEY, CAROLINE N., University of California

SCHOOLEY, JOHN C, University of California

SCHWARTZ, JAMES H., New York University College of Medicine

SMITH, GENEVA A., Mount Holyoke College

TEMIN, HOWARD M., California Institute of Technology

THEIS, ROGER ELLIOT, Harvard University

TING, ROBERT C. Y., Amherst College

WILT, FRED H., Indiana University

YAMAMOTO, ROBERT T., University of Illinois

YOUNG, ROBERT R., Yale University

ZORZOLI, ANITA, Vassar College

INVERTEBRATE ZOOLOGY

ANDREWS, FRED B., Indiana University

BAREN, CARL F., Brooklyn College

BARTOL, ROBERTA B., Dunbarton College

BINGHAM, EULA L., University of Cincinnati

BISHOP, ALISON, Cornell University

BOOHAR, RICHARD K., Drew University

BURGER, CHARLES L., University of Illinois

CHAMBERLAIN, NORMAN A., University of North Carolina

CLARK, GEORGE A., Amherst College

COLLIER, NANCY V., Goucher College

DOUGLAS, DONALD, Oberlin College

DOWLING, RICHARD A., State University of Iowa

EIGER, JOAN V., Harvard University

ELLIS, JOHN F., Amherst College

FIORE, CARL, Fordham University

FLEISCHMAN, JULIAN B., Harvard University

FORD, ELIZABETH, Washington University

FRIEDL, FRANK EDWARD, University of Minnesota

GOLDBERG, MORTON F., Harvard College

GOUDSMIT, ESTHER M., University of Michigan

GUIGNON, ERNEST F., Washington and Jefferson College

HAMMER, BEVERLY A., Randolph-Macon Woman's College

HAMMONDS, JOANNE, Chatham College

HARMON, WALLACE, Syracuse University

HARMAN, WALTER J., University of Illinois

HENDRICKS, FREDERICK B., DePauw University

HICHAR, JOSEPH K., Harvard University

HOLMES, WILLIAM F., University of Pennsylvania

HOLT, KATHLEEN, Western Maryland College

REPORT OF THE DIRECTOR 21

HUBER, IVAN, University of Maryland
JOLINE, LAURENCE T., Washington University
KANWISHER, JOAN T., Woods Hole, Mass.
KARAKASHIAN, STEPHEN, Drew University
KIRCHEN, ROBERT V., University of Michigan
KOUKIDES, MELPOMENI, University of Pennsylvania
LASSEN, IDA, Elmira College
LONDON, ABRAM M., Harvard University
McNAB, BRIAN K., University of Wisconsin
MOFFAT, GRACE H., City College of New York
MORROW, CYNTHIA J., Tufts College
PLUMB, MARY E., Vassar College
PURPLE, RICHARD, Hamilton College
RABINOWITCH, VICTOR, University of Illinois
REPPERT, JERE ANNE, Goucher College
ROBERTSON, ROBERT, Harvard University
SCHAEFER, CARL W., II, Oberlin College
SCHEUING, MARILYN R., State Medical School
SPOCK, MICHAEL, Antioch College
SULLIVAN, HELEN M., Marquette University
TAUB, STEPHEN, Indiana University
TAYLOR, ROBERT E., University of Delaware
TEFFT, EDWIN R., Fordham University
TESTER, RUTH E., Hunter College
WHARTON, THALIA J., Mount Holyoke College
YESAIR, DAVID W., Cornell University

ECOLOGY

FERSAHL, SISTER JOHN BAPTIST, Fordham University

FOSTER, WALTER S., Colby College

GATES, JOHN O., Cornell University

GILBERT, ANN C., Columbia University

HIRCHINSON, VIVIENNE, Mount Holyoke College

JONES, SARAH R., Connecticut College

STRELECKI, RAYMOND F., Drew University

VAN DYK, N. JOANNE, University of New Hampshire

WEINSTOCK, AMMON, Brandeis University

3. FELLOWSHIPS AND SCHOLARSHIPS, 1956

Arsenious Boyer Fellowship :

FATHER WM. LYNCH

Lucretia Crocker Scholarship :

ROBERT ANDERSON, Botany Course

N. JOANNE VAN DYK, Ecology Course

The Gary N. Calkins Scholarship :

ROBERT ROBERTSON, Invertebrate Zoology Course

The Edwin Grant Conklin Scholarship :

HANS LAUFER, Embryology Course

Emma Coote Drew Scholarship :

ELOISE CLARK, Physiology Course
Bio Club Scholarship :

GRACE MOFFATT, Invertebrate Zoology Course

The Edwin Linton Scholarship :

ERNEST GUIGNON, Invertebrate Zoology Course

22

MARINE BIOLOGICAL LABORATORY

4. TABULAR VIEW OF ATTENDANCE, 1952-1956

1952

INVESTIGATORS — TOTAL 306

Independent 172

Under Instruction 38

Library Readers 49

Research Assistants 47

STUDENTS — TOTAI 123

Zoology 55

Embryology 23

Physiology 27

Botany 11

Ecology 7

TOTAL ATTENDANCE 429

Less persons represented as both students and inves-
tigators 2

427

INSTITUTIONS REPRESENTED — TOTAL 149

By investigators 92

By students 57

SCHOOLS AND ACADEMIES REPRESENTED

By investigators 1

By students 3

FOREIGN INSTITUTIONS REPRESENTED

By investigators 7

By students 2

1953

310

176

37

46

51

136

55

30

31

11

9

446

446

155
90
65

15
6

1954

208

180

20

52

46

134

56

29

28

12

9

432

5

427

136

104

32

2

1

11
13

1955

250

162

9

54

25

148

56

30

30

19

13

398

398

129

95
34

3

9

5. COOPERATING AND SUBSCRIBING INSTITUTIONS, 1956
Cooperating Institutions

Amherst College

American Cancer Society

American Philosophical Society

Brooklyn College

Brown University

Bryn Mawr College

California Institute of Technology

Children's Hospital of Philadelphia

City College of New York

Colby College

College of Mt. St. Joseph on the Ohio

Columbia University

Columbia University, College of Physicians

and Surgeons
Cornell University
Cornell University Medical School
Duke University
Elmira College
Emory University
Florida State University
Fordham University
Grass Foundation
Hahnemann Medical College
Harvard University
Harvard University Aledical School

1956

304

184

20

50

50

140

55

28

30

18

9

444

2

442

130

97
33

3
1

9
6

Indiana University

Institute for Cancer Research

Institute for Muscle Research

Johns Hopkins University

Johns Hopkins University Medical School

Lalor Foundation

Eli Lilly and Company

Marquette University

Morgan State College

Mount Holyoke College

National Institutes of Health

National Science Foundation

New York University — Heights

New York University, College of Medicine

New York University — Washington Square

College

North Carolina State College
Northwestern University
Oberlin College
Office of Naval Research
Princeton University
Public Health Institute of New York
Rockefeller Foundation
Rockefeller Institute for Medical Research
Rutgers University

REPORT OF THE DIRECTOR

23

Saint Louis University

Sloan-Kettering Institute

Southwestern Medical College

State University of Iowa

State University of New York, College of

Medicine, at Syracuse
Syracuse University
Temple University
Tufts College
University of Chicago
University of Connecticut
University of Illinois

University of Maryland School of Medicine
University of Michigan
University of Minnesota

University of Pennsylvania

University of Pennsylvania Medical School

University of Pittsburgh

University of Rochester

University of Virginia, School of Medicine

University of Wisconsin

Vassar College

Washington University

Washington and Jefferson College

Wellesley College

Wesleyan University

Western Reserve University

Yale University

Yale University Medical School

Subscribing Institutions

Acadia University

Armour and Company

Brandeis University

Chatham College

City College of New York

Drew University

Albert Einstein College of Medicine

Ethicon Corporation

Falmouth Medical Associates

Goucher College

Hamilton College

House of Good Samaritan

Hunter College

Massachusetts Institute of Technology

National Agricultural College

Purdue University

Michael Reese Hospital

Smith College

University of California

University of Florida

University of Maine

University of Massachusetts

University of New Hampshire

University of Oklahoma

University of Puerto Rico

University of Vermont

Veterans Administration Hospital

Washington University School of Medicine

6. EVENING LECTURES, 1956

June 2')

DANIEL MAZIA "Processes in cell reproduction"

July 6

DONALD R. GRIFFIN "Listening in the dark"

July 13

HARRY GRUNDFEST "The different kinds of electrical responses

and their significance to the organism"
July 20

VINCENT DU VIGNEAUD "The posterior pituitary hormones"

July 27

W. D. MCELROY "Recent developments in the biochemistry

of light emission"
August 3

F. E. LEHMANN "Cytoplasmic organization and develop-
mental physiology of the egg of Tubifex"
August 10

V. B. WIGGLESWORTH "The insect cuticle"

August 17

ALBERTO STEFANELLI "The life cycle of neurons"

August 24

KEITH R. PORTER "The submicroscopic morphology of proto-
plasm"

24 MARINE BIOLOGICAL LABORATORY

7. TUESDAY EVENING SEMINARS, 1956

July 10
JOAN WOLFF and ROBERTS RUCH "The relation of gonad hormones to x-ir-

radiation sensitivity in mice"
PAUL R. GROSS "Amphibian yolk : chemistry and ultrastruc-

ture"
SYLVAN NASS "Amphibian yolk : the phosphoprotein phos-

phatase system"

8. MEMBERS OF THE CORPORATION, 1956
1. LIFE MEMBERS OF THE CORPORATION

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

DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut

DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota

GOLDFORB, 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

MACNAUGHT, MR. FRANK M., Woods Hole, Massachusetts

MALONE, DR. E. F., 6610 North llth Street, Philadelphia 26, Pennsylvania

MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts

MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Missouri

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

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
ADDISON, DR. W. H. F., 286 East Sidney Avenue, Mount Vernon, New York
ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine and Den-
tistry, Rochester, New York

ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota
ALLEN, DR. M. JEAN, Dept. of Biology, Wilson College, Chambersburg, Pennsyl-
vania
ALLEN, DR. ROBERT D., Dept. of Biology, Princeton University, Princeton, New

Jersey

ALSCHER, DR. RUTH, Dept. of Physiology, Manhattanville College, Purchase,
New York

REPORT OF THE DIRECTOR 25

AMBERSON, DR. WILLIAM R., Dept. of Physiology, University of Maryland School

of Medicine, Baltimore, Maryland

ANDERSON, DR. J. M., Dept. 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., c/o Dr. A. Lurff, Institut Pasteur, 28 Rue du Dr. Roux,

Paris XVe, France
ARMSTRONG, DR. PHILIP B., State University of New York College of Medicine,

Syracuse 10, New York

ATWOOD, DR. KIMBALL C., 68% Outer Drive, Oak Ridge, Tennessee
AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts
AYERS, DR. JOHN C., Dept. of Zoology, University of Michigan, Ann Arbor,

Michigan
BAITSELL, DR. GEORGE A., Osborn Zoological Laboratory, Yale University, New

Haven, Connecticut

BAKER, DR. H. B., Zoological Laboratory, University of Pennsylvania, Philadel-
phia, Pennsylvania
BALL, DR. ERIC G., Dept. of Biological Chemistry, Harvard University Medical

School, Boston 15, Mass.
BANG, DR. F. B., Dept. of Pathobiology, Johns Hopkins University School of

Hygiene, Baltimore 5, Maryland

BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire
BARD, DR. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland
BARRON, DR. E. S. G., Dept. of Medicine, University of Chicago, Chicago, 111.
EARTH, DR. L. G., Dept. of Zoology, Columbia University, New York City, New

York

BARTLETT, DR. JAMES H., Dept. of Physics, University of Illinois, Urbana, Illinois
BEAMS, DR. HAROLD W., Dept. of Zoology, State University of Iowa, Iowa City,

Iowa
BECK, DR. L. V., Dept. of Physiology and Pharmacology, University of Pittsburgh

School of Medicine, Pittsburgh 13, Pennsylvania
BEERS, DR. C. D., Dept. of Zoology, University of North Carolina, Chapel Hill,

North Carolina

BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana
BENNETT, DR. MIRIAM F., Dept. of Biology, Sweet Briar College, Sweet Briar,

Virginia
BERNSTEIN, DR. MAURICE, Virus Laboratory, University of California, Berkeley 4,

California

BERTHOLF, DR. FLOYD M., College of the Pacific, Stockton, California
BEVELANDER, DR. GERRIT, New York University School of Medicine, New York

City, New York
BIGELOW, DR. HENRY B., Museum of Comparative Zoology, Harvard University,

Cambridge, Massachusetts
BISHOP, DR. DAVID W., Dept. of Embryology, Carnegie Institute of Washington,

Baltimore 5, Maryland

BLANCHARD, DR. K. C., Johns Hopkins Medical School, Baltimore, Maryland
BLOCK, DR. ROBERT, c/o Biological Abstracts, 3815 Walnut Street, Philadelphia 4,

Pennsylvania

26 MARINE BIOLOGICAL LABORATORY

BLUM, DR. HAROLD F., Dept. of Biology, Princeton University, Princeton, New

Jersey
BODANSKY, DR. OSCAR, Dept. of Biochemistry, Memorial Cancer Center, 444 East

68th Street, New York 21, New York
BODIAN, DR. DAVID, Dept. of Epidemiology, Johns Hopkins University, Baltimore

5, Maryland

BOELL, DR. EDGAR J., Yale University, New Haven, Connecticut
BOETTIGER, DR. EDWARD G., Dept. of Zoology, University of Connecticut, Storrs,

Connecticut

BOLD, DR. H. C., Dept. of Botany, Vanderbilt University, Nashville, Tennessee
BOREI, DR. HANS, Dept. of Zoology, University of Pennsylvania, Philadelphia,

Pennsylvania

BRADLEY, DR. HAROLD C., 2639 Durant Avenue, Berkeley 4, California
BRIDGMAN, DR. ANNA J., Dept. of Biology, Agnes Scott College, Decatur, Georgia
BRONK, DR. DETLEV W., Rockefeller Institute, 66th St. and York Avenue, New

York 21, New York
BROOKS, DR. MATILDA M., Dept. of Physiology, University of California, Berkeley

4, California
BROWN, DR. FRANK A., JR., Dept. of Biological Sciences, Northwestern University,

Evanston, Illinois
BROWN, DR. DUGALD E. S., Dept. of Zoology, University of Michigan, Ann Arbor,

Michigan

BROWNELL, DR. KATIIERINE A., Ohio State University, Columbus, Ohio
BUCK, DR. JOHN B., Laboratory of Physical Biology, National Institutes of Health,

Bethesda, Maryland (10405 Muir Place, Kensington, Maryland)
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia
BULLOCK, DR. T. H., Dept. of Zoology, University of California, Los Angeles 24.

California

BURBANCK, DR. WILLIAM D., Box 834, Emory University, 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., Dept. of Biology, 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. A. J., Dept. of Physiology, University of Chicago, Chicago 37, Illinois
CARLSON, DR. FRANCIS D., Dept. of Biophysics, Johns Hopkins University, Balti-
more 18, Maryland

CAROTHERS, DR. E. ELEANOR, 9 Gladys Sparr, Murdock, Kansas
CARPENTER, DR. RUSSELL L., Tufts College, Medford 55, Massachusetts
CARSON, Miss RACHEL, 204 Williamsburg Drive, Silver Spring, Maryland
CATTELL, DR. McKEENE, Cornell University Medical College, 1300 York Avenue,

New York City, New York
CATTELL, MR. WARE, Cosmos Club, Washington 5, D. C.

REPORT OF THE DIRECTOR 27

CHAET, DR. ALFRED B., Boston University School of Medicine, 80 E. Concord

Street, Boston 18, Massachusetts
CHAMBERS, DR. EDWARD, Dept. of Physiology, University of Miami Medical School,

Coral Gables, Florida
CHAMBERS, DR. ROBERT, c/o W. N. Chambers, Hitchcock Clinic, Hanover, New

Hampshire
CHASE, DR. AURIN M., Dept. of Biology, Princeton University, Princeton, New

Jersey
CHENEY, DR. RALPH H., Biology Department, Brooklyn College, Brooklyn 10,

New York

CLAFF, MR. C. LLOYD, 5 Van Beal Road, Randolph, Massachusetts
CLARK, DR. A. M., Dept. of Biological Sciences, 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., Dept. of Biology, Union College, Schenectady, New York
CLARKE, DR. GEORGE L., Harvard University. Biological Laboratories, Cambridge

38, Massachusetts

CLELAND, DR. RALPH E., Indiana University, Bloomington, Indiana
CLEMENT, DR. A. C., Dept. of Biology, Emory University, Emory, Georgia
CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana
COE, DR. W. R., 183 Third Avenue, Chula Vista, California

COHEN, DR. SEYMOUR S., Dept. of Physiological Chemistry, University of Pennsyl-
vania, Philadelphia, Pa.
COLE, DR. KENNETH S., National Institutes of Health (NINDB), Bethesda 14,

Maryland

COLLETT, DR. MARY E., 34 Weston Road, Wellesley 81, Massachusetts
COLTON, DR. H. S., Box 601, Flagstaff, Arizona

COLWIN, DR. ARTHUR L., Dept. of Biology, Queens College, Flushing, New York
COLWIN, DR. LAURA H., Dept. of Biology, Queens College, Flushing, New York
COOPERSTEIN, DR. SHERWIN J., Dept. of Anatomy, Western Reserve University

Medical School, Cleveland, Ohio

COPELAND, DR. D. E., 1027 N. Manchester Street, Arlington 5, Virginia
COPELAND, DR. MANTON, Bowdoin College, Brunswick, Maine
COPLEY, DR. ALFRED L., Centre National de Transfusion Sanguine 6, Rue

Alexandre-Cobonel, Paris XVe, France

CORN MAN, DR. IVOR, Hazleton Laboratories, Box 333, Falls Church, Virginia
COSTELLO, DR. DONALD P., Dept. of Zoology, University of North Carolina, Chapel

Hill, North Carolina
COSTELLO, DR. HELEN MILLER, Dept. of Zoology, University of North Carolina,

Chapel Hill, North Carolina

CRANE, MR. JOHN O., Woods Hole, Massachusetts
CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts

CROASDALE, DR. HANNAH T., Dartmouth College, Hanover, New Hampshire
GROUSE, DR. HELEN V., Goucher College, Baltimore, Maryland
CROWELL, DR. P. S., JR., Dept. of Zoology, Indiana University, Bloomington,

Indiana

28 MARINE BIOLOGICAL LABORATORY

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., Dept. of Zoology, King's College, London, England

DAVIS, DR. BERNARD D., Dept. of Pharmacology, New York University College of

Medicine, New York 16, New York

DAWSON, DR. A. B., Harvard University, Cambridge 38, Massachusetts
DAWSON, DR. J. A., College of the City of New York, New York City, New York
DILLER, DR. IRENE C., Institute for Cancer Research, Philadelphia, Pennsylvania
DILLER, DR. WILLIAM F., 2417 Fairhill Avenue, Glenside, Pennsylvania
DIXON, DR. FRANK J., Dept. of Pathology, University of Pittsburgh School of

Medicine, Pittsburgh, Pennsylvania
DODDS, DR. G. S., West Virginia University School of Medicine, Morgantown,

West Virginia

DOLLEY, DR. WILLIAM L., Heim Road, Getzville, New York

DONALDSON, DR. JOHN C., University of Pittsburgh School of Medicine, Pitts-
burgh, Pennsylvania

DOTY, DR. MAXWELL S., Dept. of Biology, University of Hawaii, Honolulu, T. H.
DRINKER, DR. CECIL K., Box 502, Falmouth, Massachusetts
DuBois, DR. EUGENE F., 200 East End Avenue, New York 28, New York
DUGGAR, DR. BENJAMIN M., Lederle Laboratories Inc., Pearl River, New York
DURYEE, DR. WILLIAM R., George Washington University School of Medicine,

Dept. of Physiology, Washington 5, D. C.
EDDS, DR. MAC V., JR., Dept. of Biology, Brown University, Providence, Rhode

Island

EICHEL, DR. BERTRAM, Bureau of Biological Research, Box 515, Rutgers Univer-
sity, New Brunswick, New Jersey

EICHEL, DR. HERBERT J., Hahnemann Medical College, Philadelphia, Pennsylvania
ELLIOTT, DR. ALFRED M., Dept. of Zoology, University of Michigan, Ann Arbor,

Michigan

EVANS, DR. TITUS C., State University of Iowa, Iowa City, Iowa
FAILLA, DR. G., College of Physicians and Surgeons, Columbia University, New

York City, New York

FAURE-FREMIET, DR. EMMANUEL, College de France, Paris, France
FERGUSON, DR. F. P., Dept. 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, Dept. of Zoology, Newcomb College, Tulane University,

New Orleans 18, Louisiana
FISCHER, DR. ERNST, Dept. of Physiology, Medical College of Virginia, Richmond

19, Virginia
FISHER, DR. JEANNE M., Dept. of Biochemistry, University of Toronto, Toronto,

Canada

REPORT OF THE DIRECTOR 29

FISHER, DR. KENNETH C, Dept. of Biology, University of Toronto, Toronto,

Canada
FORBES, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge

38, Massachusetts
FRAENKEL, DR. GOTTFRIED S., Dept. of Entomology, University of Illinois, Urbana,

Illinois
FRIES, DR. ERIK F. B., Dept. of Biology, City College of New York, New York

City, New York

FRISCH, DR. JOHN A., Canisius College, Buffalo, New York
FURTH, DR. JACOB, 18 Springdale Road, Wellesley Farms, Massachusetts
GABRIEL, DR. MORDECAI, Dept. of Biology, Brooklyn College, Brooklyn, New York
GAFFRON, DR. HANS, Research Institutes, University of Chicago, 5650 Ellis Ave-
nue, Chicago 37, Illinois
GALL, DR. JOSEPH G., Dept. of Zoology, University of Minnesota, Minneapolis 14,

Minnesota

GALTSOFF, DR. PAUL S., Woods Hole, Massachusetts

GASSER, DR. HERBERT S., Director, Rockefeller Institute, New York 21, New York
GEISER, DR. S. W., Southern Methodist University, Dallas, Texas
GERARD, DR. R. W., Illinois Neuropsychiatric Institute, Chicago 12, Illinois
GILMAN, DR. LAUREN C., Dept. of Zoology, University of Miami, Coral Gables,

Florida

GINSBERG, DR. HAROLD S., Western Reserve University School of Medicine, Cleve-
land, Ohio

GOODCHILD, DR. CHAUNCEY G., Dept. of Biology, Emory University, Emory Uni-
versity, Georgia

GOODRICH, DR. H. B., Wesleyan University, Middletown, Connecticut
GOTTSCHALL, DR. GERTRUDE Y., 315 E. 68th Street, New York 21, New York
GOULD, DR. H. N., Biological Sciences Information Exchange, 1113 Dupont Circle

Building, Washington, D. C.
GRAHAM, DR. HERBERT, Director, Woods Hole Lab., Fish and Wildlife Service,

Woods Hole, Massachusetts
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
GRAY, DR. IRVING E., Duke University, Durham, North Carolina
GREEN, DR. JAMES W., Dept. of Physiology, Rutgers University, New Brunswick,

New Jersey

GREEN, DR. MAURICE, Dept. of Biochemistry, University of Pennsylvania, Phila-
delphia, Pennsylvania

GREGG, DR. JAMES H., University of Florida, Gainesville, Florida
GREGG, DR. J. P., Dept. of Zoology, Columbia University, New York 27, New York
GREIF, DR. ROGER L., Dept. of Physiology, Cornell University Medical College,

New York 21, New York
GROSCH, DR. DANIEL S., Dept. of Zoology, North Carolina State College, Raleigh,

North Carolina

GROSS, DR. PAUL, Dept. of Biology, New York University, University Heights,
New York 53, New York

30 MARINE BIOLOGICAL LABORATORY

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 1, Michigan

GUTTMAN, DR. RITA, Dept. of Physiology, Brooklyn College, Brooklyn, New York
GUYER, DR. MICHAEL F., University of Wisconsin, Madison, Wisconsin
HAJDU, DR. STEPHEN, U. S. Public Health Institute, Bethesda, Maryland
HALL, DR. FRANK G., Duke University, Durham, North Carolina
HAMBURGER, DR. VIKTOR, Dept. of Zoology, Washington University, St. Louis,

Missouri

HAMILTON, DR. HOWARD L., Iowa State College, Ames, Iowa
HANCE, DR. ROBERT T., Box 108, R. R. No. 3, Loveland, Ohio
HARDING, DR. CLIFFORD V., JR., Wistar Institute, University of Pennsylvania,

Philadelphia, Pennsylvania

HARMAN, DR. MARY T., Box 68, Camden, North Carolina
HARNLY, DR. MORRIS H., Washington Square College, New York University, New

York City, New York

HARRISON, DR. Ross G., Yale University, New Haven, Connecticut
HARTLINE, DR. H. KEFFER, Rockefeller Institute for Medical Research, 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
HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New

Jersey
HAUSCHKA, DR. T. S., Roswell Park Memorial Institute, 663 North Oak Street.

Buffalo 3, New York

HAXO, DR. FRANCIS T., Division of Marine Botany, Scripps Institute of Oceanog-
raphy, University of California, La Jolla, California
HAYASHI, DR. TERU, Dept. of Zoology, Columbia University, New York City, New

York

HAYDEN, DR. MARGARET A., 34 Weston Road, Wellesley 81, Massachusetts
HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts
HEILBRUNN, DR. L. V., Dept. of Zoology, University of Pennsylvania, Philadel-
phia, Pennsylvania
HENDLEY, DR. CHARLES D., 615 South Second Avenue, Highland Park, New

Jersey
HENLEY, DR. CATHERINE, Dept. of Zoology, University of North Carolina, Chapel

Hill, North Carolina
HENSHAW, DR. PAUL S., 17th floor, 501 Madison Avenue, New York 22, New

York

HESS, DR. WALTER N., Hamilton College, Clinton, New York
HIBBARD, DR. HOPE, Dept. of Zoology, Oberlin College, Oberlin, Ohio
HILL, DR. SAMUEL E., 135 Brunswick Road, Troy, New York
HINRICHS, DR. MARIE, Board of Education, Bureau of Health Service, 228 North

LaSalle Street, Chicago, Illinois
HISAW, DR. F. L., Harvard University, Cambridge 38, Massachusetts

REPORT OF THE DIRECTOR

HOADLEY, DR. LEIGH, Harvard University, Biological Laboratories, Cambridge 38,

Massachusetts
HODGE, DR. CHARLES, IV, Dept. of Zoology, Temple University, Philadelphia,

Pennsylvania
HOFFMAN, DR. JOSEPH, Dept. of Biology, Princeton University, Princeton, New

Jersey
HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia,

Pennsylvania
HOLLAENDER, DR. ALEXANDER, P. O. Box W, Clinton Laboratories, Oak Ridge,

Tennessee
HOPKINS, DR. HOYT S., New York University College of Dentistry, New York

City, New York
HUNTER, DR. FRANCIS R., Dept. of Physiology, Southern Illinois University, Car-

bondale, Illinois
HUTCHENS, DR. JOHN O., Dept. of Physiology, University of Chicago, Chicago 37,

Illinois
HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City,

New York

IRVING, DR. LAURENCE, U. S. Public Health Service, Anchorage, Alaska
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts
JACOBS, DR. M. H., School of Medicine, University of Pennsylvania, Philadelphia,

Pennsylvania
JACOBS, DR. WILLIAM P., Dept. of Biology, Princeton University, Princeton, New

Jersey

JENKINS, DR. GEORGE B., 5339 42nd Street N.W., Washington 15, D. C.
JENNER, DR. CHARLES E., Dept. of Zoology, University of North Carolina, Chapel

Hill, North Carolina
JONES, DR. E. RUFFIN, JR., Dept. of Biology, University of Florida, Gainesville,

Florida
KAAN, DR. HELEN W., National Heart Institute, National Institutes of Health,

Bethesda 14, Maryland
KABAT, DR. E. A., Neurological Institute, College of Physicians and Surgeons, New

York City, New York
KARUSH, DR. FRED, Dept. of Pediatrics, University of Pennsylvania, Philadelphia,

Pennsylvania
KAUFMANN, DR. B. P., Carnegie Institute, Cold Spring Harbor, Long Island, New

York

KEMPTON, DR. RUDOLF T., Vassar College, Poughkeepsie, New York
KEOSIAN, DR. JOHN, Dept. of Biology, Rutgers University, Newark 2, New Jersey
KETCHUM, DR. BOSTWICK, Woods Hole Oceanographic Institution, Woods Hole,

Massachusetts

KILLE, DR. FRANK R., Carleton College, Northfield, Minnesota
KIND, DR. C. ALBERT, Dept. 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

32 MARINE BIOLOGICAL LABORATORY

KISCH, DR. BRUNO, 845 West End Avenue, New York City, New York
KLEINHOLZ, DR. LEWIS H., Department of Biology, Reed College, Portland, Oregon
KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston,

Illinois
KOLIN, DR. ALEXANDER, Division of Biological Sciences, University of Chicago,

Chicago, Illinois
KOPAC, DR. M. J., New York University, Washington Square College, New York

City, New York
KORR, DR. I. M., Dept. of Physiology, Kirksville College of Osteopathy, Kirksville,

Missouri

KRAHL, DR. M. E., Dept. of Physiology, University of Chicago, Chicago 37, Illinois
KRAUSS, DR. ROBERT, Dept. of Botany, University of Maryland, Baltimore, Mary-
land

KREIG, DR. WENDELL J. S., 303 East Chicago Avenue, Chicago, Illinois
KUNITZ, DR. MOSES, Rockefeller Institute, 66th Street and York Avenue, New

York 21, New York

KUFFLER, DR. STEPHEN, Dept. of Ophthalmology, Johns Hopkins Hospital, Balti-
more 5, Maryland
LACKEY, DR. JAMES B., University of Florida, College of Engineering, Gainesville,

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
LANGE, DR. MATHILDA M., Box 307, Central Valley, New York
LANSING, DR. ALBERT I., Dept. of Anatomy, University of Pittsburgh Medical

School, Pittsburgh 13, Pennsylvania
LAUFFER, DR. MAX A., Dept. of Biophysics, University of Pittsburgh, Pittsburgh,

Pennsylvania

LAVIN, DR. GEORGE I., 3714 Springdale Avenue, Baltimore, Maryland
LAZAROW, DR. ARNOLD, Dept. of Anatomy, University of Minnesota, Medical

School, Minneapolis 14, Minnesota
LEDERBERG, DR. JOSHUA, Dept. of Genetics, University of Wisconsin, Madison 6,

Wisconsin
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
LEHMANN, DR. FRITZ, Zool. Inst., University of Berne, Berne, Switzerland
LESSER, DR. MILTON A., Dept. of Physiology, Ohio State University, Columbus,

Ohio

LEVINE, DR. RACHMIEL, Michael Reese Hospital, Chicago 16, Illinois
LEVY, DR. MILTON, Chemistry Dept., New York University School of Medicine,

New York City, New York

LEWIN, DR. RALPH A., Marine Biological Laboratory, Woods Hole, Massachusetts
LEWIS, DR. I. F., 1110 Rugby Road, Charlottesville, Virginia
LING, DR. GILBERT, Dept. of Neurophysiology, University of Illinois, Chicago,

Illinois

REPORT OF THE DIRECTOR 33

LITTLE, DR. E. P., 150 Causeway Street, Anderson Nichols & Company, Boston 24,
Massachusetts

LLOYD, DR. DAVID P. C, Rockefeller Institute, 66th Street and York Avenue,
New York 21, New York

LOCH HEAD, DR. JOHN H., Dept. of Zoology, University of Vermont, Burlington,
Vermont

LOEB. DR. LEO. 40 Crestwood Drive, St. Louis 5, Missouri

LOEB. DR. R. F.. Presbyterian Hospital. 620 W. 168 Street, New York 32, New
York

LOEWI, DR. OTTO, 155 East 93rd Street, New York City, New York

LORAND, DR. LASZLO, Dept. of Chemistry, College of Liberal Arts, Northwestern
University, Evanston, Illinois

LOVE, DR. Lois H., 4253 Regent Street, Philadelphia 4, Pennsylvania

LOVE, DR. WARNER E., Institute for Cancer Research, 7701 Burholme Avenue, Fox
Chase, Philadelphia, Pennsylvania

LYNCH, DR. CLARA J., Rockefeller Institute, 66th Street and York Avenue, New
York 21, New York

LYNCH, DR. RUTH STOCKING, Dept. of Botany, University of California. Los An-
geles 24, California

LYNCH, DR. WILLIAM, Dept. of Biology, St. Ambrose College, Davenport, Iowa

LYNN, DR. WILLIAM G., Dept. of Biology, Catholic University of America, Wash-
ington, D. C.

MACDOUGALL. DR. MARY S., Mt. Vernon Apts., 423 Clairmont Avenue, Decatur,
Georgia

McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School,
Philadelphia, Pennsylvania

MCDONALD, SISTER ELIZABETH SETON, Dept. of Biology, College of Mt. St. Joseph,
Mt. St. Joseph, Ohio

MCDONALD, DR. MARGARET H., Carnegie Institute of Washington, Cold Spring
Harbor, Long Island, New York

MACKLIN, DR. CHARLES C., 37 Gerard Street, London, Ontario, Canada

MAGRUDER, DR. SAMUEL R., Dept. of Anatomy, Tufts Medical School, 136 Harri-
son Avenue, Boston, Massachusetts

MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York

MARSHAK, DR. ALFRED, Woods Hole, Massachusetts

MARSLAND, DR. DOUGLAS A., New York University, Washington Square College,
New York City, New York

MARTIN, DR. EARL A., Dept. of Biology, Brooklyn College, Brooklyn, New York

MATHEWS, DR. A. P., Glenwood Boulevard, Schenectady, New York

MATTHEWS, DR. SAMUEL A., Thompson Biological Lab., Williams College, Wil-
liamstown, Massachusetts

MAVOR, DR. JAMES W., 8 Gracewood Park, Cambridge 58, Massachusetts

MAZIA, DR. DANIEL, Dept. of Zoology, University of California, Berkeley 4, Cali-
fornia

MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania

MEIGS. MRS. E. B., 1736 M Street N.W., Washington, D. C.

34 MARINE BIOLOGICAL LABORATORY

MEINKOTH, DR. NORMAN A., Dept. of Biology, Swarthmore College, Swarthmore,

Pennsylvania

MEMHARD, MR. A. R., Riverside, Connecticut
MENKIN, DR. VALY, Agnes Barr Chase Foundation for Cancer Research, Temple

University Medical School, Philadelphia, Pennsylvania

METZ, DR. C. B., Dept. of Zoology, Florida State University, Tallahassee, Florida
METZ, DR. CHARLES W., University of Pennsylvania, Philadelphia. Pennsylvania
MILLER, DR. J. A., Basic Science Building, Emory University, Georgia
MILNE, DR. LORUS J., Department of Zoology, University of New Hampshire, Dur-
ham, New Hampshire
MINNICH, DR. D. E., Dept. of Zoology, University of Minnesota, Minneapolis 14,

Minnesota
MOE, MR. HENRY A., Secretary General, 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., Dept. of Zoology, University of New Hampshire, Durham,

New Hampshire
MOORE, DR. JOHN W., Laboratory of Biophysics, NINDB, National Institutes of

Health, Bethesda 14, Maryland
MOUL, DR. E. T., Dept. of Botany, Rutgers University, New Brunswick, New

Jersey

MOUNTAIN, MRS. J. D., 9 Coolidge Avenue, White Plains, New York
MULLER, DR. H. J., Dept. of Zoology, Indiana University, Bloomington, Indiana
MUSACCHIA, DR. XAVIER J., Dept. 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, Dept. of Biology, Hamilton College, McMaster Univer-
sity, Hamilton, Ontario

NACHMANSOHN, DR. DAVID, College of Physicians and Surgeons, Columbia Uni-
versity, New York City, New York

NAVEZ, DR. ALBERT E., 206 Churchill's Lane, Milton 86, Massachusetts
NELSON, DR. LEONARD, Dept. of Anatomy, University of Chicago, Chicago, Illinois
NEURATH, DR. H., Dept. of Biochemistry, University of Washington, Seattle 5,

Washington

NEWMAN, DR. H. H., 173 Devon Drive, Clearwater, Florida
NICOLL, DR. PAUL A., Indiana Contract, Box K, A. P. O. 474, San Francisco,

California
OCHOA, DR. SEVERO, New York University College of Medicine, New York 16,

New York
OPPENHEIMER, DR. JANE M., Dept. of Biology, Bryn Mawr College, Bryn Mawr,

Pennsylvania
OSTER, DR. ROBERT H., University of Maryland, School of Medicine, Baltimore 1,

Maryland
OSTERHOUT, DR. W. J. V., Rockefeller Institute, 66th Street and York Avenue,

New York 21, New York

REPORT OF THE DIRECTOR 35

OSTERHOUT, DR. MARION IRVVIN, 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

PARMENTER, DR. CHARLES L., Dept. of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania
PARPART, DR. ARTHUR K., Dept. of Biology, Princeton University, Princeton, New

Jersey
PASSANO, DR. LEONARD M., Osborn Zoological Laboratory, Yale University, New

Haven, Connecticut
PATTEN, DR. BRADLEY M., University of Michigan School of Medicine, Ann Arbor,

Michigan

PEEBLES, DR. FLORENCE, 380 Rosemont Avenue, Pasadena 3, California
PERKINS, DR. JOHN F., JR., Dept. of Physiology, University of Chicago, Chicago

37, Illinois
PETTIBONE, DR. MARIAN H., Dept. of Zoology, University of New Hampshire,

Durham, New Hampshire

PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York
PLOUGH, DR. HAROLD H., Amherst College, Amherst, Massachusetts
POLLISTER, DR. A. W., Columbia University, New York City, New York
POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut
PRATT, DR. FREDERICK H., 105 Hundreds Road, Wellesley Hills 82, Massachusetts
PROCTOR, DR. NATHANIEL, Dept. of Biology, Morgan State College, Baltimore 12,

Maryland
PROSSER, DR. C. LADD, 401 Natural History Bldg., University of Illinois, Urbana,

Illinois
PROVASOLI, DR. LUIGI, Dept. of Biology, Haskins Laboratory, 305 E. 43rd Street,

New York 17, New York
QUASTEL, DR. JUDA H., Dept. of Biochemistry, McGill University, Montreal,

Canada

RAMSEY, DR. ROBERT W., Medical College of Virginia, Richmond, Virginia
RAND, DR. HERBERT W., 7 Siders Pond Road, Falmouth, Massachusetts
RANKIN, DR. JOHN S., Dept. of Zoology, University of Connecticut, Storrs,

Connecticut
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., Dept. of Biology, Emory University, Emory, Georgia
REDFIELD, DR. ALFRED C., Woods Hole, Massachusetts
REID, DR. W. M., Poultry Dept., University of Georgia, Athens, Georgia
REINER, DR. J. M., Columbia-Presbyterian Medical Center, 622 W. 168 St., New

York 32, 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

36 MARINE BIOLOGICAL LABORATORY

RICHARDS, DR. A. GLENN, Entomology Department, University Farm, University
of Minnesota, St. Paul, Minnesota

RICHARDS, DR. OSCAR W., American Optical Company, Research Center, South-
bridge, Massachusetts

RIESER, DR. PETER, Marine Biological Laboratory, Woods Hole, Massachusetts

ROCKSTEIN, DR. MORRIS, Dept. 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., Dept. of Physiology, University of Delaware, Newark,
Delaware

ROOT, DR. R. W., Dept. 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, Dept.
of Physiology, New York City, New York

ROSE, DR. S. MERYL, Dept. of Zoology, University of Illinois, Champaign, Illinois

ROSENTHAL, DR. THEODORE B., Dept. of Anatomy, University of Pittsburgh Medi-
cal School, Pittsburgh 13, Pennsylvania

Rossi, DR. HAROLD H., Dept. of Radiology, Columbia University, New York 32,
New York

ROTH, DR. JAY S., Dept. of Biochemistry, Hahnemann Medical College, Philadel-
phia 2, Pennsylvania

ROTHENBERG, DR. M. A., Chief, Chemical Laboratories, Dugway Proving Ground,
Dug way, Utah

RUGH, DR. ROBERTS, Radiological Research Laboratory, College of Physicians and
Surgeons, New York City, New York

RUNNSTROM, DR. JOHN, Wenner-Grens Institute, Stockholm, Sweden

RUTMAN, DR. ROBERT J., Dept. of Zoology, University of Pennsylvania, Philadel-
phia, Pennsylvania

RYAN, DR. FRANCIS J., Columbia University, New York City, New York

RYTHER, DR. JOHN H., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts

SANDEEN, DR. MURIEL I., Dept. of Zoology, Duke University, Durham, North
Carolina

SAUNDERS, MR. LAWRENCE, R. D. 7, Bryn Mawr, Pennsylvania

SCHAEFFER, DR. ASA A., Dept. of Biology, Temple University, Philadelphia,
Pennsylvania

SCHARRER, DR. ERNST A., Albert Einstein College of Medicine, 1710 Newport
Avenue, New York 61, New York

SCHECHTER, DR. VICTOR, College of the City of New York, New York City, New
York

SCHLESSINGER, DR. R. WALTER, Dept. 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., Dept. of Biology, Massachusetts Institute of Technol-
ogy, Cambridge, Massachusetts

REPORT OF THE DIRECTOR 37

SCHMITT, DR. O. H., Dept. of Physics, University of Minnesota, Minneapolis 14,
Minnesota

SCHOLANDER, DR. P. F., Institute of Zoophysiology, University of Oslo, Oslo,
Norway

SCHOTTE, DR. OSCAR E., Dept. of Biology, Amherst College, Amherst, Massa-
chusetts

SCHRADER, DR. FRANZ, Dept. of Zoology, Columbia University, New York City,
New York

SCHRADER, DR. SALLY HUGHES, Dept. of Zoology, Columbia University, New
York City, New York

SCHRAMM, DR. J. R., University of Pennsylvania, Philadelphia, Pennsylvania

SCOTT, DR. ALLAN C, Colby College, Waterville, Maine

SCOTT, DR. D. B. McNAiR, Dept. of Biochemistry, University of Pennsylvania
Hospital, Philadelphia, Pennsylvania

SCOTT, SISTER FLORENCE M., Seton Hill College, Greensburg, Pennsylvania

SCOTT, DR. GEORGE T., Oberlin College, Oberlin, Ohio

SEARS, DR. MARY, Woods Hole Oceanographic Institution, Woods Hole, Massa-
chusetts

SEVERINGHAUS, DR. AURA E., Dept. of Anatomy, College of Physicians and Sur-
geons, New York City, New York

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., Dept. 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, Dept. of Botany, University of Illinois, Urbana, Illinois

SLIFER, DR. ELEANOR H., Dept. of Zoology, State University of Iowa, Iowa City,
Iowa

SMITH, DR. DIETRICH C., Dept. of Physiology, University of Maryland School of
Medicine, Baltimore, Maryland

SMITH, DR. EDWARD H., Woods Hole Oceanographic Institution, Woods Hole,
Massachusetts

SMITH, MR. HOMER P., General Manager, Marine Biological Laboratory, Woods
Hole, Massachusetts

SMITH, DR. RALPH I., Dept. of Zoology, University of California, Berkeley 4,
California

SONNEBORN, DR. T. M., Dept. of Zoology, Indiana University, Bloomington,
Indiana

SONNENBLICK, DR. B. P., 40 Rector Street, Newark 3, New Jersey

SPEIDEL, DR. CARL C., University of Virginia, University, Virginia

SPIEGEL, DR. MELVIN, Dept. of Biology, Colby College, Waterville, Maine

SPRATT, DR. NELSON T., JR., Dept. of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota

38 MARINE BIOLOGICAL LABORATORY

STARR, DR. RICHARD C., Dept. of Botany, Indiana University, Bloomington, Indiana
STEINBACH, DR. HENRY BURR, Dept. of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota

STEINBERG, DR. MALCOLM S., Dept. of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota

STEPHENS, DR. GROVER C., Dept. of Zoology, University of Minnesota, Minne-
apolis 14, Minnesota

STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois
STOKEY, DR. ALMA G., Dept. of Botany, Mt. Holyoke College, South Hadley,

Massachusetts

STRAUSS, DR. W. L., JR., Johns Hopkins University, Baltimore 18, Maryland
STUNKARD, DR. HORACE W., New York University, New York City, New York
STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena 4,

California
SULKIN, DR. S. EDWARD, Dept. of Bacteriology, University of Texas, Southwestern

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

TASHIRO, DR. SHIRO, University of Cincinnati Medical College, Cincinnati, Ohio
TAYLOR, DR. WM. RANDOLPH, Dept. of Botany, University of Michigan, Ann

Arbor, Michigan

TEWINKEL, DR. Lois E., Dept. of Zoology, Smith College, Northampton, Massa-
chusetts

TRACY, DR. HENRY C., P. O. Box 54, Oxford, Mississippi
TRACER, DR. WILLIAM, Rockefeller Institute, 66th Street and York Avenue, New

York 21, New York
TRINKAUS, DR. J. PHILIP, Dept. of Zoology, Osborn Zoological Laboratories, Yale

University, New Haven, Connecticut

TWEEDELL, DR. KEN YON S., Dept. of Zoology, University of Maine, Orono, Maine
TYLER, DR. ALBERT, California Institute of Technology, Pasadena 4, California
UHLENHUTH, DR. EDWARD, University of Maryland School of Medicine, Baltimore,

Maryland

URETZ, DR. ROBERT B., Dept. of Biophysics, University of Chicago, Chicago, Illinois
DEVILLAFRANCA, DR. GEORGE W., Dept. of Zoology, Smith College, Northampton,

Massachusetts

VILLEE, DR. CLAUDE A., Harvard Medical School, Boston 15, Massachusetts
VINCENT, DR. WALTER S., Dept. of Anatomy, State University of New York

School of Medicine, Syracuse 10, New York
WAINIO, DR. W. W., Bureau of Biological Research, Rutgers University, New

Brunswick, New Jersey
WALD, DR. GEORGE, Biological Laboratory, Harvard University, Cambridge 38,

Massachusetts
WARBASSE, DR. JAMES P., Woods Hole, Massachusetts

REPORT OF THE DIRECTOR 39

WARNER, DR. ROBERT C, Dept. of Chemistry, New York University College of
Medicine, New York 16, New York

WATERMAN, DR. T. H., Osborn Zoological Laboratory, Yale University, New
Haven, Connecticut

WEBB, DR. MARGUERITE, Dept. of Physiology and Bacteriology, Goucher College,
Towson, Maryland

WEISS, DR. PAUL A., Laboratory of Developmental Biology, Rockefeller Institute,
New York 21, New York

WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania

WHEDON, DR. A. D., 21 Lawncrest, Danbury, Connecticut

WHITAKER, DR. DOUGLAS M., Rockefeller Institute for Medical Research, New
York 21, New York

WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania

WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia, Pennsylvania

WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania,
Philadelphia, Pennsylvania

WICKERSHAM, MR. JAMES H., 530 Fifth Avenue, New York 36, New York

WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia,
Pennsylvania

WIEMAN, DR. H. L., Box 485, Falmouth, Massachusetts

WIERCINSKI, DR. FLOYD J., Dept. of Physiology, Hahnemann Medical College,
Philadelphia, Pennsylvania

WILBER, DR. C. G., Medical Laboratories, Applied Physiology Branch, Army
Chemical Center, Maryland

WILLIER, DR. B. H., Dept. of Biology, Johns Hopkins University, Baltimore 18,
Maryland

WILSON, DR. J. W., Dept. of Biology, Brown University, Providence, Rhode Island

WILSON, DR. WALTER L., Dept. of Physiology, University of Vermont College of
Medicine, Burlington, Vermont

WITSCHI, DR. EMIL, Dept. of Zoology, State University of Iowa, Iowa City, Iowa

WOLF, DR. ERNST, Pendleton Hall, Wellesley College, Wellesley, Massachusetts

WOODWARD, DR. ARTHUR A., Army Medical Center, Maryland (Applied Physiol-
ogy Branch, Army Chemical Corps. Medical Laboratory)

WRIGHT, DR. PAUL A., Dept. of Zoology, University of Michigan, Ann Arbor,
Michigan

WRINCH, DR. DOROTHY, Dept. of Physics, Smith College, Northampton, Massa-
chusetts

YNTEMA, DR. C. L., Dept. of Anatomy, University of New York College of Medi-
cine, Syracuse 10, New York

YOUNG, DR. D. B., Main Street, North Hanover, Massachusetts

ZINN, DR. DONALD J., Dept. of Zoology, University of Rhode Island, Kingston,
Rhode Island

ZIRKLE, DR. RAYMOND E., Dept. of Radiobiology, University of Chicago, Chicago
37, Illinois

ZORZOLI, DR. ANITA, Dept. of Physiology, Vassar College, Poughkeepsie, New York

ZWILLING, DR. E., Dept. of Genetics, University of Connecticut, Storrs, Connecticut

40

MARINE BIOLOGICAL LABORATORY

3. ASSOCIATE MEMBERS

ALDRICH, Miss AMEY OWEN

ALTON, DR. AND MRS. BENJAMIN H.

ANTHONY, MR. RICHARD A.

ARMSTRONG, DR. AND MRS. P. B.

BARBOUR, MR. Lucius

BARTOW, MR. AND MRS. CLARENCE

BARTOW, MRS. FRANCIS D.

BARTOW, MR. AND MRS. PHILIP

BELL, MRS. ARTHUR

BENJAMIN, MR. EDWARD

BRADLEY, MR. ALBERT L.

BRADLEY, MRS. CHARLES CRANE

BROWN, MRS. THORNTON

BURDICK, MR. CHARLES L.

BURLINGAME, MRS. F. A.

CAHOON, MRS. SAMUEL

CALKINS, MR. G. NATHAN, JR.

CALKINS, MRS. GARY N.

CALKINS, MR. SAMUEL

CARLETON, MRS. WINSLOW

CLAFF, MR. AND MRS. C. LLOYD

CLARK, DR. AND MRS. ALFRED HULL

CLARK, MRS. LEROY

CLARK, MR. W. VAN ALAN

CLOWES, MR. ALLEN W.

CLOWES, MRS. G. H. A.

CLOWES, DR. AND MRS. GEORGE, JR.

COLTON, MR. H. SEYMOUR

CRANE, Miss LOUISE

CRANE, MRS. W. CAREY

CRANE, MRS. W. MURRAY

CROSSLEY, MR. AND MRS. ARCHIBALD M.

CROWELL, MR. PRINCE S.

DANIELS, MR. AND MRS. F. HAROLD

DAY, MR. AND MRS. POMEROY

DRAPER, MRS. MARY C.

DREYER, MRS. FRANK

ELSMITH, MRS. DOROTHY

ENDERS, MR. FREDERICK

EWING, MR. FREDERICK

FAY, MR. AND MRS. HENRY H.

FISHER, MRS. BRUCE CRANE

FRIENDSHIP FUND, INC.

FROST, MRS. EUGENIA

GALTSOFF, MRS. EUGENIA

GlFFORD, MR. AND MRS. JOHN A.

GlLCHRIST, MR. AND MRS. JOHN A.
GlLDEA, DR. AND MRS. E. F.

GREEN, Miss GLADYS W.
HAMLEN, MR. J. MONROE
HARRELL, MR. AND MRS. JOEL E.
HARRINGTON, MR. AND MRS. A. W.
HARRINGTON, MR. ROBERT D.
HOUSTON, MR. AND MRS. HOWARD E.
HOWE, MRS. HARRISON E.
JANNEY, MRS. WALTER C.
JEWETT, MRS. GEORGE F.
KEITH, MR. AND MRS. HAROLD C.
KIDDER, MRS. HENRY M.
KING, MR. FRANKLIN
KOLLER, MRS. LEWIS
LAWRENCE, MR. MILFORD
LEMANN, MRS. SOLEN B.
LOBB, MRS. JOHN
LOEB, DR. ROBERT F.

McCLINTIC, MRS. GUTHRIE

McKELOY, MR. JOHN

MARVIN, MRS. WALTER T.

MAST, MRS. S. O.

MEIGS, MRS. EDWARD B.

MEIGS, DR. AND MRS. J. WISTER

MEIGS, Miss MARY ROBERTS

MELLON, MRS. RICHARD K.

MISKELL, MR. JOSEPH B.

MITCHELL, MRS. JAMES McC.

MIXTER, MRS. JASON

MOORE, MRS. WILLIAM A.

MOSSER, MRS. FLORENCE M.

MOTLEY, MRS. THOMAS

NEWTON, Miss HELEN K.

NICHOLS, MRS. GEORGE

NIMS, MRS. E. D.

PACKARD, DR. AND MRS. CHARLES

PACKARD, MRS. LAURENCE B.

PARK, MR. MALCOLM S.

PECK, MR. AND MRS. SAMUEL A.

PENNINGTON, Miss ANNE H.

REDFIELD, MRS. ALFRED

REZNIKOFF, DR. PAUL

RIGGS, MRS. LAWRASON

RIVINUS, MR. AND MRS. F. MARKOE

REPORT OF THE LIBRARIAN

41

RODES, MRS. BOYLE
ROOT, MRS. WALTER
RUDD, MRS. H. W. DWIGHT
SANDS, Miss ADELAIDE G.
SAUNDERS, MRS. LAWRENCE
SINCLAIR, MR. W. R.
SMITH, MRS. EDWARD H.
STANWOOD, MRS. F. A.
STOCKARD, MRS. MERCEDES
STONE, MR. AND MRS. S. M.
SWIFT, MR. AND MRS. E. KENT
SWOPE, MR. AND MRS. GERARD, JR.
SWOPE, Miss HENRIETTA H.

TILNEY, MRS. ALBERT A.
TOMPKINS, MR. AND MRS. B. A.
VANNEMAN, DR. AND MRS. JOSEPH
WAKSMAN, MRS. SELMAN A.
WEBSTER, MRS. EDWIN S.
WHITELY, MR. AND MRS. G. W., JR.
WHITELY, Miss MABEL W.

WlCKERSHAM, MR. AND MRS. JAMES H.

WILLISTON, Miss EMILY
WILLISTON, Miss MARY D.
WILLISTON, PROF. SAMUEL
WILSON, MRS. EDMUND B.

WOLFINSOHN, MRS. WOLFE

V. REPORT OF THE LIBRARIAN

In 1956, the number of currently received journals totalled 1575 (48 new).
Of these titles, there were 471 (6 new) Marine Biological Laboratory subscriptions ;
607 (12 new) exchanges and 182 (15 new) gifts; 81 (9 new) were Woods Hole
Oceanographic Institution subscriptions ; 184 (4 new) were exchanges and 50 (2
new) were gifts.

The Laboratory purchased 60 books, received 77 complimentary copies (12
from authors and 65 from publishers), and accepted 25 miscellaneous gifts. The
Institution purchased 23 titles and received 5 gifts. The total number of new
books accessioned amounted to 190.

By purchase the Laboratory completed 14 journal sets and partially completed
13. The Institution completed four sets and partially completed three. Volumes
and numbers received by gift and by exchange completed four sets and partially com-
pleted 16 sets.

There were 6112 reprints added to the collection, of which 2326 were of current
issue.

At the end of the year, the Library contained 66,590 bound volumes and 202,201
reprints.

The Library sent out on inter-library loan 192 volumes and borrowed 74 for the
convenience of the investigators.

Dr. E. Newton Harvey's collection of reprints was processed in 1956 and sev-
eral thousand were added to the Library's collection. The duplicate material was
presented to the University of North Carolina Library.

At the close of the year, Dr. Albert I. Lansing was influential in having an
accumulation of reprints sent from the University of Pittsburgh. These will be
processed in 1957.

Grateful acknowledgment is extended to Drs. Alfred C. Redfield, Henry
Stommel, Ethel B. Harvey, Roberts Rugh, P. W. Whiting, Wm. R. Amberson,
Dorothy Wrinch, Helen W. Kaan, and to the Tompkins-McCaw Library, Medical
College of Virginia, for valuable and useful contributions of books, reprints and
old photographs.

42 MARINE BIOLOGICAL LABORATORY

During the summer, the Library Committee, fully aware of the demand for an
increase in the purchase of books, took action in securing a larger appropriation
for this purpose. A Library Advisory Committee was appointed to recommend
titles for purchase in 1957. There are 23 persons on this Committee and it is
hoped a very substantial increase in book acquisitions will be realized. Extra funds
are also expected in 1957 for the binding of back periodical volumes — work that
has been neglected in order to keep the current binding up to date. The steady
increase in the number of current periodicals now being published has also made
it necessary to request an increase in the budget for 1957.

In August, Mrs. Priscilla B. Montgomery, the former Librarian, passed away.
It is due to her foresight during the early days of the Library's rapid growth, that
the present system, initiated by her, has proved to be an adaptable and an efficient
one. The Library is a very fitting memorial to the loyal service and painstaking
work displayed by her during the years 1919—1947. (A memorial paper is con-
tained in the Laboratory's report).

Respectfully submitted,

DEBORAH L. HARLOW,

Librarian

VI. REPORT OF THE TREASURER

The combined market values of securities for the General Fund and the Library
at December 31, 1956, amounted to $1,472,265.00 as compared with the total of
$1,500,773 as of December 31, 1955. The average yield on the securities was
3.76% of market value and 5.55% of book value. The total uninvested principal
cash in the above accounts as of December 31, 1956, was $1,551.72. The securities
list held in the Endowment Funds appears in the auditor's report.

The pooled securities had a market value at the end of the year of $242,759.00
with uninvested principal cash in the amount of $430.12. The book value of the
securities in this account was $223,341.33. The average yield on market value
was 3.77% and 4.10% on book value.

The proportionate interest in the Pooled Fund account of the various Funds as
of December 31, 1956, is as follows:

Pension Fund 16.680%

General Laboratory Investments 60.619

Other :

Bio Club Scholarship Fund 1.768

Rev. Arsenious Boyer Scholarship Fund 2.161

Gary N. Calkins Fund 2.024

Allen R. Memhard Fund 392

F. R. Lillie Memorial Fund 6.824

Lucretia Crocker Fund 7.391

E. G. Conklin Fund 1.252

M. H. Jacobs Scholarship Fund 889

REPORT OF THE TREASURER 43

During the year we received a gift entitled "Frank R. Lillie Fellowship Fund"
consisting of 800 shares of Crane Company common stock. While WTC can change
the investments in this account, it must be kept intact. It was therefore not eligible
to be included in the Pooled Fund account. Accordingly, a special custodian ac-
count was established for these securities. We are also holding in this account
the securities of the General Biological Supply House.

The pledge of $8,000 par value government bonds still remains to secure the
loan of the MBL Club which has been reduced over the years from $7,000 to $2,330
at the year end.

Donations from MBL Associates for 1956 were $5,255.00 as compared with
$2,940.00 for 1955. Gifts from individuals for unrestricted use were $679.00.
Foundations, societies and companies donated $25,500.00. The $6,677.83 balance
of the $50,000 from the National Science Foundation for hurricane rehabilitation
has been used to cover delayed repairs to the plant, attributed to the hurricane.

The apartment house rentals during the winter months amounted to $2,902.30.
The cost of heating was $922.19 leaving a balance of $1,980.11 to be applied against
the general expenses of lighting, water and insurance.

We were able to purchase short term treasury bills to activate some of the cash
in the Falmouth account arising from the donations from foundations for anticipated
construction, the payment for which was scheduled at a later date. The purchase
of $50,000 U. S. Treasury bills was made in November, 1956 to run for 90 days.
The interest earned on this purchase was $330.50.

Lybrand, Ross Bros, and Montgomery have examined our books and sub-
mitted financial statements for examination.

Following is a statement of the auditors.

To the Trustees of the Marine Biological Laboratory, Woods Hole, Massachusetts:

We have examined the balance sheets of the Marine Biological Laboratory as
at December 31, 1956, and the related statements of operating expenditures and
income and of current fund for the year then ended. Our examination was made
in accordance with generally accepted auditing standards, and accordingly included
such test of the accounting records and such other auditing procedures as we con-
sidered necessary in the circumstances.

In our opinion, the accompanying financial statements present fairly the assets,
liabilities and funds of the Marine Biological Laboratory at December 31, 1956,
and the expenditures and income for the year then ended.

LYBRAND, Ross BROS. & MONTGOMERY
Boston, Massachusetts

44 MARINE BIOLOGICAL LABORATORY

MARINE BIOLOGICAL LABORATORY

BALANCE SHEET
December 31, 1956

Investments

Investments held by Trustee :

Securities, at cost (approximate market quotation $1,472,265) $ 995,086

Cash 1,552

996,638

Investments of other endowment and unrestricted funds :

Pooled Investments, at cost (approximate market quotation $242,759) 223,341

Other investments (Note A) 75,085

Cash 6,690

Accounts receivable 6,838

311,954

Plant Assets

Land, buildings, library and equipment (Note B) 2,424,492

Less allowance for depreciation (Note B) 991,930

1,432,562

Current Assets

Cash 100,045

U. S. Treasury bills, face value $50,000, due 2/15/57 temporarily invested pending ex-
penditures for rehabilitation of Supply Department building and certain labora-
tories (approximate market quotation $49,800) 50,000

Common stocks, at market value at date of gift 5,728

Accounts receivable ($12,880 from U. S. Government) 25,535

Inventories of specimens and Bulletins 58,269

Prepaid insurance and other 15,980

$2,996,711

Notes :

A — The Laboratory has guaranteed a note of approximately $2,400 of the M. B. L. Club
and has pledged as security therefor bonds with an original cost of $7,900 included
in other investments.

B — The Laboratory has since January 1, 1916 provided for reduction of book amounts
of plant assets and funds invested in plant at annual rates ranging from \% to 5%
of the original cost of the assets.

REPORT OF THE TREASURER 45

MARINE BIOLOGICAL LABORATORY

BALANCE SHEETS

December 31, 1956

Endozvinent Funds

Endowment funds given in trust for the benefit of the Marine Biological Laboratory $ 996,638
Endowment funds for awards and scholarships :

Principal $ 62,861

Unexpended income 1,420 64,281

Unrestricted funds functioning as endowment 206,378

Retirement fund 41,824

Pooled investments — accumulated gain or (loss) (529)

311,954

Plant Liability and Funds

Mortgage payable on demand, 5% 5,000

Funds expended for plant, less retirements 2,419,492

Less allowance for depreciation charged thereto 991,930 1,427,562

1,432,562

Current Liabilities and Funds

Accounts payable 13,653

Unexpended balances of gifts for designated purposes 11,540

Advance payments on research contracts 83,189

Current fund 147,175

$2,996,711

46 MARINE BIOLOGICAL LABORATORY

MARINE BIOLOGICAL LABORATORY

STATEMENT OF OPERATING EXPENDITURES AND INCOME

Year Ended December 31, 1956

Operating Expenditures

Direct expenditures of departments :

Research and accessory services $161,259

Instruction 33,186

Library, including book purchases 28,631

Biological Bulletin 15,902

238,978

Direct costs on research contracts 55,343

Administration and general 48,744

Plant operation and maintenance 76,926

Hurricane emergency repairs 6,678

Dormitories and dining services 131,464

Equipment purchased from current funds 2,850

560,983
Less depreciation included in plant operation and dormitories and dining services

above but charged to plant funds 36,424

524,559

Income

Direct income of departments :

Research fees 44,507

Accessory services (including sales of biological specimens $66,571) 94,978

Instruction fees 17,174

Library fees and income 6,625

Biological Bulletin, subscriptions and sales 18,148

181,432

Reimbursement and allowance for direct and indirect costs on research contracts 56,429

Dormitories and dining services income 108,375

346,236

Investment income 81,501

Gifts for current use 124,062

Sundry income 376

Total current income . 552,175

Excess of income $ 27,616

REPORT OF THE TREASURER

47

MARINE BIOLOGICAL LABORATORY
STATEMENT OF CURRENT FUND
Year Ended December 31, 1956

Balance January 1, 1956 $119,559

Excess of income over operating expenditures, 1956 27,616

Balance December 31, 1956 $147,175

MARINE BIOLOGICAL LABORATORY

SUMMARY OF INVESTMENTS

December 31, 1956

Securities held by Trustee :
General endowment fund :

Cost

Approximate Investment

% Market % of Income

Total Quotations Total 1956

U. S. Government bonds

$ 97052

11 7

$ 93 46'

77

$ 4 799

Other bonds

406 226

489

409 437

^3 6

8990

Preferred stocks

503,278
85788

60.6
103

502,899
71 500

41.3
58

13,289
3370

Common stocks

241,652

29.1

644 556

529

99350

830,718

100.0

1,218,955

100.0

46,009

General Educational Board endowment
fund :

U. S. Government bonds

31,037

189

30 164

11 9

1096

Other bonds

65,925

40.1

53,200

21 0

1 276

Preferred stocks

96,962
27,281

59.0
16.6

83,364
24,274

32.9
96

2,372
1 130

Common stocks

40125

244

145 672

575

5807

164,368

100.0

253,310

100.0

9,309

Total securities held by Trustee

$995,086

$1,472,265

$55,318

48

MARINE BIOLOGICAL LABORATORY

MARINE BIOLOGICAL LABORATORY

SUMMARY OF INVESTMENTS — Continued

December 31, 1956

Cost

Approximate Investment

% Market % of Income

Total Quotations Total 1956

Investments of other endowment and unre-
stricted funds :

Pooled investments :

U. S. Government bonds $37,800 16.9 $ 34,119 14.1 $ 905

Other bonds 93,936 42.1 90,628 37.3 2,527

131,736 59.0 124,747 51.4 3,432

Common stocks 91,605 41.0 118,012 48.6 5,439

223,341 100.0 $ 242,759 100.0 8,871

Other investments :

U. S. Government bonds 7,920 220

Common stocks 43,600 20,613

Real estate and mortgage 23,565

75,085 20,833

Total investments of other en-
dowment and unrestricted
funds $298,426 $29,704

Total investment income received 85,353

Custodian's fees charged thereto (412)

$84,941

THE BREEDING OF POLYCHAETOUS ANNELIDS
PARGUERA, PUERTO RICO

M. JEAN ALLEN 1

Department of Biology and Institute of Marine Biology, College of Agriculture and
Mechanic Arts, University of Puerto Rico, May agues, Puerto Rico

The writer set out to accomplish an embryological problem involving some of
the polychaetous annelids of Puerto Rico. No information on the breeding habits
of this group was available. To obtain suitable embryological material, therefore,
it was necessary to determine the breeding periods of some of the species which
were readily accessible. The information obtained is thus incidental to the main
problem. Several people, however, have expressed an interest in its publication,
since apparently little is known concerning the breeding habits of tropical marine
forms. The information may be of some value to others interested in studying
tropical or subtropical marine forms.

Observations and collections were made throughout the year. Night collections
were made at the laboratory dock on the island of Magiieyes, Parguera, Puerto
Rico, with a reflector bulb attached to an extension cord, and a dip net, or from a
boat over nearby coral reefs with the aid of a reflector light attached to a battery.
Some of the former collections were made throughout a full year ; collections over
the reefs were made from March to December, 1955. Collections made during the
day were largely from the shallow seas over coral reefs, and along the edge of
mangrove islands. The \vaters of this area of the Caribbean are relatively warm
throughout the year, being somewhat cooler during the "winter" months. The
temperature of the surface water goes as high as 87° F. at the laboratory dock
(September 1, 1955). The tides in this area show little variation in height, the
difference between the lowest low tide and the highest high tide rarely exceeding
41 cm. in any month.

Dr. Olga Hartman and Dr. Marian H. Pettibone were kind enough to identify
the species here described. I am most grateful for their generous help, and also
wish to express my appreciation to Mr. Donald Erdman who kindly assisted with
many of the night collections, and to those individuals of the College of Agriculture
who were so cooperative in putting the facilities of the Marine Institute and of the
Department of Biology at my disposal.

The polychaetes on which observations have been made are arranged by families
for convenient reference. Throughout the paper, one asterisk (*) refers to those
species identified by Dr. Hartman, and two asterisks (**) to those identified by
Dr. Pettibone.

FAMILY SYLLIDAE. Autolytns oniatns (Verrill) *. The female or sac-
conereis stage of the red-banded Antolytns may be collected at the dock at night the

1 U. S. Public Health Service Post-doctoral Research Fellow of the National Cancer
Institute, 1954-56. Present address : Department of Biology, Wilson College, Chambersburg,
Pennsylvania.

49

50 M. JEAN ALLEN

year round with the possible exception of the end of January and the beginning of
February. It was found to be most abundant during the last half of May and
June, during July (particularly during the middle of the month), and the first part
of September. Individuals rise from a depth of several feet to the surface within
the circle of light. When abundant, as many as 50 have been collected with one dip
of the net, and as many as 165 have been collected within an hour. The female
stage of this polychaete measures approximately 13 mm. However, individuals
appear about half this size as they tend to curl ventrally around an egg sac in which
developmental stages are borne. Before being released, white spherical eggs, ap-
proximately 85 micra in diameter, are packed within the coelom for the entire length
of the body. Their development will be considered in a subsequent paper. The male
(polybostrichus) stage of this syllid has been observed only twice. This was not
at the dock where the sacconereis stage collects but over a reef on the nights of July
26, when 8 specimens were obtained, and September 21, 1955, when 2 specimens
were collected.

In a series of papers written in 1951, Fauvel describes some polychaetous
annelids from the Gulf of Tadjoura (French Somaliland) caught by M. J.-L. Dantan
during night fishing with a light in the months of January, February, and March,
1933 and 1934. The Gulf of Tadjoura is closer to the equator than is Puerto Rico
so presumably the water would be very warm the year round. Fauvel (195 la)
notes that during night fishing in the gulf the syllids and nereids were the most
abundant and most interesting of the species collected although other epitokal species
were obtained too. The collecting in Puerto Rico also yielded primarily syllids and
nereids. Fauvel (1951b) describes a number of species of syllids including
Autolytus. It is interesting to note, however, that the polybcstrichus stage of
Autolytus was caught in enormous quantities (many hundreds) while the sacconereis
stage was relatively rare (Fauvel, 1951a). As described above, the reverse was
true in the Puerto Rican waters (although "enormous quantities" were not ob-
tained), the male stage rarely being observed. From a plankton collection Thorson
(1946) reported three sacconereis specimens of A. prolifer with larvae close to hatch-
ing stage in the ventral egg sac and one polybostrichus stage of the same species.

The temperature of the surface water from which A. ornatus was collected at
Parguera, Puerto Rico, probably never goes below 75° F., even in January. It is
interesting to note Bumpus (1898a) has reported that at Woods Hole, Massa-
chusetts, during the month of March, 1898, Antolytus cornutus was frequently
taken with eggs, the temperature ranging from 38° F. at the beginning of March to
43° F. at the end. Mead, in 1898, reported that Autolytns (species unspecified)
with egg clusters attached was regularly taken in his tow at Woods Hole, visually
3 or 4 at a time, during the early part of April (water temperature approximately
41 to 42° F.). Bumpus (1898b) noted that the tow-net on May 7 brought in
Autolytus but without eggs ; on May 10th he noted several egg-bearing individuals,
and on the llth a male swimming about and finally fastening to a female with his
jaw. Throughout the latter portion of May, Autolytus was abundant. In his
report for June, July, and August, Bumpus (1898c) notes that Autolytus and other
syllids which were frequently taken at Woods Hole had ventral egg sacs.

In the cold waters of Point Barrow, Alaska, both female stolons (with egg
sacs) and male stolons have been reported for several species of Autolytus (Petti-
bone, 1954). This material was collected by Dr. G. E. MacGinitie of the Arctic

BREEDING OF PUERTO RICAN POLYCHAETES 51

Research Laboratory. While the writer was still collecting A. ornatus in Puerto
Rico, the following very interesting description of Autolytus jallax from the Point
Barrow collection was published (MacGinitie, 1955, p. 138) : "The female sac-
conereis stages bearing egg sacs were taken through the ice from January 25 to May
17, 1950. On March 29, 1950, there were hundreds of these worms, with egg sacs,
swimming in the water at the 80-foot plankton hole and on April 7, 1950 (1.9 miles
from shore) when the slush ice was removed from a 4-foot- wide lead, hundreds were
welling up and swimming around. The worms were about 10 mm. long and the egg
sacs 2.5 mm. long." It thus appears that Autolytus is able to tolerate a very wide
temperature range for breeding, and that in the warm waters of Puerto Rico A.
ornatus breeds all the year round with the possible exception of late January and
early February. Hartman (1951) remarks that both sacconereis and polybostrichus
stages of Autolytus brevicirrata Winternitz may be taken in plankton during the
summer. Dales' comments (1951) on the breeding season of A. f>rolifer (A.
cornutusf — see Pettibone, 1954) indicate that the breeding habits of this species
may be similar to those of A. ornatus.

The sacconereis stage of another syllid, unidentified as yet, was collected oc-
casionally along with A. ornatus. This species is yellow in color, is not banded, and
has smaller eggs and larvae (various stages were collected) than those of A. ornatus.
Thus far it would appear that this species cannot be collected in sufficient numbers
for embryological purposes.

FAMILY NEREIDAE. Nereis riisci Grube*. Both male and female het-
eronereids of this species have been obtained in the evenings, both at the dock and
over reefs, during most of the year. No real swarming, such as has been described
for some species of Nereis, has been noted. Usually only one to a few
individuals have been observed in an evening. The largest number observed was
within an hour's time (between 8:00 and 9:00 P.M.) on March 23. Shedding does
not ordinarily occur when the worms are isolated but only after a male and female
are put together. The spherical eggs, approximately 190 micra in diameter, have
numerous oil droplets as is characteristic of other nereid eggs. Results following
attempts at artificial fertilization varied. In some cases no shedding occurred; in
others, shedding of gametes occurred without development ; in others, development
ceased during cleavage ; and in successful cases the eggs developed into larvae with
three external sets of setae within three days. Results suggest that the height of the
breeding period of this form extends from the last week in July to the beginning of
September. None were obtained during four observations throughout June, with
the exception of June 27 when one individual was observed. None were observed
during the last part of September and October, 1955 (a few were collected on
October 7, 1954). One was observed on November 7, none on November 14, and
two (one with posterior end missing) on November 22, 1955. Occasional indi-
viduals were collected throughout the other months of the year.

A number of nereids have been described by Fauval (1951a-d) from the collec-
tion from the Gulf of Tadjoura but this group did not include Nereis riisei Grube.
The heteronereid stages of N. riisei, collected at Parguera, Puerto Rico, are in size,
color, and behavior under the light, reminiscent of Nereis sitccinea (Leuckart),
commonly called Nereis liuibata (Ehlers), found at Woods Hole, Massachusetts.
N. riisei differs, however, in that the males observed were larger than the females
and no real swarming occurs. This is in even greater contrast to the spectacular

52 M. JEAN ALLEN

behavior of the Nereis snccinea occurring in eastern Canadian waters (Berkeley
and Berkeley, 1953). Swarming in the case of N. succinea (at least, in the Woods
Hole area) seems to be more closely associated with the lunar cycle (Lillie and
Just, 1913) than is the appearance of the heteronereid stage of N. riisei in Puerto
Rican waters.

Ccratoncrcis inirabilis Kinberg **. Small heteronereids of this species (approxi-
mately 7 mm. in length) were collected at night over coral reefs. They were never
observed at the dock. Night collections over reefs were begun on March 11, 1955,
when the only suggestion of real swarming of this species was observed. The
heteronereids shed their gametes when isolated. Males appear yellow in color prior
to shedding and pinkish (posterior portion) after shedding. Females appear whitish
before and pinkish after shedding. The eggs, approximately 120-125 micra in the
long diameter, are flattened spheres suggesting biconvex discs. Artificial fertiliza-
tion was attempted whenever both males and females were collected. It was always
successful. Embryos swim within 8 hours after fertilization, trochophores develop
within 12 hours after fertilization, and beautiful larvae with three bushy sets of
setae develop within 211> days. A few have been kept alive for 22 days but devel-
oped no additional setae. Collections were made from March 1 1 to December 14,
1955, but both sexes were observed only during the new moon each month with
the exception of March 11, three days after full moon, when the best batch of eggs
was obtained (to date this exception has not been explained), and three days before
the first quarter in September when a few were obtained. It should be noted that
only one sex in the heteronereid form was collected during the new moons of June,
September, and December.

Fauvel (195lc) describes several species of Ceratdncreis, including C. inirabilis,
from the night collections in the Gulf of Tadjoura. In several species, only
heteronereid males were caught. In the case of C. inirabilis, the male epitoke was
caught in numbers with a few females (mostly in fragments). Fauvel points out
that while this species is very widely distributed in all warm regions of the Atlantic,
of the Pacific and particularly of the Indian Ocean (with which the Gulf of Tadjoura
is indirectly connected), the epitokal form has been only rarely encountered, the few
females being mainly fragments. In the Puerto Rican waters the writer was ap-
parently fortunate in obtaining both sexes in the heteronereid form for embryological
purposes.

Nereis sp**. This is a small pink species, measuring approximately 2 cm. in
length, with a rather thick body. This worm was frequently collected along with
Ceratonercis and other small nereids, but insofar as can be determined only the
male heteronereids were observed. This has been the experience of other investi-
gators (see Fauvel, 1951a-d).

Platynereis dumerilii (Audouin and M. Edwards) **. This small pink het-
eronereid, flattened dorso-ventrally, is usually somewhat smaller than the preceding.
The heteronereid stage was collected frequently along with Ceratonereis and Nereis
sp. Again, only male heteronereids were detected. Fauvel (1951d) describes
several species of Platynereis but not P. dumerilii. It is interesting to note that in
the Platynereis dumerilii at Naples which breeds from October through May
spectacular swarming has been observed after the full moon in May (Just, 1929),
while only a few heteronereids were observed in Puerto Rico and these during the
last quarter of the moon and the new moon (several observations). The het-

BREEDING OF PUERTO RICAN POLYCHAETES 53

eronereid stages of P. ditinerilii were also observed between 8:00 and 11:00 P.M.
in March, 1955, during the dark of the moon (no moon) on Loch Hourn,
Scotland (Dr. L. R. Fisher, personal communication, 1957). These observations
suggest that P. dumcrilii is able to tolerate a wide temperature range for breeding.

Nereis allenae (Pettibone). Only two heteronereids of this small species (ap-
proximately 1 cm. long) have been collected, one on August 25 (first quarter of the
moon) and one on September 15 (new moon), 1955. Both were collected during
the evening from the shallow water over the reef between the Marine Laboratory
and Caballo Blanco Island, Parguera, Puerto Rico. This was the first time that the
writer had observed polychaete eggs laid in short strings resembling blue-green
algae. The eggs made up a single row of cells (approximately 30 eggs in some
strings). Within about an hour after laying, the eggs became isolated due to the
dissolution of the substance (jelly?) holding the eggs together. The eggs after
fixation measure approximately 160 micra in diameter. This polychaete is a new
species (Pettibone, 1956).

Several other species of nereids are represented by heteronereids (including egg-
laying females) in night collections but they have not, as yet, been identified.

FAMILY GLYCERIDAE. Glyccra fsphyrabrancha Schmarda **. On the
evening of October 31, 1955, between 8 and 8:40 P.M., two females, 28 cm. and
23 cm. long, respectively (when fixed), and a long male segment of this species-
were observed swimming in surface water at the dock light. They were caught in a
dip net and isolated in finger bowls, whereupon the shorter female and the male-
segment shed their gametes. The eggs (resembling flattened tops and approxi-
mately 125 micra in diameter) are whitish, with a large central germinal vesicle
(50 micra in diameter) and finely granular cytoplasm. They are liberated in two
streams, apparently from two pores about midway down the body. Two drops of
diluted very active spermatozoa were used to inseminate the eggs. At approxi-
mately 9:30 P.M. another female was observed and caught with the dip net. The
relatively few eggs suggested she may have shed already. Within 1 hour and 40
minutes after insemination a number of the eggs of the first female were at the 2- to
4-cell stage (mostly the latter) but a number had not cleaved. Within 11 hours
many ciliated swimmers were observed in the dish. Within the next 24 hours
larvae with a prominent prototroch had formed and within the next 24 hours (age,.
21/2 days) elongating larvae with a differentiating gut had developed.

EUNICIDAE. Eunice fpennata (Miiller) **. These slender worms, ap-
proximately 714 cm. long, build small sand tubes, externally covered with shell..
They are found on the under surface of coral in shallow water. When the coelom
of a female is packed with eggs, the posterior half of the body appears green due to
the pigment within the eggs. The eggs, approximately 175 X 160 micra in di-
ameter, are oval with a prominent germinal vesicle. The posterior half of males
with spermatozoa appears cream in color. Not many specimens have been col-
lected, but females with eggs have been observed in February, March, April, May
(one female was observed over a reef on the evening of May 6), September, and
November, 1955. The few attempts at artificial fertilization were unsuccessful.

Eunice (Nicidion} sp.**. This is a small species about the size of E. fpennata.
The worms are found on the under surface of coral in association with E. Fpennata
tubes, or close to them, and may occur in clumps of several individuals twined about
one another. The eggs (observed only in September) are bright pink and may be

54 M. JEAN ALLEN

observed readily through the body wall. This species has been observed only in
September, October, and November, but no definite attempts were made to look for
it. Fauvel (1951d) identified several species from the Gulf of Tadjoura collection,
including Eunice (Nicidion) cdcntuluni which he describes as a cosmopolitan and
widely distributed species living in the Indian Ocean, Pacific, and Atlantic.

Lysidice sp.**. Between 7:30 and 9:00 P.M. on the evening of December 14,
1955, a number of worms wrere observed in the circle of light swimming in the
shallow water over a reef near the laboratory and several were shedding gametes.
The adults were isolated and the eggs were inseminated artificially. In about three
hours spherical surface swimmers were observed and 16 hours after insemination
orange-red eyespots were visible. This species also was collected in November
but shedding was not observed. Fauvel (1951d) identified Lysidice collaris from
the Gulf of Tadjoura and described it as being widely distributed in warm seas.

AMPHINOMIDAE. Hennodice carnnculata (Pallas) *. This large fireworm
is rather plentiful in the Puerto Rican area and is locally referred to as a "marine
centipede." It has been observed during the daytime lying about on reefs in
shallow water or on the sandy to muddy bottoms surrounding mangrove islands.
An occasional individual was checked for gametes at least once a month from Janu-
ary through June, 1955, by making a small slit in the body wall. No gametes were
observed in this period except on January 13 when an injured female shed many
orange-colored eggs, approximately 100 micra in diameter. In November, 1955,
a few specimens were again checked for gametes. Eggs were observed (though
not very abundant) in one female on November 8, and in another on November 29.

Eurythoc coinf>lanata (Pallas) *. This is another common "marine centipede"
often collected along with H. carunculata on shallow reefs. Eggs have not been
observed but very small worms have been collected early in November (one meas-
ured 19 mm. and a second 13 mm. when fixed). Fauvel (1951a) identified one
specimen of this species taken from the Reef of Ambouli, noting that it is found
in all tropical seas. Both this species and the preceding are found in the Gulf of
Mexico along the coast of Florida ( Hartman, 1951 ) .

Notopygos crinita Grube **. This species of fireworm has been observed only
in May. On the evening of May 13, 1955, two males and one female were ob-
served swimming over a reef. When isolated in finger bowls, they shed immedi-
ately. The eggs fertilized readily, developing into much flattened, top-like trocho-
phores within 14 hours. Four days after insemination, they had developed into
weird-looking larvae with two sets of much elongated glass-like setae. The larvae
died within the next two days. On the evening of May 20 a small male was
collected similarly. It did not shed until taken to the laboratory. No other indi-
viduals were observed.

FAMILY POLYNOIDAE. Chactacanthus magnificns (Grube) *. Often one
to several of these worms were collected incidentally on coral reefs in shallow water
from September, 1954, to May, 1955, but no shedding was observed nor were
gametes seen when the body wall was pricked. Later (November 14, 1955) a few
more were collected. Upon puncturing the body wall one of these shed a few
whitish, opaque, almost spherical eggs, approximately 100 X 85 mm. in diameter.

FAMILY TEREBELLIDAE. Thelepus sctosus (Quatrefages) *. This
species is found in tubes, covered by sand and shells, on the under surface of coral
in shallow water. Pink, disc-shaped eggs (the larger measuring approximately 185

BREEDING OF PUERTO RICAN POLYCHAETES 55

micra in diameter) are readily observed through the body wall moving about within
the coelomic fluid. A number of individuals have been collected each month from
November, 1954, to November, 1955, at various phases of the moon (collections
were made during the day) but no fertilization has been attained. Individuals
when collected were isolated and checked periodically for shedding. Changing the
temperature, and also the sea water in which they have been kept, was done con-
sistently but did not prove successful in stimulating shedding. In one instance, on
March 31, a male shed active spermatozoa within 30 minutes after isolation; an
attempt at artificial fertilization was unsuccessful. On September 10, one individual
out of six shed many eggs on the day after it was collected (following two changes
of temperature and of sea water). The breeding habits of Thelepus may be similar
to those of its relative, Amphitrite ornata Verrill. Mead (1897) collected approxi-
mately 800 specimens of A. ornata from June through August and only rarely ob-
tained ripe gametes. He stated (p. 229) that it "is useless to cut the animals open,
for, if the sexual products are mature, they will be discharged, usually at about 6
o'clock in the evening, more often on the day of capture, sometimes the next day.
The rarity of ripe specimens is partly compensated for by the enormous number
of eggs which may be obtained from one female." Scott (1909) concluded that the
height of spawning for A. ornata is in July and is closely related to the spring tide.
As suggested by the above observations, no definite conclusions can be drawn, as
yet, regarding the specific breeding habits of Thelepus setosus.

Eiipolyiiiiiia crassicornis (Schmarda) *. This worm lives in a collapsible tube
of mucus reinforced by pebbles, sand, shell, calcareous algae, etc. The breeding
habits of this species probably are similar to those of Thelepus. Eggs, if plentiful
enough, appear as lavender areas seen through the body wall. The color is due to
the pigment in the ova. The larger of the disc-shaped eggs are approximately 175
micra in diameter. This species likewise has been checked the year round, from
January to December, 1955, and treated in the same manner as Thelcpns. On
March 15, at 5:00 P.M., individuals collected that day were isolated, and by 8:00
P.M. two females had shed many eggs, and one a few eggs. By then, the germinal
vesicle had broken and one male had shed active spermatozoa. Two batches of
eggs were inseminated; one showed no development and the second had an occa-
sional swimmer moving slowly within 3% hours after insemination. All of these
swimmers died within the next day and a half. Possibly the eggs were over-ripe.
On June 27, another attempt at artificial insemination was unsuccessful. On
September 1 1 , six males shed active spermatozoa. Artificial insemination was tried
although the female used had not shed normally. No development occurred. On
November 15, one out of the six worms collected that morning had shed active
spermatozoa by 6:00 P.M., and on November 29 one of two males shed at approxi-
mately 6:00 P.M.

SABELLIDAE. Sabellastarte magnifica (Shaw) **. This large feather cluster
lives in a membranous tube attached to coral in shallow water. Relatively few
specimens have been checked the year round for gametes. The rather small eggs
(approximately 55 micra in diameter) are whitish discs with a central germinal
vesicle. They tend to shrink when freed from the coelom, suggesting that they are
not osmotically balanced with the sea water. Eggs have been observed in August,
September, and October, and males with active spermatozoa have been collected

56 M. JEAN ALLEN

in September, October, and November (one male, November 23). Tbe few at-
tempts at artificial fertilization were unsuccessful.

Sabella mclanostigina Schmarda*. This species of feather duster, smaller and
more slender than the preceding, lives in a slender membranous tube (covered with
fine silt and mud) found attached to stones or shells or to mangrove roots. Indi-
viduals with eggs (most of the eggs shrivel in sea water) and motile spermatozoa
were collected from January through May. No gametes were observed in the few
specimens checked in June, July, and August. One check was made in September
- two females examined had minute eggs ; a male individual, considerably smaller
than the females, had active spermatozoa. Both eggs and active spermatozoa were
noted in October and November. The few attempts at artificial fertilization, scat-
tered throughout the year, proved unsuccessful.

SERPULIDAE. Spirobranchus tricornis (Morch) *. This small feather
duster builds a calcareous tube in the common coral, Porites porites. The eggs, ap-
proximately 80-85 micra in diameter, are flattened spheres. They are relatively
clear except for the orange pigment granules in the cytoplasm. The sex of indi-
viduals can often be determined with the naked eye if the coelom is packed with
gametes. Females have bright orange tips (basal end), as the pigment in the mass
of eggs shows through the body wall ; males have whitish or creamish tips when
packed with spermatozoa. This species has been checked for gametes in every
month from September, 1954, to November, 1955. Only a few swimmers developed
following attempts at artificial fertilization from November through February. A
number cleaved following artificial fertilization from March through October, 1955.
These developed into typical trochophores, some batches swarming with the ciliated
swimmers. A member of this genus, Spirobranchus gigantcus (Pallas), has been
described by Fauvel (195ld) in the collection from the Gulf of Tadjoura. He
notes that this species occurs in all intertropical regions of the Indian Ocean, Pacific,
and Atlantic, mainly on coral reefs.

It is hoped that the positive results recorded here will prove useful to other
investigators interested in tropical or subtropical marine forms. Negative results
should be viewed with caution.

LITERATURE CITED

BERKELEY, C., AND E. BERKELEY, 1953. Swarming of Nereis succinca (Leuckart) off the east

coast of Canada. Nature, 171: 847.
BUMPUS, H. C., 1898a. The breeding of animals at Woods Holl during the month of March,

1898. Science, 7: 485-487.
BUMPUS, H. C., 1898b. The breeding of animals at Woods Holl during the month of May,

1898. Science, 8: 58-61.
BUMPUS, H. C., 1898c. The breeding of animals at Woods Holl during the months of June,

July and August. Science, 8: 850-858.
DALES, R. P., 1951. Observations on the structures and life history of Autolytus prolifer

(O. F. Miiller). /. Mar. Biol. Assoc., 30: 119-128.
FAUVEL, P., 1951a. Annelides Polychetes du Golfe de Tadjoura recueillies par M. J.-L.

Dantan en 1933, au cours de peches nocturnes a la lumiere. Bull. Mus. Natl. Hist. Nat.,

23 : 287-294.
FAUVEL, P., 1951b. Annelides Polychetes du Golfe de Tadjoura recueillies par M. J.-L.

Dantan en 1933, au cours de peches nocturnes a la lumiere (suite). Bull. Mus. Natl.

Hist. Nat., 23 : 381-389.

BREEDING OF PUERTO RICAN POLYCHAETES 57

FAUVEL, P., 1951c. Annelides Polychetes du Golfe de Tadjoura recueillies par M. J.-L.

Dantan en 1934, au cours de peches nocturnes a la lumiere (suite). Bull Mus. Natl.

Hist. Nat., 23 : 519-526.
FAUVEL, P., 1951d. Annelides Polychetes du Golfe de Tadjoura recueillies par M. J.-L.

Dantan en 1934, au cours de peches nocturnes a la lumiere (suite et fin). Bull. Mus.

Nat!. Hist. Nat., 23 : 630-640.
HARTMAN, O., 1951. The littoral marine annelids of the Gulf of Mexico. Pnbl. Inst. Mar.

Sci., 2 : 7-124.

JUST, E. E.. 1929. Breeding habits of Nereis dnmerilii at Naples. Biol. Bull.. 57: 307-310.
LILLIE, F. R., AND E. E. JUST, 1913. Breeding habits of the heteronereis form of Nereis

liinbata at Woods Hole, Massachusetts. Biol. Bull., 24: 147-168.
MAcGiNiTiE, G. E., 1955. Distribution and ecology of the marine invertebrates of Point

Barrow, Alaska. Smithsonian Misc. Coll., 128: 1-201.

MEAD, A. D., 1897. The early development of marine annelids. /. Morph., 13 : 227-326.
MEAD, A. D., 1898. The breeding of animals at Woods Holl during the month of April, 1898.

Science, 7 : 702-704.

PETTIBONE, M. H., 1954. Marine polychaete worms from Point Barrow, Alaska, with addi-
tional records from the North Atlantic and North Pacific. Proc. U. S. Nat. Mus.,

103: 203-356.
PETTIBONE, M. H., 1956. Some polychaete worms of the families Hesionidae, Syllidae, and

Nereidae from the east coast of North America, West Indies, and Gulf of Mexico.

J, Il'ash. Acad. Sci., 46: 281-294.
SCOTT, J. W., 1909. Some egg-laying habits of Amphitrite ornata Verrill. Biol. Bull., 17:

327-340.

THORSON, G., 1946. Reproduction and larval development of Danish marine bottom inverte-
brates, with special reference to the planktonic larvae in the sound (0resund). Mcdd.

Konnn. Dan-marks Fisk.-Havundersjfgelser. Ser.: Plankton, 4: 1-523.

ENCYSTMENT STAGES OF DICTYOSTELIUM *

JOAN CORMIER BLASKOVICS 2 AND KENNETH B. RAPER
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin

The genus Dictyostelium Brefeld (1869) is representative of the Acrasieae, a
group of simple, cellular slime molds, wherein the life cycle consists of an amoeboid
vegetative phase and a plant-like fruiting phase. The vegetative phase of these primi-
tive micro-organisms is characterized by the independent movement and multiplica-
tion of free-living myxamoebae which feed by the ingestion and digestion of bacterial
cells. The fruiting phase normally begins with the exhaustion of this food supply
and is first evidenced by the coordinated inflowing of the myxamoebae to form
wheel-shaped aggregates, or pseitdoplasinodia. It attains its definitive expression
as a portion of the myxamoebae thus assembled becomes transformed into vacuolate,
parenchyma-like cells to form the upright stalk, or sorophore, whilst the remainder
differentiate into capsule-shaped reproductive cells, or spores, to form the elevated
spore mass, or sonis, of the slime mold fructification, or sorocarp. In most cultures
of Dictyostelium grown under favorable cultural conditions, virtually all of the
former vegetative myxamoebae enter directly into radiate pseudoplasmodia and
subsequently differentiate into either stalk cells or spores (Olive, 1902; Raper,
1935, 1940a, 1940b; Bonner, 1944). However, marked exceptions to this be-
havioral pattern occur in certain species and strains.

Under some conditions, not wholly understood, many of the vegetative
myxamoebae never enter the fruiting state (i.e., aggregate to form pseudo-
plasmodia) but as individual cells enter an encystment stage. Such resting cells
are termed microcysts (Fig. 1). This designation was first applied by Cienkowski
(fide Olive, 1902) to describe those myxamoebae of Guttnlina which under unfavor-
able conditions tended to form rounded protoplasmic bodies with definite ecto-
plasmic membranes. Olive widened the application to include other Acrasieae
and stated that microcyst formation occurred under unfavorable conditions such as
slow drying in hanging-drop preparations. In our investigations such individually
encysted cells have been observed from time to time and in large numbers in agar
plate cultures of Dictyostelium mucoroides Brefeld (1869), and with even greater
frequency in D. minntitni Raper (1941), D. polycephalnni Raper (1956a, 1956b),
Polysphondylium palliduin Olive (1901, 1902), and Acytostelinm Icptosomum
Raper (1956b). They are believed to occur in greater or lesser numbers in all
aging cultures.

An additional, multicellular encystment stage occurs in occasional strains of
Dictyostelium mucoroides and in many isolates of D. minutum. These relatively
complex structures arise by a morphogenetic process possibly alternative to normal

1 Research investigations reported in this paper have been aided by grants from the
National Science Foundation and the National Institutes of Health.

2 Present address : Department of Pharmacology, Marquette University Medical School,
Mihvaukee, Wisconsin.

58

ENCYSTMENT STAGES OF DICTYOSTELIUM 59

sorocarp formation. Because of their larger dimensions and their multicellular
origin and constitution, these bodies are termed macrocysts (Fig. 2) . This designa-
tion was first applied by Raper in 1951 with reference to structures seen in certain
cultures of D. minntum isolated from soil, but no description of the macrocysts
was given. More recently, Cormier and Raper (1955) and Raper (1956b) have
reported briefly concerning their formation and structure. It is of special interest
that Brefeld, in his original paper on D. mucoroides (1869), described and illus-
trated as "dwarfed sporangia" structures which are believed to have represented
macrocysts (Fig. 3).

The primary purpose of this investigation has been to extend our observa-
tions on these long neglected structures and to seek answers to three basic questions
pertaining to them, namely: How are the macrocysts and microcysts formed?
What conditions favor their development ? What are the roles of these encystment
stages in the life cycle of the Acrasieae? A resume of our present knowledge of
these matters is presented herewith.

MATERIALS AND METHODS
Micro-organisms investigated

Several cultures of Dictyosteliuni were examined relative to the production and
possible function of the macrocysts, including Dictyosteliuui mucoroides (Strains
S-28b, NC-12, and WS-47) and D. minutum (Pur-8a and WS-56-2). Dictyo-
steliuui mucoroides was the slime mold studied most intensively, particularly strain
S-28b which produces abundant macrocysts. Macrocysts were first observed and
photographed in strain NC-12 when this slime mold was isolated in 1937 (Fig. 2).

Microcysts of several species of the Acrasieae were studied, including those of
Dictyosteliuni minutum (WS-116b), D. polycephalum (S^4), Polysphondylium
pallidum (WS-116c), and Acytostelium leptosomum (FG-12a). Those of D.
polycephalum are illustrated in Figure 1.

Different bacterial associates were investigated as food sources for the slime
molds and for their possible influence upon macrocyst formation. Included among
gram-negative species were Escherichia coli (No. B-281), Aerobacter sp. (Singh's
strain), Aerobacter aerogenes (Sussman's strain), Flavobacteriitm sp. (DIF),
Serratia marc esc ens (No. B-175), and Pseudomonas fluorescens (No. B-112) ; in-
cluded among gram-positive species were Bacillus megaterium (No. B-160), B.
subtilis (Sarles' strain), and Sarcina lute a (No. B-1018). Escherichia coli, the
organism used most commonly, is a short rod which is mildly proteolytic and
ferments both dextrose and lactose ; the myxamoebae appeared to grow best when
feeding upon this bacterium.

Cultivation of the slime molds

Environmental and cultural conditions, including (1) temperature, (2) culture
media, (3) pH of substrate, (4) ammonium ion concentration, and (5) per cent
relative humidity, were varied to determine their effect upon macrocyst formation
in Dictyosteliuni.

Incubation temperatures used in most of the experiments were 10, 15, 20, 25,

60

J. C. BLASKOVICS AND K. B. RAPER
PLATE I

I

ENCYSTMENT STAGES OF DICTYOSTELIUM 61

and 30° C. The optimum growth temperature for the macrocyst-forming strains
of Dictyostelium mucoroides was about 20° C., whereas that of the D. miniituin
isolates more nearly approximated 25° C.

Many different media were employed for the cultivation of the slime molds and
the associated nutritive bacteria, which, for the most part, were patterned after
substrates previously reported by Raper (1951). An agar medium containing
0.1% lactose and 0.1% peptone (0.1 L-P agar) was used most extensively since
it provided the most reproducible growth and the most consistent macrocyst forma-
tion in D. mucoroides and in D. iiiinutmn. Horse dung-infusion and 0.05% uric
acid agars 3 were also employed because of Brefeld's report (1869) that dung
extract and uric acid induced spore germination.

The pH of the substrates employed for the conjoint growth of the slime molds
and associated bacteria was varied by buffering with KC1, potassium acid phthalate,
KH,PO4, and KSBO3 as recommended by Clark and Lubs (Clark, 1928). Bac-
teria were also pre-grown on buffered 0.1 L-P medium of varied pH and trans-
ferred to unbuffered medium prior to inoculation with the slime mold (Raper,
1951). Experiments to determine the possible effect of ammonium ions on growth
and macrocyst formation in Dictyostelium were patterned after those reported by
Cohen (1953).

Different relative humidities were obtained by placing specified concentrations
of HL)SO4 in water in desiccators, as reported by Wilson (1921). Two types of
slide cultures were employed : ( 1 ) Maximov tissue culture slides containing one
ml. 0.1 L-P agar inoculated with a mixed suspension of spores, or myxamoebae
and macrocysts, of D. mucoroides and E. coli cells ; (2) plain flat slides spread with
1.0 ml. molten 0.1 L-P agar and inoculated by on-flowing a mixed suspension of
slime mold and bacteria. The latter slides were supported in an upright position
by small wooden blocks during incubation.

Agar plate cultures were grown routinely in glass Petri dishes, to maintain a
high per cent relative humidity, and incubated in the dark at varying temperatures.
The plates were cross-streaked or completely smeared with the bacterial associate
and then inoculated at the center with spores, macrocysts, or myxamoebae of the
selected slime mold, the type of inoculum and bacterial associate being varied with
the experiment.

3 Dung decoction was made by autoclaving 100 grams of fresh horse dung/liter of water for
twenty minutes at 15 pounds' pressure. The resulting decoction was filtered, solidified with
1.5% agar, and re-sterilized (final pH 6.1-6.35). Uric acid agar consisted of a 0.05% aqueous
solution of uric acid to which 2.0% agar was added.

PLATE I

FIGURE 1. Microcysts of Dictyostelium polycephalum, representing individually encysted
vegetative myxamoebae. X 530.

FIGURE 2. Macrocysts of Dictyostelium mucoroides, Strain MC-12, showing the charac-
teristic habit and structure of these bodies ; the cellular stalk of a normal sorocarp appears at the
far left. X 250.

FIGURE 3. Illustrations from Brefeld's description of Dictyostelium mucoroides (1869),
from left to right : "Dwarfed sporangia," which are believed to be identical with the macrocysts
reported in this paper ; "small sporangia" with "rudimentary stalks" in the spore-forming plasm ;
"sporangium" with a small stalk surrounded by an enclosing membrane. < 300.

62 J. C. BLASKOVICS AND K. B. RAPER

Reagents

Several reagents were employed to establish the cellulosic nature of the walls of
the macrocysts, namely :

( 1 ) Chloroiodide of zinc solution was prepared by dissolving 30 grams of
ZnCl, 5 grams of KI, and 0.89 gram of iodine in 14 ml. of distilled water (Stevens,
1916). The material to be tested was mounted in water, after which the reagent
was applied to one edge of the cover glass and drawn under it by placing a piece
of filter paper against the opposite edge. Cellulosic material stains violet-blue.

(2) Congo red was prepared as a 0.5% aqueous solution of the dye made
alkaline by adding two or three drops of concentrated NaOH. In an alkaline
solution this reagent stains cellulose red, which when put in HC1 turns blue
(Raper and Fennel], 1952). This is a presumptive but non-specific test for cellu-
lose.

(3) Schweitzer's reagent was prepared by bubbling air through 60 ml. of
NH4OH containing 10 grams of fine copper turnings for one hour (Hodgman,
1951). Cellulose is quickly dissolved by this strong cuprammonium solution. In
practice, the macrocysts were generally mounted in water and the reagent was
applied by drawing it under the cover glass, hence diluting and advantageously
slowing its action. A 72% aqueous solution of H2SO4 was also used as a cellulose
solvent.

(4) A strong birefringence in polarized light was likewise interpreted as con-
firming the predominantly cellulosic composition of the macrocyst wall.

Nile blue sulfate was employed as a vital dye for staining myxamoebae and
progressive developmental stages as reported by Bonner (1952). It was also
incorporated into 0.1 L-P agar prior to inoculation with E. coli and the slime
mold, ca. 3 ml. /liter of a Q.5% aqueous solution being employed for this purpose.
Used directly, or when added to the growth medium, it stained vegetative
myxamoebae a very light blue, whereas aggregating myxamoebae and the cells
of developing macrocysts assumed somewhat darker shades and appeared less
granular.

EXPERIMENTAL RESULTS
Origin and morphology of macrocysts

Macrocysts are flattened, irregularly circular to ellipsoidal multicellular struc-
tures, ranging from 25 to 50 ju, in diameter. The myxamoebae which contribute
to their formation appear normal for Dictyostelium mucoroides and D. minutum
in every respect as they move and feed upon bacterial cells, re-dividing until the
available food supply is exhausted. As this occurs, the myxamoebae begin to
aggregate into pseudoplasmodia which, except for their generally limited di-
mensions, appear basically similar to other pseudoplasmodia that proceed to sorocarp
formation (Fig. 4). However, instead of producing upright sorocarps, the
myxamoebae comprising these aggregates remain in compact heaps and subse-
quently become surrounded by comparatively thick, cellulose walls. When only a
few myxamoebae aggregate to form pseudoplasmodia, small macrocysts develop
singly ; when larger numbers of cells are involved, the macrocysts are somewhat
larger and may occur in groups of varying size, ranging from small packets to

ENCYSTMENT STAGES OF DICTYOSTELIUM 63

sheet-like ribbons, depending upon the number of myxamoebae massed together.
In these larger aggregations, incipient macrocysts are delimited by the secretion
of delicate cellulosic membranes around limited groups of cells more or less regularly
spaced throughout the primary aggregate. This is followed by the subsequent
deposition of thick, predominantly cellulosic walls, mostly circular to oval in pattern,
that become the relatively rigid boundaries of the individual macrocysts. Com-
monly the secondary wall is laid down in general conformity with the primary
membrane, but not infrequently two, three or even more macrocysts develop within
an initial area of demarcation (Figs. 5-7).

Somewhat prior to the first evidence of secondary wall formation around the
nascent macrocyst, cells in the center of the previously undifferentiated mass ex-
hibit signs of modification and become surrounded individually by strongly re-
fractive membranes. This process of cellular differentiation advances outward
until all of the cells comprising the macrocyst are transformed into closely packed,
seemingly firm-walled cells, termed cndocytcs (Figs. 8-9). The constitutive
myxamoebae show no obvious orientation during the early stages of this process;
but as differentiation proceeds, the peripheral and still amoeboid cells become con-
spicuously elongate as if appressed against the surface of the steadily enlarging body
of endocytes (Fig. 6). Parallel with this progressive differentiation of the endo-
cytes, but in a manner not yet understood, the whole body of functionallv integrated
cells succeeds in building around itself a tough and relatively heavy wall that is rich
in cellulose. Significantly, the position of this wall is not determined by that of the
thin primary membrane which initially delimited the bloc of myxamoebae that
jjtif/Jit collaborate in macrocyst formation; rather it is determined by the group (s)
of cells which first differentiate as endocytes. This is clearly evident from the
examination of primary aggregates of different dimensions. If the aggregate is
small, the subsequently formed macrocyst wall will conform generally to that of the
primary membrane. If the aggregate is relatively large, several centers of endocyte
formation will arise simultaneously, and outward from these loci, cells will differ-
entiate progressively to form separate and independent macrocysts, each with its
own characteristic heavy wall but all contained within the primary membrane (Figs.
7-9. 25—27). The entire process of macrocyst formation is normally completed
within 18 to 24 hours. Successive stages in macrocyst formation are illustrated
in Figures 4 through 9.

The endocytes may be isodiametric or slightly elongate, ranging in size from
about 3.6 to 4.8 p. in diameter. As observed within the macrocyst, and when first
released by breaking the macrocyst wall, these cells normally appear polyhedral
in outline, but soon become spheroidal or ellipsoidal when no longer compressed by
adjacent endocytes (Fig. 9). For reasons still unknown they are appreciably
smaller than the myxamoebae which enter the primary aggregate, the latter usually
ranging between 6.0-8.0 p. in the unexpanded state. The smaller dimensions of
the endocytes may result from a substantial water loss during their differentiation,
or the contributing myxamoebae may possibly undergo division prior to the de-
velopment of the refractive membranes which so strikingly distinguish them from
other cells still amoeboid (Fig. 8).

Endocytes normally remain as distinct cellular entities within the macrocyst for
a period of two or three weeks (Fig. 10), after which they commonly lose their
identity and the protoplasmic content of the entire structure assumes a homo-

64 J. C. BLASKOVICS AND K. B. RAPKR

PLATE II. Origin and structure of macrocysts in Dictyostcliitin i/ntcoroidcs, Strain S-28b.

FIGURE 4. Small radiate pseudoplasmodia which lead to the formation of macrocysts.
X 80.

FIGURE 5. Completed aggregations, consisting of irregular mounds of myxamoebae, prior
to the formation of the primary membranes that delimit incipient macrocysts. K 80.

FIGURE 6. Enlarged view of a later stage in macrocyst development, showing incipient
macrocysts and the orientation of their constituent myxamoebae. < 360.

FIGURE 7. More advanced stage showing clusters of differentiated endocytes centrally
located in developing macrocysts. X 80.

FIGURE 8. Much enlarged view of two macrocysts in process of formation within a single

ENCYSTMENT STAGES OF DICTYOSTELIUM 65

geneous appearance. With further aging, from four to six weeks, the apparently
acellular content often shrinks to approximately 60-75% of its original volume,
and in the form of a compacted, brownish mass occupies a central position within
the partially empty macrocyst wall (Fig. 13). The surface of this central body
appears slightly irregular and affords no clue to the presence of a continuous
bounding membrane ; rather it suggests a plasmolyzed and shrunken mass that has
been subjected to uneven pressures and tensions during the process of contraction.

Many questions remain unanswered concerning the sequence of events leading
to the advanced acellular structures just described. We have inadequate informa-
tion concerning their true nature, and we have only incomplete knowledge of their
significance in the life cycle of those slime molds wrhere they occur. Nevertheless,
a detailed study of macrocysts of different ages, and under many conditions, has
revealed a considerable body of information concerning their development and
behavior. If an endocyte-filled macrocyst is subjected to pressure in an aqueous
mount, the heavy cellulose wall breaks, much as a hollow rubber ball, and the endo-
cytes pour out, undergoing the limited changes in shape already noted but retaining
their identity as relatively firm-walled cells. In contrast, if an older macrocyst
from which the discrete endocytes have disappeared is similarly crushed, the en-
veloping wall breaks in a comparable manner, but the entire content flows out as a
structureless fluid containing innumerable fine particles that immediately exhibit
brownian movement as they enter a more aqueous environment.

The explanation for endocyte disappearance in naturally aged macrocysts re-
mains unknowrn, but a superficially similar state can be produced artificially with
alkaline solutions. This \vas first observed when Schweitzer's reagent was applied
to preparations of young macrocysts. Upon contact with the cuprammonium so-
lution, the refractive wralls of the endocytes disappeared, the seemingly merged
content of the entire macrocyst swelled, and with the partial dissolution of the
enveloping cellulose wall, the content emerged as a homogeneous and finely granular
mass superficially resembling a large and completely undifferentiated protoplast
(Fig. 17). A comparable disappearance of endocytes was subsequently observed
in preparations following the application of NaOH (1.0 and 0.1 N), albeit the
heavy cyst walls remained intact (Figs. 10-11). The application of HC1 (1.0 N)
to the same preparations prompted a most dramatic phenomenon, for upon contact
with this reagent the endocytes reappeared in their original numbers and positions
(Fig. 12). The same phenomenon of endocyte reversal accompanied the applica-
tion of acid following treatment of young macrocysts with Schweitzer's reagent.
If the exposed macrocyst was quite young the endocytes emerged individually as the
cellulose wall dissolved, and these collected into a spreading amorphous mass within
which cell boundaries remained faintly evident, as seen in Figure 16. If the cyst
was older (but still packed with endocytes) the content emerged as an intact,
seemingly homogeneous mass following similar treatment (Fig. 17). In each case
a discrete cellular structure reappeared with the addition of acid (Fig. 18).

Macrocyst germination has been observed with the emergence of amoeboid cells
from the ruptured cyst, and it was first thought that such induced disappearance

primary envelope, showing clusters of endocytes surrounded by undifferentiated myxamoebae.
x. 800. '

FIGURE 9. A body comparable to that shown in Figure 8, compressed to release endocytes
and still undifferentiated cells. X 800.

66

J. C. BLASKOVICS AND K. B. RAPER

PLATE III. Behavior of young (endocyte-filled) and aged (homogeneous) macrocysts in
D. mucoroidcs, Strain S-285, in the presence of alkali and acid.

ENCYSTMENT STAGES OF DICTYOSTELIUM 67

of endocytes might be analogous to the natural phenomena of macrocyst maturation
and germination. However, further experiments failed to substantiate this view,
for endocyte reversals did not occur at pH levels that permit growth and other
vital activities of the slime mold. Furthermore, in the alternate presence of acid
and alkaline solutions an initially endocyte-filled macrocyst could be interconverted
repeatedly from an obviously cellular to a seemingly homogeneous state. Such
reversal undoubtedly represents a physico-chemical reaction rather than a vital
phenomenon, since macrocysts pre-killed with iodine-alcohol or Schaudinn's fixa-
tive react as do untreated structures.

The aforementioned tests in combination with certain others have provided
substantial information regarding the wall structure of the macrocysts, and speci-
mens treated alternately with alkali and acid and stained with chloroiodide of zinc,
followed by Schweitzer's reagent, have proved particularly revealing. The "wall"
of a mature, endocyte-filled macrocyst is seen to consist of three strikingly differ-
ent parts : ( 1 ) an outer, loosely fitting primary covering of indeterminate form
which characteristically surrounds one or more macrocysts, and may be continuous
with, or adherent to, comparable envelopes of adjacent structures (Figs. 24-26) ;
(2) a very much thicker secondary layer that is uniform in thickness, smooth in
contour, and usually circular or ellipsoidal in outline (Fig. 26) ; and (3) an inner
membrane formed by the endocytes prior to their disappearance as discrete cellular
entities (Fig. 22). Both the primary covering and the secondary wall contain
cellulose, apparently embedded within a matrix of more resistant material (Fig.
24-25). Both stain blue in chloroiodide of zinc and both are birefringent when
viewed with polarized light, yet neither is completely dissolved by Schweitzer's
reagent. However, each loses its birefringence upon the addition of the cupram-
monium or an aqueous solution of 72% H2SO4. The primary membrane contains
relatively little cellulose, embedded within a mucus-like material, and is reminiscent
of the slime track and the slime sheath seen in D. discoidcitin ; in contrast, cellulose
constitutes the principal building substance of the thick secondary wall (Figs. 23
and 29), just as it does in the sorophore sheath of Dictyostelimn (Raper and
Fennell, 1952). The tertiary wall, if it may be so designated, contains no cellulose
and represents a continuous film formed in a peripheral position by the mass of
differentiating endocytes. This thin, innermost layer is non-rigid and contracts or
expands with changes in the volume and character of the protoplasmic material that

PLATE III

FIGURE 10. Ten-day-old macrocysts produced on 0.1 L-P medium containing Nile blue
sulfate, untreated. X 360.

FIGURE 11. The same macrocysts following exposure to 1.0 N NaOH ; note that all
evidence of the constituent endocytes has disappeared. X 360.

FIGURE 12. The same macrocysts following the application of 1.0 N HC1 ; note how the
endocytes have reappeared. X 360.

FIGURE 13. Six-week-old macrocysts showing the typical contracted homogeneous content
of such structures, untreated. X 360.

FIGURE 14. The same macrocysts following exposure to 1.0 N NaOH; note how the
contents have swelled. X 360.

FIGURE 15. The same macrocysts, displaced in their relative positions, following the ap-
plication of acid ; note that the cyst contents have contracted somewhat, but that they show no
evidence of a cellular structure. X 360.

68 J. C. BLASKOVICS AND K. B. RAPER

PLATE IV. Behavior of young and aged macrocysts in Schweitzer's reagent.

21

FIGURE 16. Young (endocyte-filled) macrocysts following exposure to Schweitzer's re-
agent, showing how the cuprammonium solution dissolves the cellulose walls of the macrocysts
allowing the endocytes, faintly discernible, to escape. X 360.

ENCYSTMENT STAGES OF DICTYOSTELIUM 69

it envelops, functioning as the tenacious covering that confines the merged cyto-
plasmic content of the aged macrocyst when this is released by dissolution of the
heavy cellulose wall (Fig. 21). In contrast, when an aged but untreated cyst is
broken by mechanical pressure this covering obviously ruptures with the cellulose
wall which surrounds it (see above).

Similar preparations afforded equally interesting if more perplexing observa-
tions concerning the behavior of the contracted acellular protoplasmic masses pres-
ent in the older macrocysts. When exposed to NaOH the shrunken content lost
the brownish color characteristic of aged cysts and swelled to fill completely the
heavy macrocyst wall. Upon the substitution of acid, the brownish pigmentation
returned in part and the content again contracted, but not to its former dimensions
(Figs. 13-15). Treated with Schweitzer's reagent the content swelled once more
and erupted from the disintegrating thick cellulose wall as a single, seemingly
homogeneous mass. Significantly, no return to a cellular structure has been ob-
served in the content of any older cysts treated with cuprammonium or NaOH
solutions upon the addition of acid (Fig. 15). Thus, there is evidence that the
endocytes actually disappear, either by fusion or disintegration, at the time the
aging macrocyst assumes a homogeneous appearance. But why does the emergent
content remain intact following treatment with cuprammonium (Fig. 21) instead
of flowing out freely as when the wall of the untreated homogeneous macrocyst is
broken? Does the protoplasmic content represent, in fact, a plasmodium-like mass
formed by the fusion of endocytes ? Possibly so. Does the macrocyst wall actually
consist of three layers, the innermost persisting about the freed protoplasmic body,
not because of its inherently greater strength but because it is cupramrnonium-
resistant? Possibly this is true, for upon the application of slight pressure this
bounding membrane breaks, permitting the fine granular content to escape whilst
the membrane per sc remains as a delicate, irregularly wrinkled and contracted
envelope exhibiting no birefringence. Alternatively, and more plausibly, if aged
macrocysts remain viable, as observations indicate, the thin hyaline envelope thus
demonstrated may, in the living state, represent only the inconspicuous and func-
tionally modified protoplasmic membrane of the contracted central body itself.

Two lines of evidence point to the presence of such a semipermeable membrane
at progressive stages in macrocyst development. When an endocyte-filled macro-
cyst is exposed to a saturated solution of NaCl or sucrose, the content as a whole
appears to become plasmolyzed and to withdraw7 from the surrounding thick cellu-
lose wall as seen in Figure 22, plainly demonstrating the presence of a continuous;

FIGURE 17. Older macrocysts (but still endocyte-filled) following exposure to this reagent;
note how the nearly homogeneous cyst contents remain intact, and how the formerly heavy
macrocyst walls are shrunken following the dissolution of their cellulose content. >< 360.

FIGURE 18. Macrocysts of the same age as those shown in Figure 16, treated with
Schweitzer's reagent and then exposed to 1.0 N HC1 ; note how the endocytes have reappeared,
even in the freed contents of a macrocyst (a). X 250.

FIGURE 19. Two-month-old macrocysts, untreated. X 360.

FIGURE 20. The same macrocysts following treatment with Schweitzer's reagent ; note
how the outer cellulose wall is being dissolved and how the membrane-encased content is es-
caping intact. X 360.

FIGURE 21. The same preparation after an additional 10 minutes, showing the intact cyst
contents completely free of the macrocyst walls ; the latter are no longer birefringent when
viewed with polarized light. X 360.

70

J. C. BLASKOVICS AND K. B. RAPER
PLATE V. Macrocyst structure.

28

FIGURE 22. Young (endocyte-filled) macrocysts in the presence of a concentrated sucrose
solution ; note how a membrane, the "tertiary wall," surrounds the endocytes and in the process
of plasmolysis pulls away from the rigid cellulose wall which is external to it. X 360.

FIGURE 23. Macrocysts of comparable age stained with chloroiodide of zinc and then sub-
jected to pressure to release the endocytes. X 300.

ENCYSTMENT STAGES OF DICTYOSTELIUM 71

and differentially permeable membrane external to the constituent endocytes but
internal to the heavy cellulose wall. The same unitary pattern of plasmolysis is
seen in macrocysts recently turned homogeneous, and in these there is no suggestion
of persistent identity for the contributory myxamoebae or endocytes as one might
expect if they remained as indistinguishable but nonetheless discrete cells. Aged
macrocysts likewise provide contributory evidence. When placed in relatively large
volumes of distilled water such structures show variable response depending upon the
temperature of incubation. In preparations held at 10° C. evident swelling of the
contracted homogeneous content is observed within 72 hours, and after 10 days
many empty macrocyst cases, together with abundant free myxamoebae, may be
observed (Figs. 41-42). At 15° C. little swelling occurs, even after 10 days, and
only an occasional empty case may be seen. At 20 to 25° C. these responses are
almost completely lacking (Fig. 40). This behavior is interpreted to indicate a
selective permeability mediated by a low and favorable incubation temperature, and
it is most unlikely that this could be attributable to the more conspicuous cellulose
wall. The evidence would seem to point, indisputably, to the membrane that sur-
rounds the shrunken cyst content.

The formation of macrocysts is observed not infrequently in Dictydstelium
tninutujn, but their occurrence in strains diagnosed as D. mucoroides is relatively
rare. In fact, not more than a half-dozen such isolates have been encountered
among the hundreds of strains of the latter species that we have examined. In
view of this, it is surprising and noteworthy that Brefeld (1869), in his description
of D. mucoroides, reported objects believed to be similar to the macrocysts de-
scribed above. His cultures were grown on microscope slides, and these structures
developed in older preparations of that type. He described the structures as
"dwarfed sporangia," since at times there was evidence of a rudimentary stalk in
the ''spore-forming plasm." He reported this type of sporangium to be enclosed
by a comparatively thick membrane of cellulose, which upon examination was ob-
served to be stratified and was stained violet with chloroiodide of zinc. This
account agrees well with our observations of macrocysts submitted to various tests.
He made no mention of the germination of "spores" from the "dwarfed sporangia."
Rudimentary stalks such as Brefeld described and illustrated (Fig. 3) have not
been observed during these investigations, and we believe that he may have ob-
served immature macrocysts and interpreted clusters of differentiating endocytes
as representing rudimentary stalks. In our experience, endocytes first appear in a
localized central position within the developing macrocyst, but occasionally such
cells do extend to the periphery along a particular radius before comparable dif-
ferentiation occurs throughout the macrocyst. However, we must not overlook the
possibility that, in his particular isolate cultivated under different conditions, he
may have encountered stages truly transitional between macrocysts and well-formed
sorocarps comprised of sorophores and sori. Certain evidence points to such a
possibility. The basic similarity of the aggregative processes leading to sorocarp

FIGURES 24-27. Selected preparations stained with chloroiodide of zinc to show the loosely
fitting primary membranes within which 1, 2 or 4 macrocysts have developed; the bodies shown
in Figure 27 are older, hence the heavy macrocyst walls stain darkly. X 360.

FIGURE 28. Two-month-old macrocysts as viewed with normal light. X 275.

FIGURE 29. The same as seen under polarized light. X 275.

72

J. C. BLASKOVICS AND K. B. RAPER

PLATE VI. Influence of substrates and incubation temperature upon macrocyst formation in

D. mitcoroidcs, Strain S-28b.

'v ;:'-:: '.'•::. •••iV..'.t.-: •*•*.• ••/•*•';• •*!.. ?'.••:"'

•'••-• . •• . » . •'* • , '• \ V*. • ." • • , • •' »«'••.**..*

:-':C.- ••%./ ."v:"--v;:-r -;; •: c" *" l-.'?4':^ ^-'O*1

.';•"/•••".' •" • •/•".•/••* • .*V- ,; %."e •". :••"••' • ••

«* • *«•* »* «*•* ** *-J! ! *„ *, . »";*•".*•-'***•

35

FIGURE 30. "Spore-forming clone" grown in association with R. coli on 0.1 L-P medium
at 20° C. X 2.

FIGURE 31. Enlarged view of a portion of this culture showing abundant sorocarps and a
complete absence of macrocysts. X 5.

FIGURE 32. The same culture as seen in Figure 30, but growing upon a medium containing
0.1% glucose-0.1% yeast extract, at 20° C. X 2.

ENCYSTMKNT STAGES OF DICTYOSTELIUM 73

formation, on the one hand, and to macrocysts on the other is indeed striking, and
\ve have commonly observed in a single microscopic field separate pseudoplasmodia
undergoing differentiation in these two directions simultaneously. Such a situation
is illustrated in Figure 36. Unfortunately, we have no adequate explanation of the
subtle differences that underlie this contrasting behavior, but it is easily conceivable
that one might encounter individual cases where the shift from one to the other of
these morphogenetic processes would be incomplete, as suggested by some of
Brefeld's illustrations.

The formation of macrocysts may in effect represent an aberration of the normal
fruiting process, or it may represent an alternative pattern of differentiation with
implications of far-reaching significance that are yet unappreciated. The heavy
cellulose wall which it develops bears in many ways a striking resemblance to the
sorophore sheath, so essential to the construction of the normal sorocarp (Raper
and Fennell, 1952), but with this important distinction: the wall of the macrocyst
is secreted external to the whole mass of myxamoebae that contribute to the
formation of this body, whereas the sorophore sheath is produced at a critical
circular locus within the mass by a limited group of specialized cells which subse-
quently differentiate as the vacuolate cellular elements of the sorophore itself.

We have, at present, an incomplete picture of the morphology of the macrocyst.
We have convincing evidence that they can germinate and re-initiate the life cycle
of the slime mold. However, we do not know the fate of the cells which enter the
macrocyst, and we do not know the origin of those which subsequently emerge.
Until such information is at hand we cannot compare in any definitive sense the
morphogenetic processes that underlie these contrasting developmental stages. A
thorough cytological and histological study is clearly needed and will be undertaken
at the earliest possible opportunity.

Factors influencing the formation of macrocysts

The obvious factor most directly affecting macrocyst formation in Dictyosteliitm
nine oroides and D. niiniituni is the inherent genetic constitution of the particular
isolate. Within our experience, a minority of D. minutuiu strains and only an
occasional isolate of D. inucoroides have exhibited this capacity. However, once
this ability has been demonstrated for a culture, it is sometimes possible to alter
markedly the relative proportions of macrocysts and of normal sorocarps by
changing the conditions under which the slime mold is cultivated. The responses
of D. inucoroides, strain S-28b, have been studied in considerable detail and the
observations subsequently recorded apply particularly to that strain. Whereas the
various factors that influence the ratios of sorocarps to macrocysts are invariably
interrelated, certain conditions which strongly affect these balances have been
identified and investigated more or less independently.

Temperature: Second only to genetic constitution is the influence of the incuba-
tion temperature. When cultivated at 24-25° C. in association with Escherichia

FIGURE 33. "Cyst-forming clone" growing in association with E. coli on 0.1 L-P medium
at 20° C, . X 2.

FIGURE 34. Enlarged view of a portion of this culture showing abundant macrocysts and
a complete absence of sorocarps. X 7.5.

FIGURE 35. The same culture as seen in Figure 33, but growing upon a medium containing
0.1% glucose-0.1% yeast extract at 20° C. X 2.

74

J. C. BLASKOVICS AND K. B. RAPER

PLATE VII. Macrocyst formation in Dictyostelium mitcoroidcs, Strain WS-47.

,*»'

i"

f

o

O

36

FIGURE 36. Radiate pseudoplasmodia leading to macrocyst formation (a) and to sorocarp
formation (b) within the same microscopic field, as seen with low magnification. X 24.

FIGURE 37. Irregular clump of macrocysts developing from a single pseudoplasmodium
such as that shown in the preceding figure. X 125.

FIGURE 38. Detail of some of the macrocysts seen in Figure 37. X 600.

ENCYSTMENT STAGES OF DICTYOSTELIUM

75

PLATE VIII. Macrocyst germination in Dictyostellum inncoroidcs. Strain S-28b.

FIGURE 39. Two-month-old macrocysts incubated continuously at 20° C. and removed from
an agar plate immediately before being photographed ; note the single macrocyst at upper right
which still contains endocytes. X 300.

FIGURE 40. Macrocysts similar to the preceding, but removed from agar and incubated
in distilled water at 20° C. for 10 days prior to photography; note that little evident change has
taken place, x 300.

FIGURE 41. Macrocysts like the preceding, but incubated for 10 days at 10° C. just prior
to being photographed ; note the empty cyst cases and the mass of free myxamoebae which have
escaped from these. X 300.

FIGURE 42. Photomicrograph showing a second field from the same preparation shown in
Figure 41 ; note the broken walls of the empty cyst cases, also that a few myxamoebae still
remain within the germinating cyst at top center. X 300.

76 J. C. BLASKOVICS AND K. B. RAPER

coli on 0.1 L-P, hay-infusion or thin hay-infusion agars, the stock strain of S-28b
regularly produces both sorocarps and macrocysts in abundance, although individual
cultures and even different areas within the same Petri dish commonly exhibit
conspicuously disproportionate ratios of these contrasting structures. If the tem-
perature is raised as little as two or three degrees the ratio of macrocysts to soro-
carps is increased substantially ; conversely, if it is lowered to 20° C. or less this
ratio is strongly depressed.

Clonal substrains of S-281) have been isolated which exhibit temperature sensi-
tivity even more dramatically. These were obtained by heating spore suspensions
in standard salt solution (Bonner, 1947) for varying lengths of time and then,
following appropriate dilution, plating the spores in association with Eschcrichia
coli on 0.1 L-P agar. Plaques, evidenced by clearance of the bacteria, developed
after four days in plates incubated at 20° C., presumably from single spores. Some
of these subsequently developed only macrocysts, others produced only sorocarps.
By re-isolation from such contrasting areas, a "cyst-forming clone" and a "spore-
forming clone," with strikingly different temperature responses, were isolated
(Figs. 30-35). Upon continued recultivation on 0.1 L-P agar, the former
characteristically produced only macrocysts at 20° C. but developed abundant
sorocarps and scattered macrocysts at 15° C., whereas the latter typically produced
only sorocarps at 20° C. but formed both sorocarps and macrocysts at 25° C.
Cultures initiated with spores from the cyst-forming clone grown at 15° C. de-
veloped macrocysts when recultivated at 20° C., demonstrating a marked degree of
genetic specificity in the myxamoebae and spores of the clone. Thus the pattern
of cellular differentiation exhibited by different clones of S-28b results from in-
herited characteristics that are temperature-dependent for their expression.

Culture media: The production of macrocysts is strongly influenced by the
substrates upon which the slime mold is cultivated, and these structures are regu-
larly formed in greater abundance upon 0.1 L— P agar than upon media based upon
hay-infusion. Their development is even further accentuated if yeast extract is
substituted for peptone, this being used either as the sole nutrient or in combination
with lactose or glucose. For example, upon 0.1% lactose-yeast extract or 0.1 %
glucose-yeast extract agars abundant macrocysts are formed by the cyst-forming
clone incubated at 15° C. and by the spore-forming clone at 20° C., in each case
at temperatures where few or no macrocysts normally develop on 0.1 L-P agar.
An intimate association obviously exists between substrate composition and incuba-
tion temperatures as these factors affect macrocyst formation, but just how they
condition the cultural environment and how they affect the fructifying myxamoebae.
influencing them to produce either macrocysts or sorocarps, has not been determined
(Figs. 30-35).

Bacterial associate: Dictyosteliwn mucoroidcs, S-28b, can be cultivated success-
fully with a variety of bacterial associates on the nutrient-poor media employed in
this investigation. Gram-negative bacteria support better growth of the slime mold
than do Gram-positive types, and of the former Eschcrichia coli was the most
favorable species investigated. Aerobacter aerogcnes, Pseudomonas flnoresccns
and Serratia marcescens yielded satisfactory but less luxuriant slime mold growth.
The formation of macrocysts and sorocarps followed, in general, the patterns ob-
served with E. coli. The myxamoebae of strain S-28b digest or destroy the red
pigment (prodigiosin) of 6\ marcescens. hence yield uncolored sorocarps and

ENCYSTMKXT STAGES OF DICTYOSTKLIUM 77

macrocysts. In marked contrast, the cells of strain NC-12, like those of D.
discoidcitni. retain the pigment (Raper, 1937), hence produce sorocarps and
macrocysts that are pink in color. Bacillus subtilis, a Gram-positive bacterium,
supported fair growth of the slime mold and the formation of abundant macrocysts
on 0.1% lactose-yeast extract agar at 20° C. Sarcina Intca, a second Gram-positive
species, permitted only limited growth of myxamoebae and few macrocysts were
produced on any substrate. An experiment conducted to determine what effect
the age of the nutritive E. coli might have on growth and macrocyst formation in
D. iniicoroides failed to reveal any significant differences.

Hydrogen-ion concentration: A heavy suspension of E. coli cells previously
grown in 0.1 L-P broth and concentrated by centrifugation was cross-streaked on
0.1 L-P agar plates to investigate the effect of pH on slime mold growth and
macrocyst formation. The underlying agar substrate was adjusted over a range
of pH from 3.3 to 9.2 using the buffers previously cited. In these experiments
growth of myxamoebae was obtained between pH 4.5 and 8.0. Slight evidence of
aggregation was observed at pH 4.5 but no well-defined pseudoplasmodia developed,
and of course no sorocarps or macrocysts were produced. A heavy development
of sorocarps and/or macrocysts was obtained between pH 5.5 and 7.0, depending
upon the incubation temperature. No growth occurred above pH 8.0 where KoBO3
was used as buffer. Questioning whether it was the high pH or the borate buffer
which inhibited growth, we made additional tests using KH.2PO4 as buffer with
the addition of NaOH to yield pH levels from 7.5 to 9.0. Growth of the stock
culture occurred to pH 9.0, but few and abnormal sorocarps and no macrocysts were
formed. Even in the cyst-forming clone incubated at 20° C. where one would
normally expect only macrocysts, many sorocarps and very few macrocysts de-
veloped on the more alkaline substrates. The pH of the medium did not change
appreciably during the period of slime mold growth.

Ammonia concentration: Cohen (1953) reported that ammonia suppressed
normal morphogenetic development in the Acrasieae, either inhibiting growth com-
pletely, or causing various abnormalities in sorocarp formation. His procedures
were carefully followed to ascertain whether different concentrations of ammonia
would enhance macrocyst formation. It was thought that if these structures repre-
sented aberrant sorocarps, their production might be enhanced under conditions
where sorocarp formation was inhibited. However, macrocysts were not formed
in any of the ammonia concentrations employed, possibly indicating a suppressive
effect comparable to that reported for sorocarps by Cohen.

Relative Jnuniditv: The effect of per cent relative humidity on slime mold
growth and the development of macrocysts was tested by the procedures previously
indicated. Large depression and ordinary flat slides with one ml. of 0.1 L-P
agar were employed, rather than Petri dishes, to insure rapid establishment of an
equilibrium between the moisture of the agar and the atmosphere in the desiccators.
Olive (1902) attributed the encystment of myxamoebae (microcysts) to adverse
conditions such as the drying of culture substrates. If macrocyst formation should
represent a stage comparable to the encystment of individual myxamoebae, we
would have anticipated more macrocysts when the per cent relative humidity was
lowered, causing a drying of the substrate. Such did not occur in our tests.

Macrocyst formation in other strains: Attempts were made to enhance macro-
cyst formation in other strains of D. initcoroidcs (WS-47 and NC-12) and in D.

78 J. C. BLASKOVICS AND K. B. RAPER

minutum (WS-56-2 and Purdue 8a). Cultural conditions were varied much as
previously outlined for D. mucoroides, S-28b. However, in these additional slime
molds neither the type of culture medium nor the concentration of its ingredients
seemed to markedly affect the relative number of macrocysts produced. For ex-
ample, the proportion of macrocysts to sorocarps remained relatively constant upon
media containing yeast extract vs. peptone and upon nutrient-rich vs. less concen-
trated media, although total growth of the bacteria and of the slime mold varied
substantially with such changes.

A variety of culture media were buffered and adjusted between pH 6.0 and 8.5.
All supported growth of the slime mold, and in no case was there an exceptional
increase in macrocyst production.

Temperature relationships were examined carefully. Dictyostelium mucoroides,
WS^7 (Figs. 36-38), produced more macrocysts at 20° and 25° C. than at 15°
C., but this response was not so striking as in strain S-28b. A few macrocysts
developed in strain NC-12 at 20° C., but at 25° C. only irregular growth of the
slime mold occurred, indicating too high an incubation temperature. In D.
minutum, Purdue Sa, approximately equal macrocyst formation was observed at
20°, 25° and 28° C. In contrast, WS-56-2 formed no macrocysts at 20° C., only
a very few at 25° C., and grew very poorly at 28° C.

Germination of macrocysts

The possible role of the macrocysts in the life cycle of Dictyostelium engaged
our attention from the outset, since it did not seem reasonable that such structures
produced in great abundance and under seemingly optimal conditions would repre-
sent a terminal and functionless kind of differentiation. For this reason much
thought and effort have been given to their germination. In this, as in other
phases of the investigation, studies have been centered upon D. mucoroides, S— 28b,
and our researches have been greatly facilitated by the cyst-forming clones. By
capitalizing upon their unique temperature responses it has been possible to produce
at will large populations of macrocysts under cultural conditions where no spores
were formed. At the same time any vegetative myxamoebae which might have ad-
hered to the macrocysts were readily killed by heating at 42° C. for 10 minutes.
No growth of the slime mold occurred when the macrocysts were heated at a tem-
perature high enough to kill the mature spores, and such would have been present in
any macrocyst preparations taken from the parent culture. The comparative tem-
perature tolerances of the myxamoebae and spores of D. mucoroides, S-28b, are
shown in Tables I and II, respectively.

Proceeding on the assumption that the macrocysts might represent a resistant
stage in the cycle ot Dictyostelium, they were subjected to a variety of cultural en-
vironments and treatments. Plates of 0.1 L-P agar containing abundant macro-
cysts were alternately frozen at - - 10° C. and thawed at + 25° C. in an attempt
to instigate germination, but consistent results were not obtained. Growth from
macrocysts treated in this way was twice observed after ten days' incubation.
Crump (1950) had reported that raising the temperature favored the germination
of encysted free-living amoebae, but no germination ensued after heating macro-
cysts in a water bath for five minutes at 55° C.

At the suggestion of the late Dr. Charles Thorn, an attempt was made to stimu-

ENCYSTMENT STAGES OF DICTYOSTELIUM

79

late macrocyst germination by spreading a heavy inoculum of pre-grown cysts and
E. coli on sterilized soil in Petri dishes and incubating the plates at 10°, 15° and
20° C. No evidence of germination was obtained in 28 days, although we had
demonstrated previously that growth of the slime mold could take place under these
natural conditions.

Brefeld (1869) stated that germination of Dictyostelium spores took place only
in a nitrogenous medium and suggested fresh horse dung decoction and uric acid
media as substrates. Both were investigated. No evidence of macrocyst germina-
tion was observed on fresh horse dung agar plates, but growth was obtained, in
association with E. coli, on 0.05% uric acid agar after eight days incubation at
15° C.

TABLE I

Temperature tolerance of myxamoebae of Dictyostelium mucoroides, S-28b,
suspended in distilled water*

Relative amount of growth per test

Time in water bath,
minutes

Temperature, ° C.

1

2

3

0

42

+ + + +

+ + + +

+ + + +

5

0

+

+

10

0

0

0

15

0

0

0

+ + +

+
0

= Excellent growth.
= Very good growth .
= Good growth.
= Limited growth.
= No growth.

* Similar results were obtained when myxamoebae were suspended in standard salt solution.

Macrocysts were dismembered in a McShan-Erway tissue homogenizer in the
hope that the endocytes thus freed would re-initiate growth under suitable cultural
conditions. Such homogenates were mixed with E. coli and the resulting suspen-
sions spread on 0.1 L-P agar plates. No growth of the slime mold ensued, but the
possibility of serious injury to the endocytes could not be discounted.

Despite the negative results obtained, the seeming logic of this approach led
us yet again to attempt the dissolution of the macrocyst wall by other means as a
possible aid to germination and regrowth. The procedure employed was probably
ineffective per sc, but in performing the experiment the "treated" macrocysts were
incubated at a variety of temperatures which provided a clue to cultural conditions
where germination not infrequently occurred. A cellulase preparation, contributed
by Dr. Emory G. Simmons, was investigated as a means of digesting the heavy
enveloping macrocyst wall. The enzyme was employed as a \% solution in M/20
citrate solution at pH 5.0. Macrocysts were harvested from cultures where no
spores had formed, suspended in the cellulase-citrate solution, and heat-treated to
kill any adherent myxamoebae. Germination occurred in varying amounts in the
cellulase-treated macrocysts and also in controls similarly heat-treated in standard
salt solution. The earliest evidence of germination was observed after 6 days'

80

J. C. BLASKOVICS AND K. B. RAPER

incubation, and new plaques continued to develop for as long as 22 to 40 days at
varied temperatures. The percentage germination was approximately the same
for macrocysts heat-treated in the cellulase-citrate and in the standard salt solutions.
The earliest evidence and the greatest amount of germination were observed among
the macrocysts incubated at 15° C., with decreasing amounts to 25° C. Actual

TABLE II

Temperature tolerance of spores of Dictyostelium mucoroides, S-28b, suspended
in standard salt solution (Test 1) and in distilled water (Tests 2, 3, and 4)

Time in water
bath, minutes

Temperature,
°C.

Relative amount of growth per test

1

2

3

4

0

42

+ + + +

90

+ + + +

0

50

+ + + +

+ + + +

65

+

+

0

55

+ + + +

+ + + +

5

+ +

+ + +

10

0

+

15

+

+

20

0

+

25

0

0

30

0

+

35

0

0

0

60

+ + + +

+ + + +

+ + + +

+++ +

5

+ + +

+ +

+

++

10

+

+ +

+

0

15

0

+ +

0

0

20

0

+

0

+

25

0

+

0

0

30

0

0

0

0

0

65

+ + + +

+ +++

++ + +

5

+

+

0

10

0

0

0

15

+

0

0

20

0

0

0

+ + + + = Excellent growth.
+ + + = Very good growth.
+ -f- = Good growth .

+ = Limited growth.

0 = No growth.

germination of a macrocyst, or of the endocytes contained within it, was not then
observed, but in some instances one or more empty macrocyst cases were evident
where a plaque of growth occurred.

It was now hoped that even though the percentage viability was apparently
low, a few macrocysts might be seen to germinate if these were carefully isolated
and observed periodically over a period of several days. Single macrocysts of dif-

ENCYSTMENT STAGES OF DICTYOSTELIUM

81

ferent ages (3, 15 and 35 days) were selected, heat-treated to kill any vegetative
myxamoebae, and placed individually in marked squares on 0.1 L-P agar plates
smeared with E. coli. Evidence of macrocyst germination was noticed after in-
cubation at 15° C. for five days.

A series of experiments was undertaken to determine whether macrocysts of
a particular age would germinate more readily than cysts of other ages ; and since
presumptive germination had seemed to vary at different incubation temperatures,
special consideration was given to this matter. To obtain macrocysts of specific
ages, a heavy suspension of myxamoebae of the cyst-forming clone and E. coli

TABLE III

Percentage germination among macrocysts of Dictyostelium mucoroides (No. S-28b) of different ages,
and the number of days before such germination was observed at different

incubation temperatures

Age of
macrocysts,
days

Temperature of incubation

5°

10°

15°

20°

25°

Days

%

Days

%

Days

%

Days

%

Days

%

5 (1)*
(2)*

35
44

0
0

35
18

0
0.6

35
30

0
0.2

35
30

0
0.07

35
38

0
0

10 (1)
(2)

35
30

3.4

+ **

20

14

3.1

+

22
14

7.2
3.0

28

25

4.5
0.7

42
30

0
0

15 (1)
(2)

38
43

1.6
0

18
20

2.9

+

12
20

1.2

+

18
20

5.5

+

38
37

0
0

20(1)
(2)

25
38

0.5
0

18
23

1.8
1.0

12

15

1.0
0.9

12
15

1.0

1.0

32
29

0.2
0

25

38

+

15

4.0

15

+

18

1.0

30

0

35

28

+

20

+

8

0.2

8

0.1

20

0.07

* Indicates separate experiments.

** (+) Indicates germination of macrocysts, but percentage of total population could not be
calculated.

was spread over the surface of 0.1 L-P plates and incubated at 20° C. for 5, 10, 15,
20, 25 and 35 days. These macrocysts were then harvested, heat-treated to eliminate
all vegetative myxamoebae, spread on fresh agar plates with E. coli, and incubated
at 5°, 10°, 15°, 20° and 25° C. for 5 to 6 weeks. The results of these experiments
are summarized in Table III.

Substantial growth of the slime mold was obtained in certain of the above tests,
particularly in plates inoculated with 10- and 15-day-old macrocysts incubated at
intermediate temperatures, the highest percentage (7.2%) being observed in 10-day
cysts incubated at 15° C. The prevalence of empty macrocyst cases in the de-
veloping plaques, the prior heat-treatment of the cysts to kill adherent myxamoebae,
the carefully prepared and examined source plates from which the macrocysts were

82 J. C. BLASKOVICS AND K. B. RAPER

taken for these experiments, and the observed presence of empty macrocyst cases
at central locations within many plaques, all convinced us that the observed growth
must have developed from germinated macrocysts.

Nevertheless, we had not actually observed this phenomenon, and even in the
most consistent macrocyst-forming culture it is possible that a minute sorocarp
could go unobserved and that an occasional spore, which would not be killed by
heating to 42° C., might be carried over with the implanted macrocyst. An ex-
periment was carried out to determine whether an occasional spore, if present,
might have served as the initiator of the plaques of amoeboid growth in the macro-
cyst germination plates. It was known that growth from spores would eventually
occur at all of the incubation temperatures employed (5° to 25° C.) ; therefore, the
times required for plaques to develop from individual spores under cultural condi-
tions duplicating the above were determined. At 20° C. plaques were evident
within 4 days, at 15° and 25° C. within 6 days, at 10° C. within 11 days, while at
5° C. growth was not evident until 25 days. Since plaque formation is usually
optimal at 15° C. on the macrocyst germination plates, and does not become evident
until after 8 to 1 1 days, these results provided additional evidence that the observed
growth resulted from macrocysts and not from occasional contaminating spores.

Having determined the optimum cyst age and the incubation temperature that
are favorable for macrocyst germination, 10-day macrocysts in association with
E. coli were implanted on freshly poured plates and on sterile Maximov slides
containing 1.0 ml. of 0.1 L— P agar to observe the germination of the macrocysts
directly. Realization of this objective proved unexpectedly time-consuming, but
it was accomplished. Germination in this instance, as in the majority of cases
observed up to this time, was from a macrocyst filled with endocytes at the time of
implantation in the test culture. Pre-germination changes were not observed but
it is assumed that the heavy macrocyst wall was ruptured either by swelling of its
content and/or by enzymatic dissolution (see below).

Re-examination of Table III reveals that maximum germination occurred
among the 10-day-old macrocysts ; i.e., structures which were packed with endocytes
at the beginning of the tests. More significantly, appreciable germination was re-
corded for some of the older macrocysts, notably the 25-day cysts incubated at
10° C. This result is especially interesting since cysts of this age would have
already lost their discrete endocytes, in the great majority of cases, and would
have assumed the homogenous appearance that characterizes aged cysts. Thus
presumptive evidence was obtained that macrocysts of the latter type are capable
of germination — presumptive because even in preparations taken from cultures after
several weeks occasional macrocysts are seen in which the endocytes remain distinct,
and the recorded germination could have resulted from such non-homogeneous
structures. The improbability of this explanation was subsequently demonstrated.
Blocks of 0.1 L-P agar bearing abundant homogeneous macrocysts aged 6 and 8
weeks were placed in sterile Petri dishes, flooded with sterile distilled water, and
incubated at 10°, 15°, 20°, and 25° C. Within only 10 days approximately half of the
cysts of both ages incubated at 10° C., and only at this temperature, had germinated
among populations where prior examination had established that only occasional
cysts (2—4%) were still in the endocyte stage at the beginning of the experiment
(Figs. 39-42). An understanding of the intracystic events which transpire during
the progression from endocyte differentiation to their subsequent disappearance.

ENCYSTMENT STAGES OF DICTYOSTELIUM 83

and from this through the stage of seeming homogeneity and protoplasmic contrac-
tion to the eventual swelling of this mass and the reappearance of amoeboid cells
at the time of germination, must await careful cytological investigation. For the
present we can only record that in the presence of a proper aqueous environment
and at a favorable temperature the previously shrunken "protoplast" (long be-
lieved doubtfully viable) swells and gives rise to myxamoebae which escape during
cyst germination. Additionally, there is evidence that these myxamoebae, or the
parent coenocyte (?), produce cellulolytic enzymes which facilitate rupture of the
heavy cyst wall, for viewed with the polarizing microscope the empty cyst cases
contain conspicuously less cellulose than do the walls of macrocysts still ungermi-
nated.

Formation and germination of microcysts

Many members of the Acrasieae are characterized by a second, simpler type
of encystment stage where individual vegetative myxamoebae round up and become
encased by relatively thin protective membranes (Fig. 1). The walls of these
resting cells, or microcysts, like those of the macrocysts and the more resistant
spores, are predominantly cellulosic in composition. As reported by Olive (1902),
there is ample evidence that these form in response to sub-optimal growth condi-
tions. It is probable that the myxamoebae of any member of the Acrasieae may
enter such a stage temporarily, but they are most commonly encountered, and in
greatest numbers, in isolates of Dictyostelium minutum, D. polycephalmn, Poly-
sphondylium pallidum, and Acytostelium leptosomum.

Microcysts of several species of the Acrasieae were examined to determine
their method of germination. Microcysts were placed in hanging-drop slides in
thin-hay broth with killed cells of E. coli and incubated at 25° C. Within two
days many of the microcysts had germinated, as evidenced by the number of free,
feeding myxamoebae and by the empty microcyst cases from which these had
emerged. Previous workers (Olive, 1902) had not reported true excystment of
the microcysts, but had intimated that the myxamoebae absorbed the protective
covering, or wall, during germination. The emptied cases are extremely delicate
and hyaline, and some reveal a fairly obvious opening at one side through which
the myxamoeba escaped. They do not germinate by the emergence of the proto-
plast through a pore or exit tube, neither do they appear to split as do the spores
of most species ; rather, germination appears to take place by the dissolution of a
fractional portion of the microcyst wall. The cyst cases stain violet-blue with
chloroiodide of zinc and show a weak birefringence, indicating that they contain
some cellulosic material.

In Dictyostelium mucoroides microcysts are about twice the dimensions of the
endocytes that comprise the macrocysts, ranging from about 5.0 to 7.5 ^ in
diameter and being generally spheroidal.

DISCUSSION

Intriguing questions are posed by the macrocysts of Dictyostelium with regard
to their morphogenesis and their probable primary function in the life-cycle of these
slime molds. Do they represent a normal but generally unrevealed stage in the

84 J. C. BLASKOVICS AND K. B. RAPER

development history of the Acrasieae, i.e., could they be demonstrated in all members
of the group if we but knew the conditions required to evoke them? Do they
provide a resting stage whereby these micro-organisms survive otherwise im-
possible environmental conditions ? Do they perhaps constitute some unanticipated
manifestation of a sexual stage ? Or do they represent, as their superficial appear-
ance might suggest, groups of myxamoebae so thwarted in their normal morpho-
genesis that they become "captives" doomed to a type of terminal differentiation
approximating that of sterile stalk cells? Does the identity of the contributory
myxamoebae remain unchanged during the formation of the endocytes, and do the
latter in some altered form persist to once again emerge during germination as
myxamoebae capable of perpetuating the species? For certain of these questions
we have succeeded in providing partial answers.

Brefeld illustrated some "dwarfed sporangia" that contained differentiated cells
which he interpreted as representing elements of abortive stalks, and early in this
investigation we questioned whether the endocytes might not in fact reflect a type of
cellular differentiation of this type. More careful examination has established be-
yond question that such is not the case despite certain superficial similarities in
appearance. The walls of the endocytes contain no demonstrable cellulose and the
content of such cells is actually condensed, whereas the walls of true stalk cells
contain cellulose and the cell content is strongly vacuolate, occupying a peripheral
position within the semi-rigid cell. Upon treatment with alkali (e.g., 1.0 N
NaOH) the walls of stalk cells do not disappear as do those of the endocytes.

Much evidence supports the belief that macrocysts arise through an orderly
and natural morphogenetic process, and hence in no wise represent aberrant fruiting
structures. For those strains which produce them, they would appear to be no less
normal than the sorocarps which regularly develop under similar or, in some
instances, altered conditions. A measure of homology is suggested by the basically
similar aggregative process which precedes the formation of both types of structure.
The pseudoplasmodia leading to macrocyst formation are generally diminutive, but
this condition is not a necessary precedent to their formation. Additionally, the
myxamoebae entering a pseudoplasmodium destined to form macrocysts rarely
show the marked elongation characteristic of cells entering larger aggregations,
but this weak cellular response is believed to indicate degree rather than difference,
i.e., to reflect a feeble aggregative stimulus incident to, or responsible for, the small
pseudoplasmodium.

A point of similarity should be noted between the morphogenetic processes lead-
ing to the formation of gregarious sorocarps in certain species (e.g., D. minutum
and D. lacteuwi) and to clustered macrocysts in D. mucoroides, for in both situations
the magnitude of the initial pseudoplasmodium often exceeds the number of
myxamoebae that can effectively collaborate in producing a single sorocarp or
macrocyst. In the former instance, secondary centers appear soon after the over-
all pattern of the wheel-like aggregate becomes evident, and from each of these a
separate sorocarp subsequently develops ; in the latter case, multiple loci of endocyte
formation similarly appear within the initial aggregate, and from each of these later
develops a discrete and typical macrocyst.

Substantial differences characterize subsequent steps in the two morphogenetic
processes, and there is little if any evidence to suggest that the macrocysts represent
modified or abortive sorocarps. The latter can be produced in any known species

ENCYSTMENT STAGES OF DICTYOSTELIUM 85

by a variety of devices (e.g., unfavorable pH, increased temperature, etc.), but
in no observed instance have such abnormal fruits presented a pattern which is
remotely suggestive of macrocyts. Rather, they assume the form of Guttulina-
like fructifications wherein the myxamoebae produce irregular mounds and undergo
incomplete differentiation, but they never form a common protective wall about the
mass of cells so assembled.

The heavy cellulosic wall of the macrocyst bears a structural likeness to the
sorophore sheath of the normal sore-carp, but, as noted earlier, the relative positions
at which these are deposited by the constitutive cells are quite different. Further-
more, the formation of the sorophore sheath is antecedent to cellular differentiation
in sorocarp building, whereas it lags behind this phenomenon in macrocyst con-
struction. There is yet another difference which may prove highly significant.
Bonner et al. (1956) demonstrated that the sorophore sheath is secreted by an
ever-changing epithelium-like layer of myxamoebae that are oriented perpendicular
to the surface of the wall being deposited, whilst in the macrocysts the last re-
maining amoeboid cells, hence those adjacent to the developing wall, are oriented
in quite the opposite direction. Only in those cultures where seemingly identical
and intermixed pseudoplasmodia give rise either to sorocarps or to macrocysts, as
in strain WS-47, do we find evidence that the two morphogenetic pathways may be
closely allied, and in these we have at present no concept of what major or minor
organizational differences may underlie such divergence. Judging from Bref eld's
illustrations (1869), it is possible that he may have seen so-called "dwarfed
sporangia" that were transitional between sorocarps and macrocysts, but no struc-
tures of this type have been observed in our cultures. Finally, we would reiterate
that the body of evidence presently available points to macrocyst production as
representing an alternative but thoroughly normal morphogenetic pathway that is an
inherited character possessed by occasional strains of D. mucoroidcs and by many
isolates of D. niinutum. The isolation of contrasting "cyst-forming" and "spore-
forming" clones in strain S— 28b strengthens this belief, as does also our inability
thus far to induce macrocyst formation in any culture of D. mucoroides which did:
not naturally exhibit this capacity at the time of its isolation.

In contrast to this situation, the capacity to produce microcysts seems to be
generally present among the Acrasieae, and it is suspected that every isolate may,
under certain variable environmental conditions, exhibit such a resting stage. It
should be recognized, however, that this phenomenon is probably totally unrelated
to macrocyst formation. Microcysts represent the responses of single myxamoebae
to effect a transitory resting stage in the vegetative phase of these slime molds
and is perhaps strictly comparable to the encystment of certain small, free-living
amoebae. Their natural function is indisputably one of enabling the species to
survive otherwise unfavorable environments. The macrocysts, on the other hand,
arise through multicellular integration and differentiation and represent the product
of a specific morphogenetic process, just as do the sorocarps. This function is still
incompletely known.

We have obtained convincing evidence that macrocysts germinate under certain
circumstances, emitting amoeboid cells which then re-initiate vegetative growth.
But we cannot say with confidence that the macrocysts represent a vital resting
stage, as their appearance might suggest. Heat tolerance tests indicate that they
can withstand appreciably higher temperatures than vegetative myxamoebae, but

86 J. C. BLASKOVICS AND K. B. RAPER

they are in turn less resistant than true spores. While the matter has not been
explored under conditions that exist in nature, it is possible that they might be
produced under circumstances which would preclude the formation of sorocarps and
spores, e.g., in strains such as S-28b at elevated temperatures. The general appli-
cation of this premise is doubtful, however, for many macrocyst-producing strains
fail to show a comparable response. Based upon laboratory tests, we could not, at
present, conclude that they possess singular survival value.

Possibly they are endowed with other unique properties, a suggestion presently
based less upon fact than fancy. We find it difficult to dismiss lightly a structure
of multicellular origin which appears to be so highly organized as the older macro-
cyst. We cannot say with absolute certainty that its content represents a single
homogeneous multinucleate protoplast, but such tests as we have applied would
seem to support this notion. If the endocytes do actually lose their identity, as
appears to be the case, the acellular content of the aged macrocyst would represent
a coenocyte, or to use a term more commonly associated with slime molds, perhaps
a plasmodium, albeit one that is enclosed within a heavy cellulose wall. Such a
plasmodium would of course be quite unlike that which Brefeld (1869) once thought
to be present, or that which Skupienski (1920) envisioned as an accompaniment to
reported sexuality in Dictyostelimn. Needless to say, it would represent quite a
different structure from the large vegetative body that occupies so conspicuous a
place in the life-cycle of the Myogastrales. Clearly, the two could not be regarded
as homologous. The same may be said of the plasmodial stages of the Plasmodio-
phorales, for although their dimensions would be more nearly comparable, these also
are never characterized by heavy cellulose walls. Furthermore, no one has yet
reported a swimming stage, either gametic or vegetative, for any member of the
Acrasieae and such are generally precedent to the formation of plasmodia in each
of the other orders.

