Full text of "The Biological bulletin"

See other formats

| a' A|' B'| a

=, \o.\ alan,

ararur wr

ae ‘
lAIAAA\ | \f | AA alala
: AARNA AMAR ae Reena AAP
Ap AAR ‘i Oy
nnontgaronmnneat RAMAN NARA, NAAAAD
CAR AARARRS WAN ARERR RAAAAARAR

lain A AARAAAAAAA AA A
gal ala | \ \ \ a a alg \,

Walaa

al RAY NAVAS
A -AAAAAAs mnnnnnnn RARRAAAA A> | (

A RR RRGALELECOE wa

A AAA RM AAARANAAAAND

_o @& a . ai vy V¥Y Vw w wr

PAA (PS
WMS

> ao
>_ ie

“ AEE
> 2D)

>
>
>»
) >
> >» YD»
>>
»
>>
»)

a | an!
nAAAAAAAAAAAAA A

a} han

WARARAAAM Anan ANAAAWAA AAA AAR:

= ALA A ry | ‘a AVY V\ 2 Ap
AAAAA P AP NAlA AARAA AR A AAAAAA AAA VV \f APA AAAS AAy
Apr). IAAAAARAAAAAARRAAAAAAAA ioe wy
LA NAANAAPYY lala
AAA AA AAA aaa AAaaaaaaaa AA AAAAARAA ann
AA ANA \

~ i } |
A AA A Maia rYV\.¥ aN
tf | ) f
- u |

}
\ \

eo i

5 }

la AAAI AR A

la
WANA panaaanan Cara

ARO
AIAAAA AAA AAANAANAA A TAARe
PERERA AAA BRERA EE

or) Rt ae

senha ; Pe nnhih Iie,
PRAY SY ue CHE ashe

Oe yk my tay eti\.
ences, Rag iui

Seat f

THE
BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Editorial Board

Gary N. CALKINS, Columbia University E. E. JUST, Howard University

E. G. CONKLIN, Princeton University FRANK R. LILLIE, University of Chicago

E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago

SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
LEIGH HOADLEY, Harvard University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University

H. S. JENNINGS, Johns Hopkins University EDMUND B. WILSON, Columbia University

ALFRED C. REDFIELD, Harvard University
Managing Editor

VOLUME LXXIII

AUGUST TO DECEMBER, 1937

‘Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.

THE BIOLOGICAL BULLETIN is issued six timesa year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.

Subscriptions and other matter should be addressed to the
Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa.
Agent for Great Britain: Wheldon & Wesley, Limited, 2, 3 and
4 Arthur Street, New Oxford Street, London, W.C. 2.

Communications relative to manuscripts should be sent to
the Managing Editor, Marine Biological Laboratory, Woods
Hole, Mass., between June 1 and October 1 and to the Institute
of Biology, Divinity Avenue, Cambridge, Mass., during the re-
mainder of the year.

Entered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.

LANCASTER PRESS, INC., LANCASTER, PA.

CONTENTS

No. 1. AvucGust, 1937

. PAGE

THIRTY-NINTH REPORT OF THE MARINE BIOLOGICAL LABORATORY 1
SAYLES, LEONARD P., AND S. G. HERSHKOWITZ

(ERI sais Sig ty A eS RD Ba ae ee Ue tte a a aR sill
Marza, V. D., EUGENIE V. MARZA AND Mary J. GUTHRIE
Histochemistry of the Ovary of Fundulus heteroclitus with —

Special Reference to the Differentiating Odcytes........... 67
MATTHEWS, SAMUEL A.
The Development of the Pituitary Gland in Fundulus....... 93

Beams, H. W., AND R. L. KING

we SUT INGT oe ove Gat Aten eNO satel Aa MMM aD oar weet cess, al cui nh 99
Miter, E. DEWrTT
A Study of the Bacterial and Alleged Mitochondrial Content
OhtheCells-of the Clover Nodulev 28) 3 sc dees 112
Mast, S. O., AND NATHAN STAHLER
The Relation between Luminous Intensity, Adaptation to
Light, and Rate of Locomotion in Amoeba proteus (Leidy).. 126
ABRAMOWITZ, A. A.
The Réle of the Hypophyseal Melanophore Hormone in the
Chromatic Physiolosy of Funduluse 2.2 see aee ee eee 134
BUTLER, MARGARET RUTH
The Effect of its Nitrogen Content on the Decomposition of
the Polysaccharide Extract of Chondrus crispus............ 143
PAYNE, NELLIE M.
The Differential Effect of Environmental Factors upon Micro-
bracon hebetor, Say (Hymenoptera: Braconidae) and its
Host, Ephestia kiihniella Zeller (Lepidoptera: Pyralidae).

TTI Te, GEO Scan aA Ch eB Ne aa EA alia hes ie i a a ea 147
HOADLEY, LEIGH
AuLovomy mm: che brachyiran,|Uca pieiax.. 200.0...) ae 155

TYLER, ALBERT, AND HANS BAUER
Polar Body Extrusion and Cleavage in Artificially Activated
Bscrot Wnechis Cato sas. adn: eta sc eee es eee Me, ele 164

iv CONTENTS

PAGE
Boyp, WILLIAM C.
Cross-reactivity of Various Hemocyanins with Special Refer-
ence to the Blood Proteins of the Black Widow Spider...... 181

No. 2. OcToBErR, 1937

WELsH, J. H., F. A. CHAcz, Jr., AND R. F. NUNNEMACHER

The Diurnal Migration of Deep Water Animals............ 185
COONFIELD, B. R., AND A. GOLDIN

The Problem of a Physiological Gradient in Mnemiopsis

During Nesenerationeee, tat eee ki) weer es fet 2 197
GLASER, OTTO, AND GEORGE P. CHILD
ihe Hexoctalrednon and :Growthhy 4 32. ae ee ee ee 205

CARVER, GAIL L.

Studies on Productivity and Fertility of Drosophila Mutants 214
BALL, Eric G., AND C. CHESTER STOCK

The pH of Sea Water as Measured with the Glass Electrode 221
GOLDSMITH, E. D.

The Relation of Endocrine Feeding to Regeneration, Growth,

and Egg Capsule Production in Planaria maculata.......... 22
PROSSER, C. LADD, AND JOHN Z. YOUNG

Responses of Muscles of the Squid to Repetitive Stimulation

of the: GramtNerve: Fibers...) .h1 jus eee See gee eee 237
SPARROW, F. K., JR.
The Occurrence of Saprophytic Fungi in Marine Muds...... 242

WHITAKER, D. M.

Determination of Polarity by Centrifuging Eggs of Fucus

LEBEN CUIS ty S302 le seam SOO MER Wert a ge age i 249
TYLER, ALBERT, AND W. D. HuMASON

On the Energetics of Differentiation, VI. Comparison of the

temperature coefficients of the respiratory rates of unfertilized

andvok tentilized ie9es <5... occ Jae eee bee os le eee 261
KENK, ROMAN

Sexual and Asexual Reproduction in Euplanaria tigris (Girard) 280
HORSTADIUS, SVEN

Investigations as to the Localization of the Micromere-,

Skeleton and Entoderm-forming Material in Unfertilized Egg

(0) Svaicl OTC itn en MEISE ER Coo) ce 295
HORSTADIUS, SVEN

Experiments on Determination in the Early Development of

Gerebratulusilacteusan. ac occ eee taae eae ee 317
PROGRAM AND ABSTRACTS OF SCIENTIFIC MEETINGS, SUMMER

OB DOS 7k tire aaa ate harness 508 ri oe a a SEs 343

CONTENTS Vv

No. 3. DECEMBER, 1937

PAGE
WATERMAN, A. J.
Effect of Salts of Heavy Metals on Development of the Sea
WicchiiwAnbacia pUNnctMlatay a! eat Me ee hha ieee 401
REDFIELD, ALFRED C., HOMER P. SMITH, AND BOSTWICK KETCHUM
The Cycle of Organic Phosphorus in the Gulf of Maine...... 421
ANDERSON, BERTIL GOTTFRID, H. LUMER, AND L. J. ZUPANCIC, JR.
Crowth and Vanability im Daphnia pulex:...-..0......... 444

CLARKE, GEORGE L., AND DONALD J. ZINN

Seasonal Production of Zodplankton off Woods Hole with

special reference to Calanus finmarchicus.................. 464.
Lituick, Lots C.

Seasonal Studies of the Phytoplankton off Woods Hole, Massa- .

CIMMISSTNES Be: os Gk A Cae ete ne eR oe ee oa i ee 488
CAMPBELL, MILDRED L., AND ABBY H. TURNER

Serum Protein Measurements in the Lower Vertebrates. I.

The colloid osmotic pressure, nitrogen content, and refractive

IMmdexoh turtle sexum- and body Mimiday sa. .456 eos. eo 8 504
TURNER, ABBY H.

Serum Protein Measurements in the Lower Vertebrates. II.

In marine teleosts and elasmobranchs...................-. Sulil
Goopricu, H. B., AND Maurice A. SMITH

Genetics and Histology of the Color Pattern in the Normal

and Albino Paradise Fish, Macropodus opercularis L........ o2i
Darton, H. CLark, AND H. B. GoopRIcH

Chromatophore Reactions in the Normal and Albino Paradise

IS Tereyi eg Mew Psa et eciaes. gS SMe ik Sl Aa RE eR eee, aie aaa wa 535
STURTEVANT, A. H.

Autosomal Lethals in Wild Populations of Drosophila pseudo-

lose uve: Ox sete aes i eeea sits IANS NAD UNA DADA O RID Se Dette eT 542
WHITAKER, D. M., anp C. M. CLANCY

The Effect of Salinity upon the Growth of Eggs of Fucus fur-

CSAC UIS) 103 Aare pte ees RUSS Reais Naas? A LPM eons NRE MER 6 552
HEILBRUNN, L. V., AND Kart M. WILBUR
Stimulation and Nuclear Breakdown in the Nereis Egg...... Soil

Fry, Henry J.
Studies of the Mitotic Figure. VI. Mid-bodies and their sig-
nificance for the central body problem..................-- 565
Riocu, Davin McK.
A Physiological and Histological Study of the Frontal Cortex
GS TOV S1E ea Si ea 2S oe ae rk i a RE Lf 591

"Volume LXXDT quill sy : Number 1

ia ~
as “4 A ” :
‘ } 2 5 J ' a
. ' if
s! Na f a
N47; AS
“SONAL EEE

BIOLOGICAL BULLETIN

PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY

Editorial Board

GARY N. CALKINS, Columbia University E. E. JUST, Howard University

E. G. CONKLIN, Princeton University FRANK R. LILLIE, University of Chicago

E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago

SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
LEIGH HOADLEY, Harvard University T. H. MorRGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University

H. 8. JENNINGS, Johns Hopkins University EDMUND B. WILSON, Columbia University

ALFRED C. REDFIELD, Harvard University
Managing Editor

AUGUST, 1937

Printed and Issued by
LANCASTER PRESS, Ine.
PRINCE & LEMON STS.
LANCASTER, PA.

THE BIOLOGICAL BULLETIN

Tue BIoLocicaL BULLETIN is issued six times a year. Single
numbers, $1.75. Subscription per volume (3 numbers), $4.50.

Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Mass., between June 1 and October 1 and to the Institute of
Biology, Divinity Avenue, Cambridge, Mass., during the remainder
of the year. | :

INSTRUCTIONS TO AUTHORS

Preparation of Manuscript. In addition to the text matter, manuscripts
should include a running page head of not more than thirty-five letters.
Footnotes, tables, and legends for figures should be typed on separate sheets.

Preparation of Figures. The dimensions of the printed page (43¢x7
inches) should be borne in mind in preparing figures for publication. Draw-
ings and photographs, as well as any lettering upon them, should be large
enough to remain clear and legible upon reduction to page size. Illustrations
should be planned for sufficient reduction to-permit legends to be set below
them. In so far as possible, explanatory matter should be included in the
legends, not lettered on the figures. Statements of magnification should take
into account the amount of reduction necessary. Figures will be reproduced
as line cuts or’ halftones. Figures intended for reproduction as line cuts
should be drawn in India ink on white paper or blue-lined coordinate paper.
Blue ink will not show in reproduction, so that all guide lines, letters, etc.
must be in India ink. Figures intended for reproduction as halftone plates
should be grouped with as little waste space as possible. Drawings and
lettering for halftone plates should be made directly on heavy Bristol board,
not pasted on, as the outlines of pasted letters or drawings appear in the
reproduction unless removed by an expensive process. Methods of repro-
duction not regularly employed by the Biological Bulletin will be used only
at the author’s expense. The originals of illustrations will not be returned
except by special request. —

Directions for Mailing. Manuscripts and illustrations should be packed
flat between stiff cardboards. Large charts and graphs may be rolled and
sent in a mailing tube.

Reprints. Authors will be furnished, free of charge, one hundred re-
prisits without covers. Additional copies may be obtained at cost.

Proof. Page proof will be furnished only upon special request. When
cross-references are made in the text, the material referred to should be
marked clearly on the galley proof in order that the proper page numbers
may be supplied.

Bntered October 10, 1902, at Lancaster, Pa., as second-class matter under
Act of Congress of July 16, 1894.

Vol. LXXIII, No. 1

THE

August, 1937

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

rt VANE BIOLOGICAL LABORATORY

Turrty-NintH Report, FOR THE YEAR 1936—

Forty-NINTH YEAR

I. Trustees AND ExecuTIvVE ComMITTEE (As oF Aucust II,

HOR GC) ee ee tere ee aM UN Cleve, ok Ba lta 1

TEAR an © OVEN TD RE n 1s cic de apsaeinie ty eet atiencuabsreectn OS elcr enelb 3

ee NC REO HIN CORPORAI TON) (0s ents cusnay a chee ie eusoshecMicteyedeheueier <2 seve 3

wy eAWGuOn DEE | CORPORATION sets efile 5 a)2 Dinh arbre ile Bee « 4

IN eGIWE PORMOF DHE URDASURER ay shel ae te eek arene ele Gh S)

Neier PORTION CMELE STB RATAN ch viet os ella crares Ge ain sale eg seals 10

Vie PORT) Ob VMETE GOMRECHOR! ass «6. sacs see ales wire nee els 11

SEAECMTE TUES AU ey nee cere Peele ree Altes te as alate. alin areas 11
Addenda:

end Se RS teat S Oy anv rita akeer cc ck otk Pinata rates ote mua 16

2 Anvestivatons and Students, 1936) 2: 2a. 5a. 5 -). sae 18

Salalbularm Niewmolse AtreM@ance: |... vas sacs see 29

4. Subscribing and Codperating Institutions, 1936 .... 30

5a areanae ILeeamey MOTO aM athe y Go diols Golem © 46 clots 30

6. SMOcwsr Sem msneleeynatsy MEE 855. ay doe yo 5 a 4 O 31

7, General Semanune Wiseutae, WO 2. c..0sce0d8ecae 34

On Menibersrot the Corporation) se eno tae eal 38

1 IN USUaISS)

EX OFFICIO

Frank R. Lituir, President of the Corporation, The University of Chicago.

MERKEL H. JAcogs, Director, University of Pennsylvania.

Lawrason Ruccs, Jr., Treasurer, 120 Broadway, New York City.
CHARLES PACKARD, Clerk of the Corporation, Columbia University.

EMERITUS

C. Bumpus, Brown University.
G. ConKLin, Princeton University.
R. Crane, New York City.
H. Donatpson, Wistar Institute of Anatomy and es
J. GrrenMAN, Wistar Institute of Anatomy and Biology
A. Harper, Columbia University.
. M. Metcatr, Waban, Mass.
. H. Parker, Harvard University.
1

gece ie

MARINE BIOLOGICAL LABORATORY

W. B. Scort, Princeton University.
W. M. WueEE ER, Harvard University.
EavBe WILson, Columbia University.

TO SERVE UNTIL 1940

H. B. Bicetow, Harvard University.

R. Cuampers, Washington Square College, New York University.
W. E. Garrey, Vanderbilt University Medical School.

CASWELL GRAVE, Washington University.

S. O. Mast, Johns Hopkins University.

A. P. MatHeEws, University of Cincinnati.

C. E. McCune, University of Pennsylvania.

C. R. Srocxarp, Cornell University Medical College.

TO SERVE UNTIL 1939

W. C. ALLeE, The University of Chicago.

ry N. CaLxins, Columbia University.

M. Ducear, University of Wisconsin.

V. HEILBRUNN, University of Pennsylvania.

. IrnvinG, University of Toronto.

W. J. V. OsterHour, Member of the Rockefeller Institute for Medical Re-
search.

A. H. Sturtevant, California Institute of Technology.

LorANDE L. WoopruFr, Yale University.

G
Be
Ie
1D

TO SERVE UNTIL 1938

E. R. Ciark, University of Pennsylvania.
Otto C. Graser, Amherst College.

Ross G. Harrison, Yale University.

E. N. Harvey, Princeton University.

H. S. JeEnnines, Johns Hopkins University.
F. P. Knowtton, Syracuse University.
FRANZ SCHRADER, Columbia University.

B. H. Witter, University of Rochester.

TO SERVE UNTIL 1937

W. R. AmBerson, University of Tennessee.

H. B. Goopricu, Wesleyan University.

I, F. Lewts, University of Virginia.

R. S. Litire, The University of Chicago.

T. H. Morean, California Institute of Technology.
A. C. REDFIELD, Harvard University.

C. C. SpEIDEL, University of Virginia.

D. H. TENNENT, Bryn Mawr College.

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES

FRANK R. Lituit, Ex. Off. Chairman.
MERKEL H. Jacoss, Ev. Off.
LAwrason Ric6s, Jr., Ex. Off.

F. P. KNow Ton, to serve until 1937.
B. H. Writer, to serve until 1937.

ACT OF INCORPORATION 3

E. R. Cxark, to serve until 1938.
C. C. SPEIDEL, to serve until 1938.

Tue Lisprary COMMITTEE

E. G. ConxKLin, Chairman.
WILLIAM R. AMBERSON.
CaO wISELING Ll:

C. C. SPEIDEL.

A. H. STURTEVANT.
WILLIAM R. TAYLOR.

Tue APPARATUS COMMITTEE

L. V. HEILBRUNN, Chairman.
W. R. AMBERSON.
D. J. Epwarps.
W. E. Garrey.
E. N. Harvey.
L. IRVING.
M. H. Jacoss.
B. Luck.

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 Sedg-
wick 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 scien-
tific study and investigation, and a school for instruction in biology and
natural history, and have complied with the provisions of the statutes of this
Commonwealth in such case made and provided, as appears from the cer-
tificate of the President, Treasurer, and Trustees of said Corporation, duly
approved by the Commissioner of Corporations, and recorded in this office;

Now, therefore, I, HENry B. Pierce, Secretary of the Commonwealth
of Massachusetts, do hereby certify that said A. Hyatt, W. S. Stevens,
W. T. Sedgwick, E. G. Gardiner, S. Minns, C. S. Minot, S. Wells, W.
G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and suc-
cessors, are legally organized and established as, and are hereby made, an
existing Corporation, under the name of the MARINE BIOLOGICAL
LABORATORY, 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 Ejighty-
Eight.

[SEAL]
HENRY OB] Pree:
Secretary of the Commonwealth.

4 MARINE BIOLOGICAL LABORATORY

III. BY-LAWS OF THE CORPORATION OF THE MARINE
BIOLOGICAL) LABORATORY

I. The annual meeting of the members shall be held on the second
Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 11.30 A.M.,
daylight saving time, in each year, 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. There shall be thirty-two Trustees thus chosen divided
into four classes, each to serve four years, and in addition there shall be two
groups of Trustees as follows: (a) Trustees ex officio, who shall be the
President of the Corporation, the Director of the Laboratory, the Assogiate
Director, the Treasurer and the Clerk; (b) Trustees Emeritus, who shall be
elected from the Trustees by the Corporation. Any regular Trustee who
has attained the age of seventy years shall continue 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 Cor-
poration and he shall become eligible for election as Trustee Emeritus for
life. The Trustees ex officio and Emeritus shall have all 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.

II. Special meetings of the members may be called by the Trustees to
be held in Boston or in Woods Hole at such time and place as may be
designated.

III. Inasmuch as the time and place of the Annual Meeting of Members
is 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 said 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.

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

V. The Trustees shall have the control and management of the affairs
of the Corporation; they shall present a report of its condition at every
annual meeting; they shall elect one of their number President of the Cor-
poration who shall also be Chairman of the Board of Trustees; 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. They shall from time to time elect members to the Corporation
upon such terms and conditions as they may think best.

VI. Meetings of the Trustees shall be called by the President, or by
any two Trustees, and the Secretary shall give notice thereof by written
or printed notice sent to each Trustee by mail, postpaid. Seven Trustees
shall constitute a quorum for the transaction of business. The Board of
Trustees shall have power to choose an Executive Committee from their
own number, and to delegate to such Committee such of their own powers
as they may deem expedient.

AUG 21 1937

REPORT OF THE TREASURER 5

VII. The accounts of the Treasurer shall be audited annually by a
certified public accountant.

VIII. The consent of every Trustee shall be necessary to dissolution
of the Marine Biological Laboratory. In case of dissolution, the property
shall be disposed of in such manner and upon such terms as shall be de-
termined by the affirmative vote of two-thirds of the Board of Trustees.

IX. These By-laws may be altered at any meeting of the Trustees, pro-
vided that the notice of such meeting shall state that an alteration of the
By-laws will be acted upon.

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

iy vee REPORT Ob Gib KREASURER

To THE TRUSTEES OF THE MarINE BIoLoGicAL LABORATORY :

Gentlemen: Herewith is my report as Treasurer of the Marine Bio-
logical Laboratory for the year 1936.

The accounts have been audited by Messrs. Seamans, Stetson and
Tuttle, certified public accountants. A copy of their report is on file at
the Laboratory and is open to inspection by members of the Corporation.

At the end of the year 1936, the book value of the Endowment Funds
in the hands of the Central Hanover Bank and Trust Company as
Trustee, was

CT POCO ed SCCUTUIES 6. ory. Ges VR yA ORIG ieee S$ 910572.2
Grrl phe Stet Poa at. a Ne each co ah Ober eae eee ee 9,037.32
SEIS) TS a ails DS I SED TL a adie ERE gah 1,616.08
BODILY UAL ES CCUFULIESS § Aa) dls, eu cicase Anes oe Ne Seah of 172,261.84
(GEIS Ge nee MRR EMMA Dig Fo A Aas ON 21,584.91

$1,115,072.36

The income collected from these Funds was as follows:

GRC RO UD TONUCTIE AIRS cokes 6 65 Soe, ss RE RE Be le $41,941.24
LOTUS MI RU LI0 Wn Stoel rae Vet OA A Sua Pkg oe a 29132
$50,192.56

an increase of more than $2,000 over the income from these Funds in
L955:

The income due from these Funds in arrears, some of which may
never be collected, was on December 31, 1936
General Fund ....... 2 ita the WS nae ch are te is Un arr $12,605.25
[LAND SAE TET IEG IS SSS. St RE ag a Oe ee 5,050.00

$17,655.25

6 MARINE BIOLOGICAL LABORATORY

The total amount in arrears was about $325 less than on December 31,
1935.

The dividends from the General Biological Supply House have con-
tinued—the total received for the year amounting to $12,700.

Retirement Fund. A total of $4,060 in pensions was paid. The
Fund at the end of the year consisted of securities of the book value
(Gye arena chy a otio! Sear eee ane IO pe ha oe a egy $18,923.27

Gala Perea re ee ice Walesa ae 684.04 $19,607.31

Income in arrears on December 3lst was ................ $3125

Plant Assets. The land (exclusive of Gansett and Devil’s Lane
tracts), buildings, equipment and library, represented an investment
VIEW Stet, Sado 8 cy ce Aout ne $1,755,892.28
less reservenion depreciation ..-.......- 471,880.72 $1,284,011.56

Income and Expenses. Expenses including $41,782.21 depreciation
exceeded income by $6,951.86.

There was expended from current funds net $26,319.79 for plant
account.

During the year the Laboratory acquired by gift from Dr. Meigs Lot
““X,” Bay Shore property and the bathhouse on it and by purchase the
Howes property, completing the frontage on Water Street, and the
assets of the Bar Neck Corporation which included the leasehold and
buildings of the Penzance Garage and the adjacent Spindell lot. At the
end of the year the Laboratory owed $8,500 on mortgage on the Howes
property and $8,500 in notes given for the acquisition of the Bar Neck
assets. In addition it owed on notes.and accounts payable $10,855.71.
It had accounts and notes receivable of $14,367.37 and $14,773.65 in
cash and bank accounts in its current assets.

Following is the balance sheet, the condensed statement of income
and outgo and the surplus account all as set out by the accountants:

EXHIBIT A

MariINE BroLocicAL LABORATORY BALANCE SHEET,
DECEMBER 31, 1936

Assets
Endowment Assets and Equities :
Securities and Cash in Hands of Central Hanover
Bank and Trust Company, New York, Trus-
tee—-Schedulesil-agand labs. Gems .n:\<cc i eee $1,115,072.36
Securities and Cash—Minor Funds—Schedule II 8,298.06 $1,123,370.42

REPORT OF THE TREASURER

Plant Assets:

iLaraa-—Syelaveckelle IY goscandedocase $ 109,749.39
Buildings—Schedule IV .......... 1,238,562.84
Equipment—Schedule IV ......... 157,202.67
Library—Schedule IV ...........:; 250,377.38 $1,755,892.28
jEess) Resenve: Lon Wepreciation 6. .). 4.20... .- 4.4 - 471,880.72
$1,284,011.56
Cash in Dormitory Building Fund .............. 223.24
Casini iwesenvie unde & ad yam weesnotiijae esac kines 24.65
Current Assets :
(CSTR ne BY Sa Ni oP ey SROs cet $ 14,773.65
Accounts and Notes-Receivable ................. 14,367.37
Inventories :
Supply Department .......... $ 41,039.96
Biological Bulletin ........... 12,179.92 53,219.88
Investments :
Devil’s Lane Property ........ $ 43,633.13
Gansett, Property, 6. ¢4s-q-5 .4- 5,614.49
Stock in General Biological
Supply ilicusey inc: 222-2. - 12,700.00

Securities and Cash—Retire-
ment Fund—Schedule V .. 19,607.31 81,554.93

ee Dade MISUL AM Cer Mapp tsrqtiass co ce ee maar aks cera 3,293.50
ihigias im Spppemse CNG) scescoocescsoccnsoades 323.44

$1,284,259.45

$ 167,532.77

Liabilities
Endowment Funds:
Endowment Funds—Schedule III . $1,114,980.01
Reserve for Amortization of Bond

I REITIIUIMSI Me nomic cet ea oa ones 92.35 $1,115,072.36
Minor Funds—-Schedule III] ................... 8,298.06
Plant Liabilities and Funds :
Mortgage—Payable, Howes Property ........... $ 8,500.00
Notes—Payable a/c Bar Neck Property Purchase 8,500.00
Donations and Gifts—Schedule III ............. 1,032,072.61
Other Investments in Plant from Gifts and Cur-
TTA UIILCIS) oh.).s Sky Serene i ea RO Be ge! esa 235,186.84
Current Liabilities and Surplus:
PNCCOUTITS—=NayAable “saiedalaec sae pavers era esis ey oe $ . 5,317.04
INotes=sP ayalloler ys cicw vets sce es at ai sane sod mie 5,500.00
‘Woods Hole Oceanographic Institution .......... 38.67
$ 10,855.71
Gurrent) Sugplas—Exiibit (© .245..5 osc. .ce esse 156,677.06

$2,575,162.64

$1,123,370.42

$1,284,259.45

$ 167,532.77

$2,575,162.64

Income :

General Endowment Fund ...

ibrary jhund Teacn-er cece
MS trUEtTOMl Ya) steyem cielo sees
Researches sc: ema an eee
Evening Lectures .............
Biological Bulletin and Member-

Ship Duesia tera racecars
Supply Department—

Schedulem Wil nie ere ies
Mess—Schedule VII ..........
Dormitories—Schedule VIII

(Interest and Depreciation

charged to above three
Departments—See Sched-
ules VI, VII, and VIII)

Dividends, General Biological
Supply House, Inc. .........
Rents:
Danchakoff Cottages ........
Newman Cottage ...........
Janitonsmblouses ass enn sone se
Howes Property ............
Bar Neck Property .........
Sale of Duplicate Library Sets
Interest on Notes-Receivable ..
Sundries) Geter eee eee
Maintenance of Plant:
Building and Grounds .......
Chemical and Special Appa-
WEES. dic dao oonccaucooagnon
Library Department Expense
Truck Expense. j2.2....4----
Sundry Expemse ............
Workmen’s Compensation In-
SUATICE! uearnteeertM ene ee
General Expenses:
Administration Expenses ....
Endowment Fund Trustee .
BadimDebts ia tveus succes oes

Excess of Expenses over In-
come carried to Current Sur-
plus—Exhibit C ............

EXHIBIT B

Martine BrioLtocicAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1936

Total
Expense Income
$ 41,941.24
8,251.32
$ 8,176.73 10,305.00
4,146.96 14,215.00
88.95
9,133.99 9,087.00
38,262.35 43,144.91
21,588.75 20,443.21
31,984.12 11,965.84
35,320.19
12,700.00
294.76 700.00
100.96 250.00
21.76 240.00
SSE SV 40.00
2,908.74
250.79
165.00
50.90
23,393.18
13,329.83
7 193:293
910.27
173.58
509.71
14,912.47
980.29
663.53
41,782.21

MARINE BIOLOGICAL LABORATORY

$183,210.81 $176,258.95 $125,991.43

6,951.86
$183,210.81

Net
Expense Income

$ 41,941.24

8,251.32

2,128.27

10,068.04
$ 88.95
46.99

4,882.56
1,145.54
20,018.28

35,320.19

12,700.00

405.24

149.04

218.24
283.37

2,508.74

250.79

165.00

50.90
23,393.18
13,329.83
SAS)
910.27
173.58
509.71
14,912.47
980.29
663.53
41,782.21

$119,039.57

6,951.86

$125,991.43

REPORT OF THE TREASURER 9)

EXHIB -€

MARINE BroLocicaAL LABORATORY, CURRENT SURPLUS ACCOUNT,
YEAR ENDED DECEMBER 31, 1936

Ee ACG AIT Ver tem OG Onna nach ck ica alsas Scbee es ces esc cae uvees $152,246.38
Add:
Reserve for Depreciation charged to Plant Funds ............... 41,782.21
$194,028.59
Deduct :

Payments from Current Funds during Year for Plant
Assets as shown in Schedule IV,

I Geavat let ites cracls ata ares ie ta a RR RAG ARAL a $10,146.34
J BYSvIGINOVERS AMA a coat One Lat Oe Reem ADH AC 13,329.91:
EV GUI p Me tata ery deg ee cee ete Pate catna is casio hiner cts 5,421.95
Ube ilaeeutayee vec garners ere ae enact ean) Se alam wns 14,757.42
$43,655.62
Less,

Notes and Mortgage payable on
account of additions to Plant,

Hand and Buildings 2:....... $17,000.00
Received for Plant Assets dis-
POSER Okt Aen a. wero ae oes 287.50

Adjustment of Accrued Charges
on account of Library, De-

cember 43); 19350. 25s) eek 48.33 17,335.83
$26,319.79
Pensions and Allowances Paid ........... $ 4,060.00
Expenses on account of Retirement Fund
SEGUIILICS Maye apts te Sel Pits eee oie rae ee 905.43
$4,965.43
Less,
Retirement Fund Income ............ $ 845.13
Profit on Sale of Retirement Fund Se-
CUTIES SE ye eee kr uaa ude 40.42

$ 885.55 $ 4,079.88

Excess of Expenses over Income for Year as shown :
CUPOLA OEMS PestenD ASU scab HR SOE Monec otto eee bc GOSS ON NIS7. Jol. I8

Balance ecember ol, 199G— Ee xibitiNy 2 mance eo eee ates ase setae $156,677.06

Respectfully submitted,
LAWRASON RIGGS, JR.,

Treasurer

10 MARINE BIOLOGICAL LABORATORY

V2 OEE PORT OF Eis Se bik AdseAsN

A report of the budget assigned to the Library by the Executive
Committee for the year 1936 is as follows: books, $1,000 (with the
understanding that any part of this not used for books be transferred
to back sets) ; serials, $6,000; binding, $1,500; express, $300; supplies,
$500; salaries, $7,150; back sets, $2,350; total, $18,800. The sum of
$250.79 acquired by the Librarian by the sale of duplicates increased
the total budget to $19,050.79. The expenditures of the Library under
the same headings are itemized for the end of the year as follows:
books, $695.02 ; serials, $5,471.00; binding, $1,614.85 ; express, $187.10;
supplies, $386.65; salaries, $7,150; back sets, $3,478.86; total, $18,-
983.48. An unspent balance of $67.31 may be accounted due to the
sale of duplicates and the Librarian hopes that this sum in reverting
to the general accounts may be used by the Laboratory toward securing
a drying unit for the basement room in the dormitory where the Li-
brary’s duplicate reprints are stored.

The acquisitions of the Library during 1936 follow: 34 back sets
were completed and 20 partially completed; 29 of the completed sets
were for the Marine Biological Laboratory and 5 for the Woods Hole
Oceanographic Institution; 12 of the partially completed sets were for
the former and 8 for the latter ; the total number of current serial titles
received was 1,339: 376 purchased by the Marine Biological Laboratory
(19 new), 36 by the Woods Hole Oceanographic Institution (1 new),
630 by exchanges with the “ Biological Bulletin” (9 new) and 57 by
exchange for the publications of the Woods Hole Oceanographic Insti-
tution (18 new), 240 by gift, 224 and 16 respectively. The Marine
Biological Laboratory purchased 86 new books and the Woods Hole
Oceanographic Institution purchased 1, authors and publishers (authors,
8 and publishers, 26) presented 33 to the former and 1 to the latter; 30
were old books variously acquired and a fine copy of Swammerdam’s
“Book of Nature,’ 1758, was sent to the Marine Biological Laboratory
by J. C. Waller of Liverpool, England, accompanied by a letter of good
will to the Laboratory. The new reprints filed were 3,339 (675 cur-
rent and 2,664 of previous date). Uncatalogued, and therefore omitted
from this count, 580 further 1936 reprints are on hand, making the
excellent total of 1,255 current reprints received before February of the
following year. This is the highest record for any year in the history
of the Library, and justifies the innovation of last summer in the read-
ing-room display of current reprints. About 1,000 of the older reprints
filed were from the large collection of Dr. Gilman A. Drew’s reprints.
Mrs. Drew presented this collection to the Marine Biological Laboratory

AUG 21 1937

REPORT OF THE DIRECTOR 11

last summer. There remain from the valuable collection more than
6,000 reprints duplicate to the files in the Library stacks. These will
be placed in the duplicate collection.

With the final statement of this report that the Library now totals
42,287 volumes and 94,980 reprints, the Librarian is under the obliga-
tion to state that the stacks for serials are filled with an allowance of
growth space for each current set for the next four years (including
1937) and that the serial sets are now spread through the entire fifth,
fourth and second floors and the side shelves of the third (book) floor
and the first (reprint) floor. This arrangement and spacing of the
serial sets was accomplished during the fall of 1936. Space for new
“back sets’”’ can be made at one end of the “book stack” for a few
years, since the present book holdings may be crowded into two thirds of
the space they now occupy. The reprint boxes fill the first floor space
allotted to them and will be very crowded before the end of four years.
All duplicate serials and reprints have now been housed outside the
Library. During the year many duplicate serials and reprints were
sold or exchanged. This is shown in the unusual sales and filled-in
serial sets recorded above. It is necessary to explain also how 50,000
volumes, which will be the total in four years if growth occurs at the
present rate of about 2,000 annually, and 108,000 reprints if each year
adds 3,500, will completely fill space that in 1925 was estimated to be
adequate for 100,000 volumes, or 20,000 on each of five floors. The
reprint floor at once reduces the available space for volumes to a capac-
ity for 80,000. Besides this the many serial sets and books of quarto
size and over reduce the space and half of the bound serials recorded
in Our count are in reality two volumes bound together, so that the Li-
brary will at the end of the year 1940 actually be housing more nearly

75,000 volumes counted as volumes and not by the accession number,
and 108,000 reprints.

Vile thn ee PORT OF EE yD CLO

To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY :

Gentlemen: I beg to submit herewith a report of the forty-ninth ses-
sion of the Marine Biological Laboratory for the year 1936.

1. Attendance. An inspection of the tabular summary of attendance
on page 29 of this report and the corresponding summaries in earlier
reports shows that during the past 14 years the attendance of investi-
gators at the Marine Biological Laboratory has passed through two dis-
tinct phases and now seems to be entering into a third. The first phase
was marked by a steady annual increase during which the number of

12 MARINE BIOLOGICAL LABORATORY

investigators grew from 176 in 1923 to 362 in 1931, the latter figure be-
ing the largest yet reached in the history of the Laboratory. In 1932,
when the effects of the depression had become fully felt by most Ameri-
can colleges and universities, the second phase began with a sharp drop
in the attendance to 314, at approximately which level it was main-
tained with no significant changes for four years. In 1936 with a sud-
denness equalled only by that of the decrease in 1932 a large increase
brought the figure back almost to its highest previous level (359 in 1936
as compared with 362 in 1931). Indeed, since the number of students,
which fluctuates within the rather narrow limits from year to year,
happened to be at about its maximum in 1936, the total attendance of
investigators and students together, after allowing for duplications, was
in that year the largest in the history of the Laboratory (473 in 1936 as
compared with 467 in 1931). Particularly worthy of mention is the
very large number of institutions represented by students and investi-
gators in 1936 (158 as compared with the next-highest number of 143
in 1935).

At the time of the writing of this report, though accurate figures are
not yet available, it appears likely that the attendance in 1937 will sur-
pass all previous records. If, as seems possible, the Laboratory is now
entering into a new period of increasing attendance, serious considera-
tion must be given to the best means of preventing undue crowding in
the future. Since without additions to the present laboratory buildings
the maximum number of investigators that can be accommodated at one
time does not greatly exceed the figures reached in 1931 and in 1936,
further increases will be possible only by lengthening the season and
flattening the peak of attendance which now occurs early in August.
That very considerable possibilities in these directions still exist is shown
by the following tabulation of the attendance during the past ten years
at approximately ten-day intervals throughout the working season.

1927 1928 1929 1930 1931 1932 1933 1934 1935 1936

May Ohi sch eta eaemenars Tels 9 6 Ox 8 eA alc
June DO sss eeaeP mee 50). 64 55° 50 «51 54° 46° 54 as a
Pete: ZOU Ee an annex 14S 1400 139-153) 153) 127 129) 137 127 ae
SOM ett Aeon 212 240 197 208 217 172 184 196 174 190
July LO sae Scotts 247 281 238 253 258 225. 235 249° 226, 242
~ PAA eee anton iors 2A7 7 282 242 ~250 273\ 2450) 258) (250 Zoe OU)

i SO) yaar sites Sher eavent 245.272 249 253 281. 248.255 248 257 272
August OL ps aval able masgg onens 234 250 256 254 302 257 261 264 245 282
i QO) EOE I Nessa tsit 208 226 243 245 280 236 244 250 235 266
ates SMe ane A AEN 168 183 220 204 239 190 205 211 192 223
SEM MO sy scsoaneess WOM ITZ 157, 122, 136.129 OS en ote
‘ 20) exenstesstet slay she she SOQ) 43)).59) 44. (69 58 AS SS ee Zoi ena

i BO! Ee eialapeet a eteuote 2a a 1 G0) ee Ss amt Olas 20)

2. The Report of the Treasurer. While the figures given above

REPORT OF THE DIRECTOR 13

indicate that as measured by its attendance the recovery of the Labora-
tory from the effects of the depression is now substantially complete, the
same is not yet true with regard to its financial position, which though
entirely sound, is still such as considerably to restrict its scientific ac-
tivities. A study of the reports of the Treasurer for the past 6 years
shows that the chief reason for this condition is the reduced income from
the Endowment Fund. In 1931 the amount received from this source
was $57,728.26; by 1934 the corresponding figure was $46,939.97.
While it is gratifying to be able to record an increase in 1935 of
approximately $1,000 above the amount received during the preceding
year and a further increase of $2,000 in 1936, it is extremely unlikely,
because of the low rates: of interest at which funds liberated by the
maturing of securities can be reinvested, that the pre-depression income
from this source can be restored. Fortunately for the Laboratory,
special dividends declared by the General Biological Supply House dur-
ing the past two years have helped to some extent to make good the loss
of income from other sources. It is also encouraging to be able to
report a small but satisfactory increase in 1936 over 1935 both in the
gross sales and in the profits of the Laboratory’s own Supply Depart-
ment.

Next in importance to the income of the Laboratory from its endow-
ment funds is that from the fees paid by institutions and individuals for
research space. That financial recovery has in this case tended to lag
behind scientific recovery is shown by the fact that whereas the number
of investigators in attendance in 1936 was only 3 less than in 1931 the
income from the space they occupied was less by nearly $4,000. To this
extent, therefore, the burden of the depression has been transferred
from colleges and universities to the Marine Biological Laboratory at a
time when its own income from other sources has been significantly re-
duced. It is encouraging to note, however, that during the past year
there has been a substantial increase in the amount received by the Lab-
oratory for research space, the figures for the years 1935 and 1936 be-
ing $12,470.00 and $14,215.00 respectively.

3. The Report of the Librarian. During the year covered by this
report the growth of the Library has continued at a very satisfactory
rate. Particularly noteworthy are the increases in the number of jour-
nals currently received (1,339 in 1936 as compared with 1,271 in 1935),
the completion of a considerable number of back sets of journals, and
further important additions to the reprint collection. With complete
sets of almost all the more important periodicals in Biology and the
related fields accessible at all times in the library itself, and with almost
100,000 reprints available for the use of investigators in their own

14 MARINE BIOLOGICAL LABORATORY

rooms, it can now be said without exaggeration that the biologists work-
ing at the Marine Biological Laboratory enjoy library facilities which
are unsurpassed anywhere. The growth of the library since 1926 is
more completely summarized in the following table:

1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936
Serials received cur-

rently eee 628 764 874 985 1,060 1,080 1,126 1,137 1,197 1,271 1,336
Total number of

bound volumes . 18,200 22,800 26,500 28,300 31,500 33,800 36,000 37,400 38,600 40,200 42,000
Reprints ee 38,000 43,000 51,000 59,000 64,000 70,000 76,000 81,000 86,000 92,000 95,000

4. Courses of Instruction. At its last meeting in 1936 the Executive
Committee received and accepted with regret the resignation of Dr. EI-
bert C. Cole as head of the Course in Invertebrate Zoology, a position
which he had filled with conspicuous success since 1932. As his suc-
cessor the Committee appointed Dr. T. H. Bissonnette of Trinity Col-
lege, who as a member of the Staff since 1926 and as Acting Head of
the Course in 1936 is peculiarly well fitted to continue the work so ably
directed by Dr. Cole and his predecessors.

5. Lectures and Scientific Meetings. During the past summer 13
evening lectures were given and 9 evening meetings were held for the
presentation and discussion of shorter papers by investigators asso-
ciated with the three Woods Hole scientific institutions. The number
of papers so presented was 37; their titles are listed on page 31. In
addition, the General Scientific Meeting on August 27 and 28, which
was devoted exclusively to work accomplished at the Marine Biological
Laboratory during the current season, was the most successful ever
held. So large was the number of titles submitted that it was necessary
to devote two full mornings to scientific papers and an additional after-
noon to demonstrations. The program of this meeting on page 34
and the abstracts of the papers published in the Biological Bulletin
for October 1936, give a very excellent picture of the work carried on
at the Laboratory during the summer of 1936. In addition to its own
scientific activities, the Laboratory also acted as host to the Genetics
Society of America, which on September 3, 4 and 5 held in Woods
Hole a very well-attended and successful meeting.

6. Acquisitions of Property. The past year has seen three impor-
tant additions to the land and buildings owned by the Marine Biological
Laboratory. The first of these, a very generous gift to the Laboratory
by Dr. Edward B. Meigs, is the bathing beach and large bathhouse on
Buzzard’s Bay, together with the remainder of what is officially known
as Lot X of the Bay Shore Property of Henry H. and Sarah B. Fay,
having a total area of approximately 35,000 square feet. The beach,
with a frontage of approximately 200 feet on the water, is the best one

———

REPORT OF THE DIRECTOR 15)

available in the vicinity of the Laboratory, and has been used for many
years by our students and investigators, though with some uncertainty
as to whether this privilege might at some future time be withdrawn.
The generosity of Dr. Meigs has effectively put an end to all such
doubts.

A second very important acquisition, obtained through the foresight
and generosity of Mr. Charles R. Crane, to whom the Marine Biological
Laboratory is already so deeply indebted for support during its early
struggles for existence and for very substantial aid in placing it in its
present position of security, is that of the land and buildings occupied
by the Penzance Garage on Main Street and the Spindell lot adjoining
it on the southeast. This tract of over two acres extends from the
property owned by the Oceanographic Institution to the entrance into
the Eel Pond. Its fine frontage on Great Harbor offers very important
possibilities for the future development of the Laboratory.

The third addition, obtained by purchase, is the Howes property on
Main Street consisting of a house and barn and a lot 77 by 119 feet
lying between the Kidder House and the “ Homestead.” The acqui-
sition of this property completes the ownership by the Laboratory of
the entire block bounded by Main and Center Streets and East and
West Streets, respectively. Within these boundaries are the original
laboratory building erected in 1888 and most of the wooden buildings
added subsequently. The importance to the Laboratory of the posses-
sion of this unbroken tract is obvious.

7. Board of Trustees. At the annual meeting of the corporation
held on Tuesday, August 11, Professor S. O. Mast of Johns Hopkins
University was chosen to fill the vacancy in the class of 1940 created
by the earlier election at the same meeting of Dr. M. J. Greenman as
Trustee Emeritus.

There are appended as parts of the report:

Pebhe Stat 1936:

. Investigators and Students, 1936.

. A Tabular View of Attendance, 1932-1936.

. Subscribing and Cooperating Institutions, 1936.
. Evening Lectures, 1936.

. Shorter Scientific Papers, 1936.

. General Scientific Meeting, 1936.

. Members of the Corporation, August, 1936.

CON OD uf WN

Respectfully submitted,
Me ACOs;

Director.

16 MARINE BIOLOGICAL LABORATORY

1 ie SiN OSG

MERKEL H. Jacoss, Director, Professor of General Physiology, University
of Pennsylvania.
Associate Director: ——

ZOOLOGY .

I. INVESTIGATION

Gary N. Carxins, Professor of Protozodlogy, Columbia University.

E. G. Conxiin, Professor of Zodlogy, Princeton University.

CASWELL Grave, Professor of Zodlogy, Washington University.

H. S. Jennines, Professor of Zoology, Johns Hopkins University.

Frank R. Litre, Professor of Embryology Emeritus, The University of
Chicago.

C. E. McCiune, Professor of Zoology, University of Pennsylvania.

S. O. Mast, Professor of Zodlogy, Johns Hopkins University.

T. H. Morcan, Director of the Biological Laboratory, California Institute
of Technology.

G. H. Parker, Professor of Zoology Emeritus, Harvard University.

E. B. Witson, Professor of Zoology Emeritus, Columbia University.

LorAnvE L. Wooprurr, Professor of Protozodlogy, Yale University.

II. INstTRuUCTION

. H. Bissonnette, Professor of Biology, Trinity College.

. C. Cote, Professor of Biology, Williams College. (Absent in 1936.)

E. Haptey, Associate Professor of Biology, New Jersey State Teachers

College at Montclair.

R. Kite, Instructor in Zodlogy, Swarthmore College.

A. MattTHews, Associate in Anatomy, School of Medicine, University of

Pennsylvania.

O. E. Netsen, Assistant Professor of Zodlogy, University of Pennsylvania.

L. P. Sayers, Assistant Professor of Biology, College of the City of New
York.

A. J. WatEeRMAN, Assistant Professor of Biology, Williams College.

er eal 1) leslie)

Junior INSTRUCTORS

P. S. Crowe t, Jr., Instructor in Biology, Brooklyn College.
A. M. Lucas, Associate Professor of Zodlogy, Iowa State College.

PROTOZOOLOGY
I. INVESTIGATION

(See Zodlogy)

II. InstructTion

Gary N. Carxins, Professor of Protozodlogy, Columbia University.
ELi1zABETH DrumTRA, Instructor in Zodlogy, Wilson College.
G. W. Kipper, Instructor in Zodlogy, College of the City of New York.

REPORT OF THE DIRECTOR 17

EMBRYOLOGY
I. INVESTIGATION

(See Zoology)

II. INstTRUCTION

L. G. Bartu, Assistant Professor of Zoology, Columbia University.

Husert B. Goopricu, Professor of Biology, Wesleyan University. (Ab-
sent in 1936.)

BENJAMIN H. Grave, Professor of Biology, De Pauw University.

LricH Hoap.tey, Professor of Zoology, Harvard University.

CHARLES Pacxarp, Assistant Professor of Zoology, Institute of Cancer
Research, Columbia University.

Oscar Scuotreé, Assistant Professor of Biology, Amherst College.

PHYSIOLOGY
I. INVESTIGATION

Witt1am R. AmBerson, Professor of Physiology, University of Tennessee.

Harotp C. Brapiey, Professor of Physiological Chemistry, University of
Wisconsin.

WALTER FE. Garrey, Professor of Physiology, Vanderbilt University Med-
ical School.

Ratpu S. Litre, Professor of General Physiology, The University of Chi-
cago.

ALBERT P. Matuews, Professor of Biochemistry, University of Cincinnati.

II. INSTRUCTION
Teaching Staff

ROBERT CHAMBERS, Professor of Biology, New York University.

J. K. W. Fercuson, Assistant Professor of Physiology, University of West-
ern Ontario.

RupotF H6ser, Visiting Professor of Physiology, University of Pennsyl-
vania.

LAuURENCE IrvinG, Professor of Experimental Biology, University of To-
ronto.

Leonor MicHAELIs, Member of the Rockefeller Institute, New York City.

C. Lapp Prosser, Assistant Professor of Physiology, Clark University.

Junior Instructors

KENNETH FisHER, Demonstrator in Biology, University of Toronto.
F. J. M. Stcuet, Royal Society of Canada Fellow.

BOTANY
I. INVESTIGATION

C. E. Aten, Professor of Botany, University of Wisconsin.

S. C. Brooks, Professor of Zodlogy, University of California.

B. M. Ducear, Professor of Physiological and Economic Botany, University
of Wisconsin.

Ivey F. Lewis, Professor of Biology, University of Virginia.

Wm. J. Rossins, Professor of Botany, University of Missouri.

18 MARINE BIOLOGICAL LABORATORY

II. INSTRUCTION

WILLIAM RANDOLPH Tay_tor, Professor of Botany, University of Michigan.
FRANCIS DroueEt, Research Fellow, University of Missouri.
G. W. Prescott, Assistant Professor of Biology, Albion College.

GENERAL OFFICE

F. M. MacNaueurt, Business Manager.
Potty L. Crowe t, Assistant.
EpitH BILiincs, Secretary.

RESEARCH SERVICE AND GENERAL MAINTENANCE

SAMUEL E. Ponp, Technical Man- Wut11amM Hemenway, Carpenter.

ager. Lester F. Boss, Research Techni-
Oscar W. RicHarps, Chemical cian.

Service. J. D. Grauaw, Glassblower.
G. Fartta, X-Ray Physicist. P. H. LitjEstranp, Assistant.

Tuomas E. Larkin, Superintendent.

LIBRARY

PrisciLLA B. Montcomery (Mrs. Thomas H. Montgomery, Jr.), Librarian.
DrEBoRAH LAWRENCE, Secretary.
Doris ENDREJAT, Mary A. Rowan, Assistants.

SUPPLY DEPARTMENT

James McInnis, Manager. GEOFFREY Leuy, Collector.
Mitton B. Gray, Collector. WaLTER KAHLER, Collector.
A. M. Hixton, Collector. RutuH S. CrowELt, Secretary.
A. W. LeEaTtHeErs, Shipping Depart- Anna N. Hatt, Secretary.
ment.
MUSEUM

GerorcE M. Gray, Curator Emeritus.

ZEN Wa SG AT ORS: AND Sap ENG S O56

Independent Investigators

AprAmowltTz, A. A., Research Assistant, Harvard University.

Avpison, Witt1AM H. F., Professor of Normal Histology and Embryology, Uni-
versity of Pennsylvania.

Aes, W. C., Professor of Zodlogy, The University of Chicago.

AMBERSON, WILLIAM R., Professor of Physiology, University of Tennessee.

ANDERSON, RuBeErT S., Research Associate, Princeton University.

AppEL, F. W., Associate Professor of Biology, St. John’s College.

ARMSTRONG, PuHILtP B., Assistant Professor of Anatomy, Cornell University Medi-
cal College.

BatLey, Percy L., Jr., Instructor in Physiology, College of the City of New York.

Batt, Ertc G., Associate in Physiological Chemistry, Johns Hopkins University
Medical School.

REPORT OF THE DIRECTOR 19

BALLARD, WILLIAM W., Assistant Professor, Dartmouth College.

BartH, L. G., Assistant Professor of Zoology, Columbia University.

Bauer, Hans G. E., International Research Fellow, Rockefeller Foundation.

BERNSTEIN, FELIX, Professor of Biometrics, New York University.

BISSONNETTE, T. H., Professor of Biology, Trinity College.

Boning, J. H., Head of Zodlogy Department, University of Iowa.

Bozter, Emit, Fellow in Medical Physics, Johnson Foundation, University of
Pennsylvania.

Bray, CHARLES W., Assistant Professor, Princeton University.

BRINLEY, F. J., Assistant Professor of Zoology, North Dakota State College.

Bronk, D. W., Professor of Biophysics, University of Pennsylvania.

Brooks, M. M., Research Associate in Biology, University of California.

Brooks, S. C., Professor of Zodlogy, University of California.

Brown, Dueatp E. S., Assistant Professor of Physiology, New York University,
College of Medicine.

Bupineron, R. A., Professor. of Zodlogy, Oberlin College.

Burton, ALAN C., Training Fellowship, General Education Board.

Cas_e, R. M., Assistant Professor of Parasitology, Purdue University.

CaLkins, Gary N., Professor of Protozodlogy, Columbia University.

CAMERON, JOHN A., Instructor in Zodlogy, University of Missouri.

CANNAN, Ropert K., Professor of Chemistry, New York University, College of
Medicine.

Carson, J. G., Instructor in Zoology, University of Alabama.

CAROTHERS, E. ELEANOR, Research Associate, University of Iowa.

CatTELL, McKeen, Associate Professor of Pharmacology, Cornell University Med-
ical College.

CHAMBERS, Ropert, Research Professor of Biology, New York University.

CHENEY, RatpH H., Professor of Biology, Long Island University.

CuILp, GreorcE P., Research Assistant in Biology, Amherst College.

CLarK, ELEANOR L., University of Pennsylvania Medical School.

Ciark, Extor R., Professor of Anatomy, University of Pennsylvania.

CLEMENT, ANTHONY C., Assistant Professor of Biology, College of Charleston.

Crowes, G. H. A., Director of Research, Eli Lilly & Co.

Cor, W. R., Professor of Biology, Yale University.

Coxer, R. E., Professor of Zodlogy, University of North Carolina.

CoonFIELD, B. R., Assistant Professor, Brooklyn College.

CoreLAND, Manton, Professor of Biology, Bowdoin College.

Corey, H. IRENE, Research Assistant, University of Pennsylvania.

Co Tur, FRANK W., Associate Professor, New York University, College of Medi-
cine.

Cownry, E. V., Professor of Cytology, Washington University.

CROWELL, PRINCE SEARS, JR., Instructor, Brooklyn College.

Curtis, W. C., Professor of Zodlogy, University of Missouri.

Curwen, Atice O., Assistant Professor of Anatomy, Woman’s Medical College
of Pennsylvania.

Dan, Katsumaé, Research Associate, Misaki Marine Biological Station, Misaki,
Japan.

Ditpine, GLENN C., Instructor in Zoology, Northwestern University.

DILLER, WILLIAM F., Instructor in Zodlogy, Dartmouth College.

Donatpson, Henry H., Member, Wistar Institute.

DornFELp, Ernst J., Assistant in Zoology, University of Wisconsin.

Dreyer, Wit1i1AM A., Instructor in Zoology, University of Cincinnati.

DrovueEt, FrANcts, Research Fellow, University of Missouri.

puBuy, H., Research Fellow, Harvard Medical School.

Dun1rHueE, F. W., Instructor, New York University.

Ers, Harotp N., Associate Professor, Loyola University, School of Medicine.

20 MARINE BIOLOGICAL LABORATORY

Fercuson, JAMES K. W., Assistant Professor of Physiology, University of West-
ern Ontario, Medical School.

Ficce, Frank H. J., Associate Professor of Anatomy, University of Maryland
Medical School.

FLeEIsHER, Moyer S., Professor of Bacteriology and Hygiene, St. Louis University.

FRENCH, C. S., 18 Tremont Street, Boston, Massachusetts.

Fry, Henry J., Visiting Investigator, Cornell University Medical College.

Garrey, W. E., Professor of Physiology, Medical School, Vanderbilt University.

GERARD, RatpH W., Associate Professor of Physiology, The University of Chi-
cago.

GrucHrist, FRANcIs G., Assistant Professor of Zoology, Pomona College.

GOLDFELDER, ANNA, Research Assistant, Columbia University, Institute of Cancer
Research.

GoTTSCHALL, GERTRUDE G., Assistant in Biochemistry, Cornell University Medical
College.

Grave, B. H., Professor of Zoology, DePauw University.

GRAVE, CASWELL, Professor of Zoology, Washington University.

Haney, CHaries E., Associate Professor of Biology, New Jersey State Teachers
College.

Harting, H. K., Fellow in Medical Physics, University of Pennsylvania.

Harvey, ETHEL Browne, Princeton University.

HEADLEE, WittiAm H., Instructor in Biology, Purdue University.

Eiommoeny EAS J sspeinic Professor of Zoology, University of Pennsylvania.

HENSHAW, PAUL S) Biophysicist, Memorial Hospital.

Hess, WALTER N., Sigal of Biology Department, Hamilton College.

Hipparp, Horr, Associate Professor of Zodlogy, Oberlin College.

Hitt, SAMUEL E., Assistant in General Physiology, Rockefeller Institute.

Hoapiey, LeicH, Professor of Zoology, Harvard University.

HOoser, JOSEPHINE, University of Pennsylvania.

Hoser, Rupotr, Visiting Professor of Physiology, University of Pennsylvania.

HOLLAENDER, ALEXANDER, Special Investigator, Radiation Committee, National Re-
search Council, University of Wisconsin.

HOoLtTFRETER, JOHANNES, Assistant, Zoologisches Institut, Miinchen, Germany.

Hopkins, Dwicut L., Assistant Professor of Zoology, Duke University.

Horstapius, Sven, Associate Professor, University of Stockholm, Stockholm,
Sweden.

Howe, H. E., Editor, Industrial and Engineering Chemistry.

HuMMEL, KATHARINE P., Research Instructor, Cornell University.

IrRvING, LAURENCE, Professor of Experimental Biology, University of Toronto.

Jacoss, M. H., Professor of General Physiology, University of Pennsylvania.

JENKINS, GrorGE B., Professor of Anatomy, Medical School, George Washington
University.

Jouitn, J. M., Associate Professor of Biochemistry, Vanderbilt University Medi-
cal School.

Jones, E. Rurrin, Jr., Associate Professor, College of William and Mary.

Jones, RutH McC., Instructor in Zoology and Botany, Swarthmore College.

Kein, Ersa M., Assistant Professor of Zoology, Rutgers University.

KettcH, Anna K., Research Chemist, Eli Lilly & Co.

Kipper, GrorceE W., Instructor, College of the City of New York.

KiILLe, FranK R., Instructor, Swarthmore College.

Kinprep, JAMES E.., Associate Professor of Histology and Embryology, University
of Virginia, Medical School.

KiNG, JESsIE L., Professor of Physiology, Goucher College.

KLEINHOLZ, L. He Austin Teaching Fellow, Harvard University.

KNISELY, MeLvin . Fellow, General Education Board.

Knower, Henry McE., Research Associate in Biology, Yale University.

REPORT OF THE DIRECTOR Bl

KNowLTon, FRANK P., Professor of Physiology, Syracuse University, Medical
College.

Kopac, M. J., Research Associate in Biology, New York University.

Korr, Irvin M., Procter Fellow, Princeton University.

Kraamtz, C. P., Graduate Assistant in Zoology, University of Cincinnati.

Kraut, M. E., Research Chemist, Eli Lilly & Co.

KutTtner, ANN G., Resident Physician, Rockefeller Institute.

LANCEFIELD, DonaAtp E., Professor of Zodlogy, Columbia University.

LANCEFIELD, ReBecca C., Associate in Bacteriology, Rockefeller Institute.

LEHMANN, JORGEN, Rockefeller Fellow, Rockefeller Institute for Medical Re-
search.

LeviInE, Darwin S., Teacher of Biology, Theodore Roosevelt High School, New
York City, New York.

Lititz, FRANK R., Professor of Embryology, Emeritus, The University of Chi-
cago.

Lititz, Ratpu S., Professor of General Physiology, The University of Chicago.

LinpEMAN, V. F., Assistant Professor of Zoology, Syracuse University.

Loomis, W. E., Associate Professor of Plant Physiology, Iowa State College.

Lucas, Atrrep M., Associate Professor of Zoology, Iowa State College.

Lucas, Mtrtam Scott, 412 Tenth Street, Ames, Iowa.

Lucxké&, Batpuin, Professor of Pathology, University of Pennsylvania, School of
Medicine.

LuvetT, BAsILe J., Associate Professor of Biology, St. Louis University.

Lynn, W. GarpNer, Instructor, Johns Hopkins University.

MacCarpte, Ross C., Assistant Professor of Biology, Temple University.

MacDoueatt, Mary Stuart, Professor of Zodlogy, Agnes Scott College.

Mac ean, Bernice L., Instructor in Zodlogy, Hunter College.

Macers, EvizasetH, Assistant Professor of Physiology, Vassar College.

MAGRUDER, SAMUEL R., Research Associate, University of Cincinnati.

MarstaAnp, Douctas A., Assistant Professor of Biology, Washington Square Col-

lege, New York University.

Martin, Eart A., Professor of Biology, Brooklyn College.

Martin, WAtrTER E., Instructor, Purdue University.

Mast, S. O., Department of Zoology, Johns Hopkins University.

MartuHews, Apert P., Professor of Biochemistry, University of Cincinnati.

MatTHEws, SAMuEL A., Associate in Anatomy, University of Pennsylvania.

Mavor, JAMES W., Professor of Biology, Union College.

McCiune, C. E., Director, Department of Zoology, University of Pennsylvania.

McGrecor, James H., Professor of Zodlogy, Columbia University.

MicHaetts, Leonor, Member, Rockefeller Institute for Medical Research.

Mittrr, Forrest W., Research Entomologist, Amherst College.

Moment, GAtIRDNER B., Instructor, Goucher College.

MorcAn, Litrtan V., Pasadena, California.

Morean, T. H., Professor of Experimental Zoology, California Institute of Tech-
nology.

Morritz, Dr. C. V., Cornell University Medical College.

Mutter, H. J., Geneticist, Institute of Genetics, Moscow, Russia.

Nasrit, S. M., Professor of Biology, Atlanta University.

Naum, Laura J., Instructor in Biology, Flat River Junior College.

Navez, ALBERT E., Milton Academy.

NeLsen, Orin E., Assistant Professor of Zoology, University of Pennsylvania.

Nonipez, José F., Assistant Professor of Anatomy, Cornell University Medical
College.

NortHrop, JoHN H., Member, Rockefeller Institute.

NUNNEMACHER, RupotpH F., Assistant, Harvard University.

Orr, PAut R., Instructor in Biology, Brooklyn College.

D2, MARINE BIOLOGICAL LABORATORY

OsterHouT, W. J. V., Member, Rockefeller Institute for Medical Research.

PAcKARD, CHARLES, Assistant Professor of Zoology, Institute of Cancer Research,
Columbia University.

PAINTER, ELIzABETH E., Instructor in Physiology, University of Maryland, School
of Medicine.

Parker, G. H., Professor of Zodlogy, Emeritus, Harvard University.

Parpart, ARTHUR K., Assistant Professor, Princeton University.

PioucH, Harorp H., Professor of Biology, Amherst College.

Ponp, SAMUEL E., Technical Manager, Marine Biological Laboratory.

Prescott, G. W., Assistant Professor of Biology, Albion College.

Prosser, C. Lapp, Assistant Professor of Physiology, Clark University.

Puckett, WitttAM O., Instructor in Biology, Princeton University.

RAFFEL, DANIEL, Research Geneticist, Institute of Genetics, Moscow, Russia.

Rick, KENNETH S., Woods Hole, Massachusetts.

RicHArRpDS, Oscar W., Instructor in Biology, Yale University.

Rosertson, C. W., Assistant in Biology, Washington Square College, New York
University.

Root, WALTER S., Associate Professor of Physiology, Medical School, University
of Maryland.

RueH, Roserts, Instructor in Zodlogy, Hunter College.

Sastn, ALBERT B., Assistant on the Scientific Staff, The Rockefeller ene for
Medical Pesenedh

SANDowW, ALEXANDER, Instructor, Washington Square College. -

SAsLow, GeEorcE, Assistant Professor of Biology, New York University.

SAYLES, Leonarp P., Assistant Professor of Biology, College of the City of New
York.

SCHECHTER, Victor, Instructor, College of the City of New York.

SCHENTHAL, JoSEPH E., Julius Friedenwald Research Fellow, University of Mary-
land, Medical School.

Scuminpt, L. H., Research Fellow, Christ Hospital.

ScHMITT, Francis O., Associate Professor of Zoology, Washington University.

ScHorTte, Oscar E., Assistant Professor of Biology, Amherst College.

SCHRADER, Franz, Professor of Zoology, Columbia University.

SCHRADER, SALLY Hucues, Member, Natural Science Faculty, Sarah Lawrence
College.

ScHwas, JosEPH J., Assistant in Zodlogy, The University of Chicago.

Scott, ALLAN C., Instructor in Biology, Union College.

Scott, J. Paut, Associate Professor of Zodlogy, Wabash College.

SHAPIRO, HERBERT, Research Assistant, Princeton University.

SICHEL, FERDINAND J. M., Fellowship, Royal Society of Canada.

SICKLES, GRETCHEN R., Senior Laboratory Technician, New York State Depart-
ment of Health. :

SMELSER, GeorGE K., Instructor in Anatomy, Columbia University.

SmitH, Drerricn C., Instructor in Physiology, University of Tennessee.

SmitH, H. P., Professor of Pathology, University of Iowa.

SPEICHER, B. R., National Research Fellow, Columbia University.

SPEICHER, KATHRYN G., New York City, New York.

SPEIDEL, Cart C., Professor of Anatomy, University of Virginia.

STEIN, KatHryn F., Assistant Professor, Mount Holyoke College.

STEINBACH, H. Burr, Instructor in Zodlogy, University of Minnesota.

STERN, Kurt G., Visiting Lecturer in Physiological Chemistry, Yale University.

STEWART, Dorotuy R., Assistant Professor of Biology, Skidmore College.

pane Cuartes R., Professor of Anatomy, Cornell University Medical Col-
ege.

REPORDO DHE DIRECTOR 23

Srronc, Oxtver S., Professor of Neurology and Neuro-histology, Columbia Uni-
versity.

SrunKarp, H. W., Professor of Biology, New York University.

Summe_nrs, Francis M., Instructor in Biology, Bard College, Columbia University.

SZEPSENWOL, JOSEL, University of Geneva.

Tart, Cuartes H., Jr., Associate Professor of Pharmacology, Medical Branch,
University of Texas.

Tasutro, SHIRo, Professor of Biochemistry, University of Cincinnati, Medical
College.

Tayitor, WILLIAM RANpoLPH, Professor of Botany, University of Michigan.

TEWrnkEL, Lots E., Assistant Professor of Zodlogy, Smith College.

Tracy, Henry C., Anatomy Department, University of Kansas.

Tracer, WILLIAM, Assistant, Rockefeller Institute.

UnLeNHUTH, Epuarp, Professor of Anatomy, University of Maryland, Medical
College.

VARRELMAN, FERDINAND, American University.

Wa ker, Rotanp, Instructor in Biology, Rensselaer Polytechnic Institute.

Warner, E. O., Assistant Professor of Pathology, University of Iowa.

Warren, MarsHatt R., Graduate Assistant in Zodlogy, University of Cincinnati.

Waterman, A. J., Assistant Professor of Biology, Williams College.

WEISSENBERG, RICHARD, University of Berlin.

WELLts, G. P., Lecturer, University College, London, England.

Wuenpon, ArtHur D., Professor of Zodlogy and Head of Department, North Da-
kota State College.

Wuirtaker, D. M., Professor of Biology, Stanford University.

Witter, B. H., Professor and Head of Department of Zodlogy, University of
Rochester.

Witson, Epmunp B., Professor Emeritus in Residence, Columbia University.

Wintroge, Maxwett M., Associate in Medicine, Johns Hopkins University.

Wotr, E. Atrrep, Associate Professor of Biology, University of Pittsburgh.

Wotr, Ernst, Research Associate, Harvard University.

Wotr, Opat M., Lecturer, Barnard College.

Wooprurr, LoranvE L., Professor of Protozodlogy, Yale University.

Yntema, C. L., Instructor, Cornell University Medical College.

Younc, Joun Z., Fellow, Magdalen College, Oxford, England.

Younc, Rocer A., Assistant Professor of Zodlogy, Howard University.

Youncstrom, Kart A., Instructor in Anatomy, Kansas University.

Beginning Investigators

Axpaum, Harry G., Graduate Student, Columbia University.

AucirE, GLENN H., Graduate Student, University of Maryland, School of Medicine.

AwnpverscH, Marte, Assistant Professor of Biochemistry, Woman’s Medical Col-
lege, Philadelphia, Pennsylvania.

ANGERER, C. A., Instructor, University of Pennsylvania.

Artuur, J. B. M., Jr., Laboratory Assistant, Amherst College.

BisHop, Davin W., Instructor, University of Pennsylvania.

BocskeEy, STEPHEN C., Associate Professor of Biology, University of Notre Dame.

Brooks, JEANNE R., Oberlin College.

CHAMBERS, ALFRED H., Jr., Swarthmore College.

Cuurney, Leon, Instructor in Zoology, University of Pennsylvania.

Compton, ALFRED D., Jr., Graduate Student, Yale University.

CopELAND, D. Eucene, Biology Assistant, Amherst College.

Corson, Samuet A., Research Associate, Washington Square College, New York
University.

Dan, JEAN Crark, University of Pennsylvania.

24 MARINE BIOLOGICAL LABORATORY

Denny, MarrHa, Assistant in Zoology, Barnard College.

DonNELLON, JAMES A., University of Pennsylvania.

Dorpick, IsAporE, Graduate Student, University of Pennsylvania.

DrumTrRA, ELIzABeTH, Instructor, Wilson College.

Ducat, Louts-Paut, Instructor in Biology, University of Montreal.

Emerson, Henry S., Graduate Assistant, Amherst College.

FisHER, KENNETH C:, Demonstrator in Biology, University of Toronto.

FormMAN, RicHarp C., Graduate Student, Amherst College.

FRIEDMAN, SAM, Graduate Student, Washington Square College, New York Uni
versity. f

FronczAk, MicHAert J., Assistant Advisor to the Department of Biology, Seton
Hall College.

GREENFIELD, SYDNEY S., Graduate Student, Brooklyn College.

HarpreicH, Morton A., Graduate Student, University of Pennsylvania.

Harris, DaANniEL L., University of Pennsylvania.

Haw_ey, KATHARINE J., Smith College.

Henson, Marcaret, Smith College.

HersHKow!Tz, Sot. G., Student, New York University, College of Medicine.

HirscHFELD, NATHAN B., Student, Columbia University.

HoLLtIncGswortH, JOSEPHINE, University of Pennsylvania.

Hornor, HELEN B., Assistant in Zoology, Barnard College, Columbia University.

Horton, R. G., Graduate Student, Cornell University.

HucueEs, Roscoe D., Assistant in Zodlogy, Columbia University.

HunNnNINEN, ARNE V., Assistant, Johns Hopkins University.

Hunter, Francis R., Part-time Teaching Assistant, Princeton University.

Hunter, Laura N., Graduate Student, University of Pennsylvania.

Hurcuines, Lois M., Teacher of Biology, Weequahic High School.

IroH, Htpecoro, Graduate Student, Zodlogical Laboratory, University of Pennsyl-
vania.

JAILER, JosEPH W., Teaching Fellow, Washington Square College, New York
University.

JAKoBSEN, EpitH M., Science Teacher, Hawthorne High School, Hawthorne, New
Jersey.

Katiss, NATHAN, Assistant in Zoology, Columbia University.

KaurMan, ALAN L., Student, Franklin and Marshall College.

KEHOE, CATHARINE E., Research Assistant, Oberlin College.

Litty, Dantet M., Instructor in Biology, Providence College.

Lipman, Harry J., Graduate Assistant, University of Pittsburgh.

Lone, Marcaret E., Graduate Student, University of Pennsylvania.
Lorp, Rospert N., Student, Williams College.

MargQuette, WILLIAM G., Graduate Student, Columbia University.
Mayo, VirctntA, Teacher, Chairman, Department of Science, Dana Hall School.
Mazza, Dante, Harrison Fellow in Zoology, University of Pennsylvania.
McBring, T. F., Instructor Clinical Dentistry, School of Dentistry, University of
Pittsburgh.

MenpozA, GUILLERMO, Teaching Assistant, Northwestern University.
Moser, Fioyp, Graduate Student, University of Pennsylvania.

Newman, Morris, Graduate Student, University of Pennsylvania.
Novikorr, Arex B., Tutor, Brooklyn College.

O’Brien, JoHNn P., 1326 Quincy Street, N. E., Washington, D. C.
OpLAuc, THERON, Graduate Assistant, New York University.

Otson, RopNey A., Tufts College.

PALMER, CHARLES M., Assistant Professor of Botany, Butler University.
Peazopy, EL1zABETH B., Graduate Student, Radcliffe College.
RICHARDSON, EsTELLE, Graduate Student, New York University.

RoseE, S. Mervt, Assistant, Columbia University.

REPORT OF THE DIRECTOR 1)

RosENFIELD, RicHArpD E., Graduate Student, University of Pittsburgh.

ScHOENBORN, Henry W., Graduate Assistant, New York University.

ScHOEPFLE, Gorpon M., Undergraduate Assistant, DePauw University.

Scott, Brrore L., Student and Graduate Assistant, Atlanta University and Spel-
man College.

SEITCHEK, JosePH N., Graduate Student, University of Pennsylvania.

SoLBerG, ARCHIE N., University Fellow, Columbia University.

Specut, Heinz, Assistant in Physiology, New York University College of Medi-
cine.

Stock, CHarLEs C., Graduate Student in Physiological Chemistry, Johns Hopkins
Medical School.

TEITELBAUM, Harry A., Instructor in Anatomy, University of Maryland, Medical
School.

Watporn, Resecca S., Graduate Assistant in Zoology, University of Missouri.

WuHuee ter, NorMAN C., Assistant in Physiology, Purdue University.

WICHTERMAN, RALPH, Graduate Student, University of Pennsylvania.

Research Assistants

ARMSTRONG, LoursE S., Research Assistant, Cornell University Medical College.
AtcHLey, Dana W., Jr., Student, Harvard College.

Bear, Ricuarp S., Research Assistant, Washington University.

Beck, Lyte V., Varr Cott Research Fellow, Long Island College of Medicine.
BoswortH, Mirirarp W., Research Assistant, Wesleyan University.
BucHHEIT, J. Ropert, 1676 Van Buren Street, Saint Paul, Minnesota.
CARMICHAEL, J. C., Student, Vanderbilt University, Medical School.

CastLe, RutH M., New Jersey State Teachers College.

CLARK, JoHN K., Assistant, Trinity College.

Emppen, Maya, Research Assistant, University of Toronto.

ERLANGER, MarGAreET, Agnes Irwin School.

Evans, GrertrupbE, Research Assistant, The University of Chicago.

FENNELL, R. A., Research Assistant, Johns Hopkins University.

Ficcr, RosALIE YERKES, University of Maryland, Medical School.

Fiynn, Cart M., Instructor, University of Maine.

FoitcH-P1, Jorpt, Research Fellow, Rockefeller Institute.

FRANKENSTEIN, NorMAN L., Marquette University.

GLAssMAN, Haron N., Graduate Student, University of Pennsylvania.
GopricH, JAcK, Research Assistant, Columbia University.

GoFFIN, CATHERINE E., Research Assistant, Eli Lilly & Co.

Gotpin, ABRAHAM, Laboratory Assistant, Brooklyn College.

GrEEY, CONSTANCE M., University of Toronto.

GreEEY, ELizABeTH L., Research Assistant, University of Toronto.

Hin1t, Epear S., Research Assistant, Washington University.

Hosson, LAwreNceE B., Graduate Assistant in Zoology, University of Cincinnati.
Kaytor, Cornetius T., Fellow in Biology, Princeton University.

LEDERMAN, Epwarp, Assistant, University of Cincinnati.

Levin, Louts, Laboratory Assistant, DePauw University.

Marmer, Dina, Assistant, Columbia University.

Mast, ErtsaBetH T., Research Assistant in Psychology, Johns Hopkins Univer-
sity.

Mitts, KATHARINE O., Technician and Assistant, University of Missouri.
Monkg, J. Victor, Teaching Fellow, University of Tennessee, Medical School.
Mooreg, Joun A., Assistant in Zodlogy, Columbia University.

MoragueEz, V., Barcelona Medical School, Spain.

Morris, Marton C., Washington University.

NESTLER, HERBERT A., Reader, Brooklyn College.

26 MARINE BIOLOGICAL LABORATORY

Nicott, PAut A., Washington University.

O’Brien, HELEN, University of Pennsylvania.

PAPPENHEIMER, JoHN R., Research Assistant, Harvard University.

REEDER, ELIzABETH M., Instructor in Zoology, University of Missouri.

Ricca, Renato A., Research Assistant, University of Pennsylvania.

Rosertson, Kay, Research Fellow, University of Toronto.

Rogertson, Lota E., Research Assistant, New York University.

Rostnson, R. A., University of Pennsylvania.

Ross, Eart T., Milford, Iowa.

Rustin, Sotomon H., Teaching Fellow, New York University College of Medicine.

SALK, Jonas, Fellow in Chemistry, New York University.

SuHaw, Istpor, Research Assistant, Long Island University.

SmitH, Jay A., Assistant in Department of Zodlogy, DePauw University.

STANBURY, JOHN, Student, Harvard Medical School.

STEIMAN, S. E., Boston University.

Taytor, JoHN F., Johns Hopkins Medical School.

TuHompson, JAMes U., Weaver Fellow in Anatomy, University of Maryland,
Medical School.

TuHorNTON, C. S., Assistant, Princeton University.

WEISBERG, Harry F., Fellow in Vertebrate Zodlogy, College of the City of New
York.

WIGHTMAN, JoHN C., Assistant, Brown University.

Young, S. B., Technician, Rockefeller Institute for Medical Research.

Students

BOTANY

Asuton, MirtAm R., Instructor, University of British Columbia.

Gites, Grorce H., High School Teacher, Wilsonville, Nebraska.

PatMeEr, CHArRLEs M., Graduate Student, Indiana University.

Poort, Marcery, Student, Radcliffe College.

ScHEER, Beatrice A., Assistant in Botany, Connecticut College.

SHURTLEFF, RosAMOND L., Student, Wheaton College.

Tuuritow, Martua, Goucher College.

TROMBETTA, VivIAN V., Assistant in Botany, Barnard College, Columbia Univer-
sity.

VELASQUEZ, GreGoRIO T., Graduate Student, University of Michigan.

WILLIAMS, JEAN L., Botany Assistant, Wellesley College.

EMBRYOLOGY

Aves, Hartow W., Assistant, Department of Zodlogy, University of Illinois.
ArtHur, JAMES B. McK., Jr., Student, Amherst College.

Bauer, DoNnALD, Student, Dartmouth College.

BiocH, JANET, Student, Sarah Lawrence College.

Brooks, JEANNE R., Oberlin College.

CLARK, BEATRICE, Wellesley College.

Coutz, EL1zABeTH S., 377 Voss Avenue, South Orange, New Jersey.
Conant, Betsy D., Graduate Student, University of Rochester.
Curperson, Maser H., Instructor, Simmons College.

Danner, Epwin C., Assistant Instructor, University of Illinois.
Forses, THoMAS R., Fellow in Anatomy, University of Rochester.
Fow er, Warp S., Student, Swarthmore College.

FroeticH, Heten L., Science Librarian, Stephens College.

REPORT ON DHE DIRECTOR 27

FronczAk, MicHAeEt I., Department Adviser, Seton Hall College.

Gaver, H. KENNETH, Oberlin College.

Henson, Marcaret, Student, Smith College.

HuMMEL, Katuarine P., Instructor in Zoology, Mount Holyoke College.
Jounson, Davin F., Graduate Student, New York University.

Jostin, Stuart L., Wesleyan University.

KeNNeEDY, KATHLEEN M., Demonstrator, Memorial University College.
Kramer, Cuartes H., Student, Wabash College.

KriETE, BERTRAND C., DePauw University.

LipMAN, Harry J., Graduate Assistant, University of Pittsburgh.
McCarreELL, JANE D., Instructor, Vassar College.

MenpozA, GuILLERMO, Graduate Assistant, Northwestern University.
MorGAn, THEODORE J., Fellow in Biology, Washington and Jefferson College.
Pack, Viretnta L., Student, Sarah Lawrence College.

Price, Joun W., Associate Professor of Zodlogy, Ohio State University.
Reppick, Mary L., Assistant Teacher, Spelman College.

Russet, Attce M., Instructor, University of Pennsylvania.

ScHNEIDER, RutH M., Student, Skidmore College.

SmitH, Gorpon L., Undergraduate Assistant, DePauw University.
SHYKIN,; PEARL, Student, Radcliffe College.

Watzorn, Resecca S., Assistant in Zodlogy, University of Missouri.

PHYSIOLOGY

AwnverscH, Marie A., Assistant Professor of Biochemistry, Woman’s Medical
College, Philadelphia, Pennsylvania.

BALLENTINE, RosBert G., Princeton University.

Cameron, Joun A. C., Instructor in Zoology, University of Missouri.

CHAMBERS, ALFRED H., Jr., Swarthmore College.

Ducat, Louts-Paut, Instructor in Biology, University of Montreal.

GoLDWASSER, SEYMoRE, Amherst College.

GROSSMAN, JAcos, Graduate Student, Columbia University.

Lewis, Lena A., Technician, Lancaster General Hospital.

Litty, DanreL McQ., Instructor in Biology, Providence College.

MaGALHaeEs, HutpA, Graduate Assistant, Mount Holyoke College.

Macers, ExizAsetH, Assistant Professor of Physiology, Vassar College.

Marcotts, Frep, Student, University of Pittsburgh.

McDonatp, Marcaret R., Graduate Student, Rutgers University.

Mitts, KaTrHarine O., Technician and Assistant, University of Missouri.

MorAQUEZ, VICENS, Facultad de Medicina, Barcelona, Spain.

RatTnorF, Oscar D., Student, College of Physicians and Surgeons.

REED, Emerson A., Graduate Student and Teaching Assistant, University of Cali-
fornia.

RicHARDSON, EsTELLE D., Graduate Student, New York University.

RosENFIELD, RicHArD E., Student, University of Pittsburgh.

SmitH, JupirH D., Laboratory Assistant in Physiology, Wellesley College.

SmiTH, PAut E., Graduate Assistant, University of Rochester.

Woop, ALBERTA Brown, 75 East 55th Street, New York City, New York.

PROTOZOOLOGY

BERENBERG, Naomi R., Student, New Jersey College for Women.
BocsKry, STEPHEN C., Associate Professor of Biology, University of Notre Dame.
BrusH, RutH M., Hunter College.

28 MARINE BIOLOGICAL LABORATORY

Busu, AELEtA N., 723 Williams Street, Atlanta, Georgia.

Crarr, C. Lioyp, 5 Van Beal Road, Randolph, Massachusetts.
CUNNINGHAM, KATHERINE, American University.

Dewey, VirGINIA C., Technician, Harvard Medical School.

FERENBACH, Cart, Student, Princeton University.

GREENFIELD, SYDNEY S., Student, Brooklyn College.

GrossMAN, CrEcELIA M., Teacher of Biology, Abraham Lincoln High School.
Hupson, Grace P., Hunter College.

Lee, GeorcE O., Columbia University.

Lewis, WitmA M., Student Assistant, State Teachers College, Montclair.
Mectitscu, Paut A., Student, University of Illinois.

Mercatr, Isaac S. H., Assistant in Zoology, Columbia University.
MitTcHELL, Atison M., Student, New Jersey College for Women.

Mock, SarauH H., Graduate Student, Missouri University.

INVERTEBRATE ZOOLOGY

ALLEN, THomAS H., University of Iowa.

Bascock, Rutu H., Teacher, Caldwell High School, New Jersey.

Baver, JoAN E., Student, Montclair State Teachers College.

BrsHop, Davin W., Instructor, University of Pennsylvania.

Bonnet, Davin D., Harvard University.

BoweEN, WILLIAM J., Johns Hopkins University.

BurLincton, Mary, McGill University.

Busu, Arteta N., 723 Williams Street, Atlanta, Georgia.

Carson, Hampton L., Jr., University of Pennsylvania.

Cassipy, Morton H., Instructor in Biology, Hyde Park High School.

Caytor, Ricwarp L., Associate Professor of Biology, Delta State Teachers Col-
lege.

CorELAND, Donatp E., Graduate Assistant, Amherst College.

CreGAN, SISTER Mary Bertua, Professor of Biology, St. Xavier College.

CROASDALE, HANNAH T., Research Assistant, Dartmouth College.

Dawson, RALPH W., Assistant Professor, University of Minnesota.

DoyLt, WINFIELD G., Oberlin College.

Fasen, ANN REED, Goucher College.

FaARRADAY, CLAYTON L., Jr., Swarthmore College.

GRANGER, BARBARA S., Research Assistant and Graduate Student, Mount Holyoke
College.

GRAVE, CASWELL, II, Assistant, Washington University.

GropsTEIN, CLIFForD, College of the City of New York.

Harris, W. AtrreD, DePauw University.

Hit, Davin L., Graduate Student, State University of Iowa.

Hocan, Sister STELLA Marta, Professor of Biology, St. Xavier College.

Hoyt, J. SourHGcATE Y., Washington and Lee University.

HuntIncton, MaArcaret O., Swarthmore College.

JouNnson, REUBEN B., Jr., Connecticut State College.

KIMBALL, RicHArpD F., Student, Johns Hopkins University.

Kwortn, Sipyy C., Head of Science Department, Gulf Park College.

Koster, Rupotr, Graduate Student, Harvard University.

Lewis, WitMA M., Student Assistant, New Jersey State Teachers College.

Morcan, GWENDOLYNN W., Sarah Lawrence College.

Mover, Exizasetu K., Graduate Assistant, Mount Holyoke College.

Norris, CHARLES H., Hamilton College.

Potts, Hucu E., New York University.

Ray, Davin T., Teacher, Johnson C. Smith University.

REPORT OF THE DIRECTOR

Reep, Mary V., Instructor in Science and Mathematics, The Knox School.

Roxy, JoHN B., Jr., Graduate Student, Wesleyan University.
Sarin, Leon, Student, Colby College.

SasLow, Georce, Assistant Professor of Biology, New York University.
Seaton, Mary J., Pennsylvania College for Women.

SENSENIG, WAYNE, JR., Student, Haverford College.

SHELTON, MEREDITH, Sarah Lawrence College.

Spratt, Nerson T., Jr., Instructor in Biology, Emory University.
STEVENSON, JAMES H., Assistant in Zoology, Oberlin College.
Stokes, MirtaM, Graduate Assistant, Mount Holyoke College.
Stump, ALEXANDER B., Student, University of Virginia.

SwiFT, KATHARINE W., Student, Smith College.

TwicHELL, ALLEN R., Student, Wabash College.

WATERMAN, Tarzot H., Harvard University.

WEIERBACH, Lity A., University of Pennsylvania.

WEINBERG, STANLEY L., Graduaté Student, Columbia University.
Weir, ELLEN H., Student, Wilson College.

WHEELER, NorMAN C., Assistant in Physiology, Purdue University.
Woop, EvizABetH C., Montclair State Teachers College.

J LNB EAR, VE TOR SA iN DAN Cres

ENVESDIGATORS—NOtally Gi. dusayaas eos cece sera hanes 314 319 323
ad epee etnterres seve Sci cae eae lar eater ecaraiaee Dane ae 212 210 222 208
(hac eralrastinietiomiae mete eat vie eee oc os ce eines 6 73 66 49
NESE ALCHBENSSIStANItG, rca ss eisinie ele cose ote wm wletiiy eyes ate BOR AS Tn 52
S THWID BINT SMO tal lorena Med caea tate cadet ena eciauh sean aa lease, 132 118 131
LE SONG EA 8 oe HE NEN: HOO: BeBe ASO ao Pm agl eP 55) 54.54
ERROLOZOOL O Payee d uatey neyo Re Re eeIn uek He Se tid ne eas Ga
Embryology .......... iS So oh ee ae Ee LOR Oe IR 29 28 30
HTT Ol OSaye Be atss eae Eee 5 RIS eee ae ems oA en a 18 19 23
ESO Uelinny pemteenere tec Ee ys Vag ON sac ray sean at eo tee a No 14 G6 is
MRO ATAM ACIDE NANAINIGE:, jc Shcchicclelac nero a arse Shee ce Nee ee 446 437 454 445
Less Persons registered as both students and investi-
EE INOFAS G HIG Behar 8 Aan ae CEA Pe eee UN RE ALG Pra Cra Nay We 5
432 425 439 429
INSTITUTIONS REPRESENTED—Total ................... 141 120 131
By aMlnviestigatOns wie eek rene tag aso a cea eee 94 92 98
yan CU Gl Smite uel ys terete eet an pre ene He RE pera oe 76 58 75
ScHOOLS AND ACADEMIES REPRESENTED
By aelinnviesti@avOnsin os ccart as Maen cee ness Sure oases ole a - 1 1
BARS LIGL ETS raatpertacnctic Oe Meets Aeiedicc (hee cara em guanes 1 2 5
ForEIGN INSTITUTIONS REPRESENTED
aya linestietOr Samet ls sc) Siacicens tds secrete ee cee oi ender onets 8 5 4

BYyAIS EUGENES meer caer va neta a ened Neu, Wan ee alate ue. erty 1 - 1

30 MARINE BIOLOGICAL LABORATORY

4. SUBSCRIBING AND COOPERATING INSTITUTIONS
IN 1936

American University

Amherst College

Atlanta University

Bowdoin College

Bryn Mawr College

College of Charleston

Columbia University

Cornell University

Cornell University Medical College

DePauw University

Duke University

General Education Board

Goucher College

Hamilton College

Harvard University

Harvard University Medical School

Hunter College

Industrial & Engineering Chemistry,
of the American Chemical Soci-
ety

Iowa State College

Johns Hopkins University

Johnson Foundation

Eli Lilly & Co.

Long Island University

Memorial Hospital, New York City

Morehouse College

Mount Holyoke College

New York State Department of
Health

New York University

New York University
School

Northwestern University

Oberlin College

Pennsylvania College for Women

Princeton University

Purdue University

Radcliffe College

Medical

Rensselaer Polytechnic Institute
Rockefeller Foundation
Rockefeller Institute for
Research
St. Xavier College
Seton Hall College
Smith College
Swarthmore College
Syracuse University
Temple University
Tufts College
Union College
Jniversity of Chicago
Jniversity of Cincinnati

Medical

niversity of Iowa

Jniversity of Kansas

University of Maryland Medical
School

niversity of Minnesota

niversity of Missouri

niversity of Pennsylvania

niversity of Pennsylvania Medical
School

University of Pittsburgh

University of Rochester

University of Virginia

University of Wisconsin

Vanderbilt University Medical

School

Vassar College

Wabash College

Wellesley College

Wesleyan University

Wheaton College

Wistar Institute of Anatomy and

Biology
Yale University

U
U
University of Illinois
U
U

U
U
U
U

5. EVENING LECTURES, 1936

Friday, June 26

DR Ae CPREDEIELD*:odeek kes cles

Friday, July 3
Dr. H. K. HartLine

eee er eee eee

“The Ecological Significance of the

Circulation of the Gulf of Maine.”

“ Electrical Studies of Visual Mech-

anisms.”

REPORT OF THE DIRECTOR 31

Friday, July 10

ID yes \WN ge (Ge, 24 VG De nie a ea a “Recent Studies in Mass Physiology.”
Friday, July 17
ID IRS (Goa is La BA 252 “ Neurohumors as the Means of Ani-

mal Color Changes.”
Friday, July 24

ree Oey WINE INN mies cetiese Cha bs es “Problems of the Eel Grass Situa-
tion.”
Friday, July 31
DRE SVEN ELORSPADIUS) ss eaanin a. “Researches on Determination in the
Early Development of the Sea-
urchin.”

Friday, August 7
Dre jp Ko W. PerGuson.:....... Newer Views of CO, Transport
and Their Significance to Other
Physiological Processes.”
Friday, August 14
ID Res SOM UINICARD! |. 2c /2als ants “Life Cycles of Digenetic Trema-
todes.”
Wednesday, August 19
WD A/a bee RUNGE nts se ees rete wanes sas “Submicroscopical Structure of Liv-
ing Organs (Muscle, etc.) Re-
vealed by X-Rays.”
Friday, August 21

iinet cy NOOUINIG) weg cineca Ae etre thay vs “Giant Nerve Fibres in the Squid.”
Monday, August 24
IDR RS NVETISSENBERG ecu es- oes “The Lymphocystic Disease of Fishes

and Its Significance for Intracellu-
lar Parasitism: a Contribution to
the Knowledge of the Virus Dis-
eases.”

Thursday, September 3 (Under the joint auspices of the Genetics Society of
America and the Marine Biological Laboratory)

Drie ie MDORZEVANSIKY) @ wt .)2. a « “Genetic Nature of Species Differ-
ences.”
DRE IE EE RO DIGH Rs artis Se sha ess “Some Types of Waltzing and Epi-

lepsy in Mice of the Genus Pero-
myscus.” (Motion Picture.)
Friday, September 4
Dene OEVAIN MIlipORTE Giles wus e ic a3 « “ Distribution of Marine. Animals in
Relation to Their Environment.”

CA SHORD ERS SCIENTIFIC: PAPERS, 1936

Tuesday, June 30
De Niemi DA, Me BROOKS. ......... “The Effect of Methylene Blue on
the Spectrophotometric Picture of
Hemoglobin, CO-Hemoglobin and
CN-Hemoglobin.”
Mi EAR, )squEPNTAND . 2%. 426.0 ‘““The Phosphatase Content of the De-
veloping Chick Embryo.”

32 MARINE BIOLOGICAL LABORATORY

Dr. KENNETH C. FISHER AND
Dr. LAURENCE IRVING ...........

Dr: Erie G.ByAdeiee ieee nen etatene se

Tuesday, July 7
IDR, Avis) IML, ILGGAS Gocetoboese

Dre) JOSE Ws INONIDEZ a eyyleae se crn ins

DraiG ADD BROSSER je. 0 ose -
DR Re Ve (GERARDS sereiias seiine 6

Tuesday, July 14
DRE AA IKGIPARPIARD fey. ciolsieuispcrn sie

Dr. H. Burr STEINBACH .........

Mr. DAantiEL MaAziIA AND
IWR A HAN VIE CLARKS or. 5). cures ccs

Dr. Kurt G. STERN AND
Dr. DELAFIELD DUBOIS ..........

Tuesday, July 21
Ding Wi, WW, IBALL) GGG e656 con oboe

Dr. T. H. BISSONNETTE ..........

Dr. Roperts RuGH ............-.

Tuesday, July 28
Dr. GERTRUDE EVANS ..........0-

Drs (Gy IR RUNING ag aa Ee eee

DRE ee GUNDRID pect yes ose

“The Description of an Oxidative
Process Maintaining the Fre-
quency of the Heart Beat.”

“ Oxidation-reduction Potentials and
Potentiometric Determination of
Ascorbic Acid.”

“Nerve Cells Without Central Proc-
esses in the Fourth Spinal Gan-
glion of the Frog.”

“Receptor Areas in the Venae Cavae
and the Pulmonary Veins and
Their Relation to Bainbridge’s Re-
exces

“Extinction of Reflex Responses in
the Rat.”

“Factors Influencing the Electrical
Activity of the Brain.”

“The Permeability of the Erythro-
cytes of the Ground-hog.”

“ Effect of Salts on the Injury Poten-
tials of Frog’s Muscle.”

“ Free Calcium in the Action of Stim-
ulating Agents on Elodea Cells.”

“A Photoelectric Method for Record-
ing Fast Chemical Reactions: Ap-
plication to the Study of Catalyst-
Substrate Compounds.”

“Observations on Lens Regeneration
in Amblystoma.”

“Fertile Eggs from Pheasants in Jan-
uary by Night-lighting.”

“A Quantitative Analysis of the An-
terior Pituitary-Ovulation Relation
in the Frog.”

“The Relation between Vitamins and
the Growth and Survival of Gold-
fishes in Homotypically Condi-
tioned Water.”

“A Possible Endocrine Role of the
Eosinophil Leucocytes in the Fe-
male Rat.”

“An Interpretation of the Secondary
Lymphoid Nodules in the Albino
Rat.”

RELORM Of Tih DIRECTOR

Dr. LAURENCE [RVING ...........
Tuesday, August 4
Dr. ETHEL BROWNE HARVEY

i Y

CC ee ee

Tuesday, August 11
Dr. Laura J. NAHM

Dr. E. ALFRED WoLF AND
Miss Grace RIETHMILLER

eee we ee

Dr. ALEXANDER SANDOW

eee ee wee

Dr. Frank H. J. Ficce

Dr. ROBERT CHAMBERS ..........
Tuesday, August 18

Dr. H. P. Smite anp

Dr. E. D. WARNER

eee ee ee oe ee we
eee ee ee we ww

eee ee ee ee ee ee ee

Dr. ALEXANDER HOLLAENDER .....
Tuesday, August 25
Dr. E. ELEANOR CAROTHERS

eee eee

Dr.
Mr
Mr

KatsuMA Dan,
. T. YANAGITA AND
. M. SuGiyaMa

eee ee ee ee ee we

Dr.

eoe ee es

“ Physiological Adjustments to Div-
ing in the Beaver.”

‘“ Development of Arbacia Eggs with-
out Nuclei: Parthenogenetic Mero-
gony.”

“Cortical Changes in Arbacia Eggs
During Fertilization—A Moving
Picture.”

“Temperature Effects on Mitotic
Changes in Arbacia Eggs.”

“Sea-urchin Larve with Cytoplasm
of One Species and Nucleus of
Another.”

“A Study of the Cells of the Adrenal
Gland of the Ewe during Estrus
and Pregnancy.”

“Studies in Calcification: III. The
Shell of the Hen’s Egg.”

“ Diffraction Patterns of Striated
Muscle and Sarcomere Behavior
during Contraction.”

“The Effect of Some Oxidation-re-
duction Indicator Dyes (Phenol
Indophenol) on the Eyes and Pig-
mentation of Normal and Hypo-
physectomized Amphibians.”

“The Elimination of Neutral Red by
the Kidney Tubules.”

“ Quantitative Studies on Blood Clot-
ting.”

“The Basis of the Principle of the
Master Reaction in Biology.”

“ Efficiency of Photosynthesis in Pur-
ple Bacteria.”

“Some Effects of Ultraviolet Radia-
tion on Bacteria.”

“Cellular Behavior in Abnormal
Growths Produced by Irradiation
of Grasshopper Embryos.”

“The Behavior of the Cell Surface
During Cleavage.”

.““ Modified Sexual Photoperiodicity in

Ferrets, Raccoons and Quail.”

Dr. Eart A. MARTIN

eee ew ee ee eee

Mr. G. P. WELLS

eee ee ee ee we ee eo

MARINE BIOLOGICAL LABORATORY

“ Asexual Reproduction in Dodecace-
ria fimbriatus.”

“The Physiology of the Stomatogas-
tric System in Arenicola marina.”

7. GENERAL SCIENTIFIC MEETING, 1936

Thursday, August 27
Mr. F. R. HUNTER AND
Dr. E. N. Harvey

Ce re re TY

Dr. B. Lucxé,
Mr. R. Ricca AND
Dr. H. K. HartLine

Mr.

Dr.

. R. CHAMBERS

Dr. M. J. Kopac

Cr )

IDR, IP, S, IBleISEAMY Sal eeseesnoss
Miss ANNA K. KELtTcH,

Dr. G. H. A. CLowEs AND

Dr. M. E. Kran

eee ee ee ee we oe

Dr. M. E. Krad,
Dr. G. H. A. CLowEs AND
Seats WE

Mr TAYLOR

Dr.
Dr.

W. C. ALLEE AND
GERTRUDE EVANS

eee ee ee ee eo

Dr.
Dr.

PARPART AND

ih UR
M. H. Jacogs

eos ee ee ee ee oe ew

“The Effect of Lack of Oxygen on
the Permeability of the Egg of
Arbacia punctulata.”

‘Comparative Permeability to Water
and Certain Solutes of the Egg
Cells of Three Marine Inverte-
brates (Arbacia, Cumingia and
Chaetopterus ).”

‘“Permeability of Ameba proteus to
Tons.”

“A Kinetic Method of Studying Sur-
face Forces in the Egg of Ar-
bacia.”

“Experimental Studies on the Oil-
wetting Property of the Plasma
Membrane.”

“Tnterfacial Films between Oil and
Cytoplasm.”

“The Question of Recovery from
X-ray Effects in Arbacia Sperm.”

“The Respiratory Effects Exerted by
Certain Organic Compounds in
Relation to Their Molecular Struc-
tunes?

“ Action of Metabolic Stimulants and
Depressants on Cell Division at
Varying Carbon Dioxide Ten-
sions.”

“Further Studies on the Effect of
Numbers Present on the Rate of
Cleavage in Arbacia.”

“ Paradoxical Osmotic Volume
Changes in Erythrocytes.”

REPORT OF THE DIRECTOR

Dr. M. H. JAcoss,
Mr. H. N. GLassMAN AND
Dr. A. K. PARPART

Friday, August 28
DraG1C SPreDpEer

Miss Exsa M. KEIL AND .
Drei: JM. SICHEL

eee ee ee ee eo ew

«A Be IBOVAC SOU el ee let a rn ere
Dr. F. O. Scumitt,
. R. S. BEaR AND
Mr. J. Z. Younec

Mr. G. SCHOEPFLE AND
Miter Acs WOUNG Hes cha teed Geter
IDR, JEL, ING IBDN Bs asec u ool

Dr. K. C. FISHER AND
Dr. J. A. CAMERON

Ce eC er ey

Dr. G. SasLow

Dr. J. A. CAMERON AND
Miss K. O. Mitts

eee ee ee ew ew ew ow

Dr. E. R. CLARK AND
Mrs. ELEANoR L. CLARK

eee ee ee oe

Miss Laura N. HunTER

eee ee oe ew

Dr. S. Horstapius

eee ee oe ee ee eo

“Further Studies on Specific Physio-
logical Properties of Erythro-
cytes.”

“ Experiments on the Contractile Sub-
stance of Muscle Fibers.” (With
Motion Pictures.)

“ The Injection of Aqueous Solutions,
Including Acetylcholine, into the
Isolated Muscle Fiber.”

“The Double Refraction of Smooth
Muscle.”

“Some Physical and Chemical Prop-
erties of the Axis Cylinder of the
Giant Axons of the Squid, Loligo
pealii.”

“The Structure of the Eye of Pecten.”

“The Discharge of Impulses in the
Optic Nerve Fibers of the Eye of
Pecten irradians.”

“The Effect of Light on the CO-
poisoned Embryonic Fundulus
Heart

“Prevention of Edema in Frog Per-
fusions in the Absence of Serum
Proteins.”

“Behavior of Frog Tadpole Epi-
dermal Cells during Seven Suc-
cessive 24-Hour Regeneration Pe-
riods.”

“ Observations on Conditions Affect-
ing Growth of Cells and Tissues,
from Microscopic Studies on the
Living Animal.”

“Some Nuclear Phenomena in the
Trichodina (Protozoa, Ciliata,
Peritrichida) from Thyone bria-
reus (Holothuroidea).”

“Tnvestigations on Determination in
the Early Development of Cere-
bratulus.”

36 MARINE BIOLOGICAL LABORATORY

DReORSGRUGH 9 Sarin ate see ere “ Preliminary Evidence as to a Source
of the Growth and the Sex-stimu-
lating Hormones in the Bullfrog.”

Dr: Ps BeyARMSTRONG {yee aero: “Mechanism of Hatching in Fundu-
lus heteroclitus.”

Dr. B. H. GRAVE AND

NER: ) dak SIMI S t5. ore oem “ Hermaphroditism and Sexual In-

version in Mollusca.”

PAPER Sew AUD Yi an Wes:

Dre SG Muar. erento a “Separation of the Conducting and
Contractile Elements in the Re-
tractor Muscle of Thyone bria-
reus.”

Dr. A. V. HUNNINEN AND

DRE RY WTCHTERMANG |. 6 2 52 oe ee “ Hyperparasitism: A Species of
Hexamita (Protozoa, Mastigo-
phora) Found in the Reproductive
Systems of Deropristis inflata
(Trematoda) of Marine Eels.”

Dre VARS) VEACDOUGALT. 2.4... “Cytological Studies of the Genus
Chilodonella. JII. The Conjuga-
tion of Chilodonella labiata, varia-
toms

DR VACTORESCHEGHTER? =... 62: “Comparative Hypotonic Cytolysis of
Several Types of Invertebrate
Egg Cells and the Influence of

Age.”

DR Eee SMUNKARD | 4-46. 22..+ “Notes on Life Cycles of Digenetic
Trematodes.”

De AVR ESRER MOAN ope os cals ale 2 < “Tnhibition of Gastrulation in Arba-
cia with NiCl,.”

IDs PROOWVITCHUEER MAN) (coc. cee c ess “ Division and Conjugation in Nycto-

therus cordiformis (Ehr.) Stein
(Protozoa, Ciliata) with Special
Reference to the Nuclear Phe-
nomena.”

DEMONSTRATIONS

Thursday, August 27

DRE wom) PeLOIGGE | 2). ocr. sees ale “The Effect of Some Indophenol
Dyes on the Eyes and Pigmenta-
tion of Various Amphibian Lar-
Vices

DR RWRISSENBERG 202. G-05 6. os “Tntracellular Parasitism of Micro-
sporidia and of Lymphocystic Dis-
ease

Dr.

Dr. EtHeL B. Harvey

Dr. E. R. CLARK AND
Mrs. ELEANOR L. CLARK

Miss GERTRUDE GOTTSCHALL

Dr.

Mr.

Dr.

REPORT OF THE DIRECTOR 37

A. HOLLAENDER

ID), IMUNRSIC/NID) Ge oe oneeeoouens

. H. K. Hartrine

. M. N. KNIsELY

ec ee ee ee ee eee

ee ee

CC ee

ese ee ee we

eee ee ee ee oe

cee ee ee ee

eee ee

“ A Semi-portable Cold Room Provid-
ing Daylight and Automatic Ad-
justment of Temperature from
—10° C. to Room Temperature.”

“Some Nuclear Phenomena in the
Trichodina from Thyone briareus
(Vegetative Stages, Binary Fis-
sion, Conjugation (?)).”

“A Simple Intense Mercury Vapor
Lamp.”

“Cleavage of Non-nucleate Arbacia
Eggs.”

“Observations on Conditions A ffect-
ing Growth of Cells and Tissues,
from Microscopic Studies on the
Living Animal.”

“The Determination of Glutathione
in Animal Tissues.”

“A Bomb Permitting the Microscopic
Observation of Cells and Tissues
during Hydrostatic Compression.
The Effects of Compression on the
Cleavage of Arbacia Eggs.”

“ Action Currents in Nitella.”

“ Apparatus for Determining Volume
Changes of Cells under Anzrobic
Conditions.”

“Life Cycles of Digenetic Trema-
todes.”

“Anatomy and Physiology of Giant
Nerve Fibers in the Squid.”

“Method Recording Changes of Dou-
ble Refraction during Muscular
Contraction.”

“Sensitive Method of Recording Re-
sponses of Blood Vessels.”

“The Discharge of Impulses in the
Optic Nerve Fibers of the Eye of
Pecten irradians.”

“Microscopic Demonstration of Sev-

eral Living Internal Organs of
Frogs Illuminated with Fused
Quartz Rods.”

38 MARINE BIOLOGICAL LABORATORY

8. MEMBERS OF THE CORPORATION

1. Lire Memeers

Aus, Mr. E. P., Jr., Palais Carnoles, Menton, France.

ANpREWwS, Mrs. GwWENDOLEN FOULKE, Baltimore, Maryland.

Bituincs, Mr. R. C., 66 Franklin St., Boston, Massachusetts.

Conxtin, Pror. Epwin G., Princeton University, Princeton, New
Jersey.

Crane, Mr. C. R., New York City.

Evans, Mrs. GLENDOWER, 12 Otis Place, Boston, Massachusetts.

- Foot, Miss KarHerine, Care of Morgan Harjes Cie, Paris, France.

GarpIner, Mrs. E. G., Woods Hole, Massachusetts.

Jackson, Miss M. C., 88 Marlboro St., Boston, Massachusetts.

Jackson, Mr. Cuas. C., 24 Congress St., Boston, Massachusetts.

Kipper, Mr. NatHaniev T., Milton, Massachusetts.

Kine, Mr. Cuas. A.

Ler, Mrs. Freperic S., 279 Madison Ave., New York City.

Lowett, Mr. A. Lawrence, 17 Quincy St., Cambridge, Massachusetts.

McMuratcu, Pror. J. P., University of Toronto, Toronto, Canada.

Means, Dr. James Howarp, 15 Chestnut St., Boston, Massachusetts.

Merriman, Mrs. Daniet, 73 Bay State Road, Boston, Massachusetts.

Minns, Miss Susan, 14 Louisburg Square, Boston, Massachusetts.

Morecan, Mr. J. Prerpont, Jr., Wall and Broad Sts., New York City.

Morean, Pror. T. H., Director of Biological Laboratory, California
Institute of Technology, Pasadena, California.

Morcan, Mrs. T. H., Pasadena, California.

Morritu, Dr. A. D., Hamilton College, Clinton, N. Y.

Noyes, Miss Eva J.

Porter, Dr. H. C., University of Pennsylvania, Philadelphia, Pennsyl-
vania.

Sears, Dr. Henry F., 86 Beacon St., Boston, Massachusetts.

SHepp, Mr. E. A.

THORNDIKE, Dr. Epwarp L., Teachers College, Columbia University,
New York City.

TRELEASE, Pror. WILLIAM, University of Illinois, Urbana, Illinois.

Ware, Miss Mary L., 41 Brimmer St., Boston, Massachusetts.

Witson, Dr. E. B., Columbia University, New York City.

2. RecuLAR Memsers, 1936
Apvams, Dr. A. ExizasetH, Mount Holyoke College, South Hadley,
Massachusetts.
Appison, Dr. W. H. F., University of Pennsylvania Medical School,

Philadelphia, Pennsylvania. ‘

REPORT OF THE DIRECTOR 39

ApoteH, Dr. Epwarp F., University of Rochester Medical School,
Rochester, New York.

ALLEE, Dr. W. C., The University of Chicago, Citas Illinois.

Attyn, Dr. Harriet M., Mount Holyoke College, South Hadley, Mas-

sachusetts.

AmpBerson, Dr. WILLIAM R., University of Tennessee, Memphis, Ten-
nessee.

ANDERSON, Dr. E. G., California Institute of Technology, Pasadena,
California.

ARMSTRONG, Dr. Puitip B., Cornell University Medical College, 1300
York Avenue, New York City.

AustTINn, Dr. Mary L., Wellesley College, Wellesley, Massachusetts.

BAITSELL, Dr. GEorcGE A., Yale University, New Haven, Connecticut.

BaLpwin, Dr. F. M., University of Southern California, Los Angeles,
California.

BALLARD, Dr. WILLIAM W., Dartmouth College, Hanover, New Hamp-
shire.

Batt, Dr. Ertc G., Johns Hopkins Medical School, Baltimore, Mary-
land.

Barb, Pror. Puriip, Johns Hopkins Medical School, Baltimore, Mary-
land.

Barron, Dr. E. S. Guzman, Department of Medicine, The University
of Chicago, Chicago, Illinois.

Bartu, Dr. L. G., Department of Zodlogy, Columbia University, New
NWork City.

BEecKwitTH, Dr. Cora J., Vassar College, Poughkeepsie, New York.

Beure, Dr. ELinor H., Louisiana State University, Baton Rouge, Lou-
isiana.

Bennitt, Dr. Ruporr, University of Missouri, Columbia, Missouri.

BiceLow, Dr. H. B., Museum of Comparative Zoology, Cambridge,
Massachusetts.

BicELow, Pror. R. P., Massachusetts Institute of Technology, Cam-
bridge, Massachusetts.

BinForp, Pror. Raymonp, Guilford College, Guilford College, North
Carolina.

BIssONNETTE, Dr. T. Hume, Trinity College, Hartford, Connecticut.

BLANCHARD, Pror. KENNETH C., Washington Square College, New
York University, New York City.

Bovine, Dr. J. H., University of Iowa, Iowa City, Iowa.

Borrnc, Dr. Avice M., Yenching University, Peking, China.

Bozier, Dr. Emit, Ohio State University, Columbus, Ohio.

40 MARINE BIOLOGICAL LABORATORY

BrabLey, Pror. Harotp C., University of Wisconsin, Madison, Wis-
consin.

Bripces, Dr. CAtvin B., California Institute of Technology, Pasadena,
California.

BRONFENBRENNER, Dr. JACQUES J., Department of Bacteriology, Wash-
ington University Medical School, St. Louis, Missouri.

Bronk, Dr. D. W., University of Pennsylvania, Philadelphia, Pennsyl-
vania.

Brooks, Dr. S. C., University of California, Berkeley, California.

Brown, Dr. Ducatp E. S., New York University, College of Medicine,
New York City.

BuckINGHAM, Miss Epitu N., Sudbury, Massachusetts.

BupincTon, Pror. R. A., Oberlin College, Oberlin, Ohio.

BuLuincTon, Dr. W. E., Randolph-Macon College, Ashland, Virginia.

Bumpus, Pror. H. C., Duxbury, Massachusetts.

Byrnes, Dr. EstHer E., 1803 North Camac Street, Philadelphia, Penn-
sylvania.

CaLKINS, Pror. Gary N., Columbia University, New York City.

CALVERT, Pror. Puitip P., University of Pennsylvania, Philadelphia,
Pennsylvania.

CANNAN, Pror. R. K., University and Bellevue Hospital Medical Col-
lege, New York City.

Carson, Pror. A. J., The University of Chicago, Chicago, Illinois.

CaroTHers, Dr. E. ELEawnor, University of lowa, lowa City, Iowa.

CARPENTER, Dr. Russety L., College of Physicians and Surgeons, Co-
lumbia University, 630 W. 168th Street, New York City.

CarROLL, Pror. MircHer, Franklin and Marshall College, Lancaster,
Pennsylvania.

Carver, Pror. Gatt L., Mercer University, Macon, Georgia.

CaTTELL, Dr. McKeen, Cornell University Medical College, 1300 York
Avenue, New York City.

CaTTELL, Pror. J. McKEEN, Garrison-on-Hudson, New York.

CATTELL, Mr. Ware, Garrison-on-Hudson, New York.

CHAMBERS, Dr. Ropert, Washington Square College, New York Uni-
versity, Washington Square, New York City.

CHENEY, Dr. Ratpeu H., Biology Department, Long Island University,
Brooklyn, New York.

CHIDESTER, Pror. F. E., Auburndale, Massachusetts.

Cuitp, Pror. C. M., The University of Chicago, Chicago, Illinois.

CLARK, Pror. E. R., University of Pennsylvania Medical School, Phila-
delphia, Pennsylvania.

Cuiark, Dr. LEonarD B., Union College, Schenectady, New York.

REPORT OF THE DIRECTOR 4]

CLELAND, Pror. Ratpu E., Goucher College, Baltimore, Maryland.

Crowes, Dr. G. H. A., Eli Lilly and Co., Indianapolis, Indiana.

Cor, Pror. W. R., Yale University, New Haven, Connecticut.

Coun, Dr. Epwin J., 183 Brattle Street, Cambridge, Massachusetts.

Cote, Dr. Evsert C., Department of Biology, Williams College, Wil-
liamstown, Massachusetts.

Cote, Dr. KENNETH C., College of Physicians and Surgeons, Columbia
University, 630 W. 168th Street, New York City.

Coie, Dr. Leon J., College of Agriculture, Madison, Wisconsin.

CoLteTT, Dr. Mary E., Western Reserve University, Cleveland, Ohio.

Cotton, Pror. H. S., Box 127, Flagstaff, Arizona.

CooNnFIELD, Dr. B. R., Brooklyn College, 80 Willoughby Street, Brook-
lyn, New York.

CoPpELAND, Pror. Manton, Bowdoin College, Brunswick, Maine.

CosTELLO, Dr. Donatp P., Department of Zoology, University of North
Carolina, Chapel Hill, North Carolina.

CosTELLo, Dr. HELEN MILER, Department of Zoology, University of
North Carolina, Chapel Hill, North Carolina.

Cowonry, Dr. E. V., Washington University, St. Louis, Missouri.

CRAMPTON, Pror. H. E., Barnard College, Columbia University, New
York City.

Crane, Mrs. C. R., Woods Hole, Massachusetts.

Curtis, Dr. Mayniz R., Crocker Laboratory, Columbia University,
New York City.

Curtis, Pror. W. C., University of Missouri, Columbia, Missouri.

Dan, Dr. Katsuma, Misaki Biological Station, Misaki, Japan.

Davis, Dr. DonaLp W., College of William and Mary, Williamsburg,
Virginia.

Dawson, Dr. A. B., Harvard University, Cambridge, Massachusetts.

Dawson, Dr. J. A., The College of the City of New York, New York

City.

DEDERER, Dr. PauLine H., Connecticut College, New London, Con-
necticut. :

Driver, Dr. WittiAm F., Dartmouth College, Hanover, New Hamp-
shire. :

Dopps, Pror. G. S., Medical School, University of West Virginia, Mor-
gantown, West Virginia.

Do iey, Pror. WILLIAM L., University of Buffalo, Buffalo, New York.

Donatpson, Pror. H. H., Wistar Institute of Anatomy and Biology,
Philadelphia, Pennsylvania.

Donatpson, Dr. JoHN C., University of Pittsburgh, School of Medi-
cine, Pittsburgh, Pennsylvania.

42 MARINE BIOLOGICAL LABORATORY

DuBois, Dr. Eucene F., Cornell University Medical College, 1300
York Avenue, New York City.

Ducear, Dr. BENJAMIN M., University of Wisconsin, Madison, Wis-
consin.

Duneay, Dr. NEIL S., Carleton College, Northfield, Minnesota.

Durvegz, Dr. WiLtiAM R., 680 Madison Avenue, New York City.

Epwarps, Dr. D. J., Cornell University Medical College, 1300 York
Avenue, New York City.

Extis, Dr. F. W., Monson, Massachusetts.

Faur&-FREMIET, Pror. EMMANUEL, Collége de France, Paris, France.

Fercuson, Dr. JAMEs K. W., Department of Physiology, Ohio State
University, Columbus, Ohio.

FLEISHER, Dr. Moyer S., School of Medicine, St. Louis University,
St. Louis, Missouri.

Forses, Dr. ALEXANDER, Harvard University Medical School, Boston,
Massachusetts.

Fry, Dr. Henry J., Cornell University Medical College, 1300 York
Avenue, New York City.

’ Gace, Pror. S. H., Cornell University, Ithaca, New York.

GALTSOFF, Dr. Paut S., 420 Cumberland Avenue, Somerset, Chevy
Chase, Maryland.

GarrEy, Pror. W. E., Vanderbilt University Medical School, Nashville,
Tennessee.

GaTEs, Pror. R. Ruaces, University of London, London, England.

Geiser, Dr. S. W., Southern Methodist University, Dallas, Texas.

GERARD, Pror. R. W., The University of Chicago, Chicago, Illinois.

GuAseEr, Pror. O. C., Amherst College, Amherst, Massachusetts.

GotpForB, Pror. A. J., College of the City of New York, New York
City.

GoopricH, Pror. H. B., Wesleyan University, Middletown, Connecticut.

GrAHAM, Dr. J. Y., University of Alabama, University, Alabama.

GRAVE, Pror. B. H., DePauw University, Greencastle, Indiana.

GRAVE, Pror. CASWELL, Washington University, St. Louis, Missouri.

Gray, Pror. Irvine E., Duke University, Durham, North Carolina.

GREENMAN, Pror. M. J., Wistar Institute of Anatomy and Biology,
36th Street and Woodland Avenue, Philadelphia, Pennsylvania.

Grecory, Dr. Louise H., Barnard College, Columbia University, New
York City.

GuTuris, Dr. Mary J., University of Missouri, Columbia, Missouri.

Guyer, Pror. M. F., University of Wisconsin, Madison, Wisconsin.

Haptey, Dr. Cuartes E., Teachers College, Montclair, New Jersey.

Hacue, Dr. Firorence, Sweet Briar College, Sweet Briar, Virginia.

REPORT OF THE DIRECTOR 43

Hatt, Pror. FranxK G., Duke University, Durham, North Carolina.

Hance, Dr. Rosert T., University of Pittsburgh, Pittsburgh, Pennsyl-
vania.

Hareitt, Pror. Georce T., Duke University, Durham, North Carolina.

Harman, Dr. Mary T., Kansas State Agricultural College, Manhattan,
Kansas.

Harnty, Dr. Morris H., Washington Square College, New York Uni-
versity, New York City.

Harper, Pror. R. A., Columbia University, New York City.

Harrison, Pror. Ross G., Yale University, New Haven, Connecticut.

Hartiine, Dr. H. Kerrer, University of Pennsylvania, Philadelphia,
Pennsylvania.

Harvey, Dr. ErHet Browne, 2 College Road, Princeton, New Jersey.

Harvey, Dr. E. Newton, Guyot Hall, Princeton University, Princeton,
New Jersey.

HaypeNn, Dr. Marcaret A., Wellesley College, Wellesley, Massachu-
setts.

Hayes, Dr. FreperIcK R., Zoological Laboratory, Dalhousie Univer-
sity, Halifax, Nova Scotia.

Haywoop, Dr. Cuaritotre, Mount Holyoke College, South Hadley,
Massachusetts.

Hazen, Dr. T. E., Barnard College, Columbia University, New York
City.

Hecut, Dr. SEtic, Columbia University, New York City.

HEILBRUNN, Dr. L. V., Department of Zoology, University of Pennsy]l-
vania, Philadelphia, Pennsylvania.

Henvbex, Dr. EsrHer Crissry, Russell Sage College, Troy, New York.

HensuHaw, Dr. Paut S., Memorial Hospital, 2 West 106th Street, New
York City.

Hess, Pror. WALTER N., Hamilton College, Clinton, New York.

Hipparp, Dr. Hore, Department of Zoology, Oberlin College, Oberlin,
Ohio.

Hirt, Dr. Samuet E., The Rockefeller Institute, 66th Street and York
Avenue, New York City.

Hisaw, Dr. F. L., The Biological Laboratories, Harvard University,
Cambridge, Massachusetts.

Hoapbtey, Dr. Lercu, The Biological Laboratories, Harvard University,
Cambridge, Massachusetts.

Hoser, Dr. Rupotr, University of Pennsylvania, Philadelphia, Penn-
sylvania.

Hocue, Dr. Mary J., 503 N. High Street, West Chester, Pennsylvania.

44. MARINE BIOLOGICAL LABORATORY

HoLLAENDER, Dr. ALEXANDER, Biology Building, University of Wis-
consin, Madison, Wisconsin.

Hooker, Pror. Davenport, University of Pittsburgh, Pittsburgh,
Pennsylvania.

Hopxins, Dr. Dwicut L., Duke University, Durham, North Carolina.

Hopxins, Dr. Hoyt S., New York University, College of Dentistry,
New York City.

Howe, Dr. H. E., 2702 36th Street, N. W., Washington, D. C.

How .anb, Dr. Rutu B., Washington Square College, New York Uni-

versity, Washington Square East, New York City.

Hoyt, Dr. Witt1am D., Washington and Lee University, Lexington,
Virginia.

Hyman, Dr. Lippie H., 85 West 166th Street, New York City.

Irvinc, Pror. LAuRENCE, University of Toronto, Toronto, Ontario,
Canada.

Jackson, Pror. C. M., University of Minnesota, Minneapolis, Minne-
sota.

Jacoss, Pror. MERKEL H., School of Medicine, University of Pennsyl-
vania, Philadelphia, Pennsylvania.

JENKINS, Dr. GeorcE B., George Washington University, 1335 M
Street, N. W., Washington, D. C.

JENNINGS, Pror. H. S., Johns Hopkins University, Baltimore, Mary-
land.

Jewett, Pror. J. R., Harvard University, Cambridge, Massachusetts.

Jounin, Dr. J. M., Vanderbilt University Medical School, Nashville,
Tennessee.

Jorpan, Pror. E. O., The University of Chicago, Chicago, Illinois.

Just, Pror. E. E., Howard University, Washington, D. C.

KAUFMANN, Pror. B. P., University of Alabama, University, Alabama.

KEEFE, Rev. ANSELM M., St. Norbert College, West Depere, Wiscon-
sin.

Keit, Dr. Ersa M., Rutgers University, New Brunswick, New Jersey.

Kroper, Dr. Georce W., Department of Biology, College of the City of
New York, New York City.

Kriire, Dr. Frank R., Swarthmore College, Swarthmore, Pennsylvania.

Kinprep, Dr. J. E., University of Virginia, Charlottesville, Virginia.

Kine, Dr. Heten D., Wistar Institute of Anatomy and Biology, 36th
Street and Woodland Avenue, Philadelphia, Pennsylvania.

Kine, Dr. Rosert L., State University of Iowa, Iowa City, Iowa.

KincsBury, Pror. B. F., Cornell University, Ithaca, New York.

KwnapkeE, Rey. Bebe, St. Bernard’s College, St. Bernard, Alabama.

REPORT OF THE DIRECTOR 45

Knower, Pror. H. McE., Osborn Zoological Laboratory, Yale Univer-
sity, New Haven, Connecticut.

Knowtton, Pror. F. P., Syracuse University, Syracuse, New York.

Kriss, Dr. HERBERT, 250 Copley Road, Upper Darby, Pennsylvania.

LANCEFIELD, Dr. D. E., Columbia University, New York City.

Lance, Dr. MatuitpE M., Wheaton College, Norton, Massachusetts.

Les, Pror. F. S., College of Physicians and Surgeons, New York City.

Lewis, Pror. I. F., University of Virginia, Charlottesville, Virginia.

Lewis, Pror. W. H., Johns Hopkins University, Baltimore, Maryland.

Litiiz, Pror. FRANK R., The University of Chicago, Chicago, Illinois.

Lizuiz, Pror. RatpH S., The University of Chicago, Chicago, Illinois.

Linton, Pror. Epwin, University of Pennsylvania, Philadelphia, Penn-
sylvania. ;

Logs, Pror. Leo, Washington University Medical School, St. Louis,
Missouri.

LowTHer, Mrs. FLoRENCE DEL., Barnard College, Columbia Univer-
sity, New York City.

Lucas, Dr. ALtFrepD M., Iowa State College, Ames, Iowa.

Lucx&, Pror. Batpurn, University of Pennsylvania, Philadelphia,
Pennsylvania.

LuscomseE, Mr. W. O., Woods Hole, Massachusetts.

Lyncu, Dr. Ciara J., Rockefeller Institute, New York City.

Lyncu, Dr. RutH Stocxine, Maryland State Teachers College, Tow-
son, Maryland.

MacCarpte, Dr. Ross C., Temple University, Philadelphia, Pennsyl-

vania.

MacDoveatt, Dr. Mary S., Agnes Scott College, Decatur, Georgia.

Macxktin, Dr. CHartes C., School of Medicine, University of Western

Ontario, London, Canada.

MALone, Pror. E. F., University of Cincinnati, Cincinnati, Ohio.

MANwELL, Dr. Recrnatp D., Syracuse University, Syracuse, New
Work:

Marsvanp, Dr. Douctas A., Washington Square College, New York

University, New York Cie

Martin, Pror. E. A., Department of Biology, Brooklyn canes: 80

Willoughby Street, Brooklyn, New York.

Mast, Pror. S. O., Johns Hopkins University, Baltimore, Maryland.

MatuHeEws, Pror. A. P., University of Cincinnati, Cincinnati, Ohio.

MattTHews, Dr. Samuet A., Department of Anatomy, University of

' Pennsylvania, Philadelphia, Pennsylvania.

Mavor, Pror. JamEes W., Union College, Schenectady, New York.

46 MARINE BIOLOGICAL LABORATORY

McCune, Pror. C. E., University of Pennsylvania, Philadelphia,
Pennsylvania.

McGrecor, Dr. J. H., Columbia University, New York City.

Mepes, Dr. Grace, Lankenau Research Institute, Philadelphia, Penn-
sylvania.

Metcs, Dr. E. B., Dairy Division Experiment Station, Beltsville, Mary-
land.

Meics, Mrs. E. B., 1738 M Street, N. W., Washington, D. C.

Metrcatr, Pror. M. M., 94 Nehoiden Road, Waban, Massachusetts.

Metz, Pror. CHartes W., Johns Hopkins University, Baltimore,
Maryland.

MicHAcELtis, Dr. Leonor, Rockefeller Institute, New York City.

MiTrcHeELL, Dr. Puitip H., Brown University, Providence, Rhode
Island.

Moore, Dr. Cart R., The University of Chicago, Chicago, Illinois.

Moore, Pror. GEorGcE T., Missouri Botanical Garden, St. Louis, Mis-
souri.

Moore, Pror. J. Percy, University of Pennsylvania, Philadelphia,
Pennsylvania.

Morcutts, Dr. Sereius, University of Nebraska, Omaha, Nebraska.

MorriLi, Pror. C. V., Cornell University Medical College, 1300 York
Avenue, New York City.

NEAL, Pror. H. V., Tufts College, Tufts College, Massachusetts.

Netsen, Dr. Oxin E., Department of Zodlogy, University of Pennsyl-
vania, Philadelphia, Pennsylvania.

Newman, Pror. H. H., The University of Chicago, Chicago, Illinois.

NicuHots, Dr. M. Louisr, Rosemont, Pennsylvania.

Nose, Dr. GLAapwyn K., American Museum of Natural History, New
York City.

Nonipez, Dr. José F., Cornell University Medical College, 1300 York
Avenue, New York City.

OXKKELBERG, Dr. PETER, University of Michigan, Ann Arbor, Michigan.

Ospurn, Pror. R. C., Ohio State University, Columbus, Ohio.

OstErHOUT, Mrs. W. J. V., Rockefeller Institute, 66th Street and York
Avenue, New York City.

OstERHOUT, Pror. W. J. V., Rockefeller Institute, 66th Street and
York Avenue, New York City.

PacKArD, Dr. CHaArLes, Columbia University, Institute of Cancer Re-
search, 1145 Amsterdam Avenue, New York City.

Pace, Dr. IrvinE H., Rockefeller Institute, New York City.

PapPpENHEIMER, Dr. A. M., Columbia University, New York City.

Parker, Pror. G. H., Harvard University, Cambridge, Massachusetts.

REPORT OF THE DIRECTOR A7

PARPART, Dr. ARTHUR K., Princeton University, Princeton, New Jer-
sey.

PaTTEN, Dr. Braptey M., University of Michigan Medical School,
Ann Arbor, Michigan.

Payne, Pror. F., University of Indiana, Bloomington, Indiana.

PEARL, Pror. Raymonp, Institute for Biological Research, 1901 East
Madison Street, Baltimore, Maryland.

PEEBLES, ProF. FLORENCE, Chapman College, Los Angeles, California.

Pinney, Dr. Mary E., Milwaukee-Downer College, Milwaukee, Wis-
consin.

PioucuH, Pror. Harotp H., Amherst College, Amherst, Massachusetts.

PotiisTEr, Dr. A. W., Columbia University, New York City.

Ponp, Dr. Samuet E., Marine Biological Laboratory, Woods Hole,
Massachusetts.

Pratt, Dr. FrepErIcK H., Boston University, School of Medicine,
Boston, Massachusetts.

Prosser, Dr. C. Lapp, Clark University, Worcester, Massachusetts.

RAFFEL, Dr. DANIEL, Institute of Genetics, Academy of Sciences, Mos-
cow, U5 Sas. R.

Ranp, Dr. Hersert W., Harvard University, Cambridge, Massachu-
setts.

REDFIELD, Dr. ALFRED C., Harvard University, Cambridge, Massachu-
setts.

ReEEsE, Pror. Abert M., West Virginia University, Morgantown,
West Virginia.

DERENYI, Dr. GeorceE S., Department of Anatomy, University of
Pennsylvania, Philadelphia, Pennsylvania.

REZNIKOFF, Dr. Paut, Cornell University Medical College, 1300 York
Avenue, New York City.

Rice, Pror. Epwarp L., Ohio Wesleyan University, Delaware, Ohio.

RicHarps, Pror. A., University of Oklahoma, Norman, Oklahoma.

Ricuarps, Dr. O. W., Osborn Zodlogical Laboratory, Yale University,
New Haven, Connecticut.

Rices, LAwrason, Jr., 120 Broadway, New York City.

Rocers, Pror. CHARLES G., Oberlin College, Oberlin, Ohio.

Romer, Dr. ALFRED S., Harvard University, Cambridge, Massachusetts.
Root, Dr. W. S., University of Maryland Medical School, Baltimore,
Maryland. .
Rucu, Dr. Roserts, Department of Zodlogy, Hunter College, New

AtGule Giny,
SasLow, Dr. Georce, Department of Physiology, School of Medicine,
University of Rochester, Rochester, New York.

48 MARINE BIOLOGICAL LABORATORY

SayLes, Dr. LEonaArD P., Department of Biology, College of the City
of New York, 139th Street and Convent Avenue, New York City.

ScHECHTER, Dr. Victor, College of the City of New York, 139th Street
and Convent Avenue, New York City.

Scuorré, Dr. Oscar E., Department of Biology, Amherst College, Am-
herst, Massachusetts.

ScuHrApDER, Dr. Franz, Department of Zodlogy, Columbia University,
New York City.

ScHRADER, Dr. Satty Hucues, Department of Zodlogy, Coltimbia Uni-
versity, New York City.

ScuramM, Pror. J. R., University of Pennsylvania, Philadelphia, Penn-
sylvania.

Scott, Dr. ALLAN C., Union College, Schenectady, New York.

Scott, Dr. Ernest L., Columbia University, New York City.

Scort, Pror. G. G., College of the City of New York, 139th Street and
Convent Avenue, New York City.

Scott, Pror. Joun W., University of Wyoming, Laramie, Wyoming.

Scott, Pror. WitiiAm B., 7 Cleveland Lane, Princeton, New Jersey.

Seme.e, Mrs. R. Bowtrne, 140 Columbia Heights, Brooklyn, New
York.

SEVERINGHAUS, Dr. Aura E., Department of Anatomy, College of Phy-
sicians and Surgeons, 630 W. 168th Street, New York City.

SHapPIRO, Dr. HERBERT, Princeton University, Princeton, New Jersey.

SHULL, Pror. A. FRANKLIN, University of Michigan, Ann Arbor,
Michigan.

SHuMwAY, Dr. Watpo, University of Illinois, Urbana, Illinois.

SIcHEL, Dr. FErpINAND J. M., Department of Physiology, Howard
University, School of Medicine, Washington, D. C.

Srvickis, Dr. P. B., Pasto deze 130, Kaunas, Lithuania.

SmitH, Dr. DieTRIcH Conran, Department of Physiology, University
of Tennessee, Memphis, Tennessee.

Snow, Dr. Laetitia M., Wellesley College, Wellesley, Massachusetts.

So_ttMAN, Dr. Toratp, Western Reserve University, Cleveland, Ohio.

SONNEBORN, Dr. T. M., Johns Hopkins University, Baltimore, Mary-
land.

SPEIDEL, Dr. Cart C., University of Virginia, University, Virginia.

STABLER, Dr. Ropert M., Department of Zodlogy, University of Penn-
sylvania, Philadelphia, Pennsylvania.

Stark, Dr. Mary B., New York Homeopathic Medical College and
Flower Hospital, New York City.

STEINBACH, Dr. Henry Burr, University of Minnesota, Minneapolis,
Minnesota.

REPORT OF THE DIRECTOR F 49

STERN, Dr. Curt, Department of Zoology, University of Rochester,
Rochester, New York.

STEWART, Dr. Dorotuy R., Skidmore College, Saratoga Springs, New
York.

STocKARD, Pror. C. R., Cornell University Medical College, 1300 York
Avenue, New York City.

Stoxey, Dr. Atma G., Woman’s Christian College, Cathedral Post Of-
fice, Madras, India.

Stronc, Pror. O. S., College of Physicians and Surgeons, Columbia
University, New York City.

STUNKARD, Dr. Horace W., New York University, University Heights,
New York City.

STURTEVANT, Dr. ALFRED H., California Institute of Technology, Pasa-
dena, California.

SumMeErs, Dr. Francis Marion, Instructor in Biology, Bard College,
Annandale, New York.

Sumwatt, Dr. Marcaret, Department of Pharmacology, University of
Michigan, Ann Arbor, Michigan.

Swett, Dr. Francis H., Duke University Medical School, Durham,
North Carolina. |

Tart, Dr. CHartes H., Jr., University of Texas Medical School, Gal-
veston, Texas.

TasuHtiro, Dr. Sutro, Medical College, University of Cincinnati, Cin-
cinnati, Ohio.

Taytor, Dr. WittiAm R., University of Michigan, Ann Arbor, Michi-
gan.

TENNENT, Pror. D. H., Bryn Mawr College, Bryn Mawr, Pennsylvania.

TREADWELL, Pror. A. L., Vassar College, Poughkeepsie, New York.

Turner, Pror. C. L., Northwestern University, Evanston, Illinois.

Ty er, Dr. ALBERT, California Institute of Technology, Pasadena, Cali-
fornia.

UHLENHUTH, Dr. Epuarp, University of Maryland, School of Medi-
cine, Baltimore, Maryland.

Uncer, Dr. W. Byers, Dartmouth College, Hanover, New Hampshire.

VisscHER, Dr. J. Paut, Western Reserve University, Cleveland, Ohio.

Waite, Pror. F. C., Western Reserve University Medical School,
Cleveland, Ohio.

Wattace, Dr. Louise B., 359 Lytton Avenue, Palo Alto, California.

Warp, Pror. Henry B., Cosmos Club, 1520 H Street, Washington,
1D).

WarreEN, Dr. HERBERT S., Department of Biology, Temple University,
Philadelphia, Pennsylvania.

50 MARINE BIOLOGICAL LABORATORY

WaTERMAN, Dr. ALLyN J., Department of Biology, Williams College,
Williamstown, Massachusetts.

WenricH, Dr. D. H., University of Pennsylvania, Philadelphia, Penn-
sylvania.

Wuepon, Dr. A. D., North Dakota Agricultural College, Fargo, North
Dakota.

WHEELER, Pror. W. M., Museum of Comparative Zoology, Cambridge,
Massachusetts.

Wuerry, Dr. W. B., Cincinnati Hospital, Cincinnati, Ohio.

WuitakeEr, Dr. Douctas M., P. O. Box 2514, Stanford University,
California.

Waite, Dr. E. Grace, Wilson College, Chambersburg, Pennsylvania.

WuHitTinG, Dr. PHineas W., Zoological Laboratory, University of
Pennsylvania, Philadelphia, Pennsylvania.

Wuitnery, Dr. Davin D., University of Nebraska, Lincoln, Nebraska.

Wireman, Pror. H. L., University of Cincinnati, Cincinnati, Ohio.

Witter, Dr. B. H., Department of Zoology, University of Rochester,
Rochester, New York.

Witson, Pror. H. V., University of North Carolina, Chapel Hill, North
Carolina.

Witson, Dr. J. W., Brown University, Providence, Rhode Island.

Witscui, Pror. Emit, University of Iowa, Iowa City, Iowa.

Wotr, Dr. Ernst, Harvard University, Cambridge, Massachusetts.

Wooprurf, Pror. L. L., Yale University, New Haven, Connecticut.

Woops, Dr. Farris H., Lefevre Hall, Columbia, Missouri.

Woopwarp, Dr. Atvatyn E., Zodlogy Department, University of
Michigan, Ann Arbor, Michigan.

Youne, Dr. B. P., Cornell University, Ithaca, New York.

Youn, Dr. D. B., 110 Del Ray Avenue, Bethesda, Maryland.

ZELENY, Dr. CHARLES, University of Illinois, Urbana, Illinois.

PLACOID SCALE TYPES AND THEIR DISTRIBUTION IN
SQUALUS ACANTHIAS

LEONARD P. SAYLES AND \S. G. HERSHKOWITZ

(From Department of Biology, College of the City of New York, and the Marine
Biological Laboratory, Woods Hole, Mass.)

Detailed studies of the development of placoid scales were reported
by Hertwig (1874). His descriptions of certain integumentary scales
and of stomodeal denticles have been supplemented by Steinhard
(1903), Imms (1905) and Radcliffe (1916). These workers give no
indication that Squalus has more than a couple of different types of
integumentary scales or that scaleless regions occur. Most text-book
statements imply that dogfish scales all have pointed spines. For in-
stance, Wilder (1923, p. 82) says: “ The scales in the dogfish are of the
form known as placoid, each consisting of an approximately flat base
from which rises a sharp-pointed cusp, inclined in the direction of the
free edge of the scale, or posteriorly when the scale is in place.” Saka-
moto (1930), however, has reported several different shapes of scales in
five Japanese species of sharks.

The work reported here was undertaken after it had been noted that
there was considerable variation in the scales of pieces of dogfish skin
taken at random for demonstration to students. An attempt is here
made to describe the types, distribution and orientation of scales of
specimens of Squalus acanthias of 54 to 62 cm. length.

For this work, the skin was removed in relatively large pieces, the
exact orientations and positions of which were noted. In all cases the
same regions of both sides of the body were compared. Detailed studies
of 7 specimens were made. Several other fish were used as additional
checks of certain points. At first these were studied after clearing in
methyl salicylate without staining. Later several were prepared by
staining in alizarin red S and then clearing. In addition small portions
of the skin, representative of different scale-types, were macerated in
0.5 M NaOH at a temperature of about 85° C. Isolated scales were
thus obtained.

OBSERVATIONS

For purposes of convenience the integument may be considered as
belonging to the following regions, each of which will be considered

52 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

separately : trunk and tail regions of the body proper, as contrasted with
head; fins, including spine pockets and, in male, claspers; head; mouth
opening and labial pouches; ampullz of Lorenzini; olfactory sacs; eye-
lids; spiracles; external gill slits; internal gill slits; mouth cavity and
pharynx.

The placoid scale of Squalus acanthias consists of a basal plate and
spine (as noted by Hertwig and many others). The basal plate (except
in the case of stomodeals and combs) is quadrangular with two angles
extending laterally, one anteriorly and a fourth posteriorly. The sto-
modeal basal plate also usually bears four extensions, but the lateral
ones, as well as the anterior, extend anteriorly. The basal plate of the
comb type is similar to that of stomodeals but the angles are often
indefinite and the whole basal plate frequently lacks the regularity usu-
ally found in this part of other scales.

The spine may be interpreted as consisting of two main elements:

Fic. 1. Left lateral aspect of a dorsal body type scale. X 75.

Fic. 2. Right lateral aspect of a ventral body type scale. X 75.
Fic. 3. Postero-lateral aspect of a transitional type scale. XX 75.
Fic. 4. Postero-lateral aspect of a body tricuspid scale. X 75.

(1) a longitudinal plate which extends, in plan, between anterior and
posterior angles of the basal plate; and (2) a transverse plate which
extends between the lateral angles of the basal plate. The transverse
component is ordinarily tilted posteriorly and rests on the posterior part
of the longitudinal component. The anterior part of the latter extends
onto the transverse element at least to some extent. Scale types are
associated with more or less marked variations in the development and
shapes of these spine components.

It is well known that an opening on the under side of the basal plate
connects with a system of dentinal canals which extend even into the
spine. The openings:and canal systems are not shown in the drawings.

Trunk and Tail Regions of Body Proper

~ On the trunk and tail regions of the body proper there are four gen-
eral types of scales: (1) dorsal body type (helmet-scale of Hertwig) ;

|
4

DISTRIBUTION PLACOID SCALE TYPES IN SQUALUS 53

(2) ventral body type; (3) transitional type; (4) tricuspid type, show-
ing various degrees of development of the lateral cusps. These divi-
sions are not sharp, many intermediate forms occurring.

In the dorsal body type (Fig. 1) the transverse component is more
than half as wide as the basal plate; the part of the longitudinal element
anterior to the transverse element is of about the same width as the lat-
ter ; the posterior part of the longitudinal element is small ; and the entire
spine is about as high as it is wide. In the ventral body type (Figs. 2
and 14) the transverse component and the anterior part of the longi-
tudinal component are about two-thirds as wide as in the dorsal type;
the height of the spine, on the other hand, is about fifty per cent greater
than in the dorsal type. The so-called transitional type (Fig. 3) is
intermediate between dorsal and ventral types. The tricuspid scales
(Figs. 4 and 18) are of about the same proportions as the dorsal and
transitional types but bear two lateral, secondary cusps set some distance
anterior to the tip of the scale and varying greatly in size in different
scales.

Along the mid-dorsal line there are several (3-5) rows of large,
heavy scales in which tricuspids predominate but occasional dorsal body
scales are present. The dorsal and dorso-lateral surfaces bear dorsal
body type and tricuspids. The latter vary from scales with large sec-
ondary cusps to some with these so small that the scales may be inter-
preted as intermediate between tricuspids and dorsal body type. From
the level of the lateral line through the region of reduction in integu-
mentary pigmentation, the dorsal body type grades over, through the
transitional type, to the ventral body type (Fig. 16). In this transition
zone the tricuspids become more and more scarce until, in the practically
white ventral part of the body, nearly all scales are of the ventral body
type. In this transition zone the basal plates are about twenty per cent
smaller, both in length and in width, than they are either dorsally or
ventrally. This smaller size, coupled with a slightly greater scattering
of scales in the transition zone, gives the impression that there is a
relatively great reduction in the number of scales here.

Neither the type nor the arrangement of scales is particularly altered
along the lateral line (Fig. 16). Also no significant size-differences
exist between anterior and posterior levels.

Body scales all point posteriorly. Frequently several may be some-
what deflected so that the long axis is slightly oblique. The general pat-
tern of distribution shows the scales to be arranged in diagonal rows,
the scales of each row lying between and behind two of the preceding
row (as noted by Klaatsch, 1890, and many others). Under high mag-
nification, however, it is at once evident that the scales are not arranged

54 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

in a precise geometric fashion. This is due to the fact that the rows are
not exactly parallel, the scales vary in size, and “ extra” scales are quite
numerous, thus disarranging the pattern.

Fins

The pectoral fin will be described in detail first. The other ap-
pendages will then be described only in so far as they differ from the
pectorals.

The pectoral fin has a heavy anterior margin, a thinner axillary one
and a curved, filamentous distal one. The anterior and axillary extreme
margins, where the skin of the dorsal side of the fin passes over into
that of the ventral, both bear scales. The anterior border is covered by
closely arranged and frequently overlapping fin-marginal scales (Figs.
5 and 19). Each of these scales is relatively large with a large, high

Fic. 5. Lateral aspect of a fin-marginal scale. X 75.

Fic. 6. Marginal type scale from vicinity of axillary scaleless area. X 75.

Fic. 7. Paramarginal scale from region slightly farther from axillary scale-
less area than that occupied by scale in Fig. 6. X 75.

pedicel. The massive, transverse spine-component is tilted so far that
its surface is nearly parallel with the surface of the integument. The
posterior tip of this is only slightly pointed. A short, thick part of the
anterior longitudinal component projects from the anterior end of the
transverse element. Toward the distal end of the fin the spines of these
scales become smaller and somewhat less massive. Extending from the
entire length of this anterior border toward the middle of the fin there
is a zone of transition to the common fin type which is similar to the
dorsal body type but with the average scale smaller. Following through
this zone from the anterior border, we find first the fin-marginals more
disperse than on the border, then transitionals and body tricuspids ap-
pearing, and finally the common fin type. This transition zone extends
onto the body of the fish a very short distance.

The arrangement of scales on the axillary border is complicated
somewhat by the presence of a scaleless area at the base-of the fin here.

DISTRIBUTION PLACOID SCALE TYPES IN SQUALUS 55

The extent of this area is less on the fin than on the body wall. It is
shaped somewhat like a low, thick J with the long part extending along
the dorsal side of the fin-body junction, the loop around the posterior
side and the short part on the ventral side for a short distance. This
scaleless area is surrounded by several rows of marginal scales (Figs. 6
and 17). These scales have a large, rounded transverse component with
the longitudinal component reduced posteriorly and almost lacking an-
teriorly. Next to these scales are rows of paramarginals (Fig. 7) with
smaller and less rounded transverse components than have the marginals.
Both of these types are more numerous on the body than on the fin.
They are also less numerous at the anterior ends of the scaleless area
where they may even be lacking. At the anterior ends of the ventral
and dorsal parts of this area the spines point considerably away from it.
In the posterior part they return gradually to their ordinary orientation.
This arrangement is, however, subject to some variation. Several
spines may point toward the scaleless area, for example. The basal
part of the axillary border of the fin has paramarginal scales for a short
distance, always greater on the dorsal side than on the ventral, probably
associated with the difference in extent of the scaleless areas on the two
sides. The remainder of the axillary border is covered with scales simi-
lar to those on the anterior but slightly smaller. The zone of transition
toward the common fin type is narrower here than at the anterior border.

The general transition from body to fin is not associated with a
change in scale type. The dorsal body type scales, found here, are
smaller than on the body wall. This is especially true near the distal
margin of the fin. Most of the fin is covered by this common type, more
closely set than on the body wall and arranged in a more regular pattern.
The scales extend to the very edge of the filamentous border which is
covered by small transitional and common fin types and, at the very
edge, tricuspids. These tricuspids are slightly larger than other scales
here.

The scales of the anterior and axillary borders curve to follow these
borders but not exactly so. The result is that the spines at the very
edges extend off the margins. Except at the borders, the orientation on
the fin does not follow the curvature of the fin rays but is in straight
lines toward the filamentous border.

On the anterior and posterior dorsal fins the transition zone at the
anterior margin extends onto the body anterior to the spine pocket which
is covered with massive, shingling fin-marginals. The anterior margin
of the fin is devoid of scales immediately behind the basal portion of
the spine (Fig. 19). This small scaleless area is surrounded by mar-
ginal or paramarginal scales. ‘There is also a scaleless area at the poste-

56 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

rior attachment of the fin. This area is small, symmetrical, with
rounded margins, and surrounded by marginal and then paramarginal
scales.

On the pelvic fins the fin-marginal scales are less massive than on
other fins and the median borders are almost entirely covered by para-
marginal scales. The cloaca is scale-free. There is a large scaleless
area extending around the cloacal opening and continuing along the
attachments of the median borders of the fins and onto their dorsal sur-
faces which are in contact with the body wall. This area does not ex-
tend onto the ventral surfaces of the fins. Around this scaleless area are
marginal and then paramarginal scales. In the male the ventral side of
each clasper bears marginal scales at its posterior end. The anterior end
bears marginals medially, tricuspids laterally and paramarginals in tran-
sition between them on the ventral side. These scales all point medially
and are closely arranged. The dorsal side of each clasper bears no
scales, this scaleless area being continuous with that around the cloaca.

The caudal fin has no scaleless area associated with it. Transitional
and tricuspid scales are more numerous than on other fins, especially to-
ward the dorsal side.

Head

The snout is covered by a characteristic snout type scale (Figs. 8, 9
and 15). Each of these scales, like fin-marginals, is relatively large
with a large, high pedicel and with a massive transverse spine-component

Fics. 8 and 9. Different aspects of two snout type scales. X 75.
Fic. 10. Postero-lateral aspect of a head tricuspid scale. X 75.

tilted so that its surface is nearly parallel with the surface of the integu-
ment. The entire margin of this component is robust and the posterior
end is rounded. In many cases there is a distinct overlapping of these
scales. All snout scales radiate from a point at the extreme anterior tip
of the snout but slightly dorsal. Laterally this type extends farther to

2. ee Ame = Yo Egy

DISTRIBUTION PLACOID SCALE TYPES IN SQUALUS 57

the posterior and is also more numerous than on either dorsal or ventral
surfaces. These scales extend posteriorly for several millimeters where
they merge into the head type (Figs. 10 and 20). Scales of the latter
type are thick-set and have relatively short tricuspid spines with second-
ary cusps nearly as large as the main one. They become somewhat
smaller and less numerous posteriorly. Near the dorsal midline they are
oriented with the spine pointing directly toward the posterior. On either
side of this region the spines have a slightly lateral orientation. At the
level of the spiracle the head type is mixed with the dorsal body type.
The spiracle opening, however, is usually surrounded by the head type
for the most part. At the level of the first functional gill slit the tran-
sition to body type is usually complete. On the ventral side there are,
in addition to the head type, scales similar to the snout type but less
massive. The region of transition to ventral body type, on the ventral
side, is relatively broad and is complicated by the presence of the mouth
opening. The transition is completed a short distance posterior to the
lower jaw.

Mouth Opening and Labial Pockets

There is usually a very narrow scaleless area just outside the rows of
teeth. This is frequently absent near the mid-line but widens out to-
ward the angles of the jaws. In the upper jaw region the integument
adjacent to this scaleless area possesses one or two rows of marginal
scales anterior to which are several rows of the paramarginals. These
scales have interrupted those of the ordinary head type which are
prevalent anterior to them. On the lower lip is a row, or two, of scales
quite similar to the snout type, posterior to which scales of the head
type occur again. Scale orientation is not affected by the presence of
the lips.

The walls of the labial pouches are scaleless. No marginal or snout-
type scales bound this area. Instead, the same type of scale is found
here as occurs in this general region of the head, namely: paramarginals
bounding the anterior part of the pouch adjacent to the upper jaw and
head type along the posterior part. Apparently the only modification in
scales in the vicinity of these pouches is in their orientation which is
changed so that the spines follow the margins of the pouches and point
postero-laterally.

Ampulle of Lorenzim

The presence of the numerous openings of the ampullz of Lorenzini
on the head does not bring about any distinct local changes in scale-type
(Fig. 20). The spines are often shaped to curve about the margins of

58 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

the openings when parts of the basal plates would ordinarily be in the
area which the openings occupy. This condition is more common
dorsally than ventrally. Often a spine overlaps an opening.

Olfactory Sacs

There is usually no modification of scale type about the margin of
an olfactory opening. Head type scales are found here. The scales do
not end abruptly but continue into the cavity a short distance. Posterior
orientation is maintained mediad but not laterad to the opening.
Some of the scales antero-lateral to the opening are turned medially to-
ward the anterior flap, others laterally to follow the lateral margin of
the aperture. The anterior flap is covered by scales similar to the head
type but considerably smaller and more deeply serrate. They are very
closely arranged and point posteriorly. The smaller posterior flap is
covered by similar scales, somewhat larger, closely arranged and point-
ing posteriorly.

Eyelids

The dorsal and ventral eyelids do not differ as far as the scales are
concerned. ‘The scales covering the margin of an eyelid are of several
types. Part of the lid is covered by head type and part by a mixture of
small transitional and tricuspid scales. These scales are smaller than
those of the surrounding area and much more closely arranged, being
almost shingled. The scales curve about the margins, especially an-
teriorly. The scales are confined to the external margins, none being
found on the edge of the conjunctiva.

Spiracles

The external opening of the spiracle is entirely surrounded by head
type scales, smaller and more closely grouped than elsewhere in that
vicinity. At the anterior side the scales are deflected away from the
middle of the opening. In those cases where the head-body transition
extends to this level the spiracle may be surrounded by the several types.
No integumentary scales enter the opening.

The posterior wall of the spiracular cavity bears numerous typical
stomodeal denticles (Fig. 11) somewhat like those described by Hert-
wig, Steinhard, and others. Following anteriorly around the wall, along
the dorso-medial side, the denticles become less numerous up to the mid-
dorsal part, then more abundant until they are usually almost as abund-
ant in the anterior wall as in the posterior. The ventro-lateral wall is

DISTRIBUTION PLACOID SCALE TYPES IN SQUALUS 59

without denticles. There is no particular transition into this area from
either anterior or posterior sides. Instead, the denticles stop rather
abruptly. Also the denticles are less numerous near the external open-
ing which they approach very closely. The points of these scales are
directed toward the pharynx.

External Gill Slits

The scales covering the distal, exposed ends of the gill septa are of
several types, dependent on the location of the septum. The scales
covering the whole anterior surface of the first flap—in front of the
first functional gill slit—are of the type on the body wall nearby but
smaller. They are usually of dorsal body type, but sometimes tran-
sitionals. The posterior surface of the septum is scaleless nearly to the
gill filaments. A narrow area adjacent to the filaments has comb type
scales (Figs. 12 and 13) such as have already been described by Stein-
hard (1903). These are very few in number and haphazardly arranged.

The distal, exposed ends of the septa of the four holobranchs are
alike. The anterior surface, at its free border, is covered usually by
dorsal body type scales, often interspersed with transitional and tricuspid
types, sometimes with small head type or paramarginals. There is a
transition through paramarginals to marginals internally up to the point
where the septum is covered by that in front. Then there is a scaleless
region. Beyond the latter there are comb scales similar to those on the
posterior face of the first flap. The posterior faces of these septa are

Fic. 11. Stomodeal denticle from floor of pharynx. X 75.
Fics. 12 and 13. Two comb scales from wall of a gill slit. 75.

similar to that of the first. There are wider areas bearing comb scales
on the anterior than on the posterior faces, possibly associated with the
fact that anterior demibranchs do not extend externally as far as do
posterior ones. .The extra part of the smooth septum in each case bears
comb scales. No scales occur on the gill filaments.

60 LEONARD, Py SAYLES, AND S. G) BERSHKOW IZ

The posterior wall of the fifth gill pocket bears no filaments and no
comb scales near the surface of the body. A few, scattered, marginal
scales are found in the outer part of the area covered by the flap of the
last holobranch. Where exposed, really the general surface of the body
at that point, there are at first marginal scales. The latter merge pos-
teriorly into typical fin-marginal scales, probably associated with the ad-
joining pectoral fin.

Internal Gill Slits

The scales on the membrane which covers each gill arch are of two
distinct types (as reported by Steinhard, 1903). One, a typical stomo-
deal denticle, is found toward the pharyngeal side of the arch and on

PLATE I

Surface view photomicrographs of pieces of integument of a Squalus acanthias
55 cm. long. In all cases the top of the figure is the anterior end and the spines
point toward the bottom of the page. Figure 17 is about X 50, all others about
x 40.

Fic. 14. From the ventral, mid-trunk region. The basal plates show as gray
backgrounds for the spines. Compare Fig. 2.

Fic. 15. From dorsal side of snout near point from which all scales radiate.
The “heart-shaped” surface of one scale—in the lower right quarter—has been
outlined. The limits of another—near the center—have also been indicated. Over-
lapping obscures to some extent the limits of many scales. Slightly to the left and
below the center of the figure there is a scale with the anterior parts of the pedicel
and basal plate clearly outlined as they recede from the notched anterior margin

of the scale surface. Compare Figs. 8 and 9.
Fic. 16. From the lateral line area in the mid-trunk region. Two openings
of the lateral line canal show in the center.

Fic. 17. From the side of the trunk in the axillary region. At the right side
of the top of this figure the very edge of the axillary scaleless region shows. A
scale in the lower right quarter has been outlined in stipple. The solid black areas
are the basal plates. Compare Fig. 6.

Fic. 18. From the dorso-lateral part of the mid-trunk region. The two scales
in the upper corners are slightly modified dorsal body type. All others are tri-
cuspids. To the right of the center is one which has been outlined in stipple.
Compare Fig. 4.

Fic. 19. From the region immediately distal to the serlleless area behind the
base of the spine of the antero-dorsal fin. The very tip of this scaleless area shows
at the center of the top edge of the figure. Two scaleless patches also show, one
in the center and the other near the bottom. This strip shows the transition from
the ordinary marginal type of scale—at the top—to the heavy fin-marginal type.
In the lower left corner is one of the latter which shows clearly the ramifications
of the portions of the pulp cavity which extend into the “heart-shaped” outer part
of the spine. Compare Fig. 5.

Fic. 20. From the dorsal side of the head just anterior to the eyes. The
opening of an ampulla is shown at the top. Note that the spines of scales around
the opening are still oriented posteriorly. In the lower right corner a scale has
been outlined. To the left of the latter the general form of a head tricuspid shows
clearly. Compare Fig. 10 and also body tricuspids in Fig. 18.

een

PLaTE I

62 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

the gill rakers. These scales are never very numerous, but are more
abundant on the anterior than on the posterior side. Usually there are
more toward the dorsal than toward the ventral end of the arch and
more on the rakers than on the adjacent portions of the arch. The
spines point toward the pharynx. There is no distinct arrangement plan
of the scales.

The second type is the comb scale, similar to that found just external
to the filaments. These scales are smaller than the stomodeals and very
much more numerous. ‘They occur on the side of the arch toward the
filaments, being almost exclusively confined to the anterior surface along
its whole length. When present on the posterior surface, they are con-
fined to two small areas—one dorsal and one ventral—where the mem-
brane of the anterior side of one arch passes over into that of the pos-
terior side of the arch in front.

This situation holds true for all four holobranchs. The anterior wall
of the first functional gill slit bears only combs at the extreme dorsal and
ventral ends, as mentioned for the other slits. The posterior wall of the
fifth slit bears no filaments but does have many comb scales, not only in
the inner part but also scattered over much of the area which corre-
sponds to that occupied by filaments in other slits.

As with the stomodeal type, the combs all point toward the pharynx.
They are usually several rows wide (4-6), frequently with each scale
lying behind and between two others. This arrangement is not precise
as the rows are somewhat irregular, the sizes of the scales vary and
“extras ” occur.

Mouth Cavity and Pharynx

There are no scales on the roof of the mouth or pharynx. The few
scales on the dorso-lateral part of the pharynx are associated with the
membrane covering the gill arches rather than that of the pharyngeal
roof. The floor of the pharynx bears typical stomodeal denticles in a
restricted area which begins in the mid-ventral line at the level of the
first functional gill slit, or slightly anterior to it, and extends as far
posterior as the last slit. This area is usually quite narrow near its an-
terior end but is about half the width of the floor of the pharynx
throughout most of its length. The lateral quarter of the floor, on each
side of this scale-bearing region, is ordinarily scaleless. In some speci-
mens a few scales may be present in the posterior parts of these lateral
areas. These stomodeal denticles are much sparser than body scales
and are irregularly oriented, although the majority point posteriorly.
They are haphazardly arranged.

DISPRIEBUMION PLACOID SCALE DYPES IN SOUALUS 63

DISCUSSION

Most descriptions of the integumentary scales of Squalus acanthias
have been confined to two types: (1) the general body type, mainly of
the dorsal variety; and (2) the tricuspid, also called tridentate. Rad-
cliffe (1916) described but one type in the integument of this animal,
namely, the tridentate. In his Fig. 20 he showed twenty-one scales, all
obviously of this type. This is the type which would ordinarily be pres-
ent in a piece “ from the middle of the side below the first dorsal fin,”
which Radcliffe—in a later paper (1917)—suggested using for identifi-
cation purposes. ‘These scales resemble closely those of Scyllium de-
scribed by Klaatsch (1890) whose sketches have been used frequently
as a source in depicting placoid scales. The general body type of
Squalus was early described by Hertwig (1874) and later by Steinhard
(1903). We have observed scales of both of these types in considerable
numbers on the trunk and tail regions of this animal. The body type
really exists in two forms, both of which have similar basal plates. The
ventral body type, however, differs from the dorsal in having a con-
siderably higher spine, both components of which are much narrower
than in the dorsal type.

In addition to these two types, we have observed at least two other
main types. One of these is a large scale with large pedicel and massive
transverse spine-component the surface of which lies nearly parallel
with the body surface. The other is a marginal type consisting of a
stellate base, an abbreviated pedicel and a spine made up, for the most
part, of a large, circular transverse-component, the longitudinal elements
being much reduced. In addition there are scales of several other
shapes which should perhaps be regarded as intermediates between other
types since they are found in places which may be considered transition
zones.

The massive scales are apparently of two distinct varieties. One is
the so-called snout type found on the tip of the snout and on the tip of
the lower jaw. The other, the fin-marginal, is on the anterior margins
of all fins except the parts of the antero- and postero-dorsals which are
protected by the fin-spines. Both of these varieties tend to be set close
together and to overlap in many cases. It would appear that this is a
special protective type found especially on anteriorly exposed surfaces.

The ordinary marginal scales surround scaleless areas where one
part of the actual surface of the body covers another. Such areas in-
clude: those in the two axillary regions (on the body wall and on the
base of each pectoral fin along the posterior portion of the attachment
of the latter) ; those beneath the posterior portions of the two dorsal

64 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

fins; those behind the spines of these dorsal fins; those beneath the
external flaps of the gill septa; and a very large area along the dorsal
sides of the pelvic fins, on adjacent parts of the body wall and around
the cloaca. No mention of these scaleless areas has been made by other
workers, including Sakamoto (1930), who made a thorough study of
several species of Japanese sharks. On the other hand, those scaleless
areas which are inside of various invaginations, in most pores and in
other openings are usually not surrounded by special marginal scales but
instead by the type commonly found in the region in question. Included
among these places are the ampulle of Lorenzini (Fig. 20), the pores
of the lateral line canals (Fig. 16) and associated canals of the acustico-
lateral system of the surface of the head, the labial pockets, the spiracles,
the olfactory sacs, and the inpocketings to form the eyelids.

Sakamoto (1930) has depicted a number of types of scales some of
which resemble certain of these of Squalus. In describing the scales of
Cynias manazo, he reported that the “ ridges of the scales on the dorsal
side of the trunk are more massive and higher than those of the scales
on the ventral side of the same.” In Squalus there exists a similar con-
dition of the single ridge which we have called the anterior part of the
longitudinal spine-component. None of the scales of Squalus seem to
bear more than a single such ridge. Rudiments of this element are
present on most scales of Squalus. They were confined to the trunk and
caudal scales of four of the five species studied by Sakamoto. In
‘Carcharinus japonicus, ridges were also present on scales of the buccal
cavity. This worker also reported that in general in Cymas “the width
of the basal plate (is) proportional to that of the whole placoid scale.”
In Squalus such is not the case. For example, the spines of ventral
trunk scales are much more narrow than those of the dorsal type. The
basal plates, however, are of approximately the same size and shape in
both types. Also, in the lateral transition zone, where the spines are
intermediate in size, the basal plates are considerably smaller than those
of either dorsal or ventral scales.

Sakamoto also found that in Cynias “the dimension of the scale is
the largest in the trunk, larger on the head, and the smallest on the tip
of the fins.” In Squalus, on the other hand, scales of the snout type
are the largest and fin-marginals are next in size. Those on the tips of
the fins are smallest, as in Cymias.

Steinhard’s observations on the structure of the comb scales of the
gill cavities have been verified by us. On the other hand, the stomodeal
denticles described and depicted by Hertwig and by Steinhard do not
seem to match exactly those found by us in the pharynx. We agree
with Steinhard that the longitudinal element is much reduced or absent,

DISTRIBUTION PLACOID SCALE TYPES IN SQUALUS 65

giving this scale a more delicate appearance in comparison with those of
the skin. Two quite marked differences were not shown by him. The
lateral elements of the basal plate project distinctly anterior in these
denticles and the spine arises well toward the posterior end of this scale.

