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THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago

E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden

SELIG HECHT, Columbia University T jj MORGAN, California Institute of Technology

LEIGH HOADLEY, Harvard University Q H pARKER Harvard University

L. IRVING, Swarthmore College

M. H. JACOBS, University of Pennsylvania A' C' REDFffiLD, Harvard University

H. S. JENNINGS, Johns Hopkins University F. SCHRADER, Columbia University

FRANK R. LILLIE, University of Chicago DOUGLAS WHITAKER, Stanford University

H. B. STEINBACH, Washington University
Managing Editor

VOLUME 89

AUGUST TO DECEMBER, 1945

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE & LEMON STS.

LANCASTER, PA.

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

Subscriptions and similar matter should be addressed to The
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Massachusetts. Agent for Great Britain: Wheldon and Wesley,
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers, $1.75. Subscription per volume (three
issues), $4.50.

Communications relative to manuscripts should be sent to the
Managing Editor, Marine Biological Laboratory, Woods Hole,
Massachusetts, between July 1 and October 1, and to the Depart-
ment of Zoology, Washington University, St. Louis, Missouri,
during the remainder of the year.

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

LANCASTER PRESS, INC., LANCASTER, PA.

CONTENTS

No. 1. AUGUST, 1945

PAGE

ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1

WHITING, ANNA

Dominant lethality and correlated chromosome effects in Habrobracon
eggs x-rayed in diplotene and in late metaphase I 61

HARVEY, ETHEL BROWNE

Stratification and breaking of the Arbacia punctulata egg when cen-
trifuged in single salt solutions 72

PACE, D. M.

The effect of cyanide on respiration in Paramecium caudatum and
Paramecium aurelia 76

METZ, CHARLES B.

The agglutination of starfish sperm by fertilizin 84

KOZLOFF, EUGENE N.

Cochliophilus depressus gen. nov., sp. nov. and Cochliophilus minor sp.
nov., holotrichous ciliates from the mantle cavity of Phytia setifer
(Cooper) 95

SCHEER, BRADLEY T.

The development of marine fouling communities 103

SPIEGELMAN, S. AND FLORENCE MOOG

A comparison of the effects of cyanide and azide on the development of
frogs' eggs 122

No. 2. OCTOBER, 1945

KIDDER, GEORGE W. AND VIRGINIA C. DEWEY

Studies on the biochemistry of Tetrahymena. IV. Amino acids and
their relation to the biosynthesis of thiamine 131

LEFEVRE, PAUL G.

Certain chemical factors influencing artificial activation of Nereis eggs. 144

HlBBARD, HOPE AND GEORGE I. LAVIN

A study of the Golgi apparatus in chicken gizzard epithelium by means of

the quartz microscope 157

HAYASHI, TERU

Dilution medium and survival of the spermatozoa of Arbacia punctulata.

I. Effect of the medium on fertilizing power 162

KOZLOFF, EUGENE N.

Heterocineta phoronopsidis sp. nov., a ciliate from the tentacles of

Phoronopsis viridis Hilton 180

ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL

LABORATORY, SUMMER OF 1945 184

MARINE BIOLOGICAL LABORATORY

H. S. JENNINGS, University of California

C. E. McCLUNG, University of Pennsylvania

S. O. MAST, Johns Hopkins University

A. P. MATHEWS, University of Cincinnati

T. H. MORGAN, California Institute of Technology

W. J. V. OSTERHOUT, Rockefeller Institute

G. H. PARKER, Harvard University

W. B. SCOTT, Princeton University

TO SERVE UNTIL 1948

ERIC G. BALL, Harvard University Medical School

R. CHAMBERS, Washington Square College, New York University

EUGENE F. DuBois, Cornell University Medical College

COLUMBUS ISELIN, Woods Hole Oceanographic Institution

C. W. METZ, University of Pennsylvania

H. H. PLOUGH, Amherst College

E. W. SINNOTT, Yale University

W. R. TAYLOR, University of Michigan

TO SERVE UNTIL 1947

W. C. ALLEE, The University of Chicago

G. H. A. CLOWES, Lilly Research Laboratory

P. S. GALTSOFF, U. S. Fish and Wild Life Service

L. V. HEILBRUNN, University of Pennsylvania

LAURENCE IRVING, Swarthmore College

J. H. NORTHROP, Rockefeller Institute

A. H. STURTEVANT, California Institute of Technology
LORANDE L. WOODRUFF, Yale University

TO SERVE UNTIL 1946

DUGALD E. S. BROWN, New York University
E. R. CLARK, University of Pennsylvania
OTTO C. GLASER, Amherst College

E. N. HARVEY, Princeton University

M. H. JACOBS, University of Pennsylvania

F. P. KNOWLTON, Syracuse University
FRANZ SCHRADER, Columbia University

B. H. WILLIER, Johns Hopkins University

TO SERVE UNTIL 1945

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

L. G. BARTH, Columbia University

S. C. BROOKS, University of California

W. C. CURTIS, University of Missouri

H. B. GOODRICH, Wesleyan University

R. S. LILLIE, The University of Chicago

A. C. REDFIELD, Harvard University

C. C. SPEIDEL, University of Virginia

EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES

LAWRASON RIGGS, Ex officio, Chairman
E. N. HARVEY, Ex officio

D. M. BRODIE, Ex officio

ACT OF INCORPORATION

CHARLES PACKARD, Ex officio

E. G. BALL, to serve until 1946

L. G. BARTH

ROBERT CHAMBERS, to serve until 1945

WM. RANDOLPH TAYLOR

THE LIBRARY COMMITTEE
A. C. REDFIELD, Chairman
E. G. BALL
S. C. BROOKS
M. E. KRAHL
J. W. MAYOR

THE APPARATUS COMMITTEE
D. E. S. BROWN, Chairman

C. L. CLAFF
G. FAILLA
S. E. HILL

A. K. PARPART

THE SUPPLY DEPARTMENT COMMITTEE

D. A. MARSLAND, Chairman
P. B. ARMSTRONG

P. S. GALTSOFF
R. T. KEMPTON
CHARLES PACKARD

THE EVENING LECTURE COMMITTEE

B. H. WILLIER, Chairman
M. H. JACOBS
CHARLES PACKARD

THE INSTRUCTION COMMITTEE
H. B. GOODRICH, Chairman
W. C. ALLEE
S. C. BROOKS
VIKTOR HAMBURGER
CHARLES PACKARD, Ex officio

THE BUILDINGS AND GROUNDS COMMITTEE

E. G. BALL, Chairman
D. P. COSTELLO
MRS. E. N. HARVEY
ROBERTS RUGH
MRS. C. C. SPEIDEL

No. 3170

II. ACT OF INCORPORATION

COMMONWEALTH OF MASSACHUSETTS

Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T.
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells,
William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves
with the intention of forming a Corporation under the name of the Marine Biological
Laboratory, for the purpose of establishing and maintaining a laboratory or station for
scientific study and investigation, and a school for instruction in biology and natural his-
tory, and have complied with the provisions of the statutes of this Commonwealth in such

MARINE BIOLOGICAL LABORATORY

case made and provided, as appears from the certificate of the President, Treasurer, and
Trustees of said Corporation, duly approved by the Commissioner of Corporations, and
recorded in this office ;

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

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

HENRY B. PIERCE,
Secretary of the Commonwealth.

III. BY-LAWS OF THE CORPORATION OF THE MARINE

BIOLOGICAL LABORATORY

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

II. The officers of the Corporation shall consist of a President, Vice President, Di-
rector, Treasurer, and Clerk.

III. The Annual Meeting of the members shall be held on the second Tuesday in
August in each year, at the Laboratory in Woods Hole, Massachusetts, at 11:30 A.M.,
and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve
one year, and eight Trustees to serve four years, and shall transact such other business
as may properly come before the meeting. Special meetings of the members may be
called by the Trustees to be held at such time and place as may be designated.

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

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

VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by
these By-laws, no notice of the Annual Meeting need be given. Notice of any special
meeting of members, however, shall be given by the Clerk by mailing notice of the time
and place and purpose of such meeting, at least fifteen (15) days before such meeting,
to each member at his or her address as shown on the records of the Corporation.

VII. The Annual Meeting of the Trustees shall be held on the second Tuesday in
August in each year, at the Laboratory in Woods Hole, Mass., at 10 A.M. Special
meetings of the Trustees shall be called by the President, or by any seven Trustees, to be
held at such time and place as may be designated, and the Secretary shall give notice
thereof by written or printed notice, mailed to each Trustee at his address as shown on
the records of the Corporation, at least one (1) week before the meeting. At such
special meeting only matters stated in the notice shall be considered. Seven Trustees of
those eligible to vote shall constitute a quorum for the transaction of business at any
meeting.

VIII. There shall be three groups of Trustees :

(A) Thirty-two Trustees chosen by the Corporation, divided into four classes, each
to serve four years ; and in addition there shall be two groups of Trustees as follows :

(B) Trustees ex officio, who shall be the President and Vice President of the Cor-
poration, the Director of the Laboratory, the Associate Director, the Treasurer, and
the Clerk;

REPORT OF THE TREASURER 5

(C) 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 Corporation and he
shall become eligible for election as Trustee Emeritus for life. The Trustees ex officio
and Emeritus shall have all the rights of the Trustees except that Trustees Emeritus shall
not have the right to vote.

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

IX. The Trustees shall have the control and management of the affairs of the Cor-
poration; they shall elect a President of the Corporation who shall also be Chairman of
the Board of Trustees; and shall also elect a Vice President of the Corporation who shall
also be the Vice 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. The Board of Trustees shall have the power to choose an Executive Committee
from their own number, and to delegate to such Committee such of their own powers as
they may deem expedient. They shall from time to time elect members to the Corpora-
tion upon such terms and conditions as they may think best.

X. Any person interested in the Laboratory may be elected by the Trustees to a group
to be known as Associates of the Marine Biological Laboratory.

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

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

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

IV. THE REPORT OF THE TREASURER
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY:

Gentlemen :

Herewith is my report as Treasurer of the Marine Biological Laboratory for
the year 1944.

The accounts have been audited by Messrs. Seamans, Stetson, and Tuttle, certi-
fied public accountants. A copy of their report is on file at the Laboratory and
inspection of it by members of the Corporation will be welcomed.

The principal summaries of their report— The Balance Sheet, Statement of
Income and Expense, and Current Surplus Account — are appended hereto as
Exhibits A, B and C.

The following are some general statements and observations based on the
detailed reports :

/. Assets

1. Endoi^incnt Assets

As of December 31, 1944, the total book value of all the Endowment Assets,
including the Scholarship Funds, was $983,900.57, a loss for the year of $19,808.06.

6 MARINE BIOLOGICAL LABORATORY

The Scholarship Funds were increased by the gift of $5,000.00 from Bishop James
E. Cassidy of Fall River to establish the "Reverend Arsenious Boyer Burse." The
principal losses incurred were due, as in the previous year, to the foreclosure of
mortgage participations on New York City realty and the subsequent sale of the
properties. In 1944 a four story tenement at 4856 Broadway on which the Lab-
oratory held a mortgage investment of $30,057.10 was sold, the Laboratory receiv-
ing $6,026.00 cash and a new mortgage participation for $17,000, and sustaining
a loss of $7,031.10. The property at 47 Murray Street, a five story loft building,
was sold entirely for cash, at a loss of $9,569.24 on an investment of $21,928.75.

At the end of the year $803,403.76 was invested in marketable securities (bonds,
preferred stocks and common stocks) with a market value of $825,005.80. $163,-
769.79 was invested in mortgage participations on New York City real estate and
in real estate participations resulting from mortgage foreclosures. $16,727.02 was
in uninvested principal cash.

The Treasurer's estimate of the actual value of the $163,769.79 in mortgage and
real estate participations held on December 31 is $87,750.00. With the market
value of $825,005.80 on marketable securities and the $16,727.02 in cash this makes
a total current valuation of $929,482.82 compared with total book value of $983,-
900.57 and original capital value of $1,116.924.25.

2. Plant Assets

The total of Plant Assets (excluding the Gansett and Devil's Lane tracts) was
$1,333,726.48 after deduction of $656,341.78 accumulated Depreciation Reserve, a
decrease for the year of $7,699.40. Depreciation charges for 1944 were $26,-
929.31. The Reserve Fund was increased to a total of $16,895.62 by $3,529.41
transferred from current income (representing $279.41 profit on sale of Gansett
lots, the Crane Company dividends, and part of the dividends on the General
Biological Supply House stocks) and $93.99 interest received from the temporary
investment of $10,000 of the Reserve Fund in U.S.A. Treasury bonds.

3. Current Assets

Current Assets including cash, inventories, and investments not in the Endow-
ment Funds at cost, amounted to $202,239.67, an increase of $8,127.98. Current
Liabilities totalled $2,181.09. The special reserve fund for repairs and replace-
ments, made up of a portion of the 1943 income from the United States Navy
rentals, and the value of certain equipment received from the Navy in lieu of
restoration and repairs upon termination of the Navy lease, was $15.998.62 at the
end of the year. Current Surplus was $184,059.96, $4,442.14 under the total
for 1943.

II. Income and Expenditures

Total Income was $164,240.13, an increase of $4,943.19 over the 1943 income.
Total Expenses were higher, $160,013.13, including Depreciation Reserves of
$26,929.31 and special hurricane damage repairs of $2,466.17, but there was an
actual net surplus of $4,227.00 for the year.

This surplus compares favorably with the $19,323.67 surplus in 1943 which
resulted largely from the $20,150.00 rental from the United States Navy combined
with reduced expenditures, and the deficit of $17,211.93 for 1942. Some of the
reductions in 1944 income were a decline of $3,600.91 in endowment income, a

REPORT OF THE TREASURER 7

loss of $6,000 in net income from the Supply Department compared with 1943,
and a reduction of $2,286.00 in the dividends from the General Biological Supply
House. The principal gains were Mrs. W. Murray Crane's gift of Otis Elevator
stock valued at $2,325.00, and an increase of over $4,000 in net income from
Research.

The income and expense items, although more normal than in 1943, still do
not reflect what may be regarded as regular operations. Expenditures for equip-
ment and necessary improvements, for example, are still unavoidably under what
they should be to maintain the Laboratory at full efficiency. Some reserves have
been built up for a few of these expenditures, but the Laboratory needs a larger
endowment income to take care of maintenance.

EXHIBIT A
MARINE BIOLOGICAL LABORATORY BALANCE SHEET, DECEMBER 31, 1944

Assets
Endowment Assets and Equities:

Securities and Cash in Hands of Central Hanover Bank and

Trust Company, New York, Trustee $ 968,737.59

Securities and Cash in Minor Funds 15,162.98

$ 983,900.57
Plant Assets:

Land $ 1 1 1,425.38

Buildings 1,327,675.21

Equipment 186,122.42

Library 329,639.23

$1,954,862.24
Less Reserve for Depreciation 656,341.78 $1,298,520.46

Reserve Fund, Securities and Cash 16,895.62

Book Fund, Securities and Cash 18,310.40

$1,333,726.48
Current Assets:

Cash $ 27,513.52

Accounts Receivable 12,357.71

Inventories:

Supply Department $ 43,964.75

Biological Bulletin 19,498.15 63,462.90

Investments:

Devil's Lane Property $ 46,260.84

Gansett Property 1 ,900.42

Stock in General Biological Supply House,

Inc 12,700.00

Other Investment Stocks 20,095.00

Retirement Fund 12,966.30 93,922.56

Prepaid Insurance 4,184.40

Items in Suspense 798.58

$ 202,239.67
Total Assets $2,519,866.72

MARINE BIOLOGICAL LABORATORY

Liabilities

Endowment Funds:

Endowment Funds $ 967, 1 13.46

Reserve for Amortization of Bond Premiums. 1,624.13

Minor Funds.

$ 968,737.59
15,162.98

Plant Funds:

Donations and Gifts $1,172,564.04

Other Investments in Plant from Gifts and Current Funds. . 161,162.44

$ 983,900.57

Current Liabilities and Surplus:

Accounts Payable

Reserve for Repairs and Replacements.
Current Surplus (Exhibit C)

2,181.09

15,998.62

184,059.96

1,333,726.48

$ 202,239.67
Total Liabilities. , $2,519,866.72

EXHIBIT B

MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1944

Income:

General Endowment Fund

Library Fund

Donations

Instruction

Research

Evening Lectures

Biological Bulletin and Membership Dues

Supply Department

Mess

Dormitories

(Interest and Depreciation charged to

above 3 Departments)

Dividends, General Biological Supply

House, Inc

Dividends, Other Investment Stocks

Rents:

Bar Neck Property

Janitor House

Danchakoff Cottages

Rooms in Laboratory, Special

Sale of Library Duplicates and Micro Film

Microscope and Apparatus Rental

Sundry Income

Total
Expense Income

$ 27,291.16

5,718.91

2,325.00

8,485.47

5,570.00

$ 2,915.47

4,981.44

13,654.38

45.85

4,102.15

7,952.96

37,307.27

45,588.92

20,225.08

17,878.72

2,346.36

30,761.47

13,190.82

17,570.65

25,076.43

759.46
21.35

278.44

16,510.00
785.00

4,800.00
360.00
643.33
420.00
194.90

1,168.24
187.79

Net
Expense Income

27,291.16
5,718.91
2,325.00

8,672.94

3,850.81
8,281.65

25,076.43

16,510.00
785.00

4,040.54
338.65
364.89
420.00
194.90

1,168.24
187.79

REPORT OF THE TREASURER

Maintenance of Plant:

Buildings and Grounds 18,759.11 18,759.11

Apparatus Department 3,765.15 3,765.15

Chemical Department 1,681.50 1,681.50

Library Expense 6,756.08 6,756.08

Workmen's Compensation Insurance. .. 440.09 440.09

Truck Expense 327.35 327.35

Bay Shore Property 93.41 93.41

Great Cedar Swamp 20.25 20.25

General Expenses:

Administration Expense 15,275.38 15,275.38

Endowment Fund Trustee and Safe-
Keeping 1,015.28 1,015.28

Bad Debts 592.50 592.50

Special Repairs on account of 1944 Hurri-
cane Damage 2,466.17 2,466.17

Reserve for Depreciation 26,929.31 26,929.31

$160,013.13 $164,240.13 $100,999.91 $105,226.91
Excess of Income over Expense carried to

Current Surplus 4,227.00 4,227.00

$164,240.13 $105,226.91

EXHIBIT C

MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT,
YEAR ENDED DECEMBER 31, 1944

Balance January 1, 1944 $188,502.10

Add:

Excess of Income over Expense $ 4,227.00

Gain on Gansett Lots Sold 1 76.04

Bad Debts Recovered 37.64

Reserve for Depreciation charged to Plant Funds 26,929.31 31,369.99

$219,872.09
Deduct:

Payments from Current Funds during Year for Plant
Assets:

Buildings $ 3,064.00

Equipment 1,542.52

Library 5,559.12

$10,165.64
Less Received for Plant Assets Sold. 172.00

$ 9,993.64
Pensions Paid $ 3,460.00

Less:

Retirement Fund Income $223.07

Retirement Fund Gain on Securi-
ties 351.86

Retirement Fund, Recovery on

account of 1943 loss. , .51 575.44

$ 2,884.56

10 MARINE BIOLOGICAL LABORATORY

Transfers to Reserve Fund:

Portion of Dividends from General Biological

Supply House, Inc $ 2,500.00

Dividends from Crane Company 750.00

Profit on Gansett Lots for 1943. . 279.41

$ 3,529.41
Building Fixtures and Equipment Received from First

Naval District, transferred to Plant Funds. $ 7,225.00
Less Loss on Fixtures and Equipment Discarded 620.48

$ 6,604.52
Repairs and Replacements Made by First Naval District during

their occupancy of properties, set up as a Reserve 1 2,800.00

35,812.13
Balance, December 31, 1944 $184,059.96

Respectfully submitted,

DONALD M. BRODIE,

Treasurer

V. REPORT OF THE LIBRARIAN

The sum of $11,239.77 appropriated to the library in 1944 was expended as
follows: books, $760.49; serials. $2,626.99; binding, $884.00; express. $60.14;
supplies, $416.17; salaries, $6.239.77; back sets. $214.50; insurance, $50.00; sun-
dries, $2.21 ; total, $11,254.27. The cash earnings of the library reverting to the
laboratory were $194.90: from sale of duplicates, $38.73; microfilms, $144.86;
serials lists, $11.31.

Of the Carnegie Corporation of New York Fund, $2,433.69 was expended for
the completion of five and partial completion of nine back sets and two books.

The sum appropriated by the Woods Hole Oceanographic Institution for 1944
was $1,900.00. A balance of $263.08 remaining from 1943 made an available total
of $2,163.08. Of this sum $113.69 was expended on current books and journals
and $1,100.00 on salaries, leaving a balance of $949.39. A comparison of the
amount spent on current books, journals and back sets during the pre-war years
with that of the war years will show that this accumulating budget balance will be
expended when the material for which it was designated shall have become available.

During 1944 the library received 678 current journals: 248 (10 new) by sub-
scription to the Marine Biological Laboratory; 15 (none new) to the Woods Hole
Oceanographic Institution; exchanges 201 (three new) with the "Biological Bul-
letin" and 23 (one new) with the Woods Hole Oceanographic Institution publica-
tions; 186 as gifts to the former and five to the latter. The Marine Biological
Laboratory acquired 169 books: 119 by purchase of the Marine Biological Lab-
oratory ; six by purchase of the Woods Hole Oceanographic Institution ; nine gifts
from the authors, 22 from the publishers and 13 from miscellaneous donors. There
were 18 back sets of serial publications completed: ten purchased by the Marine
Biological Laboratory (five with the "Carnegie Fund") ; two secured by exchange
with the "Biological Bulletin" ; one by exchange with the Woods Hole Oceano-
graphic Institution publications; and five by duplicate material exchange and by

REPORT OF THE DIRECTOR 11

gift. Partially completed sets were 59 : purchased by the Marine Biological Lab-
oratory, 23 (nine with "Carnegie Fund") ; by exchange with the "Biological Bul-
letin," one; and by exchange of duplicate material and by gift, 35. In addition, 15
of the odd journal numbers presented by Dr. Dorothy R. Stewart (126 in all)
were fitted into gaps in our sets.

The reprint additions to the library number 2,404 : current of 1943, 401 ; cur-
rent of 1944, 58; and of previous dates, 1,945. A total of 3,957 reprints, 1,321
not duplicates of our holdings, were presented to the library: 1,378 by Mrs. G. N.
Calkins; 2.306 by Dr. Dorothy R. Stewart; 192 by Dr. Libbie H. Hyman; and 81
by Dr. D. A. Fraser.

It is with great pleasure that two very valuable gifts are acknowledged as pre-
sented to the library this year. Dr. Walter E. Garrey has presented his collection
of reprints to be incorporated in the library's reprint holdings. As yet no count
of these has been made. More detailed acknowledgment will occur in a later
report. The same delayed account will be given of the reprints from Dr. E. B.
Meigs' library, a gift of Mrs. Meigs. In addition to the reprints, Mrs. Meigs in-
cluded in her gift long runs of fourteen different journals. As a further gift from
Mrs. Meigs three of these sets will be bound and, with an appropriate book plate
inserted, will be substituted for the old volumes now in the library.

At the end of the year 1944 the library contained 52,885 bound volumes and
133,054 reprints.

VI. THE REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOOGICAL LABORATORY:

Gentlemen:

I beg to submit the following report of the fifty-seventh session of the Marine
Biological Laboratory for the year 1944.

During the year the Laboratory has coped with difficulties brought on by the
war — shortage of labor, and materials much needed for research and for the main-
tenance of the plant and the Supply Department — and with a hurricane which for-
tunately did not seriously damage our buildings. The immediate problem now
reflects the encouraging change in the general situation throughout the country ;
it is to find laboratory space for the large number of investigators who expect to
return in 1945.

1. Attendance

The anticipated increase in the number of investigators and students indicates
that we have already passed the low point in the curve of attendance. This can
be seen in the chart on page 20 which forms a part of the report prepared for the
Committee of Review. Our numbers in the five year period from 1936-1940 were
the greatest in the history of the Laboratory; in 1942 they decreased by nearly 50
per cent. The year 1943 showed a still further decline. A definite improvement
is seen in the record of 1944, as shown in the Tabular View of Attendance on page
21. The prospects for a still larger number in 1945 are excellent.

From the curves on the chart, one can see the effect on attendance of changing
conditions in the Laboratory and in the country at large. When new buildings

12 MARINE BIOLOGICAL LABORATORY

were erected here, the attendance increased sharply. The first World War re-
versed the upward trend, but only for one year. The business depression, felt from
1932-1935, affected chiefly the number of "New Investigators." The present war
has reduced our attendance to the level of 25 years ago. After 1945 there should
be a rapid rise, but with our present buildings and equipment we cannot accom-
modate more investigators and students than we had in 1940.

New Investigators are those who come here for the first time ; after the first
year they are classified among "Returning Investigators." They are chiefly "In-
vestigators under Instruction," that is, graduate students and Fellows. Over a
long period of years they have constituted nearly one third of all investigators in
attendance. Recently this proportion, as shown in the Tabular View of Attend-
ance, has grown smaller ; but actually, until the war, the number of graduate
students. Fellows, and young instructors present each summer did not diminish.
What happened was that many came as Research Assistants, probably for eco-
nomic reasons.

Since 1941 the number of beginning investigators has declined by about 75 per
cent from its previous level. The loss of so large a proportion of this important
source of new members and future supporters of the Laboratory will be felt for
many years. We fervently hope that those who are now prevented from work-
ing here will eventually return to us.

It is a pleasure to report that the number of institutions represented during the
war years has not greatly diminished, and that the list of supporting institutions
receives new additions every season.

2. Laboratory Activities

During the summer all of the usual activities of the Laboratory were carried on.
After a lapse of one year, the weekly seminars were resumed, nine being held. In
addition to these, several small groups of investigators met to discuss topics in
which all were especially interested. It was the general opinion that more meet-
ings of this kind should be held. All of the courses of instruction were given, the
total registration of students being 75, a moderate increase over the preceding year.
Dr. John B. Buck, who served for two seasons with signal success as head of the
Invertebrate Zoology course, resigned at the end of the summer. The Committee
on Instruction accepted his resignation with regret, and selected Dr. Frank A.
Brown, of Northwestern University, to succeed him as instructor in charge.

3. Associates

The Trustees, at the regular meeting this year, directed the Executive Com-
mittee to appoint a committee to consider the advisability of establishing a new kind
of membership in the Corporation, to which those interested in the welfare of the
Laboratory might be elected. Mrs. W. Murray Crane, Mr. Lawrence Saunders,
Dr. J. P. Warbasse (all of whom were elected to membership in the Corporation at
the August meeting). Dr. G. H. A. Clowes, and the Director, were asked to discuss
the matter. This committee felt that there are many people without special train-
ing in Biology, who have a sincere interest in the Laboratory, and that such friends
would appreciate a formal connection with it. Following the Committee's approval

REPORT OF THE DIRECTOR 13

of the plan, a special meeting of the Trustees was held in New York on December
9, 1944 to amend the By-laws so that this new type of membership could be made
possible. The amendment which was adopted reads as follows : "Any person in-
terested in the Laboratory may be elected by the Trustees to a group known as
'Associates of the Marine Biological Laboratory.' It is hoped that both summer
and permanent residents of Woods Hole and the vicinity may become members,
and that friends in other parts of the country may also join.

At this special meeting the opinion was voiced by several Trustees that a winter
meeting should be held regularly in order that current Laboratory problems could
be discussed.

4. The Committee of Review

When the Friendship Fund in 1924 contributed a large sum to the Laboratory
for endowment purposes, the Trustee of the endowment was directed to call once
every ten years upon a Committee of Review to make a study of the work of the
Laboratory. This Committee, which consists of nine members, includes a repre-
sentative of the National Academy of Sciences, of the National Research Council,
of the American Association for the Advancement of Science, and one professor of
Biology from each of the following universities : Chicago, Columbia, Harvard,
Pennsylvania. Princeton, and Yale. Its function is to determine whether the

Laboratory continues to perform valuable services in biological research. The
complete text of the Deed of Trust, in which the duties of the Committee are set
forth, is printed in the 26th Annual Report which appears in Vol. 47 of the "Bio-
logical Bulletin."

The Committee first met in 1934 and voted that the Laboratory was satisfac-
torily fulfilling the purpose for which the endowment was given. The second
decennial Committee met this year. Its findings, and the statement of the Presi-
dent and Director of the Laboratory regarding our activities during the years 1934—
1943 are appended to this report. The Committee, in addition to its formal vote
of approval, pointed out that in order to maintain a high level of usefulness, the
Laboratory should secure additional funds for endowment and for purposes which
are specified in their report. These recommendations, coming from a group of
biologists, the majority of whom were not connected with the administration of the
Laboratory, should be given most careful consideration.

5. The Hurricane

The September hurricane did not seriously damage our buildings. No water
came in, as happened in the 1938 storm, but roofs and windows suffered. Some
of the slate from the Dormitory and Apartment House roofs was blown off, many
pieces imbedding themselves in distant houses. Fortunately no one was struck by
these flying missiles. The Cayadetta wharf was practically demolished, and the
sea wall badly broken by the tremendous waves that tossed great stones on to the
street. The wharf has been partially restored by the . Oceanographic Institution
which has used it for the past two years. Had the full fury of the storm struck
at high tide we might well have sustained a loss, due to sea water, even greater
than that which we suffered in 1938. The wind, whose velocity far exceeded that

14 MARINE BIOLOGICAL LABORATORY

of the previous hurricane, levelled a great number of trees in the Gansett and
Devil's Lane tracts, on Dr. Clowes' property, and in the Fay Woods.

Against the destructive power of winds we can do little, but it is possible to pro-
tect the Brick Building from high water. The matter of increased protection should
be given consideration.

6. Loss by Death

This year the Corporation has lost by death Prof. William Trelease who was
elected in 1888 at the first regular meeting of the Trustees after the incorporation
of the Laboratory.

7. Gift

The Laboratory acknowledges with sincere appreciation the receipt of 100 shares
of Otis Elevator stock valued at $2,325.00, a gift of Mrs. W. Murray Crane.

8. Election of Trustee

At the meeting of the Corporation held August 8, 1944, L. G. Earth, Associate
Professor of Zoology at Columbia University, was elected to fill the vacancy caused
by the resignation of Prof. I. F. Lewis.

9. There are appended as parts of this report :

1. Memorial to Dr. Caswell Grave. •

2. The Report of the Committee of Review.

3. The Decennial Review — Submitted to the Committee of Review.

4. The Staff.

5. Investigators and Students.

6. Tabular View of Attendance, 1940-1944.

7. Subscribing and Co-operating Institutions.

8. Evening Lectures.

9. Shorter Scientific Papers.

10. Members of the Corporation. „

Respectfully suumitted,

CHARLES PACKARD,

Director

1. MEMORIAL TO DR. CASWELL GRAVE
By Prof. R. A. Budington

It is with the greatest reluctance, and with true sorrow, that today we must
include among those permanently lost to the Corporation the name of Caswell
Grave. Those who knew him, as most of us here did, will miss his genial per-
sonality, with his habit of industry, his steady, keen interest in everything biological,
his strict integrity of character ; and the Board of Trustees will be very conscious
of the absence of his sincere interest in the ongoing of the Laboratory, its policies,
and its scientific significance.

REPORT OF THE DIRECTOR 15

Grave was born a Hoosier, on a farm in Monrovia, Indiana, and was very nearly
74 years of age at the time of his death at his home in Winter Park, Florida, last
January 8th. He graduated from Earlham College in 1895, with Phi Beta Kappa
rank; his alma mater honored him with her Doctor of Laws degree in 1928. His
graduate studies were done at Johns Hopkins University, which conferred the
Ph.D. in 1899. Meanwhile, he had spent summers at the Fisheries Bureau in
Woods Hole, and at the Johns Hopkins Laboratory in Jamaica. After two further
years of study as Bruce Fellow, he was appointed to the Hopkins teaching staff,
a relation he continued for 18 years, for 13 of which he held, the rank of Associate
Professor. In 1919 he was appointed to the headship of the Zoological Depart-
ment at Washington University, St. Louis, where the new Rebstock Laboratory
had just been built. Here he gathered about him a staff of men of outstanding
competency, and put the department on a basis widely recognized for scholarship
and general efficiency.

Other responsibilities carried by Grave were : Director of the U. S. Fisheries
Laboratory, Beaufort, N. C., 1902-1906; Shellfish Commissioner of Maryland,
1906-1912. In World War I he was ranked a captain in the Chemical Warfar.e
Service. He was an active member of the AAAS ; the American Society of
Naturalists ; a member of Sigma Xi ; by turn he was Secretary-Treasurer, Vice
President, and President of the American Society of Zoologists. As for the Marine
Biological Laboratory, he was an outstandingly successful director of the Inverte-
brate Course from 1912-1917; a Trustee for 20 years, 1920-1940; thereafter,
Trustee Emeritus. Few, if any, have taken the welfare of the Laboratory more
seriously to heart than did he.

Grave's research interest embraced three quite different fields : pelecypod
mollusca as to structure, physiology, and life histories ; echinoderms, with special
reference to embryology, and intraphyla relationships ; while in later years he
attacked the problem of metamorphosis in the ascidians, with special reference to
the chemical factors retarding or accelerating it.

It is not too much to say that Caswell Grave was a wise man ; and in the truest
sense, in all that the appellations should imply, he was a "gentleman and a scholar."
We are glad to pause and offer him such honor as we may, today.

August 9, 1944

2. MINUTES OF THE COMMITTEE OF REVIEW OF THE MARINE BIOLOGICAL

LABORATORY

The Committee of Review provided for in the Deed of Trust Covering Funds
for Endowment, Friendship Fund, Inc.. and Central Hanover Bank and Trust
Company of New York, met at the Marine Biological Laboratory, Woods Hole,
Massachusetts, on August 9, 1944, at 9 :00 A.M.

Mr. Lawrason Riggs, President of the Corporation, read the Call of Meeting,
and commented on the history of the origin of the Deed of Trust, and on the duties
of the Committee.

Present :

Professor W. C. Allee — representing The University of Chicago
Professor G. A. Baitsell — representing Yale University

16 MARINE BIOLOGICAL LABORATORY

Professor A. F. Blakeslee — representing The American Association for the Ad-
vancement of Science

Professor A. B. Dawson — representing Harvard University
Professor W. K. Gregory — representing The National Academy of Science
Professor R. W. Griggs — representing The National Research Council
Professor E. N. Harvey — representing Princeton University
Professor M. H. Jacobs — representing The University of Pennsylvania
Professor Franz Schrader — representing Columbia University

Dr. Blakeslee was elected Chairman of the Committee and (by invitation) Dr.
Charles Packard, Director of the Laboratory, Secretary.

Dr. Packard presented the Decennial Review containing a brief statement of
the activities of the Laboratory for the years 1934-1943, and called attention to the
nine exhibits.

The Committee examined the exhibits, and after full discussion, unanimously

VOTED That the Marine Biological Laboratory is performing valuable services
in biological research.

It was the opinion of the Committee that it could perform a useful service to
the Laboratory by making suggestions regarding its future development.

VOTED That the Committee understands and appreciates the high quality of the
Board of Trustees of the Laboratory, but thinks it desirable that each class of
Trustees should contain at least one biologist not closely associated with the work
of the Laboratory.

Moved and seconded that a recommendation be formulated that some means
be considered for effecting more frequent changes in the Board of Trustees.

The motion was lost.

VOTED That the Chairman appoint a sub-committee of three to report to the
full Committee on the specific needs of the Laboratory.

The Chairman appointed Drs. Harvey, Dawson, and Packard.

VOTED That the Chairman appoint a sub-committee of three to draft a state-
ment in support of the first motion, this to follow in general the form of the report
of the 1934 Committee of Review.

The Chairman appointed Drs. Schrader, Jacobs, and Baitsell.

Afternoon Session.

VOTED To accept and adopt the following statement in support of the first
motion.

The Marine Biological Laboratory is performing valuable services in biological
research. Its record is especially commendable in view of the difficult conditions
experienced during the past ten years. Despite the steady decrease in income from
endowments, and the more recent handicaps involved in war conditions, the scien-
tific activities of the Marine Biological Laboratory have been maintained at a high
level.

With marked decrease in attendance due to wartime conditions, the standards
of the courses of instruction have been maintained.

The Library, already recognized as one of the foremost in its field, has on a
reduced budget been steadily improved.

REPORT OF THE DIRECTOR 17

Important research continues to be done. To compensate for a decrease in
attendance there has been some utilization of the Laboratory facilities for war work.

As in the past, one of the important features of the Marine Biological Labora-
tory has been the close association of investigators working in different fields.
Likewise, cooperation and association with the Woods Hole Oceanographic Insti-
tution, as well as with the local station of the U. S. Fish and Wild Life Service,
has increased to a laudable extent.

VOTED To accept and adopt, as amended, the following statement of the sub-
committee on Laboratory needs :

1. The committee notes that the income of the Marine Biological Laboratory
has decreased while the needs have continually mounted. The budget has
been balanced at the expense of upkeep and necessary improvements. Obvi-
ously the setting up of a sufficient reserve for future developments has been
impossible. Additional income is urgently needed for the following specific
purposes :

a. Replacement of apparatus, boats, and other equipment now becoming
obsolete.

b. Repair and renovation of buildings.

c. Payment of subscriptions to foreign journals now held in Europe.

d. Probable adjustment of salaries to meet increased cost of living.

e. Additional pensions.

f. A naturalist to replace Mr. G. M. Gray, now retired.

g. A fireproof building to replace the present wooden Laboratory build-
ings.

2. The Committee recognizes that the acquisition of funds for the above pur-
poses and for additional endowment constitutes the most important problem
confronting the Trustees of the Laboratory. In view of the anticipated in-
crease in research activity after the war, these needs appear to be immediate
and imperative.

The Committee directed the Secretary to inform the Trustees of the Laboratory
of the above matters. The condensed report will be forwarded to the Bank as
Trustee of the Endowment Funds ; the full minutes will be published in the 1944
Annual Report of the Director.

The Committee adjourned at 4:45 P.M.

CHARLES PACKARD,

Secretary

August 9, 1944

3. To THE COMMITTEE OF REVIEW

Gentlemen:

The first decennial review (1923-1933) included the period of rapid growth
of the Laboratory. The Endowment Fund was set up ; the chief building erected,
more than doubling the space available for research ; a special endowment for the

18 MARINE BIOLOGICAL LABORATORY

Library permitted a notable addition to its holdings of journals and books ; a large
amount of apparatus and other tools of research became available. As a result,
the scientific activity of the Laboratory increased greatly. Toward the end of the
period,' the economic depression brought about a temporary slowing down of
growth.

In the period now under review (1934-1943) growth was resumed. The
Library overflowed the space allotted to it and spread into the new wing, a gift of
the Rockefeller Foundation. The number of investigators increased, exceeding
all previous records. The war has temporarily ended this growth. The Library
now receives few foreign journals ; the younger investigators are in active service
or in war research ; the classes, which have been maintained without interruption,
are almost devoid of men. But the Laboratory has continued to offer all of its
usual facilities to investigators and students. The current year (1944) shows
a marked upward trend in attendance and scientific activity. The stability of the
Laboratory during these periods of w7ar and economic depression is noteworthy.

Personnel

Many important changes in personnel have occurred in the past ten years.
Dr. F. R. Lillie retired as President of the Corporation, and was elected President
Emeritus. In his stead, Mr. Lawrason Riggs, the Treasurer since 1924, was
chosen President ; and the office of Vice President, created in 1942, was filled by
Dr. E. N. Harvey.

The following changes have occurred in the Board of Trustees :

(a) Died in Office: C. R. Stockard, D. H. Tennent.

(b) Elected Trustees Emeritus (having reached the age of seventy years) :

G. N. Calkins, d. 1943 H. S. Jennings

E. G. Conklin C. E. McClung

B. M. Duggar S. O. Mast

W. E. Carrey A. P. Mathews

Caswell Grave, d. 1944 W. J. V. Osterhout

M. J. Greenman, d. 1938 G. H. Parker

R. G. Harrison W. M. Wheeler, d. 1937

E. G. Ball. Assoc. Prof. Biol. Chem., Harvard Medical School
S. C. Brooks, Prof, of Zoology, University of California

D. E. S. Brown, Prof, of Physiology, N. Y. University Dental School
G. H. A. Clowes, Director of Research, Eli Lilly Laboratory

E. F. DuBois, Prof, of Physiology, Cornell Medical College

P. S. Galtsoff, Senior Biologist, U. S. Fish and Wild Life Service

Laurence Irving, Prof, of Biology, Swarthmore College

Columbus Iselin, Director, Woods Hole Oceanographic Institution

C. W. Metz, Prof, of Zoology, University of Pennsylvania

J. H. Northrup, Member, Rockefeller Institute

REPORT OF THE DIRECTOR 19

H. H. Plough, Prof, of Biology, Amherst College
Franz Schrader, Prof, of Zoology. Columbia University
E. W. Sinnott, Prof, of Botany, Yale University

A. H. Sturtevant, Prof, of Genetics, Calif. Institute Technology
W. R. Taylor, Prof, of Botany, University of Michigan

B. H. Willier, Prof, of Zoology, Johns Hopkins University

Dr. M. H. Jacobs, appointed Director in 1926, resigned in 1937. Dr. Charles
Packard was made Assistant Director in that year, and Director in 1939. Since
1942 he has been Resident Director.

Our investigators and students are drawn from institutions widely distributed
throughout the country (cf. map, Exhibit 3). In addition to universities and col-
leges, 36 Medical Schools and Hospitals have sent representatives ; 9 Research In-
stitutes, a number of Federal and State services, and industrial laboratories are
also represented. A complete list of all of these various institutions is found in
Exhibits 4 and 5.

Statistics of attendance for the period under review are shown in Exhibit 3.
The chart indicates the annual attendance since 1888 when the Laboratory was
founded. The term "New Investigators" refers to those who work here for the
first time ; ''Returning Investigators" are those who have previously spent one or
more seasons at the Laboratory. The effect of the first world war and of the
present war ; of periods of economic depression ; and of expansion in research
facilities, can be seen.

An incomplete list of publications from this Laboratory is found in Exhibit 8.
The scientific record of students attending the courses for the years 1918-1931 is
also a part of this Exhibit since it indicates their continuing interest and success in
biological research and teaching.

The Laboratory is in full operation and is open for your inspection.

Respectfully submitted,

LAWRASON RIGGS, President
CHARLES PACKARD, Director

EXHIBITS

For the \cars 1934—1943 inclusive

1. Annual Reports

2. Annual Announcements
*3. Statistics of Attendance

*4. Institutions represented by Investigators and Students

*5. Subscribing and Cooperating Institutions

*6. Additions to the Library. Check List of Journals

7. Catalog of Investigators

8. Partial List of Publications from the Laboratory

9. The Scientific Record of Students attending the Courses

* These exhibits appear in this Report.

MARINE BIOLOGICAL LABORATORY

INSTITUTIONS

REPRESENTED BY
I IMVESTIGATOR5 AND STUDENTS'
1934-1943

1000

ZOOOmi

Geographical distribution of institutions represented at the Marine Biological Laboratory

1934-1943

500

400

300

ZOO

100

E>ldq5

ane

Wor

Ld-Warl

"XT

Total

^f:

Buslnes
De^resstor

-J"

New

Alter

<?turr

nves

ic lanc< !

World WarH.

a tors

'90 '95 1900 "05 '10 '15 '20 '25 '30 '35 1940

Attendance at the Marine Biological Laboratory 1888-1943

REPORT OF THE DIRECTOR

EXHIBIT 3
A TABULAR VIEW OF ATTENDANCE 1934-1943

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Investigators — Total

373

315

359

391

380

357

386

337

701

160

Independent

7,7.7,

708

776

756

746

713

753

197

137

Beginning

Research Ass'ts

52'

Library Readers

Students — Total

131

130

138

133

137

133

178

131

Botany

Embryology .

Physiology

Protozoology

Zoology

Total Attendance less double registrations
Institutions represented

439
131

429
143

473
158

511
165

496
151

471
167

507

148

461
144

273
176

222
116

By investigators

111

170

134

175

137

117

107

By students

Schools and Academies
By investigators

By students

Foreign Institutions .
By investigators

By students

MARINE BIOLOGICAL LABORATORY

EXHIBIT 4

INSTITUTIONS REPRESENTED BY INVESTIGATORS AND STUDENTS 1934-1943

A. UNIVERSITIES AND COLLEGES

The figures after the name of the institution refer to the year the institution was

first represented at the Laboratory

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Adelphi College

N. Y. '38

Agnes Scott College

Ala. '15

Akron, University of

Ohio '15

Alabama, University of

Ala. '20

Alabama University Medical

'37

Alabama Polytech. Inst.

Ala. '38

Albany Medical Coll.

N. Y. '31

Albion College

Mich. '92

American Internat. Coll.

Mass. '42

American University

D. C. '31

Amherst College

Mass. '13

Antioch College

Ohio '23

Arizona, University of

Ariz. '25

Assumption College

Mass. '42

Atlanta University

Ga. '34

Baldwin-Wallace Coll.

Ohio '35

Bard College

N. Y. '35

Barnard College

N. Y. '96

Baylor Univ. Medical

Tex. '42

Beloit College

Wis. '96

Bennington College

Vt. '35

Berea College

Ky. '28

Birmingham-South. Univ.

Ala. '26

Boston College

Mass. '38

Boston University

Mass. '17

Boston University Medical

Mass. '37

Boston Teachers Coll.

Mass. '37

Bowdoin College

Me. '93

Bradley Poly. Inst.

111. '96

Bridgewater State T. C.

Mass. '38

Brooklyn College

N. Y. '32

Brown University

R. I. '90

REPORT OF THE DIRECTOR
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Bryn Mawr College
Buffalo, University of

Penn. '88

N. Y. '95

5
2

Buffalo University Medical
Butler University

N. Y. '39
Ind. '16

California Inst. Tech.
California, Univ. of

•

Cal. '29
Cal. '00

3
1

3
2

4
4

2
5

Canisius College
Carnegie Inst. Tech.

N. Y. '37
Penn. '09

1
1

Catholic Univ. of Amer.
Central State T. C.

D. C. '42
Okla. '36

Centre College
Charleston, Coll. of

Ky. '37
S. C. '06

Chestnut Hill College
Chicago, University of

Penn. '41
111. '92

2
2

Chicago University Medical
Cincinnati, Univ. of

111. '40
Ohio '92

1
7

Cincinnati Univ. Medical
Clark University

Ohio '35
Mass. '88

3
2

2
1

3
1

5
2

Coe College
Colby College

Iowa '20
Me. '99

Colgate University
College of Scholastica

N. Y. '98
Minn. '39

College City of N. Y.
Colorado, Univ. of

N. Y. '94
Col. '14

7
2

2
1

Colorado Univ. Medical
Columbia University

Col. '25
N. Y. '91

1
18

Columbia University Medical
Connecticut College

N. Y. '94
Conn. '20

5
2

7
2

8
2

5
3

3
3

5
2

4
1

Converse College
Cornell University

S. C. '38
N. Y. '91

1
3

Cornell University Medical
Dartmouth College

N. Y. '09
N. H. '96

12
3

11
4

8
4

10
4

5
3

1
2

1
1

Davis and Elkins Coll.
Delaware, Univ. of

W.Va.'41
Del. '98

Delta State T. C.
DePaul University

La. '36
111. '41

MARINE BIOLOGICAL LABORATORY
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

DePauw University
Dillard University

Ind. '89
La. '42

1
1

Drew University
Drury College

N. J. '38
Mo. '43

Duchesne College
Duke University

Neb. '35
N. C. '25

1
2

Earlham College
East Carolina T. C.

Ind. '01
N. C. '38

Edgewood Park Jr. Coll.
Elizabethtown Coll.

N. C. '34
'38

Elmira College
Elon College

N. Y. '21
N. C. '40

Emory University
Emory Junior Coll.

Ga. '97
Ga. '33

— -

Fisk University
Flat River Jr. Coll.

Tenn. '31
'28

Flora Macdonald Coll.
Florida, University of

N. C. '25
Fla. '41

Florida State College
Florida Coll. for Women

Fla. '39
Fla. '29

1
1

Fordham University
Franklin Marshall Coll.

N. Y. '19
Penn. '91

George Washington Univ. Medical

D. C. '14

Georgia, University of, Medical

Ga. '37

Gettysburg College
Goucher College

Penn. '39
Md. '94

2
3

Great Falls Norm. Coll.
Grinnell College

Mont. '38
Iowa '95

Gulf Park College
Hahneman Medical Coll.

Ky. '36

N. Y. '38

Hamilton College
Harvard University

N. Y. '91
Mass. '89

2
14

2
11

2
15

1
16

1
14

Harvard University Medical
Haverford College

Mass. '89
Penn. '89

Heidelberg College
Herzl Junior College

Ohio '91
111. '43

REPORT OF THE DIRECTOR
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Hood College
Howard University

Md. '15
D. C. '09

1
1

Hunter College
Illinois, Univ. of

N. Y. '14
111. '90

7
5

3
6

4
2

4
1

3
3

Indiana University
Iowa, State Univ. of

Ind. '89
Iowa '94

1
6

2
5

2
6

Iowa, State College of
Johns Hopkins Univ.

Iowa '19
Md. '89

2
9

1
3

3
8

3
11

3
17

5
12

1
10

1
11

Johns Hopkins Univ. Medical
Kansas, University of

Md.
Kan. '90

2
1

4
2

2
1

Kansas State College
Kansas State T. C.

Kan. '26
Kan. '34

Kent State University
Kenyon College

Ohio '30
Ohio '96

Lander College
La Verne College

S. C. '38
Cal. '34

Leland Stanford Univ.
Lincoln University

Cal. '91
Penn. '01

Long Island Univ.
Long Island Univ. Medical

N. Y. '29
N. Y. '19

Loyola Univ. Medical
Maine, University of

La. '31
Me. '95

1
1

Marquette University
Maryland, Univ. of

Wis. '36

Md. '41

Maryland Univ. Medical
Mass. State College

Md. '96
Mass. '89

11
1

13
1

11
1

4
1

5
2

1
1

Mass. Inst. Technology
Miami University

Mass. '88
Ohio '91

2
3

2
2

Michigan, Univ. of
Michigan Agric. Coll.

Mich. '88
Mich. '10

Middlebury College
Middlesex College

Vt. '95
Mass. '37

Minnesota, Univ. of
Mississippi, Univ. of

Minn. '98

Miss. '99

3
1

Missouri, Univ. of
Missouri State T. C.

Mo. '95
Mo. '43

MARINE BIOLOGICAL LABORATORY

EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Moberly Junior Coll.
Morehouse College

Wis. '40
Ga. '27

Morristown College
Mt. Holyoke College

Tenn. '37
Mass. '88

Mt. Mercy College
Mt. St. Louis Coll.

Penn. '39
'34

Mundelein University
National Park Coll.

111. '39
Md. '42

Nebraska, Univ. of Medic.
New Hampshire State U.