Here the matter must rest for the present, and definitive information regarding
the true nature and ultimate significance of the macrocysts must await further
research.

The writers are indebted to Miss Mildred M. Smith for her invaluable aid in
the preparation of the illustrations used in this report.

SUMMARY

1. Two encystment stages of cellular slime molds belonging to the genus
Dictyosteliutn are described :

The first of these, termed microcysts, are unicellular and represent a transient
resting stage in the vegetative phase of these simple slime molds. If returned to a
favorable environment, microcysts germinated by excystment to re-initiate vege-
tative growth.

The second encystment stage, termed macrocysts, are multicellular and arise
through a morphogenetic process possibly alternative to normal sorocarp formation.
Myxamoebae aggregate to form typical but generally diminutive pseudoplasmodia
which, instead of forming normal sorocarps, subdivide into rounded cell masses
that become encased in relatively heavy cellulose walls. Concurrent with this de-

ENCYSTMENT STAGES OF DICTYOSTELIUM 87

velopment, the myxamoebae that comprise the nascent macrocyst undergo limited
differentiation and appear as polyhedral cells with highly refractive membranes.
After a period of 10 to 14 days these so-called endocytes generally disappear
whereupon the content of the macrocyst assumes an acellular, homogeneous appear-
ance. With further aging the protoplasmic content shrinks away from the heavy
cellulose wall and in this contracted stage retains its viability for protracted periods.
Under favorable conditions of temperature and substrates, macrocysts of different
ages germinate to release amoeboid cells which re-initiate the vegetative stage. The
sequence of cytological changes underlying this behavior has not been elucidated,
and this propagative function may or may not represent the full measure of their
significance in the life-cycle of those slime molds which produce them.

2. The ability to produce microcysts is apparently inherent in all members of
the Acrasieae, including the genus Dictyostelium. In contrast, the capacity to
produce macrocysts is more restricted, having been observed only in occasional
isolates of D. mucoroides and in many strains of D. minutum. Various environ-
mental factors influence their production, and from one strain of D. mucoroides
temperature-dependent "cyst-forming" and "spore-forming" clones have been iso-
lated.

LITERATURE CITED

BONNER, J. T., 1944. A descriptive study of the development of the slime mold Dictyostelium

discoideum. Amer. J. Bot., 31 : 175-182.
BONNER, J. T., 1947. Evidence for the formation of cell aggregates by chemotaxis in the

development of the slime mold Dictyostelium discoideum. J. Exp. Zool., 106: 1-26.
BONNER, J. T., 1952. The pattern of differentiation in amoeboid slime molds. Amer. Nat.,

86: 79-89.
BONNER, J. T., A. DUNCAN CHIQUOTNE AND MARJORIE QUICK KOLDERIE, 1955. A histochemical

study of differentiation in the cellular slime molds. /. Exp. Zool., 130: 133-158.
BREFELD, O., 1869. Dictyostelium mucoroides. Ein neuer Organismus aus der Verwandschaft

der Myxomyceten. Abhandl. Senckenbergisch. Naturf. Ges. Frankfort, a/m, 7:

85-107.
CLARK, W. M., 1928. The determination of hydrogen ions. Third Edition. The Williams

and Wilkins Co., Baltimore.
COHEN, ARTHUR L., 1953. The effect of ammonia on morphogenesis in the Acrasieae. Proc.

Nat. A cad. Set., 39: 68-74.
CORMIER, JOAN L., AND KENNETH B. RAPER, 1955. The macrocysts of Dictyostelium. Bact.

Proc. p. 36 fabst.).
CRUMP, L. M., 1950. The influence of bacterial environment on the excystment of amoebae

from soil. /. Gen. Microbiol., 4 : 16-21.
HODGMAN, C. D., ed., 1951. Handbook of chemistry and physics. Thirty-third edition.

Chemical Rubber Publishing Co., Cleveland, Ohio.
OLIVE, E. W., 1901. A preliminary enumeration of the Sorophoreae. Proc. Amer. Acad. Arts

Set., 37 : 333-344.

OLIVE, E. W., 1902. Monograph of the Acrasieae. Proc. Boston Soc. Nat. Hist., 30: 451-513.
RAPER, KENNETH B., 1935. Dictyostelium discoideum, a new species of slime mold from de-
caying forest leaves. /. Agric. Res., 50 : 135-147.

RAPER, KENNETH B., 1937. Growth and development of Dictyostelium discoideum with differ-
ent bacterial associates. /. Agric. Res., 55 : 289-316.
RAPER, KENNETH B., 1940a. Pseudoplasmodium formation and organization in Dictyostelium

discoideum. J . Elisha Mitchell Sci. Soc., 56 : 241-282.
RAPER, KENNETH B., 1940b. The communal nature of the fruiting process in the Acrasieae.

Amer. J. Bot., 27 : 436-448.

88 J. C. BLASKOVICS AND K. B. RAPER

RAPER, KENNETH B., 1941. Dictyostelium minutum, a second new species of slime mold from

decaying forest leaves. Mycologia, 33 : 633-649.
RAPER, KENNETH B., 1951. Isolation, cultivation, and conservation of simple slime molds.

Quart. Rev. Biol, 26: 169-190.
RAPER, KENNETH B., 1956a. Factors affecting growth and differentiation in simple slime molds.

Mycologia, 48: 169-205.
RAPER, KENNETH B., 1956b. Dictyostelium polycephalum n. sp., a new cellular slime mould

with coremiform fructifications. /. Gen. Microbiol., 14: 716-732.
RAPER, KENNETH B., AND DOROTHY I. FENNELL, 1952. Stalk formation in Dictyostelium.

Bull. Torrey Bot. Club, 79: 25-51.
SKUPIENSKI, F. X., 1920. Recherches sur le cycle evolutif des certains myxomycetes. Paris.

(Published by the author.)

STEVENS, W. C., 1916. Plant anatomy. Third Edition. P. Blakiston's Son & Co., Philadelphia.
WILSON, R. E., 1921. Humidity control by means of sulphuric acid solutions, with critical

compilation of vapor pressure data. /. Ind. and Enr/. Chan., 13: 326-331.

PHYSIOLOGICAL OBSERVATIONS ON STARVATION AND DESIC-
CATION OF THE SNAIL AUSTRALORBIS GLABRATUS

THEODOR VON BRAND, PATRICIA McMAHON AND M. O. NOLAN

U. S. Department of Health, Education, and Welfare, Public Health Service,

National Institutes of Health, National Institute of Allergy and

Infectious Diseases* Bethesda, Maryland

It has been shown that planorbid snails, all of which are aquatic pulmonates,
can withstand desiccation rather well both in nature and under laboratory condi-
tions (Precht, 1939; Olivier, 1956a, 1956b ; Olivier and Barbosa, 1955, 1956).
The physiology of desiccating planorbids has, however, received scant attention.
Magalhaes Neto (1954) observed that five specimens of Austrdorbis glabratus
showed a considerably decreased rate of oxygen consumption during desiccation
at an unspecified relative humidity. Desiccating aquatic snails retract into their
shells; they are unable to feed and hence come under conditions of starvation.
Since starvation decreases the rate of oxygen consumption (von Brand, Nolan
and Mann, 1948), and since the anatomical relationship of a retracted snail to the
source of oxygen is quite different from that of an active one, the following ques-
tions arise : Is the reduction in oxygen consumption due mainly to starvation, to
difficulties in securing sufficient oxygen, or to desiccation proper? These and
related questions are discussed in the present paper.

MATERIAL AND METHODS

Laboratory-reared albino Austrdorbis glabratus, derived from a normally pig-
mented Venezuelan strain, were used in preference to pigmented specimens because
the heart-beat could easily be seen through the shell. This was important, not
only because a study of the heart rate under desiccation was interesting in itself, but
also in order to establish whether a snail was alive or dead. The usual procedure
of placing a desiccated snail in water to observe whether it resumes its normal
activities could not be employed because in most of our experiments repeated
measurements with the same specimens were required, or because a chemical
determination had to be made on desiccated specimens. Most of the snails that
appeared dead, as judged by cessation of the heart-beat, were tested further by
placing them in water. Of about 200 such snails, only three revived, indicating
that our death criterion was reasonably accurate.

All snails initially weighed between 180 and 350 mg. and had fed ad libitum.
They were freed of excess moisture as described previously (Newton and von
Brand, 1955) and weighed to the nearest mg. During starvation, snails (minimum
of 28 per series) were kept individually in numbered beakers filled with dechlori-
nated tap water. They were shifted daily to fresh beakers during the first week of
starvation and thereafter twice weekly. During desiccation, snails (minimum of

1 Laboratory of Tropical Diseases.

89

90 T. VON BRAND, P. McMAHON AND M. O. NOLAN

33 per series) were put into individual dry beakers and these were kept at the same
temperature (27 ± 1° C.) as the starvation series in desiccators over water or over
saturated salt solutions giving a desired relative humidity. The solutions employed
gave the following relative humidities, as determined by an Aminco electric
hygrometer.

H,O giving 95 to 97 per cent relative humidity, average 96 per cent
ZnSO4 giving 83 to 87 per cent relative humidity, average 85 per cent
NaCl giving 72 to 76 per cent relative humidity, average 74 per cent
NaBr giving 57 per cent relative humidity, average 57 per cent
CaCL giving 28 to 31 per cent relative humidity, average 30 per cent
LiCl giving 13 to 17 per cent relative humidity, average 15 per cent

When survival, weight, heart rate, and rate of oxygen consumption were de-
termined, all snails alive on a given day were used. At the end of the determina-
tions they were returned to the desiccators, or the water-containing beakers, re-
spectively. These snails were used repeatedly until the last specimen died. When
chemical determinations were done, only the specimens to be analyzed on a given
day were used, while the others remained undisturbed until required for analysis.

The heart rate was determined by counting the heart-beats for one minute under
a dissecting microscope.

The conventional Warburg technique was used for the oxygen consumption.
The vessels contained 2 ml. of dechlorinated tap water in the starvation series and
in the experiments designed to give the pre-desiccation rate. In the desiccation
experiments the snails were put into the main compartment of the vessel without
water. In these cases the side arms of all vessels, including thermobarometer,
contained 0.3 ml. of the same salt solution that was present in the desiccators where
the snails had been kept, thus maintaining approximately the same relative humidity.
In all cases the temperature was 30° C.

Polysaccharides were determined according to von Brand's (1936) micro-
modification of Pfluger's method. Total lipids were determined by heating the
crushed snail with 30 per cent NaOH in a boiling water bath, acidifying the solution
with 7 per cent H2SO4, extracting the solution three times with ether, washing the
combined ether fractions with distilled water, evaporating the ether, and weighing
the lipids on a microbalance after drying at 80° C. For lactic acid 2 and volatile
acids the methods of Barker and Summerson (1941) and Bueding (1949) were
used, respectively.

All measurements were done on numbered individual snails, with the exception
of the volatile acid determinations, where two snails were used. All values are
expressed on the basis of the initial, pre-experimental weight of the snails.

RESULTS

1. Survival. Figure 1 shows the survival groups of starving snails at various
relative humidities and in water. It is obvious that time of survival decreased
with decreasing humidity. When the 50 per cent death times are plotted log-
arithmically against relative humidity (lower part of Fig. 1), no straight line

- We are indebted to Mr. C. Elwood Claggett for carrying out the lactic acid determina-
tions.

DESICCATION OF SNAILS

91

X
LJ

LJ

on

100
80
60
40

20

0

64

128

A Starving in Water

• Desiccation at 96% Relative Humidity
& Desiccation at 85% Relative Humidity
o Desiccation at 74% Relative Humidity
n Desiccation at 57%Relative Humidity

• Desiccation at 30% Relative Humidity
o Desiccation at 15 % Relative Humidity

I 2 4 8 16 32 64 128

DAYS REQUIRED TO REACH 50 PERCENT DEATH POINT

FIGURE 1. Survival of Australorbis glabratus starving in water or desiccating at various
relative humidities. At the beginning each desiccating series consisted of 33 specimens, the
water-starvation series of 28 specimens. The same groups of snails yielded the results shown
in Figures 2, 3 and 4.

92

T. VON BRAND, P. McMAHON AND M. O. NOLAN

results. The shape of the curve indicates that no fixed relationship between time
of survival and relative humidity exists, but that decreasing humidity leads progres-
sively to an ever more accelerated death rate. Survival during starvation in water
was only about half as long as during desiccation over water (96 per cent relative

DAYS

ID
I

UJ
01

100

80

60

40

20

0

*. Starving in Water

• Desiccation at 96% Relative Humidity
A Desiccation at 8 5 % Relative Humidity
Q Desiccation at 74% Relative Humidity
a Desiccation at 57% Relative Humidity
m Desiccation at 30%Relative Humidity
(^Desiccation at 15% Relative Humidity

I

I

I

I 2 4 8 16 32 64 128

DAYS REQUIRED TO LOSE 25% OFWEIGHT

FIGURE 2. Weight relationships of Australorbis glabratus starving in water
or desiccating at various relative humidities.

humidity). However, the 50 per cent death points of these two series were closer
together. This is due to the fact that the first deaths due solely to starvation oc-
curred later than those from the combined influences of starvation and desiccation.
2. Weight loss. Thirty fed control snails averaged 40.8 per cent dry substance.
The shells of 30 other controls, after the soft parts had been removed according to
Nolan and von Brand's (1954) procedure averaged 31.6 per cent of the total

DESICCATION OF SNAILS

93

weight. • The dry weight of the soft tissues was thus 9.2 per cent of the total body
weight. Since the shell contains practically no water, the total water found can
be ascribed to the soft tissues. Their initial over-all hydration is then calculated
as 87 per cent (Table III).

A total weight loss of 13 per cent was observed in snails starving to death in
water. It was hence greater than the total dry weight of the soft tissues initially
present and it must be concluded that it was due in part to a loss of water, perhaps
corresponding to the hydration water of the metabolized organic material.

The weight loss of desiccating snails (Fig. 2) was much more pronounced than
that of snails starving in water and was clearly dependent on the relative humidity.
The lower part of Figure 2 indicates that the relationship between humidity and
weight loss is very similar to that described above for survival. Snails desiccating
and starving at 96 per cent relative humidity metabolize during 128 days approxi-

TABLE I

Desiccation of Australorbis glabratus at 85 per cent relative humidity. The figures are per cent of the
pre-desiccation values; the figure following the ± sign is the standard error of the mean

Heart rate

Weight

Rate of O2 consumption

Days of

Per cent

desiccation

survival

A

B

A

B

A

B

1

98±2.6

98±4.2

89±0.5

90±0.7

49±2.6

47±4.5

100

2

100±4.2

107±5.2

83±0.3

85±1.7

46±2.2

47±4.4

100

3

95±3.0

93±4.8

79±0.7

81 ±0.9

33±2.7

30±3.0

100

8

105±3.7

98±3.7

69±1.0

71±1.1

27±2.1

20±2.4

100

15

93±4.6

95±3.7

61±1.1

64±1.7

28±2.5

30±3.6

81

22

81 ±9.9

81±9.9

57±1.6

57±1.6

10±2.0

10±2.0

44

28

81±2.4

57±3.7

19±5.0

16

The initial number of snails was 33.

A = Values of all snails alive at specified day.

B = Values of all snails surviving on day 22.

mately 50 to 60 per cent of their organic material (corresponding to 4—5 per cent
of the pre-experimental live body weight, see Discussion). Since in the humidity
range of 15 to 85 per cent practically all snails had died by day 20, their loss of
organic material must have been much smaller, and no appreciable error can be
introduced if the entire weight loss is here ascribed to loss of water. At these
humidities the weight had declined terminally to 60 per cent of the initial, corre-
sponding to a loss of 40/59.2 = approximately 70 per cent of the initial water.

At 96 per cent humidity, on the other hand, survival was much longer and
the weight at death was higher, amounting to 65 to 70 per cent of the initial
weight. As mentioned above, about 4 to 5 per cent weight loss must in this case
be ascribed to metabolized organic material. It is therefore clear that at this high
humidity the last animals died before being desiccated to quite the same degree as
in the other desiccation series. It is probable that in this case starvation was a
contributing factor to death.

It should be realized that the figures summarized in Figure 2 (the following
applies also to the data presented in Figs. 3 and 4) are averages for all snails

94

T. VON BRAND, P. McMAHON AND M. O. NOLAN

I I I

Starving in Water

• Desiccation at 96% Relative Humidity
A Desiccation at 85% Relative Humidity
o Desiccation at 74% Relative Humidity
o Desiccation at 57% Relative Humidify

• Desiccation at 30% Relative Humidity
9 Desiccation at 15% Relative Humidity

DAYS

FIGURE 3. Heart rates of Australorbis glabratus starving in water
or desiccating at various relative humidities.

alive at a specified day. They therefore represent the average changes occurring
in a population. Some irregularities in the curves, especially noticeable towards the
end of an experiment, are due to the summation of experimental errors and the
slightly variable behavior of the individual snails. The curves do not change
materially, however, if the data are restricted to snails which are still alive on a day
nearing the end of a given experiment, provided the number is sufficient to give
a valid average. This is illustrated for one of our series in Table I for weight and
other criteria studied.

3. Heart rate. The heart rate of snails (Fig. 3) starving in water slowed
precipitously to 63 per cent of the original rate during the first 24 hours of starva-
tion. During the remainder of the starvation period the heart rate declined slowly
further, the final value being about 40 per cent of the initial one. In snails desic-
cating at 96 per cent relative humidity there was no decline during the first 24 hours,

DESICCATION OF SNAILS

95

but thereafter the heart rate became progressively slower, reaching about the same
end-point as in snails starving in water.

A different situation prevailed in snails desiccating at all lower humidities.
Within the first few days, there was a period when the heart-beat increased in
frequency above the pre-experimental value, this period being followed by one of
more or less precipitous decline. While the heart-beat was generally full and regu-
lar in snails in water and in air at 96 per cent relative humidity, many irregularities
were observed at lower humidities, such as partial contractions of the heart, or

128

100
80

60

UJ 40

_J
UJ

a:

20

0

A Starving in Water

• Desiccation at 96% Relative Humidity
A Desiccation at 8 5% Relative Humidity
o Desiccation at 74% Relative Humidity
n Desiccation at 57% Relative Humidity

• Desiccation at 3O%Re lative Humidity
o Desiccation at 15% Relative Humidity

I 2 4 8 16 32 64 128

DAYS REQUIRED TO LOWER 02 CONSUMPTION TO 30%
OF PRE-EXPERIMENTAL VALUE

FIGURE 4. Oxygen consumption of Australorbis glabratus starving in water
or desiccating at various relative humidities.

96

T. VON BRAND, P. McMAHON AND M. O. NOLAN

cessation of the pulsations for a few seconds followed by a period of very rapid
contractions. It should be noted that at 57 per cent humidity the last value raises
the curve (Fig. 3.) This artifact is due to abnormally high rates in all three
surviving snails.

Heart rate apparently had no direct relation to survival under our conditions.
For instance, in our series of 33 snails desiccating at 96 per cent relative humidity,
7 snails with initial heart rates of 33 to 37 beats per minute survived an average
of 48 days. The other extreme was represented by 5 snails with initial rates of
50 to 63 beats/minute and an average survival of 80 days. After 128 days of
desiccation, 5 snails still survived ; their average initial rate was 42 beats/minute
with 37 and 53 as extremes.

4. Oxygen consumption. The rate of oxygen consumption (Fig. 4) declined
in all series more or less rapidly, but the decline was slower in the water-starvation
than in the desiccation series. The daily variations were more pronounced in the
former than in the latter, possibly due to the motility of the snails starving in water

TABLE II

Chemical determinations on Australorbis glabratus starving in water or desiccating at 96 per cent

relative humidity. All values have been calculated on the basis of the pre- starvation or pre-desicca-

tion live weight of the snails. The figure following the ± sign is the standard error of the mean,

the figure in parenthesis indicates the number of determinations

Days

Per cent lipids

Per cent polysaccharides

M Lactic acid

/i Volatile acid*

Desiccation

Starvation

Desiccation

Starvation

Desiccation

Desiccation

0
10
20
30

0.76 ±0.023 (24)
0.58 ±0.019 (24)
O.S4±0.016 (20)
0.52 ±0.019 (19)

0.65 ±0.036 (24)
0.43 ±0.026 (24)
0.38 ±0.011 (19)
0.36 ±0.011 (19)

1.03 ±0.10 (23)
0.77 ±0.07 (22)
0.69 ±0.07 (24)
0.56 ±0.08 (20)

1.29±0.16 (22)
1.04±0.18 (24)
0.69 ±0.14 (21)
0.59 ±0.1 2 (22)

140±27.8 (12)
117±28.1 (10)
35± 7.9 (12)
0.0 (11)

53 ±28.1 (6)
11 ± 5.4 (6)
18±12.8 (6)
23 ± 3.9 (3)

* The volatile acids are expressed as acetic acid, since this is the predominant volatile fatty acid (Mehlman and
von Brand, 1951).

as contrasted with the immobility of the desiccating specimens. In the starvation
series the final rate was about 30 per cent of the pre-experimental one, while in the
desiccation series the endpoint varied from about 20 to well below 10 per cent of
the initial value. The rapidity of decline (lower half of Fig. 4) showed a rough
correlation with degree of humidity, but it was not so close as that shown between
humidity and survival or humidity and weight loss.

A rough inverse correlation probably exists between survival and initial rate
of oxygen consumption. Taking the series of snails desiccating at 96% relative
humidity, as example, six snails had initial rates varying between 205 and 277
mm.3 O2/gm./ hr. with an average survival of 51 days. In four snails the rate
varied initially between 82 and 108 mm.3 O2/gm./hr. and their average survival was
103 days. The five snails surviving 128 days desiccation had an initial rate of 143
mm.3 Oo/gm./hr., with 106 and 171 mm.3 as extremes.

5. Chemical determinations. Chemical determinations were performed only
during the first 30 days on animals starving in water and desiccating at 96 per cent
relative humidity since snails desiccating at lower humidities died too early. In
view of the variability in storage of reserve substances, it was essential to limit

DESICCATION OF SNAILS 97

periods of exposure to experimental conditions to those tolerated by all or at least
the great majority of specimens employed, since otherwise a possible differential
death rate between snails with high and low initial reserves would make valid
conclusions impossible.

The data summarized in Table II show that starving and desiccating snails
use appreciable amounts of both polysaccharide and lipids, the consumption being
more pronounced during the first 10 days than after prolonged exposure to experi-
mental conditions. Snails starving in water used little more of these reserve
substances than did the desiccating specimens. In desiccating snails the lactic acid
initially present in the tissues disappeared completely within 30 days, while the
volatile acid content decreased only slightly.

DISCUSSION

The laboratory strain of Australorbis glabratus used in the present studies
withstood desiccation fairly well. As was expected, the snails retracted into their
shells. They were not capable of forming a true epiphragm which in many other
species is an efficient mechanism for preventing excessive loss of water, nor did
they produce complete mucus membranes across the shell aperture, an auxiliary
mechanism frequently employed (Gebhardt-Dunkel, 1953). Partial mucus mem-
branes were observed occasionally, but they did not seem to change the rate of
evaporation materially.

Marked reduction in the rate of oxygen consumption with time was charac-
teristic at all humidities studied and a loose inverse correlation with humidity
existed. This reduction was not due solely, and in the series at low humidities
not even primarily, to starvation. Snails starving in water maintained a higher
rate of oxygen consumption than the desiccating specimens ; they must therefore
have used their reserve substances at a faster rate. It would then seem that the
amount of reserve substances available to the desiccating animals would have
sufficed to maintain an equal rate of oxygen consumption if starvation alone were
involved. An altered anatomical relationship to the source of oxygen can also be
eliminated as the cause of this reduction. If difficulties in securing oxygen played
a significant role, a partial shift to anaerobiosis would have been expected. It
should be noted in this connection that a partial shift to anaerobiosis can readily
be induced in Australorbis by exposure to low concentrations of pentachloro-
phenol (Weinbach and Nolan, 1956) and that a considerable increase in lactic acid
content has been reported from aestivating Pila (Meenakshi, 1956). In our desic-
cating specimens, on the contrary, the lactic acid present initially disappeared com-
pletely and the volatile acids diminished. There is little doubt that Australorbis,
at least during desiccation at high humidity, maintained a purely aerobic metabolism
despite the deep retraction into the shell.

The lung of a contracted snail is probably largely compressed and it is prob-
lematical whether it plays a large role in the gaseous exchanges. Diffusion through
the tissues exposed to the air within the shell may have been sufficient. It should
be kept in mind that conditions are quite different when a snail retracts into its
shell in water. During desiccation the tissues are in direct contact with atmospheric
air where the absolute amounts of oxygen are much higher than in water and where
diffusion is incomparably more rapid than if the whorl is filled with water. It is

98 T. VON BRAND, P. McMAHON AND M. O. NOLAN

therefore most likely that the reduction in oxygen consumption was largely a
consequence of desiccation proper, although in the longer-lasting series starvation
may well have been a contributing factor.

That loss of water per se influences snails can be deduced also from our heart-
beat observations. Starvation in water led to a reduction in rate, and a similar
reduction, though slower to appear, was evident in snails desiccating at 96 per cent
relative humidity. At lower humidities, on the contrary, the period of decline was
preceded by one of increased rate and many irregularities in heart action were ob-
served. While the final water loss was not very different, it was more gradual
in the 96 per cent series and a difference in over-all tissue hydration was probably
present even towards the end of the experiments (see below). On the whole, the
impression was gained that at 96 per cent humidity the heart had an opportunity
to adapt itself to changed conditions, while this did not occur during the shorter
periods involved at lower humidities. It is probable that at the lower humidities
increased concentration of organic and inorganic materials accumulating in the
blood may have put a strain on the heart. It was not directly demonstrated because
Australorbis is for technical reasons not suitable for such experiments, but
Arvanitaki and Cardot (1932) had found previously a salt concentration of 0.080 N
in Helix pisana collected immediately after a rain and 0.147 N seven days after-
wards.3

In humidities of 85 per cent and below, the last snails died when they had lost
about 70 per cent of their original water.4 While this loss is very large,5 the over-
all tissue hydration does not decline to the same extent, because the remaining water
hydrates the tissues of an animal whose weight has declined. The general relations
between over-all tissue hydration and total water loss are shown in Figure 5. This
figure is drawn on the assumption that the organic material remains unchanged,
and is therefore valid only in cases of very rapid desiccation. In experiments of
long duration, such as at 96 per cent relative humidity, a considerable percentage
of tissue is lost. While no exact data could be secured, a final loss of 50 to 60 per
cent appears possible (see below). If this loss is taken into account, the over-all
tissue hydration was about the same at the beginning as at the end of the desiccation
period (Table III). Even if this should be literally true (and no such claim is
made), the physiological state of the desiccating snail probably differs from that of
snails kept in water. For instance, any loss of water, whether accompanied by a
loss of tissue or not, should result in an increased percentage of inorganic material

3 It is probably unwarranted, however, to generalize: Pusswald (1948) reported that the
blood of the slugs Arion and Limax lost 84.5 and 92.0 per cent of their initial water, respectively,
when the water loss of the entire body was 60 per cent. In Limax the percentage of water
content of the blood had at this point declined from 97.6 per cent to 77.3 per cent. In snails
with external shells the water loss of the blood seems to be less pronounced. Gebhardt-Dunkel
(1953) studied five species of terrestrial snails and found a decline in the water content of the
blood from initial values ranging in the various species from 97.7 to 98.1 per cent to final values
varying between 88.4 and 88.8 per cent shortly before death from desiccation.

4 Actually, the water loss is probably slightly higher since the water resulting from the
oxidation of food reserves has not been taken into account in this calculation.

5 This resistance to loss of water is not unique. Roots (1956) states that the earthworms
Allolobophora chlorotica and Lumbricus terrestris survive losses of body water of 75 and 70
per cent, respectively. Other invertebrates are more sensitive. According to Biancamaria
(1955), the crayfish Potamon cdnlis dies after having lost 15 to 23 per cent of the original
water. For older data on resistance to desiccation, see Hall (1922).

DESICCATION OF SNAILS

99

100

• Weight of snail

Hyd ration of soft tissues

10 20 30 40 50

PERCENT OF TOTAL WATER LOST

70

FIGURE 5. Theoretical relation between weight (water loss) and over-all hydration of soft
tissues in desiccating Australorbis glabratus. Initial water content 87 per cent.

in the remaining tissues, unless the snail is capable of incorporating the excess into
the shell, a point that was not studied. Computations from data presented by
Buck and Keister (1949, Fig. 7) show that in flies, also, considerable water loss
may occur without decrease in over-all tissue hydration. This suggests that the phe-
nomenon may be widespread.

The endogenous foodstuffs during desiccation were studied only at 96 per cent
relative humidity where a consumption of polysaccharides, lipids, lactic and volatile
acids was found. Since there was no indication of a partial shift to anaerobiosis,
total oxidation can be assumed and the oxygen required for it can be calculated.
It is also possible to calculate approximately the total oxygen consumed during the
desiccation period by graphic integration of the rates determined at the intervals
shown in Figure 4. As Figure 6 indicates, the above substances account only for

TABLE III

Calculated over-all hydration of the soft tissues of Australorbis glabratus when 50 per cent of the soft

tissues disappear during desiccation at 96 per cent relative humidity and the final total weight of

the desiccated snail is 67 per cent of the pre-desiccation value, as was found

Total weight,
mg.

Shell weight,
mg.

Weight of soft
tissues, mg.

Weight of
water, mg.

Per cent water
in complex soft
tissues +water

Pre-desiccation

100

31.6

9.2

59.2

87

Post-desiccation

67

31.6

4.6

30.8

87

100

T. VON BRAND, P. McMAHON AND M. O. NOLAN

a relatively small fraction of the total oxygen consumption, both during desiccation
and during starvation in water. In conformity with other starving organisms,
it may be assumed that proteins were the main substrate. A calculation of the total
oxygen consumed by snails desiccating at 96 per cent relative humidity for 128 days
gives approximately 56 ml. oxygen per one gram original weight. About 9 ml. are
accounted for by the oxidation of polysaccharides and lipids during the first 30
days (the additional amount for lactic and volatile acids is negligible). Since these
reserves were largely depleted at the end of this period, 47 ml. of oxygen can

MEASURED OXYGEN CONSUMPTION
CALCULATED " "

SNAILS STARVING IN WATER
SNAILS DESICCATING AT 96%
RELATIVE HUMIDITY

DAYS

FIGURE 6. Comparison of oxygen consumption calculated from polysaccharide and lipid
consumption, assuming total oxidation, and measured oxygen consumption (total oxygen
consumption obtained by graphic evaluation of determinations done at specified intervals ; see
text). The values have been calculated for one gram pre-starvation or pre-desiccation weight.

tentatively be linked with protein consumption, permitting the oxidation of ap-
proximately 48 mg. of protein. Since a one-gram snail (initial weight) contains,
on an average, 92 mg. of dried soft tissue, the calculated tissue loss would be
roughly 50 to 60 per cent. This figure appears possible, since even higher organ-
isms can lose more than 50 per cent of their weight during starvation, e.g., a dog
discussed by Putter (1911) decreased in weight from 19.65 kg. to 9.17 kg., yet
recovered upon feeding. Other examples are planarians, which decrease so mark-
edly in size during starvation that their weight loss must be far larger than 50 per
cent (Stoppenbrinck, 1905; Berninger, 1911).

DESICCATION OF SNAILS 101

SUMMARY

1. Decreasing humidity leads to a progressively more rapid decline of survival
time, body weight and rate of oxygen consumption. Snails starving in air of high
humidity survive longer than snails starving in water, but their final weight is
lower.

2. The heart rate of snails starving in water or desiccating at 96 per cent rela-
tive humidity decreases. At all lower humidities a transitory phase of increased
heart rate and many irregularities in heart action occurs.

3. During starvation in water and during desiccation, polysaccharide and lipid
stores become depleted. Lactic acid disappears completely from the tissues during
desiccation and volatile acids diminish.

4. It is concluded that the decrease in oxygen consumption is largely due to
desiccation proper but that at high humidity starvation is a contributing factor.

5. Snails desiccating at high humidity have a purely aerobic metabolism.
The relationship between the oxygen required for oxidation of polysaccharides and
lipids and the total oxygen consumed indicates that protein may be the main sub-
strate during prolonged periods of starvation in water or of desiccation.

6. The percentage of total body water lost and the percentage of water in the
tissues do not decrease at the same rate during desiccation, tissue hydration de-
clining at a slower rate. If marked tissue losses occur during long periods of
desiccation, the over-all tissue hydration may remain unchanged even if the total
water loss is very pronounced.

LITERATURE CITED

ARVANITAKI, A., AND H. CARDOT, 1932. Sur les variations de la concentration du milieu

interieur chez les mollusques terrestres. /. Physiol. et Pathol. gen., 30 : 577-592.
BARKER, B. S., AND W. H. SUMMERSON, 1941. The colorimetric determination of lactic acid

in biological material. /. Biol. Chem., 141 : 535-554.
BERNINGER, J., 1911. Ueber die Einwirkung des Hungers auf Planarien. Zool. Jahrb. Abt.

Allg. Zool., 30: 181-216.
BIANCAMARIA, E., 1955. Recherches biologiques et physiologiques sur Potamon edulis (Latr.)

(Crustace decapode brachyoure). Bull. Soc. Hist. Natur. Afrique Nord., 46: 155-168.
VON BRAND, T., 1936. A rapid working micro-modification of Pfliiger's glycogen method.

Skand. Arch, Physiol., 75 : 195-198.
VON BRAND, T., M. O. NOLAN AND E. R. MANN, 1948. Observations on the respiration of

Australorbis glabratus and some other aquatic snails. Biol. Bull,, 95: 199-213.
BUCK, JOHN B., AND MARGARET L. KEISTER, 1949. Respiration and water loss in the adult

blowfly, Phormia regina, and their relation to the physiological action of DDT. Biol.

Bull, 97 : 64-81.
BUEDING, E., 1949. Studies on the metabolism of the filarial worm, Litomosoides carinii.

J. Exp. Med., 89: 107-130.
GEBHARDT-DUNKEL E., 1953. Die Trockenresistenz bei Gehauseschnecken. Zool. Jahrb. Abt.

Allg. Zool, 64: 235-266.

HALL, F. G., 1922. The vital limit of exsiccation of certain animals. Biol. Bull., 42: 31-51.
MAGALHAES NETO, B., 1954. Agao de dessecagao e do jejum sobre a respiragao do Australorbis

glabratus. Pubs. Avulsas Instit. Aggeu Magalhaes, 2 : 5-9.
MEENAKSHI, V. R., 1956. Physiology of hibernation of the apple-snail Pila virens (Lamarck).

Current Science (India), 25: 321-322.
MEHLMAN, B., AND T. VON BRAND, 1951. Further studies on the anaerobic metabolism of some

fresh water snails. Biol. Bull., 100: 199-205.
NEWTON, W. L., AND T. VON BRAND, 1955. Comparative studies on two geographical strains of

Australorbis glabratus. Exp. Parasitol., 4: 244-255.

102 T. VON BRAND, P. McMAHON AND M. O. NOLAN

NOLAN, M. O., AND T. VON BRAND, 1954. The weight relations between shell and soft tissues

during growth of some fresh-water snails. /. Washington Acad. Sci., 44: 251-255.
OLIVIER, L., 1956a. Observations on vectors of schistosomiasis mansoni kept out of water in the

laboratory. I. /. Parasitol, 42: 137-146.
OLIVIER, L., 1956b. The location of the schistosome vectors, Australorbis glabratus and Tropi-

corbis centrimctralis, on and in the soil on dry natural habitats. /. Parasitol., 42 :

81-85.
OLIVIER, L., AND F. S. BARBOSA, 1955. Seasonal studies on Australorbis glabratus Say from

two localities in Eastern Pernambuco, Brazil. Pubs. Avulsas Inst. Aggeii Magalhaes,

4: 79-103.
OLIVIER, L., AND F. S. BARBOSA, 1956. Observations on vectors of schistosomiasis mansoni

kept out of water in the laboratory. II. /. Parasitol., 42 : 277-286.
PRECHT, H., 1939. Die Resistenz gegen Austrocknung bei Planorbiden. Zoo/. Anz., 128 :

124-135.

PUTTER, A., 1911. Vergleichende Physiologic. G. Fischer, Jena.
PUSSWALD, A. W., 1948. Beitrage zum Wasserhaushalt der Pulmonaten. Zeitschr. /. vergl.

Physiol., 31 : 227-248.

ROOTS, B. L, 1956. The water relations of earthworms. II. Resistance to desiccation and im-
mersion, and behaviour when submerged and when allowed a choice of environment.

/. Exp. Biol, 33: 29-44.
STOPPENBRINCK, F., 1905. Einfluss der herabgesetzten Ernahrung auf den histologischen Bau

der Siisswasser Tricladen. Zeitschr. f. wiss. Zoo/., 79 : 496-547.
WEINBACH, E. C., AND M. O. NOLAN, 1956. The effect of pentachlorophenol on the metabolism

of the snail Australorbis glabratus. Exp. Parasitol., 5 : 276-284.

SIMILARITIES BETWEEN DAILY FLUCTUATIONS IN BACKGROUND

RADIATION AND Oo-CONSUMPTION IN THE

LIVING ORGANISM *• 2

FRANK A. BROWN, JR., JOAN SHRINER AND H. MARGUERITE WEBB

Department of Biological Sciences, Northwestern University and Department of
Physiology and Bacteriology, Goucher College

Recent studies on fluctuations in Oo-consumption and in spontaneous activity
in conditions constant with respect to all factors known to influence organisms, have
provided strong evidence that some external fluctuating physical factors are still
exerting an influence on protoplasmic systems. The studies were made in con-
junction with an analysis of temperature-independent, solar-day and lunar-day,
cycles under constant conditions. Solar-day cycles have been known for a number
of years to be widespread among organisms, and more recently it has become evident
that lunar-day cycles also occur.

The evidence for an influence of an external factor has come from the recent
rediscovery (see Stewart, 1898, for early literature) of correlations of organismic
activities with barometric pressure and its changes (Brown, Freeland and Ralph,
1955 ; Brown, Webb, Bennett and Sandeen, 1955 ; Brown, Bennett, Webb and
Ralph, 1956). These correlations have recently also been shown to occur in two
lag-lead relationships. One is between the rates of barometric pressure change at
certain specific times of day n, n-l, and n-2 as correlated with biological activity
at an approximately corresponding time on day n. A second correlation is between
the organismic activity at a particular time of day, expressed either in absolute
terms or as deviation from the daily mean and the mean daily barometric pressure
of the second day thereafter. That these correlations are in no manner responses
to pressure changes themselves is clear not only from the lead-correlation of the
organism on barometric pressure, but also from studies in which organisms were
shielded from the normal external pressure fluctuations for as long as three con-
secutive months.

Recent work (Figge, 1947; Brown, Bennett and Ralph, 1955) has suggested
that some form of cosmic radiation might be capable of influencing organisms.
This view has been strengthened by the discovery of 27-day organismic fluctuations
(Brown, Bennett, Webb and Ralph, 1956), a frequency recently reported to exist
also in fluctuations in cosmic radiation (Simpson, 1954). As a consequence, the
following studies were undertaken to investigate in some detail any possible re-
lationships between general background radiation and organismic metabolism.

1 These studies were aided by a contract between the Office of Naval Research, Department
of the Navy, and Northwestern University, NONR-122803.

2 The authors wish to acknowledge their indebtedness to Professor H. T. Davis of the
Department of Mathematics, Northwestern University, who gave freely very valuable advice
during the course of the investigation and preparation of the material for publication.

103

104 F. A. BROWN, JR., J. SHRINER AND H. M. WEBB

MATERIALS AND METHODS

The O2-consumption of potatoes was recorded continuously from February 1
through May 31 at Evanston, Illinois by means of Brown (1954) recording
respirometers modified in such a manner (Brown, 1957), that a constant pressure
was maintained by hermetically sealing the respirometers and recording system in
rigid copper containers, the barostats, in which the pressure was kept at a constant
reduced level of 28.50 inches Hg. Five barostats, each with four respirometers
jointly providing a single continuous record of the fluctuations in rate of O2-
consumption, were in essentially continuous operation during the four-month period.

A cylindrical core of potato with an eye was placed in each respirometer.
These cores were about 2.2 cm. in diameter and l1/^ cm. high. The first lot was
prepared on January 31, and with the exception of a very few occasional single-
potato replacements continued in the respirometers until May 1 when a completely
new set of potatoes was substituted. These latter were followed through the month
of May. Therefore, the first lot of potatoes remained in constant conditions in-
cluding pressure for three months except for brief periods of 15—20 minutes once
every two to six days when the O, reservoirs were being refilled and the CO2-
absorbent renewed. The second lot remained in constant conditions, with no
replacements for one month.

Only complete, uninterrupted calendar-days of recordings were used in the
analysis. Partial days of data (days a respirometer was set up) were discarded.

Background radiation was recorded continuously during the same four-month
period by means of a 2 X 30-inch cosmic-ray counter with an appropriate sealer
and data printer. This monitoring system, located in the same laboratory with the
five barostat-respirometer ensembles, yielded a count rate of the order of 40.000/
hour. A few days of data were missed about the middle of May.

RESULTS

There were clear systematic fluctuations in Oo-consumption in the potatoes
throughout the four-month period. These were most commonly ones appearing to
possess a single major cycle a day. When 3 X 7-hour moving means of the average
of all those two to five barostats for which recordings were complete on that day
were calculated it was found that these daily fluctuations involved up to 28% in-
crease, with a mean of 13.7% from lowest to highest values for the day for 30
sample days taken at random. In view of the leveling influence of the 3 X 7-hour
sliding average, the actual range was undoubtedly substantially greater.

In Figure 1 (Nos. 1-5) are seen the mean forms of the daily fluctuation for the
four-month period for each of the five barostats. It is quite evident that two general
forms of mean daily fluctuation are apparent. Numbers 1, 3 and 4 showed a clear
major cycle with a minimum in the early morning hours and a maximum in the late
afternoon. Numbers 2 and 5 exhibited essentially a 180°-phase shift relative to
the others. These five, independent, four-month samples, from lowest to highest
values in the mean daily cycles are, respectively, 10%, 7.3%, 9.0%, 10%, and
4.8%. The average value of the five, 8.2%, is in remarkable agreement with the
value, 8.0%, obtained for a two-month period in the spring of 1955 (Brown, 1957).
All five mean cycles possess a minor peak about 6 P.M. and slight minima at
1-2 A.M. and 3 P.M., irrespective of the form of the major cycle. This fact is em-

METABOLISM AND BACKGROUND RADIATION

105

o
0

6
AM

12

6
PM

FIGURE 1. 1, 2, 3, 4, and 5 depict the mean daily cycles of fluctuation in Oa-consumption
in potatoes, expressed as deviations from daily means, for each of the five independent respirom-
eter-recording systems over the four-month period of study. 1-5 is the mean cycle for all the
data. The actual percentage of the fluctuations is given in the text.

106

F. A. BROWN, JR., J. SHRINER AND H. M. WEBB

phasized in the mean 4-month cycle for all the barostats, Figure 1, (1-5) in which
the 6 P.M. deviation from the daily mean is positive and highly significantly dif-
ferent from 0.

The mean daily cycle for all the barostats displayed no really significant evidence
of a daily cycle except for the 5-6-7 P.M. peak clearly as a consequence of the
algebraic summation of two forms of cycles, one essentially 180° out of phase with
the other. It was apparent, also, from inspection of the single monthly mean

FEB

MAR

APR

MAY

B.

PM

12

18

FIGURE 2. A. The mean daily cycles of general background radiation for each of four
months. B. The mean lunar-day cycles of general background radiation for each of the four
months. Lunar Zenith occurs at the 12th hour ; Nadir, about 0 or 24. All these cycles are three-
hour moving means of radiation. The mean percentages of the fluctuations are given in the text.

cycles for the single barostats that each of the five mean four-month cycles included
monthly cycles tending to be of the form of the final four-month mean form, or 180°
out of phase with it.

It seemed quite evident, therefore, that daily cycles of the potatoes were tending
to exhibit one or the other of two forms, with, to the present, no suggestion that
the occurrence of one type or the other is other than random.

An inspection of the fluctuations in radiation indicated there to be, in general,
a clear mean daily cycle with a maximum about 6 A.M. and a minimum about 6

METABOLISM AND BACKGROUND RADIATION

107

P.M. The mean daily cycles of three-hour moving means for each of the four
months are seen in Figure 2 A. The mean cycles for February, March, and April
are of strikingly similar form, with a gradually increasing amplitude (1.2%, 1.5%,
and 2.3% increase from lowest to highest values). The maximum amplitude for
a single day was about 10%. The cycle for May (5 days of data missing) showed
an altered form though the amplitude, 1.3%, was of the same magnitude. In-
spection of the daily data showed five days of the month (May 5, 15, 20, 23, 29) to
have their cycles shifted about 180° relative to all the other days. The mean ampli-
tude for the "typical" days was 2.2%, that for the five "shifted" days, 3.2%. Three
days of the preceding month (April 1, 2, and 15) were also "shifted" days; no
"shifted" days were present in February and March.

In view of the described presence of mean lunar-day cycles in numerous animals
and plants, the mean lunar-day cycles of radiation were determined for each of the
four months. These are seen in Figure 2 B. The cycles for February and March

50

25

0

. 0
25
50

FEB
AM

PM

V -»'

B

MAR

APR

MAY

O

o

FIGURE 3. Five-day weighted (1 :2:3:2:1) means of deviations of radiation intensity during
the 2-6 A.M. and 2-6 P.M. periods from the daily means (solid lines) and corresponding weighted
five-day means of O2-consumption in potatoes for the 4-7 A.M. and 4-7 P.M. periods (dotted
lines).

were rather similar, that of April shifted essentially 180° relative to the preceding
two months. The cycle for May, with the highest amplitude of the four, again
resembled those of February and March. There appeared to be a tendency for a
maximum, or a minimum, to occur between the time of lunar Zenith and two to three
hours afterwards, and for a maximum or a minimum to occur at the time of lunar
Nadir. The amplitudes of the four average monthly cycles are, respectively, from
minimum to maximum values, 0.57%, 0.37%, 0.60%, and 0.83%.

CORRELATION BETWEEN RADIATION AND OO-CONSUMPTION

It is quite evident that even if the potato possesses mean daily cycles of O2-
consumption, its apparent tendency towards 180° phase-shifting would obscure
much of this when large quantities of data were averaged. In view, however, of the
relatively large-amplitude, daily fluctuations of the mean rates for all those potatoes
recorded on a single day, an attempt was made to learn whether there might be
a correlation between the amplitude of the day-by-day fluctuation in the background

108

F. A. BROWN, JR., J. SHRINER AND H. M. WEBB

radiation and the amplitude of the day-by-day fluctuation in CX-consumption of the
potatoes.

In Figure 3 A is shown a weighted (1:2:3:2:1) five-day moving mean of
the deviations in intensity of radiation from its daily mean for the 2-6 A.M. period
(all were positive values), and similar deviations from the daily means for the
2-6 P.M. period (all were negative values). Plotted on different ordinate scales are

1.7
1.6

1.5

1.4

1.3
1.2

I.I
JO

O
O

.9
.8

7
6

.5
.4

.3

O

O

O

200 400 600 800
RADIATION

1000 1200

FIGURE 4. The relationship between the deviation in O2-consumption at 4-7 A.M. and
4-7 P.M. from the daily mean of day n, expressed as deviations from monthly means, and the
square of the deviation of radiation at 2-6 A.M. and 2-6 P.M. from its daily means for day n-1
expressed in the same terms (P < 1Q-6).

superimposed correspondingly weighted five-day moving means of the deviation of
Oo-consumption from the daily means for the 4—7 A.M. and 4—7 P.M. periods. It
is evident from inspection that there is a highly suggestive similarity between the
fluctuations in radiation and in Oo-consumption if one admits that the organismic
cycles display alterations in sign of their correlation from time to time.

To quantify this similarity and to obtain at least an approximate measure of the
significance of such an apparent similarity, a coefficient was determined for the

METABOLISM AND BACKGROUND RADIATION 109

correlation between the deviations of radiation from its mean monthly deviations for
each of the two times of day, on the one hand, and the deviations of CX-consumption
from their mean monthly deviations, for these times of day on the other. In this
correlation, the signs of the deviations were ignored. A study of the regressional
relationship indicated the relationship to be non-linear, and that the deviation in
CX-consumption was, instead, linearly related to the square of the deviation in radia-
tion, and that the deviation in O2-consumption on day n showed its correlations with
day n-2, and especially n-l, of radiation with a rapid drop in coefficient to days
n-3 and n. The coefficients and their errors for days n through n-3 were, re-
spectively, 0.191 ± 0.066, 0.337 ± 0.062, 0.290 ± 0.064, and 0.135 ± 0.068.

The calculated regression for CX-consumption on day n on the square of radiation
for day n-l is seen in Figure 4. The relationships between deviations in radiation
and in CX-consumption were calculated to be as follows :

Radiation QO2

0.5% 0.3%

1.0% 1.8%

1.5% 4.1%

2.0% 7.0%

The lag-lead relationship, radiation day n-l, was apparently simply the best
compromise between radiation on day n-2 when using only the 4-7 A.M. value of
O2-consumption (0.41 ±0.081), and radiation on day n using only the 4-7 P.M.
value (0.369 ±0.084).

An inspection of the form of the fluctuations in radiation and in O2-consumption
clearly suggested that in no other lag-lead relationship would correlations sig-
nificantly different from zero be found over the four-month period. However, this
was investigated more specifically. Correlations were found between radiation and
CX-consumption as follows : between the 4-7 A.M. deviation in CX-consumption on
day n from its mean 13-day deviation for this time of day, and the deviation of the
4—7 P.M. (day n-2) to 4—7 A.M. (day n-l) change in radiation from its mean
monthly change for this period the coefficient was 0.352 ± 0.087. On the other
hand, between the corresponding 4-7 P.M. deviation in CX-consumption for day n
and the corresponding 4—7 A.M. to 4-7 P.M. change in radiation of day n the
coefficient was 0.382 ± 0.083. Eleven other lag-lead relationships for each of the
times of day, sampling from day n-30 to day n + 15 for radiation failed to yield
any correlations similarly highly significantly different from zero.

DISCUSSION

The results which have just been described provide one additional kind of
evidence in support of the conclusion reached by Brown, Freeland and Ralph

(1955) and Brown, Webb, Bennett and Sandeen (1955), in a study of correlations
between metabolism of various organisms and concurrent rates and directions of
barometric pressure change, that the organism in "constant conditions" still is
responding to fluctuations in some external physical factor or factors. This view
was given further support by the studies of Brown, Bennett, Webb and Ralph

(1956) on the quahog. In more recent studies (Brown, 1957) employing baro-
stats as in the current study, correlations with barometric pressure were found

110 F. A. BROWN, JR., J. SHRINER AND H. M. WEBB

for O2-consumption in potatoes over about a l^-month period of study. In this
latter investigation, unlike in earlier ones, there was a strong indication that the
fluctuations in O2-consumption on any given day tended to display either one of two
patterns, one tending to be 180° out of phase with the other for either the whole or
part of the day.

In the last-mentioned study there was a correlation of the deviations (ignoring
sign) from the daily mean of the O2-consumption of the potato at 4—7 A.M. on day n
with the rate and direction of barometric pressure change from 2 to 6 A.M.
centered on day n-2. In another kind of analysis of the data, there was found to be
for the potato, a correlation between the 4-7 P.M. deviation from the daily mean of
O2-consumption and the mean barometric pressure on day n + 2 (Brown, Webb
and Macey, 1957). Despite these correlations, the directly effective factor, in view
of the use of barostats, could not have been pressure per se.

It is interesting that in this current four-month study there was also clearly
reproduced a positive correlation between the deviation of the 4—7 P.M. value of
CX-consumption on day n from its daily mean and the mean barometric pressure
of day n + 2 (Brown, Webb and Macey, 1957). It will be recalled that the 4—7
P.M. period was the only period of the day possessing a non-inverting cyclic
component, and the correlation with barometric pressure correspondingly remained
positive throughout the whole four-month period. In the spring of 1954, when a
comparable and relatively striking negative correlation (—0.65) was found using
the potato (not in barostats), it was similarly only the 4-7 P.M. period of the day
which possessed this property.

Another notable observation made in the current studies is that the correlation
with the deviations in radiation were maximum for days n to n-2 (n-2, for the
4—7 A.M. O2-consumption) for radiation. This is essentially the same lead-lag
relationship found for the potato in the spring of 1955, a year earlier, with rates of
barometric pressure change for comparable periods of the day, and also which
obtained during the summer of 1955 for the sea weed, Fucus (unpublished results).

It seems reasonable to postulate that the living organism is displaying a mean
one- to two-day lag response to some external factor correlated both with fluctua-
tions in barometric pressure and with daily cycles in background radiation, and
that the effective external fluctuations are in some manner correlated with the
mean daily barometric pressure on the third to fourth day thereafter. From the
standpoint of a possible significance of these external factors for the maintenance
of the precision of the many known regular daily cycles observed to persist for long
periods under constant conditions, it must be admitted that the organism can
exhibit a metabolic response to some external factor which has clear mean daily
cycles, even though with a randomly fluctuating amplitude. It has been postu-
lated earlier (Brown, 1957) that organisms, through an endogenous capacity to
oscillate, are able to maintain in many instances an endogenous cycle of the same
frequency as the external ones.

The mean lunar-day cycles of radiation are of special interest relative to lunar-
day mean cycles of biological activities which have been described (e.g., Brown,
Freeland and Ralph, 1955 ; Brown, Webb, Bennett and Sandeen, 1955 ; Brown,
Shriner and Ralph, 1956). The principal maximum (or minimum) in both the
mean lunar-day cycles of radiation and of most of those biological activities so far
described appear to occur at the times of lunar Zenith or shortly afterwards. The

METABOLISM AND BACKGROUND RADIATION 111

results encourage a more detailed study of lunar-day relationships of radiation and
activity comparable to the current one for solar-day relationships. Such a study
is in progress. Also, very suggestive in this regard is the observation that the
ratio of amplitude of the mean solar-day cycles to the lunar-day ones of radiation,
is of the same order as the ratio of the amplitude of the solar-to-lunar-day cycles of
most of the reported mean cycles for organisms, namely 2 or 3 to 1 .

SUMMARY

1. Oo-consumption of potatoes was recorded continuously through the four-
month period, February-May, 1956.

2. There were daily fluctuations in rate which, even using 3 X 7-hour moving
means, displayed a mean amplitude of about 14%.

3. Five independent respirometer-recording systems yielded mean daily cycles
for the four-month period ranging in amplitude from 4.8 to 10.0% with a mean
of 8.2%.

4. The mean cycles were of two forms, one essentially 180° out of phase with
the other.

5. Solar-day and lunar-day mean fluctuations in background radiation were
also determined for the period of the investigation.

6. A small but very highly significant correlation existed between the fluctua-
tions in amplitude of the daily cycles in radiation on day n-l and amplitude of the
daily fluctuations in CX-consumption in the potato on day n.

LITERATURE CITED

BROWN, F. A., JR., 1954. Simple, automatic, continuous-recording respirometer. Rev. Sci.

fnstr., 25: 415-417.
BROWN, F. A., JR., 1957. Response of a living organism, under "constant conditions" including

pressure, to a barometric-pressure-correlated cyclic, external variable. Biol. Bull.,

112: 288-304.
BROWN, F. A., JR., M. F. BENNETT AND C. L. RALPH, 1955. Apparent reversible influence of

cosmic-ray-induced showers upon a biological system. Proc. Soc. Exp. Biol. and Med.,

89: 332-337.
BROWN, F. A., JR., M. F. BENNETT, H. M. WEBB AND C. L. RALPH, 1956. Persistent daily,

monthly, and 27-day cycles of activity in the oyster and quahog. /. Exp. Zool., 131 :

235-262.
BROWN, F. A., JR., R. O. FREELAND AND C. L. RALPH, 1955. Persistent rhythms of O2-con-

sumption in potatoes, carrots and the seaweed, Fucus. Plant Physiol., 30 : 280-292.
BROWN, F. A., JR., J. SHRINER AND C. L. RALPH, 1956. Solar and lunar rhythmicity in the rat

in "constant conditions" and the mechanism of physiological time measurement. Amer.

J. Physiol., 184: 491-496.
BROWN, F. A., JR., H. M. WEBB, M. F. BENNETT AND M. I. SANDEEN, 1955. Evidence for an

exogenous contribution to persistent diurnal and lunar rhythmicity under so-called

constant conditions. Biol. Bull., 109 : 238-254.
BROWN, F. A., JR., H. M. WEBB AND E. J. MACEY, 1957. Lag-lead correlations of barometric

pressure and biological activity. Biol. Bull., 113: 112-119.
FIGGE, F. H. J., 1947. Cosmic radiation and cancer. Science, 105 : 323-325.
SIMPSON, J. A., 1954. Cosmic-radiation intensity-time variations and their origin. III. The

origin of 27-day variation. Physical Rev., 94 : 426-440.
STEWART, C. C., 1898. Variations in daily activity produced by alcohol and by changes in

barometric pressure and diet, with a description of recording methods. Amer. J.

Physiol., 1 : 40-56.

LAG-LEAD CORRELATIONS OF BAROMETRIC PRESSURE
AND BIOLOGICAL ACTIVITY *• 2

FRANK A. BROWN, JR., H. MARGUERITE WEBB AND ERWIN J. MACEY

Department of Biological Sciences, Nortlnvestern University; Department of Physiology
and Bacteriology, Goucher College; and the Marine Biological Laboratory,

Woods Hole, Mass.

It has been known for many years that numerous kinds of organisms repre-
senting most of the major divisions of living things exhibit under constant condi-
tions overt cyclic fluctuations of solar-day frequency of one or more of their
processes. Less well known is the fact that at least a few organisms which live in
the intertidal regions of the oceans have similarly clear cycles of tidal frequency.
These rhythms appear to be expressions of a biological-clock system upon which
the organism normally relies importantly in the adaptive regulation of its physio-
logical behavior in its rhythmic external environment. In the past few years it has
been firmly established that the frequencies of these cycles are independent of
temperature over a wide temperature range, a characteristic which is, of course,
an essential one if these cycles were to have practical adaptive value in the normal
environment with its substantial fluctuations in temperature from hour to hour and
day to day. Particularly within the past two years it has become more and more
evident that all animals and plants have average solar-day and lunar-day fluctua-
tions and that in the maintenance of these cycles, the organisms are receiving stimuli
of some character from the fluctuating external physical environment even under
conditions generally considered to be constant.

Evidence for an influence of a fluctuating external factor affecting the organisms
even under "constant conditions" has come from both 1) highly significant
correlations between hourly rates of metabolism in several organisms and concurrent
hourly barometric pressure changes, and 2) remarkable similarities in the forms
of day-to-day changes in mean rates of metabolism for the whole day, or specific
parts of a day, and the forms of the day-to-day large climatic changes in the mean
daily barometric pressures (approximated by the pressure for any arbitrarily se-
lected, restricted, time of day). The day-to-day drifting of mean barometric
pressures in a temperate-zone area appears superficially to be random, with every
given period a month or so long, exhibiting its own specific pattern. Despite this,
the forms of the day-by-day fluctuations in rates of metabolism or certain other
biological phenomena of a number of species of organisms have appeared to be
rather similar, either in a direct relationship or an inverse one, to the concurrent
fluctuations in mean daily pressures. This has been found true to such an extent

1 These studies were aided by a contract between the Office of Naval Research, Department
of the Navy, and Northwestern University, NONR-122803.

2 The authors wish to acknowledge their indebtedness to Professor H. T. Davis of the
Department of Mathematics, Northwestern University, who gave freely very valuable advice
during the course of the investigation and preparation of the manuscript for publication.

112

METABOLISM AND BAROMETRIC PRESSURE 113

as to seem more than fortuitous, though there is no generally acceptable test for
significance of correlations of such time series.

For many years relationship between barometric pressure and human physiology
has been believed to exist. As a result of the prevalence of such a view during the
first half of the last century Vivenot (1860) performed what he considered a crucial
experiment to test this view. He constructed an air-tight chamber within which an
experimenter could study pulse and respiration rates in subjects while the pressure
within the chamber was experimentally altered. With this he found no evidence
that small pressure changes had any influence upon the individual. A few years
later, Lombard (1887) described striking similarities between fluctuations in the
strength of the normal knee-jerk and concurrent fluctuations in external barometric
pressure and temperature. The correlation was positive with pressure and negative
with temperature. Later, the same investigator (Lombard, 1892) in more ex-
tensive studies reported a correlation between the rate of fatigue for voluntary
contraction of the flexor muscle of the second finger and barometric pressure.
With rising pressure there was increased capacity for work and with falling, de-
creased. The biological phenomenon retained its correlation with both the regular
daily tidal and the irregular climatic pressure changes. Furthermore, pressure
changes experimentally obtained by ascending and descending a mountain yielded
quite comparable correlations.

Some experiments with spontaneous activity of other mammals yielded com-
parable apparent relationships to barometric pressure changes. Hodge (1897),
studying the spontaneous running of two dogs through the application of special
pedometers to their collars, found a correlation between the mean daily activities
of the two dogs with respect to one another and to the concurrent mean daily
barometric pressure. With the latter the correlation was positive over the two-
month period of study. The correlation with pressure was extensively confirmed
by Stewart (1898) employing rats and certain other mammals, in activity recorders.
Gray rats exhibited a negative correlation with pressure over the 70-day period of
his study. White rats, for a 30-day period included within the previous study
period, showed a positive correlation with pressure. Stewart postulated, on the
evidence at hand, that wild mammals displayed a negative, and domesticated animals
a positive, correlation with pressure.

A widespread occurrence of a correlation between barometric pressure changes
and biological activities was found by Brown, Freeland and Ralph (1955) and
Brown, Webb, Bennett and Sandeen (1955) who were investigating fluctuations
in Oo-consumption in potatoes, carrots, seaweed, fiddler crabs, and salamanders.
All of these organisms showed correlations highly significantly different from zero
between the hourly rates of O2-consumption and the concurrent rate and direction
of barometric pressure change. Later, Brown, Bennett, Webb and Ralph (1956)
found correlations between the spontaneous opening of oysters and quahogs, and
barometric pressure changes. In these recent studies it was strongly suggested
by inspection of the data, however, that in paralleling the patterns of fluctuations,
the organisms \vere actually leading the barometric pressure changes by more than
a day.

One purpose of this report is to describe results from a simple statistical analysis
of all the results on this score obtained in our laboratories between March 1, 1954
and June 9, 1955. These point conclusively to an ability of a wide variety of living

114

F. A. BROWN, JR., H. M. WEBB AND E. J. MACEY

TABLE I

Organism and place

Dates (inclusive)

Correlation times
Organism/Bar, pressure

r

Fucus1
Woods, Hole, Mass.

July 28-Aug. 2\1954
Aug. 21-29 J

Aug. 3-20, 1954

day n / n + 2
(5-7 A.M.) (5-7 A.M.)
day n / n -\- 2
(5-7 A.M.) (5-7 A.M.)

-0.476±0.200
+0.507±0.176

Ostrea (10° C.)2
Evanston, 111.

Ostrea3

Mar. 1-April 13, 1954

June 18-July 28, 1954
July 29-Aug. 27, 1954

day n / n + 1
(av. daily) (av. daily)

day n / n + 2
(av. daily) (av. daily)

+0.411 ±0.125
+0.383±0.139
-0.658±0.104*

Rattus*

Nov 16-Dec. 3l 1954
Feb. 2-Mar. 13J 1955

Dec. 5-Feb. 1, 1954-55

day n / n -\- 7
(noon) (noon)
day n / n -f- 7
(noon) (noon)

+0.598±0.068*
-0.362±0.113

Solarium1
Evanston, 111.

Solatium6
Evanston, 111.
(5 groups)

May 12-June 9, 1954
April 1-June 8, 1955

day n / n + 1

day n / n -\- 2
(5-7 P.M.) (5-7 P.M.)

-0.650±0.110*
+0.266±0.071

Tritums*
Evanston, 111.

May 12-June 9, 1954

day n / n -\- 2
(5-7 P.M.) (5-7 P.M.)

-0.842 ±0.059*

Uca pugilator*
Woods Hole, Mass.

June 20-July 20, 1954
July 21-Aug. 27, 1954

day n / n + 2
(5-7 A.M.) (5-7 A.M.)
day n / n + 2
(5-7 A.M.) (5-7 A.M.)

-0.472±0.144
+0.595±0.104*

Uca pugnax*
Woods Hole, Mass.

June 18-Aug. 29, 1954

day n / n + 2
(5-7 P.M.) (5-7 P.M.)

-0.423±0.100

Venus3
Woods Hole, Mass.

June 18-Aug. 29, 1954

day n / n + 2
(av. daily) (5-7 A.M.)

-0.446±0.096

1 Brown, Freeland and Ralph (1955).

2 Brown (1954).

3 Brown, Bennett, Webb and Ralph (1956).

4 Brown, Webb, Bennett and Sandeen (1955).
6 Brown, Shriner and Ralph (1956).

6 Brown (1957).

* Conventionally determined standard errors for such large values of r do not provide true
measures of probabilities.

METABOLISM AND BAROMETRIC PRESSURE 115

things, ranging from lower to higher plants and from lower to higher animals, to
show a lead correlation with barometric pressure changes by one to seven days
(usually two). The only organism studied in our laboratory during this period
which was not included in this report is the carrot, which was omitted, not because
of any lack of similar type of correlation, but rather because there were some known
injury effects on the (X-consuniption during part of the single-month period of
study.

The organisms, the times of study, and the coefficients of correlation obtained
in 703 organism-days are given in Table I. These are all correlations of three-day
moving means except for the potato in 1955, in which daily mean values of O2-
consumption were correlated with three-day sliding averages of barometric pressure.
Indicated in footnotes are the references to the publication of the results obtained
with these species. In these publications it had not occurred to the authors to make
this type of analysis, and furthermore, due to the extraordinary nature of the
conclusions, it is to be doubted that a single demonstration of the phenomenon
of a lead correlation, even though shown to be statistically significant (assuming
random fluctuation in the organism), would have been credited by most physiolo-
gists.

All of the results were obtained in conditions of constant, continuous low
illumination of the order of 1 ft. c. or less, except with the white rat, Rattus, for
which two periods of continuous darkness, totalling 43 days, were included in the
data. The conditions of the experiments with the brown alga, Fucus, two species
of fiddler crabs, Uca pugnax and Uca pugilator, the salamander, Triturus and the
potato, Solanum included also very precisely regulated constant temperature, and,
in addition, for Solanum in 1955, constant pressure through the use of a barostat.

The biological process studied was the rate of CX-consumption in Fucus, Uca
pugnax and Uca pugilator, Triturus and Solanum. It was the average daily
minutes open per hour for both the oyster, Ostrea and the quahog, Venus; it was the
total distance spontaneously run per day in the case of Rattus.

To determine when the correlation would be highest, whether with the same day
(day w) of barometric pressure, the following day (day n+ 1), the second day
after (day n + 2), or some earlier or later one, tracings of the fluctuations of the
physiological process and of barometric pressure were superimposed and inspected
in various temporal relationships. In the vast majority of cases, there was clearly
only one relationship which promised high correlation with an obviously rapid drop
towards no correlation with either greater or less displacement and no other re-
lationship with nearly as high a correlation could be seen by such inspection over
the rest of the period. In two cases, Triturus and Solanum, it was not evident
from inspection, whether the correlation would be best with day n + 1, n + 2,
or day n + 3. In these cases, coefficients were determined for all three times and
the highest one was selected ; it was obvious, however, there was no other relation-
ship with these species in which the correlation would be as high or significant.

The change in sign of the correlation from time to time seems clearly to be a
biological contribution, probably to be compared superficially with the well-known
changes of sign frequently observed in animal orientation. The sign change appears
to occur abruptly and cleanly; the cause of the change is still unknown. It is
known, however, that in all those cases examined in which the sign of correlation

116 F. A. BROWN, JR., H. M. WEBB AND E. J. MACEY

changed, there was concurrently a transformation of the form of the mean daily
and lunar-day cycles to essentially their mirror images.

For all the periods the correlations ranged in size from 0.266 to 0.842. All
were significantly different from zero by ordinary tests for correlations. Both the
— 0.362 for Rattiis and the 0.383 for Ostrea appeared to include one or two very
brief periods of change of sign in the relationship. The potatoes in 1955, judging
by periodic inversions of their solar-day cycles, appeared to be changing the sign
of their correlation from time to time, and probably hence the lowest, though quite
real, correlation.

The great majority of the correlations centered on day n + 2 of barometric
pressure. Solanum in 1954 and Ostrea at 10° C., centered on day n + 1. The
potato in 1955 gave almost the same correlation for n + 2 and n + 3. The most
surprising result, on the basis of hypotheses available to account for this phe-
nomenon, was that Rattus showed by far its highest correlation with day n + 7.
The coefficient would have risen from 0.598 ± 0.088 to 0.668 ± 0.07 by the justifi-
able statistical procedure of eliminating from consideration the transitional values,
those of Dec. 1-3 and Feb. 2, which were seen clearly to contribute naturally to
neither series.

Since there appeared to be no test for the significance of correlations between
two time series that would be acceptable to all statisticians, further experiments were
performed to attempt to demonstrate the reproducibility of the phenomenon. The
first of these involved a four-month study of potatoes in Evanston, Illinois. Small
cores of potatoes bearing eyes were obtained by means of a large cork-borer, and
one was placed in each of 20 respirometer vessels (Brown, 1954). Four
respirometer vessels upon a single recording system were sealed in each of five
barostats on Feb. 1, 1956. During a three-month period, the respirometers were
opened for about 15 minutes once every two to six days, to refill the O2-
reservoirs and replace the CO2-absorbent. Very rarely a potato was replaced with
a new one during this period. On the first of May, a completely new lot of
potatoes replaced the old, and the observations were continued through May 31.
During this study the potatoes were maintained in constant conditions of tempera-
ture (19.5° C.), of light (< 0.5 ft. c.), and of all other factors known to influence
organisms. The respirometers were kept under a constant reduced pressure of
28.50 inches Hg. Approximately 56,000 organism-hours of CX-consumption were
obtained.

Inspection of three-day moving means of the mean daily barometric pressure
for the four-month period, and comparison of weighted (1 :2 :3 :2 :1) five-day mov-
ing means of the 4—7 P.M. deviation in rate of O2-consumption from the daily mean,
gave clear suggestion that just as with the potatoes in the 1954 and the 1955 ex-
periments, there was a lag correlation of barometric pressure on CX-consumption
by about two days. A scatterplot of the relationship between the barometric
pressure of day n + 2 and CX-consumption on day n is seen in Figure 1. This
yielded a correlation coefficient of 0.339 ± 0.0835, a value highly significantly differ-
ent from zero.

Figure 1, B, illustrates the various values of r obtained in various lag-lead
relationships between the two phenomena, i.e., for CX-consumption of day n
correlated with barometric pressure in various temporal relations from day n — 30 to
day n + 15, a 45-day span. In this instance not only was a correlation centered

METABOLISM AND BAROMETRIC PRESSURE

117

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FIGURE 1. A. Scatterplot of the relation between the mean barometric pressure on day
n + 2 and the 5-6-7 P.M. deviation from the daily mean of O2-consumption of the potato on day
n. B. Coefficients of correlation (ordinate) between the 5-6-7 P.M. deviation in O2-consumption
from the daily mean on day n and the mean barometric pressure on various days from n — 30 to
n + 15 (abscissa).

on day n + 2, but a correlation was also found with barometric pressure, day

n-5.

A second attempt was made in 1956 to confirm an organismic lead-correlation*
of metabolism on barometric pressure. This one was performed in Woods Hole,
Mass., between June 16 and August 1. These observations were made upon fiddler
crabs, whose CX-consumption was measured under constant conditions including
pressure in the same type of apparatus as that used for the potatoes. In this study
of the fiddler crab, the temperature was similarly very constant but at a higher

A.

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FIGURE 2. A. Scatterplot of the relation between the mean barometric pressure on day
n + 2 and the 5-6-7 P.M. value of Do-consumption of the fiddler crab on day n. B. Coefficients
of correlation (ordinate) between the 5-6-7 P.M. value of Oa-consumption from the daily mean
on day n and the mean barometric pressure on various days from n— 10 to n + 10 (abscissa).

118 F. A. BROWN, JR., H. M. WEBB AND E. J. MACEY

value, near 24° C. In this analysis, as for the experiments performed in 1954 and
1955, the actual rates of O2-consumption at the 4-7 P.M. period were used. The
scatterplot relationship between respiration on day n and barometric pressure on
day n + 2 is shown in Figure 2, A. This yielded a coefficient of 0.49 ± 0.12,
remarkably close, coincidentally, to the mean of all the values obtained in 1954 and
1955 (0.5). In Figure 2, B, are depicted the values for r together with their
deviations for this and other lag-lead correlations between day n of O2-consump-
tion and day n — 10 to n + 10 for pressure. The only real correlations in the series
are for days n + 1 and n + 2, with the latter being the more highly significant.
In these correlations with the crabs three-day moving means were used for both
pressure and metabolism.

These two series of experiments performed in 1956 would appear to confirm
in a striking manner the conclusions reached in the earlier studies.3

The explanation of the phenomenon considered here seems from a general
standpoint to be quite evident. Since the organisms cannot be determining the
barometric pressure changes which are to occur, the organisms must be responding
to some physical factors and their fluctuations which themselves exhibit a lead-
correlation on barometric pressure. The potatoes in 1955 and the potatoes and
crabs in 1956 remained in each case in barostats at 28.50 inches Hg from one to
three months, the barostats opened only for about 15 minutes once every two to
six days, and hence, the lead correlation cannot be due to any special responses to
current rates of change in pressure itself. Since organisms have been shown to
possess fluctuations in metabolic rates correlated with the rates and directions of
barometric pressure change and especially since 27-day cycles have been found,
it appears suggestive that the organism may be able to respond directly to fluctua-
tions in the intensity of some high energy radiation or of some other physical
factor with radiation-correlated fluctuation.

It is futile at the present time to do much speculating as to the external forces
involved and the manners in which fluctuations in them may interact with the now
established solar-day and lunar-day clocks and average cycles within organisms.
This is now being investigated. It may be shown eventually that the phenomenon
described in this report depends in some manner on the possession by both the
organisms and the atmosphere of a solar-day and lunar-day cyclicity, and the inter-
action of these with some less orderly cosmic factor to which both the organism
and atmosphere can react in an oscillatory fashion. But irrespective of the detailed
mechanism, correlations of the order of magnitude described here (nearly 0.50 as
the average degree of correlation for eight species of animals and plants over about
850 days), and tending very strongly to be centered on day n + 2, especially since
they cannot be correlations with an actual causative force, are to be viewed as extra-
ordinary. They force one to conclude that the living organism is clearly responsive
in an orderly way to forces not hitherto seriously considered by biologists to possess
any influence.

3 Since the manuscript was completed, a further study of the potato during October,
November and December, 1956 also yielded a lead correlation in which the highest correlation
(— 0.400 ± 0.089) was similarly found with the mean barometric pressure of day n + 2, rapidly
falling on days n + 1 and n +3 to - 0.307 ± 0.094 and - 0.328 ± 0.093, respectively, and on days
n and n + 4 to a value not significantly different from zero. From inspection there was no
other lag or lead relationship in which a significant correlation existed.

METABOLISM AND BAROMETRIC PRESSURE 119

For the biologist who is attempting to account for the remarkable capacity of
organisms to measure off with great precision under so-called "constant condi-
tions," temperature-independent cycles of the frequencies of natural external cosmic
events, it becomes highly important to know whether the conditions are truly con-
stant for the organism, and if not, what is the actual character of the fluctuations
in the effective external factor or factors.

SUMMARY

1. Eight species of living things, ranging from lower to higher plants and lower
to higher animals, in studies over a three-year period and including approximately
850 species-days have exhibited without exception a statistically significant lead-
correlation on barometric pressure with an over-all mean coefficient of about 0.5.

2. The correlation involved sometimes only the 5-6-7 A.M., sometimes only the
5-6-7 P.M., and other times the mean daily rates of CX-consumption.

3. The sign of the correlation was sometimes positive and other times negative.
Sign changes, when they occurred during a single period of study were abrupt, and
correlated with a 180° -shift in the phase relationships of the concurrent mean
solar-day cycles.

4. In twelve periods of study, ranging from one to four months each, the corre-
lation in nine cases centered on day n + 2 of barometric pressure. In twTo cases it
centered on day n + 1 and in one, on day n + 7.

LITERATURE CITED

BROWN, F. A., JR., 1954. Persistent activity rhythms in the oyster. Amer. J. Physiol, 178:

510-514.
BROWN, F. A., JR., 1957. Response of a living organism, under "constant conditions" including

pressure, to a barometric-pressure-correlated, cyclic, external variable. Biol. Bull.,

112: 288-304.
BROWN, F. A., JR., M. F. BENNETT, H. M. WEBB AND C. L. RALPH, 1956. Persistent dalfy,

monthly and 27-day cycles of activity in the oyster and quahog. /. Exp^Zool,, 131 :

235-262. ^__~

BROWN, F. A., JR., R. O. FREELAND AND C. L. RALPH, 1955. Persistent rhythms of O2-con-

sumption in potatoes, carrots and the seaweed, Fucus. Plant Physiol., 30: 280-292.
BROWN, F. A., JR., J. SHRINER AND C. L. RALPH, 1956. Solar and lunar rhythmicity in the

rat in "constant conditions" and the mechanism of physiological time measurement.

Amer. J. Physiol., 184: 491-196.
BROWX, F. A., JR., H. M. WEBB, M. F. BENNETT AND M. I. SANDEEN, 1955. Evidence for an

exogenous contribution to persistent diurnal and lunar rhythmicity under so-called

constant conditions. Biol Bull, 109: 238-254.
HODGE, C. F., 1897. Experiments on the physiology of alcohol, made under the auspices of the

committee of fifty. Pop. Sci. Monthly, 50 : 796-812.
LOMBARD, W. P., 1887. The variations of normal knee-jerk, and their relation to the activity

of the central nervous system. Amer. J. Psych., 1 : 5-71.
LOMBARD, W. P., 1892. Some of the influences which affect the power of voluntary muscular

contractions. /. Physiol., 13 : 1-58.
STEWART, COLIN C., 1898. Variations in daily activity produced by alcohol and by changes in

barometric pressure and diet, with a description of recording methods. Amer. J.

Physiol, 1 : 40-56.
VrvENOT, R. v., JR., 1860. Ueber den Einfluss des veranderten Luftdruckes auf den Menschli-

chen Organismus. Virchow's Archiv., 19: 492-522.

THE LUMINESCENCE OF THE MILLIPEDE, LUMINODESMUS

SEQUOIAE *

J. WOODLAND HASTINGS AND DEMOREST DAVENPORT

Department of Biological Sciences, Northrvestcrn University, Evanston, Illinois and Department
of Biological Sciences, University of California, Santa Barbara College, Goleta, California

The millipede Luminodesmus sequoiae was first described by Loomis and
Davenport (1951). Following this, Davenport, Wootton and Gushing (1952)
described the biology of the animal and the general nature of its luminescence.
They found that light emission originates from cells in the deeper layers of the
integument. The present paper describes a more detailed study of its luminescence.

The bioluminescent reaction in four different organisms (Cypridina, fireflies,
bacteria and Gonyaulax*) has been studied in recent years (Tsuji, Chase and
Harvey, 1955 ; McElroy and Hastings, 1955 ; McElroy and Green, 1956 ; Hastings
and McElroy, 1955; Strehler, 1955; Hastings and Sweeney, 1957). The common
feature is that the reaction involves an enzymatic oxidation with molecular oxygen.
Although it was demonstrated by McElroy that adenosine triphosphate is an abso-
lute requirement for firefly luminescence, its possible role as an energy source in
the reaction has not been clarified. In bioluminescent reactions in general it is
assumed that the energy must be derived from the oxidation of a substrate, which
is usually termed luciferin (e.g., Cypridina luciferin, firefly luciferin, etc.). None
of the products of luminescent reactions have been definitely identified and the
reactants as well as the enzymes are different in all cases studied. Such studies are
of interest for the general problem of how the living cell transforms chemical
energy into other forms of energy.

MATERIALS AND METHODS

About 1000 animals were collected by a party of five on the nights of May 10
and 11, 1956, in the vicinity of Camp Nelson, Tulare County, California. The
animals were abundant and readily visible to the dark-adapted eye by their own
light on the surface of the ground in the forest. The animals were brought back
to the laboratory and stored in glass containers with ample humus. Light in-
tensity was measured with apparatus previously described (Hastings, McElroy and
Coulombre, 1953), using a photomultiplier tube and automatic graphic recording.

RESULTS
1. In vivo luminescence

Luminescence in Luminodesmus is continuous but fluctuating. The light in-
tensity (recorded from single animals over long periods up to 24 hours) fluctuates

1 This study was supported by grants from the National Science Foundation, the Graduate
School of Northwestern University, and the Research Committee of the University of Cali-
fornia, Santa Barbara College.

120

THE LUMINESCENCE OF LUMINODESMUS

121

by 20 to 40 per cent (or occasionally more) around a relatively constant mean.
Since these light intensity changes could be detected with the eye, we are certain
that they do not result from movements of the animal. A two-hour portion from
such a recording is reproduced in Figure 1A. It can be seen that there is an
instance when the light intensity doubled, apparently spontaneously. Such a marked
increase in light intensity also occurs when the animal is handled. Indeed we found
that striking the test tube in which the animal was placed would evoke such a re-
sponse (Fig. IB). The response still occurred immediately after the animal had
been decapitated. Upon stimulation of the nerve cord which induced electrical
shocks no luminescent response was observed which could be attributed to the effect
of the electrical stimuli. The way in which the luminescent changes are brought
about in the living animal is not clear.

\ \

FIGURE 1. Reproductions of recordings of Luminodesmus luminescence. Ordinate, light
intensity; abscissa, time, to be read from right to left in both cases. Left: Luminescence of an
undisturbed animal over a two-hour interval. Time between vertical divisions, 15 minutes.
Right: Luminescence changes of an animal where the test tube containing the animal was tapped
lightly at the two instances noted by arrows. Time between vertical divisions, one minute.

Isolated pieces of the animal retain their luminescence for a long time. The
intensity decreases gradually to about one half of its original value in 8 hours.
Although fluctuations in intensity may occur for the first 15 minutes, it is essentially
a steady luminescence thereafter. An eviscerated specimen from which the first
few and last few segments are cut off behaves in essentially the same manner.
Animals removed from humus and kept in a test tube for a day or more also showed
little fluctuation in intensity. Whether or not this was due to water depletion or
to starvation was not determined. Preparations with little or no light intensity
fluctuation were used in the various experiments described below.

2. Possibility of luminous symbiotic bacteria

In some organisms luminescence arises from an association with luminous bac-
teria (see Harvey, 1952) . The possibility that this might be the case in Luminodes-
mus was investigated some years ago by W. D. McElroy and one of us (J. W. H.).
Whole animals and extracts of animals were put on agar plates containing a variety
of media. Plates with a range of salt concentrations (0, 1%, 2% and 3% sodium
chloride) were made up with both glycerol and glucose as carbon sources and
Bacto Tryptone. No growth of luminescent bacteria occurred on any plate.

122

J. WOODLAND HASTINGS AND DEMOREST DAVENPORT

It may also be noted that the effect of varying oxygen concentration upon the
luminescence of Luminodesmus (see section 5) indicates that the light is not bac-
terial in nature. In bacteria (Hastings, 1952; Shapiro, 1934) decreasing the
oxygen concentration has no effect upon luminescence unless the concentration
is less than about 0.3%.

3. Color of the light

The light emission from Luminodesmus is weak, requiring dark-adaptation on
the part of the observer to see it. The emission spectrum was determined by
placing a single animal at the entrance slit of a Bausch and Lomb Grating Mono-
chromator, and the phototube at the exit slit. A second phototube was placed by
the entrance slit to monitor any changes in the intensity of the animal during the
course of the experiment. The entrance and exit slits were both set at 1 mm., which
gives a dispersion of 12 mju, with the grating used (15,000 lines per inch). The
light intensity was measured at the various wave-length settings and then corrected
for phototube sensitivity and for monochromator efficiency. The corrected values
are plotted against wave-length in Figure 2, giving the emission spectrum with the
maximum in the green at 495 mju. This spectrum is similar to that of some of the

V
CO

z

uj 2

I-

I

o

J

I

I

1

1

460 480 500 520 540
WAVE LENGTH- MILLIMICRONS

FIGURE 2. Emission spectrum of Luminodesmus sequoiae. Ordinate, light intensity in
arbitrary units, corrected for phototube sensitivity and monochromator efficiency.

THE LUMINESCENCE OF LUMINODESMUS

123

10

20

30

40

.0034 .0036

TEMPERATURE- °C. I/T- ABSOLUTE

FIGURE 3. (A) Effect of temperature upon luminescence. Ordinate, light intensity in
arbitrary units. See text for details. (B) Data of Figure 3 A plotted according to the
Arrhenius equation. Ordinate, light intensity in arbitrary units plotted on a log scale ; abscissa,
the reciprocal of absolute temperature.

luminous bacteria, although the spectrum of bacterial emission may depend upon
the density of the suspension (Harvey, 1952).

4. Effect of temperature

The effect of temperature upon the luminescence was determined using both
intact organisms and eviscerated specimens. The results were essentially the same
in both cases. The specimen was held in place in a test tube by a cotton plug.
Temperatures were adjusted by holding the tube in a water bath, and the tempera-
tures plotted are those read from a thermometer placed in the tube beside the speci-
men. The tube was then quickly removed from the bath and placed in front
of the phototube.

The data from one experiment with an eviscerated specimen are plotted in
Figure 3, along with a plot of the data according to the Arrhenius equation. The
Q10 for the process is 1.95 between 10° and 20°; 1.73 between 15° and 25° ; and
1.55 between 20° and 30°. The sharp decrease in luminescence above the optimum
of 31.5° is most likely the result of heat denaturation of enzymes. The activation
energy for the over-all process may be calculated from the slope of the straight line
drawn in the Arrhenius plot. The value obtained in this case is about 12,000
calories.

5. Effect of varying oxygen concentration

The effect of oxygen concentration upon the luminescence was determined
quantitatively, using both whole animals and eviscerated specimens. Both gave

124

J. WOODLAND HASTINGS AND DEMOREST DAVENPORT

similar results. Luminescence was greatest in 100% oxygen and progressively
decreased at lower concentrations, being reversibly extinguished in pure nitrogen,
as reported by Davenport, Wootton and Gushing (1952). An oxygen concentra-
tion of about 6.5% decreased the intensity from that in air by about one half.

The specimen was held in place with cotton in a stoppered 10-ml. test tube,
with glass tubing to bring the gas mixtures into the test tube. Gas mixtures of the
desired oxygen concentration were prepared by mixing nitrogen with air or oxygen
at measured rates, using calibrated flow-meters. The typical effect of lowered

>

b

CO
UJ

i

4.6 %0Z

AIR

1

1

4-68
TIME- MINUTES

10

FIGURE 4. Changes in luminescence with time when a gas mixture containing 4.6% oxygen
was passed over the animal for four minutes followed by the readmission of air.

oxygen concentration upon luminescence is shown in Figure 4. The response is
essentially similar to that found in several other luminous organisms (Hastings,
1952; Hastings, McElroy and Coulombre, 1953; Hastings and Buck, 1956).
There is a characteristic "undershooting" when the animal is exposed to a lowered
oxygen concentration, and an overshoot or excess flash of luminescence, when it is
exposed to a higher concentration. This excess luminescence is greater when a
lower oxygen concentration is used during the period previous to the time when
air is readmitted. For example, in the experiment shown in Figure 4, the
luminescence was about twice the baseline level when air was admitted. With

THE LUMINESCENCE OF LUMINODESMUS

125

\°/o oxygen the luminescence was 2.5 times and with 10% oxygen it was 1.5
times the baseline level. This suggests that the substrate for the luminescent
reaction (luciferin) is the product of a series of relatively slow reactions. When
the oxygen concentration is changed the luciferin comes to a new steady-state
concentration, but only relatively slowly.

The values for light intensity versus oxygen concentration plotted in Figure
5 are the steady-state values, measured just previous to the time when the animal
was returned to air. The data plotted are the results obtained with six different
specimens. Although there was a variation in the results, the data for any given

100

80

60

LJ

\- 40

\-

O 20

_J

10

20

30

40

50

60

70

80

PER CENT OXYGEN

FIGURE 5. Effect of oxygen concentration upon the steady-state luminescence of Luminodes-
mus. Data are taken from experiments such as are illustrated in Figure 4. The luminescence,
in low or high oxygen just prior to the readmission of air, is expressed as the per cent of the
steady-state of luminescence in air.

animal fall along a smooth curve, suggesting a real difference between one animal
and another, rather than error in the method. The reason for these differences
could not be ascertained. Neither carbon monoxide nor carbon dioxide when
added to the gas mixtures in 1% concentrations had any effect upon the shape of
the curve for a given animal.

6. Fluorescence

Luminodesmus is highly fluorescent under ultraviolet light, and it was suggested
previously (Davenport et al, 1952) that this fluorescent compound might be in-

126 J. WOODLAND HASTINGS AND DEMOREST DAVENPORT

volved in the luminescent reaction. In luminous bacteria, for example, it has been
demonstrated that reduced flavin mononucleotide (FMNH2) is involved in the
luminescent reaction, possibly as luciferin (McElroy, Hastings, Sonnenfeld and
Coulombre, 1953; Strehler, Harvey, Chang and Cormier, 1954). FMN and other
flavins are highly fluorescent in the oxidized form and are not at all fluorescent in
the reduced form. Under anaerobic conditions in the intact animal the luciferin
should be essentially 100% in the reduced state. However, there is absolutely no
change in the fluorescence of Luniinodesmus during anaerobiosis. The possibility
that the fluorescence of the organism comes from a flavin compound involved in the
luminescent reaction must therefore be ruled out.

7. Luminescence in extracts

Davenport, Wootton and Cushing (1952) reported negative results in all at-
tempts to restore luminescence in filtered water extracts of Luminodesmus. Using
sensitive light-measuring equipment, we have repeated their experiments and made
additional studies with extracts. The most suitable method which we found for
preparing extracts was to remove the gut from animals and dry them overnight
in a vacuum desiccator with calcium chloride. The dry animals were then pulver-
ized to a fine powder by grinding, and extracted with cold acetone. The acetone
was removed by filtration and the dry powder kept in a vacuum desiccator. The
powder remained active in this state for a period of at least two weeks.

When this powder was mixed with water a dim luminescence occurred (visible
only to the dark-adapted eye), lasting for about 10 minutes, the half time for decay
being about two minutes. Stirring always resulted in a temporary increase in
luminescence, suggesting that leaching from participate matter was taking place.
Following stirring the light intensity returned to the original level. When such a
solution was filtered through fine sintered glass (maximum pore size, 5.5 microns),
the filtrate retained luminescence. Stirring did not affect the intensity of the
filtrate, indicating that the particles from which leaching was occurring had been
removed.

The intensity of luminescence in these extracts was dependent upon the pH of
the solution, with an optimum at about pH 8.9. The determination was made by
extracting equal quantities of the powder with 0.05 M trihydroxyaminomethane-
maleic acid buffer at various pH values and measuring the light intensity of the
solution. In all experiments described below a buffered extract at pH 8.9 was used,
and buffered reagents where needed.

In the classical luciferin-luciferase test a fraction which has been extracted with
hot water is combined with a cold water extract in which the luminescent reaction
has been allowed to run to completion. In the hot water extract the enzyme has
been destroyed, presumably leaving available substrate, or luciferin. In the cold
water extract the luciferin has all been used up leaving active enzyme, or luciferase.
The two mixed together should therefore give light, but with Luniinodesmus com-
pletely negative results were obtained. Moreover, neither the hot water extracts
nor exhausted cold water extracts, when added to a luminescing extract, had any
effect upon the light intensity.

Attempts to separate the presumed luciferase and show its activity in the re-
action were negative. Fractions were obtained by ammonium sulfate precipitation,

THE LUMINESCENCE OF LfM IXODESMUS 127

by alcobol fractionation, and by dialysis. None was active when added either to
luminescing mixtures or to hot water extracts.

A large number of compounds were tested for their ability to modify the light
intensity in luminescing extracts.2 ATP was found to have appreciable activity.
ATP added to an extract which was emitting light caused the intensity to increase
by 10 to 30 per cent. The effect was not due to a pH change since buffered ATP
solutions were used. It was effective with filtered extracts as well as unfiltered.
Other pyrophosphate compounds were not tried, so it is possible that the action of
ATP could be non-specific, similar to the effect of pyrophosphate compounds added
secondarily to luminescing firefly extracts (McElroy, Hastings, Coulombre and
Sonnenfeld, 1953). The fact that ATP would not restore luminescence to dark
extracts indicates such a non-specific role. No restoration of luminescence oc-
curred even when ATP was added to hot water extracts together with exhausted
cold water extracts. However, when MgSO4 was added following ATP addition,
there was an additional increase in light intensity, suggesting the possibility of a
more specific role for ATP. Also, 0.05 M Versene (ethylene diamine tetra acetic
acid) was found to depress luminescence, indicating the possibility that the reaction
is activated by a metal ion. All of the coenzymes listed in footnote 2 were tested
in combination with ATP. None was found to have any stimulatory effect, al-
though FMN and riboflavin slightly depressed luminescence.

The results give little clue as to the nature of the reaction. If a luciferin type
compound is involved in a classical oxidative reaction, possibly in combination with
ATP, then the luciferin must be highly unstable. In fact, such a highly unstable
and heat-labile luciferin could account for the results we have obtained. The in-
hibition by flavins might mean that some flavin compound is involved in the reaction,
although we would not expect a flavin to be particularly unstable.

SUMMARY

1. The luminescence of Liiininodcsinus is continuous, but fluctuates by 20 to 40
per cent or more. The mechanism by which light emission is controlled is not
known. No evidence was found for the suggestion that the light is bacterial in
origin.

2. The luminescence is green with a maximum emission at 495 m/A and is
optimal at a temperature of 31.5° C. Light emission is greatest in pure oxygen
and extinguished in pure nitrogen. An oxygen concentration of 6.5% decreased
the intensity from that in air by about one half.

3. Luminescence in water extracts of dried acetone powders has been demon-
strated.

4. We have not been able to restore luminescence to dark extracts either by the
classical luciferin-luciferase technique or by adding a variety of biochemical inter-

- Substances tested for activity, either singly or in combination, were : adenosine triphos-
phate (ATP), MgSO4, riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide,
oxidized and reduced diphosphopyridine nucleotide, oxidized and reduced triphosphopyridine
nucleotide, coenzyme A, beef heart extract (Armour), yeast concentrate (Sigma), liver con-
centrate (Sigma), do-decyl aldehyde, ethyl alcohol, glycerol, glucose, glucose-1 -phosphate,
thiomalate, thiooctate, glutathione, cystine, cysteine, KCN, sodium arsenite, iodoacetate, sodium
fluoride, sodium azide, p chloro-mercuro-benzoate, naphthoquinone, quinhydrone, hydroquinone,
quinone, firefly extracts, NaCl, Na_HPO:, KHPO4 and MnSO4.

128 J. WOODLAND HASTINGS AND DEMOREST DAVENPORT

mediates. We have found that if adenosine triphosphate is added to extracts while
they are luminescing an increase in light intensity occurs.

LITERATURE CITED

DAVENPORT, DEMOREST, D. M. WOOTTON AND JOHN E. GUSHING, 1952. The biology of the
Sierra luminous millipede, Lunrinodcsnuts scquoiae Loomis and Davenport. Biol.
Bull., 102: 100-110.

HARVEY, E. N., 1952. Bioluminescence. Academic Press, New York, New York.

HASTINGS, J. W., 1952. Oxygen concentration and bioluminescence intensity. I. Bacteria
and fungi. /. Cell. Coinp. Physio!., 39: 1-30.

HASTINGS, J. W., W. D. MCELROY AND J. COULOMBRE, 1953. The effect of oxygen upon the
immobilization reaction in firefly luminescence. /. Cell. Coinp. Physiol., 42: 137-150.

HASTINGS, J. W., AND W. D. MCELROY, 1955. Purification and properties of bacterial luci-
ferase. In: The luminescence of biological systems, (F. H. Johnson, ed.), pp. 257-264.
A.A.A.S. Press, Washington, D. C.

HASTINGS, J. W., AND JOHN BUCK, 1956. The firefly pseudoflash in relation to photogenic
control. Biol. Bull., Ill: 101-113.

HASTINGS, J. W., AND B. M. SWEENEY, 1957. The luminescent reaction in extracts of the
marine dinoflagellate Gonyaulax polycdra. J. Cell. Comp. Physiol.. 49: in press.

LOOMIS, H. F., AND DEMOREST DAVENPORT, 1951. A luminescent new xystodesmid milliped
from California. /. Wash. Acad. Sci., 41 : 270-272.

MCELROY, W. D., J. W. HASTINGS, J. COULOMBRE AND V. SONNENFELD, 1953. The mechanism
of action of pyrophosphate in firefly luminescence. Arch. Biochcm. Binfhys., 46:
399-416.

MCELROY, W. D., J. W. HASTINGS, V. SONNENFELD AND J. COULOMBRE, 1953. The require-
ment of riboflavin phosphate for bacterial luminescence. Science. 118: 385-386.

MCELROY, W. D., AND J. W. HASTINGS, 1955. Biochemistry of firefly luminescence. In:
The luminescence of biological systems, (F. H. Johnson, ed.), pp. 160-198. A.A.A.S.
Press, Washington, D. C.

MCELROY, W. D., AND ARDA GREEN, 1956. Function of adenosine triphosphate in the activation
of luciferin. Arch. Biochcm. Biophys.. 64: 257-271.

SHAPIRO, H., 1934. The light intensity of luminous bacteria as a function of oxygen pressure.
/. Cell. Coinp. Physiol., 4: 313-327.

STREHLER, B. L., E. N. HARVEY, J. J. CHANG AND M. J. CORMIER, 1954. The luminescent oxi-
dation of reduced riboflavin or reduced riboflavin phosphate in the bacterial luciferin-
luciferase reaction. Proc. Nat. Acad. Sci., 40: 10-12.

STREHLER, B. L., 1955. Factors and biochemistry of bacterial luminescence. /;;: The lumines-
cence of biological systems, (F. H. Johnson, ed. ), pp. 209-255. A.A.A.S. Press, Wash-
ington, D. C.

TSUJI, F. L, A. M. CHASE AND E. N. HARVEY, 1955. Recent studies on the chemistry of
Cypridina luciferin. In: The luminescence of biological systems, (F. H. Johnson,
ed.), pp. 127-159. A.A.A.S. Press, Washington, D. C.

THE ANTIMITOTIC AND CARCINOSTATIC ACTION
OF OVARIAN EXTRACTS 1

L. V. HEILBRUNN, W. L. WILSON, T. R. TOSTESON, E. DAVIDSON

AND R. J. RUTMAN

Department of Zoology, University of Pennsylvania, and the
Marine Biological Laboratory, Woods Hole, Mass.

The search for chemical substances which might have a retarding effect on
the growth of tumors has led along many paths. All sorts of substances have been
tried. Those investigators who have theorized at all have for the most part
thought in terms of some block of metabolic activity. Our own program has
sought to discover relatively non-toxic antimitotic substances of natural origin, and
in the search for such substances we have based our attack on what we believe to
be a proper theory for the initiation and suppression of mitosis. Such a theory is
discussed in some detail in a recent book (Heilbrunn, 1956). It holds that the
mitotic spindle results from a gelation of the protoplasm, the mitotic gelation. Vari-
ous substances can prevent this gelation by keeping protoplasm fluid. And be-
cause the most usual type of protoplasmic gelation and the type involved in the
mitotic gelation is a clotting similar to the clotting of blood, it is our belief that
anticlotting agents such as heparin or similar substances can prevent cell division.
This indeed they do. The protoplasmic colloid contains substances which favor
clotting and those which tend to prevent it. We have made extracts from various
tissues and have found that ovaries are especially rich in anticlotting agents. These
appear to resemble heparin and to be mucopolysaccharides. This work (Heil-
brunn, Wilson and Harding, 1951; Heilbrunn, Chaet, Dunn and Wilson, 1954;
Heilbrunn and Wilson, 1956) showed that the ovaries of various invertebrates and
fishes do actually contain antimitotic substances which prevent the mitotic gelation
and suppress cell division. In the search for some substance or substances which
might eventually prove to have clinical value, we have recently investigated the
ovaries of mammals and especially large mammals. In what follows, we will at-
tempt to show first that extracts of mammalian ovaries do have antimitotic action,
an action which is associated with a liquefying or anticlotting effect on the proto-
plasm ; and second that such extracts may possess carcinostatic activity.

MATERIALS AND METHODS

In studying antimitotic activity, the most favorable test objects and the easiest
to work with are the eggs of various marine invertebrates. Eggs such as sea urchin
eggs or those of the marine worm Chaetopterus divide synchronously following
fertilization. These eggs can be obtained in large quantity and they represent a
surprisingly constant material. In previous studies on Chaetopterus eggs, we have
described the simple techniques required (Heilbrunn and Wilson, 1948). Most of

1 This investigation was supported by research grants from the National Cancer Institute
and the American Cancer Society.

129

130 HEILBRUNN, WILSON, TOSTESON, DAVIDSON AND RUTMAN

our work was clone on Chaetopterus eggs. One experiment was done on eggs of
the clam Spisula. The technique required for the use of this egg has been described
by Allen (1953). All of our experiments were done at a controlled temperature
of 21° C. Two minutes after insemination the eggs were placed in the solutions
to be tested.

Crude extracts were prepared by cutting the ovaries into thin slices and im-
mersing these slices in solutions of acidified sea water at a pH of approximately
4.8. For each gram of tissue, one ml. of sea water was used. After extraction the
extract was brought to the pH of sea water by the addition of NaOH. In a few
cases the ovaries were homogenized before extraction, but this seemed to produce
less favorable results in our antimitotic studies. The acidification of the sea water
used in extraction was apparently not necessary and potent antimitotic extracts
could be obtained over a wide range of pH values.

The Chaetopterus egg has a very fluid protoplasm which stiffens markedly
before the formation of the mitotic spindle. There is indeed a two-fold increase
in the viscosity of the protoplasm and this can readily be followed with the
centrifuge method described in earlier studies. With a simple hand centrifuge,
turned so as to give a force approximately 2250 times gravity, for some time after
fertilization it requires only 7 seconds to move granules through the protoplasm
of the Chaetopterus egg to such an extent as to give the appearance of zones. The
number 7 is taken as a relative value for the viscosity of the protoplasm ; actually
it probably represents about twice the value of the viscosity in centipoises. At
approximately 30 minutes after fertilization, 14 seconds of exposure to a force
2250 times gravity is necessary to produce the appearance of zones in the proto-
plasm. In studying the antimitotic and the anticlotting action of our extracts we
determine the percentages of cleavage following exposure to various dilutions, and
we also determined the effect of the extracts on the viscosity of the protoplasm at
the time when the normal control eggs showed increased viscosity. These tests
had to be made rapidly, for the duration of the mitotic gelation is short.

Crude extracts prepared in the way we have indicated are very potent anti-
clotting and antimitotic agents, as our results will show, but generally speaking they
are not very effective as carcinostatic agents. Indeed these extracts contain not
only substances which tend to prevent protoplasmic clotting, they also contain sub-
stances which have exactly the opposite effect. Thus, although in one case we did
obtain a definite carcinostatic effect with such crude extracts, for the most part we
were not successful, and indeed in some instances the survival time of cancerous
mice treated with crude extracts was decreased rather than increased. Hence we
were led to try and find methods of extraction which would give us preparations
of as high a carcinostatic action as possible with a minimum of toxicity or adverse
action.

For many months, we had little or no success. Then we hit on a method of
fractional alcoholic precipitation, and this has now provided us with extracts which
possess a definite carcinostatic action. This was perhaps to be expected, for in
studying the antimitotic action of extracts of starfish ovaries, we found that the
potent substance could be precipitated by alcohol (Heilbrunn, Wilson and Harding,
1951).

The procedure we finally adopted was the following : Cow ovaries, fresh from
the slaughter-house, were ground up in a meat grinder and were then extracted

ACTION OF OVARIAN EXTRACTS 131

in a solution containing 0.9^0 sodium chloride and 0.125% sodium bicarbonate.
For eacb gram of ovarian material, 2 ml. of solution were used. The extraction
was carried out in a cold room at 5° C. with constant stirring, and was continued
for 16-18 hours. After the extract was strained through cheesecloth, it was
centrifuged at 2000 rpm in a refrigerated centrifuge. The supernatant was then
centrifuged in a Spinco centrifuge at 16,000 rpm (20,000 g) for an hour. The
resultant supernatant was then precipitated by various concentrations of alcohol
at approximately 0° C. Following each precipitation by a given percentage of
alcohol, the supernatant was decanted. The precipitates were then lyophyllized.
Our final product represented only a small fraction of the original ovarian material.
Thus typically by precipitation with 45-60% ethanol we obtained about 10 mg. from
a kilogram of ovaries.

In studying carcinostatic action, \ve used Swiss white mice. These were in-
oculated routinely with 1,500,000 Ehrlich ascites tumor cells in a volume of one
ml. of ascitic fluid. Then 24 hours after inoculation, treatment with the extracts
was begun. Each day for five days, each one of the mice to be treated was injected
with a solution containing 15 mg. of the material. In general the 15 mg. were dis-
solved in 0.5 ml. of saline solution. Details of our technique and growth studies of
our particular tumor will be published in a paper to be written by two of its
(Tosteson and Davidson).

RESULTS

In the summer of 1955, we tested the effect of crude extracts of mammalian
ovaries on cell division in the Chaetopterus egg. These extracts in every case
stopped cell division. They also prevented the mitotic gelation. Our results are
shown in Table I. In most cases the viscosity of the protoplasm at 30—40 minutes
after fertilization is given as "less than 8." It may have been decidedly less than
8, but in the short space of time available for these measurements it was not possible
to make enough tests to be sure of an exact value. However, the fact that the
viscosity was less than 8 is a sure indication that the mitotic gelation has been
suppressed, for during this gelation, the viscosity rises to a value of 14.

In addition to the data presented in the table, we have results from a few
additional experiments ; these results are entirely consistent with those shown in
the table. In most of these other experiments, we attempted to improve the
potency of the extracts by purifying them, but in every case the crude extracts
prepared as described in the previous section were superior to the "purified" prod-
uct. We also tried modifying our extraction procedures. In the results reported
in the table, the extractions were made at a pH of 4.8, but we also tried extracting
the ovarian material at pH's of 2.25, 3.90, 6.15, 7.1, and 10.0. All of the extracts
prepared at these different pH's were likewise effective. Heating the extracts did
not seem to have a very harmful effect, although our results were somewhat variable
and minor differences in procedure seemed to be important. In one experiment
an extract kept at 100° C. for three hours was still highly potent. In other ex-
periments, exposure to 100° C. for shorter periods caused some loss in potency.
Extracts made in distilled water and then lyophyllized were not effective. When
the ovaries were homogenized before being extracted, the results were less favorable.
Homogenization seemed to favor the release of thromboplastic substances into the
extraction medium.

132

HEILBRUNN, WILSON, TOSTESON, DAVIDSON AND RUTMAN

Although during the summer of 1955, we were never able to obtain any degree
of successful purification of our extracts, in the following winter one of us (R. J.
Rutman) hit upon the plan of precipitation with various concentrations of alcohol.
Some of the precipitates obtained in this way when dissolved in saline solution had
a very definite carcinostatic action, and we determined therefore to test the anti-
mitotic action of these precipitates. Chaetopterus eggs (at Woods Hole) are only
available during the summer months, so that we had to wait until the summer of
1956 before making our tests.

In making these tests, we had some difficulty, for the precipitates dissolved
scarcely at all in the sea water in which we had to use them. However what tests
we were able to make with the limited amount of material we had at our disposal
indicated that only those precipitates obtained with intermediate concentrations of
alcohol were effective. We had three fractions, of which A represented the pre-
cipitate obtained from cow ovary extracts with 0-45% alcohol, B the precipitate
obtained with 45-60% alcohol, and C the precipitate with 60-80% alcohol. Of
these three fractions, neither A nor C was very soluble. Fraction B seemed to go
into solution, but when the solution was looked at under the microscope it was seen
to have a large number of small solid particles suspended in it. We did several
experiments with this fraction B. In one of these experiments, 5 mg. were dis-
solved in one ml. of sea water and then this solution was diluted so that the resultant
dilutions contained 2.5 mg. per ml. of sea water and 1.25 mg. per ml. of sea water,
respectively. All three of these solutions prevented the mitotic gelation and in all
of them the fluidity of the protoplasm of the Chaetopterus eggs was maintained.
In the most concentrated solution only 29% of the eggs cleaved, in the middle con-
centration 19%, and in the most dilute of the three solutions, 26% of the eggs
cleaved. In the control 90% of the eggs cleaved. These counts were made 30
minutes or more after 50% of the control eggs had cleaved. The fact that the

TABLE I

Effect of ovarian extracts on the mitotic gelation and on cleavage
of the eggs of Chaetopterus

Source of
material

Duration of
extraction in
minutes

Dilution

Viscosity at
30-40 min.

% cleavage

% cleavage
in control

Cow

30

4

1

98

Cow

60

—

4

0

98

Cow

120

—

less than 8

0

100

Cow

120

i

2

less than 8

0

100

Cow

120

i

about 8

0

100

Cow

120 •

1

about 8

5

100

Cow

60

less than 8

0

99

Cow*

60

5

0

99

Cow**

60

5

0

99

Cow

120

less than 8

0

99

Cow

120

less than 8

0

100

Pig

155

less than 8

0

99

Lamb

105

less than 8

0

99

* Ovarian capsules.
** Corpora lutea.

ACTION OF OVARIAN EXTRACTS

ACTIVITY OF COLD ALCOHOL PRECIPITATES

OF AQUEOUS EXTRACTS OF OVARIAN TISSUE (COW)

ACTIVE FRACTION
INACTIVE FRACTION

133

10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 9O

% ALCOHOL
EXTRACT 2 WAS INACTIVE

FIGURE 1. Activity of cold alcohol precipitates of aqueous extracts

of ovarian tissue (cow).

various concentrations of material all acted in essentially the same way indicates
that the material was only slightly soluble so that the actual concentration in true
solution was the same in all three cases. In the very few experiments that we
did with fractions A and C. these were without effect, perhaps because their
solubility was extremely low.

In addition, the material of fraction B was tried on the eggs of the clam Spisula,
and in this case also the material exerted an antimitotic effect. Concentrations of
2.5 mg. per ml., 1.25 mg. per ml. and 0.625 mg. per ml. showed strong antimitotic
action, the cleavage being reduced from a control value of 97% to values of 31%
for what presumably was the more concentrated solution, 39% for the intermediate
concentration, and 40% for the weakest concentration.

Thus in spite of the fact that the material was soluble only slightly, the fraction
obtained by precipitation with 45-60% alcohol did actually exert an antimitotic
action, and in the case of Chaetopterus eggs this action was associated with a pre-
vention of the mitotic gelation. No attempt was made to study the effect of
fraction B on the mitotic gelation in the Spisula egg, for as yet the cycle of
viscosity changes in the Spisula egg has not yet been worked out with sufficient
thoroughness.

Clearly, our results with marine eggs indicate that fractionation of the crude
cow ovary extracts by alcoholic precipitation can preserve the antimitotic and anti-
gelating action.

Let us consider now the carcinostatic action of these alcoholic precipitates ob-
tained from extracts of cow ovaries. Up until the present, we have accumulated a

134 HEILBRUNN, WILSON, TOSTESON, DAVIDSON AND RUTMAN

large body of data. Indeed we have experimented with well over 4,000 mice.
Our results show that the precipitates obtained by treating cow ovary extracts with
intermediate concentrations of alcohol can cause survival of some 15-25% of mice
previously inoculated with a lethal tumor, a tumor which regularly kills 100%
of all mice properly inoculated with it.

Figure 1 illustrates the fact that treatment with intermediate concentrations
of alcohol in the cold can produce precipitates which have a carcinostatic action.
This figure gives the results obtained with five different extracts and is based on
experiments with about 2,000 mice. Further details of these experiments will be
presented in another paper soon to be prepared by several members of our group
(Tosteson, Davidson and Rutman). Since the data for the table wrere collected
we have obtained additional confirmatory data. Also we have been experimenting
with various other types of extraction media and with other types of fractionation.
Preliminary results lead us to the hope that extracts and fractions can be obtained
which will cause a higher percentage of survival than we have been able to obtain
with the extracts described in this paper.

DISCUSSION

Our work has been based on the idea that the protoplasm of all cells is much
alike both chemically and physically, that inasmuch as the process of mitosis is
much the same throughout the animal kingdom, the forces and agents which initiate
cell division and those which suppress cell division are also much the same. On
the basis of this pattern of thought, and on the basis also of many experiments on
the initiation and suppression of mitosis (see Heilbrunn, 1956), we have been able
to develop a new type of carcinostatic agent. At present this agent is at least as
potent for our Ehrlich ascites tumors as other long-studied agents. It is our hope
that the type of agent we are using can be perfected to give even better results.
This will require much additional work.

SUMMARY

Extracts of the ovaries of cows, pigs and sheep can suppress mitosis in eggs
of the worm Chaetopterus. This they do by keeping the protoplasm fluid and in-
hibiting the mitotic gelation which is a necessary precursor of the mitotic spindle.
The potent substance or substances in extracts of cow ovaries can be precipitated
by treating the extracts in the cold with intermediate concentrations of alcohol.
Such purified preparations have a definite antimitotic effect and they also have a
very definite carcinostatic action.

LITERATURE CITED

ALLEN, R. D., 1953. Fertilization and artificial activation in the egg of the surf-clam, Spisulu

solidissima. BioL Bull., 105: 213-239.

HEILBRUNN, L. V., 1956. The dynamics of living protoplasm. Academic Press, New York.
HEILBRUNN, L. V., A. B. CHAET, A. DUNN AND W. L. WILSON, 1954. Antimitotic substances

from ovaries. BioL Bull., 106: 158-168.
HEILBRUNN, L. V., AND W. L. WILSON, 1948. Protoplasmic viscosity changes during mitosis.

BioL Bull., 95: 57-68.
HEILBRUNN, L. V., AND W. L. WILSON, 1956. Antimitotic substances from the ovaries of

vertebrates. BioL Bull., 110: 153-156.
HEILBRUNN, L. V., W. L. WILSON AND D. HARDING, 1951. The action of tissue extracts on cell

division. /. Nat. Cancer hist., 11: 1287-1298.

CARDIAC PHYSIOLOGY OF THE SCORPION PALAMNAEUS

BENGALENSIS C. KOCH1

M. S. KANUNGO -
Department nf Zoology, Rarcushau1 Collci/c, Cuttack, India

The nature of heart-beat among arthropods has been studied by several workers
(Prosser, 1942; Needham, 1950; Krijgsman, 1952). It has been observed in
Crustacea and Insecta that the origin of heart-beat in each class is of varied types
and has no relationship with the taxonomic classification. Among the arachnids,
Liinulus has a myogenic heart-beat in the young which becomes neurogenic in the
adult (Prosser, 1942; Krijgsman, 1952) and the heart-beat of spiders is neurogenic
(Rijilant, 1933). Even though Police (1902) indicated the presence of an epi-
cardiac nerve on the heart of scorpion, the heart-beat of the scorpion, Palamnaeus
bengalcnsis has been reported by Kanungo (1955) to be myogenic. It is of interest
to find that, also in Arachnida as in Crustacea and Insecta, the nature of heart-beat
is not of a single type. The hearts of arachnids are poorly understood and the
present work is a detailed study of the physiology and pharmacology of the heart
of Palamnaeus bengalensis.

MATERIALS AND METHODS

Scorpions freshly collected from their natural habitat (Lai and Kanungo, 1953)
were used for these experiments. They were lightly chloroformed and immediately
dissected in a saline containing sodium chloride, 0.65 gm. ; potassium chloride, 0.03
gm., and calcium chloride, 0.03 gm. ; in 100 ml. of distilled water. The saline, which
was prepared fresh before the experiments, was maintained at pH 6.3 using phos-
phate buffer, as the haemolymph of the scorpion was found to be on the acid side
of neutrality in agreement with Maluf's (1939) statement. The heart was exposed
fully in situ by carefully cutting the terga at the sides and removing them. Isolated
heart preparations were made in petri dishes containing the saline. Effect of pH,
temperature and drugs on the heart-beat were observed on hearts both in situ and
isolated. Nearly 150 heart preparations have been made for various drug experi-
ments.

ANATOMY OF THE HEART

Like that of ButJuts (Parker and Haswell, 1940), the heart of P. bengalensis
is eight-chambered, spongy and muscular. It is enclosed in a pericardium and is

1 This research was supported by the Board of Scientific and Industrial Research, Orissa,
India.

1 am grateful to Dr. C. L. Prosser and Dr. J. D. Anderson, Department of Physiology,
University of Illinois, for their help in preparation of the manuscript.

2 Present address : Department of Physiology, University of Illinois, Urbana, Illinois,
U. S. A.

135

136 M. S. KANUNGO

held in its position between the two lobes of the liver by eight pairs of alary
muscles. It is 2.5 cm. long in a medium-sized scorpion with a body length of ap-
proximately 5 cm. A thin- walled anterior aorta arising from the heart bifurcates
on the oesophagus. A thin-walled posterior aorta proceeds to the tail.

GENERAL PROPERTIES OF THE HEART-BEAT

Hearts both in situ and isolated showed a high degree of automaticity ; contrac-
tions occurred simultaneously throughout the myocardium and following one an-
other in a regular uninterrupted sequence rhythmically. Simultaneous contraction
of the scorpion heart had been reported earlier (Du Buisson, 1925). When first
isolated, the rate of beat of the heart was irregular and slow, but it became normal
after about 5 minutes, and showed a little acceleration in the rate of beat as com-
pared with that of hearts in situ. The rate of beat of intact hearts was 50— 54/
minute at room temperature (25-27° C.). Cutting of the alary muscles in situ
resulted in a slight increase of the rate to 56-62/minute, which was the same as that
of isolated hearts. It appears, therefore, that even though the property of automatic
movement lies in the muscles of the heart itself, the regulation of the rate of beat
is effected by alary muscles. The rate of isolated heart preparations remained
normal for 10-12 hours after which it decreased and the amplitude fell gradually.
Mechanical stimuli like shaking the saline or pressing the heart with a needle
temporarily inhibited the heart-beat. After 2-A minutes, an acceleration in the
rate was observed. Excised pieces of the heart beat for about 4—5 minutes. In
all preparations, the anterior end stopped first and the posterior end later, sug-
gesting that the pace-maker of the heart is situated at the latter end.

COURSE OF CIRCULATION

As stated above, isolated hearts beat with the anterior and posterior ends con-
tracting and relaxing simultaneously. During diastole the heart shortens in length
and bulges and the haemolymph flows in through the paired ostia. During systole,
the heart extends lengthwise, the ostial valves close and the haemolymph is ex-
pelled at both the ends. A freshly isolated heart was placed in a dry watch glass
in such a manner that the two ends were at a higher level than the middle region.
A drop of neutral red was put at the center of the watch glass. The heart con-
tinued to beat and neutral red was seen flowing out at both the ends.

EFFECT OF TEMPERATURE

Isolated hearts in petri dishes containing the saline were kept at different tem-
peratures in an incubator and their rates were noted. An upper limiting rate of
80-85/minute was observed at 42° C., above which beating ceased permanently.
The lower limiting rate on cooling was 4— 5 /minute at 5° C. Below this tempera-
ture the heart ceased beating but recovered when the temperature was increased.

EFFECT OF pH

Separate stocks of the same saline solution were prepared by buffering with
phosphate buffer between pH 5.5 and pH 7.5 and the hearts were kept in these

CARDIAC PHYSIOLOGY OF THE SCORPION

137

salines. The heart remained active between pH 6.1 and pH 6.5. With increase
or decrease of the pH of the saline beyond this range, depression of the heart rate
occurred.

EFFECTS OF DRUGS

Fresh dilutions of 10'3, lO4, 5 X 10'4, lO'5, 5 X 1O5, and lO'6 of various drugs
were made in the saline before each set of experiments. Both intact and isolated
hearts were bathed side by side with one of the diluted drugs to compare their
effects on the heart-beat in isolated and in situ preparations. No difference between
isolated and intact hearts was observed. Mechanical shock to the heart was
avoided as far as possible. The drug was sucked out with a pipette, the heart

60

LJ

K
O
Z

2

u

CD

20

B

10
TIME IN MINUTES

15

2O

FIGURE 1. Effect of acetylcholine on the heart of P. bengalensis.
Ach., acetylcholine ; Sal., saline.

washed with the saline three times and the fresh drug added slowly. Recordings
of the heart rate were made following the methods of Jones (1954). Three re-
cordings, one minute each in length, were made one minute after adding the drug.
•Acetylcholine more dilute than 5 X 10~5 had no effect on the heart rate. Con-
centrations of 5 X 10~5 or stronger depressed the heart rate ; the time taken for
depression was inversely proportional to the concentration of the drug (Fig. 1).
There was a gradual weakening of the strength of beat, reduction in the amplitude,
rest-pauses and sporadic irregularities in 5 X 10~5 or stronger concentrations. In
no case was there any acceleration before the depression. Neither did the beat
recover if the heart was left in the drug. However, all such hearts recovered after
they were washed wdth the saline, but the normal rate of beat was never reached.
Some hearts showed tolerance to the drug up to 10~4 after prior treatment with
more dilute solutions and gradually increasing concentrations (Fig. 2D).

138

M. S. KANUNGO

80r

60

z

2

40

ui

0.

U)
CD

20

Ach id"6

10

TIME IN MINUTES

15

20

FIGURE 2. Effects of various drugs on the heart of P. bengalcnsis. Ach., acetylcholine ; Adr.,
adrenaline ; Atr., atropine ; Hist., histamine ; Physo., physostigmine ; Sal., saline.

Physostigniinc at 10~4 or stronger did not by itself show any appreciable effect
on the heart rate. However, application of acetylcholine to the heart, after treat-
ment with physostigmine, potentiated the effect of acetylcholine (Fig. 2E).

Histamine at 10~* or stronger accelerated the heart rate to a maximum of 85/
minute and this effect was reversible on washing with the saline. The time taken
for the heart to reach the maximal rate in different dilutions was directly pro-
portional to the dilutions of the drug. It antagonized acetylcholine action, and
hearts collapsing under the treatment with acetylcholine could be revived by this
drug. Such hearts also beat at 85/minute (Fig. 2F).

Adrenaline at 10~5 or stronger accelerated the heart rate to about 75/minute
and this effect was reversible.

TABLE I
Comparison of the effects of drugs on the hearts of Limulus and P. bcngalensis

Ach.

Atropine

Adrenaline

Ether

Histamine

Physostig-
mine

Chloroform

Limulus*

+

+

+

P. bengalensis

—

—

+

0

+

0

0

+ , excitation; — , inhibition; 0, no effect.
* Krijgsman, 1952.

CARDIAC PHYSIOLOGY OF THE SCORPION 139

Atropinc at 5 X 10~4 or stronger inhibited the heart rate reversibly.

Half -saturated and fully-saturated aqueous solutions of ether had no observed
effect on the heart-beat.

Chloroform had no observed effect on the heart-beat.

It was found in all the cases that the drug-treated hearts recovered after wash-
ing with the saline. A quicker recovery of the heart was attained by using warm
saline which was added slowly to the heart container. The time taken for such
recovery varied from five to fifteen minutes.

Table I gives comparatively the effects of various drugs on the hearts of
Limnlns and P. bcngalcnsis. Even though Table I does not indicate the effect of
ether on the neurogenic heart of Limulus, it may be mentioned here that ether in-
hibits neurogenic hearts in low concentrations (Needham, 1950).

HAEMOLYMPH PRESSURE

Bleeding occurred when incisions were made at pedipalpi, abdomen and tail
regions ; this indicates positive haemolymph pressure throughout the body. By
inserting capillaries in continuation writh U-tubes, actual pressure was found to be
6 mm. of saline at the pedipalpi and at the abdomen.

DISCUSSION

The pharmacology of the scorpion heart resembles that of the crustacean,
Daphnia (Baylor, 1942) and the vertebrates, and has no resemblance to that of
Limulus. According to Prosser et al. (1950), the hearts of arthropods are of non-
innervated myogenic, innervated myogenic and neurogenic types, which description
is based mainly on acetylcholine effect. Needham (1950) classified the crustacean
hearts into two categories, myogenic and neurogenic, by taking several factors into
consideration. The heart of P. bcngalcnsis in showing (1) autonomous rhythmicity
with contractions developing simultaneously throughout the myocardium, (2) in-
sensitiveness to ether, and (3) inhibition by acetylcholine indicates that the nature
of its beat or that of its pacemaker is innervated myogenic. The epicardiac nerve
reported by Police (1902) appears to be either extrinsic or regulating in function.

SUMMARY

1. The heart of P. bcngalcnsis beats continuously at a rate of 50-62/minute
at a temperature of 26° C. The contraction is developed simultaneously through-
out the muscle.

2. Acetylcholine and atropine depress the heart-beat and their actions are re-
versible. Physostigmine potentiates the effect of acetylcholine.

3. Ether and chloroform have no effect on the heart-beat.

4. Histamine and adrenaline accelerate the heart-beat and their effects are
reversible on washing with saline.

5. The haemolymph pressure is 6 mm. of saline.

6. It is concluded that the pace-maker of the heart of P. bengalensis is of the
innervated myogenic type.

140 M. S. KANUNGO

LITERATURE CITED

BAYLOR, E. R., 1942. Cardiac pharmacology of the cladoceran, Daphnia. Biol. Bull., 83 :

165-172.
Du BUISSON, M., 1925. Recherches sur la circulation sanguine et la ventilation pulmonaire

chez les scorpions. Bull. Acad. Belg., Cl. Sci., Ser. 5, 11 : 666-680.
JONES, J. C., 1954. The heart and associated tissues of Anopheles quadrimaculatus. J. Morph.,

94: 71-124.

KANUNGO, M. S., 1955. Physiology of the heart of a scorpion. Nature, 176: 980-981.
KRIJGSMAN, B. J., 1952. Contractile and pace-maker mechanisms of the hearts of arthropods.

Biol. Rev., 27: 320-346.

LAL, M. B., AND M. S. KANUNGO, 1953. Invertase in P. bengalensis. Science, 117: 57-58.
MALUF, N. S. R., 1939. The blood of arthropods. Quart. Rev. Biol., 14: 149-191.
NEEDHAM, A. E., 1950. Neurogenic heart and ether anesthesia. Nature, 166: 9-11.
PARKER, T. J., AND W. A. HASWELL, 1940. A Textbook of Zoology. Vol. I. Page 509.

Macmillan and Co., London. Sixth Edition.

POLICE, G., 1902. II Nervo del Cuore nello Scorpione. Boll. Soc. Nat. Napoli, 15: 146-147.
PROSSER, C. L., 1942. An analysis of the action of acetylcholine on hearts, particularly in

arthropods. Biol. Bull., 83: 145-164.

PROSSER, C. L., F. A. BROWN, JR., D. BISHOP, T. L. JAHN AND V. J. WULFF, 1950. Com-
parative Animal Physiology. W. B. Saunders Co., Philadelphia. Chapter 15, pp.

531-575.
RIJILANT, P., 1933. L'automatisme cardiaque chez 1'Araignee: Mygalc, Espcire, Tarentule,

etc. C. R. Soc. Biol. Paris. 133: 917-920.

A COMPARATIVE STUDY OF THE CUTICULAR STRUCTURE OF
THREE FEMALE MEALY BUGS (HOMOPTERA:

PSEUDOCOCCIDAE)

•

HARRY F. LOWER

Waitc Agricultural Research Institute, University of Adelaide,

Adelaide, Australia

The recent discovery in the arid north-west of South Australia of a new species
of the hitherto monotypic genus, Epicoccits, afforded an opportunity to examine the
cuticle of a drought-resistant pseudococcid. Members of the Pseudococcidae are
normally confined to microhabitats where they are surrounded by humid equable
conditions, and it was anticipated that considerable modification of cuticular struc-
ture would be shown by a species which has evolved in an area \vhere it is fully
exposed to the desiccating effects of high temperatures, low relative humidities, and
drying winds. The extent of such modification, if any, could be gaged only after
the cuticle had been compared with that of a typical form, of which, however, there
appears to be no published account. A study of the cosmopolitan long-tailed mealy
bug, Pseudococcus adonidnin L., was therefore undertaken as a preliminary step
in the investigation. Finally, the cuticle of the one described species of Epicoccus,
E. acaciae (Maskell) wras examined to ascertain whether any major differences in
cuticular structure occur within the genus.

The terminology is in accord with the scheme which I recently outlined (Lower,
1956).

MATERIALS AND METHODS

Specimens of P. adonidnm were obtained locally from a heavily-infested plant
of Daphne odora. Those of Epicoccus sp., were collected from Acacia aneura F.
Muell., at Yudnapinna in the north-west of South Australia while those of E.
acaciae came from the coastal strip of Western Australia, the only area in which the
species is known to exist.

The insects were first killed with cyanide, and free-hand sections of some of each
species at once stained with Sudan black B. The remainder, after fixation in
Sanfelice's fluid, were embedded, part in a water-soluble wax and part in paraffin.
Sections cut from these were similarly stained. Comparison showed that there was
no observable loss of cuticular lipoid when paraffin was used as the embedding
medium. All work wras therefore done using paraffin sections cut at 4 /j, and 1 p.,
except that when the external wax of Pscudococcns was being investigated, sections
prepared by the first two methods wrere used.

The histochemical tests and techniques applied have been described elsewhere
(Lower, 1957a).

I. THE CUTICLE OF PSEUDOCOCCUS ADONIDUM L.

The cuticle (Fig. 1) is thin, measuring in most parts between 6 /z and 8 p; only
exceptionally does it attain a thickness of 10 /j,. Its structure is relatively un-

141

142

HARRY F. LOWER

specialized and except for the brown outer layer of the epicuticle it is unpigmented.
Pore canals, if present, could not be observed with the light microscope either under
bright-field or phase-contrast conditions. A thick layer of wax (discussed later)
covers the cuticular surface.

GR

100 JJ

\

FIGUKI-: 1. I', udoniihiiii: Cuticle. A. Part of cuticle adjoining glandular duct. B. Gen-
eral cuticle, c, cuticulin layer ; en, endocuticle ; Ep, epicuticle ; GR, gland reservoir ; P, paraffin
layer; S, spinule; SS, secretory sheath; W, surface wax layer ; \YK~, wax-impregnated cuticle.

A. The Epicuticle

The epicuticle (Fig. IB) is two-layered and has a total thickness of about 1 /;,
of which the inner layer constitutes the greater part.

The inner or cuticulin layer is colorless and transparent. It responds positively
to the Millon, xanthoproteic and biuret tests, is non-argentaffin, and is unaffected
by either Sudan black B or Nile blue sulfate. It is stained red by the routine stain
and pink by Sevki's diluted Giemsa technique, but it cannot be stained either by
Mallory's PTAH or any of the iron haematoxylins. Schmorl's test shows the ab-
sence of reducing substances. It is soluble in warm concentrated solutions of
potassium hydroxide or hydrochloric acid. Its location and general chemical re-
actions indicate that it is probably homologous with the cuticulin layer of Rhodnins
(Wiggles worth, 1947).

The thinner outer layer is brown-pigmented. The color is difficult to discharge,
sections requiring about a week's immersion in \Qc/c hydrogen peroxide to effect
this. The strong positive response to Schmorl's test, together with the non-
argentaffin nature of the layer, suggests that the coloring matter is a lipofuscin.
The layer responds to none of the protein tests. It is blackened by Sudan black B,
and stained deep blue by Cain's Nile blue sulfate technique. Prolonged differentia-
tion during the latter process removes much of the blue without any red appearing
so that acidic lipoid only appears to be present. It is stained black by iron haema-
toxylins and deep violet by Mallory's PTAH. It is resistant to concentrated so-
lutions of potassium hydroxide and hydrochloric acid but fuming nitric acid or
aqua regia, when gently warmed, attacks some of its constituents and liberates a
material which is practically instantaneously soluble in cyclohexane and is quickly
and easily stained by any of the fat stains. Its anatomical position and general
chemical behavior indicate that the layer is a paraffin epicuticle (Dennell and
Malek, 1955). It differs from the corresponding layer in Sarcophaga (Dennell,

CUTICLE OF FEMALK PSEUDOCOCCIDAE 143

1946) by being non-argentaffin, in giving no response to the xanthoproteic reaction,
and in its content of lipofuscin pigment.

B. The Procuticle

Practically the whole of the procuticle is present as enclocuticle. With the ex-
ception of the spinules mentioned below, no exocuticle occurs, while the mesocuticle
is confined to the alveoli of the spinules and the small convex circular patches (ap-
pearing crescentic in transverse section) where muscles are inserted in the cuticle.

Scattered over the cuticular surface are weakly-sclerotized, colorless spinules
(Fig. IB), each secreted by a trichogenic cell which is much larger and more
granular than the adjacent hypodermal cells. A cytoplasmic process from each
trichogenic cell, after passing through the cuticle, attenuates and forms a core to the
spinule for its basal two-thirds. Both spinules and alveoli are feebly argentafnn and
are stained by the routine stain, the former a very pale pink and the latter red.

The endocuticle is laminated and closely resembles that found in other insects
with soft cuticles. Its principal constituent is the normal chitin-protein complex.

II. THE CUTICLE OF EPICOCCUS SP.

To comprehend the structure of the cuticle of Epicoccits, some knowledge of its
mode of development is essential.

At the end of a short period of wandering, the second female nymphal instar
assumes a permanent position on a twig, petiole, or leaf, moults, and enters her
third and final stadium. Rapid development of the cuticle now begins. The cells
of the dorsal hypodermis hypertrophy and their secretory activity correspondingly
increases. This gives rise to enormous allometric growth of the dorsal cuticle
as two lobes, one along either side, whose inner surfaces are in close contact with
the bark of the host. Posteriorly these do not clasp the stem but grow round until
their margins meet enclosing a small orifice (Fig. 3). While these changes have
been in progress, glands located along the parts of the lobes contiguous with the
bark have been pouring out large quantities of wax. not unlike bees-wax in colour
and texture, which cements the insect firmly to the surface of the stem. Mean-
while, its ventral surface has moved outwards away from the bark, the space so
formed constituting a brood chamber (Figs. 2 and 3) whose only means of com-
munication with the exterior is through the pore between the adpressed posterior
tips of the lobes. As a result of these changes, the greater part of the cuticle, and
the only part visible when the insect is in situ, consists of the large thickened dorsum.
The remaining much-smaller enclosed region comprises the small thin sternal
cuticle and the relatively-minute, degenerate pleural regions. The whole develop-
ment resembles much more closely that of a coccid than that of a pseudococcid.
The thickened dorsum, deeply infolded to expose the least possible area to the
atmosphere, the enclosure of the entire thin part of the cuticle, and the soiracles
opening into the brood chamber, all appear to be adaptations to minimize water loss.

The fully-developed female (Fig. 4) has an average length of about 3 mm. and
is somewhat less than this in width. Transverse sections display a great variety
of contour, the only consistent feature being their distorted U-shaped outline. Al-
though an occasional isolated female may be almost bilaterally symmetrical, the
normal condition, brought about by the gregarious mode of life, is for one or the

144

HARRY F. LOWER

H

2mm.

FIGUHE 2. Epicocciis sp. Transverse section showing spatial relation between insect and
host. BC, brood chamber ; D, dorsal cuticle ; H, host ; P, pleural cuticle ; V, ventral cuticle ;
W, cementing wax.

O

I-

mm.

FIGURE 3. Epicoccns sp. Fully developed and immature third female instars. Ventral
aspect showing brood chamber and posterior pore. D, dorsal cuticle ; Pp. posterior pore ; V,
ventral cuticle ; W, cementing wax.

CUTICLE OF FEMALE PSEUDOCOCCIDAE

145

FIGURE 4. Eficoccus sp., in situ. Pliotoi/niph courtesy of Helen M. Brookes. The fe-
males are invariably so oriented that the rounded cephalic end is directed towards the host's
region of growth. Part of the cementing wax is visible on the second insect from the top.

146

HARRY F. LOWER

other lateral lobe to be more developed than its fellow. Which of these is the
larger is determined by the proximity of neighboring females, leaf petioles or similar
obstructions, and the natural asperities of the bark encountered during growth.
Figure 2 was drawn from a section selected, not because it was typical, but because
it clearly displayed the spatial relation between insect and host.

The mouthparts excepted, the only regions of the body capable even of limited
movement are the pleura and the ventral abdominal surface, both of which are
subordinate to the functions of oviposition and defaecation.

The cuticle exhibits great diversity of thickness, structure and composition.
For descriptive purposes, four major types are here recognized: the respective
cuticles of the dorsum, the venter, the pleura, and the intersegmental membranes.
The latter are restricted to the enclosed ventral surface; externally, they are sup-
pressed by fusion of the segments and are represented only by sutures.

1. The Dorsal Cuticle

Relative to the size of the insect, the dorsal cuticle is massive, ranging in thick-
ness from a minimum of 30 p. near its junction with the pleura (Figs. 5 and 6A)
to some 40 p, in the remainder (Fig. 6C). Maximum thickness is attained in
small localized patches where muscles, particularly those in the abdomen associated
with the organs of oviposition, are inserted. In such areas thickenings of 70 \L
or more are not uncommon (Fig. 6B). It exhibits a general uniformity of struc-
ture throughout, consisting of a two-layered epicuticle overlying the procuticle.
It is interesting to note that no part of the cuticle is argentaftin.

A. The Kpicuticle

Irrespective of its location, the epicuticle displays a constancy of thickness,
structure, and composition so that what is said of it here in connexion with the

FIGURE 5. Epicoccus sp. Dorso-pleural and ventro-pleural junctions. The part of the cuticle
drawn is indicated by the small circle in the diagram at right. Symbols as for Figure 2.

CUTICLE OF FEMALE PSEUDOCOCCIDAE

147

{£?!*•;•:.

mes

FIGURE 6. Epicoccus sp. Dorsal cuticle. A and C, general cuticle ; B, cuticular ingrowth.
Pore canals are omitted from B and C for clarity, hb, heavily-staining zone ; m, margins of
heavily-staining zone; mes, mesocuticle. Other symbols as for Figure 1.

dorsal cuticle has general application. Except for the non-pigmented condition
of the paraffin layer, it is practically indistinguishable from the same structure in
Pseudococcns. It has a total thickness of about 1.5 ju. distributed between the
(outer) paraffin component which constitutes about one third of it, and the (inner)
cuticulin layer which comprises the remainder (Figs. 6 and 7).

The Cuticulin Layer:

The cuticulin layer is colorless and transparent. It gives a vigorous response
to Millon's reagent, is stained yellow by the xanthoproteic reaction, and orange-

148

HARRY F. LOWER

red by the routine stain. It is non-argentaffin, non-reducing, non-iodophil, and is
unaffected by Danielli's, Gibb's, or Mallory's PTAH techniques. It gives negative
responses to Sudan black B and Nile blue sulfate. It is easily soluble in hot
solutions of potassium hydroxide but resists for at least twelve hours the action
of 10% hydrochloric acid at 60° C. These responses indicate a composition largely
proteinaceous. The materials to which the protein is bound are such that the
usual range of tests do not serve for their identification. They also confer on it
its resistance to extraction bv hot dilute acids.

FIGURE 7. Ej>icoccns sp. Dorsal cuticle 4 /* ; stained with Sudan black B, bright-field. Hy,
hypodermis ; L, lipoid zone; mes, mesocuticle. Other symbols as for Figure 1.

The Paraffin Layer:

This layer is colorless. Its characteristic component is lipoid, as the strong
positive responses to fat stains (Fig. 7) and osmic acid indicate. Schmorl's test
shows it to possess strong reducing properties and Mallory's PTAH stains it deep
violet. It is intensely iodophil but it is not affected by the other reagents used
nor is it soluble in dilute acids or alkalis.

The Surface Secretion:

The dorsal cuticle of living insects or of those killed with cyanide has a resinous
luster (Fig. 4) which is retained when the dead specimens are dehydrated. The

CUTICLE OF FEMALE PSEUDOCOCCIDAE 149

glossy surface cannot be wetted. If freshly killed or dried specimens are treated
with fat solvents, 90% ethanol, or warm S(/o sodium or potassium hydroxide solu-
tion for five minutes, and then washed with water and dried, the luster is lost and
the entire surface becomes dull. Treatment with boiling water, hot concentrated
hydrochloric acid, or exposure to dry heat at 100° C. for twelve hours does not
affect it. Immersion of insects in molten water-soluble wax similarly leaves the
luster undimmed.

Untreated and ''dulled" insects were therefore embedded in this medium, the
sections stained with aqueous dyes in acid solution, and mounted in glycerol which
is without effect on the luster. Sections cut from both batches were indistinguish-
able nor did comparison with sections of fresh material reveal any recognizable
differences.

This scanty evidence suggests that a layer of sub-microscopic thickness (possibly
containing lipoid) may cover the paraffin layer of the dorsal epicuticle.

B. The Procuticle

The procuticle consists almost entirely of mesocuticle (Fig. 7). Exocuticle is
restricted to small widely-dispersed papillae, each bearing a minute hemi-sclerotized
seta. The endocuticle forms a narrow zone about 1 p. in thickness except at the
termination of cuticular ingrowths where its lobes and thickened portions serve as
intermediaries for muscle attachment (Figs. 6 and 7).

The E.rocnticlc:

In the sense that exocuticle is completely-sclerotized procuticle, this zone is
wanting in Epicoccus. Both papillae and setules are transparent; the former are
very pale yellow, the latter are colourless. Neither is inert to aniline stains as is
true exocuticle. The routine stain colours both of them pink and the colour deepens
as time of immersion is increased. Treatment for a few minutes with "Dia-
phanol" is sufficient to destroy the incipient sclerotization and they then stain in-
tensely and rapidly with acid dyes. Their development appears to be in a stage
intermediate between mesocuticle and exocuticle. Complete sclerotization com-
monly induces changes in the overlying cuticulin layer but the epicuticle of the
papillae differs in no respect from that of other parts of the body.

The Mesocuticle:

The mesocuticle shows little structural differentiation. When unstained sec-
tions are examined under the highest powers of the light microscope the mesocuticle
appears uniform and featureless. Under phase-contrast conditions it is trans-
versely marked with numerous, irregular dark streaks which indicate the positions
of pore canals of whose organization, however, no details are observable. The
routine stain dyes the zone red but supplies little information on canal structure,

Chemically, the mesocuticle is as complex as it is structurally simple. Millon's
reagent differentiates it into three clearly-defined sub-zones which, beginning with
the outermost, are here referred to as the A, B, and C sub-zones, respectively
(Fig. 8).

The A sub-zone is stained pink by Millon's reagent, and the distal parts of the
pore canals appear as indistinct, thin red lines which, after traversing the region,

150

HARRY F. LOWER

attenuate before they terminate at its outer surface. It is of variable width ; in
some parts it may form as much as a third of the cuticle but is generally less. Oc-
casionally it is suppressed by the outward extension of the B sub-zone (Fig. 6B).
The B sub-zone takes the form of a conspicuous, broad, bright-red band, sharply
demarcated externally and internally from the remainder of the procuticle by
narrow crimson margins (Figs. 6 and 8). It is continuous throughout the dorsal
mesocuticle, though it thins before it terminates at the dorso-pleural junctions.
This intensely-staining proteinaceous region exhibits great and sudden variations
in thickness. Its maximum development may be observed in cuticular ingrowths
of which it occupies the greater part (Fig. 6B). Where it is thinnest (Fig. 6C),
it is composed solely of the contiguous crimson margins. The distinct differences

FIGURE 8. Epicoccits sp. Dorsal cuticle, 4 fj.. Millon's reagent; bright field. A, B, and
C are the three sub-zones referred to in text. Maximum definition in the mesocuticle has been
sought ; the epicuticle is distorted through being out of focus.

in color between the central part and its marginal bands suggest either that differ-
ent proteins are present in each or that the concentration of the one protein is
higher in the margins than internally. The general appearance is reminiscent of
the result obtained by the use of the paper-chromatography technique. The great
mass of the pore canals (Fig. 9) is located within the zone and it is possible that a
protein complex in solution diffuses from them into the circumjacent cuticle there
to undergo partial separation, the chitinous matrix acting similarly to the chromato-
graphic paper.

Sub-zone C is stratified in alternate red and pale-pink layers (Fig. 8). Whether
or not these coincide with the probable original lamellate deposition of the pro-
cuticle cannot be determined since no other stain or technique used, produced
comparable differentiation. This sub-zone varies in thickness but occupies about
the inner third of the mesocuticle.

CUTICLE OF FEMALE PSEUDOCOCCIDAE 151

Of the wide range of tests applied to the mesocuticle none so clearly dis-
tinguished the sub-zones as did Millon's reagent. Other tests which responded
positively gave diffuse results, hut confirmed the fact that the concentration of
protein in the B sub-zone is higher than in any other part of the mesocuticle.
Xinhydrin colored the mesocuticle violet-pink, the greatest depth of color being
developed in the B sub-zone. The xanthoproteic reaction stained it deep orange
medially, paling to yellow towards either surface. The iodine technique (Lower,
1957b) approximately delineated the sub-zone by staining it deep purple-black,
the C sub-zone was uniformly dark red, and the A sub-zone was practically un-
stained. Mallory's PTAH produced a similar picture, the B sub-zone being-
reddish violet with deeper violet margins, the C sub-zone light red, and the A
sub-zone almost colorless.

joq//

FIGURE 9. Epicoccus sp. Pore canals in latero-dorsal cuticle. One micron ; stained by
Sevki's technique; bright-field. Note termination of pore canals at outer mesocuticular surface.
C, cuticulin layer ; P, paraffin layer ; pc, pore canals.

As is usually the case, Sevki's technique produced. the clearest differentiation of
the pore canals (Figs. 9, 10). Their thin hypodermal connections were colored
red, their thickened portions in the B sub-zone were deep purple, and their thin
terminal parts in the A sub-zone were red. Their numerosity is such that even
in sections cut at 1 p. they appear as a confused mass (Fig. 10). The only valid
conclusions that can be drawn concerning them are the following : the pore canals
are confined to the procuticle, being continuous between its outer surface and the
hypodermis ; they are extremely numerous, of highly irregular form, and the mass
of their contents, which possess a high concentration of tyrosine-containing protein,
is almost entirely located within the B sub-zone.

The mesocuticular protein is separable into two fractions. Sections were im-
mersed for twelve hours in 10% hydrochloric acid maintained at 60° C. After

152 HARRY F. LOWER

washing in distilled water it was found that the mesocuticle had lost its charac-
teristic staining properties. Millon's reagent colored it uniformly pink and the
xanthroproteic reaction pale yellow. Ninhydrin, Mallory's PTAH, and the Sevki
and iodine techniques all gave negative results. The routine stain dyed it light
red. In no section were pore canals visible and even the use of phase-contrast
failed to reveal their positions. These results demonstrate that while most of the
protein is extractable with hot dilute hydrochloric acid, there is a residual fraction
firmly bound to the chitin in such a manner that its extraction is more difficult.

There is no evidence to suggest either the mode of accumulation or functioning
of the apparently high protein concentration in the B sub-zone. The adult male
of either species of Epicoccus is unknown; the cuticles of first and second nymphal

HSBSBr

10

FIGURE 10. Epicoccns sp. Pore canals in thick part of dorsal mesocuticle.
One micron ; stained by Sevki's technique ; bright field.

instars of either sex do not differ significantly from those of the corresponding
instars of other pseudococcids. Of the known stages, therefore, the phenomenon
is confined to the third female instar in which the thickness of the sub-zone relative
to that of the cuticle as a whole increases with the age of the insect.

The Endocuticle:

As mentioned above, the endocuticle is greatly reduced. It is wanting in many
parts of the cuticle, and, where present, is never more than 1 /*, in thickness except
where it caps the cuticular ingrowths. Its structure appears normal.

The Lipoid Zone:

When the routine stain is applied to a cuticular section, a thin layer, staining
a much deeper green than does the endocuticle, can be seen to separate the latter

CUTICLE OF FEMALE PSEUDOCOCCIDAE 153

from the hypodermal cells. This layer has an average thickness dorsally of about
1 /j., and is continuous throughout the dorsal cuticle except where interrupted by
cuticular ingrowths. Laterally, it attenuates until it disappears in those parts of
the dorsal lobes which are in contact with the bark. It is absent from the cuticles
of the pleura, venter, and intersegmental membranes. In unstained sections, the
layer is indistinguishable from the endocuticle, since the structure and natural
color of both are the same. Its high content of chitin shows it to be definitely of
procuticular origin, and but for its impregnation with other materials, it would
merely represent the innermost part of the endocuticle.

It is remarkable for its high lipoid content, and to obviate additions to the
terminology, unjustifiable at this stage, it is referred to descriptively as the lipoid
zone. The lipoid zone has unusual chemical properties for a part of the procuticle,
so anatomically located. In addition to the reactions characteristic of endocuticle
generally, it is stained deep violet by Sevki's technique and Mallory's PTAH. It is
most intensely colored, however, by the fat stains.

In free-hand sections of fresh material, the zone can be deeply stained at room
temperature by short immersion in the fat stains. Sections cut from paraffin-
embedded material, or those cut from cuticles which have been repeatedly extracted
with boiling cyclohexane, cannot be stained in this manner. Nearly as intense color,
however, can be developed by immersion for several hours at 60° C. in the same
stains. The Nile blue sulfate technique shows that part, at least, of the lipoid is
neutral, since the zone is stained red after differentiation. If sections cut from ex-
tracted or paraffin-embedded material be gently warmed with fuming nitric acid,
the chitin-lipoid association is destroyed, and the previously-dispersed lipoid ag-
gregates into minute droplets which stain rapidly and intensely with fat stains.
These results indicate that a lipoid complex, rather than a single lipoid, is involved.
Part of the lipoid is free, and easily extractable ; part is bound to the chitin and the
other constituents of the procuticle of the region, and resists extraction.

Of the origin and function of the lipoid zone, nothing is known. Examination
of females in various developmental stages shows that lipoid impregnation syn-
chronizes with secretion of the procuticle, and that the zone maintains its position
relative to the hypodermis throughout the development. It is not present in the
first and second nymphal instars, and was absent from one very young third female
instar which had reached this stage only about the time that the insects were
collected. In all the other females examined, no measurable difference in its
thickness was observed, and in two exceptionally large specimens (both para-
sitized), the zone wras of normal thickness.

Only twice previously has the presence of a layer between the endocuticle and
the hypodermis been recorded in the literature. The two records are those of
Schmidt (1956) and Malek (1956).

Schmidt reported that, in certain insects which he had examined, a glyco-
protein layer, the "sub-cuticle," separated the endocuticle from the hypodermis.
As he stated categorically that the "sub-cuticle" was non-chitinous, it clearly has no
affinity with the lipoid zone of Epicoccits, and need not be further discussed here.

Malek demonstrated that when the desert locust, Schistocerca gregarla, is
moulting, the inner part of what was originally endocuticle becomes impregnated
with a lipo-protein complex, to form the ecdysial membrane of the insect.

This membrane and the lipoid zone of Epicoccus appear to be homologous struc-

154 HARRY F. LOWER

tures. Since both are derived from the innermost part of the procuticle contiguous
with the hypodermis, both occupy corresponding anatomical positions. Both are
impregnated with lipoid, and both, by reason of their derivation, contain chitin.

The stages of the life cycle in which each is present are, however, completely
reversed. In Schistocerca, the lipoid-impregnated procuticle must, by forming an
ecdysial membrane, be necessarily confined to immature stages, since the adult does
not moult. The lipoid zone of Epicoccus, on the contrary, is found in the adult
only. Few details are given in Malek's preliminary note, and it will be necessary
to await his full account, before an adequate comparison of the two structures can be
made.

2. The Ventral Cuticle

The cuticle of the venter is thin, averaging some 4 p. in thickness. Its epi-
cuticle is indistinguishable from that of other parts of the body.

A. The Procuticle

There is no exocuticle. Less than half of the procuticle consists of mesocuticle
which displays no signs of the chemical sub-zonation characteristic of the dorsal
mesocuticle. It has no visible internal structure and chemically, is typical of this
zone generallv.

The endocuticle comprises rather more than half of the procuticle, being
thicker than that of the dorsum. No lipoid zone is present. Both chemically and
structurally it resembles the endocuticle of P. adonidiiui.

3. The Pleura! Cittiele

What is here assumed to be the cuticle of the degenerate pleura covers two
narrow regions, one on either side, which connect the ventral and dorsal cuticles.
From both of these the pleural cuticle differs greatly in structure. In transverse
sections, its surfaces are irregular (Fig. 5) and its general appearance suggests that
it is in a contracted state ; it is possible that in the living insect it may be much more
extended and correspondingly thinner.

It is frequently thick, exceeding 30 /A in a few places. The epicuticle is typical.
The procuticle consists wholly of endocuticle which, when stained, shows no struc-
ture under bright-field conditions. When viewed under phase-contrast conditions,
whether stained or not, numerous, fine, approximately-transverse dark lines can be
seen. These appear to be artifacts produced by the contraction assumed to have
occurred. The change from pleural to dorsal, or pleural to ventral, cuticles is
sharp ; there is no gradation of one region into the other.

Tests reveal the presence of the normal chitin-protein complex; there are no
unusual components.

4. The Cuticle of the Interseginentctl Membranes

Functional intersegmental membranes are found uniting the ventral abdominal
segments only, so that cuticle of this kind is restricted to the ventral surface.

It is very thin and consists of the typical epicuticle overlying a procuticle rarely

CUTICLE OF FEMALE PSEUDOCOCCIDAE 155

exceeding 2 ^ in thickness and often being less. Its highly plicate condition in man)
places shows that it permits of considerable movement of the ventral region. The
procuticle, which is composed wholly of endocuticle, has no visible internal struc-
ture, and no peculiar chemical properties. There appear to be no pore canals
in it.

III. THE CUTICLE OF E. ACACIAE (MASKELL)

The cuticle of E. acaciae differs in details, only, from that of its congener. The
species is somewhat smaller than is Epicoccus sp. but relative to the size of the
insect its cuticle is still massive.

The epicuticles of both forms are indistinguishable even to the extent that
dorsally the paraffin layer of each is covered by a sub-microscopic surface secretion
of similar properties.

What has been said of the cuticles of the venter, the pleura, and the inter-
segmental membranes of Epicoccus sp., applies equally to those of the same regions
of E. acaciae, such differences as do occur being confined to the dorsal procuticle.

The endocuticle of the latter has undergone still further reduction and forms
irregular cappings to terminations of the cuticular ingrowths. It does not occur
elsewhere. The procuticle thus consists almost entirely of mesocuticle which
structurally resembles that of Epicoccus sp. ; its distinguishing characters are chemi-
cal. Millon's reagent colors it uniformly cherry-red — a much deeper shade than
is produced by the reagent in insect cuticle generally. The xanthoproteic test
stains it uniformly deep orange, the iodine technique purple-black, ninhydrin violet-
pink, and Mallory's PTAH deep purple. Sevki's technique displays the pore
canals as deep violet, filamentous tubes of approximately constant diameter through-
out their length and demonstrates their continuity between the hypodermis and the
outer procuticular surface. After twelve hours' extraction at 60° C. with \0%
hydrochloric acid, Millon's reagent stains the whole procuticle pink, and the
xanthoproteic test colors it yellow. It fails to give visible responses with the other
stains and reagents.

These results would seem to indicate that extra protein is present in the
mesocuticle. that it is uniformly distributed (in contrast to its aggregated condition
in Epicoccus sp.), and that it is differentiated into acid-extractable and acid-resistant
fractions.

A lipoid zone, whose extent and reactions are identical with those of the corre-
sponding zone of Epicoccus sp., is present. It differs in being brown pigmented
with melanin or a melanin-like product, and is hence easily recognizable even in
unstained sections.

IV. THE WAX GLANDS AND THEIR SECRETION

(a) P. adonidnni

Wax glands are conspicuous in most sections of P. adonidnni, frequently as
many as four or five, transected in various places, being visible in the one section.
They resemble those of P. niartinins as described by Pollister (1937) and it is prob-
able that a general uniformity of glandular structure prevails throughout the family.
Fundamentally, each consists of a multicellular secretory sheath enclosing a large.

156 HARRY F. LOWER

sub-spherical, central reservoir which communicates with the surface by means
of one or more ducts (Fig. 1A).

When sections of unfixed material are treated with Sudan black B, the surface
wax, the contents of reservoirs and ducts, and limited parts of the cuticle surround-
ing the ducts, are blackened. The local cuticular impregnation appears to be
brought about by diffusion of some of the wax through the duct walls. The Nile
blue sulfate technique stains the surface wax, that filling the ducts, and the im-
pregnated cuticle deep blue, but it colors the reservoir contents red. Fat solvents
readily dissolve the surface wax and that of the dvicts but have no apparent effect
on the impregnated cuticle or the reservoir material. The latter is highly resistant
to such solvents; prolonged extraction with methanol-chloroform, pyridine, ether,
or boiling cyclohexane removes part only of its lipoid. This explains why even in
paraffin sections cut at 1 /* the contents of the reservoirs are retained apparently
unaltered by the treatment they have undergone in the course of their preparation.