Steinhard (1903), Imms (1905), and Fahrenholz (1915) found
stomodeal denticles to be missing from the roof of the mouth and
pharynx but present on the lining of the gill arches and the entire
covering of the floor of the mouth between the level of the ventral ends
of the first functional gill slits and that of the corresponding parts of
the fifth pair. Cook and Neal (1921), however, reported these scales
to be present not only in the above-mentioned regions but also some-
what more anterior than the first slit and, more significant, on the roof
of the pharynx in small numbers. We have been unable to find denticles
on the roof of the mouth or pharynx medial to the region of the dorsal
ends of the gill slits. They are, however, present along inner edges of
the septa and in the floor of the pharynx. We agree with Steinhard and
Imms that these scales are ordinarily found ventrally, only between the
first and fifth slits. Our observations differ from those of others in
that we have noted that stomodeal denticles are lacking in the lateral
parts of the pharyngeal floor, except for occasional ones toward the
posterior ends of these regions.

Steinhard (1903) reported that comb scales occur in the pharyngeal
slits both internal and external to the lamella. A further study verifies
this statement. Certain details may be added. The areas of comb
scales external to the four demibranchs on the posterior walls of the
slits are considerably wider than those external to the five demibranchs
of the anterior walls. Comb scales are present iti considerable numbers
internal to the demibranchs of posterior walls but, except for the ex-
treme ventral-and dorsal ends, they are lacking internal to the demi-
branchs of anterior walls. There is, of course, no demibranch on the
posterior wall of the fifth cleft. The external part of this wall bears
no comb scales. The internal part has many of these scales and much
of the area corresponding to the positions of demibranchs of other slits
has scattered scales of this type.

The spiracular cleft does not have comb scales associated with it.
There are, however, stomodeal denticles. These are numerous on the
posterior wall, somewhat less abundant on the anterior wall, quite limited
between these regions on the dorso-medial side, and absent from the
ventro-lateral wall.

From our observations, we conclude that in the integument of
Squalus of 54 to 62 cm. length there is a quite constant pattern of dis-
tribution of scaleless areas and of scales of various types. There are

66 LEONARD P. SAYLES AND S. G. HERSHKOWITZ

at least four general types. Since three of these exist in two quite
constant subtypes which do not seem to intergrade, the number of dis-
tinct types is probably about seven. In addition, there are two types
which are almost certainly transitional. In the linings of the mouth,
pharynx and gill slits there are two additional types—stomodeals and
combs—as previously reported by Steinhard (1903) and others.

LITERATURE CITED

Coox, M. H., ann H. V. Neat, 1921. Are the taste-buds of elasmobranchs ento-
dermal in origin? Jour. Comp. Neurol., 33: 45.

FAHRENHOLZ, C., 1915. Uber die Verbreitung von Zahnbildungen und Sinnes-
organen im Vorderdarm der Selachier und ihre phylogenetische Beurteil-
ung. Jena. Zettschr. Naturwiss., 53: 389.

Hertwie, O., 1874. Ueber Bau und Entwickelung der Placoidschuppen und der
Zahne der Selachier. Jena. Zeitschr. Naturwiss., 8: 331.

Imus, A. D., 1905. On the oral and pharyngeal denticles of elasmobranch fishes.
Proc. Zool. Soc. London, 1905 (1): 41.

KiaatscH, H., 1890. Zur Morphologie der Fischschuppen und zur Geschichte der
Hartsubstanzgewebe. Morph. Jahrb., 16: 97.

RapciirFe, L., 1916. The sharks and rays of Beaufort, North Carolina. Bull.
U. S. Bur. Fish., 34 (1914) : 241.

Ranpciirre, L., 1917. Notes on the taxonomic value of dermal denticles and teeth
in identifying sharks. Copeia, 42: 25.

Sakamoto, K., 1930. Placoid scales of five species of selachians belonging to
Carchariide. Jour. Imp. Fish. Inst., 25: 51.

STEINHARD, O., 1903. Ueber Placoidschuppen in der Mund- und Rachen-Hohle
der Plagiostomen. Arch. Naturgesch., 69 (1): 1.

Wiper, H. H., 1923. The History of the Human Body. Second Edition. New
York. ;

Mist OCH vViIShRY OF DAE OVARY OF FUNDULUS
HETEROCLITUS WITH SPECIAL REFERENCE
LOPE Dib Eh ENA ING OOCYTES

V. D. MARZA, EUGENIE V. MARZA AND MARY J. GUTHRIE?

(‘rom the Histological Laboratory of the Faculty of Medicine at Jassy, Rowmania,
and the Department of Zodlogy, University of Missouri, Columbia, Mo.)

The process of differentiation in the odcytes of many animals has
been studied by histological and cytological methods. Although the
general agreement in the morphological changes is very striking, the
divergence in interpretation of the significance of the structures ob-
served and the relationships between them is more so. Konopacki and
Konopacka (1926), Konopacka (1935), and Guthrie (1925 and 1929)
have emphasized the importance of a physiological viewpoint in such
studies. The limitations of attempts to analyze the chemical contents
of regions of a cell by means of their reactions with fixing or staining
fluids are numerous, in spite of the fact that considerable security can
be felt in some instances. With histochemical methods a somewhat dif-
ferent attack can be made on the question of the significance of changes
in differentiating cells. Not only is the identification and specific locali-
zation of a variety of substances possible at different periods, but the
use of quantitative standards of comparison gives data on the shifting
concentration of materials, as Marza and Marza (1935) and Marza
(1935) have shown in the hen’s egg. The present collaboration was
undertaken in order to provide histochemical data on a form in which
cytological studies had been made, with the expectation that the se-
quences of differentiation in the oocytes would be revealed more clearly.

Observations have been made on all parts of the ovary—stroma,
interstitial cells, follicular theca, follicular epithelium, zona radiata, and
oocytes in all stages of differentiation. For purposes of reference in
the descriptions the differentiating odcytes are placed in two main groups
(Fig. 1). Those in the first part of the growth-period (Period 1)
have increasing amounts of cytoplasm, with cytosomes that may range
up to 300 microns in diameter, but no yolk vesicles (Figs. 8, 12, and 16).

1 Dr. and Mrs. Marza have made all of the histochemical analyses recorded in
this paper. Their report has been prepared for publication by Mary J. Guthrie,
who provided the specimens, through the Supply Department of the Marine Bio-
logical Laboratory, at Woods Hole, Massachusetts, U. S. A., and who has corre-
lated the histochemical observations with cytological observations on samples from
the same and many other specimens.

68 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

Period 2 begins with the appearance of vesicles (proteinaceous yolk
vesicles of Guthrie, 1928) in the odcytes and includes the remainder of
the period of differentiation. During stage A, of the second period
there are few yolk vesicles and the diameter of the cell may be as great
as 400 microns (Figs. 16,17, and 18). The increase in number of yolk
vesicles distinguishes stage A,, at the end of which only a narrow zone

Period 1

Fic. 1. Diagram showing the stages in the growth-period of odcytes in
Fundulus which are referred to throughout the text. In the cytosome the circles
which are empty represent the vesicles in which yolk may appear later; circles
which are lined represent fat deposits; and circles which are solid represent either
intra- or extravesicular yolk.

of peripheral cytoplasm is free of vesicles and cells may be 600 microns
in diameter (Fig. 14). Between the vesicles the internal cytoplasm is
seen as a reticulum. The zona radiata appears at this stage, and the
nucleus of the odcyte is increasingly eccentric. The beginning of stage
B, is marked by the initial appearance of globules of yolk (intra-
vesicular yolk) within the vesicles (Figs. 10 and 13). During stage

HISTOCHEMISTRY OF OOCYTES 69

B, globules of extravesicular yolk begin to appear directly in the cyto-
plasm between the vesicles, and the diameter of the oocyte may reach
1,000 microns (Fig. 15). The globules of yolk that appear within the
so-called proteinaceous yolk vesicles, as well as those which appear be-
tween the vesicles, constitute the fatty yolk of Guthrie (1928). As the
oocyte continues to grow, intravesicular and extravesicular yolk in-
creases in amount. The yolk globules deposited within and without the
original large vesicles become confluent and indistinguishable (Fig. 13).
Toward the end of the growth-period (stage B,), a continuous mass of
yolk is found surrounded by a cortical layer of cytoplasm (Figs. 11, 12,
and 13) in which the germinal vesicle is located. PINE oocytes may
reach 1,600 microns in diameter.

This report includes observations on the variations in amount and
distribution of iron and potassium, on the plasmal and nucleal reactions,
and on the localization and relations of the acid proteins. Other
analyses are in progress.

DETERMINATION OF [RON

Iron occurs in fish eggs in the ichthulin molecule which, like vitellin,
contains a hematogen (Walter, 1891, in the carp). A histochemical
study of iron has been made by Smiechowski (1892), Wassermann
(1910), Marza, Marza, and Chiosa (1932), and Marza (1935) in the
hen’s egg at various stages in its growth. In Fundulus heteroclitus we
have studied the localization and changes in content of iron in the
oocytes during the period of growth and yolk-formation.

The ovaries used were from fish collected at Woods Hole, Mas-
sachusetts, on June 27 and September 11, 1934. Fixation was in 96
per cent alcohol, and paraffin sections 10 microns in thickness were
studied. Since it seemed probable that both inorganic and organic iron
were present in eggs (cf. Warburg, 1914, on eggs of Strongylocentrotus),
methods for detecting inorganic and total iron content were used. Ma-
callum’s (1912) ammonium-sulfide method and Liesegang’s (1923) po-
tassium-ferrocyanide method were employed to test for inorganic iron,
while Policard’s (1924) microincineration method made possible the
identification and localization of the total iron content.

With Macallum’s method certain granules in the stroma, follicle,
and odcyte become dark brown, which presumably is evidence of their
inorganic iron content. However, some of these dark brown granules
are apparently pigment since they are seen in similar locations after the
use of Liesegang’s method, with which the presence of inorganic iron
is indicated by a homogeneous blue staining of certain regions. It ap-

70 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

pears, therefore, that Macallum’s method is not entirely trustworthy for
this material. Tests for inorganic iron have not been considered posi-
tive unless the results with the two methods were in agreement. After

TABLE I

Results of tests for inorganic iron and total iron content. Four hundred and
twenty-eight odcytes in 12 ovaries were examined. A (?) indicates that a result is
considered unreliable for reasons stated in the text.

Methods
Region

Macallum Liesegang Policard
Interstitial cells............ Ahn. + se ae
Stroma Sais eer ec oe Ge eth + (?) - —

Theca

Period Qe soe one + =

Period:2) Buss. 8 Pee: aia =F == |")
Follicular epithelium

Perloded woe Aatiiek cae

Zona radiata
Reriode2 Ara iy eet
Period. 2) Bio sence sees:

++
++
|

Odcyte cytoplasm
Period Wee aea ces sae. = >
Peripheral region
Period Je Aca. eerie.
Period 2 Bee ee ens es
Internal region
Period) 2 Alysia s seas
Period: 2 Bi. vaca «

++ ++

Yolk
Intravesicular.............
Extravesicular.............

1+
I+
++

Oécyte nucleus
Ghromatin' 0) 9. ces ok —
Niucleabie cry eae ated neta 3 + (?) _ | _

the incineration of sections according to the method of Policard the
various parts of the ovary are recognizable. Of the mineral residues
only the oxides of iron are colored orange or red; others are white or
gray. The detection of iron and its localization within the cells are

HISTOCHEMISTRY OF OOCYTES fall

thus possible. Microincinerated sections were examined against a dark
field with a Greenough microscope and, also, by means of a Reichert
oblique illuminator.

Inorganic iron is most abundant in the interstitial cells, some of
which give a more interise reaction than others, and is rarely seen in
the stroma. The nucleus of the growing oocyte does not contain iron
at any stage. During the first part of the growth-period iron has not
been demonstrated either in the cells of the follicle or in the cytoplasm
of the oocyte. However, in the second period inorganic iron is some-
times seen in the follicle cells and in the zona radiata but not through-
out the entire circumference of any one egg. Within the oocyte at this
stage a positive inorganic iron test of weak intensity is sometimes ob-
tained in the peripheral layer of cytoplasm, as well as in the internal
cytoplasm between the yolk vesicles. In the intravesicular yolk the in-
organic iron test is sometimes positive, but in the extravesicular yolk
inorganic iron has not been detected. After microincineration the fully
differentiated yolk is seen to contain iron, which must have been in
organic combination (Fig. 11). The summary of tests for iron is
given in Table I.

It should also be recorded that white ash (probably calcium) is
abundant in the cytoplasm of odcytes in Period 1 (Fig. 8). These
oocytes differ strikingly from those of the hen, which do not contain ash
at a comparable stage. White ash also occurs in both the peripheral and
internal cytoplasmic regions during Period 2 in oocytes of FPundulus
(Fig. 9). This white ash may obscure the red where it is present in
small amounts and render the negative microincineration results for
iron unreliable for certain regions. As the intravesicular yolk appears
a gray ash remains after microincineration (Fig. 10). The nucleoli of
the fish egg are very rich in white ash, and the chromatin in gray ash.

Inorganic iron apparently passes through the follicle and zona
radiata of the odcyte during the period of yolk deposition and is found
in the cytoplasm and in the yolk forming within the vesicles. During
the final differentiation of yolk the accumulated iron must be combined
in organic form, probably in the hematogen of ichthulin. The finding
of iron in both inorganic and organic form may account for certain
discrepancies in previous reports.

QUANTITATIVE DETERMINATION OF PorassIUM

The quantity of potassium exceeds the combined amounts of cal-
cium, sodium, and magnesium in ripe fish eggs. Bialaszewicz (1929)
has shown that high potassium content is characteristic of egg cytoplasm

(2, V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

of both invertebrates and vertebrates. Seventy-four to 92 per cent of
all the diffusible bases in egg cytoplasm is potassium. Chemically the
potassium content of a number of ripe and fertilized fish eggs has been
determined (Needham, 1931, p. 356), but the eggs of Fundulus have
not been studied from this point of view. A histochemical study of
potassium has not been made on any fish egg, nor have studies been
made on any differentiating odcytes.

The method used was that of Macallum (1905) as modified by
Marza and Chiosa in 1934 and 1935. By means of a comparison eye-

Fic. 2. Use of comparison eyepiece in quantitative histochemical observations.
A, arrangement of microscopes and light; B, diagram of preparation of ovary
(left, below), standard slide (right, below), and field of vision (center, above).

piece and a series of standards made of agar-agar and containing con-
centrations of cobalt sulphide calculated as potassium equivalents, it is
possible to determine the quantity of potassium in specific regions of the
ovaries (Fig. 2).2 Ovaries from twelve fish were fixed in 96 per cent
alcohol and sectioned in paraffin at 10 microns. One hundred and
thirty-six odcytes of Period 1, 63 of Period 2A, and 72 of Period 2B

2 Marza and Chiosa (1935) have given the technique for the preparation and
use of the standard slides.

HISTOCHEMISTRY OF OOCYTES (fy)

have been studied. Within these stages of differentiation the odcytes
observed have been sorted according to actual size attained.

The smallest oocytes contain more potassium in their cytosomes than
do larger ones (Figs. 3 and 12, and Table II). Since these cells, which
are from 20 to 50 microns in diameter, do not have a continuous follicu-
lar layer the concentration of potassium is not conditioned by selective
permeability of the follicle (Fig. 1). The nucleoli of such young
oOcytes are very rich in potassium but the chromatin contains little.

TABLE II

Results of tests for potassium, indicated as average percentages. Two hundred

and seventy-one odcytes in 12 ovaries were examined.

Renton Hercenteee Region Percentage
Stroma Odcyte cytoplasm
@ytoplasm..... 0. .s cys ss 0.065 RemOd lean eyes 0.160
INICLeUS eer eee ais aie: ene 0.130 Peripheral region
eniod 2 Ate se saae 0.087
Theca Period 2 B......... 0.084
Cytoplasm Internal region
Period 2 AandB..... 0.032 Reriod 20 Ay Siena. ve 0.087
Nucleus Period 2 B......... 0.109
Period 2 Aand B..... 0.087
Follicular epithelium Yolk
Cytoplasm Intravesicular.......... 0.108
Beriod 2.0 ics ee aces — Extravesicular.......... 0.075
Beriod 295A. 335 rhea: 0.066
Reriod’ 2 Bosh ass 0.057 | Odcyte nucleus
Nucleus Chromatin
Reniodydi tia. sec wines: — Period islta sncducts ues 0.098
Period! 2A. soe 0.109 Period 2 A......... 0.049
Reriod’ 2B) ic fo sie cie,s 0.116 Period’ 2) Baka... 0.065
Nucleoli
Zona radiata Period. sive a soe): 0.151
Reriodi2 vA ye. 0.054 Period 2A. oak 0.125
Period 2° Bi... 5:5). 0.057 Reriody2) Bian ou: 0.130

During the second period the theca is somewhat more conspicuous,
and the concentration of potassium is not the same in it as in the cells
of the stroma (Table II). In the follicle cells the nuclei are richer in
potassium than are the cytosomes. No concentration of potassium oc-
curs in the zona radiata. With the appearance of vesicles in the odcyte
(Period 2 A,) the concentration of potassium in the cytoplasm con-
tinues to decrease (Fig. 3). The penetration of potassium does not
keep pace with the rate of cytoplasmic increase in the odcyte. This de-

74 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

crease is noticeable, also, in the chromatin and nucleoli. During the
early stages of yolk deposition the percentage of potassium in the cyto-

0.2
0.15
\ G of
‘\ omg
a a
0.1 a ae
: uN 4
eS & bs
&e. Seu Se Vea
Ee
0.05

ee Cy oplasm

ts Mfemenge Peripheral region

iat ie Internal region

0
Diameter 0.05 01 0.3 0.6 08 10
in mm. A, A, B, Bo

Fic. 3. Variation in percentage of potassium, shown on vertical axis, in cyto-
plasm of odcytes during the growth-period.

plasm begins to rise, especially in the internal region. Yolk contains a
high percentage of potassium which again falls off as the oocyte grows

HISTOCHEMISTRY OF OOCYTES 75

(Fig. 12). A similar shift in proportion of potassium during yolk-
formation has been observed in the hen’s egg in the ground substance
of both white and yellow yolk-spheres.

The fact that the theca and follicular epithelium vary so little with
respect to potassium content throughout the growth-period is different
from the findings in the ovary of the hen (Marza and Chiosa, 1936).
There, toward the end of yolk-formation, the percentage of potassium
increases in the follicle cells. In the ovary of the fish there appears to
be little increased concentration, or temporary storage, in the follicle
cells of substances utilized in the formation of yolk. However, there is
some indication that the follicle cells determine the rate of penetration
of potassium which decreases in amount in the cytoplasm of the odcyte
following the association of follicle cells. The follicular epithelium ap-
parently undergoes changes in permeability. It seems to be more
permeable to potassium during stages A, and B, and only slightly
permeable at later stages. That the result may be conditioned by an
altered rate of entrance of other materials is also a possibility.

PLASMAL REACTION

In 1924 two color reactions were reported for cells after the ap-
plication of Schiff’s sulfurous-fuchsin reagent. The first or plasmal
reaction was observed in the cytosome, and the nuclei were uncolored
except when the reagent was applied for a long period (Feulgen and
Voit, 1924). If the sections were hydrolyzed with hydrochloric acid
before being treated with Schiff’s reagent, the color reaction was in the
nucleus ; this was called the nucleal reaction (Feulgen and Rossenbeck,
1924).

The names given to these reactions indicate that aldehydes in the
cytoplasm or chromatin were believed to be responsible for the appear-
ance of the color. Feulgen and Voit (1924) pointed out that the
plasmal reaction was related to the presence of fat in the cytoplasm.
If the tissue is dehydrated before sectioning the reaction is usually nega-
tive. Verne (1929) demonstrated that only unsaturated fats in the
process of oxidation gave a positive plasmal reaction. However, fatty
substances are not the only ones to react positively. Feulgen and Voit
(1924), Voss (1929), and Marza and Marza (1934) have reported an
intense plasmal reaction in the elastic elements of large blood vessels,
and it occurs in tissue dehydrated after fixation. Many who use this
method of Feulgen consider it to be specific for aldehydes. Feulgen
and co-workers pointed out that while Schiff’s reagent reacted with
alkalis (a red color) and bromine they considered it to be specific for

76 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

aldehydes in cells. Lison (1932) found that the color of the reaction
with various aldehydes varied from deep violet to blood red. He con-
cluded, therefore, that similar color reactions by substances other than
aldehydes should be considered as positive. With this criterion alkalis,
aliphatic ketones, certain unsaturated compounds (such as oleic acid),

TABLE III

Results of plasmal and nucleal reactions, indicated as averages of color intensity;
0.3 is the lowest positive reading.

Region Plasmal reaction Nucleal reaction
Stroma
C@ytoplasmise venenatis es es == =
INUIGIENS HAR Rear e. Bite ci Sed — 1.25
Theca
@Cytoplasmiaaenie ements oes = =
ING CTe tishe caylee Bac ach ots ce — 1.25
Follicular epithelium
CGyLOplasnie ne detsts eas ase 213) = =
Chromatin is. 3 aes cle cca ees — 1.25
INUCIEOLI YE rae eee oie Gis eee _ 1.25
Nucleoplasm.................. — 0.3
LoOnairadiat ance mone: vain wake: 0.3 0.3
Oécyte cytoplasm
Periogiilimee nse cere 0.3 0.4
Peripheral region
Reriod) 2yAvand 1B 7.0... - 0.3 0.3
Internal region
Period 2 AandB......... 0.3 0.3
Yolk
Intravesicular................. 0.75 0.75
Extravesicular................ 0.6 0.5

OGcyte nucleus
eri OG lye casa exe sig arate ae = —
PEriOd) 2eAG Set es ane htass _ —

weak salts of strong bases (such as acetates and phosphates), some
amino oxides, and certain catalytic oxidizing systems gave positive color
tests. Even though certain of these substances occur not at all or in
very minute quantity in normal cells, the presence of alkalis, phosphates,
ketones, and oxidases makes it clear that a positive plasmal reaction can-
not be considered specific for aldehydes or fats.

HISTOCHEMISTRY OF OOCYTES a

Voss (1927) first studied the plasmal reaction during the growth-
period of odcytes, using ovaries of amphibians. Hibbard (1928) and
Brachet (1929) employed it with amphibian ovaries, and Marza and

* Plasmal reaction
= Nucleal reaction
y)
j
0.50

SMS
UU

TMM
MMM

G PR. LR. LY. Bey;

Fic. 4. Plasmal and nucleal reactions in the odcytes. C, cytoplasm of oocytes
of Period 1; P. R., peripheral region of cytoplasm of odcytes of Period 2; J. R.,
internal region of cytoplasm of o6dcytes of Period 2; J. Y., intravesicular yolk;
and E. Y., extravesicular yolk. The numbers on the vertical axis refer to the
comparison color scale.

Marza (1934) with the hen’s ovary. For the reaction in Fundulus,
twelve ovaries were fixed in sublimate-acetic as recommended by Feul-
gen. Frozen sections were used in order not to lose the fatty sub-

78 V. D. MARZA, EV. MARZAVAND IM: J, GUDEHRIE

stances during dehydration. The sulfurous-fuchsin reagent was pre-
pared according to Wermel (1927),? and the short technique was fol-
lowed: thirty minutes in the reagent, rapid washing in three jars of
water turbid with sulfur dioxide, washing in distilled water, dehydra-
tion, and mounting. In order to evaluate the reaction qualitatively a
scale of color intensity was employed. An intensity designated as 0.3
indicates the least positive reaction and below that value the coloring is
the result of adsorption of the reagent; 3.0 is an extremely intense posi-
tive reaction.

A slightly positive plasmal reaction may be obtained in the cyto-
plasm of odcytes of Period 1 (Table III and Fig. 4). In this material
the so-called yolk-nucleus, as described by Hibbard (1928) in Discoglos-
sus and Marza and Marza (1934) in the hen, is not distinguishable.
The reaction of the nucleus of the odcyte is negative at all times except
for an occasional faint reaction in a nucleolus. No reaction is ob-
served in the ovarian stroma, the theca, or the follicular epithelium at
any stage. The interstitial cells give an intense positive reaction.

During the second period the zona radiata varies in reaction, ap-
parently depending on the material present in its canals at any particular
time; it is positive in about half the cases. In the peripheral and in-
ternal cytoplasm of the odcyte the reaction is variable with an average
on the positive side. Yolk forming within the vesicles gives a more
positive reaction than that formed directly in the cytoplasm. Since the
reaction is the same in sections dehydrated in alcohol before being
treated with the Schiff’s reagent, the substance responsible cannot be fat.
The difference in degree of reaction in the intravesicular and extra-
vesicular yolk parallels the difference in potassium content. It seems
possible that the plasmal reaction in the yolk is conditioned by the pres-
ence of the alkali.

NucLEAL REACTION

The nucleal reaction has been studied in the eggs of numerous in-
vertebrates and some vertebrates. Contrary to the usual positive re-
action in the nuclei of cells, the nucleal reaction becomes negative in the
nuclei of odcytes early in the growth-period and remains negative until
the period of diakinesis (Koch, 1925; Voss, 1927; Hibbard, 1928; Lud-
ford, 1928; Brachet, 1929; Gresson, 1930; Mukerji, 1930; Bauer, 1932;

3 Dissolve 1 gram of basic fuchsin in 200 cc. of boiling distilled water and
filter. (Griibler’s gelblich fuchsin was used.) Add 20 cc. of normal hydrochloric
acid. When the solution is cool add 2 grams sodium bisulfite and stir for several
minutes. After 2 hours add 0.2 cc. acetaldehyde; the solution becomes intensely
red. After 45 minutes again add 20 cc. of normal hydrochloric acid and 1 gram
of sodium bisulfite. Stir the solution for 15 minutes and set aside until the fuchsin
is decolorized. The pale amber reagent should be kept dark and cool.

HISTOCHEMISTRY OF OOCYTES 79

Marza and Marza, 1934). ‘This peculiar situation is characteristic of
only female germ cells, and the nucleal reaction is positive during the
entire course of spermatogenesis.

Ovaries of Fundulus were fixed in sublimate-acetic, and the Feulgen-
Rossenbeck technique was followed on paraffin sections. The color
scale used for the plasmal reaction records was again employed.

The cytoplasm of cells of the stroma and of the follicular epithelium
has a negative nucleal reaction (Table III). The nuclear membrane
and chromatin, but not the nucleoplasm, of cells of the stroma give posi-
tive tests. In the nuclei of the follicular epithelium the reaction is
negative in the membrane, faintly positive in the nucleoplasm, and
equally strong in the chromatin and in the nucleoli. These reactions do
not change conspicuously during the course of differentiation.

There is a positive reaction in the cytoplasm of the oocyte during the
first period of differentiation which is similar to the reaction without
hydrolysis—the plasmal reaction (Fig. 4). It seems likely that this is
to be explained on the basis of the potassium content. Even in the
smallest oocytes the nucleal reaction is negative in all parts of the
nucleus.

During the second period of differentiation the zona radiata has a
faintly positive reaction in stage B. The cytoplasm at the periphery of
the oocyte and between the yolk globules gives a faintly positive reaction.
As with the plasmal tests, the yolk gives a positive nucleal reaction of
similar intensity. Since hydrolysis does not alter the reaction, it is ap-
parent that it is not always conditioned by fat, and that it may be at-
tributed to the potassium content. In the odcyte of the hen Marza and
Marza (1934) made further tests that led them to the hypothesis that
the positive plasmal and nucleal reactions arise from the presence of po-
tassium. They found in the yolk of the hen’s egg that the plasmal and
nucleal reactions were localized in the centers of. the globules. When
the nucleoprotein test (method of Unna, 1921) was positive the locali-
zation was peripheral. The reaction for oxidases was negative. Pro-
longed extraction with ether and chloroform did not alter the reaction,
which eliminated fats as the source of aldehyde. There was no aug-
mentation of the reaction after treatment with alcohol; it had been sug-
gested that alcohol might be adsorbed by the yolk and partially changed
to aldehyde.

The nucleal reaction is negative in nuclei of all odcytes during the
second period in Fundulus, and no observations were made on the time
of shift of the reaction before meiosis. There is no evidence upon
which to offer an explanation of this negative test. Koch (1925) and
Brachet (1933) attributed it to a chemical change in the nucleic acid

80 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

during the growth-period, while Feulgen (1926) and Ludford (1928)
considered it to be the effect of dispersion or dilution of thymonucleic
acid as the nucleus of the odcyte increased in volume.

Acip PROTEINS

In order to study changes in the proteins during differentiation of
the oocyte the method of Unna (1921) for acid proteins was used.
This procedure enables one to distinguish albumins, globulins, and
nucleoproteins. It is based on differences in color reaction and solu-
bility between the groups. ‘Tissues are fixed in 96 per cent alcohol,
sectioned in paraffin, and stained for twenty minutes in the Pappenheim-
Unna reagent.* Albumins and globulins are stained red by the pyronin,
while the nucleoproteins are stained blue-green with the methyl green.
To separate the albumins and globulins advantage is taken of their
specific solubilities. Albumins and pseudoglobulins are soluble in dis-
tilled water while globulins are not. Both albumins and globulins are
soluble in salt solutions. By comparison of sections stained before and
after chromolysis in water and in salt solution the location and relative
proportions of acid proteins of the three groups can be determined.

Sections of the ovary of Fundulus were divided into three series.
One series was stained directly. In such sections all acid proteins were
shown. A second series was placed in sterile distilled water in an oven
at 39° C. for twenty-four hours before staining. This was the first
chromolysis. The difference in reaction with pyronine between these
sections and those of the first series was conditioned by the loss of
albumins (cf. Figs. 16 and 18). A third series was placed in a sterile
2 per cent solution of sodium chloride in an oven at 39° C. for twenty-
four to forty-eight hours before staining. This was the second chro-
molysis which removed both albumins and globulins. If nucleoproteins
were present in such sections they would then be stained with the
methyl green (cf. Fig. 17 with Figs. 16 and 18). To obtain results
subject to comparison, sections of the three series were handled simul-
taneously by an arrangement of forceps during the staining and dehydra-
tion. The latter process had to be completed rapidly. Results were
read on a standard color scale in which 0.3 was the least positive reaction
and 4.0 the most intense. Two hundred and sixteen oocytes have been
studied in sections from twelve ovaries.

Two varieties of interstitial cells are demonstrated with Unna’s
technique. Those which are pigmented, occur in masses, and contain

4The Pappenheim-Unna reagent used consisted of 0.15 gram of Grubler’s
methyl green (from which admixed methyl violet was removed by shaking the
crystals in chloroform), 0.25 gram of pyronine, 2.5 cc. of 96 per cent alcohol, and
100 cc. of 0.5 per cent phenol.

Se ee ee eee ee ee

yo a gee pig rede

HISTOCHEMISTRY OF OOCYTES 81

iron do not color with the stains used. Cells of the other type have
variable shapes, with pseudopodia-like histiocytes or simple elongations
like fibroblasts, or may be oval with eccentric nuclei somewhat similar

=.= Glebulins /

Albumins yi SS

2.9

1.5

0.5

: - 0.3 05 08 1.0 1.6
Diameter in mm. A A, B, Bo Bs

Fic. 5. Variation in acid proteins in the cytoplasm of follicle cells. The
numbers on the vertical axis refer to the comparison color scale.

to plasmocytes. Such cells have been described in the ovarian stroma
of the hen by Goodale (1919), Nonidez (1921), Marza (1934), and
Marza and Golaescu (1935); the analogies are not completely clear.

82 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

These cells in Pundulus are found in the endothelium of the blood
vessels, isolated in the stroma, in the tunica albuginea, and, rarely, within
the masses of pigmented cells. The cytoplasm of the non-pigmented
interstitial cells stains intensely with the pyronine.

0.3 0.6 08 1.0 16
A, As B, Bo B,
Fic. 6. Variation in acid proteins in the cytoplasm of oocytes in Period 1 and
in the peripheral region of cytoplasm in Period 2. Designations as in Fig. 5.

Diameter in mm.

Only a small quantity of irregularly localized globulin is found in
the cytoplasm of cells of the ovarian stroma, and neither globulins nor

HISTOCHEMISTRY OF OOCYTES 83

albumins occur in the nuclei which are, however, rich in nucleoproteins
(Table IV). Both globulins and albumins are found in the cytoplasm
of thecal cells during Period 2A; the amount is somewhat reduced

2.0 ‘

15
3 ‘

a ere
1.0
0.5
a)
Diameter in mm. 0.6 0.8 1.0

A, B, Bo

Fic. 7. Variations in acid proteins in the internal regions of cytoplasm in
oocytes of Period 2. Designations as in Fig. 5.

during Period 2 B. The nuclei of thecal cells are rich in nucleoproteins.
Concentrated masses of globulins are seen localized along the length
of the cytosomes of follicle cells during Period 1 (Fig. 5), but the ground

84 WS IDS IWR ZG, Ti) WG WIDER. JANIIID) IES jf, (EAU AD Teale

cytoplasm of follicle cells gives only a slight reaction (0.5). The
amount of globulins in the follicle is increased conspicuously during
Period 2, and albumins appear in stage 2B. These albumins may
occur because of the splitting of globulins in the follicle as penetration
into the odcyte begins. Globulins appear in the chromatin during stage
2 B and are found, together with nucleoproteins, in the nucleoli through-
out the growth-period. No acid proteins have been detected in the zona
radiata at any time.

EXPLANATION OF PLATE I

Microphotographs

Frc. 8. Small odcyte (Period 1) after microincineration, showing much white
ash in cytosome and some in nucleus. X 150.

Fic. 9. Odcytes of Period 2 A after microincineration. Ash is visible in the
cytoplasm but not in the vesicles. X 30.

Fic. 10. Odcyte of Period 2 B: after microincineration. White ash occurs in
the region of the cytoplasm and gray ash in the intravesicular yolk which is pres-
ent. The follicle appears two-layered because of the plane of the section. X 56.

Fic. 11. Odcyte of Period 2 Bs after microincineration. The homogeneous
central mass of yolk is somewhat shrunken from the peripheral cytoplasm and its
ash content is visible. X 30.

HISTOCHEMISTRY OF OOCYTES

Aion aN;

Results of tests for acid proteins, indicated as averages of color intensity;
0.3 is the lowest positive reading. Where a range is given it indicates a shift in
content between the smaller and larger cells of a given period.

Regions Albumins Globulins Nucleoproteins
Stroma
Gy toplasiiews sees set Se seit — 0.8 —
Ghromeatinee ss ee eke i ess ees = = 25
INFRUGISONI es aides toes 5 ctsleen sean eineemen — = 3.0
Theca
Cytoplasm
BETO GQ w Aten yee ees (see 0.4 0.6 —
Period 2° Bes... 56 5.42 Steet — 0.6 =
Chromatin
Period 2 A ame 1B. sss sen oo — — 2.0
Follicular epithelium
Cytoplasm
Remiodelisess Us sstess) sted see — 1.1 =
Period 2 Aj-As............. — 2.2-2.3 —
enmiOdiwwe Bie cates a eae 0.6 2.9 —
Period 2 Boa=B3.:........-.. 1.0-0.5 2.7-2.4 =
Chromatin
Re reOclbleamee see cl create tea — — 2.5
TRYSSEOyGI EAU Ge ae ee oe nee — — 3.0
Beriode2i Bie. ics eee cs — 3.0 3.0
Nucleoli
Reriocdswliance?) sae ee ee -~ 3.0 3.0
Oécyte cytoplasm
ReTiOd ee teins ee An eels — 2.0—4.0 —
Peripheral region
RETIOG eee AG tsk ete ater ae — 3.4 —
PenodetAcie sack ho ee 0.4 2.4 —
RETO CED Brits nee cs et nsteess 0.4 1.4 —
Period 2 B:-B3............. 0.6-0.4 1.4-0.7 —
Internal region
Periodt2 Anew caer ken ene 0.7 2.4 -=
Reriod=2"Bie <8 cc weak 0.5 1.1 —
Reriode2 Bate iy ccs oor 0.6 1.1 —
Yolk
Intravesicular
REGiOUE2R Bias, a: Sats eks oe — 0.75 0.5
Pernod 245 en nace sees — 0.5 0.5
Extravesicular
PeniOdeZ Borgo ce can eee — — 0.5
Oécyte nucleus
Chromatin
Remodsp lean een ee 0.3 0.5 —
Nucleoli
IRtesrnoals il @inGl 7. 2 ocobancco. — 3.5 —

86 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

EXPLANATION oF PLATE II

Microphotographs

Fic. 12. Group of odcytes, showing variation in potassium content; method
of Macallum. X 41. Odcyte 1 is in Period 1 and has a high potassium content.
Oécyte 2 is in Period 2 B:; 3 is in Period 2 B2; and 4 is in Period 2 B: (4 is about
1,200 microns in diameter). Note the decrease in potassium content in the yolk
in oocytes 3 and 4.

Fic. 13. Group of odcytes showing variation in total acid proteins; method
of Unna, without chromolysis.. X 17. Odcyte 1 is in Period 2 A, and gives an
intense reaction in the cytoplasm; odcyte 2 is in Period 2 Bi; 3 is in Period 2 B:;
and 4 is in Period 2 Bs with a central homogeneous mass of yolk (4 is about 1,200
microns in diameter). Note shift in reaction in yolk.

Fic. 14. Group of odcytes showing variation in total acid proteins; method
of Unna, without chromolysis. X 41. Odcytes 1, 2, and 3 are in Period 2 A,
with numerous vesicles; and 4 is in Period 2 B». Note shift in reaction in
cytoplasm.

Fic. 15. Portion of odcyte early in Period 2 Bz showing variation in acid
proteins; method of Unna, without chromolysis. X 204. Note larger hetero-
geneous vesicles containing some intravesicular yolk and the much smaller globules

of extravesicular yolk.

inn pr

HISTOCHEMISTRY OF OOCYTES 87

Acid proteins with a globulin reaction are conspicuous in the cyto-
plasm of the smallest oocytes and reach a very high concentration by
the end of Period 1 (Fig. 6). They are uniformly distributed in the
cytosome (Figs. 16 and 18). No albumins or nucleoproteins are ob-
served at this stage (Table 1V). As vesicles appear and increase in
number the peripheral layer of cytoplasm has a decreasing amount of
globulins (Figs. 6, 13, 14, and 15). Again we see in the oocyte of
Fundulus during the first period a conspicuous accumulation of sub-

EXPLANATION OF PLateE III

Microphotographs

Fic. 16. Odcytes showing intense acid protein reaction; method of Unna,
without chromolysis. X 140. Smaller odcyte is in Period 1, larger is in Period
2 A:

Fic. 17. Oocytes in Period 2 A:, showing the absence of globulins; method
of Unna, after chromolysis in salt solution. X 140. The failure to stain with
methyl green indicates the absence of nucleoproteins.

Fic. 18. Odcytes showing globulin content; method of Unna, after chromoly-
sis in water. XX 140. Smaller odcyte, of which only a part is seen, is in Period 1;
larger is in Period 2 Ax.

stances before the actual synthesis of yolk begins. Albumins appear
toward the end of stage 2 A and are found in small quantities until the
end of yolk-formation (Fig. 6). In general there is less acid protein
in the internal cytoplasm, but the albumins are somewhat more abundant
(Fig. 7). It would appear as if proteins adjacent to the places of yolk
deposition were chiefly in the form of simple albumins. However, in
the yolk deposited within the vesicles some globulins are found but no
albumins, while in extravesicular yolk neither can be demonstrated
(Fig. 15). The synthesis of simple acid proteins into ichthulin and its
combination with other groupings is apparently complete and not de-

88 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

stroyed by the method of treatment. This differs from the findings in
the hen’s egg where Marza (1935) found that globulins and pseudo-
globulins were detectable in the fully differentiated yolk. The yolk in
Fundulus contains an appreciable amount of nucleoprotein, much more
than the yolk of the hen’s egg. Konopacka (1935) has reported the
presence of nucleoproteins in the yolk of two fresh-water fishes; she
has also identified a mucoprotein in the vesicles of Period 2A.

The presence of nucleoproteins in eggs has stimulated considerable
discussion. From an analysis of the ichthulin of various fish eggs
Walter (1891), Hammarsten (1905), and Linnert (1909) concluded
that there was no nucleic acid present. However, Konig and Grossfeld
(1913) and others have isolated small quantities of purine bases or
nucleic acids from the whole eggs of several fishes. The eggs of
animals which develop in an aquatic environment contain the constituents
of nucleoproteins, while the eggs of terrestrial animals are very poor
in them. Needham (1931) has considered these facts and their im-
plications. The histochemical method of Unna (1921) reveals the pres-
ence of nucleoprotein in the growing odcyte of Fundulus which con-
firms the chemical findings in other teleosts. It may be that the positive
Feulgen-Rossenbeck (1924) reaction in yolk is conditioned in part by
nucleoprotein, but the presence of potassium in the same region confuses
the interpretation.

The nucleus of the odcyte does not give a nucleoprotein reaction at
any time (Table IV). Both globulins and albumins occur in the
chromatin masses. Nucleoli are rich in acid proteins, but only globulins
are present. In the largest nucleoli the globulin reaction at the centers
is 2.0 and at the peripheries, 4.0. Smaller nucleoli give a homogeneous
reaction like the cortices of the larger ones. The absence of nucleo-
protein has been considered in the discussion of the nucleal reaction.
Unna’s method does not provide data on which a decision can be made
concerning the reason for the absence of the nucleal reaction in the
nucleus of growing oocytes.

THe FoLiicuLar EPpitHELIUM AND ZONA RADIATA

It is very striking that the concentrations of the substances recorded
in this paper shift conspicuously in the odcytes during the growth-period.
This might be a result of changing resources in the blood stream, or of
a selective action of the follicle and zona, or, in some cases, of synthetic
activity within the odcyte in which combination altered the quantity of
a substance free to react with the testing reagent.

HISTOCHEMISTRY OF OOCYTES 89

Since variations in height of epithelial cells have been taken in many
cases to indicate degree of metabolic activity, measurements of the
width of the follicle were made. At the same time the zona radiata

ie Zona Radiata
20

Follicular Epithelium /

0.1 02 03 04 05 06 07 08 09 10141 12 13 1.6
Diameter —

in mm. A, A; B, Ba Bs

Fic. 19. Variation in height of follicular epithelium and zona radiata of the

oocyte during growth-period. The height in microns is indicated on the vertical
axis.

was measured (Fig. 19). The smallest odcytes, from 10 to 50 microns
in diameter, do not have a continuous covering of follicle cells, al-
though some squamous cells may be associated with them (Fig. 1).

90 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

When the follicle is complete, in odcytes from 100 to 150 microns in
diameter, its cells do not exceed 1.5 microns in height. During this
period the cells of the follicle exercise no inhibitory effect upon the
entrance of substances into the oocyte. The quantity of potassium
and acid proteins is relatively very great.

Maximum growth and differentiation of the follicle occurs during
Period 2 A, and the height of the cells rises to 11 microns in 2 B,. The
changes in potassium and protein content have been noted. Obviously
chemical changes are correlated with the differentiation of the follicle
cells. During Period 2 A the large vesicles which are the earliest indi-
cation of the unique differentiation of the odcytes appear, first at the
periphery and then throughout the cytosome. One may assume that
no longer are the constituents of cytoplasm being delivered in the same
relative proportions. Certain substances are accumulating in excess of
others. That this is conditioned by the composition of the follicle cells
is a simple assumption suggested by the evidence at hand. When the
deposition of yolk is occurring rapidly and directly in Period 2 B the
follicle undergoes slight change. Some decrease in height is observed
as the oocyte reaches a maximum diameter, and nuclear regression is
apparent. The amount of albumins is lowered during this stage.

In contrast to the follicular epithelium, the zona radiata continues
to increase in thickness until the end of the growth-period. Some sub-
stances can be identified in its canals, but obviously nothing is stored in
it. The observations made during this investigation do not suggest that
the zona radiata functions in the selection of materials entering the
oocyte.

SUMMARY

The plasmal and nucleal tests and the histochemical methods for
detection of iron, potassium, and acid proteins indicate that changes in
quality, quantity, and localization of substances characterize the period
of differentiation in the odcyte of Fundulus. The oocyte at the end of
its growth-period is morphologically very different from the oogonium.
Its structural changes are found to be correlated with the metabolic
activities leading to food storage. Cytosomal differentiations are con-
spicuously chemical phenomena in egg-cells.

Die RAL URE Clip.

Bauer, H., 1932. Die Feulgensche Nuklealfarbung in ihrer Anwendung auf cy-
tologische Untersuchungen. Zeitschr. f. Zellf. u. mikr. Anat., 15: 225.

BrataszEwicz, K., 1929. Recherches sur la répartition des électrolytes dans le
protoplasme des cellules ovulaires. Protoplasma, 6: 1.

HISTOCHEMISTRY OF OOCYTES 91

BracHeT, J., 1929. Recherches sur le comportement de l’acide thymonucléinique
au cours de l’oogenése chez diverses espéces animales. Arch. Biol., 39:
677.

BracHeET, J., 1933. Recherches sur la synthése de l’acide thymonucléique pendant
le développement de l’oeuf d’oursin. Arch. Biol., 44: 519.

FEuLceEn, R., 1926. Die Nuklealfarbung. Abderhalden’s Handb. der biol. Arbeits-
methoden, 5 (2): 1055.

FEuLGEN, R., AND H. RossEenBEcK, 1924. Mikroskopisch-chemischer Nachweis
einer Nucleinsaure vom Typus der Thymonucleinsaure und die darauf
beruhende elektive Farbung von Zellkernen in mikroskopischen Praparaten.
Zettschr. f. physiol. Chem., 135: 203.

FreuLcen, R., anp K. Vorrt, 1924. Uber einen weitverbreiteten festen Aldehyd.
Pfiiiger’s Arch. f. d. ges. Physiol., 206: 389.

GoopaLe, H. D., 1919. Interstitial cells in the gonads of domestic fowl. Anat.
Rec., 16: 247.

Gresson, R. A. R., 1930. Certain phenomena of Tenthredinid oogenesis as re-
vealed mainly by Feulgen’s nuclear-reaction. Quart. Jour. Micr. Sct., 73:
617.

Guturiz, Mary J., 1925. Cytoplasmic inclusions in cross-activated eggs of teleosts.
Zeitschr. f. Zellf. u. mikr. Anat., 2: 347.

GuturizE, Mary J., 1928. Further observations on the ovarian eggs of Fundulus.
Anat. Rec., 41: 64.

Guturiz, Mary J., 1929. Cytoplasmic inclusions in the oocytes of vertebrates.
Anat. Rec., 44: 231.

Hammnarsten, O., 1905. Zur Chemie des Fischeies. Skand. Arch. f. Physiol.,
17: 113.

Hispard, Hore, 1928. Contribution a l’étude de l’ovogenése, de la fécondation,
et de l’histogenése chez Discoglossus pictus Otth. Arch. Biol., 38: 251.

Kocu, A., 1925. Morphologie des Eiwachstums der Chilopoden. Zeitschr. f.
Zellf. u. mikr. Anat., 2: 293.

Konic, J., AND J. GrossFetp, 1913. Der Fischrogen als Nahrungsmittel fur den
Menschen. Biochem. Zeitschr., 54: 351.

Konopacka, B., 1935. Recherches histochimiques sur le développement des pois-
sons. I. La vitellogénése chez le goujon (Gobio fluviatilis) et la carpe
(Cyprinus carpio). Bull. Acad. Polonaise Sci. et Let. (sér. B, II), p. 163.

Konopackt, M., anv B. Konopacxka, 1926. La micromorphologie du metabolisme
dans les périodes initiales du développement de la grenouille (Rana fusca).
Bull. Acad. Polonaise Sct. et Let. (sér. B.), p. 229.

LiesecANc, R. E., 1923. Nachweis geringer Eisen- und Kupfermengen in Leinen,
Papier oder tierischen Geweben. Zeitschr. f. wiss. Mikr. u. mikr. Tech.,
40: 14.

Linnert, K., 1909. Enthalt Kaviar (Stor- resp. Hauseneier) Purinbasen? Bio-
chem. Zeitschr., 18: 209.

Lison, L., 1932. Sur la spécificité du réactif de Schiff envers les aldéhydes. Bull.
d’ Histol. Appl., 9: 177.

Luprorp, R. J., 1928. Studies in the microchemistry of the cell. I. The chromatin
content of normal and malignant cells, as demonstrated by Feulgen’s
“nuclealreaktion.” Proc. Roy. Soc. London, B, 102: 397.

Macatium, A. B., 1905. On the distribution of potassium in animal and vegetable

cells. Jour. Physiol., 32: 95.

Macatium, A. B., 1912. Die Methoden der biologischen Mikrochemie. <Abder-

halden’s Handb. der biochem. Arbeitsmethoden, 5: 1099.

Marza, V. D., 1934. Les cellules interstitielles et les histiocytes de l’ovaire de

poule. Compt. Rend. Soc. Biol., 117: 1278.

Marza, V. D., 1935. The formation of the hen’s egg. Part V. Quart. Jour,
Micr. Sci., 78: 191.

92 V. D. MARZA, E. V. MARZA AND M. J. GUTHRIE

Marza, V. D., ann L. Cutosa, 1934. Modification de le méthode de Macallum
pour la détection histochimique du potassium. Compt. Rend. Soc. Biol.,
117: 524.

Marza, V. D., anp L. Cutosa, 1935. Essai de détection histochimique quantita-
tive du potassium. Bull. d’Histol. Appl., 12: 58.

Marza, V. D., anp L. T. Cutosa, 1936. Histochimie quantitative du potassium
dans les ovules en croissance. Bull. d’Histol. Appl., 13: 153.

Marza, V. D., anp M. Goraescu, 1935. Distribution des mastocytes et des cellules
de type plasmocytaire dans lovaire de poule. Compt. Rend. Soc. Biol.,
118: 1470.

Marza, V. D., AND EucENIE V. MaArza, 1934. Réaction plasmale et réaction
nucléale au cours de la vitellogénése (oeuf de poule). Bull. d’Histol.
A ppl., 11: 65.

Marza, V. D., anp Eucénig V. Marza, 1935. -The formation of the hen’s egg.
Parts I-IV. Quart. Jour. Micr. Sct., 78: 133.

Marza, V. D., Eucenr: Marza, anp L. Cuiosa, 1932. Etude histochimique du

fer dans l’ovaire de poule. Bull. d’Histol. Appl., 9: 213.

Mukerji, R. N., 1930. The “nucleal reaction” in Apanteles sp. with special

reference to the secondary nuclei and the germ-cell determinant of the

egg. Proc. Roy. Soc. London, B, 106: 131.

NEEDHAM, J., 1931. Chemical Embryology. The Macmillan Co., London.

Noniwez, J. F., 1921. Studies on the gonads of the fowl. I. Am. Jour. Anat.,
28: 81.

Pouicarp, A., 1924. La microincinération et son intéret dans les recherches histo-
chimiques. I. Technique. Bull. d’Histol. Appl., 1: 26.

SmiecHowskI, A., 1892. Die Bedeutung der Megaspharen in der Keimscheibe
des Htthnchens. Anat. Hefte, 2: 109.

Gunna, P. G., 1921. Chromolyse. Abderhalden’s Handb. der biol. Arbeitsmetho-
den, 5 (2): 1

VERNE, J., 1929. Etude histochimique des substances aldéhydiques formées au
cours du métabolisme des corps gras. Ann. Physiol., 5: 245.

Voss, H., 1927. Beobachtungen tiber das Vorkommen des Plasmalfarbung.
Zeitschr. f. mikr.-anat. Forsch., 10: 583.

Voss, H., 1929. Das Verhalten der Arterienwand zur Plasmalfarbung. Klin.
Wochenshr., 8: 887.

Watter, G., 1891. Zur Kenntniss des Ichthulins und seiner Spaltungsproducte.
Zeitschr. f. physiol. Chem., 15: 477.

Warsure, O., 1914. Uber die Rolle des Eisens in der Atmung des Seeigeleis nebst
Bemerkungen tiber einige durch Eisen beschleunigte Oxydationen. Zeit-
schr. f. physiol. Chem., 92: 231.

WASSERMANN, F., 1910. Uber den makro- und mikrochemischen Eisennachweis
im Dotter des Huhnereies. Anat. Hefte, 42: 281.

WeERMEL, E., 1927. Untersuchungen tiber die Kernsubstanzen und die Methoden
ihrer Darstellung. I. Mitteilung. Uber die Nuklealreaktion und die
chromolytische Analyse. Zeitschr. f. Zellf. uw. mikr. Anat., 5: 400.

THE DEVELOPMENT OF THE PITUITARY GLAND
IN FUNDULUS

SAMUEL A. MATTHEWS

(From the Department of Anatomy, University of Pennsylvania, and the M arine
Biological Laboratory, Woods Hole, Massachusetts)

In discussing the structure of the pituitary gland of teleosts Stendell
(1914) remarks that in no other group of vertebrates does the hypophy-
sis present such variations, for in nearly every species within this group
a different type of structure is encountered. While these can all be re-
duced to a single generalized plan it is not easy to determine accurately
what structures are comparable in the glands of different species. This
difficulty is obvious when the glands of Esox and of Fundulus are com-
pared. According to Stendell, in the pars glandularis of the pituitary
gland of Esox there is found a small “ Hauptlappen” (a term fre-
quently used as a synonym with “pars anterior”; see Tilney, 1936)
which is restricted to the anterior dorsal part of the organ, a pars in-
termedia forming the caudal or ventro-caudal third of the entire gland,
_and between the two is a region found only in the teleosts and in
Petromyzon, an “ Uebergangsteil.” In Fundulus, however, (Matthews,
1936) the pars glandularis presents only two distinct regions, a pars
intermedia bordering processes of the pars nervosa and forming the
posterior pole of the gland, and another part which comprises the major
portion of the gland. Does this represent the pars anterior or the
Uebergangsteil of other teleosts? A comparison of the cellular struc-
ture of this region with that of the pars anterior and the Uebergangsteil
of Esox is of little value in homologizing these parts, for in Esox both
the pars anterior and the Uebergangsteil are composed of basophilic cells
in general with acidophiles restricted to the neighborhood adjacent to
blood vessels, while in Fundulus the cells of this major part of the gland
are predominantly acidophilic. In its relative size, however, this region
in Fundulus compares more favorably with the Uebergangsteil, for it is
certainly the largest part of the gland (Fig. 5) and Stendell states defi-
nitely (p. 92) that the Uebergangsteil is decidedly more voluminous
than the principal lobe. Because of this, in describing the structure of
the adult gland, I labelled this part the “transitional region,” although
the appropriateness of the term was questioned. It was thought that a
study of the development of the hypophysis in this form might demon-

94 SAMUEL A. MATTHEWS

strate which of the parts found in the pituitary gland of other teleosts
was lacking in Fundulus. Accordingly a series of embryos and young
animals ranging in age from two to thirty-eight days after fertilization
were fixed, sectioned and studied.

The rudiment of the glandular portion of the hypophysis can first be
distinguished five days after fertilization as a small, flattened plate of
ectodermal cells lying in close contact with the ventral surface of the
forebrain in the region of the future infundibulum (Fig. 1). At this
stage of development the foregut is represented by thick cellular masses
in which cavities are beginning to appear. Unicellular gland cells de-
scribed by Armstrong (1936) are already present. In animals six days
old this flat plate of ectodermal cells has increased in size, and pushed
upward away from the ectoderm so that it now lies slantwise against the
posterior ventral angle of the infundibulum which is now quite definite.
This epithelial rudiment of the hypophysis is still connected with the
ectoderm by a well-defined stalk. These same relationships are present
in animals seven days old.

Fight days after fertilization rather pronounced changes in the de-
veloping brain can be noted, chief among which is the first appearance
of fibrous material in the ventral portion. The epithelial portion of the
hypophysis is larger in size and more posterior in position, lying be-
tween the posterior wall of the infundibulum and the solid mass of cells
which later becomes the foregut and is now heavily marked with the
deep-staining unicellular gland cells mentioned above. At nine days of
age the epithelial portion of the hypophysis is still further posterior in
position (Fig. 2) and the stalk which connects it with the ectodermal
region from which it first arose is somewhat longer. The small spaces
which formed the first lumen of the gut now form a definite gut cavity
but this does not communicate with the outside. At eleven days of age.
when the foregut has extended up to the anterior end of the embryo and
its lumen is closed off by only a thin membrane, the epithelial portion of
the hypophysis lies still more posterior in position although maintaining
its relationship to the infundibulum with which it is in close contact
dorsally. It is in contact with the entoderm of the gut ventrally and a
long, thin strand of ectoderm still connects it with the ectoderm at the
anterior end of the embryo. In some cases at this age a few fibers may
be found connecting this developing glandular portion of the hypophysis
with the infundibulum, although in most cases these fibers appear in
later development. Cell boundaries in the developing gland are still in-
distinct, as they have been throughout the course of development. Be-
tween the eleventh and twelfth days of age the strand of ectoderm which
has previously connected the epithelial portion of the hypophysis with

DEVELOPMENT OF PITUITARY GLAND 95

Fic. 1. Fundulus embryo 8 days old, sagittal section of anterior end. Camera
lucida outline, X 83. EN, entoderm; HY, hypophysis; JNC, infundibular cavity ;
OR, optic recess.

Fic. 2. Embryo nine days old, sagittal section of anterior end, X 85. AY,
hypophysis; INC, infundibular cavity; OR, optic recess; G, gut.

Fic. 3. Fundulus pituitary gland thirty-two days old, X 315. INC, infundibu-
lar cavity; P, pituitary gland.

Fic. 4. Fundulus pituitary gland approximately one year old, X 155. INC,
infundibular cavity; P, pituitary gland.

Fic. 5. Fundulus pituitary gland, adult animal, 60 mm. long, sagittal sec,
x 50. INC, infundibular cavity; PA, pars anterior; PJ, pars intermedia; PN,
pars nervosa.

96 SAMUEL A. MATTHEWS

the ectoderm at the anterior end of the embryo becomes interrupted and
can no longer be traced in embryos twelve days old. The floor of the
infundibulum dorsal to the developing pars glandularis has thinned con-
siderably.

From this stage on changes in the developing pituitary gland occur
more slowly. By the time the stomodeal membrane has broken through,
which occurs in this series between the twelfth and thirteenth days of
development, the epithelial part of the hypophysis has attained its adult
position in relation to the pharynx and to the brain. It now lies on the
ventral surface of the midbrain immediately beneath the ventral wall of
the infundibulum, and fibers from the infundibulum join it to the brain.
These fibers increase in number until, thirty-two days after fertilization,
a short but fairly definite infundibular stalk can be distinguished. These
fibers occupy the dorsal border and central portion of the hypophysis,
forming the rudiment of the pars nervosa, with the cells that will pro-
duce the pars glandularis restricted to the periphery of the organ (Fig.
3). Blood vessels invade the gland between the fifteenth and sixteenth
days after fertilization.

During this development of the pituitary gland in Fundulus no cavity
is to be found at any time. The process in this animal thus differs from
that described by Haller (1898), who observed a number of small spaces
which appeared in the developing hypophysis of Salmo, these spaces
eventually coalescing to produce an hypophysial cavity.

The cells of the developing glandular portion of the hypophysis are
closely packed, with indistinct cell membranes. Although they do be-
come larger as development proceeds, in the oldest of the animals reared
in the laboratory, 38 days of age, the cells are still closely packed around
the periphery of the organ and cell membranes are indistinct, with no
secretory cells present comparable to those found in the pituitary gland
of the adult. The smallest animals in which such cells have so far been
found were collected in July, maintained in the laboratory for two
months, and measured 26 millimeters when killed in September (Fig. 4).
In the pituitary gland of these animals the cells of the pars glandularis
are still located around the periphery but they are larger and have defi-
nite granules in their cytoplasm. The pars nervosa has also increased
in size and processes extend from it into the pars glandularis. Since
these animals were collected rather than raised in the laboratory their
exact ages are unknown, but in all probability they had been spawned
the previous summer and were approximately one year old. Obviously
secretory cells first appear sometimes between the sixth week and the
twelfth month of development. The exact time of their appearance and
their coincidence with other features of development with which begin-

DEVELOPMENT OF PITUITARY GLAND 97

ning activity of the pituitary gland might be concerned must be deter-
mined in a series of animals hatched and maintained in the laboratory
for longer periods.

Stendell (p. 109) emphasizes the fact that during the development
of the pituitary gland of teleosts no cavity is formed. The cells of the
solid ectodermal outgrowth proliferate rapidly at its anterior and poste-
rior poles to form the pars anterior and the pars intermedia respectively.
As a result of this polar proliferation of cells the region between the
anterior and posterior poles, which is less active in development, presents
characteristics transitional in nature between the pars anterior and pars
intermedia, and constitutes the Uebergangsteil. But here in Fundulus
apparently not only cells at the anterior and posterior poles but all the
peripheral lying cells multiply to produce the pars glandularis. From a
comparison of the pituitary gland of year-old animals (Fig. 4) with the
developing gland in animals 32 days of age on the one hand (Fig. 3) and
with that of adult animals on the other (60 mm. in length, Fig. 5) it is
apparent that the main mass of the pars glandularis is produced by
multiplication of the peripheral cells found in the early developmental
stages. Later those cells adjacent to the pars nervosa may be distin-
guished from the others, particularly at the posterior pole of the gland
where they forma large mass. This is undoubtedly the pars intermedia.
The remainder of the pars glandularis is of uniform character. Nei-
ther in its development nor in its adult structure can it be called a transi-
tional region between two other parts of the gland. Hence there exists
no apparent reason for labelling this major part of the gland an Ueber-
gangsteil. Because of this I now believe that the choice of the term
“transitional region” for this major part of the gland of the adult is
an unfortunate and confusing one. As a matter of fact, the anterior
portion of the gland of Esox as described by Stendell differs so much
from that part of the gland in Pundulus that on the basis of structure
alone it is uncertain what parts are comparable in this region of the two
glands. Since in Fundulus this part in question forms the anterior
portion of the organ it may appropriately be called the pars anterior,
although it is doubtful whether it is homologous with Stendell’s “ Haupt-
lappen.”

SUMMARY

The epithelial portion of the pituitary gland of Fundulus arises from
ectodermal cells that proliferate to form a solid mass which comes to lie
between the infundibulum and the pharynx. No lumen occurs in this
mass of cells. After fibers from the infundibulum have entered this
mass the cells are restricted to the periphery where they multiply to

98 SAMUEL A. MATTHEWS

produce the pars glandularis of the adult gland. Those cells adjacent to
the pars nervosa can be distinguished from the rest of the glandular
portion and constitute the pars intermedia. The remainder of the pars
glandularis is of uniform character. No “transitional region” can be
distinguished in it either in the developing or in the adult gland. Since
this portion forms the anterior part of the organ it may appropriately
be called the pars anterior.

LITERATURE CITED

Armstronc, P. B., 1936. Mechanism of hatching in Fundulus heteroclitus. Biol.
Bull., 71: 407 (Abstract).

Hatter, B., 1898. Untersuchungen tiber die Hypophyse und die Infundibularor-
gane. Morph. Jahrb., 25: 31.

Martruews, S. A., 1936. The pituitary gland of Fundulus. Anat. Rec., 65: 357.

STENDELL, W., 1914. Die Hypophysis Cerebri. In Oppel’s Lehrbuch der vergl.
mikros. Anat., Teil 8.

TitneEy, F., 1936. The development and constituents of the human hypophysis.
Bull. Neurol. Inst. of N. Y., 5: 387.

iiteSUrERpoolON OF CLEAVAGE IN ASCARIS EGGS
BYE MRACENTRIEUGING*

Ee Ve DEAS ANID Ro KING

(From the Zoological Laboratory, State University of Iowa)

It has long been known that the division of the cytoplasm of the liv-
ing egg can be stopped by many external agencies (cold, lipoid solvents,
hypertonic and hypotonic solutions, mechanical agitation, etc.) without
necessarily inhibiting the division of the nucleus (Wilson, 1902; Cham-
bers, 1924 for references). Furthermore, it is also true that cyto-
plasmic cleavage may take place in the absence of nuclear material
(Harvey, 1936). Accordingly, it is evident that, although nuclear and
cytoplasmic divisions are usually correlated they may, nevertheless, be
quite independent processes, neither of which is fundamentally de-
pendent upon the other.

In some recent experiments (King and Beams, 1937) on the effects
of strong centrifugal force upon the early cleavage stages of Ascaris
eggs, particularly the diminution process, it was noted that many eggs
undergo typical nuclear division in the ultracentrifuge without the ac-
companying division of the cytoplasm. We are reporting here these
experiments together with an analysis of the results in the event that
they may have some bearing upon the problems involved in the me-
chanics of mitosis.

MATERIAL AND METHODS

The eggs used in these experiments were taken from the anterior
one-half inch of the uteri of Ascaris equorum (= megalocephala)
variety univalens,? which had been kept in a refrigerator at 5° C. for
not longer than thirty days. Microscopic examination of the eggs
taken from this portion of uteri showed them to be in the pronuclear
stage of development.

Some of the eggs from each batch were placed in the rotor of the air-
driven ultracentrifuge (Beams, Weed, and Pickels, 1933) while in the
pronuclear stage, and were centrifuged in 0.9 per cent salt solution at

1 Aided by grant from the Rockefeller Foundation for work on Cellular
ae is indeed a pleasure to express here our indebtedness to the authorities of

the Hill Packing Company at Topeka, Kansas, for kindly supplying us with the
worms.

100 H. W. BEAMS AND R. L. KING

approximately 150,000 times gravity for varying lengths of time, the
maximum being 10 days. In addition, observations were made upon
the eggs while centrifuging by means of the centrifuge-microscope re-
cently described by Pickels (1936). In other experiments the eggs
were placed under the same conditions in an electric-driven centrifuge,
which developed a force of approximately 5,000 times gravity. Part of
the eggs from each batch used in an experiment were’always kept as
controls. All the experiments were done at room temperature which
varied from 20° to 25° C.

Sealed cover-glass preparations of the normal and experimental liv-
ing eggs were studied under the oil immersion lens. It was found that
centrifuged eggs placed in 10 per cent formalin solution at 55° C. were
coagulated so that the various conditions of stratification are preserved
and may be studied at leisure. When permanent slide preparations
were made, the eggs were killed in a mixture of 4 parts of 95 per cent
alcohol and 1 part of glacial acetic acid and subsequently stained in
Heidenhain’s hematoxylin. In an effort to analyze the components of
the various stratified layers a few eggs were killed in Champy’s solu-
tion at 55° C., and afterwards impregnated in 2 per cent osmic acid for
5 days. By use of antiformin it is possible to remove the shell from
living eggs without apparent injury to them.

DESCRIPTION

After sperm entrance and the completion of maturation in Ascaris,
the eggs shrink and round up giving rise to a rather large perivitelline
space which is filled by a watery fluid and enclosed by a very resistant
shell (Plate I, Fig. 1). The odplasm of the egg at this time is hetero-
geneous in structure, being composed of small brown granules, granular
and short rod-like mitochondria (?), clear spherical vacuoles, oil
globules and an optically homogeneous cytoplasm. ‘The pronuclei are
transparent, rounded bodies located in or near the center of the egg.
Surrounding the egg is a delicate plasma membrane, the morphology of
which is not discernible even under the oil immersion lens.

Upon centrifuging at 150,000 times gravity the eggs are stratified
almost immediately into the following layers: (1) oil globules at the
centripetal pole, (2) a zone of clear vacuoles, (3) transparent cyto-
plasm, and (4) brown granules at the centrifugal pole (Plate I, Fig. 2).
The nuclei and also the mitotic spindle in dividing eggs are included in
the clear cytoplasmic layer. The mitotic spindle is oriented at right
angles to the centrifuge axis and parallel to the stratified layers. Unlike
the condition in embryonic chick cells, the elements of the mitotic spindle

ULTRACENTRIFUGING ASCARIS EGGS 101

are not distorted. This indicates that their relative specific gravity is
approximately the same as that of the cytoplasmic layer. When viewed
through the microscope-centrifuge, the eggs were observed to be some-
what elongated along the centrifuge axis, so that the centrifugal and
centripetal ends of the eggs are in direct contact with the shell, which
no doubt aids in preventing their fragmentation. Eggs in the pronuclear
stage may, in rare cases, be divided into three parts within the shell as
follows: fat at the centripetal pole, protoplasm and clear vacuoles in the
middle and brown granules at the centrifugal pole (Plate I, Fig. 3). In
other more frequent cases only the oil globules are completely rounded
off from the rest of the egg at the centripetal pole, and flattened in the
form of a disc (Plate I, Figs. 6, 7, 8,9,11). Eggs of this kind, if re-
moved from the centrifuge before cleaving, frequently develop into
normal larve, with the flattened disc of oil globules within the perivitel-
line space. However, if the eggs are removed from the centrifuge be-
fore the oil globules are completely cut off they become reincorporated
into the egg through a narrow stalk, and eventually are redistributed as
are the other layers of the egg. In Plate I, Fig. 4, is illustrated a
centrifuged egg with conjugating pronuclei that had been killed in
Champy’s solution and subsequently impregnated in 2 per cent osmic
acid. It will be noted that a dark layer, which is thought to contain the

mitochondria, appears just centripetal to the brown granules. This

layer of small granules can also be seen in the living egg under suitable
conditions. It had previously been thought that the brown granules
represent the mitochondria in Ascaris eggs (Fauré-Fremiet, 1913).
The dark granular layer just centrifugal to the fat globules may possibly
represent the Golgi material.

If the eggs are left in the centrifuge until the controls have under-
gone cleavage a typical mitotic apparatus (centrosomes, asters, spindle
and chromosomes) is formed (Plate I, Fig. 5), giving rise to two nuclei
within the cell because of the failure of the cytoplasm to cleave. (The
cytology of the mitotic figure and the chromosome behavior in such eggs
is to be reported elsewhere—King and Beams, 1938.) These two nuclei,
in turn, may form typical division figures simultaneously (Plate I, Fig.
6), giving rise to four nuclei within the uncleaved egg (Plate I, Fig. 8) ;
the four nuclei may divide in the same manner (Plate I, Fig. 11), each
division corresponding to a cleavage in the controls. In fact, the first
two nuclear divisions often take place at about the same rate as the first:
two cleavages of the controls. However, the subsequent cleavages often
become more or less irregular and out of step with the controls.

If the eggs are removed from the centrifuge after one, two or more
divisions of the nuclear material only, a curious surface phenomenon is

102 H. W. BEAMS AND R. L. KING

noted in the region of the clear cytoplasmic layer. Immediate examina-
tion of the eggs upon removal from the centrifuge often reveals many
small and a few quite large pseudopodium-like processes flowing out of
the egg; these are in many cases completely cut off (Plate I, Fig. 7).
This material which has been stratified is thought to be or to give rise to
the “ surface active material’ which breaks out and precipitates, form-
ing a film or plasma membrane. The presence of such a material in the
egg is assumed to be necessary in the regions where the cleavage fur-

PLATE I
EXPLANATION OF FIGURES

All the figures were drawn from living eggs by Mary Crosten. The amount
of the centrifugal force has been in all cases 150,000 times gravity unless other-
wise indicated. The direction of the centrifugal force in all cases except Fig. 1
was approximately toward the bottom of the plate.

Fic. 1. Pronuclear stage of a normal Ascaris egg showing the general struc-
ture of the cytoplasm and the position of the pronuclei.

Fic. 2. Egg as in Fig. 1 after centrifuging for one-half hour. Materials
stratified with fat at the centripetal pole, then a layer of clear vacuoles, next a layer
of clear cytoplasm containing the pronuclei, and finally, a layer of brown granules
at the centrifugal pole.

Fic. 3. Unusual condition of egg fragmented into three parts: fat and clear
vacuoles in the centripetal fragment, cytoplasm and pronuclei in the middle frag-
ment and the brown granules in the centrifugal fragment. Centrifuged two hours.

Fic. 4. Egg fixed in Champy’s solution immediately upon removal from the
centrifuge, and subsequently impregnated in 2 per cent osmic acid. Stratification
clearly shown. The dark layer between the nucleus and the brown granules is
thought to represent the mitochondria. The darkly impregnated layer just cen-
trifugal to the fat globules may possibly represent the Golgi material.

Fic. 5. Egg undergoing division of the nucleus without division of the cyto-
plasm. The asters are faintly shown.

Fic. 6. Prophase stage of the two nuclei which correspond to those in the
second cleavage of the controls. Small masses of “ surface active material’ shown
budded off from the egg.

Fic. 7. Drawing of egg removed from centrifuge after the controls had un-
dergone one division. The formation of the pseudopodia in the region of the
cytoplasmic layer is shown.

Fics. 8-9. Eggs that have undergone two mitotic divisions of the nuclear
material without division of. the cytoplasm in the centrifuge. Large fragments
are shown which were actively undergoing ameboid movement. The oil globules
are shown fragmented from the egg at the centripetal pole. Drawings made after
a partial redistribution of the stratified layers had taken place.

Fic. 10. Egg removed from centrifuge at the time the controls had under-
gone the first cleavage. Many pseudopodium-like processes shown at the pe-
riphery of the egg, none of which in this particular case was fragmented off.
Drawing made after partial redistribution of the stratified layers.

Fic. 11. Egg that had passed through three mitotic divisions of the nuclear
material in the centrifuge. One nucleus is seen cleaved off into a small cell of
clear cytoplasm. A redistribution of the brown granules in the egg has taken
place. Fat globules are shown at the centripetal pole.

Fic. 12. “Ball” egg that divided in centrifuge at 5,000 times gravity. Most
of the brown granules are cleaved off during the first division. The stratification
of the various materials is clearly shown.

ULTRACENTRIFUGING ASCARIS EGGS 103

Leys ane Il

104 H. W. BEAMS AND R. L. KING

rows are destined to form. The fragments show very striking and
rapid ameboid movement within the perivitelline space. In text fig. 1
(a-l) are illustrated diagrammatically the consecutive movements of a
typical fragment like that shown in Fig. 8 during an interval of 5
minutes. Such active movement has been noted to continue for four or
five hours, after which time the fragments may divide into many smaller
ones. We have not observed that these bodies subsequently fuse with
the egg.

In other less frequent cases, where the eggs were treated in a similar
way, many small pseudopodium-like processes are formed at the surface

Text Fic. 1, a-l, diagrams showing the ameboid movement of a typical frag- |
ment like that shown in Plate I, Fig. 8, during an interval of 5 minutes.

of the clear and granular layers but they do not become separated off
(Plate I, Fig. 10). Such pseudopodia upon division of the egg outside
of the centrifuge are reincorporated with the blastomeres. It is an in-
teresting fact that frequently one observes eggs (such as those shown in
Plate I, Fig. 11), where one or more small cells with clear cytoplasm
have been cleaved off in the centrifuge. This condition probably occurs
when one of the mitotic spindles is sufficiently close to the periphery of
the egg in the presence of “ surface active material.”

Those eggs that have undergone one or two divisions of the nuclei
without division of the cytoplasm in the centrifuge will, upon removal

ULTRACENTRIFUGING ASCARIS EGGS 105

from the centrifuge, frequently divide into twice as many cells as there
were nuclei present. This division, however, takes place only after
partial or complete redistribution of the stratified materials.

Ascaris eggs like those of the sea-urchin (Harvey, 1933) will di-
vide in a gravitational field of 5,000 times gravity. In most of the eggs
the cleavage is complete. However, in a few it is incomplete, as it does
not pass through, but around the brown granular layer at the centrifugal
pole, giving rise to what Boveri (1910) and Hogue (1910) have called
“ball” eggs (Plate I, Fig. 12). The fact that these eggs undergo cyto-
plasmic cleavage may be explained by assuming that the force used, al-
though sufficient to produce stratification of the visible materials, has
not been great enough to displace and stratify the “surface active ma-
terial.’ This assumption is supported by the fact that eggs centrifuged
at 5,000 times gravity while dividing do not usually form pseudopodia
as do those centrifuged at higher speeds.

No attempt has been made here to determine the absolute viscosity
of the protoplasm of Ascaris eggs. However, the relative viscosity has
been determined at room temperature by the centrifuge method, and by
observations upon Brownian movement in the eggs at various stages
in the mitotic cycle. Fauré-Fremiet (1913) has also used the centrifuge
method to study the viscosity of Ascaris eggs at different temperatures.
In our experiments the eggs, which were in different stages of division,
were placed in the electric machine and centrifuged at 5,000 times
gravity for 20 minutes. The relative position or layering out of the
brown granules was used as an index of the relative viscosity of the
eggs, in the pronuclear stage, prophase, metaphase, anaphase and telo-
phase. Brownian movement was followed in eggs that were under-
going division, as was the redistribution rate of the brown granules
after they had been displaced by the centrifuge.

The evidence from these observations indicates that the egg is rela-
tively fluid during the pronuclear and prophase stages. During meta-
phase the egg becomes progressively more rigid and: Brownian move-
ment usually ceases except perhaps at the periphery. During anaphase
and telophase the eggs show the highest degree of viscosity. At the end
of telophase when the asters begin to recede, and the reorganization of
the nuclei takes place, the viscosity gradually becomes low again.

It should be pointed out that, during the prophase, when the amphi-
aster is forming, a gelation or relatively high viscosity is present in the
region of the immature or small aster. It seems probable that the vis-
cosity is as high in this region as it will ever be, and that the following
stages, which show a higher viscosity for the egg as a whole, are due
primarily to a spreading of this gelation from the region of the small

106 H. W. BEAMS AND R. L. KING

aster toward the periphery of the egg, a condition associated with the
development and growth of the asters. (For a recent review of the
literature on viscosity studies see Fry and Parks, 1934.)

DISCUSSION

Cell division under normal conditions appears to be accomplished by
a series of complex integrated phenomena, involving both the nucleus
and cytoplasm; this becomes manifest, in part at least, through changes
in viscosity, appearance of the mitotic figure, and cleavage furrow.
These morphological changes in the dividing egg are, no doubt, the ex-
pression of what might be termed a “ mitotic field of force,’ by which
a part of the work necessary for cell division is accomplished. It is,
however, beyond the scope of this paper to discuss the whole mechanism
of cell division, but a few remarks upon the process of cytokinesis in
the light of our results seem desirable.

It should be pointed out at the beginning that the failure of the
Ascaris eggs to cleave in the ultracentrifuge can not be attributed to any
marked change in the morphology or position of the mitotic figure, as its
form is exactly comparable to that of the normal egg, and its position
in the cytoplasmic layer is the same as that in eggs which undergo cleay-
age in the centrifuge at 5,000 times gravity. Furthermore, contrary to
the views expressed by some workers (Chambers, 1924; Harris, 1935),
no evidence was observed which would indicate that the strong centri-
fugal force used in our experiments did in any way affect the normal
viscosity cycle during nuclear division. In this respect our results differ
from those of most other investigators who have inhibited cell division
by external agencies (cold, ether, hypertonic and hypotonic solutions,
mechanical agitation, etc.), in that these methods affect the viscosity of
the cell, resulting in a partial or complete disintegration of the asters,
which, in turn, inhibits cell division. In other words, “cells which
possess asters under normal conditions absolutely depend upon the pres-
ence of asters for cytoplasmic division’ (Chambers, 1924). It is evi-
dent, therefore, that the inhibition of cytoplasmic cleavage in the Ascaris
egg by the ultracentrifuge can not be explained upon the same basis as
the cases cited above.

It is a well established fact (Erlanger, 1897; Conklin, 1917; Speck,
1918; Strangeways, 1922; Chambers, 1924; Gray, 1931; Harris, 1935,
and others) that in many dividing cells, particularly the nematode egg,
there are important peripheral changes which take place in the cyto-
plasm, which seem to be associated with the development of the amphi-
aster, and which have a distinct bearing upon cell division (see Speck,

;
¢
2
;
t
7
i
‘
‘
4

ULTRACENTRIFUGING ASCARIS EGGS 107

1918 and Chambers, 1924 for literature). In general, it may be said
from the results of the above-named investigators, that when the asters
develop to the point of almost reaching the periphery of the egg, a de-
crease in peripheral viscosity becomes noticeable. In this relatively
fluid periphery it is possible to see a flow, initiated by a change in sur-
face tension, which, according to all the investigators who have de-
scribed it, is directed toward the equatorial region of the cell. It is not
possible in the Ascaris egg to observe a flowing movement in the pe-
ripheral region during division, but it is significant that here, particularly
at the equator, cessation of Brownian movement is generally last ob-
served during metaphase. Furthermore, in a few instances, it was
found that, during the normal mitosis of Ascaris eggs, a distinct “ bub-
bling ” or protrusion of a relatively clear material in the form of small.
pseudopodia was observed, especially at the equatorial region. Kautzsch
(1912), and Painter (1915) have likewise described marked conditions
of this kind in Ascaris eggs in which, apparently, the normal division
had been delayed. This activity or mobility at the surface of dividing
cells has frequently been observed, particularly in tissue culture cells
(Strangeways, 1922).

It seems not unreasonable to suppose, therefore, that the relatively
fluid peripheral material is formed during the gelation of the amphiaster,
or at the solution of the nuclear membrane, and then is forced to take
up a position at the periphery of the egg, where it seems to function in
the formation of the cleavage furrow.

The fact that the marked formation of extraovates takes place in the
region of the cytoplasmic layer of eggs, that have undergone one or
more typical nuclear divisions in the centrifuge although cleavage has
been suppressed, may be explained by the assumption that the peripheral
surface active, membrane-forming material is stratified in this region.
Our observations indicate that at each division of the nucleus more of
the “surface active material” is formed, and, if in the centrifuge, is
stratified so that when the centrifugal force is released, some of this ma-
terial, which changes surface tension, is extruded, and frequently is
completely separated from the egg. Ameboid activity ensues until all
the surface energy is dissipated. If this interpretation is correct, and,
if the surface active material is essential for film or membrane forma-
tion, as there is good evidence for believing (Heilbrunn, 1928), then one
need only assume that the inhibition of cleavage of the Ascaris eggs in
our experiments is due to a concentration of the material into a layer
parallel to the axis of the mitotic spindle, thus depleting the egg of the
membrane-forming material in the regions where the cleavage furrows
would normally form. This interpretation, we believe, is in harmony

108 H. W. BEAMS AND R. L. KING

with the highly significant work of Heilbrunn (1928) on the “ surface
active material” of Arbacia eggs. He found, after stratifying of the
visible materials in the eggs by means of the centrifuge, and subsequent
crushing, that a surface film was formed only at the centrifugal pole,
the position taken up by the pigment granules. Heilbrunn, therefore,
concluded that the “ surface active material ” had been displaced in the
centrifuge, and that it was the pigment granules or material stratified in
the same region which is or gives rise to the “ surface active material.”

There are, no doubt, some who will interpret the failure of the
cytoplasm to cleave in the centrifuge as simply mechanical, because of
the intense stratification or “ packing” of the elements in the stratified
layers. This condition of stratification may possibly be a factor; but
it has been shown by Boveri (1910), Hogue (1910), and in this paper,
that cleavage will take place in the centrifuge when the visible materials
are clearly stratified. It is only when the centrifugal force has been
very great that division fails to occur. Why the cytoplasm fails to
cleave in the region of the dividing mitotic figure is not known, but one
might assume that the “surface active material” is stratified into a
layer which does not include the mitotic spindle; or perhaps because of
its stratification, the “surface active material” has become temporarily
inactivated.

It should be pointed out that in certain biological material, the for-
mation of a plasmodium or syncytium is the normal condition. For
example, in the developing insect egg, the first divisions involve the
nucleus only; the cleavage of the cytoplasm takes place later after the
nuclei have migrated to the surface of the egg. No satisfactory ex-
planation for this condition has ever been suggested, but might it not
be because of the lack of a sufficient quantity of some material essential
for cytoplasmic cleavage, such as, for instance, the surface active, mem-
brane-forming material? Or could it be that the materials essential
for cytoplasmic cleavage can not function properly in the interior of
the egg?

In a preliminary report (Beams and King, 1936), it was shown that
eges of Ascaris suum were not killed by centrifuging at 400,000 times
gravity for 30 minutes; the same condition holds true for eggs of
Ascaris equorum. These facts are interesting in the light of the recent
work of Svedberg (1934) on strongly centrifuging non-living colloidal
systems. He found that certain of the large molecules may be thrown
out of solution at 75,000 to 200,000 times gravity. Tobacco virus pro-
teins may be likewise thrown out of solution and crystallized at 200,000
times gravity (Stanley and Wyckoff, 1937), and recently studies have
indicated that it might be possible to separate isotopes by the ultracen-

ULTRACENTRIFUGING ASCARIS EGGS 109

trifuge method (Beams and Haynes, 1936). One might assume, there-
fore, from these facts, that a similar separation of the light and heavy
molecules might take place in protoplasm at such strong forces. In
fact, Moore (1935) has recently concluded that the living elements of
a plasmodium consist of heavy and light components, which, when cen-
trifuged at 75,000 times gravity for 5 minutes, are separated, and pro-
liferation stopped. However, if allowed to rest for sufficient time, the
separated elements return to their normal spatial. relationship and pro-
liferation is resumed. Our work shows definitely that Ascaris eggs
will live and undergo nuclear division in the centrifuge at 150,000 times
gravity for at least ten days. Because of this fact we do not believe
that any marked separation of protoplasmic structure in the manner
suggested by Moore has taken place. On the other hand, it seems more
likely that forces, perhaps electro-static in nature, are sufficiently great
in Ascaris protoplasm to prevent a disruption of its architecture by such
strong centrifugal forces, or, less likely, perhaps the living units of the
protoplasm may all be of the same specific gravity.

CONCLUSIONS

1. Eggs centrifuged at 5,000 times gravity are stratified into the
following layers: (1) fat at the centripetal pole; (2) clear vacuoles;
(3) clear cytoplasm containing the pronuclei or the mitotic spindle; the
latter usually oriented with its long axis at right angles to the centrifugal
force and parallel to the layers; and (4) brown granules at the centrifu-
gal pole. When centrifuged at 150,000 or 400,000 times gravity and
subsequently fixed in osmic acid solution, two additional layers are
noted: one just centripetal to the brown granules, the other just cen-
trifugal to the fat layer; the former is believed to represent the mito-
chondria; the other may possibly represent the Golgi material. At
these high speeds the brown granules rarely become fragmented from
the egg. However, the oil globules are frequently cut off at the cen-
tripetal pole.

2. Eggs in the pronuclear or early prophase stages will, upon being
placed at 5,000 times gravity, undergo the early cleavages at about the
same rate as controls. In a low percentage of eggs the brown granules
are cut off at the first cleavage, which usually takes place at right angles
to the stratified layers.

3. Eggs in the pronuclear or early prophase stages that are cen-
trifuged at 150,000 times gravity undergo typical mitotic division of the
nucleus without the usual division of the cytoplasm. This gives rise to
two nuclei within the egg, which may in turn form typical mitotic

110 H. W. BEAMS AND R. L. KING

figures and divide at the same time, giving rise to four nuclei within the
uncleaved egg. The first two or three divisions of the nuclear material
often correspond in tempo to the first two or three cleavages of the
controls.

4. Eggs that have undergone one or more divisions of the nucleus
without the division of the cytoplasm show, upon immediate examination
after removal from the centrifuge, a curious peripheral “ bubbling,”
or the extrusion of pseudopodium-like processes in the region of the
clear cytoplasmic layer. These pseudopodia may be cut off from the
egg, in which case they undergo very rapid ameboid movement. If not
detached they are withdrawn into the egg during the redistribution of
the stratified materials and subsequent cleavage.

5. The material, which is extruded in the form of pseudopodia, is
thought to represent “ surface active material’; a material which upon
coming in contact with the surface or outside environment of the egg
coagulates, giving rise to a film or plasma membrane. It is this same
material which during normal cleavage concentrates at the periphery of
the egg and functions in the formation of the cleavage furrow.

It seems probable that the increase or growth of the “ surface active
material” is not markedly inhibited by high speed centrifuging, so that
at each division of the nucleus more of it is formed, and becomes strati-
fied. Thus, upon cessation of the centrifuging, part of this material is
extruded, a membrane is formed upon coming in contact with a foreign
environment and active ameboid movement is initiated.

6. Uncleaved eggs centrifuged at 400,000 times gravity for 30
minutes are not killed, and, if the stratified materials are redistributed
before cleavage takes place, they apparently develop normally.

7. No evidence was found which indicates a separation of the ultra-
microscopic cytoplasmic components had taken place.

LITERATURE CITED

Beams, J. W., A. J. Weep anv E. G. Pickers, 1933. The ultracentrifuge. Sci-
ence, 78: 338.

Beams, J. W., anp F. B. Haynes, 1936. The separation of isotopes by centri-
fuging. Phys. Rev., 50: 491.

Beams, H. W., anv R. L. Kina, 1936. The effect of ultracentrifuging upon chick
embryonic cells, with special reference to the “resting” nucleus and the
mitotic spindle. Biol. Bull., 71: 188.

Beams, H. W., ann R. L. Kine, 1936. Survival of Ascaris eggs after centrifug-
ing. Science, 84: 138.

Bonric, R., 1925. Die’ Determination der Hauptrichtungen des Embryos von
Ascaris megalocephala. Zeitschr. wiss. Zool., 124: 407.

Boveri, Tu., 1910. Uber die Teilung centrifugierter Eier von Ascaris megalo-
cephala. Arch. Entw.-mech., 30: 101.

ULTRACENTRIFUGING ASCARIS EGGS 111

Cuampers, R., 1924. The physical structure of protoplasm as determined by
microdissection and injection. Section 5, General Cytology (edited by
E. V. Cowdry).

Conx.in, E. G., 1917. Effects of centrifugal force on the structure and develop-
ment of the eggs of Crepidula. Jour. Exper. Zool., 22: 311.

Eriancer, R. V., 1897. Beobachtungen tiber die Befruchtung und ersten zwei
Teilungen an den lebenden Eiern kleiner Nematoden. Biol. Centralbl.,
17 152) 339:

Faur&-Fremiet, E., 1913. Le cycle germinatif chez L’Ascaris megalocephala.
Arch. d’Anat. Micros., 15: 435.

Fry, H. J., anp M. E. Parks, 1934. Studies of the mitotic figure etc. Proto-
plasma, 21: 473.

Gray, J., 1931. A Textbook of Experimental Cytology. New York.

Harris, J. E., 1935. Studies on living protoplasm. I. Streaming movements in
the protoplasm of the egg of Sabellaria alveolata (L). Brit. Jour. Exper.
Biol., 12: 65. .

Harvey, E. B., 1933. Effects of centrifugal force on fertilized eggs of Arbacia
punctulata as observed with the centrifuge-microscope. Biol. Bull., 65:
389.

Harvey, E. B., 1936. Parthenogenetic merogony or cleavage without nuclei in
Arbacia punctulata. Biol. Bull., 71: 101.

Hermsrunn, L. V., 1928. The Colloid Chemistry of Protoplasm. Berlin.

Hocus, M. J., 1910. Uber die Wirkung der Centrifugalkraft auf die Eier von
Ascaris megalocephala. Arch. Entw.-mech., 29: 109.

Kautzscu, G., 1912. Studien tber Entwicklungsanomalien bei Ascaris. Arch.
Entw.-Mech., 35: 642.

Kine, R. L., anp H. W. Beams, 1937. Effect of ultracentrifuging on the egg of
Ascaris megalocephala. Nature, 139: 369.

Kine, R. L., anp H. W. Beams, 1938. An experimental study of chromatin
diminution in Ascaris. (In press.)

Moors, A. R., 1935. On the significance of cytoplasmic structure in plasmodium.
Jour. Cell. Comp. Physiol., 7: 113.

Painter, T. S., 1915. The effect of carbon dioxide on the eggs of Ascaris.
Jour. Exper. Zool., 19: 355.

PicxeEts, E. G., 1936. Optical designs for observing objects in centrifugal fields
of force. Science, 83: 471.

Speck, J., 1918. Oberflachensponnungsdifferenzen als eine Ursache der Zellteilung.
Arch. Entw.-mech., 44: 5.

Srantey, W. M., ann R. W. G. Wycxorr, 1937. The isolation of tobacco ring
spot and other virus proteins by ultracentrifugation. Science, 85: 181.

STRANGEWAYS, T. S. P., 1922. Observations on the changes seen in living cells
during growth and division. Proc. Roy. Soc., Ser. B, 94: 137.

SvEDBERG, THE., 1934. Molecular weight analysis in centrifugal fields. Science,
79: 327.

Witson, E. B., 1902. Experimental studies in cytology. Arch. Entw.-mech., 13:
353.

A STUDY OF THE BACTERIAL AND ALLEGED MITO-
CHONDRIAL CONTENT OF THE CELES OF
THE CLOVER NODULE

E. DEWITT MILLER
(From the Laboratory of Histology and Embryology, University of Virginia)

INTRODUCTION

In a paper recently published the author expressed the view that cer-
tain granular and rod-like cytoplasmic inclusions occurring in Amaba
proteus and Arcella vulyaris belonged in the category of bacteria, prob-
ably symbiotic or commensal, while still other similar inclusions ap-
peared to be only temporary bacterial invaders of the cytoplasm. Cer-
tain of these granular and rod-like inclusions had, in the past, been de-
scribed as mitochondria. In the course of this investigation the initial
intention was to employ the clover nodule as material for making a
comparative study between certain cell inclusions, alleged mitochondria, |
as they appeared in the nodule, and similar inclusions occurring in
Ameba and Arcella. From available literature the clover nodule prom-
ised to be very valuable material for making such a comparative study.
Certain applied tests, however, induced me to discard that material for
the time, with the hope of being able later to publish my findings
separately.

The phenomenon involving bacteria and alleged mitochondria as it
occurs in the nodule of the clover is quite puzzling. The problem of
making a clear distinction between the two by present-day methods of.
technique has previously been recognized by investigators, including
Buchner (1921).

Techniques, similar to that employed in arriving at the above-men-
tioned conclusions regarding Amaba and Arcella, have likewise been
employed here in an intensive study of the contents of the clover nodule.
Since this material can easily be secured throughout the year and since
the nodules contain bacterial forms, including both rods and granules,
varying in size from minute forms which approach the limit of micro-
scopic vision to forms of relatively large size, it becomes quite evident
that such material offers an excellent opportunity for the study of
symbiotic relationship, as well as for making a comparative study of
different cell constituents.

112

BACTERIA AND MITOCHONDRIA; CLOVER NODULE 113

Mitochondria have been described in the nodule of clover in associa-
tion with the different pleomorphic forms of recognized bacteria.
Cowdry (1923) has presented characteristics which he considers serve
to distinguish between small bacterial forms and mitochondria, as they
occur in the nodule of the white clover. Differentiation between bac-
teria and alleged mitochondria has been made largely on the grounds of
staining reactions and responses to well-known lipoid solvents. It is
the purpose of this investigation to show that size, staining ability, re-
action to Janus green, and lipoid solvents are not sufficient to distinguish
bacterial forms and apparent mitochondria as they occur in the clover
nodule.

From the following observations upon many nodules, treated with
different methods of technique, smears, cultures on artificial media of
bacteria from both the nodule and from the soil, I am convinced that an
important bacterial type, or possibly types, as well as their general stain-
ing qualities, have been overlooked.

Wallin (1922) is of the opinion that a minute type of bacteria, not
unlike mitochondria in appearance, exists in the nodule of the white
clover. He designates them “ juvenile forms” and believes them to be
young bacilli which have recently entered the nodule from the soil. He
asks (p. 458) : “ What evidence may be submitted to indicate that these
bodies, generally considered cytoplasmic organs, are not bacteria that
have gained entrance to the plant through the root-hairs? The author
has not been able to find any evidence that would satisfactorily answer
these questions.”

In the present paper, it is my aim to deal chiefly with the minute
rod-like and granular inclusions occurring in the clover nodule and in the
soil and to submit evidence which further indicates that minute forms of
bacteria have been confused with mitochondria.

MATERIAL AND METHODS

Clover nodules were secured from various locations, during different
seasons of the year, and from soils varying in moisture content. While
the nodule of the white clover was chiefly used during this investigation,
nodules of crimson clover were likewise studied. Mitochondrial fixa-
tives, including Altmann’s, Regaud’s, Champy-Kull’s, Flemming’s, and
Murray’s, were employed. In addition to mitochondrial fixatives,
Schaudinn’s fluid with iron-hematoxylin was used. In some cases, prior
to fixation, nodules were treated with 95 per cent alcohol or ether for 12
to 24 hours. Still others were fixed in Altmann’s fluid to which had
been added 5 per cent of acetic acid. In order to avoid as much as pos-

114 E. DEWITT MILLER

sible variations which naturally occur in structure and content of dif-
ferent nodules, as well as in different regions of the same nodule,
nodules were severed longitudinally in nearly equal halves and the halves
subjected to different treatment. The two halves of the same nodule
were then placed side by side, embedded, sectioned, stained and mounted.

Nodules were always thoroughly washed in sterile water before
cutting. After cutting on a slide, in a drop of sterile water, smears
were made of that portion of the contents normally lost during the
cutting procedure. The smears, likewise, served in making compara-
tive studies. In addition to treating the nodule as a whole, smears of
entire nodules were made. All smear preparations were allowed to dry
completely before further treatment. While some were treated with
alcohol or ether before applying mitochondrial fixatives, others were not.

Supravital tests were made on the contents of the clover nodule by
thoroughly crushing the nodule upon a slide which had previously been
filmed with a 1-20 alcoholic solution of Janus green (Grubler’s) pre-
pared from a 1 per cent stock solution in absolute alcohol. Compara-
tive tests of the nodular contents were made by examining similarly
treated nodules which were not exposed to Janus green. For perma-
nent staining, anilin-fuchsin with methyl green or toluidin blue counter-
stain and iron-hematoxylin were employed.

In the following illustrations granules and rods which color red in
the stained material are drawn in black, while those staining blue, green,
or indefinitely are illustrated in diluted ink.

Artificial media were inoculated with contents of the nodule, as well
as with soil which had been removed from the roots. Two different
kinds of media were used for this purpose: a mannite medium, free
from combined nitrogen and containing a fermentable carbohydrate, the
formula for which will be found in Topley and Wilson (1932), p. 315,
Vol. 1; for plating a 2 per cent agar plus 1 per cent lithium chloride.

I wish to make grateful acknowledgment to Dr. H. E. Jordan of
the Department of Histology and Embryology, who placed at my dis-
posal the facilities of his laboratory, for his very valuable aid and kindly
criticism during the course of this investigation and preparation of this
paper.

OBSERVATIONS

Wallin describes and illustrates three distinct types of bacteria-like
organisms as they occur in the nodule of the white clover. One of
these types, consisting of minute rods and granules, he found located in
the distal or younger portion of the nodule. He states: “In Fig. 3 the
cells contain small bodies that are not unlike mitochondria in appearance.

o! Sey

BACTERIA AND MITOCHONDRIA; CLOVER NODULE 15

These forms, undoubtedly, are the young bacilli that have recently en-
tered the nodule, or they represent a young form that will later meta-
morphose into the mature type. They may be designated the ‘ juvenile
forms.” Cowdry, on the other hand, thinks: “that Wallin’s ques-
tions may be answered through the simultaneous and differential dem-
onstration of mitochondria and Bacillus radicicola side by side within
the same cells.” In other words, Cowdry is of the opinion that mito-
chondria and bacteria can be distinguished in the nodule on the grounds
of morphology, differential staining, arrangement in the cell, fixing with
Bouin’s fluid and 95 per cent alcohol. Cowdry also states: “ In figures
1 and 2 only mitochondria are shown. ‘They occur in about the same
number in neighboring cells and are, for the most part, rod-like, and
occasionally filamentous, not spherical, as indicated by Wallin in his
figures 6 and 7.”

Among the many nodules examined during the present investiga-
tion, much variation in nodular contents has been encountered. In
matter of fact so much variation occurs with respect to cellular struc-
ture, nature, and arrangement of cellular contents, morphological bac-
terial types, rods and granules, differential staining, that one is at a loss
to determine just what constitutes the rule. Nodules occur which have
little cellular arrangement, the cytoplasm being in a continuous, un-
limited mass, in so far as the cell walls are concerned. Minute rods
and granules sometimes occur in this cytoplasmic mass in abundance.
In some sections the large majority stain red and have the appearance
of mitochondria. In later sections of the same nodule apparently the
same inclusions sometimes show more differential staining qualities, a
lesser number staining red while more are stained green, until finally
very few, if any, present a mitochondrial appearance, but stain green.
Other nodules present relatively large cavities devoid of cytoplasm, or
cells with little or no cytoplasm, and these may be partially or com-
pletely filled with bacteria-like inclusions. These inclusions may vary
slightly in size in some cases, but many are very minute (Plate I, Figs.
1 and 2). They frequently stain red, while in some cases they show
differential staining properties.

Minute rods and granules which color red occur in some cells which
possess large metamorphic forms of Bacillus radicicola and are dis-
tributed peripherally, as Cowdry has shown, or they may be scattered
more or less uniformly throughout the cell (Plate I, Figs. 3 and 4).
Among these, one finds a certain amount of differential staining in some
cases. I have been unable to find any evidence that strict peripheral ar-
rangement of these minute inclusions in such cells is the rule, as Cowdry
has emphasized. They appear as rods, short filaments, or granules.

116 E. DEWITT MILLER

Seldom does one find two nodules, even from the same plant-root,
presenting the same picture with regard to bacterial or minute bacteria-
like content. Different metamorphic forms of B. radicicola appear in
the same nodule, but from the fact that I have never observed the entire
metamorphic series within a single nodule, I conclude that a complete
series within a single nodule is most probably an exception and not the
rule. In fact, in many nodules, one single form seems to predominate
throughout. Usually, there are several forms present.

Rods and granules, many of which are minute and approach the
limit of clear microscopic vision, are found within some of the nodules.
In some cells they are abundant, while in others they are sparsely scat-
tered (Plate I, Figs. 5 and 6). In some nodules, apparently, they are
absent, while in others only a small percentage of the cells possess them.
Their staining ability varies, even within the same cell; i.e., some will
stain red, while others stain green or blue (Plate II, Figs. 7 and 8).

The fact is well recognized that properties such as the above, alone,
are not sufficient to determine the true nature of small cytoplasmic inclu-
sions, making it necessary to seek additional evidence. One of the
recognized criteria for demonstrating mitochondria after mitochondrial
fixation is their ability to color red with anilin-fuchsin, followed by a
counter-stain of methyl green or toluidin blue. A significant fact here
is that, among the numerous nodules investigated during the present

EXPLANATION OF PLATES

All drawings were made with the aid of a Bausch and Lomb 1.8 mm. oil-
immersion objective and a 10 X ocular. All figures with the exception of Fig. 18
(Plate III) were taken from preparations treated with mitochondrial fixatives and
stained with anilin-fuchsin and counterstained with methyl green or toluidin blue.
Figure 18 was taken from a preparation treated with Schaudinn’s fluid and stained
in iron-hematoxylin. Magnification about 1,200.

PLATE |
Explanations of Figures

Fic. 1. Section of a nodule with the lumen partially filled with bacteria-like
rods which show some differential staining. Altmann’s fixation.

Fic. 2. Section of a nodule with cells and lumen containing both rods and
granules. Shows some differential staining. Regaud’s fixation.

Fic. 3. Cells from a nodule, showing mature forms of B. radicicola together
with numerous minute granules and rods located in different regions of the cell.
Shows some differential staining. Regaud’s fixation.

Fic. 4. Two cells from a nodule, showing minute granules and rods scattered
among the mature bacterial forms. Murray’s fixation.

Fic. 5. Cell from a nodule, showing minute cell inclusions. Flemming’s
fixation.

Fic. 6. Section of a nodule, the cells of which contain many rods and
granules. The greater number stain red, a few green, while some are indefinitely
stained. Champy-Kull’s fixation.

117

CLOVER NODULE

)

BACTERIA AND MITOCHONDRIA

a e\

“o 18
§,

e
owe

wi te B

—

PILING, IL

118 E. DEWITT MILLER

work, a relatively small percentage of the cells examined presented
granules or rods suggestive of mitochondria. More rarely, in compari-
son, have there been cases of cells possessing the larger metamorphic
bacterial forms and at the same time exhibiting minute rods or granules,
or both, suggesting mitochondria. In some cases where this latter con-
dition has prevailed the minute forms have shown differential staining
properties (Plate I, Fig. 3 and Plate II, Fig. 8).

Nodules which had been treated with 95 per cent alcohol or ether for
12 to 24 hours before fixation, or with Altmann’s plus 5 per cent of
acetic acid, have shown minute granules and rods, sometimes in rela-
tively large numbers. Many of these inclusions stain red with anilin-
fuchsin and toluidin blue. Like those appearing in nodules which had
not been subjected to the above-mentioned treatment before fixation,
they vary to some extent in size, sometimes in the same cell. Figures
9, 10 and 11 (Plate II) illustrate results obtained in different nodules
which had been treated in 95 per cent alcohol for 12 to 16 hours and
fixed in Murray’s fluid and stained with anilin-fuchsin and toluidin blue.
Granules and rods can be observed scattered more or less uniformly
throughout the cell. While some of these inclusions are stained faintly
red and others are almost completely overshadowed by numerous large
bacteria, still others can be observed much more readily.

In Fig. 12 (Plate II) a cell containing granules and rods stained red
can be observed and illustrates one of many cases following treatment
with 95 per cent alcohol for 12 hours and fixed in Altmann’s fluid. It
will be noted that the cell is completely filled with minute bacteria-like
forms and that there is a close likeness between the forms that stain red
and those that stain green. Figure 14 (Plate II), and Fig. 15 (Plate
III) are drawings made from two separate nodules which were treated
with ether for 12 hours before fixing with Champy-Kull’s fluid and
stained in anilin-fuchsin and methyl green. These two specimens show
much variation in number, form and arrangement of granules and rods.

Priate II

Fics. 7 and 8. Cells from different nodules, showing differential staining of
minute granules. Flemming’s and Altmann’s fixation, respectively.

Fic. 9. Cell from nodule after 12 hours treatment with 95 per cent alcohol
before fixation. Murray’s fixation.

Fics. 10 and 11. Cells from different nodules after 16 hours treatment with
95 per cent alcohol before fixation. Murray’s fixation.

Fic. 12. Cell from a section of a nodule after treatment with 95 per cent
alcohol before fixation. Altmann’s fixation.

Fic. 13. Section from nodule, showing minute granules. Altmann’s plus 5
per cent of acetic acid fixation.

Fic. 14. Section of nodule after 12 hours treatment with ether before fixation.
Champy-Kull’s fixation.

BACTERIA AND MITOCHONDRIA; CLOVER NODULE 119

ow fF o®

+
gee
*.°
eo

Feat yt
a?
@,

2 So wr ; : |

oe / dk

* = wee ."
. Bae -

IPL AVI, IL

120 E. DEWITT MILLER

Figure 13 (Plate I1) represents a specimen from a nodule which was
fixed in Altmann’s fluid to which 5 per cent of acetic acid was added,
and stained with anilin-fuchsin and methyl green.

In order that a more satisfactory comparison between alcohol and
non-alcohol-treated specimens could be made, nodules were cut and the
two halves subjected to different treatment. This procedure overcame,
in a measure, the difficulties encountered when examining different
nodules. Sections of parts of a single nodule mounted on slides in
close juxtaposition facilitated a detailed comparative study. Likewise,
there remained slight opportunity for variations in fixing and staining
procedure. Such a comparison showed a strikingly close resemblance
in the rod-like and granular cell contents. In numerous cases there
were no observable differences in the size, general arrangement, nor in
staining qualities of these inclusions in the separate halves (Plate III,
Figs. 16 and 17).

In smears of the nodule, prepared by thorough maceration and
allowing them to dry before fixation, one is frequently able to observe
minute rods and granules, sometimes in abundance, stained red with
anilin-fuchsin and toluidin blue, or methyl green (Plate III, Figs. 18,
19, and 20). The same is true if the smear has been treated with alco-
hol or ether prior to mitochondrial fixation (Plate III, Fig. 22). Simi-
lar forms of bacteria possessing identical staining qualities occur on
artificial media which had been inoculated with substance from the
nodule or from the soil. Frequently, these minute bacteria from arti-
ficial media stain red while larger forms occurring in the same media
stain blue or green (Plate III, Figs. 21, 23, and 24).

Pirate III

Fic. 15. Section of a nodule different from the one shown in Fig. 14 after
12 hours treatment with ether before fixation. Champy-Kull’s fixation.

Fic. 16 (a) and (b). Cells from approximately the same region of the same
nodule: (a) after 12 hours treatment with 95 per cent alcohol before fixation, and
(b) without alcohol treatment. Murray’s fixation.

Fic. 17 (a) and (b). Cells from approximately the same region of the same
nodule: (a) after 12 hours treatment with 95 per cent alcohol before fixation, and
(b) without alcohol treatment. Champy-Kull’s fixation.

Fic. 18. Bacteria from nodule smear. Schaudinn’s fixation and stained in
iron-hematoxylin.

Fic. 19. Bacteria from smear made from a nodule. Altmann’s fixation.

Fic. 20. Bacteria from nodule smear. Champy-Kull’s fixation.

Fics. 21 and 23. Bacteria from different nodules and cultured on lithium-
agar plate. Altmann’s fixation.

Fic. 22. Bacteria from nodule smear, dried and treated with 95 per cent
alcohol. Altmann’s fixation.

Fic. 24. Bacteria from mannite medium inoculated from the soil. Champy-
Kull’s fixation.

Wil

BACTERIA AND MITOCHONDRIA; CLOVER NODULE

PICA, IOUT

22 E. DEWITT MILLER

Freshly macerated nodules, in addition to showing large, mature
forms of bacteria, also show many minute rod-like and granular forms.
Many, due to their minute size, presented a dark appearance. Supra-
vital tests on this material with Janus green revealed nothing definite.
There were no indications that the number of dark staining rods and
granules had been increased.

Worthy of note at this point is the fact that among the different
mitochondrial methods of technique, Regaud’s method gave the least
satisfactory results, following treatment of the nodule with alcohol.
While granules and a few rod-like inclusions frequently appeared in
cells after alcohol treatment, they did not stain well nor stand out nearly
so clearly as after other methods of fixation nor in cells which had not
been subjected to alcohol. There were some indications that granular
material in the alcohol-treated nodules represented merely precipitate,
and, for that reason have been omitted from the list of illustrations.

DISCUSSION

Topley and Wilson (1932, p. 315) describe the nodule organism,
Rhizobium radicicola, as one having a life-cycle with gross changes in
morphology: “ Shows non-motile coccoid forms; very small, highly
motile, ellipsoidal form (swarmer), 0.9 X 0.2 microns; motile rods
2-3 < 0.5 microns.” ‘They state with respect to the smaller forms, p.
314: “ They are so small as to be able to pass through a Chamberland
filter: 9 ohms (1921). (Plate is. Bice 1Ob Plates Hicwloy silane
Fig. 83), gives an excellent idea of the minuteness and variation in size
of rod-like bacteria belonging to B. radicicola. It has been thought by
some investigators that other microorganisms, besides B. radicicola, may
enter the nodule of the clover. To quote Wallin (1927), p. 90: “In
the young root nodules no other organisms but the Bacillus radicicola
are present in the cells. As the nodules mature, other microorganisms
begin to invade the cells of the nodule. In an old nodule, very few if
any Bacillus radicicola can be found in the nodule. They appear to
have been replaced by a variety of parasitic microorganisms.”

In the preceding pages I have shown that minute bacteria, both rod-
like and granular, present in the nodule and in the soil are not unlike, in
their general appearance and staining properties, rod-like inclusions of
the clover nodule and described heretofore as mitochondria. Likewise,
the illustrations show how mitochondria-like inclusions appear fre-
quently in nodules which have been subjected to prolonged treatment
with alcohol, ether, or fixatives containing acetic acid. Since, in addi-
tion, minute bacterial forms of B. radicicola enter the clover rootlets and

BACTERIA AND MITOCHONDRIA; CLOVER NODULE 1S)

subsequently bring about the formation of the nodule, together with the
probability that still other bacterial microorganisms invade the nodule,
it appears likely that the minute inclusions having a mitochondrial ap-
pearance represent bacteria. For the fact that among nodules, or halves
of nodules, which have received different treatment prior to fixation with
mitochondrial fixatives (excepting perhaps Regaud’s), there have ex-
isted in many cases no observable differences in the nature of minute cell
inclusions, strongly indicates that inclusions lacking essential mitochon-
drial characteristics are present in the nodules of the clover.

A close study of the illustrations given by Cowdry, showing the
morphological forms of B. radicicola as they occur in the clover nodule,
leads me to believe that the minutest of the bacterial forms, such as I
have observed in smears of nodules, freshly macerated nodules, as well
as from cultures of bacteria from the nodule and from the soil, have not
been included in these illustrations as bacteria, but as mitochondria.
The reason for such an omission, if true, appears to lie in the fact,
largely, that Cowdry is of the opinion that the smallest of bacterial
forms found in the nodule stain green with methyl green after mito-
chondrial fixatives and not red with anilin-fuchsin. He states, p. 340:
“ A little farther up the rootlet small forms of B. radicicola are en-
countered, as represented in figure 3. They are distinguishable from
the mitochondria in the preparations by their green color, their larger
size, and their variable distribution in clumps in different cells.” As
has already been shown in preceding pages and the illustrations, bacteria
equaling in size apparent mitochondria as observed in nodules, occur in
nodules and in the soil and stain red with anilin-fuchsin and methyl
green or toluidin blue. Many of these bacteria are apparently smaller
than many red colored rods and granules observed in nodules which may
or may not have been treated with lipoid solvents before fixation.

For the reason that differential staining sometimes occurs among
these inclusions, whether in the nodule, smears, or on slides prepared
from cultures of material from the nodule and from the soil, it would
appear that the slightest variation in the chemical composition of the
inclusions, or in technique, is often all that is necessary to alter the
staining reaction of these minute forms. For the fact that, in nu-
merous cases, where nodules have been subjected to alcohol treatment,
cells occur which are completely filled, or nearly so, with minute rods
and granules, some of which stain red while others stain green, makes it
quite obvious that minute bacteria within the cell of the nodule may
stain like mitochondria.

From the above observations it would seem that there are three pos-

124 E. DEWITT MILLER

sible conclusions. Firstly, either mitochondria as such do not exist in
these nodules, but have been confused with an “invading form” of
minute bacteria from the soil; or secondly, mitochondria possess char-
acteristics which have not been fully recognized, in that they may with-
stand alcohol, ether, dilute acetic acid, maceration of the nodule, and
drying ; or thirdly, they are indistinguishable in the nodule from minute
forms of bacteria with our present-day methods of technique. Cer-
tainly, the above methods of technique have aided little in making a clear
distinction between bacteria and mitochondria, but in the case of the
clover nodule the results materially strengthen the indications that the
alleged mitochondria are merely forms of minute bacteria.

CONCLUSIONS

1. The morphology of minute cytoplasmic inclusions as they occur
in the nodule of the clover, their response to Janus green and lipoid
solvents, staining reaction, fail to produce sufficient evidence for plac-
ing them in the category of mitochondria.

2. Bacillary and granular bacteria occur in clover nodules, in nodule
smears, in cultures of material from nodules and from the soil on
artificial media, which possess staining qualities and physical features
characteristic of mitochondria.

3. Minute rods and granules, having the physico-chemical properties
of mitochondria, can be demonstrated in some nodules which have been
subjected to prolonged treatment with 95 per cent alcohol, ether and
fixatives containing 5 per cent of acetic acid.

4. Differential staining cannot be relied upon in this case to dis-
tinguish bacteria from the alleged mitochondria, since certain forms of
minute bacteria found in nodule-smears and cultures made from the
nodule and from the soil stain red with anilin-fuchsin and methyl green
after mitochondrial fixatives, just as do many rod-like and granular
inclusions in sectioned material.

5. Indications are that the slightest variation in technical manipula-
tions, or possibly chemical composition of different regions of the
nodule, is often sufficient to alter the staining reaction of otherwise
identical minute forms occurring in the clover nodule.

6. Janus green apparently does not stain any of the minute rods nor
granules found in freshly macerated nodules.

7. The results of this investigation appear to support strongly the
inference that minute bacterial forms have heretofore been misinter-
preted as mitochondria in the nodule of the clover.

BACTERIA AND MITOCHONDRIA; CLOVER NODULE 125

LITERATURE CITED

Bucuner, Paut, 1921. Tier und Pflange in intracellularer Symbiose. Berlin.

Cownpry, E. V., 1923. The independence of mitochondria and the Bacillus radici-
cola in root nodules. Am. Jour. Anat., 31: 339.

Lounts, F., 1921. Studies upon the life cycles of bacteria. Mem. Nat. Acad.
Sct., 16: No. 2. Washington.

Mitrer, E. DeW., 1937. A comparative study of the contents of the gelatinous
accumulations of the culture media and the contents of the cytoplasm of
Ameeba proteus and Arcella vulgaris. Jour. Morph., 60: 325.

ToprLey, W. W. C., anp G. S. Witson, 1932. The Principles of Bacteriology and
Immunity, Vol. 1. Wood and Co. New York.

Wat in, Ivan E., 1922. On the nature of mitochondria: A comparative study
of the morphogenesis of root-nodule bacteria and chloroplasts. Am. Jour.
Anat., 30: 455.

Watt.in, Ivan E., 1927. Symbionticism and the Origin of Species. Williams and
Wilkins. Baltimore. —

THE RELATION BETWEEN LUMINOUS INTENSITY,
ADAPTATION DO MEIGH TAN Dak Ai sO
LOCOMOTION IN AMOEBA
FROME US (LET)

S) OO; MAST VAND NATHAN STAHEER
(From the Zoological Laboratory of the Johns Hopkins University)

INTRODUCTION

Strasburger (1878), Loeb (1890), and Davenport (1897) maintain
that organisms appear to move faster in strong light than in weak light
because their path is straighter and orientation is more precise and they
conclude that the rate of locomotion is independent of intensity. This
conclusion is supported by the results obtained by Mast (1910, 1911)
on Ameba proteus and blow-fly larve, Dolley (1917) on Vanessa
antiopa, Minnich (1919) on Apis mellifera, Mast and Gover (1922) on
Phacus pleuronectes and Euglena gracilis, Folger (1925) on Amoeba
proteus, and Ullyott (1936) on Dendrocalum lacteum.

But it is not supported by those obtained by Oltmanns (1892),
Holmes (1903), and Laurens and Hooker (1920) on Volvox, Yerkes
(1903) on Gonionemus, Herms (1911) on blow-fly larve, Patten
(1917) on the whip-tail scorpion, Buddenbrock (1920) on Helix, Moore
and Cole (1921) on the Japanese beetle and Drosophila melanogaster,
Clark (1928) on Dineutes assimilis, and Welsh (1932) on Unionicola.

Clark made a more thorough study of the problem than any of the
other investigators. He found that with increase of intensity, the rate
of locomotion increases to a maximum at 0.5 m.c., then remains con-
stant until 8,000 m.c. is reached, and then decreases. Trezise (1936)
obtained similar results in observations on Dineutes nigrotr.

Mast (1907, 1927) contends that the activity of organisms in any
given luminous intensity is closely correlated with the state of adapta-
tion of the organism to that intensity. This contention is supported by
results obtained by Walter (1907) in observations on Planaria gono-
cephalus. He maintains that this organism moves faster in light than
in darkness, but the results which he presented show that if the speci-
mens are transferred from darkness to light and left there, the rate of
locomotion decreases so that after 10 minutes it is much lower than it
was in darkness.

126

LIGHT ADAPTATION AND LOCOMOTION IN AMCEBA 127

It is consequently obvious that there is great diversity in the results
obtained by different investigators concerning the relation between rate
of locomotion and luminous intensity. This is doubtless largely due to
the fact that in most of the investigations the state of adaptation was
not adequately considered.

MATERIAL AND METHODS

Amoeba proteus (Leidy) was used exclusively in the following ex-
periments. It was raised in finger-bowls containing a few grains of

TABLE I

Relation between rate of locomotion in Amoeba proteus and intensity of light.

The ‘‘total average rate’’ is the mean for all measurements at the indicated
intensities of light. The ‘‘mean maximum rate” is the mean for all measurements
made after the maximum rate wasreached. The time required to reach the maximum
rate was obtained by inspection of the graphs for each intensity of light. The
standard deviation was calculated for the rate after the maximum was reached.

: Number of Total Mean . bene Fee Standard

Intensity aes pla Morse to Teach iment son : dgsianien

meter candles micra/min. micra/min. minutes micra/min.
50 21 128.8 128.8 1 74 10.8
225 18 124.8 131.0 11 8.7 9.0
4,170 14 145.8 147.4 16 16.5 8.6
7,530 16 160.0 136.6 14 9.0 11.3
11,120 18 176.2 198.4 11 12.0 9.3
13,140 18 191.3 215.7 15 10.8 6.4
15,000 19 198.1 219.3 8 16.8 11.4
17,000 20 176.0 211.1 17 10.8 11.4
20,000 23 170.0 200.2 Dp) 14.7 43
24,000 Dy 160.9 205.7 23 12.4 14.2
26,600 21 151.8 195.6 22 Wo 9.8
40.000 23 120.7 150.2 30 20.4 8.5

rice and numerous chilomonads in Hahnert solution, which consists of
pure water (1 liter), CaCl, (4 mg.), NaHCO, (4 mg.), CaH,(PO,),
(2 mg.), KCl (4 mg.), Mg,(PO,), (2 mg.). The cultures were kept
at a temperature of 24°—26° and at a luminous intensity of about 20
meter-candles.

All the experiments were performed in a darkroom. The light used
was produced by a monoplane filament stereopticon bulb mounted in a
projection lantern which was in turn enclosed in a box, light-tight, ex-
cept for an opening 25 cm. square. A parallel-sided glass vessel 8 cm.

128 S. O. MAST AND NATHAN STAHLER

4170 mc. 11120 m.c.

O
Oo O
ooo
yee Oe ig ; ; een
0 0) LX) 4
10 aE ee 5 ee!
on Te ODO Oe,

CG oD Sorc

j 20000 m.c.

196

141

Rate of locomotion, micra per minute

196

141

ies 15 22.5 7.5 15 22.5 30
Time in light in minutes

Fic. 1. Relation between adaptation to light of different intensities and rate
of locomotion in Amoeba proteus.

Each point in the figures represents the average for one measurement on each
of 14 to 23 specimens (see Table I). The time in light is the time from the
beginning of movement after exposure until the measurements were made.

LIGHT ADAPTATION AND LOCOMOTION IN AMCEBA 129

thick, filled with distilled water, was placed in the box in front of the
lamp, to absorb the heat waves.

Three stereopticon bulbs were used (300, 500, and 1,000 W.,
120 V.). The voltage on the line was maintained at 110 + 2 volts by
means of a rheostat. The desired intensities of light were obtained by
means of these bulbs and Eastman Kodak neutral-tint filters.

205

175

145

Rate of locomotion, micra per minute

115

10,000 20,000 30,000
Intensity of light in meter-candles

Fic. 2. Relation between intensity of light and rate of locomotion in Amoeba
proteus.

Upper curve, mean maximum rate; lower curve, total average rate.

Each point on the lower curve is the average for 420 to 920 measurements on
14 to 23 specimens. Each point on the upper curve represents the average for the .
same number of measurements in the lowest intensity but progressively fewer as
the intensity increases (see Table I and Fig. 1).

The beam of light was reflected up through the stage of a compound
microscope by means of the substage mirror. The microscope had no
substage condenser. The intensity on the stage was measured directly
by means of a Weston photometer.

A Pitts warming stage (Pitts, 1932) was fastened to the stage of
the microscope. This consists essentially of a beaker in a metal box so
arranged that water of any desired temperature can be passed through

130 S. O. MAST AND NATHAN STAHLER

the box and around and under the beaker.t. The bottom of the beaker
consisted of smooth Pyrex glass.

The observations were made as follows: A large number of amcebe
were taken from a vigorous culture and put into a small glass dish con-
taining 10 cc. redistilled water and left ten minutes. The water was
then changed four times. Then about 60 large specimens with few food
vacuoles were selected and put into a dish containing redistilled water
and left without food 15 hours or longer in darkness at a temperature
of 24° to 26°. Then 5 to 10 of the specimens were transferred to a
dish containing .001 N CaCl, and immediately transferred again, with
CaCl, solution, to a vaseline enclosure on the bottom of the beaker in
the Pitts warming stage and covered with a cover-glass. The solution
in the enclosure was so deep that the amcebz did not touch the cover-
glass. The temperature in the beaker was maintained at 24 + 0.5°.

They were then subjected to light of 50 m.c. for five minutes, then to
darkness for 30 minutes. Then a weak red light was turned on and a
specimen which was monopodal or nearly so was selected, brought to
focus, and exposed to white light of the desired intensity, from the pro-
jection lantern.

The posterior end of the image of the amceba, projected on black
paper by means of a camera lucida, was then outlined with a yellow
pencil, once a minute for 30 or 40 minutes, and the rate of locomotion
calculated. If the specimen under observation came in contact with
other specimens or with the vaseline ring during this time the record
was discarded. The whole process was then repeated with other speci-
mens in light ranging from 50 to 40,000 meter candles. The results ob-
tained are presented in the following paragraphs, Table I, and Figs. 1
and 2.

RESULTS

In all but the lowest intensities movement ceased shortly after the
specimens were exposed, and in the highest intensities it did not begin
again until 2-7 minutes later. In these intensities movement usually
began in a pseudopod on the upper surface of the amceba. This pseudo-
pod often extended until it contained nearly all the substance in the
amoeba, then a pseudopod formed at the base and extended in contact

with the slide. This frequently occurred several times during the proc-

ess of adaptation to light.
In the lower intensities the specimens were fairly consistently and
firmly attached to the substratum and monopodal in form. In the

1 Small pieces of glass 5 mm. thick were cemented to the inner base of the
warming stage so that a constant depth of water under the beaker obtained.

or es

7 hans a Ri nO WIE

LIGHT ADAPTATION AND LOCOMOTION IN AMCG&BA 131

higher intensities they were much less consistently monopodal and at-
tachment to the substratum was more sporadic.

Figure 1 shows that, in all except the lowest intensity, as the time of
exposure to light increased, the rate of locomotion rapidly increased to
a maximum and then remained constant; and that, as the intensity in-
creased, the time required for the rate to reach maximum decreased
from about 11 minutes at 225 m.c. to a minimum of about 8 minutes at
15,000 m.c., and then increased to about 30 minutes at 4,000 m.c. This
. proves that the rate of locomotion in Amoeba proteus is closely corre-
lated with the state of adaptation to light and it indicates that, except in
the very low intensities, the time required for light-adaptation is least
at 15,000 m.c.

Table I and Fig. 2 show that as the intensity to which the amcebe
were exposed increased, the rate of locomotion after the amcebe were
fully light-adapted, increased from 128.8 + 10.8 micra per minute at
50 m.c. to a maximum of 219.3 + 11.4 micra per minute at 15,000 m.c.,
and then decreased to 150.2 - 8.5 micra per minute at 40,000 m.c. This
proves that the rate of locomotion in Amoeba proteus is very definitely
correlated with the intensity of the light, and that the optimum intensity
of light is 15,000 m.c. Figure 2 shows also that the total average rate is
correlated with the intensity of the light in essentially the same way as
the mean maximum rate.

Table I shows that the percentage of lobose forms increased with in-
crease in luminous intensity, and that the standard deviation for the
mean maximum rate is relatively low.

The average rate of locomotion of 20 amcebe for 30 minutes each in
the red light used in the experiments described above, was 134.7 + 8.9
micra per minute. There was no indication of decrease in rate in any
of the specimens after exposure to the red light. The intensity of the
red light was not measured but the rate of locomotion in it was some-
what higher than the rate of locomotion in white light of 225 m.c. in
which there was marked retardation (Fig. 1). The facts that the rate
in red light (in which there was no retardation) was higher than the
rate in white light (in which there was marked retardation) and that
blue is very much more efficient than red in inducing retardation (Har-
rington and Leaming, 1900 and Mast, 1910) strongly indicate that re-
tardation and decrease in rate of locomotion with increase in intensity
(in the higher intensities) observed in white light, is due largely to the
action of blue and other short waves, and that increase in rate of loco-
motion with increase in intensity (in the lower intensities) is due largely
to the action of red and other long waves.

132 S. 0. MAST AND NATHAN STAHLER

There is no evidence which indicates the nature of the action of the
longer waves of light, but it is well known that the shorter waves tend
to induce gelation. It is therefore highly probable that retardation in
rate of movement is due to the gelating effect of these waves.

SUMMARY

1, The rate of locomotion in Amoeba proteus is definitely correlated
with the intensity of the light to which it is exposed and the state of
adaptation.

2. In light of any given constant intensity, as adaptation to light in-
creases, the rate of locomotion increases to a maximum and then re-
mains constant, but in constant light of different intensities, the time
required for adaptation decreases from about 15 minutes at 225 m.c. to
a minimum of about 7 minutes at 15,000 m.c. and then increases to about
30 minutes at 40,000 m.c. and the rate of locomotion increases from
128.8 + 10.8 micra per minute at 50 m.c., to a maximum of 219.3 + 11.4
micra per minute at 15,000 m.c., and then decreases to 150.2 + 8.5 micra
per minute at 40,000 m.c.

3. Increase in rate of locomotion with increase in intensity to
optimum at 15,000 m.c. is largely due to some unknown action of the
longer waves of light. Decrease in rate with increase in intensity be-
yond the optimum is probably due to the gelating action of the shorter
waves of light.

REFERENCES CITED

Buppensrock, W., 1920. Versuch einer Analyse der Lichtreaktionen der Helici-
den. Zool. Jahrb., Abt. allgem., 37: 313,

Crark, L. B., 1928. Adaptation versus experience as an explanation of modifica-
tion in certain types of behavior (circus movements in Notonecta).
Jour. Exper. Zool., 51: 37.

Core, W. H., 1922. Note on the relation between the photic stimulus and the rate
of locomotion in Drosophila. Science, 55: 678.

Davenport, C. B., 1897. Experimental Morphology. New York. Vol. 1.

Dotiey, W. L., Jr., 1917. The rate of locomotion in Vanessa antiopa in inter-
mittent light and in continuous light of different illuminations, and its
bearing on the “continuous action theory” of orientation. Jour. Exper.
ZOol 232 507

Fotcer, H. T., 1925. A quantitative study of reactions to light in Amceba. Jour.
Exper. Zo6l., 41: 261.

Harrincton, N. R., anp E. Leamrine, 1900. The reaction of Amceba to lights of
different colors. Am. Jour. Physiol., 3: 9.

Herms, W. B., 1911. The photic reactions of sarcophagid flies, especially Lucilia
cesar Linn. and Calliphora vomitoria Linn. Jour. Exper. Zool., 10: 167.

Hoimes, S. J., 1903. Phototaxis in Volvox. Biol. Bull., 4: 319.

Laurens, H., anp H. D. Hooxer, Jr., 1920. Studies on the relative physiological
value of spectral lights. II. The sensibility of Volvox to wave-lengths
of equal energy content. Jour. Exper. Zool., 30: 345.

LIGHT ADAPTATION AND LOCOMOTION IN AMCEBA _ 1133

Logg, J., 1890. Der Heliotropismus der Thiere und seine Ubereinstimmung mit
dem Heliotropismus der Pflanzen. Wiurtzburg.

Mast, S. O., 1907. Light reactions in lower organisms. II. Volvox. Jour. Comp.
Neur. and Psych., 17: 99.

Mast, S. O., 1910. Reactions in Ameceba to light. Jour. Exper. Zo6l., 9: 265.

Mast, S. O., 1911. Light and the Behavior of Organisms. New York.

Mast, S. O., 1927. Reversal in photic orientation in Volvox and the nature of
photic stimulation. Zeitschr. vergl. Physiol., 5: 730.

Mast, S. O., Anp M. Gover, 1922. Relation between intensity of light and rate
of locomotion in Phacus pleuronectes and Euglena gracilis and its bearing
on orientation. Biol. Bull., 43: 203.

Minnicu, D. E., 1919. The photic reactions of the honey bee, Apis mellifera L.
Jour. Exper. Zool., 29: 343.

Moore, A. R., anp W. H. Core, 1921. The response of Popillia japonica to light
and the Weber-Fechner law. Jour. Gen. Physiol., 3: 331.

Mttier, A., 1925. Uber Lichtreaktionen von Landasseln. Zettschr. vergl.
Physiol., 3: 113.

Ottmanns, F., 1892. Uber die photometrischen Bewegungen der Pflanzen.
Flora, 75: 183.

Patten, B. M., 1917. Reactions of the whip-tail scorpion to light. Jour. Exper.
Zool, 23 251 .

Pitts, R., 1932. Constant temperature apparatus adapted for use on the micro-
scope stage. Science, 76: 626.

STRASBURGER, E., 1878. Wirkung des Lichtes und der Warme auf Schwarmsporen.
Jena Zeitschr., 12: 551.

TreEzIsE, W. J., 1936. Sensitivity to light in the beetle, Dineutes nigroir (Roberts).
Dissertation, Johns Hopkins University.

Uttyort, P., 1936. The behavior of Dendrocoelum lacteum. Brit. Jour. Exper.
Biol Wor 253s

Watter, H. E., 1907. The reactions of planarians to light. Jour. Exper. Zodl.,
5135:

We tsa, J. H., 1932. Photokinesis and tonic effect of light on Unionicola. Jour.
Gen. Physiol., 16: 349.

Yerkes, R. M., 1903. A study of the reactions and reaction time of the Medusa
Gonionemus murbachii to photic stimuli. Am. Jour. Physiol., 9: 279.

THE ROLE OF THE HYPOPHYSEAL MELANOPHORE
HORMONE IN THE CHROMATIC PHYSIOLOGY
OL EON DULY Ss

A. A. ABRAMOWITZ

(From the Marine Biological Laboratory, Woods Hole, Mass., and the Biological
Laboratories, Harvard University)

The endocrine control of melanophore reactions of vertebrates has
been extended within the past twenty years to the majority of animals
which exhibit color changes. It has long been generally conceded that
the pituitary gland is the sole regulator of chromatic activity in the
Amphibia (Smith, 1916; Allen, 1916; Atwell, 1919; Swingle, 1921;
Hogben, 1924). Within recent years, the cyclostomes (Young, 1935),
the elasmobranchs (Lundstrom and Bard, 1932), and certain reptiles
(Noble and Bradley, 1933; Kleinholz, 1936) have been found to react
to hypophysectomy by complete pallor due to the concentration of the
pigment in the cutaneous melanophores. It is also generally acknowl-
edged that such hypophysectomized specimens remain indefinitely pale
regardless of certain environmental conditions which in normal speci-
mens would induce melanophore expansion. Furthermore, these hypo-
physectomized specimens cannot normally exhibit the dark phase of
their coloration unless the melanophore hormone of the pituitary is
administered in one form or another to them.

The teleosts are, as far as I am aware, the only color-changing
vertebrates thus far investigated which do not behave to hypophysectomy
like those already mentioned. The melanophores of the teleosts have
long been known to be controlled by the sympathetic nervous system
(von Frisch, 1911), but the importance of the pituitary gland has not
been satisfactorily evaluated except perhaps in one case, Ameturus
(Parker, 1934a; Abramowitz, 1936).2 In Fundulus, the relation be-
tween the pituitary and the chromatic function is uncertain. Parker
(1935) is of the opinion that the hypophysis is without functional sig-
nificance as a normal means of controlling color change in Fundulus,
although later (1936) he writes that certain responses call for further

1 Aided in part by a grant from the Rockefeller Foundation, administered by
F. L. Hisaw.

2 Recently Veil (1937) has reported that Ameiurus reacts to hypophysectomy

as the selachians do. Her paper is probably a preliminary report since very little
or no data are given in support of this conclusion.

134

~ DAE E GE AOLC i EAAT AB

HYPOPHYSEAL HORMONE IN FUNDULUS 135

elucidation. This paper is intended to define the role of the hypophyseal
melanophore-expanding hormone in the chromatic physiology of
Fundulus.

Desmond (1924) was apparently the first to investigate the effect
of the pituitary on the melanophore responses of Fundulus, but his work
has been overlooked by the subsequent investigators of this problem.
He found that hypophysectomy was without effect on the melanophore
reactions of young Fundulus. This negative effect following hypophy-
sectomy could not be attributed to the absence of the melanophore hor-
mone in the pituitary of Fundulus, for he found that while pituitary
transplants from adult fish or amphibians into Fundulus were without
effect on the melanophores of the host, similar transplants made into
hypophysectomized axolotls or tadpoles produced the typical darkening
of these hosts. It thus appeared that the pituitary of Fundulus con-
tained the melanophore-expanding hormone which was effective on
amphibian melanophores but ineffective on its own black pigment cells.
Matthews (1933) also observed that hypophysectomy did not affect the
melanophores of adult Fundulus. The melanophores expanded nor-
mally when pituitaryless specimens were adapted to a black background
and contracted promptly when adapted to a white background. How-
ever, when Matthews immersed isolated scales in a water extract of
Fundulus pituitaries, the scale melanophores always contracted. On
the belief that the pituitary contained a melanophore-contracting hor-
mone, Matthews reasoned that it might conceivably affect the contrac-
tion of denervated melanophores in a caudal band (Mills, 1932). How-
ever, denervated trunk or tail areas faded in the same time in hypophy-
sectomized as in intact animals during prolonged white-background
adaptation.

Thus the situation became complicated, for Matthews’ work indi-
cated that a contracting hormone was present in the pituitary of
Fundulus, and yet the melanophores, innervated or denervated, con-
tracted normally in the absence of the pituitary. The presence of a
contracting principle was disputed by Kleinholz (1935), who showed
that Fundulus pituitaries could darken normal white-adapted frogs and
catfishes, and hypophysectomized lizards, and furthermore that injec-
tions of pituitrin or extracts of crushed Fundulus pituitaries were fol-
lowed by a darkening of a pale denervated caudal band in normal white-
adapted Fundulus. The latter experiment is significant in relation tc
the neurohumoral theory (Parker, 1932) for it raises the possibility
that the black background response (the expansion of contracted dener-
-vated melanophores in a normal Fundulus when adapted to a black
background) might be due, not to a dispersing neurohumor as Parker

136 A. A. ABRAMOWITZ

(19340) has maintained, but to the melanophore-expanding hormone of
the pituitary (Kleinholz, 1935).

The responses of teleost melanophores to various hypophyseal ex-
tracts are by no means clarified. Spaeth (1917), Hewer (1926),
Odiorne (1933) and Matthews (1933) have reported melanophore con-
traction following treatment with certain hypophyseal extracts. The
significance of these reports is enhanced by Hogben and Slome’s
(1936) recent work with amphibians where, in Xenopus, evidence for
a dual pituitary control by melanophore-expanding (“black”) and con-
tracting (“white”) hormones has been accumulated. Thus it may be
possible that the pituitary gland of Fundulus contains a contracting
melanophore principle. The existence of a melanophore-expanding hor-
mone in the teleost pituitary rests on a more substantial basis, for this
principle has been shown to be present in the pituitary glands of all
chordates from cyclostomes through the primates, including man. The
complexity of melanophore responses to pituitary extracts was increased
by the work of Przibram (1932), who showed that the injected dosage
was a factor determining the direction of melanophore response. He
found that low dosages of a commercial pituitary extract resulted in
an expansion of amphibian melanophores, while when higher dosages
were employed the melanophores contracted. This result he offered
as an explanation of the conflicting reports of the effect of pituitary
extracts upon the melanophores of the same fish (Phoximus) by Abolin
(1925) and by Hewer (1926). There is thus a great difference of
opinion regarding the responses of teleost melanophores (intact, iso-
lated from the body, or isolated experimentally—denervated—within
the body) to various pituitary extracts.

METHODS

Large healthy specimens of Fundulus heterochitus were used through-
out this work. A stock supply of fish was maintained in a large
aquarium of running sea water and fed daily with generous portions
of macerated clams and live shrimp. Background responses were elicited
by the customary white or black vessels, three of each kind being placed

3 Przibram’s experiments were carried out with a commercial pituitary ex-
tract (Infundin), which is not primarily prepared for the melanophore hormone
and which is probably contaminated with preservatives, and other posterior pitui-
tary hormones. When high concentrations of Infundin were employed, the pres-
ence of the extraneous substances might have conceivably complicated the response.
My results with hypophysectomized frogs do not substantiate Przibram’s explana-
tion for many such specimens injected with high dosages of a relatively pure
melanophore hormone (1 gram equivalent of whole sheep pituitary powder poten-
tiated 25 times by alkalinization) always became black and remained so for two

weeks.

HYPOPHYSEAL HORMONE IN FUNDULUS Si

on a lead table and arranged so that a constant current of sea water
flowed through all the dishes. All six vessels were illuminated by two
60-watt lamps. Hypophysectomies were performed by the opercular
approach (Abramowitz, 1937). Slightly over 200 hypophysectomies
were performed and an equal number of normal specimens kept as
controls. Since the operator can determine whether the entire gland is
being removed, histological examination of the hypothalamus was
deemed unnecessary. When the gland was seen to fragment during
an operation, the animal was immediately discarded. Twelve hypo-
physectomized Fundulus were selected at random and the region of the
hypothalamus dissected under an operating binocular but no trace of
pituitary tissue could be found. Denervating operations in the base
of the tail were carried out by Wyman’s technique (1924). The width
of these bands was in every case 2 mm., since all cuts were made with
the same instrument. The technique of extracting the melanophore-
expanding hormone from the blood was somewhat similar to that
described by Jores (1933) for man.

RESULTS

I. Effect of Hypophysectomy on the Responses of Denervated
Melanophores to Backgrounds

A denervated band of melanophores was established in the base
of the tail of each of 26 Fundulus. Twelve of these specimens were
hypophysectomized, and these and the remaining 14 unoperated animals
placed in illuminated white vessels. Three days later the denervated
bands which during this time appear black due to melanophore expan-
sion become completely pale (melanophore contraction) in both unop-
erated and hypophysectomized groups. The pituitary then does not
affect the contraction of denervated melanophores during prolonged
white background adaptation immediately following denervation, as
first demonstrated by Matthews (1933). Then, both sets were trans-
ferred to an illuminated black background. Twelve hours later, 11 of
the 14 normal animals exhibited fully dark bands; the remaining 3
showed partly dark bands. In the hypophysectomized series, 6 showed
faintly dark bands and 5 exhibited completely pale bands.* After 36
hours of black-background adaptation, there was no change in either
of the two series of animals.

#Tn all of the experiments cited, microscopical examination of the melano-
phores was made. In the absence of a quantitative treatment of melanophore
responses, the following descriptive terms will be employed: fully dark—complete
melanophore expansion; partly dark—slightly more expanded than intermediate ;

faintly dark—melanophores more contracted than intermediate; pale—complete
melanophore contraction.

138 A. A. ABRAMOWITZ

Thus the pituitary seems to be indispensable for the complete ex-
pansion of denervated melanophores following black-background adap-
tation. Since this experiment is important from the standpoint of the
neurohumoral theory (Parker, 1932) it was repeated in different ways.
Nineteen Fundulus were hypophysectomized, and denervating opera-
tions induced in their caudal fins. These were placed on a white back-
ground for 13 days. By this time, the cut nerves in the tail fin have
completely degenerated (Parker and Porter, 1933; Abramowitz, 1935).
The bands are also completely pale due to extreme melanophore con-
traction. Thirteen additional animals to serve as control were treated
in the same way except that they were not hypophysectomized. At the
end of 13 days, both sets were transferred to a black background for
24 hours. The control animals showed fully black bands but in the
hypophysectomized group, only 1 band was fully black, 12 were faintly
dark, and 6 completely pale.

This black-background experiment was again repeated in a third
manner. In 30 Fundulus, caudal bands were established and the ani-
mals white-adapted for 5 days. Ten animals were then changed to a
black background for 24 hours. The remaining twenty were hypo-
physectomized and also transferred to a black background for 24 hours.
All 10 normal animals exhibited fully dark bands. In the hypophy-
sectomized group, 11 showed faintly dark bands, 1 fully dark, and 8
completely pale. The experiment was continued, and the specimens
examined after 40 hours of black-background adaptation. The normal
group still showed fully dark bands. Half of the hypophysectomized
evidenced faintly dark bands, and half showed completely pale bands.

Thus, it appears that the melanophore (expanding) hormone of the
pituitary is necessary for complete expansion of denervated contracted
melanophores in the change from white to black background. In this
respect, it is in agreement with Ameturus (Abramowitz, 1936). So far
nothing has been said of the reactions of the innervated melanophores
following hypophysectomy. In all of the experiments, the innervated
melanophores contracted promptly when the fish was exposed to a white
background in both normal and hypophysectomized. Both sets of ani-
mals also showed expansion of the innervated melanophores when ex-
posed to a black background. These statements are confirmatory of
Matthews’ observations. However, my hypophysectomized animals
never became quite as dark as normal animals when exposed for equal
periods of time to a black environment.

These experiments, as well as those of Matthews, indicate that the
hypophysis has no effect on the contraction of either innervated or
denervated melanophores. The latter must be due to some factor

HYPOPHYSEAL HORMONE IN FUNDULUS 139

(neurohumoral) other than nervous action or a possible hypophyseal
melanophore-contracting hormone. The full expansion of contracted
denervated cells, however, does not occur in the absence of the melano-
phore-expanding hormone, and the expansion of the innervated melano-
phores is only slightly retarded in hypophysectomized animals. It would
seem, therefore, that Parker’s conception of the neurohumoral theory
as applied to Fundulus may be modified to include the aid of the
melanophore-expanding hormone of the pituitary.

II. Presence of the Melanophore (Expanding) Hormone in the Blood
of Fundulus

If these results are correct it should be possible to obtain the melano-
phore hormone from the blood of Fundulus, especially the black-adapted
specimens. Previous attempts to detect the presence of hormones in
the blood of Fundulus have been unsuccessful. Mills (1932) injected
blood from a dark Fundulus into a pale one, but without effect on the
melanophores of the latter. The reverse experiment, blood from a
pale fish injected into a dark one, was equally negative. Parker (1934)
also reported similar experiments and similar negative results. Ob-
viously, these experiments indicate that the blood of a dark Fundulus
is ineffective in eliciting expansion of the innervated melanophores of
a pale animal, but they do not prove that the blood of Fundulus does not
contain the melanophore-expanding hormone. The fact that blood of
a dark fish does not cause a pale fish to darken is due, as shown in the
next section, to the insensitivity of the innervated melanophores of
Fundulus to the melanophore hormone. Consequently, the presence of
the latter could not be demonstrated by the experiments already cited.

The test was therefore made on the hypophysectomized frog, which
in my experience is the most reliably sensitive object for the biological
detection of the pituitary melanophore hormone. Specimens of Rana
pipiens were totally hypophysectomized by a direct oral route, and
within the following three hours these specimens became totally pale.
Hypophysectomized males (30-40 grams in weight) were used through-
out. Blood was drawn from 10 black-adapted specimens and extracted
with 10 cc. of 50 per cent ethanol by boiling for 2-3 minutes. The
soluble portion was separated by centrifugation, dried, extracted with
70 per cent ethanol. The soluble portion was separated, taken up in
0.5 cc. distilled water, and injected into the dorsal lymph sac of hypo-
physectomized frogs. These became dark within an hour and remained
dark for 5 hours, after which they became again pale. A similar test
with the blood of 10 white-adapted fish also gave a positive response

140 A. A. ABRAMOWITZ

but less intense, inasmuch as the frogs remained dark for 3 hours.
Repetition of the experiment with blood from white-adapted hypophy-
sectomized or black-adapted hypophysectomized specimens (hypophy-
sectomized 7 days previously) did not produce any response in the
pituitaryless frogs.

III. Quantitative Studies on the Melanophore Hormone of the
Pituitary of Fundulus

Having demonstrated the presence of the melanophore-expanding
autocoid in the blood, and the inability of the denervated melanophores
to expand fully in the absence of this hormone from the circulation, it
was necessary to determine quantitatively whether the pituitary could
elaborate sufficient hormone to affect the denervated cells. Thirty white-
adapted hypophysectomized specimens containing pale denervated bands
were injected intraperitoneally in groups of 6 animals, each with various
known amounts of melanophore hormone prepared from commercial
sheep pituitary powder. All experiments in this section with sheep
melanophore hormone were performed with dilutions of the same stock
solution. Quantitative treatment is obtained by expressing the injected
dosage in terms of gram equivalents of commercial pituitary powder.
Fish of equal weight (approximately 10 grams) were chosen and the
specimens after injection were, of course, returned to the white back-
ground for the duration of the experiment. The minimal dosage neces-
sary to darken a pale band was found to be 1 mg. equivalent. In no
case did the innervated cells expand although as much as 1 gram
equivalent was injected. Doses higher than this amount were some-
times followed by death, and the usual darkening of the entire animal.
Thus the melanophores of a white-adapted fish, maintained in a con-
tinually contracted state by efferent motor impulses from the C.N.S.,
seem to be absolutely insensitive to the melanophore-expanding hormone.
Intraperitonal injections of various known dilutions of the same stock
solution into hypophysectomized frogs disclosed that the minimal dose
necessary to cause the beginning of melanophore expansion in the web
of the legs was 0.01 mg. equivalents. The minimal effective dose for
the denervated tail melanophores of Fundulus is thus 100 times greater
than that for the melanophores of the frog. This difference in sensi-
tivity is especially marked when the weights of the two animals are
compared (frog 35-40 grams, Fundulus 10 grams) and when the num-
bers of reacting cells are compared. The two-millimeter denervated
caudal band contains roughly 7,000 melanophores, while the entire skin
of the frog, although no computations were made, contains easily many

HYPOPHYSEAL HORMONE IN FUNDULUS 141

times this figure. These experiments seem to explain the negative
results of Mills and Parker.

The pituitary of an ordinary sized Fundulus (10 cm.) when ex-
tracted with distilled water contains approximately four frog units.
(We define the frog unit as the amount of hormone contained in 0.2
ec., which when injected into the dorsal lymph sac of hypophysectomized
frogs produces a reaction (melanophore expansion) during 3 hours.
The period of 3 hours is measured as the time intervening between the
time of injection and the time at which the animals regain their condi-
tion of pallor.) However, if a pituitary is extracted with a small
amount of N/10 NaOH, boiled, and neutralized to pH 7 with N/10
HCl, the gland assays at 100 frog units. The potentiation of the
melanophore hormone by alkali is thus about twenty-five fold, in agree-
ment with the results of Stehle (1936). The equivalent of 1 Fundulus
hypophysis, extracted with distilled water, is ineffective in darkening
a pale denervated band of a normal white-adapted animal. However,
the band may be made fully dark with the equivalent of 4%—-o of a
Fundulus pituitary when extracted with alkali. Thus there appears to
be sufficient hormone in the pituitary of this fish to aid in the normal
process of expansion of denervated melanophores. Similarly, blood of
one dark Fundulus, when extracted with 50 per cent and 70 per cent
ethanol, and then boiled with 1.0 cc. of N/10 NaOH, and neutralized
to pH 7 is also effective in evoking a darkening of a pale band in a
normal white-adapted fish. Thus, there appears to be sufficient melano-
phore hormone circulating in the blood of Fundulus to establish it as a
normal agency in the chromatic physiology of this fish.

SUMMARY

1. After hypophysectomy in Fundulus, denervated melanophores
cannot exhibit the normal black-background response. Normally inner-
vated melanophores are only slightly affected.

2. The inability of the denervated melanophores in hypophysec-
tomized animals to expand completely is due to the absence of the hypo-
physeal melanophore hormone from the blood.

3. There is sufficient melanophore hormone in the pituitary and in
the circulating blood of Fundulus to establish it as a significant agency
in the melanophore responses of this teleost.

BIBLIOGRAPHY

Asotin, L., 1925. Arch. mikr. Anat., 104: 667.
Asramowi7z, A. A., 1935. Proc. Nat. Acad. Sci., 21: 137.
AsraAmowirz, A. A., 1936. Biol. Bull., 71: 259.

142 A, A. ABRAMOWITZ

AsrAMowltz, A. A., 1937. Science, 85: 609.

Auten, B. M., 1916. Science, 44: 755.

ATWELL, W. J., 1919. Science, 49: 48.

Desmonp, W. F., 1924. Anat. Rec., 29: 103.

Hewer, H. R., 1926. Brit. Jour. Exper. Biol., 3: 123.

Hocsen, L. T., 1924. The pigmentary effector system. Edinburgh.

Hocsen, L., anp D. Stomeg, 1936. Proc. Roy. Soc., 120: 158.

Jores, A., 1933. Zeitschr. exper. Med., 87: 266.

Kuetnuouz, L. H., 1935. Biol. Bull., 69: 379.

Kue1nuorz, L. H., 1936. Proc. Nat. Acad. Sci., 22: 454.

Lunpstrom, H. M., anp P. Barp, 1932. Biol. Bull., 62: 1.

Matruews, S. A., 1933. Biol. Bull., 64: 315.

Mus, S. M., 1932. Jour. Exper. Zool., 64: 245.

Nosie, G. K., anp H. T. Braptey, 1933. Biol. Bull., 64: 289.

OpiornE, J. M., 1933. Proc. Nat. Acad. Sci., 19: 745.

Parker, G. H., 1932. Humoral Agents in Nervous Activity with Special Refer-
ence to Chromatophores. Cambridge, England.

Parker, G. H., 19340. Jour. Exper. Zool., 69: 199.

Parker, G. H., 1934b. Jour. Exper. Biol., 11: 81.

Parker, G. H., 1935. Quart. Rev. Biol., 10: 251.

Parker, G. H., 1936. Color Changes in Animals in Relation to Nervous Activity.
Philadelphia, Pa.

Parker, G. H., anp H. Porter, 1933. Jour. Exper. Zool., 66: 303.

PrzipraM, H., 1932. Zeitschr. vergl. Physiol., 17: 565.

Sir, P. E., 1916. Science, 34: 280.

SparetH, R. A., 1917. Jour. Pharm. and Exper. Ther., 9: 356.

SreH te, R. L., 1936. Jowr. Pharm. and Exper. Ther., 57: 1.

SwincLe, W. W., 1921. Jour. Exper. Zool., 34: 119.

VEIL, C., 1937. Compt. Rend. Soc. Biol., 124: 111.

von Friscu, K., 1911. Arch. ges. Physiol., 138: 319.

Wyman, L. C., 1924. Jour. Exper. Zool., 39: 73.

Youn, J. Z., 1935. Brit. Jour. Exper. Biol., 12: 254.

Pat owthCh On wt S NITROGEN CONTENT ON THE
DECOMPOSIMON OF Et POLYSACCHARIDE
PGW Ci Or HON DRUS CRISPUS

MARGARET RUTH BUTLER

(From the Woods Hole Oceanographic Institution, Woods Hole, Massachusetis 1)

It has been frequently pointed out (Keys, Christensen and Krogh;
Johnson; Waksman and Carey, et al.), that the bacterial decomposition
of organic matter in stored sea water is limited by the supply of avail-
able nitrogen. A rather striking illustration of this arose from an
experiment in which the organic material used was the purified poly-
saccharide extract of the marine alga, Chondrus crispus, which provides
as well a natural source of nitrogen.

METHOD

Oxygen consumption was used as the index of decomposition. The
customary procedure of storing glass-stoppered bottles (of approxi-
mately 200 ml. capacity) under water, at room temperature (20-25° C.),
in the dark, was followed. The oxygen remaining was determined by
the Winkler titration method, at various intervals. The results given
are averages of at least two bottles, more often of three.

EXPERIMENTAL

In a preliminary experiment, the rate of decomposition of the ex-
tract was compared with that of the plant itself. The results, given
in Table I, show the whole plant to be more readily decomposed than
the extract. This fact was attributed to the lower quantity of nitrogen
in the latter. For instance, plants containing 1.20 per cent nitrogen,
on a dry weight basis, might yield an extract having only 0.30 per cent
of nitrogen (Butler, 1936). The method of preparing the extract was
described in a previous paper (Butler, 1934).

Table II shows the effect of various amounts of nitrogen, added as
nitrate, on the decomposition of the extract.

It is evident from this table that the decomposition was increased
in proportion to the amount of nitrogen added, up to a certain point

1 Contribution No. 144.
143

144 MARGARET RUTH BUTLER

only; other factors then apparently become limiting. In seven days the
decomposition occasioned with the addition of 0.042 mgm. of nitrate
nitrogen was as great as with 0.140 mgm., at least as measured by
oxygen consumption. The fact that, with the lower quantity of nitrate,
seven days were required to bring about the same amount of decomposi-
tion that was accomplished in three days with the larger quantity, sug-
gests a possibility of the nitrogen being utilized more than once. This,
however, is not likely, for von Brand, Rakestraw and Renn (1937)
have shown that decomposition stops only after an interval varying

TABLE I

Decomposition of Chondrus crispus and its Polysaccharide Extract

Oxygen consumed (ml. per liter)

Mg.
Material added per
Boe 1 day 2 days 3 days 5 days 6 days
Sea water only....... — 0.09 0.24 0.36 — 0.57
Ghondruss52.-0- bee 3 1.11 1.71 1.89 — 4.32
Oa erase Ade, 5 — 1.26 — 3.48 —
POA ese ea nema 10 1.38 3.60 4.32 — 5.46*
eu ye A ek ee 20 — 3.18 — 5.88* —
SXtractenscc no tater 25 0.18 0.54 0.81 — 1.47
Ae tithe ihe See ee eee 5 — 0.81 — 1.92 —
ee Are cu ee bean eee 10 0.66 1.17 1.68 — 3.36
Sle ip eee Rae aa 20 — 1.68 — 5.31 —

* All oxygen consumed.

from 8-20 days. A more probable explanation is that the bacterial
population supported by the smaller quantity of nitrogen has a much
lower total metabolism than that of the abundant population when
nitrogen is more plentiful.

The bacterial numbers given in Table I] were furnished by Dr.
Margaret Hotchkiss at the Woods Hole Oceanographic Institution, and
are simply included here as a matter of record, but will not be discussed.

Having seen the effect on the breakdown of the Chondrus extract
of adding various quantities of inorganic nitrogen, the effect of different
quantities of organic nitrogen became of interest. Since extracts from
different collections of Chondrus contain varying amounts of nitrogen
(Butler, 1936), it was a relatively simple matter to study the effect of
this naturally occurring organic nitrogen on the decomposition of the
polysaccharide complex. For the purpose, a series of bottles was pre-
pared in which each group contained the extract from a different monthly
collection of Chondrus plants. As each extract from the plants col-

N CONTENT AND DECOMPOSITION IN CHONDRUS 145

-lected at monthly intervals contained a different quantity of nitrogen,
a natural series was provided, in which each member had a different
nitrogen content. Furthermore, this nitrogen is in that form in which
it most probably occurs under natural conditions, in the plant itself.
Great care was taken to make up each extract in exactly the same
concentration, 25 mgm. of extract per liter of sea water. This insured
that any individual sample was directly comparable with all the others
in the series. Oxygen determinations were made after one day of
storage. The results are given in Table III along with the percentage

TABLE II

Effect of Added Nitrate on Decomposition of Chondrus Extract

Oxygen consumed No. bacteria
Extract added Nitrate nitrogen (Gaul ssa) We
(mg. per bottle) (mg. per bottle)
3 days 7 days 3 days
0 0 0.53 0.74 —
0 0.014 0.55 0.80 90,000
0 0.042 0.55 1.24 —
5 0 1.32 4.07 560,000
5 0.014 3.27 5.02 980,000
5 0.042 4.28 5.28* —
5 0.140 5.28* 5.28* 3,200,000

* All oxygen consumed.
In this experiment the sea water was enriched by the addition of 1 mg. KzHPO,

per liter.

of nitrogen in each sample. It is readily seen that a correlation exists
between the two and is most direct where nitrogen is low. This seems,
therefore, to furnish definite evidence of the limiting effect of nitrogen
on the decomposition of, at least one type of, organic material such as
occurs in the sea. It suggests also that the low level of nitrogen during
the summer may be partially responsible for the slow rate of decomposi-
tion.

While in the present discussion it is assumed that the decomposition
is brought about by bacteria, the possibility that the oxygen is consumed
by other organisms is not excluded. The water used may, undoubtedly,
have contained larger organisms. It was surface water collected in
Vineyard Sound and filtered through a No. 25 net of bolting silk. The
bacterial considerations cannot be discussed here although evidence was
obtained for the bacterial nature of the decomposition. However, the
important fact remains that the consumption of oxygen on storing the

146 MARGARET RUTH BUTLER

polysaccharide extract of Chondrus in sea water is correlated with the
amount of nitrogen which it contains naturally.

JOABiEE es LED

Oxygen Consumption of Various Extracts of Chondrus crispus *

: Oxygen consumption
Extract Nitrogen occasioned in one day

(per cent dry weight) by addition of extract
(ml. per liter)

Januanycccicucmt eset ies & snes 1.92 0.92
We bruat ys cose cus eee sarees 2.30 1.05
Marchiss: 490 tepeee eee cee: 2.40 1.21
TNO) gl tem wpuenaerncs es anid sc ail Aone, aa 2.18 este
1M EE Genero Niele so Bis gis aN Ae eee 1.40 1.09
June. nese 2 Pa SN eR ee Che cC cine ate 0.82 —

a 2 seas oer rece Nerant fa le 0.34 0.23
HSU fea 1) ene OR TENG vy © 2, RCRD ay eR 0.37 0.27
September soe ace no cusses 0.32 0.20
OCOD A eae hn eee eek 0:39 0.24
INGVvembertten vs aenmeee eek 1.28 0.65
November. 3 he eerie aie 0.80 0.51
January. o de eer er ee toes 2.34 0.80

* The sea water used in this experiment showed an oxygen consumption of 0.55
ml. per liter in one day.

SUMMARY

The decomposition of Chondrus crispus, as measured by oxygen con-
sumption, has been shown to be more readily accomplished on storing
in sea water than that of its polysaccharide extract. This has been
attributed to the higher nitrogen content of the former.

Inorganic nitrogen added to the polysaccharide extract of Chondrus
increased its decomposition.

Samples of the extract containing different quantities of nitrogen
have been found to decompose in direct proportion to the amount of
nitrogen which they contain.

REFERENCES

Butter, M. R., 1934. Biochem. Jour., 28: 759.

Butter, M. R., 1935. Biochem. Jour., 29: 1025.

Butter, M. R., 1936. Biochem. Jour., 30: 1338.

Jounson, F. H., 1936. Jour. Bact., 31: 547.

Keys, A., E. H. Curistensen, AND A. Krocu, 1935. Jour. Mar. Biol. Assn, 20:
181.

von Branp, T., N. W. RaxkestRAw, AND C. E. Renn, 1937. Biol. Bull., 72: 165.

Waxsma\, S. A., C. L. Carey, AnD H. W. Reuszer, 1933. Biol. Bull., 65: 57.

Waxsma\, S. A., AnD C. L. Carey, 1935. Jour. Bact., 29: 531.

THE DIFFERENTIAL EFFECT OF ENVIRONMENTAL FAC—
TORS UPON MICROBRACON HEBETOR SAY
(HYMENOPTERA: BRACONIDAE) AND ITS
HOST, EPHESTIA KUHNIELLA ZELLER
(LEPIDOPTERA: PYRALIDAE)

II]. Errect oF THE STING OF THE PARASITE AND OF Two CHEMICAL
AGENTS ON THE RESPIRATORY RATE AND QUOTIENT OF
THE Host Larva (E. KUHNIELLA ZELLER)

NELLIE M. PAYNE

Many species of parasitic Hymenoptera paralyze their hosts by sting-
ing them, after which they lay eggs in or near the host. Although the
gross effect of the sting on the host has long been known, yet the precise
effect has been little studied. In this paper the change in oxygen con-
sumption of the host larva after being stung by the parasite is meas-
ured and these results compared with those produced by ether and by
orthodichlorbenzene.

Metnmops AND MATERIALS

The respiratory exchange of full-grown larve of Ephestia kiihniella
Zeller was measured with the manometer described by Krogh (1914)
and modified by Bodine and Orr (1925). Baskets of copper gauze
lined with cotton were hung on the glass hook which extended from the
open end of the manometer. Larve placed in them were unable to
move about but may have developed some muscular tension by struggling
against the cages. Since the larve tended to be sluggish when un-
disturbed, they probably developed but little tension. Manometers were
calibrated by weighing the mercury required to fill them. They were
run for periods of two and one-half hours.

Larve to be paralyzed were exposed to adult females of Micro-
bracon hebetor Say which pierce the host larve with their ovipositors,
then lay eggs on or near them. Since the parasite does not begin to
feed on the host until these eggs hatch, the host larve suffer no loss of
material from the parasitic attack. Occasionally a host larva will
writhe and struggle when the wasp is stinging it and for some time
afterwards. Stung larve were placed in the manometer only when
they were quiet.

147

148 NELLIE M. PAYNE

Larve were also paralyzed by ether and by orthodichlorbenzene. A
dosage of the reagent that would paralyze one-half the larve used and
from which recovery took place in at least twelve hours was chosen.
Since there were wide differences between the susceptibility to chemical
agents even among larve hatched from the eggs of the same female,
considerable experimentation was required to find a suitable dose.
Larvee were given sufficient anesthetic to paralyze but not to kill them.
Such an amount may be designated as the median-paralytic dose.
Larve to be paralyzed by a chemical agent were placed in lots of twenty
in a cylindrical wire cage made of screening. These cages were
fastened to a hook blown onto a glass tube extending through the
ground-glass stopper of a fumigation flask. The reagent was intro-
duced into the flask through a glass tube which extended but a short
distance below the stopper. Both this tube and the longer one on which
the insects were hung were furnished with ground-glass stopcocks. The
flasks were shaken from time to time.

After the median-paralytic dose had been established for both ether
and orthodichlorbenzene, individual insects were transferred directly
from the fumigating flasks into the manometers, where oxygen con-
sumption was determined. The results obtained directly after the larve
were treated are recorded under “ Period I” in the tables. A second
group of determinations, made six hours after treatment, is recorded
under Period II. Larve removed from the flasks directly and those
which had been in the manometer were kept in individual cages to see if
they recovered. Only those measurements obtained from larvz able to
resume normal movement and feeding and finally to pupate appear in
the tables. Thus respiration of paralyzed larve but not of dying ones
was measured.

Oxygen consumption was measured at seven different temperatures
namely al? "Cm G@ae 2027 @..925° Ce 27°C. 30" Caran ome
Manometers were placed in water baths which controlled temperatures
to 2210. 5 25©:

Determinations were repeated until results from three larve, all of
which recovered from the anesthetic, were obtained. Oxygen consump-
tion of three individual larve paralyzed by parasitic sting was measured.
Determinations on the oxygen consumption of three normal larve, un-
treated with anesthetics and not exposed to the attacks of the parasite
were used as control. Carbon dioxide production was determined for
each of the larve used and the respiratory quotients calculated. Re-
sults in the tables are averages of three determinations each. The
temperature coefficient or Q,, was calculated between the intervals of
temperature at which determinations were made.

EFFECT OF FACTORS UPON MICROBRACON HEBETOR. III 149

RESULTS

Oxygen consumption of normal untreated larve increased directly
with the temperature until 32° C., at which point it decreased sharply.
The respiratory quotient, however, showed little or no change until the
temperature rose to 32° C. Between 30° C. and 32° C. the respiratory
quotient dropped from 0.91 to 0.83 (see Table I). Since the full life
cycle of the Mediterranean flour-moth cannot be completed at tempera-
tures much above 32° C., it would appear that the lowering of the

WARD I

Comparative data on oxygen consumption of normal full-grown Ephestia larve and
Ephestia larve stung by Microbracon hebetor

an ee ca Say Qio Respiratory quotient
in degrees

CERN gals Normal Stung Normal Stung Normal Stung

larvee larve larve larve larve larve

10 24.2 18.6 0.91 0.92

15 36.4 18.2 2.18 1.00 0.92 0.89

20 42.0 21 1.73 1.12 0.89 0.91

25 53.6 25.3 *1.47 *1.32 0.92 0.88

(2.54) (2.04)

27 61.5 Os) 71.04 71.72 0.93 0.93

30 72.3 31. 1.72 1.476 0.91 0.87

32 54.7 26.3 0.83 0.81

* Between 15° C. and 25°C. ( ) Between 20° C. and 25° C.
+ Between 20° C. and 27° C.

respiratory quotient into a range indicating protein oxidation indicates
some degree of injury.

Larve treated with ether were not only motionless but appeared soft
and flaccid. The relaxation produced by the ether was apparently re-
flected by the lowered oxygen consumption of the etherized larvee com-
pared with the normal. As the larve recovered they used more oxygen.
At all temperatures their oxygen consumption was less than that of un-
treated larve, but at low temperatures this difference was not as marked
as at high. Etherized larve showed a similar decrease in oxygen con-
sumption between 30° C. and 32° C. to that of untreated larve (see
Table II). After six hours oxygen consumption had returned nearly
to normal levels. After twenty-four hours etherized larve could not be
distinguished from untreated by either their appearance or by their
oxygen consumption. Etherized larvae from which the records were
made were able to spin cocoons and to emerge as normal adults.