Neb. '34
N. H. '00

New Jersey College for Women

N. J. '28

New Jersey State T. C.
New Rochelle, Coll. of

N. J. '31

N. Y. '34

2
1

New York University
New York University Medical

N. Y. '96

N. Y. '25

4
6

3
2

10
6

7
10

7
7

2
3

5
9

7
5

2
7

. 6

New York University Wash. Sq.
Newark, University of

N. Y. '24
N. J. '41

8
1

Newark State T. C.
North Carolina, Univ. of

N. J. '41
N. C. '99

1
3

North Carolina Coll. for Negroes

N. C. '40

N. C. State College
N. C. Womens College

N. C. '31
N. C. '22

2
1

N. Dakota State Univ.
N. Dakota Agric. Coll.

N. D. '93
N. D. '23

1
1

N. Texas Agric. Coll.
Northwestern Univ.

Tex. '38
111. '93

1
1

Notre Dame Univ.
Oberlin College

Ind. '21
Ohio '90

1
8

1
7

1
4

Ohio State Univ.
Ohio University

Ohio '90
Ohio '14

4
2

Ohio Wesleyan Univ.
Oklahoma, Univ. of

Ohio '91
Okla. '09

2
1

Oklahoma City, Univ. of
Pennsylvania, Univ. of

Okla. '37
Penn. '91

1
31

1
28

1
26

1
32

1
27

Pennsylvania, Univ. of Medical
Penn. Coll. for Women

Penn.
Penn. '01

3
3

5
1

5
2

8
2

3
1

REPORT OF THE DIRECTOR
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Penn. State Coll.
Pittsburgh, Univ. of

Penn. '07
Penn. '21

7
1

Placer Junior College
Pomona College

Cal. '40
Cal. '24

Princeton University
Providence College

N. J. '90
R. I. '35

7
1

12
2

Purdue University
Queens College

Ind. '28

N. Y. '28

5
1

1
1

3
4

3
3

Radcliffe College
Randolph-Macon Coll.

Mass. '95
Ya. '89

5
1

Reed College
Rensselaer Poly. Inst.

Ore. '39

N. Y. '36

Rice Institute
Richmond, College of

Tex. '16
Va. '13

1
1

Rochester, Univ. of
Rochester, Univ. of Medical

N. Y. '92

N. Y. '35

7
2

5
2

5
1

Russell Sage College
Rutgers University

N. Y. '20
N. J. '14

2
2

2
1

1
2

5
2

St. Francis Xavier Coll.
St. Johns College

111. '14
Md. '34

2
1

St. Louis University
St. Louis University, Maryville

Mo. '03
Mo. '37

3
1

St. Thomas College
St. Vincent College

Minn. '35
Penn. '24

Sarah Lawrence College
Seton Hall College

N. Y. '32
N. J. '35

5
2

Seton Hill College
Simmons College

Penn. '29

Mass. '07

1
1

Skidmore College
Smith College

N. Y. '22
Mass. '92

1
4

J. C. Smith University
Southern California, University of

N. C. '34
Cal. '96

Southern Oregon State Normal

Ore. '38

Southwestern Univ.
Spring Hill College

Tenn. '22
Ala. '38

Springfield College
Stephens College

Mass. '40
Mo. '36

MARINE BIOLOGICAL LABORATORY
EXHIBIT 4 — Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Swarthmore College
Sweet Briar College

Penn. '88
Va. '09

Syracuse University
Syracuse University Medical

N. Y. '94
N. Y. '99

1
2

2
1

1
2

1
1

Temple University
Tennessee, Univ. of

Penn. '97
Tenn. '95

2
3

Tennessee, Univ. of Medical
Texas, University of

Tenn. '35
Tex. '95

Texas, University of Medical
Texas Christian Univ.

Tex. '36
Tex. '21

Toledo, Univ. of
Tougaloo College

Ohio '17
Miss. '39

2
1

Trinity College
Tufts College

Conn. '00
Mass. '92

2
1

2
2

1
1

1
2

Tulane University
Tulane Newcomb Coll.

La. '16
La. '13

1
1

Union College
Union College

N. Y. '15
Ky. '39

2
1

Ursinus College
Vanderbilt University

Penn. '95
Tenn. '91

1
1

Vanderbilt University Medical
Vassar College

Tenn. '34
N. Y. '88

4
4

3
2

3
3

4
6

3
3

2
6

1
6

Vermont, University of
Vermont, University of Medical

Vt. '15
Vt. '38

1
1

Vermont State Normal
Villanova College

Vt. '40
Penn. '39

1
6

1
1

Virginia, Univ. of
Virginia, Univ. of Medical

Va. '91
Va.

2
2

Virginia State T. C.
Virginia Interment Coll.

Va. '34
Va. '41

Wabash College
Washburn College

Ind. '07
Kan. 40'

2
1

Washington University
Washington University Medical

Mo. '00
Mo.

2
2

12
6

10
2

8
3

5
1

Washington, Univ. of
Washington and Jefferson College

Wash. '15
Penn. '11

1
2

REPORT OF THE DIRECTOR
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Washington and Lee

Va. '17

Wayne University

Mich. '34

Wellesley College

Mass. '88

Wesleyan University

Conn. '89

West Virginia Univ.

W.Va.'Ol

Westbrook Junior Coll.

Conn. '40

Western College

Ohio '98

Western Reserve Univ.

Ohio '95

Wheaton College

Mass. '18

Whitman College

Ore. '43

William and Marv

Va. '22

Williams College

Mass. '92

Wilson College

Penn. '24

Wisconsin, Univ. of

Wis. '98

\Vomens Medical College

Penn. '92

Wooster College

Ohio '13

Wright Junior College

111. '41

Wyoming, Univ. of

Wyo. '29

Yale University

Conn. '91

Yale University Medical

Conn. '38

B. HIGH SCHOOLS, ACADEMIES, ETC.

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Abraham Lincoln H. S.

N. Y.

Agnes Irwin School

N. Y.

Annapolis H. S.

Md.

Berkshire School

Mass.

Birmingham H. S.

Ala.

Boston H. S.

Mass.

Bronxville H. S.

N. Y.

Caldwell H. S.

N.J.

Central H. S.

D. C.

Chicago H. S.

111.

Choate School

Conn.

Dana Hall

Mass.

Darrow School

N. Y.

Deerfield Academy

Mass.

MARINE BIOLOGICAL LABORATORY
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

Eastern District H. S.
Emma Willard School

N. Y.

N.Y.

Exeter Academy
Galesburg H. S.

N. H.
111.

Grand Falls H. S.
Groton School

Can.
Conn.

Hallahan H. S.
Hawthorne H. S.

Penn.

N.J.

Hyde School
Hyde Park School

Mass.

111.

Jenkintown H. S.
Knox School

Penn.

N. Y.

Lawrenceville School
Los Angeles H. S.

Mass.
Cal.

1
1

Milton Academy
Nativity H. S.

Mass.

5
1

Negaunee H. S.
Oradell H. S.

Mich.
N.J.

Pennsgrove School
Potomac School

Penn.
D. C.

St. Andrews School
St. Catherine School

Del.
Va.

St. Joseph's Seminary
St. Mary of the Lake Sem.

N. Y.
111.

Scott, H. S.
Society of the Divine Word

Ohio

Mass.

Straubenmiller H. S.
Theo. Roosevelt H. S.

N. Y.

1
1

Union H. S.
Union City H. S.

N.J.
Tenn.

1
1

University H. S.
Vineland Training School

Minn.
N.J.

1
1

Walton H. S.
Washington H. S.

N.J.
D. C.

Weequahic H. S.
Westtown Friends School

N.J.
Penn.

REPORT OF THE DIRECTOR

EXHIBIT 4— Continued
C. INSTITUTES, FOUNDATIONS, ETC.

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

American Mus. Nat. Hist.
Arlington Chemical Co

N. Y. '09
'31

1
1

Barnard Skin and Cancer Hospital

Mo.

Beth Israel Hospital
Biol. Institute, Phil.

N. Y. '42
Penn. '39

Carnegie Institute
Cold Spring
Washington

N. Y. '14
D. C. '15

Frick Education Comm.
Guggenheim Foundation
Guggenheim Dental Clinic

'42
'40
'43

Journ. Industrial and Engineering
Chemistry

'28

Internal. Cancer Research Founda-
tion

'37

Eli Lilly Company
Marine Studios, Inc.

Ind. '19
Fla. '42

Memorial Hospital
Mt. Sinai Hospital

N. Y. '26
N. Y. '40

2
2

1
1

Nat. Cancer Institute
Nat. Research Council

Md. '39
D. C.

N. Y. State Agricult. Station
N. Y. State Dept. Health

N. Y. '18
N. Y. '19

Overly Biochemical Research Found.

N. Y. '43

Phila. Acad. Nat. Sci.
Rockefeller Institute

Penn. '89
N. Y. '11

1
13

1
14

Rockefeller Foundation Fellowship

Russell Sage Institute of Pathology

N. Y. '34

St. Luke's Hospital
LT. S. Dept. Agriculture

N. Y. '40
D. C. '99

U. S. Dept. Public Health
U. S. Fish and Wild Life Service

D. C. '20
D. C. '42

Wistar Institute
Woods Hole Oceanographic Inst.

Penn. '08
Mass. '43

MARINE BIOLOGICAL LABORATORY

EXHIBIT 4— Continued
D. INSTITUTIONS OUTSIDE THE UNITED STATES

1934

•35

'36

'37

'38

'39

'40

'41

'42

'43

Austria

Univ. of Innsbruck

Univ. of Vienna

Belgium

Univ. of Ghent

Univ. of Liege

Belgian-American Educ. Founda-

tion

British

Queens Coll. Belfast

Isles

Cambridge University

Trinity Coll. Dublin

Univ. of Edinburgh

Univ. of Leeds

Univ. of London

Univ. Coll. London

Univ. Coll. Nottingham

Oxford University

British Fish. Service

Canada

Acadia University N. S.

Univ. British Columbia

Dalhousie University, N. S.

McGill University, Ont.

Univ. of Manitoba

Univ. of Montreal

Memorial University, N. F.

Coll. Ste. Marie, Montreal

Univ. of Toronto

Univ. West. Ontario

Royal Soc. Canada

Far East

Womens Medical College, Madras,

India

Judson Coll. Rangoon, Burma

China

Peking Union Med. Coll.

Colombia

Cotton Res. Station

Denmark

Carlsberg Laboratory

Univ. of Copenhagen

Egypt

Egyptian Educ. Commission

REPORT OF THE DIRECTOR
EXHIBIT 4— Continued

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

France

Pasteur Institute, Paris

University of Paris

University of Strasbourg

University of Strasbourg Medical

Germany

University of Berlin

Neurolog. Inst. Frankfurt

University of Munich

Hungary

University of Debrescen

Budapest Univ. Medical

Franz Joseph University

Italy

University of Padua

Japan

Misaki Biolog. Inst.

Norway

University of Oslo

Peru

Guano Administration

Poland

University of Lwow

Russia

Moscow, Inst. Genetics

Serbia

Belgrade Medical Coll.

Spain

Barcelona Medical Coll.

University of Lund

Sweden

Karolinska Inst. Stockholm

University of Stockholm

Switzer-

Physiological Inst. Berne

land

Zoological Inst. Berne

University of Geneva

Uruguay-

Ministry of Pub. Health

Cuba

University of Havana

Summary

Universities and Colleges
High Schools and Academies
Institutes, Foundations, etc.
Foreign Institutions

1923-33

1934-43

246

269

101

433

409

MARINE BIOLOGICAL LABORATORY

EXHIBIT 5
SUBSCRIBING AND COOPERATING INSTITUTIONS

A cooperating institution is one that has subscribed for the two preceding
years, or that announces its intention of subscribing regularly. A subscribing
institution is one that pays for one or more tables or rooms.

1934

'35

'36

'37

'38

'39

'40

'41

'42

'43

American University

Amherst College

Atlanta University

Barnard College

Belgian-Amer. Educ. Found.

Bell Telephone Laboratory

Berea College

Beth Israel Hospital

Biological Institute, Phila.

Bowdoin College

Brooklyn College

Brown University

Bryn Mawr College

Buffalo University Medical

Butler University

C. R. B. Educational Found.

California Inst. Technol.

Canisius College

Carnegie Inst. Washington

Catholic Univ. of America

Chicago, University of

Chicago, University of Medical

Children's Hospital Cincinnati

Chinese Educational Mission

Christ Hospital, Cincinnati

Cincinnati, University of

Columbia University

Columbia University Medical

Commonwealth Fund

Connecticut College

Cornell University

Cornell University Medical

Dalhousie University

Dartmouth College

De Pauw University

Drew University

REPORT OF THE DIRECTOR
EXHIBIT 5 — Continued

1934

•35

'36

'37

'38

'39

'40

'41

'42

'43

Duke University

Eli Lilly Research Lab.

Elmira College

Fisk University

Fordham University

Frick Educational Comm.

General Education Board

Georgia, Univ. of Medical

Goucher College

Hamilton College

Harvard University

Harvard University Medical

Heidelberg College

Howard University

Hunter College

Illinois, University of

Indiana University

Industrial and Engin. Chem.

Iowa, State Univ. of

Iowa, State College of

Johns Hopkins University

Johns Hopkins University Medical

Johnson Foundation

Josiah Macy Foundation

Julius Rosenwald Fund

Kansas, University of

Kenyon College

Leland Stanford Univ.

Lincoln University

Long Island University

Loyala Univ. Medical

McGill University

Markle Foundation

Maryland, Univ. of Medical

Marine Studios, Inc.

Mass. General Hospital

Mass. State College

Memorial Hospital, N. Y.

Michigan, University of

Minnesota, University of

Missouri, University of

Morehouse College

MARINE BIOLOGICAL LABORATORY
EXHIBIT 5— Continued

1934

'35

'36

'37

'38

•39

'40

'41

'42

'43

Mt. Holyoke College

Mt. Sinai Hospital

Mundelein University

National Research Council

N. Y. Dept. of Health

New York University

New York University Medical

New York University Wash. Square

N. Carolina Coll. for Negroes

Northwestern University

Notre Dame University

Oberlin College

Ohio State University

Ohio Wesleyan University

Penn. College for Women

Pennsylvania, Univ. of

Pennsylvania, Univ. of Medical

Pittsburgh, Univ. of

Princeton University

Purdue University

Radcliffe College

Rensselaer Poly. Inst.

Rice Institute

Rochester, University of

Rochester, University of Medical

Rockefeller Foundation

Rockefeller Institute

Royal Egyptian Foundation

Russell Sage College

Rutgers University

St. Elizabeth, College of

St. Francis Xavier College

St. Johns College

Sarah Lawrence College

Seton Hall College

Seton Hill College

Smith College

J. C. Smith University

Spring Hill College

Springfield College

Swarthmore College

Sweet Briar College

REPORT OF THE DIRECTOR
EXHIBIT 5— Continued

1934

•35

'36

•37

'38

'39

'40

'41

'42

'43

Syracuse University

Syracuse University Medical

Temple University

Toledo, University of

Tufts College

Tulane University

Tulane University Newcomb Coll.

Union College, N. Y.

Union College, Ky.

U. S. Fish and Wild Life Serv.

Vanderbilt University

Vanderbilt University Medical

Vassar College

Vermont, University of

Villanova College

Virginia, University of

Wabash College

Washington University

Washington University Medical

Wellesley College

Wesleyan University

Western Reserve University

Wheaton College

William and Mary College

Williams College

Wilson College

Wisconsin, University of

Wistar Institute

Woods Hole Oceanographic Inst.

Yale University

Yale University Medical

MARINE BIOLOGICAL LABORATORY

EXHIBIT 6
ADDITIONS TO THE LIBRARY

Bound
volumes

Reprints

Sets
completed

Partially
completed

New
journals

Classics

Budget

1934

1,138

5,028

$20.325

1935

1,622

4,478

22.444

1936

2,107

3,339

22.510

1937

1,155

7,042

22.029

1938

1,455

6,905

19.515

1939

1,239

3,850

22.149

1940

1,561

3,528

17.923

1941

1,482

3,321

16.964

1942

1,758

3,097

161

15.332

1943

1,008

7,927

11.047

Total added

14,525

48,515

250

468

227

Total in

51,945

129,723

1800

600

Libr.

approx.

4. THE STAFF, 1944

CHARLES PACKARD, Director, Marine Biological Laboratory, Woods Hole, Massachusetts.

SENIOR STAFF OF INVESTIGATION

E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University.

FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of Chicago.

RALPH S. LILLIE, Professor of General Physiology, Emeritus, The University of Chicago.

C. E. McCLUNG, Professor of Zoology, Emeritus, University of Pennsylvania.

S. O. MAST, Professor of Zoology, Emeritus, Johns Hopkins University.

A. P. MATHEWS, Professor of Biochemistry, Emeritus, University of Cincinnati.

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

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

ZOOLOGY

I. CONSULTANTS

T. H. BISSONNETTE, Professor of Biology, Trinity College.
L. L. WOODRUFF, Professor of Protozoology, Yale University.

II. INSTRUCTORS

J. B. BUCK, Assistant Professor of Zoology, University of Rochester, in charge of course.

T. H. BULLOCK, Instructor in Neuro Anatomy, Yale University.

W. D. BURBANCK, Associate Professor of Biology, Drury College.

C. G. GOODCHILD, Associate Professor of Biology, Southwest Missouri State Teachers

College.

JOHN H. LOCH HEAD, Instructor in Zoology, University of Vermont.
MADELENE E. PIERCE, Assistant Professor of Zoology, Vassar College.
W. M. REID, Assistant Professor of Biology, Monmouth College.
MARY D. ROGICK, Professor of Biology, College of New Rochelle.

REPORT OF THE DIRECTOR 39

III. LABORATORY ASSISTANTS

GENE LEHMAN, Teaching Fellow Zoology, University of North Carolina.
MARY E. BANKS, Washington University.

EMBRYOLOGY

I. CONSULTANTS

L. G. BARTH, Assistant Professor of Zoology, Columbia University.
H. B. GOODRICH, Professor of Biology, Wesleyan University.

II. INSTRUCTORS

VIKTOR HAMBURGER, Professor of Zoology, Washington University, in charge of course.
DONALD P. COSTELLO, Aesistaat Professor of Zoology, University of North Carolina.
RAY L. WATTERSON, Assistant Professor of Zoology, University of California.

PHYSIOLOGY

I. CONSULTANTS

WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of

Medicine.

HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin.
WALTER E. GARREY, Professor of Physiology, Vanderbilt University Medical School.
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania.

II. INSTRUCTORS

ARTHUR K. PARPART, Associate Professor of Biology, Princeton University, in charge

of course.

ROBERT BALLENTINE, Lecturer in Zoology, Columbia University.

ARTHUR C. GIESE, Associate Professor of Biology, Stanford University (absent in 1943).
RUDOLF T. KEMPTON, Professor of Zoology, Vassar College.

BOTANY

I. CONSULTANTS

S. C. BROOKS, Professor of Zoology, University of California.

B. M. DUGGAR, Professor of Plant Physiology, University of Wisconsin.

II. INSTRUCTORS

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

course.
HANNAH CROASDALE, Technical Assistant, Dartmouth College.

EXPERIMENTAL RADIOLOGY

G. FAILLA, Memorial Hospital, New York City.

L. ROBINSON HYDE, Phillips Exeter Academy, Exeter, N. H.

LIBRARY

PRISCILLA B. MONTGOMERY (MRS. THOMAS H. MONTGOMERY, JR.), Librarian
DEBORAH LAWRENCE MRS. ELON H. JESSUP MARY A. ROHAN

40 MARINE BIOLOGICAL LABORATORY

•

APPARATUS DEPARTMENT

E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager

J. D. GRAHAM

CHEMICAL DEPARTMENT
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager

SUPPLY DEPARTMENT

JAMES MC!NNIS, Manager

RUTH CROWELL GRACE M. KENNERSON

M. B. GRAY W. E. KAHLER G. LEHY

A. M. HILTON A. W. LEATHERS F. N. WHITMAN

GENERAL OFFICE

F. M. MACNAUGHT, Business Manager
POLLY L. CROWELL MRS. CARLOTTA I. COWIN

GENERAL MAINTENANCE

T. E. LARKIN, Superintendent

W. C. HEMENWAY G. T. NICKELSON, JR.

R. W. KAHLER T. E. TAWELL

A. J. PIERCE

THE GEORGE M. GRAY MUSEUM
GEORGE M. GRAY, Curator Emeritus

5. INVESTIGATORS AND STUDENTS
Independent Investigators, 1944

ABELL, RICHARD G., Assistant Professor of Anatomy, University of Pennsylvania.

ADDISON, WILLIAM H. F., Professor of Normal Histology and Embryology, University of

Pennsylvania.

ALLEE, W. C., Professor of Zoology, University of Chicago.
ANDERSON, RUBERT S., Assistant Professor, University of Maryland.
BALLENTINE, ROBERT, Instructor, Columbia University.
BARTH, L. G., Assistant Professor of Zoology, Columbia University.

BARTLETT, JAMES H., Associate Professor of Theoretical Physics, University of Illinois.
BELCHER, JANE C., Instructor, Sweet Briar College.

BERGER, CHARLES A., Director, Biological Laboratory, Fordham University.
BERGMANN, WERNER, Associate Professor, Yale University.
BERTHOLF, LLOYD MILLARD, Dean of Faculty and Professor of Biology, Western Maryland

College.

BROWN, DUGALD S., Professor of Physiology, New York University.
BROWNELL, KATHARINE A., Research Associate, Ohio State University.
BUCK, JOHN B., Assistant Professor of Zoology, University of Rochester.
BUDINGTON, ROBERT A., Professor of Zoology, Emeritus, Oberlin College.
BULLOCK, THEODORE H., Instructor in Neuro-Anatomy, Yale University.
BURBANCK, W. D., Associate Professor of Biology and Chairman of Department, Drury

College.

REPORT OF THE DIRECTOR 41

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

CLARK, ELEANOR L., Voluntary Research Worker, University of Pennsylvania, Medical School.

CLARK, ELIOT R., Professor and Head of Department of Anatomy, University of Pennsylvania,
Medical School.

CLEMENT, A. C., Associate Professor of Biology, College of Charleston.

CLOWES, G. H. A., Director of Research, Lilly Research Laboratories, Eli Lilly and Company.

CONKLIN, EDWARD G.. Professor Emeritus of Biology, Princeton University.

COPELAND, MANTON, Professor of Biology, Bowdoin College.

COSTELLO, DONALD P., Professor of Zoology, University of North Carolina.

CRAMPTON, H. E., American Museum of Natural History.

CROASDALE, HANNAH T., Technical Assistant in Zoology, Dartmouth College.

CUTKOMP, LAURENCE K., Research Fellow in Zoology, University of Pennsylvania.

ELLIOTT, S. D., Visiting Investigator, Rockefeller Institute.

FAILLA, G., Professor of Radiology, College of Physicians and Surgeons, Columbia University.

FROEHLICH, ALFRED, Associate, May Institute for Medical Research, Cincinnati, Ohio.

GALTSOFF, PAUL S., Senior Biologist, U. S. Fish and Wildlife Service.

CARREY, W. E., Professor of Physiology, Vanderbilt University, School of Medicine.

GIESE, ARTHUR C., Associate Professor of Biology, Stanford University of California.

GLASER, OTTO C., Professor of Biology, Amherst College.

GOODCHILD, C. G., Associate Professor of Biology, State Teachers College.

GORBMAN, AUBREY, Instructor in Biology, Wayne University.

GRAND, C. G., Research Associate, New York University.

GRELL, SISTER MARY, Student and Investigator, Fordham University, New York.

HALLOCK, FRANCES A., Associate Professor, Hunter College.

HAMBURGER, VIKTOR, Professor of Zoology, Washington University.

HARNLY, MORRIS H., Associate Professor, Washington Square College, New York University.

HARRIS, DANIEL L., Research Associate, University of Pennsylvania.

HARTMAN, FRANK A., Professor and Chairman of Department of Physiology, Ohio State Uni-
versity.

HARVEY, E. NEWTON, Professor of Physiology, Princeton University.

HARVEY, ETHEL BROWNE, Independent Investigator, Princeton University.

HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania.

HIATT, EDWIN P., Assistant Professor of Physiology, New York University.

HOPKINS, HOYT S., Associate Professor of Physiology, New York University, Dental College.

JACOBS, M. H., Professor of General Physiology, University of Pennsylvania, Medical School.

KEMPTON, RUDOLF T., Professor of Zoology, Vassar College.

KNOWLTON, FRANK P., Professor of Physiology, College of Medicine, Syracuse University.

KRAHL, MAURICE E., Research Biological Chemistry, Lilly Research Laboratories.

LANCEFIELD, REBECCA C., Associate Member, Rockefeller Institute for Medical Research.

LAVIN, GEORGE L, In Charge of Spectroscopic Laboratory, Rockefeller Institute for Medical
Research.

LAZAROW, ARNOLD, Senior Instructor, Western Reserve University.

LEVY, MILTON, Assistant Professor, New York University College of Medicine.

LEWIS, MARGARET REED, Research Associate, The Carnegie Institution of Washington.

LEWIS, WARREN H., Member, The Wistar Institute of Anatomy and Biology.

LIEBMAN, EMIL, Research Fellow, Princeton University.

LILLIE, RALPH S., Professor Emeritus of Physiology, University of Chicago.

LITTLE, ELBERT P., Instructor in Science, Exeter Academy, Exeter, New Hampshire.

LOCH HEAD, JOHN H., Instructor in Zoology, University of Vermont.

McCLUNG, C. E., Professor of Zoology, Emeritus, University of Pennsylvania.

MCELROY, WILLIAM D., Research Associate, Princeton University.

MACLEAN, BERNICE L., Assistant Professor, Department of Biological Sciences, Hunter College.

MCMASTER, PHILIP D., Associate Member, Rockefeller Institute.

MARSLAND, DOUGLAS A., Associate Professor, New York University, Washington Square
College.

MAST, S. O., Professor of Zoology, Emeritus, Johns Hopkins University.

MATHEWS, ALBERT P., Professor of Biochemistry, Emeritus, University of Cincinnati.

42 MARINE BIOLOGICAL LABORATORY

MEMHARD, ALLEN R., Crescent Road, Riverside, Connecticut.

METZ, CHARLES B., Instructor in Biology, Wesleyan University.

METZ, C. W., Director of Zoological Laboratory, Chairman of Department of Zoology, Univer-
sity of Pennsylvania.

MICHAELIS, LEONOR, Member Emeritus, Rockefeller Institute for Medical Research.

NACHMANSOHN, DAVID, Research Associate in Neurology, Columbia University.

OPPENHEIMER, JANE M., Assistant Professor of Biology, Bryn Mawr College.

OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research.

PARKER, GEORGE H., Professor of Zoology, Emeritus, Harvard University.

PARPART, ARTHUR K., Associate Professor, Princeton University.

PIERCE, MADELENE E., Assistant Professor of Zoology, Vassar College.

POLLISTER, ARTHUR W., Associate Professor of Zoology, Columbia University.

REID, W. MALCOLM, Assistant Professor of Biology, and Department Head, Monmouth College.

RENN, CHARLES E., Associate Sanitary Biologist, Massachusetts Department of Health.

REYNOLDS, J. PAUL, Professor of Biology, Birmingham Southern College.

RICHARDS, A. GLENN, JR., Instructor in Zoology, University of Pennsylvania.

ROGICK, MARY DORA, Professor of Biology, College of New Rochelle, New Rochelle, New York.

RUGH, ROBERTS, Associate Professor of Biology, New York University.

SCHAEFFER, A. A., Professor of Biology, Temple University.

SCHALLEK, WILLIAM B., Teaching Fellow, Harvard University.

SCHARRER, ERNST A., Assistant Professor of Anatomy, Western Reserve University School of
Medicine.

SCHMITT, FRANCIS O., Professor of Biology, Massathusetts Institute of Technology.

SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College.

SHAPIRO, HARRY H., Assistant Professor, Department of Anatomy, College of Physicians and
Surgeons, Columbia University.

SICHEL, F. J., Associate Professor of Physiology, University of Vermont, College of Medicine.

STEINBACH, H. B., Associate Professor of Zoology, Washington University.

STEWART, DOROTHY R., Research Fellow in Physiology, University of Pennsylvania.

STOKEY, ALMA G., Professor Emeritus of Plant Science, Mount Holyoke College.

STUNKARD, HORACE W., Professor of Biology, Head of Department, New York University.

TAFT, CHARLES H., Associate Professor of Pharmacology, Medical Branch, University of Texas.

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

TsWiNKEL, Lois E., Associate Professor of Zoology, Smith College.

TROEDSSON, PAULINE H., Instructor in Biology, Brooklyn College.

WAINIO, WALTER W., Assistant Professor of Physiology, New York University.

WATTERSON, RAY L., Assistant Professor of Zoology, University of California.

WENRICH, D. H., Professor of Zoology, University of Pennsylvania.

WHITING, ANNA R., Associate Professor of Zoology, Swarthmore College.

WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania.

WICHTERMAN, RALPH, Assistant Professor, Temple University.

WITKUS, ELEANOR RUTH, Instructor in Botany and Bacteriology, Fordham University.

WRINCH, DOROTHY, Lecturer in Physics, Smith College.

YNTEMA, CHESTER L., Assistant Professor of Anatomy, Cornell University, Medical College.

ZWEIFACH, BENJAMIN W., Research Associate in Biology, New York University.

Beginning Investigators, 1944

EDELMAN, ABRAHAM, Graduate Student, Columbia University.

HERMANSON, VIRGINIA, Graduate Student, Ohio State University.

JAEGER, LUCENA, Graduate Student, Columbia University.

KEISTER, MARGARET L., Instructor, Wheaton College.

KOOPMAN, KARL F., Graduate Student, Columbia University.

KRUGELIS, EDITH J., Research Assistant and Graduate Student, Columbia University.

LEHMAN, GENE, Teaching Fellow, University of North Carolina.

Low, RUTH H., Graduate Student, Zoology Department, Columbia University.

MORTENSEN, EDITH, Assistant Professor of Zoology, The George Washington University.

REPORT OF THE DIRECTOR 43

Sen NEVER, LEON H., Instructor, New York University College of Dentistry.
SISSELMAN, CHARLOTTE B., Research Student, Columbia University.

Library Readers, 1944

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

ANDERSON, THOMAS F., Associate, Johnson Foundation, University of Pennsylvania.

ARMSTRONG, PHILIP B., Professor of Anatomy, College of Medicine, Syracuse University.

BEVELANDER, GERRIT, Associate Professor of Anatomy, New York University.

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

BODIAN, DAVID, Associate in Epidemiology, Johns Hopkins University.

CAHEN, RAYMOND L., Research Assistant, Yale University, Medical School.

CASSIDY, HAROLD G., Assistant Professor of Chemistry, Yale University.

CHOUCROUN, NINE, Cornell University Medical College.

CLARKE, ROBERT W., Research Assistant, Yale University, Medical School.

Cox, EDWARD H., Professor of Chemistry, Swarthmore College.

DISCHE, ZACHARIOUS, Department of Biochemistry, College of Physicians and Surgeons, Colum-
bia University.

FAUST, ERNEST C., Professor of Parasitology and Director Department of Tropical Medicine,
Tulane University.

FRIDEMANN, ULRICH, Chief of Division of Bacteriology, Brooklyn Jewish Hospital.

FURTH, JACOB, Associate Professor of Pathology, Cornell University Medical College.

GATES, R. RUGGLES, Emeritus Professor, University of London.

GOODRICH, H. B., Professor of Biology, Wesleyan University.

GUREWICH, VLADIMIR, Assistant Visiting Physician, New York College of Medicine and Belle-
vue Hospital.

HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College.

JOHLIN, J. M., Associate Professor of Biochemistry, Vanderbilt University.

KELLER, RUDOLPH, Prague, Czechoslovakia.

KEYES, DONALD B., Professor of Chemical Engineering, University of Illinois.

KRASNOW, FRANCES, Head of Department of Biological Chemistry-Related Basic Sciences,
Guggenheim Dental Clinic.

LANDIS, EUGENE M., Professor of Physiology, Harvard University, Medical School.

LEE, RICHARD E., Research Assistant, Columbia University, College of Physicians and Surgeons.

LOEWI, OTTO, Research Professor, New York University.

MAVOR, JAMES W., Provessor of Biology, Union College.

MENKIN, VALY, (Fellow), Guggenheim Research Foundation.

MEYERHOF, OTTO, Research Professor of Biochemistry, University of Pennsylvania.

MOLDAVER, JOSEPH, Research Associate in Neurology, Columbia University.

MOSCHOWITZ, ELI, Assistant Professor of Chemical Medicine, Columbia University.

O'BRIEN, JOHN A., JR., Instructor in Biology, Catholic University of America.

OCHOA, SEVERO, Research Associate in Medicine, New York University, College of Medicine.

OSEASOHN, ROBERT, Student, Long Island College of Medicine.

PERLMANM, GERTRUDE E., Research Fellow, Harvard University, Medical School.

POWDERMAKER, HORTENSE, Assistant Professor of Anthropology, Queens College.

RUNNER, MEREDITH, Instructor, University of Connecticut.

SHEN, SHIH-CHANG, Member, National Institute of Physiology, China.

SHWARTZMAN, GREGORY, Head Bacteriologist, The Mount Sinai Hospital.

SINGER, MARCUS, Harvard University, Medical School.

SMELSER, GEORGE K., Assistant Professor of Anatomy, Columbia University.

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

STERN, KURT G., Chief Research Chemist, Overly Biochemical Research Foundation, New York
City.

TASHIRO, KIYOSHI, University of Cincinnati, College of Medicine.

WAGNER, CARROLL E., Research Assistant, Histology, Naval Medical Research Institute.

WEIDENREICH, FRANZ, Honorary Director, Cenozoic Research Laboratory, China.

WEINER, NATHAN, Director of Research, Endo Products, Inc.

44 MARINE BIOLOGICAL LABORATORY

WILLIER, B. H., Professor of Zoology, Director of Biological Laboratories, The Johns Hopkins

University.

WOODWARD, ALVALYN E., Assistant Professor, University of Michigan.
ZORZOLI, ANITA, Teaching Fellow, New York University.

Research Assistants, 1944

ABRAMSKY, TESSIE, Technician, Rockefeller Institute for Medical Research.

BRUNELLI, ELEANOR L., Research Assistant, New York University Dental School.

DEFALCO, ROSE H., Research Assistant-Secretary, University of Pennsylvania.

DZIORNEY, LEON, Research Assistant, New York University.

FRUMIN, M. R., Research Assistant, University of Pennsylvania.

GOLDIS, BERNICE R., Research Assistant, University of Pennsylvania.

GREGG, JOHN R., Graduate Student, Princeton University.

HIRST, SHIRLEY M., Research Assistant, University of Pennsylvania.

HONEGGER, CAROL, Temple University.

HOPKINS, AMOS, Junior Engineering Aide, Massachusetts State Health Department.

LAWLER, H. CLAIRE, Research Assistant, New York University.

LEFEVRE, LINDA, Research Assistant, University of Pennsylvania.

LEFEVRE, PAUL G., Research Assistant, University of Pennsylvania.

LEVY, BETTY, Laboratory Technician, Rockefeller Institute.

MARKS, MILDRED H., Assistant Research Worker, University of Pennsylvania.

MORTON, JANE W., Technical Assistant in Zoology, University of Pennsylvania.

PRICE, WINSTON HARVEY, Research Assistant, University of Pennsylvania.

QUINN, GERTRUDE P., Research Assistant, New York University.

WILSON, WALTER L., Research Associate, University of Pennsylvania.

WOODWARD, ARTHUR A., Research Assistant, University of Pennsylvania.

Students, 1944
BOTANY

CHEW, ROBERT M., Student, Washington & Jefferson College.
DEVINE, VERONA, Student, Hunter College.
GUZMAN, JULIA, Student, Washington University.
HOSKINS, BARBARA, Student, Wellesley College.
MITTLACHER, HELEN, Student, Wheaton College.

EMBRYOLOGY

ANDERSON, JOAN C, Student, McGill University.

COURANT, GERTRUDE E., Student, Swarthmore, College.

CULLEN, SISTER MARY URBAN, Graduate Student, Yale University.

DAVIDSON, MARGARET E. M., Student, McGill University.

FARFANTE, ISABEL PEREZ, Student, Cambridge, Massachusetts.

FINKELSTEIN, GRACE, Teaching Fellowship, New York University.

GETZ, CHARLOTTE E., Undergraduate Student, University of Chicago.

GODWIN, DORIS RUTH, Graduate Assistant, University of North Carolina.

HENLEY, CATHERINE, Graduate Assistant, University of North Carolina.

HONEGGER, CAROL MARIE, Student, Temple University.

KELLEY, ELLEN MARY, Student, New Jersey College for Women.

KIVY, EVELYN, Instructional Staff, Brooklyn College.

LANDAU, CAROL, Student, Goucher College.

LANTZ, ELSIE JEAN, Student, Washington University.

McGovERN, BEULAH H., Teaching Fellow, New York University.

MURRAY, HELEN ERNESTINE, Student, Emmanuel College.

POTTS, ELLA ELIZABETH, Student, Sarah Lawrence College.

ROTH, OWEN H., Instructor in Biology, St. Vincent College.

REPORT OF THE DIRECTOR 45

SCHNELLER, SISTER MARY BEATRICE, Professor, Saint Joseph College for Women.

STRONG, HELEN MARGARET, Teaching Fellow, Smith College.

VISHNIAC, WOLF, Student, Brooklyn College.

WILLIAMSON. FRANCES ALICE, Student, New Jersey College for Women.

WILLIS, MARIAN, Student, Iowa State College.

PHYSIOLOGY

BERNSTEIN, JEANE, Graduate Student, New York University.

CARSON, GWENETH, Student, University of Toronto.

CREGAR, MARY, Demonstrator in Physiology, Bryn Mawr College.

KEISTER, MARGARET LOUISE, Instructor in Zoology, Wheaton College.

MCLEAN, DOROTHY JUANITA, Graduate Student, University of Toronto.

PARTRIDGE, JUDITH ANN, Assistant in Physiology, Vassar College.

PEPPER, BILLIE BARBARA, Student, Radcliffe College.

REICH, EVA, Student, Barnard College.

TAYLOR, BABETTE, Student, Washington University.

THERIEN, MERCEDES, Assistant Research, Montreal University.

ZOOLOGY

AUSTIN, JANE, Student, Randolph-Macon Woman's College.

BANKS, MARY ELIZABETH, Research Assistant, Washington University.

BARROWS, SHIRLEY LOUISE, Student, University of Rochester.

BENSON, ELEANORE BIE, Student, University of Pennsylvania.

BUTT, FERDINAND H., Instructor, Cornell University.

CALKINS, JANET ELIZABETH MORSE, University of Chicago.

CONRAVEY, JUNE ROSE, Student Assistant, Newcomb College, Tulane University.

DEREVERE, JOAN BROOKS, Undergraduate Student, Wilson College.

DOUGLIS, MARJORIE B., Assistant in Zoology, Chicago University.

DUNBAR, SALLY, De Pauw University.

FAIRFIELD, JANET, Student, Russell Sage College.

FALKNER, ETTA, Instructor, American Museum of Natural History.

FOGERSON, VIRGINIA LEE, Drury College.

GOSFORD, BARBARA, Duke University.

HABERT, YVONNE A., High School Teacher, City of Boston.

KOOPMAN, KARL FRIEDRICH, Graduate Student, Columbia University.

LANGMAN, IDA K., University of Pennsylvania.

LAUTHERS, ROSEMARY ANN, Student, Oberlin College.

LEDUC, ELIZABETH HORTENSE, Graduate Assistant, Wellesley College.

LLOYD, MARY REMSEN, Vassar College.

MARKS, MILDRED HELEN, Graduate Student, University of Pennsylvania.

McCLiNTOCK, MARY, Instructor, Bemidji, Minnesota.

NEAL, LUCY LEE, Drury College.

RANDALL, NANCY Lois, Student, Swarthmore College.

REESE, JEAN, Goucher College.

ROOT, OSCAR M., Instructor, Brooks School.

ROTH, OWEN HAROLD, Instructor in Biology, St. Vincent College.

SCHMID, LEO A., Baltimore, Md.

SLAVIN, ALICE CECILIA, Student, Seton Hill College.

SOUTHWELL, VIOLET M., Student, Wilson College.

STEENBURG, ISABELLA, Student, Vassar College.

STEKL, ELEANOR B., Science Teacher, N. Tonawanda High School.

SWEENEY, PATRICIA GEORGIA, Student, Oberlin College.

VAN GEYT, VIRGINIA, Student, University of Rochester.

VIOSCA, MIRIAM A., Student Assistant, Newcomb College, Tulane University.

WELLER, DORIS A., Undergraduate, Radcliffe College.

WARNER, ROSE ELLA, Teacher of Biology, Frick Educational Commission.

MARINE BIOLOGICAL LABORATORY

6. TABULAR VIEW OF ATTENDANCE

1940 1941 1942 1943 1944

INVESTIGATORS — Total 386 337

Independent 253 197

Under instruction 62 59

Library readers 31

Research assistants 71 50

STUDENTS— Total 128 131

Zoology 55 55

Protozoology (not given after 1940) 7

Embryology 34 37

Physiology 22 24

Botany 10 15

TOTAL ATTENDANCE 514 468

Less persons registered as both students and investi-
gators 7 7

507 461

INSTITUTIONS REPRESENTED — Total 148 144

By investigators 112 102

By students 79 72

SCHOOLS AND ACADEMIES REPRESENTED

By investigators 1 5

By students 2 2

FOREIGN INSTITUTIONS REPRESENTED

By investigators 2 3

By students . 1 1

7. SUBSCRIBING AND COOPERATING INSTITUTIONS

1944

201

160

132

275

228

273

222

126

116

—

193
112
11
50
20
75
37

276

275

106

1
2

Amherst College

Barnard College

Bowdoin College

Brooklyn College

Bryn Mawr College

Catholic University of America

Columbia University

Cornell University

Cornell University Medical College

Duke University

Fish and Wild Life Service, U. S. Dept. of the

Interior

Fordham University

Henry C. Frick Educational Commission
Goucher College
Harvard University
Hunter College
Industrial and Engineering Chemistry, of the

American Chemical Society
Johns Hopkins University
The Lankenau Hospital Research Institute
Eli Lilly and Co.

Massachusetts Department of Health
Massachusetts Institute of Technology
Mount Holyoke College

H. Sophie Newcomb College

New York University

New York University College of Medicine

New York University Washington Square

College

Oberlin College
Ohio State University
Princeton University
Radcliffe College

Rockefeller Institute for Medical Research
Russell Sage College
St. Joseph College for Women
Smith College
State University of Iowa
Syracuse University
Syracuse University Medical School
Temple University
Tufts College
University of Chicago
University of Cincinnati
University of Illinois
University of Maryland Medical School
University of Pennsylvania
University of Pennsylvania School of Medicine
University of Rochester

REPORT OF THE DIRECTOR 47

Vassar College Western Reserve University

Villanova College Wheaton College

Washington University Wilson College

Wayne University Wistar Institute

Wellesley College Woods Hole Oceanographic Institution

Wesleyan University Yale University

8. EVENING LECTURES, 1944

Friday, June 30

DR. T. H. BISSONNETTE "Some Recent Studies on Photoperiodicity

in Animals, particularly Fur-bearers."
Friday, July 7

DR. ETHEL BROWNE HARVEY "Some Results of Centrifuging the Arbacia

Egg."
Friday, July 14

DR. A. C. GIESE "Ultraviolet Radiations and the Life Activi-
ties of Cells."
Friday, July 21

DR. H. J. MULLER "Evidence for the Meticulousness of Adap-
tation."
Friday, July 28

DR. CARL C. SPEIDEL "Experimental Studies of Special Sensory

Organs and Nerves."
Thursday, August 3

DR. ERNEST CARROLL FAUST "Problems of Tropical Medicine in the

United States."
Friday, August 4

DR. A. K. PARPART "Blood Preservation : A Problem in Cellu-
lar Physiology."
Thursday, August 10

MR. G. G. LOWER "Local Invertebrates."

Friday, August 1 1

DR. W. C. ALLEE "Social Orders Among Vertebrates."

Wednesday, August 16

DR. RALPH TURNER "Rehabilitation of Scientific Institutions in

Devastated Europe."
Friday, August 18

DR. A. W. POLLISTER "The Centriole Problem."

Friday, August 25

PROF. G. H. PARKER "Animal Coloration, Fixed and Changeable."

9. SHORTER SCIENTIFIC PAPERS, 1944

Tuesday, July 18

DR. B. H. WILLIER "Melanophore Control of Sexual Dimor-
phism in Feather Pigmentation of the
Barred Rock Fowl."

DR. VIKTOR HAMBURGER "The Effects of Peripheral Factors on

Motor Neuron Differentiation in the Chick
Embryo."

DR. W. H. LEWIS "The Superficial Gel Layer and Its Role in

Development."

48 MARINE BIOLOGICAL LABORATORY

Tuesday, July 25

DR. LEONOR MICHAELIS "Ferritin and Iron Metabolism."

DR. ARNOLD LAZAROW "The Chemical Organization of the Cyto-
plasm of the Liver Cell."
DR. LEONOR MICHAELIS "Theory of Metachromatic Staining."

Tuesday, August 1

DR. DOROTHY WRINCH "The Native Protein in Crystalline Form."

DR. OTTO MYERHOF "The Role of Adenylpyro-Phosphatase in

Alcoholic Fermentation of Yeast."
DR. ERNEST SCHARRER "The Naples Station Still Lives."

Tuesday, August 8

DR. A. M. SHANES "Application of Bio-electricity to the Study

of Functioning in Nerve."

DR. DAVID NACHMANSOHN "On the Energy Source of the Nerve Ac-
tion Potential."

DR. T. H. BULLOCK "Oscillographic Studies on the Giant Nerve

Fiber System in Lumbricus."

DR. PAUL WEISS "Evidence for the Perpetual Proximo-distal

Growth of Nerve Fibers."
Tuesday, August 15

DR. L. V. HEILBRUNN "A Toxic Substance from Protoplasm."

DR. D. L. HARRIS

DR. P. G. LEFEVRE

DR. W. H. PRICE

DR. W. L. WILSON

DR. A. A. WOODWARD, JR.

DR. D. L. HARRIS "The Chemical Nature of a Toxic Substance

DR. W. H. PRICE from Protoplasm."

DR. L. V. HEILBRUNN

DR. G. 'I. LAVIN "Recent Developments in Ultraviolet Mi-
croscopy."
Tuesday, August 22

DR. B. W. ZWEIFACH "The Peripheral Circulation in Traumatic

Shock."

DR. W. R. AMBERSON "Recent Experience with Hemoglobin-saline

Solutions."

DR. R. G. ABELL "Gelatin as a Plasma Substitute."

DR. W. M. PARKINS

Thursday, August 24

DR. C. A. BERGER "Experimental Studies on the Cytology of

Allium."

DR. VALY MENKIN "Studies on the Chemical Basis of Fever."

DR. MIRIAM F. MENKIN "In Vitro Fertilization of Human Ova."

Tuesday, August 29

DR. L. M. BERTHOLF "Studies on Metamorphosis in the Tunicate."

E. MORTENSEN "Behavior and Tube Building Habits of

DR. P. S. GALTSOFF Polydora ligni."

DR. J. B. BUCK "The Click Mechanism of Elaterid Beetles."

REPORT OF THE DIRECTOR 49

Thursday, August 31

DR. G. K. SMELSER "Orbital Changes in Experimental Exoph-

thalmos."

DR. A. GORBMAN "Radioactive Iodine Absorption in Lower

Chordates and the Problem of Homology
of the Thyroid Gland."

DR. D. L. HARRIS "Phosphoprotein Phosphatase, a New En-
zyme from the Frog Egg."

10. MEMBERS OF THE CORPORATION, 1944

1. LIFE MEMBERS

ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France.

ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Maryland.

BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York.

BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts.

CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania.

COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin.

CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey.

COWDRY, DR. E. V., Washington University, St. Louis, Missouri.

EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Massachusetts.

FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France.

JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts.

JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts.

KING, MR. CHAS. A.

KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York.

LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland.

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

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

MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pa.

MORGAN, MRS. T. H., Pasadena, California.

MORGAN, PROF. T. H., Director of Biological Laboratory, California Institute of

Technology, Pasadena, California.
NOYES, Miss EVA J.

PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania.
SCOTT, DR. ERNEST L., Columbia University, New York City, New York.
SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts.
SHEDD, MR. E. A.
THORNDIKE, DR. EDWARD L., Teachers College, Columbia University, New York

City, New York.

TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, New York.
TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois.
WAITE, PROF. F. C., 144 Locust Street, Dover, New Hampshire.
WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California.

2. REGULAR MEMBERS

ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts.
ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia,
Pennsylvania.

50 MARINE BIOLOGICAL LABORATORY

ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New
York.

ALBAUM, DR. HARRY G., 3115 Avenue I, Brooklyn, New York.

ALBERT, DR. ALEXANDER, Biological Laboratories, Harvard University, Cambridge,
Massachusetts.

ALLEE, DR. W. C., The University of Chicago, Chicago, Illinois.

AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland,
School of Medicine, Lombard and Greene Streets, Baltimore, Maryland.

ANDERSON, DR. RUBERT S., University of Maryland School of Medicine, Depart-
ment of Physiology, Baltimore, Maryland.

ANDERSON, DR. T. F., University of Pennsylvania, Philadelphia. Pennsylvania.

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

AUSTIN, DR. MARY L., Wellesley College, Wellesley, Massachusetts.

BAITSELL, DR. GEORGE A., Yale University, New Haven, Connecticut.

BAKER, DR. H. B., Zoological Laboratory, University of Pennsylvania, Philadelphia,
Pennsylvania.

BALLARD, DR. WILLIAM W., Dartmouth College, Hanover, New Hampshire.

BALLENTINE, DR. ROBERT, Columbia University, Department of Zoology, New York
City, New York.

BALL, DR. ERIC G., Department of Biological Chemistry, Harvard University Medi-
cal School, Boston, Massachusetts.

BARD, PROF. PHILIP, Johns Hopkins Medical School, Baltimore, Maryland.

BARRON, DR. E. S. GUZMAN, Department of Medicine, The University of Chicago,
Chicago, Illinois.

EARTH, DR. L. G., Department of Zoology, Columbia University, New York City,
New York.

BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana,
Illinois.

BEADLE, DR. G. W., School of Biological Sciences, Stanford University, California.

BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa
City, Iowa.

BECK, DR. L. V., Hahnemann Medical College, Philadelphia, Pennsylvania.

BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana.

BERTHOLF, DR. LLOYD M., Western Maryland College, Westminster, Maryland.

BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts.

BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Massa-
chusetts.

BINFORD, PROF. RAYMOND, Guilford College, North Carolina.

BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut.

BLANCHARD, PROF. K. C., Johns Hopkins Medical School, Baltimore, Maryland.

BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City,
Iowa.

BORING, DR. ALICE M., Yenching University, Peking, China.

BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wisconsin.

BRODIE, MR. DONALD M., 522 Fifth Avenue, New York City, New York.

BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Washington Uni-
versity Medicaf School, St. Louis, Missouri.

REPORT OF THE DIRECTOR 51

BROOKS, DR. MATILDA M., University of California, Department of Zoology, Berke-
ley, California.

BROOKS, DR. S. C, University of California, Berkeley, California.

BROWN, DR. DUGALD E. S., New York University, College of Dentistry, 209 East
23d Street, New York City, New York.

BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern University,
Evanston, Illinois.

BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.

BUCK, DR. JOHN B., Department of Zoology, University of Rochester, Rochester,
New York.

BUDINGTON, PROF. R. A., Winter Park, Florida.

BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.