These results suggest that the material in the reservoir is "protowax" con-
sisting of neutral lipoid (indicated by the Nile blue sulfate technique) so bound to
other substances as to render its extraction extremely difficult. Alternatively the
protowax may be secreted as an emulsoid whose finely divided lipoid micelles are
dispersed in an inert medium. By some means at present unknown, the lipoid
is freed and passed along the ducts. On its way to the surface the neutral lipoid
becomes acidic, in which form it is deposited as wax on the surface. This in-
terpretation of the course of events is purely speculative. An important obstacle to
its acceptance is the failure of any of the wide range of tests applied, to demonstrate
the presence in the reservoir of anything except lipoid.

There is some evidence suggesting that the outer surface of the wax layer may
be covered by a sub-microscopic layer of protective material.

(a) If insects are killed with cyanide and then immersed in a solution of Sudan
black B, the surface wax is stained only where it has been damaged during
manipulation. If they are first lightly brushed, the areas so treated stain rapidly.

(b) If the insects have a prior immersion for fifteen minutes in 10% hydro-
chloric acid at 35° C. before being put in Sudan black B, staining of the wax is rapid
and complete. Moreover, from insects so pre-treated, cold cyclohexane dissolves
the wax rapidly whereas it acts much more slowly on untreated insects.

(c) If the insects are dropped into water at 70° C., the wax oozes away forming
a surface film on the water. If such insects are then embedded in paraffin and
sectioned, the epicuticle, when viewed under phase-contrast conditions, has an
outer surface which cannot be sharply focussed. If the same sections are then
treated with warm ICK/r hydrochloric acid for ten minutes, and re-examined, the
epicuticle has a well-defined outer boundary.

Should an outer layer be present, the wax and its protective layer would afford
an interesting analogy to the two outer layers of the four-layered type of epicuticle.
They would occupy the same anatomical position relative to the two inner layers,
and apparently perform the same function of limiting water loss as do the wax and
cement layers. They differ in that the wax at least is a glandular product, they
are not secreted until after moulting, and finally, some of the wax is used by many
species as a covering for egg masses.

CUTICLE OF FEMALE PSEUDOCOCCIDAE 157

(1)) Epicoccus

In Epicoccus most of the wax glands have atrophied. Their orifices are still
open, but either their ducts are internally sealed off by the growth of procuticle
across them, or their secretory cells are small and produce little wax. Glands of the
latter kind are confined to the dorsum where they open in small groups in deep
infolds of the cuticle adjoining muscle insertions. They secrete little more wax
than is needed to plug the ducts and keep them filled. Their orifices are marked
by small white spots at the surface (Fig. 4).

Typical glands are confined to the lateral extremities of the lobes in contact with
the bark. The gland contents and the cementing wax respond similarly to stains
and tests as do those of P. adonidum. The principal differences between the
waxes of the two species are that that of Epicoccus has a higher melting point, and
dissolves easily and quickly in all fat solvents. It would appear that the wax having
ceased to function as an agent for reducing desiccation, no protective layer covers it.

V. DISCUSSION

The results of this investigation demonstrate that notwithstanding the funda-
mental similarity of cuticular structure which prevails throughout the Pseudo-
coccidae, great morphological differences distinguish the two genera studied.

A. The Epicuticle

The epicuticles of all three species consist of the essential cuticulin and paraffin
layers. The paraffin layer of Epicoccus is overlain by a sub-microscopic covering
of apparently lipoidal material which probably corresponds to the wax layer of more
complex epicuticles, as a cement layer in this anatomical position would be ab-
normal. There is no indication of the presence of any such layer in Pscndococcus
in which it is replaced by a thick layer of wax secreted by hypodermal glands;
this surface wax itself appears to possess an extremely tenuous protective covering.

The effectiveness of either of these systems as a means for restricting water loss
is probably slight. Much of the surface wax of Pseiidococcns, for example, is dis-
posed in long filaments which would have little value in this regard, more especially
for a species which inhabits a humid micro-environment. Dead insects of either
genus, after removal of the surface layer, do not lose water to a dry atmosphere at
a significantly greater rate than do dead intact ones.

B. The Procuticle

The procuticles of the two genera differ as greatly as do the environments in-
habited by each. The thin procuticle of Pseudococcus, consisting almost solely of
endocuticle, displays little specialization. That of Epicoccus, on the contrary, is
highly specialized both structurally and chemically.

In absolute thickness it is comparable with those of large sclerotized forms
such as Periplaneta, while relative to the individual's size, there are probably few
other insects which can match it. It consists almost wholly of mesocuticle.

Among insects whose cuticles have been described, that of Epicoccus is unique
in the high proportion of protein in the procuticle. aggregated in a well-defined zone.

158 HARRY F. LOWER

Its possession of a deep-seated lipoid zone in the procuticle is. so far as is known,
shared only by the desert locust. Scliistoccrca yregaria.

All this specialization betokens a long period of evolution under conditions
adverse to insect life generally, and the acquisition of a massive cuticle, together
with the loss of mobility by the adult female, are presumably closely linked with
this. Incapacity for locomotion may be disadvantageous; but failure to cope with
a hostile environment spells extinction.

The collected material came from a single half-dead plant, not over ten feet
in height. Fully-exposed as the insects were, they not infrequently had to contend
with direct sun temperatures of 60° C., or even more, accompanied by relative
humidities often below 10r/ , in a situation commonly swept by parching winds.
The brood chambers of mature females contained eggs, and first and second
nymphal instars. These immature forms were found nowhere else. Apart, there-
fore, from ensuring survival of the mother, the thick leathery cuticle functions
equally well in protecting those stages in the life cycle most vulnerable to the environ-
mental conditions, thereby making it possible for the species to maintain itself in
a region relatively-poor in the higher forms of insect life.

I wish to thank Miss Helen M. Brookes of this department for supplying the
material used and for allowing publication of the photograph reproduced in Fig-
ure 4.

My thanks are also due to Mr. Keith P. Phillips (in charge of Photographic
Department) who is responsible for all the photography.

SUM MARY

1. The cuticular structure of three female pseudococcids, Pscudococcns adoni-
dnui L., Epicoccus sp., and E. acociac (Maskell). has been investigated.

2. The cuticle of P. adonidnui consists of a two-layered epicuticle, overlying
a thin procuticle. almost all of which is endocuticle.

3. The cuticle of Efiicoccns sp., is highly specialized. Its epicuticle closely re-
sembles that of P. adonidntn. The dorsal cuticle is relatively thick, and is much
modified chemically. Most of it consists of mesocuticle in which Millon's reagent
delimits three well-defined zones which differ greatly in their reactions to stains
and histochemical reagents. The endocuticle is much reduced. A thin layer of
procuticle between the hypodermis and the endocuticle is impregnated with lipoid to
from a "lipoid zone."

4. The cuticle of E. acaciac is thick. It differs from that of Epicoccus sp.
principally in that there is no chemical zonation of the procuticle, and the lipoid
zone is melanin-pigmented.

5. Wax glands are numerous in the cuticle of P. adonidnui. The contents of
their reservoirs ("protowax") differ chemically from the surface wax. In Epi-
coccus, many of the glands have atrophied ; typical glands are confined to lateral
parts of the cuticle in contact with the host plant, and these secrete large quantities
of wax which fixes the insect permanently in position.

6. The specialized cuticle of Epicoccus appears to have evolved over a long
period, during which the insects have been exposed to adverse environmental con-
ditions.

CUTICLE OF FEMALE PSEUDOCOCCIDAE 159

LITERATURE CITED

DENNELL, R., 1946. A study of an insect cuticle: the larval cuticle of Sarcoplun/n jalcitlata

Pand. (Diptera). Proc. Roy. Soc. London, Scr. B, 133: 348-373.
DENNELL, R., AND S. R. A. MALEK, 1955. The cuticle of the cockroach Pcriplaneta anicricana

II The epicuticle. Proc. Roy. Soc. London, Scr. B, 143: 239-257.
LOWER, H. F., 1956. The terminology of the insect cuticle. Nature, 178: 1355-1356.
LOWER, H. F., 1957a. The acellular coverings of the immature stages of Aphodius hoivitti

Hope (Coleoptera: Scarabaeidae). /. Morph. (in press).

LOWER, H. F., 1957b. lodophil components of insect cuticle. Stain Tech., 32: 127-129.
MALEK, S. R. A., 1956. An ecdysial membrane in the locust cuticle. Nature, 178: 1185-1186.
POLLISTER, P. F., 1937. The structure and development of wax glands of Pseudococcus

inaritiinus ( Homoptera, Coccidae). Quart. J. Micr. Sci., 80: 127-148.
SCHMIDT, E. L., 1956. Observations on the subcuticular layer in the insect integument.

/. Morph., 99: 211-226.
WIGGLESWORTH, V. B., 1947. The epicuticle in an insect, Rhodnius proli.rus (Hemiptera).

Proc. Roy. Soc. London. Scr. B, 134: 163-181.

PHENYLTHIOUREA TREATMENT AND BINDING OF RADIOACTIVE

IODINE IN THE TADPOLE

W. GARDNER LYNN 1 AND JAMES NORMAN DENT -

Biology Division, Oak Ridge National Laboratory,3 and Departments of Biology,
The Catholic University of America and University of Virginia

In a study of the distribution of I131 after administration to tadpoles, Dent
and Hunt (1952) demonstrated not only the expected concentration of this sub-
stance in the thyroid gland but also significant accumulations in several other
regions. Notable among these were the thymus, the horny teeth, the melanophores
of the skin, and the pigmented layer of the retina. The same pattern of distribution
was observed in tadpoles of Hyla, Rana, and Bufo in various stages of development
and was not altered by thyroidectomy. It was suggested that, since tyrosine is a
precursor of melanin, the localization of I131 in pigmented tissues may be attributable
to the binding of I131 to tyrosine that must be present in those tissues. The
hypothesis was also advanced that perhaps the same enzymes that bring about
oxidation of tyrosine to melanin are able to facilitate the union of iodine and tyro-
sine. Gennaro and Clements (1956a, 1956b), studying the binding of I131 in the
skin of the adult frog, provided evidence to support these views. Our experiments
were undertaken as a further investigation of the association of iodine with pig-
mented tissues and as a further test of the hypothesis cited.

It is known that administration of certain derivatives of thiourea results in an
inhibition of melanin formation. This has been demonstrated for mammals
(Richter and Clisby, 1941; Dieke, 1947), fishes (Frieders, 1954), and several
different amphibians (Lynn and de Marie, 1946; Lynn, 1948; Blackstad, 1949;
Millott and Lynn, 1954). In amphibians the results are most striking when these
substances are given to embryos or young larvae before any melanophores have
appeared. Such individuals do not develop black pigment so long as the treat-
ment is continued. Cessation of treatment is followed by rapid melanogenesis.
Since it has been demonstrated (Bernheim and Bernheim, 1942; Paschkis,
Cantarow, Hart and Rakoff, 1944; Dubois and Erway, 1946) that thiourea deriva-
tives inhibit tyrosinase activity in vitro, it is assumed that their role in preventing
melanin formation in frog embryos is the inhibition of tryosinase activity. In our
experiments, larvae of several different ages were treated with one of these tyrosi-
nase inhibitors, phenylthiourea, to obtain unpigmented tadpoles or tadpoles with
reduced pigmentation. The pattern of iodine uptake in these animals was com-
pared with that in untreated controls at various times after the beginning of treat-
ment.

MATERIALS AND METHODS

The animals used for this experiment were tadpoles of Hyla versicolor versicolor
LeConte hatched from eggs collected in a small temporary pool near Oak Ridge,

1 Address : Department of Biology, The Catholic University of America.

2 Address: Department of Biology, University of Virginia. Work supported in part by
A. E.G. Contract AT- (40-1) -2000.

3 Operated by Union Carbide Nuclear Company for the U. S. Atomic Energy Commission.

160

PHENYLTHIOUREA AND IODINE BINDING 161

Tennessee. A few hours after hatching, the larvae were distributed in groups of
fifteen in finger bowls, each containing 200 ml. of spring water. On the first,
fourth, and fifteenth days after hatching, experimental series were established in
this way : Larvae were transferred to six finger bowls, fifteen larvae to each bowl ;
two contained 200 ml. of 0.01% phenylthiourea in spring water, two contained
200 ml. of 0.005% phenylthiourea in spring water, and two contained spring water
alone (controls). The tadpoles, kept at laboratory temperatures (21°-23° C.),
were fed crumbled pellets of Purina rat chow and, occasionally, a strained beef-
and-liver soup prepared for infants. The culture fluids were changed daily and
there was no mortality. At four-day intervals three animals from each experi-
mental and control group were selected at random and examined under the
binocular microscope. Records were kept of the gross changes in pigmentation
and of the developmental stage reached. The system of staging devised by Taylor
and Kollros (1946) for Rana pipiens was used and adapted with minor variations
for Hyla versieolor.

At three ages (4, 24, and 29 days after hatching), animals were removed from
the experimental and control series and used in the preparation of autoradiograms.4
The procedure was as follows : Five larvae from each bowl were put in 50 ml. of a
solution of one part per million of stable sodium iodide 5 and enough radioiodine
to give an activity of one /xc./ml. at the beginning of the immersion period. For
each experimental group, one set of five animals was put in a radioiodine solution
made up in spring water and another was put in a radioiodine solution made up in
the same phenylthiourea solution in which the animals had been raised. After
24 hours in the radioiodine solution, the larvae were passed through two baths of
spring water and left in a third bath of spring water, or the appropriate phenyl-
thiourea solution, for another 24 hours. All were then fixed in a 1 :1 mixture of
Bouin's fluid and Cellosolve. After 8 hours' fixation, they were dehydrated in
Cellosolve, embedded in paraffin, and sectioned at 10 micra. The mounted and
dried sections were passed through two changes of xylol, transferred to absolute
alcohol, and then dipped in a 1.0% solution of collodion in ether-alcohol, and dried.
The slides were attached by stationer's binder clips against the emulsion of Eastman
medium contrast lantern slide plates in the darkroom and left for 8 days. Finally,
the lantern slide plates were developed and the sections themselves were stained
with Harris' haematoxylin and Ponceau de xylidine-orange II (Gray, 1952).

RESULTS
1. Gross effects of treatment u'itJi phenylthiourea

During ovogenesis (Kemp, 1953) melanin granules are laid down in the cortical
region of most anuran ova. Superficially, the pigmented area extends from the
animal pole to the presumptive germ ring. These granules are retained within

4 Joftes and Warren (1956) have recommended the substitution of the term "radioautogram"
for "autoradiogram." It is felt that since the semantic basis offered by Joftes and Warren for
the use of the former term does not appear to be much stronger than the etymological basis
presented by Boyd (1955) for the use of the latter and since the latter term has become well
established, its use should be continued.

5 There is some danger of introducing errors by use of carrier-free isotopes because of
their tendency to adhere to glassware. The stable Nal was added with the view of eliminating
that effect.

162

W. GARDNER LYNN AND JAMES NORMAN DENT

••";

4

78 9 10

All sections shown are from 26-day-old larvae of Hyla vcrsicolor that had been immersed
in a solution of one /uc of Ii:il/rnl. for 24 hours and fixed 24 hours after removal from the
solution.

FIGURE 1.
phenylthiourea

FIGURE 2.

FIGURE 3.

FIGURE 4.

FIGURE 5.
then in spring

FIGURE 6.

FIGURE 7.

FIGURE 8.

Photograph of living tadpoles 12 days old. Upper animal raised in 0.01%
; lower animal raised in spring water.

Section through the eye of a control tadpole.

Autoradicgram prepared from the section shown in Figure 2.

Section through the eye cf a tadpole kept in 0.01 'A phenylthiourea continuously.

Section through the eye of a tadpole kept in 0.019^ phenylthiourea for 24 days,
water for two days.

Autoradiogram of the section shown in Figure 5.

Section through the mouth of a control tadpole showing the horny teeth.

Autoradiogram of the section shown in Figure 7.

PHENYLTHIOUREA AND IODINE BINDING 163

the presumptive ectoderm and mesoderm but disappear as melanophores begin to
differentiate and form melanin.

As was anticipated on the basis of previous experiments with other amphibians,
the larvae that had been put in phenylthiourea solutions within a few hours after
hatching stopped forming melanin and after only 24 hours were noticeably paler
than controls. After 48 hours, and at all later stages, the experimental animals
were completely unpigmented, the embryonic pigmentation having disappeared
(Fig. 1). Larvae in which treatment was delayed until four days after hatching
had already developed much melanin in both skin and eyes and exhibited no differ-
ence from the controls for at least three days. After this they gradually paled,
however, and by the tenth day of treatment their skin was without pigment. Pig-
mentation of the eyes was lost much more slowly and even when the experiment
was terminated (51 days after hatching), the eyes of these animals still showed
some pigment, though far less than those of controls. Larvae that were first put
into phenylthiourea solutions 1 5 days after hatching showed no blanching of the skin
until near the end of the experiment, and the pigmentation of the eyes never became
grossly different from that of controls. Examination of the living animals under
the binocular microscope and later study of the sectioned material revealed no
significant pigmentary difference between animals treated with 0.01% phenyl-
thiourea and those treated with 0.005%. It appears that both concentrations are
fully effective in inhibiting melanin formation.

Since phenylthiourea is one of the well-known thyroid-inhibiting drugs, the
experimental animals not only differed from the controls in degree of pigmentation
but also in their failure to exhibit definitive metamorphic changes. No significant
differences in development were noted for the first 20 days of the experiment.
At this time both treated and untreated tadpoles were in late limb bud stages
(Stages IV and V). Later, however, the experimental animals showed definite
inhibition in development. By 24 days the controls were in Stages VI and VII
whereas the tadpoles in phenylthiourea solutions remained at Stages IV and V.
The controls continued to differentiate steadily, most specimens reaching Stage IX
by the 28th day, Stage XV by the 40th day, and late metamorphic stages (Stages
XVIII to XXV) by the 48th day. As is usual with anuran larvae, there was a
considerable variation in developmental rate among the controls, a few specimens
being retarded and others exceptionally advanced. Thus, although forelimb emer-
gence occurred in one control 45 days after hatching, and had occurred in more than
half the surviving controls by 51 days, at this latter time there was still one animal
at Stage VI and several at Stage XIII. Among the tadpoles placed in 0.01%
phenylthiourea, either immediately after hatching or 4 or 15 days later, none
advanced beyond Stage VI and most remained at Stage IV or Stage V. Tadpoles
raised in 0.005% phenylthiourea advanced somewhat beyond those in the higher
concentration ; most of those kept to the end of the experiment reached Stage VI
or Stage VII and a few specimens differentiated to Stage VIII. There is thus
some indication that the lower concentration is not fully effective in inhibiting
thvroid activity.

j J

FIGURE 9. Section through the mouth of a tadpole treated continuously with 0.01%
phenylthiourea.

FIGURE 10. Autoradiogram of the section shown in Figure 9.

164 W. GARDNER LYNN AND JAMES NORMAN DENT

As noted previously, the larvae used for the preparation of autoradiograms were
put in a radioiodine solution for 24 hours, and this was followed by another 24-hour
period without radioiodine, which allowed for elimination of excess iodine before
fixation of the tadpoles. Autoradiograms were made from two sets of phenyl-
thiourea-treated animals, one in which the phenylthiourea treatment was continued
until fixation and one in which the radioiodine solution and the subsequent 24-hour
bath were without phenylthiourea. The latter thus had a 48-hour period of re-
covery from phenylthiourea treatment immediately preceding fixation. It has been
demonstrated that in amphibian embryos melanin reappears very rapidly after
phenylthiourea treatment has stopped (Millott and Lynn, 1954). In the present
experiments the animals put in radioiodine solutions containing no phenylthiourea
showed a well-defined darkening of the eyes within 12 hours ; and at 24 hours, when
they were removed from the radioiodine solutions, a scattering of pigmented
melanophores was also visible in the skin. Larvae kept in phenylthiourea solutions
throughout, of course, showed no such pigmentary change.

2. Effects of phenylthiourea treatment on radioiodine bindintj in the pigmented
epithelium of the retina

The first group tested for radioiodine binding consisted of animals on which
the phenylthiourea treatment started on the day of hatching and continued for
four days only. The second and third groups tested included animals on which
treatment was begun on the day of hatching and some in which treatment was
started later (4 and 15 days after hatching). Since the results were similar in all
three groups, detailed consideration will be given for only one, the second experi-
mental group, which was given radioiodine on the 24th da}' after hatching and
contained animals under treatment for 24, 20, and 9 days.

Photomicrographs and corresponding autoradiograms of the eye region in
typical animals of this group are shown in Figures 2-6. Figures 2 and 4 illus-
trate the conditions found in control and experimental animals of the series in
which phenylthiourea treatment was started at hatching. The pigmented epithelium
of the retina is quite dark in the controls but is entirely without melanin in the
animals given continuous phenylthiourea treatment. The autoradiogram prepared
from the control (Fig. 3) shows that there was a marked concentration of radio-
iodine in the pigment epithelium. On the other hand, an autoradiogram prepared
from the eye shown in Figure 4 was entirely blank and is not shown. It has been
pointed out that some melanin formed in the animals allowed a recovery period
from treatment with phenylthiourea. Plates prepared from these specimens show
faint but definite autoradiograms, indicating that some binding of radioiodine oc-
curred both in the skin and in the retina (Figs. 5, 6). On the basis of these
results it might be concluded that the binding of radioiodine depends on the presence
of melanin and varies directly in amount with the amount of melanin present.
However, study of the larvae in which phenylthiourea treatment was initiated at
later ages reveals that this is not the case. It will be remembered that pigment
was lost from the eyes only very slowly in the series started at 4 days and not at all,
so far as could be seen externally, in the series started at 15 days. Thus these
latter tadpoles, even though under continuous treatment with phenylthiourea. still
had much melanin in the pigment epithelium. Nevertheless the autoradiograms

PHENYLTHIOUREA AND IODINE BINDING 165

prepared from this series exhibited the same variations as those previously de-
scribed. Control animals showed high radioiodine level, animals treated continu-
ously with phenylthiourea showed no radioiodine binding, those treated with
phenylthiourea and then allowed two days' recovery showed a very low radioiodine
level. Yet the degree of pigmentation of the members of all groups was about the
same. It is therefore clear that the binding of radioiodine does not depend on the
amount of formed melanin present.

3. Effects of phenylthiourea treatment on radioiodine binding by the horny teeth

In all larvae given radioiodine a significant localization was found in the horny
teeth. The 4-day series (fixed at 6 days after hatching) shows relatively little
cornification of the teeth. Nevertheless the autoradiograms indicate that radio-
iodine was bound in these structures, not only in the controls but also, and ap-
parently to the same extent, in the phenylthiourea-treated animals. The 24- and
39-day series both exhibit extensive cornification of the teeth, and in these there is
an indication that phenylthiourea did lessen the binding of radioiodine without,
however, completely halting it. Photomicrographs and corresponding autoradio-
grams of the teeth in control and experimental animals of the 24-day series will
serve to illustrate this inhibitory effect (Figs. 7-10). It was found that whereas
the autoradiograms of control tadpoles (Fig. 8) and those of treated animals
allowed a 48-hour recovery period show equally dense spots, representing the
teeth, autoradiograms of larvae subjected to continuous treatment with phenyl-
thiourea show definite but much less intense darkening (Fig. 10). This result was
consistent in all specimens of the older series.

4. Effects of phenylthiourea treatment on the thyroid and thymus

Although these experiments were not primarily concerned with the thyroid
gland, some observations on the thyroid response are of interest. In most of the
control larvae of the 4-day series the thyroid proved to be at a stage of early
follicle formation. Only one or two follicles were present in each thyroid, the rest
of the gland consisting of irregular cords of cells. The formed follicles had a
cuboidal epithelium that still retained some of the pigmentation characteristic of
the anuran thyroid rudiment. The lumina were very small and contained no
stained colloid. Two of the five control specimens had no organized follicles at
all, the entire thyroid being composed of clumps and cords of epithelial cells. In
the experimental animals of this series, there were some with glands lacking
organized follicles ; these showed no histological differences from the two controls
just mentioned. On the other hand, the phenylthiourea-treated larvae in which
follicles had appeared showed a sharp contrast to controls in that the follicles were
larger, had a flattened epithelium, and contained a relatively large amount of
homogeneous basophilic colloid. The autoradiograms of the control thyroids and
those allowed 48 hours' recovery from phenylthiourea showed evidence of binding
of radioiodine, whereas the animals treated continuously with phenylthiourea did
not. It is noteworthy that the thyroids of all 5 of the controls and of all 5 of the
recovery series produced clear autoradiograms but in some of them no organized
follicles were yet present. The effect of phenylthiourea treatment on thyroid

166 W. GARDNER LYNN AND JAMES NORMAN DENT

physiology is thus detectable histologically as soon as follicles are formed and
physiologically even before follicles are formed.

The thyroids of controls fixed at 26 days after hatching showed a quite uni-
form appearance. The follicular epithelium was cuboidal and the colloid acido-
philic, usually with some chromophobe droplets. Experimental animals differed
consistently in having many more chromophobe droplets and slightly higher
epithelium. The different lengths of treatment with phenylthiourea resulted in
no histologically observable differences. There was also no significant difference
in the appearance of the thyroids of animals treated continuously and those allowed
two days' recovery before fixation. Autoradiograms prepared from the sections of
this series showed a high level of radioiodine in the control thyroids, a very low
level in the thyroids of larvae treated continuously with phenylthiourea, and a high
level, apparently as high as that of controls, in those of larvae removed from
phenylthiourea at the time of their exposure to radioiodine solution.

The controls of the series fixed at 41 days after hatching were all in metamorphic
stages and, as would be expected, their thyroids gave indications of high activity.
The epithelium tended to be columnar and chromophobe droplets were abundant.
The experimental animals, on the other hand, had enlarged follicles with a mark-
edly flattened epithelium and much homogeneous colloid. The autoradiograms of
this series are similar to those for the 24-day series.

The concentration of radioiodine in the thymus gland observed by Dent and
Hunt (1952) was confirmed in these experiments and proved to lie affected by
phenylthiourea treatment in exactly the same way as is radioiodine concentration
in the thyroid. Autoradiograms made from control tadpoles of the 24-day series or
from tadpoles allowed a recovery period show high concentration of radioactivity
in the thymus; those made from tadpoles under continuous treatment with
phenylthiourea show no radioactivity in this region.

DISCUSSION

In these experiments the administration of phenylthiourea to early larvae of
Hylct vcrsicolor vcrsicolor resulted in the production of completely unpigmented
tadpoles. Both concentrations tested (0.01 and 0.005%) proved equally effective
and neither gave any indications of toxicity. The gradual blanching of the skin
produced by treatment with phenylthiourea has sometimes been referred to as a
depigmentation effect. It is probable, however, that the drug has no effect on any
pigment already present when treatment is begun but acts entirely by preventing
the formation of new pigment. The results of our experiments are in accord with
this view for, as has been pointed out, the blanching of the skin (and of the eyes)
occurred rapidly in larvae treated immediately after hatching, more slowly in those
in which treatment was delayed until 4 days after hatching, and very slowly indeed
in those in which treatment was begun 15 days after hatching. It must be assumed
that in all these animals the phenylthiourea treatment effectively blocked melano-
genesis and that the rate of "depigmentation" depended on the rate of loss of the
melanin already present. In fact, it would appear that this rate of blanching after
treatment with phenylthiourea should furnish an indication of the normal rate of
metabolic turnover of melanin at various ages. Our results indicate that turn-
over is rapid at early ages but much slower in older animals. In fact, it seems

PHENYLTHIOUREA AND IODINE BINDING 167

likely that some of the formed melanin persists indefinitely after a certain age is
reached.

These experiments also demonstrate that phenylthiourea affects the binding of
radioiodine by the tapetum nigrum. Only the untreated animals show a significant
concentration of I131 by this structure. Tadpoles given phenylthiourea and then
removed from the solution exhibit an ability to bind iodine within the first 24
hours after cessation of treatment. In all experiments, however, the autoradio-
grams, though they do not give quantitative information, indicate clearly that I131
is not taken up by the pigmented epithelium of the eye in direct proportion to the
amount of melanin present. Tadpoles for which phenylthiourea treatment is begun
at 15 days after hatching retain much pigment in the eye yet show no tendency to
bind I131. This indicates that the binding of iodine in the pigmented epithelium
of the eye (and doubtless in chromatophores as well) takes place only while
melanin is actually being formed and is dependent on some enzymatic activity that is
inhibited by phenylthiourea. Since this substance is known to inhibit tyrosinase
activity in vitro and since tyrosine must be present where melanogenesis is going
on, it is natural to suspect that tyrosinase is the enzyme involved. These views
are supported by the findings of Gennaro and Clements, who extracted radioactive
mono- and diiodotyrosine from discs of frog skin that had been incubated in Ringer
solution containing Ii:u and from the skins of intact frogs injected with I131
(1956a). They also showed that pretreatment of the discs with thiourea decreased
the degree of incorporation of I131 in the melanized areas (1956b). According to
the concept outlined, tyrosinase would be active in pigment-forming tissues both
in the oxidation of tyrosine to melanin and in the oxidation of iodide to iodine to
permit the production of mono- and diiodotyrosine and possibly iodinated proteins.
Inhibition of tyrosinase activity would thus be expected to result simultaneously
in cessation of melanogenesis and failure to bind I131. Whether these findings can
be directly related to the goitrogenic effects of phenylthiourea is not certain. The
mechanism by which iodination of tyrosine occurs in the thyroid is not well under-
stood (Roche and Michel, 1955). There is no evidence of the presence of
tyrosinase in the mammalian thyroid (Pitt-Rivers, 1950). Fawcett and Kirkwood
(1954) have hypothecated a "tyrosine iodinase" as a catalyst for the process.
There are many varieties of tyrosinase (Lerner and Fitzpatrick, 1950), however,
and it may be that amphibian tyrosinase has a special property of oxidizing
iodine or, on the other hand, that our experiments may offer the key to a better
understanding of iodination of tyrosine in the thyroid itself.

The accumulation of iodine in the horny teeth of larval anurans was first
reported by Dent and Hunt (1952). Association of iodine with similar hard
structures is known to occur in a number of invertebrates. Noteworthy examples
are the hypodermis of Drosophila larvae (Wheeler, 1950), the setae and pharyn-
geal teeth of polychaetes (Swan, 1950), and the exoskeleton of Daphnia (Gorbman,
Clements and O'Brien, 1954). Gorbman (1955) has discussed this matter in
some detail and notes that in all these cases the localization of radioiodine is in
scleroprotein. The present experiments indicate that phenylthiourea treatment, if
given over a sufficiently long period, has an inhibitory effect on the binding of
radioiodine here although it does not completely prevent it.

The effects of derivatives of thiourea on the functioning of the thyroid gland
have been widelv studied in mammals and in several amphibians. The histological

168 W. GARDNER LYNN AND JAMES NORMAN DENT

changes seen in the thyroids of the animals studied in our experiments are in agree-
ment with those previously reported and need not be discussed. The autoradio-
graphic analysis of the thyroids of the control and experimental animals also gave
results that would be expected on the basis of what is known of the effects of this
drug. Larvae under continuous treatment with phenylthiourea showed an extremely
low ability to bind IK;1. However, treated larvae recovered this ability very rapidly
after treatment was discontinued. This rapid rate of recovery is in contrast with
the slower rate observed in the pigmented regions and may be indicative of a
difference in the mechanism of iodine binding.

The basis for the accumulation of radioiodine by the thymus gland is not known.
It was first reported by Dent and Hunt (1952), and it is clearly demonstrated in
our material. It is completely inhibited in animals under continuous treatment
with phenylthiourea but cessation of treatment is followed by prompt recovery of
the animal's ability to concentrate iodine. It appears, then, that the binding of
iodine in the thymus is more closely related to that process as it occurs in the
thyroid than as it occurs in the melanophore.

Earlier observations on various vertebrates (see citations in Dent and Hunt,
1954) have all been to the effect that the onset of iodine accumulation by the
thyroid does not occur until discrete follicles make their appearance. It is of
some interest, then, that in the animals studied here iodine concentration began
while the cells of the thyroid rudiment were still arranged in cords at four days
after hatching. Moreover, such animals, when given continuous phenylthiourea
treatment, showed no ability to concentrate I131. Thus the iodine-concentrating
activity of thyroid tissue, and also the ability of phenylthiourea to inhibit this ac-
tivity, are evidenced well before the appearance of follicles or colloid.

SUMMARY

1. Larvae of Hyla versicolor were immersed in solutions of phenylthiourea at
0, 4, and 15 days after hatching. At 4, 24, and 39 days after hatching, I131 was
administered and contact autoradiograms were prepared from serially sectioned
representative specimens.

2. The tadpoles treated with phenylthiourea from the time of hatching became
completely unpigmented. The blanching of the second series was slower and never
complete. The third series became very little lighter during the course of the
investigation. This indicates that the metabolic turnover of melanin goes on at a
decreasing rate as the larvae increase in age.

3. From the autoradiograms, evidence was obtained to confirm earlier findings
of the binding of iodine in pigmented areas, to show that the binding is apparently
not associated with formed melanin, and to support the view that the same enzyme
or enzymes that catalyze melanogenesis can catalyze the binding of iodine (pre-
sumably with tyrosine).

4. The accumulation of iodine by the horny teeth was inhibited to some degree
by phenylthiourea treatment.

5. Accumulation of radioiodine by the thymus gland was confirmed and was
found to be completely inhibited by phenylthiourea treatment.

6. The thyroid rudiment acquires the facility for concentrating iodine even
before follicle formation begins, and at that time it also responds to the inhibitory
action of phenylthiourea.

PHENYLTHIOUREA AND IODINE BINDING 169

LITERATURE CITED

BERNHEIM, F., AND MARY L. C. BERNHEIM, 1942. The action of phenylthiocarbamide on

tyrosinase. /. Biol. Clicin., 145: 213-217.
BLACKSTAD, T. W., 1949. Depigmentation in Rana temporaria tadpoles as a result of methyl-

thiouracil treatment. /. Endocrinol., 6 : 23-27.

BOYD, G., 1955. Autoradiography in biology and medicine. Academic Press, Inc., New York.
DENT, J. N., AND E. L. HUNT, 1952. An autoradiographic study of iodine distribution in

larvae and metamorphosing specimens of Anura. /. Exp. Zool., 121 : 79-97.
DENT, J. N., AND E. L. HUNT, 1954. Radiotracer techniques in embryological research. /.

Cell Coinp. Physiol., 43: 77-101.
DIEKE, SALLY H., 1947. Pigmentation and hair growth in black rats, as modified by the

chronic administration of thiourea, phenylthiourea and alpha-naphthyl thiourea.

Endocrinol., 40: 123-136.
DuBois, K. P., AND WILMA F. ERWAY, 1946. Studies on the mechanism of action of thiourea

and related compounds. II. Inhibition of oxidative enxymes and oxidations catalyzed

by copper. /. Biol. Chan.. 165: 711-721.

FAWCETT, D. M., AND S. KIRKWOOD, 1954. Tyrosine iodinase. /. Biol. Clicin., 209: 249-256.
FRIEDERS, F., 1954. The effects of thyroid-inhibiting drugs on some tropical fish. Catholic

Univ. of America Biol. Studies, 31 : 1-37.
GENNARO, J. F., AND MARGARET M. CLEMENTS, 1956a. The incorporation of I131 in the frog

skin in vitro and in vivo. Anat. Rcc., 124: 294.
GENNARO, J. F., AND MARGARET M. CLEMENTS, 1956b. Iodine concentrating mechanism in

skin of the frog. Fed. Proc., 1 : 72.
GORBMAN, A., 1955. Some aspects of the comparative biochemistry of iodine utilization and the

evolution of thyroidal function. Physiol. Rev., 35 : 336-346.
GORBMAN, A., MARGARET M. CLEMENTS AND R. O'BRIEN, 1954. Utilization of radioiodine by

invertebrates, with special study of several Annelida and Mollusca. /. Exp. Zool.,

127: 75-92.

GRAY, P., 1952. Handbook of basic microtechnique. Blakiston Company, Philadelphia.
JOFTES, D. L., AND S. WARREN, 1956. Terminology. Science, 124: 1155-1156.
KEMP, N. E., 1953. Synthesis of yolk in oocytes of Rana pipiens after induced ovulation. /.

Morph., 92: 487-511.
LERNER, A. B., AND T. B. FITZPATRICK, 1950. Biochemistry of melanin formation. Physiol.

Rev., 30: 91-126.
LYNN, W. G., 1948. The effects of thiourea and phenylthiourea upon the development of

Eleutherodactylus ricordii. Biol. Bull., 94: 1-15.

LYNN, W. G., AND SISTER ALFRED DE MARIE, 1946. The effect of thiouracil upon pigmenta-
tion in the tadpole. Science, 104: 31.
MILLOTT, N., AND W. G. LYNN, 1954. The effect of phenylthiourea on pigmentation by melanin

in the developing frog, Eleutherodactylus inartinicensis. Quart. J. Micros. Sci., 95:

17-24.
PASCHKIS, K. E., A. CANTAROW, W. M. HART AND A. E. RAKOFF, 1944. Inhibitory action

of thiouracil, thiocarbamide and other compounds on melanin formation by tyrosinase.

Proc. Soc. Exp. Biol. Mcd., 57: 37-39.
PITT-RIVERS, ROSALIND, 1950. Mode of action of antithyroid compounds. Phvsiol. Rev., 30:

194-205.

RICHTER, C. P., AND K. H. CLisBY, 1941. Greying of hair produced by ingestion of phenyl-
thiocarbamide. Proc. Soc. Exp. Biol. Med., 48 : 684-687.
ROCHE, J., AND R. MICHEL, 1955. Nature, biosynthesis and metabolism of thyroid hormones.

Physiol. Rev., 35: 583-610.
SWAN, E. F., 1950. Calcareous tube secreting glands of the serpulid polychaetes. /. Morph.,

86: 285-314.
TAYLOR, A. C., AND J. J. KOLLROS, 1946. Stages in the normal development of Rana pipiens

larvae. Anat. Rcc., 94: 7-23.
WHEELER, BERNICE M., 1950. Halogen metabolism of Drosophila gibbcrosa. Part I. Iodine

metabolism studied by means of I131. /. Exp. Zool., 115: 83-107.

ENDOPARASITIC POLYCHAETOUS ANNELIDS OF THE FAMILY
ARABELLIDAE WITH DESCRIPTIONS OF NEW SPECIES1

MARIAN H. PETTIBONE

Department of Zoology, University of Neiv Hampshire, Durham, New Hampshire

Among the polychaetes, which for the most part are free-living, crawling,
burrowing, and tube-dwelling, commensalism is rather common but internal para-
sitism is rare indeed. Among the relatively few cases that have been reported are
some lumbrinerid-like polychaetes belonging to the superfamily Eunicea, which
includes the families Eunicidae, Onuphidae, Lumbrineridae, Arabellidae, Lysareti-
dae, and Dorvilleidae (sometimes considered as subfamilies belonging to the family
Eunicidae). Some of the parasitic euniceans invade other members of the same
superfamily and may attain an enormous size in proportion to the host. All of
the known lumbrinerid-like parasites belong to, or show affinities to, the family
Arabellidae as defined by Hartman (1944).

In connection with a study of polychaete material from various sources, in-
cluding that in the United States National Museum, some arabellids were found
living as endoparasites in other polychaetes of the related family Onuphidae.
Two new species are described herein, the types of which are deposited in the
United States National Museum. Some small specimens of a third species, living
parasitically in the onuphid, Diopatra, are thought to be the young stages of the
arabellid, Notocirrns spinifents (Moore). Since this interesting type of parasitism
is not widely known and has not received the attention it no doubt deserves, the
relatively few known cases of lumbrinerid-like species living in other polychaetes
and echiuroids are reviewed and the chief characteristics of the Eunicea and Arabel-
lidae to which the parasites belong are summarized.

Superfamily EUNICEA

All the members of the Eunicea are equipped with a complex series of strong,
dark, chitinous or horny jaws. The pharynx is capable of protrusion and is pro-
vided with a pair of ventral plates, called mandibles, and a more dorsal bilaterally
arranged series of plates, called maxillae. The prostomium is distinct, with or
without eyes or appendages. Typically the first two segments are apodous. The
parapodia are essentially uniramous, the upper lobe or notopodium represented at
most by a few embedded notoacicula and a rudimentary papilla-like lobe. The
members of the Eunicea vary from those of minute size with a moderate number of
segments to some very large ones with very numerous segments, some of which are
among the largest of the polychaetes. They are essentially free-living, predaceous,
and carnivorous. They secrete abundant mucus, which may aid in burrowing or
forming temporary or more or less permanent tubes.

1 This study was aided by a grant from the National Science Foundation (NSF-G 2012).

170

ENDOPARASITIC POLYCHAETOUS ANNELIDS

171

Family ARABELLIDAE Hartman

The arabellids show a superficial resemblance to the members of the Lumbri-
neridae, differing from them in setal and pharyngeal characters. They have the
body elongate, cylindrical, of nearly uniform width, and tapering slightly anteriorly
and posteriorly. The prostomium is reduced to a simple, conical or flattened
spatulate lobe, without appendages, with or without eyespots on the posterior
margin. The first two segments are distinct, apodous, and without appendages.
The parapodia are essentially uniramous ; the notopodia sometimes represented by a
minute papillar lobe (sometimes referred to as a reduced dorsal cirrus) with em-
bedded notoacicula ; the neuropodia are unequally bilobed, with shorter rounded and
longer digitiform postsetal lobes. The neurosetae are simple (not compound),
limbate, and taper to fine tips ; in addition they sometimes have projecting thick

TABLE I

Parasite

Host and Distribution

References

Notocirrus sp. (young)

Eunicidae: Marphysa sanguinea (Montagu). Mediter-
ranean

Koch, 1847. Ehlers, 1868,
p. 364. See below w*

Nolocirrus Jspiniferus
(Moore) (young)

Onuphidae: Diopatra cuprea (Bosc). Woods Hole region,

Massachusetts

Allen, 1952. See below

Haematodeptes terebellidis
Wiren

Terebellidae: Terebellides slroemii Sars. Off Sweden,
130 meters

Wiren, 1886

Labrorostratus parasiticus
Saint-Joseph

Syllidae: Odontosyllis ctenostoma Claparede, Syllis pro-
"lifera Krohn, Eusyllis monilicornis Malmgren, Piono-
syllis lamelligera Saint-Joseph, Grubea clavata (Clapa-
rede)

Saint-Joseph, 1888. Caul-
lery and Mesnil, 1916.
Fauvel, 1923, p. 440

Oligognathus bonelliae
Spengel

Echiuroidea: Bonellia viridis Rolando. Mediterranean

Spengel, 1882. Fauvel, 1923,
p. 442

Oligognathus parasiticus
Cerruti

Spionidae : Spio mecznikowianus Claparede. Mediter-
ranean

Cerruti, 1909. Fauvel, 1923,
p. 442

Drilonereis parasiticus
(Caullery)

Terebellidae: Genus and species? Near Timor, Dutch
East Indies, 73 meters

Caullery, 1914. See below

Drilonereis forcipes
(Hartman)

Eunicidae: Eunice sp., possibly E. antennata Savigny.
San Benito Island, Lower California, 66-81 fathoms

Hartman, 1944. See below

Drilonereis benedicti n. sp.

Onuphidae: Onuphis magna (Webster). Tampa Bay,
Florida, 12 fms.

See below

Drilonereis caulleryi n. sp.

Onuphidae: Onuphis (Nothria) conchylega Sars. Off
Massachusetts to off Virginia, 101-317 fms.

See below

acicular setae or acicula (without hooded hooks as in the Lumbrineridae). The
parapodia have no dorsal or ventral cirri or branchiae. The eversible proboscis is
equipped with strong, chitinous, black jaw pieces: usually with a pair of ventral,
flat plates, the mandibles; with 4 or 5 pairs (may be fewer in parasitic forms) of
more dorsal maxillae arranged in parallel rows, with a pair of long filiform carriers
to which a shorter median unpaired piece is attached on the ventral side. The
arabellids are essentially a burrowing, predaceous, carnivorous group. They bur-
row readily but rather slowly in sand or mud. They secrete a good deal of mucus,
which probably serves to lubricate the burrow.

Some of the arabellids are parasitic in other polychaetes (eunicids, onuphids,
syllids, terebellids) and in echiuroids (Bonellia), living inside the body cavity or
vascular body wall or even in the vascular system of the host, at least during their

172

MARIAN H. PETTIBONE

early developmental stages. The eight previously reported parasitic arabellids,
along with the two species described herein, are summarized in Table I and in the
illustrated Key to the genera and species.

ILLUSTRATED KEY TO THE GENERA AND SPECIES OF
PARASITIC ARABELLIDAE

[Figures are copied from the original descriptions, a, anterior end; b, parapodium ; c, setae;

d, mandibles; e, maxillae and maxillary carriers; f, maxillae]
A1. Numerous specimens in single host.

B1. Parasitic in body cavity of Marpliysa sanguined . . . Notocirrus sp. (young).

(See below under TV. spiniferus.)
B2. Parasitic in body cavity and vascular body wall of Diopatra cuprea

Notocirrus fspinijcrus (young). (See Figs. 4, g and 5.)
A2. One parasite per host.

C1. One to three pairs of rudimentary maxillae, with single elongate rodlike maxillary

carrier. Mandibles present.

D1. Maxillae a single pair of curved rods, each curved tooth at base. Setae and acicula
not projecting from lobe. Mandibles paired, triangular. Prostomium without
eyes. (Up to 25 mm. long, about 200 segments. Colorless.) Parasitic in peri-
intestinal blood sinus of Tcrebcllidcs stroemii. . . . Genus Haematocleptes Wiren

H. tercbcllidis Wiren

D2. Two or three pairs of maxillae. Setae projecting from parapodial lobe.

E1. Maxillae two pairs of very small denticled pieces. Mandibles wing-shaped,
each with a spine. Setae all of one kind, limbate, smooth, tapering to long
flexible tips. Prostomium with four eyes in transverse row. (Up to 70
segments.) Parasitic in body cavity of syllids, Odontosyllis ctenostoma,
Syllis prolijera, Eusyllis monilicornis, Pionosyllis lamelligera, Grubea clavata.
Also found free among calcareous algae, Lithothamnion.
Genus Labrorostratus Saint-Joseph L. parasiticus Saint-Joseph

ENDOPARASITIC POLYCHAETOUS ANNELIDS

173

E2 Maxillae three pairs recurved unidentate hooks. Mandibles U-shaped, with
two wing-like pieces or rods united by transverse band. . . . Genus
Oligognatluts Spengel

F1. Setae of one kind, simple, arched, limbate, striated. Prostomium with four
eyes. (Up to 100 mm. long, more than 200 segments. Bright orange
yellow.) Parasitic in body cavity of echiuroid, Bonellia riridis.
O. bonclliae Spengel

O

s

F2. Setae of two kinds: capillary, flexible and stouter, wide, tapering to fine
tips. Prostomium without eyes. (Up to 8 mm. long. 50 segments.
Colorless, transparent.) Parasitic in body cavity of spionid, Spio
mecznikoivianus.
O. parasiticits Cerruti

MARIAN H. PETTIBONE

C-. Four pairs of well developed maxillae, with pair of elongate rodlike maxillary carriers
and shorter unpaired piece (basal or maxillae I large, strong, hooked, forceps-like ;
maxillae II rectangular plates, sometimes denticled ; maxillae III and IV each a
single strong thorn-like tooth). Mandibles absent. Parapodia with heavy acicula
or acicular setae, the tips of which usually protrude. Prostomium without eyes.

Genus Drilonci'ds Claparede (includes Labidognathus Caullery ; see below).
G1. Parasitic in peri-intestinal blood sinus of terebellid (unidentified). With bilimbate

setae and single stout acicular seta extending out of parapodial lobe. Maxillae

II edentate ( ?, incompletely observed). (More than 100 segments). . . . D.

parasiticus (Caullery)

G2. Parasitic in body cavity of branchial fragment of eunicid, Eunice sp., possibly
E. antcnnata. With bilimbate setae and single stout yellow aciculum, the latter
not extending out of parapodial lobe. Maxillae II flat plates, practically without
teeth. (More than 140 segments, more than 30 mm. long.). . . . D. forcipcs
(Hartman)

ENDOPARASITIC POLYCHAETOUS ANNELIDS 175

G3. Parasitic in body cavity of onuphids. Maxillae II rectangular plates, each with
four distinct teeth.

H1. Parasitic in branchial fragment of Onuphis magna. Without setae or acicular
setae visible externally (even in a specimen of more than 1200 crowded
segments and more than 240 mm. long). . . . D. benedicti n. sp. See Fig.
1, a-g.

H2. Parasitic in anterior fragment of Onuphis (Nothria) conchylega. With
limbate setae and single stout acicular seta extending from lobe (except in
smaller specimens; up to 400 segments, 110 mm. long). . . . D. caulleryi
n. sp. See Fig. 2, a-o.

The parasitic arabellids may be separated into two main groups. The first in-
cludes those species of which numerous specimens are found in a single host.
Within a single host, they may be found in varying stages of development, from
small specimens, with few segments, no eyes, and no jaws, to larger ones, with
numerous segments, eyes, developing jaws, parapodia, and setae. The two re-
ported cases are thought to be the young stages of species of Notocirrus. When
first observed by Koch (1847), they were reported as a lumbrinerid-like stage of
the young of the presumably viviparous eunicid host, Marphysa sanguinea; this
species lives in a loose mucous tube, irregularly encrusted with rocks, shells, and
such. Similar forms have since been found in the onuphid, Diopatra cuprea,
which lives in a parchment-like tube, one end of which is buried in the sand or mud,
the other end sticking out of the substratum and covered with shells, plant debris,
etc. The parasites evidently penetrate the host at an early stage, just how early
and the mechanism for penetration being unknown. They grow and develop within
the host to an advanced stage, when they evidently leave the host and perhaps con-
tinue to grow and mature, taking on a free-living existence.

The other type of parasitism is the condition where a single parasite is found in
a host and where the parasite may attain enormous dimensions in comparison to
the host, becoming nearly as large or larger than the host or host fragment. They
evidently penetrate the host at an early stage also and grow to an advanced stage,
perhaps even completing their growth within the host. The parasite may be com-
pletely enclosed in the host or part of it may protrude. Perhaps it matures after
leaving the host, as sex products have not been observed in the parasites found in
the host.

The parasitic arabellids appear to be rare. Most of the records of the different
species have been based on single specimens. In a long study from 1875 to 1888 at
Dinard, Saint- Joseph (1888) observed only 14 examples of Labrorostratus para-
siticus in the body cavities of several species of syllids. After numerous years of
microscopical examination of numerous syllids, Caullery and Mesnil (1916) ob-
served only a single parasite of the same species. Considering that the syllids are
small and somewhat transparent and that the parasites may be as much as three-
fourths of the length of the host, the presence of the parasites would probably be

176 MARIAN H. PETTIBONE

noted. Where the host is larger and opaque, the parasites would be observed only
accidentally in fragmented or dissected specimens.

Four of the parasitic species belong to the genus Drilonereis, which also includes
free-living species. The maxillae and maxillary carriers are well developed. In
D. caulleryi n. sp. the parapodial armature develops gradually ; first the heavy
aciculum appears, then the setae, only the tips of which project at first, then the
setae project further and the tips of the heavy acicula finally protrude. In D.
benedicti n. sp. the acicula and setae are rudimentary and do not project from the
parapodial lobes, even in a specimen of more than 1200 crowded segments. The
parasitic genera, Oligognathus Spengel, Labrorostratus Saint-Joseph, and Haemato-
cleptes Wiren, have the maxillae more rudimentary than in Drilonereis, with a
single elongate rodlike maxillary carrier, darker toward the outside. Haetnatocleptes
terebellidis shows the most rudimentary condition, having only a single pair of
maxillae and the setae and acicula not projecting from the parapodial lobe.

Genus Drilonereis Claparede, 1870

Type (by original designation) : D. filum (Claparede, 1868).

Labidognathus Caullery 1914; type (by monotypy) : L. parasiticus Caullery, 1914.

Diagnosis. Prostomium conical to spatulate, flattened ventrally, usually with
central depression dorsally, without eyes or appendages. First two segments
apodous and achaetous, first sometimes partially fused dorsally to prostomium.
Parapodia with dorsal lobe or notopodium usually small, rudimentary, with a
few embedded notopodial acicula ; neuropodium with two unequal lips, supported
by acicula. Setae all simple, of two kinds: (1) bilimbate or winged, tapering to
fine tips, smooth or faintly striated (not denticled) ; (2) 1-2 stout acicular setae
with tips protruding from parapodial lobe; (in some parasitic forms, setae may be
rudimentary, not extending out of parapodial lobes). Pharynx with mandibles
or lower jaws rudimentary or absent; maxillae or upper jaws 4—5 pairs, sym-
metrical, dark, chitinous, supported by a pair of long slender maxillary carriers and
a shorter unpaired piece ; basal maxillae I large, heavy, falcate pincers or forceps ;
maxillae II rectangular plates, usually denticled; maxillae III and IV with one to
few teeth ; maxillae V rudimentary or absent.

Remarks. Labidognathus is herein referred to Drilonereis. The type species
of the former, L. parasiticus Caullery (1914, p. 490), was found living as a parasite
in a terebellid (not yet described) near Timor, Dutch East Indies; the parasite was
found in the peri-intestinal blood sinus, coiled in a complicated manner around the
intestine of the host. According to Caullery, both the host and parasite were in
rather poor condition; the jaw apparatus of the parasite was not studied completely.
Hartman (1944, p. 180) noted the affinities of Labidognathus with Drilonereis and
described a new species, L. jorcipes, found in the body cavity of a fragment of a
species of Eunice from San Benito Island, Mexico. In addition two new species
are described below. Because of the scarcity of material of the parasites, it is
difficult to work out the developmental stages. As indicated below for the four
specimens of Drilonereis caulleryi n. sp., parasitic in Onuphis conchylcga, there are
differences in the development of the jaws and parapodia in different stages of
growth. The four parasitic species of Drilonereis show essentially the characters

ENDOPARASITIC POLYCHAETOUS ANNELIDS

177

of the genus. They all lack mandibles and have four pairs of maxillae, of which
maxillae I are stout falcate hooks and maxillae III and IV each a single stout
conical hook. The parasites were found singly, one to a host. The hosts, at least
when collected, were either anterior or middle fragments, with the parasites pro-
truding and exposed in part.

Drilonereis benedicti n. sp.
Fig. 1, A-G

The species is known from a single specimen, incomplete posteriorly (U.S.N.M.
No. 28637), found in a fragment of 18 segments from the branchial region of

I

6
E
o

•

o

E
rO
O

FIGURE 1. Drilonereis benedicti n. sp. : A, Lateral view anterior end; B, dorsal view
anterior end; C, left parapodium from setiger 10, anterior view; D, same from about setiger 300;
E, right parapodium from about setiger 600, anterior view ; F, four pairs maxillae and maxillary
carriers, dorsal view (more ventral unpaired piece not shown) ; G, maxillae II, enlarged.

Onuphis magna (Andrews), North Channel into Tampa Bay, Florida, 12 fms.,
Fish Hawk Sta. 7108, 1901, Dr. J. E. Benedict, collector. The parapodia of the host
fragment were compared with those of an incomplete specimen of 0. magna found at
the same station. The middle part of the parasitic Drilonereis extended through
the body cavity of the host fragment, the greater part of the anterior and posterior

178

MARIAN H. PETTIBONE

N

FIGURE 2.

ENDOPARASITIC POLYCHAETOUS ANNELIDS 179

ends of the parasite being exposed. It is named for the collector, who evidently
put it aside to be worked up later.

Description. Length of incomplete specimen 240 mm., greatest width 1.5 mm.,
segments more than 1200. Body cylindrical, with segments slightly longer an-
teriorly becoming very short and crowded posteriorly, colorless (in alcohol), shiny
iridescent anteriorly, dull posteriorly. Prostomium (Fig. 1, A, B) conical, rounded
anteriorly, flattened ventrally, with a longitudinally depressed area mid-dorsally.
First two segments achaetous, subequal to the following, first with mid-dorsal
nuchal notch. Parapodia (Fig. 1, C-E) similar along length of body, small, un-
equally bilobed, with shorter rounded and longer thick digitiform lobes. No setae
exposed external to lobes; internally few notopodial acicula extending into base
of rudimentary low notopodial lobe and larger group of neuropodial acicula and
setae with tips extending into the short neuropodial lobe ; one of the acicular group
is much stouter, probably corresponding to the stout acicular seta characteristic of
Drilonereis. Proboscis without mandibles and with four pairs maxillae. Maxillae
(Fig. 1, F, G) well developed, dark, with a pair of long, dark filiform maxillary car-
riers and short oval unpaired piece, dark anteriorly, light amber-colored posteriorly ;
basal maxillae I stout, falcate, hooked; maxillae II rectangular plates, each with
four distinct teeth and slight indication of a fifth; maxillae III and IV each a single,
large, thorn-like tooth.

Remarks. D. benedicti differs from the other parasitic species of Drilonereis in
the complete absence of exposed setae, even in a specimen of more than 1200 seg-
ments. The host normally lives in a parchment-like tube. Perhaps the host frag-
ment moves along in the tube carrying the parasite with it. Possibly the parasite
at this stage feeds for itself, as the anterior and posterior ends were exposed.
There is also the possibility that the host fragmented at the time it was collected.

Drilonereis caulleryi n. sp.
Fig. 2, A-0

The species is represented by four specimens, each of which was found living
parasitically in anterior fragments of Onnphis (Nothria) conchylega Sars, collected
by the Fish Haivk and Albatross off Martha's Vineyard, Massachusetts, and off
Cape Henry, Virginia, from 1880 to 1883. Two of the four specimens are small,
showing different developmental stages. The specimen designated as the type
(U.S.N.M. No. 12867) is complete and was found coiled inside a host of about 40
anterior segments. The largest paratype (U.S.N.M. No. 8987) consists of an
anterior end of 25 mm. and a posterior end of 10 mm. protruding from the posterior

FIGURE 2. Drilonereis caulleryi n. sp. : A, Habit sketch of parasite and host (2) ; B, dorsal
view anterior end (1) ; C, dorsal view posterior end (1) ; D, lateral view anterior end, proboscis
partially extended (2) ; E, ventral view same ; F, left parapodium from setiger 20, anterior
view (1) ; G, right parapodium from middle of body, anterior view (1) ; H, limbate setae
from same ; I, acicular seta from same ; J, four pairs maxillae and maxillary carriers, dorsal view
(more ventral unpaired piece not shown) (2) ; K, right maxillae II-IV, enlarged; L, dorsal view
anterior end of slightly coiled smaller paratype, proboscis partially extended (3) ; M, parapodium
from setiger 10 of same; N, slightly lateral view anterior end of smallest paratype (4) ; O, para-
podium from setiger 12 of same. (1) type specimen; (2) largest paratype specimen; (3)
smaller paratype specimen; (4) smallest paratype specimen.

180 MARIAN H. PETTIBONE

end of the host fragment of 18 segments, the middle part of the parasite being
inside the host (Fig. 2, A). A smaller paratype (U.S.N.M. No. 28636) occupied
setigers 8-18 of an anterior fragment of a host of 18 segments ; the posterior end
of the parasite protruded from the posterior end of the host fragment. A very small
paratype (U.S.N.M. No. 28635) was found in a host fragment of 17 segments; the
anterior end of the parasite was sticking out dorsally between setigers 15 and 16,
the posterior end was protruding ventrally between the same segments. The
species is named for Dr. Maurice Caullery, who described the first parasitic
drilonereid (as Labidognathus} .

Description. Length up to 110 mm., greatest width up to 1 mm., segments
up to 400 or more. Body cylindrical, shiny iridescent. Prostomium (Fig. 2, B,
D, E) conical, rounded anteriorly, flattened ventrally, with a mid-dorsal depressed
area. First two segments apodous and achaetous, subequal to the following,
first with mid-dorsal nuchal notch. Anal end (Fig. 2, C) short, cylindrical, tapering
to pair of short bulbous lobes (no distinct anal cirri). Parapodia (Fig. 2, F, G)
similar along length of body, short, unequally bilobed, with shorter rounded and
longer, thick digitiform lobes. Two larger specimens with two kinds of setae
projecting from parapodial lobe : 4—5 bilimbate, straight and curved setae, tapering
to slender tips, faintly striated (not denticled ; Fig. 2, H) ; also single yellowish
stout, pointed acicular seta (Fig. 2, I) ; with additional internal neuropodial acicula
as well as few notopodial acicula, the tips of which extend into short bulbous rudi-
mentary notopodium ; posterior end of body with tips of limbate setae only project-
ing from parapodial lobes. Smaller specimen (12+ mm. long, 0.6 mm. wide) with
tips of limbate setae only extending out of lobe, stout acicular seta being visible
inside lobe (Fig. 2, 1, M). Smallest specimen (5 + mm. long, 0.3 mm. wide) with
no setae projecting, single stout acicular seta being visible inside parapodial lobe
(Fig. 2, N, O). Proboscis, when partially extended, appearing as three bulbous
lobes (Fig. 3, D, E) ; no mandibles; maxillae four pairs, well developed, dark, with
pair of long filiform carriers and shorter wide oval unpaired piece, dark anteriorly,
lighter more posteriorly (Fig. 2, J, K) ; maxillae I stout, falcate, forceps-like; maxil-
lae II rectangular plates, each with four distinct teeth ; maxillae III and IV each a
single large hooked thorn-like tooth. In smaller specimen, maxillary forceps and
slender carriers visible through transparent body wall (Fig. 2, L). In smallest
specimen (Fig. 2, N) only maxillary carriers visible.

Remarks. D. caulleryi differs from the other parasitic species of Driloncreis
as indicated in the key above. As shown in the three developmental stages found
in the four specimens, the maxillary carriers develop first, then the stout maxillary
forceps ; in the parapodia, the stout acicular seta appears first, then the limbate setae,
the tips of which may protrude, the stout acicular seta not protruding (this would
correspond to the condition described for D. forcipes by Hartman) ; finally the
limbate setae protrude further and the tips of the stout acicular setae protrude.
The host, Onuphis conchylega Sars, occupies a flat parchment-like tube encrusted
with flattened pieces of rocks and shells. All four host specimens are anterior
fragments. The parasite may be completely enclosed in the host fragment or a
portion of the parasite may stick out, revealing its presence. The parasite perhaps
gets into the host by encouraging it to fragment; none of the fragments showed
any signs of regeneration, although 0. conchylega fragments and regenerates read-
ily.

ENDOPARASITIC POLYCHAETOUS ANNELIDS 181

Material examined. Type: off Martha's Vineyard, Massachusetts, 39° 53' N.,
69° 47' W., 317 fms., soft green mud, Fish Hawk Sta. 1096, 1882. Paratypes:
40° 02' N., 70° 23' W., 115 fms., mud, fine sand, Fish Hawk Sta. 871, 1880; 40°
02' N., 70° 37' W., 101 fms., grey mud, fine sand, Fish Hawk Sta. 1108, 1882;
off Cape Henry, Virginia, 37° 19' N., 74° 26' W., 102 fms., green mud, shell,
Albatross Sta. 2004, 1883.

Distribution. Atlantic, off Massachusetts to off Virginia. 101 to 317 fathoms.

Genus Notocirrus Schmarda, 1861, emend. Ehlers, 1868
Type (designated by Ehlers, 1868, p. 406) : N. chilensis Schmarda, 1861.

Diagnosis. Prostomium conical, without appendages, usually with 4 eyespots.
First two segments apodous and achaetous. Parapodia with dorsal lobe or noto-
podium represented by small rudimentary lobe and few notopodial acicula; neuro-
podium unequally bilobed, supported by stout acicula, the tips of which project
(except in young developing stages). Setae all simple, bilimbate or winged,,
tapering to fine tips, striated and finely to coarsely denticled along limbate border.
Pharynx with pair of wing-shaped, dark chitinous mandibles or lower jaws;
maxillae or upper jaws 4—5 pairs, dark, chitinous, denticled, supported by pair of
long filiform maxillary carriers and shorter unpaired piece; maxillae I and II
asymmetrical, maxillae I dentate throughout entire length or only slightly falcate
or with short hook distally.

Remarks. Notocirrus has affinities with Arabella, having similar prostomia and
pharyngeal jaws ; both have limbate setae with denticled border. Notocirrus also
has affinities with Drilonereis, both having stout acicula or acicular setae which
project from the parapodial lobes. A species of Notocirrus is herein reported to be
parasitic in an onuphid (Diopatra) during its early development.

Notocirrus spiniferus (Moore, 1906)

Figs. 3—5
Arabella spinifera Moore, 1906, p. 501, pi. 19, figs. 1-7.

The species was described from a single specimen which was dredged on muddy
bottom in the middle of Buzzards Bay, Massachusetts. No additional records have
been reported. In working over a good deal of material collected in New England
and vicinity, four additional free-living specimens were found. The species appears
to be rare but, due to its superficial resemblance to the more common Arabella
iricolor, it may be confused with that species in collections. The four specimens
were obtained from the following sources : Buzzards Bay, Massachusetts, 1956,
H. Sanders, collector; off Cape Henry, Virginia, Chesapeake Bay, 9 fms., shelly
and sand, Fish Hawk Sta. 8838, 1920 ; Isle of Wight Bay above Ocean City, Mary-
land, 1953, S. McDowell, collector; Beaufort, North Carolina, 1951, E. Cole,
collector.

In addition two of the specimens found living parasitically in the body cavity
of Diopatra cuprea (Bosc), collected by M. Jean Allen at Hadley Harbor,
Nonamesset Island, Woods Hole region, Massachusetts, were examined. They ap-

182

MARIAN H. PETTIBONE

FIGURE 3. Notocirrus spiniferns. Drawn from specimen from Buzzards Bay: A, Dorsal
view anterior end; B, same, ventral view; C, parapodium from setiger 10, posterior view; D,
limbate seta from same; E, limbate seta from setiger 100; F, tip of one of acicula from same;
G, mandibles, ventral view ; H, left maxillae, I-IV, dorsal view ; I, right maxillae, I-IV, dorsal
view; J, left maxillae spread apart; K, right maxillae spread apart.

pear to be the young of a Notocirrus, possibly that of N. spinijerus. In a note
recording the find of more than 50 parasitic specimens in a single specimen of
Diopatra, Allen (1952) indicated that they might be the young of Arabella iricolor
but the parapodia differ from that species as indicated below.

ENDOPARASITIC POLYCHAETOUS ANNELIDS

183

Description of specimens found free-living. Length 40-110+ mm., width 1-4
mm., segments 140-220+ . Body cylindrical, tapering slightly anteriorly and
posteriorly, stiff, wiry. Prostomium (Figs. 3, A, B; 4, A, B) subconical, rounded
anteriorly, slightly depressed dorsoventrally but not greatly flattened as in Drilo-
nereis; a pair of faint longitudinal grooves ventrally and four eyes in transverse row
at posterodorsal border, rather than two eyes as reported by Moore. First two seg-
ments achaetous and apodous, first with mid-dorsal nuchal notch. Parapodia
(Figs. 3, C; 4, D) prominent, similar along length of body, with small but distinct
notopodium supported internally by few notoacicula; neuropodium bilobed, with

FIGURE 4. Notocirrus spiniferns. Drawn from specimen from Isle of Wight Bay (A-F) :
A, Dorsal view anterior end ; B, lateral view same ; C, dorsal view posterior end ; D, parapodium
from middle of body ; E, mandibles, ventral view ; F, five pairs maxillae and maxillary carriers,
dorsal view (more ventral unpaired piece not shown). G, Dorsal view anterior end of small
specimen of Notocirrus fspiniferus, living parasitically in Diopatra cuprea, Woods Hole region.

short rounded setal lobe and longer, prominent digitiform postsetal lobe (some-
times referred to as ventral cirrus or cirriform branchial organ), within which is a
vascular loop. Setal lobe with 1-3, usually 2, stout, deep yellow acicula, the tips
of which project out of lobe (thus differing from Arabella, which has no stout
projecting acicula) ; acicular tips bluntly pointed, tapered abruptly to short fine tips,
or sometimes obviously broken (Fig. 3, F). Setal lobe with 4-8 bilimbate, doubly
curved setae with fine tips, with wings wide, striated and finely denticled along
border, sometimes with a few coarser denticles near base of wing (Fig. 3, D, E).
Anal end (Fig. 4, C) tapered to pair of short bulbous lobes, each with very short
rudimentary anal cirrus.

184

MARIAN H. PETTIBONE

FIGURE 5.

ENDOPARASITIC POLYCHAETOUS ANNELIDS 185

Proboscis, when partially extended, appears as bulbous bilobed tongue with
pair of lateral lobes. Ventral mandibles (Figs. 3, G; 4, E) well developed, brown
or black, wing-shaped, sometimes with exposed white tips. Five pairs of dorsal
jaws or maxillae (Figs. 3, H-K; 4, F), with pair of long slender carriers, thickened
distally and subdistally, and shorter unpaired piece ; fifth pair, consisting of single
tooth, easily confused with the fourth, to which it is closely allied. First two or
basal pairs of maxillae asymmetrical. Right maxilla I longer than left, with up to
10 denticles along length of inner border and without distal hook; left maxilla
I with up to six basal teeth and distinct distal hook. (Moore, in figure of the type,
showed distal hooks on both first maxillae and fewer teeth.) Left maxilla II much
larger than right, completely overlapping left maxilla I and extending down to
maxillary carriers, with up to 12 or 13 teeth; right maxilla II partially overlapping
right maxilla I, with up to 8 or 10 teeth. Maxillae III to V symmetrical, III each
with 6 teeth, IV each with 5 teeth, and V each a single tooth, without basal ex-
tension as in other maxillae. (The last may appear to blend in with maxillae IV
and this may have led to Moore's count of only four pairs of maxillae.) Color
(in alcohol) yellowish to brownish, iridescent.

Description of two young specimens of Notocirrus ?spiniferus, living parasiti-
cally in Diopatra cuprea. The larger parasitic specimen is 25 mm. long, 1.5 mm.
wide, and consists of almost 200 segments. The smaller specimen is 12 mm. long,
0.5 mm. wide, and contains about 150 segments. Prostomium of smaller one
(Fig. 4, G) conical, bullet-shaped, that of larger one (Fig. 5, A-C) more elongated,
bulbous basally; both show four eyes in transverse row. Parapodia of smaller
specimen showing characteristic bilobed form, without setae projecting except for
first two setigerous segments in which a single seta projects; each of setal lobes
provided with stout aciculum (appears dark basally). In larger specimen, para-
podia of anterior region (Fig. 5, E) with four limbate setae and single stout aciculum ;
those of middle region (Fig. 5, H) with two limbate setae and tip only of a third
one projecting; those of posterior region (Fig. 5, K) with single limbate seta and
tip of a second one projecting; notopodia small but distinct, with tip of notaciculum
extending into lobe. Limbate setae with long fine tip, curled backward, distinctly
denticled along limbate border (Fig. 5, F, I) ; stout projecting acicula with fine short
tips (Fig. 5, G, J). Thus parapodia essentially as in larger free-living specimens of
Notocirrus spiniferus except for fewer acicula and setae. Anal end of small speci-
men tapering gradually to cylindrical posterior end, without distinct anal cirri ; that
of larger specimen (Fig. 5, D) essentially as in N. spiniferus. Pharynx, when
partially extended, appears as three-lobed structure, the middle lobe rounded,
tongue-like, consisting of pair of lobes (Fig. 5, B). Jaw parts of smaller specimen
(Fig. 4, G) not dissected but darker paired mandibles and maxillary carriers visible
through the somewhat transparent body wall, maxillary carriers appearing as single
elongated rod. Jaw parts of larger specimen, when dissected out, showing well

FIGURE 5. Young Notocirrus fspiniferus, parasite taken from body cavity of Diopatra
cuprea, Woods Hole region : A, Laterodorsal view anterior end ; B, ventral view same ; C, lateral
view same; D, dorsal view posterior end; E, parapodium from setiger 10; F, limbate seta from
same; G, aciculum with projecting tip from same; H, parapodium from middle of body; I,
limbate seta from same; J, aciculum with projecting tip from same; K, parapodium from
posterior part of body; L, mandibles, ventral view; M, developing maxillary carriers and
maxillae, dorsal view.

186 MARIAN H. PETTIBONE

developed mandibles (Fig. 5, L), and incompletely developed maxillae (Fig. 5, M)
with elongated rodlike maxillary carriers (appearing as single rod but darker toward
the outside), and developing denticled maxillae, indicated by slightly darker amber-
colored areas on walls of pharynx.

Remarks. The smallest parasites reported by Allen were composed of seven
segments ; some specimens of 30 segments showed no eyes or setae ; the largest
specimen reported was 50 mm. long and was composed of about 200 segments.
These parasites had not emerged naturally from the host. Thus they live a para-
sitic life for a considerable period. The smaller specimens were in the body wall
of the host near the parietal blood vessels, the larger ones were free in the body
cavity. A few large parasites were observed emerging from the body cavity of
Diopatra by Dr. Frank Brown (Allen, 1952), but the lengths and the developmental
stages of these specimens were not indicated. It may be that the parasites remain
in Diopatra until the jaw parts are completely developed; they were not completely
developed in the largest specimen I was able to examine. It is unknown how the
parasites get into the host. Perhaps the eggs of Notocirrus are laid and fertilized
within the tube of Diopatra. Notocirrus, being a burrowing form, could enter the
parchment-like tube of Diopatra on the buried end which, as far as has been ob-
served, is open. In some way, the fertilized eggs or young at a very early stage
get into the body cavity of Diopatra. The posterior end of Diopatra is soft and
flaccid and fragments easily; perhaps the young are able to bore into the broken
fragmented end of the host. Diopatra regenerates readily also. One host, found
by Allen, contained about 30 small parasites, composed of from 7 to approximately
30 segments ; another host contained over 50 parasites of varying sizes, some up to
50 mm. in length.

In this connection, it may be of interest to mention the observation made by
Koch (1847) of filamentous lumbrinerid-like forms crawling out of a broken
truncated posterior end of a strongly contracting specimen of Marphysa sanguinea,
which was dredged at considerable depth in the Mediterranean. On further exami-
nation of the Marphysa, he found numerous young specimens in the body cavity,
in various stages of development ; the smallest were small roundish microscopic
forms with only slight indication of a few segments and without eyes ; a more ad-
vanced stage of 25-30 segments showed a distinct prostomium with 2 eyes, para-
podia with stout aciculum only, and jaw apparatus in early stages of development;
a still more advanced stage of 50-100 segments showed a distinct prostomium with
4 eyes in a transverse line, parapodia with 2 stout acicula and a few setae confined to
the parapodia; a still later stage of 100-120 segments showed the parapodia with a
short rounded setal lobe and a longer postsetal lobe, with stout dark yellow acicula,
the tips of which appear from Koch's figure to project, as in Notocirrus or Drilo-
nereis, and the jaw apparatus well formed (probably not completely formed).
Koch thought that the specimens he observed were the young stages of a viviparous
Marphysa sanguinea and that during its development, the young pass through a
lumbrinerid-like stage. Ehlers (1868, p. 364), commenting on the observation
of Koch, indicated that Koch had more likely observed parasitic forms of a
lumbrinerid-like species living in M. sanguinea and that the latter was not vivi-
parous; he stated that it was unreasonable to think that a young specimen of M.
sanguinea of more than 100 segments would not show some of the characteristics
of the adult, that of antennae, branchiae, characteristic setae, etc. In consideration

ENDOPARASITIC POLYCHAETOUS ANNELIDS 187

of the long jaw pieces (maxillary carriers) and the four eyes, Ehlers concluded that
it might be a parasitic species of Arabella. The figures and description given by
Koch suggest to me that the young developing stages in Marphysa that he observed,
were the parasitic young stages of a species of Notocirrus, as indicated especially
by the long maxillary carriers, the stout parapodial acicula, and the four eyes in a
transverse row.

LITERATURE CITED

ALLEN, M. JEAN, 1952. An example of parasitism among polychaetes. Nature, 169: 197.
CAULLERY, MAURICE, 1914. Labidognathus parasiticus n.g., n. sp. Cas neuveaux d'endopara-

sitisme evolutif chez les Euniciens. C. R. Soc. Biol. Paris, 77 : 490-493.
CAULLERY, MAURICE, AND FELIX MESNIL, 1916. Notes biologiques sur les mares a Litho-

thamnion de la Hague. 1. Presentation d'un Labrorostratus parasiticus S.J., parasite

interne d'Odontosyllis ctenostoma Clpd. Bull. Soc. Zool. Paris, 40: 160-161.
CERRUTI, ATTILIO, 1909. Oligognathus parasiticus n. sp., endoparassita dello Spio meczm-

kowianus Clpd. Arch. Zool. Napoli, 4 (2) : 197-209.
EHLERS, ERNST, 1868. Die Borstenwiirmer (Annelida Chaetopoda) nach systematischen und

anatomischen Untersuchungen dargestellt, Abteilung II: 269-748.
FAUVEL, PIERRE, 1923. Polychetes errantes. Faune de France, 5: 1-488.
HARTMAN, OLGA, 1944. Polychateous annelids. Part V. Eunicea. Allan Hancock Pac.

Exped., 10 (1) : 1-236.
KOCH, HEINRICH, 1847. Einige Worte zur Entwicklungsgeschichte von Eunice. Denkschr. Allg.

Schiveis. Ges. Naturw. Neuenberg, 8:1-12.
MOORE, J. PERCY, 1906. Descriptions of new species of Polychaeta from the southeastern coast

of Massachusetts. Proc. Acad. Nat. Sci. Phil, 58: 501-508.
SAINT-JOSEPH, BARON ANTOINE DE, 1888. Les Annelides polychetes des cotes de Dinard.

Part 2. Ann. Sci. Nat. Zool, Paris, ser. 7, 5 : 141-338.
SPENGEL, J. W., 1882. Oligognathus bonelliac eine schmarotzende Eunicee. Mitt. Zool. Stat.

Neapel, 3: 15-52.
WIREN, AXEL, 1886. Haematoclcptcs tcrcbellidis, nouvelle annelide parasite de la famille des

Euniciens. Bihang Kongl. Sven. Vet. Akad. Hand!., 11 (12) : 3-10.

A DIURNAL ACTIVITY RHYTHM IN PLETHODON CINEREUS

AND ITS MODIFICATION BY AN INFLUENCE

HAVING A LUNAR FREQUENCY

CHARLES L. RALPH 1
Department of Biological Sciences, Northivestern University, Evanston, Illinois

It has become increasingly clear that many of the physiological processes in
organisms do not occur at constant rates, even when the organism is in a constant
laboratory environment. These fluctuations in rates are often of regular recurrence
and may be designated as rhythms.2 Various manifestations of these changes taking
place within the organisms may be observed. Among them are rhythms of O2-
consumption and CO,-production, locomotor activity, chromatophore pigment dis-
persal and body temperature changes.

Judging from the number of contributed works in the field of biological rhythms,
locomotor activity has been more often utilized as an index to rhythmic behavior
than any other kind of biological process. The simplicity of automatic recording
devices needed, the long span of time over which animals may be used for such
studies, and the minimal interference with the animals' normal functioning, are some
of the reasons why activity studies have been so popular.

Among the earliest investigators of activity rhythms was Szymanski (1918),
who demonstrated them in a variety of animals. Subsequently, studies of the
activity rhythms of a host of animals have been made. Among them, to name only
a few representative ones, are those of Ralph (1957) on the earthworm, Harker
(1956) on Periplaneta americana, Brown (1954) on the oyster, Marx and Kayser
(1949) on lizards, and Aschoff (1952) on mice.

The diurnal rhythm is the one most commonly encountered in terrestrial organ-
isms (reviewed by Welsh, 1938, and Kleitman, 1949). Among marine organisms
both diurnal and tidal rhythms have been found associated together (Brown,
Fingerman, Sandeen and Webb, 1953), but sometimes a tidal rhythm may be the
only one apparent (Rao, 1954). Lunar periodicities in marine animals are well
known (Korringa, 1947).

That lunar influences may also be significant in the metabolic rhythms of ter-
restrial organisms has been indicated by recent works (Brown, Freeland and Ralph,
1955; Ralph, 1957). The present study was undertaken in order to examine the
activity behavior of a terrestrial animal, the salamander, Plethodon cinereus, and
to analyze it for lunar influences.

1 Present address : Entomology Branch, Directorate of Medical Research, Army Chemical
Center, Maryland.

2 A rhythm, as used here, is defined as a definitely persisting, regularly recurring, quanti-
tative change that continues after external stimuli are withdrawn. Rhythms can be roughly
divided into (a) those of 24 hours (diurnal) or less, and (b) those of more than 24 hours (e.g.,
lunar). They generally have a causal relation to external factors, but are to be distinguished
from periodicities which are of extrinsic origin and which vary directly with environmental
factors.

188

ACTIVITY RHYTHM IN SALAMANDERS 189

EXPERIMENTAL PROCEDURE

Twenty-four adult Plethodon cinereus were collected on May 8, 1955, in a
beech-maple forest near New Buffalo, Michigan. The animals were brought to the
laboratory and placed in two 9-inch crystallizing dishes, the bottoms of which were
covered with a moist sand layer and bits of wood debris. Small earthworms and
sowbugs were provided as food and these were replenished frequently during the
experiment. The dishes were placed in a slowly-moving stream of cold tap water.

All work was carried out in a room designed as a photographic darkroom.
There was one opening to the room, a door fitted with a light-baffle. Thermograph
records of the air temperature a few inches above the running water in which the
animals were kept throughout the experiment were taken between May 10th and
26th. These showed no diurnal temperature variations, but rather only slow changes
requiring several days.

Continuous records of the locomotor activity of seven animals were obtained
by the use of the same apparatus, and data were translated from the experimental
records to tabular form in the same manner, as described by Ralph (1957) for the
study of the earthworm. The recording device consisted essentially of seven
rectangular platforms that rocked freely upon knife-blade bearings. Upon each
platform was mounted a chamber consisting of a 3%-inch Petri dish enclosing the
bottom half of a 234-inch Petri dish, thereby forming a circular track one-half inch
in width. Movement of the animal around this track resulted in different degrees
of tipping of the platform. Platform movements were transmitted via a thread
to a recording pen system that reproduced them on a sheet of paper moving at a
rate of two centimeters per hour. The recording apparatus occupied a position
adjacent to the dish of reserve animals and the activity chambers were suspended
about two inches above the water's surface in order to keep the animals in the
same cool air layer.

From May 9th to 13th all the animals were exposed to alternating light, from
6 A.M. to 6 P.M., and darkness, from 6 P.M. to 6 A.M. The light source during this
period was two 7%-watt opalescent, incandescent lamps suspended about four feet
above the animals. The form of the diurnal activity cycle under simulated day and
night conditions was determined during this period.

During the afternoon of May 13th a light-proof box was mounted over the
water table in which the reserve animals were kept so that they could be main-
tained in constant low illumination. The box was equipped with a light source at
the top so that diffuse light of less than one foot-candle reached the salamanders.
Also, the transparent glass covers of the activity chambers were replaced by black-
painted covers in order to exclude all light. Thus, when the animals were in the
reserve dishes they were under continuous and constant low illumination, and when
placed in the recording apparatus they were in darkness. Twenty-nine days of
continuous records for seven animals at a time were obtained under these con-
ditions.

A regular sequence of replacement was established at the outset of the experi-
ment, so that only one or two animals were replaced daily. As the animals were
removed from the chambers they were placed in one of the crystallizing dishes, while
the replacement animal was randomly selected from the other dish. When the
supply of animals was exhausted from one dish, replacement was started from the

190

CHARLES L. RALPH

dish that had been receiving the animals. Thus, each of the 24 animals participated
in the study at least twice for approximately four days each time.

In order to minimize any effects that the placing of a fresh animal in the ap-
paratus might have on the data, the time of replacement was varied over the day
from 8 A.M. to midnight. As a further precaution, the first three hours of the
record produced by a fresh animal were not included in the data, since the animals
tended to be hyperactive for several minutes after being placed in a chamber.

RESULTS
Diurnal rhythm

The mean activity for the animals in alternating light and darkness is shown
by Figure 1, A.3 It will be seen that the animals were most active during the

z -

FIGURE 1. (A) The average activity of seven salamanders for about five consecutive days
while exposed to light from 6 A.M. to 6 P.M. and darkness from 6 P.M. to 6 A.M. (B) The
average activity of seven salamanders for 29 consecutive days. Their activity was recorded
while they were in darkness. (C) The influence of the lunar-day frequency on the activity of
the salamander, as demonstrated by analysis for lunar effects. (See text for explanation.) The
times of zenith for the 29-day period were synchronized in column 6 for this analysis.

dark hours and least active during the light hours. A more complete analysis of
the exact form of this curve will be presented in the discussion.

Table I shows the mean activity when the animals were in dark chambers.
These data represent hourly determinations for 29 consecutive days. The mean
values for seven animals for each hour were placed in the table under the hour on
which the determination ended.

3 All graphs in this paper are plotted from sliding averages of three adjacent values. For
example, the averages of the 2, 3, and 4 p. M. columns were averaged to give the 3 P.M. value.
This technique is useful for smoothing curves and shows trends more clearly.

ACTIVITY RHYTHM IN SALAMANDERS

191

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192 CHARLES L. RALPH

The data were first analyzed to find the mean activity for each hour of the day.
All 24 columns were averaged and the results are plotted in Figure 1, B. The
form of this variation is similar, in a general way, to that obtained when the animals
were exposed to alternating light and darkness. There are conspicuous differences
between them, however, and these will be discussed later.

Lunar analysis

The data of Table I were inspected for a lunar influence before they were sub-
jected to a detailed analysis. First the data for each day were plotted, each day
under the preceding one, and then a line representing lunar zenith was drawn
across the plots, intersecting the abscissae at the time of zenith. Since the moon
reaches zenith for any given locality approximately 50 minutes later each day, the
line representing zenith intersected the time scales about 50 minutes later with
each successive day.

Upon close examination of these plots, it was seen that the activity pattern for
each day had certain unique variations, but generally bore a similarity to the mean
pattern for the 29 days. However, it was noted that when zenith occurred at
times of usually moderate or high activity the level of activity was generally low
for a few hours before and after the time of zenith. That is to say, when zenith
occurred during the "night hours," which were usually the times of greatest ac-
tivity, depression of activity resulted.

On the starting day of this 29-day study, May 14th, the moon was in third, or
last, quarter and thus the time of zenith was approximately 6 A.M. On May 21st
new moon occurred with the time of zenith at noon. First quarter, with zenith
at approximately 6 P.M., was on the 28th of May and full moon, with zenith at mid-
night, occurred on June 5th.

It may be postulated that the mean activity for any one day would tend to be
high if zenith occurred during the times of normally low activity and would tend
to be lower if zenith occurred during the times of high activity. Therefore, if one
plots the mean activity for each day for one lunar month, the resulting curve should
be essentially the inverse of the mean hourly activity curve, provided the lunar effect
is operative in the postulated manner. A comparison of Figure 2, which shows the
daily means for a lunar month, with Figures 1, A or 1, B, the hourly mean curves,
bears out this hypothesis. This would then suggest strongly that a lunar zenith
effect is operative ; it is a depressive effect, and the time at which zenith occurs
does indeed appear to determine to some extent the mean activity for any one day.

A second method of demonstrating the presence of an apparent lunar modifica-
tion of the diurnal rhythm was applied in the following manner. If the hourly
averages of the days between third quarter and new moon are found, a pattern of
activity variation essentially like the average for the entire 29 days should result,
since little depressive influence should be in evidence during this time. Figure 3,
A, shows the means for each hour of the day for the seven days of that period, and it
will be seen that this pattern is very similar to that for the entire 29-day period.
Likewise, the hourly means for the succeeding seven days, May 21st to 27th, that is,
from new moon to first quarter, should also be similar to the means for the entire
29 days and similar to the pattern for the preceding seven days. Upon examina-
tion of Figure 3, B, it will be seen that this is true.

ACTIVITY RHYTHM IN SALAMANDERS

193

3RD

LUNAR PHASES
NM IST

FM

i i i i i i i

FIGURE 2. The average activity per day for seven salamandecs over a 29-day period.
The approximate times of the lunar quarters are indicated.

194

CHARLES L. RALPH

During the next eight days, however, the time of zenith moved from about 6
P.M. to near midnight. Any zenith-associated depressive influence should be very
evident during this period. Figure 3, C shows that for these days the lowest levels
of activity occurred between 6 P.M. and midnight. Evident, also, in this figure is

8 -

6
10

8

tr

x

X

LL)

O

10

8

D

i i i i i

j i i i

6
AM

12

6
PM

12

FIGURE 3. The average hourly activity of seven salamanders. A. May 14-20 (3rd quarter-
New Moon). B. May 21-27 (New Moon-lst quarter). C. May 28- June 4 (1st quarter-Full
Moon). D. June 5-11 (Full Moon-3rd quarter).

ACTIVITY RHYTHM IN SALAMANDERS 195

a low at 6 A.M. This was largely caused by a low average value of 2.9 at 6 A.M.
Consequently, the sliding averages in the region around 6 A.M. are affected. Its
significance is unknown.

Finally, the hourly averages for the last quarter of the lunar month are shown
in Figure 3, D. Once again, depression appears evident, this time between 1 and
6 A.M., the range over which zenith occurred during the final seven days of this
study.

The third method for showing the presence of a lunar rhythm is that which
has been employed with much success by Brown (Brown, Bennett and Webb, 1954).
For the purposes of this analytical technique, one may visualize the times of lunar
zenith, and other corresponding lunar positions, as diagonals running downward
and from left to right across the daily rows of data in Table I. As pointed out
earlier, the time at which lunar zenith occurs for any one location is later with each
successive day by about 50 minutes. Thus, the lunar day is of about 24.8 hours
duration. A given lunar position completes one diagonal crossing of all the 24-hour
vertical columns in about 29 days.4

Any lunar-associated influence may be made apparent if the corresponding lunar
positions are aligned in vertical columns. Such a manipulation will serve also to
"neutralize" the diurnal rhythm. To accomplish this, the day-by-day data of Table
I were shifted to the left an average of about 50 minutes with respect to the pre-
ceding day. That is, day 1 was left in its normal hourly relationship, as was day
2, also. Then days 3 to 7 were each moved one hour to the left with respect to
the clock hours of the preceding days. The data of day 8 were kept synchronized
with those of day 7, but days 9 to 13 were each shifted one hour further to the left,
and so on throughout the 29 days of data. Twenty-four vertical columns were
retained by transposing, in sequence, the data which extended to the left beyond the
first value of day 1 to the right side of the table.

Figure 1, C, shows the results of this analysis. Since zenith occurred at ap-
proximately 6 A.M. on the initial day of this experiment and all zeniths of the suc-
ceeding days were aligned with it, the position of zenith is indicated in the sixth
column in Figure 1, C. This analysis provides further evidence that a depressive
effect, which modulates the diurnal rhythm, is associated with the time of lunar
zenith.

DISCUSSION

Upon comparison, it will be observed that the amplitude of the average cycle
represented in Figure 1, A is about twice that shown by Figure 1, B. This differ-
ence may be explained upon the basis of two possible reasons, both of which may
apply. When the animals are in alternating light and darkness (Fig. 1, A), the
persistent rhythm may be amplified due to a direct influence of light intensity on
locomotion. In continuous darkness the rhythm may fail to attain fullest expres-
sion, being "damped," but maintaining essentially the same frequency.

A second contributing cause for the difference in amplitude, and one which, in
addition, may explain the occurrence of the minor minimum around midnight in
Figure 1, A, could be that, in the period represented, only the first half of the A.M.
hours, approximately, were subjected to the postulated depressive lunar influence,
whereas all hours of the period represented by Figure 1, B were exposed to this

4 The synodical lunar month, the period from one new moon to the next, has a mean length
of 29 days, 12 hours, 44 minutes, and 2.8 seconds.

196 CHARLES L. RALPH

influence. Thus, Figure 1, B presents the average diurnal rhythm with the lunar
influence "neutralized," but nevertheless with lowered amplitude caused by the
lunar depression. Figure 1, A is possibly distorted, in part, by the lunar influence,
but most of its values were little affected by lunar depression.

The rhythm of locomotor activity, as determined in this experiment under con-
stant laboratory conditions, is undoubtedly similar to the variations in activity of
this salamander in nature. Park, Lockett and Myers (1931) note that in the
forest this salamander apparently passes the day beneath logs and stones and be-
comes active by 8 :45 P.M.

The possession of a rhythm that assists in regulating the activity of the sala-
mander, as described here, may be of supreme importance for the survival of the
animal. As we have seen, it appears to have an activity rhythm that is determined
by two components : ( 1 ) the diurnal activity pattern, which tends to keep activity
minimal during the daylight hours and maximal during the night hours, and (2)
the lunar modulation, which alters the diurnal pattern so as to minimize activity
on moonlit nights. Thus, the inference is that the animal forays out of its hiding
niche on nights when there is little or no moonlight and, hence, when there is less
exposure to predators.

Since the period of study extended through only one lunar cycle, it cannot
be unequivocably stated that the lunar-frequency modulation constitutes a lunar
rhythm, but due to its close correlation with the lunar cycle and the similarity of
this modulation with lunar rhythms that have previously been described (cf. Brown,
Freeland and Ralph, 1955), it appears very likely that it is a lunar rhythm.

Though there appears to be a causal relationship between the lunar cycle and the
lunar modification, there need be no direct inductive influence of the moon affecting
the organisms. Diurnal rhythms show a causal relationship to the day-night cycle,
but, as several studies have shown, the phase relationships of rhythms to the day-
night cycle need not be fixed. Thus, the apparent "influence" of the moon on the
activity rhythm may be only a persisting behavioral pattern that continues after
the inductive influence of the moon is removed. Just as the 24-hour solar cycle
may be impressed upon the activity pattern, likewise the 24.8-hour lunar cycle may
also be impressed upon the pattern. The two frequencies together would appear
largely to determine the overt expression of activity.

The author wishes to express his appreciation to Dr. F. A. Brown, Jr., for his
helpful suggestions concerning the manuscript.

SUMMARY

1. The salamander, Plethodon cincrcus, shows a diurnal rhythm of locomotor
activity, both in alternating 12-hour periods of light and darkness, and in continuous
darkness.

2. The diurnal rhythm is strongly modified by a depressive influence that is
apparently associated with the time of lunar zenith.

3. The activity of the animal at any given time is a function of the diurnal and
lunar influences operative at that time.

4. The significance of the rhythm to the animal in nature is discussed.

ACTIVITY RHYTHM IN SALAMANDERS 197

LITERATURE CITED

ASCHOFF, J., 1952. Frequenzanderungen der Aktivitatsperiodik bei Mausen im Dauerlicht und

Dauerdunkel. P finger's Archiv, 255: 197-203.
BROWN, F. A., JR., 1954. Persistent activity rhythms in the oyster. Amer. J. Physiol, 178:

510-514.
BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB, 1954. Persistent daily and tidal rhythms

of Go-consumption in fiddler crabs. /. Cell. Comp. Physiol., 44 : 477-505.
BROWN, F. A., JR., M. FINGERMAN, M. I. SANDEEN AND H. M. WEBB, 1953. Persistent diurnal

and tidal rhythms of color change in the fiddler crab, Uca pugnax. J. Exp. Zool., 123 :

29-60.

BROWN, F. A., JR., R. O. FREELAND AND C. L. RALPH, 1955. Persistent rhythms of O2 con-
sumption in potatoes, carrots and the sea-weed, Fucus. Plant Physiol., 30 : 280-293.
HARKER, J. E., 1956. Factors controlling the diurnal rhythm of activity of Periplaneta ameri-

cana L. /. Exp. Biol, 33: 224-234.

KLEITMAN, N., 1949. Biological rhythms and cycles. Physiol. Rev., 29: 1-30.
KORRINGA, P., 1947. Relations between the moon and periodicity in the breeding of marine

animals. Ecol. Monogr., 17: 347-381.
MARX, C., AND C. KAYSER, 1949. Le rhythme nycthemeral de 1'activite chez le lezard (Lacerta

agilis, Lacerta muralis). C. R. Soc. Biol., 143: 1375-1377.
PARK, O., J. A. LOCKETT AND D. J. MYERS, 1931. Studies in nocturnal ecology with special

reference to climax forest. Ecology, 12 : 709-727.
RALPH, C. L., 1957. Persistent rhythms of activity and (Vconsumption in the earthworm.

Physiol. Zool, 30: 41-55.
RAO, K. P., 1954. Tidal rhythmicity of rate of water propulsion in Mytilus, and its modifiability

by transplantation. Biol. Bull, 106: 353-359.
SZYMANSKI, J. S., 1918. Die Verteilung von Ruhe- und Aktivitatsperioden bei einigen Tierarten.

Pfliiger's Archiv, 172: 430-448.
WELSH, J. H., 1938. Diurnal rhythms. Quart. Rev. Biol., 13: 123-139.

STUDIES ON THE EFFECTS OF IRRADIATION OF CELLULAR

PARTICULATES.1 II. THE EFFECT OF GAMMA RADIATION

ON OXYGEN UPTAKE AND PHOSPHORYLATION

HENRY T. YOST, JR. AND HOPE H. ROBSON

Department of Biology, Amherst College, Amherst, Massachusetts

It is now well established that dilute solutions of many enzymes are readily
inactivated by ionizing radiation and that the presence of solute molecules, other
than enzyme, decreases the effect of the radiation (Barren, 1954). Since the
classical experiments of Dale (1940, 1942), it has been possible to explain the effects
of low dosages of radiation on the basis of interactions between the protein molecule
and the ionization products of water. These findings have led to the discovery of
important facts about radiation damage and about the nature of enzymes. However,
an important question is raised by this work; that is, are we able to draw valid
conclusions about the biological effects of radiation from such studies ? It is evident
that cellular enzymes are not in a pure state, nor are they as dilute as is necessary
to achieve effects in some cases. This makes it necessary to investigate the effects
of radiation on enzymes under conditions which approximate those of the cell.

There are two obvious ways to do this. The most usual method is to radiate
a whole organism (or cell) and then determine the enzymatic activity after radia-
tion. The results of such work indicate that the damage to enzymes by lethal
doses of radiation may be negligible (LeMay, 1951 ) . However, there are a number
of obvious difficulties in such work ; and since the organisms do die eventually and
do show loss of respiration in some cases (Barren, 1954), one is left with an un-
satisfied feeling. For this reason, it seemed advisable to study the effects of
ionizing radiation on cellular participates (Yost, Robson and Spiegelman, 1956).
The participates of intermediate size (mitochondria) offer several interesting possi-
bilities for such investigations : they can be isolated from the cell in good condition ;
they contain a large number of vital enzymes ; they have a definite structure to
which some of the enzymes are attached ; the enzyme studied would always be in
an environment similar to, if not exactly the same as, that in which it finds itself
within the cell ; and most important, the participate is sufficiently large that one
might assume that a major part of the damage done to enzymes within the particu-
late would result from the passage of the ionizing "particle" through the mito-
chondrion itself. This would mean that the effects of the radiation on the enzyme
studied would be the same whether the mitochondrion was extracted or within the
cell.

The experiments reported in this paper had a two-fold purpose : the establish-
ment of a dose-inactivation curve for an enzyme known to be closely associated with
the structure of the particulate, and the determination of the effect of ionizing
radiation on the phosphorylation mechanism. Cytochrome oxidase was chosen as the

1 This work was supported by a grant. No. C-2154, from the National Institutes of Health.

198

RADIATION OF CELLULAR PARTICULATES 199

test enzyme since it is known to be closely bound to the particulate and therefore
might be an indicator of effects of the radiation on the particulate as a whole, and
since it is of vital importance to the electron transport system. Furthermore, the im-
plication of the cytochrome system in the production of mutations by ionizing radia-
tion (Haas et al., 1954) suggests this enzyme as an excellent starting point for the
investigation of the effects of radiations on cells.

MATERIALS AND METHODS

White laboratory rats were starved overnight and sacrificed by a blow on the
head. The liver was removed and placed in cold 0.85% KC1, where much of the
blood was washed free. The liver was weighed and pressed through a bronze screen
to remove connective tissue. The resulting mash was then suspended in 50 ml. of
cold 8.5% sucrose containing 0.005 M disodium versenate and homogenized in a
glass homogenizer with a "Teflon" pestle driven by a cone-drive stirring motor.
The mitochondria were then separated from the rest of the homogenate by the
method of differential centrifugation (Schneider, 1948). The mitochondrial frac-
tion alone was kept.

In the studies of the effect of gamma radiation on the activity of cytochrome
oxidase, the mitochondria were suspended in 2.5 ml. of sucrose-versenate per gram
of original liver. For irradiation, a sample of the suspension was diluted 1 in 20
with distilled water. Five milliliters of the dilute preparation were put in a glass
cup and irradiated in the beam of a 440-curie Co60 source. The radiation was fil-
tered by a half-inch of lucite to remove beta radiation ; the intensity of the radiation
was 1000 r per minute. Controls were kept in a sheltered alcove outside the
radiation room under the same conditions as the radiated material. The Warburg
assays were run with the diluted preparations.

Treatment of the preparation for the determination of the effect of gamma
radiation on phosphorylation differed from the above in some respects. The
initial preparation was made by suspending the mitochondria in one ml. of sucrose-
versenate per gram of liver. This suspension was then diluted 1 in 20 and radiated
in a 25-ml. "Lusteroid" centrifuge tube. The controls were treated in the same
manner, with the exception of the exposure to the radiation. After the radiation,
control and treated suspensions were centrifuged, and the mitochondria re-suspended
in one ml. This final suspension was assayed for phosphorylation.

The cytochrome oxidase activity was estimated manometrically by the method
of Hogeboom, Claude and Hotchkiss (1946). The main compartment of each
vessel contained: 0.35 ml. of mitochondrial suspension, 0.1 ml. Sorenson phosphate
buffer (pH 7.4), 1 ml 1.3 X 10~* M cytochrome-c (Sigma, horse-heart) in
0.85% NaCl. and 0.15 ml. 0.005 M A1C1,. The center well contained 0.1 ml.
5 N KOH, and the side arm held the reducing agent, 0.15 ml. 0.228 M sodium
ascorbate.

Estimation of phosphorylation was concluded by a modification of the method
of Maley and Lardy (1954), using succinate as the substrate. The main compart-
ment of the vessel contained: 0.3 ml. (30 /JVI) phosphate buffer (pH 7.4), 0.3 ml.
0.1 M sodium succinate, 0.8 ml. 8.5% sucrose, 0.1 ml. (0.3 /*M) cytochrome-c, 0.3
ml. (6 /*M) ATP (Schwartz, neutral), 0.1 ml. (30 /xM) MgSO4, 0.1 ml. (40
KF, and 0.5 ml. of the mitochondrial suspension. The center well contained

200

HENRY T. YOST, JR. AND HOPE H. ROBSON

0.1 ml. 5 N KOH, and the side arm held 0.5 ml. (20 mg.) of hexokinase (Pabst).
To assure that the final pH of the reaction would be 7.0 or higher, the pH of
some of the more acid reactants was adjusted with NaOH before addition to the
flasks. Failure to do this results in lowered oxygen uptake and lowered phosphory-
lation. Readings of the oxygen uptake were taken for 30 minutes, after which
time the reactions were stopped with TCA and the phosphate determined by the
Lowry-Lopez method as presented by Click (1949).

Assays of oxidase activity were made at 38° C. ; assays of phosphorylation were
made at 25° C. Assays of oxygen uptake were made in triplicate; assays of
phosphorylation were made in duplicate. All experiments were repeated at least
three times.

RESULTS

Table I presents the data obtained from radiation of mitochondrial preparations
of differing age. The preparation labeled "Day 1" was radiated on the same day

TABLE I
Inactivation of cytochrome oxidase by gamma radiation

Day 1

Day 2

Day 3

Dose r

No. runs

Per cent

inactivation

No. runs

Per cent
inactivation

No. runs

Per cent
inactivation

2,500

2

1.8±4.1

6

6.4±3.8

6

5.7±6.0

4,000

8

4.8±2.4

18

18.9±2.9

4

8.7±2.2

5,000

13

9.4±2.9

18

8.8±2.1

12

10.2±4.4

10,000 6

10.5±2.2

10

6.5±1.8

9

10.5±2.6

12,500

8

16.7±2.4

8

25.7±4.0

10

27.1±3.2

15,000

14

17.4±2.9

17

26.7±2.0

7

26.5±1.6

20,000

19

29.9±2.4

16

29.7±2.3

16

26.7±2.1

30,000

5

41.3±3.S

11

34.6±2.5

6

31.0±6.5

40,000

6

49.1±2.8

10

42.8±4.5

12

34.1 ±3.3

that it was extracted ; the preparation labeled "Day 2" was radiated on the following
day; etc. These data show that cytochrome oxidase is extremely resistant to
gamma radiation. This is in accord with the earlier studies of Barren ct al.
(1949). Nevertheless, although the data are extremely erratic below 10,000 r,
some effect is achieved with doses as low as 2500 r. There is little indication that
a maximum has been reached at 40,000 r. It is necessary to comment on the
variability shown by the data. The figures are averages of several runs done with
different rats. We have found that preparations from different rats give different
results. In the case of the data obtained with 4000 r, Day 2, the inactivation
varied from 12.1 per cent to 36.5 per cent. Whether this intrinsic variability is the
result of differences in age, sex, or physiological condition, we are unable to judge
at this time. In addition, there is always the problem of variation in the con-
centration of the preparations. All dilutions are made from suspensions made up
as 2.5 ml. per gram of liver extracted. There is no reason to suppose that the
number of mitochondria will be the same in each case. Until studies are done in

RADIATION OF CELLULAR PARTICULATES

201

TABLE II

The effect of aging on the inactivation of cytochrome oxidase

Morning

Afternoon

Y\

No. runs

Per cent inactivation

No. runs

Per cent inactivation

5,000

4

0.0±2.0

4

16.9±2.0

15,000

4

7.4±1.5

4

21.9±2.4

20,000

4

24.5±4.5

4

39.6±1.8

which the number of mitochondria in each sample are the same, no conclusions can
be drawn about the variability between rats.

The data in Table I indicate that the preparations become more sensitive to low
doses of radiation with time. On the first day, a dose of 2500 r produces little or
no inactivation ; on the second day, it produces about 6 per cent. This is shown in
a much more striking manner in Table II. In this case the data from some runs
done on the first day are broken down into those done in the morning and those
done in the afternoon. In all cases, a preparation was radiated in the morning and
afternoon, the only difference being the age. It is evident that when the mito-
chondria are first extracted they are much more resistant to radiation, particularly
at the lower doses. At high doses, Table I indicates that there is a progressive
decrease in sensitivity with age. Intermediate doses are erratic. As a result of

50

40

20

10

40

FIGURE 1. Inactivation of cytochrome oxidase by gamma radiation. Points represent
means, with standard errors shown as limits. Preparation radiated the same day it was ex-
tracted. The dotted line represents the best fit of a regular curve.

202

HENRY T. YOST, JR. AND HOPE H. ROBSON

this aging effect, the inactivation curve shown in Figure 1 was constructed on the
basis of the first-day results only.

The data presented in Table III show that the oxidative phosphorylation
mechanism is far more sensitive to radiation than cytochrome oxidase. All runs
were done with a fresh preparation. There is no appreciable difference in the age
of the preparations. In this case, each run represents a different rat, so that the
variability results from this alone. It can be seen that there is little effect of the
radiation upon the oxygen uptake in any case. The stimulation of phosphorylation
by 2500 r is slightly greater than would be expected on the basis of increased

TABLE III

Inactivation of phosphorylation by gamma radiation

Phosphate uptake:

Dose

Per cent decrease

No. treated

Ch uptake,
% decrease

Controls

Treated

2,500

6.6±1.0

6.8±0.64

-3.0

10

-9.3

5,000

8.0±1.3

7.5±1.1

6.2

10

7.9

10,000

6.7±1.1

5.1±0.62

23.8

10

6.5

15,000

9.1±1.4

6.4±0.47

29.6

11

5.3

20,000

9.2±2.5

4.4±1.3

52.2

3

7.5

30,000

11.8±2.6

4.9±1.0

58.5

5

5.4

40,000

6.2±0.93

1.7±0.36

72.6

6

8.3

oxygen uptake, when calculations are made using a theoretical P :O ratio of 2
for succinate.

In all tables the data are shown as means ± standard error.

DISCUSSION

The data presented in this paper indicate that the inactivation of cytochrome
oxidase by gamma radiation follows an irregular course. In all cases, there is a
plateau reached at 5000 r to 10,000 r, followed by a sharp increase between 10,000
r and 12,500 r. The plateau is not lost upon aging (if anything, it is intensified) ;
nor is it absent in fresh preparations. Table II indicates that in very fresh prepa-
rations there is a sharp increase between 15,000 and 20,000 r. It appears that
the age of the preparation merely increases the dose necessary to cause the
"jump." This observation requires special consideration. In the oxidation of
cytochrome-c by ionizing radiation, it has been shown that the effects of radiation
doses below 10,000 r are completely reversible (Barren, 1954). It is possible that
part of the curve (Fig. 1) between 10,000 r and 15,000 r represents a shift from
indirect to direct effects upon the particulate. Since we are radiating the entire
particulate system, we can expect that the terminal oxidase of this system is con-
stantly being reduced by substrate (either the supplied cytochrome-c or internal
metabolites). This continual reduction may protect the cytochrome oxidase from
the effects of the radiation, either during the time of radiation, when it must draw
upon its internal supplies, or during the assay procedure, when it is supplied with
a reducing agent which may effect post-irradiation recovery. Support for this idea
conies from the aging effect shown in Table II. If the oxidase were being pro-

RADIATION OF CELLULAR PARTICULATES 203

tected by materials within the mitochondrion at the time of radiation, we would
expect that aging the preparation would lead to depletion of the internal stores with
a consequent lowering of the protective effect. This is what is observed. At
higher doses this protective effect is apparently negligible compared to the dose
administered. Thus the effect of the radiation is reduced with age as might be
expected from the protective effect of particulates whose oxidase has been inacti-
vated by causes other than radiation.

It may be argued that the protection is merely the result of the various solute
molecules which are not removed by washing during the extraction. It is difficult
to decide whether there is a specific type of protection resulting from the genera-
tion of "reducing power" from the substrate, or whether there is a non-specific
protective effect of additional substrate (Dale, 1942). It is clear that the effective
substance is lost during aging ; therefore, it seems sure that the protective substance
must be within the particulate to be effective. If the substance were merely lost
from the mitochondrion, it should be in the suspending medium which is diluted
before radiation. The same concentration of substance would be present in either
case. Such considerations raise the question of whether the effects of the radiation
are independent of the concentration of the mitochondria. It might be suspected
that only those ionizations produced within a mitochondrion would affect the en-
zymes within the structure. The particulate is sufficiently large to justify such
an assumption. However, this does not prove to be the case. It was first estab-
lished that preparations diluted in sucrose were less easily inactivated than those
diluted in distilled water. This suggests that the ionization products of water are
acting on the mitochondrion and that the sucrose is acting as a non-specific pro-
tective agent. Unfortunately this is not a clear test. The sucrose might be caus-
ing a change in the osmotic condition of the particulate, so that the protective sub-
stance (normally lost by aging) is more concentrated within the mitochondrion, or
is not lost as readily. However, the data presented in Table III indicate that there
is a dependance upon dilution. It can be seen that in a preparation 2.5 times as
concentrated as that used to obtain the data in Table I, 40,000 r produce 8.3 per
cent inactivation of oxygen uptake, whereas they produce 49.1 per cent in the
dilute preparation. Clearly, 49.1 per cent is much greater than 2.5 times 8.3
per cent. Studies of intermediate dilutions bear this out. It is necessary to con-
clude that the effect of radiation upon the particulates is indirect in the sense that
any solute molecule outside the mitochondrion will exert some protective effect
upon the enzymes which are internal. Therefore, we must further conclude that
the effect of the protective substance which is lost upon aging must be the result of
some action it has -within the mitochondrion, and that the loss which occurs with
age probably results from the destruction of the substance by the particulates. As
pointed out above, it is impossible to be sure of the mode of action of this protective
agent, but the generation of materials which keep the enzymes in a reduced condi-
tion seems a likely mechanism.

A second explanation of the sudden "jump" in inactivation is that at doses over
10,000 r the particulate structure is undergoing severe change. This might result
in the freeing of enzyme molecules, with consequent dilution of protective sub-
stances or in the loss of function of one part of the system with its release from
another part. This seems a very unlikely mechanism. The fact that cytochrome
oxidase can be freed from the rest of the particulate and still retain its activity

204 HENRY T. YOST, JR. AND HOPE H. ROBSON

(Eichel et al. 1950) makes such an hypothesis difficult to maintain. Examination
of the preparations by phase contrast did not show any radical changes ; however,
any alterations of ultrastructure could only be detected by other methods.

The dilution effect deserves additional comment. These experiments were
originally designed in the hope that we would be able to approximate the biologi-
qal condition. Radiation studies with dilute solutions of pure enzymes are in-
formative with regard to radiation problems, but somewhat off the point for
biological problems. No cellular system exists which is a single molecular species.
Attempts to show that enzyme damage is the cause of radiation death in cells have
been relatively unsuccessful (LeMay, 1951). On the other hand, the cell is so
complex that it may die from many different causes, and it is difficult to be sure that
one is investigating the right system in any particular case. It seemed necessary
to investigate the problem in a system which had biological characteristics but
which was not quite so diffuse as a whole cell. The participates appear to offer
such a system. It is possible to extract a "package" of enzymes, each of which
has a relationship to other enzymes in the "package." The "package" resembles
a cell in many aspects, but it is much simpler in its total organization. Further-
more it seemed that it was of sufficient size that only those ionizations produced
within the mitochondrion would have any effect on the internal enzymes. This
would be of great importance to the radiobiologist since it would indicate that
there are sub-cellular bodies which can be considered to be separate from the rest
of the cell, with regard to radiation damage. The chromosomes are frequently
considered to be bodies of this type. Unfortunately this does not seem to be the
case. The data presented in this paper indicate that ionizations external to the
mitochondrion may cause internal damage, in a system containing only participates
and distilled water. It would appear that, at the doses studied, the ionization
products of water are capable of producing their effects over relatively great dis-
tances, or that the effects of these products on the surface of the mitochondrion are
capable of reducing the activity of the enzymes which are internal. This is of
special interest as it has been suggested that the major effect of radiation on cyto-
chrome-c is produced by hydroxyl radicals alone (Barren, 1954). Knowledge of
the exact position of the oxidase in the participate would be necessary to any final
conclusion about these effects. However, the difficulties encountered in the ex-
traction of cytochrome oxidase (Eichel et al., 1950) suggest that the enzyme is
internally bound.

From the foregoing discussion, it seems evident that we cannot conclude that
cytochrome oxidase is damaged to any great extent by radiation doses used in most
biological studies. It is difficult to know the concentration of the participates in
any cell, but it seems that the final suspension used in the phosphorylation studies
(one ml. per one gm. liver) would best approximate the natural condition in liver
cells. At no time was radiation given to participates at this dilution. The strong-
est preparation ever used (during radiation) was 20 times diluted. At this dilu-
tion (Table III) there is little inactivation at 40,000 r. It is interesting that the
effect on the oxidase seems to be the same for a wide range of doses. It is possible
that in any dilution some small fraction of the activity would be lost, but this seems
to be an unlikely cause of cell death. It is necessary to note that many cells do not
have the high concentration of particulates which liver has. In these cases the
effective doses necessary to inactivate cytochrome oxidase might fall within the

RADIATION OF CELLULAR PARTICULATES 205

limits of biological experimentation, even with the protective substance of the
cytoplasm present. In highly organized forms, the failure of one part may result
in the death of the whole, so that damage to this system might in some cases result
in death. This is particularly true of the forms which require fantastic doses of
radiation to induce lethal changes.

When one considers the data in Table III, it becomes apparent that the oxygen
uptake may be a faulty criterion for estimate of the health of a cell. These data
show quite clearly that the phosphorylation mechanism is much more sensitive than
the oxidase. Here we have a case in which over 70% of the ability to conserve
energy as organic phosphate is gone with no apparent effect on the oxygen uptake.
Considering the vital role of this system in the life of the cell, it seems quite probable
that disruption of vital processes would result from the loss of 25 per cent (or less)
of the ability to phosphorylate. We do not know how prevalent this loss is in the
whole system. In these studies only the uptake which resulted from the oxidation
of succinic acid was measured. It is possible that the whole phosphorylation
mechanism of the participates is damaged. Studies to determine the extent of the
damage and to determine whether phosphorylation is carried out by a single system
for all substrates are now in progress. In any case, it is evident that the phos-
phorylation mechanism is subject to destruction by ionizing radiation and that in-
activation is achieved in relatively concentrated preparations which are very fresh.
Although such preparations must be high in the concentration of the protective
substance found for cytochrome oxidase, there seems to be little protection of the
phosphorylation mechanism. This may be an indication that the protection
mechanism is specific for the electron transport system (if not for cytochrome oxi-
dase itself) or that the phosphorylation mechanism is extremely sensitive to radia-
tion.

These data suggest an explanation for several different phenomena which have
radiation as their sole common element. The phenomena are: induced crossing-
over, induced tumor formation, and the general protective effect exerted by reducing
compounds of the cysteine type. It has been suggested that one basis for the
changes in genetic crossing-over induced by radiation is the alteration in the avail-
ability of phosphate linkages within the chromosome (Yost and Benneyan, 1957).
It is evident that alterations in the phosphate pool of the cell must result from the
type of damage described in this paper. Indeed, it is to be expected that such
changes will have drastic effects on the chromosome structure, as many studies
have already indicated (Haas ct at., 1954). It is also possible that some radiation-
induced tumors are the result of the uncoupling of the oxidative metabolism of the
cell. This could result in a situation similar to that which Warburg has suggested
several times (Warburg, 1956). In cases in which oxygen uptake alone is meas-
ured, there is no assurance that the phosphorylation mechanism is functioning.
Lastly, the general effect of reducing agents may be more than the maintenance of
vital sulfhydryl groups. It is possible that the actual utilization of these compounds
as reducing agents in the general metabolism will result in the protection of many
non-sulfhydryl systems of the cell.

SUMMARY

Data are presented which indicate that the phosphorylation mechanism is much
more sensitive to gamma radiation than cytochrome oxidase. It is suggested that

206 HENRY T. YOST, JR. AND HOPE H. ROBSON

the utilization of substrates by enzymes within the particulate may protect these
enzymes against ionizing radiation. Various consequences of these findings are
discussed.

LITERATURE CITED

BARRON, E. S. G., 1954. The effect of X-rays on systems of biological importance. Radiation

Biology, A. Hollaender, ed., Vol. 1, Chapter 5. McGraw-Hill Book Co., New York.
BARRON, E. S. G., S. DICKMAN, J. A. MUNTZ AND T. P. SINGER, 1949. Studies on the

mechanism of action of ionizing radiations. I. Inhibition of enzymes by X-rays.

/. Gen. Physiol, 32 : 537-552.

DALE, W. M., 1940. The effect of X-rays on enzymes. Biochem. J., 34: 1367-1373.
DALE, W. M., 1942. The effect of X-rays on the conjugated protein d-amino acid oxidase.

Biochem. J., 36 : 80-85.

EICHEL, B., W. WAINO, P. PERSON AND S. COOPERSTEIN, 1950. A partial separation and char-
acterization of cytochrome oxidase and cytochrome-6. /. Biol. Chem., 183 : 89-103.
CLICK, D., 1949. Techniques of Histo- and Cytochemistry. Interscience Publishers, Inc.,

New York.
HAAS, F. L., E. DUDGEON, F. E. CLAYTON AND W. S. STONE, 1954. Measurement and control

of some direct and indirect effects of X-radiation. Genetics, 39: 453-471.
HOGEBOOM, G., A. CLAUDE AND R. HOTCHKISS, 1946. The distribution of cytochrome oxidase

and succinoxidase in the cytoplasm of the mammalian liver cell. /. Biol. Chem., 165 :

615-629.
LEMAY, M., 1951. Effect of X-radiation on succinoxidase of rat kidney. Proc. Soc. Exp.

Biol. Med., 77 : 337-339.
MALEY, G. F., AND H. A. LARDY, 1954. Phosphorylation coupled with the oxidation of reduced

cytochrome-c. /. Biol. Chem., 210: 903-909.
SCHNEIDER, W., 1948. Intracellular distribution of enzymes. III. The oxidation of octanoic

acid by rat liver fractions. /. Biol. Chem., 176: 259-266.
WARBURG, O., 1956. On the origin of cancer cells. Science, 123 : 309-314.
YOST, H. T., JR., AND R. N. BENNEYAN, 1957. The effects of combined radiations on crossing

over in Drosophila melanogaster. Genetics, (in press).
YOST, H. T., JR., H. H. ROBSON AND I. M. SPIEGELMAN, 1956. Studies on the effects of

irradiation of cellular particulates. I. Inhibition of cytochrome oxidase by ultraviolet.

Biol. Bull, 110: 96-106.

Vol. 113, No. 2 October, 1957

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE GENERAL FORM OF EXCRETION IN THE LOBSTER,

HOMARUS *

J. WENDELL BURGER
Trinity College and The Mount Desert Island Biological Laboratory

While there exists an extensive literature on the regulation of inorganic ions in
the higher Crustacea (Krogh, 1939; Robertson, 1949, 1953; Prosser et al., 1950),
the experimental study of nephridial function has received little attention, due ap-
parently to a lack of a method for securing repeated samples of urine. This report
gives such a method and it is an attempt to develop an integrated picture of the
regulation of the internal environment of a single species, Homarus americanus
(a lobster), through the experimental study of a variety of organic and inorganic
substances.

Comprehensive inorganic analyses of lobster blood have been made by Cole,
and Smith in Cole (1940), and of blood and urine by Robertson (1939, 1949, 1953),
and by Robertson and Webb (1939). The nephridial anatomy has been described
by Marchal (1892), Waite (1899), and Peters (1935). Historically there is no
structure corresponding to the vertebrate glomerulus. The study by Cuenot (1895)
of the differential concentration of dyes by various organs in different decapods is
of considerable interest.

MATERIALS AND METHODS

Because of the large number of substances studied and of experiments per-
formed, individual techniques will be described in context. Over two hundred lob-
sters were studied. These were largely from the Mt. Desert Island region, but a
few were from Nova Scotia. For all experiments the animals were given at least
twenty-four hours to equilibrate to the sea water at the Laboratory.

Urine from the nephridial bladders was secured at will without catheters by a
technique shown to us by Dr. P. R. Wilder of the Atlantic Biological Laboratory,
St. Andrews, N. B. If one's thumb is placed between the bases of the pereiopods
with the ringers over the carapace, preferably with the tail flexed, squeezing the
hand results in two jets of urine from the nephridiopores which can be collected in
test tubes held by the other hand. This technique works best with so-called "hard-
shelled" lobsters. The value of the technique was tested critically with twelve lob-
sters. After expressing the urine, the carapace was cut open and the bladders were
examined directly. In eight animals the bladders were empty ; the highest residuum

1 Aided by a grant from the New York Heart Association.

207

208 J. WENDELL BURGER

of the remaining four was 10% of the expressed volume. Thus the technique, while
not perfect, is serviceable. Since not all lobsters from commercial pounds form
urine, and since individual animals differ in the ease with which urine can be ex-
pressed, another critical study of twelve lobsters was made which showed we could
differentiate accurately the anuric specimens. As with many other techniques,
judgments depend more on practice than on formal rules. In general, however, if
the animal has good muscle tone, if the opercular flaps of the nephridiopores are
elevated on squeezing, and if the lobster is fresh from sea water, the animal is pro-
ducing urine. It is necessary sometimes to flick the opercular flaps with the finger-
nail to start the flow of urine.

Safe occlusion of the nephridiopores can be made by placing a wide rubber band
across the pores, crossing the band over the dorsal carapace, and then securing the
band on the posterio-ventral margin of the gill covers.

Test substances can be injected into the hemocoele, or more safely can be pi-
petted through the mouth into the stomach from which they are absorbed. Blood
is most easily withdrawn from the ventral surface of the abdomen. The only re-
liable method we found for the prevention of clotting was to whip or shake the blood,
filter it several times (Cole and Kazalski, 1939), and then preferably dilute it.

RESULTS

The data 2 are presented in three sections : Organic Substances, Inorganic Sub-
stances/Intake and Output, with the discussion pertinent to each in situ.

Organic substances

1. Inulin. Single injections into eight lobsters of inulin were followed up to 28
hours with up to 5 sampling periods. The analytic method of Schreiner (1950)
was used. For blood levels which ranged between 17 and 1.4 mg. %, the urine-
plasma (U/P) ratios were essentially one (1.0-1.1), with the concentrations in the
blood and urine falling with the same slope. Forster and Zia-Wohlrath (1941),
working with higher blood inulin levels (192-65 mg. %), also found inulin U/P
ratios of one. In the lobster, inulin is not secreted by the nephridium as reported
for the crayfish (Maluf, 1941). Since the inulin U/P ratio is substantially one,
the U/P ratios alone of other test substances should offer a reasonably accurate
guide for determining the partitioning ability of the nephridium, and there is no
need to compare the concentration of a test substance with the urinary concentration
of inulin in subsequent experiments, as would be necessary if there were a differ-
ential separation of water and inulin.

2. Vertebrate hemoglobin and plasma proteins. To test the permeability of the
nephridium to large molecules, solutions of hemoglobin prepared from hemolyzed
red cells of the dogfish (Squalus acanthias) were injected into the hemocoele of
eight lobsters. The normally clear urine promptly became pink and remained so
for several days. Plasma proteins from the dogfish were injected into four lobsters.
On gentle evaporation, the urine jelled, an unnatural event. While it was not de-

2 The following technical assistance is acknowledged gratefully : Dr. E. L. Becker, freezing
point depression; Dr. Klaus Brunn and Xenia Boysen, urea; Dr. Roy P. Forster, inulin and
PAH ; Drs. Henry Heinemann and Wilbur Sawyer, chloride ; Drs. Martin Rubin and Frederick
Berglund, magnesium and calcium; Dr. Charles G. Zubrod, glucose.

LOBSTER EXCRETION

209

termined that the original molecules were recovered in the urine in their original
state, it does appear that the nephridium of the lobster is permeable to molecules of
the size found in the blood of fish. The natural urine of the lobster is protein-free
(Forster and Zia-Wohlrath, 1941). Urine treated with the standard protein-
precipitating agents shows no increased Tyndall effect or clouding.

3. Glucose. The Hagedorn-Jensen sugar titration method (Peters and Van
Slyke, 1946) gave in mg. % urine/blood values of: 0/22; 0/24; 0/27; 0/28; 0/32;
0/37; 0/39; 0/40. Two urine values were slightly positive: 2/31 ; < 5/31. It is

u,

Z
CD ro

I I

I I

0

8 10

DAYS

FIGURE 1. Concentrations of phenol red in urine (Ui, U2) and blood (P) in two lobsters
with 6 and 4 mg. of dye pipetted into the stomach. The blood levels were so similar that they
are given as one curve, scarcely distinguishable from the horizontal axis. At these blood levels
the dye is obviously secreted by the nephridia, but leaves the animal slowly for reasons explained
in the text.

uncertain whether these are genuinely positive values since the method involves sub-
tracting from the titrated value the value of a blank. Morgulis (1922) reported
previously blood "glucose" levels of 19-26 mg. % for Homarus, with a considerable
variability in other decapods (1922, 1923). That the test was measuring sugar and
not some other reducing substance is indicated by the fact that in lobsters with the
heart destroyed, the blood was free from reducing substance fifteen minutes later.
With the injection of exogenous glucose, the lobster's nephridium behaves like
the vertebrate kidney. Up to blood levels of about 100 mg. % the urine remains
free of glucose. Urine-plasma ratios were: 0/81 ; 0/84; 0/94; 0/106. With fur-
ther elevation, glucose spilled into the urine, e.g., 30/202; 30/210, and at blood con-
centrations of 400-500 mg. % the U/P ratio approached one. The report by For-

210

J. WENDELL BURGER

ster and Zia-Wohlrath (1941) of a glucose U/P ratio of one in Homarus is due
undoubtedly to the high level of glucose employed.

Phlorizin resulted in glycosuria with or without priming by sub-liminal exoge-
nous glucose (10 lobsters). In short, under normal conditions there seems to be
an active mechanism which excludes glucose from the urine, a mechanism which can
be poisoned by phlorizin.

4. Phenol red. Phenol red was extracted from the blood by acid alcohol, read
colorimetrically at 440 in//,, alkalized and read again at 550 m/t. Urine and stomach
fluid were treated similarly. It was found desirable to use control blanks from in-

ro
O

ro

0

24

48
HOURS

72

FIGURE 2. Urinary concentrations (U) of phenol red after injection into the hemocoele,
over wider blood levels (P) than shown in Figure 1. Note the concentration of the dye in the
digestive fluid (S). The isolated point is a urine value.

dividual lobsters, and not to rely on a generalized control zero. Over thirty animals
were studied.

The pattern of nephridial excretion of phenol red is seen in Figures 1 and 2.
At low plasma concentrations (Figs. 1, 2), the dye is clearly concentrated by the
nephridium (the U/P ratio is a valid criterion; see paragraph on inulin). As the
plasma level is raised, the U/P ratio approaches one (cf. Fig. 4). This is the pat-
tern for all substances "secreted" by the nephridium ; concentration is evident at low
plasma concentrations but is apparently swamped by high plasma concentrations.
It is to be remembered that the lobster's kidney is the elaboration of only one pair
of nephrons.

Phenol red is not lost or absorbed through the gills or body covering. Four
lobsters were injected with 5-10 mg. and were placed for 18 hours in volumes of

LOBSTER EXCRETION

211

water sufficiently small to detect a small fraction of the injected dye. No phenol
red was detected in the external medium. Four animals were placed for 18 hours
in sea water containing 300 mg. % phenol red. No dye was detectable in the blood,
urine, or digestive gland. Phenol red placed in the stomach is absorbed. After
injection of phenol red, this substance was detectable in the cells of the gills. The
uptake of dye by branchial cells is seen more dramatically with Evans blue. After
a single injection of Evans blue, the gills become a bright blue, and so remain for at
least a month, when no dye is detectable in the blood. The dye obviously entered

8-

4-

o

u

8

16 24 32
HOURS

\ \

40 48

FIGURE 3.

Concentrations of para-aminohippurate in urine (U) and blood
stomach infusion. Nephridial secretion is evident.

after a 4-mg.

the cells of the gills, but did not move easily either out to the sea water or back to the
blood. One can speculate that for certain substances passage through the branchial
cells involves at least two steps : cellular uptake or penetration, and extrusion.

Fecal loss of phenol red is minute. The dye, however, is taken up by the di-
gestive gland. Extracts from the gland show phenol red when after injection the
blood level has subsided to insignificant amounts. The dye absorbed by the di-
gestive gland is secreted with the digestive juice at concentrations greater than those
of the urine (Fig. 2). Whether the individual cell of the digestive gland can con-
centrate more than the nephridial cell requires further study, since the mass of the
digestive gland is greater than that of the kidney, and the movement of water
through these two types of cells probably is not the same.

The dye secreted with the digestive juice is not lost with the feces or voided by

212

J. WENDELL BURGER

mouth, but is reabsorbed, and cycles back and forth between the stomach and the
digestive gland, slowly being lost with the urine (Fig. 1). The nephridia seem,
therefore, the principal port of exit for phenol red.

5. Suljobromophthalein (bromsulfalein). This dye, analyzed colorimetrically,
was explored over blood concentration of 0.02-100 mg. % in eight lobsters. The
U/P ratios were between one and two with no tendency to rise at the lower plasma
concentrations. It is concluded that bromsulfalein is not concentrated by the ne-
phridium. In the digestive juice, however, bromsulfalein is concentrated more

O
O

o

•

V
o\

Ol

o

I I

12

84 36 48

HOURS

60

FIGURE 4. Urine (U) blood (P), and digestive juice (S) values following a 400-mg. dose
of PAH, placed in the stomach. At high blood levels the U/P ratio approaches one. As the
blood level falls, the U/P ratio increases. This sort of curve is characteristic for substances
secreted by the nephridia.

strongly than phenol red. Without further quantitation, a numerical comparison
is dangerous, but with comparable doses (10, 25 mg.) of dyes into lobsters of com-
parable weight, the digestive juice bearing bromsulfalein showed about four times
the concentration of dye as that bearing phenol red. The differential ability of the
hepato-pancreas to concentrate this dye like the vertebrate liver indicates that the
name for this organ is more than an anatomical appellation. Like phenol red, the
loss from the lobster of bromsulfalein is very slow.

6. Para-aminohippurate (PAH). Using the analytic method of Smith ct al.
(1945), nephridial concentrations were explored in eight lobsters with blood levels

LOBSTER EXCRETION 213

of 120-0.5 mg. %. At low blood levels (Fig. 3), PAH is concentrated in the urine
and the U/P ratio rises as the blood level falls. As the blood concentration is
raised the U/P ratio approaches one (Fig. 4). The digestive gland does not con-
centrate PAH ; rather, the stomach juice concentration is below the blood concen-
tration.

7. Urea and nitrogen. Analyses for urea and volatile ammonia were made by
the method of Seligson and Seligson (1951). For non-protein nitrogen (NPN)
sulphuric acid digests were nesslerized directly. Urea and volatile ammonia were
undetectable in blood arid urine (10 animals). The natural range for NPN was
5-32 mg. %. Morgulis (1922) found a range of 12.5-13.3 mg. % for Homarus,
with a wider range in other decapods. No constant relationship was observed be-
tween blood and urine NPN. The U/P ratios varied from 0.7-6.0 in ten animals.

Exogenous urea is lost rapidly from the blood through the gills. Four lobsters
with occluded nephridiopores were injected with 500 mg. urea, then placed in
measured volumes of sea water. After one hour sea water/blood concentrations
in mg. % fell between 12.8/12.65 and 19.6/14.25. Since the external volume was
greater than that of the animals, most of the urea was in the external medium. In
four other animals injected with 500 mg. urea and with occluded nephridiopores,
no urea was detectable in the blood after the animals were 24 hours in running sea
water. Morgulis (1922) found a rapid disappearance of blood urea and ammonium
sulphate without nephridial occlusion in Panulirus. While currently there is no evi-
dence that urea is the main nitrogen excretory product, the above experiments in-
dicate a high in-out permeability to urea by the gills, and that this loss can occur
without nephridial participation. The presence of a substantial blood NPN must
indicate a low branchial permeability to certain nitrogenous compounds.

Inorganic substances

Chemical analyses for sea water, blood, and urine are given in Table I. In the
paragraphs following are data on individual ions. The purpose of this study is to
understand the range of capacities for dealing with various ion's and their ports of
entrance and exit. Robertson's studies (1939, 1949, 1953; Robertson and Webb,
1939) have defined at a non-experimental level the natural partition ratios for
Homarus vulgaris. Robertson (1949) has criticized correctly the presentation of
blood values in millimols/liter, and has emphasized the need for a correction for
the presence of blood proteins. In working up his ratios he seems to have used a
single average value of 29 g./liter protein for Homarus, although for a variety of
decapods he found values ranging from 29-80 g./liter. Allison and Cole (1940)
got values of 17.1-31.2 g./liter of hemocyanin for Mt. Desert Island lobsters. Our
data from over thirty Mt. Desert Island lobsters show a very high variability in
what we are somewhat arbitrarily calling blood protein. Values ranged from 11-
62 g./liter, with about 50 g./liter as a generalized usual figure. .The above values
were secured, following one of the methods of Robertson (1949), by drying weighed
calibrated volumes of serum to constant weight at 100° C. From the residual
weight was subtracted the weight of electrolytes (Na, K, Ca, Mg, Cl, PO4, SO4),
an arbitrary value for the water of crystallization derived from dried sea water,
known organic substances such as NPN and glucose. In the absence of any evi-
dence in the literature of massive amounts of some unknown substance, and since

214

J. WENDELL BURGER

known organic substances occur in fractions of a gram/liter, the values here pre-
sented while perhaps not entirely pure, do seem to offer a fair picture of the order
of magnitude and of the natural range of blood proteins. It perhaps should be
emphasized the values presented represented a selection of living lobsters some of
which were lethargic and sub-standard. The range for so-called normal lobsters
was 35-62 g./liter.

It is obvious that unless the non-electrolyte concentration of the blood is de-
termined for each animal, molal expressions for electrolytes are not entirely precise.

TABLE I

Analyses of lobster blood, urine, and of sea water

Sea water

Blood

Urine

pH*

(7.6-8.0) [10]

(7.45-7.6) [16]

(7.4-7.55) [26]

Dried solids,** g./liter

(35.5-35.9) [10]

(71.2-98.6-^47) [22]

(32.7-35.9) [8]

Organic solids, g./liter

• — •

50(35-62-*!!) [22]

( <0.4) [8]

Water, g./liter of blood

— •

950(935-959-^976) [22]

—

Milliosmols

(915-920) [12]

(920-952) [20]

(918-950) [20]

Sodium, mM/liter

440(428-445) [15]

472(451-488) [20]

474(454-486) [22]

Potassium, mM/liter

(9-10) [10]

(6-11) [14]

(4-10) [16]

Magnesium, mM/liter

(50-52) [8]

6.8(5.4-8.6) [15]

11.4(7.2-17.6) [15]

Calcium, mM/liter

(9-10) [8]

15.6(13.1-18.6) [15]

12.7(5.6-16.3) [15]

Chloride, mM/liter

503(476-515) [27]

470(465-490) [27]

505(490-520) [27]

Phosphate, g./liter

—

0.016(0.008-0.018) [8]

0 [16]

"Glucose," g./liter

—

(0.22-0.40) [10]

(0-0?) [10]

Non-protein nitrogen,

g./liter

—

(0.05-0.32) [10]

(0.05-0.32) [10]

Volatile ammonia

0 [4]

0 [10]

0 [12]

Urea

0 [4]

0 [10]

0 [12]

Figures within parentheses give the range of values. These fluids are to be considered as
having a natural variability and are not of constant composition. The above ranges exceed the
experienced variability of the methods. Figures outside of parentheses are average values.
Figures to the right of arrows are unusual values. Figures in brackets show the number of
samples (sea water) or the number of animals analyzed. The various zeros are zeros for the
method used. Analyses were done in duplicate or triplicate.

* Measured by Beckman and Cambridge meters. Sea water from the Bay was about 8.0;
the running laboratory water was variable.

** Includes water of crystallization.

Some of these data were secured over several years, some in a single summer season, and some
in shorter periods. For this reason they should be viewed only as general parameters. For
critical quantitative work complete work-ups of individual animals should be made.

For this reason we have presented our data in Table I in the raw form of mM/liter.
A crude generalized correction can be made by taking the blood protein as 50
g./liter.

1. Phosphate. Fiske-SubbaRow phosphate (method given by Hawk et al.,
1947) was absent from sixteen urines. Blood levels of eight lobsters were about
1.6 mg. % (range: 0.8-1.8). If exogenous inorganic phosphate (sodium salts
mixed to a slightly alkaline pH) is injected, phosphate spills over into the urine.
With blood levels of about 8 mg. %, phosphaturia occurred. The nephridium be-
haves toward inorganic phosphate as it does to glucose.

LOBSTER EXCRETION 215

2. Magnesium and calcium. An ethylenediamine tetraacetic acid (EDTA) ti-
tration, developed by Dr. Martin Rubin (personal communication), was used.
Calibration studies indicated that the method is serviceable if one discards all sam-
ples which do not titrate sharply. The method gives initially combined magnesium
and calcium, then magnesium. Calcium is obtained by subtraction.

The analyses of Cole, Smith in Cole (1940) showed there is a marked partition-
ing between sea water and blood in Homarus. Robertson (1953) found a molal
magnesium U/P ratio of 1.8, and a calcium U/P ratio of 0.64. Our data on fif-
teen lobsters give the following molar ratios (see above) : blood/sea water: Mg +
Ca, 0.37; Mg, 0.13; Ca, 1.68. Urine-plasma ratios were: Mg + Ca, 1.16 (range:
1-1.3) ; Mg, 1.7 (range: 1-2.6) ; Ca, 0.81 (range: 0.53-1). In most animals mag-
nesium is concentrated in the urine and calcium is reduced. There are instances,
however, where each ion is not affected (U/P ratio of one), and there was one ani-
mal where both the magnesium and calcium U/P ratio was one. In dilute sea
water, the magnesium U/P ratio falls, and may drop to 0.8. In short, some quali-
fication must be placed on the idea that the lobster's nephridium concentrates mag-
nesium in the urine, and conserves calcium, although this is the usual situation.

The gills and carapace are relatively impermeable to magnesium. There was
no elevation in blood or urinary magnesium in four lobsters placed for twelve hours
in baths made of half sea water with magnesium chloride added to return the water
to near its original equivalence. In lobsters naturally anuric, blood magnesium is
at normal levels. There seems no tendency in these animals for magnesium to
build up in the blood as might be expected if the gills were permeable inwardly to
magnesium.

Since the lobster usually produces urine, it is losing magnesium with the urine.
The daily urinary loss approximately equals the magnesium found in 5 ml. of sea
water. The port of entrance seems to be the stomach. With urinating lobsters
sea water is drunk intermittently with the food or on an empty stomach (see sec-
tion on Intake and Output), and this sea water is absorbed. The stomach normally
contains a concentration of magnesium greater than that of the blood, although in
unfed lobsters empty stomachs are found frequently. Sea water placed in the
stomach is slowly absorbed, and as shown below, the stomach fluid does not furnish
the bulk of the fluid for the urine (24 mL/diem). It must not be thought that
there is a steady rapid movement of magnesium-laden fluid into the blood from the
stomach which would result in high levels of blood magnesium. The anuric ani-
mals mentioned above had empty stomachs.

With urinating lobsters, injected magnesium sulphate or chloride results in an
increased magnesium excretion although the U/P ratios do not rise above those
found normally ; magnesium sulphate has a marked diuretic and then an anesthetic
effect. Exogenous magnesium placed in the stomach is absorbed (see Intake and
Output). In dilute sea water lobsters conserve magnesium. Four animals kept
for 24 or 48 hours in 60-70% sea water had urinary levels lower than plasma levels.
Blood levels remained within 1 mM/liter of the levels found with full sea water.
Under these same conditions blood calcium fell more obviously, 2-4 mM, and the
nephridium did not conserve calcium more than it did in full sea water.

It is obvious from Table I and from the above data that the nephridium has very
modest powers in partitioning these two ions (Mg, Ca). Magnesium is lost more
through the volume of urine flow than by nephridial concentration. Calcium is only

216 J. WENDELL BURGER

weakly conserved. It should be noted that all the lobsters used were hard-shelled
animals near the end of an inter-molt period.

Levels of blood magnesium can be changed without nephridial participation, at
least on a short term basis. Blood levels were raised up to 33 mM/liter by placing
magnesium chloride solutions in the stomach of anuric animals. Over a subsequent
11-hour period, blood magnesium fell to 10-21 mM/liter without urine production
or without a rise in stomach magnesium. During this experiment, blood calcium
levels remained unchanged. Either the magnesium was taken up by the tissues
and carapace or it passed out through the gills.

3. Sulphate. Analyses were semi-quantitative. Known volumes of fluid were
treated with known volumes of barium chloride in an ice bath, and the amount of
precipitate was measured. The general picture for this ion is like that for mag-
nesium. The gills and carapace are relatively impermeable to sulphate. After 18
hours, there was no elevation of blood or urinary sulphate in six lobsters placed in
baths with elevated sulphate (Na, Mg). Blood and urine sulphate was elevated
when sulphates were placed in the stomach. In 18 normal lobsters, the sulphate
U/P ratio varied between one and two, but rose to four with small amounts of in-
jected sulphate. With increased dosage up to blood levels of 300 mg. %, the U/P
ratio fell toward one. Increased sulphate was markedly diuretic to urinating lob-
sters. Following the elevation of blood levels, the return to normal was slower than
for magnesium ; as with phenol red, days were required to effect normal blood levels.
This seems to indicate that sulphate is lost primarily through the nephridia.

4. Monovalent ions. Natural values are given in Table I. Analyses for sodium
and potassium were done by flame photometry. Chlorides were done by electrical
conductivity measurements of silver nitrate titrations, or by a mercuric nitrate ti-
tration with s-diphenyl-carbazone as a visual indicator.

On the basis of a generalized calculated molality, using 50 g./liter protein, chlo-
ride ratios of sea water/blood and urine/blood are essentially one (1-1.01). This
same ratio holds for serum dialyzed through Visking membranes against full
strength and 60% sea water. Cole's data (1940) and Robertson (1949) give this
same ratio. Under normal conditions of undiluted sea water the distribution of
chloride in the blood and urine seems to be a passive one. This might be expected
when one considers that the blood proteins have a negative charge and that there
is no heavy concentration of a non-diffusible cation in the blood.

Cole (1941) has presented evidence that in dilute sea water the lobster can se-
crete chloride inwardly. Twelve lobsters whose blood/sea water molar ratio av-
eraged 0.95 (sea water, 504 ± 9 mM/liter) were placed in dilute running sea water
which did not exceed 394 mM/liter. Between 37 and 118 hours in this diluted sea
water, and following an initial hemodilution, the blood/sea water ratio rose to 1.05-
1.11 in five lobsters sampled.

In twenty-seven analyses of sea water, blood, and urine for normal lobsters, we
found without exception that the molar blood/sea water ratio was below one, and
the urine/blood ratio was above one. The millimolar differences between sea water
and blood were about 25-35. Dialysis of serum of three lobsters (La, b, c)
against sea water for 24 hours through Visking membranes gave the following
chloride values in mM/liter: sea water, 511, 512, 518; La, 478, 478; Lb, 480, 485;
Lc, 483, 487. Dialysis of serum of three lobsters (Ld, e, f ) against dilute sea water
gave the following values: dilute sea water, 308, 308, 311; Ld, 287, 287; Le, 288,

LOBSTER EXCRETION 217

291 ; Lf, 287, 292. These values show that there is no capacity of the blood which
tends to elevate blood chloride in situations where the blood is separated from full
and dilute sea water by an inert membrane. Under dilute conditions, any marked
rise in blood chloride can not be due to the blood itself or to an inert membrane
effect.

Cole's experiment was repeated with seven lobsters (Ll-7). For three lobsters
(Ll-3) pre-dilution values in mM/liter averaged: sea water, 510 ± 5; serum, 470
± 5. Running dilute sea water varied between 310-338. After 72 hours, serum
chloride values were : 370, 370; 357 ± 3 ; 355 ± 3. For L4—5. initial control values
were: sea water, 500 ± 2; serum, 485 ± 5. Dilute sea water was 358-371. After
96 hours, serum values were 376, 380 ; 380, 384. For L6-7, control values were :
sea water, 503 ±7; serum, 485, 485; 477 ± 1. Dilute sea water was 378-392.
After 116 hours in diluted sea water, serum values were: 420, 424; 412, 417. It
should be noted that during the first twenty-four hours of dilution blood chloride
falls below the above values, and subsequently rises.

In all the above animals in dilute sea water, the blood/sea water ratio was above
one, an event which does not occur in full sea water or after dialysis with an inert
membrane. It would seem that this elevation in blood chloride is due to some ac-
tive process.

In all the above animals in dilute sea water, the urine chlorides were 20-33
mM/liter higher than the blood chlorides. When calculated on a molal basis, this
difference disappears (see above). These data indicate that the nephridium is
losing chloride at the elevated blood levels and the nephridium is not participating
in the elevation of blood chloride nor is it helping to conserve chloride. In other
Crustacea, nephridia are known to conserve chloride and the gills are known to
secrete chloride inwardly. Here in the lobster, the nephridia clearly do not play
such a role. While our data do not irrefutably establish the secretion of chloride,
the most likely site for the agency effecting the elevation of blood chloride is the
gills. The persistent elevated chlorides after 116 hours would seem to indicate that
the higher blood chloride is not due to tissue chloride which has come out of cells
but has not been carried away by the urine, diffusion, etc.

While it is clear that in full- strength sea water the distribution of chloride can
be accounted for on a passive basis, the sodium picture is a bit more complex. On
a molal basis there are about 60 equivalents more sodium in the plasma than in sea
water. The sum of the equivalents of the major electrolyte cations of the blood
(Na, K, Mg, Ca) accounted for about 99% of the cations of sea water. Since the
data were worked up on an average basis the percentage figure should not be taken
too literally, but merely to indicate there is not an unforeseen discrepancy between
the cationic sum of blood and sea water (the blood and sea water are roughly iso-
tonic; see below and Table I). The extra sodium of the blood can be accounted
for on the basis of the exclusion of most of the magnesium of sea water from the
blood. If one groups sea water magnesium and calcium, subtracts plasma mag-
nesium and calcium and the equivalents bound to blood sulphate (arbitrarily taken
as 10 mM/liter from data of Robertson and Webb, 1939 and Cole, 1940), one has
about 50-60 equivalents which is the excess of sodium in the blood. Blood and sea
water potassium are very close to each other (Table I).

The U/P partition is not so easily explained. There seems on a molal basis to
be a persistent deficit (10-30 mEq.) of combined urinary sodium and potassium,

218 J. WENDELL BURGER

largely sodium. Urinary potassium tends irregularly to be several milliequivalents
lower than plasma (not higher) ; combined magnesium and calcium add only several
milliequivalents to urine as compared to blood. Blood/urine potassium, magne-
sium, and calcium thus tend to cancel each other. Robertson (1949) gives a molal
U/P) ratio for sodium of 0.99, indicating again a sodium deficit. It may be that
since the urine is isotonic with the blood, there is an increased activity in the urine
where the cations are free from the depressing influence of the blood proteins.

The above natural data and the following experimental data indicate that sodium
chloride is freely mobile across the gill-blood and blood-urine barriers. On placing
lobsters in dilute sea water (60, 70, 75, 80, 90%) the osmotic uptake of water, as
judged by the gain in body weight, is completed in less than one hour, with a gain
of 1-2% of body weight (18 animals). While this uptake results in hemodilution,
the blood sodium chloride continues to fall, approaching the concentration of the
external medium in twelve hours or less. The blood concentration after the first
fifteen minutes of dilution falls in an almost linear fashion with a slope varying with
the amount of external dilution. Within the first three hours the bulk of the sodium
chloride to be lost is lost. Blood sodium chloride begins to asymptotically approach
the external concentration but remains superior to the external concentration on a
molar basis. On returning the lobsters to full-strength sea water, the concentration
curves are not the reverse of the dilution curves. During the first hour the blood
concentration rises very steeply and asymptotically approaches normal values in
three to six hours. After injecting sodium chloride sufficient to raise blood levels
15-20 mM/liter, the blood returns to normal values in three hours. Under these
altered sodium chloride loads, the urine reflects the blood concentration, rising or
falling in sodium chloride concentration with blood concentration or dilution. In
anuric lobster, blood sodium chloride adjusts to control values following the injec-
tion of hypo- or hypertonic saline within about three hours.

The above data suggest that while under conditions of external dilution, the
lobster has the capacity to actively raise its blood chloride, under normal conditions
of undiluted sea water, sodium chloride is passively distributed between sea water,
blood, and urine. That is, normally there is no active process which is acting on
sodium chloride as such. The amount of sodium in the blood can be explained as
the amount in sea water plus the equivalents replaced for the sea water magnesium
which is excluded from the blood. The discrepancy between blood and urine so-
dium is too small to support the idea that some active partitioning of sodium is being
effected by the nephridium. Indeed, the dilution experiments indicate that the ne-
phridium does not have a chloride-conserving mechanism.

Intake and output

While it is obvious that in the natural environment, the intake of organic chemi-
cals is by mouth, it is not recognized that in the lobster sea water enters by mouth.
The stomach capacity is about 10 ml. for a 500-gram animal. On eating, the food
is diluted with a good deal of sea water. But even lobsters with empty stomachs
will fill the stomach with sea water. This drinking is not due to fright or experi-
mental handling. If the stomach is emptied by pipette, some animals will imme-
diately fill the stomach with sea water. The stomach contents are never regurgi-
tated unless the animal is in great distress. The stomach contents are always com-

LOBSTER EXCRETION 219

pletely absorbed. The above observations come from dozens of lobsters made over
five years and are not quick impressions. Many lobsters were followed at intervals
for several days. It is not to be inferred that all non-feeding lobsters keep their
stomachs full of sea water. The drinking is an intermittent affair, whose cause was
not determined.

Since all substances mentioned in this report, with the exception of fish protein
and lobster NPN which were not studied for this purpose, were found experimen-
tally to be absorbed from the gut when placed in the stomach, the diet and imbibed
sea water contribute to the internal electrolytic and non-elecrolytic content of the
lobster. Since, too, the gills and carapace were found to be relatively impermeable
to magnesium and sulphate, the stomach seems, to be the principal port of entrance
for these substances. Possible branchial uptake of calcium and phosphate was not
studied, but these obviously enter with the diet. While water and monovalent ions
do enter from the stomach, this route does not appear as the principal route (see
below). The principal route seems to be the gills.

The nephridia and the gills seem to form the principal points of exit for the
substances under discussion. The feces were not studied critically. Their fluid
volume is small and only minute amounts of phenol red or bromsulfalein are lost
with the feces. There seems little likelihood that the feces play any great role with
the chemicals here discussed.

The concentrating powers of the nephridium have been discussed. The daily
chemical loss through the nephridium depends not only on the nephridium's parti-
tioning powers but also on the urine flow. For so-called normal lobsters the urine
output is about 1 ml./hr./0.5 kg. (determined by hourly collections over 12-hour
periods), with flows up to 4 ml./hr. with extreme diuresis, e.g. after injected mag-
nesium sulphate. There are various degrees of oliguria and anuria; lobsters may
be completely anuric for at least a month. Nephridial occlusion is not fatal to pre-
viously urinating lobsters over two- and three-week periods.

Contrary to possible supposition, the fluid volume of the lobster is not regulated
by the volume containable within the exoskeleton. Fifteen to twenty-five ml. can
always be injected into a normal animal (c. 0.5 kg.). The volume increase between
emptiness arid distension of the stomach (c. 10 ml.) and the nephridial bladders
(c. 6 ml.) requires internal space into which these organs can expand. If one draws
blood repeatedly, one sees directly a loss of blood volume which is not repaired in
a few hours or even days to the initial volume. A lobster may be mobile and active,
and yet from it blood can be drawn only slowly in contrast to the normal situation
where blood can be drawn rapidly. Through the transparent ventral surface of the
tail one can see the reduced blood volume.

In the normal animal there are about 24 ml., principally a sodium chloride solu-
tion, flowing through the animal and out as urine. This is at least several times
the amount that is absorbed from the stomach. Indeed, urine at the above rate can
be formed with lobsters with empty stomachs. It would appear that this sodium
chloride solution must enter through the gills. Experiments with injected sodium
chloride solutions (hypo- or hypertonic) with nephridial occlusion showed that
water and sodium chloride can pass quickly (several hours) in either direction
through the gills. Iodine also passes in either direction through the gills. Isotopic
flux studies are necessary to define these parameters. The minimal net flux of
water and sodium chloride is the urine flow, and this flow means that a sodium

220 J. WENDELL BURGER

chloride solution is moving inwardly across the gills faster than it is moving out-
wardly across the gills.

The inward direction of this flow of a sodium chloride solution seems to be gov-
erned by the blood proteins. Anuria and oliguria were associated with low blood
protein as determined by the copper sulphate specific gravity method or by the
weight per volume of blood. A specific gravity below 1.029 accompanied poor
urine formation. So-called normal specific gravities were in the 1.032-1.033
range. In some anuric lobsters, erratic transitory flows of urine could be induced
by the infusion of hypo- or hypertonic solutions (Na, Mg, Cl, SO4). The transfu-
sion, however, of 10-15 ml. of serum from urinating lobsters into anuric lobsters
induced permanent urine flows, i.e., the regular production of urine over a subse-
quent one week's test period (8 animals). Osmolar measurements by the Ther-
mistor method (Table I) always gave isotonic or slightly hypertonic, never hypo-
tonic, values for the blood as compared with sea water. The withdrawal of large
amounts of blood from urinating lobsters, e.g. 0.1 of the blood volume (see Burger
and Smythe, 1953), did not check urine formation, apparently because the blood did
not become markedly diluted. With a 1200-gram lobster with a blood specific
gravity of 1.032, the removal of 24 ml. of blood did not check urine formation nor
was the specific gravity of the blood lowered 1.5 hours later. With a 650-gram
animal, blood protein sp. gr. 1.0335, the specific gravity one and five days after the
removal of 10 ml. of blood was 1.032.

Urine formation could not be correlated with hemocoelar blood pressure, a rise
in which is accompanied by a rise in arterial pressure (Burger and Smythe, 1953).
Hemoconcentration, resulting from keeping lobsters in the air, suppresses urine
formation. In view of the above transfusion experiments, it would appear that
the non-diffusible molecules of the blood draw in water principally through the gills,
and this water is bailed out by the nephridium as urine. With this water moves
sodium chloride. Ions to which the gills are not readily permeable, as well as
some sodium chloride solution, enter from the stomach and leave through the ne-
phridium. The nephridium keeps the blood volume below the fluid capacity of the
shell. The constant nephridial removal of water from the blood should slightly
raise the osmotic pressure of the blood. Since the blood circulates rapidly through
the gills (Burger and Smythe, 1953), a very slight gradient should be enough to
extract water from the external medium. The nephridium, however, is not merely
an organ for maintaining fluid volume. It is capable of secreting some organic and
inorganic substances and of restraining completely or partially other constituents of
the blood. In short, it acts like a kidney. The all-over pattern seems to be one
where substances are carried away by a high urine flo\v rather than by a powerful
concentration of secreted substances. Only with phenol red, and only with very
low blood concentrations was nephridial concentration marked. Since anuric lob-
sters live for at least a month without eating or drinking and since electrolytes such
as magnesium do not build up in the blood, there is presumably no net inward flow
of fluid as is found in the urinating animal. If the nitrogenous waste is ammonia,
this can be lost through the gills. While there is no evidence that the lobster forms
urea, exogenous urea is lost through the gills, and not through the nephridium.
The absence of any volatile ammonia in the urine and the lack of any clear U/P
difference in NPN make it dubious that the kidney is concerned with primary ni-
trogen excretion, although the occurrence of a persistent blood NPN indicates ni-

LOBSTER EXCRETION 221

trogenous compounds which are not readily diffusihle through the gills and which
are removed in the urine. The gills, stomach, and nephridia form an interlocking
system of entrance and exit, each with individual capacities.