150 NELLIE M. PAYNE

Directly after treatment with orthodichlorbenzene, larve of Ephestia
were tense and appeared to be in a state of rigor. They held their
heads stiffly upright in a sphinx-like position from a tense motionless
body. Even six hours after treatment the larve appeared somewhat
stiffer than normal. In contrast to the larve treated with ether, those
treated with orthodichlorbenzene showed a greater oxygen consumption
than did normal larve, over the temperature range from 10° C. to 30° C.
inclusive. (Compare Table I, Column 2 and Table II, Column 2.) At
32° C. the oxygen consumption of larve treated with orthodichlor-

‘LABEL

Oxygen consumption of full-grown Ephestia larve treated with orthodichlorbenzene
(1 mgm. per liter), and with ether (.568 gm. per liter)

Oxygen consumption in cc. per gm. per hr. Qio
Temper- :
ature in Period I Period II Period I Period II
degrees
Centi-
grade Larve Larve Larve Larve Larve Larve Larve Larve

exposed exposed exposed exposed exposed exposed exposed exposed
to ether | to CsH4Cle}| to ether | to CeHsCle | to ether | to CeHsCle} to ether | to CeHuCle

10 DMS) 24.3 23.4 252
15 S185 39.3 Sail 37.1 BO 2.12 3.04 3.2
20 37.4 47.1 44.1 43.5 1.677 1.98 1.84 1.8
25 42.7 58.5 S203 Saath *1.304 | *1.49 71.46 1.4
2.4
2
1.6

(2E28) 548). 152239) ae
27 45.6 67.4 61. 60.9 1295 72.04 lel
30 52.1 80.5 69.9 Teil 1.39 1.73 1.58
32 45.8 46.7 51.6 53.8

* Between 15° C. and 25°C. () Between 20° C. and 25° C.
+ Between 20° C. and 27° C.

benzene was less than that of the untreated: This greater decrease in
oxygen consumption may indicate that larve exposed to orthodichlor-
benzene are more susceptible to injury by high temperatures than are
normal larve.

Since larve exposed to fatal doses of orthodichlorbenzene showed
a gradual decrease in oxygen consumption during the period they were
in the manometer, they could be distinguished from larve exposed to a
non-fatal dose. A marked decrease in oxygen consumption during the
time that larve were in the manometer was interpreted as a measure-
ment of a death process rather than of the specific effect of orthodichlor-
benzene. )

The respiratory quotient of larve treated with orthodichlorbenzene
did not differ significantly from that of normal larve. In both cases the

EFFECT OF FACTORS UPON MICROBRACON HEBETOR. III 151

respiratory quotient dropped from the carbohydrate to the protein range
at 32° C. Both normal larve and those treated with orthodichlorben-
zene showed a marked decrease in oxygen consumption at the same
point as that at which the respiratory quotient decreased.

Larve stung by Microbracon closely resembled those treated with
ether. In both cases the larve were soft, relaxed and motionless.
They consumed less oxygen than either normal larve or etherized larve
throughout the range of temperature used. As measured by oxygen
consumption, the sensitivity to temperature change of stung larve was
far less than for normal larve. Thus between 10° C. and 15° C. there
was no increase in oxygen consumption by the stung larve. At 30° C.
the oxygen consumption of-the stung larve was about that of normal
larvee at 15° C. (see Table I). At 32° C. the oxygen consumption of
the stung larvae decreased from what it had been at 30° C. Also at
32° C. the respiratory quotient showed a marked decrease.

The level of oxygen consumption of the stung larve remained ap-
proximately constant for at least a week after they were stung since
measurements taken a week after their exposure to Microbracon were
nearly the same as those taken directly after their paralysis. The mag-
nitude of change produced by the Microbracon was far greater than that
caused by either of the chemical agents used. In the experiments re-
ported here, both ether and orthodichlorbenzene produced a reversible
change while the sting of Microbracon produced an irreversible change.
If the dosage of ether or of orthodichlorbenzene be increased to the
point of irreversible change the larve die rather than remain quiescent
as they do when stung by Microbracon.

A few experiments were tried to see if either orthodichlorbenzene or
ether would affect larve stung by Microbracon. As far as could be
detected from measurements on respiratory rate and quotient neither
orthodichlorbenzene nor ether had any effect on larve stung by Micro-
bracon. Furthermore, the stung larve treated with orthodichlorbenzene
did not stiffen nor those treated with ether become more flaccid. Ap-
parently the Microbracon sting had already destroyed or damaged the
mechanism on which these drugs could act.

DISCUSSION

Levels of respiratory metabolism of Ephestia larve stung by Micro-
bracon are so low and so insensitive to temperature change as to suggest
those of cells in diapause such as the “ blocked’ embryonic grasshopper
cells reported by Bodine (1934). The resting condition of the Ephestia
larve differed, however, from that of the embryonic grasshopper cells in

152 NELLIE M. PAYNE

that the rest of the grasshopper embryos could be broken with greater
ease than could that of the larve stung by Microbracon. ‘The author
has never observed an Ephestia larva recover from Microbracon sting.
Mr. H. C. Donohoe, in an unpublished communication, informed the
author that he had seen Ephestia larve recover from the sting of the
parasite but that recovery was very rare.

Although temperature rise from 10° C. to 15° C. did not change the
respiratory rate of Ephestia larve stung by Microbracon and tempera-
ture change from 15° C. to 30° C. increased the rate in stung larve only
one-third as compared to twice in normal larve, still temperature rise
from 30° C. to 32° C. was associated with a drop in respiratory rate for
the larve stung by Microbracon, as it was so associated with normal
larve. Thus the stung larve, while insensitive to changes in tempera-
ture through the range through which normal development occurs, are
susceptible to injury from high temperature.

Throughout the temperature range used in the experiments witn
Ephestia larve, the respiratory quotient of the larve stung by Micro-
bracon did not differ from that of normal larve. Both were in the
carbohydrate range from 10° C. to 30° C.; both dropped to the protein
range at 32° C. In so far as the respiratory quotient indicates the type
of metabolism, a lack of difference between the respiratory quotient of
stung larve and of normal larve would indicate the same type of
metabolism in both. Differences not measured by the respiratory quo-
tient might well occur. ,

Neither the effect of ether nor that of orthodichlorbenzene duplicated
that of the Microbracon sting. Ether did produce flaccidity in the larve
and did lower the respiratory rate. It was impossible to obtain as low a
rate with ether as the Microbracon sting produces, without killing the
larve. Orthodichlorbenzene produced a rigidity in the larve and also
increased respiratory metabolism. Neither chemical agent affected the
respiratory quotient.

Hartzell (1935) found that lesions produced by the killer wasp,
Specius speciosus Dru., in the nervous system of the cicada, Tibicen
pruinosa Say, were similar to those found in insects killed with tri-
orthocresyl phosphate and the pyrethrins. Injections of formic acid
and of acetic acid in meal worms (Tenebrio molitor L.), however, failed
to produce the lesions characteristic of paralysis.

The finding of Hartzell (1935) of lesions sufficient to be demon-
strated microscopically in the nervous system of a cicada stung by a
wasp may be correlated with the profound differences in the muscular
tonus between normal larve and stung larve. In turn these alterations

2 celia dia

EFFECT OF FACTORS UPON MICROBRACON HEBETOR. III 153

of the nervous system associated with marked changes in respiratory
rate may point to an intimate connection of the nervous system with
respiration.

SUMMARY

1. Larve of Ephestia kiihmella Zeller, when stung by the parasite
Microbracon hebetor Say, become soft, flaccid and motionless.

2. Associated with the flaccidity of the stung larvae is a greatly low-
ered respiratory rate through the temperature range used, namely 10° C.
HOMO eC.

3. Not only does the Microbracon sting lower the respiratory rate
but it decreases the sensitivity of Ephestia larve to change in tempera-
ture to such an extent that between all temperature intervals used the
Q,, of larve stung by Microbracon was less than that of normal larve.
Between 10° C. and 15° C., the Q,, was 1.

4. Orthodichlorbenzene in a median paralytic dosage (just sufficient
to cause a reversible paralysis in half the larve) produced stiffness and
rigidity in Ephestia larve. Associated with this stiffness was a marked
increase in respiration rate beyond that of untreated larvee except at
32° C. where it fell below that of normal larve.

5. Ether in a median paralytic dosage produced flaccidity in Ephestia
larve. Associated with this flaccidity was a marked decrease in respira-
tory rate, but at no temperature was the decrease produced by ether of
the same magnitude as that produced by the Microbracon sting.

6. Unlike the respiratory rate, the respiratory quotient was not af-
fected by the parasite sting nor by either of the two chemical agents
used.

7. Rise in temperature to the point at which the rate of development
of Ephestia decreases, namely 32° C., was associated with a drop in
respiratory rate and a decrease in respiratory quotient from the range
indicating carbohydrate metabolism to that indicating protein. Not only
normal larve, but also those treated with ether or orthodichlorbenzene or
stung by the parasite, showed a decrease both in rate and quotient at
O27 1G

ACKNOWLEDGMENTS

Oxygen consumption and respiratory quotient of the larve were
determined at the Zoological Laboratory of the University of Pennsyl-
vania during the years 1931-1933. Calculations and redeterminations
of some of the results were made at the Division of Entomology, Uni-
versity of Minnesota. Thanks are due to Dr. C. E. McClung for the
use of laboratory facilities at the University of Pennsylvania and to
Dr. William A. Riley for the same courtesy at Minnesota.

154 NELLIE M. PAYNE

LITERATURE CITED

Bovine, J. H., 1934. The effect of cyanide on the oxygen consumption of normal
and blocked embryonic cells (Orthoptera). Jour. Cell and Comp. Physiol.,

4: 397.
Bovine, J. H., anp P. R. Orr, 1925. Respiratory metabolism. Physiological t
studies on respiratory metabolism. Biol. Bull., 48: 1. }
Hartze.1, A., 1935. Histopathology of nerve lesions of cicada after paralysis by
the killer-wasp. Contr. Boyce Thompson Inst., 7: 421. . |
;
F

Krocu, A., 1914. Ein Mikrorespirationsapparat und einige damit aus gefihrte
Versuche tiber die Temperatur- Stoffwechselkurve von Insektenpuppen.
Biochem. Zeitschr., 62: 266.

Payne, N. M., 1934. The differential effect of environmental factors upon Micro-
bracon hebetor Say (Hymenoptera: Braconidae) and its host, Ephestia
ktthniella Zeller (Lepidoptera: Pyralidae). II. Ecological Monographs,
vol. 4. i?

AUTOTOMY IN THE BRACHYURAN, UCA PUGNAX

LEIGH HOADLEY

(From the Biological Laboratories, Harvard University)

A study of the extent to which the anomuran Porcellana platycheles
will autotomize its legs on single stimulation of successive appendages
has shown (Hoadley, 1934) that there is a clear-cut and fundamental
difference between the behavior of males and of females bearing eggs.
This form is ideal for such experiments in that gently seizing the leg
between the points of blunt forceps will ordinarily be followed by auto-
tomy. Males tested in the above fashion autotomized an average of
5.57 + legs per animal (69.6 per cent), the distribution of the autoto-
mies being 4 for the first four trials and 1.57 -+ for the second four.
Quite in contrast to this, the gravid females cast an average of two legs
in the eight trials (25 per cent), the distribution being 1.5 and 0.5 in the
first and second four trials respectively. Unfortunately but one female
not bearing eggs was available at the time of the examination of this
material, but a test of that animal showed five legs autotomized (62.5
per cent) (distribution 4 and 1), a figure which compares more favor-
ably with the condition encountered in the male than with that of the
remaining females. The suggestion was made, therefore, that the func-
tional state of the animal bearing eggs was different from that of the
male and that of the non-gravid female and that this difference was
directly reflected in the extent of the autotomization. The present re-
port is based on an examination of autotomy by males, egg-bearing
females, and females not carrying eggs, in the brachyuran Uca pugnax.

The occurrence of autotomy in the decapod Crustacea and the meth-
ods by which this is accomplished have been examined in detail by Wood
and Wood (1932). That of the fiddler crab, Uca pugnax, has been
found by them to be a true autotomy in contrast to autospasy and auto-
tilly, which may be of great importance in other forms. When the
autotomy of Uca is compared with that of Porcellana described above,
however, it is at once evident that while Porcellana casts its legs on the
slightest provocation and hence care in handling the animal is essential,
Uca casts its legs only after injury so that a relatively harsh treatment
is necessary to induce the reflex.

The animals used in the following experiments were obtained from

155

156 LEIGH HOADLEY

the shore of Rhode Island* and brought immediately to the laboratory
where the experiments were performed. Two lots of animals were
worked with, one obtained in August of 1934 when the females were
not bearing eggs and the other in June of 1935 when over half of the
females were carrying eggs attached to the abdomen. All of the animals
used in the experiments were mature. It was necessary in some of the
experiments to use a few animals which had already cast one of the legs
but in no case was an animal with more than one leg lacking employed.

Autotomy was induced by injuries of two sorts. In the first a limb
was grasped and crushed between the jaws of a pair of plier forceps
with a quick motion in order that the leg should actually be held as short
a time as possible. Such injuries were generally made in the carpus
though the exact position on the segment varied slightly and in several
instances the injury was in the distal portion of the meros. It was soon
found that holding the animal either by the body or by the leg tested
facilitated the autotomy of the member so that the animal was not held
during the infliction of the injury. That fact, together with the effort
to accomplish the injury in as short a time as possible, led to slight
variations in the actual extent of the trauma. In the male, the large claw
of the cheliped was crushed across the middle at the base of the dactylus
and consequently the injury there was relatively great.

The second type of injury inflicted to induce autotomy was cutting.
Legs were cut with a heavy pair of scissors at or near the joint between
meros and carpus, removing the carpus and propus. The claw of the
cheliped of the male had to be cut by means of a small pair of bone
forceps, the cut being located in the same region as the injury by crush-
ing mentioned above. Both types of experiment were carried out in
clean moist crystallizing dishes; in addition, crushing experiments were
performed in crystallizing dishes, the bottoms of which were covered by
filter paper. Only females bearing eggs and males were used in the
cutting experiments.

The results of the experiments have been summarized in Table I.
The letters following the year indicate the order in which the experi-
ments for that year were done. While every effort was made to per-
form the experiments in the same way, it is but natural that those done
in the same year should be most similar and that those done successively
in any one year should be most nearly alike in execution. Thus, of the
experiments in which appendages were crushed on glass, 34A (dg’s) and
34B (9’s) are comparable, as are also 35A (9’s with eggs) and 35B
(3’s) on the one hand, and 35G (9’s with eggs), 35H (9’s without

1] wish to thank Dr. F. C. Chace for the Uca which he was so kind as to
collect for me.

AUTOTOMY IN UCA PUGNAX ey

eggs) and 35I (0’s) on the other. It is evident when these are com-
pared that the stimulation in 35A and 35B was not as effective as in 35G,
35H and 351. This will be dealt with in some detail below. The results
themselves seem to be comparable, however, for when the number of
legs autotomized by the males is compared with the number autotomized
by the female bearing eggs it is found to be very similar in the two cases

Sec-| series| ani- 5 5 total

trials | tri- Q P
ond 5 mals | trials | trials

als

a 41 23 | 64 1.81—| 34A 9 |4.56— | 2.56—] 7.1+ o/ 2 =1.22+ EAE s.
o’s=

2 37 21 | 58 | 1.76+] 34B il | 3.7 2.1 5.8 Q’s= 6.65
pl = —]| 2 eggs=4.4

QOwith | 15 5 | 20} 3.0 385A 5 |3 1 40 |oc/2 eges=1.60
|
° eggs
28 ase ies
=| a ot 19 13 | 32 1.46 35B 5 | 3.8 2.6 6.4
& Owith | 16 8 | 241 2.0 35G 5 | 3.2 1.6 4.8 o/ 9 =1.04

eggs I o/ 2 eggs=1.62

9 16 | 14 | 30] 1.144) 35H

oi 23 | 16 | 39 | 1.44—/ 351 5
foil 21 | 15 | 36] 1.40 | 34C 5 | 4.2 3.0 7.2 o/ 9 =0.94

|
|

Sie 9 20 | 17 | 37] 1.18—| 34D A SHE | Ge

bf ASA el a aN aa ea

= | Qwith| 15 | 9 | 24] 167 | 358 Slo Snel ats 3/2 =1.16¢

58 egps N.B. anterior appendages first

o# ——|—-— — —— in every case
o | 17 | 11 | 38] 154+] 35F*| 5 134 [22 | 5.6

2 Galeton |i 5) |i laulmae2) ae) isan eqn oroea ll iiom mist

g cf ah ere ar ce Al een sc LS |e Mee aD

a |Qwith) 11 | 1] 12/110 | 35C] 5 /22 2 124 Io/2 eggs=15

Bic eges ag | S| al fae Ral it a

2 oe 12 | 6/18] 20 | 35D} 5 |24 1/12 3.6

Average
o/ 2 =1.067—
o/ 9 Eggs=1.57 +

* The large claw was injured first in each of the experiments of this series;
see discussion in text.
+ Omitted in the average.

being expressed by the ratios 1.60:1 and 1.62:1 respectively. When
the average total number of legs autotomized by the three classes of ani-
mals is examined, it is found that, with one exception, the largest num-
ber is cast by the males; the females without eggs come next; the fe-
males with eggs cast the smallest number. It will also be noted that the
females without eggs cast a relatively smaller number of legs in August

158 LEIGH HOADLEY

than they did in June. Compared with the males, the index for such
animals is 1.22 in August as compared with 1.04 in June, the behavior
at the latter time being similar to that of the males. It appears quite
evident from the tabulated data that the female bearing eggs behaves
quite differently in autotomizing its legs from the male and from the
female not bearing eggs. It is also evident that the greater difference
is to be found in the second five trials. This can be appreciated most
easily by examination of the column in which the relation between the
number of legs lost in the first five trials is compared with the number
lost in the second. If the number lost in each series is approximately
the same the ratio will approach 1 while if the number diminishes mate-
rially it will rise. In all of the first group the higher quotients are to be
found in animals which are females and carry eggs.

The second type of experiment differed from the first only in that
the bottom of the dish in which the animals were placed during the ex-
periment was covered with filter paper. Examination of the totals
would appear to indicate that the behavior in this case is similar to that
in the preceding. The exception is found in the case of the males of
35F which showed an average total of 5.6 which is well below that of
the previous series and also well below those of the males and of the
females without eggs used in 34C and 34D. Reference to the protocol
of the experiment shows, however, that in this series (35F) the large
claw was the first to be injured and this has evidently had a marked ef-
fect on the subsequent behavior of the individuals. This might be ex-
plained either on the basis of the extent of injury or on the basis of some
aid in autotomy rendered by the large claw. While the large claw is
used directly to rid the animal of the injured member at times, this is by
no means usual and could not, I believe, account for the discrepancy in
the results. It is also of interest to note that when the ratio between
the autotomies in the first five trials and the second five is examined in
this entire group it is found that the number of autotomies is relatively
high in the second five and that hence the index is lowered. Again, this
is not true of the males in 35F. While no quantitative data are available
on this point it should be mentioned that on the filter paper, the amount
of bleeding which takes place is greater than when the animal is in the
clean glass dish. The possible significance of this will be discussed be-
low, more particularly in relation to the cutting experiments. As in the
previous group of experiments, the relation between the total number of
autotomies by males and by females not carrying eggs is approximately
the same.

Only three sets of experiments were made cutting the appendages of
the animals upon glass. Two of these were done in 1935 and one in

AUTOTOMY IN UCA PUGNAX 159

1934. Comparing the males of 34E with those of 35D it is found that
in both cases the number of legs autotomized is very low, being (as
averages) 3.2 in the first instance and 3.6 in the second. These figures
are very similar and are characteristic of the individuals of each series
of experiments. The females bearing eggs showed a similar low ay-
erage of 2.4. The immediate result of the experimental procedure dif-
fers from that recorded above in that there is far more bleeding after
cutting than after the crushing previously discussed. When the males
of the 1935 series are compared with the females it is found that the
relation approaches that of the crushing experiments, being 1.5. When
the number of autotomies in the first five trials is compared with the
number in the second five, it is found that for the males the quotient is
relatively high, being 2.2 and 2.0; but this is low compared with that for
the females bearing eggs, which is 11.0, the highest encountered in any
of the experiments. The relation between the males of the two years is
1.11, which is close agreement.

The data obtained in the experiments may be dealt with in two ways.
It is possible to compare the total number of legs cast by each group ac-
cording to the animals composing it, or the number of legs cast in the
first five trials may be compared with the number cast in the second five
and the quotients examined. The results of each method yield informa-
tion which is interesting and hence will be considered separately.

The relation between the total number of legs autotomized by com-.
parable groups of animals will be considered first. The most casual
examination of the table will show that the males and the females with-
out eggs consistently cast more appendages than did the gravid females.
The difference is so great that when the males are compared with fe-
males bearing eggs which were tested at the same time the ratio is as
1.57 + (average) is to 1. For the reasons stated in the table the re-
sults of tests 35E and 35F have been omitted from this average. It is
of especial interest to note that while the method employed may have a
great effect on the totals obtained it has not modified the relation between
the totals (see “ cutting on glass ”’).

When the autotomies of females not bearing eggs are compared with
those of the males it is found that in the 1935 tests (June) the results
for the two sexes are essentially the same. In the tests of the 1934
series it might at first be thought that the averages in 34A and 34B
showed a difference in behavior. A comparison with the results of 34C
and 34D which are also comparable leads to the conclusion that the
discrepancy in the former case is the result of unequal stimulation rather
than of a difference in the animals. This is not unlikely for 34A and
34B were the first Uca to be tested. When the results obtained in all

160 LEIGH HOADLEY

of the tests are averaged it is found that the relation between autotomy
by the male and by the non-gravid female is as 1.067 — is to 1, the
difference not being significant. The variation in results between more
widely separated tests of the same year and between tests of different
years is to be explained on the basis of manipulation.

A comparison of the number of autotomies in the first five trials
with the number in the second five should prove most interesting and
should yield important information, for by this method groups of stimu-
lations of individuals are dealt with and these represent tests which are
most similar in every respect. Several general statements may be made
based on the results of this comparison. It may be stated definitely that
in comparable experiments the relative number of autotomies in the
first five trials in females bearing eggs is consistently higher than in the
remaining females or in the males. On the other hand, the differences
encountered in this ratio between animals of this second class (34A and
B; 35B, H, and 1) of the first group alone is fairly large as. is the dif-
ference between the gravid females (35A and G). While both the
latter values are above those of the first class, the difference between
the highest value of the first class and the lower value of the second is
relatively small. The ratio in itself becomes more significant after the
two components are examined. Insofar as the first five trials are con-
cerned the results are much the same whether the crushing be on filter
paper or on glass; as for the second five, the average on the filter paper
is in all cases high. This would lower the value. The reverse is par-
ticularly marked in the cutting experiments where in males the average
of the first five trials is approximately half that in the crushing experi-
ments while that of the second five is nearer one-third. The females
with eggs behaved much as the males in the first five trials but in the
second five autotomy was practically suppressed so that the relation be-
tween the two rises to a value of 11.0. In this connection it should be
stated that the results of a few experiments on cutting on filter paper
indicate that in that case also there is a similar reduction in the number
of autotomies in males. In 35F we find that both the first and the sec-
ond set have been reduced, the second more than the first. This results
in a value for first/second of 1.54 + which approaches that of the fe-
male bearing eggs as did the total number of autotomies (5.6 av.).

It has already been mentioned above that relatively harsh treatment
is necessary to induce autotomy in Uca. Sensory receptors associated
with hairs are present in the cuticle covering the limbs. The tactile
stimulation of the appendage of Porcellana is sufficient to induce the
casting of a leg but in Uca the actual injury of the member is essential.
This, in turn, must result in much more violent stimulation of afferent

AUTOTOMY IN UCA PUGNAX 161

sensory nerves. As may be seen in the data presented above, the degree
of stimulation in different experiments differs and with this variation
there is an accompanying variation in the number of autotomies. Evi-
dence of the same sort may be derived from the comparison of cutting
experiments and crushing experiments. In the former the number of
autotomies is far lower than in the latter. Similarly it might be ex-
pected that the extent of stimulation both in degree and in duration
would be greater in the case of crushing than in the case of cutting.
This probably does not account completely for the discrepancy in the
two cases as the amount of bleeding observed differs.

The blood of the Crustacea coagulates very readily. Ordinarily
there is relatively little loss of blood following traumatic injury to the
limbs. It has been stated above that there is a greater loss of fluid
when the appendage is crushed on filter paper than when crushed on
moist glass. The loss is far greater when the appendage is cut at or
near the base of the carpus. With successive stimulation the latter type
of injury eventually results in the loss of considerably more blood than
does the crushing. This may contribute to the difference in the be-
havior of the two types of material.” Similarly the extent of the injury
to the large claw on crushing must result in the loss of more of the body
fluid than would injury to other appendages. This may in part account
for the reduction in the total number of autotomies in series 35F, The
amount of bleeding following autotomy, i.e. at the breaking joint, is
relatively small so that the act in itself tended to restrict the loss of body
fluid by the injured animals in the majority of the crushing experiments.

The autotomy reflex of the Crustacea as generally conceived involves
the transmission of stimuli along the sensory neurones and transmission
of motor impulses by the motor neurone to at least the autotomizing
muscle. The path is presumably completed centrally in the ventral
ganglion. There are very few motor nerve fibers in the segmental
nerves of the crab, indicating that whole muscles and possibly even dif-
ferent muscles may be innervated by collaterals of a single neurone. In
the crab the fifth to the thirteenth lateral nerves all have their origin in
a common ventral ganglionic mass, the ninth to the thirteenth innervat-
ing the five appendages as well as the muscles of the body wall of these
segments. In the experiments reported here as well as those on Porcel-
lana referred to above, the reflexes which are activated by successive
stimulations have a cumulative effect and lead to an inhibition and
eventual failure of the response even when the stimulations involve re-

2 Dr. John H. Welsh informs me that some collectors bleed crabs in order to

prevent their casting appendages when put into przservatives. He himself has
used this method successfully.

162 LEIGH HOADLEY

ceptors situated in different appendages. In the male of Porcellana the

inhibition first became evident after the fifth successrve appendage was
stimulated ; in the male of Uca the inhibition is usually noted after the
fourth stimulation. In females bearing eggs, inhibition appears be-
tween the first and the second trials in Porcellana, and after the third in
Uca. Females not bearing eggs appear to behave as males in Uca and
the indications are that the same is true for Porcellana. ‘That there is
actually an inhibition and that it is gradually built up is demonstrated by
the fact that without exception the number of autotomies in the first five
trials is greatly in excess of those in the second five. It is also apparent
that the inhibition becomes effective more rapidly in the female bearing
eggs than in the male for the relation between the number of legs cast
in the first five trials and in the second five yields a higher quotient in
the first case. These facts suggest strongly the formation of a block
most easily explained in terms of a chemical inhibitor. This inhibition
may be effected in the periferal mechanism or in the fused central
ganglionic region. The fact that bleeding tends to reduce the number
of autotomies also leads us to the conclusion that the inhibitor is of a
chemical nature and that it is readily eliminated through the blood, ac-
cumulating more rapidly when the amount of blood in the body is
reduced.

One additional observation deserves mention in that it, too, suggests
a mutual effect, though in this instance the effect is in terms of stimula-
tion rather than of inhibition. In several instances a delayed autotomy
has been observed which took place on the stimulation of another ap-
pendage. This is well illustrated by a case in which the third leg stimu-
lated failed to be cast at the time but gave a second response which was
successful when the fifth successive leg was stimulated. Both reflexes
were typical and the leg was eventually autotomized in the usual fashion.
Apparently the nerve impulses in the neighboring units were responsible
for the autotomy, possibly through the production of diffusable humoral
agents which, in turn, effect the activation of previously stimulated
components.

In conclusion it should be emphasized that the evidence for the exist-
ence of inhibiting and stimulating substances is indirect and that they
have not been demonstrated. However, the behavior of both Porcellana
and Uca may be most readily understood on some such basis. The
method of performing the experiment has a definite influence on the
kind of result obtained. Not only should a study be made of the com-
ponents of the blood, but, recognizing the presence of the fused ventral
central ganglia, similar experiments should be performed on the crayfish
or some like form in which the ganglionic enlargements from which the

AUTOTOMY IN UCA PUGNAX 163

walking legs are innervated are segmentally arranged and discrete, and
where, therefore, mutual influence in the central mechanism would be
less likely. The experiments do demonstrate very clearly that, in re-
spect to the extent to which legs are autotomized, there is a funda-
mental difference between females bearing eggs, on the one hand, and
females without eggs and males on the other.

LITERATURE CITED

Hoaptey, Letcu, 1934. Autotomy in the anomuran, Porcellana platycheles (Pen-
nant). Biol. Bull., 67: 494.

Woop, F. D., anp H. E. Woon, II, 1932. Autotomy in decapod Crustacea. Jour.
Exper. Zool., 62: 1.

POLAR BODY EXTRUSION AND CLEAVAGE IN
ARDIEMICTAMILY ACMV AE DenGGSsO@k
URECHIS (CAUPR®

ALBERT TYLER AND HANS BAUER?

(From the William G. Kerckhoff Laboratories of the Biological Sciences, Cali-
forma Institute of Technology, Pasadena, California)

INTRODUCTION

In recent articles, Hiraiwa and Kawamura (1935, 1936) report some
parthenogenesis experiments on eggs of Urechis unicinctus. As their
results differ in some respects from those of Tyler (1931a, b, 1932a, b)
on Urechis caupo and as one of us (B.) intended to continue the un-
finished studies of Bélar on the cytology of parthenogenesis, we decided
to test the methods of the Japanese authors on the Californian Urechis.
The principal point of the mentioned difference is that, in the experi-
ments of Hiraiwa and Kawamura, high percentages of cleavage were
obtained in batches of eggs most of which had extruded polar bodies,
whereas, in the experiments of Tyler, eggs that extruded polar bodies
almost invariably failed to divide. Hiraiwa and Kawamura used heat,
hypo- and hypertonic sea water, KCN sea water, and, most success-
fully, ammoniacal sea water, for which method alone they present the
data on polar body extrusion and cleavage. Tyler had used only
anisotonic sea water.”

It was considered advisable to isolate the various types of eggs ac-
cording to their behavior in the maturation divisions, a precaution which
Hiraiwa and Kawamura failed to take. This becomes of especial im-
portance in view of the fact that after certain treatments and at certain
stages blisters may appear on the surface of the egg that may easily be
mistaken for polar bodies. The Japanese authors do not mention these
blisters though they show them in their drawings. Also it is possible,
as was pointed out by Hiraiwa and Kawamura, that when two polar
bodies are present on the egg, they may represent the first and second

1 Fellow of the Rockefeller Foundation.

2 The Japanese authors erroneously attribute to Tyler the view that cleavage
cannot occur in eggs which have extruded both polar bodies. Although it was
found that such eggs failed to divide, the possibility was admitted that “by means

of other agents or by additional treatment the eggs that extrude two polar bodies
may be made to develop” (Tyler, 193la, p. 209).

164

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 165

‘

polar bodies or the divided first polar body or two “second” polar
bodies. Isolation of the eggs helps to decide these points.

It may be stated at the outset that the conclusion of Hiraiwa and
Kawamura could be confirmed that, after certain treatments with am-
moniacal sea water, eggs divide after polar body formation. However,
certain reservations must be made, since only low percentages of cleav-
age were obtained, and also the same exposure that gives cleavage gen-
erally produces abnormalities in the maturation divisions, so that it is
not certain that the divided eggs with two polar bodies are haploid.
This and related points will be cleared up in the cytological investiga-
tion. Where one or both maturation divisions are submerged, we find
that cleavage almost invariably follows and the type of cleavage is re-
lated to the polar bodies produced.

Hiraiwa and Kawamura also report that, by means of a second treat-
ment with ammoniacal sea water applied after the extrusion of the
second polar body, eggs which ordinarily would not do so, may be made
to divide. Repetition of this experiment on Urechis caupo failed to
confirm the result. They report too that the “ poorly activated” eggs
described by Tyler did not appear in their experiments. Using the am-
moniacal sea water treatment with U. caupo, however, we find again
this type of egg after certain treatments, and its behavior is the same as
in the hypotonic sea water experiments.

A new agent was also tried in the experiments reported here, namely
ammoniacal dilute sea water which gave high percentages of cleavage,
without polar body formation. The kind of cleavage, however, differs
in an interesting manner from that obtained by the ammoniacal sea
water treatment. Acidified sea water was also used in some experi-
ments with results that were essentially the same as in the hypotonic sea-
water experiments.

TREATMENT WITH AMMONIACAL SEA WATER

Urechis eggs may be activated by sea water containing ammonia in a
wide range of concentrations. Solutions ranging from 0.02 molar to
0.002 molar NH, in sea water were employed. The minimum exposure
necessary to obtain 100 per cent activation is, of course, shorter the more
concentrated the solution. A typical run is presented in Table I. The
solution employed in this experiment is the same as that used in some of
the experiments of Hiraiwa and Kawamura, and the activating ex-
posures are roughly the same as in their experiments. At other con-
centrations the results with Urechis caupo also approximate those with
U. unicinctus when allowance is made for the temperature differences.

166 ALBERT TYLER AND HANS BAUER

Also the exposures giving cleavage are in part similar in both cases.
But certain differences are evident. In the U. unicinctus experiments
no cleavage was obtained after short exposures to the activating solu-
tion, whereas we obtain cleavage after exposures too short to activate
all of the eggs. The eggs that divide after the short treatment are all
of the “ poorly activated’ type previously described and figured (Tyler,
193la, Figs. 27 to 30). They produce no polar bodies. Hiraiwa and
Kawamura state that they could not find any such eggs in Urechis
unicinctus. After somewhat longer exposures, ranging in the tabulated
case from 2 to 7 minutes, 100 per cent activation is obtained but none

TABLE I

Activation with ammoniacal sea water. Solution = 0:0in NH; in sea water,
temperature = 20° C.

Polar bodies First cleavage

Length Total Poorly
of acti- acti-
a we veo valle 0 1 2 or 3 2-cell 3-cell 4-cell
minutes per cent per cent per cent | per cent | per cent | per cent | per cent | per cent
Vy 40 70 75 5 20 60 3 2
7) 90 30 — — — 20 0 0
1 100 15 15 0 85 15 1 0
2,3,5,and7| 100 0 @) 0 100 0 0) 0
10 100 0 10 20 70 20 10 2
15 100 0 40 30 30 10 40 45
20 100 0 = == = 10 45 25
D5 100 0 60 15 DS) 5 20 15
30 100 0 — — — 5 10 10
40 100 0 100 0 0 0 0 0

of the eggs divide. This confirms roughly the cleavage-activation rela-
tion obtained with hypotonic sea water (Tyler, 1931b).* But upon still
longer exposures cleavage is obtained, rising to about 90 per cent, and
then dropping again to zero while the activation remains 100 per cent.
Data on the number of polar bodies extruded are also given in Table I.
It is at once apparent that after 2 to 7 minutes exposure, which gives
100 per cent activation but no cleavage, both polar bodies are extruded
by all the eggs. After the longer exposures giving increasing percent-
ages of cleavage, the proportion of eggs that extrude two polar bodies
decreases. From the figures in the table it would appear that at these
exposure times eggs with two polar bodies may divide. However, it
must be pointed out that these counts are only approximate, and also
that it is especially after such exposures that blisters very much re-

8 See also Dalcq, Pasteels and Brachet (1936).

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 167

sembling polar bodies may appear. In Fig. 3 a blistered egg is shown
and in Fig. 4 the same egg, ten minutes later, shows the blisters gone
and no polar bodies present. Some eggs become covered with blisters
so that polar bodies, if present, are completely obscured. The blister-
ing usually appears at some time between extrusion of the second polar
body and cleavage, and often reappears during cleavage. In many cases
the blisters might easily be mistaken for polar bodies. Preserved eggs
would, of course, be better material on which to make the counts, but the
isolation experiments reported below give a satisfactory basis for the
conclusions.

CLEAVAGE OF Eccs IsoLATED ACCORDING TO NUMBER OF POLAR
Bopitrs PRODUCED

The eggs that divide after short exposures to the ammoniacal sea
water all fail to extrude polar bodies just as in the hypotonic sea water
experiments. One hundred such eggs were isolated from a batch

Tasce II

Polar body extrusion and cleavage. Eggs treated for 15 minutes with 0.01 n
NH; in sea water. Isolated 60 to 80 minutes later. Eighty-five to 90 per cent
cleavage in main batch.

Number of eggs Number cleaved
No polar bodies................... 47 47
One polar body................... 12 12
Two polar bodies.................. 74 43

treated for 114 minutes with 0.005 n NH, in sea water. All of the
eggs divided into two cells at 2 to 214 hours after treatment. No signs
of submerged maturation spindles occurring were noted in the living
egg, and it is likely that the first cleavage division is the equivalent of
the first maturation division as in the case of eggs ‘‘ suboptimally ”’
activated with hypotonic sea water (Tyler, 1932a).

The eggs that divide after the longer exposure behave differently.
The first cleavage may give two, three or four cells. There are two
types of three-cell cleavage. One type produces three approximately
equal-sized cells directly (Fig. 10). The other first attempts to go into
four cells, then the cleavage plane between two of the cells disappears
giving a three-cell egg in which one of the cells is equal in size to both
of the others (Fig. 14). This type should be considered a four-cell
cleavage. There are also two types of two-cell cleavage, one of which

168 ALBERT TYLER AND HANS BAUER

is derived from an attempted three-cell cleavage. The different types
of cleavage correspond to differences in polar body extrusion as will be
shown below.

In order to determine the relation between polar body extrusion and
cleavage, eggs that were given a treatment with ammoniacal sea water
sufficient to induce a high percentage of cleavage were isolated according
to whether they produced two, one or no polar bodies. Table II con-
tains data of this sort from four experiments. The eggs were isolated
at about 20 to 40 minutes after the time of appearance of the second
polar body. It may be seen from the table that all of the eggs with
no polar bodies and with one polar body divide and also more than half

TasLe III

Polar body extrusion and cleavage. Eggs treated for 15 minutes with 0.01n NH;
in sea water. Isolated immediately after appearance of first polar body and again
at time of appearance of second polar body. Seventy-five to 90 per cent cleavage .
n main batch.

Cleavage at Cleavage at

Polar Num- 13 to 23 hours 33 to 5 hours

bodies ber of

From eggs with no polar None 39 4 | 10 | 24 0} 0 0 | 38
bodies 1 9 Dy 6 1 0} 1 0 8

2 12 11 1 0 0} 0 1 | il

From eggs with one polar 1 2 1 1 0 O} @ 0 2
body 2 43 3 2 2 20 lee See etal

3 89 8 4 @) | dik | § 3) 2

of those with two polar bodies. However, it is not certain here that the
two polar bodies represent the first and second polar bodies. To check
this point more carefully the isolation was begun at the time of appear-
ance of the first polar body, eggs with no polar bodies being separated
from those with one. Then, at the time of appearance of the second
polar body, eggs with two, one, or none were separated from the first
group, and eggs with three, two or one from the second. The results of
five experiments are summarized in Table III. Of 60 eggs that at first
showed no polar bodies, nine later produced one polar body and twelve
produced two. Of 134 eggs that at first had one polar body, 43 later
showed two and 89 showed three. Actually some of the eggs isolated
as having two polar bodies may later have formed three, since this was
not carefully checked in each case. However, there are no significant

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 169

differences in the behavior of these latter two classes.* It may be seen
from the table that at 114 to 2% hours after the start of the treatment in
the first group cleavage is obtained in 38 out of the 39 eggs with no
polar bodies, in all of the 9 eggs with one polar body and in all of the
12 eggs with two polar bodies. In the second group the two eggs with
one polar body both divide at this time, but only 7 out of the 43 with two
polar bodies and 12 out of the 89 with three polar bodies. Later, at 344
to 5 hours, more cleavage is obtained in these last two classes, 17 out of
the 43 with two polar bodies and 39 out of the 89 with three polar bodies.
Thus, while practically all of the eggs with no polar bodies in the first
group divide, only 14 per cent of the eggs with two and three polar
bodies in the second group divide at the same time. Considerably later
42 per cent of the latter divide, but it is questionable whether, in most
of these, anything like real cleavage had occurred (see below).

The type of cleavage that the eggs undergo is also given in Table III.
It may be seen that most of the eggs that extrude no polar bodies divide
at once into four cells. The four eggs, listed in the table, that divided
into two cells and the ten that went into three cells were all eggs that
had attempted to go into four cells. These eggs all show in the living
condition, shortly after the time at which the second polar body should
appear, four distinct nuclei (Figs. 13 and 15), which quite evidently
result from submerged maturation divisions. The nuclei are visible for
about 10 minutes, but it is apparent that the four centers for the first
division arise from the centers associated with each of these nuclei.
These centers evidently do not divide before the first cleavage, since the
division is into four, not eight cells.

The eggs that extrude one polar body divide into three cells, the two
eggs listed that went into two cells being derived from an attempted
three-cell cleavage and the one egg that went into four being an excep-
tion. Since this type of egg is isolated from eggs that showed no polar
body formation at the time when the first should appear, the single polar
body is evidently the second polar body. In the living condition (Figs.
9 and 11) three nuclei are seen shortly after the time of extrusion of
the second polar body.

The eggs of this group that extrude two polar bodies divide into two
cells, one exception having gone into three. The two polar bodies of
these eggs are both seen to arise simultaneously at the time of second
polar body extrusion and evidently represent two “second polar”
bodies. That is, the first maturation division was submerged, then each
of the nuclei produced a polar body at the time of second polar body

: 3 In normally fertilized eggs of Urechis the first polar body often fails to
ivide,

170 ALBERT TYLER AND HANS BAUER

extrusion. The two polar bodies of such an egg (Fig. 5) are usually
further apart than in the case of a first and second polar body (Fig. 2).
Two nuclei later appear in these eggs. If their history were not known
such eggs might be assumed to have normal first and second polar bodies,
or to have simply a divided first polar body.

In the second group, two of the 134 eggs that had extruded the first
polar body failed to extrude the second. ‘These were isolated rather late
and are doubtful cases. It appears from this as well as other observa-
tions that when the first polar body is extruded, the second almost im-
mediately follows. Those eggs of this group that divided went for the
most part into two cells at once. Many of them stop in the two, three or
four-cell stage and in most of those listed in the table as having more
than four cells, cleavage had occurred in only one of the original cells.
Very often, too, it becomes difficult to decide in the living condition
whether cleavage or some sort of fragmentation or lobulation had oc-
curred. About two-thirds of the “two and three polar body” eggs
that were listed as cleaved at the later time in Table III were actually
in the condition illustrated in Fig. 12, showing small lobules. A more
detailed study of these eggs would be necessary in order to decide
whether cleavage had actually occurred. The number of lobes present
is generally considerably greater than the number of cells to be expected
at that time.

PLATE |
Explanation of Figures

Artificially activated eggs of Urechis; eggs of Figs. 1 and 2 treated for 5
minutes with 0.01n NH; in sea water; those of Figs. 3 to 6 and 9 to 16 treated
for 15 minutes with the same solution; those of Figs. 7 and 8 treated for 5 minutes
with 0.004n NH: in 40 per cent sea water.

Fic. 1. Showing first polar body extruded at the normal time.

Fic. 2. Same egg with second polar body.

Fic. 3. Blisters appearing on an egg at the time of polar body appearance.

Fic. 4. Same egg photographed in the same position ten minutes later, show-
ing disappearance of blisters.

Fic. 5. Showing two polar bodies extruded simultaneously at the time when
the second polar body should normally appear; the two nuclei present in this type
of egg not visible here. The two polar bodies are further apart than in the case
of a first and second polar body.

Fic. 6. First cleavage of the same egg shown in Fig. 5.

Fic. 7. “ Poorly activated” type of egg.

Fic. 8. First cleavage of the same egg shown in Fig. 7.

Fic. 9. Showing one polar body extruded at the time when the second polar
body should normally appear; the three nuclei remaining in the egg are visible.

Fic. 10. First cleavage of the same egg; polar view.

Fic. 11. Another egg showing three nuclei present after the time when the
second polar body normally appears; a single polar body is present but not in focus.

Fic. 12. ‘“ Lobulation” of an egg in which polar body extrusion was appar-
ently normal; taken when other eggs of the same lot were in 16 to 64 cells.

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS lof

14
PALAIS, IL

Fic. 13. Egg with four nuclei present after the time when the second polar
body normally appears; no polar bodies are present but the pole is indicated by
the slight dent in the membrane.

Fic. 14. First cleavage of the same egg, polar view; the three cells result
from a disappearance of the cleavage plane between two cells.

Fic. 15. Another egg showing four nuclei present after the time when the
second polar body normally appears.

Fic. 16. First cleavage of another egg of this type.

ii ALBERT TYLER AND HANS BAUER

TIME OF PoLAR Bopy ExTRUSION

The time of appearance of the polar bodies varies with the length
of exposure. This is illustrated in Table IV, in which the time of ap-
pearance of the first polar body is given. The time from the start of
the treatment becomes increasingly greater, the longer the exposure to
the activating solution. If the length of exposure is subtracted, we
might expect the same value for all lengths of treatment. This, how-
ever, is not the case, as the last column in Table IV shows. The time
first decreases with increasing length, then rises slightly. We may
interpret this to mean that about four to five minutes of developmental
progress is made during the sojourn in the solution. Thus, for ex-
posures up to five minutes, the first polar body appears at approximately

TABLE LV
Time of extrusion of first polar body. Eggs treated with 0.01n NH; in sea water.
Length of Time from Start Time from Removal
Exposure of Treatment from Solution
minutes minutes minutes
Ieee eae abet cee a ce ee ee 344 334
Drees Ra hrs fine ORRIN ar 0205: SN 2g 34 32
Sitemeter cited Mer a tee ape 34 31
Pe ania ie Winey MM reine eae oe 35 30
RR Me eRe aE NL ies a 36 29
INQ) cai eiaree es A ne este, ay rer aie he 384 284
1S cantata RESIS et meta oro NERS 44 29
(Deg ateicpet ego ener ran arte Morn. 50 30

the same time from the start, and for longer exposures the delay in
appearance of the first polar body corresponds to the additional time
of treatment. This differs from the results with dilute sea water in
which, if allowance is made for the treatment, the time of polar body
extrusion is the same for all exposures (Tyler, 193la). However, no
developmental changes take place in the hypotonic sea water, whereas
in the ammoniacal sea water breakdown of the germinal vesicle and
membrane elevation is seen to occur. These changes occur in the solu-
tion at about the same rate as in normally fertilized eggs. It appears
then that in the case of the longer exposures the observed changes oc-
curring in the solution correspond to considerably more than the four
or five minutes necessary to account for the delay in appearance of the
first polar body. This means then that while certain changes such as
membrane elevation proceed at a normal rate in the solution, other proc-
esses leading to polar body extrusion are blocked after four or five
minutes. Without elaborating on this, it may be pointed out that a

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 7S

possible explanation of the increasing percentage of eggs that fail to
extrude polar bodies after increasing lengths of exposure may well be
the lack of synchrony of the early changes.

It may also be suspected that in those cases in which cleavage is
obtained after polar body extrusion the maturation divisions do not
proceed normally, but that some of the chromosomes may fail to reach
the maturation spindles or that the polar bodies may not receive a full
complement. Evidence of this is contained in the work of Hiraiwa
and Kawamura, who find often more than the haploid number of chro-
mosomes on the first cleavage amphiaster and also polar bodies devoid
of chromosomes. To establish this point, however, it would be neces-
sary to show that no normal maturation divisions occur after treatments
giving 100 per cent cleavage, or, where less cleavage is obtained, that a
corresponding percentage of abnormal maturation divisions occurs. It
may be possible to determine this in the cytological investigation.

DousBLE TREATMENT

After certain lengths of exposure to ammoniacal sea water 100 per
cent activation with normal polar body extrusion is obtained. No
cleavage, however, occurs (Table I). Hiraiwa and Kawamura report
for Urechis unicinctus that if such eggs are given a second treatment
with ammoniacal sea water immediately after the extrusion of the
second polar body, as much as 37 per cent cleavage may be obtained.
We have tried to repeat this but with no success. In Table V three
sets of re-treatment experiments are presented. In the first set the
second series of exposures is given at various times ranging from 2 to
64 minutes after extrusion of the second polar body. With the excep-
tion of the first series of second exposures, less cleavage is obtained than
by the single treatment. The slightly higher percentage of cleavage
obtained in the series started at two minutes may be significant. The
time of extrusion of the second polar body is taken when about 50 per
cent have reached that stage. Eggs developing in an unshaken vessel
show considerable spread in the time of polar body extrusion, which
at 20° C. may be as much as four or five minutes and much more at
lower temperatures. It is likely then that in the series begun at two
minutes, a number of the eggs had not extruded the second polar body.
In the second set in Table V the second treatment is begun at 11 minutes
before the extrusion of the second polar body. In this case 5 per cent
cleavage is obtained after 15 minutes of second treatment whereas the
first treatment gave no cleavage. In the third set hypertonic sea water
is used for the second treatment, but no increase in cleavage is obtained.

174 ALBERT TYLER AND HANS BAUER

Three other sets of experiments similar to the first of Table V and
using 0.003n, 0.005n and 0.01n NH, in sea water for the second treat-
ment, gave similar results.

That cleavage may be obtained after a second treatment applied
before the extrusion of the second polar body was shown in the hypo-
tonic sea water experiments (Tyler, 19325). But in those experi-
ments, too, a second treatment applied after polar body extrusion gave

TABLE V

Double treatment. Solution A = 0.005n NH; in sea water. Solution B = 0.0in
NHs3 in sea water. Solution C = 3.0 g. NaCl in 100 cc. sea water. Temperature
20.1 + 1°C.

Time after second
Treatment Solus Exposure Activation Be oe nee Cleavage
of treatment
minutes per cent minutes per cent

First. A 5 99 — 2
Second...... A 5 to 40* 2 2to4
Second...... A 5 to 40* 6 1 to 2
Second...... A 5 to 40* 12 1
Second A 5 to 40* 20 1
Second A 40 64 1
EMTS Er Sonnet B 2 100 = 0
Second...... B 1 to 53 —11 0 to 0.1
Second...... B 10 —11 2
Second...... B 15 —11 5
Bitsteccus scl OA 53 99 — 0.5
Second...... C D 3 0.5
Second...... C 5 to 40* 3 0.5 to 0
Second......| C 2 15 0.3
Second......| C 5 to 30* 15 0.2 to 0

* Five-minute intervals.

no cleavage. Negative results, of course, do not prove very much.
It is entirely possible that U. caupo and U. unicinctus respond differ-
ently to identical treatments, and that by varying such factors as the
time treatment, the length of exposure, the temperature, the concen-
tration and the kind of activating agent, cleavage might also occur in
U. caupo. It is also possible that in the experiments of Hiraiwa and
Kawamura some of the eggs had not given off the second polar body
when the second treatment was begun.

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 175

TREATMENT WITH AMMONIACAL-DILUTE SEA WATER

Hypotonic sea water is not always effective in activating Urechis
eggs. It generally fails to activate eggs that have stood in a dish for
more than two hours, or that have been taken from animals kept in the
laboratory more than a week. If, however, ammonia is added to the
dilute sea water 100 per cent activation and a high percentage of cleavage
may be obtained. An experiment with 0.004n NH, in 40 per cent sea
water is given in Table VI. After five minutes exposure 100 per cent

TABLE VI

Ammoniacal hypotonic sea water. Solution = 0.004n NH; in 40 per cent
sea water. Temperature = 20° C.

Polar bodies Cleavage
Exposure Activation
0 1 2 or 3 2-cells 3-cells 4-cells
minutes per cent per cent per cent per cent per cent per cent per cent
3 90 98 1 il 85 0.1 0.1
4 95 95 2 $i 95 1 0.5
5 100 97 1 2 90 0.2 0.1

activation is obtained and 90 per cent of the eggs divide. Very few
(3 per cent) of the eggs extrude polar bodies and it is apparently only
the “no polar body” eggs that divide. The first cleavage, however,
is into two cells (Fig. 8). The eggs resemble the “ poorly activated ”
eggs obtained by the short treatment with ammoniacal sea water or the
straight dilute sea water treatment. These eggs, too, show rather poor
membrane elevation until the time of first cleavage (Fig. 7). With
other agents, this type of egg is obtained principally when the total
activation is low. Here, however, with treatment giving 100 per cent
activation, practically all of the eggs are of that type.

ACTIVATION OF “ BLocKED” EGcGs

It has been shown (Tyler and Schultz, 1932) that fertilization may
be reversed in Urechis by treatment with acidified sea water within
three minutes after insemination. The spermatozoa remain in such
eggs, but they can be re-inseminated and thereupon they behave as
polyspermic eggs. If, instead of re-inseminating, the eggs are given
the kind of treatment with dilute sea water by which polar body extru-
sion but no cleavage is obtained, they develop as normally fertilized
eggs. This experiment was repeated using ammoniacal sea water and
the results are shown in Table VII. The control eggs (B) are placed

176 ALBERT TYLER AND HANS BAUER

in sea water removed from the blocked eggs in order to check the pos-
sibility that the ammoniacal sea water merely stimulates the extra
sperm present in the dish. The results show that this is not the case,
since no cleavage is obtained after the four minutes exposure of the
control eggs. From the blocked eggs, however, 80 per cent cleavage
and 60 per cent normal development are obtained. With the longer
treatment 10 per cent of the control eggs divide and 60 per cent of the
blocked ones. The percentage of normal development is lower in this
case, indicating some deleterious action of the longer treatment.

These results show that the treatment giving 100 per cent activation

TaBLe VII

Activation of ‘‘ Blocked” Eggs

Set A. Eggs placed in acid sea water (0.2 cc. of 0.5n HCI in 100 cc. sea water)
at 1 minute after insemination. Removed after 20 minutes.

Set B. Unfertilized eggs placed in sea water taken from A. Both sets treated
with 0.0in NH; in sea water.

Set Exposure Activation Cleavage Normal embryos
minutes per cent per cent per cent
A 4 100 80 60
B 4 100 0 0
A 10 100 60 20
B 10 100 10 0

and polar body extrusion but no cleavage actually provides sufficient
stimulus for development. The ability of the blocked egg to divide is
not due to any additional stimulus by the spermatozoon present but
rather to the ability of the sperm aster to form an amphiaster.

Another kind of experiment shows the non-additive nature of the
stimuli provided by the sperm and by artificial activation. If after
treatment with dilute sea water or ammoniacal sea water sufficient to
induce 100 per cent polar body extrusion but no cleavage, the eggs are
transferred to a sperm suspension, fertilization, normal maturation, and
normal development occurs. The combined action of the activating
agent and the sperm is therefore not equivalent to a prolonged treatment
with the activating agent, but simply equivalent to insemination of
untreated eggs.

ACTIVATION WITH ACIDIFIED SEA WATER

Lefevre (1907) activated eggs of Thalassema by means of acid and
reported obtaining cleavage with or without polar body extrusion.

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS Meser

The solutions employed by Lefevre were tried on Urechis eggs and were
found to be strong enough to kill or injure the eggs in one or two
minutes. Using weaker solutions good activation could be obtained.
Im Table VIIL an experiment with-0.25 ce. of 1.0n HCl in 100 ce. sea
water is presented. This is just about the lowest concentration of acid
that gives activation, 0.2 cc. being too weak even after long exposures.
It may be seen from the table that the activation rises rapidly with
length of exposure and at the same time the percentage of cleavage
drops off. It is almost exclusively the “ poorly activated” type of egg
that divides. These eggs, of course, produce no polar bodies. With

Tasle VIII

Activation with acidified sea water. Solution = 0.25 cc. of 1.0n HCl in 100 cc.
sea water. Temperature = 20°C.

other acids and other concentrations the results are similar. If three
times the above concentration of acid is used, 100 per cent polar body
extrusion with no cleavage is obtained after one-half-minute exposure.

DISCUSSION

It should be pointed out that we do not consider it impossible to
obtain cleavage and normal development after extrusion of both polar
bodies in Urechis. The point to be made, however, is that when eggs
with no polar bodies and with two polar bodies are isolated from the
same treated lot, practically all of the former divide whereas very few
of the latter show real cleavage. In the “no polar body ” eggs obtained
by prolonged exposure to ammoniacal sea water, it is clear from the
presence of four nuclei that submerged maturation divisions had oc-
curred. In the “no polar body ” eggs obtained by short exposure to
ammoniacal sea water or to ammoniacal-dilute sea water the behavior
is similar to that reported for dilute sea-water activation, no maturation

178 ALBERT TYLER AND HANS BAUER

divisions occurring until the first cleavage. The first type divides into
four cells at once, the second type into two.

The behavior of the chromosomes during cleavage in the first type
of egg has not yet been worked out. In the second type both haploid
and diploid cleavages have been found, although the embryos examined
show only the diploid number (Tyler, 1932a). In a recent article on
artificially activated frog’s eggs Parmenter (1933) lists among the
possible methods of regulation to diploidy this utilization of what is the
equivalent of a maturation division spindle for the first cleavage. But
it should be noted that if reduction proceeded normally in the first two
cleavages only the haploid number of chromosomes would result. Since
diploid as well as haploid later cleavages are observed, regulation must
occur but this type of behavior certainly does not insure diploidy.

The same treatment that results in both maturation divisions occur-
ring submerged also gives eggs in which only the first spindle is sub-
merged. These eggs may produce either one or two polar bodies at
the time of second polar body extrusion, and practically all the eggs
cleave. When one polar body is produced three nuclei remain in the
egg (visible for a short time) and the first cleavage is into three cells.
If two polar bodies are produced two nuclei remain in the egg and the
first cleavage is into two cells. Eggs that produce the first polar body
almost invariably extrude the second. One should expect this to be
the case from the manner of treatment. With the shorter exposures
the polar bodies are extruded normally in all the eggs. With longer
exposures changes are evidently produced in the egg that prevent polar
body extrusion, even when the treatment ends before the time of first
polar body formation. If the first polar body appears it simply means
that these changes have not been produced or that the egg has recovered
before that time. The second polar body should therefore follow. The
extrusion of one or two second polar bodies after a submerged first
division would mean a recovery of the egg in the interim. On this
basis eggs in which the first division is submerged should occur after
lengths of treatment that are intermediate between those giving only
normal polar body formation and those giving only submergence of
both maturation divisions. This is in fact the case.

The eggs that extrude two polar bodies have evidently received an
adequate stimulus for development, as the experiments with “ blocked ”
eggs show. That they generally fail to divide in our experiments is
probably due to the inability of the centrosome left in the egg after
polar body extrusion to divide. Those cases of cleavage after polar
body extrusion may then result from division of this centrosome. In
the four-cell stage of the “no polar body ” eggs where the polar spindles

POLAR BODY EXTRUSION AND CLEAVAGE URECHIS 179

appear to serve for cleavage, the centrosome of one of the cells is
equivalent to the one left in the egg after normal polar body extrusion.
Yet in this case it continues to divide. The difference may be merely
one of position in the cell. Centrifugation, in view of the experiments
of Morgan and Tyler (1935), might be of use in examining these
questions.

In the dilute sea-water experiments the type of “no polar body” e
with submerged maturation divisions was not obtained. The difference
in the reaction of the egg while in the dilute sea water and in the am-
moniacal sea water may account for this. With the former agent no
developmental changes occur until removal to sea water. With the
latter, development starts in the solution. Short exposures to the latter
agent give results similar to those by dilute sea water. Prolonged ex-
posures allow the development of the polar spindle to proceed, but either
by modifying the surface or in some way preventing the movement of
the spindle to the surface do not result in polar body extrusion. This
effect is not produced by dilute sea-water activation since no develop-
mental changes occur in the agent. The interest in activation by dilute
sea water concerned primarily this fact. With all other partheno-
genetic agents, continued exposure after the maximum of activation is
reached soon results in injury and death. With dilute sea water in
certain concentrations, continued exposure after the maximum of ac-
tivation results in a falling-off of the activation to zero without injuring
the eggs or affecting even their fertilizability.

SUMMARY

1. Urechis eggs activated by prolonged exposure to ammoniacal sea
water give a high percentage of cleavage as Hiraiwa and Kawamura
showed, but such exposures also give a high percentage of polar body
suppression.

2. Isolation of eggs of known polar body history shows that prac-
tically all those eggs divide that fail to extrude the first or the first and
second polar bodies, but only a small percentage of Enos eggs that
extrude the first and second polar body.

3. Eggs that fail to extrude the first polar body may produce two,
one or no polar bodies at the time when the second polar body should
appear. Later, two, three or four nuclei are visible in the egg corre-
sponding to whether two, one or no polar bodies are present. The
first cleavage of these three types gives two, three or four cells
respectively.

4. Eggs that extrude the first polar body almost invariably produce
the second.

180 ALBERT TYLER AND HANS BAUER

5. Determinations of the time of polar body appearance after various
lengths of exposure indicate that not more than four or five minutes
progress toward polar body extrusion is made during sojourn in the
solution, whereas other developmental changes progress further.

6. Eggs that had been given the kind of first treatment that results
in 100 per cent normal polar body formation but no cleavage could not
be induced to divide by a second treatment applied at various times after
the extrusion of the second polar body. Cleavage was, however, ob-
tained when the second treatment was started before the time of extru-
sion of the second polar body.

7. The “poorly activated” type of egg that divides without polar
body formation or evidence of submerged maturation division is ob-
tained by short exposures to ammoniacal sea water, to ammoniacal-
dilute sea water, or to acid sea water, the second agent giving the highest
percentages. The first cleavage is into two cells.

8. Normal development is obtained when “ blocked” fertilized eggs
are activated with the treatment that ordinarily results in 100 per cent
polar body formation but no cleavage. Also superposition of fertiliza-
tion after such treatment results in normal development.

LITERATURE CITED

Datcg, A., J. PASTEELS AND J. BrAcHET, 1936. Données nouvelles (Asterias
glacialis, Phascolion strumbi, Rana fusca) et considerations theoriques
sur l’inertie de l’oeuf vierge. Mem. Mus. Roy. d’Hist. Nat. Belg., 2™°
Série, 3: 881.

Hiraiwa, Y. K., anp T. Kawamura, 1935. Studies on the artificial partheno-
genesis of Urechis unicinctus (von Drasche). Jour. Sct. Hiroshima
Univ., 4: 35.

Hiratwa, Y. K., anp T. KAwAmura, 1936. Relation between maturation division
and cleavage in artificially activated eggs of Urechis unicinctus (von
Drasche). Biol. Bull., 57: 344.

LeFeEvreE, G., 1907. Artificial parthenogenesis in Thalassema mellita. Jour. Exper.
Zool., 4: 91.

Morcan, T. H., anp A. Tyrer, 1935. Effects of centrifuging eggs of Urechis
before and after fertilization. Jour. Exper. Zool., 70: 301.

PARMENTER, C. L., 1933. Haploid, diploid, triploid, and tetraploid chromosome
numbers, and their origin in parthenogentically developed larve and frogs
of Rana pipiens and R. palustris. Jour. Exper. Zool., 66: 409.

Tyer, A., 193la. The production of normal embryos by artificial parthenogenesis
in the echiuroid, Urechis. Biol. Bull., 60: 187.

Tyier, A., 1931b. The relation between cleavage and total activation in arti-
ficially activated eggs of Urechis. Biol. Bull., 61: 45.

Tyrer, A., 1932a. Chromosomes of artificially activated eggs of Urechis. Buol.
Bull., 63: 212.

Tyrer, A., 1932b. Production of cleavage by suppression of the polar bodies in
artificially activated eggs of Urechis. Biol. Bull., 63: 218.

Tver, A., AND J. ScHuLtz, 1932. Inhibition and reversal of fertilization in eggs
of the echiuroid worm, Urechis caupo. Jour. Exper. Zodl., 63: 509.

CROSS-REACTIVITY OF VARIOUS HEMOCYANINS WITH
SPECIAL REFERENCE TO THE BLOOD PROTEINS OF
DEH BLACK WIDOW: SPIDER

WILLIAM C. BOYD

(From the Boston University School of Medicine, and Evans Memorial,
Massachusetts Memorial Hospitals)

It was reported by Graham-Smith in Nuttall’s well known book
(1904) that an antiserum against the blood of Limulus reacted with
spider serum, even more strongly than with crab serum, but neither the
species of spider nor any further details were given. Since Nuttall’s
sera were prepared by injecting rabbits with the whole blood serum of
the species studied, it remained uncertain if the cross-reaction of
Limulus and the spider were due to serologically similar blood pigments
or to small amounts of some other protein.

I have found that an antiserum prepared by injecting the carefully
purified hemocyanin of Limulus into rabbits (Hooker and Boyd, 1936)
reacted with the diluted serum of the black widow spider. The only
other species tested, outside of the injected antigen, which reacted with
this antiserum was crab (Cancer irroratus), which gave a weaker reac-
tion. The cross-reactions of a number of hemocyanins are set forth
in Table I.

It will be noted that no other antiserum reacted definitely with the
spider serum, and that the anti-crab serum did not react definitely with
the Limulus hemocyanin, so that the relationship between these two
antisera and antigens is not reciprocal, as is often the case. Of the
other hemocyanins, Cancer and Homarus cross-react to a considerable
degree, as do Vivipara and Busycon, paralleling their relationship.

Absorption of the anti-Limulus serum with the optimal (Dean and
Webb, 1926) dose of spider serum removed all reactivity to spider but
left considerable reactivity to Limulus ; the limiting titer of the antiserum
against Limulus remaining about the same. By determination of the
optimal proportions ratio, which provides an index of the antibody con-
tent (the larger the ratio, the lower the content of antibody), it was
possible to show that considerable antibody had been removed (the
ratio rose from 780 to 2970). This phenomenon of partial cross-reactiv-
ity in related species is familiar (Hooker and Boyd, 1934), and could
probably be made the basis of a quantitative measure of serological rela-

131

182 WILLIAM C. BOYD

tionship. The complete removal of all reactivity to one antigen, leaving
some to another, presumably indicates a common or closely related anti-
genic determinant in the two proteins, although in the present case it
must be remembered that ultracentrifugal analysis of Limulus hemo-
cyanin has revealed three distinct proteins with different molecular
weights, and possibly only one or two of these might be concerned in
the present cross-reaction. In the case of species as closely related as
the -hen and duck, absorption of the antiserum for one crystalline egg
albumin with the other in most cases hardly affected the optimal ratio
against the injected antigen (Hooker and Boyd, 1934) ; a greater effect
would have been expected.

Determinations of copper and nitrogen (for methods, see Hooker
and Boyd, 1936) on the diluted spider serum showed the presence of

TABLE [|

Cross-reactivity by the precipitin test of various hemocyanins

Antigen: Hemocyanins of:

Antiserum against

purified hemocyanin of: Black Snail
, : nai
ee Limulus Homarus Cancer (Vivipara) Busycon
TEUIUTUS cone culee cee eee + ++ — + — =
LOMOTUS HR aan ae = — ++ + — =
Camicen in stinks B ? + +4 - —
VEEGQUPDs ca0566e0006 _ — _ = abe +
IBUSYCON cae. 2 toe — — = = oe ete

The tests were made by the interfacial (ring) technic. The symbol — means no
reaction, ? means doubtful reaction, -+- moderate reaction, +--+ very strong reaction.

0.522 mg. of N per cc., and 0.0054 mg. Cu per cc. This gives a ratio
nitrogen to copper of 96.7, not very different from the ratio of nitrogen
to copper of 103 which follows from the values for Limulus hemocyanin
(Redfield, 1930, Redfield et al., 1928). In some Limulus preparations
I have found values as low as this or slightly lower. Montgomery
(1930) says “In the case of Limulus, the hemocyanin appears to ac-
count for about 95 per cent of the protein of the serum.” It would
seem that the above data are consistent with the view that the serum
protein of the black widow spider is chiefly or entirely a hemocyanin
having about the same copper and nitrogen content as that of Limulus,
which it closely resembles serologically. According to D’Amour et al.
(1936), however, others have reported the presence of hemolysins in
the legs of spiders, which presumably means that there may be present
in the blood small amounts also of these substances, probably proteins.

CROSS-REACTIVITY OF HEMOCYANINS 183

Unfortunately it was impossible to estimate accurately the dilution
of spider serum which resulted from bleeding the spiders used in this
experiment. -It was probably around 1:10 or 1:15. This would im-
ply that the blood of the black widow spider contains 3 to 5 per cent of
hemocyanin.

The author wishes to express his best thanks to Dr. F. E. D'Amour
for the gift of one hundred black widow spiders for this work.

BIBLIOGRAPHY

D’Amour, F. E., F. E. Becker, anp W. V. Riper, 1936. The black widow spider.
Quart. Rev. Biol., 11: 123.

Dean, H. R., anp R. A. Wess, 1926. The influence of optimal proportions of
antigen and antibody in the serum precipitation reaction. Jour. Path. and
Bact., 29: 473.

Hooker, S. B., anp WiLttAm C. Boyp, 1934. The existence of antigenic deter-
minants of diverse specificity in a single protein. II. In two natural pro-
teins; crystalline duck egg albumin and crystalline hen egg albumin.
Jour. Immunol., 26: 469.

Hooker, S. B., anp Witit1Am C. Boyp, 1936. Two antigens of high molecular
weight: hemocyanins of Limulus polyphemus and Fulgur caniculatus.
Jour. Immunol., 30: 33.

Montcomery, H., 1930. The copper content and the minimal molecular weight
of the emer renas of Busycon canaliculatum and of Loligo pealei. Biol.
Bull., 58: 18.

Nurratt, G. H. F., 1904. Blood Immunity and Blood Relationship. Cambridge
University Press,

RepFietp, A. C., 1930. The equilibrium of oxygen with the hemocyanin of
Limulus polyphemus determined by a spectrophotometric method. Biol.
Bull., 58: 238.

RepFIELD, A. C., T. CooLipce, AND M. A. SHorts, 1928. The respiratory pro-
teins aE the blood. I. The copper content and the minimal molecular
weight of the hemocyanin of Limulus polyphemus. Jour. Biol. Chem.,
1G20185:

BIOLOGICAL MATERIALS
She

INCE 1890 the Supply Department of the Marine Biologi-
cal Laboratory has been furnishing both living and
preserved specimens to schools and colleges. It is the desire
_ of the Laboratory to continue this service in an efficient and
satisfactory manner, and we would be pleased to quote on
your needs for the coming school year.

All our materials are freshly collected each season and are
carefully prepared by men of long experience.

A new catalogue will be in circulation within a few weeks
and this issue will list a general reduction in prices.

All specimens are of the first quality, and we guarantee
them to give absolute satisfaction.

CAKKD
SUPPLY DEPARTMENT

Est. 1890

MARINE BIOLOGICAL LABORATORY
Woods Hole, Mass.

CONTENTS
THIRTY-NINTH REPORT OF THE MARINE BIOLOGICAL LABORA-

_ SAYLES, LEONARD P., AND S. G. HERSHKOWITZ

Placoid Scale Types and their Distribution in Squalus
ACATIUHIAS 62 ei eee SRL at ABS Meu At Bn Rog ean aeah JS

“MARZA, V. D., EUGENIE V. Manze AND Mary J. GUTHRIE.

Page |

Histochemistry of the Ovary of Fundulus heteroclitus with ©

Special Reference to the Differentiating Odcytes..........

MATTHEWS, SAMUEL A.
The Development of the Pituitary Gland in Fundulus......

BEAMS, H. W., AND R. L. KING

The Suppression of Cleavage in decent Babe by Ultracentri-
FUP IMe e Pie S Ae ae LNs aE Bg AO USO aru int a ae

MILLER, E. DEWITT

A Study of the Bacterial and “Atleeed! Mitochondrial Content
of the Cells of;the Clover Nodule. . 2.0.0.0. .402 00.0,

Mast, S. O., AND NATHAN STAHLER

The Relation between Luminous Intensity, Adaptation to
Light, and Rate of Locomotion in Amoeba proteus (Leidy). .

ABRAMOWITZ, A. A. .
The Role of the Hypophyseal Melanophore Hormone in the
Chromatic Physiology of Fundulus.............. CUES ee

BUTLER, MARGARET RUTH

The Effect of its Nitrogen Content on the Decomposition of

the Polysaccharide Extract of Chondrus crispus:..........

PAYNE, NELLIE M,

The Differential Effect of Environmental Factors upon Micro-
bracon hebetor Say (Hymenoptera: Braconidae) and its
Host, Ephestia kiihniella Zeller (Lepidoptera: Pyeeae),
100%

ee 8 ww we et we we we 8 kw ee ee we 8 we 8 ee oe 8 8 8 8 wh we ee we ele we wwe Ht
/

HOADLEY, LEIGH
Autotomy in the Brachyuran, Uca pugnax ANN RUE A a CRNA 78

TYLER, ALBERT, AND HANS BAUER

Polar Body Extrusion and Cleavage in Artificially Activated
Eessof Urechis:caupo 2 24 Re Oe an Ue UN NG Bere

' BOYD, WILLIAM C.

Cross-reactivity of Various Hemocyanins with Special Refer-
ence to the Blood Proteins of the Black Widow Spider.....

112
126.

134

143

147

155

164

Volume LXXIII . Number 2

poe HE. ‘
BIOLOGICAL BULLETIN

PUBLISHED BY ;
THE MARINE BIOLOGICAL LABORATORY

(

| Editorial Board

GARY N. CALKINS, Columbia University _ __E. E. JUST, Howard University
E, G. CONKLIN, Princeton University — FRANK R, LILLIE, University of Chicago
in E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago

SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
‘ LEIGH HOADLEY, Harvard University T. H. MORGAN, California Institute of Technology
Z M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University

H. S. JENNINGS, Johns Hopkins University EDMUND B. WILSON, Columbia University

ALFRED C. REDFIELD, Harvard University
Managing Editor

OCTOBER, 1937

Printed and Issued by

ae LANCASTER PRESS, Inc.

i | PRINCE & LEMON STS.
LANCASTER, PA.

NEW BLAKISTON BOOKS
FOR THE BIOLOGIST

PRACTICAL PHYSIOLOGICAL CHEMISTRY,
lith Edition.

By HAWK anp BERGEIM (1937) $8.00

The new edition of this laboratory textbook continues to
represent. the best in methods, theories and conclusions de-
veloped by the leading biochemists throughout the world.

TEXTBOOK OF GENERAL PHYSIOLOGY.
By T. CUNLIFFE BARNES _ (1937) $4.50

“A book that will be useful to advanced students and re-
search workers as well as to beginning students.”

LABORATORY MANUAL OF GENERAL

PHYSIOLOGY.

By T. CUNLIFFE BARNES (1937) $1.00
A carefully selected collection of experiments covering the

various fields in general physiology.

COMPARATIVE ANATOMY.

By NEAL anp RAND (1936) $4.75
An eminently successful foundation text by two experienced

teachers.

RECENT ADVANCES IN CYTOLOGY,
2nd Edition.

By C. D. DARLINGTON (1937) $6.00

A presentation of the recent studies of the nucleus and
chromosomes.

— EE

P. BLAKISTON’S SON & CO., INc.

1012 WALNUT ST. PHILADELPHIA, PA.

ete

="
“mh

Vol. LXXIII, No. 2 October, 1937

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

THE DIURNAL MIGRATION OF DEEP-WATER ANIMALS

je Eee Vie St Eee OEE Hy | NiO ke Eo NUNN E MACHER

(From the Woods Hole Oceanographic Institution 1 and the Biological
Laboratories, Harvard University)

The diurnal vertical migration of planktonic organisms in the sea
is a well-known phenomenon (Russell, 1927; Clarke, 1933; 1934).?
Many more of the actively swimming animals, such as copepods, are
found nearer the surface at night than during the day. For example,
the level of maximum numbers of adult Calanus, in the Gulf of Maine,
may be around 40 meters during the night while during the day it may
be below 120 meters (Clarke, 1934). It is generally agreed that the
most important external factor which regulates these daily movements
is light. The greatest depth to which light may have an effect, how-
ever, is not known, since almost all studies have been confined to rela-
tively shallow water. Obviously it would depend on the locality and
the organisms being investigated, for an animal living at a considerable
depth in a region where the water was very transparent might be influ-
enced by the penetrating light, while another organism at the same
depth in a region of low transparency would be unaffected. If diurnal
migrations occur in deep water they must be considered in any study of
vertical distribution. The lowest level at which they occur might be a
satisfactory measure of the lower limit of the photic zone. These and
other problems of the biology of the deeper water organisms have a
direct relation to the possible vertical movements which may occur at
levels deeper than any which have been adequately investigated. For
these reasons a study was begun of diurnal migrations in deep water
during a cruise of “ Atlantis ” to the Sargasso Sea in 1936.

During the cruise of the “ Michael Sars” enough data were obtained
from hauls with open nets to indicate that certain of the deep-water
fishes and decapod crustaceans were to be found at higher levels during
the night than during the day (Murray and Hjort, 1912). The
“ Michael Sars” data, however, did not indicate how deep such move-

1 Contribution No. 145.
2 A rather complete list of references may be found in these papers.

185
— ee WO
\ WI Pp Low

186 WELSH, CHACE AND NUNNEMACHER

ments might occur, and are conclusive for only a few species of crusta-
ceans and fishes.

The Sargasso Sea was selected for the present investigation since
collections with closing nets were being made in this region, and because
of the transparency of the water. Helland-Hansen (1931) had found
that photographic plates exposed at a depth of 1,000 meters in waters
between the Canary Islands and the Sargasso Sea were blackened after
an exposure of 80 minutes. Clarke (1936) had calculated from data
obtained by Clarke and Oster (1934) that there is sufficient light to
enable deep sea fishes to see objects at a depth of 430 meters or deeper
in the Sargasso Sea, depending on certain assumptions regarding their

TABLE I

Showing eDiles time of day and duration of hauls made with closing nets at ‘‘ Atlantis”
Station 2607.

Haul Depth Date Nets Open Nets Closed

meters

iL ae Sept. 6 6:52 a.m. des2eabite

2 a Sept. 7 4:07 a.m. 5:10 a.m,

3 aoe Sept. 7 6:57 a.m. (25 esa

4 He Sent. 7 Seen eu 11:31 a.m.
400

5 300 Sept. 7 1:18 p.m. 3:18 p.m.
400

6 300 Sept. 7 5:10 p.m. 7:10 Dan:
ae Sept. 7 8:57 p.m. 10:57 p.m.

8 400 Sept. 8 12:39 a.m. 2.39 a.m.
800 Sept. 8 Net attached improperly, failed to fish.

visual acuity and spectral sensitivity. Light of an intensity sufficient to
affect the general activity of an animal would be found below the level
at which objects could be seen.

Since it was desirable to make as many hauls as possible in a twenty-
four-hour period only two closing nets were used and they were set to
tow at 400- and 800-meter levels. The location of the station (2667)
was 35° 40’ N. and 69° 36’ W. ‘The duration of each haul and the time
of day may be seen in Table I. One haul was made on September 6
with the nets open at 6:52 A.M. and closed at 7:52 A.M. A rough sea
prevented further use of the nets that day but tows were resumed on
September 7. The first three hauls were each of one hour duration, but

DIURNAL MIGRATION DEEP WATER ANIMALS 187

the absence of salps and other forms which sometimes prevent longer
tows, and the relatively small catches made it possible and desirable to
increase the time of towing of subsequent hauls to two hours. In order
to compare the catches the numbers of organisms taken in hauls 1, 2
and 3 have been doubled. 7
Since a modification of the closing nets developed and described by
Leavitt (1935) were used for these hauls, and since they worked most
successfully they will be briefly described and figured. The method of

Fic. 1. Net closed, descending.
Fic. 2. Net open, towing.
Fic. 3. Net closed, ascending.

a. Fetter rope. gg’. Light string stops.
b. Single towing rope. h. Small iron ring.

c. Three-point towing bridle. m, First messenger.

d. Attachment of bridle on two-meter ring. mM Second messenger.
e. Attachment of pursing rope. t,. Primary release.

f. Pursing rope around belly-band of net. t.. Secondary release.

opening and closing was the chief feature of this net which differed from
the one used by Leavitt. The releasing devices were the same. The
changes were made by Mr. Nunnemacher with the advice of Dr. H. B.
Bigelow.

The accompanying figures show a net: (1) closed, descending;
(2) open, towing; (3) closed, ascending. The net itself has a diameter
of 2 meters at the mouth and is laced to a strong galvanized iron ring of
2 meters diameter. ‘The sides of the net are made of stramin (6 threads

188 WELSH, CHACE AND NUNNEMACHER

per cm.) and are parallel for the first 2 meters and then sewed to a 60
centimeter wide, heavy canvas belly-band to which six 5-centimeter brass
rings are fastened at equal distances over the lower half of the band.
From the rear edge of the belly-band the net tapers from a 2-meter
diameter to one of 25 centimeters over a distance of 7 meters. The end
is finished off with another canvas band. Into the end of the net a silk
net of corresponding taper is sewed so that it lines the last meter of the
stramin and prevents excessive chafing of the catch.

On lowering the net (Fig. 1) a short fetter rope (a) fastened at its
mid-point to the bottom of the belly-band tightly encircles the folded
canvas, passes through the small iron ring (4) which is attached to the
rim of the net and both ends of the fetter rope are hooked into the
primary release. The releasing devices were those previously used by
Leavitt (1935) and they proved entirely satisfactory. The loose towing
and pursing ropes are “stopped” (loosely tied with cotton string) at
points g and g’ to make fouling impossible. After lowering the net to
the desired depth, the primary release is opened by the first messenger
and the fetter rope easily falls out of the way below the net. The net
in opening merely unfolds, the loop of the pursing rope having been
previously opened to the diameter of the net at the belly-band and then
folded up with it. Figure 2 shows the net being towed by the strong
three-point towing bridle and the pursing rope loosely surrounding the
belly-band and “ stopped ” to the towing bridle. A bucket is also shown
tied to the end of the net to prevent the tail from fouling in the eddies
behind the closed net. After the required time of towing the second
messenger releases the towing bridle which falls out of the way. The
pursing rope, securely fastened to the cable, now takes the strain, con-
stricts the net at the belly-band and effectively closes the net (Fig. 3).

At each haul an open net was attached 50 meters below the lower
closing net. Since the level of maximum numbers of certain of the red
prawns and blackfish is near 850 meters in this region of the Sargasso
Sea, at least during the day, large catches were always taken by the open
net-and this material is being used in connection with other studies such
as the eyes of deep sea crustaceans (Welsh and Chace, 1937). With the
method we used to determine the depth at which a closing net fishes it
is impossible to be accurate within about + 100 meters at a depth of 800
meters. One can keep the level of the nets relatively constant by so con-
trolling the speed of the ship as to maintain a constant wire angle. But
it is not possible to know very accurately the level of fishing because of
certain unknown forces which affect the catenary of the wire. We used
the calculations made by Leavitt for setting the nets at 400- and 800-
meter levels. ‘These were based on the length of towing cable out, the

DIURNAL MIGRATION DEEP WATER ANIMALS 189

pull of the nets when open, the weight at the end of the towing cable and
the wire angle at the surface.

After sorting the hauls it was evident that some forms had not been
taken in sufficient numbers to allow any definite conclusion to be drawn
regarding their behavior. Others have not yet been identified. But
several of the prawns of the families Acanthephyridz and Sergestide,
one of the chetognaths, and the copepods and fishes were present in
considerable numbers and the data for these will be presented. The
copepods were present in such very large numbers in some hauls at the
400-meter level that their numbers were estimated in those hauls and
no attempt was made to separate and identify the several species.
Fishes at 800 meters were also present in large numbers and counts were
made of these in each haul but again they were not separated into genera
or species. In spite of dealing with mixed populations of copepods and
fishes, the data readily convince one that the more abundant species be-
have in a consistent manner and that their movements are obviously
related to day and night. A remarkable correlation in the numbers of
copepods and fishes in the separate hauls at the 400-meter level will be
discussed later. .

In the graphs the total number of animals of a group such as cope-
pods, or of a species, where separations into species were made, taken in
any haul is shown by a vertical bar. Haul 1 made on September 6 from
6:52 A.M. to 7:52 A.M. serves as a check on the consistency of catches
from day to day as another haul was made on September 7 from 6:57
A.M. to 7:57 A.M. The lined bars represent the September 6 catches.
When no representatives of a given species were present the word
“none” appears in place of a bar. The net at the 800-meter level at
the last haul made on September 8 from 12:39 A.M. to 2:39 A.M. failed
to work as it was improperly attached; hence no data are available for
this time and depth. The time of sunrise was 5:37 A.M., of sunset
6:19 P.M. at the position of this station on September 6. A heavy line
at the bottom of each graph indicates the hours between sunset and
sunrise, but it should be remembered that at any considerable depth the
period of relative darkness would be longer than at the surface (Clarke,
1934). It should also be noted that haul number 6 began before sunset
and continued some time afterward. Further remarks concerning a
particular species or group of animals may best be made under separate
headings.

Acanthephyride (Fig. 4)

The complete absence of any of the large red prawns from hauls
made during the day at 400 meters was the first reassuring evidence that
our results would add more convincing weight to the conclusions reached

190 WELSH, CHACE AND NUNNEMACHER

by Murray and Hjort (1912). Systellaspis debilis and Acanthephyra
purpurea had been taken previously in this region of the Sargasso Sea
(Welsh and Chace, 1937) but in these earlier collections with closing

A. PUPUREA

400M

A. PUPUREA

800M

S. DEBILIS
400M

S. DEBILIS
800M

SUNSE |] 2S ae ee ee ae)
4AM

ME SUNRISE
4AM 8AM 12N 4PM 8PM 12M

Fie. 4.

DIURNAL MIGRATION DEEP WATER ANIMALS 191

nets little attention had been paid to the time of day when the hauls were
being made. At 800 meters the largest catch of A. purpurea was made
in the early morning before sunrise, none were taken in the haul made
nearest midday. On the other hand, S. debilis was taken at 800 meters
only during daylight hours. Since it is known that S. debilis occurs at
a somewhat higher level than A. purpurea a comparison of the catches of
these two forms at the two levels strongly suggests that during the day
the majority of S. debilis descend to a depth around 800 meters and the
majority of A. purpurea to a somewhat greater depth.

Both A. purpurea and S. debilis have well-developed eyes. In con-
trast another acanthephyrid, Hymenodora glacialis, with degenerate eyes,
has never been taken with closing nets from “ Atlantis” at levels higher
than 1,000 meters. It is an example of a form which has become
adapted to a region below that at which penetrating sunlight has an effect
on behavior and the development of the eyes. The upper limit of its
distribution suggests that 1,000 meters is approximately the depth to
which a biologically significant amount of sunlight can penetrate in water
as transparent as that of the Sargasso Sea.

Sergestide (Fig. 5)

Of the sixteen species of Sergestes recorded from the North Atlantic,
thirteen were taken at Station 2667. Of these thirteen, three species
were taken in numbers large enough for statistical study. Sergestes
sargassi appeared only in hauls at the 400-meter level and then only in
those made during daylight hours. It was most numerous in the hauls
made soon after sunrise. As this is known to be a comparatively shal-
low water species, it appears that it moves toward the surface during the
night and returns to a depth around 400 meters during the day.

S. atlanticus was taken at the 400-meter level in two night hauls and
in the haul which extended from about an hour before sunset until an
hour after. None were taken at this level during the day. For some
reason no sergestids of any species were taken in haul 7 at the 400-meter
level in the late evening. At 800 meters S. atlanticus appeared in con-
siderable numbers in the hauls made during the day, but except for one
specimen which was taken in haul 2, none appeared in the other night
hauls. Evidently this species migrates to deeper water during the day
than does S. sargassi. It should be added that S. atlanticus has been
taken in surface hauls made during the night.

The catches of S. corniculum at 400 meters were very much like
those of S. atlanticus as regards relative numbers and times of appear-
ance. At 800 meters, however, S. corniculum was found in relatively
large numbers in all hauls during both day and night. It would appear

192 WELSH, CHACE AND NUNNEMACHER

SERGESTES

SARBASSI

400M

OLLI ALAA AMAL LLAMA LLL

S._ATLANTICUS

400M

S. ATLANTICUS

40 800M

S._CORNICULUM

4 400M -

S._CORNICULUM

40 800M

GS SUNRISE SUNSC] i= aa re er D
4AM 8AM 12N 4PM 8PM 12M 4AM

Jae,

DIURNAL MIGRATION DEEP WATER ANIMALS 193

that it was a form that migrated less extensively than some of the other
species of Sergestes. That there are sergestids which are found below
a level to which a sufficient amount of light penetrates to cause any di-
urnal vertical movements is known from our records on S. mollis. This
species has been taken with closing nets only at depths of 1,600 meters
and greater. Thus we see in this series of sergestids that the level of
maximum numbers depends on the species and the time of day or night;
also that the average depth at which a given species may be found de-
pends on the extent of daily migration, if it occurs at all.

Copepods and Fishes (Fig. 6)

Because of the number and variety of fishes they have not been sep-
arated into species and neither have the copepods. These two distinctly
different types of animals show, at the 400-meter level, a very similar
type of distribution at different times of day and night, and this correla-
tion may be seen if the graphs in Fig. 6 are examined. The largest
catches were taken at the 400-meter level in the haul made just before
sunrise and the one which extended from about an hour before until an
hour after sunset. Most of the other catches when compared with these
were small. It is probable that the large catches were taken as the
migrating copepods and fishes passed the 400-meter level on their ascent
and descent. The very large catches before sunrise must mean that
these animals anticipate the return of dawn and start downward before
there is any increase in light intensity. When the numbers of fishes
taken at 400 and 800 meters are compared it is first obvious that many
more were taken at the deeper level, and then that many more were
taken during the day than during the night. It is probable that this
depth is not very far from the daytime level of maximum numbers of
deep-water fishes of this region. The striking correlation in numbers
of copepods and fishes at the 400-meter level suggests that the fishes
move with the copepods and thereby stay near their chief food supply.

Sagitta hexaptera (Fig. 7)

One large, easily recognized chetognath, Sagitta hexaptera,®? was
separated and counted and will serve as another example of a form
which was taken only at 400 meters. On examination of the graph of
the distribution of this form it is seen that the largest catches were made
near midday, the smallest in the late afternoon and early evening. It
would seem that the level of maximum numbers of this species was in

3 Tdentified by Miss Alice Beale.

194 WELSH, CHACE AND NUNNEMACHER

CoPEPODS
400M

VILLLLLLLLLLILLL LLL LEO

FISH
400M
200
100
A
FISH
800M
600
400

200

" UUCZ LLL LLL LLL CLL LEE

WEES SUNRIS
4AM 8AM 12N 4PM 8PM 12M 4AM

DIURNAL MIGRATION DEEP WATER ANIMALS 195

water shallower than 400 meters during the night and that they de-
scended to near this level during the day.

Day to night differences in the numbers of certain species and groups
of organisms, at depths of 400 and 800 meters, indicate not only that the
animals of the deeper water of the Sargasso Sea migrate as do those
of the shallow water, but that for certain species these daily migrations
may be over a considerable distance. Light is probably the only day to
night variable at a depth of 800 meters, or approximately one half mile;
hence differences in light intensity must be largely responsible for these
movements. There is, however, some indication that deep-water animals
anticipate sunrise as Clarke (1934) found certain shallow water forms
such as Metridia to do. This suggests that a physiological rhythm may

SAGITTA

400M

N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N

GHEY SUNRISE SUNSET RASA AES a pa a a TR TEED
4AM 8AM 12N 4PM 8PM 12M 4AM

Fic. 7.

in part be responsible for their behavior. Diurnal rhythms are known
to persist for considerable lengths of time in the absence of regularly
recurring changes in light intensity (Welsh, 1936). An internal cycle
resulting in periodic changes in activity would account for the downward
movement which begins before there is an increase in light intensity.
Many questions have arisen which can be answered only by further
investigations. It will be of interest to determine the greatest depth at
which daily migrations occur and to correlate these movements with
measurements of light intensity. It will be necessary to make collections
at many more levels before final conclusions can be drawn regarding the
behavior of the organisms discussed in this paper, and of the many
which have been omitted from this discussion. Possible differences in

196 WELSH, CHACE AND NUNNEMACHER

the behavior of animals with and without light organs should be investi-
gated. The lower limit of the penetration of light of an intensity sig-
nificant in determining the vertical distribution of animals should be
correlated with the rather marked changes in temperature, salinity, oxy-
gen and phosphates which occur at depths near 1,000 meters in certain
parts of the Sargasso Sea. A combination of factors is doubtless
responsible for keeping the majority of the macroplankton above 1,000
meters in this region, but it would be of interest to know which one, if
any, is most important.

The collection and analysis of data of this kind is inevitably slow, but
the vertical movement of organisms in the sea is of practical as well as
of theoretical importance since the nightly aggregation of organisms near
the surface increases greatly the potential food supply of surface fishes.
Further work in the Sargasso Sea is planned and it should be possible to
obtain a much more complete idea of the biology of deep-water or-
ganisms than we now possess.

LITERATURE CITED

CiarkE, G. L., 1933. Diurnal migration of plankton in the Gulf of Maine and its
correlation with changes in submarine irradiation. Biol. Bull., 65: 402.

CLARKE, G. L., 1934. Further observations on the diurnal migration of copepods
in the Gulf of Maine. Biol. Bull., 67: 432.

CiarkeE, G. L., 1936. On the depth at which fish can see. Ecology, 17: 452.

CLARKE, G. L., anp R. H. Oster, 1934. The penetration of the blue and red com-
ponents of daylight into Atlantic coastal waters and its relation to phyto-
plankton metabolism. Biol. Bull., 67: 59.

HELLAnD-Hansen, B., 1931. Physical oceanography and meteorology. Rept.
Michael Sars N. Atlantic Deep-sea Exp., 1910, 1: 43.

Leavitt, B. B., 1935. A quantitative study of the vertical distribution of the larger
zooplankton in deep water. Biol. Bull., 68: 115. .

Morray, J., AnD J. Hyort, 1912. The Depths of the Ocean. Macmillan and Co.

Russet, F. S., 1927. The vertical distribution of plankton in the sea. Biol. Rev.,
74 ONS).

WetsH, J. H., 1936. Diurnal movements of the eye pigments of Anchistioides.
Biol. Bull., 70: 217.

WetsH, J. H., anp F. A. CuHAcs, Jr., 1937. Eyes of deep-sea crustaceans. I.
Acanthephyride. Biol. Bull., 72: 57.

ib eROBEEM Oh A PHY SIOL@GICAL GRADIENT IN
MNEMIOPSIS DURING REGENERATION?

B. R. COONFIELD AND A. GOLDIN

(From the Department of Biology, Brooklyn College, and the Marine Biological
Laboratory, Woods Hole, Massachusetts)

Experiments on certain ctenophores have shown the presence of
physiological gradients in these animals. According to Child (1933)
these animals show a decreasing susceptibility to toxic agents from the
aboral to the oral end of a plate row. There are also definite indica-
tions that Mnemiopsis leidyi exhibits an aboral dominance in certain
aspects of its activity (Coonfield, 1934 and 1936a). In addition to
these reactions, it is known that this animal will regenerate readily
(Coonfield, 1936). All of these evidences suggested the advisability
of making certain experiments on Mnemuopsis leidyi to test for a physio-
logical gradient during regeneration and in response to grafts.

The experiments reported herein were of three types. In one, the
time of regeneration following the removal of the aboral zone was
recorded; in another, the time of regeneration in the plate rows was
observed; and, in a third experiment, the reactions of Mnemiopsis to
grafting of an apical organ was followed. Similar experiments have
been used previously by Watanabe (1935) on Euplanaria dorotocephala
and by Child (1935) on Corymorpha. ‘The method of keeping Mnemi-
opsis during the experiments and of observing these animals was the
same as reported by Coonfield (19360). The temperature of the sea
water in which the experimental animals were immersed ranged from
19.0° to 20.5° C. Each type of experiment is described and the results
are given according to the headings: regenerating oral pieces, regen-
erating rows, and absorption of apical organ grafts.

REGENERATING ORAL PIECES

The purpose of these experiments was to record the time at which
oral pieces of Munemiopsis would regenerate organs which had been
removed by a single cut across the body. These cuts were made at
four levels: 4, B, C, and D (Fig. 1). By these transverse cuts certain
organs (Fig. 1) were removed along with the aboral portion of the
body. We have assumed that regeneration was complete just as soon

1 Contribution No. 23 from the Department of Biology, Brooklyn College.
197

1982 B. R. COONFIELD AND A. GOLDIN

as the lost organs had been reformed. At this time the experimental
animals had not regained their former size, but we believe that only
growth is necessary for this to be accomplished. ‘The results of these
experiments are shown in Table I.

REGENERATING Rows

In the experiments reported here, the time required by rows to
regenerate removed sections was recorded. A section including ten
plates was removed from each of the four adtentacular rows at four
different levels of the body. Level 1 begins at the apical organ and
extends aborally ; level 2 begins at the apical organ and extends orally ;
level 3 begins at the oral end of level 2 and extends orally; and level 4
begins at the oral end of level 3 and extends orally. Levels-1 and 2
were removed from the left and right rows respectively on one side,
while levels 3 and 4 were removed from the left and right rows respec-
tively on the other side of the animal. The healing and regenerating

AWARE al

Time in days required for aboral regeneration

Numb Numb Numb A
bevels operaHions sunnved reeenerated Ge
A 40 36 36 4.0
B 30 28 28 3.0
C 55 39 39 3.0
D 59 46 46 Soll

processes which followed the operations have been described adequately
by Coonfield (1937a). The results of these experiments are shown
in Table IT.

ABSORPTION OF APICAL ORGAN GRAFTS

In previous experiments (Coonfield, 1936a), it was found that
apical organs which had been grafted to the surface of Mnemuiopsis be-
came absorbed by the host within a few days. The response of the
host in these experiments suggested that this animal might be examined
for a gradient by transplanting an apical organ to its body at different
levels and observing the times at which the grafts are absorbed. There-
fore a piece of an animal containing an apical organ, a portion of the
infundibular funnel, and a part of the adradial canals was transplanted
to the surface of another animal between its adtentacular rows. ‘The
levels are shown in Table III as 1, 2, and 3. Level 1 is just above the

PHYSIOLOGICAL GRADIENT IN MNEMIOPSIS 199

apical organ ; level 2 is mid-way between the apical organ and the bases
of the auricles; and level 3 is near the bases of the auricles. Usually
these grafts established connections with certain canals of the host as
described by Coonfield (1937b). In these the order of connections was
with the adtentacular, the tentacular, the paragastric canals, and with the
stomodeum (Fig. 2, 4). In some, however, this order in connecting

Fic. 1. This is a diagram of Mnemiopsis showing levels A, B, C, and D
at which cross cuts were made. The organs as shown are: AE, adesophageal row; —
AP, apical organ; AT, adtentacular row; AU, auricles; L, lobes; M, mouth; S,
stomodeum.

was not followed (Fig. 2, B). The results of these experiments are
shown in Table III.

Since the apical organ is believed to exert a dominant influence over
the other parts of the body of Mnemiopsis (Coonfield, 1936a), it is
possible that this organ might be chiefly responsible for the absorption
of the graft and would interfere with the detection of a physiological
gradient. Therefore the aboral region including the apical organ was
cut from specimens at the time that the transplantations were made.

200 B. R. COONFIELD AND A. GOLDIN

TABLE II
Time in hours required for regeneration
Numb Number ~ Solid Holl N
Levels Sperntiens Saaived pod oualy ines
1 20 20 2.3 5.0 42.2
2 20 20 2.3 5.6 40.1
3 20 20 2.35 5.8 42.6
4 20 20 2.35 5.6 43.7

Controls were observed to detect any variation within groups of speci-
mens. The results of the experiments are shown in Table IV. The

levels indicated in this table correspond to the similar ones shown in
Mable sak

Tasce III

Time in days required for absorption of apical organ grafts. (The host was
intact except for the transplantation.)

Numb Numb Numb A
“eel eee eee pe ee pe
1 17 8 8 9.25
2 17 14 14 12.25
3 16 8 8 10.50
TABLE IV

Time in days required for absorption of apical organ grafts. (Apical organ of the host
was removed at time of transplantation.)

Regeneration No regeneration No absorption No absorption
Namber No regeneration
Level opera- Num-
tions Number | Average | Number | Average ber Average
absorbed| time absorbed] time ieee Time Nien Time
2 24 7 11.6 5 11.4 2 10 10 7.1
3 15 2 12.0 2 9.0 3 6 8 8.1
DISCUSSION

Mnemiopsis will regenerate organs that have been removed and
many of the changes taking place during regeneration can be observed
easily (Coonfield, 19360). Should a gradient be present during re-
generation, there should be a difference in the time at which organs will

PHYSIOLOGICAL GRADIENT IN MNEMIOPSIS 201

regenerate. In making this test specimens were cut across at four
levels and the regenerating oral pieces were observed. The progressive
changes following these cuttings at these levels were very similar. This
resemblance is illustrated in Figs. 3 to-10 where the arrows which point
to a plate in a similar location on each of a series show that it is pos-
sible to believe that in Figs. 3 to 6 are represented four different stages
of one animal. These figures represent four different animals at four
different stages during regeneration. In Figs. 7 to 10 four other speci-
mens which were cut at a lower level than those in Figs. 3 to 6 are

Fic. 2. These are photographs of the adtentacular surfaces of two specimens
to which apical organs were grafted at level 3 (Table III). A shows the graft
connected to two adtentacular canals of the host. B shows the graft connected to
the tentacular canal (at arrow) of the host. Both photographs were taken five
days after the operations.

shown. ‘The results of these experiments (Table 1) show clearly that
there is no difference in the time of regeneration at the four levels of
the body.

Following the removal of sections from plate rows the remaining
ends of the rows pulled together, fused, stretched, and formed solid
cords which later became hollow as described by Coonfield (1937a).
Later new plates formed in the healed region and in between the old
plates which had been pulled apart by the stretching. The results of
these experiments are shown in Table II]. Here the average time in

202 B. R. COONFIELD AND A. GOLDIN

hours required for the formation of the solid cord of cells, the hollow
canal, and the new plates is shown. There was no significant difference
in the time of formation of these structures at the four levels of the
body. This experiment is significant since the same organs of an in-
dividual were regenerating at the same time and at different levels.
Furthermore there was no appreciable variation in the time of regen-
eration of these parts of rows when the results of experiments on
individual animals were compared with each other.

Two methods of testing for a gradient by the absorption of apical
organ grafts were employed. In one where the specimens were intact,
only the time of absorption was involved. In the other where the apical
organ of the host was removed at the time of the transplantation, the
possible influence of this organ on the absorption of the graft was
eliminated. The results obtained by the first method show that the
grafts near the apical organ were absorbed at a slightly shorter time
than those which were farther away from this organ. The difference
in the time, however, is not enough to be significant. In those animals
from which the apical organs had been removed at the time of the
grafting (Table IV) the results show that there is no appreciable
difference in the time of absorption of the grafts at different levels. It
is clear also that a lower percentage of grafts was absorbed when the
influence of the apical organ was removed. We believe that this low
percentage of absorption was due to the absence of the influence of the
apical organ. ‘This influence is demonstrated by the delayed regenera-
tion of an apical organ in those specimens on which the grafts persisted.
We point out that in a relatively high percentage of these specimens
the grafts persisted and there was no regeneration. We believe that
the failure to regenerate was due to the inhibitory influence of the graft.
In these specimens the grafts joined the various canals exactly as a
normal apical organ is associated with the canals of the animal.

We conclude from the results of these experiments that a physiologi-
cal gradient that has been shown to influence the times of regeneration

EXPLANATION OF PLATE I

Fires. 3, 4, 5, and 6 are photographs of four specimens regenerating. They
were all cut across at the same time and at level A (Fig. 1). They were photo-
graphed at 13, 39, 48, and 90 hours respectively after the apical ends had been
removed. The arrows are pointing at similar plates on the four specimens to
show the similarity of the regenerating processes.

Fies. 7, 8, 9, and 10 are photographs of four specimens regenerating. They
were cut across at the same time and at level B (Fig. 1). They were photographed
at 13, 39, 48, and 90 hours respectively after the apical ends had been removed.

PLATE I

204 B. R. COONFIELD AND A. GOLDIN

and the absorption of organs in other animals is not present in Mnemi-
opsis leidyi.

CONCLUSIONS

1. A physiological gradient which would influence the times of
regeneration and absorption of grafts is not present in Mnemiopsis.

2. The time of regeneration of oral pieces following transverse
cuts at four levels of the body is the same at all of these levels.

3. The time of regeneration following the extirpation of sections
of plate rows at four levels of the body is the same at all of these levels.

4. The time of absorption of apical organ grafts is the same at three
different levels of the body.

5. Retained apical organ grafts either inhibit or delay the regenera-
tion of an apical organ by the host.

IPIMES OANA URI, (CIAL TID)

Cuitp, C. M., 1933. The swimming plate rows of the ctenophore, Pleurobrachia.
as gradients: with comparative data on other forms. Jour. Compar.
Neurol., 57: 199.

Cuitp, C. M., 1935. Dominance of hydranths induced by grafts in Corymorpha.
Jour. Exper. Zool., 71: 375.

CoonFIELD, B. R., 1934. Coordination and movement of the swimming-plates of
Mnemiopsis leidyi, Agassiz. Biol. Bull., 66: 10.

CoonFIELD, B. R., 1936a. Apical dominance and polarity in Mnemiopsis leidyi,
Agassiz. Biol. Bull., 70: 460.

CoonFieELp, B. R., 1936b. Regeneration in Mnemiopsis leidyi, Agassiz. Biol.
Bull., 71: 421.

CoonFiELp, B. R., 1937a. The regeneration of plate rows in Mnemiopsis leidyi,
Agassiz. Proc. Nat. Acad. Sci., 23: 152.

CoonFIELD, B. R., 1937b. Symmetry and regulation in Mnemiopsis leidyi, Agassiz.
Biol. Bull., 72: 299.

WatTAnase, Y., 1935. Rate of head development as indicated by time of appear-
ance of eyes in the reconstitution of Euplanaria dorotocephala. Physiol.
Zool., 8: 41.

THE HEXOCTAHEDRON AND GROWTH?

OTTO GLASER AND GEORGE P. CHILD
(From the Biological Laboratories of Amherst College)

Kelvin’s (1887) analysis of the conditions under which soap bubbles
assume hexoctahedral form and together incorporate space without
remnant, applies to tissue cells whenever these are surrounded and
compressed on all sides by units essentially similar to themselves (Lewis,
1923, 1926, 1933). In all cases the more generalized the tissue and
the more exact its quantification, the more closely the cell approximates
a solid whose thirty-six equal edges bound eight hexagons and six
non-contiguous squares.

JUL

Since cells and nuclei contain emulsions, we may anticipate, under
appropriate conditions, either orthic or rhombic hexoctahedra among
various kinds of protoplasmic inclusions. Indeed, Lewis (1926, p. 23)
has verified Pouchet’s (1847) discovery that polyhedra of this type
occur among the yolk granules of a hardboiled egg. Here, despite much
fortuity, we find practical realizations of the ideal form shown in Fig. 1.
As Lewis has not depicted these polyhedra and Pouchet’s figures are not
easily available, a selection of coagulated yolk granules is reproduced
inal laite, A,

Comparable shapes, sometimes indistinguishable from orthic hexoc-
tahedra, can be observed in dried cornstarch (Fig. 3). Many illus-
trations in Reichert’s (1913) monograph indicate the same form, more
or less modified, in other types of starch.

III

Translucent polyhedral bodies fill and often burst the nuclei in
various tissues of “ wilted” caterpillars (Fig. 4). In any given nucleus

1 We are indebted to Professor George W. Bain of Amherst College for photo-
graphs of the polyhedral bodies of the silkworm. Dr. Bain first drew our atten-
tion to the fact that these bodies do not polarize light. We wish to express our
appreciation also to Dr. R. W. Glaser of the Rockefeller Institute for material
sent in 1936 and for the privilege of studying the photographic plates made in
connection with the original Glaser, and Glaser and Chapman investigations on
the polyhedral bodies of insects.

205

206 OTTO (GEASER AND GHORGE PAG HiiED

the polyhedra are remarkably uniform in size; in different nuclei they
range from 0.5 to 15 in diameter (Glaser and Chapman, 1916).

A B Gc

Fic. 1. Models of the hexoctahedron. In the originals, made of alumimum,
the edges are exactly 2 cm. in length. A, with hexagonal facet toward the ob-
server and lying on the hexagon, opposite. B, with square facet toward the ob-
server and lying on the square opposite. C, interhexagonal edge or dihedral angle
toward the observer and resting on the interhexagonal edge opposite.

Fic. 2. Granules of coagulated egg-yolk under various illuminations and
magnifications. No granule is completely in focus throughout.

A, B. Quadrilateral facets produced by truncation insufficient to realize equi-
lateral edges.

C. A group of granules sufficiently uniform to illustrate practically ideal stack-
ing. Focused on the planes of contact. The three dots are “ waste” space.

Ocular recognition of form in three dimensions depends on optical
sections combined with significant surface views. For the interpreta-
tion of these, oriented models (Fig. 1) are indispensable.’

2 Useful models can be constructed after the patterns of Matzke (1931).
Matzke has also published a careful analysis of the orthic tetrakaidecahedron and
its aggregation without waste of space (1927).

HEXOCTAHEDRON AND GROWTH 207

Experimentation shows that hexoctahedra, in air, are most stable
when resting on their hexagonal faces. In this position, the surface
nearest the observer is also hexagonal and between this proximal and
the basal hexagon are two alternating tiers in each of which three lateral
hexagons alternate with three squares (Fig. 1, 4). Ina second posi-
tion, most frequent in viscous liquids, the polyhedron is supported by
the overhanging edges of the lateral hexagons and comes to rest on a
square face. Now the uppermost surface is also square and separated
from its basal counterpart by two tiers of four hexagons each and an
equatorial tier of four non-contiguous squares (Fig. 1, B).

To interpret microscopic polyhedra requires patience. Even though
the facets are true planes, we cannot assume that every individual resting
on a square or hexagon is also level. A specific polyhedron may deviate

Fic. 3. Granules of dried cornstarch photographed by reflected light under
various magnifications.

A. Upper left granule showing a clearly quadrilateral face; lower right,
showing square facet, vaguely.

B. Lateral view of cornstarch granule showing two hexagonal facets; the
lower one with a glitter point. To the right a quadrilateral face.

C. Similar to B but showing hila in hexagonal facets.

from the ideal, or have its basal facet tilted by neighbors or fragments
of foreign material. Thus, an optical section may not conform with
expectations based on simple square or hexagonal orientation.

What then is required to establish the hexoctahedral form? Clearly,
the object must be three-dimensional; must possess facets; and these,
when not hexagonal, must be square. Squares cannot be contiguous.
Facial angles, when not 120°, must be 90° and all edges must be equal.
We should expect sections in which the hexagons are not equilateral and
sections indicative of more than six sides. Octagons, decagons and
dodecagons are all possible. Fragmentary outlines of the higher poly-
gons are not easily intelligible.

Photographs, based on the two stable orientations of the models in
Fig. 1 and including only samples of the typical surface views and
optical sections most frequently encountered with the polyhedral bodies

208 OTTO GLASER AND GEORGE P. CHILD

of the silk worm, are shown in Fig. 5. These records enable us to
postulate the hexoctahedral form for the polyhedral bodies of “ wilt.”

If sufficiently familiar with the form, the observer can recognize
the three-dimensional character of the polyhedra and indications of the
two types of facet even at the lower magnifications shown in Fig. 4.
To these views, we add the evidence secured by dark field and unilateral
illumination. In favorable instances such lighting picks out some of

B €

Fic. 4. Nuclei of wilted army worm and gipsy moth caterpillars.

Polyhedral bodies in three dimensions with numerous indications of facets in
black or white and sharp dihedral edges. A, polyhedra in half the nucleus of a
fat cell of an infected army worm X 1500: enlarged from the photographic plate
reproduced in Glaser and Chapman (1916) as Fig. 3, Plate II. B, half of smaller
nucleus of same X 1416. C, polyhedra in nucleus about tracheal tube of gipsy
moth caterpillar X 1875: enlarged from the original reproduced as Fig. 2, Plate
II, in Glaser (1915). As now printed, the above enlargements are reduced one-
half.

HEXOCTAHEDRON AND GROWTH 209

the trihedral angles. These appear as minute points from which may
radiate three slightly luminous edges. Photographs in which the ob-
jects are vaguely outlined by means of illuminated facets, trihedral
angles, and edges are shown in Fig. 6. The hexagonal distribution of
the trihedral angles and the square facets, each outlined by four luminous
points, are especially noteworthy.

Fic. 5. Hexagonal and square orientations of polyhedral bodies.

The large hexagon is viewed by transmitted light. It is equilateral and the
facial angles are 120°. The square orientations demonstrate equal sides with 90°
angles. In many instances the upper levels of the adjacent lateral hexagons are
partially lighted. The octagonal marginal outlines are visible whenever the optical
sections cut hexagonal facets parallel to their diameters and square faces parallel
to their diagonals.

Fic. 6. Polyhedral bodies of the silk worm at various magnifications showing
facets, trihedral and dihedral angles, under lateral illumination.

IDV!

For our immediate purposes it is unnecessary to know the chemical
composition of the intra-nuclear hexoctahedra of insects, or whether
these objects are crystals (cf. Glaser and Chapman, 1916). It is im-
portant that bodies with this shape stack without waste space; that
slightly rhombic forms can be treated as if they were orthic (Kelvin,

210 OTTO GLASER AND GEORGE P. CHILD

op. cit.) ; and that the properties of cellular emulsions and the pressures
to which they may be subjected are such that hexoctahedra of all pos-
sible orders of magnitude and all degrees of perfection may be postu-
lated. Many of these may be only rough temporary approximations ;
others may be expected whenever the emulsions of which they were
parts are properly compressed and undergo fixation. Conceivably even
true crystals may appear under conditions that would impose the same
form on non-crystalline materials.

Systems of this sort may not be as regular throughout as the poly-
hedra within a given insect nucleus; nevertheless even yolk contains
regions of essentially orthic units constant in size and presumably con-
stant in weight. Whether aberrancies cancel out in sufficiently volum-
inous masses or whether the polyhedra or their precursors in a given
system may deviate from the hexoctahedral ideal by constant fractions,
the fact remains that irregularities prove relatively unimportant.

Stacked in 1 layers about a central unit, the total number of indi-
viduals, Sn, in an isogonic aggregate of orthic hexoctahedra is 4n°
+ 6n? + 4n+ 1 (Marvin, 1936). This statement is identical with
(2n + 1)

Sn + 2—05 =
Essentially, therefore,

log Su=3 log (2n + 1) —0.3010.

With this transformation hexoctahedral aggregation assumes a formal
identity with organic growth,

log w=k log (2t+1)+C,

in which weight is equated against time (Glaser, 1938, in press).
Hence, if at some level within the cell, organic growth is controlled by
hexoctahedral aggregation, layer number in such aggregates must be
proportional to time.

This proportion suggests an absolute standard for heterogonic cor-
relations since the heterogony constant k; in y= 0x", is the ratio be-
tween the growth constants for x and y (Huxley, 1932; Glaser, 1938).
Without identifying x or y with the units that control their magnitudes,
it is possible to attribute the serial changes in x or y to a summation with

HEXOCTAHEDRON AND GROWTH Balik

properties indistinguishable from those of the hexoctahedral aggregation
series. Accordingly, this series must be able to replace either organic
correlative in the heterogonic equation.

When the ages of x or y are unknown, proper adjustment between
the hexoctahedral series and the organic correlates may be possible by
trial and error; when the organic weights are dated, the proportionality
between layer number and time is decisive.

4.50

4.00

3.50

Log Hexoctahedral Aggregation Series

3.00 F

2.50 |
0.00 1.00 2.00 3.00 4,00
Log Mg.

Fic. 7. Heterogonic plot. Two organic weight series against the ideal
hexoctahedral aggregation series. A, the quantitatively organized chlorides in the
chick embryo, based on Table I, Murray (1926, p. 794). B, fresh weight of
chick embryos based on the original analyses of Murray (1926; Table III, p. 410).

Substitutions of the dated variety are illustrated in Fig. 7. Curve
A shows the relation between the quantitatively organized chlorides of
the chick embryo and the Marvin series; B, the relation between this
series and the weight of the entire chick. In both instances the linear
relationship implicit in y = b+", is fairly realized. Moreover, since the

growth constant for the chick is 3.76 (Glaser, op. cit., in press) the

hexoctahedral heterogonic constant should bes = 0.797. The inde-

DAD, OTTO GLASER AND GEORGE P, CHILD

pendent graphic determination yields 0.80. For the chlorides with a
growth constant of 3.53 (Glaser, op. cit.) the constant for curve A

should be Oo. Independent graphic determination indicates

5:53)
0.89.

Many other quantitative relationships of the types here illustrated
can be predicted and, where the data permit, verified. For this reason
we may consider the hexoctahedral aggregation series as an instrument
which enables us to penetrate to analytical levels not attainable without
reference to the geometrical foundations of organic cumulative growth.

SUMMARY

1. Certain starch grains and the polyhedral bodies of insects are
hexoctahedra of the type described by Lewis for tissue cells and coagu-
lated yolk, and by Kelvin in systems of soap bubbles.

2. Compression of units visible in cellular emulsions results in aggre-
gates of hexoctahedra. Under the same conditions the same form is
postulated as either imminent or actual, at levels below the limits of
visibility.

3. If this extension applies to materials that control the dimensions
of organisms, Marvin’s hexoctahedral aggregation series should be ca-
pable of replacing either organic correlative in the heterogonic equation
y = bx", Evidence is afforded by heterogonic curves in which the
Marvin series is plotted against the chick embryo and the chlorides of
the chick embryo.

4. The success of this test depends on two facts: (a) the heterogonic
constant is the ratio between two growth constants; and (0) when iso-
gonic aggregates of hexoctahedra are produced by organic growth, the
number of layers in which the resulting units stack about a central unit,
is proportional to time.

REFERENCES

Guaser, R. W., 1915. Wilt of gipsy moth caterpillars. Jour. Agr. Res., 4: 101.

GuaseER, O., 1938. Growth, time, and form. Biol. Rev., 13: 1.

Graser, R. W., AND J. W. Cuapman, 1913. The wilt disease of gipsy moth cater-
pillars. Jour. Econ. Entomol., 6: 479.

GuAser, R. W., AND J. W. Cuarpman, 1916. The nature of the polyhedral bodies
found in insects. Biol. Bull., 30: 367.

Huxtey, J. S., 1932. Problems of Relative Growth. Methuen and Co., London.

KELVIN (Sir W. THompson), 1887. On the division of space with minimum par-
titional area. Pil. Mag., Series 5, 24: 503.

Lewis, F. T., 1923. The typical shape of polyhedral cells in vegetable parenchyma
and the restoration of that shape following cell division. Proc. Am.
Acad. Arts and Sct., 58: 537.

HEXOCTAHEDRON AND GROWTH DANG)

Lewis, F. T., 1926. A further study of the polyhedral shapes of cells. Proc.
Am. Acad. Arts and Sci., 61: 1.

Lewis, F. T., 1933. The significance of cells as revealed by their polyhedral
shapes, with special reference to precartilage, and a surmise concerning
nerve cells and neuroglia. Proc. Am. Acad. Arts and Sci., 68: 251.

Marvin, J. W., 1936. The aggregation of orthic tetrakaidecahedra. Science, 83:
188.

Matzke, E. B., 1927. An analysis of the orthic tetrakaidecahedron. Bull. Torrey

Bot. Club, 54: 341.

Matzke, E. B., 1931. Modelling the orthic tetrakaidecahedron. Torreya, 31:

129.

Murray, H. A., 1926. Physiological Ontogeny, A. Chicken embryos. VII. The

concentration of the organic constituents and the calorific value as func-

tions of age. Jour. Gen. Physiol., 9: 405.

Murray, H. A., Jr. 1926. Physiological Ontogeny, A. Chicken embryos. XI.
The pH, chloride, carbonic acid, and protein concentrations in the tissues
as functions of age. Jour. Gen. Physiol., 9: 789.

Poucuet, F. A., 1847. Théorie positive de l’ovulation spontanée. Paris.

RetcHert, E. T., 1913. The Differentiation and Specificity of Starches in Rela-
tion to Genera and Species. Carnegie Institution of Washington, Wash-
ington, D. C. Publication 173.

SIUIDIITS QIN TIXOIDUIC INDY NE JUNI) TIBI IMU ITION Ou
DROSOPHILA MUTANTS

(GuANIUL, Ibs (CAROYIOIS

(From Mercer University, Macon, Ga.)

INTRODUCTION

From his tests of the mutant “ Truncate’ in various crosses, Hyde
(Jour. Exper. Zool., 17: 173) concluded that the degree of fertility of
each gamete was directly dependent upon the genes carried by that
gamete, irrespective of the zygote producing it. On the other hand,
the crosses carried out by Morgan (Zeitschr. Abst. Vererb., 7: 323)
with the sex-linked recessive “ rudimentary ’’ had led to the hypothesis
that “prematuration” effects of the zygote upon the developing germ
cells explained the peculiar partial sterility of the rudimentary females.
Accordingly, a study of the effects of gametic versus zygotic constitution
on the productivity and fertility of various mutants was undertaken.
These tests were started at Columbia University but were mainly made
at Woods Hole during five summers there. I wish to express my
sincere thanks to Professor Morgan and Dr. Bridges for their advice
and help in this analysis.

PRODUCTIVITY OF VESTIGIAL

The first mutant investigated was the second chromosome recessive
‘vestigial wings’ which had given evidence of low productivity not
complicated by obvious sterility of any sort. In order to avoid high
or aberrant mortality through overcrowding or underfeeding of the
growing larvae, the individual females and their mates were transferred
every few days to fresh culture bottles of large size liberally supplied
with food. The total productivity of each female was obtained by
continuing the transfer as long as the female lived. Table I gives sum-
maries of the productivities of the wild stock control and of the crosses
involving vestigial.

The most striking relation evident in the result is that the produc-
tivity is dependent primarily on the zygotic type of the mother. The
cultures from vestigial mothers averaged 344 (51 vg @ with 17,538 off-
spring) not much more than half the average of 581 from non-vestigial
mothers (72 non-vg 2 with 41,897 offspring). But the average length
of life of the vestigial mothers was 28 days while the average life of

214

PRODUCTIVITY AND FERTILITY DROSOPHILA MUTANTS 215

the non-vestigial mothers was 51 days. That the difference in pro-
ductivity was mainly due to this difference in life-span was shown by
the fact that the daily productivity averages 12.3 for the vestigial and
11.4 for the non-vestigial mothers.

In the results of Table I, a second striking relation is shown, viz.:
that productivity rises with outcrossing to diverse strains. Thus, while
the productivity of the females of the inbred Falmouth strains aver-
aged 534, the productivity rose to 612 when outcrossed to another wild
strain and to 611 when outcrossed to vestigial males. Similarly, the

productivity of vestigial females of the inbred cross was only 283, but
females of this same stock gave a productivity of 426 when outcrossed
to Falmouth males and our intermediate productivity of 373 when out-
crossed to F, heterozygotes. That the improvement with outcrossing

TABLE I

Crosses testing productivity of vestigial and wild

Average

Q rofl Pairs aim +o Vg 2 Ve oS) Total | Produc-
tion
alex Ballin e chv.fotein coe 37 10,053 | 9.677 — = 19,730 | 534
ale Weed... 9 DSI. G6S il (== at 5,491 | 612
FMS Wore Sek eu ee 10 BHO) 200850) as 6,105] 611
AG kee SG ieee a at 16 2,742 | 2,611 | 2,678 | 2,540 | 10,571 | 661
es ee 21 at SE AS00418 2193878 | 504 283
Te I eee eee 10 PS se onto? lho _ 4,259 | 426
ee ee ee 11 1,022 | 1,033 | 1,057 988 | 4,100| 373
Faye X Fuve.......-.. 9 ae ee e645: | 1.5982 0 1st2 380 econ

is due to the genetic constitution of the hybrids as compared with the
parental races seems clear and is analagous to the “heterosis” effect
observed in corn strains. Further evidence of genetic influence upon
productivity is seen in the fact that vestigials extracted from the F,
gave an average productivity of 360—higher than the productivity of
the vestigial stock. The improvement seen in the extracted vestigials
must be due to genes that assort separately from the vestigial gene.

Tue FERTILITY OF EGGS FROM THE VESTIGIAL CROSSES

Besides length of life and average daily output of eggs, another
factor in productivity is the percentage of eggs that give adults the
fertility of the eggs. Hyde had introduced the method of isolating
counted eggs and observing how many adults these produced. Simi-
larly, I studied fertility by isolating each female and her mates in a

216 GAIL L. CARVER

bottle and collecting her eggs upon a strip of blotting paper thickly
spread with fermented banana. These strips were changed twice daily,
the eggs counted and transferred to well-fed culture bottles where larve
developed. By additional food supplied as the larve grew, mortality,
in the post-egg stage, was minimized and the differences in egg fertility
brought out clearly. In Table II are summarized the results of the
fertility studies on crosses involving vestigial and the wild stock
Falmouth.

TABLE II

Crosses testing fertility of vestigial and wild

exe Eggs Adults Fertility

per cent
Rial wale eyeee er uate les ale. 3,505 2,080 59
Male oxen NSicgesa ts mite 1,995 1,456 73
Paleo ee asin ees is che 2,619 1,868 71
SIVA ETA eee ete ae 1,801 1,475 82
NON SH OO Areca ds 3,240 ' 2,070 64
STAVE VEDI get coher esc hsscealane 2,066 1,441 70
siege > heal LE tae eae 1,617 1,069 66
Vig Date Noy a ae ect Gas 1,973 1,164 59
UAE CO AUACAG es Gaga Cm are Ee ete angie 4,149 1,897 46
leone S< ISS col bobo oo bos 640 465 55

The significant feature of the results of Table II is the parallelism
shown between the fertilities and the productivities of Table I. Thus
the fertility of the Falmouth inbred stock was 59 per cent which was
raised to 71 per cent and 73 per cent when these same females were
outcrossed to vestigials or to heterozygotes. Similarly, the fertility of
the vestigial females was raised from 41 per cent to 66 per cent and
59 per cent when outcrossed to Falmouth and to heterozygous males—
the increase in fertility being similar in amount to the increases in pro-
ductivity and presumably accounting for their occurrence. The fertility
of the heterozygous females mated to wild males was markedly better
(82) than that of the wild females (59) or vestigial females (66) mated
to wild males. Finally, the fertilities of the F, extracted vestigials (55)
was better than that of the inbred stock (46) and was matched by the
increased productivity noted in Table I.

Hence, it may be concluded that the primary genetic difference
between the vestigial and wild stock (the vestigial gene) was responsible
for the curtailment of productivity of vestigial females to approxi-

PRODUCTIVITY AND FERTILITY DROSOPHILA MUTANTS 217

mately half that of wild through its determination of a phenotype not
able to function for much more than half the normal span of life. The
two original stocks differed in secondary gene pairs which (1) height-
ened the productivity of crosses by improved viability of the hybrids
in the egg stages, (2) heightened the productivity of hybrids (heterosis
effect) and which (3) were able to redistribute with respect to the
vestigial pair and bring about the improved fertility and consequent
improved productivity of the F, extracted vestigials.

FERTILITY OF THE HIGH-PRODUCING “ Harry” STRAIN

Unusually high production had been noted for a few strains, notably
for the heterozygotes of hairy. A study of the fertility of hairy
(Table III) showed that the outcross of hairy by Falmouth gave an
increased fertility of 64 per cent as compared with the 59 per cent of
Fal. X Fal. and the 60 per cent of hairy < hairy. The hybrids them-
selves gave an even higher fertility of 73 per cent corresponding to the
unusually high productivity originally observed.

‘Cassie JOM

Fertility of hairy and wild

©) 3s Ct Eggs Adults Fertility

: per cent
Epa all gees she Peat 3,505 2,080 59
IR eo Eva eee ut ta a ili a 1,157 743 64
J? SC lnk en cer eRe Bee ee ae ane On 1,706 1,032 60
States ose a) Osea evens 679 494 73
TG OS ONL ay erry perder 4,527 2,747 61
Delpy OV Sa) saa feo rath ee ere coe 2,625 1,459 56

Tue FERTILITY RELATIONS OF SINGED AND SINGED?

Among the mutant strains at Columbia there were three which
showed complete or nearly complete sterility of the females but for
which there existed allelomorphic strains similar in phenotypes but with
approximately normal fertility of the females.

One such set of allelomorphs was singed and singed’, sex-linked
recessive found by Mohr. In both mutants, the bristles and hairs are
tightly curled or crumpled, but singed* has normal fertility while singed
females lay few eggs all abnormal in shape and filaments.

As shown in Table IV, the abnormal eggs laid by young singed
females averaged only about 7 per day as compared with 20 per day

218 GAIL L. CARVER

for singed* females and 25 per day for their hybrids. None of the
eggs of the singed females gave offspring while the fertility of the eggs
of singed* females was 33 per cent with brothers and 53 per cent in
crosses to singed males. Fertility of the hybrids was 38 per cent and
37 per cent when mated to singed and singed* males—better than that
of either pure race.

TABLE IV
Fertility of singed and singed 3

>< Cf Eggs Eggs Daily Adults Fertility

per cent
STU SGESTI Gn eer Medan te 754 6.6 0 0
SI MUSTO ea sian he nees 1,023 7.8 0 0
STIG OC STION EEE ettn aa: 3,272 20.4 1,085 33
SNUG ISH eae eee 3,269 19.2 1,718 53
SYST SK Stso esol sace 2,903 24.4 1,093 38
sn/sn? X sn?.......... 3,034 25.9 iL il?) 37

FertTItiry RELATIONS OF REDUCED AND SCRAGGLY

The second—chromosome-recessive allelomorphs “reduced” (rd)
and “scraggly”’ (rds) both found by Bridges, are characterized by
irregular and variable reduction in number and size of the bristles.
Scraggly is of normal fertility but females of reduced lay only a few
eggs which almost never give offspring though they are normal in
appearance.

AV ABER OVS

Fertility of reduced and scraggly

SS Ct Eggs : Adults Fertility

per cent
TiC Clase ats evil Role a eat ge 460 0 0
TROL CEG Ey 6S) a ee ee 540 4 1
== /fidle> <iik6 I eee ee 385 89 23
eine bee Gera leurs wegen es an See oat 1,305 563 43
POS aT ere eeaae nen 5. gk 1,299 526 40
OSM onal eens ereicnome eet ani nals 688 315 46
Taide Dede ye ke 6G 3,983 1,162 29
TOY TOS XG Ge ees eeyers Ghee nice: 4,560 996 DD;

As shown in Table V, the eggs from reduced @ x reduced ¢
gave no offspring, while reduced 9 out-crossed to scraggly § gave only
4 offspring. That the poor fertility of reduced is semi-dominant seems

PRODUCTIVITY AND FERTILITY DROSOPHILA MUTANTS 219

probable from the sub-normal fertility of heterozygous rd/+ 99 X rd
did (23 per cent) and X rd* dif (43 per cent), though, as usual, the
outcross gave the higher fertility. Similarly, the semi-dominance of
the poor fertility of reduced may account for the fact that the rd/rdé
females gave only 29 per cent and 22 per cent fertilities, lower than the
fertility of scraggly females (40 per cent inbred and 46 per cent out-
crossed). Within each fertility test involving reduced or scraggly, a
high degree of variability appeared, with a tendency for the fertilities
of individual pairs to fall into three groups—one below 5 per cent, one
centering around 20 per cent and one around 50 per cent.

FERTILITY OF RUDIMENTARY’ AND RUDIMENTARY“

The original mutation rudimentary had been lost, but several new
allelomorphs had been found by Bridges. Among them, rudimentary
had the very short wings and female sterility characteristics of the rudi-
mentary, while rudimentary’ had wings only-slightly shorter than nor-
mal and fertility not obviously reduced? The eggs of both strains are
normal in appearance.

TasLe VI

Fertility of Rudimentary’ and Rudimentary“

YS ot Eggs Eggs Daily Adults Fertility
per cent
er Se hese See AA aN 2,390 13 0 0
Tein ee epttn cus sialon 6 2,485 14 3 o+
FPS les AR ROR eA eae 3,016 18 1,449 48
5B Soe aC ic 2,194 18 1,263 58
tei ptes eee ee ait ee 6,562 40 2,883 44
cy ites ee ol Gan ea 3,413 35 1,886 55

As seen in Table VI, rudimentary™ 99 laid an average of 13.5 eggs
per day, though none gave offspring in the mating to rudimentary™* jig
and almost none in the outcrosses to rudimentary’ ¢¢. Rudimentary’
2 gave more eggs per day (18) and these had the nearly normal fer-
tility of 48 per cent upon inbreeding and 58 per cent upon outcrossing
to cudimentary-. Ihe r/c hybrids gave egg outputs of 39 =: per
day, higher than either parent. The fertility of the hybrids, 44 per
cent, and 55 per cent, show only a little lowering from the rudimentary’
standard through the substitution of the infertile rudimentary’* gene
whose effects are nearly recessive.

220 GAIL L. CARVER

CORRELATED VARIABILITY OF PHENOTYPE AND FERTILITY

The phenotype variability of vestigial, hairy, singed and singed?®
was very slight under the stable culture conditions of the experiments
and correspondingly the productivity and fertility were highly uniform |
for the different individual mating within each type of cross.

The reduced and scraggly phenotypes showed considerable varia-
bility both in the bristly size and number and in the fertilities. Even
more variable in phenotypic expression are rudimentary’ and rudi-
mentary™, and the greatest variability in wing size and venation re-
duction was shown in the r‘/r1* hybrids. Corresponding fairly close
to the degree of phenotypic departure was the reduction in productivity
of the rudimentary’ and r7/r™ hybrids. Nine hybrids, selected for their
extreme shortness of wings, gave 626 eggs from which only one off-
spring emerged—thereby resembling their rudimentary** parent in both
phenotypes and productivity.

DISCUSSION

The results of the experiments are all compatible with the view that
fertility of gametes is independent of their genic content but is deter-
mined by the phenotype of the zygote producing them. Even in nor-
mal races, a surprisingly high proportion of eggs, about 40 per cent,
may fail to produce adults. It would seem that the stages of fertiliza-
tion and early embryonic development must be highly unstable and pre-
carious. ‘That the genetic composition of the zygote influences strongly
the early process of development is evident from the practically uni-
versal improvement in fertility and productivity brought about by out-
crossing. Other evidences of the rdle of inheritance in the fertility—
increased fertility of the hybrids and changed fertility of extracted types
—point to a situation for Drosophila analagous to that in maize, where
inbred strains differ by large numbers of genes with slight individual
but larger cumulative effects.

Hep Oh SAE R NS MEASURED! WIT TEs
CES Seale CPR OMT

ERIG{G, BAEL AND C. CHESTER STOCK

(From the Marine Biological Laboratory, Woods Hole, Mass., and the Department
of Physiological Chemistry, the Johns Hopkins University
School of Medicine, Baltimore, Maryland)

Most methods available for the determination of pH must be sub-
jected to critical study when applied to sea water. The reason lies in
the fact that sea water is a relatively weakly buffered medium with a
high salt content. For instance, the application of the colorimetric
method to sea water has required the evaluation of rather large correc-
tion factors for the so-called salt error. In the case of the quinhydrone
electrode, the determinations of sea water are not only subject to a salt
error but accuracy is also impaired by the inherent instability of quinone
at the pH values encountered. The method of choice would appear to
be the glass electrode since it is highly suitable to unbuffered solutions.
It is, however, well known that the glass electrode is susceptible to salt
effects particularly in the alkaline range. It is the purpose of this paper
to show that the salt content of sea water does not interfere with the
application of the glass electrode to the determination of its pH and
that such determinations may be made with a fair degree of accuracy.

PROCEDURE

The method of reference in all pH determinations is the hydrogen
electrode. It would therefore appear that the simplest procedure for
checking the accuracy of the glass electrode would be parallel determina-
tions on sea water by both procedures. Such a study is unfortunately
open to experimental difficulty by reason of the fact that the CO, ten-
sion of sea water and thereby its pH is altered while developing the
atmosphere of hydrogen necessary to the use of the hydrogen electrode.
Though this experimental difficulty is surmountable by the use of spe-
cial apparatus of the type described by McClendon (1917), it was felt
that a simpler though valid test of the effect of sea salt on the glass
electrode might be furnished by parallel determinations by both methods
on sea water deaerated with hydrogen. Even though the pH of sea
water so treated is altered toward more alkaline values a sample uni-
formly treated should serve for an adequate comparison of the two

222 IRIE, (Ce BVAEIL, MIND) (Es MCISUS|S MESS SOK

methods. It was, however, impossible to obtain with the hydrogen
electrode consistent values upon sea water so treated. The poor buffer-
ing capacity of such samples may be responsible. An indirect attack
upon the problem not unlike that used by Sgrensen and Palitzsch (1910)
in their standardization of the colorimetric method for use with sea
water was therefore adopted. This involved the addition of an amount
of dry sea salt to various buffer solutions so as to give a concentration
of sea salt equal to that found in sea water. The pH values of the
buffer solutions were determined both before and after the addition of
the sea salt using both the. hydrogen and the glass electrode. ‘The sea
salt was obtained by evaporation of sea water on a water bath, any
organic matter that precipitated out during the heating was filtered off.
The salt so obtained was dried at 130° C. A liter of sea water yielded
33.1 grams of dry salt.

The hydrogen electrode employed in the determinations was the Clark
type shaking vessel. A saturated KCl-calomel half-cell was used which
was standardized against a standard acetate buffer, which in turn had
been checked against a 0.1 molal KCl-calomel half-cell. The tentative
standard of potential proposed by Clark (1928) was used. The hy-
drogen used was deoxygenated by passing it over heated (850° C.)
platinized asbestos. The potentiometer circuit was the orthodox one,
a galvanometer being used as a null point instrument.

Two types of glass electrodes were used. What will be designated
hereafter as Type I was a commercial apparatus known as the Beckman
pH meter. In this glass electrode the glass membrane separates a
quinhydrone electrode from the sample to be measured which in turn
makes a liquid junction with a saturated KCl-calomel half-cell. The
amplifier and potentiometer circuit were not available from the manu-
facturer. The outfit was used without any modifications. In the other
apparatus (Type IL) we employed a Leeds and Northrop, No. 7673
vacuum tube amplifier in conjunction with the potentiometer and gal-
vanometer of the hydrogen electrode circuit. The glass electrode, which
was blown from Corning 015 glass, separated a Ag-AgCl half-cell from
the sample and a saturated KCl calomel half-cell. The junction be-
tween the sample and saturated KCl was made at a stopcock of the
type employed by Stadie, O’Brien, and Laug (1931).

All determinations were carried out in a room which was automat-
ically kept at a relative humidity of 50-60 per cent. Rigid temperature
control was not maintained though the variation during a series of read-
ings was never more than 0.1° C. The control of humidity was found
to be an important item in the reliable use of glass electrodes of the type
employed under the climatic conditions of the seashore.

pH OF SEA WATER AS MEASURED WITH GLASS ELECTRODE 223

RESETS

In Table I are summarized the results obtained on buffer solutions
with and without the addition of sea salt. Each pH value given for
the hydrogen electrode is the average of triplicate determinations while
the glass electrode results are the average of duplicates. The hydrogen
and glass electrodes agreed within the experimental error in all cases
except that of the borate buffer with added sea salt. Here the glass
electrode readings tended to fall a little below those of the hydrogen elec-
trode. This may be the result of a salt effect but it should be noted that
the pH has shifted toward more alkaline values where the glass electrode

TasBLe [

The pH of buffer solutions as affected by the addition of sea salt.
Temperature, 25° C.

Buffer Hydrogen Glass Bledrade Glass Blectnode:
TEU Cl Ae act eee Le Co eae ee 1.04 1.02 1.04
GIR Ensaio 2 hein eae a ott ile 1.11 1.12
ENCEGALC Re oor Ne tay iain a eat d achat 4.60 4.59 4.61
INCE ater Salts ccc Lise eee oars 4.58 4.59 4.61
TeThveisyol van coy A an a ta 6.99 6.99 6.99
Phosphate -+-salt*... 0 -..6.s5..05 6.72 6.70 6.70
Ona tet tie, Gnesi AAA errand channel 9.13 9.13 9.13
Borate crsalttan ys ee eoe nee oe 9.51 9.46 9.47

* Dried sea salt added to a known volume of buffer in amounts equal to that
present in the same volume of sea water.

usually begins to deviate below the theoretical. The pH shift produced
by the addition of sea salt to the borate and phosphate buffers is partly
to be attributed to the precipitation of some of the buffer components
by the Ca and Mg salts present in the sea salt. This precipitate was
filtered off before making the measurements recorded here. Another
contributing factor to this pH change is undoubtedly the change in ac-
tivity coefficients brought about by the higher ionic strengths of the
solutions. This factor and the volume changes that may have occurred
on addition of the dry salt to the buffer solutions, are largely responsible
for the pH shift in the case of the acetate and HCl buffers since no
precipitates occurred in these solutions.

224. BRICIG BALE AND VGyCHESTER SiOGk

We feel that these results justify the direct use of the glass electrode
for the measurement of the pH of sea water. In drawing this conclu-
sion we are mindful of the fact that the precipitation of some Ca and
Mg salts in the two most alkaline buffers has somewhat distorted the
ion balance from that found in sea water. It is doubtful, however,
that the presence or absence of such small amounts of these large di-
valent ions would modify perceptibly the behavior of the glass electrode
when much larger quantities of the smaller univalent sodium ion have
no pronounced effect. Dole (1937) has stated that “the cation error is
greatest with those cations that most easily penetrate the glass, sodium
and lithium being the worst offenders, while large ions, such as potas-
sium, and also divalent ions have only a small influence.”

TABLE II

The pH of sea water as measured with the glass electrode. All samples
collected September 10, 1936, Woods Hole, Mass.

Sample No. Source T pe ee S pH
car

1 Great Harbor * 20.0 8.13
Laboratory Dock 8.14

2 Tank supply e200 8.05
(Room 333) 8.05

3 Tank supply 20.0 8.05
(Room 110) 8.05

4 Great Harbor * 20.0 8.14
Laboratory Dock 8.14

5 Eel pond * 21.5 7.99

* Surface water.

In Table II are summarized a few determinations on different sam-
ples of sea water. These results were obtained with the Type I glass
electrode. Of interest is the difference in pH between samples drawn
from the tank system supplying the laboratories, the water for which is
pumped from the harbor, and samples collected directly from the harbor.
This difference is well outside the maximal experimental error (=: 0.02).
Since all samples were collected in the order given, this difference cannot
be attributed to a sudden change in electrode behavior because Samples
1 and 4 are in good agreement. Moreover, the agreement obtained on

pH OF SEA WATER AS MEASURED WITH GLASS ELECTRODE 225

Samples 2 and 3 collected in different parts of the laboratory building
would indicate that local contamination of the system was not responsible.
Since the feed pipes are mainly of lead it is possible that the acid swing
is due to the removal of carbonate as an insoluble lead salt. This seems
unlikely, however, in view of the fact that such lead feed pipes after
continued use accumulate a protective coating of such a precipitate which
should prevent further interaction. Another possible explanation for
the difference may be the fact that though the two sets of samples are
taken from the same locality, one is pumped from a depth and the other
is surface water. ‘Though the two samples registered the same tempera-
ture at the time of collection, the water from the greater depth may
have possessed a lower temperature before its passage through the stor-
age and feeder system. If so, a more acid pH would be encountered due
to the lag in the readjustment of the carbon dioxide equilibrium as the
temperature increased, a process which Irving (1925) has shown to be
slow. The greater acidity of the water collected from the eel pond is
to be expected in view of the more stagnant condition of this body of
water.

The glass electrode has been used by Mitchell and Taylor (1935)
to measure the pH of sea water at different dilutions in order to deter-
mine the dissociation constant of cresol red in sea water of different
salinity. This work was confirmed and extended and, in addition, data
upon phenol red were obtained by Mitchell, Buch, and Rakestraw. Since
these authors used a glass electrode with a thin film membrane in con-
trast to the heavier type employed by us, we wish to emphasize the fact
that our proof of the validity of the glass electrode values on sea water
applies to this type of membrane only. It would be desirable to have a
similar study made with the thin film type of membrane.

SUMMARY

The hydrogen electrode and the glass electrode registered approx-
imately the same pH values for various buffer solutions in which dry sea
salt was dissolved to give a concentration similar to that found in sea
water. A direct comparison of the two methods on sea water was not
possible because the behavior of the hydrogen electrode was erratic in
this poorly buffered medium. It is concluded that the salt content of
sea water does not interfere with the application of the glass electrode
to the determination of the pH of sea water and that such determinations
may be made with a fair degree of accuracy (+ 0.03 pH). The pH
value of several samples of sea water have been determined and their
differences discussed.

226 ERIC G. BALL AND C. CHESTER STOCK

BIBLIOGRAPHY

CLarkK, W. M., 1928. The Determination of Hydrogen Ions. Williams and
Wilkins, Baltimore, 3rd edition.

Dote, M., 1937. Measuring pH with the Glass Electrode. Maywood, Illinois.

Irvine, L., 1925. Jour. Biol. Chem., 63: 767.

McCrenpon, J. F., 1917. Jour. Biol. Chem., 30: 265

MircHeEtt, P. H., anp I. R. Tayzor, 1935. Jour. Conseil. intern. exploration mer.,
10: 169.

MircHett, P. H., K. Bucn, anp N. W. Raxestraw, 1936. Jour. Conseil. intern.
exploration mer., 11: 183.

S@rensen, S. P. L., anv S. Parirzscu, 1910. Biochem. Zeitschr., 24: 387.

StapiE, W. C., H. O’Brien, anp E. P. Laue, 1931. Jour. Biol. Chem., 91: 243.

THE RELATION OF ENDOCRINE FEEDING TO REGEN-
ERATION, GROWTH AND EGG CAPSULE PRO-—
DUCRON UN SPEAN RIA VMACULAT A

BD GOLDSMITE

(From the Department of Biology, Washington Square College, New York
University and the College of the City of New York)

An attempt was made to determine whether the time required for
head regeneration in Planaria maculata following a series of decapita-
tions would be altered by feeding beef endocrines. In view of the re-
sults of Wulzen (1923) on P. maculata and Castle (1928) on P. velata
a record of the size of the individuals was kept. A preliminary report
(Goldsmith, 1935) summarized the results; a more complete account
follows. .

MATERIAL AND METHODS

From a stock of Planaria maculata originally collected at Woods
Hole, Massachusetts and kept in the laboratory for three months, 204
individuals were selected for experimentation. They were divided into
two groups of large and small specimens; those of the large size group
(Group L) measuring 9-12 mm., those of the small size group (Group
S) measuring 5-8 mm. in length. The animals of each of the groups
were distributed into twelve sets—those of Group L containing eight
individuals each, and those of Group S nine individuals each.

The sets were kept in finger-bowls containing 100 cc. of tap water,
and were placed on the following diets:

1. Liver, fresh gland.

2. Liver, aqueous extract.

3 and 4. Anterior pituitary, fresh gland.

5. Anterior pituitary, aqueous extract.

6 and 7. Thyroid, fresh gland.

8. Thyroid, aqueous extract.

9 and 10. Anterior pituitary + thyroid, fresh gland.
11. Anterior pituitary + thyroid, aqueous extract.

The animals in set 12 were not given any food and set 12 is hereafter
called the “ starved ” set.
Fresh beef liver, pituitary and thyroid glands were obtained weekly

227

228 E. D. GOLDSMITH

at the slaughter house. The first feeding was made within two hours
of the obtainment of the material. It was then frozen, so kept for a
week during which time it was given to the planarians, and was then re-
placed by fresh material.

Small pieces of liver and small pieces of thyroid and anterior pitui-
tary, which had been minced, were placed in the dishes and left there for
several hours. The food was then removed, the dishes cleaned and
fresh tap water added. Feedings were on alternate days and in some
experiments daily. In the anterior pituitary + thyroid set the anterior
pituitary and thyroid were given on alternate feeding days. The fre-
quent feedings ensured an adequate food supply for the experimental
animals.

Another method of treatment was the addition of filtered aqueous
tissue extracts to the tap water. Not only were the animals of the
aqueous extract sets exposed to the extract but they also ingested some
of it as was indicated by the movements of their pharynges.

The thyroid material was tested throughout by treating frog tadpoles
with it. Thyroid glands frozen two weeks retained their potency in that
tadpoles feeding on this tissue or exposed to aqueous extracts of it of
much lower concentration than those used on the planarians showed
greatly accelerated metamorphosis.

Using a binocular dissecting microscope the length and width of the
animals were measured as they moved on a glass cover-slip mounted
over a piece of millimeter cross-section paper. By regulation of the
light source the animals could be made to move along the rulings.

REGENERATION

Stevens (1901) and others have reported that the eyes in planarians
are regenerated more rapidly at an anterior than at a posterior level. It
is also known that eyes regenerate more rapidly in a form such as P.
maculata possessing high regenerative potentialities than in Procotyla
fluviatilis possessing, in general, low regenerative potentialities.

In the work which follows the time for eye appearance after decapi-
tation was used as an index of the regeneration rate under the influence
of different diets. Following each operation the ventral and dorsal as-
pects of the animals were carefully examined for the presence of eye
pigment. |

The anterior portions of all animals were removed five times. The
specific data of each decapitation are given below.

after the previous decapitation.

capitation No. 4; e, decapitation No. 5

ENDOCRINE FEEDING AND REGENERATION

229
Group I
Decapitation No. 1—Thirty-six days after their first feeding the ani-
mals were measured. Two days later four animals from each dish were

decapitated by a transverse cut directly posterior to the auricles (Fig.
1, a) and placed in an additional set of finger-bowls.

The operated animals comprise Group I.

Those which were not
operated upon were left in their original aquaria and comprise Group II.

Mess --x
Va XY
i .
lf
7 \
/ \
LOO
\ /
t t
i {
! 1
i
X--}--------+---X

aes

Decapitations in Group 1.

Broken lines indicate tissue regenerated
4a-% indicates level of cut.

All figures diagram-
a, decapitation No. 1; b, decapitation No. 2; c, decapitation No. 3; d, de-

230 E. D. GOLDSMITH

Decapitation No. 2.—Fourteen days after the first decapitation the
animals were fed. These feedings were continued during a twenty-
four-day period, and the regenerated heads were removed as in decapita-
tion No. 1 (Fig. 1,0). Food was placed in the dishes and left there for
several hours each day throughout the regeneration period.

Frc. 2. Decapitations in Group 2. a, decapitation No. 1; b, decapitation No.
2; c, decapitation No. 3 producing A and B pieces; d, portion B resulting from de-
capitation No. 3; e, decapitation No. 4 directly anterior to pharynx of portion A
resulting from decapitation No. 3; f, decapitation No. 5

ENDOCRINE FEEDING AND REGENERATION 231

Decapitation No, 3—Twenty-one days after the second decapitation
the anterior ends were removed by a transverse cut approximately 1 mm.
anterior to the pharynx (Fig. 1, c). As above, food was placed in the
dishes throughout the regeneration period.

Decapitation No. 4.—Twenty-five days after the third decapitation
the entire prepharyngeal region was removed (Fig. 1, d).

Decapitation No. 5—The animals were starved for nine days after
the fourth decapitation and then all were placed on a liver diet for thirty-
five days. Two days after the last feeding the entire prepharyngeal
region was removed (Fig. 1, e).

Group II

Decapitation No. 1.—Sixty-three days after their first feeding those
animals which had not been decapitated previously (Group II) were
measured and on the following day were decapitated by a transverse
cut directly posterior to the eyes (Fig. 2, a).

Decapitation No. 2—Three days after the first decapitation feeding
was recommenced and continued for fifteen days. On the following
day the heads were again removed by a cut similar to the first (Fig. 2,
Dy.

Decapitation No. 3—Feeding was begun the day after the second de-
capitation and continued for nineteen days. The following day a trans-
verse cut was made directly posterior to the pharyngeal pore (Fig. 2, c).
The posterior portions of the worms (B, Fig. 2, c) were placed in ad-
ditional dishes and were observed for eye and head regeneration (Fig.
2, da). The anterior portions (4, Fig. 2, c) possessing the pharynges
were fed.

Decapitation No. 4:—Twenty-three days after the third decapitation
a transverse cut was made directly anterior to the pharynx (Fig. 2, e)
in the anterior pieces arising as a result of the previous decapitation (A,
ie) 25.6):

Decapitation No. 5.—During the eight days following the previous
decapitation the animals were not fed. Then they were fed liver on
alternate days during a thirty-six-day period. Two days after the last
feeding the entire prepharyngeal region was removed by a transverse
cut directly anterior to the pharynx (Fig. 2, f).

The regeneration rate following the first four operations appeared
not to be influenced by the type of food fed if the quantity were suf-

232 E. D. GOLDSMITH

ficient. Following each decapitation the rate of regeneration of a num-
ber of the animals varied slightly. The variation in rate within a set of
worms on the same diet was as great or greater than the variation among
the individuals on different diets (exception noted below).

At the time of the fourth decapitation, 124 days after the first treat-
ment, the animals were in great part composed of tissues built from ma-
terials supplied by the particular food to which they were restricted.
Notwithstanding this, the results did not differ from those of the pre-
vious decapitations.

The exception mentioned above is that of the Liver L (gland) set
in Group I. Following each of the four operations several worms in
this group regenerated more slowly than did any in the other gland sets.
In order to ascertain whether this difference in rate was to be attributed
to the difference in diet or to an innate tendency of these particular indi-
viduals, all of the experimental animals were starved for eight days and
then fed only liver for thirty-six days. The anterior ends were then
removed (fifth decapitation). Several planarians in the original liver-
fed set in Group I, L, still regenerated more slowly than the others.
Since these animals still lagged behind the others when all individuals in
all sets were on the same liver diet, and since the animals in the Liver L
and Liver S (gland) sets in Groups II and those in the Liver S (gland)
set in Group I did not lag following any of the decapitations, it would
appear that the retarded rate was not to be regarded as an indication
that thyroid or anterior pituitary glands accelerated or that liver re-
tarded the rate of regeneration. Rather, it would seem that the slowly
regenerating animals did so because of innate individual differences.

In Group II, S, the animals starved for sixty-four days regenerated
more slowly than any of the others. At a temperature of 21° C. eyes
made their first appearance as listed:

Solid foods, 140 hours
Aqueous extracts, 160 hours
Starved, 190 hours

In Group II, L, the animals starved for sixty-four days regenerated
at about the same rate as the others, 142 hours. This may have been
due to the larger animals having more of a food reserve.

In Group I, S, following the first decapitation thirty-six days after
treatment was begun the rate was the same in all groups. Following
the second decapitation, at which time the animals had been treated
for seventy-five days, the individuals given aqueous extract lagged about
twenty hours behind the animals maintained on the fresh gland. The
starved forms lagged slightly behind the “ extract ” forms.

ENDOCRINE FEEDING AND REGENERATION 233

The starved animals and the extract-treated ones which also showed
starvation symptoms were fixed and preserved. Those in Group I (L
and S) were fixed after regeneration had been observed after their
second decapitation and those in Group II (L and S) after their first
decapitation.

Retardation after starvation is well known and has been described
in planarians by Morgan and in Triton cristatus by Morgulis (Morgulis,
1223)

GROWTH

Measurements made as described in the earlier part of this paper are
recorded in Table I.

TABLE I

The effects of feeding liver and thyroid glands and of starvation upon the growth
of Planaria maculata

A B (Gz
Beginning 36 days after A 27 days after B
2/19 3/27 4/23
Food
Average | Average | Average | Average | Average | Average
length width length width length width
iver Glancdtow.qyose etme 7.7 11.109) 7} 10.4 | 1.2(9)
10.2 WAG) | Wes ea)
Iver Gland fb. .. Vee vedo. 106 AG) | Wes ae)
13.5 | 1.6(4) 15.5 1.9(4)
Thyroid Gland S............ : 8.0 | 1.0(17) 8.9 | 1.1(17)
8.8 |1.1(9) 9.8 | 1.4(9)
Thyroid Gland L............. 10.0 |1.4(16)} 11.6 | 1.3(16)
11.4 | 1.3(8) 11.6 1.5(8)
StaiVicPom enw oneee wa the ema 7.7 | 1.1(9) 6.1 |0.6(9)
5.7 | 0.6(5) 3.8 | 0.4(5)
S Hae lease oc nie re daco-any eens 10.7 | 1.6(8) 8.9 |0.9(7)

8.6 |0.9(3) 6.6 | 0.7(3)

* A number of animals in each set were decapitated on 3/29. The unoperated
animals were again measured on 4/23.
} Figures in parenthesis indicate number of animals in set.

It is clear that the liver-fed animals show the greatest increase, and
that thyroid gland, while not causing as much of an increase as liver,
certainly does not cause the animals to become smaller than starved in-
dividuals as is the case in P. velata (Castle, 1928).

234 E. D. GOLDSMITH

A number of worms newly emerged from their capsules were also
found to increase in length and width when given only fresh thyroid
gland for periods ranging from 15-65 days. |

Further evidence that P. maculata does not respond to thyroid gland
treatment as does P. velata is presented by those animals which were
on an exclusive thyroid diet for 124 days. During this period their
anterior ends had been removed four times with typical regeneration
resulting. The animals were in excellent condition, and almost indis-
tinguishable from the liver-fed worms. The only noticeable difference
was that the latter were slightly thicker dorso-ventrally.

These results are in agreement with the data presenter by Wulzen
(Table II, p. 178, 1923).

Ecc CAPSULE PRODUCTION

During the latter part of March, egg capsules appeared in some of
the dishes. Records of the numbers produced are presented in Table II.

TABLE ID

Number of egg capsules produced by Planaria maculata on indicated diets

Number Number of Number of
fo) Food capsules to capsules to
animals 4/22*
9 alee vierieulann alin gre est clo Ce ee kee ee greene 68 87
18 Anterior pituitary gland................ 36 40
17 Anterior pituitary-thyroid gland......... 55 62
18 Whiyvrotd-alamd ies oh 714 je keto oes 18 20
4 iver agueonsiextract 2 0.2.46. + ine iL ; 1
Ieiveraqueous extract iS \.... vaste. ees 0 0
9 Anterior pituitary aqueous extract....... 0 0
9 Anterior pituitary-thyroid aqueous extract. 0 0
9 thyroid aqueous extract.» > ss. eae 0 0
9 SSLHA N23 ce nM PER ERTE cooh Ba Asc ie 0 0

* The animals were decapitated on 4/23. They continued to produce capsules
to 5/12.

Comparison of the extremes of the series, the liver-fed with the
starved and the extract-treated forms, leads one to believe that low cap-
sule production may be due to a food deficiency. Of the starved and
extract-treated forms which were becoming smaller, only Liver Aqueous
Extract L was productive—a single capsule was produced. ‘The animals
in this set were slightly larger than the starved and other extract-treated
animals, and smaller than any of those which were fed fresh glands.

The thyroid-fed animals, which were least productive of the gland-

ENDOCRINE FEEDING AND REGENERATION 235

fed animals, were in good condition and increasing in size but were
smaller than those on the liver diet. Although the planarians fed an-
terior pituitary and anterior pituitary-thyroid glands approximated the
liver-fed ones in all dimensions, they produced a smaller number of
egg capsules than the liver-fed individuals.

It is realized that the evidence is too scanty to permit a definite con-
clusion. It is interesting to note that Greenberg and Schmidt (1936)
described an ether-soluble factor in liver which acts as a growth-
promoting agent for Planaria maculata. Smith and Seegers (1934)
found a principle in liver which acts as a growth-promoting agent and
which is, in some way, concerned with the typical functioning of the
reproductive mechanism in the albino rat.

The writer is indebted to Professor Robert Chambers for his criti-
cism of the manuscript and to Professor F. Gudernatsch for his counsel
throughout the course of this work.

SUMMARY

1. Individuals of Planaria maculata were fed on abundant, exclusive
diets of beef liver, anterior lobe of the pituitary and thyroid glands.
Others were given aqueous extracts of the glands or were completely
starved.

2. No significant differences were noted in the head regeneration
time of the gland-fed animals following each of five amputations of the
anterior region.

3. Decapitated animals which were starved and those which were
kept on the aqueous extract diet and which showed starvation symptoms
regenerated more slowly than those which were fed the fresh glands.
There appeared to be a correlation between the rate of regeneration and
the initial size of the animals.

4. Thyroid-fed individuals increased in size but to a lesser extent
than the liver and pituitary-fed forms. All were in excellent condition
following four decapitations. Individuals newly emerged from capsules
also increased in size when fed thyroid gland exclusively.

5. Liver-fed animals produced a greater number of egg capsules than
any of the others. The starved and extract-treated forms, with the
exception of a single capsule by the Liver L Aqueous Extract set, pro-
duced no capsules.

236 E. D. GOLDSMITH

BIBLIOGRAPHY

CastLeE, W. A., 1928. An experimental and histological study of the life-cycle
of Planaria velata. Jour. Exper. Zool., 51: 417.

GotpsmitH, E. D., 1935. Regeneration and growth in Planaria maculata under
the influence of endocrine feeding. Anat. Rec., 64 (Supp. No. 1): 78.

GREENBERG, L. D., ann C. L. A. Scamp, 1936. Studies on the properties of a
growth-promoting factor for Planaria maculata. Jour. Exper. Zool., 73:
375.

Morcutts, S., 1923. Fasting and Undernutrition. E. P. Dutton Co., N. Y.

SmitH, H. G., ann W. H. Seecers, 1934. The nutritive value of animal tissues
in growth, reproduction, and lactation. J. Alcohol extracted beef liver.
Jour. Nutrition, 7: 195. II. The presence of a new dietary principle in
liver. Jour. Nutrition, 7: 209.

Stevens, N. M., 1901. Notes on regeneration in Planaria lugubris. Arch. f.
Entw.-mech., 13: 396.

Wut1zen, R., 1923. A study in the nutrition of an invertebrate, Planaria maculata.
Univ. Calif. Pub. Physiol., 5: 175.

RESON Ss OF MUSCLES OF THE SOUID TO REPETITIVE
STIMULATION OF THE GIANT NERVE FIBERS

CHEADD PRO SSE Re AND) a OLIN 2. YOUNG
(From the Marine Biological Laboratory, Woods Hole, Mass.)

INTRODUCTION

Investigation of the response of muscles to repetitive stimulation of
their nerve fibers has shown that there are great variations among dif-
ferent muscles and different animals in the possibilities of facilitation
at the neuromuscular junction. Although the response of an intact
muscle fiber, normally activated, is probably always maximal (all-or-
nothing), yet in some muscles, for instance those of Crustacea, increase
in the frequency of stimulation often increases the tension developed on
account of the fact that, some muscle fibers are not activated by single,
or even by few impulses. On the other hand, in vertebrate striped mus-
cle, unless drugged or fatigued, a single nerve impulse excites all the
muscle fibers which it reaches (Lucas, 1909; Adrian and Lucas, 1912).

The range of muscles which have been investigated from this point
of view is still small, especially among invertebrates, and we have accord-
ingly investigated the muscular response to repetitive stimulation of the
giant axons in the stellar nerves of the squid, Loligo pealii. These
fibers have been shown to innervate the circular muscle fibers of the
mantle, and a single condenser discharge, unless of great intensity or
duration, sets up in the axon a single impulse which is capable of ac-
tivating all of the muscle fibers which it reaches (Young, 1937). The
great stellar nerve, containing a single giant axon, was prepared for
stimulation in the manner described elsewhere (Young, 1937), and the
contractions of a portion of the muscles which it innervates were re-
corded by attachment to a semi-isometric lever.

Repetitive stimulation was applied by condenser discharges through
a thyratron circuit. With the intensity constant and supraliminal the
frequency was varied up to approximately seventy stimuli per second.
Fatigue set in very rapidly at higher frequencies. Sixteen experiments
in which the giant fiber of the great stellar nerve was stimulated yielded
consistent results.

1 Assisted by a Fellowship of the Rockefeller Foundation.
237

238 C. LADD PROSSER AND JOHN Z. YOUNG

RESULTS

A typical experiment is shown in Fig. 1. Development of tension
is registered by a downward deflection in these records. The first stim-
ulation was at 13 stimuli per second and the second at 24 per second.
Incomplete relaxation between twitches occurred in both, and the max-
imum tension at 24 per second was very slightly greater than at 13.

GE -

NM
Ol
(e%)
Ol

im
|
a

|
5
(e)

Fic. 1. Record of an experiment in which the giant fiber of the great stellar
nerve was stimulated repetitively and the contraction of the mantle muscle was
recorded semi-isometrically. Records in order in which they were taken. Num-
bers indicate frequencies of stimulation.

With stimulation at 4 per second there was complete relaxation between
stimuli. In general, complete relaxation between twitches accompanies
frequencies of stimulation up to approximately 8 per second. The prep-
aration was then stimulated at 20 and at 25 per second, and showed very
slightly increased tension. At 35 stimulations per second fusion of
contraction was complete. At 50 per second, as at 35, there was no in-
crease in maximum tension above that at 25 but at 50 the tension de-
clined rapidly, indicating fatigue. The effects of this fatigue were
shown when stimulation was returned to 20 per second, where a lower
tension was recorded. Thereafter (6 and 40 stimuli per second) the
tension varied with the frequency.

Figure 2 shows similar results in three other preparations. In each
of these experiments, as in most of the others, there was a slight increase

RESPONSES SQUID MUSCLE TO STIMULATION 239

in tension (5-10 per cent) as the frequency increased during the range
of incomplete relaxation, an effect which may be ascribed to the mechani-
cal properties of the muscle.

25 5O 15
Frequency

Fic. 2. Plots of maximum tension developed by the mantle muscle against
frequency of stimulation of the giant fiber in the great stellar nerve in three prep-
arations. Stimulation proceeded from low to high frequencies. In C the lowest
point (15 per second) represents response after fatiguing at highest frequency
(75 per second).

In Experiment 4, Fig. 2, the periods of stimulation were brief and
there was no increase in tension with increasing frequency of stimula-
tion; this is the general result when no fatigue occurs.

Preparation B showed fatigue at 33 stimulations per second, and
Preparation C at 75 per second. Thereafter the tension fell off and

240 €: LADD PROSSER AND JOHN,Z: YOUNG

varied with the frequency of stimulation, the higher frequencies eliciting
a greater response than the lower.

The preparation is extremely sensitive to strong excitation and high
frequency stimulation during one second causes irreversible fatigue.
The failure of the response is parallelled by a growing opacity of the
mantle.

Discussion

It is evident that there is normally no facilitation at the junction be-
tween the endings of the giant fiber and the muscles. Increased fre-
quency of stimulation produces no increase in tension, indicating that all
the muscle fibers are activated by each single impulse which reaches
them. Young (1937) observed similar results with increasing intensity
of stimulation. In the state of fatigue, however, changes occur, prob-
ably at some stage in the contractile mechanism, so that summation oc-
curs and greater tensions are produced at the higher frequencies.

This condition is closely similar to that found in the striped muscles
of the frog (see Adrian and Lucas, 1912), but contrasts sharply with
that of Crustacea, where a single impulse often elicits no mechanical re-
sponse (Pantin, 1934, 1936). Thus Katz (1936) found that the tension
produced by the flexor muscle of the claw of Maza increases nearly ten
times when the frequency of stimulation is raised from 50-200 per sec-
ond. Recent observations by Mr. Grossman in the Physiology Course
at the Marine Biological Laboratory indicate that the tension developed
by the claw of Limulus increases from 25 to 650 grams with a rise
of frequency from 1-50 per second.

The absence of such facilitation at the neuromuscular junctions of
the giant nerve fiber system of the squid is correlated with the function
which the system serves in the animal, namely to produce the contrac-
tions by which a jet of water is expelled suddenly from the mantle.
Once the contraction has occurred the mantle cavity must enlarge again
before further work can be done, and there would be no use for sus-
tained or gradually increasing tensions. The expulsion of each jet of
water is a single unitary act, performed in an all-or-nothing manner,
and any gradation in speed or distance of propulsion must be obtained
by variation in the number of contractions set up.

SUMMARY

With increasing frequency of stimulation of a giant nerve fiber in
the squid, Loligo pealti, the only increase in the tension developed by the
circular muscle fibers of the mantle is a small amount (5 to 10 per cent)
over the range of incomplete relaxation. The absence of any increased

RESPONSES SQUID MUSCLE TO STIMULATION 241

response at higher frequencies shows that in the fresh muscle a single
nerve impulse is capable of activating every muscle fiber which it reaches.