BURBANCK, DR. WILLIAM D., Department of Biology, Drury College, Springfield,
Missouri.

BURKENROAD, DR. M. D., Yale University, New Haven, Connecticut.

BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Pennsylvania.

CANNAN, PROF. R. K., New York University College of Medicine, 477 First Ave-
nue, New York City, New York.

CARLSON, PROF. A. J., Department of Physiology, The University of Chicago, Chi-
cago, Illinois.

CAROTHERS, DR. E. ELEANOR, 134 Avenue C. East, Kingman, Kansas.

CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massachusetts.

CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster, Pennsyl-
vania.

CARVER, PROF. GAIL L., Mercer University, Macon, Georgia.

CATTELL, DR. McKEEN, Cornell University Medical College. 1300 York Avenue,
New York City, New York.

CATTELL, MR. WARE, 3609 Military Road, N. W., Washington, D. C.

CHAMBERS, DR. ROBERT, Washington Square College, New York University, Wash-
ington Square, New York City, New York.

CHASE, DR. AURIN M., Princeton University, Princeton, New Jersey.

CHENEY, DR. RALPH H., Biology Department, Long Island University, Brooklyn,
New York.

CHIDESTER, PROF. F. E., Auburndale, Massachusetts.

CHILD, PROF. C. M., Jordan Hall, Stanford University, California.

CHURNEY, LT. LEON, 28th Alt. Tng. Unit, HAAF, Harlingen, Texas.

CLAFF, MR. C. LLOYD, Department of Biology, Brown University, Providence,
Rhode Island.

CLARK, PROF. E. R., University of Pennsylvania Medical School, Philadelphia,
Pennsylvania.

CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New
York.

CLARKE, DR. G. L., Woods Hole Oceanographic Institution, Woods Hole, Mas-
sachusetts.

CLELAND, PROF. RALPH E., Indiana University, Bloomington, Indiana.

CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indian; .

COE, PROF. W. R., Yale University, New Haven, Connecticut.

COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts.

52 MARINE BIOLOGICAL LABORATORY

COLE, DR. ELBERT C., Department of Biology, Williams College, Williamstown,
Massachusetts.

COLE, DR. KENNETH S., University of Chicago, Chicago, Illinois.

COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio.

COLTON, PROF. H. S., Box 601, Flagstaff, Arizona.

COOPER, DR. KENNETH W., Department of Biology, Princeton University, Prince-
ton, New Jersey.

CQPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine.

COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina,
Chapel Hill, North Carolina.

COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro-
lina, Chapel Hill, North Carolina.

CRAMPTON, PROF. H. E., American Museum of Natural History, New York City,
New York.

CRANE, JOHN O., Woods Hole, Massachusetts.

CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts.

CROASDALE, HANNAH T., Dartmouth College, Hanover, New Hampshire.

CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio.

CURTIS, DR. MAYNIE R., 377 Dexter Trail, Mason, Michigan.

CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri.

DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan.

DAVIS, DR. DONALD 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, New
York.

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

DEMEREC, DR. M., Carnegie Institution of Washington, Cold Spring Harbor, Long
Island, New York.

DILLER, DR. WILLIAM F., 1016 South 45th Street, Philadelphia, Pennsylvania.

DODDS, PROF. G. S., Medical School, University of West Virginia, Morgantown,
West Virginia.

DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York.

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

DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York Avenue,
New York City, New York.

DUGGAR, DR. BENJAMIN M., c/o Lederle Laboratories Inc., Pearl River, New
York.

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

DURYEE, DR. WILLIAM R., Surgeon General's Office, Washington, D. C.

EDWARDS, DR. D. J., Cornell University Medical College, 1300 York Avenue, New
York City, New York.

ELLIS, DR. F. W., Monson, Massachusetts.

EVANS, DR. TITUS C., College of Physicians and Surgeons, 630 West 168th Street,
New York City, New York.

FAILLA, DR. G., College of Physicians and Surgeons, 630 West 168th Street, New
York City, New York.

REPORT OF THE DIRECTOR 53

FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France.

FAUST, DR. ERNEST C., Tulane University of Louisiana, New Orleans, Louisiana.

FERGUSON, DR. JAMES K. W., Department of Pharmacology, University of Toronto,
Ontario, Canada.

FIGGE, DR. F. H. J., 4636 Schenley Road, Baltimore, Maryland.

FISCHER, DR. ERNST, Department of Physiology, Medical College of Virginia, Rich-
mond, Virginia.

FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, To-
ronto, Canada.

FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto,
Canada.

FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachu-
setts.

FRISCH, DR. JOHN A., Canisius College, Buffalo, New York.

FURTH, DR. JACOB, Cornell University Medical College, 1300 York Avenue, New
York City, New York.

GAGE, PROF. S. H., Lock Box 70, Interlaken, New York.

GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Mary-
land.

GARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee.

GATES, DR. REGINALD R., Woods Hole, Massachusetts.

GEISER, DR. S. W., Southern Methodist University, Dallas, Texas.

GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois.

GLASER, PROF. O. C., Amherst College, Amherst, Massachusetts.

GOLDFORB, PROF. A. J., College of the City of New York, Convent Avenue and 139th
Street, New York City, New York.

GOODCHILD, DR. CHAUNCEY G., State Teachers College, Springfield, Missouri.

GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut.

GOTTSCHALL, DR. GERTRUDE Y., 1630 Rhode Island Avenue, N.W., Washington,
D. C.

GRAHAM, DR. J. Y., Roberts, Wisconsin.

GRAND, CONSTANTINE G., Biology Department, Washington Square College, New
York University, Washington Square, New York City, New York.

GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana.

GRAY, PROF. IRVING E., Duke University, Durham, North Carolina.

GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City,
New York.

GUDERNATSCH, DR. J. FREDERICK, New York University, 100 Washington Square,
New York City, New York.

GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri.

GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin.

HAGUE, DR. FLORENCE, Sweet Briar College, Sweet Briar, Virginia.

HALL, PROF. FRANK G., Duke University, Durham, North Carolina.

HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St.
Louis, Missouri.

HANCE, DR. ROBERT T., The Cincinnati Milling Machine Co., Cincinnati 9, Ohio.

HARGITT, PROF. GEORGE T., Department of Zoology, Duke University, Durham,
North Carolina.

54 MARINE BIOLOGICAL LABORATORY

HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kansas.

HARNLY, DR. MORRIS H., Washington Square College, New York University, New
York City, New York.

HARPER, PROF. R. A., R. No. 5, Bedford, Virginia.

HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut.

HARTLINE, DR. H. KEFFER, University of Pennsylvania, Philadelphia, Pennsylvania.

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

HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jer-
sey.

HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey.

HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachusetts.

HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University, Halifax,
Nova Scotia.

HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts.

HECHT, DR. SELIG, Columbia University, New York City, New York.

HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.

HENDEE, DR. ESTHER CRISSEY, Russell Sage College, Troy, New York.

HENSHAW, DR. PAUL S., National Cancer Institute, Bethesda, Maryland.

HESS, PROF. WALTER N., Hamilton College, Clinton, New York.

HIATT, DR. E. P., New York University, 100 Washington Square, New York City,
New York.

HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio.

HILL, DR. SAMUEL E., Department of Biology, Russell Sage College, Troy, New
York.

HINRICHS, DR. MARIE, Department of Physiology and Health Education, Southern
Illinois Normal University, Carbondale, Illinois.

HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts.

HOADLEY, DR. LEIGH, Harvard University, Cambridge, Massachusetts.

HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Pennsylvania.

HODGE, DR. CHARLES, IV, Temple University, Department of Zoology, Philadelphia,
Pennsylvania.

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

HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Laboratory of In-
dustrial Hygiene, Bethesda, Maryland.

HOPKINS, DR. DWIGHT L., Mundelein College, 6363 Sheridan Road, Chicago, Illi-
nois.

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

HOWLAND, DR. RUTH B., Washington Square College, New York University,
Washington Square East, New York City, New York.

HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia.

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

IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsylvania.

ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts.

REPORT OF THE DIRECTOR 55

JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsylvania, Phila-
delphia, Pennsylvania.

JENKINS, DR. GEORGE B., 30 Gallatin Street, N.W., Washington, D. C.

JENNINGS, PROF. H. S., Department of Zoology, University of California, Los An-
geles, California.

JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville, Tennessee.

JONES, DR. E. RUFFIN, JR., College of William and Mary, Wllliamsburg, Virginia.

KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor, Long Island,
New York.

KEMPTON, PROF. RUDOLF T., Vassar College, Poughkeepsie, New York.

KIDDER, DR. GEORGE W., Brown University, Providence, Rhode Island.

KILLE, DR. FRANK R., Swarthmore College, Swarthmore, Pennsylvania.

KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia.

KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, 36th Street and
Woodland Avenue, Philadelphia, Pennsylvania,

KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa.

KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York.

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

KORR, DR. I. M., Department of Physiology, New York University, College of Medi-
cine, 477 First Avenue, New York City, New York.

KRAHL, DR. M. E., College of Physicians and Surgeons, 630 West 168th Street,
New York 32, New York.

KRIEG, DR. WENDELL J. S., 303 East Chicago Ave., Chicago, Illinois.

LANCEFIELD, DR. D. E., Queens College, Flushing, New York.

LANCEFIELD, DR. REBECCA C., Rockefeller Institute, 66th Street and York Avenue,
New York City, New York.

LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.

LAVIN, DR. GEORGE L, Rockefeller Institute, 66th Street and York Avenue, New
York City, New York.

LEWIS, PROF. I. F., University of Virginia, Charlottesville, Virginia.

LILLIE, PROF. FRANK R., The University of Chicago, Chicago, Illinois.

LILLIE, PROF. RALPH S., The University of Chicago, Chicago, Illinois.

LITTLE, DR. E. P., Phillips Exeter Academy, Exeter, New Hampshire.

LOCH HEAD, DR. JOHN H., Department of Zoology, University of Vermont, Bur-
lington, Vermont.

LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri.

LOEWI, PROF. OTTO, 155 East 93d Street, New York City, New York.

LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York
City, New York.

LUCAS, DR. ALFRED M., Regional Poultry Research Laboratory, East Lansing,
Michigan.

LUCKE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Pennsylvania.

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

LYNCH, DR. RUTH STOCKING, Maryland State Teachers College, Towson, Mary-
land.

56 MARINE BIOLOGICAL LABORATORY

LYNN, DR. WILLIAM G., Department of Biology, The Catholic University of Amer-
ica, Washington, D. C.

MACDOUGALL, DR. MARY S., Agnes Scott College, Decattir, Georgia.

MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa-
chusetts.

McCLUNG, PROF. C. E., 417 Harvard Avenue, Swarthmore, Pennsylvania.

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

MCGREGOR, DR. J. H., Columbia University, New York City, New York.

MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario,
London, Canada.

MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Bos-
ton, Massachusetts.

MALONE, PROF. E. F., College of Medicine, University of Cincinnati, Department
of Anatomy, Cincinnati, Ohio.

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

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

MARTIN, PROF. E. A., Department of Biology, Brooklyn College, Bedford Avenue
and Avenue H, Brooklyn, New York.

MAST, PROF. S. O., Johns Hopkins University, Baltimore, Maryland.

MATHEWS, PROF. A. P., Woods Hole, Massachusetts.

MATTHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College,
Williamstown, Massachusetts.

MAVOR, PROF. JAMES W., Union College, Schenectady, New York.

MAZIA, DR. DANIEL, Department of Zoology, Gowen Field, Boise, Idaho.

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

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

MENKIN, DR. VALY, Duke University, School of Medicine, Durham, North Caro-
lina.

METZ, PROF. CHARLES W., University of Pennsylvania, Philadelphia, Pennsylvania.

MICHAELIS, DR. LEONOR, Rockefeller Institute, 66th Street and York Avenue, New
York City, New York.

MILLER, DR. J. A., Division of Anatomy, College of Medicine, University of Ten-
nessee, Memphis, Tennessee.

MINNICH, PROF. D. E., Department of Zoology, University of Minnesota, Minne-
apolis, Minnesota.

MITCHELL, DR. PHILIP H., Brown University, Providence, Rhode Island.

MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois.

MORGAN, DR. ISABEL M., Poleomyelitis Research Center, 1901 E. Madison Street,
Baltimore 5, Maryland.

MORGULIS, DR. SERGIUS, University of Nebraska, Omaha, Nebraska.

MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue,
New York City, New York.

MULLER, PROF. H. J., Amherst College, Amherst, Massachusetts.

NACHMANSOHN, DR. D., College of Physicians and Surgeons, 630 W. 168th Street,
New York City, New York.

REPORT OF THE DIRECTOR 57

NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Milton, Massa-
chusetts.

NEWMAN, PROF. H. H., 173 Devon Drive, Clearwater, Florida.

NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania.

NONIDEZ, DR. JOSE F., Cornell University Medical College, 1300 York Avenue,
New York City, New York.

NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey.

OCHOA, DR. SEVERO, New York University, College of Medicine, 477 First Avenue,
New York 16, New York.

OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn
Mawr, Pennsylvania.

OSBURN, PROF. R. C., Ohip State University, Columbus, Ohio.

OSTERHOUT, PROF. W. J. V., Rockefeller Institute, 66th Street and York Avenue,
New York City, New York.

OSTERHOUT, MRS. MARIAN IRWIN, Rockefeller Institute, 66th Street and York
Avenue, New York City, New York.

PACKARD, DR. CHARLES, Marine Biological Laboratory, Woods Hole, Massachu-
setts.

PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio.

PAPPENHEIMER, DR. A. M., Columbia University, New York City, New York.

PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts.

PARMENTER, DR. C. L., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.

PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jersey.

PATTEN, DR. BRADLEY M., University of Michigan Medical School, Ann Arbor,
Michigan.

PAYNE, PROF. F., University of Indiana, Bloomington, Indiana.

PEEBLES, PROF. FLORENCE, Lewis and Clark College, Portland, Oregon.

PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York.

PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin.

PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts.

POLLISTER, DR. A. W., Columbia University, New York City, New York.

POND, DR. SAMUEL E., 1203 Enfield Street, Thompsonville, Connecticut.

PRATT, DR. FREDERICK H., Boston University, School of Medicine, Boston, Massa-
chusetts.

PROSSER, DR. C. LADD, University of Chicago, Chicago, Illinois.

RAND, DR. HERBERT W., Harvard University, Cambridge, Massachusetts.

RANKIN, DR. JOHN S., Zoology Department, University of Connecticut, Storrs,
Connecticut.

REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massachusetts.

RENN, DR. CHARLES E., Harvard University, Cambridge, Massachusetts. •

RENSHAW, PROF. BIRDSEY, Rockefeller Institute for Medical Research, 66th Street
and York Avenue, New York City, New York.

DERENYI, DR. GEORGE S., Department of Anatomy, University of Pennsylvania,
Philadelphia, Pennsylvania.

REZNIKOFF, DR. PAUL, Cornell University Medical College, 1300 York Avenue,
New York City, New York.

RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio.

58 MARINE BIOLOGICAL LABORATORY

RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma.

RICHARDS, PROF. A. G., Department of Zoology, University of Pennsylvania, Phila-
delphia, Pennsylvania.

RICHARDS, DR. O. W., Research Department, Spencer Lens Company, 19 Doat
Street, Buffalo, New York.

RIGGS, LAWRASON, JR., 120 Broadway, New York City, New York.

ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio.

ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York.

ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts.

ROOT, DR. R. W., Department of Biology, College of the City of New York, Con-
vent Avenue and 139th Street, New York City, New York.

ROOT, DR. W. S., College of Physicians and Surgeons, Department of Physiology,
630 West 168th Street, New York City, New York.

RUEBUSH, DR. T. K., Dayton, Virginia.

RUGH, DR. ROBERTS, Department of Biology, Washington Square College, New
York University, New York City, New York.

SASLOW, DR. GEORGE, Washington University Medical School, St. Louis, Missouri.

SAUNDERS, LAURENCE, President, Saunders Publishing Company, Philadelphia,
Pennsylvania.

SAYLES, DR. LEONARD P., Department of Biology, College of the City of New York,
139th Street and Convent Avenue, New York City, New York.

SCHAEFFER, DR. ASA A., Biology Department, Temple University, Philadelphia,
Pennsylvania.

SCHARRER, DR. ERNST A., Western Reserve University, School of Medicine, 2109
Adelbert Road, Cleveland 6, Ohio.

SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street and Con-
vent Avenue, New York City, New York.

SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.

SCHMITT, PROF. F. O., Department of Biology and Public Health, Massachusetts
Institute of Technology, Cambridge, Massachusetts.

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

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

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

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

SCOTT, DR. ALLAN C, Union College, Schenectady, New York.

SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey.

SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College, Greens-
burg, Pennsylvania.

SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York.

SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and
Surgeons, 630 West 168th Street, New York City, New York.

SHANES, DR. ABRAHAM M., New York University, College of Dentistry, New
York.

SHAPIRO, DR. HERBERT, Radiation Laboratory, Massachusetts Institute of Technol-
ogy, Cambridge, Massachusetts.

REPORT OF THE DIRECTOR 59

SHELFORD, PROF. V. E., Vivarium, Wright and Healey Streets, Champaign, Illinois.

SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan.

SHUMWAY, DR. WALDO, University of Illinois, Urbana, Illinois.

SICHEL, DR. FERDINAND J. M., University of Vermont, Burlington, Vermont.

SICHEL, MRS. F. J. M., 35 Henderson Terrace, Burlington, Vermont.

SINNOTT, DR. E. W., Osborn Botanical Laboratory, Yale University, New Haven,
Connecticut.

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

SMITH, DR. DIETRICH CONRAD, Department of Physiology, University of Mary-
land School of Medicine, Lombard and Greene Streets, Baltimore, Maryland.

SNYDER, PROF. L. H., Ohio State University, Department of Zoology, Columbus,
Ohio.

SOLLMAN. DR. TORALD, Western Reserve University, Cleveland, Ohio.

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

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

STARK, DR. MARY B., 1 East 105th Street, New York City, New York.

STEINBACH, DR. H. BURR, Department of Zoology, Washington University, St.
Louis, Missouri.

STERN, DR. CURT, Department of Zoology, University of Rochester, Rochester,
New York.

STERN, DR. KURT G., Polytechnic Institute, Department of Chemistry, 85 Living-
ston Street, Brooklyn, New York.

STEWART, DR. DOROTHY R., University of Pennsylvania Medical School, Depart-
ment of Physiology, Philadelphia 4, Pennsylvania.

STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts.

STRONG, PROF. O. S., College of Physicians and Surgeons, Columbia University,
New York City, New York.

STUNKARD, DR. HORACE W., New York University, University Heights, New
York.

STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena,
California.

SUMMERS, DR. FRANCIS MARION, R.F.D. Route 2, Box 507-A, Dinuba, California.

TAFT, DR. CHARLES H., JR., University of Texas Medical School, Galveston, Texas.

TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio.

TAYLOR, DR. C. V., Leland Stanford University, Leland Stanford, California.

TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michigan.

TEWINKEL, DR. L. E., Department of Zoology, Smith College, Northampton,
Massachusetts.

TURNER, DR. ABBY H., Wilson College, Chambersburg, Pennsylvania.

TURNER, PROF. C. L., Northwestern University, Evanston, Illinois.

TYLER, DR. ALBERT, California Institute of Technology, Pasadena, California.

UHLENHUTH, DR. EDUARD, University of Maryland, School of Medicine, Balti-
more, Maryland.

VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, Ohio.

60 MARINE BIOLOGICAL LABORATORY

WALD, DR. GEORGE, Biological Laboratories, . Harvard University, Cambridge,

Massachusetts.

WARBASSE, DR. JAMES P., Woods Hole, Massachusetts.
WARD, PROF. HENRY B., 1201 W. Nevada, Urbana, Illinois.

WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Pennsylvania.
WATERMAN, DR. ALLYN J., Department of Biology, Williams College, Williams-
town, Massachusetts.
WEISS, DR. PAUL A., Department of Zoology, The University of Chicago, Chicago,

Illinois.

WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania.
WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North Dakota.
WHITAKER, DR. DOUGLAS M., P. O. Box 2514, Stanford University, California.
WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania.
WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania,

Philadelphia, Pennsylvania.

WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Nebraska.
WICHTERMAN, DR. RALPH, Biology Department, Temple University, Philadelphia,

Pennsylvania.

WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio.
WILLIER, DR. B. H., Department of Biology, Johns Hopkins University, Baltimore,

Maryland.

WILSON, DR. J. W., Brown University, Providence, Rhode Island.
WITSCHI, PROF. EMIL, Department of Zoology, State University of Iowa, Iowa

City, Iowa.
WOLF, DR. ERNST, Biological Laboratories, Harvard University, Cambridge,

Massachusetts.

WOODRUFF, PROF. L. L., Yale University, New Haven, Connecticut.
WOODWARD, DR. ALVALYN E., Zoology Department, University of Michigan, Ann

Arbor, Michigan.

WRINCH, DR. DOROTHY, Smith College, Northampton, Massachusetts.
YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medical College,

1300 York Avenue, New York City, New York.
YOUNG, DR. B. P., Cornell University, Ithaca, New York.
YOUNG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland.

DOMINANT LETHALITY AND CORRELATED CHROMOSOME

EFFECTS IN HABROBRACON EGGS X-RAYED IN

DIPLOTENE AND IN LATE METAPHASE I x

ANNA R. WHITING
University of Pennsylvania

INTRODUCTION

If oviposition is prevented in well-fed females of the parasitic wasp Habrobracon
by witholding them from their host they continue to produce mature eggs until the
egg sacs are filled. These stored eggs may number as many as twenty per female
and are in late metaphase of the first meiotic division (metaphase I). Their re-
tention in this stage for four days has no effect on their hatchability which is 96
per cent in the wild type stock used for the experiments herein described.

When unmated females with stored eggs are x-rayed and allowed to oviposit
at 30° C. all eggs laid during the first six hours after treatment will have been
irradiated in late metaphase I. Eggs laid during the seventh and eighth hours
after treatment consist of a variable mixture treated in metaphase I and in late
diplotene (including all eggs in diakinesis) and are, therefore, of no use in the
present study. Eggs laid during the ninth to twelfth hours after treatment will
have been post-synaptic with their diffuse chromosomes in a relatively quiescent
condition when irradiated. These are designated as late and early diplotene.

An advantage in the use of these eggs for the detection of injuries lies in the
fact that they develop parthenogenetically if unfertilized and so indicate directly
the effects of treatment on a haploid set of chromosomes. Disadvantages are the
large number (;; == 10) and small size (less than 1 /j. in diameter) of their chromo-
somes. The details of oogenesis appear to be orthodox and so the results should
be universally applicable to forms with comparable type of meiosis. Failure to
hatch and cytological changes in stages immediately following treatment have been
the criteria of injury. Preliminary results were first published in 1938 (Whiting,
1938). Details of technique and hatchability effects, as well as extensive bibliog-
raphy, are given elsewhere (Whiting, 1945) ; cytological effects and their correla-
tion with mortality and dose are presented here in detail.

DOSE-HATCHABILITY RELATIONSHIPS

Hatchability effects may be summarized briefly. No correction for control
hatchability is made since it is so close to 100 per cent. Eggs x-rayed in diplotene

1 This investigation has been aided by a grant (to P. W. Whiting) from the Rockefeller
Foundation, for apparatus and technical assistance. The work was done at the Zoological Lab-
oratory of the University of Pennsylvania and at the Marine Biological Laboratory, Woods
Hole, Massachusetts. To these institutions the author is grateful for the use of laboratory
facilities and of x-ray equipment. Valuable assistance was also given by the American Onco-
logic Hospital of Philadelphia through the use of x-ray equipment.

ANNA R. WHITING

and allowed to develop parthenogenetically give a dose-hatchability curve which
appears to be linear at low doses and to become "mixed" at high doses ; they have
50 per cent mortality at 1 2,000 r and 100 per cent at about 45 ,000 r; they showed
no significant change in hatchability in preliminary and inadequate tests of time-
intensity differences. Those treated in early diplotene (laid during the eleventh
and twelfth hours after treatment) show no change in hatchability at any dose with
fractionated treatment, those treated in late diplotene (laid during the ninth and
tenth hours after treatment) show a significant increase at high doses with frac-
tionated treatment. The dose-hatchability curve for combined diplotene owes much
of its mixed character at high doses to late diplotene, early diplotene response being
more nearly linear.

Eggs x-rayed in late metaphase I and allowed to develop parthenogenetically
show a linear decline in hatchability with increasing dose and have 50 per cent
mortality at 375 r, 100 per cent at about l,400r; they show no change in dose-
hatchability relationships with aging between treatment and oviposition, time-in-
tensity differences or fractionation of dose.

When Habrobracon females are mated, about two-thirds of the eggs are fer-
tilized. If treated females are mated to untreated males, the survival of any ap-
preciable number of eggs through the aid of normal spermatozoa would increase
percentage of hatchability thereby indicating the presence of recessive lethal effects
by comparison with hatchability of eggs from treated unmated females. Table I

TABLE I

Hatchability percentages for eggs of treated females, unmated and
mated to untreated males

Stage treated

Dose in
r units

Unmated females

Mated females

Number
of eggs

Hatchability
percentage

Number
of eggs

Hatchability
percentage

Metaphase I

560

319

39.8±2.7

318

40.5±2.7

Prophase I

5,600
22,400

137
100

71.5±2.6
19.0±3.9

126

182

70.6±4.0
19.2±2.9

Controls

127

98.4±1.1

363

98.6±0.6

demonstrates that most, if not all, of the lethal effects induced in these stages by
x-rays are dominant, at least in respect to hatchability. This is rather surprising
at first glance but in treated metaphase I, as pointed out below, chromosomal de-
letions appear to be relatively large and in either stage, it is possible that deletions
small enough to act as recessives in fertilized eggs may not kill the individual until
after hatching in unfertilized eggs. Lethals which are recessive in diploids may be
due to such minute losses as to exert their effects only after hatching in haploids.
Perhaps viable deficiency heterozygotes are so rare that hatchability of irradiated
eggs is not perceptibly altered by fertilization with untreated spermatozoa. In
any case, conditions are well suited to an analysis of dominant lethal ratios induced
by x-rays in identifiable stages of meiosis and, although the chromosomes present

X-RAYS AND DOMINANT LETHALITY 63

difficulties, the eggs themselves are easily handled, fixed and stained for observa-
tion.

About 40,000 eggs were collected and observed for hatchability. Records were
kept of the results from individual females in all cases so that aberrant behavior
in eggs from any individual could be recognized. Such behavior was extremely
rare.

From the work of Sax (1938, 1940), Faberge (1940) and others on dose-
chromosome injury curves, certain tentative conclusions were drawn concerning
cytological effects before study of chromosomes was begun. For diplotene it was
assumed that the great majority of chromosome breaks must undergo restitution ;
that broken ends of chromosomes within the same cell increase as dose increases,
permitting complicated reunions (translocations, large interstitial deletions) so
that lethal individual chromosome changes tend to be due increasingly to more than
one ionization, especially in late diplotene ; that bridges can be formed in either
meiotic division or in both, due to lateral fusion of the broken ends of chromatids
whenever two adjoining chromatids are broken by a single ionization. From the
work of Sturtevant and Beadle (1936) and of McClintock (1941) it was thought
that bridges in division I might be permanent or delayed in breakage and might
offer an explanation for some, at least, of the high resistance of this stage to
irradiation.

Concerning metaphase I it was assumed, because of the linear relationship of
hatchability to dose, that injuries were in the form of terminal deletions or of
minute interstitial deletions, in other words, injuries due to single ionizations.
The high sensitivity of this stage suggested that most injuries must be permanent.
It was doubted that a single ionization would break two chromatids due to the degree
of separation in late metaphase I and so the occurrence of bridges in either division
from this cause seemed improbable. There was also the possibility that high
metaphase sensitivity might be due to "physiological" effects, stickiness, etc.,
which would result in fusion bridges, delay in division or death.

CYTOLOGICAL OBSERVATIONS

Cytology of controls. The cytology of the stages before metaphase I has not
been studied in detail, either in control or irradiated material, because of the small
size and large numbers of chromosomes and of their elongate and diffuse condition.
Synapsis occurs in very young oocytes and the subsequent behavior through con-
densation appears to have nothing exceptional about it. Changes take place slowly
and are not obvious in character until just before condensation of chromosomes
(diakinesis) when tetrads move to periphery of the nucleus. Most students of
hymenopteran cytology would question the conclusion that the stored oocyte is in
an orthodox and identifiable stage, late metaphase I. They state that the chromatin
has reverted to a resting stage or has formed an abortive spindle, a compact clump
or a composite body, etc. Speicher (1936) finds that the most advanced eggs in
the Habrobracon egg sac are in "early anaphase of the first maturation" which the
author prefers to call late metaphase. Speicher's observations that distinct chromo-
somes are present, are in the form of tetrads (Fig. 1) and are ten in number has
been repeatedly checked by the author and cannot be questioned. They show the

64 ANNA R. WHITING

forms expected for tetrads and each resolves immediately into two pairs of dyads
upon completing division I. The conclusion must he drawn either that Habro-
bracon differs from many other Hymenoptera in having orthodox oogenesis or
that its chromosomes retain more easily their individuality when fixed.

FIGURE 1. Three tetrads from one late metaphase I spindle. Untreated. Drawn from whole
mount of egg with aid of a camera lucida. Semi-diagrammatic. X 4,625.

The stages of normal oogenesis following oviposition, as described by Speicher,
are briefly as follows. During the process of oviposition the maturation spindle
is moved from dorsal to ventral side of the egg. It then passes into telophase I.
The second division follows immediately. The four groups of chromosomes (la,
Ib, 2a, 2b) lie in a row roughly perpendicular to the egg surface. During anaphase
II polar nuclei la and 2a remain stationary, Ib moves close to 2a, and 2b (func-
tional nucleus) sinks deeper into the egg, a membrane forming as it moves.
Nucleus la soon disintegrates, Ib and 2a unite and form a metaphase plate which
divides and then disintegrates. Cleavage is of the usual insect type, with nuclei
moving about until blastoderm formation when cell membranes first appear. The
stages following oviposition are the ones which were studied after irradiation.

No evidences of displaced chromosomes or of aberrant conditions resembling
those observed in irradiated eggs were found by Speicher or by the author in
large numbers of control eggs studied.

Cytology of irradiated eggs. In experiments concerned with cytological
effects, eggs from control and treated females were incubated according to standard
schedules, dropped into fixative (formalin-acetic-alcohol), punctured at the pos-
terior end to facilitate fixation, treated with the Feulgen technique and mounted
whole in balsam. Control hatchability tests were made of eggs treated at the same
time as those fixed. Slides were made of about 2,500 eggs.

After treatment in diplotene acentric fragments, dicentrics or both may occur
in division I (Fig. 2a, b, c) or in division II or in both divisions. Bridges in
division I may be permanent and can be seen bulging at the side when nucleus Ib
moves towards 2a, indicating that chromatin bridges are tensile but not elastic
(Fig. 2c). Acentric fragments remain visible throughout both divisions. No
evidences of stickiness or of clumping of chromatin (Fig. 2a, b, c) or of retarda-
tion of meiosis are apparent for doses up to lethal (45, 000 r). Of eggs treated in
diplotene with 44,800 r, 1.1 per cent died at first cleavage, 30.4 per cent with a
few nuclei, 54.3 per cent with many nuclei and 14.2 per cent at blastoderm.

X-RAYS AND DOMINANT LETHALITY 65

Immediately after irradiation in late metaphase I (Fig. 1), chromosomes show
no apparent change but at telophase I acentric fragments are left within the spindle
and these remain visible throughout division II (Fig. 2d, e). They are often
almost as large as entire chromosomes and can usually be identified as double struc-
tures. No bridges have been seen in division I in over 1,500 eggs observed. In

FIGURE 2. Illustrations were drawn from whole mounts of eggs with the aid of a camera
lucida. a, b, and c, eggs were irradiated in late diplotene with 44,800 r ; a, telophase I. X 1,500,
b, metaphase II. X 875 ; c, telophase II. X 1,250; d, c, and /, eggs were irradiated in late
metaphase I with 2,000 r; d, metaphase II. X 875 ; c, telophase II. X 1,250; /, third cleavage,
telophase. X 4,550.

division II bridges occur and after heavy treatment (2,000 r) several may be seen
in each second division spindle (Fig. 2e). Small fragments occasionally appear
in division II spindles. No evidences of stickiness or of clumping (Fig. 2d, e) or
of retardation of meiosis occur in development following treatment with lethal dose
of this stage which, except for absence of bridges in division I, behaves cytologically
as treated diplotene. Percentages of eggs with fragments in division I and mean
number of fragments per treated increase linearly with increased dose (Whiting,
1945). All eggs exposed in late metaphase I to 2,016 r undergo some develop-
ment. 2.4 per cent die in first cleavage, 7.2 per cent with a few nuclei, 71.4 per
cent with many nuclei and 19.0 per cent at blastoderm stage. In spite of their high
sensitivity, some eggs treated in metaphase I developed to the fifth cleavage (ex-
pected) after 15,000 r, to metaphase II after 25,QOO r, to pronucleus after 35,000 r
and one to anaphase II after 200,000 r. No records were kept of rate of develop-
ment at these higher doses.

66 ANNA R. WHITING

The similar patterns of stage at death for both diplotene and metaphase I at
their respective lethal doses indicate that, in spite of the great difference in sensi-
tivity between the stages, cause of death is of the same nature in both. These data
on time of death check what has often been noted, especially in respect to mature
spermatozoa, that so-callecl lethal doses are not actually lethal to the treated cell
itself but, instead, to its descendents. The fact that the oocyte continues to func-
tion normally and that death does not occur until it becomes an embryo, supports
the argument that cytoplasmic injury is not at the basis of mortality. It is due,
rather, to loss of parts of chromosomes during meiosis following irradiation and
to resulting incomplete chromosome complements in every cleavage nucleus.

Bridges occur in cleavage I after treatment in either stage indicating that, if
chromatids are already split when treated, there occurs lateral fusion of broken
ends of half -chromatids. If they are not split when treated the split occurring in
the first mitosis must have been incomplete in the broken chromatid or have re-
sulted in lateral fusion of broken ends of daughter chromosomes. Bridges appear
in subsequent cleavages. Fragments, which also occur in cleavage, could not be
explained at first until it was noted that they are tapering at the ends and that they
result from double breaks in a bridge which releases a thickened middle portion
(Fig. 2f). Fragments were not observed after every mitosis in the same embryo
although bridges, if present, appear in all cleavage figures.

CORRELATION OF INJURY WITH CHROMOSOME FORM WHKN TREATED

It is perhaps unwise to devote much time and space to the subject of the cor-
relation of the nature of the injuries and the form of the chromosomes when treated
in view of the small size of Habrobracon chromosomes and the disagreement of
investigators in this field. Obviously, there is a correlation. The studies of Sax
(1938, 1940), Faberge (1940) and McClintock (1938) will be used as a basis of
a brief discussion, since the results of these investigations are consistent with their
theories.

Chromosome injuries fall into two classes, those caused by single ionizations
and those caused by more than one. The former consist of terminal deletions and
minute interstitial deletions. Two identical terminal deletions can be induced by
a single ionization if two chromatids are sufficiently close together. When this
happens, lateral fusion of broken ends occurs resulting in a dicentric, from parts of
the two chromatids still attached to spindle fibers, and an acentric, from the re-
leased and fused ends. Single terminal deletions can be induced by single ioniza-
tions and this appears to be the rule when chromatids are widely separated. An
acentric is ultimately lost and a dicentric forms a bridge when its two spindle fiber
attachment points (centromeres) are pulled apart. If the bridge does not break,
an entire chromosome may be missing from a daughter cell. If it does break, the
resulting chromosomes are incomplete and each daughter cell will have an incom-
plete chromosome and, therefore, an incomplete set of genes. Such a terminally
incomplete chromosome may continue to form a bridge in each subsequent division,
either by failing to split completely or by a lateral fusion of the broken ends after
splitting. This appears to be the general rule but McClintock (1941) has found
that when such an incomplete chromosome occurs in the sporophyte tissue of maize,
it forms no bridge.

X-RAYS AND DOMINANT LETHALITY 67

An interstitial deletion caused by a single ionization in a chromosome would
mean the loss of genes and would be lethal if they were numerous or of sufficient
importance but it would not be cytologically apparent in subsequent divisions be-
cause of its small size.

Injuries which must be due to more than one ionization since they involve
breaks in chromosomes too far apart to be caused by a single ionization are large
inversions, large interstitial deletions and tfanslocations. Inversions would not be
apparent, either cytologically or in effect on viability of the embryo receiving them
in the present study since they would be induced after synapsis and crossing over
and the inversion of a block of genes would probably have no lethal effect. Large
interstitial deletions would have a lethal effect but could not be identified in ma-
terial used in these experiments. Translocations might be lethal and would be
visible as bridges should centric parts of non-homologues become attached to each
other. Such bridges cannot be distinguished cytologically from those resulting
from double terminal deletions in this material.

The nature of the hatchability curves suggests that most injuries in early
diplotene and late metaphase I at all doses and in late diplotene at low doses are
caused by single ionizations, that many injuries in late diplotene at high doses
are caused by more than one ionization. Since there is no reason to suppose that
the nature of original breaks would be changed by higher doses it is presumed that
the number of single breaks per cell increases with high dose and allows greater
opportunity for new combinations because of increased number of broken ends
available at any one time. This would take for granted the breaking of single
chromatids per ionization for if two were broken the lateral fusion of broken ends
would prevent translocations, fusion with more distant chromosomes. The reduc-
tion in injury by fractionation of dose is explained on the grounds that, with re-
peated smaller doses, fewer free ends are available at any given time for new com-
binations and the intervals between treatments afford an opportunity for restitution
or changes in broken ends to occur so that they are no longer capable of joining
with other broken ends formed by later treatments.

Three conditions seem to be of importance, then, in determining response of
the chromosomes here studied to irradiation. These are (1) relation of tetrads
to each other in the nucleus, (2) degree of separation of adjoining chromatids
within a tetrad and (3) nature and degree of tension on chromosomes. Each of
the three stages will be discussed briefly from these points of view.

In early diplotene the tetrads are evenly distributed within the nucleus, sister
chromatids are in contact, homologues separated except at chiasmata, and neither
traction of the spindle fibers nor terminalization has begun. Most breaks will be
temporary because of lack of tension and relaxed state of the chromosomes. Trans-
locations should be possible but the majority of breaks will involve both sister
chromatids with the production of acentrics and dicentrics. Permanent double
breaks can occur either between centromeres and proximal chiasmata (with pro-
duction of bridge in division II) or distal to chiasmata (producing bridge in divi-
sion I if distal to "odd" chiasmata. in division II if distal to "even") since the
slight tension which exists is equally exerted everywhere along the length of the
chromosome.

In late diplotene the tetrads move peripherally but are still widely separated,
terminalization (movement of chiasmata towards ends of tetrads) has begun, as

68 ANNA R. WHITING

well as movement of centromeres away from each other, and chromatids are not
so closely associated, especially toward ends of chromosomes. Single and double
breaks will occur (the latter nearer the centromere) and more of them will be
permanent because of new tensions. Bridges should be less frequent in division
I than in the case of early diplotene but this has not been checked. This stage
will be somewhat more sensitive and will exert its lethal effects through trans-
locations and large interstitial deletions as well as through double terminal deletions.

In late metaphase I, the tetrads are isolated from each other and stable in posi-
tion on the spindle so that interchanges between them would not be expected.
Centromeres are pulled far from each other and chiasmata resist further terminal-
ization (Fig. 1) so that tension exerted between centromeres and proximal
chiasmata is very great, tension exerted distal to chiasmata not so great. loniza-
tions will cause double breaks near centromeres where sister chromatids are closely
approximated and these will all be permanent because of the extreme tension.
They will result in large double fragments (acentrics) in division I, bridges (di-
centrics) in division II. Breaks induced towards ends of chromosomes, and espe-
cially distal to chiasmata, will be less likely to be permanent and more likely to
be single. There will be few or no bridges in division I and single fragments will
appear in division I or division II (McClintock, 1938).

Any injury to a tetrad which results in a single bridge in division II reduces the
chance of hatching of the egg by fifty per cent ; in division I the effect is the same
if the bridge breaks promptly. If it is delayed in breaking or does not break the
hatchability of the egg is not affected, since an incomplete chromatid is thereby
restrained from entering the ootid nucleus. A single terminal deletion reduces
the chance of hatching by twenty-five per cent.

With ten tetrads of the diverse forms found in Habrobracon, combinations of
changes induced by single ionizations can become very complex. If added to these
are the complication of translocation and of large interstitial deletions (character-
istic especially, perhaps, of late diplotene) the great resistance of diplotene is to be
wondered at. The author (Whiting, 1945) has reviewed the data here re-
ported in the light of the numerous theories devised to explain differential sensi-
tivity of chromosomes to x-rays and has found that the only one which applies is
that put forth by Goodspeed in 1929. He suggested tension as the important fac-
tor. It seems highly probable that numerous breaks do occur in the evenly dis-
tributed, diffuse, slowly moving chromosomes of diplotene but that the majority
of them is temporary. Healing or restitution must take place quickly for there is
always some movement and these chromosomes ultimately go through the same
stresses as those treated in late metaphase I and, in addition, those attendent upon
condensation and complete terminalization. Their response to fractionation also
argues for relatively rapid restitution.

The development of individuals, normal in appearance and in reproductive
activity and with normal descendents, after treatment in diplotene with 35,000 r,
illustrates graphically the resistance of this stage to permanent injury by ioniza-
tions.

Sax (1942) summarizes the information available on "physiological" effects of
x-rays, one of which is stickiness of chromatin. It has been found that condensed
chromosomes are most sensitive in respect to stickiness, that such effects are
temporary, delay subsequent division, have a threshold dose, are lethal only after

X-RAYS AND DOMINANT LETHALITY 69

very high doses and result in "fusion" bridges if the cell divides before recovery.
The stage in the present study most likely to show the effects of stickiness in the
form of fusion bridges is division I after treatment of metaphase I. This is the
only division which shows bridges of no kind even after doses much higher than
lethal. A delay of twenty-four hours between treatment and resumption of meiosis
does not increase hatchability, meiosis is not appreciably delayed after irradiation,
there is no threshold effect (down to 50 r). It should be emphasized again that,
wide apart as are the lethal doses for diplotene and metaphase I, at their respec-
tive lethal doses, the pattern of stages at death is the same, the same percentage
dies at first cleavage, at blastoderm, etc.; in other words, 45,000 r has no more
drastic effect on development of treated diplotene than l,400r on metaphase I.
All evidence indicates that cause of death is of the same order for both stages and
that "physiological" effects are of every minor importance, and not appreciably
different in the two stages.

Sturtevant and Beadle (1936) failed tp recover an expected genetic type of
chromosome aberration correlated with a dicentric in division I. They suggested
that in a form like Drosophila where the four meiotic nuclei lie in a row and where
a terminal one alone functions, a bridge in division I might fail to break, or might
be delayed in breaking, thereby tying together injured chromatids and allowing
uninjured ones to pass to the terminal nuclei. McClintock (1941) also offers as
explanation for the failure to obtain expected genetic results correlated with
bridges in division I of maize, the selective effect of these bridges on broken
chromatids. Terminal nuclei (one of which becomes the functional megaspore)
tend to receive the uninjured chromatids. Figure 3c demonstrates that bridges
in division I in Habrobracon eggs do not break, at least in some cases.

In divisions following treatment of diplotene to which this selection of injured
chromatids for elimination would apply, the chances of having bridges in the second
division are as frequent as in the first or more so and selection through permanence
of bridges would apply, therefore, only in the simplest kind of injury and that to
but one or very few tetrads since any number of breaks would be certain to produce
some bridges in division. This selection, although it undoubtedly occurs, cannot
explain more than a small amount of resistance of diplotene. It would be expected
to apply especially with low doses when but a single break occurs in a single tetrad.

The wide difference in size of lethal doses (45,000 r-1, 400 r) of such closely
related stages of the same cell, the unreduced Habrobracon egg, confirms the truth
of the conclusion made long ago (1906) by Krause and Ziegler in an extensive
and critical study of tissue injury by x-rays, that it is less the kind of cell than its
stage at the time of treatment which determines sensitivity.

The facts and theories just presented are of interest in connection with a dis-
cussion of dominant lethals by Pontecorvo (1942). He explains dominant lethal
effects in Drosophila spermatozoa by assuming that single chromosome breaks are
produced by radiations at a rate proportional to radiation dose and that these
neither undergo restitution nor participate with other breaks in the same nucleus in
rearrangements. "Chromosomes with broken ends give rise to a cycle of breakage-
fusion-bridge phenomena in development." He also writes, "It is therefore an
open question whether sister unions are so frequent as to cause a considerable
portion of dominant lethality. Should this be the case, the trend of the curve of
dominant lethality could be explained. Most dominant lethality would be de-

70 ANNA R. WHITING

termined by single-break sister unions at low dosages and as the dosage increased
lethal changes of the other two types (translocations and deletions) would come
to play an increasing part." Translocations and deletions would not be produced
actually until syngamy since breaks appear to remain open in the sperm chromo-
somes until that time.

Broken chromosome ends do undergo restitution or participate with other
broken ends in the egg very soon after treatment but the final contribution to the
zygote may be the same as that made by the irradiated sperm-, viz., a chromosome
with a broken end which will give rise to the breakage-fusion-bridge cycle in the
first cleavage as well as in subsequent ones. Most dominant lethality in the pres-
ent study is, without much doubt, caused by single ionizations and only in late
diplotene at high doses does treatment appear to cause a high percentage of death
from the cooperation of two or more ionizations.

It is of interest in this connection to note that the dose-injury curve for domi-
nant lethality in the spermatozoa of Drosophila (Sonnenblick, 1940; Demerec and
Fano, 1944) and for Habrobracon (Heidenthal, 1945) is of the same nature as
that for late diplotene.

CONCLUSION

1. (Tentative) The majority of dominant lethals induced in late diplotene by
low doses (to ll,000r) and in early diplotene and in late metaphase I by all doses
through lethal, in Habrobracon eggs, is caused by single ionizations which break
adjoining chromatids. Lateral fusion of broken ends results, followed by con-
tinued breakage-fusion-bridge phenomena in cleavage. With doses above 1 1,000 r
in late diplotene an increasing number of lethal changes arises from two or more
ionizations (translocations, large interstitial deletions). 2. Lethal doses are not
lethal to the treated cell (oocyte) itself but to its descendents (embryo). Frag-
mentation of chromosomes is not lethal, loss of fragments is. 3. The nature and
degree of chromosome injury can be correlated with the form of the chromosome
and with forces acting upon it during and immediately following treatment. 4.
The kind of cell is less important than its stage in determining sensitivity to x-rays.
5. Tension is the main factor in determining permanence of breaks caused by ioniza-
tions, chromosome form and movement in determining the nature of the new com-
binations of broken ends. 6. The chromosome phenomena here dealt with are
common ones in the majority of animals and plants and it is predicted that, when
metaphase and anaphase are sufficiently studied in other forms, they will be found
to be the stages most sensitive to x-rays. This has proved to be the case for Sciara
(Metz and Bozeman, 1940; Reynold's, 1941) and for Trillium (Sparrow, 1944).

SUMMARY

Unlaid Habrobracon eggs x-rayed in diplotene (lethal dose about 45,000 r) and
allowed to develop parthenogenetically, show fragments, bridges or both in division
I ; either or both in division II. Bridges in division I may be permanent.

Unlaid eggs x-rayed in late metaphase I (lethal dose about l,400r) show frag-
ments but no bridges in division I ; bridges, fragments or both in division II.

An explanation of difference in cytological effects of x-rays on these stages and
of the differences between them in sensitivity and in nature of survival curves is
attempted through comparison with studies on forms with larger chromosomes.

X-RAYS AND DOMINANT LETHALITY 71

LITERATURE CITED

DEMEREC, M., AND U. FANO, 1944. Frequency of dominant lethals induced by radiation in

sperms of Drosophila melanogaster. Genetics, 29 : 348-360.
FABERGE, A. C., 1940. An experiment on chromosome fragmentation in Tradescantia by X-rays.

Jour. Genetics, 39 : 229-248.
GOODSPEED, T. H., 1929. The effects of X-rays and radium on species of the genus Nicotiana.

Jour. Hcrcd., 20: 245-259.
HEIDENTHAL, GERTRUDE, 1945. The occurrence of X-ray induced dominant lethal mutations in

Habrobracon. Genetics, 30: 197-205.
KRAUSE, P., AND K. ZIEGLER, 1906. Experimented Untersuchungen iiber die Einwirkung der

Rontgenstrahlen auf tierisches Gewebe. Fortschr. a. d. Gcb. d. Rontgenstr., 10: 126-

182.
McCLiNTOCK, BARBARA, 1938. The fusion of broken ends of sister half-chromatids following

chromatid breakage at meiotic anaphases. Missouri Agric. Exp. Sta. Bull., 290 : 1-48.
McCLiNTOCK, BARBARA, 1941. The stability of broken ends of chromosomes in Zea mays.

Genetics, 26 : 234-282.
METZ, C. W., AND M. L. BOZEMAN, 1940. Further observations on the mechanism of induced

chromosome rearrangements in Sciara. Proc. Nat. Acad. Sci., 26: 228-231.
PONTECORVO, G., 1942. The problem of dominant lethals. Jour. Genetics, 43 : 295-300.
REYNOLDS, J. P., 1941. X-ray induced chromosome rearrangements in the females of Sciara.

Proc. Nat. Acad. Sci., 27 : 204-208.

SAX, KARL, 1938. Chromosome aberrations induced by X-rays. Genetics, 23: 494-516.
SAX, KARL, 1940. An analysis of X-ray induced chromosomal aberrations in Tradescantia.

Genetics, 25 : 41-68.

SAX, KARL, 1942. The mechanism of X-ray effects on cells. Jour. Gen. Pliysiol., 25 : 533-537.
SONNENBLICK, B. P., 1940. Cytology and development of the embryos of X-rayed adult

Drosophila melanogaster. Proc. Nat. Acad. Sci., 26: 373-381.
SPARROW, A. H., 1944. X-ray sensitivity changes in meiotic chromosomes and the nucleic acid

cycle. Proc. Nat. Acad. Sci., 30: 147-155.
SPEICHER, B. R., 1936. Oogenesis, fertilization and early cleavage in Habrobracon. Jour.

Morph., 59: 401-421.

STURTEVANT, A. H., AND G. W. BEADLE, 1936. The relations of inversions in the X chromo-
some of Drosophila melanogaster to crossing over and disjunction. Genetics, 21 : 554-

604.
WHITING, ANNA R., 1938. Sensitivity to X-rays of stages in oogenesis of Habrobracon. Rcc.

Genetics Soc. Am., 7 : 89.

WHITING, ANNA R., 1945. Effects of X-rays on hatchability and on chromosomes of Habro-
bracon eggs treated in first meiotic prophase and metaphase. Amer. Naturalist, 79 :
193-227.