This report gives no new information on the mechanism of urine formation.
That the blood and urine are absolutely isosmotic (Table I) would be expected
when one sees the extreme delicacy of the walls of the large nephridial bladders.
Despite this delicacy, substances such as phenol red can be held at higher than
blood concentrations. Pressure from the nephridial fluid must distend these blad-
ders and one can imagine a small isosmotic filtration from bladder to blood, or the
reverse when the blood pressure is raised.

From the work of Peters (1935) it is assumed that urine is formed by nitration.
The ready passage of fish blood proteins and of inulin supports the filtration idea.
Histologically, the nephridium seems more comparable to the aglomerular fish kid-
ney where filtration does not occur. The nephridial arteries lie behind a wall of
glandular epithelium and there seems to be no glomerulus-like structure or arrange-
ment. This problem is worthy of further study.

SUMMARY

1. A method is given for safely securing repeated evacuations of the nephridial
bladders of the lobster, Homarus, thus permitting experimental analysis of ne-
phridial function.

2. Routine chemical analyses for blood, urine, and sea water are given.

3. Experimental analysis shows that the nephridium can concentrate in the
urine phenol red, para-aminohippurate, magnesium, and sulphate. At normal blood
levels, it completely excludes glucose and Fiske-SubbaRow phosphate. Phlorizin
blocks glucose retention. With high blood levels, the secretory or exclusion powers
of the nephridium are swamped. Calcium is partially excluded from the urine.
The nephridium is indifferent to inulin, bromsulfalein, dogfish hemoglobin and
plasma protein, sodium and chloride. The ability of the digestive gland to con-
centrate phenol red and bromsulfalein in the digestive juice is noted.

• 4. Exogenous urea is lost through the gills.

5. The gills and carapace are relatively impermeable to magnesium and sul-
phate, and to phenol red, but are freely permeable to water, sodium chloride, and
to exogenous urea.

6. In full-strength sea water, the distribution of sodium chloride between sea
water, blood, and urine seems to be passive. In dilute sea water, experiments
indicate that blood chloride is elevated in some active fashion, presumably by the
gills. The nephridium, however, does not aid in the conservation of chloride.
Chloride is lost at the elevated blood level. The nephridium does not seem to
have powers to deal actively with sodium chloride.

7. The normal urine flow is approximately 1 ml./hr./0.5 kg., with wide varia-
tions. Water and sodium chloride for this flow enter largely through the gills
although there may be intermittent contributions from the stomach. Magnesium
and sulphate enter largely through the stomach. Calcium, in addition to that of
sea water, is in the food. The lobster intermittently drinks sea water with food
or on an empty stomach. The stomach contents are absorbed completely but
not rapidly enough to furnish the fluid for the bulk of the urine flow. The daily

222 J. WENDELL BURGER

urinary excretion of magnesium roughly ecjuals the magnesium found in 5 ml.
of sea water. All test substances entered the blood from the stomach. In any
study of sea water and blood, the gastric contribution to the chemistry of the
blood, even for electrolytes, must be considered.

8. Lobsters from commercial pounds are frequently oliguric or completely
anuric. Inability to form urine is not lethal, at least over a one-month period.
Erratic transitory urine flows can be induced in anuric lobsters by the injection of
various saline solutions. Normal urine flow can be induced in anuric lobsters
by the transfusion of serum from one lobster to another. Apparently, substances
such as blood proteins which are not diffusible through the gills draw in water
which is bailed out by the nephridia. Osmolar measurements found the blood
isotonic or slightly hypertonic, never isetonic, to sea water.

9. While the nephridium has a range of capacities for dealing with individual
substances (secretion, exclusion, partial exclusion, and a lack of partition power),
its secretory capacities are not great and are masked easily by elevated blood
levels. Nephridial removal of substances from the blood depends more on a
flush-out principle, using large urine flows, than upon secretory powers. Some
substances such as exogenous urea are lost by the gills and not by the nephridium.
Together, the nephridia and the gills form an excretory system, each with in-
dividual capacities.

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dog and man. /. Clin. Invest., 24 : 388.
WAITE, F. C., 1899. The structure and development of the antennal glands in Homanis

americanus Milne-Edwards. Bull. Mus. Comp. Zool., Harvard College, 35 : 151-204.

BODY GROWTH VERSUS SHELL GROWTH IN
BALANUS IMPROVISUS x

JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT

Duke University Marine Laboratory, Beaufort, N. C.; and Department of Zoology,

Duke University, Durham, N. C.

The acorn barnacles are unique among the arthropods in that the body is per-
manently enclosed by, and separated from, the outer shell of calcareous plates. The
chitinous exoskeleton of the body, the inner chitinous lining of the mantle adjacent
to the shell-secreting tissues, and the opercular hinge are shed at regular intervals
of two to three days in Balanus improvisus (Costlow and Bookhout, 1953) and in
Balanus amphitrite niveus (Costlow and Bookhout, 1956). The calcareous shell,
which grows continuously, is not shed and apparently is not affected by the molting
cycle of the body.

Numerous papers have appeared which deal with the secretions of the endocrine
organs in relation to molting of Malacostraca. These have been reviewed recently
by Knowles and Carlisle (1956). Other research workers have been concerned
with the subdivision of the molting cycle (Carlisle and Dohrn, 1953; Drach, 1936,
1939; Hiatt, 1948; and Kincaid and Scheer, 1952). Most of these studies have
been confined to two groups, the Brachyura and Astacura, and have not been
primarily concerned with actual growth. Relatively few investigators have con-
sidered the increase in size following ecdysis. Tait (1917) studied molting in the
isopod, Ligia oceanica, and makes reference to size after molting. Olmsted and
Baumberger (1923) worked on the form and growth of grapsoid crabs. Marshall
(1945) noted that molting can occur without growth in Panulnris argus maintained
in live cars and Dawson and Idyll (1951) confirmed these observations from
studies on tagged individuals. In all of these studies direct measurements of the
exoskeleton could be made, a procedure which could not be followed in the barnacles
without removing the body and thereby killing the animal. It is not surprising,
therefore, that most studies on growth in barnacles have been confined to the shell.

The rate of shell growth has been studied for several species of barnacles, under
both natural and experimental conditions. Barnes (1955) described the growth
rate of Chthamalus stcllatus and Barnes and Powell (1953) studied the effect of
varying conditions of submersion on shell growth in Balanus balanoides and Balanus
crenatus. Crisp (1954) described morphological changes in the shell which are
associated with differences in yearly growth rates of Balanus porcatus. The rela-
tionship between the daily increments of the continuously growing shell and the
growth rate of the body, following a series of consecutive molts, is not known for
any of the acorn barnacles. In order to obtain data over a series of consecutive
molts of the same individuals it is theoretically possible to measure the exuvia, or

1 These studies were aided by a contract between the Office of Naval Research, Department
of the Navy, and Duke University NR 163-194.

224

BODY VERSUS SHELL GROWTH IN BALANUS

225

-•>] I - :•;'.'. ' , ; • j -; . t 3 f

I" :!t t •:•:•;, • J ' j %'.?•• / ,-1 ,:

K^^mAmiM

FIGURE 1. Sagittal view of a typical barnacle, with left side of shell removed, showing
relationship between internal chitinous covered body and external calcareous shell.

Abbreviations
c — cirri of body

ca — carina : posterior plate of shell
b — basis

g —gill

r — rostrum : anterior plate of shell

sc — scutum : anterior opercular plate

sr — shell ring: calcareous ring to which opercular

plates are attached by opercular hinge
tr — tergum : posterior opercular plate

some portion of it, following ecdysis. Since the shed exoskeleton tends to wrinkle,
accurate measurements of total length or width are not possible. The mandibles
and maxillae, however, are sufficiently rigid to retain their shape and size and, as
will be demonstrated, reflect the per cent increase of the entire body at molting.

The objectives of this study were three-fold : one, to determine whether or not
body growth always accompanies an ecdysis ; second, to follow the increase in body
size, as represented by two mouth parts, through consecutive molting periods;
and, third, to compare the relative increase in size of the body with that of the shell.

The authors wish to thank Dr. R. J. Monroe, Professor of Experimental Sta-
tistics, North Carolina State College, for reviewing the statistical portion of this
paper, and Dr. Henry Kritzler for providing the original plate for Figure 1.

METHODS

Balanus improvisus which had metamorphosed from the cyprid during the
previous 24 hours were obtained from pyralin plates suspended on racks beneath

226 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT

the laboratory dock. Twenty barnacles were maintained individually in plastic
compartment boxes containing Chlamydomonas sp. at 25° C. under daylight
fluorescent lights. Each compartment was examined twice daily for the presence
of molts. When exuviae were found they were removed and mounted on glass
slides. In young barnacles the mouth parts, maxillae and mandibles, were dis-
sociated from the remainder of the exoskeleton by repeated manipulation of the
cover-slip. In older barnacles it was necessary to dissect out these parts with
insect pins or glass needles. The width of the mouth parts, at the base of the
spines or teeth, was then measured with an ocular micrometer mounted in a com-
pound microscope. If the actual molting process was observed, the exuvia was
removed as soon as possible, the mouth parts measured, and then returned to a
depression slide containing the culture medium. Twenty-four hours later the
maxillae and mandibles were measured again to determine if any changes in size
had occurred.

Daily measurements were also made of the barnacle shell, rostro-carinal length
and lateral-lateral width, with an ocular micrometer mounted -in a dissecting micro-
scope. The barnacles were observed and measured over a period of 60 days, an
average of 20 molting periods.

A second series of 20 barnacles, of varying size and age, was obtained from the
harbor. The entire body was removed from the shell and the widths of the thorax,
maxillae, and mandibles were measured. The thorax was measured to determine
if, in living barnacles, a relationship exists between the size of the mouth parts and a
major body dimension, or if growth of the mouth parts is differential.

RESULTS AND DISCUSSION

The body of a sessile barnacle is so completely enclosed by a calcareous shell
(Fig. 1) that direct measurements of the body cannot be made over a series of molts.
To obtain data on body growth in relation to molting it was necessary, therefore,
to resort to a measurement of mandibles and maxillae of a consecutive series of
exuviae. It must be established, however, whether the increase in size of an
appendage, or a distinct portion of the animal, reflects the increase of the body
or represents a differential growth rate which is restricted to a specific part. The
results obtained from measurements on whole bodies and mouth parts of 20 living
barnacles taken at random from the harbor indicate that there is a definite correla-
tion between the width of the thorax and the width of the maxillae. A ratio of
6.271 ± 0.104 was found in barnacles of varying size and age. A simple correlation
coefficient of 0.922, significant at the 1 per cent level, was determined from the
sample. This value exceeds that given by the standard tables at 18 degrees of
freedom and indicates that 85 per cent of the variability in thorax size is associated
with concomitant variability in maxillae size, leaving 15 per cent attributed to other
factors including chance. Thus the maxillary size, as well as per cent increase at
molting, is not differential but reflects the increase in size of the body.

Even though the barnacles were examined every 12 hours, it was not possible
to observe the actual time of molting for each barnacle. Thus there was the pos-
sibility that changes in size of the exuviae could occur from the time of ecdysis until
actual measurement. To determine if shrinking or swelling of the shed mouth
parts occurred, they were measured immediately after molting, when the process was

BODY VERSUS SHELL GROWTH IN BALANUS

227

25

kl

cc
u,5

o

MAXI LL A
[] MANDIBLE

I. Ill ll

3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22

MOLTS

FIGURE 2. Per cent increase of maxillae and mandibles of one Bahuuis iinproi'isus from the

2nd to the 22nd molt.

observed, and again after 24 hours' immersion in the culture medium. No change
in size was found in the mouth parts after 24 hours and, inasmuch as all measure-
ments were made within 8 to 12 hours after each ecdysis, the recorded size accurately
indicates the width of the mouth parts prior to molting.

4.8 -
4.6 -
4.4 -
4.2 -
4.0 H
3.8 -
3.6 -
3.4 -
3.2
3.0
2.8
2.6
2.4
2.2
2.0

I .8

I .6

I .4

I .2

1.0
0.8
0.6
0.4
0.2
.0

0

BODY GROWTH

10

15

20

25 30
DAYS

35

40

45

50

55

FIGURE 3. Comparison of shell growth and body growth over 23 molting periods for one
Balamts iinprovisus. Figures for maxillae size, representing increase in body size; X 10 -1.

228 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT

The mandibles, while more difficult to measure accurately, are approximately
twice the width of the maxillae. This ratio is also maintained in barnacles of
varying size and further supports the hypothesis that the size increase of the
maxillae is not differential but reflects the size increase of the entire body.

Body growth. Figure 2 compares the per cent increase in size of the maxillae
and mandibles from the second to the twenty-second molt. It will be noted that the
per cent increase corresponds closely at each molt. That is, if the mandible increases
greatly following one molt, the maxillae increase approximately the same per cent
and similarly, if one shows no increase at molting, no increase is found in the
other. Figures 2 and 3 indicate that increase in size of the maxillae does not
necessarily accompany molting. There are several periods, ranging from one to
three consecutive molts, when no measurable increase in maxillae size occurred
(Fig. 2, molts 11, 13, 18, and 22; Fig. 3, molts 21, 22, and 23). Tait (1917) re-
ported that no discernible increases in size occurred when Ligia occanica molted, but
he did not actually measure the pre- and postmolt animals. Carlisle (1956) found
this to be true only when the animals were starved. Marshall (1945) reported
the absence of growth after molting in Panuliris argus maintained in live cars and
Travis (1954) also found that increase in length did not necessarily accompany
molting in P. argus maintained in the laboratory. That this condition cannot be
attributed solely to sub-optimal environment of the live cars and the laboratory is
evidenced by the work of Dawson and Idyll (1951). Their study of tagged in-
dividuals, recovered from the area around the Florida Keys, showed an absence of
growth in some individuals and 7 cases of "negative growth." The latter, how-
ever, were attributed to faulty measurements. Lloyd and Yonge (1947), working
with female Crangon vulgaris under laboratory conditions, found that normally
there is an increase in size after molting. No increase occurred, however, at the
molt leading into the egg-laying condition. Figure 3 gives the increase in maxillae
size for one of the 20 D. improvisus taken at random. Molting without growth,
however, was observed in each of the 20 barnacles studied, although not necessarily
at the same time or molt. This was true even though the same quantity of food
was supplied each day.

As shown in Figure 3, the increments in maxillae size over a period of 20 molts
are variable. In some instances, a slight increment was followed by a molt which
produced considerable increase in size. Olmsted and Baumberger (1923) found
that Pachygrapsus crassipcs does not increase by the same fraction at each molt
and that the increment gradually diminishes as the crabs become larger. Lloyd
and Yonge (1947) also noted a decrease in size increment at molting with increase
in over-all size but attributed it to egg production. These observations were ap-
parently made on molts of various sized individuals and not consecutive ecdyses
for the same animals. It does apply, however, to size increase in the maxillae of
B. improvisus. Olmsted and Baumberger (1923) attribute some reduction in
increment at molting to the decrease in molting frequency with age in P. crassipes.
Costlow and Bookhout (1953) found that the frequency of molting in B. improvisus
continued to be on the average of two to three days through the first 40 molts.
Thus, reduction of molting frequency with age is not a factor which influences the
gradual reduction in size increment observed for B. improvisus during the first 20
molting periods.

BODY VERSUS SHELL GROWTH IN BALANUS

229

Shell growth. In contrast to the body, which may grow only at the time of
molting, the calcareous plates of the shell grow continuously. Figure 3 compares
the growth rates of shell and maxillae for one barnacle. As indicated previously,
there were periods up to three molts when no increase in maxillae size occurred.
During this same interval of time the shell continued to grow. Costlow and
Bookhout (1953), comparing shell growth of B. improvisus in the laboratory with
that found in the natural environment of the harbor, found that while the two rates
followed the same general curve, growth in the laboratory was approximately one-

4.8

4.6

4.4

4.2

4.0

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

.0

BODY GROWTH

10

15

20

25 30

DAYS

35

40

45

50

55

FIGURE 4. Comparison of average shell growth and average body growth, as indicated by
measurements of the maxillae, for 20 Balanus improvisus over a period of 20 molts. Maxillae
size X 10 -1.

third that found in nature. Thus, while not optimal, laboratory conditions will
support growth of the shell and the absence of body growth cannot be attributed to
starvation.

Figure 4 shows the average increase in size of maxillae and shell for 20 barnacles.
It will be noted that the initial ratio between maxillae and shell sizes is not main-
tained over any great length of time. As the body can grow only by molting, it
might be expected to show an increase in size which would correspond with shell
growth for that particular intermolt period. As shown in Figure 5, however, the
per cent growth of the maxillae rarely equals the per cent accumulated shell growth
during that intermolt period. With the exception of the third molt, the total per

230

JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT

cent shell growth during the two- to three-day intermolt period was always higher
than that exhibited by the maxillae. If this trend were to continue it would result
in a large shell enclosing a relatively small body. The changing ratio shown in
Figures 3 and 4 is compensated in part by changes in opercular plate level at the
time of molting. In a newly set barnacle the opercular plates occupy an extreme
apical position. At each molt a new opercular hinge is secreted and the opercular
plates gradually become more basal. While this tends to reduce the internal volume
of the shell, a large shell and relatively small body are not totally incompatible when
certain other factors are considered.

20

I 5

o
cr

I 0

5-

-

I

ACCUMULATED SHELL GROWTH

7

D

DAILY SHELL GROWTH

4

\,

\

-

I

s

\

\
s,

1

BODY GROWTH

_

X

3

s
s

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5

s

_

;

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s

\

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»

\

y

•

s

\

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s

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s

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s

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s
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6 [:

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s

\

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8

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-

!

\
\

;

\
\

s

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s

9

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s

.

s

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-

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:

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\
\ _

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,

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s

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s

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3

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_

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s

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: | , \ S • ; 19

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—

,

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'-

2

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10

15

20

25 30

DAYS

35

40

50

55

FIGURE 5. Comparison of average daily per cent shell growth, average accumulated per
cent shell growth during each intermolt period, and average per cent body growth for 20
Balanus improvisus over 20 consecutive molting periods.

B. improvisus retains the fertilized eggs within the mantle cavity until the time
of hatching, when the nauplii are expelled through the opercular orifice. While
actual figures are not available, Bousfield (1955) has estimated that B. improvisus
retains from 1,000 to 10,000 eggs during each breeding period. These embryos,
forming lamellae on both sides of the body, require space in the mantle cavity be-
tween the body and the shell-secreting mantle. In a young barnacle the mantle,
covering the internal surface of the developing shell, occupies considerable space
and, with the body, leaves little room for the mantle cavity. Because of the intricate
construction of the shell (Costlow, 1956) it has not been possible to accurately
measure the volume which is actually utilized by the body at different ages. If the

BODY VERSUS SHELL GROWTH IN BALANUS 231

original ratio between the body and shell sizes were maintained, however, the size
of the mantle cavity might not be sufficient to allow retention of the large numbers
of embryos.

SUMMARY AND CONCLUSIONS

From daily measurements of shell and portions of the shed exoskeletons of 20
Balanus iinprovisus over 20 molting periods, and comparison of mouth part size
and body size of barnacles taken from the harbor, the following conclusions may
be made :

1. A ratio of 6.271 ±0.104 exists between the width of the thorax and the width
of the maxillae in barnacles of varying size and age taken from the harbor. Thus,
the maxillae, which retain their form and size in the shed exoskeleton, are not
subject to differential growth and may be used as an index of body size increase.
The mandibles, approximately twice the width of the maxillae, may also be used
as an index of body size.

2. Growth of the body, as indicated by measurements of maxillae and mandibles,
does not necessarily accompany ecdysis. The absence of growth in mandibles and
maxillae occurs frequently and may extend over periods of three molts.

3. The increase in size of the mouth parts is variable and tends to become smaller
as the over-all size of the barnacle increases.

4. Growth of the calcareous shell occurs even when there is no increase in size
of the mouth parts.

5. The accumulated shell growth during the two- to three-day intermolt period
is usually greater than the size increment of the maxillae and mandibles at molting.
The original ratio between body size and shell size is not maintained as growth
occurs.

6. The hypothesis is presented that the resulting large shell and relatively
smaller body of an adult barnacle provide a mantle cavity sufficiently large to accom-
modate the developing embryos.

LITERATURE CITED

BARNES, H., 1955. The growth rate of Chthamalus stellatus (Poli). /. Mar. Biol. Assoc.,

35 : 355-361.
BARNES, H., AND H. T. POWELL, 1953. The growth of Balanus balanoides (L.) and Balanus

crenatus Brug. under varying conditions of submersion. /. Mar. Biol. Assoc., 32 :

107-128.
BOUSFIELD, E. L., 1955. Ecological control of the occurrence of barnacles in the Miramichi

Estuary. National Museum of Canada, Bulletin No. 137, Biological Series No. 46.
CARLISLE, D. B., 1956. Studies on the endocrinology of isopod crustaceans. Molting in Ligia

oceanica (L.). /. Mar. Biol. Assoc., 35: 515-521.
CARLISLE, D. B., AND P. F. R. DOHRN, 1953. Studies on Lysmata seticaudata Risso (Crustacea

Decapoda). II. Experimental evidence for a growth and moult accelerating factor

obtainable from eyestalks. Pubbl. Stas. Zool. Napoli, 24 : 69-83.
COSTLOW, JOHN D., 1956. Shell development in Balanus improvisus Darwin. /. Morph., 99 :

359^15.

COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT, 1953. Moulting and growth in Balanus im-
provisus. Biol. Bull., 105 : 420-433.
COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT, 1956. Molting and shell growth in Balanus

amphitrite niveus. Biol. Bull, 110: 107-116.
CRISP, D. J., 1954. The breeding of Balanus porcatus (DaCosta) in the Irish Sea. /. Mar.

Biol. Assoc., 33 : 473^96.

232 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT

DAWSON, C. E., AND C. P. IDYLL, 1951. Investigations on the Florida spiny lobster, Panulirus

argus (Latreille). Florida Board of Conservation, Technical Series, No. 2.
DRACH, P., 1936. Le cycle parcouru entre deux mues et ses principales etapes chez Cancer

pagurus Linne. C. R. Acad. Sci., Paris, 202 : 2103-2105.
DRACH, P., 1939. Mue et cycle d'intermue chez les Crustaces decapodes. Ann. hist. Occanogr.

Paris, 19 : 103-391.
HIATT, R. W., 1948. The biology of the lined shore crab, Pachygrapsits crassipes Randall.

Pacif. Sci., 2 : 135-213.
KINCAID, F. D., AND B. T. SHEER, 1952. Hormonal control of metabolism in crustaceans. IV.

Relations of tissue composition of Hcmigrapsus nndus to intermolt cycle and sinus

gland. Physiol. Zool, 25: 372-380.
KNOWLES, F. G. W., AND D. B. CARLISLE, 1956. Endocrine control in the Crustacea. Biol.

Rev., 31 : 396-473.
LLOYD, A. J., AND C. M. YONGE, 1947. The biology of Crangon vulgaris L. in the Bristol

Channel and Severn estuary. /. Mar. Biol. Assoc., 26: 626-661.
MARSHALL, N., 1945. The molting without growth of spiny lobsters, Panulirus argus, kept in

a live car. Trans. Amer. Fish Soc., Vol. 75.
OLMSTED, J. M. D., AND J. P. BAUMBERGER, 1923. Form and growth of grapsoid crabs.

/. Morph., 18 : 279-294.
TAIT, J., 1917. Experiments and observations on Crustacea. II. Moulting of isopods. Proc.

Roy. Soc. Edinburgh, 37 : 59-68.
TRAVIS, D. F., 1954. The molting cycle of the spiny lobster, Panulirus argus (Latreille). I.

Molting and growth in laboratory-maintained individuals. Biol. Bull., 107 : 433-450.

FACTORS AFFECTING TERMINAL GROWTH IN THE
HYDROID CAMPANULARIA 1

SEARS CROWELL AND CHARLES WYTTENBACH 2

Department of Zoology, Indiana University, Bloomington, Indiana, and the
Marine Biological Laboratory, Woods Hole, Mass.

The experiments reported here pertain to the limitation of terminal growth in
the hydroid Campanularia flexuosa. The general problem of growth limitation,
and specifically of terminal growth, has been examined for many different organisms.
A review is impossible in a brief paper. Few general unifying concepts seem to
have been established clearly : we are still in the stage of assembling the necessary
facts.

Three considerations led to these particular experiments. First, the fact that
each species of hydroid has a stem of characteristic and fairly definite height sug-
gests the presence of limiting factors which must either vary from species to species
or have a variable effectiveness. Secondly, an earlier experiment (Crowell, 1957)
had shown that terminal growth is very sensitive to nutritional level, and also that
the effects of lowered nutrition are more striking the older the stem. This assured
us that the terminal growth zone is not autonomous but has a dependence upon
events and conditions beyond itself. Thirdly, there was a paradox in our observa-
tions : well-fed young colonies added one hydranth per day. The internodes or
distances between adjacent hydranths are (almost exactly) one mm. long. A stem
35 days old should be expected to have 35 hydranths and be 35 mm. tall. Yet
our older colonies in the laboratory had only 20 hydranths at the most, and were
only about 20 mm. high. Likewise, in nature, stems of this species are rarely as
tall as 30 mm. and usually are much shorter.

This discrepancy and the general considerations led to the following questions.
Is a slowing down inevitable? Is it gradual or abrupt? Does the ability to grow
finally cease entirely? Can experimental procedures be used which eliminate the
inhibiting factors? Clear answers should give clues to the nature of such factors.

Figure 1 and the following remarks will enable a reader who is unfamiliar with
hydroids to visualize the pattern of growth and understand the terminology em-
ployed. A colony has an attached branching stolon system from which stems arise.
The first hydranth of a stem, 1 in Figure 1, is produced by upward growth from
the stolon. An apical growing zone produces an internode of the main stem, then
a pedicel, and finally a hydranth bud. After this there is renewed proliferation
from the first internode to produce the next internode, the pedicel of hydranth 2,

1 Contribution Number 631 from the Department of Zoology, Indiana University. This
investigation was supported by a research grant (H-1948) from the National Institutes of
Health, Public Health Service ; and by a grant-in-aid from the American Cancer Society upon
recommendation of the Committee on Growth of the National Research Council.

2 Address of second author is Department of Embryology, The Carnegie Institution of
Washington, Baltimore, Maryland.

233

234

SEARS CROWELL AND CHARLES WYTTENBACH

and hydranth 2. This sequence is continued as hydranth after hydranth is added
terminally. The increasing height is due not to continuous apical growth but rather
to a renewal of proliferative activity by a succession of zones, each of which, at
the time of its activity, is in a location just proximal to the most distal hydranth,
(GZinFig. 1).

FIGURE 1. A young upright showing the pattern of growth and the location of the terminal
growth zone. GZ, terminal growth zone ; S, secondary growth which may produce a branch
or a gonangium ; 1 — 5 the designations of the nodes and of the primary hydranths or positions.

In Campanularia both gonangia and lateral branches develop later from some
of the crotches (S in Fig. 1). Also, hydranths live for only about a week, after
which they undergo regression and are replaced by a new hydranth at the same
location (Crowell, 1953). These secondary developments may be relevant to the
problem of apical growth since they must be in competition with the apical growth
zone for available nutrition, and perhaps also in less obvious ways. In this report,
however, they have been disregarded.

The term stem (or hydrocanlns) , strictly speaking, means only the stem itself.
There is no technical term for the stem plus all the hydranths, side branches, and

TERMINAL GROWTH IN CAMPANULARIA 235

gonangia which it bears. We call these uprights: that which grows up from, or
away from, the stolon. An approximate botanical equivalent is shoot. The term
position is used to indicate the primary hydranths or nodes. Each is numbered as
in Figure 1. Distances along an upright are described in number of positions.

The colonies used for experimentation were developed from cuttings from cul-
tures originally obtained at Woods Hole two years earlier. These grew on micro-
scope slides, in running filtered sea water cooled to 19° C. Colonies were fed twice
a day by placing them in a dense suspension of newly hatched brine shrimp
(Artemia) for 5-10 minutes, a procedure which provides nutrition for maximal
growth.

THE RATE OF TERMINAL GROWTH

Since terminal growth is not continuous, but is a consequence of renewal of
activity by tissues just proximal to the last hydranth, the best measure of rate
which we have been able to devise is the time in hours from the cone stage of the
terminal hydranth to the same stage of the next terminal hydranth. The cone stage
may be thought of as a hydranth bud ; it represents a point in the development of
a hydranth at which cellular proliferation is about finished and differentiation is
commencing. During one three-day period, observations were made every three
hours to determine exactly the time difference between each of the developmental
stages of a hydranth. With this information it was possible, in the principal experi-
ments, to make observations once each day and then convert these to the time at
which the cone had been or would be present. Since this method depends upon
the assumption that once development of a hydranth is initiated it develops at a
constant rate, the evidence for this needs to be set forth.

In the series of close observations made to establish the normal times for develop-
ment, we found no significant differences between old and young uprights except
in the period of delay prior to the beginning of development. The same is true
when specimens at different nutritive levels are compared (Crowell, 1957). Re-
grettably, the earlier report of Lund (1923) was overlooked in that paper. Lund
gives detailed measurements and states (p. 86) that "The sequence in the formation
of polyps on isolated internodes of Obelia is not due to a difference in rate of re-
generation, but is due to a difference in the period of delay between cutting and
initiation of the regeneration process. The rate of regeneration (growth) of
polyps is the same in basal and apical internodes." Obelia is almost identical with
Campanularia in its growth. An analogous condition is reported by Steinberg
(1955) in Tiibiilaria, a hydroid quite different from Campanularia. He finds that
the difference in time of hydranth regeneration under varied conditions is in the
preparatory phase, but not in later stages.

In the first experiment several colonies with a large number of uprights were
used. Each day a record was made of the stage of development of the terminal
hydranth of each upright. Every 2-3 days new stolons were removed so that each
colony consisted of only about 20 of the oldest uprights. At the end of 5 weeks
this trimming was stopped to permit the growth of a "crop" of young uprights.
When the latter had reached a height of about 10 positions, both they and the older
uprights were used for the second series of experiments described later on.

On two occasions during the experiment, when the Artcmia supply was known
to be sub-maximal, old uprights which had been adding a new position every

236

SEARS CROWELL AND CHARLES WYTTENBACH

30-35 hours would suddenly require from 72-144 hours. The older the upright,
the more pronounced was this slowing down. With equal suddenness, however, as
soon as they were returned to maximum feeding, the time shortened to 30-35 hours.
Large values obtained during times of food depletion were not included in the
averages.

The rate of terminal growth, measured in hours, from cone stage to cone stage,
is shown for each position on the uprights in Table I and in Figure 2. Rate,
strictly speaking, should be expressed as quantity per unit time, or reciprocal of
hours. The conversion seems unnecessary here. A few uprights were kept be-

TABLES I-III

Hours required for the development of a new terminal hydranth at each position on an upright. In

each table is shown the position, the number of cases, the mean, and the standard

deviation. The mean values are plotted in Figure 2

Table I. The main series of observations;
— • in Figure 2.

Table II. The old controls (0:con) during
the experimental period; — O in Figure 2.

Position

n

M

SD

Position

n

M

SD

1

48

24.9

4.1

31

10

28.6

3.5

2

50

23.8

4.9

32

10

32.0

4.4

3

50

22.1

3.7

33

10

30.0

3.1

4

53

23.6

4.8

34

9

32.6

4.9

5

54

23.8

5.4

35

8

30.5

4.0

6

53

24.5

4.7

36

7

32.9

2.7

7

51

24.4

4.2

37

6

31.8

4.4

8

52

24.9

4.7

38

4

32.0

2.5

9

47

26.2

4.2

10

42

27.3

3.7

11

36

26.1

3.6

Table III. Young controls (N:con) before

12

37

28.6

4.0

and during the experimental period ;

13

42

29.2

4.2

— X in Figure 2.

14

43

30.0

5 7

15

45

30.8

*J • 1

4.5

Position

n

M

SD

ifi

39

?0 8

A Q

1 \J

17

o y

31

J\J.O

31.3

^.s

4.3

1

12

27.3

5.3

18

33

32.4

5.5

2

17

23.4

4.9

19

38

33.4

6.0

3

18

21.6

3.4

20

35

35.6

5.1

4

18

21.7

4.1

21

39

35.6

5.8

5

18

22.5

3.1

22

36

32.0

4.9

6

18

22.0

2.0

23

41

33.8

6.6

7

18

22.4

3.4

24

47

32.4

4.5

8

18

22.4

2.4

25

49

32.6

6.0

9

18

23.3

3.0

26

48

33.9

7.7

10

17

22.9

2.0

27

45

32.8

10.7

11

18

25.4

3.7

28

36

32.9

5.8

12

18

25.1

2.8

29

28

31.6

6.1

13

17

27.1

3.9

14

18

29.1

4.5

15

17

29.0

5.4

16

16

28.6

3.3

17

16

30.1

3.8

18

12

30.1

4.4

TERMINAL GROWTH IN CAMPANULARIA 237

24

CO

x x

x x

•.xx*

• X X °

• •

32

36

8 12 16 20 24 28 32 36

POSITION

FIGURE 2. The rate of development of terminal hydranths, expressed as number of hours
from one cone stage to the next, for each position on a stem. • , main series of observations.
O, a few of the same stems continued (0: con). X — a second series of observations (N : con).
Data in Tables I-III.

yond the 30th position and used as controls in the subsequent experiments. These
are plotted as 0 in Figure 2 and the data are given in Table II. Also, a second
series of values was available for young uprights and these are shown by X in
Figure 2 and are presented as Table III. The slightly higher values obtained in
these later observations are probably a reflection of some slight, but unrecognized,
improvement in conditions. In Figure 2 three periods are identifiable : During the
first 8-10 days one new hydranth is added each 24 hours ; then the rate becomes
slower until about 20 positions are established ; after this there is a period of steady
rate at about 33 hours. How long this might continue no one knows. The oldest
stems, at about 60 days, were taller than any we have seen either in nature or in
our ordinary cultures.

AMPUTATED UPRIGHTS AND SECTIONS OF UPRIGHTS

The following experiments were designed to show the terminal growth rate
when the relationship of the terminal growth zone to the rest of the upright and to
the colony is altered. The uprights whose history had been closely followed for
5 weeks, and the newer uprights which were 8-13 positions high, were cut according
to the scheme shown in Table IV and Figure 3. Those sections which were de-
tached were held in place at the edge of slides by a thread. Their proximal ends,
as well as the basal ends of any lateral branches, were ligatured with a single strand
of Dacron to prevent growth at all but the distal end.

Measurements were made for the time from cone stage to cone stage for the
four hydranths produced terminally on each upright of the control groups. In the
experimental groups the hydranth develops from the cut end of the upright and the
time is measured from the moment of cutting to the cone stage. The times for the
three successive hydranths, which develop from the zone of prospective growth,
are measured as before, from cone stage to cone stage.

238

SEARS CROWELL AND CHARLES WYTTENBACH

In these experiments the distal cut end sometimes failed to produce a hydranth,
but instead produced an indeterminate growth similar to a stolon. These are
called free (i.e., not attached) stolons. When they developed we cut them off.
Following such amputation, a free stolon sometimes developed again and sometimes
the new growth was a hydranth. This complicates the analysis and makes it
necessary to report both the rate of hydranth development when it did occur and
also the incidence of hydranth development as compared with that of free stolons.

: Con 0:I8

0:8

N; Con

-y

N'2

0:22-26

Q'13-17

N'Ti p

0=3-7

N'3-7

FIGURE 3. Diagrams to illustrate the plan of the experiments as outlined in Table IV.

TERMINAL GROWTH IN CAMPANULARIA 239

TABLE IV

Plan of experiment, shown also in Figure 3. The abbreviations at the left are
the designations given the experimental groups

From older series of uprights, 0:

0:con Controls with about 30 positions, connected with rest of colony
0:18 Trimmed to leave 18 positions, connected with rest of colony

0 : 8 Trimmed to leave 8 positions, connected with rest of colony

0:2 Trimmed to leave 2 positions, connected with rest of colony

0:3-7 Sections consisting of positions 3 to 7
0: 13-17 Sections consisting of positions 13 to 17
0:22-26 Sections consisting of positions 22 to 26

0:tip Sections with 5 positions including the uninjured terminal position (approximately
32-35)

From younger series of uprights, N :

N:con Controls with about 10 positions, connected with rest of colony
N : 2 Trimmed to leave 2 positions, connected with rest of colony

N:3-7 Sections consisting of positions 3-7

N:tip Sections with 5 positions including the uninjured terminal position (approximately
12-15)

Since the development of a hydranth involves more differentiation and organization
than the production of a free stolon, we think of the former as more "difficult" as
well as more effective in respect to the normal growth of the colony.

Both in describing the results and in the discussion the word significantly has
been used only when differences are significant at the 1% level as determined by
"t" test.

The data are presented in Table V, A. The young controls, N :con, have a
value of 26.6 hours for positions 12-15. This is significantly faster than the rate
for the old controls at 30.7 hours. The old uprights cut near the base, 0 : 2 and
0 : 8, have rates well above 40 hours. They are significantly slower than old and
young controls and also slower than the young uprights, N : 2, similarly amputated.
The poorest rate is seen for the few uprights which produced hydranths when cut
at the nineteenth internode, 0:18:56.4 hours (significantly slower at the 5%
level than 0:2, in spite of the small number of cases).

The times for the development of the first hydranth are shown in Table V, A in
a separate column. Since once development starts, its rate is the same for all
groups, differences among them must be a reflection of the time required for healing
and preparation for proliferation. The O : — groups are nearly identical one with
another but slower than the younger N : 2 group.

Table V, B shows the percentages of cases in which the trimmed uprights
produced hydranths rather than free stolons at the cut surface. In one column
is shown the percentage of cases in which hydranths were produced at the first
opportunity, in the last column the percentage of cases which finally produced a
hydranth after one or more removals of the stolon which had grown at first. The
groups stand in the same relationship one to another whichever column is used.
Comparison of the values among the groups in Table V, B with those of Table V, A
shows that the uprights which produce hydranths most rapidly are the same ones
which more often produce hydranths rather than free stolons.

In the "effectiveness" or "efficiency" of terminal growth the experimental groups

240

SEARS CROWELL AND CHARLES WYTTENBACH

stand in the following order : Tips of unamputated young uprights — N : con ; tips
of unamputated old uprights - - O : con ; young uprights amputated near their
base — N : 2 ; old uprights amputated at the third or ninth internode --0:2 and
0:8; and old uprights amputated at the nineteenth internode — 0 : 18.

TABLE V

The development of hydranths and free stolons from amputated uprights

Experimental
group

Position
of new
hydranths

Hours for hydranth production

Hydranths produced instead of
free stolons

Number of
cases

Cone to cone

Cut to cone

Number of
cases

% the first
time

% at any
time

Trimmed Uprights

0:2

3
4-6

A

B

14
38

43.9 db 12.2

37.9 ± 6.0

31

16

74

0:8

9
10-12

9

25

47.6 ± 12.8

35.9 ± 4.9

16

19

75

0:18

19
20-22

7
5

56.4 ± 20.3

37.1 ± 5.4

12

8

58

0:con

32-35

39

30.7 ± 4.2

N:2

3
4-6

26
69

35.2 ± 8.1

32.5 ± 2.3

33

52

97

N:con

12-15

71

26.6 ± 4.0

Isolated Sections

0:3-7

8
9-11

C

D

9
21

43.9 ± 12.4

29.3 ± 7.1

19

16

32

0:13-17

18
19-21

4
11

44.1 ± 10.1

33.8 ± 7.1

15

13

27

0:22-26

27
28-30

9
26

36.2 ± 6.1

30.2 ± 4.2

16

56

68

0:tip

32-35

45

35.6 ± 8.3

N:3-7

8
9-11

17

48

43.0 ± 12.4

32.4 ± 7.1

19

74

95

Nrtip

12-15

60

36.0 ± 9.2

TERMINAL GROWTH IN CAMPANULARIA 241

Before considering the results of the measurements on the isolated sections of
uprights, it is necessary to point out that the values for them cannot be directly
compared with those of the specimens still attached to the rest of the colony. Because
there was a good deal of regression of hydranths in segments isolated from lower
levels of the uprights and because it seemed essential to be able to compare the
group one with another, we fed each specimen two brine shrimp a day for each
position present (whether a hydranth was at each position or not). This gives
as constant a nutritional intake as can be achieved. It is at a level known to be
adequate for moderate, but not maximal, growth (Crowell, 1957).

As before, the times are calculated separately for the first hydranth to be pro-
duced (from the time of cutting) and for the subsequent three. Data are presented
in Table V, C and D.

The time required for the development of the first hydranth is nearly equal for
the four groups.

The growing tips of new and old colonies are almost identical in rate although
they are at levels 20 positions apart. That their rates are somewhat slower than
those of intact uprights is interpreted as due to the lower quantity of food received
(discussed above). Levels farther from the growing tips have slower rates. The
level nearest the tip, 0 : 22-26, is essentially equal to the tip, and significantly better
than the lower two levels, 0:13-17 and 0:3-7 which are alike. N : 3-7, even
though it came from a young upright, and was only about 7 positions below the
growing tip, is also slow.

In the case of the amputated uprights it will be recalled that there was a close
parallelism between rate of hydranth production and the tendency to produce
hydranths rather than free stolons. The same is true for the isolated sections
(Table V, D), with one exception: the N: 3—7 group had a slow rate but rarely
produced stolons.

DISCUSSION AND CONCLUSIONS

The continuous record for 60 days of the terminal growth of uprights (Fig. 2)
shows that when nutrition is optimal, the growth rate is maximal for about 10 days,
then gradually reaches a new and lower level which it maintains thereafter. One
can postulate that whatever factors reduce the rate from about 24 to 33 hours,
these have no further effect, and, at the somewhat slower rate, terminal growth
could continue forever. In nature a long stem would, of course, eventually be
broken off. In the laboratory one could cut off the distal portion and follow its
history, an experiment which we have not yet carried out. The "immortal" hydras
described by Brien and Reniers-DeCoen (1949), and the successful indefinite
asexual reproduction of some oligochaetes and turbellarians (e.g. Stenostomum,
Sonneborn, 1930) are examples of growth without limitation somewhat comparable
to the situation described here.

Earlier experiments (Crowell, 1957) have shown that the height or age of an
upright determines quite precisely the extent to which lowered nutritive level affects
the rate of terminal growth. This effect of age (or height) is evident in stems
when they are only a few positions high or a few days old and indicates that at
least one factor which can influence growth is accumulating long before it can be-
come effective in well-fed colonies.

242 SEARS CROWELL AND CHARLES WYTTENBACH

The experiments involving the trimming of uprights and isolation of sections
at different levels were designed with some prejudice in favor of a correlation be-
tween the rate of terminal growth and the age of the tissues. Although the terminal
growth zone is always young in actively growing colonies, it is still possible that
there occurs an inherent slowing down with time, independent of factors external
to the terminal growth region itself. In the absence of this condition it may be
possible that the older tissues below the terminal zone, as they become older, are the
source of factors which adversely affect terminal growth.

Analysis of the data has made it necessary to consider four possibilities. 1 )
Correlation with age of tissues as discussed above. 2) Inhibitory substances arising
from hydranths already present, but not correlated with the age itself of these
hydranths — perhaps a process of like inhibiting like. 3) Deficiency of nutrition
as the distance from the stolon becomes greater. This might merely be a con-
sequence of less efficient hydroplasmic streaming in the distal portion of tall up-
rights. 4) Any combination of the above.

The factors suggested are not the only possible ones. Hydranth regression,
production of lateral branches and the development of gonangia may have effects,
but this question has not yet been examined. There are, of course, still other
possibilities.

If slowing of terminal growth were inherent to the growing terminal portion
of the upright, one would not expect this to be expressed during only the production
of the tenth to twentieth positions. Tips of old and young uprights, different in
length by 20 positions, when isolated showed the same rates of terminal growth
(0 : tip and N : tip in Table V, C) .

When we compare groups 0: 3-7, 0: 13-17, 0: 22-26, and 0: tip, the similarity
of rate between the latter two suggests that no marked slowing has occurred 7
positions below the tip. However, those about 15 positions below the tip, 0: 13-17,
show a significantly slower rate. The oldest group, 0 : 3-7, is the same as 0 : 13-17.
It should be emphasized that each of these specimens received the same amount of
food ; hence differences cannot be accounted for on the basis of nutritional intake.
Further evidence of a correlation with age is seen in the comparison of groups
N : 3-7 and N : tip. The former, comprising tissue about 7 positions back from
the tip, shows a significantly slower rate than the tip. Comparisons in Table V, A
between N : 2 and N : con and between N : 2 and 0 : 2 also show a correspondence
between rate and age of tissues.

It is evident that there is no aging at the growing tip itself. It is also clear
that the rate of hydranth formation at increasing distances from the tip and in
older tissues is slower. The tissues do not express the consequence of aging until
about 7 hydranths have been produced beyond them, that is, about 10 days after
the tissue had been first established. At this time this effect increases sharply ;
soon it becomes maximal and thereafter there is no further decrease in rate related
to the increasing age of the tissues. The similar rates shown by 0 : 3-7 and
0: 13-17 support this last statement.

Not all of the results are consistent with the idea that aging effects explain
the slowing of terminal growth. The experiments do not distinguish between the
role of possible inhibitors and that of the efficiency of circulation. With increasing
length of upright there is not only an increase in the number of hydranths to
produce an inhibition, but also a greater length of coenosarc separating the proliferat-

TERMINAL GROWTH IN CAMPANULARIA 243

ing zone from the basal stolon. The chief indication that one or both of these
factors are operating is seen in Table V, A, particularly for the old uprights.
With increasing distance from the stolon (decreasing age of tissues) there is an
increase in the time required for hydranth production. Statistically, 0 : 2 with
the faster rate is not significantly different from 0:8, but is different at the 5%
level from 0 : 18. These results are the opposite to those expected as a consequence
of aging and indicate that some f actor (s) exert an effect opposite to that of aging.

The frequency with which the cut specimens developed free stolons was higher
the slower the rate of hydranth production. To this generalization there was one
exception : the N : 3-7 group which produced relatively few free stolons but had
a slow rate for hydranth production. This same group has been mentioned earlier
in connection with the effect of age of the tissues. It had a slower rate than
tissue of about the same age belonging to older uprights, the 0 : 22-26 group.
We are unable to decide whether we should regard this case as anomalous in respect
to its rate or in respect to the frequency of free stolon production. Its significance
may lie in the hint which it gives that the factors which influence the production
of free stolons may be different from those which control rate.

One result, so far mentioned only incidentally, is shown in Table V, A and C.
For those cases in which the distal tip had been severed, the rate of replacement
of the first hydranth, that is the one arising from the cut surface, is tabulated
separately from the rates for successive hydranths. For all of the groups there
is little difference in the rates at the cvit surface but marked differences for
hydranths subsequently produced. Lund (1923) found differences in replacement
of hydranths of Obelia correlated with the distance from the growing tip. The
absence of such differences in our specimens is unexpected.

Although the rates for the production of the hydranth from the cut surface are
similar for all our cases there was great variation in the frequency of free stolon
production by these same cut ends. Apparently if a hydranth is to develop, it
can begin to do so with about the same speed regardless of the level of the cut or
age of tissue. A correlation with age is seen in the frequency of free stolon
production, but the effect of age applies to hydranth production only after the first
has been produced.

The general conclusion from these many considerations and comparisons is
that there is a period of maximal rate of terminal growth expressed by uprights
only during their first 10 days ; after this the zones of prospective terminal growth
become adversely affected by factors external to themselves. Although the age
of cut sections of stems is one factor correlated with slower hydranth production
and with the development of free stolons instead of hydranths, many of the results
cannot be explained on this basis. What these other factors may be can only be
conjectured until further experiments are carried out.

SUMMARY

1. When terminal growth rate was measured for stems of well-fed colonies
of the hydroid Campanularia fle.ruosa for a period of 60 days, it was found that
this rate is constant for about ten days, becomes progressively slower for the next
10 to 15 days, and then remains constant.

2. Isolated sections of stems of different ages differ one from another both in

244 SEARS CROWELL AND CHARLES WYTTENBACH

respect to the time required for the production of additional terminal hydranths,
and in their ability to produce hydranths rather than free stolons. In general the
same experimental groups which most readily produce hydranths following cutting
produce them at fairly high rates.

3. Older levels of stems are in general less efficient in the rate of terminal
hydranth production and in the ability to produce hydranths rather than free stolons.
Not all of the results can be explained on the basis of an effect of aging. The
possible role of inhibitors and of differences in efficiency of circulation must be
considered.

LITERATURE CITED

BRIEN, P., AND M. RENIERS-DECOEN, 1949. La croissance, la blastogenese, 1'ovogenese chez
Hydra fusca (Pallas). Bull. Biol. de France et Belg., 83: 293-386.

CROWELL, S., 1953. The regression-replacement cycle of hydranths of Obclia and Campanularia.
Physiol. Zool, 26: 319-327.

CROWELL, S., 1957. Differential responses of growth zones to nutritive level, age, and tempera-
ture in the colonial hydroid, Campanularia. J. E.vp. Zool., 134 : 63-90.

LUND, E. J., 1923. Experimental control of organic polarity by the electric current. III.
Normal and experimental delay in the initiation of polyp formation in Obelia inter-
nodes. /. Exp. Zool, 37 : 69-87.

SONNEBORN, T. M., 1930. Genetic studies on Stcnostonuun incandatum (Nov. Spec.). /. Exp.
Zool., 57 : 57-108.

STEINBERG, M. S., 1955. Cell movement, rate of regeneration, and the axial gradient in
Tnbularia. Biol. Bull., 108: 219-234.

RESPIRATORY METABOLISM OF THE FIDDLER CRAB UCA

PUGILATOR FROM TWO DIFFERENT

LATITUDINAL POPULATIONS

NOELLE DEMEUSY 1
Biological Laboratories, Harvard University, Cambridge, Massachusetts

By definition a poikilotherm is an animal whose internal state fluctuates with
temperature changes in its environment, a lowering of environmental temperature
resulting in a reduction of metabolism or activity. Accordingly, it is to be expected
that a poikilotherm living in colder habitats and subjected seasonally to low tempera-
tures would have a lower metabolic rate than would the same species inhabiting
a region of higher temperatures. Nevertheless, considerable information indicates
that many cold-blooded animals living in colder habitats operate at higher rates
than those we could expect by deduction from their rate-temperature curve. This
is supported by the observation that many species of marine invertebrates are as
active in colder seas as those in warmer waters. This phenomenon of compensatory
adaptation of rate of metabolism or activity has been recently reviewed by Bullock
(1955) and Prosser (1955).

Compensatory adaptation has been studied in three different ways : ( 1 ) by
experimental acclimation of animals at various temperatures; (2) by observation
of animals at various seasons; and (3) by comparisons of groups of individuals
varying in latitude.

It has been observed, especially among the aquatic poikilotherms, that certain
species from the northern and southern regions may show rather similar rates of
functions when measured at their own habitat temperature. Many animals from
higher latitudes tend to show higher rates than animals from lower latitudes, when
measured at any given temperature.

With respect to latitudinal temperature compensation, we find numerous in-
vestigations. Different species of a genus or different individuals of a given species
show about the same rate functions in Greenland, the North Sea and the Mediter-
ranean (Sparck, 1936; Thorson, 1936; Wingfield, 1939). Thorson (1956) says
(p. 695) : ". . . an arctic Macoma community at 0° C. shows, roughly, a similar
metabolic rate, a similar rate of growth, and similar feeding habits, as a boreal
Macoma community at 8° C., or a Mediteranean community at about 12° C., or a
tropical community at a still higher temperature."

The more recent investigations deal, in large part, with studies of the same
species from different latitudes, and the results obtained by Roberts (1952) and
Rao (1953) on crabs and mussels along the west coast of the United States are
also in favor of a certain adaptation of metabolism or activity to temperature in
different populations.

In 1948, Dr. Dorothy Bliss (personal communication) undertook a few experi-

1 Present address : Institut de Biologic, Nancy, France.

245

246 NOELLE DEMEUSY

ments concerning the tolerance to low temperatures of two species of Uca from
Woods Hole, Massachusetts, namely U. pugnax and U. pugilator, and of two
latitudinal populations of Uca pugilator from Woods Hole and from Florida.
These experiments suggested first, a very obvious difference in the resistance to
cold between the two species of Uca from Woods Hole, and secondly, a noticeable,
although less impressive, difference in the behavior of the northern and southern
populations of Uca pugilator when very low temperatures were reached, that is
to say, between 1° and 3° C.

The purpose of this present investigation was to continue with this problem of
physiological variations and compensatory mechanisms, perhaps resulting from the
different latitudes at which Woods Hole and Florida crabs are living. Uca
pugilator (Bosc), the sand fiddler, according to Rathbun (1918) and Crane
(1943), ranges from Boston Harbor to Galveston, Texas. Up to now, a single
species has been described, and Jocelyn Crane (personal communication) states
that the taxonomic problem of Woods Hole and Florida Uca pugilator has not
yet been investigated.

We have been concerned, first, with comparisons at a given temperature of the
metabolism of these animals, measured as oxygen uptake, and, in addition, with
observations of their activity and their resistance to cold.

This work has been carried out during a sojourn at Harvard University while
holding a Smith-Mundt Fulbright travel grant. It was supported, in part, by
Research Grant B-623 from the National Institute of Neurological Diseases and
Blindness, Public Health Service. I am happy to express my thanks to Dr. J. H.
Welsh for his kind hospitality, for his constant and valuable support and for all
material facilities he provided. I am also much indebted to Dr. Dorothy Bliss who
helped me with the use of the respirometers and showed a constant interest in the
course of my work.

MATERIALS AND METHODS

Crabs from Woods Hole (latitude 41° N) and from Florida (vicinity of
Englewood, latitude 28° N) were shipped periodically to Cambridge, Massachu-
setts. As soon as they arrived, they were placed in containers with a small amount
of sea water and kept at room temperature, that is to say, at an average of 20° C.
They were fed fish twice a week. Water was changed regularly. Some experi-
ments dealt with animals which were kept for various lengths of time at 10° C.
Throughout the work, only male crabs were used.

For the determinations of the rates of oxygen consumption, Dixon volumetric
respirometers were employed. Carbon dioxide was absorbed either by Ascarite
or by 20% KOH. In some experiments, in order to absorb any excreted ammonia,
a cupric chloride solution was added in the apparatus containing KOH. No
significant differences in readings were noted. Two temperature baths were used
in measuring oxygen consumption; one was maintained at 15° C., the other at
1.4° C.

A period of time varying between one-half hour and three-fourths of an hour
was devoted to equilibration of the apparatus. Every experiment was run at the
same time of the day (between one P. M. and six P. M.) to avoid fluctuations due
to the diurnal variations in metabolism. Readings were made at convenient inter-

RESPIRATION OF FIDDLER CRABS

247

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NOELLE DEMEUSY

vals. The first hour's readings were discarded, for we observed an excessive
oxygen consumption at 1.4° C. and at 15° C. After one hour, more uniform
uptake occurred. (Recently, Grainger (1956) has observed a similar phenomenon
with Artemia.} Readings were recorded during the following three hours. Re-
sults have been corrected to standard temperature and pressure.

The observations on cold tolerance were made on the animals after they had
been taken out of the respirometers.

RESULTS

I. Rate of oxygen consumption as a function of latitude
1. Measurement at 1.4° C.

According to the previously mentioned experiments of Dr. Bliss, we chose the
temperature of 1.4° C. for our comparisons between the rates of oxygen consump-
tion of Woods Hole and Florida Uca.

The measurements were begun on the first day after arrival for southern crabs
and on the tenth for the northern populations. Starting on September twenty-
seventh, they were continued for an eight-week period for Florida crabs and a
seven-week period for those from Woods Hole. The stocks of crabs of both regions
were kept at room temperature (20° C.). Each day the rates of oxygen con-
sumption of two crabs of each population were measured and then the animals were
discarded. In no case was an animal used twice.

a) The results, as seen in Table I, indicate that there is an obvious difference
between the rates of oxygen consumption of the two populations when recorded
at this temperature and converted to standard conditions. We tested the signifi-
cance of the differences between these rates in using the t test of significance after

10
9
8

or?

o
„ 4

la

2
1

• •

o o

O O O

o o

o o

O O o 0->0

o o

j i I

j I I

5 1O 15 2O 25 3O 35 4O 45 5O 55 6O

TIME IN DAYS

FIGURE 1. Oxygen consumption of Uca pugilator from Florida (O) and from Woods Hole
(•), as a function of time spent at 20° C. Measurements were made at 1.4° C.

RESPIRATION OF FIDDLER CRABS

249

TABLE II

Respiratory rates of Uca pugilator measured at 15° C.

Wet body weight (g.)

Qo2 (mmVg./hr.)

Populations
from:

Size of
sample

Significance of
difference between
Q02

arithm.

arithm.

standard

range

mean

range

mean

deviation

Woods Hole

12

2.32-3.85

2.33

29.3-16.8

25.0

4.60

t-l.lS

P>0.10

Florida

14

1.30-3.17

3.15

37.3-16.0

22.2

6.90

no signif .

Chambers (1952). This indicates clearly the possesion of a compensatory mecha-
nism by Uca pugilator in relation to its habitat temperature, the northern group
showing the higher rate.

b) During their sojourn at a common temperature of 20° C. no significant
change in the rate of oxygen consumption could be noticed either in Florida Uca
or in Woods Hole Uca. A straight horizontal line could represent the steady rate
of oxygen consumed over the period of seven or eight weeks (Fig. 1). Therefore,
the same amplitude of divergence still exists between the two groups at the end
of their sojourn at 20° C.

c) A comparison between the two series of values obtained shows that there
is« a greater spread in the values of Qo2 for Woods Hole animals than for those
from Florida.

2. Measurements at 15° C.

Determinations of the resting metabolism of the two populations made at the
common higher temperature of 15° C. gave a mean value of 25 mm3/g./hr. for
Woods Hole Uca pugilator and a mean value of 22.2 mm3/g./hr. for the Florida
group. The difference is not statistically significant (Table II).

3. Temperature coefficient

It would be reasonable to expect to find an alteration of the sensitivity of
metabolism to temperature changes among the cold-adapted population from Woods
Hole. This would mean that the northern animals could withstand large tempera-
ture variations and show less change in their oxygen consumption than those
living in the south. In fact, such findings have been revealed by different investi-
gators for various physiological activities in many different species (Rao, 1953;
Dehnel, 1955; Tashian, 1956). Rao and Bullock (1954), in their review of the
relation of Q10 to the temperature at which the animal is adapted, give good
evidence of a decrease in Q10 with high latitude, in spite of contrary results reported
by Scholander et al. (1953).

From our metabolism studies on Uca pugilator from Woods Hole, we find,
between 1.4° C. and 15° C., a Q10 of about 2.47. If we compare the rates of
Florida Uca at the same temperatures, we get a Q10 of 3.52. This shows a decrease
in Q10, with increasing latitude. Recently, Tashian (1956) found a Q10 of 2.6
for Uca speciosa (weight 0.3-1.4 g.) from Key Biscayne, Florida, for temperatures
between 14.8 and 24.5° C. If we calculate the Q10 from his results on Uca pugnax,

250 NOELLE DEMEUSY

which were about the same weight as our Uca pugilator (3 g.), we find that it is
about 3.0 for Florida U. pugnax and 1.3 for New York U. pugnax. But the com-
parison of his results with those of the present writer is not entirely valid because
of the different choice of temperature range for determination of the rates (see
Rao and Bullock, 1954).

II. Activity

During the experiments on oxygen consumption, we noticed a difference be-
tween the two populations of Uca pugilator in their ability to withstand low tempera-
ture. After a five hours' stay at 1.4° C., specimens of Uca from Florida were
entirely relaxed and motionless as though dead. Not a single sensitive region of
the body responded to touch. As they recovered, most of them were extremely
spastic with occasional movements of their legs. This condition lasted as long
as six days. Some appeared as if they were shivering. In October, of forty-two
Florida crabs subjected to this temperature, eighteen were found dead on the
next day.

Individuals of Woods Hole Uca appeared much more resistant under the same
conditions and behaved in a different way. Immediately after removal from their
stay at 1.4° C., they were also very quiet, but one could notice a few very sluggish
movements of their legs, and generally the eyes responded to touch. They re-
covered rapidly, and sometimes some of them were moving around a quarter of
an hour after they had been returned to room temperature. No spasticity at all
could be observed. Of forty-two crabs subjected in October to 1.4° C. for five
hours, only two were found dead on the next day.

No difference at all could be found between representatives of the two popula-
tions after they had been kept at 15° C. for six hours.

III. Experimental acclimation at low temperature

Oxygen consumption measurements were also undertaken at 1.4° C., after
the animals of the two regions had been kept at a common lower temperature of
10° C., to determine if there was any adaptation of metabolism and activity of the
animals at such a low temperature.

Florida Uca showed a slight decrease in oxygen consumption for the first
seven days of the experiment (in comparison with the measurements made with
crabs maintained at room temperature). By the end of the first week, a slight
increase in the amount of oxygen uptake was noticed, but may not be really
significant. Nevertheless, at this time, a change in the behavior of the Florida
crabs was noticeable. Crabs transferred from room temperature to 10° C. became
quickly inactive. Only when strongly disturbed did they respond by very slow
and sluggish movements. They remained in the water all of the time. This
behavior could be observed for five days, after which they gradually began to move,
and on the seventh day all of them were found standing up on their legs. Now,
much more sensitive, they were disturbed even by a threat of catching them, and
many moved out of the water. This recovery of activity, clearly a sign of a certain
amount of acclimation, coincided with the observed slight increase in respiration.

It was also observed that after this previous stay at 10° C. the animals were
more resistant to a five-hour stay at 1.4° C. ; no spasticity was apparent when they

RESPIRATION OF FIDDLER CRABS 251

became active after exposure at this low temperature. Even after one day at
10° C., they appeared to be able to see and to move their eyes and legs very slowly
during their exposure to 1.4° C. After three to eight days at the previous tempera-
ture of 10° C., certain individuals recovered within a few minutes after removal
from the 1.4° C. bath.

Woods Hole Uca kept at 10° C. and compared with animals kept at 20° C.
showed an important decrease in their metabolism for the first ten days and then
an appreciable increase, but in any case the rate of oxygen consumption was higher
than when measured after sojourn at room temperature. After thirty days at
10° C. it decreased again, perhaps because the animals were eating very little at
this low temperature.

DISCUSSION

This work is an example to be added to those showing the existence of a
latitudinal compensatory mechanism within representatives of a given species.
Our results are partially in agreement with those of Roberts (1952), obtained with
Pachygrapsus crassipes. This author compared the metabolism of four populations
differing in latitude along the coast of California. He observed that when the
oxygen consumption was measured at a common temperature of 16° C. during the
winter, the more northern the populations, the more oxygen they consumed.

They also agree with the latitudinal compensatory differences found in rates
of ciliary pumping of water in the mussel Mytilus californianus (Rao, 1953).
The rates of pumping for unit weight in Mytilus of similar weights from Los
Angeles, Fort Ross and Friday Harbor are, at any given temperature, much higher
for animals from higher latitudes than in those from lower latitudes. However,
we must not forget that our own experiments bear upon only a very restricted
life span of Uca pugilator. Animals of both populations were considered as adult
crabs, and their weights did not vary outside the range 1.05 g.-4.73 g. Therefore
we could not study the fate of Qo2 and Qio over an extensive size range but we
can satisfactorily and precisely compare these coefficients between our two groups.

The higher rate of metabolism shown by Uca pugilator from Woods Hole as
compared with Florida individuals was very obvious at the low temperature of
1.4° C. At 15° C. the difference was not significant any longer. This temperature
must be very close to the environmental temperature of Woods Hole crabs in
October. One might predict that at their own habitat temperature the rates of
Florida crabs would be slightly higher than those of Woods Hole crabs. Recently,
Tashian (1956) found that in comparing the metabolism of the Florida and New
York Uca pugnax, at a temperature of 24° C., the rates were quite similar. The
measurements having been done during the summer, it happens that 24° C. cor-
responds to the environmental temperature during the collecting periods. This
author deduces that at their normal habitat temperatures the rates of the two
populations are similar. If the measurements had been done during the winter
when there is a marked difference between the water temperature of the two
regions, the results would probably have been different.

After keeping Uca for seven or eight weeks at a common temperature of 20° C.,
we could not, in contrast to Roberts, notice any decrease in the amount of oxygen
consumed by the representatives of each locality. The fact that our animals were
regularly fed may be responsible for this steady rate of metabolism. Moreover,

252 NOELLE DEMEUSY

the difference noticed at the beginning of the experiments between the metabolism
of the two populations could not be abolished by a sojourn of eight weeks at a
common temperature. Roberts showed that Pachygrapsus crassipes from different
localities become adjusted to the new temperature of 16° C., at which they have
been placed for six weeks, and that the original metabolic differences become
doubtfully significant. This is not the case for Uca pugilator, at least from Woods
Hole and Florida.

From our experiments, it appears as if the two populations of Uca pugilator
have acquired their own metabolic rates which have become fixed and not sus-
ceptible to modification, at least when only one factor of their environment has
been changed. The two populations, apparently good examples of "distance isola-
tion," have found conditions of existence quite different at their own habitats, and
several ecologic factors are certainly responsible for their differences. According
to the quite important difference in latitude, temperature is no doubt the most
significant factor.

Whether the physiological differences in metabolism and tolerance to low
temperature in Uca pugilator of different latitudes are more than phenotypic, we
cannot say. Work needs to be done with breeding tests. Knowledge of growth
rates, time of sexual maturity, and behavior of the animals in each locality would
be very useful.

As we have already noted, no information about differences in morphology of
the two populations could be found. After a comparative examination of the
individuals, we can say that not very striking morphological characters occur.
Nevertheless, after becoming familiar with the animals, we can easily separate the
two forms by their color. Woods Hole Uca, always darker than those from
Florida, are generally bluish-gray. Florida Uca are reddish-yellow. The shape
of the claw is also slightly different, with longer dactylopodite and propodite for
the individuals from Florida.

Thus, the extreme range of Uca pugilator does not seem to be accompanied by
great morphological modification. In our comparison between Carcinus maenas
from Atlantic and Mediterranean French coasts, the morphological differentiation
appeared more pronounced (Demeusy and Veillet, 1953). But Uca, physio-
logically, is succeeding very well, and it may provide an example of a physiological
adaptation previous to important morphological speciation (Crane, 1943).

By their physiological features (rates of oxygen consumption, sensitivity to
low temperatures), as well as by a few morphological characteristics, the popula-
tions of Uca pugilator from Woods Hole and from Florida might be separated
into two subspecies.

SUMMARY

Determinations of the respiratory rates of Uca pugilator from two different
latitudes (Woods Hole and Florida) have been made at 1.4° and 15° C.

1 . Woods Hole Uca pugilator show a higher rate of metabolism at low tempera-
ture than do specimens of the more southerly populations.

2. Uca pugilator from the higher latitude are less sensitive to temperature
changes than Uca pugilator of same weight from a southern latitude. This has
been shown by a lower Q10 for the Woods Hole population.

3. Woods Hole Uca are more resistant to low temperature than Florida Uca.

RESPIRATION OF FIDDLER CRABS 253

4. The same experiments made after the animals have been left at a common
temperature of 20° C, show that a stay of seven or eight weeks under similar
conditions does not abolish the metabolic differences observed between populations.

5. These physiological characteristics and some morphological ones might be
used to distinguish two subspecies of Uca pugilator.

LITERATURE CITED

Bosc, L. A. G., 1801-2. Manuel de 1'histoire naturelle des Crustaces contenant leur description

et leurs moeurs ; avec des figures dessinees d'apres nature. Vol. I, an. X.
BULLOCK, T. H., 1955. Compensation for temperature in the metabolism and activity of

poikilotherms. Biol. Rev., 30: 311-342.
CHAMBERS, E. G., 1952. Statistical calculation for beginners. Cambridge at the University

Press.
CRANE, J., 1943. Display, breeding and relationships of fiddler crabs (Brachyura, Genus Uca)

in the northeastern United States. Zoologica, 28: 217-223.
DEHNEL, P. A., 1955. Rates of growth of gastropods as a function of latitude. PhysioL

Zoo/., 28: 115-144.
DEMEUSY, N., AND A. VEILLET, 1953. Sur 1'existence de deux populations de Carcinus macnas

Pennant et sur les caracteres morphologiques qui les distinguent. C. R. Acad. Sci..

236: 1088-1090.
GRAINGER, J. N. R., 1956. Effects of changes of temperature on the respiration of certain

Crustacea. Nature, 178: 930.

PROSSER, C. L., 1955. Physiological variation in animals. Biol. Rev., 30: 229-262.
RAO, K. P., 1953. Rate of water propulsion in Mytilus californianus as a function of latitude.

Biol. Bull, 104: 171-181.
RAO, K. P., AND T. H. BULLOCK, 1954. Qin as a function of size and habitat temperature in

poikilotherms. Amer. Nat., 88: 33-44.
RATHBUN, M. J., 1918. The grapsoid crabs of America. Bull. U. S. Nat. Mus., No. 97, xxn,

1-461.
ROBERTS, J. L., 1952. Studies on thermal acclimatization in the lined shore crab, Pachygrapsus

crassipes Randall. Ph.D. thesis, Univ. of Calif., Los Angeles.
SCHOLANDER, P. F., W. FLAGG, V. WALTERS AND L. IRVING, 1953. Climatic adaptation in

arctic and tropical poikilotherms. Physiol. Zool., 26 : 67-92.
SPARCK, R., 1936. On the relation between metabolism and temperature in some marine

lamellibranchs and its zoogeographical significance. Biol. Medd., 13 : 1-27.
TASHIAN, R. E., 1956. Geographic variation in the respiratory metabolism and temperature

coefficient in tropical and temperate forms of the fiddler crab, Uca pugnax. Zoologica,

41 : 39-47.
THORSON, G., 1936. The larval development, growth and metabolism of arctic marine bottom

invertebrates compared with those of other seas. Medd. Gr0nland, Vol. 100, No. 6.
THORSON, G., 1956. Marine level-bottom communities of recent seas, their temperature

adaptation and their "balance" between predators and food animals. Trans. N. Y.

Acad. Sci,, 18 : 693-700.
WINGFIELD, C. A., 1939. The activity and metabolism of poikilothermal animals in different

latitudes. IV. Proc. Zool. Soc. London, Ser. A, 109: 103-108.

A QUANTITATIVE EXAMINATION OF TESTICULAR GROWTH
IN THE WHITE-CROWNED SPARROW 1

DONALD S. EARNER AND A. C. WILSON

Laboratories of Zoophysiology, Department of Zoology, State College of Washington,

Pullman, Washington

Since the pioneer investigations of Rowan (1925, 1926) and Bissonnette (1930)
on Junco hy emails and Sturnus vulgaris, respectively, there has been accumulated
an abundance of experimental evidence which indicates that artificial elongation
of the daily photoperiod in winter can cause testicular growth and development in
a substantial number of temperate zone species of birds. Much of the available
information has been reviewed or cited by Bissonnette (1937), Wolf son (1945,
1952), Benoit (1950), Burger (1949), Hammond (1954), Aschoff (1955) and
Schildmacher and Rautenberg (1953). In general these studies contain relatively
little information on the actual rates of testicular development as functions of the
length of the daily photoperiod, although the investigations of Burger (1948,
1953) on 6". vulgaris, Bartholomew (1949) on Passer domestlcus, and Winn
(1950) on /. hy emails are exceptions in this respect. Despite the sparsity of data
on rates of testicular development the investigations of the photoperiodic stimula-
tion of testicular growth and development have provided the basis for widely
accepted theories in which the increasing vernal day-length is regarded as the
primary stimulator and timer in the testicular cycle. These theories are supported
by the observations that, in at least most species studied, retention at winter day-
lengths results in no development or relatively slight development. Consequently
an appraisal of the photoperiodic theories indicates them to be qualitatively rational
and logical, at least for many temperate zone species, but deficient with respect to
quantification. Because of this deficiency it is not possible to evaluate, either in-
dividually or collectively, the roles of other variables in the natural course of
testicular development. It is the function of this paper to present approximate
quantifications of the function of day-length in testicular development and conse-
quently to indicate the approximate magnitude of the roles of other variables in
natural testicular development in a population of the white-crowned sparrow,
Zonotrichla leucophrys gambelll. The analyses to be presented here suggest argu-
ments against the objections of Blanchard (1941) and Marshall (1952, 1955) to the
photoperiodic theories. The data on which our analyses are based were obtained
in the course of an extensive series of experiments on the mechanism of photo-
stimulation of testicular development.

MATERIALS AND METHODS

Our experimental birds were captured with Japanese mist nets from a wintering
population in the Snake River Canyon in southeastern Washington (Mewaldt and

1 The data on which this paper is based were obtained in investigations supported by the
Office of Naval Research (Contract Nonr-1520) and by funds made available for biological
and medical research through State of Washington Initiative Measure No. 171.

254

TESTICULAR GROWTH IN ZONOTRICHIA 255

Farner, 1953). Prior to the beginning of the experiments they were held in large
outdoor aviaries. For experimental lighting they were placed, one or two per
cage in small cages (8^ X 10 X 16 inches), or three or four per cage in larger
cages (12 X 24 X 18 inches). Illumination was provided with incandescent lamps
at an intensity of 40-60 foot candles which is substantially above the maximal
intensity (i.e., minimum intensity at which maximum rate of development occurs)
of about four foot candles. The birds were fed a vitamin- and mineral-enriched
chick-starter mash prepared according to a formula developed by the Department
of Poultry Science of the State College of Washington. Food and water were
available at all times. Except as otherwise noted the experimental birds were
held at 20-24° C.

Immediately after removal, testes were placed in acetic acid-formaldehyde-
ethanol fixing mixture. After five days they were transferred to 70% ethanol
and five days thereafter weighed with a Roller-Smith torsion balance.

We are indebted to Drs. Richard A. Parker and Morris S. Knebelman for
suggestions and criticisms concerning the mathematical analyses and interpretation
of the data. Drs. I. O. Buss and Albert Wolf son have made criticisms and sug-
gestions concerning the manuscript. Some of the data are from experiments con-
ducted by Dr. L. R. Mewaldt while he was a member of our research group.

TESTICULAR WEIGHT AS A FUNCTION OF TIME WITH A CONSTANT

DAILY PHOTOPERIOD

An examination of the weights of the testes taken at intervals during a period
of treatment with constant daily photoperiods of stimulatory duration suggests a
relationship which approximates a logarithmic growth curve until the combined
testicular weights reach about 200 mg. If this is a good approximation then the
relationship between time and testicular weight may be expressed as

logio Wt = logio W0 + kt, (1)

where W0 is the initial testicular weight in milligrams, Wt is the weight at t days,
and t is the period of treatment in days. As an illustration and test of this rela-
tionship, data from three experiments have been combined in Figures 1 (first-year
birds) and 2 (adults). These three experiments involved treatments with 15-hour
daily photoperiods at 0° C. mean temperature, 20-hour photoperiods at 20° C.,
and 15-hour photoperiods at 22° C., respectively. Since the rate constants (&),
with t expressed in days, are different, t in Figures 1 and 2 is expressed in arbitrary
relative units (1 = time required to attain 100 mg. combined testicular weight).
On this basis there were no apparent differences among the three groups; there-
fore, they are not differentiated in Figures 1 and 2. An inspection of Figure 1
indicates that, for first-year birds, there is a reasonably good linear relationship
between the logarithms of testicular weight and time up to about 200 mg. (log]0 =
2.3) and hence a reasonably good conformance with equation (1). For adults
(Fig. 2) it appears possible that a weak S-relationship may exist between t and
log Wf. However, for purposes of comparison of rates of development among
experimental groups, it appears that no useful purpose can be effected in seeking

256

DONALD S. EARNER AND A. C. WILSON

logW

first year

0

time

FIGURE 1. Combined testicular weights (W) of first-year white-crowned sparrows as a
function of time with constant daily photoperiods of stimulatory duration. Time in arbitrary
units (see text). Closed circles represent developing testes; open circles represent regressing
testes. Broken lines represent upper and lower 95%-fiducial limits of the slope.

TESTICULAR GROWTH IN ZONOTRICHIA

257

logW

time

FIGURE 2. Combined testicular weights (W) of adult white-crowned sparrows as a
function of time with constant daily photoperiods of stimulatory duration. Time in arbitrary
units (see text). Closed circles represent developing testes ; open circles represent regressing
testes. Broken lines represent upper and lower 95% -fiducial limits of the slope.

258

DONALD S. FARNER AND A. C. WILSON

a more precise relationship. Because of the less precise relationship between t and
Wt for adult birds, it is now our policy to perform all critical experiments with
first-year birds. The solid lines in Figure 1 and 2 are drawn according to the values
of k obtained by the procedure outlined by Mood (1950, Chapter 13) ; similarly

0.10

0.05

i

0 A

llf

r

*

4
I
/

8 12 16 20

daily photoperiod in hours

24

FIGURE 3. The rate of testicular development as a function of the duration of the daily
photoperiod (/>). See text for definition of rate constant (k). The shaded area encloses the
upper and lower 95% -fiducial for all points. Open circles represent samples of first-year
birds; closed circles represent adjusted means for samples of adults.

the broken lines represent the upper and lower 95% fiducial limits, also obtained
according to Mood. In order to minimize the effect of possible non-linear relation-
ships between log Wt and t it is now a practice in our laboratory to kill birds when
the testes have attained a combined weight of about 100 mg.

TESTICULAR GROWTH IN ZONOTRICHIA 259

THE TESTICULAR GROWTH RATE CONSTANT (K) AS A FUNCTION OF THE
LENGTH OF THE CONSTANT DAILY PHOTOPERIOD

An examination of k as a function of the length of a constant daily photoperiod
(/>) has been effected by analysis of 18 series of data (11 first-year and 7 adult)
for photoperiods varying in length from 8 to 24 hours. For each of these an esti-
mation of k and the approximate 95% fiducial limits for k were obtained according
to Mood (1950). These are given in Figure 3. In the calculation of the 95%
fiducial limits for k the same W0 sample was used for all groups with the same
W0 date ; the fiducial limits thus obtained are therefore to be regarded as estimated
limits rather than true fiducial limits. As indicated above, k is greater for first-
year birds than for adults, approximately 1.16 times as great for the series of data
used here. Therefore in plotting the data in Figure 3, the adult values, both for
estimates of k and the approximate fiducial limits, were multiplied by 1.16. The
broken line, suggesting a functional relationship between p and k, has been drawn
by inspection. The shaded zone on either side encloses all of the estimated fiducial
limits for the individual estimates of k. It must be emphasized that this shaded
zone does not represent the fiducial limits for the slope of the curve. The linear
portion of the curve (between 10 and 18 hours) may be represented by

lst-yr.

= 0.009 (P - 9.1). (2)

Obviously there is an uncertainty in the nature of the functional relationship
between p and k for values of p less than 10 hours. The relatively small changes
in W with respect to the natural variability of W and the errors in measurement
involved with small values of W make this a difficult problem. Fortunately, as
it will become evident subsequently, the values of k in this range are sufficiently
small so that the calculations based on equation (2) are not serious even when it is
extrapolated linearly to k — 0.

THE ROLE OF ENVIRONMENTAL TEMPERATURE IN THE RATE OF
TESTICULAR DEVELOPMENT

For Z. /. gambelii it has been demonstrated earlier (Farner and Mewaldt, 1952,
1953) that the rate of testicular development with a fixed daily photoperiod of
stimulatory duration is a function of environmental temperature between 0° and
22° C. It should be emphasized, however, that elevation of environmental tempera-
ture has no effect when the photoperiod is below stimulatory level (Farner and
Mewaldt, 1953). Assuming, for purposes of estimation, that the effect of tempera-
ture is linear with respect to k, the following relationship should hold :

kB/kA = 1 + c(TB - TA). (3)

We have estimated the value of c on the basis of two groups of birds subjected to
15-hour daily photoperiods, one at a mean environmental temperature of 1° C., the
other at 22° C. On the basis of mean values of k for these two groups, c has a
value of about 0.009 degrees"1. Substitution of this value for c in equation (3)
indicates that the role of temperature must be relatively minor. It must be ob-
served here, however, that the responses of the lower temperature group were
sufficiently variable so that, were the true k near the lower 95% fiducial limit, the

260

DONALD S. EARNER AND A. C. WILSON

value of c would be approximately 0.019. Engels and Jenner (1956) have ex-
amined the effect of environmental temperature on the rate of testicular develop-
ment in /. hyemalis subjected to daily photoperiods of 10-12 hours. Although
differences in methods and analyses do not allow a direct comparison, it appears
that c for this species must be of the order of 0.02-0.03. Similarly, from the data

log W

FIGURE 4. A comparison of predicted curves of testicular development with natural de-
velopment. Open circles represent combined testicular weights for first-year birds taken
from the Snake River Canyon population ; closed circles represent combined testicular weights
for adults from the same population. These groups cannot be distinguished after the prenuptial
molt which occurs in late March and early April. The four curves were calculated according
to equation (4) including the adjustment for temperature effects indicated in equation (3).

of Burger (1948) for 5". vulgaris we have estimated c to be about 0.02. For these
three species, then, it appears that the role of environmental temperature in testicular
development is of a small, although similar, order of magnitude.

A PREDICTED COURSE OF TESTICULAR DEVELOPMENT UNDER NATURAL CONDITIONS
CALCULATED ON THE BASIS OF LABORATORY-ESTABLISHED RATES

Using the empirical relationships presented above, an attempt has been made
to "predict" the course of vernal testicular development. Since environmental

TESTICULAR GROWTH IN ZONOTRICHIA 261

temperature is an irregularly fluctuating variable, two temperature values, 0° C.
and 20° C., were used for the construction of two predicted curves. For the most
part the environmental temperatures fall within these limits although some sub-
zero nights occur in January and February. The four curves in Figure 4 are
based on values of k from Figure 3 assuming that k is a continuous positive func-
tion of p between the limits of 0 and 24 hours, k being insignificantly (< 0.002)
small for all values of p below 9.1 hours. Calculations were begun with 21 Decem-
ber (day-length about 8.8 hours) when the mean logarithms of the testicular
weights for adults and first-year birds were + 0.15 ± 0.10 and — 0.17 ± 0.16,
respectively. As p increases, the correspondingly larger values of k were em-
ployed ; the operation may be represented by

n

logic Wn = logio W0 + Z kt, (4)

where n is the number of days after the initial date in the calculation.

If the relationship between p and k assumed here is correct, then the day-lengths
following the termination of the refractory period in mid-November (Farner and
Mewaldt, 1955) would be stimulatory. Accordingly the curves in Figure 3 have
been extrapolated back into November. This extrapolation indicates weights for
early November which are within the range of one standard deviation of December
weights and well within the range of early November weights.

An alternative interpretation of the functional relationship between p and k is
that expressed by equation (2). Four curves (adults at 0° C. and 20° C., first-
year birds at 0° C. and 20° C.) were therefore constructed using the equation

logic Wn = logio W0 + nki...n (5)

in which,

k = 0.009^- - - 0.082. (6)

n

The curves obtained thus were not detectably different from those obtained by the
operation noted in equation (4) and plotted in Figure 4. In the use of equations
(4), (5), and (6), p has been assigned the value of the time between sunrise and
sunset. Obviously this is arbitrary because of fluctuations in effective day-length
as a consequence of differences in meteorologic conditions. That such fluctuations
may affect the rate of development is obvious from our unpublished data on k as a
function of light intensity. These indicate that the maximal intensity is about four
foot candles whereas the minimal intensity, if such exists, is somewhat less than
one foot candle. There is the additional variable of the amount of cover about the
bird early in the morning and late in the evening. Our measurements of light
intensities in the Snake River Canyon suggest that the period between sunrise and
sunset is about as satisfactory an approximation as we can select. A discussion
of the approximate magnitude of error which could be attributable to this selection
of values for p is included subsequently.

262 DONALD S. EARNER AND A. C. WILSON

A COMPARISON OF THE ACTUAL AND PREDICTED CURVES OF VERNAL

TESTICULAR DEVELOPMENT

A comparison of the predicted and natural courses of vernal testicular develop-
ment is effected in Figure 4 by plotting testicular weights of birds taken in the
Snake River Canyon up to the time of migration during the springs of 1952, 1953,
1955, and 1956. These data indicate that the calculations based on laboratory-
established rates predict the attainment of 100-mg. testicular weights about 10 days
prior to the actual time of attainment in nature. This relatively close conformance
is consistent with the hypothesis that the increasing vernal photoperiod is the
primary timer in the annual development of the testicular cycle. However, it is
of importance to examine the possible bases for this relatively small discrepancy
between the calculated and actual curves. The possible bases are both statistical
and natural.

( 1 ) The statistical factors. A readily apparent possible cause of the differences
between the calculated and actual curves in Figure 4 is the statistical nature of the
initial testicular weights employed in the calculations, 0.7 mg. for first-year birds
and 1.4 mg. for adults. These are mean values for birds taken in the field during
the appropriate period. The logarithms are — 0.17 ± 0.16 and + 0.15 ± 0.19,
respectively. An inspection of Figure 4 indicates that a good conformance would
have occurred had the selected initial weights been about one standard deviation
below the estimated means. Also important is the possible error in weighing small
testes since the balance and method used in weighing have a combined error of
± 0.1 mg.

Another possible factor is the statistical nature of the slope of the line relating
k as a function of p. The curve in Figure 3 has been drawn by inspection with
attention both to the means and their approximate 95% fiducial limits. It can
be noted that a reasonable conformance between the predicted curve and the natural
data would require the slope constant a in Figure 3 and equation (2) to be 0.007
instead of 0.009. Since this appears somewhat improbable, an error in a, although
possibly contributory, cannot be regarded as the primary cause of the difference.
It is also possible that the functional relationship between W and t with constant
daily photoperiods may differ further from the relationship in equation (1) than
our data suggest. An examination of models indicates that it is very unlikely
that this could be the primary source of the difference. Obviously it could be
contributory.

A further possible source of error is in the value of the minimal stimulatory
day-length (q), or in our assumptions concerning the relationship of k to p and
the value of p where k becomes insignificantly small. However, as noted above,
since very small values of k are involved such errors affect the calculated curve
only slightly.

There is also the possibility of error with respect to the effect of environmental
temperature as indicated by c in equation (3). As noted previously the IOWT-
temperature group which was used in the estimation of c showed considerable
variability with relatively wide fiducial limits for k. If the true k were close to
the lower 95% fiducial limit, c would be about 0.019 and the 0° C. curves in Figure
4 would reach the 100-mg. level about 18 days later than the 20° curves, or about
nine days later than shown in Figure 4. This would give a better, although not

TESTICULAR GROWTH IN ZONOTRICHIA

263

a complete, conformance. In other words, the statistical nature of c is such that
it could contribute to the discrepancy although it is highly unlikely that it could
be the sole basis for it.

(2) The natural factors. A possible source of the difference between the
predicted curve and the natural data, as indicated above, lies in our selection of
values for the effective day-length, p. The complexity of this problem has been
discussed well by Bartholomew (1949). As a test of the magnitude of this source
of error, we have assumed that p for each day is 30 minutes less than the period
between sunrise and sunset. This alteration is almost sufficient to make the pre-
dicted curve coincide with the natural data. However, our observations on light
intensities in the Snake River Canyon indicate that this assumption is unreason-
able, for only rarely would p be as much as 30 minutes less than the period from
sunrise to sunset ; more frequently it would be somewhat greater than this period.

TABLE I

A comparison of body weights of male white-crowned sparrows in large outdoor
aviaries at Pullman with those taken from the Snake River Canyon

Snake River Canyon

In cages at Pullman

Period

No.

Weight,
grams

Standard
Deviation

No.

Weight,
grams

Standard
Deviation

1-15 January

37

27.8*

±1.5

43

27.1*

±2.1

16-31 January

128

28.0

±1.6

51

27.8

±1.9

1-14 February

67

27.8**

±1.4

53

26.6**

±1.5

15-28 February

38

26.9

±1.2

45

27.5

±1.6

Prenuptial molt

55

27.8

±1.5

28

28.0

±2.8

* Significantly different, P = ca. 0.04.
** Significantly different, P < 0.001.

Therefore, although our definition of p may be the source of some error, it is
neither of sufficient magnitude nor in the right direction to account for the differ-
ence.

The estimates of k were affected with birds which had a nutritionally adequate
food constantly available. The possibility exists that a nutritional difference could
be involved in the differences between the calculated and actual courses of testicular
development. That this possibility warrants consideration is evident from the
demonstrated reduction of production of gonadotropic hormones by mammals in
nutritionally inadequate states, as summarized by Ershoff (1952). Whether
periods of nutritional insufficiency occur for white-crowned sparrows in the Snake
River Canyon is obviously difficult to ascertain. However, a comparison can be
made between the body weights of birds retained for a month or more under out-
door conditions in large aviaries at Pullman and those of birds taken from the
natural population in the Snake River Canyon. The former received the same
food ad libitum as did the experimental birds from which our laboratory-established
rates were obtained. These data are summarized in Table I ; it is obvious that
they in no way support a hypothesis of poorer nutritional state among the wild
birds. A further argument against an effective difference in nutritional state comes

264 DONALD S. FARNER AND A. C. WILSON

from a series of 29 males held in large outdoor aviaries at Pullman. The course
of testicular development for this group shows no apparent difference from that
of the natural population despite the constant availability of nutritionally adequate
food. Although we cannot reject completely the possibility of an interaction be-
tween low temperature and poor nutritional state, certainly we find no evidence
in our data to support it.

DISCUSSION

It is patent that the greatest of caution must be applied in any inductive extra-
polation of our experience with a single population of a single species. Never-
theless it may be of some value to consider briefly some possible bases for variations
among the temporal patterns in the initiation of the testicular cycles of the temperate-
zone passerine species in which day-length is the primary timer. In this discussion
it is assumed that the basic relationships described here for Z. /. yainbclii constitute,
in a general way, a typical scheme.

It then becomes desirable to consider the general form of equation (2), relating
day-length (/>), the minimum effective day-length (q), and the rate constant (&) :

k = a(p - q). (7)

For arguments presented here, days (in winter) where p < q must be treated as
though p = q and k = 0. This may be unrealistic, the true situation possibly
being, as suggested above, that k may be a continuous positive function of p be-
coming insignificantly small as p becomes somewhat smaller than q. Our data
are not inconsistent with this possibility. The data of Burger (1953) for S.
vulgaris suggest this possibility. Although the effective difference between these
two interpretations of the functional relationship between p and k is relatively
trivial for Z. I. gambelii this may not necessarily be the case for other species. It
is obvious that one way in which testicular maturation can be attained earlier in
the year is with a lower value of q, or with a stronger curvilinear relationship be-
tween p and k in the lower tail of the curve. It is also obvious that when q is less
than the shortest winter day, or, in the alternative interpretation of the relationship
between p and k, when k has an appreciable value at the lowest winter values of p,
the time of termination of the refractory period may become important with respect
to the curve of testicular development. It seems possible that this could be ap-
plicable to certain non-migratory British species in which abortive fall and mid-
winter sexual activity has been noted (Marshall, 1949, 1952). An essential part
of such a hypothesis would be a more marked relationship between k and the tem-
perature coefficient (c).

The literature actually contains 'very little useful data on the minimum effective
day-length (q). For 5\ vulgaris it appears to be less than 8.5 hours (Burger,
1949). A recalculation of the data of Bartholomew (1949, Fig. 15) suggests that
q is about nine hours for P. domesticus. However, because, in another experiment
his data (p. 444) contain two cases of at least some histologic development at 8-
hour photoperiods, it is possible that the alternative interpretation of the relationship
between p and k may hold. In considering the matter of the minimum effective
day-length (q), it is necessary to bear in mind the possibility of the existence of
an internal timer as Miller (1955) has suggested for Zonotrichia coronata and

TESTICULAR GROWTH IN ZONOTRICHIA 265

Zonotrichia leucophrys nuttalli. The data of Benoit ct al. (1955) suggest a similar
possibility in ducks.

Another way in which the time of maturation of the testes may be affected is
by the rate of testicular growth (&) once stimulatory day-lengths occur. As indi-
cated in equation (7), a greater slope constant a will result in a more rapid develop-
ment. From the data of Bartholomew (1949) for P. domesticus it has been
possible to calculate a curve relating k to />. In this calculation it has been assumed
that the rate of testicular growth under the influence of daily photoperiods of
constant length is in accordance with the logarithmic curve expressed by equation
( 1 ) . Values of k for several different photoperiods have been similarly calculated
from data obtained for this species in our laboratory and from Vaugien (1952).
These values are consistent with the curve derived from Bartholomew's data.
The curve is similar to that of Z. I. gambelii (Fig. 3) ; however, the linear part is
steeper, a being 0.013 compared to 0.009 and 0.008 for first-year and adult Z. I.
gambelii. It appears, then, that the greater value of a for P. domesticus correlates
well with the earlier testicular development of this species in nature. It should
be observed that direct comparisons of a for different species are meaningful only
when the differences between the logarithms of the resting and developed testes are
very similar. This is essentially true for Z. 1. gambelii and P. domesticus.

It should also be noted that a greater adjustment to the conditions of the develop-
ing season could be obtained with a greater sensitivity to environmental temperature
as indicated in equation (3). As c becomes greater the time of maturation would
fluctuate more as a function of environmental temperature. This would be par-
ticularly valuable to an early breeding, non-migratory species, but also of some
value to species whose migratory route is confined to a relatively restricted range
of temperate-zone latitude. As noted above, it would be very interesting to in-
vestigate the British species discussed by Marshall (1949, 1952) with respect to
temperature sensitivity, the termination of the refractory period, and the functional
relationship between p and k.

It is obvious that variations in the values of a, c, q (or the nature of the lower
tail of the curve relating k to />), and the terminal dates of the refractory periods
could produce a wide range of times at which testes mature. However, it must
be re-emphasized that these arguments are derived almost exclusively by extra-
polation from experiments on a single population of Z. I. gambelii. It would be of
great interest to examine additional species similarly.

SUMMARY

1. The rate of testicular development in Zonotrichia leucophrys gambelii has
been examined quantitatively as functions of day-length, light intensity, and environ-
mental temperature.

2. From these laboratory-established relations a predicted curve for testicular
growth under natural conditions was calculated and compared with data obtained
from a natural population. The predicted curve indicates the attainment of 100-
mg. combined testicular weight about ten days earlier than its occurrence in the
natural population. Although this relatively small discrepancy may be reasonably
explained on a statistical basis, it is not possible to rule out minor effects by environ-
mental variables other than daily photoperiod and temperature.

266 DONALD S. FARNER AND A. C. WILSON

3. The calculations, and the relatively close agreement with the observations of
the natural population, emphasize quantitatively the overwhelming importance of
the daily photoperiod as the primary timer in the testicular cycle for this population
of Zonotrichia leucophrys gambelii. Other environmental factors, as they operate
in the Snake River Canyon, appear to be responsible for fluctuations with a com-
bined maximum possible magnitude of the order of ten days to two weeks.

LITERATURE CITED

ASCHOFF, J., 1955. Jahresperiodik der Fortpflanzung bei Warmblutern. Stndiuin Generate,

8: 742-776. "
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BENOIT, J., 1950. Reproduction — caracteres sexuels et hormones — determinisme du cycle

sexuel saisonnier. In: Trait e de Zoologie, edited by Pierre-P. Grasse, Vol. XV.

Masson et Cie., Paris, pp. 384-478.
BENOIT, J., I. ASSENMACHER AND E. BRARD, 1955. Evolution testiculaire du canard domestique

maintenu a 1'obscurite totale pendant une longue duree. C. R. Acad. Sci., 241 : 251-

253.
BISSONNETTE, T. H., 1930. Studies on the sexual cycle in birds. I. Sexual maturity, its

modification and possible control in the European starling (Stunnts vulgaris). Amer.

J. Anat., 45 : 289-305.

BISSONNETTE, T. H., 1937. Photoperiodicity in birds. Wilson Bull., 49 : 241-270.
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seaboard: environment and annual cycle. Univ. Calif. Pitbl. Zool., 46: 1-178.
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starling. /. Exp. Zool., 109 : 259-266.
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birds. Wilson Bull, 61 : 211-230.
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male starling, Stnrnus vulgaris. J. Exp. Zool., 124: 227-239.
ENGELS, W. L., AND C. E. JENNER, 1956. The effect of temperature on testicular recrudescence

in juncos at different photoperiods. Biol. Bull., 110: 129-137.
ERSHOFF, B. H., 1952. Nutrition and the anterior pituitary with special reference to the

general adaptation syndrome. Vitamins and Hormones, 10: 79-140.
FARNER, D. S., AND L. R. MEWALDT, 1952. The relative roles of photoperiod and temperature

in gonadal recrudescence in male Zonotrichia leucophrys gambelii. Anat. Rec., 113:

612.
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and diurnal photoperiods in gonadal activation in male Zonotrichia leucophrys gambelii

(Nuttall). Expericntia, 9: 219-221.
FARNER, D. S., AND L. R. MEWALDT, 1955. The natural termination of the refractory period

in the white-crowned sparrow. Condor, 57: 112-116.
HAMMOND, J., 1954. Light regulation of hormone secretion. J'itamins and Hormones, 12:

157-206.
MARSHALL, A. J., 1949. Weather factors and spermatogenesis in birds. Proc. Zool. Soc.

London, 119: 711-716.

MARSHALL, A. J., 1952. The interstitial cycle in relation to autumn and winter sexual be-
haviour in birds. Proc. Zool. Soc. London, 121 : 727-740.

MARSHALL, A. J., 1955. Reproduction in birds : the male. Mem. Soc. Endocrinol., 4 : 75-93.
MEWALDT, L. R., AND D. S. FARNER, 1953. The composition of a wintering population of white-
crowned sparrows in southeastern Washington. Condor, 55 : 313-314.
MILLER, A. H., 1955. The expression of innate reproductive rhythm under conditions of winter

lighting. Auk, 72: 260-264.
MOOD, A. M., 1950. Introduction to the Theory of Statistics. McGraw-Hill Book Company,

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TESTICULAR GROWTH IN ZONOTRICHIA 267

ROWAN, W., 1925. Relation of light to bird migration and developmental changes. Nature,

115: 494-495.
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5) : 345-351.
VAUGIEN, L., 1952. Sur 1'activite testiculaire, la teinte du bee et la mue du moineau domestique

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407.
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male gonadal growth in the slate-colored junco. Doctoral Dissertation, Northwestern

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WOLFSON, A., 1945. The role of the pituitary, fat deposition, and body weight in bird migra-
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191-200.

A BEHAVIORAL MECHANISM FOR OSMOTIC REGULATION
IN A SEMI-TERRESTRIAL CRAB

WARREN J. GROSS

Division of Life Sciences, University of California, Riverside, California

It is generally known that an animal can regulate osmotically by possessing a
relatively impermeable integument and by utilizing specialized organs which by
active metabolic processes control the flux of salts and water between animal and
its external medium. However, there is evidence that animals are capable of
assisting the osmo-regulatory mechanism by their behavior.

Barnes (1935, 1938, 1940) demonstrated that the isopod, Ligia baudiniana,
prefers distilled water to normal sea water when the choice is offered in the form
of moistened filter paper. Gross (1955) revealed that the land crab, Birgus latro,
can control the osmotic pressure of its body fluids by selection of appropriate sea
water concentrations. Krijgsman and Krijgsman (1954) have produced evidence
of an osmo-receptor in the spiny lobster Jasus which apparently serves to guide the
animal with respect to salinities.

Pachygrapsus crassipes is a crab of semi-terrestrial habits, most often found in
exposed, rocky shore situations, but also occasionally found in protected bays in
muddy burrows. Typically, it does not inhabit waters deviating much in salinity
from normal sea water, but it can regulate osmotically in both dilute and con-
centrated sea water (Jones, 1941; Gross, 1955; Prusser et a/., 1955). Gross
(1955) raised the question as to the adaptive significance of strong osmo-regulatory
powers in this crab which is extremely agile and easily capable of reaching normal
sea water should it by chance venture from the sea into an osmotic stress such
as would be afforded by an isolated, evaporated tide pool, or one diluted by rain
w-ater.

In Southern California, situations affording such osmotic stresses are rare.
However, there undoubtedly are regions in this crab's range where osmotic stresses
are readily enough available so that selection would favor the development of an
osmo-regulatory mechanism. On the other hand, should Pachygrapsus be capable
of detecting salinities deviating from normal sea water and should it shun abnormal
salinities, then the adaptive significance of osmotic regulation in this species could
remain in doubt.

The present investigation will show that Pachygrapsus does show preference for
sea water of normal salinity.

MATERIALS AND METHODS

Crabs of the species Pachygrapsus crassipes, collected at Laguna and Newport,
California, were placed singly in a box in which there were four containers, each
containing sea water of a different salinity. The containers were placed in each
corner of the box and sunk so that their rims were flush with the platform which

268

SALINITY PREFERENCE IN A CRAB

269

constituted a second floor of the box. In each container was a treadle which could
be depressed by about 15 grams. The surface of the treadle was barely below the
water level in the container.

As a crab entered a given medium, it was necessary for it to depress the treadle.
This caused the deflection of a signal magnet which was recorded on a kymograph
drum, rotating at a known rate. Thus a crab placed in the box had the choice of
remaining on the platform in the air or entering any of the containers in each of
the four corners of the box. The kymograph record then would indicate the move-
ments of the crab while it remained in the box, what container it entered, when it
entered and how long it remained.

The selection box was placed in a darkened, quiet, temperature-controlled room
(20° C.). As a control against selection for any given corner, rather than salinity,
the respective sea water concentrations were rotated in position with each successive
test specimen. In most cases only three containers were filled with water ; the
fourth remained empty to check on the preference for corners. Each crab re-
corded, remained in the box for at least 40 hours.

Sodium and potassium concentrations of the blood were determined in a few
cases by means of a Beckman flame photometer.

RESULTS

Table I summarizes the results of the following experiments :
1. Crabs freshly removed from normal sea water were given a choice of 50,
100, or 150% sea water (seven crabs).

TABLE I

Salinity preference in Pachygrapsus crassipes

Treatment

Mean time spent in selected salinities

50% sea water

75% sea water

100% sea water

125% sea water

150% sea water

Time
spent out
of water
(%)

%
time

time (hr.)

%
time

time (hr.)

%
time

time (hr.)

%
time

time (hr.)

%
time

time (hr.)

No. visits

No. visits

No. visits

No. visits

No. visits

Normal
(7 animals)

6.2

3
32

29.7

16

55

3.7

2
29

59.7

Normal
(5 animals)

6.1

3
12

39.6

17
49

6.9

3

24

47.5

Acclimated to
50% sea water
(5 animals)

7.7

4
42

29.8

15

37

7.3

4
33

55.4

Acclimated to
150% sea water
(5 animals)

13.5

7
32

16.1

9

22

10.1

6

21

60.3

Desiccated
(6 animals)

11.4

8
51

47.6

29

54

3.6

2
32

37.5

Legend : Normal = animals freshly removed from normal sea water ;
% time = % total time spent in chamber.

270 WARREN J. GROSS

2. Crabs freshly removed from normal sea water were given a choice of 75,
100 or 125% sea water (five crabs).

3. Crabs acclimatized to 50% sea water 2—7 days were given a choice of 50,
100 or 150% sea water (five crabs).

4. Crabs acclimatized to 150% sea water 1-3 days were given a choice of 50,
100 or 150% sea water (five crabs).

5. Crabs desiccated 2-4 days were given a choice of 50, 100 or 150% sea water
(six crabs).

Of the above 28 specimens studied in the selection chamber, 25 spent more
time in 100% sea water than any salinity offered, thus indicating that Pachygrapsus
does show a decided preference for normal sea water. Two of the above exceptions
were among the group which had been acclimated to 150% sea water. The third
was one of those which had been desiccated. Although Table I shows that animals
first acclimatized to 150% sea water average more time in 100% sea water than
any other salinity, the histories of the individuals do not show such a preference.

All six of the crabs which were desiccated before being placed in the box re-
mained in water longer than in the air, which might be expected. However, of
the remaining 22 test crabs, 17 spent more time out of the water than in all salinities
offered, thus suggesting the degree of the aerial habit in this marine form.

Considering individual histories, 14 crabs spent more time in the hypotonic
medium than in the hypertonic medium. Six spent more time in the concentrated
media and eight showed no significant preference between the two types of stresses.
It is interesting, however, that of the 14 preferring dilute to concentrated media,
four had been previously acclimated to 150% sea water, only one of which preferred
dilute to normal sea water. Three of the above 14 had been desiccated previously,
one of which showed a preference for dilute sea water over normal sea water.
With the sample at hand, nothing can be said concerning the preference of dilute
and concentrated sea water or vice versa.

In only one of the 24 cases where one of the four containers was not filled with
water did a crab spend more time in the empty container than it did on the platform
between the containers, i.e.. out of the water. Thus, there does not seem to be any
particular preference for corners, at least in an aerial situation.

Of 25 crabs showing preference for 100% sea water, judging by the time they
spent in this salinity, 14 made more visits to normal sea water than any other
offered aqueous medium. On the other hand, 11 made more or an equal number
of visits to other than 100% sea water. Thus, with the sample at hand, nothing
can be resolved precisely as to the learning process involved.

Table II shows the blood sodium and potassium concentrations of some of the
crabs which had spent at least 40 hours in the selectivity chamber. Here it can be
seen that the blood sodium of one of the five crabs which was first acclimated to
150% sea water (number 4) was definitely above the normal concentration. It
should be pointed out, however, that this particular specimen was somewhat
weakened by its previous treatment in 150% sea water and this was the only crab
that spent more time in concentrated sea water than in any of the other salinities.
Thus, four out of five specimens in the group attained close to normal blood con-
centrations after a period in the selectivity chamber. The blood sodium concentra-
tion of two out of the three of the desiccated group sampled was below the normal
concentration range. This is particularly interesting in the case of specimen num-

SALINITY PREFERENCE IN A CRAB

271

TABLE II

Blood sodium and potassium concentrations in Pachygrapsus following
period in selection chamber

Specimen No.

Treatment

Blood concentration after treatment*

Na (meq/1)

K (meq/1)

1

Normal

484

6.35

2

Acclimatized to 50% sea water

498

—

3

481

8.08

4

Acclimatized to 150% sea water

544

7.72

5

477

6.62

6

477

7.66

7

495

8.81

8

498

8.06

9

Desiccated

417

7.35

10

479

8.50

11

414

10.4

95% fiducial limits**

478-489

7.19-7.67

* Concentration choices available for all specimens were 50, 100, and 150%.

* Calculated from observations on 36 crabs freshly removed from the sea.

her 9 which spent 51% of its time in 100% sea water while it was in the chamber.
Specimen number 11, however, spent more time in 50% sea water than in any
other offered salinity. The remaining cases shown in Table II are within or close
to the normal blood concentrations with respect to sodium.

Considering the intrinsic error in determining potassium concentrations by the
methods used in this investigation (about 10% of the concentration of normal
blood), only the blood potassium of specimen 11 (Table II) seems to vary from
normal. This, however, would have little effect on the osmotic pressure of the
blood.

The activity in the selection chamber of the above crabs also was considered
with respect to the time of day and phase of the tide, but no periodicity could be
resolved.

DISCUSSION

Using duration of immersion as a criterion, Pachygrapsus prefers normal sea
water to hypotonic or hypertonic media varying at least 25% from 100% sea
water. It thus seems that in a natural situation, this crab probably would not
remain long in an osmotic stress, but soon would return to the salinities of normal

272 WARREN J. GROSS

sea water. Pachygrapsus possesses a relatively impermeable integument (Gross,
1957) and this means that there would be a strong passive resistance to salt-water
exchanges, should the crab enter an osmotic stress. The question is posed then
as to the need for osmotic regulation in an animal which is rarely exposed to such
stresses and which would attempt to escape, and could escape to the comfort of the
nearby sea should the occasion arise.

The strong preference for normal sea water, it would seem, would be a powerful
factor tending to restrict this species to the intertidal and subtidal zones of the sea.
For example, a period of desiccation, such as might be encountered by remaining
out of the water, would stimulate the return to the water, but as suggested above,
not just to any water, but to normal sea water. Again, in an estuarine situation,
where a sharp salinity gradient would be available, an animal responding thus to
osmotic stresses would tend to avoid brackish water, even though it possessed the
ability to regulate strongly in such stresses. Even in a case where a population
were temporarily trapped in a dilute situation for an extended period, this period
of acclimation, as shown above (Table I), does not seem to diminish the preference
for normal sea water.

Of course, the distaste for brackish water possibly could be overcome by other
factors, e.g., food source and retreat from predators.

Acclimation to 150% sea water apparently breaks down the preference for
normal sea water (Table I), although the fact that four out of five of the specimens
thus treated were able to achieve normal blood concentrations suggests effective
salinity selection of a sort. This preference breakdown perhaps is correlated with
the ability of Pachygrapsus to regulate more strongly in dilute than in concentrated
media (Jones, 1941 ; Gross, 1955; Prosser et al., 1955). In such case, a period of
acclimation would necessitate a greater alteration on the physiological condition
of the organism and consequently a greater effect on its behavior. Then, too, a
precise "knowledge" of external salinities possibly is necessary for adequate regula-
tion. Should a period of immersion in 150% sea water reduce the accuracy of the
appropriate receptors, whatever they may be, and efficiency of such receptors were
necessary for regulation, then it would follow that Pachygrapsus would show rela-
tively weak regulation in the concentrated sea water.

On the other hand, as shown above, acclimation to 50% sea water does not
appreciably affect the preference for normal sea water. This may be simply be-
cause Pachygrapsus can regulate strongly in dilute media and the consequent
physiological alteration in the crab would be held at a minimum. Or the hypotonic
media may not reduce the efficiency of the osmo-receptors and the organism, being
"aware" of the external salinity, could regulate accordingly.

It seems, then, that should a population of Pachygrapsus be confined to a hyper-
tonic medium for an extended period, by losing its preference for normal sea water,
it might remain in such concentrated environments, assuming all other biological
requirements were satisfied. However, it is difficult to imagine natural hypertonic
situations capable of isolating Pachygrapsus from the sea.

Table II demonstrates the general tendency for Pachygrapsus to achieve normal
blood sodium concentrations after a period in the selectivity chamber. This seems
to be true even when blood concentrations were forced away from normal by
acclimation to 50% and 150% sea water. Two exceptions, however, were crabs

SALINITY PREFERENCE IN A CRAB 273

previously desiccated. The final blood concentrations in these cases were lower
than normal, thus suggesting over-compensation.

It has been demonstrated numerous times that organisms immersed in stress
media metabolize more rapidly than when they are in their normal media ( Schlieper,
1929; Schwabe, 1933; Flemister and Flemister, 1951). This increased metabolism
has been interpreted often as added osmotic work. However, doubts have been
thrown on this interpretation (Krogh, 1939; Wikgren, 1953; Potts, 1954).

Gross (1957) reports that crabs immersed in stress media apparently attempt
to escape and thus become more active. He suggested, then, that increased oxygen
consumption in increased osmotic stresses was merely the reflection of the attempt
to escape an uncomfortable medium. The preference for normal sea water estab-
lished quantitatively by the present investigation corroborates this suggestion.

With the exception of those crabs first desiccated, the experimental animals
spent about half their time out of water. This suggests a high degree of adaptation
to the aerial habit, but not so much compared with the land crab Birgus which
under experimental conditions spends only about one hour per day visiting water
(Gross, 1955).

These studies were aided by a contract between the Office of Naval Research,
Department of the Navy, and the University of California NR 163-309. I am
pleased to express my gratitude for the able technical assistance of Mr. Paul
Holland. Also, I wish to thank Professor Theodore Holmes Bullock for his critical
reading of the manuscript.

SUMMARY

1. The shore crab Pachygrapsus crassipes prefers 100% sea water to 50, 75,
125 and 150% sea water.

2. This preference could not be altered by first desiccating the animal or by
acclimating the animal for several days in 50% sea water. The preference could
be altered somewrhat by acclimating to 150% sea water.

3. The preference for normal sea water by Pachygrapsus suggests a mechanism
which tends to restrict this crab to the intertidal and subtidal zones of the sea.

4. Pachygrapsus under the experimental conditions of the present investigation
spends about 12 hours per day visiting water. This compares to one hour per
day for the land crab Birgus.

5. Pachygrapsus tends to maintain normal sodium and potassium blood con-
centrations when given free choice of salinities, including 100% sea water. Normal
concentrations are generally achieved under the same conditions even when the
blood has been forced away from normal by acclimation in 50 or 150% sea water.
However, animals previously desiccated may over-compensate when offered a
choice of media varying in salinity and consequently achieve blood sodium con-
centrations below the normal range.

LITERATURE CITED

BARNES, T. C., 1935. Salt requirements and orientation of Ligia in Bermuda III. Biol. Bull.,

66: 259-268.
BARNES, T. C., 1938. Experiments on Ligia in Bermuda V. Further effects of salts of heavy

sea water. Biol. Bull, 74: 108-116.

274 WARREN J. GROSS

BARNES, T. C, 1940. Experiments on Ligia in Bermuda VII. Further effects of sodium and

magnesium. Biol. Bull., 78: 35-41.
FLEMISTER, L., AND S. FLEMISTER, 1951. Chloride ion regulation and oxygen consumption in

the crab Ocypode albicans (Bosq). Biol. Bull, 101: 259-273.
GROSS, W. J., 1955. Aspects of osmotic regulation in crabs showing the terrestrial habit.

Amer. Nat., 89 : 205-222.
GROSS, W. J., 1957. An analysis of response to osmotic stress in selected decapod Crustacea.

Biol. Bull., 112: 43-62.
JONES, L. L., 1941. Osmotic regulation in several crabs of the Pacific Coast of North America.

/. Cell. Coinp. Physiol., 18: 79-92.
KRIJGSMAN, B. J., AND N. KRIJGSMAN, 1954. Osmorezeption in Jasus lalandii. Zeitschr. f.

vergl. Physiol., 37: 78-81.

KROGH, A., 1939. Osmotic regulation in aquatic animals. Cambridge at the University Press.
POTTS, W. T. W., 1954. The energetics of osmotic regulation in brackish and fresh water

animals. /. Exp. Biol., 31 : 618-630.
PROSSER, C. L., J. GREEN AND T. CHOW, 1955. Ionic and osmotic concentrations in blood and

urine of Pachygrapsus crassipes acclimated to different salinities. Biol. Bull., 109 :

99-107.
SCHLIEPER, C., 1929. Ueber die Einwirkung nieder Salzkonzentrationen auf marine Organismen.

Zeitschr. f. vergl. Physiol.. 9: 478-514.
SCHWABE, E., 1933. Ueber die Osmoregulation verschiedener Krebse (Malacostracen).

Zeitschr. f. vergl. Physiol.. 19: 183-236.
WIKGREN, B., 1953. Osmotic regulation in some aquatic animals with special reference to

the influence of temperature. Acta Zool. Fcnnica, 71 : 1-102.

FREEZING AND DRYING IN INTERTIDAL ALGAE *

JOHN KANWISHER
Woods Hole Oceanographic Institution, Woods Hole, Mass.

The intertidal region is an environment characterized by widely fluctuating
conditions. It will be shown in this paper that in high latitudes the algae in this
zone are exposed to extensive freezing and drying. These two aspects of im-
mersion are considered together, since they have the common feature of cellular
dehydration. In particular, their separate effects on the metabolism of algae have
been investigated.

First will be discussed the effect of low temperatures in freezing a large amount
of water in certain algae. Next, the natural dehydration that is caused by evapora-
tion in several of the same species will be described. Finally, measurements will be
reported which show a greatly depressed respiration in both the frozen and dried
states. Some observations on the winter survival of Fucits in the Arctic are also
included.

I. FREEZING

In the Woods Hole region in winter there are a number of macroscopic brown,
green, and red algae exposed to freezing temperatures by the tide. They fre-
quently feel brittle to a degree which suggests ice in them. Some of them have
been observed imbedded in the ice at temperatures as low as -- 20° C. Conditions
are even more rigorous in the Arctic where FUCKS is a prominent intertidal alga.
It may spend six months or more frozen in the ice at temperatures which go below
— 40° C. Since these plants contain 70 to 80 per cent water, it seemed pertinent
to determine how much of this water, if any, is frozen at these extreme temperatures.
Bieble (1939) reported that several intertidal algae would survive being frozen,
but he made no quantitative determinations of the ice.

1. Method

Water gives off heat and also expands when it changes to ice. The expansion
has been measured directly in a dilatometer by Moran (1935), Gortner (1937),
and others to determine the amount of ice. Scholander ct al. (1955) devised a
flotation method to measure specific gravity and found as much as 90 per cent
frozen water in the lichen Cctraria richardsonii. Volumetric methods were un-
suitable here because of the difficulty of the dissolved gases that come out of solu-
tion on freezing. This would cause a density change which could not be separated
from the same effect due to ice formation. Calorimetric determinations of ice,
based on the conveniently large heat of fusion of water, have been used by Great-
house (1935), Ditman et al. (1942), and others. Scholander et al. (1953) have
recently reviewed these two methods.

1 Contribution Number 887 from the Woods Hole Oceanographic Institution.

275

276 JOHN KANWISHER

There has been no previous quantitative work on ice in algae. This author
has described a simplified calorimetry for small animals (Kanwisher, 1955) which
has been used here on several of the large intertidal algae. The calorimeter vessel
is an ordinary Thermos bottle. The temperature of the water inside is read to
0.01 ° C. by a mercury thermometer through the stopper. The sensitivity is varied
by changing the amount of water in the Thermos. Weighed pieces of ice are used
for calibration.

A piece of the alga to be frozen, usually a few grams, is sponged free of excess
water with filter paper, weighed, and placed in a small vial in a cold chamber set
to the desired temperature. At least four hours is allowed for phase equilibrium
between the water and ice in the alga. A check weighing at this point usually
showed less than 1 per cent loss of water by evaporation.

The Thermos is thermally equilibrated with the desired amount of water and
the temperature noted. Then the vial is removed from the cold chamber and alga
immediately shaken into the open Thermos. The vial prevents transfer of heat
from the hands to the frozen alga. The stopper is replaced quickly and the
Thermos is shaken. During the next few minutes the lowest temperature is noted.
The measurement is completed by weighing the alga after drying it for two hours
in an oven set at 100° C. The amount of water and dry substance in the initially
frozen material is then computed.

If no ice is formed the number of calories supplied to the alga is proportional
only to its weight and the temperature interval through which it is warmed. In
the absence of any change of state the specific heat is nearly constant with tempera-
ture. If ice is present the calorimeter must supply 80 additional calories to melt
each gram.

The calories supplied to the alga by the calorimeter are equal to the temperature
drop observed times the heat capacity which has been determined by calibration
with ice. Part of these calories go to warm the dry substance and the water from
the cold box temperature Tt up to the final calorimeter temperature T2. This is
equal to

(T2 - TJ X (0.3 X dry wt. + wt. of water),

where 0.3 has been separately determined to be the specific heat of the dry substance.
That of water is 1.0. The remainder of the calories melt any ice that is present.
This is converted to grams of ice. Since the water in the starting sample is known,
the fraction of it frozen at the temperature Tl has been determined. A small
correction is necessary because the specific heat of ice is only half that of water
(Ditman** a/., 1942).

2. Results

Figure 1 is typical of the data thus obtained. It is a plot of the per cent of
water that is converted to ice at various freezing temperatures in Fucus vesiculosus.
Similar curves were obtained for Ascophyllum nodosum, Chondrus crispus, and
Ulva lactuca. The following table shows the percentage of water as ice at — 15° C.,
the lowest temperature used.

It is evident that a large fraction of algal water is readily frozen at temperatures
which frequently occur in nature. The large surface area of Ulva would t^nd

FREEZING AND DRYING IN ALGAE

277

a:
LU

FUCUS VESICULOSUS

o

100

LU 75

M
O

50

25

0

TEMP. C

FIGURE 1. Freezing curve of Fucns.

to absorb heat during the transfer to the calorimeter. This may account for its
having the least ice in Table I.

II. WATER CONTENT IN ALGAE NATURALLY DRIED

Exposed upper intertidal algae become very obviously dried on a windy day
when the relative humidity is low. Isaac (1933) measured a 68 per cent loss
in weight in Pclvetia canaliculata during normal exposure on the short. He did
not determine the dry wyeight. Feldmann (1937) noted that Bangia juscopurpurea
can remain out of the water for periods as long as 15 days and still survive.
Zaneveld (1937) found only a 30 per cent weight loss in drying conditions. A
series of measurements have been made here to determine how much water is
normally lost under such conditions.

When the algae looked very dry at low tide on a windy day, samples were taken
and weighed immediately. They were then immersed in sea water for several
hours and weighed again. Some of them were used to demonstrate photosynthesis
by a method described later. Finally, oven drying and weighing gave the necessary

TABLE I

Species

Fucus vesiculosus
Ascophyllum nodosum
Chondrus crispus
Ulva lactuca

Per cent ice at

82
76
74
69

-15° C.

278

JOHN KANWISHER

TABLE II

Species

Fucus vesiculosus
Chondrus crispus
Ulva lactuca
Enteromorpha linza

Per cent of water lost

91
63

77
84

data to compute the water present when immersed and also in the dried state.
Table II gives the maximum dehydration values found.

To measure the rate of loss and reabsorption of water, pieces of Fucus were
exposed to room air of 22° C. and 40 per cent relative humidity. Such values are
not uncommon in nature. They were weighed at intervals and then replaced in
sea water and their weight again followed. From Figure 2 it is clear that severe
drying can take place during the length of time of tidal exposure. The rate was
increased several fold when a breeze was simulated with an electric fan. Less
than an hour after re-immersion most of the lost water has been regained. Thin
forms such as Ulva probably dry even more rapidly. Where the algae grow in many
overlapping layers probably only the uppermost are dried very much. The interest
here, however, is only in the maximum drying that can be tolerated.

III. RESPIRATION IN FROZEN AND DRIED ALGAE

Method

Respiration is awkward to measure at freezing temperatures with conventional
methods such as the familiar manometric technique. The volume change resulting
from water turning to ice cannot readily be separated from that due to oxygen
consumption. The Winkler method of following the disappearance of dissolved

100

80 -

DRYING IN INTERTIDAL
FUCUS VESICULOSUS

u_
O

I

0

.5 I 1.5

TIME IN HOURS

FIGURE 2. Drying curve of Fucus.

FREEZING AND DRYING IN ALGAE 279

oxygen cannot, of course, be applied to frozen or dried material. Oxygen con-
sumption in the region of freezing is so sharply temperature-dependent that any
method which requires part of the time at higher temperatures is open to gross
errors. Scholander et al. (1953), in reviewing the literature on respiration in
frozen material, concluded that many of the techniques were inadequate for the
problem at hand. They used a method of gas analysis which has been applied
here with slight modification.

The material is enclosed in a syringe writh a known amount of air and kept in
the dark. Samples of gas are withdrawn at intervals and analyzed for oxygen.
Respiration is computed from the rate of oxygen decrease in the gas phase. Since
blanks run in the same way give negligible values, the utilization of oxygen can
only be attributed to the frozen or dried algae. The method is specific for oxygen
and does not rely on volume decrements which are assumed to result from oxygen
being used.

For the low temperature values the algae were placed in darkened hypodermic
syringes in a cold bath set at the desired temperature. At least six hours was
allowed for the ice-water equilibrium to be reached. The syringes were flushed
with chilled outside air and sealed. The tips of the syringes extended above the
surface of the coolant. Samples of the gas could be removed without taking the
syringes from the bath. The plunger was free to move up as a sample was with-
drawn. By thus avoiding any differential pressure when sampling, the danger
of leaks is reduced.

The oxygen was measured with the %-cc. gas analyzer of Scholander (1947)
to 0.02 per cent accuracy. Duplicate analyses were made. When a respiratory
period was started by flushing in outside air, the initial concentration was assumed
to be 20.94 per cent. Repeated measurements varied between 20.93 and 20.96.
Because of the possibility of rate of oxygen consumption depending on tension,
oxygen was never depleted below 18 per cent. Two readings were usually taken.
The time between these varied from 1 to 200 hours. The slope determined by
these points was used to compute the rate of oxygen consumption. The figures are
in units of mm.3 02 per gram of dry weight of alga per hour.

For the dried algae a fresh sample was weighed fully moist and dried by
exposing it to air. It was then weighed again and placed in a syringe. The air
in the syringe becomes saturated and no additional water is lost. The readings
were made by the same method as for the frozen material. The temperature was
kept at 15° C.

Fully moist respiration measurements above 0° were made with volumetric
respirometers (Scholander et al., 1952). At 0° these checked with the gas analysis
method.

2. Results

Respiration in Fucus above and below 0° is plotted in Figure 3. Similar curves
were found for Chondrns and Ulva. The respiration drops sharply below 0°. In
in the interval from 0 to — 10 the apparent Q10 is 17. For the same interval above
0° it is 2. The other species had a Q10 of 15 and 23 below 0°, respectively, and
close to 2 above. At — 15° it was necesary to wait 7 to 8 days for the oxygen in
the syringe to decrease by a large enough amount to insure an accurate deter-
mination.

280

JOHN KANWISHER

When the algae were dried, the oxygen consumption again decreased. Figure 4
shows the respiration of Fucus related to the degree of dehydration. When 80
per cent of the normal water was lost, the metabolism was down to one-sixth of
its normal value. If a sample was allowed to regain water by soaking, the metab-
olism increased. The solid points were taken consecutively on the same piece of
material and showed the reversible nature of the phenomenon. Chondrus and Ulva
showed the same decreased respiration when dried and also recovered the higher
rate when re-immersed.

lOOOr-

100

CJ

O
re

10

o

o
o

o
o
o

8

o
I

I

I

8
8

RESPIRATION OF
FUCUS VESICULOSUS

-15° -10° -5°

10° 15°

0" 5W
TEMP. °C

FIGURE 3. Oxygen consumption vs. temperature of Fucus.

Loss of water in freezing or drying must increase the salinity of the remaining
fluids. Although part of this fluid is in the inter-cellular space, it is in equilibrium
with the interior of the cells. Thus loss of any algal water will raise the salinity
inside the cells. To determine whether salinity had a specific effect on metabolic
rate, higher salinities were made by draining the brine from frozen sea water.
Pieces of Fucus, Chondrus, and Ulva were immersed in these for 12 hours. The
tissue chloride concentration was measured by acid digestion and titration. It was
always proportional to that of the external medium. These species show no
evidence of regulating chloride. Respiration rates were measured at the various

FREEZING AND DRYING IN ALGAE

281

salinities. In all cases oxygen consumption decreased less than 30 per cent when
the salinity was increased by three times. This concentration is produced at
- 8° C. by freezing out of water. At such a temperature the respiration is de-
creased 10- to 15-fold below that at 0°. Salinity is clearly not the primary respira-
tory depressant when water is lost from the cells by either freezing or drying.

1500

1000

O
ro

500

0

DRYING VS RESPIRATION
IN FUCUS VESICULOSUS

\ o

\

1

1

0 20 40 60

% WATER LOST

FIGURE 4. Oxygen consumption at various degrees of dehydration.

80

IV. DISCUSSION

Previous attempts have been made to determine respiratory gas exchange in
frozen plants. Scholander ct al. (1953) measured a precipitous drop in oxygen
consumption of several Arctic phanerograms and lichens below 0° C. Ice determina-
tions by a floatation method showed that at -- 20° C., more than 90 per cent of the
water in some lichens was frozen. They thought it likely the drop in metabolic
rate was due to cellular dehydration. They have reviewed the literature and point
out that the techniques used previously to measure low temperature respiration
were inadequate for the low rates that occur. At freezing temperatures they found
a Q10 of 20 to 50, while above 0° the same material showed the usual two- to four-
times change in oxygen consumption over a ten-degree interval.

Smyth (1934) found a linear relationship between water content and respiration

282

JOHN KANWISHER

in lichens. The respiration of air-dried Acacia seeds was measured by White
(1909) to be only 1/10,000 that of moistened seeds. In dry Ricinus seeds he
could detect no oxygen consumption. Spores of single-celled forms are dehydrated
and are known to show very low oxygen consumption. Respiration in dried algae
does not seem to have been previously studied.

The intertidal algae used in these experiments are normally exposed to both
freezing and drying. It has been shown here that their respiration under either

LU
N
O

LJ

I

P

100

80

60

40

20

0

SEA WATER

ASCOPHYLLUM

0'

-5'

-10'

-15'

TEMP. °C

FIGURE 5. Curves showing the freezing of a greater fraction of sea water as

compared to algal water.

of these conditions is sharply reduced. In the region of freezing temperatures
their Q10 is about two times what would be expected from dehydration alone. In
going from 0° to - - 10°, 75 to 80 per cent of the water is frozen. This water
loss alone should cause a decrease of six to ten times in oxygen consumption.
When the straight temperature effect of a Q10 of two is also included, the total
apparent Q10 should be in the range of 12 to 20. This is in reasonable agreement
with those measured directly in the frozen algae.

At progressively lower temperatures calorimetric ice determinations become
less accurate. More heat must be supplied to warm the material over the increased
temperature range. The calories representing the melting of ice become a smaller

FREEZING AND DRYING IN ALGAE 283

fraction of the total measurement and are thus subject to a larger percentage error.
It appears from Figure 1, however, that 15 to 20 per cent of the water is resistant
to freezing. The same technique of calorimetry has been applied to a vial of sea
water to find the amount of ice at various temperatures. It is compared with
Ascophyllum nodosuin in Figure 5. A larger fraction of the water in sea water
freezes at all temperatures than in the algae.

It appears that part of the water in algal cells is unavailable for freezing.
Many authors have presented evidence for unfreezable water in gels and also in
plant and animal material (Moran, 1926; Greathouse, 1935; review of the literature
by Scholander ct al., 1953). Moran was unable to freeze all of the water in a
gelatin, even at — 40° C. The water molecule has a large dipole moment and may
well be subject to forces less powerful than conventional bonding but still strong
enough to prevent its being frozen. However, Grollman (1931) has rejected the
idea of bound water in colloidal systems.

White (1909) found evidence for binding of part of the water in plants at
ordinary temperatures. In Acacia seeds three per cent remained even after drying
over calcium chloride.

Roualt's law expresses a linear relationship between the concentration of a
solution and its freezing point depression. If one can assume that the ratio of
dry matter to water is equivalent to a concentration, this quantity should increase
linearly below freezing. Scholander ct al. (1953) found this to be so in a Chirono-
mus larva. Sea water is nearly linear as would be expected of a solution of
crystalloids. The ratio in Ascophyllum tends towards a constant value at low
temperatures. This could happen if part of the water were bound in such a way
that it would not freeze. It would also result from any of the dissolved substances
coming out of solution. The concentrations increase as water is frozen out while
at the same time the solubility must decrease with temperature. In a frozen alga
at — 15° C., the solubility of sodium chloride has been exceeded. The cells can
either maintain a supersaturation or must be able to actually cope with internal
salt crystals.

Siminovitch and Briggs (1949) measured an increased mobility of water in
the frost-hardy cells of the black locust, Robinia. They thought this was necessary
to allow a more rapid exit of water from the cells when intercellular freezing occurs.
Such is likely the case with the algae used here when they undergo rapid freezing
and drying. It is generally believed that internal freezing is lethal to cells, prob-
ably by the physical disruption of ice crystals in the protoplasm. Direct observation
will be necessary, however, to determine the locus of this ice.

The lowered respiration observed in the frozen and dried algae may be of
value to them in surviving these periods of stress. There can be little growth at
such times since the usual supply of nutrients from the sea water is not available.
When the alga is frozen, the light available for photosynthesis is usually limited,
such as during the Arctic winter. The slowing-down observed here represents a
less serious drain on the food stores. The ability of these algae to survive in the
intertidal zone may, however, be merely a case of their not being injured by the
freezing and drying that are inevitable in such a location.

Respiration has been called the flame of life. In algae at low temperatures it
burns very low but is never entirely out.

284 JOHN KANWISHER

V. OBSERVATIONS OF ARCTIC Fucus

The author was a member of an expedition to Hebron in northern Labrador,
sponsored by the Arctic Institute of North America in 1954. On arrival early
in July, Fucus was abundant in the intertidal zone along much of the coast. Large
reproducing plants were common although the ice had been gone only a month.
It seemed certain they had not grown this much in the brief period of open water.
Yet during the winter the ice is several feet thick and is solid to the bottom along
the shore. One was led to believe the Fucus was frozen solid in the ice during
the entire winter.

During this summer visit, pieces of Fucus were cooled to — 13° C. in a vial
immersed in a salt and ice mixture. Calorimetry showed that % of the water was
frozen at this temperature. About two grams wet weight of recently frozen Fucus
were put in a 20-cc. syringe with sea water. The syringe was placed in the sun
and kept close to 0° by a snow and water mixture. One-mi, samples were removed
and analyzed for dissolved oxygen by a gasometric method (Scholander et al.,
1955). In 30 minutes the oxygen rose from 3.6 to 8.4 mm3/cc. Soon after this,
bubbles formed indicating supersaturation of the dissolved oxygen. Thus the
sample of Fucus was still able to photosynthesize actively, even directly after being
unnaturally frozen during the summer.

Fortunately it was possible to return to the same spot in March and check on
the winter condition of the Fucus. The ice was many feet thick along the shore.
In places tidal stresses had buckled it and the projecting sheets contained some
of the algae frozen into the ice when it formed. This Fucus was fully exposed to
the air temperatures which, during the brief visit, were as low as -- 26° C. Earlier
in the winter they had dropped to — 40° C. or lower.

Pieces of ice with Fucus in them were chipped free and thawed. The melted
water contained only about 0.3 per cent salt. The alga was again checked for
photosynthesis with the same positive result. The winter respiration rate was
found to be close to that in the summer. There was no sign of a large oxygen
debt from the long period in the ice. The Fucus is apparently ready to start active
growth again in the spring where it left off in the fall.

These studies were aided by a contract between the Office of Naval Research,
Department of the Navy, and Arctic Institute of North America. Reproduction
in whole or in part is permitted for any purpose of the United States Government.
I am deeply indebted to Dr. Per Scholander for advice and encouragement. My
wife has been of considerable assistance both in the field and laboratory.

SUMMARY

1. As much as 80 per cent of the water in intertidal marine algae is frozen when
exposed to the low air temperatures that regularly occur in nature.

2. The same species may lose 90 per cent of their water by ordinary drying
during tidal exposure.

3. Metabolism is greatly depressed in both the frozen and dried states.

4. The ability to withstand drying may be related to freezing hardiness.

5. Some extreme conditions in the Arctic are described. Fucus spends many
months frozen into the sea ice at temperatures down to""40° C., yet it is capable of
photosynthesis immediately upon being thawed out.

FREEZING AND DRYING IN ALGAE 285

LITERATURE CITED

BIEBLE, R., 1939. tJber die Temperaturresistenz von Meeresalgen verschiedener Klimazonen

und verschieden tiefer Standorte. Jahrb. Wiss. Bot., 88: 389-420.
DITMAN, L. P., G. B. VOGT AND D. R. SMITH, 1942. The relation of unfreezable water to

cold hardiness of insects. /. Economic Entomology, 35 : 265-272.
FELDMANN, J., 1937. Recherches sur la vegetation marine de la Mediterranee. La cote des

Alberes. Rev. Algologique, 10: 1-399.
GORTNER, R. A., 1937. Selected topics in colloid chemistry. Cornell Univ. Press, Ithaca, New

York.
GREATHOUSE, GA. A., 1935. Unfreezable and freezable water equilibrium in plant tissues as

influenced by sub-zero temperatures. Plant Physiol., 10 : 781-788.
GROLLMAN, A., 1931. The vapour pressure of aqueous solutions with special reference to the

problem of state of water in biological fluids. /. Gen. Physiol., 14 : 661-683.
ISAAC, W. E., 1933. Some observations and experiments on the drought resistance of Pclvetia

canaliculata. Ann. Bot., 47 : 343-348.

KANWISHER, J. W., 1955. Freezing in intertidal animals. Biol. Bull., 109: 56-63.
MORAN, T., 1926. The freezing of gelatin gel. Proc. Roy. Soc. London, Ser. A, 112: 30-46.
MORAN, T., 1935. Bound water and phase equilibria in protein systems : egg albumin and

muscle. Proc. Roy. Soc. London, Ser. B, 118: 548-559.
SCHOLANDER, P. F., 1947. Analyzer for accurate estimation of respiratory gases in one-half

cubic centimeter samples. /. Biol. Chem., 167 : 235-250.
SCHOLANDER, P. F., C. L. CLAFF, J. R. ANDREWS AND D. F. WALLACH, 1952. Microvolumetric

respirometry. /. Gen. Physiol., 35 : 375-395.
SCHOLANDER, P. F., L. VAN DAM, C. L. CLAFF AND J. W. KANWISHER, 1955. Micro-gasometric

determination of dissolved oxygen and nitrogen. Biol. Bull., 109: 328-334.
SCHOLANDER, P. F., W. FLAGG, R. J. HOCK AND L. IRVING, 1953. Studies on the physiology

of frozen plants and animals in the Arctic. /. Cell. Comp. Physiol., 42 : 1-56.
SIMINOVITCH, D., AND D. R. BRiGGS, 1949. The chemistry of the living bark of the black locust

tree in relation to frost-hardiness. Arch. Biochem., 23: 8-17.
SMYTH, E. S., 1934. A contribution to the physiology and ecology of Peltigcra canina and

P. polydactyla. Ann. Bot., 48 : 781-818.
WHITE, J., 1909. The ferments and latent life of resting seeds. Proc. Roy. Soc. London, Ser.

B, 81 : 417-442.
ZANEVELD, J. S., 1937. The littoral zonation of some Fucaceae in relation to desiccation. /.

Ecology, 25 : 431-468.

SOUND PRODUCTION IN THE SPINY LOBSTER PANULIRUS

ARGUS (LATREILLE)1'2

JAMES M. MOULTON
Bowdoin College, Brunsivick, Maine

That crustaceans contribute significantly to marine sound is well-known. A
list of marine sound producers prepared by the United States National Museum
as early as 1942 included members of 17 crustacean families (Fish, 1954). The
significance of crustacean sounds to crustacean behavior and to the behavior of
other marine animals is, however, largely unknown. There is some evidence that
spiny lobsters can detect the vibrations of the sounds they produce (Lindberg,
1955; Dijkgraaf, 1955). Cohen (1955) rejects the statocysts of the lobster,
Homarus americanus, as auditory organs in the sense of responding to pressure
waves in the water, but suggests that statocyst vibration receptors may detect
substrate vibrations accompanying sounds.

During June, July and August of 1956, while a guest of the Lerner Marine
Laboratory of the American Museum of Natural History, I studied the acoustical
behavior of the West Indian spiny lobster, Panulirus argus. A study was made
of the anatomy of its sound-producing mechanism, and recordings were taken of
the sounds produced by this species under various conditions. Through direct
observation and motion pictures, data were obtained on the behavior of this spiny
lobster in relation to sound production. The study was performed on North
Bimini Island, the site of the Lerner Marine Laboratory.

Listening and recording equipment used in the investigation consisted of two
Rochelle salt hydrophones, one an AX-58-C, the other undesignated, a Woods
Hole Suitcase amplifier or a modified Heathkit amplifier Model A-7C, and an
Ekotape tape recorder Model 205. Recordings were made at speeds of 3% and
7% in./sec., and were analyzed on a Vibralyzer vibration frequency analyzer at
the Woods Hole Oceanographic Institution. Sound-generating equipment em-
ployed in the experiments consisted of a Hewlett-Packard audio oscillator Model
LAJ or the Ekotape tape recorder, a Craftsman C550 amplifier, and a QBG
transducer.

THE MECHANISM OF SOUND PRODUCTION

Stridulation by spiny lobsters (Palinuridae) has been described by a number
of authors (Mobius, 1867; Kent, 1877; Goode, 1878; Parker, 1878; Heldt, 1929;
Parker and Haswell, 1940, p. 455; MacGinitie and MacGinitie, 1949, p. 282;
Dijkgraaf, 1955; Lindberg, 1955) and reference to it is found in classical literature
(Yonge, 1854, p. 537). The characteristic rasp of palinurids is produced by an

1 Contribution No. 914 from the Woods Hole Oceanographic Institution.

2 The work was performed at the Lerner Marine Laboratory of the American Museum
of Natural History and at the Woods Hole Oceanographic Institution, under grants of the
Institution and of the Bowdoin College Faculty Research Fund established by the Class of 1928.

286

SOUND PRODUCTION IN PANULIRUS 287

intricate stridulatory mechanism which has been partially described by Parker
(1878) and Dijkgraaf (1955) in a Mediterranean species (Palinnrus vulgaris).
The following description is based on the mechanism as it exists in both males
and females of Panulirus argus, and adds detail to descriptions previously pub-
lished of other species.

The acoustic mechanism of P. argus consists in part of a toothed ridge, orange-
colored in life, rising from the surface of the carapace medial to each antennal base
and extending anteriorly from beneath each stalked eye (Fig. la). A medial
process of the basal segment of each antenna fits over the corresponding ridge ; as
an antenna is raised, a chitinous longitudinally-ridged membrane, the stridulatory
membrane (Fig. 2c), forming part of the undersurface of the medial process,
moves proximally over the toothed ridge, in the manner described by Dijkgraaf
(1955) for P. vulgaris, so that a sound may be produced; the sound varies with
the rate and force of raising of the antenna.

That a sound is produced only when the stridulatory membrane moves prox-
imally over the toothed ridge — that is, when the antennae are raised — -is due to a
combination of factors. The medial process of the antenna is itself jointed, the
process being divided into a basal flange (Figs. Ic, 2a) firmly united with the basal
antennal segment, and a terminal portion (Figs. Ib. 2d) joined to the posterior
edge of the flange. This terminal portion, which Dijkgraaf describes as being a
freely-projecting skin-fold in P. vulgaris, is covered on its upper surface in P. argus
with typical exoskeleton. Most of its under surface is formed by a soft chitinous
membrane covered with a dense mat of minute setae, the setae becoming sparse
along the leading edge of the stridulatory membrane. The under surface of the
joint between the basal flange and the terminal portion is the site of the stridulatory
membrane.

As a result of the arrangement described, when an antenna is suddenly swept
back, the jointed edge of the basal flange bears down on the stridulatory membrane
so that the latter is forced to rub proximally over the toothed, orange ridge and
a sound is created. Lowering of the antenna relieves pressure on the membrane.

The second factor which determines the effective direction of antennal movement
in producing sound is the presence on the portion of the orange ridge over which
the stridulatory membrane moves of anteriorly projecting microscopic teeth (Fig.
5) against which the stridulatory membrane is forced when the antennae are
swept back. These minute teeth are arranged in shingle-like fashion, teeth of
adjacent rows alternating with each other, and the teeth of one row appearing to
project from beneath those of the row behind. Anteriorly on the toothed ridge, the
leading edges of the teeth form an angle of approximately 60 degrees with the
surface of the ridge ; in the posterior third of the stridulatory portion of the ridge,
the edges are more nearly vertical. Anteriorly the edges of the teeth are slightly
serrated; posteriorly they are smooth. In a narrow transition zone (transverse
groove between a and b in Fig. 4) between the stridulatory (Fig. 4b) and non-
stridulatory (Fig. 4a) parts of the ridge, the teeth give way to blunter microscopic
projections, irregularly distributed, which are characteristic of the general carapace.

The presence of the teeth lends to the stridulatory portion of the orange ridge
a dull appearance when the ridge is dry ; the posterior portion of the ridge, from
which the teeth are lacking, possesses a shiny surface when dry. The presence
of the teeth can be detected by drawing the tip of the finger posteriorly along the

288

JAMES M. MOULTON

4

Stridulatory mechanism of Panulirus argus.

FIGURE 1 : dorso-lateral view of head region ; a) toothed portion of orange ridge, b)
terminal portion of medial antennal process, c) basal flange. X 1.3.

FIGURE 2: under surface of medial process of right antenna; a) basal flange, b) guiding
knob, c) Stridulatory membrane, d) terminal portion. X 11.

FIGURE 3 : cross-section of Stridulatory membrane. X 650.

FIGURE 4: lateral view of right orange ridge; a) smooth portion, b) toothed portion, c)
groove receiving guiding knob. X 7.

FIGURE 5 : shingle-like teeth of dorsal surface of right orange ridge (anterior to the right) .
X500.

SOUND PRODUCTION IN PANULIRUS

toothed portion of the orange ridge. No difference to the touch between tooth-
bearing and toothless portions of the orange ridge can be detected when the finger
tip is drawn anteriorly.

The region of transition between the toothed and toothless regions of the ridge
is marked by a shallow transverse groove (Fig. 4) which is the posterior limit
reached by the stridulatory membrane when an antenna is raised. The action of
the stridulatory membrane is guided by the presence of a small knob (Fig. 2b)
projecting from the ventrolateral surface of the basal flange adjacent to the lateral
end of the stridulatory membrane ; the knob during raising and lowering of the
antenna runs in a well-defined groove (Fig. 4c) on the lateral surface of the orange
ridge. This groove extends as far posteriorly as the transverse groove already
described. Behind the transverse groove, the orange ridge gradually flattens to
the contour of the general carapace.

That the mat of short setae covering most of the under surface of the terminal
portion of the medial antennal process becomes very sparse adjacent to the proximal
edge of the stridulatory membrane allows one to observe that the surface from
which the setae arise bears a polygonal, usually hexagonal, configuration. In this
area individual setae spring from individual polygons ; elsewhere, two to several
setae arise from each polygon, and the dense mat of setae resulting obscures the
underlying membrane.

Parker (1878) has figured a cross-section of the ridges of the stridulatory
membrane in P. vulgaris. In P. argus, this membrane is constructed as follows
(Fig. 3) : there is a basal stratified layer of squamous epithelial cells. From this
layer outward, the membrane is vertically striated to the level of the grooves be-
tween ridges, in a pattern which suggests that the major portion of the membrane
is comprised of many fused setal processes. The ridges themselves comprise
approximately % of the thickness of the membrane, and within the ridges, the
striations radiate outward to the surface of each ridge. The whole surface of the
stridulatory membrane is covered with a thin cuticular layer following the contour
of the membrane. In surface view, the ridges are sculptured in a finely polygonal,
usually hexagonal, pattern, the size of the polygons decreasing in the direction of
the basal flange. Within the grooves between ridges the cuticular border is some-
what folded and the appearance of surface granulation of the ridges is absent.
The stridulatory membrane is somewhat flexible, the relatively stiff ridges yielding
a rasping sound when the tip of a dissecting needle is drawn across them.

The stridulatory membrane, as seen in cross-section, is also striated horizontally,
in a pattern reminiscent of growth lines in skeletal parts of other animals (e.g.,
tooth enamel, fish scales and otoliths). These horizontal lines continue into the
ridges where they are curved in the contour of the ridges.

If the stridulatory membrane and the terminal portion of the medial antennal
process are removed, raising of the antenna by hand produces only a slight squeak-
ing sound as the joint edge of the basal flange bears directly on the toothed part
of the orange ridge. The spiny lobster itself produces no sound on raising of an
antenna so treated, as Parker (1878) also observed.

A specimen of P. argus which shed in one of the cement pools of the Lerner
Marine Laboratory during the night of August 11-12, 1956, stridulated as usual
in the morning, although the general exoskeleton was still soft. No animal from

290 JAMES M. MOULTON

which the stridulatory mechanism had been removed shed during the summer, so
that regeneration of the mechanism cannot be stated as fact.

THE SOUNDS OF PANULIRUS ARGUS

The observation of Kent (1877), questioned by Goode (1878), that a shrill
squeaking sound is produced by the spiny lobster (Palinurus quadricornis) by
rubbing together of abdominal segments was probably accurate. During abdominal
contractions, after P. argus is taken from the water, the abdomen is at times held
tightly for a few moments under the cephalothorax, and a rather intense vibration
is felt throughout the hand-held animal. At such times a squeaking sound, higher
pitched than any antennal noise, may occasionally be heard. The body vibration
accompanying this action is similar to that sometimes produced by the lobster,
Homarus americanus, freshly removed from a trap or tank ; while the latter does
not produce an audible sound, vibration is at times so intense that an inexperienced
person may drop the animal. This matter requires further study.

The antennal sounds of P. argus most frequently heard are either a rasp or a
slow rattle of longer duration. The slow rattle, recorded during my study only
when several animals were confined together in a live car, is seen after vibration
analysis to consist of 5 or 6 pulses of sound spanning approximately .5 to 3.3 kc.,
the rattle lasting about % second. The pulses are produced at an average rate
of 27/second, varying in several cases from 24 to 31 /second. The greatest intensity
of each of these pulses lies at approximately .6 kc.

The antennal rasp, which usually accompanies abdominal contractions when a
specimen is held in the hand in air or water, is a single burst of sound in which
the individual pulses of sound cannot be distinguished. The sound lasts slightly
over .1 second, and spans frequencies from below .04 kc. to approximately 9 kc.
The zones of greatest intensity lie at .8 kc. and in a rather broad band between
approximately 2.5 and 4.7 kc.

As a hand-held animal slows or ceases its abdominal contractions, the antennae
may be swept back alternately rather than together. In sounds created by this
action, individual pulses can be distinguished on vibration analysis. They are
produced at rates varying between 56 and 133/second, with intensity peaks at the
levels of the antennal rasp.

The rasp of P. argus can be roughly duplicated through moving the antennae
upward by hand. The vibration analyses of sounds thus produced are like those of
the rasp, except that instead of a single burst of sound there are from four to
several bursts at somewhat irregular intervals ; the upper peak intensity is more
diffuse than in the normally produced rasp, and the lower peak (.8 kc.) of the
normally produced sound is lacking. It has not been possible by this method
to duplicate the slow rattle recorded from confined animals undisturbed by the
observer.

SOUND PRODUCTION AND THE BEHAVIOR OF PANULIRUS ARGUS

That the production of sound by the spiny lobster is a response to definite stimuli
is indicated by the observations of Lindberg (1955) and Dijkgraaf (1955). The
antennae of P. argus are frequently moved without the production of sound, even

SOUND PRODUCTION IN PANULIRUS 291

when the basal segment of the antenna is involved. Aquarium-confined spiny
lobsters trained to accept food offered by hand from above during the summer of
1956 characteristically raised themselves on their anterior legs, reaching their an-
tennae up toward the proffered food. At such times, no sound was produced.
Similarly, observations on live-car confined animals disclosed no antennal sounds
produced during listening by hydrophone at times when the majority of animals were
moving their antennae.

Although conditions surrounding production of the slow rattle are obscure,
the sound bursts characteristic of the rasp frequently accompany strong, rapid
abdominal contractions which are characteristic of the hand-held or net-captured
animal. During these contractions, whether the restrained P. argus is struggling
in the water or in air, the antennae are held back over the body and each forward
thrust of the rapidly and forcefully contracting abdomen is accompanied by a brief
rasp as the stridulatory membrane rubs over the orange ridge. Sounds inter-
mediate between the slow rattle and the rasp are produced by antennal movements
in the absence of abdominal contractions; although these intermediate sounds are
difficult to distinguish by ear from the rasp, vibration analysis distinguishes them.

It is evident that the sounds produced by P. argus and the behavior which they
accompany differ with circumstances. Dijkgraaf describes production of the rasp
during struggling between two individuals of P. vulgaris. Circumstances under
which the rasp is produced in P. argus (grasping in the hand, capturing in a net,
injury) suggest that this sound is related to defensive behavior. This relationship
is further emphasized by the fact that during production of the rasp, the spiny
antennae are held over the back, tightly depressed against the carapace or against
the grasping hand. During abdominal contractions, the forward-directed spines
of the carapace are driven against the hand, and the hand is driven against the
backward-directed spines of the antennae ; blood is frequently drawn. At the same
time, the abdominal contractions provide an efficient scissor-like action through
the sharp edges of the abdominal exoskeleton, and the fingers caught within the
scissors may be cut. Presumably the action described would also be performed
against natural captors in the sea.

Gradual subsidence of abdominal contractions and of antennal sound by the
hand-held animal will take place. A sudden movement of the hand will, however,
re-initiate the whole process, and again a gradual subsidence, usually through a
period of alternate antennal stridulations, will occur. The response gradually
diminishes.

At the Lerner Marine Laboratory, up to several dozen spiny lobsters are con-
fined at a time in a live car of screen and boards adjacent to the Laboratory dock
in Bimini Harbor. When a hydrophone was lowered into this live car and left
hanging in it during the daytime, the slow rattle already described was frequently
heard. The individual pulses of the slow rattle, probably produced as the stridula-
tory membrane skips along the orange ridge, can be detected. The sound does
not necessarily accompany marked activity of the animals to judge from visual
observations, and is not recorded from a highly active animal. This slow rattle
seems to be more in the nature of a conversational sound, as compared with implica-
tions of argument suggested by circumstances under which the rasp is produced.

292 JAMES M. MOULTON

The slow rattle could not be heard with the equipment employed 30 feet from the
live car, although the rasp was clear over this distance underwater.

That the slow rattle is not produced by accidental movements of the antennae
is suggested by the following observations : if the spiny lobsters in the live car were
stirred up with an oar neither the slow rattle nor the rasp was heard, and after
this procedure it was some time before the slow rattle was heard again ; nearly
constant antennal movements by isolated animals moving about in glass aquaria did
not result in production of any sound, nor was the slow rattle recorded from any
isolated animal ; the slow rattle was recorded only during daylight, not at night
when the spiny lobsters were moving about the live car ; if an animal were suddenly
clamped to the bottom with the edge of a dip net, only the rasp was heard to
accompany the animal's activity, never the slow rattle which might have been
anticipated if it were an accidental sound.

During the daytime, undisturbed P. argus tended to remain heaped in the
corners of the live car ; at night the animals were more obviously active, moving
about the screen forming the sides of the car. Yet the slow rattle was heard only
during the daytime. Thus, during 1% hours of listening between 0900 and 1100
the morning of 21 June, the slow rattle was heard 76 times, whereas it was not
heard at all during comparable time between 1950 and 2130 the evening of 20 June
under an approximately % full moon. These observations were made when the
live car contained between two and three dozen animals.

The stridulatory mechanism is best rendered inoperative by bilateral removal
of the medial process of the antenna, at the junction of the basal flange and basal
segment of the antenna. It is difficult effectively to remove the stridulatory mem-
brane without removing the whole medial process, since even a remnant of the
ridges will produce considerable sound.

A male spiny lobster with the stridulatory mechanism bilaterally removed was
confined in an aquarium with a normal male of similar size during July of 1956.
The normal animal was the dominant individual, frequently approaching the
operated animal head-on with extended anterior walking legs and slightly raised
antennae, walking forward as the operated animal retreated to a corner. At such
times, no sounds were heard.

Numerous attempts to influence the behavior of spiny lobsters by playing re-
cordings of the rasp and of the slow rattle into the live car and into aquaria contain-
ing individual specimens were unsuccessful, nor did recorded sounds of spiny
lobsters played underwater noticeably alter the distribution of various fishes confined
with them. The same was true if pulsating signals generated with the audio
oscillator set at 17 to 40 cps. (Moulton, 1956) were played into the water.

DISCUSSION

The production of two characteristic sounds by Panulirus argus and the be-
havior which the sounds accompany, parallel a situation occurring among fishes
in sea robins (Prionotus spp.) which when free during the breeding season produce
a staccato call, but which when disturbed in various ways produce a vibrant grunt
of other frequency characteristics (Moulton, 1956). Like the staccato call of the
sea robins, the slow rattle of the crayfish has not been recorded from hand-held
or otherwise disturbed specimens. As the grunt and the staccato call of the sea

SOUND PRODUCTION IN PANULIRUS 293

robins relate to two different patterns of behavior, thus the rasp and the slow
rattle of the spiny lobster, P. argus, do also.

The significance of their sound production to the survival of spiny lobsters is
unknown. Lindberg (1955, pp. 178-179) observed that P. interruptus did not
stridulate unless it and an attacking fish touched each other. Since the sounds
produced by P. argus span the frequency sensitivity of various fishes which have
been tested (Kleerekoper and Chagnon, 1954), it is not impossible that a combina-
tion of sound production and injurious strokes of the abdomen may combine as a
deterrent to predators. Unfortunately, information is lacking on this point. My
own observations concur with those of Lindberg in denying an obvious effect of
the rasp on fish behavior. A mutilated specimen of P. argus passing, while vigor-
ously rasping, through approximately 25 feet of clear water at Bimini on the
west side of Turtle Rocks, did not immediately nor during several minutes of ob-
servation through glass panels at the surface attract any of the numerous reef
fishes feeding within the zone of observation, nor did visible fishes which might be
anticipated to feed on injured spiny lobster noticeably alter their behavior.

Dijkgraaf's (1955) observation that stridulation could be induced in a highly
excited P. vulgaris by imitating the rasp adjacent to the aquarium is compatible with
Lindberg's observation that P. interruptus moved away from other animals forced
to stridulate nearby but out of sight. Lindberg observed that stridulation occurred
only upon impending conflict and thus considers the sound "a threat rather than
an alarm." Several P. argus contained in aquaria at Bimini moved rapidly away
from a net or grasping hand, but did not stridulate until grasped or captured in
the net — that is, the rasp was not necessarily produced during rather violent escape
maneuvers.

The failure of fishes to respond to the sounds of P. argus is consistent with
previous observations on the responses of free fishes to sound generally (Moulton
and Backus, 1955; Moore and Newman, 1956). Since, however, several kinds of
fishes will respond initially by quickened swimming movements — startle reactions —
to sounds which later will fail to affect their behavior, it is possible that a sudden
rasp by a mouth-held palinurid might cause a preying fish to release its hold and
thus its prey, particularly if the sound were accompanied by strong abdominal
contractions. This is to suggest that the rasp may act as a double assurance
mechanism.