However, the isolated muscle very readily becomes fatigued when
stimulated at high frequency and thereafter greater tension is produced
at the higher rates. In the normal animal there would be no use for
peripheral facilitation and each contraction of the mantle is produced
as an all-or-nothing response.

REFERENCES

ApriANn, E. D., ann K. Lucas, 1912. On the summation of propagated dis-
turbances in nerve and muscle. Jour. Physiol., 44: 68.

Katz, B., 1936. Neuro-muscular transmission in crabs. Jour. Physiol., 87: 199.

Lucas, K., 1909. The “all-or none” contraction of the amphibian skeletal
muscle fibre. Jour. Physiol., 38: 113.

Pantin, C. F. A., 1934. On the excitation of crustacean muscle. I. Jour. Exper.
Biola s ll

Pantin, C. F. A., 1936. II. Neuromuscular facilitation. Jour. Exper. Biol., 13:
Tae

Pantin, C. F. A., 1936. III. Quick and slow responses. Jour. Exper. Biol., 13:
148.

Youne, J. Z., 1937. The functioning of the giant nerve fibres of the squid. In
press.

THE OCCURRENCE OF SAPROPHYTIC FUNGI IN MARINE
MUDS

F. K. SPARROW, JR.

(From the Woods Hole Oceanographic Institution1 and the Botany Depariment
of the University of Michigan 2)

INTRODUCTION

Since it has been recognized for many years that fungi play an im-
portant and significant role in the disintegration of organic materials
in land soils, it is natural to suspect that they might perform a similar
function in sea bottoms. Previous papers (Petersen, 1905; Sparrow,
1934, 1936) have shown that in the littoral of certain localities in north-
ern Europe and eastern United States there are true marine fungi which
are active in initiating the destruction of living, autophytic marine plants
and in certain cases also in aiding in their disintegration. As no sys-
tematic study of off-shore localities for the presence of wholly sapro-
phytic fungi in the muds had been reported, the preliminary investigation
described in this paper was undertaken during July-August, 1936, at
the Woods Hole Oceanographic Institution.

STATIONS

The following are the locations of stations selected for study and
from which the mud cores were obtained. All were in localities marked
“sticky ” or “sand and mud” on the hydrographic charts.

Station 1. One-half mile N.W. of Weepecket Rock Buoy, Buzzard’s
Bay. Depth: 18.0 meters.

Station 2. Western entrance to Vineyard Sound, 314 miles E. of Vine-
yard Sound Whistling Buoy, 514 miles W. by S., % S. on
Gay Head Light. Depth: 32.7 meters.

Station 3. Gulf of Maine, 45°35’ N., 69°11’ W. (Atlantis Station No.
2640). Depth: 163.6 meters.

Station 4. Gulf of Maine, 42°19’ N., 69°20’ W. Depth: 220 meters.

Portions of two cores collected by Dr. Henry Stetson from the Gulf
of Maine and labelled “ Canyon B” (depth 1127.2 meters; Station No.
5, Table I) and “ Canyon E”’ (depth 718.0 meters; Station No. 6, Table

1 Contribution No. 73.

2 Contribution No. 630.

242

SAPROPHYTIC FUNGI IN MARINE MUDS 243

I) were obtained from Dr. S. A. Waksman. Since these had previously
been partly used for bacteriological purposes and had been stored for
some days they were considered questionable sources of data.

METHODS AND RESULTS

In order to obtain any significant information in the limited time
available, the methods outlined below were considered most practical
even though they were subject to very definite limitations.

Collection of Cores

The apparatus commonly used in the collection of stratified mud
cores for bacteriological purposes was employed. This is a modification
of the instrument used by Moore and Neil (1930) in the Clyde Sea
area. To eliminate as far as possible contamination during collection,
all parts of the sampling apparatus in contact with the sterilized glass
tube were thoroughly swabbed with 10 per cent formalin immediately
before use. However, trials with swabbed and unswabbed apparatus
showed little difference in the number of colonies of fungi obtained.
All the usual precautions were employed to keep the cores free from
contamination after collection. The lengths of the cores varied but
averaged about 12 cm.; their diameter was 18 mm.

Recovery of Fungi

After the tubes containing the cores were brought into the laboratory,
the upper cork was flamed, the free water removed, and the supernatant
liquid immediately above the mud put in a sterile container for future
use. The core was then “ blown” under sterile conditions into a steril-
ized Petri dish and the outside surface and ends removed with a hot
scalpel as an additional precaution against contamination during col-
lection.

Two methods were followed in recovering the fungi. The first,
practised extensively in the study of fresh-water fungi, involves the use
of water cultures. Five jars of sterilized sea water were prepared into
each of which was placed a core from Station 2, a site at which cores
were readily obtainable. The cores were broken up in the water, the
mixture allowed to settle, and 20-30 dead Calanus finmarchicus and
Saggita from Station 2 were added. Three jars were kept at room
temperature (23° C.) in the light and two in the dark at about 6° C.
Bits of “shiner ”’ (a small marine fish) removed aseptically were also
dropped into the cultures. After 10 days no fungi were found on any
of this “ bait.”

244 Bike SPARROW).

All the significant data were obtained by the plate method. The
medium used was the following:

DexctioS@mrn tm ket ec een aiareee 10.0 grams
PEpLOMeuee cmt tera Nace aise as ltrs 2.0 grams
ANGHEN Er Altus Poayinaal Neca een St ea 15.0 grams
SCAMVATC IUCR Tae ia oe teas isa 1000.0 cc.

This gave a reaction of pH 6.1.

In plating out, samples were taken from the supernatant water, the
surface, the middle, and the bottom of the core. The water was dis-
tributed, undiluted, among five dishes and the medium added. Five
samples were taken from each of the three regions of the core. These
were mixed with 1 cc. of sterile water and plated out in the usual man-
ner. Each of the mud samples was about the size of a pea and weighed
200-250.0 mg. In many cases pieces of mud were also laid on the
solidified medium, but these dishes merely told whether or not fungi
were present on or near the face of the sample. Flasks of unsolidified
media were also tried but these became too heavily overgrown with
bacteria and protozoans to be of value. As controls for each core, five
plates of media were used to each of which was added the contents of
a 1 cc. sterile water blank. While these were the actual controls, it
will be seen that the ten dishes containing material from the middle and
bottom of the core also acted as checks against laboratory contamination.

Table I gives the results of the plating out of the mud samples.

The fungi recovered from the muds by the methods outlined all be-
longed to genera commonly found in land soils and easily recovered in
spore form from the atmosphere. A large majority were species of
Penicillium, while others belonged to such genera as Cephalosporium,
Trichoderma, Aspergillus, Chetomium, Alternaria, Cladosporium, and
even Rhizopus. It was soon evident that no fungi which could be con-
sidered characteristically marine were being recovered by these methods
and interest in them, qualitatively at least, was greatly diminished.

A preliminary experiment was also carried out to determine whether
such fungi might be associated with decaying phytoplankton. Using a
sterilized net, two sterilized jars were filled with a heavy concentration
of phytoplankton from Vineyard Sound. ‘The material was distributed
equally between two sterilized battery jars, both of which were then
placed under running sea water in the laboratory. By allowing the water
to flow very gently into one jar (““A”’) the mass of diatoms soon rested
on the bottom. Since the control jar (“ B”’) was subjected to a stronger
stream, the material was soon washed out. After four days, during
which time the phytoplankton had gradually disintegrated, five 1 cc.
samples were plated out from the bottom of each of the jars. Five

245

SAPROPHYTIC FUNGI IN MARINE MUDS

‘QUOTe PNUI 9deJINS JO sadaId asn 0} O—F SUOTIEIS WO] [PI1o}eUT JUSTOYJNS JOU SeM DIB, ,

‘suOT]vUTUIe}UOD Ajqeqoid 919M PU YSIP 9Y} JO 9Spa ay} 7e 919M SaTUOTOD Vsaq} JO OMT ¢

*paiins00 pey UOT}eUTUTe}UOD }eY} AjTIqissod ay} Jo esnedaq PepAPOSIP SPA 9UO 4N Pe}II]JOO 919M S2I0I OMT ;z
"JI BAOGK 10}VM UT SB [Jam se 1aAe] doy ay} Wor r8uny sjueseides A[qeqoid pue Appnul A1VA 1938 ;

G sie) 14 sf) Vv s9 ev $9 LE 0¢ 6S 09 Gls Bieeee ee sjpeIOL
0 $ 0 s io ‘I C if es a 0 s 1 OST 9
0 $ 1 $ 0 a 0 S = rs 0 cl T C LCV “|
0 Ol ! Or 0 Or i! Or = —— @8 s raul! 0°0¢7 v
0 Or 0 Or 0 Ol CC Or 91 Or t Ly OT (4 Oreo £
I 07 i! 0¢ I! 0¢ 6S 0c 0c st a 07 v LCE (4
L ST i “il! 0 “il! 67 “i! i! ¢ (6 ST € 081 i
S4a}aUL
Isuny soysip Isuny so[dues Isuny sojdures Isunj sojdures Iduny sojdures Isuny so]dures $9100
jo jo jo jo jo jo jo jo jo jo jo jo jo yideq UO1}24S
2) 180) (0h) ‘ON SoTUO[OD ‘ON, iS) 1000) (o}@) “ON SOTUO[OD, ‘ON, SOIUO[OD ‘ON SoIuUO[OD, ‘ON ON
soysIp yquelq O2H 99 T + yuelq OF 99 T + yuelq OFH 99 T + 2109 2109 sojdures
jorqu08D 2109 jo u10}}0g 2109 JO 3[PplN 9109 JO 99ejINS jo 90RjInNS 2vAOge 19,2 JO UISIIO

“padanorad wun, {0 Sa1U0709 [Oo LaQUinU aY] PUD UId1AO 41aY4

‘pasn sajquips fo 4IQUnU AY] SD [Jam SD SUOYIDIS AY] Mos, UaYD] Sa409 fo Aaquinu puD YIdap ay] BUIMOYS

I aIavy

246 F, K. SPARROW, JR.

dishes of medium alone acted as controls. Sixteen colonies of fungi
were recovered from jar “ A” which contained the phytoplankton, and
none from jar “ B” which contained only the running water. The con-
trol plates remained sterile. It was apparent in this case, at least, that
there existed a definite relationship between the presence of disintegrat-
ing phytoplankton and the moulds. Again, as in the muds, the fungi
recovered were all common dust-borne species.

DISCUSSION

From the foregoing, which it must be emphasized is a very prelim-
inary investigation intended more to stimulate further inquiry than to
arrive at definite conclusions, certain points seem worthy of further
consideration.

If truly marine fungi of a filamentous type exist in the marine muds
studied and if there is nothing radically wrong with the methods and
type of medium employed, then these organisms must be extremely rare.
The types of fungi recovered, i.e., species of Aspergillus, Penicillium,
Trichoderma, etc., have cast doubts on their being concerned to any great
extent in disintegrating processes in the sea. Proof of their presence
in marine muds does not mean that they are in an active vegetative state
and hence, a working factor in the cycle of decomposition. To the
writer’s mind, large numbers of direct microscopic observations of my-
celium in mud seem the only positive method of demonstrating this
important point. The failure of the water cultures to produce sapro-
phytic fungi is also in line with the negative aspects of the data. How-
ever, the scanty evidence afforded by the plates of disintegrating phyto-
plankton presents a more positive picture of relationship. Furthermore,
in direct microscopic observations on this disintegrating algal material -
where freedom from opaque, inorganic matter greatly facilitated the
search, active septate mycelium was found in several instances. !

Since only land and dust-borne fungi have been recovered it is nat-
ural to ask whether they are only contaminants or whether they are actu-
ally in the mud. In the collection of the cores every precaution against
outside contamination possible with the apparatus was employed and
before using the cores the mud surfaces in contact with the tube and
the water were cut away. Contamination during culture procedure does
not seem likely since mud from the middle and bottom of the cores
yielded under identical laboratory manipulation a total of eight colonies
in 130 samples and the 65 control plates two. When we consider that
239 colonies developed in the 155 dishes containing samples of super-
natant water or mud from the top layer of the core it would seem fairly
conclusive that the fungi recovered were actually present in the mud.

SAPROPHYTIC FUNGI IN MARINE MUDS 247

Two methods whereby the moulds may have reached the muds are
suggested. Either they have been washed from land soils into the sea,
or they have been blown, in spore form, from land onto the surface of
the water where they have gradually sunk to the bottom. While the
stations studied were not in the littoral zone, none was sufficiently off-
shore to be free from the possibility of obtaining spores from sedimen-
tary material of land origin. Spores are known to be everywhere in the
lower atmosphere, and in the immediate vicinity of a continent would
be particularly abundant. Many of these would eventually reach the
surface of the sea where they might ultimately sink to the bottom. In
this connection, qualitative studies (unpublished) by Miss Lois Lillick
of the phytoplankton of Vineyard Sound and the Gulf of Maine show
that in the former locality fungous spores occur generally throughout
the year in the samples, and that they also occur in the shallower waters
of the Gulf of Maine, particularly during April and May. It is entirely
possible, therefore, that either or both methods of conveyance have con-
tributed fungous material to the marine muds.

Perhaps the most interesting feature of the present investigation
has been the information added to our knowledge of the vertical dis-
tribution of these ubiquitous moulds. Many types have been recovered
from the atmosphere both over land masses and the ocean and even in
the stratosphere. Land soils and fresh water have yielded fungi and
now they have apparently been recovered in some viable form from the
surface of the ocean floor at depths up to 220 meters. Such hardihood
is not surprising to those who have observed the capacity of certain of
these fungi to withstand adverse environmental circumstances and even
to produce under these conditions active vegetative growth.

Finally, it might be emphasized that while no typically marine fungi
were found there is strong evidence for believing that certain soil and
dust-borne fungi can exist in the surface muds although it has not been
shown in what form they occur or that they take an active part there in
the disintegration of organic materials.

SUMMARY

Stratified samples of marine bottom were collected under as sterile
conditions as possible from four stations which varied from 18 to 220
meters in depth. These stations were located in Buzzard’s Bay, Vine-
yard Sound, and the Gulf of Maine and varied considerably in their
distances from land. Attempts were made to recover saprophytic fungi
from these cores. Two methods were used: (1) water cultures
“baited” with suitable material of marine origin; and (2) the plating
out of samples from (a) the water immediately above the surface of

248 F. kK. SPARROW, JR.

the mud core, (b) the surface of the core, (c) the middle, and (d) the
bottom of the core. Suitable controls were maintained. No fungi were
found in the water cultures. By the plate method using a nutrient
medium made up in sea water, 239 colonies were formed in dishes con-
taining water from just above the surface of the core and from the sur-
face of the core itself, eight colonies, in dishes containing material from
the middle and bottom of the core, and two colonies in the controls.

A preliminary experiment to determine whether or not fungi were
associated with decaying phytoplankton showed definitely that such was
the case.

The fungi obtained by the plate method were all common dust and
wind-borne forms. Since the methods used in the recovery of fungi
did not show in what form they existed on the sea-bottom and since no
species which might be called typically marine were recovered, it is
doubtful in the present state of our knowledge whether these organisms
play an active part in the disintegration of organic materials present in
the mud.

BIBLIOGRAPHY

Moorg, H. B., anv R. J. Netix, 1930. An instrument for sampling marine muds.
Jour. Mar. Biol. Ass’n., 16: 589.

Petersen, Hennine E., 1905. Contributions a la connaissance des Phycomycetes
marins (Chytridineee Fischer). Oversigt K. danske vidensk. Selsk. For-
handl., 1905 (5): 439.

Sparrow, F. K., Jr. 1934. Observations on marine Phycomycetes collected in
Denmark. Dansk. bot. Ark., 8 (6): 1.

Sparrow, F. K., Jr., 1936. Biological observations on the marine fungi of Woods
Hole Waters. Biol. Bull., 52: 236.

DETERMINATION OF POLARITY BY CENTRIFUGING
BEGCS, Oh PUCUS HUREADUS *

D. M. WHITAKER

(From the School of Biological Sciences, Stanford University, California)

INTRODUCTION

The visible inclusions in the protoplasm of many animal eggs have
been segregated within the cell into zones or strata by means of the
centrifuge. In smaller, less yolky eggs in which the protoplasm is quite
fluid, the inclusions, such as yolk and oil droplets, pigment granules,
mitochondria (Arbacia, Harvey, 1936) etc., segregate in the protoplasm
in accordance with their relative densities. In other cases there is
sometimes a rotation or dislocation of regions of the cytoplasm which
move as a whole.

The results of numerous earlier experiments on animal eggs are
reviewed in such standard texts as Morgan’s “ Experimental Embryol-
ogy” (1927) and Wilson’s “The Cell in Development and Heredity ”
(1925), and therefore no exhaustive review of the literature will be
undertaken here. It should be noted, however, that in general the
mere displacement of the visible inclusions has had surprisingly little
effect on the development of most of the eggs. The primary polar axis
is not determined by the axis of stratification, although Runnstr6m
(1927) has shown that dorso-ventrality may be determined in the sea
urchin egg. The general conclusion has been that primary polarity
depends on factors in the transparent hyaloplasm rather than upon
particles large enough to be moved readily by the centrifugal forces
employed.” By centrifuging unfertilized Urechis eggs at 4,800 X g.
for very long periods (up to 18 hours), Taylor (1931) was able to
determine or shift the primary polar axis considerably, as shown sta-
tistically, but it did not come to coincide precisely with the axis of
stratification in most of the eggs. Morgan and Tyler (1935) found
that when fertilized Urechis eggs are centrifuged, the polar bodies may

1 This work has been supported in part by funds granted by the Rockefeller
Foundation.

2 Particles moved by the ordinary type of centrifuge may have very pronounced
physiological effects, however. Shapiro (1935) has shown that fragments of
centrifuged Arbacia eggs which contain the heavier granules respire at nearly twice
the rate of transparent fragments, and Navez and Harvey (1935) have found twice
the indophenol oxidase activity in the fragments containing these granules.

249

250 D. M. WHITAKER

be set free in regions determined by the axis of centrifugation, but in
this case the developmental axes of the eggs do not shift with the polar
bodies, but instead remain unaltered.

Conklin (1931) found that a dislocation of organs takes place in
ascidian eggs when whole regions of the cytoplasm have been displaced
or dislocated by centrifuging. Other alterations of the normal devel-
opment of animal eggs have been brought about by the mechanical
consequences of centrifuging, such as twinning (Tyler, 1930; Harvey,
1933,0 9SaNetes):

Eggs of the marine brown alge belonging to the Fucacee are more
labile than most animal eggs. A number of environmental agents may
determine polarity in the Fucus egg, including unilateral light (Rosen-
vinge, 1889) (Kniep, 1907) (Hurd, 1920), especially in the short end
of the visible spectrum (Hurd, 1920), direct electric current (Lund,
1923), the presence of neighboring eggs (Rosenvinge, 1889) (Kniep,
1907) (Hurd, 1920) (Whitaker, 1931), especially in acidified sea water
(Whitaker, 1937), a pH gradient (Whitaker, 1935) and a temperature
gradient (Lowrance, 1937). Knapp (1931) believes that the entrance
point of the sperm determines the point of rhizoid origin and the
polarity in the egg of Cystosira, but the effect of such environmental
agents as directed light, if applied at an appropriate time, will supplant
this determination and establish a new polarity. The effect of the
entrance point of the sperm has not been tested in Fucus, but if it de-
termines polarity, this is readily altered by such means as those just
indicated. Knapp found further in Cystosira that rhizoids form cen-
trifugally when the eggs are centrifuged either before or after fertiliza-
tion. Schechter (1934) found that rhizoids develop toward the positive
pole when pieces of the red alga Griffithsia are reared in a direct electric
current. Lund (1923) earlier had found that Fucus eggs form rhizoids
toward the positive pole. Schechter observed that chromatophores mi-
grated toward the positive pole in the Griffithsia, and he tried moving
the chromatophores by means of the centrifuge (1934, 1935). The
chromatophores and other bodies were moved, and accumulated cen-
trifugally under relatively low centrifugal force (150 X g.) so that the
cell-materials stratified, but the place of rhizoid origin was unaffected.
Polarity was altered, however, in that shoots formed in the regions to
which heavier materials had been thrown.

METHOD AND RESULTS

Eggs of Fucus furcatus were obtained from the same locality and
by methods which have been described previously (Whitaker, 1936).

DETERMINATION OF POLARITY IN EGGS OF FUCUS 251

Experiments were performed in February and March, 1937. Since this
species of Fucus is hermaphroditic, and sheds egg and antheridial cap-
sules at the same time,’ fertilization takes place when the egg capsules
break down. This can readily be observed, and eggs were used from
capsules breaking down during a time-span of 20 minutes or less. The
mid-point of this time-span was counted as the average time of fertili-
zation. The eggs were centrifuged at 3,000 x g. An International
electric centrifuge was used which tended to warm up during a 20-
minute run, and therefore the sea water in the centrifuge tube and the
water around the tube were cooled at the start. The temperature of
the eggs during the centrifuging rose from about 8° or 10° to 20° C.
At all other times the eggs were kept in a humid, dark, constant tem-
perature room at 15+%4° C. After being centrifuged they were
briefly exposed only to red light which does not affect the polarity.
The pH of the sea water used in the experiments ranged from 7.9 to
8.0, as measured by means of a glass electrode. In order to rule out
the effects of neighboring eggs on each other, no egg lying within 5 egg
diameters of another was counted in the results (see Whitaker, 1937).

THE First SERIES OF EXPERIMENTS

In the first series of experiments, the eggs were centrifuged in sea
water beginning from 12 to 26 minutes after the average time of fer-
tilization, and were then reared in sea water in Petri dishes. Pre-
liminary survey showed that after being centrifuged for 5 minutes the
eggs were quite definitely stratified, but not so sharply so as after being
centrifuged for 15 to 20 minutes (see Fig. 1, 4). It was also found,
when the eggs were observed about 24 hours later, that the stratified
material had redistributed much more markedly if the eggs were cen-
trifuged for only 5 minutes. In all of the experiments to be cited, the
eggs were centrifuged at 3,000 X g. for either 15 or 20 minutes (usu-
ally 20). Most of the eggs remained spherical and nearly all of those
that were distorted by neighbors in the centrifuge tube rounded up again
in the Petri dish.

Figure 1, A shows a typical egg 23 minutes after being centrifuged
for 20 minutes. Three principal zones are sharply demarcated. At
the centripetal end, to which the least dense materials are thrown, there
is a cap of globules which presumably are oil or fat. Next to this is a
dark brown zone in which the chloroplasts are concentrated, and within
which the nucleus 1s concealed. The remainder of the ege, including

* Most of the antheridial capsules dissolve first so that antherazoids, or sperm,

are swimming about the ege capsules when they dissolve and set the eggs free in
the sea water.

SE) D. M. WHITAKER

the centrifugal pole, is essentially transparent, although a few plastids
may remain scattered especially in the cortical region, and the general
texture appears slightly granular. Figure 1, B shows the same egg 15
minutes later, and material of the dark zone is already seen to be moving
back toward the center of the egg. Soon afterward the nucleus migrates
out of the dark band, moving toward the center of the egg. It is usually

Fic. 1. Photomicrographs of typical developing centrifuged and normal Fucus
eggs. (A) shows an egg 23 minutes after being centrifuged for 20 minutes at
3,000 X g. The centrifuging began shortly after fertilization, and the centrifugal
end is below. Chloroplasts are concentrated in the dark centripetal band. (B)
shows the same egg 15 minutes later, and already inclusions in the dark band are
seen to be diffusing back to some extent. (C) shows another egg from the same
sample 3 hours and 10 minutes later. The nucleus has emerged from the dark
stratum within which it was earlier concealed. (2D) shows another egg from the
same sample 13 hours and 25 minutes later. It may be seen, especially by re-
ferring to the remnant of the centripetal cap, that the rhizoid protuberance has
formed at the centrifugal end of the egg. (A’) and (D’) are normal non-cen-
trifuged eggs in stages corresponding to (A) and (D) respectively. All eggs
were reared in the dark at 15° C. at pH 7.9-8.0 (see text).

closely followed, and sometimes it is flanked along the sides or sur-
rounded, by redistributing dark material. Figures 1 C and 1 D show
other eggs from the same dish at later times. In Fig. 1, D it can be
seen by referring to the reduced remnant of the oil cap that the rhizoid
has formed quite precisely at the centrifugal pole. Some of the eggs

DETERMINATION OF POLARITY IN EGGS OF FUCUS Zs)

do not remain stratified but redistribute completely. These will be
referred to later.

In six similar experiments of the first series, large numbers of eggs
were reared in Petri dishes after being removed from the centrifuge, and
the final records were taken 20 to 24 hours later. When centrifuged
eggs are placed in a dish of sea water, most of them rotate and lie so
that the heavy side is down. When the rhizoid extends downward it
lifts the cell body which usually rolls over to one side. The strata can
then be seen. Each egg was further rolled over by hand with a glass
needle if necessary to observe the stratification when the results were
recorded.

Sample counts of over 1,000 eggs indicated that the vast majority
of the eggs which remained stratified, so that the axis of centrifugation
could be identified, formed rhizoids at or near the ends to which heavier
materials had been thrown. In three experiments more precise esti-
mates were made of the positions of the rhizoids. A random count of
490 eggs showed that 96 per cent formed rhizoids within 10° of the
centrifugal pole, and the remaining 4 per cent formed them between
10° and 22° from the pole. In addition to the eggs which remained
stratified (Fig. 1, D), and could be analyzed as above, some eggs re-
distributed the stratified materials so completely that the axis of cen-
trifugation could not be identified. The proportion of these was usually
small, about 4 to 10 per cent, although in one set of eggs, for reasons
which are not clear, approximately 50 per cent of the eggs had redis-
tributed at 24 hours. There was usually a sharp distinction between
eggs that remained clearly stratified and those that had completely
redistributed, with relatively few transitional cases.

From the experiments cited above, it was concluded that the rhizoid
forms at the end of the egg to which heavier materials are thrown, at
least in the eggs which remain stratified after 24 hours. The Fucus
egg is spherical and has no early identifying mark of polarity (Fig.
1, A’). It is conceivable, however, that the egg actually has a pre-
determined rhizoid pole which is also the heaviest part of the egg. It
so, in centrifuging, the egg would be thrown to the bottom of the tube
in an oriented position. This does not appear very probable, but if it
were true the stratification would conform to the oriented position and
might itself be irrelevant in the determination of polarity. A second
series of experiments was therefore designed to test this and other points.

THE SECOND SERIES OF EXPERIMENTS
In the second series, eggs were embedded in 1.5 per cent agar-sea
water before centrifuging, after centrifuging, and without being centri-

254 D. M. WHITAKER

fuged. Eggs develop normally in this medium, and observations on
stratified eggs in various positions indicate that they are firmly held and
that few 1f any move or rotate within the agar-sea water after it has
cooled and jelled, if it is undisturbed. If the agar is cut with a razor,
eggs lying close to the cut may rotate as a consequence of the mechanical
disturbance. One and a half per cent agar-sea water is a firm jelly at
15° C., and the surface of a fertilized Fucus egg attaches to it firmly.

Eggs were embedded by pipetting a concentrated mass of eggs in a
minimum of sea water into agar-sea water at 38° C. The agar-sea water
gelates below this temperature. The mixture was taken in and out of
the agar pipette once or twice to assure mixing and the dish was put
on ice to cool rapidly and shorten the exposure of the eggs to high
temperature.

When a population of fertilized eggs which are not centrifuged is
embedded in agar-sea water and reared in the dark, the rhizoids form
at random in all directions as seen from above. The upward and down-
ward components are not equal, however, when the population is con-
sidered statistically. Rhizoids are found in every possible position, from
straight down to straight up, but more are found with a downward com-
ponent than with an upward component. The average position is some-
what downward from the horizontal plane. For example, in a popula-
tion of 413 eggs, 14 per cent of the rhizoids formed upward (+: 45°),
41 per cent to the side (+ 45°), and 45 per cent downward (+ 45°).
In other populations more rhizoids formed to the side than downward.
It has been shown elsewhere (Whitaker, 1937) that the downward
component increases with acidity of the sea water and with increased
concentration of eggs. It cannot be said at the present time whether the
downward component is due to gravity or to concentration gradients of
substances (e.g. CO,) which diffuse through the upper boundary of the
medium but cannot diffuse through the bottom of the dish.

When eggs were first centrifuged, and then mixed at random into
agar-sea water, it was found by analyzing 600 eggs statistically after
the agar had solidified that the eggs tended to be oriented with the heavy
side downward. This orientation, while appreciable, was on the whole
not pronounced. It presumably took place just before the agar solidi-
fied, as a result of the strong density gradients which had been created
in the eggs by stratification in the centrifuge. The results from these
eges which bear on the determination of polarity will be considered after
the principal results of the second series have been taken up. These
are derived from eggs first embedded in agar-sea water and then cen-
{rifuged.

In six similar experiments of the second series, eggs were embedded

DETERMINATION OF POLARITY IN EGGS OF FUCUS 250

at random soon after fertilization in 1.5 per cent agar-sea water in
moulds which formed castings which fitted the bottoms of the centrifuge
tubes. The castings were centrifuged, and then small blocks or strips
of agar, bearing stratified eggs, were cut with a razor and placed in
Petri dishes of sea water. All eggs in a block were oriented the same
way with respect to their stratification, except for some eggs, especially
those near the edge, which were mechanically disturbed and rolled when
the block was cut and handled. These blocks were oriented in three
different positions: with the centrifugal side (1) straight down, (2) to
the side, and (3) straight up. In each experiment, blocks of agar cui
from the same centrifuged piece were placed in either two or three of
the positions.

After the eggs in these blocks of agar-sea water had developed in
the dark for 20 to 24 hours, they were inspected with the microscope
and sample counts were made of the positions of rhizoid origin. When-
ever necessary, eggs were rolled over within the agar with a glass needle.
The eggs were classified as having formed rhizoids downward within
45°, or to the side within 45°, or upward within 45°. The positions of
the rhizoids with respect to stratification were noted, and whether or not
redistribution had taken place so that stratification was no longer dis-
cernible. The results of the separate experiments were essentially sim-
ilar, and therefore the averaged results will be considered.

Of 423 eggs in agar-sea water blocks placed so that the centrifugal
sides of the blocks were downward, 46 or less than 11 per cent had re-
distributed, while the remaining 89 per cent remained stratified. Ninety-
nine per cent of the eggs which remained stratified formed rhizoids within
10° of the centrifugal pole (see Fig. 1, D), while 1 per cent formed them
farther away from the centrifugal pole, but still on the centrifugal
hemisphere. Ninety-five per cent of the eggs which remained stratified
had retained their position in the agar, without rotating after being cen-
-trifuged, so that their centrifugal poles were downward, while 5 per
cent had been rotated so that their centrifugal poles were to the side
within 45°. Of the 46 eggs which had redistributed, 3 formed rhizoids
downward, 25 to the side, and 18 upward. The distribution of these
rhizoids is strikingly different from those on the eggs which remained
stratified. Relatively many more are to the side and upward, which
suggests either that the determination of rhizoid formation at the cen-
trifugal poles is largely lost when eggs redistribute, or else that a large
proportion of the redistributed eggs had been oriented with their cen-
trifugal poles to the side or upward (as a result of rotating when the
block was cut), and that such orientations favor redistribution. Evi-

256 D. M. WHITAKER

dence that both of these factors operate is provided by eggs reared with
their centrifugal poles upward.

Eggs reared in agar-sea water blocks placed so that the centrifugal
sides of the blocks were upward were held with the heaviest materials
at the top and the lightest at the bottom. This is the exact opposite
of the position which the eggs tend to assume when free in a dish, and
it should tend to favor redistribution since the effect of gravity is added
to that of diffusion. Of 488 eggs which developed in blocks in this
position, 166, or 34 per cent had redistributed (compared with 11 per
cent in blocks placed centrifugal side downward), and of these redis-
tributed eggs 130 formed rhizoids downward, 33 to the side, and 3 up-
ward. In other words, the percentage of redistribution was trebled, and
the redistributed eggs formed rhizoids more nearly in the general pattern
found in a population of eggs which have never been centrifuged. Even
more rhizoids formed downward than in the average population of non-
centrifuged eggs. Three hundred and twenty-two eggs remained strati-
fied, and 93 per cent of these formed rhizoids within 10° of the cen-
trifugal pole. The remaining 7 per cent formed them farther away
from the centrifugal pole, but not more than 90°. Two hundred and
seventy-two eggs formed rhizoids upward, and 99 per cent of these had
remained stratified and formed rhizoids within 10° of the centrifugal
pole. ‘Twenty-six of the 488 eggs remained stratified but had been ro-
tated so that the centrifugal pole was to the side (+ 45°), and in 27 it
was downward (+ 45°).

Eggs reared in agar-sea water blocks placed so that the centrifugal
sides of the blocks were to the side gave results which are on the whole
intermediate between those from eggs in blocks in up and in down posi-
tions. Four hundred and twenty-five eggs were analyzed. Sixteen per
cent had redistributed, compared with 34 and 11 per cent, respectively,
in the other two positions. These redistributed eggs formed 1 rhizoid
upward, 39 to the side, and 27 downward (all + 45°). Three hundred
and fifty-eight eggs remained stratified and 333 of these (93 per cent)
formed rhizoids within 10° of the centrifugal pole. The remaining
7 per cent formed them between 10° and 90° from the centrifugal pole.
Of the 333 eggs which formed rhizoids within 10° of the centrifugal
pole, 314 (94 per cent) developed rhizoids laterally (i.e., they retained
their original orientation in the agar blocks).

As earlier mentioned, eggs were also centrifuged first and then em-
bedded in agar-sea water. The axes of stratification lay at various
angles with respect to the horizontal plane. The results obtained by
analyzing 600 eggs selected at random were entirely in harmony with
those just cited.

DETERMINATION OF POLARITY IN EGGS OF FUCUS Dash

Discussion

Most of the centrifuged eggs remain stratified long after the rhizoid
protuberances have formed, although the stratified materials begin to
diffuse back to some extent quite early (see Fig. 1). The results show
that, regardless of the position in which they are held and reared, the
centrifuged eggs which remain stratified form rhizoids on their cen-
trifugal hemispheres. With very high incidence, they do so quite pre-
cisely at their centrifugal poles to which heavier materials have been
thrown. Thus, of 1,057 eggs which remained stratified in blocks of
agar-sea water placed so that the centrifugal sides were downward, to
the side, or upward (comparable numbers of eggs being in each of these
3 positions), 1,007 or more than 95 per cent formed rhizoids within
10° of their centrifugal poles as marked by the strata. The remainder
formed them between 10° and 90° from the centrifugal poles. These
eggs were embedded in random positions in firm agar-sea water before
being centrifuged, and presumably could not orient in the centrifuge in
accordance with any pre-determined polarity. The developmental po-
larity is therefore determined by the axis of centrifugation. If there is
an earlier polarity (as found by Knapp (1931) in Cystosira; see intro-
duction), it is completely altered.

Some of the eggs do not remain stratified, but instead the visible
inclusions have redistributed so completely when they are observed after
the rhizoid protuberances have formed that they are indistinguishable
from eggs which have never been centrifuged. When eggs are held in-
verted in agar-sea water so that the centrifugal poles are uppermost, the
proportion of the eggs which redistribute is considerably increased, al-
though the redistributed eggs still remain in the minority. When in-
verted eggs redistribute, the tendency to form rhizoids at their centrifu-
gal poles disappears. They form rhizoids more nearly in the positions
in which they would have formed them if they had never been centri-
fuged. In fact, both in the blocks placed centrifugal side upward and
in those placed centrifugal side downward, the redistributed eggs formed
even more rhizoids on the sides of the eggs away from the centrifugal
sides of the agar blocks than non-centrifuged eggs would have been
expected to do in their place.

_The fact that only some of the eggs redistribute, while most others
held in the same position do not, must be attributed to variations in the
properties of different eggs in regard to factors such as viscosity, etc.,
and perhaps to the fact that some of the eggs had been fertilized as
much as 20 minutes longer than others at the time of centrifugation.

Knapp (1931) found that rhizoids form at the centrifugal poles of

258 D. M. WHITAKER

Cystosiva eggs, in high percentages if the eggs have been visibly strati-
fied. The principal results on Fucus eggs are thus similar. Schechter’s
(1934, 1935) results on pieces of the red alga Griffithsia are similar in
that the location of organs was determined by centrifuging, but in this
case shoots, not rhizoids, formed at the centrifugal pole. The place of
origin of rhizoids was unaffected.

It is possible that a special rhizoid-forming substance accumulates
at the centrifugal pole of the Fucus egg as a result of centrifugation.
However, since gradients of a number of factors which affect the rate
of activities such as respiration, CO, production, etc. (e.g. temperature,
pH, etc.; see Introduction) determine the point of rhizoid origin in
this egg, it is also possible that polarity is determined in the stratified
egg by gradients of such activities caused by the asymmetrical distribu-
tion of cell inclusions. Moreover, these factors may influence the mi-
totic figure which in turn may affect the polarity. The present experi-
ments do not provide a basis for choosing between these possibilities.

SUMMARY

1. When eggs of Fucus furcatus are centrifuged at 3,000 X g. for
15 or 20 minutes, beginning between 12 and 37 minutes after fertiliza-
tion, the visible cell inclusions are thrown centripetally (see Fig. 1).

2: Most of the eggs remain visibly stratified until long after the rhi-
zoid protuberances have formed. Ninety-three to ninety-nine per cent
of the eggs which remain stratified form rhizoids quite precisely at the
centrifugal pole (within 10°; see Fig. 1), even when they have been em-
bedded in random positions in firm agar-sea water before being centri-
fuged so that they could not orient in the centrifuge in accordance with
any earlier polarity. The remainder also form rhizoids on the cen-
trifugal hemisphere of the egg, but farther removed from the centrifugal
pole.

3. In eggs which remain stratified, the place of rhizoid origin and
the developmental polarity are therefore determined by the axis of cen-
trifugation. ‘This is true regardless of the position in which the strati-
fied eggs are held in agar-sea water during development. If there is
an earlier polarity in the egg it is completely supplanted by the effects of
centrifugation.

4. In a smaller proportion of the eggs, the cell inclusions have re-
distributed so completely when the eggs are inspected after the rhizoid
protuberances have formed that, in high contrast to the eggs which
remain stratified, they are visibly indistinguishable from eggs which have
never been centrifuged. Such redistribution is more prevalent when
eggs are reared heavy side up in agar than when they are reared heavy

DETERMINATION OF POLARITY IN EGGS OF FUCUS LSS)

side down. The determination of rhizoid formation at the centrifugal
pole is lost in inverted eggs which have redistributed. Under the con-
ditions of the experiments, the determination therefore appears to cor-
relate with the distribution of visible inclusions.

The author is indebted to Mr. C. W. Clancy for assistance in carry-
ing out the experiments.

BIBLIOGRAPHY

CoNKLIN, E. G., 1931. The development of centrifuged eggs of ascidians. Jour.
Exper. Zool., 60: 1.

Harvey, E. B., 1933. Effects of centrifugal force on fertilized eggs of Arbacia
punctulata as observed with the centrifuge-microscope. Biol. Bull., 65:
389.

Harvey, E. B., 1935. Some surface phenomena in the fertilized sea urchin egg as
influenced by centrifugal force. Bzol. Bull., 69: 298.

Harvey, E. B., 1936. Parthenogenetic merogony or cleavage without nuclei in
Arbacia punctulata. Biol. Bull.,71: 101. —

Hurp, A. M., 1920. Effect of unilateral monochromatic light and group orienta-
tion on the polarity of germinating Fucus spores. Bot. Gaz., 70: 25.

Knapp, E., 1931. Entwicklungsphysiologische Untersuchungen an Fucaceen-Fiern.
1. Zur Kenntnis der Polaritat der Eier von Cystosira barbata. Planta
(Abb. E), 14: 731.

Kniep, H., 1907. Beitrage zur Keimungs-Physiologie und Biologie von Fucus
Jahrb. wissensch. Bot., 44: 635.

Lowrance, E. W., 1937. Determination of polarity in Fucus eggs by temperature
gradients. Proc. Soc. Exper. Biol. and Med. (in press).

Lunp, E. J., 1923. Electrical control of organic polarity in the egg of Fucus.
Bot. Gaz., 76: 288.

Morcan, T. H., 1927. Experimental Embryology. Columbia University Press,
New York.

Morcan, T. H., ann A. Tyrer, 1935. Effects of centrifuging eggs of Urechis
before and after fertilization. Jour. Exper. Zool., 70: 301.
Navez, A. E., ann E. B. Harvey, 1935. Indophenol oxidase activity in intact
and fragmented Arbacia eggs (abstract). Biol. Bull., 69: 342. |
Rosenvince, M. K. L., 1889. Influence des agents extérieurs sur l’organisation
polaire et dorsi-ventrale des plantes. Rev. Gen. Bot., 1: 53.

Runnstrom, J., 1927. Experimentelle Bestimmung der Dorso-ventral-achse bei
dem Seeigelkeim. Arkiv. for Zoologi., 18: (No. 4), 1.

Scuecuter, V., 1934. Electrical control of rhizoid formation in the red alga,
Griffithsia bornetiana. Jour. Gen. Physiol., 18: 1.

ScuHecuTer, V., 1935. The effect of centrifuging on the polarity of an alga,
Griffithsia bornetiana. Biol. Bull., 68: 172.

Sapiro, H., 1935. The respiration of fragments obtained by centrifuging the
egg of the sea urchin, Arbacia punctulata. Jour. Cell. and Comp. Physiol.,
6: 101.

Taytor, C. V., 1931. Polarity in normal and centrifuged eggs of Urechis caupo
Fisher and MacGinitie. Physiol. Zo6l., 4: 423.

Tver, A., 1930. Experimental production of double embryos in annelids and mol-
lusks. Jour. Exper. Zool., 57: 347.

Wuiraxer, D. M., 1931. Some observations on the eggs of Fucus and upon their
mutual influence in the determination of the developmental axis. Bvol.
Bull., 61: 294.

260 IDS IE NaVles Adie OSC DIRS

Wuitaker, D. M., 1935. Induction of polarity in Fucus furcatus by a localized
concentration of hydrogen ions. Proc. Soc. Exper. Biol. and Med., 33:
472.

Wuiraker, D. M., 1936. The effect of white light upon the rate of development
of the rhizoid protuberance and the first cell division in Fucus furcatus.
Biol. Bull., 70: 100.

Wuirtaker, D. M., 1937. The effect of hydrogen ion concentration upon the in-
duction of polarity in Fucus eggs. 1. Increased hydrogen ion concen-
tration and the intensity of mutual inductions by neighboring eggs of
Fucus furcatus. Jour. Gen. Physiol., 20: 491.

Witson, E. B., 1925. The Cell in Development and Heredity. The MacMillan
Co., New York.

ON THE ENERGETICS OF DIFFERENTIATION, VI

CoMPARISON OF THE TEMPERATURE COEFFICIENTS OF THE RESPIRATORY
RatTES OF UNFERTILIZED AND OF FERTILIZD Eccs ?

ALBERT TYLER AND W. D. HUMASON

(From the William G. Kerckhoff Laboratories of the Biological Sciences,
California Institute of Technology, Pasadena, California)

The results of these experiments show principally that the tempera-
ture coefficients of the rates of respiration are the same for unfertilized
eggs as for fertilized eggs over most of the temperature range investi-
gated. They diverge somewhat at the lower temperatures, the ferti-
lized eggs giving higher values.

‘THEORETICAL PART

An unfertilized egg is generally considered as a resting cell, pre-
sumably being concerned merely with keeping itself alive. It should
thus exhibit simply maintenance (basal) metabolism. The maintenance
metabolism of an organism is measured by the heat production or the
respiration under “resting” conditions. The respiration of an unferti-
lized egg may then be taken as a measure of its maintenance require-
ments.

A fertilized egg is also considered as having a maintenance require-
ment, but in addition there are requirements for processes termed
growth and differentiation. It is conceivable that by such an agent as
change in temperature, these three processes might be affected differ-
ently. But from experiments on the effect of temperature on the rate
of development and the rate of respiration of fertilized eggs (Tyler,
1936), this does not appear likely, unless these changes are compensa-
tory. The cited experiments had shown that there is the same total
oxygen consumption in reaching a given stage of development at one
temperature as at another. Thus the temperature coefficients of rate of
respiration and development are the same and there is no temperature
within the normal range at which development is accomplished with a
minimum of respiration.

It was to be expected, then, that the rate of respiration of unferti-
lized eggs should give the same temperature coefficients as that of de-

1 This investigation was supported in part by a grant from the Penrose fund
of the American Philosophical Society.

261

262 TYLER AND HUMASON

veloping eggs. An investigation of this sort was made by Rubenstein
and Gerard (1934) on the sea-urchin egg. They found much higher
coefficients for the unfertilized than for the fertilized eggs, the average
values for Q,, being 4.1 for the former and 1.8 for the latter. An ex-
amination of their data shows a number of errors in the calculations
which, while not greatly affecting the average values, make their con-
clusions somewhat less convincing. Considering also the difficulties in-
volved in measuring the respiration of unfertilized eggs, it seemed ad-
visable to repeat these experiments on the sea-urchin and in addition to
investigate other forms.

EXPERIMENTAL PART
Material and Methods

The eggs used were those of the sea-urchin, Strongylocentrotus pur-
puratus; the sand-dollar, Dendraster excentricus; the echiuroid worm,
Urechis caupo,; and the ascidian, Ciona intestinalis.

The manometric method of Warburg was employed for measuring
the respiration. Since one of the principal difficulties with unfertilized
eggs is their relatively low rate of respiration per unit mass, special ves-
sels were constructed which would tend to make the measured pressure
changes as large as possible, at the same time insuring adequate gas ex-
change. Considering the various factors involved, the cylindrical type
of vessel previously described (Tyler, 1936), but with calibration vol-
umes of 18 to 20 cc. and capable of taking 8 cc. of egg suspension, was _
employed. ‘The vessel constants are of the order of 1.0. The eggs are
pipetted into the vessels with special automatic pipettes. Errors in cal-
ibration of the vessels and of delivery from the pipettes amount to less
than 0.1 and 0.3 per cent and are therefore negligible. The amount of
material employed was determined from the nitrogen content of the eggs,
obtained by means of a modified Kjeldahl method. The error here
depends upon the amount of material employed, but even for the most
dilute egg suspension it does not exceed one per cent. In some instances,
noted as “no Kjeldahl” in the tables, no nitrogen determinations were
made, but the eggs were simply pipetted from a uniform suspension.
The manometers are read to the nearest 0.5 mm., so the reading error |
will be determined by the magnitude of the pressure change. In gen-
eral no coefficients were calculated for pressure changes of less than 10
mm. and in most cases the readings used were between 30 and 80 mm.
In cases in which only dilute suspensions of eggs are available, the runs
must be continued over longer periods of time to get sufficient pressure
change to reduce the reading error. This involves another difficulty.

RESPIRATORY RATE OF UNFERTILIZED EGGS 263

One of the chief difficulties that was encountered is the variation
in rate of respiration during the progress of arun. ‘The rate of respira-
tion of the unfertilized egg does not remain constant, as is usually as-
sumed, but rises after a shorter or longer period of time. This rise has
been previously noted by Warburg (1915) and by Runnstrom (1930).
We find that the unfertilized eggs of the different animals we have stud-
ied vary in regard to the rate of rise. ‘This is illustrated in Fig. 1. It

/' hour//m

/ a
ee) abe
as

eee |
Hours (after removal or intentitized eggs ue after fasemmigation for fertilized eggs)

Rate of Oxygen Consumption (cmm. 05

EXPLANATION OF FIGURES

Fic. 1. Rates of oxygen consumption of unfertilized and fertilized eggs of
Ciona, Urechis, Dendraster and Strongylocentrotus. The unfertilized rates for
the two latter rise much more rapidly with time than for the other two.

may be seen that the eggs of Dendraster and Strongylocentrotus show
a much more rapid rise than do the eggs of Ciona and Urechis. In fact,
in Dendraster and in Strongylocentrotus the unfertilized egg respiration
rate rises almost as rapidly as does the fertilized egg respiration rate.
This rise in respiratory rate appears to be correlated with the loss of
fertilizability of the eggs. Without presenting quantitative data at this
time, it may be pointed out that Ciona and Urechis eggs remain fertil-
izable after standing 24 to 48 hours in sea water whereas Dendraster
and Strongylocentrotus eggs are no longer fertilizable after 5 to 8 hours.
Different batches of eggs differ somewhat in the rate of rise, but the

264 TYLER AND HUMASON

curves of Fig. 1 are typical. The unfertilized eggs of all the forms in-
vestigated show sooner or later a rise in respiratory rate.”

Occasionally in some runs (e.g. Experiment XI of Table 1) a de-
crease in rate is manifested in the early part of a run. This appears to
be due to the sticking of the unfertilized eggs to the walls of the vessels.
In some cases a considerable amount (perhaps 5 per cent) of the eggs
stick to the walls. The stuck eggs are presumably unaffected while
under the sea water, but when the shaking is stopped for a reading some
of the stuck eggs are left well above the water level in the vessels. Such
eggs, if partially or completely cytolyzed, would give an abnormal res-
piratory rate. In our experiments on cytolyzing eggs with dry ice or
with distilled water we find an initially high respiration followed after
about an hour by a very low rate. If this holds for the stuck eggs we
would expect to get slight decreases in rate as well as slight initial in-
creases, since most of the sticking occurs at the start of a run.

Due to the variations in rate of respiration with time the temperature
coefficients cannot be determined simply by placing a vessel first at one
temperature then at another. It is necessary in addition to run eggs
from the same batch simultaneously at the two temperatures. The ex-
periments were therefore performed in the following manner. Usually,
four vessels were prepared from the same batch of eggs as soon as pos-
sible after removal from the animal and two of them were placed in
each of two different temperature baths. After a certain number of
readings were made the vessels in the high temperature bath were ex-
changed with those in the low temperature bath. After another set of
readings, the vessels were replaced in the original baths and the readings
continued. At least fifteen minutes was allowed for temperature equi-
librium to be attained.

The temperature coefficients were then calculated in two ways;
first, from the oxygen consumption in the vessels run simultaneously
at the two temperatures; secondly, from the oxygen consumption in
one vessel run alternately at the different temperatures. or the first
type of coefficient it is important to know the quantities of eggs in the
different vessels or to have identical samples of a uniform suspension
in each. For the coefficients calculated in the second manner this is, of

2 The rise is not due to conditions in the respiration vessels, as an experiment
with Urechis illustrates. Two samples of eggs, run continuously for 20 hours at
22°, gave Xo,’s of 3.05 and 3.00 cu. mm. in the first hour, and 5.12 and 5.35 cu.
mm, in the twentieth hour. Two more samples of eggs from the same batch,
that had stood for twenty hours and washed before using, gave Xo,’s of 4.81 and
5.06 cu. mm. in the first hour. Subsequent fertilization was 85 to 100 per cent.
The capacity for fertilization is not lost until after a considerable rise in respira-

tion is manifest. In some experiments 100 per cent fertilization was obtained
after an almost two-fold rise in the unfertilized rate.

RESPIRATORY RATE OF UNFERTILIZED EGGS 265

course, unnecessary. Errors in the determinations of the quantity of
eggs therefore do not enter into the coefficients calculated in the second
manner. However, variations in rate of respiration with time will sig-
nificantly affect the coefficients calculated in the second manner. To
compensate for this the average of two values is taken. For a vessei
starting in the high temperature bath, one value is obtained by dividing
the initial rate of oxygen consumption by the rate during the ensuing
period in the low temperature bath. The other value is gotten by di-
viding this same low temperature rate into the rate during the following
period in the high bath. Where the rate rises, as it generally does, the
first value will be lower than the second but the average of the two will
be nearer the true value. -For a vessel starting in the low temperature
bath the two values are obtained in a similar manner. In this case an
increasing rate will make the first value too high and the second too low,
but again the average will be nearer the true value. The principle in-
volved is the same as in determining the rest point of a balance.

In the tables, the coefficients calculated in the second manner are
not given for each individual vessel, but the average for each pair of
temperatures is listed in the line “ average for individual vessels.” The
respiration values from which the coefficients are readily determined are
given under the headings Xo,. The probable errors are given in cases
in which fifteen or more values are averaged. For this purpose, the
average for each vessel, determined as mentioned above, is considered as
a single value. The last two columns of the tables give the coefficients
determined in the first manner; that is, for oxygen consumption during
the same period of time at the two temperatures. Where duplicates
are run, the coefficient is the ratio of the average oxygen consumption
in the two vessels at each temperature. The mean of these values for
each pair of temperatures is given in the line marked “average ” in the
tables. In determining this average an experiment with only two ves-
sels is weighted one-half (rather than one-quarter); that is, two ex-
periments with two vessels are considered the same as one experiment
with four vessels. The probable errors are again given for cases in
which fifteen or more values are averaged.

In most of the experiments only unfertilized eggs were run, a con-
siderable number of coefficients for fertilized eggs having been obtained
in previous work (Tyler, 1936). These values for fertilized eggs are
listed in the lines “average from previously published data” in the
tables. In some experiments (e.g. VI, X and XI of Table I) two ves-
sels with fertilized eggs were run along with two vessels with unfertilized
eggs from the same batch. The figures in parentheses in the tables are
for fertilized eggs. In some experiments (I, V and VI of Table V)

266 TYLER AND HUMASON

after an unfertilized run, the eggs were inseminated in the vessels and
the measurements continued. The fertilized eggs give in general much
more consistent values, so relatively fewer experiments, in addition to
those previously published, were required. ‘The coefficients for the ferti-
lized eggs were calculated here in the same two ways as for the unferti-
lized eggs. The values taken from previously published data were ob- »
tained in a somewhat different manner (loc. cit.) which automatically
took into account the rising rate of respiration of the fertilized eggs.
However, since the rate rises very slowly at the start and since the
methods of calculation also allow for the rising rate, comparable values
are obtained for short runs. In prolonged runs, values for fertilized
eggs obtained in this manner would tend to deviate because the rate of
rise increases somewhat with time.

TABLE I

Eggs of Urechis. Xo, = mm.? O2 consumed per mg. egg nitrogen; figures in
parentheses are for fertilized eggs, the rest are for unfertilized eggs. Quo = ratio of
Xo, values (average Xo, where duplicates are run) for the same time intervals at the
two temperatures.

Vessels A B C D
Experi- Tem- Tem-
ment Time] pera- | XO» XO, | pera- | XOe XO» Quo unfert. Quo fert.
No. ture ture
hours| °C. 2G
I 1 DD, 1.99} 1.90} 12 0.82] 0.83 2.37
1 12 0.84] 0.91 D2, 2.09] 2.17 2.43

1 2, DPA Sh |e Dee sl eel 0.97 | 0.96 2.41

22 6.10} 6.00) 12 2.30} 2.04 2.79

II 2
2 WZ DES 4 | 249 D2 6.43 | 6.28 Das)
2 22 GAT OO 1 2.19 | 2.04 2.98
III 22 4.35| 4.24 | 12 1.79 | 1.80 2739

IV 2 Di 4.77| 4.60 | 12 2AO 2 ern Os 2.30

VI 1 DD 1.38 | (3.38)} 12 0.67 | (1.43) 2.06 PSII
1 12 Ore @32) 22 1.64 | (3.38) 2.65 2.56
1 22 1.64 | (3.14)| 12 0.87 | (1.52) 1.88 2.07

RESPIRATORY RATE OF UNFERTILIZED EGGS 267

TaBLE I (Continued)

Vessels A B G D
Experi- Tem- Tem-
ment Time| pera- | X02 KO, | pera- | XO» XO» Quo unfert. Qo fert.
No. ture ture
hours| °C. HG
VII 1 D2 S50) o-35) |) ke ORS TAS, 2.74
3 12 3.87 | 3.93 | 22 | 10.91 }11.28 2.85
3 22 |10.14/10.28 | 12 3.99 | 4.17 2.50
VIII 1 DD 4,49 12 Load 2.54
2 12 3.00 22 8.23 2.74
3 22 A AOLST a 12 3.35 3.24
IX } 22 7.38 12 2.90 255
2 12 2.58 22 6.27 2.43
2 22 6.44 12 2.30 2.80
x 1 22 4.42 | (4.50)| 12 1.82 | (1.65) 2.43 DS
1 12 1207) (270) 22 4.46 | (4.75) SATA 2.79
1 2D, 3.69 | (4.69)| 12 1.82 | (1.68) 2.03 2.79
XI 1 22 5.06 | (4.75)| 12 1.96 | (1.62) 2.69 2.93
1 12 155) Clas) 922 5.12 | (4.80) 3.30 2.70
1 22 4.33 | (4.88)| 12 1.82 | (1.72) 2.39 2.84
EMV ETAC CMA TN CT tae EN vase eu rts Oe a Lore 2.57 + 0.03 | 2.64
Average for Experiments II, V and VII............... 2.70
Average for individual-vessels;. .: J..2046 0 oes be aes eee 2.57 + 0.04 | 2.65
Average for individual vessels, Exp. II, V and VII...... 2.77
Average for fertilized eggs from previously published
LeU trata everett Poe MRR va Dm Meurn CAE Me LAR CHa Je oh 2 2.79 + 0.02

Samples of the eggs from the vessels were inseminated at the end
of each run and gave in most cases 90 to 100 per cent fertilization. The
experiments in which it was less are as follows: Table I, I, 85, 75, 90,
OOF INES) D5, oo) oo); Lable UME My 707S5-0be 80/070; 90,85; Vable
INGO CVn 7): lable Vile. 20. 3550. ie; 10,50; 207 (Phere
is no evident relation between the low fertilization in these cases and
values of the corresponding temperature coefficients, as an examination
of the tables shows.

It is clear from temperature experiments on biological material that
coefficients determined at different parts of the temperature scale are not
alike. They generally increase as the temperature range is lowered.
In other words, Q,, (and even p of the Arrhenius equation) is not a
constant. We must therefore make our comparisons for the same tem-
perature intervals and not use the average of values obtained from all

268 TYLER AND HUMASON

parts of the temperature scale. We have concentrated on one pair of
temperatures in attempting to obtain consistent values and supplemented
with fewer experiments at other temperatures.

Urechis caupo

Seventeen sets of experiments were run with the eggs of Urechis;
eleven at the temperatures 22° and 12°, three at 20° and 10°, two at 18°
‘and 8° and one at 15° and 5°. The experiments at 22° and 12° are
listed in Table I. The average of the coefficients for the unfertilized
eggs is 2.57 by both methods of calculation. For the fertilized eggs
(Experiments VI, X and XI) the average Q,,’s are 2.64 and 2.65 re-
spectively by the first and by the second methods of calculation. From
previous data the value for fertilized eggs is 2.79. Considering the
variation in the individual values, we can only conclude that there is no
significant difference between the coefficients for the unfertilized and
the fertilized eggs in this temperature range.

Comparison of the first and last values of Xo, in each experiment
gives the change in rate of respiration. In some instances (e.g. Ex-
periment IV) the unfertilized rate is greater during the last period than
at the start. In other cases (e.g. Experiment II) it is fairly constant
and in others (e.g. Experiment IX) it drops somewhat. But since the
direction of change and relative miagnitude is the same at both tem-
peratures, the O,,’s calculated in the first manner are not very greatly
affected. Thus for Experiment IV we have 2.30, 2.22 and 2.35. The
coefficients calculated in the second manner also are not greatly affected
where the rate changes roughly uniformly. Thus in Experiment IV
we have the O,, values 2.36, 2.40, 2.19 and 2.24 for the individual ves-
sels. It would, of course, be better to consider only cases in which
there is very little change in rate. Taking Experiments II, V and VII
as such, we get average Q,,’s of 2.70 and 2.77 which are closer to the
values for fertilized eggs. We could not, however, find any criterion,
such as the extent of agreement between duplicate vessels, the behaviour
of the eggs upon fertilization, etc. that would justify the exclusion of
any of the experiments listed.

The agreement between the coefficients calculated in the two ways
described simply means that, where the rate of respiration changes, the
change is fairly uniform. It does not, of course, measure the accuracy
of the values.

The experiments at 20° and 10°, 18° and 8°, and 15° and 5° are
listed in Table II. At these temperatures, the unfertilized eggs give

RESPIRATORY RATE OF UNFERTILIZED EGGS

TABLE IT

Eggs of Urechis. Same description as Table I.

269

Vessels A B Gs D
Experi- Tem- Tem-
ment Time} pera- XO, XO pera- XO XOo Qio unfert. | Qio fert.
No. ture ture
hours| °C. 2 a q
XII 1 20 1.74) 1.84 10 0.73) 0.68 2.55
1 10 0.93} 0.88 20 1.99} 2.10 2.26
1 20 DIX) | Baile 10 1.00; 1.02 2.16
XIII 1 20 1.95 1.87 10 0.81} 0.84 2.30
1 10 0.77| 0.74 20 2.03} 2.07 2.71
1 20 1.89] 1.87 10 0.78| 0.79 2.40
XIV 1 2 Oa S223 (223) 10 | 12.3 | (18.2) 2.63 2.87
(no Kjeldahl) | 1 10 | 14.9 | (20.4) 20 | 37.0 | (52.0) 2.48 2.55
1 20 | 31.8 | (54.6) 10 | 11.9 | (16.4) 2.67 3.33
AN GESTENEOG a ov: © SS CHET Re die aPC SETI aR CEE Ee ree eye 2.46 2.92
Averacetorindivicduall vessels ap saan cas eae see oe 2.47 2.82
PMVCIAPeMOmpclea VAG el Gata es. sed es Meng wine ye ole 2 ciel 3.30
2 18 2.01 1.97 8 ss Os i es 0) 1.81
XV 2 18 DIES DPX) 8 1.05} 0.90 2.32
2 18 2.92} 2.80 8 0.83} 1.00 3.13
XVI il 18 1.42} 1.42 8 0.60) 0.67 2.24
1 8 0.83 | 0.79 18 1.52) 1.48 1.85
1 18 1.34} 1.28 8 0.73] 0.78 1.73
INS TETTEIIS DPE repair Rade eh aa ha ECR era naar pe ie ey 2.18
Averageton imdividuall vesselsijaans. ©. 242s. e ae aceon ae 1.94
XVII 2 15 | 46.4 | (37.9) 5 | 16.9 | (10.9) Pret) 3.48
(no Kjeldahl)| 2 5 | 19.5 | (11.2) 15 | 49.7 | (36.0) 2.55 3.23
2 15 | 55.5 | (37.9) By eSgs) Guiles) 3.58 3.29
JASH TRIS, & seer ash REG ebro eI at AOR Bt Poon ee aN Me Rta ava 2.96 938)
2.84 3.30

Average for individual vessels

consistently lower Q,,’s than do the fertilized eggs.
the experiments were fertilized eggs run.
tion experiments were available from previous work, but the coefficient
for cleavage (3.30) which should be the same as for respiration (Joc.
cit.) is listed. For 18° and 8° and for 15° and 5° the cleavage value is

In only two of

For 20° and 10° no respira-

270 TYLER AND HUMASON

Asie MUL

Eggs of Strongylocentrotus. Same description as Table I.

Vessels A B i C D
Experi- . Tem- 2 Tem-
ne | Be | Pee) Aon | nee | Moe | Oe rer ae
hours) °C %G (aa

I 2 DY 3.39) 3.60) 12 130) Sess 2.63

2 12 1.23] 1.48] 22 BAS) || SoA 2.39

2 22 Soi) 4220) “12 1.46] 1.58 2.61
Il D 22 4.74|(16.4) | 12 1.65 |( 6.8) Neel 2.42
Pualnton ol03 \( 7-2)-) 22) 5.03 \GneD) 2.57 2.97
2 2D 5.46 |(22-1))| 12 2.44 | (10.6) 2.24 2.08
Ill 1 22 |30.0 |(64.7)| 12 | 11.81 |(29.5) 2.54 2.19
(no Kjeldahl) | 1 12 ASS NGO 2 SAS) (oS) 2.37 2.08
Henao 13400 (66.4) |) 2) sh isetie \GoM) 2.60 DOA
LNA ESTENERE a: 8.05 0! ois e528 AG a! 9 SLO CAE CRA Ce ROE ne iatee A iciahe 2.54 2.33
AV eErAgCeMOnmmciviclialyviesselsis ci, cus Nek sb ek byt oe seme 2.53 2.39

IV 2 20 Dre 552-65) 10 0.67} 0.70 3.84

2 10 EQ 2 e224 20 Papen Wie A) O)5) 2.24

2 20 POG S225, TESSa eal DS2.
V 1 AQ | 2263 WEBS WO) 8.9 |(10.9) 2.49 2.08
(no Kjeldahl) | 1 LO LO GOL PAO PSs (PS?) 2.46 2.14
1 20 125-4 23.2) 10> se 12 saa Gisas) 1.99 1.69
VI 1 20 | 24.4 |(62.9)] 10 (24.3) 2.59
(ro Kjeldahl) | 1 10 | 10.6 |(29.0)| 20 (64.4) DD,
1 20 | 26.7 |(77.3) | 10 (Qi) 2.81

VII 1 20 1.48} 1.38] 10 0.49] 0.51 2.86

1 20 1F6O) 4-537 © 0.48} 0.51 3.16

1 20 1GO 1262) tO 0.54} 0.51 S407

VIII 1 20 1.03} 1.00} 10 0:33 |) 0:30 3.22

10 0.40} 0.42} 20 1.20] 0.92 2.59

1 20 1.09} 0.96; 10 0.55] 0.39 2.18

IX 1 20 eT 10) 0.42} 0.52 2.67

2D 10 PSO eA PAD 2.89 | 2.88 2-35

1 20 Led2 |) ew) Al@ 0.65} 0.67 2.67

x 1 20 PP eS al) 0.59} 0.33 2.56

1 20 tes Any ealle2S) esd 0 0.78| 0.41 2.20

1 20 1.41] 1.34] 10 0.95 | 0.39 2.05
PANNA Oa Net Shes BAN Hf a ese oA Lr RY oa ROM tA 2.63 + 0.06| 2.26
Averavenotandivaciall viesselsij.cis. 19s toe 64 gies eee 2-57 30105) 2-28
Average from previously published data.................... 2.30

RESPIRATORY RATE OF UNFERTILIZED EGGS Dia

not given because Urechis eggs do not divide at 5° and only occasion-
ally do so at 8°. By themselves, the experiments at these temperatures
cannot be taken to demonstrate a difference between the coefficients for
the unfertilized and fertilized eggs. They are, however, consistent with
the results on the other forms investigated in giving at the lower tem-
peratures somewhat lower values for the unfertilized eggs.

TABLE IV

Eggs of Strongylocentrotus. Same description as Table I.

Vessels A B G D

Experi- Tem- Tem-
ment Time] pera- | XO, XOg pera- | XO, XO_ Qio unfert.| Qio fert.

Number ture ture

hours} °C. ew SG: Rear

XI 1 18 |38.5] (48.2)| 8 16.0} (16.7) 2.41 2.88
(no Kjeldahl) | 1 | 8 |15.5| (19.5)| 18 |38.5| (48.6)| 2.48 | 2.50
(eS 40) 7055-3) 18) tG Sil CSE 246) «| 9.39

XII LS 2a ee (ONES) NS OFS)" (239) 2.61 2.56
(no Kjeldahl) 1 See OOS eS) el Semmi |Z | (Os-0) Desi 2-31
1 ;

PAW CAG Chaney MGs ear Wen Maral el ep Mae ls tecneyssge wiencaahab anes aiohaue & 2.58 2.69
PAVerAae Lom ANGiviGd Wal Vessels. os Oi ieli ss | Sanh eae oe oe ce Shoes 2.59 Dei

XIII ES AO COLO ere I ae
(no Kjeldahl) | 2 | 7.5 |18.3| (38.4)! 17.5 147.7 | (106.3) | 2.60 De
DN tes \SA oil d224)\" saeshl2ont

ESUSEDIECO RE 0 ae hel Rea Ne ire ee NTL en ean eg et RG Re 2.67 2.79

PV erase tor individual vesselsiac wise andes Melani eyed anee oe 2.69 2.80
QO1s O1s

XIV 2 320) 137-5 N@2to) |) SialitO-81| 128-0) |) G47 4.21
(no Kjeldahl) |-2 | 5 |12.0| (31.3)| 20 [44.6] (129.0)} 3.72 4.12
AN ES ee) SS) Aa OD) TE VGk eZee

PNVSRNEDA oiblesd 6s 8 yea Win he LRM Ces Mane tye Sees. Bin gk Ane eke 3.61 4.22
Averare ror indivacualiyessels. sas yaee ese ane een oe ale ae 3.74 4 24

It is of some interest to compare the absolute values for the rate of
oxygen consumption in different experiments. Considering only the
first period in each experiment, we see in Table I that the rate varies
from as low as 1.38 cu. mm. O, per hour per mg. N, as in Experiment
VI, to as high as 5.06 cu. mm. as in Experiment XI. The differences

272 TYLER AND HUMASON

are roughly correlated with the length of time that the animals were
kept in the aquaria before the eggs were used, eggs from freshly col-
lected animals giving higher absolute rates of respiration. Animals from
different localities vary, but considering, for example, Experiments VI
to XI in which the animals were collected at one time in one locality,
the times after collection for numbers XI, VIII, X, IX, VII and VI
are 14, 3, 4, 9, 10 and 40 days respectively, and the absolute rates of
respiration decrease in just about that order. Eggs from the same
animal were used in Experiments VII and XI. We shall not attempt
an explanation at this time, but we may point out that Urechis does not
store its eggs in the ovary (if there is a definitive ovary) but in “ neph-
ridial”’ sacs. Also, it is evident that simply aging the eggs in sea water
produces the reverse effect, namely a rise in the absolute rate.

With the fertilized eggs we find no marked differences in the ab-
solute rate of respiration. In addition to the figures of Table I we have
ten more values for the absolute rate of oxygen consumption during the
first hour after fertilization, all of which lie between 4.2 and 4.5 cu. mm.
We find thus in Urechis cases in which the rate of respiration rises two
or three-fold after fertilization, cases of no change in rate and cases
(e.g. Experiment X1) of a decrease in rate. Whitaker (1933) showed
that in different species the rate of respiration may rise, fall or remain
unchanged after fertilization, the absolute rates for the fertilized eggs
tending toward the same level'in all. Here we have one animal exhibit-
ing all three types of behavior.

Strongylocentrotus purpuratus

Tables III and IV give the respiration data and temperature coef-
ficients for fourteen experiments with eggs of Strongylocentrotus.
There are three at 22° and 12°, seven at 20° and 10°, two at 18° and
Sosone at l/c and 7.5°, and) one at) 20° andia 2a) tn) all ontnenisie
rate of oxygen consumption of the unfertilized eggs shows a rise during
the experiment. The shorter runs (e.g. Experiment III of Table IIT)
show a smaller rise than do the longer runs (e.g. Experiment IT).

In all of the experiments the temperature coefficients for the unferti-
lized eggs are fairly close to those for the fertilized eggs. For the
temperatures 22° and 12°, the difference is small. For the temperatures
20° and 10°, it is somewhat greater. But if we omit the high values
3.84 and 3.22 of Experiments IV and VIII, the average Q,,, by the
first method, is 2.27, which is much closer to the value for the fertilized
eggs. An examination of the corresponding respiration figures shows
that there is some justification for omitting these values, since, in both
these experiments, vessels C and D give oxygen consumption figures
that are evidently too low during the first period.

RESPIRATORY RATE OF UNFERTILIZED EGGS Daf)

TABLE V

Eggs of Ciona. Same description as Table I.

Vessels A B Cc D
Experi- Tem- Tem-
ment | Time] pera- XO_ XO» pera- XO, XO, Qio unfert. Oro fert.
No. ture ture
hours| °C. bay Gos
I 2 25 16.02 | 14.84 15 9.32 1.66
1 15 4.64 4.04 25 9.77 Dey)
1 25 8.71 8.28 15 4.76 1.79
I 2 DOA (2ONA2) 1 29236) US (S257) | (ALOT) 2.14
1 15 | ( 7.37)| ( 7.63)| 25 | (14.03) | (14.24) 1.88
1 25 ALO) 19:09) Sasi C esl) NG 7-07) 2.65
NG SARS. S S58 0 SHO MOOS CeO BER EERON GILEAD eo net eae ae 1.90 Dela
Averace ton individual vessels.);. 222... 2c won uh cee Oe ewan 1.92 2.14
Average from previously published data................... 2.27
-| hours
II lz || 22 il72 | Wileul 12 7.35 6.04 1.74
1 12 3.25 4.07 22 7.97 E22. 2.08
ily) 22 9.78 | 11.28 12 4.19 5.47 2.18
III 2 D2) 19.88 | 21.93 12 10.72 | 11.03 1.92
2 12 8.39 8.34 22 16.88 | 15.28 1.92
D DD 17.64 | 18.28 12 7.04 7.54 2.46
IV 1 22 7.43 6.30 12 4.06 3.40 1.84
$| 12 3.16 4.55 YD 8.89 8.28 2.23
1 22 6.29 5.06 12 4.68 4.86 1.19
San 200 et 2D AASO3" a tO 75) 10) (65.85 2.03
2 12 7.61 5.92 22 12.85 1.90
2 DY 13.48 | 12.30 12 5.86 2.20
V 2 22 | (22.50) | (22.39)| 12 | ( 9.64)| ( 9.89) DD)
2) 12 | ( 9.93)| ( 9.56)}| 22 | (20.94) | (20.41) DAD
2 22 | (25.00) | (25.47)| 12 | ( 8.84) ¢ 8.47) 2.92
VI 1 22 7.26 6.42 12 3.62 3.85 1.83
1 12 3.77 3.49 22 6.55 6.57 1.81
1 22 6.22 6.72 12 3.17 3.15 2.05
VI 1 22 |( 9.55)|( 9.30)| 12 | ( 4.16)| ( 3.67) 2.41
1 12 | ( 3.58)| ( 3.49)| 22 | ( 9.82)| ( 8.97) 2.66
1 22 | (10.90) | (10.04)} 12 | ( 4.08) | ( 3.85) 2.64
LESH ESREN SON etd SAMS 1 Oe Oty oc StL RO AS TA Me ee cea I 2 AG 1.96 + 0.05] 2.50
Average for individual vessels..................--+0+0-0-- 1.98 +0.06} 2.47
Average from previously published data................... 2.84

274 TYLER AND HUMASON

For the temperatures 18° and 8° and 17.5° and 7.5° there are also
no significant differences between the coefficients for the unfertilized
and the fertilized eggs, as the figures in Table IV show. One experi-
ment at 20° and 5° shows some difference which may be significant.
The unfertilized eggs give lower coefficients (Q,, in this case) than do
the fertilized, which is consistent with the results on Urechis at the
lower temperatures. The coefficients for cleavage which could be ob-
tained more accurately than those for respiratory rate, are not given
here because the eggs do not divide at 5° C.

TABLE VI

Eggs of Dendraster. Same description as Table I.

Vessels A B C D
Experi- Tem- Tem-
ment | Time| pera- XO. XO, pera- XO, XOo Qio (unfert.) | Quo (fert.)
No. ture ture
hours| °C. Es
I 2 22 6.04 5.98 7 2.19 2.30 2.68
2 12 3.46 3.28 DY 7.73 8.00 2.34
2 22 11.08 | 11.02 1 3.96 4.08 Dele
II 2 22 4.40 4.05 12 1.96 2.08 2.09
2 iD DD 2.31 D2, 6.10 6.19 2.44
2 2D 10.80 9.51 12 3.39 4.17 2.69
Ill 2 22 7.46 U5) 12 3.11 3.07 2.40
2 22 10.59 | 10.13 12 3.55 3.98 Dells)
2 DD, 12.56 | 11.82 12 4.26 4.66 2.74
IV 2 DY 7.83 12 3.01 2.60
2 22 12.58 12 4,29 2.93
PENNIES 5 1'e ti ests ERD Bah Ro ERR RU era Skeets te Mare on, 2.57 + 0.04
Aver ce nonindividialevessels. j\0u. 2.00... 22) oe eee 2.71
Average from previously published data................... 2.80

In the experiments of Tables III and IV marked “no Kjeldahl”
the quantities of eggs employed were not determined, so the figures in
these experiments cannot be compared with the absolute rates in the
others. Also we cannot compare unfertilized and fertilized rates in
those experiments, since the suspensions of unfertilized and of fertilized
eggs were not of the same concentration. In the other experiments,
if we compare the absolute rates of respiration for the unfertilized eggs
during the first period of a run, we see no such differences as were ob-
tained with Urechis. Here, the eggs used came from freshly collected

RESPIRATORY RATE OF UNFERTILIZED EGGS 27s

animals. Whether keeping Strongylocentrotus in an aquarium tank
would affect the rate of respiration of the unfertilized eggs was not
determined.

Ciona intestinalis

Certain difficulties in handling Ciona eggs were previously (1936)
pointed out. In addition it may be noted that to secure large quantities
of unfertilized eggs it is necessary to use many individuals and to wash
the eggs thoroughly before mixing them, in order to remove the sperm
that almost unavoidably comes out with the eggs. Since Ciona eggs
tend to float rather than sink to the bottom of the dish, it generally
takes about ten or more washings to remove the sperm.

Six experiments were run with eggs of Ciona, one at 25° and 15°
and five at 22° and 12°. The results are given in Table V. At the
temperatures 25° and 15° there are no significant differences between
the O,,'s for the fertilized and the unfertilized eggs. In this experi-
ment the vessels 4, B and C were removed at the end of the unferti-
lized run, the eggs inseminated, and the measurements continued on
the fertilized eggs. In addition a fourth vessel, D, of freshly insem-
inated eggs was added. Fertilization was 100 per cent in all. At the
temperatures 22° and 12° the average Q,,’s are somewhat less for the
unfertilized than for the fertilized eggs. Considering the magnitude
of the difference in relation to the probable error and the fact that it
agrees with Ureciis and Strongylocentrotus in giving at the lower tem-
peratures * lower coefficients for the unfertilized eggs, we are inclined
to regard it as significant.

Comparison of the oxygen consumption figures for the unfertilized
and fertilized eggs shows at most a less than two-fold rise upon ferti-
lization. This is much lower than in the case of the sea-urchin egg.
The rise is more of the order of that found with Nereis eggs (Whitaker,
LOS: Barrom, 1932).

Dendraster excentricus

Four sets of experiments were done on Dendraster eggs, all of them
dtiezZ and I22nC, Nhe =results are presented ine Vable Wie “nenone
were large enough quantities of eggs obtained to get usable oxygen con-
sumption values in less than two hours at each temperature. Dendraster
shows a fairly rapid rise with time in the rate of respiration of the un-
fertilized egg. The rate rises even more rapidly than in the case of
Strongylocentrotus as Fig. 1 illustrates. In the last two hours of a six-
hour run we may have more than twice the oxygen consumption ob-

3 Ciona eggs are adapted to a higher temperature range than the others; they
fail to divide below 12° and above 26° C.

276 TYLER AND HUMASON

tained in the first two hours, as in Experiment II. The temperature
coefficients, however, are not particularly affected by the rise. This
means simply that the relative rise is about the same at different tem-
peratures.

The average Q,,’s for the unfertilized eggs by the two methods of
calculation are 2.57 and 2.71 respectively. These are slightly lower
than the previously determined value of 2.80 for fertilized eggs. The

Acie. Wall

Average temperature coefficients (Qio) for unfertilized and fertilized eggs. Probable
errors given where sample consists of 15 or more values. Coefficients under A are
determined from the respiration of eggs in different vessels run simultaneously at
different temperatures. Coefficients under B are determined from the respiration of
eggs in one vessel run consecutively at different temperatures. Coefficients under C
taken from previously published data.

Unfertilized eggs Fertilized eggs
Tempera-
tures No. of No. of
4 ves- A B ves- A B C
sels sels
Strongylocen- | 22° and 12° 8 2.54 2.53 4 | 2.33 | 2.39
trotus 20° and 10° 23 |2.63 + 0.06/2.57 + 0.05) 4 2.26 | 2.23 | 2.30
18° and 8° 4 2.58 2.59 4 2.69 | 2.57
17.5° and 7.5° 2 2.67 2.69 2 2.79 | 2.80
20° and 5° y) 3.61F 3.747 D 4.224) 4.24+
Urechis 22° and 12° 34 |2.57 + 0.03/2.57 + 0.04) 6 2.64 | 2.65 | 2.79
22° and 12° 12* 2.70 DT
20° and 10° 10 2.46 2.47 2 2.92 | 2.82 | 3.30
18° and 8° 8 2.18 1.94
15° gyavel 52 2 2.96 2.84 2 3.33 | 3.30
Ciona 25° and 15° 3 1.90 1.92 4 DDN BAlah |) DLA

22° and 12° | 19 |1.96 +0.05/1.98 + 0.06] 8 | 2.50 | 2.47 | 2.84

Dendraster 22° and 12° 14 |2.58 + 0.04 Dial 2.80

* Selected experiments, included in line above.

t Qis values.
difference, however, cannot be taken as significant. The two average
values for the unfertilized eggs differ more here than in the previous
cases. That is because in two of the experiments (III and IV) coef-
ficients could not be determined by the second method (from individual
vessels) since the vessels remained at one temperature throughout the
run.

DISCUSSION

The average values of the temperature coefficients for the eggs of the
four animals investigated are listed in Table VII. As was pointed out

RESPIRATORY RATE OF UNFERTILIZED EGGS DT

in considering the individual cases, there are no large differences be-
tween the unfertilized and fertilized eggs. At the higher temperatures
in each case, there are certainly no significant differences. At the lower
temperatures, the consistently lower values for the unfertilized eggs of
the different animals incline us to regard the difference as significant.
With fertilized eggs, or for that matter most biological material (see
Belehradek’s review), Q,, increases as the temperature interval is low-
ered. Here, it appears that for the respiration of the unfertilized eggs,
Q,,) remains a constant or increases only slightly at lower temperatures.
Thus, with Strongylocentrotus we get the values 2.54 at 22° and 12°
aude aroonatliZe sande 7.50. Nath Omeehis) the @ 5s ane: 2.57 at 22°
and 12° and 2.90 at 15° and 5°. We are not, however, particularly
concerned here with the constancy of Q,,. Any other convenient meas-
ure of variation in rate with temperature would serve for comparing
unfertilized and fertilized egg respiration.

We had expected to find the same values for unfertilized as for ferti-
lized eggs. At the higher temperatures that appears to be the case.
But if we accept the divergence at the lower temperatures as significant,
then it would seem that one of the assumptions, upon which this expecta-
tion was based, must be wrong. This might well be the assumption that
an unfertilized egg is a resting cell exhibiting only maintenance. Other
processes besides what we term maintenance may be involved. It would
seem important then to determine with certainty whether real differences
exist at the lower temperatures. We do not, however, consider it likely
that with the present material and methods simply expanding the ex-
periments will improve the data very much. Besides, there now appear
to be other ways of getting at the questions involved.

The unfertilized eggs of all the forms investigated exhibit a rising
rate of respiration. In Strongylocentrotus and Dendraster the rate rises
much more rapidly than in Urechis and Ciona (see Fig. 1), the differ-
ence being correlated with the time of loss of fertilizability on the part
of the eggs. Runnstrom (1928, footnote p. 4) has likewise noted that
the sea-urchin eggs lose their capacity for fertilization after exhibiting
a spontaneous rise in respiration. There are several agents that have
been reported to prolong the fertilizable life of the egg; namely, cyanide
(Loeb, 1912) thyroxin (Carter, 1931), alcohol and dextrose (Whitaker,
1937). It would be of interest to know whether these agents would
prevent the rise in respiratory rate of the aging unfertilized egg. Under
such conditions it is quite possible that different values for the tempera-
ture coefficients would be obtained.

4 Cyanide and CO suppress this rise according to Runnstrom (1930), but the
concomitant fertilization test is not given.

278 TYLER AND HUMASON

Rubenstein and Gerard (1934) reported in Arbacia average values
of 4.1 and 1.8 for the QO,,’s of unfertilized and fertilized eggs respec-
tively. They point out, then, that as the temperature is increased the
rise in respiration that occurs upon fertilization in the sea-urchin egg
diminishes. By extrapolation they show that at 32° C. there would be
no rise. In the sea-urchin, Strongylocentrotus, that we have used, as
well as in the three other forms, no such differences in the coefficients
are evident. At the lower temperatures, there are possibly significant
differences, but in the reverse direction from what the above investigators
find. However, if it is assumed that Q,, for the fertilized eggs de-
creases as the temperature rises, while O,, for the unfertilized eggs re-
mains constant, the unfertilized eggs would presently give the higher
values. By extrapolation, then, if there is any point to it, we would find
that the temperature, at which there would be no rise in respiration upon
fertilization in Strongylocentrotus, approaches that of boiling sea water.

SUMMARY

1. The effect of temperature on the rate of oxygen consumption of
unfertilized and fertilized eggs of Urechis, Strongylocentrotus, Ciona
and Dendraster was investigated.

2. The unfertilized eggs exhibit a rising rate of respiration with time
in all four species. The rise is much more rapid in Strongylocentrotus
and in Dendraster than in Urechis and in Ciona. ‘This rise appears to
be correlated with the loss of fertilizability on the part of the eggs.

3. Methods of determining the temperature coefficients in such a
way as to take into account the general rise (which is a significant factor
in prolonged runs) and other variations are described.

4. Only the temperature coefficients for the same temperature in-
tervals are compared, the respiration being determined at two tempera-
tures in each experiment. With Urechis and with Strongylocentrotus
eggs, experiments were run at temperatures between 22° and 5°; with
Ciona, between 25° and 12°; and with Dendraster, 22° and 12°. The
experiments with Urechis and Strongylocentrotus thus included low
temperatures at which the fertilized eggs fail to cleave.

5. No significant differences between the temperature coefficients of
the respiratory rate of unfertilized and of fertilized eggs of the four
animals investigated are found over most of the temperature range in
which development is possible. At the lower temperatures, there are
differences that are possibly significant, the unfertilized eggs giving con-
sistently lower values.

6. Comparison of the absolute rates of respiration of the unfertilized
and fertilized eggs shows in Strongylocentrotus and Dendraster the rise

RESPIRATORY RATE OF UNFERTILIZED EGGS 279

in respiration upon fertilization typical of the echinoids; in Ciona a less
than two-fold rise is manifest; in Urechis, the rate may rise consider-
ably, remain constant or decrease slightly, depending upon the particular
batch of eggs employed. Eggs from animals kept some time in cap-
tivity give lower unfertilized rates and manifest a rise upon fertilization ;
eggs from freshly collected animals give higher unfertilized rates and
no rise or even a slight decrease upon fertilization.

IIMA IN UNees, (CMU SB)

Barron, E. S. G., 1932. Studies on cell metabolism. I. The oxygen consumption
of Nereis eggs before and after fertilization. Biol. Bull., 62: 42.
BELEHRADEK, J., 1935. Temperature and living matter. Protoplasma Mono-
graphien, vol. 8. ;

Carter, G. S., 1931. Iodine compounds and fertilization. III. The fertilizable
life of the eggs of Echinus esculentus and Echinus miliaris. Jour. Exper.
Biol., 8: 194.

Logs, J., 1912. The Mechanistic Conception of Life. University of Chicago Press.

RuBENSTEIN, B. B., anp R. W. Gerarp, 1934. Fertilization and the temperature
coefficients of oxygen consumption in eggs of Arbacia punctulata. Jour.
Gen. Physiol., 17: 677.

RunwNstroM, J., 1928. Die Oxydationserhohung bei der Entwicklungserregung
des Seeigeleies. Arkiv. Zool., 20 (Art. 3): 1.

Runnstrom, J., 1930. Atmungsmechanismus und Entwicklungserregung bei dem
Seeigelei. Protoplasma, 10: 106.

Tyzer, A., 1936. On the energetics of differentiation. IV. Comparison of the
rates of oxygen consumption and of development at different temperatures
of eggs of some marine animals. Bziol. Bull., 71: 82.

Warsure, O., 1915. Notizen zur Entwicklungsphysiologie des Seeigeleies. P/flii-
gers Arch., 160: 324.

Wuitaker, D. M., 1931. On the rate of oxygen consumption by fertilized and
unfertilized eggs. III. Nereis limbata. Jour. Gen. Physiol., 15: 191.

Wuirtaker, D. M., 1933. On the rate of oxygen consumption by fertilized and
unfertilized eggs. V. Comparison and interpretation. Jour. Gen. Physiol.,
16: 497.

Wuitaxker, D. M., 1937. Extension of the fertilizable life of unfertilized Urechis
eggs by alcohol and by dextrose. Jour. Exper. Zod6l., 75: 155.

SEXUAL AND ASEXUAL REPRODUCTION IN
EUPLANARIA TIGRINA (GIRARD)

ROMAN KENK

(From the Miller School of Biology, University of Virginia, and the
Department of Zoélogy, University of Ljubljana, Yugoslavia)

Several authors have reported on the reproduction of Euplanaria
tigrina (Synon. Planaria maculata Leidy), one of the commonest fresh-
water planarians of North America. It has been known for a long
time that this species often reproduces by fission. Curtis (1902),
in his valuable paper on the life cycle of this form, also states that, at
least in several localities, the animals grow sexually mature and
deposit egg capsules in the early summer. Much material on the
processes of fission has been gathered by experimental workers,
particularly by Child and his co-workers. From these we know that
the rate of fission may be controlled by external factors, such as temper-
ature, the amount of food, and chemical properties of the water.

Nevertheless, the relations between the two manners of reproduc-
tion, the sexual and the asexual one, are as yet little known. The
only extensive observations with regard to this question were under-
taken by Curtis on material from the vicinity of Woods Hole, Massa-
chusetts. This author investigated the life history of the species in
four different localities through the course of three years and sum-
marized his results as follows: “‘In some localities the species seems to
have reproduced exclusively, so far as the observations go, by fission,
in others only by the sexual process, while there are still others where
both processes occur at different seasons”’ (1902, p. 556). In a later
communication (Curtis and Schulze, 1924, p. 105) he writes: “It may
also be noted that the differing habits of reproduction, originally
reported . .. for the P. maculata in four different localities near
Woods Hole, Massachusetts, have been confirmed by all our subsequent
collecting in these localities.”’

These differences in the manner of reproduction in certain places
suggested two possible interpretations: (1) there might be some differ-
ences in the physical and/or chemical characteristics of these localities,
which influence the life cycles of the animals living there; or (2) there
might be more than one physiological race of Euplanaria tigrina,
showing different habits of reproduction. If the latter be the case,

280

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 281

the manner of reproduction would be determined, chiefly, by internal
factors.

It is the purpose of this paper to report on a series of experiments
performed to analyze several factors that might control these processes.
Most of the work has been carried out at the Miller School of Biology,
University of Virginia. I wish to express my indebtedness to the
Rockefeller Foundation which made this investigation possible, as
well as my sincere gratitude to Professor William A. Kepner and
Professor Ivey F. Lewis for the privileges extended to me during my
stay at the University of Virginia.

MATERIAL AND METHODS

The animals used in the experiments were collected in four locali-
ties: (1) Sinclair’s Pond, Park Street, Charlottesville, Virginia; (2)
Big Spring, near Kerr’s Creek, Rockbridge County, Virginia; (3)
Mary’s Lake, Naushon Island, Massachusetts; and (4) Pond south of
Main Street, behind the Episcopal Church, Falmouth, Massachusetts.

In the experiments on asexual reproduction, the influence of
temperature, hydrogen-ion concentration, amount of food, and ir-
radiation with ultra-violet rays upon the rate of fission were in-
vestigated. The cultures were run in tap water.

Temperature experiments were performed in three series of cul-
tures: at indoor temperature, low and high temperatures. The low
temperature cultures were kept in electric, thermostatically controlled
refrigerators, while high temperatures were obtained in simple, electri-
cally heated ovens.

The experiments on the influence of the hydrogen-ion concentration
in the culture water were carried out at indoor temperature. In
order to keep the pH constant, small quantities (1:20) of buffer
mixtures were added to the water. The buffers used were mixtures
of KH,PO, and NaOH having a pH of 6.4, 7.0, and 7.6, prepared after
Clark’s formule. Their addition to the tap water kept the media at
a fairly constant acidity of respectively 6.5, 7.0, and 7.5.

The animals were fed with beef liver at regular intervals; it has
been proved by various workers that cultures of fresh-water planarians
may be run for years on this food. The liver was freely taken.

Ultra-violet irradiation was carried out by means of a Hanovia
mercury-arc lamp kindly placed at the writer’s disposal by the Com-
mittee on Effects of Radiation, National Research Council. These
experiments did not yield any results bearing upon the process of re-
production; a more detailed description of the technique employed
may, therefore, be omitted.

282 ROMAN KENK

EXPERIMENTS ON ASEXUAL REPRODUCTION

For these experiments animals from Sinclair’s Pond and from Big
Spring were used. Each experimental series comprised at least three
cultures containing, at the beginning, 5 to 10 specimens each. When
fissions occurred, the posterior pieces were eliminated from the cul-
tures. It is well known that the two pieces derived from fission, the
head and the tail piece, behave differently in the regeneration of the
missing parts. The head piece does not undergo any considerable
change, its prepharyngeal part remains almost the same size, while the
posterior end appears to grow out of it; the head piece, therefore,
regenerates chiefly by epimorphosis. The tail piece, however, shows
decided morphallaxis, i.e. it rearranges its proportions and a large
part of the old tissues is directly transformed into parts of the missing
prepharyngeal region (cf. Curtis, 1902, p. 529). Moreover, in a short
time, before the regeneration is completed, the tail piece may redivide
spontaneously. In head pieces, the intervals between two fissions are
more regular. We may, with the necessary caution, take these inter-
vals as indicative of the rate of reproduction. We must, however,
realize that an absolute regularity in fission cannot be assumed.
Although we select specimens of the same origin, the same age, the same
size, and keep them in the same aquarium, yet a great variety of fission
intervals will be observed. This is obvious in view of the fact that we
cannot control all the factors concerned in the induction of
fission. Among these, the nutrition of the individual specimens and
their locomotory activity play important réles. To eliminate the
fluctuations of the fission intervals as much as possible, a sufficient
number of specimens should be used for each experimental series and
the cultures maintained for a sufficiently long time.

In order to express the frequency of fission in a convenient way,
two data were calculated for each experimental series: the average
interval between two consecutive fissions, and the average fission rate.
The latter is the number of fissions per day, calculated for a lot of 100
specimens. It is a disagreeable fact that, in cultures running for a
long period of time, the numbers of specimens can hardly be main-
tained constant, despite careful handling of the animals. The animals
are apt to crawl up the wet walls of the culture dishes and then dry up
if not noticed in time. Occasionally, individual specimens are lost
while one is feeding and changing water. Therefore, the fission inter-
vals and the fission rates were determined for shorter periods (usually
10 days) and from these data the average intervals and rates cal-
culated for the entire length of observation.

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 283

Temperature Experiments

The indoor temperature varied considerably during the course of
the experiments. In general, it fluctuated between 20 and 30° C.,
occasionally, for a short time, dropping below these limits (minimum
13.8° C., March 7, 1932) or rising above them (maximum 32.6° C.,
August 8, 1932). The results obtained in parallel cultures at indoor
temperature and in the refrigerator are shown in Table I. At low
temperatures (10—12° C.) the processes of fission were greatly inhibited
or even entirely suppressed for several months. In a culture of 20
specimens (the number decreased, during the experiment, to 18)
running 330 days, only five fissions occurred, the first of them on the
267thday. This shows that at least 13 specimens for almost 11 months

TABLE I

Low-temperature experiments. Average fission rate and fission interval in
cultures kept in the refrigerator (10—-12° C.) compared with those of control cultures
at indoor temperature.

Number of
specimens Duration Average Average
Origin of material Temperature | of experi- fission fission
ce ee ment rate interval
Initial | Final
a. days days
Sinclair’s Pond......... 20 18 | Refrigerator] 330 .084 1189
Sinclair’s Pond......... 15 4 | Indoor 341 5.9 16.9
IBIOP SPRING a) Wis a oike es oi 18 18 | Refrigerator] 329 all5 658
PISO PLUG ys ciate erie ss 18 2 | Indoor 329 UP 13.9
Biker Syoyinlers 4.4 sede oe ae 15 14 | Refrigerator} 174 041 2436
Bree Spring x22... < -ais'a.e\s 15 11 | Indoor 174 6.0 16.7

did not display fission at all. The animals were well fed, so the de-
crease of the fission rate could not be due to starvation. On the con-
trary, in the culture referred to, the length of the animals was from
5 to 6 mm. at the beginning of the experiment, while at the end lengths
of 16 to 20 mm. were measured. The control animals, raised at indoor
temperature, generally remained smaller, since they divided before
attaining the size of the refrigerator specimens. Another factor that
might be suspected of preventing fission is the decreased locomotory
activity in the refrigerator culture. The stimulating effect of light
was eliminated in both series of cultures by keeping the aquaria in the
dark. There is, however, a decrease of the activity caused directly
by the low temperature. Nevertheless, since the animals in the re-
frigerator were often seen to move about in the aquaria, this difference

284. ROMAN KENK

can hardly be responsible for the extreme rarity of divisions at low
temperature.

Several cultures were run at high temperature which varied be-
tween 29 and 34.5° C., in the main amounting to about 32°C. They
showed a significant change in fission frequency during the course of
the experiment (Table II). At the beginning, the fission rates were

TABLE II

High-temperature experiments. Fission rate in cultures kept in the thermostat
(about 32° C.), compared with those in control cultures at indoor temperature.

Number of Fission rate
specimens
Origin of material Temperature
Initial | Final 1st-—18th day | 19th—36th day} 37th—55th day
Big Spring..... 14 8 | Thermost. 4.8 o2.8 81
Big Spring..... 2 11 Indoor 4.2 2.3 4.3

similar to those of the control cultures at indoor temperature. Soon
the divisions became rarer and finally, after 43 days, no fissions
occurred any more. Parailel with this decline in reproduction was
a reduction of the size of the animals. The lengths of the specimens
decreased from 7-13 mm. at the beginning to 3-5 mm. when the
experiment was discontinued (after 55 days).

Unfortunately, I had no opportunity to determine exactly the
optimum temperature for asexual reproduction. Nevertheless, ob-
servations on cultures at indoor temperature indicated that fissions
were most frequent when the water temperature was about 25° C.
or a little over.

Hydrogen-ion concentration

The observation of fissions in media of different acidity comprised
only a small range of pH, viz. 6.5, 7.0, and 7.5. The addition of buffer
mixtures to the culture water had no injurious effects on the animals.
The reproduction continued in a normal way. No significant differ-
ences in the fission rates were observed in cultures of different con-
trolled acidity, nor between them and the cultures in tap water (Table
III). It may be noted, however, that the range of pH used was
relatively narrow. It is known that planarians tolerate a considerable
variation of acidity, towards the acid as well as towards the alka-
line side.

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 285

TABLE III

pH experiments. Average fission rate and fission interval in buffered culture media
and in tap water.

Number of
specimens Duration Average Average
Origin of material pH of experi- fission fission
Pegnren Pee Tok ment rate interval
Initial | Final

days days
Bigg Spree taste eces-.- 15 11 6.5 168 8.2 12.3
[Biter Syormtnes ce oocaceaaas 15 13 7.0 168 6.3 15.9
BICC OPRING. | fe esse eee 15 10 US 174 7.4 13.5
IBICES PRINS acon his ses ss tS 11 tap w. 174 6.0 16.7

Irradiation with Ultra- Violet Light
A number of specimens were exposed to the light of a quartz-
mercury lamp on several consecutive days. During irradiation, the
animals were kept in shallow dishes with little water. The animals so
treated showed a slight decrease of the fission rate (Table IV). On

TABLE IV

Fission rates and fission intervals in cultures exposed to different doses of ultra-
violet radiation and in control cultures not irradiated.

Number of
specimens Duration Average Average
Origin of material Dose of experi- fission fission
Eno ment rate interval
Initial | Final
days days
Big Spring..... 15 7 Single 174 4.4 22.8
Big Spring..... 15 2. Double 174 4.6 Pill
Big Spring..... 15 11 —— 174 6.0 16.7

the other hand, the irradiation apparently exerted a general injurious
effect on the animals, which resulted in a high mortality in these
cultures. I am, therefore, inclined to attribute the decrease of the
fission frequency to the weakening of the animals rather than to a
specific effect of the ultra-violet irradiation.

Starvation

It is well known that planarians can stand long periods of starvation
very well. In prolonged starvation they grow smaller and, to a certain
extent, simplify their anatomical structure. Their physiological
condition becomes that of young animals. It has been proved re-

286 ROMAN KENK

peatedly that starvation causes asexual (as well as sexual) reproduction
to cease. Thisisshown very clearlyin Table V. For our experiments,
well-fed specimens of a length of 12 to 15 mm. were taken. During
the first week of starvation, fissions took place at an almost normal
rate; later on, the frequency of division decreased rapidly. The last
fission occurred on the twenty-second day after the starting of the
experiment. On the sixty-fifth day the culture was discontinued.
The size of the animals had decreased to 344-6 mm.

I wish to point out that no traces of sexual reproduction or maturity
were ever observed in any of the cultures of Euplanaria tigrina from

TABLE V

Fission rates in cultures starving and in control cultures well fed.

Number of ele
specimens Fission rate
Origin of oe
astavial Nutrition -
te . 1st—10th 10th—20th 20th—30th 30th-65th
Initial | Final day day dace aay
Big Spring | 30 21 | Starving Os 2.4 a3 .0
Big Spring| 30 | 26 | Fed 12.0* 4.0 4.3 5.1

* The high fission rates during the first days were due to the fact that fully grown
specimens had been selected for the experiment.

Sinclair’s Pond and Big Spring, either in the stock aquaria or among the
specimens subjected to various external conditions.

EXPERIMENTS ON SEXUAL REPRODUCTION

Since sexuality could not be induced in material from two localities
in Virginia, animals were procured from those places where Curtis
(1902) had made his observations on the reproduction of the species.
In July, 1932, asexual animals were collected in Mary’s Lake; at the
same time specimens brought in from the Pond in Falmouth proved
to be sexually mature and laying egg capsules. Animals from these
localities were kept in separate culture dishes, but under external condi-
tions as identical as possible. They were raised at indoor temperature
and fed on beef liver.

As a result, the two lots retained, in the main, their characteristic
manners of reproduction also in the laboratory. The animals from the
asexual locality continued to undergo fission. There were no decided
seasonal cycles of reproduction observed, apparently because of the

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 287

relatively favorable temperature all the year round. No sex organs
developed in the course of 5 years.!

The animals from the sexual locality continued to propagate sexu-
ally in more or less regular periodical cycles. Though a definite coin-
cidence of the periods of reproductive activity in the laboratory cul-
tures with those observed in the field need not be expected, neverthe-
less the periodicity conformed, in the main, with Curtis’ records of the
life history in the natural habitat.

The duration of the egg-laying periods in indoor cultures varied
within wide limits. Usually the animals started depositing the first
egg capsules in the late fall or in winter (middle of November to
February). The breeding-reached its height in March and April, then
declined, and generally ceased in June. Only once did I observe, in
one culture, a breeding season extending from July till the beginning
of September. Each specimen deposited several cocoons during one
season. The size of the body gradually decreased from about 18-24
mm. to 13-15 mm. After the breeding season was over, the animals
were rather sensitive, as if exhausted. The rate of mortality seemed
to be higher at that time in the cultures. Nevertheless, most of the
animals survived and recovered completely, if well attended to, and
in the next egg-laying season again proceeded to produce cocoons.
The individual lifetime of this species may be estimated at several
years. Curtis’ data on the degeneration of the sex organs between
two seasons of sexual activity were confirmed.

Temperature Experiments

Like the fission of the asexual form, the rate of breeding in the
sexual form is influenced by the temperature of the medium. This is
clearly shown in Table VI. At low temperature the laying of cocoons
proceeded very slowly; it was almost inhibited; at high temperature
the breeding rate increased. On the other hand, the breeding season
lasted much longer at low temperatures than at high ones. In the
refrigerator culture referred to in Table VI, maintained at 10° C.,
single egg capsules were deposited at long intervals and the animals
remained in the sexual state for at least one year.

1One single seeming exception was noticed when, in March, 1933, one sexual
specimen appeared in the asexual culture and laid two cocoons. This specimen was
subsequently eliminated from the aquarium. Since then and up to now (May, 1937)
no second case of sexuality has occurred. I am inclined to assume that this one and
only mature animal had, by careless handling, been transferred to this vessel from
the sexual culture while the dishes were being cleaned. Even though this assumption
were not correct, the occurrence of one sexual individual among many hundreds of

asexual animals would not be significant, and could not essentially affect the result
of the experiment.

288 ROMAN KENK

Besides reproduction by cocoons, the animals from the sexual
locality also showed asexual reproduction by fission. Fission did not
occur in all animals of the cultures and was confined to a short season
of the year, from June to August. As this season followed the season
of egg-laying, fissions were usually observed in animals which had
previously deposited egg capsules. Young animals, hatched from
cocoons in the preceding spring, likewise occasionally divided asexually,
provided they had already reached a sufficient size and had not yet de-
veloped sex organs.

From the fact that the fissioning season coincides with the warmest
season of the year, we may conclude that fission requires a high temper-
ature. Temperature, however, is not the sole decisive factor: the
animals must, at the same time, be in the state of sexual inactivity.
If they are subjected to high temperature during the breeding season,
they continue to deposit cocoons and do not divide.

TABLE VI

Temperature experiments on material from the pond at Falmouth. Daily breeding
rate (calculated for 100 specimens) and breeding interval in cultures kept at indoor
temperature, compared with those kept at lower and higher temperatures.

Number Duration Number Average Average
Temperature of of of breeding breeding
specimens | observation cocoons rate interval
days days
Indoor (about 20° C.).. 20 31 29 4.68 21.4
Refrigerator (about 10° C. yes 19 147 10 36 279.3
Thermostat (about 28.6° C.) 9 35 98 31.1 3.21

Two tail-pieces of specimens which had fissioned after they had
stopped laying egg capsules were studied anatomically. In both of
them remainders of the copulatory organs were found, but in a state of
apparent degeneration. The genital pore, parts of the atrial cavity,
and the penis could still be identified, though their structure differed
from that of the organs in function. Bursa and bursa stalk had
entirely disappeared. In various places in the parenchyma of the
genital region there were patches of brown substance, apparently the
product of disintegration of tissues. In short, the pieces exhibited
that typical picture of degeneration of sex organs which had been
described by Curtis (1902, pp. 546-550) as occurring after the egg-
laying.

I should like to emphasize again that fission, in the laboratory
cultures, did not occur in the case of every individual specimen. The
animals that did undergo fission usually divided only once. After

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 289

the tail-piece had grown to a certain size, it occasionally divided a
second time if the conditions were still favorable. In any case,
fission plays only an insignificant réle in the life history of the planarian
from the pond at Falmouth.

The tendency to divide can be easily controlled by external factors,
particularly those that influence the fission rate in the asexual form
from Big Spring and Sinclair’s Pond, i.e., temperature and nutrition.
The following experimental series may illustrate this statement:

April 27, 1936, three cultures of 20 specimens each were prepared.
The animals measured 18 to 24 mm. in length and were at the height of
the breeding period.

Culture 1. Kept at indoor temperature, fed twice a week with
beef liver. Egg-laying ceased June 12, fissioning started June 19 and
lasted until August 3. Thirteen animals had divided, 7 remained
undivided. These, measured September 21, were from 13 to 15 mm.
in length. They entered a new breeding season on March 8, 1937.

Culture 2. Kept at indoor temperature, not fed. The last egg
capsule was deposited on May 11; fissions occurred between June 30
and July 22. Only 3 specimens had fissioned, 15 were undivided, 2
had died. September 21, the animals measured 5 to9 mm. From
that time on they were regularly fed. The new breeding period started
February 18.

Culture 3. Kept in a refrigerator at 10° C. Cocoons were laid at
long intervals,—only 10 cocoons up to September 21, when the animals
measured 15 to 21 mm. The egg-laying continued.

It is interesting to note that sexually active animals which had
been kept at low temperature for a long time showed an extraordinary
increase of the breeding rate when transferred to indoor temperature.
This appeared in the continuation of the refrigerator experiment re-
ferred to in Table VI. Ten specimens which had been exposed to a
temperature of 10° C. for 147 days had shown an average breeding rate
of .36 (breeding interval of 279.3 days). Transferred to indoor temper-
ature, they laid during the course of two months no less than 65
cocoons. This corresponds to a daily rate of 10.5 cocoons per hundred
specimens or to a breeding interval of 9.5 days. Animals constantly
kept at indoor temperature propagate at a much slower rate (see
Table VI). From this experiment it would appear that low tempera-
tures, while retarding the deposition of egg capsules at the time, do not
inhibit the maturing of the germ cells to the same extent. Brought
into normal temperature conditions the animals react to the accumula-
tion of egg cells (and yolk material) in the body by an increased
breeding activity.

290 ROMAN KENK

DISCUSSION AND CONCLUSIONS

From the foregoing paragraphs it is evident that there must be at
least two physiologically different races of Euplanaria tigrina. They
differ, mainly, in their respective habits of reproduction. We may
call them, shortly, the ‘‘sexual” and the ‘‘asexual’”’ race. The differ-
ent life cycles of this species in different localities near Woods Hole,
Massachusetts, reported first by Curtis (1902 and 1924), are due
chiefly to differences inherent in the animals themselves, not to physico-
chemical properties of the environment. The asexual race lives in
Sinclair’s Pond, Big Spring, and Mary’s Lake; the sexual one in the
pond at Falmouth (see p. 281).

Asexual reproduction has been observed in a relatively large number
of fresh-water triclads. An excellent review of the material gathered
has been given by Vandel (1921, pp. 370-374), who also assumed the
existence of different races of Euplanaria tigrina, in which the manners
of reproduction were hereditarily different. Few observations, how-
ever, have so far been made on the entire life cycles of fissioning
planarians and little has been done to investigate the processes of
reproduction in an experimental way. Nevertheless, it is a striking
fact that in almost all species subjected to closer examination, differ-
ences in the incidence of sexual and asexual reproduction have been
revealed in different localities (cf. Benazzi, 1936, p. 364). We know
such physiological races in all European species of fissioning planarians:
Polycelis felina (Dalyell) (Vandel, 1921, pp. 478-479; Thienemann,
1926, pp. 298-300), Crenobia alpina (Dana) (Vandel, 1921, pp. 478-
479), Euplanaria gonocephala (Dugés) (Vandel, 1925; Benazzi, 1936),
and Fonticola vitta (Dugés) (Beauchamp, 1932, pp. 285-294). Among
the American species, sexual and asexual forms are known in Eu-
planaria dorotocephala (Woodworth) (Kenk, 19356, p. 451). The
same phenomenon is now confirmed in Euplanaria tigrina.

There is no reason to distinguish these races as separate taxonomic
units, e.g. species or subspecies. Small morphological differences
between them may occur, particularly in the shade of coloration, the
size, and the proportions of the body; they are, however, of little tax-
onomic value. Some of these characteristics, such as the size and
shape of the animals, may be directly correlated with the mode of
reproduction.

The reproduction of the asexual form of Euplanaria tigrina is
exclusively asexual. In this the field investigations of Curtis and the
experiments described in this paper agree. External factors may
accelerate the processes of propagation, retard, or even inhibit them,
but they cannot change the process to a sexual one. The main factors

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 291

influencing the rate of fission are temperature and nutrition. At low
temperatures division is suppressed, it takes place only at about 10° C.
or more, provided the animals have attained a certain minimum size.
The interval between two fissions is shorter, the higher the temperature.
During this interval, the anterior piece at least has to regenerate the
posterior portion and therefore the nutrition of the animal plays an
important réle. The optimum temperature, though not exactly
established, appears to be between 25 and 28° C. Above this temper-
ature, the fission rate decreases. This decline probably is not the
result of a reduced tendency to fission, but a sign of defective nutrition:
at high temperatures the metabolism of the body is increased and the
food taken does not suffice to cover the energy needs of both mainte-
nance of body size and regenerative growth necessary to prepare the
animal for the next fission. The animal is therefore in a state of
inanition. This is seen also in the rapid decrease of the size. The
maximum temperature tolerated continuously is a little above 30° C.
(Lillie and Knowlton, 1898, determined this temperature for ‘‘ Planaria
torva”’ to be 32° C.; the species used in their experiments was probably
identical with Euplanaria tigrina.)

There appears to be a correlation between the temperature and the
size of the body at which fission takes place: at low temperatures the
animals do not divide until reaching a considerable length, while at
higher temperatures fission occurs already in smaller animals. This
seems to apply to all fissioning planarians so far investigated. This
phenomenon has not yet been subjected to a comparative study on a
larger scale. Castle (1928, p. 420) e.g., observed it in another Ameri-
can planarian, Fonticola velata.

In their natural habitats, the life history of the asexual race of
Euplanaria tigrina is simple: no reproduction in winter, fission during
the warmer seasons. This cycle has been observed by Curtis (1902,
p. 517) in ‘‘locality 4." Moreover, Taliaferro’s (1920, p. 63) planarian
from an abandoned ice-pond near the University of Virginia, in which
reproductive organs were not observed for nine years, apparently
belongs to the same race. This applies also to Hyman’s (1920, p. 405)
material from ‘‘the lagoon in Jackson Park in the city of Chicago”’
(p. 404).

The sexual race of Euplanaria tigrina, on the other hand, is capable
of both sexual and asexual reproduction. Generally speaking, it
develops reproductive organs and deposits egg capsules. Even if
conditions be favorable throughout the year, sexual activity is not
continuous, but occurs in certain physiological cycles. These cycles,
in the field as well as in the laboratory (at indoor temperature),

292 ROMAN KENK

correspond to the seasons of the year. Apparently the yearly fluctua-
tions of temperature are the decisive factor. The egg-laying season
lasts several weeks or months. It goes on until the animals, especially
their sex organs, are exhausted. Then follows a period of sexual
inactivity during which the complicated copulatory apparatus first
degenerates, to be newly reconstructed before the next breeding season. —
A detailed account of these processes has been given by Curtis (1902).

During the period of sexual inactivity the animals may undergo
fission. This occurs only during the warm season of the year and is
not at alla regular phenomenon. Many specimens remain undivided.

The regular life cycle can be influenced, particularly by the tem-
perature. Low temperature retards the laying of cocoons, high
temperature accelerates it. The formation of egg capsules appears
to demand a certain minimum temperature, which lies not far below
LOC i

These data derived from laboratory cultures agree, in the main,
with Curtis’ observations in the field. Curtis (1902, p. 517) reports
that Euplanaria tigrina in Locality 1 near Woods Hole, Massachusetts,
lays egg capsules during May and June. After that, reproduction
ceases and the reproductive organs degenerate. In August and Sep-
tember, i.e. in the season when the water is warmest, the animals are
found reproducing by fission. Then reproduction is again suspended
and a regeneration of the reproductive organs takes place during the
winter.

In Locality 2, according to this author, the life cycle is similar to
that in Locality 1, except that no fissions were observed there. There
are several possible explanations of this fact: (1) the temperature may
not rise so high as in Locality 1, i.e. it may not reach the minimum
temperature necessary for fission in the asexual state; (2) the popula-
tion in this place may belong to a physiologically different race with a
fission temperature higher than that of the race from Locality 1 or even
with the tendency to fission suppressed; and (3) there may be less food
available in Locality 2, wherefore the animals may not reach the
necessary size for fission. It would be desirable to investigate this
question on the spot.

It is interesting to compare the data concerning Euplanaria tigrina
with those observed in regard to other fissioning planarians. In cases
where both manners of reproduction, the sexual and asexual, alternate
according to the seasons of the year, the asexual phase always coincides
with the warmer season. The majority of the species concerned
develop sex organs preferably in the winter and early spring: Polycelis
felina (Dalyell) and Crenobia alpina (Dana) (according to various

SEXUAL AND ASEXUAL REPRODUCTION, EUPLANARIA 293

authors); Euplanaria dorotocephala (Woodworth) (cf. Hyman, 1925,
p. 65); E. gonocephala f. subtentaculata (Draparnaud) (see Draparnaud,
1801, p. 101, and Vandel, 1925, p. 502); Fonticola morgani (Stevens and
Boring) (see Kenk, 1935a, p. 102); F. velata (Stringer) (see Castle, 1928,
p. 419). In the natural habitats this rule is often obscured by two
facts: First, certain localities have an almost constant temperature
all the year round, e.g. springs, deep lakes, and subterranean waters;
in these habitats often no alternation of reproduction takes place,
though the animals in other surroundings would be capable of both
sexual and asexual propagation. Secondly, there often occur physio-
logical races, in which the tendency to sexual or asexual reproduction
differs to a considerable extent (cf. Vandel, 1921, p. 478) and either one
of them may be entirely suppressed; these latter forms, of course, are
not considered here, since they have no alternation of reproduction.

The sexual race of Euplanaria tigrina appears to be different from
the other fissioning forms in so far as it becomes sexually active in the
warm season of the year. This difference is, however, only apparent.
Here, as well as in the others, fission takes place at a higher temperature
(August, September) than sexual activity requires (May, June).
Only, the minimum temperatures necessary for either kind of repro-
duction are comparatively high.

It is a matter of further investigation to decide whether this analysis
of the processes of reproduction, carried out for two races of Euplanaria
tigrina, is valid for other forms of this species as well. It may be as-
sumed that additional material will furnish a still greater variety of
physiological characteristics.

SUMMARY

1. Euplanaria tigrina occurs in at least two physiological races
which differ in the manner of reproduction: a sexual and an asexual
race.

2. The asexual race, according to observations covering several
years, reproduces exclusively by fission. Temperature and nutrition
control the rate of fission but do not induce sexuality.

3. The sexual race periodically develops reproductive organs and
lays cocoons. After the breeding period has ceased, the sex organs
degenerate and fission may occur at high temperature. The in-
dividual animal can outlive several periods of sexual activity.

4. In nature, all planarians that have alternating (sexual and
asexual) reproduction, appear to propagate sexually during the colder
season and asexually during the warmer season of the year.

294 ROMAN KENK

LITERATURE CITED

BEAUCHAMP, P. DE, 1932. Biospeologica. LVI. Turbellariés, Hirudinées, Bran-
chiobdellidés. Deuxiéme série. Arch. Zool. expér. gén., 73: 113.

BenaAzzi, M., 1936. Razze fisiologiche di Euplanaria gonocephala differnziate dalla
diversa attitudine alla scissiparita. Rend. R. Accad. Lincet (Roma), Cl.
Sc. fis., 23: 361.

CasTLE, W. A., 1928. An experimental and histological study of the life-cycle of
Planaria velata. Jour. exper. Zool., 51: 417.

Curtis, W. C., 1902. The life history, the normal fission, and the reproductive
organs of Planaria maculata. Proc. Boston Soc. Nat. Hist., 30: 515.

Curtis, W. C., anp L. M. Scuuuzz, 1924. Formative cells of planarians. Anat.
Rec., 29: 105.

DRAPARNAUD, J., 1801. Tableau des Mollusques terrestres et fluviatiles de la
France. Montpellier et Paris, (1801).

Hyman, L. H., 1920. Physiological studies on Planaria. IV. A further study of
oxygen consumption during starvation. Am. Jour. Physiol., 53: 399. ~

Hyman, L. H., 1925. The reproductive system and other characters of Planaria
dorotocephala Woodworth. Trans. Am. micros. Soc., 44: 51.

KENK, R., 1935a. Studies on Virginian triclads. Jour. of the Elisha Mitchell Scient.
Soc., 51: 79.

Kenk, R., 1935d. A morphological proof of the existence of zooids in Euplanaria
dorotocephala. Physiol. Zool., 8: 442.

LiLLiz, Fr. R., AND F. P. KNowiTon, 1898. On the effect of temperature on the
development of animals. Zool. Bull., 1: 179.

TALIAFERRO, W. H., 1920. Reactions to light in Planaria maculata, with special
reference to the function and structure of the eyes. Jour. exper. Zool., 31:
59.

THIENEMANN, A., 1926. Hydrobiologische Untersuchungen an den kalten Quellen
und Bachen der Halbinsel Jasmund auf Riigen. Arch. Hydrobiol., 17: 221.

VANDEL, A., 1921. Recherches expérimentales sur les modes de reproduction des
Planaires triclades paludicoles. Bull. biol. France Belgique, 55: 343.

VANDEL, A., 1925. Planaria subtentaculata Drap. n’est qu’une race asexuée de
Planaria gonocephala Dugés. Bull. biol. France Belgique, 59: 498.

INVESTIGATIONS AS TO THE LOCALIZATION OF THE
MICROMERE-, THE SKELETON-, AND THE
ENTODERM-FORMING MATERIAL IN THE

UNFERTILIZED EGG OF ARBACIA
PUNGRULA:

SVEN HORSTADIUS

(From the Zoétomical Institute, University of Stockholm, and the Marine
Biological Laboratory, Woods Hole, Massachusetts)

I. INTRODUCTION

The available information about the localization of micromere-,
entoderm- and skeleton-forming material, and about the stability of
the egg-axis in the uncleaved egg of the sea urchin is not consistent.
Driesch (1896, 1898, 1899, 1900) studied the cleavage of fragments (ob-
tained by shaking) of unfertilized and fertilized eggs of Echinus. He
came to the conclusion that the formation of micromeres is due to the
cytoplasm, not to the nucleus. By way of explanation he suggested
‘ein polar-bilaterales Gerichtetsein der Teilchen ”’ of the whole egg,
but he also spoke of a certain local specific structure of the cytoplasm.
Morgan (1894) found in part a migration of pigment, in part a forma-
tion of micromeres at the vegetative pole of Arbacia, even when the
furrows were formed in an atypical sequence. Boveri (19010)
called attention to the vegetative polar cap and its specific qualities,
and Horstadius (1928, p. 14) confirmed the results of Boveri: vegeta-
tive and meridional halves of Paracentrotus may show a typical cleav-
age, whereas the animal half does not form any micromeres. By
removing vegetative fragments of different sizes from the unfertilized
Paracentrotus egg, Horstadius (loc. cit., p. 18) proved that the po-
tentiality of forming micromeres gradually decreases from the vegeta-
tive pole towards the animal and ceases about halfway between the
normally micromere-forming area (unpigmented) and the equator.
The micromere-forming region is thus restricted to the unpigmented
pole-cap and the lower half of the pigment ring material in Para-
centrotus. (Boveri (19010, p. 155) also saw a pigmented micromere.)
Furthermore, Hoérstadius (loc. cit., p. 15) found, by aid of the pigment
band, that the cleavage axis in fragments remained unchanged, the
micromeres both in meridional and vegetative fragments being formed
from the unpigmented pole-cap.

Whereas Driesch (1900 etc.) in his early papers, spoke of all parts of

295

296 SVEN HORSTADIUS

the sea urchin or starfish egg as equipotent, Zoja (1895), Terni (1914),
v. Ubisch (1925) and Hoérstadius (1928) found that animal halves of
cleavage stages could not gastrulate, nor form any skeleton. Boveri
(19010, p. 158, 1902) used the pigment ring of Paracentrotus to study
whether the polarity remains unchanged in fragments of unfertilized
eggs. Animal fragments (without pigment) did not gastrulate. One
vegetative and one meridional fragment gave larve in which the pig-
mented and unpigmented regions were differentiated in such a way as
to show that the polarity of the fragments was not altered. Haér-
stadius (1928, p. 33) confirmed these results. Animal halves of both
fertilized and unfertilized eggs of Paracentrotus developed in the same
ways as animal halves of 8- or 16-cell stages. They do not gastrulate,
nor do they form skeleton. Meridional and vegetative fragments, on
the other hand, invaginate archentera and produce spicules. Thus
not only the micromere-forming, but also the archenteron- and skele-
ton-forming material is restricted, in the unfertilized Paracentrotus egg,
to the vegetative half. Isolation of animal and vegetative fragments
of the 64-cell stage in a plane corresponding to the middle of the pig-
ment ring region (between veg: and vege, Hérstadius, 1935, p. 319)
demonstrated that the upper level of the skeleton-forming area cor-
responds roughly to that of the micromere-forming material in the
uncleaved egg. This upper limit of the skeleton material was not
determined in detail in the unfertilized egg (cf. above as to the micro-
mere-forming material). By aid of the pigment ring Ho6rstadius
(19362) also showed in agreement with Boveri, that the polarity of
_ fragments of the unfertilized eggs does not change.

These investigations on Paracentrotus have proved that both the
micromere-, entoderm-, and the skeleton-forming material is localized
in the vegetative part, roughly speaking, the vegetative quarter, of the
unfertilized egg, and that the polarity of the egg remains unchanged in
fragments, both as regards the cleavage axis and the differentiation
axis.

Some results obtained by other investigators on different material
conflict considerably with this view. Harnly (1926) found the cleavage
pattern of fragments of unfertilized eggs of Arbacia dependent, not
upon the orientation of the plane of fragmentation in relation to the
egg-axis, but in relation to the nucleus. Boveri (1901a) stated that
the pronucleus of the mature egg of Paracentrotus may have any posi-
tion in relation to the egg-axis. Harnly (1926) and Hoadley (1934)
found the same thing in Arbacia. Thus neither the animal, nor the
vegetative half is noticeably preferred. From the different cleavage
patterns resulting on dividing eggs in different planes with relation to

iti

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 297

the nucleus, Harnly comes to the conclusion that the micromere-
forming material prior to fertilization lies in the region between the
pronucleus and the center of the egg. A nucleated fragment as large
as half an egg, or larger, cleaved as does the whole egg. When an egg
was divided into two equal halves in the plane shown in Fig. 2, the non-
nucleated fragment did not form any micromeres. But when the egg
was cut in a plane close to the nucleus, it was the non-nucleated frag-
ment that cleaved as a whole egg. Shortly after fertilization Harnly
found a different arrangement. Now only the half containing the egg
nucleus will divide. Both this and the uncleaved half remained inside
the intact fertilization membrane. Fragments that must have been
meridional and vegetative cleaved as whole eggs, and other fragments
segmented as animal halves. These results from fertilized eggs agree
with those of Boveri and Ho6rstadius, although Harnly’s interpretation
is somewhat different (see p. 309),—Harnly studied only the cleavage
pattern; he did not follow the further development.

Taylor and Tennent (1924), Taylor, Tennent and Whitaker (1926),
and Tennent, Taylor and Whitaker (1929) obtained essentially differ-
ent results for Lytechinus (Toxopneustes) variegatus. The polar bodies
and the funnel (micropyle, made visible by octopus ink) served as
landmarks for orientation. These authors state that a new polar axis
is established in fragments. With a few exceptions, the first two
planes of cleavage were at right-angles to the surface of section, regard-
less of the orientation of the cut, and the micromeres were formed on
the cut side at the end of the intersections of these two planes. Nor
is there a complete segregation of the presumptive primary mesen-
chyme or entoderm in the vegetative part of the mature, unfertilized
egg. Their experiments indicated that, prior to fertilization, a
mesenchyme-entoderm-forming substance has a uniform distribution
through about 19/20 of the egg; the animal polar cap alone seemed to
contain only ectoderm material. In the larger part of the egg, how-
ever, the presumptive entoderm and mesenchyme was supposed to be
restricted to the interior, surrounded by a superficial layer of ectoderm-
forming material, as small superficial fragments never gastrulated.
Thus both animal, vegetative, meridional and oblique halves of
Lytechinus could form not only micromeres, but also mesenchyme,
and could develop to plutei. In most cases a new polar axis was
established, perpendicular to the cut side.

In August, 1936, when spending the summer as a fellow of the
Rockefeller Foundation at the Marine Biological Laboratory, Woods
Hole, I had the opportunity of testing the localization of the micro-
mere-, the entoderm-, and the skeleton-forming material in the egg of

298 SVEN HORSTADIUS

Arbacia punctulata. Because of the short time available for this in-
vestigation the material examined was scanty, but as the results of
the observations are very definite, I think they are worth publishing.
They indicate that localization in the Arbacia egg does not differ from
that in Paracentrotus.

The eggs were cut with a fine glass needle manipulated by hand.
In order that the eggs should not slip away during the sectioning they
were placed in a little scratch in a piece of celluloid (H6rstadius, 1928).
The cut was orientated either in relation to the pronucleus (Fig. 2),
or to the micropyle, which was made visible by ink from the squid
(Loligo) (Fig. 1).

Fic. 1. Cleavage and differentiation of animal and vegetative halves of the
unfertilized egg of Arbacia punctulata. Orientation by means of the micropyle (a, 6).
The egg nucleus may lie in the animal or the vegetative half (a, 0). c. Equal cleavage
of the animal half. d. The vegetative half has formed macro- and micromeres, the
latter at the original vegetative pole, antipolar to the cut side, where a cytoplasmic
lobe has protruded through a slit in the fertilization membrane. e. Differentiation of
the animal halves into blastulz with enlarged apical tuft. f, g. The vegetative halves
gastrulate and form skeleton, developing to ovoid larve (g) or plutei (f). The results
indicate a segregation of the micromere-, the entoderm-, and the skeleton-forming
material in the vegetative part of the egg.

II. IsoLateED ANIMAL AND VEGETATIVE HALVES, AND
MERIDIONAL HALVES

Twelves eggs were divided equatorially. At the operation the
pronucleus in some cases lay close to the plane of section, in other
cases far away from it. In 7 pairs the egg nucleus lay in the animal
half, in 5 in the vegetative (Figs. 1a, 6). The animal and the vegeta-

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS DY

tive halves of each egg were reared separately and kept in pairs. Of
the twelve pairs only one vegetative fragment died early.

A. Differentiation

Zoja (1895), Boveri (19016), Terni (1914), and others have stated
that animal fragments do not gastrulate and possess a very enlarged
apical tuft. This phenomenon was studied in greater detail by
Horstadius (1935, pp. 283-314).

None of the twelve animal halves gastrulated. They all developed
considerably enlarged apical tufts, thus differentiating completely in
conformity with animal fragments in Paracentrotus (Fig. 1e). On the
other hand, all the vegetative fragments gastrulated and formed
skeletons. Some of them developed into good plutei with mouth and
arms (Fig. 1f), the others showed a more vegetative type with ovoid
body shape, no mouth, and a poor skeleton (Horstadius, 1928, 1935)
(Fig. 1g).

As controls for the animal and vegetative halves four pairs of
meridional halves were also isolated. In two pairs only one partner
developed. The other six larve (two pairs and two single larvz) all
gastrulated and formed skeletons, developing into more or less typical
plutei, or only ovoid larve.

B. Cleavage

After fertilization of egg fragments a fertilization membrane forms
in the usual way. In Arbacia the membrane is not raised from, but
lies close to the surface of the egg. When, on cleavage, the volume of
the egg increases because of the division into blastomeres, the mem-
brane often bursts on the cut side, and a lobe of cytoplasm protrudes
through the opening (Harnly, 1926; Tennent, Taylor and Whitaker,
1929, p. 18; Hérstadius, 19360, p. 820). Sometimes this cytoplasmic
bud is nearly cut off by the edges of the membrane, but very often it is
connected with the egg by a rather broad base. Nuclei may migrate
into the lobe, which may thus be divided into cells. In both cases the
bud indicates the cut side.

Of the 12 vegetative halves 11 formed micromeres. Morgan
(1894) found that the red pigment in the Arbacia egg in the 4-cell,
seldom in the 2-cell stage, migrates away from the vegetative pole.
As a consequence the micromeres are lighter and whiter than the other
blastomeres. The micromeres in our vegetative halves were of this
whitish type. But there were not always 4 micromeres (only in seven
cases). In one case there was only 1, in two cases 2, and in one case 3
micromeres. One egg showed an irregular cleavage,—it was the vege-

300 SVEN HORSTADIUS

tative half that died. It is a well-known fact that the formation of
micromeres may be suppressed, even in an entire egg, without any
influence on the later development. Thus the primary mesenchyme
is not dependent on the presence of micromeres during the cleavage.
The suppression of micromeres is often caused by a slight mechanical
disturbance (Boveri, 19016; Hérstadius, 1928, p. 124). The absence
of one, two, or three micromeres is probably due to such secondary
influences. .

The essential point is the relation of the cytoplasmic bud to the
micromeres, i.e. the position of the micromeres in relation to the egg-
axis. In three eggs no lobe was formed, in all the other eight cases
the cytoplasmic bud was antipolar to the micromeres (Fig. 1d). This
shows, without any exception in the cases where the landmark was
visible, that the micromeres were formed at the original vegetative
pole. '

The cleavage pattern of the meridional halves was not so regular.
In one pair both halves had cells of somewhat varying sizes, but no
real micromeres were observed. In another pair one fragment had all
cells of equal size, those of the other being slightly irregular. In the
remaining two pairs, one half had no small cells, the other two either
light micromeres close to the bud or only one small cell, the character
of which is uncertain. (In the absence of the cytoplasmic bud its
position could not be determined.)

Eleven of the 12 animal halves showed cells of equal, or slightly
varying sizes, but no micromeres (Fig. 1c). The twelfth fragment
possessed two pairs of small cells, but they cannot be regarded as real
micromeres, as they were not lying close together.

C. CONCLUSIONS

In dealing with the often irregular cleavage patterns of fragments,
we have to define what is meant by a micromere. Not every small
cell is a micromere. Not even every small, whitish cell. As we shall
find below, small whitish cells can be formed, owing to particular
factors, so that we cannot speak of real micromeres. We only define
as micromeres those small, whitish cells which have been formed by a
process of fundamentally the same character as in the normal egg.

The isolation of animal and vegetative halves of the unfertilized
egg of Arbacia gave exactly the same results as with Paracentrotus.
Animal halves show partial cleavage (no macro- and micromeres are
formed, Fig. 1c), and differentiate as isolated animal halves typically
do, with enlarged apical tuft, and without gastrulation (Fig. 1e).
The vegetative halves segment as whole eggs, and without any rotation
of the egg-axis (Fig. 1d). The micromeres are formed at the original

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 301

vegetative pole, opposite the cut (equatorial) side, thus not on the cut
side as in Lytechinus. The vegetative halves of Arbacia also differ-
entiate as isolated vegetative halves of other species do, giving ovoid
larve or plutei (Fig. 1f, g). This characteristic cleavage and differ-
entiation of animal and vegetative halves takes place irrespective of
whether the larve are haploid or diploid, and irrespective of the posi-
tion of the pronucleus of the egg in relation to the plane of section.
The results thus indicate a segregation of the micromere-, the ento-
derm- and the skeleton-forming material in the vegetative part of the
egg.

The meridional halves also support this view. All surviving frag-
ments gastrulated and formed spicules. It is important to note that
micromeres were not formed very regularly. In the only positive case
did they lie close to the cytoplasmic bud, thus close to the cut side, as
one would expect. This absence of micromeres is probably due to the
fact that the mechanism of micromere formation—especially in the
unfertilized egg—is very sensitive to mechanical injury (Ho6rstadius,
1928, pp. 19, 124), and that the cut passes through the supposed
micromere-forming region. In Paracentrotus I obtained micromeres
also in meridional halves in most cases, but there the membrane is less
tight, or perhaps the vegetative cytoplasm less sensitive. It is to be
regretted, that more cases were not available for study.

III. FRAGMENTS OF EQUAL SIZE ISOLATED BY A CUT
AS FAR AWAY FROM THE NUCLEUS AS POSSIBLE

To test Harnly’s results, unfertilized eggs were divided into two
approximately equal halves by a cut as far from the nucleus as possible.
Eggs were chosen in which the nucleus lay fairly close to the periphery.
The plane of section was perpendicular to a line through the nucleus
and the center of the egg and passing through the latter (Fig. 2a).
All the micromere-forming material which, according to Harnly, lies
between the nucleus and the center of the egg, should be found in the
fragment containing the egg nucleus. The plane of section thus is at
random in relation to the egg-axis. Fifty-one pairs were reared.

A. Differentiation

Not all the fragments started to develop after fertilization; some
remained undivided. A few happened to get lost. But in 35 pairs
both partners reached full differentiation (Fig. 2). In 13 only one of
the two fragments developed. In 18 pairs both fragments gastrulated
and formed skeletons, developing into plutei or ovoid larve. In 5
the diploid partner differentiated as an animal half (enlarged apical
tuft, no invagination), the haploid as a vegetative (archenteron,

302 SVEN HORSTADIUS

skeleton, ovoid larva or pluteus), and in 6 pairs I found the reverse, the
haploid fragment behaving as an animal half. In addition there were
3 cases in which the diploid had only a small archenteron, with or with-
out skeleton, but the haploid a typical or large archenteron, and skele-
ton, whereas in another 3 pairs the reverse was the case (the haploid
larve with a small invagination—Fig. 2). The 13 single larve devel-
oped as follows: 7 diploid, 2 haploid larve with more or less typical
archentera and skeletons; 1 diploid gastrula with too small invagina-
tion; 1 diploid and 2 haploid blastule with enlarged apical tuft.

eG Og
® OLYO@

Fic. 2. Diagram of the differentiation of halves of the unfertilized egg of
Arbacia isolated by a cut perpendicular to the line nucleus-center of the egg. The
cut thus at random in relation to the egg-axis. In 18 pairs both partners gastrulated
and formed skeleton, developing like approximately meridional halves (6). In 11
‘pairs the one larva differentiated as an animal half, which in 5 of those cases was
diploid (c), in 6 haploid (d). In 3 pairs the diploid, in 3 the haploid fragment devel-
oped into a gastrula with too small archenteron (e, f). The results demonstrate a
localization of entoderm- and skeleton-forming material in a part of the unfertilized
egg that must be smaller than half the egg, and independent of the position of the egg
nucleus.

B. Cleavage

We have already seen that the cleavages of the meridional halves
were somewhat atypical, as the micromere formation was often in-
hibited. Moreover, the blastomeres may often vary more or less in
size. In the halves isolated by a cut at random in relation to the egg-
axis, we find all sorts of cleavage patterns.

It has been emphasized above that probably not all small cells
observed are micromeres. On the contrary, small cells evidently may
be formed as a result of factors fundamentally different from those
leading to the formation of the typical micromeres. One has to be
very careful regarding the interpretation of the nature of the small
cells. Sometimes the cytoplasmic bud, protruding through the open-
ing of the fertilization membrane—although not containing any nuclei

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 303

or real cell membranes—may at the first glance look like a micromere,
or a group of micromeres. In some cases the cell membranes at one
or two divisions may also pass through the cytoplasmic lobe, but the
small ‘‘cells’’ formed in that way will still lack nuclei. Very often,
however, a nucleus migrates out into the bud and subsequently divides
there. After two divisions, the lobe is divided into four small (if
the lobe was small) cells, which are similar to micromeres, in that they
are lighter than the other cells (the cytoplasmic bud is often not so
markedly pigmented as the other part of the egg). When the lobe
does not protrude much outside the membrane, these cells have a
striking similarity to micromeres. But we can also find small cells,
not micromeres, inside the membrane, but formed in connection with
the bud. If the cytoplasmic bud is pressed out in the 2- or 4-cell
stage, one of the quarter-blastomeres may be much smaller than the
other ones. After two further divisions its descendants will constitute
four small cells inside the fertilization membrane (or two or three, if
one nucleus has migrated into the lobe, Fig. 3a). The bud may remain

a b c

Fic. 3. a. Formation of small cells at the cut surface, because one of the earlier
blastomeres has lost the greater part of its cytoplasm by a cytoplasmic protrusion.
At the following divisions the descendants of the part left inside the membrane
naturally were much smaller than the other blastomeres. 0, c. Formation of small
cells under the edge of the membrane, single (c) or in groups (8) at a distance from each
other.

or fall off. This group of apparent micromeres on the cut side are
thus formed by a process essentially different from that of the normal
formation of micromeres. Occasionally the first cleavages in fragments
may produce blastomeres of more or less unequal size with or without
the formation of a bud. In that way, after several divisions, the
smallest blastomere may give rise to a quartet of small cells, without
any particular relation to the egg-axis, or to the cut side. The small
cells of probably non-micromere origin may be of any number from one
to five or six, or even more. It seems to me, moreover, that the pres-
sure of the edges of the membrane on the base of large buds produces
small cells, not only as descendants from a small half- or quarter-
blastomere, but as a result of the pressure at the cleavage leading to

304 SVEN HORSTADIUS

the 8- or 16-cell stage. This appears more probable in that several
times small cells were found just under the edge of the membrane,
and not always as a single group, but separated from each other
(Hicense DMG):

Thus there are several possibilities of obtaining small cells, often
of a lighter color than the other blastomeres, and these small cells
are not real micromeres. One would expect from this that small cells,
micromeres and atypical small cells, would occur much more fre-
quently in fragments than normally. But the contrary is the case.
As mentioned above (pp. 300 and 301), micromere formation is very
sensitive to mechanical injury; e.g. after shaking, an egg may show an
equal cleavage, but the differentiation will not be influenced. In
Arbacia fragments the micromere-formation seems to be inhibited very
often.

As a result of these experiences, we have to deal with the following
possibilities when dividing the unfertilized egg at random, if we assume
the same arrangement of micromere material as in Paracentrotus.
Vegetative and meridional halves ought to give whole cleavage,
animal halves equal. But any fragment may cleave equally, i.e. if
the micromere formation has become inhibited. Animal halves, and
the other types of halves with inhibited micromere formation may
show a more or less irregular pattern, the cells varying slightly in size.
Any kind of fragment may form small cells which are not micromeres,
either from the bud itself, close to the bud, or at any other place.
It is naturally not easy to draw any conclusions from experiments
where there are so many possibilities! According to Taylor, Tennent
and Whitaker in Lytechinus all micromeres should appear on the cut
side. According to Harnly, only the diploid halves form micromeres.

One diploid and 3 haploid fragments showed whole cleavage, with
a bud opposite the micromeres. They thus behaved like the vegeta-
tive halves (p. 300 and Fig. 1d). Two diploid and 2 haploid fragments
cleaved as whole eggs, having a lobe more or less on the side: they cor-
respond to more or less meridional halves. Six diploid and 4 haploid
fragments formed typical macro- and micromeres, as far as I could
judge, but without any bud that made possible an orientation. Nine
diploid and 5 haploid fragments cleaved equally, with or without bud.
In 6 diploid and 4 haploid fragments the size of the blastomeres varied
more or less, but no cells were as small as micromeres. In 7 diploid and
10 haploid fragments I found one or several small cells, the nature of
which is open to question. They were in most cases formed either
from or near the lobe, but sometimes not close to the lobe, or at a
distance from each other.

These observations are at variance with those of Taylor, Tennent

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 305

and Whitaker on Lytechinus, as micromeres were sometimes formed
at a distance from or even opposite the cut side, and they are not in
agreement with the results of Harnly on Arbacia, as of the cases re-
garded as typical cleavage 9 were diploid while the same number, 9,
were haploid.

Let us now examine the relations between cleavage patterns and
differentiation. We speak below of three kinds of larve (Fig. 2).
One corresponds to the animal halves of Paracentrotus and of Arbacia
as we have seen in the preceding chapter, a blastula with enlarged
apical tuft. One constitutes a gastrula with a large archenteron and
with skeleton, derived from vegetative, meridional, and oblique
vegetative-meridional halves. It is not essential to mention whether
the larva later becomes a typical pluteus or only an ovoid larva. The
important point is whether it gastrulates or not. The third type is a
gastrula with a too small archenteron, such as is derived from oblique
animal-meridional halves (Fig. 2, e, f).

Of the 18 fragments above characterized as showing whole cleavage,
17 developed to gastrule with large archentera and skeletons; one
gave a blastula with enlarged apical tuft. The 24 fragments with
equal (14) or slightly unequal (10) cleavages differentiated into 6
blastule, 2 gastrule with small, and 16 with large archentera. This
result is what would be expected, as I consider that these two cleavage
types correspond to animal halves and other types of half with sup-
pressed micromeres. The 17 dubious cases must be described more in
detail. In one pair one fragment showed two, the other one small
cell inside the membrane, near the bud, but not quite in contact with
it. Both developed into gastrule. I am inclined to interpret those
as real micromeres, the halves as meridional fragments. In another
case, some small light cells were formed at some distance from the bud,
about 45°. The fragment differentiated into a gastrula. The nature
of the cells is unknown. In one case the lobe itself divided into two
small cells and one large cell. The former are certainly not micro-
meres, as the latter lay between them. The development led to a
blastula with enlarged apical tuft. Three fragments had three or four
small cells lying inside the membrane, just under the bud (Fig. 3a).
I assume that these cells were so small because the expulsion of the
bud deprived one of the quarter-blastomeres of a large part of its
cytoplasm. Another reason for believing that these small cells are
not micromeres is that the other blastomeres cannot be classified as
meso- and macromeres. All three fragments gastrulated, but one of
them had a slightly enlarged apical tuft and only a small archenteron.
In four cases a large part of the fragment had protruded through the

306 SVEN HORSTADIUS

opening in the fertilization membrane and divided, just as well as the
part within the membrane. In all four fragments small cells were
formed, and in all they were situated under the edge of the membrane
(Fig. 36, c). I do not believe that these small cells are comparable
to micromeres, as in three of the four fragments they were found single
or in groups at a distance from each other (Fig. 30, c). One developed
into a gastrula, two into blastule of the animal type, and one died.
The remaining six fragments had one or several small cells, but the
sizes of the other cells were so varied, and the position of the ‘“‘micro-
meres’? sometimes so scattered, that we cannot speak of a regular
cleavage pattern with micromeres. In one case a close examination
showed that the two apparent micromeres were not cells at all, only
two small buds without nuclei. Three of these six fragments gastru-
lated, two developed into blastule with enlarged apical tufts, and
one died.

C. Conclusions

In this experiment we divided the egg with a random section in
relation to the egg-axis, the plane of section being oriented in relation
to the egg-nucleus (Fig. 2). The entoderm- and the skeleton-forming
material cannot be limited to the region between the egg nucleus and
the center of the egg as both the diploid and the haploid larve form
archentera and skeletons with about equal frequency (in 27 and 26
cases respectively, from the pairs) and as the blastule with enlarged
apical tufts were derived not only from haploid (6 cases) but also from
diploid fragments (5 cases). Nor can the micromere-forming material
be localized between the egg-nucleus and the center of the egg, as
Harnly stated. Of the 18 cleavage stages that were considered to
possess typical micromeres, 9 were diploid and just as many—9—
haploid. Fifteen diploid and 9 haploid fragments formed no micro-
meres, showing equal or slightly irregular cleavage. In 7 diploid and
10 haploid fragments, small cells were found, the nature of which has
been discussed in detail.

As 5 diploid and 6 haploid fragments of the pairs and 1 diploid and
1 haploid of the single halves failed to gastrulate, the entoderm- and
skeleton-forming material cannot have the same widespread distribu-
tion in Arbacia as in Lytechinus, in which, according to Tennent,
Taylor and Whitaker, it occupies 19/20 of the volume of the egg.
In that case every healthy fragment of the size of a half-egg would have
gastrulated. Furthermore, the micromeres were not always formed
on the cut side. In 4 cases they were found antipolar to the cyto-
plasmic bud, in 4 this bud was situated laterally in relation to the

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 307

cleavage axis. In 24 fragments no micromeres were formed. In some
of the 17 dubious cases we could show that the small cells on the cut
side were not real micromeres.

On the contrary, our results with one exception are in conformity
with the view that both the micromere-forming and the entoderm- and
skeleton-forming material is localized in a part of the Arbacia egg that
is smaller than half the volume and comparable to the vegetative
quarter of the Paracentrotus egg. Both diploid and haploid halves
could gastrulate or differentiate into blastule of the animal type.
Both diploid and haploid halves could form micromeres. These could
appear on the cut side, opposite the cut side, or on any other side.
With one exception, all fragments that formed “real’’ micromeres
gastrulated. Fragments with equal or slightly irregular cleavage
gave all kinds of larve, in accordance with the view that they com-
prise the animal halves and any other kind of halves with suppressed
micromeres. Of the dubious cases, some with small cells developed
into blastule with enlarged apical tuft, i.e. as animal halves; but it
was shown above that in all probability those small cells were not real
micromeres, as either the whole cleavage stage was very irregular, or
the small cells were located at some distance from each other. The
only case that does not fit in with the assumption of a localization of
both the micromere-forming and the entoderm- and skeleton-forming
material in the vegetative part of the egg was one fragment which was
supposed to have a typical 16-cell stage but differentiated into a blas-
tula with large apical tuft (p. 305). Was that a case of micromere
formation of an animal half, or was the interpretation of the cleavage
stage wrong?

IV. Discussion

Although the micromere- and the skeleton-forming material occupy
the same region in the Paracentrotus egg,—roughly speaking, the most
vegetative quarter—the factors causing the formation of the micro-
meres and those causing the formation of the primary mesenchyme
of the skeleton-forming cells are not identical. As many authors
have pointed out, normal primary mesenchyme is formed even if the
micromeres are suppressed. i

Harnly (1926) studied only the localization of the micromere
material, but we have also paid attention to the differentiation of the
fragments which were isolated in order to test his results. The out-
come of his experiments, that the micromere-forming material in
Arbacia prior to fertilization lies between the nucleus and the center
of the egg, is contradicted by the two series of experiments described
above. Animal halves did not form any micromeres, whereas the

308 SVEN HORSTADIUS

vegetative fragments did, and in both cases irrespective of the position
of the egg nucleus: the animal and the vegetative fragments could be
diploid or haploid; the egg nucleus could at the operation lie close to
the plane of section or at some distance from it. When the egg was
divided into two equal halves by a section as far from the nucleus as
possible, Harnly (see his Table 3 and Fig. 1a) obtained normal cleavage
only in the nucleated fragments (23), whereas 28 haploid fragments
showed ‘‘two tiers of eight equal cells.” The pairs described above
formed (in the fairly clear cases) micromeres in 9 diploid and 9 haploid,
and no micromeres in 15 diploid and 9 haploid fragments.

In the light of my experiences, I have difficulty in understanding
Harnly’s results. He found in his experiment (Table 1) that “in no
case did a nucleated half that divided normally through the first three
cleavages give other than a normal fourth cleavage.” Of 132 diploid
fragments 30 showed normal fourth cleavage, 38 irregular and partial
first cleavage, 50 endoplasmic buds (not studied), and 9 were undeter-
mined. In Tables 3-5 no buds and no irregular and partial first
cleavage are recorded. When the eggs were fragmented, as in our
Fig. 2a, practically all diploid fragments segmented normally, but the
haploid formed ‘“‘two tiers of eight equal cells.’ When the eggs were
divided into two equal halves by a section close to the nucleus, all the
fragments that were determined, diploid or haploid, segmented in a
normal way, except a few haploid ones, which had only two or three
macro- and micromeres. As we have seen, I have not found that
fundamental difference between the diploid and haploid fragments
cut as in Fig. 2a. Both kinds could form micromeres, and both kinds
could show equal or slightly irregular cleavage. When equal, the
blastomeres were hardly ever arranged as regularly as in two tiers of
eight cells, as Harnly states for his 28 enucleated halves. Harnly also
isolated non-nucleated fragments containing two-thirds or more of the
material of the egg. One fragment was undetermined, 18 segmented
normally. Harnly’s explanation is that these large haploid fragments
contained all the micromere-forming material lying between the nucleus
and the center of the egg. A more or less typical cleavage would in
this case be expected also when assuming a localization of the micro-
mere material in the vegetative quarter of the egg, since only very few
of the fragments would be completely devoid of such material.

Harnly (1926) also divided fertilized eggs into two equal halves.
According to Harnly, the fusion nucleus is to be found in the egg-axis,
just above the equator; Harnly tried to cut equatorially. The fertil-
ization membrane could not be divided, the two halves remained
inside the membrane, flattened against each other. Only the half

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS 309

containing the egg nucleus segmented. Three types of cleavage were
found: whole cleavage with the micromeres opposite the cut side,
whole cleavage with the micromeres close to the cut side, and equal
cleavage. Harnly concludes from his experiments that, within five
minutes after fertilization, the micromere-forming material changes
its position. Both the nucleus and the micromere material are now
oriented with regard to the primary axis. Harnly does not expressly
state where along the axis the micromere material is now located, but
he seems to indicate a central position. As far as I can see, his results
accord just as well with the view that the micromere material is
restricted to the vegetative part of the egg. The equal cleavage (the
largest number, as the nucleus in most cases was lying above the
equator) belongs to the animal halves; the whole cleavage with the
micromeres opposite the cut side corresponds to the cases in which the
nucleus came to lie in the vegetative half; and the two fragments with
the micromeres close to the plane of division are probably, as Harnly
himself presumes, meridional halves.

Tennent, Taylor and Whitaker (1929, p. 66) state that their results
“have compelled the conclusion that in the unfertilized egg of Lytechi-
nus there is an animal polar cap and a superficial layer of ectoderm-
forming material surrounding a core of undifferentiated material that
is potential endoderm, primary and secondary mesenchyme, and
mesoderm.’ The presence of an animal cap of ectoderm-forming
material, occupying 1/20 of the volume of the egg (loc. cit., p. 66) was
concluded from the fact that gastrule were obtained from vegetative
fragments of 1/11 the volume of the egg (but there is only one of that
size, No. 1926 253), while none of the larve from fragments of equal
size from the region of the animal pole developed beyond the stage of
blastule with mesenchyme (No. 1926 276, 279; see also pp. 47, 63, 64,
66). The smallest fragment that developed into a blastula was 1/21
of the volume of the egg. These results point, as it seems to me, rather
to the size 1/11 than 1/20 for the animal polar cap. The reason for
the conclusion that there is a superficial layer of presumptive ectoderm
also in the other parts of the egg is not given in the paper in question,
asfarasI can find. But in the preliminary report by Taylor, Tennent
and Whitaker (1926), the same conclusion has been drawn from the
fact that small superficial fragments—with a diameter of one-fifth of
that of the egg—only gave blastule with or without mesenchyme,
while larger, but still small fragments gastrulated. A fragment with
a diameter of one-fifth of that of the egg has a volume that is only
1/125 of that of the egg. Nobody has so far been able to rear gastrule
from such small fragments. The smallest blastomere that has been

310 SVEN HORSTADIUS

observed to gastrulate is a half-macromere (Horstadius, 1936a). Its
volume is larger than 1/32 but smaller than 1/16 of the egg. Morgan
(1895) and Driesch (1900, 1902) estimated the smallest egg fragment
that could gastrulate at 1/40-1/60 and 1/32 respectively of the volume
of the egg. But these results are very uncertain, as the size of the
fragment was concluded from the size of the larve. In the full report,
Tennent, Taylor and Whitaker (1929, p. 62) state, contrary to the
preliminary note, that the smallest fragment that gave a gastrula had
1/11 the volume of the egg, the smallest that gave a blastula 1/21 (cf.
above). Under such circumstances I cannot see how the idea of asuper-
ficial layer of ectoderm-forming material round the whole egg can be
supported. Very small fragments probably do not gastrulate because
they are too small, not because of lack of presumptive archenteron
(if derived from a vegetative part of the egg). As the smallest blas-
tule obtained were derived from fragments measuring 1/21 of the
volume of the egg, no blastule seem to exist that could prove the
presence of a superficial layer of ectoderm-forming material outside
the entoderm-mesenchyme-forming substance that, in Lytechinus, is
supposed to occupy 19/20 (10/11?) of the egg.

We now turn to a comparison of the facts leading to the idea of an
animal cap of ectoderm material, occupying only 1/20 (1/11?) of the
egg in Lytechinus, with the facts demonstrating that in Paracentrotus
and Arbacia the presumptive ectoderm is distributed over more than
half the egg. Animal halves of the unfertilized egg of Paracentrotus,
oriented at the operation by means of the pigment ring, never gastru-
lated—cf. p. 296. None of our 12 animal halves of Arbacia (orientation
by means of the micropyle) gastrulated, and all showed an enlarged
apical tuft. Of the halves cut at random, many differentiated in the
same way (Fig. 2). Tennent, Taylor and Whitaker (1929, p. 57) report
the history of 27 pairs of fragments isolated by horizontal sections.
But of these pairs, both fragments reached gastrulation age only in 9
cases. If the presumptive entoderm- and mesenchyme-forming ma-
terial occupies 19/20 (10/11?) of the egg, every horizontal fragment
larger than 1/20 (1/11?) should gastrulate. This was the case in 6
pairs, the relative size of the fragments varying from 1:1 to 1 : 3.4
(No. 1924 8, 42, 44, 54, 61, 1926 230). In one case both gastrulated
with the size difference 10:1 (No. 1926 253), the animal fragment
being the larger. In two pairs, however, the animal partner did not
gastrulate. In the one pair its volume was only 1/17 of that of the egg
(No. 1926 229), but in the other the fragments were of approximately the
same size (1 : 13, No. 1926 231). The animal fragment formed a blas-
tula without mesenchyme. In addition, 3 of the single larve, all of half

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS Sill

size (No. 1924, 5, 45, 79) remained as blastule, while 11 gastrulated.
These results, with 4 fragments of approximately half size differentiat-
ing only to blastule, are at variance with the assumption that the pre-
sumptive entoderm and mesenchyme occupies 19/20 or even 10/11
of the egg.

Of 21 vertical sections in Lytechinus both partners developed to
gastrulation age only in 5 pairs. Here we should expect gastrulation
in every fragment of sufficient age (10 hours), and it occurred in 4 pairs
(No. 1924 40, 53, 56, 80). But in the fifth pair (No. 1924 60) one frag-
ment only formed a “blastula with mesenchyme,” while the other
developed to a pluteus. These two fragments were of equal size.
Furthermore, not less than 3 (No. 1924 23, 25, 1926 267) of the other
10 vertical halves (only one partner living) that lived longer than 10
hours failed to gastrulate.

Seven pairs of fragments obtained by diagonal sections (Tennent,
Taylor and Whitaker, 1929, p. 58) were reared (No. 1926 275, 279-
283, 285). In four cases the small, oblique animal fragment, of size
1/6, 1/11, 1/11, 1/21, formed a blastula with mesenchyme, while the
larger vegetative fragment gastrulated. In two cases the animal frag-
ment was the larger (5:1, 4:1); both partners gastrulated. In the
seventh pair also both developed to gastrule, the size of the oblique
animal fragment being 1/7 of that of the vegetative.

The blastule obtained from the animal fragments are said to have
a thickened posterior wall and often a number of mesenchyme cells.
No mention is made of the size of the apical tuft in these blastule.
Nevertheless, I get the impression from the drawings of such blastule
(Figs. 44, 47) that they are typical animal fragments. Thus the
thickened wall would correspond, not to the posterior, but to the
animal and ventral side (cf. Lindahl, 1933; Hérstadius, 1935, p. 286,
1936a, p. 56). This interpretation is supported by the fact that the
““mesenchyme’”’ cells in the figures are located further towards the
thin wall than towards the thickened wall. Furthermore, I very
much doubt whether all the cells reported as mesenchyme cells have
that character. In fragments, very often some cells pathologically
migrate into the blastoccel. Many isolated animal halves of Para-
centrotus may have a few, or a large number of cells free in the interior
of the blastula. They have nothing to do with real mesenchyme cells.
They may, of course, also appear in meridional and vegetative frag-
ments, together with primary mesenchyme cells. It requires an inti-
mate knowledge of the material to recognize the real mesenchyme
cells from these pathological cells, and also to interpret correctly the
animal blastulz. The cells in the blastule (Figs. 44 and 47) are rather

312 SVEN HORSTADIUS

varied in size. This indicates, it seems to me, that here we have not
to deal with real primary mesenchyme. Furthermore, the assumption
that the mesenchyme material occupies 19/20 of the egg is contra-
dicted by the fact that Tennent, Taylor and Whitaker report several
blastule of half size without mesenchyme (No. 1924 5, 23, 25, 1926 231).
The fact that 4 of the horizontal and 4 of the vertical fragments of
half-size did not gastrulate is incompatible with the conception of the
presumptive entoderm occupying 19/20 (10/11?) of the egg. It rather
shows that it is restricted to less than the half of the egg. Also the
small diagonal fragments illustrate the same thing, as in some cases
they contain material from the equator region without gastrulating.
The fact that both partners did gastrulate in 7 of the 9 animal-
vegetative pairs, and that 4 of 20 vertical fragments did not gastrulate,
seems to indicate that the orientation was not always that desired.
When discussing the localization of the entoderm and mesenchyme
material in Arbacia with some colleagues at Woods Hole in 1936, I
said that one ought to obtain blastule of animal type from, roughly
speaking, 15 per cent of the halves isolated at random, if the same
conditions as in Paracentrotus prevailed. My material of random
sections on Arbacia numbers 14 blastule with enlarged apical tuft
and 65 larve that gastrulated. Thus 18 per cent did not gastrulate.
This indicates that the presumptive entoderm occupies less than half
of the egg, otherwise the animal blastulz would have been much fewer.
Hight of the Lytechinus fragments of approximately half-size remained
in the blastula stage, while 40 developed into gastrule or plutei. Thus
17 per cent did not gastrulate. These figures of all the horizontal
and vertical sections together raise some doubts as to the accuracy of
the orientation of the cut, particularly as 4 of 20 meridional fragments
only formed blastule. Has the plane of section often been different
from that desired? One possibility is that the egg slipped or turned
during the operation. The three authors state (1929, p. 6) that the
operator could control this. The micropyle and the polar bodies
served as landmarks. Are they reliable? Tennent, Taylor and
Whitaker (1929, p. 12) regarded it as possible that the egg might
rotate in its jelly and in that manner make the micropyle of uncertain
value as a landmark. ‘‘No evidence of rotation was found. Careful
examination showed the polar bodies lying at the base of the micropyle.
In one instance only were they out of line as much as five degrees.”
But in the preliminary report (1926) it is stated that ‘‘our preliminary
observations having convinced us that the egg might be rotated in its
jelly, we felt that our orientation of the egg was dependable only when
the polar bodies could be found at the base of the micropyle.” But

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS ol

is it not possible that the egg can rotate inside the jelly, the polar
bodies being held by the micropyle? (Hérstadius, 1928, p. 91.) If
so, the egg-axis may lie at any angle to the axis indicated by the
micropyle and the polar bodies. Or has the whole egg rotated during
the slow sectioning with the micromanipulator?

From the fact that, in certain fragments obtained by vertical sec-
tion, the first cleavage amphiaster lies temporarily in the short axis
of the cell, and that in those cases the size of the cleavage cells is
atypical, Tennent, Taylor and Whitaker (1929, pp. 41-48) conclude
that there is a prelocalization of an entoderm-forming substance. I
can but support the reservation of Dr. Taylor (loc. cit., p. 47), who
doubts the significance of cell-size as indicative of a segregation of
entoderm-forming material in the unfertilized egg. In Paracentrotus,
under certain circumstances, the four animal cells of the 8-cell stage
may be much larger than the four vegetative (vorzeitige Mikromeren),
or the spindles of the second and third division may stand obliquely,
or the spindle of the first division in an animal-vegetative direction
instead of equatorial, or a vegetative half may show a typical whole
cleavage, all without any change of the position or amount of not only
the micromere-, but also the entoderm-forming material (see Hoér-
stadius, 1928, pp. 10, 14). As far as I can see, the results in question
only permit conclusions as to the cleavage factors, not as to the
localization of presumptive entoderm.

Both Harnly (1926) and Tennent, Taylor and Whitaker (1929)
state that the first two furrows in fragments stand at right angles to the
surface of section, irrespective of the orientation of the egg. The
last-mentioned authors maintain that a new polar axis has been es-
tablished (loc. cit., p. 66). But, as we have seen above, the position
of the first two furrows may be entirely independent of the polar axis
of the egg and larva. In order to determine whether a new polar axis
has been established, one has to ascertain by local vital staining whether
the cleavage axis coincides with the axis apical organ—blastopore in
the gastrula. In Paracentrotus the micromeres are always formed at
the vegetative pole, irrespective of the position of the first two furrows,
and differentiation takes place in accordance with the original egg-
axis (HGrstadius, 1928, p. 10; 1936a, p. 53). Also, if micromeres are
formed on the cut surface at the intersection of the first two cleavage
planes, as stated for Lytechinus, this does not necessarily mean a change
of polarity, as the factors for micromere formation and skeleton and
entoderm formation are not identical (cf. the paragraph above and
my S07).

The statement that the micromeres are always formed on the cut

314 SVEN HORSTADIUS

side in Lytechinus is surprising. Tennent, Taylor and Whitaker have
studied the cleavage of fragments in a great many cases, and the
drawings seem convincing, although they look rather schematized.
But the statements are perhaps not entirely conclusive, as the micro-
meres in our vegetative Arbacia fragments were formed opposite the
cut side and as the three authors (1929, pp. 36-39) also made some
experiments on Arbacia at Woods Hole,'which do not agree with those
presented here. Only 14 of 45 operations were either fully or partially
successful. If we count all the fragments that developed, single and
pairs, 1-4 micromeres were found in 6 vegetative and 6 animal frag-
ments, while 2 vegetative and 1 animal fragment cleaved irregularly,
and 5 animal and 2 vegetative divided equally. Nothing is said as to
the position of the micromeres in relation to the cut surface. These
results contrast markedly with mine: 11 of 12 vegetative fragments
showed micromeres, all opposite the cut side, while the animal halves
did not form any real micromeres. Tennent, Taylor and Whitaker
do not discuss any of the possible sources of error regarding the
micromere formation. Moreover, perhaps the free hand is a better
instrument for cutting the eggs than the micromanipulator, which may
work so slowly that a rotation of the egg after the final orientation is
possible. A renewed investigation on Lytechinus, as well as on Ar-
bacia, with attention payed particularly to the orientation of the cut
and the possible formation of false micromeres, with a large number of
larvee that reach full differentiation, and with local vital staining to
study the relation of the axis of the egg to that of the fragment and the
relation of the micromeres to the cut surface, would be very desirable.

The critical discussion may be summarized as follows. Harnly’s
assumption (1926) that the micromere-forming material in the un-
fertilized egg of Arbacia is situated between the nucleus and the center
of the egg does not hold, as vegetative halves formed micromeres, and
animal halves showed no micromeres, in both cases irrespective of the
position of the nucleus, and as nucleated and non-nucleated halves
isolated by a cut perpendicular to the line nucleus-center of the egg
(Fig. 2a) formed micromeres with equal frequency. The conclusions
of Tennent, Taylor and Whitaker (1929) that the egg of Lytechinus
has an animal polar cap occupying only 1/20 (1/11?) of the volume of
the egg, and a superficial layer of ectoderm-forming material are
contradicted by their own results. The conclusion is drawn from the
differentiation of one animal and two vegetative fragments of the size
1/11. But there are diagonal fragments of the same size, or larger,
reaching down to the equator, that do not gastrulate. Moreover,
several animal and meridional fragments of half size and sufficient

LOCALIZATION IN UNFERTILIZED ARBACIA EGGS oS

age did not gastrulate. These facts indicate that the presumptive
ectoderm occupies at least half the egg. The fact that several merid-
ional halves also differentiated as an animal fragment arouses the
suspicion that the plane of section has not always been that desired,
which would explain many of the discrepancies between Lytechinus
and Paracentrotus. It is questionable whether some of the cells de-
scribed as mesenchyme cells are not pathological. The position of the
first two cleavage planes does not necessarily indicate the position
of the axis of the larva. In fragments of Lytechinus eggs the micro-
meres are reported by Tennent, Taylor and Whitaker to be formed on
the cut side. In Arbacia I found the micromeres formed at the vegeta-
tive pole, irrespective of the position of the section. The fact that the
results of Tennent, Taylor and Whitaker as to the localization of the
micromere-forming material in Arbacia do not agree with mine, which
in turn confirm those on Paracentrotus, makes a reinvestigation of
Lytechinus also desirable.

V. SUMMARY

Animal halves of unfertilized eggs of Arbacia punctulata segmented
equally and developed into larve of animal type: blastule with en-
larged apical tuft. Vegetative halves formed micromeres antipolar
to the cut side, and gastrulated, differentiating into ovoid larve or
plutei. Meridional halves all gastrulated. The type of development
was independent of the presence or absence of the egg nucleus (Fig. 1).

Unfertilized eggs were divided into approximately equal halves
by a section perpendicular to the line nucleus-center of the egg (Fig.
2a), the plane of section thus being laid as far away from the nucleus as
possible, and at random in relation to the egg-axis. Nucleated and
non-nucleated fragments formed micromeres and gastrulated with
equal frequency. Some of the fragments differentiated as animal
halves (Fig. 2). Atypical small cells, particularly on the cut side,
which may be mistaken for micromeres are often formed.