STRATIFICATION AND BREAKING OF THE ARBACIA PUNCTU-

LATA EGG WHEN CENTRIFUGED IN SINGLE

SALT SOLUTIONS

ETHEL BROWNE HARVEY

Marine Biological Laboratory, Woods Hole, and the Biological Laboratory,

Princeton University

A study lias been made of the comparative rate of stratification and breaking of
the Arbacia egg in single salt solutions, when subjected to centrifugal force. It
might lie expected that when more rapid stratification occurs, the eggs would break
apart more readily. This was, however, found not to be the case when the eggs
were centrifuged in hypo- and hypertonic sea water, but this is probably due to the
change in volume of the eggs (E. B. Harvey, 1943). With the increased surface
area of the eggs in hypotonic sea water the tension at the surface is increased
(Cole, 1932) and the eggs are more difficult to break apart. In the present experi-
ments with pure salt solutions the surface area remained constant.

The solutions used in the following experiments were those routinely used at
Woods Hole as isotonic with the sea water there, and found by me to be isosmotic
on measuring the eggs after immersion, namely: 0.52 m NaCl, 0.53 m KC1, 0.34 m
CaCL, and 0.37 m MgCl2. The pH of the solutions was found to be respectively,
5.54, 5.44, 5.53, and 6.31. It was determined, however, that the pH in itself, at
least of sea water, has no effect on the stratification and rate of breaking. Sea
water was made up of pH ranging from 5 to 9 by adding HC1 or NaOH ; eggs
kept in these solutions and centrifuged in them at the same time as those in normal
sea water showed no difference in stratification or breaking. This was found also
by Barth (1929) for stratification in sea water, though he did find an effect in
NaCl. However Heilbrunn (1928, 1943) finds that Na definitely increases vis-
cosity. The eggs were not injured by the pure salt solutions as they could be fer-
tilized on removal to sea water after 40 minutes in the solutions and produced
normal plutei. However, the eggs cannot be fertilized while in the solutions;
the sperm are immotile in all except NaCl, and here no fertilization membrane
was seen.

Arbacia pitiictnlata eggs were placed in 50 cc. of the isosmotic salt solution for
20 minutes and this was replaced by a fresh salt solution for another 20 minutes.
Three tubes of experimental eggs (in different salt solutions) and one tube of con-
trol eggs (in sea water) were centrifuged at the same time; isosmotic sugar solu-
tion was placed in the bottom of each tube to keep the eggs suspended. Care must
be taken that exactly the same amount of sugar solution is used in each tube and
exactly the same amount pf egg suspension placed on top, so that the eggs in each
tube are thrown to the same level and are subjected to exactly the same amount of
centrifugal force. For stratification the force used was about 3,000 X g for two
minutes, and for breaking 10,000 X g for four minutes. Each experiment was re-
peated many times. A single batch of eggs was always used in each experiment.

EGGS CENTRIFUGED IN SINGLE SALT SOLUTIONS 73

Stratification NaCl (KC1) Breaking

^ * ^

KXLJLANATION OF PLATE

Stratification of Arhacia pitncliilulu eggs centri filled at .i,()(l() X g for two minutes in (1)
NaCl, (3) sea water, (5) MgCl.. Breaking apart of eggs at 10,000 X g for four minutes in (2)
NaCl, (4) sea water, (6) MgCL KC1 acts much like NaCl and CaCl, much like MgCl,.

74 ETHEL BROWNE HARVEY

The experiments were carried out at approximately 23° C., so that the temperature
effect observed by Costello (1934, 1938) was not involved.

It was found that in the monovalent salts, NaCl and KG, the rate of stratifica-
tion is less than in sea water, and in the bivalent salts, CaCU and MgClL,, the rate
of stratification is greater than in sea water (Photographs 1, 3, 5). The viscosity,
then, is increased in NaCl and KC1 and decreased in CaCL and MgCL. In the
effect on the rate of stratification the series runs, from most to least : Ca > Mg
> S.W. > Na > K. This is similar to the series given by Heilbrunn (1923,
1928) in a slightly different experiment with Arbacia eggs, except that Na and K
are reversed. This is possibly due to a difference in the tonicity of the solutions
used. His series for S tent or is the same as my series for Arbacia.

In ease of breaking with centrifugal force, the series runs in the reverse order.
Eggs in KC1, where the stratification is least in a given time, break most readily,
and those in CaCl2, where the stratification is greatest, break least readily. Eggs
in the monovalent salts, NaCl and KG, break more readily than those in sea
water while the eggs in the bivalent salts, MgCL and CaCL, break less readily than
those in sea water (Photographs 2, 4, 6). In ease of breaking, the series runs,
from greatest to least: K > Na > S.W. > Mg > Ca. The ease of breaking has
been judged by the percentage of eggs broken in a given time with a constant force,
rather than by the time for a definite percentage to break, since the experiment can
be carried out more accurately when experimental and control eggs are centrifuged
at the same time. An average experiment gives the following figures for per-
centage of eggs broken when centrifuged for four minutes at 10,000 X g.

KC1 NaCl Sea water MgCl2 CaCl2

99% 90% 50% 20% none

There was no measurable difference in the relative size of the two "halves" in any
of the pure salt solutions; the white and red "halves" were the same size as those
obtained when eggs were kept and centrifuged in sea water.

There is considerable variation in ease of breaking in different lots of eggs with
the same centrifugal force, and even the same batch varies slightly after being kept
in sea water for several hours. In one experiment 98 per cent were broken in sea
water, and 40 per cent in CaCl., ; in another experiment, 50 per cent were broken
in NaCl and 20 per cent in sea water. In every experiment, however, the eggs
in the solutions broke in the order named. It was thought that possibly the jelly
surrounding the eggs might be influenced by the salt solutions and be responsible
for the difference in ease of breaking. Jelly was found to be present on the eggs in
all the solutions. Eggs from which the jelly was removed by addition of 0.2 cc.
N/10 HC1 to 50 cc. sea water, and then well washed in sea water broke in the
solutions in the same order as those with jelly.

Since the experimental results are contrary to the expectation that the interior
viscosity is the controlling factor in breaking of the eggs, we are led to the con-
clusion that the salts affect the ''tension at the surface." Despite the increased
interior viscosity in pure NaCl and KG, the surface forces resisting the pulling
apart of the eggs are actually decreased. In CaCL and MgCL they are increased
though the interior viscosity is decreased. Heilbrunn (1923, 1943) has pointed
out that in Amoeba, and apparently also in Arbacia eggs, the cortical protoplasm

EGGS CENTRIFUGED IN SINGLE SALT SOLUTIONS 75

reacts differently from the interior protoplasm, and Brown (1934) has found a
difference in cortical and interior protoplasm in response to hydrostatic pressure on
fertilized Arbacia eggs.

An effect on the surface forces without any effect on the interior viscosity is
given by eggs in Ca-free sea water. Unfertilized eggs kept and centrifuged in
Ca-free sea water stratify at the same rate as those in sea water, as shown in
previous experiments with a double image centrifuge microscope (E. B. Harvey,
1933). They break apart more readily in Ca-free sea water than in normal sea
water — at about the same rate as those in NaCl alone. The fertilized eggs also
break more readily in Ca-free sea water than in normal sea water, as shown previ-
ously. The absence of calcium therefore tends to decrease the surface forces and
the presence of calcium alone tends to increase them. That calcium has an effect
on the surface layers of eggs is well known, and has been especially emphasized
by Heilbrunn (1928, 1943). A very good example is given by the classic experi-
ments of Herbst (1900) in separating blastomeres due to the dissolution of the
ectoplasmic (hyaline plasma) layer in Ca-free sea water.

SUMMARY

When unfertilized Arbacia pnnctnlata eggs are centrifuged in isosmotic single
salt solutions, they stratify with decreasing readiness (indicating increasing vis-
cosity) in the following order: CaCL > MgCL > S.W. > NaCl > KG. ^They
break into "halves" with decreasing ease in the reverse order, those in CaCU which
stratify best, break least readily. In the bivalent salts they stratify better and
break less readily than in sea water, and in the monovalent salts they stratify less
and break more readily than in sea water. The ease of breaking must be de-
termined by an effect of the salts on the surface layers rather than by their effect
on the interior viscosity.

LITERATURE CITED

EARTH, L. G., 1929. The effects of acids and alkalies on the viscosity of protoplasm. Proto-

plasma, 7 : 505-534.
BROWN, D. E. S., 1934. The pressure coefficient of "viscosity" in the eggs of Arbacia punctu-

lata. Jour. Cell, and Coinp. Pliysiol., 5 : 335-346.

COLE, K. S., 1932. Surface forces of the Arbacia egg. Jour. Cell, and Comp. Pliysiol., 1 : 1-9.
COSTELLO, D. P., 1934. The effects of temperature on the viscosity of Arbacia egg protoplasm.

Jour. Cell, and Comp. Pliysiol.. 4: 421-433.
COSTELLO, D. P., 1938. The effect of temperature on the rate of fragmentation of Arbacia eggs

subjected to centrifugal force. Jour. Cell, and Comp. Pliysiol.. 11 : 301-307.
HARVEY, E. B., 1933. Effects of centrifugal force on fertilized eggs of Arbacia punctulata, as

observed with the centrifuge microscope. Biol. Bull., 65: 389-396.
HARVEY, E. B., 1943. Rate of breaking and size of the "halves" of the Arbacia punctulata eggs

when centrifuged in hypo- and hypertonic sea water. Biol. Bull., 85: 141-150.
HEILBRUNN, L. V., 1923. The colloid chemistry of protoplasm. I. General considerations.

Amer. Jour. Pliysiol.. 64: 481-489.

HEILBIU-XX, L. V., 1928. The colloid chemistry of protoplasm. Monograph. Berlin.
HEILBRUNN, L. V., 1943. .In outline of general physiology, 2nd edition. Saunders Co.
HERBST, C., 1900. Uber das Auseinandergehen von Furschungs- und Gewebzellen in kalk-

freiem Medium. Arch. f. Enhc. Mccli., 9: 424-463.

THE EFFECT OF CYANIDE ON RESPIRATION IN PARAMECIUM
CAUDATUM AND PARAMECIUM AURELIA l

D. M. PACE

Department of Physiology and Pharmacology, Collei/e of Pharmacy,
University of Nebraska. Lincoln. Nebraska

In some ciliates the presence of a cytochrome-oxidase system has been estab-
lished. Pitts (1932) claimed that Colpidinin campylitm showed an ititial sensi-
tivity to HCN but that the oxygen consumption soon increased until it even sur-
passed normal consumption. Lwoff (1934) also found an initial inhibition followed
by an acceleration in respiration in another ciliate. Glaucoma pyriformis, when it
was exposed to KCN. Hall (1941) definitely established that HCN inhibits
respiration in Colpidinin ca-inpylitin and Baker and Baumberger (1941) found that
HCN inhibits respiration in Tetrahymena gclcii.

Paramecium is usually cited as one of the several exceptions to the rule that
most animal cells are sensitive to HCN. In fact, ciliates as a group have been
regarded by some investigators as being insensitive to cyanide, although very few
species have been tested. Lund (1918), Shoup and Boykin (1931), and Gerard
and Hyman (1931) found that Paniiiiechtin candatnm was resistant to cyanide.
However, Child (1941) refers to unpublished data obtained by Hyman, in which
she found a considerable decrease in O., consumption of Parainccinin in KCN.
Dr. Hyman - has also informed the author by personal communication that she

1 These investigations were partly supported by a grant-in-aid received from the Society of
Sigma Xi. The Barcroft-Warburg apparatus was purchased by a grant furnished by Mr.
Arthur S. Raymond of the Lincoln Drug Co., Lincoln, Nebraska.

- Dr. Libbie H. Hyman has granted me the privilege of using the following communication
which she sent to me at my request : "Some years ago, being skeptical of Lund's failure to find
any cyanide-sensitive respiration in Paramecium, I spent a great deal of time and effort in
testing the action of cyanide on the oxygen consumption of Paramecium, using Winkler's method.
I met with so many difficulties that I never published the results ; chief among them were the
impossibility of measuring equal suspensions of Paramecium from a volumetric pipette because
the animals adhere to the glass, and the toxicity to Paramecium of all waters except the culture
water, which in itself has high oxygen consuming powers. However, my results indicated that
starved Paramecium have no cyanide-sensitive respiration, in agreement with the finding of
Lund, but non-starved ones have about 35 per cent such respiration. After giving up the work
as impractical by my methods, I sought the help of Dr. Gerard. Dr. Gerard kindly consented
to test the matter on his manometers but failed to find any depressing action of cyanide on non-
starved Paramecium. As I played no role in this work except that I furnished the Paramecium,
I feel that Dr. Gerard was over-generous in making me co-author. I was not satisfied with
these results, first, because successive manometric readings were highly variable, and second,
because the buffer solution used was toxic to Paramecium, depressing oxygen consumption by
about 50 per cent in itself.

"As a cyanide sensitivity of the extra oxygen consumption caused by feeding was indicated
in my experiments, it became interesting to know the nature of this extra respiration. I there-
fore attempted to compare the effects on oxygen consumption of the ingestion by Paramecium
of particles without food value (carbon suspension) and of particles with food value (yeast).
Here, again, I met with insuperable difficulties. 1 could never get any sample of yeast, no

EFFECT OF CYANIDE ON RESPIRATION 77

found an inhibition of O., consumption in P. caudatinn when it was exposed to
HCN.

Sato and Tamiya (1937) claimed that they found cytochrome a and c in
Paramecium. If this is true, then it is difficult to understand the insensitivity of
the respiratory mechanism of this species to HCN. Because of these observations
and of the unpublished results of Hyman, and since studies have not been made on
the sensitivity of Paramecium to cyanide when proper KOH-KCN mixtures are
used as absorption media (Krebs, 1935), the following investigation was carried
out.

MATERIAL AND METHODS

Two species were used in this work, Paramecium caitdatnin and Paramecium
aurelia. The culture solution used was highly buffered and was the same as was
used later in the flasks of the Barcroft- Warburg apparatus for testing. The solu-
tion consisted of ICHPO4.H,O -- 80 mg., KH,.PO4--80 mg., CaCU - - 104 mg.,
Mg,POj--2 mg., and redistilled water to make one liter.

In making up the stock culture, 15 gms. of timothy hay were boiled in 500 ml.
of this solution for one-half hour, after which the solution was made up to its
original volume by the addition of distilled water. This "broth" was then diluted
further by the addition of the above buffered solution to make 4000 ml. The
hydrogen ion concentration was held at pH 7.0 ± 0.2.

This culture solution, along with approximately 3 gms. of sterile hay, was put
into chemical bottles with 500 ml. capacity and moderately narrow necks (3-4 cm.
in diameter). About 4000 paramecia were added to each container. Within 5
days they became extremely numerous, especially in the neck region of the bottle
whence they could be removed easily in large numbers.

The Barcroft- Warburg apparatus was used for ascertaining rate of oxygen
consumption. The shaking mechanism was adjusted to operate at 110 complete
cycles per minute. Because of the possibility of NH3 production (Specht, 1934),
a 0.3 ml. portion of 0.3 N HC1 was added to the side arm (onset) of each ma-
nometer flask.

During the course of these investigations, various test solutions were made up
containing different concentrations of KCN as follows: 0, 10~r', 10~4, and 10~3 M.
Corresponding KOH-KCN absorption solutions were made up for each concen-
tration of test solution according to Krebs (1935), and 0.4 ml. of the proper mix-
ture (Pace and Belda, 1944) was added to the inner well (inset) of each flask con-
taining organisms in KCN. To the inset of each of the flasks in which the test
solution contained no KCN, a 0.4 ml. portion of M KOH was added.

A typical test was made in the following manner : Paramecia were drawn off
from the top of the bottles in which they were cultured and placed in 15 ml. cen-
trifuge tubes in which they were washed several times in fresh solution by careful
centrifugation. The only time the organisms were subjected to centrifugation was

matter how many times boiled and centrifuged, that did not have high oxygen consuming
powers, and all carbon suspensions also remove oxygen from the medium. However, there
were indications that ingestion of a non-nutritive substance can cause as great an increase in
oxygen consumption as does ingestion of food. This suggests that the extra respiration of feed-
ing does not result from an oxidation of the food material."

78 D. M. PACE

during the washing process and this was carried out with great care hy means of a
hand centrifuge. An attempt was made to have between 2000 and 3000 P. aurclia
or 1000 and 2000 P. candatitin in each 5 ml. sample. A count was always made
of the organisms in each flask at the end of an experiment.

In all the tests reported here, those organisms designated "young" paramecia
were taken from 5-7 day-old cultures; those designated "old" paramecia, from 15-
20 day-old cultures ; those designated "starved" paramecia were "old" organisms
that had been placed in inorganic buffer solution without food material for 2 or 3
days. The "young" paramecia had much more food material present in the form
of food vacuoles than the "old" paramecia.

RESULTS
Effect of cyanide on respiration in Paramcciiiin aurclia

Parameciitin aurclia was the first species studied. It is a much smaller form
than P. caiidatuin, but its rate of respiration per unit volume is similar to the latter
(Pace and Kimura, 1944).

A number of tests were made at various KCN concentrations. Organisms
that were taken from cultures 15-17 days after they had been started (i.e., "old"
paramecia) were used in most of the tests. They were washed by centrifugation
in the solution given above, and then divided into two portions. KCN was added
to one of these portions in the concentrations designated in the table. Several
tests were also carried out on starved paramecia and young paramecia. The re-
sults are presented in Table I.

P. aurclia was found to be sensitive to KCN in all the tests made, except where
starved individuals were used. The normal average oxygen consumption for or-
ganisms taken from the 15 or 17 day-old cultures was 6.31 nmr per hour per mm3
of cell substance at 25° C. This compares favorably with the results of Pace and
Kimura (1944) who found that P. aurclia consumed oxygen at the rate of 6.16
mm3 per hour per mm3 of cell substance at 25° C.

The presence of food material may have something to do with the fact that in
all the tests made, the younger paramecia showed a much greater sensitivity to
cyanide than the older. In fact, starved specimens were insensitive to cyanide.
When exposed to KCN at a concentration of 10"* M, respiration in the young
organisms was inhibited on the average by about 40 per cent. The respiration of
old organisms showed an average inhibition of 22 per cent to the same concentra-
tion of KCN. At KCN concentrations of 10~3 M, inhibition of respiration was
greater than with the lower concentration, but the results were similar insofar as
young and old organisms are concerned. In young paramecia, the average (X
consumption (1318 mm3 O.2 per hour per million) in the buffered solution without
KCN was about twice that in old organisms. An average O.2 consumption of 640
mm3 was found for the young paramecia when they were exposed to 10~3 M KCN.
Thus the cyanide at this concentration results in a 50 per cent inhibition in respira-
tion in P. aurelia.

Effect of KCN on respiration in Parameciitin caudatuni

Paraineciuin caitdatiiin has been studied to a much greater extent than P.
aurclia and, as brought out previously, all the work (except for unpublished early

EFFECT OF CYANIDE ON RESPIRATION

TABLE I

The effect of KCN on respiration in Paramecium aurelia. *, starved specimens; 5-7 day
cultures, young specimens; all others, old specimens. Temperature, 25° C.; pH, 7.0 ± 0.2.
Average volume of one million paramecia, 121.4 mm.3 (this does not include the volume of starved
specimens). Each figure in columns 4 and 5 represents the average for 3 tests.

Molar
concentration
of KCN

Age of culture
in days

Duration of test
in hours

Average O2 con-
sumption in mm.3
per hour
per million

Average Oi con-
sumption in mm.3
per hour per mm.3
of cell substance

Per cent
inhibition

17*

462

None

io-4

484

746

6.14

19.9

io-4

598

4.92

680

5.60

io-4

485

3.99

709

5.84

36.1

io-4

453

3.73

808

6.65

io-1

665

5.47

841

6.92

io-4

747

6.15

906

7.46

28.5

io-4

657

5.42

1360

11.20

10"4

788

6.49

16*

520

None

io-3

511

818

6.73

io-3

557

4.58

1516

12.48

io-3

605

4.98

1120

9.22

io-3

677

5.57

results of Dr. Libbie H. Hyman) indicates that P. coiidatniii is insensitive to
cyanide. One great difference in the work reported here and previous investiga-
tions carried out on the effect of cyanide on Paramecium is that in these experi-
ments suitable KCN-KOH absorption mixtures rather than pure KOH were used
in the manometer flasks to prevent absorption of HCN from the test solution.

The same procedures were followed here as for P. aurelia. The results are
presented in Table II.

As indicated by the results, much variation was found in the action of KCN
on Paramecium caiidatiim. In the first few tests very great difficulty was experi-

D. M. PACE

TABLE II

The effect of KCN on oxygen consumption in Paramecium caudatum. *, starved specimens;
5 day cultures, young specimens; all others, old specimens. Temperature, 25° C.; pH, 7.0 ± 0.2.
Average volume of one million paramecia, 591 mm.3 Each figure in columns 4 and 5 represents
the average for 3 tests.

Molar
concentration
of KCN

Age of culture
in days

Duration of test
in hours

Average Ch con-
sumption in mm.3
per hour
per million

Average O> con-
sumption in mm.3
per hour per mm.3
of cell substance

Per cent
inhibition

16*

1565

None

IO-5

1518

3273

5.53

15.5

10~5

2734

4.62

3734

6.33

io-5

3181

5.37

4420

7.47

io-5

2650

4.48

3040

5.14

None

10-'

3010

5.09

2700

4.56

io-4

1978

3.34

3787

6.40

io-4

2243

3.80

4270

7.22

10~J

2475

4.18

16*

1190

None

io-3

1280

3580

6.05

IO-3

2072

3.50

4590

7.76

io-3

1560

2.63

4170

7.05

io-3

2380

4.02

encecl. chiefly because some apparently minor details in manipulation were over-
looked and this may have had a very noticeable effect on the results. It was
suspected from the results of the first few tests that food played an important part
in the degree of sensitivity of these organisms to KCN. For this reason several
tests were conducted on this species under the same type of conditions as was used
for P. aitrelia, namely: (1) young paramecia (5 day cultures), (2) old paramecia
(15 to 19 day cultures) and (3) starved paramecia.

The results indicate that although there was great variation in some of them, the
young specimens show a greater sensitivity to KCN. The starved specimens
proved to be non-sensitive. In some tests there appeared to be an actual accelera-

EFFECT OF CYANIDE ON RESPIRATION

tion of CX consumption when starved P. candatiiin was put into KCN solutions but
the results may have been due to experimental error. They are not included in
the table. In one test (included in table) which was made upon old organisms,
there was no evidence of cyanide sensitivity ; no explanation can be given for this
exception.

The average inhibition of (X consumption found in old P. can-datum exposed to
solutions containing 10~r' M KCN was approximately 15 per cent; in solutions con-
taining 10~4 AI, 33 per cent; and in solutions containing 10~3 M, 42 per cent. In
young P. candatiiin exposed to 10~r' M KCN, respiratory inhibition was approxi-
mately 30 per cent; in solutions containing 10~4 M KCN, 42 per cent; and in solu-
tions containing 10 3 M, approximately 66 per cent. Thus, inhibition of oxidative
metabolism increases with increase in KCN concentration, and the degree of sensi-
tivity to cyanide seems to depend upon the quantity of food material present. This
is in agreement with the results of Hyman. Higher concentrations than 10~3 M
KCN were attempted but the results are meaningless because of such extreme
variations and for this reason they have not been included in this report.

Effect of dextrose on the degree of inhibition hv c\anidc

Many workers have reported that one of the factors in the sensitivity of the
respiratory mechanism to cyanide is the degree of carbohydrate saturation in the
cell. Keilin (1932) suggests that perhaps the most important factor concerned
with cellular sensitivity to cyanide is the concentration of carbohydrate. Com-
moner (1939) working with bakers' yeast, Emerson (1927) with Clilorclla, and
Hall (1941) with Colpidinui cmnpylinn, all found either a greater inhibition with
cyanide when dextrose was present or no inhibition without dextrose.

Since it is highly probable that a large portion of the food material of
Paramecium is carbohydrate and since it was found that the greatest sensitivity to
cyanide occurred when the greatest quantity of food was present, it was thought
advisable to run respiration tests with the organisms in a dextrose solution.

Old paramecia were selected and washed in the buffered test solution contain-
ing 0.01 M dextrose. Then the solution containing the paramecia was divided

TABLE III

The effect of KCN on Paramecium caudatum in a 0.01M dextrose-buffer solution. All the
organisms were taken from 16 to 19 day-old stock cultures. Temperature, 25° C.; pH, 7.0 ± 0.2.
Average volume of one million paramecia, 580 mm.3 Each figure represents the average for
3 tests.

Molar
concentration
of KCN

Age of culture
in days

Duration of test
in hours

Average O-i con-
sumption in mm.3
per hour
per million

Average O« con-
sumption in mm.3
per hour per mm.3
of cell substance

Per cent
inhibition

4550

7.84

10~4

2360

4.06

3860

6.65

io~4

1890

3.25

4120

7.10

10~4

1895

3.26

82 D. M. PACE

into two portions. To one portion, KCN was added to 10~4 M ; the other portion
was used as control. This experiment was repeated twice and the results are pre-
sented in Table III.

The results show that the rate of respiration in Parauicciiiin can datum is in-
creased with the addition of dextrose to the test solution. The average rate of
respiration in the dextrose-buffer solution for all tests without KCN added was
4170 mm3 per hour per million organisms as compared to an average 3470 mm3
in the same type of organisms tested in the buffer solution without dextrose (Table
II). They also show that there was an average inhibition of 51 per cent in O2
consumption in 10~4 M KCN in the dextrose-buffer solution which is much greater
than the average inhibition in 10~4 KCN without dextrose. The average inhibi-
tion for two experiments in which the latter solution was used, was 33.5 per cent;
in one of the experiments there was no inhibition whatever, but this has not been
included in the average.

DISCUSSION

Many factors may have contributed to the failure of earlier investigators to
find inhibition in respiration in Parauicciiiin when exposed to cyanide. Consider-
able error must have been caused by the absorption of free HCN by the KOH used
as absorption fluid. The initial inhibitory effect followed by an increase in oxygen
consumption noted in the results of Pitts (1932) and Lwoff (1934) is evidently
due to the fact that little attention was given to the rapid absorption of cyanide
(via distillation of HCN) by the absorption fluid. Hall (1941), using suitable
KOH-KCN mixtures as absorption media, proved conclusively that respiration in
Colpidium was cyanide sensitive.

In the investigations reported here, care wras taken to prevent distillation of
HCN over into the absorption fluid. However, there is another factor that may or
may not have been realized by these earlier workers, namely, the food content of
the paramecia with which they worked. It is possible that the organisms used by
them were taken from "old" cultures and hence had comparatively little food ma-
terial in them. If this be true, it explains their failure to find inhibition in respira-
tion, for, as reported above, sensitivity seems to depend, at least partly, upon the
food content of Paranieciiun. This very important factor was noted some twenty
years ago by Dr. Libbie Hyman (see footnote 2).

In these experiments, the organisms were taken from the culture solution,
washed, and placed in fresh test solution, and then put into manometer flasks, all
within 10-15 minutes. Thus in most of the tests the organisms were actually in
inorganic solution without food for 3.5 hours; in some tests 4.5-5.5 hours, but
rarely longer than this. During this time, very little change could be noted in food
vacuole content or size. It was also noted that respiration varied very little, if at
all, from the beginning to the end of a test. In other words, the decrease in food
content is so slight within this short period of time that there was no noticeable
change in rate of respiration.

Carbohydrate makes up a great portion of the food of Parameciitin. One of
the most important factors in the degree of sensitivity of respiration to KCN, etc.
is the concentration of carbohydrate in the cell. Thus when dextrose was added
to the buffer solution in which the respiration of Paramccinm candatnni wTas tested,
the per cent inhibition was greater than in the buffer solution without dextrose.

EFFECT OF CYANIDE ON RESPIRATION 83

SUMMARY

1. The oxygen consumption in Paramecium candatnm and Paramecinm aurclia
is partially inhibited by potassium cyanide.

2. The extent of inhibition by cyanide is dependent upon the food content of the
organisms as well as upon the concentration of cyanide in the solution.

3. In P. aurclia, starved specimens are insensitive to cyanide; old specimens are
not as sensitive as young. In 10~4 M KCN respiration in the old organisms was
inhibited by approximately 22 per cent while in the young organisms it was in-
hibited by approximately 40 per cent.

4. In Paraiucchtui candatnin, starved specimens were non-sensitive to KCN;
old specimens exposed to 10~3, 10~4, and 10 5 M KCN show, respectively, a 42, 33,
and 15 per cent inhibition in respiration. Young specimens, exposed to 10~3, 10~4,
and 10~ r> M KCN show, respectively, a 66, 42, and 30 per cent inhibition.

5. The inhibition in the rate of respiration in P. caiidatitin was greater in buffer
solution plus dextrose (0.01 M) than in the same solution without dextrose.

6. The effect of cyanide on respiration in Paramecium depends upon the degree
of saturation of the respiratory mechanism with carbohydrate.

LITERATURE CITED

BAKER, E. G. S., AND BAUMBERGER, J. P., 1941. The respiratory rate and the cytochrome con-
tent of a ciliate protozoan (Tetrahymena geleii). /. Cell, and Comp. Ph\sioL, 17:
285-303.

CHILD, C. M., 1941. Patterns and Problems of Development. University of Chicago Press,
Chicago, 111. 811 pp.

COMMONER, B., 1939. The effect of cyanide on the respiration of bakers' yeast in various con-
centrations of dextrose. /. Cell, and Comp. Physiol., 13 : 121-138.

EMERSON, R., 1927. The effect of certain respiratory inhibitors on the respiration of Chlorella.
/. Gen. Physiol., 10 : 469-477.

GERARD, R. W., AND HYMAN, L. H., 1931. The cyanide sensitivity of Paramecium. Amer. J.
Physiol., 97: 524-525.

HALL, R. H., 1941. The effect of cyanide on oxygen consumption of Colpidium campylum.
Physiol. Zool, 14 : 193-208.

KEILIN, D., 1932. Cytochrome and intracellular respiratory enzymes. Ergeb. der Ensymfor-
schung, Bd. 2: 239-271.

KREBS, H. A., 1935. Aletabolism of amino-acids. III. Deamination of amino acids. Biochcin.
J.. 29: 1620-1644.

LUND, E. J., 1918. Rate of oxidation in P. caudatum and its independence of the toxic action
of KCN. Amer. J. Physiol.. 45: 365-373.

LWOFF, M., 1934. Sur la respiration du Cilie Glaucoma piriformis. C. R. Soc. Biol., Paris,
115: 237-241.

PACE, D. M., AND BELDA, W. H., 1944. The effects of potassium cyanide, potassium arsenite,
and ethyl urethane on respiration in Pelomyxa carolinensis. Biol. Bull., 87 : 138-144.

PACE, D. M., AND KIMURA, K. K., 1944. Effect of temperature on respiration in Paramecium
caudatum and Paramecium aurelia. /. Cell, and Comp. Physiol., 24: 173-183.

PITTS, R. F., 1932. Effect of cyanide on respiration of the protozoan, Colpidium campylum.
Prof. Soc. E.vp. Biol N. Y., 29 : 542.

SATO, T., AND TAMIYA, H., 1937. Uber die Atmungsfarbstoffe von Paramecium. Cytologia.
Fujii Jubilee Volume, pp. 1133-1138.

SHOUP, C. S., AND BOYKIN, J. T., 1931. The sensitivity of Paramecium to cyanide and effects
of iron on respiration. /. Gen. Physiol., 15: 107-118.

SPACHT, H., 1934. Aerobic respiration in Spirostomum ambiguum and the production of am-
monia. /. Cell, and Comp. Physiol., 5 : 319-333.

THE AGGLUTINATION OF STARFISH SPERM BY FERTILIZIN 1

CHARLES B. METZ 2

William G. Kcrckhoff Laboratories of the Biological Sciences,
California Institute of Technology. Pasadena, California

Agglutination of starfish sperm by specific egg water (supernatant sea water
from egg suspensions) has never been clearly demonstrated. Glaser (1914) and
Woodward (1918) reported a strong agglutination of Asterias forbesii sperm by
homologous egg water, but Just (1930) was unable to confirm this work. At-
tempts to demonstrate agglutination of sperm by egg water in other species of
starfish have failed. Thus Loeb (1914) observed no reaction in Asterias (prob-
ably Pisaster) ochraccns. and Tyler (1941) had a similar result with Patina
mini at a. From this it might appear that fertilizin is not present in starfish egg
water. However, Tyler found that treatment of Patina sperm with egg water
lowered the fertilizing power of the sperm. Tyler (1941, 1942) interpreted this
as support for his view that fertilizin may exist naturally in a non-agglutinating
"univalent" form. An individual molecule of such univalent fertilizin should have
but one combining group capable of reacting with groups (antifertilizin) on the
sperm surface. On the basis of the Marrack-Heidelberger (1938) lattice theory,
univalent fertilizin should therefore combine with but not agglutinate these cells.
Tyler suggests that such univalent fertilizin may be present quite generally in
forms showing no agglutination of sperm by egg water. He therefore supports the
belief held by Lillie (1919) and Just (1930) that fertilizin occurs universally.

In view of the concept of univalent fertilizin and the provisional status of the
starfish with respect to sperm agglutination by egg water, it is of some interest
that sperm of certain starfish agglutinate when mixed with homologous egg water
and an "adjuvant." The first adjuvant found was lobster (Paniilints) serum.
The agglutination reaction was discovered accidentally in the course of studies on
the natural agglutinins in lobster serum (Tyler and Metz, 1944). In an attempt
to separate natural agglutinins for Patiria eggs and sperm, the serum was treated
with eggs and then titrated for sperm agglutinins. The treatment with eggs in-
creased the sperm agglutinin titer several fold. Investigation of this unexpected
result showed that sperm absorbed lobster serum (freed of natural sperm agglu-
tinins), when mixed with Patiria egg water, agglutinated Patiria sperm. Tests on
other material showed the presence of adjuvant in hen's egg white. A preliminary
report (Metz, 1944) on this work has already appeared. The studies confirm
Tyler's view that fertilizin is present in Patiria egg water. However, the experi-
ments indicate that this fertilizin is multivalent. Data are given which suggest
that normal Patiria sperm is "univalent" with respect to exposed antifertilizin
groups, but that more of these groups are "exposed" by the adjuvant.

1 Submitted to the Graduate School of the California Institute of Technology in partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

- Present address : Shanklin Laboratory of Biology, Wesleyan University, Middletown,
Conn.

AGGLUTINATION OF STARFISH SPERM 85

MATERIAL AND METHODS

The Pacific webbed star, Patiria ininiata, was used as standard material. The
Pacific star Pisaster ochraccus, the Pacific sand star Astropecten sp. and the At-
lantic Asterias forbesii were used in confirmatory and specificity tests.

Egg and sperm suspensions were prepared from ripe extirpated gonads. These
organs were minced in a measured volume of sea water and then filtered through
bolting cloth to remove the gonadal tissue. The difference in volume of the filtrate
and the sea water initially added gives the volume of "dry" (undiluted) material.
Egg and sperm dilutions were reckoned from this "dry" volume. Egg water solu-
tions were obtained by drawing off the supernatant from standing egg suspensions
(25-50 per cent of dry eggs iiT sea water), or by heating such suspensions and
filtering or centrifuging off the eggs.

Lobster (Panulirus interruptus) serum was obtained by drawing blood from
the heart and allowing it to clot. After syneresis the serum was drawn off. Since
Panulirus serum contains natural heteroagglutinins for sperm of various organisms
(Tyler and Metz, 1944) including Patiria, Pisaster and Astropecten. it is im-
practical to use the untreated serum. By absorption with Patiria sperm the natural
agglutinins for Pisaster and Astropecten as well as Patiria sperm can be removed.
Such absorbed serum was used as the adjuvant for sperm of all three species. For
reasons of economy both in material and time, hen's egg white was used as the
adjuvant in the later experiments. This material was made isotonic by adding one
volume of concentrated (1.73 X ) sea water. It was then diluted to 20 per cent with
normal sea water and filtered to remove the mucin, chalazae and other insoluble
.material. This diluted egg white was usually heated to 100° C. and filtered or
centrifuged since this procedure increased its activity several fold. Hen's egg
white does not contain natural agglutinins for Patiria. Pisaster or Astropecten
sperm. Thus, initial absorption with starfish sperm was not necessary.

Assays of unknown egg water were made by diluting the unknown solution in
twofold steps with sea water and then adding constant amounts of adjuvant-treated
sperm to each dilution of unknown egg water. Adjuvant was titrated in a similar
manner. However, when titrating adjuvant, constant amounts of sperm suspen-
sion were added to the dilutions of unknown adjuvant. Subsequently, constant
amounts of egg water were added to each adjuvant dilution. Less satisfactory re-
sults are obtained if any other order of mixing is employed in this test. In all
cases the presence or absence of agglutination was determined by microscopical
examination one to several minutes after mixing. Titers were recorded as the
highest dilution of unknown showing agglutination of the test sperm.

The apparatus and methods used in ultraviolet irradiation have been described
in a previous article (Metz, 1942).

ACTIVATION AND AGGLUTINATION OF STARFISH SPERM

In these studies no definite agglutination of Patiria, Pisaster, Astropecten or
Asterias sperm was observed following the addition of homologous egg water.
However, Patiria sperm suspensions frequently appeared "granular" after this
treatment. These microscopic "granules" consisted of two or three sperm fixed
together and represent a plus-minus agglutination reaction. Various devices such

86 CHARLES B. METZ

as centrifugation were employed in an attempt to bring this reaction to a distinct
agglutination, but all of them failed.

The starfish sperm in dilute (0.5 to 1.0 per cent) sea water suspension were
virtually immobile. The cells did not respond to treatment with fresh sea water
(lowering CO., tension) or with homologous egg water. Starfish sperm thus
differ from Arbacia and Nereis sperm which become more active when diluted
with sea water (Lillie, 1913; Just, 1930), and from Arbacia (Lillie, 1913), Strongy-
hcentrotits (Tyler, 1939) and Mcgathnra sperm (Tyler, 1940) which become in-
tensely motile when mixed with homologous egg water.

Patiria, Pisastcr and Astropecten sperm, although refractory to treatment with
sea water and egg water, nevertheless became intensely active when treated with
isotonic hen's egg white or the serum of the lobster (Panulirus), fish (Crassius),
hen, or rabbit. Furthermore, adjuvant-treated sperm of these starfish agglu-
tinated strongly on addition of homologous egg water. Astcrias was tested on
three successive seasons. The sperm became intensely active when treated with
isotonic hen's egg wrhite. Weak agglutination sometimes occurred after addition
of homologous egg water to the sperm egg white suspension. Unfortunately, the
agglutination was so weak and occurred so irregularly that quantitative studies
could not be made.

In Patiria the agglutination resulting from treatment of sperm with adjuvant
and egg water was exclusively head to head. Each clump consisted of a central
mass of sperm heads tightly bound together, and a peripheral region of free tails
which projected out radially from the central mass of heads. Patiria thus differs
from Megathura, since sperm of the latter agglutinate tail to tail as well as head to
head (Tyler, 1940). The clumped Patiria sperm soon became immobile even
though the free sperm remained active for an hour or more. The spontaneous
reversal of agglutination so characteristic of the sea urchin occurred to a limited
extent only after the free sperm had become inactive.

PROPERTIES OF PATIRIA FERTILIZIN

Fertilizin may be defined by the following properties: (1) it combines with
(but does not necessarily agglutinate) sperm, (2) it is highly specific in this re-
action, and (3) it is obtained primarily from eggs. Studies on the role of egg
water in the agglutination of treated sperm show that Patiria egg water has these
properties.

Absorption of Patiria egg water by sperm. A direct combination between sea
urchin fertilizin and sperm may be demonstrated by absorption of egg water with
sperm, or by neutralization of egg water with appropriate sperm extract (Lillie,
1913; Frank, 1939). Similarly, it may be shown that sperm-absorbed Patiria
egg water will no longer agglutinate treated sperm. Indeed, complete exhaustion
of the egg water may be attained even in the absence of adjuvant. In a typical
experiment 20 drops of Patiria egg water were mixed with 22 drops of concentrated
(25-50 per cent) Patiria sperm. The mixture was set aside to allow for reaction.
Twenty drops of the same egg water were mixed with 22 drops of sea water to serve
as a control. After centrifugation the fluid of both tubes was titrated with adjuvant-
treated (0.5-1 per cent) sperm. The undiluted absorbed egg water did not ag-
glutinate the sperm, whereas the control unabsorbed egg water clumped the sperm

AGGLUTINATION OF STARFISH SPERM

even at a dilution of 1/256 of full strength. Other controls showed that sperm
without adjuvant were not agglutinated by control or absorbed egg water, or by
the adjuvant (Patina sperm-absorbed Panitlirus serum). Thus a substance (fer-
tilizin) is present in Patiria egg water which will combine with specific sperm in-
dependently of the adjuvant. The adjuvant is required only for agglutination.

Specificity of starfish fertilizin. The reaction between sperm and fertilizin is
characterized by a high order of specificity (Tyler, 1940). Cross tests between
Patina. Pisaster and Astropecten sperm and fertilizin show that these starfish are
not exceptional in this respect. Sperm suspensions of the three species were
treated with Patiria sperm-absorbed Pannlirus serum, and then cross tested with
the egg waters of the three species. The data are given in Table I.

TABLE I

Specificity of Patiria, Pisaster and Astropecten egg waters

Patiria
sperm

Pisaster
sperm

Astropecten
sperm

Patiria

egg +
water

adjuvant

+ + +

—

sea water

—

Pisaster

egg +
water

adjuvant

—

+ + +

—

sea water

—

+ +

—

A stropecten

egg +
water

adjuvant

—

sea water

—

adjuvant
+
sea water

—

Patiria
sperm supernatant

—

It will be seen that Patiria and Pisaster egg waters agglutinated only homol-
ogous sperm. Thus the species specificity rule holds for these two forms. In
this experiment Pisaster egg water clumped homologous untreated sperm. This
reaction did not occur with predictable regularity. The reaction between Astro-
pecten egg water and homologous sperm was doubtful. This may be ascribed to
neutralization of the Astropecten egg water by the Patiria sperm supernatant pres-
ent in the adjuvant solution. The relationship here is somewhat involved. With
the exception of the reaction between Patiria sperm supernatant and Astropecten
egg water, the reactions were species specific.

The source of Patiria fcrtilizin. Only egg water prepared from suspensions of
Patiria eggs possessing their normal gelatinous coats agglutinated species sperm
in the presence of the adjuvant. Blood from female animals did not have this
effect. Thus it may be concluded that a specific substance is obtained from star-
fish eggs which will react with and under certain conditions agglutinate species

CHARLES B. METZ

sperm. This then gives clear and direct support to Tyler's (1941) view that
fertilizin exists in the Patiria egg water.

THE NATURE OF PATIRIA FERTILIZIN

Tyler (1941) concluded that Patiria fertilizin was univalent for combining
groups complementary to sperm. It follows from the lattice theory that such
univalent fertilizin must become multivalent to agglutinate the sperm. The ad-
juvant should then convert the natural univalent Patiria fertilizin to a multivalent,
agglutinating form. However, another possibility in accord with the lattice theory
is that the fertilizin is multivalent but the sperm is normally univalent. The re-
sults of the following experiments favor this latter view.

Effect of ultraviolet light on Patiria fertilizin. Sea urchin fertilizin can be con-
verted to the univalent form by proper exposure to heat, enzymes, x-radiation and
ultraviolet light (Tyler, 1941; Metz, 1942). Such treated fertilizin will not ag-
glutinate sperm but it will combine with sperm rendering the sperm unagglutinable
by untreated fertilizin. To test for the possibility of a similar action, Patiria fer-
tilizin was exposed to ultraviolet irradiation. It was found that irradiated Patiria
fertilizin will not agglutinate adjuvant-treated homologous sperm, and normal fer-
tilizin will not subsequently agglutinate the sperm that has been treated with
irradiated fertilizin. Thus it is possible that the natural fertilizin is multivalent
and the irradiated material is true univalent fertilizin. The data from a typical
experiment are given in Table II.

TABLE II

Destruction of agglutinating power of Patiria fertilizin by ultraviolet light and agglutination

inhibiting properties of this fertilizin

Irradiated

Solution

Irradiated

Control

fertilizin

sea water

fertilizin

control

fertilizin

Reaction of adjuvant-

—

+ + + +

—

+ + + +

treated sperm

Two stender dishes each containing 5 cc. of a Patiria fertilizin solution and one
dish containing 5 cc. of sea water were irradiated for 220 minutes. The control
fertilizin sample was screened from the ultraviolet light by a "noviol C" filter.
After the irradiation the control and irradiated fertilizin samples were tested for
agglutinin activity by mixing 2 drops of hen's egg white treated sperm ( 1 % ) with
2 drops of each fertilizin solution. At the same time 2 drops each of the sperm
and irradiated sea water were mixed. It will be seen that the irradiated fertilizin
was inactive whereas the control strongly agglutinated the sperm. After this ex-
amination one drop of unirradiated test fertilizin was added to the irradiated fer-
tilizin-adjuvant-sperm mixture and one drop to the irradiated sea water-adjuvant
sperm. In this test for inhibition of agglutination it will be seen that sperm treated

AGGLUTINATION OF STARFISH SPERM 89

with irradiated fertilizin did not agglutinate upon subsequent addition of normal
fertilizin, whereas the sperm treated with irradiated sea water reacted strongly.

Agglutination of adjuvant-free Patina sperm b\ fertilizin. More definite evi-
dence for the multivalent nature of starfish fertilizin was obtained from a study of
the effect of adjuvant on sperm. Adjuvant was added to Patina sperm and then
removed. Such adjuvant free sperm agglutinated on addition of natural fertilizin.
Twenty drops of 0.5 per cent sperm were mixed with 10 drops of isotonic hen's
egg white. A control sample consisted of 20 drops of 0.5 per cent sperm plus 10
drops of sea water. Both samples were centrifuged and the packed sperm was
resuspended in 20 drops of sea water. Two drops of sperm from each were tested
with Patina fertilizin. The control suspension did not react whereas the sperm
centrifuged from the egg white agglutinated moderately. The suspensions were
recentrifuged and the supernatants tested and found free of adjuvant. The sperm
masses were resuspended in 16 drops of sea water after the second centrifugation
and tested. The control sperm did not react to fertilizin whereas the sperm previ-
ously treated with adjuvant agglutinated weakly.

This experiment was not confirmed with Astropecten. Astropecten sperm
after centrifugation from hen's egg white solution were not agglutinated by homol-
ogous fertilizin alone, although this sperm reacted strongly when both egg white
and fertilizin were added.

It seems clear then that Patina fertilizin will agglutinate sperm after the ad-
juvant has been removed from the sperm. It may therefore be concluded that the
natural Patina fertilizin is multivalent.

UNIVALENT SPERM

Evidence has just been presented to show that natural Patiria fertilizin is
multivalent and capable of agglutinating sperm. It follows that the normal sperm
is incapable of agglutination. The adjuvant must then convert the sperm to an
agglutinating condition.

It seems unlikely that stimulation of the normally immobile sperm to intense
activity is of any considerable importance in this adjuvant-fertilizin agglutination
of Patiria sperm since immunological doctrine does not require motility of cells
for agglutination. Thus non-motile bacteria and erythrocytes agglutinate strongly
when mixed with specific antibody. Furthermore, heat killed sea urchin sperm
agglutinate strongly on addition of fertilizin. However, heat killed Patiria sperm
did not react when mixed with fertilizin and adjuvant. The deficiency of the
normal sperm must then involve the antigenic structure of the cell surface. For
agglutination to occur the area of the sperm surface containing groupings com-
plementary to fertilizin must be rather extensive. If this region of the sperm
surface were limited in extent and contained but a few or even a single antifertilizin
group, the sperm could be considered "univalent" for this particular antigen. Such
sperm should not agglutinate when mixed with complementary agglutinin (fer-
tilizin). At best only two or three sperm could clump together. This condition
is occasionally observed when untreated Patiria sperm and fertilizin are mixed.
It has been described as the "granular" reaction.

Absorption of Patiria fertilisin by treated and normal sperm. If normal Patiria
sperm are "univalent" with respect to exposed antifertilizin groups, the cells must

CHARLES B. METZ

be made multivalent before they can be expected to agglutinate. The adjuvant is
believed to effect such a conversion to the multivalent form by "exposing" latent
or unreactive antifertilizin present on or near the sperm surface. Treated sperm
then should bind more fertilizin than the normal "univalent" sperm. One of three
absorption experiments demonstrating this is recorded in Table III.

TABLE III

Absorption of fertilizin by sea water and egg white treated Patiria sperm

Absorbing Mixtures

Tube I

Tube II

Tube III .

Tube IV

0.5 cc. sea water
0.5 cc. fertilizin
0.5 cc. sperm

0.5 cc. egg white
0.5 cc. fertilizin
0.5 cc. sperm

0.5 cc. egg white
0.5 cc. fertilizin
0.5 cc. sea water

0.5 cc. sea water
0.5 cc. fertilizin
0.5 cc. sea water

Titration of absorbed fertilizin solutions

Dilution of
absorption
supernatant

1/2

1/4

1/8

1/16

1/32

1/64

1/128

1/256

1/512

1/1024

1/2048

Tube I

Tube II

Tube III

Tube IV

Four absorption tubes were prepared as indicated in the table. Fifty per cent
sperm was used in the absorption and raw isotonic hen's egg white was employed as
adjuvant. The tubes were refrigerated for nine hours to allow for complete re-
action, and then centrifuged. The supernatants were then titrated for fertilizin
with one per cent treated sperm. In absorption tubes III and IV sea water was
substituted for the sperm added to tubes I and II. No adjustment was made in
the titration for the volume of absorbing sperm removed from I and II by cen-
trifugation. This is justified since the titration was made on a comparative basis
and tubes III and IV represent controls for neutralization of fertilizin by egg white.
Furthermore, the error in absolute values introduced by this involves something
less than % of a dilution and therefore is well within the error of the method.
Likewise, no adjustment was made in the supernatant of tube I for the adjuvant
present in the absorption supernatant of tube II. Such adjustment was apparently
unnecessary since the titers of the control tubes III and IV were the same (1024)
The titers of these tubes also show that the egg white does not neutralize fertilizin.
Comparison of tubes I and III shows that the sea water-sperm mixture caused an
8- to 64-fold drop in fertilizin concentration. However, in tube II (titer 0)

AGGLUTINATION OF STARFISH SPERM

the adjuvant-sperm mixture completely exhausted the fertilizin. The striking
difference in the titers of tubes I and II (128 and 0 respectively) demonstrates
clearly that treated sperm has a greater fertilizin binding capacity than normal
sperm.

EFFECT OF THE ADJUVANT ON THE FERTILIZING POWER OF PATIRIA SPERM

Since the adjuvant increases the fertilizin binding power of sperm and also the
motility of these cells, it seemed likely that treated sperm would be unusually
effective in fertilization. Several experiments comparing the treated and normal
sperm in this respect showed this to be the case. The results of one such experi-
ment are given in Table IV.

TABLE IV

The effect of hens egg white on the fertilizing power of Patiria sperm

Egg white

Sea water

Egg white +

Sea water +

treated sperm

Patiria eggs

Sperm

% cleavage

1/2

94% (75)*

38% (95)*

0.7% (152)*

0.0% (118)*

1/4

95% (66)

0% (58)

1/8

89% (45)

7.4% (54)

1/16

88% (50)

6.8% (74)

1/32

89% (53)

2.0% (65)

* Total number of eggs counted.