Vibration analyses of various fish sounds recorded from identified species at
Woods Hole and Bimini indicate that sounds produced by several kinds of fishes
under duress of various types are more like the rasp of the spiny lobster than like
the slow rattle of undisturbed P. argus or than like the staccato call of free sea
robins during the breeding season. The burst of sound characterizing the spiny
lobster rasp also characterizes the sounds produced, during handling, by sea robins
(Prionotus spp.), grouper and hind (Epinephalus spp.), angelfish (Poinacanthus
spp. and Angelichthyes ciliaris), squirrelfish (Holocentrus ascensionis), triggerfish
(Balistes vetula and Melichthyes piccus), swellfish (Sphcroides spp.), porcupine-
fish (Diodon hystrix}, a jack (Caranx hippos), and grunts (Haemulon spp.) —
defensive behavior of all of these includes production of sounds lacking sharply
defined individual pulses within the limits of the analyses. Thus sounds produced
in a variety of ways — by stridulation of skeletal parts, by muscle contraction against
an air bladder or adjacent tissue, by pounding of pectoral fins against the body —

294 JAMES M. MOULTON

but under similar circumstances, present similar, although not identical, vibration
analyses. These observations, combined with observations on the slow rattle of
P. argus and the staccato call of sea robins, suggest that while sounds produced
by undisturbed marine fishes and crustaceans are likely to be sounds comprised
of individual pulses of brief duration, sounds of marine fishes and crustaceans under
duress are more likely to be bursts of sound of longer duration in which individual
pulses are obscured or absent.

Another category of marine sound, comparable in its vibration analyses to
sounds produced under duress by fishes and to the rasp of the spiny lobster, is the
feeding noise of such fishes as wrasses (Labridae), porcupinefish, swellfish, trigger-
fish, and angelfish. If sound bursts comprise threats or alarms, as conditions of
their production would suggest, noisy eating by these organisms may serve to repel
rather than to attract predators.

Exceptional in producing sounds of sharp individual pulses during defensive
behavior are representatives of two crustacean groups in the Bimini area, the
stomatopod (Gonodactylus oerstedi) and several kinds of snapping shrimp (Al-
pheidae), especially Alpheus armatus and Synalpheus spp. (Pearse, 1950). Al-
though produced in a different way, the sound of the mantis shrimp is like that
of the snapping shrimp (Goode, 1878; Johnson, Everest and Young, 1947; Fish,
1954), and their vibration analyses are indistiguishable. Both are either burrowers
or symbionts of organisms providing highly protected situations (Pearse, 1950;
Townsley, 1953; Banner, 1953; Clarke, 1955). Further, the sounds produced are
the by-products of accompanying movements which have a distinctly protective
value — the decisive closure of the snapping shrimp large chela with its accompany-
ing squirt of water (Schmitt, 1931, p. 192; Johnson et al.} 1947) and the stinging
extension of the raptorial appendage of the mantis shrimp.

That some sounds of marine arthropods are other than accidental is, however,
clearly indicated by the intricate stridulatory mechanism found among the spiny
lobsters (Palinuridae). The usefulness of the sounds produced, as of the sounds
produced by many kinds of fishes, is yet to be determined.

I am indebted for constructive criticism to Mr. William Schevill who read the
manuscript of this paper, and for careful execution of the drawings to Mr. Ernest R.
Powell, an undergraduate of Bowdoin College.

SUMMARY

1. The intricate stridulatory mechanism of the West Indian spiny lobster,
Panulirus argus, and the sounds it produces are described. The sounds are related
to various patterns of behavior.

2. On the basis of behavioral evidence it is suggested that a slow rattle is
characteristic of spiny lobsters when contained in groups, and that a rasp is a usual
component of defensive behavior.

3. The characteristics of the sounds of P. argus are compared to those of other
marine sounds of biological origin. On the basis of this comparison, an attempt
is made to generalize (a) the type* of sound which accompanies defensive behavior
of marine fishes and crustaceans, and (b) the type of sound stemming from marine
fishes and crustaceans when undisturbed.

SOUND PRODUCTION IN PANULIRUS 295

4. It is concluded that the intricacy of the sound-producing mechanism of P.
argns, and of other palinurids, justifies a conclusion of a significance of sound to
the biology of spiny lobsters. While certain suggestions of a possible value of the
rasp to survival of spiny lobsters are presented, a consistent effect of the rasp on
the behavior of other spiny lobsters and on predator organisms has yet to be
demonstrated.

LITERATURE CITED

BANNER, A. H., 1953. The Crangonidae, or snapping shrimp, of Hawaii. Pacific Science, 7 :

3-144.
CLARKE, W. D., 1955. A new species of the genus Heteromysis (Crustacea, Mysidacea) from

the Bahama Islands, commensal with a sea-anemone. Amer. Museum Nov., No. 1716 :

1-13.
COHEN, M. J., 1955. The function of the receptors in the statocyst of the lobster Homarus

americanus. J. Physiol., 130: 9-34.
DIJKGRAAF, S., 1955. Lauterzeugung und Schallwahrnehmung bei der Languste (Palinunts

vulgaris). Experientia, 11: 330-331.

FISH, M. P., 1954. The sonic marine animal problem. Research Reviews, Dec. : 13-18.
GOODE, G. B., 1878. The voices of crustaceans. Proc. U. S. Nat. Mus., 1 : 7-8.
HELDT, H., 1929. Rapport sur la langouste vulgaire (Panulirus vulgaris Latreille). Rapports

et Proces-Verbaux des Reunions, Commission Internationale pour ['Exploration Scien-

tifique de la Mer Mediterranee, 4: 113-126.
JOHNSON, M. W., F. A. EVEREST AND R. W. YOUNG, 1947. The role of snapping shrimp

(Crangon and Synalplicus} in the production of underwater noise in the sea. Biol.

Bull., 93 : 122-138.

KENT, W. S., 1877. Sound-producing arthropods. Nature, 17: 11.
KLEEREKOPER, H., AND E. C. CHAGNON, 1954. Hearing in fish, with special reference to

Semotilus atromaculatus atromaculatus (Mitchill). /. Fish. Res. Bd. Can., 11: 130-

152.
LINDBERG, R. G., 1955. Growth, population dynamics, and field behavior in the spiny lobster,

Panulirus interruptus (Randall). Univ. Calif. Publ. Zool., 59: 157-247.

MACGINITIE, G. E., AND N. MACGINITIE, 1949. Natural history of marine animals. McGraw-
Hill Book Co., Inc., New York.
MOBIUS, K., 1867. Ueber die Entstehung der Tone, welche Palinurus vulgaris mit den ausseren

Fiihlern hervorbringt. Arch. f. Naturgeschift., 33: 73-75.
MOORE, H. L., AND H. W. NEWMAN, 1956. Effects of sound waves on young salmon. Spec.

Sci. Report, Fisheries No. 172, U. S. Fish and Wildlife Service.
MOULTON, J. M., 1956. Influencing the calling of sea robins (Prionotus spp.) with sound.

Biol. Bull, 111: 393-398.
MOULTON, J. M., AND R. H. BACKUS, 1955. Annotated references concerning the effects of

man-made sounds on the movements of fishes. Fisheries Circ. No. 17, Dep't of Sea

and Shore Fisheries, Augusta, Maine.
PARKER, T. J., 1878. Note on the stridulating organ of Palinurus vulgaris. Proc. Zool. Soc.

London, 1878: 442-444.
PARKER, T. J., AND W. A. HASWELL, 1940. A text-book of zoology, 6th ed., rev. by O. Lowen-

stein. Macmillan and Co., Ltd., London.
PEARSE, A. S., 1950. Notes on the inhabitants of certain sponges at Bimini. Ecology, 31 :

149-151.

SCHMITT, W., 1931. Crustaceans. Smithsonian Scientific Series, 10, Part 2: 192-199.
TOWNSLEY, S. J., 1953. Adult and larval stomatopod crustaceans occurring in Hawaiian waters.

Pacific Science, 7 : 399-437.
YONGE, C. D., 1854. Translation of The Deipnosophists or Banquet of the Learned of Athen-

aeus, Vol. 2. Henry G. Bohn, London.

ISOLATION AND ASSAY OF THE NEMATOCYST TOXIN OF
METRIDIUM SENILE FIMBRIATUM

JOHN H. PHILLIPS, JR.1 AND DONALD P. ABBOTT

Hopkins Marine Station of Stanford University, Pacific Grove, California

The study of the immune mechanisms of marine invertebrates is inhibited by
our lack of knowledge of the infectious diseases of these organisms. However,
the many commensal relationships which exist between a variety of organisms and
members of the Phylum Coelenterata suggest that a study of induced immunity to
nematocyst toxins would at least yield information pertinent to the development
of an understanding of antitoxic immunity in marine invertebrates.

There have been a number of attempts at the purification and description of
these toxins (Cosmovici, 1925; Cantacuzene, 1926; Cantacuzene and Damboviceanu,
1934a, 1934b; Richet and Portier, 1936; Sonderhoff, 1936; Welsh, 1955). In all
cases the material isolated represents extracts of the whole animal or some of its
organs, e.g., tentacles or acontia. As has been pointed out by Hyman (1940) in
no case can it be certain that the material isolated is actually from nematocysts, the
stinging capsules, and is not some toxic tissue component which normally does not
play a role in the defensive or food gathering activities of the animal.

These present studies were carried out to develop a method of obtaining purified
suspensions of nematocysts from sea anemones (Actiniaria) in order to obtain a
toxic preparation which could be considered to be nematocystic in origin and could
be used in studies on the antitoxic response of a variety of marine invertebrates.

ISOLATION OF NEMATOCYSTS

The entire anemone was used as a source of nematocysts. Attempts to isolate
nematocysts by enzymatic digestion of the surrounding tissues with pepsin, trypsin,
ficin, and papain always resulted in damage to these structures, but the physical
methods of separation described below have yielded suitable material. A number
of variations in the method of preparation have been used, and the properties of
the resulting materials have varied with the method. A few of these variations are
included here since they illustrate species differences with regard to ease of purifica-
tion and nature of the nematocysts, and these variations in method may be of help
in similar investigations on other members of the phylum.

The anemones were first cleaned of adherent debris by placing them for a few
days in a coarse wire mesh basket in an aquarium with running sea water. Ap-
proximately 500 grams, wet weight, of the animals were macerated in a Waring
Blendor with 500 ml. of 1 M sucrose in sea water. An additional 500 ml. of
this suspending medium was added, and the material was passed through a series
of graded screens with openings of 1.168, 0.589, 0.295, and 0.147 mm. with the aid

1 American Cancer Society Fellow. Present address : Institute of Microbiology, Rutgers
University, New Brunswick, N. J.

296

NEMATOCYST TOXIN OF METRIDIUM 297

of suction. Tyler Standard Screens fastened to a Buchner funnel with masking
tape were used. These screens remove the large particles of tissue from the sus-
pension and allow the nematocysts to pass through, along with fine tissue debris,
dissolved tissue components, and very fine sand. Filtrates from anemones, whose
tissues contain symbiotic algae, bear these organisms as an additional contaminant.
Upon centrifugation at 1000 rpm. for 15 minutes, the nematocysts were collected
along with the sand, fine tissue debris, and algal cells if these were present. The
sediment was washed free of dissolved tissue constituents by repeated re-suspension
in the sucrose solution and re-centrifugation. This procedure also removed a
considerable amount of the fine tissue debris. The nematocysts were purified
further by differential centrifugations of 15 minutes and 15 seconds at top speed
in a small International Clinical Centrifuge with a bucket head. The longer
centrifugation left most of the fine tissue debris in suspension while the nematocysts
were collected in the sediment. The shorter centrifugation left the majority of
nematocysts in suspense but removed the sand. Five or six pairs of centrifugations
were usually sufficient.

Three criteria for the success of any method were employed, i.e., purity of the
suspensions, susceptibility of resulting nematocysts to artificial discharge, and
toxicity of the material released on discharge. Particularly good results were ob-
tained in the case of Metridium senile fimbriatum. Characteristics of these prepara-
tions are discussed below. However, the treatment of Anthopleura .vanthogramica
or Anthopleura elegantissima in this fashion resulted in unsuitable material. A.
vanthogramica is infected with zooxanthellae which could only be removed by
shaking the nematocyst suspensions with ether. Upon centrifugation the nem-
atocysts were found in the sediment and the algal cells along with any remaining
fine tissue debris stayed in the ether phase which had a gelatinous consistency.
With this modification highly purified suspensions could be obtained, but the
nematocysts could not be artificially discharged. While it proved possible to
obtain, in protected, darkened areas, specimens of A. elegantissima which did not
contain symbiotic algae, the nematocysts obtained from these animals were also
not susceptible to artificial discharge.

CHARACTERISTICS OF NEMATOCYST SUSPENSIONS FROM METRIDIUM

SENILE FIMBRIATUM

Approximately 0.5 gram, dry weight, of nematocysts was obtained from 500
grams, wet weight, of this species. The material was all but completely free of
tissue debris and sand when examined microscopically. Continued differential
centrifugation neither increased the per cent hexosamine content of 3.1-3.2% after
hydrolysis nor decreased the total nitrogen content which was 10.2-10.4%. The
per cent composition of different batches of nematocysts agreed within experi-
mental error. A dried preparation could be obtained by washing suspensions with
a solution of glycerine and distilled water, 1 : 1 by volume, followed by 95% ethanol
and ether and drying in a desiccator.

The half-life of a purified nematocyst suspension appears to depend at least
partly upon the osmotic pressure exerted by the suspending medium. When kept
in the refrigerator the time required for discharge of one half of the nematocysts
was 12 hours in 1 M sucrose in sea water, 7 days in 1 : 1 glycerine and distilled

298

JOHN H. PHILLIPS, JR. AND DONALD P. ABBOTT

water, at least three months in 95 % ethanol, and over 6 months for dried material.
These observations are in agreement with those of Glaser and Sparrow (1909).
The dried material would probably keep indefinitely (Weill, 1926). However,
neither alcoholic suspensions nor dried material exhibits any toxicity. Both ether
and alcohol effectively detoxify the nematocysts.

The spectrum of nematocyst types, cnidom, for M. senile fimbriatum has been
described recently by Hand (1955). Table I gives the differential count for each
of the types found in the suspensions. Some of the types have been divided into
size categories which represent approximate mean dimensions. This variation
in size with respect to a particular type makes a physical separation of one type
from another extremely difficult. Until it can be determined that the toxin of

TABLE I
Differential count of nematocysts in suspensions from Metridium senile fimbriatum

Nematocyst type

Size (microns)

Counted

Per cent

Microbasic b-mastigophore

60 X 5

15

7.4

30X4

6

2.9

10X3

6

2.9

Microbasic p-mastigophore

20 X 3

2

1.0

10 X 5

14

6.9

Microbasic amastigophore

70X7

10

4.9

30 X 5

21

10.4

10 X 4

8

3.9

Basitrich

20X4

20

9.8

13 X 2

16

7.8

Atrich

24-47 X 7-15

2

1.0

Holotrich

13-23 X 4-6

7

3.4

Spirocysts

12-30 X 4

76

37.4

Totals

203

99.7

one type is the same or different from the toxin of another type, it seems desirable
to present such counts as a part of the description of the material whose toxicity
is under investigation.

NEMATOCYST TOXIN FROM METRIDIUM SENILE FIMBRIATUM

The purified suspensions contain 37-39 7« discharged nematocysts. The re-
mainder can be artificially discharged by treatment with distilled water, methylene
blue, weak acid, weak base, sodium thioglycolate, or sodium taurocholate. In
order to obtain the maximum release of toxin, the nematocysts were placed in
distilled water for 12 to 18 hours. After such a period all but approximately \%
are discharged. Such "normal" discharge was found to be just as effective a
means of obtaining the toxin as grinding and extraction. Discharge released
21.8% of the dry weight of the nematocysts. Grinding with carborundum in a
mortar and extraction with distilled water removed 21.7%. Discharge in a test
tube may be followed with the naked eye. The tubes everted from the nematocysts
become entangled and eventually form a slimy, cottony sediment.

Some information as to the chemical nature of the toxin has been obtained

NEMATOCYST TOXIN OF METRIDIUM

299

(Phillips, 1956). Hydroxy-indoles were detected on paper chromatograms. In
an attempt at isolation of these substances from large amounts of nematocysts,
the content of 5-hydroxy-indoles was followed quantitatively, using the method of
Mitoma et al. (1956) during the purification of the nematocysts. As the suspension
became more and more free of tissue components, the level of 5-hydroxy-indoles
dropped steadily. This would suggest that these substances are not a part of the
toxin but instead represent a soluble tissue component.

Various marine invertebrates were tested for their susceptibility to extracts
of the nematocysts of Metridium senile fimbriatum. The animal found to be the
most convenient for assay purposes was Littorina plana.vis, a small snail from the
high intertidal zone. This animal normally has no contact with coelenterates of
any sort, at least during its post-larval and adult stages. When placed upside down
in sea water it rapidly rights itself and moves out of the water to a relatively dry

TABLE II

Per cent inhibition of the righting response of Littorina planaxis by distilled water
extracts of the nematocysts of Metridium senile fimbriatum

Dose,

Time, minutes

Hours

Days

micrograms

5

10

20

40

1

2

4

8

1

2

4

8

150.0

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

80%*

75.0

100

100

100

100

100

100

100

100

80

80

70

40**

37.5

100

100

100

100

100

100

100

100

70

30

30

0

18.75

100

100

100

100

100

90

80

50

0

0

0

0

9.375

100

90

80

80

80

80

40

20

0

0

0

0

7.5

100

100

90

30

20

0

0

0

0

0

0

0

0.75

100

80

40

20

0

0

0

0

0

0

0

0

0.00

70

60

40

0

0

0

0

0

0

0

0

0

* 8/10 dead.
** 4/10 dead.

place. It was found that the time required for this righting and withdrawal from
the water could be prolonged by addition of the toxin and the length of inhibition
is dependent upon the concentration of the toxin. With very high doses of the
toxin the time was infinitely extended since there was a resulting death of the snail.
The titrations of toxicity were carried out as follows : The various doses of
toxin were prepared in 1 ml. of sea water and placed in flat bottomed tubes, 25 X 95
mm. Ten tubes of each dose were prepared so that ten snails per dose could
be tested simultaneously. The snails were dried with a towel, and the water in
the mantle cavities was removed by gentle pressure on their opercula with a towel-
covered probe. They were then dropped into the toxin and shaken so that the
snails were upside down and the cup of the shell was filled with toxin. One milli-
liter of the toxin dilution was insufficient to cover the snail, so that the amount
of diluted toxin actually involved in the test was the amount which was contained
in the cup of the shell and was ultimately drawn into the mantle cavity. The time
at which the snails were first dropped into the diluted toxin was noted, and the time
at which they righted themselves was noted. Table II shows the results obtained

300

JOHN H. PHILLIPS, JR. AND DONALD P. ABBOTT

with various doses of the toxin. The dose is expressed in micrograms dry weight
of toxin contained in 1 ml. of sea water. For convenience, 1% hours was taken
as the period of observation in subsequent titrations.

Since Littorina is exposed to fresh water in the form of rain at not infrequent
intervals, it seemed unlikely that the righting response would be affected by dilution
of the sea water with distilled water. However, since the toxin was obtained by
discharge of the nematocysts in distilled water, the effect of the dilution of the sea
water by the addition of toxin was determined. No effect due to dilution could
be found over the range used in the test.

The snails employed in the titrations show a considerable variation in size. On
the basis of body weight, including shell, they vary from 1.15 to 0.33 grams. Yet
their response to the toxin did not appear to be correlated with body weight. In
order to explain this observation a group of snails was weighed independently
before and after the removal of the fluid from the mantle cavity. In this way the
volume and weight of the effective dose received by the snails of various weights

TABLE III

Per cent inhibition of the righting response of Littorina planaxis by toxin of Metridium
senile fimbriatum obtained by discharge of nematocysts in two different media

Dose (micrograms/ ml. of sea water)

30.0
15.0

7.5
3.75
1.875
ED 50

Toxin obtained by discharge in:
Distilled water Sodium thioglycolate

95% confidence limits of ED6o

% Inhibited

90
80
20
10

0
12

1.52

7.9-18.2

% Inhibited

100
50
40

0

0
11.5

1.75

6.5-20.4

were determined. It was found that the dose received by each snail is proportional
to its body weight. The volume of sea water that contains the toxin and is drawn
into the mantle cavity varies from 0.10 to 0.01 ml., depending on the size of the
snail. This means that the effective dose of toxin is %0 to Vioo °f the amount
present in the milliliter of toxin dilution.

The toxin was assayed using five doses and ten snails per dose. The titrations
were terminated after 1% hours. Since there was a possibility that the toxin was
sensitive to oxygen, toxin obtained by discharge of the nematocysts in 0.05% sodium
thioglycolate was compared with toxin obtained by discharge in distilled water.
The method of Litchfield and Wilcoxon (1949) was used to obtain: (a) the median
effective dose, ED50; and (b) the factor, fEDr,0, for obtaining the 95% confidence
limits of the ED50. The curves were found to be parallel within experimental
error. Table III shows the results of these titrations and includes the parameters
mentioned above. It does not appear that the toxin is particularly oxygen-labile.

SUMMARY

1. A method of obtaining purified suspensions of nematocysts has been de-
veloped, and a method of obtaining the toxin they contain has been described.

NEMATOCYST TOXIN OF METRIDIUM 301

2. A method of assay, using inhibition of the righting response of Littorina
plana.ris, has been shown to be applicable for toxicity titrations.

3. Further work on the chemical and immunochemical characterization of the
toxin is planned, as well as the use of the toxin for studies of antitoxic immunity
in marine invertebrates.

LITERATURE CITED

CANTACUZENE, J., 1926. Activation des poisons de I'Adamsia palliata par la lecithine et pourvoir

hemolytique. C. R. Soc. Biol, Paris, 95: 118-120.
CANTACUZENE, J., AND A. DAMBOVICEANU, 1934a. Caracteres biologiques de 1'extrait des

acconities d'Adamsia palliata apres deproteinisation. C. R. Soc. Biol., Paris, 117:

136-138.
CANTACUZENE, J., AND A. DAMBOVICEANU, 1934b. Caracteres physiochimiques du poison des

acconities d'Adamsia palliata. C. R. Soc. Biol., Paris, 117: 138-140.
COSMOVICI, N. L., 1925. Les poisons de 1'extrait aqueux des tentacules et des nematocystes

d'Adamsia palliata sont-ils detruits par 1'ebullition? Essais d'adsorption. C. R. Soc.

Biol., Paris, 92 : 1373-1374.
GLASER, O. C., AND C. M. SPARROW, 1909. The physiology of nematocysts. /. Exp. Zool., 6 :

361-382.
HAND, C., 1955. The sea anemones of Central California. Part III. The acontiarian anemones.

Wasmann J. Biol, 13 : 189-251.
HYMAN, L. H., 1940. The Invertebrates : Protozoa through Ctenophora. McGraw-Hill Book

Co. Inc., New York.
LITCHFIELD, J. T., JR., AND F. WiLcoxoN, 1949. A simplified method of evaluating dose effect

experiments. /. Pharm., 96: 99-113.
MITOMA, C., H. WEISSBACH, AND S. UDENFRIEND, 1956. 5-Hydroxytryptophan formation and

tryptophan metabolism in Chromobacterium violaceum. Arch. Bioch. Biophys., 63:

122-130.
PHILLIPS, J. H., 1956. Isolation of active nematocysts of Metridiiun senile and their chemical

composition. Nature, 178: 932.
RICHET, C., AND D. PORTIER, 1936. Recherches sur la toxine des coelenteres et les phenomenes

d'anaphylaxie. Res. Camp. Sci., Monaco, 95 : 3-24.
SONDERHOFF, R., 1936. Uber das Gift der Seeanemonen I. Em Beitrag zur Kenntnis der

Nesselgiftes. Liebig's Ann., 525: 138-150.
WEILL, R., 1926. Une technique permettant d'obtenir la devagination des nematocystes au

ralenti, d'une quantite prealablement determinee et fractionnee. La conservation des

nematocystes. C. R. Soc. Biol., Paris, 94: 1328-1329.
WELSH, J. H., 1955. On the nature and action of coelenterate toxins. Papers in Marine

Biology and Oceanography. Suppl. to vol. 3 of Deep Sea Research, pp. 287-297.

STUDIES ON THE LIFE-HISTORY OF ALLOCREADIUM
ALLONEOTENICUM SP. NOV. (ALLOCREADIIDAE-

TREMATODA)

DONALD M. WOOTTON
Marine Biological Laboratory, Woods Hole, Mass.

The systematics of the trematode genus Allocreadium has been confused because
of studies which reported two cercarial types from members of the genus. The
cercariae which have been reported are not only morphologically dissimilar, but
the molluscan hosts include both sphaeriid clams (Looss, 1894; Dollfus, 1949;
and Peters, 1957) and prosobranch snails (Seitner, 1951).

Looss (1894) and Dollfus (1949) described an ophthalmoxiphidiocercaria de-
veloping from rediae in sphaeriid bivalves as the cercarial stage of the type species
A. isoporum (Looss 1894). Peters (1957) also described a similar ophthal-
moxiphidiocercaria from rediae developing in sphaeriid bivalves as the cercarial
stage of A. neotenicum Peters 1956. These three authors did not demonstrate
the life cycles of the parasites experimentally but based their conclusions on morpho-
logical similarities of the cercariae and adults. The ecological evidence presented
in each case supported their conclusions.

Seitner (1951) described the larval forms of A. ictaluri Pearse 1924, from
Pleurocera acuta (Say), a prosobranch gastropod. In this case, the cercaria was
described as a gymnocephalous biocellate form, bearing setae in symmetrically
arranged papillae on the body and tail. Seitner pointed out that it is extremely
unlikely that species in the same genus would be morphologically different and that
they would have such widely different molluscan hosts. Since Seitner's work
was supported by experimental evidence he correctly regarded the earlier con-
clusions of Looss and Dollfus as inconclusive. Peters (1957) pointed out, how-
ever, that there are several reasons to question whether the cercaria described
by Seitner is actually the larva of A. ictaluri. Peters further showed that morpho-
logical and ecological data tended to prove that Seitner was probably dealing with
the larval stages of Skrjabinopsolus manteri (Cable 1952), a lepocreadioid, instead
of A. ictaluri.

Mathias (1937) reported on the life-history of Allocreadium angusticolle (Haus-
mann) but this trematode has since been placed in the genus Coitocaecum by
Dollfus (1949).

Evidence from controlled experiments in the present study supports the morpho-
logical and ecological observations of Looss, Dollfus and Peters, since the molluscan
hosts are sphaeriid clams which liberate ophthalmoxiphidiocercariae. These pene-
trate into caddis fly larvae and become precociously mature in the haemocoel of
these hosts.

METHODS AND MATERIALS

Materials used in this study were obtained from the clam, Pisidiwn abditmn
Haldeman, and from caddis fly larvae belonging to the genus Limnephilus collected

302

LIFE-HISTORY OF ALLOCREADIUM 303

at two localities near Falmouth on Cape Cod, Mass. One collecting area was a
spring-fed pond draining into the Coonamessett River just off Sandwich Road
and the other was an extensive cranberry-ditch area along the Quashnet River
just off Highway 28. Other caddis fly larvae and various beetle larvae and adult
beetles occurring in the type localities were never infected in nature and were
refractive to experimental infection. Living material, from both fingernail clams
and caddis fly larvae, was used for the study of the excretory system and other
morphological details.

A caddis fly larva harboring adult flukes (Fig. 1) could be determined by
forcing the larva partially out of the case and observing the appendages and ab-
dominal area for the presence of the eggs of the parasite. The infected larvae
were characteristically darker brown than uninfected ones because of the presence
of the eggs. If eggs were present in the larvae, at least one adult fluke was present.
In a few cases, the fluke had degenerated so that they appeared as a blackish,
internally amorphous mass which, however, still retained the characteristic external
shape of the worm. Various degenerative stages of the flukes were observed to
form an uninterrupted series so that it was evident that this was not an isolated or
abnormal occurrence. These stages were more often found in caddis fly larvae
harboring many flukes. (Some contained up to 25 worms.)

Worms and eggs were obtained by carefully detaching the head from the larvae,
dissecting the last two segments from the abdomen and withdrawing the intestine,
thus allowing space for the escape of the flukes from the haemocoel.

Specimens were fixed by squirting them into Gilson's fluid at 60° C. Whole
mounts were stained with Semichon's aceto-carmine and Harris' haemotoxylin.
Sections of uninfected and infected caddis fly larvae with worms in situ were
stained with Delafield's haematoxylin.

Cercariae were studied alive with the aid of aqueous vital stains such as neutral
red. orange G, brilliant cresyl blue, and Nile blue sulphate and also as fixed and
stained specimens.

Two methods were used to obtain miracidia for study. One was to wash out
as many eggs as possible from dissected material, then to remove the remains of
the larvae and wash the eggs. Such "clean" eggs did not hatch even when filtered
water from finger bowls containing an abundance of dead leaves and other organic
material was substituted for stream water. When numerous miracidia could be
observed actively moving within the eggs, small snails, Aplexa hypnorum (L.),
from Coonamessett pool were added. The snails readily ingested the eggs and
as eggs appeared in the feces of the snail, the miracidia began to hatch. This
process was observed under the dissecting microscope. The other method of
obtaining miracidia, which gave equal results as far as numbers of miracidia was
concerned, was to dissect the caddis fly larvae but to leave the remains in the
container with the eggs and to add filtered water rich in organic acids. This
method had the disadvantage of encouraging bacterial and protozoan growth.

Miracidia obtained by these two methods were studied, alive by the use of vital
stains, and also as fixed and stained specimens. For the study of the epidermal
plates, the miracidia were impregnated using the silver nitrate method of Goodchikl
(1948) and mounted in glycerine jelly.

All measurements used in this study are in millimeters. Length is given first,
followed by width.

304

DONALD M. WOOTTON

EXPLANATION OF PLATE I

FIGURE 1. Limnephilns sp. (10 mm. in length) ; lateral view with Allocreadium al-
loneotcnicum adult in haemocoel. Eggs shown only in middle thoracic segment. Entire figure
diagrammatic.

FIGURE 2. Adult (3.5 X 1.3 mm.), ventral view.

FIGURE 3. Cercarial stylet (0.0238 mm. in length).

FIGURE 4. Worn stylet of adult (0.023 mm. in length).

FIGURE 5. Female complex, lateral view.

FIGURE 6. Miracidium (0.095 X 0.045 mm.), dorsal view.

FIGURE 7. Miracidial epidermal plates (0.061 X 0.044 mm.), dorsal view.

LIFE-HISTORY OF ALLOCREADIUM 305

DESCRIPTIONS OF STAGES IN THE LIFE-CYCLE

Allocreadiwn alloneotenicutn n. sp. : The specific name alloncotenicum ("an-
other" neotenicum) was chosen because of the similarity in neotenous development
to A. neotenicum Peters 1957.

Host: The caddis fly larva Limnephilus sp.

Site : Haemocoel.

Incidence: 40 of 120 larvae (30%).

Type locality : Coonamessett River, Barnstable County, Cape Cod, Massachu-
setts, U. S. A.

Type specimens : A type and paratype will be deposited in the Helminthological
Collection of the United States National Museum.

Adult (Fig. 2) :

Body elliptical, 1.38-4.42 X 0.59-1.41 mm., thick but slightly flattened dorso-
ventrally, anterior end bluntly pointed ; usually with posterior indentation at ex-
cretory pore ; cuticle smooth. Well developed eyespots, located at level of posterior
margin of oral sucker, present in all stages of development. Oral sucker sub-
terminal 0.12-0.20 X 0.08-0.19 mm. (average 0.16 X 0.14 mm.). Stylet present
(Fig. 4), imbedded in dorsal lip of oral sucker in relatively same position as in
cercaria at an angle of 45°-60° from the longitudinal axis of the body. Ventral
sucker 0.13-0.22 X 0.10-0.22 mm., (average 0.175 X 0.156 mm.), "in anterior
one-sixth or one-seventh of body. Prepharynx short, usually not evident but
distinguishable in sections. Pharynx spherical, 0.06-0.11 mm. in diameter,
posterio-dorsal to oral sucker. Esophagus as long as, to twice as long as, pharynx ;
intestinal bifurcation at level of anterior edge of ventral sucker, caeca simple, some-
what inflated, extending dorsally almost to posterior end of body. Excretory
bladder elongate, sac-shaped with well defined lumen, extending anteriorly to
middle of posterior testis. Excretory pore terminal within indentation at posterior
end of body. Main excretory tubules extend anterio-laterad from bladder in a
somewhat tortuous pattern, but without recurrent loop to level of mid-anterior
testis where each receives an anterior and posterior secondary tubule. Each
secondary tubule drains three groups of flame cells, exhibiting considerable varia-
tion both in numbers and position. Apparent flame cell formula is 2 [(6 + 6 + 6)
+ (6 + 6 + 6)].

Ovary oval, 0.21-0.37 X 0.19-0.46 mm. (average 0.28 X 0.31 mm.), posterior
to ventral sucker, sometimes overlapping the latter. Oviduct extends mediad a
short distance, receives the common duct of receptaculum seminis and Lauer's
canal (Fig. 5), then turns anteriad. Receptaculum seminis variable in shape,
usually about one-fourth size of ovary. Sinuous Lauer's canal opens dorsally,
posterio-median to ovary. Oviduct extends to form the ootype which is surrounded
by a prominent Mehlis' gland ; from ootype the uterus extends anteriorly composed
of few loops, usually confined to area on right side of body bounded posteriorly
by anterior testis and medially by ovary, but occasionally overlapping these struc-

FIGURE 8. Miracidial epidermal plates (0.044 mm. in diameter), anterior view.
FIGURE 9. Miracidial epidermal plates (0.044 mm. in diameter), posterior view.
FIGURE 10. Egg (0.096 X 0.056 mm.), lateral view.

306 DONALD M. WOOTTON

tures. Uterus sometimes with a loop or two anterior to the ovary on left side of
body. Metraterm present, opening into genital pore. Shallow genital pore ventro-
median, near level of pharynx.

Vitelline follicles extend posteriad from anterior edge of ovary, more or less
confluent in post-testicular half of body on ventral side, reaching almost to posterior
end of body. Right and left vitelline ducts unite to form the vitelline reservoir
in the angular space between the testes and ovary.

Testes oval or irregular in outline, never lobed, subeqtial, anterior testis,
0.19-0.46 X 0.24-0.44 mm., (average 0.29 X 0.35 mm.) ; posterior testis, 0.19-
0.51 X 0.19-0.59 mm., (average 0.31 X 0.39 mm.). Testes diagonal, contiguous
or separated by a short distance, ventral in position and confined to anterior one
half of body. Vasa efferentia arise on anterior margin of testes, proceed anteriorly
and join at posterior end of cirrus sac to form a very short vas deferens which enters
cirrus sac forming a vesicula seminalis. Cirrus sac, 0.09-0.22 X 0.12-0.34 mm.
(average 0.14 X 0.22 mm.), median or submedian to left of mid-line, between
pharynx and ventral sucker ; vesicula seminalis convoluted, pars prostatica tubular,
prostate cells numerous and well developed.

Eggs number up to 45, measuring 0.092-0.108 X 0.055-0.060 mm. (average
0.0975 X 0.0577 mm.) with a small antopercular knob (Fig. 10), shell thin, light
golden brown.

Miracidium (Fig. 6) :

When first laid, the eggs are segmented. Development of the eggs is extremely
variable. Within 48 hours, motile miracidia can be observed in some eggs, in others
movement of miracidia can not be observed for several days. Upon hatching, the
miracidium swims in a slightly zig-zag path, rotating slowly. Miracidia positively
phototactic. converging either on light side of shallow dish or on opposite side
where light rays are concentrated after passage through the water. Body of
miracidium either elongate or pear-shaped when swimming, terrebratorium some-
times protruded. Miracidia were infective to both laboratory-reared young Pisid-
ium abditum and Musculium partumeium (Say). No attraction to clam hosts
was observed but infections were present within 24 hours after clams were added
to the container with miracidia.

In using the Goodchild (1948) modification of Lynch's (1933) silver nitrate
method of delineating epidermal plates, it was found that if the 1.0% silver nitrate
solution was warmed slightly, there was less distortion of the miracidium. Silver
nitrate-treated miracidia measured 0.061 X 0.044 mm.

The epidermal cell formula is 6-6-4—2 (Figs. 7, 8, 9). The same formula was
observed by Peters (1957) for A. neotenicum. The miracidia of both these
neotenous forms are quite similar, agreeing closely in most respects. The anterior
and second tiers consist of two ventro-lateral, two dorso-lateral and two lateral
cells lined up essentially end-to-end. The four cells in the next tier are arranged
with a ventro-lateral and a dorso-lateral cell on each side, while the posterior tier
consists of a dorsal and a ventral cell. The conspicuous double eyespots are
situated dorsally about one-third of the length from the anterior end in living
miracidia. The nervous system surrounds the eyespots and sends branches laterally
to sensory pores and posterior branches diagonally to the middle of the lateral ciliary
plates in the second tier. The anterior fourth of the body is occupied by the apical

LIFE-HISTORY OF ALLOCREADIUM 307

gland which is without a stylet, and with 3-4 nuclei along its posterior margin.
Granular structures representing the "penetration" glands of other authors are
difficult to see clearly. They occupy most of the lateral portions of the miracidium.
Observations of miracidial penetration clearly show four discrete unicellular glands.
Two flame cells, usually not at same level, lie near the middle of the miracidium.
Five to nine germinal cells are scattered in the center of the body posterior to the
eyespots. Living miracidia measure 0.068-0.108 X 0.036-0.052 mm.

Miracidial penetration into the molluscan host has been observed by several
workers, notably Thomas (1883) in Fasciola hepatica, Barlow (1925) in Fasciolop-
sis buski, Bennett (1936) in Cotylophoron cotylophorum, Rees (1940) in Parorchis
acanthus and Goodchild (1948) in Gorgodera cnuplicava. Barlow suggested that
the apical gland secreted an "erosive fluid" while Goodchild considered it to secrete
an adhesive substance. Several authors have observed droplets of fluid at the
pores of the "penetration" glands and have assigned penetration functions to the
secretions from these glands. Bennett pointed out that as transformation of the
miracidium took place, it gradually changed shape and a very thin cuticula was
formed around the outside of the body, but he did not give its origin.

In order to study the process of pentration and transformation of the miracidium
into the sporocysts, an excised gill from a small uninfected P. abditum was added
to a drop of water containing several miracidia on a slide. The actively swimming
miracidia came into contact with the gill many times before they started to penetrate
into the gill tissue. No attraction to the tissue was evident. The process of pene-
tration was observed under high magnification of a compound microscope.

When penetrating into the gill, initially there is a rotatory movement combined
with extreme prolongations of the anterior end of the larva, followed by progressive
swelling from anterior to posterior, which draws the miracidium into the tissue.
After approximately 15 minutes, rotary movements cease and the miracidium
begins a rhythmical contraction and elongation of the body. Four small distinct
droplets of granular material (Fig. 11) are extruded from the pores of the "pene-
tration" glands. These pores are not symmetrically arranged although they each
open in the anterior space between the first tier of ciliary plates. On the left of
the miradicium one pore opens in the space between the dorso-lateral plates, while
the other opens between the dorso-lateral and the lateral plates. On the right side
of the body, however, one of the pores opens between the two ventro-laterals and
the other between the ventro-lateral and lateral plates. The droplets coalesce into
two droplets (Fig. 12). At this time granular material lighter in color and more
fluid in consistency begins to flow from pores on the terrebratorium draining the
apical gland. This material appears to be histolytic in function since there is a
progressive breakdown and liquefaction of the clam tissue anterior to the miracid-
ium. As additional granular material is extruded from the pores of the "penetra-
tion glands," the droplets fuse into an apical cap covering the anterior end. It
could not be clearly observed if the secretion of the apical gland was pushed ahead
of the forming cap or if it mixed with the cap material. At least some of the
apical gland secretion stays anterior to the apical cap. As the apical cap becomes
more extensive (Fig. 13), the sporocyst begins to extend into it from the miracidial
covering by way of the terrebratorium.

The process of sporocyst emergence (Figs. 14-18) requires over three hours.
During this time the apical cap becomes increasingly thicker until it extends 0.015

308

DONALD M. WOOTTON

PLATE II

LIFE-HISTORY OF ALLOCREADIUM 309

mm. anterior to the miracidium. The sporocyst gradually emerges into the apical
cap material from the miracidial covering by a rhythmical series of anterior ex-
tensions and expansions. It is always covered by either the miracidial covering
or the cap material since the latter gradually extends more posteriorly. As the
granular material extends posteriad it appears to force the epidermal plate cells
ahead of it. The epidermal cells become quite evident, each with a well defined
nucleus. When the cap material covers about three-fourths of the emerging
sporocyst, the epidermal cells are almost spherical and their cilia are perpendicular
to the cell membrane and are still actively beating. A similar appearance of
epidermal plate cells was described by Thomas (1883) and by Barlow (1925).
Three hours after the sporocyst begins to emerge, the cap material completely covers
the body of the sporocyst. This cuticle is more evident on the sides of the sporocyst
than it is on the ends, being twice as thick on the former as on the latter. As the
cuticle is fully formed, the rounded epidermal cells break away and are moved
about by their beating cilia.

The apical gland thus appears to secrete a histolytic substance which aids in
penetration and the "penetration" glands do not seem to aid in penetration other
than perhaps passively by filling the space eroded by the material from the apical
gland. Since the "penetration" glands do secrete material which forms a cuticle
for the sporocyst, it would be more correct to call these "cuticle-producing" glands
and assign the penetration function to the apical gland.

Sporocysts:

Newly formed sporocysts measure 0.061-0.068 X 0.029-0.031 mm. Eyespots
are still contiguous and the apical gland and cuticle-forming gland remnants are
confined to the anterior end of the sporocysts.

Additional development of the sporocysts was followed mainly in infections in
P. abditiim but M. partnmeiitm infections were followed for two weeks and develop-
ment in the two clam hosts was parallel. Presumably the infection in M. partu-
meium will also develop to the cercarial stage, but limited numbers of small clams
of this species did not allow further study of development.

Much of the variation observed in the following developmental stages is due to
the fact that clams were left in containers with hatching miracidia to insure infection,
and since miracidia continued to hatch for several weeks, superimposed infections
were common.

Four days after infection, the smallest sporocyst observed measured 0.08 X 0.051
mm., developing in the gill of the clam. Two eyespots were present, one in the
middle of the anterior end and one almost midlength on the side of the body. No
evidence of a sucker was found. Nine developing germinal cells occupied most of

EXPLANATION OF PLATE II.

FIGURES 11-14. Penetrating miracidium, showing secretions from "penetration" and apical
glands and formation of apical cap (cilia omitted).

FIGURES 15-18. Formation of cuticle by gradual posterior progression of cap material
forcing ciliated epidermal cells away from new sporocyst.

FIGURE 19. Sporocyst (0.42 X 0.082 mm.).

FIGURE 20. Mother redia (0.60 X 0.120 mm.).

FIGURE 21. Daughter redia (0.090 X 0.25 mm.).

FIGURE 22. Cercaria (body 0.341 X 0.15 mm., tail, contracted, 0.30 X 0.063 mm.), ventral
view. Detail shown on the left and excretory system on the right of body.

310 DONALD M. WOOTTON

the body space. The two flame cells were situated diagonally in the middle of
the body.

Seven days after infection, clams yielded several sporocysts that measured
0.108-0.136 X 0.035-0.08 mm., with pigment sometimes diffuse but usually present
as distinct eyespots, variable as to their location.

Eighteen days after infection, sporocysts measured 0.103-0.274 X 0.047-0.072
mm. In all sporocysts at this stage of development, eyespots were still present,
and two flame cells were easily seen, one posterior and one anterior. Usually
one germinal ball (sometimes two) had increased in size, and one was usually
three or four times as large as the other germ balls.

By the 25th day the sporocysts were widely distributed in the tissues of the
clam, being present in the gills, foot, digestive gland and mantle. These sporocysts
ranged in size from 0.244-0.494 X 0.072-0.108 mm. At this stage eyespot pigment
was usually diffuse or absent. An identifiable redia, with its conspicuous globular
sucker, was usually present in each sporocyst. By the time the infection was 33
days old, rediae could be found in the tissues of the clam but their escape from
sporocysts was not observed. Sporocysts (Fig. 19) increased very little in size
beyond 0.50 X 0.12 mm.

At this stage, rediae surpassed the sporocysts in size, varying from 0.288 X
0.057 to 0.63 X 0.075 mm. The redial sucker is large, reaching 0.055 X 0.048 mm.
The mother rediae (Fig. 20) are morphologically the same as the daughter rediae
but their germinal cells give rise only to rediae and not to cercariae. Mother rediae
usually contained several developing rediae, one of which was typically larger than
the other.

In infections from 40 to 50 days old, sporocysts appeared to be absent while
mother rediae with a maximum size of 0.73 X 0.094 mm. were still producing
daughter rediae. There appeared to be only one generation of mother rediae, since
daughter rediae began to differentiate identifiable cercariae when the infection was
50 days old.

Mature daughter rediae (Fig. 21) are elongate, thin- walled sacs without
locomotory processes. The sucker is spherical, 0.04-0.057 mm. ; the intestine
reduced and inconspicuous. The birth pore is just anterior and lateral to the
sucker, not clearly visible, but cercariae were observed escaping, one by one from
the pore.

Twelve cercariae with definite eyespots were the maximum number observed
in any redia. Combinations of developing cercariae with eyespots, and germinal
masses numbered up to 18. Rediae containing eyed-cercariae measured 0.52-1.16
X 0.12-0.30 mm. (average size 0.87 X 0.20 mm. ; average number of eyed-cercariae
7). Occasional rediae contained eyed-cercariae, germinal masses and daughter
rediae. Rediae were also observed containing germ balls and daughter rediae.
This would indicate that asexual multiplication is a continuous process and possibly
continues for the life of the infection. Flame cells were four in number and
appeared to be paired into two homologous systems, each with an anterior and a
posterior flame cell. However, the ducts were difficult to distinguish.

Cere aria:

The cercaria is an ophthalmoxiphidiocercaria (Fig. 22), ellipsoidal in outline,
from two to three times as long as wide, slightly depressed dorso-ventrally with

LIFE-HISTORY OF ALLOCREADIUM 311

unarmed cuticle. Anterior sucker (0.047-0.05 mm. in diameter) equal to, or
slightly larger than, ventral sucker. A stylet (Fig. 3) is present in the dorsal lip
of the oral sucker, oriented at approximately a 45°-60° angle from longitudinal
axis of the body. Stylet quite constant in size within each of the two populations,
0.023-0.024 mm. in cercariae from Coonamessett River and 0.021-0.0235 mm. in
cercariae from Quashnet River. Stylet with lateral projections curved slightly
upwards, about one-fourth the length of stylet from anterior end. Ventral sucker
(0.042-0.048 mm. in diameter) located at about middle of the body, pedunculate,
external margin bearing numerous serrate papillae. Prepharynx short; pharynx
globular, 0.019-0.020 mm. in diameter, located mediad or slightly anteriad to the
eyespots. Esophagus two to three times as long as the pharynx, bifurcation of
the intestine just anterior to the midlength of the body. Caeca incompletely
developed, usually reaching latero-posteriorly only to anterior edge of ventral
sucker. Nervous system composed of a transverse band at the level of the pharynx
with fibers extending to the eyespots. Eyespots well developed, brownish black.
Three pairs of non-lobed penetration glands lie lateral and anterior to the ventral
sucker. Each of the anterior pair of glands is drained by a duct which runs anteriad
between the eyespots, while each of the two posterior pairs of glands has a duct
which extends side by side anteriad between the body and the eyespots. Posterior
and some\vhat dorsal to the ventral sucker are the primordia of the genital organs
abutting against the anterior end of the excretory bladder. Cystogenous glands
appear to be absent and since no cyst is formed in caddis fly larvae, this might be
expected.

The sac-shaped excretory bladder extends anteriorly almost to the ventral
sucker. The wall of the bladder is thick, composed of numerous cells. Anteriorly
two excretory canals enter laterally, proceeding along a sinuous path, from a point
about mid-level to the ventral sucker where the posterior and anterior excretory
ducts join. Ascending and descending ducts each drain three groups of flame cells :
each group composed of four flame cells. The flame cell pattern is thus 2
[(4 + 4 + 4) + (4 + 4 + 4)].

The tail is attached slightly ventrally and is variable in length. Usually it is
a little longer than the body but it can be extended to over twice the length of the
latter. When fully contracted, it is shorter than the body but it is never as wide as
the body, thus differing from the cercaria of A. isoporum as described by Looss
(1894) and by Dollfus (1949).

Measurements of the tails of several cercaria killed by pipetting them into hot
formalin solution ranged from 0.20-0.34 mm. in length and 0.036-0.042 mm. in
width. Both Looss and Dollfus pointed out that the tail of the cercaria of A.
isoporum possessed an inner medullary portion, containing the nuclei, and a clear
outer transparent cortical zone. The tail of the the cercaria of A. alloneotenicum
also has a medullary and a cortical layer very similar to A. isoporum.

Measurements of both the body and the tail of the cercariae are extremely vari-
able in living as well as in preserved specimens. No method of killing and fixing
the cercaria was found which gave consistent results, so that morphological features
such as sucker size, stylet shape and size, and size and extent of the excretory
bladder are more reliable descriptive characteristics.

A single precocious cercaria which was 0.81 X 0.30 mm., with a tail 0.17 mm.
long, was present in a redia 0.968 mm. long. Immature gonads were clearly de-

312 DONALD M. WOOTTON

fined. The stylet was absent in this precocious cercaria. Since the stylet was
observed to aid in the escape of normal cercariae from rediae, this might account
for the retention of the cercaria. The increase in size and development of gonads
while still in the clam host is surprising.

Juvenile worms:

No cysts are formed although occasionally young worms became isolated in the
gills of the caddis fly larvae and thus appeared cyst-like. Such worms seem to be
prevented from reaching the haemocoel by the accumulation of detached tracheal
vessels in the proximal part of the gill. Isolated worms typically cause the deposi-
tion of dark brown pigment by the larvae, which is a common response to any
mechanical injury at any place in their bodies. .

Experimental infections of caddis fly larvae isolated with individual clams
liberating A. alloneotenicum cercariae resulted in the presence of numerous juvenile
worms. One hundred caddis fly larvae were brought into the laboratory from
sources thought to be free of infection ; 50 were dissected and found to be negative,
25 were used in infection experiments and the remaining 25 were kept as controls
and found to be negative upon dissection at the conclusion of the experiment.

In infections up to a week old, the worms varied from 0.27-0.33 X 0.13-0.17
mm. The pharynx was 0.02-0.021 mm. in diameter; the oral sucker 0.057-0.058
mm. and the ventral sucker 0.047-0.050 mm. In the larger specimens the genital
primordium had begun to differentiate into identifiable reproductive organs. In
later stages the testes had developed more rapidly than the ovary, similar to the
development of these structures in other trematodes.

Eggs are present in infections 24 days old, but are few in number for an addi-
tional 14 days during which the worms continue to increase in size. When eggs
are first produced, the worms are usually 1.5 X 0.62 mm. in size but the number
of worms present in the larvae influences this size as well as the ultimate sizes.

DISCUSSION

Peters (1957) reviewed the genus Allocreadiurn and emended the generic diag-
nosis, retaining 16 of the 31 species described in the genus. He further listed 5
as species dubiae and transferred the remaining 10 species to other genera or left
them as species inquirendae because of inadequate descriptions.

A. alloneotenicum conforms to the genus Allocreadium as emended by Peters
(1957). It can be separated from the other species in the genus, however, by
the extreme anterior position of the ventral sucker (within the anterior one-sixth
of the body), and in the position of the testes (within the first half of the body).
It specifically differs from A. ictaluri Pearse 1924, and A. pseudotritoni Rankin
1937 in lacking vitellaria in the forebody ; by ventral sucker being larger than the
oral sucker it differs from A. handiai Pande 1937, A. nicolli Pande 1938a, A. kosia
Pande 1938a, and A. mahaseri Pande 1938b. A. alloneotenicum also differs from
A. transversale (Rudolphi 1802) Szidat 1939, A. schisothorcis Pande 1938b, A.
lobatum Wallin 1909, and A. hasu Ozaki 1926 in having the ventral sucker well
within the anterior fourth of the body, and in the shape and position of the gonads.
It differs from the type species A. isoporum (Looss 1894), and from A. nemachilus
Kaw 1950 and A. thapari Gupta 1950 in the size and distribution of the vitelline

LIFE-HISTORY OF ALLOCREADIUM 313

follicles, the position of the cirrus sac, extent of the uterus and number and size
of eggs.

The original descriptions of A. markewitchi Koval 1949 and A. dogieli Koval
1950 were not available for comparison but from the description of these species
in Markevich (1952), it is apparent that A. alloneotenicum is distinct from these
species.

A. alloneotenicum corresponds most closely to A. neotenicum Peters 1957. It
differs in the shape of the body (always at least twice as long as wide), the posterior
extent of the vitellaria and caeca, in the extent of the excretory bladder (which
only reaches mid-level of the posterior testis instead of to under the anterior testis),
and in the relative position of the ovary complex and the cirrus sac. A. neotenicum
and A. alloneotenicum are unique in that they are the only two species within the
genus known which apparently develop to sexual maturity in insects.

The clam, P. abditum, from Coonamessett also contained an infection of
Crepidostomum sp. The cercariae from this infection were also ophthalmoxiphidio-
cercariae developing from rediae. They encysted both in nature and experimentally
in the amphipod, Gammants sp. Crepidostomum infections could be differentiated
in the redial stages by the larger number of cercariae (usually approximately 36
being present) as well as by morphological differences.

The Crepidostomum cercariae possess a slightly smaller stylet, 0.017-0.019
mm., which has more of a median keel and slightly different lateral projections.
They also possess 44-48 clearly denned cystogenous glands, 12-14 anterior and
32-34 posterior to the ventral sucker. The three pairs of penetration glands tend
to be lobed. The excretory bladder does not extend as far anteriorly and the
genital primordium is more extensive.

No fish were collected from this pool during the period November, 1956 to
April, 1957. Attempts to infect various fish, including Eitcalia inconstans, Fundu-
lus heteroclitus, and Sah'clinus jontinalis, with infected amphipods yielded only a
limited number of juvenile Crepidostomum from the trout.

Infection experiments using the same three species of fish, feeding them caddis
fly larvae known to be infected with juvenile A. alloneotenicum, were all negative.
The worms were digested with the caddis fly larvae and portions of both could be
recovered on the second day from the posterior portion of the gut of the fish. It
might be possible that eggs would remain viable after passage through the intestine
of a fish, but this was not investigated.

It appears, from the large number of eggs produced by the adult worms (up to
1200 eggs being recovered from a larva containing a single worm), that the infection
in caddis fly larvae is the normal one for this species of AUocreadium. Infected
larvae are never as active as uninfected ones; they are usually smaller and their
cases show signs of neglect. The presence of numerous eggs throughout the body
of the caddis fly larvae, including the appendages and the head capsule, plus the
erosion and decrease in numbers and size of the fat-bodies make it extremely un-
likely that an infected larva is ever able to pupate and reach adulthood. The re-
covery of the remains of dead larvae, still within the cases, enclosing empty egg
shells of A. alloneotenicum, substantiates this view.

Peters (1955, 1957) stated that AUocreadium neotenicum from aquatic beetles
from Michigan (possibly identical with species found by Crawford (1940) in
aquatic beetles from Colorado) was a progenetic form that did not require a

314 DONALD M. WOOTTON

vertebrate host in order to complete its life cycle. He presented ecological evidence
which supported his supposition. Buttner (1950, 1955) reviewed the progenetic
trematodes and proposed four degrees of development of this characteristic. Peters
(1957) added A. neotenicum to the fourth group of Buttner, that is, to the group
in which the development of the gonads, the genital activity and the fecundity rivals
that of the true adult. The example of this group cited by Buttner was Paralepo-
dernia brumpti, a plagiorchid. A. alloneotenicum should be added to this group
also. Both of these species of Allocreadium differ from the example cited by
Buttner, however, since they develop to maturity in invertebrate rather than in
vertebrate hosts.

The presence of ophthalmoxiphidiocercariae developing from rediae in sphaeriid
clams in A. alloneotenicum supports the systematic scheme proposed by Dollfus
(1949). Seitner's work (1951) should be re-investigated in the light of the results
of the present study before final acceptance of the scheme of Dollfus. The con-
trolled experiments on the life-history of A. alloneotenicum, supporting the morpho-
logical and ecological data presented by Looss (1894) and Dollfus (1949) for
A. isoporum and by Peters (1957) for A. ncotcnicum, indicate a close relationship
of the genera Allocreadium, Crepidostomwn, and Megalonia. They all have
ophthalmoxiphidiocercariae developing in rediae from sphaeriid bivalves thus form-
ing a natural group.

The author wishes to express his appreciation to the Director of the Marine
Biological Laboratory, Woods Hole, Mass., for the use of facilities ; to Dr. Nathan
W. Riser for reading the manuscript and offering helpful suggestions ; and to Mr.
Lewis Peters for making his material on A. neotcnicum available for comparison
and for the opportunity of reading his Master's thesis.

SUMMARY

Allocreadium alloneotenicum sp. nov. is described from the haemocoel of
Limnephilus sp., caddis fly larvae, from Cape Cod, Massachusetts. The life-cycle
is demonstrated both in natural and experimental infections. The normal clam
host is Pisidium abditum. Miracidia hatch in the debris from dead larvae or after
ingestion and passage in the feces of the snail, Aplexa hypnorum. The process
of miracidial peneration was observed. Secretions from the apical gland are
histolytic in action, facilitating penetration, while the "penetration" glands produce
the cuticula of the sporocyst. Sporocysts give rise to one generation of mother
rediae which in turn liberate daughter rediae. Daughter rediae give rise to
ophthalmoxiphidiocercariae and also produce occasional rediae. The cercariae
penetrate caddis fly larvae (as many as 25 of them being found in natural infec-
tions). They mature in the haemocoel and a single worm was found to have
laid 1200 eggs. Experimental infections of fish with infected caddis fly larvae were
negative.

LITERATURE CITED

BARLOW, C. H., 1925. The life history of the human intestinal fluke, Fasciolopsis buski

Lankester. Amcr. J. Hyg., Monogr. Ser., 4 : 1-98.
BENNETT, H. J., 1936. The life history of Cotylophoron cotylophorum, a trematode from

ruminants. Illinois Biol. Monogr., 14 (4) : 1-119.

LIFE-HISTORY OF ALLOCREADIUM 315

BUTTNER, A., 1950. La progenese chez les trematodes digenetiques. Sa signification. Ses

manifestations. Contributions a 1'etude de son determinisme. Ann. Parasitol., 25

(5-6): 376-434.
BUTTNER, A., 1955. Les distomes progenetiques sont-ils des pre-adultes ou des adultes veri-

tables? Valeur evolutive de la progeneses chez les Digenea. C. R. Soc. Biol., 169:

267-272.
CRAWFORD, W. W., 1940. An unusual case of a sexually mature trematode from the body

cavity of a diving beetle. /. Parasitol., 32, Suppl. : 32.
DOLLFUS, R. PH., 1949. Sur une cercaire ophthalmoxiphidiocerque Ccrcaria isopori A. Looss

1894 et sur la delimitation des Allocreadioidea. Ann. Parasitol., 24: 424-435.
GOODCHILD, C. G., 1948. Additional observations on the bionomics and life history of Gorgodera

amplicava Looss, 1899 (Trematoda, Gorgoderidae). /. Parasitol., 34 (5) : 407-427.
GUPTA, S. P., 1950. On a new trematode, Allocreadium thapari n. sp. of the sub-family

Allocreadiinae Looss, 1899 from the intestine of a fresh-water fish, Rita rita (Ham).

Indian J. Helminth., 2: 17-22.
KAW, B. L., 1950. Studies in helminthology : Helminth parasites of Kashmir. Part I.

Trematoda. Indian J. Helminth., 2 : 67-126.
Looss, A., 1894. Die Distomen unserer Fische und froche neue Untersuchungen iiber Bau

und Entwickelung des Distomenkorpers. Biblioth. Zool. Stuttgart. 6 (16) : 1-296.
LYNCH, J. E., 1933. The miracidium of Heronimits chclydrae MacCallum. Quart. J. Micros.

Sci., 76: 13-33.

MARKEVICH, A. P., 1952. Parasite fauna of fresh-water fish of Ukrainian SSR. Kiev.
MATHIAS, P., 1937. Cycle evolutif d'un trematode de la famille Allocrcadiidac Stossich

(AUocrcadium angusticolle (Hausmann). C. R. Acad. Sci. Paris, 205 (15) : 626-628.
OZAKI, Y., 1926. On some new species of trematodes of fresh water fish from Japan (Pre-
liminary report.). D abuts. Zasshi, Toyko, 38: 124-130. (In Japanese).
PANDE, B. P., 1937. Morphology and relationships of a new digenetic trematode from an

Indian freshwater fish, Ophiocephalus punctatus. Ann. Mag. Nat. Hist., Scr. 10, 20 :

415-421.
PANDE, B. P., 1938a. The trematode genus Allocreadium in north Indian freshwater fishes.

Proc. Indian Acad. Sci., 7 (B) : 54-60.
PANDE, B. P., 1938b. On two new trematodes from Indian cyprinoid fishes with remarks on

the genus Allocreadium Looss. Proc. Nat. Acad. Sci., India, 8: 110-115.

PEARSE, A. S., 1924. Observations on parasitic worms from Wisconsin fishes. Trans. Wis-
consin Acad. Sci., 21 : 147-160.
PETERS, L. E., 1955. Morphology of the adult and the miracidium of a progenetic species of

AUocrcadium from water beetles of the family Dytiscidae. /. Parasitol., 41, Suppl.: 36.
PETERS, L. E., 1957. An analysis of the trematode genus Allocreadium Looss with the

description of Allocreadium neotenicum sp. nov. from water beetles. /. Parasitol.,

43: 136-142.
RANKIN, J. S., 1937. New helminths from North Carolina salamanders. /. Parasitol., 23:

29-42.
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Nicoll. Part II. Structure of the miracidium and germinal development in the larval

stages. /. Parasitol., 32 : 372-391.
SEITNER, P. G., 1951. The life cycle of Allocreadium ictaluri Pearse, 1924 (Trematoda:

Digenea). /. Parasitol., 37: 223-244.
SZIDAT, L., 1939. tJber AUocrcadium transvcrsale Rud. 1802 aus Misgurnus fossilis L.

Zeitschr. f. Parasitenk., 10 : 468-475.

THOMAS, A. P., 1883. The life history of the liver fluke. Quart. J. Microsc. Soc., 23 : 90-133.
WALLIN, I. E., 1909. A new species of the trematode genus Allocreadium. Trans. Amer.

Micros. Soc., 29 : 50-66.

ABSTRACTS OF PAPERS PRESENTED AT
THE MARINE BIOLOGICAL LABORATORY

1957

ABSTRACTS OF SEMINAR PAPERS

JULY 2, 1957

Differentiation of cortical cytoplasm and extra- cellular membranes of oocytes, in-
cluding changes at fertilisation. NORMAN E. KEMP.

The cortical cytoplasm of the growing oocyte not only functions in selecting and transport-
ing raw materials for new protoplasm and stored inclusions but also cooperates with surrounding
follicle cells in the synthesis of the extra-cellular membranes, the vitelline membrane and chorion.
It is not known whether the cortical cytoplasm of the oocyte actually synthesizes the materials
for the vitelline membrane or merely serves as a form on which materials of follicular origin are
deposited. Electron micrographs of developing oocytes of Rana pipiens and Fundulus hetero-
clitus have revealed fine details of the intimate morphological relationships between inwardly
directed protoplasmic processes of follicular epithelial cells and outwardly directed protoplasmic
processes of the oocyte. In the frog a layer of microvilli greatly increases the surface area of
the oocyte, and some follicular processes extend into the layer of microvilli. Follicular processes
within the microvillous layer may possibly connect with the surface of the oocyte, but connec-
tions have not been found at stages after oocyte and follicle cells are well separated. In the fish,
protoplasmic processes extend from the oocyte through the zona radiata, and these processes
either adjoin or are continuous with follicular cell processes within the subfollicular space. The
student of fertilization would like to know (1) what membranes or jelly layers surround the egg
and how they develop, (2) how sperm cells get through these membranes, and (3) how the
cortical cytoplasm of the egg reacts to sperm entrance.

Lytic and other activities of the individual spermatozoon during the early events
of sperm entry (Hydroides, Saccoglossus, and several other invertebrates') .a
LAURA HUNTER COLWIN AND ARTHUR L. COLWIN.

In a number of invertebrate species the early events of sperm entry fall into two phases. It
is suggested that during the first phase, which lasts only a matter of seconds, the following events
occur: (a) the spermatozoon arrives at or near the jelly or membrane (whatever serves as a
barrier around the egg) ; (b) the spermatozoon undergoes the acrosome reaction (of Dan) and
sends its acrosome filament to or into the egg proper; (c) the acrosome filament delivers a stimu-
lus to the egg; (d) the egg begins to react (i.e., the fertilization reaction is initiated). It is not
known how the filament succeeds in spanning the barrier or in what way the filament delivers
the stimulus to the egg. The possibility that an enzyme (Bowen) or some other substance is
carried into the egg by the acrosome filament should be examined. During the second phase,
which in a number of species requires several minutes for completion, the acrosome filament and
its attached sperm head, acting as one unit, move through the barriers and pass into the egg.
Pits and spaces which appear in the egg membranes of Saccoglossus and Hydroides, as seen in
living material and electron micrographs of thin sections, are interpreted as areas of erosion caused
by egg membrane lysin emanating from the spermatozoon. It is suggested that the individual

1 Supported in part by a grant (RG-4948) from the National Institutes of Health, U. S.
Public Health Service.

316

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 317

spermatozoon uses its own lysin to enable its relatively large head to pass through the barrier
membranes, which the acrosome filament has initially spanned during the first phase of sperm
entry.

JULY 9, 1957

Membrane potential changes and ion movements in frog muscle. WILLIAM K.
STEPHENSON.

Frog sartorius muscles soaked in potassium-free saline for 11 to 18 hours lost % their origi-
nal fiber potassium and increased ca. 5 times their fiber sodium concentrations. Upon transfer to
10 mM KC1 saline the fibers reaccumulated potassium and extruded sodium in equi-equivalent
amounts of 31 mEq/liter fiber water within 50 minutes (recovery period). The mean electrical
membrane potential remained constant at 40 mV during the recovery period.

Sodium extrusion is not directly responsible for the observed membrane potential since, in
individual muscles, neither membrane potentials nor membrane potential changes were correlated
with the amounts of sodium extruded. Two lines of evidence suggest that the observed potentials
are riot potassium diffusion potentials : (1) there was no correlation between potential changes and
potassium accumulated during recovery, and (2) log fiber potassium plotted against membrane po-
tential does not give the straight line relationship predicted by the Nernst equation. Results from
individual muscles also indicate that the accumulation of potassium during recovery is an active
process since: (1) the membrane potential during recovery was not large enough to cause the
net passive influx of potassium, and (2) membrane potentials were not positively correlated with
log fiber potassium.

Coupling of membrane potential to contraction in muscle. G. HOYLE.

The current tendency is to regard contraction as coupled to the membrane potential. In
crustacean muscle this hypothesis can be tested probably more satisfactorily than in any other
kind. There are two or more motor nerve fibers which effect different rates of contraction in
the same muscle and there are also inhibitory fibers which can uncouple the excitatory action at
the muscle.

The problem has been studied in conjunction with C. A. G. Wiersma. Muscles of eight spe-
cies of decapod Crustacea were studied with the aid of intracellular recording. The nerve fibers
were isolated and stimulated separately.

In many of the muscles, tension was associated with summating junction potentials which
achieved a plateau of depolarization. The tension was closely related to the height of the plateau.
Inhibitory nerve stimulation caused the level of the plateau to fall, the membrane potential re-
turning towards the resting level. These observations therefore support the hypothesis.

But in muscles in the "paradox" state a contraction occurred at a low frequency of "slow"'
fiber stimulation. The largest responses were less than 0.5 mV and they were too widely spaced
to summate. It did not seem possible that the depolarization was adequate to be the activating
agent. This was supported by the fact that in the same muscle fiber the "fast" nerve fiber did
not evoke a contraction at the same frequency of stimulation although it gave rise to large
(12 mV) depolarizations.

Thus the contraction is coupled to some membrane "occurrence" which can be evoked (by
"slow" fiber stimulation) or abolished (by inhibitory stimulation) more or less directly. The
"occurrence" cannot be equated to membrane potential change. However, an adequate lowering
of membrane potential has a similar effect.

Evidence for electrical inexcitability of neuron soma. W. H. FREYGANG, JR.

Grundfest has suggested that the portion of the neuron soma which is excited by synaptic
transmitter substances does not produce a spike in response to an electrical depolarization. The
dendrites and most of the somatic membrane of neurones in the mammalian central nervous sys-
tem, as well as the cat's anterior horn cell, are covered with synaptic endings and, therefore, may
not be electrically excited. If the intracellularly recorded action potential caused by antidromic

318 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

excitation is produced by the spike of the axonal initial segment followed by the spike of a small
area of electrically excited membrane on the soma, the impedance of most of the soma-dendritic
membrane will not be altered. Since the resistance and capacity, as well as the transient voltage
change across the soma-dendritic membrane, are known, the membrane current can be calculated.
The calculated membrane current has a time course that is very similar to the externally re-
corded current, as measured from an external micropipette with its tip practically in contact with
the soma-dendritic membrane. This finding supports the hypothesis that most of the soma-den-
dritic membrane is not electrically excited.

JULY 16, 1957

Further studies of the antimitotic and carcinostatic action of ovarian extracts. T. R.
TOSTESON, S. A. FERGUSON AND L. V. HEILBRUNN.

The earlier work of Heilbrunn and Wilson showed clearly that saline extracts of the ovaries
both of vertebrate and invertebrate animals could prevent mitosis in eggs of the worm Chae-
topterus by keeping the protoplasm fluid and preventing the mitotic gelation. We then tried to
apply this knowledge in the hope of finding a new type of carcinostatic agent. However, our
early experiments on mice inoculated with Ehrlich ascites tumors were only occasionally suc-
cessful and various attempts were made to purify the extracts so as to improve their efficacy.
Finally after many unsuccessful experiments, we were able by fractional alcoholic precipitation
in the cold to obtain preparations which markedly increased the survival time of mice inoculated
with a highly lethal ascites tumor. The control of untreated mice, when properly inoculated, all
died within 30 days. In experiments on thousands of mice we regularly were able to obtain a
30-day survival of about 20% of the mice injected intraperitoneally with the extracts. A 30-day
survival is essentially equivalent to indefinite survival, for the mice that survive for this length
of time were found on autopsy to be free from tumors. The extracts obtained by alcoholic pre-
cipitation are still highly impure. We are now able further to purify the crude extracts, and in
some of our latest experiments 30-40% survival was obtained. The potent fractions derived
from the crude extracts contain protein, lipid and carbohydrate. They are almost wholly in-
soluble in sea water, and probably for this reason their antimitotic action on Chaetopterus eggs
is slight. However, even in very dilute solution they do tend to prevent the mitotic gelation.

The action of insulin on living cells. L. V. HEILBRUNN, FRANCIS T. ASHTON,
CARL FELDHERR AND WALTER L. WILSON.

As yet, in spite of many attempts to show some effect of insulin on the enzymes extracted
from living cells, no great success has been obtained. It is possible, therefore, that the primary
action of insulin is on protoplasm and that the changes in the protoplasm affect the enzymic ac-
tivity. This is rendered all the more probable by the many-sided evidence that colloidal changes
in the protoplasm do markedly influence enzymic activity. This evidence is reviewed in a recent
book, The Dynamics of Living Protoplasm (Academic Press, 1956). In ameba, sol-gel changes
are constantly occurring, and this organism is therefore favorable for study. We used the giant
ameba Chaos chaos. The surface precipitation reaction in ameba was found to be prevented by
very dilute solutions of heparin. However if a dilute solution of insulin is added to the heparin
solutions, the surface precipitation reaction is not inhibited. Using a solution of relatively zinc-
free insulin, kindly supplied through the courtesy of G. H. A. Clowes by the Eli Lilly Company,
we were able to demonstrate a very definite antagonism between insulin and heparin. Such an
antagonism is further indicated by the fact that solutions of insulin prevent the metachromatic re-
action of heparin with toluidine blue. Moreover although normally the amebae give a strong
metachromatic reaction with toluidine blue, after they have been immersed for some time in solu-
tions of insulin they no longer give this reaction. If, as we believe, the heparin or heparin-like
substances of protoplasm act as a brake on the enzymic reactions of the cell, it is easy to under-
stand why insulin would promote these reactions. In many experiments, we have also attempted
to show an effect of insulin on the permeability of marine egg cells to glucose. As yet we have
not been able to demonstrate any significant effect.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 319

Magnetic studies on cells and protoplasm. F. T. ASHTON.

Magnetic forces can be used to study some of the properties of cells and protoplasm. Blood
cells can be moved by strong magnets. When a dilute suspension of human blood cells in a
small Petri dish was placed between the polepieces of a magnet with a field strength of 5202
gauss, within 12 hours most of the erythrocytes had migrated to a point between the polepieces.

Giant amebae (Chaos chaos) if fed paramecia with ingested iron, after digesting the proto-
plasm of the paramecia, will have vacuoles containing iron. If these are in the interior proto-
plasm, but not in the cortex, they can be moved by a magnet which gives a force of 980 dynes at
a distance of one centimeter. Measurement of the force was accomplished by determining the
distance at which the weight of small particles of iron on the pan of a microbalance was doubled.
Using Stokes' law with necessary corrections, it was possible to measure the viscosity of the
flowing protoplasm, although not as yet with any precision. A value of approximately 11 centi-
poises was obtained for the flowing protoplasm as a whole, or approximately 3 centipoises for
the granule-free protoplasm. The interior protoplasm of the ameba gives no evidence of
elasticity.

Attempts were made to shoot iron particles into the interior of sea urchin eggs by placing a
magnet under a centrifuged mass of the eggs. Usually the eggs are smashed by this procedure,
but in one case, an egg was made to contain a tiny iron rodlet. This could readily be twisted by
a magnetic field, almost as readily as the magnetic rodlets outside the egg. On removing the
magnet, both the rodlets inside and outside of the egg pointed to the magnetic north like tiny
compasses.

The metachromatic reaction in various types of protoplasm. CARL FELDHERR.

At the present time, it is commonly believed that heparin is found only in mast cells. This
opinion is due largely to earlier work which has shown that when sections of liver or lung are
stained with toluidine blue, only the mast cells show a metachromatic reaction such as is given
by heparin.

However it can readily be shown that many types of protoplasm give a metachromatic re-
action with toluidine blue. Thus a very strong reaction is given by the cortex of the giant
ameba, Chaos chaos. Likewise isolated nerve fibers of the lobster give a violet color when
stained with dilute solutions of toluidine blue. Other types of cells which ordinarily show no
metachromatic reaction may contain heparin or other metachromatic substances combined with
substances which inhibit the metachromatic reaction. Thus frog muscle fibers stain blue with
toluidine blue, but if they are previously treated with alcohol, if they are heated, if they are ex-
posed to distilled water or if they are aged, they can be seen to give a metachromatic reaction.
Similarly, the protoplasm of sea urchin eggs, ordinarily not metachromatic, becomes strongly
metachromatic when the eggs are placed in distilled water. Nematocysts of hydra after treat-
ment with alcohol also give a metachromatic reaction.

Electrophoretic mobility studies on irradiated fibrinogen. PETER RIESER.

Previous studies demonstrated that irradiation of purified bovine fibrinogen with 500 r causes
a clotting delay upon the addition of thrombin. A reduction of 30% in the liberation of fibrino-
peptide also occurs. Thus, a reduction in the generation of charged sites can be expected to
occur. In order to determine whether a charge loss has occurred, an electrophoretic mobility
curve of the irradiated and unirradiated protein was obtained. The conditions of electrophoresis
were: protein concentration 12-15 g./l., dialysis against 0.1 ionic strength buffers containing
3.33 M urea. The mobility of fibrinogen irradiated with 500 r was decreased both above and
below the isoelectric point. Since it is not likely that these results are due to an increase in the
frictional ratio, the change in mobility is probably a consequence of a decrease in net charge.
The difference in mobility may be converted into differences in net charge. A plot of Ah against
pH shows that the curve levels off at 2.5, meaning that irradiated fibrinogen has lost that many
negative charges. No leveling off appears to occur on the acidic side, but the charge difference
cannot be much larger than 10 because of the relatively small shift in the isoelectric point. The
latter is shifted in the acidic direction because the loss of cationic groups exceeds the loss of

320 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

acidic groups. The cationic groups lost are probably imidazoles which have been shown by
Mihalyi to take part in the polymerization.

Mechanisms of sol-gel transformations in the cytoplasm.'1 PAUL R. GROSS, SYLVAN
NASS AND WILLIAM PEARL.

Certain in vitro properties of a Ca++-initiated nucleoprotein aggregation reaction, obtainable
in breis of sea urchin eggs or perfused rat liver, resemble closely the biochemical characteristics
of the sol-gel transformation occurring in the cytoplasm in vivo. Fractionation experiments with
both the marine egg material and liver homogenates show that the material which aggregates
upon addition of small amounts of Ca++ is a nucleoprotein particle originating in the heteroge-
neous "microsome" fraction. A second fraction, ordinarily found among the soluble macro-
molecules in differential centrifugation, also precipitates under the influence of Ca++, but this re-
action differs kinetically and with respect to mechanism from that involving the microsomal
nucleoprotein particles. The soluble phase particle, also a nucleoprotein, precipitates instantane-
ously with Ca++, even at levels of 0.001 M Ca++ and less, but the reaction shows a primary kinetic
salt effect, ceasing altogether at total ionic strengths lower than those probably obtaining in the
living cell. Electron microscopic evidence is presented which suggests that the sensitive micro-
somal particle is identical with the "dense RNA particles" described by Palade and others in the
cytoplasm of many cell types. A discussion of mitotic spindle structure is given, with micro-
graphs which show that in the mitotic apparatus of the sea urchin egg, fixed by conventional
buffered OsOi or with cold 30% ethanol and studied in ultrathin sections, the "Palade particles"
comprise a major structural component.

JULY 23, 1957
Uricase inactivation by urea.2 AURIN M. CHASE.

The inhibiting effect of urea on the oxidation of uric acid catalyzed by uricase (Worthing-
ton's semipurified preparation) was studied spectrophotometrically (\ = 300 m/u) in 0.1 M, pH 9
glycine buffer at 26° C.

Uricase undergoes an immediate, completely reversible inactivation by urea, similar to that
reported by Osborne and Chase for Cypridina luciferase (J. Cell. Comp. Physiol., 1954) and by
Chase and Krotkov for yeast invertase (same journal, 1956). Whereas, however, luciferase is
completely inactivated by 1.5 M urea and invertase by about 3 M, the inactivation of uricase re-
quires much higher urea concentrations and varies considerably according to the experimental
conditions. For example, quite different results are obtained when borate buffer is used than
with phosphate or glycine buffer.

In 0.1 M glycine buffer of pH 9 at 26°, no significant inactivation of uricase by urea occurs
at concentrations lower than 3 M. Activity becomes increasingly less, however, as the urea con-
centration is raised above this value, and it is abolished at a concentration of about 8 M.

In addition to the immediate, reversible inactivation of uricase, the enzyme also undergoes an
irreversible loss of activity when exposed to urea concentrations greater than about 4 M. This
irreversible process apparently involves a primary, relatively rapid reaction and a secondary,
slow one. The former is very dependent upon the urea concentration, its rate increasing about
one hundred-fold between 4.5 and 7.5 M concentrations of urea.

The irreversible loss of activity of uricase during exposure to urea greatly resembles the
situation observed by Simpson and Kauzmann for the effect of urea on the change in optical ro-
tation of ovalbumin solutions (J. Amer. Chem. Soc., 1953), and the underlying mechanisms may
well be similar.

Clotting of blood: A study in the polymerization of proteins. L. LORAND.

When fibrinogen is clotted by thrombin in the presence of calcium ions and the fibrin-stabi-
lizing factor (FSF) of plasma, a clot is obtained which resembles the "plasma clot" in every re-

1 Supported by a grant from the American Cancer Society.

2 Aided in part by a grant from the National Science Foundation.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 321

spect. It cannot be dissolved in urea or monochloroacetic acid, in contrast to the soluble and
mechanically weaker clot formed in the absence of the factor. Thus, the insoluble clot is re-
garded as a covalently cross-linked network, whereas the soluble kind is thought to be held to-
gether by secondary forces only (Lorand, 1954).

The fibrin-stabilizing factor has now been prepared from bovine plasma in a highly purified
(> 300-fold,) form, as one of the globulin fractions giving virtually a single sedimenting boundary
in the ultracentrifuge. The activity of the purified factor is protected by cysteine, but the latter
cannot substitute for it. Fibrin-stabilizing factor contains titratable sulfhydryl groups, and all
the evidence indicates that these may be essential for biological activity.

The insoluble clot, which in the case of the human and bovine species is probably the only
naturally occuring one, was shown to be a co-polymer of fibrin and the fibrin-stabilizing factor in
definite stoichiometric proportions. The co-polymerization of two different proteins, as illustrated
by the above example, should be considered as a possible pattern for the biogenesis of other
protein fibers.

This work was aided by a grant from the National Institutes of Health.

JULY 30, 1957
Effect of plant hormones on sea weeds. LUIGI PROVASOLI.

Bacteria-free cultures of Ulva lactuca were obtained from small pieces of thallus treated with
an antibiotic mixture. In a sea water medium enriched with vitamins and organic compounds,
the zoospores formed filaments which never developed in the typical foliaceous thallus.

The filaments, after reaching various lengths, stop grownig, bleach almost completely, leav-
ing a few intensely green dots, sparsely arranged. Upon transfer to a fresh medium, new fila-
ments arise from these islands of pigmented cells.

Adenine, kinetin, indolacetic acid, and gibberellin, which affect morphogenesis in land plants,
were then tried in the hope of obtaining the development of the typical thallus. This goal was
only partially reached but preliminary experiments show a clear effect of the plant hormones on
the germlings of Uh'a.

Adenine, kinetin, and indolacetic acid favor the initiation of more filaments and affect the
length of the new germlings. Adenine and indolacetic acid seem antagonistic at certain con-
centrations. Gibberellin dramatically promotes the elongation of filaments.

The response of a relatively simply organized sea weed to plant hormones promises that these
morphogenetic determinants will be important factors for complex sea weeds. This response
links even more tightly the green algae to the higher plants.

The variety of evolutionary steps towards increased morphological complexity in the algae
offers the opportunity to choose appropriate organisms which, because of their relative morpho-
logical simplicity, may permit a better understanding of the mode of action of the plant hormones.

Sequence of changes in nucleic acids in synchronised cultures of Escherichia coli.
DWIGHT McNAiR SCOTT.

Synchronization of division in cultures of E. coli in synthetic medium was achieved by
chilling the cells at 6° for 45 minutes and returning them to 37°. The past history of the cul-
ture determined the possibility and time of synchronous division. Growing cultures, containing
glucose, divided almost immediately after return to 37°. Growing cells, chilled in medium with-
out glucose, divided about 60 minutes after glucose was added. Cultures which had exhausted
glucose divided synchronously at 120 minutes, but only if they had been depleted at least six
hours. During depletion, turbidity and DNA (by diphenylamine) remained constant, the count
approximately doubled and RNA (by orcinol) decreased.

Methods of extraction of nucleic acids were investigated. Treatment of acid-washed cells
with 5% perchloric acid at 4° for 17 hours extracted one half the RNA and left one half RNA
and all the DNA in the residue (core). A variable proportion of DNA was made unreactive
to diphenylamine by acid. Just before division the effect was slight but at midcycle approxi-
mately one half the whole cell DNA was affected. Acid treatment increased the color developed
with orcinol (RNA), not exactly equivalent to the DNA decrease. These findings may indicate
an unstable intermediate in DNA svnthesis.

322 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

Frequent samples of synchronized cultures were analyzed to determine the time and extent of
changes in nucleic acid components after glucose addition. Of interest are the following observa-
tions: 1) Increase at a logarithmic rate of bases as measured by absorption in the UV. 2) An
immediate increase, up to 100% in 10 minutes in core RNA, then variations around a plateau
and another rise before division. 3) A delayed increase in extractable RNA. 4) A variable
time and percentage of rise in DNA. 5) Certain reciprocal relationships between core RNA
and bases, core RNA and DNA, and core and extractable RNAs.

Sonic observations on the ribonudease system in rat liver. JAY S. ROTH.

The supernatant fraction of rat liver, obtained by centrifugation of a 1:10 homogenate of rat
liver in water at 60,000 G for 90 minutes, contains a ribonuclease (RNase) inhibitor and an in-
active RNase (i-RNase). These substances show the characteristics of proteins and have been
purified and separated by the use of salt precipitation, calcium phosphate gel absorption and heat
treatment. The RNase inhibitor is absorbed by calcium phosphate gel under optimum condi-
tions ; i-RNase is not, allowing 95-100% separation. The properties of the purified inhibitor and
i-RNase have been investigated. RNase inhibitor is easily inactivated by heating, dilution with
water, dilute acid and sulfhydryl reactants such as p-chloromercuribenzoate (CMB) and Pb++.
The inactivation by Pb++, but not CMB, is reversible with hydrogen sulfide. Inactive RNase is
stable to heating at pH 5 and 65° C. for 5 minutes, and is activated by this treatment as well as
by treatment with hydrogen ion, pH 2 for 10-20 minutes at 25° C. Activation of i-RNase may
also be accomplished by sulfhydryl reactants, the order of decreasing effectiveness being Pb++,
CMB, phenyl arsine oxide, iodoacetamide. The activation of i-RNase by sulfhydryl reactants is
easily reversible with hydrogen sulfide. RNase inhibitor reacts competitively with yeast RNA
and RNase. A new assay system has been developed which allows accurate simultaneous deter-
mination of i-RNase and RNase inhibitor. This is essential for purification studies where activa-
tion or inactivation of either component may occur during the purification process. The data
suggest that RNase inhibitor is monovalent, that is, combines with one molecule of RNase.
Thus, i-RNase is probably a complex of RNase inhibitor and alkaline RNase. (Aided by grants
from the National Institutes of Health, Damon Runyon Memorial Fund and the American Cancer
Society.)

AUGUST 6, 1957
Thymidine incorporation into the macronucleus of Euplotes.1 JOSEPH G. GALL.

The hypotrich ciliate Euplotcs has a single very large U-shaped macronucleus and a much
smaller spherical micronucleus. Prior to macronuclear division a so-called reorganization band
appears at the tip of each arm of the U and works its way slowly to the middle of the elongated
nucleus. As seen in Feulgen-stained preparations these bands are composed of a lightly staining
zone distal to a darker transverse line. In parts of the nucleus through which the reorganiza-
tion band has passed, the Feulgen reaction shows an increased concentration of desoxyribose
nucleic acid (DMA). To check the hypothesis that the reorganization band might reflect a
progressive wave of DNA synthesis, rapidly dividing individuals were placed in a solution con-
taining 5.5 /iC/ml. of tritium-labeled thymidine. Thymidine is known to be a specific precursor
of DNA and the tritium permits high autoradiographic resolution. After 8 hours growth in the
label, the animals were squashed on glass microscope slides, stained by the Feulgen reaction, and
covered with autoradiographic stripping film. After an appropriate exposure period the films
were developed and made into permanent preparations. Radioactivity was found solely in those
parts of the macronucleus through which a reorganization band had passed ; thus there is a radio-
active area distal to each reorganization band and a single non-radioactive zone between the
approaching bands. The incorporation of thymidine into only those areas showing increased
Feulgen staining suggests that the reorganization band represents the leading edge of a wave of
DNA synthesis. Since reorganization bands are found in other Protozoa, the phenomenon de-
scribed here is probably of general significance.

1 Supported by funds from the National Science Foundation and the Graduate School of the
University of Minnesota.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 323

Interactions between chromosomes and cytoplasm during early embryonic develop-
ment in Sciara (Diptera). C. W. METZ.

As indicated in earlier papers, Sciara exhibits an extraordinary series of phenomena which
reflect apparently clear cut interactions between cytoplasm and chromosomes. One sequence in-
volves specific chromosomal (genie) influences on the cytoplasm of the growing ovarian oocyte,
such that later this cytoplasm acts selectively on particular individual chromosomes, inhibiting
their mitotic activity and causing their elimination at specific times during cleavage stages in
the fertilized egg. The work in question was done in our laboratory, largely by Anne Marie
DuBois, M. Louise Schmuck (Mrs. Philip Armstrong), R. O. Berry and the writer. The basic
genetic and cytological data are essentially complete. Since the problem is important and the
material may be exceptionally favorable, present attention is focused on interpretations and on
further extension of experiments to ascertain more about the underlying nature of the respective
"influences."

In the fertilized egg of Sciara coprophila Lintner, the prospective soma includes all but the
germinal "pole-plasm" at the posterior end. Somatic chromosome eliminations ordinarily occur
in a peripheral clear zone of cytoplasm. The initial interpretation of the cause of these elimina-
tions was based on Boveri's classical findings in Ascaris (pre-localization theory) by assuming
a pre-localization of elimination-inducing materials in the clear zone in Sciara. However, more
recent unpublished evidence of the writer indicates that elimination can occur normally in cen-
tral areas of the egg and that all somatic eliminations may be under control of progressive physio-
logical changes taking place uniformly throughout the cytoplasm. The distinction between the
interpretations is important in relation to further work and our problem is to devise experiments
which will eliminate one alternative or the other. The seminar report was designed to stimulate
discussion of techniques and further procedures. Chromosome elimination in the germ line, by
an entirely different process, was also considered.

AUGUST 13, 1957

Uptake of P3- in bcnthic algae in relation to primary productivity. EUGENE P.
ODUM, EDWARD J. KUENZLER AND SISTER MARION XAVIER BLUNT, SNJM.

The rate of uptake of P32, net productivity, respiration and gross productivity of large inter-
tidal benthic algae were measured simultaneously in light and dark bottles suspended in a run-
ning sea water aquarium under controlled light and temperature. One microcurie of P32 and ap-
proximately one gram (dry weight) of alga were placed in each 500-ml. bottle of sea water under
450 ft. candles illumination at 21° C. for 3.5 hours. Uptake of P32 was measured by determining
activity of the medium at intervals during the experiment, while productivity and respiration were
determined from the initial and final oxygen concentration. Per cent uptake of P32 per hour per
gram dry weight was similar in the light and dark for a given species, but markedly different
between species. Rate of uptake was lowest (6-10%) in Fucus vesiculosis which also had the
lowest gross production (0.8 ml. O2 per gram per hour) and respiration rates and the lowest
surface-to-volume ratio (approx. 22 cm2 per cm3). Uptake was highest in Ceramium rubrum
and Ulva lactuca (32-52%) which had much larger surface-to-volume ratios and were four times
as productive. Gross production/respiration (P/R) ratios were between 3 and 4 for all species
under conditions of the experiment. It was evident that where environmental concentration of
phosphorus is low, as is usual in sea water, tracer amounts of P32 are taken up at a rate charac-
teristic for a given species and independently of light. Ability of a species to absorb the tracer
appears to be related to structural features of the alga, high surface-to-volume ratio increasing
the rate of uptake as well as increasing the ability of the plant to fix and utilize energy under
favorable light conditions.

AUGUST 20, 1957

Biochemical studies of relaxation in glycerinated muscle. L. LORAND, J. MOLNAR
AND C. Moos.

Rabbit psoas fibers kept at 0° C. in 50% glycerol for two days and extracted further in
20% glycerol at 20° C. for 4-8 hours, develop maximal isometric tension on addition of 4 mM

324 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

adenosinetriphosphate, and no spontaneous relaxation follows. Relaxation occurs, however, on
adding 10 mM phosphoenolpyruvate (PEP) to the fibers, the diameter of which may be less than
80 fj. ("single fibers"). Such relaxation is enhanced by K+ similarly to the unique activation of
pyruvate phosphokinase. Since the fibers contain this enzyme, PEP is believed to cause relaxa-
tion through transphosphorylating to adenosinediphosphate. But transphosphorylation alone is
not sufficient, for fibers extracted in 20% glycerol for about 24 hours cannot be relaxed simply
by adding PEP, in spite of the fact that the pyruvate phosphokinase is still present. Muscle
contains an additional relaxation factor which is purified as follows. Minced rabbit muscle is
homogenized in the cold with three volumes of KC1 (0.15 M) , and centrifuged at 1000 G. for
20 minutes. The supernatant is centrifuged at 1200 G. for 20 minutes to remove the mitochon-
dria which have no relaxing activity. The "microsomes" are sedimented by a subsequent run at
41000 G. lasting one hour; the sediment is washed with KC1 and finally suspended in KC1. On
long extracted fibers, neither the transphosphorylase system nor "microsomes" give rise to re-
laxation, but jointly the two are very effective. The "microsomal" principle is thermolabile, is
unaffected by ribonuclease, but is inactivated by trypsin, urea or desoxycholic acid. Since the
latter does not affect the adenosinetriphosphatase of the particles, this enzyme cannot be identi-
cal with the relaxing factor. Apart from this, our findings are in agreement with those of
Ebashi and Kumagai who studied isotonic relaxation with creatine phosphokinase and myokinase.

All relaxation studies to date (including the prevention of unloaded shortening of myofibrils
by Portzehl) were carried out in the presence of a transphosphorylating system. The interac-
tion of "microsomes" and the transphosphorylating system might give rise to a product which
relaxes the fiber. As found for liver microsomes (Kenney, Colowick and Barbehenn), inorganic
pyrophosphate, which is known to cause relaxation, might conceivably be produced under these
conditions.

This work was aided by a grant from the Muscular Dystrophy Associations of America, Inc.

The dependence of creatine phosphate and adenosine triphosphate breakdozvn on
work in iodoacetate poisoned muscles. FRANCIS D. CARLSON AND ALVIN SIGER.

Net creatine phosphate (CrP) and adenosine triphosphate (ATP) breakdown were deter-
mined, using the analytical methods of Ennor, and Strehler and McElroy, respectively, in an-
aerobic, iodoacetate-poisoned frog sartorius muscle at 0° C. (no rigor) as a function of the work
done in a series of isotonic twitches under a 5-gm. load. Creatine phosphate breakdown varied
linearly with work and no net adenosine triphosphate breakdown occurred until the creatine
phosphate concentration dropped by 60% or more. Under a 5-gm. load .350 micromole of crea-
tine phosphate was split for each millicalorie of work done, and the work done in a single
maximal isotonic twitch was .821 millicalories per gm. of muscle. These data give a value of
.287 micromole of creatine phosphate split per gram of muscle in a single isotonic twitch. On
the basis of the heat studies of Hill it is possible to estimate the total energy output (heat plus
work) for a single maximal isotonic twitch under a 5-gm. load. The value obtained is 3.54
mcal./gm. Since the work done is degraded to heat during relaxation the total energy released
in a twitch appears as heat and so an estimate of the heat of hydrolysis of creatine phosphate in
muscle at 0° C, pH 7.1, can be obtained. A value of 12,300 cal./mole results. Using a myosin
content of 12%, a molecular weight of 440,000 for myosin, and the figure of .287 micromole of
creatine phosphate split per gram in a single twitch, it is calculated that 1.05 creatine phosphate
molecules are split per myosin molecule in a single maximal isotonic twitch with a 5-gm. load.

GENERAL SCIENTIFIC MEETINGS
AUGUST 26-29, 1957

Abstracts in this section (including those of Lalor Fellowship Reports) are ar-
ranged alphabetically by authors under the headings "Papers Read," "Papers Read
by Title," and "Lalor Fellowship Reports." Author and subject references will also
be found in the regular volume index.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 325

PAPERS READ

The influence of the branchial nerve and of 5-hydroxytryptaniine on the ciliary ac-
tivity of Mytihis gill. EDWARD AIELLO.

When the gill of Mytilus is excised, ciliary activity on the lateral epithelium of the gill fila-
ments declines gradually for about one hour, at most, and then stops. A similar result is ob-
tained by cutting the branchial nerve in situ, just distal to its origin at the visceral ganglion.
Ciliary activity on the gill of the opposite, uncut side continues unabated for many hours. Cilia
made quiescent in this way can be stimulated to renewed activity by crushing a small section of
the gill filament. This resumption of activity spreads for about one mm. in all directions and
lasts about five minutes. A hot water extract of gill tissue also activates quiescent lateral cilia
and accelerates the beating of cilia that are already active. Veratrine sulfate 10~6 M and 5-
hydroxytryptamine 10"7 M have similar stimulating effects. Gill tissue extracts are presently
being assayed for 5-HT. These experiments suggest that lateral ciliary activity might be de-
pendent on the release of some activating substance by tonic discharge of the branchial nerve.

Supported in part by a Predoctoral Fellowship from the National Institutes of Health and
a grant to Dr. T. Hayashi from the Muscular Dystrophy Associations of America.

Motility in developing teleost embryos.^ PHILIP B. ARMSTRONG.

The development of aquatic lomotion in Amciurus nebnlosus closely parallels that described
for Amblystoma by Coghill. The earliest contractions of the skeletal muscle occur in the an-
terior segments of the body which is on the dorsum of the yolk and produces passive movements
of the tail as a whole. Finally all of the muscle segments participate, a wave of contraction
passes down one side of the embryo, a coil is formed as the tip of the tail passes over the dorsum
of the embryo. There is no set and continuous regularity of alternation of these early contrac-
tions. None of the above movements are propulsive. They are seen prior to hatching.

At the time of hatching, propulsive contractions appear abruptly in which a wave of con-
traction extending down one side of the animal is so quickly followed by a wave down the op-
posite side that a coil cannot form. A rapid succession of alternate contraction waves produces
forward motion.

Fwidulus heteroclitus and Opsanits tail show a similar development of motility with some
differences. Ameiurus is much the most active embryo showing considerable spontaneous ac-
tivity, Fundulus considerably less. Opsanus is relatively lethargic.

Patterns of response and neural organisation of supramedullary neurons of puffer
(bloTvfish'} , Spheroides maculatus. M. V. L. BENNETT, S. M. GRAIN AND H.
GRUNDFEST.

The supramedullary neurons of puffer respond in unison to stimulation of spinal cord, cranial
nerves, dorsal (but not ventral) roots. This is shown by simultaneous intracellular recordings
from random pairs of cells (see abstract by Grain, Bennett and Grundfest, this issue). With
threshold stimuli or with stronger stimulation that evokes complex responses of many spikes at
variable intervals, all cells fire nearly in synchrony. Direct intracellular excitation or stimula-
tion of a cell with closely applied extracellular electrodes does not generally activate other cells.
Irregularly in some cells, however, a direct spike is followed by a small potential. When this
potential occurs a spike also appears in each of the other cells. These results are inconsistent
with spread of excitation ephaptically or through protoplasmic bridges, and suggest synaptic
mechanisms.

Cord section immediately above or below the cell cluster does not abolish synchronization.
Division of the cluster by section involving the dorsal half of the cord eliminates synchrony be-
tween the caudal and rostral halves, but not within each half. Although responsiveness to stimuli
distal to the section is lowered, both halves respond to rostral and caudal stimulations.

Activity of the cluster is followed by efferent impulses in cranial nerves and dorsal (but not

1 This investigation was supported in part by NIH grant B-643.

326 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

ventral) roots. By tests of threshold, conduction velocity, and collisional extinction the efferent
fibers belong in the same group as the afferents exciting the cluster. Since indirect stimulation is
never observed to excite an individual cell, either the efferent impulses are transmitted across a
synapse, or antidromic invasion of the cells is impossible.

No obvious functional loss follows chronic removal of the cluster.

While functional and anatomical relations of the supramedullary cells remain unknown, the
electrophysiological data demonstrate that they cannot be sensory, as presumed by anatomists.

A morphological color change controlled by molting hormone in Lepidoptera.
DETLEF BUCKMANN.

A morphological color change preceding metamorphosis has been investigated in Cerura
v'mula. The larvae are green. When they stop feeding to spin their cocoon, they turn dark red
because a red pigment is formed in the epidermal cells. After this, red pigment is formed in the
fat body and the gut, too. The processes of pupal molt in the epidermis begin only 5 days after
color change. Ligaturing experiments show that also only at this time the molting hormone is
distributed in sufficient amount to cause pupation. The pupal molt is completed only 10 days
after color change.

The color change can be prohibited by a ligature. Only the part of the body anterior to the
ligature will redden. Evidently a factor causing color change is formed in the thorax, its dis-
tribution being prevented by the ligature. Extracts of the molting hormone Ecdyson, kindly
provided by Dr. Karlson, Munchen, injected into green abdomina of ligatured larvae in large
dose (3000 Calliphora units) caused pupation without color change. Smaller doses caused red-
dening of fat body only. Very small doses (50 units) caused reddening of the epidermis only.
So the normal course of events may be brought about by slowly increasing hormone concen-
tration.

Thus it has been shown that Ecdyson not only causes molting in the epidermis. In small
concentration it has quite a different effect. It acts on some metabolic processes leading to the
formation of red pigments in epidermis, fat body and gut. The nature of the pigments is being
investigated. They are ommochromes. The way ommochromes are formed in insects is well
known. They are a product of trytophane metabolism. Ommochrome production preceding
pupation has been found in other species, too. Presumably it is a characteristic of changes in
metabolism preparing metamorphosis. Recent experiments tend to show that juvenile hormone
also acts on ommochrome production.

Further studies in experimental hypothermia. C. LLOYD CLAFF, FREDERICK N.
SUDAK AND MARVIN H. CANTOR.

Twelve-hour fasted male white rats injected (intramuscular) with Thorazine (25 mg./kg.)
responded, in a cold environment of 6.5-7.5° C., with a fall in body temperature of 5° C. and a
rise of 91% in metabolic activity as measured by CO2 production. Animals treated with 2,4 di-
chlorophenoxyacetic acid (200 mg./kg., subcutaneously) responded to cold with a drop in rectal
temperature of 3.0° C. but only a 54% increase in CO2 output. Rats treated with Thorazine and
2,4-D in combination responded under the same conditions with a decrease in rectal temperature
of 7.0° C. while CO2 production increased only 15%.

At room temperature, rats treated with Nembutal (30 mg./kg., intraperitoneal) showed a
decrease in rectal temperature as well as a lower CO2 output. However, the peak metabolism
of these animals, brought about by placing them in a cold environment (6.5-7.5° C.), was 102%
above basal and body temperature fell 2° C. The peak CO2 production and rectal temperature
response of animals injected with Thorazine and Nembutal in combination were the same as those
receiving either injection separately. Animals treated with a combination of Nembutal (30
mg./kg.) and 2,4-D (200 mg./kg.) responded to cold with a 10° C. drop in body temperature
and no change in metabolic activity.

Studies made in an isothermic environment of 30.0° C. showed that the action of Thorazine
and Nembutal causes a slight decrease in CO2 production and no significant change in rectal tem-
perature. On the other hand, animals treated with 2,4-D increase their metabolism 33%% while
the temperature response was the same as a control animal. It was noted that the metabolism of

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 327

2,4-D treated animals was highly sensitive to any change in ambient temperature for a period of
seven hours after injection.

Tissue transplantation in Pecten irradians.1 JOHN E. GUSHING.

This work is part of a study initiated on sipunculids (Triplett, Gushing and Durall, unpub-
lished data) concerned with the responses of invertebrates to transplants. The purpose is to
learn more as to whether or not invertebrates synthesize antibodies. Pecten was selected be-
cause, while little work has been done on transplantation in mollusks, Butcher (1930) grafted
eyes to gonads in this genus. As he gave few details, efforts were made to confirm and extend
his observations. Transplantations were performed by removing mantle strips of a few mm.,
splitting the integument of the female portion of the gonad with the tip of a syringe needle, and
tucking the strip ends under the edges of the cut, leaving the center portion exposed. Half the
grafts so made took after 48 hours, whereas none took if completely buried, placed in the male
portion of gonad, or under the mantle. Explants died in 48 hours, but established autografts have
retained eyes, tentacles and contractility for over a month. A comparative series of auto and
homo grafts survived as follows : start A 31, H 31 : two days A 17, H 14: three days A 15, H 12 :
five days A 12, H 11 : eleven days A 10, H 7 : fourteen days A 9, H 5. This small series does
not permit conclusions to be drawn, but does show the potential value of Pecten for transplanta-
tion studies. Of further value is the fact that grafts can be placed so that they deteriorate in a
few days, permitting second graft series to be made.

Uptake of a radiomercury labeled diuretic (chlormerodrin) by the nephridia of
Phascolosoma gouldi. ROGER L. GREIF.

The injection of chlormerodrin labeled with Hg203 into the coelomic fluid of Phascolosoma
gouldi results in radiomercury accumulation in the nephridia in much higher concentration than
in other tissues. This accumulation occurs when animals are kept in running sea water at 22° C.,
but if the worms are cooled to 5° C. for 12 hours prior to injection and are maintained thereafter
at the lower temperature, the concentration of radiomercury in the nephridia will remain low.
Upon transfer of the injected cooled animals to the higher temperature, accumulation of Hg203 in
the nephridia begins. Diffusion of chlormerodrin within the coelomic cavity at 5° C. is not limit-
ing. Dimercaprol (BAL) appears to decrease the affinity of the nephridia for mercury at 22° C.
The relation of these findings to mercurial diuresis will be discussed.

Electron microscope observations on the cytoplasm of sea urchin eggs.2 PAUL R.
GROSS, DELBERT E. PHILPOTT AND SYLVAN NASS.

Fertilized, unfertilized, centrifuged, uncentrifuged, "normal,'' and injured eggs of Arbacia
punctnlata have been examined in thin sections with the electron microscope. Of several fixa-
tives employed, the best for general purposes was found to be 1% OsOi in a veronal -buffered
isotonic balanced salt solution, pH 7.4. Some elements of the mitotic apparatus are more easily
studied in cold alcohol-fixed specimens, however. Among the structures studied were: pigment
vacuoles, yolk particles, cytoplasmic vesicles of varying diameter, oil globules, mitochondria,
double membrane systems in the cytoplasm, nuclear and outer limiting membranes, cortical
granules, and small, dense particles such as described by Palade in mammalian tissue cells.
These last are of average diameter 170 A, with variation between 140 and 200 A. The mito-
chondria show double outer membranes and "cristae," with osmiophilic layers 60 A thick and
total width of 150 A. Pigment and yolk granules are surrounded by 60 A membranes, but the
yolk particles were occasionally seen surrounded by a double membrane of 150 A thickness with
osmiophilic outer components of 60 A. The limiting membrane of the cell is peripheral to the
cortical granules and about 100 A thick, while the nuclear membrane is composed of a pair of

1 Supported in part by the Penrose Fund, American Philosophical Society, and the Office of
Naval Research.

2 Supported by grants from the American Cancer Society, the National Science Foundation,
and the Graduate School of New York University.

328 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

87 A dense layers separated by a distance of 100 A, although this latter dimension varies greatly
to produce annuli, seen in tangential sections. In the centrifuged eggs, the classical descriptions
of particle distribution hold, except that mitochondria are always found trapped in the centripetal
oil layer as well as in the layer immediately centripetal to the yolk.

The effect of ions on the response of smooth muscle to cooling. RITA GUTTMAN
AND SAMUEL Ross.

The anterior byssus retractor of Mytilus cditlis possesses both phasic and tonic systems. The
slow tonic responses to rapid cooling were investigated by simultaneously recording tension and
resting potential changes after ruling out the nervous elements by soaking the muscle in 1U~* M
Banthine. Similar results were obtained with Phascolosoma and Thy one.

In these muscles cooling is not an effective stimulus unless the tissue is treated with sub-
threshold concentrations of potassium. The quantitative relations between the amount of cooling
and the amount of associated depolarization necessary for contraction at various concentrations
of potentiating potassium were established. The results can be expressed in a family of curves
(one curve for each potassium concentration). The plateaus of the curves for sea water and
potassium-free sea water were beneath the depolarization value necessary for contraction so that
it is clear that no amount of cooling with sea water alone or with potassium-free sea water would
ever be effective.

The effects of high and low sodium and of high and low calcium were also investigated.
When the muscle is treated with subthreshold amounts of potassium and rapidly cooled in vari-
ous concentrations of sodium ion and calcium ion, respectively, the sodium and calcium have no
effect whatsoever upon the response. Acetylcholine, in subthreshold amounts, has a potentiating
effect but, unlike potassium and cooling, acts through the nervous apparatus.

These results suggest that this muscle will respond to cooling with tonic contraction when-
ever a critical threshold amount of depolarization is achieved. Cooling alone cannot trigger the
contraction since it cannot bring about sufficient depolarization. Cooling can result in contrac-
tion, however, if used in conjunction with some other subthreshold depolarizing agent. It is
concluded that cooling affects the contractile mechanism indirectly by first causing membrane
breakdown and depolarization.

Schooling behavior in mud snails in Barnstable Harbor leading to the formation of
massive aggregations at the completion of seasonal reproduction.1 CHARLES E.
JENNER.

During the present summer, as in the preceding one (Jenner, 1956), snails on an extensive
sand-mud flat in Barnstable Harbor, Massachusetts, underwent a striking change in distribution
pattern — from a dispersed distribution, in which the snails were present over extensive areas of
the flat, to an aggregated distribution, with snails occurring in massive aggregations. In both
years the change took place at the same stage of reproductive activity within the snail popula-
tions. The timing of the termination of reproductive activity can be followed with great precision
by making adequate samples and recording for females the per cent of those containing a formed
egg case and for males the per cent having a fully developed copulatory organ (Jenner, 1956).
The change in distribution pattern occurred during the first half of the transition period between
the active reproductive phase (females with egg cases, males with developed copulatory organs)
and the post-reproductive phase (females without egg cases, males with resorbed copulatory or-
gans). It would appear that this change from dispersed to aggregated distribution is a regular
aspect of the seasonal activity of these snails and is related to the state of reproductive activity
within the population.

No concerted effort has been made to follow the history of the aggregations once they have
been established, but it is now clear that the snails within these groups display schooling behavior,
snails in any one part of the aggregation often moving in mass in the same direction. Such oc-
currences, often involving thousands of snails, present a dramatic sight. While such factors as

1 Aided by grants from the National Institutes of Health, U. S. Public Health Service
(E-356) and from the University Research Fund, University of North Carolina.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 329

currents, presence of food, and other physical and chemical stimuli are of importance in orienta-
tion in these snails, their schooling behavior cannot be accounted for solely in these terms.
Clearly, social factors are involved.

Protoplasmic bridges between follicle cells and developing oocytes of Fundulus
licteroclitus. NORMAN E. KEMP AND EMERSON HIBBARD.

Continued work with the electron microscope permits us to amend the report presented by
Kemp and Allen at the General Scientific Meetings of the Marine Biological Laboratory in 1956.
We can now assert that in Fundulus hctcroditiis there are direct protoplasmic connections be-
tween oocyte and follicular cells. What we formerly called microvilli within the zona radiata
and subfollicular space are in reality not microvilli which simply adjoin the branching processes
of follicular epithelial cells. Instead, these processes within the zona radiata are regional parts
of intercellular bridges which are continuous from oocyte to follicle cells. The bridges form
when follicle cells first separate from the surface of the oocyte, and they lengthen with increasing
thickness of the zona radiata and subfollicular space. In view of this clarification of the nature
of the protoplasmic bridges, we must reconsider the concept (Kemp and Allen, 1956) that the
zona radiata is a product of the oocyte and that the chorion internum is therefore anatomically
a vitelline membrane. It is possible that the materials for the construction of the zona radiata
come at first from the follicle cells as they pull away from the oocyte. The zona radiata ap-
pears to thicken by apposition of fibrous matrix internally, i.e., on the side next to the oocyte.
It may be, however, that the materials for the matrix are transported inward from the follicle
cells, by way of the protoplasmic bridges, rather than outward from the cortex of the oocyte. If
the follicle cells are the primary source of the proteins used in constructing the zona radiata,
then the chorion internum is not anatomically a vitelline membrane.

An effect of calcium-deficient Ringer on intact frog muscle. R. P. KERNAN AND
A. CSAPO.

When frog toe muscle is soaked for about 10-15 minutes in Ca-deficient Ringer solution
(0.18 mM/1.) and is then stimulated (in a 60 c/s, longitudinal a.c. field, for % second), the tetanus
tension does not fall immediately when the stimulus is withdrawn but continues for several sec-
onds. It is of interest to determine whether prolonged relaxation is accompanied by prolonged
membrane activity or is due to a delayed cessation of the active state. Isometric tension and
electrical activity of the membrane were recorded simultaneously on a dual beam oscilloscope so
that the duration and amplitude of tension and action potentials could be compared directly.
During electrical recording the muscle was removed from the bath and was stimulated through
contact electrodes, placed at one end of the preparation. The pick-up electrodes were placed
about 4 mm. apart at the other end of the muscle and were connected via a preamplifier (gain
X 60) to the oscilloscope. The conditions in normal and Ca-deficient muscles were compared.
Action potential of 13-17 mV accompanied tension in the normal muscle during stimulation. The
electrical activity ceased at the end of the stimulus before the tension declined. When the muscle
was stimulated after 10 to 15 minutes of soaking in Ca-deficient Ringer, the cessation of stimula-
tion was followed by prolonged relaxation and also by action potentials of 4-7 mV declining
slowly with tension decrement. Prolonged relaxation can be explained, therefore, by continued
membrane activity without assuming a genuine prolongation of the active state.

Potassium contracture in frog twitch muscles. R. P. KERNAN AND A. CSAPO.

Tension and its rate of development were measured in the sartorius and toe muscles after
immersion in high potassium Ringer solution. Maximum tetanus tension, elicited by a longi-
tudinal electric field (Vz sec., 60 c/s, a.c.), was used as a standard of comparison. Barkan-
Boyle-Locke fluid in which the K level was varied from 24 to 120 m.eq./l. (substituted for Na)
was introduced suddenly into the muscle bath to produce contracture. When contracture was
completed the modified Ringer was replaced by normal Ringer. The contractures produced
showed two phases, a rapid and a slow one. The initial fast phase could be abolished by curare

330 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

or by pretreatment with 12 mM/1. K indicating that this phase is associated with propagated re-
sponse and end plate activity. The second phase, which was smaller than the first, was un-
affected by this treatment but its amplitude was found to be directly proportional to the external
[K]. This suggests that diffusion through the interspace is a limiting factor in the K contrac-
ture. This point is further supported by the fact that for a given [K], smaller muscles developed
relatively greater tension. In order to eliminate the effect of diffusion, toe muscles were reduced
to small fiber bundles (approximately 15 fibers) and these were immersed in 96 to 120 mM/1. K
Ringer. Contracture tensions then became almost equal to that of a normal tetanus. In small
fiber bundles, tension and its rate of rise were directly proportional to the log of the external
[K], suggesting that tension in K contracture is a function of the degree of depolarization of the
fiber membrane.

Adaptation to salinity and temperature in a euryJialine hydroid. OTTO KINNE.

The brackish water hydroid Cordylophora caspia (Pallas) is able to endure salinities from
fresh water to nearly pure sea water (salinity optimum 15%c-17#f) and temperatures from about
4 to 25° C. In extremely suboptimal salinities (e.g., fresh water) and in extremely supraoptimal
salinities (e.g., 30%e) the form of the colonies and especially the form of the hydranths alter
considerably. Colonies with identical genotype consist in fresh water mainly of stolons ; hydro-
cauli are short and unramified. In I5%e and in 30%e, hydrocauli are longer and ramified. The
most important alterations take place in the hydranths, the site of exchanges of water and ions,
of oxygen uptake and excretion, of propagation, growth, etc. In suboptimal salinity and in supra-
optimal salinity the hydranths become shorter and bear fewer and shorter tentacles. As the
breadth is greatest in fresh water and decreases with increase of salinity, the hydranths are in
fresh water somewhat globular, surface-volume relation of hydranths is low and increases with
salinity. Surface-volume relation depends also on temperature; it is higher at 20° C. than 10° C.
There occur remarkable alterations at the cellular level : fresh water hydranths consist at 20° C.
of about 22,000 cells; these are columnar (high and narrow) and have a large nucleus. Epithe-
lium of fresh water hydranths seems to be physiologically highly active and less permeable;
30%e-hydranths, on the other hand, consist of only 4-5,000 cells which are squamous (flat and
broad) and have a small nucleus. Epithelium of 30/ce-hydranths seems to be physiologically less
active and more permeable. All these alterations caused by salinity are stronger at 20° C. than
at 10° C. Cell-number is greater and nuclei are larger at low temperature.

Inorganic pyropJiosphatase activity of glycerinated muscle. C. Moos AND L.

LORAND.

In the course of investigating relaxation in glycerinated muscle fibers, a study was under-
taken of the interaction of inorganic pyrophosphate (POP) with glycerinated muscle, and in
particular, of the pyrophosphatase (POPase) activity of this material. Samples of glycerinated
muscle, prepared by extraction in cold 50% aqueous glycerol as described by Szent-Gyorgyi, are
homogenized in 0.1 M KC1 containing 4 mM MgCl2 and buffered at pH 7.0 with 10 mM imid-
azole. Upon addition of POP to the suspension, orthophosphate is liberated at roughly a con-
stant rate until the POP substrate is nearly exhausted. This rate is, in rough order of magni-
tude, about 1% of the rate of hydrolysis of adenosine triphosphate (ATP) under similar condi-
tions. It has been found that POPase activity is retained in glycerinated muscle stored at — 20°
C. for as long as 18 months. The activity is completely extracted from the muscle upon homog-
enization in the above buffered salt solution, but homogenization in water leaves a major fraction
of the activity in the insoluble residue. The POPase activity of the homogenate is inhibited by
low concentrations of calcium; using 1.0 mM POP, 0.2 mM CaCU causes more than 60% in-
hibition, and 1 mM CaCl2 inhibits over 90%. Salyrgan also inhibits POPase, but only partially;
all concentrations of the drug above 0.2 mM reduce the activity by only about 70%, while ATP
hydrolysis is more than 99% inhibited under these conditions. Utilizing these facts, the effect of
added ATP on POPase activity was investigated, and it was found to have no effect. Adenylic
acid, which could be studied without salyrgan, was also without effect.

This work was aided by a grant from the Muscular Dystrophy Associations of America, Inc.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 331

Metabolic consequences of a genetic block between a-ketoglutarate and succinate in
Escherichia coli. ARNOLD L. NAGLER, ELIZABETH S. MINGIOLI AND BERNARD
D. DAVIS.

In a search for auxotrophic mutants of Escherichia coli, using ultraviolet irradiation and
selection by means of penicillin, a new kind of mutant was obtained. This strain (309-1) grew
under anaerobic but not under aerobic conditions when the mineral -glucose medium was supple-
mented with certain biosynthetic products of the 4-carbon dicarboxylic acids : lysine plus me-
thionine or threonine. (A biosynthetic precursor of these compounds, aspartate, was inhibitory.)
Succinate supported even more rapid growth and was also effective, alone or with glucose, under
aerobic conditions. Glutamate was inactive. Furthermore, this mutant, growing on glucose
plus succinate, heavily fed a strain that responds to only a-ketoglutarate or glutamate. These
findings suggested that mutant 309-1 is blocked between a-ketoglutarate and succinate. This
conclusion was established by showing in a Warburg respirometer that non-growing cell suspen-
sions of mutant 309-1 failed to oxidize glutamate to COa, whereas the wild type carried out this
conversion almost quantitatively. In contrast, succinate was oxidized at a similar rate by the
two strains.

These results throw further light on the metabolic role of the tricarboxylic acid cycle in
E. coli. Previous studies (Gilvarg and Davis, 1956) have shown that a mutant blocked between
oxalacetate and citrate requires glutamate but not any direct biosynthetic products of the dicar-
boxylic acids. The present findings show that when these 4-carbon acids are being further
metabolized to a-ketoglutarate without being regenerated from that compound, the rate of their
formation from glucose is not sufficient to meet the biosynthetic needs of the organism.

Contractility of the Iryaline layer of Arbacia pitnctulata. A. K. PARPART AND
JULIEN CAGLE.

The hyaline layer of fertilized Arbacia punctulata eggs is probably a polysaccharide and is
contained in a polymerized state in the cortical granules prior to fertilization. Fertilization or
parthenogenic agents cause rapid and explosive depolymerization followed by slow polymeriza-
tion, to form the hyaline layer, about 2 /* thick, closely surrounding the egg.

This hyaline layer exhibits remarkable contractile properties when the pH of the environ-
ment is decreased. At pH 4.0 slight contraction occurs. However, at pH 2.4 a rapid and
strong contraction occurs. It shrinks down to a thin line tightly compressing the egg and is
capable of pulling two- and four-cell stage blastomeres tightly together. Several acids added to
sea water cause this. The force of contraction is so great that the plasma membrane and cyto-
plasm of the egg are forced through numerous minute apertures in the hyaline layer to form
ca. 3 to 5 fj. blebs over most of the egg surface. The contraction of the hyaline layer is reversed
in sea water and this contraction and relaxation can be repeated a number of times.

The blebbing that follows active contraction is not due to the acidity of the environment.
This was established by depolymerizing the hyaline layer in isosmotic sucrose and then exposing
the egg to isosmotic sucrose brought to pH 2.4. This induced a mild degree of polymerization
of the hyaline polysaccharide but no blebbing.

The hyaline layer is not stainable by methyl green, which suggests it is not a sulfate-poly-
saccharide. It is stained in vivo by methylene blue, toluidine blue, pyronin and neutral red,
which suggests it may be an acid-polysaccharide. It is not stainable by phenol red or Janus
green B. Methylene blue, added to depolymerized hyaline layer in vivo, causes a partial poly-
merization. This suggests the polysaccharide may be related to the lichenins.

Observations on the histology and o.ri dative metabolism of gill cartilage from Limn-
lus polyphemus. PHILIP PERSON AND ALBERT FINE.

Limnlus gill cartilage, a mesenchymally derived endoskeletal tissue, is histologically very
similar to vertebrate hyaline cartilage. Gill cartilage growth and development involves differ-
entiation, maturation, and degeneration of cells, such as are encountered in the life history of a
typical vertebrate cartilage. It is of interest that there may be found, in this invertebrate tissue,

332 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

sequences of cell development typically associated with vertebrate endochondral ossification, with
the exception that Limit! us cartilage is never replaced by a process of ossification. In homog-
enates and slices prepared from cartilage from larger animals, six inches in length or more, it
was not possible to demonstrate direct oxygen uptake in the presence of substrates such as glu-
cose, succinate, ascorbate, and hydroquinone, using as much as one gram, wet weight of tissue,
per Warburg vessel. A wide variety of suspending media was used in air and oxygen atmos-
pheres. Inability to demonstrate terminal oxidase activity by manometric and spectrophotometric
methods was not considered unusual in view of the severe degenerative changes seen in the cells
of cartilage taken from larger animals. In specimens approximately one and one half to three
inches in length, the gill cartilages resemble early embryonic cartilage, histologically. Homog-
enates prepared from such tissue demonstrate aerobic utilization of succinate and hydroquinone.
Malonate, equimolar with succinate, inhibited oxygen uptake 100%. Hydroquinone utilization
was inhibited 56% by 10"4 M cyanide and 32% by 10"* M azide (final concentrations). Spectro-
photometrically, such homogenates could reduce cytochrome c in the presence of succinate and
10~3 M cyanide ; and could oxidize reduced cytochrome c. This marks the first demonstration of
succinoxidase and cytochrome oxidase activity in cartilage tissue from any source.

Studies on the distribution and properties of tlie ribonuclease system in marine
forms. JAY S. ROTH AND DOROTHY BACHMURSKI.

Previous studies on the ribonuclease (RNase) system in rat liver have demonstrated the
presence of two active enzymes, an enzyme inhibitor and an enzyme-inhibitor complex. The oc-
currence of different RNase systems in lower forms, correlated with other biochemical factors,
may give some insight concerning the physiological purpose of these systems which occur, appar-
ent!}', in most animal cells.

Various tissues were homogenized in ice-cold water and a supernatant fraction prepared (ex-
cept with sperm) by centrifugation at 60,000 G for 30 minutes. The supernatant fraction was
assayed for RNase activity. RNase inhibitor and inactive RNase (i-RNase, RNase-inhibitor
complex) and the homogenate was assayed for RNase activity at pH 5.6, 6.4, 7.0 and 7.8, and
after heating for 5 minutes at pH 5.6 and 7.8. All assays were by previously published methods
using ABC buffer.

In a series of experiments on marine eggs and sperm, i-RNase but no RNase activity was
found in the supernatant fractions. With starfish nucleoli (supplied by Dr. Walter Vincent) the
specific activity of both RNase and i-RNase was the highest encountered, which is of consider-
able interest in view of the rapid RNA metabolism in this particulate. RNase inhibitor was de-
tected, in small amounts, in Chaetoptems eggs or sperm, the greatest activity being measured at
pH 7.0 or 7.8.

When the supernatant fractions from some higher forms were examined, all contained from
moderate to large amounts of alkaline RNase activity in contrast to rat liver, which contains
little. This RNase activity was strongly inhibited in squid gill and starfish gonads by 4 X 10"4
M p-chloromercuribenzoate indicating that the RNase was of a sulfhydryl nature as contrasted
to mammalian enzymes which are not. No i-RNase was detected, but RNase inhibitor was wide-
spread, and both acid and alkaline RNase activity were detected in most specimens. (Supported
by grants from National Institutes of Health, American Cancer Society and Damon Runyon
Memorial Fund.)

The effect of sugars on gastrulation of Chaetopterus embryos. DWIGHT B. Mc-
NAIR SCOTT.

During the course of investigation in previous summers on the carbohydrate metabolism of
the developing Chaetopterus embryo we attempted to measure the production of labelled CO2
from glucose-1-C" and found that the presence of glucose in the sea water had an adverse effect
on the subsequent development of the larvae. Therefore the effects of different concentrations of
glucose, fructose and sucrose, added at different times before and after fertilization, have been
investigated. Fertilized Chaetopterus eggs hatch at about 7 hours and the trochophores invagi-
nate at 33-36 hours. Glucose (0.0055 M), added before fertilization and at times up to 9 hours
after fertilization, caused a slight delay in the time of gastrulation which otherwise was normal.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 333

Glucose (0.014 M) delayed the onset of gastrulation, decreased the size and functions of the
alimentary tract, decreased the proportion of embryos completing gastrulation and prevented
later growth of the larvae in size. Glucose (0.028 M) added at any time up to 7 hours (hatch-
ing) prevented gastrulation; and addition at 9 hours and at 10 hours delayed gastrulation to the
fourth day and the third day, respectively. These larvae did not increase in volume beyond that
of the egg. Concentrations 0.045 M and above prevented any development beyond the trocho-
phore and produced abnormal forms. Transfer of the embryos from glucose to sea water after
three hours or more did not lead to normal gastrulation. Sucrose, 0.03 M, produced no effect
on the time or course of larval development. Thus these results are not comparable to those re-
ported by K. Dan and A. R. Moore with Dendraster or Arbacia eggs. Their sucrose concentra-
tions were higher and produced abnormal invagination and exogastrulation by osmotic effects.

The effects of glucose are more comparable to the effects of amino acids on marine egg's as
reported by Mathews, or H. D. King.

Studies on the interactions of the bound nucleotide of actin. RICHARD C. STROH-

MAN.1

An investigation was carried out on the changes in the bound nucleotide of actin associated
both with actin-actin and actin-myosin interaction. The possibility that the bound nucleotide was
also available for interaction with creatine phosphate (CP) was also studied.

It was found that a reversible depolymerization of actin can be obtained by dialysis against
CP in the presence of creatine phosphokinase. During depolymerization the ADP of the F-actin
was converted to ATP. If, however, F-actin is reacted with CP but under conditions where the
actin remains polymerized then no such transphosphorylation takes place.

The possibility that the classical depolymerization of actin with ATP might also occur
through a transphosphorylation, where ATP is the phosphate donor, was subsequently investi-
gated. Depolymerization was run using Ci4 ATP and it was found that there was no incorpora-
tion of radioactivity into the bound nucleotide. Further studies are in progress on this system
but the tentative conclusion is that the mechanism of nucleotide change is one involving transfer
of the terminal phosphate of ATP to the ADP of the actin.

It is possible to show that G-actin forms a complex with H-meromyosin in which the ATP
of the actin is converted to ADP. The nucleotide of the complex is still able to interact with
the creatine-phosphokinase system since regeneration of ATP is observed when CP is added
under the proper conditions. In F-actin-H-meromyosin complexes the nucleotide is unable to
interact with CP under the conditions used. G-actin-H-meromyosin complexes are thus able to
carry out a turnover of the terminal phosphate of the actin nucleotide in the presence of a phos-
phate donor. No such turnover can as yet be demonstrated for F-actin-H-mero-myosin com-
plexes.

Electrical recording in the living squid. ROGER E. THIES.

The resting potential and spontaneous activity of the giant axon were measured in Loligo
pealii. Animals were maintained for many hours strapped under the lid of a Incite box contain-
ing oxygenated sea water. The axon was observed near the rostral margin of the fin with
transmitted light. To insure visibility the ink sac duct was ligated and the skin removed locally.

Spontaneous activity of two animals was recorded between two glass-insulated silver wires
plunged into the mantle muscle just above the axon and one centimeter apart. Irritation by-
poking the head caused firing of the giant fiber. The resultant axon potentials of 0.2-0.4 milli-
volts were followed after 2-3 milliseconds by muscle potentials of 1 to 5 millivolts. The poten-
tials occurred singly or in bursts of up to eight. The longer trains began at frequencies of 140
per second and declined to 50 per second, with the total duration never exceeding 100 milli-
seconds. During continuing irritation such bursts recurred at one- to five-second intervals.

Resting potentials were measured with the squid in a closed system of circulating sea water
cooled to 8° C. The resulting decreased rate of mantle contraction minimized movement of the
axon during penetration with KCl-filled micropipette electrodes. Removal of muscle from above

1 Supported by fellowship from U. S. Public Health Service to the author and by a grant to
Dr. T. Hayashi from the Muscular Dystrophy Assn. of America.

334 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

the fiber allowed penetration of the axon membrane under direct observation. Values of 60 to
111 millivolts were measured in sixteen penetrations of six axons, with a mean value of 77 milli-
volts. Three axons gave values of 52 millivolts and below.

The high resting potentials may be due to the elimination of the mechanical trauma associ-
ated with removal of the giant axon, as well as preservation of all the animal's functions.
Prosser and Young showed that single impulses in the giant axon give maximal contraction.
Yet apparently the squid frequently uses short bursts to insure effective contraction.

Glucuronidase and sulfatase of mollusks. WALTER TROLL.

Glucuronidase and sulfatase are useful reagents in the study of mammalian metabolism, since
a number of compounds such as steroids and aromatic amines are excreted as glucuronides and
sulfate esters in the urine. Recently a number of English workers have reported that mollusks
occurring at their sea coast are excellent sources of these enzymes. We have confirmed this ob-
servation with several mollusks obtainable here. A good source appears to be the liver of
Mactra homogenized with water. Both glucuronidase and sulfatase are readily purified by am-
monium sulfate fractionation yielding enzyme preparations with 50-fold the activity of the most
purified mammalian preparations. The optimum pH for glucuronidase using phenolphthalein
glucuronide as the substrate is pH 4. The optimum pH for the sulfatase using p-nitrophenol
sulfate as the substrate is 5.6. The sulfatase is inhibited by phosphate and sulfate ions. All
these properties are identical with the ones reported by the English workers for a variety of
molluskan enzymes. These observations can be interpreted to indicate that these enzymes are
characteristic constituents of mollusks.

Proteins of starfish nucleoli. W. S. VINCENT.

Among the many unknown things about nucleoli is where the nucleolar materials come from.
As about 95% of the nucleolar materials are protein, an analysis of this component might sug-
gest some answers to this particular basic problem.

The major protein component of isolated starfish nucleoli proved to be insoluble in reagents
which might yield solutions suitable for ultracentrifugal or electrophoretic analysis, so the tech-
nique of end group labelling with dinitrofluorobenzine was used. When nucleoli were reacted
with this reagent, the hydrolysate, upon chromatographic separation, yielded only a single amino
acid (as yet unidentified) labelled with the dinitrophenol. A control reaction on acetone powder
of whole starfish eggs yielded some half-dozen labelled amino acids.

Although the possibility is not completely excluded that the free amino end groups of some
of the protein species may not be available to the reagent, the simplest interpretation of these ex-
periments is that the nucleolus consists of a single protein. If this simple explanation is true,
then it is likely that the nucleolar protein is the product of a single genetic "site" rather than
being the accumulated products of many different chromosomal regions.

Survival of marine invertebrate cells in tissue culture (Limulus and Ostrea). ANNE
WARWICK AND F. B. BANG.

Limulus amoebocytes were cultured in varying dilutions of Limulus serum in roller tubes
with 100 units of penicillin and 100 units of streptomycin at room temperature. As Loeb has
shown, the amoebocytes maintain their oval, granular appearance in undiluted Limulus blood.
In this medium a prompt loss of granules and change in shape of the cells were obtained with a
bacterial toxin within 15 minutes. With renewals of medium, Limulus cells survived more than
30 days in 10% Limulus serum in artificial sea water. Similar observations were made in 25,
50 and 100% Limulus serum. In both living and Giemsa-stained preparations no mitotic figures
were observed. In all of the cultures there was a gradual decrease in the mass of cell material.
Attempts at transfer by explantation of recently formed clots, by trypsinization, in Ca-free sea
water and Versene treatment failed. Oyster amoebocytes obtained by cardiac puncture survived
in their own serum for 5 days. Mantle tissue was explanted in artificial sea water, 10% and
25% Limulus serum in artificial sea water with 10 mg% glucose, and in hemocyanin-free Limu-
lus serum. Following the early migration of amoebocytes from the mantle tissue, in the sea
water there was a degeneration of all of the cells by two days. The addition of the Limulus

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 335

serum was accompanied by the organization of mantle epithelial cells, the formation of ciliated
cysts within the mantle and ciliated borders. Such ciliated cysts remained spinning for 8 to 10
days and the amoebocytes persisted for 12 days. Again no mitotic figures were seen and the
cultures gradually degenerated. Some muscle cells apparently also persisted since contraction of
the explant was observed as late as 10 days.

Method of analysis of a "gene" in Mormoniella. P. W. WHITING.

A restricted region of the germ plasm, a gene so-called, may be studied by the interaction of
mutant allcles in the compounds. The allelic series is likely to be complex, affecting more than
one factor. The different mutant states of the factors constitute sub-series of alternatives within
the series of pleiotropic allelic genes. These states cause impairment of function of greater or
less severity. Alleles may be produced by irradiation of wild type and isolated by subsequent
crossing to a stock with a recessive marker. Thus, irradiated wild-type Mormoniella males have
been crossed to females with the .R-locus eye color peach-333.5. A mutant-type daughter will
be a compound of peach and a new 7?-locus mutant gene. Wild-type daughters indicate either
no mutation, mutation at loci other than R, or 7?-locus mutation in factors other than those
affected by. the marker gene. Peach-333.5 is a triple recessive gene, mutant in eye-color factors
O, S and TV but not in M. Mahogany-846 is recessive in M alone. The compound female is,
therefore, wild type — o.s. + .«/+ . + .m. +. There have also been found in this region at least
three factors, A, B and C, affecting viability and sterility. Mutations in A may be female-
sterile, fsa, or lethal, la. The compound, fsa/la, is a viable but sterile female like the homo-
zygote, fsa/fsa. Some of the TtMocus alleles suppress crossing-over with purple body, pu, eleven
map units distant. It is postulated that a similar method of analysis applied to other regions
also would produce pleiotropic alleles and convert these regions into loci by suppressing crossing-
over within them and thus integrating their factors into segregating units, genes.

PAPERS READ BY TITLE
Energy metabolism and ciliary activity of Mytilus gill. EDWARD AIELLO.

In 1924 James Gray reported that veratrine stimulated both ciliary activity and oxygen up-
take of excised Mytilus gill to about 150% normal and suggested a direct effect of veratrine on
energy metabolism. It has been found that veratrine sulfate 0.01% causes no significant change
in the oxygen uptake or ciliary activity of gills whose cilia have been totally inhibited by 2,4-
dinitrophenol 10"3 M or high salt (3 X normal osmolarity) or partially inhibited by KCN 0.01
M. It does, however, almost totally restore the oxygen uptake and ciliary activity after inhibi-
tion by sodium azide 0.01 M. Azide, in turn, only slightly inhibits the oxygen uptake of gills
in DNP or high salt but greatly inhibits it, and ciliary activity, in natural sea water. The ab-
solute values of oxygen uptake in DNP or high salt with azide present are higher than those
with azide alone although ciliary activity is only present, but weak, in the latter. Veratrine does
not restore ciliary activity that has been inhibited by lack of oxygen. The simplest explanation
seems to be that veratrine effects oxygen uptake only indirectly through its effect on ciliary ac-
tivity ; also, that azide effects oxygen uptake directly only to a small degree and mainly by a
direct inhibition of the ciliary mechanism. This latter action is the only one reversed by vera-
trine. A further implication is that, depending on circumstances, the degree of ciliary activity
regulates oxygen uptake, e.g. with veratrine stimulation, or the rate of oxygen uptake determines
the degree of ciliary activity, e.g. with cyanide inhibition.

Supported in part by a Predoctoral Fellowship from the National Institutes of Health and
a grant to Dr. T. Hayashi from the Muscular Dystrophy Association of America.

Reaction to injury in the oyster. F. B. BANG.

Amoebocytes of the oyster readily phagocytize a variety of marine bacteria in vitro. How-
ever, many strains of marine bacteria were not phagocytized under the same conditions. When
phagocytosis took place it was preceded by clumping of bacteria on the amoebocytes and fre-
quently by the sticking of the bacterial flagella on the processes of the amoebocytes (electron

336 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

microscopy). The formation of an extracellular clot with oyster amoebocytes was demon-
strated frequently in blood obtained from well-fed oysters which had been kept from feeding dur-
ing the preceding 6 to 12 hours. This extracellular clot entrapped bacteria and spread from the
amoebocytes throughout and around the cellular clot. This extracellular clot was also observed
in vivo. Intra vascular clotting (white cell clumping) was observed in the vessels of the living
oyster following trauma directly to the oyster or in badly traumatized oysters opened on the half
shell. It was produced regularly by the intracardiac injection of an extract of oyster tissue
(gill). This intravascular clotting consisted of clumping of white cells, the sticking of these
clumps to the walls of the vessels and the contraction and thrombosis of large vessels which
lasted for one to two hours. Control injections of sea water and carmine failed to produce these
effects. Heated extract and bacterial suspensions produced temporary effects (10 to 15 minutes).

Lethal irradiation of Tillina nmgna in its active and encysted states. JOSEPHINE
BRIDGMAN.

The ciliate Tillina nun/na, in its active state, is somewhat more sensitive to x-irradiation than
are many other protozoa for which lethal doses are known, but like the other protozoa tillinas
vary considerably among themselves in susceptibility to radiation. This is particularly con-
spicuous if they are irradiated at different times in their life cycles.

Tillina, like the better known Colpoda, is very easily induced to form cysts. These are
smaller than the active form and in them the animal is de-differentiated. In a series of tests
active tillinas and tillina cysts, in groups of twenties, were irradiated at intensities of 175, 200
and 225 kr, doses chosen because they are on the borderline of survival for these animals. It is
well known from earlier work on Tillina that animals which initially survive a dose of irradia-
tion often go subsequently into an abnormal cyst from which there is no revival. The fate of
the active animals irradiated at these doses was often such a cyst. The effect of irradiation on
a normal cyst is not immediately apparent. However, unless such a cyst, under circumstances
favorable to excystment, becomes active within twenty-four hours, it is unlikely that it is alive.

Examination, after twenty-four hours, of the animals treated as described above indicated in
every case a higher percentage of survival in the animals irradiated as cysts than as active ani-
mals. In one typical experiment active animals irradiated at 225 kr showed 10% survival after
24 hours and cysts treated at the same time to the same dose had a 75% survival. Similar dif-
ferences between susceptibilities of active and encysted forms were shown at the other doses.
The experiments as a whole show clearly that active animals are more susceptible to x-irradia-
tion than animals in resting cysts.

Work done with the support of the A. E. C. under contract No. AT-(40-1 )-1818, with
Agnes Scott College.

The inliibition of the cardiac ganglion of Lininlus polypJiemus by 5-hydroxytryp-
tamine. A. S. V. BURGEN x AND S. W. KUFFLER.

The rhythmic bursts of activity in the isolated cardiac ganglia of Liinulus polyphemns were
slowed by 5-hydroxytryptamine (5-HT) 5 X 10~8 g./ml. and arrested by 2-3 times this concentra-
tion. The inhibitory effect of 5-HT could be prevented by roughly equal concentrations of
bromlysergic diethylamide (BOL) which by itself had no striking effect on the activity of the
ganglion. Gamma-aminobutyrate had a similar action to that of 5-HT but was about 100 times
less active.

Larval development of Streblospio benedicti Webster. MILDRED A. CAMPBELL.

Early developmental stages of Strcblospio benedicti occur within a protected area on the
dorsal surface of the middle segments of the adult. In the living material these segments appear
to form a brood chamber with dorsolateral folds of the body wall, but definite information must
wait upon histological sections. The eggs are blue-green in color, as are the larvae at the time
of hatching. Larvae are released into the plankton at the 3- or 4-segmented stage. They are
typical spiomorphic larvae.

1 Fellow of the Lalor Foundation.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 337

At the time of hatching the larvae have a well developed prototroch and telotroch, finely
serrated provisional setae, strong sensory cilia on the prostomium, and two pairs of brownish-red
eyes. The pygidium has four anal cirri, which look as though they contain bacillary glands.

In the later larvae gastrotrochs develop on the posterior segments, and the neurotroch ap-
pears to extend to the posterior border of the first segment. The provisional setae are replaced
by the adult type and the pygidium loses the anal cirri. The prostomium and peristomium are
fused to form a bell-shaped anterior end. The lateral lips of the vestibule are fused anteriorly.
There are posterio-lateral palp-like projections of the vestibule wall which are capable of great
extension. Diffuse light brown pigment develops in the wall of the vestibule, as well as on the
prostomium and pygidium. Palps and branchiae do not develop before the 9-segmented stage.
From the 10-segmented stage on, the larvae appear to be bottom dwellers. In nature meta-
morphosis must occur at about the 13-segmented stage, since one collection contained a specimen
13 segments long which did not possess any larval characteristics.

Electrical stimulation of light emission in fireflies. ]. F. CASE AND JOHN BUCK.

To provide further data on the control of flashing in the fireflies Photinus pyralis and
I'liotiiris pennsylvanica, the effects of variation in strength, duration and frequency of electrical
stimuli were studied. Pulses from a Grass P4 stimulator were applied either to the thoracic
ventral nerve cord or directly to photogenic organs with central connections severed. The photo-
genic responses of both species resemble those of known neuro-effector systems in showing a
well defined stimulus intensity threshold, facilitation and adaptation. Both species give evidence
of complex neuro-effector organization in that areas or sub-units of the light organs may re-
spond asynchronously to the same external stimulus.

The two species differ in spontaneous light emission, P. pennsylvanica producing shorter
flashes which are characteristically double peaked, a small inflection occurring on the ascending
limb of the major peak. This suggestion of more precise neural control of flashing by P. penn-
sylvanica is confirmed by the considerably higher stimulus frequencies to which its light organ
can respond with discrete flashes (at least 20 per second compared with less than 5 per second
for P. pyralis) and by its considerably shorter response latency to high frequency stimulus trains.

In P. pennsylvanica response latency to direct organ stimulation is about 70 msec, and about
200 msec, to thoracic cord stimulation. Since only a small fraction of these delays can reason-
ably be accounted for in terms of synaptic delay and neural transmission time it appears that the
control of light emission must involve either some rather unconventional type of neuro-effector
linkage or considerable delay within the photogenic tissue.

The nature of electrical responses of doubly-innervated insect muscle fibers.
]. CERF, H. GRUNDFEST, G. HOYLE AND F. V. McCANN.

Depolarizing pulses applied to muscle fibers of Romalea microptcra through one inserted
microelectrode evoke responses at another (recording) microelectrode at a low level of de-
polarization. As the pulse strength is increased larger, spike-like responses are obtained. These
are, however, always graded events. Sustained applied depolarization produces repetitive pulsa-
tile responses, but successive potentials become smaller and may die out. The maximum magni-
tude of the responses is about 35 mV.

Keeping the pulse strength constant and recording at different distances from the stimulating
electrode, the spike-like responses are seen to die out very rapidly. At 3 mm. distance even the
largest are barely detectable. The graded responses are therefore decrementally propagated.

Junction, or postsynaptic potentials (psp's) evoked by stimulating "fast" or "slow" nerve
fibers decrease with increasing depolarization of the muscle fiber. When the membrane potential
is reversed to a sufficient extent, the psp's reverse in sign. With increasing membrane polariza-
tion the psp's increase in amplitude. These findings suggest that psp's are generated in electri-
cally inexcitable membrane.

The "fast" psp's evoked during strong membrane hyperpolarization continue to produce spike-
like responses. The latter often appear to be overshooting and may be initiated at lower levels
of depolarization than in normally polarized fibers.

338 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

These data can account for the different types of activity evoked in one muscle fiber by
stimulation of its "fast" or "slow" nerve. The psp's generated synchronously at multiple sites of
innervation evoke graded, decrementally propagated pulsatile responses. Different combinations
of the two response components, large "fast" psp's evoking large graded responses ; small "slow"
psp's producing smaller graded activity, result in the characteristically different mechanical re-
sponses of these doubly-innervated muscle fibers.

Neuromuscular transmission in the grasshopper Romalea microptera. J. CERF, H.
GRUNDFEST, G. HOYLE AND F. V. McCANN.

Some muscle fibers of insects receive double motor innervation, one fiber giving rise to large
electrical responses and powerful twitch contractions, the other evoking smaller potentials and
weaker contractions. This is also the case in flexor and extensor muscles of the meso- and
metathoracic legs of Romalea. The extensor muscle of the jumping leg receives a single "fast"
and a single "slow" axon which leave the ganglion in separate nerve trunks homologous to those
designated 3b and 5 in locusts (Hoyle, 1955).

Intracellular recording reveals that almost all the muscle fibers receive branches from the
"fast" axon, although some have been found without "fast" responses. These were fibers with
particularly large "slow" responses. The resting potentials were 50-70 mV in magnitude.
"Fast" responses typically involved a small overshoot. Return to the resting potential was often
almost as fast as the rising phase but in many muscle fibers the later part was much slower
than the first. This was attributed to the presence of persistent junction (postsynaptic) poten-
tials. "Fast" responses are followed by a refractory period for the spike-like component.

Responses to stimulation of the "slow" nerve were found particularly in a muscle bundle
situated at the proximal border of the extensor. These fibers have a richer tracheal supply than
purely "fast" innervated ones. The "slow" responses ranged widely in magnitude. The smaller
ones had a long time-course and summed to give a plateau of depolarization during repetitive
stimulation ; they showed no refractoriness. The larger ones gave rise to graded secondary re-
sponses and had a shorter time-course, though the postsynaptic potential was quite long in some
cases.

The responses to both kinds of nerve stimulation were similar when recorded simultaneously
at different points along the fiber, reflecting the multiterminal nature of the innervation.

Larval development of the mud crab Ncopanopc tcxana sayi (Smith).1 NORMAN
A. CHAMBERLAIN.

Larvae of Ncopanopc tcxana sayi have been successfully reared in the laboratory through
the second crab stage. Two egg-bearing females were collected from Great Pond, Falmouth,
Massachusetts, on July 22, 1957, and maintained in the laboratory. The larvae hatched as
prezoeae and developed through four zoeal stages and one megalopal stage before metamorphosing
into the first crab stage. The larvae were cultured in 20-liter glass jars containing 15 liters of
sea water which was aerated continuously. Cultures of larvae in isolation were also maintained.
The temperature of the cultures remained at 24.0° C. ± 1.0° C. All prezoeae shed within five
minutes to become first zoeae. Duration of the later stages in days for the two cultures was,
respectively: first zoea, 12, 8; second zoea, 3, 2; third zoea, 1, 1; fourth zoea, 2, 2; megalops,
7, 7; first crab, 7, 7. Thus the planktonic stages lasted 25 and 20 days, respectively.

Zoeal stages were fed Phacodactylum tricornutum (200,000/ml.) and Dunaliclla euchlora
(20,000/ml.). Early larvae of Arenicola cristata were fed to the zoeal and megalopal stages
with the exception of the first zoeae of the first culture which were fed only Phacodactyhim
tricornutum for the first six days of their development. This fact probably accounts for the
observed difference in the durations of the first zoeal stages. Megalopal and crab stages were
fed Artemia salina nauplii. Crab stages were also fed small pieces of clam gill and mantle.

These data indicate that duration of planktonic stages can be shorter than has been esti-
mated from plankton studies.

1 This study was aided by a summer fellowship from the Woods Hole Oceanographic Insti-
tution.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 339

Fertilisability of Arbacia eggs after pretreatment in trim e thy lated xanthine deriva-
tives. RALPH HOLT CHENEY.

A viability time table for Arbacia punctulata gametes in relation to time was noted by the
author in 1950. The effect of M/400 di- and trimethylated xanthines was published in 1956.
Concurrently with a study of inhibitory effects of derivatives of 2:6 dioxypurines on mitosis and
growth of this species, the insemination process and appearance of the fertilization membrane
were observed.

Eggs were shed into equal volumes of sea water, and into M/200 and M/400 concentrations
in SW of trimethylated xanthine (caffeine), 8-methoxycaffeine, 8-ethoxycaffeine, and 8-chloro-
caffeine. Eggs were mixed with non-treated, fresh sperm in SW after eggs were pretreated for
4, 10, 24, and 48 hours.

Delay in insemination time and appearance of the fertilization membrane was only slight un-
less the egg pretreatment period exceeded four hours although a 30-minute immersion does affect
subsequently the mitotic process and early growth. Order of increasing delay or complete in-
ability of the egg to produce the FM after 10, 24, and 48 hours in M/200 or M/400 showed the
same sequence ; namely, 8-MOC ; 8-EOC ; C ; and 8-CC.

All of these compounds have been reported to penetrate the plasma membrane readily in
plant cells but only EOC penetrates the nuclear "envelope." Centrifugation studies by the author
(1949) demonstrated an egg surface-action effect of the trimethylated xanthine. Unpublished
data with respect to sperm potency after subjection to these compounds indicate that the egg re-
mains normal longer than sperm in identical molarities. It is suggested that the variation of the
physico-chemical surface forces induced by these compounds may account for the differences in
the insemination time-period and the lifting of the fertilization membrane.

Dioxypurine derivatives as mitotic and growth inhibitors. RALPH HOLT CHENEY.

Dimethylated (theophylline) and trimethylated (caffeine) 2:6 dioxypurines, and a methoxy-
group (OCH3) substituted for H at C8 (methoxycaffeine), an 8-ethoxy radical (OCH2CH3) the
8-ethoxycaffeine, and a Cl substitution 8-chlorocaffeine, were employed in M/200 (5 mmol/L)
and M/400 (2.5 mmol/L) concentrations. Test cell was the egg of Arbacia punctulata. Non-
treated, fresh sperm were mixed in sea water (SW) with eggs pretreated 30 minutes in the ex-
perimental solutions : SW alone, TpSW, CSW, 8-MOCSW, 8-EOCSW, and 8-CCSW.

Based upon (1) degree of delay and induced abnormalities of the mitotic process and cell
division, and (2) observation of the maximal growth at death in equivalent molarities, the order
of increasing inhibitory effectiveness of the number, position, and nature of the radicals was de-
termined. In M/200, the order of increasing effectiveness indicated by relative mitotic inhibition
was Tp ; 8-MOC ; 8-EOC ; C ; 8-CC. An identical sequence was demonstrated by the death
series. Using M/400, a similar order was noted except that the mitotic inhibition results sug-
gested a possible reversal in the relative effectiveness of 8-EOC and C.

Analyses of sequences demonstrate an inhibitory increase associated with an increase in N-
bound methylation, still greater inhibition with ethoxy- radical compared with the methoxy-
group, and a maximal effect with the chlorine substitution for H at C8. Relative inhibitions indi-
cate a significant relationship between the molecular structure involving CH3, OCH3, OCH2CH3)
and Cl, the relative penetration power of these compounds at the nuclear border, and possibly the
electro-negative property of chlorine. Results regarding the relative inhibitory effect of C and
8-CC are in general agreement with the report regarding these same dioxypurine derivatives
upon plant cells in Allium root tip. See Kihlman, B. (Symb. Bot. Upsal, 11, No. 2: 1-40
(1951); 11, No. 4: 1-96 (1952)).

The demonstration of histamine in heparin- containing invertebrate cells.1 ALFRED
B. CHAET AND WILLIAM R. CLARK, JR.2

This study of invertebrate eggs resulted from a consideration of the mammalian mast cell
which has been shown to contain heparin and more recently to be a major source of histamine.

1 Supported in part by Parke, Davis Company.

2 Lederle Medical Student Research Fellow.

340 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

There exists the possibility that a single substance within the cell binds both heparin and hista-
mine, and the work, in vivo and in vitro, of other investigators suggests that there may in fact
be a heparin-histamine complex. With these studies in mind, it seemed worthwhile to search
for histamine in other cells known to contain heparin. Since blood clotting experiments and/or
the metachromatic reaction have indicated the presence of heparin in eggs of the clam (Spisula
solidissima) , the sea urchin (Arbacia punctulata), and the annelid (Chactoptcms pergamcn-
taceus), we assayed these eggs for histamine using the micro-chemical technique of Lowry et al.
The results suggest that these eggs are rich in histamine. Our analyses show that the Spisula
egg contains 2.0 X 10~° Mgm histamine base per egg and that 4.8 X 10~6 yugrn histamine is present
in Arbacia. Preliminary experiments indicate 9.6 X 10~8 /xgm histamine per Chaetopterus egg.
These figures are slightly lower than those calculated by others for rat mast cells (6.0 X 10~G
Mgm histamine per cell). Disruption of the cell membrane is apparently sufficient to liberate
most of the histamine within the egg since homogenization, freezing and thawing, or hypotonic
solutions result in histamine release. Since compound 48/80 releases histamine from mast cells
in vivo, we have attempted to liberate histamine from Spisula and Arbacia eggs using various
concentrations of 48/80 (20-200 mg%) ; however, in no case did it act as a liberating agent.
These experiments are complicated by the fact that 48/80 interferes with the histamine deter-
mination. Further studies on the mechanism of histamine release by histamine liberators, as well
as by heparin liberators, are being carried out.

A tecJiuic for preparing whole mounts of veliyer larvae. A. C. CLEMENT AND J. N.
GATHER.

Excellent, almost life-like whole mounts of the young veliger larvae of Ilyanassa obsoleta
and Anachis avara have been prepared by the following relatively rapid and simple technic.
Larvae are first immobilized by placing them in a mixture of one part saturated aqueous solution
of chloretone and two parts sea water. Three or four minutes exposure to this mixture is ade-
quate ; prolonged exposure may be damaging. The larvae are then fixed for one hour in a solu-
tion of 10% formalin in sea water. In a satisfactory proportion of the cases the fixed larvae will
show the velum fully expanded. The larvae are next dehydrated in ethyl alcohol ( 10 minutes in
each of the following: 35%, 70%, 95%, and two successive baths of 100%) and mounted in
Euparal under coverslips supported by strips of filter paper. In larvae in which the yolk has
been largely or wholly absorbed, structural details of the digestive tract, velum, foot and other
parts may be seen with great clarity in these preparations. The shell and velar cilia are well
preserved. The natural pigments are preserved initially, but some deterioration of the velar pig-
ment has been observed after a month. A light tinting of the tissues with Orange G in 95%
alcohol enhances the value of the preparations for some purposes and provides a pleasing con-
trast with the natural pigments.

The atninopeptidase and catheptic activity of the egg of Ilyanassa obsoleta. ]. R.
COLLIER.

The activities of two proteolytic enzymes, leucine aminopeptidase and cathepsin C, were
identified in the egg of Ilyanassa obsoleta. These enzymes were characterized by 1) their
specificity toward synthetic substrates, 2) their pH optimum, and 3) their requirements for
activation.

The aminopeptidase activity was determined by measuring the ammonia liberated after the
enzymatic hydrolysis of leucinamide. At a substrate concentration of 0.05 M , optimal hydrolysis
of leucinamide occurred at pH 8.2. The addition of 0.02 M MnCl» (final concentration) gave a
three-fold increase in the hydrolysis of leucinamide. Leucine aminopeptidase is the only enzyme
known to hydrolyze leucinamide under these conditions.

The activity of cathepsin C was identified by the fact that the Ilyanassa egg hydrolyzes
glycyl-1-tyrosinamide at the amide bond. Fruton has shown that this substrate is hydrolyzed
only by chymotrypsin and, at an acid pH, by an intracellular proteinase which he has designated
as cathepsin C. At a substrate concentration of 0.05 M, optimal hydrolysis of glycyl-1-tyrosin-
amide by the Ilyanassa embryo occurred at pH 6.0. This enzyme was not activated by 0.01 M

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 341

cysteine. A second pH optimum, at pH 7.5, may be due to the activity of an enzyme similar to
chymotrypsin.

The activity of both leucine aminopeptidase and cathepsin C showed a marked increase dur-
ing the course of embryonic development. This increase in activity occurred at about the fourth
day of development. The relative activity of aminopeptidase was found to be about 25 to 30
times greater than the activity of cathepsin C.