The results indicate that the micromere-forming and the entoderm-
and the skeleton-forming material in Arbacia is located in the most
vegetative part of the unfertilized egg, occupying less than half the
volume of the egg. The results of Harnly (1926) on Arbacia and of
Tennent, Taylor and Whitaker (1929) on Lytechinus are critically
discussed.

LITERATURE CITED

Boveri, THEODOR, 190ia. Die Polaritat von Ovocyte, Ei und Larve des Strongy-
locentrotus lividus. Zool. Jahrb., Abt. Anat. u. Ont., 14: 630.

Boveri, THEODOR, 19016. Uber die Polaritat des Seeigel-Eies. Verhandl. d. phys.-
med. Gesellschaft. Wurzburg, 34; 145.

316 SVEN HORSTADIUS

Boveri, THEODOR, 1902. Uber mehrpolige Mitosen als Mittel zur Analyse des
Zellkerns. Verhand. d. phys.-med. Gesellschaft Wiirzburg, 35: 67.

DriescH, Hans, 1896. Betrachtungen iiber die Organisation des Eies und ihre
Genese. Arch. Entw.-mech., 4: 75.

DriescH, Hans, 1898. Uber rein miitterliche Charaktere an Bastardlarven von
Echiniden. Arch. Entw.-mech., 7: 65.

DriescH, Hans, 1899. Die Lokalisation morphogenetischer Vorgange. Arch
Entw.-mech., 8: 35.

DriescH, Hans, 1900. Die isolirten Blastomeren des Echinidenkeimes. Arch.
Entw.-mech., 10: 361.

DriescH, Hans, 1902. Neue Ergainzungen zur Entwickelungsphysiologie des
Echinidenkeimes. Arch. Entw.-mech., 14: 500.

Harnty, Morris H., 1926. Localization of the micromere material in the cytoplasm
of the egg of Arbacia. Jour. Exper. Zodl., 45: 319.

Hoab ey, Lreicu, 1934. The relation between the position of the female pronucleus
and the polar bodies in the unfertilized egg of Arbacia punctulata. Bzol.
Bull., 67: 220.

HOrstTapius, SVEN, 1928. Uber die Determination des Keimes bei Echinodermen.
Acta Zoologica, Stockholm, 9: 1.

HOrstTapius, SVEN, 1935. Uber die Determination im Verlaufe der Eiachse bei
Seeigeln. Pubbl. Staz. Zool., Naples, 14: 251.

HORSTADIUS, SVEN, 1936a. Weitere Studien tiber die Determination im Verlaufe
der Eiachse bei Seeigeln. W. Roux. Arch. Entw.-mech., 135: 1.

HoOrstapius, SVEN, 1936b. Studien iiber heterosperme Seeigelmerogone nebst
Bemerkungen iiber einige Keimblattchimaéren. Mém. Mus. d’ Hist. Nat.
de Belgique, Ser. 2, Fasc. 3: 801.

LinpaH1, P. E., 1933. Uber ‘‘animalisierte” und “vegetativisierte” Seeigellarven.
W. Roux. Arch. Entw.-mech., 128: 661.

MoreGan, THomas Hunt, 1894. Experimental studies on Echinoderm eggs. Anat.
Anzeig., 9: 141.

Morean, THomas Hunt, 1895. Studies of the “partial” larve of Sphzrechinus.
Arch. Entw.-mech., 2: 81.

Taytor, C. V., AND D. H. TENNENT, 1924. Preliminary report on the development
of egg fragments. Carnegie Inst. Wash. Yearbook No. 23.

Taytor, C. V., D. H. TENNENT AND D. M. WHITAKER, 1926. Investigation on
organization of Echinoderm egg. Carnegie Inst. Yearbook 25. .

TENNENT, D. H., C. V. TAyLor anp D. M. WuiTaKeEr, 1929. An investigation on
organization in a sea-urchin egg. Carnegie Inst. Wash. Publ. No. 391.

TERNI, T., 1914. Studio sulle larve atipiche (blastule permanenti) degli Echinoidi.
Mitt. Zool. Stat. Neapel, 22: 59.

von Usiscu, L., 1925. Entwicklungsphysiologische Studien an Seeigelkeimen.
Zettschr. wiss. Zool., 124: 361.

Zoya, R., 1895. Sullo sviluppo dei blastomeri isolati dalle uova di alcune meduse
(e di altri organismi). Arch. Entw.-mech., 2: 1.

”

EXPERIMENTS ON DETERMINATION IN THE EARLY
DEVELOPMENT OF CEREBRATULUS LACTEUS

SVEN HORSTADIUS

(From the Zodtomical Institute, University of Stockholm, and the Marine
Biological Laboratory, Woods Hole, Massachusetts)

I. INTRODUCTION

The eggs of nemerteans, annelids, gastropods, lamellibranchs and
scaphopods show the spiral type of cleavage. Most of these eggs,—
those of the annelids and mollusks,—are assigned to the mosaic type
of determination. Their isolated blastomeres have been found to
develop in accordance with their prospective significance. None of
these eggs has, however, been investigated in such detail as the sea
urchin egg, of which we know that some parts may develop into a
complete larva (Driesch, 1891, etc.) and that other parts may differen-
tiate less typically than in the case of normal development, owing to the
fact that interactions have to take place between different parts of the
egg, bringing the formation of some organs (HGrstadius, 1928, 1935,
1936). [von Ubisch (1936) has denied the réle of interactions in the
sea urchin egg. However, new isolation and implantation experi-
ments, as well as constriction experiments (unpublished), completely
confirm my earlier results as to the interactions.| The scope of this
investigation was to see whether parts of an egg of the mosaic type
might not show traces of interaction when transplanted atypically upon
each other. As I could not find any annelid or mollusk egg so suitable
for isolations and transplantations as the Cerebratulus egg, I had re-
course to the latter. This egg shows a spiral cleavage but is not wholly
a mosaic egg. It belongs to the regulation type insofar as fragments
of the uncleaved egg and isolated half- and perhaps quarter-blastomeres
may give rise to harmonic dwarf larve. But, on the other hand, the
investigations of E. B. Wilson (1903), Zeleny (1904) and Yatsu (1910)
indicate that the animal and vegetative parts of cleavage stages
differentiate as fragments. Thus in that respect we seem to have to
deal with a mosaic.

The cell-lineage of the nemertean egg has never been worked out.
For that reason, in order to be able to interpret rightly the results of
the isolation and transplantation experiments, we had first to in-
vestigate the prospective significance of the cell layers. As the experi-
ments of the authors mentioned above are rather incomplete, we also
have had to repeat and extend the study of isolated fragments. The
results of previous investigators will be mentioned below in connection
with each corresponding experiment.

sili

318 SVEN HORSTADIUS

II. MATERIAL AND METHODS

The investigation was undertaken in July and the first part of
August, 1936, at Woods Hole. The worms (Cerebratulus lacteus
Verrill) were sent in several deliveries in ice-cooled jars from Lynn,
north of Boston, and kept in an ice-box. In order to obtain the eggs,
a piece of a female was put into a piece of cheesecloth in sea water.
The cheesecloth retains the slime but allows the eggs to fall through.
Coe (1899) has shown that the germinal vesicle gradually fades away,
when the eggs come out in the sea water and the first polar spindle is
formed, but the spindle remains in the metaphase until fertilization
occurs. In order that uniform conditions as regards maturation and
fertilization might be secured, the sperms were added half an hour
after the shedding of the eggs (Zeleny, 1904). The egg forms no
fertilization membrane but is surrounded by a jelly and a soft mem-
brane, which are easily removed by gentle shaking.

The blastomeres of the 2-, 4-, and 8-cell stages are easily separated
with glass needles. The separation of the four layers of the 16-cell
stage is more difficult, as the blastomeres from two adjacent layers
lie partly between each other (Fig. 1, B). But it can be done by

fa-Id
1A -1D

A B
Fic. 1. Eight-cell stage (A) and 16-cell stage (B) of Cerebratulus.

cutting before the fourth cell division is quite completed, because at
that moment the cells have not yet sunk in between each other.

For transplantation the fragments were placed one on top of the
other in a cavity of appropriate size, made in a celluloid plate, and a
small glass sphere placed on top of the upper fragment to produce
pressure (H6rstadius, 1928). In some cases the fragments fused after
being made to adhere by a brief, gentle pressure of the needle. Local
staining (of a single blastomere or of a layer of blastomeres) was made
by leaning the cell or cells in question against a piece of agar (Vogt,
1923, 1925; v. Ubisch, 1925) or by staining an isolated cell layer and
then transplanting it (Hérstadius, 1928, 1935).

The number of experiments is rather limited. This is due partly
to the fact that the termination of the breeding season already in early
August cut short my experiments before the completion of an adequate
study, partly to the fact that in many series nearly all larve died.

DETERMINATION IN CEREBRATULUS EMBRYO 319

The reason for this was not a pathological condition of the larve.
They looked very healthy until they suddenly disappeared by getting
caught and expanded, just as if exploded, by the surface tension. The
death ratio was considerably reduced when the larve were reared
in a large drop of water between a slide and a coverslip on plasticine
feet and protected from evaporation by vaseline.

III. NorMAL DEVELOPMENT. NOMENCLATURE

The following facts regarding the normal development have to
be borne in mind. The Cerebratulus egg is very dark brown, opaque.
But the polarity of the just-laid egg may be accurately determined
partly by a conical protuberance at the vegetative pole, partly by a
clear area (the fading germinal vesicle) at the animal pole. Later the
polar bodies at the animal pole give a good landmark. They are also
present in the cleavage stages.

The first two furrows are meridional, the blastomeres of the 4-cell
stage all being of the same size. Thus we have no predominance of
the D-quadrant in the nemertean egg. The third cleavage is dexio-
tropic, forming four micromeres (1a—id) and four macromeres (1A—
1D), but the micromeres are larger than the macromeres, as Coe (1899)
first described (Fig. 1, A). At the next (leiotropic) cleavage, the
micromeres (the first quartet of micromeres, 1a—1d) are divided into
two layers each of four cells (1a'-1d! and 1a?-1d?), and the macromeres
bud off a second quartet of micromeres (2a—2d). The macromeres
are now called 2A—2D (Fig. 1, B).

The use of the words micro- and macromeres at the 8-cell stage is
rather confusing, as the micro- are larger than the macromeres. In
the following account we prefer to call them the animal and the vegeta-
tive cells of the 8-cell stage. Instead of speaking of the two layers
derived from the first quartet of micromeres (1a‘1d!, 1a?-1d?), of the
second quartet of micromeres (2a—2d), and of the macromeres (2A—2D)
of the 16-cell stage, we may simply designate the four layers as ani,
an, veg, and vege (Fig. 1, B), as has been done with the four layers in
the sea urchin egg (Horstadius, 1931, 1935). The composition of a
larva may then be expressed by a formula, e.g., an; + any + veg;
means a larva from which vege has been removed, etc.

The gastrula is still rather opaque, but the fully differentiated
larva, the pilidium, is quite transparent. The blastopore forms the
mouth, leading into an cesophagus and stomach; there is no intestine
or anus (Fig. 2, A). One or two days after gastrulation two lappets
grow out on the left and right sides of the mouth. The whole surface
of the pilidium is ciliated, but the lappets are bordered by a special

320 SVEN HORSTADIUS

ciliated band where the cilia are longer, more concentrated, and beat
in a characteristic way as compared with the cilia of the rest of the
ectoderm. The cells of the ciliated band are more yellowish than the
other ectoderm cells. At the animal pole we find an apical organ which
is a sense organ, a long flagellum in a thickened pit of the ectoderm.
The flagellum is not a single structure but is composed of a bundle
of fine threads.

The right and left sides of the pilidium evidently are those which
bear the lappets where the lappets are formed. As there is no anus it
is difficult to say what corresponds to the dorsal, what to the ventral
side. In the gastrula we call that axis dorso-ventral which is per-
pendicular both to the animal-vegetative and the right-left axes, but
in the pilidium the dorso-ventral axis probably, as the ventral side
presumably is so short, only forms an acute angle with the egg (animal-
vegetative) axis. After metamorphosis the egg axis corresponds to the
dorso-ventral axis of the worm.

Fic. 2. A. Normal pilidium. 8B, C. Pilidia from the isolated two half-
blastomeres of one egg.

IV. THE PROSPECTIVE SIGNIFICANCE OF ani, ame, vegi, AND vege

The four animal cells of the 8-cell stage were stained by leaning
them against a piece of agar. The whole ectoderm down to the
ciliated band was blue, and there was also an abundance of blue
granules in the band. When the four vegetative cells of the 8-cell
stage were stained, the stomach, the cesophagus, the inner sides of the
lappets, and the ciliated band were blue. Thus also in this case, we
found the ciliated band markedly stained. When the animal cells

DETERMINATION IN CEREBRATULUS EMBRYO oa

were stained, the upper, most animal part of the whole band (with
one probable exception, see below) contained blue granules, and when
the vegetative cells had been stained, the color was restricted more or
less to the lower, vegetative part of the band. It is very difficult to
trace the limit of the stained area with certainty in Cerebratulus, but
I think it is evident that the ciliated band is composed of material
both from the animal and the vegetative cells.

It was observed in several cases that on one day the young pilidium
showed the ectoderm, including the ciliated band, bluish (staining
of the animal cells); the next day the stomach too had turned blue.
It was noted, however, that the cesophagus and the inner sides of the
lappets were still unaffected by the Nile blue. Thus it would appear
that we are not dealing with a general diffusion of the stain. It seems
probable that at this stage the stomach acts as an excretory organ, as
is the case with the digestive tract of turbellarians (Westblad, 1923).

Fic. 3. In the 16-cell stage the four most animal cells, an:, were isolated, vitally
stained and transplanted back on the an2 cells. The animal fragment was probably
rotated 180° at the transplantation: note the little patch of ciliated band too high up
on the posterior side. Normally it probably constitutes the most anterior part of
the band. Stained area stippled.

Staining of the four vegs-cells of the 16-cell stage resulted in a blue
stomach. Whether a small part of the vego-material is or can be used
to form a part of the cesophagus I cannot tell,—the limit of the stained
area was not sufficiently sharp.

Also in staining the four an;-cells I had great difficulties in observing
the colored line. It seems to me that these four cells form the greater
part of the pretrochal ectoderm down to about the equator of the
pilidium. Thus ang would form the ectoderm on the outside of the
lappets and a part of the ciliated band. I have the impression that
_the an;-material in the anterior part of the larva goes down to the
ciliated band and contributes to the formation of its foremost part,
whereas posteriorly it does not reach so far down. This view is sup-
ported by the following result. For the purpose of obtaining a sharper
limit of the stain, the four an; cells were cut off, stained, and replaced.
In one case they probably happened to be rotated 180°, because the

Se SVEN HORSTADIUS

larva showed a short piece of ciliated band way up from the ciliated
band at the posterior end of the pilidium, and the stain border line ran
from this isolated piece of band to the band at the anterior end of the
larva (Fig. 3). This would indicate that the part of the ciliated band
that is normally derived from the an;-material at the front end of the
larva has undergone self-differentiation too high up on the posterior
side.

The prospective significance of the four layers thus seems to be as
follows: an; gives the ectoderm of the upper half of the pilidium,
including a very small piece of the ciliated band. ane forms the rest
of the outside ectoderm (the lappets) and a great part of the ciliated
band. veg; also contributes to the ciliated band and forms, moreover,
the insides of the lappets, and the cesophagus. vege constitutes the
material for the stomach and perhaps a small part of the cesophagus
as well.

V. THe POosITION OF THE FIRST FURROW

In order to interpret the results of isolating the first two blasto-
meres, it is necessary to know the position of the first furrow, whether
it has a fixed position in relation to the median plane of the larva
or may form any angle to this plane. In 34 2-cell stages one blasto-
mere was stained by leaning it against a piece of agar. Owing to the
large size of the blastomeres, the stain seldom penetrated the whole
blastomere. Thus we did not get a very exact staining, with a sharp
border line along a meridian of the egg. The stain rather indicated
that side of the blastomere which was opposite the furrow. In 9 of
the larvee the color was too weak or too diffuse. In the other 25 the
following parts of the larvee were found to be blue: dorsal 5, ventral 3,
left 6, right 3, dorsal-left 3, dorsal-right 4, ventral-right 1.

The number of cases is small and the method not very exact.
Nevertheless, I think these results show that the first furrow is not
confined to a certain plane, but probably may have any position in
relation to the median plane. Isolating half-blastomeres, we shall
thus obtain dorsal and ventral or right and left or oblique meridional
halves.

VI. ISOLATION EXPERIMENTS

A. Isolation of Animal and Vegetative Halves
of Unfertilized Eggs
Wilson (1903), Zeleny (1904), and Yatsu (1904, 1910a) found that
fragments from any part of the unfertilized Cerebratulus egg may be
fertilized and may segment as whole eggs. Zeleny and Yatsu found
that fragments obtained during the stages between the fertilization
and the completion of the first cleavage show a progressive specification

DETERMINATION IN CEREBRATULUS EMBRYO 323

of cleavage factors: just before initiation of the first cleavage the
fragments segment as halves. Wilson followed the further develop-
ment of fragments from unfertilized eggs and found that, if of sufficient
size (not smaller than one-quarter), they may produce normally
formed dwarf pilidia. According to Yatsu (19106) even an animal
half of an egg that has already given off the polar bodies is able to
gastrulate and develop into a pilidium.

As the cleavage of fragments has been studied in such detail, I
only wanted to repeat the experiments on the localization of gastrula-
tion potencies in fragments of the unfertilized egg. Animal and vege-
tative fragments were isolated from the beginning of the fading of the
germinal vesicle up to the formation of the first polar spindle (as far
as the maturation proceeds before fertilization). The eggs used were
not very satisfactory for they were the last of the season and the worms
had lived for a long time in the ice-box. Eighteen animal and 20
vegetative fragments were fertilized. Five of the animal and 10 of the
vegetative fragments died. The remaining 13 animal and 10 vegeta-
tive fragments all gastrulated. The best of the vegetative fragments
developed into pilidia, whereas the animal ones died before that stage.

Our finding that not only the vegetative, but also all animal
fragments gastrulate and that vegetative fragments may form apical
organs supports Wilson’s results (1903) that fragments from any part
of the unfertilized egg are able to produce dwarf pilidia.

B. Isolation of Half-Blastomeres

Charles B. Wilson (1900) was the first to obtain two pilidia from
one egg by isolating the half-blastomeres. E. B. Wilson (1903),
Zeleny (1904) and Yatsu (1910a@) found that the isolated half-blasto-
meres segment, not as wholes, but typically as if the missing blasto-
mere were present, thus a strictly partial cleavage. In the following
development, sometimes closed and spherical, sometimes cup-shaped
or flat, plate-like forms were observed. All these types of young blas-
tule could produce pilidia, but those from the plate-like forms were
usually asymmetrical. The best-developed dwarf larve seemed to
be typical in every respect. But some abnormalities were frequent
among the other pilidia. The apical organ was sometimes displaced
towards the anterior end (Wilson). However, Wilson never found
this organ duplicated in half-larve. Zeleny observed no constant
defect except possibly in the case of the lappets. But he generally
killed his larvz before the lappets could have developed fully. Yatsu
(19105) obtained some perfect pilidia, some with slight abnormalities,
among them not infrequently double apical organs. He concludes

324 SVEN HORSTADIUS

that bilaterality of egg substances cannot be detected at the 2- or 4-
cell stage.

As the segmentation of half-blastomeres has been studied in detail
by E. B. Wilson, Zeleny, and Yatsu, I restricted my observations to the
differentiated larve. The two half-larve derived from one egg were
reared together in order that the development of the bilateral sym-
metry in the pairs might be studied. The lappets were formed in four
days.

Some larve developed into perfect dwarf pilidia. Others were
more cylindrical in shape, with a short ciliated band, not forming any
lappets. Some looked spherical from the side, namely when the main
part of their bodies had the typical form, but the ciliated band was
very short, forming only a narrow ring round the mouth. In some
cases a lappet was formed on one side only. In many larve the apical
organ was displaced towards the anterior end, as Wilson (1903) found,
but a displacement to the posterior side was also observed. Contrary
to Wilson’s statement, in several cases I saw the apical organ doubled .
in the half-larve. Not only was the flagellum split up in two, but
larve with two separated ectodermal pits were found.

If we now turn to the pairs, there were 6 pairs with both larve
typical, symmetrical, thus each larva with two lappets (Fig. 2, B, C).
The largest group, 13 pairs, is characterized by one typical and one
cylindrical larva. In the latter the shape of the cesophagus-stomach
generally indicates a bilateral symmetry, which is, however, hardly
expressed in the ectoderm. In 6 cases both partners were cylindrical.
In 3 pairs one larva was typical, the other irregular and difficult to
interpret. One larva had a lappet only on the right side, the partner
being cylindrical. In one pair the two pilidia showed supplementary
deficiencies: one had a left, the other a right lappet. This is what
would be expected if we had a rather definite bilateral organization of
the egg in the 2-cell stage. But against this one case were two where
both partners had the lappets better developed on the same side, the
right. .

In the sea urchin, Hérstadius and Wolsky (1936) have demon-
strated that the larve derived from isolated half-blastomeres, from
halves of the 16-cell stage, and even halves of the unfertilized egg,
show characteristic differences, according to their origin from the left
and right, or dorsal and ventral parts of theegg. In Cerebratulus I was
not able to refer the half-larve to certain parts of the egg. We know
that the larve obtained may correspond to right and left, or dorsal
and ventral, or oblique meridional halves of the egg (cf. above). But
only in one case of 29 did we find a bilateral asymmetry that would
indicate right and left halves. At the same time there were two pairs

DETERMINATION IN CEREBRATULUS EMBRYO S25)

with the right side better developed in both partners, thus detracting
from the value of the supplementary deficiencies in the first-mentioned
case. Furthermore, I was not able to detect any features that would
incline me to ascribe one partner to the dorsal, the other to the ventral
part of the egg. There is no sign of the cylindrical larve arising from
a dorsal, or ventral fragment. Thus the abnormalities in the cylin-
drical, rounded, or irregular larve do not, as far as I can judge, tell us
anything about the origin of these larve. Our conclusion, then, would
be like that of Yatsu, that the Cerebratulus egg in the 2-cell stage is not
yet so markedly bilaterally organized that the half-larve show
deficiencies on the side where material has been removed.

C. Isolation of Quarter-Blastomeres

The quarter-blastomere also undergoes a fractional cleavage
analogous to that characteristic of the half-blastomere (Wilson, 1903;

Fic. 4. Four larve from the isolated quarter-blastomeres of one egg.

Zeleny, 1904). It gives rise to a much larger proportion of abnormal
forms, and even at best the larvee were never normal in every respect
(Wilson, 1903; Yatsu, 19106). Zeleny (1904) killed his quarter-
larvee before they were fully differentiated. The apical organ was
generally markedly displaced towards the anterior end (Wilson,
Yatsu), or it might be lacking (Yatsu). The archenteron, also, could
be abnormally formed, displaced towards the posterior end (Wilson).

Seven quartets of quarter-blastomeres were isolated, the blasto-
meres being reared in separate dishes and kept in such order of sequence
as in the 4-cell stage, so that we know, for instance, that No. 4 had-had
Nos. 3 and 1 as neighbors. The larve were all more or less irregular
(Fig. 4). The apical organ was generally displaced, or might be
absent; the archenteron was not always well differentiated. The
position of the ciliated band was in most cases more or less oblique.
But, as in the half-blastomeres, it was not possible to find any definite

326 SVEN HORSTADIUS

relation between these asymmetries and a supposed bilateral organiza-
tion of the egg.

D. Isolation of the Four Animal and the Four Vegetative
Cells of the Eight-Cell Stage

Zeleny (1904) found that larve developing from the upper quartet
have an apical organ, but no archenteron, those from the lower
quartet have an archenteron, but no apical organ, while those from
lateral four-cell groups have both apical organ and archenteron.
Zeleny drew the conclusion that certain organ-forming materials are
definitely separated by the third furrow and that the larve from the
upper and the lower quartet have not the power of replacing the
material lacking. The meridional halves, however,’ possess both kinds

Fic. 5. Larve from the isolated four animal cells of the 8-cell stage.

of material. Zeleny preserved his larve before they were fully differ-
entiated. Yatsu (19100) isolated 7 animal quartets, six of which gave
a blastula with apical organ, with 1 to 3 flagella, and, to judge from the
drawings, a ciliated field or band at the opposite pole. Thus no
gastrulation occurred. Strange to say, the seventh larva developed
into an almost perfect pilidium (Yatsu, 19100, Fig. 18, D). Of the 3
vegetative quartets Yatsu mentions two as ‘‘defective.’’ The third
is an almost normal pilidium (loc. cit., Fig. 18, J).

My material numbers 10 pairs of animal and vegetative quartets,
and moreover 9 animal and 4 vegetative fragments of the same kind,
but of which the corresponding vegetative and animal quartet respec-
tively are missing. None of the 19 animal fragments gastrulated.
They gave a blastula with a pretrochal pavement epithelium, a
thickened vegetative ciliated band or field, and one, or several, apical

DETERMINATION IN CEREBRATULUS EMBRYO Sat

organs (Fig. 5). Ten of these blastule had but one normal apical
organ, 4 had two, and 3 had three flagella. In most cases, perhaps
in all, every flagellum had its own ectodermal pit. In one case the
apical organ was entirely lacking. One blastula was very peculiar,
with seven flagella, three of them on the pretrochal part, the other four
growing out from the ciliated band. The shape of the animal larve
is shown in Fig. 5. It is rather rounded or ovoid, the ciliated band
occupying the broader or the narrower end. The “ciliated band”
has more the character of a ciliated field of high cells, with here and
there some irregular protrusions. This ciliated area has the yellowish
color of the ciliated band, and the cilia wave in the same typical way.
Thus, on the whole, the animal quartet develops just as it would have
done in the normal larva, giving rise to the same kind of tissues:
pretrochal ectoderm with apical organ, and ciliated band tissue. A
difference lies in the frequent duplication of the apical organ.

Fic. 6. Larve from the isolated four vegetative cells of the 8-cell stage.

The vegetative quartets also (Fig. 6) differentiate in accordance
with their prospective significance. An archenteron invaginates, but
it evidently corresponds only to the stomach of the pilidium, as it
differentiates in the same way, containing the small crystals character-
istic of the stomach. The “ectoderm” of the gastrula corresponds to
the oesophagus and the insides of the lappets, and at the animal pole
of this vegetative fragment we find a ciliated field, presumably derived
from the material that normally contributes to the formation of the
ciliated band. This field is, however, often divided into two or more
ciliated patches of the same kind of tissue as the ciliated band.

All the animal and vegetative fragments thus developed in accord-
ance with their prospective significance. When Yatsu (cf. above)
found that one animal and one vegetative fragment developed into
almost perfect pilidia, he must have isolated two meridional halves
instead of one animal and one vegetative. The orientation of the
8-cell stage is not always easy, unless the polar bodies are clearly seen.

328 SVEN HORSTADIUS

E. Isolation of any

In two cases Zeleny (1904) successfully separated the four animal
cells of the 16-cell stage (ani) from the twelve lower (an + veg; +
veg»). The two anj-larve possessed an apical plate but were not
reared long enough to show the differentiation in detail.

Twenty-five an; were isolated. One did not develop very well.
Fourteen disappeared altogether (cf. p. 319), and the remaining 10
developed into blastule with apical organs (Fig. 7). Five of them
had one flagellum, 3 had two flagella, and 2 none. I had the impression
that the pit in some cases was missing or not so pronounced as in
normal pilidia, but this observation may be erroneous. It may be
mentioned that Yatsu (1904), in the uncleaved egg, localized the basis

Fic. 7. A-—D. Larve from the isolated four most animal cells of the 16-cell
stage:ani. . Isolated veg: (the four most vegetative cells of the 16-cell stage):

of the apical organ, not in the animal pole, but in a broad zone a little
above the equator. Wilson (1903) also observed flagella without
ectodermal pit, namely in larve from vegetative egg fragments. There
is need of further elucidation of the question whether the absence of
the ectodermal pit in an;-larve, if this takes place, has any relation to
the localization suggested by Yatsu. If so, it would indicate a trace
of dependent differentiation.

The ectoderm of these an;-blastule was ciliated in the usual way.
At the pole opposite the apical organ they had small fields of ciliated
band tissue (high, yellowish cells). In some cases this field had the
form of a protrusion, with the ciliated band like a girdle round it (Fig.
7, D). Jinterpret this to mean that the patch of ciliated band tissue
in an; corresponds to that part of the normal band which, on the basis
of our staining and transplantation experiments, we ascribed to the
an,-cells (See Fig. 3).

DETERMINATION IN CEREBRATULUS EMBRYO 329

F. Isolation of ang + vegi + vege

The best one of two fragments of this kind that Zeleny obtained
(cf. above) formed a ciliated rotating embryo, with a large solid arch-
enteron filling up the cavity of the blastoccel. Neither apical organ
nor lappets were present.

Of the five larve only one developed really well. In the early
gastrula stage the archenteron seemed to fill up the entire blastoccel,
as Zeleny found. Also in the early pilidium the archenteron looked
very large in comparison to the ectoderm. A day later this was no
longer the case to such an extent as one would have expected after
removal of such a large part of the presumptive ectoderm (Fig. 8, A).

A B C

Fic. 8. A. any + vegi + veg. B, C._am + an, + vegi.

The larva now appears as a small pilidium, save that the apical organ is
missing. The stomach is of normal size. The cesophagus is smaller
than normal but probably contains as much material as in the normal
pilidium, as its wall is thicker. The ciliated band and the pretrochal
ectoderm are too small, which is easily understood in that the larger
part of the ectoderm has been removed. That, in spite of this, the
ectoderm does not look still narrower is probably due to an extreme
stretching and thinning of this material.

G. Isolation of vege

This region constitutes the material for the stomach (perhaps
also a small part of the cesophagus). When isolated it does not live
long. On the first day after fertilization these fragments were gener-
ally all dead. Those which lived the longest formed a mass of cells,
as seen in Fig. 7, E. Only in one case were some cilia developed,
indicating an initial differentiation.

330 SVEN HORSTADIUS

H. Isolation of any + ang + veg

Only three fragments of this type were studied, but they all devel-
oped in a similar way. They formed, much as the isolated four animal
cells of the 8-cell stage, a blastula with an apical organ and a ciliated
band (Fig. 8, B, C). Thus no real deep gastrulation took place,
although the vegetative end of the larva at one stage may be a little
curved inwards, as in a normal larva just at the beginning of the
gastrulation. This part later differentiated into an epithelium which
mostly looked like ectoderm, but perhaps was a little more densely
ciliated. In the larva from the upper quartet of the 8-cell stage the
“ciliated band”’ rather was a ciliated field of high cells, occupying the
lower part of the larva (Fig. 5). Our present larve differ in the respect
that we here have a real band, surrounding the ectoderm-like area just
mentioned, which in turn must correspond to the inside of the lappets
and the cesophagus. In one of the larve this area first curved inwards
and was later turned out again (Fig. 8, B), whereas in the other two
it remained as a very shallow invagination; the ectoderm formed like
a lappet on one side (Fig. 8, C). Thus also a larva from which veg:
has been removed seems to develop in accordance with the prospective
significance of the material.

I. Isolation of anz + veg,

The same holds for the fragments ang + veg:. A great many
larvee disappeared. The 9 surviving ones differentiated, although
varying in shape, into blastule with a broad, ciliated band around the
equator (Fig. 9). As the ectoderm does not expand, the ciliated band

Fic. 9. Larvee from the two middle layers of the 16-cell stage: any + vegi.

is very thick and broad. The pretrochal, animal epithelium which
corresponds to the thin ectoderm formed by ane is generally thinner
than the vegetative epithelium of the band (corresponding to the inside
of the lappets and the oesophagus). Some larve are more or less
massive, and irregular in shape (Fig. 9, C, D).

DETERMINATION IN CEREBRATULUS EMBRYO Sol

VII. TRANSPLANTATION EXPERIMENTS
A. any, + veL2

The most animal and the most vegetative of the cell layers of the
16-cell stage were fused for the purpose of studying whether organs
that normally arise from the middle part of the egg could be formed by
regulation. The larve gastrulated, the vege-material being invagi-
nated. The ectoderm appears identical with that of the isolated an,
having an apical organ and a small patch of ciliated band tissue (cf.
Figs. 10 and 7). In some cases the archenteron differentiated into
just a stomach, very characteristic with its crystals in the wall. Butin
other cases I also obtained what looked like a small part of an cesoph-
agus (Fig. 10, C). This might be a structure formed by regulation,

Fic. 10. A-—C. Larve composed of the most animal and the most vegetative

layers of the 16-cell stage: an; + vega. D. an; + er -+Vveg.

but, as pointed out on p. 321, it is possible that veg, normally also forms
a part of the csophagus. Iam not sure that the third furrow always
lies at the same level. I have the impression that the differences in
size between the animal and the vegetative cells of the 8-cell stage
may vary. As a consequence probably the cleavage plane vegi—vegs
also may vary in relation to the limit between the presumptive cesoph-
agus and presumptive stomach. This would explain the slightly
different results (Fig. 10, A—C).

B. any + + vege

In three cases vege was added to an; + 2 ane-cells. This increase
in ectoderm and ciliated band material gave a larva of essentially the
same type as an; + veg, but with a larger ectoderm and a larger
amount of ciliated band tissue, which now forms a complete ring
(Bie OD):

332 SVEN HORSTADIUS

C. Fusion of an Animal and a Meridional Fragment of
the Eight-Cell Stage

The problem is whether the entoderm material of a meridional
fragment of the 8-cell stage can bring about any entodermization of
adjacent presumptive ectoderm from an animal fragment, and whether
a regulation will take place also regarding other organs (apical organ,
ciliated band), so that a harmonic individual arises. The animal
fragment of the 8-cell stage was vitally stained before transplantation.
At the fusion the polarity of this fragment stands at right angles to
that of the meridional one (Fig. 11, A).

Fic. 11. A. Fusion of a meridional half and a vitally stained (stippled) animal
half of the 8-cell stage. B, D. The larve have differentiated conformably to their
prospective significance. C, E. Commencing regulation by redifferentiation.

The 5 larve all gave the same result (Fig. 11, B, D). Each com-
ponent differentiated in the same way as it would have done normally;
there seemed to be no interaction between them whatsoever. At
gastrulation none of the adjacent blue animal material was invaginated.
The meridional partner developed an apical organ at its animal pole and
half of a ciliated band, embracing the mouth like a horseshoe. The
animal component clearly has a polarity at right angles to that of the
meridional one. The apical organ of the animal fragment is situated

DETERMINATION IN CEREBRATULUS EMBRYO 333

at its animal pole; its ciliated band forms a girdle round its base, at
right angles to the horseshoe of the meridional component. In the
larva, Fig. 11, D, the apical organ of the meridional fragment is not
situated close to the stained ciliated band. The position of the two
ciliated bands gives the explanation. They are not at right angles to
each other. The fusion of the two components has occurred obliquely.

Thus both halves differentiated according to their prospective
significance. It is now highly interesting to note that, in spite of the
lack of interaction in the period of embryological differentiation, we
find a regulative interaction in the following development. Several
days after the differentiation of the organs of the pilidium, the ciliated
band of the animal component gradually disappeared. Only a short
part of itremained to form, together with the horseshoe of the merid-
ional part, a complete ciliated ring (Fig. 11, C; cf. also E). Thus
we here witness a step towards the individualization of this hetero-
geneous larva by means of redifferentiation. The larve did not live
long enough to show any further changes.

VIII. CoNcLUSIONS AND DISCUSSION

By means of vital staining the prospective significance of the
animal and the vegetative cells of the 8-cell stage, and of the four cell
layers ani, ane, vegi, and vege of the 16-cell stage, was studied. The
isolation experiments showed that, on the whole, the fragments isolated
by cuts vertical to the egg-axis differentiated in accordance with the
prospective significance of such parts interpreted through the presence
of different organs, e.g., apical organ, ectoderm pavement epithelium,
ciliated band, epithelium of the inside of the lappets, cesophagus, and
stomach. There is some doubt, however, as to whether all fragments
differentiate in every detail according to their prospective significance,
as the latter could not be determined with accuracy in all cases. The
staining experiment was not quite conclusive as to the fate of am,
but the transplantation experiment (Fig. 3), too, indicates that ani
normally forms a piece of the ciliated band, as the isolated fragment
always does (Fig. 7). It is more uncertain whether veg: normally
contributes to the cesophagus. We must thus leave open the question
whether the small cesophagus in some of the larvae an: + vege (Fig. 10)
was formed from presumptive cesophagus or by regulation. The
rather extensive ectoderm in the larva ane + veg: + veg may result
from an unusual stretching of the material. The mosaic development
of the larve in Fig. 11 supports the view that the apparent deviations
from the prospective significance in the differentiation of the cases
just mentioned are more apparent than real.

334 SVEN HORSTADIUS

It was suggested as possible that the apical organ is not definitely
determined in an;. An interesting feature is the appearance of several
apical organs in fragments (not only is the flagellum subdivided but
there may also be several ectodermal pits). Is that a sign that the
organ was not determined at the time of operation? Not necessarily,
as it may, on the contrary, indicate a mosaic differentiation. If the
apical organ is normally formed from all four quadrants, a slight rela-
tive change of position of the animal parts of the blastomeres at or
after the operation may cause the parts of the presumptive apical
organ to become separated by other ectoderm. In my experiments
I never got more than two apical organs in half-larve, only one in the
quarter-larve, and, with one exception, not more than two or three in
animal fragments, thus never more than the number of quadrants
present. On the other hand, the exception, an animal fragment with
seven separate flagella (p. 327) seems to show, as well as the possible
lack of ectodermal pit in an;-larve (see p. 328), that the determination
of the apical organs in fragments is more complicated. The problem
must be left unsolved.

The scope of this investigation was to compare an egg with mosaic
development with that of the sea urchin. An animal fragment of the
sea urchin egg (am, or an; + ane of so-called equatorial eggs (HG6r-
stadius, 1935, p. 309)) forms less organs (no ciliated band, no stomo-
deeum) than it would have done in normal development, whereas a
vegetative half, by regulation, often forms more organs than the same
material would normally have yielded (the vegetative halves may have
apical tuft, mouth, oral arms). An isolated vegs-layer develops into
a larva with both more animal (ectoderm) and more vegetative differ-
entiations (skeleton) (loc. cit., p. 423) than it would have given rise to
in normal development (archenteron). Another way of demonstrating
the changes of differentiation that may occur in the sea urchin is to
add a vegetative fragment to an animal fragment, e.g., to add the
four micromeres to an; or to an animal half (an; + ane). As a result
of an interaction between the animal and the vegetative qualities a
complete dwarf pluteus develops (Joc. cit., p. 330).

Our experiments with Cerebratulus have shown that there is proba-
bly no change in differentiation in animal and vegetative fragments of
cleavage stages. If any changes take place they are so slight that we
have not been able to detect them with certainty. At all events, there
is no regulation to compare with that of a vegetative half, or a vege, in
the sea urchin, and, in the Cerebratulus fragments, there is no failure
of some organs to appear (in comparison to the prospective significance)
as in the isolated an; or an; + ane of the sea urchin. We have several

DETERMINATION IN CEREBRATULUS EMBRYO 339

directly comparable experiments which show this very clearly. In
the sea urchin it is possible to obtain two plutei from one egg, after
cutting at right angles to the egg-axis, i.e., if we cut twice and put the
two polar parts together (Hérstadius, 1936a). The middle part will
thus give more animal and more vegetative differentiations than it
would have done normally, the animal and vegetative fragments
(an; + the micromeres, or an; + ang -+ the micromeres) will, by
regulation, form the organs characteristic of the excised middle part
of the egg (archenteron, etc.). The equivalents to these experiments
are our isolations of ane + veg; (Fig. 9) and fusion of an; + vege (Fig.
10) in Cerebratulus. As far as we can judge, these larvee show mosaic
development. The second experiment implies the adding of an
animal half to a meridional half, at right angles to each other as regards
polarity. This larva differentiates in the sea urchin into a perfect
pluteus. The animal half becomes completely incorporated: a part
of it is entodermized, now taking part in the formation of the archen-
teron, and the ectoderm, too, acts in all details (ciliated band, stomo-
dzeum) as a part of the new individual (Horstadius, 1928, 1935). In
Cerebratulus this is not true (Fig. 11). We find a strict mosaic develop-
ment, without any trace of entodermization or accommodation of the
ectoderm of the animal fragment to that of the meridional half.

I recall the fact that the ciliated band of the animal component
of these larvae may disappear. We witness an initial change of the
heterogeneous larva into a more harmonic individual. It has already
been pointed out (p. 333) that this change is brought about by a re-
differentiation after the embryological differentiation is already com-
plete. In this connection we must mention a similar observation by
Yatsu (19106). Whereas an apical organ is not formed in larve
which in the cleavage stages were deprived of their most animal part,
Yatsu found that the apical organ regenerated, when it was removed
from late gastrule or young pilidia.

We have to remember that the mosaic development of animal and
vegetative parts mentioned above concerns the layers of the 8- and
16-cell stages. Our isolation of animal and vegetative halves prior
to fertilization confirms the results of Wilson (1903) and Yatsu (19100),
—that any fragment of the unfertilized egg may develop into a pilid-
ium. Thus the transition from a regulation to a mosaic type takes
place between the beginning of maturation (before fertilization, cf.
p. 322) and the 8-cell stage. Yatsu (19100) observed that an animal
half of even a mature egg could form a pilidium. Removal of the most
animal part of the egg in the first cleavage of the two blastomeres
of the 2-cell stage had no effect, but when the most vegetative material

336 SVEN HORSTADIUS

was cut off, the archenteron was missing or too small (Yatsu). Thus
at that time the localization of the presumptive entoderm seems to
have advanced a great deal. When 2-cell stages were compressed so
that the second furrow came to lie equatorially, instead of meridionally,
and the two animal cells were separated from the vegetative, the former
did not gastrulate, whereas the vegetative gastrulated but formed no
apical organ. Thus already at the 4-cell stage we seem to have exactly
the same animal-vegetative localization as in the 8-cell stage.

Wilson (1903) and Zeleny (1904) have assigned the location of the
entoderm material to the vegetative part of the egg and the deter-
mination of the apical organ to the rearrangement of material at the
breaking down of the germinal vesicle. Yatsu (1904) observed, in
sections, that a segregation of egg material does actually take place at
that period, the yolk accumulating in the lower hemisphere while the
clear and more finely granulated protoplasm collects at the animal pole
of the egg. Some of the results presented by Yatsu as to the time at
which determination has taken place are contradictory. Further in-
vestigations are desirable as to how much the determination becomes
fixed along with the visible rearrangement of materials, and how much
is a progressive process going on after the completion of this rear-
rangement, and hence a process of a different character.

The rearing of fragments of blastule has given startling results.
It would seem as if these fragments were richer in potencies than
fragments of cleavage stages. Wilson (1903), Zeleny (1904) and
Yatsu (19106) found that animal halves of blastulae could gastrulate,
although the archenteron was generally smaller than that of the
corresponding vegetative fragment. This result is in conflict with
everything we know of the progressive embryological determination
which leads to gradual restriction of potencies in every zone. (We
recall the fact that the regeneration of an apical organ in the young
pilidium and the redifferentiation in our larve meridional half +
animal half took place only after the completion of the embryological
differentiation—cf. p. 335.) Wilson is inclined to explain at least some
of his results on the grounds of oblique sections. In view of this,
Zeleny (1904) took special care in determining the orientation, but
nevertheless he found in his two cases that the blastula fragments had
greater regulative power than those of the 8- and 16-cell stages. One
of Yatsu’s animal fragments had a small gut, and the vegetative was
provided with an apical organ. Until these results have been con-
firmed by means of very careful experiments, I am, like Wilson, in-
clined to explain them on the grounds of oblique sections. It is much
more difficult to cut a blastula in a desired plane, than a cleavage

SS.

DETERMINATION IN CEREBRATULUS EMBRYO 33H

stage. In the latter you follow a furrow all through the egg. More-
over, if you start cutting a blastula equatorially with the correct
orientation, the result may be oblique halves, for the knife may pass
obliquely in animal or vegetative direction through the egg. This
can only be detected by a careful examination of the fragments from
all sides. The variability of the results on blastule, and the uni-
formity of those on 8- and 16-cell stages speak in favor of this assump-
tion. As the fragments of the uncleaved egg are more or less equi-
potent, oblique cutting is most readily detected in the blastula stage.

If we turn to the fragments in which all the layers are present in
the same proportion as in the egg (isolated half and quarter-blasto-
meres), we find that they are able to develop into more or less typical
pilidia. All the organs are present, but the larvee may be more or less
asymmetrical, especially the quarter-larve. On the other hand,
several pairs of half-larvee showed a perfect bilateral symmetry (Fig.
2, B, C). These pairs probably correspond to both right-left, dorsal-
ventral, and obliquely separated meridional halves (cf. p. 322).
Furthermore, the pairs with asymmetrical larve did not show from
which parts of the egg the halves originated. This seems to indicate
that the bilateral symmetry is not fixed at an early cleavage stage
when the animal-vegetative layers are already determined. In this
respect a comparison with the sea urchin is of great interest. It has
not been possible to study experimentally the unripe egg of the sea
urchin. In the mature egg, before fertilization, we find not only an
animal-vegetative segregation of presumptive ecto- and entoderm, but
also slight traces of a bilateral organization. These traces are more
marked in the cleavage stages. Right and left halves show comple-
mentary deficiencies of the skeleton on the cut side. Ventral halves
develop their ventral side faster and better than dorsal halves. It is
very interesting that the dorso-ventral axis in the dorsal halves is
inverted (Horstadius and Wolsky, 1936). I regret that time did not
allow me to study in a similar way (by staining the cut side of the two
isolated half-blastomeres) the relation of the median planes of the
half-larve to that of the egg of Cerebratulus.

Thus the animal-vegetative determination in Cerebratulus seems to
take place between fertilization,—at the beginning of maturation,—
and an early cleavage stage. In the sea urchin we find a localization
of presumptive ecto- and entoderm already in the mature, unfertilized
egg but the same degree of determination is not reached as in the 8-cell
stage of Cerebratulus until much later; in the case of some organs not
until the beginning of gastrulation (Horstadius, 1936b). On the
other hand, the unfertilized, but mature sea urchin egg already seems
to have a bilateral organization, which can be traced in meridional frag-

338 SVEN HORSTADIUS

ments, whereas a bilateral organization of the Cerebratulus egg is not
possible to detect, even in the early cleavage stage. It has many
times been pointed out in the literature that there is no fundamental
difference between the mosaic and the regulation eggs. In some eggs
the determination sets in at an earlier stage than in others. This
relative displacement in time is strikingly illustrated in these cases.
In the Cerebratulus egg the animal-vegetative determination is ac-
complished much earlier; the determination of the bilateral symmetry
later than in the sea urchin egg.

The formation of mesenchyme has not yet been mentioned. It
would have been of great interest to determine whether, in fragments,
mesenchyme can be budded off from other parts of the egg than those
under normal conditions. In the figures, mesenchyme cells have been
drawn in those larve in which they were clearly seen. In many iso-
lated fragments we find cells in the blastoccel which are not real mesen-
chyme cells, but pathological. Those are more rounded, and often
larger than the true mesenchyme cells. We find typical mesenchyme
cells in the half- and quarter-larve (Figs. 2 and 4), in the isolated
vegetative cells of the 8-cell stage (Fig. 6), in an; + vege (Fig. 10),
and in the meridional half + the animal fragment (Fig. 11). Many
of the an; (Fig. 7, B, C) and the an; + ano-larve (Fig. 5, A, B) were
empty, others had some cells of the pathological type in the blastoccel
(Fig. 7, A, D, 5, C, D). Only in one case was just one single cell
observed which looked like a true mesenchyme cel] (Fig. 5, C). The
any + veg)-larve had some cells in their interior, but I could not
determine their nature. To summarize, the larve with vege present
show typical mesenchyme cells (Figs. 2, 4, 6, 84 cells not drawn, 10,
11). The animal fragments (Figs. 5 and 7) have with one exception
no typical mesenchyme cells. As regards the réle of veg:, we cannot
say anything with certainty. The character of the cells in Fig. 9
(ane + vegi) is unknown, and as to the larve an; + ane + veg: (Fig. 8,
B, C), I have no records regarding the mesenchyme cells. Even with
more detailed observations on the occurrence of the mesenchyme in
fragments, it would be difficult to state whether the prospective potency
as regards mesenchyme formation exceeds the prospective significance,
as observations on normal mesenchyme and mesoderm formation are
varied and contradictory. Coe (1899) traces the mesenchyme to the
divisions of a large posterior pole cell, as in annelids, and to some of the
entoderm cells. Charles B. Wilson (1900) speaks of micro- and macro-
mesencytes, both derived from large entoderm cells close to the ecto-
derm. E. B. Wilson (1903) describes two symmetrically placed
mesoblast cells which, just before invagination, pass into the cleavage
cavity near one end of the embryo, and from them smaller mesenchyme

DETERMINATION IN CEREBRATULUS EMBRYO 339

cells are budded forth, without, however, giving rise to definite meso-
blast bands, as in the annelid embryo. FE. B. Wilson finds it probable
that the first two mesoblast cells do not arise from a single cell. All
these statements refer to the genus Cerebratulus. Nusbaum and Oxner
(1913) compare the observations of previous investigators with their
own and come to the conclusion that the mesenchyme in the genus
Lineus has a double origin: ‘‘(1) Aus Mesoblastzellen, die sich sehr
friih aus den Mikromeren des vierten Quartetts differentieren und (2)
aus nachtraglich schon im Gastrulastadium aus dem Entoderm sich
abtrennenden Zellen.”” According to Nusbaum and Oxner (1913)
the micromere 4d in Lineus divides into two teloblasts (Urmesoderm-
zellen), whereas Lebedinsky (1897), in Tetrastemma and Drepanophorus,
speaks of four mother cells for four mesoblast bands. In Malacobdella
Hammarsten (1918) also traces the mesoderm to four mother cells,
2a—2d. Thus the observations diverge widely, even within one genus,
and even within one species. A renewed study of the mesenchyme
formation in Cerebratulus, combined with isolation experiments, would
be of great value. One problem is whether veg; normally and in
fragments buds forth any mesenchyme. As half-larve (which may be
right-left as well as dorsal-ventral halves) and quarter-larve always
have mesenchyme cells, a second problem is whether the mesenchyme
normally arises in all four quadrants of the egg, or the mesenchyme
in some of the meridional fragments is formed by regulation. It may
be added that the mesenchyme of the pilidium corresponds to both
the larval mesenchyme (ectomesoderm) and the entomesoderm of the
trochophore, as a part of the mesenchyme cells in the pilidium preserve
an indifferent character until the formation of the worm.

It would lead us too far to consider here all experiments on other
eggs with spiral cleavage. A full review of the literature on annelids
and mollusks has been given by Schleip (1929); see also Huxley and de
Beer (1934, Chapter V). The main difference between the nemertean
egg and those of the other groups is the equality of the four quadrants
of the egg in the former. In the uncleaved eggs of annelids and mol-
lusks we are dealing with specific substances, more or less visible,
which are of an ‘‘organ-forming”’ nature, the pole-plasms. They are
precociously chemo-differentiated (Huxley and de Beer) to varying
degrees in different species, and, moreover, the time at which they
become specifically localized varies. Very often a polar lobe is formed
temporarily during the first cleavage divisions. Removal of this lobe
leads to an absence of the apical organ and to a defective post-trochal
region; no somatoblasts will be formed. Because of this the isolated
quarter-blastomeres develop in a very different way. Only the D-
quadrant—the one that has the polar lobe—can produce somatoblasts

340 SVEN HORSTADIUS

(which give rise to the ectodermal and mesodermal (entomesoderm)
germ-bands. The A-, B-, and C-blastomeres cannot develop into
complete larve. They may have larval mesenchyme (ectomesoderm),
but are devoid of entomesoderm. As regards the apical organ, the
conditions are, however, not so uniform. In Patella, for example,
(Wilson, 19040) all four quadrants acquire an apical organ, in Dentalium
(Wilson, 1904a) only the D-quadrant, whereas in Sabellaria (Hatt,
1932) the factor determining the apical organ goes to the C-quadrant.

In mosaic development we thus find (at varying times) precociously
differentiated substances distributed to particular regions of the egg,
and in varying degrees by different forms, and subsequently isolated
by the cell divisions. If the vegetative pole-plasm be equally distrib-
uted to the first two cells (Tubifex, by heat or deprivation of oxygen,
Penners, 1924; Chetopterus, Nereis, and Cumingia by compression,
high or low temperature, centrifuging, or anaerobiosis, Titlebaum,
1928, Tyler, 1930) double monsters are formed, or the blastomeres
may, if isolated, give rise to more or less complete larve. In isolated
normal half-blastomeres we thus find a self-differentiation in AB, on
the one hand, differing from that of CD on the other, but when the
vegetative pole-plasm is evenly distributed, both are capable of pro-
ducing all organs. An interesting problem still remains unsolved.
The isolated normal blastomeres differentiate as fragments, but would
the same blastomeres behave in the same way if placed in contact
with each other atypically? Although the ‘‘organ-forming’’ sub-
stances are now separated by cell-walls, could any kind of interaction
be detected, any kind of change of the prospective significance of the
material be brought about? Penners (1926, 1934) obtained, after
killing teloblasts of Tubifex at different stages, a development in many
respects of mosaic character, but also detected a certain dependence
of the ectodermal and mesodermal components of the germ-bands on
each other, and on the entoderm of the germ-bands, etc.

In Cerebratulus we also have an early rearrangement of substances,
namely at the time of the breakdown of the germinal vesicle. But
these substances are not, as far as we know, unevenly distributed to
the quadrants. As has been pointed out above, we do not yet know
with certainty or in detail at what time the progressive animal-
vegetative determination is accomplished. The potencies of the
blastula are still particularly obscure. Nor do we know how much
of that determination is due to a rearrangement of substances, or to
another metabolic process. A close study of mesenchyme formation
in Cerebratulus, both in normal development and in animal-vegetative
and meridional fragments, would be of special interest in rendering
possible a comparison with the conditions in annelids and mollusks.

DETERMINATION IN CEREBRATULUS EMBRYO 341

IX. SUMMARY

1. The prospective significance of the animal and the vegetative
cells of the 8-cell stage, and of the layers an, ane, vegi, and vege of the
16-cell stage (Fig. 1) was studied. an; forms the greater part of the
pretrochal ectoderm, including (probably) the most anterior part of
the ciliated band (Fig. 3). ame gives rise to the rest of the pretrochal
ectoderm, and a great part of the ciliated band. veg) also partakes in
the formation of the ciliated band, and, moreover, differentiates into
cesophagus and the insides of the lappets. veg, corresponds to the
stomach. The boundary between the layers could not, however,
always be determined with complete accuracy.

2. The first furrow may form any angle to the median plane of the
larva.

3. The two isolated half-blastomeres from one egg may develop
into pilidia with perfect bilateral symmetry (Fig. 2B, C). When the
isolated half- and quarter-blastomeres are less typical, their abnormal-
ities do not show from which part of the egg the dwarf larve come.
The bilateral symmetry is not determined in the early cleavage stage.

4. Animal and vegetative fragments differentiate, as far as we can
judge from comparison with our results concerning the prospective
significance, in the same way as they would have done in normal
development: the animal (Fig. 5), and the vegetative (Fig. 6) cells of
the 8-cell stage; an; (Fig. 7, A—D) ; vege (Fig. 7, £); ane + veg: + vege
(Fig. 8, A); an; + ane + veg, (Fig. 8, B, C); ane + veg: (Fig. 9).

5. Larve composed of an; + vege did not form any of the organs
normally derived from the excised middle part of the egg (Fig. 10).
Fusion of an animal with a meridional half of the 8-cell stage also
showed a complete mosaic development (Fig. 11).

6. The results are discussed and compared with those with sea
urchins, annelids and mollusks. The desirability of further investiga-
tions is emphasized.

LITERATURE CITED

Cor, W. R., 1899. The maturation and fertilization of the egg of Cerebratulus.
Zool. Jahrb., Anat., 12: 425.

DrigEscH, Hans, 1891. Entwickelungsmechanische Studien. I. Zezischr. f. wiss.
Zool., 53: 160.

HAMMaARSTEN, OLoF D., 1918. Beitrag zur Embryonalentwicklung der Malacobdella
grossa (Miill.). Inaugural-Dissertation Stockholm. Uppsala 1918: 1.
Also in Arbeten fr. Zootomiska Institutet, Stockholm, 1.

Hatt, PIERRE, 1932. Essais expérimentaux sur les localisations germinales dans
l’oeuf d’un Annélide (Sabellaria alveolata L.). Arch. d’Anat., Micr., 28: 81.

HOrsTApius, SVEN, 1928. Uber die Determination des Keimes bei Echinodermen.
Acta Zoologica, Stockholm, 9: 1.

Horstapius, SvEN, 1931. Uber die Potenzverteilung im Verlaufe der Eiachse
bei Paracentrotus lividus Lk. Arkiv. Zool., Stockholm, 23: No. 1.

342 SVEN HORSTADIUS

Horstapius, SvEN, 1935. Uber:die Determination im Verlaufe der Eiachse bei
Seeigeln. Pubbl. Stazione Zoologica, Naples, 14: 251.

HORSTADIUS, SVEN, 1936a. Weitere Studien iiber die Determination im Verlaufe
der Eiachse bei Seeigeln. W. Roux’ Arch. Entw.-mech., 135: 1.

Horstapius, SVEN, 19366. Uber die zeitliche Determination im Keim von Para-
centrotus lividus Lk. W. Roux’ Arch. Entw.-mech., 135: 1.

HORsSTADIUS, SVEN, AND ALEXANDER Wo tsky, 1936. Studien tiber die Determina-
tion der Bilateralsymmetrie des jungen Seeigelkeimes. W. Roux’ Arch.
Entw.-mech., 135: 69.

HUXLEY, JULIAN S., AND G. R. DE BEER, 1934. The Elements of Experimental
Embryology. Cambridge University Press.

LEBEDINSKY, J., 1897. Beobachtungen iiber die Entwicklungsgeschichte der
Nemertinen. Arch. mikr. Anat., 49: 503.

Nussavum, J., AND M. Oxner, 1913. Die Embryonalentwicklung des Lineus ruber
Miill. Zettschr. wiss. Zool., 107: 78.

PENNERS, A., 1924. Experimentelle Untersuchungen zum Determinationsproblem
am Keim von Tubifex rivulorum Lam. I. Arch. mikr. Anat. u. Entw.-
mech., 102: 51.

PENNERS, A., 1926. Experimentelle Untersuchungen zum Determinationsproblem
am Keim von Tubifex rivulorum Lam. II. Zeitschr. f. wiss. Zool., 127: 1.

PENNERS, A., 1934. Experimentelle Untersuchungen zum Determinationsproblem
am Keim von Tubifex rivulorum Lam. III. Zeztschr. f. wiss. Zool., 145: 220.

SCHLEIP, WALDEMAR, 1929. Die Determination der Primitiventwicklung. Leipzig,
Akad. Verlags.

TITLEBAUM, A., 1928. Artificial production of Janus embryos of Chaetopterus.
Proc. Nat. Acad. Sct., 14: 245. '

TyLer, A., 1930. Experimental production of double embryos in annelids and
mollusks. Jour. Exper. Zodl., 57: 347.

von Upsiscu, L., 1925. Entwicklungsphysiologische Studien an Seeigelkeimen, I.
Zeitschr. wiss. Zool., 124: 361.

von Usiscu, L., 1936. Uber die Organisation des Seeigelkeims. W. Roux’ Arch.
Entw.-mech., 134: 599.

Voet, WALTHER, 1923. Morphologische und physiologische Fragen der Primitivent-
wicklung, Versuche zu ihrer Lésung mittels vitaler Farbmarkierung. Siéz.-
ber. d. Ges. f. Morph. u. Physiol. Miinchen, 35: 22.

Voct, WALTHER, 1925. Gestaltungsanalyse am Amphibienkeim mit Ortlicher
Vitalfarbung. I. Arch. Entw.-mech., 106: 542.

WESTBLAD, EINAR, 1923. Zur Physiologie der Turbellarien. Kungl. Fysiograf.
Sdllsk. 1. Lund Handlingar, N. F., 33: No. 6.

Witson, CHARLES B., 1900. The habits and early development of Cerebratulus
lacteus (Verrill). Quart. Jour. Micros. Sct., 43: 97.

Witson, Epmunp B., 1903. Experiments on cleavage and localization in the
nemertine egg. Arch. Entw.-mech., 16: 411.

Witson, Epmunp B., 1904a. Experimental studies on germinal localization. I.
The germ-regions in the egg of Dentalium. Jour. Exper. Zodl., 1: 1.

Witson, Epmunp B., 1904+. Experimental studies on germinal localization. II.
Experiments on the cleavage-mosaic in Patella and Dentalium. Jour.
Exper. Zoél., 1: 197.

Yartsu, N., 1904. Experiments on the development of egg fragments in Cerebratulus.
Biol. Bull., 6: 123.

Yatsu, N., 1910a. Experiments on cleavage in the egg of Cerebratulus. Jour.
Coll. Sct. Tokyo, 27: No. 10.

Yartsu, N., 19106. Experiments on germinal localization in the egg of Cerebratulus.
Jour. Coll. Sci. Tokyo, 27: No. 17.

ZELENY, CHARLES, 1904. Experiments on the localization of developmental factors
in the nemertine egg. Jour. Exper. Zoél., 1: 293.

PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRE-
SENTED AT THE MARINE BIOLOGICAL LABORATORY

Jumy, 6.1937

Parthenogenetic merogony in the Naples sea-urchins. Ethel Browne
Harvey.

Parthenogenetic merogony, or development of non-nucleate parts of eggs
which have been artificially activated, has been studied in the four species of sea-
urchin commonly occurring at Naples. The non-nucleate fractions were obtained,
as previously, by breaking the eggs apart with centrifugal force.

In Arbacia pustulosa, which is similar to the Arbacia at Woods Hole, asters
and cleavages occur in the non-nucleate half; when activated, similar to what has
been described for Arbacia punctulata.

In Parechinus (Echinus) microtuberculatus and Paracentrotus (Strongylo-
centrotus) lividus, the fertilization membrane in activated non-nucleate halves is
well separated from the cell surface, just as in normally fertilized whole eggs of
these species. A large monaster is formed, an amphiaster and there are one or two
cleavages. The cleavage planes frequently disappear and some time afterward the
egg breaks up into a number of pieces. These become progressively smaller and
more numerous until what resembles a blastula is formed. This breaks through
the fertilization membrane, but no further development has been observed. The
non-nucleate halves of Sphaerechinus granularis can also be activated and they
break up into small pieces in a similar way.

It might appear that this breaking up of the egg is a form of degeneration or
cytolysis. But the same spontaneous breaking up of the cell has been observed
also in normal whole nucleate eggs artificially activated. These sometimes gave
rise to typical blastulae which became free swimming and looked normal in every
respect.

It has also been found that by treating the immature egg with parthenogenetic
agents, there is formed a very definite layer on the periphery of the cell similar to
the ectoplasmic layer in the fertilized or activated mature egg. This is formed
both on the half containing the germinal vesicle and the heavier half separated
from it by centrifugal force. Another evidence of activation is the failure to
respond to sperm by the formation of blisters on the surface. The treated, im-
mature egg, after some time, pinches off a small piece, always at the part most
distant from the germinal vesicle in elongated eggs. Later there are two, three or
more pieces and then a great number.

A recent critical examination of prepared slides of the parthenogenetic mero-
gones of Arbacia punctulata shows the presence of asters, usually in pairs, but no
chromosomes. The Feulgen reaction, in the procedure of which I was assisted
by Dr. Jean Brachet, is entirely negative. The parthenogenetic merogones show
no red-staining nuclear material, whereas the fertilized merogones which were
used as control material, show it very clearly.

Some oxidative properties of isolated amphibian germinal vesicles.
Jean Brachet.

Amphibian germinal vesicles isolated from full-grown odcytes in a saline
solution absorb oxygen and eliminate carbon dioxide during several hours; the

343

344 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

oxygen consumption was measured by means of a modified Garard-Hartline micro-
respirometer using Triturus pyrrhogaster as material while the COz excretion of
Rana fusca nuclei was estimated by a titrimetric micro-method. The QOs2
(cu. mm. O2)
—_____________—_ of the isolated nuclei (the approximate wet weight being
grams wet weight X hour
calculated from the volume and the density) is about 13 while the full-grown
odcytes have a QO: of 37. The respiration of one single isolated nucleus is only
1-1.5 per cent of the amount of gas exchanged by one full-grown odcyte deprived
of its follicular epithelium. Removal of the germinal vesicle does not affect
markedly the CO: elimination of full-grown odcytes during several hours. Ad-
dition to the nuclei of glucose or of cytoplasm removed from the odcytes with a
micropipette does not increase their respiration. Presence or absence of Ca ions
(which, according to W. R. Duryee, greatly affect the physical properties of the
germinal vesicle) does not affect appreciably the metabolic rate of the isolated
nuclei.

Influence of temperature and other agents on the respiration and devel-
opment of marine eggs. Albert Tyler. (Most of the data given in
this report appear in an article on p. 261 of this issue of the Biolog-
ical Bulletin under the title, “On the energetics of differentiation,
VI.” The report has also been abstracted in the Collecting Net of

July 10.)
aoe wants

Effects of fatigue due to muscular exercise on the Purkinje cells of the
cerebellum of mice at various ages. Warren Andrew.

The problem of morphological changes in nerve cells as the result of func-
tional activity has been studied by a number of investigators. Nevertheless, the
question still remains open not only as to what changes occur due to activity but
as to whether any such changes do occur.

Among the factors to be taken into account in such work is that of age.
The present experimental work is based on earlier work in which the Purkinje
cells of mice and rats were studied from the time of their differentiation up to
and including extreme senility of the individual.

Sixteen black mice were used in the experimental work on fatigue. “ Fa-
tigue”’ means complete exhaustion brought about by running in a motor-driven
rotary cage. For each fatigued animal, a control animal of the same brood was
killed at the same time and the tissues from the two animals carried through the
technical processes together.

The animals represent a range of ages including pairs of 23 days, 25 days, 43
days, 46 days, 98 days, 101 days, 746 days, and two animals with marked signs of
senility—of 744 and 746 days, killed without fatiguing. In each case 100 cells
were examined.

The major conclusions to be drawn from the present work are: 1. There are
morphological changes in nerve cells as the result. of fatigue carried to exhaustion,
consisting primarily in a loss of Nissl material, an increased basophilic property
of the nucleus, and an increased average cell size. 2. In senile animals there is
also a loss of Nissl substance and an increase in the basophilic properties of the
nucelus. The binucleate condition of the Purkinje cell is a phenomenon of senility.
The differences between the Purkinje cells of senile and of young animals are far
- more marked than are those between exhausted and fresh animals of the same age.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 345

Localization in the oculomotor nuclei of the goldfish. Zareh Hadidian,
Milton S. Dunn and Roland Walker.

The position of cells innervating the individual eye muscles was studied to see
whether there is any pattern in the oculomotor nuclei which might be correlated
with the type of eye coordination, which differs from that in animals with bi-
nocular vision.

The method was a study of chromatolysis in the oculomotor cell bodies after
cutting a peripheral nerve or its muscle, since these operations gave similar re-
sults. In normal animals there was a negligible proportion of cells showing ad-
vanced degeneration, while in animals with both eyes removed practically all the
cells showed chromatolysis. There were no changes in the nuclei studied after re-
moval of the contents of an eyeball.

When the contents of one orbit were removed, thus insuring complete
damage to all fibers of III and IV on that side, about half of all the oculomotor
and trochlear cells were altered.. Chromatolysis in the trochlear nucleus was con-
tralateral, while the degenerate oculomotor cells were about 70 per cent homo-
lateral, the contralateral ones being mostly in the ventromedial portion.

Chromatolysis after damage to the inferior oblique muscle or its nerve was
about 80 per cent homolateral, with a slight tendency toward ventromedial local-
ization. For the anterior rectus degeneration was about 60 per cent homolateral
with no localization. The inferior rectus showed about 85 per cent homolateral
degeneration with definite localization in the dorsolateral nucleus anteriorly,
changing gradually toward the ventromedial nucleus posteriorly. The superior
rectus had only about 20 per cent homolateral cells, and the contralateral group
was localized in the ventromedial nucleus.

A consideration of these results leads to the conclusion that since there is
poor definition of cell groups, and scattering of cells for any one muscle through-
out the nucleus, any merely anatomical studies of oculomotor localization are in-
adequate for the understanding of the type of eye codrdination in the goldfish.

Some new observations on the secretory activity of neurones. E.
Scharrer. (Followed by Demonstration. )

Nerve cells having more or less the appearance of secretory cells have a wide
distribution. Among the invertebrates they have been found in annelids, molluscs,
crustaceans and insects; among the vertebrates, in the diencephalon of selachians,
teleosts, amphibia, reptiles and mammals, including man. In the case of the bony
fishes, gland-nerve cells have also been found in the nucleus of the nervus ter-
minalis, in the midbrain and in the caudal region of the spinal cord; the latter
being especially well developed in selachians (see Speidel, 1919, 1922). All stages
can be found, from typical nerve cells containing only a few granules in the cyto-
plasma to cells with a spectacular formation and storage of droplets of a col-
loid-like substance. There are even cases, such as that of the Mediterranean fish
Cristiceps, where the nerve cells in the so-called “diencephalic gland” are trans-
formed into gland cells lacking any nervous character. A marked nuclear poly-
morphism is also typical for many gland-nerve cells and pericellular as well as
endocellular blood capillaries are often observed in the secretory diencephalic
nuclei of vertebrates.

Observations of this kind, even when based on such extensive material, would
not suffice to prove the glandular function of the cells in question. It must be
demonstrated that there is a functional cycle in the production of the colloid ma-
terial by the nerve cells in the neuro-secretory regions of the nervous system.
This has been done with the diencephalic gland (nucleus praeopticus) of the
American toad (Bufo americanus). Sections through this gland show all stages,

346 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

from the first appearance of fine granules in the cytoplasma of the cells, which
stain brilliant orange in Azan preparations, to larger droplets, which finally are
extruded and lie as colloid masses among the cells. This cycle can be shown
clearly and the identity of those processes in nerve cells with the different stages
of secretory activity in gland cells seems sufficiently demonstrated. The physio-
logical meaning of the gland-nerve cells is still unknown and a wide field is
opened for future investigation.

Synaptic transmission in the sixth abdominal ganglion of the crayfish.
C. Ladd Prosser.

Transmission through the sixth abdominal ganglion of the crayfish was
studied by simultaneous recording of impulses entering and leaving the ganglion
in response to stimulation of caudal sensory hairs. Flexion of one hair gives
rise to one sensory impulse. To set off one efferent unit, however, summation of
two to four afferent impulses in different fibers is necessary. No efferent neurone
is excited by one incoming impulse. When several efferent units are excited more
afferent impulses are required to activate the first efferent unit in a response than
to activate later ones. Thus both convergence and overlap play a part in conduc-
tion through this ganglion.

Ganglionic delays range from 3 to 30 milliseconds as measured from the
time the first sensory impulse enters the ganglion. Most of the fastest units show
delays of 5-6 milliseconds, and later ones fall in groups which are multiples of the
first. This multimodal distribution of delays is interpreted as indicating the ex-
istence of internuncial neurones. A given unit may show fluctuation of 2-3 milli-
seconds in synaptic delay. The afferent neurones respond to stimuli separated by
intervals as short as .01 second, whereas the efferents show recovery times of
.05 to .1 second. This synaptic recovery time is longer than that of the fibers,
hence no relatively refractory period can be detected in the responses of the indi-
vidual efferent units.

There are no connections from tactile receptors across the ganglion to contra-
lateral efferent nerves of the sixth segment.

Acetylcholine and eserine have no facilitating action upon the synapse. Eser-
ine is toxic in high concentrations. Nicotine blocks conduction through the syn-
apse. Excess potassium reduces action potentials in the afferent fibers and may
block them before affecting the synapse. Adrenaline acts similarly to excess
potassium.

It is concluded that those humoral agents which mediate transmission in some
mammalian ganglia do not have a similar action in this crayfish ganglion.

Jwiex, 20
Chemical stimulation of the amphibian ectoderm. L. G. Barth.

Further work on the chemical nature of the amphibian organizer indicates that
the formation of the neural plate is due to some general stimulus imparted to the
cells by a variety of substances. Earlier experiments using cephalin as an in-
ductor showed that cytolysis occurred in the region of the implant. Following
this other cytolytic agents such as digitonin, acids and bases were used and plates
of neural cells were induced in the presumptive epidermis. Digitonin in concentra-
tions of .5 to .05 °/o in powdered egg albumen and buffers at pH 3 and 10 have
given positive results by implantation into the blastocoel of Amblystoma mexi-
canum.

With regard to the naturally occurring organizer, it was found impossible to
extract it completely with fat solvents. In comparing the action of ether-alcohol
extracts with protein residues, the protein residue gave better neural tubes. The

PRESENTED AT MARINE BIOLOGICAL LABORATORY 347

ether-alcohol extracts were relatively weak in inducing power. The experiments
suggest that the naturally occurring organizer is in the protein residue.

Limb bud transplantation in chick embryos. Viktor Hamburger.

Wing and hind-limb primordia of chick embryos, incubated 2-3 days (25-30
somites) were transplanted to the lateral trunk region or into the coelomic cavity
of host embryos of the same stages. In a considerable number of experiments,
the anterior-posterior and the dorso-ventral axes were inverted with respect to the
axes of the host embryo. The transplants showed complete self-differentiation
with respect to form, size and axial pattern.

Transplants located near the spinal cord of the host were supplied by trunk
nerves or by nerves branching from the brachial or the lumbo-sacral plexus of
the host. The spinal ganglia contributing to the innervation of the transplant
reacted always to even small increase in their peripheral fields by hyperplastic
growth. In several cases, the number of the motor neurones in the level of the
spinal cord which supplied the transplant was likewise found to exceed the number
of motor neurones of the normal side, the hyperplasia ranging from 14 per cent
to 30 per cent.

Transplants located far ventrally were not innervated by spinal nerves. They
showed, nevertheless, normal development and differentiation. The embryonic
development of the limbs of the chick is therefore independent of innervation.

Adult organizers and their action in adult tissues. Oscar E. Schotté.

The development of a salamander, Amblystoma punctatum. L.S. Stone
(motion picture. )

The development of the common black, yellow spotted salamander, Ambly-
stoma punctatum, has been recorded in detail on one motion picture reel showing all
the stages from the one-cell egg to the time the larva begins feeding. Rate in de-
velopment was recorded at various speeds from a few hundred to several hundred
times in order to analyse various stages more carefully.

The detailed study in development extends over a period of about four weeks
during which time a constant temperature of about 70° Fahrenheit was maintained.

During segmentation one can see clearly the movement of blastomeres, the
regional waves of cell division and movement of egg mass due to shifts in the
center of gravity. The formation of the blastopore and the development of the
neural plate and closure of the neural folds are shown in detail. All the changes
in surface development are shown throughout the tail-bud stages from the time
at which cilia on the surface of the embryo first come into action to the period
when motor activity begins. Occasional local quivering movements in various parts
of the embryo are also recorded.

Following these periods in development, dorsal, ventral and lateral views of
embryos are shown which carry the growth up to the time the larva begins feeding.
The formation of many external features as well as the beginning of peristaltic
movements in the intestines are clearly seen.

In appropriate parts of the film are shown clusters of eggs as they are nor-
mally laid, circulation in the tail fin, and a view of the larva taking its first meal.
The picture begins with a view of a typical habitat and ends with a view of several
stages in development from the egg to the adult animal in order to give a compari-
son of sizes during growth.

348 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

Jury 27

The effect of standing on the carbon dioxide content of alveolar air and
total ventilation volume. J. K. W. Ferguson and F. A. Hitchcock.

A study of the respiratory exchange during consecutive ten-minute periods
of reclining and standing, has revealed in every experiment a decrease of 7-15
per cent in the CO: content of alveolar air (confirming Higgins, 1914, Turner,
1927 and Main, 1937). This fall, however, cannot be attributed to overventilation
in the usual sense of the word, because: (1) the R.Q. fell during standing in 21
out of 24 experiments, (2) the CO. output fell in 12 out of 24 experiments, (3)
the total ventilation fell in 9 out of 24 experiments.

When four consecutive periods, with the subject alternately reclining and
standing, were studied, it was found that the Oz consumption and the CO. output
were greater during the second reclining period than during the preceding stand-
ing period. This is interpreted as evidence of oxygen debt and COs: retention
during standing. It seems probable that this disequilibrium, during standing, be-
tween metabolic requirements and the respiratory exchange is due not to inade-
quate pulmonary ventilation but to inadequate circulation in the dependent parts
of the body resulting from the erect posture.

The mechanism of the loss of heat from the human body. Eugene F.
DuBois and James D. Hardy.

The three main channels of heat loss are radiation, convection and vaporiza-
tion. The respiration calorimeter of the Russell Sage Institute of Pathology in
New York Hospital measures the vaporization by weighing the moisture that
comes from the skin and lungs. It measures radiation plus convection in a stream
of cool water that flows through coils in the top of the calorimeter. Radiation is
then determined independently by means of a Hardy radiometer pointed in rapid
succession at 20 different places on the surface of the body. The total surface is
measured and the effective radiating surface calculated as 80 per cent of the
total. Convection is then estimated by difference. It has been possible for the
first time to separate the factors of radiation and convection in persons exposed
to varying atmospheric conditions such as are found indoors.

Two normal men were studied naked at temperatures between 22 and 35°C.
Their basal metabolism was uniform throughout this range not rising until a few
minutes before the onset of shivering which occurred after exposures of two
hours at 22°C. At the higher temperatures there was profuse sweating and
practically all of the heat was lost through vaporization. The percentage of heat
lost in radiation decreased steadily with rising temperatures. In the neutral zone
of 28 to 32°C. convection accounted for 10-15 per cent of the heat loss and radia-
tion 50-60 per cent. Convection was markedly increased by slight movements of
the body. With moderate exercise or shivering it accounted for 25-30 per cent
of the total loss. An electric fan raised the percentage to 33. Two athletes
playing violent squash for 36 minutes showed a 2°C. rise in rectal temperature
and about an equal fall in average surface temperature. The total heat lost
through radiation increased but little, the percentage fell to 15. Convection dur-
ing the exercise and recovery period accounted for 5-15 per cent. Vaporization
dissipated 70-80 per cent of the heat.

Peripheral inhibition of smooth muscle. Emil Bozler.

The antagonism of vasoconstrictor and vasodilator nerves was studied using
perfused frog legs and rabbit’s ears and recording the vascular responses by a
sensitive flow-meter. Stimulation of the vasodilators of the dorsal roots blocks
the action of single volleys of impulses of the vasoconstrictor fibers, whereas re-

PRESENTED AT MARINE BIOLOGICAL LABORATORY 349

petitive stimulation produces'a response. Likewise acetyl choline blocks entirely
the action of the first few impulses produced by repetitive stimulation. In an
attempt to explain these results a difficulty was encountered in the observation
that neither vasodilator stimulation nor acetyl choline antagonise the action of
adrenaline. The simplest explanation seems to be the assumption that during in-
hibition the passage of impulses from vasoconstrictor fibers to the muscle is
partially blocked, thereby preventing the formation of the adrenaline-like mediator.
It is suggested that the increase of polarisation, which has been observed in other
cases of inhibition, is the immediate cause of the partial block. The mediator
produced by the vasodilator fibers may be the cause of the change of polarisation
of the muscle fibers and, therefore, indirectly also of the partial block produced
by the activity of these nerve fibers.

The relationship of tissue chloride to blood chloride. William R. Am-
berson, Thomas P. Nash, Arthur G. Mulder and Dorothy Binns.

A number of previous investigators have found that amphibian muscles, placed
in, or perfused with, isotonic sucrose solutions, rapidly lose their chloride almost
completely, while retaining almost all of their potassium and phosphate. When
such muscles are soaked in solutions of varying chloride content, the tissue chlo-
ride varies directly with that of the external fluid. Such observations have led to
the conclusion that muscle chloride is extra-cellular, a view reinforced by its. low
concentration in this tissue.

Other students have recently attempted to extend this concept to other tissues,
and to the mammalian body. In studying this literature it occurred to us that it
would be useful to know whether the chloride of the mammalian tissues can be
diminished when the plasma chloride is lowered. We have found it possible to
produce very radical diminutions in the plasma chloride by a modification of the
method of total plasmapheresis described by Stanbury, Warweg, and Amberson
(Am. Jour. Physiol., 1936, 117, 230). We make up our artificial plasma with
sulfates instead of chlorides, adding chloride-free gum acacia, and suspending in
this plasma ox red blood corpuscles which have been rendered chloride-free by
many washings through sulfate-Ringer-Locke solution.

By long perfusion of the mammalian body we are able to remove most of
the chloride before death ensues. In some tissues, such as skeletal muscle, liver
and kidney, the tissue chloride falls off in direct proportion to the plasma chloride,
as perfusion proceeds. In other tissues the straight line through all the points
does not pass through the origin, but shows a y-intercept, suggesting that a portion
of the chloride is indiffusible, and presumably intra-cellular. The stomach and
spleen have particularly large intercepts of this character.

The central nervous system resists removal of chloride, whereas most of
the chloride of peripheral nerve may be washed away. ‘The brain and cord chlo-
ride is held either (1) because it is largely intra-cellular, or (2) because the
sulfate ion is unable to penetrate into the brain tissue.

Certain tissues, such as tendon and lung, have chloride concentrations so
high that it is impossible to explain it all by allotting it to extracellular fluid.

AuGust 2

The use of diatoms from geological excavations at Clovis, New Mexico,
as indicators of water conditions. Ruth Patrick.

Mammoth Pit lies between Clovis and Portales in the Staked Plains region
of New Mexico. The stratigraphy is as follows: The lowest stratum is coarse
gravel devoid of diatoms. Above this is a stratum of speckled sand, in which is
a diatom flora which seems to indicate a fresh to brackish water condition by the

350 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

dominance of Anomoconeis sphaerophora (Kiitz.) Pfitzner, Amphora ovalis Kiitz.,
and Amphora ovalis var. pediculus Kiitz. Horse skeletons are found in this level.
The next stratum is a “bluish clay” of about the same constituents as the
speckled sand plus considerable carbonized vegetable matter. In the lower part
of this stratum, a very rich diatom flora consisting mainly of fresh water species
such as Eunotia arcus Ehr., Cymbella affinis Kitz. Fragilaria brevistriata Grun.
and Synedia ulna (Nitzsch.) Ehr. was laid down. The change in abundance and
also in the kind of species from the previous level points to a freshening of the
water. This agrees with other geological evidence that this was a period of
much higher precipitation than now exists in New Mexico. Mammoth skeletons
are most abundant in this level of the stratum. Passing from bottom to top of
this stratum the typical fresh water species disappear. The dominant species near
the top are the brackish or alkaline water types such as Epithemia argus Kiitz.,
Rhopalodia gibba (Ehr.) Mill., and R. gibberula (Ehr.) Mill. Coincident with
the change in diatom species, the mammoth bones disappear and bison skeletons be-
come much more numerous. The top stratum consists of brown dune sand devoid
of diatoms. It is in the bottom of this layer that the bison bones disappear. Thus
the change in water conditions as indicated by the diatoms seems to coincide with
a change in the fauna as shown in this stratigraphy.

Gemmuipary in Kalanchoe rotundifolia and other Crassulaceae. Harry
N. Stoudt.

The phenomenon of vegetative propagation in Crassulaceae has received much
attention from students or morphology and physiology. A comparison of the
morphological development of plantlets of this species with other members of the
family should aid in understanding more adequately the phenomenon so character-
istic of the group.

Yarbrough (1936) reports that apparently mature parenchyma cells in the
base of the petiole of Sedum Stahlii resume mitotic activity to form plantlets when
the parent leaf is removed from the plant.

Stoudt (1934) published an account of vegetative propagation in Byrnesia
Weinbergu. In this species a plantlet forms at the base of the sessile leaf from
a residual meristem that is undifferentiated into plantlet rudiments.

Freeland (1933) discusses this phenomenon in Bryophyllum crenatum in which
plantlets develop from residual meristems in the notches of the parent leaf. He
finds that the amount of differentiation of plantlet rudiments varies. A stem
primordium only may be formed by the time the parent leaf is mature.

A nine-millimeter leaf of Kalanchoe rotundifolia reveals a meristematic region
on the adaxial surface of the petiole. This region is undifferentiated into plantlet
rudiments but when the leaf attains its maximum size, three to four centimeters in
length, leaf and stem primordia have formed. The bud then remains dormant
until the leaf is severed from the parent plant.

Root, stem and leaf primordia are usually visible in the notches of the mature
leaves of Bryophyllum calycinum according to Naylor (1932) etc. while in Kalan-
choe daigremontiana and K. tubiflora plantlets consisting of root, stem, and leaf
primordia are visible macroscopically even before the parent leaf has attained
maximum size.

Thus there is a definite sequence into which the various species arrange them-
selves in respect to the degree to which the meristematic cushion becomes dif-
ferentiated by the time the parent leaf is mature. Their greatest differences are
in the stage of development attained by the meristem, or organ rudiments derived
from it, at this time.

PRESENTED AT MARINE BIOLOGICAL LABORATORY Sol

Pollen analysis of the air in relation to hay-fever. A. O. Dahl.

It is essential for the successful diagnosis and treatment of hay fever (“ pol-
lenosis”’) that detailed data concerning the concentration of pollen in the air of
any species at a given time or locality be made available. Records of the pollen
content of the air have been obtained by exposing each day an oil-coated slide out
of doors for 24 hours. The pollen grams observed in 25 systematically distri-
buted low-power fields are identified and counted. The approximate number of
pollen grains per cubic yard of air can be calculated by use of physical formulae.
The pollen grains involved in hay fever in Minnesota vary from approximately
15 to 80 micra in diameter.

Hay fever is a regional problem and atmospheric data from one locality will
not apply in a detailed manner to another place. In Minnesota, pollen concen-
trations of clinical interest occur between late March and early November. For
purposes of clinical convenience, the pollens found in the air during the entire
season have been placed into 16 groups. Thus, in diagnosis, one scratch-test for
each group will test the patient’s sensitivity to all pollens present during the entire
season. Prefaced by such procedure, successful therapeutic measures can be insti-
tuted. (The complete report is to be published in joint authorship with Dr. C. O.
Rosendahl and Dr. R. V. Ellis, under whose direction the study has been carried
on for the last 5 years at the University of Minnesota).

Avueust 3

A convenient test of physical agents as producers of dominant lethals.
P. W. Whiting.

Dominant lethals occurring in the spermatozoa cause failure of development
of zygotes. Since in bisexual reproduction eggs also fail to develop unless fer-
tilized, the two types of male sterility, due to (1) lack of sperm and (2) dominant
lethals, cannot be statistically distinguished. In Habrobracon, reproducing males
by haploid parthenogenesis, matings with males lacking sperm result in as many
progeny per day (gd) as cultures from unmated females (4.73 gd) or as matings
with untreated males (1.21 fd, 3.30 99). If fathers are X-rayed, males per day
are not increased while females per day are decreased (to 2.22 with 2,500 R, 0.62
with 5,000 R, 0.16 with 7,500 R, 0.07 with 10,000 R, and 0 with 20,000, 40,000 or
75,000 R). Since neutrons induce dominant lethals and ultra-short (one-meter)
radio waves do not, only the former may be expected to induce recessive lethals
and visibles.

Cytological observations on colchicine. Bernard R. Nebel.

The action of the alkaloid colchicine on mitosis was studied in the following
material: .

Stamen hairs of Tradescantia, roots and shoots of Zea, Vicia, Tomato,
Tagetes, Antirrhinum, Trifolium, Papaver, Dianthus, Solanum, and Lilium,—
testes of Podisma, eggs of Asterias and Arbacia.

In Tradescantia all stages of mitosis are easily seen in life. The cells will
continue to divide in salt sugar solutions to which drugs may be added.

Colchicine in concentrations of 5 X 10* to 5 X 10° molar will stop mitosis
during metaphase. In concentrations of 5 X 10° to 10° molar it tends to produce
binucleate cells. When blocked in metaphase the plate in Tradescantia is char-
acteristically tilted, since a true spindle is not developed. A comparative study of
phenyl-, amyl-, propyl-, ethyl urethane and chloral-hydrate showed that these
anesthetics within their respective reversible concentration ranges will only oc-
casionally produce binucleate cells. There is no particular evidence of a metaphase

352 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

block with the urethanes. In studying the action of colchicine on the developing
egg of Arbacia punctuiata it was necessary to use fixed material to determine the
nuclear stage accurately. Colchicine applied 10 minutes after insemination will
block the first cleavage metaphase in concentrations above 10* molar. Between
6.5 X 10° and 3.5 X10° molar nuclear divisions of abnormal type proceed while
cleavage is impeded. The rhythm of nuclear division may persist so that when
controls are in third cleavage metaphase the eggs to which colchicine was added
10 minutes after fertilization will also show approximately 4 groups of chromatin,
but the plates are hypoploid, often containing only one to six separate chromosomes,
which may partly represent fused units. The micronuclei which form are not far
apart from one another and their resting stage is relatively short; with lower con-
centrations the rhythm of division is not affected in the early cleavages, but the
subsequent development of the larvae is markedly stunted even at 10° molar.
Colchicine in increasing concentrations thus first interferes with the normal course
of cleavage, then astral rays are prevented from forming, next the spindle is re-
duced in size and finally obliterated. Meanwhile chromosomes become pycnotic
during a prolonged metaphase; they fail to divide orderly; the abnormal division
may separate entire chromosomes rather than split halves.

In the plants, in which root and shoot growth was studied (Ruttle) cuttings and
seedlings respectively were subjected to colchicine treatments by immersion in
aqueous solutions primarily. The active concentration ranges were found to be
the same as in Arbacia and Tradescantia. All genera showed marked reactions,
the tomatoes being the least sensitive. Where plant meristems were treated, the
resulting tissues showed markedly irregular growth—incised and crumpled leaves
as well as chlorophyll defects. Cytological investigation showed necrotic cell
lineages and multinucleate cells in varying degrees. The drug is being studied
further as an agent which may induce mutations and polyploidy.

In cooperation with other investigators, some physiological effects of col-
chicine were investigated. Respiration was tested on Arbacia eggs with col-
chicine 1.8 X 10° to 7.5 X 10°. Respiration did not vary from the control in any
of these concentrations (Tyler).

Pectinmethoxylase (pectase) from tomato juice showed inhibition which is
of doubtful significance since with higher concentrations of colchicine a precipita-
tion was observed in the reaction mixture (Kertesz).

The nitroprussiate reaction for S— H=S—S groups gives a color reaction
in stamen hair cells of Tradescantia, the color being located in the chromonemata
and in certain plasmatic granules. Cells under colchicine gave the same reaction
(Medes).

No significant influence was observed on the action of carbonic anhydrase
from blood.

No inhibition occurred of the reduction of methylene-blue by yeast or blood
with and without admission of air.

No sensitization of Arbacia eggs to X-rays was obtained by the addition of
colchicine, when nuclear dbnormalities were used as a criterion.

Demonstration of vital staining preserved in paraffin sections of lamprey
embryos (Bismark Brown method). R. Weissenberg.

In 1929 I recommended Bismark-brown for localized vital staining of the egg
of lamprey because it is very easy to preserve these stained areas for paraffin
sections.

The quick-working method which I am using now in my studies of localiza-
tion on lamprey egg is a very simple one. It is based on the surprising fact that
Bismark-brown as a vital dye is alcohol-proof without further treatment in con-
trast to Nile-blue sulfate.

a ee

PRESENTED AT MARINE BIOLOGICAL LABORATORY Soh)

I fix the embryos in a mixture of 1.5 parts of absolute alcohol and 0.5 part of
acetic acid for ten minutes, wash in absolute alcohol for a few minutes, and trans-
fer them directly into cedar oil.

The stained areas are very well preserved by this simple method and the
eggs can remain in the cedar oil for years without any loss of the dye. They can
be studied in the cleared condition in the cedar oil as total preparations, or they
can be imbedded in paraffin at any time and cut with excellent preservation of the
vital stain within the sections.

The slides demonstrated are balsam preparations seven years old and still
give a true representation of the stained areas of the living organism. The
preservation obtained by this method is complete also in the finer localization of
the staining. Carriers of the vital staining in the egg of lamprey are chiefly the
yolk granules because here, in contrast to most amphibian eggs, pigment granules
are missing in the earlier stages of the embryo.

Microfilm on some experiments on isolated amphibian germinal vesicles.
William R. Duryee.

The film shows colloidal changes in the frog ovocyte nucleoplasm, nucleoli,
and chromosomes brought about by relatively slight changes in the Na, K, Ca
chloride concentrations of the medium. H* ions reverse the normal negative
charge on the nuclear components to positive. When this change is gradual
enough, as with 0.003 N HCl, a dark converging “ring” forms from flocculating
particles in the approximate pH region of 4.0 to 5.0.

Ca, Mg, Cu, Hg and basic dyes behave similarly to H* ions in causing a phase
separation and an appearance of chromosomes from a previously transparent nu-
cleus. On the other hand, K and Na and especially OH tend to disperse the
nuclear colloids, thus stretching and separating the chromosome pairs, and at the
same time making the nucleus transparent. Within narrow limits these changes
are reversible.

In Triturus pyrrhogaster, the “ Binnenkorper,’ or first maturation spindle
anlagen situated at the center of the germinal vesicle, can be made to swell and
separate the chromosomes radially, but not in typical bipolar directions. Jana
fusca appears unique in having a differentiated coagulable capsule around the
chromosomes, which may be important in forming the denser portions of the
spindle. In R. pipiens the contraction of this material under the influence of
calcium is less striking.

Similar changes, including phase separation and violent contraction of the
chromosomes, occur when acid fixatives are added or when the nuclei are exposed
to their disintegrating cytoplasm. Hence this latter effect may be termed auto-
fixation. Prominent differentiated areas or sac-like projections of the nuclear
membrane reversibly swell and shrink in bases and acids respectively. Such struc-
tures are obliterated by fixatives. It is concluded that in the forms studied Dar-
lington’s assertion as to the absence of a nuclear membrane must be modified.
With merely fine forceps and pipette it is easily possible to isolate various com-
ponents of these giant nuclei (0.8 mm. diameter),— e.g. nuclear membrane, spindle
anlagen, nucleoli, and chromosome pairs (100-200“)—during the fall, winter and
early spring months of the year.

Cortical cytolysis of the echinoderm egg. Robert Chambers. (Motion
picture. )

Motion pictures were taken of those experiments designed to demonstrate the
physical properties of the cellular cortex particularly in marine ova. Nearly all
experimentally induced effects on living cells are extremely transitory. A later
study of the motion picture record permits of a more careful and complete analysis

354 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

of these phenomena. For example, the film may be slowed down, stopped at any
point or reversed at will. The film presented at the seminar is a compilation of
many of these experiments.

Included in the film is a scene showing the spontaneous coalescence of a de-
nuded Arbacia egg with an oil drop. From such a record it is possible to measure
with considerable precision the diameter of the oil drop at the instant of coalescence.
These data are essential if the coalescence phenomena are to be treated quantita-
tively, yet it is practically impossible to obtain the oil drop sizes in any other way.

Another scene shows the natural elevation of the cellular surface and the re-
action of this surface to mechanical injury. Over-insemination of an immature
Arbacia egg results in the formation of many large insemination cones. Gentle
manipulation with a microneedle will cause these cones to run together, thus bring-
ing about an elevation of a continuous film which is separated from the granular
cytoplasm by a liquid space. If this elevated film is ruptured at one spot with a
microneedle, the entire film will rapidly disintegrate. The underlying cytoplasm
becomes exposed and it quickly becomes converted into an irreversible coagulum.

The following phenomena, inter alia, are also demonstrated: cytolysis of star-
fish eggs in hypotonic sea water, effects of tearing eggs in pure salt solutions
isotonic with sea water and the shrinking of the cortex of one blastomere following
the puncture of the other.

The photography was done by Mr. C. G. Grand.

Aucust 10

Some aspects of normal and regulative development in the colomal ciliate,
Zoothamnium alternans. F. M. Summers.

A remarkable number of studies on metazoan “ organizers” have already demon-
strated the importance of extrinsic factors for determination in specific parts. It
was felt that additional information about these factors could be gained by ap-
plying operative techniques to an animal type in which, presumably, the relation-
ships of parts have not attained so great a degree of complexity. Zodthamnium
alternans is a protozoan colony whose cells collectively possess in some degree
many of the attributes of an individual organism. It is admirably adapted to this
type of work for many reasons, particularly by virtue of the precision with which
the characteristic colonial pattern develops.

One of the most important consequences of this study of more than 200 normal
and operated colonies is the demonstration of qualitatively different physiological
relations between cells at different locations on the colonial framework. Under
normal conditions a specific pattern unfolds. When the cell at the apex of the
frond-like colony is cut away some cell of a lower order, one whose complete
developmental potentialities are never otherwise expressed, assumes the dominant
generative functions and the normal colonial pattern perseveres in the parts re-
generated by it. These results are intelligible in terms of what Child (1929) calls
physiological correlation: the relations of dominance and subordination between
parts. Apical control appears to be continuous and quantitative.

In this organism the transformation of the apical cell into an ex-conjugant
initiates a developmental phase which furnishes another clue to the general nature
of apical control. About four days after the union of gamonts, even before ex-
conjugant generations are produced, the normal developmental relations are upset
in an unusual way. The first three or four branches below the conjugant level
begin to develop out of all proportion to the normal expectations. Each branch
develops almost as an individual colony. Its cells divide precociously, forming
secondary and even tertiary branch strains. The greatest effect obtains on the
branch nearest the conjugant and diminishes basally as a gradient ; the basal
branches are apparently unaffected.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 355

Under varying physiological conditions in the apical cell the coordinating in-
fluences exerted upon the mitotic activity of neighboring cells may be inhibitory
(as shown by the regulative response after the apical cell is removed) or excita-
tory (when the apical cell is transformed into an ex-conjugant). The precocious
development does not occur when the apical cell is present or when it is dissected
away; it appears to be effected by some new quality in the coordinating mechanism
arising in consequence of conjugation activities in one particular cell—the apical
cell. These results invite the conclusion that the integrative factors in a colony
of Zoothamnium are qualitative and discontinuous.

Morphology, behavior and reproduction in Type A and Type B of Chaos
chaos Linnaeus, the giant multi-nucleate amoeba of Roesel. A. A.
Schaeffer.

Chaos chaos Linnaeus 1767,.the first amoeba to be discovered, was described
by Roesel von Rosenhof, a painter of miniatures, in 1755, in Germany. It has been
seen 5 times since then: in 1900 by H. V. Wilson, North Carolina; in 1902 by E.
Penard, Switzerland; in 1916 by W. A. Kepner in Virginia and by A. A. Schaeffer
in Tennessee; in 1936 by A. A. Schaeffer in New Jersey.

The general morphology and behavior of this amoeba are so much like those of
the common laboratory amoeba, Chaos diffluens Miller, 1786, that there is strong
probability that both amoebas belong to one and the same species. Conclusive
evidence of such relationship is, however, still lacking and therefore, until such
evidence is found, the two taxonomically specific names, C. chaos and C. diffluens,
will be used to avoid confusion.

C. chaos falls into at least two types which are distinct in some morphological
details and also in antigenic reactions. Type A has discoid nuclei and divides
usually while freely rolling around on the substrate. Type B has broadly ellipsoid
nuclei, and divides while fastened to the substrate. The nuclei of type A are larger
than those of type B. With the collaboration of Dr. J. A. Harrison, preliminary
antigenic tests were made in which it was found that there is a marked difference
between the two types, type B standing closer to diffiluens than to type A.

Three of the most striking differences between chaos and diffluens are: in size,
chaos being from 50 to 500 times as large as diffluens; in number of nuclei, chaos
being multinucleate (up to 1,000 or more) ; in reproduction, chaos dividing at any
single division, into 2, 3, 4, 5 or 6 daughters, with a strikingly marked mode at 3
daughters.

Pieces cut from chaos grow up to full size, whether the piece contains one or
more nuclei.

Both types give off, when crushed, a strong cucumber-like odor which can
readily be detected when only one amoeba is crushed on a slide. In diffluens this
odor is also present but to a much smaller degree.

aH ea j

Observations upon the chemical composition and the metabolism of a
larval parasitic nematode. Theodor von Brand.

The experiments were performed with an immature Eustrongylides from
Fundulus heteroclitus. The red color of the worms is due to the presence of hemo-
globin in the body fluid. With its low fat and high glycogen content the general
chemical composition resembles that of the adult Ascaris. The worms consume
per unit weight much less glycogen than Ascaris, if kept in saline at 37° C. under
aerobic conditions. They are able to keep their glycogen level high, even if their
hosts starve for a long time and lose during this starvation period more than half of
their polysaccharide stores.

356 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

Observations and experiments on sex change in the adult American
oyster, Ostrea virginica. Paul S. Galtsoff.

Sex of oysters was determined by inducing ovulation or ejaculation by in-
creased temperature and chemical stimulation. During the summer of 1936 indi-
vidual spawning records of 203 adult oysters were obtained and the discharged
products microscopically examined. Oysters were measured, carefully marked by
numbers engraved on the shell of each, and transferred to Milford, Connecticut, to
be kept in tidal tanks through the winter. The same procedure was repeated dur-
ing the summer of 1937.

Results showed that 9.7 per cent of the oysters reversed their sex; the per-
centage of reversals being higher among females (13.1 per cent) than among males
(8.0 per cent). Mortality during the year was only 7.04 per cent and failures to
react, due to lack of gonad development, was only 1.07 per cent, indicating healthy
conditions under which oysters were kept.

In the sex-reversed males the physiological set-up of the organism changes
with the sex; typical female reaction, characterized by the rhythmicity of con-
tractions of adductor and maintenance of a constant tonus level, develops in the
former male. In several instances development of this reaction lagged and the
newly-formed female retained physiological characteristics of the male, i.e, eggs
were discharged through the cloaca instead of through the gills and rhythmical
contractions of adductor were absent. All sex-reversed females reacted as true
males.

It is concluded that female reaction developed as a secondary adaptation
providing mechanism for dispersing eggs throughout the water. From the
simultaneous occurrence of sex reversal in the oyster population the conclusion is
made that Orton’s theory of metabolism change (protein to carbohydrate) as
the sex-determining factor in O. edulis is not applicable to O. virginica.

Avucust 17

A sea water buffer for marine eggs. Albert Tyler and Norman H. Hor-
owitz. (This paper has already appeared in full in Science, July 23,
vol. 86, pp. 85-86. )

The effect of CO, upon the oxygen capacity of the blood of some fresh-
water fish. Edgar C. Black and Laurence Irving.

Conditions of respiration for fish differ from the respiratory conditions for
mammals... The respiration of fresh-water fish must proceed in a medium in
which the pressure of oxygen is always less and the pressure of carbon dioxide
usually greater than in atmospheric air. At different levels in the water, pres-
sures of gases are altered by the temperature changes. Types of blood which
are suitable for the transport of CO, and oxygen under one set of conditions
might be quite unsuitable under another set. The characteristics of the blood of
the carp (Cyprinus carpio L.) and the common sucker (Catastomus commersonit)
show examples of two types of blood, each suited for a different and limited
range of pressures of oxygen and CO..

Oxygen dissociation curves obtained from. those two species are not as
sigmoid as are those for mammalian blood. The presence of 5 or more mm. CO,
(for the carp 10 or more) prevents the complete saturation of whole blood, even
at high partial pressures of oxygen. This effect of CO, is quite different from
the familiar effect of CO. upon mammalian blood. In the presence of relatively
high pressures of CO, the blood of the carp is suitable for the transport of
small quantities of oxygen, while the blood of the sucker would be quite useless.

PRESENTED AT MARINE BIOLOGICAL LABORATORY Si

At very low pressures of CO, and high pressures of oxygen in the water the
blood of the sucker can serve to transport much more oxygen than that of the
carp.

Hemolysis by the addition of saponin to the blood of the sucker, carp and
bowfin (Amia calva) abolished the effect of CO, upon the oxygen capacity at
high pressures of oxygen (150 mm.). The CO, effect is in part at least de-
pendent upon the integrity of the corpuscles.

Oxidative mechanisms in the resting and fertilized sea-urchin egg.
Irvin M. Korr.

Since cyanide inhibits certain iron-containing systems, and since pyocyanine,
a bacterial respiratory pigment, functions as a “hydrogen-carrier,” it was possible
to vary the relative proportions of respiration going through simple, non-ferrous
carrier and that going through the cytochrome-indophenol oxidase system; by
adding KCN and pyocyanine, separately and in various combinations, to sea urchin
eggs. The respiratory rates of the untreated fertilized and unfertilized sea
urchin eggs, and those in which the mechanisms had been altered as above, were
measured at different temperatures. These experiments were designd (1) to
give the temperature relations of the two types of respiration, (2) to throw fur-
ther light upon the factors determining temperature coefficients of cellular
respiration and (3) upon the change in oxidative rate and mechanism that occurs
upon fertilization of the sea urchin egg.

It was found, first (in partial confirmation of Rubenstein and Gerard, 1934),
that the temperature coefficient of unfertilized eggs was much higher than that
of the fertilized egg. The effectiveness of KCN was found to increase with
temperature.

Increasing the respiration of the fertilized egg with added hydrogen-carrier
did not appreciably change its temperature coefficient. Fertilized eggs, in which
the iron system had been maximally inhibited and the respiration restored to or
above normal with pyocyanine, also had the same temperature coefficients as the
untreated fertilized eggs.

In the unfertilized egg, whose respiration is cyanide-stable, the addition of
carrier not only increases the rate of respiration, but also, above a certain con-
centration, lowers the temperature coefficient. A concentration of pyocyanine
which increases the respiratory rate to that of the fertilized egg also lowers the
coefficient to that of the fertilized egg.

The results, in conjunction with older work, lead to the conclusions that
(1) respiration through a simple non-ferrous carrier and that through the cyto-
chrome system do not, per se, have different temperature coefficients, that (2)
these H-transfer mechanisms are the rate-controlling link in the respiration of
the fertilized and unfertilized egg. (3) Temperature coefficients are largely de-
termined by the ratio of the rate at which H-atoms are transferred, from sub-
strate to oxygen, to the maximum rate at which they can be produced by the
substrate-dehydrogenase systems. The more nearly the H-transfer rate ap-
proaches the maximum H-production rate, the lower the temperature coefficient,
and vice versa—within the limits set by the fertilized and unfertilized egg.

Methods for the study of rapid chemical reactions and thew application
to the kinetics of enzyme-substrate and enzyme-tnhibitor compound
formation. Kurt G. Stern and Delafield DuBois.

The observation of spectroscopically defined enzyme-substrate and enzyme-

inhibitor compounds, made in the course of recent studies on catalase and peroxi-
dase, offers an experimental approach to the detailed analysis of the mechanism of

358 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

action of these enzymes. A photoelectric method for the recording of such proc-
esses and some preliminary results were reported last year (K. G. Stern and D.
DuBois, J. Biol. Chem., 116, 575 (1936)). This method has since then been im-
proved by replacing the single photoelectric cell by a differential photometer con-
taining two photocells. The technique of mixing by injecting one reactant into
the solution of the other with a spring gun has been retained. The new apparatus
is quite insensitive to any but color changes. In addition, a simple spectro-
graphic method has been developed which permits the recording of fast reactions
without the use of photocells or electric instruments. This is achieved by
replacing the plate holder of a spectrograph by a falling plate camera. While the
plate is falling, the trigger of the spring gun is released and mixing is complete
within 8 to 27 milliseconds. The time is recorded by a rotating time marker or
a Neon tube flash circuit. With this method the reaction of catalase and
methemoglobin with hydrogen peroxide, ethyl hydrogen peroxide, hydrocyanic
acid, and hydrofluoric acid has been studied. The reaction rate is greatly de-
pendent on the concentration of the reactants and on their ratio, as would be
expected from a bimolecular process. The reactions studied appear to be slower
than the reactions of hemoglobin or hemocyanin with oxygen or carbon monoxide
which have been measured by Hartridge, Roughton, and G. A. Millikan with the
flow method. Only when a considerable excess of substrate or inhibitor over the
catalysts is employed does the rate of the reactions studied by the present authors
approach the length of the mixing time which is, of course, the limiting factor
in such experiments. Inasmuch as the rate of fall of the photographic plate may
be varied within wide limits (0.3 to 29 cm. per second), a wide range of reaction
rates may be studied. The use of supersensitive panchromatic plates permits the
recording of changes of light absorption in the red and green region of the
spectrum at rates of fall of the plate corresponding to an exposure time of less
than 0.002 second for an individual spectrum. The continuous strip of spectra
recorded on the plates corresponds to 350 individual spectra.

Aucust 24

Mechanism of cellular death by freezing. B. Luyet. (The essentials
of the paper were published in the August issue of Biodynamuca.)

Binding and penetration of bivalent cations in Elodea cells. Daniel
Mazia.

The fact that Elodea cells contain soluble oxalates in their vacuoles makes it
possible to study by a direct method the binding of Ca, Sr, and Ba ions. By
subjecting the cells to strong electric currents or to ultraviolet radiation or to
plasmolysis and deplasmolysis, one can set free the bound Ca in their protoplasm,
which can then be observed as a precipitate of distinct calcium oxalate crystals
in the vacuole. The Ca must come from the protoplasm, for the cells are kept
immersed in distilled water or non-electrolyte solution.

That the Ca actually is bound in living cells is indicated by the fact that it
cannot be washed out by prolonged immersion (up to 14 days) in distilled water,
although it can easily be removed by a few minutes of washing in a citrate
solution and replaced then by a few seconds of immersion in a 0.01 M CaCl,
solution. Further studies on leaves from which the Ca has been removed by
citrate show that it can be rebound to a maximum level from Ca solutions as
dilute as 5 X 10° M, the time required increasing with the dilution. This binding
is influenced by Na and also by K ions, so that when the Na/Ca ratio is greater
than about 100, the binding of Ca is largely prevented.

It is possible to substitute Sr and Ba in the place of Ca in the protoplasm.
They are bound in the same way as Ca. Cells in which they have been substituted

PRESENTED AT MARINE BIOLOGICAL LABORATORY 359

for Ca seem to function normally, but when subjected to agents regularly causing
the release of Ca show characteristic crystals of strontium oxalate or barium
oxalate in their vacuoles.

The Sr ion penetrates the protoplasmic layer easily, even when there is no
concentration difference between the Sr inside and outside or even a higher con-
centration inside. It can be demonstrated that this transport requires first the
binding of the Sr in the protoplasm. I{ Na or K is added to an Sr solution in
sufficient concentration to prevent the binding of Sr in the protoplasm, the pene-
tration of Sr does not occur, whereas, in the control, a pure SrCl, solution of the
same concentration, the transport does occur, and crystals of strontium oxalate can
be seen in the vacuole.

Factors governing cellular responses to nitro and halo phenols. G. H.
A. Clowes, A. K. Keltch and M. E. Krahl.

A recent comparison of respiratory stimulating and cell division blocking
effects of three closely related compounds having pK values ranging from 4.1 to
4.5, mononitrocarvacrol, dinitrocarvacrol and dinitrothymol, shows that the first
and third substances exert little or no effect on respiration but block division at
the extraordinary dilution of 2 X 10° M. The second increases respiration almost
four-fold to a peak at 10° M, at which concentration division is blocked. A
fourth structurally related substance, 2,4-dinitro-o-isopropyl phenol, having a pK
of 3.0, produces a moderate effect, giving a respiratory peak and cell division
block at 10° M. These results lend further support to the conclusion previously
reached that, while the substituted phenol anion undoubtedly exerts a significant
effect inside the cell, it is quite impossible, from a knowledge of only the dissocia-
tion constants of such substituted phenols, to predict with certainty the range of
concentrations, if any, in which a given compound will affect either cell respira-
tion or division or both.

From experiments conducted in 1935 and confirmed in 1936, in which varying
numbers of eggs were used in a constant volume of sea water medium, certain
substituted phenols were observed, contrary to general experience with anes-
thetics, to block division at greater dilution when larger numbers of eggs were
employed and vice versa. This was believed to be attributable to a rise in intra-
cellular acidity due to CO,. At last year’s meeting it was demonstrated that
with varying CO, tensions, incapable in themselves of blocking division, the
division-blocking effect of substituted phenols was greatly enhanced.

In an attempt to evaluate the relative role of undissociated substituted phenol
molecule and anion, the dissociation constants of some thirty substituted phenols
have been determined during the past winter and used in analyzing the respiratory
stimulating and cell division blocking effects obtained with the compounds in
question at fixed exterior and varying interior acidities. These will be reported in
the following paper.

On the assumption that the substituted phenols penetrate the living cell only
as undissociated molecules, for a given total concentration of the phenol, the
intracellular concentration of phenol molecule and phenol anion in an intracellu-
lar aqueous phase may be calculated for any levels of extracellular and intra-
cellular acidity.

The possible role of acidic dissociation in the physiological effects pro-
duced by nitro and halo phenols. M.E. Krahl, G. H. A. Clowes and
A. K. Keltch.

From experiments performed during the summer of 1935, using a constant
extracellular pH of 7.5 and a constant intracellular pH approximating the normal

360 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

6.8, the intracellular concentrations of phenol molecule and phenol anion necessary
to give 50 per cent reversible inhibition of division and approximately optimum
respiratory effect (where this effect was present) were calculated for 30 substi-
tuted phenols. The necessary concentration of phenol molecule was found to
vary from 3X10" M for 2,4-dinitro-o-cyclohexyl phenol to 3 X 10* M for
m-nitrophenol. The necessary concentration of phenol anions likewise varied
over a wide range, from 1.0 X 10° M for 2,4-dinitro-o-cyclohexyl phenol to
2X 10% M for 2,4-dinitro-o-isopropyl phenol. In confirmation of our previous
work and contrary to the results of Tyler and Horowitz (Proc. Nat. Acad. Sci.,
23: 369, 1937), picric acid and o-nitrophenol, when free of impurities, produce
no reversible stimulation of respiration or reversible cell division block. It is
clear that no final conclusion regarding the precise roles played by the phenol
anion and undissociated phenol molecule can be reached from such experiments,
and that there is some as yet undetermined factor involved. The conclusions of
Tyler and Horowitz, who used a limited number of compounds which happened
to fall, for the most part, in the middle of the above series, are therefore not
justified by the evidence at present available.

With a constant extracellular pH and a decreasing intracellular pH, the
ratio of intracellular anion to intracellular substituted phenol molecule decreases,
while the concentration of phenol molecule is independent of intracellular pH
and dependent only on the total extracellular concentration of phenol and the
extracellular pH. In experiments on fertilized eggs of Arbacia punctulata, it
was found, as anticipated, that at constant extracellular pH of 6.7, the concentra-
tions of 2,4-dinitrophenol, 4,6-dinitro-o-cresol, 2,4,5-trichlorophenol, 2,4-dichloro-
phenol, and m-nitrophenol necessary for 50 per cent division block were not af-
fected by decrease in the intracellular pH, while the optimum levels of respiratory
stimulation were decreased with decreasing intracellular pH, this decrease in
respiratory optimum being largest with 2,4-dinitrophenol and 4,6-dinitro-o-
cresol which have pK values of 4.1 and 4.4 respectively, and small or negligible
with 2,4-dichlorophenol and m-nitrophenol which have pK values of 7.7 and 8.3
respectively.

Depolarization of muscle and nerve membranes by organic substances.
Rudolf Hober and Bernard R. Nebel.

It is fairly generally accepted that some surface film of muscle and nerve
fibers is the seat of a polarized state resulting from the high content of the in-
terior of the fibers in free K ions and from the selective permeability of this film
to cations. Furthermore, it is believed that the negative electric wave sweeping
along the fibers after excitation is indicative of a local and reversible propagated
depolarization due to an increase of ion permeability. This increase would be
the result of an electro-chemical reaction, which involves a structural alteration
of the surface film. Since the excitation process has been shown to be con-
nected with an increased metabolic activity of the fibers, it seemed worthwhile
to study whether organic compounds either identical or more or less related to
normal constituents of the fibers would bring about depolarization.

Experiments were performed on sartorius muscles and sciatic nerves of the
frog, complemented in coOperation with Dr. M. Andersh, by studying nerves of
the spider crab. Injury potentials were measured, following the usual pro-
cedure.

The experimental result is this, that not only certain organic cations, com-
parable to the normally penetrating K ions, but also certain organic anions and
non-electrolytes are enabled to depolarize the surface membrane, as disclosed by
the arising electronegativity. The active cations concerned are those of higher
dialkylamines and of alkaloids, the anions those of higher fatty acids and bile
acids, the non-electrolytes anesthetics and saponin-like compounds. All these

PRESENTED AT MARINE BIOLOGICAL LABORATORY 361

substances are likewise significant by their cytolytic power, which is associated with
a polar structure of their molecule, with surface activity and lipoid-solubility.
Lytic effects frequently appear to be irreversible. But under certain conditions,
e.g., with the fatty acids by shifting the pH from a more acid to a more alkaline
reaction, the depolarized state can be returned to the normal polarization.

These statements tempt one to raise the question whether reversible cytol-
ysis may play a rdéle in producing the traveling negativity, the propagated re-
versible disturbance of the surface film of the excited fibers. Support may be
lent to such an assumption by the facts that the phosphatides, characteristic con-
stituents of the plasma membranes, are containing surface-active higher fatty
acids and that electric currents, in passing artificial membranes, comparable to
the action currents accompanying excitation, have been demonstrated to alter
the ion concentrations, particularly the H ion concentrations in the electrolyte
solutions bordering the membranes. Under such conditions, in the membranes
various physico-chemical or chemical events could be released.

GENERAL SCIENTIFIC MEETING
Aucust 26

On some conditions determining sub-cooling in plant tissues. B. J.
Luyet and E. L. Hodapp.

Organisms found in frozen water or on the frozen ground are said to be
sometimes killed by a slight concussion incapable otherwise of harming them.
This would be attributable to the sudden freezing of the sub-cooled tissues under
the action of the concussion. In the present experiments we studied the condi-
tions determining sub-cooling and freezing after sub-cooling, in the potato tuber.
Sub-cooling was found to occur with about the same frequency in living and in
dead tissue. The temperature to which the material was heated before being
cooled had an evident influence on preventing sub-cooling. Freezing of the sub-
cooled tissues can definitely be induced by concussion, although the relatively
high percentage of inefficient shocks indicates that some unknown factors, un-
influenced by concussion, hold the system in the sub-cooled condition.

On the double freezing point of some living tissues. B. J. Luyet and
Sister P. M. Gehenio.

Some plant tissues have been found to present sometimes two freezing points,
one a few tenths of a degree below zero and the other about a degree lower.
In the present work we investigated the conditions in which one obtains the double
freezing-point in the potato tuber. From a large number of determinations it
results that it is a character of living tissue and that a congelation of the material
at the first freezing-point does not kill it, while at the second it does. The presence
of the double plateau in the freezing curve, and its shape, have been studied in
terms of the cooling velocity, of the occurrence of sub-cooling, of the size of the
piece of tissue, of its degree of desiccation or of imbibition, and of the type of
thermometric device used. The possible factors responsible for the double
freezing point are discussed.

Transverse electric impedance of the squid giant axon. H. J. Curtis and

Ke SGCole.

The transverse electric impedance of the giant axon of the lateral mantle
nerve of the squid has been measured by means of a Wheatstone bridge over a
frequency range from 200 to 2,500,000 cycles per second. The bridge current was

362 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

at all times kept well below that necessary to stimulate, and excitability was
tested at the end of each run.

The phase angles measured for this axon ranged from 70° to 85° which
indicates that the membrane impedance is of the polarization type. These phase
angles are considerably higher than those found for nerve bundles from the
same animal, so it seems very likely that part of the low phase angles found for
nerve bundles may be due to a statistical distribution of fiber diameters and
membrane capacities. In several cases, impedance runs were taken both before
and immediately after the fiber lost excitability, and none of the impedance char-
acteristics of the axon changed when this occurred. Some time later, however,
the membrane impedance dropped to zero which indicated the death of the cell.
Membrane capacities found for this axon average 0.42 ui/cm.” at 100,000 cycles,
which is in good agreement with the values previously found for nerve bundles.

Electric impedance of suspensions of unfertilized and fertilized Arbacia
COGS ieee olerands)ia vies Spencer,

The alternating current resistance and capacity of suspensions of Arbacia
eggs in sea water, measured at frequencies from one thousand to ten million
cycles per second, give average membrane capacities, with 90° phase angles, of
0.84 wi/em.? for the unfertilized, and 3.5 ui/cm.’ for the fertilized eggs. Some
slightly lower phase angles were probably indications of abnormalities. The
previously reported additional capacity element in the fertilized eggs disappeared
as the season progressed. It was found that the plasma membrane enclosed
volume averaged 1.7 per cent less than the non-conducting volume for the un-
_ fertilized egg and 2.5 per cent less for the fertilized egg, while the fertilization
membrane enclosed volume averaged 32 per cent greater than the non-conducting
volume. Thus the plasma membrane lies very close to the non-conducting mem-
brane in both the unfertilized and fertilized egg, and the fertilization membrane
is practically perfectly conducting. It is then probable that, on fertilization, the
plasma membrane capacity increases to some four times its unfertilized value.

Electric impedance of single Arbacia eggs. K.S. Cole and H. J. Curtis.

The end of a two or three-mm. thin-walled glass tube is heated until it
closes down to a short capillary about 50 in diameter. The tube is partially im-
mersed in sea water, and when an egg, dropped in the upper open end, settles to
the top of the capillary, the water level in the tube is raised until the egg is
pushed into the middle of the capillary. Impedance measurements are then made
between electrodes placed in the tube and in the outside solution. The low fre-
quency resistance of a 48m tube rose from 24,000 ohms, when filled with sea
water, to 840,000 ohms with an unfertilized egg in place. This increase might
be due to a membrane resistance of 20 ohm cm.’ but this value is no more than a
lower limit since a layer of sea water 0.25 thick between the egg and the glass
would produce the same result. The low frequency resistance for a fertilized
egg was equivalent to a 4.2 space, which is larger than the membrane elevation.
The observed low frequency capacities and the higher frequency data give average
membrane capacities of 0.8 uf/cm.’ for the unfertilized, and 2.8 ufi/cm.’ for the
fertilized eggs. These ‘results are in agreement with these obtained from sus-
pensions and the technique may be used for several problems which are not other-
wise possible.

The effect of NaCl on potentials in Nitella. Samuel E. Hill.

The normal action current in Nitella requires about 15 seconds for com-
pletion, including recovery. After the cells have soaked for 30 minutes or more

PRESENTED AT MARINE BIOLOGICAL LABORATORY 363

in 0.01 M NaCl the action currents may become very brief, lasting not more than
1 or 2 seconds. The form of the action curve changes, showing 1 peak instead
of 2, and the amplitude is usually less.
After 24 hours in 0.01 M NaCl the action curve tends to become normal
again. ,
The cells show no signs of injury after 24 hours or more in 0.01 M NaCl.

The coalescence of a plant cell with oil drops. M. J. Kopac.

The young aplanospores of Valonia ventricosa are essentially naked proto-
plasts and coalescence with oil drops readily occurs. As the aplanospores become
older, the tendency to coalesce with oil drops becomes decreased. Coalescence
with oils of a high interfacial tension against sea water (paraffin oil, tension ca.
40 dynes* cm.*) is inhibited in aplanospores over 3% hours old. This decreasing
tendency to coalesce with oil drops is believed to signify the building up of ex-
traneous coats by the protoplast. Ultimately the aplanospores become coated
with a cellulose wall.

In aplanospores about 1%4 hours old, coalescence with low tension oils (oleic
acid in paraffin oil, tension ca. 3 dynes* cm.*) occurs rarely. A small drop of this
oil may be placed in contact with an aplanospore without coalescence occurring.
If a few seconds later a small drop of a higher tension oil (oleic acid, tension ca.
10 dynes* cm.*) is applied to the opposite side, coalescence between it and the
aplanospore immediately takes place. From % second to several minutes later,
the first drop snaps into the aplanospore. In these young aplanospores no cellu-
lose cell wall has been formed. The inhibition of coalescence with a low tension
oil may be due to the preliminary solidification at the cell surface prior to the
formation of a cellulose wall. Coalescence with oleic acid apparently induces a
peripheral disorganization at the cell surface which then permits the protoplast
to coalesce with a low tension oil. This disorganization in the case of the
aplanospore may actually be a disintegrative action at the cell surface. Addi-
tional evidence for this point is shown by the release of chloroplasts from the
protoplast following coalescence with the two drops. These investigations were
started at the Tortugas Laboratory this summer and are being continued at the
Marine Biological Laboratory.

The influence of length, tension, and tone upon the birefringence of
smooth muscles (Phascolosoma and Thyone). Ernst Fischer.

The retractor muscles of Phascolosoma and Thyone respond to direct stimu-
lation with a twitch-like contraction. After indirect stimulation involving the
ganglion the quick contraction of both muscles is followed by a sustained tonic
contraction. Besides this “contractile tone” both muscles show marked “ vis-
cosoidal tone.” A muscle stretched by a load is constantly lengthening, and
when the muscle is released later on, it shortens only to a small extent. By
alternatingly loading and releasing a muscle with increasing weights, the bire-
fringence of the muscle can be measured for the same muscle length under no
tension and under a well-determined tension. When no tension is exerted, the
birefringence of the muscle is about proportional to the square root of the
length, as found by Bozler for a smooth snail muscle under comparable condi-
tions. For the same muscle length under tension the birefringence is higher, the
increase being proportional to the tension present,—a true “ photoelastic effect.”
In consequence, when a muscle is stretched quickly, the birefringence increases at
a steadily growing rate until suddenly the birefringence diminishes and the muscle
tears through.

Under isometric conditions the tonic sustained contraction increases the double
refraction under all conditions, while for the twitch-like contraction, as shown

364 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

previously, the direction of the birefringence change is dependent on length and
tension. This indicates that “normal contraction” and “tonic contraction” are
fundamentally different processes. For both muscles investigated the same re-
sults were obtained with the only difference that in Thyone the birefringence is
merely 60 per cent of that in Phascolosoma.

The mechanism of salt penetration in Amoeba—some micromanipulative
data. Samuel A. Corson.

Addition of inorganic acids (to a pH of 4.5 or lower) to K or Na salt
solutions in which the amoebae were immersed prevented the decrease in proto-
plasmic viscosity and the cessation of movement which occurred when Ameba
proteus (Chaos diffluens) was immersed in neutral or alkaline solutions of these
salts. The same acids also prevented the marked increase in protoplasmic vis-
cosity produced by immersion in neutral or alkaline Ca salt solutions. Utilizing
the Chambers micromanipulator and a new method which permits quantitative
injections (the method consists essentially of enclosing the solution between a
layer of heavy and one of light oil and measuring the quantity of solution drawn
into the pipette by means of a calibrated ocular micrometer) it was shown that
while KCl injections produced the same effects as in the immersion experiments
(a fact observed previously by R. Chambers), K,SO, injections failed to liquefy
the protoplasm though they did inhibit locomotion. These effects, just as the effects
of CaCl, injections, were not influenced by acidification of the injected solutions
(to a pH of 4.2-3.0). Since in the immersion experiments the K effect was the
same irrespective of the anion used, these results support the previously suggested
hypothesis that the plasma membrane of this amoeba is selectively permeable to
cations and relatively impermeable to anions.

The efficiency of monochromatic ultraviolet radiation in the activation of
Arbacia eggs. Alexander Hollaender.

During an investigation of the effects of ultra-violet radiation on the eggs
of Arbacia punctulata, it was observed that when the eggs are exposed for as
short a time as 1/10 of a second to the entire radiation of a water-cooled high
pressure quartz capillary mercury vapor lamp, a large percentage of the eggs
went through one or more cleavages without fertilization. Exclusion of the
infra-red did not inhibit while exclusion of the radiation below 3,000A inhibited
the effect.

The eggs were then irradiated with measured quantities of monochromatic
radiation of nine different wave-lengths from 2,260 to 3,650A. Special care
was taken to develop a method which would make certain that not only each egg
within the dish but each part of each egg received an equivalent amount of energy.
This was done by rotating a small dish in which the eggs were suspended in
3 mm. of sea water and blowing an air current against the water surface. The
eggs were removed after irradiation to a larger volume of sea water and kept
at 24° and 10° C.

Three types of controls were handled in each series of experiments: (1)
unirradiated, unstirred eggs, (2) eggs stirred in the usual manner but protected
against the radiation, (3) sea water irradiated with the wave-lengths most
effective in producing activation to which unirradiated eggs were afterwards
added. In none of these controls could any activation be recognized if the
original eggs were in good condition.

The energy was measured with a standardized vacuum thermopile-gal-
vanometer set up and the incident energy per egg calculated taking into account
the total energy entering the dish, the time of exposure, the diameter of the egg
and the fact that the eggs were exposed interruptedly ignoring for the present the
energy reflected and scattered by the eggs.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 365

Three to five hours after irradiation a high percentage of the eggs (up to
98 per cent) were found activated (one or more cleavages), if certain wave-
lengths and energies per egg were used. The wave-length found most effective
was 2,260A, and the efficiency decreased with the increase in wave-length, be-
coming negligible around 2,500A; 0.13 to 0.25 erg of incident energy per
organism at 2,260A produce the highest percentage of activation. A plot of the
percentage cleaving against the energy applied for each effective wave-length
shows a typical S-shaped curve, a definite plateau, and finally a decreasing rate
of cleaving with further increase of energy.

A plot of the efficiency of radiation against wave-lengths with its most pro-
nounced maximum at 2,260A and its low efficiency at 2,600A suggests interesting
comparisons with the ultra-violet inactivation spectra of urease and the virus of
typical tobacco mosaic, and the absorption spectra of other protein-like sub-
stances.

Activation of centrifuged whole eggs of Arbacia and their fractions by
monochromatic ultra-violet radiation. Ethel Browne Harvey and
Alexander Hollaender.

Whole Arbacia eggs which have been stratified and elongated by centrifugal
force (10,000 x g for 3 minutes) are activated by the same ultra-violet radiation
as the whole uncentrifuged eggs; full arc 1/10-5 seconds, monochromatic 2,350-
2,480A for 2-8 minutes. They behave in exactly the same way as when activated
by other parthenogenetic agents such as hypertonic sea water but pass through
only a few cleavages.

White half-eggs (nucleate) are activated by the same radiation but are much
more sensitive to slight variations from an optimum treatment, and fewer cleave.

Red half-eggs (non-nucleate) are activated by the same radiation and also
by a band of longer wave-length, 2,650-3,050A for 4-12 minutes, which does not
affect the whole eggs and the white halves. Fertilization membranes are formed,
some large asters occur, the egg often becomes aspherical and somewhat amoeboid.
A notch frequently appears at the equator of the more spherical red halves, indi-
cating the beginning of a cleavage plane, and this usually completes itself. Stages
with 8-12 cells have been observed usually unequal in size. There occur later
on some eggs filled with many small asters, a possible precursor to a blastula.

Yolk quarters (non-nucleate) are activated in just the same way as the red
halves and by the same wave-lengths and dosage, and some 8-12-celled stages
have been observed. The pigment quarters (non-nucleate) show some evidence
also of being activated, since they form an ectoplasmic layer and become some-
what amoeboid.

Ultra-violet radiation, therefore, acts upon the whole eggs and their frac-
tions obtained by centrifugal force exactly like other parthenogenetic agents such
as hypertonic sea water. Since the radiation affects the non-nucleate fractions as
well as the nucleate, the action must be on the cytoplasm, but since many abnormal
and irregular mitoses are observed in stained sections of later cleavages, the
action must be also on the nucleus.

The cytology of Arbacia punctulata actwated by monochromatic ultra-
violet radiation. B.R. Nebel, Ethel Browne Harvey and Alexander
Hollaender.

Unfertilized eggs of Arbacia treated in the summer of 1936 with the full
output of a high pressure mercury vapor lamp for a few seconds, showed activa-
tion in all the eggs and fairly normal first cleavages following the normal nuclear
changes in 80 per cent of the eggs, but the cleavage was delayed by two or more

366 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

hours. A very characteristic feature of late prophase and metaphase stages in
later cleavages as shown in prepared sections is the presence of small rings or
spheres among the chromatic material. Occasionally the spindles of more than
two plates appear united at various angles. Large cytasters are observed. Regu-
lar dicentric spindles, typically non-astral, occur but the most frequent type is a
monaster surrounding the chromosomes, which as the division proceeds appears
to form two polar half-asters. The eggs developed usually only to about an
4 to 8-cell stage.

Unfertilized eggs treated in the summer of 1937 with measured intensities
of monochromatic ultraviolet of 2,350 and 2,260A for 4 to 6 minutes show up to
98 per cent activation indicated by a fertilization membrane and ectoplasmic layer and
after 3-4 hours cleavage. Nuclear divisions are monastral and irregular, normal
dicentric asters not being formed. The first division is frequently of the restitu-
tion type leading to diploidy without cell or nuclear division. The length of the
spindle is diminished. During subsequent stages the products of successive
chromatic multiplication may be separated. Thus irregular blastomeres may be
formed containing no chromatin, approximately haploid, diploid or polyploid
nuclei. The average activation does not produce more than 4 to 6 nuclei con-
taining approximately variable multiples of the haploid chromosome number,
distributed irregularly in 2 to 8 asymmetrical blastomeres.

Unfertilized eggs treated with the same and larger total energy given as ultra-
violet \ = 2,480, 2,650 and 3,050A showed no marked activation.

Fertilized eggs treated with ultraviolet of the short wave-length (2,260 and
2,350A) soon after insemination showed slight inhibition. Fertilized eggs treated
with ultraviolet of longer wave-length (2,650 and 3,050A) showed marked in-
hibition. In both treatments occasional nuclear irregularities were observed.

The relationship of sperm extracts to the fertilization reaction in Arbacia.
John A. Frank. ,

A specific egg-agglutinin, previously reported, is present in the filtrate from
boiled Arbacia sperm suspensions. This substance is present in the fat-free resi-
due on extracting sperm suspensions with alcohol and ether. It is not found in
the lipid extract.

Eggs fertilized in sperm extracts show a marked drop in fertilizability when
compared with eggs fertilized in sea water. Experiments were performed to de-
termine whether this inhibition of fertilization is due to the action of sperm ex:
tracts on the egg alone, spermatozoon alone, or on both gametes.

Sperm suspensions exposed for varying lengths of time to sperm extracts ex-
hibit a marked loss in fertilizing power. Sperm extracts thus block fertilization
by a rapid direct action on sperm.

The fertilizability of jellyless eggs exposed to sperm extracts and subse-
quently fertilized with fresh sperm decreases markedly. Sperm extracts there-
fore exert an inhibitory effect on the cortex.

When sperm extract is added to egg water containing fertilizin, the mixture
will not agglutinate sperm. Some substance in the sperm extract has inactivated
fertilizin. Sperm extracts inactivate fertilizin in definite quantitative proportions.
The capacity for fertilizin inactivation varies directly with the concentration of
sperm extract. Thus sperm extracts contain a substance which resembles Lillie’s
anti-fertilizin.

On ageing, the fertilizing power of sperm suspensions is lost concurrently
with the capacity of extracts of these suspensions to inactivate fertilizin and to
ageglutinate eggs.

Evidence at present indicates that sperm extracts contain substances related
in some way to the fertilization reaction.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 367

Stimulation and nuclear breakdown in the Nereis egg. L. V. Heilbrunn
and Karl M. Wilbur.

Recent studies of stimulation in diverse types of protoplasmic systems have
indicated that one of the primary effects is a breakdown of a calcium proteinate
gel in the cell cortex and a release of free calcium into the cell interior. As yet
these studies of stimulation have thrown no light as to the rdle the nucleus may
play when a cell is stimulated to divide. In the egg of the worm Nereis, various
types of stimulating agents cause a breakdown of the germinal vesicle. Thus,
such an effect is produced by heat, ultraviolet radiation, and Roentgen rays, as
well as by various chemicals. If our theory is correct, one of the initial steps in
the series of processes that result eventually in nuclear breakdown is a calcium
release from the cortex of the cell. Our experiments lend support to this theory.
If Nereis eggs are exposed for 6 or more minutes to an isotonic citrate solution,
subsequent treatment with ultraviolet radiation causes no nuclear breakdown; al-
though on return to sea water-the eggs again show a typical response following
irradiation. Similarly previous treatment with citrate prevents the nuclear break-
down which otherwise occurs very beautifully when eggs are placed in isotonic
solutions of sodium or potassium chloride. The sodium and potassium ions appear
to be capable of provoking a release of calcium ions from the cell cortex, and
these calcium ions induce changes which eventually lead to a breakdown of the
nuclear membrane.

The movement of the egg nucleus in relation to the sperm aster in Ly-
techinus and Echinarachnius. Edward L. Chambers.

The observations indicate first that the egg nucleus is moved to the center of the
sperm aster by centripetal currents of cytoplasm. This is in conformity with the
early observations of Conklin. He held that the approach and union of the two
nuclei were determined by protoplasmic currents in the odplasm.

The granules in the cytoplasm move along with the egg nucleus.

The curvature of the path of the egg nucleus is caused by the continual change
in direction of the cytoplasmic currents due to the progressive movement of the
sperm aster towards the center.

The increasing acceleration of the egg nucleus indicates the existence of more
and more intense centripetal currents of cytoplasm as the pronucleus migrates into
the aster.

These observations indicate, second, that the aster is a jellied mass. This con-
firms R. Chambers’ conclusion.

The diminishing acceleration of the egg nucleus as it moves down the ever-
narrowing cytoplasmic path extending from the margin to the center of the aster
demonstrates a resistance to movement due to the presence of a jellied material
around the path through the aster.

The deformation (ellipsoidal) of the egg nucleus as it moves along this path
demonstrates that the path is gradually tapering cone of fluid cytoplasm in the
jellied mass of the aster.

The physical state of the wall of the furrow in a dividing cell. Robert
Chambers.

Evidence is accumulating that the wall of the furrow is solid and constitutes
the most solid part of the cortex of the cell. The advance of the furrow displaces
the interior toward the poles which bulge because of the relative weakness of the
polar cortex.

No symmetrical arrangement of the surface seems to be essential since strands
of cortical protoplasm may be dragged out either at the equatorial or polar regions

368 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

without affecting the cleavage. The strands at the equatorial region are much
stiffer than those at the poles. In tissue cultures fibroblasts often retain extended
strands especially at the poles while the furrow forms at the equator.

Conditions within the cell interior need not affect the division once cleavage is
under way. A dividing epithelial cell sometimes shows a rhythmic back-and-fortk
flow of the internal contents through the narrowing stalk connecting the twe
daughter cells. This ceases only when the constriction of the stalk has completed
the division. In the sea-urchin egg undergoing division in calcium-free sea water,
the contents of one blastomere, when torn, pour out while the furrow continues
to deepen. The continued pinching-down of the furrow on the connecting stalk
frequently rescues the other blastomere from disintegration.

The thickness of the cortex of the advancing furrow and the force with which
it advances has been determined by injecting an oil drop into the equatorial region.
The oil drop tends to come to lie in the central region at the equator so that the
advancing furrow closes down on it and constricts it in two. This occurs when
the surface of the floor. of the furrow is some distance from the surface of the
oil-drop.

The advance of the wall of the furrow must be considered as a growth
phenomenon—material being added progressively to the gelated cortex of the fur-
row analogous to the apposition of material along the plane of division of a plant
cell.

AvucGust 27
Chromosome studies in sundew (Drosera). A. Orville Dahl.

For purposes of comparison with chromosomes in certain members of the
Saxifragaceae, cytological material of Drosera filiformis Raf., D. longifolia L.,
and D. rotundifolia L. has been locally collected. A preliminary examination of
root-tip meristem in aceto-carmine demonstrates 20 chromosomes in D. filiformis
which is consistent with Levine’s (Mem. N. Y. Bot. Gard., 6: 125-147, 1916) re-
port of 10 chromosomes in pollen mother-cell material from Lakehurst, N. J.
Rosenberg (Ber. der Deutschen Bot. Ges.. 21: 110-119, 1903) found 20 chromo-
somes in cells of root-tips, stems, leaves, and flowers of D. rotundifolia collected in
Germany, Norway, and Sweden. A comparative study of the early somatic meta-
phase in D. filiformis from Mashpee, Mass. and D. rotundifolia from North Fal-
mouth, Mass. shows that the chromosomes of the former are approximately 1.49
times longer and 1.38 times wider than those of the latter. The metaphase chromo-
somes are of comparatively small size, those of D. rotundifolia being about 1.90 u
in length while those of D. filiformis are 2.82 in length. A visibly four-parted
structure, with the distance between the half-chromatids 0.3 » to 0.4 4, could be de-
tected at mid-prophase, late prophase (at which time the nucleus has a diakinetic
appearance), early metaphase, and late telophase.

Mitosis in the giant amoeba, Chaos chaos Linnaeus. M. Catherine
Hinchey.

Chaos chaos is from 50 to 500 times as large as C. diffluens and has from 50
to over 1,000 discoid nuclei. The chromatin granules are arranged in a layer
underneath the nuclear membrane in the living amoeba.

When mitosis begins, the nuclei become spherical and the chromatin granules
congregate in a thick equatorial plate. Then most of the granules seem to dis-
appear, leaving a spherical nucleus, with chromatin granules distributed in a thin
plate. What appear to be spindle fibers become visible at this stage. These fibers
can be seen in the living amoeba under the micro-compressor.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 369

Following this stage, the nucleus shortens along the polar axis and becomes
wider at the equator, so that a ladder of fibers is seen, in optical section, with the
chromatin distributed along the mid-points of the ladder-rungs.

The chromatin then separates into two plates which move along the spindle fibers
toward the poles, until the plates are three times their diameter apart. At this
stage fibers can be plainly seen in both stained and living material, extending be-
tween the plates and from the plates to the poles.

Protoplasmic streaming next moves the plates apart. The fibers between the
plates become twisted and disappear, but the fibers going to the poles persist for
some time longer. The chromatin plate becomes thinner, and so homogeneous that
it is extremely difficult to follow in living material, but in fixed material, a con-
centration of chromatin occurs along the inner edge of the plate. The daughter
nuclei next become wider and-more granular. This process continues by gradual
steps until the interphase stage is reached.

Cytoplasmic division—usually into 3 daughters—occurs during the reorganiza-
tion of the daughter nuclei into interphase nuclei.

The striking features of the nuclear division are: 1. All the nuclei divide at
the same time. 2. Practically every stage of mitosis can be seen in the living
amoeba under the micro-compressor. 3. Although the total number of nuclei
doubles during mitosis, cytoplasmic division usually results in three daughters.

Some effects of oxygen on polarity in Tubularia crocea. James A.
Miller. |

A chamber was constriicted by means of which the two ends of Tubularia
stems could be exposed to different agents or to different concentrations of the
same agent. This consisted of a double chamber with a partition which separated
the solutions but which had perforations through which the stems could be passed.
Using this apparatus preliminary studies were made upon the effects of high and
low oxygen tensions on polarity. By placing the stems in alternating orientations
each experiment served as its own control. Oxygen determinations by the Winkler
method were made in all but preliminary experiments.

When oxygen was bubbled on one side of the partition and boiled sea water
was placed on the other, hydranths developed only on the side with high oxygen.
One half of these were distal and the other were proximal hydranths. Similar
results were obtained when oxygen was bubbled on one side of the partition and
standing sea water (with 4.1 to 5.0 cc. Oz per liter) was on the other. That these
results were caused primarily by the oxygen differential and not by a possible
accumulation of carbon-dioxide was demonstrated when nitrogen was bubbled on
one side of the partition and oxygen on the other. Here again there was a re-
versal of polarity in all stems with their proximal ends exposed to the oxygen.

The importance of circulation of the medium to sessile forms such as Tubu-
laria was illustrated by experiments in which 95 per cent of the stems developed
proximal hydranths in running aerated water when the distal ends were exposed
to standing water, while only 6.3 per cent developed proximal hydranths when the
conditions were reversed. Oxygen determinations in two of these experiments -
showed a difference of only 0.1 cc. per liter in each case.

Some effects of strychnine on reconstitution of hydranth primordia in
Tubularia crocea. Faith Stone Miller and James A. Miller.

Miller (1937) found that pieces of planarians regenerating in strychnine
showed no evidence of stimulation. Since in Tubularia the size and time of de-
velopment of hydranth primordia can be measured, uals form was used to con-
tinue the study of the effects of strychnine.

370 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The behavior of unoperated individuals placed in solutions of strychnine
sulphate in sea water was undistinguishable from that of controls anesthetized by
magnesium sulphate.

In reconstitution experiments stems selected for uniform appearance and
diameter were used. Ten-millimeter pieces were cut with the distal end five milli-
meters from the base of the hydranth. Continuous exposure resulted in a de-
crease in size of hydranth primordia and increase in time of development. Con-
centrations used ranged from M/20,000 which showed very little effect to M/5,000
in which few stems survived. Temporary exposures to M/1,000 for periods of %
to 8 hours showed similar but less consistent results. The frequency of the oc-
currence of bipolar forms was decreased in strychnine and none developed in the
higher concentrations.

The results obtained with strychnine on Tubularia are similar to those with
inhibitory agents and indicate that it produces a definite depression. By bubbling
oxygen through the solutions it was possible to antagonize the strychnine effect.

The life cycle of Moniezia expansa. Horace W. Stunkard.

Anoplocephaline cestodes are common parasites of herbivorous animals and
one species is recorded from man. They are worldwide in distribution, and the
group has been intensively studied for more than fifty years. The final hosts
harbor sexually mature worms in their intestines, eggs of the parasites are voided
with the feces of the hosts, but what occurs in the interval before the cestode re-
appears in the intestine of the primary host has remained quite unknown.

Stunkard (1934) published results of experiments which demonstrated that
the final hosts could not be infected with eggs of the parasite and that an inter-
mediate host is necessary for the completion of the life cycle.

Experiments have been continued, using species of Momiezia from sheep and
Cittotaenia from rabbits. Various minute, terrestrial invertebrates, chiefly in-
sects, have been used in attempts to discover the intermediate hosts of these ces-
todes. In the spring of 1936, tyroglyphid mites were fed eggs of Momiezia and
onchospheres were recovered from the body cavity three days later. The struc-
ture, habits, and life history of these mites indicate that they would not be suit-
able intermediate hosts of Momiezia. The oribatid mites, however, appeared to be
admirably suited, and representatives of this family were employed. Galumna sp.
are abundant in regions where Moniezia occurs and specimens of this mite were
collected from areas in which there were no sheep. These mites were fed eggs
of both Moniezia and Cittotaenia and onchospheres of both species were recovered
from the body cavity. During the past year thousands of Galumna sp. have been
fed eggs of Moniezia expansa and a series of developmental stages, from the
onchosphere to the mature cysticercoid, have been recovered from them.

A new method for studying the pH of the intercellular substance in the
living mammal. Richard G. Abell and Eliot R. Clark.

This method involves the installation of phenol red within a transparent moat
chamber in the ear of a rabbit. Such a chamber contains a thin space, called the
‘bay, into which living tissue, continuous with the subcutaneous tissue of the ear,
grows through two small entrance holes at the proximal end. The bay has a glass
bottom and a mica top, and is only 504 to 100 deep. Consequently the arterioles,
capillaries, and venules, and other constituents of the tissue within it can be seen
clearly with the microscope. At its distal end the bay opens directly into a reser-
voir, called the ‘moat.’

Following the introduction into the moat of a 0.4 per cent solution of phenol
red, made isotonic with rabbit’s blood by the addition of NaCl, the indicator
diffuses into the bay, and there colors diffusely the intercellular substance of the

PRESENTED AT MARINE BIOLOGICAL LABORATORY 371

tissue for a distance of approximately 1 mm. proximal to the most advanced capil-
laries. It is not concentrated by the cells, and it is not toxic.

The color of the indicator in the intercellular substance of tissue with an active
circulation is pink, the shade of pink matching that of a phosphate buffer at pH
7.2, to which phenol red has been added, seen with a microscope in the counting
chamber of a hemocytometer under the same conditions of illumination as used
for the tissue.

When the circulation is cut off, by compressing the main artery of the ear, the
color of the intercellular substance changes to the orange-yellow of a buffer at pH
6.8 within 10 to 15 minutes, indicating accumulation of acid metabolites. Within
1 to 2 minutes after the artery is released and the circulation once more becomes
active, the color of the intercellular substance changes from orange-yellow back
to pink.

By means of the present method, the pH of the intercellular substance can
be studied under a variety of experimental conditions.

The behavior of living mammalian arterioles, capillaries, and venules
when exposed to CO,. Richard G. Abell and Eliot R. Clark.

The experiments to be described were performed in a transparent moat cham-
ber in the ear of a rabbit. The behavior of the vessels was studied with the micro-
scope, and changes in the pH of the intercellular substance detected by means of
the indicator method presented above.

When CO: is passed through the moat, the color of the phenol red in the inter-
cellular substance of the tissue in the bay turns from pink (pH 7.2) to yellow
(pH 6.8-6.6). No increase in stickiness of the endothelium occurs when the
amount of CO2 employed is small. If the endothelium is sticky toward leukocytes
before CO: perfusion is started, it reverts to a state in which the leukocytes roll
freely along the vessel walls.

No increase in the diameter of the arterioles, capillaries, or venules occurs as
long as the circulation continues to pass through them. The arterioles in these
experiments were not supplied by nerves.

The color of the indicator in the intercellular substance of the proximal tissue
turns from pink to yellow more rapidly when the circulation is sluggish than when
it is rapid. Such color change is followed shortly by thickening and vacuolization
of the endothelium of the arterioles.

If CO: perfusion is stopped at this stage, the vacuoles disappear, and the endo-
thelium resumes its normal thickness. If perfusion is continued, a marked in-
crease in permeability of the arterioles, and also of the capillaries and venules,
occurs. The plasma passes through the walls of the vessels, leaving within their
lumina only the formed elements of the blood. During this process extensive
crenation of erythrocytes occurs. In vessels containing concentrated cells, the
flow of blood is blocked.

No increase in diameter of the arterioles, capillaries, or venules occurs at the
time of onset of plasma hemorrhage, but may take place shortly thereafter, indi-
cating a softening of the endothelium. These changes are reversible if CO: per-
fusion is stopped when plasma hemorrhage first occurs.

The control of peripheral circulation in the mammal. Eliot R. Clark
and Eleanor Linton Clark.

It has been possible, in transparent chambers introduced in the rabbit’s ear,
to watch the mode of formation and behavior of extra-endothelial cells, with
the following results:

Fibroblast-like cells from outside the endothelium become flattened out on the
wall of the capillary at a very early stage in capillary formation—often during

372 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

sprout formation. If the vessel remains a capillary, there may be no increase in
their number and they may remain permanently as sparsely distributed inert,
oval cells. If the capillary becomes a portion of an artery the number of out-
side cells increases rapidly—in part at least by mitotic division—the axis of the
cells changes quickly from a longitudinal to a transverse position and muscle
cells develop which show typical contractility in case they receive a nerve supply.

If the capillary becomes a part of a venule or small vein, the number of
adventitial cells increases slightly, their long axis remains longitudinal and they
do not develop contractility.

Persistent observations of the living vessels made under a great variety of
conditions has corroborated earlier findings that, in the mammal, neither the
endothelium nor the adventitial cells, as found on the capillary or venule, mani-
fests active contractility. The control of the peripheral flow resides in the
muscle cells of arteries, arterioles and certain of the veins—providing the muscle
cells are under nerve control.

While there may be changes in the caliber of capillaries and bulgings into
the lumen of endothelial nuclear thickenings or of adventitial cells, all such
changes are apparently passive, secondary to a variety of factors, chief of which
are changes in internal pressure and rate of flow, produced by contraction or
dilatation of supplying arteries or arterioles, and changes in outside pressure
occasioned either by variations in the amount of fluid accumulation in the inter-
vascular spaces or by the elasticity of the enclosing wall.

The structure of the liver lobule. Louis Loeffler.

The liver of the pig is the only one which shows clearly defined lobules.
By that is meant that the lobules have a connective tissue membrane separating
one lobule from another. The capillaries, also, of an individual lobule are
separated from the capillaries of the adjacent lobules. All the other livers of
mammals, reptiles and fishes, so far as has been investigated, show no separating
membranes and also show anastomosing capillaries throughout the whole organ.
Nevertheless, one is justified to speak of liver lobules, because the vessels, sit-
uated in regular distance from one another, form figures similar to the pig
liver lobules. It is shown, however, that there are no so-called sub-lobular veins,
because such veins, next in size to the central veins within each lobule, function
as central veins quite the same. Hepatic veins come to lie outside the lobules,
not before. The hepatic veins reach a diameter half or much more than that
of a liver lobule. A collecting lobe of about 6 or 8 lobules around a sub-lobular
vein as usually found in the diagrams of anatomical textbooks does not exist or
is quite arbitrary. The pig’s liver should be explained on the basis of a physio-
logical liver cirrhosis.

A preliminary note on the innervation of the swim-bladder of the sea-
robin. John B. Gaylor and Ernst Scharrer.

This communication deals with the peripheral innervation of the sea-robin
as investigated by Dr. Gaylor at Woods Hole; a combined paper will be published
later when Dr. Scharrer has worked out the central connections.

The swim-bladder of the sea-robin consists of a two-lobed sac in the ab-
dominal cavity. It possesses intrinsic skeletal muscle which subserves the func-
tion of noise production and, in the interior of the cavity, a gas gland which
is in the form of a “rete mirabile” covered with secretory epithelium. A branch
of the vagus on either side affords a motor supply to the striated musculature.
Free endings, knob endings and ring terminations have been observed. There is
no apparent difference in the mode of termination in the swim-bladder muscle from

PRESENTED AT MARINE BIOLOGICAL LABORATORY 373

that of the usual somatic musculature. Ganglia in the striated muscle are sparse;
the disparity between the wealth of fiber and the number of ganglia argues a double
innervation to the skeletal muscle—one direct from the vagal nucleus and one
relayed through peripheral ganglion cells. The presence of muscle-spindle organs
in the region where the muscle takes origin from the fibrous sac is suspected but
not yet definitely established.

The “rete mirabile” contains fine non-medullated fibers which enter along
with the vessels and which appear to be sympathetic. Between the vessels there
is a large number of multipolar ganglion cells which are presumably parasym-
pathetic relays.

The origin and development of the thyroid in Eleutherodactylus, an
anuran with no tadpole stage. W. Gardner Lynn.

The Jamaican tree-toad Eleutherodactylus nubicola lays its eggs on land and
the young hatch after about twenty-four days with a definitive body form. During
the embryonic development some of the larval characters which are usually found
in frogs appear very transiently but others, such as the formation of external
and internal gills, are entirely lacking. A study has been made of the thyroid
in embryos preserved at twenty-four-hour intervals throughout the period of
development. The thyroid takes its origin from the pharynx at the sixth or
seventh day and the cells exhibit signs of secretory activity almost as soon as
the thyroid anlage is definitely recognizable. Throughout the succeeeding stages
there is a steady increase in the amount of stored colloid. Intracellular vacuoles
are abundant in the follicle cells at all stages. Vacuolation of the colloid mass
is most striking during a period of about four days extending from the tenth
to sixth day before hatching. There is no evidence of a sudden release of any
large amount of colloid into the blood stream at any time during the embryonic
history. The indication is, rather, that a regular release occurs even from the
early stages. This would agree with the regular course of bodily differentiation
and the absence of any striking metamorphic pattern. However, certain of the
unusual features of development in this frog, such as the absence of external
gills, cannot be attributed to precocious thyroid functioning. Thus it appears
that while the evolutionary changes which have brought about the atypical life
history of the species are some of them changes in the development and functioning
of the gland complex, still others are changes which cannot be attributed to hor-
monal influence but are direct alterations in the developmental potentialities of
the tissues themselves.

Some effects of mammalian follicle-stimulating and luteimizing hormones
in adult female urodeles* Virginia Mayo.

During October adult salamanders (Triturus viridescens) were subjected to
preliminary tests on their ovulatory response to a single intraperitoneal injection
daily of the following mammalian pituitary extracts made according to H. L.
Fevold’s method: (a) physiologically pure follicle-stimulating hormone (F.S.H.) ;
(b) physiologically pure luteinizing hormone (L.H.); (c) F.S.H. with a trace
of L.H.; (d) an unfractionated extract containing both F.S.H. and L.H. Each
injection contained 1/20 gram-equivalent acetone-dried sheep pituitary powder.
The results indicated that fractions containing L.H. induced egg-laying whereas
F.S.H. alone was almost entirely ineffective. Injections given both normal and
hypophysectomized newts in November—December, May-June, and June—July-
August corroborated the October findings.

*This work was begun at the Biological Laboratories, Harvard University,
and continued at the Marine Biological Laboratory.

374 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

Counts of eggs released by groups of 10 animals treated during the breeding
season for 40 days gave the following: hypophysectomized L.H.-injected animals
ovulated 801 eggs; normal L.H.-injected, 582; hypophysectomized F.S.H.-in-
jected, 18; and normal F.S.H.-injected, 25.

At the onset of the June-July—August series the newts’ ovaries were almost
completely emptied of large eggs. By early August the ovaries of F.S.H. and
F.S.H. + L.H.-treated animals were filled with yolked eggs, while those of L.H.-
treated individuals were only slightly stimulated. Average ovarian weights of
7-14 animals were: untreated controls, 28 mg.; hypophysectomized + L.H., 69
mg.; normals + L.H., 88 mg.; hypophysectomized + F.S.H., 119 mg.; normals +
F.S.H., 134 mg.; hypophysectomized + F.S.H. and L.H., 150 mg.; and
normals + F.S.H. and L.H., 173 mg.

On the basis of these results it seems that F.S.H. brings about a striking
enlargement of the ovary while the L.H. is primarily responsible for egg release

The relation of melanophore responses to vascular disturbances. G. H.
Parker.

It is difficult to cut nerves in experiments on melanophore control without
at the same time cutting blood-vessels or at least introducing vasomotor disturb-
ances. Does the stimulation of melanophore nerves thus brought about excite
vasomotor changes which in turn excite responses in melanophores or do the
nerves act directly on the melanophores? In the killifish FPundulus and in the
catfish Ameiurus the melanophores have a double innervation, concentrating
nerve-fibers inducing a blanching of the fish through a concentration of pigment
in its melanophores and dispersing fibers darkening the fish through a dispersion
of this pigment. In the dogfish Mustelus there are only concentrating fibers, the
dispersion of the pigment being accomplished through a pituitary neurohumor in
the blood.

In Fundulus and Ameiurus when melanophore nerves are cut the dispersing
nerve-fibers are stimulated and dark areas or bands result. In these two fishes
and in Mustelus when the melanophore nerves are stimulated electrically the
concentrating fibers are excited and the fishes blanch locally.

One way of finding an answer to: the question under consideration is to
ascertain whether these responses will occur or not in the absence of an active
circulation of blood. To this end the ventral aorta of a given fish was
ligated just anterior to the heart and the circulation of blood thus brought to a
complete standstill. The melanophore nerves of such a fish were then subjected
to electric stimulation or were cut. In all such instances there was either local
blanching or darkening according to the stimulus. These responses, though less
marked than in normal fishes, showed unquestionably that the complete loss of
circulation was not accompanied by a loss of the power of color response and that
therefore vasomotor or other vascular changes could not form any essential
part in the chain of events between nerve and melanophore.

Some effects of chloroform on the respiratory systems of yeast. E. P.
Hiatt and J. K. W. Ferguson.

The rate of reduction of methylene blue in suspensions of yeast is increased
by small amounts of chloroform (0.05 per cent by weight). With larger amounts
of chloroform, acceleration up to 40 times was obtained. No stage of inhibition
was reached with fresh yeast. Dried yeast and yeast extracts, which have a much
faster rate of reduction than equivalent amounts of fresh yeast, were retarded
by chloroform. The same effects can be demonstrated with other oxidation-re-
duction indicators, e.g., pyocyanin, thionin, and anthraquinone.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 375

When chloroform was exerting its maximal acceleration, the cytochrome
bands showed a characteristic change. Only the C band was visible and it could
not be made to disappear on shaking with air. Subsequently, different amounts
of chloroform were found to affect the cytochrome spectrum differently. With
concentrations up to 0.1 per cent by weight, the time for the appearance of all
three bands (rate of reduction), was shortened. At about 0.3 per cent the A
band disappeared, the B and C bands appearing slowly and remaining fixed. At
about 0.5 per cent the B band disappeared.

Similar but less marked effects were obtained with ether and 95 per cent
ethyl alcohol. It is interesting to note that carbon tetrachloride has little effect.

The oxygen consumption of the chloroform-treated yeast, as determined by
the Warburg technique, was also greatly increased;—up to 20 times. Maximal
acceleration was obtained in a medium of phosphate buffer at pH 6.6. Less
effect was noted at pH 7.0 and little or none at 8.5. A similar acceleration was
observed with fertilized and unfertilized Arbacia eggs.

In view of the prevalent idea that narcotics act by depressing cellular respira-
tion, it seems significant that these accelerating effects on respiration were obtained
with concentrations of the same order of magnitude as are effective in producing
general anesthesia.

The oxygen consumption of activated and fertilized eggs of Chaetopterus.
Jean Brachet.

Unfertilized Chaetopterus eggs undergo activation when they are treated
with 5 per cent isotonic KCl in sea water; maturation is followed by a series of
monasterian cycles leading to the formation of unicellular larvae resembling
gastrulae and trochophores (F. R. Lillie’s differentiation without cleavage).
The oxygen consumption of these activated eggs has been compared with the
respiration of unfertilized and of fertilized eggs during 7 hours (Warburg’s
method). Activation is followed by a considerable drop in the oxygen consump-
tion (49 per cent); and fertilization has exactly the same effect, as was
shown first by Whitaker. The O, uptake increases then, but at a much slower
rate in the activated eggs than in the fertilized ones: while these resume their
initial respiratory rate after 3144 hours, the activated eggs need 6 hours to reach
that level. The respiration of the unfertilized eggs remains constant for 7
hours. Control experiments showed that isotonic KCl in the concentration of
5 per cent used has no significant effect on the metabolism of Chaetopterus eggs:
the slope of the curve is not changed if the KCl treated eggs have been re-
peatedly washed or when KCl is added to the fertilized eggs. The reduced
metabolic activity of the activated eggs must thus be linked to either their slower
development or to the fact that they remain unicellular.

Influence of respiratory inhibitors on stimulation of metabolism by nitro
and halo phenols. M. EK. Krahl, Anna K. Keltch and G. H. A.

Clowes.

At-the 1934 meeting, experiments reported from this laboratory showed that
the respiratory stimulation produced by 4,6-dinitro-o-cresol in fertilized eggs
of Arbacia punctulata was progressively inhibited and could be completely
abolished by increasing concentrations of potassium cyanide and that the division-
blocking effects of the two reagents were additive. During the past three seasons
these experiments with cyanide and 4,6-dinitro-o-cresol have been extended and
similar experiments made with other respiratory inhibitors.

The following concentrations of inhibitors have been found, with eggs in
sea water at pH 8, give a suppression of normal respiration which is just measur-
able (ie. 5 to 20 per cent): CO, 94CO:60,; ‘Amytal’ (Iso-amyl Ethyl Bar-

376 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

bituric Acid, Lilly), 2 X 10° M; malonic acid, 10° M; and, at pH 6, iodoacetic
acid, 10* M. At these same respective concentrations of the inhibitors there is
a progressive decrease in the extent to which the various inhibitors suppress the
respiration stimulus by 4,6-dinitro-o-cresol, the first two members of the series
giving almost complete, and the last two members almost negligible suppression
of the stimulated respiration. With the exception of phenyl urethane and
malonic acid, with which such experiments were not made, all inhibitors used
gave complete suppression of stimulation by 4,6-dinitro-o-cresol when tried in
sufficiently high concentrations. With partial inhibition by CO or KCN, the
optimum stimulation of the residual respiration was produced by concentrations
of 4,6-dinitro-o-cresol larger than those required in the absence of CO or KCN.
With partial inhibition by the other inhibitors, optimum stimulation of the re-
sidual respiration was produced by concentrations of 4,6-dinitro-o-cresol equal to
or less than those required in the absence of inhibitor.

In this series of experiments, the concentration of 4,6-dinitro-o-cresol re-
quired, in the absence of inhibitors, to give 90 to 100 per cent block to division
was 8 X 10° M. The concentrations of 4,6-dinitro-o-cresol required in the pres-
ence of the concentrations of inhibitors mentioned above, which alone gave little
or no block to division, were 4 X 10° M in CO; 8 X 10° M in low oxygen ten-
sion; less than 10° M in KCN; 8 X 10° M in malonic acid.

Substituted phenols as inhibitants of the fertilization of Arbacia and of
ciliary movement of Arenicola larvae. G. H. A. Clowes, M. E.
Krahl and Anna K. Keltch.

It has already been demonstrated for a considerable series of nitro and halo
phenols that the point of concentration required for maximum stimulation of
respiration corresponds approximately with the point at which cell division is
blocked in fertilized sea urchin eggs. In an attempt to throw further light on the
nature of the mechanism involved, the concentrations were determined at which
certain representative nitro and halo phenols blocked the fertilization of Arbacia
eggs by sperm and anesthetized Arenicola larvae. The block to fertilization and
anesthesia of larvae occurred at about the same concentration for each individual
compound, but these concentrations differed in certain cases very greatly from
the concentrations at which the respiration peak and cell division block occurred.

In the case of 2,4-dinitrophenol and 4,6-dinitro-o-cresol, having pK values
of 4.1 and 4.4 respectively, the ratios of concentration required for anesthesia to
that required for internal cell division block were found to be 137:1 and 228: 1.
That for 2,4,5-trichlorophenol, having a pK of 6.9, was found to be 19:1, whilst

‘Concentrations (moles per liter X 10°) of substituted phenols required to inhibit
various physiological functions. pH 8.0.

eal meciieeten IV :

Compound pK Moreen agar Breton I a TV
Arenicola Treated Arbacia
Sperm

2,4-Dinitrophenol............... 4.1 205 410 3.0 137:1
4,6-Dinitro-o-cresol............. 4.4 205 205 0.9 228:1
2,4,5-Trichlorophenol...........| 6.9 13 26 1.4 191

o-Nitrophenolwnrn 42.4 7.2. | Noeffect | No effect | No effect —
2,4-Dichlorophenol.............. dedi 51 26 26.0 esi
m-Nitrophenol.......:.....:..: 8.3 205 205 51.0 4:1

PRESENTED AT MARINE BIOLOGICAL LABORATORY 377

those for 2,4-dichlorophenol, having a pK of 7.7 and m-nitrophenol, having a pK
of 8.3, were found to be 1:1 and 4:1. It is particularly interesting to note that
orthonitrophenol, which although proved to enter the cell, had no effect on cell
division or oxidation, had also no effect on the fertilization process or on ciliary
movement of Arenicola. It appears advisable to refrain from speculation re-
garding these results until further data are available.

Stimulation of the rate of cell division of Arbacia eggs by carcinogenic
hydrocarbons. Anna K. Keltch, M. E. Krahl and G. H. A. Clowes.

As a part of an investigation into the mechanism by which certain polycyclic
hydrocarbons produce cancer, a study has been made, during the seasons of 1935
and 1936, of the effects produced by three carcinogenic and two closely related
non-carcinogenic hydrocarbons on cell division of fertilized eggs of Arbacia
punctulata, using each hydrocarbon in the form of its water-soluble choleic acid,
these being addition compounds of the hydrocarbon with desoxycholic acid.

Unfertilized eggs were exposed to varying concentrations of each choleic
acid in sea water solution for varying periods of time. They were then fertilized,
and left in the same respective solutions and at the same respective temperatures
during the periods of pretreatment, fertilization and division, with the single ex-
ception that at 5° C., the eggs were raised to 15° for approximately five minutes in
order to allow fertilization to take place and then returned immediately to 5° C.,
the controls in every case being subjected to treatment identical with that given
the experimental material.

Typical representative results obtained with a five-hour pretreatment are
presented in the accompanying table. In these data it is desired to emphasize—
(a) that the choleic acids of the three hydrocarbons which produce cancer in
mice also produce a shortening of division time and that the choleic acids of the
two closely related hydrocarbons which produce no cancer in mice do not pro-
duce a shortening of division time; (b) that, with optimum concentration of
6-methyl cholanthrene choleic acid, there is a progressively smaller relative de-
crease in the division time as the temperature is raised from 5° to 15° C.