A fresh one per cent sperm suspension was divided into two parts. One part
was diluted serially (in twofold steps) with boiled isotonic hen's egg white. The
other part was diluted similarly but with sea water. Sperm dilutions are given as
the dilution of one per cent sperm added to the eggs. One drop of each sperm
suspension was added to twelve drops of Patiria eggs in 6 cc. of sea water. To
control for parthenogenesis one drop of egg white was added to one dish of eggs
and a drop of sea water to a second dish. The eggs were examined for cleavage
three hours after addition of sperm.

Although the number of eggs counted was small it can readily be seen that the
egg white treatment greatly increased the fertilizing power of the sperm. Even
at the lowest dilutions the treated sperm was twice as effective as the untreated
cells. At high dilutions the treated sperm fertilized nearly 90 per cent of the eggs
whereas the normal sperm fertilized less than 10 per cent. Gray (1915) has re-
ported a similar result with alkali treated Asterias glacialis sperm.

SPECIFICITY OF THE ADJUVANT

Although no exhaustive search was made for different sources of adjuvant, a
number of unrelated preparations were encountered which stimulated Patiria sperm
and rendered it agglutinable by fertilizin. These preparations included Pannliriis,
rabbit, fish (Crassius), and hen sera, and hen's egg white. Thus the source of the
adjuvant is not highly specific.

92 CHARLES B. METZ

PROPERTIES OF THE EGG WHITE ADJUVANT

The adjuvant action can not be attributed to the high pH of raw egg white
(Needham. 1931) since the material is active at sea water pH. Therefore, pre-
liminary attempts were made to characterize an "active principle" in the hen's egg
white. The agent is quite heat stable. Its activity was retained even after several
hours at 100° C. In fact heating increased the activity of the egg white several
fold. Ultraviolet light had a similar effect. This suggests the release of inactive
bound agent. The "active principle" was quite nondialyzable both before and
after heating. It was soluble in saturated ammonium sulfate, but insoluble in
strong acetone and alcohol. Thus it is probably neither ordinary protein nor
lipoid.

DISCUSSION

From the evidence presented it is concluded that fertilizin is obtained from
Patiria eggs, and that this fertilizin, although it does not agglutinate normal sperm,
is a multivalent agglutinin that reacts with the normal sperm. It is further be-
lieved that the exposed antifertilizin of normal Patiria sperm is limited to a small
area of the sperm surface and contains only a few or even a single combining group
complementary to fertilizin. For practical purposes such sperm may be consid-
ered "univalent." It is necessary to assume that some antifertilizin is exposed on
the normal sperm to explain the absorption of fertilizin by such sperm and to
account for the "granular" agglutination reaction. This then is a reversal of
Tyler's (1941, 1942) view. He believed that the normal Patiria fertilizin was
"univalent" and that the sperm was multivalent.

The various adjuvant solutions stimulate the sperm to intense motility and
presumably expose more antifertilizin on the sperm surface. The latter effect is
believed to be the essential one in rendering the sperm agglutinable. This action
of the adjuvants bears a superficial resemblance to the "transformation" of human
erythrocytes by an enzyme present in certain bacterial filtrates (Thomsen effect).
Any human serum will agglutinate these transformed cells. There are several im-
portant differences between the process of erythrocyte transformation (Friedenrich,
1930) and the action on starfish sperm. The transformation requires a consider-
able period of time (15 minutes to several hours), is irreversible, and involves a
fixation and subsequent release of the transforming principle: The action on
Patiria sperm takes place very rapidly, the process reverses slowly when the ad-
juvant is removed, and it involves no fixation of the adjuvant. Repeated attempts
failed to show any neutralization or absorption of egg white adjuvant by sperm
or sperm-fertilizin mixtures. Friedenrich (1930) believes that a new agglutinogen
is developed which is not present in latent or unreactive form on the normal ery-
throcyte. However, the case of Patiria sperm is more easily explained by assum-
ing that a considerable amount of antifertilizin is in latent form on or near the cell
surface.

Di Macco's (1923) "coagglutination" of sheep erythrocytes by mixtures of
ricin and guinea pig serum also resembles the fertilizin-adjuvant agglutination of
Patiria sperm. Neither ricin nor guinea pig serum alone agglutinated the sheep
cells. Absorption of the separate solutions with cells failed to remove the active
agents. Agglutination failed to occur if the ricin and guinea pig serum were mixed

AGGLUTINATION OF STARFISH SPERM 93

first and the sheep cells added subsequently. Thus neither of the necessary agents
reacted directly with the cells. Di Macco concluded that agglutination of sheep
cells resulted from a reaction between the cells and an evanescent ricin-serum
complex formed at a critical stage in the reaction between these substances. It is
apparent, then, that the mechanism of the coagglutination is fundamentally differ-
ent from the fertilizin-adjuvant agglutination of Patiria sperm.

The striking difference in fertilizing power of normal and adjuvant-treated
sperm can be explained by the motility of the cells. Furthermore, this effect should
be expected, regardless of motility, from the recent views of Tyler (1941). He
has shown that fertilizin treatment lowers the fertilizing power of Patiria sperm
and explained this by assuming that at fertilization a union occurs between anti-
fertilizin on the sperm and fertilizin at the egg surface. If all of the sperm anti-
fertilizin is bound by free fertilizin, then no reaction can occur between sperm and
the surface of the egg. It follows from this that the normal univalent sperm
would have much less chance of reaching the egg surface in an unsaturated con-
dition than would the multivalent sperm. At present it is impossible to judge
the relative importance of the intense motility and the multivalency of the ad-
juvant-treated sperm in this fertilization effect. If this increased fertilizing power
should be found in species that regularly give low percentages of fertilized eggs,
it might be useful for technical purposes.

SUMMARY

I. Starfish sperm does not ordinarily agglutinate when treated with homologous
fertilizin. However, when the sperm of some species (Patiria miniata, Pisastcr
ochraccus, Astropecten sp.) is treated with certain adjuvants the cells become
intensely active and agglutinate when fertilizin is added. This reaction provides
a means for studying the relationship between starfish sperm and fertilizin.

II. Patiria sperm will combine with homologous fertilizin and remove it from
solution even in the absence of the adjuvant.

III. Cross tests between Patiria, Pisastcr and Astropecten sperm and fertilizin
solutions revealed no cross agglutination reactions.

IV. It is concluded that Patiria fertilizin is multivalent, since irradiated fer-
tilizin will not agglutinate treated sperm but will inhibit the agglutination of such
sperm by normal fertilizin ; and since normal fertilizin will agglutinate sperm which
has been freed of adjuvant.

V. It is suggested that normal Patiria sperm possesses but a single antifertilizin
combining group and that more such groups are exposed on the sperm surface
through the action of the adjuvant. Experiments which show that the fertilizin
binding power of sperm is increased by the adjuvant support this view.

I am most grateful to Dr. Albert Tyler for the aid and encouragement he has
given throughout the course of the work.

LITERATURE CITED

Dr MACCO, G., 1923. Ueber die coagglutinierende und prazipitierende Wirkung des Rizins.
Zeitschr. f. Immunit., 38 : 467-488.

FRANK, J. A., 1939. Some properties of sperm extracts and their relationship to the fertiliza-
tion reaction in Arbacia punctulata. Biol. Bull., 76 : 190-216.

94 CHARLES B. METZ

FRIEDENRICH, V., 1930. The Thomscn Hemagglutination phenomenon. Levin and Munks-

gaard, Copenhagen.
GLASER, O., 1914. A quantitative analysis of the egg secretions and extracts of Arbacia and

Asterias. Biol Bull., 26: 367-386.
GRAY, J., 1915. Notes on the relation of spermatozoa to electrolytes and its bearing on the

problem of fertilization. Quart. Jour. Microscopical Science, N.S., 61: 119-126.
HEIDELBERGER, M., 1938. Chemistry of amino acids and proteins, Chap. XVII. C. C. Thomas,

Springfield, 111.
JUST, E. E., 1930. The present status of the fertilizin theory of fertilization. Protoplasma, 10:

300-342.
LILLIE, F. R., 1913. Studies of fertilization. V. The behavior of the spermatozoa of Nereis

and Arbacia with special reference to egg extractives. Jour. E.vp. Zoo/., 14: 515-574.
LILLIE, F. R., 1919. Problems of fertilization. University of Chicago Press, Chicago.
LOEB, J., 1914. Cluster formation of spermatozoa caused by specific substances from eggs.

Jour. Exp. Zoo/., 17: 123-140.
MARRACK, J. R., 1938. The chemistry of antigens and antibodies. Medical Research Council,

Special Report Series, No. 230, London.
METZ, C. B., 1942. The inactivation of fertilizin and its conversion to the "univalent" form

by x-rays and ultraviolet light. Biol. Bull., 82 : 446-454.
METZ, C. B., 1944. Agglutination of starfish sperm by homologous egg water. Anat. Rec., 89 :

559.

NEEDHAM, J., 1931. Chemical embryology, Vol. I. Macmillan Co., New York.
TYLER, A., 1939. Crystalline echinochrome and spinochrome : their failure to stimulate the

respiration of eggs and sperm of Strongylocentrotus. Proc. Nat. Acad. Sci., 25 : 523-

528.
TYLER, A., 1940. Sperm agglutination in the keyhole limpet Megathura crenulata. Biol. Bull.,

78: 159-178.
TYLER, A., 1941. The role of fertilizin in the fertilization of eggs of the sea urchin and other

animals. Biol. Bull., 81 : 190-204.
TYLER, A., 1942. Specific interacting substances of eggs and sperm. Western Jour. Surgery,

Obstetrics and Gynecoloyy, 50 : 126-138.
TYLER, A., AND C. B. METZ, 1944. Natural heteroagglutinins in lobster serum. Anat. Rec., 89 :

568.
WOODWARD, A. E., 1918. Studies on the physiological significance of certain precipitates from

the egg secretions of Arbacia and Asterias. Jour. E.vp. Zoo/., 26 : 459-497.

COCHLIOPHILUS DEPRESSUS GEN. NOV., SP. NOV. AND COCHLIO-

PHILUS MINOR SP. NOV., HOLOTRICHOUS CILIATES FROM

THE MANTLE CAVITY OF PHYTIA SETIFER (COOPER)

EUGENE N. KOZLOFF
Department of Zoology, University of California

INTRODUCTION

Examination of specimens of the pulmonate snail Phytia sctijer (Cooper) *
from salt marshes bordering San Francisco Bay disclosed the presence of two
closely related species of flattened holotrichous ciliates within the mantle cavity.
A new genus, Cochliophilus, is proposed to include these ciliates, which will be
described herein as Cochliophilus deprcssns gen. nov., sp. nov. and Cochliophilus
minor sp. nov.

I wish to express my appreciation to Professor S. F. Light and Professor
Harold Kirby for their interest and helpful advice during the progress of this
investigation.

TECHNIQUE

Phytia sctijer occurs under matted vegetation and debris in salt marshes and in
the vicinity of brackish water ponds on the Pacific Coast of central and northern
California. Material for this study was collected at several localities along the
east shore of San Francisco Bay at Oakland and Berkeley.

For observation of the living ciliates the shell of the snail was carefully re-
moved and the anterior part of the animal crushed in a drop of sea water on a
slide. Fixation of the organisms for permanent preparations was accomplished
by liberating them in this manner on a coverglass and then dropping the cover-
glass smear-side down onto the surface of the fixative in a Petri dish.

Staining with iron hematoxylin gave good results following the fixatives of
Schaudinn, Champy, Bouin, and Heidenhain ("susa"). For a study of the ciliary
system the method devised by Bodian (1936, 1937) for impregnation with activated
silver albumose (protargol) was used after fixation in Hollande's cupric-picro-
formol mixture. The Feulgen nuclear reaction was tried with success on material
fixed in a saturated aqueous solution of mercuric chloride with 5 per cent of glacial
acetic acid.

DESCRIPTION OF SPECIES

There is no agreement among protozoologists in regard to the orientation for
descriptive purposes of compressed ciliates in which the cytostome is situated
along the margin of the flattened body or displaced to the surface opposite that in

1 Dall (1921) has implied that the species described by Cooper (1872) is distinct from
Phytia myosotis (Drap.) of Europe and the Atlantic Coast of North America. No conclu-
sive evidence has been presented to support or to refute this contention.

96 EUGENE N. KOZLOFF

contact with the substrate. Hentschel (1924), writing of Entodiscus (Crypto-
chilum} borealis, stated that "since convention dictates that the side on which the
mouth is situated shall be called ventral, we must say that the animal is flattened
from side to side." Reichenow (1927-29) applied this scheme to Conchophthirus,
as did also Kahl (1931, 1934) and Raabe (1932, 1934b).

De Morgan (1925), in his description of Kidderia (Conchophthirus} mytili,
considered the concave under-surface to be ventral and the position of the cytostome
to be lateral. Kidder (1933b) recognized the oral surface of Kidderia mytili as
the "physiological ventral surface," but for purposes of clearness accepted De
Morgan's plan of orientation. In the present paper I will follow De Morgan and
Kidder in referring to that surface of the body most often found in contact with the
substrate as ventral. The lateral margin on which the cytostome is situated will
be referred to as the oral margin, and the opposite side as the aboral margin.

Cochliophilus depresses gen. nov., sp. nov. (Figs. 7 and 2)

The body outline as seen from the dorsal or ventral aspect is ovoid, often some-
what truncate at the posterior end. A view from the oral or aboral margin shows
this ciliate to be much flattened, the ventral surface being slightly concave and the
dorsal surface convex. In some individuals the curvature of the dorsal surface
appears to be less regular than in others.

Twenty living individuals taken at random ranged from 70 ju, to 107 /JL in length
and from 47 ^ to 77 ju, in width, averaging about 93 /A by 63 p.. The thickness varied
from 11 /A to 16 fji. The relation of the length to the width is not the same in all
specimens. Fixation of the organisms on coverglasses produced some shrinkage
and frequently also distortion of shape due to compression.

The elongated peristomal area is situated in the posterior fourth of the body.
Specialized ciliary elements wrhich will be described presently extend from the an-
terior end of the peristomal indentation to the cytostome. That part of the peri-
stomal area lying posterior to the cytostome is naked.

A well-defined pharynx is not present. I prefer to regard the irregular tubular
structure which passes from the cytostome into the cytoplasm as the gullet. This
gullet is difficult to see in living individuals, but in fixed material is demonstrable
following staining in iron hematoxylin. As it approaches the macronucleus the
gullet widens out and its boundaries become inexact.

A thin pellicle covers the body. Flexure of the pellicle in this ciliate is rarely
noted, and then only when the animal comes in contact with solid obstructions in
its path of movement. Trichocysts are absent.

The cilia of the body are disposed in 52 to 56 longitudinal rows and beat meta-
chronously. The cilia on the dorsal and ventral surfaces are somewhat longer
than those along the margin. The ventral cilia are thigmotactic, but not strongly
so. On the ventral surface at the anterior end is a transverse suture (anterior
field) from which the ventral rows of cilia extend backward, and from which the
dorsal rows curve upward and continue posteriorly. Most of the dorsal rows con-
verge in a characteristic pattern towards the posterior end. A definite unciliated
area is evident between the longer dorsal rows and the ventral rows which curve
upward a short distance over the posterior end.

ULIATES FROM PHYTIA SETIFER

FIGURE I. Cochliophllus deprcssus gen. nov., sp. nov. Dorsal aspect. Heidenhain's fixative
("susa")-iron hematoxylin. Drawn with aid of camera lucida. X 900.

FIGURE 2. Cochliophilus depressus gen. nov., sp. nov. Distribution of ciliary rows. Hol-
lande's fixative-protargol. Drawn with aid of camera lucida. A. Dorsal aspect. Though dis-
torted somewhat due to compression, this individual shows well the arrangement of ciliary rows
entering the peristomal indentation. X 670. B. Ventral aspect. X 950.

EUGENE N. KOZLOFF

Three ventral rows of cilia close to the oral margin turn dorsally near the end
of their course to delimit the naked part of the peristomal area posteriorly. The
first of these rows is ordinarily seen to ramify into an incomplete double or triple
series of cilia. The post-peristomal extensions of the ventral rows and the terminal
part of the dorsal row which borders the peristome above bear cilia which are two
to three times as long as the peripheral cilia elsewhere on the body.

The specialized peristomal cilia arise from two series of closely-set basal
granules, each of which is seen to be a continuation of two rows of peripheral cilia
essentially lateral in position, lying between the three ventral rows of cilia and one
dorsal row marking off the peristomal area. The cilia of the upper peristomal row
are appreciably longer than those of the lower row and appear in living individuals
to form a membrane-like structure which beats up and down as a unit. The cilia
of the lower row are much thicker and do not beat synchronously. The activity
of the peristomal cilia ceases soon after the organism is dissociated from the host.

The cytoplasm is colorless. Greenish granules appearing as highly refractile
bodies are distributed through the cytoplasm. These are most numerous around
the macronucleus and following fixation stain intensely with iron hematoxylin.

The macronucleus is centrally located. In outline it varies from oblong to
round, and in life is conspicuous as a clear granular body surrounded by food
inclusions and cytoplasmic granules. The micronucleus is greenish in color and
difficult to detect in living individuals. It is easily demonstrated by iron hema-
toxylin or the Feulgen nuclear reaction. The micronucleus is commonly situated
close to the macronucleus, between the latter and the oral margin. Upon fixation
it shrinks considerably and draws away from the membrane by which it is invested.

The contractile vacuole lies in the posterior fourth of the body behind the gullet,
and apparently opens to the exterior at a point between the convergence of the
shorter dorsal ciliary rows. I have been unable to distinguish a permanent open-
ing in the pellicle.

When free in water, Cochliophilus deprcssus swims actively, generally in circles
and with its concave ventral surface in contact with the substrate. Occasionally,
however, it follows an erratic course, rotating on its longitudinal axis. The trans-
verse anterior field is always at right angles to the direction of movement. In the
presence of pieces of tissue from the host Cochliophilus depressus will sometimes
seek refuge among them or cling to epithelial surfaces by means of its ventral
thigmotactic cilia.

I have found Cochliophilus depressus to be present in the mantle cavity of
nearly all specimens of Phytia setifcr which I have examined. It occurs in small
numbers and is usually less common than the following species.

Cochliophilus minor sp. nov. (Figs. 3 and 4}

The shape of this species resembles in general that of Cochliophilus depressus,
except that the posterior end is rather pointed, never truncate, and the dorso-
ventral dimension in relation to the length and breadth is comparatively greater.
In addition, the curvatures of the ventral and dorsal surfaces are more pronounced
in Cochliophilus minor.

CILIATES FROM PHYTIA SETIFER

FIGURE 3. Cochliophilns minor sp. nov. Dorsal aspect. Heidenhain's fixative ("susa")-iron
hematoxylin. Drawn with aid of camera lucida. X 1250.

FIGURE 4. Cochliophilus minor sp. nov. Distribution of ciliary rows. Hollande's fixative-
protargol. Drawn with aid of camera lucida. A. Dorsal aspect. X 1250. B. Ventral aspect.
X 1250.

100 EUGENE N. KOZLOFF

Twenty living individuals taken at random ranged from 51 /x to 80 /x in length
and from 33 //, to 56 p. in width, averaging about 63 /* by 45 p. The thickness
varied from 11 /A to 18/x.

The peristomal area is situated in the posterior fourth of the body. Two rows
of specialized cilia extend from the anterior end of the peristomal indentation to
the cytostome. That part of the peristomal area posterior to the cytostome is
naked.

An irregular gullet may sometimes be traced a short distance from the cyto-
stome, but it is not as easily discerned as the comparable structure in Cochliophilus
depressus.

The cilia are disposed in 36 to 38 longitudinal rows and beat metachronously.
The cilia on the ventral and dorsal curvatures are slightly longer than those along
the margin. The ventral cilia are weakly thigmotactic. The ventral rows extend
from an anterior transverse suture to the posterior tip of the body. The basal
granules of most of the ventral rows come to lie farther apart towards the posterior
end, while those of three or four rows near the oral margin lie closer together.
The dorsal rows of cilia pass from the transverse suture over the anterior end of
the body and continue backward to terminate in a conformation homologous with
that found in Cochliophilns depressus. The posterior dorsal unciliated area of
C. depressus has no exact homologue in this species. There exists, nevertheless,
an unciliated area between the converging dorsal rows and the dorsal row bordering
the peristomal area above.

One or two rows of cilia following the oral margin curve dorsally near the end
of their course to delimit the naked part of the peristomal area posteriorly. These
extensions and the terminal part of the most nearly lateral dorsal row on the oral
side bear exceptionally long cilia.

The peristomal ciliary apparatus consists of a membrane-like structure of long,
fine cilia which curves downward over a row of closely-set, rather thick cilia ex-
tending from the anterior end of the peristomal indentation to the cytostome. The
membrane-like structure appears to be non-motile and to function as a funnel
directing food particles into the cytostome.

The cytoplasm is colorless. Refringent cytoplasmic granules are present, but
to a lesser extent than in Cochliophilns depressus.

The size and shape of the macronucleus are highly variable. In living as well
as fixed individuals it is nearly always seen to be ramified, although ovoid or round
macronuclei are occasionally noted in this species. Reorganization stages in which
two or more smaller and round macronuclei are present are not infrequently met
with. The micronucleus ordinarily occupies a position between the macronucleus
and the oral margin. In fixed and stained preparations it is considerably shrunken.

The contractile vacuole is situated anterior to the cytostome. It opens to the
exterior between the convergence of the shorter dorsal ciliary rows. I have not
detected a permanent opening in the pellicle.

When separated from its host Cochliophilus minor swims in circles or proceeds
forward rotating on its longitudinal axis. Its movements are in general slower
than those of Cochliophilus depressus.

Cocliliophiliis minor is found in association with Cochliophilus depressus in the
mantle cavity of Phytia setter. It is usually more numerous than C. depressus.

CILIATES FROM PHYTIA SETIFER 101

SYSTEMATIC POSITION

On the basis of certain features of the morphology of the two species of
Cochlio philips which I have described it may be justifiable to allocate this genus to
the sub-order Thigmotricha Chatton and Lwoff, although in view of the deficiencies
of the systems of classification of holotrichous ciliates currently recognized I must
defer a conclusive statement with regard to its position. The organization of the
peristome of Cochliophilus hints its affinity with Kidderia Raabe, represented by
K. inytili (De Morgan) from Mytilns cditlis. Raabe (1936) retained Kidderia in
the family Conchophthiridae Reichenow,2 but removed to the family Thigmo-
phryidae Chatton and Lwoff Myxophyllum and Conchophyllum, genera created by
him to accommodate, respectively, Stein's species Conchophthirus steenstrupi, com-
mensal on various terrestrial pulmonate molluscs, and Conchophthirus caryoclada
Kidder, from the bivalve Siliqua patula. It is interesting to note, in passing, that
a specific character of Conchophyllum caryoclada is its branched macronucleus,
of which the macronucleus of Cochliophilus minor is reminiscent.

The presence of a membrane-like structure in the peristome of Cochliophilus
could be the basis for objections to the inclusion of this genus in the Thigmotricha.
Very similar ciliary elements have been observed, however, in certain species of the
family Ancistrumidae Issel. Raabe (1932, 1934b) has stressed the presence of an
undulating membrane in Conchophthirus, although Kidder (1934), after studying
species of Conchophthirus from fresh water mussels in this country, was unable to
corroborate Raabe's findings, and suggested that Raabe may have mistaken the
fibers of the peristomal basket for an undulating membrane.

Gciuts Cochliophilus gen. nov.

Diagnosis : Flattened holotrichous ciliates, ovoid in outline as seen in dorsal or
ventral view. The peristomal area is elongated and is situated on the right lateral
margin in the posterior fourth of the body. A membrane-like structure of fine cilia
overlies a series of thick cilia extending from the anterior end of the peristomal
indentation to the cytostome ; that part of the peristomal area posterior to the
cytostome is naked. The peripheral cilia are disposed in longitudinal rows extend-
ing from a ventral transverse suture at the anterior end of the body. The dorsal
rows converge in a characteristic pattern posteriorly. Thichocysts are absent.
The macronucleus is centrally located ; the micronucleus is usually situated near
the macronucleus, between the latter and the oral margin. The contractile vacuole
opens to the exterior between the convergence of the shorter dorsal ciliary rows ; no
permanent opening in the pellicle is discernible. Genotype: Cochliophilus dc-
prcssus gen. nov., sp. nov. Two species, commensal in the mantle cavity of Pliytia
setifer (Cooper).

Cochliophilus deprcssns gen. nov., sp. nov.

Diagnosis : Average size about 93 /A by 63 /*, the thickness being about one-sixth
the length. The ciliary rows are 52 to 56 in number. The peristomal membrane-
like structure is motile. The macronucleus is round or oblong. Syntypes are in
the collection of the author.

2 Reichenow (1927-29) was apparently the first to use the name Conchophthiridae, although
Raabe credits Kahl (1931) with establishing this family.

102 EUGENE N. KOZLOFF

Cochliophilus minor sp. nov.

Diagnosis : Average size about 63 ju. by 45 /x, the thickness being about one-
fourth the length. The ciliary rows are 36 to 38 in number. The peristomal
membrane-like structure is apparently immobile, serving as a funnel directing food
particles into the cytostome. The macronucleus is characteristically ramified.
Syntypes are in the collection of the author.

LITERATURE CITED

BODIAN, D., 1936. A new method for staining nerve fibers and nerve endings in paraffin sec-
tions. Anal. Rec., 69 : 89.
BODIAN, D., 1937. The staining of paraffin sections of nervous tissue with activated protargol.

The role of fixatives. Anal. Kcc., 69: 153.
COOPER, J., 1872. On new Californian Pulmonata, etc. Proc. Acad. Nat. Sci. Philadelphia, 24:

143.
BALL, W., 1921. Summary of the marine shellbearing mollusks of the northwest coast of North

America, from San Diego, California, to the Polar Sea. U. S. Nat. Mus. Bull. 112.
DE MORGAN, W., 1925. Some marine ciliates living in the laboratory tanks at Plymouth, with

a description of a new species, Holophrya coronata. Jour. Mar. Biol. Assoc. United

Kingdom, 13 (n.s.) : 600.
HENTSCHEL, C, 1924. On a new ciliate, Cryptochilum boreale, sp. nov., from the intestine of

Echinus esculentus Linn., together with some notes on the ciliates of echinoids.

Parasitology, 16: 321.
KAHI., A., 1931. Urtiere oder Protozoa. I: Wimpertiere oder Ciliata (Infusoria). 2. Holo-

tricha. In Dahl, F. : Die Tierwelt Deutschlands, 21 Teil. Gustav Fischer, Jena.
KAHL, A., 1934. Ciliata entocommensalia et parasitica. In Grimpe, G., and E. Wagler : Die

Tierwelt der Nord- und Ostsee, Lief. 26 Teil II C,. Akademische Verlagsgesellschaft,

Leipzig.
KIDDER, G., 1933a. Conchophthirius caryoclada sp. nov. (Protozoa, Ciliata). Biol Bull., 65:

175.

KIDDER, G., 1933b. Studies on Conchophthirius mytili De Morgan. I. Morphology and di-
vision. Arch. Protistcnk., 79: 1.
KIDDER, G., 1934. Studies on the ciliates from fresh water mussels. I. The structure and

neuromotor system of Conchophthirius anodontae Stein, C. curtus Engl., and C. magna

sp. nov. Biol. Bull., 66: 69.
RAABE, Z., 1932. Untersuchungen an einigen Arten des Genus Conchophthirus Stein. Bull.

int. Acad. Cracovie, Cl. Sci. math, not., B (II), 1932: 295.
RAABE, Z., 1934a. Uber einige an den Kiemen von Mytilus edulis L. und Macoma balthica

(L.) parasitierende Ciliaten-Arten. Ann. Mus. zool. polon., 10: 289.
RAABE, Z., 1934b. Weitere Untersuchungen an einigen Arten des Genus Conchophthirus Stein.

Mem. Acad. Cracovie, Cl. Sci. math, not., B (II), 1934: 221.
RAABE, Z., 1936. Weitere Untersuchungen an parasitische Ciliaten aus dem polnischen Teil

der Ostsee. I. Ciliata Thigmotricha aus den Familien : Thigmorphryidae, Concho-

phthiridae und Ancistrumidae. Ann. Mus. zool. polon., 11: 419.
REICHENOW, E., 1927-29. Lehrbuch der Protozoenkunde. 5th ed. Gustav Fischer, Jena.

THE DEVELOPMENT OF MARINE FOULING COMMUNITIES

BRADLEY T. SCHEER
Wni. G. Kerckhoff Marine Laboratory. California Institute of Tcchnolofiv, Corona del Mar

This paper constitutes an examination of the sedentary communities found on
float bottoms and other submerged objects in Newport Harbor, California. Par-
ticular attention has been paid to the changes in composition of such communities
with time.

The basic problem in the development of a sequence of communities in a limited
environment is that of distinguishing between seasonal progression and true suc-
cession. Seasonal progression results fundamentally from differences in breeding
seasons of various organisms. This type of development was noted at Beaufort,
N. C. by McDougall (1943). Most of the organisms observed by McDougall had
short life cycles and short breeding seasons. As a result, most of the organisms
which settled in the winter months were dead or moribund by spring, and were
replaced by organisms breeding in the latter season.

Succession, in contrast to seasonal progression, involves definite relations be-
tween organisms, Shelford (1930) has suggested the following criteria for the oc-
currence of succession : ( 1 ) Early forms must drop out, and be replaced by later
forms, and (2) Some of the earlier forms must be essential for the establishment
of the later forms. The use of the word "essential" in this connection is perhaps
unfortunate. It would be nearly impossible, in most cases, to prove that one organ-
ism is essential for the establishment of another. On the other hand, the presence
of one organism might well provide conditions favoring the establishment of an-
other, and certainly such favorable conditions would suffice to insure the displace-
ment of early settlers by later arrivals.

The phenomena of ecological succession are well known in terrestrial com-
munities. In littoral marine communities, it has sometimes been stated that true
succession does not occur, or is of little importance (Shelford, 1930; McDougall,
1943). The clearest case of succession in intertidal communities is that reported
by Hewatt (1935). In the Mytilus californianits community characteristic of ex-
posed rocky coasts along the entire Pacific coast of the United States, the estab-
lishment of a climactic condition requires more than two and one-half years, and
involves a definite sequence of organisms. The reports of Kitching (1937),
Moore (1939) and Moore and Sproston (1940) also give some indication that
recolonization of intertidal rock surfaces is a slow process. It appears that the
first event is ordinarily a heavy settlement of algae, and that many animal forms
appear 'only after the plants have become established. Kitching (1937) provides
evidence of a succession of algal forms on rocky intertidal ledges.

The sedentary organisms inhabiting floats, pilings, boat bottoms and similar
s.ructures have been the subject of many investigations. The literature in this
field has been reviewed recently by McDougall (1943) and need not be cited ex-

103

104

BRADLEY T. SCHEER

tensively here. The most thorough investigations dealing with the Pacific forms
are those of Coe (1932) and Coe and Allen (1937). These studies, covering a
period of nine years, have provided invaluable information regarding the biology of
the organisms concerned. The data reported in the current study have been
accumulated between February 1943 and March 1945.

THE FLOAT-BOTTOM COMMUNITIES OF NEWPORT HARBOR

Field observations on float bottoms and similar structures in Newport Harbor
disclosed the existence of five or six rather definite communities. For convenience,
throughout this paper, these communities will be referred to by designations in-
dicating the most abundant organisms in the community. In this way, we may
designate (a) algal, (b) bryozoan, (c) Ciona, (d) Stycla, (e) Mytilus, and (f)
Balanus communities. These communities were not all sharply marked off, one
from another, and communities intermediate in composition between algal and
bryozoan, bryozoan and Stycla, Stycla and Mytilus, bryozoan and Mytilus, and
Ciona and Mytilus have been observed. The various communities showed no rela-
tion to the position of the floats in the harbor, and indeed several different com-
munities were found within a distance of a hundred feet on different floats. Evi-
dence will be presented that this results from a definite succession, and that the
composition of the community on any particular float bottom depends on (a) the
length of time during which the float has been in the water, and in part on (b) the
season during which the float was first immersed. We shall first consider the com-
position of the various communities.

The bottoms of floats were examined with the aid of a periscopic device involv-
ing an ordinary underwater viewing glass with a mirror attached (Fig. 1). Or-
ganisms were also removed from floats with a long-handled scraper.

FLOAT

MIRROR

FIGURE 1. Apparatus for the examination of float bottoms.

The algal community. When a clean surface was placed in the bay, the first
settlers were bacteria, algae, protozoans, and, during the cooler months of the year,
hydroids. The algae included small sedentary diatoms which have not been iden-
tified in the present study (see Coe, 1932; and Coe and Allen, 1937), colonial
diatoms of the genus Licmophora, and one or more species of Ectocarpus, notably
E. granulosoidcs. In addition, Enteromorpha sp., Lophosiphonia villmn, and
Ptcrosiphonia bipinnata were frequently noted. The sedentary protozoans in-

MARINE FOULING COMMUNITIES 105

eluded a form similar to Zoothauinimn, and the suctorian Ephelota. There were
seven or eight species of hydroids ; these were not identified, but Obelia dichotomy
was usually conspicuous. Bryozoans were found in this community, sometimes
in abundance. On float bottoms. Bitgnla ncritina may be an important member
of the community, and Membranipora titbcrculata was observed in one instance
on glass plates. Encratea clavata, a small semi-erect bryozoan, occasionally oc-
curred in considerable numbers on glass plates. Finally, young colonies of a
number of other species of bryozoans appeared after a time. These will be dis-
cussed in more detail later.

The bryozoan community. A good many floats supported a very heavy growth
of bryozoans. The principal organisms involved were the encrusting bryozoans
Schisoporella nniconiis, Cryptosula pallasiana, Rhynchozoon tumuhsinn and
Holoporclla apcrta. The erect bryozoans were less constant in occurrence, but
were quite abundant in some cases. Bngula ncritina w^s less frequent in this
community than among the algae, while Encratea clavata was more frequently
found among the encrusting bryozoans than among the algae. Crisnlipora occi-
dcntalis and Scrnpoccllaria diegcnsis were usually present and often very abundant
among the bryozoans. Four or five other species of erect bryozoans occurred less
frequently.

Although the bryozoans by far outnumbered the other members of this com-
munity in most cases (Table VII), other organisms were often quite abundant.
Notable are the serpulid worm Eupomatits gracilis, and the colonial amphipod
Erichthonius brasiliensis. Eupomatus was almost always found, with its wind-
ing calcareuos tubes, between the colonies of encrusting bryozoans. Occasionally,
it was very abundant, the tubes making a more or less solid mass. Erichthonius
was irregular in occurrence. During 1943, it did not appear in quantity, but in
1944 it was extremely abundant during July and August, the mud tubes often
covering as much as half of the area of a glass plate. Coe and Allen (1937) noted
a similar variation at La Jolla. The ascidians Styela barnharti, Halocynthia
johnsoni , and Ciona intestinalis. and the mussel Mytilns sp. were found among the
bryozoans in many cases, but since they were more characteristic of other com-
munities, they will be dealt with later. Many crustaceans, annelids and other
motile forms used the bryozoan clumps for shelter.

The Ciona community. The previous paragraphs have dealt with communi-
ties in which several species were abundant and the proportions of each species
showed considerable variation in different communities of the same type. Most
of the Ciona communities, in contrast, were composed almost wholly of specimens
of Ciona intestinalis. This was particularly true during the summer and fall, when
these communities were at their peak of development. Many float bottoms pre-
sented a solid mass of Ciona, with only a few other organisms present. These
latter were usually colonial ascidians, growing on the tests of the Ciona, and such
crustaceans and annelids as might have taken refuge among the stalks.

The Styela community. This was a poorly defined community, intermediate
in composition between the bryozoan and Mytilns communities. The encrusting
bryozoans noted earlier were usually present, forming a substratum for the stalks
of Styela, while the erect bryozoans were often found among these stalks. Small
specimens of Mytilns were often attached to the stalks in large numbers. Large

106 BRADLEY T. SCHEER

sponges, which have not been identified, were also frequently present, sometimes
in such quantity as to dominate the community. It might indeed he preferable
to refer to a Styela-Spouge community.

The My til us community. Mytilus was without question the most abundant
dominant on the float bottoms in Newport Bay during the period of this study.
This has not always been the case, according to reliable observers (G. E. Mac-
Ginitie, A. M. Strong, personal communications) ; during several previous years,
Mytilus has not been abundant in the bay. The exact identity of this mussel re-
mains in doubt. It is probably the same form which has been recorded infre-
quently from this area as M. edulis. However, conchologists are not entirely
agreed that this is the proper designation. It is certainly not M. calif ornianus.
The Mytilus communities sometimes were observed on a substratum of old and
badly decayed bryozoans ; at other times they wefe attached directly to the float
bottom. Old specimens of Styela or dona were often present among the mussel
clumps, and various types of sponge were often quite abundant.

The Balanus community. Communities in which Balanns is the dominant or-
ganism \vere not observed on float bottoms in Newport Harbor, although they are
frequently observed on experimental surfaces exposed in the open sea at La Jolla.
Indeed, Balanus tintinnabulum is probably the principal dominant at La Jolla (Coe,
1932). One experimental panel exposed at this laboratory developed a Balanus
community comparable to those observed at La Jolla, however.

CHANGES IN FLOAT-BOTTOM COMMUNITIES

Eight floats, all located along the mainland side of the channel between Balboa
Island and Corona del Mar, and within a distance of 100 yards of one another,
were selected in September of 1944, and kept under observation for a period of six
months. The results of this study are presented in Table I. At intervals of about
one month, the bottom of each float was examined with the viewing glass, and
samples of the population removed by hand and with the scraper for later examina-
tion in the laboratory.

Float number one had been immersed in the bay for only about one week
previous to the first examination. It had at that time (Sept. 21) a typical algal
community, with a few specimens of Bugula. In October, examination showed in-
creased numbers of Bugula, and a few small colonies of other erect bryozoans. In
November, Bugula and the encrusting bryozoan Holoporella had displaced the
algae, and a number of small specimens of dona were present. The float was
then covered with a typical bryozoan community. During the remainder of the
period, until March, the encrusting bryozoans continued to increase in numbers and
size.

Floats 2 and 3 supported typical bryozoan communities in September. In ad-
dition to the bryozoans, a number of specimens of dona were observed, and several
small Mytilus. During the period of observation, Mytilus grew at the expense of
the bryozoans and ascidians, becoming very abundant in December, and largely
dominating the community by February. The two float populations were very
similar in composition in September, but the presence of Styela on float 3 in Octo-
ber appears to have favored the earlier establishment of Mytilus on this float. The
presence of sponges on this float may also be related to Styela. Float 4, in Sep-

MARINE FOULING COMMUNITIES

107

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BRADLEY T. SCHEER

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MARINE FOULING COMMUNITIES 109

tember, had a population similar to that observed on float 2 in November, with
relatively large numbers of Mytilus and Ciona on a bryozoan substratum. Within
a month, Mytilus had largely displaced the bryozoans, and within three months,
Ciona had also disappeared.

The Ciona community of float 5 remained virtually unchanged from September
to February. By this time, however, the Ciona, began to show signs of deteriora-
tion. They were heavily covered with algae and hydroids, and had many small
Mytilus about their bases. In a few places, the ascidians had fallen from the
float, to be replaced by encrusting bryozoans. In March, this change had pro-
gressed so far that Mytilus and the bryozoans could be regarded as the dominant
organisms.

Floats 6 and 7 supported two types of sponge-Styela communities. These
were rather rapidly displaced by Mytilus, however. Float 8 represented a well-
developed Mytilus community and showed no change in composition during the six
months of regular observation.

These observations suggest strongly that succession is operating here. The
algal community is replaced by the bryozoans, and these in turn by Mytilus.
Ciona and Stycla communities are likewise replaced by Mytilus, but the Mytilus
community is relatively stable. Further information bearing on this conclusion
is available from the experimental studies to be reported in the next section.

EXPERIMENTAL OBSERVATIONS WITH GLASS AND METAL SURFACES

Experimental observations were made using glass plates, and supplementary
information was available from a series of aluminum panels immersed for another
purpose. The fact that the changes observed on the glass plates were entirely
similar to those observed on wooden floats and metal plates suggests that the
changes reported here are not dependent on the nature of the submerged surface.
Coe (1932) and Coe and Allen (1937) concluded that the seasonal variations in
abundance of populations or of different groups of organisms were the same on
glass, concrete, and wood surfaces. They did find significant differences in the
numbers and types of organisms on the different surfaces, however.

The glass panels used were four by nine inch rectangles of ordinary window
glass in most cases ; in a few experiments three by five inch panels were used. The
metal plates were five by eight inch rectangles of aircraft aluminum (Alclad ST-
37). The glass panels were at first exposed in a horizontal frame (Fig. 2) of
redwood weighted with concrete. The frame was suspended from the laboratory
pier, situated in the entrance channel to Newport Harbor about one-half mile from
the outer end of the jetties protecting the harbor entrance. A rapid tidal flow
passes this point twice daily, carrying with it abundant larvae from both the quiet-
water fauna of the harbor and the open shore fauna of the jetties and adjacent
rocks. In the second year of this study, with the glass plates, and throughout the
work with the metal plates, a vertical suspension was used to facilitate handling
of larger numbers of plates. The plates were suspended in slotted redwood crates,
with a distance of one inch between plates. As the growth on the plates became
heavier, this distance was increased to two inches. The plates were always sus-
pended one or two feet below the level of the lowest tides.

110

BRADLEY T. SCHEER

All of the plates were examined regularly at intervals of two weeks, and then
returned to the bay. A count was made, in most instances, of the numbers of each
of the larger species on one surface (always the same for any plate). An estimate
was also made of the area covered by each of the more abundant types of organism.
Usually, this was done by a direct count of ten or more low-power microscopic
fields distributed over the surface. When a plate was finally removed from the
water, the organisms were carefully removed, sorted and weighed.

o o

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The development of the algal community, and its transition to the bryozoan
community could be followed very well on these plates. The first settlers were
bacteria, diatoms, protozoans and, in the cooler months, hydroids. These were
followed by the multicellular algae, especially Ectocarpus.

In the first months of this study it was observed that the larvae of bryozoans
usually settled on the plates in quantity only after the second week of exposure,
and sometimes did not settle until the fourth to sixth week. In order to verify
this observation, careful counts were made during 1944 of the number of bryozoan
colonies on each plate at two-week intervals. In this way, the minimum number
of new settlers during any two-week period could be determined. Data obtained
in this way are tabulated in Tables II, III, and IV for encrusting bryozoans, erect
bryozoans and Eiiponmtiis. The tabulation for the erect bryozoans omits the fig-
ures for the small semi-erect Eucratea clavata; representatives of this species set-
tled in great numbers at irregular intervals, showing a behavior in this respect
which was not at all comparable to the settlement of the other forms. The colonies
were, moreover, rather short lived, dying often within a month of the original
settlement.

MARINE FOULING COMMUNITIES

111

During the first two weeks of exposure of any plate, the number of bryozoan
and tubeworm settlements was usually less than during subsequent two-week
periods. The preliminary period of light settlement was followed by a very heavy
settlement in most cases. The growth of the earlv settlers, and in many cases, the

o J j

large number of organisms settling during the maximal period, combined to reduce
the available surface, and there was in consequence a very definite decrease in the
number of organisms on the plate. By the time this decrease became evident, the
plate was completely covered with bryozoans and tubeworms.

TABLE II

Number of new settlements of encrusting bryozoans on glass plates
during successive two-week periods, 1944

Date

examined

Date of original exposure

Dec.
20

Jan.

Jan.
17

Jan.
31

Feb.
14

Feb.

Mar.
12

Mar.
27

Apr.
27

May
9

June
8

July
6

Aug.
1

Sept.
11

Oct.
10

Jan. 5

Jan. 17

Jan. 31

Feb. 14

Feb. 28

Mar. 12

—

Mar. 27

Apr. 8

—

Apr. 26

120

May 8

123

May 24

June 7

June 21

July 6

July 17

July 31

197

Aug. 14

Aug. 28

105

Sept. 11

Sept. 25

Oct. 10

Oct. 23

112

BRADLEY T. SCHEER

Figure 3 represents data derived from a metal plate first exposed March 28,
1944, and shows the changes in area covered by the algae, bacteria and hydroids on
the one hand, and bryozoans on the other. The major increase in area occupied by
the bryozoans occurred after the period of maximum settlement ; the heaviest
settlement occurred between the sixth and eighth weeks, while the rapid increase
in area began between the tenth and twelfth weeks. This was in part the result of
the manner of growth of bryozoan colonies. The number of new zooids formed
increases directly with the number of zooids composing the colony, so that the rate
of growth increases exponentially until crowding prevents further increase in the
size of the colony.

TABLE 111

Number of new settlement.-, of erect bryozoans (exclusive of Eucratea cluvata) on glass plates

during successive two- week periods, 1944

]

Date of

original

exposur

examined

Jan.
17

Jan.
31

Feb.

Feb.
28

Mar.
12

Mar.
27

Apr.

May
9

June
8

July

Aug.
1

Sept.
11

Oct.
10

Mar. 27

Apr. 8

Apr. 26

May 9

May 24

—

June 7

June 21

—

July 6

July 17

July 31

Aug. 14

Aug. 28

Sept. 11

Sept. 25

Oct. 10

Oct. 23

The length of time required for this sequence of events varied with the season
of the year, but the character of the sequence did not vary. Thus, the plate ex-
posed December 20 did not reach "saturation" with encrusting bryozoans until
April, while the plate exposed May 9 had become "saturated" before the end of
June (Table II). If we consider any particular two-week period, however, it is

MARINE FOULING COMMUNITIES

113

TABLE IV

Number of new settlements of Eupomatus on glass plates
during successive two-week periods, 1944

Date
examined

Date of original exposure

Dec.
20

Jan.

Jan.
17

Jan.
31

Feb.
14

Feb.

Mar.
12

Mar.
27

Apr.

May
9

June
8

July
6

Aug.
1

Sept.
11

Oct.
10

Jan. 5

Jan. 17

Jan. 31

Feb. 14

Feb. 28

Mar. 12

Mar. 27

Apr. 8

Apr. 26

May 8

—

May 24

—

June 7

June 21

July 6

11
4

July 17

July 31

Aug. 14

—

Aug. 28

Sept. 1 1

Sept. 25

—

Oct. 10

Oct. 23

evident from Talles II to IV, that in general, the most recently exposed plates
received lighter settlements of the three types of organisms concerned than did those
which had been in the water somewhat longer. Evidently changes occurred follow-
ing immersion vhich rendered the plate more suitable for settlement of bryozoans
and tubeworms than was the clean surface. These changes occurred more rapidly
in the warmer nonths.

Two experin/Mits were performed to test this hypothesis, and to throw more
light on the nature of the changes involved. ZoBell and Allen (1935) and Coe and

114

BRADLEY T. SCHEER

100

0 WEEKS 5

ABHo —

BRYOZOANS x

FIGURE 3. Relative areas, 'it, per cent, covered by algae, bacteria, and hydroids (A B H),
and bryozoans on an aluminum panel exposed March 28, 1944. The figures along the abscissa
represent number of new settlements of bivozoans in each two-week period.

Allen (1937) have suggested that bacterial film is an important feature in the
establishment of sedentary forms on a submei<jed surface. In the first experi-
ment (Table V), ten three by five inch glass pl^es were sterilized. Two were
then exposed in the bay, two were left in sterile sea \\ater, two in a sterile solution
of 0.1 per cent peptone in sea water, and two were placed in a solution of 0.1 per
cent peptone in water freshly drawn from the bay. Aftei four days, by which time
a vigorous bacterial population had developed in the ba; water solution, all ten

TABLE V

Settlement of organisms on pretreated glass plates, June 6-10, 1944. Duration of treatment,
4 days. Figures represent number of organisms or cdonies

Treatment:

First series (3 days immersion)

Second seies (4 days immersion)

Hydroids

Bryozoans

Ascidians

Hydroids

Bryozoans

Ascidians

Sterile sea water

Sterile sea water + 0.1%
peptone

Bay water + peptone
(bacterial)

150

174

Immersal in bay 4 days

Sterile plate

MARINE FOULING COMMUNITIES

115

plates were placed in the bay. The results are presented in Table V. The
hydroids evidently settled more abundantly on the bacteria-coated plates then on
the others, but the bryozoans and ascidians were not influenced by the bacterial
coating. Rather, they settled more abundantly on the plates which had been in
the bay longest ; these plates had a more abundant diatom population than did
the others.

A second similar experiment was carried out in the fall, with daily observations
during several weeks of exposure, and careful determinations of the bacterial and
algal populations. Diatoms appeared on the plates in small numbers within the
first' two to four days in the bay (Table VI). For a period of two to three weeks,
however, the diatoms covered less than 5 per cent of the surface. This period was

TABLE VI

Settlement of organisms on treated panels, October 21 to November 24, 1944. The figures
represent the per cent of the area of one side of the panel covered by bacteria, diatoms, and proto-
zoans respectively, and total number of organisms or colonies on one side of the panel for the
larger organisms (bryozoans, annelids, ascidians).

Duration of treatment:

5 days

18 days

Sterile

Fresh

Sterile

Fresh

Treatment:

Bay

Sterile

sea
water +

bay
water +

Bay

Sterile

sea
water +

bay-
water +

peptone

Days after
treatment

Organism

Bacteria

0.3%

0.2%

0.3%

11%

Diatoms

80%

0.3%,

Protozoans

0.3%

Bryozoans:

Membranipora

•j

Other encrust-

ing forms

Annelids

Ascidians

Bacteria

Diatoms

35%

11%

16%

51%

18%

Protozoans

0.1%

Encrusting

bryozoans exc.

Membranipora

Annelids

Ascidians

Time of

Diatoms

maximum

Encrusting

increase,

bryozoans exc.

days after

Membranipora

immersion

in bay

116 BRADLEY T. SCHEER

followed by a relatively sudden increase in the number of diatoms, until 25 per cent
to 80 per cent of the plate was covered. The reason for this delay is not clear.
During the first two to three weeks of exposure, bacteria and protozoans as well
as diatoms settled on the plates. That bacteria were not concerned in the eventual
diatom outburst is indicated by the fact that the bacteria-coated plates (bay water
and peptone) showed no difference from the other plate in the time of the outburst.
About 5 per cent of the area of the plate which had been immersed in bay water
plus peptone for five days was covered with bacteria when the plate was immersed
in the bay; the eighteen day plate was covered to an extent of about 11 per cent.
This coating was largely lost within a few days, however. It is noteworthy that,
although the time of maximum increase of the diatoms was not affected by the
presence of bacteria, larger populations of diatoms eventually developed on the
bacteria-coated plates than on the other plates. Algae other than diatoms were
not important in this experiment ; Ectocarpus, Enteromorpha, Cladophora, Scyto-
siphon, Pterosiphoiiia and Lophosiphonia were noted, but did not appear in quan-
tity until some time after the diatom increase.

The data available from this study are not sufficient to establish a succession
within the algal community. Wilson (1925), however, has reported a definite
sequence, involving diatoms, hydroids and filamentous algae, on rocks at La Jolla.
It is quite possible that careful study over a longer period would reveal a similar
situation here.

It appears that the relatively heavy growth of diatoms on the bay water plus
peptone plates is correlated with a correspondingly heavy settlement of bryozoans.
Whether there is a direct causal relation between diatom growth and bryozoan
settlement is uncertain. However, the maximum period of bryozoan settlement
never preceded, and usually followed, the period of maximum diatom increase in
the experiment of Table IV.