Egg membrane lysis by a sperm extract in Hydroidcs he.vagonus (Annelida) .*
ARTHUR L. COLWIN AND LAURA HUNTER COLWIN.

Eggs of Hydroidcs hcxagomis were immersed in solutions of sea water extracts of frozen-
thawed sperm of the same species. The principal substance of the vitelline membrane appeared
to swell and then dissolve ; a very thin inner portion appeared to remain as a capsule close to
the egg ; a very thin outer portion became elevated from the egg and was frequently ruptured
and shed. Possibly the outer portion represents the outer border layer, or some part of that
layer, which can be seen in electron micrographs of thin sections. Following treatment, the
outer portion can be caused to wrinkle and often some part comes to lie directly against the
inner capsule. However, if a treated egg is compressed, globules of exudate will pour out
through the inner capsule and push the outer portion away. In cases where the outer portion
has been shed, eggs may be pushed together so that the inner capsule of one egg directly touches
the inner capsules of adjacent eggs. These facts are interpreted as indicating that the substance
between the inner and outer portions of the vitelline membrane becomes liquefied.

Essentially the same results were obtained with fertilized and with unfertilized eggs.

It is concluded that the sperm of Hydroidcs he.vagonus contains a lysin or lysins which can
dissolve the principal material of the vitelline membrane but cannot attack the inner and outer
portions. This conclusion lends strength to the recently expressed view that the individual
spermatozoon of Hydroidcs hcxagomis exerts lytic action at the site of its passage through the
vitelline membrane (Colwin, Colwin and Philpott, 1957).

Observations of sperm entry during re-fertilisation in Saccogtossus kowalevskii
(Enteropneusta) .* LAURA HUNTER COLWIN AND ARTHUR L. COLWIN.

In this egg normal sperm entry is marked by the formation of a prominent fertilization cone
(Colwin and Colwin, 1954). After the fertilization membranes and other surrounding layers
had been dissected away from fertilized eggs, fresh insemination led to the entry of additional
spermatozoa into these eggs. Re-fertilization was observed to occur as late as the early blastula
stage. The entry of these additional spermatozoa is also marked by formation of a cone or cone-
like structure, but this structure is less prominent than the fertilization cone of normal sperm
entry.

Spermatozoa also were observed to enter artificially activated, but unfertilized, eggs .provided
the membranes had been dissected away. Cones similar to those observed in re-fertilization were
formed.

The cortical response of the Nereis ovum to activation after centrifuging.- D. P.

COSTELLO.

If uninseminated eggs of Nereis limbata are centrifuged at 6000 to 200,000 G for appropriate
periods of time (60 minutes for low or moderate, 10 minutes for higher accelerations) and in-
seminated shortly after removal from the centrifuge tubes, an asymmetrical perivitelline space is
produced and asymmetrical jelly outflow occurs. Since the eggs must be "cushioned" during

1 Supported by a grant (RG-4948) from the National Institutes of Health, U. S. Public
Health Service.

'- Aided by a grant from the National Institutes of Health, RG-5328.

342 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

the centrifuging, in isopycnotic sucrose, this is washed from the eggs before inseminating. The
asymmetry of the cortical response of the centrifuged egg is obviously due to displacement of the
jelly-precursor granules, which accumulate at the centrifugal pole of the egg, to a degree de-
pendent upon the intensity and duration of the applied centrifugal force. Parthenogenetic agents
produce the same asymmetry of cortical response in the strongly centrifuged eggs, as do
spermatozoa.

If unfertilized Nereis eggs are centrifuged sufficiently to sediment the cortical jelly-precursor
granules, and then treated with alkaline NaCl at pH 10.5, the vitelline membranes elevate to an
exaggerated degree, with a marked asymmetry. The perivitelline space formed is widest at the
centrifugal pole and lacking at the centripetal pole. With continued exposure to the alkaline
NaCl, the jelly in the asymmetrical perivitelline space swells, forcing the egg against the cen-
tripetal region of the vitelline membrane. Eventually the membrane ruptures at this point, and
the egg is extruded; it invariably emerges with the centripetal oil cap forward, due to the con-
tinued pressure of the swelling jelly in the wide centrifugal region of the perivitelline space.

This provides further evidence that the cortical response to activation in the Nereis egg is
basically similar to that in the echinoderm egg.

Electrical activity of supramedullary neurons of puffer (bloivfisli) Spheroidcs
maculatiis. S. M. GRAIN, M. V. L. BENNETT AND H. GRUNDFEST.

Supramedullary neurons of puffer, 300-500 /j. in diameter, are readily visualized and pene-
trated with microelectrodes. Their resting potentials range to 70 mv. Spikes up to 100 mv. in
amplitude and 3-5 msec, in duration are evoked directly by stimulation with an intracellular elec-
trode or with external electrodes close to the cell, and indirectly by scratching the skin, or by
electrical stimulation of spinal cord, dorsal (but not ventral) roots, or cranial nerves. Indirect
spikes have an inflection on their rising phase at 20-30 mv. depolarization, which is also threshold
for direct spikes. During refractoriness or hyperpolarization with another intracellular electrode,
indirect stimuli may fail to produce full-sized spikes, failure occurring at the inflection. The re-
maining small potential cannot be graded by varying stimulus strength.

When recording extracellularly close to the cell, electrode negativity indicates inward cur-
rent flow and active membrane under the electrode ; positivity denotes outward flow and active
membrane distant from the recording site. At the supramedullary cell's dorsal surface, extra-
cellular recording reveals a large positivity associated with the first component of the indirect
spike and variations from small positivity to large negativity associated with the bigger second
component. Therefore, the active membrane producing the second component neighbors or in-
cludes the cell's dorsal surface ; that producing the first is distant.

As the interval is shortened between a direct and a succeeding indirect spike, the second
component of the latter fails to fire. The first component, although reduced in size during the
observed post-spike reduction of membrane resistance, cannot be abolished. Moreover, it decays
with a time constant greater than that of the membrane. These data (see also abstract by
Bennett, Grain, and Grundfest in this issue) suggest that the first component of the indirect spike
is a postsynaptic potential.

The inhibition by a series of nitro- and haloplicnols of glucosc-6-phosphate dehy-
drogenase from Arbacia eggs and yeast. ROBERT K. CRANE, HOWARD H. HIATT
AND G. H. A. CLOWES.

The influence of a series of substituted phenols on the activity of glucose-6-phosphate dehy-
drogenase from Arbacia eggs (A) and yeast (Y) and of 6-phosphogluconate dehydrogenase
from Arbacia eggs (PGD) was determined with the usual spectrophotometric techniques. The
enzymes differed greatly in the degree to which they were inhibited. In general, A was strongly
inhibited at low concentrations of certain phenols ; Y and PGD were inhibited to a lesser extent
at much higher concentrations. The data suggest the possibility that the inhibition of A differs
in character from the inhibitions of Y and PGD. This possibility is being explored with enzyme
preparations from rapidly dividing cells other than Arbacia eggs. Also, the relative inhibition
of A by the individual phenols shows a pattern markedly different from that previously estab-

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 343

lished for inhibition of cell division and of aerobic phosphorylation. It will thus be possible to
investigate the respective roles of glucose-6-phosphate oxidation and of aerobic phosphorylation
in cell division and other cellular processes. The data from the present experiments are re-
corded below. The numbers given for each of the enzymes, A, Y, and PGD, are the per cent
of inhibition which occurred at the stated concentration of substituted phenol. The compounds
are listed in descending order of their inhibition of A. 2,4,5-trichlorophenol, 0.000066 M, A-55,
0.00033 M, A-86. Y-0, PGD-0; 4,6-dinitro-o-cresol, 0.00017 M, A-S8, Y-0, PGD-6, 0.0003 M,
A-80, Y-16, PGD-29, 0.000033 M, A-19, Y-0, PGD-0 ; 2,4-dinitro-o-cyclohexylphenol, 0.0002 M,
A-70, Y-17, PGD-18; 2,6-dinitro-4-chlorophenol, 0.00033 M, A-73, Y-0; 2,4-dichlorophenol,
0.00033 M, A-47, Y-0 ; 2,4,6-trinitrophenol, 0.00033 M, A-37, Y-0 ; 2,4-dinitrothymol, 0.00033 M,
A-23, Y-0, PGD-0 ; 2,4-dinitrophenol, 0.00033 M, A-13, Y-0, PGD-7 ; 4,6-dinitrocarvacrol, 0.00033
M, A-ll. Y-0, 0.001 M, A-54, Y-0, PGD-28; 2,6-dinitrophenol, 0.00033 M, A-8, Y-16, PGD-0,
0.0017 M, A-72, Y-16, PGD-1S ; p-nitrophenol, 0.00066 M, A-18, Y-0, PGD-0; o-nitrophenol,
0.002 M, A-55, Y-57, PGD-49.

Preliminary studies on the incorporation of glucose-U-C*4 into the poly sac charide
of Arbacia and Mactra larvae and its inhibition by 4,6-dinitro-o-cresol. ROBERT
K. CRANE, ANNA K. KELTCH, C. PATRICIA WALTERS AND G. H. A. CLOWES.

Twenty-four-hour swimming forms of Arbacia and of Mactra were incubated in sea water
containing added glucose-U-C14. After an initial period of incubation, various concentrations of
4,6-dinitro-o-cresol were added to some of the vessels and the incubation was continued. At
termination of incubation, the larvae were recovered by centrifugation and their acid-soluble
components were separated by paper chromatography. The two-dimensional solvent system of
Bandurski and Axelrod was used. The areas of the developed papers containing C14 were
identified by contact autoradiography. Three or four well-defined spots, which have not been
identified, could be made out. However, the greatest amount of radioactivity was found to re-
main at the origin, suggesting that significant incorporation into polysaccharide had occurred.
The radioactivity of this spot was markedly less when 4,6-dinitro-o-cresol had been added.
Polysaccharide identification of this spot was not attempted. Instead, "glycogen" was isolated
from duplicate samples of larvae by the conventional alkaline digestion-ethanol precipitation pro-
cedure. Assay of this "glycogen" for radioactivity by the usual counting techniques and for total
carbohydrate by the anthrone method revealed that glucose-U-C14 was indeed incorporated dur-
ing incubation. The specific activity of the "glycogen" in the control larvae increased during
prolonged incubation. In the "glycogen" of larvae to which 4,6-dinitro-o-cresol had been added,
the specific activity decreased. The experiments were not completed, owing to the scarcity of
viable eggs, and they will be continued next season.

The preservation of intact erythrocytes of marine vertebrates for blood group re-
search.1 ]. E. GUSHING, G. J. RIDGWAY AND G. L. DURALL.

The glycerol -freezing technique (cf. Chaplin et al., 1954) appears to be a promising method
for preserving the erythrocytes of marine vertebrates collected in the field. The following points
serve as a guide for further study. Cells of several species of fish and one humpback whale have
been treated. Whole blood from vessels in the gill, tail or other areas is allowed to run into
screwcap bottles, as much as 25 to 50 ml. being taken. An equal volume of a solution containing
glycerol (40%) and 5% trisodium citrate (60%) is added and the mixture placed in a freezer
(approximately —20° C.) after thirty minutes.

Cells are recovered from a few ml. aliquots of thawed blood by reducing the glycerol con-
tent of their milieu stepwise at five-minute intervals, using cool solutions. For example, albacore
cells were recovered by first adding an excess of 20% glycerol in 2% NaCl and then reducing
the glycerol, by dilution with 2% NaCl, to 14, 8.5, 6.5, 5.0, 4.0, 3.0, 2.0 and 1.5 per cents; 1.5%
NaCl was substituted and the glycerol reduced to 0.7, 0.35 and finally 0.0%. (Reasonable devia-

1 Supported in part by the Penrose Fund, American Philosophical Society, and the Office of
Naval Research.

344 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

tions from this schedule were also successful.) The cells obtained kept well and could be used
in agglutination and absorption studies.

While the degree to which different antigens remain unchanged during prolonged storage
probably varies, as in humans, cells of the whale, shiner seaperch, goosefish and three species of
salmon retained antigenic specificities during preservation. Absorption experiments suggest in-
dividual differences in the antigens of preserved albacore cells that appear to resemble those of
other tuna.

The effect of nervous system extracts on inhibition and excitation in single nerve
cells. C. R. ELIOT, A. KAJI, P. SEEMAN, E. UBELL, S. W. KUFFLER AND A. S.

V. BURGEN.1

Stretch receptors in lobster and crayfish contain a sensory neuron on whose dendrites an
efferent nerve fiber forms inhibitory synapses. On this system extracts from acetone-dried
lobster nerve cords and leg nerves were tested. First the physiological action of crude extracts
and then of chromatographically purified fractions was determined. The following effects on the
nervous structures were studied :

1. Change of discharge rate, largely reflecting the state of the dendrites of the sensory cell.

2. Changes in resting potential and in inhibitory synaptic potentials.

3. Changes in effectiveness of inhibitory transmission.

Three distinct kinds of action were seen, namely, slowing (inhibition) of sensory discharge,
acceleration of sensory discharge, and reduction in effectiveness of inhibitory transmission. Each
of these effects could be obtained separately from crude extracts, depending on concentration and
source of extracts, leg nerve extracts having little inhibitory effect on the discharge rate. In
chromatographic fractions run in phenol-ammonia-water or in chloroform-methanol-HCl, the
fractions with relatively low mobility mainly blocked inhibitory transmission and slightly de-
polarized the nerve cell. The most mobile fractions had a purely excitatory effect. Some of the
intermediate fractions inhibited the sensory discharge in a manner similar to gamma-amino-
butyric acid, causing mainly a small hyperpolarization or no appreciable membrane potential
changes. The activity of the excitatory material decreased after acid hydrolysis.

The mating type system in variety nine of Tetrahymena pyriforniis. ALFRED M.
ELLIOTT AND GORDON M. CLARK.2

Matings involving parental clones TC 105, TC 110, TC 156, TC 160, TC 148, and TC 89
were used, as well as their ¥•>, F3, F4, and F5 progeny. This variety differs from the other
varieties studied so far in that (1) the old macronucleus migrates anteriorly after nuclear ex-
change has occurred, (2) conjugation is delayed for 18 to 36 hours after cultures have been
mixed (25° C.), (3) no detectable immaturity period exists, and (4) this variety has only been
found in the tropics to our knowledge.

The lack of an immaturity period complicates the picture in that no check can be made as
to whether nuclear exchange has occurred. Since pairs are isolated when 70-80% are in the
anlagen stages, it is felt that a high percentage of cultures obtained are from pairs in which true
nuclear exchange has occurred. Variety nine possesses five mating types. No new ones have
been derived. A cross of any two mating types yields either of the parental types in 98% of
the cases. Mating types other than the parental, selfing caryonides, and non-reactive clones make
up the remaining 2%. Non-reactive clones may possess an immaturity period the length of
which may be genetically determined. These clones might be used in the future to obtain clones
with an immaturity period to serve as a check as to whether nuclear exchange has occurred,
which also can be checked by inbred lines using the serine mutant as a marker. Cytoplasmic
exchange occurs but may not be a universal phenomenon in the variety. Time of separation of
the pairs, after the anlagen stages are reached, is highly variable. The data to date would sup-
port a mating type system analogous to the B-system of Paramecinm aurclia with multiple mat-
ing types involved.

1 Fellow of the Lalor Foundation.

2 This investigation was supported in part by a research grant (PHSE 1416) from the Na-
tional Institutes of Health, Public Health Service.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 345

X-radiation effects during conjugation of Tetrahyinena pyrifonnis. ALFRED M.
ELLIOTT AND GORDON M. CLARK.1

An attempt was made using x-rays to induce back mutations to the serine and pyridoxine
requirement. Conjugating F3 progeny (var. 9) serine non-requirers and conjugating F3 progeny
(var. 2) pyridoxine non-requirers were x-radiated during early prophase. If no back mutation
occurred, the serine F4's should not require serine and the pyridoxine F4's pyridoxine. Dosages
from 200 kr-600 kr at 100-kr intervals were used (dose rate 4720 r per minute). Forty pairs
were isolated into peptone for each cross and for each dosage, with half the pairs incubated at
11° C. for 24 hours and then returned to 25° C. The remainder were incubated at 25° C. Suit-
able controls were used. Clones obtained were checked for growth on peptone, complete,
pyridoxine and serine-deficient media.

Vegetative cells survived dosages of 600 kr as opposed to 300 kr for conjugants. No sig-
nificant differences in survival were observed for pairs incubated at 11° C. or 25° C. In the
pyridoxine cross viability was 17.7% for controls, 27.5% for 300 kr and 40% for 200 kr. The
serine control gave viabilities of 62.5%, 12.5% at 300 kr and 25% at 200 kr (pooled data for
both temperatures). Serine conjugants are more sensitive to x-radiation than the pyridoxine
ones. The apparent higher viability in the x-rayed versus the control pyridoxine cross may be
due to separation of pairs before nuclear exchange has occurred. From the pyridoxine work 27
irradiated clones were obtained. Eighteen grew without pyridoxine, six required and three
would grow only on peptone. In the serine study 15 clones were obtained, 12 of which were
serine non-requirers, two gave variable results and one would grow only on peptone.

Prophase is a radiosensitive stage in conjugating forms with bridges and fragments observed
at anaphase stages.

Post x-radiation effects of temperature on vegetative cells of Tetrahyinena pyri-
fonnis. ALFRED M. ELLIOTT AND GORDON M. CLARK. x

Variations exist in the x-ray sensitivity for the various varieties of Tetrahyinena. Strains
in varieties 1, 2, 5 and 6 survive 500 kr and 3, 4, 7 and 8 stand 400 kr. Strains TC 89 (serine
mutant) will not survive above 300 kr although inbred F4 lines survive 600 kr.

Using starved cultures of TC 89 (var. 9), the effects of various post x-radiation tempera-
tures on fission rate and recovery after incubation at 11° C., 15° C., 20° C., 25° C., 32° C.
and 35° C. were studied. Dosages of 100, 200, and 300 kr were employed (dose rate 4720 per
minute). Forty vegetative cells were isolated onto peptone (25° C.) and incubated at the vari-
ous temperatures for 24 hours and then returned to 25° C. Suitable controls were used.

A stimulatory effect of 100 kr, as reflected by an increased rate of fission over controls, was
seen at all temperatures. Above 100 kr suppression of fission by x-rays increases with increas-
ing dose. Survival of cells after x-radiation is somewhat higher at lower temperatures, with
variable results at 11° C. and 20° C. No irradiated cells survived a post-irradiation tempera-
ture of 35° C. For those cells surviving the higher temperatures, recovery was more rapid and
seemingly temperature-dependent, increasing with increasing temperatures.

Abnormal cells, characterized by budding and blistering of the pellicle, as well as fusion of
fission products to form multinucleated masses, were common at 300 kr. A delay in appearance
of these forms was evidenced at lower temperatures. Incubation for longer periods of time after
x-radiation at various temperatures may produce in the future studies with more striking results.

The effects of some aniino acids on the perfused lobster heart. P. E.'S. ENGER AND

A.'S. V. BURGEN.2

The response of the perfused heart of the lobster Hoinanis americanus to various amino
acids has been tested. Aspartate and glutamate were both found to have a marked stimulant

1 This investigation was supported in part by a research grant (PHSE 1416) from the Na-
tional Institutes of Health, Public Health Service.

2 Fellow of the Lalor Foundation.

346 PAPERS PRESENTED AT MARINE .BIOLOGICAL LABORATORY

effect on the rate and amplitude of the heart beat, effective down to a few micrograms. Aspartate
had a greater action on the rate than had glutamate. Both substances in higher concentrations
produced an increase in diastolic tone. Asparagine was ineffective, but glutamine showed a very
weak stimulatory action possibly due to contamination with traces of glutamate. L and D
glutamate were equally effective. A powerful inhibition of the heart was produced by 7-amino-
butyrate. LTsually this action was purely on rate but occasionally some depression of force of
contraction was also produced. A similar but weaker effect was produced by /3-guanidopro-
pionate, /J-alanine, 7-guanidobutyrate, 5-aminovalerate, and S-guanidovalerate in descending order
of activity. No significant response was obtained with glycine, alanine, leucine, serine, lysine,
arginine, cystine, methionine, tyrosine, histidine, hydroxy-proline, /3-aminobutyrate, «>-amino-
octanoate, or the peptides diglycine, tryglycine, and carnosine. Aqueous alcohol extracts of
lobster nerve cord and leg nerves produced regular stimulation of the heart rate and amplitude
and in some dilutions a transitory inhibition. Preliminary separation of these two types of ac-
tivity was obtained by paper chromatography with a phenol-water-ammonia system. These
fractions were also tested on the segmental stretch receptors of the lobster and crayfish.

Electron microscope studies of the flagella of Chlamydomonas. SARAH P. GIBBS,
DELBERT E. PHILPOTT AND RALPH A. LEWIN.

Ultrathin sections of Chlamydomonas inoeivusii were studied to determine normal flagellar
structure, the structure of the flagella in paralyzed mutants, and the nature of the protoplasmic
bridge formed between mating cells. Each flagellum reveals the characteristic 9 double tubular
filaments surrounding a central pair, all imbedded in a structureless matrix and enclosed by a
thin sheath continuous with the plasma membrane of the cell. The two central fibrils extend
slightly beyond the peripheral ones, forming a mucronate tip. Slightly distad from the cell
surface, a diaphragm traverses the flagellum, and below this the 9 peripheral fibrils continue
to form the walls of the basal body (blepharoplast). Extending slightly above and below the
diaphragm, there is a short cylinder (possibly two appressed half -cylinders) of osmiophilic
material in the center of the flagellum and the blepharoplast. The central fibrils appear to
end just above this cylinder. In this species of Chlamydomonas the two flagella arise from
opposite sides of an anterior papilla and the blepharoplasts are continuous basally. Anteriorly
the fused basal bodies send forward a conical median projection into the papilla.

The flagella of five paralyzed mutants were found to be identical to normal flagella in all
respects. Two mutants had stubby flagella, 2-4 ^ instead of 12-24 /x long, but otherwise normal
in structure. Two mutants which through the light microscope appear flagella-less were found
to have very short flagella 0.6-1. 0,u long. Their structure is under investigation.

Sections of mated pairs show that the intergametic bridge is formed by an extension of the
anterior projections of the blepharoplasts. Whether the anterior projections from both cells
grow out and fuse with each other, or whether only one grows out, invades the other cell, and
connects to its blepharoplast, is presently being studied.

Effect of sodium fluoride on the development of Arbacia punctulata. SYLVIA S.
GREENBERG AND ARTHUR GREENBERG.

Fluoridation of community water supplies, as a caries preventive measure, is a widespread
practice. The optimum concentration of sodium fluoride has been determined to be one to two
parts per million. Hypotheses have been advanced as to the nature of the chemical reaction
between the fluoride ion and bone and tooth structure. Most of the experimental work has
been done with rats and hamsters fed increasing concentrations of fluoridated water. Subsequent
sectioning and ashing of hard tissues have indicated the extent of incorporation of fluoride ion.
In the present experiments, the eggs of the sea urchin, Arbacia punctulata, were fertilized and
allowed to develop in sea water containing varying concentrations of sodium fluoride. Skeletal
changes in the developing plutei were followed. At concentrations of one and ten parts per
million, differences between experimental and control animals were not detectable with the
light microscope. At fifty parts per million, approximately 60% of the animals exhibited
skeletal aberrations. These included plutei with one arm, no arms, partially developed arms,
as well as completely skeletonless bodies. At seventy-five parts per million, most of the cultures

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 347

were entirely free of skeletal spicules. When these amorphous forms were placed in non-
fluoridated sea water, skeletal development was stimulated in two days.

The intercellular cement of sodium fluoride-treated embryos was noticeably weakened at
concentrations of fifty and seventy-five parts per million. Free blastula cells were present as
well as exogastrula forms. The affected embryos resembled those grown in calcium-free sea
water. The reduction in adhesiveness was not as great, however, since many animals were
able to develop to more advanced stages. The data suggest that high concentrations of sodium
fluoride in the growing media remove available calcium and phosphate ions, which are necessary
for normal development of Arbacia punctulata embryos.

Stimulation of the taste receptors of the rat with organic salts. C. W. HARDIMAN.

The responses of the chemoreceptors of the tongue were studied by integrating the spike
potentials elicited in the chorda tympani nerve bundle. A constant area of the tongue was
bathed with the various stimulating solutions.

The concentration/response curves were determined for a number of organic salts. The
response increases as the concentration increases until 0.5 M ; the response is then almost
maximal and doubling this concentration gives only a slightly larger response. The concentra-
tion at which the maximum or saturated response level occurred varied with the anion within
a given cation series. The order of the ability of the anions to stimulate within a sodium
series was : chloride > oxalate > citrate > tartrate > succinate > formate > salicylate acetate
> glutamate > propionate > butyrate > oleate. The response curves of many of these same
anions were determined using Li, NH4, Alg, and K. The relative order of the anions remained
the same within each of the cation series although the cation species determines to a great
degree the magnitude of the maximum response.

An enhanced response of NaCl occurred after an application of lithium citrate. This
enhancement was time-dependent, in that the response of NaCl shortly following lithium citrate
was much greater, whereas it declined to the standard response within a few minutes. Other
citrate salts did not show this enhancement, nor did oxalates or mono-sodium glutamate.

Potassium benzoate below 0.25 M did not stimulate the salt receptor, but indeed inhibited
the small amount of spontaneous background activity. As the solution was washed from the
tongue, there was a large response to water. Concentrations above 0.25 M responded as a
typical salt. Neither KC1 nor other benzoate salts produced this effect.

The spectral energy curve of Cypridina and other luminous organisms. E. N.
HARVEY, A. M. CHASE AND W. D. MCELROY.

Thanks to the loan of a spectrophotofluorometer by the American Instrument Company,
spectral energy curves for the bioluminescence of colorless perfectly transparent aqueous
solutions of Cypridina luciferin and luciferase have been recorded with an accuracy of ± 5 m//,.
The maximum emission of Cypridina is at 470 m/x and extends from 410 to 620 m/j.. The
luminous bacterium, Achromobacter fischeri, has a maximum at 495 m/j. and extends from
440 to about 600 m/j.. Two new strains of luminous bacteria, isolated at Woods Hole from
flounder and from squid, have essentially the same spectral distribution. The ctenophore,
Mnemiopsis leidyi, squeezed through cheesecloth and with water added to the transparent
suspended material, luminesced with a maximum at 490 m/t and its emission extended from 430
to 610 m/i. A pale yellow solution of luminol plus H2O9 excited to luminesce with catalyst
A exhibited an emission with a maximum at 460 m^, and extending from 380 to 610 m^. All
curves are slightly unsymmetrical, with the maximum nearer the blue than the red end of the
emission spectrum.

Neuromuscular transmission in Limulus. G. HOYLE.

The electrical and mechanical events associated with neuromuscular transmission have been
studied particularly in the closer muscle of the claw of the walking legs, with the aid of
intracellular recording electrodes. The mean resting potential is about 50 mV. This muscle

348 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

is innervated by both a "slow" and a "fast" nerve fiber. The mechanical response to single
excitation of the former is very small. At frequencies of about SO per second a smooth tonic
contraction results. The electrical response consists of a small junction potential. This shows
a moderate degree of facilitation. At about 50 per sec the maximum height is about 5 mV
depolarization. The duration is about 50 msec.

The mechanical response to stimulation of the "fast" nerve fiber consists of a sharp twitch,
though this does not completely close the claw. The electrical responses to a single shock
vary in different fibers from about 2 to 8 mV. Their duration is about 50 msec. The smaller
ones have the characteristic shape associated with a pure postsynaptic potential. Some of the
larger ones show a departure ; there is a small, secondary response of about 4—5 mV magnitude.

The responses show no refractoriness. During repetitive stimulation there is a small
amount of growth (facilitation) in the "fast" responses. They summate completely. If they
are sufficiently closely spaced the fifth or sixth may give rise to a secondary response which
reaches or just overshoots the zero baseline.

The major part of the response thus consists of a small synaptic potential which never-
theless gives rise to a twitch contraction. Repetitive stimulation can evoke secondary, graded
electrical responses which possibly cause an augmentation of the mechanical response.

Comparative distribution of radioactive alloxan, thiocyanate, and urea in islet and
other tissues of the toad fish (Opsanus tan). ARNOLD LAZAROW AND S. J.

COOPERSTEIN.

As part of a project designed to determine the mechanism by which alloxan selectively
kills the insulin-producing cells of the islets of Langerhans, we have studied the uptake of
radioactive alloxan by islet and other tissues of the toadfish. Since the radioactivity of the
various tissues measured includes the radioactivity of contained blood, extracellular fluid, and
intracellular fluid, we have attempted to differentiate between these components by comparing
the distribution of radioactive alloxan with that obtained for sodium thiocyanate and urea.
Sodium thiocyanate is distributed throughout the extracellular fluid, but it does not enter the
cells. On the other hand, urea is distributed throughout both the intracellular and extracellular
phases.

The data obtained to date indicate that alloxan and thiocyanate have similar patterns of
distribution in the various tissues (with the exception of kidney) at all times following in-
jection. On the other hand the amount of urea present in the various tissues is much greater
than that observed for either thiocyanate or alloxan. Within 2-5 minutes after injection, the
amount of alloxan and thiocyanate present in the islet tissue represents about 20% of the blood
value. However, within 5 minutes after injection, the amount of urea in islet is equal to 62%
of the blood value. By the end of 30 minutes, both the alloxan and thiocyanate have reached
a value equal to 42% of blood whereas the urea reached a value equal to 85% of blood.

These preliminary results suggest that the injected alloxan is distributed primarily in the
extracellular fluid. However, further work is necessary before any final conclusions can be
drawn. If this observation proves to be correct it would suggest that alloxan may produce its
effect by acting on a system at the surface of the beta cell.

A further study on the induced furrowing reaction in Arbacia punctulata. DOUGLAS
MARSLAND 1 AND WALTER AUCLAIR.

Premature furrowing, starting as early as 40 minutes ahead of normal schedule (at 20° C.),
can be induced when fertilized eggs are centrifuged for 2-5 minutes at high force (40,000-
50,000 G) and at high pressure (6,000-12,000 lbs./in.2).

The induction of the furrowing reaction appears to depend upon the centripetal displacement
of material derived from, or affiliated with, the nucleus, as is indicated by the following
considerations.

1) Eggs treated prior to prophase of first cleavage never display induced furrowing unless
the nuclei are broken by the treatment. Conversely, when the nuclei are broken, induced

1 Work supported by the National Cancer Institute, Grant Series C-807.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 349

furrowing always occurs in a considerable number (5-95%) of the eggs, starting some 3-6
minutes after the treatment.

2) The unbroken nuclei, in the centrifuged non-pressurized controls, come to lie in the
hyaline zone, close to the oil cap, but in cells with broken nuclei, one finds 1-3 relatively small
Feulgen-positive fragments lying in or near the mitochondrial zone. No trace of the other
nuclear material, which presumably is thrown in a centripetal direction, has been found. Asters
and spindles are not found when furrow induction precedes the first mitosis.

3) The plane of induced furrows is always at right angles to the axis of centrifugation,
whereas that of non-induced furrows (in control eggs centrifuged without pressure) is always
parallel. Induced furrows tend to be displaced toward the light pole, quite drastically when
higher pressures and forces are used.

4) Eggs treated during early prophase, just before the nuclear membrane disappears,
require less pressure and force for breaking the nucleus and inducing furrows.

5) With eggs treated during late prophase, metaphase or anaphase, centrifugation alone,
without pressure, suffices to induce furrowing.

Shortening of potassium depolarised muscle in different electric fields. HIDENOBU
MASHIMA AND ARPAD CSAPO.

Muscles were marked off into several segments along their longitudinal axis by a fluorescent
dye (Zn Cd S Phosphor), and were illuminated by ultraviolet light. A continuously moving
film recorded the movement of these marks during shortening of the loaded muscle. Stimula-
tion was made by electric fields set up in the perfusion Ringer, using 60 c/s a.c., rectified a.c.,
constant current and repeated square pulse d.c.

Then the muscle of the frog was rendered non-propagating by excess K, 16 mM/1. (sub-
stituted for Na). In the longitudinal a.c. field at 2 V/cm. the ends and the middle portion of
the muscle contracted whereas the regions adjoining them did not. When relative shortening

(non-propagating \

propagatin°- ' / was P^otte^ against the position of the segments along the length of

the muscle, a W-shape curve was obtained. Increasing the field strength resulted in a gradual
smoothening effect on the W-shaped curve as the segments close to the ends increasingly
participated in the shortening resulting in a convex curve. At 8 V/cm. the convex curve was
smooth. In d.c. fields only the cathodal end shortened.

When the longitudinal a.c. field was stepwise rotated so as to become eventually transverse,
shortening along the length of the muscle became gradually more uniform and in our plot of
relative shortening transverse stimulation yielded a straight horizontal line. In d.c. transverse
field there was no shortening whatever.

Contraction at the ends of the non-propagating muscle in the longitudinal a.c. field or
shortening of the whole muscle in the transverse a.c. field may be explained by the depolarizing
effect of the applied current. But shortening in the middle portion, when the segments close
to the ends are uncontracted, cannot be explained by the depolarizing action of the external
longitudinal a.c. field.

Uterine strips from pregnant rabbits were rendered non-propagating by excess K = 120

mM/l.( jp-= 1 ). Shortening was more excessive in the longitudinal than in the transverse
a.c. field. In the longitudinal d.c. field the strip shortened along its whole length.

Cytophysiology of ultracentrifuged normal and neoplastic frog kidney cells.1 G. M.
MATEYKO.

Viable cell populations of normal kidney cells and tumor cells (Lucke renal adenocarcinoma
of Rana pipiens) were obtained by mechanical means or by trypsin treatment. To effect

1 This investigation was supported in part by a research grant (C-3490) from the National
Cancer Institute, U.S.P.H.S.

350 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

intracellular displacement and stratification of the subcellular components, the cells were sub-
jected to high centrifugal fields (Spinco model L centrifuge). To prevent crushing, an
isopycnotic cushion was established, the most satisfactory one being an isotonic, isosmotic
stabilized colloidal silica with a density gradient maximum of 1.19. Most of the tumor cells
had a density (ca. 1.123) greater than that of normal cells.

For the neoplastic population incipient stratification became evident, in a few cells, after
centrifugation for 60 minutes at 60,000 G at 10° C, but was clearly defined only after ultra -
centrifugation at 110,000 G. A typical stratified cell was deformed into an ovoid shape with an
agranular centripetal pole and a centrifugal end packed with small to large granules, with
the nucleus shifted to the sub-equatorial or centrifugal zone. Normal cells showed an incipient
stratification after two hours of ultracentrifugation at 110,000 G. Although they eventually
stratified after four hours at 110,000 G, they never exhibited the range of subcellular displace-
ment (centripetal lipid droplets, packing of granules into layers with sharply defined boundaries,
pulled out and elongated nuclei, and extranuclear nucleoli) that was characteristic of malignant
cells. Other cytophysical differences became exaggerated under high accelerations ; for example,
nucleolonemata remained undisplaced, but optically homogeneous (phase contrast observations)
nucleoli exhibited a centrifugal shift within nuclei.

Relocation of the displaced components occurred imperceptibly in normal cells and rapidly
in malignant cells. In the latter, within ten minutes after cessation of centrifugation, vigorous
cytoplasmic turbulence was observed. The original cytoarchitectural configuration was gen-
erally restored within two hours. Ciliary movement in normal cells continued even after ex-
posure to high centrifugal fields. Accordingly, cytoplasmic consistency and density differ in
normal and malignant cells.

Cytochemical studies will complete the identification of stratified subcellular constituents,
already partially determined by supravital dye techniques.

Studies on the accelerator cleavage factor recovered in homogenatcs of Arbacia
punctulata ovaries. VALY MENKIN, LOUISE MENKIN AND RICHARD S.

HEILMAN.1

The aqueous homogenate of the ovaries of Arbacia after centrifugation at 510 G for 10
minutes yields a supernatant fraction which in turn is centrifuged at 1150 G for 10 minutes.
The supernatant (Si), as a rule, contains predominantly the retarding factor. However, this
fraction can be dialyzed for several days against distilled water in a refrigerator. The diffusate
is found to contain the accelerator cleavage factor. Thus, by differential centrifugation followed
by dialysis, the accelerator factor can be dissociated from any admixed retarding factor.
Eventually the retarding factor will also tend to diffuse outward, but apparently this is a slower
process, presumably owing to its larger molecular size. Following centrifugation to obtain
the above described Si in the supernatant, the sediment in 0.25 M sucrose is centrifuged for 20
minutes at 8,170 G. The supernatant freed of mitochondrial particles is centrifuged one hour
at 21,600 G in a Servall angle centrifuge. The soluble phase (Sz of the accelerator factor) is
then dialyzed against water in the cold for several days to eliminate some of the admixed
retarding factor. In 9 out of 12 experiments, the accelerator factor has been recovered in the
diffusate. In the Si diffusate, 6 out of 8 experiments revealed the presence of the accelerator
factor, the average number of ova in the first blastomeric division being 44.5% in the experi-
mental group and 22% in the controls. In the succeeding cleavage the averages were 46% and
26.5%, respectively. Absorption measurements of this active Si diffusate fraction by Dr. J. S.
Roth have indicated a peak at 265 to 270 millimicra. Test for the presence of ATP by Dr.
W. D. McElroy has been negative. The fraction is yellow in color, and in the visible range
manifests a very tiny prominence at 390 millimicra. Studies, at the suggestion of Dr. O.
Lindberg, by the phenol-water-ether extraction method indicate that the purified accelerator
factor is a dinucleotide. Since uracil per se induces similar accelerating effects on cleavage,
the possibility that the factor is a uracil dinucleotide is suggested.

1 Aided by grants from the Society of the Sigma Xi and the National Cancer Institute,
U. S. Public Health Service.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 351

Studies on the nature of the retarding cleavage factor in homogenates of sea urchin
ovaries. VALY MENKIN, LOUISE MENKIN AND RICHARD S. HEiLMAN.1

The aqueous homogenate of ovaries of Arbacia punctulata was purified by differential
centrifugation as follows : centrifugacion of the homogenate at 510 G for 10 minutes yielded a
sediment, in turn treated with 0.25 M sucrose. The suspension was centrifuged at 21,600 G
for 10 minutes. The supernatant was then centrifuged at the same speed for one hour. The
resulting soluble phase (S2), devoid of mitochondrial particles and probably of some of the
microsomal fraction, was a clear yellow fluid which was very active in inducing a retardation
in the cleavage development of ova. Usually the ova were exposed to the material for 20
minutes prior to fertilization. In two experiments the ova were first fertilized and then the
S2 fraction added. The results were similar in both procedures. From 0.5 to 1 ml. of the
S2 fraction were employed. It was found in 18 experiments, about 44 minutes following
fertilization, that an average of 38.9% of control ova were in the first blastomeric division
as compared with an average of only 2.9% in the experimental series. The retarding effect
was likewise reflected in the second or further advanced cleavages, the average being 30.4%
in the controls and 18.9% in the experimentals. The retarding factor was found to be heat-
stable, withstanding boiling for 30 to 45 minutes. The active principle was diffusible through
cellophane tubing of size 20/32. Absorption measurements by Dr. J. S. Roth and Mrs. Laura
Inglis with a DU spectrophotometer indicated a peak at 265 to 270 millimicra. These facts
suggest the nucleotide nature of the retarding factor. ATP and ADP were found present in
one fraction but not in another active one by Dr. W. D. McElroy, by utilizing the firefly test.
In the visible range there is also a peak at 400 millimicra. The nature of the yellow pigment
is not yet clear. Addition of ascorbic acid induces temporary disappearance of the color, to be
soon replaced by a deep orange color. This suggests that an oxidation-reduction system may
also be present in the purified material.

Study of diatom populations on sand and mud flats in the Woods Hole area. E. T.
MOUL AND DAVID MASON.

The presence of large populations of animals on and in mud flats of Barnstable harbor has
raised the question of the source of primary production. Since few quantitative studies have
been made of the diatom flora of mud flats, this study was undertaken.

Duplicate mud cores 2.5 cm. in diameter and 6 cm. deep were taken in plastic tubes from
the mud flats of Barnstable harbor. The tubes were frozen and sectioned into layers. The
samples from one tube were diluted with sea water and counts made of living diatoms. The
duplicate sample was treated with acetone to extract chlorophyll. The number in the surface
2.5 cm. of mud varied from 9000 cells per cubic millimeter of mud in June to 500 cells per
cubic millimeter in August. Living diatoms were found in the mud to a depth of 6 cm., de-
creasing markedly, however, below the surface layer. The number in the mud 5 to 6 cm.
deep varied between 40 cells to 0 cells per cubic millimeter. The total calculated number of
cells in a column of mud beneath a square meter of surface in one collection, for example, was
9.8 X 109 cells. These counts should be regarded as a relative index of abundance, since many
of the diatoms grew tightly appressed to individual sand grains affording a possible source of
error.

Chlorophyll was determined colorimetrically and presented in graph form along with the
counts. There was general correspondence between the curves given by the two methods. In
the example given above, the chlorophyll present in the column of mud was .948 gram per
square meter. The order of magnitude of cells and chlorophyll present is in general agreement
with that found for phytoplarikton in a column of water beneath a square meter of sea surface
in coastal waters.

1 Aided by grants from the Society of the Sigma Xi and the National Cancer Institute,
U. S. Public Health Service.

352 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

The effect of varying concentrations of ribonucleic acid on the development of some
marine embryos. M. C. Niu AND STEVEN D. DOUGLAS.

It has been demonstrated recently by the senior author that potency of isolated ribonucleic
acid (RNA) from animal tissue is related to concentration. For in vitro differentiation of
presumptive ectoderm, the concentration was 20-40 /ug per milliliter in modified Holtfreter
solution. A ten-fold concentration or dilution eliminated this effect upon differentiation. The
present experiment was conducted to investigate the possible role of RNA concentrations in the
development of some marine embryos.

Embryos of Styela partita (two-cell stage), obtained by artificial fertilization, were em-
ployed, as were those of Arbacia pimctulata. They were grown in pasteurized sea water con-
taining known amounts of RNA, isolated from the liver of Raia crinacca. Chemical tests
for polysaccharide and protein in the RNA were negative.

At concentrations of 700 or more /ug per milliliter, RNA arrested development at cleavage
stage, 16-64 cells in Styela. At approximately 150-450 /j.g per milliliter rate of development was
decreased ; in this series, when the tadpoles hatched, the body became round and abnormal, a
conspicuous otolith was present, and cross-sections revealed the reduction of neural tissue,
particularly the cerebral vesicle. With concentration lowered from 100 to 5 /ug per milliliter
the rate of development increased, with a maximum, equal to or occasionally faster than the
control. However, at concentrations lower than 3 /j,g per milliliter no appreciable effect was
observed.

The effect of lower concentration is more striking in experiments with Arbacia embryos :
approximately 4-16 t^g per milliliter accelerated development. Thus, in a typical series, after
treatment with RNA (4 /ug per milliliter), for twenty-six hours, at room temperature (22° C.),
most embryos attained the young prism to young pluteus stage, while in the control they were
predominantly in the gastrula to prism stage.

In both forms the rate of development was influenced by RNA isolated from the liver of
Raia. At optimal concentration of RNA, when the embryos hatched, neural structure in
Styela appears to have been reduced.

Production of permanent lesions in living protoplasm. W. J. V. OSTERHOUT.

Colorless root cells of Nitclla were used on account of their transparency.

Acid fuchsin did not enter the cell unless it was injured. Brilliant cresyl blue entered the
vacuole more rapidly the higher the external pH value but as soon as the cell was injured the
dye entered independently of the external pH values. These results were used as criteria of
injury.

When a living cell was bent at the center, a lesion was formed which stained red with acid
fuchsin but the rest of the cell remained colorless. When the cell was transferred to a solution
containing no acid fuchsin, the dye escaped from the lesion and the cell lived for a long time in
spite of the presence of the lesion.

The cyclosis stopped on bending but soon it was resumed and took a normal course unless
the condition of the lesion prevented it.

In bending, cytoplasmic masses were often forced into the vacuole and they moved slowly
and often revolved in the sap.

Sometimes the vacuole separated at the point of bending into two vacuoles but they later
coalesced to form one vacuole again. If the cell was transferred to a solution of brilliant cresyl
blue, the dye entered the vacuole more rapidly the higher the external pH value.

The vacuole stained red with neutral red separated into two red vacuoles at the point of
bending but the space between them was colorless. Later the red vacuoles coalesced to form
one red vacuole again.

A few cells freshly collected were found with some lesions. Their behavior toward acid
fuchsin and brilliant cresyl blue was similar to that of the cell which had a lesion after bending.

These results indicate that permanent lesion can be present at one or more points on a cell
without affecting the rest of the cell.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 353

Selective permeability in relation to movement of water into living cells. W. J. V.
OSTERHOUT.

The purpose of the experiments was to determine if the movement of water controlled the
rate of entrance of solutes into living cells.

A living cell was divided into two parts, A and B, by means of a vaseline seal. Water was
placed at A and 0.025 M sucrose solution was placed at B. The water rapidly moved into the
cell and travelled inside the cell from A to B, carrying with it particles which collected at B.
No injury occurred.

When the water at A was replaced by a solution of acid fuchsin the water entered rapidly
as before but the dye did not enter the cell unless it was injured. The acid fuchsin did not enter
the living cell immersed in the dye solution unless it was injured. These experiments indicate
that the movement of water into the cell does not control the rate of penetration of the solutes.

This was confirmed by experiments with basic dye, brilliant cresyl blue. If the water at A
was replaced by brilliant cresyl blue at pH 5, in which the dye was chiefly in the form of ions, no
dye entered the cell though the water entered rapidly as before. But the dye penetrated rapidly
into the vacuole from a solution at pH 9 in which the dye was chiefly in the form of undis-
sociated molecules, though the rate of movement of water remained unchanged. The dye was
largely dissociated in the sap at pH 5.5. The dye moved from A to B and collected at B when
the sucrose solution was at pH 9 but the dye escaped from the cell at B when the sucrose solu-
tion was at pH 5. When the cell was immersed in the dye solution the dye penetrated more
rapidly at pH 9 and escaped from the cell more rapidly into the solution at pH 5 containing
no dye.

Vital staining of eggs of Spisula solidissima by methylene blue.'1 LIONEL I.
REBHUN.

Washed eggs of Spisula solidissima are stained over a period of hours in solutions of
methylene blue in sea water. Initial concentrations of dye used are one part in 1,000,000 and
final ones, one part in 250,000. Small (%-micron) particles appear in the cytoplasm and be-
come larger and darker with increased dye concentration and time of staining. After fertiliza-
tion, these particles migrate to the asters. By the time the first polar body spindle is formed,
the particles outline the asters, although many particles remain in other parts of the egg. By
the first polar body stage, the particles are usually tightly organized about the asters and spindle.
The particles may move rapidly (up to 2-3 microns per second) into the aster. They surround
the female pronucleus when it is formed and then form a ring in the plane tangent to the
pronuclei when these come into contact. The ring divides into two masses, each surrounding
an aster, and each mass is distributed to one blastomere at cleavage. Usually the larger blasto-
mere receives more particles. The particles gather at the peripheral cap of the re-formed
blastomere nucleus and just before the next division this cap divides in two, each half surround-
ing an aster. After division, the peripheral cap forms again on each nucleus. The sequence of
dividing into two before cleavage and cap reconstitution after cleavage is followed by the particles
into late cleavage stages.

Electron micrographs of normal and stained eggs indicate that the dye causes the forma-
tion of vacuoles in the cytoplasm. These vacuoles appear to be swollen, distorted mitochondria.
If so, only certain mitochondria appear susceptible and it is these which undergo the described
changes in localization.

The biochemical basis for positive photokinesis of the starfish, Astcrias forbesi.2
MORRIS ROCKSTEIN AND MELVIN RUBENSTEIN.

Pigments were extracted from the dorsal skin and "eyespots" of dark-adapted animals
through acid buffer and into alkaline 2% digitonin solutions and their absorption spectra de-

1 Aided by a grant from The American Cancer Society.

2 This research was supported in part by a grant from the Sigma Xi-RESA Research Fund
and by a grant from the National Science Foundation.

354 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

termined before and after exposure to light of wave-lengths from 300 to 700 m/j.. Both types
of extracts showed maximum absorbance at 495 rmx, with a primary minimum at 725 and a
secondary minimum absorbance at 395 mfj.. Exposure to sunlight, ultraviolet and visible light
up to 450 m/j. bleached either type of extract in a similar fashion, without altering visibly its
natural violet color ; maximum density loss occurred at 585 m/i, minimum loss occurred at 485
m/u; no change (isosbestic point) occurred at 520 m/j.. Minimal changes in the entire absorp-
tion spectrum were observed for exposures at 500 and 550 m/j. (i.e., near the isosbestic point),
whereas at 600 to 700 m/i, changes in absorption spectra for either kind of extract were exactly
reciprocal of those obtained earlier at lower wave-lengths ; i.e., maximal density loss at 480 and
minimal at 580 mp., with the same isosbestic point of 520 rmx. The consistent occurrence of an
isosbestic point at 520 m/j,, as well as the similarity of their respective "difference spectra," indi-
cates a common photosensitive biochemical component in the diffuse skin and more highly or-
ganized "eyespot" receptors of this animal. Their possible respective roles in the positive light
orientation of this animal are being further explored in the form of correlative behavioral and
pigment sensitivities to different intensities of different wave-lengths of light.

The initiation and inhibition of cleavage of the Chaetopterus egg by ethyl urethane.
HERBERT SCHUEL.

Because many of the same chemical and physical agents that initiate cell division also
inhibit it, experiments were conducted for the past two summers on the effect of ethyl urethane
on the cleavage of the Chaetopterus egg. Eggs placed in a 1% solution of urethane in sea water
5 minutes after insemination did not cleave, and viscosity studies made with the hand centrifuge
indicated that the mitotic gelation, without which the spindle cannot form, did not occur.
Similarly, fertilized eggs placed in 2% or 3% solutions did not cleave, and the mitotic gelation
was again absent. Return of these eggs to normal sea water after the controls had cleaved
showed the treatment with a 3% solution to be irreversible while the treatment with the 2%
solution was only partly reversible. Almost all the eggs treated with a 1% solution cleaved
within 15 minutes after being returned to sea water; a rapid and sharp increase in viscosity cul-
minated in cleavage.

Unfertilized eggs exposed to a 3% solution for periods ranging from 10 to 60 minutes be-
gan to cleave and develop, with a peak of activity evident at about 30 minutes exposure. Opti-
mum cleavages ranged from 20% to 88%. Viscosity studies showed an increase of the same
order of magnitude as the mitotic gelation. A 2% solution would only occasionally initiate
cleavage and development, indicating it to be a threshold concentration.

Larvae produced either from unfertilized eggs treated with 2% or 3% solutions or from
fertilized eggs treated with 1% or 2% solutions appeared to be morphologically abnormal, and,
instead of swimming about, rotated rapidly in place on the bottom of the dish.

Preliminary studies of tlic ontogeny of schooling behavior in the silversides, Menidia
menidia. EVELYN SHAW.

The development of schooling was studied to try to clarify some of the factors involved in
the interaction of fish to fish when the first school is formed. It was found that schooling does
not appear immediately after hatching but develops gradually as the fish matures. Sixty five
individual fish, swimming freely in glass bowls, were observed in their responses to other fish.
No schooling occurred in fish younger than 9 days. Between 10 and 15 days of age, schooling
was brief and sporadic after a long period of no response. On the 17th day all of the fish re-
sponded instantly and schooling continued throughout the observation. At this time the fry
had grown from 5 mm. at hatching to 12 mm., the size of the smallest schooling fishes found
in the field and in our laboratory aquarium.

The initial stimulus to schooling appears to be a visual one. This was tested by placing a
fish in a sealed glass tube with a fish who was swimming freely in a bowl. Fifteen out of twenty
fish attempted to line up with the fish in the tube. After one or two minutes schooling ceased
and the fish vibrated near the tube, suggesting that additional stimuli may be necessary to the
continuance of schooling.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 355

In another series of experiments eighteen fish were deprived of early social contact with
species mates to see if this environmental condition would influence the appearance of schooling.
On the 17th day of development species mates were introduced into their bowls. Nine of the
separated fish schooled within the first minute while the remaining nine did not school during
the entire twenty minute observation. After twenty four hours, however, schooling was noted
in all of the test fish. Evidently lack of contact with species mates does not alter the pattern
of schooling, but initially inhibits the number of fish participating in this behavior.

This work was supported by an ONR contract with the MBL.

Effects of x-ray irradiation on two strains of Tetrahymena corlissi.1 CARL CASKEY
SPEIDEL.

Two strains of a recently named new species, Tetrahymena corlissi, have been subjected
to severe successive x-ray treatments. Strain "W" was found by Speidel at Woods Hole, Mas-
sachusetts, as a facultative parasite in tadpoles of Bitfo. Strain "C" was found by Thompson at
Charlottesville, Virginia, in a moribund larva of Pseudotriton. Structurally and functionally
the two strains seemed identical. A micronucleus was present. The tetrahymenae reproduced
rapidly by binary fission and by reproductive cysts. Conjugation was not seen. During the
summer of 1956 strain C was subjected to a series of 9 x-ray treatments over a period of 60
days. The separate doses ranged in strength from 400-600 kr ; the cumulative dose totaled 4217
kr. During the summer of 1957 strain W was subjected to a similar series of 9 x-ray treatments
over a period of 57 days. The cumulative dose for this strain totaled 4500 kr. Surviving
tetrahymenae in each strain gave rise to progeny containing many amicronucleate individuals.
Clones of amicronucleate tetrahymenae were readily established from both x-rayed strains.
During the summer of 1957 after a 10-month interval of recovery, cultures of amicronucle-
ate tetrahymenae derived from strain C were subjected to 5 additional x-ray treatments totaling
2900 kr over a period of 41 days. The separate doses ranged in strength from 500-700 kr. The
total cumulative dose for both summers received by this strain amounted to 7017 kr. The sur-
viving progeny exhibited no marked difference in radiosensitivity or radioresistance as com-
pared with normal tetrahymenae. There was no conspicuous lasting effect on the capacity and
speed of reproduction.

Enzymatic dissociation of sponge cells.'2 MELVIN SPIEGEL AND CARROLL METCALF.

The effect of several enzymes on sponge reaggregation was studied in an effort to prepare
completely dissociated cell suspensions. The following enzyme solutions in artificial sea water
(Tyler) were used: 2% crude protease, pH 7.6; 0.2% crystalline trypsin, pH 7.7; 0.2% crystal-
line chymotrypsin, pH 7.7; 2% crude papain containing 100 mgm% cysteine, pH 7.0; 2% steap-
sin, pH 7.0. The stomach juices of the green crab, Carcinus maenas, and of the blue crab,
Callinectcs sapidus, were also used. Two-tenths ml. of the expressate obtained by pressing 2 gm.
of the sponge Microciona prolifera through bolting cloth into 20 ml. of artificial sea water was
then added to 3 ml. of each enzyme solution. Three ml. of artificial sea water served as control.

Trypsin, protease, chymotrypsin, and steapsin had no effect on dissociation. Aggregates
formed, rounded up within 6-8 hours, and adhered to glass but did not exhibit the flattening and
spreading out which normally occur 24 hours after dispersal. In papain, aggregates were formed
which failed to round up with the cells loosely adhering to one another. Attempts to obtain
complete dissociation by passing the papain-suspension through a fine pipette led to cytolysis.

In stomach juice of the green crab, at first large aggregates were formed but the peripheral
cells were in loose contact and 18 hours after dispersal many small aggregates were formed.
No rounding up occurred. In blue crab stomach juice little reaggregation took place. In the
center of the largest aggregrates which were formed condensations of closely packed cells were
noted with loosely connected cells between these condensations. Eighteen hours after dispersal
a good cell suspension could be obtained by passage through a pipette.

iThis investigation was supported by a research grant (PHS RG-4326 R) from the Na-
tional Institutes of Health, Public Health Service.

2 Supported by Research Grant E-1365 from the National Institute of Allergy and In-
fectious Diseases.

356 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

Viability of dissociated frosen-tJunvcd sponge cells.1 MELVIN SPIEGEL AND
CARROLL METCALF.

A cell suspension of the sponge Microciona prolijcra was prepared by pressing 2 gm. of
sponge through bolting cloth into 20 ml. of filtered sea water. Two-mi, aliquots were trans-
ferred to 8 ml. of varying concentrations of ethylene glycol or glycerine in filtered sea water,
frozen at — 12° C. ; after 1-24 hours rapidly thawed at 18° C. and washed 3 times with filtered
sea water. A control suspension in filtered sea water was treated in identical fashion. Cells
frozen in ethylene glycol did not survive the treatment. After freezing in 10% glycerine, ap-
proximately 30% free cells were noted but the amoebocytes exhibited no pseudopodial move-
ment and no collar cells with a beating flagellum were noted. No reaggregation of cells oc-
curred. Alter freezing in 20% and 30% glycerine, only 10% free cells were noted with no cell
movement. In 40% glycerine the amoebocytes showed pseudopodial movement and a few col-
lar cells with beating flagellum were noted. Twenty-four hours later the normal rounding up
of aggregates had not occurred with no aggregate adherence to glass. Dialysis vs. filtered sea
water did not improve survival. Further reaggregation did not occur after culture for 7 days.
Similar results are obtained with unfrozen filtered sea water-glycerine suspensions. After thaw-
ing of filtered sea water suspensions the amoebocytes exhibit normal psudopodial movement
and adhere to glass. No collar cell movement was noted. Normal reaggregation occurs during
the first 4 hours after thawing but within 8 hours cytolysis sets in and reaggregation ceases.
Varying the time for freezing and for thawing has not furthered survival. The results show
that sponge cells can survive freezing and thawing in glycerine-filtered sea water solutions and
in sea water alone. Neither glycerine nor ethylene glycol offers any protective advantage com-
pared to filtered sea water.

The reaggregation of Microciona cells in culture medial MELVIN SPIEGEL AND
CARROLL METCALF.

Although aggregates of sponge cells which have been dissociated by passage through
bolting cloth into sea water can easily regenerate to form a new sponge when placed in a
live-car, attempts to rear such aggregates in culture media, synthetic or otherwise, have failed.
In an effort to circumvent this difficulty the following culture media were used : A. 1-leucine, 31
mgm. ; dl-phenylalanine, 10 mgm. ; dl-tryptophane, 8 mgm. ; 1-cysteine, 2 mgm. ; 1-arginine, 16
mgm. ; dl-methionine, 26 mgm.; glucose, 1.7 gm. ; 200 ml. artificial sea water (Tyler). B. 1%,
0.5%, and 0.1% glucose in artificial sea water. C. 1%, 0.5%, and 0.1% egg albumen in artificial
sea water. D. Medium A minus glucose. E. 1%, 0.5%, and 0.1% galactose in artificial sea
water. F. 1%, 0.5%, and 0.1% levulose in artificial sea water. G. 0.5% boiled dried yeast in
artificial sea water. All solutions were adjusted to pH 8.2. Filtered sea water and artificial
sea water served as controls. Two ml. of a cell suspension, obtained by pressing 2 gm. of
Microciona prolijcra through bolting cloth in 20 ml. of artificial sea water, were added to 8 ml.
of each medium.

In media A-F reaggregation was impeded. More numerous but smaller aggregates were
formed than in the controls. These did not regenerate further. In medium G a functional
sponge was formed with canals and chambers lined with beating collar cells and raised oscula.
When yeast cells stained with neutral red were added to this sponge within two hours the entire
regenerate was stained. In control media canals and chambers were formed but neither water
currents nor oscula were observed.

Uptake of amino acids from sea water by ciliary-mucoid filter feeding animals.
G. C. STEPHENS AND R. A. SCHINSKE.

Several reports in the literature concerning filter feeding animals indicate that some may
retain small quantities of dissolved protein. The following observations were undertaken to

1 Supported by Research Grant E-1365 from the National Institute of Allergy and Infectious
Diseases.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 357

determine whether ciliary-mucoid filter feeders are capable of taking up small organic molecules.
An initial set of observations was made with 2 mM. glycine (150 mg./liter sea water) using
the slipper limpet, Crepidula fornicata; the mussel, Mytihis cdulis; and the coral, Astrangia
danac. Single mussels, or several Crepidula, or an Astrangia colony, were placed in approxi-
mately 250 ml. of solution. This solution was sampled periodically and the amino acid concen-
tration measured colorimetrically using a ninhydrin reaction. Control samples were run in
parallel, and colorimetric determinations were done in triplicate. Considerable variation in rate
of uptake was observed, particularly in the case of Mytilus and Crepidula. However, the fol-
lowing results may be cited as typical. Mytilus in one experiment removed 10% of the glycine in
five hours and 45% in twenty-four hours, while Crepidula removed 15% and 65% and Astrangia,
20% and 97%.

Astrangia was chosen as a suitable animal for additional experiments. Five readily solu-
ble amino acids were selected to cover a range of isoelectric points. These were glutamic acid,
methionine, glycine, alanine and arginine whose isoelectric points are, respectively, 3.08, 5.7,
6.06, 6.1 and 10.76. Uptake in all cases was roughly comparable to that for glycine. These
observations were made by sampling at intervals about one liter of the solution in which several
colonies had been immersed. Various controls included rocks from which the colonies had been
removed but which still included masses of the sponge, Cliona, and rocks together with the
crushed coral organisms. These did not show significant uptake.

Observations were also made on the rate of removal in concentrations from 0.4 mM. to 10.0
mM. No striking difference in rate of uptake was apparent over this 25-fold concentration
difference.

The effect of 2,4-dichloropheno.ryacctic acid on o.vygcn consumption in Uca pugnax.
FREDERICK N. SUDAK AND C. LLOYD CLAFF.

The lethal concentration of 2,4-dichlorophenoxyacetic acid, as determined by injection di-
rectly into the hemocoel, was found to be 400 mg./kg. wet body weight. The L.D. 50/24 hrs.
was determined as 325 mg./kg. wet body weight. Symptoms resembling myotonia, found in
homeothermic vertebrates treated with this compound, were produced with concentrations be-
tween 200 and 275 mg./kg. wet body weight. Muscular movements were initiated after some
delay but once movement was started, the animal continued to move without any apparent
difficulty.

Oxygen consumption of crabs treated with 2,4-D was measured at 17.5° C. using a modified
Scholander respirometer for aquatic animals. A decrease in O2 consumption of an average of
78% occurred within 60 minutes after injection of 200 to 250 mg./kg. into the ventral hemocoel.
Oxygen consumption returned to pre-injection levels 10-15 hours later. Control animals, in
simultaneous experiments, were injected with equal volumes of filtered sea water. O2 consump-
tion was increased for 30 minutes after injections followed by a recovery to pre-injection levels.

Metabolic responses of albino rats treated with 2,4-dlchlorophenoxyacetic acid to
changes in ambient temperature. FREDERICK N. SUDAK, C. LLOYD CLAFF AND
MARVIN H. CANTOR.

Twelve hour fasted male albino rats injected with 300 mg./kg. of 2,4-dichlorophenoxyacetic
acid increased their CO2 output 43% with no change in body temperature when kept in an iso-
thermic environment of 30° C. for 40 to 60 minutes after injection. Carbon dioxide production
increased 85% accompanied by a rise of 2° C. in body temperature when the ambient tempera-
ture was increased to 33° C. Animals treated with this compound died within 20 minutes in an
environmental temperature of 35° C. Rectal temperatures taken at the time of death were above
40° C. Control animals injected with an equal volume of physiological saline decreased their
CO2 production 14% at both isothermic and 33° C. temperatures while rectal temperatures re-
mained constant. Carbon dioxide production returned to the control levels taken at room tem-
perature and the rectal temperatures increased 1° C. when the ambient temperature was increased
to 35° C. Both groups of animals perspired freely at this temperature.

Myotonia induced in albino rats with subcutaneous injections with 2,4-dichlorophenoxyacetic
acid persists for 18 to 24 hours after injection but the duration of the metabolic response to in-

358 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

creased ambient temperature is much shorter, i.e., seven hours. Animals injected two hours
earlier died within 20 minutes when the ambient temperature was increased from 30° C. to
35° C. The rectal temperature of these animals increased from 37° C. to above 40° C. Ani-
mals injected five hours earlier survived the 20-minute exposure to 35° C. heat. CO2 produc-
tion increased 40% and rectal temperature rose 2° C. Metabolic and body temperature re-
sponses were the same as those of control animals when the ambient temperature was raised
to 35° C. seven hours after an injection of 2,4 D.

Metabolic activity of rats treated with 2,4 D behaved much like that of a poikilothermic ani-
mal for seven hours after injection. The calculated Q,n of these animals was 3.5 in heat and 1.0
when they were placed in a cold environment.

Expansion of the pre-placcntal yolk-sac in Mustclus canis. Lois E. TEWINKEL.

Yolk-sac folds and villi were mentioned by ten Cate-Hoedemaker and Ranzi in detailed ac-
counts of the placenta of Mitstelits lac-ris, but the extent of yolk-sac growth was not emphasized,
undoubtedly because they had no late pre-placental stages. Mahadevan, in several Indian
Selachii (Scoliodon), noted elaborate yolk-sac growth, but made no measurements.

Scarcity of pregnant smooth dogfish at Woods Hole has prevented a thorough-going study,
but, when yolk-sacs of 40 mm. embryos are compared with those of 55, 65, and 109 mm. embryos,
it is clear that in Mustelus canis the yolk-sac grows enormously as yolk is depleted. Spreads
of yolk-sacs, dried and measured on millimeter paper, show an increase from an initial area of
1260 mm.' for a 40 mm. embryo (where the yolk-sac has not begun expansion) to 4474 mm.1 for
a 65 mm. embryo, and over 10,000 mm.2 for a 109 mm. specimen.

Although yolk is still present in all sacs and in vitelline ducts of embryos 50 mm. and longer
in the stages studied, progressive growth in surface area of the sac as yolk decreases, suggests
that, in addition to yolk absorption, material is being taken in from fluid diffusing into the egg-
case cavity from uterine sources. That absorption of water occurs in these pre-placental stages
is shown in a comparison of wet and dry weights of embryos plus yolk-sacs when embryos
measured approximately 17, 34, and 60 mm. The following figures for each class represent an
average of three specimens. Wet weights were: 2.10, 2.722, and 4.22 grams, respectively; and
dry weights: 1.016, 1.19, and 1.395 grams. Thus, in a 60 mm. embryo and yolk-sac, water
content is 2.6 times that in a 17 mm. specimen and has risen to 67% of the wet weight as com-
pared with 52%. Ash determinations have not yet been made.

The hexose monophosphate shunt in marine invertebrates. CLAUDE A. VILLEE,
JANET LORING AND FREDERICA WELLINGTON.

The hexose monophosphate shunt provides a path for the metabolism of glucose which is an
alternative to the Embden-Meyerhof glycolytic cycle. Glucose-6-phosphate is oxidized to 6-phos-
phogluconic acid which is then oxidatively cleaved to CO2 (from carbon 1) and the pentose,
ribulose-5-phosphate. An estimate of the fraction of glucose metabolized by each path is ob-
tained by comparing the rates of metabolism of glucose-6-C14 and glucose-1-C14 to C14O2 (Ge/G,)-
The rate of carbon 1 of glucose to CO2 is taken as the sum of the two paths and that of carbon
6 is taken as the glycolytic path alone. Krahl had shown that the monophosphate shunt is an
important path in the metabolism of developing Arbacia eggs and that it could account for
essentially all of the oxygen consumed. We have confirmed his results and extended the study
to a variety of excised tissues from other echinoderms, annelids and molluscs. In our experi-
ments the GS/G! ratio (mean of 12 experiments) for unfertilized Arbacia eggs was 0.07, for
fertilized eggs (12 hours after fertilization) was 0.20 and for sperm was 0.23. This confirms
Krahl's finding that the glycolytic path becomes more important as development proceeds.
From the G6/G! ratio it appears that the hexose monophosphate shunt is an important metabolic
pathway in Busycon red retractor muscle and digestive gland, in Thyone respiratory tree and gut,
and in Chaetopterus and Arenicola muscle and gonads. In marked contrast, the Ge/Gi ratio
was unity in Pecten gill and mantle and in Loligo gill. The gills of these molluscs have high
rates of oxygen consumption but it would appear that all of the glucose is metabolized via a
glycolytic system.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 359

X-irradiation of the giant multinucleate ameba, Chaos chaos. RALPH WICHTERMAN.

Specimens from the original 1936 Schaeffer strain were irradiated singly with doses from
20,000 r to 80,000 r in steps of 10,000 r and with 120,000 r — the LD 50, 72 hours. A single
ameba was placed in either a one- or two-cc. closed Lucite chamber free of air and containing
only 10 parts of spring and 90 parts of glass-distilled water. Five or 10 of the isolated amebas
were irradiated at one time. Immediately after irradiation, the water was replaced and
Paramecium multimicronucleatum and Chilomonas added as food. The amebas were maintained
in culture until either their death or until successful mass clonal cultures were established.
When an irradiated ameba produced a clone of at least 30 specimens, it was considered a success-
ful mass culture. Occasionally some of the clones were allowed to produce several hundred
amebas.

This multinucleate organism may divide commonly into two or three, occasionally more,
daughters. As a more reliable measure, all progeny of a divided specimen were kept in the
same container and totalled since it was found that some early daughters of an irradiated
ameba may die before dividing later, while others may survive to divide again and produce
successful mass clonal cultures.

As has been observed for a number of other Protozoa, the greater the dosage, the greater
the delay in the first division following irradiation. Thereafter — if the cell is to divide again—
the time between the next and succeeding divisions is progressively shorter. There is no con-
sistency in the number of daughters produced by either a single unirradiated control specimen or
an irradiated one when observed daily over a period of at least 2 weeks.

After irradiation with 40,000 r, some isolated specimens produced as many as 10 or less
amebas which died while others yielded clones of hundreds. On the other hand, a similarly ir-
radiated specimen may live for at least 23 days without dividing.

With 50,000 ?- and 60,000 r, it may require as long as 10-17 days before the ameba first
divides to eventually yield a mass clonal culture. Generally a cell which divides earlier than
others similarly irradiated is one that is more likely to produce a mass culture.

Successful mass cultures have been established after the irradiation of single specimens with
from 20,000 r to 70,000 r.

Some physiological characteristics of the fish heart. CHARLES G. WILBER.

In mammals it is well known that the average heart-rate varies inversely with body size.
In the present work fish, which varied in size from pipefish to striped bass, were studied electro-
graphically to ascertain whether a similar relation was true. It was found that the average
resting heart-rate in fish varies inversely with body size and an equation expressing the rela-
tionship has been derived. Teleosts fit the plotted curve very well ; elasmobranchs do not, al-
though they apparently fit a special curve for themselves. Various drugs were tested with the
intact hearts of different species of fish. Fundulus heart responds readily to atropine with an
increase of rate. Darstine has a similar but more pronounced effect. Atropine is not very
effective in the toadfish. However, darstine in relatively large doses brings about complete
A-V dissociation ; an idioventricular rhythm is established ; atrial rate is slightly depressed.
The interpretation of the results is not easy. Preliminary results have been obtained which in-
dicate that in the sea robin an increase in temperature of the fish brings about an increase of
blood pressure as measured directly from the first gill vessel. The heart rate in the intact fish
also increases with temperature but in our work a temperature coefficient of 2 or 3 has not been
routinely found. This is in accord with our data for alligators. These studies are supported
by the National Science Foundation.

LALOR FELLOWSHIP REPORTS

Accessory fiber synoptic excitation of squid stellar giant axons. S. H. BRYANT.

In the excised squid (Loligo pealii) stellate ganglion preparation equilibrated in oxygen-
saturated medium, it is possible to observe excitatory behavior of the proximal (accessory)
synapses of the stellar giant axons.

360 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

The large pre-ganglionic axon to the giant (distal) synapses was removed from the nerve
trunk near the ganglion for stimulation. The accessory fiber and other smaller giant axons
were stimulated in the nerve trunk 2 to 3 cm. from the ganglion. Selective stimulation of the
remaining pre-ganglionic axons indicated that the proximal synapses were excited mainly by the
accessory fiber (Young), occasionally by one or more of the small giants, but not by the small
axons. There was no evidence of giant synapse inhibition by stimulation of the pre-ganglionic
giant axons.

The proximal synapses are more sensitive to oxygen lack and can be fatigued independently
of, and earlier than, the giant synapses. Intracellular recording from the last stellar axon near
its inflection in the ganglion reveals both the proximal and giant synapse excitatory post synap-
tic potentials (e.p.s.p.). The proximal e.p.s.p. was preceded by a synaptic delay and had a
temporal form similar to that of the giant synapse. Depolarization levels for spike initiation
were nearly the same in spite of different initial slopes.

When the e.p.s.p. of the proximal synapses is timed to arrive shortly after the giant synapse
is excited, it can abolish the undershoot and add to the middle of the falling phase of the spike.
If it arrives about 1 to 2 msec, later, it can give rise to a second spike, depending upon the re-
fractory period of the post axon. Parallel results were obtained when the proximal e.p.s.p. ar-
rived before that of the giant synapse.

In preparations where both sets of synapses were critically fatigued it was possible to get
addition of excitation when the e.p.s.p.'s were added in their early rising phase. If they were
added later there was addition of depolarization but not necessarily excitation.

The site of origin of the nerve impulse in the lobster stretch receptor. J. F. CASE,
C. EDWARDS/ R. GESTELAND AND D. OTTOSON.

The cell body and axon of the lobster stretch receptor organ are clearly visible with dark
field illumination. Thus, microelectrodes may be placed on visually well defined sites to re-
cord potential changes when the cell is activated by stretch or by antidromic stimulation.
Simultaneous recordings have been made with two glass micro-electrodes of potential changes in
the cell body and at various places along the axon of the lateral seventh thoracic stretch receptor
immersed in saline. An analysis of latencies and potential configurations was made to locate
the site of origin of the conducted impulses.

In a lightly stretched receptor the potential change of the soma starts with a positivity fol-
lowed by the negative spike. In the axon the impulse lacks the initial positive phase if re-
corded from a point near the cell body. The start of the positive phase of the cell body re-
sponse is synchronous with the start of the negative phase of the potential change in the axon ;
the cell body responds with an impulse 0.1-0.3 msec, later than the initial part of the axon. If
antidromic spikes are recorded with the electrodes at the same position, the potential changes in
the axon start with a positive phase while the cell body response is identical to that set up by
stretch. The soma impulse follows the axonal spike by about the same interval whether set
up by stretch or by antidromic stimulation. On the basis of these observations the conclusion
is drawn that the impulse set up by stretch starts in the axon near to the cell body and then
propagates out along the axon as well as back into the cell body.

Phosphoarginine and arginine phosphokina.se from Homarus americanus. L.
LORAND.

Enzyme systems capable of producing adenosinetriphosphate from adenosinediphosphate
seem to be involved in the relaxation of muscle, as suggested by previous studies with creatine
phosphate and phosphoenolpyruvate. It would be of interest to test the effect of an invertebrate
phosphate donor system, such as phosphoarginine, on vertebrate muscle. As a preliminary to
these studies, phosphoarginine and the enzyme arginine phosphokinase had to be prepared.

Since phosphoarginine cannot be synthesized, its isolation from Homarus americanus was
attempted. The tail muscle was homogenized without prior freezing in liquid nitrogen, other-
wise the procedure of Ennor, Morrison and Rosenberg (Biochemical Journal, 1956), given for

1 Fellow of the Lalor Foundation.

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 361

Jasus lalandii, was followed. Phosphoarginine could be obtained from the American lobster,
although in considerably lower yield than from the Australian species.

The enzyme arginine phosphokinase was also purified from Homanis amcricanus by the
method given for Jasus verraitxi by Morrison, Griffiths and Ennor (Biochemical Journal, 1957).

The effect of the invertebrate phosphate donor system on the relaxation process is now
being studied.

This work was carried out during the tenure of a Lalor Fellowship.

Incorporation of labeled iron into hemerythrin. MARTIN P. SCHULMAN.

Hemerythrin is an iron-containing respiratory pigment that occurs in nucleated coelomic
corpuscles of Phascnlnsoma gouldii. It combines reversibly with O2, is not an iron-porphyrin
compound, and can be crystallized readily (Florkin, 1932). When coelomic cells or hemolysates
of these cells were incubated with Fe59Cl3, the isolated oxyhemerythrin contained Fe59. The
incorporation of Fe'"'9 into oxyhemerythrin in a hemolysate was fifteen times greater than in an
intact cell preparation, suggesting low permeability of these cells to ferric iron. Although the
incorporation of Fe59 into hemerythrin in hemolysates was not linear past one hour incubation,
the uptake of Fe69 after five hours was still considerable. Experiments that ruled out a non-
enzymatic exchange of Fe59 with hemerythrin were: (1) at 0° intact cells and hemolysates did
not incorporate Fe59 into hemerythrin ; (2) incubating Fe59CU for varying lengths of time with
oxyhemerythrin or hemerythrin reduced by deoxygenation did not result in any uptake of Fe59
by the pigment. Oxyhemerythrin was crystallized four times to rid the pigment of ionic Fe5!> ;
at this stage the specific activity of oxyhemerythrin that contained Fe59 as a result of incubation
was constant. Oxyhemerythrin containing Fe59 was assayed by the following procedure : a
known volume of pigment in 0.35 M NaCl was assayed for radioactivity in a scintillation well
counter, while another aliquot was measured spectrophotometrically to determine the concen-
tration of hemerythrin. The purity of each sample was determined by measuring the optical
densities at 280 m/*, 330m/i and 500 m/u and calculating the following ratios : 280/330 and 330/500
which were 5.27 and 3.16 ±1%, respectively, for the best preparations. The specific activity was
expressed as counts per minute per optical density unit at 330 rn.fi. It is believed that the in-
corporation of Fe59 into hemerythrin is enzymatic and represents a biosynthesis of the molecule.

Aided by AEC contract AT (30-1) 1343 at Marine Biological Laboratory, Woods Hole,
Mass.

Heme synthesis in peripheral blood of marine fishes. MARTIN P. SCHULMAN AND
GEORGE A. LAMB.

Twenty-five per cent suspensions of twice-washed erythrocytes in saline containing Fe69Cl3
were shaken in air for two hours at 22° C. The cells were then washed with saline, hemo-
globin carrier added, and hemin isolated and purified by Fisher's procedure. Red cells were
stained supravitally with brilliant cresyl blue according to Dawson (1933), who found that
bloods of different fishes varied in their percentages of immature cells. Our study showed
that the incorporation of Fe68 into heme varied with the degree of reticulation in each blood.
For example, the red cells of the toadfish (Opsanus tau) had less than 0.5% immature forms
and incorporated negligible Fe69 into heme. Other teleosts, such as the common sea robin
(Prionotus carolinus), scup (Stcnotomns chrysops), king mackerel (Scomberomorus regalis)
and bonito (Sarda sarda}, had 5 to 10% immature red cells and incorporated Fe59 in proportion
to the degree of reticulation. In the elasmobranchs studied — smooth dogfish (Mustehts cams'),
spiny dogfish (Squalus acanthias) , spotted skate (Raja diaphanes), dusty shark (Carcharhinus
obscurus) — 25 to 30% of the red cells were immature and the uptake of Fe69 was considerable.
Since the bloods of the elasmobranchs contained about four times as many immature cells as did
the teleost bloods, it was expected that the incorporation of Fe59 would also be increased four-
fold. However, the incorporation was much greater and may be explained by the increased den-
sity of reticulum per immature cell of the elasmobranchs. Additional studies on red cells
of Mustelus showed that Fe59 incorporation was linear during the incubation, addition of gly-
cine and succinate, precursors of the protoporphyrin moiety of heme, did not stimulate the
uptake of Fe69, and Fe59 incorporation into heme was markedly lowered when cells were in-
cubated in serum.

362 PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY

Aided by USPHS grant C-1852 and AEC contract AT (30-1) 1343 at Marine Biological
Laboratory, Woods Hole, Mass.

The effects of metabolic inhibitors on ion distribution and membrane potential in
muscle fibers of the green crab, Carcinides maenas. WILLIAM K. STEPHENSON.

Fiber ion compositions were determined in muscle fibers from the flexor in the meropodite
of the green crab, Carcinides maenas. Sodium and potassium analyses were carried out by
flame photometry, and chloride was measured with a mercurimetric titration. Fresh muscle
has an average fiber potassium concentration of 156 mEq/liter fiber water (Ifw) (17 determina-
tions), a sodium concentration of 72 mEq/lfw (17 determinations), and a chloride concentra-
tion of 94 mEq/lfw (13 determinations). In calculating fiber ion concentrations an extracellular
space value of 5% (of wet weight), as determined by the inulin method, and a dry weight of
22% were used. Microelectrode determinations of membrane potentials on fresh fibers bathed
in blood gave an average value of 58 mV for 43 fibers from 5 crabs. Upon exposure to natural
sea water or to MBL-formula sea water the potential rises to an average of 69 mV over a pe-
riod of 30-60 minutes.

If MBL sea water is perfused through the removed, but unopened, leg segments, the fiber
potassium falls less than 5% over a 6-hour period. 5 mM iodoacetic acid (IAA), 5 mM cya-
nide (CN), and 0.2 mM dinitrophenol (DNP), added to MBL sea water, were similarly per-
fused through the preparation and their effects on the ion balance were observed. In terms of
increasing the loss of potassium the effects of the inhibitors were in the following order : CN <
IAA < DNP ^ DNP + CN ^ IAA + CN < DNP + IAA. The effects of inhibition on sodium
and chloride concentrations were too equivocal to include here. The inhibitors also produced
a decrease of membrane potential in the following order: CN <DNP^DNP + CN < IAA <
DNP + IAA (the effect of CN IAA was not clear). IAA thus produces a rapid effect on
membrane potential without markedly altering the potassium, sodium, or chloride distributions.

In vitro studies on intestinal absorption of fish. T. HASTINGS WILSON.

The ability of fish intestine to transport sugars and amino acids across the wall against a
concentration gradient was tested with an in -vitro technique. A tied sac of everted small in-
testine was filled with saline (usually 2 ml.), placed in a 50-ml. Erlenmeyer flask containing 3 ml.
of saline and gassed with 100% oxygen. The saline composition was as follows : NaCl (0.21 M) ,
MgSO4 (0.002 M), NaHCO3 (0.002 M), K2HPO4; pH 7.0 (0.01 M).

Proline and glycine were actively transported by the intestine of the puffer (Spheroides
maculatus). In a representative experiment L-proline (50 mg%) was added to the saline
on each side of the intestinal wall. At the end of one hour of incubation at 26° the concentra-
tion of proline on the mucosal side fell to 24 mg% while that on the serosal side rose to 65
mg%. There was a net loss of proline from the system which could be partially accounted for
by the appearance on the mucosal side of an amino acid chromatographically similar to glutamic
acid.

In contrast to the results obtained with amino acids, glucose was not transported across the
intestinal wall against a gradient by in vitro preparations from any of the following fish : sea
robin (Prionotus carolinus), scup (Stenotomus chrysops), toadfish (Opsamis tan) or puffer.
Galactose and 6-deoxy-D-glucose were also tested with the puffer gut preparation and were not
transported.

During the in -vitro incubation the mucosal side of puffer and sea robin intestine became
progressively alkaline (from pH 7.0 to about 7.6). This alkaline intestinal secretion was con-
firmed in vivo in the case of the sea robin.

Phylogenesis of plasma proteins and plasma cells. I. Starch gel zone electrophoresis
of sera from marine invertebrates and fishes. KENNETH R. WOODS AND RALPH
L. ENGLE, JR.

The method of zone electrophoresis in starch gel described by O. Smithies in 1955 has
been used to separate proteins from the sera of several marine invertebrates, cartilagenous and

PAPERS PRESENTED AT MARINE BIOLOGICAL LABORATORY 363

bony fishes, and a few amphibians, reptiles, and lower mammals. From six to twelve speci-
mens of each form were analyzed. The resulting serum electrophoretograms exhibited a high
degree of intraspecies reproducibility and a high degree of interspecies specificity.

Closely related decapod Crustacea sometimes gave similar serum electrophoretograms, but
exceptions were found. More than one major protein fraction was always obtained from hemo-
cyanin-containing sera. This finding was attributed to the dissociation of hemocyanin into
non-identical components at the high pH (9.0) of the starch gels. None of the invertebrates
examined yielded serum proteins with the electrophoretic properties of human gamma-globulin.

Each of seven species of the elasmobranchs produced its own unique serum electrophoreto-
gram. These sera contained fractions which migrated into the cathodic region of the gel in a
fashion similar to the movement of human gamma-globulin. Bony fishes gave highly specific
patterns, but proteins corresponding electrophoretically to human gamma-globulins were either
greatly reduced or absent. Among the bony fishes the occurrence of a protein corresponding to
albumin and of another with a slightly slower mobility than beta globulin were consistently ob-
served. Other proteins of random mobilities and distributions were present, giving each bony
fish species a characteristic pattern.

Despite the few exceptions which might prove to have important biological significance,
these studies indicate that serum electrophoretic patterns of closely related species have a re-
markable degree of specificity. These findings suggest that starch gel electrophoresis of serum
proteins may contribute valuable information to population studies, comparative immunology,
taxonomic problems, and to other considerations of biochemical individuality.

Phylogenesis of plasma proteins and plasma cells. II. Observations on the occur-
rence of plasma cells in marine invertebrates and fishes. RALPH L. ENGLE, JR.
AND KENNETH R. WOODS.

Microscopic examinations were conducted to determine whether or not plasma cells are
present in invertebrates and cold-blooded vertebrates. Plasma cells in mammals are recognized
by their eccentric nucleus and basophilic granuloplasm usually containing a paranuclear clear
zone. Cells in lower forms having the same appearance were considered to be plasma cells.

Invertebrate blood was obtained by puncturing a blood vessel and allowing blood to drip
directly onto a siliconed glass slide. The smear was fixed immediately in osmic acid or formalin
vapor. Blood of vertebrates was smeared on glass slides and dried. Spleens and bone marrows
were sectioned and exposed surfaces lightly touched to glass slides which were dried. All
preparations were stained with Wright-Giemsa and examined using oil immersion lens. A few
wet preparations were examined with the phase contrast microscope. One to five specimens of
each form were studied.

The following invertebrates were examined: Arenicola cristata, Loligo pealii, Cambanis
limosus, Homarus americanus, Cancer borealis, Libinia emarginata, Limulus polyphemus, and
Hadrnrus arisonensis. No cells recognizable as plasma cells were found in the blood of any of
these.

Among vertebrates, the elasmobranchs and teleosts examined were Mustelus canis, Squalus
acanthias, Carcharhinus obscurus, Raja ocellata, Torpedo nobiliana, Dasyatis centroura, Echeneis
naucratcs, Lophins piscatorius, Tantoga onitis, and Psendoplcuronectes americanus. Plasma
cells were detected in spleens but not in blood of all species.

Two amphibia were studied. No plasma cells were found in blood or spleens of Rana
catesbiana or Rana pipiens. However, bone marrow of Rana catesbiana did contain plasma
cells. Satisfactory marrow was not obtained from Rana pipiens.

These observations demonstrate that cells having morphologic characteristics of human
plasma cells exist in lower forms. The phylogenic occurrence of these cells is being correlated
with serum electrophoretic studies in an attempt to determine relationships between plasma cells
and gamma-globulin.

Vol. 113, No. 3 December, 1957

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

CHEMICAL ANALYSES OF ANTERIOR AND POSTERIOR BLAS-
TOMERES OF CIONA INTESTINALIS

WILLIAM E. BERG
Department of Zoology, University of California, Berkeley 4, California

Segregations of specific cytoplasms occur during early cleavage of the ascidian
egg (Conklin, 1905) of which the most obvious is the localization of the myoplasm,
presumptive for larval musculature, in the posterior cells at the four-cell stage.
Cytological (Meves, 1913; Duesberg, 1915; Conklin, 1931) and cytochemical (Ries,
1937) studies indicate the localization of granules, presumably mitochondria, within
the myoplasm. Recently Reverberi (1956) followed the distribution of mito-
chondria during development, using the vital stain, Janus green. The quantitative
measurements of cytochrome oxidase in anterior and posterior blastomeres (Berg,
1956) gave a biochemical confirmation of the above studies as regards the localiza-
tion of mitochondria.

The present study is a continuation of quantitative chemical analyses of anterior
and posterior blastomeres from the four-cell stage of dona, choosing constituents
which might be expected to be located on cellular particles. The minute amounts of
cytoplasm available require the use of microchemical methods and limit the number
of substances which may be studied ; however, differences in activities of succinic de-
hydrogenase, apyrase, acid phosphatase, and ribonucleic acid have been found in
homogenates of the two types of cells.

METHODS

Chorions were digested off unfertilized dona eggs in 3 per cent protease in sea
water. The "naked" eggs were washed thoroughly, fertilized, and transferred to
agar-coated dishes. At the end of the first cleavage the blastomeres were separated
in large numbers by agitation and as each of these cleaved in turn, the anterior and
posterior cells were separated with the tip of a fine braking pipette and segregated
into different dishes.

The posterior blastomere is recognizable by an elongated shape and a clear cyto-
plasmic cap (Castle, 1896). These characteristics are transitory and the period for
separation is critical ; separation before completion of the cleavage furrow results
in cytolysis whereas shortly after cleavage identification becomes increasingly diffi-
cult due to sphering of the posterior cell. This period was extended by lowering
the temperature to 8-10° C. after completion of cleavage, thus prolonging the elon-
gated state of the posterior cell. Furthermore it was discovered that with oblique

365

366 WILLIAM E. BERG

lighting the posterior cell, even after sphering, exhibits a bright crescentic rim of
cytoplasm which is the remnant of the clear cytoplasmic cap. This persists for
some minutes after cleavage and greatly extends the length of time during which
identification is possible.

Separation of blastomeres was begun as soon as possible after cleavage, using the
difference in shapes for identification. As the posterior cells began to round up, an
oblique lighting was adopted and separation continued, using the bright rim of the
posterior cell as a marker. In this manner several hundred blastomeres could be
isolated, a considerable improvement over initial attempts where a maximum of
30-40 were obtained (Berg, 1956).

The desired number of segregated blastomeres was counted and transferred to
0.1 -ml. centrifuge tubes by means of a braking pipette. After light centrifugation
excess sea water was removed, a few [A. of homogenizing solution were added and
the cells homogenized by drawing them in and out of a fine-bore pipette. The ho-
mogenization was carried out at 1-2° C., attained by placing the centrifuge tube in a
previously chilled copper block. All micromethods were spectrophotometric, using
the Beckman spectrophotometer adapted for the use of microcuvettes (Lowry and
Bessey, 1946).

Succinic dehydrogenase activity in homogenates was measured by the method of
Cooperstein et al. (1950). The blastomeres were homogenized in 1 //,!. of 0.19 M
sodium succinate ; 5 /J. of 0.19 M succinate and 2 p\. of 0.03 M sodium cyanide were
added and the mixture transferred to 45 /*,!. of 2 X 10~5 cytochrome c contained in a
microcuvette. All solutions were buffered with 0.04 M phosphate buffer at pH 7.4.
The reduction of cytochrome c was followed at 550 m/j. for three minutes at the end
of which time a few grains of sodium hydrosulfite were added to completely reduce
the cytochrome c. A semi-logarithmic plot of the readings, after substraction of the
optical density of reduced cytochrome c, gave a straight line from which a velocity
constant, (Alog ferricytochrome c/AT), could be calculated. Succinic dehydroge-
nase activities were expressed as velocity constants for rates of reduction of
cytochrome c.

Acid and alkaline phosphatases and apyrase were measured by micromethods
described by Lowry et al. (1954). For acid phosphatase 10 /*,!. of substrate (8 mM
disodium p-nitrophenyl phosphate in 0.05 M succinate buffer, pH 5) were mixed
with the homogenate. After thirty minutes of incubation at 25° C., 45 /A. of 0.1 N
NaOH were added, with immediate mixing, and read at 410 mju. within thirty
minutes.

Alkaline phosphatase was measured in a similar manner except that a buffer (2
amino-2 methyl- 1-propanol) at pH 10 was used with an incubation period of one
hour. It was not necessary to carry out protein precipitations in determinations of
either acid or alkaline phosphatase. Blanks were prepared by separate incubation of
substrate and homogenate with mixing just before addition of NaOH.

Apyrase measurements were made by homogenizing the cells in 0.75 per cent
sodium desoxycholate, a procedure which considerably increases the enzyme activity
presumably due to particle breakdown. Ten /xl. of substrate (2.5 mM adenosinetri-
phosphate in 0.05 M tris hydroxy-amino methane at pH 8.0) were added to the
homogenate and the mixture incubated for one hour at 25° C. Protein was pre-
cipitated by adding 2 p\. of 30 per cent trichloracetic acid and, after centrifugation,
the supernatant was transferred to another tube with 100 p\. of molybdate-ascorbic

ANALYSES OF CIONA BLASTOMERES 367

acid reagent and read at 870 m^. Blanks were prepared by separate incubation of
substrate and homogenate with mixing at the time of addition of trichloracetic acid.
Both apyrase and acid phosphatase activities were expressed as optical densities after
subtraction of blank values.

Ribonucleic acid was determined by a micromethod based on the procedure of
Ogur and Rosen (1950). Cells were extracted with 60 //.I. of cold 70 per cent al-
cohol for 5-10 minutes followed by extraction with 60 /u.1. of warm alcohol-ether
(3 : 1). After a few minutes extraction with cold 0.1 M perchloric acid, a final ex-
traction with 45 /*!. of 1.0 M perchloric acid was carried out for 48 hours.

A typical absorption curve for ribonucleic acid was obtained with the latter ex-
tract and the optical density at 260 m/x was used as a measure of the amount. Re-
peated test extractions with 70 per cent alcohol, alcohol-ether, and 0.1 M perchloric
acid demonstrated that these solutions removed, within a few minutes, all amino
acids, polypeptides and acid-soluble substances which absorb at 260 m/t. Although
most extractions were carried out for a longer time, nearly all the ribonucleic acid
was removed within 24 hours.

Protein was measured by the method of Lowry et al. (1951). The cells were
placed in a 0.5-ml. test tube and 100 /*,!. of alkaline copper solution added and mixed.
After 10 minutes, 10 /*!. of diluted Folin reagent were added with immediate mixing
and the sample read at 750 m//.. Addition of the Folin reagent is critical and the
reliability of color development depends upon the rapidity and effectiveness of the
mixing. On a micro scale this is difficult to control and the resulting variability
seriously limits this method.

Calibration curves (Fig. 1) were made for each of the micromethods. Ferti-
lized eggs, with the chorions removed, were used for these tests and as shown in
Figure 1 the optical density measurements, or velocity constants for succinic dehy-
drogenase, are proportional to enzyme activities as represented by the number of
eggs. The optical densities at 260 m/x of perchloric acid extracts, representing
amounts of ribonucleic acid, are proportional to the number of eggs extracted.

RESULTS

Anterior and posterior blastomeres of Ciona were separated and kept at 1° C.
in agar-coated dishes until ready for counting and transference to the reaction tubes.
From the calibration curves (Fig. 1 ) an estimation was made of the number of blas-
tomeres necessary in each test for reliable measurements of the constituent. Thirty
to forty cells in each tube were sufficient for enzyme and protein measurements,
whereas nearly one hundred were required for a reliable measure of ribonucleic acid.

All determinations of enzymes, ribonucleic acid, and proteins in anterior and
posterior blastomeres were paired ; i.e., two reaction tubes were used, one containing
anterior cells, the other containing an equal number of posterior cells. The anal-
yses were then carried out simultaneously. Due to the variability of results ob-
tained with microchemical methods it was necessary to repeat the analyses a number
of times and, as each experiment was on eggs from different animals and carried out
under slightly different conditions, the data are expressed as ratios of activities or
concentrations for each paired experiment. The average of these ratios is used to
summarize the results although statistical significance was calculated directly from
the paired series.

368

WILLIAM E. BERG

0.8

0.7

0.6

0.5"

UJ
Q

0.3

<
o

O 0-2"

O.I-

Acid phosphatase

Succinic

dehydrogenase

10
NUMBER OF EGGS

15

20

FIGURE 1. Acid phosphatase (activity expressed as optical density), apyrase (optical den-
sity), succinic dehydrogenase (A log ferricytochrome c/3 minutes) and ribonucleic acid (optical
density) in homogenates of Ciona eggs.

The data for eighteen paired measurements of acid phosphatase in anterior and
posterior cells are summarized in Table I. The anterior cells contain 12 per cent
more of this enzyme than posterior cells, a difference which is significant at the one
per cent level. A number of these experiments were carried out using sodium de-
soxycholate in the homogenization medium, with no detectable increase in enzyme
activity.

TABLE I

Acid phosphatase, expressed as optical densities, in homogenates of anterior

and posterior cells of Ciona

Number of tests

Acid phosphatase
in anterior cells

Acid phosphatase
in posterior cells

Average ratio of paired
determinations (ant. /post.)

18
(25-40 cells for
each test)

0.318

0.287

1.12 ± 0.03

ANALYSES OF CIONA BLASTOMERES

369

A homogenate of thirty or more Ciona eggs was necessary to obtain even a de-
tectable activity of alkaline phosphatase and accordingly it was not feasible to meas-
ure the activity of this enzyme in isolated blastomeres. The very low alkaline phos-
phatase activity in early cleavage stages is also characteristic of the mollusk Mytilus
edulis and the sea urchin Strongylocentrotus purpuratus.

TABLE II

Succinic dehydrogenase, expressed as velocity constants for reduction of cytochrome c,
in homogenates of anterior and posterior blastomeres

Number of cells

Succinic dehydrogenase
in anterior cells

Succinic dehydrogenase
in posterior cells

Ratio (ant./post.)

45

0.048

0.071

0.68

40

.033

.062

.53

45

.025

.070

.36

40

.043

.071

.61

45

.042

.089

.47

Average 0.53 ± 0.05

As would be expected on the basis of earlier measurements of cytochrome oxi-
dase (Berg, 1956), succinic dehydrogenase activity is greater in posterior cells.
The average of five experiments shows that homogenates of posterior cells contain
twice as much of this enzyme as anterior cells (Table II).

TABLE III

Apyrase, expressed as optical densities, in homogenates of anterior and

posterior blastomeres

Number of blastomeres

Apyrase in
anterior cells

Apyrase in
posterior cells

Ratio (ant./post.)

44

0.286

0.342

0.84

66

.334

.451

.74

50

.164

.260

.63

60

.230

.352

.65

30

.229

.342

.67

30

.269

.328

.82

30

.269

.412

.65

30

.169

.240

.70

40

.282

.471

.60

Average 0.70 ± 0.03

Apyrase measurements, summarized in Table III, show that the activity of this
enzyme in homogenates of anterior cells is 70 per cent that in homogenates of poste-
rior cells. In adult tissues (Frank et al., 1950) and in amphibian embryos (Earth
and Jaeger, 1947) apy rases with different pH activity characteristics have been ex-
tracted and thus for quantitative measurements of apyrases from different sources

370

WILLIAM E. BERG

it is necessary to determine pH-activity curves. Accordingly an attempt was made
to determine the effect of pH on apyrases from anterior and posterior cells.

Succinate, tris-maleate, and ammediol buffers were used to cover the pH range
from 5 to 9.3 ; pH values were checked with a glass electrode on mixtures prepared
on a macroscale exactly as used for microanalyses. Apyrase activities were deter-
mined simultaneously at four different pH's in a paired series with thirty anterior
or posterior blastomeres in each reaction tube. It was not feasible to cover the en-
tire pH range in any one test ; accordingly it was necessary to overlap pH values in

0.4

> 0.3

H
O

C/)

<

rr

0.2

CL

< O.I-

posterior cells

anterior cells

6 7 8 9 10

PH

FIGURE 2. Apyrases in homogenates of anterior and posterior blastomeres ; pH activity curves.

different experiments. The considerable variability of determinations was probably
in part due to the necessity of constructing the curves from data obtained on differ-
ent batches of eggs.

The pH-activity curves of apyrases from anterior and posterior cells (Fig. 2)
are very similar although the optimum for posterior cell apyrase seems more alkaline.
The difficulties of obtaining a pH-activity curve on such minute amounts of enzyme
are so considerable that this slight difference cannot be considered significant.

The data for paired ribonucleic acid extractions, listed in Table IV, indicate that
anterior cells contain 9 per cent less ribonucleic acid than posterior cells, a difference

ANALYSES OF CIONA BLASTOMERES

371

which is significant. This was at first thought to be due to a slight volume differ-
ence between anterior and posterior cells. Visual observation of the four-cell stage
often gives the impression that the posterior cells are larger. However, careful
measurements of volumes by different methods failed to show any significant differ-
ence in size.

Diameters of blastomeres, with subsequent cleavages prevented by KCN or fixa-
tion in trichloracetic acid, were measured with an ocular micrometer. The average
volume of 85 anterior blastomeres, as calculated from diameter measurements with
cleavage inhibited by 3 X 10~4 M KCN, was 97 per cent that of 75 posterior cells.
The average volume of 134 anterior cells, after fixation in 2 per cent trichloracetic
acid, was 98 per cent that of 178 posterior cells. Although with both types of meas-
urements the average volume of the anterior cells was slightly less, statistical anal-
yses of the data failed to show significant differences of these values.

TABLE IV

Ribonucleic acid, expressed as optical densities at 260
anterior and posterior blastomeres

, extracted from

Number of blastomeres

RNA in anterior cells

RNA in posterior cells

Ratio (ant./post.)

130

0.285

0.308

0.93

140

.230

.272

.85

114

.216

.240

.90

94

.202

.234

.86

112

.302

.301

1.00

64

.160

.170

.94

70

.154

.200

.77

130

.268

.295

.91

75

.221

.215

1.03

100

.140

.146

.96

120

.271

.320

.85

100

.237

.252

.94

Average 0.91 ± 0.02

An additional determination of volumes was made indirectly by measuring rela-
tive amounts of protein in the two types of cells using a micromethod (Lowry et al.,
1951). Twenty-four paired measurements of protein, using 15^40 cells in each
test, gave an average protein ratio (ant./post.) of 0.99 ± 0.046. The difference in
ribonucleic acid content thus cannot be due to a volume difference and must repre-
sent a slight localization of this constituent.

DISCUSSION

A localization of indophenol blue oxidase, presumably cytochrome oxidase, and
succinic dehydrogenase in the ascidian embryo has been demonstrated by cyto-
chemical methods (Ries, 1937; Reverberi and Pitotti, 1939; Mancuso, 1952). The
microchemical measurements of cytochrome oxidase (Berg, 1956) and those of
succinic dehydrogenase in the present study confirm the above cytochemical obser-
vations as to the localization of these enzymes in an early cleavage stage.

372 WILLIAM E. BERG

Information on the intracellular localization of enzymes in the ascidian egg is
lacking ; however a possible interpretation of the distribution of cytochrome oxidase,
succinic dehydrogenase, and apyrase is that they are localized in mitochondria which
are unequally distributed in the early cleavage blastomeres. There is considerable
evidence that these enzymes are intracellularly located in mitochondria of a variety
of cells (reviewed by Schneider and Hogeboom, 1951, 1956). A localization of
mitochondria in the myoplasm of the ascidian egg has been demonstrated by cyto-
logical methods (Meves, 1913; Duesberg, 1915) and more recently by vital staining
with Janus green (Reverberi, 1956). Reverberi (1957a) also presents evidence
that cytochrome oxidase is intracellularly localized in the Janus green-staining
mitochondria.

It is doubtful that the granules described by Duesberg (1915) and Conklin
(1931), classified by them as mitochondria according to morphological and staining
criteria, are responsible for the unequal distribution of enzymes. The granules in
Ciona and "yellow mitochondria" in Styela are displaced by centrifugation to the
centripetal pole (Conklin, 1931) whereas in centrifuged eggs the oxidase reactions
are lacking in this region (Ries, 1939). The yellow granules in Styela may be dis-
placed by light centrifugation without altering the localization of the oxidase reac-
tions (Ries, 1942). In centrifuged homogenates of Ciona eggs cytochrome oxidase
is found in the heavier fraction (Berg. 1956). Furthermore Reverberi (1957a) de-
scribes rod-like mitochondria, stainable with Janus green, which, in the centrifuged
egg, collect at a different location than the granules described by Duesberg and
Conklin. It appears probable, as Reverberi (1957a) also suggests, that several
types of mitochondria are localized in the myoplasm of the ascidian embryo.

The intracellular localization of apyrase is probably to some extent within mito-
chondria, as has been shown for other types of cells (Schneider and Hogeboom,
1951, 1956), and the higher apyrase content of posterior cells most likely results
from the segregation of mitochondria into these cells. The average ratio of activi-
ties in the two types of cells differs significantly from those for cytochrome oxidase
or succinic dehydrogenase, which is interpreted as due to a more heterogeneous
intracellular localization of this enzyme as compared to the oxidative enzymes.

Barth and Jaeger (1947) demonstrated that apyrases with different pH activity
curves are present in several protein fractions of the amphibian embryo. A similar
fractionation of proteins and measurements of the associated apyrases were not pos-
sible on Ciona blastomeres, due to the minute amounts of material available for anal-
yses. The measurements made therefore represent total apyrase activities of the
homogenates and, although there may be qualitatively different apyrases, the simi-
larity of the pH-activity curves for anterior and posterior blastomeres indicates that
segregation of this enzyme at the second cleavage is mainly quantitative.

Although acid phosphatase has been found to be intracellularly localized in small
mitochondria of adult cells (Appelmans et al., 1955), in the present experiments the
opposite distribution of oxidative enzymes and acid phosphatase suggests a non-
mitochondrial localization of the latter. It is possible that the localization of acid
phosphatase may be a consequence of an unequal distribution of mitochondria.
Thus a 2 : 1 distribution of mitochondria, as indicated by enzyme analyses, might
cause displacement of a non-mitochondrial constituent into anterior cells.

Without information on the intracellular localization of ribonucleic acid in the
ascidian egg, little can be said regarding the higher ribonucleic acid content of the

ANALYSES OF CIONA BLASTOMERES 373

posterior cells. A factor other than mitochondrial segregation may be involved
since mitochondria have a low content of ribonucleic acid (Schneider and Hoge-
boom, 1951, 1956).

The results are not due to volume differences since extensive measurements, dis-
cussed previously, of diameters and total protein in the two types of cells failed to
demonstrate any significant differences in volumes. A differential solubility of ribo-
nucleic acid in anterior and posterior blastomeres might lead to erroneous results;
however, there is no indication of this, in that continuous extraction for four days
with perchloric acid did not change the ratio of amounts extracted.

These quantitative analyses do not, of course, give any information as to the
significance of the chemical differences in subsequent differentiation. A few pre-
liminary experiments of rearing embryos in graded concentrations of KCN failed to
show, by visual observation of whole embryos, any obvious differential effects on
differentiation of anterior and posterior cells. Recently, however, Reverberi
(1957b) has shown specific effects of sodium azide, malonate, and selenite on differ-
entiation of the musculature of ascidian larvae, an effect presumably due to blocking
activities of mitochondrial enzymes. Previously Ries (1939), by displacement of
cytoplasmic areas with centrifugation, had concluded that the presence of the oxi-
dative enzymes was essential for muscle differentiation.

Although localization of oxidases may be of significance in subsequent differen-
tiation of ascidian embryos and several other mosaic forms ( Tubijex, Lehmann and
Wahli, 1954; Nereis, Reverberi and Pitotti, 1940; Myzostoma, Pitotti, 1947), this
is not a common process during cleavage of all mosaic eggs.

Quantitative measurements of cytochrome oxidase in AB and CD blastomeres
of Mytilns editlis indicated no unequal distribution of the enzyme in this mosaic egg
(Berg, unpublished). First-cleavage blastomeres were isolated by previously de-
scribed methods (Berg, 1950) and cytochrome oxidase measured by a microspectro-
photometric method (Berg, 1956). Twelve paired measurements of the enzyme in
homogenates of isolated blastomeres gave an average ratio of enzyme activity for the
two types of cells (AB/CD) of 0.98 ± 0.03 after correction for volume differences.

Furthermore cytochemical tests for oxidative enzymes failed to reveal segrega-
tion of these enzymes during early cleavage of Sabellaria (Raven et al., 1950),
Chaetopterus and Pomatocerus (Ries, 1937), Hydroides (Reverberi and Pitotti,
1940) and Limnaea (Raven, 1946).

I am indebted to Professor Martin W. Johnson of the Scripps Institution of
Oceanography, University of California, La Jolla for generously providing space
and facilities in his laboratory where a portion of this work was carried out.

SUMMARY

Anterior and posterior blastomeres of the four-cell stage of Ciona were separated
for quantitative microchemical analyses of succinic dehydrogenase, apyrase, acid
and alkaline phosphatases, and ribonucleic acid. Larger amounts of succinic dehy-
drogenase, apyrase and ribonucleic acid were found in homogenates of posterior cells
whereas acid phosphatase activity was higher in anterior cells. The pH-activity
curves of apyrases from anterior and posterior cells are similar, indicating a quan-
titative segregation of this enzyme. The unequal distribution of succinic dehydroge-
nase, apyrase and, possibly indirectly, acid phosphatase, is probably the result of a
segregation of mitochondria.

374 WILLIAM E. BERG

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ANALYSES OF CIONA BLASTOMERES 375

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THE EFFECTS OF SOME DEVELOPMENTAL INHIBITORS ON THE
PHOSPHORUS BALANCE OF AMPHIBIAN GASTRULAE

JOHN R. GREGG AND MARGIT KAHLBROCK

Zoology Department, Columbia University, New York 27, Nezv York x

Considering the available evidence, it is possible that the morphogenetic move-
ments of gastrulating amphibian embryos are energetically coupled to exergonic
metabolic processes, and it is reasonable to assume that the coupling is mediated
by energy-rich phosphate-bonds. On this basis, it is possible to propose explana-
tions for the well-known inhibitory effects upon gastrular development of such
agents as anaerobiosis, azide and dinitrophenol (Ornstein and Gregg, 1952; Gregg
and Ornstein, 1953), for all of these are believed to dissever or restrict energetic
couplings: anaerobiosis by switching out the aerobic exergonic processes of the
Krebs cycle, azide perhaps by promoting the immediate remineralization of newly
esterified phosphorus in the Embden-Meyerhof system (Spiegelman, Kamen and
Sussman, 1948), and dinitrophenol perhaps by promoting the catalytic reminerali-
zation of esterified phosphorus by mitochondrial dephosphorylases (Hunter, 1951)
or by direct "quenching" of energy-rich phosphate bonds (Middlebrook and Szent-
Gyorgyi, 1955). If the energetic demands of gastrular movements are at all appre-
ciable, then embryos treated with such agents might be expected to exhibit decreases
in their stores of esterified phosphorus, accompanied by corresponding increases of
their inorganic phosphorus contents. The experiments reported in the sequel are
intended to test this proposal.

METHODS

Obtaining and rearing embryos. Fertilized eggs were obtained by stripping
eggs from gravid Rana pipiens females into suspensions of active sperm (R. pipiens
or R. sylvatica) . After about two hours, the clutches of embryos were cut with
scissors into small groups, dispersed thinly among several finger bowls, and reared
at a temperature of 14—15° C. until required for use. The medium in the bowls,
changed daily, was 10% amphibian Ringer's solution without phosphate or bicar-
bonate. Just before use, the embryos were freed of their jelly-coats with forceps.
Their developmental stages were determined by reference to the tables of Shumway
(1940).

Treatment with inhibitors. Solutions of sodium azide and 2, 4 dinitrophenol
were prepared by dissolving weighed samples in aliquots of the same medium in
which embryos were reared. The treatment consisted in placing 25 or 30 Stage 10
embryos in a covered stender dish containing 5 or 10 ml. of inhibitor solution for
24 hours at 14—15° C. At the end of this period their developmental stages were
noted, then they were washed rapidly with distilled water and lyophilized (see
below). Afterward, they were dry-stored in the freezing compartment of a re-
frigerator until phosphorus analyses could be made, usually within two or three days.

1 This investigation was supported in part by a research grant, No. A-1082, from the Public
Health Service.

376

GASTRULA PHOSPHORUS BALANCE 377

Anaerobiosis. For each experiment, 25 or 30 Stage 10 embryos were put into
an Erlenmeyer flask fitted with a two-hole rubber stopper carrying a gas-inlet tube
dipping into the medium in the flask (50 ml. of 10% amphibian Ringer's solution,
without phosphate or bicarbonate) and a gas-outlet tube. Nitrogen alone, or 95%
N2 : 5% CO2 (both previously deoxygenated over hot copper), or hydrogen, was
then bubbled through the flasks for one hour, after which the inlet and outlet tubes
were closed off with pinch clamps. Controls were prepared similarly, except that
air, instead of nitrogen or hydrogen, was bubbled through the medium. After 24
hours (at 14-15° C), the embryos were removed from the flasks, quickly staged,
washed in distilled water and lyophilized. These steps preliminary to freeze-drying
were carried out as rapidly as possible to prevent the occurrence of aerobic recovery
processes. As before, frozen-dried embryos were stored for a short time, if neces-
sary, in the freezing compartment of a refrigerator.

(The authors are grateful for extensive assistance from Dr. Sasha Malamed in
this part of the work.)

Lyophilising. Embryos were dried in vacua in the frozen state with the help of
an all-metal apparatus of conventional design built by Mr. Andrew Pfeiffer, Old
Lyme, Connecticut. Washed embryos were placed with a minimum amount of dis-
tilled water in 10 X 75 mm. Pyrex test tubes which were then partly immersed in
a Cellosolve-dry ice slush. After one or two minutes, the tubes were transferred to
the drying apparatus. Drying was usually complete in three hours. Samples for
chemical analysis were weighed out as rapidly as possible in a closed balance con-
taining a silica-gel desiccant wafer, since lyophilized amphibian embryos are quite
hygroscopic.

Phosphorus analyses. Total phosphorus (PT), total acid-soluble organic phos-
phorus (PAO) and inorganic phosphorus (Pj) were estimated, in micro- and semi-
micro amounts, using the methods of Lowry ct al. (1954). Since we made no es-
sential departures from their recommendations, the reader is referred to their paper
for procedural details. The results of analyses are expressed as micrograms P per
milligram dry weight of embryo.

DISCUSSION OF RESULTS

Aside and dinitrophenol. At a temperature of 14-15° C., normal R. pipiens em-
bryos will develop from Stage 10 (early gastrula) to Stage 12 (late gastrula) in
about 24 hours. In the presence of inhibitors, morphogenesis may be reduced in
amount or abolished altogether, with varying degrees of recovery following the ces-
sation of inhibitory treatment (Table I).

After 24 hours in 10~5 M azide, R. pipiens embryos immersed at Stage 10 will
have reached early Stage 12, with no observable after-effects. Embryos similarly
treated with 10'* to 10~3 M azide will have gastrulated only partially, with after-
effects ranging from slight to severe. In 10~2 M azide, no gastrulation occurs, and
the after-effects are very severe.

After 24 hours in 10~7 M 2, 4 dinitrophenol, R. pipiens embryos immersed at
Stage 10 exhibit no developmental peculiarities, and there are no detectable after-
effects of this treatment. In 10~6 to 10 5 M dinitrophenol development is usually
retarded in various degrees and the after-effects range from moderately severe to
complete failure of recovery. Dinitrophenol in concentrations of 10~4 M, or greater,
inhibits gastrulation altogether, and no recovery has yet been observed.

378

JOHN R. GREGG AND MARGIT KAHLBROCK

TABLE I

Development of R. pipiens embryos after treatment with azide or dinitrophenol, 14°-15° C. Stage
10 jelly-free embryos were immersed in inhibitors for 24 hours, then washed daily in 10% amphibian
Ringer's solution until controls reached Stage 21. Sew = stage of treated embryos after 24 hours
when controls are in Stage 12. Sczi = stage of treated embryos when controls are in Stage 21

Inhibitor

Molar
concentration

Sci2

SC21

Morphological condition

10~s

12-

21

Normal

10-"

10|-12

20

Mostly normal. Gills underdeveloped

but with circulation

Azide

10-3

10-11

18-20

Thickened tail buds, swellings on flanks,

underdeveloped gills with circulation

ID-2

10

18-19

Large yolk plugs, spina bifida, swellings

on flanks, muscular response

10-'

12

21

Normal

io-6

11-12

11-18

Most in Stage 17-18, fairly normal, some

with spina bifida

2,4 Dinitrophenol

io-5

10+-11-

10+-16

Most in Stage 10+-11~. Others mis-

shapen, with large yolk plugs, whitish

"bloom" on surface coat

io-«

10

10

"Bloom"

IO-3

10

10

"Bloom"

Thus, it would seem that embryos which have been totally blocked with azide
can afterwards attain a considerable degree of morphological maturity, something
which is apparently denied embryos similarly blocked with dinitrophenol. Just pos-
sibly, however, this difference between azide and dinitrophenol may be less related
to specific differences in their chemical activities than to differences in their separate
abilities to pass outwards through the vitelline membrane. For, we found that even
repeated washing fails to remove all of the dinitrophenol from treated embryos ; part
of it, at least, remains visibly concentrated in the perivitelline fluid. Whether there
is a similar retention of azide we do not kno\v, since solutions of this inhibitor are
colorless.

We turn now to discuss the effects of such treatments upon the phosphorus bal-
ance of amphibian gastrulae.

TABLE II

Developmental stage and phosphorus balance of R. pipiens gastrulae after 24 hours exposure to
sodium azide, 14—15° C. For meanings of column headings, see section on Methods. Numerals
following ± designate standard deviations. Parenthesized numerals designate numbers of experi-
ments upon which values are based

Molar
cone, azide

Shu m way
stage

PT

PAO

Pi

0

10

13.0 ± 0.4 (3)

0.59 ± 0.09 (3)

0.12 ±0.02 (3)

0

12-12+

12.9 ± 0.7 (3)

0.59 ± 0.11 (3)

0.13 ± 0.02 (3)

io-5

12-

13.0 ± 0.3 (2)

0.65 (1)

0.16 ± 0.03 (2)

io-4

10J

12.8 ±0.5 (3)

0.43 ± 0.02 (2)

0.23 ± 0.02 (3)

10-3

10+

13.1 ± 0.4 (2)

0.44 ± 0.04 (2)

0.29 ± 0.05 (2)

IO-2

10

13.2 ± 0.5 (2)

0.44 ± 0.01 (2)

0.30 ± 0.01 (2)

GASTRULA PHOSPHORUS BALANCE

379

Table II shows that the total phosphorus content of R. pipiens gastrulae, exposed
for 24 hours to 10~5 to 10~2 M azide, is the same as that of untreated control gastru-
lae. This is an important point because (together with the data in Table I) it sug-
gests that such embryos are practically undamaged, otherwise a certain amount of
leakage of phosphorus might be expected. The other two phosphorus fractions,,
however, exhibit changes, for as the concentration of environmental azide is in-
creased, there is an increase of the inorganic phosphorus content of treated embryos,
accompanied by a quantitatively similar decrease in the level of acid-soluble organic
phosphorus. Furthermore, as the concentration of environmental azide is progres-
sively elevated, there is a parallel increase in the severity of gastrular retardation.

The data summarized in Table III show that a similar set of results may be ob-
tained with 2, 4 dinitrophenol at concentrations of 10~5 M, or lower. At concen-
trations greater than 10~5 M, however, gastrulae are damaged to such an extent that
they begin to leak phosphorus ; this is clearly shown by a correlated decline in the
levels of all three phosphorus fractions.

Finally, it should be noted that the gastrulation of untreated control embryos is
unaccompanied by any significant alterations of phosphorus balance.

TABLE III

Developmental stage and phosphorus balance of R. pipiens gastrulae after 24 hours exposure to
2,4 dinitrophenol (DNP), 14-15° C. For meanings of column headings, see section on Methods.
Numerals following ± designate standard deviations

Molar
cone. DNP

Shumway
stage

PT

PAO

PI

No. expts.

0

IQ--IO

13.1 ±0.7

0.59 ± 0.07

0.13 ± 0.01

2

0

12

13.1 ±0.1

0.58 ± 0.04

0.13 ± 0.01

2

10-«

12

12.9 ± 0.1

0.60 ± 0.09

0.17 ±0.05

2

10-5

10+-11-

12.6 ± 0.2

0.45 ± 0.01

0.31 ± 0.02

2

5 X 10-6

10-

11.9

0.34

0.22

1

io-4

IQ--IO

12.4 ± 0.3

0.19 ± 0.09

0.08 ± 0.08

2

Those results suggest the following interpretation. If the movements of gas-
trulation of normal untreated embryos demand an available supply of phosphate-
bond energy, the resulting draughts upon esterified phosphorus are immediately re-
imbursed, and the phosphorus balance is steadily maintained. This result is in full
agreement with the data of Earth and Jaeger (1947), summarized in their Table 1.
It is also consistent with the view that gastrulation is a complex of morphogenetic
movements whose execution requires no expenditure of energy. The results with
azide and dinitrophenol suggest the contrary, however, because of the correlation
between the presence of these inhibitors, the reduction of esterified phosphorus, the
elevation of inorganic phosphorus, and the retardation of gastrular movements.
For it is difficult to explain this correlation except by assuming that in the presence
of inhibitors the production of esterified phosphorus is uncoupled from its utiliza-
tion as a source of morphogenetic energy because it is made available to enzymes
catalyzing its remineralization (see the remarks at the beginning of this paper).
Of the complex of movements, Gregg and Ornstein (1953) have presented evidence
suggesting that epiboly is the most sensitive to treatment with dinitrophenol, while

380

JOHN R. GREGG AND MARGIT KAHLBROCK

TABLE IV

Developmental stage and phosphorus balance of R. pipiens gastrulae after 24 hours anaerobiosis,
14°-15° C. For meanings of column headings, see section on Methods. Numerals following ±
designate standard deviations. P values in A and B are listed separately in order that the former
may be compared with those obtained from hybrid embryos prepared from the same five clutches of
eggs (Table V)

Treatment

Shumway
stage

PT

PAO

PI

No. expts.

Control
A Control
Nitrogen

10

12

11

13.5 ± 0.4
13.2 ± 1.0
13.5 ± 1.1

0.58 ± 0.04
0.55 ± 0.03
0.56 ± 0.05

0.13 ±0.02
0.13 ± 0.01
0.15 ±0.01

5
5
5

Control
B Control
Nitrogen

10+
12
11

12.4
13.00
12.9

0.58
0.52
0.52

0.12
0.13
0.18

1
1
1

Control
C Control
Hydrogen

10
12-
11-

12.4
12.5
12.5

0.58
0.60
0.64

0.13
0.13
0.18

1
1
1

epiboly and notochordal elongation are most affected by the presence of azide ; but
the exact relations of their results to the present ones are yet to be worked out.

Anaerobiosis. Table IV summarizes the results of experiments designed to
show the effects of 24 hours anaerobiosis on the phosphorus balance of R. pipiens
gastrulae. Embryos are morphologically retarded under these conditions, but, in
general, there is no alteration of the phosphorus balance other than a slight elevation
of the inorganic phosphorus level. The total phosphorus and acid-soluble organic
phosphorus levels are unaffected. There is thus a considerable morphogenetic ef-
fect of anaerobiosis, apparently unaccompanied by a decrease in the stored phos-
phate-bond energy potentially available. This conclusion is not in agreement with
that of Barth and Jaeger, who found that anaerobioses of 10 to 22 hours duration
resulted in a considerable decrease of ADP— ATP phosphorus. Their fractionation
procedure is not strictly comparable with ours, however, and the apparent discrep-
ancy cannot be resolved without further investigation.

TABLE V

Developmental stage and phosphorus balance of R. pipiens 9 X R. sylvatica c? gastrulae after
24 hours anaerobiosis, 14-15° C. For meanings of column headings, see section on Methods.
Numerals following ± designate standard deviations. These values should be compared with those
in part A of Table IV, obtained from R. pipiens embryos prepared from the same five clutches of eggs

Treatment

Shumway
stage*

PT

PAO

PI

No. expts.

Control

10

13.2 ± 1.0

0.57 ± 0.04

0.12 ± 0.01

5

Control

12

13.7 ±0.7

0.55 ± 0.05

0.12 ± 0.01

5

Nitrogen

11

13.6 ± 1.0

0.56 ± 0.05

0.14 ± 0.02

5

* The stages assigned are those of simultaneously developing R. pipiens control embryos
(Table IV, A).

GASTRULA PHOSPHORUS BALANCE 381

Embryos in the hybrid R. pipiens $ X R. sylvatica $ fail to gastrulate, but re-
main alive during the whole time required for control R. pipiens embryos to reach
the hatching stage (Moore, 1946; see review by Gregg, 1957). They are charac-
terized by low respiratory rates and by low rates of aerobic and anaerobic glycolysis.
The expectation that they might therefore find it more difficult than normal embryos
to maintain esterified phosphorus stores under the stress of anaerobiosis was con-
firmed by the data of Earth and Jaeger. Our own experiments do not bear out this
expectation, for they show (Table V) that hybrid embryos under anaerobiosis do
not alter their phosphorus balance to a greater extent than R. pipiens controls. But
it is not clear that these results are in genuine disagreement with those of Barth and
Jaeger, for the reason stated at the end of the preceding paragraph.

SUMMARY

1. Rana pipiens gastrulae treated with non-damaging concentrations of sodium
azide or 2, 4 dinitrophenol for 24 hours at 14-15° C. exhibit a reversible retardation
of morphogenetic movements, a diminished store of acid-soluble organic phosphorus,
an elevated content of inorganic phosphorus and an unaltered total phosphorus
content.

2. Anaerobiosis for 24 hours at 14-15° C. does not alter the phosphorus balance
of R. pipiens gastrulae, or of gastrula-arrested hybrids of R. pipiens 5$ with R.
pipiens ($($, beyond a slight elevation of the inorganic phosphorus level.

3. These results are discussed briefly in respect to the energy-requirements of the
morphogenetic movements of gastrulation.

LITERATURE CITED

BARTH, L. G., AND L. JAEGER, 1947. Phosphorylation in the frog's egg. Physiol. Zool., 20:
133-146.

GREGG, J. R., 1957. Morphogenesis and metabolism of gastrula-arrested embryos in the hybrid
Rana pipiens $ X Rana sylvatica d1. In: The Beginnings of Embryonic Development,
edited by A. Tyler, R. C. von Borstel and C. B. Metz, Publ. No. 48, A. A. A. S., Wash-
ington, D. C.

GREGG, J. R., AND N. ORNSTEIN, 1953. Explant systems and the reactions of gastrulating am-
phibians to metabolic poisons. Biol. Bull., 105 : 466-476.

HUNTER, F. E., JR., 1951. Oxidative phosphorylation during electron transport. In: Phospho-
rus Metabolism, Vol. I, edited by W. D. McElroy and B. Glass, The Johns Hopkins
Press, Baltimore.

LOWRY, O. H., N. R. ROBERTS, K. Y. LEINER, M.-L. Wu AND A. L. FARR, 1954. The quantita-
tive histochemistry of brain. I. Chemical methods. /. Biol. Chem., 207 : 1-17.

MIDDLEBROOK, M., AND A. SzENT-GvoRGYi, 1955. The action of iodide on oxidative phosphory-
lation. Biochim. et Biophys. Acta, 18: 407^410.

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, N., AND J. R. GREGG, 1952. Respiratory metabolism of amphibian gastrula explants.
Biol. Bull, 103 : 407-420.

SHUMWAY, W., 1940. Normal stages in the development of Rana pipiens. Anat. Rec., 78:
130-147.

SPIEGELMAN, S., M. D. KAMEN AND M. SUSSMAN, 1948. Phosphate metabolism and the dis-
sociation of anaerobic glycolysis from synthesis in the presence of sodium azide. Arch.
Biochem., 18 : 409-436.

RESPIRATION OF HOMOGENIZED EMBRYOS: RANA PIPIENS
AND RANA PIPIENS $ X RANA SYLVATICA <$ 1

JOHN R. GREGG AND FRANCES L. RAY
Zoology Department, Columbia University, Nciv York 27, Neii' York

Embryos belonging to the hybrid R. pipicns $ X R. sylvatica <$ cleave and blas-
tulate normally, but the normal sequence of gastrulation movements does not occur
(Moore, 1946). Such embryos remain alive, but suspended in a morphological
state superficially similar to that of a very young gastrula. Before the occurrence
of the developmental block, the respiration of hybrid embryos is quantitatively simi-
lar to that of normal R. pipicns controls, increasing exponentially with age ; but for
most of the period following its occurrence, their respiration is characterized by a
function whose value is a constant (Earth, 1946). For an account of the attempts
that have been made to analyze the biochemical and morphogenetic peculiarities of
this hybrid, the reader is referred to the review by Gregg (1957).

The problem of explaining the respiratory peculiarities of gastrula-blocked hy-
brids is of course closely connected with that of constructing a theory to account for
the exponential respiratory increase characterizing the pre-hatching development of
normal embryos. In connection with this latter problem, suggested explanations
have tended to fall into at least two classes : ( 1 ) those which postulate an increas-
ingly rapid developmental synthesis of respiratory enzymes or substrates, and (2)
those which assume a progressive increase in the structural availability of respira-
tory enzymes to their substrates. Each of these is supported by at least some of the
available evidence. On the assumption that one or both of those types of explana-
tion is well-founded, it is plausible to suggest two corresponding sorts of explana-
tion to account for the post-blastula deficiencies of hybrid respiration: (i) that there
is a failure to continue the synthesis of respiratory enzymes or substrates at a suffi-
cient rate, or (ii) there is a failure of some developmental process which normally
continues to increase effective contact of respiratory enzymes and their substrates.

In this paper we report the results of some simple homogenization experiments
which it is hoped will have some bearing on these various questions.

METHODS

Fertilizing, rearing and staging embryos. For fertilizing and rearing embryos,
the following routine was adopted. Two separate sperm suspensions, one of R.
pipiens sperm and one of R. sylvatica sperm, were prepared simultaneously in two
fingerbowls, N and H. Half of the ripe eggs from a gravid R. pipiens were
stripped into N and half into H. After two or three hours, the R. pipiens embryos
in N were separated with scissors into small groups and distributed among several
fingerbowls N1; . . . Nn ; the hybrid embryos in H were similarly dispersed among
several bowls Hb . . . Hn. Embryos in pairs of bowls (Ni; HI) were reared at

1 This investigation was supported in part by a research grant, No. A-1082, from the Public
Health Service.

382

HOMOGENATE RESPIRATION

383

similar temperatures, ranging from 8° C. to 25° C., as convenient. Thus, the nor-
mal embryos in bowl NI served as controls for the hybrids in the corresponding bowl
Hi. The medium in the bowls, 10% amphibian Ringer's solution without phosphate
or bicarbonate, was changed daily. At the desired stages, obtained by reference to
the tables of Shumway (1940). embryos were freed of their jelly coats with jewel-
er's forceps, re-staged, and homogenized. Hybrid embryos were assigned the stage-
numbers characterizing the developmental stages of normal control embryos.

4-1
*»

e

S3 H

^ -

Intacr

75

»00

(S°C-

12$

875

FIGURE 1. Respiration of homogenized R. pipiens embryos. Lower curve for intact R. pipiens

embryos constructed from data of Moog (1944).

Preparation of homogenates. Cell-free breis were prepared by homogenizing
batches of n jelly-free embryos in 0.05 w-ml. aliquots of suitable ice-cold media, using
a high-speed homogenizer manufactured by the Lourdes Instrument Corporation.
Two sorts of media were used routinely :

(a) 0.01 M phosphate buffer made up in 10% amphibian Ringer's solution with-
out phosphate or bicarbonate, pH 6.8-7.0 Breis prepared in this medium will be
called plain-breis.

(b) Medium (a) with the addition of 0.2% sodium deoxycholate (DOC).
Breis prepared in this medium will be called DOC-breis. (We are indebted to Dr.
W. H. Berg for suggesting the use of deoxycholate.)

384

JOHN R. GREGG AND FRANCES L. RAY

Measurement of brei respiration. Within 10 minutes after preparation, 1.0-ml.
aliquots of cold homogenate (about 20 embryos) were pipetted into 7-ml. Warburg
flasks rigged for the measurement of oxygen uptake. After a period of temperature
equilibration in the respirometer bath (about 10 minutes), manometer readings were
begun and continued at 6-minute intervals for 45-60 minutes. The temperature of
the respirometer bath was controlled at 24° C. The flasks were shaken continu-
ously at a rate of 100-110 complete cycles per minute, at an amplitude of 6-8 centi-
meters. The rates of oxygen uptake were calculated from the readings taken during
the first 30 minutes and are expressed as microliters (/A!.) of oxygen per hour per
embryo.

Treatment of data. In Shumway's tables, each stage s is correlated with a
unique time t(s), namely, the time required for a normal embryo to develop from
fertilization to that stage, at 18° C. Furthermore, each t(s) falls in exactly one
of the successive 25-hour intervals following the moment of fertilization. We have
made use of these correlations in presenting the results of measurements of the respi-
ration of breis made from embryos with different environmental (temperature) his-

TABLE I

Effect of buffer concentration on respiration of plain- and DOC-breis, R. pipiens, stage 10. After

equilibration period in respirometer, deoxycholate in appropriate buffer, pH 6.8-7.0,

added from side-arm. Final brei concentration, 20 embryos per ml.

Plain-breis

Buffer concentration, molar

0.025

0.05

0.075

0.1

0.15

0.2

n\. O2 per hour per embryo

0.35

0.23

0.43

0.43

0.27

0.19

DOC-breis,
0.2% DOC

Buffer concentration, molar

0

0.01

0.02

0.04

0.07

0.08

/xl. O2 per hour per embryo

7.8

8.0

7.9

6.4

6.6

6.9

tories. Thus, to construct Figure 1 and Figure 2, we have averaged for each 25-
hour interval the respiratory rates of breis made from embryos whose stage s has a
t(s) in that interval and have plotted the resulting average against the interval's
midpoint.

RESULTS

R. pipiens, plain-breis. Reference to Figure 1 will show that the average respi-
ratory rate of plain-breis increases exponentially with the age of the embryos used
in their preparation from an initial value of about 0.2 /*!. O2 per hour per embryo to
a maximum value in the fourth 25-hour interval of about 4.5 /*!. O2 per hour per
embryo, and thereafter tends to decline.

This result is in sharp contrast to that of Spiegelman and Steinbach (1945),
who reported that the endogenous respiration of plain-breis prepared from embryos
shortly after fertilization is already at a maximum. We are at a loss to explain the
discrepancy, for we have been able to elevate the respiration of breis only by treat-
ment with a detergent (see next paragraph), but not by altering the buffer concen-
tration (Table I), nor, in preliminary experiments, by adding glycogen, glucose,
hexose diphosphate, adenylic acid, magnesium, or various combinations thereof.

HOMOGENATE RESPIRATION

385

TABLE II

Effect of sodium deoxycholate concentration on respiration of homogenized R. pipiens embryos,
stage 10-11. Breis and deoxycholate made up separately in 0.07 M phosphate buffer, pH 6.8-7.0.
After equilibration period in respirometer bath, deoxycholate added from side-arm. Final brei con-
centration, 20 embryos per ml.

Final concentration of sodium deoxycholate,
per cent

0

0.1

0.2

0.4

0.8

/il. O2 per hour per embryo

0.12

2.8

6.6

5.6

4.4

R. pipiens, DOC-breis. The maximum respiratory rate attained by plain-breis,
i.e., that exhibited by embryos homogenized during the fourth 25-hour period of de-
velopment, can be matched by that of deoxycholate-treated breis prepared from em-
bryos of any lesser age (Fig. 1). Plain-breis and DOC-breis prepared from em-
bryos older than this, however, respire at the same rate. The degree of respiratory
elevation obtained earlier is a function of the concentration of DOC, 0.2% being the
optimal concentration (Table II).

5 -

1 -

I -

Intatt ...

25

59 ^S 100

hours Development I8*C-

FIGURE 2. Respiration of homogenized gastrula-blocked hybrid embryos (R. pipiens $ X R.
sylvatica c?). Lower curve for intact embryos constructed from data of Earth (1946) and
Moog (1944).

386 JOHN R. GREGG AND FRANCES L. RAY

The curve for DOC-breis shown in Figure 1 is very similar to that reported for
plain-breis by Spiegelman and Steinbach, although the general respiratory level is
much higher and begins to decline somewhat later. We do not know if the stimu-
latory effect of DOC can be obtained with other detergents. However, we have
found that plain-breis of early embryos respire at unusually high rates if they are
prepared in Waring Blendor vessels freshly washed in Alconox. The effect wears
off after several preparations without intervening cleansings with Alconox. This
suggests that detergent is trapped in the bearings and leaks out slowly during the
preparation of breis, but a systematic study of the question has not been made.

Hybrids, plain-breis and DOC-brcis. The results of our measurements of the
respiratory rates of plain- and DOC-breis made from hybrid embryos (Fig. 2) can
be summarized very briefly : the respiration of such breis is quantitatively similar to
that of corresponding breis made from normal control embryos, except perhaps in
the seventh 25-hour period when the hybrids are moribund.

DISCUSSION

There is no need to postulate a developmental synthesis of respiratory enzymes
or substrates to account for the exponential rise of respiratory rate characterizing
the development of normal amphibian embryos; for, as the high respiratory rates
of DOC-breis show, there is from the outset of development enough respiratory ma-
chinery to support oxidation-rates greater than any exhibited by intact pre-hatching
embryos. Similarly, there is no need to assume a synthetic failure to account for
the abnormal constancy of post-blastula hybrid respiration, because the experiments
with DOC-breis have shown that hybrid embryos at nearly all stages are potentially
capable of as much respiration as normal controls.

For normal embryos, we adopt the conclusion of Spiegelman and Steinbach,
namely, that the exponential respiratory increase is causally related to morpho-
genetic changes which progressively facilitate the union of respiratory enzymes and
their substrates. Correspondingly, to explain the respiratory constancy of aging
post-blastula hybrids, we assume that those changes somehow have been brought to
a halt at the commencement of the gastrula stage. What sorts of changes might be
involved is at present unknown. The elevation of embryonic respiration obtained
by homogenizing in plain phosphate buffer, increasingly extensive as development
proceeds, suggests that some cellular structures are becoming increasingly sensitive
to mechanical disturbance or to alterations of chemical milieu, and the maximal
respiration obtained at all stages by the treatment of breis with deoxycholate sug-
gests a cellular component sensitive to detergent action : in both cases the mitochon-
dria come immediately to mind because of their close association with oxidative en-
zymes of the Krebs cycle and of the hydrogen transport system, and because they
are sensitive to isolative procedures, especially in the presence of deoxycholate
(Siekevitz and Watson, 1956). It is reasonable to suppose that the extensive frag-
mentation of mitochondria by deoxycholate is only a more thoroughgoing version
of what happens at any developmental stage to the mitochondria in plain-breis. In
any case, it would be very interesting to study the mitochondria of differentiating
embryos, and to compare those of hybrids with those of normal embryos.

The decline in the respiration obtainable from breis of late embryos may be ex-
plained by assuming, as Spiegelman and Steinbach do, a depletion of endogenous

HOMOGENATE RESPIRATION 387

respiratory substrates. In this connection, it is known that only half of the stored
carbohydrate available at the time of fertilization remains in normal embryos at
the time of hatching, although more than this remains in hybrids of the same age
(Gregg, 1948).

SUMMARY

1. The respiration of phosphate-buffered cell-free homogenates made from
R. pi pi ens embryos increases exponentially with the age of the embryos up until
the time at which they are in the tailbud stage, after which the rate declines.

2. Addition of 0.2% sodium deoxycholate elevates the respiration of homoge-
nized embryos at any pre-tailbud stage to that of tailbud-breis, but has no effect upon
that of breis of post-tailbud embryos.

3. The respiration of plain- or deoxycholate-treated breis is at all stages greater
than or equal to that of intact embryos.

4. The respiration of breis (plain- and deoxycholate-treated) made from gas-
trula-arrested R. pipiens $ X R. sylvatica <$ hybrid embryos is at all non-moribund
stages quantitatively the same as that of control breis of normal embryos.

5. The implications of these findings are briefly discussed.

LITERATURE CITED

BARTH, L. G., 1946. Studies on the metabolism of development. /. Exp. Zool., 103 : 463-486.

GREGG, J. R., 1948. Carbohydrate metabolism of normal and of hybrid amphibian embryos.
/. Exp. Zool, 109: 119-134.

GREGG, J. R., 1957. Morphogenesis and metabolism of gastrula-arrested embryos in the hybrid
Rana pipiens ? X Rana sylvatica <$. In: The Beginnings of Embryonic Development,
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.

MOOG, F., 1944. The chloretone sensitivity of frog's eggs in relation to respiration and de-
velopment. /. 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.

SIEKEVITZ, P., AND M. WATSON, 1956. Cytochemical studies of mitochondria. I. The separation
and identification of a membrane fraction from isolated mitochondria. /. Biophys. Bio-
chem. Res., 2 : 639-652.

SHUMWAY, W., 1940. Normal stages in the development of R. pipiens. Anat. Rec., 78: 139-
149.

SPIEGELMAN, S., AND H. B. STEINBACH, 1945. Substrate-enzyme orientation during embryonic
development. Biol. Bull., 88 : 254-268.

A RATIONAL APPROACH TO THE PROBLEM OF CANCER

CHEMOTHERAPY *

L. V. HEILBRUNN AND W. L. WILSON

Department of Zoology, University of Pennsylvania, Philadelphia, Pa.; Department of

Physiology and Biophysics, University of Vermont, Burlington, Vt.; and the

Marine Biological Laboratory, Woods Hole, Mass.

In spite of the fact that over a long period of time, many workers in various
parts of the world have been studying the curative effect of various chemical agents
on cancer, there has been no very great effort to determine why these agents act
as they do, and there is at present but little pertinent information as to the type of
action these agents have on the cancer cell.

The problem is complicated by the fact that many of the very agents that have
a definite curative effect can also act as carcinogenic agents. This was first noted
by Haddow in 1935, and he and his collaborators have written a number of papers
on the subject. In his very useful book on the "Biochemistry of Cancer," Green-
stein (1954) frequently refers to this phenomenon as the "Haddow paradox."
But neither Haddow nor Greenstein has any interpretation of the paradox. In 1951,
Haddow in writing about carcinogens and substances which have a therapeutic
effect on cancer writes (p. 264) : "But in no case — a striking fact — do we know the
place in the cell at which they act — whether the cell surface, the cytoplasm, the
nuclear membrane, the nucleus itself — or the nature of the receptors with which they
combine." And all that Greenstein has to suggest when he considers the problem
is that (p. 278) "The capacity for intellectual flexibility combined with scientific care
is one of the demands in the field."

In the past, most of the work that has been done on the chemical treatment of
cancer and the theory back of such treatment has been done by chemists. And
whatever efforts they have made toward interpretation have for the most part been
inspired by chemical concepts built around the idea of some disturbance of metabo-
lism. But some of the most powerful chemotherapeutic agents do not affect the
growth of the cell. Thus in the presence of nitrogen mustard or its oxygen deriva-
tive, Nitromin, cells increase in size but do not divide (Bodenstein, 1947; Frieden-
wald, Buschke and Scholz, 1948; Sato, Belkin and Essner, 1956).

When a tumor arises in an organ or tissue the appearance of the neoplasm is
always accompanied by a great increase in the number of cells that are dividing.
Thus for example in the brain, there are normally no mitoses, but in a brain tumor
there are great numbers of dividing cells. It is of course possible to believe that
some increase in metabolism, or some change in metabolism, is the primary cause
that started the tumor to develop. But it is just as possible to assume that the pri-
mary factor is an initiation of mitosis and that metabolic changes are a result rather
than a cause.

1 Supported by grants from the National Institutes of Health and from the American Cancer
Society.

388

RATIONALE OF CANCER CHEMOTHERAPY 389

At the present time, we have a considerable body of information concerning the
initiation of cell division, and there is also a satisfactory theory as to why and how
cells can be made to divide. Moreover we have information as to the suppression of
cell division and the reasons for it. Both the initiation and the suppression of cell
division can be understood in terms of the behavior of the protoplasmic colloid, and
the changes that occur in it that lead to the formation of the mitotic spindle. For
a survey of this knowledge, see Heilbrunn's "Dynamics of Living Protoplasm"
(1956). One of the strange facts about protoplasm is that the same agents which
suppress its activity can also under certain conditions arouse it to activity. We thus
have a parallel to the Haddow paradox, and indeed the initiation of cell division can
be regarded as one example of the response to stimulation.

Can we hope therefore to interpret the chemotherapy of cancer and the Haddow
paradox on the basis of a colloidal theory ? Surely such an approach is worth look-
ing into, especially in view of the fact that other types of interpretation have not as
yet been successful.

In a long series of papers, most of them referred to in the book already cited,
Heilbrunn and his co-workers have investigated the problem of the initiation and
suppression of cell division from the standpoint of the colloidal properties of proto-
plasm and the changes that the protoplasmic colloid must undergo in order to form
a mitotic spindle. The basic colloidal reaction of protoplasm is a clotting reaction
similar in many respects to the clotting of blood. The protoplasm is in a state of
equilibrium between the various factors which favor or induce clotting and those
which prevent it. Before a mitotic spindle can form, a gelation must occur in the
protoplasm. This is the mitotic gelation, and it can be induced either by a release of
calcium from a bound state in the outer cortex of the cell or by the entrance into
the cell of thrombin-like substances. Many agents prevent the mitotic gelation —
among them, substances of a heparin-like nature.

Starting out from this point of view, we thought to investigate the action of cer-
tain chemotherapeutic agents which have been used in the experimental study of
cancer as well as in clinical practice. The first question to be answered is whether
or not these agents prevent the mitotic gelation. Then later we will consider the
question of the Haddow paradox.

MATERIALS AND METHODS

The cell we used as a test object was the egg of the worm Chaetopterus perga-
wientaccus. The eggs of this worm can readily be obtained at Woods Hole during
the summer months. When the eggs are shed into sea water they are in the germi-
nal vesicle stage. In 7 minutes (at 21° C.) the germinal vesicle breaks down and
the first maturation division proceeds as far as the metaphase. Then all mitotic
activity ceases until the egg is fertilized. Following fertilization the first maturation
division is completed and the second maturation division immediately follows the
first. As a result two polar bodies are given off and the egg then prepares for its
first cleavage division. The mitotic spindle for this division appears at forty min-
utes after insemination (at 21° C.) and 50% of the eggs cleave at 56 minutes after
insemination. Before the appearance of the mitotic spindle, the viscosity of the pro-
toplasm increases markedly ; this constitutes the mitotic gelation. Simultaneously
with the appearance of the spindle the protoplasm becomes more fluid again so that

390 L. V. HEILBRUNN AND W. L. WILSON

at the metaphase the viscosity is again low. Details of these changes are given by
Heilbrunn and Wilson (1948). One advantage in using the Chaetopterus egg is
that in any given lot the course of events in different individual eggs varies but
little, and there is almost perfect synchrony.

Viscosity measurements were made with a hand centrifuge. At the present time
it is not possible to buy suitable hand centrifuges. The ones we use are made for
us by Mr. J. A. Appenzeller, technician of the Zoology Department of the University
of Pennsylvania. They are adapted from a hand centrifuge sold by Sears, Roe-
buck and Co. and intended for the separation of cream from milk. In order to use
these cream separators for our purposes it is necessary to fit them with a head
which will hold glass tubes. The tubes we use have an outside diameter of 4 or 5
millimeters. When our centrifuges are turned at the rate of one turn of the handle
per second, they give a force approximately 9,000 times gravity. We prefer to
turn the handle once every two seconds ; this gives a force one-fourth as great,
that is to say, 2,250 times gravity. With a force of this magnitude, for most of the
time between insemination and cleavage, it requires 7 seconds to move the granules
in the Chaetopterus egg sufficiently so as to give the impression of zones. (The
heavier granules move centrifugally and the lighter granules centripetally.) The
number of seconds required to produce zoning is taken as an arbitrary viscosity
value. At about 27 minutes after insemination (at 21° C.) the viscosity of the pro-
toplasm begins to increase, and by about 30 or 32 minutes the viscosity has increased
until it is approximately twice what it was before this mitotic gelation began. The
viscosity then stays high until the spindle appears, a matter of about 8 minutes. It
is during this time that tests of viscosity must be made if we are to discover if a
given substance keeps the protoplasm fluid and prevents the mitotic gelation. These
tests must therefore be made rapidly so that it is not possible to obtain definite
values. But we can be sure that in the controls mitotic gelation has occurred if the
viscosity is high enough so that it requires more than 8 seconds of centrifugal turn-
ing to cause an appearance of zones. Actually the viscosity during normal mitotic
gelation is 14 in our arbitrary units. In the tables we record the viscosity of the
control eggs as more than 8 ; it almost certainly is 14.

The nitrogen mustard used in our experiments was obtained from Sharp and
Dohme in the form of a commercial preparation called Mustargen. This prepara-
tion comes in separate sealed vials, each of which contains 10 mg. of nitrogen mus-
tard, that is to say methyl-bis(beta-chloro-ethyl) amine hydrochloride plus 100 mg.
of NaCl. The contents of each vial were hastily dissolved in sea water, but be-
cause of the presence of the NaCl it was not possible to prepare solutions of high
concentration, for such solutions would have been hypertonic and might have
masked the effect of the nitrogen mustard.

Through the kindness of Dr. Edward S. Essner we were able to obtain Nitromin,
an oxide derivative of nitrogen mustard manufactured by the Yoshitomi Pharma-
ceutical Industries of Osaka in Japan and distributed by Takeda Pharmaceutical
Industries, also of Osaka. Nitromin is methyl-bis(beta-chloroethyl)amine-N-
oxide hydrochloride. This compound is less toxic than nitrogen mustard and has
been claimed to have better therapeutic value.

In using 6-mercaptopurine, we had difficulty. This substance is scarcely soluble
at all in sea water. In order to obtain a solution we dissolved it first in a small
amount of normal NaOH. Then strongly acidified sea water was added until a pH

RATIONALE OF CANCER CHEMOTHERAPY

391

TABLE I
Effect of Mustargen on fertilized Chaetopterus eggs

%

pH

Viscosity at 32-38 min.

% Cleavage

0 (control)

>8

94

0.1

7.5

8 or less

18

0.05

8 or less

20

0.025

8 or less

16

0 (control)

>8

94

0.1

7.5

8 or less

27

0.05

8 or less

32

0.025

8 or less

19

a little higher than that of sea water was reached. At this pH, microscopic obser-
vations showed the solution to be full of suspended material, so that we could not be
at all certain as to how much of the substance remained in solution. In all of our
experiments we were dealing with a saturated solution of unknown concentration,
and we have the impression that in sea water 6-mercaptopurine is barely soluble.

RESULTS

When nitrogen mustard is dissolved in sea water the resultant solution has a
much lower pH than does sea water. In our first experiments we made no attempt
to neutralize the acid in our solutions, and the results of these experiments were
therefore discarded. Table I gives the results of two experiments in which the
Mustargen solution was made more alkaline by the addition of NaOH solution. In
both experiments, the solutions were brought to a pH of 7.5 and were thus still
somewhat less alkaline than sea water. However, in preparing dilutions from the
0.1% solution of nitrogen mustard, the dilutions were of course made with sea
water so that in the lower concentrations of the drug, the pH was not very different

TABLE II

Effect of Nitromin on fertilized Chaetopterus eggs

%

pH

Viscosity at 31-35 min.

% Cleavage

0 (control)

>8

100

0.4

7.9

8 or less

0.5

0.3

8 or less

4

0.2

8 or less

9

0 (control)

>8

97

0.5

8.0

8 or less

0

0 (control)

>8

97

0.4

7.8

8 or less

0

0.3

8 or less

0

0.2

8 or less

2

392

L. V. HEILBRUNN AND W. L. WILSON

from that of sea water. Moreover, a pH of 7.5 has but little effect on the proto-
plasm of Chaetopterus eggs.

The experiments with nitrogen mustard indicate that this substance keeps pro-
toplasm fluid and prevents the mitotic gelation. It thus acts in the same way as do
various other antimitotic substances previously studied by us (Heilbrunn and
Wilson, 1950a, 1950b, 1956; Heilbrunn. Wilson and Harding, 1951; Heilbrunn,
Chaet, Dunn and Wilson, 1954). The mechanism of this action will be discussed
later.

Our experiments with Nitromin gave more striking results than those with ni-
trogen mustard. Like Mustargen, Nitromin when dissolved in sea water causes a
substantial reduction in the pH. As before, we added enough NaOH to bring back
the solution to a pH like that of sea water. The results obtained with Nitromin are
shown in Table II. They show conclusively that this derivative of nitrogen mustard
keeps protoplasm fluid and completely prevents the mitotic gelation. Very few of
the eggs exposed to rather dilute solutions of Nitromin ever cleave.

As pointed out in the section on Materials and Methods, 6-mercaptopurine is
very sparingly soluble in sea water, and sometimes we wondered if any of it went

f

TABLE III

Effect of 6-mercaptopurine on Chaetopterus eggs

%

pH

Viscosity at 30-35 min.

% Cleavage

0 (control)

>8

97

0.1

8.25

8 or less

49

0 (control)

>8

98

0.3

8.1

8

99

0.3 (exposure 30 min.

before fertilization)

8.1

8

70

into solution at all. Our results with this substances are not very impressive ; they
are shown in Table III. The results we did obtain indicate clearly enough that
6-mercaptopurine tends to keep the protoplasm of the Chaetopterus egg fluid. This
effect is more pronounced when the solution is a little more alkaline, presumably
because at the higher alkalinity more of the substance stays in solution. Also at
the higher pH the inhibition of cleavage was greater.

Nitrogen mustard, Nitromin and 6-mercaptopurine thus all have the same sort
of effect on the protoplasmic colloid. All of them tend to keep the protoplasm fluid
and prevent the mitotic gelation. In the past it has been shown many times that the
same agents which prevent gelation may, in other concentrations, have quite the
opposite effect (for references and discussion, see Heilbrunn, 1956). Fat solvent
anesthetics, which keep protoplasm fluid and thus prevent response to stimulation,
may in certain concentrations act as stimulating agents and when they do they
induce a clotting or gelation of the protoplasm. Now it is proper to consider the
prevention of cell division by agents which do not kill the cell as a form of anesthesia
or narcosis, and indeed various anesthetic agents do prevent cell division. And
the initiation of cell division can be regarded as a response to stimulation. All this
being true, might it not be possible to show that with other concentrations of nitrogen

RATIONALE OF CANCER CHEMOTHERAPY 393

TABLE IV

Effect of ethyl urethane on fertilized Chaetopterus eggs

Viscosity 30-33 min.
% Urethane after fertilization % Cleavage

0 (control)
2
1.5
1

>8
8 or less
8 or less
8 or less

100
0
0
0

mustard or Nitromin a gelation of the protoplasm could be induced and perhaps
also an initiation of cell division ? If we could show this, we would have a way of
interpreting the Haddow paradox.

We did not attempt to do this experiment with nitrogen mustard, for the prepa-
ration of this drug that was available to us — Mustargen — contains ten times as
much NaCl as it does nitrogen mustard, and if we made relatively concentrated solu-
tions we would arrive at concentrations of salt which would in themselves cause the
initiation of cell division. However, with Nitromin this difficulty does not exist.
Accordingly, we tried the effect of a \% and a 0.5% solution of Nitromin on un-
fertilized Chaetopterus eggs. Both of these solutions caused a marked increase in
the viscosity of the protoplasm. In the weaker solution this increase (after 80 min-
utes) was at least two-fold; in the stronger solution the viscosity increase was even
greater and the protoplasm seemed quite solid. In both cases the drug caused a
vacuolization of the protoplasm. This is the type of reaction which Loeb (1913)
called cytolysis, and it is a reaction commonly produced by agents which initiate
division in marine eggs when these agents are used in too strong a concentration or
for too long an exposure. However, in the one experiment we tried, we were not
able to obtain any initiation of cell division. In this experiment the eggs were ex-
posed to \% and to 0.5% Nitromin for periods of 1, 2, 5, 10, 20, 30 and 60 minutes.
Our failure to obtain initiation of cell division with the Nitromin solutions \vas not
surprising, for although in every case when egg cells are stimulated to divide, the
viscosity of the protoplasm in the interior of the cell is markedly increased, the re-
verse is not true ; for an agent which tends to gel or clot the protoplasm may be too
toxic to permit cell division to proceed. Thus in the work on Nitromin, there is only
a partial explanation of the Haddow paradox. For though it is true that Nitromin
can produce opposite effects on the protoplasmic colloid, we know only that it can
suppress cell division and not that it can initiate it.

TABLE V
Effect of ethyl urethane on unfertilized Chaetopterus eggs

Time of exposure
to 3% urethane Viscosity

20 min. 11

32 13

54 13

Control (untreated) 8

394

L. V. HEILBRUNN AND W. L. WILSON

TABLE VI

Effect of 3% ethyl urethane in initiating cell division of unfertilized Chaetopterus eggs

Exposure time
in minutes

Exp. A:
% cleavage
after 3$ hours

F.xp. B:
% cleavage
after 6 hours

Exp. C:
% cleavage
after 8 hours

5

0

0

3

10

0

16

24

15

0

16

50

20

15

40

55

25

26

80

50

30

37

88

45

35

26

29

40

20

34

45

11

20

50

13

23

55

10

22

60

4

6

Control

0

0.05

9

We thought therefore to try the effect of urethane ; for this substance, which is
known to act both as a carcinogen and as a chemotherapeutic agent for tumors, is
presumably less toxic than Nitromin. In relatively weak concentrations, ethyl ure-
thane suppresses cell division in the Chaetopterus egg. This is shown in Table IV.
This table also shows that in concentrations which suppress cell division, the urethane
keeps the protoplasm fluid and prevents the mitotic gelation.

Higher concentrations of urethane have quite the opposite effect. Thus when
unfertilized eggs are placed in a 3% solution of urethane, the protoplasmic viscosity
increases sharply, as is shown in Table V.