Minutes to 50 Per Cent
First Cleavage
Tem- Carcinogenic
Choleic Acid pera- eae tap ah See Activity
ture Minimum in Mice
Control with
Hydrocarbon
© Ce
6-methyl cholanthrene........... 5 1717 1482 Positive
6-methyl cholanthrene........... 10 235 221
6-methyl cholanthrene........... 15 109 100
10-methyl-1,2-benzanthracene..... 15 122 116 Positive
1,2,5,6-dibenzanthracene......... 15 126 123 Positive
phenanthrene ec a. 2. see oot sees 15 LUST 118 Negative
Fluoranthene................... 15 115 115 Negative

The molecular species concerned in the action of substituted phenols on
marine eggs. Albert Tyler and N. H. Horowitz.

In a recently published article (Tyler and Horowitz, 1937) the view was
expressed that the substituted phenols penetrate as the undissociated molecule,

378 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

but exert their respiratory stimulating and reversible block to cleavage effects as
the anion. The evidence for this rests on the fact that with any one of these
compounds the concentration required for maximum effect varies with the pH
of the solution, but the calculated concentration of the undissociated form present
is the same at all pH’s. This might mean that the undissociated form is the
active species. However, when the various compounds are compared, the con-
centration of undissociated form at maximum effect shows enormous differences.
On the other hand, when the comparison is made on the basis of calculated con-
centrations of the anion present inside the cell the various substances give values
of the same order of magnitude. The compounds investigated include the three
mononitrophenols, 2,4- and 2,6-dinitrophenol, 2,4,6-trinitrophenol, the three mono-
chlorophenols, 2,4-, 2,5-, 2,6- and 3,5-dichlorophenol, 2,4,6-trichlorophenol and
2,6-dichloro-4-nitrophenol. Four of these; namely, o-nitrophenol, trinitrophenol,
o-chlorophenol, and 2,6-dichlorophenol, show large deviations, but these substances
are actually inactive or only slightly active in stimulating respiration.

Extension of the length of fertilizable life to more than twice the control is
also obtained with dinitrophenol, confirming the findings reported by Clowes
and Krahl. This prolongation occurs at high concentrations giving no respira-
tory stimulation. Determinations of the temperature coefficient of the respiration
of unfertilized eggs shows it to be the same in dinitrophenol as in sea water.

Ovoverdin, a pigment chemically related to visual purple. Kurt G.
Stern and Kurt Salomon.

The eggs of the lobster (Homarus americanus) owe their green color to a
pigment belonging to the class of carotenoid-proteins. According to G. Wald?
visual purple is another member of this widely distributed group of chromo-
proteids.

The carotenoid contained in the lobster egg pigment is astacin which is
esterified with an as yet unidentified organic acid.” * This “ovoester” is in turn
linked up with a protein of albuminoid character. The name ovoverdin is pro-
posed for the native pigment complex.

Ovoverdin may be obtained in solution by grinding the eggs with sand and
extracting them with distilled water. Treatment with an equal volume of sat-
urated ammonium sulfate removes oil globules containing carotine and small
amounts of globulins. The solutions may be further purified by repeated pre-
cipitation of ovoverdin in saturated ammonium sulfate solution or by dialysis at
low temperature.

Ovoverdin has two absorption bands in the visible, at 6,400 and 4,700 A.; in
addition it shows the typical protein absorption in the ultraviolet. The molecular
weight, according to the rate of sedimentation in the ultracentrifuge as measured
by Dr. R. W. G. Wyckoff, is of the order of 300,000. The isoelectric point is at
pH 6.7.

Organic solvents and weak acids liberate the orange red carotenoid by virtue
of denaturation of the protein carrier. When solutions of ovoverdin or the
whole lobster eggs are rapidly brought to 65 to 70° the color turns from grass
green to bright orange red. The red form shows increased light absorption at
4,700 A and greatly diminished absorption at 6,400 A as compared with the green
form. When the heated material is rapidly cooled, the green color returns.
This reversible thermal dissociation is different from an irreversible dissociation
which takes place upon longer exposure to these temperatures or upon raising the
temperature to the vicinity of the boiling point. At the latter point coagulation

*Wald, G., 1935-36. Jour. Gen. Physiol., 19: 351.
* Kuhn, R., and E. Lederer, 1933. Ber. Deutsch. Chem. Ges., 66: 488.
*Karrer, P., L. Loewe, and H. Huebner, 1935. Helv. Chim. Acta, 18: 96.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 379

of the protein occurs. The sequence of events and a photoelectric study of the
color-temperature-time curves indicates a lower energy requirement of the first,
reversible stage of dissociation as compared with the later, irreversible stages.

It has been suggested** that the bleaching of visual purple by light is a
disruption of the purple carotenoid-protein complex; the orange carotenoid,
retinene, is liberated and the protein is denatured. This assumption, however, is
open to the objection that the energy content of the effective wave-lengths of
light is smaller than the energy required for inactivation of the visual purple
complex. The present observations would suggest that the reversible bleaching of
the retinal pigment does not involve a denaturation of the protein component but
is rather of the type of the thermal phenomenon here observed. This hypothesis
appears to receive support from the fact that the energy levels at which the latter
occurs are lower than those at which protein denaturation takes place and secondly
that the process observed here is rapidly reversible whereas protein renaturation
in general is a time-consuming process and therefore not well suited for the
regeneration requirements of visual purple during vision.

The increase of CO, and decalcification in certain pelecypods. Louis-
Paul Dugal and Laurence Irving.

In three forms of pelecypods, Venus mercenaria, Ostrea sp. and Elleptio
complanatus, the mantle cavity fluid gains CO, when they are kept out of water.
The change is from 5 or 8 (in fresh ones) to 90 ml. per 100 ml. of fluid (for
individuals kept out of water about 5 or 6 days). When the mollusks begin to
die, the total CO, decreases. Return to water before death restores the CO2 to
normal. The accumulation of CO, probably results from a disturbance of respira-
tion.

In Venus, which was most carefully studied, the total CO, of the M.C.F.
increases rapidly. The pH decreases only from 7.4 to 7.2 and the Poo, increases
only from 3 to a maximum of 25 mm. Hg, so that it is evident that the buffering
capacity increases.

The shell is eroded during these changes and a few analyses showed that
the mantle cavity fluid gained Ca, so that it is easy to guess that the buffering
is provided by the solution of CaCO; from the shell.

The shell erosion is localized in the central inner part; the mantle tissue and
no others, gain CO.. This indicates that the buffering is effected by the activity
of a special tissue.

The effect of pH and ionic strength on the activity of carbonic anhydrase.
J. K. W. Ferguson and E. C. Black.

The manometric method of Meldrum and Roughton for determining the ac-
tivity of carbonic anhydrase by following the rate of evolution of CO2 from a
mixture of phosphate and bicarbonate solutions, has been favored, because of its
simplicity, for use in physiological and pathological studies. The reaction used in
this method takes place in a medium of changing ionic strength and pH. As yet
no adequate analysis of the effect of these variables on the activity of the enzyme
has appeared.

In this preliminary study the COz output method has been used and con-
sequently the variation of pH was limited to the range from pH 5.7 to pH 8.0.
The pH and ionic strength taken as corresponding to a certain activity was the
mean pH and ionic strength of the range traversed by the stage of the reaction
used in calculating the activity (usually from the beginning to two-thirds com-
pletion).

* Mirsky, A. E., 1936. Proc. Nat. Acad. Sci. (Washington), 22: 147.

380 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

Between pH 6.0 and 8.0 an eight-fold increase in enzymic activity was found,
the activity increasing with pH. Between ionic strengths of 0.5 and 0.1 at constant
pH a 2.5-fold increase in enzymic activity was found, the activity increasing as
the ionic strength decreased.

The distribution of carbon dioxide in dogfish blood. J. K. W. Fergu-
son, S. M. Horvath, and J. R. Pappenheimer.

After unsuccessful attempts to apply to fish bloods the chemical and kinetic
methods of estimating carbhemoglobin, evidence concerning the state of CO, in
erythrocytes of fish was sought by studying the distribution of COs between red
cells and plasma. In this study smooth dogfish (Mustelus canis) and a few speci-
mens of spiny dogfish (Squalus acanthius) were used. Defibrinated or heparinized
blood was used exclusively after it was found that both oxalate and fluoride grossly
affected the distribution of electrolytes between cells and plasma.

The bloods were found to fall into two groups as regards total COz2 capacity.
During June and early July the CO: capacities at a CO2 pressure of 40 mm. Hg.
were on the average twice as great as in the bloods obtained during late July and
August. In the early part of the season the curves were steeper, indicating
greater buffer power. This difference could not be attributed to differences in
hemoglobin content. The degree of oxygenation has no effect on the CO, capacity
of dogfish blood.

Usually the concentration of CO2 in the red cells (per units H2O) is greater
than in the corresponding plasma. This is different from the situation in mam-
malian blood and inconsistent with the view that the bulk of the CO: in the cells
is in the form of active bicarbonate ions. When chloride distributions were
studied the contrast was striking. The ratio of intracellular to extracellular con-
centration of chloride, (rci), is always much smaller than the similar ratio for
COs, (rco,). In mammalian blood rco, = € 1.25 Xrci. In these bloods rco, = 1.7
to 3 Xrce. If the intracellular CO. which is in excess of the amount to be ex-
pected from the chloride ratios is assumed to be “ non-bicarbonate”’ COs, this
“non-bicarbonate”” CO2 is found to comprise about two-thirds of the CO, in the
red cell or about one-sixth of the CO, in the whole blood.

The influence of certain alcohols on the permeability of the erythrocyte.
M. H. Jacobs and A. K. Parpart.

Low and moderate concentrations of the so-called indifferent narcotics have
frequently been found to decrease cell permeability. Anselmino and Hoenig
(Pfliigers. Arch., 225: 56, 1930) have reported such an effect in the case of the
penetration of human erythrocytes by several non-electrolytes including glycerol.
The present study, involving the erythrocytes of a number of species of mammals
and of some other vertebrates shows somewhat more complicated conditions. Thus,
while n-butyl alcohol in concentrations from 0.0156 M to 0.25 M may greatly de-
crease the permeability of the erythrocytes, not only of man but also of the rat,
rabbit, guinea pig, groundhog and several birds, the opposite effect is obtained
with the erythrocytes of the ox, sheep, pig, horse, dog, cat and several reptiles and
fishes. In general, these two groups of species are the same as those already
distinguished by other properties of their erythrocytes (Jacobs, Glassman and
Parpart, Jour. Cell. Comp. Physiol., 7: 197, 1935). In several cases involving de-
creased permeability, the order of effectiveness of a series of alcohols is: methyl<
ethyl <propyl<butyl<amyl. With erythrocytes of the groundhog the effective-
ness of n-butyl alcohol increases with increasing molecular weight of the penetrat-
ing substance in the order: ethylene glycol<glycerol<erythritol<mannitol. As
contrasted with glycerol and related substances to which n-butyl alcohol increases
the permeability in some species and decreases it in others, thiourea, under the

PRESENTED AT MARINE BIOLOGICAL LABORATORY 381

same conditions, always shows an increased permeability. The same was found to
be true of lipoid-soluble substances such an monoacetin and ammonium salts of
weak acids, which by hydrolysis give rise to NH, and lipoid-soluble acids. On
the contrary, permeability to the ammonium salts of strong acids, where the pene-
tration of the cell by ions is presumably involved, was in all species found to be
greatly decreased.

Tonic exchanges of erythrocytes inferred from volume changes. A. K.
Parpart, M. H. Jacobs and A. J. Dzienmian.

The exchange of SO, for Cl— across the erythrocyte surface leads to a
volume change of the erythrocyte which may be measured photoelectrically. Cells
shrink when placed in isosmotic NazSO, solutions. The process is reversible since
cells which have had the normal Cl— of their interior replaced by SO,= will swell
when placed in isosmotic NaCl solutions. The exchange of Cl— for SO is
relatively slow in comparison with the osmotic shifts that accompany it. The rate
of this ionic exchange varies for the erythrocytes of different species of mam-
mals in the order rat>guinea pig>rabbit >beef > pig, cat.

In view of the slowing effect that butyl alcohol, ammonium salicylate and
ammonium benzoate have on hemolysis in ammonium chloride it appeared of in-
terest to study the effect of these substances on the exchange of Cl— for SO.
In all of the species studied the above substances were found to slow the rate of
this ionic exchange. Control experiments indicated that these substances, in the con-
centrations used, had little or no effect on the volume of erythrocytes suspended
in isotonic NaCl.

PAPERS READ BY TITLE

Oxygen as a controlling factor in the regeneration of Tubularia. L. G.
Barth.

Preliminary experiments in which the distal end of the stem of Tubularia
was inserted into a glass tube gave complete inhibition of regeneration of this
end with a marked increase in rate at the opposite end. This indicated that low
oxygen inhibited regeneration.

Following this the oxygen tension of sea water was varied and rate of
regeneration was calculated by dividing the length of the primordia in micra by
the number of hours necessary for its formation. Results of nine complete ex-
periments agree in showing that from 4.0 to 21.6 cc. of oxygen per liter the rate
of regeneration increased linearly with the logarithm of the oxygen tension.
Below 3.0 cc. of oxygen per liter the rate of regeneration drops off to 0 at
.35 cc. and the shape of the curve depends somewhat on the way in which the
experiment is carried out. The lower limit for regeneration is between .35 cc.
and 1 cc. of oxygen per liter. At .35 cc. complete inhibition results, which is
reversible when stems are returned to high oxygen.

The results are interpreted as showing that regeneration of JTubularia is
closely dependent on the amount of oxygen which the tissues receive. It is
suggested that the perisarc of Tubularia is relatively impermable to oxygen and
that the stimulus for regeneration is the admission of oxygen due to cutting of
the perisarc.

The effects of different drugs on the melanophores of Fundulus hetero-
clitus. Sinisha B. Bogdanovitch.

In the following experiments the effects on melanophores of different drugs
alone and in combination were studied. Isolated scales of Fundulus heteroclitus

382 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

were used and the drugs studied were atropine, pilocarpine, physostigmine, epine-
phrine, acetylcholine, mecholyl and deuterium oxide. The technical procedures
were the same as those described in a previous paper. A summary of the results
obtained follows:

Atropine: Expands melanophores and also does so after they had been pre-
viously contracted by epinephrine, deuterium oxide, acetylcholine or mecholyl.
Ergotized melanophores were also expanded by atropine.

Epinephrine: Contracts melanophores and also does so after they had been
previously expanded by pilocarpine, physostigmine or atropine. Ergotized
melanophores are, as is well known, expanded by epinephrine.

Physostigmine: Expands melanophores and also does so after they had
been previously contracted by epinephrine, acetylcholine, or mecholyl. Well
ergotized melanophores are not expanded by physostigmine.

Pilocarpine behaves as physostigmine, but appears to be slightly more ef-
fective.

Acetylcholine contracts melanophores and also does so with melanophores
previously expanded by physostigmine or pilocarpine. However, with melanophores
previously expanded by atropine acetylcholine produces either very slight con-
tractions or none at all. Ergotized melanophores are contracted by acetylcholine.
Ergotized melanophores are, of course, already contracted, so that additional con-
traction is sometimes quite slight. Mecholyl behaves as acetylcholine but the
effects appear to be more lasting.

Deuterium oxide contracts melanophores after they had been previously ex-
panded by atropine, pilocarpine or physostigmine. Other effects of deuterium
oxide on melanophores were described in a previous paper.

Scales removed from fish and kept in balanced solutions for two hours or
more showed an initial expansion in epinephrine followed by contraction. Such
a result, while possibly due to a pathological change, may explain contradictory
results of previous investigators and offer a clue as to the nature of the inner-
vation of melanophores.

Further investigations on the effect of tissue on different drugs. Sinisha
B. Bogdanovitch.

In a former paper it was established that epinephrine and acetylcholine are
destroyed by tissue (in this case scales of Fundulus heteroclitus), but that deu-
terium oxide protects these substances from such destruction. Using the same
technique as in former experiments atropine, pilocarpine, physostigmine and
mecholyl (acetyl 6-methylcholine) were similarly investigated with the following
results.

Solutions of atropine (sulphate) which had previously expanded melano-
phores, no longer did so or at most only slightly, after treatment with fish scales
for 24 to 48 hours. However, atropine solutions to which scales and deuterium
oxide were added for the same length of time, showed no change in expanding
melanophores. Physostigmine (sulphate) and pilocarpine (chloride) solutions
were not affected by fish scales even after 48 hours. Solutions of mecholyl
(chloride) were also practically unchanged by similar treatment.

It thus appears that atropine is destroyed by fish scales as was epinephrine
and acetylcholine, but that physostigmine, pilocarpine and mecholyl were not af-
fected. In the former paper it was suggested that the destruction of epinephrine
and acetylcholine by fish scales may be due to the action of enzymes such as
acetylcholinase and some oxidase. In support of this view I observed that scales
which had been heated at 100° C. for 10 minutes did not destroy atropine even
after 48 hours. Similar experiments on epinephrine and acetylcholine gave the
same results. This thermolability of the “destructive principle” in fish scales
for these three substances is consistent with the enzyme hypothesis.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 383

A quantitative study of the staiming of marine eggs by neutral red.
Barry Commoner.

Unfertilized eggs of Chaetopterus and Nereis limbata were stained in sea
water containing various concentrations of neutral red at 14.0°, 15.5°, 17.0°,
18.4°, and 19.5° . The pH was maintained at 7.6.

Eggs were suspended in sea water acidified to pH 7.6. The density of eggs
per ml. of suspension was determined and a known quantity of neutral red added.
The same certified stain of 61 per cent dye content was used throughout. Con-
centrations are expressed as mgm. of this preparation.

Samples were removed periodically from the suspension and centrifuged to
concentrate the eggs. The eggs were washed in sea water to remove unbound
stain, again concentrated and shaken in 10 ml. of a neutral red extractant. (One
volume of N/1 HCl and 9 volumes of 95 per cent ethyl alcohol.) The neutral
red content of the egg sample was then determined by comparing (in a Duboscq
colorimeter) the intensity of the extract with a standard neutral red solution.
The neutral red content per unit volume of eggs was then calculated. (Maximum
error; 3 per cent.)

Curves were plotted indicating the neutral red content of the eggs after
various periods of time in the stain. Curves of similar shape were obtained for
Chaetopterus and Nereis eggs. The maximum staining rate occurs during the
first 20 minutes. Thereafter, the velocity of the process decreases until after
60-80 minutes an equilibrium is reached and no further change in neutral red con-
tent occurs.

At concentrations below .02 mgm./ml. the staining rate is proportional to the
concentration of neutral red in the suspension. The stain content of the eggs
at equilibrium is also proportional to the concentration of neutral red.

The initial staining velocity and the quantity of neutral red bound at
equilibrium increase with temperature. Within the temperatures noted (con-
centration: .01 mgm./ml.) the Q,;, obtained from the initial velocities of the
staining curves of Chaetopterus eggs was 4.4.

The adaptation of Paramecium to sea water. John A. Frisch, S.J.

In cultures of hay and wheat infusions, in which the concentration of sea
water was gradually increased by evaporation, many individuals survived and
divided until a concentration between 35 and 40 per cent was reached; all died
before a concentration of 45 per cent was reached. Daily observations of a
culture in which the concentration of sea water increased to 40 per cent in 20
days showed that the average rate of pulsation and the average rate of feeding
were lower day for day than in fresh water cultures; that both rates varied
from day to day as in fresh water cultures, and that the rate of pulsation varied
with the rate of feeding, increasing or decreasing from day to day, as the rate of
feeding increased or decreased, just as in fresh water cultures. As the salt con-
centration increased the animals decreased in length and volume and became
emaciated. Addition of nutrient medium or of bacteria to ‘the cultures always
resulted in an increase in the number and in the volume of the individuals, and
in the rate of feeding and the rate of pulsation, except when the concentration of
sea water had reached 40 per cent. The data indicate that the decrease in the
rate of pulsation is not due to the increase in osmotic pressure of the medium,
but to the decrease in the rate of feeding; that the decrease in the rate of feeding
is due to a shortage of bacteria in the higher salt concentrations; that death is
due to an increase in the viscosity of the protoplasm and to other toxic effects
of the salts taken in by the cytostome, which result in the vacuolization of the
protoplasm.

384 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The water and fat content of skeletal muscle in marine fishes. Charlotte
Haywood and Abby Turner.

In the course of a study of water balance in fishes of different life habits
and phylogenetic relationships a series of determinations was made of both the
water and fat content of skeletal muscle. This work was done in part at Bergens
Museum Biologiske Stasjon, Herdla, Norway, and in part at the Marine Biologi-
cal Laboratory, Woods Hole.

Percentage of Water in

No. of Percentage
Indi- of
viduals Fats Whole Fat-free
Muscle Muscle
Elasmobranchs:
Mustelus canis.............. 4 Trace 78.4 78.4
Pristiurus catulus*...........- 4 — 80.0 —
Galeus glaucus..............- 1 Trace 81.0 81.0
Carcharias Tauruse 2-2... 2 0 77.4 77.4
Squalus acanthias*........... 1 — 78.4 —
Squalus acanthias*........... 7 5.4 75.0 79.4
Spinax niger*............--- 4 — 80.6 —
ARO OMAUOPWOITES arias ee eis 2 eh 1 0 Sail oe
IRI G SHOLODUOS = ds go Oo aoa 2 0 76.5 76.5
IROGO WOMORUG” 65 eo cba haahoo€ 1 — 79.0. —
Raja oxyrhynchus*........... 3 — 81.1 —
Narcacion nobilianus......... 3 0.4 83.9 84.2
Dasybatus marinus........... 2 Trace 717.9 717.9
Chimaera monstrosa*......... 3 —_ 81.3 —
Teleosts:

Anguilla vulgaris*........... 3 — 66.4 —_
Anguilla rostrata............. 1 4.7 73.3 ee
Scomber scombrus*........... 6 — 66.5 —
Scomber scombrus*........... 8 5.4 70.5 74.5
Centropristes striatus......... 11 i.) Useth 79.5
Cynoscion regalis.......5.5-- 2 Soll 76.7 79.2
Tautoga onitis............... 4 Trace 79.8 79.8
Cyclopterus lumpus*.......... 1 — (BL —
Prionotus strigatus........... 2 0 79.9 79.9
Echenets naucrates........... 1 3.7 73.1 76.2
OPSUTIS UD si c0c0 en cco cone os 2 Trace 80.2 80.2
Anarhichas lupus*........... 6 — 84.0 —
Gadus callarias*............. 8 — 80.3

Gadus callartas*............. 6 0 81.7 81.7
Gadus pollachius*............ 7 — 79.4

Brosmius brosme*............ 6 — 80.0 =
Molva molva*................ Z — 80.3 =
Lophius piscatorius*.......... 5 — 84.3 —
Lophius piscatorius*.......... 2 0 87.1 87.1

* Determinations made in Norway.

Reactions to light of different intensities in Dolichoglossus kowalevsky.
Walter N. Hess.

For studying the sensitivity of Dolichoglossus to different light intensities a
100-watt Tungsten bulb, supplied with neutral tint Wratten filters, was used and

PRESENTED AT MARINE BIOLOGICAL LABORATORY 385

light of 115, 11.5, 1.15, and .115 m.c was thus obtained. For studying the rela-
tive photosensitivity of different regions of the body a fused quartz rod was sus-
pended so that its base was illuminated by an arc lamp. The distal end, which
was used in testing photosensitivity of small areas, was drawn out into a very
small blunt point.

The animals reacted negatively to ordinary intensities of light but at .115 m.c.
a rather large majority of positive responses occurred. By means of the pin-
point light it was shown that the animal is photosensitive over its entire body
though certain regions are more light-sensitive than others. The tip of the
proboscis is the most sensitive to light. In general, the dorsal and lateral body
surfaces are more photosensitive than the ventral surface.

Mucous cells occur on the abdomen in patches interspersed with non-mucous
cells. Very little response was obtained by illuminating the areas of mucous cells
especially on the ventral surface, yet if the areas of non-mucous cells were
illuminated the animals responded quickly.

These results show that the photoreceptor cells must be widely distributed
and that they must be more numerous or more specialized in certain regions
than in others. Cells of a certain type which correspond in their distribution to
the relative photosensitivity of the animal have been identified. In keeping with
the early chordate characteristics of this animal these cells resemble in their
general morphology the retinal cells of vertebrates.

The hatching of the squid. Hope Hibbard.

The hatching gland of Loligo pealit is the so-called Hoyle’s organ, a Y- or
T-shaped gland which lies on the dorsal posterior surface of the larva, the arms
of the gland extending out on the fins. It appears very early (about stage
12 of Naef in L. vulgaris), gradually matures, and disappears entirely shortly
after the larva hatches. At the height of its development the gland protrudes
slightly. A similar hatching gland has been described by Wintrebert and by
Jung in several other cephalopods found in Europe. Staining the eggs or young
embryos, even very heavily, with neutral red does not impede their normal de-
velopment, and the organ in question can be very readily observed since it does
not stain and appears as a white streak against a reddened mantle.

In early development the larva rotates inside its shell by ciliary action, push-
ing the posterior end up until gravity causes it to lose balance, and fall to the
lower side of the shell whence rotation recommences. But as hatching approaches
the animal adheres very firmly to the shell as if stuck by a secretion, in a disc-
shaped area around the hatching gland. The head is directed downward, and
rhythmic contractions of the free part of the mantle are constant. The gland
itself appears to wriggle and squirm from time to time, due to underlying muscle
cells, pushing ever closer to the shell. In some cases the weak place bulges con-
siderably before giving way. Finally the shell appears to dissolve away and the
animal swims out backward through a neat round hole. There is no tearing,
and after the larva has emerged the round hole remains in the empty shell. .

The cells of the gland are very long, slender ones with the nucleus at the
proximal end deeply embedded in the mantle. The secretion is conspicuously
granular and resembles well-fixed zymogen granules in a pancreas cell. Pressure
on the cells forces the granules out, and they maintain their individuality out-
side. After hatching, the cells remain for a few hours, but the distinctness of
the unsecreted granules disappears, the material becomes more fluid and runs
together. Further cytological examination of the evolution of these cells is
under way.

386 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The vacuole system of the marine Amoeba, Flabellula mira. D. L. Hop-
kins.

In this study the vacuole system was observed when the amoebae were under
normal conditions; slightly abnormal conditions; unstained; stained with the vital
dyes, neutral red, Nile blue sulfate and Janus green B; and after impregnation
with osmium tetroxide.

Mitochondria which stain with Janus green B are not present in normal fully
active amoebae, but appear and may be stained with Janus green B when the
amoebae are placed under slightly abnormal conditions. The mitochondrial sub-
stance in fully active amoebae appears to be contained in very small vacuoles.
These vacuoles arise de novo from the protoplasm, grow, fuse with each other
and engulf food. The food vacuoles and the vacuoles containing no food fuse
with each other, forming larger and larger vacuoles, until finally a large fusion
vacuole, the cloacal vacuole, is formed. The fluid and solid contents of this
cloacal vacuole after a period of digestion are voided to the outside.

Nile blue sulfate stains all these vacuoles blue from the time of their origin
until after their entrance into the cloacal vacuole where the blue fades out, in
some cases becoming clear of color directly, while in others it becomes pink or
red and then clear. This indicates that fatty acids are present in all vacuoles,
and perhaps some neutral fat. Both of these types of substances disappear from
the vacuoles before the residue is voided.

The substance stained by neutral red is present in all the smaller vacuoles
but disappears entirely before they enter into the cloacal vacuole.

Neutral red staining, and osmium tetroxide impregnation of amoebae pre-
viously treated with neutral red show that the smaller vacuoles and the cloacal
vacuoles are in a more highly oxidized, or in other words, in a less reduced con-
dition than are the vacuoles of intermediate size.

Effect of electrical shocks upon the division rate of Stylonychia pustulata
as measured by the interdivisional period. Lois Hutchings.

The normal interdivisional period of Stylonychia pustulata in summer was
found to be six to ten hours. Most frequently the control animals underwent
division once every eight hours. Every animal used in these experiments came
from one protozoon. Isolation dishes were used. The culture medium was
filtered hay-tea in which mass cultures had lived previously for five to twenty
days, average pH 7.4. A piece of dry outmeal, one-millimeter square, was placed
in each dish when the medium was changed each day.

Two new dry-cell batteries were connected in series to the primary circuit of
an inductorium, Harvard type. The secondary coil was kept at the eight-cen-
timeter mark. The current which flowed through the secondary circuit had a
non-smooth alternation of 106 times a second, known as tetanizing current. A
foot switch closed the circuit at will. From the secondary circuit were two
insulated copper wires to whose tips were soldered one-inch lengths of No. 30
platinum wire. Each point electrode was so manipulated by hand that a con-
stant distance of twice the body-length between it and the animal was maintained.
Even when the protozoon moved it was usually possible to keep the interelec-
trodal distance constant.

Each Stylonychia received treatment hourly. For several reasons physiologi-
cal indices of sufficient treatment were adopted. In order of importance these
were: degree of swelling, loss of lateral orientation to the current, and rapid
spinning. The length of treatment varied from 10-75 seconds.

Disregarding the five cases in which the treated Stylonychia had the same
length of interdivisional period as the control animals, the results may be simply
stated. Fifteen treated animals underwent an average shortening of the inter-

PRESENTED AT MARINE BIOLOGICAL LABORATORY 387

divisional period by 2 hours, but 102 animals had an average lengthening of the
interdivisional period by 634 hours. The range of shortening was %-3%4
hours and the range of lengthening was 14-23 hours. In other words, although
in 13 per cent of the cases application of electrical shocks caused an apparent
acceleration of the division rate, in far the greater number of cases, 87 per cent,
such shocks caused a slowing down of the division rate.

Further comparative studies on the permeability of the erythrocyte. M.
H. Jacobs and H. N. Glassman.

In earlier papers (Proc. Am. Philos. Soc. 70, 363, 1931; Jour. Cell. Compar.
Physiol. 7, 197, 1935) certain characteristic differences in the permeability of the
erythrocytes of different species of vertebrates to dissolved substances were reported.
The results previously obtained by means of the hemolysis method were not
very satisfactory in the case of many species having nucleated erythrocytes be-
cause of the failure of suspensions of the latter to become completely transparent
on hemolysis. By taking advantage of the fact that this difficulty is largely
removed by the addition to the hemolytic solutions of 0.001 M NaHCOs, without
greatly altering the time of hemolysis in the case of most of the species studied,
further data have been obtained on the permeability to ethylene glycol, glycerol,
urea, and thiourea, of the erythrocytes of 9 species of elosmobranchs, 14 of
teleosts, 2 of frogs, 6 of turtles, 4 of snakes and 4 of birds. In addition to
various highly characteristic specific differences the following general peculiarities
of the larger groups—subject to some exceptions which cannot here be men-
tioned—are of interest. Fishes: permeability to ethylene glycol greatest; that to
urea and glycerol highly variable from species to species; permeability to thiourea
usually greater than that to urea. Amphibia: (data as yet too scanty to permit
generalizations). Birds (gull, tern, English sparrow, pigeon): permeability to
ethylene glycol and glycerol both very great and nearly equal; that to thiourea
much less and to urea least of all. Earlier experiments showed similar conditions
in the starling but not in the duck and the chicken. Reptiles: permeability to urea
relatively great, followed by that to ethylene glycol and at a much greater dis-
tance by that to thiourea. Permeability to glycerol slight as compared with that
to the other substances. Mammals: (for comparison) permeability to urea ex-
tremely great; that to ethylene glycol much less and to thiourea still less; per-
meability to glycerol in some species greater and in some species less than that
to thiourea.

The attenuation of toxins by interfacial adsorption. J. M. Johlin.

In view of the fact that some toxins, upon standing, are changed into toxoids,
the writer thought it likely that similar attenuations might be brought about
more speedily by methods which induce interfacial adsorption, and has applied
such a method to the attenuation of ricin, tetanus toxin and snake venom. This
speedy attenuation may be regarded as the result of the catalytic influence of the
interface in bringing about an increased surface concentration of properly oriented
molecules of the toxin at the interface. Adsorption was brought about by
emulsification of the toxin with a volatile inert liquid such as ether or chloroform
which could be easily removed by evaporation under reduced pressure afterwards.
Ricin, thus attenuated, was found to be ten thousand times less active when
injected intracutaneously into rabbits. One thousand M.L.D.’s of tetanus toxin,
when attenuated, could be injected subcutaneously into mice repeatedly at two-
day intervals without killing them or causing any apparent prolonged discomfort.
Multiple lethal doses of mocassin and rattlesnake venom could also be injected
into mice without causing death or producing the usual signs of damage caused
by the untreated venoms. In such immunization experiments with rabbits as

388 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

have been carried out it was found that the attenuated toxin had retained its
antigenic property and could be injected daily in large amounts without producing
visible symptoms of any kxind.

The action of acetylcholine on the skeletal muscle fibers of the frog.
_Elsa M. Keil and F. J. M. Sichel.

In a previous report by the present authors (Biol. Bull. 71: 402, 1936) it was
shown that the application or injection of very small amounts (50004°) of
acetylcholine in concentrations ranging from 1 in 10* to 1 in 10* had no effect on
the isolated single fiber (adductor muscle of frog’s leg, Rana pipiens), beyond
the effects attributable to the medium in which the acetylcholine was dissolved.
Recent experiments with acetylcholine on single muscle fibers isolated from the
sartorius of the frog confirm this. However, if the sartorius be dissected from
the frog, and even if both the tibial and pelvic ends be cut across, then acetylcho-
line will cause a propagated twitch when applied as a small droplet with a micro-
pipette to the dorsal surface of the muscle in the region of the nerve twigs.
This twitch is noticed in only those muscle fiber bundles which are in the
vicinity of the point of application of the acetylcholine. If either the pelvic
or tibial half of the sartorius is split lengthwise into two parts, one containing
about twenty fibers, the other the rest of the fibers, then acetylcholine applied
to the surface of either of these bundles also causes a similar propagated twitch
response. If the smaller bundle of fibers is progressively split into still smaller
groups, a degree of subdivision will eventually be reached such that even dilute
acetylcholine will no longer evoke twitches in fibers still irritable to electrical
stimulation. This apparently occurs when the subdivision has interfered with
the nerve supply, the fine nerve twigs being injured by the process of dissection.

We conclude that these experiments offer further evidence in support of our
previous statement that the action of acetylcholine is not upon the contractile
mechanism of the muscle directly. We further conclude that acetylcholine evokes
twitches in the sartorius muscle of the frog only when the terminal nerve supply
or possibly some junctional tissue or receptor is intact. These conclusions are
in accord with Garrey’s experiments on turtle and Limulus heart, and with Arm-
strong’s experiments on embryonic Fundulus heart.

The effect of pituitary on nuclear changes in the egg of the frog. John
A. Moore.

In an effort to separate the processes of maturation and ovulation in the
frog, Rana pipiens, mature females were given homoplastic anterior lobe injec-
tions during the middle of the summer, at which time it has not been possible to
stimulate the release of eggs. Parts of the ovary were removed before and
after injection so the same animal serves as a control and as an experimental.
It was found that by thirty to forty hours after injection (at 25-27° C.) the
germinal vesicle had moved from its position in the center of the egg to the
surface at the animal pole. This movement is found only in the large eggs with
evenly distributed yolk. By one hundred and twenty hours the eggs are degen-
erating and have been heavily invaded by phagocytes. This latter condition is
similar to that found by Miss King in the few mature eggs that remained in the
ovary of the toad after the breeding season.

The effect of urea upon the surface of unfertilized Arbacia punctulata
eggs. Floyd Moser.

Following the procedure of Moore (Protoplasma, 1930) Arbacia punctulata
eggs were treated with molar urea solutions. After a total exposure of no

PRESENTED AT MARINE BIOLOGICAL LABORATORY 389

more than two minutes, the eggs were transferred to sea water. Some of them
were then inseminated and others served as uninseminated controls.

Within three or four hours irregularly cleaving eggs (two to eight cells)
may be found among both the treated inseminated eggs and the treated lunin-
seminated eggs. At the same time apparently normal blastulae with membranes
perhaps a little thinner than normal may be found among the treated inseminated
eggs, but not among the uninseminated eggs. In some cases it is possible to
demonstrate very thin and but slightly elevated membranes in these irregularly
cleaving eggs.

If a small drop of sea water containing centrifuged or uncentrifuged eggs
be placed on a slide and a larger drop of molar urea solution be caused to flow
into the drop of eggs while the latter are being observed under the high power of
the microscope, a breakdown of cortical granules occurs. Immediately following
this cortical response membrane elevation occurs with the formation of a very ©
wide perivitelline space. Within a few seconds the elevated membrane begins
to recede toward the egg surface, becoming thinner as it moves, until finally, in
some cases, no vestige of the membrane remains. The cortical response secured
in these urea-treated eggs is essentially like that obtained upon stimulation with
sperm cells or with agents which induce parthenogenesis such as saponin. Similar
cortical responses are secured in molar thiourea and molar glycerine solutions.

It is evident, therefore, that these non-electrolyte solutions do not prevent
membrane elevation in Arbacia punctulata eggs (either irreversibly or otherwise),
but may actually stimulate the egg to cortical response and subsequent membrane
elevation. Indeed, delayed and irregular cleavage often follows the cortical re-
sponse after treatment with these agents.

The cortical response of Arbacia punctulata eggs to direct current.
Floyd Moser.

In a previous abstract (Biological Bulletin, 1935) it was noted that a layer
of granules located in the cortex of the unfertilized Arbacia egg breaks down
when the egg is subjected to a number of different stimulating agents, as well as
to normal insemination. The present experiments with direct current extend the
list of stimulating agents reported earlier.

A non-polarizable system of Cu-CuSO, electrodes with agar bridges was used.
The eggs were placed in small glass tubes both ends of which were plugged with
2 per cent agar made up in sea water. The current intensity varied from 1.5 to
20.0 milliamperes.

As soon as the circuit is closed the eggs begin to move toward the anode.
Within a few seconds cortical layer granules on the anodal side of the egg break
down, releasing the fertilization membrane from that portion of the egg over
which the response has taken place. When the current is reversed the same re-
sponse takes place on the opposite side of the egg. Generally some of the cyto-
plasmic granules beneath the cortex also exhibit this breakdown phenomenon.
Indeed, when the current flows for too long a time the cortical response is
followed or accompanied by complete cytolysis.

In the uncentrifuged eggs the cortical response stops when the circuit is
broken, but generally completes itself in wave-like fashion in the centrifuged egg.
The so-called fifth layer usually breaks down in the centrifuged eggs.

If the eggs are treated with M/4 ammonium oxalate, 0.3 M potassium
oxalate or 0.35 M potassium citrate there is no granule breakdown or membrane
elevation upon exposure to direct current.

The observations here recorded are in general agreement with the point of
view developed by Heilbrunn and his students in their studies of stimulation.

390 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The histology of the retractor muscles of Thyone briareus Lesueur.
Magnus Olson.

A decided gap exists in our knowledge of the comparative histology of in-
vertebrate muscle. This fact has made desirable a more comprehensive study of
invertebrate muscles particularly since a considerable number of these have been
employed in recent physiological research.

The only extensive paper on the histology of holothurian muscle (Hall,
1927 on Cucumaria) misinterprets their essential structure and has been responsible
for some erroneous conclusions by workers in muscle physiology.

The present study has shown that the retractor muscles of Thyone consist
of enormously long narrow unstriated fibers imbedded in a connective tissue
network. The fibers occur in bundles of 2-15 and in each bundle are arranged
circularly in a single layer. Each bundle is surrounded by a comparatively dense
layer of connective tissue which penetrates only sparingly into the interior of the
bundle. Hall interprets the bundles of fibers as being single cells each of which
contains many enormously large fibrils. That these so-called fibrils of Hall are
actually the muscle fibers can be determined readily not only by the presence of
connective tissue within the fiber bundles but also by a study of the arrangement
of fiber nuclei. Critical examination of this arrangement reveals a peculiar pic-
ture. In the contracted fiber the nucleus lies on the outside of the fiber and may
be connected to it only by a narrow protoplasmic strand. In the extended con-
dition of the fiber the nucleus is elongated in the axis of the fiber and lies closely
pressed against it. Occasionally nuclei are found directly within the fibers. As
far as it has been possible to determine, the fibers appear to be uninucleate. Con-
nective tissue nuclei occur not infrequently within the bundles of fibers.

The fibers are extremely extensible. The muscles may vary in length from
about 5 mm. in the contracted condition to 5 or 6 cm. in normal extension or
after anaesthetization with magnesium chloride. The fiber diameter varies from
5-10 mu in a contracted condition to 2-4 mu in an extended condition.

From teased preparations it may be seen that although the fibers are ex-
tremely long, they do not extend the full length of the muscle. The terminal
portions of the fibers are long and tapering. The fibers are spherical or hemi-
spherical in cross-section in a contracted condition, laterally compressed in an
extended condition. The connective tissue fibers appear to run at right angles
to the muscle fibers and form a dense network at the periphery of the muscle.

Strength-duration curves of nerve fibers in the squid. C. Ladd Prosser
and A. H. Chambers, Jr.

Giant nerve fibers and fin nerve fibers of the squid were stimulated by con-
denser discharges through calomel electrodes and strength-duration curves were
obtained. Excitation constants (Hill’s characteristic time, k) range from .33
to .60 milliseconds and average .47 milliseconds for the isolated giant fibers. The
time constants are essentially the same (characteristic time averages .42 milli-
seconds) for the giant fiber intact in the stellar nerve as for the isolated fiber,
but the whole strength-duration curve is shifted upward with the intact nerve.
Hence the effect of other fibers in a nerve trunk upon the excitation of one fiber
is to serve as a shunt for the stimulating current.

Decreasing the electrode separation from 12 to 3 mm. shifts the strength-
duration curve diagonally upward and to the left, with the result that the time
constants are shorter at the smaller separation. As the preparation deteriorates
the threshold rises and the characteristic times become shorter, i.e. the change in
the strength-duration curve is similar to the change resulting from shortening in-

PRESENTED AT MARINE BIOLOGICAL LABORATORY 391

terelectrode distance. With electrodes 7 mm. in diameter the time constants are
approximately 1.5 times as long as with electrodes less than 0.6 mm.

The diameters of the giant nerve fibers are approximately 100 times those of
the fin nerve fibers. The fin nerve fibers have characteristic times approximately
twice as long as the giant fibers (.88 millisecond with electrode separation of 12-
14 mm.).

New structures induced by implants of adult nerve cord in the polychaete,
Clymenella torquata. Leonard P. Sayles.

For several summers a study has been made in which a piece of adult nerve
cord, removed from one Clymenella, was transplanted into another. All implants
were of one to two segments in length. They were placed in the body wall in a
dorso-lateral region to minimize the chance of injuring the nerve cord of the host.
No bud has been formed in any case in which the nerve cord slipped into the
coelom free from the body wall.

To date 63 buds have been induced by this type of implant. These include 12
heads, 29 tails, 2 double buds each consisting of a head and a tail, and 20 irregular
or weakly developed buds. No head buds have appeared posterior to the tenth
segment of the host. One of the double structures was formed at the fourth,
the other at the eighth.

Both the source of the implant and the region of implantation in the host
seem to influence the type of bud to be formed. Many more cases must be secured
before any conclusions can be drawn concerning this point. The possibility that
orientation of the implant may also be a factor has not been fully eliminated.

Cytoplasmic division in type B of the giant amoeba Chaos chaos Lin-
naeus. A. A. Schaeffer.

Although type A and type B of Chaos chaos are very closely similar, morpho-
logically, to the common laboratory amoeba, Chaos diffluens, there are certain
characteristics which set them apart very definitely. One of these characteristics
is the striking phenomenon of frequent division into three daughters instead of
two, which occurs in both types of chaos. During division the amoebas of type
A round up into a mulberry-like shape, while those of type B, which are also berry-
shaped at first, soon flatten out somewhat, so that one cen predict to a certain
extent the number of daughters about to be produced, by counting the well-defined
lobes of the flattened-out amoeba. The number of daughters may be 0, 2, 3, 4, 5,
or 6, and the number of lobes formed before separation is well correlated with the
number of daughters produced. The amount of undigested food in the amoeba
influences the number of daughters to be formed, to a markedly greater extent
than it influences the number of lobes formed during division. In both cases the
number is reduced by large quantities of undigested food.

In type A, more divisions result in 3 daughters than in all the other classes
put together, while in type B, in a culture with food in excess of needs, as many
amoebas divide into 2 as into 3. But the number of amoebas showing 3 lobes prior
to division, under these cultural conditions, is much greater than those with 0, 2
and 4 lobes combined. The average variation in size between twins from a 3-
lobed parent is also greater than that between twins or triplets whose parents
showed 2 and 3 lobes respectively before separation of daughters. More sex-
tuplets occur than would be expected on pure chance, although not enough data are
at hand to warrant the conclusion that the curve of frequency rises again to some
degree at 6 daughters.

Something akin to a trigonal field of force in an apparently turbulent medium
exists here which stands in striking contrast to the common halving type of cyto-
plasmic division.

392 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The sense of taste in the free fin rays of the sea robin (Prionotus).
Ernst Scharrer.

Experiments were carried on to show that the free fin rays of the sea robin
are chemoreceptors as well as organs of locomotion. Blinded sea robins react
after 3-4 weeks of acclimatisation in the aquarium with snapping reactions when
the juice of a spider crab or of a clam is gently poured on the free fin rays by
means of a pipette. A water current caused by a pipette never is followed by a
reaction. The behavior of the sea robin is therefore identical with that of the
Mediterranean genus Trigla (Scharrer, 1935) and it might be concluded that all
Triglidae use the three free rays of the pectoral fins as chemoreceptors in search
for food.

Calcium and magnesium in relation to longevity of Mactra, Nereis and
Hydroides egg cells. Victor Schechter.

Experiments on the relation of calcium to longevity of unfertilized Arbacia
egg cells (Biol. Bull., 72: 366) were extended to the cells of the mollusk, Mactra,
and to those of Nereis and Hydroides. The Mactra egg, which in change in rate
of hypotonic cytolysis with age (Biol. Bull., 71: 410) follows the Arbacia egg’
closely, also exhibits prolongation of life in low calcium most clearly. This ac-
tion, as in Arbacia, seems to be one rather specific to calcium as a decrease in
magnesium alone does not produce it nor does lowering of both magnesium and
calcium have an added effect. Increase of magnesium above the normal concen-
tration in sea water did result in an additive effect on longevity.

The two phenomena need not, however, be related. The first sign of deteriora-
tion in the Mactra egg is usually the breakdown of the germinal vesicle. Insemina-
tion also causes germinal vesicle breakdown. In eggs which cannot be activated
by sperm, cytoplasmic cytolysis may occur with age while the germinal vesicle is
still intact. With such eggs, increasing the magnesium concentration retarded
cytoplasmic disintegration; and the eggs then lived longest if calcium was low.

Therefore, in so far as the phenomena of aging may be partitioned between
the various cellular components, the effect of low calcium is tentatively regarded
as one which retards deteriorative changes in the nucleus, whereas the action of
high magnesium may be primarily in the nature of an anaesthesia. Magnesium
may act chiefly on the cell membrane since with increased magnesium the egg cells
retain their spherical shape beyond the time when controls appear plasmolyzed.

Preliminary experiments with Hydroides eggs show prolongation with low
calcium.

In one-sixth the normal calcium concentration the reaction of Nereis eggs was
variable. This is possibly due to egg condition and it may be that with different
calcium and magnesium concentrations prolongation of life in these cells will
also be obtained consistently.

A convement method for the measurement of nerve respiration. Francis
O. Schmitt and Otto H. Schmitt.

For the determination of the effect of electrical stimulation on the oxygen con-
sumption of single nerves it is desirable that readings be made over short in-
tervals of time (every 2-3 minutes) and that the variations between individual
readings be small (ca 2-5 per cent). Even with differential volumeters of small
volume this requires that temperature fluctuations between the two vessels be re-
duced to a minimum. This may be accomplished without the use of a precision
thermostat by immersing the vessel in a mercury bath, the latter in turn being en-
closed in a well insulated box. The mercury is contained in a copper box lined
inside and outside with insulating material and provided with a glass window which

PRESENTED AT MARINE BIOLOGICAL LABORATORY os)

permits following the movement of the index droplet with a traveling microscope,
the capillary being only partially immersed in mercury and bent so that the vessels
are submerged well below the surface of the mercury. Wires from the electrodes
are brought out through rubber tubes. The capillary is illuminated by filtered
light brought in by a glass rod fastened on the carriage of the travelling micro-
scope. The copper box is surrounded by kapok or eiderdown contained in a well-
insulated box which also carries the traveling microscope. The entire unit is
easily portable and, at least for work at room temperature, the method has been
found quite as satisfactory as the conventional method which requires a precision
thermostat of special design.

Temporal relations in the excitation of the isolated muscle fiber. F. J.
M. Sichel and C. Ladd Prosser.

The possibility of spatial summation in the excitation of the isolated muscle
fiber has been reported previously (Sichel and Prosser, Biol. Bull., 69: 343, 1935).
In the present case the excitatory effects of two stimuli separated by a varying
interval have been studied. The stimuli were obtained from two condensers, each
controlled by a gas triode, discharging into a common resistance. The interval
between the two shocks was varied by a Lucas’ spring rheotome in the grid cir-
cuits of the gas triodes. The time-constant for the discharge of each condenser
was 1 millisecond.

The fiber (adductor longus, Rana catesbiana) was isolated and mounted in a
manner previously described (Sichel, Jour. Cell. Compar. Physiol. 5: 21, 1934) and
the isometric tension recorded by a micro-lever (Brown and Sichel, Jour. Cell.
Compar. Physiol. 8: 315, 1936). The electrodes were silver-silver chloride, and
were sufficiently large to make the spreading of the field at the ends of the fiber
unimportant.

The tension developed falls continuously as the interval between the shocks
increases, rapidly at first, then more and more slowly toward a steady value reached
when the interval between shocks is about 20 milliseconds. The tension is then
one-third to three-quarters of the tension developed when the stimuli are simul-
taneous.

If a similar experiment is done on the whole sartorius using maximal stimuli,
as the interval between stimuli increases the tension at first remains constant, this
being related to the absolute refractory period, then increases rather abruptly to a
new level. With submaximal stimuli there is an initial fall to the constant level
followed by a subsequent rise.

The absence in the case of the isolated fibers of the low constant level of
tension for short intervals, and the smooth nature of the tension-interval curve
we attribute to the absence of an absolute refractory period in the excitation of
these fibers.

Electrolytes in Phascolosoma muscle. H. Burr Steinbach.

The retractor muscles of the marine annelid Phascolosoma gouldi consist of
closely packed long smooth muscle fibers. Muscles freshly excised from the
animal contain the following average amounts of inorganic elements, expressed as
milliequivalents per hundred grams wet weight: Na 10.4, Cl 9.1, K 10.6, Ca 0.85.
The body fluid surrounding the muscles contains approximately: Na 38, Cl 43,
K 3.5, Ca 2.0. On immersion for two hours or more in sea water, the tissues gain
weight by 11 per cent, chloride increases by 16 per cent, Na by 14 per cent, Ca by
' 13 per cent while 11 per cent of the K is lost. These changes can be partly ac-
counted for by assuming an extra-cellular chloride space which alone is involved
in the tissue swelling and the substitution of sea water for body fluid. Both Cl
and Ca increase somewhat more than would be expected on this simple hypothesis.

394. PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

The Na increase is less than would be expected while a large part of the K loss
must involve the fibers themselves.

Muscles were soaked for two hours or more in artificial sea water containing
known concentrations of K or Ca, or in sea water diluted with isotonic sucrose
solution.

Na, Cl and Ca contents of the muscle bear a simple linear relationship to
the concentrations of these ions in the immersion fluid. All of the Na and Cl ap-
pears perfectly free and diffusible, the slope of the curves Na-tissue/Na-fluid and
Cl-tissue/Cl-fluid indicating about 30 per cent chloride (extra cellular) space at
all concentrations. About half of the initial Ca content does not diffuse into Ca-
free sea water or isotonic sucrose, but using that figure as zero concentration of
Ca in the muscle, the curve for Ca-tissue/Ca-fluid is linear and has a slope indi-
cating about 40 per cent diffusion space. Eighty per cent of the K does not diffuse
into a K-free medium. As the K content of the fluid is increased the K of the
muscle increases, rapidly at first then more slowly until finally a linear relation-
ship is shown between K-tissue and K-fluid at concentrations between 0.05 M and
0.25 M K in the fluid.

Leaf development and vegetative propagation in Polystichum plasch-
nickianum. Harry N. Stoudt.

Material for this work was collected by the writer in the rain forests along
the Vinegar Hill Trail, Jamaica, B. W. I. in the summer of 1936 and the investi-
gation was undertaken at the suggestion of the late Professor Duncan S. Johnson.

Leaves of this plant are produced from segments of the apical cell of the
rhizome. The leaf grows, apparently, by means of a bifacial initial which persists
in the mature leaf. This cell continues to cut off segments to form the first em-
bryonic leaf of the plantlet at the apex of the leaf. An apical cell is cut out from
a marginal cell of one of the last segments of the parent leaf to form the shoot
of the plantlet. Later leaves are formed from segments cut off from this cell.
A meristematic cushion forms at the apex of the parent leaf and adventitious roots
arise endogenously in this region and grow by means of a tetrahedral initial. By
this time the roots penetrate the moist soil and the plantlet matures. Simultaneous
with plantlet development the parent leaf develops wings that encircle the plantlet.
Further investigation is needed to understand the origin and development of these
wings. When several leaves and roots have formed the plantlet becomes inde-
pendent and this region of the parent leaf decays. The remaining portion of the
mother leaf persists to carry on spore formation.

Morphological and experimental cytology of lobster spermatozoa. (Pre-
liminary note.) T. Terni.

In Woods Hole I have begun to microdissect the spermatozoa of decapods,
most appropriate material because of its immobility and large size. I refer here
only to my preliminary results.

1. The three long rays of the head have a permanent shape which is slightly
curved. They are elastic, and if deformed by the needles, they immediately resume
their original form. The rays are, on the other hand, neither plastic nor extens-
ible if pulled with the needle. The mechanical stimulation does not induce visible
contraction of the ray-like filament. Movements of these, if really existing, are
in all cases so slow that only the microcinematographical technique will reveal them.

2. If with the tip of the needle one exerts considerable pressure, although not
sufficient to injure, on the anterior part of the spermatozoa, there always appears
the phenomenon of the so-called “explosion” (Koltzoff). The process seems to
be irreversible; I have followed for 20 hours the fate of the exploded spermatozoa
without changes. In order that the explosion should take place after pricking, it
is necessary that the spermatozoa be freshly removed from the spermaduct.

PRESENTED AT MARINE BIOLOGICAL LABORATORY 395

3. In the posterior part of the small tube contained in the chitin capsule of the
tail there exists before the explosion material strongly acidophilous. After the
explosion the capsule and its content become suddenly basophilous while the nu-
clear chromatin loses its strong basophily, perhaps because it diffuses in the capsule.

4. With microdissection it is easy to follow the very rapid process of invagina-
tion of the anterior part of the spermatozodn into the capsule, which simul-
taneously becomes swollen. The examination of good preparations (ac. osmic-iron
hemat.) shows the fact that the basis of the cephalic rays are pulled into the tail
portion like an umbrella that closes itself into a sheath. Simultaneously from the
posterior pore of the tube there flows out a basophilous body, perhaps the anterior
centrosome.

Observations on arterial pressure in marine fishes. Abby H. Turner
and Charlotte Haywood.

In an effort to determine representative values for the arterial pressure sup-
plying the organs of fishes the. anterior mesenteric artery was employed for can-
nulation. Pressures were measured in centimeters of isotonic saline, using a manom-
eter. Respiration was well maintained by a stream of water over the gills, but
obvious disadvantages in the method are that the fish is not entirely submerged in
water and that the body cavity must be opened.

A number of the determinations were made in the summer of 1936 at Bergens
Museum Biologiske Stasjon, Herdla, Norway.

No. of Range of Values Average Values
Individuals in cm. Saline in cm. Saline

Elasmobranchs:

WVustelus CONUS). 6.3 ce ens ss 4 11. —25.5 16.7

Carcharius taurus. 75-5. 05..0- 2 16. —19. Wes

Squalus acanthias............ 7 8.5-18.8 foal

Raja dia phranes a.) 25... -- 1 Me

RFC SIQUULLT ONES: o-oo ecient 2 12°5—2 5 Iie

OD OXNTIN WENUSE on ee 1 8.3

Narcacion nobtlianus......... 3 Extremely low

Dasybatus marinus........... 2 9. -10.1 9.5
Teleosts:

Centropristes striatus......... 1 37.

VAOTTAGHOSILPUS.. fa. 4 9.7-15. HES

Gadus callartas. 4:0) e458 oe 12 10.9-43. ISTE

- Gadus pollachius............. 2 ID D2 WHY 12.2
Lophius piscatorius.......... 6 9. -23.7 16.7

Respiratory rate and length of fertilizable life of unfertilized Arbacia
eggs under sterile and non-sterile conditions. Albert Tyler, Nelda
Ricci and N. H. Horowitz.

The respiratory rate of unfertilized eggs of marine animals does not remain
constant with time, but sooner or later shows a marked rise. This rise occurs
at the time when the eggs lose their fertilizability (Tyler and Humason, 1937).
Measurements of the respiration of Arbacia eggs in 2 per cent alcohol or other
agents that are known to prolong the fertilizable life (Whitaker, 1936) show that
the rise is correspondingly delayed, while the initial absolute rate is unaffected.

396 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

By plating out for marine bacteria from the respiration vessels, it is found that
the bacteria increase as the rate of respiration rises both in sea water and in
alcohol. When sterile or almost sterile cultures of eggs are run it is seen that
the respiratory rate remains low and shows even a slight drop. Such eggs have
been run for four days without showing a rise in respiratory rate, whereas under
ordinary conditions the rise begins at about 10 hours.

Culturing eggs under sterile conditions in sealed dishes prolongs their fer-
tilizable life as Gorham and Tower (1902) found, some of the eggs lasting as
long as 20 days. Under such conditions alcohol appears to shorten the fertilizable
life. Addition of bacteria accelerates somewhat the disintegration of the eggs.
Alcohol in ordinary sea water or in the presence of small amounts of bacteria
considerably prolongs the life of the egg. In the presence of large quantities of
bacteria, 2 per cent alcohol is toxic within a few hours, such cultures showing a
drop in pH to as low as 4.60.

The results evidently mean that bacteria contribute to the destruction of the
eggs only after certain disintegrative changes have occurred. The agents that
prolong the life do not act bactericidally, but by retarding these initial spontaneous
changes on the part of the egg.

The effect upon gastrulation and differentiation in Arbacia of NiCl,,
CuCl,, and Na,SiO, in modified artificial sea water. A. J. Water-
man.

A formula for artificial sea water consisting of 390 cc. Im NaCl, 9 cc. lm
KG 23:35) ce. im MeCL” 25.5 ce) lm MesO,, 9:35)ce) In (Ca@l, about On vearand
NaHCoOs, additional water to make one liter (Chambers), and corrected for pH
supports development of Arbacia from the blastula stage in a typical manner.
Modifications and omissions of salt proportions have been studied in relation to
effects on gastrulation and embryonic differentiation (Runnstrom, Lindahl,
Dalcq). The addition of NiCl, and CuCl, to the above has been studied and
comparison made with previous observations with these heavy metals in sea water.
Increase, decrease or omission of Mg, SO,, K, and Ca influence type and rate of
effect of the metals. Exogastrulation occurred in some experiments and gas-
trulation could be temporarily inhibited. The blastula stage was used in all
experiments.

Na.SiOs was tested in relation to respiratory and cell division rates of the
embryos in sea water and in the above modifications. In relatively large concen-
trations silicon is non-toxic and does not affect gastrulation. Normal cleavage
rate is increased, top-swimming blastulae appear earlier, plutei mature sooner,
and gastrulation may be accelerated. There is no noticeable differential accelera-
tion of germ-layer differentiation. This has also been studied in artificial media
where the salt balance has been disturbed or metallic salts added. Toxicity may
be increased. Addition of silicon (Mast and Pace) to cultures lacking sulphur
may or may not influence developmental rate.

Compounds of silicon are very abundant in nature and present in all waters
(Reynolds, 1909). Both animals and plants use silicon and it is found in animal
tissues. According to Mast and Pace, 1937, it increases rates of respiration and
division in Chilomonas and has a catalytic action on synthesis of complex organic
compounds.

Studies on living conjugants of Paramecium caudatum. Ralph Wich-
terman.

A new method of approach to a better understanding of the problem of sex-
uality in Paramecium is presented. By means of a precision micro-compressor, it

PRESENTED AT MARINE BIOLOGICAL LABORATORY 397

is possible to observe in the living condition, the behavor of nuclear phenomena
and establish time relationships during conjugation.

With a micro-pipette, a single pair of recently joined conjugants is placed
on the glass slide of the compressor in a small drop of the culture medium and
studied at 26° C. The metal top holding the glass cover-slip is screwed down
while observations continue with the microscope. The animals are prevented
from spiraling between the two pieces of glass and allowed to move around slowly.
Adjustments within a few microns make this possible.

The divisions of the micronuclei and their behavior have been seen and
photographed in the living condition. The micronucleus of each conjugant is
seen to leave its place near the macronucleus and increase in size. While this
enlargement takes approximately four hours, the end of the division (including
the anaphase and telophase stages) is more rapid requiring only about 18 minutes.
The “crescent” prophase stage and long anaphasic-telophasic separation spindle
so characteristic of this division are clearly shown. The swelling at the center
of the separation spindle becomes separated and is passed into the cytoplasm.
Cyclosis moves it about until it ultimately degenerates. Each product of the
first division enters into the second where again two long spindles in each con-
jugant are visible. The second division requires 50 minutes for completion from
the time the first division products are formed. The anaphase and telophase
stages are still more rapid, requiring only nine minutes. Degeneration of three
of the four products of the second division is observed. Behavior of the pro-
nuclei after the third division is being more carefully studied in an effort to
record their movements accurately. The pronuclei of a given conjugant have
been seen to fuse and form a synkaryon in the same individual. In a number of
observations, definite evidence has been obtained to show that crossing-over of
pronuclei does not occur. Too, no evidence has been obtained to show that
crossing-over of pronuclei takes place.

In view of these observations, any conclusions, genetical or otherwise, based
on the assumption that there is crossing-over of pronuclei, are open to grave
question.

Photographs are being made of the living conjugants and will be included in
the full paper. It is hoped also to make a motion picture of the entire process.

Conjugation in Paramecium trichium Stokes (Protozoa, Ciliata) with
special reference to the nuclear phenomena. Ralph Wichterman.

A cytological study was made of conjugation in a race of Paramecium
trichium Stokes. The preconjugants, which are smaller than the vegetative
individuals, fuse along their oral grooves. The centrally located oval macro-
nucleus undergoes complete fragmentation involving the formation of a twisted
ribbon which becomes thinner and longer resulting in small irregular rod-like
elements. These result in spherical bodies which disappear in the cytoplasm after
exconjugant reorganization.

The deeply staining oval-shaped micronucleus divides three times. The first
pregamic division results in two micronuclear products; the second division,
four products, three of which degenerate. The remaining micronuclear product
enters into the third division to produce the pronuclei. The developing third
pregamic spindle is commonly seen to press against the opposite conjugant
where a cone is clearly visible. There is strong evidence to believe that the
pronuclei cross approximately in the mid-region of the conjugants. This point
is being investigated further by observing living conjugants under the micro-
compressor. Pronuclei fuse to form a synkaryon which divides three times to
produce eight products. Two of these divisions occur while the conjugants are
together; the third division occurs in the exconjugant. Exconjugants with four

398 PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS

macronuclear anlagen and a single micronucleus are commonly seen. This re-
duction in number from eight nuclear products of the synkaryon to five, is being
studied further. Variations occur in regard to the distribution and fate of the
eight nuclear products. Individuals with four macronuclear anlagen and one
micronucleus divide with two anlagen going to each daughter while the micro-
nucleus divides mitotically. In exconjugants with two anlagen and one micro-
nucleus, a final division distributes a single macronuclear anlage to each daughter
as the micronucleus again divides mitotically thus restoring the original nuclear
condition to each animal.

This work confirms Diller’s observations as reported in his SEES: in
Anatomical Record, 1934.

Mitotic activity of stimulated rat adrenals measured by colchicine tech-
mique. Opal M. Wolf.

In connection with a recent investigation it was noted that daily injections of
an alkaline extract” of the anterior lobe of the pituitary into rats at 1 cc. and
2 cc. levels gave evidence of activity in the adrenals as measured by the colchicine
technique. Control and injected animals were etherized the morning following
the 2nd, 4th, 6th, and 10th days of injection. Nine to fifteen hours before the
animals were killed, 0.3 mg. (per 100 grams of body weight) of colchicine °
dissolved in distilled water was injected sub-cutaneously.. At autopsy tissues were
fixed in Bouin’s, dehydrated by the aniline oil-dioxan method, cut at 10 micra and
stained in Delafield’s haematoxylin. Mitoses were counted with an oil im-
mersion lens.

Preliminary observations were made of every 20th section in order to esti-
mate the effect of colchicine on the organs. A 171 gram control male showed
the following total counts:

sDhiy Olden areca eek cere 157
Parathyroid. c)ih scarce cere 213
TANGENT Meme ieee oat wish ww 625 (highest count for controls)

An average count for the three largest sections of the adrenal, 45 in the con-
trols, was used as a basis for comparison. Counts of both adrenals varied by
11-17 mitoses. Injected animals showed slight stimulation after 2 days, and
greater stimulation after 4 days, but the differences were not marked for the
two levels. The greatest stimulation occurred after 6 days of injection. One
series of males weighing 170 grams gave:

Control. oases 21
1h, CS HRB eRe ie che 90
CCN c/a se kh CR 232 (2149—total for every 20 sections)

With one exception (126 mitoses on 2 cc. level), after 10 days injection
the animals showed a count of 44. Nine to eleven hours after colchicine many
of the cells were in early prophase, at 15 hours the greatest number were counted
in the later stages. Most of the mitoses occurred in the outer part of the
zona fasciculata, but a few cells in the zona glomerulosa and reticularis were
dividing. No late stages of mitosis were observed in the medulla. In several
instances very large nuclei appeared to be in early prophase.

* The experimental work was performed at Goucher College, the sections were
cut and studied at the Marine Biological Laboratory.

* The growth extract was purchased from E. R. Squibb and Sons.

* Colchicine U.S.P. was purchased from Merck and Co. in the form of a
powder.

PRESENTED AT MARINE BIOLOGICAL LABORATORY Oey

The effects of the drug on the adrenal cells were similar to the descriptions
by Ludford, Allen et al, and Nebel. Apparently normal stages of mitosis as well
as stages leading to multi-nuclear cells were observed. The latter were probably
caused by the varying concentration of the drug due to the rate of absorption and
elimination. It was noted that the level of dosage had a toxic effect on some rats,
others apparently showed no ill effects.

)

Induced breeding reactions in isolated male frogs, Rana pipiens
Schreber.1 Opal M. Wolf.

Adult male frogs give a characteristic call, show ridged epidermal thumb-
pads with large, active mucous glands on the first finger of the fore limbs, de-
velop amplexus, normally in the presence of the female, and shed sperms at, the
time of reproduction. In earlier work it was shown that the male as well as
the female could be stimulated. by the injection of frog anterior lobe to repro-
duce normally as early as the latter part of October and throughout the winter
months. It seemed of interest to report experiments on individually isolated
mature male Rana pipiens.

In the first experiments injections of frog anterior lobes from Rana pipiens,
an extract of whole pig pituitary, a purified extract of the gonad-stimulating
fraction of horse anterior lobe* and an extract of pregnant mare’s serum, at
times other than the breeding season and the summer, caused the shedding of
sperm. Inspection of the cross-section of the testes showed empty seminiferous
tubules and were comparable to the picture of the testes of frogs which were
known to have fertilized eggs following pituitary stimulation. Following in-
jection of the extract of pregnant mare’s serum sperm were recovered as they
passed from the red and swollen cloacal opening and stained preparations were
made. In some cases the characteristic call or trill was heard 8 to 9 hours
following the injection of as small an amount as one frog anterior lobe. The
call was not heard following the injection of the extracts. The thumb-pads
showed active mucous glands but the effect on the epidermal ridges was not so
clearly shown because the thumb-pad of the adult male is ridged during the winter
season.

Examination of the thumb-pads of adult males in the middle of July showed
a smooth surface with slight indication of ridging and small mucous glands.
A small piece from the thumb-pad of the experimental animals was removed as a
control and the individually isolated animals were injected daily for 9 days with
the anterior lobe from male Rana catesbiana. The injected animals gave the
characteristic call of the breeding season, sperm were shed, active spermatogenesis
was going on, the thumb-pads had developed ridges and the mucous glands were
active. The thumb-pads of the experimental animals resembled those prepared
from male Rana clamitans caught in the field and prepared immediately for
histological examination. This animal breeds during the summer from June to
the middle of August while Rana pipiens breeds from the first of April to the
middle of May depending on the season and latitude.

It appears from an examination of the testes of the isolated experimental
animals killed at varying times after prolonged pituitary treatment that the mature
sperm are shed and spermatogenesis induced by frog anterior lobe and the extracts
used in these experiments.

1Part of the experimental work was performed at the University of Wis-
consin Department of Zoology and part at the Marine Biological Laboratory, 1936.

* The extract of horse anterior lobe, Prephysin A, prepared by Chappel Bros.,
and the other extracts prepared in Dr. Hisaw’s laboratory were obtained through
the courtesy of Dr. Frederick L. Hisaw.

Experimental
Biology
Monographs

eA unique cumulative reference

library for biologists, providing full accounts of both the
experimental and the theoretical aspects of all significant
current research work in the various biological sciences.

NEW VOLUMES

Autonomic Neuro-Effector Systems

By Walter B. Cannon and Arturo Rosenblueth.
The first complete summary of the nature of the chemi-

cal mediation of nerve impulses.

$4.00

Phytohormones By F. W. Went and Kenneth

V. Thimann.

An inclusive, critical survey of experi-

mental work, covering experimental techniques and a-
large number of new and unpublished experiments. $4.00

PUBLISHED AND FORTHCOMING VOLUMES

PACEMAKERS IN RELATION TO
ASPECTS OF BEHAVIOR
By Hupson Hoaguanp. $3.00
NEUROEMBRYOLOGY, AN
EXPERIMENTAL STUDY
By Samuget R. DetwiterR. $3.75
THE EGGS OF MAMMALS
By Grecory Pincus. $3.75
THE RECEPTOR PROCESS IN
VISION
By Srenig Hecut
GEOTROPISM: A STUDY OF
DETERMINISM IN BEHAVIOR
By W. J. Crozier anp GREGORY PINCUS
THE BIOLOGY AND CHEMISTRY
OF OVARIAN HORMONES
By Grorce W. CorNER AND
Wituarp M. ALLEN
BIOLOGICAL OXIDATIONS
By E. 8. Guzman Barron
THE HYPOPHYSIS
By J. B. Couuie, D. L. THomson,
anp H. SELYE
NITROGEN METABOLISM IN
ANIMALS
By Henry Borsooxk

TEMPERATURE
CHARACTERISTICS
By W. J. Crozizr
CYTO-GENETICS AND PLANT
PHYLOGENY
By Ernest B. Bapcock

BIOELECTRIC PHENOMENA IN
PLANTS

By L. R. Buinks

PERIODICITY IN ANIMAL
BEHAVIOR
By T. J. B. Sripr

THE GENETIC ANALYSIS OF
SPECIES DIFFERENCES
By T. H. DopzHansky

DYNAMICS OF INFLAMMATION:
AN INQUIRY INTO THE MECH-

ANISM OF INFECTIOUS

PROCESSES
By Vary MENKIN

The Macmillan Company
60 Fifth Avenue New York

CONTENTS |
| Page
WELSH, J. H., F. A. CHACE, JR., and R. F. NUNNEMACHER |
185

The Diurnal Migration of Deep Water Animals...........

COONFIELD, B. R. and A. GOLDIN

The Problem of a Physiological Gradient in Mnemiopsis
During Regeneration: 109. oat hs Wh eel ke he oa al

GLASER, OTTO and GEORGE P. CHILD

The Hexoctahedron and Growth. ..............000..-5.5.

CARVER, GAIL L.
Studies on Productivity and Fertility of Drosophila Mutants.

BALL, ERIC G. and C. CHESTER STOCK
The pH of Sea Water as Measured with the Glass Electrode

GOLDSMITH, E. D.
The Relation of Endocrine Feeding to Regeneration, Growth,
and Egg Capsule Production in Planaria maculata.........

PROSSER, C. LADD and JOHN Z. YOUNG )
Responses of Muscles of the Squid to Repeutive Stimulation
of the Giant Nerve Fibers................. ieee ARNE

SPARROW, F. K., JR- !

The Occurrence of Saprophytic Fungi in Marine Muds

WHITAKER, D. M.

227

237

242

Determination of Polarity by Centrifuging Eggs of Fucus 7

PITCATIS ce Oe Re ee Tea a uM ep eT ne ee

TYLER, ALBERT and W. D. HUMASON
On the Energetics of Differentiation, VI. Comparison of the
temperature coefficients of the respiratory rates of unfertilized
and of fertilized eggs SL Tae PONTE Mette | age Sac otal, deeta fee
KENK, ROMAN
Sexual and Asexual Reproduction in Euplanaria tigris
(Garay i) RAR TE LOC ISTHE yu Aah or a Lt da a
HORSTADIUS, SVEN |

Investigations as to the Localization of the Micramere=,
Skeleton and Entoderm-forming Material in Unfertilized Egg
Of ArbAatidiy eR) ee See i pal a he atten een ae

HORSTA DIUS, SVEN

i Experiments on Determination in the Early Peverpiaeu of

GETEDFA tS HACTELLSS ee ee Oe ON Ti A eth Fk ac

PROGRAM AND ABSTRACTS OF SCIENTIFIC MEETINGS, SUMMER
OB LOR ee ee ORM e 21g 1c Pa eare MAS be al Pecalerk Soa

249

261

280

205,

317

Eee Oe
Rae en ee ee

ee a

- Volume LXXIII 3 : Number 3

THE
BIOLOGICAL BULLETIN

| PUBLISHED BY
THE. MARINE BIOLOGICAL LABORATORY

Editorial Board

GARY N. CALKINS, Columbia University E. E. JUST, Howard University
E. G. CONKLIN, Princeton University FRANK R. LILLIE, University of Chicago
E. N. HARVEY, Princeton University CARL R. MOORE, University of Chicago
SELIG HECHT, Columbia University GEORGE T. MOORE, Missouri Botanical Garden
LEIGH HOADLEY, Harvard University T. H. MORGAN, California Institute of Technology
M. H. JACOBS, University of Pennsylvania G. H. PARKER, Harvard University
H. 8S. JENNINGS, Johns Hopkins University EDMUND B. WILSON, Columbia University
ALFRED C. REDFIELD, Harvard University
Managing Editor

DECEMBER, 1937 |

Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.

NEW BLAKISTON BOOKS

The Microtomist’s
Vade Mecum. 10th Edition

Includes Both Animal and Plant
Microscopic Anatomy and Technique

11 Illustrations. Appendices. 784 Pages. Washable Sturdite Covers.
$9.00

In this new edition an important change has been made by the inclusion of
plant technique. This section has been written by Dr. D. G. Caicheside of
London University. There are new chapters on Frozen Section Staining;
Vital Staining. Dr. Conn has recast the whole chapter on Staining. Dr.
Duthie has done the same for the article on Blood and Glands. Other sec-
tions which have been thoroughly revised are those on Celloidin Imbedding
by Mr. Richardson; Fats by Drs. Kay and Whitehead; Protozoological
Technique by Dr. Helen Pixell-Goodrich; Nervous System by Drs. Green-
field and Stern.

Dr. Theophilus Painter has contributed a masterly article on Animal Chro-
mosome Technique.

The authors of this edition are:

Editors: J. Bronte Gatenby, M.A., Ph.D., Sc.D. GfTrinity College,
Dublin), and Theophilus S. Painter, M.A., Ph.D. (Texas University).

Contributors: D. G. Catcheside, Sc.D. (London Univ.), Harold J. Conn,
Ph.B. (N. Y. Agric. Station), E. S. Duthie, M.Sc., M.B., Ph.D. (Sheffield
Royal Hosp.), Helen Pixell-Goodrich, M.A., Sc.D. (Oxford Univ.), J. G.
Greenfield, M.D., F.R.C.P. (Nat. Hosp. Lond.), W. W. Kay, M.Sc., M.B.
(Manchester Univ.), Reginald: Ludferd, Ph.D., Se.D. (Cancer Research
Lab. Lond.), K. C. Richardson, M.Sc. (Univ. College, Lond.), Ruby O.
Stern, M.D. (St. Andrew’s Hosp. Northampton), Raymond Whitehead,
M.Sc., M.D. (Manchester Univ.).

Barnes— Textbook of General
Physiology

166 Illustrations. 554 Pages. Washable Cloth Covers. $4.50
By T. Cunliffe Barnes, Sc.D. (Asst. Prof. of Biology, Yale Univ.)

P. BLAKISTON’S SON & CO.,INC. ‘“Svindcinnia Pan

Vol. LXXIII, No. 3 December, 1937

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

EFFECT OF SALTS OF HEAVY METALS ON DEVELOPMENT
OF THE SEA. URCHIN, ARBACIA PUNCTULATA

A. J. WATERMAN

(From the Marine Biological Laboratory, Woods Hole, and the Thompson Biology
Laboratory, Williams College)

In previous experiments an examination has been made of the
effects on gastrulation and embryonic differentiation in Arbacia of
ether, changes in temperature, alcohols, X-rays, hypertonic and
hypotonic sea water, and of the disturbance of the salt balance in
sea water. Disturbance of the salt balance of sea water and the
action of certain alkaline earth metals etc., have been tested on the sea
urchin (Paracenirotus lividus) by Runnstrém (1935), Lindahl (1936),
Lindahl and Stordal (1937) and others in relation to gastrulation,
ectodermization and endodermization of the embryo and specifically
to differentiation. References to other workers have been given in
earlier papers. These studies have been concerned with the un-
fertilized and fertilized egg rather than with the blastula stage.

At gastrulation physical movements take place that result in an
invaginated endodermal tube from which arises much of the meso-
dermal material. These movements may be delayed temporarily,
retarded or inhibited, and they also may be influenced independently
of differentiation. In the latter case an exogastrula may or may
not form. What initiates the movements is another problem, but at
least it has been shown that a host of factors, producing quite similar
effects, can interfere with gastrulation; disturb the proportions of
potential ectoderm and endoderm; and influence the differentiation of
skeleton, body form, ectoderm and endoderm.

As regards embryonic differentiation, the effect has been in general
very much the same for these various environmental modifications.
Some types of differentiation, taking place as development proceeds,
can be slowed down to a greater extent than others. Ectoderm, and
to a lesser extent mesoderm, may grow and differentiate when endo-
derm formation is entirely inhibited. Furthermore, the ectoderm is

401

402 A. J. WATERMAN

the most resistant to environmental modifications. Different cultures
may vary under apparently identical conditions; cultures from one
female may vary from those of another; while individuals in the same
culture always vary in their susceptibility. The exact condition of
early development, i.e. fertilized egg to early gastrulation, seems of
minor importance, since the results are somewhat similar under the
conditions of the experiments.

Some metallic salts, which are a natural constituent of sea water,
are more toxic to animal life than are others. On the whole, it is
probable that the concentration of a particular metal changes very
slightly throughout the ocean. The possible exception may be
where large bodies of water empty into the ocean causing dilutions of
certain constituents of sea water and concentrations of others,
especially metals. What concentrations can the developmental stage
of a littoral animal such as the sea urchin, Arbacia punctulata, with-
stand and what is the effect of increased concentration of different
metals on such a developmental process as gastrulation? Finally, are
the effects on development comparable to those secured by other
experimental methods?

In this study ZnCl, ZnSO., Zn(C2H3O02)2, FeCl, PbCle, CuCl,
HegCle, AlCls3, NiCle and CdCle have been used in various concentra-
tions. As in previous studies, the blastula stage was selected for
study and exposure was made for long periods of time.

Reference to Tables I-IV will show the approximate concentra-
tions which produced certain results. The concentrations are ex-
pressed as proportions and represent the amount of the metallic salt
which was added to the sea water. No estimate was made of the
normal amount of the metal in sea water. As nearly equal numbers of
embryos as possible, without actually counting them, were used in each
culture. By mixing the embryos secured from several females more
or less similar cultures were secured. All results were based on
random sampling from three places in the culture. The exposure
time was varied, and in certain cases where developmental arrest had
occurred without death, the culture was washed and returned to
fresh sea water for recovery. In some instances samples were trans-
ferred at intervals from a lethal solution to fresh sea water to study
the rate of the toxic action and the recovery ability of the embryos.

It is said that many of the heavy metal salts are precipitated by
sea water. The concentrations specified in this study may represent
initial rather than final concentrations and some of them may have
changed in the course of the experiment. At this point it may be

EFFECT OF HEAVY METALS ON DEVELOPMENT 403

said that those experiments were discarded in which visible precipita-
tion had occurred. The whole subject of heavy metal action is
obscure.

This study was made at room temperature during the months of
July and early August. Hence there was some variation as some of
the experiments ran for several days. To avoid repetition only the
results secured from the exposure to ZnCle will be described, but
comparisons with the other metals will be made. The data for the
others are included in the tables.

Ill

A survey of the more recent literature has revealed numerous
references to the effects of heavy metal salts on animal development
and embryonic differentiation. The effect on gastrulation has ap-
parently not been tested.

Hammett and Wallace (1928) found that the lead ion retarded
growth, and differential development of the head and optic regions
was markedly inhibited in chick embryos. Child (1929) has used
CuSO, in the study of physiological gradients in the chick embryo.
Féré (1893) obtained monsters following the injection of lead nitrate
into developing chick embryos (see also Franke, 1936, for selenium
salts).

Galtsoff (1932) has shown that marine invertebrate animals
can concentrate different metallic elements in their bodies. Certain
groups concentrate zinc, others copper, etc. Copper salts affect
oyster larve by inducing attachment (see also Prytherch, 1931), and
by initiating metamorphosis. Metallic silver causes sperm of the sea
urchin to lose their fertilizing power, and paralyses plutei (Drzewina
and Bohn, 1926).

Hoadley (1923) has studied the effects of the salts of the heavy
metals on the fertilization reaction in the sea urchin, Arbacia. The
inhibiting concentration varied for the different metals tested. Gold
chloride was most toxic for membrane elevation and cleavage and
cadmium or cobalt chloride least. Other salts tested included CuCl,
ZnClz, LaCls3, AICl3, PtCls, PbCl, NiCl, in the order of the toxicity.
HgCl, differed from the others in that membranes elevated at a con-
centration of 1 part in 600,000, which was toxic for cleavage. Con-
centration of these metals varied slightly for different batches of eggs
and thus showed the influence of a time factor.

HgCl, has a harmful effect on cleavage at different concentrations
following very short exposures (Hoadley, 1930). It affects the cortical
region resulting in membrane elevation. After longer exposures it
affects the pigment which has mercury-avid properties. The pigment

404 A. J. WATERMAN

may be extruded and such an egg may develop if not injured. A con-
ceivable mechanism is thus available by which the mercuric ion, which
has entered the egg, may be bound and removed.

Copper salts are known to have an injurious effect on many |
organisms. In a very low concentration which inhibits fertilization,
sperm may be still active. Inhibition is marked in a concentration as
low as 1 part in 2,500,000 parts of sea water and is reversible provided
the eggs are not injured too much. Copper appears to injure the
vitality of eggs and acts as a slow poison (Lillie, 1921). A concentra-
tion of 1/62,500 is necessary to suppress cleavage. The effect of
HgCl, is different from that of copper: initial stages of fertilization are
little affected, susceptibility increases as fertilization progresses,
fertilized eggs show the effect more rapidly than unfertilized eggs, and
the movement of sperm is suppressed at great dilutions. It produces
membrane elevation alone and favors it in fertilization. The effects
are reversible if exposure is not continued too long. Lillie concludes
from these studies that the effect of mercury and copper on fertilization
following membrane formation may be due to enzyme poisoning.
The effect on the initial stages of the fertilization reaction does not
correspond so well to the enzyme analogy. _

Glaser (1923) shows that in the egg of Arbacia copper becomes
concentrated in the chorion, vitelline membrane and cortex. It is
diffused throughout the cytoplasm. Copper occurs normally in egg
pigment, membrane chorion and cortex associated with lipolysin.
Normal eggs secrete copper compounds as well as removing copper
sulphate from sea water. Parker (1924) has shown that marine
animals will grow upon any heavy metal plate provided the metal does
not liberate ions or soluble compounds. ‘The effect of CuCl, MnCh,
and FeCl; upon cardiac explants cultured in vitro has been studied by
Hetherington and Shipp (1935). The survival time was tested.
Other interesting studies could be given but the above are sufficient
to indicate the type of work which has been done.

NiCl,.—In an experiment to test the effect of this metallic chloride
upon the development and differentiation of the germ layers after
their formation had been initiated, oval blastule to early gastrulz
were exposed to various dilutions of a 1 per cent stock solution made up
in sea water. The cultures were examined after 21-42 hours of
exposure and in some instances the embryos were returned to fresh
sea water. NiCl, gave the best and most numerous examples of
exogastrulation of all the metallic salts which were tried. Further-

EFFECT OF HEAVY METALS ON DEVELOPMENT 405

more, the exogastrulz underwent further differentiation. The lowest
concentrations employed gave also marked inhibition of development.

After 21 hours exposure in a concentration of 1 part in 60,000,
development had only progressed to a large triangular stage with
skeletal spicules or short rods and initial flap formation. A few simple
exogastrulz with tri-radiate spicules were seen in the culture. During
the same length of time the control embryos had advanced to a medium
pluteus stage. A 42-hour exposure killed most of the experimental
embryos although a few large blastulae and triangular stages showed
slow movement.

In concentrations of 1/40,000, 1/20,000, 1/14,285, development was
progressively inhibited but the number of exogastrule increased
markedly. The embryos were all on the bottom of the culture dish.
Both the evaginated and invaginated endodermal tubes failed to
differentiate although some growth occurred, while the mesenchyme
either formed small spicules or failed to show any skeleton-forming
activity. The ectoderm was the least affected and continued growth.
It acquired its characteristic appearance and the apical plate appeared.
In such cases where the formation of the endoderm was entirely
inhibited, large circular or oval ectoblastule were seen. A longer
exposure killed the embryos, while those which had been transferred
to fresh sea water showed increased activity but only slightly ad-
vanced development, especially in the case of the ectoderm and
skeleton. The endoderm of these embryos showed no further change.
The ectoderm in the region of the vegetal pole tended to be more or
less irregular and the shape of the body was often lumpy.

When transferred to fresh sea water after 21 hours exposure, the
embryos either died or failed for the most part to continue develop-
ment. The activity of the survivors increased. Blastule and exo-
gastrule with spicules, stomodeum and apical plate were seen.
Some of the survivors showed no formation of endoderm while others
contained a simple endodermal tube but no skeleton. Thus the
embryos were unable to overcome in sea water the poisonous effects of
the metal. It acts in very low concentrations (Table I) and within a
relatively short time. The effects are not reversible and embryos in a
culture react differently. The various abnormal types seen in the
cultures are the result of differential susceptibility among the embryos
and also in the developmental processes involved. This has been a
common observation in previous studies of gastrulation in the embryos
of Arbacia.

The number of exogastrule decreased in concentrations from
1/10,000 to 1/6,000. Differentiation was further inhibited while

A. J. WATERMAN

406

“asueyo

ON
"DAT

*asueyo

oN
“OAT?

LS8‘Z/T

*SOxO *saynoids
MaJ ‘sayndids| ON ‘soxa
YUM Jepnsue |Jomoaq “wn

-11} 0} wpnyserg | -sejq [[euIS

“SOX9

*sOxe | 1YSI[S Aue

ii (o) a ‘x[nayses

-Eynajses ON | = [eIUL May

000'S/T

999'9/T

*sOxa *x[N14se3 [e1} | "sOxO MIJ
Auey ‘sojn | -1ul Maj ‘soxa| *uWI07}30q

“aA0qe oY} Ul sayn | -oIds YIM May | a10][ ‘Sie[NOIs | UO Aso]

-o1dg ‘soxe Auey] |‘e[n1jse3 a]duris -0A xINIselg *Jo][eus
*x[NJse]q [[eUIS 0} e[NAse[g | *WO}30q UO ][V |JeYMoWIOS

w]NI4seyy [EIU] 0} wjnj4se[q 93e7
*UOTJEULIOJ
UWepopus Iepnse1 *SOXO
-1I Io ‘soxe aid a]duiis a31e]

-WIS Josi1e] Aue Md ‘“JaMOysS ‘uO}IIOyS | wnzWses
‘Uu0l}e[N.AYses 3e JUSUIZAOPY | ON ‘xNsWse3 94] 02
sjdu19}7e Jamey | ‘x[nqselq a10y{ | 97e] 0} w[nNAsepgq | wmIpeyy

x[Nqselg [eotteayds 07 [eAC
000'01/T S8'PT/T 000'02/T 000°0#/T

*SOx9

MOT “SaTpITeUr
-I0UGE BUIOS
“U0J9]94S YA

Je[NSsuels as.1e7

sgjnoids
YIM siejnsue
-I1} 0} w[n41}se5

000‘09/T

Toqnjd

wUNIpeTl

1aynjd
UINIpsUl 07
wznijsey

Jorqu0g

‘UOI}VEIJUSJEHIp MuUOAIqUIa PUL UOT}E[NAYses UO Z{DIN JO SUOT}eIQUaDUOD JUSIAYIP Jo poy”

[| A14vL

EFFECT OF HEAVY METALS ON DEVELOPMENT 407

growth and movement became slower. The ectoderm was the most
resistant. In a concentration of 1 part in 5,000, there occurred some
increase in size but no development. After 21 hours in a 1/2,000
concentration there were few intact individuals left which had not
progressed beyond the state at which they were placed in the solutions.

In an effort to see how quickly the embryos would be affected,
samples from a series of different concentrations were transferred at
certain intervals to fresh sea water. These were examined about 24
hours later. The results are summarized in Tables II and III. They
show that a relatively strong concentration. of NiCl, which inhibits
any further development in the experimental solution acts very
quickly upon the embryo. For example, 6-7 hours exposure to a
1/2,000 concentration was fatal to further differentiation but the
embryos lived and increased in size (growth) in fresh sea water.

In the tables, comparative data are given for concentrations
ranging from 1/10,000 to 1/1,666. In a 1/2,000 concentration endo-
derm formation was entirely inhibited following an exposure of about
6 hours. In a 1/10,000 concentration the gastrulation processes first
showed inhibitory effects after 7 hours in some individuals of a culture.
Up to this time, the effect had been upon differentiation and growth.
In several repetitions of this experiment, a peculiar result was seen.
After 3-4 hours exposure followed by transfer to fresh sea water for
18-19 hours, quite normal appearing embryos were found. Longer
exposure times likewise gave more active and better differentiated
embryos than did the shorter exposures.

This result is surprising since it was expected that continued
exposure would eventually result in death for all individuals. Is it
possible that over a long exposure period the embryos lost their
sensitivity to the toxic effect of the salt and were able to differentiate
normally or that the salt was taken out of solution in some way?
This result was observed in three out of the five repetitions. In a
1/5,000 concentration development was progressively inhibited up to
about 43 hours while after 63 hours there was a marked advance in
differentiation. In a 1/2,857 concentration this ‘‘pick up” was ob-
served after 44 hours exposure. In a 1/2,000 concentration the
improvement was noticed in cultures exposed to the salt for 3¢ hours
or longer.

In other experiments, Tables II and III, the above phenomenon
was not observed. These show a progressive depression of develop-
ment in the higher concentrations. In Table III it can be seen that
the capacity to gastrulate was destroyed in a 1/5,000 concentration
after about 8 hours exposure, and no recovery was made in fresh sea

A. J. WATERMAN

*3n3 OU SIe[NoIsa/A,

"WI0}}0q UO [|e JsOW]y

“Apoq 3404S

*sajnords uojajeys ‘3n3
YUM Sie[NSuelI} VIO[\
*‘soAIqUIO Ie[NdISaA

][euls pue o9.1e] ore Iso

*saseo

awios ul Adwin] wisp
-0104 ‘“Sajnoids pue 3n3
YIM wnaysey ‘iegnyid
suno< 0} dn sad4} [|v

*4ns sAey

MO ‘SIe[NIISO/A

*4n3 ou

YM IP[NISIA 31
QWIOS ‘soynords yIIM
Jen suels 1e[ndoIt1]

yns YIM
SUIIOJ JE[NIISa/

*]eulIOU
-Ge Ud}JO U0}2]94S

*“sajnoids pue

ns YIM wnIYWse3
pue siejnsuers
][eWs Moy ‘uoTs
-a1 ajod ]e1930A
Je[Nsei11 10 pause
Shey MENUS ae
‘soAIquis 1e[nN
-JISaA ‘adie] AUeT

“suse

peseds Ajeprm
pue Apog 3104s
YIM t1agnjd sunoA
07 dn sedAQ [TV

‘aseis Iejnsuelsy
dy} payoval savy
J9}}2] VY} JO Jama
‘SNISes JOM]
pue x[nyseI[q s10;\[

uO}I[a4s [eulIouqe
YIM iegnjd sunoA
0} SUIIO} pozUNIS

‘ent

-Ses0x9 o]duIIs
Ma, ‘seynords
Yum x[N1}

-ses Jejnsuelsy
0} e[NIAse[q
jeoreyds osie7T

*Jasu0] SUIe

pue 1931e]
Apog ‘uo1
-epiejai sso]

OPeUl SEA OSULYD VOY S[eAIOIUI INOF{

“Ie[nsat

-4I U9}JO U0}IJB4S
‘ans yyIM soAiquia
Je[NSsuel.y od1e7

*sdey wie

[elit yim ouos
0} U0}JI]OYs pue yns
YIM Ie]NISaA Os[y
“UOT}epse}a1 BUIOG

€

“repn3uers ‘asieqT | 00s‘z/T
“sdey wie
JO suIIe JOYS YIM

awio0g ‘1eynjd Bunox | e¢e'e/T

JO1}U09 O¥X1T | 000‘OT/T

wornjos

*ase1S

408

' *709]J9 DIXO} JO vaIZap pue 9301 ay} MOUS e}ep IT,
snajnjd WNIpsul & }e SAA [O1]UOD 9Y} 9UIT] SITY JY JOP] SINOY G[-Q] Poulurexy “319}eM Las YSoIJ 0} S[eAIOJUI }e paAoutel sajdures
pue uoljnjos ur posed xjnysejq ‘eutt) Jo sported polseA JO} 2{DIN Jo suoI}e1]UBDUOD JUSIEIpP 0 e[Nyse][q Jo simsodxa jo WeyyA

Il S1avL

409

EFFECT OF HEAVY METALS ON DEVELOPMENT

wo1}e13
-9}UISIP paMmoys IUIOG

*e[Nyses
-oxo podeys Ajiepnsei1
pue pazejUusIoyIpun Mj

VY ‘ouljjno ul rejnso1
-II WO1de1 ojod [eIad0/
‘4nd [eUtoJUI ON “YSsIp

jO W10}30q UO [Je jsO;,

e]ngseiq [IV

"sary

-IJe[Nseiit [eusto}
-X9 1O ‘S][99 Jo sseur
10 ‘aqn} Wiepop
-u9d OU MOYS ISO
*Ie[NIIsaA Ae]
-nseii soAIqUIy

“UOT}e[N14SeS [eIzIUI
pemoys Moy Aro/

‘zxnyseiq AjIsoy

“xn I4Se3

JaMa,J “UOJIPIYS ON
*QUI]INO UT IejNse1
-1I pue Iejnsisa/,
AS UES ACES ING

sain

-o1ds ou ‘e[n13
-ses UINIpeul
0} zjnyseIg

*sdey

UII [eIQIUI ON
"sojniods |[eus
‘xen I4Sse3
Te[NSIsvA 910Ul
pue Jayyeus

SPEUI SBM OsUeYD VOYM S[eAIOIUT IMOF{

‘w2][N.1QSes0xe o[durIs
Moy ‘Sopnords 4AM
X[NIses repnsuersy

0} KINASE] [[eUIS

sojnords yAIM
X[NI4ses 99] [[eUIS

‘uoT}eWI10; dey-urse
[eIUI pamoys VUIOG
‘sojnoids |[ewis pue
asiey “Je[NoISaA
IOJ, “JOT[CUIS

Beis |

*paueq

-yey Apog ‘Auvur
UI pessel UIIEpo}
“uor}eUlIoy dey
-WIe [eI}IUI pamoys
auI0S ‘saynords
Je[nse111 10 [euliou
pue yn3 YIM soA1q

“Wd Ie[NSuUeII} as1e7]

999'T/T

000°2/T

wornjos

PenuyuUoy)—_j{] ATaV LL

A. J. WATERMAN

410

wz[nqseiq

[eAo 03 [eortayds

Je[NIISaA IIOJ\[

[01}U09 ayxTT

“Z]
—nijses eI}

“SOX9

Md, “soznords

do} uo /y}1M Jepnsuery

JOMaT “aules

0} JeNdIsa/\,

yee)
-njd sunoé
eee

wI0}}0q uO
[l@ 2[nYses
Jepnsuersy
0} xN\selq

eee‘e/T

x[nyseyq | 2[nysejq

aie YSOJ | 912 JSOT[

“sain

-o1ds YAM Ie]
—NSUPII} MJ
‘xen IYWse3
pue 1e[noIsoA
0} x[NAselq

auou
Jo sojnaids
UOT} epsejOI
2IOW IUIOG

u0j2]94S
YIM 1e[ns
-UPII} [[eWIsS
0} e[Nz4se3
Jen se11]

quoul
-9AOU 97}
“HWY = ~eynay
Ses [CIIUI
07} eNjse[g

aures

UO0J9[OyS YIM
Je[Nsuel}
0} z[NI4ser)

-IUT 0} xn SIE]NISAA
-sejq ‘do OBIE] 0} X]
DUES uo 9UON | -Ny4se]q s107\,
"u049] XS
ONG
*x[N4] | -nyse[q s107q
“ses JoMoy “QUITINO UT
“Iejnoisaa |Aduin] pue 1e]
Jeorseyds | [eorteyds jaroy, “WOR |-nSerzy =" W077
-10q UO |[V | -30q uO JsoTJ
sojnords
Taynjd | epnsqses 07 YIM 1eins
poor | sze[noisa, | -uerI} [[eUIS
6 EL 9

{01

mie

JO}EM LOS YSolj 0} OPEUI SEAA OSULYO USYM S[eAIOzUT INOFT

Toynjd
[[ews 09
Jen suersy,

$]0.1]U09

SPN en fa

UOI}NIOS Ut
UT 3J9] eSOyL,

000'S/T

000‘OT/T

wolnjos

*Ja}e] SINOY FZ JNoGe pauturexs soinj4jnd vsey J,
ul ynd xjnjsejq [eAo 03 jeol1ayds

*{99]J9 OIXO} 9Y} JO d1Sep puv 9301 ay} MOYsS e}ep ay, “1a3N,[d pamoys jo1}U0>7

II] adv LL

"Ja Jed1IY} S[PAJOIUI 72 JoJeM IS YSIIJ 0} PSAOUIII UsYy} PUe OUT UTe}I00 & Ye SUOTINIOS
‘aUII} JO spotted palizeA JOJ *{DIN JO suor}eI1}UBNUOD JUdIBYIp 0} wjnyse[q Jo sinsodxe jo Wey”

411

EFFECT OF HEAVY METALS ON DEVELOPMENT

aules
qynoqy QUIS
QUIES x[nqselgq
xz[n1}
“ses YSIS

aules | 0} e[Nyse[q

“UOIJEUISEAUI
[Reg es aese al
‘x[NIselg

‘gyn

-IdsON “&]
-NIjSes 99]
0} w[NIse[g

uO} eUl
-SeAUL JY SIIS
JO x[N}se|q
dIOJ, “WOR
OC LLOn TIN,

eco) al
-€139}UISIp
dUIOG “WIO}

-J0q uO ]TV

*u0}}0q UO
TV “9utes

auIesS

"u0}9]94S
ON ‘9ules

*sajnoids

YUM xn}
-Se3 9}P] 0} &]
-nysejq “uwloy
-jJoq uo Aueyy

“u0y
Jays YUM
Jejnsuelsy
0} 2[Nyselq
*W10930q

uO }SOJ

u0}29]94S
YIM Jens
-UPII} 99°]

0} 2[Nyselq

sojn

-o1ds yyIM
re[nsue
-11} 0} e[N1
-SeS 9307]

*u01}e.139)
-UISIP 9UIOS
‘xNAselgq

xz[nqselq

wzny
-SE]q [PAO
03 jeotrayds

999'T/T

000‘2/T

00S‘2/T

sodeys
SNOLIGA JO
aues | xniselg
*W01}2.139]UISIP
DUIOG ‘aes aUIeS
aues | xniselg
rat #01

JO}EM aS YSolj 0} OPEUI SEM OSULYD USA S[eAIOIUT INOF,

panuyuoj—{] ATAv

uOlyNjos ur
eT SsOuL

WOINjoS

A. J. WATERMAN

412

*S][20

Jeoleyds jo} ‘worje1s9qUISIpP

E2jbal
-ses sunoA

wx[niqses
WINIpour

Livohls) E>: (5)
{NOYIIM siTe[nsueiy

Sossep, “peesq|yonyL ‘“x[nIselg 0} enysel[g| 0} x2[nyselq 0} E[nayses Apieq
000‘000'S/T 000'000'8/T 000‘000‘9T/T |000°000‘ZE/T 000‘000‘84/T
“sul
-JsIsiod soueiq
*ai10d XI “Ul 2] :
-O}SE[G os1e7T| -n1ses [eI -nijses sunoAé lojnjd sunoA uonepie,
‘oures Jnoqy 0} wlnyselg aUlOS 0} wnyselg ejnaqse5) 0} x[N14se5) -o1 WSIS [O1}U09 OyIT
“peep
BUOS “XI
-Nijses SunoA uory
aules ynoqy 0} xinIselg aUleS w]Niqse45 SIe[NSUeI | -eprejor JUSS] [01}U0D oxTT [O1}U0D oyIT
“41S
-1od soueiquioul *uo1}e[NI1} E2jabal *uI0}30q UO “JUS sie[ns
Aqdwuq ‘xT -Ses [CIUT -Ses sUNOA!| JSsO;, ‘“x[nI -9AOU I9MOT[S -UCII} Vd1P]
-nysejq Asopy |’ 2[nyselq s1077 0} X[Nysejq| -Ses UMIpeyy| “ewes ay} YON] x[nzWses 97e7]] 0} L[n1se5 [01}U09 oy]
*110}}0q uO =
sO. ‘“Soueiq *U10}0q
*WI0}}0q UO} -WoUl UL [[Ss\ul Aue; *3UTqSIS
“u01je13 IlV ‘Seueiq) Aueyy ‘x[n13) -19d souviquiour *soynoIdsS
“HONCIZIIUISIP] -d}UISIP BWIOS|-UoU ul AUST, -SPS8 [III] BWIOG *xX[NIWsed w[nijses| ‘srepnsueisy
eo, ‘“aues| ‘A[UO x[NIse[g)| “A[UO ~z[nyse[g 0} L[nyse[q] SuUNoA 0} [eI}IUT] 923e] 0} BuNOA] 07 K[NIWSe5 JO13U09 oy]
000°‘002/T 000‘0SZ/T 000‘00¢/T 000°00F/T 000'00S/T 000‘008/T 000°009'T/T 000'002'‘¢/T
[O19U09 94} WOI} sdUSTOIp ou AyjeonoeIg
e[niy wnt}
“Sed IoMoj pue] -ses WINIpew wz[ni4se3 siepnsueny yo19
x[Nqse[q sop, 0} L[NIse[g] 93e] 0} sunoy, 0} L[N14Sse4)|UOTJeprejoeI YSIS JO1}U09 ayI7T -U09 o¥IT
000‘0S/T 000‘09/T 999'99/T EES‘ Es/T 000‘00T/T 0S8'CPT/T 000'00Z/t | 000‘00#/T
*Sopnords sojn
ON ‘siey] -o1ds 471M
“WO1}e139}UL “4sIsiod wnt} -nsueldy sie[ns
-SIq ‘UWI03}0q SoueIquIsUI “SIOUIIMS w[ngse[q UOe[N14Ses| -Ses UINIPsU|/x[n414ses 9}e]}] Ajies 03) -UeIIZ 07
UI [JV “eules) suIOG ‘“aUIeS| May ‘“eWIeS} 9}e] 07 SUNOA| [eWIUI [eUOISeDDO 0} w[nIselg 0} Ssuno,| w[N1WseyH} wN4IWses
*s][29 *e[niqse3 “SIOUIWIIMS doz
JO SeSSeUI [eDI] [eIIUI [euOIs MOT “xL[NI17 x[nijse3 Teyny{d
uoneis -IaydS ‘uone]] -e20Q ‘xn we[n14Sse3 -Sses [CI}IUT Ooze] AIOA aures| SuNOoA 07
-9}UISIP BUIOS eules] -e[Nniyses ON| -selq ATIsOyT [equ Tome 0} X[Nyse[q| 0} x[nIse[g qnoqy| e[naqses
000°S/T 000'9/T 999'9/T eee's/T 000'0T/T C8e PT/T 000'02/T 000'0F/T | 999‘99/T

“sIq
ct

“SI
EET

“Siu
FET

ainsod

-xo Jo
yjsueT

To3njd

sunoj,

Toynj{d
suno,

jeynyjd
suno x,

Teqnjd

suno jx,

leqynjd
sunoj,

Toyn[d
suno jx

Toyn{d
suno x

Teqnyd
sunoj

reqnyid

sunoj,

yor
-u09

%10°0
73H

2(@O&H2D)UZ
%1'0

%1'0
(‘OSUZ)

10
OLYA

%10
Ono

%V0
JOe4

VO
104d

%O't
40) 30)

%O't
TOV

Te}

"Ia}eM aS UI JUasaId Apeai[e VSOY} 0} S}][eS OI|[eJOUI JUSJEYIp Jo sJUNOWe snoliea Jo uOIIIPpe ay} Jo JusuIdOjeAep uodn yayq

Al 314V.L

EFFECT OF HEAVY METALS ON DEVELOPMENT 413

water. The length of time necessary to destroy the ability to gastru-
late varies according to the concentration employed; Tables II
and III. The results summarized in the tables represent single
experiments, but they are indicative of what was secured by several
repetitions of each.

Al,Clg.—In dilutions of a 1 per cent stock solution of Al,Cle,
gastrulation in all individuals was practically inhibited at a 1/8,333
concentration during an exposure of 133 hours. Table IV shows that
the first indication of the inhibition of gastrulation in some of the
individuals was observed at a concentration of 1/20,000. Further
inhibition of more individuals occurred in higher concentrations until
at a 1/6,666 concentration no gastrulation took place during the
experimental period. Samples transferred to fresh sea water from
concentrations between 1/14,285 to 1/8,333 after this interval re-
covered. Gastrulation and development to the pluteus stage took
place. Abnormalities of arm and skeleton formation appeared in the
sample from the 1/8,333 culture. Samples transferred to sea water
from the 1/6,666 culture died.

To test the rapidity of the toxic effect, samples were transferred
from a 1/6,666 concentration, at certain intervals, to sea water.
Those transferred after $ to 23 hours of exposure gave plutei. The
longer exposures caused progressive retardation, and more gastrule
and fewer plutei were found for the same length of time. After 5
hours exposure, a few gastrulated but most died in the sea water;
after 63 hours exposure, all died without any development.

When the toxic effect of a 1/5,000 concentration was tested, an
exposure of $ hour markedly retarded development, 1 hour inhibited
almost all gastrulation and 24 hours killed the embryos.

CdCl.—Table IV includes the effect of this metallic chloride in
different concentrations. Over a 133 hour period, gastrulation was
inhibited in a 1/8,333 concentration. In sea water, these blastule
grew in size, became irregular in outline, but did not gastrulate or
form skeleton. Samples from a 1/10,000 concentration formed large,
vesicular, irregular blastule and gastrule in sea water.

PbCl,.—Lead chloride is slightly more toxic than the three metallic
chlorides just mentioned, and hence a 0.1 per cent stock solution was
used. Table IV includes part of the data secured in one experiment
which may be considered typical. Slight retardation became evident
in a concentration of 1 part in 100,000. In aconcentration of 1/60,000
gastrulation was inhibited in some individuals. The latter increased
in number in a concentration of 1/50,000 after which little change was
noted in the cultures until a concentration of 1/35,714 was reached.

414 A. J. WATERMAN

One result was the persistence of the fertilization membranes even
after the embryos had escaped. Thus this salt causes the membranes
to harden and prevents their dissolution during the experimental
period.

In the 1/35,714 solution late gastrula to triangular stages were
found, while in a 1/33,333 concentration even young plutei occurred.
This condition was found in all cultures up to a concentration of
1/22,222. No higher concentrations were tried. This peculiar
change may perhaps be accounted for by the precipitation of lead
seen in the bottom of the container, during the experimental period.
The precipitation did not appear when the experimental solutions
were made up, but only after standing. Enough was left in solution
to retard development slightly.

FeCl,.—This metallic chloride is apparently not toxic to any ex-
tent. Very little if any difference was observed in graded cultures
up to a concentration of 1 part in 14,285.

CuClh.—This metallic salt is far more toxic for development than
PbCle. A 0.1 per cent stock solution was used. Table IV summarizes
the results of a typical experiment. Gastrulation was inhibited in
most individuals in a concentration of one part in 400,000 and entirely
in a 1/300,000 concentration. The fertilization membranes showed a
hardening in these concentrations and failed to dissolve. This must
occur rather quickly because in these and higher concentrations more
of the embryos failed to escape from the membranes. The same
persistence of the membranes was also seen in the PbCl, solutions but
to a lesser extent. In the latter case the embryos had escaped so
the hardening occurred more slowly. This lack of digestion of the
membranes in both the PbCl. and CuCl, solutions may be accounted
for by enzyme-poisoning which is characteristic of some of the heavy
metal salts. As will be seen below, the higher concentrations of the
Zn salts likewise produced persistent membranes.

After transfer to fresh sea water, the original 1/500,000 culture
continued development to the pluteus stage. Also large vesicular
blastulz without skeleton were found, indicating growth of the ecto-
derm but inhibition of endoderm and mesoderm formation. Most of
the individuals in the 1/400,000 concentration gastrulated in fresh
sea water and in 24 hours formed triangular embryos with spicules.
More large blastulee were present as well as a few exogastrule showing
tri-partite gut, anal opening, and apical plate. Thus in some indi-
viduals this metallic salt inhibits the gastrulation processes without
inhibiting endoderm formation.

As seen in Table IV, no gastrulation occurred in a 1/300,000

EFFECT OF HEAVY METALS ON DEVELOPMENT 415

concentration during the experimental period. However, in sea water
some did gastrulate and a very few formed simple exogastrule. No
skeleton was seen in any individual, indicating the inhibition of
mesoderm formation or the formation of skeleton during the period
of observation. The endoderm did not differentiate. Only a few
individuals from a 1/250,000 concentration recovered in sea water.
Large, spherical and oval blastulz were found as well as a few initial
gastrule. The individuals from a 1/200,000 culture failed to gastrulate
even after 48 hours; many died; and many failed to get out of the
membranes.

The time required for recovery varies of course with the concentra-
tion. Embryos which did not gastrulate or in which no skeleton
formed during a 24-hour period in sea water, did so after 48 hours.
Since it is known that certain marine organisms may store copper in
their tissues (oysters) this inhibition may be due to the concentration
of the toxic metal, and the slowness of recovery and differentiation be
due to the slowness with which the metal left the cells and again
reéntered the sea water in such a dilute concentration as not to inhibit
further development. The metal could not have irreversibly injured
the protoplasm, but rather inhibited its function.

Angerer (1935) has found that after Arbacia eggs were exposed to
CuCl, an interval of time elapsed during which the metal produced
no visible effect in the protoplasm as regards its viscosity. This
time interval is a function of the concentration of copper in solution.
At the conclusion of this latent period, there ensues a sharp rise in
viscosity values resulting in an irreversible gelation (coagulation) of
the protoplasm. In the case of Arbacia egg protoplasm, there is no
concentration at which gelation is reversible.

In the light of Angerer’s results the temporary inhibition of
differentiation cannot have been due to gelation, otherwise recovery
would not have occurred. An irreversible effect was seen first in the
concentration of 1/200,000 where no recovery occurred even during
48 hours in sea water.

To test the rate of lethal action of CuCl, a concentration of 1
part of stock solution in 150,000 parts sea water was used, which
inhibits all development and causes complete disintegration in an
18-hour period of exposure. Embryos were removed from this solution
to fresh sea water at various intervals. An exposure of 3} hours
resulted in some inhibition and many persistent membranes from which
numerous embryos had failed to escape. After 94 hours in the solution
only a few embryos gastrulated during the following 28 hours in sea
water. Those which did not escape from the membranes, or were only

416 A. J. WATERMAN

partially successful in it, did not differentiate. A concentration of
1/250,000 produced no inhibitory effects during 8 hours exposure.
Membranes hardened while the embryos which did not escape differ-
entiated slightly.

HgCl,.—This metallic chloride is very toxic. A concentration of 1
part in 48,000,000 parts of sea water retarded development while a
concentration of 1/8,000,000 prevented gastrulation during a 15-hour
exposure (Table IV). After return to sea water, only very few of
the more hardy individuals were able to gastrulate, but they developed
no further. In a concentration of 1/5,000,000, the swollen cells
showed a clumping of the pigment granules.

To test the rate of action of HgCl:, spherical to oval blastulez were
placed in a concentration of 1/2,500,000. The embryos transferred
to sea water after 5 minutes exposure were able to recover with
only slight evidences of retardation in 24 hours. A 10-minute ex-
posure visibly retarded development. More gastrula and triangular
stages were found, while the control showed medium plutei. After
15-minute exposure, fewer individuals were able to gastrulate. These
decreased in numbers following exposures of 20 to 45 minutes. During
this interval more of the embryos were killed and movement became
progressively slower. An exposure of 55 minutes killed most of the
embryos, though a few survivors showed attempts to gastrulate.

This metallic salt does not appear to affect the gastrulation process
independent of other developmental processes. As long as any sur-
vived they still attempted to gastrulate. Development went no
further, however, during the period of observation. The most pro-
nounced toxic effect on the majority of the embryos occurred within
the first 15 minutes exposure.

ZnClo.—This metallic salt is slightly less toxic than CuCl. Gastru-
lation was inhibited in most individuals when the concentration was
about 1/200,000. The most pronounced inhibitory effects appeared
in concentrations up to 1/400,000. Persistent membranes appeared in
the higher concentrations, and embryos failed to escape. Also an
increasing number of embryos were killed in concentrations of 1/120,000
and higher. In these concentrations only an occasional attempt at
gastrulation was seen; while in a 1/100,000 concentration, no attempt
at gastrulation was seen and many were dead. On return to fresh
sea water, the surviving individuals formed large globular structures
without gut or skeleton. The ectoderm was often irregular and lumpy
in appearance and movement was lethargic.

To test the rate of action of ZnCl, samples were transferred to
fresh sea water from a 1/10,000 dilution every five minutes following

EFFECT OF HEAVY METALS ON DEVELOPMENT A17

their introduction at 6:50 P.M. and were examined the following
morning at 8-9 A.M. The control at this time showed young plutei.
A 5-minute exposure gave blastule to late gastrule. Membranes
persisted and some of the embryos failed to escape completely. The
latter had not gastrulated. A 15-minute exposure gave blastule to
young gastrule. Many had died and disintegrated or failed to escape
from their membranes. The toxic effect of this metallic chloride is
manifested very quickly after exposure. The most pronounced effects
occurred within the first 10-15 minutes. After this the noticeable
changes occurred very gradually. Thus even after one-half hour of
exposure, gastrulation of many of the surviving embryos did occur
during the experimental period. Movement, however, was very
lethargic. After 1-hour exposure no normal gastrule were found,
although some had attempted it.

ZnSO,.—A stock solution of 0.1 per cent was used. As seen in
Table IV, a concentration of 1/500,000 caused conspicuous retardation.
In a 1/250,000 concentration, some individuals failed to gastrulate and
those which did gastrulate were still in the young gastrula stage
after 15 hours exposure. In a concentration of 1/175,000 most failed
to gastrulate and some died. The hardened membranes failed to
dissolve. The few individuals which attempted to gastrulate showed
very broad invaginating regions. This effect was also observed in
the ZnCl, study. In a 1/100,000 concentration only an occasional

individual showed an attempt to gastrulate.

Zn Acetate.—This salt gave results quite comparable to the other
zinc salts used (see Table IV). It caused the membranes to persist
and the initial gastrule in the higher concentrations showed broad
blastopores. The blastoccel was packed with cells. Gastrulation
seemed to be inhibited at concentrations of 1/100,000 and higher.

It is well known that cyclic changes in the distribution of marine
invertebrates are often accompanied by changes in the chemical
composition of the sea water. Reproduction, embryonic development
and growth are dependent on the presence of various constituents but
the relative proportions of the various necessary elements may vary
without any essential detrimental results. The absence cf a necessary
element or its presence in a non-utilizable form naturally disturbs
development while the presence of too much of the element (experi-
mental study) will also bring about developmental changes. The
latter probably does not play as important a part in marine life as
does the lack of the necessary amount of the element, but nevertheless

418 A. J. WATERMAN

the study furnishes information on the specific effect of the element
upon development.

The chlorides of some heavy metals, especially NiCle, whose effects
on gastrulation and subsequent differentiation in the sea urchin,
Arbacia punctulata, have been described in this study, may be added
to the growing list of physical and chemical agents which may provoke
exogastrulation. As is well known from studies by others, some
metals are more toxic than others (Table IV) and the more toxic
ones exert their toxic effect on gastrulation very quickly. The
relative toxicity of the metallic salts seems to be as follows: HgCle
> CuCl, > ZnClo, ZnSOu, Zn(CeH302)e > PbCl, > AleCle, CdCh,
NiCl. > FeCle. In the case of most of the salts employed, the
initial effect is upon growth and differentiation. Retardation and
inhibition become increasingly more conspicuous in progressively
higher concentrations, while the gastrulation process is the last to
be effected. Finally it has been found that certain of the metals,
especially NiCle, give a larger number of exogastrule at certain con-
centrations than in some of the previous studies where other types of
environmental modifications were made, but they are not as effective
in provoking exogastrulation as LiCl, (Runnstré6m). It would appear,
therefore, that such different physiological processes as gastrulation,
differentiation and growth have different thresholds of inhibition for
the same toxic substance.

Information is given on the concentrations which inhibited gastrula-
tion. Providing the exposure has not been long enough to injure the
embryo fatally, gastrulation and even further development will take
place in fresh sea water. The rate at which some recovery takes
place depends upon the concentration employed and the length of
exposure. No cases of fused embryos were found.

As seen in previous studies (cf. Runnstrém, Lindahl), differentia-
tion of the ectoderm and even of mesoderm, within limits, may take
place independently of gastrulation or of the formation of the gut
tube. Of the three germ layers, ectoderm is the most resistant to
injury. In general the types of inhibitory or retardational effects
are similar to those produced by other environmental modifications
and by other workers. These include the behavior of the skeleton
forming mesenchyme, the development of body form and size, the
relative proportions of the potential ectoderm and endoderm, the
inhibition of the gastrulation process, etc. The various metals give
quite similar results but at different concentrations.

Information is also given on the rate of action of a lethal solution
of each metallic chloride tested. The more toxic the metal, the

EFFECT OF HEAVY METALS ON DEVELOPMENT 419

quicker are inhibitory effects shown even in very dilute concentra-
tions. In higher concentrations of certain heavy metals like Zn,
Cu and Pb, the fertilization membrane does not disappear. In such
cases, and even where partial escape has occurred, the embryos do
not differentiate. This lack of digestion of the membrane may be
due to enzyme-poisoning which is so characteristic of some of the
heavy metal salts (Ishida, 1936).

Although the differentiation of organs and tissues in both gastru-
lated embryos and in those where exogastrulation has occurred has
been of interest to numerous workers, the effect of environmental
modifications on the relative proportions in the amount of ectoderm
and endoderm has attracted much study (Runnstrém, Lindahl,
Lindahl and Stordal, and others). By appropriate stimulation of the
egg the embryo can be animalized or vegetalized and the differentiation
followed. Furthermore, this phenomenon can be inhibited by various
means. For example, Li salts will vegetalize the embryo while SO,
deficiency will animalize (ectodermize) the embryo.

Chlorides of certain heavy metals, acting on the blastula stage,
likewise produce ectodermal embryos which lack gut or any endo-
dermal material as far as can be seen. All transitions between this
and typical gastrulation occur. The ectodermal embryos differentiate
skeletal spicules or rods, apical plate and sometimes stomodzum.
If treatment has not been too severe, the exogastrulz likewise differ-
entiate these structures. Arms never develop but oral and anal
flaps may appear and the body tends towards typical shape which,
however, is modified by the abnormal distribution of skeletal material.
In extreme cases only large globular, ectodermal embryos are found in
which the skeleton does not go beyond the spicule stage and neither
stomodzum or apical plate differentiates.

Interpreted in the light of Runnstrém’s hypothesis, the heavy
metals employed may effect in a differential manner the ectodermal
and endodermal gradients which he believes to exist in the fertilized
ege (ectodermization and endodermization of the embryo, Lindahl,
1936). It has been shown previously that these same gradients may
be present at the blastula stage and hence the limits of the endoderm
and ectoderm are not definitely established even at the oval blastula
stage which immediately precedes gastrulation. It is doubtful if the
limits are established even at gastrulation. Lindahl (1936) considers
that the two gradients have different metabolic rates and also that
hydrocarbon metabolism dominates the animal pole while protein
metabolism dominates the vegetal pole. The heavy metals used in
this study may therefore exert their toxic action upon these metabolic

420 A. J. WATERMAN

processes or produce substances which give the same effect which
would account for some of the various developmental abnormalities
and inhibitory effects described above.

LITERATURE CITED

ANGERER, C. A., 1935. The action of cupric chloride on the viscosity of Arbacia egg
protoplasm. Anat. Rec., Supplement No. 1, 64: 81.

CHILD, C. M., 1929. The physiological gradients. Protoplasma, 5: 447.

Drzewina, A., AND G. Bonn, 1926. Action de l’argent métallique sur le sperme et
les larves d’Oursin. Compt. Rend. Acad. Sct., 182: 1651.

Féreé, M. Cu., 1893. Note sur l’influence des injections de liquides dans |’albumen
sur l’incubation de l’oeuf de poule. Compt. Rend. Soc. Biol., 45: 787.

FRANKE, K. W., ET AL., 1936. Monstrosities produced by the injection of selenium
salts into hens’ eggs. Anat. Rec., 65: 15.

GatsorfF, P. S., 1932. The life in the ocean from a biochemical point of view.
Jour. Wash. Acad. Sci., 22: 246.

GLASER, O., 1923. Copper, enzymes, and fertilization. Bzol. Bull., 44: 79.

Hammett, F. S., AND V. L. WALLACE, 1928. Biology of metals. VII. Influence of
lead on the development of the chick embryo. Jour. Exper. Med., 48: 659.

HETHERINGTON, D. C., AND Mary E. Suipp, 1935. The effect of cupric, manganous,
and ferric chlorides upon cardiac explants in tissue culture. Bvzol. Bull., 68:
215%

Hoab.ey, L., 1923. Certain effects of the salts of the heavy metals on the fertiliza-
tion reaction in Arbacia punctulata. Biol. Bull., 44: 255.

Hoan.ey, L., 1930. Some effects of HgCle on fertilized and unfertilized eggs of
Arbacia punctulata. Biol. Bull., 58: 123.

IsHipA, J., 1936. An enzyme dissolving the fertilization membrane of sea-urchin
eggs. Annot. Zodl. Japon., 15: 453.
Lire, F. R., 1921. The effects of copper salts on the fertilization reaction in
Arbacia and a comparison of mercury effects. Biol, Bull., 41: 125.
LinDAHL, P. E., 1936. Zur Kenntnis der physiologischen Grundlagen der Determi-
nation im Seeigelkeim. Acta Zoologica, 17: 179.

LINDAHL, P. E., UND A. StorDAL, 1937. Zur Kenntnis des vegetativen Stoffwechsels
im Seeigelei. Arch. f. Entw.-Mech., 136: 44.

PaRKER, G. H., 1924. The growth of marine animals on submerged metals. Bzol.
Bull., 47: 127.

PRYTHERCH, H.F., 1931. The réle of copper in the setting and metamorphosis of the
oyster. Science, 73: 429.

RUNNSTROM, J., 1935. An analysis of the action of lithium on sea-urchin develop-
ment. Biol. Bull., 68: 378.

THE CYCLE OF ORGANIC PHOSPHORUS IN THE GULF
OF MAINE

ALFRED C. REDFIELD, HOMER P. SMITH AND BOSTWICK KETCHUM

(From the Woods Hole Oceanographic Institution’ and the Biological Laboratories
Harvard University)

It is generally recognized that the fertility of the sea depends upon
a cycle in which carbon, nitrogen, phosphorus and other substances are
assimilated under the influence of photosynthetic processes in surface
waters and are set free again by processes of digestion or decay. The
total organic productivity of a region is limited to the rate at which this
cycle is completed. While it is apparent that in many localities the
principal limiting factor is the rate of restoration of the inorganic
products of decay (NO3, PO.) to the surface or photosynthetic zone,
little is known concerning the exact locus within the sea at which
decomposition actually sets these substances free, or of the rate at
which the cycle as a whole or in part is completed.

The concentration of the ultimate products of decomposition, such
as NO; and POu,, tell us little about these points since they are stable
substances capable of accumulating over a long period of time to high
concentrations, and of being transported far from their place of origin.
The actual site of decomposition is better indicated by the presence of
intermediate products of decay such as ammonia, nitrite, and organic
compounds of nitrogen and phosphorus.

The present paper is an account of the distribution of phosphorus
throughout the year at a standard station in the Gulf of Maine. At all
depths the amount of phosphorus present has been measured in three
forms: (1) inorganic phosphate (PO,), (2) dissolved organic phosphorus,
and (3) particulate organic phosphorus. The analysis of these data
gives some indication of the magnitude of the synthetic and dis-
integrative processes undergone by phosphorus compounds in different
parts of the water column at different times of year, and of the extent to
which phosphorus is transported from one depth to another.

The station chosen for study was located in the deeper portion of
the western basin of the Gulf of Maine, 30 miles northeast of Highland
Light. The surface water in this region is apparently relatively free
from strong non-tidal currents, being sufficiently far offshore to avoid
the coastal drift which accompanies the freshening of the water along

1 Contribution No. 127.
421

422 REDFIELD, SMITH, AND KETCHUM

the margin of the Gulf. In this part of the Gulf, as the result of
freedom from strong currents, there develops each summer maxima!
surface temperatures and maximal stability of the water column. In
this location one also finds relatively deep water (230-270 meters) free
from strong non-tidal currents. Preliminary measurements made by
Dr. E. E. Watson with current-meter indicate maximal tidal velocities
of 11 cm. per second at 40 cm. above the bottom, and 14 cm. per second
at 177 cm. above the bottom. The oxygen content in the deep water
below 200 meters is low, varying from 4 to 4.5 cc. per liter. The point
chosen for study thus presents conditions in which there exists above
the bottom a considerable column of water too poorly illuminated to
permit of photosynthesis, which terminates at a depth of 40 to 50
meters (Clarke and Oster, 1934) and in which decomposition occurs in
sufficient magnitude to maintain a low oxygen concentration, and one

TABLE [|

Station positions and dates.

Atlantis Depth
Station Date Location of
Number water
meters
2440 May 18, 1935 42° 22’ N. 69° 35’ W 249
2468 August 20, 1935 42° 20’ N. 69° 32’ W 232
2493 November 8-9, 1935 42° 21.5’ N. 69° 32’ W 256
2495 February 26, 1936 42° 22’ N. 69° 33’ W 270
2558 May 14, 1936 42° 27’ N. 69° 31.5’ W 254

as well chosen as may be to avoid disturbances due to the non-tidal
drift of the water.

Stations were made on five occasions separated by intervals of three
months, thus completing one yearly cycle. Their positions and dates
are given in Table I. The approximate position is indicated by a
circle in Fig. 1.

ANALYTICAL PROCEDURE
Inorganic Phosphate

Phosphate samples were collected in black bottles and analyzed for
inorganic phosphate at room temperature on shipboard by the Deniges-
Atkins method, except that the solution of stannous chloride used
contained 0.1 gram SnCle.2H.2O in 10 ml. of 1 to 10 hydrochloric acid.
Salt effect correction factor as measured was 1.35. Corrections for
salt error and reagent blank were both applied.

CYCLE OF PHOSPHORUS IN GULF OF MAINE 423

Particulate Organic Phosphorus

Samples of water of about 300 ml. volume were filtered on shipboard
as soon after collection as possible.

The procedure by which particulate organic phosphorus was
determined is as follows. The particulate matter is filtered out by
suction on a precipitate of barium sulfate on a 3G4 Jena sintered glass
funnel with polished surface. The barium sulfate precipitate is

Fic. 1. The distribution of inorganic phosphate, PO., throughout the Gulf of
Maine in May, 1934, at the surface and at the depths of 60, 120, 180 meters. The
circle indicates the position of the stations at which the present investigations were
made.

prepared by stirring 0.6 ml. of normal barium chloride into about 10 ml.
of hot water containing excess sulfuric acid. It is poured over the
funnel, the liquid sucked through, and filter and flask washed thor-
oughly with distilled water. The sea water sample is run through the

424 REDFIELD, SMITH, AND KETCHUM

filter, and the volume of the filtrate measured. Plankton and barium
sulfate are washed off the filter into a 125-ml. Erlenmeyer flask with a
stream of distilled water. The sides and bottom of the filter are
cleaned with a rubber policeman and all the precipitate transferred to
the flask to insure removal of the plankton. The funnel is cleaned by
reverse suction, treatment with sulfuric and chromic acids and thorough
washing.

After the addition of 2 ml. of 38 per cent (by volume) sulfuric acid
to the plankton, the flask is evaporated on the steam-bath to charring
of the organic matter. The flask is ignited to fumes of sulfuric
anhydride, and a drop of phosphate-free hydrogen peroxide (Cooper,
1934) (prepared by vacuum distillation of concentrated hydrogen
peroxide) is added. A few seconds heating without loss of sulfuric
anhydride fumes suffices to destroy the organic matter. The excess
hydrogen peroxide is decomposed by heating the sample at 120° C. for
one hour on an oil-bath. The cooled samples are diluted with ca.
50 ml. of distilled water and warmed on a steam-bath before filtering
through a sintered glass funnel to remove the barium sulfate. The
filtrate is diluted to 100 ml. in a glass-stoppered bottle. After the
addition of 2 ml. of 23 per cent ammonium molybdate each sample is
shaken. Standards of similar phosphate content as potassium
dihydrogen phosphate are made up with 2 ml. of 38 per cent sulfuric
acid, 2 ml. ammonium molybdate and distilled water to 102 ml. and
shaken. Two drops of stannous chloride solution containing 2.5
grams SnCl».2H.2O in 100 ml. of 1 to 10 hydrochloric acid are added to
each sample and standard with immediate shaking. After five
minutes the samples are compared with the standards in a colorimeter
with about 30 cm. depth of solution. Phosphate in the reagents for the
development of the color (designated as Blank A) is determined by
intercomparison of dilute standards. Phosphate introduced in the
treatment of the unknowns (designated as Blank B) is determined by
carrying through the procedure on a barium sulfate precipitate not
treated with sea water. Blank A is added to the standards to give
their true phosphate content. Both Blanks A and B are subtracted
from the phosphate found in the unknowns. The net phosphorus in

1,000
ml. sample
the plankton phosphorus in grams X 10~* per liter. Errors in the
analysis due to loss of phosphorus by volatilization or change in acidity
by loss of sulfuric anhydride are shown to be negligible by carrying
through blank analyses with known amounts of added phosphorus.

A procedure employing nitric acid as the oxidizing agent (Robinson

grams X 10-* found in the unknowns multiplied by gives

CYCLE OF PHOSPHORUS IN GULF OF MAINE 425

and Kemmerer, 1930), in which the nitric acid was evaporated off and
the last traces removed by an evaporation with hydrochloric acid gave
check results with the hydrogen peroxide procedure but a poor color
match with the distilled water standards. It might be possible to use a
procedure similar to that of von Brand (1935) for separation of the
diatoms from sea water. No effort was made to differentiate between
phosphorus and arsenic in the plankton phosphorus determinations.
It was found, however, that the procedure of Zinzidze (1935) using
bisulfate and sulfuric acid will bring about the complete reduction of at
least 9 X 10~® grams of arsenic to the limit detectable by the cerulio-
molybdate method (2 X 10-’ grams) and make possible a distinction
between phosphate and arsenic in plankton analyses.

Dissolved Organic Phosphorus

The phosphorus in solution in organic combination was obtained by
subtracting the inorganic phosphorus from the total phosphorus in the
filtrate from which the particulate matter had been separated. The
following procedure for the determination of total phosphorus in a sea
water sample was devised with a view to avoiding the interference of
pentavalent arsenic with the ceruleo-molybdate phosphorus determi-
nation and to insure the complete destruction of organic matter. In
the more rapid methods of analysis which have been used, in which sea
water is treated directly with oxidizing agents, arsenic in the sample is
oxidized and not subsequently reduced, while organic matter is not
completely destroyed in solutions containing large amounts of chloride.

Fifty-milliliter samples of sea water are treated with 3 ml. of
concentrated sulfuric acid (arsenic-free) in a 125-ml. Erlenmeyer flask.
After evaporation on a steam-bath under a hood to carbonization of the
organic matter, each sample is evaporated to the formation of fumes
of sulfuric anhydride with swirling to avoid bumping. A drop of
phosphorus-free 5 per cent hydrogen peroxide (prepared by vacuum
distillation of 30 per cent hydrogen peroxide) is added. The flask is
heated in the same manner without loss of sulfuric anhydride for half a
minute more to make the solution colorless. If necessary, more
hydrogen peroxide may be added. About 60 ml. of distilled water is
added to the cooled sample, which is set on the steam-bath for complete
solution of the precipitated salts. After transfer to a 500-ml. Erlen-
meyer and addition of 8.5 ml. of concentrated ammonia water, the
excess of ammonia is boiled off and the volume of solution reduced to
about 30 ml. The sample is transferred to a small ground-glass-
stoppered bottle and warmed on the steam-bath, open, with 2.47 ml. of
38 per cent (by volume) sulfuric acid. Four milliliters of 10 per cent

426 REDFIELD, SMITH, AND KETCHUM

sodium sulfite (anhydrous, or hydrated salt in double the concen-
tration) is added, and the stopper held in with a clip to prevent the
escape of sulfur dioxide. Excess hydrogen peroxide is immediately
destroyed, but eight hours heating on the steam-bath is necessary to
reduce pentavalent arsenic to the trivalent form. The sample is
transferred to a 500-ml. Erlenmeyer, boiled for five minutes to remove
sulfur dioxide, cooled, and diluted to 100 ml. in a ground-glass-
stoppered bottle. The residual acid in the sample is 2 ml. of 38 per cent
sulfuric. After the addition of 2 ml. of 2.5 per cent ammonium
molybdate the sample is shaken. Standards which will develop about
the same color intensity as the unknowns are made up from a potassium
dihydrogen phosphate solution with the addition of 2 ml. 38 per cent
sulfuric acid, 2 ml. of 2.5 per cent ammonium molybdate solution, and
distilled water to 102 ml. These are shaken. Each standard and
sample is again immediately shaken after the addition of two drops of a
solution containing 2.5 grams of SnCle 0.2H2O in 100 ml. of 1 to 10
hydrochloric acid. After four or five minutes for the development of
the color the samples are compared with the standards in a colorimeter
with about 30 cm. depth of solution.

Intercomparison of dilute standards in distilled water is made to
estimate the phosphate in the reagents for producing the color (desig-
nated Blank A). A blank determination carried through with
reagents alone gives the phosphate introduced by reagents in the
treatment of the sample (designated Blank B). Blank A is added to
the standards to give their true strength in calculating the phosphate in
the unknowns. Both Blanks A and B are subtracted from the
phosphate found in the unknown. The salt effect correction has been
determined as 0.885 for unknowns by analysis of the same sea water
with varying amounts of phosphate added. The slope of the graph of
phosphate added against phosphate recovered is the salt effect cor-
rection. No variation in salt effect correction was found with salinity
from 31.5 to 38.5. A similar procedure gave a salt effect correction of
0.81 in determining Blank B. Change of salt effect correction with
temperature was not studied but extremes of temperature were
avoided in making the analyses.

Since several samples of sea water gave identical analyses with or
without the addition of as much as 165 mg. per cubic meter of arsenic
(about eight times the amount found in sea water), arsenic was assumed
to be reduced completely by the procedure used. Reduction of
arsenic in solutions for determining Blank B was found to be much
slower than in sea water samples and quantitative only for amounts of
added arsenic equivalent to 50 mg. per cubic meter or less. The

CYCLE OF PHOSPHORUS IN GULF OF MAINE 427

difficulty was avoided by the use of reagents containing negligible
amounts of arsenic. Schering-Kahlbaum “pro analysi’’ sulfuric acid
was found suitable. The method can easily be adapted to use with a
photometer although this was not attempted.

Table II, containing representative data from the analysis of
samples of sea water, illustrates the procedure used in computing the
dissolved organic phosphate, and gives some indication of the de-
pendability of the methods. The total phosphorus in the samples
obtained by combining the phosphorus in the filtrate with the par-
ticulate phosphorus retained by the filter (column IV) is compared with
the total phosphorus in the water obtained by direct analysis without
filtration (column V). It is evident that some small losses result from
filtration, but these do not greatly exceed the normal variation of the
Deniges-Atkins method. The values for the particulate phosphorus
are small, but are consistent within themselves. The smaller values do
not greatly exceed that of the blanks employed and in one set of data
(for Station 2468, August 20, 1935) the values for the particulate
phosphorus are in doubt owing to uncertainty in the value of the blank.
The values for dissolved organic phosphorus in these samples are
consistently positive and larger than the apparent errors of the
method. During a large part of the year, as will be shown, much less
soluble organic phosphorus is present in the water. Since this fraction
is obtained by taking the difference of two large measures, each subject
to considerable errors, it sometimes eventuated at such times that
negative values were obtained for this fraction. The methods em-
ployed evidently do not give an exact measure of the distribution of the
phosphorus fractions, but they do serve to indicate the general magni-
tudes of the quantities in which each occurs.

The meaning of the particulate and dissolved fractions of organic
phosphorus depends upon the properties of the barium sulfate filter.
When a suspension of diatoms, Nitzschia closterium, containing a known
amount of phosphorus is filtered, the phosphorus may be recovered
quantitatively from the filter. The refiltration of a filtrate leaves no
detectable amount of phosphorus upon the filter. Evidently diatoms
and microorganisms of similar size are completely retained in the
particulate fraction. Dr. Charles E. Renn has kindly tested the filter
for the retention of bacteria. After filtering sea water containing some
400 bacteria per milliliter the filtrate contained about one-sixth that
amount. The particulate organic phosphorus fraction probably con-
tains the phosphorus of all the larger phytoplankton and smaller
zooplankton and the greater portion of the bacteria and detritus as
well. The dissolved organic phosphorus fraction may include a small

1)$)

428 REDFIELD, SMITH, AND KETCHUM

TABLE II

Specimen of data on phosphorus fractions in samples of sea water. Concentrations
expressed as milligrams PO, per cubic meter.

I II III IV Vv VI VII
Depth Total Total Organic
in Inorganic | Particulate | Phosphorus phos- phosphorus. phos-
meters phos- phosphorus in filtrate phorus Direct Difference phorus
phorus Il + Ill analysis Ill —I
1 45 10 64 74 62
70 80 86
68 78
67 77 71 + 6 22
10 27 13 64 Hil 68
60 73 86
64 77
63 76 Hdl — 1 36
20 39 14 66 80 86
70 84 86
45 59
60 74. 86 —12 21
60 110 6 116 130
116 — i 6
122 123
100 110 4 126 142
142
130 142 —12 — 16
125 115 5 132 138
146
137 142 — § 17
160
156
150 111 5 142 147 158 —11 31
200 123 5 152 154
162
157 158 — 1 29
225 108 8 148 162
158

156 160 — 4 40

CYCLE OF PHOSPHORUS IN GULF OF MAINE 429

portion of the bacterial flora, perhaps some minute nannoplankton,
and detritus which has been reduced to the smallest dimensions in
addition to organic compounds of phosphorus in colloidal form or in
true solution. Renn (1937) has estimated that a bacterial population
of 100,000 cells per milliliter would represent only 2.9 mg. PO, per
cubic meter. It is evident that the much larger values of dissolved
organic phosphorus obtained in our analyses can be due in only
negligible part to the presence of bacteria. We believe, consequently,
that our measurements represent chiefly the presence of phosphorus
compounds in solution.

TABLE III

Inorganic phosphorus. Concentrations expressed in milligrams PO, per cubic
meter. Depths not corrected for wire angle, which in no case would reduce by
more than 4 per cent.

Depth in May 18 August 21 November 8-9 February 26 May 14
meters 1935 1935 1935 1936 1936
1 35 5 45 105 14
10 28 Dall 27 102 10
20 34 53 39 103 15
30 59 96 78 99 82
40 70 92 73 100 95
50 — — 77 96 97
60 97 93 110 88 116
80 105 110 89 99 117
100 108 109 110 95 120
125 114 107 115 99 129
150 122 122 111 96 147
175 138 132 123 131 152
200 146 137 123 141 146
225 161 136 106 143 157
250 170 139 139

The distribution of phosphorus in the three forms into which it has
been separated is recorded in Tables III, IV, V and VI. Inorganic
phosphate represents by far the greater quantity of phosphorus in
the water amounting to 72 to 92 per cent of the total at different
times. It is rather uniformly distributed at depths greater than
80-100 meters throughout the year, the concentrations increasing
somewhat with depth. In the superficial layers, as has been frequently
observed elsewhere, the inorganic phosphate becomes greatly reduced
in quantity in the spring and is restored to concentrations characteristic
of greater depths during the winter.

Particulate organic phosphorus, representing organisms and
detritus, is in general the smallest of the three fractions, amounting
to about 5 per cent of the total. It occurs in greatest quantity in the

430 REDFIELD, SMITH, AND KETCHUM

upper layers—above 40 meters—corresponding to the observed
distribution of phytoplankton (Gran and Braarud, 1935). The quanti-
ties in these layers are highest in spring; in midwinter the quantity in
surface water is scarcely to be distinguished from that in deep water.
The values obtained in August are subject to doubt. Below the
photosynthetic zone the distribution of filterable organic phosphorus is
on the whole very uniform, amounting to about 5 y PO. per liter. The
distribution and magnitude of the concentrations of filterable phos-
phorus agree well with that of particulate nitrogen observed by von
Brand (1937) in these waters in the summer of 1936.

TABLE [IV

Particulate organic phosphorus. Concentrations expressed in milligrams PO,
per cubic meter. Depths not corrected for wire angle, which in no case would
reduce by more than 4 per cent.

Depth in May 18 August 21 November 8-9 February 26 May 14
meters 1935 1935 1935 1936 1936
1 20 13 10 6 17
10 18 14 13 5 13
20 15 15 14 5 20
30 20 7 9 4 12
40 12 8 7 4 0
50 — = 5 4 8
60 5 § 6 6 9
80 8 6 4 4 12
100 5 5 4 4 3
125 4 3 5 3 2
150 3 = 5 3 3
175 5 3 13 3 wa |
200 4 3 5 3 7
225 4 4 8 4 7
250 6 5

In May dissolved organic phosphorus occurred in only minimal
quantities in the water of all depths except near the surface. During
the summer the concentration increases markedly until November,
when over 20 per cent of the total phosphorus is in this form. In the
early winter there is a rapid disappearance of this form of phosphorus,
associated in time with the increase in concentration of inorganic
phosphate in the upper waters. The appearance of organic phosphorus
commences in May at the surface, and the concentrations appear to
grow from the surface downward. Not until November are high
concentrations observed near the bottom. These observations suggest
that considerable decomposition is taking place throughout the water
column and in particular in those depths where phyto- and zoéplankton

CYCLE OF PHOSPHORUS IN GULF OF MAINE 431

are known to exist in greatest numbers; and that at this station
decomposition at the bottom may be relatively unimportant. The
findings concerning organic phosphorus are somewhat similar to those
of Kreps and Osadchik (1933), who made studies in Barents Sea.
They found organic phosphorus to show a gradual increase from
August to January reaching concentrations of some 40 mg. P.O; per
cubic meter and actually exceeding the inorganic phosphorus during the
latter month. Their observations, which did not cover the earlier part
of the year, showed the greatest concentrations in the deeper waters.
As will be pointed out in a subsequent publication, the seasonal
distribution of soluble organic phosphorus has some resemblance to

TABLE V

Dissolved organic phosphorus. Concentrations expressed in milligrams POs,
per cubic meter. Depths not corrected for wire angle, which in no case would
reduce by more than 4 per cent.

Depth in May 18 August 21 November 8-9 February 26 May 14
meters 1935 1935 1935 1936 1936
1 30 49 21 — 2 12
10 15 36 35 0 0
20 —1 50 29 De, 35
30 —4 14 7 14 18
40 3 24 36 — 3 3
50 —_— = 43 5 3
60 8 36 7 1 26
80 0 24 50 9 55
100 2 30 24 Zyl 22
125 —3 29 20 21 22
150 y) 6 38 WM 9
175 —6 20 20 4 —11
200 —7 22 30 6 8
BOS) —2 17 43 — 4 10
250 8 —10

that of ammonia, which may be considered to be a somewhat analogous
stage in the nitrogen cycle.

The data recorded in Tables III, IV and V have been submitted to
further analysis with a view to determining in so far as possible, just
what alterations take place in the phosphorus cycle at various depths
and at different times of year.

The column of water is considered to be virtually a closed system in
which every exchange with the surroundings is exactly balanced by an
equal and opposite exchange. By dividing the column into a number
of segments lying at different depths, in which the quantity of phos-
phorus in the different forms is recorded from time to time, and by

432 REDFIELD, SMITH, AND KETCHUM

observing certain general biological and hydrographic principles, it
becomes possible to estimate to what extent changes in the concen-
trations of each form of phosphorus may be derived from processes
taking place in situ, and to what extent vertical movements of phos-
phorus from one segment or layer to another must be postulated.

To obtain a workable body of data, Table VI has been drawn up
recording the quantity of phosphorus present in each of the three

TABLE VI

Summary of distribution of phosphorus fractions.

Depth in | May 18 | Aug. 20 | Nov. 8 | Feb. 26 | May 14
meters 1935 1935 1935 1936 1936

Total Pas grams PO: per sq. m. 0-240 | 28.7 31.0 32.9 29.9 34.4

Percentage of total phosphorus 0-60 16.4 20.6 18.0 22.0 14.9
in each 60-meter layer 60-120 | 23.7 23.2 25.0 23.0 28.0
120-180 |. 27.0 26.3 28.0 25.0 27.9

180-240 | 32.9 29.9 29.0 30.0 29.2

Total 100.0 | 100.0 | 100.0 | 100.0 | 100.0
Inorganic phosphorus as per- 0-60 11.9 12.6 11.3 19.7 10.6
centage of total 60-120 | 22.1 16.8 18.7 19.4 20.8
120-180 | 26.0 7 ess) 21.6 21.1 25.0
180-240 | 32.0 25.6 Dee, RST 26.5

Total 92.0 ies 72.8 88.9 82.9

Dissolved organic phosphorus 0-60 1.5 6.2. 5.0 1:4. \> 23
as percentage of total 60-120 0.4 5.4 5.4 Dea 6.0
120-180 0.2 3.2 5.0 3.3 2.5

180-240 0.0 3.7 6.4 0.6 1.7

Total 2.1 18.5 21.8 8.0 1225

Particulateorganicphosphorus| 0-60 3.0 1.8 ier 0.9 2.0
(organisms and detritus) as| 60-120 1.2 1.0 0.9 0.9 1.2
percentage of total 120-180 0.8 0.6 1.4 0.6 0.4

180-240 0.9 0.6 1.4 0.7 1.0

Total 5) 4.0 5.4 Sell 4.6

forms for each of four layers each of 60 meters depth at each time of
observation. ‘The values are obtained by graphical integration and are
expressed as percentages of the total phosphorus in a water column of
240 meters depth at each time of observation. In analyzing these data
the following premises are held:

1. The horizontal exchange due to the drift of water past the station
may be neglected. ‘This premise is not justified on the ground that the

CYCLE OF PHOSPHORUS IN GULF OF MAINE 433

station is located in a region of minimal drift. There can be little
doubt that water is constantly drifting past the station. The observed
changes in salinity demonstrate this. The total phosphorus recorded
varies + 7 per cent from the mean value throughout the year. Since
this variation shows no seasonal sequence, the total phosphorus being
lowest in May, 1935 and maximal in May, 1936, there evidently is
some variation in the character of the water occupying the station at
different times. These differences are eliminated by expressing the
phosphorus fractions as percentages of the total, a procedure which
imposes artificially the character of a closed system upon the set of
data. The justification of this procedure lies in the relatively small
differences in total phosphorus observed from time to time, and in the
fact that on the whole the horizontal distribution of phosphorus
throughout the Gulf at any time, at least to judge by inorganic POs, is
much more uniform than is the vertical distribution. The horizontal
distribution of PO, at various depths throughout the Gulf as observed
in May, 1934 are shown in Fig. 4 and illustrate this fact. The general
character of the phosphorus cycle may be supposed to be similar in all
parts of the basin.

2. Phosphate present as zodplankton and nekton and not sampled by
the water bottle may be ignored. A large number of vertical zodplankton
hauls made throughout the years 1933-34 in all parts of the Gulf
yielded an average catch of 40 cc. dry plankton per square meter of
surface. It may be estimated from analyses made on such material
that this would contain about 0.4 per cent of the total phosphorus in
the water from which it was strained. Since the particulate organic
phosphorus amounts to about ten times this quantity, it may be seen
that the neglect of this fraction does not introduce a significant error.

3. All synthesis of particulate or soluble organic phosphorus com-
pounds from inorganic phosphate takes place in the upper layer. This
is justified by Clarke’s measurements on the penetration of light into
the Gulf of Maine and on determinations of the compensation point in
photosynthesis by diatoms in bottle experiments at different depths
(Clarke and Oster, 1934).

4. All downward movement of phosphorus is due to the sinking of
organisms (particulate organic phosphorus). ‘This is the only fraction
affected by gravity. It is also the only fraction displaying a well-
marked concentration gradient decreasing downward—a condition
essential for downward dispersal by eddy conductivity.

5. All upward movement of phosphorus is due to the transport of
inorganic PO. by eddy conductivity. The gradient of concentration of
inorganic PO, increases downward and is well marked except in mid-

434 REDFIELD, SMITH, AND KETCHUM

winter. The soluble organic phosphorus never develops a strong
gradient in this direction. Since it is present in much smaller concen-
trations than is the inorganic phosphate, it may safely be ignored in
considering vertical transport by eddy conductivity.

6. All observed transformations in any layer are attributed to processes
occurring in that layer, except so far as vertical transport must be postulated
to account for the transformation. ‘This premise is introduced since
without it a unique solution cannot be obtained. It implies that all
values arrived at for vertical exchange are minimal.

7. The portion of the exchange in which the cycle runs to completion 1s
necessarily ignored. All values for the magnitude of the exchange are
ea minimal.

Fic. 2. See text.

A consideration of this limitation may serve to make clear the
general basis of the analysis. If we start with the system in a steady
state and if, during the period between two sets of observations the
cycle has proceeded without change in the relative velocity of the
processes in any part, the distribution of the fractions of phosphate in
all parts of the system will be the same at the end as it was in the
beginning. This does not mean that no exchanges of phosphate
between different parts of the system have taken place, rather that all
exchanges are exactly compensated. While the system is in a steady
state an unobservable quantity of phosphate is undergoing transfor-
mation from each stage in the cycle to the next stage. If the system is
disturbed, as through seasonal changes in the physical conditions, then

CYCLE OF PHOSPHORUS IN GULF OF MAINE 435

transformations of one sort may proceed more rapidly than those of
another with the result that differences in the distribution of phos-
phorus are observed, and from these differences the magnitude and
nature of the processes which have caused the differences may be
deduced. The observations tell us nothing, however, of the basal level
of activity on which the differences are superposed.”

Figure 2 illustrates the principle of the method. At any time there
will be a basal level of activity represented by the transformation of an
unobservable quantity of phosphorus, x, through each stage in the
cycle. This quantity will have been transported upward from layer 2
into layer 1 to be synthesized into particulate organic form. If the
system is to remain unchanged, the equivalent of this material must
have sunk back into the deeper layers and been decomposed, passing
through the soluble organic form, to exactly replace that which was
transported upward. Portions of x, designated as x, ¥2, etc. may sink
to deeper layers before undergoing transformations from organic to
inorganic form. ‘The general conditions are that the quantity of x
entering and leaving any part of the system shall be equal, that « move
upward as inorganic phosphate (Postulate 5), and downward as
filterable organic phosphorus (Postulate 4), that it represent synthesis
of filterable organic phosphorus only in the upper layer, and that it
represent a transformation of organic into inorganic phosphorus in any
layer.

If the system is disturbed between observations, then changes in the
quantity of phosphorus, AY, in any form and part of the system may be
observed. These changes may be accounted for only by additional
exchanges between the various parts of the system. The problem is to
determine the minimal additional exchanges of this sort, Y1, Y2, Ysetc.,
which will account for the change in each part of the system in ac-
cordance with the postulates laid down above. The general conditions
are that AY, the change in any fraction in any layer shall equal the
difference in Y;, Yo, Y3 etc., the amounts of phosphorus entering or
leaving that fraction and layer during the period between observations.
Furthermore, AY must be accounted for so far as possible by exchanges
taking place within the layer in question (Postulate 6).

An attempted analysis of the changes in phosphorus distribution is
presented in Tables VII, VIII and IX.

February to May (Table VII) represents the period in which the

2The method is analogous to the integration of a differential equation, the
unobserved basal activity corresponding to the constant of integration. It is only in
proportion as the system undergoes great seasonal fluctuation that the partial effects

observed approach the total exchanges taking place. The method is applicable
consequently particularly to studies made in high latitudes.

436 REDFIELD, SMITH, AND KETCHUM

great spring flowering of phytoplankton occurs. During this period an
amount of phosphorus equivalent to 9.1 per cent of the total disappears
from the inorganic phosphate of the upper layer. Of this only 2.0
per cent can be accounted for as an increase in particulate and soluble
organic phosphorus remaining in that layer. Seven and one-tenth per
cent must have sunk to the deeper layers following its synthesis into
organic matter. Only one-eighth of the phosphorus absorbed in

TABLE VII

Balance sheet of phosphorus exchanges February 26 to May 14, 1936. Numbers
represent the change in the phosphorus fractions as percentages of total phosphorus
in water column.

: Soluble Particulate
I 5 :
Depths phosphorus ahgchare Shosphorae
Photosynthesis —9.1 —> +9.1
Decomposition +0.9<— —0.9
0-60 0 =< Qo
meters Exchange with layer below 0 0 —7.1
Net change —9.1 +0.9 +1.1
Exchange with layer above 0 0 +7.1
60-120 Decomposition +4.7 <— —-4.7
meters +1.4 <— —1.4

Exchange with layer below 0 en) — eal
Net change +1.4 +3.3 +0.3
Exchange with layer above 0 0 +2.1
120-180 Decomposition +0.9 <— —0.9

meters +1.7 <— —-1.7
Exchange with layer below +2.2 0 —1.4
Net change +3.9 —0.8 —(0.2
180-240 Exchange with layer above —2.2 0 +1.4
meters Decomposition 0 +1.14<— —-1.1
Net change aed +1.1 +0.3

photosynthesis has remained as particulate matter in the upper layer.
This observation accords with the conclusion of Harvey (1934) that
several times more vegetation is produced during the spring flowering
of diatoms in the English Channel than is found there at the time of its
maximum.

To account for the increasing concentrations of inorganic phos-
phorus in the deeper layers after allowing for the greatest possible
decomposition in situ at least 2.1 per cent must sink past the 120-meter

CYCLE OF PHOSPHORUS IN GULF OF MAINE 437

level and 1.4 per cent past the 180-meter level. The phosphorus
removed from inorganic form in the upper layer by photosynthesis is
redistributed during the spring through considerable depths by the
sinking of particulate matter. It is unnecessary to assume that any of
the particulate matter sinks beyond the lower level before undergoing
decomposition, though it is possible that this may be the case.

May to November (Table VIII) includes the greater part of the

TABLE VIII

Balance sheet of phosphorus exchanges May 18, 1935 to November 8, 1935.
Numbers represent the change in the phosphorus fractions as percentages of total
phosphorus in water column.

: Soluble Particulate
I c :

a phosphorus | ,,o°85RiC, | organs

0-60 Photosynthesis —19.2 — > +19.2

meters Decomposition 4+3.5<— —3.5
0 =< oO

Exchange with layer below +18.6 0 —17.0

Net change —0.6 +3.5 —1.3

60-120 Exchange with layer above —18.6 0 +17.0

meters Decomposition +5.0 <— —5.0
0 < 0

Exchange with layer below +15.2 0 —12.3

Net change —3.4 +5.0 —0.3

120-180 Exchange with layer above —15.2 0 +12.3

meters Decomposition +4.83<— —4.8
0 < 0

Exchange with layer below +10.8 0 —6.9

Net change —44 +4.8 +0.6

180-240 Exchange with layer above —10.8 0 +6.9

meters Decomposition +64<— -—64

Net change —10.8 +6.4 +0.5

growing season. The important feature of this period is the appear-
ance of large quantities of dissolved organic phosphorus at all depths.
One-fifth of all the phosphorus in the water is in this form in November.
This material can have been produced only by photosynthetic processes
taking place in the upper layer. It must have been set free for the
most part by decomposition of the particulate fraction in the layer in
which it is observed (Postulate 5). In order to account for the

438 REDFIELD, SMITH, AND KETCHUM

quantities of dissolved organic phosphorus observed, a large vertical
movement of inorganic phosphorus upward through all depths must be
postulated as well as an equivalent sinking of organisms to the sites at
which the soluble organic phosphate appears. Over 17 per cent of the
total phosphorus in the water must pass through the zone of photo-
synthesis in the course of the six summer months. Since the account is
balanced without supposing any phosphorus to pass from the organic

TABLE IX

Balance sheet of phosphorus exchanges November 8, 1935 to February 26, 1936.
Numbers represent percentages of total phosphorus in entire water column.

: Soluble Particulate
I : -
ash phosphorus | oa. | pram
Photosynthesis 0 ==> 0
Decomposition +0.6<— —0.6
0-60 +4.2 <— —4.2
meters Exchange with layer below +4.2 0 —0.2
Net change +8.4 —3.6 —0.8
Exchange with layer above —4,2 0 +0.2
Decomposition 0 <— 0
60-120 +2.7 <— —-2.7
meters Exchange with layer below +2.2 0 —0.2
Net change +0.7 —2.7 0
Exchange with layer above —2.2 0 +0.2
120-180 Decomposition 0 <— 0
meters +1.7 <— —-1.7
Exchange with layer below 0 0 —1.0
Net change —0.5 —1.7 —0.8
Exchange with layer above 0 0 +1.0
180-240 Decomposition +1.7<— -—-1.7
meters +7.55 <— —7.5
Net change +7.5 —5.8 —0.7

back to the inorganic form, a process which must certainly be taking
place, this figure may be far below that actually obtaining.

November to February (Table IX) is marked chiefly by the re-
generation of inorganic phosphorus, which increases by 16 per cent of
the total, and by the equalization of the concentration of this fraction
throughout the water column. The table shows that this regeneration
is made to a large extent at the expense of the soluble organic phos-

CYCLE OF PHOSPHORUS IN GULF OF MAINE 439

phorus, that the transformation takes place throughout the entire
_ range of depths, though greatest near the bottom. The decomposition
of organic phosphorus compounds in situ and the vertical transport of
inorganic phosphate are about equally important in effecting the
equalization of the concentration of the latter throughout the water
column.

THE MECHANISM OF VERTICAL TRANSPORT

The foregoing analysis indicates that very considerable exchanges of
phosphorus take place between various depths of water. At the same
time these exchanges appear to diminish in extent as the depth in-
creases. Downward movement has been attributed to the sinking
of particulate matter under the influence of gravity. There appears to
be no difficulty in considering that in depths of a few hundred meters
organized particles of the dimensions of diatoms would sink to the
bottom before undergoing decomposition. If such were the case very
large quantities of phosphorus would be withdrawn from the water
during each growing season. This may be the case in shallow waters,
but it does not appear to be happening in the western basin of the Gulf
of Maine. If so the total phosphorus in the water should show a
marked seasonal change. The situation is probably complicated by
biological considerations. Harvey (1934) has presented evidence that
the stock of phytoplankton is grazed down by zodplankton during the
summer. This conclusion suggests that the zodplankton are important
active agents in converting particulate organic phosphorus into its
decomposition products. Since these animals, and particularly the
copepods, make extensive diurnal vertical migrations, and since some
time must elapse between the taking of food near the surface and its
elimination as waste products, they provide an agency for a limited
vertical transport of organic material. From this viewpoint the
zooplankton become an important agency in maintaining the fertility
of the water for phytoplankton, since they hasten the conversion of
bound nutrients into inorganic form and prevent these nutrients from
becoming unavailable by the sinking of particulate matter to great
depths or to the bottom.

The vertical transport of inorganic phosphate is simpler since it
can be effected only by the mixing of the water. It is pertinent to
inquire whether the conditions are such as to permit of the amounts
of transport deduced during the various periods of observation. The
amount of a constituent, Q, passing through unit horizontal surface in
unit time depends upon the gradient of concentration of the constituent

440 REDFIELD, SMITH, AND KETCHUM

dc/ds and the coefficient of eddy conductivity, A (Austausch coeffi-
cient).

QO = Adc/ds

The coefficient of eddy conductivity, A, represents the volume of water
exchanged through each horizontal unit surface in unit time.

The gradient of phosphate concentration observed in February,
May and November is shown in Fig. 3. Between May and November,
when large vertical movements have been deduced, a well-marked
gradient exists particularly in the upper layers, as is required for such
movements. In February this gradient has disappeared, and the

fo)
30
60
90
120
150 =
180

210

NOVEMBER FEBRUARY
240

60 80 100 120 140 160 80 100 120 140

Fic. 3. Distribution of inorganic phosphate concentration with depth in
November, 1935 and February and May, 1936 at standard station in the western
basin of the Gulf of Maine. Depths in meters measured downward along the ordi-
nate; concentrations in milligrams PO, per cubic meter along the abscissa.

concentration of phosphate is equal at all depths down to 150 meters.
No amount of mixing can effect a change in its vertical distribution.
Since this condition must exist during a considerable portion of the
winter, it is not surprising that the vertical transports deduced from our
data between November and May are smaller than those observed in
the summer.

A knowledge of the coefficient of eddy conductivity, A, is the key
to understanding the nutritive conditions in deep bodies of water.

CYCLE OF PHOSPHORUS IN GULF OF MAINE 441

Methods of estimating its value are so indirect that little is known of
its magnitude under any circumstances. Although our data are
admittedly very unprecise and yield only minimal values for the
exchange, it is nevertheless of some interest to use it in estimating
the coefficient required to account for the transport of phosphate in the
Gulf of Maine. The gradient of phosphate concentration is sufficiently
uniform throughout the period May—November to permit a single
value to be taken at any level as representative of the entire period,
This is not the case during the remainder of the year. We have

(0)
30 MAY

(e)

NOVEMBER
90
120
150
180

210

240 FEBRUARY

25.0 25.5 26.0 265 27.0 26.0 265 270

Fic. 4. Distribution of density, o:, with depth in November, 1935, and Febru-
ary and May, 1936, at standard station in the western basin of the Gulf of Maine.
Depths in meters measured downward along the ordinate; density, o:, measured along
the abscissa.

calculated the value of A for the boundary of each of the layers, using
the data presented in Table VIII for estimating Q and the slopes of
the curves in Fig. 3 for dc/ds. The result is shown in Table X. The
values of A are minimal and are less reliable at the greater depths.
The values obtained are not unreasonable. Seiwell considered A to
equal 2 C.G.S. units in the thermocline of the tropical Atlantic, whereas
values increasing to 50 C.G.S. units have been obtained in various
waters (Seiwell, 1935).

442 REDFIELD, SMITH, AND KETCHUM

The value of the coefficient of eddy conductivity depends upon the
forces responsible for mixing and varies inversely with the stability,
oo of the water. If K represent the mixing forces,
| des

Seiwell considers that K = 4.73 X 10-4 in the thermocline of the
North Atlantic. Figure 7 shows the distribution of density, o:, with
depth at the station in the Gulf of Maine in May, 1935 and in No-
do t
ds
in Table X are taken. The values of K given by multiplying =

K=AX

vember. From these curves the representative values of entered

by A are of the order obtained by Seiwell. It is concluded that the

TABLE X

Estimation of coefficient of eddy conductivity in Gulf of Maine,
May to November, 1935.

Depths Q Felis A dox|ds Haran et
per cent in mg. mg. ml. grams
meters 3 months cm.2 X sec. ml. cm. cm. X sec. | ml. X cm.
60 >18 >3.5X10- | 6.610%] > 5.2 | 1.01074) >5.2x10~4
120 >15 >2.4<107-8 | 2.0X10-9 | >12.0 | 0.51074 | >6.010%
180 >10 >2.010-8 | 3.0X1079 | > 6.6 | 0.4X104 | >2.6x104

vertical transport of inorganic phosphate deduced from the seasonal
change in the distribution of the various fractions of phosphorus
compounds does not require unreasonable assumptions concerning the
magnitude of the eddy conductivity.

SUMMARY

1. Methods are described for the determination of the phosphorus
present in particulate form and of the total phosphorus in a sample of
sea water.

2. The distribution of phosphorus present as inorganic phosphate,
as dissolved organic compounds, and as particulate matter (detritus
and microérganisms) has been determined at all depths throughout
the year at a station in the western part of the Gulf of Maine.

3. In late winter over 90 per cent of the phosphorus is in inorganic
form and three-quarters of the remainder is present as soluble organic
compounds.

CYCLE OF PHOSPHORUS IN GULF OF MAINE 443

4, In the spring—February to May, inorganic phosphorus is con-
verted to organic form by photosynthesis in the upper layer of water.
Most of this fraction sinks to considerable depths before undergoing
decomposition.

5. During the summer—May to November, large quantities of dis-
solved organic phosphorus appear at all depths, indicating a very con-
siderable transport of inorganic phosphate from deep water to the
surface and the sinking of an equivalent amount of phosphorus in
particulate form to the depths in which organic compounds are
liberated by decomposition. Decomposition appears to take place
throughout the water column.

6. During the winter—November to February, the organic phos-
phorus compounds are converted to inorganic phosphate. This and
vertical mixing of preformed phosphate are about equally important in
bringing about the equalization of phosphate concentrations through-
out the depth of water.

7. A method is described for analyzing quantitatively the factors
producing a seasonal change in the distribution of a compound such
as phosphorus. It is shown that the vertical transport of material
within the water mass demanded by such an analysis may be accounted
for reasonably by the hydrographic conditions obtaining.

8. Values of the coefficient of eddy conductivity at several depths
are obtained.

BIBLIOGRAPHY

VON BRAND, T., 1935. Bol. Bull., 69: 22.

Von BRanp, T., 1937. Biol. Bull.,'72: 1.

CLARKE, G. L., AND R. H. OsTER, 1934. Biol. Bull., 67: 59.

Cooper, L. H. H., 1934. Jour. Mar. Biol. Ass’n, 19: 755.

Gran, H. H., anp T. BRAARuD, 1935. Jour. Biol. Bd., Canada, 1: 279.

Harvey, H. W., 1934. Jour. Mar. Biol. Ass’n, 19: 755.

Kreps, E., AND M. Osapcuik, 1933. Intern. Rev. Hydrobiol. und Hydrograph., 29:
Beile

RENN, C. E., 1937. Biol. Bull., 72: 190.