The encrusting bryozoan Mcinbranipora tnbcrcnlata, which normally inhabits
the stipes of kelp, occurred on the experimental plates only on one occasion, and
remained only a short time. Most of the colonies fell from the plate within the
space of a month. Unlike the other bryozoans, however, this species showed
definite preference for the bacteria-coated plate.

These experiments suggest, then, that the important change which occurs on
plates favoring bryozoan settlement, is the growth of diatoms. In view of this
conclusion and of the fact that the bryozoan community, throughout the course of
this study, has been observed to form only on surfaces previously supporting an
algal growth, we may say that a definite succession is established.

The observations on the glass plates provide little evidence concerning the other
communities, but two instances are worthy of mention. The development of a
Styela community from a bryozoan community was noted in one instance, on a
plate exposed horizontally in March 1943. The algal coat which developed upon
exposure was displaced by bryozoans before the end of June. In September, how-
ever, specimens of Styela which had settled in July had become so large as to domi-
nate the community. The remaining bryozoans gradually lost ground and fell
from the plate, leaving Styela as the principal organism present. The fact that
Styela was always found growing out of a substratum of old bryozoan colonies on
the floats examined in the course of this study indicates that this sequence prob-
ably occurs frequently.

MARINE FOULING COMMUNITIES 117

The second instance concerns the formation of a Balanns community. The plate
concerned was exposed horizontally in August 1943. The algal community formed
very rapidly, and in addition, within two weeks, larvae of Eupomatus, encrusting
bryozoans, Pccten and Balanns had settled in large numbers. In the ensuing
competition for space, the barnacles emerged victorious. In September, there were
more than two hundred barnacles on the exposed side of the plate, covering the
surface almost completely. An equal number of Eupomatus occupied the spaces
between the barnacles, but encrusting bryozoans were not abundant. During
subsequent months, however, growth of the bryozoans was continuous, and by
December, the barnacles were almost completely covered by the rapidly growing
bryozoans.

DISCUSSION AND CONCLUSIONS

In order to make a satisfactory analysis of the phenomena described in the pre-
ceding sections, it would be necessary to know (a) the breeding seasons of the
organisms involved, (b) the normal duration of life of each of the important or-
ganisms, (c) the length of the free-swimming larval period in each case, and (d)
the nature of the surfaces to which such free-swimming larvae wrill attach. We
do not have such information in most cases. Nevertheless, it is possible to make
some interpretations on the basis of the available information.

It was stated at the outset that the basic problem is that of distinguishing be-
tween seasonal progression and true succession. Several examples of progression
\vere noted at Beaufort, N. C. by McDougall (1943). The organisms which settled
during the winter were, for the most part, dead or moribund by spring, and were
consequently replaced by organisms breeding chiefly in the spring. There is some
reason to expect that seasonal progression may be less important in Newport
Harbor than at Beaufort. The annual range of monthly mean temperatures at
Beaufort is 23° C., from 5.5° in February to 28° in July. The annual range in
Newport Harbor is only 5° C., from a low of 14.1° in February to a high of 19.2°
in July. The breeding seasons of most of the organisms involved in the sequences
described here extend through most of the year. Certainly algae, bryozoans and
mussels have been observed to settle during every month of the two years covered
by this study.

In the present study, it seems probable that the algal community, and most
probably the diatoms comprising the basis of that community, provide favorable
conditions for the settlement of bryozoans. Bryozoans settled in quantity only
after the development of a fairly vigorous algal community. Moreover, in the
experimental test described in Table VI, bryozoan settlement was definitely cor-
related with the settlement and growth of diatoms. There remains the possibility
that some common factor favored settlement of both diatoms and bryozoans, the
former remaining "dominant" only until the slower but persistant growth of the
latter displaced them. It is difficult to rule out such common factors, but it ap-
pears unlikely that chemical alterations in the glass on exposure to sea water are
involved. The plates used in this particular experiment had previously been
immersed in the bay for two months. They were then scrubbed with a brush in
tap water, wrapped in paper and sterilized in an autoclave. The experimental
plates were soaked in three liters of solution for several days as noted in the table,

118

BRADLEY T. SCHEER

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4-1
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Serpulids

tn
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Balanus

tn
a!

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CJ
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Ascidians

MARINE FOULING COMMUNITIES

119

while the control plates were placed directly in the bay. The same sequence of
organisms is apparent on both experimental and control plates, and the periods of
time involved are not significantly different.

The data in Table VII are of interest in this connection. It is apparent that the
organisms listed fall into four classes : ( 1 ) Those which appear in abundance on all
plates, but most abundantly on those exposed for the shortest periods (algae, etc.).

(2) Those which appear only on plates exposed more than four weeks, and most
abundantly on plates exposed twenty weeks or longer (bryozoans, serpulid worms).

(3) Those which appear in measurable quantities only on plates exposed twenty
weeks or longer, and are not abundant even on plates exposed as long as thirty-six
weeks (Mytilus, Sa.i'icava, sponges, ascidians). (4) Those which appear irregu-
larly, without relation to the duration of exposure (annelids, Balanns, Erichthonius
and other crustaceans, Pecten).

It is particularly significant that the dominant organisms of the primary com-
munities involved in the sequence described earlier — viz. algae, bryozoans, ascidians
and mussels — fall into separate categories on this basis, and that the sequence here
is the same as that observed in the sequence of communities. It appears that the
settlement of ascidians certainly and mussels probably is favored by the existence of
a thriving bryozoan community.

In any event, there is no evidence that seasonal progression is involved to a
significant extent in the algae-bryozoan-My/'z'/!^ sequence. A plate exposed in
December went through the same sequence as did one exposed in March or April ;
the time relations varied, but the sequence did not. And in the absence of a
seasonal progression, it is difficult to avoid the conclusion that true succession is
involved.

CLEAN SURFACE,

BACTERIA

ALGAE
HYDROIDS

BALANUS

X
X

BRYOZOANS
.SERPULIDS

CIONA

STYELA
SPONGES

MYTH-US
FIGURE 4. Sequence of dominant organisms on surfaces exposed in Newport Harbor.

In many studies of the life histories of sedentary organisms, estimates of the
season of settling have been based on the number of new settlers on a plate ex-
posed for a brief period. It is evident from the results reported here that such

120 BRADLEY T. SCHEER

estimates may be unreliable if succession is involved in tbe settlements under con-
sideration. Thus, plates exposed for four weeks or less in Newport Harbor in
the winter months would receive few or no settlements of bryozoans, despite the
fact that settling larvae are present in the water during these months. It is im-
portant, therefore, that studies of this sort take into consideration the question
whether succession is occurring.

With the evidence presented in this paper, we can make a number of sugges-
tions as to the possible factors involved in the events described. The sequence is
depicted in Figure 4. A newly exposed surface is first colonized by bacteria, algae
and, in some seasons, hydroids. The development of these forms provides a
favorable surface for establishment of bryozoan colonies, and also for the settle-
ment of serpulid larvae. The vigorous growth of the bryozoans eventually dis-
places the algae and hydroids. The resulting bryozoan community in turn pro-
vides a favorable basis for the attachment of Mytilus larvae. The growth of the
mussels effectively covers the whole surface of the bryozoan community, the mem-
bers of which eventually perish from lack of food, oxygen, etc. Seasonal factors,
involving the settlement of ascidian or barnacle larvae in tremendous numbers, may
introduce variations into this sequence. Ciona may sometimes colonize a clean
surface, or one covered with algae, to such an extent that the bryozoans are unable
to maintain their foothold. Stycla apparently settles only on bryozoan substrata,
but may become established before Mytilus, and hence a community dominated by
Styela may follow the bryozoan stage. Sponges are frequently associated with
Styela. Both Ciona and Styela communities are eventually displaced by Mytilus
which at present represents the climax in the float-bottom associations of Newport
Harbor.

SUMMARY

1. The sedentary communities characteristic of float bottoms in Newport Har-
bor, California, are described.

2. The most important communities at present are dominated, respectively,
by algae, bryozoans, Ciona intestinalis, Stycla sp. and Mytilus sp.

3. These communities represent stages in an ecological succession.

4. The algal community appears first on freshly exposed surfaces, to be fol-
lowed usually by a bryozoan community.

5. The bryozoans prominent in the bryozoan community settle more readily
on surfaces supporting a vigorous growth of diatoms and other algae than on clean
surfaces.

6. The community dominated by Mytilus constitutes the climax at present.

7. Mytilus has been observed to settle only on surfaces bearing a bryozoan,
Ciona or Styela community.

8. The establishment of Ciona or Styela communities appears to depend in
part on seasonal factors.

ACKNOWLEDGMENT

I should like to express my appreciation of the helpful criticisms and suggestions
of Professors W. R. Coe, M. W. Johnson, G. E. MacGinitie and A. C. Redfield.
Miss Margaret L. Campbell rendered invaluable technical assistance during a part

MARINE FOULING COMMUNITIES 121

of this work. I am indebted to Dr. Raymond C. Osburn for identification of the
bryozoans.

LITERATURE CITED

COE, W. R., 1932. Season of attachment and rate of growth of sedentary marine organisms at

the pier of the Scripps Institution of Oceanography, La Jolla, California. Bull. Scripps

lust. Occanogr. Tech. Scr., 3: 37-86.
COE, W. R., AND W. E. ALLEN, 1937. Growth of sedentary marine organisms on experimental

blocks and plates for nine successive years at the pier of the Scripps Institution of

Oceanography. Bull. Scripps Inst. Occanogr. Tech. Scr., 4: 101-136.
HEWATT, W. G., 1935. Ecological succession in the Mytilus calif ornianus habitat as observed in

Monterey Bay, California. Ecology, 16: 244-251.
KITCHING, J. A., 1937. Studies in sublittoral ecology II. Recolonization of the upper margin

of the sublittoral region. /. Ecology, 25: 482-491.
McDouGALL, K. D., 1943. Sessile marine invertebrates at Beaufort, North Carolina. Ecol.

Monogr., 13 : 321-374.
MOORE, H. B., 1939. The colonization of a new rocky shore at Plymouth. /. An. Ecologv, 8:

29-38.
MOORE, H. B., AND N. G. SPROSTON, 1940. Further observations on the colonization of a new

rocky shore at Plymouth. /. An. Ecology, 9: 319-327.
SHELFORD, V. E., 1930. Geographic extent and succession in Pacific North American intertidal

(Balanus) communities. Publ. Pugct Sound Biol. Sta., 7: 217-224.
WILSON, O. T., 1925. Some experimental observations of marine algal successions. Ecology, 6 :

303.
ZoBELL, C. E., AND E. C. ALLEN, 1935. The significance of marine bacteria in the fouling of

submerged surfaces. /. Bacterial., 29: 239-251.

A COMPARISON OF THE EFFECTS OF CYANIDE AND AZIDE ON
THE DEVELOPMENT OF FROGS' EGGS l

S. SPIEGELMAN 2 AND FLORENCE MOOG

Department of Zoology, Washington University, Saint Louis 5, Missouri

Loeb's (1895) observations that the eggs of Fuuditlns heteroditus are capable
of considerable development under anaerobic conditions has since been extended
to various amphibian embryos. Brachet (1934), in confirming the possibility of
anaerobic development for Rana temporaria. eggs, reported also that cyanide is
similar to anaerobiosis in its effects on embryogenesis. Eggs placed in cyanide
immediately after fertilization were arrested in the late blastula, but those placed in
cyanide after gastrulation had begun would continue to the formation of a com-
plete blastopore. Later stages were increasingly sensitive to cyanide. Although
it has generally been thought that the arrests of development caused by cyanide are
due to inhibition of the cytochrome oxidase of the Warburg-Keilin system (Keilin,
1933), it might be inferred from the recent work of Holtfreter (1943) that the
repressive effects of cyanide solutions result merely from their alkalinity. It will
be shown in this paper however that only post-mortem effects are influenced by the
pH of the cyanide solution, the actual stoppage resulting from the presence of the
toxic radical itself.

In 1936 Keilin reported in detail on another specific inhibitor of cytochrome
oxidase, sodium azicle (NaN3). On the basis of these experiments NaN3 and
NaCN have in some cases been used interchangeably. Philips (1940), in com-
paring the developmental sensitivities to anaerobiosis of pelagic and non-pelagic
fish eggs, employed both NaCN and NaN... He found that Fundulus eggs before
the end of gastrulation are capable of extensive development in concentrations of
both cyanide and azide which completely and almost immediately inhibit pelagic
eggs. Except for the higher concentrations required in the case of NaN3 he could
demonstrate no difference between the effects of the two reagents. Recently
Barnes (1944) tested the same reagents on the development of Rana piplcns. The
results with cyanide confirmed the earlier observations of Brachet (1934). While
no detailed data are given, the effects of azide were apparently found to completely
parallel those of cyanide, for Barnes states: "Eggs exposed to M/100 NaN3 at pH
7.0 are able to develop to the gastrula stage. Gastrulation never occurs in the
presence of azide." Lower concentrations (M/1000) did not stop gastrulation
although the eggs developed at a slower rate.

The present authors (Moog and Spiegelman, 1942). while investigating the
relation between regeneration and metabolic activity, demonstrated a specific differ-
ence between the effects of azide and cyanide on hydranth reconstitution in Tubu-
laria. Azide could inhibit regeneration at concentrations which did not sensibly

1 Aided by a grant from the Rockefeller Foundation.

2 Present address : Department of Bacteriology and Immunology, Washington University
Medical School, Saint Louis, Missouri.

122

EFFECTS OF CYANIDE AND AZIDE ON FROGS' EGGS 123

affect respiration whereas cyanide caused parallel depressions in rates of regenera-
tion and of respiration. Subsequent analysis (Spiegelman and Moog, 1944) of
the differential effects of these two agents on the mass and time of appearance of
the new hydranth emphasized the difference in their activities.

In the fall of 1941 the authors undertook a comparison of the effects of NaCN
and NaN3 on the development of Ran a pipicns.3 The results obtained disagree in
certain respects with those reported by Barnes (1944). Azide was found to be
completely effective in stopping morphogenesis at all stages of development, in-
cluding those between fertilization and gastrulation which are not inhibited by
cyanide. In an effort to discover the cause for the disagreement these experiments
were repeated recently under conditions closer to those employed by Barnes. Our
earlier results were confirmed and the discrepancy remains unresolved. No direct
comparison with the findings of Philips (1943) is possible, not only because of the
difference in material but also because the highest concentration he employed was
below the one we found to give consistent inhibitions.

The results will be detailed and the difference obtained between the effects of
azide and cyanide will be discussed in the light of recent findings on azide inhibi-
tions of anaerobic synthetic processes.

GENERAL METHODS AND MATERIALS

Eggs of Rona l^lpicns, obtained by injection of pituitary glands, were expressed
and artificially fertilized. After swelling of the jelly the eggs were cut up into
small groups in 10 per cent Ringers solution adjusted to the desired pH with
phosphate buffer. Stages were determined according to the schedule of Pollister
and Moore (1937) and are so numbered in the present paper. The eggs were
stripped from the jelly with fine forceps before being immersed in the experimental
solutions.

All hydrogen ion concentrations were determined with a glass electrode after
the reagents were added. Where temperature control is indicated the designated
temperature was held within ± 0.2° C. Other experimental details will be found
in the appropriate places of the text.

EXPERIMENTAL RESULTS
The effects of azide and cyanide on development

Kfeilin (1936) as well as subsequent investigators demonstrated the critical in-
fluence of pH on the effectiveness of azide as a respiratory inhibitor. Using the
isolated Warburg-Keilin system as well as yeast cells Keilin obtained maximal
effects at about pH 6.3 when the azide was used in concentrations of 0.001 and
0.002 molar. In the experiments to be described in the present section azide solu-
tions were adjusted to pH 6.6. The concentration chosen for study was 0.005
molar, since parallel experiments on the effects on respiration (see Spiegelman
and Steinbach, 1945) indicated maximal effects at this concentration on respiratory
rate. The same can be said for development, for 0.005 M azide yields completely
effective inhibition. All controls for the azide experiments were similarly adjusted

3 These studies were carried out in the laboratories of the Department of Zoology, Columbia
University, New York.

124

S. SPIEGELMAN AND FLORENCE MOOG

to pH 6.6. In the case of cyanide both experimental and controls were run at
pH 8.4. The controls at pH 8.4 did not differ detectably in rate of development
from those at pH 6.6. Every experimental set had its own control and both were
thus handled exactly the same number of times and in the same fashion. This
avoided the relatively more frequent handling and examination of the controls
which would have been necessary if one set of eggs were the controls for a larger
number of experimentals. For convenience in observations all of the present ex-
periments were done at 15.2° C. in a cold room. To avoid the accelerating and
decelerating effects of changing temperatures during development (see Ryan, 1943)
the eggs were kept at 15.2° C. in 10 per cent Ringers until they reached the stage
it was desired to test. They were then transferred to the approximate solutions
previously brought to the same temperature. The cyanide solutions were freshly
prepared and renewed every 12 hours during the course of an experiment ; the
experimental solutions were kept in stacked fingerbowls, with an empty bowl cover-
ing the top member of the stack.

For the purposes of comparison with the azide experiments, the results with
cyanide in the early stages are reproduced in Table I. They do not differ in essen-
tials from those reported by Brachet (1934). Eggs placed in cyanide early in
development, although delayed as compared with controls, continue to develop up
to gastrulation. The later the stage at which they are subjected to cyanide the
closer is the approach to gastrulation ; they do not however actually begin to gas-
trulate. Eggs in early stage 9 will continue to segment until the cells at the vegetal
pole are quite minute but will evidence no signs of dorsal blastopore lip formation.
If however the invagination has already started cyanide will not immediately stop
it and the eggs may proceed to the formation of a complete blastopore before ceas-
ing activity. Later stages become increasingly sensitive to cyanide.

TABLE 1

The effects of cyanide on development at pH 8.4 at 15.2° C. These experiments were done
in 1941-2 on material obtained from Vermont. The numbers represent the developmental stages
as described under Methods.

Hours after immersion

Stage at start
of experiment

Solution

No. of
eggs

120

Uncleaved

0.001 M

210

Uncleaved

Control

210

0.001

200

Control

200

0.001

160

Control

160

0.001

200

Control

200

0.001

200

Control

200

EFFECTS OF CYANIDE AND AZIDE ON FROGS' EGGS

125

The results obtained with azide are summarized in Table II. It is immediately
evident that all stages are azide sensitive, including the early ones which are not
effectively inhibited by cyanide. It might be noted that under these experimental
conditions the cessation of developmental activity on immersion in azide solution
is, as far as can be determined, abrupt and immediate. This was easily ascertained

TABLE II

The effect of azide on development at pH 6.6 at 15.2° C.; 1941-2, material from Vermont.
The numbers represent the developmental stages as described under Methods

Stage at
beginning

Solution

Hours after immersion

No. of
eggs

120

Uncleaved

0.005 M

100

Uncleaved

Control

100

0.005 M

150

Control

140

0.005 M

Control

0.005 M

110

Control

110

0.005 M

160

Control

160

0.005 M

Control

0.005 M

105

Control

105

0.005 M

120

Control

120

0.005 M

Control

0.005 M

110

Control

110
60

0.005

Control

in the early cleavage stages since no further cleavage was observed. Although the
observations are more difficult in the later stages, careful examination failed to
reveal any development subsequent to treatment with azide. If the eggs are re-
moved within 30 minutes after being placed in the azide solution and thoroughly
washed they can proceed with their development.

Barnes' (1944) experiments with azide were done at higher temperatures, con-
centrations, and pH than those described above. Accordingly when the azide ex-

126

S. SPIEGELMAN AND FLORENCE MOOG

periments were repeated they were done at room temperature (ca. 25° C.). at pH
7.4 and 8.3 (i.e. with and without added hydrochloric acid), and with concentra-
tions up' to 0.01 M. The results of these experiments are given in Table III. At
both hydrogen ion concentrations, 0.01 M azide caused immediate arrest in all pre-
gastrular stages. The 0.005 M concentration used in the early experiments was
retested under these conditions and found to give exactly the same results as previ-
ously obtained. Controls kept in Ringers buffered at the experimental pH de-
veloped normally in all cases, and are not reported in the table.

TABLE III

The effect of azide on development at 25° C.; 1944-5, material from Wisconsin

Stage at immersion

Cone. (Molar)

Stage at arrest

0.001

7.4

0.001

8.3

0.005

7.4

0.005

8.3

0.01

7.4

0.01

7.4

0.01

8.3

0.005

7.4

0.01

7.4

0.005

7.4

0.005

7.4

11 +

0.01

8.3

12-

0.01

8.3

* There was no delay in reaching this stage.

It is clear that we can offer no support to Barnes' statement that at the concen-
tration and pH she employed, azide, like cyanide, permits eggs to develop to gas-
trulation.

The effect of NaCN on development at different pH values

Holtfreter (1943) presented evidence showing that the disruptive effects of
strong cyanide solutions (0.1 M to 0.0015 M) can be imitated by potassium hy-
droxide solutions of equal pH. Although the author did not specifically claim that
the oxidation-repressing effects of cyanide are to be regarded as completely ir-
relevant to its influence on development, it nevertheless seemed advisable to us to
clarify the points which were left in an indecisive state by Holtfreter's work. This
we did, in our 1944-1945 series of experiments, both by comparing the effects of
NaCN solutions brought to pH 7.2 with HC1 with those at pH 9.6-9.8, and by de-
termining the effects of solutions of either NaOH or KOH at pH 9.8. The tests
were made at about 25° ; the cyanide solutions were changed three times daily, the
hydroxide solutions once daily.

The results of the NaCN tests completely confirmed our earlier findings (Table
IV). The stage in which development was stopped, and the speed with which
that stage was reached, was in all cases the same in solutions of equal concentration
at both low and high pH. Only after the egg had been in an arrested stage for

EFFECTS OF CYANIDE AND AZIDE ON FROGS' EGGS

127

more than 12 hours did a difference between the two pH's become evident. At
high alkalinity the pigment became streaked, the surface disintegrated, and the egg
was in the majority of cases reduced to a loose, fuzzy mass of cells within 36 hours ;
at low alkalinity the surface was only moderately eroded after 72 hours.

TABLE IV

The effects of NaCN at pH 7.2 and 9.8; 1944-5, material from Wisconsin

Stage at
immersion

Cone.
(Molar)

pH 7.2

pH 9.6-9.8

Stage
at
arrest

Later effects

Stage
at
arrest

Later effects

1
1

0.003
0.006

Not tested
Not tested

7
7

Egg swollen and surface
severely depigmented after
36 hrs.

7
7

0.003
0.006

9
9

Marked depigmentation
after 40 hrs.

9
9

Depigmentation after 20
hrs., surface disintegrated
after 36 hrs.

9
9

0.003
0.006

11
11

Blastopore lip disappeared
within 20 hrs. after forming

11
11

Blastopore lip also disap-
peared. Surface completely
disintegrated after 24 hrs.

0.004

Surface became mottled
but did not disintegrate
within 72 hrs.

Complete disintegration
within 24 hrs.

0.004

Egg swelled to twice its
normal diameter but did
not disintegrate within 96
hrs.

Complete disintegration
within 24 hrs.

0.004

Surface became mottled
and egg swelled somewhat,
but did not disintegrate
within 88 hrs.

Surface became mottled
within 24 hrs., complete
disintegration within 38
hrs.

The studies with hydroxides revealed that Rana pipiens eggs can develop from
fertilization to the stage of tail-fin circulation (stage 22, at which they were dis-
carded) at pH 9.8 (i.e., 2.5 X 10~4M). Stage 22 was also achieved uneventfully
if the eggs were immersed in the hydroxide solutions at the stage of the morula
(S7), late blastula (S9), mid-gastrula (Sll), neurula (S14), muscular movement
(S18) ; in the last two cases the vitelline membrane was removed before the em-
bryos were placed in the alkali solutions. In complete contradiction to Holtfreter's
finding that eggs disintegrate in the morula stage in KOH solutions of pH 9.0 to
9.4, we did not observe either retardation or abnormality of development. In
three experiments with NaOH and two with KOH, we obtained identical results.
Thus we may conclude that the suppressive action of NaCN (or KCN) on living
egg is due to the poisonous effect of the CN component.

128 S. SPIEGELMAN AND FLORENCE MOOG

DISCUSSION

Both azide and cyanide are effective inhibitors of respiration in the early as
well as in the later stages of development (Barnes, 1944; Spiegelman and Stein-
bach, 1945). The fact that cyanide cannot inhibit at any stage before gastrulation
whereas azide can inhibit at all stages, cannot be explained on a respiratory basis.
This is even more pointedly demonstrated by the capacity of eggs to develop to
gastrulation under anaerobic conditions. The ability of cyanide to depress respira-
tory rates at all stages clearly proves that it gets into the cells of the early embryos,
and consequently a difference in permeability cannot be invoked to explain the
difference between the effects of azide and cyanide on development. It is clear from
these experiments that, at least in the early stages, NaN3 is inhibiting some cyanide-
insensitive process necessary for development.

Recent work has served to question conclusions drawn from Keilin's earlier
experiments that azide and cyanide are essentially equivalent inhibitors of the
Warburg-Keilin system. Stannard (1939) showed that cyanide inhibited the
respiration of both resting and active muscle while azide affected active muscle
only. Armstrong and Fisher (1940) demonstrated that azide and cyanide behave
differently in inhibiting the enzymes controlling the frequency of the embryonic
fish heart-beat. Differences in cyanide and azide inhibitions of tissue respiration
led Korr (1941) to postulate the existence of different pathways of respiration in
resting and stimulated tissues. Ball (1942) suggested different oxidation-reduc-
tion potentials for the Atmungsferment-cyanide and Atmungsferment-azide com-
pounds as an explanation of the different effects of the two inhibitors. Winzler
(1943), after subjecting the kinetics of the respiratory inhibition by cyanide and
azide in yeast to a careful examination, came to the conclusion that cyanide in-
hibited yeast respiration by three different pathways: (1) by combining with
oxidized Atmungsferment ; (2) by increasing the apparent KO2 of reduced At-
mungsferment ; and finally (3) by combining with the enzyme which controls the
rate-limiting step of the rate of respiration. Azide on the other hand exhibited
only one type of inhibition, namely, combination with oxidized Atmungsferment.

Aside from these studies on respiration, others have been made on assimilatory
activity of microorganisms. Barker (1936) and Giesberger (1936) showed that
suspensions of bacteria could under certain circumstances synthesize carbohydrate
from various substrates. Clifton (1937) studied the effect of azide on these
syntheses and found them to be completely inhibited. In the presence of azide
external substrate was completely oxidized. Clifton and Logan (1939) extended
these findings and showed that it was possible to differentially inhibit assimilatory
processes with both NaN3 and 2, 4-dinitrophenol. Winzler (1940), working with
acetate assimilation in yeast, showed that low concentrations of azide, cyanide, or
2, 4-dinitrophenol prevented assimilation. Azide was also shown by Winzler
(1944) to prevent the anaerobic assimilation of glucose by yeast without interfering
with its fermentation. Winzler, Burk, and du Vigneaud (1944) found that azide
in concentrations of 10^4 and 10~3 molar inhibits completely the anaerobic assimila-
tion of ammonia.

These experiments show that azide, and in certain instances cyanide, can in-
hibit synthetic processes which are essentially anaerobic in nature and not con-
nected with the Warburg-Keilin system. It seems most probable that it is this

EFFECTS OF CYANIDE AND AZIDE ON FROGS' EGGS 129

sort of inhibitory activity which is involved in the ability of azide to stop embryonic
development. Unfortunately, with the exception of Winzler's (1940) study of
acetate assimilation no detailed comparison between the effects of azide and cyanide
on synthetic processes has been published. In view of the results reported in the
present paper one would venture to predict that such differences will be discovered.
It may be noted that one such difference has been found in the case of adaptive
enzyme formation in yeast, which is azide sensitive but is not inhibited by cyanide
(Spiegelman, 1945). A suggestive finding has been reported recently by Meyer-
hof (1945), who prepared a solution of adenylpyrophosphatase from yeast by
supersonic vibration and found it insensitive to cyanide but highly sensitive to
azide. This enzyme, involved as it is in transphosphorylation, might conceivably be
a part of the azide sensitive anaerobic synthetic processes.

SUMMARY

Previous observations that amphibian eggs can develop up to the beginning of
gastrulation in cyanide solutions have been confirmed on eggs of Rana pipiens.
The effect of cyanide is independent of pH, and eggs can develop into tadpoles in
2.5 X 10-4 molar NaOH or KOH solutions at pH 9.8.

Azide has been found to arrest development immediately in all stages from
fertilization to tail-bud formation. The effect is the same from pH 6.6 to pH 8.3.

These differences are discussed in the light of recent studies on the effects of
azide and cyanide on respiratory, assimilatory, and phosphorylative processes.

LITERATURE CITED

ARMSTRONG, C. W. J., AND K. C. FISHER, 1940. A comparison of the effects of respiratory

inhibitors azide and cyanide on the frequency of the embryonic fish heart. /. Cell.

Comp. PhysioL, 16: 103-112.
BALL, E. C., 1942. Oxidative mechanisms in animal tissues. A Symposium on Respiratory

Enzymes. Wisconsin, University Press.
BARKER, H. A., 1936. The oxidation metabolism of the colorless alga, Prototheca zopfi. /.

Cell. Comp. PhysioL, 8 : 231-250.
BARNES, M. R., 1944. The metabolism of the developing Rana pipiens as revealed by specific

inhibitors. /. £.r/>. Zoo/., 95: 399-417.
BRACKET, J., 1934. fitude du metabolisme de 1'oeuf de grenouille (Rana fusca) au cours du

developpement. I. La respiration et la glycolyse, de la segmentation a 1'eclosion.

Arch, de Biol, 45: 611-727.
CLIFTON, C. E., 1937. On the possibility of preventing assimilation in respiring cells. Enzy-

mologia, 4 : 246-253.

CLIFTON, C. E., AND W. A. LOGAN, 1939. On the relation between assimilation and respira-
tion in suspensions and in cultures of Escherichia coli. /. Bact., 37 : 323-540.
GIESBERGER, G., 1936. Beitrage zur Kentniss der Battung Spirillum Ehbg. Dissertation,

Utrecht.
HOLTFRETER, J., 1943. Properties and functions of the surface coat in amphibian embryos.

/. Exp. Zool, 93 : 251-323.
KEILIN, D., 1933. Cytochrome and intracellular respiratory enzymes. Eng. Ensymjorschg., 2 :

239-271.
KEILIN, D., 1936. The action of sodium azide on cellular respiration and on some catalytic

oxidation reactions. Proc. Roy. Soc., London, B, 121 : 165-173.
KORR, I. M., 1941. The relation between cellular metabolism and physiological activity. Am.

J. PhysioL, 133 : 167.
LOEB, J., 1895. Untersuchungen iiber die physiologischen Wirkungen des Sauerstoffmangels.

P fliiger's Arch., 62 : 249-295.

130 S. SPIEGELMAN AND FLORENCE MOOG

MEYERHOF, O., 1945. The origin of the reaction of Harden and Young in cell-free alcoholic

fermentation. /. Biol. Chcm., 157: 105-119.
MOOG, F., AND S. SPIEGELMAN, 1942. Effects of some respiratory inhibitors on respiration and

reconstitution in Tubularia. Proc. Soc. Exp. Biol. and Mcd., 49 : 392-395.
PHILIPS, F. A., 1940. Oxygen consumption and its inhibition in the development of Fundulus

and various pelagic fish eggs. Biol. Bull., 78 : 256-274.
POLLISTER, A. E., AND J. A. MOORE, 1937. Tables for the normal development of Rana sylvatica.

Anat. Rcc., 68: 489-496.
RYAN, F. J., 1941. Temperature changes and the subsequent rate of development. /. Exp.

Zool, 88 : 25-54.

SPIEGELMAN, S., 1945. The effect of cyanide and azide on adaptive enzyme formation. Un-
published.
SPIEGELMAN, S., AND F. MOOG, 1944. On the interpretation of rates of regeneration in

Tubularia and the significance of the independence of mass and time. Biol. Bull., 87 :

227-241.
SPIEGELMAN, S., AND H. B. STEINBACH, 1945. Substrate-enzyme orientation during embryonic

development. Biol. Bull, 88 : 254-268.
STANNARD, J. N., 1939. Separation of the resting and activity oxygen consumption of frog

muscle by means of sodium azide. Am. J. Physiol., 126: 196-213.
WINZLER, R. J., 1940. The oxidation and assimilation of acetate by baker's yeast. /. Cell.

Comp. Physiol., 15 : 343-354.

WINZLER, R. J., 1943. A comparative study of the effects of cyanide, azide, and carbon mon-
oxide on the respiration of baker's yeast. /. Cell. Comp. Physiol., 21 : 229-252.
WINZLER, R. J., 1944. Azide inhibition of anaerobic assimilation of glucose by yeast and its

application to the determination of fermentable sugar. Science, 99 : 327-328.
WINZLER, R. J., D. BURK, AND V. DU VIGNEAUD, 1944. Biotin in fermentation, respiration,

growth, and nitrogen assimilation in yeast. Arch. Biochcin., 5 : 25-47.

Vol. 89, No. 2 October, 1945

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

STUDIES ON THE BIOCHEMISTRY OF TETRAHYMENA.

IV. AMINO ACIDS AND THEIR RELATION TO THE

BIOSYNTHESIS OF THIAMINE

GEORGE W. KIDDER AND VIRGINIA C. DEWEY 1

Arnold Biological Laboratory, Brown University,
Providence, Rhode Island

It was reported earlier (Kidder and Dewey, 1942) that two species of Tetra-
hymena were able to carry out the synthesis of thiamine, if provided with a sub-
stance found mainly in the leaves of plants. This substance was called Factor S
and was found in highest concentration in alfalfa leaf meal but could not be demon-
strated from materials of animal origin. Factor S was characterized by its solu-
bility in water and alcohol (up to 75 per cent), insolubility in ether and acetone,
stability to prolonged heat in the presence of either alkali or acid, and its stability
to ultraviolet radiation. It was shown to be dialyzable through cellophane and not
to be precipitated by the salts of heavy metals. It was shown that Tetrahymena
gave optimal growth in a medium consisting of 'Vitamin-free" casein, salts and a
heat- and alkali-treated water extract of alfalfa meal. Very little growth occurred
in the absence of the alfalfa extract and the addition of thiamine, riboflavin, pyri-
doxine, pantothenic acid, nicotinic acid, pimelic acid, z-inosital, uracil, or />-amino-
benzoic acid either singly or in combination had no significant effect. Inasmuch as
the heat- and alkali-treated alfalfa extract wras certainly free of thiamine it was con-
cluded that Tetrahymena could synthesize the thiamine required for its metabolic
needs when supplied with Factor S. It was suggested that Factor S possibly acted
as a catalyst necessary for the synthesis of the thiamine molecule.

It was recognized that the alfalfa extract used contained Factors I and II
(Dewey, 1941 ; 1944) and we now know that the casein base contained Factor III
(Kidder and Dewey, 1945a).

This work was criticized by Hall and Cosgrove (1944) on the basis that the
"vitamin-free" casein used for the base medium was not free of thiamine. They
reported growth of their strain of Tetrahymena in heat- and alkali-treated casein
in the presence of thiamine and not in its absence. This criticism was shown to
be invalid (Kidder and Dewey, 1944) when an extension of the earlier studies was
carried out, using heat- and alkali-treated base media (casein, casein hydrolysate,
gelatin, gelatin hydrolysate). It was then found that heat and alkali treatment of

1 Aided by grants from the Morgan Edwards Fellowship Fund, the Manufacturers Research
Fund for Bacteriology and Protozoology of Brown University.

131

132 GEORGE W. KIDDER AND VIRGINIA C. DEWEY

whole casein produced toxic substances which could not be overcome by thiamine
addition for T. geleii W but could to a slight extent for T. gcleii H. In trypto-
phane-supplemented gelatin (Harris), however, indefinitely transplantable growth
was possible after all of the thiamine had been destroyed. The addition of thiamine
did not affect the generation time but did increase significantly the maximum yield
and survival. The addition of heat- and alkali-treated alfalfa extract decreased the
generation time and raised the maximum yield to optimal for the species, and the
addition of thiamine had no significant effect. This was interpreted as meaning
that gelatin possessed low concentrations of Factors I, II, and S and that the final
cessation of growth was due to the depletion principally of Factor S, as the ad-
dition of thiamine did raise the maximum yield.

One of the difficulties encountered in the earlier work was the separation of
Factor S from Factors I and II. The heat and alkali treatment of peptones seemed
to destroy the Factor I activity, but toxic substances were produced which rendered
the medium inferior for our tests. Nevertheless, it was possible to show that lead
acetate precipitate (containing no factor S) from plant material could replace the
heat- and alkali-destroyed fraction only if thiamine was added. This was taken to
mean that peptone contained no Factor S but did contain Factor II which was stable
to the treatment used for dethiaminization, and Factor I which was unstable. It
was recognized that little more could be done until active preparations of Factors I
II could be obtained which wrere essentially free of both Factor S and toxic ma-
terials.

Recently we have been able to obtain such a preparation and it has been possible
to test the activity of Factor S. This work, to be reported here, while confirming
our earlier conclusions on thiamine synthesis, has forced us to alter our original
theory concerning the role of Factor S in the metabolic activities of Tetrahymena.

MATERIAL AND METHODS

The organism used in the present study was the ciliated protozoan Tetrahymena
geleii W, which is the strain used in the previous studies on thiamine synthesis
(Kidder and Dewey, 1942; 1944). All work was done with pure (bacteria-free)
cultures. The ciliates were grown in 2 ml. quantities of media in Pyrex tubes ac-
cording to the technique described elsewhere (Kidder and Dewey, 1945b). All
media, made with water twice distilled over permanganate in an all-Pyrex still,
were adjusted to give a final pH of 6.8-7.0 and sterilization was by autoclaving.
Serial transplants were made and results are recorded only after the third trans-
plant. Transplants were made at 72 hour intervals using a bacteriological loop de-
livering approximately 0.008 ml. of fluid. Incubation was at 25° C. Population
densities were determined by the direct counting technique (Kidder, 1941). All
glassware used in this investigation was made chemically clean with sulfuric-di-
chromate solution, thoroughly rinsed and air dried before use.

In order to eliminate the possibility of cotton fibers contributing substances to
the medium, Pyrex wool plugs were used extensively. It was found helpful to
flame the protruding ends of the plugs until a thin crust had formed to eliminate
the annoying strands inevitably present in this type of plug. This treatment fuses
enough of the Pyrex strands to cause the plugs to hold their shape and increases
appreciably the ease with which they may be handled.

BIOCHEMISTRY OF TETRAHYMENA IV 133

Two types of base media were used for most of this work. One was 0.5 per
cent hydrolyzed Eastman purified calfskin gelatin (Lot no. 144). This hydroly-
sate was prepared by refluxing 100 gr. of gelatin in one liter of 25 per cent HoSO4
for 5 hours, removing the sulfate as BaSO4 and reducing to the required concentra-
tion. Hydrolysate prepared with HC1 was also used and the two were similar in
every way. The gelatin hydrolysate was supplemented in all cases with 0.01 per
cent /( — )-tryptophane and (with one exception to be noted later) with 0.02 per
cent d/-valine. This base medium will be referred to as EGH.

The second type of base medium employed was a mixture of the eleven amino
acids found to give optimum growth for this strain of Tetrahymena gclcii (Kid-
der and Dewey, 1945a). These amino acids with the concentration in mg. per
cent of each were as follows: /-( + )-arginine monohydrochloride — 82; /( — )-histi-
dine monohydrochloride — 10; c?/-isoleucine — 35; (//-leucine — 35; o?/-lysine — 60; dl-
methionine — 34; (//-phenylalanine — 14; c?/-serine — I; rf/-threonine — 20; /( — )-tryp-
tophane — 10; of/-valine — 20. This base medium will be referred to as 11 AA.
The sources of the amino acids used have been given elsewhere (Kidder and Dewey,
1945b).

Inasmuch as our primary concern was with thiamine all media were made up to
contain other known growth factors, minerals and sugar to insure against limiting
factors outside the scope of this investigation. Accordingly to our base media the
following were always added :

mg./ml.

Difco bacto dextrose ........................... 2.00

MgSO4-7H2O ................................. 0.10

K2HPO4 ...................................... 0.10

CaCl2-2H2O .................................. 0.05

FeCl3-6H2O ................................... 0.00125

MnCl2-4HoO .................................. 0.00005

ZnCl2 ........................................ 0.00005

Micrograms/ml.

Biotin methyl ester ............................ 0.00005

Calcium pantothenate .......................... 0.10

Nicotinamide ................................. 0.10

i-Inositol .................................. ... 1.00

Choline chloride ............................... 1 .00

^-Aminobenzoic acid ........................... 0.10

Pyridoxine hydrochoride ....................... 0.10

Uracil ....................................... 0.10

Folic acid2 .................................... 0.01

Riboflavin .................................... 0.10

The sources of the salts and growth factors have been given earlier (Kidder and
Dewey, 1945b).

Our preparation containing Factors I, II, and III was made from Liver Frac-
tion L.3 Fifteen grams of Liver Fraction L was dissolved in 750 ml. of distilled
water, adjusted to pH 4.5, and extracted continuously for 96 hours in a liquid-
liquid extracting apparatus (Wilson, Grauer, and Saier, 1940) with 750 ml. of 11-
butyl alcohol. The extracted material was freed of butyl alcohol, neutralized and

2 Folic acid concentrate with a "potency" of 5000, furnished through the courtesy of Dr.
R. J. Williams.

3 Furnished through the courtesy of Dr. David Klein and the Wilson Laboratories.

134

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

the volume reduced to 300 ml. This was designated 12L, and was found to con-
tain adequate amounts of Factors I, II, and III. The pH of this preparation was
adjusted to 9.5-10.5 with NaOH and heated in the autoclave at 123° C. for one
hour for dethiaminization. This preparation will be designated 12L1, which was
found to be free of Factor S activity. 12L1 was used as a supplement in a final
concentration of 1 : 20.

Preparations containing Factor S were obtained from alfalfa meal. Water ex-
tract of alfalfa, as previously described (Kidder and Dewey, 1942; 1944), was
heated in the autoclave at 123° C. for one hour at pH 9.5-10.5 to insure the de-
struction of thiamine. This preparation, designated A, was used in a final con-
centration of 1 : 10.

RESULTS

When Liver Fraction L is heated with alkali to destroy thiamine, changes take
place which make it inferior as a source of supplementary factors for Tetrahymena.
The addition of thiamine does not completely overcome these toxic effects, although
the inhibition is less than that produced when proteose-peptone is dethiaminized.
It was found, however, that toxic materials were not produced upon heating pro-
vided the Liver Fraction L was extracted previously with butanol. The butanol
extraction was used originally for the removal of pyridoxin and riboflavin (to be
reported in detail later).

TABLE I

Growth in EGH and 11 AA with and without added Factor S from dethiaminized alfalfa
extract (A) and with and without added thiamine. All tubes contain 12L1 and the numbers
represent organisms per ml. in the third serial transplant after 72 hrs. of growth.

Additions

Thiamine

A + Thiamine

EGH

3,100

305,000

75,000

290,000

11AA

120,000

310,000

165,000

300,000

It was found that optimum growth resulted when a gelatin hydrolysate medium
(with tryptophane, valine, and ten known growth factors), referred to as EGH,
was supplied with 12L1 and thiamine, and very low growth occurred when the
thiamine was omitted. The 12L1 was low in Factor S yet contained adequate
amounts of Factors I, II, and III. This offered the opportunity to test the mode
of action of Factor S, which could now be supplied from plant material without
reference to the amounts of essential growth factors. Accordingly tests were set
up using both EGH and 11 A A as base media, both supplemented with 12L1. To
these base media were added various combinations of dethiaminized alfalfa extract
(A) and thiamine. The results which were expected, namely the failure of growth
unless either thiamine or Factor S was present, were not realized in 1 1 AA. Table
I shows that very little growth occurred in the media based on EGH unless thi-
amine or Factor S was supplied but relatively good growth was obtained in the
amino acid mixture in the absence of both. It will also be noted that thiamine is
much more stimulatory, under these conditions, than is Factor S.

BIOCHEMISTRY OF TETRAHYMENA IV

135

It was apparent from the foregoing results that the ability of Tetrahymena to
synthesize thiamine was not dependent on the presence of Factor S when 11 AA
was used as the base medium. This led to the conclusion that either some amino
acid or combination of amino acids in the gelatin hydrolysate was blocking the
synthetic mechanisms or that materials in the 12L1 were causing the block, the
latter block being removed by some combination of the pure amino acids not pres-
ent in the gelatin hydrolysate. The first of these possibilities was tested by making
up an amino acid mixture based exactly on the published analysis for gelatin, but
adding both tryptophane and valine (indispensable for this species). The ciliates
behaved in this synthetic gelatin hydrolysate just as they had in 11 AA, so it was
apparent that the first of the possibilities was untenable. The only known differ-
ence between the synthetic gelatin hydrolysate and EGH from a qualitative point of
view was the inclusion in the former mixture of synthetic unnatural isomers (in
the dl form, because of availability) of a number of the amino acids.

The addition of 11 A A to EGH plus 12L1 resulted in good growth without the
addition of either thiamine or Factor S. This led us to test the effect of omitting
each of the 11 amino acids singly from the 11 AA added to EGH. These results
were inconclusive as fair growth occurred in all tubes. This was taken to mean
that more than one of the 1 1 amino acids could counteract the inhibition to thiamine
synthesis.

TABLE II

Growth of EGH with the addition of varying concentrations of racemic mixtures of amino
acids. All tubes contain 12L1. The numbers represent organisms per ml. in the third serial
transplant after 72 hrs. of growth.

Concentration of amino acid added (mg./ml.)

Amino acid

0.1

0.3

0.5

0.8

1.0

Control,
nothing added

dl-phenylalanine

84,000

80,000

82,000

94,000

101,000

3,800

dl-methionine

92,000

8,600

8,000

6,000

7,800

dl-serine

58,000

82,000

97,000

114,000

110,000

dl-norleucine

31,000

11,500

dl-aspartic acid

21,000

46,000

51,000

87,000

62,000

dl-isoleucine

11,500

56,000

70,000

97,500

60,000

dl-lysine monohydrochloride

4,500

15,000

58,000

61,000

78,000

dl-threonine

6,000

31,000

42,000

66,000

81,000

dl-homcystine

10,500

33,000

11,000

8,000

6,400

dl-alanine

3,000

4,200

26,000

11,000

4,500

dl-glutamic acid

6,500

7,500

12,500

21,000

37,000

The next set of experiments was designed to determine whether or not the ad-
dition of single amino acids to the gelatin hydrolysate medium could counteract the
inhibition to thiamine synthesis. Arbitrary amounts of each of nineteen amino
acids were added to EGH. Thiamine synthesis occurred to a marked degree in
some of the tubes, moderately in others and very little in some. In all cases where
the inhibition was not removed the amino acid used was in its natural form while
those amino acids which were most effective were synthetic.

136

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

This set of experiments was repeated using varying concentrations of the syn-
thetic amino acids and some of the results are given in Table II. It will be seen
that the effectiveness of the amino acids in releasing the inhibition of thiamine
synthesis varied with the amino acid and the concentration. Phenylalanine was the
most effective throughout the range of concentrations used while methionine was
most effective in the lowest concentration. Norleucine was moderately effective at
a concentration of 0.1 mg. per ml. but was toxic at 0.5 mg. per ml. or higher. These
results indicated that the unnatural isomers were in some way able to release the
inhibition of thiamine synthesis. It seemed more probable that the ratio between
the two isomers was not the explanation, as some release of inhibition was found
with some of the nonsynthetic amino acids. It is known that in the preparation of
amino acids from natural sources some racemization is likely to occur and this
might account for the small amount of activity.

TABLE III

Comparison of the effect of the natural isomer (1 + ) and the unnatural isomer (1 — ) of iso-
leucine, added to EGH + 12L1. Numbers represent organisms per ml. in the third serial
transplant after 72 hrs. of growth.

Amino acid

Concentration of amino acid added (mg./ml.)

0.1

0.3

0.5

0.8

1.0

Control,
nothing added

l(+)-isoleucine
1( — )-isoleucine

7,800
42,000

4,100
91,000

6,300
81,000

9,800
68,000

11,500
21,000

3,100

This was shown to be the probable explanation by two sets of experiments. We
had samples of natural /( + )-isoleucine, unnatural /( — )-isoleucine and synthetic
c//-isoleucine. A comparison of the figures for dHsoleucine in Table II with those
in Table III shows that /( — )-isoleucine is effective in approximately one half the
required concentration of rf/-isoleucine. This is what is to be expected if only the

TABLE IV

Comparison of the effect of the natural isomer (1 — ) and the racemic mixture (dl) leucine,
added to EGH + 12L1. Numbers represent organisms per ml. in the third serial transplant
after 72 hrs. of growth.

Concentration of amino acid added (mg./ml.)

0.1

0.3

0.5

0.8

1.0

Control,
nothing added

1( — )-leucine
dl-leucine

2,500

4,100

3,500
5,500

10,500
26,000

14,000
29,000

13,500
31,000

3,800

unnatural isomer is effective in the removal of thiamine synthesis inhibition. The
effectiveness of /( + )-isoleucine is low and increases with the concentration. This
could be due to the occurrence of some racemization during its preparation.

When natural leucine was compared to (//-leucine the former was found to be
less effective in the release of the synthesis inhibition (Table IV). The difference

BIOCHEMISTRY OF TETRAHYMENA IV

137

here, however, was not as marked, as the natural form appears to contain a con-
siderable quantity of racemic mixture and the synthetic leucine is rather low in ac-
tivity. It should he noted that we used Kahlhaum c?/-leucine as this was found
previously (Kidder and Dewey, 1945b) to be free of isoleucine, a common con-
taminant of many brands of synthetic leucine (Hegsted and Wardwell, 1944).

Inasmuch as EGH contained added rf/-valine it was thought advisable to deter-
mine whether the unnatural isomer of this ammo acid might be responsible for the
ability of the ciliates to grow at all without added thiamine, Factor S or unnatural
isomers of amino acids (see controls in Tables I-IV). Accordingly EGH minus
valine was tested with varying concentrations of dl-valme with and without thi-
amine. Table V shows that without thiamine, very little growth occurs with no

TABLE V

Effect of the addition of dl-valine to EGH (minus valine). All tubes contain 12L1. Numbers
represent organisms per ml. in the third serial transplant after 72 hrs. of growth.

Concentration of dl-valine (mg./ml.)

0.05

0.1

0.3

0.5

0.8

1.0

Minus thiamine
Plus thiamine

150
190,000

2,500
210,000

4,000
265,000

7,500
310,000

16,000
305,000

37,000
325,000

45,000
315,000

added valine, and that the inhibition to thiamine synthesis is counteracted more
effectively the higher the concentration of added rf/-valine. With added thiamine,
however, the addition of valine had little effect. This indicates that the sample of
gelatin used differs from our previous sample of Eastman de-ashed gelatin in that
it contains nearly optimum amounts of natural valine for this species. It had previ-
ously been found (Kidder and Dewey, 1945b) that Eastman de-ashed gelatin would
not support growth of Tctrahyincna gclcii W without added valine, even in the
presence of thiamine. The fact that transplantable, though very low, growth occurs
without the addition of any unnatural isomers of amino acids may mean that the
inhibition to thiamine synthesis is never complete or that some racemization of the
amino acids has occurred during hydrolysis.