Moreover exposure to 3% urethane can, in a high percentage of cases, cause the
egg cells to divide. We have done some experiments of our own to show this, but
experiments done by Mr. Herbert Schuel are more complete than ours and we prefer
to present them. They are shown in Table VI.

DISCUSSION

Clearly, then, one and the same agent in different concentrations can cause either
initiation of cell division or suppression of cell division and these opposite effects are
readily correlated with the action the reagent has on the colloidal state of the pro-
toplasm in the interior of the cell.

The facts as we have reported them are so clear cut that they scarcely require
additional comment. Therapeutic agents commonly used in the treatment of cancer
can prevent cell division by keeping the protoplasm fluid. Some of these agents,
when used in different concentrations, can have opposite effects both on the physical
state of the protoplasmic colloid and also, in the case of urethane, on the end result.
Here, then, we have a way of interpreting the Haddow paradox, and we are able to
supply the information Haddow was so concerned about, namely on which part of
the cell these agents act and what they do.

But the question immediately arises as to the mechanism of the paradoxical ac-

RATIONALE OF CANCER CHEMOTHERAPY 395

tion. In the case of urethane, the answer is rather obvious in the light of what
we know concerning the way various anesthetic or narcotic agents act on protoplasm.
This subject is discussed at some length by Heilbrunn (1956). Suffice to say here
that these agents liquefy the cortical protoplasm and release calcium from it; the
calcium thus released enters the interior of the cell and there causes a clotting reac-
tion, which may lead either to excitation or to a complete vacuolization of the pro-
toplasm and death of the cell. But fat solvent anesthetics not only tend to free cal-
cium from the cortex, they also tend to prevent calcium in the cell interior from
causing a clotting reaction. There is a large and growing body of evidence in sup-
port of these statements (see Heilbrunn, 1956). In addition it should perhaps be
noted that ether, which in low concentrations keeps the protoplasm of sea urchin
eggs fluid (Heilbrunn, 1920, 1925), can in higher concentrations induce cell di-
vision (Mathews, 1900; McClendon, 1910) ; in these higher concentrations it causes
a clotting reaction in the protoplasm.

It is to be hoped that other investigators will join with us in approaching the
problem of cancer chemotherapy from the standpoint of the colloid chemistry of
protoplasm. At the present time there are so many excellent and well trained
workers interested in the metabolic approach, and scarcely anyone concerned with
the reasons why the protoplasmic colloid changes in such a way as to form a mitotic
spindle. And yet, without mitosis there can be no cancer, and if there are rela-
tively non-toxic ways of preventing mitosis, certainly this is a field that should
be investigated on a large scale. Insofar as we know at present, neither the initia-
tion nor the suppression of mitosis depends on any particular metabolic pathway
and it certainly does depend on colloidal changes in the protoplasm. Antimitotic
substances such as can be extracted from ovaries can indeed be used to cure mice
inoculated with a lethal ascites tumor (Heilbrunn, Wilson, Tosteson, Davidson and
Rutman, 1957) and in their therapeutic action on this tumor they are at least as
effective as nitrogen mustard or Nitromin. Indeed more recent experiments have
shown them to be decidedly more effective.

SUMMARY

1. Nitrogen mustard, Nitromin, 6-mercaptopurine, and urethane suppress cell
division in Chaetopterus eggs.

2. This inhibition of mitosis is due to the fact that these agents keep the proto-
plasm fluid and prevent the mitotic gelation.

3. In relatively high concentration, both Nitromin and urethane cause a gela-
tion of the protoplasm and in these concentrations, urethane can initiate cell division
in a high percentage of the eggs.

4. An interpretation is given of this paradoxical action of reagents in causing
either liquefaction or gelation, either suppression or initiation of cell division.

5. The results are believed to provide an explanation of the Haddow paradox.

LITERATURE CITED

BODENSTEIN, D., 1947. The effects of nitrogen mustard on embryonic amphibian development.

/. Exp. Zool, 104: 311-341.
FRIEDENWALD, J. S., W. BUSCHKE AND R. O. SCHOLZ, 1948. Effect of mustard and nitrogen

mustard on mitotic and wound healing activities of the corneal epithelium. Bull.

Johns Hopkins Hasp., 82: 148-160.

396 L. V. HEILBRUNN AND W. L. WILSON

GREENSTEIN, J. P., 1954. Biochemistry of Cancer, Second Ed. Academic Press, New York.
HADDOW, A., 1935. Influence of certain polycyclic hydrocarbons on the growth of the Jensen

rat sarcoma. Nature, 136: 868-869.
HADDOW, A., 1951. Advances in the study of chemical carcinogenesis. Proc. Roy. Soc. Mcd.,

44: 263-266.
HEILBRUNN, L. V., 1920. An experimental study of cell division. I. The physical conditions

which determine the appearance of the spindle in sea-urchin eggs. /. E.rp. Zool., 30 :

211-237.

HEILBRUNN, L. V., 1925.The action of ether on protoplasm. Biol. Bull., 49 : 461-476.
HEILBRUNN, L. V., 1956. The Dynamics of Living Protoplasm. Academic Press, New York.
HEILBRUNN, L. V., A. B. CHAET, A. DUNN AND W. L. WILSON, 1954. Antimitotic substances

from ovaries. Biol. Bull, 106: 158-168.
HEILBRUNN, L. V., AND W. L. WILSON, 1948. Protoplasmic viscosity changes during mitosis

in the egg of Chaetopterus. Biol. Bull, 95 : 57-68.
HEILBRUNN, L. V., AND W. L. WILSON, 1950a. Effect of bacterial polysaccharide on cell

division. Science, 112: 56-57.
HEILBRUNN, L. V., AND W. L. WILSON, 1950b. The prevention of cell division by anti-clotting

agents. Protoplasma, 39: 389-399.
HEILBRUNN, L. V., AND W. L. WILSON, 1956. Antimitotic substances from the ovaries of

vertebrates. Biol. Bull, 110: 153-156.
HEILBRUNN, L. V., W. L. WILSON AND D. HARDING, 1951. The action of tissue extracts on

cell division. /. Nat. Cancer Inst., 11: 1287-1298.
HEILBRUNN, L. V., W. L. WILSON, T. R. TOSTESON, E. DAVIDSON AND R. J. RUTMAN, 1957.

The antimitotic and carcinostatic action of ovarian extracts. Biol. Bull., 113: 129-134.
LOEB, J., 1913. Artificial Parthenogenesis and Fertilization. University of Chicago Press,

Chicago.
MATHEWS, A. P., 1900. Some ways of causing mitotic division in unfertilized Arbacia eggs.

Amer. J. Physio!., 4: 343-347.

McQ-ENDON, J. F., 1910. On the dynamics of cell division. II. Changes in permeability of de-
veloping eggs to electrolytes. Amer. J. Physiol, 27: 240-275.

SATO, H., M. BELKIN AND E. ESSNER, 1956. Effect of Nitromin on mitosis and cytoplasmic vol-
ume in the cells of two mouse ascites tumors. /. Nat. Cancer Inst., 17: 421-433.

FURTHER STUDIES IN THE BEHAVIOR OF COMMENSAL

POLYCHAETES

JOHN F. HICKOK AND DEMOREST DAVENPORT

. of Biological Sciences, University of California, Santa Barbara College, Golcta, Calif.

To date a number of studies have been made on the response specificity of com-
mensal polychaetes (Davenport, 1950, 1953a, 1953b; Davenport and Hickok, 1951 ;
Bartel and Davenport, 1956). In these studies a number of techniques to discern
the presence of chemical responses to host have been employed. The subject of
specificity and behavior in animal partnerships has recently been reviewed (Daven-
port, 1955).

During the summer of 1956 further investigations of the behavior of a number
of polychaete commensals were conducted at the Friday Harbor Laboratories of the
University of Washington. The authors wish to express their appreciation to the
Director and staff of the Laboratories for their continued interest and support in
these researches. The studies are currently continuing in the Marine Biological
Laboratory of Santa Barbara College and have been supported since 1955 by a con-
tract from the Office of Naval Research.

The preliminary investigations cited above had indicated the necessity to com-
pare the behavior of populations of single species of facultative and obligate com-
mensals of diverse host-habit, and to determine whether these populations showed
different response specificity. The following studies were directed to that end.

THE FACULTATIVE COMMENSAL PODARKE PUGETTENSIS JOHNSON
Material

The hesionid polychaete Podarke pugettensis provides a most interesting subject
for behavioral studies. The worm is a facultative commensal, and there appears to
be no discernible morphological difference between free-living and commensal mem-
bers of the species. In the free state the species occurs in great numbers under cer-
tain conditions; one may at times collect as many as 15—20 per square yard on the
mudflats of Garrison Bay, San Juan Island, Washington. In the Southern Cali-
fornia region it may be collected as it settles out of the plankton by suspending open-
mouth jars under floats in San Pedro Harbor (D. J. Reish, personal communica-
tion), wrhile numbers of adults may be taken by scraping the under surface of floats
in the same locality and in Santa Barbara Harbor. It may also be collected by re-
moving large pieces of the growth from pilings, where it occurs near the wood surface
deep among the shells of the gastropod Aletes and the pelecypod Chama. It occurs
among the byssus threads of Mytilus on pilings. Under these conditions the worms
do not appear to be associated with any particular organism, but they certainly seem
to thrive in environments of extremely rich organic content. Free-living indi-
viduals will be found sporadically in many sorts of environments, particularly where
there is rich mud, in the inter-tidal and subtidal. During the summer of 1956 ripe

397

398 JOHN F. HICKOK AND DEMOREST DAVENPORT

swarming adults were taken at the night light at Friday Harbor for the first time ;
whether these had been free-living or had come from hosts could not be determined.
Swarming has never been observed by us in Southern California.

In California, these worms are commonly associated with the web-star Patiria
ininiata (Brandt), on one individual of which as many as 15-20 may occur. In the
Pacific Northwest they are equally common on the mud-star Luidia joliolata Grube.
There may be considerable variation in the size of worms on both hosts, indicating
repetitive colonization by different age classes. In the Puget Sound- Vancouver
Island region they may occur on the cushion-star Pteraster tcsselatus Ives and to-
gether with Nereis cyclurus Harrington commensal with hermit crabs (Berkeley and
Berkeley, 1948). Steinbeck and Ricketts (1941) list the species as commensal with
the starfish Oreastcr occidcntalis Verrill in the Gulf of California. That it may
occur occasionally with Pisaster ochraceus (Brandt) is indicated by a single speci-
men in the collection of Dr. Olga Hartman, taken by Dr. S. F. Light at Dillon Beach,
California. In spite of examining numerous specimens of the common starfish,
from Puget Sound to Southern California, we have never found commensal poly-
chaetes of any sort associated with it ; there would appear to be a likelihood that the
above case was fortuitous.

Method of investigating responses
A.

A choice-apparatus has been designed for the investigation of the possible role of
chemical attractants in the regulation of partnerships, such as that between Podarke
and Patiria, in which we have been unable to demonstrate in the individual com-
mensal partner any sharply defined, objectively recordable response to the host
(Bartel and Davenport, 1956). The apparatus consists of an aquarium with a cen-
tral chamber surrounded by and connected by passages with six radially arranged
chambers. It may be constructed out of latex as described in the above citation.
Our use of the apparatus was as described except that a cover of plywood was added
to reduce the possible effects of light. The latter factor was eliminated from con-
sideration in any series of tests by the random selection of test chambers from the
possible six. In all the experiments using this apparatus described below, the pres-
ence or absence of an attractant factor in one of the chambers among the radial six
(the "critical" chamber) is indicated when probabilities, using the null hypothesis
that distribution into the six chambers is the result of chance, indicate that either a
significant or insignificant number of worms have moved from the central chamber
into the critical chamber. Tests averaged from 8 to 12 hours.

Between each test in a series in any experiment the apparatus was washed.
Host animals were generally housed during tests directly in one of the radial cham-
bers, but in certain tests indicated below, when host animals were very large, they
were housed in a clean, redwood and glass aquarium and the water therefrom si-
phoned into a radial chamber of the choice-apparatus.

B.

Prior to employing the above described choice-apparatus, Bartel and Davenport
(1956) had found, by the simple expedient of placing in dishes large numbers of
free-living and commensal Podarke together with Patiria, that toward this host

BEHAVIOR OF COMMENSAL POLYCHAETES

399

there is a marked difference in behavior in the two populations ; the commensals
gathered on the star while none of the free-living worms did. It seemed wise to re-
peat this experiment in Puget Sound, using both the free-living worms and those
commensal with the common host of that region, Luidia foliolata. As an experi-
mental animal in behavior studies this starfish presents difficulties ; it readily au-
totomizes its arms when handled or placed in a confined space and hence is not well
suited to the latex choice-apparatus. At the same time it is so large that one cannot
readily place it in a dish or tray with commensals. We therefore employed a large
cement water table (internal dimensions 3' X 5' X 3") in which the starfish could
wander freely and "pick up" commensals or in which one could confine the star to
a limited space so that the commensals had to "find" it (Fig. 1). In order to so
confine the starfish we simply placed a plywood "T" in the table as shown, which
would allow free movement of water or worms under its parts but which would

FIGURE 1. Plan of water table.

trap the star in one corner. Water was introduced at a very slow flow in one cor-
ner and drained out at the point shown. One introduced experimental worms at
random at the lower end of the table.

This apparatus lent itself well to the study of the specificity of response in part-
nerships in which evidence for a chemical attraction effective at a considerable dis-
tance from the host had already been presented (Arctonoc-Evasterias, etc. — Daven-
port, 1950), and also made it possible to conduct tests concurrently with those using
the latex apparatus, likewise testing the responses of a large sample of worms in a
single test run.

Experiments

Experiment No. 1. Will commensal worms gather on the host Luidia when
both have the freedom of the water table ?

400 JOHN F. HICKOK AND DEMOREST DAVENPORT

After a time duration of approximately eight hours, 23 out of 36 introduced
worms (59%) had moved onto the starfish.

Experiment No. 2. Will commensals, introduced at random at the lower end
of the table, find the host if it is trapped at the opposite end ?

After a time duration of approximately nine hours, out of 23 worms introduced
13 had found the starfish (56%).

Experiment No. 3. Will free-living Podarke (Garrison Bay) gather on Luidia
when both have the freedom of the water table ?

After a time duration of approximately eight hours, out of 32 worms introduced
none had moved onto the starfish.

Experiment No. 4. Can an attraction for commensal worms be demonstrated
in the latex choice-apparatus if water is siphoned from a large aquarium containing
the host Luidia into one of the six radial chambers ?

Six Luidia were placed in a large redwood aquarium and three tests followed a
control. In the control test with no starfish water in the system, 18 out of 23
worms made a choice and the distribution in the radial chambers was random. In
the first test to starfish water, out of 25 worms introduced, 23 made a choice and of
the 23, 14 entered the critical chamber (P < .001). In the second test all of 20
worms made a choice and of these 8 entered the critical chamber (P < .01). In
the final test out of 25 worms 11 made a choice, of which 9 entered the critical
chamber (P < .001).

It is clear that an attraction can be demonstrated with the Podarke-Lnidia part-
nership in the latex apparatus. The above data compare very well with those ob-
tained by Bartel and Davenport (1956) with the Podarke-Patiria partnership in
California, in which two tests gave probabilities of < .001 (24 out of 68 and 23 out
of 53 entering the critical chamber).

Experiment No. 5. Are free-living worms attracted to a radial chamber into
which water from an aquarium containing Luidia is siphoned ?

In a control test with no starfish water in the system, 16 out of 22 worms made
a choice and the distribution was random. In four tests in which 15 out of 25, 16
out of 20, 17 out of 20 and 17 out of 24 made a choice when starfish water was in
one of the chambers, the distribution was still purely random. In a single test
against starfish water when 16 out of 24 worms made a choice, 8 entered the critical
chamber (P < .01).

Since the above results were not consistent, further tests were indicated to de-
termine whether or not the release of metabolites in test chambers may occasionally
cause free-living worms to distribute themselves unequally in the choice-apparatus,
in spite of the fact that under conditions more nearly approaching natural ones, they
do not gather on Luidia (Experiment No. 3 above).

Experiment No. 6. Can closer propinquity to starfish (and therefore possibly
greater concentration of metabolites) perhaps be the answer to the unequal distri-
bution that may occur when free-living worms are tested in the choice-apparatus
against Luidia? With considerable difficulty a single small Luidia was obtained for
testing and one test completed with the starfish directly in one of the radial chambers
before it autotomized its arms. In this test when 23 out of 30 worms made a choice,
8 entered the critical chamber (P < .01).

BEHAVIOR OF COMMENSAL POLYCHAETES 401

Experiment No. 7. A further series of tests were conducted at a later date to
see whether free-living worms from a different environment than those used in Ex-
periments 5 and 6 might distribute themselves in a non-random fashion when a host
was in the system. Free-living Podarkc from harbor floats in Santa Barbara were
tested against the host starfish of California, Patina miniata. In four out of five
tests of this kind the distribution was random, but in one, when 19 out of 27 worms
made a choice, 10 entered the critical chamber (P < .001).

Experiments 5, 6 and 7 have all given an indication that under certain conditions
the behavior of free-living worms may be so affected by the presence of a host star-
fish in the system (perhaps by some metabolite) that their distribution will be non-
random. However, they certainly do not respond positively as consistently as their
commensal relatives.

Experiment No. 8. It has been demonstrated that commensal Podarke show a
positive response to the host Luidia (Experiments 1, 2, and 4). Will worms from
Luidia respond to the alternate host Pter aster tesselatus?

In two tests in which water from a redwood aquarium containing a single large
Pter aster was siphoned into one of the six chambers, samples of 19 and 20 worms
distributed themselves in a random fashion. But when a smaller Pteraster was
placed in a radial chamber directly, in one test when 18 out of 23 worms made a
choice, 9 entered the critical chamber (P < .01) and in the second test when 31
out of 36 worms made a choice, 16 entered the critical chamber (P < .001).

Here again propinquity may be a factor, and perhaps the great secretion of
mucus produced by handling this starfish may have been a factor in preventing a
response in the first two tests, when the starfish was at a greater distance.

Experiment No. 9. How specific is the response of commensals from Luidia
in the choice-apparatus ? Will the commensals respond to non-host starfish ?

In a test against Mediaster aequalis Stimpson, when 24 out of 31 worms made a
choice, 9 entered the chamber containing the host (P < .01). In a test against
Pisastcr ochraceus (Brandt) when 9 out of 19 worms made a choice, 5 entered the
critical chamber (P < 1.0) and in a test against Evasterias troschelii (Stimpson)
when 17 out of 27 worms made a choice, 11 entered the critical chamber (P < .001).

Apparently no response specificity can be demonstrated in the latex choice ap-
paratus when one tests Podarkc commensal with Luidia in Puget Sound.

Experiment No. 10. Do California Podarke commensal with Patina show a
similar non-specific response in the latex choice-apparatus ?

In a series of six control tests against the host alternated with tests against non-
host stars, distributions giving probabilities of < .001 were obtained in five, while
in one test the distribution was random. In the series of 15 tests against P. ochra-
ceus (Brandt), P. gigantcns (Stimpson), Pycnopodia hclianthoidcs (Brandt) and
Dermasterias imbricata (Grube), when samples of from 10 to 44 commensals were
used in a single test, all but one test gave completely random distributions. In one
test against P. gigantcns 19 out of 33 worms making a choice entered the critical
chamber (P < .001).

There would appear to be a marked difference in the response specificity demon-
strable in the choice apparatus between worms commensal with Luidia in Puget
Sound and worms commensal with Patiria in California, the latter demonstrating a
greater specificity.

402 JOHN F. HICKOK AND DEMOREST DAVENPORT

THE OBLIGATE COMMENSAL, ARCTONOE FRAGILIS (BAIRD)
Material and methods

The polynoid commensal Arctonoe fragilis has been studied previously by the
authors (citations above). In the first experiments in which it was demonstrated
that a marine commensal would respond positively to sea water which housed its
host, a Y-tube olfactometer was used, but no detailed studies of response-specificity
were made. Such apparatus does not lend itself readily to investigations of speci-
ficity of response, since large samples of commensals cannot be tested at once. As
it had already been demonstrated that these worms showed an ''overt" response to
sea water from their host even at a distance, there appeared to be no advantage in
employing the latex choice-apparatus. The use of similar water-table tests as de-
scribed above (Fig. 1) was in order.

A. fragilis has been listed as commensal by Pettibone (1953) with the following
asteroid hosts : Evasterias troschelii; Leptasterias aequalis and L. hexactis; Ortha-
sterias koehleri; Pisaster ochraceus; Solaster dazvsoni and Stylasterias foreri. We
collected and used in the experiments below a large number of worms commensal
with Evasterias, a few with Orthasterias and one (?) with Solaster dazvsoni. It is
unfortunate that cross-specificity studies are made difficult by the fact that it is al-
most impossible to collect a working sample of commensals from any other host than
Evasterias. The Berkeleys tell us that at Nanaimo large numbers of Orthasterias
koehleri can be collected in the inter-tidal zone in winter and early spring ; in sum-
mer they can only rarely be so collected. Our few specimens of Orthasterias were
taken in dredges and with the aqualung. It may in fact be possible in the future to
compare the behavior of populations of A. fragilis from Evasterias and Orthasterias,
by conducting winter experiments. The value of making a thorough comparison of
the behavior of two or more separate populations of a single commensal species
which inhabits several hosts is obvious. The brief preliminary tests presented below
give evidence that the results of such experiments would be most interesting.

Experiment No. 11. Prior to running cross-specificity tests with the two popu-
lations of Arctonoe available, it was necessary to run a control experiment to deter-
mine whether under the conditions of the water table, Arctonoe fragilis (commensal
with Evasterias) would show a response to non-host stars. In four control tests
against Evasterias, run in alternation with tests against non-host stars, fifteen worms
were used in three and fourteen in one. Tests had a duration of not less than nine
hours. In the first three 12 out of 15 (79.9%), 10 out of 15 (66.6%), and 10 out
of 15 "found" the "trapped" host. In the one test using 14 worms, 13 "found"
the host (92.8%). In single tests using fifteen worms against Pisaster ochraceus,
Luidia foliolata, Mediaster aequalis, Hippasteria spinosa and Dermasterias inibri-
cata no worms "found" or moved onto the "trapped" non-host.

Commensal Arctonoe, therefore, demonstrate a rather precise response specificity
in the water-table.

Experiment No. 12. It appears that commensals from Evasterias do not, as one
might expect, demonstrate in the water-table a response to stars with which the
species is not associated, but what sort of behavior would the worms show in rela-
tion to alternate hosts? Will the worms, regardless of host habit, respond to al-
ternate hosts?

BEHAVIOR OF COMMENSAL POLYCHAETES 403

To answer this question we presented mixed samples of Arctonoe, some from
Evasterias and some from Orthasterias koehleri, with opportunities to "find" each
host in the water table. Will each host "sort out" the correct commensals? Un-
fortunately, because of the above-mentioned difficulty of finding Orthasterias we
were able to collect only two specimens of Arctonoe from this host. In the follow-
ing experiments these worms were lightly stained in indulin in order to distinguish
them from the sample collected from Evasterias.

In a series of three water-table tests of this mixed population against Evasterias
a total sample of 16 worms were used in each. In the first two tests 15 Evasterias
commensals "ran against" one Orthasterias commensal. At the end of nine hours
in both tests 10 (75%) of the Evasterias commensals had "found" their host while
the single Orthasterias commensal was still wandering free. In the third test 14
Evasterias commensals were "run against" two Orthasterias commensals. At the
end of nine hours all but one of the Evasterias commensals had "found" the host
while the two Orthasterias commensals were still wandering free.

In two tests against Orthasterias a mixed sample of 15 worms was used in the
first and of 17 worms in the second. In the first, out of 14 worms from Evasterias
13 were still wandering free after nine hours, while the single Orthasterias worm
had "found" its host. In the second test at the end of the same time, out of 15
Evasterias worms none had moved onto the Orthasterias, while of the two Ortha-
sterias worms, one had "found" the host.

These preliminary experiments against alternate hosts were conducted with a
much smaller sample of worms than one would desire and it is hoped that at some
time such tests can be repeated with balanced samples. But the tests give an indi-
cation of what may be a significant fact. There may, if such responses are not con-
ditioned during development, be good physiological or behavioral races inhabiting
different hosts within single commensal polychaete species. That this may be the
case was further indicated by a brief experiment in which we tested a mixed popu-
lation from Evasterias and Solaster. Accurate identification of the three species of
Solaster with their two commensal species of Arctonoe (S. stitnpsoni and 6". endeca
with A. vittata and ^S". dawsoni with A. jragilis}, may be difficult. This is particu-
larly true in the case of the worms, in which two species inhabiting closely related
hosts may resemble each other greatly ; identification can at times only be made by
dissection which renders the animals useless for behavior experiments. However,
we believe our identification of a single A. jragilis on 5\ dawsoni to be correct.
When a mixed population consisting of 16 A. jragilis, one from Solaster and 15 from
Evasterias, were tested in the water-table against Solaster, not one of the Evasterias
worms moved to the star and yet the single Solaster commensal quickly "found"
its host.

THE OBLIGATE COMMENSAL ARCTONOE VITTATA (GRUBE)
Material and methods

The polynoid Arctonoe vittata, closely related to A. jragilis, has perhaps the most
interesting variation in host-habit of all the members of the genus. It colonizes cer-
tain asteroids, amphineurans, gastropods and polychaetes and within these groups
shows a rather precise specificity (Pettibone, 1953). For this reason one might
suppose that there could hardly be a commensal better suited to studies of response

404 JOHN F. HICKOK AND DEMOREST DAVENPORT

specificity. Unfortunately, however, as with Arctonoe fragilis, it is very difficult to
obtain large enough numbers of commensals from each host to make good studies of
cross-specificity. In addition to the difficulty in collecting diverse populations of
this commensal, one faces the problem of the general inactivity of the worm, which
makes studies using a Y-tube or an open water-table tedious in the extreme. It was
found, however, that a sample of animals distributed themselves well overnight in
the latex choice-apparatus. The following questions were asked and to a certain
extent answered, using a single population of worms from the key-hole limpet
Diadora in the latex apparatus, according to the technique described above.

Experiment No. 13. Will commensals from Diadora show a response to the
host in the choice-apparatus ?

In eight tests samples of from 11 to 29 worms were tested against a group of six
limpets in a radial chamber. In two of the eight tests the worms distributed them-
selves in a random fashion but in six of the tests enough worms chose the critical
chamber to give probabilities of < .001, < .01, < 1.0, < 1.0, < .001, and < .1.

Experiment No. 14. Do worms commensal with Diadora show a response to
alternate hosts? Sample alternate hosts tested were the starfish Luidia foliolata
Grube, Solastcr stiinpsoni Verrill, and Dermasterias imbricata Grube; the chiton
Cryptochiton stelleri Middendorf; and the gastropods Acmaea mitra Eschscholtz
and Fusitriton orcgonense (Redfield). Large hosts (Luidia, Solaster, Dermaste-
rias, Cryptochiton} were housed in a clean redwood aquarium and the water si-
phoned from this into one of the radial chambers of the choice-apparatus. Small
hosts (Acmaea, Fusitriton, small Cryptochiton and Dermasterias} were placed di-
rectly in a radial chamber.

In thirteen tests against these alternate hosts using samples in each of from 13
to 27 commensals the worms distributed themselves in the radial chambers in a ran-
dom fashion. In a single test of the three against Fusitriton, when 20 worms made
a choice, 8 entered the critical chamber (P < .1), while in the other two tests the
distribution, although in both cases the greatest number of worms making a choice
entered the critical chamber, gave probabilities > .1.

This series of tests indicates that the population of worms commensal with Dia-
dora shows under these experimental conditions a rather precise response speci-
ficity. Unfortunately, the time duration of this experiment precluded our going
further than analyzing the response to an array of available hosts. Certainly, a
longer series of tests should be made against Fusitriton to determine whether toward
this animal, which in some places occupies the identical environment from which the
host Diadora may be collected, the worms show a constant response.

DISCUSSION

Since the initiation of the study of the specificity of response of polychaete com-
mensals in the summer of 1949, a number of different forms have been investigated.
It has been our continued aim to try to correlate this response specificity with the
known host specificity of the species or races. In our effort to make comparisons
we have been continually faced with difficulties, some of which have been insur-
mountable. Among these is the fact that it is extremely difficult to collect large
enough samples of worms for such studies in those most interesting species which
show within themselves a diversity of host habit ; in most such species the worms

BEHAVIOR OF COMMENSAL POLYCHAETES 405

will, in one locality, occur commonly on one host but very rarely on others. A dif-
ficulty encountered in making comparisons between the behavior of different species
has been that, as one might expect, not all species exhibit the same sort of response,
some showing as individuals a marked or overt response to factors from the host
coming from a distance and others merely "accumulating" on or near the host after
a passage of time. With such forms as the latter it has been necessary to design
special techniques quite different from those used in studies on the former to discern
whether or not there is in actuality any response to chemical factors coming from
the host. The use of entirely different techniques has made a comparison of results
difficult. Some differences in response specificity may turn out to be more apparent
than real when some technique has been developed which lends itself equally well to
the study of the responses of forms which appear to differ in behavior. Recently
we have begun an analysis of the behavior of individual polychaete commensals
when under the influence of host factor, using apparatus which may give us some
truly comparable data even when studying animals of greatly differing activity or
sensitivity.

However, it may be possible at this time, in spite of the above-mentioned diffi-
culties, to make some brief general observations on response specificity in commen-
sal polychaetes.

There would appear to be different categories of response specificity. There is
a range of behavior, from the sort which is exhibited by species or populations within
species that respond to their host alone, to the sort in which the commensals appear
to have no chemical discernment and respond, at least under experimental condi-
tions, to many non-host animals. Specificity of host habit is by no means an indi-
cation of specificity of response in experimental apparatus. As an example of the
first category which exhibits precise response we have Arctonoc fragilis and its be-
havior in relation to Erastcrias, Orthasterias and Solaster. But there are also spe-
cies in which populations from one host may give a similar precise response to some,
but not necessarily all, alternate hosts, regardless of the absence of any taxonomic
affinity between the hosts to which they do respond (Hannothoc lunulata from the
brittle-star Acrocnida brachiata vs. its host and the alternate eunicid Lycidicc ninetta
—Davenport, 1953b). Among such species of diverse host habit there may be a
population occurring on one of the array of hosts which responds to its host alone,
in spite of the fact that other populations of the same species respond to several al-
ternates (Hannothoc lunulata from Lcptosynapta inhacrens). A further category
consists of those species which respond with the same intensity to the known alter-
nate hosts but with reduced intensity to a number of non-host relatives of their hosts
(Acholoc astcricola from Astropecten irrcgitlaris vs. its host and the alternate Litidia
ciliaris, as well as non-host stars — Davenport, 1953a). Finally we have a category
which, though somewhat unexplainable, can be demonstrated to exist even \vhen
using a standard technique. In some facultative commensals there appear to be
populations (Podarke on Patina) which show a precise response specificity to their
host alone and others (Podarke on Litidia) which seem unable to discern the differ-
ence between their host and other non-host animals.

It is therefore quite apparent that it is pressing to determine, particularly in
forms such as Podarke. whether responses are inherited or conditioned. Although
it would seem difficult to imagine a mechanism whereby such a host response could
be conditioned in forms such as Podarke, the early stages of which (in the labora-

406 JOHN F. HICKOK AND DEMOREST DAVENPORT

tory) remain in the plankton for some 30 days, nevertheless only successful breeding
and settling experiments will give us the answer.

SUMMARY

1 . A new water-table test apparatus for the investigation of commensal response
behavior is described.

2. Evidence is presented that the two populations of the facultative commensal
Podarke pmjcttcnsis ( Polychaeta : Hesionidae) which may be termed "commensal"
and "free-living" differ markedly in their response to host animals, the commensal
worms showing a strong tendency to respond positively to the host and the free-
living worms not doing so.

3. Commensals with the starfish Lnidia in Puget Sound appear to respond with
almost equal intensity to other non-host animals (the response is not specific), while
commensals of the star Fatiria in Southern California show a more precise and spe-
cific response. This behavioral difference remains unexplained.

4. The behavior of three populations of the obligate commensal Arctonoc fragilis
(Polychaeta: Polynoidae) was compared. Evidence is presented that each popula-
tion (one commensal with the star Evastcrias, one with the star Orthastcrias and
one with the star Sohistcr) shows a response to its host alone.

5. The response behavior of Arctonoc rittata (Polychaeta: Polynoidae), an ob-
ligate commensal of diverse habit, was investigated in relation to a number of its
alternate hosts.

LITERATURE CITED

BARTEL, A. H., AND D. DAVENPORT, 1956. A technique for the investigation of chemical re-
sponses in aquatic animals. Brit. J. Aniin. Bchav., 4: 117-119.

BERKELEY, E., AND C. BERKELEY, 1948. Canadian Pacific Fauna, 9. Annelida. 9b(l) Poly-
chaeta Errantia. Fisheries Research Board of Canada, Toronto, 100 pp.

DAVENPORT, D., 1950. Studies in the physiology of commensalism. I. The polynoid genus
Arctonoe. Biol. Bull.. 98: 81-93.

DAVENPORT, D., 1953a. Studies in the physiology of commensalism. III. The polynoid genera
Acholoe, Gattyana and Lepidasthenia. /. Mar. Biol. Assoc.. 32: 161-173.

DAVENPORT, D., 1953b. Studies in the physiology of commensalism. IV. The polynoid genera
Polynoe, Lepidasthenia and Harmothoe. /. Mar. Biol. Assoc. 32 : 273-288.

DAVENPORT, D., 1955. Specificity and behavior in symbioses. Quart. Rev. Biol., 30: 29-46.

DAVENPORT, D., AND J. F. HICKOK, 1951. Studies in the physiology of commensalism. II. The
polynoid genera Arctonoe and Halosydna. Biol. Bull., 100: 71-83.

PETTIBONE, MARION H., 1953. Some scale-bearing polychaetes of Puget Sound and adjacent
waters. Univ. of Wash. Press, Seattle, 89 pp.

STEINBECK, J., AND E. F. RICKETTS, 1941. Sea of Cortez. The Viking Press, N. Y., 598 pp.

ON THE MORPHOLOGY OF THE NEPHRIDIUM OF NEREIS

VEXILLOSA GRUBE T

MEREDITH L. JONES

/'r/1/. of Zoolof/v. ['nh'crsitv of California, Berkeley, California2

It has been well established that certain of the Nereidae are capable of surviving
in waters of low salinity. In the field this is demonstrated by their invasion of
brackish and even fresh waters (Johnson, 1903; Hartman, 1938; Smith, 1950, 1953,
1956), and has also been suggested in the experimental work of some investigators
(Schlieper, 1929; Nomura, 1930; Jurgens, 1935; Sayles, 1935; Beadle, 1937; Ellis,
1937; Topping and Fuller. 1942; Krishnan, 1952; Smith, 1955). In spite of the
fact that many physiological studies have been carried out on various nereids, only
a few morphological descriptions of the presumed osmoregulatory organ of these
annelids, the nephridium, are to be found in the literature.

The first detailed description of nephridial morphology was made by Goodrich
(1893) on ATcrcis dircrsicolor. He found three sections along the length of the
nephridial tubule, each grading into the next. The sections varied in respect to the
presence or absence of cilia, the diameter of the tubule lumen, and the extent of
convolution of the tubule.

Fage (1906) studied Pcrincrcis cultrijcra, confining his work to living material.
He also found areas of ciliation, but these differed from those observed by Goodrich
in X . dirersicolor. Much later, in his extensive review of observations of nephridia
and genital ducts, Goodrich ( 1945) re-stated his earlier findings but added little to
them. In his work on Lycastis indica. Nereis chilkacnsis, and Perinereis nitntia,
Krishnan (1952) made a study of the nephridia of each species and compared them
with respect to vascularization and size, relative to body size^

Because of the paucity of adequate morphological studies on nereid nephridia, it
was felt that further study was in order, to provide a better basis for physiological
work, and for later studies of comparative functional morphology.

MATERIALS AND METHODS

Specimens of Nereis t'e.rillosa utilized in this study were obtained from a break-
water at the Berkeley Yacht Harbor, in San Francisco Bay, California. Although
no salinity determinations were made at this time, the annual salinity range for this
area is from 26.3 to 32.4/^r (approximately 73 to 90% sea water), according to
Sumner ct al. ( 1914) and Miller ct al ( 1928).

The worms were relaxed by gradual addition of 30% ethyl alcohol, fixed in
Benin's fixative and serially sectioned at eight micra. They were then stained with

1 Representing a portion of a thesis submitted in partial satisfaction for the degree of Master
of Arts in Zoology at the University of California at Berkeley.

- Present address : United States Naval Mine Defense Laboratory, Panama City, Florida.

407

408

MEREDITH L. JONES

Harris1 haematoxylin and counterstained with eosin Y. Other fixatives and stains
were utilized, but these gave poor results.

In order to obtain a graphic representation of the canal as it passed through the
nephridial mass, a plaster-of-Paris reconstruction was made. Camera lucida draw-
ings were transferred to sheets of paraffin of proper thickness, and as the replica was
built up, the lumen of the canal was hollowed out. Later, the canal was filled with
plaster, and the surrounding paraffin was melted away.

NEPHRIDIAL MORPHOLOGY

The nephridia of Xcrcis I'c.villosa are paired organs in the coelomic cavity of
each segment, just lateral to the ventral longitudinal muscle bundles, near the base
of each parapodium. Within the broad base of attachment of the nephridium to the
body wall, the nephridial canal opens to the exterior by way of the nephridiopore

SEP

VSV

EG

NPR

FIGURE 1. Schematic diagram of a nephridium of Nerds :'c.rill/isa.

(Figs. 1, 2, 6, NPR). The internal opening of the nephridial canal, the nephro-

stome (Figs. 1, 3, 5, NST), is found at the end of an anterior extension of the canal

(the post-septal canal, Figs. 1, 3, PSC) which leaves the mass of the nephridium and

passes anteriorly, through the inter-segmental septum (Figs. 1, 3, 5. SEP), to the

next segment.

Externally, the nephridium is a discrete mass of tissue, varying from globose or
pyriform to irregular in shape. The surface may be ridged to some extent, because
of the passage of the nephridial canal close to the surface. In mature worms of from
55 to 70 segments (ca. 70 mm. long when relaxed) nephridia were approximately
.250 micra at their widest and about the same dorso-ventrally. They measured
nearly 200 micra through their antero-posterior axis, exclusive of the post-septal
•canal and nephrostome which, in themselves, were about 300 micra in length.

In section, the convoluted nephridial canal is seen as many perforations in the

MORPHOLOGY OF THE NEREID NEPHRIDIUM 409

nephridial tissue (Figs. 1, 2, 3, 4, NC). A fairly discrete wall often lines the tubule,
although, usually, the boundaries of these cells are difficult to resolve (Fig. 4). The
surface of the nephridium is covered by a single, very thin layer of squamous co-
elomic epithelium cells (Fig. 2, EPI). It is well to point out that, as far as is
known, all nereid nephridia have this general configuration, i.e., a convoluted canal
in the nephridial mass. An apparent contradiction to this fact occurs in a recent
text (Prosser ct a!., 1950, pp. 17-18) where it is stated that ". . . the nephridium
of AT. cultrifcra is a simple sac." This is justified by a figure modified from Jiirgens
(1935) which had been redrawn from the work of Fage (1906). Page's original
figure was a surface view of the nephridium of Nereis cultrifera, and in subsequent
copyings, the delicate shadings which showed surface texture were lost, and the
figure evolved to that of an optical section of the nephridium. The fate of the figure
notwithstanding, Fage (1906, p. 338) described the nephridium as a "... masse
spongieuse, perforce en tons sens par un grand nombre de canaux" ; therefore, it is
certainly not a simple sac. The matrix of the nephridium is a highly vacillated,
syncitial, network of loose connective tissue, which serves to bind the convolutions
of the nephridial canal.

Xuclei are of two types, a smaller kind, rich in chromatic material (3 X 5 /A), and
a larger, clearer kind (5 X 10 /j.). Nuclei of both types are usually found in or near
the canal walls, and only occasionally are they seen isolated in the matrix tissue
(Figs. 2. 3, 4).

No blood vessels have been noted within the nephridial mass, and in only two
places is the nephridial system approached by vascular elements. One blood vessel
passes over the anterior face of the nephridium (Fig. 3, BV) and another, the ven-
tral segmental vessel, lies along the post-septal canal (Figs. 3, 5, VSV). In neither
case is the association intimate, and there is little opportunity for the transfer of
materials from one structure to the other. Although neither Goodrich (1893) nor
Fage (1906) mentions the relationships between the nephridium and the vascular
system, Krishnan (1952) has indicated that in Lyeastis indica, Xercis chilkacnsis,
and Perinereis uuntia, blood vessels are found in close association with nephridia.
He also points out that the extent of nephridial vascularization is inversely related
to the salinity of the environment.

Occasionally, eosinophilic granules have been seen in the tubule walls and the
matrix tissue (Fig. 4, EG). There is no special distribution along the length of the
canal, and no special accumulations in the nephridial mass. Goodrich (1893) men-
tioned minute granules in the cells of the tubule wall in N. divcrsicolor and con-
sidered these to be composed of excretory materials. Fage (1906) observed that
with the addition of neutral red to the sea water bathing the freshly-dissected ne-
phridia of Perinereis cultrifcra, granules were formed which were similar to those
which were observed in untreated nephridia, and which Fage terms, excretory gran-
ules (grains d'excretion).

The post-septal canal (Fig. 3, PSC) is produced anteriorly as an extension of
the nephridial canal, and is covered by the same thin squamous layer which invests
the nephridium. Nuclei are uniformly scattered along its length and are concen-
trated in a band at the level of the septum (Fig. 5, NB). Anterior to the septum,
the post-septal canal enlarges and gives rise to the funnel-shaped nephrostome.
The lateral margin of the nephrostome is slightly recurved, and around the entire
rim, there are numerous cytoplasmic processes (Fig. 5, CP).

410

MEREDITH L. JONES

L_5OMICRA

200

50 MICRA

FIGS. 2-7.

MORPHOLOGY OF THE NEREID NEPHRIDIUM 411

At the terminal end of the nephridial canal, the wall of the lumen thins and be-
comes continuous with the invaginated cuticle of the outer surface to form the ne-
phridiopore (Fig. 6, NPR). In this respect, the structure of this area differs from
that of N. dii'crsicolor and A', clullcacnsis, for Goodrich (1893) states that in N.
dii'crsicolor the wall of the tube pierces the epidermis, and Krishnan (1952) presents
a figure showing the same condition in N. cltilkacnsis.

By means of camera lucida drawings, the entire course of the convolutions of the
nephridial canal was followed from the nephrostome to the nephridiopore. It was
then possible to ascertain the extent of ciliation within the lumen of the canal (Fig.
4, CIL). It was seen that the ciliation of the nephrostome is extremely heavy, and
forms a tightly wound swirl in the throat of the nephrostome (Fig. 5. CM). The
heavy ciliation is maintained throughout the rest of the post-septal canal, and gives
a characteristic "star" or "wagon wheel" aspect to transverse sections of this struc-
ture. The ciliation of the portion of the canal included within the nephridial mass
is constant, but not uniform. No obvious areas of heavy or sparse ciliation, such
as were noted by Goodrich (1893) in N. dii'crsicolor, have been observed here, and
in general, the midportion is only slightly more heavily ciliated than either end. In
the region of the nephridiopore (Fig. 6), the canal is devoid of cilia for about the
last 40 micra of its length. It has also been noted that in Ar. z'c.villosa the cilia are
never attached on only one side of the lumen as Goodrich reported in N. dircrsi-
color. In addition, no tufts of cilia, such as Fage ( 1906) has described in the ne-
phridial canal of Pcrincrcis cultrifcra, have been seen here.

The plaster reconstruction (Fig. 7) shows that after the post-septal canal joins
the nephridial mass, the canal is thrown into fairly tight, somewhat spiraled convo-
lutions ( I, Fig. 7). It then winds back and forth along the medial surface, roughly
parallel to the antero-posterior axis (II, Fig. 7). Next, it passes to the mid-lateral
portion of the mass and is once more tightly convoluted (III, Fig. 7). This condi-
tion gives way to a relatively straight length which terminates at the nephridiopore
(Fig. 7, NPR).

In addition to affording a three-dimensional view of the path of the canal through

Key to lettering: BV, blood vessel; CIL, cilia; CM, mass of cilia; EG, eosinophilic gran-
ules; EPI, coelomic epithelium; NB, band of nuclei of nephrostome; NC, nephridial canal: NPR,
nephridiopore; NST, nephrostome; CP, cytoplasmic processes of nephrostome; PSC, post-septal
canal; SEP, intersegmental septum; VSV, ventral segmental blood vessel; I, II, III, first, sec-
ond, and third regions of the nephridial canal, respectively.

FIGURE 2. General view of a nephridium, transverse section (8 /*, Harris' haematoxylin
and eosin ; the cavity extending internally from the area of the nephridiopore, NPR, is a longi-
tudinal fold of the body wall ; the ventral nerve cord is to the left of the figure and the para-
podium is to the lower right).

FIGURE 3. View of nephridium and its associated nephrostome, frontal section (8 n, Harris'
haematoxylin and eosin ) .

FIGURE 4. Detailed view of nephridial tissue (8 /JL, Harris' haematoxylin and eosin).

FIGURE 5. Detailed view of the nephrostome of Figure 3.

FIGURE 6. Detailed view of the nephridiopore, transverse section ( 8 /j., Harris' haema-
toxylin and eosin ; the large cavity extending toward the upper left of the figure is a longitudinal
fold of the body wall ).

FIGURE 7. Plaster reconstruction of the nephridial canal, view of the anterior face. (The
consecutive numbers indicate the course cf the canal; section I, 1-18; section II, 19-30- section
III, 31-55.)

412 MEREDITH L. JONES

the nephridium, the reconstruction shows three regions which merge gradually into
one another. After the narrow post-septal canal joins the nephridium, the canal he-
comes slightly enlarged through the first region of convolution (I, Fig. 7). The
canal is then further enlarged to its maximal diameter as it passes to the medial sur-
face (II, Fig. 7). It becomes narrowed in the second series of tight convolutions
(III, Fig. 7), and it is at its minimal diameter as it passes to the nephridiopore.
This condition is reflected to some extent in Figure 8, which is a graph of the inner
diameter along the length of the canal (a measurement of the outer diameter, which
would show the thickness of the canal wall, was not possible, due to the poor defi-
nition of the cells of the wall). It is of interest to mention the regions of the tubule
within the nephridium, as determined by Goodrich and Krishnan, although such
differences may well be due to observations of different species. In N. dh'crsicolor,
Goodrich (1893) found a much convoluted portion with few cilia, into which the
post-septal canal led. The next region was very narrow, and the cilia here were
confined to one side of the canal. The last section was short, less convoluted, mod-
erately wide, and without cilia. In N. chilkaensis, Krishnan (1952) found that the
first portion of the canal, as it enters the nephridium, is convoluted and ciliated.
The next portion is wider, without cilia, and longer than the preceding section.

PSC

FIGURE 8. Graphic representation of the inner diameter of that part of the nephridial canal
within the nephridial mass (reconstructed by measuring the shortest diameter of elliptical sec-
tions of the nephridial canal).

This latter portion gives way to a canal which leaves the body of the nephridium
and terminates at the nephridiopore.

The mean diameter of the nephridial canal upon which the reconstruction (Fig.
7) and the graph (Fig. 8) were based, was 24 /*, and the over-all length of the canal
within the nephridial mass (not including the nephrostome or the post-septal canal)
was approximately 1.7 mm.

The author wishes to acknowledge advice, criticisms, and suggestions from Dr.
Ralph I. Smith and Dr. Kenneth B. DeOme of this Department, and from Dr. Wil-
lard D. Hartman, of the Peabody Museum, Yale University.

SUMMARY

1. The morphology of the nephridia of Nereis vexillosa Grube is described.

2. Comparisons are made with the morphology of the nephridia of certain other
nereids and differences are noted. Chief among these are, in N. vc.rillosa:

a. that ciliation extends along the whole length of the nephridial canal, with the
exception of a short region just preceding the nephridiopore;

MORPHOLOGY OF THE NEREID NEPHRIDIUM 413

b. that three general regions of the nephridial canal are noted, on the basis of the
diameter and the amount of convolution ;

c. that the wall of the nephridiopore appears to be inserted on the invaginated
surface cuticle.

3. A reconstruction of the nephridial canal is presented in which the course of
the canal is readily seen.

LITERATURE CITED

BEADLE, L. C., 1937. Adaptation to changes of salinity in the polychaetes. I. Control of body
volume and of body fluid concentration in Nereis diversicolor. J. Exp. Biol., 14 : 56-70.

ELLIS, W. G., 1937. The water and electrolyte exchange of Nereis diversicolor (Miiller). /.
Exp. Biol, 14 : 340-350.

FACE, L., 1906. Recherches sur les organes segmentaires des annelides polychetes. Ann. Sci.
Nat. Zool, Ser. 9, 3 : 261-410.

GOODRICH, E. S., 1893. On a new organ in the Lycoridea and on the nephridium in Nereis di-
versicolor, O. F. Miiller. Quart. J. Micr. Sci., 34 : 387-402.

GOODRICH, E. S., 1945. The study of nephridia and genital ducts since 1895. Quart. J. Micr.
Sci,, 86: 113-392.

HARTMAN, O., 1938. Brackish and fresh-water Nereidae from the northeast Pacific, with the
description of a new species from central California. Univ. Calif. Publ. Zool., 43 :
79-82.

JOHNSON, H. P., 1903. Fresh-water nereids from the Pacific Coast and Hawaii with remarks
on fresh-water Polychaeta in general. Mark Anniversary Volume, Art. 10; 205-223.

JURGENS, O., 1935. Die Wechselbeziehungen von Blutkreislauf, Atmung und Osmoregulation bei
Polychaten (Nereis diversicolor O. F. Mull.). Zool. Jahrb., Abt. allg. Zool. u. Physiol^
55 : 1-46.

KRISHNAN, G., 1952. On the nephridia of Nereidae in relation to habitat. Proc. Nat. Inst. Sci.
India, 18 : 241-255.

MILLER, R. C., W. D. RAMAGE AND E. L. LAZIER, 1928. A study of physical and chemical con-
ditions in San Francisco Bay especially in relation to the tides. Univ. Calif. Publ.
Zool, 31 : 201-267.

NOMURA, S., 1930. A note on the physico-chemical conditions of the habitat of Nereis japonica,
Izuka. Tohoku Imp. Univ. Sci. Rep., 4th ser., 5 : 549-553.

PROSSER, C. L., F. A. BROWN, JR., D. W. BISHOP, T. L. JAHN AND V. J. WULFF, 1950. Com-
parative Animal Physiology. W. B. Saunders Co., Philadelphia.

SAYLES, L. P., 1935. The effects of salinity changes on body weight and survival of Nereis
virens. Biol. Bull, 69 : 233-244.

SCHLIEPER, C., 1929. tiber die Einwirkung niederer Salzkonzentrationen auf marine Organis-
men. Zeitschr. f. vergl. Physiol, 9 : 478-514.

SMITH, R. I., 1950. Embryonic development in the viviparous nereid polychaete, Neanthes lighti
Hartman. /. Morph., 87: 417-455.

SMITH, R. I., 1953. The distribution of the polychaete Neanthes lighti in the Salinas River Es-
tuary, California, in relation to salinity, 1948-1952. Biol Bull, 105 : 335-347.

SMITH, R. L, 1955. Comparison of the level of chloride regulation by Nereis diversicolor in
different parts of its geographical range. Biol Bull, 109 : 453-474.

SMITH, R. L, 1956. The ecology of the Tamar Estuary. VII. Observations on the interstitial
salinity of intertidal muds in the estuarine habitat of Nereis diversicolor. J. Mar. Biol
Assoc., 35 : 81-104.

SUMNER, F. B., G. D. LOUDERBACK, W. L. ScHMiTT AND E. C. JOHNSTON, 1914. A report upon
the physical conditions in San Francisco Bay, based upon the operations of the United
States Fisheries steamer "Albatross" during the years 1912 and 1913. Univ. Calif. Publ
Zool, 14: 1-198.

TOPPING, F. L., AND J. L. FULLER, 1942. The accommodation of some marine invertebrates to
reduced osmotic pressures. Biol Bull, 82 : 372-384.

STUDIES ON THE ISOLATED ISLET TISSUE OF FISH.1 II. THE
EFFECT OF ELECTROLYTES AND OTHER FACTORS ON
THE OXYGEN UPTAKE OF PANCREATIC ISLET
SLICES OF TOADFISH, USING THE CAR-
TESIAN DIVER MICRORESPIROMETER

ARNOLD LAZAROW, S. J. COOPERSTEIN, D. K. BLOOMFIELD AND

C. T. FRIZ

Departments of Anatomy, University of Minnesota, Minneapolis 14, Minnesota; Western
Reserve University, Cleveland, Ohio; and the Marine Biological Laboratory,

Woods Hole, Mass.

We have undertaken a detailed characterization of the metabolism of islet tissue
because we believe that these studies may provide the basis for understanding the
factors which control insulin synthesis and the mechanism by which alloxan and
other toxic agents selectively kill the insulin-producing cells (Lazarow, 1949). In
approaching this problem we have found it convenient to use the toadfish as an ex-
perimental animal. Whereas in mammals the islet tissue is distributed throughout
the pancreas in a million or more individual islets of Langerhans totaling only 1%
of the pancreatic mass, in the toadfish the islet cells are segregated into one or more
discrete bodies which are located in the mesentery and which are called the principal
islets (Diamare, 1899; Rennie, 1905). The pancreatic acinar tissue in the toadfish
does not form a definite organ ; rather, it is diffusely scattered throughout the mesen-
tery, along the bile ducts, and within the liver.

In a previous study (Lazarow and Cooperstein, 1951) we have measured the
activity of certain specific enzymes (cytochrome oxidase and succinic dehydroge-
nase) in normal toadfish islet tissue homogenates. However, in order to character-
ize the over-all metabolic pathways in islet tissue, it is important to measure the
endogenous oxygen uptake as well as that following the addition of specific exoge-
nous substrates. We have therefore studied the endogenous respiration of islet
tissue slices and in the present paper we are reporting the effect of varying pH,
tonicity, electrolyte composition, and other factors. By means of these studies we
have been able to define the conditions under which maximal respiration of the islet
tissue slices occurs. This should provide a base-line for subsequent work, which
will include a study of (a) the effect of various substrates known to play a role in
intermediary metabolism, (b) the effect of various inhibitors and, (c) the effect of
hormones and other agents which influence the blood sugar level and/or insulin
secretion.

METHODS

Mature toadfish, Opsanits tan, weighing 200 to 600 gm. were used. During the
summer months the animals were kept in a running sea water tank, and they were

1 Aided by a grant from the National Science Foundation (G-1928) and from the National
Institute for Arthritis and Metabolic Diseases of the U. S. Public Health Service (#A-720).

414

ELECTROLYTES AND ISLET METABOLISM 415

killed within several weeks of the time that they were caught. During the fall and
winter months the toad fish were kept in a live car for varying periods of time
(.1-6 months) after which they were shipped inland by air express from the Marine
Biological Laboratory, Woods Hole, Massachusetts. The fish were stored for pe-
riods up to one week in an aerated sea water tank (30-gallon crock). The tem-
perature of the sea water was maintained below 20° C. by circulating cold water
through the inside of submerged lead coils.

The cartesian divers used were of the cylindrical type without a bulb and they
were silicone-treated prior to use. Before the fish were killed, the divers were filled
with the various liquid media to be studied ; they were completely filled except for a
1-2 mm. air bubble at the very bottom of the diver. The divers were then cooled to
0° C. by placing them in a cooling block. During the intervals between manipulative
procedures, the divers were stored in this cold block. After the toadfish were
decapitated, the islets were dissected from the mesentery and placed on a piece of
Parafilm. The connective tissue capsule surrounding the islet was removed. A
petri dish cover, containing a piece of wet filter paper, was placed over the tissue;
this served as a moist chamber. The razor blade used for cutting the islet slices was
previously cleaned with sodium hydroxide (to remove all traces of oil), thoroughly
rinsed in tap water, and finally washed in distilled water. In general the islet ob-
tained from one toadfish was used for each day's experiment ; it was cut into eight
pieces, each weighing approximately 0.1 to 0.2 mg. wet weight. A slice of islet
tissue was then placed at the lower air-liquid meniscus in each of eight divers with
the aid of a fine stainless steel needle. Most of the liquid medium was then re-
moved from the diver using a micro pipette ; however, a cylindrical segment of
medium, about 1 mm. in length, was left behind to form the tissue seal containing
the islet slice. With the aid of the cartesian diver filler (Lazarow, 1950), the neck
seals of sodium hydroxide, oil and flotation medium were successively placed, in
the stated order, above the tissue sample. The divers were then transferred to a
thermostatically controlled water bath maintained at 25° C. ± 0.01° C. The pres-
sure was measured using a Wallace Tiernan gauge (Belleville, New Jersey) which
was initially suggested and used by Claff (personal communication, 1948). This
gauge was calibrated in millimeters of Brodie's solution. A compensating device
(Lazarow and Bloomfield, unpublished) connected to the outer chamber of the
gauge was used to minimize the effect of changes in barometric pressure. An ini-
tial reading was taken after a 20-minute equilibration period. Subsequent readings
were taken at 20-minute intervals during the next hour. Thermobar divers usually
showed a pressure change of less than 4 mm. per hour, whereas experimental divers
showed a change up to 100 mm. per hour. At the end of the experiment the divers
were removed from the water bath and the sodium hydroxide, oil and flotation
medium seals were removed. The tissue slice plus the tissue seal were transferred
from the diver to a smaller model of the conical-tipped micro homogenizer (Lazarow
and Portis, 1951), using a capillary pipette. The tissue was homogenized in 100
/A!, of water and three 25-/xl. aliquots were removed for protein estimation. The
protein was determined by a modification of the method of Lowry, Rosenbrough,
Farr and Randall (1951). Twenty-five /*!. of the sample were mixed with 250 /*!.
of the protein reagent; the mixture was allowed to stand at 45° C. for ten minutes
and, at the end of this time, 25 /xl. of the diluted phenol reagent were added. The
absorption was read after 15 minutes at room temperature in the Beckman spectro-

416

ARNOLD LAZAROW ET AL.

photometer at 700 m/x. The amount of protein in the sample was determined by
comparing the extinction with that obtained using a standard serum albumin solu-
tion. The metabolic activity was expressed as millimicroliters (m/xl.) of oxygen
taken up per microgram of protein per hour.

RESULTS

At the time this study was begun the osmotic pressure of toadfish blood (sample
obtained from the gill) had been determined and found to be equivalent to
0.19 M NaCl (Green, personal communication). In the first group of ex-

TABLE I

Effect of phosphate buffer on islet tissue respiration

m/il. Oz/Vg. protein/hr. in

m^l. Oa/Mg- protein/hr. in saline

saline-phosphate

(0.19 M NaCl)

(0.033 M Na2HPO<-KH2PO«,

pH 7.4 + 0.144 M NaCl)

Series

P**

No.

No.

determi-

Aver.

<7*

determi-

Aver.

CT*

nations

nations

1

11

1.42

0.80

10

2.24

0.72

.012

2

17

0.78

0.42

14

1.97

0.89

<.001

3

17

1.54

0.41

17

2.20

0.70

.001

4

—

—

—

15

2.62

1.00

—

5

—

—

—

8

2.63

0.84

—

6

—

—

• —

9

2.26

1.00

—

7

—

—

. —

19

2.08

0.99

. — .

8

—

—

. —

24

2.12

0.95

—

9

—

—

—

16

2.72

0.78

—

10

—

—

—

15

2.32

0.97

—

11

• —

—

—

14

2.14

0.79

—

Aver.

45

1.22f

—

161

2.28f

0.37f

(deviations from mean)2

N

Difference

/ 2 2

** Calculated from the formula \/— + — •

\Ni N2

| Each series taken as one figure.

periments shown in Table I, the respiration of islet tissue in 0.19 M NaCl
was compared with that observed in a saline-phosphate buffer mixture (con-
taining 0.144 M saline and 0.033 M Na2HPO4-KH2PO4 buffer, pH 7.4). In
three series of experiments the oxygen uptake of islet tissue in the saline-phosphate
mixture was 43 to 153% greater than that observed in 0.19 M saline. The p values
indicated that the differences were all highly significant. Table I also shows that
in a group of 1 1 experiments in which the saline-phosphate mixture was used, there
was excellent reproducibility in the average oxygen uptake for the individual series.
On the other hand, in the three series of experiments in which 0.19 M NaCl was

ELECTROLYTES AND ISLET METABOLISM

417

OXYGEN 2.0
UPTAKE
m>j* 02 PER

>jg PROTEIN
PER HOUR

1.5

1.0

1

1

0.2 0.4 0.6 0.8 1.0

WIDTH OF TISSUE SEAL (mm)

1.2

FIGURE 1. Effect of cartesian diver tissue seal width on the respiration of islet slices sus-
pended in 0.19 M NaCl. All of the points on the graph in which the tissue seal width was 0.8
mm. or less represent an average of 19 or more individual determinations ; the other points repre-
sent the average of 5 to 13 individual determinations.

used, the reproducibility was not as good as that observed in the saline-phosphate
mixture. A possible explanation of this is shown in Figure 1. The volume of the
tissue seal varied considerably from experiment to experiment and in Figure 1 the
oxygen uptake of islet slices in 0.19 M saline was plotted against the volume of tissue
seal in the diver, i.e., the volume of fluid medium surrounding the tissue slice. The
metabolic activity of the islet slices in 0.19 M saline decreased as the volume of the
tissue seal increased. Moreover, this did not occur when phosphate was used ;
therefore, this greater metabolic variability in saline may be the result of variable
dilution of the phosphate extracted from the tissue. Since phosphate addition stim-
ulated the respiration of islet, one would expect that the oxygen uptake would be
greater in the divers with small tissue seals.

The oxygen uptake of the islet tissue slices, when expressed as m/A. of oxygen per

TABLE II

Effect of different proportions of Na+ to K+ on islet tissue respiration. All media contained 0.033 M

phosphate buffer pH 7.4 + 0.144 M NaCl or KCl. The proportions of Na+ to

K+ were changed by varying the buffer and saline cations

Ratio Na+/K+
in medium

No.
determinations

Aver,
m^l. O2/Mg. protein/hr.

<7

100/0
96.5/3.5*

16
15

2.77
2.62

1.26
1.00

* Saline-PO4 mixture as described in Table I.

418

ARNOLD LAZAROW ET AL.

TABLE III

Effect of different proportions of Na+ to K+ on islet tissue respiration. All media contained 0.033 M

phosphate buffer pH 7.4 + 0.144 M NaCl or KCl. The proportions of Na+ to

K+ were changed by varying the buffer and saline cations

Ratio Na+/K+

No.

Aver.

in medium

determinations

m^l. O2/Mg- protein/hr.

100/0

16

2.77

1.26

80/20

16

3.24

1.46

0/100

17

3.26

1.33

/xg. of tissue protein, appears to be independent of the size of islet tissue slice
used.

Since the stimulation observed in the saline-phosphate buffer medium could be
due either to (a) the addition of potassium ion (contained in the buffer), (b) buf-
fering action, or (c) a specific phosphate ion effect, experiments were carried out
in order to determine which factor was responsible.

The effect of varying the potassium and sodium ion concentrations of the medium
is shown in Tables II and III. In the usual saline-phosphate buffer medium there
are three and one half parts of potassium to ninety-six and one half parts of sodium.
When an all-sodium phosphate buffer was used (prepared by mixing sodium mono-
basic and sodium dibasic phosphates), there was no change in metabolic activity of
the islet slices. When higher potassium ion concentrations were used (Table III),
the activity was about 20% higher than that observed with an all-sodium medium.
However, analysis of these results showed that this difference was not statistically
significant. Since the addition of phosphate buffer increased the metabolic ac-
tivity of islet slices by 43 to 153%, it may be concluded that this stimulation is not
due to the addition of the potassium ion contained in the buffer.

The effect of pH. Various saline-phosphate-buffer mixtures were prepared in
which the pH of the phosphate buffer was varied. Table IV shows that there were
no significant differences in the metabolic activity of islet slices when the pH was
varied from 6.2 to 8.0; the p values were all greater than 0.3. This finding is in
keeping with the studies previously reported by other investigators (Elliott and
Birmingham, 1949) who suggest that the internal pH of tissue slices is maintained
fairly constant over a wide range of external pH. This finding therefore suggests
that the stimulatory effect observed with phosphate buffer addition (Table I) was

TABLE IV

Effect of pH on islet tissue slice respiration. In all cases the medium consisted of
0.144 M NaCl + 0.033 M NasHPOi-KHsPO* buffer

pH of medium

No. of
determinations

Aver.
mftl. OS/MS- protein/hr.

a

6.2

11

2.39

0.67

6.8

14

2.46

0.89

7.4

14

2.14

0.79

8.0

15

2.53

1.25

ELECTROLYTES AND ISLET METABOLISM

419

due to a specific phosphate ion effect rather than to the ability of the phosphate
buffer to maintain the pH of the medium.

The effect of buffer type. The metabolic activity of islet slices was studied in
phosphate, tris (trishydroxyaminomethane), and veronal buffers at pH 7.4 and the
results are shown in Table V. The highest metabolic activity was observed with
phosphate buffer. With an equimolar concentration of tris buffer, the oxygen up-
take was lower than with phosphate buffer (p value < .001), but it was of the same
order of magnitude as with 0.19 M saline (Table I). With veronal buffer the me-
tabolic activity was 86% lower than with phosphate and much lower than with 0.19
M saline. Thus veronal buffer inhibits the metabolic activity of islet slices. Since
in the presence of 0.033 M tris plus 0.033 M phosphate buffer the (X uptake was
the same as in phosphate, the low activity in tris buffer was not due to inhibition,
but rather to the absence of phosphate.

The effect of phosphate concentration. A large series of experiments were car-
ried out in which varying concentrations of the Na2HPO4-KH2PO4 buffer (pH 7.4)

TABLE V

Effect of buffer type on islet tissue slice respiration. In all cases the medium contained 0.033 M
buffer, pH 7.4; the medium containing both tris and phosphate buffer was 0.033 M with respect to
each. Enough NaCl was added to each medium to maintain a tonicity equivalent to 0.19 M NaCl

Series I

Series II

Buffer in medium

No.

Aver.

No.

• Aver.

determi-

m/ul. Ot/pg.

a

determi-

mn\. O*/fi.g.

<r

nations

protein/hr.

nations

protein/hr.

Na2HPO4-KH,P04

15

2.32

0.97

19

2.08

0.99

Tris

14

1.29

0.64

18

1.60

0.47

Veronal

6

0.33

0.13

—

—

—

Tris Na2HPO4-KH2PO4

—

—

—

19

2.02

0.52

were added and the tonicity of the medium was maintained equivalent to 0.19 M
NaCl by adjusting the NaCl concentration. These results are shown in Figure 2.
There was a progressive increase in the oxygen uptake as the phosphate ion con-
centration was increased. Maximal stimulation of metabolic activity was observed
at a phosphate ion concentration of 0.066 M. The stimulation observed in the
presence of 0.066 M phosphate was highly significant. A comparison of the ac-
tivity in 0.066 M phosphate with that observed in the absence of phosphate, or in
the presence of 0.002 M or 0.008 M phosphate, gave a /> value < 0.001. The dif-
ference between 0.066 M and 0.033 M phosphate is also probably significant (/>
value = 0.057). At a phosphate ion concentration greater than 0.066 M there was
no further stimulation; in fact the value at 0.136 M is actually 5% lower than at
0.066M.2 This difference, however, is not significant (p - 0.6).

- Later studies on the effect of tonicity showed that at the high concentrations of phos-
phate one would have expected a large decrease in metabolic activity due to the increasing tonicity
of the medium. In the presence of 0.136 M phosphate, this decrease should have been much
larger than the 5% actually observed. The fact that a decrease of only 5% was observed using
0.136 M phosphate suggests that the higher concentration of phosphate is effectively stimulating
the metabolic activity but that this stimulation is masked by inhibitory effects of increasing

420

ARNOLD LAZAROW ET AL.

The effect of tonicity. In order to study the effect of tonicity, the oxygen up-
take of islet slices was measured in media of varying salt concentrations. However,
since the phosphate buffer concentration that gives maximal stimulation (0.066 M)
has a tonicity equivalent to 0.093 M NaCl, it was necessary to reduce the phosphate
ion to a suboptimal value if lower tonicities were to be used. Therefore additional

OXYGEN 4.0
UPTAKE
m/j I 02 PER
>jg PROTEIN
PER HOUR

3.0

2.0

1.0

I

.02 .04
PHOSPHATE

.06 .08 .10 .12

CONCENTRATION (M/*)

.14

FIGURE 2. Effect of phosphate ion concentration on the respiration of toadfish islet slices.
The tonicity of the medium was maintained equivalent to that of 0.19 M NaCl; pH = 7.4.
Each of the points represents the average of 32 to 161 individual determinations.

studies were carried out with lower phosphate concentrations. Where necessary
the tonicity was adjusted by adding NaCl. Table VI shows that the oxygen uptake
increased with decreasing tonicity. When the tonicity of the medium was equivalent
to 0.093 M NaCl, the metabolic activity of islet slices was 60% greater (p = 0.006)

tonicity. Thus the appearance of a maximum at a phosphate concentration of 0.066 M may be
more apparent than real.

ELECTROLYTES AND ISLET METABOLISM

421

than that observed when the tonicity was equivalent to 0.19 M NaCl. When the
tonicity was decreased further, the oxygen uptake diminished. Furthermore it
should be noted that at a tonicity equivalent to 0.093 M NaCl, a progressive decrease
in the phosphate ion concentration from 0.066 to 0.033 to 0.017 M gave a slight but
progressive decrease in metabolic activity of the islet slices. This decrease is of the
order of magnitude that would be expected from the phosphate curve (Fig. 2).

In order to explore more fully the effect of the tonicity of the medium, a series
of experiments were carried out in which the phosphate ion concentration was main-
tained at 0.033 M (a. slightly sub-optimal phosphate level) and in which the tonicity
was varied between a sodium chloride equivalent of 0.048 M and 0.50 M. The re-
sults are shown in Figure 3. The maximum activity was observed at a tonicity
equivalent to 0.075 M NaCl. At higher or lower tonicities the oxygen uptake was
less than this optimal value. On statistical analysis the differences between the
oxygen uptake at a tonicity equivalent to 0.075 M NaCl and those at the following

TABLE VI

Effect of varying tonicity on islet tissue slice respiration in phosphate buffer. The media contained

varying concentrations of Na^HPO^KH^POt buffer, pH 7.4; the proper tonicity

•was attained by adding the appropriate concentration of NaCl

Tonicity

PO4

concentration

(M)

of medium
(equivalent
NaCl con-
centration)

No.
determi-
nations

Average
protein/hr.

a

M/l

0.066

0.76

8

1.84

0.93

0.066

0.380

8

2.37

0.92

0.066

0.190

8

2.68

1.18

0.066

0.093

8

4.43

0.91

0.033

0.190

8

2.62

0.84

0.033

0.093

8

4.10

1.28

0.033

0.047

8

3.16

1.05

0.017

0.093

8

3.35

0.93

tonicities were found to be significant: 0.047 M (p = 0.036), 0.147 M (p = 0.036),
0.190 M (p = 0.03). Although the differences between the oxygen uptake at
0.075 M and those at the other tonicity values were not significant on statistical
analysis, these values nevertheless fall into a smooth curve (Fig. 3). Although a
more precise localization of the maximum could be determined if a larger number of
studies were carried out, there is little doubt that maximum stimulation occurs at or
near a tonicity equivalent to 0.075 M NaCl.

Since 0.066 M phosphate buffer has a tonicity equivalent to 0.093 M NaCl, it is
obvious that one cannot simultaneously achieve the conditions for both optimal phos-
phate and optimal tonicity. In order to achieve the most effective compromise, we
have compared the respiration of islet tissue slices at pH 7.4 and at optimal phos-
phate sub-optimal tonicity (0.066 M Na2HPO4-KH2PO4 buffer, tonicity equivalent
to 0.093 M NaCl) with that obtained at sub-optimal phosphate-optimal tonicity
(0.054 M Na2HPO4-KH2PO4 buffer, tonicity equivalent to 0.075 M NaCl). The
oxygen uptake in 0.054 M phosphate buffer was 3.54 m/xl. per /xg. protein per hour :

422

ARNOLD LAZAROW ET AL.

OXYGEN 5.0
UPTAKE
nryj I 02 PER
jg PROTEIN

PER HOUR

4.0

3.0

2.0

1.0

ISOTONIC

I I

I I

.05

TONICITY
(EQUIVALENTS

.10 .15

OF MEDIUM
OF NaOf IN MAO

.20

FIGURE 3. Effect of tonicity of the medium on the respiration of islet slices suspended in
0.033 M Na,HPO4— KH2PO4 buffer, pH 7.4. Each of the points represents the average of 8
to 10 individual determinations. The measurements at zero tonicity were carried out in distilled
water and therefore this point represents the respiration in the absence of phosphate.

this was 5% greater than the activity in 0.066 M phosphate buffer. Although this
difference is slight, it does agree with the value that would be expected on the basis
of data shown in Figures 2 and 3. A decrease in the phosphate concentration from
0.006 M to 0.054 M would bring about a 3% decrease in the metabolic activity
(Fig. 2), whereas a change in the tonicity of the medium from a sodium chloride
equivalent of 0.093 M to 0.075 M would bring about a 13% increase in the meta-
bolic activity. Therefore in the subsequent studies we used a 0.054 M Na2HPO4-
KH,PO4 buffer, pH 7.4, which has a tonicity equivalent to 0.075 M NaCl.

ELECTROLYTES AND ISLET METABOLISM 423

The effect of calcium. Media containing a constant amount of phosphate buffer
(0.054 M) and varying amounts of calcium chloride ranging from 0.002 M to
0.00005 M were prepared. Since the final tonicity of these solutions ranged from
a sodium chloride equivalent of 0.075 M to 0.077 M, the expected changes in meta-
bolic activity as a consequence of tonicity changes would be insignificant. The ad-
dition of calcium ion at a concentration of 0.002 M produced a 49% inhibition (p <
0.001) of oxygen uptake; at 0.001 M it produced a 25% inhibition (p = 0.02) of
oxygen uptake ; at concentrations of 0.0005 M and 0.00005 M it did not affect the
metabolic activity.

The effect of magnesium. The effect of magnesium ion addition was tested
both in the presence and absence of added calcium. Magnesium, in concentrations
ranging from 0.01 M to 0.002 M, did not affect the metabolism of the islet tissue
slices either in the absence or presence of calcium chloride (1 X 10~* M CaCl2).

The effect of trace metals. A mixture of trace metal salts, constituting the mini-
mal trace element requirements for Neurospora growth (Beadle, 1945), was added
to 0.054 M phosphate buffer. The final concentrations of the trace elements per
liter of medium were : boron 0.01 mg., molybdenum 0.02 ing., iron 0.2 mg., manga-
nese 0.02 mg., and zinc 2.0 mg. These metals were added as the following salts :
Na,B4O7, (NH4)2MoO4, FeQ3, MnCl,, and ZnCL. In addition, two other media
were prepared in which the concentrations of the trace metals were 10 times and 100
times greater, respectively, than those listed above. The addition of the above trace
metals at the minimal concentration did not affect the respiration of the islet tissue
slices. When the concentration of each trace metal was increased 10-fold there was
a 15% inhibition (p = 0.44) of the oxygen uptake; when increased 100-fold, there
was a 30% inhibition (p = 0.05).

The effect of pyrophosphate. The respiration of islet slices was studied in a
medium containing 0.005 M pyrophosphate plus 0.054 M phosphate buffer. The
added pyrophosphate can act as a chelating agent and thus remove trace metal ions.
Its addition, however, did not affect the respiration of islet tissue slices.

The effect of serum. Samples of toadfish blood were drawn from the gill by
venipuncture and the serum separated by centrifugation. The oxygen uptake of
islet slices in serum was compared with that in 0.054 M phosphate buffer and found
to be 34% lower (p = 0.007). On the other hand, when the serum was previously
dialyzed against three liters of 0.054 M phosphate buffer for 18 hours at 0° C, the
oxygen uptake almost equaled (95%) that in the 0.054 M buffer. Part of this dif-
ference may be due to a phosphate ion effect. However, since the tonicity of toad-
fish serum is considerably greater than the tonicity of 0.054 M phosphate (equiva-
lent to 0.075 M NaCl), the low values obtained in serum, and the higher values
obtained in dialyzed serum, can in part be due to tonicity differences. From the
results obtained in Figure 3 one would expect that an increase in tonicity to that
found in serum would produce a 50% decrease in the oxygen uptake, whereas a
35% decrease was actually found. These results suggest that the addition of serum
protein per sc does not materially affect the respiration of islet slices.

DISCUSSION

It should be noted that maximal stimulation of islet respiration was observed at
a tonicity equivalent to 0.075 M NaCl ; this is considerably lower than the tonicity

424 ARNOLD LAZAROW ET AL.

of toadfish blood.3 Similar stimulation of the oxygen uptake of brain homogenates
when suspended in hypotonic media has been reported by Elliott and Libet (1942).
The activity of the succinic oxidase system is likewise increased in hypotonic media
and this stimulation is believed to be a direct effect on the enzyme complex (Tyler,
1954).

The stimulation of toadfish islet metabolism by phosphate is of interest. The
addition of phosphate increases the oxygen uptake of brain homogenates (Elliott
and Libet, 1942) ; it also increases the activity of certain isolated enzyme systems
(Kearney, Singer and Zastrow, 1955; cf. Koeppe, Boyer and Stulberg, 1956). It
has been suggested that it may also play a role in the control of respiration (cf.
Lardy and Wellman, 1952). It would therefore be of interest to determine whether
the oxygen uptake of slices of other toadfish tissues is similarly stimulated by phos-
phate addition, or if this stimulation is limited to islet tissue. Since islet tissue con-
tains large amounts of zinc (Okamoto, 1942), and since high concentrations of the
trace metal ions inhibited the oxygen uptake of the islet tissue, the phosphate stimu-
lation could be the result of zinc chelation. It would therefore be of interest to see
if pyrophosphate or other chelating agents can substitute for phosphate.

Islet tissue contains about 11% 4 protein and therefore the oxygen uptake of
islet tissue slices (3.5 rm/.l. of oxygen consumed per p.g. of protein per hour) would
be equivalent to 0.39 cc. per gram of tissue (wet weight) per hour. This observed
value is about equal to the reported value for brain brei and greater than that re-
ported for liver slices. Vernberg (1954) found that the oxygen uptake of toadfish
brain brei was equal to 0.41 cc./gm./hr. ; the Qo2 of toadfish liver slices was equal
to 0.27 cc./gm./hr. The Qo2 of the toadfish islet tissue is about 10 times greater
than the oxygen utilization by the intact animal. Hall (1929) has reported that the
oxygen consumed by the toadfish varies directly with the oxygen tension (between
a partial pressure of 0 and 115 mm. of oxygen). At the atmospheric oxygen con-
tent he found that the toadfish utilized 0.038 cc. of oxygen per gram of fish per
hour. Thus the toadfish islet tissue is very active metabolically compared to the
fish as a whole and to the other tissues.

SUMMARY

1. The metabolic activity of toadfish islet slices was measured in a cartesian
diver microrespirometer under varying experimental conditions. The effects of
pH, specific electrolytes, tonicity, trace metals, and protein addition were studied.

2. The metabolic activity was not affected by varying the pH of the medium
between 6.2 and 8.0. The addition of phosphate ion stimulated the respiration.
The maximum stimulation was observed when the external medium contained
0.066 M phosphate.

3. The respiration of islet slices was increased when the tonicity of the sus-
pending media was reduced ; optimal respiration was observed in a hypotonic me-

3 Green and Hoffman (1953) have measured the osmotic pressure of blood samples obtained
from the heart and found them to be equivalent to 0.14 M NaCl ; the osmotic pressure of blood
samples obtained from the gill were equivalent to 0.19 M NaCl. These authors consider the
tonicity values of the heart blood samples to be the more accurate, for the gill blood samples may
have been contaminated with sea water.

4 The protein content of 5 samples of islet tissue was measured and found to contain 11.2%
protein.

ELECTROLYTES AND ISLET METABOLISM 425

dium with a tonicity equivalent to 0.075 M NaCl ; this corresponds to a phosphate
buffer concentration of 0.054 M. This is slightly sub-optimal with respect to phos-
phate ion concentration. Since the metabolic activity of islet tissue slices suspended
in 0.054 M phosphate buffer (optimal tonicity but sub-optimal phosphate) is slightly
greater than in 0.066 M phosphate buffer (optimal phosphate but sub-optimal
tonicity), subsequent studies were carried out using 0.054 M phosphate buffer.

4. High concentrations of calcium (0.001-0.002 M) inhibited the respiration of
islet slices. The addition of serum protein, lower concentrations of calcium ion
(0.0005 M), magnesium ion (0.0002-0.01 M}, and small amounts of trace metals
(boron, molybdenum, iron, manganese, zinc) did not stimulate the respiration of
islet tissue slices.

LITERATURE CITED

BEADLE, G. W., 1945. Genetics and metabolism in Neurospora. Physiol. Rev., 25 : 643-663.
DIAMARE, V., 1899. Studii comparativi sulle isole di Langerhans del pancreas. Internal.

Monatschr. J. Anat. v. Physiology, 16: 155-201.
ELLIOTT, K. A. C., AND M. K. BIRMINGHAM, 1949. The effect of pH on the respiration of brain

tissue; the pH of tissue slices. J. Biol. Chem., 177: 51-58.
ELLIOTT, K. A. C., AND B. LIBET, 1942. Studies on the metabolism of brain suspensions. /.

Biol. Chem., 143 : 227-246.
GREEN, J. W., AND J. F. HOFFMAN, 1953. A study of isotonic solutions for the erythrocytes

of some marine teleosts and elasmobranchs. Biol. Bull., 105 : 289-295.

HALL, F. G., 1929. The influence of varying oxygen tensions upon the rate of oxygen con-
sumption in marine fishes. Amer. J. Physiol., 88: 212-218.
KEARNEY, E. B., T. P. SINGER AND N. ZASTROW, 1955. On the requirement of succinic dehy-

drogenase for inorganic phosphate. Arch. Biochem. Biophys., 55 : 579-581.
KOEPPE, O. J., P. D. BOYER AND M. P. STULBERG, 1956. On the occurrence, equilibria, and

site of acyl-enzyme formation of glyceraldehyde-3-phosphate dehydrogenase. /. Biol.

Chem., 219 : 569-583.

LARDY, H. A., AND H. WELLMAN, 1952. Oxidative phosphorylations : Role of inorganic phos-
phate and acceptor systems in control of metabolic rates. /. Biol. Chem., 195: 215-224.
LAZAROW, ARNOLD, 1949. Factors controlling the development and progression of diabetes.

Physiol. Rev., 29 : 48-74.
LAZAROW, ARNOLD, 1950. Microanalysis : Respirometer ; Cartesian Diver. Medical Physics,

II. Edited by O. Glasser. Yearbook Publishers, Inc., Chicago, pp. 490-496.
LAZAROW, ARNOLD, AND S. J. COOPERSTEIN, 1951. Studies on the isolated islet tissue of fish. I.

The cytochrome oxidase and succinic dehydrogenase content of normal toadfish

(Opsanus tan). Biol. Bull, 100: 191-198.
LAZAROW, ARNOLD, AND RICHARD A. PORTIS, 1951. Micro-conical tipped homogenizer and its

use in analytical procedures. /. Lab. Clin. Med., 38 : 773-776.
LOWRY, O. H., N. J. ROSENBROUGH, A. L. FARR AND R. J. RANDALL, 1951. Protein measurement

with the folin phenol reagent. /. Biol. Chem., 193 : 265-275.
OKAMOTO, K., 1942. Biologische Untersuchungen der Metalle. VI. Histochemischer Nach-

weis einiger Metalle in den Geweben, besonders in den nieren, und deren verander

Ungen. Tr. Soc. Path-Jap., 32 : 99-105.
RENNIE, J., 1905. The epithelial islets of the pancreas in teleostei. Quart. J. Micr. Sci., 48 :

379-406.
TYLER, DAVID B., 1954. The effect of osmotic pressure on succinoxidase activity. /. Biol.

Chem., 209: 893-900.
VERNBERG, F. J., 1954. The respiratory metabolism of tissues of marine teleosts in relation to

activity and body size. Biol. Bull., 106 : 360-370.

EVIDENCE FOR HORMONE-CONTAINING GRANULES IN SINUS
GLANDS OF THE FIDDLER CRAB UCA PUGILATOR

MARIA DOLORES PEREZ-GONZALEZ 1
Biological Laboratories, Harvard University, Cambridge 38, Mass.

It is known that in neurosecretory systems the products of secretion are stored
in axon terminations where they aggregate in particles or granules (Scharrer and
Scharrer, 1954; Welsh, 1955). '

Some recent electron microscope studies have shown a constancy in the ap-
pearance of such structures in the neurohypophysis of different vertebrates (Dun-
can, 1955, 1956). The size of these granules varies from 0.1 to 0.3 micron, and in
the neurohypophysis of the rat they seem to be bounded by a delicate membrane
(Palay, 1955).

Through the differential centrifugation technique for isolation of mitochondria
and other particles of the cells, Hillarp, Lagersted and Nilson (1953) and Blaschko
and Welch (1953) could obtain a fraction of granules which is responsible for 80
to 90% of the total adrenaline and noradrenaline present in the adrenal medulla of
cattle. Further, Hillarp and Nilson (1954) and Blaschko, Hagen and Welch
(1955), doing physiological experiments with the separated granular fraction, ob-
tained information concerning the nature of the granules containing the catechol
amines. Similar results were obtained for the granules containing vasopressin and
oxytocin in the posterior pituitary of the rat (Pardoe and Weatherall, 1955). The
observations of the several authors, above cited, strongly support the assumption
that the granules have a semipermeable membrane of a lipid or lipo-protein nature.
The granules, which are stable in isotonic solutions of saline or sucrose, release
their hormone when treated by agents which are known to damage biological
membranes.

In the invertebrates, especially among insects and crustaceans, some neuro-
secretory systems are very well known, and the study of the granules in these
systems might give valuable information concerning such storage particles. A good
structure for these studies is the "sinus gland" of the crustaceans. A sinus gland
in each eyestalk is the storage-release organ for several neurohormones of the crus-
taceans. They are formed by the axon terminations of neurosecretory cells local-
ized in the X-organ, in the brain and in other parts of the central nerve system
(Passano, 1951a, 1951b; Bliss and Welsh, 1952; Bliss, Durand and Welsh, 1954).
The axon terminations in sinus glands are filled with granules 0.1 to 0.3 micron in
diameter (Potter, 1956) which can be seen in living preparations (Passano, 1952).

The aim of the present work was to show that the granules in sinus glands are
really the depots of neurosecretory materials and that they behave like similar struc-
tures found in the neurosecretory systems of vertebrates.

1 Rockefeller Foundation Fellow, from Department of General and Animal Physiology,
Fac. Fil. Cien. Letras, Universidade de Sao Paulo, Brasil.

426

HORMONE-CONTAINING GRANULES IN UCA 427

MATERIAL AND METHODS

For this purpose the study of one of the chromatophorotropic hormones stored
in sinus glands was chosen. According to the usual technique for isolation and
preservation of mitochondria (Hogeboom, Schneider and Palade, 1948) the sinus
glands were homogenized in isotonic solutions of sucrose. The homogenates be-
fore and after several treatments were injected into test animals for an estimation
of the activity of the hormone in the different cases.

Preparation of the homogenates

Sinus glands of the fiddler crab, Uca pugilator, from Florida, were used in these
experiments. With the aid of a dissecting microscope the sinus glands were iso-
lated from the adjacent tissues immediately after cutting the eyestalk of the crabs,
and were placed in solutions of cold 1.3 M sucrose, which, according to Abramo-
witz and Abramowitz (1938), is isosmotic with the blood of Uca. In each experi-
ment four or more sinus glands were homogenized in one ml. of isotonic sucrose,
in the Elvehjem homogenizer for three minutes. After homogenization more su-
crose was added according the requirement of the experiment. A part of the ho-
mogenate was then kept at 2° C. until the moment of the experiment, and the re-
mainder was submitted to different treatments. Before being injected into the test
animals all homogenates were diluted in isotonic sucrose, or sea water to make the
same final concentration. All the procedures were carried out in the cold at 2° C.
For details, see below.

Assays

The activity of the black chromatophore-dispersing hormone in the different
homogenates was tested in isolated legs of Uca pugna.v. It was observed that in
legs of Uca pugilator when they are separated from the body, the black chromato-
phores disperse gradually. Such dispersion may be explained by a direct effect of
light on the chromatophores, since legs isolated and kept in sea water in the dark
do not show this phenomenon. A direct effect of light on the chromatophores of
Uca pugilator has been already observed by Brown and collaborators (Brown and
Sandeen, 1946, 1948; Brown, Guyselman and Sandeen, 1949). For this reason in
the present experiments the legs of Uca pugna.v which do not show this behavior
were used.

Uca pugna.v were destalked 24 hours before the experiment so that at the time
of the experiment the black chromatophores were in the stage of maximal concen-
tration. The legs were cut off at the level of the ischial segment, and were placed
in 5 ml. of sea water in Syracuse dishes. Each leg received an injection of 0.01 ml.
of homogenate, and the dispersion of the black chromatophores was observed every
ten minutes for one hour. Uca pugilator was used for experiments in which the
homogenates were tested in the whole animal. In these cases, each animal re-
ceived 0.1 ml. of homogenate and the stages of the chromatophores were observed
for several hours.

Preliminary attempts were made to remove granules from the homogenates by

centrifugation.

All the results are presented in graphs according to Hogben and Slome (1931),

428

MARIA DOLORES PEREZ-GONZALEZ

where 1 represents maximal concentration of the chromatophores, 5 maximal dis-
persion, and 2, 3 and 4, intermediate stages.

RESULTS
/. Hormone in granules and in cytoplasm

The homogenate of 4 sinus glands in 1 ml. of 1.3 M sucrose was divided; one-
half of the suspension received 4.5 ml. of distilled water and was kept at 2° C. ;
the other half was kept undiluted at the same temperature. After one-half hour,
the homogenate in sucrose was diluted with sucrose, and that with added distilled

LU

CL
o

o

(T

10 20 30 40 50
TIME IN MINUTES

60

FIGURE 1. Response of the black chromatophores of isolated legs of Uca pugnax to injections
of homogenates of sinus glands of Uca pugilator : •, homogenates in distilled water ; O, homogenates
in 1.3 M sucrose. Each point in the graph represents the average of 20 experiments.

water was diluted with sea water to make a final concentration of 0.02 sinus gland
per ml. Every time that distilled water was added to the homogenates, the final
dilution was made in sea water; these homogenates throughout the paper will be
called "homogenates in distilled water," to shorten the explanation.

As one can see in Figure 1, the homogenate in distilled water caused maximal
dispersion of the chromatophores in the legs of Uca, and that in sucrose exhibited
only a small effect. These results show that in isotonic sucrose the black chromato-
phore-dispersing hormone is present in large part in a state in which it cannot act ;
whereas in distilled water it seems to be free in solution and able to induce the dis-

HORMONE-CONTAINING GRANULES IN UCA

429

persion of the chromatophores. This evidence supports the view that the hormone
is contained in granules, which in isotonic sucrose remain intact and in distilled
water release the hormone into the solution.

If that were the case it would be possible to separate a fraction of granules con-
taining hormone, using the usual technique of differential centrifugation. To avoid
the high density of a medium like 1.3 M sucrose, 16 sinus glands were homogenized
in one ml. of a mixture of 25% of 1.3 M sucrose and 75% of sea water, which has
been demonstrated to be as effective in preserving the granules as pure sucrose.
The homogenate, after dilution to make 10 ml., was centrifuged at 800 X gravity

UJ

CD/

LU

<r

i2

o:
o

O

10 20 30 40 50 60
TIME IN MINUTES

FIGURE 2. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in isotonic sucrose after centrifugation: •, "supernatant A" (low speed) ; C, "supernatant
B" (high speed) ; O, "sediment C" (high speed).

for 30 minutes for separation of unbroken cells, nuclei, etc. No visible sediment
was observed after this slow-speed centrifugation, so the whole solution was de-
canted. Part was set aside, as "solution A," and the rest was centrifuged at 20,000
X gravity for 30 minutes. No sediment was observed this time either. The whole
solution was decanted carefully and taken as "solution B." Then one ml. of dis-
tilled water was added to the centrifuged tube and was stirred and the tube walls
were scraped with a spatula. After 15 minutes 9 ml. of sea water were added to
make 10 ml., and this solution was called "solution C." Part of the solution A and
B was treated with distilled water and all three solutions were finally diluted to the
concentration of 0.02 sinus gland per ml. Both solutions A and B showed the same

430

MARIA DOLORES PEREZ-GONZALEZ

effect on the chromatophores of legs of Uca pugna.v. No significant loss of activity
was observed in solution B after the high speed centrifugation. However, solution
C, the suspension of a presumably invisible sediment, caused a small effect on the
chromatophores (Fig. 2). This fact is indicative of some sedimentation of gran-
ules and from these results it is not possible to infer how much of the hormone is
present in granules and how much is found free in the homogenate. It is prob-
able that for a complete sedimentation a longer and higher-speed centrifugation is
necessary.

An indication of the percentage of hormone contained in granules in isotonic
sucrose is given by the analysis of the activity of homogenates in isotonic sucrose
and distilled water after a series of dilutions. Figure 3 shows the results of injec-
tions of 0.01 ml. of homogenates of 2 sinus glands in 1 ml. of 1.3 M sucrose and in
1 ml. of distilled water diluted 10, 100 and 1000 times in 1.3 M" sucrose and sea

(T

§3

CL
O

I2

<r

x
o

I

c- — ®

Q — 9

10 20 30

40
TIME

50 60

IN

10 20
MINUTE S

30 40 50 60

FIGURE 3. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in distilled water (left) and in 1.3 M sucrose (right) in different concentrations: e, 0.2;
C), 0.02; and O, 0.002 sinus glands per ml.

water, respectively. It appears that in isotonic solution only less than 10% of the
hormone is found free in the suspension, since the effects of the homogenates in
sucrose are smaller than those of the homogenates in distilled water, ten times more
diluted.

//. Effect of several treatments on the release of the hormone

1. Effect of the tonicity of the medium. From the homogenates of 8 sinus
glands in 2 ml. of 1.3 M sucrose, 7 samples of 0.25 ml. each were separated. The
addition of 9.75 ml. of 0.9, 0.8, 0.7, 0.65, 0.32 M sucrose was made to a series of 5
tubes and to a sixth, 4.75 ml. of distilled water were added. After one hour at
2° C., the solutions were diluted in 1.3 M sucrose to the final concentration of 0.02
sinus gland per ml. and were tested on isolated legs. Figure 4 illustrates the ac-
tivity of the hormone in the different solutions. As the tonicity of the medium de-

HORMONE-CONTAINING GRANULES IN UCA

431

creases there is a liberation of hormone which, to some extent, is proportional to
the concentration of sucrose, from 1.3 to 0.7 M. In 0.65 and 0.32 M solutions there
seems to be a complete release since the activity of the hormone in these two latter
concentrations is as great as that of homogenate in distilled water. The action of
distilled water after 15 minutes standing is as effective as after 30 minutes. This
fact shows that the release in distilled water is rapid. The granules in this respect,
like red blood cells and mitochondria under the same conditions, appear to act as
osmometers.

2. Effect of different solutions. One group of experiments was performed to
determine whether the granules containing the hormone are also stable in isotonic

LJ

U

or
o

CL
O

o'

<r

o

10 20 30
TIME IN

40 50 60
MINUTES

FIGURE 4. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in different concentrations of sucrose solutions: +, 1.3; O, 0.9; O, 0.8; ©, 0.7; 3, 0.65; and
C, 0.32 M; •, homogenate in distilled water.

solutions of electrolytes. Samples of 0.5 ml. of homogenates in 1.3 M sucrose were
held for 30 minutes at 2° C. with the addition of 4.5 ml. of the following: distilled
water, sea water, sodium chloride and potassium chloride. The sodium chloride
was either isotonic with sea water (0.54 M) or isotonic with 1.3 M sucrose (0.78
M). Figure 5 shows that sea water and isotonic salt solutions produce a large and
rapid release of hormone. This fact indicates that the simple dilution in isotonic
electrolyte solutions is sufficient to provoke alterations in the granules very similar
to those observed by lowering the tonicity of the medium. However, in electrolyte

432

MARIA DOLORES PEREZ-GONZALEZ

solutions to which an equal part or a fourth part of isotonic sucrose is added, the
granules remain largely intact. The activity of the hormone in these media (Fig.
5) is comparable to that in isotonic sucrose.

In another group of experiments an effort was made to find the best medium
for preservation of the granules. Watanabe and Williams (1953) have shown that
2.5% bovine plasma albumin in isotonic potassium phosphate buffer at pH 7 is a
good medium to preserve mitochondria of insect muscles. In the following experi-
ments, besides the 1.3 M sucrose, a mixture of 25% 1.3 M sucrose and 75% sea
water was also used, as well as 2.5% bovine plasma albumin in 0.54 M potassium

Oty
LU

o:

I2

a:
o

10 20 30 40 50 60
TIME IN MINUTES

FIGURE 5. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in different solutions: O, distilled water; f>, sea water; O, 0.78 and 0.54 M NaCl; o, 0.54
M KC1 ; e, G, and 3, NaCl, KC1 and sea water in a mixture with 25% of 1.3 M sucrose.

phosphate at pH 7. The homogenates of sinus glands in these three media were
kept at 2° C. and at different times were diluted in 1.3 M sucrose and assayed using
legs of Uca (Fig. 6). After 6 hours of incubation in these media, the activity of
the black chromatophore-dispersing hormone is insignificant, and after 24 hours
only a slight effect was observed. That the hormone was preserved in the granules
was shown by the following procedure. After 24 hours the homogenates were
heated for 5 minutes in boiling water and diluted in sea water. After this treat-
ment all the solutions produced a maximal dispersion of the chromatophores, com-
parable to that caused by homogenates in distilled water. Thus, the three different
media used seem to be equally efficient in keeping the granules intact.

HORMONE-CONTAINING GRANULES IN UCA

433

3. Effect of heat, and of freezing and thawing. A release of hormone from the
granules was observed when homogenates of sinus glands in isotonic sucrose were
kept at room temperature for several hours. However, homogenates in 1.3 M su-
crose when heated for 5 minutes at 70° C. or in boiling water showed only a slightly
greater activity than the original homogenate without this treatment (Fig. 7).

Freezing at — 10° C. and thawing to room temperature three times in succes-
sion was more effective than heating, but even so, the release of the hormone was
not the same as when homogenate was merely diluted in distilled water (Fig. 7).

4. Effect of detergents and digit onin. Detergents and digitonin did not give a
complete release of hormone from the granules. Samples of 0.25 ml. of homoge-

5-

LU

e>
<

LU

or
o

CL

o

i-

o
a:

T.
O

I

oa — on

on

10 20 30 40 50
TIME IN MINUTES

60

FIGURE 6. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in different media. Circles, homogenate in 5% bovine plasma albumin in 0.54 M potassium
phosphate buffer; squares, homogenate in 1.3 M sucrose; triangles, homogenate in 25% 1.3 M
sucrose plus 75% sea water. • D A, homogenates kept 6 hours and C), C, A, 24 hours at 2° C.;
O, D, A, after being kept 24 hours at 2° C., the homogenates were heated and diluted in sea water.

nates in 1.3 M sucrose were maintained for one hour at 2° C. with 1.75 ml. of 10~3
M concentration of the following substances: sodium lauryl sulfonate (Duponol) ;
sodium desoxycholate, saponin and digitonin, in 1.3 M sucrose. After the required
dilution of the homogenates for the bio-assays, the concentration of the detergents
and digitonin was 10~5 M. When control legs of Uca or the whole control animal
received injections of the detergents and digitonin in such concentration, no effect
on the chromatophores was observed. Therefore, the dispersion following the in-
jections of homogenates in sucrose plus detergents is attributed to the hormone
present in the solutions.

434

MARIA DOLORES PEREZ-GONZALEZ

The detergents employed and digitonin provoked only a partial release of hor-
mone (Fig. 8). Part of the homogenate plus desoxycholate after one hour of in-
cubation was heated and diluted in sea water, and greater activity was seen after
this treatment.

///. Inactivation of the hormone

In some experiments the homogenates of sinus glands in distilled water were
injected into the whole crab (Uca pugilator). Figure 9 shows the degree and the

UJ

UJ

a:
o

a_
o

cr

i
o

I

10 20 30 40 50
TIME IN MINUTES

60

FIGURE 7. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in isotonic sucrose before, +, and after heating at 70° C., O, and in boiling water, C; and
after freezing and thawing, O. Homogenate in distilled water, •

duration of the dispersion of chromatophores in relation to the concentration of the
homogenates. The injection of 0.1 ml. of a homogenate of 0.2 sinus gland per ml.,
i.e., the injection of an amount corresponding to 0.02 sinus gland, is enough to cause
a maximal dispersion of the chromatophores in almost 30 minutes and only four
hours later have the chromatophores reached the stage of complete concentration
again. It is interesting to notice that the time required for normal dark Uca to
become pale after eyestalk removal is three to four hours. At all concentrations of
homogenates dispersion was found to require less time than concentration of pig-
ment within the chromatophores. The elimination of the hormone seems to be a
very slow process. Even an injection corresponding to 0.001 sinus gland (open

HORMONE-CONTAINING GRANULES IN UCA

435

circles in Fig. 9) induces an effect which disappears completely only after three
hours.

In order to obtain some information about the inactivation of the hormone,
homogenates of sinus gland in distilled water were incubated with extracts of
hepatopancreas, hypodermis and muscle and with one ml. of blood of Uca. The
extracts were prepared by homogenizing one hepatopancreas, the muscle of one
claw, and the hypodermis of the branchiostegites separately, in one ml. of sea
water. The blood was removed at the junction of the body and the fourth walking

KT 20
TIME IN

^4CT 50
MINUTES

FIGURE 8. Response of black chromatophores of Uca to injections of homogenates of sinus
glands in isotonic sucrose before, +, and after treatment with detergents and digitonin. Q,
Duponol; 9, saponin; O, sodium desoxycholate ; O, digitonin; •. sodium desoxycholate plus heat
and dilution in sea water.

leg, with the aid of a glass pipette. The only extract which caused a complete in-
activation of the black chromatophore-dispersing hormone was that of hepatopan-
After one hour of incubation with extracts of hypodermis or muscle, or with

creas.

blood, at room temperature, no decrease in the activity of the hormone was observed
(Fig. lOa).

The enzyme in the hepatopancreas responsible for its action might be a proteo-
lytic one, since the same effect was obtained when homogenates of sinus glands in
distilled water were incubated at 37° C. for one hour with some crystals of chymo-
trypsin (Fig. lOb). These results suggest that the black chromatophore-dispersing

436

MARIA DOLORES PEREZ-GONZALEZ

LU
O

ts*

LU
(T
O

£3

02

CE
O

3

HOURS

FIGURE 9. Response of black chromatophores of the whole Uca pugilator to injections of 0.1
ml. of homogenates of sinus glands in distilled water, in different concentrations: •, 0.2 ; f), 0.02 ;
O, 0.001 sinus gland per ml.

1

TIME

2
IN H

UJ
O

UJ

o

cr
x
o

C C C «

b.

© © O ©•

10 20 30 40 50 60 10 20 30 40 50 60

TIME IN .MINUTES

FIGURE 10. Response of black chromatophores of Uca to injections of homogenates of
sinus glands in distilled water before and after incubation with extracts of different tissues for one
hour at room temperature and after incubation with chymotrypsin for one hour at 37° C. : o.
muscle; O, blood; 3, hypodermis; C, hepatopancreas ; 0, distilled water; and O, chymotrypsin.

HORMONE-CONTAINING GRANULES IN UCA 437

hormone is a polypeptide, but the acceptance of this hypothesis depends upon fur-
ther experiments.

DISCUSSION

The experiments in section I indicate that in the crab, Uca pugilator, the black
chromatophore-dispersing hormone is stored in sinus glands within the granules.
This assertion is supported by the following observations. First, homogenates of
sinus glands in isotonic sucrose have only a small effect on the chromatophores of
legs of Uca pugna.v. These homogenates diluted in distilled water cause a maximal
dispersion of the chromatophores, indicating a more or less complete release of the
hormone. Second, a sedimentable fraction containing hormone was obtained by
centrifugations at the speed of 20,000 X gravity. This centrifugation caused only
a partial sedimentation of granules. However the analysis of the activity of ho-
mogenates in isotonic sucrose and in distilled water after a series of dilutions shows
that the homogenates in sucrose are as effective as those in distilled water ten times
more diluted, indicating that only 10% or less of the total amount of hormone is
present free in the solution, the other 90% remaining in the granules. Whether
this free hormone is already present in sinus glands in vivo, or whether it is the ef-
fect of the disruption of some granules during the process of homogenizing, is not
known. Hillarp, Lagersted and Nilson (1953) have observed that at increased
duration of homogenization the catechol content of the granules of the adrenal
medulla cells decreases. Berthet and De Duve (1951) have also found that a
partial damage to the mitochondria containing acid phosphatase is caused by the
process of homogenizing liver tissue. This may be the case with the homogenates
of sinus glands.

The effect observed by lowering the tonicity of the medium reinforces the evi-
dence of the presence of the chromatophore-dispersing hormone in granules, and
suggests the existence of a semipermeable membrane for the granules. The rapid
release of hormone observed when the tonicity of the medium decreases suggests that
there is a lysis of the granules, by rapid entrance of water.

The membrane of the granules seems to be freely permeable to ions like sodium
and potassium, because the solutions of isotonic sodium chloride, potassium chlo-
ride, or sea water cause an immediate and marked release of hormone from the
granules. Hillarp and Nilson (1954) have found that the granules of the adrenal
medulla can be suspended in sucrose or in certain isotonic electrolyte solutions with-
out a considerable release of catechol amines. Blaschko, Hagen and Welch (1955),
however, have observed that in NaCl or KG an appreciable liberation of adrenaline
occurs. Pardoe and Weatherall (1955) also have obtained liberation of vasopres-
sin and oxytocin from granules of the posterior pituitary of rats, by simple dilution
in saline of the suspensions of granules in isotonic sucrose. Isotonic saline solu-
tions have been demonstrated to afford only transient osmotic protection for mito-
chondria of the rat liver (Berthet, Berthet, Appelman and De Duve, 1951 ; Appel-
man and De Duve, 1955) and for mitochondria of insect muscle (Watanabe and
Williams, 1953). The authors above cited observed also that in media where part
of the saline is replaced by isotonic sucrose, the mitochondria are very stable.
Similarly, the granules of sinus glands are equally stable in pure sucrose, in a mix-
ture of 25% isotonic sucrose and 75% isotonic salines, and in 2.5% bovine plasma
albumin in 0.54 M potassium phosphate buffer at pH 7.

438 MARIA DOLORES PEREZ-GONZALEZ

Heating, freezing and thawing, and the action of detergents have been proved
efficient treatments to release physiologically active substances from granules (Hil-
larp and Nilson, 1954; Pardoe and Weatherall, 1955). In the case of granules of
sinus glands, all these treatments induce a more or less appreciable release of hor-
mone but none of them is sufficient to cause a complete liberation of hormone from
the granules.

The hormone in the homogenates in isotonic sucrose after heating, freezing and
thawing and after the action of detergents is still present either inside the granules
or in such combination that it can not be active. This was proved by the experi-
ments in which parts of the homogenates after these treatments were diluted in sea
water, and greater activity was then observed.

These observations may suggest the following hypothesis : that inside the gran-
ules the chromatophore-dispersing hormone is found in two forms, bound to a large
molecule and as free small molecules. By heating, freezing and thawing and by the
action of detergents, the membrane of the granules suffers some disruption, per-
mitting only the passage of the small molecules to the solution. In hypotonic and
saline media, which cause a lysis of the granules, all the molecules are present free
in the solution. One has to admit also that the hormone is active in both forms,
or that once free in the solution, the large molecules disintegrate into the smaller
ones. This could explain the different activity of the homogenates of sinus glands
in isotonic sucrose after these different treatments.

It is interesting to discuss here the results of Knowles, Carlisle, and Dupont-
Raabe (1955) with the chromactivating substances of sinus glands and post-
commissure organs of Lcandcr serratus, and corpora cardiaca of Carassius. By
electrophoresis of extracts of these organs they detected the presence of a substance,
the "A-substance," which is relatively immobile at pH 7.5 and does not pass
through cellophane membranes. This substance concentrates all the red chromato-
phores of Lcander. When the extracts are left standing several hours at room tem-
perature, the A-substance disintegrates into others, the a-substances, which have
high mobility at pH 7.5 and pass freely through a dialysis membrane. The a-
substances affect only the small red chromatophores of Leander. They observed
also that only the a-substances are released by electrical stimulus of the commissure
when the post-commissure organ is in a saline bath.

So, it is reasonable to believe that the dispersing hormone of Uca can also be
found as large and small molecules and both be active on black chromatophores.
But, of course, this is an assumption which depends upon further experiments in
this subject.

Heating in boiling water does not cause loss in the activity of the chromatophore-
dispersing hormone of Uca. Inactivation of the hormone can be achieved, however,
by incubation of the homogenates of sinus glands with extracts of hepatopancreas
and by the action of the enzyme chymotrypsin. These results suggest that the hor-
mone is a polypeptide.

Carstam (1951) has found that extracts of hepatopancreas of crustaceans and
molluscs, and extracts of liver of the guinea pig inactivate the pigment-concentrating
hormone of Leander adspcrsiis, but he could not obtain the inactivation of the hor-
mone with trypsin. However, Knowles, Carlisle and Dupont-Raabe (1956) have
obtained a complete inactivation of the "A-substance" from sinus glands and post-
commissure organ of Leander, by a crystalline preparation of trypsin and also by a

HORMONE-CONTAINING GRANULES IN UCA 439

prolonged acid hydrolysis. Ostlund and Fange (1956) have suggested that a
chromactivating substance from the eyestalk of Pandalus could be an aromatic
amine, but in personal communication to Knowles and Carlisle (1956) they have
stated that their more recent work indicates that this hormone may possibly be a
polypeptide. So far, the studies concerning the nature of the chromactivating sub-
stances of crustaceans indicate that they are polypeptides. Porath, Roos, Land-
grebe and Mitchell (1955) have isolated a melanophore-stimulating peptide from
the pig-pituitary gland. Thus, also in vertebrates the chromatophorotropins seem
to be peptides.

Carstam (1951) has also obtained the inactivation of the pigment-concentrating
hormone by an enzyme present in the hypodermis of Leander. In Uca pugilator,
in hypodermis as well as in the blood, there was not found an inactivating enzyme
for the chromatophore-dispersing hormone.

The experiments where the homogenates of sinus glands in distilled water were
injected into the whole Uca pugilator show that the response of the black chromato-
phores is a function of the concentration of the hormone. These results give also
an idea about the amount of hormone liberated and its way of action in normal
crabs. Insignificant amounts of the hormone (corresponding to 0.02 sinus gland)
are enough to induce a maximal dispersion of the chromatophores for a long time.
This shows that the elimination or destruction of such small quantities of hormone
is a slow process. Stephens, Strickholm and Friedl (1956) have also observed
that the dispersing hormone in Uca was present in the circulating blood of destalked
assay animals in discernible amounts for approximately three hours after injection.
Hence, it is reasonable to believe that the dispersing hormone is liberated into the
blood in small quantities and eliminated by excretory processes without the inter-
ference of special enzymes for its inactivation.

This work has been supported in part by a Rockefeller Foundation fellowship to
the author, for a stay in the laboratory of Prof. John H. Welsh, at Harvard Uni-
versity ; and in part by Research Grant B-623 from the National Institute of Neuro-
logical Diseases and Blindness, National Institutes of Health. I wish to express
my sincere appreciation to Prof. John H. Welsh for his skillful assistance during
the course of this investigation. I am grateful to Dr. I. Ringler for his help in
running the high speed centrifugation experiments.

SUMMARY

1. Homogenates of sinus glands in isotonic sucrose cause little dispersion of
black chromatophores when injected into legs or whole Uca. A liberation of hor-
mone occurs when homogenates of sinus glands in isotonic sucrose are diluted in
distilled water. A fraction, sedimentable by high speed centrifugation, when re-
suspended in distilled water and injected into the test animals, induces a dispersion
of the chromatophores. These results support the view that the black chromato-
phore-dispersing hormone is contained within granules in sinus glands.

2. The release of the hormone from the granules, obtainable by lowering the
tonicity of the medium or by dilution in isotonic saline solutions, suggests that the
granules possess a semipermeable membrane.

440 MARIA DOLORES PEREZ-GONZALEZ

3. The release of the hormone from the granules is increased by heating, by
freezing and thawing, and by the action of detergents and digitonin.

4. The black chromatophore-dispersing hormone may be a polypeptide, since it
is inactivated by extracts of hepatopancreas and by chymotrypsin.

5. The rate of disappearance of the hormone from the blood of the crab is very
slow.

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APPELMAN, F., AND C. DE DUVE, 1955. Tissue fractionation studies. III. Further observations
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pressor substances in the pituitary glands of rats. /. Physiol., 127 : 201-212.
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Ill: 502.
PASSANO, L. M., 1951b. The X-organ, a neurosecretory gland controlling molting in crabs.

Anat. Rec., Ill: 559.
PASSANO, L. M., 1952. Phase contrast observations on living neurosecretory cells of Sesarma.

Anat. Rec., 112: 460.
PORATH, J., D. Roos, F. W. LANDGREBE AND G. M. MITCHELL, 1955. Isolation of a melano-

phore-stimulating peptide from pig-pituitary gland. Biochem. et Biophys. A eta, 17 :

598-599.
POTTER, D. D., 1956. Observations on the neurosecretory system of portunid crabs. Ph.D.

Thesis, Harvard University, Cambridge, Mass.
SCHARRER, E., AND B. SCHARRER, 1954. Hormones produced by neurosecretory cells. Recent

Progress Hormone Res., 10 : 183-240.
STEPHENS, G. C, A. STRICKHOLM AND F. FRIEDL, 1956. The rate of disappearance of the

melanophore-dispersing hormone from the blood of the fiddler crab Uca. Biol. Bull,

111: 313.
WATANABE, M. L, AND C. M. WILLIAMS, 1953. Mitochondria in the flight muscles of insect.

II. Effects of the medium on the size, form and organization of isolated sarcosomes.

/. Gen. Physiol, 37 : 71-90.
WELSH, J. H., 1955. Neurohormones. In: The hormones, 3: 97-151. Academic Press, Inc.,

New York.

THE METABOLISM OF STRONTIUM-90 AND CALCIUMS

BY LEBISTES

HAROLD L. ROSENTHAL

Division of Biochemistry, Department of Pathology, Rochester General Hospital,

Rochester, New York

That fishes accumulate mineral elements from the water in which they swim
and incorporate these elements into body tissues has recently been demonstrated by
the use of calcium-45 and strontium-89 in fresh water and marine fishes of various
species (Prosser et al., 1945; Rosenthal, 1956; Lovelace and Podoliak, 1952;
Boroughs et al., 1956; Alexander et al., 1956). The rate of incorporation of cal-
cium-45 into the total body and tissues is linear for Lebistes and Salmo sp. (Rosen-
thai, 1956; Lovelace and Podoliak, 1952), but bone and osseous tissues incorporate
the nuclide at a greater rate than either visceral organs or muscle. In Lebistes,
the loss of incorporated calcium-45 from the whole body may be described by at
least three separate first-order reactions varying from very fast to very slow, prob-
ably reflecting the rate of turnover of visceral organs, muscle, and osseous tissues,
respectively. In marine fishes, Boroughs et al. (1956) have shown that strontium-
89, placed in water, is rapidly incorporated into body tissues and the distribution in
tissues is similar to that following oral dosage of the nuclide. These investigators
also showed that the rate of excretion of a single oral dose of strontium-89 is rapid
during the first few days of the experiment. However, the isotope remaining in
the body after the first few days persisted at a constant level for a long time.

In view of the reports that small laboratory mammals (Alexander et al., 1956;
Comar et al., 1955), man (Turekian and Kulp, 1956) and marine fishes (Boroughs
et al., 1957) discriminate against strontium relative to calcium, and since strontium
is chemically similar to calcium, it was of interest to determine the uptake and turn-
over of strontium-90 by Lebistes and to compare this information with that previ-
ously obtained with calcium-45. The results of this study form the basis for this
report.

MATERIALS AND METHODS

Adult male wild-type guppies, averaging 125 mg. in weight, were obtained from
commercial sources. The experimental design and the treatment of animals and
tissues for analysis has been described in detail in a previous publication (Rosen-
thai, 1956).

All samples for radioactivity assay for strontium-90 were counted after a wait-
ing period of 20 days to permit equilibrium between strontium-90 and its yttrium-90
daughter nuclide. The samples were counted with a windowless gas flow counter
to less than a 5 per cent statistical error. Corrections for self-absorption of stron-
tium-90 were made when necessary but the 28-year half-life of this nuclide obviated
decay corrections. Assay of calcium-45 was performed as previously described,
with the same counting assembly.

442

Srno AND Ca4:' METABOLISM BY LEBISTES

443

No attempt was made to differentiate between strontium-90 and its yttrium-90
daughter, and the use of the term strontium-90 throughout this report refers to the
combined activities of strontium-90 and yttrium-90 at equilibrium. The efficiency
of the counter for strontium-90 and calcium-45 was determined to be 1.5 X 1010 and
1 X 109 counts per minute per millicurie, respectively. The strontium-90 and cal-
cium-45 were obtained from the Oak Ridge National Laboratories in the form of
carrier-free salts.

o> I

E

O
O

a.
o

0
I

LU 0
^

< 5

0

i

I

T

xlO'

A

xlO'

B

xlO'

0

10

15

DAYS

FIGURE 1. Uptake of strontium-90 by male Lebistes versus days in water containing the
isotope. Each point represents two to four fish. The water activity for Curve A, 1 X 108
cpm/ml., Curve B, 1.7 X 105 cpm/ml, Curve C, 1.1 X 104 cpm/ml.

RESULTS

The rate of uptake of strontium-90 by male Lebistes from the water in which
they swim was determined by placing the fish in glass aquaria containing 500 milli-
liters of aged tap water containing the isotope. Distilled water was added daily to
compensate for losses of water by evaporation and to maintain the isotope activity
of the water within ± 5 per cent during the experimental period.

444 HAROLD L. ROSENTHAL

The results obtained from these experiments demonstrate the rapid incorpora-
tion of strontium-90 into the body of the fish (Fig. 1). The incorporation is linear
during a 10- or 15-day experimental period for all concentrations of isotope thus
far studied, and the uptake of strontium-90 is similar to the data previously obtained
with calcium-45 (Rosenthal, 1956). The similarity between the uptake of stron-
tium-90 and calcium-45 is further shown by calculation of a "concentration factor"
which relates the logarithm of the rate of incorporation of the isotope in the body
of the fish to the logarithm of the activity of isotope in water (Table I). It is ap-
parent that the concentration factors for both calcium-45 and strontium-90 are sur-
prisingly similar within experimental error. These data are markedly different
from those obtained for marine fishes by Boroughs et al. (1957), who found that
Tilapia discriminate against strontium-89 relative to calcium. It is conceivable that

TABLE I

Relationship between rate of uptake and water activity for various nuclides

by the body of male Lebistes

Water
activity
Isotope cpm/ml. Concentration factor* ± S.E.

Strontium-90 8.25 X 10« 0.70 ± 0.007 (10)**

1.72 X 10B 0.72 ± 0.003 (16)

1.00 X 106 0.82 ± 0.004 (13)

Weighted Average 0.75

Calcium-45 8.52 X 10" 0.72 ± 0.007 (18)

9.42 X 104 0.80 ± 0.005 (20)

7.37 X 106 0.78 ± 0.010 (13)

Weighted Average 0.77

/ Uptake m count/mm, per 100 mg. per day \

* Concentration Factor = log I — ^ -1—. ; — : — — - — : — - } .

\ Water activity in count/mm, per ml. /

* The numbers in parentheses indicate number of animals analyzed.

/ S d2 V

S.E. = Standard error = I — I .

\ n(n — I)/

(n-1).

these differences are due to entirely different mechanisms involving the osmotic
physiology of fresh water and marine fishes.

The various organs of the body such as the spine, head, viscera, and muscle also
take up strontium-90 in a linear fashion during a 10-day experimental period, but
the rate of uptake differs for each organ, as shown in Figure 2. Highly mineralized
tissues such as the spine and head accumulate strontium-90 at a greater rate than
the total body while soft tissues (muscle and viscera) accumulate less of the isotope.
The accumulation of strontium-90 by the tissues of Lebistes is qualitatively similar
to that of calcium. Although the total body accumulates the same amount of
calcium-45 and strontium-90, the ratio of organ isotope concentration to total body
isotope concentration for strontium-90 differs significantly from that of calcium-45
in all of the organs studied (Table II). Thus it is apparent that the spine, head

Sr90 AND Ca15 METABOLISM BY LEBISTES

445

E
O

o

\

2

Q.
O

1.5

1.0

</> 0.5

LU

o

SPINE

I.5xl0-

HEAD 7.2xl03
TOTAL 5.9 xlO3

VISCERA I.6xl03
MUSCLE 3.0xl02.

0

DAYS

FIGURE 2. Uptake of strontium-90 by various tissues of male Lcbistcs versus days in water
containing 9 X 104 counts per minute per milliliter. Values for each tissue represent rate of up-
take of strontium-90 in terms of counts per minute per 100 milligrams per day. Each point
represents 6 values.

and viscera accumulate significantly more strontium-90 than calcium-45 on a con-
centration basis. Muscle tissue, on the other hand, tends to incorporate somewhat
less strontium-90 than calcium-45. A comparison of the total distribution of stron-
tium-90 and calcium-45 in the various tissues of the body following 10 days of up-
take of the isotope from water (Table III) is consistent with the data based on

TABLE II
Relative uptake of strontium-90 and calcium-45 by tissues of male Lebistes

Tissue

Strontium-90*

Calcium-45*

"t"

"P"

Carcass

1.00 ± 0.026** (16)***

1.00 ± 0.045**

(20)

—

—

Head

1.28 ±0.051 (18)

1.07 ± 0.039

(19)

3.29

<0.01

Viscera

0.96 ±0.118 (18)

0.59 ± 0.087

(21)

2.49

<0.02

Muscle

0.061 ± 0.020 (17)

0.102 ±0.024

(20)

3.95

<0.01

Spine

2.28 ±0.10 (15)

1.87 ±0.10

(16)

3.62

<0.01

The values represent the ratio

cpm/100 ing. tissue

cpm/100 mg. carcass
the water activity varied from 104 to 106 cpm/ml. for each isotope.

* Standard error.

* The numbers in parentheses indicate number of fish analyzed.

derived from 3 to 5 experiments in which

Students "t" value =

Mi - M2

/ sth2 + sd22 y

\Ni + X, - 27

[7 NtN2 V]
L\ Ni + No 7 J '

446

HAROLD L. ROSENTHAL

TABLE III

Distribution of strontium-90 and calcium-45 in tissues of male Lebistes
after 10 days in isotopic water

Tissue

Isotope distribution ± S.E.
(per cent of total)

"t"

"P"

Strontium-90

Calcium-45t

Carcass
Head
Viscera

100.0 ± 10.11 (13)*
29.9 ± 0.97 (13)
13.5 ± 1.87 (13)

100.0 ± 2.92 (15)*
21.3 ± 1.12 (14)
7.3 ± 0.48 (14)

5.71
3.34

<0.01
<0.01

Muscle***
Spine
Remainder**

2.8 ± 0.29 (12)
6.9 ± 0.61 (12)
46.9 ± 2.24 (12)

3.7 ± 0.31 (14)
6.2 ± 0.36 (14)
61.5 ± 2.32 (13)

2.12
1.02
4.36

<0.05
<0.01

" The numbers in parentheses indicate number of animals analyzed.
** Calculated by difference.

*** Muscle tissues estimated to comprise 40 per cent of body weight,
t From Rosenthal (1956).

concentration shown in Table II. The apparent discrepancy for the similarity be-
tween the distribution of calcium-45 and strontium-90 in the spine (Table III) and
the relative uptake of the two isotopes by the spine on a concentration basis is due,
in all probability, to our inability to always remove the entire spine from these small
fishes. This unavoidable error introduces some uncertainty into the distribution
data for the spines.

The head and "remainder" (skin, scales and fins) account for 77 per cent of
the total body strontium-90, while these tissues account for almost 83 per cent of
the total body calcium-45. Muscle contains the smallest proportion of the total
body strontium and calcium (2.8 and 3.7 per cent, respectively) while occupying
about 40 per cent of the total body weight. The spine, representing less than 3 per
cent of the body weight, contains between 6 and 7 per cent of the total calcium and
strontium nuclides, respectively. This comparison between the distribution of
strontium-90 and calcium-45, under the same experimental conditions, indicates
that the head and viscera incorporate, respectively, 25 per cent and 46 per cent
more strontium-90 than calcium-45, while muscle and the remaining tissues accu-

TABLE IV
Relative proportions of tissues of male Lebistes

Tissue

No. of
determinations

Per cent of body weight

Head

39

20.6 ± 0.22*

Viscera

38

12.3 ±0.37

Spine
Remainder**

21
39

2.8 ± 0.07
24.4 ± 0.25

Muscle

—

40.0 (estimated)

* Standard error.

* Remainder includes skin, fins, scales and is calculated by difference assuming that muscle
comprises 40 per cent of body weight.

Sr"° AND Ca43 METABOLISM BY LEBISTES

447

mulate about 25 per cent less strontium-90 than calcium-45. These differences in
tissue uptake of the two nuclides are statistically significant and further accentuate
subtle differences in the metabolism of these two elements.

Since the relationship of the weight of organs to body weight has not, to my
knowledge, been previously determined or published, for Lebistcs, the relative pro-
portions of the various tissues analyzed in this study are shown in Table IV.

150

LJ
O

o:
LU

Q_

O

o:

CO

o:
LJ

o

o:

ID

r-

0.5

0.3

DAYS

FIGURE 3. Turnover of strontium-90 by various tissues of male Lebistes versus days in
water containing no isotope. Each point represents 4 to 11 values obtained from 3 experiments.
©, spine: O, body: •. muscle: CD, head: ©, viscera. The fish contained about 104 cpm/100 mg.
on day zero of turnover.

The rate of turnover of strontium-90 by the body and tissues of Lebistcs was
determined by first placing the animals in isotope-containing water for 10 days in
order to incorporate sufficient radioactivity into the tissues. After this period the
fishes were transferred to isotope-free water which was changed periodically and
they were sacrificed at suitable intervals previously described in detail (Rosenthal,

448

HAROLD L. ROSENTHAL

1956). During a 50-day experimental period, the loss of strontium-90 from the
total fish could be resolved into two components that may be described by first
order reactions (Fig. 3). The first component, turning over rapidly with a bio-
logical half-life of about 8 days, represents loosely-bound strontium-90 in visceral
tissues of the body. This is somewhat longer than the three-day half-life for the
rate of turnover of the fast component with calcium-45 (Rosenthal, 1956). The
loss of strontium-90 by the viscera is extremely rapid, so that 92 per cent of the
radioactivity is lost during the first two days of the experiment. The second com-
ponent has an exceedingly long half -life of about two years or more. During a
similar experimental period with calcium-45 (Rosenthal. 1956) three components
with biological half-lives of 3 days, 137 days, and 309 days were apparent which
reflect the turnover rates of visceral tissues, muscle and carcass, respectively. The
absence of an intermediate component for strontium-90 is primarily due to the very
slow turnover rate of strontium-90 by muscle tissue, and to a lesser extent, the head.

TABLE V

Distribution of strontium-90 and calciinn-45 in tissues of male Lcbistes after
40-50 days of turnover in non-isotopic water

Isotope distribution ± S.E.

(per cent of total)

Tissue

"t"

"P"

Strontium-90 (SO days)

Calcium-45

(40 days)t

Carcass

100.0 ±

11.2 (5)*

ibo.o ±

5.48 (8)*

—

—

Head

42.7 ±

5.16 (5)

34.4 db

2.08 (7)

1.69

>0.15

Viscera

0.2 ±

0.06 (5)

0.5 ±

0.04 (5)

2.12

>0.05

Muscle***

3.0 ±

0.87 (5)

2.6±

0.46 (5)

0.56

NS

Spine

21.0 ±

4.47 (5)

19.4 ±

1.04 (7)

0.41

NS

Remainder*

33.1 ±

7.79 (5)

43.1 ±

2.38 (6)

1.33

>0.20

* The numbers in parentheses indicate number of animals analyzed.
** Calculated by difference.

* Muscle tissue estimated to comprise 40 per cent of body weight.
f From Rosenthal (1956).

It is of interest to note that muscle tissue strontium-90 with a biological half-life
of about two years as calculated from the last 25 days of the experiment, is lost in a
manner similar to that of the total body. This is in contrast to the biological half-
life for muscle of 137 days as determined previously for calcium-45, and it would
appear that the metabolism of the two elements differs in muscle tissue. The ex-
ceptionally slow turnover of strontium-90 in muscle tissue of marine fishes has re-
cently been observed by Boroughs ct a!. ( 1956).

The spine, which consists not only of mineral matter but also of intervertebral
cartilage, tendon, and organic bone matrix, continues to incorporate the isotope for
about 10 days after the fish is placed in isotope-free water. The additional nuclide
must be derived from a redistribution of isotope from soft tissues such as viscera.
Similar data were obtained with calcium-45 (Rosenthal, 1956). The accumulated
isotope does not remain fixed in the spine, however, but is subsequently lost and a
new equilibrium consistent with that of the mineral component of bone becomes

Sr"' AND LV:> METABOLISM BY LKBISTKS 449

established. The biological half-life of strontium-90 in the spine, calculated during
the last 25 days of the experiment, may be estimated to exceed two years, a value
consistent with the biological half-life of calcium-45 previously determined (Rosen-
thai, 1956). The additional increase of the strontium-90 and calcium-45 in the
bone and its relatively rapid loss may represent a rather labile binding-site (T1/; =
about 50 days) for bone formation.

The distribution of strontium-90 in various organs and tissues of the body after
50 days in isotope-free water is compared with the distribution of calcium-45 after
40 days in isotope-free water (Table V). It is interesting to note that the distri-
butions of both isotopes at the end of 40 days of turnover for calcium-45 and 50
days for strontium-90 are not significantly different. A comparison of the rate of
turnover of strontium-90 by the tissues, shown in Figure 3 of this report, with the
rate of turnover of calcium-45 previously published (Rosenthal, 1956) indicates
that the similarity of distribution of both isotopes at these particular time intervals
is coincidental. Extrapolation of the turnover rates for both isotopes indicates that
the head, muscle and spine would retain a greater proportion of the body strontium-
90, while the viscera and "remainder" would contain less strontium-90 throughout
the life of the fish.

DISCUSSION

It is apparent from these studies that fresh water fishes accumulate strontium-90
from the water in which they swim and that the rate of uptake is similar to that of
calcium-45. Moreover, we have recently shown that the rate of uptake of stron-
tium-90 and calcium-45 by other fresh water fishes (Danio and Tanichthys} is simi-
lar to the data we have obtained with Lcbistcs. These data differ from the studies
of Boroughs ct al. (1957), who found that marine fishes discriminate against stron-
tium-89. This apparent disagreement may be due to marked differences in osmotic
regulation between marine and fresh water fishes. On the other hand, discrimina-
tion of strontium isotopes relative to calcium by small laboratory mammals appears
to be well documented (Comar ct al., 1955 ; Turekian and Kulp, 1956; Comar ct al.,
1956). Comar ct al., (1956) have indicated that the processes of major discrimina-
tion, in rats, are decreased absorption of strontium from the intestinal tract and in-
creased urinary strontium excretion, processes which cannot be measured directly
in small fishes. These two processes would tend to limit the quantity of strontium
entering the body and its retention in the body, but in fishes, the gills play a major
role for the absorption and excretion of mineral elements. The similarity of the
"concentration factor" (Table I) for both nuclides by the total body of the fish in-
dicates no discrimination of strontium-90 by these fishes. It is possible that subtle
differences between strontium-90 and calcium-45 uptake by fishes may become ap-
parent by the use of differential methods in which both nuclides are present in the
same medium. These and other aspects of the problem are under investigation.

The rate of excretion of strontium-90 by the total body and tissues (except vis-
cera) of Lcbistcs is slower than that of calcium-45. The data appear to be in con-
trast with the report by Lengeman (1957), who showed that rat bones, in vitro,
lose more strontium-90 than calcium-45. Since the accumulation and retention of
mineral elements into bone depend on the rate of bone formation and sequestration
of elements into slowly exchanging bone matrix, comparisons between such diverse
biological systems may be hazardous. Nonetheless, tissues of Lcbistcs rich in cal-

450 HAROLD L. ROSENTHAL

cium, such as the spine and head, accumulate and retain a -larger percentage of the
total body strontium-90 than that of calcium-45, in accord with the studies of Lenge-
man (1957) and Comar ct al. (1956) for rats. Although no explanation is offered
at this time concerning the mechanism of incorporation of alkaline earth elements
from water by fishes, the similarity of uptake of calcium-45 and strontium-90 by
fresh water fishes (Rosenthal, 1957) indicates a fundamental and essentially simi-
lar process.

I am grateful to Atari Lou Pfluke, Helen Cundiff and Paul Myers for their
technical assistance and to E. Pfluke, Pfluke's Aquarium and Pet Shop, Rochester,
N. Y. for animals and supplies. This study was aided by a grant, contract No.
AT (30-1)-1712, from the Atomic Energy Commission.

SUMMARY

1. The uptake of strontium-90 by male Lcbistcs from the water in which they
swim is linear with time for the total carcass and tissues studied. Tissues con-
taining high concentrations of calcium accumulate more strontium-90 than soft
tissues. The rate of turnover of the nuclide varies from very fast to very slow ac-
cording to the type of tissue. The whole body, head and spine retain strontium-90
for long periods of time (T1^ == 600 days) \vhile viscera loses the isotope rapidly
(Ti/2==Sdays).

2. A comparison between strontium-90 and calcium-45 uptake and turnover by
male Lchistcs are qualitatively similar but significant quantitative differences are
apparent.

LITERATURE CITED

ALEXANDER, G. V., R. E. NUSBAUM AND N. S. MACDONALD, 1956. The relative retention

of strontium and calcium in bone tissue. /. Biol. Chan., 218: 911-919.
BOROUGHS, H., S. J. TOWNSLEY AND R. W. HIATT, 1956. The metabolism of raclionuclides by

marine organisms. I. The uptake, accumulation, and loss of strontium*9 by fishes.

Biol. Bull., Ill: 336-351.
BOROUGHS, H., S. J. TOWNSLEY AND R. W. HIATT, 1957. The metabolism of radionuclides

by marine organisms. III. The uptake of calcium45 in solution by marine fish.

Limnology and Oceanography, 2 : 28-32.
COMAR, C. L., I. B. WHITNEY AND F. W. LENGEMAN, 1955. Comparative utilization of dietary

strontium-90 and calcium by developing rat fetus and growing rat. Proc. Soc. E.rper.

Biol. Mcd.. 88: 232-236.
COMAR, C. L., R. H. WASSERMAN AND M. M. NOLD, 1956. Strontium-calcium discrimination

factors in the rat. Proc. Soc. E.rpcr. Biol. Mcd., 92: 859-863.
LENGEMAN, F. W., 1957. Comparative metabolism of strontium -89 and calcium-45 by bone

grown in ritru. Proc. Soc. E.rpcr. Biol. Mcd., 94: 64—66.
LOVELACE, F. E., AND H. A. PODOLIAK, 1952. Absorption of radioactive calcium by brook

trout. Proc/. Pish. Cult., 14 (4) : 154-158.

PROSSER, C. L., W. PERVINSEK, J. ARNOLD, G. SVIHLA AND P. C. TOMPKINS, 1945. Accumula-
tion and distribution of radioactive strontium, barium-lantharnum, fission mixture and

sodium in goldfish. U. S. Atomic Energy Coinin. Tech. Inform. Serv. MDDC.-496.
ROSENTHAL, H. L., 1956. Uptake and turnover of calcium-45 by the guppy. Science, 124: 571-

574.
ROSENTHAL, H. L., 1957. Uptake of calcium-45 and strontium-90 from water by freshwater

fishes. Science, 126: 699-700.
TUREKIAN, K. K., AND J. L. KULP, 1956. Strontium content of human bones. Science, 124:

405-406.

THE MOLTING CYCLE OF THE SPINY LOBSTER, PANULIRUS

ARGUS LATREILLE. IV. POST-ECDYSIAL HISTOLOGICAL

AND HISTOCHEMICAL CHANGES IN THE HEPATO-

PANCREAS AND INTEGUMENTAL TISSUES 1

DOROTHY F. TRAVIS -

Bermuda Biological Station 3 and The Biological Laboratories, Harvard University and

Radcliffe College, Cambridge 36, Mass.

Following molt, the major tasks which confront the crustacean are growth of
the soft tissues and the continued accretionary growth and hardening of the skeleton
by deposition of mineral salts therein. In spiny lobsters of 80-89 mm. carapace
length, weight stability is not achieved until 28-35 days following molt (late Stage
C) (Travis, 1954). This is a period at which the skeleton is fully hardened, water
content is normal, and presumably growth of the tissues is fairly stable. During
the early postmolt period, however, when rapid accretionary growth and calcifica-
tion of the skeleton are occurring, marked changes are observed in the hepato-
pancreas and integumental tissues. Accordingly, the present paper will be con-
cerned with those marked changes in the hepatopancreas and integumental tissues
which occur concomitantly with the development and calcification of the post-exuvial
layers of the skeleton.

MATERIALS AND METHODS
Animals

Male and female spiny lobsters ranging in carapace length from 80—89 mm. were
obtained and handled as previously described (Travis, 1954).

Designation of stages in the molting cycle

Stages of the molting" cycle were designated by time intervals, in days, as previ-
ously described (Travis, 1955a) and by the method of Drach (1939). For
Pan H lints argns. Stage A through C encompasses a period of approximately 51 days
during the summer months. Stage A. immediately following molt and the stage
in which the principal layer begins to be deposited, has a duration of approximately
24 hours or one day. Stage B, beginning calcification, continued thickening of the
principal layer and preliminary hardening of the skeleton, is approximately six days

1 This work was supported in part by an E. L. Mark Fellowship from Harvard Uni-
versity, a Mees Fellowship from the Bermuda Biological Station, and an Atomic Energy Com-
mission Pre- and Postdoctoral Fellowship. The author wishes to express her sincere apprecia-
tion to Dr. John H. Welsh, Dr. E. E. Mortensen, and Dr. A. B. Dawson for their most helpful
criticism and suggestions regarding this manuscript.

2 Present address : Department of Zoology, University of New Hampshire, Durham, New
Hampshire.

3 Contribution No. 233.

451

452

DOROTHY F. TRAVIS

FIGS. 1-6.

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 453

in length, existing from two through seven days following molt. Stage C, a stage
in which the principal layer and the new membranous layer are completed and in
which the skeleton is completely hardened, is the longest period of the molting cycle
(44 days), existing from approximately the eighth day through the fifty-first day
following molt.

Histological and histochemical methods

For the histological and histochemical studies, pieces of integument and integu-
mental tissues were removed from the carapace of Panulirus (see Figure 1 ; Travis,
1955a) on each of eight consecutive days following molt. Likewise the right pos-
terior lobe of the hepatopancreas was removed on each of seven consecutive days
following molt. Tissues from three animals were used to represent each of these
days with the exception of the first, fourth, and eighth day following molt for the
integumental tissues. In these cases, tissues from one animal were used.

Most integumental tissues were embedded in celloidin and cut at 10 p.. The pos-
terior lobe of the hepatopancreas was embedded in paraffin and cut at 8 /JL, with the
exception of hepatopancreatic tissues fixed and embedded for lipid detection.

Portions of the integument and hepatopancreas fixed in Helly's and alcoholic
Bouin's fluid were stained by the following methods :

1. Mallory's triple stain

2. Periodic acid Schiff (PAS) of McManus, as described by Lillie (1948)

3. Bensley and Bensley's method (1938), for demonstrating muco- or glycoprotein

by means of toluidine blue (see Travis, 1955a).

For detection of calcium deposits, portions of the skeleton were fixed in nine
parts of 95% alcohol and one part of 40% formaldehyde, and were stained with
the following :

1. Mallory's triple stain

2. Schmorl's purpurin (Lillie, 1948)

FIGURE 1. A reserve cell, "mulberry"-like in appearance, of the sub-epidermal connective
tissue from an animal three days following molt. Note lumpy or stainable balls of material
(arrow) of mucopolysaccharide as well as calcium. 800 X.

FIGURE 2. Large oval reserve cells of the hepatopancreas showing the presence of large
vacuoles (arrows), some of which contain flaky or granular-like stainable material while
others appear clear, a condition observed from five through seven days following molt and
possibly correlated with a marked decrease in mucopolysaccharide and calcium. 800 X.

FIGURE 3. X-ray diffraction photograph taken of dry powder obtained from triturated
pieces of the area of softening. The presence of calcite lines reveals that calcium carbonate is
present in the spiny lobster skeleton as calcite, not aragonite or amorphous calcium carbonate.

FIGURE 4. Glycogen distribution in the epidermis of the outer integument and sub-epidermal
connective tissue at one day postmolt. Note small number of glycogen granules localized in
the proximal half of the outer epidermal cells. At this same time abundant amounts are
concentrated at the bases of the epidermal cells of the inner integument. 760 X.

FIGURE 5. Note that at two days postmolt glycogen has completely disappeared from the
epidermis of the inner integument and is not observed again in this tissue during the entire
postmolt period. 760 X.

FIGURE 6. The heavy concentration of glycogen observed in the distal half of the outer
epidermal cells during a period of two through four days following molt. Little glycogen at
this time is present in the sub-epidermal connective tissue. 760 X.

454 DOROTHY F. TRAVIS

3. Alizarin red S (Manigault, 1939)

4. Von Kossa's method (Lillie, 1948). Before following this procedure, tissues

were washed in 5% aqueous KNO3 for five minutes or more to remove the
chloride present. With Von Kossa's method, the silver from the silver ni-
trate is precipitated as phosphate on the surface of calcium phosphate granules.
The silver phosphate is reduced in the presence of light to metallic silver,
forming a black crust on the surface of calcium phosphate granules. Mallory
(1942) states that calcium carbonate granules become coated with silver
carbonate, which in sunlight gives off CO2 and leaves a black silver oxide on
the surface of these granules. These reactions may not occur in the presence
of organic substances (Lison, 1953) nor in the presence of quantities of chlo-
ride (Lee, 1946).

Parts of the epicuticle, pigmented layer and principal layer appear either
black or brown with Von Kossa's method. Cameron (1930) pointed out that
the most recent calcium deposits in teeth appear dark brown by this method,
while the older calcified layers appear lighter brown. Some calcified areas,
he noted, did not stain at all. It should be pointed out that this method has
the great advantage over alizarin and purpurin in that it enables one to visu-
alize calcium deposits in granular form. It is, therefore, excellent for the de-
tection of skeletal deposits whereas the latter methods are not.

5. Microincineration (Scott, 1933) was used to confirm the presence of calcium de-

posits detected by the stains mentioned above. The white calcium ash vmder
dark-field illimination appeared in the same areas as indicated by the stains.
To confirm this as being calcium ash, the gypsum test was used.

For detection of calcium in the hepatopancreas, tissues were fixed in the
same fashion as the integumental tissues and were stained with alizarin red S.

For detection of alkaline phosphatase, integumental and hepatopancreatic tis-
sues were fixed in cold 80% alcohol, and embedded in paraffin. Alkaline phospha-
tase was determined by the method of Gomori ( 1941 ) . Control sections were made
using the incubating medium without added substrate. This method is extremely
useful for the detection of calcium deposits. In control sections which do not
show the presence of alkaline phosphatase, calcium deposits, if present, show up
remarkably well.

Only the hepatopancreas was used for the detection of lipids. Portions of the
tissue, in this case, were fixed in 10% neutral formalin and were imbedded in car-
bowax (method of Blank and McCarthy, 1950), cut at 10 and 15 ^ and stained for
lipids with Sudan black B.

OBSERVATIONS

THE POSTMOLT ANIMAL (STAGE A AND B)
1. The integument and integumental tissues
a. Tissues

During the early post-ecdysial period (Stage A and B) pieces of exoskeleton
with attached integumental tissues were removed from the lateral portion of the
carapace (see Fig. 1; Travis, 1955a). The lateral portions of the carapace of

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 455

Crustacea, as one will recall, are folded in such a way that there is an outer epi-
dermis and integument, (the outer integument being in contact with the surround-
ing sea water) as well as an inner epidermis and integument (the inner integument
facing the gills, in contact with the sea water in the branchial chamber). Sections
of the exoskeleton with attached integumental tissues indicate that the epidermal
cells of the outer integument remain extremely long and attenuated and indeed fibril-
lar in nature. This condition is similar to that observed in the late premolt ani-
mal. The markedly fibrillar nature of the outer epidermis, however, is apparent
in all stages of the molting cycle. Nuclei in these epidermal cells of the outer
integument are central (Fig. 4) whereas those of the inner integument are more
distal in location (Fig. 5). The inner epidermal cells, also somewhat fibrillar in
nature, remain about half the length of the outer epidermal cells during all stages
of the molting cycle (see figures from Travis, 1955a).

Both the outer and inner epidermis, during the early postmolt period (Stage A
and B). show a gamma metachromasia (pink-purple) with toluidine blue, indicating
the presence of a glyco- or mucoprotein. The presence of phosphatase, glycogen,
and calcium in these tissues during the early post-molt period will be discussed in a
subsequent section of this paper.

As was pointed out (Travis, 1955a), the sub-epidermal connective tissue is of
a loose spongy type.

The large oval reserve cells, described as "protein cells" by Cuenot (1893) and
resembling Leydig Cells, Type I (Kiikenthal, 1926-1927). constitute by far the
most prominent and most interesting cell types within this sub-epidermal connec-
tive tissue. These reserve cells vary greatly in structural appearance during the
molting cycle. When storing reserves they become greatly swollen and may take
on a "mulberry" appearance (Fig. 1). When devoid of reserves they may de-
crease in size with their vacuoles becoming clear or containing flaky or granular-
like stainable material. Since the reserve cells are found within the tissue spaces
among other Leydig cells, they should not, perhaps, be considered as permanent
structures within this tissue. This has become somewhat clearer from the work
of Sewell (1955), in which he points out that the origin of these reserve cells,
which he calls "lipo-protein cells," in Carcinns is from amoebocytes and that pos-
sibly they revert to amoebocytes following molt. This suggestion could account
for their cyclic peaks and declines in size and abundance, and changes in structural
appearance at daily intervals during the early postmolt period of Paniilints, as indi-
cated below.

For the sake of comparison, the reserve cells in intcrmolt animals (late Stage C)
constitute the most prominent cell-types within the connective tissue. They are
large oval cells, vesicular in nature, with a capsule-like envelope of cytoplasm and
a peripheral nucleus (see figures; Travis, 1955a). They range in size from 24-
51 /x with an average size of 32 p.. After alcoholic Bouin's fixation they stain blue-
gray with Mallory's triple stain, the vacuoles in this case containing blue-gray flaky
or granular-like material ; with the PAS method the entire cell is a deep pink-
purple color ; with toluidine blue these cells stain either blue-gray or green-gray.
After A-F (alcoholic formaldehyde) fixation they similarly stain blue-gray with
Mallory's triple stain, but do not stain with alizarin red S, purpurin or the Von
Kossa method. With these three latter stains the reserve cells could be easily
overlooked.

456 DOROTHY F. TRAVIS

Similarly, for the sake of comparison reserve cells in premolt animals (Stage D)
range in size from 30-51 p. with an average size of around 40 /JL. Structurally, they
maintain their oval appearance but stainable material within the cells is lumpy and
might well be described as consisting of rather discrete spheres (Fig. 1). After
alcoholic Bouin's fixation, the cells again stain blue-gray with Mallory's but do not
show clearly the speres of stainable material ; with PAS they again stain deep pink-
purple and show clearly the discrete balls of material ; with toluidine blue the balls
of material are yellow-green in color and refractile in nature. Following A-F fixa-
tion, the cells again fail to stain with alizarin red S, purpurin and the Von Kossa
method.

From one through seven days following molt ( Stage A and B ) , the reserve cells
appear to undergo cyclic peaks and declines in size and abundance, and the storing
of reserves.

At one dav following molt (Stage A) the reserve cells remain approximately
the same size as those observed in the premolt animal, a range in size from 32-
48 p. and an average size of 36 p.. However, there would appear to be a slight de-
crease in number. Following alcoholic Bouin's fixation, they stain in much the
same fashion as that observed in the premolt animal, although the spheres of stain-
able material are not as apparent. They fail to stain, following A-F fixation, with
the same stains mentioned in the premolt animal.

On the second dav following molt (beginning of Stage B) the reserve cells
have greatly decreased in number and size. They range in size from 12-29 p. with
an average of around 19 p.. When observed, their vacuoles are clear and vesicular,
lacking the lumpy balls of material, with the exception of very small spheres at their
periphery. They stain in a similar manner to those observed on the first day with
the exception of the fact that a few show a very small number of calcium granules
after the Von Kossa method.

On the third day the reserve cells again are present in great numbers, compara-
ble to the condition observed in the premolt animals. They range in size from
32^1-5 p. with an average size of around 38 p., which compares favorably with the
average size observed in the pre- or intermolt animal. The reserve cells at this
time take on an irregular "mulberry" appearance by enclosing large stainable spheres
of material within their vacuoles (Fig. 1). Further, their staining properties
change markedly. Following Bouin's fixation, they again stain blue-gray with
Mallory's, deep pink-purple with PAS and yellow-green with toluidine blue.
Following A-F fixation, they stain for the first time, a brilliant orange-red with
Mallory's scarlet with alizarin red S and purpurin, and yellow-brown with Von
Kossa's method. This indicates that not only is muco-polysaccharide, possibly
muco- or glycoprotein, bound by these cells at this time but that they are filled
with calcium, which is distinctly apparent with stains used for this purpose.

Interestingly enough, by the fourth da\< the reserve cells are hardly apparent.
The large stainable balls of material are lacking and the few cells present are smaller
in size, ranging from 19—29 p. with an average size of about 26 p.. In the few ap-
parent cells, there is little evidence of either mucopolysaccharide material or calcium.
On the fifth day, these cells again reach a peak in abundance and size. With
all stains and fixatives used, they are similar in every way to the three-day condition,
with the exception of the fact that the mucopolysaccharide present stains only
faintly with PAS, possibly indicating a decrease in concentration of the muco-

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 457

polysaccharide material or the unavailability of its reactive groups to PAS. The
cells range in size from 29-42 ^ with an average of around 34 /j., are "mulberry-
like" in appearance, and are again filled with calcium which shows up after appropri-
ate fixation and staining.

By si.r days, the cells are fewer in number, somewhat smaller (range 17-27 /j.,
average 23 /.i), but similar in staining properties to the fifth-day condition.

On the seventh day (end of Stage B) the cells are few in number but are some-
what larger in size (35-40 /A, average about 37 //.), and are detected after Bouin's
fixation and toluidine blue staining and following A— F fixation and Mallory's. ali-
zarin red S. purpurin, and Yon Kossa's method, indicating again that these cells
are loaded with calcium.

By eight days following molt (beginning of Stage C), the reserve cells are again
not apparent.

It is, therefore, evident that the reserve cells even within the early postmolt pe-
riod. Stage A and B, undergo, at daily intervals, cyclic peaks and declines in size
and abundance, changes in structural appearance, and staining properties. The
polysaccharide material which is distinctly evident up to the fifth day following
molt is always diastase-fast and colors deep pink-purple with PAS but does not show
gamma metachromasia with toluidine blue. Pearse ( 1953) has suggested that poly-
saccharide material staining in this way with PAS and frequently failing to show-
gamma metachromasia with toluidine blue probably indicates the presence of either
a muco- or glycoprotein. As has been mentioned previously (Travis, 1955a) the
reserve cells of late Stage C and Stage D animals stain with PAS in the same man-
ner. This polysaccharide material probably represents reserve substances for the
new skeleton and may. as was pointed out by Travis (T955a), during the premolt
period represent breakdown products from the old skeleton.

None of the integumental tissues of Panulirns, unfortunately, wrere fixed for the
detection of lipicls. As was pointed out by Travis (1955a) the reserve cells within
the connective tissue of the hepatopancreas contain much lipid and it would like-
wise be expected that the reserve cells of the sub7epidermal tissue also store it.
Sewell (1955) has definitely shown that these reserve cells beneath the connective
tissue of Ca rein us do indeed store lipoprotein. These lipoprotein reserves reach
a maximum in C4 (late Stage C) and early D (Dt) and then begin to decrease, ap-
parently as lipid content of the epidermis increases. These cells, as Sewell sug-
gests, begin to disappear after the pre-exuvial layers of the skeleton are deposited
and completely disappear by the end of Stage B. However, reserve cells of
Pannlints become filled with calcium on the third, fifth, sixth, and seventh day fol-
lowing molt whereas on the first, second, and eighth day either no calcium or very
little was apparent in the reserve cells. This would suggest to the present author
cyclic peaks in calcium storage alternating with cyclic release to the epidermis as
calcification of the skeleton occurs. The present author would expect this cyclic
process (peaks and declines in size and abundance, changes in structural appearance,
and calcium binding and release) to continue throughout early and middle Stage C,
since calcification of the skeleton is not fully completed for at least three weeks fol-
lowing molt. If, therefore, the reserve cells arise from amoebocytes, as Sewrell
(1955) suggests, and possibly revert to amoebocytes after they have discharged
their reserves, such a situation could clearly account for the cyclic peaks in size
and abundance at varying daily intervals within a single stage of the molting cycle.

458 DOROTHY F. TRAVIS

In this sense, the present author would be inclined to accept Sewell's suggestion
that the reserve cells represent phases of activity of the amoebocyts with peaks not
only before molting, as Sewell suggests, but following molt as well. The reserve
cell cycle would then correspond more closely with the oenocyte cycle, being present
throughout the molting cycle but reaching peaks at various phases of it. It might
be pointed out that days in which reserve cells are scarcely apparent (2, 4, and 8
days following molt), large numbers of amoebocytes are apparent in the sub-epi-
dermal connective tissue.

b. The integument

During the early postmolt period ( Stage A and B ) the post-exuvial layers of the
skeleton are deposited. Of the post-exuvial layers of the outer integument, only
the principal layer or calcified zone is progressively thickened during Stages A
and B.

The amount of skeletal material, in total thickness, deposited per day, in the
area of carapace from which sections of the integument were cut, during Stages A
and B, varies from 14-72 ^ with an average of around 38 /*. The thickness of the
principal layer when fully formed varies from around 460-550 ^ in animals used
in this investigation (80-89 mm. carapace length). If one assumes that a con-
stant amount of skeletal material is deposited daily in the principal layer, for exam-
ple 38 p., one can, by using this figure and the total thickness of the fully formed
principal layer, roughly calculate the time in days when this layer is completed.
By using these values, the calculated time at which the principal layer is completed
would be around 15 days following molt. This time, however, is in actuality closer
to 20 days following molt. Therefore, the membranous or non-calcified layer would
not apparently begin to be deposited before the third week following molt (Stage
C. of Drach, 1939). By the fourth week following molt (late Stage C or C4 of
Drach) the membranous layer is fully formed (Travis, 1955a).

The pigmented layer (toluidine blue-staining) shows the presence of a muco-
or glycoprotein one through five days following molt but by the end of this period
the tinctorial properties of this layer have decidedly changed. A deep purple
rather than a pink-purple is given with this stain, indicating that the properties of
the protein and closely associated chitin units of the pigmented zone have been
changed by quinones. The newly formed principal layers initially show a green
coloration with toluidine blue but shortly take on a pink coloration. The mem-
branous layer, when fully formed, is light green in color with toluidine blue, pos-
sibly indicating differences in the organic nature or composition of this and the
principal layers.

The inner integument, bordering the gill chamber, undergoes little if any thick-
ening after the second day following molt. It attains a total thickness of approxi-
mately 30 /A, that of a late Stage C animal, by the second day following molt, indi-
cating that the inner integument is completed during a period of three days preced-
ing molt and two days following molt (Travis, 1955a).

As the post-exuvial layers are deposited during the early postmolt period ( Stage
A and B), concomitant hardening of these layers occurs by the deposition of mineral
salts therein. It is evident from the analyses of small pieces of the area of soften-
ing (Travis, 19551) ) that the most abundant mineral constituent in the exoskeleton

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 459

is calcium. In order to determine what salts were deposited in the skeleton, further
chemical analyses were carried out on the entire area of softening from late Stage
C animals. The results of these analyses are indicated in Table I. From these
results it is apparent that most of the calcium present in the skeleton is in the form
of calcium carbonate. In order to determine whether this is deposited in the or-
ganic matrix of the skeleton as amorphous calcium carbonate or as crystalline arago-
nite or calcite, x-ray diffraction photographs (kindly made by Dr. C. Frondel, De-
partment of Minerology, Harvard University), were taken of dry powder obtained
from triturated pieces of the area of softening. These photographs (Fig. 3) indicate
that calcium carbonate exists in the spiny lobster skeleton as calcite.