Roginson, R. J., AND G. KEMMERER, 1930. Trans. Wisconsin Acad. Sci., 25: 117.

SEIWELL, H. R., 1935. Papers in Physical Oceanography and Meteorology. Vol. 3,
No. 4, pp. 1-56.

ZINzIDZE, Cu., 1935. Ind. and Eng. Chem., Anal. Ed., 1: 227.

GROWTH AND VARIABILITY IN DAPHNIA PULEX

BERTIL GOTTFRID ANDERSON, H. LUMER AND L. J. ZUPANCIC, JR.

(From the Biological Laboratory, Western Reserve University)

INTRODUCTION

The aim of this study is to determine the number of pre-adult
instars, growth, relative growth, and variability of individually reared
female Daphnia pulex De Geer.

Numerous studies on growth of Cladocera have been made. Most
of them are based on size-frequency distributions in natural popula-
tions. Some are based on experiments with individually reared
animals. These studies have been carried on largely by Woltereck
and his students, especially Rammner. Size-frequency distribution
methods are inadequate for growth determinations as will be brought
out more fully later. Previous studies carried out by means of indi-
vidually reared animals deal with small numbers of organisms, a
short part of the possible life span of an individual, or both. Recently
Banta and his co-workers have carried out intensive studies on large
numbers of individually reared Daphnia longispina which lived for
long periods. In the present study a large number of animals has
been observed for a relatively long time.

PROCEDURE

Essentially the same procedure was used as that employed by
Anderson (1932) for Daphnia magna. Individual female Daphnia
pulex of a single clone were isolated within eight hours after their
release from their mothers and reared in manure-soil medium (Banta,
1921). Each individual was placed in a separate glass vial containing
20-25 cc. of the medium. Semi-weekly one-half of the total volume
was replaced with fresh medium. The animals were kept at room
temperature (15°—22° C.).

At the time of isolation and daily thereafter, each individual
animal was placed in a watch glass together with a few drops of
culture medium. Just enough saturated chloretone solution was
added to bring about cessation of movement. Measurements as
shown in Fig. 1 were made by means of an ocular micrometer. The
animals were never in the chloretone solution for more than five

minutes at any one time.
444

GROWTH AND VARIABILITY IN DAPHNIA 445

Immediately before the daily mensurations note was taken of cast
carapaces and the number of young released. These were removed
at the time of their discovery.

RESULTS AND DISCUSSION
Longevity
Some 82 animals were observed in these experiments. Figure 2 is
a survival curve. Fifty-one animals lived for twenty instars and of
these, 3 continued to the twenty-fifth instar. Bourguillaut de Ker-
herve (1926) observed two Daphnia magna for nineteen instars. Each
one released nineteen clutches of young and since not more than one
clutch of young is released each instar and at least five instars precede
the release of the first clutch of young, these two Daphnia magna lived

Fic. 1. Diagram showing method of making measurements. T, total length,
longest dimension of animal exclusive of spine. C, carapace length, longest dimension
of the carapace exclusive of spine. H, height, the shortest distance between two
lines tangent to the carapace and parallel to the line of T. This measure of height is
affected very little by the number of young in the brood chamber.

for twenty-four instars or more. He cites 4 other individuals which
must have reached the twenty-second, the thirteenth, the tenth, and
the sixth instar, respectively. Anderson (1932) observed some 30
Daphnia magna for fourteen instars, 32 for thirteen instars, and others
for a smaller number of instars, all from the time of release from the
mothers. Rammner (1928) observed one Scapholeberis mucronata for
seventeen instars and another for nine. Rammner (1929) cites others
that have been observed for shorter times. Ingle, Wood, and Banta
(1937) have observed individually reared Daphnia longispina for over
twenty-five instars.

Of the 82 animals observed in the present study, 71 were
primiparous during the fifth instar and 9 during the sixth, while the
remaining 2 died during the second and third instars. The number of

446 ANDERSON, LUMER, AND ZUPANCIC

pre-adult instars is therefore variable in this species as well as in others
(Anderson, 1932). The minimum number of pre-adult instars for
Daphma pulex is probably four.

Of the 71 animals primiparous during the fifth instar, 47 lived
through the twentieth instar. The data from these 47, summarized
in Table I, are the basis for the growth studies which follow.

Absolute Growth

Figure 3 is a group of growth curves in terms of total length,
carapace length, and height. The curves are similar in shape. The

ap pe Too] pe To

NUMBER OF INDIVIOUAL S
w
S

a 10 15 £0 £5
INSTAR

Fic. 2. Survival curve for the eighty-two animals observed in these experiments.

point of inflection in each curve comes immediately before the instar
during which the animals are primiparous. Attempts have been made
to fit the Robertson and Gompertz equations to the curves. In
neither instance was the result judged satisfactory. The time unit
used in this work is the instar. Adult instars have been considered
as equivalent physiological time units (Anderson, 1933). At room
temperature, each of the first three pre-adult instars lasts approxi-
mately one day, the fourth or last pre-adult instar one and a half days,
the first adult instar about two days, and each subsequent instar
becomes increasingly longer. The use of the instar as a time unit

GROWTH AND VARIABILITY IN DAPHNIA 447

may be the cause of unsatisfactory results in attempting to fit the
equations to the present data.

When the logarithms of total length are plotted against time in
instars, a curve is obtained which may be broken up into three distinct
segments each of which approximates a straight line. The first seg-
ment takes in the first five instars, the second segment includes the
next five, and the third segment embraces instars eleven to twenty.

Figure 4 shows the growth increments in terms of total length,
carapace length, and height. The increments increase up to the fourth
instar then gradually decrease until the eleventh instar, after which
they remain much the same.

It is of interest to determine to what degree such characteristics of

MM.

Ae
7

20
C

1S
Hf

5 /0 (5-29
WSTAR

Fic. 3. Growth curves based on data from the 47 animals which were pri-
miparous in the fifth instar and lived a twenty instars or more. JT, total length;
C, carapace length; H, height.
the growth process as initial size, final size, duration of growth, and
initial velocity of growth are interdependent. As measures of these
quantities, total length in the first instar (a), total length in the
twentieth instar (A), the number of instars required to attain a length
of approximately 0.8A (¢), and the increment between the first and
second instars (7), respectively, were employed. For these the follow-
ing coefficients of correlation were obtained:

toa = 0.2309 = 0.0931

fai = — 0.4481 + 0.0792
ra; = — 0.1483 + 0.0963
fat = 0.1641 + 0.0957

— Wiss) se WH038S

=
fe
I

448 ANDERSON, LUMER, AND ZUPANCIC

It is apparent that only 7,; and ra; are significantly different from
zero. ‘There is thus an indication of an inverse relationship between
initial size and initial velocity of growth, and between duration of
growth and final size. On the other hand, there is no evident relation-
ship between initial and final size, between initial size and duration
of growth, or between initial velocity and final size.

Similar coefficients of correlation have been computed for Daphnia
longispina by Wood and Banta (1936). These authors found that with
unlimited food early growth tends to be inversely related to size at

mm. rv par ed

025

020

QI5
0.10

O05

5 Ogee Ti 1.20
INSTAR

Fic. 4. Growth increment curves for the same animals as in Fig. 3. T, total
length; C, carapace length; H, height. —

the time of release, but that when the quantity of food is limited
these factors vary independently. They found also that initially
larger animals tend to become larger adults, which is contrary to the
results obtained from our data.

A negative correlation between initial size and initial growth rate
has been found to occur also in certain mammals. Thus it has been
observed that in man (Hammett, 1918) and in the cat (Hall and
Pierce, 1934) the percentage increase in body weight in the period
immediately following birth is inversely proportional to birth weight.

GROWTH AND VARIABILITY IN DAPHNIA 449

Hammett (1918) interprets these results as indicating that differences
in birth weight correspond to differences in physiological age at the
time of birth, the physiologically younger individuals having the
greater percentage growth rate.

In multiparous mammals the situation is often complicated by
variation in litter size and attendant differences in food supply of the
sucklings. Crozier and Enzmann (1935) showed that in inbred
albino mice there is a hyperbolic relationship between birth weight and
litter size, also that individuals in large litters grow more slowly at
first than those in small litters, due to the decreased quantity of
available milk per animal. After the suckling period, however, the
growth of the smaller individuals is accelerated so that they eventually
catch up with the others, and all attain the same adult weight regard-
less of birth weight.

In order to eliminate differences in nutrition, Kope¢ (1932),
working with a non-inbred stock of mice, reduced all litters to the
same size. He found that birth weight varied inversely with litter
size, and that individuals born in large litters exhibited a higher initial
percentage growth rate than those born in small litters. When the
data were seriated on the basis of birth weight without regard to litter
size, however, there was found to be no correlation. Kope¢, who
regards the different size groups in the second case as representing
different genetic types, concluded that genetic differences in birth
weight have no effect on subsequent growth, as do environmental
differences. The validity of this argument seems open to question.
It would be interesting in this connection to determine whether or not
the same results occur with an inbred stock, also to investigate similarly
the effects of brood size in Cladocera.

The occurrence of a significant negative correlation between dura-
tion of growth and final size in the present case appears to be in line
with the results of McCay, Crowell, and Maynard (1935). These
authors observed that in white rats, individuals whose growth period
has been prolonged by partial starvation attain on adequate feeding a
final size lower than that of controls given sufficient food throughout.
Ingle, Wood, and Banta (1937), however, in a similar experiment on
Daphnia longispina, found practically no difference in final size between
the experimental animals and the controls. Also Merrell (1931)
found that in rabbits there is no significant correlation between the
time required to reach one-half the adult weight and the adult weight
attained. On the other hand, Merrell’s observation that initial growth
rate is not associated with adult weight is in agreement with our
results.

450 ANDERSON, LUMER, AND ZUPANCIC

Reproduction

Figure 5 gives the average number of living young released during
each instar. The number increases until the tenth instar followed by
a gradual decrease. The period of increase corresponds to the time
when the growth is falling most rapidly. The period of decrease
corresponds to the time when growth has practically ceased.

The number of young released by a daphnid during any one instar
has been used as an index of its condition (Anderson, 1933). The
data available in the present study may serve as a test for this assump-
tion. The young released in any instar develop from eggs produced
during the instar before. Consequently, the coefficient of correlation
must be computed from the growth increment with respect to total

£9)

NUMBER OF YOUNG
S S SS

o /0 15 £0
INSTAR

Fic. 5. Curve of the average number of living young released during each instar
by thesame animals as in Fig. 3.

length for instar five and the number of young released in instar six, etc.
The coefficients for the fifth, sixth, and tenth instars were determined.
These are .690 + .025, .649 + .028, and .645 + .030, respectively.
Hence one may conclude that the number of young produced may
serve as a valid index of the condition of a daphnid.

Relative Growth

Relative growth studies show certain characteristics of this species.
In Fig. 6 the logarithms of carapace length were plotted against those
of total length, those of height against those of total length, and those
of carapace length against those of height.. The relation of carapace
length to total length can be expressed satisfactorily on this log log

GROWTH AND VARIABILITY IN DAPHNIA 451

plot by three straight lines. The first of these can be drawn through
the points for the pre-adult instars (1-4), the second through the
points for the next eight instars (5-12) and the third through the last
eight instars (13-20) represented. The relations between height and
total length are similar to those between carapace length and total
length. The relations between carapace length and height are dif-
ferent. Two lines may be used, one for the first ten instars and a
second for the last ten (11-20). These linear relations can be ex-

pressed as
Ve DI

& ON Ste S

J SD IO Nh ALLE. LS A 3
mm.

Fic. 6. Double logarithmic plots of the relations between carapace length
and total length C/T, height and total length H/T, and carapace length and height
C/H during each instar for the same animals as in Fig. 3.

where 6 is a constant—the initial growth index, a is the equilibrium
constant, and x and y are values of two parts (Huxley and Teissier,
1936). The values of the constants are given in Table II. These
values were calculated by the least squares method.

The relationships shown above are much the same as those secured
for Daphnia magna by Anderson (1932). No very marked change in
proportions occurs as may be seen in Fig. 7. The marked change in
relative growth that occurs at the twelfth and thirteenth instars as
indicated in Fig. 6 can hardly be recognized by inspecting Fig. 7 alone.
The lines representing the thirteenth to the twentieth instars in Fig. 6
are very short since growth is very much retarded during this period.

452 ANDERSON, LUMER, AND ZUPANCIC

TABLE [

Mean values of total length, carapace length, and height and their probable errors
in millimeters for each instar. Also the standard deviation (c) and the coefficient of
variation (V) for total length.

Standard Coefficient
deviation of variation

1 | 0.5665 +0.0034 | 0.0347 +0.0024 | 6.1340.43} 0.4194 +-0.0027| 0.3056+0.0021
2 |0.7214+0.0038 | 0.0389 +0.0027 | 5.39-0.37| 0.5361 +0.0034| 0.4015 +0.0027
3 | 0.9609 =+0.0056 | 0.0568 0.0040 | 5.92 +0.41] 0.7244 +0.0048) 0.5433 +0.0039
4 | 1.2653+0.0092 | 0.0934+0.0065 | 7.38+0.51] 0.9848 +.0.0078} 0.7346+0.0054
5 | 1.6079=+0.0095 | 0.0964-+-0.0067 | 6.00+0.42) 1.2850+0.0082) 0.9597 +0.0059
6
7
8
9

Instar Total length Carapace length Height

1.8134+0.0106 | 0.1081 0.0075 | 5.96+0.41| 1.4572 +0.0097| 1.1015 0.0084

1.9332 0.0117 | 0.1196+0.0083 | 6.19+0.43| 1.5591 =+0.0110) 1.1778 0.0094

2.0571 +0.0130 | 0.1316+0.0092 | 6.40-+-0.45| 1.6615 +0.0111) 1.2445 +-0.0096

2.1673 +0.0126 | 0.1275 +0.0089 | 5.88--0.41| 1.7587 +0.0110} 1.3213+0.0090
10 | 2.2466+0.0116 | 0.1174+0.0082 | 5.23+0.36) 1.8200+0.0103] 1.3738--0.0088
11 | 2.3061 +0.0108 | 0.1095 +0.0076 | 4.75+0.33] 1.8653 0.0093) 1.3976+0.0083
12 | 2.3401+0.0099 | 0.1007 =-0.0070 | 4.31--0.30| 1.8891 +0.0088} 1.4131+0.0071
13 | 2.3705 0.0092 | 0.0939 +0.0065 | 3.96+0.28) 1.9034+0.0081| 1.4227 0.0066
14 | 2.3853+0.0095 | 0.0962 +0.0067 | 4.03+-0.28) 1.9135 0.0081} 1.4262 0.0071
15 | 2.4032+0.0092 | 0.0935 0.0065 | 3.89+0.27/ 1.9326+0.0075| 1.4274+0.0065
16 | 2.4223+0.0085 | 0.0866-+0.0060 | 3.58+0.25} 1.9433+0.0072| 1.4274+0.0053
17 | 2.4461+0.0072 | 0.0736=-0.0051 | 3.01+0.21] 1.9594 +0.0055} 1.4340 +0.0047
18 | 2.4604+0.0072 | 0.0730+0.0051 | 2.97+0.21] 1.9689 0.0060} 1.4340 0.0046
19 | 2.4835+0.0072 | 0.0730+0.0051 | 2.94--0.20| 1.9862 0.0061) 1.4435 +0.0049
20 | 2.4890+0.0072 | 0.0731-0.0051 | 2.94+0.20) 1.9928+-0.0061| 1.4429 +0.0047

Variability in Body Size
Variability in body size and its fluctuations during growth have
been studied in other forms by a number of investigators. The present
data are particularly suitable for such a study, since it may safely be
assumed that the animals are genetically homogeneous and that
genetic differences are thus eliminated as a source of variation.

TABLE II

Values of the constants 6 and a@ for the data shown in Fig. 7.

Relation Instars b a

Carapace length y.............. 1-4 0.762 1.06

Total length COR ie ues ee 5-12 0.790 1.03
13-20 0.814 0.985

Height CURR Kati ee kode? 1-4 0.417 1.09

Total length BG aie Gye heEOtete re 5-12 0.530 1.03
13-20 12.49 0.315

Carapacellengthivacs. i . fs sede: 1-10 1.333 0.99

Height Bereich A ele tie «js 11-20 0.848 2.34

GROWTH AND VARIABILITY IN DAPHNIA 453

DELO GG nie

eed ee |

Fic. 7. Camera lucida outline drawings of a single animal for each of the first
eighteen instars. Arabic numerals designate pre-adult instars; Roman numerals—
adult instars.

454 ANDERSON, LUMER, AND ZUPANCIC

Total length was employed as the best available measure of body
size. Its use in this way is justified by the fact that there is apparently
very little change in the shape of the body during growth (see the
preceding section). The standard deviations and coefficients of
variation for total length are given in Table I and are plotted against
instar number in Fig. 8. It may be noted that both these quantities
tend to increase at first, but that after the eighth instar they fall off
rather steadily until the seventeenth instar, after which they remain
approximately constant. Hence as the growth process reaches com-
pletion, body size becomes less variable both absolutely and relatively.

fort op er rp hy

o) /0 1S 20
INSTAR

Fic. 8. Standard deviation and coefficient of variation of total length in
relation to instar number.

A comparison of the curve for the coefficient of variation with the
increment curve (Fig. 4) suggests that these two quantities are corre-
lated. When the coefficient of variation is plotted against the loga-
rithm of the increment (Fig. 9), the points tend to group themselves
along a straight line, indicating that there is roughly a linear relation-
ship between the relative variability and the logarithm of the growth
rate. Whether or not any general significance can be attached to the
precise character of the relationship as indicated here is open to ques-
tion; however, there is no doubt that a distinct relationship exists.

These observations are essentially in agreement with those of
previous investigators. A trend in the coefficient of variation similar

GROWTH AND VARIABILITY IN DAPHNIA 455

to that obtained here has been found to occur in the case of both
stature and body weight in man by several workers, including Bowditch
(1877), Thoma (1882), Porter (1894), Boas (1897), Boas and Wissler
(1904), and others. A summary and discussion of the data of Bow-
ditch, Boas, and Boas and Wissler is given by Thompson (1917, pp.
78-80). Essentially the same trend has been observed for body weight
in the albino rat by Jackson (1913), King (1915), and Hanson and
Heys (1927), in the Norway rat by King (1923), and in the cat by
Hall and Pierce (1934). King (1918, 1919) has shown also that the
trend persists in albino rats inbred for as many as twenty-five genera-

mime Tee yo ype ay eS
400

200

100

.060
.040

020

0/0

006

J 4 rr) 6 Uf
V

Fic. 9. Graph of the coefficient of variation of total length plotted against the
logarithm of the increment. The values of the coefficient employed are averages of
successive pairs in Table I.

tions, although the coefficient of variability in such rats is consistently
lower than that in non-inbred animals of the same average body
weight. Weymouth and McMillin (1930) have found that in the
Pacific razor clam, Siliqua patula, relative variability in shell length,
as measured by the ratio interdecile range: median, decreases steadily
with increasing length.

Several of these investigators have remarked also the existence of a
correlation between the coefficient of variation and the growth rate
(cf. Thoma, 1882; Porter, 1894; Boas, 1897; Boas and Wissler, 1904;
King, 1915, 1923). Jackson (1913), on the other hand, states that his
data show no evident correlation between these quantities.

456 ANDERSON, LUMER, AND ZUPANCIC

Thompson (1917) computed from the data of Boas the coefficients
of variation for annual increments in human stature between the ages
of five and eighteen, and found that these increased steadily with age.
He attributes this to increasing differences in phase of growth among
individuals of the group as growth proceeds. Analogous coefficients
of variation computed for the present data fluctuate somewhat
irregularly, but show on the whole a marked upward trend. In fact,
from the thirteenth instar on, the values of the coefficients are con-
siderably higher than 100 per cent. These values, however, are of
doubtful significance, since the mean increments for these instars are
small. When the value of the mean is close to zero, as Philiptschenko
(1927) has pointed out, the coefficient of variation becomes unreliable
as a measure of variability. As a matter of fact, one would expect
that as the growth process comes to a close, the differences in phase of
growth would tend to decrease (i.e. the increments for all individuals
would ultimately become zero), and that variability in growth rate
would correspondingly decrease.

The similarity of results obtained in the cases thus far investigated
suggests that the relationships described above are of rather general
occurrence, and that it may be worthwhile to seek a general explanation
for them. In attempting to find such an explanation, it is necessary
first of all to consider the sources of the observed variability, and the
relative importance of each. There are in all four possible sources,
namely errors in measurement, genetic differences, environmental
differences, and the fundamental nature of the growth process itself.

It does not seem likely that the observed trend in variability can be
accounted for to any significant degree on the basis of error in measure-
ment. Assuming that the distribution of errors is Gaussian, we
should expect that in measurements of length or weight, the absolute
error, as indicated by the standard deviation, would remain constant
or would, in some cases, increase with increasing magnitude of the
object being measured. In the former case, the relative variability
would decrease with growth; in the latter its behavior would vary,
depending on the particular conditions. There is, however, no evident
feature of such observational error which would account for a trend
in the standard deviation such as that shown in Fig. 8, nor for the
existence of a relationship between the coefficient of variability and
the growth rate.

Some information concerning the rdéle of genetic differences can be
obtained from the data of King (1919) on the growth of inbred and non-
inbred albino rats raised under identical laboratory conditions.
Although the trend in the coefficient of variation is essentially the same

GROWTH AND VARIABILITY IN DAPHNIA 457

in both stocks, the degree of correlation between the coefficient and
the growth rate, as indicated by the scatter of points on a graph, is
much higher for the inbred rats than for the others. This may be
interpreted to mean that variability due to genetic factors tends to be
largely independent of the velocity of growth.

On the other hand, variability due to environmental differences
- would be expected to vary with the growth rate, for an individual in a
state of rapid growth is relatively highly sensitive to the action of
environmental agents, and a given fluctuation from the norm would
produce a greater deviation in growth than it would in an individual
with a low growth rate. This has been recognized by Plunkett (1932)
in connection with developmental processes in general. He points
out that as such a process asymptotically nears completion, the
organisms tend to become more stable and less sensitive to the effects
of environmental factors such as, for example, temperature. Hence
the variability with respect to the particular character involved will
decrease. However, he erroneously assumes that the converse is also
true, namely that if one group of organisms is less variable in a certain
respect than another group, it is therefore nearer to the completion of
the underlying developmental process. This may or may not be
generally true, but it clearly requires further demonstration.

It has generally been assumed that variability in growing organisms,
aside from that introduced through errors in measurement, must be
due to either genetic or environmental differences. According to a
recent theory proposed by Rahn (1932), however, such variability
arises at least in part from the physico-chemical nature of the growth
process itself. The theory is briefly as follows. The division of a
cell must be preceded by the doubling of all its genes. Let us consider
a particular gene in each of a number of unicellular organisms which
are identical with respect to genetic and environmental factors.
If we assume that the doubling of this gene in the various cells con-
forms to the law of mass action, then it follows that it will not double
simultaneously in all the cells, but will do so over a definite time
interval. If the same is true of each of the remaining genes, there
will be a variability in division time of the cells.

Rahn has developed a simple mathematical formulation of the
theory which he has applied to data on the division rate of bacteria.
For multicellular forms, he has succeeded in analyzing only the case
of a hypothetical organism in which all the cells are alike and in which
the percentage rate of cell division is constant. He has shown that
such organisms will vary with respect to the time required for the
completion of a given number of cell generations. Moreover, as the

458 ANDERSON, LUMER, AND ZUPANCIC

number of generations increases, the frequency curves become flatter
while the relative spread of variation decreases. In other words, there
will be an increasing standard deviation and a decreasing coefficient
of variation. If the theory is valid, it would follow that variability
in cell number (and in body size insofar as it depends on cell number)
in successive time intervals should behave in a similar fashion.
Obviously these conclusions are not directly applicable to growth
in actual multicellular organisms for a number of reasons. First of all,
such growth is generally characterized by a decreasing percentage
growth rate; secondly, it involves increase in cell size, as well as in
cell number; and thirdly, the average reproductive rates of different
types of cells are not equal. Before the conformity of the theory with
the observed results can be tested, the former must receive a much

a 8

NUMBER OF INDIVIDUALS
S

4 . Lana IEA
\N Ss \\ fF H L \

5 ‘ y \ * IN \ H : Nae
PN BON SP ia ase 4 MIN 5 AA 6A MIAN
IN /\ AN te Ne
/\ yy) Ui NG HENNE / 4 INF x vA \\
6 8 10 12 14 16 18 2.0 Ge,

Fic. 10. Size frequency distributions during the first seven instars for all
eighty-two animals observed. Broken lines designate the individual instars. The
solid line is a composite curve for all instars. The vertical bars near the upper edge
of the figure represent the mean total lengths for each of the first seven instars.

more elaborate mathematical formulation, and a theoretical analysis
must be made in which these factors are taken into account.

It may therefore be tentatively concluded that the observed trend
in variability in body size with growth and its relation to growth rate
are explicable largely in terms of the action of environmental factors,
and perhaps also in part in terms of Rahn’s theory.

Size-frequency Distribution
Most studies of growth in Cladocera have been made by analysis
of size-frequency distributions in natural populations. These have
been of two types (see Woltereck, 1929). One consists of plotting
the number of individuals in a size class against size. In the graph
that results a number of size modes appear which are taken as repre-

GROWTH AND VARIABILITY IN DAPHNIA 459

sentative of the growth stages or instars. A second type consists of
plotting the value for one dimension of an individual against another
dimension of the same individual for each animal in a population.
Usually the points fall into groups which are more or less distinct.
Each group of points is taken as representative of a single instar.

No one so far as we are aware has attempted a comparison between
the results secured by these methods and those secured by observations
on individually reared animals. To make such a comparison, Figs.
10 and 11 were constructed. First the values for total length fre-

mm. 6 ro 10 hé 14, 16 /8 20 Qe
TOTAL LENGTH

Fic. 11. Size frequency distributions in two dimensions during the first seven
instars for all 82 animals observed. Each instar is represented by a different type of
circle. The area of each circle is directly proportional to the number of animals of the
particular dimensions represented by the circle. The crosses are the mean values for
each of the first seven instars.

quency during the first instar for all 82 animals observed were plotted.
Then those for the second instar and so on to the seventh instar.
After that a composite curve for all the values was plotted. The
separate instar curves are shown in broken lines and the composite
curve in a solid line in Fig. 10. The vertical lines in the upper part
of the figure represent the mean total lengths for each instar up to
and including the seventh. It is obvious that each mode in the com-
posite curve does not represent the mean total length for an instar.
The first three distinct modes are near the mean values. No distinct
mode is apparent for the fourth instar. The mean value for the fifth

460 ANDERSON, LUMER, AND ZUPANCIC

instar falls near a mode but the means of the sixth and seventh instars
fall midway between modes. Further, considerable overlapping
occurs between the values for various instars. The greatest value for
the first instar is larger than the smallest for the second. Similar are
the cases for the second and third instars, the third and fourth, and
the fourth and fifth. Some of the values for the fifth are greater than
the lowest for the seventh instar. This size-frequency method may
be of some use for the pre-adult instars, but certainly not for adult
instars.

Figure 11 was constructed by plotting the values of height against
that of total length for all 82 animals for the first instar, followed by
those of the second instar, and so on through the seventh. The area
of each circle is directly proportional to the number of individuals
represented. Where values for two or more instars fall on a point
separate circles are used for each instar. The mean height and the
mean total length for each instar are represented by crosses. This
method differs from that described by Woltereck (1929) in that each
individual was represented by a point on the graph while in the present
case if more than one individual is of the same size the circle at point
on the graph is larger and its area proportional to the number of indi-
viduals represented.

In analyzing Fig. 11, it will be noted that a band of circles is secured
but no distinct groups. The circles of the greatest area may be con-
sidered to represent the instars. For the first three instars, the circles
of greatest area fall near the mean values for the instars. No con-
spicuously large circle is found to represent the fourth instar. The
cross for the fifth instar falls near a large circle but the crosses for the
sixth and seventh instars do not. In most plots of natural populations
(Woltereck, 1929) the points scatter more widely, i.e., they form a
wider band than is the case in Fig. 11. Here again it is obvious that
size-frequency distribution does not lend itself to a satisfactory study
of growth.

Studies on growth by means of individually reared animals are
advantageous over the above methods in several respects. The
individual life histories are fully known. Genetic constancy can be
maintained since diploid parthenogenesis is the only means of repro-
duction. Selection of young from one clone insures genetic constancy
except for mutations. The environment can be maintained fairly
constant. By natural population analysis methods the individual
histories are unknown, the genetic constancy cannot be controlled,
and the environment is subject to considerable fluctuation.

GROWTH AND VARIABILITY IN DAPHNIA 461

SUMMARY

Eighty-two individually reared female Daphnia pulex were ob-
served from the time they were released from the brood chamber of
their mothers until they died. Measurements of total length, carapace
length, and height were made daily on each animal. The number of
young released during each instar was recorded.

Seventy-one animals were primiparous during the fifth instar and 9
during the sixth. Consequently the number of pre-adult instars is
variable, the minimum being four.

Data from the 47 animals which were primiparous during the fifth
instar and which lived for twenty instars or more were used in con-
structing growth curves.. Growth in the three dimensions studied is
sigmoid. The point of inflection in all curves comes during the fourth
instar, the last pre-adult.

The Robertson and the Gompertz equations do not fit the data
satisfactorily. This may be due to the time unit employed, which in
this case is the instar.

The growth increment is greatest during the fourth instar. The
increments increase up to the fourth instar, then decrease gradually
until the eleventh instar, after which they remain low and relatively
constant.

A significant negative correlation exists between initial body size
and initial growth rate, also between duration of growth and final body
size. Other characteristics of the growth process investigated appear
to vary independently.

The number of young released during the adult instars increases to
a maximum at the tenth instar followed by a gradual decrease.

The number of young released during any adult instar is sig-
nificantly correlated with the growth increment for the instar preceding
the one during which the young are released.

Relative growth in the dimensions studied may be expressed satis-

factorily by the equation

: i OE

No marked changes occur in the relations between total length and
carapace length. Marked changes in the relations between carapace
length and height and between total length and height occur at the
thirteenth instar.

The standard deviation and coefficient of variation of total length
tend to increase somewhat during the early instars, but after the eighth
instar both decrease rather steadily.

Relative variability in body size, as measured by the coefficient of

462 ANDERSON, LUMER, AND ZUPANCIC

variation of total length, is roughly directly proportional to the
logarithm of the growth rate. The existence of this relationship is
ascribed largely to the mode of action of environmental factors during
growth. It may perhaps also be explicable in terms of Rahn’s theory
of the physico-chemical origin of variability in growth rate.

Size-frequency analyses of natural populations as methods of
studying growth are shown to be inferior to the method using indi-
vidually reared animals.

LITERATURE CITED

ANDERSON, B. G., 1932. The number of pre-adult instars, growth, relative growth,
and variation in Daphnia magna. JBzol. Bull., 63: 81.

ANDERSON, B. G., 1933. Regeneration in the carapace of Daphnia magna. I. The
relation between the amount of regeneration and the area of the wound
during single adult instars. Biol. Bull., 64: 70.

Banta, A. M., 1921. A convenient culture medium for daphnids. Science, 53: 557.

Boas, F., 1897. The growth of Toronto school children. Rept. U. S. Comm. of Ed.
2 (1897): 1541.

Boas, F., anp O. O. WissLER, 1904. Statistics of growth. Rept. U. S. Comm. of
Ed. 1 (1904): 25.

Bownitcu, H. P., 1877. The growth of children. Rept. Mass. State Board of Health,
1877, p. 278.

Crozier, W. J., AND E. V. ENzMAnn, 1935. On the relation between litter size,
birth weight, and rate of growth, in mice. Jour. Gen. Physiol., 19: 249.

Hatt, V. E., AND G. N. PiERcE, JR., 1934. Litter size, birth weight, and growth to
weaning inthecat. Anat. Rec., 60: 111.

Hammett, F. S., 1918. The relation between growth capacity and weight at birth.
Am. Jour. Physiol., 45: 396.

Hanson, F. B., AND F. Heys, 1927. Differences in the growth curves of albino rats
born during the four seasons of the year under uniform laboratory conditions.
Anat. Rec., 35: 83.

Hux.ey, J. S., unp G. TetssiER, 1936. Zur Terminologie des relativen Gréssen-
wachstums. Biol. Zentralbl., 56: 381.

INGLE, L., T. R. Woop, AnD A. M. Banta, 1937. A study of longevity, growth,
reproduction, and heart rate in Daphnia longispina as influenced by limi-
tations in quantity of food. Jour. Exper. Zoél., 76: 325.

Jackson, C. M., 1913. Postnatal growth and variability of the body and of the
various organs in the albino rat. Am. Jour. Anat., 15: 1.

KERHERVE, J. B. DE., 1926. La descendance d’une Daphnie (D. magna) ou ses
millions de germes en une saison. Ann. de Biol. Lacustre, 15: 61.

Kine, H. D., 1915. Growth and variability in the body weight of the albino rat.
Anat. Rec., 9: 751.

Kine, H. D., 1918. Studies on inbreeding. I. The effects in inbreeding on the
growth and variability in the body weight of the albino rat. Jour. Exper.
Zool., 26: 1.

Kine, H. D., 1919. Studies on inbreeding. IV. A further study of the effects of
inbreeding on the growth and variability in the body weight of the albino
rat. Jour. Exper. Zoél., 29: 71.

Kine, H. D., 1923. The growth and variability in the body weight of the Norway
rat. Anat. Rec., 25: 79.

Kopsé, S., 1932. Die prozentuelle Wachstumsgeschwindigkeit der Mduse in bezug
auf das Gewicht der Neugeborenen. Arch. Ent.-mech., 126: 575.

GROWTH AND VARIABILITY IN DAPHNIA 463

McCay, C. M., M. F. CRowELL, AND L. A. MAYNARD, 1935. The effect of retarded
growth upon the length of life span and upon the ultimate body size. Jour.
Nuirition, 10: 63.

MERRELL, M., 1931. The relationship of individual growth to average growth.
Human Biol., 3: 37.

PHILIPTSCHENKO, J., 1927. Variabilitat und Variation. Gebriider Borntraeger,
Berlin.

PLUNKETT, C. R., 1932. Temperature as a tool of research in phenogenetics:
methods and results. Proc. Sixth Int. Cong. Genet., 2: 158.

PorTER, W. T., 1894. The growth of St. Louis children. Trans. St. Louis Acad.
Sct., 6: 263.

Raun, O., 1932. A chemical explanation of the variability of the growth rate.
Jour. Gen. Phystol., 15: 257.

RAMMNER, W., 1928. Uber die postembryonale Entwicklung der Cladocere Scaphole-
beris mucronata. O. F. Miller (Ergebnisse aus Einzelzuchten). Arch.
Ent.-mech., 113: 287. -

RAMMNER, W., 1929. Uber periodische Erscheinungen am Cladoceren-Individuum.
Int. Rev. Hydrob. Hydrog., 21: 402.

THomMA, R., 1882. Untersuchungen iiber die Grésse und das Gewicht der anato-
mischen Bestandtheile des menschlichen K6rpers im gesunden und im
kranken Zustande. F.C. W. Vogel. Leipzig.

TuHomeson, D. A. W., 1917. On Growth and Form. University Press, Cambridge.

WEymouTH, F. W., AnD H. C. McMILLin, 1930. Relative growth and mortality of
the Pacific razor clam (Siliqua patula, Dixon) and their bearing on the
commercial fishery. Bull. Bur. Fish., 46: 543.

WOLTERECK, R., 1929. Technik der Variations- und Erblichkeitsanalyse bei
Crustaceen. Abderhalden Handb. biol. Arbettsm., Abt. 1X, Teil 3.

Woop, T. R., anp A. M. Banta, 1936. Interpretations of data on growth and
reproduction (Daphnia longispina) (abstr.). Anat. Rec., 67:55.

SEASONAL PRODUCTION OF ZOOPLANKTON OFF WOODS
HOLE With SPECIAL REFERENCE TO
CALANUS FINMARCHICUS !

GEORGE L. CLARKE AND DONALD J. ZINN

(From Harvard University, Cambridge, Mass., and Rhode Island State College, Kingston,
Rhode Island) —

The purpose of the work described in this paper was the investiga-
tion of the distribution of the plankton in the waters around Woods
Hole during the summer months and the study of the seasonal changes
in the plankton at one locality throughout an entire year. Our
primary object specifically was to ascertain whether the copepod,
Calanus finmarchicus—a form used extensively for laboratory experi-
ments—breeds in this region, and if so during which months and with
what success. It was proposed also to include measurements of the
physical and chemical factors in the environment, and the collection
of nannoplankton and phytoplankton, in order that the sequence of
biological events from season to season in this locality might be
followed. Cause and effect relationships in the growth of the various
plankton forms might thus be unraveled.

Since copepods play a prominent réle in the economy of the sea,
it is important to know what conditions promote their growth and
what factors tend to reduce their numbers (Clarke, 1934). To this
end a series of laboratory experiments was initiated in which the
nutrition of copepods, particularly of Calanus finmarchicus, was
investigated (Clarke and Gellis, 1935; Fuller and Clarke, 1936; and
Fuller, 1937). The nearest point from which copepods of this species
could be obtained in sufficient numbers was in the deeper water off
Gay Head. The population of Calanus at this locality was found to
persist throughout the summer, but we had no knowledge either of
the subsequent fate of these copepods or of the time and conditions
of their appearance here. In our studies of nutrition we had assumed
that the Calanus found in this area were living in equilibrium with
their environment and that the smaller organisms found in the same
body of water were adequate both qualitatively and quantitatively
for their food supply. If, however, these copepods had been produced
in another locality and transported hither by currents, the possibility
existed that the conditions at our point of observation were not suitable

1 Contribution No. 112, Woods Hole Oceanographic Institution.
464

SEASONAL PRODUCTION OF ZOOPLANKTON 465

for the growth or even the maintenance of Calanus and that the
copepods found here were destined shortly to die off. The proposed
field observations were therefore especially desired and when worked
out in conjunction with further laboratory experiments should
eventually yield information on the factors controlling copepod pro-
duction which would have a general application.

Let it be understood, however, that the work described in this
paper was of the nature of a reconnaissance program. The locality
chosen for the observations throughout the year was a point as far
offshore as could be reached within a day’s sail from Woods Hole,
but it probably is not entirely removed from fluctuating land in-
fluences. However, evidence will be given below for believing that
this station is typical of a considerable area.

LOCATION OF STATIONS AND HYDROGRAPHY

During the summer of 1935 the five stations in the waterways
around Woods Hole shown in Fig. 1 were visited about twice a week.
Buzzards Bay is a broad, shallow body of water with a temperature a
few degrees higher and a salinity slightly lower than Vineyard Sound.
A strong current flows in Vineyard Sound, and offshore water seems
to be carried into the Sound much more readily than is the case with
Buzzards Bay (Haight, 1936). Since access to the open sea from
Woods Hole is most direct through the western entrance of Vineyard
Sound, a line of stations was run in that direction to study the transi-
tion between inshore and offshore conditions and populations.

Station 3, which was selected for the continuation of the observa-
tions throughout the year, was visited once a month during the
autumn and winter and at least twice a month from April to Sep-
tember. The authors are indebted to Dr. Renn, Mr. Iselin, Mr:
Woodcock, and Mr. Butcher for assistance which made these trips
possible. A rough sea is almost always encountered at the exposed
location of Station 3, and during the extremely cold weather when
spray froze on the deck and rigging, work was extremely difficult.

Station 3 is about eight miles from the nearest points of land on
the north and east, and twenty-five miles from Block Island on the
west. On the south the continental shelf water extends without
obstruction. Since at Station H2 (see Fig. 1) the flood tide flows
NNE and the ebb tide flows WSW, the main body of water debouching
from Vineyard Sound and Buzzards Bay probably passes largely to
the west of Station 3. However, the currents in this region are
extremely complex and unfortunately the measurements of the U. S.
Coast and Geodetic Survey (Haight, 1936) do not extend as far out

GEORGE L. CLARKE AND DONALD J. ZINN

466

“pueysy]
jayonjueN JO sepis y30q uO vas ay} 0} Uddo SI puNog jJeyxONJUeN] SeoIOyM ‘deur ay} JO WU] ay2 Jo y}10u jsnf pus ue 0} sautOD
Aeg spiezzng ‘peazy Aes) Jo yyNos ay} 0} deur ay} Yo qsnf sol] puey] suey] ON JO purysi jews eyy, “AeAing o1Jepoar) pue
ysv0Z *S ‘(A 243 Aq pardnoo0 ZF] uol}eIS Jo puke ‘epeul orm s[neYy uozyZued yoIyM Ye G—] SUOT}eIS JO UOT}VIOT “]T “Oly

@YVAINIA
v4

JUNE SWE Y A\UG SEP

JUNE JULY AUG SEPT

Fic. 2. Percentage distribution of Calanus finmarchicus copepodid stages III,
IV, and V and adult @ and o& taken with the scrim net at Station 3 during the
summer of 1935. The open circles represent catches made on an incoming tide
(E. 1 hour—-W. 1 hour) and the solid circles represent catches made on an outgoing
tide (W. 1 hour-E. 1 hour).

468 GEORGE L. CLARKE AND DONALD J. ZINN

as our station. Our plankton catches during the summer of 1935 at
the stations nearer shore showed a considerable difference depending
upon whether they were made on an incoming or an outgoing tide.
There is some evidence that the effect of the tide was felt as far out as

JUL AUG SEP OCT NOv DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

Yoo) SAL

dy
Nn)

inv}

JUL AUG SEP OCT NOV DEC JAN FEB MAR APR Maéy. J UL

Fic. 3. The salinity and temperature at depths of 0, 15, and 30 meters at
Station 3 during 1935-6. The vertical markers along the abscissa indicate the dates
on which observations were made.

Station 2, but at Station 3 no consistent difference due to the phase of
the tide was observed, as may be seen for the Calanus population from
Fig. 2. Transparency measurements similarly indicated the presence

SEASONAL PRODUCTION OF ZOOPLANKTON 469

of typical offshore water at this station (Clarke, 1938). However,
for the sake of uniformity, our observations throughout the year were
made as far as possible only during the periods when the “‘east,’’ or
flood tide, had flowed for at least one hour, and before the ‘‘west”’
tide had flowed for more than one hour.

These tidal oscillations are superimposed upon the much larger
but slower coastal current which flows in a southwesterly direction
over the continental shelf all along this coast. Water is moving
continuously from Nantucket Shoals on toward New York. It is
presumably from the inner edge of this current that the water at our
station is derived. Yet there must be a certain admixture of inshore
water, since the salinity value at Station 3 (see Fig. 3) was slightly
lower at all seasons of the year than that characterizing the water
farther offshore? and was slightly higher than that found within
Buzzards Bay and Vineyard Sound, according to determinations which
we carried out during the summer of 1935. The prevailing wind,
which is from the southwest, would tend to blow offshore water in
toward our station, but gales from this direction or from other points
of the compass probably disturb the normal hydrographic situation
rather profoundly.

The fluctuations in the salinity at Station 3 from July, 1935 to
September, 1936, were for the most part confined between 31.5 and
32.5 Joo and generally uniform at the depths of 0, 15, and 30 meters
(Fig. 3). On several occasions, however, a noticeable freshening of the
surface layer was observed, and on December 29, 1935, an unusually
great difference in salinity existed between the surface and the bottom.
Since a pronounced transparency anomaly occurred on the same day
(Clarke, 1938), we may assume that an abnormal interchange of water
masses took place on this occasion.

The temperature change at Station 3 during the year was ex-
tremely great for a marine environment. The maximum value,
observed in August, was over 23° C. higher than the minimum value,
in February (cf. also Allee, 1919). During the summer months the
water was highly stratified, for a difference of 8° or 10° was usually
found between the surface and the bottom. This would seem to
indicate that no great stirring was produced by wind or tide in these
months, but it is conceivable that the temperature difference could
be brought about by some persistent differential water movement.
In September the surface water began to cool off rapidly and to mix
more effectively with the bottom water, thus raising the temperature
of the latter. By the first of November the temperature had become

2 At Station ‘‘Martha’s Vineyard I.” See H. B. Bigelow and M. Sears (1935).

470 GEORGE L. CLARKE AND DONALD J. ZINN

uniform from top to bottom. This condition persisted throughout
the autumn and winter indicating strong stirring action during this
period. In the spring months warming took place more rapidly in
the upper layers than in the deeper with the result that a progressively
greater difference in temperature was found between the surface and
bottom until by June or July a pronounced stratification was re-
established.

METHODS

During the summer of 1935 10-minute horizontal hauls for
zooplankton were made using the scrim closing nets described by
Clarke (1933). At Stations 1, 4, and 5 hauls were made at depths of
2 meters below the surface and 2 meters above the bottom; at Stations
2 and 3 an additional haul was made at a depth mid-way between the
other two. Water samples were taken and temperatures determined
at each of these depths. A fraction of each water sample was turned
over to Miss Lois Lillick for qualitative and quantitative analysis of
the phytoplankton. Other fractions of the water samples were
bottled for determination of salinity, and of phosphate and nitrate
content. ‘The authors are indebted to Mr. Alfred Woodcock, Mr.
Bostwick Ketchum, and Mr. Homer Smith, for these chemical analyses.

For the work at Station 3 from October, 1935 to September, 1936,
the zodplankton was taken in ‘‘oblique”’ hauls in which the net was
lowered to 30 meters (just over the bottom), towed for 1 minute, then
raised 1 meter and towed for 1 minute, then raised another meter and
so on, until the surface was reached. A double-action hand plankton
pump was added to our equipment in order that the eggs, nauplii, and
younger copepodid stages of the copepods, which were too small to be
retained by the scrim net, might be caught whenever spawning took
place. The free end of the hose was attached to the cable about 2
meters above the weight and then lowered to the bottom. The
outflow from the pump was piped directly to a 1’’ Hersey Water
Meter and then discharged into the top of a No. 20 silk phytoplankton
net hung vertically in a metal barrel. Ten gallons (about 38 liters)
were pumped at each meter from the bottom to the surface.

At 30, 15, and 0 meters bottles were filled from the hose for the
chemical analyses and for quantitative phytoplankton counts by
Miss Lillick. At 20, 10, and 0 meters the pump was stopped and
the contents of the silk net drawn off and bottled separately, thus
dividing the catch into three parts representing three strata in depth.
On each occasion temperatures were taken and light penetration was
measured as is described elsewhere (Clarke, 1938). When time

SEASONAL PRODUCTION OF ZOOPLANKTON 471

permitted, a second oblique zodplankton haul and pumping operation
were carried out to compare with the first. The net hauls and the
pump catches were analyzed by suitable dilution and subsampling
(Clarke, 1933). Our thanks are-due Mr. David Bonnet for assistance
in this task and in the preparation of the tables and diagrams.

GENERAL FEATURES OF THE ZOOPLANKTON

The number of species of zodplankton represented in our catches
by one or more specimens is enormous (cf. Fish, 1925)—too numerous
for detailed analysis in an investigation devoted primarily to the

TABLE I
' Total number of Calanus finmarchicus tn hundreds calculated on a basis of a 30-minute
haul.
Date
Station 1 Station 2 Station 3 Station 4 Station 5
1935
sf) 2A ik es 6 106 90 — ~-
RLS Oe 17 65 142 5 37
Sle Saeeneeee 8 90 109 — 0
SS) caf Steam x 91 = BK 15
I rarer x 108 216 0 4
Se aca aks 11 152 123 — —
ifile os Le 14 , 74 124 xi me
21) Ae ae 6 20 209 — x
DS) aa ae 14 288 147 Xx x
Deis 5 hh 9 529 267 5 3
JNU al as reais 84 385 232 3 0
Din esate 5 52 171 6 OF
Lo) SN i aa vt 25 72 — —
aera es 3 92 94 Bre x
TOR crc: 8 137 50 Xx 0
2 OW adie: 0 28 113 0 0
Average....... 13.6 140.1 143.9 Sil tee

— indicates station omitted.
x indicates less than 100.

study of the production of a few important forms. Even a cursory
glance at the material, however, reveals that a considerable difference
exists in the plankton at the stations near shore and at those farther
out. At Stations 1, 4, and 5 large numbers of larval forms of both
bottom-living and pelagic species were encountered mixed in with a
varying number of mature individuals of truly planktonic types.
The composition of this inshore population changed almost daily in
contrast to the more gradual fluctuations which took place offshore.

At Stations 2 and 3 fewer types were found in the plankton and,

472 GEORGE L. CLARKE AND DONALD J. ZINN

although quantities of immature specimens appeared at certain
seasons, these belonged mainly to the same species as the adult indi-
viduals. The difference in the population offshore is well illustrated
in Table I, in which the total numbers of Calanus finmarchicus at
each station are set forth. Calanus was consistently scarce at Stations
1, 4, and 5 and, although the average number at Station 3 was only
slightly greater than that at Station 2, a population of at least moderate
dimensions was always to be found at the offshore station.

TABLE II

Total zoéplankton taken in scrim net at Station 3, 1935-36. Thirty-minute oblique
hauls with 75 cm net. Approximate volumes after settling one month.

Date Haul Vol. cc Av Date neu Vol. cc Av
Septyesie. 63 181 <10 Whey 25.52 o00c 204 240
; 182 <10 <10 205 160 | 200
Oct. 1 185 105 Junev ids sees: 206 270
186 70 88 207 215 | 246
Noa 2 (Ge, Dall ter 215 June 25....... 208 105
188 160 188 209 140 | 123
ID Yeroge (kis Saeco 189 140 140) uly OF 210 70
Deer ZO ee ee: 191 105S 211 90 80
192 1OO'S) 134 afuly e225 see 2D 45
[ta eA ena el etre 193 105 S 213 40 43
194 185S} 145 | July 30....... 214 240 | 240
Rely 2 Sine nose 195 700S| 700] Aug. 6....... 215 210 | 210
IMME Ye ATi tee ecdena 197 10 10 } Aug. 14....... 216 70
pie S)\ 32. {ae 198 40 217 70 70
199 65 58) || Abe ZO) os 65 5 218 210
IN pie Ore seca 200 115 219 160 | 185
201 115 115 | Aug. 31....... 221 70
Wiens) celia We eer o 202 65 BRE 40 55
203 90 Hou Septeslom rere 223 <10
224 10 10

Note: Hauls mostly copepods except those marked ‘S’” which were mostly
sagittee. The net used catches effectively animals as large as, or larger than, cope-
podid Stage IV of Calanus finmarchicus. Many smaller forms are not retained by
the net.

The seasonal variation in the total population may be traced
from the approximate volumes of the zodplankton taken at Station 3
throughout the year (Table II). Plankton was scarce during Sep-
tember and October, but increased in November and December.
The mid-winter hauls were characterized by large quantities of
sagitte. After this plankton again became scarce, but during the
spring months volumes increased reaching a maximum early in June

SEASONAL PRODUCTION OF ZOOPLANKTON 473

The plankton remained abundant through the summer with few
exceptions until in September a sudden reduction was encountered.

At all the stations Crustacea—usually copepods—formed the
bulk of the catch with the exception of a few occasions on which
large numbers of medusz or, at the offshore stations, of sagitte
were encountered. The most common copepods, in addition to
Calanus finmarchicus, which will be treated in a separate section, were
the following (cf. also Fish, 1925):

Centropages typicus......... July to Dec. Numerous in August
Centropages hamatus........ May to July, and Numerous in June and
in Sept. and De- July
cember
Pseudocalanus minutus... .. .Jan.to Oct. Numerous Mar. to Aug.
Paracalanus parvus......... July to Sept. and Numerous in Aug.
in Feb.
Acariia tonsd.............. April to Dec.
Otthona similis............ Aprilto Sept. and probably throughout the year
Labidocera @stiva.......... Oct. to Dec.

Many of these copepods appear to breed in this region because
immature specimens of most of the list were found on one occasion
or another. The copepodid stages of Centropages (species not de-
termined) were particularly numerous during the summer months,
and the locality is a veritable nursery for Pseudocalanus as judged by
the large numbers of eggs, nauplii, and copepodites taken in the pump
catches, particularly in the spring.

Sagitta elegans was represented by at least a few specimens in
almost every haul at Station 3 and the large catches of sagittz re-
ported on several occasions consisted entirely of this species. Sagitta
enflata occurred in small numbers in October and November, 1935,
and in August, 1936. Sagitia serratodentata was taken from Sep-
tember to December, 1935, and in August, 1936, and was abundant
on only one occasion. It was noticed that the size of Sagitta elegans
varied greatly from month to month and that the smallest individuals
appeared in January, May, July, and September. Since these months
agree almost exactly with the four periods of the year during which
Russell (1932-33) believes the main breeding of Sagitta elegans to
take place at Plymouth, we may conclude that the seasonal production
of this species is approximately the same on both sides of the Atlantic.

THE PRODUCTION OF CALANUS FINMARCHICUS

Comparison of Net and Pump Catches
The numerical analysis of our zo6plankton hauls was limited almost
entirely to Calanus finmarchicus. The numbers of this species taken in
the scrim net ran into the tens of thousands. If we assume 100 per cent

474 GEORGE L. CLARKE AND DONALD J. ZINN

straining efficiency, the scrim net filtered 564,000 liters of water during
the standard 30-minute haul. In the standard pump operation, on the
other hand, the total volume of water delivered to the phytoplankton
net in pumping from 30 meters to the surface amounted to 1134 liters,
One copepod in the pump catch would theoretically correspond to
about 500 copepods in the net haul provided that the two methods are
equally effective in catching all stages of Calanus. This was not
expected to be the case, but since a few of the older copepodites and
adults were usually to be found in the pump catches, these older stages
were counted as well as the younger stages in order that the sampling
efficiency of the pump and the net might be compared.

The total number of each stage of Calanus in the pump catches
throughout the year is shown by the solid areas in Fig. 4. All the
nauplius stages have been lumped together because the duration of
each of these stages is short compared to the intervals between our
observations. Very few of the earlier nauplius stages were taken.
Certain eggs, which may have been those of Calanus, were found, but
the number of these was so trifling as not to be worth plotting. The
scarcity of eggs and early nauplii suggests that the actual spawning
ground may be farther offshore than our station.

The numbers of Calanus taken in the net hauls is indicated by
the superimposed single line (Fig. 4), but using a different scale. The
magnitude of the scale which has been used for the net hauls was
determined by finding the ratio between the two sets of data for
copepodid Stage IV. This stage was chosen for the comparison of the
numbers taken with the pump and with the net because it seems to
have been caught effectively by both methods. Since the number of
Calanus in this stage was high both on July 30 and August 6, the
average number taken by the net on these two days was divided by the
average number taken with the pump. The resulting quotient was
very nearly 100. Accordingly all the totals for the net hauls were
divided by 100 before plotting. The scrim zodplankton net should
theoretically remove animals from 500 times as much water as the
pump. Since the volume of water passing through the pump was
accurately measured, we reach the conclusion that the straining
efficiency of the scrim net is 20 per cent.

The two curves for Stage IV which have been brought together at
one point in the manner described are seen to correspond very roughly
for the rest of the year. For copepodid Stage V and for the adults,
however, the curves for the pump catches consistently lie below those
for the net hauls. This indicates that the latter method is relatively
more effective than the former for these larger individuals either

OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

200

NUMBER OF COPEPODS

fe)

400

200

(eo)

- 600

400

200

‘0;

800

600

400

200

()
O
OCI NOV DEC AN GES em ViAh Arh MAY OUN i 00a AUG an SE

Fic. 4. Thesolid black area indicates the total numbers of Calanus nauplii (N),
copepodid stages I-V, and adult 9 and @ taken with the pump per standard haul
(see text). The superimposed line shows the numbers of Calanus taken with the
scrim net per standard haul, but for these the scale must be multiplied by 100.

476 GEORGE L. CLARKE AND DONALD J. ZINN

because fewer slip through the mesh of the net or because more escape
the mouth of the hose. The opposite situation is seen in the case of
copepodid Stage III in which it appears that a disproportionate number
of this group fails to be caught by the net.

Seasonal Changes in Abundance

Nauplii occurred in significant numbers on only three occasions,
namely March 27, April 13, and May 11. Copepodid Stages I and II
have peaks of abundance on April 13, May 11, and June 25. Stage III
was numerous also on April 13 and May 11 and a third period of
abundance extended from the end of June to the first part of August.
The numbers of individuals in Stage IV were moderate in April and
May, increased rapidly in June, and reached a maximum early in
August. The net caught representatives of Stage V in every haul
throughout the year, but with the exception of December 29, numbers
were extremely low from September until the end of April. This stage
was particularly abundant at the end of May and again in August, but
never equalled the maximum of Stage IV. Adult specimens of
Calanus were most numerous in May and June, but at all times were
relatively scarce, particularly the males. Taking the species as a
whole, we may conclude that the population is of very small dimensions
during the autumn and winter. By the first of April reproduction has
begun and from then on through the summer Calanus is abundant.
At some time after the end of August the species suddenly becomes
depleted and the population is not restored again until the following
spring.

Succession of Generations

To determine the number of generations? which succeed one
another during the year recourse is best made to the percentage dis-
tribution of the various age stages within each of the hauls from season
to season because this procedure removes the confusing effect of fluctu-
ations in the size of the total catch and allows the progression from
stage to stage to stand out in relief. The percentage diagram for the
pump catches (Fig. 5) shows that on the first of October the total catch

’The term “generation” is used here instead of ‘‘brood,” which has been
employed by others in this connection, because the former word expresses correctly
the relationship of the two groups of copepods which appeared during the course of
the year. The term ‘‘brood” should be limited to its strict sense in order to avoid
confusion. Nicholls (1933) regards it possible for each female copepod to produce
more than one brood of ova. Accordingly there may be circumstances in which the
young of the second generation exist contemporaneously with a late second brood of
the first generation. The necessity for using these terms with their exact meanings
thus seems obvious.

SEASONAL PRODUCTION OF ZOOPLANKTON 477

Uf OCT NOV DEC JAN FEB. MAR APR

OG NOV DEG JAN BEB S MAR

Fic. 5. Percentage distribution of Calanus nauplii, copepodid stages I-V and adult
@ and & taken with the pump at Station 3 during 1935-6.

consisted of 66 per cent Stage V and 34 per cent Stage IV. During
November and December practically 100 per cent of the species
occurred as Stage V. But in January some of these individuals
matured into adult males and in February an even larger proportion of

478 GEORGE L. CLARKE AND DONALD J. ZINN

females appeared. A month later the males and females had largely
disappeared and Stage V was reduced to 1 per cent, while nauplii
comprised 60 per cent of the catch and copepodid Stage I amounted to
30 per cent. During April other copepodid stages appeared in suc-
cession, but these gave rise to only a very small percentage of adult
females and practically no males. In May asecond series of peaks is to
be found progressing up through the copepodid stages and culminating
in a peak for the adult males on May 25 and a prominent peak for the
females on June 11.

The relationship between these two series of peaks in April and
May is obscure. Although we know that in other areas two or more
generations may follow one another in rapid succession during the
spring and summer, the time interval between our two series appears to
be too short for them to represent successive generations. In labora-
tory experiments Nicholls (1933) found that 27 days was the minimum
time possible from the shedding of the ova to the appearance of the
adults at the temperature at which he worked (apparently 11—14° C.).
The observations of Ruud (1929), Lebour (1916) and Fish (1936)
indicate that a longer time than this is required for the entire develop-
ment of Calanus. Marshall, Nicholls, and Orr (1934) state that “the
eggs appear after two to four weeks, spawning may last for several
weeks, and the adults then die out.’”’ Taking fourteen days as the
minimum time for the maturation of the eggs and 27 days as the
minimum for development, it appears that 41 days is the shortest
interval which can exist between the spawning of one generation and
the spawning of the next—or between the two generations at any
corresponding stage of development. At certain stages in our April
and May series the peaks are less than 30 days apart and the tempera-
ture of the water at the time was only about 6-8° C. It seems very
doubtful, therefore, that the copepodites appearing in May were
produced by the individuals which were found as copepodites in April.
It is possible that the May group is a late second brood from the same
parents as the April group. Adult specimens of Calanus were present
in the water throughout April although their numbers were low.
Another possible explanation of the situation is that at the end of
April an unusually extensive movement of water along the coast swept
away the population which had reached Stage IV on April 25 and
brought in an entirely distinct population of copepods whose develop-
ment was two or three weeks behind that of the first group. The
reduction in the abundance of the first group when it reaches Stage V
and its practical disappearance thereafter support this idea (cf. Fig. 4).

Another group of Stage I copepodites appeared late in June and

SEASONAL PRODUCTION OF ZOOPLANKTON 479

continued into July, but in lesser numbers. In the succeeding weeks
the older copepodid stages became numerous one after another until by
August 20 a prominent peak had been built up in Stage V. This
summer generation was undoubtedly produced by the individuals
which we observed as mature adults in May and June, although the

JUN , JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

100

gal

JUN JUL AUG SEP OCT NOv DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

Fic. 6. Percentage distribution of Calanus copepodid stages III, IV, and V and
adult 2 and @ taken with the scim net at Station 3 during 1935-6.

eggs and nauplii which should have appeared in June were not found.
Evidently the spawning took place during the two-week’s interval
between our visits to the station in June, or else at a point farther off-
shore. There is some indication in the diagram that this summer

480 GEORGE L. CLARKE AND DONALD J. ZINN

generation consisted of more than one brood, but the irregularities
were so great that no definite conclusion can be reached. It seems
clear, however, that Stage IV moulted only slowly into Stage V during
August and that these individuals did not mature into adults im-
mediately, but formed the population destined to carry over the
autumn and winter as Stage V.

The catches with the scrim zodplankton net have similarly been
calculated on a percentage basis to serve as a check on the conclusions
reached with the pump catches (Fig. 6). The numbers of the older
stages of Calanus taken with the pump were low and the possibility
existed that the larger individuals escaped the 1-inch opening of the
hose, whereas loss from this cause would be much less in the case of the
scrim net. Since the net hauls were begun four months earlier than the
work with the pump, we have a record for two summers and the
intervening winter. At the end of June, 1935, relatively large numbers
of adult males and females were found. The spawning of these
produced the summer generation which appeared as Stage III
copepodites in July. This generation formed the winter stock which
did not mature until January, February, or March. Individuals in
Stage III appearing in the net hauls in April (with a subsidiary group in
May) constituted the spring generation of 1936. This generation
matured and spawned in June and we find the summer generation as
Stage III in July, thus nicely confirming the net haul results of the
previous year. As before, these individuals grew slowly during the
summer, and when work was discontinued in September, the population
was about equally divided between Stage IV and Stage V.

From both the pump catches ° and the net hauls we conclude that
at Station 3 a short-lived generation of Calanus occurs during the
spring and is followed by a long-lived generation which has its origin in
the early summer and lasts through the following winter. One or
more subsidiary groups were found during the spring and summer
which may represent second broods from the same adults, or immi-
grants in a different stage of development, or possibly additional
generations of very short duration.

These two main breeding periods correspond closely with those
observed by Fish (1936) in the Gulf of Maine for the ‘“‘western stock”’

4 During the summer of 1937, however, incidental observations showed that the
Calanus population was extremely small in this region and consisted mostly of
Stage V. Evidently annual variations may be considerable.

5 The fact that the pump catches obtained through a 1-inch hose gave the same
general picture of the production of Calanus as the net hauls suggests that a device
with a relatively small opening is adequate for plankton studies and could be more
widely used (cf. Hardy, 1936). Such a device would have the important advantage
that the volume of water strained could be accurately measured.

SEASONAL PRODUCTION OF ZOOPLANKTON 481

of Calanus. Fish believes that this stock forms the principal source of
supply for the Gulf and he reports that these animals breed chiefly in
March-April and in June-July, but possibly also in September. The
developmental period of two and one-half months which Fish calcu-

SEP OCT NOV DEC JAN FEB MAR APR MAY. JUN JUL AUG SEP’

CEPHALOTHORAX LENGTH

SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP

Fic. 7. Cephalothorax length of Calanus copepodid stages III, 1V, and V and
adult 9 at Station 3 during 1935-6. The shaded area indicates the range and the
heavy line the mean of 25 measurements. Since males were sufficiently numerous to
measure on only five occasions, the limits of the range and the mean are indicated by
the co’ symbols placed on the upper diagram. It will be noted that the scales of the
upper and lower diagrams overlap to a certain extent.

lated for his Calanus agrees well with the approximate interval between
our spring and summer generations. Marshall, Nicholls, and Orr
(1934) found that in the Clyde Sea three generations of Calanus
occurred during the course of a year. The spawning periods for these

482 GEORGE L. CLARKE AND DONALD J. ZINN

generations took place in March, May, and June. Our observations
differ only in that the batches of nauplii appearing off Woods Hole
from March to May seemed to belong to the same generation. Bogorov
(1934) reports that three generations occur in the Plymouth area. He
believes spawning to take place in February-March, May, and
intermittently from June to August. Other investigators (Paulsen,
1906; Bigelow, 1926; Farran, 1927; Russell, 1928; Ruud, 1929; and
Sdémme, 1934) have found a similar succession of two or more genera-
tions in their respective areas, but since thorough reviews have been
made by Fish (1936) and by Nicholls (1933), their results will not be
discussed here.®

Variation in Size

The cephalothorax length of 25 specimens of copepodid Stage V was
measured for each haul throughout the year and for Stages III and IV
and for the adults on those occasions when these groups were suffi-
ciently numerous (Fig. 7). The range of the measurements for each
group in each haul was greatest for the adults and progressively less for
the younger stages. The average length of Stage V did not fluctuate
widely but was found to be somewhat greater from January to May
than during the rest of the year. Stage IV similarly diminishes in size
after the spring months have been passed.- The adult males and
females averaged about 2.65 mm. in length from January through >
April, but in May a sudden drop occurred to about 2.45 mm. and this
smaller size persisted through the summer.

The abrupt reduction in the size of the adults in May coincides with
the appearance in the hauls of the mature males and females of the new
spring generation (Figs. 4and 5). These individuals were spawned at
the end of April or early in May when the temperature ranged between
5° and 9° C. We have already seen that the generation which was
produced in June at temperatures of 11° to 15° C. apparently did not
reach maturity until the following January or February. Conse-
quently, the summer generation resulted in larger adults than the
spring generation although the latter group was spawned at a tempera-
ture about 6° C. lower. This relationship with temperature is just the
reverse ’ of that reported by Marshall, Nicholls, and Orr (1934) and by
Bogoroy (1934). These investigators found that the individuals of the
spring generation were the largest of the year and Bogorov reported

6 Results of a similar nature have been reported by Wimpenny (1936) in a
publication which appeared since this paper went to press.

’ The adult females which were taken in April and which were of large size may

possibly have been spawned in March and, if so, present a case of correlation of large
size and low temperature during the spawning period.

ae - ee

SEASONAL PRODUCTION OF ZOOPLANKTON 483

that the females of this generation were 1.2 times larger than those of
the autumn-winter generation.

Comparison with Phytoplankton and Nannoplankton

The phytoplankton obtained in the water samples taken at 2, 15,
and 30 meters on each occasion has been analyzed by Lillick (1937).
She reports that during the summer of 1935 the phytoplankton was
dominated first by Chetoceros and associated forms and later by
Rhizosolenia. On October 1 the diatoms reached a maximum.®
During the rest of the autumn and early winter the phytoplankton was
extremely poor. Latein January numbers increased somewhat but by
the end of February the phytoplankton was scarce again. On no
occasion was a typical spring diatom maximum encountered but Miss
Lillick cites evidence for believing that a great flowering of diatoms
took place early in February at a time between two visits to the station.
During the late spring and summer of 1936 the phytoplankton re-
mained at a low ebb, with Guinardia appearing as the dominant form,
until August 9 when for a period of four days a flowering of Rhizosolenia
and Guinardia occurred.

Diatoms were the most prominent element of the flora at all times
in this region, but dinoflagellates appeared in smaller numbers through-
out the year especially in summer and fall. In addition Miss Lillick
lays stress upon a group of flagellated and pigmented forms which
appeared in all the samples in very large numbers. Although this
group was made up entirely of exceedingly small forms, she states that
their frequency was so great as to make them significant in the
phytoplankton population and she believes that “‘they must doubtless
play an important part in the general food cycle of the region.”’

Special water samples for the study of the bacterial population were
taken by Dr. C. E. Renn at 5-meter intervals from the surface to the
bottom on each trip to Station 3 from October 1, 1935 to May 26, 1936.
The numbers of cells per cc. as determined by plate counts ranged from
30 to 350 during the first three months but from February to May no
counts greater than 33 were obtained (Renn, 1937). Although the
actual numbers of bacteria in the sea may have been a thousand times
greater than this, we have shown elsewhere that populations of these
dimensions cannot be a significant source of food for Calanus (Fuller
and Clarke, 1936). Besides the bacteria, and the pigmented forms

8 Our observations on the zooplankton were not made at sufficiently short inter-
vals to determine conclusively whether or not this flowering coincided exactly with a
marked reduction in the number of grazing animals (cf. Harvey, Cooper, Lebour, and

Russell, 1935). In 1935 a sharp drop in the number of Calanus occurred on Nov. 2;
in 1936 a similar diminution of numbers took place on August 31,

484 GEORGE L. CLARKE AND DONALD J. ZINN

mentioned above, to which Miss Lillick confined her attention, other
nannoplankton types undoubtedly existed—possibly in significant
quantities. The presence of great numbers of these, during the
summer months at least, has been demonstrated by Lackey (1936).
In most routine investigations many of the smaller and more delicate
forms are probably overlooked because the usual preservatives destroy
them or render them unrecognizable.

Since Marshall, Nicholls, and Orr (1934) found that in Loch
Striven the periods of diatom increases coincided with the three main
spawning periods of Calanus, it is interesting to examine our data for
similar correlations. For this purpose observations should have been
taken at much shorter intervals than was possible in the present

TABLE III

Phytoplankton at Station 3, March to June, 1936. The number of cells per liter at 2,
15, and 30 meters have been averaged.

Mar. 27 Apr. 13* oP May 11* ey. Jute June 25

Diatomseeee ee ae 33 9,450 600} 4,700 {1,467 |2,300 700
Dinoflagellates.... 67 100 1,300 0 67 0 3,767
“Flagellates’..... 300 5,750 {1,433 950 {2,633 |7,533 6,767
Other forms...... 0 350 OF uae 67| 100 167

MOA o eee 400 iS}G50N 3,059 5,650 4,234 |9,933| 11,401
Calanus

Nauplii Abundant |Abundant Abundant

Copepodid

ESN eve sacra Abundant Abundant Abundant

* Average of two depths only.

investigation. Conclusions are also made difficult in our case because
of our lack of information in regard to the state of the fauna and flora in
contiguous areas and the effect which horizontal movements of water
masses would produce. It is clear, however, that whatever diatom
flowering may have occurred in the early spring months between two
visits to the station, this could not have served as a food supply for our
Calanus since nauplii did not appear until March 27. On this date
and throughout the ensuing four months diatoms were not abundant.
Similarly no production of Calanus was observed at the times of the
secondary diatom maximum in August or October. Thephytoplankton
which did exist during the breeding periods of Calanus may be studied
from Table III. Diatoms were relatively numerous on April 13 and
May 11, occasions when nauplii and early copepodites were abundant,

SEASONAL PRODUCTION OF ZOOPLANKTON 485

but they were scarce on March 27 when the first nauplii of the season
appeared and on June 25 when the early copepodites of the second
generation came into prominence. On this latter date dinoflagellates
were especially numerous. The forms designated as ‘‘flagellates”’
were abundant throughout this period.

Although our data are not sufficient to allow us to conclude which
are the essential food organisms for Calanus nor in what quantity they
are required, nevertheless we have obtained certain facts which limit
the possibilities. We have seen that the spawning periods of Calanus
occurred at times which did not coincide with diatom maxima. We
know from Miss Lillick’s analysis that throughout the year the
diatoms and dinoflagellates appeared suddenly at intervals of a week or
two, flourished for a few days only, and then disappeared again. The
“flagellates,” on the other hand, although small in size, were found in
relatively large numbers on all occasions. If Calanus feeds chiefly on
diatoms, we must conclude either that the small number of cells
always present as a minimum is sufficient for their nutrition, or that the
food obtained by the copepods at times of local flowerings can be
converted into reserve tissue (e.g. oil) which will tide them over until
the next period of diatom abundance. The number of diatoms present
between flowerings seems too low to fulfill the nutritive requirements of
Calanus (Fuller and Clarke, 1936; Fuller, 1937) but further investi-
gations may show this not to be the case. As regards the second
possibility, we know that Calanus, in the later copepodid and adult
stages at least, can live for a week or two without food, but is unable to
moult successfully under these conditions (loc. cit.). The copepods
might thus be able to survive without moulting from flowering to
flowering—and this may be the actual situation during the autumn
and winter—but rapid growth such as took place from April to July
would probably be impossible unless adequate nutriment was con-
tinuously available. A third possibility which our present observations
suggest is that the nannoplankton is important as food for copepods.
_ The “‘flagellates’’ which were found at Station 3 at all times in large
numbers would represent only a small amount of substance because
of their minute size, but if, as seems likely, these pigmented forms are
only a small fraction of the total nannoplankton, the latter may turn
out to be significant as a food source.

SUMMARY

1. The seasonal production of zodplankton, particularly of Calanus
jinmarchicus, was investigated by means of scrim nets and a plankton
pump at five stations in the vicinity of Woods Hole during the summer

486 GEORGE L. CLARKE AND DONALD J. ZINN

of 1935 and at one offshore station throughout the ensuing year.
Collections of phytoplankton and of nannoplankton and measurements
of temperature, salinity, phosphates, nitrates, and illumination were
carried out at the same time.

2. The zodplankton consisted largely of copepods, but medusz,
sagitte, and, at the stations near shore, larve of both benthonic and
planktonic forms occurred irregularly. Sagitta elegans exhibited four
main breeding periods during the year.

3. The numerical analysis of the Calanus population revealed the
presence of nauplii in significant numbers in March, April, and-May,
and of early copepodites in April-May and June-July. Stage V was
found at all seasons. Adult specimens were relatively scarce at all
times, but most abundant in May and June. The species as a whole
was reduced to small numbers during the autumn and winter.

4. The pump catches and the net hauls agree in indicating that a
short-lived generation of Calanus occurs during the spring and that
this is followed by a long-lived generation. The latter has its origin in
the early summer, passes through the autumn and winter as Stage V,
and matures early the following spring to give rise to the next short-
lived generation.

5. The measurement of the cephalothorax length of 25 specimens
in each stage older than copepodid Stage II showed that the average
lengths of the various groups did not fluctuate widely during the year
except in the case of the adults which exhibited a sudden drop in size
in May.

6. The spawning periods of Calanus did not occur at times of
diatom maxima, and therefore the two phenomena are not directly
related in the present case. In regard to the nourishment of this
copepod we must conclude (a) that the small number of diatoms always
present as a minimum is sufficient, or (0) that the animals build up
sufficient reserve on occasions of small local flowerings of diatoms,
which occur at intervals of a week or two, to tide them over the
intervening periods, or (c) that ‘‘flagellates,’’ which were found
continually in large numbers, and other types of nannoplankton are
important as a food source.

BIBLIOGRAPHY

ALLEE, W. C., 1919. Note on animal distribution following a hard winter. Biol.
Bull., 36: 96.

BiceLow, H. B., 1926. Plankton of the offshore waters of the Gulf of Maine. Bull.
U. S. Bur. Fish., 40 (Part 2): 1924. (Doc. No. 968).

BiGELow, H. B., AND M. Sears, 1935. Studies of the waters on the continental
shelf, Cape Cod to Chesapeake Bay. II. Salinity. Papers in Physical
Oceanography and Meteorology, 4 (1): 1.

Bocoroy, B. G., 1934. Seasonal changes in Biomass of Calanus finmarchicus in the
Plymouth Area in 1930. Jour. Mar. Biol. Ass’n., 19: 585.

SEASONAL PRODUCTION OF ZOOPLANKTON 487

CLARKE; G. L., 1933. Diurnal migration of plankton in the Gulf of Maine and its
correlation with changes in submarine irradiation. Biol. Bull., 65: 402.

CLARKE, G. L., 1934. The rdle of copepods in the economy of thesea. Fifth Pacific
Science Congress. Vancouver, B. C. AS, p. 2017.

CLARKE, G. L., 1938. Seasonal changes in the intensity of submarine illumination
off Woods Hole. cology (in press).

CLARKE, G. L., AND S. S. GELLIS, 1935. The nutrition of copepods in relation to the
food-cycle of the sea. Biol. Bull., 68: 231.

FarrAn, G. P., 1927. The reproduction of Calanus finmarchicus off the south coast

of Ireland. Jour. du Conseil, 2: 132.

Fis, C. J., 1925. Seasonal distribution of the plankton of the Woods Hole region.
Bull. Bur. Fish., 41: 91 (Doc. 975).

Fisu, C. J., 1936. The biology of Calanus finmarchicus in the Gulf of Maine and
Bay of Fundy. JBzol. Buil., 70: 118.

FULLER, J. L., 1937. Feeding rate of Calanus finmarchicus in relation to environ-
mental conditions. Biol. Bull., '72: 233.

Futter, J. L., anp G. L. CrarxKe, 1936. Further experiments on the feeding of
Calanus finmarchicus. Biol. Bull., '70: 308.

Hatcat, F. J., 1936. Currents in Narragansett Bay, Buzzards Bay and Nantucket
and Vineyard Sounds. U. S. Dept. Commerce. Coast and Geodetic
Survey, Special Pub. No, 208.

Harpy, A. C., 1936. Thecontinuous plankton recorder. Discovery Reports, 11: 457.
Harvey, H. W., L. H. N. Cooper, M. V. Lesour, AND F.S. Russgex, 1935. Plank-
ton production and its control. Jour. Mar. Biol. Ass’n., 20: 407.

Lackey, J. B., 1936. Occurrence and distribution of the marine protozoan species in
the Woods Hole area. Biol. Bull., '70: 264.

LeBour, MARIE V., 1916. Stages in the life history of Calanus finmarchicus (Gun-
nerus), experimentally reared by Mr. L. R. Crawshay in the Plymouth
Laboratory. Jour. Mar. Biol. Ass’n., N.S., 11: 1.

Liuicx, L., 1937. Quantitative and qualitative studies of the phytoplankton of
Vineyard Sound, Mass. Biol. Bull., 73: 483.

MarsHALL, S. M., A. G. NICHOLLS, AND A. P. Orr, 1934. On the biology of Calanus
finmarchicus. V. Seasonal distribution, size, weight and chemical compo-
sition in Loch Striven in 1933, and their relation to the phytoplankton.
Jour. Mar. Biol. Ass’n., 19 (2): 793.

NicHotts, A. G., 1933. On the biology of Calanus finmarchicus. I. Reproduction
and seasonal distribution in the Clyde Sea area during 1932. Jour. Mar.
Biol. Ass’n., 19; 83.

PAULSEN, O., 1906. Studies on the biology of Calanus finmarchicus in the waters
around Iceland. Meddel. fra Komm. for Havunders, geleser, 1 (4): 1.

Renn, C. E., 1937. Conditions controlling the marine bacterial population and its
activity inthesea. (Abstract.) Jour. Bact., 33: 86.

RussELL, F. S., 1928. The vertical distribution of marine macroplankton. VII.
Observations on the behavior of Calanus finmarchicus. Jour. Mar. Biol.
Ass’n., 15 (2): 429.

RussELL, F. S., 1932-33. On the biology of Sagitta. Pts. I-IV. Jour. Mar. Biol.
Ass’n., 18: 131.

Ruup, J. T., 1929. On the biology of copepods off Mére, 1925-27. Rapp. Proc.
Verb., Cons. Internat. Explor. Mer., 56: 1-84.

SgmmE, J. D., 1934. Animal plankton of the Norwegian coast waters and the open
sea. I. Production of Calanus finmarchicus (Gunner) and Calanus
hyperboreus (Froyer) in the Lofoten Area. Fisk. Skrtft., Ser. Havunderso-
kelser, 4: 1.

Wimpenny, R. S., 1936. The distribution, breeding and feeding of some important
plankton organisms of the south-west North Sea in 1934. Part I. Fusher-
tes Invest., Ser. II, 15: 1-34.

SEASONAL STUDIES OF THE PHYTOPLANKTON OFF
WOODS HOLE, MASSACHUSETTS}?

LOIs C. LILLICK

(From the Department of Botany, University of Michigan, and the Woods Hole Oceano-
graphic Institution, Woods Hole, Mass.)

During the summer of 1935 certain investigations of the plankton
in the waters near Woods Hole, Massachusetts, were undertaken by
workers at the Oceanographic Institution. The zodplankton studies
have been described in the preceding paper by Clarke and Zinn (1937);
the observations on phytoplankton form the subject of the present
paper. At the outset five stations were established for regular obser-
vations, four in Vineyard Sound, and one in Buzzard’s Bay. For the
phytoplankton work water-bottle samples were taken at each station
twice weekly, and at three depths, surface, mid-depth, and bottom.
Along with these, determinations of the temperature, salinity, nitrate
and phosphate content of the water were made.

During the first two months the water at all five stations was found
to be so homogeneous in nature with regard to the phytoplankton,
that it seemed unprofitable to continue observations at all stations.
Therefore all subsequent collections were made at Station 3 alone,
which is located at the Whistle Buoy near the western entrance to
Vineyard Sound, Latitude 41° 17’ 35’’, Longitude 71° 0’ 0’” (see Clarke
and Zinn, 1937, for map). This station was chosen because conditions
there, more nearly than at any of the others, simulate those of open
waters, as will be seen later. From September, 1935, through the
winter months samples were taken once a month; from April through
July, fortnightly; during August, weekly. The final phytoplankton
data were collected on August 20, 1936, giving a range of fourteen
months for the entire survey. Samples were taken at the 2-meter, 15-
meter, and 30-meter levels, the bottom being at 32 meters.

In this paper only such forms have been included as fall naturally
within the plant classes, the chlorophyll-bearing or food-producing
groups, which include the Cyanophycex, Diatomacex, Dinoflagellate,
Stlicoflagellate, Coccolithinex, and certain of the pigmented flagellated
groups. All of the true protozoan classes have been omitted.

In making the quantitative determinations of the phytoplankton

1 Papers from the Department of Botany of the University of Michigan, No. 629;
Contribution No. 156 of the Woods Hole Oceanographic Institution.

488

PHYTOPLANKTON OFF WOODS HOLE 489

for the majority of the samples Gran’s centrifuging method was used,
modified somewhat by having the organisms concentrated over a mem-
brane filter, and then centrifuged. Since by this method others have
found that there may be a 10 per cent loss of organisms, an original
sample of 1,100 cc. was used instead of 1,000 cc. to allow for this error.
For the winter and spring samples the precipitation method of Nielsen
and von Brand (1934) was used chiefly because the amount of sample
available was not sufficient to permit the use of the other method.
Most of the collections were made by Mr. Donald Zinn and the crew
of the Oceanographic Institution boat Asterias. Chemical determina-
tions were made by Dr. Homer P. Smith and Mr. Bostwick Ketchum.
The writer is indebted to Dr. H. B. Bigelow and Dr. F. K. Sparrow, Jr.,
for certain guidance at the Woods Hole Oceanographic Institution,
and to Dr. William R. Taylor and Professor H. H. Bartlett under whose
direction the work was completed at the University of Michigan.

HYDROGRAPHIC OBSERVATIONS

Certain physical features of the water, currents, tides, temperature,
and salinity, play an undoubtedly important part in the distribution
of plankton. The observations made on these factors during the
present investigation have been presented by Dr. Clarke (Clarke, 1937;
Clarke and Zinn, 1937). Although the control of phytoplankton
species distribution, at least in part, must be attributed to the influence
of temperature and salinity, no obvious correlations could be made
between the variations in these factors and in the seasonal distribution
of phytoplankton.

Nitrate determinations ranged from 4 milligrams of N. as NOs per
cubic meter of sea water at 15 meters on October 1, to 62 milligrams
at 2 meters on December 29 (see Figs. 1, 2, and 3). Through the
summer months of the first year, the nitrate content of the upper
strata of water was low, exhibiting slight fluctuations. Somewhat
higher values were obtained in the lower strata. During the winter
nitrates were gradually built up throughout the entire water mass to
their highest concentration in early January. It is noteworthy that
there is a decided lag in this accumulation of nitrates behind that of
phosphates, a feature which is consistent with Cooper’s theory (1933)
based on observations in the English Channel, that the phosphates
are returned directly to the water upon the decomposition of the
plankton; whereas the nitrates are regenerated only after a series of
intermediate steps. Terminating the winter accumulation, there was
a sudden dropping off in the amount of nitrates in late January and
early February, followed by a more gradual decrease throughout March
and April. The summer was marked by small amounts of nitrates with

490 LOISJ@G. LILLIcCk

PHOSPHATES

PHYTOPLANKTON

TEMPERATURE

Fic. 1. Distribution of nitrates, expressed as milligrams of N as NO; per cubic
meter of water; phosphates, as mg. of PO, per cubic meter; phytoplankton, in
thousand cells per liter; and temperature, in degrees centigrade, at 2 meters depth,
Station 3, from July, 1935, to Aug., 1936.

PHYTOPLANKTON OFF WOODS HOLE 491

PHOSPHATES

PHYTOPLANKTON

TEMPERATURE

Fic. 2. Distribution of nitrates, expressed as milligrams of N as NO; per cubic
meter of water; phosphates, as mg. of PO, per cubic meter; phytoplankton, in
thousand cells per liter; and temperature, in degrees centigrade, at 15 meters, Station
3, from July, 1935, to Aug., 1936.

492 BOISTE, VILLI CK

PHOSPHATES

PHY TOPLANKTON

TEMPERATURE °

Fic. 3. Distribution of nitrates, expressed as milligrams of N as NO; per cubic
meter of water; phosphates, as mg. of PO, per cubic meter; phytoplankton, in
thousand cells per liter; and temperature, in degrees centigrade, at 30 meters, Station
3, from July, 1935, to Aug., 1936.

PHYTOPLANKTON OFF WOODS HOLE 493

only slight fluctuations. In August there was an accumulation to ©
form a second lesser peak, which again lagged behind the phosphates,
and which was followed immediately by a decrease in concentration.
Variations with depth were never extremely pronounced. However,
there was usually some degree of stratification. From May through
August, the nitrates tended to become concentrated at the bottom.
By September they were more or less uniformly distributed, and
remained so until December, when the surface waters became very
rich in nitrates, the bottom waters much lessso. This general relation-
ship continued through April.

The phosphate distribution differed somewhat from that of nitrates.
The total amounts found in the water were always much higher, |
though the total range was greater, from none in the surface waters
through most of August and September, to 112 mg. PO. per cubic
meter at the surface in December. The seasonal phosphate cycle was
as follows: low through July and August of the first summer; increasing
through September and October; reaching a maximum during the
winter months of November, December and January; decreasing from
the end of January through February and March, and dropping to
the lowest point for the year in April, remaining low through May
and June; in July and August gradually increasing, with another
drop at the end of August. Stratification of phosphates was frequently
much more pronounced than that of nitrates. From June through
August the phosphates were found to be more concentrated at the
bottom. In October the waters became more nearly uniform through-
out, and remained so until spring, when the amounts at the surface
increased over those at lower layers, this relationship remaining until
June. The possible correlation of phytoplankton production with
nitrates and phosphates will be discussed later.

PHYTOPLANKTON
(See Tables I and II)

The larger part of the phytoplankton in our collections was made
up of diatoms, dinoflagellates, and flagellates. During certain
seasons of the year members of the Silicoflagellatee and Coccolithinez
became rather numerous, and stray members of the Cyanophycez
appeared in the collections on occasion. More than 100 different
species appeared during the year, divided among the several groups
as follows:

Diatompacez tee. Vey Ae prone sprees. co sue 57
Dinoflagellatzes che yy See res oe ae 31
Silicoflagellates cers tan pews recone atin aes 3
Coccolithinesss7 8 ales Saeco ees 2
Cyanophy ceceintrteiectac seisenohe ae oii dens « 22

Blagellatesac cerita ae cte ee a adie ?

EOS (Gr LILLick

494

000°6€ | 00E*4'|00Z | 00S | 000'8Z

oos'e |o0e |ooe | ‘°'* | 008.
Ce ee ea Oe eee Oe

000‘0¢
oe Sie ee 00 Gees ,005
ign teal ans
So. Lapa ye
eees ro 45 00E'T
008‘S | 009‘¢ | 009‘LT

00€ |009 | 099
weve [ores | gga
OOL em OO each

00¢ | 009 | 00T

007‘2
006
00S
00E*Z

0L0°7 | 000'TZ

006 | 189s | see's

6IE | 996
cos‘z | ces'z

PLL | P8T'e
eee eee ialeca
Uo cckal Were ices

cop | 942

9 | coEz

Se0'T | 784'97

89€ 90€'FT
82¢ | 801'¢

$96'CP
PL3'F
| | 968'T
8F0'7 | 8OL‘9¢

967'T | SET

oF | H8I
00S | 781
82Z | 86S

49} Ted sijap

O00'TT | OFZ'T | OOF

000°9€T| 000'SE] 000‘9T

000‘681 Sadi: OxcteOmoms oN. Oubed cacr bec siajou 7 7e [eIO],

cae e
Pe
g9e

Ue oes ee

961'T

O&¢ eo
90S Dia alecee
“tes | og

ele

081 oO

Tsny | sc A[nf | cy Aine

¢ 4qnf

SIZ ‘T SBOoooCKn HOO oe re roo o Doo oo oo 6 8 oie} oie) (|
Oy OPO. ROR O Or OD Onde C86 OUDaeteto OF ad Og OG elteydsoor1AG

Gro \[Skolo oNduo prOsted ssenen Okottdkekead’o aro IUTYI]0I90-)
een i pinta CceO-Ceamehig a chnmose.5 unjnrads snunydaysiq
sploWeiherye oll buset(emtensentertaigeiee fale al Taree Seeks eateries LL[aseyooris
eieimommeltane d ltetomencsyretienohicNsircMteWm oyiais sh tcerep Licata ee WNIaINIS *
OST 9 Oat) DeO Oo Deu"o-Osoeod Nooo WNnUAM WUNMUBIOLOAT
Bats pam | DM ov A owalecichkctaice -felmriin eesoeome: Doering a9 3 eto xajqdulas "if
GAO OOOO OOOO MOO OO Od ‘dds mnMpLday

O6e en ee ee: SISUAADULD] XDINDKU0D
Stn 0 one hook osnadernT eaten ny tiso cece oseke coos 2 e][eseyourq
CC CRISES ES LUPIOLYSUIPAO NT DAISOUWSDIDY T,
$96 1 os * SapLovyISajvU DUAUOISSDIDY T,
G: beoNG. — Io Saonqstieg a O20 .neb oF NOC 40 UINIDISOD DULAU0IA1095
OL aes en eee oars purgsauas DIDIDGaY “W
S000 |loo-Dobodopaoan oo oU Oo OU DUassyIsDAL AV)

60 00 0856 ono O-O6.p.0.8 4 00.00 0-0 SUA “T
&%.d Ml'S-dun-piGa 0, a woes snovunp snapuyir0jdaT
OOM OOO Gd God wOO-o-0 6.04 ppvony, DIPADUIN®)
COU D-o Ose Oo cob Oo D-o5 ‘Dooe So XUAISKY UOAYJOL07Z)

020900 OSG D608. 000 8G OA. ON SOS sSnui pip ‘YD
OLZ Cono oe on iano Goo ood O00 0 suardigap YD

BOO modo gonoOn moon ooo oo a5 $110940Q YD
Ce i i i ) dds SOLBI0JLY 7

a ey 1haq4Darg DIJAU01AAIS V
Fe ee ew eee ee ee VIOCUIO}LLIG]

soloed

ee ee on ae, Se
SS 00888 SS OOOO OOOO

‘OC6T “Isnsny ysno1yy ‘cesT ‘A[N[ ‘¢ uorzeIS ‘siaj0ur ST 32 sotsads uo0jyuejdozAyd jueqsodur aiour ay} Jo uoOlyNqI4ysIp Teuoseas

[ HIV

PHYTOPLANKTON OFF WOODS HOLE

008 |00€ | 000‘ZT| 00E'T | 006'S | O0E'E | 0026 | OOO'TT| 00Z'S
000'Z | 008°9 | 00¢ 000'Z | 006'2 | 000'TT| OO0'T | * 006‘'T | 000'6Z| O02
OST'L | 008‘Z | 009'Z | 00S‘z | 000‘9Z | 009‘ST | O0S‘Z | 002 008‘z | 00¢'z | 00S
OOF'T | 007‘ 00Z‘T | 00Z‘T | 00Z‘OT | 000'T 008‘T | 00S‘6
000'T OO ae ae visite 009
00z2 | 002 OOT
OOT | 008 002 | 002
Ae es MG | 007
00s 00S | 00¢
000'T bo lee 00e 00r
004 | 004
004 | 008. 009
00z't | 008 | | 008. 008'81

Ee:

FI sny | 9sny | zz Aint | of Anf | sz cunt

JayWT Jed sjjad

Wee,

sJ9joul Z 1 [BIO],

S00 0CU bo GO CO OU DOO 0.00 K6 HO 2IETaSeLy
Oto m.obdad inp o ooo Doorn uo tod on elieydsoorsAG
O08 CO Cdio-o co Aum DMO BI OOO IUTY}I]OII07,
Gath 020 OXOLD On G-Olo, G@-anc"o wunjnoads snunydasiq
OD OOOO 10 520.5000 0,5 oo ho Ud BLIL][ISEYOOT|IS

quid! Ds BAOsoScNOSONO. OG; Qudvbed-o-n O60 ago) O40 mnyyaInIS “I
OTR Lood eerie cere pti. 0 WNUWAUIU WNAJUZIOLOAT

Gg Gi 4 GO HeOuost yO. 10107010 dU OU Oxon OO xarduas oa
Sonogo Gove co og a0 60000 0g dds UnwMepiagd
Aro OL0-00-0 O10 do O60 SISUaADULD] XDINDKUOL)
nel (eed sukents’ (ot offs igculsiiey loi celNe geen isiesiateiate x e[[eseyourq

poOOoOoONO oOo GoD oOo od GS WN4DISOD DUAU0IA7IIIS
Gaps semtnib. . ChoNo: Dy Geomo dno purdsuuas DIDIDGaY ‘
Domo OO Od 0-00 000 6 0-0-5010 -400 DUASSYUBDAL ‘YW

MUCMOOUOOOoDOoODoOoOmMDoDoooOo OG SHUM "T
GOOD CCoOr on Doo Ot 5 snaMnp SnApUYKIoIgaT
GoOoenhooOoD O00 O00 GO Oo ppi9py DIPADUINE)
Too 6 Oo 0 OR oe oo Oo Oo Oo Oo XIAISKY UOLYIALOD

pOUoHoOoCoOoGDOOUDDOnBOoOo odo ow ao SHU pUp 79)
Se suaidvap ‘YD

OOO AHDDOO OOD oO moOo GO bobo oo oo $1409409 Yd
DOoOe DOD OndoOo DODO oe ooo dds SO4990JXYID

SOO O06, 6 0) 0.6.0 6-H oho 18314 DI1g DIJIUOLAAS
epretieiieCiuute tamiel (aie siren iatisitcitpitemtel ie) {altatiotes ies) LIICUIOILIG,

sotoedsS

panuyuoj—] ATAV I,

496 EOS Ney ELeLICkK

TABLE II

Complete list of the species found at Station 3 during the survey of 1935-1936.

DIATOMACEZ

Achnanthes teniata

Asterionella Bleakley1
japonica

Bacteriastrum hyalinum

Cerataulina Bergonii

Chetoceros spp.

affinis
borealis
borealis var. concavicornis
compressus
constrictus
curvisetus
debilis
decipiens
didymus
gracilis
laciniosus
teres

Cocconeis placentula

Corethron hystrix

Coscinodiscus centralis

excentricus
lineatus

Grammatophora marina

Guinardia flaccida

Hemiaulus Hauckii

Leptocylindrus danicus

MInImus

Lichmophora abbreviata

Melosira moniliformis
sulcata

Navicula spp.

Nitzschia Closterrum
longissuma
sertata

Pleurosigma Normanti

Rhabdonema adriaticum

Rhizosolenia alata

calcar-avis

fragullisima

hebatata var. semispina
imbricata var. Shrubsolet
setigera

styliformis

Sceletonema costatum

Striatella unipunctata

Surirrella Gemma ?

Synedra Gallionti ?
Thalasstonema nitzschioides
Thalasstosira decipiens
gravida
Nordenskioldit
Thalasstothrix Frauenfeldi
longissima
DINOFLAGELLAT
Cerattum Fusus
lineatum
longipes
longipes var. oceanicum
macroceros
Tripos
Dinophysis acuminata
norvegica
Ovum
Exuviella baliica
Glenodinium trochoideum
Gonyaulax tamarensis
Mesoporos asymmetricus *
Minusculus bipes
Noctiluca miliaris
Peridinium spp.
breve
Cerasus
conicotdes
conicum
denticulatum
depressum
gracile
Granti
simplex
Phalocroma irregulare ?
Prorocentrum micans
minimum
Scutellum
SILICOFLAGELLAT
Dictyocha Fibula
Distephanus speculum
Fbria tripartiia
COCCOLITHINE
Rhabdosphera tubulosa
Syracosphera spp.
FLAGELLATE
CYANOPHYCEE
Gleocapsa
Oscillatorta

PHYTOPLANKTON OFF WOODS HOLE 497

Both oceanic and neritic forms occurred with several important
species belonging to either class. Neritic forms included both pelagic
and bottom types; and occasional tychopelagic forms appeared.

The summer flora (see Table I) of 1935 was broadly typical of
the area for that season. In July various species of Chextoceros were
important. This is the normal summer flora for regions lying to the
north of Woods Hole, and to be expected here. This genus was
accompanied by Corethron hystrix, Guinardia flaccida, Leptocylindrus
danicus and WNitzschia seriata, all temperate or boreal forms. In
August these forms were replaced by species of Rhizosolenia (R. calcar-
avis, R. alata, and R. setigera) and Thalassionema nitzschioides, a
temperate flora. The summer maximum was reached during August,
and consisted of Rhizosolenia and Guinardia. These are such large
forms that the numbers recorded give a false impression of the actual
state of the water. Plankton nets drawn through the waters during
one of these maxima became clogged within a few minutes with
Rhizosolenia. ‘This summer maximum is very typical of the general
area, and is similar to that found by Fish (1925). The entire flora

2 Since the name Porella given by Schiller (1928) to a genus of the family
Prorocentracez, Dinoflagellatz, is identical with that of the well known liverwort
genus Porella [Dill.] L., order Jungermanniales, and since this latter group antedates
Schiller’s genus by a great many years, it seems necessary to change the name of the
dinoflagellate group. For this genus the name Mesoporos is offered here in substi-
tution for Porella Schiller. Since Schiller’s generic name was not established by a
Latin diagnosis, one is given here for the nomenclatorial validation of the genus, in
accordance with the International Rules.

Mesoporos gen. nov.

Porella Schiller non Porella [Dill.| L.

Cellula ovalis plusminusve lateraliter applanata. Flagella per fenestram
edentatam poriferem exserta. Hemitestae poro singulo mediano intus projecto
praeditae, si lateraliter viso invaginationem conicam obtusam apice perforatam
formanti. Chromatiphora dua vel tria flava vel brunneo-flava ad hemitestas
applanata, absque pyrenoideis. Species 5, a cl. Jos. Schiller sub nomen Porellam
descriptae, Arch. f. Protistenkunde 61:54. 1928.

Mesoporos bisimpressus (Schiller) comb. nov.
Exuviella bisimpressa Schiller, Arch. f. Protistenk. 38: 257. 1918. Porella
bisimpressa ibid., 61: 54. 1928.

Mesoporos asymmetricus (Schiller) comb. nov.

Porella perforata (Gran) Schiller, Arch. f. Protistenk., 61: 55. 1928. Porella

asymmeirica Schiller, Dinoflagellata, Bd. 10, 3 Abt., S. 29, in Rabenhorst’s

Kryptogamen-flora. 1931. :

Mesoporos globulus (Schiller) comb. nov.

Porella globula Schiller, Arch. f. Protistenk., 61:56. 1928.
Mesoporos adriaticus (Schiller) comb. nov.

Porella adriatica Schiller, Arch. f. Protistenk., 61:56. 1928.
Mesoporos perforatus (Gran) comb. nov.

Exuviella perforata Gran, 1915, Bull. plankt. for 1912. Cons. Perm. int. p.

l’explor. de la Mer, Copenhagen.

498 IQS), (C,, EINE IKONS

for this season was much more characteristic than that found in the
following summer.

The winter flora, following the fall maximum, was extremely poor
(see Table I). Collections made on October 1 showed a relatively
large number of diatom species, occurring in considerable abundance.
These collections were very similar to Fish’s (1925) typical winter
flora and indicate that the season of 1935 resembled those of 1922
and 1923 at that time. In October 1935 the species which Fish had
found to be dominant were again dominant, namely, Rhizosolenia
alata, Sceletonema costatum, and Leptocylindrus danicus. Certain of
the forms which he mentions as occurring in abundance were found in
our collections to be important here in October and at no other time
throughout the winter (Chetoceros decipiens and Rhizosolenia setigera).
This similarity is surprising in the face of the total lack of similarity
throughout the rest of the winter.

For the rest of the winter months our collection showed that the
plankton as a whole was extremely poor. The four important species,
and at times nearly the only species found, were Thalassionema
nitzschioides, Rhizosolenia hebatata var. semispina, Thalassiosira
Nordenskiéld1i, and Nitzschia seriata. All are boreal forms, the first
two oceanic, and the last two neritic. Nearly all of the supplementary
species were likewise boreal. Along with these forms there was a
relatively high percentage of flagellated forms, a group which appeared
throughout the year and which will be discussed later. These results
are in the main very different from those of Fish (1925), who found a
definite winter ‘‘maximum”’ consisting of a greater variety of abundant
species than the summer.

It is extremely unfortunate that the great difficulty in making
these collections necessitated the spacing of the trips rather far apart.
During much of the year, because of the relative stability of the flora,
this should have made little difference. Nevertheless it has resulted
in our failing to observe any especial spring flowering of diatoms which
may have occurred and which is so characteristic of the region north
of Cape Cod. It is well known from the work of Bigelow, Gran and
Braarud, and others that at some time during the early spring, and for
a period of short duration, there is a flowering of diatoms dominated
by Thalassiosira Nordenskidldii in the Gulf of Maine, Bay of Fundy,
and Massachusetts Bay, which results in phytoplankton numbers far
in excess of those of any other season of the year. Fish seems not
to have found a similar flowering, though he mentions T. Nordenskiéldii
as occurring throughout January, February and March. However,
the conditions with which he dealt at the Bureau of Fisheries’ dock

per

PHYTOPLANKTON OFF WOODS HOLE 499

were far different from those at our more off-shore station. He does,
however, record the occurrence of a winter maximum beginning in
November or December and lasting into March, for Vineyard Sound
and Long Island Sound. Whether or not a flowering of either type
occurred at our station during the spring of 1936 must remain
problematic; certainly no such conditions appeared in our collections.
It is entirely possible that in the interval between any two of our
spring collections such a flowering could have burst forth and dis-
appeared again without showing up at all in our collections. The
weight of evidence which we have at hand seems to indicate that if
such a flowering occurred during the spring of 1936, it could not have
happened in all probability later than early February. Collections
taken at the end of January show that the phytoplankton had in-
creased decidedly over the winter months, yet was not abundant
enough to be considered as a “‘flowering.’’ Further, there was a
decided increase in the number of cells of T. Nordenskiéldii. Other
species which began to appear or to become prominent were Riizo-
solenia fragillissima, Nitzschia seriata, Thalassiosira decipiens, Thalasst-
onema nitzschioides, and certain of the peridinians, all boreal forms of
the neritic or oceanic type.

By the end of January the nitrates and phosphates in the water
had decreased markedly from their winter maximum concentration
in December. Similar sudden depletions of these salts have been
found by others in the English Channel, Loch Striven, and elsewhere
(Harvey, 1928; Atkins, 1927; Marshall and Orr, 1929) to be coincident
with the outburst of spring phytoplankton. It seems logical to infer
that such an explanation may account for the sudden decrease in
these salts at our station. The collections of late February and the
following months show a marked falling off of the phytoplankton,
and of the critical species, a condition which would normally follow
the spring maximum. All of these arguments combine to indicate
that any spring flowering of diatoms which may have occurred at
our station must have done so between the end of December and the
earlier part of February.

Following February the plankton became exceedingly scarce, and
entered the summer period, which for 1936 was the poorest for the
entire year. A few species of Chextoceros, which usually make up a
large portion of the summer flora in this latitude, and which were
prominent the year before, appeared early in the season but died out
as the year progressed. The important species throughout the season
was Guinardia flaccida, a temperate neritic form. The flagellates also
became prominent for a time in June, and then decreased. ‘Temperate

500 ILIOUES) (Ce IEIMEI BINS Ie

forms became more common; whereas boreal forms tended to die out.
Certain of the smaller dinoflagellates appeared during the summer,
though never in great numbers. The summer maximum, which is of
very short duration, did not show up in our collections. However,
we do have other information which gives the time and nature of this
summer flowering. Collections made in another connection at the
Oceanographic Institution showed that during a period of three or
four days, from August 9 to August 11 or August 12 the waters in
Vineyard Sound were teeming with a diatom flora which excluded
nearly everything else, coloring the water a deep olive green. An
examination showed this flowering to be made up almost entirely of
the two large diatom species, Rhizosolenia calcar-avis, and Guinardia
flaccida, both of which were prominent in the summer maximum of the
year before. This growth appeared very suddenly and disappeared
just as rapidly. As can be seen from our collections of August 6 and
August 14, both species were present in the waters, but in very small
amounts. Table I shows the general occurrence and abundance of
the more important species found throughout the fourteen months
at a depth of 15 meters.

Diatoms were obviously the most important element of the flora
throughout the year. Dinoflagellates appeared throughout the year,
more frequently in summer and fall, but their numbers were never
sufficient to outweigh the diatoms. A group of forms which were
exceedingly small, flagellated and pigmented, which therefore must be
considered in a treatment of phytoplankton, kept appearing in all
of our collections throughout the year, and in very large numbers.
The formaldehyde used to preserve the collections apparently so
distorted these forms that identification was impossible. There is no
question that many widely separated organisms appeared in this
class, which have been lumped together indiscriminately here under
the term “Flagellatz.’’ At times certain of the forms were obviously
swarm spores of certain of the diatoms and peridinians. It is quite
possible that others may have been spores of other algal groups.
Some were evidently independent flagellate forms. Much difficult
work will be necessary before this general group can be clearly under-
stood. No mention of such a prominent group as we have found
seems to have gotten into the literature of this general region, probably
because much of the previous work has been done with a no. 20
plankton net, which would allow these small types to slip through
the meshes. Poor though our general grouping of the forms may be,
it seems desirable to include them in this account because they doubt-
less play an important part in the general food cycle of the region.

PHYTOPLANKTON OFF WOODS HOLE 501

In Table II is given a complete list of the phytoplankton found to
occur at Station 3, during the survey.

Broadly speaking, then, our results correspond well with those of
Fish, whose work is the only one of a similar nature conducted in a
nearby region. Making allowances for the great differences between
the two localities, and for the limits placed upon our collections, the
seasonal cycle of the two surveys corresponds remarkably well.
Comparison of the species given in Table II with those listed by Fish
shows that a certain number of littoral or bottom forms appeared in
his collections and not in ours. A number of these species appeared
in our collections made during the first summer at the more littoral
stations. Other features characteristic of more offshore waters are
shown by our station. Along with stratification of the water column
is seen a certain degree of stratification of phytoplankton. One of
the outstanding characteristics of Fish’s station is the homogeneity
of the water from surface to bottom. The seasonal cycle at Station 3,
if we consider that we have sufficient data from which to judge, more
nearly resembles that of offshore waters, with its spring and fall
maxima, and summer and winter lulls, than it does the neritic waters.
Conditions here must be considered to be somewhat intermediate
between the other two types.

For a long time oceanographers have held to the theory that
phytoplankton production is tied up with and dependent upon the
amounts of nitrates and phosphates found in the water in any given
latitude. Ina broad sense, although this does not seem to be the final
factor which controls the cycle, there must be, certainly, some close
correlation between the two. Some such broad relationship can be
seen in the results of our survey. An examination of Figs. 1, 2, and 3
will show that the nitrates and phosphates were relatively plentiful
during July and early August of 1935, and that they dropped suddenly
during August. They gradually accumulated during the fall and rose
to their highest point during the winter months. During January
there was a sudden drop to a summer low which continued until July,
when the salts began to accumulate again to a lower peak in August.
Another decrease occurred here. Considering the curves for the plank-
ton, it can be seen that there are certain points of correlation, assuming
that the spring flowering occurred, as has been indicated earlier,
before the beginning of February, and the summer one in the middle of
August. When the phytoplankton was low, the salts began to
accumulate. As the phytoplankton began to increase, the salts
dropped off suddenly, implying that they were used up in the plant
cells. The dying off of the phytoplankton was again followed by a

502 LOIS "@, LIELICK

gradual accumulation of salts. These were no doubt supplied in
part by the decomposition of dead organic matter, and in part by new
water entering the Sound. There are minor differences between the
curves throughout, but in the major phases this general relationship
holds. Nitrates seem to be somewhat more critical for phytoplankton
in these particular waters than phosphates. There was always more
PO, in the water column than NO3, probably always enough for
some plant growth. It is with the curve for NO; that the phyto-
plankton shows the greatest correlation, and it may be that only
when the PO, curve agrees with that of NO; in trend that it can be
considered related to the plankton production. There is a much more
pronounced relationship between the phytoplankton and the salts at
2 meters and 15 meters than at the bottom, the 15-meter level showing
the clearest correlation. The general ratio of nitrates to phosphates
which is shown by Redfield (1934) to exist in the open oceans, and
which corresponds in general to the proportions in which these occur
in plankton organisms, does not hold at our station. Our data reveal
that throughout most of the year the phosphates exceeded the nitrates,
a condition which is contrary to that normally found.

Quantitatively, a greater number of organisms were found near
the surface than at the bottom. The greatest development of phyto-
plankton is usually found to be in the lower strata of water, but at
our particular station the amounts of detritus found in the water at
all levels was so noticeably greater than that found in more open
waters, that it is quite possible that throughout much of the year
light is not available at the lower levels for photosynthesis. It
happened very frequently that the number of cells at the bottom
greatly exceeded that at 15 meters. This is probably explainable by
the fact that cells sink rapidly once they become inactive, hence the
active cells are those at the surface; those at the bottom have become
inactive or are dead. In general the important species found at all
three depths at any one time were the same. No counts obtained
during the entire survey were high, the highest number recorded being
214,000 cells per liter at the surface in October, a number which is
normally much exceeded during the spring maximum at other localities.
Doubtless the numbers during the spring flowerings which occur in
this region would exceed 200,000 many times. The lowest number
recorded was 200 cells per liter at 30 meters in December, and at 15
meters in February. Collections made in March for the entire water
column were lower than at any other time of year.

This survey, incomplete though it is, and lacking two important
phases of the phytoplankton cycle, adds definitely to our knowledge

PHYTOPLANKTON OFF WOODS HOLE 503

of the waters near Woods Hole. It is the first study of the sort in
this region of a station which is far enough from land to show some
of the hydrographic features of the open sea. The quantitative work
gives a reasonably accurate idea of the numbers of phytoplankton cells
which occur in these waters at the different seasons; and the possible
relationship between the plankton and the concentration of nitrates
and phosphates is indicated.

LITERATURE CITED

Atkins, W. R. G., 1927. The phosphate content of sea water in relation to the
growth of the algal plankton. Part III. Jour. Mar. Biol. Ass’n., N. S.,
14: 447.

BicELow, H. B., 1914. Oceanography and plankton of Massachusetts Bay and
adjacent waters, Nov., 1912—May, 1913. Bull. Mus. Comp. Zodl., 58: 385.

BicGELow, H. B., 1926. Plankton of the off-shore waters of the Gulf of Maine. Bull.
U. S. Bur. Fish. (Part II), 40: 1.

Caxins, G. N., 1902. Marine Protozoa from Woods Hole. U. S. Fish. Comm.
Bull., 1901: 413.

CLARKE, G. L., anD D. J. ZINN, 1937. Seasonal production of zodplankton off Woods
Hole with special reference to Calanus finmarchius. Biol. Bull., 73:
464.

Cooper, L. H. N., 1933. Chemical constituents of biological importance in the
English Channel, November, 1930 to January, 1932. I. Jour. Mar. Biol.
Ass’n., N. S., 18: 677.

Fisu, C. J., 1925. Seasonal distribution of the plankton of the Woods Hole region.
Bult. U. S. Bur. Fish., 41: 91.

Gran, H. H., 1908. Diatomeen, in K. Brandt u. C. Apstein, Nordisches Plankton..,
19: 1.

Gran, H. H., AND T. BRAARUD, 1935. A quantitative study of the phytoplankton
in the Bay of Fundy and the Gulf of Maine. Jour. Biol. Board, Canada, I:
279.

Harvey, H. W., 1926. Nitrateinthesea. I. Jour. Mar. Biol. Ass’n., N. S., 14:71.

Harvey, H. W., 1928. Nitrate in thesea. II. Jbid., 15: 183.

Hustent, F., 1930. Die Kieselalgen Deutschlands, Oscseeing, und der Schweiz.
iRalbannoes? s Kryptogamen-flora. Bd. 7, Leipzig.

MARSHALL, S. M., AND A. P. Orr, 1929. A sends of the spring diatom increase in
Loch Striven. Jour. Mar. Biol. Ass’n., N. S., 6: 853.

NIELSEN, E. STEEMANN, AND T. H. v. BRAND, 1934. Quantitative Zentrifugen-
methoden zur Planktonbestimmung. Rapp. Cons. Explor. Mer., 89
(Part III, App.) (1933-1934), p. 99.

PAULSEN, O., 1908. Peridiniales, in K. Brandt u. C. Apstein, Nordisches Plankton.,
18: 1.

REDFIELD, A. C., 1934. On the proportions of organic derivatives in sea water and
their relation to the composition of plankton. James Johnstone Memorial
Volume, Univ. of Liverpool.

SCHILLER, J., 1928. Die planktischen Vegetationen des adriatischen Meeres.
Arch. f. Protist., 61: 45. i;

SCHILLER, J., 1930. Coccolithineze Deutschlands, Osterreichs, und der Schweiz.
eepenhorss s Kryptogamen-flora. Bd.10,2 Abt. Leipzig.

SCHILLER, J., 1931. Dinoflagellate. Jbid., Bd. 10, 3 Abt.

SERUM PROTEIN MEASUREMENTS IN THE LOWER VER-
TEBRATES) 1h) THEVCOLLOID OSMOTIC PRESSURE,
NITROGEN CONTENT, AND REFRACTIVE
INDEX OF TURTLE SERUM AND
BODY FLUID

MILDRED L. CAMPBELL AND ABBY H. TURNER

(From the Physiological Laboratory, Mount Holyoke College,
South Hadley, Massachusetts)

The significance of the blood serum proteins in the maintenance of
the fluid balance between blood and tissues was first suggested by
Starling (1), who postulated that the osmotic pressure they exert is
the force which, acting in opposition to the hydrostatic pressure of the
blood, prevents the loss of excessive amounts of fluid to the tissues.
The numerous studies of the serum colloid osmotic pressure which
have since been made in man and the lower mammals have indicated
that for mammals Starling’s hypothesis holds true, if not completely,
at least as a major factor. A comprehensive review of the subject,
with bibliography, is that of Landis (2). A recent modification of
method is that of Wies and Peters (3).

The question of the applicability of this hypothesis to conditions
which obtain in the lower vertebrates has been investigated very little.
Data on the amphibia are given by Landis (2) and also by Drinker
and Field (4). The most recent work has been that of Keys.and Hill
(5), who studied six species of fish. It has seemed important to us to
make a series of studies of the colloid osmotic pressure of the serum in
several of the lower vertebrates, correlating the findings whenever
possible with other determinations on the serum proteins. In this
paper we present the results of a study of a reptile, the common
“slider’’ turtle. Determinations were made of the colloid osmotic
pressure, nitrogen content, and refractive index of the serum and of
the body fluid of normal animals and of those subjected to long-
continued starvation at two different temperatures.

METHODS

Fourteen adult, healthy, female turtles of the species Malacoclemmys
geographica, Les. were obtained in October at about the end of the
period of summer activity and feeding. ‘Two of these were studied at
once as normal fall animals and the remainder divided into four experi-

504

SERUM PROTEIN MEASUREMENTS IN LOWER VERTEBRATES,I 505

mental groups, of which one was kept during the succeeding months
under each of the following conditions: (1) room temperature, 19°—20°
C., with feeding, (2) room temperature without feeding, (3) winter
temperature, 7°-10° C., with feeding, (4) winter temperature without
feeding. The food consisted of common garden worms ad libitum; all
animals were kept constantly in large tanks of fresh water. During
April at the end of the winter period of hibernation and fasting, two
fresh specimens were obtained for use as spring controls.

Blood was drawn by syringe from the aorte and sinus venosus and
allowed to clot in tubes. After centrifuging, the supernatant serum
was removed by pipette. A clear, colorless fluid which was frequently
found in considerable amounts in the body cavity was removed by
syringe without contamination by blood. It is this fluid which is
referred to as body fluid.

Colloid osmotic pressures were obtained by the second method of
Krogh and Nakazawa (6), using the modifications suggested by
Turner (7). The membrane was Du Pont cellophane no. 450, the
outer liquid 0.6 per cent sodium chloride. For every determination
three or more osmometers were set up. The figure taken for the
colloid osmotic pressure of any sample was the average of values given
by osmometers which conformed to the requirements of the Krogh
technique. Determinations of the total and non-protein nitrogen
concentrations were made by the micro-Kjeldahl method combined
with direct Nesslerization as given by Peters and Van Slyke (8).
The difference between total and non-protein nitrogen gave the con-
centration of protein nitrogen. Total refractive indices were deter-
mined by a Zeiss dipping refractometer. Whenever the quantity of
serum available made it possible, a determination was made of the
refractive index of an ultrafiltrate prepared by the use of a Thiessen
ultrafiltration apparatus with cellophane 450 as membrane. The total
refractive index minus that of the ultrafiltrate gives the refractive
index of the colloid fraction.

RESULTS

The protein data on sera and body fluids are given in Table I. The
serum colloid osmotic pressure in animals of the warm, fed group was
within the range of the normal fall values for a period of three months.
The progressive decrease in pressure observed in all the other groups
may be explained as due to the effects of starvation modified by the
metabolic rates of the animals. In the warm, starved group total
starvation plus a relatively high metabolic rate resulted in marked and
immediate changes in the serum proteins as shown by the lowered
colloid osmotic pressure. Edema was expected in these animals, but

506 MILDRED L. CAMPBELL AND ABBY H. TURNER

was not found either in this or in any other group. Since the turtles
in the cold, fed group ate very little, both cold groups may be regarded
as starved. Due apparently to the depression of the metabolism by
the low temperature, the colloid osmotic pressure in these animals
decreased more slowly than in the warm, starved group and the total
fall was less. That the rate of metabolism in animals when subjected
to different environmental conditions is so affected was shown by

TABLE I

Protein data from blood serum and body fluid, Malacoclemmys geographica.
Body fluid was found only in the animals indicated. Protein nitrogen was obtained
by subtracting the analytically determined non-protein nitrogen from the total
nitrogen. The refractive index of the colloid fraction was obtained by subtracting
the refractive index of the ultrafiltrate from the refractive index of the whole serum.
An asterisk after the colloid refractive index indicates that in this case no ultra-
filtrate was available. The figure was obtained as stated in the text.

Colloid 5
e Length] osmotic Bees ie pene Refractive index
tatus of pressure
of Date | experi-
animal killed | mental

period | Se- | Body | Se- | Body | Se- | Body} Se- | Colloid | Body
rum | fluid | rum | fluid | rum | fluid rum |fraction] fluid

mm.| mm. |\mgm./| mgm./|\mgm./| mgm.|

months H20 | H20 | cc. CC. CC. CC.

Normal, fall. ..| Oct. 30} 0.0 81 81 — — —_ —_— 1.34350] .00768 | 1.33586
Be “',..| Nov. 7} 0.0 2s — — — — | 1.34453) .00886*| —
Warm, fed....| Jan. 4] 2.0 98 | 46 | 6.54} 0.75 | 6.29 | 0.61 | 1.34364] .00799 | 1.33586
of (a5 co|| es Al S40) 99} — | 683} — | 660] — | 1.34399] .00809 —

Warm, starved | Dec. 21] 1.75 58 | — | 6.24] — | 602} — | 1.34508 00907 —_—
oe 2 Jan. 17] 2.5 58} 28 | 5.85 |} 0.83 | 5.45 | 0.51 | 1.34291] .00758 | 1.33632
oe as Mar. 1] 4.0 39 | 36 | 6.58] 0.79 | 6.34 | 0.58 | 1.34342 | .00779 | 1.33636
Cold, fed...... Dec. 14} 1.5 94) 50 | 5.64} 1.77 | 5.51] 1.65 | 1.34341] .00773 | 1.33659
Be erie era Jane Lon|2e5 77| — |695 | — | 6.73} — | 1.34474} .00907*| —
SS ees ayer Feb. 12} 3.5 72! — | 6.14] — | 604] — | 1.34339] .00766 —_—
Cold, starved..} Dec. 11] 1.3 102 | 65 | 5.37 | 0.66 | 5.19 | 0.54 | 1.34356] .00789*| 1.33604
45 oll eins 1) || 583 57] 41 4.70 | 1.23 | 4.55 | 1.07 | 1.34291] .00724*| 1.33519
My we soll Jer, ZA We7/ 58 34 6.51 | 1.23 | 6.27 | 1.00 | 1.34350}| .00760 | 1.33580
a ool ads 7] Ses 66} — | 5.74} — | 5.56} — | 1.34548} .00981*) —
Normal, spring | Apr. 7] 0.0 68} — | 4.18} — | 4.02 | — | 1.34208] .00671 —
He > Apr. 9] 0.0 48 | — |5.11} — | 490] — | 1.34235] .00699 —

Hand (9) in his compilation of the results of inanition on the higher
groups of animals.

The occurrence of amounts of body fluid adequate for determina-
tions was irregular and not limited to any group or groups nor to any
particular time in the experimental period. Although the variations
in the colloid osmotic pressure were wide, the value in each case was
found to be lower than that of the corresponding serum except for one
instance of equality. No proportional relation between the two
pressures was discernible.

SERUM PROTEIN MEASUREMENTS IN LOWER VERTEBRATES, I 507

Table I shows total nitrogen and protein nitrogen figures, the latter
obtained by subtracting the non-protein nitrogen from the total
nitrogen. The non-protein nitrogen content was low and varied
irregularly in the sera from .10 to .40 mgm./cc., with eleven of the
fourteen values between .15 and .25 mgm./cc. In the seven analyses
of body fluid the variation of non-protein nitrogen was from .12 to .32

TABLE II

A table showing percentage changes as the experiment progressed. The two
normal fall animals are taken as 100 per cent. In the case of the nitrogen values,
since no analyses were made on the fall animals, the 100 per cent is furnished by
the two warm, fed animals whose other findings showed a negligible decrease as the
months went by. The two spring animals were fresh from the collector, after a
hibernation period of unknown duration probably much longer than the duration of
the experiment.

Length of Colloid

Status of animal expenmental pempite ae Rehacuye
months per cent per cent per cent
INGOT IGS gina peo uosen ee 0.0. 100 — 100
\Wanmmesteds oa 2 bo tee see 2.0 101.5 100 96.5
a SC HUE SE Sa aA 3.0 102.5 100 98
Warinm Starved: ss... 62. sls fe 60 93.5 110
oe PU 4 bok 8S ARS ai ee ae DES 60 84.5 92
oe Cie) Ni aie ie Me eae 4. 40.5 98 94
Cold Miedier sau ease ea eae o. 1.5 97.5 85.5 93.5
ne a Ce ya Nu A te 2.5 80 104 LQ)
ue SRS ey ciate Seth a8) 2 cael ae yet 3.5 75 94 93
Goldiictarved =) Aya sas 163 106 80.5 95.5
of Ce ae Ed aaah ied Pea A DES 59 70.5 87.5
i Ri reece SRE a ary Rae eh Dall 60 97 92
of RSM SEER LD ae. Be AUN 3.25 68.5 86 118
INormialiispring:. 94/511. fh fae —_— 70.5 62 81
a ibid nige Maelo pe ar — 50 75.5 84.5

mgm./cc. The concentration of protein varied widely yet it seemed
clear that starvation in this species fails to cause a fall in the amount
of protein parallel to the fall in colloid osmotic pressure. Table II
gives the values in percentages, thus showing the lack of correspondence
between the effect of starvation on colloid osmotic pressure and on
protein content. The average of the two fall specimens is taken as
100 per cent for the colloid osmotic pressure series. It will be noted
that the warm, fed animals show no fall. Nitrogen concentrations

508 MILDRED L. CAMPBELL AND ABBY H. TURNER

were not determined for the two fall animals but in the absence of that
standard it seemed permissible to use the level of the two warm, fed
animals as 100 per cent for protein comparisons. Determinations of
total protein, albumin, and globulin in the sera of mammals have been
reported by several observers (10, 11, 12, 13, 14) who have shown that
during starvation a reversal of the mammalian albumin-globulin
ratio occurs, indicative of a relatively higher globulin concentration.
Because of the larger size of the globulin molecules this shift results
in a marked fall in colloid osmotic pressure without a corresponding
decrease in total protein concentration. From our observations it
seems possible that a similar shift in albumin-globulin relations may
occur in the turtle. The two spring specimens which had doubtless
spent the entire winter in hibernation showed a fall in total protein
more nearly approaching that in colloid osmotic pressure. The con-
centration of protein in the body fluid was much lower than in the
serum and showed wide variations. No direct correlation with the
colloid osmotic pressure was apparent.

“The total refractive index of normal, fall turtles averaged 1.34377.
With two exceptions the figures for animals with lowered colloid
osmotic pressure were below this average, falling even to 1.34222 as
an average for the two spring controls. The range of the refractive
indices for the ultrafiltrates was narrow, with an average of 1.33569.
Where no ultrafiltrate could be prepared, this average figure was used
to calculate the probable value for the colloid fraction. (See Table I.)
When the refractive index of the colloid fractions was plotted against
the colloid osmotic pressure a good correspondence was evident,
though there is a closer correspondence between protein concentration
and refractive index of colloid fractions. The refractive index of the
body fluid was consistently lower than that for the corresponding
‘serum and higher than that of the serum ultrafiltrate. Since the
crystalloids of serum and body fluid are probably closely alike, this
difference between body fluid and ultrafiltrate from serum would
indicate a colloid fraction in the body fluid, as verified by analysis.

DISCUSSION

Our interest has centered upon the inferences which may be drawn
from these determinations as to the significance of the serum proteins.
Certain points stand out: first, the fall in colloid osmotic pressure with
prolonged starvation, especially at the higher temperature; second, the
much greater stability shown during starvation in the quantity of
serum protein as compared with its colloid osmotic pressure; third, the
presence in the body fluid of substances exerting a colloid osmotic

SERUM PROTEIN MEASUREMENTS IN LOWER VERTEBRATES,I 509

pressure far from negligible though the protein percentage is low.
While the relation of plasma and true tissue fluid—meaning by this
the fluid which has just made its exit through capillary walls—may
not be that shown by the comparison of the serum and body fluid, it is
nevertheless probable that capillary permeability to proteins, at least
those of the smaller molecular size, is indicated by the very perceptible
colloid osmotic pressure of the body fluid. Such proteins as may be
outside capillary walls will, of course, lessen the effective colloid osmotic
pressure of the plasma. Further, the marked decline of the colloid
osmotic pressure in starvation without evident edema speaks against
it as a major controlling factor in water balance between blood and
tissues in this species, while the persistence of the protein content of
the serum at a relatively high level may indicate the importance of
plasma proteins for some other function than the maintenance of
colloid osmotic pressure, possibly as nutritional reserves.

SUMMARY

The average serum colloid osmotic pressure of two normal fall
turtles of the species Malacoclemmys geographica, Les. was 96 mm.
water pressure. This pressure was lowered by starvation, especially at
laboratory as compared with winter temperature, but the serum
protein concentration was much less affected. A body fluid obtainable
from about half of the animals showed a colloid osmotic pressure of
more than half the serum pressure in these individuals, though the
protein content was low. These findings are interpreted as indicating
that in this species the colloid osmotic pressure may not be the only
controlling factor in the water balance between blood and tissues,
and that other functions of plasma proteins may be of importance.

BIBLIOGRAPHY

1. STARLING, E. A., 1896. On the absorption of fluids from the connective tissue
spaces. Jour. Physiol., 19: 312.

2. Lanpis, E. M., 1934. Capillary pressure and capillary permeability. Physzol.
Rev., 14: 404.

3. Wiss, C. H., AND J. P. PETERS, 1937. The osmotic pressure of proteins in whole
serum. Jour. Clin. Invest., 16: 93.

4, DRINKER, C. K., AND M. E. FieELp, 1933. Lymphatics, Lymph, and Tissue
Fluid. Baltimore.

5. Keys, A., AND R. M. Hitx, 1934. The osmotic pressure of the colloids in fish
sera. Jour. Exper. Btol., 11: 28.

6. Krocu, A., AND F. Nakazawa, 1927. Beitrage zur Messung des kolloid-

_ osmotischen Druckes in biologischen Fliissigkeiten. Bzochem. Zeitschr.,

188: 241.

7. Turner, A. H., 1932. The validity of determinations of the colloid osmotic
pressure of serum. Jour. Biol. Chem., 96: 487.

510 MILDRED L. CAMPBELL AND ABBY H. TURNER

8.
Sh

10.

11.

12,

14,

PETERS, J. P., AND D. S. VANSLYKE, 1932. Quantitative Clinical Chemistry,
Vol. II. Methods. Baltimore.

Hanp, H. M., 1934. Concentration of serum proteins in the different types of
edema. Arch. Internal Med., 54: 215.

Friscu, R. A., L. B. MENDEL, AND J. P. Peters, 1929. The production of
edema and serum protein deficiency in white rats by low protein diets.
Jour. Biol. Chem., 84: 167.

BRuCKMAN, F.S., L. M. D’Esopo, ann J. P. PETERS, 1930. The plasma proteins
in relation to blood hydration. IV. Malnutrition and the serum proteins.
Jour. Clin. Invest., 8: 577.

SHELBURNE, S. A., AND W. C. Eciorr, 1931. Experimental edema. Arch.
Internal Med., 48: 51.

WEEcH, A. A., AND S. M. Line, 1931. Nutritionaledema. Observations on the
relation of the serum proteins to the occurrence of edema and to the effect of
certain inorganic salts. Jour. Clin. Invest., 10: 869.

WEEcH, A. A., E. GOETTSCH, AND E. B. REEVES, 1935. Nutritional edema in the
dog. I. Development of hypoproteinemia on a diet deficient in protein.
Jour. Exper. Med., 61: 299.

SERUM PROTEIN MEASUREMENTS IN THE LOWER VER-
TEBRATES. II. IN MARINE TELEOSTS
AND ELASMOBRANCHS

ABBY H. TURNER

(From the Woods Hole Oceanographic Institution; 1 Bergens Museum Biologiske Stasjon,
Bergen, Norway; and the Physiological Laboratory, Mount Holyoke College,
South Hadley, Massachusetts)

The reasons for studying the plasma proteins of the lower verte-
brates as shown in the first paper of this series (1) have to do with
the validity of Starling’s hypothesis for these forms and with the
possible relation of the protein fraction of the plasma to nutritional
needs. References to contributions in this field relating to the higher
vertebrates will not be repeated. For the lower vertebrates, the list
given by Drinker and Field (2) summarizes the information available
up to their date of publication. A brief paper by Keys and Hill (3)
gives the only findings for the colloid osmotic pressure of fish sera
except those in two preliminary notes to this paper (4,5). Interésting
data allied in various ways are to be found in references (6) to (15)
inclusive. The last two papers (14, 15) have excellent bibliographies.

The serum of various species of marine teleosts and elasmobranchs
was studied at the Woods Hole Oceanographic Institution during the
summers of 1933 and 1934, and during the summers of 1935 and 1936
at the Bergens Museum Biologiske Stasjon on the island of Herdla off
the west coast of Norway. ‘To both of these institutions most sincere
thanks are due for the assistance generously rendered by them.
A grant in 1936 from the Tracy McGregor Fund helped to defray
expenses in that year.”

The determinations made were as follows: (1) colloid osmotic pres-
sure, on all samples; (2) refractive index of the whole serum, on all
samples; (3) refractive index on an ultrafiltrate, on many samples of
the Norwegian series, whenever quantity of serum permitted; (4) total
nitrogen and non-protein nitrogen in 1934, 1935, and 1936, a con-

1 Contribution No. 148.

2 The nitrogen determinations of the Woods Hole series were made by Catherine
Goffin and those of the Norwegian series by Cand. mag. Francis Wolff, of Bergens
Museum. Acknowledgment is made of the careful and helpful work done by both.
At Woods Hole the Marine Biological Laboratory made its facilities available to Miss
Goffin for her work; at Bergen the Norwegian Fiskeriforséksstasjon allowed the use of
its equipment for cold storage and for chemical work. Without this help it would
have been impossible to make the nitrogen analyses.

511

912 ABBY H. TURNER

siderable number of determinations, whenever quantity permitted.
In all, 121 individual samples representing 19 species of teleosts were
studied and 48 individual samples from 11 species of elasmobranchs.
A few of these samples were assembled from two or more small fish
each. ‘There were in addition some scattered determinations on other
species.
METHODS
Obtaining the Fish

The fish whose blood is to be studied should be in a healthy con-
dition and must be uninjured, to prevent access of the surrounding
medium to the blood. To secure fish that meet these standards is
difficult. The ordinary methods of fishing are extremely rough when
viewed from the standpoint of obtaining true samples of the circulating
blood. The effect of the severe struggling often incident to capture
may not be negligible. Grafflin (8) has told in detail of his care in
this matter of reliable sampling. The fish included in this study
have been progressively better and none are included in the figures
reported about which there is appreciable doubt. ‘Two alternatives
present themselves. Blood may be drawn the moment the fish is
taken and this is for some species, for example those from deep water
which will not live in tanks, the only possible method; or the fish may
be allowed to rest for some hours or longer in cars or tanks of water
suitable in temperature and aeration. Feeding is possible in some
cases where the fish live well in captivity. The effect of the initial
struggling is thus eliminated and the state of the fish is steadier. At
least four of the most consistent sets of determinations in this study
have been made mainly on fish thus treated, Opsanus tau, Tautoga
onitis, Anarhichas lupus, and Raia erinacea. In certain species the
skin is so delicate that it is probably best not to use them in such
studies as these unless unusual care can be taken. It is possible that
the two pleuronectids in the Woods Hole series belong in this group.

Enough is known about the relation existing between the blood
and tissues of fishes and the demands of the breeding season to make
avoidance of this period desirable in a preliminary survey. However,
the length of this season is often far beyond the months mentioned in
books so that it has not always been possible to avoid it without
sacrificing the use of fish otherwise very suitable. The depression in
the breeding season well-known to trout hatchery men, when the fish
will eat nothing or very little, is of indeterminate length and not easily
avoided. ‘The breeding fish used in this series are so described. When
it is not mentioned that a species was apparently ripe it is to be under-
stood that as far as known the samples were not from breeding fish.

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 513

Blood Sampling

The blood was taken in 1933 by syringe from gill vessels or heart;
in the other three summers, in most cases from tail vessels, occasionally
from the heart. For the suggestion of taking blood from the tail,
the author is indebted to Dr. Homer W. Smith. The fish is wrapped
in a cloth, laid in a deeply grooved board, ventral side up, restrained
by lacing tapes across the board, and held by an assistant. The
syringe needle is inserted in the mid-ventral line, somewhat behind the
anus, often along the anal fin, at an angle which will take it between
the hzemal spines. It is pushed in until the vertebral column is felt,
is slightly withdrawn and pushed in again quickly, with a good chance
of puncturing a tail vessel from which blood can be withdrawn with a
minimum of injury and a maximum of ease. If the tail vessels are too
small for use or are covered by too firm a membrane between the
spines, the heart is exposed, the ventral aorta clamped off by a hemo-
stat, and blood withdrawn from the bulbus or venous sinuses. The
blood must be handled gently when discharged from the syringe into
the centrifuge tubes else hemolysis may occur. The same thing may
happen if the vessel used is not entered quickly and neatly. Heparin
has been used in both syringe and centrifuge tubes, but some clotting
is usually evident after centrifuging, hence the term serum has been
thought more appropriate than plasma.

The blood was centrifuged three or four hours after being with-
drawn, not earlier because in that case delayed and repeated clotting
sometimes occurs and is very troublesome if in osmometer tubes.
After centrifuging, the serum was set up at once in the osmometers or
kept in a very cold refrigerator until next day. Because it was
impossible in Norway to have the nitrogen determinations made on
the first day, various preservatives were tried, but all resulted in the
appearance of a precipitate. Serum kept at about 0° C., on the other
hand, remains clear for a period up to at least three weeks, and re-
fractometric measurements show little or no alteration. At rather
low room temperatures, 16°-19° C., osmometers containing teleost
serum have held steady as long as three days. In the case of elasmo-
branch blood, the occasional rather warm intervals at Woods Hole,
with room temperatures up to 25°, in a few instances caused the
appearance of a thin whitish film on the membrane in the osmometer,
but the colloid osmotic pressure in these cases seldom varied from the
usual range. These films were not seen at the temperature of the
Herdla laboratory, usually 15°-18° C.

A sterile technique was not attempted. The place where the
needle was to be pushed through the skin was carefully wiped with

514 ABBY H. TURNER

cotton. Syringes, needles, centrifuge and transfer tubes, and all
other parts of apparatus coming into contact with the serum were
cleaned systematically as follows. They were washed thoroughly
with water, with soap when needed; they were rinsed thoroughly
first with tap water, then with distilled water, finally with 70 per cent
alcohol; they were dried in an oven at 70°-80° C. Contamination
was not apparent except in the elasmobranch samples of the Woods
Hole series in the hottest weather.

In general, then, while the securing, care, and preservation of the
blood have presented difficulties, the samples included in this report

TABLE [|

Protein determinations on serum of Opsanus tau, toadfish. Fish all of one lot,
kept in aquarium up to 10 days, fed. One or two months after the breeding season.
The length of the specimens varied from ten to twelve inches. Nos. 1 and 6 were
females, the others males. C.O.P. Colloid osmotic pressure in mm. water pressure,
determined from one or two osmometers. N-P.N. Non-protein nitrogen.

No. IN CiOnle= SRG ag Total N. N-P. N. Protein N.
mm./water mg.|cc mg.|cc mg.|cc
1 104 1.34401 — — —
2 119 1.34498 8.13 .866 7.26
3 76 1.34498 9.28 .809 8.47
4 100 1.34470 6.73 494. 6.24
5 95 1.34375 6.77 497 6.27
6 127 1.34369 6.55 419 6.13
a 125 1.34457 5.63 423 5.21
8 111 1.34386 7.44 368 7.07
9 97 1.34509 — — —
10 84 1.34468 — —_ —
Averages 104 1.34443 7.22 554 6.67

are considered reasonably good. However, it is to be said that the
history of the individual fish is in many important points entirely
unknown. Its age, except as indicated roughly by its size, its recent
activity when the fish has been bled immediately on being taken, its
nutritive condition except that it may or may not have food in the
digestive tract, all these factors of importance are not known. A con-
siderable range in findings is to be expected.

The Identification of Species

The identification of species was made at Woods Hole by the use of
Bigelow and Welsh (16) and in Norway by Otterstrém (17). In both
places much help was received from various persons who know the

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 515

fishes of the region. Especial mention should be made of the Director
at the Herdla laboratory, Professor August Brinkmann, and his son,
and of the Assistant-Director, Dr. Rustad.

Methods of Testing

Colloid osmotic pressures were determined by the method of Krogh
and Nakazawa (18) somewhat modified (19). The membrane used
was cellophane no. 450. Refractive indices were determined by the
dipping refractometers of Bausch and Lomb and Zeiss on both whole
serum and its ultrafiltrate. The latter was secured from samples of

TABLE II

Protein determinations on serum of Anarhichas lupus, wolf-fish, 1935. Blood
taken a few hours or a day after fish was brought to laboratory. Not near the
breeding season. The length of the specimens varied from 18 to 30 inches. C.O.P.
Colloid osmotic pressure in mm. water pressure. Derived from two to four osmom-
eters in all cases except where only one osmometer was used, Fish No. 7. N-P. N.
Non-protein nitrogen.

1 119 1.34530 | 1.33532 | 0.00998 6.15 5 5.90
2 178 1.35588 | 1.33609 | 0.01979 10.94 51 10.43
3 147 1.35051 | 1.33609 | 0.01442 7.96 oo 7.44
4 152 1.35128 | 1.33636 | 0.01492 8.55 .62 7.93
5 145 1.34702 | 1.33545 | 0.01157 8.12 ati 7.A1

6 153 1.34832 | 1.33551 | 0.01281 — — —

7 150 1.34415 | 1.33551 | 0.00864 5.82 slit 5.05
8 177 1.34984 | 1.33578 | 0.01406 8.30 ATT 7.53
Averages 153 1.34916 | 1.33576 | 0.01328 7.98 69 7.38

the Norwegian series by a Thiessen apparatus, with care as to uni-
formity of technique. Two cubic centimeters of serum were placed
in the apparatus with cellophane no. 450 as membrane and subjected
to a pressure of some three atmospheres for a period of 12 hours. At
the end of this time a clear, limpid filtrate was obtained, always protein-
free. The residue on the membrane at this time was small in amount
and slimy in character. Tests for chloride and reducing sugar showed
them to be present in the ultrafiltrate. It was assumed that the mem-
brane was permeable to practically all the serum crystalloids.
Nitrogen determinations were made in 1934 at Woods Hole by the
micro-Kjeldahl method with direct Nesslerization. For the non-
protein determinations the precipitant was trichloracetic acid. In

516 ABBY H. TURNER

1935 and 1936 the micro-Kjeldahl method was by titration. The
precipitant was metaphosphoric acid. All methods and reagents were
suitably controlled and tested.

TABLE III

The colloid osmotic pressure of the sera of marine teleosts. Arranged in order
of height of osmotic pressure, in two series.

Number of C.O.P, C.O.P.

Name of species individuals Range Average

mm. water | mm. water

Woods Hole Series

Sarda sarda, Bloch. Bonito................. 1 233 233
Echenets naucrates, Lin. Remora*........... 1 216 216
Hantoga ons MinkwMautog*.. 5 ase ).s- 4. - 24 79-193 129

Paralichthys dentatus, Lin. Summer flounder. . .
Pseudopleuronectes Americanus, Wal. Winter

ko tnauakere rs dst eee-B eee alee eo ae rena ae lee ee 6 79-180 120
Prionotus Carolinus, Lin. Common robin*.... 7 86-122 106
Prionotus strigatus, Cuv. & Val. Red-winged

LOD Ula ee ee RO Pere ney epee Mev oe.) Su evan 7 87-117 102
Opsanus tau, Lin. Toadfish................. 10 76-127 104

Norwegian Series

Scomber scombrus, Lin. Mackerel............ 3 samples 164-212 198
fr. 11 fisht

Anguilla vulgaris, Tur. Eel................. 4 samples 167-204 189
fr. 13 fisht

Belone acus, Risso. Hornfish................ 2 162, 184 173

Anarhichas lupus, Lin. Wolf-fish............ 13 85-181 147

Brosmius brosme, Asc. Cusk}............... 8 105-210 139

Molva molva, Flem.}..............0.000005. 4 108-177 139

Gadus morrhua, Lin. Cod.................. 11 70-192 117

Gadus pollachius, Lin. Poellack.............. 8 samples 31-172. 94
3 fr. 6 fisht

Cyclopterus lumpus, Lin. Lumpfish*......... 3 75-87 81

Lophius piscatorius, Lin. Goosefish.......... 8 25-60 42

Gadusiurrens Meter tebe eee vcys gers, see a ance ais 1 fr. 2 fisht 40 40

* Breeding season.

{| From deep trawl, 300-400 meters. Fish with swim-bladders protruding or
burst and with organs extruded in some cases on arrival at surface. Blood samples
taken immediately on board the collecting boat.

{Samples from more than one fish because of small size of samples available
from single individuals. The refractive indices were found to be similar before
samples were combined.

RESULTS

The results of the study are presented in a series of annotated
tables. The determinations on teleosts are given first. To show the
range of variation among individuals of a single species Opsanus tau,
Lin., toadfish, was chosen from the Woods Hole series of 1934, Table I,

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 517

and Anarhichas lupus, Lin., wolf-fish, Table II, from the Herdla series
of 1935. The toadfish was chosen as a sluggish fish, much studied at
Woods Hole laboratories, and the wolf-fish as a more vigorous species
much studied in Norway.

TABLE IV

Refractive indices of sera and ultrafiltrates from marine teleosts. Arranged
in order of height of C.O.P. Figures in parentheses indicate the number of indi-
viduals used for the following average, when this is less than the total number.

Name of species Aner Oe ny Serum Ps mee
Woods Hole Series ~
SOR SUS aobeian su0 co hws ean og bb. il 1.35220 = —
WIGHENCUS NAUCKOLES Mersin eon 2 1.35158 = —
TE OSXGD. OBES A Van Gio Cee eo Sor 24. 1.34629 = —
Paralichthys dentatus...............
Pseudopleuronectes Americanus...... 6 1.34313 — —
Prionotus Carolinus.............-.- 7 1.34329 — —
LEO OWS SH ALOUND org beaiule Wie tele alee if 1.34205 = —
CO PSUTLUSELILIER ON Det rain it eke ers Sclaye 10 1.34443 = —
Norwegian Series
ISGOMIDCTASCOMORUS eae eiels ar saeco 3 samples | 1.35000 |(2)1.33690 | 0.01306
fr. 11 fish*
Alon io, SOVGT IS yds = Bele as On cee 4samples | 1.35152 1.33552 | 0.01600
fr. 13 fish*
JBOD EO ICSE ance ae ie ee 2 1.35084 1.337247] 0.01360
Anarhichas lupus... .......0.0000.. 13 1.34852 1.33577 | 0.01275
Brosmius brosme.... 1.6.2... eevee 5 1.34703 1.33574 | 0.01169
VIG UCIT OLUA ih een pe Acie eae caraee 4 1.34597 | (2)1.33601 | 0.00912
(GODS GUAT DE pega hence ORC 11 1.34421 1.33588 | 0.00834
Gadus pollachius.............00005. 8 1.34401 1.33594 | 0.00807
3 of these
fr. 6 fish*
CNG G PICTUS ANU DUS A oasis «sos eee oe 3 1.34388 1.33535 | 0.00853
Lophius piscatorvtus.............05. 8 1.34168 1.33545 | 0.00623
(ROIS, TEAZ ISS Rae ATOR ee 1 sample 1.34158 1.33559 | 0.00599
fr. 2 fish*

* See note on Table III.

{+ Samples used for obtaining ultrafiltrates in these cases were only one cubic

centimeter instead of two as usual.

This may explain the aberrant value.

Table III summarizes the data on colloid osmotic pressure from

the teleosts of Woods Hole and Herdla; Table IV, the data on refractive
indices; Table V, the nitrogen determinations. The fish from Woods
Hole and Herdla are listed separately, since the procedures were more
complete in the later series. The order of names in all tables is that
of the relative heights of the colloid osmotic pressure, even though the

518 ABBY H. TURNER

order of refractive indices and of nitrogen content does not follow
exactly the same course.

In a similar manner Tables VI-IX present the data for elasmo-
branchs, Table VI showing the variation among individuals of a single
species, Raia erinacea, Lin., small skate, and Tables VII-IX the
colloid osmotic pressure, refractive indices, and nitrogen findings re-
spectively for the several species of elasmobranchs.

An effort was made to study Myxine glutinosa, Lin., hagfish, but it

TABLE V

Nitrogen determinations on sera of marine teleosts.

Number of Total Protein
N. N-P. N. N.

Name of species san Ferra Fle

Woods Hole Series

WECITEIVEUS LOM CTOLOS PD nine n tm tele re chetis of ekc Soa 1 6.68 PAID 6.41

MGS ULO ZA ON IS Ra ee Ney ten ieee he cs iesdere ee «63 6 6.62 498 | 6.12

Paralichthys dentatus......-0..0.20eeeeeee

Pseudopleuronectes Americanus............ 3 5.48 452 5.03

IPetONOLUS COV OIINUS Mee een eecares see aa eh os 1 7.90 747 Ua

ESOL TEPID we a.ceoasa 8 Ate Nicks eRe 7 7.22 554 | 6.67

Norwegian Series

SS COMMUCHESCOMUOIAUS een a tedets Welel Ac\aiel fete +)6%- 2samples | 8.30 65 7.66
fr. 9 fish*

Mpaontts SACS 33 ae 60 8 6 On 8 on Soe re Ree 3 samples | 8.47 | 1.19 7.28
fr. 11 fish*

PAHOA RUS [TALIS Vee o 00 oaks SOR aoe 10 8.11 .70 7.48

> IEOSTTRIS HADES oi, SG 6 30 0k a DO oe ae 1 6.95 7) 6.38

Gadusnmornhugaereoee eon ae eee: 5 5.93 .60 5.33

GMb Usp POUAChiUse Ow eee tee See ei sarns 3 samples | 6.11 Ay {ae Ls
fi Salishin t

Ciclo Piers Pus) Vie wert) he a Nokes che a oie 3 Sasi 125 5.09

WO PIAS PUSGOLOTIUS |: due wiry Ae eco. oe ess: 8 4.85 44 4.41

(CHGS TARA ES 55 GAO Old Be ee Dieters ene 1 sample 3.99 39 3.60
fe.) 2 fish*

* See Table III.

was found very difficult to secure blood samples entirely uncon-
taminated by slime. The individuals were not large enough to yield
more than about one cubic centimeter of blood, often less, and there-
fore of necessity the determinations were made on mixed samples,
though the refractive indices were taken before mixing and only
samples of similar indices combined. From one such mixed sample
two osmometers gave an average of 24 mm. c.o.p., the total serum
showed a 7p of 1.35073, the ultrafiltrate mp 1.33923, thus giving a
colloid index of 0.01150. The total N of the serum was 5.88 mgm./cc.

TABLE VI

Protein determinations on serum of elasmobranch, Rata erinacea, small skate.
Fish of different lots, kept in aquarium not more than three or four days. Height
of breeding season, several eggs laid in aquarium. The length of the specimens
varied from 17 to 19 inches. Nos. 2, 3 and 9 were males, the rest females.

C.O.P. Colloid osmotic pressure, determined from one to three osmometers,
usually two.

N-P. N. Non-protein nitrogen.

No. AvaGO Pam echves |! “Total N. N-P. N. Protein N.

mm.|water mg.|cc. mg.|cc. mg.|cc.
1 63 1.34694 16.83 — ==
2 73 1.34886 16.95 — =
3 14 1.34665 — — =
4 50 1.34581 — — —
5 47 1.34895 — — —
6 71 1.34765 — — =
a 17 1.34661 15.91 = a
8 16 1.34661 14.78 13.55 1.23
9 14 1.34849 13.13 13.02 0.11
10 34 1.34683 16.39 13.19 3.20
lal 19 1.34618 16.39 13.71 2.68
12 29 1.34640 16.75 13.88 2.87

Averages Sif 1.34715 15.497 13.47} 2.02

{ Five only used for average.

TABLE VII

The colloid osmotic pressure of the sera of elasmobranchs, arranged in the order
of the height of the osmotic pressures.
C.O.P. Colloid osmotic pressure.

: Number of COLE: C.O.P.
Name of species individuals Range Average

mm.|water | mm./water

Woods Hole Series

Carchartas taurus, Raf. (sp.?) Shark.......... 1 46 46
Mustelus canis, Mitch.{ Smooth Dogfish....... 4 33-58 41
Raia erinacea, Mitch.} Small Skate........... 12 14-73 37
Dasybatus marinus, Klein. Stingaree.......... 1 36 36
Norwegian Series

IROOE: HOUND PAIR, NODA ON Re Gale Bis ere a Ge 2 14, 49 32
Chimexra monstrosa, Lin.t.................... 5 * 10-40 27
Galeus vulgaris, Flem. Shark................. 1 Dil 27
Squalus acanthias, Lin.{ Spiny Dogfish........ 2 De, oil 27
Prastiurus catulus, Gun.t.s..1. ssc e 7 5-59 26
ROG ORM APSARA LIS, NUM ha ah oe bs aca ch eb asnee 6 11-45 22
HSE O PLer US SPEMas leitledy anise oie Aor eee ieee 7 samples

fr. 14 fish* | -—8-34 17

* Individual fish small, about 12-14 inches. Samples were therefore combined,
but only after refractive indices had shown them to be in the same range. Two

negative values may indicate the error of the method when the pressure is extremely
low.

{ Breeding season.

t From deep trawl, 300-400 meters. No outward sign of injury on coming to
the surface. LE. spinax and R. fullonica lived often as long as 24 hours in laboratory
tanks.

519

520 ABBY H. TURNER

TABLE VIII

Refractive indices of sera and ultrafiltrates from elasmobranchs. The number
in parentheses (4) indicates that the following ~p was derived from 4 individuals
only. See notes on Table VII.

Name of species muntabee Gt np Serum foe 1 Colloid
Woods Hole Series
Carcharias taurus.............. 1 1.34601 = —
Mustelus canis}............... 4 1.34618 — —
Rava erinaceay............+..- 12 1.34715 — —
Dasybatus marinus............ 1 1.34682 — —
Norwegian Series
Ratenulanico eee eee 2 1.34852 1.34079 | 0.00774
Chimexra monstrosat........... 5 1.34596 | (4) 1.34063 | 0.00486
Galeusiaulearis sone. che 1 1.34422 1.34113 | 0.00309
Sgualuscacanthiasiten. = ck veureae 1 1.34671 1.34096 | 0.00575
Pristuurus catulust............. 7 1.34423 1.34076 | 0.00331
Raia oxyrhynchus} t........... 5 1.34412 1.34070 | 0.00342
Himopreris spinaxy i)... 50s o. a. 7 samples | 1.34353 1.34055 | 0.00288
fr. 14 fish*

the N-P.N., 0.22 mgm./cc. These figures do not seem concordant,
but are reported since data on this form are few.

Body fluid was present in one instance only in quantity sufficient
for collection and study. This was in a wolf-fish of medium size and

TABLE IX

Nitrogen determinations on sera of elasmobranchs. See notes on Table VII.
Number in parentheses (1) indicates that in only one instance was there serum
enough for the determination of the N-P. N.

‘Name of species Buber ier Total N N-P.N. Protein N.
a mg.|cc. mg.|cc. mg.|cc.
Woods Hole Series
RO TONCLI ACEO ee ea er 5 15.49 13.47 2.02
Dasybatus marinus. ..........0.0.05. 1 16.85 11.81 5.04

Norwegian Series

ROTOR ON TCU IIR ten peice oe tes 5 3 17.50 12.16 5.34
Chimera monstrosat................. 1 13.91 11.15 2.76
Galeuswuul carts era hie ers. 1 13.76 11.47 2.29
aS COUTTS Ss, oe 6 bein Geel aie 1 14.98 12.68 2.30
Pristvurus catulusi.................. 3 | 12.72 9.58 3.14
(ROTANOGN HIN ICIS LR ey re ee ce 6 15.07 12.31 2.76
Etim pleres SPrTann. mate lee ase = A - 4samples | 12.18 | (1) 10.07 1.60

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 521

medium serum values. There was nothing to indicate that the fish
was not in a healthy condition. The data are given in Table X. The
body fluid showed a c.o.p. nearly four-fifths that of the serum, and
while the refractive index did not show quite as high a colloid fraction,
yet it is clear that this fluid contained a considerable amount of
protein, an amount comparable to that found in the body fluid of the
‘‘slider’’ turtle (1).

DISCUSSION OF RESULTS

Regarding the results on teleosts, it is apparent that the findings
for a single species show a range proportionately wider that we expect
in mammals. Such a lack of constancy has been found by other
workers on fish blood. - (See references in the first paragraph of this
paper.) Whether this is really true or due, in spite of efforts to the
contrary, to the use of unsuitable specimens is not known. Studies
are in progress on fresh-water species living in the controlled environ-

TABLE X

Comparison of serum and body fluid from one specimen of Anarhichas lupus
from which enough body fluid was obtainable for colloid osmotic pressure and re-
fractometer tests though not for nitrogen analyses.

A n, Serum n,, Ultra- .
Hae ea or fluid filtrate tore es
mm./water
Blood serum......... 129 1.34707 1.33563 0.01144
Body fluid........... 98 1.34184 1.33532 0.00652

ment of fish hatchery ponds to see if the variation from individual to
individual is as great as that seen in the toadfish, 76-127 mm. or in
the wolf-fish, 119-178 mm. water pressure. Also the yearly rhythm
is being followed to see whether colloid osmotic pressure shows the
effect of elaboration of sex products and of spawning depression.
There is obviously a wide difference between the colloid osmotic
pressure of the goosefish, 42 mm. and that of the mackerel, 198 mm.
This latter value is similar to that found by Keys and Hill (3) for
Anguilla, the European eel, and verified in this series. The ranges
for individuals of these species are seen to be mutually exclusive, but
those for species nearer the middle of the total range overlap freely
and some are indistinguishable, as for instance the two species of
Prionotus. To what this characteristic range for a species is related
is not known though various suggestions have been made. Arterial
pressure determinations on fish are few and reliable determinations still
fewer (20), while determinations of capillary pressure have not been

522 ABBY H. TURNER

made at all. The degree of habitual activity has been suggested as a
clue, for Lophius is proverbially sluggish and Scomber very active, while
the extensive migrations of Anguilla are well-known. ‘The series is as

SPECIES Fs

a D

TELEOSTS
SCOMBER SCOM BRUS
ANGUILLA VULGARIS
BELONE ACUS
ANARHICHAS LUPUS
BROSMIUS BROS ME
MOLVA MOLVA
GADUS MORRHUA
GADUS POLLACHIUS
GYCLOPTERUS LUM PUS

LOPHIUS PISGATORIUS

GADUS VIRENS

ELASMOBRANCHS

RAIA FULLONICA

CHIMAERA MONST ROSA

GALEUS VULGARIS

SQUALUS ACANTHIAS

PRISTIURUS CATULUS

RAIA OAY RHYNCHUS

ETMOPTERIS SPINAX

CuHarT 1. Refractive indices of colloid fraction in Norwegian series of teleosts
and elasmobranchs. The left end of each line shows the refractive index of the
ultrafiltrate, the right end that of the whole serum. The length of the line therefore
shows the index of the colloid fraction. The ultrafiltrates in all of the teleosts except
two are closely grouped, indicating relative constancy in the non-colloid components
of thesera. The two exceptions are in fish which were represented by limited samples
only, in the case of Belone by samples for ultrafiltration of only half the usual size.
The high position of the ultrafiltrate index for all elasmobranchs is related to their
high non-protein nitrogen fraction.

yet too short to give an answer here. The possibility of other sub-
stances which may add to the colloid osmotic pressure of the serum,
e.g. fats (21, 22) is not to be excluded particularly when one occasion-

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 523

ally sees globules of fat at the top of a centrifuged tube of blood and
infers the presence of associated substances in solution in appreciable
amounts. Also the wide variation in the fat content of fish is a matter
of common knowledge. No determinations on serum fats have been
made. The albumin-globulin ratios in fish are said by Lepkovsky

—— HA "sate
222% COLLOID

ex @ PROTEIN N tb hes Ps
SCOMBER SCOMBRUS
ANGUILLA VULGARIS

BELONE ACUS

ANARHICHAS LUPUS

BROSMIUS BROSME

MOLVA MOLVA

GADUS MORRHUA,

GADUS POLLACHIUS

CYCLOPTERUS LUMPUS

LOPHIUS PISCATORIUS

GADUS VIRENS

Cuart 2. Comparison of colloid osmotic pressure, refractive index of colloid
fraction, and protein nitrogen in Norwegian teleost series. The several ranges have
been adjusted so that the maximum values are shown by lines of approximately equal
length. The relative values of the three measurements thus appear, with a range
much wider in c.o.p. than in np or protein N. This is probably to be explained by a
variation in the albumin-globulin ratio in the direction of large molecules when the
total quantity is small.

(11) to vary widely. Such variations, with their attendant contrast
in the size of the protein molecules, may help to explain not only
differences between species but irregularities in the relationships be-
tween colloid osmotic pressure and other protein data. A few pre-
liminary studies on plasma protein regeneration after depletion by

524 ABBY H. TURNER .

hemorrhage seem to indicate that protein may come back rather
quickly, perhaps more quickly in total quantity than in colloid osmotic
pressure, which may be the same as saying that the globulins re-
generate—or are gotten into the blood—more rapidly than the
albumins. This obviously suggests the experiments on mammalian
protein regeneration from the laboratories of Whipple and Weech, to
whose papers only partial references are given (23, 24, 25).

As the refractive indices are scrutinized, it is apparent that the
range for the ultrafiltrates is narrow, indicating constancy in the non-
protein fraction of the plasma, while the refractive index for the colloid
fraction varies widely from species to species and accompanies the c.o.p.
though the correspondence is not too close. (See Chart 1.) The
analyses for total and non-protein nitrogen similarly give values for the
serum proteins which vary approximately with the c.o.p. but follow
more closely the refractive indices of the colloid fractions. These
correspondences and differences are brought out in Chart 2. It is to
be noted that the range of c.o.p. from the species of low to those of
high values is much greater than the range in protein content. This
fact may be associated with the ready appearance of protein molecules
of large size mentioned above. It is possible that in the life economy
of teleosts it is of especial importance to hold the total protein content
of the serum at a definite level while the assortment of proteins may
vary. It would follow from this that a rigid maintenance of colloid
osmotic pressure is impossible or unnecessary.

For the elasmobranchs it is to be said that all averages for colloid
osmotic pressure are low, though occasional individuals have been
found as high as. 70 mm. water pressure. As expected, the total and
non-protein nitrogen findings, the refractive indices for total sera and
for ultrafiltrates are all high, reflecting the high urea or trimethylamine
oxide content of elasmobranch blood. The protein nitrogen figures
are low with two exceptions, one specimen of Dasybatus marinus,
stingray, and three of Raia fullonica, a Norwegian skate. Differences
in the physiological make-up of elasmobranchs and teleosts thus
include the protein blood picture.

SUMMARY AND CONCLUSIONS

1. In a study of 121 individuals of 19 species of marine teleosts it
has been found that the range of colloid osmotic pressure shown by
the blood serum is wide for a single species; that the c.o.p. shows a
characteristic range in each species; and that for the species studied
the c.o.p. varies from an average of 42 mm. water pressure in Lophius
piscatorius, Lin., goosefish, to about 200 mm. in Scomber scombrus,

SERUM PROTEIN MEASUREMENTS LOWER VERTEBRATES, II 525

Lin., mackerel, Anguilla vulgaris, Tur., eel, and probably others.
Intermediate values for averages within species were found most
commonly, from about 100 to 150 mm.

2. The refractive indices for the colloid fractions of the sera and
the protein nitrogen values follow the same general distribution as the
c.o.p. though correspondence is not perfect. The c.o.p. seems to
vary through a much wider range than the protein N or the refractive
index of the colloid fraction.

3. All protein values for the 48 individuals of 11 species of elasmo-
branchs were found to be low, distinctly lower than in all save the very
lowest of the teleosts.

4. The refractive indices of the ultrafiltrates from the sera in all
teleosts were very constant as compared with the wide range of the
colloid figures. ‘This constancy, reflecting the crystalloid status of the
sera, is also characteristic of the elasmobranch ultrafiltrates though
the absolute level is materially higher in the latter group, a fact
associated obviously with the high non-protein nitrogen content of
elasmobranch blood.

BIBLIOGRAPHY

1. CAMPBELL, M. L., AnD A. H. Turner, 1937. Serum protein measurements in
the lower vertebrates. 1. The colloid osmotic pressure, nitrogen content,
and refractive index of turtle serum and body fluid. Bzol. Bull., 73: 504.
Preliminary note in Am. Jour. Physiol., 116: 24 (Proc.).

2. DRINKER, C. K., ano M. E. FIeLp, 1933. Lymphatics, Lymph, and Tissue
Fluid. Baltimore.

3. Keys, A., AND R. M. Hitt, 1934. The osmotic pressure of the colloids in fish
sera. Jour. Exper. Biol., 11: 28.

4. Turner, A. H., 1935. The colloid osmotic pressure of the blood plasma in fishes.
Am. Jour. Physiol., 113: 132 (Proc.).

5. TurNER, A. H., 1936. Serum protein of fishes: colloid osmotic pressure and
other data. Am. Jour. Physiol., 116: 155 (Proc.).

6. Dents, W., 1913. Metabolism studies on cold-blooded animals. II. The blood
aril nice of fish. Jour. Biol. Chem., 16: 389.

7. Denis, W., 1922. The non-protein eens constituents in the blood of marine
fish. Jour. Biol. Chem., 54: 693.

8. GRAFFLIN, A. L., 1935. @ilecde and total osmotic pressure in the blood of
marine teleosts. Biol. Bull., 69: 245.

9. GraFFLIN, A. L., 1936. Renal function in marine teleosts. III. The excretion
of urea. Biol. Bull., 70: 228.

10. Keys, A., 1933. The mechanism of adaptation to varying salinity in the common
eel and the general problem of osmotic regulation in fishes. Proc. Roy. Soc.,
Ser. B., 112: 184.

11. LeprKovsxy, S., 1930. The distribution of serum and plasma proteins in fish.
Jour. Biol. Chem., 85: 667.

12. Smitu, H. W., 1929. The composition of the body fluids of elasmobranchs.
Jour. gia Chem., 81: 407.

13. Smitu, H. W., 1929. The composition of the body fluids of the goosefish (eve
aisea terre). Jour. Biol. Chem., 82: 71.

526 ABBY H. TURNER

14.
5
16.
il7fe

18.

iQ).
20.
alle
Dyup

US).

24.

25%

SmitTH, H. W., 1932. Water regulation and its evolution in the fishes. Quart.
Rev. Biol., 7: 1.

Situ, H. W., 1936. The retention and physiological réle of urea in the Elasmo-
branchii. Biol. Rev., 11: 49.

BIGELow, H. B., AnD W. W. WEtsu, 1924. Fishes of the Gulf of Maine. Bull.
U.S. Bur. Fish., 40 (Part 1): 1.

OTTERSTROM, C. V., Fisk. Danmarks Fauna. 3 vols. No. 11, 1912; No. 15, 1914;
No. 20, 1917. Copenhagen.

Krocu, A., AND F. Nakazawa, 1927. Beitrage zur Messung des kolloid-
osmotischen Druckes in biologischen Fliissigkeiten. Biochem. Zeitschr.,
188: 241.

TurRNER, A. H., 1932. The validity of determinations of the colloid osmotic
pressure of serum. Jour. Biol. Chem., 96: 487.

VON SKRAMLIK, E., 1935. Uber den Kreislauf bei den Fischen. Ergebn. d. Biol.,
11: 1.

FisHBERG, E. H., 1929. The relations of serum proteins and lipids to the
osmotic pressure. Jour. Biol. Chem., 81: 205.

Man, E. B., AND J. P. PETERS, 1933. Permeability of capillaries to plasma
lipoids. Jour. Clin. Invest., 12: 1031.

MADDEN, S. C., P. M. WinsLow, J. W. HowLanp, AND G. H. WHIppPLe#, 1937.
Blood plasma protein regeneration as influenced by infection, digestive
disturbances, thyroid, and food proteins. A deficiency state related to
protein depletion. Jour. Exper. Med., 65: 431.

KnutTl, R. B., C. C. Erickson, S. C. MappEn, P. E. REKERs, AND G. H.
WuHippLeE, 1937. Liver function and blood plasma protein formation.
Normal and Eck fistula dogs. Jour. Exper. Med., 65: 455.

WEECcH, A. A., E. GOETTSCH, AND E. B. REEVEs, 1935. Nutritional edema in the
dog. I. Development of hypoproteinemia on a diet deficient in protein.
Jour. Exper. Med., 61: 299.

CENPMES SAND ei aISiOlOG i tOr Trl COLOR 2 Adan
IN THE NORMAL AND ALBINO PARADISE FISH,
MACROPODUS OPERCULARIS L.!