When thiamine was added (0.1 micrograms per ml.) to any of the above de-
scribed combinations, growth was always raised to approximately 300,000 ciliates
per ml. Thiamine, therefore, although it can be synthesized by the ciliates, is very
active as a stimulatory substance. It was of interest and importance to determine
the amount of stimulation produced by different concentrations of thiamine when
added to EGH plus 12L1 ; EGH plus 12L1 and one of the active amino acids;
EGH plus 12L1 and Factor S; and 11 AA plus 12L1. Figures 1-4 show a sum-
mary of the activity of various concentrations of thiamine. The lowest concentra-
tion tested was 0.005 millimicrograms per ml. and in every case this amount gave
significant stimulation. The stimulation was roughly proportionate to the concen-
tration up to 0.001 micrograms per ml. In all cases, after this point, the amount
of growth was increased more gradually but reached approximately the 300,000
level at 0.01 micrograms per ml. of thiamine when inhibition to thiamine synthesis
was absent or removed. Ten times this amount of thiamine was required to raise

300

250

200

150

100

0.0005

0.001 0.0015

MICROORAMS OF THIAMINE PER ML.

0.002 0.005 0.01

FIGURE 1. Curve of population densities at various concentrations of thiamine hydrocloride
with gelatin hydrolysate (EGH) and dethiaminized butanol extracted Liver Fraction L (12L1)
as base. The concentration of organisms was determined from the third transplant after 72 hrs.
of growth.

300

250

§200
tc

'150

100

0.0005

0.001 0.0015

MICROGRAMS OF THIAMINE PER ML.

0.002 0.005 0.01

FIGURE 2. Curve of population densities at various concentrations of thiamine hydrochloride
with EGH, 12L1 and d/-serine (0.5 mg./ml.) as base. Third transplant determinations after
72 hrs. of growth.

138

300

250

§200

Sioo
o

0.0005

0.001 0.0015

MICROORAMS OF THIAMINE PER ML.

0.002 0.005 0.01

FIGURE 3. Curve of population densities at various concentrations of thiamine hydrochloride
with EGH, 12L1 and dethiaminized alfalfa extract (A) as base. Third transplant determina-
tions after 72 hrs. of growth.

300

250

200

150

m
H

o 100

0.0005

0.001 0.0015

MICROORAMS OP THIAMINE PER ML.

0.002 0.005 0.01

FIGURE 4. Curve of population densities at various concentrations of thiamine hydrochloride
with the amino acid mixture (11 AA) and 12L1 as base. Third transplant determinations after
72 hrs. of growth.

139

140

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

the population to 300,000 per ml. where inhibition was pronounced (Fig. 1). These
results show that Tetrahymena is far more sensitive to thiamine below a concentra-
tion of 0.001 micrograms per ml. than to higher concentrations.

An interesting and perhaps important point to be noted in the data shown in
Figure 1 is the inflection which occurs in the curve above the 0.001 microgram per
ml. level. The reasons for this inflection are not clear, although it seems possible
that thiamine may be performing a double role where inhibition is pronounced. It
may be supplying the vitamin needs of the organisms at the lower levels and acting
to remove other inhibitions to growth as the concentrations increase.

Only the intact molecule of thiamine is capable of giving optimum stimulation.
When the pyrimidine portion of thiamine (2-methyl-5-ethoxymethyl-6-amino py-
rimidine) 4 or the thiazole portion (4-methyl-5-beta-hydroxyethyl thiazole) 4 were
added separately or together some release of inhibition occurred. Table VI shows

TABLE VI

Growth in EGH plus 12L1 with varying concentrations of the thiazole and pyrimidine
components of the thiamine molecule. Numbers represent organisms per ml. in the third serial
transplant after 72 hrs. of growth.

Concentration (micrograms/ml.)

0.001

0.005

0.01

0.1

0.5

Control,
nothing added

Thiazole
Pyrimidine
Thiazole and pyrimidine
(total cone.)

3,000
1,200
2,500

8,400
5,300
1 1 ,000

20,000

17,500
24,000

1,400
3,200
2,400

1,000
2,600
2,500

2,900

the results of these experiments. Both thiazole and pyrimidine produce stimula-
tion in low concentrations but are mildly toxic at concentrations of 0.1 micrograms
per ml. or higher. Thiazole and pyrimidine behave much the same as Factor S or
the unnatural isomers of the amino acids, although to a less degree. They appear
to cause the release of the thiamine synthesis inhibition and are themselves inhibitory
in high concentrations.

DISCUSSION

It appears from the foregoing results that there are substances present in natural
materials which can block the synthetic mechanisms of Tetrahymena. Under the
conditions of our experiments this blocking occurred specifically in the mechanism
or mechanisms for the synthesis of the thiamine molecule. That this ciliate can
synthesize thiamine, as was pointed out earlier (Kidder and Dewey, 1942; 1944)
cannot be doubted, when the blocking substance is absent or the block is released.
In our earlier work (Kidder and Dewey, 1942; 1944) where dethiaminized alfalfa
extract was used as the supply of Factors I and II (Factor III was present in the
casein and gelatin preparations; Kidder and Dewey, 1945a), it might be questioned
whether the growth obtained in the absence of thiamine might be the result of no
inhibitory substance rather than the presence of Factor S. However, it must be re-

4 Both the thiazole and the pyrimidine used were furnished through the courtesy of Dr.
George W. Lewis and Merck and Co.

BIOCHEMISTRY OF TETRAHYMENA IV 141

membered that the addition of alfalfa extract to EGH plus 12L1 (which contains
the inhibitory substance) removed the block. Whatever Factor S is, it is able to
release the block to thiamine synthesis. But it is also seen that the unnatural
isomers of the amino acids can act in a similar manner, so this reaction is far from
specific as to counteracting substances. It was formerly proposed (Kidder and
Dewey, 1942) that Factor S might act as a catalyst to the reaction wherein the
thiamine molecule was synthesized. This hypothesis appears to be no longer
tenable.

It does not seem likely that Factor S is, in reality, nothing more than racemic
amino acids, for two reasons. If enough racemization occurred during the heat
treatment of the alfalfa extract to account for the activity found then the same
amount of racemization should have taken place in the heat treatment of 12L to
produce 12L1. It was found, moreover, upon assaying the alfalfa extract for the
indispensable amino acids for Tetrahymena that it did not contain enough of any
one of the ten to support growth, when used in the concentration employed here.
But a similar assay of 12L1 demonstrated almost optimum amounts of lysine; ap-
proximately half optimal amounts of arginine, threonine, and valine ; and traces of
histidine, isoleucine, leucine, and phenylalanine. It seems at present that Factor S
represents some material present in alfalfa and the leaves of other plants (Kidder
and Dewey, 1942), the activity of which is shared by the unnatural isomers of many
of the amino acids.

The relation of amino acids to the ability of organisms to synthesize vitamins
has been pointed out before. Snell and Guirard (1943) showed that alanine could
replace pyridoxine for Streptococcus fccalis R (S. lactis R) and that alanine func-
tioned to counteract the toxicity of glycine. It does seem strange, however, that
the unnatural isomers appear to function in the release of thiamine synthesis inhibi-
tion for Tetrahymena. In nature this organism, being largely a bacteria feeder,
probably would never be called upon to use its thiamine synthesis mechanism. The
use of its ability to synthesize thiamine, therefore, is admittedly the result of arti-
ficial environmental conditions, as is also the very contact with the unnatural
isomers of the amino acids.

It is apparent that, although Tetrahymena does possess the ability to synthesize
thiamine, this vitamin is a potent stimulant to reproduction, size (Kidder and
Dewey, 1944), and longevity (Johnson and Baker, 1943). Thiamine must, there-
fore, be included in complete media for this ciliate, but the amount needed appears to
be less than has been previously used (Hall and Cosgrove, 1944; Kidder and
Dewey, 1942; 1944).

It has been stated previously (Lwoff and Lwoff, 1938; Kidder and Dewey,
1942; 1944; Hall and Cosgrove, 1944) that heating peptones or proteins with alkali
renders the media inferior for the growth of Tetrahymena. This condition could be
partially counteracted for some strains by the addition of thiamine. The explana-
tion appears now to rest in the partial destruction of serine, for we have found that
if 11 AA is heat- and alkali-treated growth (with added 12L1) is very low but re-
turns to normal with the addition of serine. Increased growth results with the ad-
dition of thiamine alone, however, indicating that this vitamin can replace serine.
Or that serine (a dispensable but highly stimulatory amino acid in the presence of
thiamine; Kidder and Dewey, 1945b), is one of the necessary factors for the syn-
thesis of vitamin B

142 GEORGE W. KIDDER AND VIRGINIA C. DEWEY

The relationship which exists between the concentration of thiamine and the
concentration of ciliates (Figures 1—4) might suggest that this organism would be
useful for assay purposes. It would be difficult, however, to assay natural prod-
ucts for thiamine in a base medium composed of EGH plus 12L1, the only com-
bination which gives a low blank, because of the likelihood of the introduction of
Factor S or other materials of like nature with the substance to be assayed. Al-
though we have not attempted to do this, it might be possible to arrange conditions
so that 11 AA (Fig. 4) could be used and the values calculated as differences. Ex-
periments directed to this end might prove valuable as the present microbiological
methods are not entirely satisfactory. The majority of organisms used are stimu-
lated by the thiamine components as well as by the whole molecule (Sarett and
Cheldelin, 1944), require complex base media (Williams, 1942), or require many
days of growth before results can be obtained (Robbins and Kavanagh, 1937).

SUMMARY

1. In Eastman gelatin hydrolysate (EGH) and Factors I, II, and III from
Liver Fraction L (heat- and alkali-treated to destroy thiamine) the ciliate Tetra-
hymena geleii W grows very poorly without added thiamine.

2. A mixture of amino acids (11 AA) with the dethiaminized liver fraction
supports fair growth without added thiamine.

3. There appear to be substances in the liver fraction or the gelatin hydrolysate
or both which specifically block the mechanism for the biosynthesis of thiamine.

4. This block can be released by Factor S from alfalfa extract or by the un-
natural isomers of a number of amino acids.

5. Some release of the inhibition to thiamine synthesis is produced by a few of
the natural amino acids but this is probably due to the presence of low concentra-
tions of unnatural isomers which result from racemization during preparation.

6. The unnatural isomer of isoleucine (the only unnatural isomer available for
testing) was found to be active in approximately one half the concentration of the
dMsoleucine.

7. Thiamine is extremely stimulatory in low concentrations.

8. The thiazole and pyrimidine components are slightly stimulatory but this
stimulation appears to be due to their ability to cause some release of the thiamine
synthesis inhibition.

LITERATURE CITED

DEWEY, V. C., 1941. Nutrition of Tetrahymena geleii (Protozoa, Ciliata). Proc. Soc. Exp.

Biol. Mod., 46 : 482-484.
DEWEY, V. C., 1944. Biochemical factors in the maximal growth of Tetrahymena. Biol. Bull.,

87 : 107-120.
HALL, R. P., AND W. B. COSGROVE, 1944. The question of the synthesis of thiamin by the ciliate,

Glaucoma piriformis. Biol. Bull., 86: 31-40.
HEGSTED, D. M., AND E. D. WARDWELL, 1944. On the purity of synthetic d/-leucine. Jour.

Biol. Chcm., 153: 167-170.
JOHNSON, W. H., AND E. G. S. BAKER, 1943. Effects of certain B vitamins on populations of

Tetrahymena geleii. Physiol. Zoo/., 61 : 172-185.
KIDDER, G. W., 1941. Growth studies on ciliates. V. The acceleration and inhibition of ciliate

growth in biologically conditioned medium. Physiol. Zoo/., 14 : 209-226.
KIDDER, G. W., AND V. C. DEWEY, 1942. The biosynthesis of thiamine by normally athiamino-

genic microorganisms. Grozvth, 6: 405-418.

BIOCHEMISTRY OF TETRAHYMENA IV 143

KIDDER, G. W., AND V. C. DEWEY, 1944. Thiamine and Tetrahymena. Biol Bull, 87 : 121-133.
KIDDER, G. W., AND V. C. DEWEY, 1945a. Studies on the biochemistry of Tetrahymena. II.

Factor three. Arch. Biochetn., 6: 433-437.
KIDDER, G. W., AND V. C. DEWEY, 1945b. Studies on the biochemistry of Tetrahymena. I.

Amino acid requirements. Arch. Biochcm., 6 : 425-432.
LWOFF, A., AND M. LWOFF, 1938. La specificite de 1'aneurine, facteur de croissance pour le cilie

Glaucoma piriformis. C. R. Soc. Biol., 127: 1170-1172.
ROBBINS, W. J., AND F. KAVANAGH, 1937. Intermediates of vitamin Bi and growth of Phyco-

myces. Proc. Nat. Acad. Sci., 23: 499-502.
SARETT, H. P., AND V. H. CHELEIN, 1944. The use of Lactobacillus fermentum 36 for thiamine

assay. Jour. Biol. Chan., 155: 153-160.
SNELL, E. E., AND B. M. GUIRARD, 1943. Some interrelationships of pyridoxine, alanine and

glycine in their effects on certain lactic acid bacteria. Proc. Nat. Acad. Sci., 29: 66-73.
WILLIAMS, R. J., 1942. Studies on the vitamin content of tissues II., Univ. Texas Publ. No.

4237 : 7-13.
WILSON, D., R. C. GRAUER, AND E. SAIER, 1940. A simplified continuous extractor for estrogens

and androgens. Jour. Lab. Clin. Med., 26 : 581-585.

CERTAIN CHEMICAL FACTORS INFLUENCING ARTIFICIAL
ACTIVATION OF NEREIS EGGS1-2

PAUL G. LEFEVRE
Marine Biological Laboratory and Zoological Laboratory, University of Pennsylvania

INTRODUCTION

Stimulation must involve physicochemical changes within cells, and the nature
of such changes has been the subject of much investigation, both experimental and
speculative. The process of fertilization, and the closely related process of activa-
tion in artificial parthenogenesis, have attracted special attention ; and evidence has
been presented for a number of interesting interpretations of this type of activation.
This report concerns a group of experiments indicating a peculiar relation of picric
acid to the artificial activation of the eggs of Nereis. The proper interpretation of
these experiments might contribute to the understanding of the stimulatory process.
The experiments described developed from incidental observations in connection
with heat-activation, during investigations concerned with the more general question
of the mode of action of heat on protoplasm.

The peculiarities of heat-activation of the unfertilized Nereis egg were first de-
scribed by Just (1915), who was able to interpret all his data in harmony with
Lillie's "fertilizin" theories. In particular, Just attributed the gradual loss of sensi-
tivity to heat, in eggs left standing in sea water, to the diffusion from them of some
fertilizin-like substance, essential to the activating process. Heilbrunn (1925) took
exception to this notion, in suggesting a "colloid chemical" interpretation of heat-
parthenogenesis ; he believed the decrease in sensitivity to heat might be due to the
gradual loss of CO2 from the medium, resulting in alkalinization of the intracellular
fluid. Heilbrunn described three experiments in which the addition of 2—^ volumes
per cent of n/10 HC1 to old insensitive egg-suspensions restored their original sensi-
tivity to heat.

To reveal a possible general relation between intracellular acidity or carbon
dioxide concentration and the response of cells to increased temperatures, these
three observations were extended. Heilbrunn's findings were in part confirmed ;
but with the accumulation of large numbers of experiments, considerable variation
was encountered in the response of the heat-sensitivity of the eggs to increased CO2
concentration through acidification of the sea water. Though such pronounced ef-
fects as described by Heilbrunn were often repeatable, as many batches of eggs
seemed totally unresponsive to the same treatment. In the course of testing several
organic acids in this connection, however, the anomalous properties of picric acid
(2, 4, 6-trinitrophenol) came to light. Extension of these properties to processes
of activation by means other than heat was then attempted.

1 This study was carried out under the direction of Dr. L. V. Heilbrunn. I gratefully ac-
knowledge his helpful suggestions throughout the investigations, and his valuable assistance in
interpreting the results.

2 A dissertation submitted to the faculty of the Department of Zoology of the University of
Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

144

CHEMICAL FACTORS IN EGG ACTIVATION 145

MATERIALS AND METHODS

Ripe females of the heteronereis form of Nereis limb at a were captured between
8 and 10 p.m., as described by Lillie and Just (1913), and kept singly or in pairs
in about 200 ml. of sea water in finger bowls overnight. These were kept cool
either on a salt water bench in a stream of sea water, in a refrigerator, or in a room
maintained at 15° C. The last was found by far the most satisfactory in maintain-
ing the worms with eggs intact, with apparently no ill effects. A few worms shed
their eggs during the night even at this reduced temperature ; these were discarded.
All experiments were begun by the transfer of one or two Nereis to a Stender
dish containing 25 ml. of sea water. The animals were cut transversely to release
the eggs, and the carcasses were quickly removed. The eggs were then concen-
trated toward the center of the dish by gentle rotation. Either a small quantity of
an especially dense suspension of eggs was then removed to another dish, or nearly
all of the supernatant fluid was withdrawn by suction, and replaced by fresh sea
water. All eggs were washed in this manner through at least another change of
sea water, before use. In the earliest work, samples were always tested for nor-
malcy by treatment with sperm from males caught in the same swarm. Only very
rarely was any egg ever found which did not become normally fertilized, and all
samples showed well over 99 per cent germinal vesicle breakdown. Since this de-
pendability of Nereis eggs is well known, and since all danger of accidental con-
tamination of eggs with sperm was to be avoided, no such tests were made in most
of the later work. Experiments were always begun on the day following capture,
so that the lapse of time between capture and the first treatment was never more
than 20 hours, and was only rarely over 15 hours.

All transfers of eggs were made with ordinary medicine droppers. All treat-
ments and exposures, unless otherwise indicated, were made in a volume of 25 ml. ;
the egg-suspensions were of such a density that, upon settling of the eggs to the
bottom, no more than half, and usually much less, of the bottom of the container was
covered with a single layer. Stender dishes of about 35 ml. capacity were used
except for the exposures to high temperatures ; the latter were carried out in 50
ml. beakers, in which the thermal insulation is much reduced. The beakers were
immersed in a small deKhotinsky constant-temperature bath to a depth 2-3 mm.
above the surface of the inside liquid. The temperature of the fluid within the
beakers was brought to equilibrium (at slightly less than half a degree lower than
the bath temperature) before the addition of 0.3-0.5 ml. of the egg-suspension.
The activating temperature used varied between 33° and 35° C., as in Just's work
(1915), but was held constant to within 0.1 of a degree for any single series of
tests.

Since it soon became evident that the degree of stirring had a considerable effect
on the response to heat, a standard policy in this regard was always followed : upon
deposition of the eggs in the warm beakers, the pipette was filled and emptied ten
times successively within 4-5 seconds. This was repeated 4 minutes after the be-
ginning of the exposure ; and the beaker was removed after 5 minutes of exposure,
at which time a sample of 5-8 ml. was removed to a Syracuse watch glass. In some
of the earlier work, the second stirring was performed at 15 minutes, the beakers
removed at 20 minutes. This exposure, which is approximately Just's optimum,
yielded a better percentage of swimmers, but the shorter exposure was found to

146 PAUL G. LEFEVRE

produce the maximal amount of germinal vesicle breakdown, and was much more
convenient in extended series of tests. A few tests indicated that further stirring
and longer exposures led to no increase in the percentage of activation. A further
trial showed that the immediate removal to Syracuse dishes was not essential ; when
the beakers were allowed to cool of their own accord, the residual heat did not affect
the percentage of activation.

For counts of activation, 5-8 ml. of- each egg-suspension were examined in a
Syracuse watch glass at a magnification of about 100 X. In certain cases involving
a doubtful response, compression of the eggs between a slide and coverslip, as sug-
gested by Heilbrunn and Wilbur (1937), and a higher magnification were neces-
sary. The counts were made on the basis of the breakdown of the germinal vesicle,
a reaction which normally occurs soon after fertilization. Counts were begun at a
minimum of 2 hours after the application of the treatment in question. The advan-
tages of the nuclear criterion are its rapidity of onset, its definite character (ordi-
narily admitting of easy and certain classification in counting), and its ready sus-
ceptibility to quantitative expression ; the criterion is well established in work on
artificial activation of this form. However, the fact should not be overlooked that
the mere breakdown of the germinal vesicle in response to stimulation is seldom
followed by development even approaching the normal, and there is rarely any cleav-
age at all. Various types of monsters are produced, mostly of the type described
as due to "differentiation without cell-division," common in annelids. All of the
types of stimulation used were capable of producing at least a small percentage of
swimming forms, though seldom was anything like a normal trochophore seen. All
counts were of 100 or 200 eggs selected by random movement of the watch glass
on the stage of the microscope.

RESULTS

Upon standing in sea water, almost all batches of eggs showed a gradual loss
of sensitivity to heat, as described by Just (1915) ; a few, however, showed a very
definite increase in sensitivity, after washing and long standing. This might per-
haps be attributable to the washing away of inhibitors in the body fluids (Just,
1915) ; but the most pronounced of these exceptions was in a special batch in which
the eggs stood in a deep layer at the bottom of a narrow container. Thus the re-
sponsible factor may have been the high CO, tension, in accordance with Heil-
brunn's views (1925). Of several organic acids tested, however, only picric acid
produced a consistent and pronounced reversal of this loss of sensitivity to heat.
After a batch of eggs had become nearly or quite heat-insensitive, a bath of 15
minutes or more in sea water to which picric acid had been added to a concentra-
tion of about M/1000 (pH 6.6) was sufficient to elicit a significant response to the
subsequent heat treatment in sea water. Yet the presence of the acid in the heat-
treated suspensions completely prevented the activation of the eggs ; if a response
was to be obtained, the eggs had to be transferred back to sea water for the heat
treatment.

These aspects of the action of picric acid were then tested in connection with
activating agents other than heat. The agents used were ultra-violet irradiation
(Heilbrunn and Wilbur, 1937), mixtures of sea water and isotonic (0.53 M) KC1
(Wilbur, 1939), and mixtures of sea water and isotonic (0.35 M) sodium citrate

CHEMICAL FACTORS IN EGG ACTIVATION

147

(Wilbur, 1941). Mixtures of KC1 or citrate with sea water are denoted after the
terminology of Wilbur (1941) ; thus a mixture of one volume of isotonic citrate
and four volumes of sea water is called a "20 per cent sodium citrate mixture."

Experiments shozving inhibition by picric acid oj -various types of activation

Heat — Of 15 experiments on the effect of picric acid on the sensitivity of
eggs to heat, only one was inconsistent with the thesis that the acid inhibits the heat-
activation. In these experiments, M/1000 picric acid was used, made up in sea
water. Eight experiments proved useless, as the control percentages were too low
to test any possible inhibition by the acid ; the heat-sensitivity of these eggs is
notoriously very variable between batches from different animals. The average of
the seven experiments in which over 10 per cent of the control eggs responded is
included in Table I, and shows a marked inhibition of the response by picric acid.

TABLE I

Inhibition by picric acid of activation of Nereis eggs by various agents

Activating agent

No. of expts.

Per cent activation
in absence of
picric acid

In picric acid, M/1000

Per cent
activation

Per cent
with incipient
activation*

Heat

KC1 mixtures

Sodium citrate mixtures

* As described on p. 147.

KC1 mixtures — In fourteen experiments in which eggs were left indefi-
nitely in a 25 per cent KC1 mixture, and four similar experiments with a 50 per cent
KC1 mixture, almost always there was nearly 100 per cent activation in the absence
of picric acid. When the acid was added to a concentration of M/1000, such acti-
vation occurred in only one instance ; this case was distinctly unusual, as 74 per cent
of the eggs were activated. Table I includes the averages for these experiments.
However, in only 5 of the 18 tests was the breakdown of the germinal vesicle com-
pletely prevented. In the others, ordinary methods of observation (at 100 X mag-
nification) did not reveal any certain change in appearance from the germinal vesicle
stage, but a distinct nuclear outline could not be made out in many eggs. Compres-
sion of the eggs and higher magnification were necessary in counting these batches ;
the criterion employed was the visibility of a definite interface between the spherical
nucleus and the cytoplasm. In the absence of this interface, the germinal vesicle
was said to be broken down, even though no real alteration in the appearance of the
egg was evident ; the average percentage of the eggs so classified is presented in the
last column of Table I. In these cells, the central nuclear region remained clear,
the granular cortical opacity was retained, the oil droplets remained discrete and
failed to migrate as in the activated eggs. None of the eggs of this type ever devel-
oped to a motile condition, or cleaved, or differentiated in any way. The appear-
ance was as though nuclear breakdown had just barely begun when inhibition set in.

148 PAUL G. LEFEVRE

Sodium citrate mixtures — Complete or nearly complete inhibition of acti-
vation by picric acid was found in 12 of 19 experiments with citrate mixtures of
10-25 per cent. Of the other seven, two showed effective inhibition beyond the
earliest stages of nuclear breakdown, as with the KC1 mixtures (last column of
Table I) ; one showed only moderate inhibition; only 4 of the 19 failed to show any
significant inhibition. The averages are included in Table I. In these experiments,
as in those with the KC1 mixtures, the eggs were left in the activating agents indefi-
nitely ; counts were made with the eggs still in the various mixtures.

Ultra-violet irradiation — Only in relation to activation by ultra-violet rays
did picric acid fail to exhibit an inhibitory effect. The presence of the acid
(M/1000) in the sea water bathing the eggs did effectively prevent their activation
by irradiation, but this action cannot be attributed to the effect of the acid on the
eggs. Reduction of the depth of the egg-suspension to under 0.5 mm., so that the
eggs are barely covered, permitted of ready activation by the rays, even in the pres-
ence of picric acid. The apparent inhibition in deeper samples is due to the absorp-
tion of the rays by the acid ; the absorption spectrum of picric acid and picrates in
salt solutions near neutrality (Eisenbrand and v. Hal ban, 1930; v. Halban and Lit-
manowitsch, 1941) is such that in any appreciable depth and concentration the
supernatant fluid would prevent most of the active radiation from reaching the eggs,
which always settle to the bottom of the dish. This interpretation is corroborated
by the fact that a shield of picric acid in a quartz dish prevents any effect of ultra-
violet rays on an underlying suspension of eggs in sea water.

Fertilization by sperm — Normal fertilization is completely inhibited in the
solutions of acid used for the experiments above (in the range of M/1000). Addi-
tion of alkali to pH 8.0 did not affect this inhibition of fertilization. However, the
removal of normally fertilized eggs to picric acid solutions within five minutes after
fertilization (whether or not such solutions were alkalinized) did not appear to
interfere with the normal development of the embryos; excellent survival and dif-
ferentiation were obtained in the acid. Nevertheless, such embryos exhibited one
outstanding anomaly : failure of the normal coalescence of the oil droplets. The oil
in embryos growing in picric acid remained scattered as numerous discrete droplets ;
while under normal conditions these soon merge to form only a few, almost always
four. The usual localization of the oil by migration (and segregation in cleavage)
was not, however, altered in the course of development in picric acid solutions.

Experiments shoeing synergism between various activators and the removal jrom
picric acid to ordinary sea water

Heat — Over 50 experiments tested the effect of baths in picric acid prior
to exposure to heat in sea water. These showed a pronounced enhancement of the
effects of the heat after the acid bath; not one showed a greater activation in the
sample from sea water than in that from the acid. This relation between heat and
removal from picric acid baths is shown in Figure 1. The synergistic action is
evident only following the shorter baths, up to about 6 hours ; since, after longer
exposures to the acid, the mere removal to sea water was in itself sufficient to acti-
Yate many eggs. The broken line curve in Figure 1 is made up from the combined
data of all experiments involving removal of eggs from picric acid to sea water
without further treatment. The other two curves on the same figure, however,

CHEMICAL FACTORS IN EGG ACTIVATION

149

cover data from paired samples of eggs, and compare the effects of heat on eggs
previously bathed in picric acid (in sea water) and on eggs from the same source
not so treated.

The synergistic action was evident over a wide range of concentration of picric
acid: from 10"4 to just over 10~3 M. The effects increased with increasing concen-
tration, but above M/1000 the results became less reliable, so that M/1000 was used
regularly, and is the only concentration for which data are reported. It is evident

lOOr

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8

LOG OF DURATION OF BATH, IN MINUTES

FIGURE 1. Relation of previous baths in picric acid to heat-activation of Nereis eggs.

Solid line connecting circular points — eggs heated after bath in M/1000 picric acid in sea
water.

Solid line connecting square points — eggs heated after bath in sea water.

Broken line — eggs removed, unheated, from bath in M/1000 picric acid in sea water.

Each point is the average of all experiments performed in the logarithmic time interval de-
noted at the base-line. See text for further explanation.

from Figure 1 that the unfertilized eggs survived in the acid about twice as long as
in sea water. Removal from sea water to the acid just prior to the expected onset
of cytolysis (about 30 hours after removal from the animal) preserved the eggs as
well as, but no better than, storage in the acid from the beginning.

KC1 mixtures — The synergistic action of KC1 mixtures and removal from
picric acid to sea water was tested in 14 experiments, summarized in Figure 2(a).
After 2-8 hours in the acid solutions, samples of eggs were removed to sea water and

150

PAUL G. LEFEVRE

to 5 per cent KC1 mixtures ; a control sample of the same batch of eggs kept in sea
water was simultaneously exposed to the 5 per cent KC1 mixture. This concentra-
tion of KC1 is just below that necessary to induce regularly an appreciable per-
centage of response in ordinary eggs. Though the combined treatment was not in
every case sufficient to activate the eggs, most experiments showed a pronounced
synergism, and none showed a difference in the opposite direction. The response

100

90
80

z 70

- 60

^ 50

. (a)

(b)

2.0 2.2 2.4 2.6 2.8

LOG OF DURATION OF BATH. IN MINUTES

FIGURE 2. Relation of previous baths in picric acid to activation of Nereis eggs by
(a) KC1 mixtures, (b) sodium citrate mixtures.

Solid line connecting circular points^eggs treated after bath in M/1000 picric acid in sea
water.

Solid line connecting square points — eggs treated after bath in sea water.

Broken line — eggs removed, untreated, from bath in M/1000 picric acid in sea water.

Each point is the average of all experiments performed in the logarithmic time interval de-
noted at the base-line. See text for further explanation.

of the eggs in these experiments, upon removal from picric acid baths to sea water,
was somewhat less than average ;' thus the broken lines in the graphs in Figure 2
differ somewhat from the similar curve in Figure 1.

Sodium citrate mixtures — The same action was demonstrated with 10 per
cent sodium citrate in place of the 5 per cent KC1 mixture; 14 of 19 experiments
showed a decided synergism. Five failed to show any significant difference be-
tween control and experimental. These failures were all among the shorter expo-

CHEMICAL FACTORS IN EGG ACTIVATION 151

sures to the acid ; the longer baths always resulted in increased sensitivity of the
eggs to the citrate. This is illustrated clearly in Figure 2(b), which includes the
data from all 19 experiments.

Ultra-violet irradiation — All attempts to show synergism between ultra-
violet irradiation and removal from picric acid baths failed. A Uviarc mercury-
vapor lamp, operating at 110 volts, 60 cycles, was used. The intensity of the radi-
ation at the point at which the eggs were exposed was on the order of 6000 micro-
watts per square centimeter.3 Under such conditions, no significant differences
could be found between the response to irradiation of eggs just removed from picric
acid baths and those from sea water.

DISCUSSION

The data illustrate three aspects of the action of picric acid in relation to activa-
tion of the eggs :

(1) in the presence of certain concentrations of picric acid, heat-activation and
chemical activation are prevented ;

(2) removal from the same concentrations of picric acid to sea water, after a
short stay in the acid, acts synergistically with other activating agents in causing
nuclear breakdown ;

(3) removal from the acid to sea water after longer stays in the acid leads to
activation without assistance from other agents.

That fertilization and maturation of marine eggs is inhibited by acids is a com-
mon observation (Clowes and Greisheimer, 1920; Smith and Clowes, 1924; Tyler
and Schultz, 1932; Tyler and Scheer, 1937) ; so that the inhibition by picric acid of
artificial activation is not surprising. Similarly, the preservation of the unfertilized
egg in picric acid against cytolysis and death is in accordance with many observa-
tions of this action of acids ; some treatments were reported far more effective in
this respect than picric acid appeared to be (Carter, 1931 ; Just, 1920; Smith and
Clowes, 1924; Tyler and Horowitz, 1937a; Tyler and Dessel, 1939). The sugges-
tion has even been made (Tyler, Ricci, and Horowitz, 1938) that the greater life-
span of eggs in alcohol, dextrose, anoxic media, etc. (Gorham and Tower, 1902;
Loeb, 1902; Loeb and Lewis, 1902; Lillie, 1931 ; Whitaker, 1937), can be explained
in each case by the production of acids. The only odd aspect of the action of picric
acid in this regard is that eggs stored in it for some time are subsequently over-
sensitive to stimulators, and eventually are activated merely by removal to sea
water. This was observed after a stay in the acid of as much as 70 hours. This
is entirely dissimilar to the acid activation of starfish eggs, as investigated exten-
sively by Lillie (1926, 1927, 1934, 1941). Lillie's exposures were of only a few
minutes' duration, and the eggs were visibly altered while in the acid ; a slightly
prolonged exposure destroyed the eggs altogether. In picric acid, however, the eggs
remain apparently unchanged for days, but immediately respond when removed to
sea water.

This fact leads to the postulate that picric acid may react with, or in some way
inactivate, an activating agent produced within the egg. Above a certain concen-
tration, this agent would lead to activation of the egg ; in still greater concentration,
or under other conditions, to cytolysis. This agent is apparently being constantly

3 Thanks are due to Dr. A. C. Giese for this measurement.

152

PAUL G. LEFEVRE

produced, and either diffuses from the egg, or is gradually destroyed as it is pro-
duced. But when picric acid is present within the egg, this agent is retained by the
acid in an inactive form ; when the egg is removed to sea water, the picric acid
diffuses away, in turn releasing any acid bound with the activating agent. Thus
the inhibition is removed, so that there is a sudden release of the accumulated acti-
vator within the egg, causing a response it" the accumulation has been great enough.
Such a suggestion is in harmony with the synergism found between other acti-
vating agents and the removal from picric acid after exposures of lesser duration,
and with the temporal pattern of the development of this synergism, as shown in
Figures 1 and 2. The activating agents may be supposed to act by accelerating the
production of the hypothetical activating substance ; subliminal doses of these agents
may then produce enough of the substance so that the added quantity released from
the picric acid suffices to produce the response. That a still greater concentration
may lead to cytolysis is indicated by the fact that less activation, with considerable
cytolysis, is found when eggs are heated after a very prolonged exposure to picric
acid, than when they are simply removed from the acid at the same time to sea
water, without heating (Figure 1).

TABLE II

Synergism between various activating agents in stimulation of Nereis eggs

Activating agents

Per cent activation

No. of expts.

A alone

B alone

Both

Heat

Sodium 5%

citrate 10%

KC1 5%

Sodium 6%

citrate 8%

10%

100

Heat, without usual

Stirring

stirring

The synergistic action indicates that at least to some extent activation is brought
about through the same channels by all four agents : heat, KC1, sodium citrate, and
removal from picric acid to ordinary sea water. Added evidence in this direction
was obtained in experiments showing pronounced synergistic action between heat
and citrate mixtures, and between KC1 mixtures and citrate mixtures (Table II).
As previously mentioned, stirring during exposure to heat had a pronounced en-
hancing action on the stimulatory effect of the heat, but stirring did not appear to
act similarly in connection with the chemical activators. Mathews (1901) reported
that Loeb and Fischer had been able to activate Nereis eggs by mechanical agitation
alone, but all attempts in this direction failed.

Attempts to show synergism between ultra-violet irradiation and sodium citrate
mixtures or removal from picric acid all failed ; this is in keeping with the failure
of picric acid to inhibit activation by ultra-violet rays. This may indicate that the
radiation acts through a different mechanism than that involved in stimulation with
the other agents. But under the conditions of the experiments the duration of

CHEMICAL FACTORS IN EGG ACTIVATION 153

the exposures to ultra-violet was on the order of 30-60 seconds, much less than
with the other types of activation ; this difference in the rate of activation may be
the entire explanation for the non-conformance of the experiments with this type
of activation.

Heilbrunn (1925), Heilbrunn and Wilbur (1937), and Wilbur (1939, 1941)
have presented several lines of evidence indicating that the breakdown of the ger-
minal vesicle in the Nereis egg involves a reaction of calcium ions with the colloids
of the protoplasm, and an associated set of changes in viscosity. Heilbrunn pro-
posed that stimulating agents act by freeing calcium ions from combination (with
lipoprotein) in the cell cortex, so that the calcium may react with the inner proto-
plasm ; this interpretation of stimulation has been applied not only to the eggs of
Nereis, but to cells in general. If such a mechanism is actually involved in the
response of the Nereis egg. it might be expected that a penetrating acid would in-
hibit activation. The picric acid might acidify the protoplasm to the extent that
the amphoteric protein molecules would become predominantly cations, with less
Ca-binding capacity than previously. This interpretation would perhaps also ex-
plain the activation found upon removal of eggs from picric acid baths to sea water ;
the calcium freed from the cortex by the acid could react with the cell interior upon
removal of the acid. Thus the same agent would act, in a sense, both as activator
and as anesthetic. A serious difficulty with this explanation of the data lies in the
fact that the eggs must be left in the acid for several hours, if they are to respond
upon removal to sea water. This would require the assumption that the liberation
by the acid of calcium ions from the cortex is a very slow process ; or else that the
acid continues to accumulate within the egg over a period of hours, quickly rising
to the inhibitory concentration, but only after hours attaining the concentration
active on the cortex. Neither of these assumptions is impossible, but both are rather
involved.

If the action of picric acid were due to this proposed effect on the Ca-binding
properties of proteins, other acids might be expected to act similarly. The action
of other acids similar to picric, as regards pK and penetrating ability, has not yet
been investigated ; however, acetic, boric, and tannic acids have been used in experi-
ments similar to those performed with picric acid. Acetic acid was used in concen-
trations from M/6000 to M/300; boric acid, from M/105 to M/5 ; and tannic acid,
from M/106 to M/100 ; the upper limits of concentrations used were factors of the
solubility and the effects of the acids on the eggs. Over M/1000, acetic acid often
injured the eggs irreversibly, so that they were not fertilizable ; this makes it diffi-
cult to evaluate cases of inhibition by acetic acid of activation, in the absence of
tests for reversal of the effect. Ten to twenty experiments were performed with
each acid in attempts to demonstrate synergism with heat, in the manner of picric
acid ; the duration of the baths ranged from 30 minutes to 24 hours. On a few oc-
casions, acetic acid in concentrations around M/500 (concentrations not always in-
nocuous) showed the synergistic action, but as often acted in the opposite manner
(probably because of injury to the eggs). On one batch of eggs, M/10-M/20 boric
acid also showed some synergistic action with heat, but this did not recur in similar
experiments with other batches of eggs.

A further corroboration of the interpretation in terms of an activator-substance
was sought in several attempts to accumulate the activator more rapidly by heating
the eggs in picric acid, with subsequent release to sea water. In only 3 of 16 such

154 PAUL G. LEFEVRE

experiments was there markedly more activation in the eggs so treated than in
those similarly exposed to the acid without application of the heat. However, none
showed differences in the other direction ; no data of any experiment thus far per-
formed militates against the suggested scheme.

The completely reversible inhibitory action of picric acid is similar to the action
of isotonic citrate in the experiments of Heilbrunn and Wilbur (1937) and Wilbur
(1941). Since the citrate is presumed to act by removing calcium ions from solu-
tion by the formation of calcium citrate, there is a suggestion that perhaps calcium
picrate is a similarly weakly dissociated salt. However, a few measurements of
the electrical resistance of calcium picrate solutions showed that the equivalent con-
ductance increased only slightly with dilution over the range n/100-n/10,000.
(The increase was in proportion to that found with CaCU in the same concentra-
tions ; the equivalent conductance of calcium citrate increased enormously with dilu-
tion over this range of concentration.) Thus the inhibitory action of picrate cannot
be explained on the same basis as that applied to citrate inhibition.

In their extensive experiments on the peculiar action of many substituted phenols
on the eggs of the sea-urchin, Clowes and Krahl (1936), Krahl and Clowes (1936,
1940), and Tyler and Horowitz (1937b, 1938) found picric acid one of only two
or three inactive members of this group of compounds. Inhibition of cleavage was
encountered only at concentrations around M/100 or more, and the stimulation to
respiration characteristic of this chemical group was lacking altogether. The calcu-
lations of Tyler and Horowitz showed that the concentration of dissociated picrate
inside the cells was about 100 X that at which the related substances showed similar
effectiveness. The same sort of relation was found for the other relatively inactive
phenols. The latter should be tested in experiments similar to those with picric
acid reported here. Such investigations might aid in deciding whether the action of
picric acid is to be attributed to its acidity or to its particular molecular configuration.

Perhaps picric acid is unique in its combination of a low pK and a rapid rate
of penetration into cells. The other phenols found to be exceptional (as regards
inhibition of cleavage and stimulation to respiration) may share this combination of
properties. Additional experimentation involving alteration of the picrate/picric
acid ratio in the solution (through addition of HC1 or NaOH) is also suggested;
such data might strongly indicate whether the acidity or the picrate itself is the active
factor. On either basis, the present data clearly show that, while anesthetizing the
eggs, this active factor constantly renders them increasingly sensitive to the removal
of the anesthetization, and to subsequent stimuli. Proper interpretation of this fact
might lead to a significant contribution to the understanding of the nature of
stimulation.

SUMMARY

1. Germinal vesicle breakdown in Nereis limbata eggs, brought about by heat,
or addition of KC1 or sodium citrate to the sea water, was inhibited by the addition
of picric acid at about M/1000.

2. After immersion for a few hours in M/1000 picric acid in sea water, germinal
vesicle breakdown occurred upon application of subliminal doses of heat, KC1, or
sodium citrate.

3. After immersion for 6-70 hours, removal of the eggs from picric acid to ordi-
nary sea water caused germinal vesicle breakdown.

CHEMICAL FACTORS IN EGG ACTIVATION 155

4. Activation by ultra-violet irradiation did not conform in these relations to
picric acid, under the conditions of the experiments.

5. These results are interpreted on the basis of a hypothetical activating sub-
stance produced within the egg, and inactivated or bound by picric acid.

6. The relation of picric acid to the calcium ion and the combination of calcium
with protoplasmic proteins is considered, in an alternative explanation of the results.

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A STUDY OF THE GOLGI APPARATUS IN CHICKEN GIZZARD
EPITHELIUM BY MEANS OF THE QUARTZ MICROSCOPE

HOPE HIBBARD AND GEORGE I. LAVIN

Oberlin College, Oberlin, Ohio, and The Rockefeller Institute for Medical Research,

Ne^v York City

The only fact about the Golgi apparatus that is universally accepted is that it is
a cytoplasmic constituent of most cells which, after special fixation, blackens with
silver nitrate or osmic acid. Controversies as to its structure, composition, func-
tion, and even its existence in the living cell, have been carried on continuously ever
since Golgi originally described such a cellular constituent in 1898, totaling consid-
erably over 2000 published papers. Mere descriptions of the blackened apparatus
appear to be no longer fruitful. Furthermore, since cells must differ in order to
carry on their specialized functions, warnings have been voiced against making hasty
generalizations about all cells from studies on particular cells. The work reported
in this paper pretends to nothing further than an analysis of certain features in one
type of cell.

The Golgi apparatus in the lining of the chicken gizzard near its junction with
the proventriculus is unusually spectacular and easily demonstrated. It can practi-
cally always be clearly shown after the usual osmic acid or silver nitrate techniques.
Moreover, its size is enormous (Fig. 5). For these reasons chicken gizzard mate-
rial is excellent for the study of the nature of the peculiar region of the cytoplasm
where this network appears.

Previous studies have followed its changes in form during embryonic develop-
ment of the gizzard (Hibbard, 1942). It can be demonstrated only in fixed material
after appropriate impregnations. It can never be seen in living cells or in cells
otherwise well fixed but not fixed by the usual methods for the Golgi apparatus.
The only cytoplasmic inclusions which are rendered visible by methods other than
silver and osmic impregnations in the general zone occupied by the Golgi apparatus,
are vacuoles which may be stained vitally or postvitally with neutral red, and occa-
sional filamentous mitocondria. Hibbard (1942) has suggested that these vacuoles
might be the antecedents of the typical Golgi network.

In an interesting series of papers, Worley (1943, 1944) has pointed out the
high susceptibility of cytoplasmic inclusions, in many types of cells, to displacement
or to changes in form and appearance with very slight changes in salt concentration
in the surrounding fluid. Within ten seconds such distortions may take place so
that quite different bodies from the original ones may be formed. Worley suggests,
as Parat did nearly twenty years ago, that the fixed picture as it appears in sections
may not at all resemble the living conditions.

Analysis of the Golgi apparatus by special types of illumination is not new.
Monne in 1939 published two papers dealing with the appearance of the Golgi appa-
ratus in Helix spermatocytes, using polarized light in one case and dark field illu-
mination in the other, in order to demonstrate physical characteristics of difference

157

158 HOPE HIBBARD AND GEORGE I. LAVIN

between the Golgi bodies and other cytoplasmic constituents. It must be remem-
bered that his results apply to spermatocytes only and great care should be taken
not to generalize them to apply to all Golgi bodies unless similar observations are
made on other types of cells. There is a great difference between the spermatocyte
Golgi apparatus and that in the glandular cells described in this paper, in staining
reactions, susceptibility to deformation and the variety of methods by which it may
be seen at all (Hibbard, 1945).

The present study was undertaken to determine, by means of ultraviolet micro-
photography, something about the nature of the cytoplasmic zone which becomes a
complex network of blackened material after impregnation. It is not a study of the
living cell, and may therefore be the analysis of an artifact. But if so it is one of
extremely uniform occurrence in the chicken gizzard and one which undoubtedly
has some precursor substance or some physical state of the material in the living
cell which produces the localized black network in the fixed sections in perfectly
regular fashion.

The technique employed was to make 5 /x sections, either in paraffin or frozen,
of material fixed in 7 per cent neutral formalin or in Da Fano, one of the fixatives
nearly always successful in demonstrating the Golgi apparatus in gizzard epithelium.
These sections were mounted on quartz slides under quartz coverslips and photo-
graphed with the quartz microscope using the 2537 A line of mercury as the light
source. The sections were unstained. Lavin (1943) has described the technique
of ultraviolet microphotography.

Examination of the microphotographs shows that most cells have no peculiar
tone variation in the zone in question (Fig. 3). However, in those cells with any
apparent difference in the Golgi zone as compared with the rest of the cytoplasm,
the Golgi zone appears somewhat paler (Figs. 1, 2, and 4).

It is known that nucleic acid has an absorption maximum of 2600 A and pro-
teins which contain tyrosine and tryptophane have an absorption maximum at 2800
A. Nucleoproteins will have a maximum at some point intermediate. In order to
show that these materials absorb in the ultraviolet region of the spectrum while in
the solid state, microphotographs of globulin and of nucleic acid pellets were taken
with the quartz microscope. They were cut and treated as if they were blocks of
tissue. These photographs are reproduced in Figure 7 and Figure 8. It will be
noted that while the nucleic acid is dark, the globulin remains pale. In a similar
way microphotographs taken in the ultraviolet should demonstrate in tissues, with-

FIGURE 1. Microphotograph, ultraviolet illumination; 5 /* paraffin section after Da Fano
fixation; no stain.

FIGURE 2. Microphotograph, ultraviolet illumination; 5 M paraffin section after 7 per cent
formalin fixation ; no stain.

FIGURE 3. Microphotograph, ultraviolet illumination; 5 M frozen section after 7 per cent
formalin fixation ; no stain.

FIGURE 4. Microphotograph, ultraviolet illumination ; 5 M paraffin section after 7 per cent
formalin fixation; no stain.

FIGURE 5. Microphotograph, visible light with green filter ; 5 p- paraffin section after Da
Fano fixation followed by silver nitrate impregnation and reduction ; no further stain.

FIGURE 6. Same as Figure 5.

FIGURE 7. Microphotograph, ultraviolet illumination ; section of a pellet of globulin.

FIGURE 8. Microphotograph, ultraviolet illumination ; section of a pellet of nucleic acid.

(In all figures, G — Golgi zone and N — nuclear region.)

GOLGI APPARATUS IN EPITHELIUM

159

FIGURES 1-8

160 HOPE HIBBARD AND GEORGE I. LAVIN

out the necessity of staining, the presence of any substance of appropriate absorption
maximum. Since the absorptive capacities of many organic tissue components are
known and thus this method of demonstrating cell inclusions is a reflection of their
chemical composition, we may say that the photographs present evidence that the
Golgi zone does not contain appreciable amounts of nucleoproteins, nucleic acids or
proteins containing tryptophane and tyrosine. The position of the Golgi apparatus
in most cells is characteristically in close proximity to the nuclear surface, a position
of possible physiological importance. This evidence that the region does not con-
tain nucleoproteins or nucleic acid in large amounts if at all, is of some importance,
particularly in view of the fact that most histological stains are in no sense chemical
tests.

Our results also shed some light on the further question, which is of some in-
terest : why does special fixation have to be practised before the subsequent impreg-
nation will "take?" Examination of Figures 1, 2, and 4 will show that the Golgi
zone is paler than the rest of the cytoplasm in many of the cells. In all probability
this clear zone corresponds to the area blackened by silver nitrate. The curious
fact is that this paler area may appear after formalin fixation whether the sections
are imbedded in paraffin or frozen (Figures 2, 3, and 4), and also after Da Fano
fixation without subsequent silver impregnation (Fig. 1). Silver impregnation ap-
plied after Da Fano fixation wrill produce a spectacular type of Golgi apparatus as
shown in Figures 5 and 6, while exactly similar impregnation after neutral formalin
as the fixative will produce only miscellaneous black granules throughout the cell
with no greater blackening of the Golgi zone than of other regions. This seems to
indicate that the Da Fano fixative either preserves some constituent lost in the
formalin, or else adds itself to material already there, to make it reduce the silver
in the conspicuous network. The identical absorption capacities of the region to
ultraviolet light, whether the fixative be formalin or Da Fano, suggests a similarity
in the quality of the fixed protoplasm. Why the Da Fano fixative should create a
focal point exactly in the Golgi zone for the subsequent reduction of the silver, is
not so clear. It may be that the Golgi zone is the site of aqueous vacuoles in the
living cell, possibly containing highly dispersed materials such as proteins and
lipoids, as found by Simpson (1941) after the freezing-drying technique. Both the
work of Simpson and our own would indicate far less concentration of proteins in
the Golgi zone than in the surrounding cytoplasm. During the process of fixation
there may be a distortion of the zone as Parat thought, and more recently, Worley ;
and the distorted "apparatus" may be fixed by any fixative that coagulates the cyto-
plasm around it. The similar appearance of the apparatus as shown by ultraviolet
photography, whether the fixative be formalin or Da Fano, shows that the reduction
of the silver on or in the apparatus subsequent to fixation depends, in all probability,
not on the fidelity of the whole cell's fixation but on the character of the fixative
used.

In conclusion, these studies of cells by means of ultraviolet photography give
certain negative information as to the material in the Golgi zone : it does not appear
that it is nucleoprotein or nucleic acid, except possibly in greater dilution than in
the rest of the cytoplasm. They also suggest that successful silver impregnation
after one fixative and not after another may be due, not to less faithful fixation of
the cell, but to a more direct relation between the fixative and the silver. Finally it

GOLGI APPARATUS IN EPITHELIUM 161

must be remembered that these studies were made exclusively on fixed material and
there is much evidence that the morphology of the cellular constituents in such mate-
rial does not coincide with that in living cells.