Since the basic organic components of the crustacean exoskeleton are chitin and
protein, which are firmly associated with one another, Trim (1941), Stacy (1943)
and Haworth (1946) regard the arthropod cuticle as a mucopolysaccharide because
of the firm combination of carbohydrate-containing amino sugars (chitin) with the
protein. Further, since calcium is the most abundant, if not the most important,
mineral constituent within the crustacean skeleton, an emphasis will be placed on
the abundance and distribution of glycogen, phosphatase, and calcium in the integu-
mental tissues. These three constituents, among others, are of extreme importance
in the development and calcification of the new skeleton.

c. Localization of glycogen, phosphatase and calcium

(Hvcogen: At one dav following molt (Stage A), glycogen granules are scat-
tered throughout the sub-epidermal connective tissue. Abundant amounts are
concentrated at the bases of epidermal cells of the inner integument while the epi-
dermal cells of the outer integument show little glycogen, which is localized in the
proximal half of the cells ( Fig. 4).

On the second day following molt, glycogen completely disappears from the
inner epidermis (Fig. 5) and is not observed again in this tissue during the entire
postmolt period. From two through four days following molt, little glycogen is ap-
parent in the sub-epidermal connective tissues. Much heavier concentrations, on
the other hand, are observed in the distal half of the outer epidermal cells (Fig. 6).
By the fifth day glycogen accumulates in large amounts in the sub-epidermal tis-
sues while the outer epidermis becomes almost depleted of it (Fig. 7). From the
si.vth through the seventh day most of the glycogen disappears from the sub-epi-
dermal connective tissue and moves again to the outer epidermis where it is heavily
concentrated in the distal portion of this tissue.

Phosf>hatase : Alkaline phosphatase becomes localized in the outer epidermis,
being more heavily concentrated in the distal rather than the proximal half of the
cells. Furthermore, the enzyme is heavily concentrated in the integument immedi-
ately above the epidermis (Fig. 8). It appears to be concentrated at this site in
the region of the proximal portions of the innumerable pore canals. The localiza-
tion of phosphatase in these sites is evident on the first day following molt, before
calcification of the branchial integument begins, and remains in this localization
throughout Stages A, B and very early C. In addition to its presence at these sites
alkaline phosphatase is observed rather evenly distributed in the reserve cells during
the entire period of observation. Control treated sections indicate that not only
the enzyme but calcium as well are present in all of these sites from the second

460

DOROTHY F. TRAVIS

*." 0

o

FIGS. 7-12.

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 461

through the eighth day, although purpurin and alizarin red S did not show the
presence of calcium within the reserve cells hefore the third day following molt. It
might also he pointed out that the enzyme is likewise heavily concentrated around
newly developing bristles.

Calcium: Calcification of the new outer integument of the branchiostegites begins
on the second day following molt (Stage B). Before calcification begins, however,
the epidermis begins to concentrate calcium in markedly evident amounts (detectable
by purpurin. alizarin red S, Von Kossa's method, Gomori's (1941) method for
alkaline phosphatase and confirmed by microincineration) . Before discussing the
calcification of the skeleton the author would like to point out that of these methods
used for the detection of calcium. Von Kossa's method and Gomori's (1941)
method for alkaline phosphatase are the most useful for showing actual stages in
calcification of the integument. Furthermore, these two methods show calcium
salts or complexes in granular form.

At one day following molt and one day before calcification of the integument
begins, the distal ends of the epidermal cells begin to show calcium. No reserve
cells of the integumental tissues, as will be recalled, bind calcium at this time.

By the second day, the distal portions of the epidermal cells are filled with
calcium. The tissue spaces or sinuses at the base of the epidermis and the blood
channels, sometimes observed between the epidermal cells, show the presence of
calcium. Although only a few reserve cells are apparent, a few show scattered
granular deposits of calcium. At this same period a narrow band of calcium gran-
ules appears in the new principal layer immediately distal to. but paralleling, the
epidermis (Fig. 9). Likewise, immediately underlying the epicuticle and within
the pigmented zone a heavy concentration of rows of granules is observed.

By the third day the epidermal cells as well as the reserve cells of the sub-epi-
dermal connective tissue are completely filled with calcium (Figs. 10. 11) detected
by all methods used for this purpose. The heaviest concentrations of calcium gran-
ules in the epidermis are observed in the distal half of the cell. Here they are ob-
served to be extruded from the distal ends of the epidermal cells in two distinct ways
(Travis, 1951a, 1951c). As the post-exuvial layers are deposited, masses of cal-

FIGURE 7. At five days following molt glycogen has disappeared from the outer epidermis
but is heavily concentrated by the sub-epidermal connective tissue. 760 X.

FIGURE 8. One day postmolt, before calcification begins in the branchial integument.
Localization of alkaline phosphatase in the distal portion of the outer epidermis and in the
integument immediately above the epidermis (region of the proximal portions of the innu-
merable pore canals, arrows). Phosphatase remains concentrated here through Stages A. B
and early C. 420 X.

FIGURE 9. At two days postmolt calcification of the integument begins. Note that the
distal portions of the outer epidermal cells (E) are filled with calcium and the narrow band
of calcium granules (arrow) which appear in the newly forming principal layer immediately
distal to, but paralleling, the epidermis. 2000 X. Van Kossa's method.

FIGURE 10. By the third day postmolt the epidermal cells (arrow) and the reserve cells
(arrow) of the sub-epidermal connective tissue are filled with calcium. 90 X.

FIGURE 11. Higher magnification showing that the epidermal cells (E) and reserve
cells (R) of sub-epidermal connective tissues, at three days postmolt, are filled with calcium.
Note lumpy stainable balls of material in the reserve cells. 760 X. Alizarin red S staining.

FIGURE 12. As the post-exuvial layers are deposited ( three days following molt ) masses of
calcium granules are simultaneously extruded from the epidermal cells (E) to form narrow
calcified bands paralleling the epidermis (arrow). 2000 X. Van Kossa's method.

462

DOROTHY F. TRAVIS

FIGS. 13-18.

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 463

cium granules are simultaneously extruded to form narrow bands paralleling the
epidermis (Fig. 12). In the pre-exuvial layers of the skeleton, deposited before
molt, calcium granules are observed in uniform vertical rows. These rows of
granules are particularly evident in the pigmented layer (Fig. 13) and can be seen
to emanate from the epidermis (Fig. 14). The rows of granules correspond in lo-
cation to the pore canals or vertical striae. Calcification of the pre-exuvial layers
occurs after their formation. Hence, calcium has to be transported and deposited
some distance from the epidermis. This transport, therefore, appears to occur
through the pore canals.

From four through scz'cn cla\s calcium in the epidermis remains equal in amount
to that observed on the third day.

Calcification continues to occur as the post-exuvial layers are deposited. As a
consequence, the horizontal bands of granules paralleling the epidermis have thick-
ened considerably by the end of Stage B (Fig. 15). By the seventh day portions
of the inner integument are completely calcified. Calcification of the inner integu-
ment appears to occur in an identical fashion to that observed in the pre-exuvial
layers of the outer integument.

2. The liepatopancreas
a. Tissues

The hepatopancreas, as discussed by Travis (1955a), is the major storage depot
of organic and mineral reserves during Stage D and is, consequently, the major or-
gan from which these reserves are mobilized when needed by other tissues during
the postmolt period.

For fi-ve days following molt (Stage A and part of Stage B), the epithelial tis-
sue of the hepatopancreatic tubules is predominantly of the absorbing type. This
epithelial tissue consists of long tall columnar cells without large vacuoles and
which may have either a central or basal nucleus (the so-called B: or R cells of
Hirsch and Jacobs, 1928, 1930) as discussed by Travis (1955a). A few of the
epithelial cells are of the secretory type (the vesicular or B2 cells of Hirsch and
Jacobs). These are enlarged swollen cells which enclose large vacuoles, some of

FIGURE 13. In the pre-exuvial layers of the skeleton, deposited before molt, calcium
granules are observed in uniform vertical rows corresponding in location to the pore canals.
2000 X. Von Kossa's method.

FIGURE 14. Note narrow vertical rows of calcium granules, corresponding in location to
the pore canals (arrow) emanating from the epidermis (E). Also note the rather wide band
of calcium granules (C) in a newly formed portion of the principal zone paralleling the epi-
dermis. 2000 X. Von Kossa's method.

FIGURE 15. Horizontal bands of calcium granules paralleling the epidermis (E) con-
tinue to be deposited as the post-exuvial layers are deposited and a thick calcined band (C)
in the new principal layer is noted by the end of Stage B. 2000 X. Von Kossa's method.

FIGURE 16. By seven days following molt (end of Stage B) the epicuticle (arrow) and
most of the pigmented layer (P) appear to be fully calcined except in certain areas where this
is not complete. 2000 X. Von Kossa's method.

FIGURE 17. Note the heavy concentration of glycogen at the basal and distal ends of
the absorption cells and at the periphery of the vacuoles of secretory cells in the tubular epi-
thelium of the hepatopancreas. One day following molt (Stage A). 200 X.

FIGURE 18. At seven days following molt (end of Stage B) the tubular epithelium of the
hepatopancreas is virtually devoid of glycogen. 200 X.

464 DOROTHY F. TRAVIS

which contain stainable material. These cells (Travis, 1955a) undergo apocrine
breakdown in Pamtlirns. Their vacuolar contents plus adjacent cytoplasm are
discharged into the lumen of the hepatopancreatic tubules, leaving only the
basal region and nucleus of the cell intact. However, none of these observed se-
cretory cells, during this period of five days following molt, show any sign of apo-
crine breakdown. This would be expected because the animals are still under-
going a period of inanition (Travis, 1954, 1955a, 1955b). Stainable material in
the large vacuoles of the secretory cells frequently indicates the presence of lipid and
mucopolysaccharide.

By the sixth and seventh day following molt, the predominant cell types ob-
served in the epithelial tissue of the tubules are the secretory cells. On the seventh
day there is much evidence of apocrine secretion within these cells. In general,
most animals begin to feed again on the seventh day. This would not only ac-
count for the numerous secretory cells but also for the apocrine breakdown of many
of these cells.

The large oval reserve cells are likewise apparent in the connective tissue be-
tween the individual tubules of the hepatopancreas. They similarly show, as do
the reserve cells of the integumental tissues, the presence of phosphatase, mucopoly-
saccharide and calcium. Much lipid is also present during Stage A and B. From
the fifth through the seventh day the reserve cells of the hepatopancreas show the
presence of large vacuoles (Fig. 2), some of which contain flaky or granular-like
stainable material while others appear to be clear. This is possibly correlated with
a decrease in mucopolysaccharide, lipid, and calcium content in these cells. The
reserve cells of the hepatopancreas, like those of the integumental tissues, undergo
cyclic peaks and declines in size and abundance, changes in structural appearance,
and the storing and apparent release of reserves as indicated below.

During the intcnnolt period (late Stage C) they are numerous and large,
ranging in size from 17-38 //, with an average size of 30 p.. They show markedly
evident amounts of mucopolysaccharide. A similar situation prevails during the
premolt period. The cells range in size from 17-38 p. with an average size of 32 /*.
and show large stainable spheres of material. At both of these stages much muco-
polysaccharide is present. Calcium is likewise apparent in large amounts in the
hepatopancreatic reserve cells, while the integumental tissue reserve cells are de-
void of it during these periods.

By the first day following molt, there are few reserve cells present in the hepato-
pancreas and these have undergone a general decrease in size (8-29 p, average size
16 p.), a situation opposite to that observed in the integumental tissues. Of the
reserves, however, little calcium seems to be apparent and there is no evidence of
spheres of material found within them. On the second day (beginning of Stage B)
the cells remain few in number but have increased in size (range 24—38 p., average
size 31 p) , again a situation opposite to that observed in the integumental tissues,
although no large spheres of material are evident. They again show the presence
of small amounts of calcium. At three days, as in the integumental tissues, the
reserve cells become numerous and remain large (range 24-48 p., average size 30 p.).
They take on a distinct "mulberry" appearance and show the presence of mucopoly-
saccharide and markedly evident amounts of calcium. A similar situation prevails
on the fourth day. The cells in the hepatopancreas remain numerous and large
(range 26-43 p., average size 35 p.) and likewise show abundant mucopolysaccharide

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 465

and calcium. By the fifth day few cells are apparent and they are slightly smaller
in size (21—48 /A, average size 28 /A). These cells become highly vacuolated, al-
though some balls of material are present within the vacuoles, and show only a
slightly pink coloration with PAS, a condition similar to that observed in the re-
serve cells of the integumental tissues. Little calcium is apparent. Except for the
increase in number and slight increase in size (29-38 p., average size 32 /A) the re-
serve cells on the si.vth day remain similar to the fifth day condition though they con-
tain large single vacuoles with no stainable balls of material. They remain numer-
ous on the seventh day but the average size is slightly smaller (24—32 /A, average
size 29 /A). Except for these changes they are similar to the fifth day condition with
respect to reserves. It should be added that at all stages of the observation period,
phosphatase and lipid are present.

Since different groups of animals were used for the study of hepatopancreatic
and integumental tissues (fixed in different years), it is difficult to determine
whether or not the cycles may be slightly out of phase at the sites of observation.
The information at hand (Sewell, 1955; Travis, 1955a) strongly suggests that
much more information is needed on these highly interesting and obviously impor-
tant reserve cells.

b. Localization of glycogen, phosphatase, calcium and lipid

Glycogcn: Although the concentration of glycogen within the tubular tissue on
the first day following molt (Fig. 17) would compare with the premolt condition
(see Figure 30; Travis, 1955a), there is a progressive decrease in abundance from
the first through the seventh day. A decidedly marked decrease occurs by the sixth
day and by the seventh day hardly a single granule of free glycogen can be detected
within the tubular tissue (Fig. 18). During Stages A and B, glycogen is more
heavily distributed in the distal and basal ends of the absorption cells. When ma-
ture secretory cells are observed during the early phase of Stage B, glycogen gran-
ules are localized at the periphery of the vacuoles and are sometimes observed
within the lumen of the tubules.

The disappearance of glycogen from the hepatopancreas by the seventh day
might be expected because of the need of this constituent in the synthesis of the
new skeleton. The integumental tissues, among others, therefore, accumulate and
use large amounts of glycogen at the expense of the hepatopancreas. This is evi-
dent in both the premolt and postmolt period.

Phosphatase: During the early postmolt period alkaline phosphatase is almost
absent from the striated borders of the tubular tissue but remains localized around
the calcospherites (Fig. 19), which disappear progressively as calcification of the
skeleton occurs. From the sixth through the seventh day when many large se-
cretory cells are present, the enzyme is localized around the periphery of small and
large secretory vacuoles. It is at all times present in the reserve cells.

Calcium phospliatc: As in the premolt animals, the tubular epithelium of the
postmolt animal is marked by the presence of innumerable calcospherites in the
apical ends of the absorbing cells, very few being apparent in the small number of
secretory cells. In the postmolt animal, however, these calcospherites disappear
progressively as the skeleton is calcified. They have markedly decreased by the
fifth day and by the seventh day (Fig. 20) hardly a single calcospherite can be de-

466

DOROTHY F. TRAVIS

FIGURE 19. The tubular epithelium of the hepatopancreas at one day following molt
(Stage A). Note alkaline phosphatase localized around innumerable calcospherites (arrow)
at the apical ends of absorbing cells. 200 X.

FIGURE 20. At seven days following molt, note that there is almost a complete absence
of calcospherites and the enzyme alkaline phosphatase in the tubular epithelium of the hepato-
pancreas, a situation that occurs progressively as the integument is calcified. 160 X.

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 467

tected. The complete disappearance of the calcospherites by the seventh day would
be expected in Paniilirus because these animals have to rely completely on this
stored phosphate, stockpiled during the two-week premolt inanition period, as a
source of phosphate for incorporation into the new skeleton. Since, as was pointed
out previously (Travis, 1954, 1955a, 19551>), phosphate is obtained primarily from
food, the stockpile of stored phosphate in the hepatopancreas would readily be
depleted by the sixth or seventh day of starvation following molt. From low
blood values following molt (Travis, 1955b) it is evident that the normal intermolt
blood concentration within the body would not be replenished for at least three or
four weeks following molt. Although the reserve cells of the hepatopancreas show
the presence of calcium from one through seven days following molt, from the fifth
through the seventh clay little is apparent.

Lipid: For the entire observation period (Stages A and B) droplets of lipid
are found throughout the epithelial tissue of the hepatopancreatic tubules. There
would appear, by the sixth and seventh day, to be a decrease over that observed in
the premolt animal, although much is still apparent. When secretory cells are ap-
parent in great numbers (6 and 7 days) lipid droplets are frequently observed
within the vacuoles (Figs. 21. 22). On the seventh day, when apocrine breakdown
is evident, and when some of the animals begin to feed a little, lipid material be-
comes quite apparent within the lumen of the tubules, a condition which would be
correlated with extracellular digestion of this constituent, as Van Weel (1955) has
shown.

The reserve cells contain considerable quantities of lipid for the entire postmolt
observation period (Fig. 23). There does appear to be a decrease over that ob-
served during the intermolt (late Stage C) and the premolt period. Because of the
presence of considerable quantities of lipid within the hepatopancreas at this time,
a histochemical or a qualitative difference in amount is difficult, with certainty, to
detect.

DISCUSSION

The continued accretionary growth and hardening of the post-exuvial layers of
the skeleton imposes upon the epidermis two major tasks, namely the synthesis and
elaboration of the organic matrix and the simultaneous or accompanying elaboration
of constituents for hardening the skeleton which may or may not alter the proper-
ties of the basic organic components, chitin and protein. The complexities of
these two functions cannot be over-emphasized. Although the epidermis takes
the lead in the performance of these tasks, the importance of other tissues, such
as the hepatopancreatic and subepidermal tissues, cannot be under-estimated.

As the principal layer of the skeleton in Panulirus is deposited during the early
postmolt period of observation (Stages A and B), the outer epidermis shows, tinc-
torially, that considerable amounts of a glyco- or mucoprotein are concentrated or

FIGURE 21. The distribution of lipid droplets within secretory cells of the hepatopancreas
(arrows). The secretory cells become apparent in great numbers at six and seven days fol-
lowing molt. 200 X.

FIGURE 22. Seven days following molt. Note lipid droplets (arrow) within the vacuoles
of the secretory cells and within the lumen of the tubules (arrow). 200 X.

FIGURE 23. Note that considerable quantities of lipid are bound by the reserve cells (ar-
rows) of the hepatopancreas. 100 X.

468 DOROTHY F. TRAVIS

synthesized by this tissue. Similarly, during Stage A and most of B, the pigmented
layer, one of the two pre-exuvial layers formed before molt, shows the presence of
this same mucopolysaccharide. By the fifth day the pigmented layer no longer in-
dicates a positive reaction for this constituent. There is a tinctorial change from
gamma metachromasia (pink-purple) to beta metachromasia (deep purple) with
toluidine blue, which indicates that the properties of the basic organic components,
chitin and protein, have been altered. Possibly this is caused by considerable
impregnation with calcium salts at this time or, more certainly, by quinones which
form cross-linkages with the native protein phase of the cuticle (Pryor, 1940).
The net result of this combination is the formation of a highly stable and insoluble
product. As Krishnan (1951) pointed out, the tanning by quinones of the pig-
mented layer occurs in Carcinns macnas shortly following molt and this is followed
by pigmentation at a slightly later period. It is, therefore, possible that both of
these related processes are completed by the fifth day in Paniilints, thus causing
this change in tinctorial properties.

The presence of muco- or glycoprotein in the epidermis for the entire postmolt
observation period (Stages A and B) is doubtlessly related to the secretion and
development of the principal layer, which is the only post-exuvial layer deposited
during this period. This layer, like the pre-exuvial pigmented layer and post-
exuvial membranous layer (formed and completed from the third through the fourth
week following molt in Pannlirns}, consists as in insects of the basic organic com-
ponents, chitin and protein, which are closely associated with one another. The
firm combination of these two organic constituents has led Trim (1941), Stacy
(1943) and Haworth (1946) to regard the arthropod cuticle as a mucopolysac-
charide because of the firm combination of the carbohydrate-containing amino
sugars (chitin) with the protein. Richards (1951) has pointed out that the con-
sideration of the arthropod cuticle as a double set of layers (the outer set being
composed of lipoprotein and the inner set being composed of glyco- or mucopro-
teins) is advantageous. This consideration emphasizes that the major cuticular
components seem to be formed and secreted as conjugated proteins and not as
separate components.

The principal layer in Pannlirns always shows, tinctorially, the presence of
muco- or glycoproteins with the exception of the fact that immediately after each
layer is deposited a green, rather than a pink, coloration with toluidine blue occurs.
This suggests that the reactive groups are slightly altered following their immediate
formation. Although the early stages in the formation of the membranous layer
were not followed, the completed layer, in contrast to the principal layer, shows a
green rather than a pink coloration, which indicates that this layer is, in some way,
different from the principal layer. This is further revealed by the fact that calcium
salts are never bound in this layer.

During the deposition of the principal layer of the outer integument glycogen
accumulates in large amounts in the epidermis. There is a periodic shift of gly-
cogen from the sub-epidermal tissues to the outer epidermis (Travis. 1951a, 1951c,
1955a) and in turn a shift from the hepatopancreatic tissues to these integumental
tissues (see Observations). During the postmolt period of accumulation and
utilization of glycogen by the integumental tissues (Stages A and B), hepatopan-
creatic glycogen progressively disappears. Although it is abundant in the tubular
tissue on the first day following molt, there is a marked decrease in glycogen by the

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 469

fifth day and hardly a single granule of free glycogen is present by the seventh day.
The rhythmical accumulation by, and disappearance of, glycogen from the sub-
epidermal tissues and similarly its accumulation by the epidermal cells and disap-
pearance from the sub-epidermal tissues suggests a rhythmical cycle of accumula-
tion and utilization in the epidermis, at the expense of sub-epidermal tissue gly-
cogen. Likewise, the rhythmical accumulation of glycogen by the sub-epidermal
tissue and progressive disappearance from the hepatopancreas similarly suggests
that there are marked cycles of accumulation and utilization. These cycles of ac-
cumulation and utilization stem from the epidermis, which takes the lead in the
elaboration of the post-exuvial layers of the integument, but also involves the hepato-
pancreas, which serves as the major storage organ from which such reserves can
be mobilized and upon which the epidermis is ultimately dependent for the success-
ful completion of its tasks. Since feeding begins on the seventh day following molt
in the summer months, a constant supply of glycogen would be available to the epi-
dermis until the integument is completed (late Stage C), but is not stockpiled in the
hepatopancreas again until Stage D. The stockpiling of glycogen during this period
of inanition would suggest strongly that the source of this constituent is from the
large quantities of lipid reserves, likewise present in the hepatopancreas at this
time. By the conversion of some lipid, through its glycerol moiety, to carbohydrate,
the latter being stored as glycogen, the peak glycogen concentration could be
achieved during Stage D. As was pointed out by Travis (1955a), evidence sug-
gests (Renaud, 1949) that during periods of inanition (Stages D, A and B), lipids
likewise serve as a major source of energy by playing a principal role in oxidative
metabolism.

The periodic accumulation and utilization of glycogen by the epidermis as the
post-exuvial layers are deposited suggests that glycogen is a necessary precursor
for chitin formation. This possibility, as discussed by Travis (1955a), has been
suggested by Verne (1924, 1926), Mataczynska-Suchcitz (1948), Renaud (1949),
Travis (1951a, 1955a) and Schwabe et al. (1952). Glycogen may likewise serve as
a ready energy-source for the synthesis and elaboration of the organic constituents
of the integument. This possibility has been suggested by Bradfield (1951). He
found an abundance of glycogen in regenerating epidermis of the vertebrates. As
the outermost cells keratinized, glycogen disappeared. He attributed this disap-
pearance to the utilization of glycogen for the supply of energy in keratin synthesis.
Glycogen may further serve indirectly as added substrate for phosphatase action
after its hydrolysis and phosphorylation by phosphorylases. In this way, it has
been postulated as one of the necessary mechanisms in calcification of bone and
teeth of vertebrates (Robison and Soames, 1924; Harris, 1932; Clock, 1940; Horo-
witz, 1942; Engel, 1948; Marks and Shorr, 1950 and others). It is more likely,
however, that glycogen participates in all of these functions and possibly others
that have not been mentioned.

During the entire postmolt observation period (Stage A and D), alkaline phos-
phatase is heavily concentrated in the distal ends of the outer epidermal cells and
is observed in the integument immediately distal to but paralleling the epidermis.
The localization of the enzyme in this latter site is distinctly apparent by the first
day following molt before any calcification begins in the branchial region of the
integument. Krugler and Birkner (1948) noted a similar localization of the
enzyme in the integument of the crayfish during premolt. In Panulints this is a

470 DOROTHY F. TRAVIS

strategic location for the enzyme during the postmolt period because it is in a
region of high activity as the deposition and hardening of the post-exuvial layers
occur. Further, it would appear that the enzyme may be specifically localized in
the proximal portions of the pore canals. Because of its heavy concentration
along this entire region of the integument, however, its specific localization in the
pore canals is difficult to determine with certainty. The enzyme is likewise heavily
concentrated in the integument around newly developing bristles. In the sub-epi-
dermal tissues it is observed in the reserve cells.

In the hepatopancreas the most marked localization of the enzyme is seen around
the innumerable calcospherites in the distal portions of the absorption cells. The
enzyme is observed in the striated borders of these cells and on the sixth and seventh
day, when a predominance of secretory cells is evident, it is observed at the periph-
ery of small and large vacuoles. The reserve cells within the blood or tissue
spaces between the tubules of the hepatopancreas likewise show the presence of the
enzyme.

As in the premolt animal, phosphatase is localized around the calcospherites in
the absorption cells of the hepatopancreas and since these disappear progressively as
calcification of the post-exuvial layers occurs, it is possible that the enzyme par-
ticipates in the mobilization of this reserve for transfer to the integument. It may
do so by dephosphorylating, in some way, the precipitated complex. In this role,
it would be serving in resorption at this site and could at the same time be involved
in mediating the synthesis of other phosphoric esters to be conveyed via the blood
from the hepatopancreas to the integumental tissues. That this indeed may be an
important function of phosphatase in bone resorption has been suggested by McLean
and Urist (1955). Further, its localization around the periphery of small and large
vacuoles on the sixth and seventh day would suggest that the enzyme is possibly
involved, in some way, with the synthesis of secretory products or the transfer of
these products from the adjacent cytoplasm into the secretory vacuoles. Phospha-
tase localization at the striated borders of the absorption, as well as the secretory,
cells would suggest that when these cells are active the enzyme would likewise serve
the function of participating in transfer reactions by producing molecules which
enter or leave the cells more readily. Such a function has been suggested by Moog
(1946).

The concentration of the enzyme at the distal ends of the outer epidermal cells
and in the integument immediately distal to, but paralleling, the epidermis suggests
its extremely important functions in the deposition and hardening of the post-
exuvial layers. The periodic accumulation and utilization of glycogen by the epi-
dermis, as was pointed out earlier, would suggest that possibly this constituent, gly-
cogen, is used as a precursor in chitin formation. If this is so, and if the synthesis
of chitin occurs, as Renaud (1949) suggested, by the hydrolysis of glycogen and
dephosphorylation of glucose phosphate to glucose, this step being followed by sub-
sequent steps to yield chitin, phosphatase would play an important role in this chain
of events by its dephosphorylation of glucose phosphate to glucose, a possible start-
ing point for chitin formation. Likewise, if glycogen were used as an energy-
source for the synthesis and elaboration of the organic matrix, phosphatase would
be intimately involved in these reactions. Glycogen, as suggested earlier, could
serve indirectly as added substrate for phosphatase action. The distribution of
phosphatase and mucopolysaccharide in the epidermis and its distribution in the

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 471

region of the newly forming post-exuvial layers of the integument, immediately
above the epidermis, suggests that it may play a very important part in the forma-
tion of the ground substance (mucopolysaccharide) of the post-exuvial layers.
Furthermore, the enzyme is thought to play an important role in the manufacture
of fibrous proteins, thus participating in the formation of the ground substance of
bone (McLean and Urist, 1955). Moog and Wenger (1952), however, have sug-
gested that since the enzyme and mucopolysaccharide are frequently found together
in fibrous structures, the mucopolysaccharide constitutes part of a cytoskeletal
mechanism to which the enzyme is bound.

The appearance of alkaline phosphatase in the integument immediately distal to
and paralleling the epidermis one day before calcification begins, likewise suggests
to the author that the enzyme is intimately involved in calcification of the integu-
ment. In such a localization it could provide a mechanism for the production of a
local high concentration of phosphate ions. In the presence of calcium ions, trans-
ferred across the cell membranes of the epidermis, phosphate could then unite to
form the calcium salt, calcium phosphate, which constitutes about 3% of the total

TABLE I

Analyses to indicate the amount of mineral and organic matter in the entire
area of softening of a late Stage C animal

Substance
analyzed

Per cent present in the
area of softening

Calculated % of
salts present

CaO

24.64

CaCO3

42.39

MgO

1.98

MgC03

4.04

P205

1.40

Ca3(P04)2

3.05

C02

21.39

Carbonates

unaccounted for

2.22

Per cent mineral as Ca,

P, and Mg oxides

28.02

Per cent organic matter

71.98

mineral salt of the integument (Table I). The almost certain presence of phos-
phorylases at these sites, although not specifically determined, would likewise be
expected to be important in the calcification of the integument, by synthesizing po-
tential substrates for phosphatase action in zones of calcification.

Hardening of the crustacean skeleton occurs by quinone tanning and calcification.
Hardening by quinones is a result of the oxidation of polyphenols to quinones,
which form cross linkages with the native protein of the cuticle. The net result of
this combination is a highly stable and highly insoluble product. Of the pre-
exuvial layers, the epicuticle of Carcinus maenas is hardened by quinones shortly
after its formation, whereas subsequent hardening of the pigmented layer occurs
soon after molt (Krishnan, 1951). As was suggested in a previous section of the
discussion, changes in tinctorial properties of the pigmented layer suggest that the
process of hardening by quinones is complete by the fifth day following molt.
Quinone tanning, although the primary cause of hardening in the exoskeleton of
insects, plays a much smaller role in Crustacea (Dennell, 1947), calcification being
the major cause of hardening.

472 DOROTHY F. TRAVIS

Calcification begins on the second day following molt in Panulirus and occurs
thereafter simultaneously with or immediately accompanying the elaboration of
layers of the principal zone. Further, the additional task posed to the epidermis
is that of calcifying the pre-exuvial layers; calcification, in this case, is of course a
process that is accomplished long after their formation but during the same time
at which the post-exuvial layers are being calcified.

In the distal region of the epidermal cells, where calcium becomes most heavily
concentrated, extrusion of calcium from this tissue occurs in two distinct ways
(Travis, 1951a, 1951c). As the post-exuvial layers are deposited, masses of cal-
cium granules are simultaneously extruded, thus forming narrow bands paralleling
the epidermis. In the pre-exuvial layers, on the other hand, calcium granules are
observed in uniform vertical rows. These rows, as pointed out earlier, are particu-
larly evident in the pigmented layer, and are likewise observed to emanate from the
epidermis. They correspond in location to the pore canals or vertical striae, proto-
plasmic extensions of the epidermis. Thus, in the case of the pre-exuvial layers,
calcification occurs after their formation. Hence, calcium must be transported and
deposited some distance from the newly forming pre-exuvial layers. This trans-
port occurs through the pore canals, thus enabling the epidermis to act at these
distant sites.

Calcification of the integument continues and is almost entirely completed by
the seventh day (end of Stage B) in the epicuticle and most of the pigmented layer.

While calcification of the integument occurs, the reserve cells in the sub-
epidermal tissues undergo what appear to be cyclic peaks in calcium storage, pos-
sibly alternating with cyclic release to the epidermis. If calcium in these cells is
used periodically by the epidermis, which it probably is, the reserve cells could serve
as reservoirs for providing additional calcium during periods of concentration by
the epidermis. At no time during Stage A and B, however, are the epidermal cells
depleted of calcium. This might be expected because the concentration of calcium
in the blood (Travis, 1951b, 1955b) is sufficiently high to provide a continued sup-
ply of this element to the epidermis. Previous reference to the reserve cells has
already been made as to their cyclic peaks and declines in abundance and size,
change in structural appearance and in the binding and release of reserves, other
than calcium (see Observations) and will not therefore be discussed in this section.

As calcification of the integument occurs there is a progressive decrease in num-
ber of calcospherites — spherules of calcium phosphate — present in the absorption
cells of the hepatopancreas. Though abundant on the first day following molt they
progressively decrease in number as calcification of the new integument occurs and
by the seventh day hardly a single calcospherite is to be detected. The calcospher-
ites, premolt storage depots of reserve phosphate from the old skeleton (Travis,
1955a), represent a major source, therefore, from which phosphate can be mobilized
for hardening of the new skeleton during Stage B, a time at which the animals do
not feed. Since the spiny lobster obtains most of its phosphorus from food and
since the animals do not feed for two weeks before molt, when resorbed phosphorus
from the old skeleton is being stockpiled in the hepatopancreas (Travis, 1955a),
one would expect a depletion of this mineral reserve during the first week following
molt as calcification of the new integument occurs. This depletion is also evidenced
by the low blood-phosphorus levels following molt (Travis, 1955b).

It is interesting that the reserve cells of the hepatopancreas show the presence

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 473

of calcium from one through seven days following molt but from the fifth through
the seventh day little is apparent. The fifth through the seventh day is a period
when these cells become vacuolated, which may be correlated with the apparent de-
crease in mucopolysaccharide, lipid and calcium. It is possible that as the calcium
phosphate is mobilized from the calcospherites it is immediately transferred from the
absorption cells to the reserve cells and from these to carriers in the blood, possibly
organic acids of oxidative and glycolytic metabolism. The evidence at hand, how-
ever, is not sufficient at this time to determine whether there is an actual movement
of the reserve cells from the hepatopancreatic tissues to the integumental tissues.

Little has been said about the development and hardening of the inner integu-
ment during Stages A and B. It will be recalled that glycogen is observed in
abundance at the bases of the epidermal cells of the inner integument on the first
day following molt (Stage A) but completely disappears from the inner epidermis
by the second day, not to be observed again at this site for the entire observation
period. Similarly, no further thickening of this integument occurs after the second
day following molt, indicating that the development of the inner integument of the
branchiostegites is completed during a period of three days preceding molt and two
days following molt (Travis, 1955a). This does not mean, however, that harden-
ing by calcification is completed at this time. Calcification of the inner integument,
as in the pre-exuvial layers of the outer integument, occurs after its formation. By
the fourth day the uniformly staining endocuticle of the inner integument has begun
to calcify and by the seventh day portions of it have completely calcified. Calcifica-
tion in the inner integument occurs in the same fashion as that observed in the pre-
exuvial layers of the outer integument, i.e., via the port canals.

The two major tasks which must be achieved by the epidermis, namely, the
synthesis and elaboration of an organic matrix and the simultaneous or accompany-
ing elaboration of calcium salts for hardening of the newly developing skeleton, are
not completely accomplished before the fourth week following molt during the sum-
mer months. Calcification is probably completed by the third week following molt,
a time at which the membranous or non-calcified layer begins to be formed. This
layer is not fully completed before the fourth week following molt (late Stage C or
C4 of Drach, 1939). Approximately three weeks following the completion of the
integument the epidermis is again confronted with the preparation for growth in
size of the animal (Travis, 1955a).

The cells of the epidermis, like the osteoblasts of bone, synthesize and elaborate
the organic matrix of the skeleton and, unlike the osteoblasts of bone, they actually
concentrate and secrete the mineral constituents, principally calcium, which are pre-
cipitated in the matrix. Furthermore, the epidermis, like the osteoclasts of bone,
participates intimately in processes of resorption of the integument. It elaborates
the proteinases and chitinases which break down the organic matrix. It likewise
resorbs these organic breakdown products along with the mineral constituents and
also participates in their transfer across its cell membranes to the blood for further
handling (Travis, 1955b).

Following molt the transfer of calcium ions from the blood across the cell mem-
branes of the epidermis and the concentration of calcium by this tissue is truly re-
markable. Within the epidermal cells, the calcium is doubtlessly immobilized ion-
ically by the binding capacity of weakly acidic groups of protein, succinate, lactate,
bicarbonate, phosphate, citrate or by other anionic groups. On release of calcium

474 DOROTHY F. TRAVIS

to the exterior of the cell, an alteration in the binding capacity of anionic groups is
necessary. After release from the epidermis, calcium is precipitated as salts, by
various mechanisms, in the organic matrix of the integument.

The distribution of alkaline phosphatase in the distal portion of the epidermis
and particularly in the integument immediately distal to and paralleling the epider-
mis suggests that this enzyme would provide a mechanism for the local high con-
centration of phosphate ions, which in the presence of some of the calcium released
from the epidermis could account for the precipitation of the 3% calcium phosphate
of the integument (Table I).

Calcium carbonate, however, is the principal salt of the spiny lobster skeleton,
and Crustacea in general, and constitutes approximately 42% of the total mineral
deposited in the skeleton of Panulirus. It is, therefore, of interest to point out a
related and possibly important enzyme involved in calcification of the skeleton.
This is the enzyme, carbonic anhydrase. Sabotka and Kann (1941) found that
this enzyme was not present in the gills of Panulirus argus. Because of this, they
suggested that elimination of CO2 is not confined to the gills, but that the bicarbon-
ate formed may be eliminated in the skeleton by precipitation as CaCO3. Maluf
(1940) found that considerable quantities of carbonic anhydrase were present in
the epidermis and skeleton of the crayfish, Carnbarus clarkii and the American lob-
ster, Homarus americanus. The fact that alkaline phosphatase is found in the dis-
tal ends of the epidermal cells and integument immediately above and paralleling
the epidermis would suggest that the hydrogen ion concentration is low and the pH
is on the alkaline side (between 8—10). At this pH, dissociation of bicarbonate into
CO3= and H+ would be expected. In the presence of calcium ions released from the
epidermis, the precipitation of calcium carbonate could occur. Thus, again the
mechanism and conditions exist for the production of local high concentrations of
bicarbonate and carbonate ions in the epidermis and integument of Crustacea.

The manner in which calcium carbonate is precipitated, i.e., as calcite rather
than aragonite or amorphous calcium carbonate, is undoubtedly determined by con-
ditions inherent in the organic matrix which favor calcite precipitation. Prenant
(1927), however, pointed out that the condition determining the state of calcium
carbonate precipitation is the proportion of phosphates to carbonates, as indicated
by the P2O5/CO2 ratio. If the ratio is more than 0.015, calcium carbonate is de-
posited in amorphous form. If the ratio is 0.105 or less, the calcium is deposited
in crystalline form. The P2O5/CO2 ratio in Panulirus is 0.0657. Calcium carbon-
ate in Panulirus is precipitated as calcite, which is consistent with the idea proposed
by Prenant. This would not mean, however, that conditions in the organic matrix
do not favor calcite, rather than aragonite, precipitation.

That the calcification mechanism in Panulirus may be influenced by the presence
of organic acids such as lactic, succinic, and citric acid at the sites of calcification
has yet to be investigated. These acids have a marked propensity for forming
weakly ionized salts with calcium. It is highly likely, therefore, that, as in the cal-
cification of bone, citric acid plays an important role in the calcification mechanism
of Crustacea. It is known from the work of Dickens (1941) that more than 70%
of the citric acid of the human body is in the skeleton and that as much as 1 % of
the fresh weight of bone may be accounted for as citrate. It is not definitely known
whether citrate is present as an ion or precipitated as a calcium citrate complex.
Bones examined for enzymes of the citric acid cycle have shown that by comparison

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 475

with other tissues, such as the kidney or liver, citrogenase and aconitase activities
are much greater than those of isocitric dehydrogenase (McLean and Urist, 1955).
As these authors point out, the mechanism for the production of local high concen-
trations of citric acid exists in bone. Other than the work of Thunberg (1949),
however, as quoted from Thunberg (1953) and Steinhardt (1946), nothing has
been done with the role of citric acid in the calcification process of the Crustacea.
Thunberg found that at least 0.8% of the gastroliths of a European crayfish was
constituted of citric acid. This would suggest strongly that citric acid is involved,
as in bone, in some way in the calcification process. Steinhardt (1946) has further
pointed out that in structures such as bone and gastroliths, where high concentra-
tions of citric acid are found, the phosphorus and calcium content is also high. In
such cases, citrate probably exists in a complex form in which calcium, phosphoric
and citric acid enter.

It is highly likely, therefore, that within the integument of Crustacea, as in the
gastroliths of the crayfish, the mechanism for the production of local high concen-
trations of citric acid exists. If so, the citrate produced, having a marked propen-
sity for forming weakly ionized salts with certain cations such as calcium, could
combine with calcium released from the epidermis and could enter into the mineral
complex of calcium salts of the integument. If this is the case, increases and de-
creases of citrate formed enzymatically within tissues may be one of the regulators
of ionic calcium activity. Furthermore, in normal metabolism, the normal activity
of tissues may be controlled, in part, by the interaction of ionic calcium with citrate
reached via the tricarboxylic cycle. Such a possibility has been suggested for
the mammal by Peters (1950).

It is apparent from the foregoing discussion that the continued growth of the
skeleton in an accretionary manner and the hardening of it by calcification, two of
the major tasks confronting the crustacean from Stage A to late Stage C, are indeed
complex. Although these extraordinary duties are put to the epidermis, which
takes the lead in the performance of them, the importance of the other tissues, namely
the hepatopancreatic and sub-epidermal tissues, should not be under-estimated.

SUMMARY

1. During the early postmolt period (Stages A and B) as rapid accretionary
growth and calcification of the skeleton are occurring, changes are observed in the
hepatopancreas and integumental tissues.

2. As the principal layer of the skeleton in Panulirus is deposited during Stage
A and B, the outer epidermis concentrates or synthesizes a considerable amount of
glyco- or mucoprotein, which is probably related to and involved in the secretion
and development of this layer. Similarly, the pigmented layer, one of the pre-
exuvial layers formed before molt, shows the presence of this same mucopolysac-
charide. Near the end of Stage B, however, the properties of the basic organic
components in the pigmented layer have been altered, possibly by considerable im-
pregnation with calcium salts or by quinones.

3. During the deposition of the principal layer of the outer integument, glycogen
accumulates in large amounts in the epidermis. There is a periodic shift of gly-
cogen from the sub-epidermal tissues to the outer epidermis and in turn a shift
from the hepatopancreatic tissues to the integumental tissues. During this period

476 DOROTHY F. TRAVIS

of accumulation and utilization by the integumental tissues (Stages A and B),
hepatopancreatic glycogen progressively disappears and by the end of Stage B
none remains.

4. The possibility of lipid conversion to carbohydrate, and the storing of this as
glycogen in the hepatopancreas during Stage D, is discussed. The utilization of
glycogen by the epidermis during Stages A and B, periods of inanition, is also
discussed.

5. During Stages A and B alkaline phosphatase is heavily concentrated in the
distal ends of the outer epidermal cells. It is observed in the integument in the
region of the proximal portions of the pore canals, even before calcification begins.
This is a region of high activity as deposition and hardening of the post-exuvial
layers occurs. The enzyme is likewise found in the reserve cells of the sub-
epidermal cells. In the hepatopancreas, the most marked localization of the enzyme
is seen around the innumerable calcospherites in the absorption cells and in the
striated border of these cells. The reserve cells of the hepatopancreas likewise show
the presence of the enzyme. Functions of phosphatase in these sites are suggested.

6. Calcification begins the second day following molt and occurs thereafter si-
multaneously with, or immediately accompanying, the elaboration of layers of the
principal zone. Calcification of the pre-exuvial layers, formed before molt, is a
process accomplished long after their formation but during the same period at
which the post-exuvial layers, formed after molt, are being calcified.

Calcium, heavily concentrated in the distal region of the epidermal cells, is ex-
truded from this tissue in two distinct ways. As the post-exuvial layers are de-
posited, masses of calcium granules are simultaneously extruded, thus forming nar-
row bands paralleling the epidermis. In the pre-exuvial layers, on the other hand,
calcium granules are observed in uniform vertical rows which emanate from the
epidermis. These vertical rows of calcium granules correspond in location to the
pore canals. Since calcification of the pre-exuvial layers occurs after their forma-
tion calcium must be transported and deposited some distance from the newly form-
ing post-exuvial layers. This transport occurs through the pore canals, protoplas-
mic extensions of the epidermis, thus enabling this tissue to act at these distant sites.

7. While calcification of the integument occurs, the reserve cells in the sub-
epidermal tissues undergo what appear to be cyclic peaks in calcium storing alter-
nating with cyclic release to the epidermis. The reserve cells in this capacity could
serve as reservoirs for providing additional calcium during periods of concentration
by the epidermis. Furthermore, these interesting reserve cells, during the early
postmolt period ( Stages A and B ) , undergo at daily intervals, cyclic peaks and de-
clines in size and abundance, changes in structural appearance, and staining prop-
erties and the storing of reserves other than calcium. The mucopolysaccharide ma-
terial, either muco- or glycoprotein, in the reserve cells disappears near the latter
part of Stage B. This indicates a decrease in the concentration of the material and
suggests that the mucopolysaccharide stored by the reserve cells represents reserve
material for the construction of the new skeleton.

The reserve cells of the hepatopancreas, like those of the integumental tissues,
undergo during the early postmolt period (Stages A and B) cyclic peaks and de-
clines in number, size and the storing and apparent release of reserves. They simi-
larly show the presence of phosphatase, mucopolysaccharide, calcium and much
lipid.

HISTOCHEMISTRY OF POSTMOLT LOBSTERS 477

8. As calcification of the integument occurs there is a progressive decrease in
number of calcospherites — spherules of calcium phosphate — present in the absorp-
tion cells of the hepatopancreas. These calcospherites, abundant preceding molt
and on the first day following molt, progressively decrease in number as calcifica-
tion of the new integument occurs and by the seventh day (end of Stage B) hardly
a single calcospherite can be detected. The calcospherites, premolt storage depots
of reserve phosphate from the old skeleton, probably represent a major source from
which phosphate can be mobilized for hardening of the new skeleton during Stage
B, a time at which the animals do not feed.

9. Development of the inner integument of the branchiostegites is completed in
Pannlirus during a period of three days preceding molt and two days following molt.
Calcification of the inner integument, as in the pre-exuvial layers of the outer in-
tegument, occurs after its formation via the pore canals, and portions of this integu-
ment are completely calcified by the seventh day following molt (end of Stage B).

10. Calcium carbonate, the principal salt of the spiny lobster skeleton, constitutes
approximately 42% of the total mineral deposited and is precipitated in the organic
matrix as calcite, rather than aragonite or amorphous calcium carbonate.

11. The roles of carbonic anhydrase and citric acid in the calcification of the
integument of Crustacea are discussed.

12. Continued accretionary growth of the skeleton and the hardening of it by
calcification are two major tasks confronting the crustacean from Stage A to late
Stage C. Although the epidermis takes the lead in the performance of these duties,
the importance of the other tissues, namely the hepatopancreatic and sub-epidermal
tissues, should not be under-estimated.

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DEVELOPMENTAL MODIFICATIONS IN THE SAND DOLLAR

CAUSED BY COBALTOUS CHLORIDE IN COMBINATION

WITH SODIUM SELENITE AND ZINC CHLORIDE 1

OLIN RULON

Dept. of Biological Sciences, Northivestern University, Evans ton, Illinois,
and The Hopkins Marine Station, Pacific Grove, California

Studies on the effects of chemical agents on early developmental patterns in
echinoderms have shown that similar modifications may often be caused by entirely
different agents. Some groups of totally different substances will cause entodermi-
zation of the young embryo while others will produce the opposite effect (ectodermi-
zation) if concentrations and exposure periods are carefully controlled. Some will
increase the area of the ventral field while others will decrease it. In recent studies
(Rulon, 1952, 1953, 1955, 1956) it has been found that compounds such as sodium
selenite, nickelous chloride, zinc chloride, and cobaltous chloride will all cause the
loss of bilaterality and a polar elongation of the larva. Such larvae differentiate
with reference to the new pattern and are very similar to each other, irrespective
of the compound used.

When two unlike substances produce similar effects certain questions may be
asked : ( 1 ) Do these agents affect identical reaction systems in the morphogenetic
process? Or (2) are different loci in the reaction complex affected in such a way
that the final results are the same ? A definite answer to either question is difficult
to make but it is believed that at least a partial answer may be suggested by the use
of combinations of the different agents.

In our experiments it has been customary to submit the eggs and early embryos
to a wide range of concentrations when testing the effects of any particular agent.
This range usually extends from a concentration that is lethal in a few hours to one
that has little or no effect. The range is commonly set up so that the succeeding
steps are each one-half the concentration of the preceding (i.e., M/100, M/200,
M/400, M/800, M/ 1,600, M/3,200 . . .). When newly fertilized eggs from the
same lot are distributed throughout the various concentrations, interesting compari-
sons may often be made. The stronger solutions commonly give proportionate in-
hibition— that is, all structures are grossly inhibited. With decreased concentra-
tions the inhibition may be disproportionate (differential) in that certain processes
or structures are strongly affected while others are affected slightly if at all. The
differential inhibition of one structure often provides for the physiological release
of another to the extent that the structure not inhibited increases in size beyond the
normal.

The present work deals chiefly with combinations of agents. Since previous in-
vestigations have shown that cobaltous chloride, sodium selenite, and zinc chloride

1 This investigation was supported by the Graduate School of Northwestern University.
The writer is also much indebted to Dr. L. R. Blinks, Director of the Hopkins Marine Station,
Pacific Grove, California, for many considerations.

480

MODIFICATIONS IN THE SAND DOLLAR 481

all cause the development of sand dollar embryos that are quite similar, it seemed
important to determine if these agents could replace one another in inhibitory solu-
tions. Would the effects of these different agents be antagonistic, additive, or
possibly synergistic?

MATERIAL AND METHODS

This work was conducted at the Hopkins Marine Station, Pacific Grove, Cali-
fornia, during the summers of 1954—55. The adult sand dollars (Dendraster ex-
centricus) were dredged from Monterey Bay and maintained in the laboratory in
running sea water. Ovaries were exposed by removing the oral surfaces of the
animals. The bright red eggs, exuding in droplets, were washed into finger bowls.
After several washings in sea water the ova were fertilized by the addition of a few
drops of sperm suspension. Only ova that were over 95 per cent fertilizable were
used. All test solutions were made up in sea water and the controls and tests were
always from the same batch of eggs. All eggs developed under uncrowded condi-
tions in finger bowls, out of direct sunlight and under the moist conditions of the
aquarium room where the temperature varied by no more than one degree from 18
degrees C. and smoking was not permitted.

EXPERIMENTAL

1. Continuous exposure of newly fertilised eggs to single and combination solu-
tions of cobaltous chloride and sodium selenite. It would be repetitious to report
here all of the modifications caused by solutions of only cobalt and selenium (see
Rulon, 1952, 1956). The object of this paper is to make comparisons between the
effects of solutions containing the ions singly and in combinations at critical con-
centrations of the range. Accordingly, only the effects of the following solutions
will be discussed although additional data have been obtained from various other
concentrations and combinations :

Solution 1 — Sea water control

2 — M/SOO cobaltous chloride

3 — M/1,600 cobaltous chloride

4 — M/800 sodium selenite

5 — M/1,600 sodium selenite

6 — M/1,600 cobaltous chloride — M/1,600 sodium selenite (50 cc. of

M/800 CoCl2-6H2O plus 50 cc. of M/800 Na2SeO3)
7— M/3,200 cobaltous chloride — M/3,200 sodium selenite (50 cc. of

M/1,600 CoCl2-6H2O plus 50 cc. of M/ 1.600 Na2SeO,)

After 48 hours almost all (98 per cent) in solution 1 (control) had developed
into normal free-swimming bilateral plutei with well-differentiated oral and anal
arms and with full skeletal development (Fig. 1). In solution 2 all were radial in
symmetry and almost all (95 per cent) showed polar elongation (Figs. 2-A}.
Most showed differentiation of an apical lobe (Figs. 2, 4). All had thickened basal
regions and commonly a basal circle of cilia was to be seen. Exogastrulation was
present in 20-30 per cent and skeletal development was inhibited in all. All were
slow-moving bottom forms. In solution 3, which was one-half the strength in co-
baltous chloride of solution 2, less than 10 per cent resembled the radial elongated

482

OLIN RULON

forms of solution 2. Instead, approximately 90 per cent were bilateral, free-
swimming larvae (as Figs. 5-7). The majority of these larvae showed well-
differentiated entera and stomodaea but no differentiation of skeleton or arms.
There were some indications of polar elongation but they were not nearly so pro-
nounced as in the larvae that had developed in solution 2. Approximately 20 per
cent showed exogastrulation.

After 48 hours in solution 4 almost all (95 per cent) were radial forms with very
little movement (Figs. 8-11). Over 75 per cent showed considerable polar elonga-
tion (Figs. 10-11) with differentiation of apical and basal lobes. No skeleton dif-
ferentiated and many had basal ciliated bands. Only a few exogastrulae appeared.
Approximately 75 per cent of the eggs developing in solution 5 resulted in bilateral

FIGURES 1-7. Figure 1, normal 48-hour larva,
exposed continuously to M/800 cobaltous chloride,
tinuously to M/1,600 cobaltous chloride.

Figures 2-4, 48-hour larvae that have been
Figures 5-7, 48-hour larvae exposed con-

plutei with short anal arms containing skeleton and at increased angles (Figs. 12-
13). Oral lobes were broad but poorly differentiated. The remainder of the lar-
vae in solution 5 graded from slightly bilateral forms without skeleton (Fig. 14)
into the polar elongated radial larvae of higher concentrations (Figs. 10-11). Only
an occasional exogastrula was seen.

Almost 100 per cent of the eggs developing in solution 6 became radial or near
radial larvae (Figs. 15-18). Of these, over 50 per cent showed polar elongation
with oral and basal lobes (Figs. 15-17). Approximately 10 per cent were exo-
gastrulae. In solution 7, there was only an occasional elongated radial form while
approximately 90 per cent were bilateral free-swimming larvae with ventral ciliated
bands but no skeleton or arms (Figs. 19-21). Approximately 10 per cent were
exogastrulae but the remainder had well-differentiated entera with stomodaea.

MODIFICATIONS IN THE SAND DOLLAR

483

From these experiments it was shown that when the eggs of D. excentricus were
exposed to a combined solution of cobalt and selenite (M/1,600 CoCl2-M/ 1,600
Na2SeO3) they differentiated according to a pattern of radial symmetry very much
as if they had been exposed to single solutions of double strength of either agent.
That is, while M/ 1,600 cobalt alone caused 10 per cent radial and M/ 1,600 selenite
alone caused 25 per cent radial, together they caused 100 per cent radial (approxi-
mately 100 per cent radial forms are caused by M/800 of either agent). In the
case of polar elongation the combined solution gave a considerably higher percentage
than did the single solutions (M/ 1,600 CoCl, or M/1,600 Na2SeO3) but not as high
as single solutions of double strength. Exogastrulation was highest in the cobalt,

8

14

FIGURES 8-14. Figures 8-11, 48-hour larvae that have been exposed continuously to M/800
sodium selenite. Figures 12-14, 48-hour larvae that have been exposed continuously to M/1,600
sodium selenite.

next in the combination, and least in the selenite solutions. The data have demon-
strated that the actions of these two different agents are additive in most respects
in affecting developmental pattern but that there are also some effects caused by
one agent but not (at least to an appreciable degree) by the other at the concen-
tration used.

2. Continuous exposure of newly fertilised eggs to single and combination solu-
tions of cobaltous chloride and sine chloride. As in the preceding experiments,
eggs from the same lot were placed in wide ranges (single and combination) of
concentrations. Since the single effects have previously been reported (Rulon,
1955. 1956) only the effects of selected solutions on development shall be reported
here. The solutions were as follows :

484

OLIX RULOX

Solution 8 — Sea water control

9 — M/3,200 cobaltous chloride
10— il//80.000 zinc chloride
11— M/ 160.000 zinc chloride

12— M/3,200 cobaltous chloride— M/ 160,000 zinc chloride (50 cc. of
M/1,600 CoCL-6H,O plus 50 cc. of 3//80.000 ZnCl.)

After 48 hours the eggs developing in the sea water control (solution 8) were prac-
tically 100 per cent normal free-swimming bilateral plutei with good development
of oral and anal arms. In solution 9 all were slightly flattened bilateral larvae

16

17

18

19

20

21

FIGURES 15-21. Figures 15-18, 48-hour larvae that have been exposed continuously to a
combination solution (If/1,600 cobaltous chloride-.V/l,600 sodium selenite). Figures 19-21, 48-
hour larvae that have been exposed continuously to a combination solution (.V/3,200 cobaltous
chloride-3//3,200 sodium selenite).

which were either actively swimming or moving about near the bottom of the cul-
ture (Figs. 22-24). Approximately 20 per cent had short anal arms with skeletal
spicules and 10-20 per cent had undergone exogastrulation. "While these larvae
were definitely in advance of those treated with cobalt solutions of twice the strength
(solution 3), they still showed markedly the effects of the agent.

In solution 10 all of the larvae (Figs. 25-27) were slow-moving and radial with
a large differentiated gut which commonly extended to the apical end (Fig. 25)
although there were approximately 10 per cent which showed exogastrulation.
These larvae had no skeleton and there was an excess of internal cells. Most
showed apical thickenings or extensions although the polar elongation fell short of

MODIFICATIONS IX THE SAXD DOLLAR

485

25

FIGURES 22-33. Figures 22-24, 48-hour larvae that have been exposed continuously to
.17/3,200 cobaltous chloride. Figures 25-27, 48-hour larvae that have been exposed continuously
to .V/80,000 zinc chloride. Figures 28-30, 48-hour larvae that have been exposed continuously
to -V/160.000 zinc chloride. Figures 31-33, 48-hour larvae that have been exposed continuously
to a combination solution (lf/3,200 cobaltous chloride-Af/160,000 zinc chloride).

that noted in certain effective solutions of cobalt and selenium. In solution 11
(which was one-half the concentration of zinc chloride as solution 10) over 90 per
cent of the larvae were bilateral (Figs. 28-30) and over 50 per cent of these had
short anal arms with skeleton. The remaining 10 per cent approached radial sym-
metry and there was approximately 5 per cent exogastrulation.

In solution 12 (combination solution) over 90 per cent were elongated radial
forms with neither skeleton nor arms (Figs. 31-33). Slight bilaterality was evi-
denced in the remaining 10 per cent. Most had well-developed entera but 20-30
per cent had undergone exogastrulation.

486 OLIN RULON

These experiments show that zinc chloride causes modifications similar to those
caused by cobaltous chloride but in concentrations that are I/ 100th those of the
latter. The addition of M/3,200 cobalt to M/160,000 zinc gives an effect which is
approximately that of a single solution of zinc of twice the concentration (M/80,000)
but not that of a single solution of cobalt of twice the strength (M/1,600). In-
stead the effect is that of a cobalt solution of four times the concentration (M/800).

DISCUSSION

Previous work by the author has shown that several substances are effective
in causing polar elongation and differentiation around a radial symmetry in the
developing sand dollar embryo. The present data show that cobalt and selenite
at the same concentrations will produce such modifications. They also show that
the effects of these two different agents were additive in causing radial symmetry
and almost additive in polar elongation and exogastrulation.

Other work has indicated that the radial symmetry described here is the result
of a process of ventralization (see Rulon, 1949) rather than a direct inhibition of
the factors which give bilaterality to the normal embryo. It has been shown (see
Child, 1941) that the ventral side of the early blastula has greater indophenol oxi-
dase (cytochrome oxidase) activity than the dorsal side, even though visible mor-
phological differences are not apparent. Neither cobalt nor selenite appears to in-
hibit the activity of ventral as much as dorsal regions. The ventral area therefore
spreads until it encircles the entire embryo. It is suggested that the similar effects
of cobalt and selenite may be related to their known inhibitory action of thiol groups
(see Rulon, 1955) and that enzymes important in symmetry relationships bear ac-
tive sulfhydryl radicals. It is further suggested that these enzymes are more dor-
sally located at the stage of development preceding visible bilaterality. Cytochrome
oxidase, whose greater activity at the ventral side has been proven, does not possess
an active sulfhydryl radical (Sumner and Somers, 1953, p. 9).

Polar elongation seems to have certain factors in common with ventralization.
Child (1941) also showed a polar gradient of cytochrome oxidase activity in these
eggs with the highest activity at the animal pole. If it is assumed that cobalt and
selenite are inhibiting activities other than cytochrome oxidase and more basally
located, then it would follow that the apical end would grow and become extended
at the expense of basal regions.

Zinc was highly effective in causing ventralization and polar elongation, and at
a concentration far below that of the other two agents. In other words, it was more
effective in inactivating, or partially inactivating, certain of the factors or processes
concerned with bilaterality and polarity. It may not be unreasonable to suggest
that the affinity of the thiol groups of certain enzymes for this ion may be much
greater than for cobalt or selenite ions. However, the picture may be more com-
plicated, as shown by the fact that a solution of zinc (M/160,000) which is effec-
tive in causing 10 per cent radial forms will, when administered along with M/3,200
cobalt, which causes no radial forms, cause 90 per cent radials. This would seem
to suggest a synergistic action although the complexity of the phenomenon does not
lend itself to an easy interpretation.

Cobalt was much more effective in the concentration used than was selenite in
causing exogastrulation and skeletal inhibition. This may be because the differ-

MODIFICATIONS IN THE SAND DOLLAR 487

entiation of gut and skeleton is of a more specific nature and therefore subject to
more specific influences than is either ventralization or polar elongation.

SUMMARY

1. Newly fertilized eggs of D. c.vcentricus were allowed to develop in single and
combined sea water solutions of cobaltous chloride, sodium selenite, and zinc
chloride.

2. Combination solutions of cobalt and selenite were additive in causing the de-
velopment of radial larvae and almost additive in causing polar elongation of the
larvae.

3. In solutions of equivalent strength the development of exogastrulae was
highest in cobalt, next in the combination, and least in the selenite solutions.

4. Solutions of zinc caused effects that were similar to those of cobalt but in
concentrations that were 1 /100th the latter.

5. Combination solutions of cobalt and zinc gave effects that indicated syner-
gistic action.

6. It is suggested that the effects of cobalt, selenite, and zinc are through their
reaction with the thiol groups of certain enzymes and that the greater effect of zinc
is because of a greater affinity for such groupings.

LITERATURE CITED

CHILD, C. M., 1941. Formation and reduction of indophenol blue in the development of an

echinoderm. Proc. Nat. A cad. Sci., 27 : 523-528.
RULON, O., 1949. The modification of developmental patterns in the sand dollar with maleic

acid. Physio!, Zool, 22: 247-261.
RULON, O., 1952. The modification of developmental patterns in the sand dollar by sodium

selenite. Physiol. Zoo!., 25 : 333-346.
RULON, O., 1953. The modification of developmental patterns in the sand dollar with nickelous

chloride. Anat. Rcc., 177: 615.
RULON, O., 1955. Developmental modifications in the sand dollar caused by zinc chloride and

prevented by glutathione. Biol. Bull., 109: 316-327.
RULON, O., 1956. Effects of cobaltous chloride on development in the sand dollar. Ph\siol.

Zool., 29 : 51-63.
SUMNER, J. B., AND G. F. SOMERS, 1953. Chemistry and methods of enzymes. Third Edition.

New York, Academic Press, Inc.

NOTES ON THE LIFE-CYCLE OF AZYGIA ACUMINATA GOLD-
BERGER, 1911 (AZYGIIDAE-TREMATODA)

DONALD M. WOOTTON 1
Marine Biological Laboratory, Woods Hole, Mass.

Studies of trematodes belonging to the genus Asygia Looss, 1899 from North
American fresh-water fish, date from the work of Leidy (1851). He described
Distoma longum from the stomach of Esox ester LeSueur, 1818 from near Cleve-
land, Ohio. Since that time many workers have added to knowledge of the North
American species belonging to this genus. Manter (1926) gave a systematic re-
view of the family Azygiidae and stated (p. 57) that "Azygia is the only genus of
the family showing taxonomic confusion in its species." He further pointed out
that these forms are all very muscular and highly contractile, which not only alters
the general shape of the worm but also changes the relative position of such struc-
tures as the acetabulum and the reproductive organs. He also mentioned that size
of the eggs and distribution of the vitellaria vary considerably within a species and
cannot be relied on as taxonomic characters. Manter recognized three valid North
American species: A. acuminata Goldberger, 1911, A. angusticauda (Stafford,
1904) and A. longa (Leidy, 1851).

Van Cleave and Mueller (1934) endorsed the action of Manter in reducing the
number of species in North America and felt that A. acuminata should also be re-
duced to synonymy with A. longa.

Stunkard (1956) gave a chronological account of the genus Asygia and noted
(p. 266) " — discordant observations and divergent opinions," concerning the pro-
posed specific and generic names for members of the genus. This thorough ac-
count need not be repeated here. Stunkard recognized A. sebago as a distinct spe-
cies and suggested that the European A. lucii may also be present in North Amer-
ica and that it possibly is distinct from A. longa.

None of the species have been studied to determine the extent of variation that
normally occurs as the result of early development in a wide variety of paratenic
hosts (small fishes and planarians) and further development in varied definitive
hosts (large fishes). Until such studies are undertaken, the taxonomic picture of
the group will remain confused.

It is evident, in reviewing the literature, that species proposed by various North
American authors and since placed in synonymy may indeed be valid species. In
such worms, which are very muscular and highly contractile, size and shape can not
be relied on as taxonomic characters except in a very general way. The fact that
these worms become sexually mature while relatively small and continue to grow
throughout life further complicates the taxonomic picture. A. angusticauda is the
only North American species which can be readily distinguished from the other
species described from this continent. It can be separated because of the more

1 Present address : Chico State College, Chico, California.

488

LIFE-CYCLE OF AZYGIA ACUMINATA 489

posterior position of the acetabulum and the presence of the gonads in the posterior
one-sixth of the body (usually within the posterior one-seventh to one-eighth of
the body). The rest of the species comprise a complex composed of A. longa,
A. sebago, A. lucii and A. acuminata. This complex may perhaps include as yet
unrecognized species, or species which at the present time are represented as
identical to A. longa.

The descriptions of the cercaria of A. longa by Sillman (1953a, 1953b), of the
cercaria of A. sebago by Stunkard (1956), and the description of the cercaria of
A. acuminata in the present report complete the life-histories of all of the recognized
species of Asygia in North America with the possible exception of A. lucii and
A. angusticauda. Dickerman (1937) named Cercaria angusticauda as a new spe-
cies, but did not describe the larva.

Moreover, in discussing the cystocercous cercariae of North America Horsfall
(1934) pointed out that C. wrighti Ward, 1916, C. anchoroides Ward, 1916, and C.
brookoveri Faust, 1918 appear to be typically azygiid in morphology and should
logically develop into adult azygiids. One of these cercariae may develop into A.
lucii, but if so it is morphologically distinct from the European larva.

Szidat (1932) showed that Cercaria mirabilis Braun is the larva of A. lucii in
Europe. Sillman (1953a) described the larva of A. longa from the snail, Amnicola
limosa, on the basis of experimental and limited natural infections. Sillman
(1953b) added additional information concerning the life-cycle of A. longa which
was reported from both the mud pickerel, Esox vermiculatus and Amia calva in the
vicinity of Ann Arbor, Michigan. Sillman also assigned worms from the bowfin,
Amia calva, to the suppressed species, A. acuminata. He was unable to experi-
mentally obtain infections of Amnicola from eggs of A. acuminata.

Stunkard (1950) identified larval distomes from the pharyngeal pockets of
planarians, Dugesia tigrinum, as immature azygiids. Planarians serve as para-
tenic hosts since further development of the distomes does not occur in this host.
Stunkard (1956) reported on the life-cycle of Azygia sebago and experimentally
showed that larval stages develop in Amnicola limosa; further that the cystocercous
cercaria is distinct from that reported by Sillman (1953a, 1953b) from the same
snail host. Stunkard also reported that these cercariae were ingested by small
fish (guppies and small blue gill sunfish, Lepomis macrochirus} and also were ob-
tained experimentally and naturally from the pharyngeal pockets of planarians,
Dugesia tigrinum. He stated (p. 265), "How the larvae reach the pharyngeal
pockets is not clear." On the basis of known feeding habits of planarians, he cor-
rectly suggested the manner in which the distomes enter the pharyngeal pockets but
did not observe it.

In preliminary observations during the present study, cystocercous cercariae
belonging to both A. sebago from Amnicola limosa and A. acuminata from Campe-
loma decisum (Say) were placed in finger bowls with Dugesia tigrinum. The
planarians reacted to close proximity of both species of larvae by raising the an-
terior end of the body and, when the swimming cercaria came in contact with the
under surface of the planarian, actively enclosing the cercaria against the glass
container. The pharynx was then extruded and the cercaria was sucked into the
protruded pharynx. Usually the tail entered first, always so in cercaria of A.
acuminata. The distome portion of A. sebago was sucked into the pharynx where
it was gradually divested of the tail and the distome became active and eventually

490

DONALD M. WOOTTON
PLATE I

FIGURE 1. Asyyia acuminata, adult (5.1 mm. in length), ventral view, collected from
Ameiurus ncbulosus.

FIGURE 2. Asyyia acuminata, miracidium in egg (0.064 mm. in length), from sketches
made of living larvae.

FIGURE 3. Azygia acuminata, redia (2.52 mm. in length), showing recognizable pharynx,
annulations of the body and developing cercariae.

LIFE-CYCLE OF AZYGIA ACUMINATA 491

crawled out of the pharynx into the pharyngeal pocket. Up to six A. sebago larval
distomes were seen to thus enter a single planarian. The larvae of A. acuminata
were also enfolded by the planarians. The tail was sucked in but the larger size of
the distome prevented both its entrance into the pharynx as well as into the aper-
ture to the pharyngeal pocket. Occasionally a distome would be sucked part way
into the pharynx and would remain so for several hours before finally becoming
detached. Observations indicated that D. tigrinum could and probably does
serve as a normal paratenic host for A. sebago but not for A. acuminata.

The cercaria of A. acuminata was found in Campeloma decisum obtained from
the Santuit River, a small stream near the settlement of Santuit, Cape Cod, Mass.
Small fishes, sticklebacks, Eucalia inconstant (Kirtland), Fundulus heteroclitus,
the trout, Salvclinus fontinalis (Mitchill), and small eels, Anguilla rostrata Le-
Sueur were obtained from streams known to be free of azygiid infections and were
utilized as paratenic hosts. Specimens of each lot were examined and found to be
free of azygiids and the remainder used, after infection, in feeding the definitive
host fishes. These were found to be bull-heads, Ameiurus nebulosus LeSueur ;
blue gill sunfish, Lepomis macrochirus Raf . ; chain pickerel, Esox niger LeSueur ;
and yellow perch, Pcrca flavescens (Mitchill). Yellow perch do not occur in this
stream. These hosts were collected from a small pond known to be free of azygiids
and were experimentally infected.

Large numbers of snails, Campeloma decisum, were kept in shallow, well aerated
aquaria. In some instances liberated cercariae were pipetted from the aquarium
and fed to small fish, and in other cases small fish were kept in the same aquarium.
Heavy infections resulted in both cases since the swimming cercariae were readily
ingested by the fish.

The young distomes differed in their relationship to the different species of
small fish. Young trout and Fundulus heteroclitus retained an infection for only
six to seven days. During this time the worms became less active and were found
further and further posterior in the intestine of the fish. Sticklebacks and eels re-
tained most of their infection in the stomach region. Worms removed from the
latter species of fish were more active and their caeca were crowded with ingested
material. Sticklebacks retained up to ten worms in the anterior portion of the in-
testine for as long as two weeks, whereas eels were found to have the stomach
crowded with as many as twenty-four distomes.

Small fish were ingested by the larger fish used in the feeding experiments. The
pectoral and dorsal spines of the sticklebacks were clipped before placing them in
the aquarium with the definitive hosts.

STAGES IN THE LIFE-CYCLE
Adult (Fig. 1)

The original description of A. acuminata by Goldberger (1911) emphasized
taxonomically-unimportant points such as zigzag caeca, rounded cephalic and

FIGURE 4. Azygia acuminata cercaria, naturally emerged, from sketches of living speci-
men (4.20 mm. in length). Flame cell pattern of the tail, transposed from sketches made from
other cercariae, shown on right side of tail. Detail of musculature shown on the left, omitted
on the right side of the tail.

FIGURE 5. Asygia acuminata, juvenile worm (1.18 mm. in length), experimental infection,
from stomach of small eel, Anguilla rostrata.

492 DOXALD M. WOOTTOX

pointed caudal ends, uninterrupted vitellaria and constricted neck region in his
specific diagnosis.

Planter (1926} studied Goldberger's material, also material identified by A. R.
Cooper, and specimens from H. B. Ward's collection. He suggested that certain
similarities in the material showed A. acuminata to be a valid species. Manter
stated (p. 61). "The most distinguishing specific characters were found to be: rela-
tively wide body, anterior extent of the vitellaria, egg size, and poorly developed
condition of the internal parenchyma muscles. It should be realized that the na-
ture of all of these features is of somewhat precarious standing in this group.
Probably no one of them, unless very marked, would justify recognition of a sepa-
rate species. Only because of the general association of all of these characters can
the forms be separated from the other common American species."

Van Cleave and Mueller (1934) regarded A. aciuninata and A. bulbosa of
Goldberger (1911) as synonyms, and since Manter had regarded A. bulbosa as a
synonym of A. longa. they added A. acuminata to the long list of synonyms of
A. longa. They pointed out that while the anterior extent of vitellaria was an im-
portant taxonomic character in the genus, the comparative width, constriction of the
neck, and insignificant differences in egg size did not justify maintaining A. acumi-
nata as a valid species. They stated that the less highly developed condition of the
internal muscles was a dubious character. On the basis of the present study it ap-
pears evident that Van Cleave and Mueller were not justified in reducing A. acumi-
nata to synonymy with A. longa; that A. bulbosa should be considered synonymous
with A. acuminata; and the species should be maintained in the genus Azygia.

En: ended description of the adult

Characters of the genus, body. 4—10 mm. long, width usually less than one-fifth
of the length. Body characteristically slightly constricted anterior to the aceta-
bulum. Position of constriction variable depending on state of contraction of the
worm. Acetabulum one-fourth to one-third total length from anterior end of body.
Genital pore and cirrus sac median in position immediately cephalad of acetabulum.
Oral sucker 0.25-0.90 mm. in diameter, acetabulum slightly larger, 0.28-0.95 mm.
in diameter. Gonads contiguous. Posterior testis larger than anterior testis.
Yitellaria extra-caecal, extending from level of posterior margin of acetabulum to
level midway between posterior testis and caudal end of body. Eggs consistently
larger than in other described azygiid species. 0.64-0.69 X 0.30-0.34 mm.

Host: Anna cah-a, Lcpomis macrochirus, and Ameiurus nebulosus.

Habitat: Stomach.

Localities : Illinois, Michigan, and Cape Cod, Massachusetts.

Miracidin

Miracidia representing all genera of the family Azygiidae have been described
except for the genus L. --irus. Azygiid miracidia lack cilia and are provided

with bristle plates or plaques. The miracidium of A. acuminata is morphologically
similar to the miracidium of A. sebago described by Stunkard (1956), to that of
Proterometra macrostoau: as reported by Hussey (1945), and to the earlier de-
scriptions of that of A. longa by Schauinsland (1883) and Looss (1894).

Eggs must be ingested by the proper snail hosts before hatching will occur.

LIFE-CYCLE OF AZYGIA ACUMIXATA 493

Attempts to hatch the eggs by placing them with material from the digestive tract
of large Campeloma decision were unsuccessful.

Miracidia were studied both alive in the egg (Fig. 2) and in stained sections of
gravid worms. The miracidium almost completely fills the egg-shell. Radiating
from the anterior end are five plaques bearing fine bristles arranged in chevron
fashion with the apices anterior in position. These plaques extend posteriad about
one-third of the length of the larva. A short distance from the posterior end of the
miracidium, four other bristle plaques extend anteriad with a tendency to spiral,
which may be due to movements of the larva.

Internally, occupying almost the anterior one-third of the miradicium. are
four unicellular gland-like structures, the so-called primitive gut of earlier authors,
which Stunkard considered probably secrete substances which aid in penetration.
"Wcotton (1957) demonstrated that in Allocreadhim alloneotenicum this group of
glands does aid in penetration and that the penetration glands of earlier authors
serve in forming the cuticle of the sporocyst. Attempts, in crushed snails, to
observe the action of the anterior gland in the miracidium of A. a-cuminata were
unsuccessful. It is possible since ciliary plates are lacking in azygiid miracidia that
the miracidial covering serves also as the covering of the sporocyst, thus explaining
the absence of cuticle-forming glands. Up to twelve germinal cells are visible in the
posterior two-thirds of the miracidium. Paired flame cells lie near the middle of
the body, one on each side, each with a duct leading caudad. The ducts could not
be clearly traced to their pores.

Sporocyst

Young snails were removed directly from the uterus of an uninfected female
Campeloma decision. The young snails readily fed in a layer of clean sand. Eggs
of A. acuminata, each containing an active miracidium. were added to the sand in
which the snails were feeding. Snails were dissected at the end of one, two, and
three weeks but no infection was found. It is probable that the snails must be
larger before infection will occur.

Rediae

Infections with larvae of A. acioninata were present only in females of Campe-
loma decision. Infections of male Campeloma were never observed. The redial
stages were present, usually with unencysted metacercariae identified as Leuco-
chloridiomorpha constantiae (Mueller), in the uterus of the snail. The loci of in-
fection did not extend into the digestive gland, the usual site of infection for larval
trematodes. Up to eighty rediae were dissected from the uterus of a single snail.
These, plus almost as many metacercariae of L. constantia-e, completely filled the
uterus. Embryo snails, which were usually found in the uterus of uninfected fe-
males, were absent in infected snails. In a few instances partially empty embryo
snail shells were found in the uterus, but it was evident that infection with larval
A. acuminata adversely affected normal development of the young snails. The
redial stages undoubtedly derived their nourishment from the developing embryo
snails within the uterus. Young snails develop normally when associated only
with metacercariae of L. constantiae. Thus, infection of the snail with larvae of

494 DONALD M. WOOTTON

A. acuminata caused the degeneration of the young snails. Rediae (Fig. 3) when
fixed averaged 2.15 X 0.71 mm.

The vermiform rediae are very active and are capable of extending to a length of
over 5 mm. When contracted, the external body wall is formed into regularly
spaced annular rings, giving it a wrinkled appearance. Even when fully extended,
these rings persist as fine annular projections. The body wall is 0.043 mm. in
thickness in sectioned material. The small pharynx is not easily observable in
stained whole mounts, but is visible in sections as a rudimentary structure (0.11
mm. in diameter) with a well defined lumen. No recognizable birth pore can be
seen, either in sectioned materials or in stained whole mounts or rediae. It is prob-
able that cercariae escape through the pharynx.

Up to twelve recognizable cercariae are present in each redia, with seven
to ten being the usual number. Other developing cercariae are present as germ-
balls, and a maximum number of twelve developing cercariae present with six
germ-balls was observed. The redial stage is very similar to that reported by Szi-
dat (1932) for A. lucii and by Stunkard (1956) for A. sebago, varying only in
size and in number of developing cercariae.

Cercariae

The development of the cercaria is typical of that reported for cystocercous
larvae. The tail becomes evident in development when the larva reaches a length
of only about 0.1 mm. When the larva reaches a total length of 0.5-0.6 mm. the
furcal buds appear as oval projections. At this stage the suckers are recognizable,
and the primordia of one testis and the cirrus sac are also visible as deeper staining
areas. The tail increases more rapidly than the distome during further develop-
ment. The largest cercaria observed within a redia as a stained whole mount
measured 2.22 mm. in total length. The distome measured 0.74 X 0.37 mm.

Cercariae appear to undergo additional development in the uterus of the snail
before escaping from the female genital pore. After emergence, they are quiescent
for a brief period before actively swimming in a typical cystocercous fashion. Cer-
cariae normally emerge between 12 :00 P.M. and 4 :00 A.M. Standard Time. Lim-
ited numbers escape during daylight hours. It appears, however, that the ma-
jority emerge during hours of darkness and are either ingested by small fish at this
time or in the early hours of daylight. They live for only ten to twelve hours, be-
coming less active as they age. Cercariae while still within the uterus of the snail
do not have the distome portion enclosed by the tail. Upon coming into contact
with the water, the anterior tail bulb absorbs water, expands rapidly anteriorly thus
enclosing the distome, as has been described for other cyctocercous cercariae.

Mature, normally liberated cercariae (Fig. 4) measure 3.21-4.69 mm. (averaged
4.22 mm.) in total length when infected snails are first brought into the laboratory.
The size of the cercariae gradually decreases in snails kept in captivity, undoubtedly
a result of deficient nutrition of the hosts. The tail stem is round in cross-section
at the bulb-like anterior end enclosing the distome. This portion measures 0.69-
0.96 mm. in diameter (average 0.78 mm.). Just posterior to the more or less rigid
anterior bulb enclosing the distome, the tail decreases slightly to a diameter of 0.52-
0.82 mm. (average 0.66 mm.). From the constricted neck-portion the tail gradu-
ally flattens and widens to a width of 0.62-1.11 mm. (average 0.84 mm.) and then
tapers gradually to an average width of 0.79 mm. just anterior to the furcal branches.

LIFE-CYCLE OF AZYGIA ACUMINATA 495

The furci are broadly lobed structures, 1.11-1.28 mm. in length (average 1.21
mm.) and 0.89-1.09 mm. in width (average 0.94 mm.). Each furca has a terminal
papilla on which the excretory pore opens and small regularly arranged scale-like
marginal protuberances. The tail of the cercaria is colorless, slightly opaque and
devoid of protuberances, spines and mammulations characteristic of C. mirabilis
Braun, 1891, C. niacrostoma Faust, 1918, C. splendens Szidat, 1932, C. anchoroides
Ward, 1916. and C. sebago Stunkard, 1956. The cercaria of A. acuminata dif-
fers in size and in the proportionate size of the distome when compared with the
tail length from other cystocercous cercariae which characteristically do not possess
papillae. It differs from A. hodgesiana Smith, 1932 since the genital organs are not
functional as they are in the latter; from A. stephanocauda Faust, 1921 in size and
shape of the tail; from C. ivrighti Ward, 1916 in size; and from C. pekinensis
Faust, 1921 in proportionate size of the distome.

The cercaria of A. acuminata is most like C. brookoveri Faust, 1918 and C.
anchoroides Ward, 1916, but is over twice as large. C. brookoveri was originally
described from crushed Campeloma sp. and the free-swimming larva was rediscov-
ered by Dickerman (1937) from the same snails. Unfortunately Dickerman did
not further describe the species. C. anchoroides was collected only in plankton tows
from Lake Erie. The size and obvious similarities in structure of the two forms,
as well as geographic proximity of the type localities, caused Horsfall (1934) to
think that they will prove to be synonymous when the life-cycles are known.

The enclosed distome measures 0.66 -0.79 X 0.37-0.47 mm. in living material.
It usually lies with the oral sucker at the anterior end of the tail-bulb. It is flat-
tened and varies as to its orientation to the width of the tail, sometimes being at
right angles and at others with its width the same as the width of the tail. The
excretory system is continuous with that of the tail, extending down the tail as a
common excretory canal bifurcating at the bases of the furci and opening at the
small points of the furci.

The structure of the larva when forced from the tail-bulb is typically azygiid.
The preacetabular region bears many papillae which decrease in size and number,
and are absent behind the mid-acetabular region. Living specimens, flattened
slightly under a cover glass, measure from 0.98—1.38 mm. (average 1.18 mm.) in
length and from 0.37-0.54 mm. in width (average 0.48 mm.). The oral sucker
varies from 0.22-0.25 mm. in length and from 0.20-0.23 mm. in width. It is sub-
terminal, opening ventrally. The pharynx measures 0.090-0.098 mm. in length and
0.49-0.61 mm. in width. The acetabulum varies from 0.22-0.25 mm. in length and
from 0.25-0.29 mm. in width. It is about midlength in the body. The digestive
caeca are filled with opaque material and extend almost to the posterior end of the
larva. The excretory bladder extends anteriorly to the region just posterior to the
testes where it branches into two main collecting ducts. These extend median to
the caeca, crossing laterad as the caeca turn mediad to join the pharynx. After
crossing under the caeca, the ducts pass laterally and antero-laterally to the oral
sucker, continuing almost to the anterior end of the body, but they do not join.
Anterio-lateral to the oral sucker each duct doubles backward and extends posteriad,
lateral to the caeca, giving off eleven branches.

The first branch is located lateral to the oral sucker, the second at the level of
the pharynx, the third and fourth anterior to the acetabulum, the fifth at the anterior
edge of the acetabulum, the sixth lateral to the acetabulum, the seventh and eighth

496 DONALD M. WOOTTON

are close together just behind the acetabulum, the ninth and tenth are about equally
spaced in the intervening region, while the last branch continues to the posterior end
of the body lateral to the excretory bladder. Each branch divides three times in a
dichotomous fashion, thus forming two primary, four secondary, and eight tertiary
branches. Each tertiary branch drains four flame cells. Thus the flame cell
formula is 2 (11 X 32) or 704 flame cells.

The number and arrangement of the flame cells is in agreement with those re-
ported by Looss (1894) for Asygia terreticolla ( = A. lucii) and by Stunkard
(1956) for A. scbago. While the numbers of branches and flame cells agree with
these earlier descriptions, the positions of the branches are different in A. acuminata
due to the relatively more posterior position of the acetabulum.

The excretory system of the tail is equally complex (Fig. 4). In addition to
the common excretory canal extending down the center of the tail and bifurcating
into each furca, two paired accessory canals paralleling the main canal were ob-
served. One duct and its branches drained the right side of the tail and the right
furca, and the other the left side and the left furca. Each duct collected from five
branches but dichotomous bifurcation of the ducts was not as clearly evident as in
the distome portion.

Each of the five branches, however, did drain from 32 flame cells, arranged in
groups of fours. The first branch turned anteriad from just caudad of the distome,
draining the enclosing bulb area, the second and third branches joined the collecting
duct close together in about the middle of the tail, the second turning anteriad and
draining that area, the third posteriad collecting from the third quarter of the tail.
The fourth branch joined the collecting ducts about three-fourths the length of the
tail and drained the final quarter of the tail. From the fourth branch, the duct ex-
tended into a furca draining from 32 flame cells. The formula for the tail is thus
2 (5 X 32) or 320 and the entire cercaria has a formula of 2 (16 X 32) or 1,024
flame cells.

The connection of the accessory ducts of the tail to the rest of the excretory sys-
tem was not resolved. The dense protoplasm at the tip of the tail in immature cer-
cariae freed from rediae and the congested area at the base of distomes enclosed in
the tail of normally liberated cercariae made observations impossible. These
ducts are 0.011 mm. in diameter compared to the common duct which is 0.055 mm.
in diameter. Faust (1921) reported that in C. pekinensis, the tail had only 32 flame
cells and connected to the excretory system of the distome as the eighth branch.
He further reported only seven branches in the distome portion of C. pekinensis.
The flame cell pattern of this form should be examined in the light of the observa-
tions of the cercariae of A. lucii Looss (1894), A. sebago Stunkard 1956, and the
present observations of A. acuminata, since C. pekinensis would appear to also
develop into an azygiid.

Young worms

Worms increased very little in size and did not undergo further development
while in the stomach of small fishes. A young distome (Fig. 5) from the stomach
of a small eel differed from one newly forced from the cercarial tail only in the
size of the caeca. In worms taken from paratenic hosts, the caeca were enlarged
with food particles. No measureable differences in worms from various hosts

LIFE-CYCLE OF AZYGIA ACUMINATA 497

were found. Worms from the sticklebacks and young eels were more active and
appeared healthier than worms from other small fish.

Manter's synopsis and key to the genus Azygia can be revised to include A,
sebago and the cercaria of each species can be noted as follows :

KEY TO THE SPECIES OF AZYGIA FROM NORTH AMERICA

1 (2) Vitellaria not extending appreciably posterior to the last testis. Length 6-54 mm.

(C. mirabilis Braun, 1891 ) A. lucii (Mueller)

2 (1) Vitellaria extending posteriad at least half the distance between posterior testis and end

of body 3

3 (4) Acetabulum near middle of body, gonads in posterior one-sixth of body (C. angiisti-

cauda Dickerman, 1937) A. angusticauda (Stafford, 1904)

4 (3) Acetabulum within anterior one-third of body, gonads more anterior 5

5 (6) Body width usually one-fifth the total length, vitellaria extending posteriad from close

behind acetabulum, internal parenchyma muscles relatively weak. Eggs 0.064-0.069 X
0.30-0.34 mm. (Cercaria acuminata, present paper) A. acuminata Goldberger, 1911

6 (7) Body width proportionately less than one-fifth the length, vitellaria begin some distance

posterior to acetabulum, internal parenchyma muscles strongly developed, eggs variable
in size but smaller than A. acuminata 7

7 (8) Body length not over 15 mm., usually smaller, body robust in appearance (Cercaria

sebago Stunkard, 1956) A. sebago Ward, 1910

8 (7) Body often extremely elongate, vitellaria beginning proportionally more posteriorly

(Cercaria longa Sillman, 1953a) A. longa (Leidy, 1851).

The writer wishes to express his appreciation to the Director of the Marine
Biological Laboratory, Woods Hole, Mass., for the use of facilities. Further, he is
particularly grateful for the helpful suggestions offered by H. W. Stunkard through-
out this investigation and for his criticism of the manuscript.

SUMMARY

1. Stages in the life-cycle of Azygia acuminata are described and figured.
Cystocercous cercariae develop from rediae in the snail, Campeloma decisum. The
cercaria is morphologically distinct from other described cystocercous cercariae.
Rediae are similar to the same stage described for other members of the genus, but
are unique since they develop in the uterus of female Campeloma decisum.

2. The excretory system of the cercaria is complex, showing a formula of 2 (11
X 32) or 704 flame cells for the distome portion and 2 (5 X 32) or 320 flame cells
in the tail. The excretory formula of the cercariae is thus 2 (16 X 32) or 1,024
flame cells.

3. Attempts to experimentally infect small snails, taken from the uterus of a
Campeloma decisum, by feeding them eggs of A. acuminata were not successful.

4. Various small fishes were utilized as paratenic hosts by the young distomes.
Infections in sticklebacks, Eucalia inconstans, and small eels, Anguilla rostrata, re-
sulted in more active and vigorous worms than did infections from other paratenic
hosts.

5. The variation that normally occurs in members of the genus Asygia due to
development in a wide variety of hosts is not known. Consequently diagnostic
characters of mature worms can not be relied on to distinguish species. On the
basis of this report the suppressed species A. acuminata is regarded as a valid species
and should be retained in the genus Azygia.

6. A. acuminata, previously reported only from Amia calva, was found occur-

498 DONALD M. WOOTTON

ring naturally in bullheads, Auiciunis ncbulosus, blue gill sunfish, Lcpomis mu-cro-
chirus, and chain pickerel, Esox niger, from Santuit River, Barnstable County, Cape
Cod, Massachusetts. Experimental infections were also obtained in these fishes
and in the yellow perch. Pcrca flarescens.

7. A revised key for the genus Azygia is presented, listing the recognized spe-
cies and the described cercarial stages.

LITERATURE CITED

BRAUX, M.. 1891. Die sogenannte "freischwimmende Sporocyste." Zentralbl. BakterioL, 10:
215-219.

DICKERMAX. E. E., 1937. Cystocercous cercariae of the mirabilis group from Lake Erie
snails. /. Parasitol.. 23 (6) : 566.

FAUST, E. C, 1918. Two new cystocercous cercariae from North America. /. Parasitol., 4:
148-153.

FAUST, E. C.. 1921. The excretory system in Digenea (Trematoda). IV. A study of the de-
velopment of the excretory system in a cystocercous larva, Cercaria pekinensis, nov. sp.
Parasitol., 13 : 205-212.

GOLDBERGER, JOSEPH, 1911. Some known and three new endoparasitic trematodes from Ameri-
can fresh-water fish. Bull. Hyg. Lab., 71 : 7-35.

HORSFALL, MARGERY \V., 1934. Studies on the life history and morphology of the cystocercous
cercariae. Trans. Aincr. Micros. Soc., 53: 311-347.

HUSSEY, KATHLEEX L., 1945. The miracidium of Proteromctra macrostoma (Faust) Horsfall,
1933. /. Parasitol., 31: 269-271.

LEIDY, JOSEPH, 1851. Contributions to helminthology. Proc. Acad. Nat. Sci. Philadelphia, 5:
205-210.

Looss, A., 1894. Die Distomen unserer Fische und Fr<")sche. Biblioth. Zoo/., Stuttgart, 6 (16) :
1-296.

Looss, A., 1899. YVeitere Beitrage zur Kenntnis der Trematoden-Fauna Aegyptens, zugleich
Versuch einer naturlichen Gliederung des Genus Distomum Rctzius. Zoo/. Jahrb.
Syst.. 12: 521-784.

MAXTER, H. W., 1926. Some North American fish trematodes. Illinois Biol. Monogr., 10
(2) : 1-138.

SCHAUIXSLAXD, H., 1883. Beitrag zur Kenntnis der Embryonalentwicklung der Trematoden.
Jenaischcn Zeitschr. Xaturin.'., 16 : 465-527.

SILLMAX, E., 1953a. The lite history of Azygia longa (Leidy, 1851) (Trematoda: Azygiidae).
/. Parasitol.. 39 (suppl.) : 15.

SILLMAX, E., 1953b. Morphology and life history studies on Azygia longa (Leidy, 1851) and
Azygia acuminata Goldberger, 1911, with taxonomic considerations in the genus
Azygia Looss, 1899 (Trematoda: Azygiidae). Thesis: University of Michigan, Ann
Arbor, Mich.

SMITH, SEPTIMA C., 1932. Two new cystocercous cercariae from Alabama. /. Parasitol., 19:
173-174.

STAFFORD, J., 1904. Trematodes from Canadian fishes. Zoo/. Anz., 27: 481-495.

STUXKARD, H. W., 1950. Larval trematodes from the planarian, Dugesia tigrinum. Biol.
Bull., 99 : 347-348.

STUXKARD, H. W., 1956. The morphology and life-history of the digenetic trematode, Azygia
scbago Ward, 1910. Biol. Bull., Ill : 248-268.

SZIDAT, L., 1932. Ueber cysticerke Riesencercarien, insbesondere Cercaria mirabilis Braun und
Cercaria splcndcns n. sp., und ihre Entwicklung im Magen von Raubfischen zu Tremato-
den der Gattung Azygia Looss. Zeitschr. f. Parasitenk., 4 : 477-505.

VAN CLEAVE, H. J., AXD J. F. MUELLER, 1934. Parasites of Oneida Lake fishes. III. A bio-
logical and ecological survey of the worm parasites. Roosevelt Wildlife Annals. 1 :
161-334.

WARD, H. B., 1916. Notes on two free-living larval trematodes from North America. /.
Parasitol., 3: 10-20.

WOOTTOX, D. M., 1957. Life-history of Allocrcadium alloneotenicum, n. sp. (Allocreadiidae —
Trematoda). Biol. Bull, 113: 302-315.

INDEX

A TP, effect of on luminescence of millipede
extracts, 120.

ABBOTT, D. P. Sec J. H. PHILLIPS, JR., 296.

Abstracts of papers presented at the M. B. L.,
316.

Acclimatization of crabs, 268.

Acetylcholine, effect of on scorpion heart-beat,
135.

Acorn barnacle, body and shell growth in, 224.

Acrasiae, encystment in member of, 58.

Actin, bound nucleotide of, 333.

Action of ovarian extracts on mitosis, 129.

Activity, correlation of with barometric pres-
sure, 112.

Activity of fiddler crabs as affected by tem-
perature, 245.

Activity of Pachygrapsus, in relation to time
of day and tidal phase, 268.

Activity rhythm in salamander, 188.

Adaptive significance of activity rhythm in
salamander, 188.

Adaptive significance of osmoregulation in
crab, 268.

Adenosine triphosphate, effect of on lumines-
cence of millipede extracts, 120.

Adrenaline, effect of on scorpion heart-beat.
135.

Aging in relation to terminal growth in Cam-
panularia, 233.

AIELLO. E. Energy metabolism and ciliary
activity of Mytilus gill, 335.

AIELLO, E. The influence of the branchial
nerves and of 5-hydroxytryptamine on the
ciliary activity of Mytilus gill. 325.

Albino snails, starvation and desiccation of, 89.

Alga, correlation of oxygen consumption of,
with barometric pressure, 112.

Algae, freezing and drying in, 275.

Alkaline phosphatase activity in spiny lobster
exoskeleton, 451.

ALLEX, M. J. Breeding of polychaetous an-
nelids near Parguera, 49.

Allocreadium, life-history of, 302.

Amino acids, effects of on perfused lobster
heart, 345.

Amino acids, uptake of by ciliary-mucoid fil-
ter-feeders, 356.

Amphibian, activity rhythm in, 188.

Amphibian, correlation of oxygen consumption
of, with barometric pressure, 112.

Amphibian gastrulae, phosphorus balance of,

376.
Amphibian tadpole, phenylthiourea treatment

of, 160.

Anaerobiosis in Rana gastrulae, 382.
Analyses of Ciona blastomeres, 365.
Anatomy of Allocreadium, 302.
Anatomy of Azygia, 488.
Anatomy of Nereis nephridium, 407.
Anemones, sea, nematocyst toxin of, 296.
Annelid, morphology of nephridium of. 407.
Annelid eggs, effects of nitrogen mustards on,

388.
Annelid esgs, effects of ovarian extracts on,

129.

Annelids, commensal, behavior of, 397.
Annelids, endoparasitic, 170.
Annelids, polychaete, breeding habits of, 49.
Annual Report of the M. B. L.. 1.
Antimitotic action of ovarian extracts, 129,

318.

Antitoxic response to nematocyst toxin, 296.
Anuran, phenylthiourea treatment of, 160.
Anuria in lobsters, 207.
Apyrase activity of Ciona blastomeres. 365.
Arabellidae, endoparasites of, 170.
Arctic Fucus, oxygen consumption of, 275.
Arctonoe as commensal, 397.
ARMSTRONG, P. B. Motility in developing

teleost embryos. 325.

Arrested gastrulae, phosphorus balance of, 376.
Artemia as food for Campanularia, 233.
Arthropod, body and shell growth in, 224.
Arthropod, cardiac physiology of. 135.
Arthropod, excretion in, 207.
Arthropod, luminescence of, 120.
Arthropod, molting cycle of, 451.
Arthropod, osmoregulation in, 268.
Arthropod, respiratory metabolism of. 245.
Arthropod, sound production in, 286.
Arthropod cuticle, structure of, 141.
Ascidian blastomeres, chemical analyses of,

365.

Ascophyllum, freezing and drying in. 275.
ASHTOX, F. T. Magnetic studies on cells and

protoplasm, 319.

ASHTOX, F. T. Sec L. Y. HEILBRUXX. 318.
Assay of Metridium nematocyst toxin, 296.
Atropine, effect of on scorpion heart-beat. 135.
AUCLAIR, \Y. .SVr D. MARSLAXD, 348

499

500

INDEX

Australorbis, starvation and desiccation of, 89.
Autolytus, breeding habits of, 49.
Autoradiograms of Hyla tadpoles, 160.
Azide, sodium, effects of on Rana gastrulae,

382.
Azygia, life-cycle and morphology of, 488.

gACHMURSKI, D. See J. S. ROTH, 332.

Balanus, body and shell growth in, 224.
BANG, F. B. Reaction to injury in the oyster,

335.

BANG, F. B. See A. WARWICK, 334.
Barnacle, body and shell growth in, 224.
Barometric pressure as correlated with bio-
logical activity, 112.

Behavior of commensal polychaetes, 397.
Behavior of spiny lobsters, 286.
Behavioral mechanism for osmotic regulation

in crab, 268.

BENNETT, M. V. L., S. M. GRAIN and H.
GRUNDFEST. Patterns of response and
neural organization of supramedullary
neurons of Spheroides, 325.
BENNETT, M. V. L. See S. M. GRAIN, 342.
BERG, W. E. Chemical analyses of anterior and

posterior blastomeres of Ciona, 365.
Binding of radioactive iodine by tadpoles, 160.
Biological activity, correlation of with baro-
metric pressure, 112.
Bioluminescence in millipedes, 120.
Birds, testicular growth in, 254.
Bladder, urinary, of lobster, 207.
BLASKOVICS, J. C., AND K. B. RAPER. Encyst-

ment stages of Dictyostelium, 58.
Blastomeres, Ciona, chemical analyses of, 365.
Blood, crab, sodium and potassium concentra-
tions in, 268.

Blood analyses of lobster, 207.
Blood of Uca, hormone level of, 426.
BLOOMFIELD, D. K. Sec A. LAZAROW, 414.
BLUNT, SISTER M. X. Sec E. P. ODUM, 323.
Body growth in Balanus, 224.
Body weight of snails, 89.
BOOKHOUT, C. G. Sec J. D. COSTLOW, JR.,

224.

VON BRAND, T., P. MCMAHON AND M. O.
NOLAN. Physiological observations on
starvation and desiccation of the snail
Australorbis, 89.

Breeding habits of polychaetes, 49.
BRIDGMAN, J. Lethal irradiation of Tillina in

its active and encysted states, 336.
Brine shrimp as food for Campanularia, 233.
Bromsulfalein, effect of on lobster excretion,
207.

BROWN, F. A., JR., J. SHRINER AND H. M.
WEBB. Similarities between daily fluctua-
tions in background radiation and oxygen
consumption in the living organism, 103.

BROWN, F. A., JR., H. M. WEBB AND E. J.
MACEY. Lag-lead correlations of baro-
metric pressure and biological activity,
112.

BRYANT, S. H. Accessory fiber synaptic exci-
tation of squid stellar axons, 359.

BUCK, J. See J. F. CASE, 337.

BUCKMANN, D. A morphological color change
controlled by molting hormone in Lepi-
doptera, 326.

Buffer action, effect of on fish islet tissue
metabolism, 414.

Bugs, cuticle of, 141.

BURGEN, A. S. V., AND S. W. KUFFLER. The

inhibition of the cardiac ganglion of Limu-

lus by 5-hydroxytryptamine, 336.
BURGEN, A. S. V. See C. R. ELIOT, 344;

P. E. S. ENGER, 345.
BURGER, J. W. The general form of excretion

in the lobster, Homarus, 207.

(CADDIS flies as hosts of Allocreadium, 302.

CAGLE, J. See A. K. PARPART, 331.

Calcareous plates of barnacles, 224.

Calcification of spiny lobster exoskeleton, 451.

Calcium, effect of on lobster excretion, 207.

Calcium, effect of on metabolism of fish islet
tissue, 414.

Calcium, role of in electrical responses of frog
muscle, 329.

Calcium, role of in hardening of spiny lobster
exoskeleton, 451.

Calcium-45, metabolism of by Lebistes, 442.

Calorimetric measurements of algae, 275.

Campanularia, terminal growth in, 233.

CAMPBELL, M. A. Larval development of
Streblospio, 336.

Cancer, ovarian extracts in treatment of, 129.

Cancer chemotherapy, 388.

CANTOR, M. H. See C. L. CLAFF, 326; F. N.
SUDAK, 357.

Carcinostatic action of ovarian extracts, 129.

Cardiac physiology of fish, 359.

Cardiac physiology of scorpion, 135.

Caribbean annelids, breeding habits of, 49.

CARLSON, F. D., AND A. SIGER. The depend-
ence of creatine phosphate and ATP
breakdown on work in iodoacetate-
poisoned muscles, 324.

Cartesian diver, use of in study of fish islet
tissue, 414.

CASE, J. F., AND J. BUCK. Electrical stimu-
lation of light emission in fireflies, 337.

INDEX

501

CASE, J. F., C. EDWARDS, R. GESTELAND AND
D. OTTOSON. The site of origin of the
nerve impulse in the lobster stretch re-
ceptor, 360.

GATHER, J. N. See A. C. CLEMENT, 340.

Cell division, effects of nitrogen mustards on,
129, 388.

Cell division, effects of ovarian extracts on,
129.

Cellular particulates, irradiation of, 198.

Centrifugation, differential, of Uca sinus gland
granules, 426.

Centrifuged Arbacia eggs, premature furrow-
ing in, 348.

Centrifuged Nereis egg, cortical response of,
341.

Ceratonereis, breeding habits of, 49.

Cercaria of Allocreadium, 302.

Cercaria of Azygia, 488.

CERF, J., H. GRUNDFEST, G. HOYLE AND F. V.
McCANN. The nature of electrical re-
sponses of doubly-innervated insect muscle
fibers, 337.

CERF, J., H. GRUNDFEST, G. HOYLE AND F. V.
McCANN. Neuromuscular transmission
in the grasshopper Romalea, 338.

CHAET, A. B., AND W. R. CLARK, JR. The
demonstration of histamine in heparin-
containing invertebrate cells, 339.

Chaetacanthus, breeding habits of, 49.

Chaetopterus eggs, effects of nitrogen mus-
tards on, 388.

Chaetopterus eggs, effects of ovarian extracts
on, 129.

Chamberlain, N. A. Larval development of
the mud crab Neopanope, 338.

CHASE, A. M. Uricase inactivation by urea,
320.

CHASE, A. M. See E. N. HARVEY, 347.

Chemical agents, effects of on sand dollar de-
velopment, 480.

Chemical analyses of Ciona blastomeres, 365.

Chemotherapy of cancer, 388.

CHENEY, R. H. Dioxypurine derivatives as
mitotic and growth inhibitors, 339.

CHENEY, R. H. Fertilizability of Arbacia
eggs after pretreatment in trimethylated
xanthine derivatives, 339.

Chloride regulation in lobster, 207.

Chondrus, freezing and drying in, 275.

Chromatography of Metridium nematocyst
toxin, 296.

Chromatophorotropic hormones in Uca, 426.

Chromosome-cytoplasm interactions in devel-
opment of Sciara, 323.

Ciliary activity of Mytilus gill, 325.

Ciona blastomeres, chemical analyses of, 365.

Citric acid, role of in calcification of lobster
integument, 451.

CLAFF, C. L., F. N. SUDAK AND M. H.
CANTOR. Further studies in experimental
hypothermia, 326.

CLAFF, C. L. See F. N. SUDAK, 357.

Clam, correlation of oxygen consumption of,
with barometric pressure, 112.

CLARK, G. M. See A. M. ELLIOTT, 344, 345.

CLARK, W. R., JR. See A. B. CHAET, 339.

Cleavage-accelerating factor in Arbacia ovary
homogenates, 350.

Cleavage of Chaetopterus eggs, effects of
ethyl urethane on, 354.

Cleavage of Chaetopterus eggs, effects of ni-
trogen mustards on, 388.

Cleavage products, Ciona, chemical analyses
of, 365.

Cleavage-retarding factor in Arbacia ovary
homogenates, 351.

CLEMENT, A. C., AND J. N. GATHER. A tech-
nic for preparing whole mounts of veliger
larvae, 340.

Clotting of blood, 320.

CLOWES, G. H. A. See R. K. CRANE, 342, 343.

Cobalt-60 irradiation of rat liver mitochondria,
198.

Cobaltous chloride, effects of on sand dollar
development, 480.

Coelenterate, adaptation to salinity and tem-
perature in, 330.

Coelenterate, nematocyst toxin of, 296.

Coelenterate, terminal growth in, 233.

Cold, effects of on intertidal algae, 275.

Cold, effects of on metabolism of fiddler crabs,
245.

Cold-treatment of rats, 326.

COLLIER, J. R. The aminopeptidase and
catheptic activity of the egg of Ilyanassa,
340.

Colonial hydroid, growth in, 233.

COLWIN, A. L., AND L. H. COLWIN. Egg
membrane lysis by a sperm extract in
Hydroides, 341.

COLWIN, L. H., AND A. L. COLWIN. Lytic
and other activities of the individual
sperm during the early events of sperm
entry, 316.

COLWIN, L. H., AND A. L. COLWIN. Observa-
tions of sperm entry during re-fertilization
in Saccoglossus, 341.

Commensal polychaetes, behavior of, 397.

Comparative study of mealy bug cuticle, 141.

Cooling of muscle, effect of ions on, 328.

COOPERSTEIN, S. J. See A. LAZAROW, 347, 414.

Correlations in barometric pressure and bio-
logical activity, 112.

Cortical cytoplasm of oocytes, 316.

502

INDEX

Cortical response of Nereis egg, 341.
Cosmic radiation, similarities between oxygen
consumption fluctuations of potato and,
103.

COSTELLO, D. P. The cortical response of the
Nereis ovum to activation after centrifug-
ing, 341.

COSTLOW, J. D., JR., AND C. G. BOOKHOUT.
Body growth versus shell growth in Bala-
nus, 224.
Crab, fiddler, hormone-containing granules in,

426.

Crab, fiddler, respiratory metabolism of, 245.
Crab, osmoregulation in, 268.
GRAIN, S. M., M. V. L. BENNETT AND H.
GRUNDFEST. Electrical activity of supra-
medullary neurons of Spheroides, 342.
GRAIN, S. M. Sec M. V. L. BENNETT, 325.
CRANE, R. K., H. H. HIATT AND G. H. A.
CLOWES. The inhibition by a series of
nitro- and halophenols of glucose-6-phos-
phate dehydrogenase from Arbacia eggs
and yeast, 342.

CRANE, R. K., A. K. KELTCH, C. P. WALTERS
AND G. H. A. CLOWES. Preliminary stud-
ies on the incorporation of glucose-U-C
into the polysaccharides of Arbacia and
Mactra larvae and its inhibition by 4,6-
dinitro-o-cresol, 343.

Crassostrea, correlation of oxygen consump-
tion of, with barometric pressure, 112.
CROWELL, S., AND C. WYTTENBACH. Factors
affecting terminal growth in the hydroid
Campanularia, 233.

Crustacean, correlation of oxygen consump-
tion of, with barometric pressure, 112.
Crustacean, excretion in, 207.
Crustacean, hormone-containing granules of,

426.

Crustacean, molting cycle of, 451.
Crustacean, osmoregulation in, 268.
Crustacean, respiratory metabolism of, 245.
Crustacean, sound production in, 286.
CSAPO, A. Sec R. P. KERNAN, 329; H. MA-

SHIMA, 349.
GUSHING, J. E. Tissue transplantation in

Pecten, 327.

GUSHING, J. E., G. J. RIDGWAY AND G. L.
DURALL. The preservation of intact
erythrocytes of marine vertebrates for
blood group research, 343.
Cuticle of mealy bugs, 141.
Cycle, molting, of spiny lobster, 451.
Cycles of oxygen consumption in potato, 103.
Cyclic changes in integument of spiny lobster,

451.
Cystocercous cercariae of Azygia, 488.

Cytochrome oxidase, sensitivity of to gamma

irradiation, 198.
Cytoplasmic segregation in Ciona, 365.

T~)AILY fluctuations in background radiation

and oxygen consumption in potatoes, 103.

Darkness, effect of on activity of salamander,

188.
DAVENPORT, D. Sec J. W. HASTINGS, 120;

J. F. HICKOCK, 397.

DAVIDSON, E. Sec L. V. Heilbrunn, 129.
DAVIS, B. D. Sec A. L. NAGLER, 331.
Day-length in relation to testicular growth in

sparrow, 254.
Dehydration in ?nails, 89.

DEMEUSY, N. Respiratory metabolism of fid-
dler crabs from two different latitudinal
populations, 245.
Dendraster, developmental modifications in,

480.

DENT, J. N. See W. G. LYNN, 160.
Deoxycholate, effect of on respiration of ho-
mogenized frog embryos, 382.
Desiccation of crabs, 268.
Desiccation of intertidal algae, 275.
Desiccation of snail, 89.
Detergents, effects of on hormone-containing

granules of Uca, 426.

Developing frog embryos, respiration of, 382.
Development of Arbacia, effect of fluoride on,

346.

Development of Campanularia, 233.
Development of Ciona, 365.
Development of Neopanope, 338.
Development of phenylthiourea-treated tad-
poles, 160.

Development of polychaetes, 49.
Development of Sciara, chromosome-cytoplasm

interrelations in, 323.
Developmental inhibitors, effects of on Rana

gastrulae, 376, 382.
Developmental modifications of sand dollar,

480.

Diadora commensals, 397.
Diatoms, ecology of, 351.
Dictyostelium, encystment in, 58.
Differential growth in barnacles, 224.
Digitonin, effects of on hormone-containing

granules of Uca, 426.
Dinitrophenol, effects of on frog gastrulae,

376.

Diopatra, endoparasites of, 170.
Dissociated sponge cells, reaggregation of,

356.

Dissociation of sponge cells, 355, 356.
Distribution of endoparasitic annelids, 170.
Diurnal activity rhythm in salamander, 188.

INDEX

503

Diver, cartesian, use of in study of fish islet

tissue, 414.
Dose-inactivation curve for irradiated rat liver

mitochondria, 198.

DOUGLAS, S. D. See M. C. Niu, 352.
Drilonereis, morphology of, 170.
Drugs, effects of on scorpion heart-beat, 135.
Drying in intertidal algae, 275.
DURALL, G. L. See J. E. GUSHING, 343.
Dyes, distribution of during lobster excretion,

207.

J7CDYSIS of barnacles, 224.
Echinoderm, developmental modifications in,
480.

Echiuroids, endoparasites of, 170.

Ecology of algae, 323.

Ecology of crabs, in relation to salinity, 268.

Ecology of diatoms, 351.

Ecology of hydroid, 330.

Ecology of intertidal algae, 275.

Ecology of marine commensals, 397.

Ectodermization of sand dollar embryos, 480.

EDWARDS, C. Sec J. F. CASE, 360.

Effects of developmental inhibitors on Rana
gastrulae, 376.

Egg, cortical response of after centrifuging,
341.

Egg, Ilyanassa, enzymes of, 340.

Egg membrane lysis by sperm extract, 341.

Eggs, Chaetopterus, effects of nitrogen mus-
tards on division of, 388.

Eggs, Spisula, vital staining of, 353.

Electrical activity of supramedullary neurons
of Spheroides, 342.

Electrical inexcitability of neuron soma, 317.

Electrical recording in the living squid, 333.

Electrical responses of doubly-innervated in-
sect muscle fibers, 337.

Electrical stimulation of firefly light emission,
337.

Electrolyte constituents of lobster blood, 207.

Electrolytes, effects of on respiration of fish
islet tissue, 414.

Electron microscopy of Chlamydomonas, 346.

Electron microscopy of Fundulus oocytes, 329.

Electron microscopy of sea urchin egg cyto-
plasm, 327.

Electron microscopy of Spisula egg, 353.

ELIOT, C. R., A. KAJI, P. SEEMAX, E. UBELL,

S. W. KUFFLER AND A. S. V. BuRGEN.

The effect of nervous system extracts on
inhibition and excitation in single nerve
cells, 344.

ELLIOTT, A. M., AND G. M. CLARK. The mat-
ing type system in variety nine of Tetra-
hymena, 344.

ELLIOTT, A. M., AND G. M. CLARK. Post x-
radiation effects of temperature on vege-
tative cells of Tetrahymena, 345.
ELLIOTT, A. M., AND G. M. CLARK. X-radia-
tion effects during conjugation of Tetra-
hymena, 345.
Embryological modifications in sand dollar,

480.

Embryos, effect of ribonucleic acid on, 352.
Embryos, frog, phosphorus balance of, 376.
Embryos, frog, respiration of, 382.
Embryos, teleost, motility in, 325.
Embryology of Ciona, 365.
Encystment stages of Dictyostelium, 58.
Endocytes of Dictyostelium, 58.
Endogenous oxygen uptake of fish, 414.
Endoparasites of arabellids, 170.
Energetics of amphibian development, 376.
ENGER, P. E. S., AND A. S. V. BURGEN. The
effects of some amino acids on the per-
fused lobster heart, 345.

ENGLE, R. L., JR., AND K. R. WOODS. Phylo-
genesis of plasma proteins and plasma
cells. II., 363.

ENGLE, R. L., JR. See K. R. WOODS, 362.
Enteromorpha, desiccation in, 275.
Entodermization of sand dollar embryos, 480.
Environment, role of in testicular growth in

sparrows, 254.

Enzyme activity of Ciona blastomeres, 365.
Enzyme studies on irradiated rat liver, 198.
Enzymes (glucuronidase and sulfatase) of

molluscs, 334.
Epicoccus, cuticle of, 141.

Erythrocytes of marine vertebrates, preserva-
tion of, 343.

Escherichia, changes in nucleic acids in, 321.
Escherichia, genetic block in metabolism in,

331.

Eunice, breeding habits of, 49.
Eunice, endoparasites of, 170.
Eupolymnia, breeding habits of, 49.
Eurothoe, breeding habits of, 49.
Evasterias as host for polychaete commensal,

397.
Excitation, synaptic, of squid stellar ganglion,

359.

Excretion in lobster, 207.

Exogastrulation in echinoderm embryos, 480.
Exogenous urea, loss of through lobster gills,

207.
Extracts, ovarian, antimitotic action of, 129,

318.

Exuviae of barnacles, 224.

Eye pigmentation in phenylthiourea-treated
tadpoles, 160.

504

INDEX

P ACTORS affecting growth in Campanu-
laria, 233.

Facultative commensals, 397.

FARNER, D. S., AND A. C. WILSON. A quan-
titative examination of testicular growth
in the white-crowned sparrow, 254.

FELDHERR, C. The metachromatic reaction in
various types of protoplasm, 319.

FELDHERR, C. See L. V. HEILBRUNN, 318.

Female mealy bugs, cuticle of, 141.

FERGUSON, S. A. See T. R. Tosteson, 318.

Fertilizability of Arbacia eggs after treatment
with xanthine derivatives, 339.

Fibrinogen, irradiated, electrophoretic mobility
of, 319.

Fiddler crab, correlation of oxygen consump-
tion of with barometric pressure, 112.

Fiddler crab, hormone-containing granules of,
426.

Fiddler crab, respiratory metabolism of, 245.

FINE, A. See P. PERSON, 331.

Fish, metabolism of strontium-90 and calcium-
45 by, 442.

Fish, studies on isolated islet tissue of, 414.

Fish as hosts for Azygia, 488.

Flatworm, life-history of, 488.

Flatworm, parasitic, life-history of, 302.

Florida fiddler crabs, metabolism of, 245.

Fluke, life-history of, 302.

Fluorescence of millipede extracts, 120.

Fluoride, effect of on Arbacia development,
346.

Food, relation of to growth in Campanularia,
233.

Freezing and drying in intertidal algae, 275.

FREYGANG, W. H., JR. Evidence for electrical
inexcitability of neuron soma, 317.

FRIZ, C. T. See A. LAZAROW, 414.

Frog embryos, homogenized, respiration of,
382.

Frog gastrulae, phosphorus balance of, 376.

Frog tadpoles, phenylthiourea treatment of,
160.

Fucus, correlation of oxygen consumption of,
with barometric pressure, 112.

Fucus, freezing and drying in, 275.

/TJALL, J. G. Thymidine incorporation into

the macronucleus of Euplotes, 322.
Gamma irradiation of cellular particulates,

198.
Ganglion, cardiac, of Limulus, inhibition of,

336.
Gastropods as hosts of polychaete commensals,

397.
Gastrulae, amphibian, phosphorus balance of,

376.

Gastrulation of Chaetopterus embryos, effect
of sugars on, 332.

Gelation changes, effects of ovarian extracts
on, 129.

Genetic analysis of Mormoniella, 335.

GESTELAND, R. See J. F. CASE, 360.

GIBBS, S. P., D. E. PHILPOTT AND R. A.
LEWIN. Electron microscope studies of
the flagella of Chlamydomonas, 346.

Glucose, effects of on lobster excretion, 207.

Glucose utilization by Arbacia and Mactra
larvae, 343.

Glycera, breeding habits of, 49.

Glycogen accumulation in spiny lobster exo-
skeleton, 451.

Glycoprotein, role of in spiny lobster ecdysis,
451.

Granules, hormone-containing, in Uca, 426.

GREENBERG, S. S., AND A. GREENBERG. Effect
of sodium fluoride on the development of
Arbacia, 346.

GREGG, J. R., AND M. KAHLBROCK. The ef-
fects of some developmental inhibitors on
the phosphorus balance of amphibian gas-
trulae, 376.

GREGG, J. R., AND F. L. RAY. Respiration of
homogenized embryos : Rana pipiens and
Rana pipiens $ X Rana sylvatica c?, 382.

GREIF, R. L. Uptake of Chlormerodrin by
Phascolosoma nephridia, 327.

GROSS, P. R., S. NASS AND W. PEARL. Mech-
anisms of sol-gel transformations in the
cytoplasm, 320.

GROSS, P. R., D. E. PHILPOTT AND S. NASS.
Electron microscope observations on the
cytoplasm of sea urchin eggs, 327.

GROSS, W. J. A behavioral mechanism for
osmotic regulation in a semi-terrestrial
crab, 268.

Growth, terminal, in Campanularia, 233.

Growth of body and shell in Balanus, 224.

Growth of testes in sparrow, 254.

GRUNDFEST, H. See M. V. L. BENNETT, 325 ;
J. CERF, 337, 338; S. M. GRAIN, 342.

Guppy, metabolism of strontium-90 and cal-
cium-45 by, 442.

GUTTMAN, R., AND S. Ross. The effect of
ions on the response of smooth muscle to
cooling, 328.

"HADDOW Paradox'" 388-
Haemolymph pressure of scorpion, 135.
HARDIMAN, C. W. Stimulation of the taste
receptors of the rat with organic salts,

347.

INDEX

505

HARVEY, E. N., A. M. CHASE AND W. D.
MCELROY. The spectral energy curve of
Cypridina and other luminous organisms,
347.

HASTINGS, J. W., AND D. DAVENPORT. The
luminescence of the millipede, Lumino-
desmus, 120.

Heart-beat of scorpion, 135.

Heart-beat of snail, 89.

HEILBRUNN, L. V., AND W. L. WILSON. A
rational approach to the problem of can-
cer chemotherapy, 388.

HEILBRUNN, L. V., F. T. ASHTON, C. FELD-
HERR AND W. L. WILSON. The action of
insulin on living cells, 318.

HEILBRUNN, L. V., W. L. WILSON, T. R.
TOSTESON, E. DAVIDSON AND R. J. RUT-
MAN. The antimitotic and carcinostatic
action of ovarian extracts, 129.

HEILBRUNN, L. V. See T. R. TOSTESON, 318.

HEILMAN, R. S. See V. MENKIN, 350, 351.

Heme synthesis in marine fishes, 361.

Hemerythrin, incorporation of labelled iron
into, 361.

Hemoglobin, vertebrate, effect of on lobster
excretion, 207.

Hepatopancreas of spiny lobster during ecdy-
sis, 451.

Hermodice, breeding habits of, 49.

Hesionid polychaete, commensalism of, 397.

Hexosamine content of Metridium nematocyst
toxin, 296.

HIATT, H. H. See R. K. CRANE, 342.

HIBBARD, E. See N. E. KEMP, 329.

HICKOK, J. F., AND D. DAVENPORT. Further
studies in the behavior of commensal
polychaetes, 397.

Histamine, effect of on scorpion heart-beat,
135.

Histamine in invertebrate cells, 339.

Histochemistry of lobster molting cycle, 451.

Histochemistry of mealy bug cuticle, 141.

Histological changes in lobster hepatopancreas
during molting, 451.

Histology of Nereis nephridium, 407.

Histology of phenylthiourea-treated tadpole
thyroids, 160.

Histology of spiny lobster stridulatory mem-
brane, 286.

Homarus, excretion in, 207.

Homogenized frog embryos, respiration of,
382.

Homopteran, cuticle of, 141.

Hormone-containing granules of Uca, 426.

Hormone control of color change in Lepidop-
tera, 326.

Hormones, plant, effects of on Ulva, 321.

Host-commensal relations, 397.

HOYLE, G. Coupling of membrane potential

to contraction in muscle, 317.
HOYLE, G. Neuromuscular transmission in

Limulus, 347.

HOYLE, G. See J. CERF, 337, 338.
Humidity, role of in metabolism of snail, 89.
Hyaline layer of Arbacia egg, contractility of,

331.

Hybrid frog embryos, respiration of, 382.
Hybrid frog gastrulae, phosphorus balance of,

376.

Hydration in snails, 89.

Hydrocaulus, growth of in Campanularia, 233.
Hydroid, terminal growth in, 233.
Hydroxy-indoles in Metridium nematocyst

toxin, 296.
Hyla tadpoles, phenylthiourea treatment of,

160.
Hypothermia in rats, 326.

IMMUNITY in relation to nematocyst toxin,

296.

Indian scorpion, cardiac physiology of, 135.
Influence of lunar frequency on activity

rhythm of Plethodon, 188.
Inhibitors, developmental, effects of on Rana

gastrulae, 376.
Insect, cuticle of, 141.
Insulin, effect of on Chaos, 318-
Integumental tissue of spiny lobster during ec-

dysis, 451.

Intertidal algae, freezing and drying in, 275.
Intertidal crab, salinity preferences of, 268.
Intestinal absorption of fish, 362.
Inulin, effect of on lobster excretion, 207.
Iodine, radioactive, binding of in tadpoles, 160.
Irradiation of cellular particulates, 198.
Isolation of Metridium nematocyst toxin, 296.
Islet tissue of fish, 414.
Isotope treatment of Hyla tadpoles, 160.
Isotopes, metabolism of by Lebistes, 442.

JONES, M. L. On the morphology of the
nephridium of Nereis vexillosa, 407.

M. See J. R. GREGG, 376.

KAJI, A. See C. R. ELIOT, 344.

KANUNGO, M. S. Cardiac physiology of the
scorpion Palamnaeus, 135.

KANWISHER, J. Freezing and drying in inter-
tidal algae, 275.

KELTCH, A. K. See R. K. CRANE, 343.

KEMP, N. E. Differentiation of cortical cyto-
plasm and extra-cellular membranes of
oocytes, including changes at fertilization,
316.

506

INDEX

KEMP, N. E., AND E. HIBBARD. Protoplasmic
bridges between follicle cells and develop-
ing oocytes of Fundulus, 329.

KERNAN, R. P., AND A. CSAPO. An effect of
calcium-deficient Ringer on intact frog
muscle, 329.

KERNAN, R. P., AND A. CSAPO. Potassium
contracture in frog twitch muscles, 329.

Key to species of endoparasitic annelids, 170.

Key to species of North American Azygia,
488.

KINNE, O. Adaptation to salinity and tem-
perature in a euryhaline hydroid, 330.

KUENZLER, E. J. Sec E. P. OouM, 323.

KUFFLER, S. W. Sec A. S. V. BURGEN, 336;
C. R. ELIOT, 344.

£^AG-lead correlations in barometric pres-
sure and biological activity, 112.

LAMB, G. A. See M. P. SCHULMAN, 361.

Larvae, frog, phenylthiourea-treatment of, 160.

Larvae, Ilyanassa, method for preparing whole-
mounts of, 340.

Larval development of mud crab Neopanope,
338.

Larval development of Streblospio, 336.

Latitudinally-different populations of fiddler
crabs, 245.

LAZAROW, A., AND S. J. COOPERSTEIN. Com-
parative distribution of radioactive al-
loxan, thiocyanate, and urea in islet and
other toadfish tissues, 348.

LAZAROW, A., S. J. COOPERSTEIN, D. K.
BLOOMFIELD AND C. T. FRIZ. Studies on
the isolated islet tissue of fish. II., 414.

Lebistes, metabolism of strontium-90 and cal-
cium-45 by, 442.

LEWIN, R. A. See S. P. GIBBS, 346.

Life-cycle of Azygia, 488.

Life-cycle of slime mold, 58.

Life-history of Allocreadium, 302.

Light, effect of on activity of salamander, 188.

Light, emission of by millipede, 120.

Light, role of in testicular growth in sparrow,
254.

Limitation of growth in Campanularia, 233.

Limnephilus, as host for Allocreadium, 302.

Lipoids in mealy bug cuticle, 141.

Littorina, susceptibility of to Metridium ne-
matocyst toxin, 296.

Liver, rat, effects of gamma irradiation on
mitochondria of, 198.

Lobster, excretion in, 207.

Lobster, spiny, molting cycle in, 451.

Lobster, spiny, sound production in, 286.

Locomotor activity rhythm in salamander, 188.

LORAND, L. Clotting of blood : A study in the
polymerization of proteins, 320.

LORAND, L. Phosphoarginine and arginine
phosphopkinase from Homarus, 360.

LORAND, L., J. MOLNAR AND C. Moos. Bio-
chemical studies of relaxation in glycerin-
ated muscle, 323.

LORAND, L. Sec C. Moos, 330.

LORIXG, J. See C. A. VILLEE, 358.

Low temperature, effect of on intertidal algae,
275.

Low, temperature, effect of on metabolism of
fiddler crabs, 245.

LOWER, H. F. A comparative study of the
cuticular structure of three female mealy
bugs, 141.

Luidia as host for polychaete commensals, 397.

Luminescence of millipede, 120.

Luminous orgaiiisms, spectral energy curve of,
347.

Lunar-day fluctuations- in cosmic radiation,
103.

Lunar influence on activity rhythm of sala-
mander, 188.

LYNN, W. G., AND J. N. DENT. Phenylthio-
urea treatment and binding of radioactive
iodine in the tadpole, 160.

Lysidice, breeding habits of, 49.

MACEY, E. J. See F. A. BROWN, JR., 112.

Magnesium, effect of on lobster excretion, 207.
Magnetic studies on cells and protoplasm, 319.
Malignant tumors, chemotherapy of, 388.
Mammalian ovary extracts, effects of on cell

division, 129.

Mandibles of barnacles, 224.
Mantle cavity of barnacle, 224.
Marine eggs, effects of nitrogen mustard on

division of, 388.
Marine eggs, effects of ovarian extracts on

division of, 129.
Marine fish, studies on isolated islet tissue of,

414.

Marine organisms, sound production in, 286.
MARSLAND, D., AND W. AUCLAIR. A further

study on the induced furrowing reaction

in Arbacia, 348.
MASHIMA, H., AND A. CSAPO. Shortening of

potassium depolarized muscle in different

electric fields, 349.
MASON, D. Sec E. T. MOUL, 351.
Massachusetts fiddler crab, metabolism of, 245.
MATEYKO, G. M. Cytophysiology of ultracen-

trifuged normal and neoplastic frog kidney

cells, 349.
Mating types in variety 9 of Tetrahymena,

344. "

Maxillae of barnacles, 224.
McCANN, F. V. See J. CERF, 337, 338.

INDEX

507

MCELROY, W. D. See E. N. HARVEY, 347.

MCMAHON, P. Sec T. VON BRAND, 89.

Mealy bugs, cuticle of, 141.

Mechanism of osmoregulation in crab, 268.

Mediaster as host for polychaete commensal,
397.

Melanin formation in phenylthiourea-treated
Hyla tadpoles, 160.

Melanin of mealy bug cuticle, 141.

Membrane potential changes in crab muscle
fibers, 362.

MENKIN, V., L. MENKIN AND R. S. HEIL-
MAN. Studies on the accelerator cleavage
factor recovered in homogenates of Ar-
bacia ovaries, 350.

MENKIN, V., L. MENKIN AND R. S. HEIL-
MAN. Studies on the nature of the re-
tarding cleavage factor in homogenates of
sea urchin ovaries, 351.

Metabolic pathways in Arbacia eggs, 342.

Metabolic responses to temperature of 2,4-D-
treated albino rats, 357.

Metabolism and ciliary activity of Mytilus
gill, 335.

Metabolism in frozen and dried algae, 275.

Metabolism of fiddler crabs, 245.

Metabolism of fish islet tissue, 414.

Metabolism of glucose in marine invertebrates,
358.

Metabolism of homogenized frog embryos, 382.

Metabolism of Limulus gill cartilage, 331.

Metabolism of snails, 89.

Metabolism of strontium-90 and calcium-45 by
fish, 442.

Metachromasy in protoplasm, 319.

Metamorphosis in phenylthiourea-treated tad-
poles, 160.

METCALF, C. See M. SPIEGEL, 355, 356.

Metridium, nematocyst toxin of, 296.

METZ, C. W. Interactions between chromo-
somes and cytoplasm during early em-
bryonic development in Sciara, 323.

Microchemical analyses of Ciona blastomeres,
365.

Microcysts of Dictyostelium, 58.

Microrespirometer, use of in study of fish
islet tissue, 414.

MINGOLI, E. S. Sec A. L. NAGLER, 331.

Miracidia of Allocreadium, 302.

Miracidia of Azygia, 488.

Mitochondria, rat, effects of gamma irradia-
tion on, 198.

Mitosis, effects of nitrogen mustards on, 388.

Mitosis, effects of ovarian extracts on, 129.

Mitotic inhibition by dioxypurine derivatives,
339.

Modification of diurnal rhythm in Plethodon,
188.

Modifications of development in sand dollar,
480.

Mollusc, correlation of oxygen consumption
of, with barometric pressure, 112.

Mollusc, starvation and desiccation of, 89.

Molluscan host of Allocreadium, 302.

Molluscan host of Azygia, 488.

MOLNAR, J. Sec L. LORAND, 323.

Molting of barnacles, 224.

Molting cycle of spiny lobster, 451.

Moos, C., AND L. LORAND. Inorganic pyro-
phosphatase activity of glycerinated mus-
cle, 330.

Moos, C. Sec L. LORAND, 323.

Mormoniella, genetic analysis of, 335.

Morphogenesis in Dictyostelium, 58.

Morphogenesis of frog embryos, 376, 382.

Morphology of Allocreadium, 302.

Morphology of annelid endoparasites, 170.

Morphology of Azygia, 488.

Morphology of Nereis nephridium, 407.

MOUL, E. T., AND D. MASON. Study of di-
atom populations on sand and mud flats
in the Woods Hole area, 351.

MOULTON, J. M. Sound production in spiny
lobster, 286.

Mouthparts of barnacles, 224.

Mouthparts of endoparasitic annelids, 170.

Mucoprotein, role of in spiny lobster ecdysis,
451.

Muscle, effect of ions on response of, to cool-
ing, 328.

Muscle, glycerinated, inorganic pyrophospha-
tase activity of, 330.

Muscle, glycerinated, relaxation in, 323.

Muscle, iodoacetate-poisoned, creatine phos-
phate and ATP breakdown in, 324.

Muscle, potassium depolarized, shortening of
in electric fields, 349.

Myxamoebae, encystment in, 58.

, distribution of in lobster blood and
urine, 207.

NAGLER, A. L., E. S. MINGOLI AND B. D.
DAVIS. Metabolic consequences of a
genetic block between alpha-ketoglutarate
and succinate in Escherichia, 331.

NASS, S. See P. R. GROSS, 320, 327.

Nematocyst toxin of Metridium, 296.

Neoplasms, chemotherapy of, 388.

Neoplastic frog kidney cells, 349.

Nephridia of Phascolosoma, uptake of Chlor-
merodin by, 327.

Nephridial function in lobster, 207.

Nephridium of Nereis, morphology of, 407.

Nereis, breeding habits of, 49.

Nereis vexillosa, nephridium of, 407.

508

INDEX

Nerve cell, effect of nervous system extracts

on, 344.

Nerve impulse in lobster stretch receptor, 360.
Neural organization of puffer supramedullary

neurons, 325.
Neuromuscular transmission in grasshopper,

338.

Neuromuscular transmission in Limulus, 347.
Neurosecretion in Uca, 426.
Nitrogen analyses of lobster urine, 207.
Nitrogen content of Metridium nematocyst

toxin, 296.
Niu, M. C, AND S. D. DOUGLAS. The effect

of varying concentrations of ribonucleic

acid on the development of some marine

embryos, 352.

NOLAN, M. O. Sec T. VON BRAND, 89.
Notocirrus, morphology of, 170.
Notopygos, breeding habits of, 49.
Nuclear incorporation of thymidine in Eu-

plotes, 322.

Nucleic acid content of Ciona blastomeres, 365.
Nucleoli, starfish, proteins of, 334.
Nuclide, metabolism of by Lebistes, 442.
Nutrition, relation of to growth in Campanu-

laria, 233.

Nutrition, role of in testicular growth in spar-
rows, 254.

QBLIGATE commensals, 397.

ODUM, E. P., E. J. KUENZLER AND SISTER
M. X. BLUNT. Uptake of P-32 in benthic
algae in relation to primary productivity,
323.

Oliguria in lobster, 207.

Onuphids, endoparasites of, 170.

Oocytes of Fundulus, electron microscope
studies of, 329.

Opsanus, studies on isolated islet tissue of, 414.

Osmolar measurements in lobster, 207.

Osmoregulatory organ of Nereis, 407.

Osmotic pressure in relation to toxicity of
Metridium nematocyst toxin, 296.

Osmotic properties of hormone-containing
granules of Uca, 426.

Osmotic regulation in crab, 268.

OSTERHOUT, W. J. V. Production of perma-
nent lesions in living protoplasm, 352.

OSTERHOUT, W. J. V. Selective permeability
in relation to movement of water into
living cells, 353.

OTTOSON, D. Sec J. F. CASE, 360.

Ova, Chaetopterus, effects of nitrogen mus-
tards on, 388.

Ova, marine, effects of ovarian extracts on
division of, 129.

Ovarian extracts, antimitotic action of, 129.

Oxygen, effect of on luminescence of millipede,
120.

Oxygen consumption of algae, 275.

Oxygen consumption of organisms as corre-
lated with barometric pressure, 112.

Oxygen consumption of potatoes, 103.

Oxygen consumption of snails, 89.

Oxygen uptake of fiddler crabs, 245.

Oxygen uptake of fish islet tissue, 414.

Oxygen uptake of homogenized frog embryos,
382.

Oxygen uptake of rat liver mitochondria, ef-
fects of gamma irradiation on, 198.

Oxygen uptake of Uca, effect of 2,4-D on, 357.

Oyster, correlation of oxygen consumption of,
with barometric pressure, 112.

Oyster, injury-reaction in, 335.

pACE-maker of scorpion heart-beat, 135.

Pachygrapsus, osmoregulation in, 268.

Palamnaeus, cardiac physiology of, 135.

Pancreatic islet tissue of fish, 414.

Panulirus, molting cycle of, 451.

Panulirus, sound production in, 286.

Paper chromatograpliy of Metridium nemato-
cyst toxin, 296.

Para-amminohippurate, effect of on lobster
excretion, 207.

Parasite, life-history of, 302.

Parasites of arabellids, 170.

Paratenic hosts of Azygia, 488.

PARPART, A. K., AND J. CAGLE. Contractility
of the hyaline layer of Arbacia, 331.

Particulates, cellular, enzyme assay of, 365.

Particulates, cellular, irradiation of, 198.

Patiria as host for commensal polychaete, 397.

PEARL, W. See P. R. GROSS, 320.

PEREZ-GONZALEZ, M. D. Evidence for hor-
mone-containing granules in sinus glands
of the fiddler crab, Uca, 426.

Permeability of crab integument, 268.

Permeability of lobster gills and exoskeleton,
207.

Permeability of Nitella, in relation to injury,
352, 353.

PERSON, P., AND A. FINE. Observations on
the histology and oxidative metabolism of
Limulus gill cartilage, 331.

PETTIBONE, M. H. Endoparasitic polychae-
tous annelids of the family Arabellidae
with descriptions of new species, 170.

pH, effect of on metabolism of fish islet tissue,
414.

Pharmacology of scorpion heart-beat, 135.

Phenol red, effect of on lobster excretion, 207.

Phenylthiourea treatment of tadpoles, 160.

INDEX

509

PHILLIPS, J. H., JR., AND D. P. ABBOTT. Iso-
lation and assay of the nematocyst toxin
of Metridium, 296.
PHILPOTT, D. E. Sec P. R. GROSS, 327 ; S. P.

GIBBS, 346.

Phlorizin, effect of on lobster excretion, 207.
Phosphatase activity of Ciona blastomeres,

365.

Phosphate, effect of on lobster excretion, 207.
Phosphate ion, effect of on fish islet tissue

metabolism, 414.
Phosphoarginine and arginine phosphokinase

from lobster, 360.

Phosphorus balance of Rana gastrulae, 376.
Phosphorylation in rat liver mitochondria,

effects of gamma irradiation on, 198.
Photoperiod in relation to testicular growth in

sparrow, 254.

Photosynthesis in Arctic Fucus, 275.
Physiological variations in fiddler crabs, 245.
Physiology, cardiac, of scorpion, 135.
Physiology of desiccation of snails, 89.
Physostigmine, effect of on heart-beat of scor-
pion, 135.

Pigment extracts from Asterias, 353.
Pigmentation in phenylthiourea-treated Hyla

tadpoles, 160.
Pisaster as host for polychaete commensal,

397.

Pisidium as host for Allocreadium, 302.
Planorbid snails, starvation and desiccation of,

89.

Plasma proteins, effects of on lobster excre-
tion, 207.
Plasma proteins and cells, phylogenesis of,

362, 363.

Platyhelminth, life-history of, 302, 488.
Platynereis, breeding habits of, 49.
Plethodon, diurnal activity rhythm in, 188.
Podarke, commensalism of, 397.
Polychaetes, breeding habits of, 49.
Polychaetes, commensal, behavior of, 397.
Polychaetes, endoparasitic, 170.
Polymerization of blood proteins, 320.
Population differences as a factor in commen-
salism, 397.
Population differences in metabolism of fiddler

crabs, 245.
Post-ecdysial changes in lobster hepatopan-

creas, 451.

Potassium concentrations of crab blood, 268.
Potassium contracture in frog twitch muscles,

329.

Potatoes, fluctuations in oxygen consumption
of, as compared with background radia-
tion, 103.

Potential, membrane, and muscle contraction,
317.

Potential changes and ion movements in frog

muscle, 317.

Pressure, barometric, as correlated with bio-
logical activity, 112.
Pressure effects on Arbacia eggs, 348.
Production of sound in spiny lobster, 286.
Productivity of algae, as studied with P-32,

323.

Protease digestion of Ciona chorion, 365.
Protection effects against gamma irradiation

of rat liver mitochondria, 198.
Protochordate blastomeres, chemical analyses

of, 365.
PROVASOLI, L. Effect of plant hormones on

sea weeds, 321.

Pseudococcidae, cuticle of, 141.
Pteraster as host for polychaete commensals,

397.
Puerto Rican annelids, breeding habits of, 49.

QUAHOG, correlation of oxygen consump-
tion of, with barometric pressure, 112.
Quantitative examination of testicular growth
in sparrow, 254.

D NA content of Ciona blastomeres, 365.

Radiation, background, as compared with oxy-
gen consumption of potatoes, 103.

Radioactive alloxan, uptake of by toadfish
tissues, 348.

Radioiodine, binding of, in tadpoles, 160.

Radiostrontium and radiocalcium, metabolism
of by Lebistes, 442.

RALPH, C. L. A diurnal activity rhythm in
Plethodon and its modification by an in-
fluence having a lunar frequency, 188.

Rana embryos, homogenized, respiration of,
382.

Rana gastrulae, phosphorus balance of, 376.

RAPER, K. B. See J. C. BLASKOVICS, 58.

Rat, correlation of oxygen consumption of,
with barometric pressure, 112.

Rat mitochondria, effects of gamma irradia-
tion on, 198.

Rate of growth in Campanularia, 233.

Rate of oxygen consumption of fiddler crabs,
245.

RAY, F. L. See J. R. GREGG, 382.

Reaggregation of dissociated sponge cells, 356.

Reconstruction of Nereis nephridium, 407.

REBHUN, L. I. Vital staining of Spisula by
methylene blue, 353.

Rediae of Allocreadium, 302.

Rediae of Azygia, 488.

Re-fertilization of Saccoglossus eggs, 341.

Reproduction of polychaetes, 49.

Respiration in frozen and dried algae, 275.

510

INDEX

Respiration of fiddler crabs, 245.

Respiration of fish islet tissue, 414.

Respiration of homogenized frog embryos, 382.

Respiration of organisms as correlated with
barometric pressure, 112.

Respiration of potatoes, 103.

Respiration of snails, 89.

Retardation of development of Rana embryos,
382.

Rhythm, diurnal activity, in salamander, 188.

Ribonuclease in rat liver, 322.

Ribonuclease system in marine forms, 332.

Ribonucleic acid, effect of on developing em-
bryos, 352.

Ribonucleic acid content of Ciona blastomeres,
365.

RIDGWAY, G. J. See J. E. GUSHING, 343.

RIESER, P. Electrophoretic mobility studies
on irradiated fibrinogen, 319.

ROBSON, H. H. Sec H. T. YOST, JR., 198.

ROCKSTEIN, M., AND M. RUBENSTEIN. The

biochemical basis for positive photokinesis

of Asterias, 353.
ROSENTHAL, H. L. The metabolism of stron-

tium-90 and calcium-45 by Lebistes, 442.
Ross, S. See R. GUTTMAN, 328.
ROTH, J. S. Some observations on the ribo-

nuclease system in rat liver, 322.
ROTH, J. S., AND D. BACHMURSKI. Studies

on the distribution and properties of the

ribonuclease system in marine forms, 332.
RUBENSTEIN, M. See M. ROCKSTEIN, 353.
RULON, O. Developmental modifications in

the sand dollar, 480.
RUTMAN, R. J. See L. V. HEILBRUNN, 129.

gABELLA, breeding habits of, 49.

Salamander, correlation of oxygen consump-
tion of, with barometric pressure, 112.

Salamander, locomotor activity rhythm in, 188.

Salinity, effect of on respiration of frozen and
dried algae, 275.

Salinity preferences of crab, 268.

Sand dollar, developmental modifications in,
480.

SCHINSKE, R. A. Sec G. C. STEPHENS, 356.

Schooling behavior in Menidia, 354.

SCHUEL, H. The initiation and inhibition of
cleavage in the Chaetopterus egg by ethyl
urethane, 354.

SCHULMAN, M. P. Incorporation of labelled
iron into hemerythrin, 361.

SCHULMAN, M. P., AND G. A. LAMB. Heme
synthesis in peripheral blood of marine
fishes, 361.

Scorpion, cardiac physiology of, 135.

SCOTT, D. B. M. The effect of sugar on gas-
trulation of Chaetopterus embryos, 332.

SCOTT, D. M. Sequence of changes in nucleic
acids in synchronized cultures of Esche-
richia, 321.

SEEMAN, P. Sec C. R. ELIOT, 344.

Segregation, cytoplasmi^, in Ciona, 365.

Selenite, effects of on sand dollar development,
480.

Semi-terrestrial crab, osmoregulation in, 268.

Serum addition, effect lof on fish islet tissue
metabolism, 414.

Sexual reproduction of polychaetes, 49.

SHAW, E. Preliminary studies on the ontog-
eny of schooling behavior in the silver-
sides, Menidia, 354.

Shell growth in Balanus, 224.

SHRINER, J. See F. A. BROWN, JR., 103.

SIGER, A. Sec F. D. CARLSON, 324.

Sinus glands of Uca, hormone-containing
granules in, 426.

Size increase in Balanus, 224.

Skeletal development of sand dollar embryos,
480.

Skeletal development of sea urchin embryos,
346.

Skin pigmentation in phenylthiourea-treated
tadpoles, 160.

Slime mold, encystment in, 58.

Snail, starvation and desiccation of, 89.

Snail, susceptibility of to Metridium nemato-
cyst toxins, 296.

Snails as intermediate hosts of Azygia, 488.

Sodium concentrations of crab blood, 268.

Sodium selenite, effects of on sand dollar de-
velopment, 480.

Sol-gel transformations in cytoplasm, 320.

Solanus, oxygen consumption of, 103.

Solar-day fluctuations in cosmic radiation, 103.

Sorocarp formation in Dictyostelium, 58.

Sound production in spiny lobster, 286.

Sparrow, white-crowned, testicular growth in,
254.

Species of polychaete endoparasites, 170.

Specificity of commensalism, 397.

SPEIDEL, C. C. X-ray effects on Tetrahymena,
355.

Sperm entry, lytic activity of sperm during,
316.

Sperm entry in Saccoglossus, 341.

SPIEGEL, M., AND C. METCALF. Enzymatic
dissociation of sponge cells, 355.

SPIEGEL, M., AND C. METCALF. The reaggre-
gation of Microciona cells in culture
media, 356.

SPIEGEL, M., AND C. METCALF. Viability of
dissociated frozen-thawed sponge cells,
356.

INDEX

511

Spiny lobster, molting cycle of, 451.

Spiny lobster, sound production in, 286.

Spirobranchus, breeding habits of, 49.

Spisula eggs, effects of ovarian extracts on,
129.

Spore-formation in Dictyostelium, 58.

Sporocysts of Allocreadium, 302.

Sporocysts of Azygia, 488.

Stages of encystment in Dictyostelium, 58.

Starfish hosts of polychaete commensals, 397.

Starvation of snail, 89.

Stem growth in Campanularia, 233.

STEPHENS, G. C, AND R. A. SCHINSKE. Up-
take of amino acids from sea water by
ciliary-mucoid filter feeding animals, 356.

STEPHENSON, W. K. The effects of metabolic
inhibitors on ion distribution and mem-
brane potential in muscle fibers of the
green crab, Carcinides, 362.

STEPHENSON, W. K. Membrane potential
changes and ion movements in frog mus-
cle, 317.

Stridulatory mechanism of spiny lobster, 286.

STROHMAN, R. C. Studies on the interactions
of the bound nucleotide of actin, 333.

Strontium-90, metabolism of by Lebistes, 442.

Structure of mealy bug cuticle, 141.

Studies on irradiation of cellular particulates,
198.

Substrate, effect of on encystment of Dictyo-
stelium, 58.

Succinic dehydrogenase activity of Ciona blas-
tomeres, 365.

SUDAK, F. N., AND C. L. CLAFF. The effect
of 2,4-D on oxygen consumption in Uca,
357.

SUDAK, F. N., C. L. CLAFF AND M. H.
CANTOR. Metabolic responses of albino
rats treated with 2,4-D to changes in am-
bient temperature, 357.

SUDAK, F. N. Sec C. L. CLAFF, 326.

Sugar, role of in lobster excretion, 207.

Sulfate, effect of on lobster excretion, 207.

Sulfobromophthalein, effect of on lobster ex-
cretion, 207.

Synergistic action of chemical agents on sand
dollar development, 480.

Systematics of trematodes, 302, 488.

'"TADPOLES, phenylthiourea treatment of,
160.

Taste receptors of rat, 347.

Taxonomy of Azygia, 488.

Taxonomy of endoparasitic annelids, 170.

Taxonomy of trematodes, 302, 488.

Teeth, accumulation of radioiodine in, in tad-
poles, 160.

Teleost, studies on isolated islet tissue of, 414.

Temperature, effect of on encystment of Dic-
tyostelium, 58.

Temperature, effect of on hormone-containing
granules of Uca, 426.

Temperature, effect of on luminescence of
millipede, 120.

Temperature, effect of on post x-irradiation
effects in Tetrahymena, 345.

Temperature, role of in metabolism of fiddler
crabs, 245.

Temperature, role of in testicular growth of
sparrows, 254.

Temperature in relation to metabolism of
2,4-D-treated albino rats, 357.

Terminal growth in Campanularia, 233.

Testicular growth in sparrow, 254.

TEWINKEL, L. E. Expansion of the pre-
placental yolk-sac in Mustelus, 358.

Thelepus, breeding habits of, 49.

THIES, R. E. Electrical recording in the liv-
ing squid, 333.

Thiol groups of enzyme, importance of in
modifications of sand dollar development,
480.

Thymus, effects of phenylthiourea-treatment
on, in tadpoles, 160.

Thyroid accumulation of radioiodine in tad-
poles, 160.

Tissue-accumulation of isotopes by fish, 442.

Tissue culture of marine invertebrate cells,
334.

Tissue transplantation in Pecten, 327.

Toadfish, studies on isolated islet tissue of,
414.

Tolerance to cold of fiddler crabs, 245.

Tonicity, effects of on fish islet tissue metabo-
lism, 414.

TOSTESON, T. R., S. A. FERGUSON and L. V.
HEILBRUNN. Further studies of the anti-
mitotic and carcinostatic action of ovarian
extracts, 318.

TOSTESON, T. R. Sec L. V. HEILBRUNN, 129.

Toxin, nematocyst, of Metridium, 296.

Trace metals, effects of on fish islet tissue
metabolism, 414.

TRAVIS, D. F. The molting cycle of the spiny
lobster, Panulirus. IV., 451.

Trematode, life-history of, 302, 488.

Triturus, correlation of oxygen consumption
of, with barometric pressure, 112.

TROLL, W. Glucuronidase and sulfatase of
molluscs, 334.

Tumor regression, effects of ovarian extracts
on, 129.

Tunicate blastomeres, chemical analyses of,
365.

Tyrosine iodination in phenylthiourea-treated
tadpoles, 160.

512

INDEX

, E. See C. R. ELIOT, 344.

Uca, correlation of oxygen consumption of,

with barometric pressure, 112.
Uca, hormone-containing granules of, 426.
Uca, respiratory metabolism of, 245.
Ultracentrifuged frog kidney cells, 349.
Ulva, freezing and drying in, 275.
Uptake, oxygen, of fish islet tissue, 414.
Uptake of isotopes by fish, 442.
Urethane, effects of on Chaetopterus eggs, 388.
Uricase inactivation by urea, 320.
Urinary output of lobster, 207.

YEGETATIVE stages of Dictyostelium, 58.

Veliger larvae, method for preparing whole-
mounts of, 340.

Venus, correlation of oxygen consumption of,
with barometric pressure, 112.

Vernal testicular development in sparrow, 254.

VlLLEE, C. A., J. LORING AND F. WELLINGTON.

The hexose monophosphate shunt in ma-
rine invertebrates, 358.

VINCENT, W. S. Proteins of starfish nucleoli,
334.

^ ALTERS, C. P. See R. K. CRANE, 343.
WARWICK, A., AND F. B. BANG. Survival of

marine invertebrate cells in tissue culture,

334.

Water loss in algae, 275.
Wax glands of mealy bugs, 141.
WEBB, H. M. See F. A. BROWN, JR., 103, 112.
Weight, body, of snails, 89.
WELLINGTON, F. See C. A. VILLEE, 358.
West Indian spiny lobster, sound production

in, 286.
White-crowned sparrow, testicular growth in,

254.
WHITING, P. W. Method of analysis of a

"gene" in Mormoniella, 335.
WICHTERMAN, R. X-irradiation of the giant

multinucleate ameba, Chaos, 359.

WILBER, C. G. Some physiological character-
istics of the fish heart, 359.

WILSON, A. C. See D. S. FARNER, 254.

WILSON, T. H. In vitro studies on intestinal
absorption of fish, 362.

WILSON, W. L. Seie L. V. HEILBRUNN, 129,
318, 388.

WOODS, K. R., AND R. L. ENGLE, JR. Phylo-
genesis of plasma proteins and plasma
cells. I., 362.

WOODS, K. R. See R. L. ENGLE, JR., 363.

Woods Hole fiddler crabs, metabolism of, 245.

WOOTTON, D. M. Studies on the life-history
of Allocreadium, 302.

WOOTTON, D. M. Notes on the life-cycle of
Azygia, 488.

Worm, annelid, morphology of nephridium of,
407.

Worm, flat, life-history of, 302, 488.

Worm eggs, effects of nitrogen mustards on,
388.

Worms, annelid, breeding habits of, 49.

Worms, annelid commensal, behavior of, 397.

Worms, endoparasites of, 170.

WYTTENBACH, C. See S. CROWELL, 233.

X-IRRADIATION of ameba, 359.

X-irradiation of conjugating Tetrahymena,

345.

X-irradiation of Tillina, 336.
X-irradiation of vegetative Tetrahymena, 345,

355.

VOLK-sac, expansion of, in Mustelus, 358.

YOST, H. T., JR., AND H. H. ROBSON. Studies
on the effects of irradiation of cellular
particulates. II., 198.

VINC chloride, effects of on sand dollar de-
velopment, 480.
Zonotrichia, testicular growth in, 254.

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Annual Report of the Marine Biological Laboratory.- 1

ALLEN, M. JEAN

The breeding of polychaetous annelids near Parguera, Puerto Rico. 49

BLASKOVICS, JOAN CORMIER, AND KENNETH B. RAPER

Encystment stages of Dictyostelium 58

VON BRAND, THEODOR, PATRICIA MCMAHON AND M. O. NOLAN

Physiological observations on starvation and desiccation of the snail
Australorbis glabratus 89

BROWN, FRANK A., JR., JOAN SHRINER AND H. MARGUERITE WEBB
Similarities between daily fluctuations in background radiation and
O2-consumption in the living organism 103

BROWN, FRANK A., JR., H. MARGUERITE WEBB AND ERWIN J. MACEY

Lag-lead correlations of barometric pressure and biological activity. . 112

HASTINGS, J. WOODLAND, AND DEMOREST DAVENPORT

The luminescence of the millipede, Luminodesmus sequoiae 120

HEILBRUNN, L. V., W. L. WILSON, T. R. TOSTESON, E. DAVIDSON AND

R. J. RUTMAN

The antimitotic and carcinostatic action of ovarian extracts 129

KANUNGO, M. S.

Cardiac physiology of the scorpion Palamnaeus bengalensis C. Koch 135

LOWER, HARRY F.

A comparative study of the cuticular structure of three female mealy
bugs (Homoptera : Pseudococcidae) 141

LYNN, W. GARDNER, AND JAMES NORMAN DENT

Phenylthiourea treatment and binding of radioactive iodine in the
tadpole 160

PETTIBONE, MARIAN H.

Endoparasitic polychaetous annelids of the family Arabellidae with
descriptions of new species 170

RALPH, CHARLES L.

A diurnal activity rhythm in Plethodon cinereus and its modification
by an influence having a lunar frequency 188

YOST, HENRY T., JR., AND HOPE H. ROBSON

Studies on the effects of irradiation of cellular particulates. II. The
effect of gamma radiation on oxygen uptake and phosphorylation 198

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