H. B. GOODRICH AND MAURICE A. SMITH
(From Wesleyan University and the Marine Biological Laboratory, Woods Hole, Mass.)

The paradise fish is well known to fish fanciers. It is thought to
have been the first ‘‘fancy”’ tropical fish to be bred in Europe, having
been first introduced in Paris by Carbonier in 1868. Specimens of the
albino variety utilized in this investigation were obtained from dealers
in New York City during the fall of 1934. These were at that time a
novelty and it is stated by Innes (1935) that they were first imported
from German fanciers in 1933.

The paradise fish is one of the labyrinth fishes and is referred to
Order Labyrinthici, Family (368) Osphronemidz in the classification
by Jordan (1923). It is also known in the literature as Macropodus
viridi-auratus. A description is given by Regan (1909). This fish is
found in the lowland streams of China, Formosa and Cochin China.
It is well fitted to live in stagnant waters because its accessory re-
spiratory apparatus, the labyrinth organ, allows it to use atmospheric
oxygen as well as to breathe by gills. On this account almost no
attention needs to be given to aeration of water in an aquarium and it
may be kept in very small containers.

The paradise fish is a “bubble nest’’ builder. The male, pre-
sumably by aid of some oral secretion, makes a floating structure of
bubbles into which he shoots the eggs as they are laid by the female.
The mating and spawning activities, in our experience, take place at
temperatures ranging from 26° C. to 29° C. Certain fish dealers, how-
ever, state that they will breed at as low a temperature as 20° C.
Studies made by Goodrich and Taylor (1934) on a species Betta
splendens, a related genus, showed a remarkably precise limitation of
the breeding activities to a temperature of about 26°C. It is neces-
sary, under aquarium conditions, to remove the female after laying, as
she may eat the eggs, and to remove the male after hatching as he may
eat the young fish. The young fry are first fed from Paramecia
cultures and later with Daphnia. The adults are fed with enchytraeids

1 This paper is published as a part of a research program at Wesleyan University
supported by the Denison Foundation for Biological Research.

The senior author wishes to acknowledge his indebtedness to his assistant, Miss
Priscilla Anderson, for carrying certain parts of this work to completion.

527

528 H. B. GOODRICH AND MAURICE A. SMITH

(the white worms of dealers), minced earthworms, liver and dry pre-
pared fish foods. Further details of the care of the fish are given by
Innes (1935). The albino variety is much more difficult to rear. It
is less viable and takes food less readily. It seems to avoid light more
than the dark variety and possibly for this reason it is less likely to
discover food.

GENETIC EXPERIMENTS

The dark or normal colored paradise fish when crossed with the
albino variety produced all dark type in the F; generation. The F2 in
two separate crosses gave the results shown in Table I. The results

TABLE I

Fy from cross between normal and albino varieties

Date of spawning ee of Number. of Ratio
OES =3 Susie esate ees 204 62 3.29/1
IEDAES GAG ct weet ase see Sais 438 145 3.02/1

F, from cross between heterozygous dark and albino

Date of spawning Supe of ee of Ratio
OE ES Spe te ok rn. wire 226 221 1.02/1
SoIDeS On eee rn eare hoes ine OE 552 551 1.002/1

Counts in both cases made eight days after spawning.

of the back cross between the F; and the albino are also shown in
Table I. Similar results were independently obtained by Kosswig
(1935) though with somewhat greater deviation from the expected
ratios. Our counts were made eight days after spawning and before
the onset of the high mortality which is unfortunately frequent in fish
cultures. These results clearly indicate that the normal and albino
types are a pair of mendelian allelomorphs in which the normal is
dominant over the albino.

THE COLOR PATTERN

The normal or wild type paradise fish (Fig. 1) is strikingly marked

on the sides with vertical stripes which are referred to in this paper as
green and red stripes. The color, however, varies with environmental
conditions. ‘The striping may largely disappear when the fish is placed
against a white background. The green stripes may be more accurately
described as being a metallic blue-green when viewed from an angle
which strongly reflects light. From other angles these stripes may

COLOR PATTERNS IN THE PARADISE FISH 529

appear much darker, giving a mottled blue and black appearance. The
red stripes are of an orange-red hue. The markings in the male are
more brilliant than in the female, especially at the breeding period.
The albino (Fig. 2) is light colored and has pink eyes but shows faint
orange and blue-green stripes. As will be shown later in detail, and as
has been mentioned by Kosswig (1935), the black pigment is entirely
absent.

HISTOLOGY OF THE COLOR PATTERN

The cells involved in the production of the color pattern are the
black cells or melanophores, yellow cells or xanthophores, red cells or
erythrophores and those containing reflecting crystals or iridocytes.
There is some evidence for considering that the erythrophores develop
from the xanthophores in this fish. The xanthophores contain yellow
pigment granules
soluble in 95 per
cent alcohol in
about two hours. F1¢. 1. Normal ©
The erythrophores ee
contain orange-red
granules less read-
ily soluble in alco- Fic. 2. Albino

‘ paradise fish. The
hol but sometimes pictures of the two

xanthophores are fish were taken
observedwhichcon- simultaneously in
tain a few granules C7> 79°
similar to those in

the erythrophores. The erythrophores appear later in development
and old fish show more red pigment than young fish.

We recognize three zones or areas of disposition of chromatophores
in relation to the individual scales. All of these are probably dermal as
they lie beneath the stratified epithelial layer of the epidermis. The
relationships of these areas are illustrated by Figs. 3 and 4 which show
diagrammatic reconstructions of median longitudinal sections of a series
of scales taken perpendicular to the body surface. ‘These diagrams are
based upon microtome sections made by three freezing methods and
upon dissections of the body wall made under a dissecting binocular
microscope. ‘The first or superficial zone (a) (Figs. 3 and 4) lies on the
upper outer surface of the scale and is connected by the second and
upward sloping intermediate zone (6) with the third or deep zone
(c) located on the underside of the next anterior overlying scale. This
deep zone also lies above a stratum of fat cells (d) which separates it
from the inner upper surface of the underlying scale.

aK ———— . .

|
|
1
|
|
|

Tics

aoe

|
S|

Sf
nano
\ETAUINAAT

LT}
es

9
aa

ao
ava
Pissscee
GEEZ
H

mal |
a

0)
EY)
ED
=e

WIRIAAT Al ba

ESAS

VS OQWES
FIG. 3
NORMAL

GIRIZIEIN) SWRulee
GIRIEZIEIN) SWRI

AWA

eavnau'

ALBINO

mae
CS 2p

HEX

a0
BY VYYT eT

(US

RED STRIPE
Rizo) Siyailrte*

DAP eT

SAY
BOD:

R
LD
AY
IRIDOCYTE

Lat

@ MELANOPHORE

PAR
bese;
wy,

zy
LF
Woe

xz
=
anvanes200s8428

rs
OSE
a

ue (0
8 8

Fic. 3. Normal dark fish. Diagram of median longitudinal section of a series
of scales. Magnification < 80. (A) Superficial zone of chromatophores; (B) inter-
mediate zone of chromatophores; (C) deep zone of chromatophores; (D) stratum of
fat cells; (Z) connective tissue layer; (F) stratified epithelium; (G) scale; (H/) muscle.
Chromatophores reduced to about 75 per cent of actual number for clarification of
diagram.

Fic. 4. Albino fish. Details as in Fig. 3.

530

COLOR PATTERNS IN THE PARADISE FISH 531

TABLE II

Dark-colored fish. Counts of chromatophores in area of 0.054 sq. mm.
M, melanophores, X, xanthophores, E, erythrophores, I, iridocytes.

Superficial zone

Green stripe Red stripe
M x E I M x E I
2D, 20 0 6 15 26 0 3
23 18 0 3 12 20 0 D,
17 17 0 5 13 20 0 1
18 24 0 4 14 18 0 2
19 ‘23 0 3 18 21 0 5
19 17 0 3 19 20 0 0
25 19 0 4 20 19 0 6
15 22 0 2 21 26 0 3
18 22 0 4 21 23 0 6
19 18 0 0 15 25 0 7
Average first 10....] 19.5 | 20 0 3.4 | 16.8 | 20.6 0 3.5 | Total
Average first 20....| 21.2 | 20.2 0 3.6 | 18.5 | 20.8 0 3.6 | 87.9
Deep zone
Green stripe Red stripe
M x E I M x E I
43 0 34 19 11 3 4
42 0 40 19 5 5 2
38 0 36 17 9 3 2
25 0 25 15 3 3 0
36 0 36 18 5 3) 1
30 @) 20 17 14 3 3
27 0 27 18 8 5 0)
28 0 28 15 15 3 1
38) 0 35 18 9 3 3
37 0 37 14 5 3 3
Average first 10....| 34.1 0 SiS Waly 8.5 | 3.4 1.9 | Total
Average first 20... .| 34.1 0 32.5 | 18.5 That Nero) 2.8 | 99.2

Only the first ten of twenty counts are tabulated but the average of all twenty
is given.

Counts of iridocytes have a greater possibility of error than that of other chro-
matophores because it is difficult to determine the boundaries of individual cells.

Counts of the xanthophores from the deep zone of the dark fish (Table II) are
not tabulated because they were so covered by melanophores and iridocytes that
no accurate count could be made, but they are few in number or absent.

532 H. B. GOODRICH AND MAURICE A. SMITH

In the dark or normal type of paradise fish (Fig: 3) the upper zone
contains a fairly uniform distribution of melanophores, xanthophores
and iridocytes. The iridocytes tend to be superficial to and often

TABLE III

Albino fish. Counts of chromatophores as in Table II.

Superficial zone

Green stripe Red stripe
M xX E I M x E I
0 20 0 14 0 30 0 16
0 27 0 13 0 28 0 19
0 26 0 17 0 30 0 14
@) 29 0 16 0 31 0 17
0 25 @) 14 0 31 0 18
0 27 0 18 0 29 0 10
@) 23 0 10 0 26 0 14
0 24 0 11 0 30 0 15
0 28 0 14 0 23 0 10
0 31 0 17 0 24 0 19
Averace first 10s Oe 26.8 0 faa ON E2802. iO, 15.29) Total
Average first 20....| 0 27.4 0 16.4 0 27.6 0 Tots eves
Deep zone
Green stripe Red stripe
M x E I M xX E I
0 17 0 20 0 13 4 18
0 18 0 23 0 7 3 17
0 12 0 19 0 8 6 10
0 15 0 20 0 6 5 10
0 20 0 17 0 10 3 21
0 15 0 16 0 4 5 11
0 Bil 0 22 @) 13 3 22
0 34 0 23 0 6 4 20
0 31 0 26 0 11 6 15
0 33 0 34 0 10 4 18

8.8 | 4.3 | 16.2 | Total

Average first 10.... : : 0
AMNeTh 0 Dae 0 8.2 | 4.4 | 15.4 | 72.8

Average first 20....

directly above melanophores in all zones. Table II shows the relative
numbers of these cells in both the green and the red stripes. The
counts in the table were made on scales removed from the body and
treated with adrenalin to cause a concentration of pigment in the cells

COLOR PATTERNS IN THE PARADISE FISH 533

and so to help to distinguish one cell from another. The counts were
of cells seen within one quadrant of an ocular counting disc. It is
calculated that the area so outlined is 0.054sq.mm. These counts will
therefore when multiplied by the factor 18.4 give the approximate
number of cells per square millimeter. It will be noticed that there is
probably no significant difference between numbers of cells of the upper
zone in the green and in the red stripes. It is the deep zone which
provides the basis of the striping. Counts were made on the deep zone
after removal of scales and after washing the side of the body with an
adrenalin solution. It will be noted in Table II that in the deep zone
the green stripes, in contrast with the red stripes, show a significant
excess of melanophores and of iridocytes and but few or no xanthophores
or erythrophores and that these latter are found in the red stripes.
The intermediate zone very seldom contains any erythrophores and
there are also fewer iridocytes than in the outer zone. It is much the
same whether located in a green or a red stripe and therefore is, like the
outer zone, neutral in regard to striping.

No melanophores are found in either stripe in the albino fish (Fig. 4
and Table III). Tests with adrenalin to concentrate pigment (cf.
Goodrich, 1927) or use of the ‘“‘dopa”’ reaction (cf. Goodrich, 1933)
failed to indicate the presence of any ‘‘colorless’’ melanophores.
Otherwise the disposition of cells is similar to that in the normal type
except that there are more xanthophores and iridocytes.

DEVELOPMENTAL HISTORY

The melanophores first appear at about the 16-somite stage
-(nineteen hours in our cultures). These are at first rather irregularly
distributed and there is no indication of pattern formation until the
fish is from nine to twelve days of age (about 3 mm). At this time
from six to eight spots appear on both the mid-dorsal and mid-ventral
aspects of the body and tail region. ‘The striping first becomes visible
in the 10 to 14 mm. fish. The number of stripes is comparable
with the number of earlier spots but no certain relationship has been
demonstrated.

DISCUSSION

Histological studies on the light color phases of various fish have
shown that different cellular complexes may produce these light types.
In A plocheilus (Oryzias) latipes a double recessive (Aida, 1921) owes its
absence of color to a reduction of amount of melanin produced in
melanophores and of xanthine in xanthophores (Goodrich, 1927).
This circumstance in other animals has given rise to the term ‘‘color-

534 H. B. GOODRICH AND MAURICE A. SMITH

less’’ chromatophores. In this case the cells seem incapable of
producing the normal amount of chromogen although sufficient oxydase
is present (Goodrich, 1933). In the goldfish it is found that the light
type, the transparent shubunkin, owes its condition to the early
disintegration of melanophores and erythrophores (Goodrich and
Hansen, 1931). In the paradise fish as described above no melano-
phores of any type have been seen at any stage of development. This
latter form is a true albino, as no pigment is present in the eyes in
contrast with the other types named above. The senior author of this
paper has unpublished observations on an albino trout which has no
pigment in the eyes but does have melanophores with reduced pigment
as in Oryzias. In each case among fish so far investigated in histolo-
gical detail a different developmental mechanism is concerned in
producing the light phase.

SUMMARY

1. In the paradise fish, Macropodus opercularis, the dark-colored or
normal type is a Mendelian dominant to the albino.

2. A description is given of the cell groupings which form the basis
of the color pattern.

3. The melanophores are entirely absent from the albino but all
other types of chromatophores are present.

LITERATURE CITED

Apa, TATuo, 1921. On the inheritance of color in a fresh-water fish, Aplocheilus
latipes Temmick and Schlegel, with special reference to sex-linked in-
heritance. Genetics, 6: 554. ;

Goopricu, H. B., 1927. A study of the development of Mendelian characters in
Oryzias latipes. Jour. Exper. Zo6l., 49: 261.

Goopricu, H. B., 1933. One step in the development of hereditary pigmentation in
the fish Oryzias latipes. Bzol. Bull., 65: 249.

Goopricu, H. B., AND I. B. HANsEN, 1931. The postembryonic development of
Mendelian characters in the goldfish, Carassius auratus. Jour. Exper. Zodl.,
59: 337.

GoopricHu, H. B., AND HoytC. Tayior, 1934. Breeding reactions in Betta splendens.
Copeia, December 31, p. 165.

INNES, WILLIAM T., 1935. Exotic Aquarium Fishes. Philadelphia. :

Jorpan, D.S., 1923. A classification of fishes, including families and genera as far as
known. Stanford Univ. Pudl., Univ. Ser., Biol. Sct., 3: 77.

Kosswic, Curt, 1935. Uber Albinismus bei Fischen. Zool. Anzeig., 110: 41.

REGAN, C. TATE, 1909. The Asiatic fishes of the family Anabantide. Proc. Zodl.
Soc. London. Nov. 9: 767.

CHROMATOPHORE REACTIONS IN THE NORMAL AND
ALBINO PARADISE FISH, MACROPODUS
OEE RGULARIS VE:

H. CLARK DALTON AND H. B. GOODRICH 1!
(From the Shanklin Laboratory of Biology, Wesleyan University)

The paradise fish, Macropodus opercularis L., was chosen for this
study because there are two varieties, the normal dark type and the
albino. It was thought that the albino would afford special oppor-
tunity for studying the erythrophores and xanthophores because these
are not hidden by the melanophores, as they are in the dark type.
These two varieties would then give a good basis for comparing the
reactions of the three types of chromatophores.

The paradise fish is well known to fish fanciers. Its native habitat
is the shallow coastal streams of southeastern Asia. Details in regard
to the characteristics and care of this fish may be found in the book on
exotic aquarium fish by Innes (1935). The albino variety apparently
was first imported into this country from Germany in 1933. This
variety has been shown by Kosswig (1935) to be a Mendelian recessive
to the normal dark form. The histological details of the color pattern
of the two varieties are described by Goodrich and Smith (1937).

CoLoR REACTIONS

The adaptations of the paradise fish to various colored backgrounds
have been studied by methods used by previous investigators (cf.
Mast, 1916; Connolly, 1925, and bibliography by Parker, 1930). The
fish were subjected to tests in small aquaria with white, black, red,
yellow, and blue paper jackets and uniform illumination from above.
Macroscopic observations on the normal dark type showed typical
color adaptations to each environment. The color responses to the
red and the yellow backgrounds were relatively similar but yet seemed
to be definitely distinguishable. Thealbino fish, in which melanophores
are entirely lacking (Kosswig, 1935; Goodrich and Smith, 1937),
showed differing responses against black, white, red and yellow
backgrounds. The greatest contrast, however, was between the very
pale appearance, when placed against the white background, and the

1 This paper is published asa part of the research program of Wesleyan University
supported by the Denison Foundation for Biological Research.

535

536 H. CLARK DALTON AND H. B. GOODRICH

various slightly differing combinations of yellow, orange and red given
in the reactions to the black, yellow and red backgrounds.

The results of microscopic examinations of the changes in the
chromatophores are summarized in Table I. Twenty normal dark
fish were available but only four albinos. Every one, however, of
these albinos was tested against each background. It will be noted
that in the normal dark fish the black, yellow and red cells react
essentially alike to environments of black or white or red by showing
the dispersed pigment in the black and red environments and concen-
trated pigment in the white environment. On the yellow background,
however, the pigment in the black cells is concentrated, while that in
the yellow and red tends to be dispersed. This gives the fish a light
yellow color. The reverse situation occurs in blue surroundings, where
the yellow and red cells show concentrated pigment and the black cells

TABLE [|

Chromatophore behavior in the paradise fish

D = dispersed pigment; C = concentrated pigment; J = intermediate state.

Dark variety Albino variety
Mel. Xan. Ery. Mel. Xan. Ery.
Bilan mata kas eee D D D and C — D D
AVAL C= a sees a eos ins C C Ge — I Cand I
Rede hemes D D DandC — D D
Mellowey Struss eee ee: C D ITandC — D D
Beng nse ce ee D C C — Cand J | Cand J

dispersed pigment. The reactions of the melanophores are more rapid
than those of the other two types. The erythrophores behaved in
general similarly to the xanthophores, except that they were slightly
slower and less extensive in their reactions.

EXPERIMENTS WITH DENERVATED CHROMATOPHORES

After the study of the color changes and their relation to the
activities of the chromatophores, attention was next turned to the
problem of the physiological control of the chromatophores. The
methods used by G. H. Parker (19340) were adopted.? The nerves
supplying a part of the caudal fin are severed by making a cut across
one of the fin rays. This operation leaves a small area between the
cut and the edge of the fin without central nervous control. The

2 Kamada (1937), whose paper has just come to hand, has also induced caudal
bands in Macropodus opercularts.

CHROMATOPHORE REACTIONS IN PARADISE FISH 537

normal dark fish lay quietly in the dish when the incision was made, but
the albino required an anaesthetic, for which purpose ether was used.
The experiments, except as otherwise noted, were carried out at room
temperature, which remained relatively uniform, the water tempera-
ture averaging 23.5° C.

Immediately upon sectioning the nerve of a dark-colored, or normal
fish the melanophores in the area between the cut and the outer edge
of the caudal fin began to disperse their pigment. A clearly-defined
dark band formed within one minute and it extended laterally to
include half of the area between the cut ray and the two adjoining rays.
When the pigment of the melanophores is dispersed, it is more difficult
to observe other types of pigment cells. Nevertheless, it could be seen
that the pigment in the xanthophores was also dispersed, but the
reaction was somewhat slower than that of the melanophores. Three
to five minutes was required for a degree of dispersion comparable to
that shown in one minute by melanophores. The erythrophores lagged
slightly behind the xanthophores in dispersing their pigment. These
observations were confirmed later on albinos, where no melanophores
obscure other types of cells.

When fish with these dark caudal bands were placed in white
porcelain dishes illuminated from above, the dark bands faded in about
ten hours, as the denervated melanophores gradually assumed the
punctate condition. The reverse reaction, of denervated melanophores
assuming the dispersed phase after concentration, is much more rapid,
taking place in two to three hours.

As noted above, the xanthophores and erythrophores can be easily
observed in the albino. The sectioning of a nerve was followed in one
or two minutes by a detectable dispersion of pigment in these cells,
which, however, take several minutes to complete the reaction. This
band, although it contains no melanophores, is nevertheless striking
because the rest of the fin with punctate xanthophores and erythro-
phores is very light. Figure 1 shows the edge of such a caudal band in
the albino fish. Although these albino fish remained under the same
conditions as the normal ones, there was a marked difference in that the
caudal bands faded in about four hours, or less than half the time
required by those in the dark-colored fish.

Certain other experiments devised by G. H. Parker (1934c), which
have special bearing on the neurohumoral hypothesis, were tried
with especial reference to the reactions of the erythrophores. The
material was limited, but the results seem to be clear. Dark fish were
operated on to produce a caudal band in each and then placed in white
dishes until the bands were completely faded. Other cuts were then

538 H. CLARK DALTON AND H. B. GOODRICH

made producing two fresh dark bands, one on either side of the distal
half of the faded band. The chromatophores in the proximal half of
the faded band remained concentrated, as were those in other parts of
the tail (the new dark bands excepted). In the distal half, however,
the chromatophores became dispersed, corresponding to the condition
of their immediate neighbors in the fresh dark bands. The dispersal
occurs first and to the greatest extent at the sides of the faded band.
It will be recognized that these observations parallel those made by
G. H. Parker on Fundulus and so support the theory of neurohumors
as he has applied it to explain the reactions of chromatophores in fish.
The humor is thought to spread from the new dark band, where it has

. tae io

Fic. 1. Photomicrograph of edge of caudal band in albino fish. The pigment
cells are erythrophores. 95 X.

been recently liberated, to the faded band and to stimulate there the
dispersion of pigment in the melanophores.

The same experiment was tried on albinos to test the occurrence of
a similar dispersing neurohumor governing the erythrophores. A
definite expansion was obtained in the distal half of the flanked bands
in two albinos in which both lateral bands were successfully induced.
It did not occur in two cases where only one flanking band was
produced.

The reactions of the denervated melanophores differ from those of
the xanthophores and erythrophores. This was shown by observations
made when normal dark paradise fish with faded caudal bands were
transferred from a white dish to a yellow environment. The melano-

CHROMATOPHORE REACTIONS IN PARADISE FISH 539

phores remained concentrated, but the xanthophores and erythrophores
became gradually dispersed. Each type of chromatophore assumed
the same condition of pigment distribution as the corresponding type of
innervated pigment cells in the surrounding area.

TEMPERATURE EXPERIMENTS

As a further test of the neurohumoral hypothesis, an experiment
was carried out to determine the effect of temperature on the rate of
disappearance of bands produced by cutting nerves in the caudal fins of
normal fish. If the fading of such bands is indeed due to the dispersion
of an oil-soluble substance from cell to cell, as Dr. Parker’s theory
indicates, it might be expected that the dispersion should occur more
rapidly at a higher temperature than at a lower one, in accordance
with laws of diffusion. Accordingly, fish were operated on as previ-
ously described for the formation of dark caudal bands and isolated in
white porcelain dishes illuminated from above. Two determinations
were made with each fish, one at a temperature of 20° C. and the other
at about ten degrees higher. The bands were considered faded when
they could no longer be distinguished by the unaided eye when the
fish were observed in a paraffin-lined Petri dish with a white surface in
the background. The following table summarizes the data obtained:

TABLE II

Effect of temperature on rate of caudal band disappearance in normal fish

Observations 1-4: Low = 20° C., high = 29° C. Observation 5: Low = 23° C.,
high = 27°C. Figures are elapsed hours after operation when bands became so
faint as to be practically indistinguishable.

Low High
TN e a SaaS 18 hrs. 8 hrs.
DR Aa RR 10 5
ES Oi ote 10 5
AR OS Charo), 10 5
Bio SAG eee US 3

The results of this experiment are what would be expected on the
basis of the neurohumoral hypothesis; i.e., that the dark bands
disappear more rapidly at a higher temperature than at a lower one.
Furthermore, it appears that with a rise in temperature of approxi-
mately ten degrees, the time required for disappearance is reduced very
nearly one half.

DISCUSSION

The action and control of chromatophores in the paradise fish are
found to be similar to that reported for certain other species. The
-rate of reaction of these processes, however, is strikingly different.

540 H. CLARK DALTON AND H. B. GOODRICH

This disparity shows particularly in the rate of disappearance of dark
bands produced by denervating chromatophores in the caudal fin.
Caudal bands in Fundulus (Parker, 1934a) fade in twenty-two to
ninety-six hours, while those in the catfish (Parker, 19345) fade only
after two or three days and may last as much as seven days. In
contrast to these conditions, bands in the normal paradise fish fade in
approximately ten hours, and in the albino, in about four hours. The
activity of the concentrating neurohumor, then, is more rapid than in
the fish previously reported.

This greater speed of action applies also to the dispersing neuro-
humor. Parker (1934a) reported that in Fundulus light bands,
produced by transferring fish with faded caudal bands from white
surroundings to black, fade in seventeen to twenty-seven hours. In the
paradise fish, however, similar bands fade in only two and a half hours.

That caudal bands in the albino should disappear in approximately
half the time taken by fading bands in the normal fish is a puzzling
problem. Conceivably the neurohumor (or humors) produced in
regulating the xanthophores and erythrophores might be more soluble
than that for controlling melanophores and, consequently, become
dispersed more readily than the latter. At any rate, a difference in
chemical or physical properties of the two substances seems to be
indicated.

The independence of action of melanophores and xanthophores is
similar to that described by Abramowitz (1936) for Fundulus majalis.
In the paradise fish, however, the erythrophores have also been studied
and their reactions are found to be similar to but a trifle slower than
those of the xanthophores. This gives additional evidence of the pos-
sible close relationship between xanthophores and erythrophores as
suggested by Goodrich and Smith (1937).

The results obtained which show that an increase of temperature
brings about an increase of rate of disappearance of the dark caudal
bands may have bearing on the theory of neurohumoralism. It
supports the concept that the effect is produced by a diffusing chemical
substance.

SUMMARY

1. The normal paradise fish adapts itself by appropriate color
changes to environments of black, white, red, yellow, and blue.

2. Analogous but less adaptive changes occur in the albino.

3. The reactions of melanophores, xanthophores and erythrophores
which produce these color changes are described.

4. Dark caudal bands, formed by cutting chromatophore nerves in
the caudal fin of normal paradise fish fade in approximately ten hours.
Similar bands in the albino fish fade in about four hours.

CHROMATOPHORE REACTIONS IN PARADISE FISH 541

5. The evidence indicates the presence of independent dispersing
neurohumors for melanophores and for erythrophores.

6. The rate of caudal band disappearance is directly proportional
to the temperature, and a rise of about ten degrees in temperature very
nearly doubles this rate.

LITERATURE CITED

ApraAmowitz, A. A., 1936. The non-identity of\the neurohumors for the melano-
phores and the xanthophores of Fundulus. Am. Nat., 70: 372.

ConnotLy, C. J., 1925. Adaptive changes in shades and color of Fundulus. Bzol.
Bull., 48: 56.

Goopricu, H. B., anp M. A. Smitu, 1937. Genetics and histology of the color
pattern in the normal and albino paradise fish, Macropodus opercularis L.
Biol. Bull., '73:527..

InnEs, W. T., 1935. Exotic Aquarium Fishes. Innes Publishing Co., Philadelphia.
463 pp.

KamapA, TAKEO, 1937. Parker’s effect in melanophore reactions of Macropodus
opercularis. Proc. Imp. Acad. Tokyo, 13: 217.

Kosswic, Curt, 1935. Uber Albinismus bei Fischen.. Zool. Anz., 110: 41.

Mast, S. O., 1916. Changes in shade, color, and pattern in fishes, and their bearing
on the problems of adaptation and behavior, with especial reference to the
flounders Paralichthys and Ancylopsetta. Bull. U. S. Bur. Fish., 34: 173.

ParKER, G. H., 1930. Chromatophores. Bzol. Rev., 5: 59.

Parker, G. H., 1934a. Cellular transfer of substances, especially neurohumors.
Jour. Exper. Biol., 11: 81.

ParkKER, G. H., 1934. The prolonged activity of momentarily stimulated nerves.
Proc. Nat. Acad. Sct., 20: 306.

Parker, G. H., 1934c. Neurohumors as activating agents for fish melanophores.
Proc. Am. Phil. Soc.,'74: 177.

AUTOSOMAL LETHALS IN WILD POPULATIONS OF
DROSOPHILA PSEUDOOBSCURA

A. H. STURTEVANT

(From the William G. Kerckhoff Laboratories, California Institute of Technology,
Pasadena, California)

INTRODUCTION

It has been recognized for some years, following the work of Muller,
that lethals are especially convenient material for the study of muta-
tion rates. This is because they occur with a frequency that is great
enough to be measured, and because their occurrence can be detected
by a technique that is independent of the personal equation of the
observer. These same two advantages apply to the use of lethals in
the study of the constitution of wild populations. Two other ad-
vantages are also to be noted in this field. Owing to the extensive
studies on the mutation rates of lethals, there is available a large body
of data on the frequency of the occurrence of new lethals under a
variety of conditions. On the other hand, the rate of elimination of
lethals from a population furnishes the minimum possible difficulty of
quantitative estimation. It is, therefore, not surprising that there is
a rapidly increasing literature in the field—to which the present
paper belongs.

MATERIAL AND METHODS

A large series of wild strains of Drosophila pseudodbscura have
been studied in this laboratory (see, for example, Dobzhansky, 1935;
Dobzhansky and Boche, 1933; Sturtevant and Dobzhansky, 1936).
Tests have been carried out on a series of these, to determine the fre-
quency with which autosomal lethals occur. Most of the work has
been with the third chromosome, though a few tests on the second will
be described below.

Wild males, or a single son from each of a series of wild females
(i.e., females already fertilized when trapped), are mated individually
to females carrying the recessive gene for orange eyes. From each
such mating a single son is mated to females of the multiple stock
orange Scute (dominant, bristle reduction) purple (recessive, eye
color). All the Sc, not-or, offspring of this mating carry one particular

542

AUTOSOMAL LETHALS IN DROSOPHILA 543

third chromosome from the wild specimen; when they are mated

ale a
OSG pre Or Sc pr
for one of the two third chromosomes of the original wild male. If this
chromosome carries a lethal, the not-Sc class is absent.

The difficulty with the method is that, as just outlined, it neglects
the possibility of crossing over. This possibility exists only in the
females used in the final test generation, since everywhere else the
tested chromosome is kept in males, in which crossing over is absent
(or at most negligible in frequency). Even in the females in question,
it happens that in the majority of cases crossing over is not a serious
source of error. As reported by Sturtevant and Dobzhansky (1936),
there is a wide variety of sequences in this chromosome. The or Sc pr
stock has the Standard sequence; and females heterozygous for
Standard and any other sequence give few crossovers. The presence
of the three mutant genes in the experiment makes it possible to
distinguish between Standard, on the one hand, and all the remaining
sequences, on the other hand, in the tested third chromosome. Incase
the tested chromosome does not have the Standard sequence (and the
majority of them do not have it) the presence of a lethal is at once
apparent from the absence or extreme rarity of wild-type flies in the
test generation. Most of the sequences do give occasional crossovers
in one region or another when tested against Standard; for this reason
special tests are required to distinguish between semi-lethals and
lethals that cross over with Sc. These have not, in general, been
carried out in the present experiments; but it is clear that very few
semi-lethals are present, since in most cases the wild-type class is either
wholly absent or approximately half as numerous as the heterozygous
Scute class. This results from the fact that few of the lethals happened
to lie in the sections that give crossovers with Scute.

If the test chromosome has the Standard sequence, lethals can
still be detected, though it is necessary to make more extensive counts
to be certain no lethal is present. This is due to the fact that Sc lies
near the middle of the chromosome (about 30 units from the left end,
40 from the right), so that any lethal shows appreciable linkage with it.
Here, however, the distinction between lethals remote from Sc, and
low-viability genes close to it, requires special tests. The test,
applied in most cases, has been to use a chromosome carrying the
dominant Emarginate (eye shape) and the Arrowhead sequence.

Em J Em |
ots an

outlined above are obtained.

together ( ) the not-Sc offspring are homozygous

From the mating , results comparable to the Sc test

544 A. H. STURTEVANT

In the case of the second chromosome, the test procedure is similar.
Wild males or single sons of wild females are mated to glass (recessive,
eye structure) females, and a single male from each such mating is
mated to Bare (dominant, bristle form) glass females. The resulting

Bare offspring (= ) are mated together, and counts are made of

ae
their offspring. This test is less efficient than that used for the
third chromosome, since there is more crossing over. No inversions
are present in the material tested, and Bare is about 60 units from the
left end of the chromosome, 40 from the right. Lethals even at this
distance do cause a disturbance of the ratios; and all suspected lethals
were verified by making a series of matings of Ba X Ba from sibs.
In every case these tests agreed in showing a lethal to be present.

TABLE I

Frequency of lethals

Lethals

Chromosomes
tested
Number Percentage

Chromosome II

LOS OP ae cr nee ane een Ce Se 21 4 19.0

Oldkstocks ee Rees een sae 16 3 18.8
Chromosome III

OSS ise Sit te RR Rance oho 25. 67 13 19.4

LOS ORE Se acne reach aks 120 23 19.2

Oldtstocks skeen ee 29 7 24.2

However, there is no test for distinguishing lethals and semi-lethals;
and the technique is more laborious than that used for the third
chromosome. For these reasons relatively few tests were made on the
second chromosome.

The mutant strains available did not permit reasonably efficient
tests for lethals in chromosomes IV or V, and none was attempted. In
the case of the X chromosome no detailed tests were made, but obser-
vations on the sex ratio from wild females and their daughters indicated
that no lethals were present in any of the wild strains studied.

It should be pointed out that what is being studied here is lethal-
bearing chromosomes, rather than lethals. That is to say, if a
chromosome carries more than one lethal the technique used does not
make the fact evident. With frequencies of lethals as high as those

AUTOSOMAL LETHALS IN DROSOPHILA 545

found it seems likely that such duplications of lethals in a single
chromosome do in fact occur. The net result will be that the fre-
quencies recorded are too low. Further complications arise in con-
nection with the tests for identity of lethals, but since few cases of
identity were found this does not seem a serious source of error.

The results obtained are shown in summary form in Table I.

The material studied in 1935 came largely from the Rocky Moun-
tains and from the Mexican plateau; that in 1936 largely from southern
California. The ‘‘old stocks’’ were, many of them, from the Pacific
Northwest. These latter had been kept in the laboratory for at least
a year before the lethal tests were begun. The striking thing about
the table is that, in spite of these differences, there was little variation
in the frequencies found for a given chromosome or even as between
the second and third chromosomes. ‘Table II, giving the data for the

WABrE UT
Lethals
Chromosomes
tested
Number Percentage
Chromosome III
San Gabriel Canyon, Azusa, Calif........ Diff 8 29.6
airilicrnby @alliteewee eters et tus se e eieeee os 21 3 14.3
Sam Goreonio Nite Calif... ose. eel. oo 7 3 17.6
Samia, Cavs liscl, Caltissosceubeapoodsoue 16 3 18.8

most-studied individual localities (included in Table I) shows a
similar relative constancy of the frequency of lethals. All these results
are from Race A; 5 Race B third chromosomes (all from the Olympic
Peninsula of Washington) were tested, and one lethal was found,
giving a frequency of 20 per cent.

As reported by Sturtevant and Dobzhansky (1936), the third
chromosomes of this species exist in a wide variety of sequences. This
phenomenon does not appear to be related to the occurrence of lethals.
Lethals have been found in the Standard, Arrowhead, Pikes Peak,
Santa Cruz, Chiricahua, Cuernavaca, and Klamath sequences. Their
frequency seems to be essentially the same in regions such as southern
California or southern Mexico, where several different sequences exist
in fairly large numbers within each population, and in the area near
the common corner of Utah, Colorado, Arizona, and New Mexico,
where only the Arrowhead sequence is found. Fifteen third chromo-

546 ated > URE VAN

somes tested from this pure Arrowhead area gave 3 lethals—exactly 20
per cent. The data on Chromosome II also indicate that diversity of
sequences is not important, since all the strains from the regions con-
cerned in these tests had the Standard Race A sequence in that

chromosome. .

TESTS FOR ALLELOMORPHISM

If two lethals, different in origin, are kept in stocks of the form
lethal
or Sc pr
If two distinct lethals are concerned, wild-type offspring will appear
in numbers approximately equal to half the size of the heterozygous
Scute class; if the two lethals are identical, no wild-type offspring
will be produced except as a result of crossing over in the female used,
and this will be infrequent if the lethal-bearing chromosome does
not have the Standard sequence. Numerous tests of this kind have

been carried out.

Fifteen of the 1935 and ‘‘old stock” lethals were tested in all
possible combinations; the result showed 14 lethals to be present. One
from Metaline Falls, Washington (1934) was identical with one from
Florence, Texas (1935). Thirteen of the 1936 lethals were tested
against each other, and were found to include 12 different ones. One
of three tested from Julian was found to be identical with one of eight
from San Gabriel Canyon. The Metaline 1934 lethal was tested
against the 12 different 1936 lethals, and was found to be allelomorphic
to one from Barton Flats, on the slopes of Mt. San Gorgonio, Calif.
The San Gabriel-Julian duplicate was different from all the seven
surviving members of the 1935 series with which it was tested. These
tests, with a few miscellaneous others, total 225 crosses between
separately found lethals, with 3 cases of allelomorphism. That is, an

, it is easy to test their identity by crossing the two strains.

average of about = = 1.3 per cent of the lethals found may be

expected to be allelomorphic to any given one selected at random.
Or, since the total frequency of lethals in this chromosome in wild
stocks is just under 20 per cent, the average frequency of a given
lethal may be estimated at about one-fourth of 1 per cent.

It may be observed that no cases of recurrence have been found
within a locality. That is to say, the same lethal has not been re-
covered from any two specimens collected in the same place. Asshown
above, one lethal was found both at Julian and at San Gabriel Canyon,
in southern California, in 1936. While the two localities are only about

AUTOSOMAL LETHALS IN DROSOPHILA 547

100 miles apart, the populations of their respective mountain ranges
(San Jacinto and Sierra Madre) appear to be distinct in that the former
contains the Santa Cruz sequence while in the latter Tree Line occurs
(along with Standard, Arrowhead, and Chiricahua, common to both
regions—see Dobzhansky and Sturtevant MS in press—Genetics).
The other recurrent lethal was found in stocks from Metaline Falls,
Washington (1934), Florence, Texas (1935), and Mt. San Gorgonio,
Calif. (1936). It seems clear in the latter case, and probable in the
former, that we are dealing with recurrent mutations rather than
with the persistence of a lethal gene in heterozygous form.

RATE OF ORIGIN OF NEw LETHALS

A preliminary lethal-accumulation experiment has been carried out.
The technique used is not essentially new, and the results are not yet
extensive. It seems sufficient at present, therefore, to record that 2
new lethals occurred in a total of 120 chromosome-generations, giving
a frequency of 1.7 per 100—with a large probable error.

PROPERTIES OF NON-LETHAL CHROMOSOMES

The frequency of lethals found in the tested wild stocks is un-
expectedly high. A possible explanation of this result seemed to be
that the species is in a condition approximating that of balanced
lethals. That is to say, that most or all its third chromosomes are so
constituted that flies homozygous for any one of them cannot compete
with heterozygotes. Such an assumption amounts to supposing that
the phenomenon of heterosis is well developed within a single pair of
chromosomes. Under these conditions it might result that the rate of
elimination of a lethal would be greatly decreased; since lethals are
eliminated only when homozygous, and the assumption is that
homozygotes play little part in the perpetuation of the species, so
that their properties are unimportant.

The simplest way to test this hypothesis is to study the properties
of individuals homozygous for a series of non-lethal chromosomes, from
the same wild populations as those that contained lethals, in com-
parison with individuals heterozygous for two such chromosomes.
There is another question that can conveniently be investigated at
the same time—are the lethals completely recessive? This latter
question is of importance in any calculation of the probable rate of
elimination of lethals from wild populations.

Only one characteristic of the flies concerned has been investigated
—namely their viability. It is clear that fertility, length of life, re-

548 AEE Sa Ewa Ve

sistance to unfavorable conditions, and other properties, would all be
of importance in determining the frequencies found in natural popula-
tions; but the labor involved in studying a series of combinations for
such a variety of characteristics has seemed prohibitive. It also seems
probable, a priori, that there will be, in general, a rough correlation
between all these properties, such that data on one of them will give a
satisfactory picture of the general situation.

The technique used was the same as that used in the detection and
testing for identity of lethals—in fact many of the cultures served both
purposes. For the study of lethals partial counts were adequate, and
were used in many cases. However, if complete counts are made on
a culture there results a measure of the viability of the wild-type
anes
or Sc pr’
the absence of crossing over, three types are expected in the ratio
1:2:1. The homozygous or Sc pr is usually present in rather small
numbers, and has been neglected in making comparisons, since the
numbers seem to be very much influenced by minor environmental
differences and are often so low as to be inconveniently sensitive to the
error of random sampling. The index of viability actually used is the
number of wild-type + the number of heterozygous Scute X 100—i.e.,
the percentage that the wild type is.of the Scute. Evidently, with
equal viability of all classes the index will be 50.

Several objections may be raised to this method. ‘The most serious
is that each of the classes with which we are concerned is compared,

chromosome concerned. From the mating together of in

not with a common standard type, but with a class that

or Sc pr
varies in constitution from one test to another. In the case of a test
of a homozygote the comparison concerns a heterozygote of that same

chromosome; in the test of a heterozygote the cee class is made up

Se
of heterozygotes for each of the two + chromosomes, in approximately
equal numbers. In a few cases tests were repeated, and the result
shows a definite, though far from complete, correlation between suc-
cessive tests of a given combination. ‘The use of this technique can,
therefore, not be considered as giving more than an indication of the
situation, and accordingly it does not seem desirable to present the re-
sults obtained in detail.

In general, the homozygotes for non-lethal third chromosomes from
wild stocks are viable and fertile in both sexes, with the result that
homozygous stocks are easily established. The test outlined above

AUTOSOMAL LETHALS IN DROSOPHILA 549

indicates that some homozygotes are definitely below par, others
apparently at no disadvantage. The average viability index of 21
tested chromosomes was 45.91, the minimum 19.4 (weighted average
of three tests—16.3, 21.6, 33.3).

The tests of heterozygotes for two-third chromosomes from wild
strains were made chiefly with lethal-bearing chromosomes. Twenty-
five different combinations of lethal X lethal gave an average index of
53.0—.e., more viable than the separate lethals heterozygous for
or Sc pr. The lowest index among the 25 was 38.3.

The most probable conclusions are that
(1) lethals are wholly recessive;

(2) homozygotes for non-lethal chromosomes are slightly less viable, on
the average, than heterozygotes;

(3) the viability of homozygotes is quite variable, some being definitely
low, others apparently as high as the heterozygotes.

COMPARISON WITH RESULTS FROM D. MELANOGASTER

The frequency of lethals in wild populations of D. melanogaster has
been studied by several authors. The most extensive work is that of
Dubinin and co-workers (1934, 1936) on material from the Caucasus.
These studies were carried out on a large scale. Altogether 4819
second chromosomes are recorded, with 470 (= 9.8 per cent) lethals.
The frequencies range from 0 (in 92 tested, from Delizhan) to 16.1
per cent (161 tested, from Ordzhonikidze). From one locality,
Gelendzhik, tests were made in three successive years, giving 7.98
per cent (877 tested), 12.86 per cent (616 tested), and 8.78 per cent
(797 tested). Tests were also carried out for identity, but only within
regions. The average value here was 2.2 per cent—i.e., an average of
2.2 per cent of the lethals found in one year at one locality may be
expected to be allelomorphic to any one lethal found in that year at
that locality. The average frequency for a given single lethal is 0.22
per cent (.022 X .098). There are no tests for identity of lethals from
different localities, but those found at Gelendzhik in successive years
were so tested, with the result that ‘‘among the 33 lethals of 1933 and
the 55 lethals of 1934, nine lethals were common to both years.”” The
conclusion is drawn that this represents survival of these lethals.
The results reported in the present paper throw some doubt on this
conclusion, for a comparable frequency of identity was found in
pseudoobscura for all years and all localities. One may surmise that,
in melanogaster also, any two series of lethals would show a fairly large
proportion of common members—i.e., that recurrent mutations are
relatively frequent.

550 A. H: STURTEVANT

The absolute frequency of lethals found in the Caucasian melano-
gaster populations is only about half that in the American pseudo-
obscura, though the chromosome concerned (II) is roughly twice as long
as the III of pseudodbscura and includes the same material plus that of
the pseudodbscura IV (Donald, 1936; Sturtevant and Tan, 1937). The
preliminary experiment described above suggests a higher lethal
mutation rate in pseudodbscura; but until more data are available on
this point it is scarcely possible to decide on the relative rates of
elimination of lethals in the two species.

One difficulty in evaluating the data of Dubinin and his co-workers
may be pointed out. The results are recorded by localities and years;
but we are nowhere given more specific data. How large an area is
included in a single “‘locality’’? Over how long a period were speci-
mens taken in one year? What kinds of places were collected—woods,
grocery stores, garbage dumps, fruit orchards? These questions are
of importance in judging the size of the populations sampled and the
probable degree of relationship of the tested individuals.

SUMMARY

1. Approximately ‘20 per cent of the third chromosomes found in
wild populations carry lethals.

2. Less extensive data indicate a similar frequency for the second
chromosome.

3. On the average, about 1.3 per cent of the lethals found may be
expected to be allelomorphic to any given one.

4. The average frequency for any one lethal is about one-fourth
of one per cent of the chromosomes of wild strains.

5. The lethals, so far as studied, are completely recessive.

6. Study of flies homozygous for non-lethal third chromosomes
shows a considerable variation in their viability. Some are at a
definite disadvantage, others apparently not. On the average they -
are not quite as viable as flies carrying two different third chromosomes.

LITERATURE CITED

DospzHAnsky, TH., 1935. The Y chromosome of Drosophila pseudodbscura.
Genetics, 20: 366.
_DoszHansky, TH., AND R. D. Bocue, 1933. Intersterile races of Drosophila
pseudodbscura Frol. Bzol. Zentralbl., 53: 314.
Dona.p, H. P., 1936. On the genetical constitution of Drosophila pseudodbscura,
Race A. Jour. Genet., 33: 103.
Dusinin, N. T., ET AL., 1934. Experimental study of the ecogenotypes of Drosophila
melanogaster. Bzol. Zhurn., 3: 166.

AUTOSOMAL LETHALS IN DROSOPHILA Sol

Dusinin, N. T., et al., 1936. Genetic constitution and gene-dynamics of wild popu-
lations of Drosophila melanogaster. Bzol. Zhurn., 5: 939.

STURTEVANT, A. H., AND TH. DoOBZHANSKY, 1936. Inversions in the third chromo-
some of wild races of Drosophila pseudodébscura, and their use in the study
of the history of the species. Proc. Nat. Acad. Sct., 22: 448.

StTuRTEVANT, A. H., anp C. C. Tan, 1937. The comparative genetics of Drosophila
pseudodbscura and D. melanogaster. Jour. Genet., 34: 415.

THE EFFECT OF SALINITY UPON THE GROWTH OF EGGS
OPSBUCUS FURCATRUS?Z

D. M. WHITAKER AND C. W. CLANCY
(From the School of Biological Sciences, Stanford University)

INTRODUCTION

The effect of salinity upon fertilization and early development in 3
species of Fucus (F. serratus, F. vesiculosus, and F. spiralis) was studied
by Kniep (1907) on the Norwegian coast. He investigated especially
the tolerance for dilution of the sea water and found it to differ con-
siderably in the different species. Kniep was able to correlate the
tolerance with the distribution of the species (and, by inference, of
additional species of Fucus and other related marine alge as well)
especially in the Baltic Sea and in near-by regions in which brackish
water communicates with the ocean, thus establishing gradients of
salinity.

The present investigation is an attempt to measure the effect of
increased and decreased salinity upon the percentage germination and
the growth rate of the fertilized eggs of Fucus furcatus f. luxurians
throughout the range of salinity in which germination and growth take
place, and to do so under strictly controlled conditions. The eggs were
transferred abruptly into sea water of altered salinity, and therefore,
any effect which a prolonged gradual adaptation might have on the
tolerance is eliminated from the measurements. ‘The data are useful in
conjunction with other experimental investigations of the development
of this egg in which the salinity of the medium varies.

MATERIAL AND METHOD

Fruiting tips of this hermaphroditic Fucus were collected at Moss
Beach, California in May, June and July, 1937. The material was
cared for in the manner described in an earlier paper (Whitaker, 1936).
Sea water was collected at the same place and was filtered twice upon
arriving at the laboratory. In each experiment, eggs from the same
batch were reared in this sea water and also in samples of the same sea
water with artificially altered salinity. Salinity was decreased by
adding triple glass-distilled water. It was increased by boiling under

1 This work has been supported in part by funds granted by the Rockefeller

Foundation.
552

SALINITY AND GROWTH OF FUCUS EGGS 5S)

vacuum so that the temperature did not rise enough to cause pre-
cipitation of salt. By this method the volume of sea water was
reduced to half without precipitation. The measure of salinity which
has been used is the total salt content per unit volume expressed as a
percentage of the total salt content per unit volume of normal sea
water. In all cases hydrometer readings were made which confirmed
the salinity as originally determined volumetrically. The specific
gravity of the normal sea water used was 1.027.

The pH of the samples of normal sea water, as determined with
a glass electrode, ranged from 7.9 to 8.2. The pH tended to rise
slightly when the salinity was artificially increased, and to fall when it
was decreased. Such shifts were usually not great, being of the order
of a few tenths of a pH unit or less in most experiments. In the most
extreme cases the shift amounted to 0.5 units. The maximum pH
range of the media in the experiments upon which Fig. 1 is based was
7.7 to 8.5. In some of the experiments, all of which gave essentially
similar results, the range was considerably less. This pH range is
much narrower than the limits of normal development for this species
of Fucus (Whitaker, 1937), and it is small enough so that it appears
safe to conclude that it is a minor factor in the present results.

Since Fucus furcatus is hermaphroditic, and sheds male and female
capsules together at the same time, it is not feasible to inseminate a
population of eggs at any precise moment. The male capsules dissolve
in the sea water first, however, so that fertilization takes place when the
egg capsules dissolve and liberate the individual eggs into the sperm
bearing sea water. By selecting eggs only from capsules which break
down during a limited period, it is possible, in effect, to confine fertiliza-
tion to this period. In these experiments eggs were used which were
fertilized during a 10-minute period.

In order, for the present purpose, to rule out the effects of altered
salinity upon fertilization and entrance of the sperm, eggs were
fertilized in normal sea water in all experiments. Twenty minutes
after the end of the fertilization period samples were transferred with a
negligible amount of normal sea water into the media of altered
salinity. Eggs had thus been fertilized 20-30 minutes when they were
removed from normal sea water. They were thinly seeded in covered
Petri dishes which were stored in a moist chamber in a dark, humid,
constant temperature room at 15°C. Fertilization and all subsequent
development took place in this constant temperature room. The eggs
were observed only with red light until the end of the experiment.

554 D. M. WHITAKER AND C. W. CLANCY

RESULTS

The fertilized Fucus eggs develop in a relatively great range of
salinity. As the limits of tolerance are approached, all eggs in a
population do not have the same end point, and the results are repre-
sented graphically by the blocks in Fig. 1 to show the percent of the
eggs in the populations which form rhizoids. Each block in the figure
represents the average of the results of from 4 to 10 experiments, each
involving counts on 300 or 400 eggs. The counts were made two days
after fertilization.

The blocks in Fig. 1 show that very high percentages of the eggs

100

70
¢o
50
40
30

CURVE: LENGTH RATIOS IN PER CENT
BLOCKS: PER CENT OF EGGS WITH RHIZOIDS

[Sa
20 30 40 50 60 70 80 90 100 110 120 130 140 150 IGO 170 180 190
SALINITY OF SEA WATER IN PER CENT

Fic. 1. Blocks: The percentages of eggs which formed rhizoids in normal sea
water, in diluted sea water and in concentrated sea water, as observed after two days.

Curve: The average over all length of embryos at 92-96 hours, reared in sea
water of the salinity indicated, expressed as a percentage of the average length of
control embryos reared in normal sea water.

In all cases the eggs were fertilized and reared in the dark at 15°C. The specific
gravity of the normal (100 per cent) sea water was 1.027 (see text).

develop in sea water ranging in salinity from 50 or 60 to 150 per cent of
that of normal sea water. Beyond these limits the percentage drops
off rapidly, although some eggs develop rhizoids when the salinity is as
extreme as 30 per cent or 180 per cent. Beyond these limits no
rhizoids are formed. In the more dilute sea water (10 per cent and
20 per cent) eggs burst and cytolyze at once, and many of the eggs
which fail to form rhizoids in 30 per cent or 40 per cent sea water also
cytolyze. In the concentrated sea water, on the other hand, most of
the eggs which fail to form rhizoids do not cytolyze, at least in the
course of several days. Eggs which have remained for two days in
sea water of twice the normal salinity form rhizoids after being returned

SALINITY AND GROWTH OF FUCUS EGGS So)

to normal sea water. The suppression at high salinity thus bears some
resemblance to anesthesia.

The Fucus egg is spherical until a bulge forms on one side, about 16
hours after fertilization (Whitaker, 1936). This bulge extends in
filamentous fashion by elongation and cell divisions until its length is
many times the diameter of an egg. Ultimately the rhizoid of the new
plant forms from this filamentous structure, while the remainder of the
embryo gives rise to the thallus. The rate at which early development
takes place, as indicated by the rate of extension of the rhizoid filament,
is a function of the salinity of the medium. In both extremes of the
range of salinity, in which many or most of the eggs form no rhizoids
at all, the rate of extension is greatly retarded in those eggs which do
form rhizoids. Within the ranges of salinity (50 or 60 to 150 per cent,
see blocks, Fig. 1) in which practically all of the eggs form essentially
normal rhizoids, the salinity affects the rate of extension as shown by
the curve in Fig. 1.

The curve in Fig. 1 shows the average full length of the embryos
reared in media of the salinities indicated, expressed as a percentage of
the average full length of the control embryos from the same batches of
eggs reared in normal sea water. The lengths were measured with an
occular micrometer 92-96 hours after fertilization. Each point was
obtained by averaging the results of 4 to 10 experiments, each involving
counts of 50 or 100 eggs, except that the point at 90 per cent salinity is
based on only 3 experiments. The results of the separate experiments
are very similar.

It is seen from the curve in Fig. 1 that the embryos grow as rapidly
in 90 per cent as in 100 per cent sea water. The average absolute
length of the embryos in these optimum salinities was 303 microns at
92-96 hours. Since the embryos were growing in the dark in the
absence of photosynthesis, no nutrients were available to the eggs
except those stored in the unfertilized eggs. It is therefore doubtful if
much new protoplasm was synthesized, although internal conversion of
stored foodstuffs may have supported some protoplasmic synthesis.
The growth or extension observed is presumably largely a develop-
- mental elaboration, i.e., an extreme change of form.

SUMMARY

1. Fertilized eggs of Fucus furcatus f. luxurians have been reared
in the dark at 15° C. in diluted and concentrated sea water.

2. When the salinity of the medium is between 60 per cent and 150
per cent that of normal sea water (sp. gr. 1.027), practically all of the
eggs in a population form rhizoids and develop. The rate of elongation

556 D. M. WHITAKER AND C. W. CLANCY

of the embryos, as measured at 4 days, is the same when the salinity is
90 per cent or 100 per cent. When the salinity is greater or less, the
growth rate is retarded as shown in the curve in Fig. 1.

3. As the salinity is reduced below 60 per cent or is increased above
150 per cent, the percentage of eggs which form rhizoids declines
rapidly as shown in the blocks in Fig. 1. In 10 per cent and 20 per cent
sea water the eggs burst and cytolyze. In concentrated sea water
which inhibits development the eggs do not cytolyze and the develop-
mental inhibition may be reversible.

BIBLIOGRAPHY

KwniEp, Hans, 1907. Beitrage zur Keimungs-Physiologie und -Biologie von Fucus.
Jahrb. f. wiss. Bot., 44: 635.

WHITAKER, D. M., 1936. The effect of white light upon the rate of development of
the rhizoid protuberance and the first cell division in Fucus furcatus.
Biol. Bull., 70: 100.

WHITAKER, D. M., 1937. The effect of hydrogen ion concentration upon the induc-
tion of polarity in Fucus eggs. I. Jour. Gen. Physiol., 20: 491.

STIMULATION AND NUCLEAR BREAKDOWN IN THE
NER EIS PEGG

L. V. HEILBRUNN AND KARL M. WILBUR

(From the Zoological Laboratory, University of Pennsylvania)

When a protoplasmic system is exposed to a so-called stimulating
agent, a series of changes occurs which results eventually in activity
of the protoplasm. Various opinions have been expressed as to the
physico-chemical basis of stimulation. In our laboratory for several
years we have been interested in proving that stimulating agents all
cause a release of calcium from a calcium proteinate gel in the cell
cortex. This calcium is then supposed to initiate a protoplasmic
clotting similar in some ways to blood clotting. The evidence on
which this theory is based has been summarized in a recent book
(Heilbrunn, 1937, Chapter 37), and the details of the theory are also
given there.

Although in previous studies various types of animal and plant
material have been used, and the theory has thus been applied to
cells very different morphologically, as yet no attempt has been made
to determine what effect stimulation may have on the cell nucleus.
Certainly in a dividing cell, the nucleus plays an all-important réle.
It is of interest, therefore, to inquire what effect stimulation may
have in those cases in which the primary result of stimulation is cell
division. As is well known, the egg cells of many marine invertebrates
are sensitive to a wide variety of stimulating agents, and following
stimulation, division is usually initiated.

Typically, during mitosis the nuclear membrane breaks down.
The mechanism of this breakdown has scarcely been investigated
experimentally, although an understanding of the causes of such
muclear breakdown would be an important link in any complete theory
of cell division. In the case of many marine invertebrates, the egg is
released into the sea water in an immature condition, and it contains
a large nucleus or germinal vesicle. Sometimes contact with sea water
is in itself sufficient to cause a breakdown of the germinal vesicle and
an initiation of the maturation or polar body divisions. This is true
of the starfish egg. In other instances, the egg remains immature and
retains its large germinal vesicle until insemination. Then the

1 This work has been aided by a grant from the Radiation Committee of the

National Research Council.
557

558 L. V. HEILBRUNN AND KARL M. WILBUR

entrance of sperm into the egg, or its contact with the egg cortex,
provides the stimulus for nuclear breakdown and maturation divisions.
The egg of the annelid worm Nereis limbata has a large germinal vesicle
when it is shed into sea water, and this is broken down after insemina-
tion. The Nereis egg has been widely used both in studies of normal
fertilization (Lillie, 1911), as well as in studies of artificial partheno-
genesis (Just, 1915; Heilbrunn, 1925). It is readily obtainable at
Woods Hole, and is an easy object to study. Following activation,
the breakdown of the germinal vesicle can readily be detected in the
living egg, especially if the egg is somewhat compressed between slide
and coverglass.

Some years ago, Just showed that the Nereis egg could be artificially
activated by heat (Just, 1915; see also Heilbrunn, 1925). Later, Just
(1933) found that ultra-violet radiation could also cause activation.
In general, it is believed by radiologists that Roentgen rays can produce
the same effect as ultra-violet radiation. Accordingly an attempt
was made to discover if activation could be produced by Roentgen
rays. It was found that in order to obtain results, extremely high
dosage was necessary. In three experiments in which eggs were
exposed to 7600 r units per minute for 75 minutes, the percentage of
eggs activated was found to be 74, 52, and 5. Three other experiments
gave negative results. The activation was not the result of heat, for
the dishes containing the eggs were packed in ice to keep them from
becoming overheated.

The Nereis egg may be activated by chemical agents as well as by
physical means. It was found that isotonic solutions of sodium
chloride cause a high percentage of germinal vesicle breakdown.
Similar effects may also be obtained with isotonic solutions of
potassium chloride. Doubtless various other chemical agents are also
effective, but no attempt was made to list all the effective reagents.
However, it may be noted that a dilute ether solution (3 per cent in
sea water) is somewhat effective in causing germinal vesicle breakdown.
Other fat solvents doubtless act in similar fashion.

The literature on artificial parthenogenesis contains many papers
in which activation of marine eggs has been found to follow various
types of treatment. Our concern has not been to prepare an ex-
haustive list of the effective agents for the Nereis egg. We have
attempted to discover the nature of the effect of several physical and
chemical activating agents, and we have assumed that if we can throw
light on the true mechanism of activation in two or three cases, such
information may perhaps be applied to all types of activation.

In our experiments we have studied the action of ultra-violet

STIMULATION AND NUCLEAR BREAKDOWN 909

radiation and of isotonic salt solutions, and we have tried to discover
whether the calcium release theory of stimulation will hold for these
cases. Our experimental procedure was very simple. First we de-
termined the percentage of germinal vesicle breakdown following
exposure to the stimulating agent. Then we immersed eggs in'*citrate
solutions, to discover if previous exposure to citrate would prevent
activation. As is well known to students of blood clotting, citrate
solutions tend to prevent the action of calcium. Opinions as to the
mechanism of the effect differ. (Compare Hastings, McLean, Eichel-
berger, Hall and DaCosta, 1934.) Some unpublished work of D.
Mazia indicates that when sea-urchin eggs are immersed in citrate

TABLE I

Nuclear Breakdown following Irradiation

Percentage breakdown Immersion Percentage breakdown
after 60 seconds . time in after 60 seconds
irradiation in 0.35 M Na irradiation in
sea water citrate Na citrate

minutes
OOPS hits sextet. 4 0
OO eer iene ec 4 il
98... 4 7
MOORE O eee ice ei ane 4 2
NOOBS ees set hc aha y 4 6
AND Gaetan eye aaa epee 4 5
TOO) sed ea tea rt rel rts 1 4 D,
NOOR eax u ake ccals, 3 4 37
Oe ear peak ai sie NS 4 11
OE Riper nee veritas ea: 4 5
HOO) eiamaanenearn ts in ee enh 5 0
NO Oper arta tot ctse 5 5
T1010) ea pees Hie een eee 5 0
OU ilapte eh ee ty ayn cch Se 5 19
CO [os EUR ut EE 29 0

solutions, a large percentage of the calcium normally present in the
cell cortex is removed. If citrate solutions are able to remove calcium
from the cell cortex, or if they prevent release of calcium to the cell
interior by some other mechanism, then citrated eggs should be
incapable of activation. This was found to be the case.

Two types of experiments were tried. In the first place, eggs were
exposed to ultra-violet radiation, both in sea water and in sodium
citrate solutions. Ultra-violet radiation was obtained from a Uviarc
Laboratory Outfit. This type of lamp is well known and its charac-
teristics have been described in a paper by Buttolph (1930). The
lamp was operated at 160 volts and 5 amperes. In our work we were
not interested in the energy output of the lamp nor in separating

560 L. V. HEILBRUNN AND KARL M. WILBUR

different wave-lengths of radiation. A few preliminary tests soon
showed that when Nereis eggs were placed in a small quantity of sea
water and exposed to the mercury arc at a distance of 26 cm. for 60
seconds, all of the eggs, or at least more than 90 per cent of them,
showed activation. Shorter exposures (e.g. 30 seconds) were not
quite so successful. On the other hand, following exposures of 3 or 4
minutes, slightly lower percentages of activation were obtained. It is
thus clear that under the conditions of our experimentation, a 60-second
exposure to the Uviarc lamp was an effective stimulus, and we have
reason to believe that other Uviarc lamps act approximately as ours
did, unless of course, the lamps have suffered very serious deteriora-

TABLE II
Nuclear Breakdown in Isotonic NaCl
Immersion time Percentage breakdown
Percentage breakdown in 0.35 M Na in NaCl following
in NaCl citrate citrate treatment
minutes
OO pte een scribe era 4 4
D Oe AD aeebane hse ea! 4 4
OOF tac Pate cei nicat cac ane 4 7
O18) od alas UR a i a eles et 4 24
SIO Chala ac ne Me RL 5 0
SO agement: My aa cc tligesn e VaN 6 2
Coates cee ee aa Seen eee hee 6 0
GOREN ee ee crane wae, 6 0
SO RP eMac sh iy eta a 6 0
O10) wished ices Ahn een ee 6 0
SIO ONS eke ae es ae ais 6 1
OO ME Preteen cea ces cere e 6 0
OS ee eee cece pee rs 6 0
QAP Means ae amete sk stag Sh 6 1
OOO ett Feet Cy ac A ant a 6 0
WON O ea ies oats eek oka bus rere ae 8 2

tion. In any case, it is a simple matter to determine the exposure
necessary to obtain activation, for there is a wide range over which
this occurs.

Table I shows the percentages of nuclear breakdown following
irradiation in sea water, as compared with the percentages obtained
following irradiation in citrate solutions. The difference is striking.
In sea water typically, practically all the eggs respond by nuclear
breakdown. On the other hand in the citrate solution, there is almost
no response.

Similarly, when eggs are exposed to isotonic sodium or potassium
chloride solutions (0.53 molar), there is a high percentage of response,
but if the eggs are first immersed in 0.35 molar sodium citrate and then

STIMULATION AND NUCLEAR BREAKDOWN 561

exposed to the sodium or potassium chloride solutions, the eggs fail
to respond. This is shown in Tables II and III. From these tables
it will be noted that although a 4-minute immersion in the citrate
solution is sufficient to prevent most of the eggs from showing nuclear
breakdown, occasionally there is some response following such im-
mersion. Thus in the fourth experiment of Table II, 24 per cent of
the eggs were activated after 4 minutes of citrate treatment. If the
- eggs are allowed to remain in the citrate solution for 6 minutes before
‘being transferred to the sodium chloride or the potassium chloride
solution, there is almost no nuclear breakdown. Apparently, the

TABLE III
Nuclear Breakdown in Isotonic KCl
Percentage Immersion time Percentage breakdown
breakdown in 0.35 M Na in KCl following
in KCl citrate citrate treatment
minutes
AWE yeas Severe eck 18
CETL IaneE cheat Geren me 4 10
II OKO eee tener vere cee 4 Some eggs broken up. Count impossible.
OO er Papers srs moines 4 Some eggs broken up. Count impossible.
Dis cee aed tahaea 5 0
DSi ey rede Mt nee i a 6 1
DISH IN egy nM ge nes 6 0
IAs eet ate eaves eile | Seat 6 0
UE Meet) Sa trer tes tagral ts 6 0-2
BAER Gen casieyaiet eae ier 6 0
ILS Same to cere are agrees 6 0
218) bela ls. a aaa MeN ue 6 0
OOM i ceo cen een 6 0
OO Ree rcamicume- chee 6 0
O10) hE aire nearer 6 0
MOORE Sree -ctein cate es 8 4

citrate solution must act 5 or 6 minutes if it is to be completely effective
in preventing response to stimulation.

In these studies of activation, our attention was focussed on the
nuclear breakdown, and no attempt was made to study the cortical
changes which occurred in the eggs. These changes are interesting,
but they were not considered in the present study.

It might well be urged that the effect of the citrate in preventing
the stimulating effect of ultra-violet radiation and of sodium and
potassium solutions was essentially due to injury. Thus, if eggs are
fixed in formalin or some other poison, they presumably would not
respond to any type of stimulation. This objection is very easily met.
If citrated eggs are returned to sea water, they immediately become

562 L. V. HEILBRUNN AND KARL M. WILBUR

sensitive to radiation again. This is clearly shown in Table IV.
Eggs exposed to citrate for 6 minutes and then returned to sea water
show nearly 100 per cent of germinal vesicle breakdown on irradiation.
In this connection, it should be pointed out that even without irradia-
tion, a certain percentage of eggs may show germinal vesicle breakdown
after transfer from the citrate solution to sea water. This is an
interesting fact and will be considered again later.

As a whole, our results show that both physical and chemical
stimulation of the Nereis egg, as evidenced by the breakdown of the
germinal vesicle, are ineffective if the eggs are first treated with citrate.
There is thus support for the theory that stimulation to be effective
must involve a calcium release from the cell cortex. It is rather easy
to understand why ultra-violet radiation might cause a breakdown of
a calcium proteinate gel with consequent release of calcium, for this

TABLE IV
Nuclear Breakdown resulting from Irradiation in Sea Water following Citrate Treatment
Immersion time Immersion time Percentage breakdown
in 0.35 M Na in sea water following

citrate before irradiation irradiation .
minutes minutes

GRU etre shensieeb ae are 4 92-95

(C203 6 is oir Coe teeoe 4 98

POE Eins ee 8 4 98

(Ore ot ner Me a aaa 10-1034 99

(Deeg Gee aan 10-103 100

(55 tail ne ARE Re aK 10-104 98

(CEN Sa 4 ee anne ROT 10-1034 100

GO omrnie eipseel con anes 5 97

is consistent with various types of earlier work both on protoplasm and
on proteins (for a review of this literature, see Heilbrunn and Mazia,
1936). On the other hand, it is rather paradoxical to assume that
immersion of a cell in pure sodium or potassium chloride solution would
cause a release of free calcium to the cell interior. However, there is
real evidence that this type of phenomenon does actually occur (see
Heilbrunn, 1937, Chapter 33).

If Nereis eggs are immersed in isotonic calcium chloride solutions
(0.29 molar) there is no breakdown of the germinal vesicle. Mag-
nesium solutions are also without effect. This apparently means that
the calcium ions are unable to penetrate the cell with any rapidity,
although this is not the only possible explanation. In the case of the
clam Mactra, some unpublished experiments of Miss R. A. Young
show that activation (which in this egg also involves germinal vesicle

STIMULATION AND NUCLEAR BREAKDOWN 563

breakdown) follows immersion in calcium chloride solutions. (See
also earlier work of Dalcq, 1925, 1928; Hérstadius, 1923; Hobson,
1928; Pasteels, 1935.) From these studies with marine eggs we can
conclude that for some, calcium chloride solutions are stimulating
agents, whereas for others calcium chloride solutions are without any
very apparent effect. This is entirely comparable to the situation
with respect to smooth muscle. Thus Tate and Clark (1922) showed
that calcium caused a contraction of the uterine smooth muscle of
the rabbit and cat, but had no effect on similar muscle in the guinea-pig
and rat.

In the case of Nereis, the eggs become sensitive to calcium chloride
solutions and to a lesser extent to sea water, following immersion in
sodium citrate solutions. Thus if eggs were immersed in 0.35 molar
sodium citrate for 60—65 minutes and then placed in 0.29 molar calcium
chloride, 80 to 100 per cent of them showed germinal vesicle breakdown
(four experiments gave percentages of 80, 100, 92, 82). It almost
appears as though the loss of calcium from the cell cortex makes it
possible for calcium to enter the cell more freely. It is possible that
there may be a relation between this phenomenon and the calcium
paradox as observed in invertebrate hearts (see, for example, Chao,
1934).

Our results lead us to conclude that the breakdown of the germinal
vesicle in the Nereis egg is the result of a release of calcium from the
cortex. Previously Dalcq had suggested that the calcium ion was
responsible for the breakdown of the germinal vesicle in the starfish
egg, but he offers no clear explanation as to why sodium or potassium
should act like calcium, either in the starfish egg or in other eggs.
(See Dalcq, 1925, 1928; also Pasteels, 1935.) Our results offer an
interpretation of this difficulty. As to the details of the mechanism
involved in the disappearance of the nuclear membrane, we are still
at a loss. Presumably, calcium ion initiates a series of changes and
these changes eventually cause nuclear breakdown. There is obviously
more than a mere action of calcium on the nucleus, for when eggs
are crushed in sea water, the germinal vesicle does not break down.

SUMMARY

When the Nereis egg is stimulated either physically by ultra-violet
radiation, or chemically by immersion in sodium or potassium chloride
solutions, the germinal vesicle breaks down. This response to stimula-
tion is prevented if the eggs are previously immersed in sodium citrate
solutions. The results are in agreement with the calcium release
theory of stimulation.

564 L. V. HEILBRUNN AND KARL M. WILBUR

LITERATURE CITED

Buttovpy, L. I., 1930. The electrical characteristics of commercial mercury arcs.
Rev. Sci. Instruments, 1: 487.

Cuao, I., 1934. Paradox phenomena in the cardiac ganglion of Limulus polyphemus.
Biol. Bull., 66: 102.

Daca, A., 1925. Recherches expérimentales et cytologiques sur la maturation et
l’activation de l’oeuf d’Asterias glacialis. Arch. de Biol., 34: 507.

Datcg, A., 1928. Leréle du calcium et du potassium dans l’entrée en maturation de
l’oeuf de pholade (Barnea candida). Protoplasma, 4: 18.

Hastines, A. B., F. C. McLEAn, L. EICHELBERGER, J. L. HALL, AND E. DACosta,
1934. The ionization of calcium, magnesium, and strontium citrates.
Jour. Biol. Chem., 107: 351.

HEILBRUNN, L. V., 1925. Studies in artificial parthenogenesis. IV. Heat partheno-
genesis. Jour. Exper. Zodl., 41: 243.

HEILBRUNN, L. V., 1937. An Outline of General Physiology. Philadelphia.

HEILBRUNN, L. V., AND D. MaziA, 1936. The action of radiation on living proto-
plasm. Duggar’s Biological Effects of Radiation. Vol. 1, p. 625. New
York.

Hosson, A. D., 1928. The action of isotonic salt solutions on the unfertilised eggs of
Thalassema neptuni. Brit. Jour. Exper. Biol., 6: 65.

Horstapius, S., 1923. Physiologische Untersuchungen iiber die Eireifung bei
Pomatoceros triqueter L. Arch. f. mikr. Anat. u. Entw.-mech., 98: 1.

Just, E. E., 1915. Initiation of development in Nereis. Bzol. Bull., 28: 1.

Just, E. E., 1933. A cytological study of effects of ultra-violet light on the egg of
Nereis limbata. Zeitschr. f. Zellforsch. u. mikr. Anat., 17: 25.

Lituig, F. R., 1911. Studies of fertilization in Nereis. I. J. Morph., 22: 361.

PASTEELS, J. J., 1935. Recherches sur le déterminisme de |’entrée en maturation de
l’oeuf chez divers Invertébrés marins. Arch. de Biol., 46: 229.

TATE, G., AND A. J. CLARK, 1922. The action of potassium and calcium upon the
isolated uterus. Arch. intern. de pharm. et de thérap., 26: 103.

STUDIES OF THE MITOTIC FIGURE. VI. MID-BODIES
AND LHEIRV SIGNIFICANCE FOR THE CENTRAL
BODY PROBLEM

HENRY J. FRY

(From the Department of Anatomy, Cornell University Medical College, New York City,
and the Marine Biological Laboratory, Woods Hole, Mass.)

INTRODUCTION

Purpose of the Study

Mid-bodies have received less attention than any other component
of the achromatic figure. In many animal cells they appear, during the
time of division, as deeply staining thickenings on the fixed spindle
fibers, in the equatorial plane. They are generally regarded as in
some way comparable to the cell-plate of plants—possibly a vestigial
homolog (Wilson, 1928, pp. 144 and 159; Sharp, 1934, p. 169).
Flemming (1891) first suggested this concept.

In a previous study of central bodies in brain cells of Squalus
embryos, in which considerable attention was also given to the mid-
bodies, I reached the conclusion that in this case they are nothing but
accumulations of dye at places where the spindle materials have been
pinched together by the division furrow, and have no existence as an
individualized cell component. The question was raised: ‘‘ Whether or
not mid-bodies generally will eventually be explained in terms of
focalization phenomena remains to be seen”’ (1933a, pp. 177-178).
This problem is the subject of the present paper.

Discussion of the Term “ Focal Body”’

Before presenting the data it is advisable to clarify further the
concept focal body or focalization phenomenon which I have discussed
previously (1932, pp. 181-182; 1933a, p. 177; and 19330, pp. 233—235).1
These terms are used to describe a structure of the achromatic figure
which appears to be produced as a result of the convergence to a
common center of either spindle materials, aster materials, or both.
If this assumption is correct, there are three possible classes of focal
bodies.

Living Focal Bodies.—These probably exist in the living cell as
minute pools of spindle or aster materials at centers of focalization, and
are preserved by fixation. The situation here may be somewhat like

1 Some of these studies, referred to here and later, were carried out with the as-
sistance of other investigators, as indicated in the bibliography.
565

566 HENRY J. FRY

that which occurs when thin lines of a substance of relatively low
viscosity are placed upon a piece of glass in a converging configuration:
a pool of material forms where the lines coalesce at the center, the size
of the focal body thus produced depending upon the number and
thickness of the lines, and the viscosity of the substance. Such a body
is non-radial and more or less demarked, but although it looks very
different from the surrounding radial area and appears to be an indi-
vidualized structure, it is actually only the inner ends of the lines which
have fused.

Coagulation Artifact Focal Bodies.—According to the hypothesis
presented here and in earlier papers, these do not exist in the living cell
but are created by the act of coagulation. It is suggested that the
process of fixation may effect a breakdown of the converging materials
at the focal center and form a body, the structure of which is de-
‘termined largely by the coarseness of the fixed rays and fibers. As I
have shown in previous studies, the comparative coarseness of such
materials is modified by the technique—the fixative used or the
environmental condition prior to fixation. For example, in eggs of
Echinarachnius (1928, 19296, and 1929c) and Chetopterus (1932 and
19330) there is a definite relationship between the structure of bodies at
centers of focalization and the coarseness of the fibers and rays, as
determined by the technique. An analogy to this situation is found in
a model similar to the one described above, but employing material of
such a composition that the converging lines do not coalesce at the
point where they meet. Upon subjecting this model to a given
treatment (the equivalent of the fixation procedure), the character of
the material is so changed that the lines coalesce at their meeting-point
and a focal body is formed.

Staining Artifact Focal Bodies.—These are only accumulations of
dye at the focal centers of some fixed mitotic figures, such as I demon-
strated in brain cells of Squalus embryos (1933a). An analogous
situation occurs if a model is made in which a large number of delicate
threads are attached to the edge of a circular frame and brought
together at the center. When such a model is dyed, the focal area
takes more stain than the peripheral region, owing to the aggregation of
threads there. If this model is viewed at the proper distance (dupli-
cating the effect produced when stained mitotic figures are studied
with a microscope) the central region may in some cases look like a
sharply demarked body, depending upon the number of threads, the
chemical nature of both threads and dye, whether they are thick or
thin, their surface rough or smooth, and their paths separate or
interwoven.

MID-BODIES AND CENTRAL BODIES 567

Assuming that the suggestion is correct that the focalization of
spindle and aster materials may result in the production of focal
bodies, we still know too little about the structure of living spindles and
asters, the effects of coagulation, and the staining process, to explain
adequately in physico-chemical terms exactly how the bodies are
produced. For these reasons the models just described are not
intended to give exact parallels of what occurs when focal bodies are
formed in mitotic figures; they only illustrate how focalization phe-
nomena may be involved in any configuration of converging substances.

Depending upon the type of cell used and the technique employed,
such bodies may be minute or large, demarked sharply or vaguely,
granular or homogeneous or vesicular in structure, and they may differ
in shape. Such variations can often be demonstrated in the same
material when different techniques are used.

Focal bodies, however, have certain characteristics by which they
can be surely differentiated from typical cell components. They exist
only at places where spindle and aster materials come to a focus.
They arise only as such places of focalization are formed, and disappear
when such areas disintegrate; they therefore do not maintain conti-
nuity from cell to cell. They change shape as the focal area changes
shape, and often become double when the focal area elongates. They
may vary widely in structure from one cell cycle to another in the
same organism, or they may vary in the same cell cycle in closely
related organisms; sometimes, on the same slide, they show differences
from cell to cell which are identical as to species, cell cycle, mitotic
phase, and the method used. They are usually unstable in structure
and easily modified by variations in the technique. In general
their morphology is related to the size of the mitotic figure and the
coarseness of the fixed focalized materials.

In all of these respects focal bodies differ markedly from such cell
components as the centriole-blepharoplasts of spermatocytes and
Protozoa, the diplosomes of vertebrate cells, and other similar struc-
tures, which have undoubted existence in the living cell as typical
individualized cell components, and in some cases can be seen in the
living condition. They exist regardless of whether areas of focalization
are present or absent, and they exhibit great stability of structure
despite wide variation in the technique.

Hence the suggestion is made that at centers of focalization,
structures are formed which are often minute, homogeneous, sharply
demarked, and look like typical cell components, but are actually
nothing more than the result of the convergence of spindle and aster
materials to focal areas, i.e., focal bodies or focal phenomena.

568 HENRY SER Y

METHODS

The behavior of mid-bodies was studied in eggs of Cerebratulus,
Cumingia, Nereis, Chetopterus, and Asterias, during the formation of
both polar bodies, during first cleavage, and also in cells of blastule;
in Arbacia eggs, during the first three cleavages as well as in blastule;
in brain cells of Squalus embryos; and in primary spermatocytes of
Romalea.

Many fixatives were used and the cells were exposed to various
environmental modifications prior to fixation. They were sectioned
at a thickness of 5 uw and stained with Heidenhain’s hematoxylin. In
the case of eggs mid-bodies were studied only in those which happened
to be sectioned in a plane passing through both the long axis of the
mitotic figure and the animal pole, since only in such sections are the
relations between the structure of mid-body and spindle shown
clearly. In each case an adequate number were examined to ascertain
the extent of variation occurring in mid-body structure and associated
spindle remnant.

In order to check the conclusions reached by these original studies I
also examined a considerable portion of the pertinent literature—
almost two hundred papers—to be sure that no important types of
mid-bodies described by other workers should be overlooked. In most
of these invéstigations the author was not interested in the mid-bodies;
in many cases they are not even mentioned in the paper, although they
are shown in the drawings. In only a few studies do the figures
delineate the detailed changes from their origin to their disappearance.

The illustrations of the present paper, whether original or repro-
duced from the literature, show all types of mid-bodies found, typical as
well as atypical.”

THE BEHAVIOR OF MID-BODIES

Conditions Under Which Mid-bodies Are Present

Mid-bodies arise only when a remnant of the spindle still exists
when cell division occurs; this is necessarily pinched together by the
advancing furrows. No mid-bodies are found if, as often happens, the
spindle disappears prior to division.

Such contrasting conditions may occur in the same cell type of
closely related species, as illustrated in cleaving eggs of Echinoidea. In
a series of experiments, done in collaboration with Dr. G. H. A. Clowes

2 Those illustrations taken from the literature (Figs. 13-69) were for the most
part reproduced by photographing the drawings of the original papers. In four cases,

however (Figs. 14, 62, 65, and 66), photographs were made of illustrations, redrawn
from the originals, in Wilson’s The Cell in Development and Heredity.

MID-BODIES AND CENTRAL BODIES 569

and Dr. M. E. Krahl, I studied Arbacia eggs under about 75 experi-
mental conditions, in an attempt to analyze the relation between
modifications of respiration and the behavior of the mitotic figure.
Usually the eggs were fixed in Bouin’s fluid, but a number of other
reagents were used. Mid-bodies are present (Fig. 5) with a spindle
remnant (with rare exceptions when the material is very abnormal and
both are absent). Boveri (1901) illustrates a similar condition in
cleaving eggs of Echinus (Fig. 59). On the other hand, Echinarachnius
eggs were also studied, using about 20 modifications of the technique,
and the situation is in contrast to that in Arbacia eggs: the spindle
disintegrates before division occurs and mid-bodies are not found.
Toxopneustes eggs (Wilson and Leaming, 1895) also show neither body
nor remnant. Here, therefore, are eggs of four species of Echinoidea,
two with mid-bodies associated with a focalized spindle remnant, and
two with neither.

The same relation between the presence and absence of a spindle
remnant and the presence and absence of a mid-body may exist even in
the same material, under different experimental conditions. When
Arbacia egg-sets are run at various temperatures, those which cleave at
temperatures from 10° to 25° C. show both mid-body and spindle
remnant, but at 30° neither is present.

As just noted, a mid-body does not arise unless a remnant of the
spindle is present when division occurs. Conversely, when a remnant
is present, a mid-body is practically always formed, but a few exceptions
to this are reported in the literature. For example, in the spermato-
gonia of Enteroxenos (Fig. 55), the remnant is present without the body.
Occasional instances like this are not surprising in view of the fact that
the behavior of mid-bodies is so variable, depending upon species, cell
cycle, fixation, and depth of stain. Scarcely a single generalization
can be made about them to which some exceptions cannot be found.

History of Mid-bodies

There are a number of divergent structural types of mid-bodies, and
their history differs. One type is illustrated in Arbacia eggs. In this
case first cleavage begins with the appearance of a furrow at the animal
pole (Fig. 1), but no mid-body arises until the furrow has advanced far
enough into the egg to impinge upon the spindle; and the body which
then forms at its tip is just a faint thickening (Fig. 2). Another furrow
appears at the opposite pole about a minute later, and a similar body
arises at its tip when it too presses upon the spindle. Meanwhile the
first furrow has advanced more deeply and its body enlarges. By the
time the two furrows are ready to touch each other, a mid-body is

570 HENRY J. FRY

present at the end of each, and when contact is made these bodies join
to form a dumbbell-shaped structure which undergoes no further
change until it disappears when the spindle remnant fades (Figs. 3-5).

Another type is illustrated in the brain cells of Squalus embryos, in
which division occurs from one side only. A mid-body appears at the
point where the spindle is first compressed by the approaching furrow.
As it advances, other bodies arise, their size and number differing from
cell to cell, but the end result in all is a single body at the opposite side
of the cell, where the spindle is finally focalized. It too disappears
when the spindle disintegrates (Figs. 6-11).

Fics. 1-11. Successive stages in the formation of mid-bodies. Figs. 1-5:
Arbacia eggs at first cleavage. Figs. 6-11: brain cells of Squalus embryos (Fry and
Robertson, 1933).

Several studies in the literature illustrate the history of other types.
In erythrocytes of duck embryos (Heidenhain, 1907) a group of
nondescript thickenings are aggregated into a centriole-like body
(Figs. 16-20). In smooth muscle cells of Amblystoma (Pollister, 1932)
an atypical elongate mid-body undergoes progressive condensation
(Figs. 21-26). And in Arion eggs (Lams, 1910) slender elongate
thickenings which arise at the middle of the spindle fibers are pinched
together into a dense mass (Figs. 27-34).

MID-BODIES AND CENTRAL BODIES 571

Although these types differ as indicated, they all have in common a
gradual aggregation of mid-body material associated with an increasing
focalization of the spindle brought about by the division process.

The final position of the fully formed mid-body is determined by the
point where the division furrows make contact. For example, in red
blood cells of the duck, the furrows meet at the middle of the cell, and
the mid-body lies there (Fig. 19). In Arbacia eggs during first cleavage
one furrow arises about a minute before the other; their meeting point is
therefore considerably off center, and the body lies there (Fig. 5). In
Squalus brain cells there is only a single furrow arising at one side
which presses the spindle against the opposite cell membrane, and the
body lies there (Fig. 11).

In most instances mid-body and spindle remnant disappear
simultaneously. This is generally true of the materials examined in
the present study. It is also shown in the illustrations in the literature,
where telophase figures which have both body and remnant are usually
succeeded by a drawing of the next mitotic phase without either. In
some cases, however, the body persists for a short time after the
remnant has disappeared. This point was studied in Arbacia eggs at
first cleavage. After division is completed, all cells have both mid-
bodies and spindle remnants, but about fifteen minutes later, just
before the next prophase figure makes its appearance, the remnant is
doubtful or absent in about 25 per cent (count of 50), although the
bodies are still present in all cells. By the time the prophase figure
arises mid-bodies have, with rare exceptions, disappeared. A few cases
are also found in the literature where the mid-body is shown as
persisting after the spindle remnant has faded, as for example, in red
blood cells of the duck (Fig. 20). In other words, if one considers the
converging configuration as a whole, the focal area is obviously the
most condensed part, and hence this may explain why it occasionally
persists longer than the surrounding region of the converging fibers,
although it may have been originally created as a result of their
becoming focalized.

Configurations of M1id-bodies

The configurations which mid-bodies show during their formation,
when cell division is not yet completed and spindle fibers are only
partially pressed together, must be distinguished from their final
structure, when the aggregation of fibers is fully accomplished. The
data here presented refer only to fully formed mid-bodies; the limited
number of atypical ones will be described later.

Many mid-bodies are round, smooth, and centriole-like (Figs. 11,

Sy} REN Re ERY

19, 46, 58, and 62); or their surface may be rough (Figs. 56, 57, and 60).
Another common type is elongate: it may be ovoid and smooth (Fig.
47), ovoid and rough (Figs. 45 and 59), dumbbell-shaped (Figs. 5 and
61), or irregular (Figs. 48 and 49); it may lie parallel to the major axis"
of the spindle (Fig. 61) or at right angles to it (Figs. 5, 45, 47, 48, 49,
and 54).

In addition to these common forms, other types are found oc-
casionally. The body may be ring-shaped (Figs. 29, 36, and 37), cone-
like (Figs. 30 and 52), linear in form (Fig. 26), or it may be composed of
thickenings which arise at the middle of the spindle and are then
aggregated loosely (Fig. 51) or tightly (Fig. 33). A specialized typeisa
large irregularly outlined area containing a small vesicle (Fig. 43).

In general the above types are found only when the division process
has focalized the spindle to a point or to a very limited area. But
associated with a specialized kind of remnant (mitosome) which is not
focalized to a point, and persists as a relatively broad band of fibers, is
a very different type of mid-body, composed of a row of granules
(Fig. 14). In the studies examined this type was found only in
spermatogonia and spermatocytes, and not in all of them. Of 15
investigations of spermatogenesis taken at random from the literature,
13 show this unusual remnant and the other two have a fully focalized
remnant with a single mid-body (e.g., Enteroxenos, Figs. 56 and 57).

In cleaving eggs of Arion the mid-body is also a broad band of
aggregated spindle fibers, but no granules are present (Fig. 33); and in
the polar body cycles of this species there is a similar band associated
with a ring-shaped mid-body (Fig. 29). ;

It is significant that the mid-body which is composed of a row of
granules is found only in the specialized broad spindle remnants of male
germ cells, whereas the other types which are usually more aggregated,
are found with remnants which are focalized to a greater degree.

In some cases the very configuration of the mid-body indicates that
it is focalized spindle substances. In cleaving Arion eggs, for example,
thickenings which appear at the mid-region of the spindle are pressed
together by the division furrow into a bundle, which is later bent into a
U-shaped figure when the nuclei move from their former position
toward the surface of the egg (Figs. 32-34). In cone-shaped bodies,
the very form shows that they are spindle substances which have been
pinched together (Figs. 30 and 52). And when the atypical, elongate
mid-body of smooth muscle cells of Amblystoma makes its first appear-
ance it is obviously a condensation of spindle materials (Fig. 22).

The other types—dots, dumbbells, ovoids, rings, lines, and vesicles
—which do not appear to the eye to be focalized spindle substances,

MID-BODIES AND CENTRAL BODIES Ss

nevertheless behave like those which do. In general, all types arise
after focalization of the spindle has begun, become more aggregated as
focalization is completed, and disappear as the spindle remnant
disappears. A series of structural groups could be arranged, beginning
with the centriole-like type, and passing by gradations to those which
are clearly aggregations of spindle materials.

Relation Between Structure of Mid-bodies and Coarseness of Spindle Fibers

When mitotic figures are subjected to various experimental con-
ditions and fixed with different reagents, mid-bodies often vary in size
and shape, and the fibers vary in coarseness. The relation between the
physical structure of the mid-body and the distinctness of the fixed
fibers was studied in primary spermatocytes of Romalea and in cleaving
eggs of Arbacia.

TABLE [|

The relation between the size of the granules composing the mid-bodies in primary
spermatocytes of Romalea microptera and the coarseness of the spindle fibers. In general,
the coarser the fibers, the larger are the granules, but there are various exceptions to
this generalization. Forty-eight modifications of the technique were used, and the
depth of stain was similar in all. The table shows the percentages of each type of
association of mid-body and spindle remnant.

Coarseness of Spindle Fibers
Size of Granules Composing

Mid-bodies
Vague or Delicate Coarse Very Coarse
per cent per cent per cent
INGER eae thie waite teeta sae 8 — 3
Snell (OL ee eae ee 18 13 21
Medium (1.0u)............ — 18 15

anoen(lesis)i. se) e.cee sees A — — 4

Romalea spermatocytes were subjected to 48 experimental modifi-
cations, differing both as to environmental factors prior to coagulation
and the fixatives employed. They were all stained to the same depth
with Heidenhain’s hematoxylin. Under usual conditions the mid-
body is composed of a row of practically contiguous granules, each
about 1 w in diameter, and the spindle fibers have a certain degree of
distinctness. The same situation obtains in spermatogonia (Fig. 14).
After modifications of the technique, however, the granules may be
absent, small, medium, or large, and they may vary in shape; the fibers
may be vague, delicate, coarse, or very coarse.

The relation between variation in size of granules and coarseness
of fibers is reported in Table I. In general, the more delicate the fibers

574 HENRY J. FRY

are, the smaller the granules. But the presence of very coarse fibers
does not guarantee large granules, and those of small size are associated
with fibers of all degrees of coarseness.

In Arbacia eggs, in the 75 experiments previously noted, the
dumbbell-like mid-body is in most cases of usual length—about 1.5 y»—
and the small spindle remnant is composed of delicate fibers. But in 6
of the experiments the fibers are unusually coarse and the bodies are
larger. In this material too, however, there are a few exceptions: the
remnant may have the usual delicate appearance and yet be associated
with exceptionally large bodies, or the fibers of the remnant may be
very coarse while the bodies are of usual size.

In general, therefore, the size of the mid-body is directly pro-
portional to the coarseness of the spindle fibers, but there are ex-
ceptions. These are probably explained by the uncertainties of
focalization phenomena—which is another way of saying that we do
not know what the structure of the living spindle is, and we know even
less about the effects of coagulation upon it, especially at points of
focalization. Furthermore, when we are dealing with areas of focaliza-
tion, we must also remember that slight differences in depth of stain
may produce marked differences in appearance.

Variations in Mid-body Structure from One Cell Cycle to Another in the
Same Species

Within the cell types of any one species—o6gonia, o6cytes, sperma-
togonia, spermatocytes, large blastomeres, small blastomeres, and the
innumerable kinds of somatic cells—there may be wide variation in
mid-body structure. .

In eggs of Cerebratulus, Cumingia, Nereis, Chetopterus, and Asterias,
the mid-bodies of the first polar body figure are larger than those of the
second. In Arbacia eggs, also, during the first three cleavages, the
average length of the dumbbell-shaped bodies is successively 1.5 u,
1.4 uw, and 1.2 » (25 measurements in each case). And in Cumingia the
diameter of the mid-body at first cleavage is 1.6 u, but at the second it
is 1.0. In all these cases Bouin’s reagent was employed. It is
probable that differences in the size of the mitotic figure may explain
why mid-bodies are larger in the first polar body figures than in the
second, and larger in first-cleavage figures than in those following.

In Nereis eggs, during the first polar body cycle, the mid-body is
round and relatively large, with a rough surface (Fig. 60); during the
second cycle it is dumbbell-shaped and small, with a smooth surface
(Fig. 61); at first cleavage, however, it is absent (Bouin’s fixation).
This is probably explained by the fact that a spindle remnant is

MID-BODIES AND CENTRAL BODIES SS)

present when the polar bodies are pinched off, but not when the egg
divides.

Crepidula eggs (Conklin, 1902) show striking differences in mid-
body structure from cycle to cycle. In first polar body figures it is a
ring, which appears as two dots when seen in cross-section (Figs. 35 and
36) ; in the second, it is a smaller ring with a dot in it (Fig. 37), or just a
dot (Fig. 38); in first-cleavage figures it is a large area containing a
vesicle (Fig. 43); in third-cleavage figures it is a small irregular blob
(Fig. 45), and in fourth-cleavage figures, a minute dot (Fig. 46). The
difference in size of the mid-bodies is apparently related to the size of
the mitotic figures concerned. The factors which produce rings in
some cells and vesicles or dots in others are not as yet understood.

Variation in the structure of mid-bodies is also exhibited in
Enteroxenos cells in different cycles (Figs. 47-57).

B00B80 868
080008 08u
[00880889

Fic. 12. Variation in mid-body structure in Arbacia eggs at first cleavage.
Twenty-five bodies selected at random from a single slide are shown in outline and
without their accompanying spindle remnants. The unit of the scale is 0.5. All
eggs are in late telophase just after cleavage is completed. Bouin’s fixation.

Instability of Mid-body Structure

Mid-bodies vary in size and shape from cell to cell on the same slide.
Outline drawings of mid-bodies in Arbacia eggs at first cleavage (Fig.
12) illustrate the variation in size and contour which may occur in cells
at the same stage treated identically and lying side by side on the same
‘slide. The extent of this variation is typical of the materials reported
in this paper.

Mid-bodies of odgonia (Figs. 47-49) and those of the second polar
body figures (Figs. 52-54) in Enteroxenos cells also exhibit variation in
shape.

576 HENRY J. FRY

Mid-bodies vary in structure due to the use of different fixatives. In
some instances a wide modification of the technique has but little effect
upon the structure of the mid-body, as in Arbacia eggs at first cleavage
(Fig. 5), but in others the effects are considerable, as in primary
spermatocytes and spermatogonia of Romalea (Fig. 14 and Table I).
As a general rule, if fixation demonstrates coarse spindle fibers, the mid-
bodies are large; if the fibers are delicate, the bodies are smaller.

Mid-body structure is varied by modifications of environmental
factors. In Arbacia eggs the size of the mid-body at first cleavage is
modified by temperature. At temperatures from 25° to 20° C. the
length is 1.5 uw; at 15° C. it increases to 2.8 uw; at 10° and 7.5° it again has
an average length of 1.5 4. There is, however, much greater variation
at the lower temperatures than at the higher ones. (Bouin’s reagent
used; counts of 25 in each case.)

When Cerebratulus eggs cleave at 20° C., mid-bodies are present
only in about 2 per cent of the cells (Boveri’s picro-acetic reagent), and
are less than 1 » in diameter; but when these eggs are allowed to develop
at 15° C., and are fixed in the same manner, mid-bodies over 2 wu in
diameter are present in about 10 per cent of the eggs. (Counts of
50 eggs.)

Mid-bodies may be present in some cells and absent 1n others prepared
im an identical manner. A given egg-set may be fixed under usual
laboratory conditions and run up in the regular manner, yet cells on the
same slide and at the same mitotic phase may show mid-bodies and
remnants in some cases and neither in others. For example, Cere-
bratulus eggs, as just noted, have only 2 per cent with mid-bodies and
spindle remnants at first cleavage. Cumingia eggs at first division
show 18 per cent (Bouin’s fixation), and Arbacia eggs show 30 per cent
when dividing at 7.5° C. (Bouin’s reagent). These percentages are
based on counts of 50 eggs. Various similar examples could be cited.

But no matter how the experimental technique is modified, a mid-
body is present only when there is a spindle remnant, with the rare
exceptions previously mentioned (pp. 569 and 571). How shall we
explain, then, the presence of a spindle remnant in some eggs and its
absence in others which are at the same mitotic phase, the two kinds
lying side by side on the same slide? The probable cause of this
variation is an uncontrollable factor in the fixation process: eggs are
added to the reagent in a certain amount of sea water, necessarily
causing some dilution of the fixative. Each egg is coagulated within
the first second after exposure to the reagent (based on a study of
Chetopterus eggs (Fry, 1932, pp. 173-176)), but several seconds are
required for the egg suspension and the fixative to mix completely.

MID-BODIES AND CENTRAL BODIES S00

Some of the eggs are thus coagulated by the reagent at full strength
and others at various degrees of dilution. In Chetopterus eggs such
uncontrollable dilutions of the fixative produce marked differences in
the structure of astral rays and central bodies (Fry, 1932, pp. 161-167).
This factor may also operate in the case of mid-bodies and their
associated spindle remnants, and may explain their instability of
structure, their presence in some cells and absence in others on the
same slide and their differences in size and contour from cell to cell.’

Mid-bodies in Protozoa

Mid-bodies are usually absent in Protozoa. In the majority of
species mitosis is intranuclear, the nucleus separating into two parts
while the membrane remains intact. The morphological details of
the process of separation vary widely: in many cases the connecting
strand, prior to the final separation, is long and thin, while in others it
is relatively broad; in some the spindle fibers are visible up to the
time the break occurs, and in others they disappear before then. But
in most species there is no special point of focalization and no mid-body.
There are a few exceptions, however, where the mode of nuclear
constriction aggregates the spindle fibers to a point or a limited area,
and these may have mid-bodies, as in Cerattum (Lauterborn, 1895) and
Coccidium (Schaudinn, 1900).

In the occasional instances where the nuclear membrane disappears
and the division figure lies in the cytoplasm, a mid-body may arise as
the spindle is focalized by the process of cell division, exactly as in eggs
of Metazoa. This is illustrated in Acanthocystis (Schaudinn, 1896).
But if cell division does not occur at the time when such a cytoplasmic
figure is present, and the spindle is hence not focalized, there is no
mid-body, as shown in Monocystis (Muslow, 1911).

Atypical Mid-bodies

The mid-bodies thus far described arise without exception only as
spindle fibers are focalized. There are, however, several cases illus-
trated in the literature where thickenings appear either just prior to the
process of focalization or in its complete absence.

3 With the assistance of Dr. George Child, an attempt was made to produce
mid-bodies artificially. Somewhat in advance of the time when focalization of the
spindle occurs naturally, eggs of Arbacia and Cheiopiterus were individually con-
stricted with a glass hair in a plane passing through the middle of the late anaphase
spindle, in order to focalize it artificially. They were fixed instantaneously while the
needle was still in position, and then run up individually. The experiment failed,
because the protoplasm was rendered completely hyaline by the manipulation, in the
plane through which the needle passed, and no structure of any kind could be seen.

578 HENRY J. FRY

In Arion eggs (Lams, 1910), both in polar-body and first-cleavage
figures, thickenings arise at the middle of the spindle during anaphase,
before division has begun. Thereafter these are aggregated in an
orthodox manner into a bundle-like body when division occurs (Figs.
27-34). A similar situation is found in spermatogonia of Blaps
(Nonidez, 1920).

In spermatocytes of Llaveia (Hughes-Schrader, 1931) the spindle is
composed of tubes, each associated with a tetrad or a dyad. Soon
after the chromosomes separate, during anaphase, the central part of
each tube shrinks to a cord-like structure. At this time in some tubes,
or a little in advance of the shrinking process in others, mid-bodies
make their appearance—one in tubes associated with dyads, two in
those with tetrads. A little later the tubes are pressed together by the
division furrows and coalesce, the mid-bodies still maintaining their
identity (Fig. 63).

In developing Drosophila eggs (Huettner, 1933) mid-bodies arise at
the middle of the disintegrating spindle during telophase, in the
complete absence of cytoplasmic division (Fig. 64). This also occurs
in maturating eggs of Aspidiotus (Schrader, 1929).

These rare cases which, with the exception of Arion, I found only in
cells of insects, do not invalidate the fact that the great majority of
mid-bodies arise only in connection with the focalization of spindle
fibers. No explanation can be given for these exceptions, but such
behavior is not surprising in disintegrating gelatinous material.

Certain other cases reported in the literature might be regarded as
atypical, but they are only additional examples of the instability of
mid-bodies. For example, Buchner (1915, p. 28) illustrates in Ascaris
eggs a mid-body of irregular shape without any spindle remnant,
whereas Carnoy and Lebrun (1897, Plate II), who studied the same
material and used a similar, though not identical reagent, illustrate
neither body nor remnant. Such different results may be due to
differences in the reagents employed, or, if the mid-body here is a
staining artifact, to variations in the depth of stain. Furthermore,
where Buchner shows it without an associated spindle remnant, this
may be one of those occasional cases where the body persists after the
remnant has disappeared.

DISCUSSION
The Nature of Mid-bodtes

The foregoing facts indicate that mid-bodies are phenomena of
focalization. They do not arise in cells in which the spindle dis-
integrates prior to the time of division; and in those in which the spindle

MID-BODIES AND CENTRAL BODIES 579

is still present at that time, mid-bodies appear only as the fibers are
focalized by the process of cell division; they disappear, with rare
exceptions, when such areas of focalization disintegrate. Their wide
variability as to mode of formation is associated with the manner in
which the spindle fibers are aggregated. Their final position in the
cell is determined by the point at which the spindle remnant is pinched
together by the division furrows. They often show variability in
appearance from species to species, from cell cycle to cell cycle, and
even from cell to cell on the same slide. They are frequently modified
by differences in the fixatives employed, or variations in environmental
factors prior to fixation, and such differences are usually related, with
exceptions, to the size of the mitotic figure concerned, and the coarse-
ness of the spindle fibers. In many cases their structure is unques-
tionably nothing but aggregated spindle substances; and it appears
that such focalization phenomena may take many forms, sometimes
even simulating centrioles.

Three classes of focal bodies were mentioned earlier: living focal
bodies, coagulation artifact focal bodies, and staining artifact focal
bodies. A number of experiments were carried out to determine to
which of these categories mid-bodies belong. Some of them un-
doubtedly exist as a delimited structure in the coagulated cell. In
these cases, when the materials are slowly destained, the result being
closely observed at every stage by the use of a high power water-
immersion objective, it is seen that the bodies retain the dye
(Heidenhain’s haematoxylin) to a greater extent than any other cell
component, but they too finally yieldit. During the destaining process
they grow lighter in color but show no change in size. When the fully
destained slides are run up and studied with an oil immersion objective,
the bodies are still seen distinctly as refringent structures, although
they are colorless. Examples of this class of focal bodies are found in
the first polar-body cycle in Nereis (Fig. 60) and in cleaving Arbacia
eggs (Fig. 5). Whether they existed in the living cells as minute pools
of fiber material at the focal center (living focal bodies), or the living
point of focalization had no such structure and the body was produced
by fixation (coagulation artifact focal bodies) cannot be determined
because no structure can be seen in the living spindle.

Other mid-bodies, though sharply demarked and sometimes looking
exactly like centrioles, are clearly nothing but accumulations of dye at
focal points. If the materials are heavily stained with Heidenhain’s
heematoxylin, the bodies are relatively large; if stained in the ordinary

580 IBIS WN IROSZ ifs IISSNY

manner, they are smaller. If these bodies are gradually destained,
while watching the process, they do not lose their color while main-
taining their characteristic size, but throughout the process they
remain intensely black and become progressively smaller until they
disappear. When such completely destained preparations are run up
and studied in the usual way they show fibers which come to a point
without the presence of a body. This is the case for example, in brain
cells of Squalus embryos which have a centriole-like mid-body (Fig. 11).

The usual hypothesis that mid-bodies of animal cells are related
in some way to the cell-plate of plant cells is not consistent with the
behavior of mid-bodies as here described. The cell-plate is associated
with a broad spindle which has distinct fibers in the mid-region
(Fig. 13), whereas the mid-body is associated with a disintegrating
spindle, and only after its fibers have been focalized. In most in-

Fries. 13-15. Resemblances between the cell-plate of plant cells and mid-bodies
of animal cells. Fig. 13: cell plate in pollen mother cells of Fritillaria (Strasburger,
1888). Fig. 14: fully formed mid-body in Romalea spermatogonia (Wilson, 1928).
Fig. 15: early stage during formation of mid-body in epithelial cells of the salamander
lung (Flemming, 1891).

stances, regardless of their diversity in shape, mid-bodies are single
structures which do not resemble cell-plates in any way. In certain
cases, however, there is enough resemblance to have given rise to the
hypothesis. The mid-body which is composed of a row of granules
simulates the fixed cell-plate to some extent, but the differences are
marked (Figs. 13 and 14). Also, there is occasionally a brief phase
during the formation of a single mid-body when several granules are
present (Fig. 15) that calls to mind the cell-plate. In general, however,
it seems that the mid-body and the cell-plate have nothing in common.

But regardless of the relation of the mid-body to the cell-plate, the
question may be raised as to whether or not the mid-body is a true cell
component playing some réle in the process of cell division and the
consequent focalization of the spindle. May it be a causative factor

MID-BODIES AND CENTRAL BODIES 581

and not just an effect of focalization? The findings of this study do not
support such an interpretation. (1) In some cases the mid-body is
only an accumulation of dye. (2) Arbacia eggs cleave whether mid-
bodies are present (at temperatures from 10° to 25° C.) or absent (at
30° C.), indicating that they play no essential réle. (3) This con-
clusion is also supported by the fact that mid-bodies are present in
cleaving eggs of some species of Echinoidea but notin others. (4) And
all the other data presented in this investigation make it highly
improbable that we are here dealing with a cell component which plays
a causative role.

Cells exhibit other areas of focalization—aside from central bodies which will be
discussed in a moment—that behave much like mid-bodies. In his classic study of
Crepidula eggs Conklin (1902) illustrates an unusual case. During anaphase of the
second maturation division the minute centriole becomes a vacuole, while the centro-
some enlarges. Fora very brief period, during middle anaphase, a number of minute
bodies appear, each associated with a bundle of spindle fibers (Figs. 67-69). No such
bodies arise during the first polar-body cycle in this species. In Chetopterus eggs,
however, such phenomena occur during the first maturation division but not in the
second (Mead, 1898, Plate 17). In Ascaris eggs (Carnoy and Lebrun, 1897, Plate I)
they occur in both cycles. Beélar (1928, p. 35) illustrates a somewhat similar situation
in Monocystts.

Another possible phenomenon of focalization is shown when protoplasm and
certain artificial emulsions are fixed with reagents which demonstrate foam structure
(Biitschli, 1894). Minute bodies are frequently found at the points where the lines
of the foam structure meet (Figs. 65-66) and these may be focal bodies.

A still different phenomenon of focalization is illustrated in telophase of the first
polar body cycle of Arion eggs (Fig. 30). By the time the center of the old aster has
disintegrated two new central bodies and asters have appeared within it. The rays
of the old aster are most aggregated about this center, where they form a dark,
diffuse, ring-like area of focalization.

The Significance of Mid-bodies for the Central Body Problem

If focalization of the middle of the spindle may result in the
formation of structures which in some cases simulate individualized
cell components, it is in order to examine the situation at the ends of
the spindle, where similar focalization areas occur, of both rays and
fibers, or fibers alone.

In certain cases the similarity in appearance between mid-bodies
and central bodies is so marked that it compels attention. For
example, Conklin (1902, p. 43) 4 notes the similarity in first-cleavage

4 In using this quotation I have taken the liberty of substituting modern termi-
nology (according to Wilson, 1928, p. 675) for that used in the original paper. The
modern term centriole is used instead of the old term centrosome to indicate a minute,
sharply demarked body; the modern term centrosome is used instead of the old term
sphere to describe a larger less sharply demarked area which surrounds the centriole or

exists alone; the term central body is a general one which includes either or both of the
others.

582 Ta EIN[IRNE IJ LEM ENC

figures of Crepidula eggs: ‘‘This mid-body is for all the world like a
centriole with its surrounding centrosome and aster, and recalls
Watasé’s (1893) comparison of the mid-body to an intercellular
centriole. This apparent resemblance is still further supported by the
fact that the mid-body in this case becomes a hollow sphere before it
finally disappears, just as the centriole does. The mid-body is sur-
rounded by a darkly staining substance which resembles the centrosome
substance”’ (Figs. 39-44). In short, the structural changes which
occur in cleaving Crepidula eggs at the ends of the spindle during the
early history of the mitotic figure are later repeated at the middle of
the spindle when it is focalized by the division process. Furthermore,
mid-bodies and central bodies are again identical in appearance at
fourth cleavage, though here both are minute dots (Fig. 46). Such a
similarity of appearance occurs frequently: in Squalus brain cells
(Fig. 6), in leucocytes (Fig. 58) and connective tissue cells (Fig. 62) of
the salamander, in erythrocytes of the duck (Fig. 19), in cleaving
Echinus eggs (Fig. 59), and in many other cases.

There are numerous instances, however, where the two bodies differ
completely in appearance. The sharply demarked, minute, dumbbell-
like mid-body in cleaving Arbacia eggs (Fig. 5) bears no resemblance to
the large non-demarked granular central body. The elongate mid-
body in the smooth muscle cells of Amblystoma is unlike the typical
centriole (Figs. 21 and 26). In the first maturation division of Arion
eggs the mid-body is a band of fibers surrounded by a ring, in contrast
to the minute dot-like centrioles (Fig. 29); during first cleavage,
however, it is a similar band but without a ring, whereas the central
body is a large area concentrically differentiated and without centrioles
(Fig. 33). Crepidula eggs during maturation (Figs. 35-38) and
Enteroxenos cells at various cycles (Figs. 47-57) also exhibit differences
in appearance between mid-bodies and central bodies, and many other
examples could be cited.

The uncertainties of focal phenomena may explain why mid-bodies
and central bodies look alike in some cases and different in others, the
instances of dissimilarity being somewhat more numerous. But the
significant point is not whether mid-bodies and central bodies look
alike, but whether or not they behave alike.

In my studies of central bodies in various cell cycles of several
species, some of which were used in the present investigation of mid-
bodies, I demonstrated that the structure of the central body in these
cases is related to the structure of the rays or fibers. In cytasters of
artificially activated Echinarachnius eggs (1928), where no spindle is

MID-BODIES AND CENTRAL BODIES 583

present, a central body occurs only when rays reach the center,
regardless of how distinct the rays are peripherally. When a spindle is
present without asters, as in Squalus brain cells (1933a), the structure
is again related to the coarseness of the fibers. When the history of the
central body is followed from the beginning to the end of a mitotic
cycle, as it was in cleaving Echinarachnius eggs (1929a) and Squalus
brain cells (1933a), it is apparent that the central body undergoes
changes related to the coarseness of the converging rays and fibers, and
their general configuration. The continuity of the bodies from one
cell cycle to another cannot be demonstrated; they arise as areas of
focalization arise, and disappear as such areas disintegrate, even though
the peripheral region still has distinct rays. When, at any given
mitotic phase, rays and fibers are modified by the use of different
fixatives or environmental factors, the structure of the central bodies
is changed, as shown in Echinarachnius eggs at metaphase of the
first-cleavage figure (19296 and 1929c). This relationship is also
shown with unusual clearness in Chetopterus eggs (1932 and 19330).
Here, furthermore, when supposedly typical centrioles are demon-
strated, it is found that they differ in size and contour from cell to cell,
at the same mitotic phase, on the same slide. Hence the conclusion
was reached that in these cases the supposed central bodies are
phenomena of focalization: staining artifacts in Squalus brain cells, and
coagulation artifacts in the others.

The technique used in studying the mid-bodies reported in the
present paper was the same as that employed in the investigations of
central bodies just noted. Many fixatives were used to modify the
structure of the fibers, in order to determine the effects of such modifi-
cations upon the structure of the bodies; for the same reason, cells were
also subjected to abnormal environmental conditions prior to fixation.
In each case the sample studied was large enough to determine the
extent of structural variation, and all classes were reported and
considered in arriving at the conclusion.

If focalization phenomena are involved in the formation of both
mid-bodies and central bodies, it is rather surprising that in mitotic
figures which have asters, those bodies occurring at the middle of the
spindle where only fibers are present, should ever look like those
occurring at the spindle-ends where both fibers and rays are concerned.
But, as previously mentioned, in some cases they may be identical in
appearance, and in others completely dissimilar. At present it is as
impossible to explain this fact as it is to explain why, for example, the
mid-bodies of Crepidula eggs are ring-like during polar body formation,
but vesicle-like at first cleavage, and centriole-like at fourth cleavage

584 HENRY Vann

Fics. 16-34. Successive stages in the formation of mid-bodies.
erythrocytes of duck embryos (Heidenhain, 1907). Figs. 21-26: non-striated muscle
cells of Amblystoma larvae (Pollister, 1932). Figs. 27-34: first polar bodies and first
cleavage in Arion eggs (Lams, 1910).

MID-BODIES AND CENTRAL BODIES 585

Fics. 35-46. Mid-body structure and central body structure in various mitotic
cycles of Crepidula eggs (Conklin, 1902). Figs. 35-36: first polar bodies. Figs.
37-38: second polar bodies. Figs. 39-44: first cleavage. Fig. 45: third cleavage.
Fig. 46: fourth cleavage.

Fics. 47-69. Mid-bodies in various mitotic cycles of Enteroxenos cells (Bon-
nevie, 1906). Figs. 47-49: odgonia. Figs. 50-51: first polar bodies. Figs. 52—54:
second polar bodies. Fig. 55: spermatogonia. Fig. 56: primary spermatocytes.
Fig. 57: secondary spermatocytes. Mid-bodies of various cells. Fig. 58: salamander
leucocytes (Béla¥, 1928). Fig. 59: cleaving Echinus eggs (Boveri, 1901). Figs.
60-61: first and second polar-body figures of Nereis eggs (original). Fig. 62: con-
nective tissue cells of the salamander lung (Flemming, 1891). Fig. 63: primary
spermatocytes of Liaveia (Hughes-Schrader, 1931). Fig. 64: developing Drosophila
eggs (Huettner, 1933). Bodies occurring at the junction of the walls of alveoli.
Fig. 65: fixed eggs of Sphaerechinus (Biitschli, 1894). Fig. 66: artificial emulsion of
olive oil and NaCl (Biitschli, 1894). Multiple bodies, in addition to central bodies,
occurring at spindle-ends during middle anaphase in the second maturation division
of Crepidula eggs (Conklin, 1902). Fig. 67: early anaphase without bodies. Fig. 68:
middle anaphase with bodies. Fig. 69: late anaphase without them.

MID-BODIES AND CENTRAL BODIES 587

(Figs. 35-46). If, however, the focalization of fibers or rays may cause
the production of focal bodies, it is not surprising that there is variation
in their structure according to the size of the configuration as a whole
and the coarseness of the converging fibers. But our knowledge of the
physical chemistry of such systems is not sufficient for us to explain the
structural diversity in focal bodies, from species to species, and cell
cycle to cell cycle, in mitotic figures of the same size and distinctness.

If the conclusion of the present study is valid, we cannot accept the
presence of a sharply demarked body in an area of focalization—whether at
the middle or the ends of the sbindle—as evidence that we have demonstrated
a true cell component. This is, however, the generally accepted practice.
Structures which appear to the eye to be as individualized as chromo-
somes or plastids may nevertheless be merely ephemeral transient
phenomena of focalization—sometimes nothing but focal accumulations
of dye, as in both mid-bodies and centrioles in brain cells of Squalus
(Fig. 6) (Fry and Robertson, 1933). The eye sees such a structure,
and the mind accepts it on the basis of its appearance and attempts
to imagine its function.

The fact that bodies do not arise at the middle of the spindle until
it is focalized by the division furrows should make us alert to possible
pitfalls—errors in interpretation—when structures are found at points
where fibers or rays or both converge. In this connection it is sig-
nificant that bodies are not present at the ends of anastral spindles
unless the tips are sharply focalized. The blunt, anastral spindles
found in some odcytes and spermatocytes do not have them (with
possibly a few exceptions in the latter when centriole-blepharoplasts
are present).

If the conclusion of this investigation is correct, the current central
body hypothesis must be reevaluated. Focal bodies have probably
been confused with such cell components as centriole-blepharoplasts
of male germ cells, diplosomes of vertebrate cells, and similar struc-
tures of Protozoa and other cell types, which for the most part are
concerned with the formation of axial filaments and flagella, no matter
what their rdle in mitosis. The great majority of cell types have no
such components, and it is yet to be ascertained to what an extent their
supposed central bodies are actually artifacts of focalization.®

5 The data of the present study suggest that the spindle, despite its homogeneous
appearance in the living cell, has some kind of linear organization. The very fact
that bodies appear in the plane where the division furrow exerts pressure on the
spindle indicates that there are differentiated materials there. If the spindle were
actually homogeneous, it is probable that its materials would flow to one side or the

other when it is pinched together, in which case no focalization bodies would be
formed.

588 HENRY J. FRY

RESUME

Mid-bodies were studied in various cell cycles in a number of
species; many fixatives were used and cells were subjected to different
environmental modifications prior to fixation, to modify the structure
of both bodies and fibers in order that the relation between them might
be analyzed.

Mid-bodies are found only in cells in which the spindle is still
present when division occurs; they are absent in the numerous cases
where the spindle disappears before that time. They arise only as the
spindle fibers are gradually brought to a focus by the advancing
division furrows; they usually disappear simultaneously with the
spindle remnant. Their final position is determined by the point
where the division furrows meet. There are many structural types:
centriole-like dots, blobs, ovoids, dumbbells, rings, cones, lines, rows of
dots, and other configurations. Some are obviously nothing but
aggregated spindle materials, whereas others look like individualized
cell components. Different structural types may occur in the same
species in successive cell cycles. They may vary in size and contour
from cell to cell on the same slide. They are generally unstable and
easily modified by the use of various environmental factors or different
fixatives. Such structural modifications are usually related to the
coarseness of the fibers.

Mid-bodies are generally regarded as a vestigial homolog of the
cell-plate of plants. The present study, however, interprets them as
phenomena of focalization or ‘‘focal bodies.”” It is suggested that these
are produced as the result of the concentration of spindle or aster
materials at points where they are focalized. In some cases mid-
bodies are only an accumulation of dye at the focal area; in others they
are probably produced by the process of fixation.

If bodies which look like typical cell components can be formed
at the middle of the spindle, as the result of the focalization of its
materials brought about by the advancing division furrows, similar
phenomena may occur at the areas of focalization at the spindle ends
where both fibers and rays may converge. Previous studies of central
bodies, in some of the same cells used for the present study of mid-
bodies, indicate that they too are produced as a result of the focalization
of spindle and aster materials. It is, therefore, suggested that in the
formulation and development of the current central body hypothesis,
focal bodies may have been confused with true cell components such as
centriole-blepharoplasts, diplosomes, and similar structures.

MID-BODIES AND CENTRAL BODIES 589

BIBLIOGRAPHY

Bear, Kari, 1928. Die cytologischen Grundlagen der Vererbung. Gebriider
Borntraeger, Berlin.

BoNNEVIE, KrisTINE, 1906. Untersuchungen iiber Keimzellen. I. Beobachtungen
an den Keimzellen von Enteroxenos 6stergreni. Jena. Zeitschr. Naturwiss.,
41: 229.

BovERI, THEODOR, 1901. Zellen-Studien, Heft4. Ueber die Natur der Centrosomen.
Gustav Fischer, Jena.

BucHNER, PAUvL, 1915. Praktikum der Zellenlehre. Gebriider Borntraeger, Berlin.

BitscuHii, O., 1894. Investigations on Microscopic Foams and on Protoplasm.
(Translated from the German work of 1892 by E. A. Minchin.) Adam and
Charles Black, London.

Carnoy, J. B., AnD H. Lesprun, 1897. La fécondation chez !’Ascaris megalocephala.
La Cellule, 13: 63.

ConkKLIN, Epwin G., 1902. Karyokinesis and cytokinesis in the maturation,
fertilization and cleavage of crepidula and other gastropoda. Jour. Acad.
Nat. Sct. Phila., Ser. 2, 12: Part 1.

FLEMMING, W., 1891. Neue Beitrage zur Kenntniss der Zelle. II. Arch. mikros.
Anat., 37: 685.

Fry, Henry J., 1928. Conditions determining the origin and behavior of central
bodies in cytasters of Echinarachnius eggs. Bzol. Bull., 54: 363.

Fry, Henry J., 1929a. The so-called central bodies in fertilized Echinarachnius
eggs. I. The relationship between central bodies and astral structure as
modified by various mitotic phases. Biol. Bull., 56: 101.

Fry, Henry J., 19296. The so-called central bodies in fertilized Echinarachnius
eggs. II. The relationship between central bodies and astral structure as
modified by various fixatives. Bzol. Bull., 57: 131.

Fry, Henry J., MATTHEW JACoBs, AND H. M. Lies, 1929c. The so-called central
bodies in fertilized Echinarachnius eggs. III. The relationship between
central bodies and astral structure as modified by temperature. Bzol.
BU Sdicnlode

Fry, Henry J., 1932. Studies of the mitotic figure. I. Chetopterus: central body
structure at metaphase, first cleavage, after picro-acetic fixation. Bzol.
Bull., 63: 149.

Fry, Henry J., anD C. W. ROBERTSON, 1933a. Studies of the mitotic figure. II.
Squalus: the behavior of central bodies in brain cells of embryos. Anat.
Rec., 56: 159.

Fry, Henry J., 19336. Studies of the mitotic figure. III. Chztopterus: central
body structure at metaphase, first cleavage, after using diluted fixatives.
Biol. Bull., 65: 207.

HEIDENHAIN, Martin, 1907. Plasma und Zelle. Gustav Fischer, Jena.

HvETTNER, ALFRED F., 1933. Continuity of the centrioles in Drosophila melano-
gaster. Zeitschr. Zellfor. mikr. Anat., 19: 119.

HUGHES-SCHRADER, SALLY, 1931. A study of the chromosome cycle and the meiotic
division-figure in Llaveia bouvari—a primitive coccid. Zettschr. Zellfor.
mtkr. Anat., 13: 742.

Lams, H., 1910. Recherches sur l’oeuf d’Arion empiricorum. Mém. Acad. roy.
Belg., Classe Sci., Deuxiéme Série, 2: 1.

LAUTERBORN, R., 1895. Protozoenstudien. I. Kern- und Zelltheilung von Ceratium
hirundinella. Zeztschr. wiss. Zool., 59: 167.

Meap, A. D., 1898. The origin and behavior of the centrosomes in the annelid egg.
Jour. Morph., 14: 181.

Mustow, Kart, 1911. Uber Fortpflanzungserscheinungen bei Monocystis rostrata,
n.sp. Arch. Protist., 22: 20.

NOoNIDEZ, José F., 1920. The meiotic phenomena in the spermatogenesis of Blaps,
etc. Jour. Morph., 34: 69.

590 HENRY) Jo ERY

POLLISTER, ARTHUR W., 1932. Mitosis in non-striated muscle cells. Anat. Rec., 53:
11.

ScHAUDINN, F., 1896. Uber das Centralkorn der Heliozoen, ein Beitrag zur Centro-
somenfrage. Verhandl. Deutsch. Zool. Ges., 6: 113.

ScHAUDINN, F., 1900. Untersuchungen iiber den Generationswechsel bei den
Coccidien. Zool. Jahrb. Abt. Anat., 13: 197.

SCHRADER, FRANZ, 1929. Notes on reproduction in Aspidiotus hederze (Coccide).
Psyche, 36: 232.

SHARP, LESTER W., 1934. Introduction to Cytology. Third Edition. McGraw-
Hill Book Co., Inc., New York.

STRASBURGER, ED., 1888. Histologische Beitrage. I. Ueber Kern- und Zelltheilung
im Pflanzenreiche usw. Gustav Fischer, Jena.

WATASE, S., 1893. Homology of the centrosome. Jour. Morph., 8: 433.

‘Witson, Epmunp B., AND Epwarp LEAmMING, 1895. An Atlas of the Fertilization
and Karyokinesis of the Ovum. Macmillan Company, New York.
WItson, EpmunpD B., 1928. The Cellin Development and Heredity. Third Edition,

with corrections. The Macmillan Company, New York.

A PHYSIOLOGICAL AND HISTOLOGICAL STUDY OF THE
PRONDAL, CORTES Of VTHE, SEAL
(PHOCA DY FOULINA) +

DAVID McK. RIOCH
(From the Department of Anatomy, Harvard Medical School)

The so-called ‘“‘motor areas’”’ of the cortex of the cat, dog and
bear have been investigated recently with the combined methods of
electrical stimulation and histological examination (Langworthy, 1928;
Smith, 1933, 1935, etc.). The general pattern, both functional and
anatomical, of these three fissiped carnivores is quite similar, although
minor variations are distinct. It was therefore of interest to make a
comparative study of the harbor seal (Phoca vitulina), a pinniped
carnivore which is functionally adapted to an aquatic existence and
in which the trunk and extremities are very considerably modified
(Howell, 1928).

I should like to express my thanks to the Superintendent, Mr.
Thomas H. Dorr, and his assistants, particularly Mr. E. C. Barter, at
the Boothbay Harbor Station of the United States Bureau of Fisheries,
for obtaining the animals and assisting in handling them.

METHODS

Experiments were successfully completed on 6 seals approximately
4 to 5 months old. The animals were in excellent condition and
weighed between 18 and 25 kgm. Satisfactory anesthesia was ob-
tained with Dial fluid (Ciba) 0.3 cc. per kgm. intraperitoneally,
supplemented by a few whiffs of ether during the opening of the
skull. It was found preferable to keep the body of the seal sub-
merged in a tank of water during the early stages in order to assist
respiration.2 The thin skull was readily removed with trephine and
rongeurs. Since the electrically excitable cortex was located behind
the eye, the latter was enucleated and the orbital roof removed in

1The seals were obtained and the physiological observations made at the
Boothbay Harbor Station of the U. S. Bureau of Fisheries.

2 When on land the seal normally breathes rhythmically and regularly. Five to
ten minutes after the injection of the dial, respirations became intermittent. Four to
five deep breaths were taken, ending in inspiration, following which the nostrils were
tightly closed and the breath held for 15 to 40 seconds, the cycle being then repeated.
The administration of ether by a cone during three or four of these cycles resulted in
regular, slow deep respiration.

591

592 DAVID McK. RIOCH

the later experiments. The rectal temperature of the animals was
kept between 37° and 40° C. by pouring cold sea water over them
occasionally.

Stimulation was bipolar by means of a pair of silver-silver chloride
electrodes with an interpolar distance of 2mm. Current was supplied
by the 60 cycle a.c. line through a potentiometer. The effective

voltage varied in different animals from } to 5 volts. For single

Fic. 1. Outline drawing of a lateral view of the brain of seal 5. X 1.5.
The position of the electrically excitable cortex is indicated by the shaded area.
an, S. ansatus. co, S. coronalis. cr, S. cruciatus. e lat, S. endolateralis. Jat, S.
lateralis. Jat p, S. lateralis posterior. olf, Bulbus olfactorius. p cr, S. post-
cruciatus. pr cr, S. precruciatus. pr s, S. presylvius. pro, S. proreus. ri,
Fissura rhinalis. s,S. pseudosylvius. ssa, ssp, sss, S. suprasylvius anterior, posterior
and superior.

shocks a Harvard inductorium, with 3 volts in the primary circuit
and the secondary coil set at 7 to 9 cm., was employed. Between
observations the cortex was kept moist by application of cotton
pledgets wet with warm Ringer’s solution.

At the end of the experiment the brains were removed and pre-
served in formalin. Serial sections in the sagittal plane were cut at
35 w of the frontal poles of the left hemispheres of seals 3 and 4, and

STUDY OF FRONTAL CORTEX OF THE SEAL 593

in the horizontal plane of the right hemisphere of seal 4. Every
twentieth section was stained with thionine and mounted.

RESULTS

In conformity with the rounded shape of the skull, the high
position of the nostrils and the microsmatic habits of the seal, the
brain tends to a more spherical form than in the terrestrial carnivores.

fF Fic. 2. Outline drawing of a frontal view of the brain of seal 4. X 1.5.

The sulci are labelled as in Fig. 1.

F The electrically excitable cortex is indicated on the left hemisphere. The
numbers are referred to in the text.

The histological areas are indicated on the right hemisphere. fr. agr, Area
frontalis agranularis. fr. gr, Area frontalis granularis. g pyr pc, Area giganto-
pyramidalis postcentralis. g pyr prc, Area gigantopyramidalis precentralis. gr pc,
Area granularis postcentralis (the caudal boundary of this area is not defined in the
figure).

The general pattern of the sulci, however, was found to be similar,
as illustrated in Figs. 1 and 2. A number of additional shallow sulci
were present, which resulted in a reduplication and subdivision of the
gyri, a condition more pronounced in other aquatic mammals (cf.
Langworthy, 1932, 1935). Certain features in which the frontal lobe
differed from that of other carnivores may be noted. The sulcus
cruciatus was relatively small and was quite shallow laterally, but

594 DAVID McK. RIOCH

medially became deeper and, behind the olfactory bulb, ran into
the well-developed sulcus postcruciatus. The sulcus ansatus was well
marked and entered the S. suprasylvius anterior. In some instances
it extended medially as a shallow groove into the S. postcruciatus,
but in no case into the S. lateralis. The S. coronalis was very shallow,
appearing merely as a slight depression in some of the hemispheres.
In one case it extended ventrally as far as the S. precruciatus, which
was also shallow. The large Gyrus proreus was divided into three
parts by the S. proreus and the thin olfactory stalk.

PHYSIOLOGICAL OBSERVATIONS

The boundaries of the electrically excitable cortex under the
present experimental conditions were as follows. The posterior limit
was defined by the S. cruciatus. Lateral to this a small area giving
movements of the tail extended onto the G. sigmoideus posterior,
medial to the S. coronalis. The medial and ventromedial boundaries
were sharply defined at the lateral margin of the olfactory bulb and
by the S. presylvius. The lateral and ventro-lateral boundaries did
not follow the superficial markings except along the posterior third of
the S. coronalis, but extended well onto the G. suprasylvius anterior
(see Fig. 2).

In two of the animals movements of the hind limb were obtained
by stimulation of high intensity (10 volts) from the posterior lip of
the S. cruciatus. In no other case, however, were striped muscle
responses evoked by superficial stimulation outside of the designated
area.

The motor cortex was readily subdivided, on the basis of the
movements evoked, into the following areas: (1) tail area, (2) tail
and hind-flipper area, (3) fore-flipper area, (4) neck area, and (5) face
area. These are correspondingly numbered in Fig. 2.

The tail area (1) was small and sharply defined. The only move-
ment obtained consisted of elevation and deflection of the tail to the
contralateral side.

EXPLANATION OF FIGURES 3-6

Fies. 3, 4, 5 and 6. Microphotographs of portions of a sagittal section of the
left hemisphere of seal 4 in the plane indicated by arrow A in Fig. 2. Thionine stain.
35. X 35.

3. Area gigantopyramidalis precentralis, caudal to the S. precruciatus (hind-
flipper area).

4. Area gigantopyramidalis precentralis, rostral to the S. precruciatus (fore-
flipper area).

5. Area frontalis agranularis, rostral to the S. presylvius.

6. Area granularis postcentralis, through the S. ansatus.

SHPUONS Ola WINONA (COKINa.< Ols Islay Sis se, 595

Sent

s
i

Set

6G sat NR

Sa ae
. -

ae er

oe “ee : ee
SN re a

OS ate

FIGURES 3-6

596 DAVID McK. RIOCH

The tail and hind-flipper area (2a, 2b, 2c) was the most extensive
and merged into the fore-flipper area (3a, 3b) in a zone (2-3) from
which various combinations of movements of the contralateral fore-
flipper, both hind-flippers and tail were evoked. From 2a the tail
and contralateral hind-flipper were elevated and deflected contra-
laterally. From 26 and c similar movements resulted of the tail
and both hind-flippers, together with ventral arching of the back so
as to raise the hindquarters of the animal off the table. From 6 the
flippers were flexed at the digits, whereas from c they were extended.

The line of demarcation between the fore-flipper and neck areas
was much sharper than that between the fore- and hind-flipper
areas. Movements of the contralateral, but not of the ipsilateral,
fore-flipper were obtained. From 3a there was extension of the
digits, moderate extension at the elbow and adduction at the shoulder.
From 30 there was flexion of the digits and elbow with abduction at
the shoulder.

The boundaries of the neck area (4) were moderately well defined.
Movements of the shoulder girdle, drawing the shoulder forward, and
of the neck, with deviation of the head to the contralateral side, were
obtained either alone or in combination. The shoulder was localized
posterior to the neck.

The face area (5) was somewhat larger than the tail area, but
much smaller than those for the flippers. Retraction of the contra-
lateral angle of the mouth and closure of the contralateral eye, usually
separately, occasionally together, were the only movements elicited.

The boundaries between areas 3 and 4, and 4 and 5 could be
defined within approximately 1 mm. in a single series of stimulations,
but there was a shift of the boundary of as much as 2 mm. backwards
or forwards in successive series of observations depending on whether
the previous stimuli had been applied in front or behind respectively.
Similarly, the predominating movements evoked from the zone 2-3
depended on whether the preceding stimulation had been of the hind-
or of the fore-flipper areas.

The most excitable area of the motor cortex was found in the central
region of the hind-flipper area (the region of the largest giganto-
pyramidal cells, see below). Here, in four of the seals, an intensity
of 0.5 volts with a.c. stimulation evoked strong movements and in
one case a single break-shock (Harvard inductorium, 7 cm.) resulted
in a jerky contraction of the contralateral hind-flipper. Epileptiform
after-discharge of varying duration was regularly elicited by a.c.
stimulation of 3—5 volts for 10 to 20 seconds from any of the areas,
but was most marked from areas 2 and 3.

STUDY OF FRONTAL CORTEX OF THE SEAL 597

Rete ay

Ot
“se
oa = tage *

ot sles! pad os "
Pais oe eS Moles. J
— 2 . ; . ae oacas

Fics. 7 and 8. Microphotographs of portions of sagittal sections of the left
hemisphere of seal 4. Thionine stain. 35. 35.

7. Transition from the Area gigantopyramidalis precentralis (face area) to the
Area frontalis agranularis, the lower border of the electrically excitable area in the
plane of arrow B, Fig. 2.

8. Area gigantopyramidalis postcentralis and the transition to Area granularis
postcentralis above, just rostral to the S. ansatus in the plane of arrow A, Fig. 2.

598 DAVID McK. RIOCH

HISTOLOGICAL OBSERVATIONS

The exposed cortex of the frontal lobes was well developed and
the several cell layers clearly defined. The average thickness was
approximately 2mm. In the depths of the sulci, however, it became
much thinner, at the expense of the lower layers, and less well differen-
tiated. This was particularly marked in the cruciate, precruciate and
presylvian sulci.

The histological areas conformed in general to the pattern and
structure described by Smith (1935) in the dog. Their extent on the
exposed surface of the frontal lobe is shown in Fig. 2. Certain charac-
teristics of the classical six-cell layers of the several areas, as seen in
sagittal and horizontal sections, may be summarized as below.

Area gigantopyramidalis precentralis—(Figs. 3, 4, 7, 9 and 10.)
Layer I was better developed than in the other areas, but Layer II
was relatively thinner and less dense, with many small to medium-
sized pyramidal cells. Layer III was very broad, with numerous
medium-sized pyramidal cells. Layer IV, absent. Layer V _ con-
tained typical giant pyramidal cells which tended to be arranged in
groups between fiber bundles. This layer showed more variation
than did the others in different parts of the area. The cells were
largest in the upper, central region (Fig. 1) (the central portion of
the hind-flipper area), becoming smaller toward the periphery (Figs. 7,
9, 10) Gncluding the tail, neck and face areas). There was a rather
abrupt change from the posterior to the anterior lip of the precruciate
sulcus (cf. Fig. 3 with Fig. 4), but elsewhere the transition was gradual.
These cells were also considerably smaller around the sulci, particularly
in the lower half of the cruciate sulcus, although a narrow band of
gigantopyramidal cells again appeared in the posterior lip of this
sulcus. Layer VI was broad, with numerous small, fusiform cells.

The myeloarchitecture was prominent throughout the area and
the cells of layers III, V and VI appeared to be grouped between
radiating bundles of fibers. Around the bottoms of the sulci, however,
the orientation of the cells seemed to be determined by U fibers.

The boundary of the Area gigantopyramidalis precentralis and the

EXPLANATION OF FIGURES 9 AND 10

Fics. 9 and 10. Microphotographs of portions of a horizontal section through
the right hemisphere of the brain of seal 4 in the plane indicated by arrow C, Fig. 2.
Thioninestain. 35yu. XX 35.

'9. Transition from the Area gigantopyramidalis precentralis (right), to the Area
frontalis agranularis (left), at the lateral margin of the Bulbus olfactorius (upper
left).

10. Transition from the Area gigantopyramidalis precentralis (extreme left)
through the Area gigantopyramidalis postcentralis (center) to the Area granularis
postcentralis (extreme right) between the S. coronalis and the S. suprasylvius
anterior.

LOSS Nae

ind CE

as 8 SECURIT Oe :
eae SE Ge nat Raat eS
: WEES) ma eh

: fo gece i Be
Pipa ° Wd SL

Cu sme A te
a ~

: Pre eat
FT Rg i

Spies es

eis

aries 5
oe

s . ae

$F Se,
fd ee a

FIGURES 9 AND 10

599

600 DAVID McK. RIOCH

Area frontalis agranularis was quite sharp (Figs. 7 and 9). The
transition, however, to the Area gigantopyramidalis postcentralis
(Fig. 8) and to the granular cortex of the G. suprasylvius anterior
was very gradual (Fig. 10).

Area frontalis agranularis.—(Figs. 5 and 7.) In comparison with
the precentral gigantopyramidal cortex this area showed the following
characteristics. Layer I was approximately half as thick. Layer II
contained more cells, but with a smaller proportion of pyramidal
forms. Layer III also showed an increase in number of cells. Layer
IV was absent. Layer V varied in thickness at different regions and
the cells varied in size from slightly smaller to slightly larger than
those of layer III. Occasional cells contained heavily staining Nissl
substance, but the majority were relatively pale. Layer VI was
similar to that in the area gigantopyramidalis. The myeloarchitecture
was less prominent, although in places the radiating arrangement of
cells between fiber bundles was noticeable.

This type of cortex extended over a wide area, including most of
the G. proreus and the ventral portions of the G. genualis where a
transition to a granular type of cortex occurred posteriorly. Anterior
to this area, between it and the undifferentiated cortex of the basal ol-
factory areas, was the Area frontalis granularis. The latter was poorly
defined and small in extent. It was somewhat thicker, but the cell
types were similar with the addition of the granular cells of layer IV.

Area gigantopyramidalis postcentralis—(Figs. 8 and 10.) The
following changes occurred through the transition zone between this
and the Area gigantopyramidalis precentralis. Layer I was reduced
to about one-third as thick. Layer II increased in density and
breadth, most of the cells being rounded in form. Layer III was
considerably reduced and the cells became smaller. Layer IV ap-
peared as a narrow, ill-defined band of granular cells. The giant
pyramidal cells of layer V persisted, but greatly reduced in numbers
and size. Layer VI showed little change. The radiating bundles of
fibers through the lower layers remained prominent.

This area covered the greater part of the exposed surface of the
G. sigmoideus posterior, the transition to the Area granularis post-
centralis being sharply defined at the postcruciate and ansate sulci
(Fig. 9). A similar, though narrower, zone extended round the lateral
border of the Area gigantopyramidalis precentralis, between it and
the granular cortex of the G. suprasylvius anterior (Fig. 10).

Area granularis postcentralis.—(Figs. 6 and 8.) This area differed
strikingly from those described above. Layer I was narrow. Layer
II was broad and dense, the cells being mainly small and rounded.
It appeared to fuse with layer IV, which contained similar cells.

STUDY OF FRONTAL CORTEX OF THE SEAL 601

Layer III showed as a band of scattered, small to medium-sized,
pyramidal cells amongst the granular cells at the junction of layers
II and IV. Layer V was sharply defined, consisting chiefly of fibers,
with a few cells of the small, pyramidal type. Layer VI was well
developed and for the most part contained rounded cells with occa-
sional fusiform and triangular elements.

The decrease in thickness of this type of cortex round the base of
the sulci was much less marked than in the other areas and was mostly
due to diminution in layer VI.

This area extended caudally over the G. lateralis and rostro-
laterally over the G. suprasylvius anterior. In the latter location
layer V was better developed than it was caudally. Laterally it
was bounded by an area in the anterior wall of the S. suprasylvius
anterior which showed a poorly developed layer III, a narrow, irregular
layer IV, and sparsely scattered, but very large, pyramidal cells in
layer V. Rostral to the Area gigantopyramidalis precentralis the
transition to the Area frontalis agranularis was relatively sharp.

DISCUSSION

The present experiments were performed under a single form of
anesthesia and on animals otherwise normal. Because of these
limitations it is obvious that conclusions with regard to correlations
between so-called electrical excitability and the cytoarchitecture of
the different cortical areas are of little or no significance (cf. Tower,
1936; Rioch and Rosenblueth, 1935). It is of interest, however, that
the lowest threshold was found in the region of the largest Betz cells;
and the observation that a strong movement could be evoked by a
single shock in this area is unusual in carnivores under dial anesthesia.

The extent and differentiation of the Area gigantopyramidalis, on
the basis of both the physiological and anatomical observations,
compare favorably with these features in other carnivores as ob-
served by the present author under similar conditions of anzesthesia
(dog and cat) and as described in the literature (cf. Langworthy, 1928;
Smith, 1933, 1935, etc.). In contrast with this high degree of develop-
ment of the central representation stands the apparently simpler and
less differentiated form of the extremities of the seal. It may be
concluded that the extent of central representation is to be correlated
with function and not with form. Further evidence for this hypothesis
is furnished by the following considerations. In the terrestrial
carnivores electrical stimulation of the cortex evokes movements of the
extremities which resemble certain phases of the normal activity of
the animal when initiating locomotion, feeding, seizing prey, etc.
In the seal, however, the movements of the trunk and extremities

602 DAVID McK. RIOCH

elicited from the cortex resembled parts of the normal swimming
actions, which are finely and swiftly executed, and not the clumsy
movements of locomotion of that animal on land.

The rostral position of the gigantopyramidal area and the shallow-
ness of the S. cruciatus may be due in part to the relatively small
size of the olfactory brain, allowing more room for superficial develop-
ment of the frontal lobes of the hemispheres.

The cerebellum, particularly the lateral lobes, and the pons were
found to be very large in the seal as compared with the dog and cat,
resembling the development of these structures in other aquatic
mammals (Langworthy, 1932, 1935). This is probably to be corre-
lated with the wide extent of the Area frontalis agranularis. The
teleological explanation which suggests itself is the necessity for par-
ticularly fine codrdination of movements controlling the position of
the animal in three-dimensional space.

SUMMARY

The exposed cortex of the frontal lobes of the left hemispheres of
six young seals (Phoca vitulina) under dial anesthesia was stimulated
electrically. Movements of the tail, both hind-flippers and the
contralateral fore-flipper, neck and face were evoked. The cortical
localization is charted in Fig. 2.

Histological examination (thionine stain) of three frontal lobes
revealed a well-developed cortex divisible into cytoarchitectural areas
structurally similar to those in other carnivores, but differing in their
extent and their relation to the sulci (Fig. 2).

On the basis of a comparison of the seal with the terrestrial carni-
vores it is concluded that the degree of differentiation of the cortical
representation is to be correlated with function and not with form.

BIBLIOGRAPHY

Howe tt, A. B.,1928. Contribution tothe comparative anatomy of the eared and ear-
less seals (genera Zalophusand Phoca). Proc.U.S. Nat. Museum, 73: Art. 15.

LanGwortTHy, O. R., 1928. The area frontalis of the cerebral cortex of the cat, its
minute structure and physiological evidence of its control of the postural
reflex. Bull. Johns Hopkins Hospital, 42: 20.

LanGwortHy, O. R., 1932. A description of the central nervous system of the
porpoise (Tursiops truncatus). Jour. Compar. Neurol., 32: 437.

LanGwortTsxy, O. R., 1935. Thebrain of the whalebone whale, Balznoptera physalus.
Bull. Johns Hopkins Hospital, 57: 142.

Riocu, D. McK., anp A. ROSENBLUETH, 1935. Inhibition from the cerebral cortex.
Am. Jour. Physiol., 113: 663.

SmiTH, W. K., 1933. Motor cortex of the bear (Ursus americanus). A physiologic
and histologic study. Avch. Neurol. and Psychiat., 30: 14.

SmitH, W. K., 1935. The extent and structure of the electrically excitable cerebral
cortex in the frontal lobe of the dog. Jour. Compar. Newurol., 62: 421.

TOWER, SARAH S., 1936. Extrapyramidal action from the cat’s cerebral cortex:
motor and inhibitory. Braim, 59: 408.

INDEX

A BRAMOWITZ, A. A. The réle of
the hypophyseal melanophore hor-
mone in the chromatic physiology of
Fundulus, 134.

Amoeba, rate of locomotion, as affected
by luminous intensity and adapta-
tion to light, 126.

ANDERSON, BERTIL GOTTFRID, H. LUMER
and L. J. Zupancic, Jr. Growth
and variability in Daphnia pulex,
444,

Arbacia, localization of micromere-, skel-

eton-and entoderm-forming material

in unfertilized egg of, 295.

, effect of salts of heavy metals on
development, 401.

Ascaris eggs, suppression of cleavage by
ultracentrifuging, 99.

Autosomal lethals in wild populations of
Drosophila pseudoobscura, 542.

Autotomy in brachyuran, Uca pugnax,

155.

BACTERIAL content of clover nodule
cells, 112.

BALL, Eric G., AND C. CHESTER STOCK.
The pH of sea water as measured
with the glass electrode, 221.

BAvER, Hans. See Tyler and Bauer,
164.

Beams, H. W., AND R. L. Kinc. The
suppression of cleavage in Ascaris
eggs by ultracentrifuging, 99.

Blood and body fluid, of turtle, colloid
Osmotic pressure, nitrogen content
and refractive index, 504.

Boyp, WILLIAM C. Cross-reactivity of
various hemocyanins with special
reference to the blood proteins of
the black widow spider, 181.

BuTLER, MARGARET RUTH. The effect
of its nitrogen content on the de-
composition of the polysaccharide
extract of Chondrus crispus, 143.

ALANUS finmarchicus, seasonal pro-
duction off Woods Hole, 464.

CAMPBELL, MILDRED L., AND ABBy H.

TURNER. Serum protein measure-

603

ments in the lower vertebrates. I.
The colloid osmotic pressure, nitro-
gen content, and refractive index of
turtle serum’ and body fluid, 504.

Carver, Gar L. Studies on productiv-
ity and fertility of Drosophila
mutants, 214.

Centrifuging, determination of polarity
by, in eggs of Fucus, 249.

Cerebratulus lacteus, determination in
early development of, 317.

Cuace,F.A.,JR. See Welsh, Chace and
Nunnemacher, 185.

Cuitp, GeEorGE P. See Glaser and
Child, 205.

Chondrus crispus, effect of nitrogen con-
tent on decomposition of polysac-
charide extract, 143.

Chromatic physiology of Fundulus, réle
of hypophyseal melanophore hor-
mone, 134.

Chromatophore reactions in normal and
albino paradise fish, 535.

Ciancy, C. M. See Whitaker and
Clancy, 552.

CLARKE, GEORGE L., AND DONALD J.
ZINN. Seasonal production of zo-
oplankton off Woods Hole with spe-
cial reference to Calanus fin-
marchicus, 464.

Cleavage and polar body extrusion in
artificially activated eggs of Urechis
caupo, 164.

, suppression of, in Ascaris eggs by

ultracentrifuging, 99.

Clover nodule, bacterial and alleged mito-
chondrial content of cells of, 112.

Color pattern, genetics and histology, in
normal and albino paradise fish, 527.

CooNFIELD, B.R., AND A. GOLDIN.
The problem of a _ physiological
gradient in Mnemiopsis during re-
generation, 197.

Cortex, frontal, of seal, physiological and
histological study of, 591.

DALTON, H. CLarkK AND H. B. Goop-
RicH. Chromatophore reactions in
the normal and albino paradise fish,
535.

604

Daphnia pulex, growth and variability
in, 444,

Determination, in early development of
Cerebratulus lacteus, 317.

Differentiation, energetics of, VI, 261.

Diurnal migration, deep water animals,
18520»

Drosophila mutants, fertility and pro-

ductivity, 214.

pseudodbscura, autosomal lethals in

wild populations of, 542.

NDOCRINE feeding, relation to re-
generation, growth and egg capsule
production in Planaria, 227.

Entoderm-forming material, localization
of, in unfertilized egg of Arbacia,
295.

Environmental factors, differential effect
of, on Microbracon hebetor Say and
its host, Ephestia kuhniella Zeller,
147,

FERTILITY and productivity of Dro-
sophila mutants, 214.

Fry, Henry J. Studies of the mitotic
figure. VI. Mid-bodies and their
significance for the central body
problem, 565.

Fucus furcatus, effect of salinity on
growth of eggs of, 552.

Fundulus, chromatic physiology of, 134.

, development of pituitary gland, 93.

, histochemistry of ovary, 67.

Fungi, saprophytic, occurrence in marine
muds, 242.

ENETICS and histology of color
pattern in normal and albino para-
dise fish, 527.

GLASER, OTTO AND GEORGE P. CHILD.
The hexoctahedron and growth, 205.

Gotpin, A. See Coonfield and Goldin,
197.

Go tpsmiTH, E.D. The relation of endo-
crine feeding to regeneration, growth,
and egg capsule production in
Planaria maculata, 227.

GoopricH, H. B., ann Maurice A.
SmiTH. Genetics and histology of
the color pattern in the normal and
albino paradise fish, Macropodus
opercularis L., 527.

GoopricH, H.B. See Dalton and Good-
rich, 535.

Growth and the hexoctahedron, 205.

INDEX

and variability in Daphnia pulex,
444,

, effect of salinity on, eggs of Fucus,
ozs

, relation of endocrine feeding in
Planaria to, 227.

GuTHRIE, Mary J. See Marza, Marza
and Guthrie, 67.

HEILBRUNN, L. V. AND Kart M.
WILBUR. Stimulation and nuclear
breakdown in the Nereis egg, 557.

Hemocyanins, cross-reactivity of, with
special reference to blood proteins of
black widow spider, 181.

HeERSHKOWITZ, S. G. See Sayles and
Hershkowitz, 51.

Hexoctahedron and growth, 205.

Histochemistry of ovary of Fundu-
lus heteroclitus and differentiating
odécytes, 67.

HoapLey, LeicH. Autotomy in the
brachyuran, Uca pugnax, 155.

Hormone, hypophyseal melanophore,
role in chromatic physiology of
Fundulus, 134.

HO6rstTapiIus, SVEN. Experiments on
determination in the early develop-
ment of Cerebratulus lacteus, 317.

—, . Investigations as to the
localization of the micromere-skele-
ton and entoderm-forming material
in unfertilized egg of Arbacia, 295.

Humason, W. D. See Tyler and Huma-
son, 261.

ENK, ROMAN. Sexual and asexual
reproduction in Euplanaria tigris
(Girard), 280.

KetcHuM, Bostwick. See
Smith and Ketchum, 421.
Kine, R.L. See Beams and King, 99.

Redfield,

ETHALS, autosomal, in wild popula-
tions of Drosophila pseudodbscura,
542.

Light, adaptation to and rate of locomo-
tion in Amoeba, 126.

Litiick, LorisC. Seasonal studies of the
phytoplankton off Woods Hole,
Massachusetts, 488.

Localization of micromere-, skeleton and
entoderm-forming material in un-
fertilized egg of Arbacia, 295.

Locomotion and relation to luminous
intensity and adaptation to light in
Amoeba, 126.

INDEX

Lumer, H. See Anderson, Lumer and
Zupancic, Jr., 444.

ARINE Biological Laboratory,
thirty-ninth report of, 1.

Marza, V. D., EUGENIE V. MARZA AND
Mary J.GuTHRIE. Histochemistry
of the ovary of Fundulus heteroclitus
with special reference to the differ-
entiating odcytes, 67.

Mast, S. O., AND NATHAN STAHLER. The
relation between luminous intensity,
adaptation to light, and rate of loco-
motion in Amoeba proteus (Leidy),
126.

MattTHEews, SAMUEL A. ‘The develop-
ment of the pituitary gland in
Fundulus, 93.

Microbracon hebetor Say and _ host,
Ephestia kithniella Zeller, differen-
tial effect of environmental factors,
147.

Micromere-forming material, localization
of, in unfertilized egg of Arbacia, 295.

Mid-bodies and their significance for the
central body problem, 565.

Migration, diurnal, of deep water ani-
mals, 185.

Mitter, E. Dewitt. A study of the
bacterial and alleged mitochondrial
content of the cells of the clover
nodule, 112.

Mnemiopsis, physiological gradient dur-
ing regeneration, 197.

Muscles, responses of, in squid to repeti-
tive stimulation of giant nerve fibers,
23s

Mutants, in Drosophila, fertility and pro-
ductivity, 214.

NEREIS egg, stimulation and nuclear
breakdown in, 557.

Nitrogen content, colloid osmotic pres-
sure and refractive index of turtle
serum and body fluid, 504.

, effect on decomposition on
polysaccharide extract of Chondrus
crispus, 143.

Nucleus, breakdown, and stimulation in
Nereis egg, 557.

NUNNEMACHER,R.F. See Welsh, Chace
and Nunnemacher, 185.

VARY, histochemistry of, Fundu-
lus heteroclitus, and differentiat-
ing odcytes, 67.

605

PARADISE fish, chromatophore reac-
tions in normal and albino, 535.

, genetics and histology of color
pattern, in normal and albino, 527.

Payne, NELLIE M. The differential
effect of environmental factors upon
Microbracon hebetor, Say (Hymen- -
optera: Braconide) and its host,
Ephestia kiihniella Zeller (Lepidop-
tera: Pyralidz). III, 147.

pH of sea water, measured with glass
electrode, 221.

Phosphorus, organic, cycle of, in Gulf of
Maine, 421.

Phytoplankton, seasonal studies of, off
Woods Hole, Massachusetts, 488.

Pituitary gland, development of, in
Fundulus, 93.

Placoid scale types and their distribution
in Squalus acanthias, 51.

Polar body extrusion and cleavage in
artificially activated eggs of Urechis
caupo, 164.

Polarity, determination of, by centrifug-
ing eggs of Fucus furcatus, 249.
Productivity and fertility of Drosophila

mutants, 214.

Prosser, C. LADD, AND JOHN Z. YOUNG.
Responses of muscles of the squid to
repetitive stimulation of the giant
nerve fibers, 237.

REDFIELD, ALFRED C., Homer P.
SMITH AND Bostwick KETCHUM.
The cycle of organic phosphorus in
the Gulf of Maine, 421.

Regeneration, and physiological gradient

during, in Mnemiopsis, 197.

, relation of endocrine-feeding to, in

Planaria, 227.

Reproduction, sexual and asexual, in
Euplanaria tigris, 280.

Respiratory rates, comparative tempera-
ture coefficients, unfertilized and
fertilized eggs, 261.

Rroco, Davin McK. A physiological
and histological study of the frontal
cortex of the seal, 591.

SALINITY, effect on growth of eggs of
Fucus furcatus, 552.

Salts, of heavy metals, effect on develop-
ment of sea urchin, 401.

SAYLES, LEONARD P., AND S. G. HERSH-
KowiTz. Placoid scale types and
their distribution in Squalus acan-
thias, 51.

606

Seal, physiological and histological study
of frontal cortex of, 591.

Serum protein measurements in marine
teleosts and elasmobranchs, 511.

Sea urchin, development, effect of salts of
heavy metals on, 401.

Sea water, pH, of measured with glass
electrode, 221.

Skeleton-forming material, localization
of, in unfertilized egg of Arbacia, 295.

SmitH, Homer P. See Redfield, Smith
and Ketchum, 421.

SmitH, Maurice A. See Goodrich and
Smith, 527.

SPARROW, F. K., Jk. The occurrence of
saprophytic fungi in marine muds,
242.

Squalus acanthias, placoid scale types
and their distribution, 51.
STAHLER, NATHAN. See Mast

Stahler, 126.

Stimulation and nuclear breakdown in

Nereis egg, 557.

, repetitive, of giant nerve fibers,

responses of muscles in squid to, 237.

Stock, C. CHESTER. See Ball and
Stock, 221.

STURTEVANT, A. H. Autosomal lethals
in wild populations of Drosophila
pseudoodbscura, 542.

and

‘TEMPERATURE coefficients of res-
piratory rates of unfertilized and
fertilized eggs, 261.

Thirty-ninth report of the
Biological Laboratory, 1.

TURNER, ABBY H. See Campbell and
Turner, 5G4.

——— —. Serum protein measure-
ments in the lower vertebrates. II.
In marine teleosts and elasmo-
branchs, 511.

Turtle serum and body fluid, colloid os-
motic pressure, nitrogen content and
refractive index of, 504.

Marine

INDEX

TYLER, ALBERT, AND HANS BAUER.
Polar body extrusion and cleavage in
artificially activated eggs of Urechis
caupo, 164.

TYLER, ALBERT, AND W. D. HuMAson.
On the energetics of differentiation,
VI. Comparison of the tempera-

_ture coefficients of the respiratory
rates of unfertilized and of fertilized
eggs, 261.

UA pugnax, autotomy in, 155.

Ultracentrifuging, suppression of cleav-
age in Ascaris eggs by, 99.

Urechis caupo, artificially activated eggs
of, polar body extrusion and cleav-
age in, 164.

V ARIAB ILITY and growth in Daphnia
pulex, 444.

WATERMAN, A.J. Effect of salts of
heavy metals on development of the
sea urchin, Arbacia punctulata, 401.

WE sH, J. H., F. A. CHACE, JR., AND
R. F. NUNNEMACHER. The diurnal
migration of deep water animals,
185.

Wauitaker, D. M. Determination of
polarity by centrifuging eggs of
Fucus furcatus, 249.

» — —, AND C. M. Criancy. The

effect of salinity upon the growth of
eggs of Fucus furcatus, 552.

Wivpur, Kart M. See Heilbrunn and
Wilbur, 557.

YOUNG, Joun Z. See Prosser and
Young, 237.

INN, Donatp J. See Clarke and
Zinn, 464.
Zooplankton, seasonal production of, off
Woods Hole, 464.
Zupancic, L. J., Jr. See Anderson,
Lumer and Zupancic, 444.

Experimental

Biology
Monographs

eA unique cumulative reference
library for biologists, providing full accounts of both the
experimental and the theoretical aspects of all significant
current research work in the various biological sciences.

PUBLISHED: AND FORTHCOMING VOLUMES

AUTONOMIC NEURO-EFFECTOR
SYSTEMS
By Watrer B. CANNON AND
ArtuRO RosENBLUETH. $4.00

PHYTOHORMONES
By F. W.. Went AND
Kennets V. Tarmann. $4.00

PACEMAKERS IN RELATION TO
ASPECTS OF BEHAVIOR
By Hupson Hoacuanp. $3.00

NEUROEMBRYOLOGY, AN
EXPERIMENTAL STUDY

$3.75

' THE EGGS OF MAMMALS
By GreGcory Pincus. $3.75

THE RECEPTOR PROCESS IN
- WISION

By Seria Hecur
GEOTROPISM: A STUDY OF
DETERMINISM IN BEHAVIOR

By W. J. CRozIER anp GREGORY PINCUS

THE BIOLOGY AND CHEMISTRY
OF OVARIAN HORMONES
By Grorge W. CoRNER AND

Wiuarp M. ALLEN

BIOLOGICAL OXIDATIONS
By EK. 8. Guzman Barron

THE HYPOPHYSIS
By J. B, Coup, D. L. THomson,
AND H. SELYE

By Samuet R. DETWILER.

NITROGEN METABOLISM IN
ANIMALS

By Henry Borsoox

TEMPERATURE
CHARACTERISTICS

By W. J. Crozier

CYTO-GENETICS AND PLANT
PHYLOGENY

By Ernest B. Bascock

BIOELECTRIC PHENOMENA IN
PLANTS

By L. R. Brinks

PERIODICITY IN ANIMAL
: BEHAVIOR

By T. J. B. Srrer
THE GENETIC ANALYSIS OF
SPECIES DIFFERENCES
By T. H. DospzHansky
DYNAMICS OF INFLAMMATION:
AN INQUIRY INTO THE MECH-

ANISM OF INFECTIOUS
~ PROCESSES

By Vaty MEmnxKIN

The Macmillan Company

60 Fifth Avenue New York

CONTENTS

: Page

WATERMAN, A. J.
Effect of Salts of Heavy Metals on Development of the Sea

Urchin; Arbacia:punctulata )y40 3) arse eek ee Se 401
REDFIELD, ALFRED C., HOMER P. SMITH, AND BOSTWICK

KETCHUM

The Cycle of Organic Phosphorus in the Gulf of Maine. .... 421

ANDERSON, BERTIL GOTTFRID, H. LUMER, AND L. J. ZUPANCIC,

JR.
Growth and Variability in Daphnia pulex................. 444

CLARKE, GEORGE L., AND DONALD J. ZINN
Seasonal Production of Zooplankton off Woods Hole with
special reference to Calanus finmarchicus................. 464

LILLICK, Lois C.
Seasonal Studies of the Phytoplankton off Woods Hole,
Massachusetts: a CenicS ty Wes SON rea 488

CAMPBELL, MILDRED L., AND ABBY H. TURNER
Serum Protein Measurements in the Lower Vertebrates.
I. The colloid osmotic pressure, nitrogen content, and re-
fractive index of turtle serum and body fluid.............. 504

TURNER, ABBY H.
Serum Protein Measurements in the Lower Vertebrates.
II. In marine teleosts and elasmobranchs.....:.......... 511

GOODRICH, H. B., AND MAURICE A. SMITH
Genetics and Histology of the Color Pattern in the Normal
and Albino Paradise Fish, Macropodus opercularis L....... 527

DALTON, H. CLARK, AND H. B. GOODRICH
Chromatophore Reactions in the Normal and Albino Paradise
1 5 Ce arg MARE GAS Sam ANAC ay aia are ete | o\ 535

STURTEVANT, A. H.
Autosomal Lethals in Wild Populations of Drosophila pseudo-
ray eS) by Ob of BS CON gee MN Op eR ee ce me Vaan ME Ocala Sic Gacwa on. 542

WHITAKER, D. M., AND C. M. CLANCY
The Effect of Salinity upon the Growth of Eggs of Fucus

PUT CAS 5. Si car Pie ba Shy aoe Gy ee SMa Peg eee 552
HEILBRUNN, L. V., AND KARL M. WILBUR
Stimulation and Nuclear Breakdown in the Nereis Egg..... 557

FRY, HENRY J.
Studies of the Mitotic Figure. VI. Mid-bodies and their
significance for the central body problem................. 565

RIOcH, DAVID McK.
A Physiological and Fiuctalosicnl Study of the Frontal Cotter
of the’ Seale a res et a eee 591

ya)

wate

en Van aN ~{7 = NAA >V VAN
nN g AA

Aa SaAWAN Anns agen

ARR RAR ARE

AAAA AA
tess Aa AAAAAA AAR [ \y
aRANARA AAA Anna sAAARAARIR

DB 3

oN NN al aol WAN

naa Mra AR AAA ms SA,
AAAA, AAA @Aary NAAAAY AAAAA \- = 2 Aa
pacer\- 2 aa ARARARERRARARA AABN i
AAAAAAGIAS - cass aaceenoaaannnce

y $3 2

PANAAAAAAAAAAAae Ai |
OOO AARAAARCE CTY YA, -e Ana

2
py»

| Na RAREARRARR AA ARRR RARERARR ARRAY AY ANA AR
: A |
AAAnaal|.|\eleae\\\\ AAAAas pas

VAAAAAAAANAA AR

AAA Bs, VAY AAA AN
ARARAARAARAAAAAAmAn
AAPAAAAARAARARAAAA

AAAAA AA ARARA BARBARA AeA NAAN
Aaa eases pa

soeternacnnes AAA,

AAA
AARAAAAS

Anas

Awan

penn alaalenN AA \"

| As

oauctet at atiaee
AAPAPAARARAAMMAG STA aAAAAAAAAARWA\,

y B/N!
v

la ye Is
pit AARA ARAL ARARAAR wp ~

Aap panei Me
BRIA

‘wiitiiiiin

088 01228 1564

Internet Archive Audio

Featured

Top

Images

Featured

Top

Software

Featured

Top

Books

Featured

Top

Video

Featured

Top

Mobile Apps

Browser Extensions

Archive-It Subscription

Save Page Now

Full text of "The Biological bulletin"

See other formats