LITERATURE CITED

HIBBARD, HOPE, 1942. The "Golgi apparatus" during development in the stomach of Callus

domesticus. Jour. Morph., 70 : 121-150.
HIBBARD, HOPE, 1945. Current status of our knowledge of the Golgi apparatus in the animal

cell. Quart. Rev. Biol, 20: 1-19.

LAVIN, GEORGE I., 1943. Simplified ultraviolet microscopy. Rev. Sci. Instr., 14 : 375-376.
MONNE, LUDWIK, 1939a. Polarizationsoptische Untersuchungen uber den Golgi-Apparat und

die Mitochondrien mannlicher Geschlechtzellen einiger Pulmonaten-Arten. Proto-

plasma, 32 : 184-192.
MONNE, LUDWIK, 1939b. tiber die Farbenveranderung der Mitochondrien und des Golgi-

Apparates im Dunkelfeld. Arch. cxp. Zclljorsch., 23 : 157-168.
PARAT, MAURICE, 1928. Contribution a 1'etude morphologique et physiologique du cytoplasme.

Arch. d'Anat. Microsc., 24 : 74-357.
SIMPSON, WILLIAM L., 1941. The application of the Altmann method to the study of the Golgi

apparatus. Anat. Rec., 80 : 329-343.
WORLEY, LEONARD G., 1943a. The structure and function of the Golgi system in the living cells

of developing molluscs. Proc. Nat. Ac ad. Sci. Wash., 29 : 225-228.
WORLEY, LEONARD G., 1943b. The relation between the Golgi apparatus and "droplets" in cells

stainable vitally with methylene blue. Proc. Nat. Acad. Sci. Wash., 29: 228-231.
WORLEY, LEONARD G., 1944a. Studies of the vitally stained Golgi apparatus. II. Yolk formation

and pigment concentration in the mussel Mytilus californianus Conrad. Jour. Morph.,

75 : 77-101.

WORLEY, LEONARD G., 1944b. Studies of the vitally stained Golgi apparatus. III. The methy-
lene blue technique and some of its implications. Jour. Morph., 75 : 261-289.
WORLEY, LEONARD G., AND E. K. WORLEY, 1943. Studies of the supra-vitally stained Golgi

apparatus. I. Its cycle in the tectibranch mollusc Navanax inermis (Cooper). Jour.

Morph., 73 : 365-399.

DILUTION MEDIUM AND SURVIVAL OF THE SPERMATOZOA

OF ARBACIA PUNCTULATA.* I. EFFECT OF THE

MEDIUM ON FERTILIZING POWER

TERU HAYASHI
Department of Zoology, University of Missouri, Columbia, Missouri

INTRODUCTION

The investigations in sperm physiology may be roughly divided into two prin-
cipal aspects. First, there is the problem of the role of the sperm cell in fertiliza-
tion. Second, there is the problem of the survival of spermatozoa as a fundamental
condition for the survival of the species. These two aspects each have their own
long lists of investigations.

The study of the sperm cell in fertilization has produced one outstanding theory.
This is the Fertilizin Theory of Lillie (1914). The course of investigations, past
and recent, shows that this theory, although not completely confirmed and prob-
ably in need of modification, has been found useful by many workers in the field.
According to the theory, the male germ cell is the carrier of a substance, the "sperm
receptor," which is functional in the fertilization process. This substance is thought
to combine with "fertilizin," an egg secretion. The complex of sperm-receptor-
fertilizin then reacts with an "egg receptor" to form a three-way complex in the
egg. The formation of this ternary complex initiates the fertilization reactions of
the egg.

The study of sperm senescence, in contrast to the above, has yielded results
which are, at best, unsatisfactory. Gray (1928a and b), who investigated the
changes in metabolism of sperm under various conditions, reported that sperm in
highly concentrated condition have a very low rate of respiration. If diluted, the
sperm show a burst of metabolic activity. The greater the dilution, the more in-
tense is this burst of action, although of shorter duration. Gray advanced the hy-
pothesis that a large part of the sperm cell's internal supply of fuel was used up in
the first burst of energy, so that the greater its intensity, proportionately shorter be-
came the life of the spermatozoon. The initial burst of activity was in turn deter-
mined by the available "free space" in which the sperm cell could move, that is, by
the dilution. In the limited space available to each cell in the concentrated sus-
pensions, the sperm cell was only incompletely activated, and, hence, its life was
prolonged.

This explanation cannot be applied without certain limitations. If it were, a
single spermatozoon placed in an infinitely large volume of diluent would end its
metabolism instantly. Further, "mechanical crowding" as an explanation is ap-
plicable only to the translatory or vibratory activity of the sperm and not to the
respiratory activity. With all the known variables, such as oxygen and carbon
dioxide tensions, rigidly controlled, Gray's evidence shows that when "free space"
is available the rate of sperm respiration increases. "Mechanical crowding" is thus

* Work done as part of the requirement for the degree of Doctor of Philosophy.

162

DILUTION MEDIUM AND FERTILIZATION 163

not an explanation for the changes in respiratory rate but a description of the con-
ditions under which the respiratory rate is low. That is, it is logical to state that
sperm are quiescent because they are forced to be immobile, but it is not logical to
state that sperm respire at a high rate because they are no longer forced to be im-
mobile. Such a statement has implications of teleology. There must exist an un-
known factor which, under conditions of dilution, brings about the increased res-
piration of sperm. Undiluted sperm, therefore, must be a system composed of the
cells plus the unknown factor. Dilution of the system, not the dilution of cells
alone, brings about the respiratory activity of the spermatozoa.

The foregoing review showrs that Lillie's fertilization studies have indicated the
existence of a substance that determines the fertilizing power of the sperm cell.
The review shows, too, that Gray's work has neglected one variable, the sperm cell
medium, or a factor in that medium which affects the duration of metabolic activity
of the sperm cell. It is the purpose of this research to present evidence for the ex-
istence of a single factor that influences the conservation of fertilizing power by
sperm and the respiratory activity of sperm. The work is presented in two sec-
tions, the first section dealing with the fertilizing capacity of sperm, and the second
with the respiratory activity of sperm.

The author is greatly indebted to Dr. Daniel Mazia for his guidance and help-
ful suggestions.

MATERIALS AND METHODS

The materials used in the series of experiments to be described were the germ
cells of the Atlantic sea-urchin, Arbacia piinctulata. The general methods and pre-
cautions outlined by Just (1939) were followed carefully. To obtain the germ
cells, the urchins were thoroughly washed in running sea water and running tap-
water, after which they were dried carefully with clean cheese-cloth. A cut around
the oral region disclosed the sex of the animal. If male, the sperm exuding from
the genital pores were received in a dry stender dish ; if female, the animal was al-
lowed to shed the eggs into a stender dish filled with sea water.

The sperm suspensions for the earlier experiments were made according to the
"drop" method of Lillie (1913). For greater precision in later experiments, sperm
were "packed" by centrifugation at 3500 r.p.m. for 30 minutes. These packed
sperm cells were drawn into a calibrated capillary tube. The tip of the capillary
was wiped clean, and the contents were used to make the sperm suspension. The
capillary was calibrated by taking up the same volume of re-distilled mercury and
weighing the mercury accurately.

As a check on the constancy of this method, sperm counts were made. A unit
quantity of packed sperm was suspended in one cc. of sea water, shaken thoroughly,
and 0.01 cc. of Bourn's fixative added. This suspension was diluted one hundred
times, and the number of sperm present counted in a haemocytometer chamber.
The results are given in Table I, and it was found that the greatest deviation from
the average was in the order of 6 per cent, a constancy not attainable by the "drop"
method.

The seminal fluid used in the experiments»was collected simply by drawing off
the supernatant fluid from the packed "dry" sperm after centrifugation.

The egg suspensions were made by washing the eggs several times in sea water
and allowing them to settle in the dish by force of gravity. Equal samples of the

164

TERU HAYASHI

TABLE I

Sperm count, using 0.00155 cc. packed sperm per cc. of sea water, diluted 100 times

Suspension

Number squares
counted

Total counted

Average number
per sq.

Cone, of packed sperm
per cc.

No. 1

386

12.0

3.06X1012

No. 2

346

10.8

2.76X1012

No. 3

379

11.8

3.02 X1012

No. 4

360

11.2

2.86 X1012

No. 5

390

12.1

3.09X1012

No. 6

359

11.2

2.86X1012

Average

2.94X1012

settled eggs were diluted in varying amounts of sea water, mixed to give homo-
geneity, and aliquots were removed with a calibrated pipette. From the number of
eggs present per unit length, the total number of eggs could be calculated. One
drop of a suspension of suitable egg-concentration was placed in 5 cc. of sea water.
Generally, the number of eggs in one insemination test was 750-1000. As Lillie
(1915a) had shown, variations of this order in the total number of eggs used in
the inseminations do not affect the final results appreciably. Since a fresh egg
suspension was used for the insemination tests at any one time, the tests at two dif-
ferent times used different suspensions whose concentrations varied somewhat, so
that the results were possibly not comparable. Those tests run at any one time
used the same egg suspension, and, therefore, the results were comparable to each
other.

For the insemination, a unit quantity of the sperm suspension in a pipette was
carefully squeezed out over the eggs, and the whole dish gently and uniformly
stirred. For determination of fertilizing power, the percentage of eggs activated
was calculated by counting a minimum of 200 eggs.

Widely diverging types of experiments were made in the course of this investi-
gation, each type entailing its own methods and techniques. Because of this, other
methods and techniques will be described in connection with particular experi-
ments. Each typical experiment to be presented in the following section was one
of a minimum of five experiments all giving similar results.

EXPERIMENTS AND RESULTS
The seminal fluid factor and the survival of sperm

Past researches have shown that sperm in the undiluted condition freshly-exuded
from the testes are immobile, and that the sperm manifest intense activity upon
dilution with sea water. Subsequently, the fertilizing power of the sperm cells de-
clines sharply and within a relatively short time. Workers in the past had diluted
sperm fresh from the testes with sea water. Since the medium seemed to be a
variable in this type of dilution, and since sperm cells in the testes were suspended
in a liquid medium, a factor influencing the fertilizing capacity of sperm cells was
sought in the seminal fluid.

DILUTION MEDIUM AND FERTILIZATION

165

To examine the effect of the seminal fluid on the fertilizing power of sperm, a
series of experiments was done using sperm suspensions of the same concentration
in seminal fluid and in sea water. These suspensions were then tested at different
time intervals for their fertilizing power. In the experiment shown in Table II, a
0.4 per cent sperm suspension (according to the terminology of Lillie) was used.
One drop of the suspension was used to inseminate 750-1000 eggs. The formation
of the fertilization membrane was used as the index of activation of the egg.

The effect of the seminal fluid in promoting the survival of the sperm was ap-
parent even after five hours, and after 12 hours, when the sperm in sea water were
completely non-functional, a large number of those in the seminal fluid were still
capable of bringing about activation. At each test, microscopic observation re-
vealed that the per cent activation of eggs was approximately directly proportional
to the number of motile sperm.

TABLE II

Activation of eggs by sperm suspensions of 0.4 per cent concentration in seminal fluid and sea water

Per cent activation

10 a.m.

11 a.m.

3 p.m.

5 p.m.

8 p.m.

10 p.m.

Sea water

100

0-2

Sem. fluid

100

The maintenance of fertilizing power of the sperm cells was a function specific
for the seminal fluid. Experiments were made using the perivisceral fluid as the
suspension medium. The perivisceral fluid was found to have a toxic effect on the
retention of fertilizing power by sperm.

It seemed clear that in the seminal fluid an unknown factor was enabling the
sperm to retain their fertilizing power for a long period of time. In view of the
work of Cohn (1918), a check of the effect of pH became necessary. The pH of
the seminal fluid was measured electrometrically with McGinnis' electrode. A
number of such measurements showed the pH of seminal fluid to vary between 7.6
and 7.9. x Experiments were done comparing the survival of sperm in seminal
fluid and sea water acidified to the same pH as the seminal fluid sample.

In the same experiments, another chemical property of the seminal fluid was in-
vestigated, namely, the heat-sensitivity. A sample of the seminal fluid in a test
tube \vas heated at 100° C. for ten minutes, the seminal fluid allowed to cool to
room temperature, and this heated seminal fluid was tested for its effect on the
survival of sperm.

The results of experiments are summarized in Table III. The dilution used was
one drop of centrifuged sperm to 5 cc. of medium. The pH of this seminal fluid
sample was 7.72; the sea water (pH 8.0) was acidified to 7.7 by the addition of 11
drops of 0.1 N HC1 to 100 cc. of sea water. All the suspensions were made at
5 p.m.

The results showed that acid sea water maintained the fertilizing power of the
sperm only slightly longer than normal sea water and not nearly so long as the

1 Done by Mr. M. E. Smith, of the MBL staff.

166

TERU HAYASHI

TABLE III

The effects of pH, heated seminal fluid on the survival of sperm,
as shown by time measurements of the fertilizing power

Per cent activation

5 p.m.

9 p.m.

10:30 p.m.

4 p.m.

10 p.m.

Sea water

100

Sem. fluid

100

Heated fluid

100

Acid s.w.

100

seminal fluid. The heated seminal fluid, on the other hand, had clearly lost the
function of promoting the survival of the sperm cells. It was evident that pH was
not the effective factor in the seminal fluid and that the effective factor was heat-
sensitive.

This heat sensitivity led to the suspicion that the unknown factor was protein.
To test this hypothesis, the seminal fluid was saturated with ammonium sulfate. A
faintly rose-colored precipitate resulted from this treatment. This precipitate was
filtered off, and the residue on the filter paper dissolved in a volume of sea water
equal to the original volume of seminal fluid. The sea water containing the residue
was then dialyzed against fresh changes of sea water in the refrigerator for 30 hours.
The dialyzing membrane was commercial sausage skin (Cenco). This treatment
removed the ammonium sulfate. The liquid inside the dialysis bag, essentially an
artificial seminal fluid, was then used as the suspending medium for the sperm.

As controls for this experiment, various other media were used to suspend
equal concentrations of the same sperm sample. For the first of these, the filtrate of
the seminal fluid (seminal fluid minus the precipitated material) wTas also dialyzed
against sea water for the same length of time as the residue solution, and this
"dialyzed filtrate" was used as a suspending medium for the sperm. Normal sea
water, acid sea water, and natural seminal fluid were also run as controls. The
dilution used was one drop of centrifuged sperm to 10 cc. of medium, and the pH
was carefully checked in each case.

The results (Table IV) showed that spermatozoa in the "artificial seminal fluid"
retained their fertilizing power nine hours longer than did the sperm in sea water.
From the data, it was concluded that the seminal fluid factor was precipitable with
ammonium sulfate and non-dialyzable. The earlier conclusion as to the negligible
effect of pH was confirmed in this experiment.

The idea of the seminal fluid factor's being protein seemed to be borne out and
warranted an analysis of the seminal fluid for its protein content, along with deter-
minations of other physical and chemical properties. For determination of protein,
Folin's micro-Kjeldahl with direct Nesslerization was used, the solutions being
compared in a photoelectric colorimeter. The results showed 2.5 mg. protein per
cc. of 100 per cent seminal fluid. The pH of the seminal fluid was found to vary
between 7.6 and 7.9 as. compared to the pH of sea water, which varied from 7.9 to
8. 1.2 The freezing point of seminal fluid was --1.715° C. as compared to that of

2 Done by Mr. M. E. Smith, of the MBL staff.

DILUTION MEDIUM AND FERTILIZATION

167

TABLE IV

The effects of various media on the survival of sperm, as shown by insemination tests

Per cent activation

Medium

0.5 hrs.

5.5 hrs.

10.0 hrs.

15.5 hrs.

24.0 hrs.

26.0 hrs.

Sea water

8.0

Acid sea water

7.7

Sem. fluid

7.6

100

Dial, residue

7.8

Dial, filtrate

7.8

Eggs tested

100

sea water, which was -1.892° C.3 Chloride analysis showed the sea water to
contain 0.508 moles per liter, while the seminal fluid contained 0.590 moles per
liter.4 Analysis for glucose (reducing sugar) showed the seminal fluid to contain
less than 10 gamma in 5 cc.

These results suggested as one possibility that the action of seminal fluid on the
sperm could be attributed to the osmotic pressure difference between the seminal
fluid and the sea water. The demonstrated heat-sensitivity of the seminal fluid
factor, however, ruled this possibility as unlikely, as did the prolonged dialysis of
the last experiment given, for such treatment would equalize the osmotic pressure
of the seminal fluid with that of the sea water.

The difference in chloride content between the seminal fluid and sea water was
not considered as a factor in prolonging the fertilizing power of the sperm cells.
The prolonged dialysis described earlier would have equalized the chloride con-
centration of the sea water and the "artificial seminal fluid" of Table IV, yet these
two media had markedly different effects upon the sperm cells. Also, the demon-
strated heat sensitivity of the seminal fluid factor indicated that it was not chloride.

The effective seminal fluid factor therefore seemed to be protein, but protein, by
its presence, would establish a colloidal osmotic pressure which might be the agency
acting on the sperm.

In Table IV, it may be noted that the "dialyzed residue" was not as effective as
the natural seminal fluid. There are several possible explanations. First, during
the prolonged dialysis, some of the protein may have been denatured, a point to be
checked in future investigations. Second, the concentration of the factor in the
"artificial seminal fluid" was probably not equal to that in the natural medium, due
to some loss of protein in handling, and difficulties in volume control in dialysis.

At this point, attention should be called to the fact that still another possibility
existed as to the manner in which the seminal fluid functions. This was the ques-
tion of nutrition of the sperm by the seminal fluid. This question will, however,
be taken up in the discussion.

There remained one mode of action of the seminal fluid factor hitherto unin-
vestigated. The results of the experiments already described validated the as-

3 Done by Dr. Jay A. Smith, of the MBL staff.

4 Done by Mr. J. Weissiger, of the MBL staff.

168 TERU HAYASHI

sumption that the seminal fluid factor acted in some manner upon the surface of the
sperm cells.

Observations made during attempts to measure sperm activity in a capillary
tube showed spermatozoa to be positively thigmotropic to glass surfaces. At the
instant of contact, the spermatozoon lost a large part of its activity and rotated slowly
about its point of contact. The observation seemed to show the presence of a sur-
face active substance on the head of the spermatozoon. This fact, previously ob-
served by Duller (1902), led to the following experiment.

Three suspensions of sperm of equal concentration were made in sea water.
Suspension No. 1 was left untreated. Glass powder was added to suspensions No.
2 and No. 3. All three suspensions were shaken simultaneously and placed in the
refrigerator, where the powdered glass was allowed to settle for three hours. In-
semination tests were run to determine the relative sperm populations in these three
suspensions. Qualitative microscopic observations on sperm population were also
made at each dilution of the original suspensions as a check.

As shown in Table V, the results indicated that the sperm population in the
second and third suspensions was greatly reduced, a result confirmed by micro-
scopic observation. It was possible that the glass powder injured a large part of
the total sperm population, but the absence of significant numbers of injured sperm
seemed to indicate that the glass powder removed the missing sperm by adhesion.

TABLE V

Activation of eggs by progressive dilutions of sperm suspensions treated with glass powder

as compared to untreated sperm suspension

Suspension

Undiluted

1:1 Dilution

3:1 Dilution

No. 1

100

No. 2

No. 3

100

A similar experiment was made to test the surface activity of seminal fluid pro-
tein, since the proposed surface-action implied the identity of sperm-surface-sub-
stance and seminal fluid protein. A sample of seminal fluid was divided into three
portions. Portion No. 1 was left as the untreated control. Glass powder was
added to portion No. 2, the portion shaken thoroughly, and the glass powder filtered
off with Whatman No. 5 filter paper. Portion No. 3 was shaken three times,
each time with fresh glass powder and filtered free of glass each time. These semi-
nal fluid portions were then used to make sperm suspensions of equal concentra-
tion and tested for the maintenance of the fertilizing power. The results are given
in Table VI.

Clearly, the glass powder removed the sperm-longevity factor from the seminal
fluid, so that seminal fluid protein, too, seemed to be surface-active on glass. Al-
though the experiments of Tables V and VI did not completely establish the
identity of the seminal fluid factor and the substance on the surface of the sperm,
they did show that both substances were apparently surface-active.

In furtherance of this line of thought, experiments were made to learn whether
sperm in sea water gave off their surface substance into the surrounding medium.

DILUTION MEDIUM AND FERTILIZATION

169

TABLE VI

Removal of the factor from seminal fluid with glass powder

Per. cent activation

0.0 hrs.

4.0 hrs.

9.5 hrs.

12.0 hrs.

14.5 hrs.

24.0 hrs.

28.0 hrs.

Sea water

100

Portion No. 1

100

Portion No. 2

100

Portion No. 3

100

Eggs tested

100

In one type of experiment, a heavy suspension of sperm in sea water was allowed
to stand for several hours. The sperm were then removed by centrifugation and
the supernatant fluid tested as a sperm medium. In another type of experiment,
the above procedure was repeated several times, the supernatant fluid used to sup-
port a fresh sample of sperm after each centrifugation. After the final centrifuga-
tion, the supernatant fluid was tested for its effect on fresh sperm. In all cases, the
results were negative. Such "sperm washings" had neither a detrimental nor fa-
vorable effect on the maintenance of the fertilizing power of the sperm.

There remained one other point of investigation in the survival time of sperma-
tozoa. Observations had shown that seminal fluid protein, even in low concentra-
tion, was effective in maintaining spermatozoa. Gray (1928a) had postulated a
"mechanical crowding" effect as the primary factor in the survival of sperm. Since
he used "dry" sperm, which was composed of about 60 per cent seminal fluid, there
arose the possibility that the longer survival of the more concentrated sperm had as
its cause, not "mechanical crowding," but the larger amounts of seminal fluid pro-
tein carried over in the "dry" sperm. A test of this possibility followed.

A sperm suspension in seminal fluid was made by suspending 0.025 cc. of
packed sperm in one cc. of seminal fluid. A second suspension was made by taking
0.2 cc. of the first suspension and adding it to another one cc. sample of seminal
fluid. This serial dilution was repeated twice more, to make four sperm suspen-
sions, all in seminal fluid. The operation was carried out quickly, the last suspen-
sion made within a minute of the first. The final concentrations of the four sus-
pensions were, in Lillie's terminology, approximately 5 per cent, 1 per cent, 0.2 per
cent, and 0.04 per cent, since packed sperm contained approximately twice the
amount of sperm per unit volume as did the "dry" sperm used by Lillie. The in-
semination tests were made at the same dilution, each of the more concentrated sus-
pensions being diluted to the lowest concentration of 0.04 per cent. One drop of
this final suspension was used to inseminate the eggs. The results are given in
Table VII.

A study of these results as compared to those of Gray showed that, even though
Gray's results might be partly explained as the action of seminal fluid protein, "me-
chanical crowding" did seem to play a part in determining the life-span of the sper-
matozoa. However, it may be pointed out that this "crowding effect" seems to be
non-linear in relation to the concentration, and is most apparent at extreme dilu-
tions.

170

TERU HAYASHI

TABLE VII

The effect of concentration on the survival of sperm in seminal fluid

Per cent activation

Suspension concentration

0.5 hrs.

4.0 hrs.

7.5 hrs.

18.0 hrs.

23.0 hrs.

30.0 hrs.

5 per cent

1 per cent

0.2 per cent

0.04 per cent

The seminal fluid factor and its role in fertilisation

In the course of the preceding experiments, sea water suspensions of sperm used
to test the eggs showed a contrasting behavior as to fertilizing power. The indi-
vidual spermatozoon in seminal fluid appeared to have a greater fertilizing power
than the spermatozoon in sea water. An experiment was devised to investigate this
more closely.

A volume of 0.025 cc. of packed sperm was suspended in one cc. of seminal fluid.
Immediately after the suspension was made, one drop of the suspension was used
to inseminate approximately 1000 eggs. Serial dilutions were made as for the
previous experiment, but as each new suspension was made, one drop was used to
inseminate approximately 1000 eggs. A sea water control was run, dilution and
inseminations being made in the same way (Table VIII).

TABLE VIII

A comparison of the fertilizing power of sperm in seminal fluid and sperm in sea water

Per cent activation

1st dilution

2nd dilution

3rd dilution

4th dilution

Sea water
Sem. fluid

100
100

97
99

37
100

12
81

The results proved that there was a strong difference in the fertilizing power of
the sperm cells in seminal fluid as compared to those cells in sea water. This dif-
ference became even more pronounced when the original concentrated suspensions
were allowed to stand for ten hours, as shown in Table IX. Only the most concen-
trated suspensions in the seminal fluid and the sea water were kept. The dilutions
were made anew.

The interpretations of these results were rather complex and will be discussed
in a later section.5

The apparent increased fertilizing power of the sperm in the seminal fluid indi-
cated that seminal fluid factor might be directly concerned with the fertilization
process. It was recalled that Lillie (1915) had given as one of the criteria for the
"sperm receptor" the power to "bind" agglutinin from the egg. Lillie meant by this

5 See page 175.

DILUTION MEDIUM AND FERTILIZATION

171

TABLE IX

A comparison of the fertilizing power of sperm in seminal fluid and sea water after 10 hours

Per cent activation

A/T A'

1st dilution

2nd dilution

3rd dilution

4th dilution

Sea water

100

Sem. fluid

100

that if the agglutinin were treated with the "sperm receptor" solution (here the
seminal fluid, presumably), the action of the agglutinin on the sperm would be
greatly reduced. This experiment was done, with the expectation that, if the semi-
nal fluid factor and the "sperm receptor" were one and the same, the agglutinating
action of the egg secretion would be reduced.

A series of dry watch glasses was arranged. In the first, two drops of seminal
fluid and two drops of egg-water were thoroughly mixed. Two drops of this mix-
ture were then removed to the next watch glass and diluted with two drops of sea
water. This treatment was repeated down the series. For the control, sea water
was used instead of seminal fluid. For the test, a drop of a standard sperm suspen-
sion (0.00155 cc. packed sperm per cc. of sea water) was placed in the watch glass,
out of contact with the mixture. The watch glass was then placed under the objec-
tive of the microscope, the two liquids (sperm suspension and sea water-seminal
fluid mixture) shaken together, and the reaction of the sperm noted. In the follow-
ing table, + indicates a positive agglutination, - - a negative agglutination, and ±
uncertain. The number of + symbols indicates the intensity of the reaction.

TABLE X

The agglutination reaction induced by dilutions of egg-water-seminal fluid mixtures,
as compared to those induced by egg-water-sea water mixture
of the same dilutions

Sea water
egg-water

Sem. fl.
egg-water

Dilution
1

1/2

1/4

1/8

1/16 + + +

1/32 ± +

1/64 +

1/128 ±

Instead of having its action on the sperm reduced, the results revealed that egg-
water treated with seminal fluid had, if anything, a more powerful agglutinating
power than the sea water-treated egg-water. In any event, the agglutinating power
was not reduced. The only conclusion possible from these results seemed to be
that the seminal fluid factor was not the "sperm receptor" of Lillie.

However, the data given indicated that the sperm reaction in the seminal fluid-
egg-water mixture was more intense than the corresponding reaction of sperm in
the egg-water sea water mixture. This phenomenon was put to a quantitative test.

172 TERU HAYASHI

Standard suspensions of sperm were made in seminal fluid and in sea water.
The concentration was 0.00155 cc. packed sperm per cc. of medium. These suspen-
sions were allowed to stand at room temperature (25° C.). At intervals, a drop
from either one or the other of the suspensions was placed on a watch glass, out of
contact of a mixture of one drop of egg-water and two drops of sea water. The
liquids were shaken together under the microscope, and the time of agglutination
(from onset to reversal) was taken with a stop-watch. The results are summarized
in Table XL

TABLE XI

The agglutination time of sperm suspended in seminal fluid as compared
to that of sperm suspended in sea water

Time Agglutination time in seconds

Tested Sperm in sea water Sperm in seminal fluid

p.m.

3:00 90 120

3:06 63 69

3:10 61 115

3:18 82 98

3:40 49 111

3:44 53 95

3:50 47 91

3:57 63 90

4:05 61 97

4:40 91 86

4:50 86 75

5:15 34 120

5:30 71 115

7:30 75 76

The data show that, on the average, the sperm in the seminal fluid remained
agglutinated for a longer time than the sperm in sea water. Although the results
showed wide variation, the contrast between the two sperm suspensions was quite
striking. From the results, it seemed reasonable to conclude that seminal fluid had
changed the sperm surface in such a way as to bring about a stronger reaction with
agglutinin.

DISCUSSION
The seminal fluid factor and sperm motility

Gray (1928a) observed that the motility of the sperm of Echinus miliaris was in
no way impaired when suspended in seminal fluid, and he stated conclusively that
the seminal fluid possessed no chemical or physical properties inhibiting sperm mo-
tility. He prepared the seminal fluid, which he called "testicular plasma," by strong
centrifugation of the "dry sperm," the same method employed in this investigation.

The experiments and observations of the present study confirm Gray. The
earlier results of the work, given in a preliminary note (Hayashi, 1940), showed
that the sperm of Arbacia punctulata were motile in seminal fluid, with an intensity
of movement at least equal to that exhibited by sperm in sea water. Moreover, this
motility persisted for a longer time in the former medium. That sperm are active
in seminal fluid was confirmed by respiration studies (results to be given in a subse-
quent report), for it was found that the respiratory activity of sperm was maintained

DILUTION MEDIUM AND FERTILIZATION 173

at a higher level for a longer time in seminal fluid than in sea water. Therefore,
it may be stated conclusively that sperm cells of A. punctulata and E. miliaris are
fully active in seminal fluid.

The observations and conclusions of Southwick (1939a) were found to be in
conflict with these results. This worker found that sperm of Echinometra sub-
angularis were immobile when suspended in the seminal fluid of the same species.
He concluded that there was present in the seminal fluid a substance which inhibited
the activity of the sperm.

Hartman (1940) and Hartmann, Schartau, and Wallenfels (1940) confirmed
Southwick on the presence of the inhibiting factor not only in the seminal fluid, but
also in the sea water that had contained large numbers of spermatozoa. Their
work, however, was done with the sperm of Arbacia pustulosa. In addition to con-
firming Southwick, Hartmann et al. stated that the function of the inhibiting factor
was to neutralize echinochrome A, a sperm-activating substance from the egg.

For several reasons, the conclusions of these workers do not seem to be justified.
In the first instance, Southwick's own observations reveal that freshly-exuded "dry
sperm" possess an intense vibratory activity, an apparent contradiction to his own
conclusion. This activity is lost after a few minutes. A number of investigators
have published observations pertinent to these phenomena. Thus, Harvey (1930)
showed that sperm of Arbacia punctulata in oxygen-free sea water were immobile ;
when oxygen was introduced the sperm regained their motility. Lillie (1913)
demonstrated that sperm of Nereis and Arbacia lost their motility in the presence
of carbon dioxide. Dungay (1913), using Nereis and Arbacia, Fuchs (1914) with
dona intestinalis, and Cohn (1918) with A. punctulata proved that acid media had
a deleterious effect on sperm. Finally, Carter (1931) working with Echinus escu-
lentus and Echinus miliaris, and Taylor and O'Melveny (1941) with Strongylocen-
trotus purpuratus and Lytcchinus anamcsus obtained experimental proof that acid
conditions lowered the respiratory activity of sperm.

In view of the results of these investigators, the brief activity of the sperm noted
by Southwick seems to be attributable to the newly-made contact of the sperm with
oxygen upon shedding. The subsequent inactivation of the sperm has its probable
explanation in the acid conditions induced by the carbon dioxide production of the
sperm.

Furthermore, the papers of Southwick and the Hartmann school yield no figures
on the pH of the media used by these workers, nor do their texts give any evidence
that this factor had been controlled. In addition, the conclusions of Hartmann et al.
concerning the effect of echinochrome have not been confirmed by the experiments
of Tyler (1939b) and Cornman (1940, 1941). The former worker found that
neither echinochrome nor spinochrome would stimulate the respiration of sperm
of S. purpuratus. The latter showed that crystalline echinochcrome did not increase
the motility of the sperm of A. punctulata. The paradoxical results of Tyler and
Cornman as opposed to Hartmann et al. may be attributed to species difference.
However, it is clearly possible that echinochrome does not activate sperm. The
non-existence of a sperm-activating function by echinochrome seems to weaken the
argument for the existence of a substance neutralizing that activating factor.

Because of these considerations, the concept of a sperm-inhibitor in the seminal
fluid seems to be questionable. In the light of parallel experimental results as re-

174 TERU HAYASHT

gards sperm motility and respiratory activity (Hayashi, unpublished), it is con-
cluded that there is no inhibitor of sperm motility in the seminal fluid of A. punc-
tulata. This conclusion does not deny the inhibiting effects of hydrogen ions, the
influence of which on the increase of the life-span of the sperm has been shown to
be insignificant. To restate the conclusion : excluding the hydrogen ion factor,
there is no inhibitor of sperm motility in the seminal fluid of A. punctulata.

The seminal fluid factor in its relation to the activating capacity of the sperm

Various experiments have proved that spermatozoa suspended in seminal fluid
retain their capacity to activate eggs longer than sperm cells suspended in sea water
(Tables II, III, IV). The factor in the seminal fluid responsible for the effect is
not found in the perivisceral fluid, the factor is not the pH of the medium, and the
factor is heat-sensitive (Table III). The seminal fluid factor is also non-dialyzable
and precipitable with ammonium sulfate (Table IV). On the basis of these results,
it may be tentatively stated that the seminal fluid factor is protein. However, the
usual protein tests have not been made, so that this conclusion cannot be drawn
with any finality, even though the conclusion is strongly supported by positive
micro-Kjeldahl analyses indicating protein content in the order of 2.5 mg. protein
per cc. of seminal fluid.

The seminal fluid factor, if protein, may act on the sperm cells in several ways.
The factor may serve as a source of nutrient for the sperm, it may act on the sperm
through the agency of the colloidal osmotic pressure which its presence establishes
in the seminal fluid, or it may act through adsorption on the sperm surface. It is
necessary to consider these possibilities carefully, if the mechanism of the action of
the seminal fluid factor is to be clarified.

The possibility of the seminal fluid factor's acting as a nutrient will be taken up
more fully in a later publication on the effect of the seminal fluid on the respiration
of sperm. The statement can be made here that these studies indicate that the
factor does not act as a nutrient for the sperm. Likewise, the probable protein
nature of the factor argues against the idea of nutrition, for the large size of the
molecule would prevent its absorption by the sperm. The fact that seminal fluid
contains no reducing sugar is further support for the belief that the seminal fluid
affords no nutritive elements for the sperm cells.

The question of the effect of colloidal osmotic pressure in prolonging the func-
tional life of the sperm cell is unsettled. Although the further experimental results
on the surface activity of seminal fluid substance validate the conclusions drawn, it is
admitted that the effects of colloidal osmotic pressure on sperm longevity is still an
open question.

The experimental results given in Tables V and VI constitute support for the
idea of surface-action of the seminal fluid factor. The data show that both the
sperm surface and the seminal fluid factor are surface-active on glass, and they indi-
cate the possible identity of the sperm-surface-substance and the seminal fluid factor.

The foregoing considerations point strongly to the conclusions that the seminal
fluid factor is protein and that it is present both in the seminal fluid and on the
sperm surface. Since the seminal fluid factor enables the sperm to retain their fer-
tilizing function, it seems logical to infer that the seminal fluid protein plays a part
directly in the fertilization process. The data of Tables VIII and IX give support

DILUTION MEDIUM AND FERTILIZATION 175

to this. idea. The experiment of Table VIII reveals the fact that, with the same
amounts of sperm, a higher percentage of activation of eggs is achieved by sperm
in seminal fluid than by sperm in sea water. Since the experiment was so arranged
that the insemination tests were made immediately after the mixing of each solu-
tion, the possibility that a large number of sperm in the sea water were immobilized
seems unlikely. The conclusion most compatible with the results is that the indi-
vidual spermatozoon in seminal fluid possesses a greater fertilizing capacity than his
fellow in sea water. The mere act of dilution in sea water, therefore, seems to have
removed a large part of the activating substance from the surfaces of the sperm cells,
reducing their individual activating power.

The idea of variation of the activating power of the individual sperm cell was
first expressed by Glaser (1915). He diluted sperm serially in sea water, and
found that several sperm cells were required to activate one egg cell, even though
only one spermatozoon was required to bring about the biparental effect. Lillie
(1915) found that when he used the same method of dilution as did Glaser, the
fertilizing power of the suspension was far less than an equal concentration of sperm
in a suspension diluted in one step. Although these two workers disagreed in
their conclusions, their results point to the validity of Glaser's interpretation, which
is confirmed in the present study.

Table IX shows the relative fertilizing powers of suspensions in sea water and
seminal fluid after they had been aged for 10 hours. If the results are compared
to those of Table VIII, it will be seen that the fertilizing capacity of the seminal
fluid sperm suspension was not affected by the aging period but that the fertilizing
capacity of the sea water suspension was markedly reduced. There are two pos-
sible explanations for the enhanced difference in the activating power of the two
suspensions, both of which probably contribute to the effect. It is possible that in
the 10-hour aging period, large numbers of the sperm cells in the sea water suspen-
sion were immobilized, so that they could not penetrate the jelly envelope surround-
ing each egg. Thus, the number of sperm cells making actual contact with the egg
surface was reduced. The final result would be a decreased percentage of activa-
tion. The second possibility is the conclusion derived from the analysis of Table
VIII, namely, that a substance functional in activation was removed from the sperm
surface. During the 10-hour period, this removal presumably continued, so that
the activating power of the individual sperm cell was further reduced. Therefore,
even if all the spermatozoa remained motile and capable of making contact with the
egg, more sperm cells per egg would be required for activation, and the end results
would be a decreased percentage of activation. The experiment, therefore, tends
to support the idea of an egg-activating substance on the sperm surface, and, also,
shows the close relationship between the motile activity and the activating power
of the sperm cell.

Many investigators have postulated the existence of a substance on the surface
of the sperm and considered that it was protein in nature. Buller (1902), from
observations of the sperm of various echinids, reported that the sperm surface was
surface-active, not only on glass, but also on air bubbles. Lillie (1913) discovered
that in the presence of egg secretions, the male germ cells of Arbacia and Nereis
became agglutinated. He concluded that the surface of the sperm cell was "sticky."
The marked similarity of the agglutination phenomenon to an immunological reac-

176 TERU HAYASHI

tion may be taken to be a strong indication for the protein nature of the responsible
agent on the surface of the sperm cell.

More direct evidence came from the work of Popa (1927). Using histochemi-
cal technique, this worker concluded that the surfaces of Nereis and Arbacia sperm
were covered with a layer of lipo-protein.

Mudd, Mudd, and Keltch (1929) investigated the surface charge of the sperm
cells of various echinids. Using the cataphoresis chamber, they reported that the
sperm surface was negatively charged. This negativity they found to be increased
after agglutination with egg-water. They concluded that their method made pos-
sible the detection of substances on the sperm surface.

Henle (1938) and Henle, Henle, and Chambers (1938) found that heat-
sensitive antigens existed on the surface of sperm heads. Their work was done
with mammalian sperm. Tyler and O'Melveny (1941) obtained rabbit anti-serum
by injection of whole sperm of 6". purpuratus and L. anainesus. The anti-serum
was found to agglutinate the sperm of these species. These immunological studies
again pointed to the protein nature of the sperm-surface-substance.

The evidence cited is not, perhaps, a complete list. The investigations provide
enough experimental data, however, to warrant the tentative conclusion that the
sperm-surface-substance is protein in nature.

This sperm-surface-substance and the seminal fluid factor may possibly be identi-
cal (Tables V and VI). A strong indication of identity could be established if it
were shown that the seminal fluid factor alone can activate eggs. Experiments have
been started to investigate this possibility, but as yet no conclusive results have
been obtained. Comparable work in this direction has not been done. The effect
of protein extracts on egg surfaces was investigated by Favilli (1932) and by Hart-
mann, Schartau, and Wallenfels (1940), while Sampson (1926) reported the acti-
vation of eggs by dialysates and filtrates of sperm suspensions. Since her activating
factor was dialyzable, and therefore non-protein, it cannot be compared to the semi-
nal fluid factor. In addition, the remarks of Just (1922, 1928, 1929a and b) criti-
cizing the auto-parthenogenesis of Glaser (1914) and Woodward (1921) are
equally applicable to the work of Sampson.

Another possible method of establishing the identity of seminal fluid protein
and the sperm-surface-substance is to obtain rabbit anti-serum by the injection of.
seminal fluid. If the anti-serum thus obtained had the power to agglutinate sperm,
the results would constitute substantial evidence for the argument that seminal fluid
protein and protein on the sperm surface were the same substance. The experi-
ment, however, was not done.

The identity, therefore, is not established, although there is some evidence in
this direction (Tables V and VI). Aside from this point, however, there are ex-
perimental results throwing light on the origin of and possible relation between the
seminal fluid factor and the sperm surface substance. It would be interesting to
know whether these substances are secreted by the sperm cells or not, and whether
the sperm-surface substance establishes the seminal fluid substance by passing off
into the seminal fluid, or whether the seminal fluid substance establishes the sperm-
surface substance by adsorption on the sperm surface.

Numerous investigators have reported that sea-urchin sperm give off a substance
into the surrounding sea water (Lillie, 1914 and 1915 ; Southwick, 1939; Hartmann,

DILUTION MEDIUM AND FERTILIZATION 177

1940 ; Hartmann, Schartau, and Wallenfels, 1940) and that this substance showed
protein characteristics (Frank, 1939; Tyler and O'Melveny, 1941). All investi-
gators agreed that the substance showed the properties of "antifertilizin" or the
power to "bind" the agglutinin of egg-water so that the agglutinating effect on
sperm was reduced.

The question here posed is : does this substance from the sperm cells have the
properties of the seminal fluid substance? The point was tested by an experiment
in which a sea water suspension of sperm was allowed to stand for several hours,
the sperm cells removed by centrifugation, and a fresh sample of sperm suspended
in the medium. The results were negative. This medium was not effective in
prolonging the fertilizing capacity of sperm, and therefore did not have the proper-
ties of the seminal fluid substance.

There is the converse question : Does the seminal fluid substance have the anti-
fertilizin property of the substance coming off the sperm cell? Again, the results
were negative (Table X).

The substance coming off the sperm cell does not have the properties of the
seminal fluid substance. The results of these experiments indicate, therefore, that
a sperm substance does not establish the seminal fluid substance, so that the seminal
fluid factor does not have its origin in the sperm cell. The same negative answer
as to the origin of the sperm-surface substance cannot be given.

However, the fact that the substance coming off the sperm surface has different
properties from the seminal fluid substance signifies nothing regarding the proper-
ties of the sperm substance while on the sperm surface. This substance on the
surface of the sperm cell is surface-active on glass, as is the seminal fluid substance
(Tables V and VI). The seminal fluid factor also enables the sperm cell to main-
tain its fertilizing capacity longer (Tables II, III, IV) and seems to enhance the
fertilizing power of the individual spermatozoon. In addition, the seminal fluid
factor affects the surface of the sperm so that the time of agglutination, or the reac-
tion with agglutinin, is increased (Table XI).

These facts point to a tentative explanation of the relation between the seminal
fluid factor and the sperm-surface substance. It is possible that a protein sub-
stance, originally present in the seminal fluid, is adsorbed on the surface of the
sperm cell, thus influencing the fertilizing power of the sperm cell, as well as render-
ing the surface of the sperm cell surface-active. By this adsorption also, the sperm
surface is rendered capable of greater reactivity with fertilizin. The subsequent
loss of this substance from the sperm cell results in the loss of fertilizing power and
the presence of antifertilizin in the sperm medium. The antifertilizin would be,
according to this scheme, a substance changed in certain properties from the original
seminal fluid substance.

SUMMARY

1. A factor is present in the seminal fluid of Arbacia punctulata which prolongs
the fertilizing capacity of the sperm cells of the same species.

2. The factor, which is not found in the coelomic (peri visceral) fluid, is heat-
sensitive, precipitated by saturation with ammonium sulfate, non-dialyzable, and
surface-active on glass. Since micro-Kjeldahl analysis of the seminal fluid gives
positive results corresponding to 2.5 mg. protein per cc. of seminal fluid, it is tenta-
tively suggested that the factor is protein.

178 TERU HAYASHI

3. Seminal fluid has a pH range of 7.6 to 7.9, its osmotic pressure is approxi-
mately 10 per cent lower than sea water, and its content of reducing sugar is
negligible.

4. In equivalent concentration and immediately after suspension the fertilizing
capacity of the individual spermatozoon is greater in seminal fluid than in sea water.

5. Seminal fluid does not contain antifertilizin since it does not neutralize the
agglutinating action of egg-water ; indeed, this action is intensified.

6. A tentative mechanism, based on the adsorption of a fertilizing substance and
its removal from the surface of the sperm cell, is suggested to explain the experi-
mental results. It is proposed that the seminal fluid factor is this fertilizing sub-
stance before adsorption and while on the surface of the sperm ; it becomes changed
upon removal from the sperm surface.

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DILUTION MEDIUM AND FERTILIZATION 179

JUST, E. E., 1922. Initiation of development in the eggs of Arbacia. I. Effect of hypertonic
sea-water in producing membrane separation, cleavage, and top-swimming plutei. Biol. -
Bull., 43 : 384-400.

JUST, E. E., 1928. Initiation of development in Arbacia. V. The effect of slowly evaporating
sea-water and its significance for the theory of auto-parthenogenesis. Biol. Bull., 55 :
358-368.

JUST, E. E., 1929a. Initiation of development in Arbacia. VI. The effect of sea-water precipi-
tates with special reference to the nature of lipolysin. Biol. Bull, 57 : 422-438.

JUST, E. E., 1929b. The fertilization reaction in eggs of Paracentrotus and Echinus. Biol. Bull,
57 : 326-331.

JUST, E. E., 1939. Basic methods for experiments on eggs of marine animals. Philadelphia.
P. Blakiston's Son and Co. 89 pp.

LILLIE, F. R., 1913. The behavior of spermatozoa of Nereis and Arbacia with special reference
to egg extractives. Jour. E.vp. Zool, 14: 515-574.

LILLIE, F. R., 1914. Studies of fertilization. VI. The mechanism of fertilization in Arbacia.
/. Exp. Zool, 16: 523-590.

LILLIE, F. R., 1915. Studies of fertilization. VII. Analysis of variations in the fertilizing
power of sperm suspensions of Arbacia. Biol Bull., 28: 229-251.

LILLIE, F. R., 1919. Problems of fertilization. Chicago. The University of Chicago Press.
278 pp.

MUDD, E. B. H., S. MUDD, AND A. K. KELTCH, 1929. Effect of echinid egg waters on the sur-
face potential difference of the sperm. Proc. Soc. Biol Mcd., 26: 392-394.

POPA, G. T., 1927. The distribution of substances in the spermatozoon (Arbacia and Nereis).
Studies by intra vitam stains and by stains of lipoids according to the methods of Schu-
macher. Biol. Bull, 52: 238-258.

SAMPSON, M. M., 1926. Sperm filtrates and dialyzates : Their action on ova of the same species.
Biol. Bull., 50 : 301-338.

SOUTHWICK, W. E., 1939. Activity-preventing and egg-sea-water neutralizing substances from
spermatozoa of Echinometra subangularis. Biol Bull, 77: 147-156.

TYLER, A., 1939a. Extraction of an egg-membrane-lysin from sperm of the Giant Keyhole
Limpet. Proc. Nat. Acad, Sci., 25 : 317-323.

TYLER, A., 1939b. Crystalline echinochrome and spinochrome : Their failure to stimulate the
respiration of eggs and of sperm of Strongylocentrotus. Proc. Nat. Acad. Sci., 25 :
523-528.

TYLER, A., AND K. O'MELVENY, 1941. The role of anti-fertilizin in the fertilization of sea urchin
eggs. Biol. Bull, 81 : 364-375.

WOODWARD, A. E., 1921. The parthenogenetic effect of echinoderm egg-secretions on the eggs
of Nereis limbata. Biol. Bull, 41 : 276-279.

HETEROCINETA PHORONOPSIDIS SP. NOV., A CILIATE FROM
THE TENTACLES OF PHORONOPSIS VIRIDIS HILTON

EUGENE N. KOZLOFF
Department of Zoology, University of California, Berkeley

INTRODUCTION

The infestation of the tentacles of Phoronopsis viridis Hilton by a small ciliate
of the family Ancistrocomidae Chatton and Lwoff * (order Holotricha, suborder
Thigmotricha) was called to my attention by Professor Harold Kirby. A prelimi-
nary study of this ciliate, from slides prepared in his laboratory from material col-
lected in Bodega Bay, California, in November, 1943, disclosed that on the basis of
the organization of the ciliary system it appeared to be most closely related to spe-
cies of the genus Heterocineta Mavrodiadi, ectoparasitic on fresh water mussels,
prosobranchs, and pulmonates (Jarocki; 1934, 1935).

In June, 1945, I collected additional material of Phoronopsis viridis ~ in an inter-
tidal mud flat in Tomales Bay. From observations of the living ciliates it was de-
termined that this new species, which will be described herein as Hcterocineta pho-
ronopsidis sp. nov., differs fundamentally from other species of Heterocineta in
having a groove-like depression originating on the left side of the body near the
anterior end and extending posteriorly along the dorsal surface close to the left
margin. I have studied a species of Hcterocineta ectoparasitic on PJiysa cooperi
Tryon from a locality near Mt. Eden, California, which agrees perfectly with the
description of Heterocineta janickii given by Jarocki (1934). This ciliate, like H.
phoronopsidis, has eight ciliary rows, but these are restricted to a more narrow area
on the ventral surface. There is no dorso-lateral groove in H. janickii. In none
of Jarocki's descriptions of ciliates of the genus Heterocineta, which apparently were
based to a large extent upon living material, is there any mention of such a groove.

TECHNIQUE

For observation of the living ciliates the tentacles of Phoronopsis viridis were
detached from the rest of the body by means of forceps and comminuted in a drop
of sea-water on a slide. Fixation of the organisms for permanent preparations was
accomplished by preparing smears in this manner on coverglasses and dropping
them onto the surface of the fixative in a Petri dish. For a study of the general

1 Chatton and Lwoff (1939) proposed the family Ancistrocomidae to include those ciliates
formerly assigned to the family Hypocomidae Biitschli which differed from the type genus of the
latter (Hypoconia Gruber) in having the suctorial tentacle disposed terminally rather than sub-
terminally and the ciliary rows arranged singly rather than in pairs.

2 Professor W. A. Hilton of Pomona College has kindly identified the phoronid species from
Tomales Bay as Phoronopsis rind is Hilton (1930). It should here be noted, however, that no
systematic revision of the phoronids from the Pacific Coast has been given in the literature and
it is not impossible that P. viridis will later be shown to be identical with one of the species
described earlier.

180

HETEROCINETA PHORONOPSIDIS 181

morphology, staining with iron hematoxylin gave good results on material fixed in
Schaudinn's fluid. Differentiation of the ciliary system by impregnation with acti-
vated protein silver (protargol) was successful following fixation in Hollande's
cupric-picro-formol mixture and Schaudinn's fluid, but this method was no more
satisfactory than staining with iron hematoxylin. The Feulgen nuclear reaction
was used after fixation in Schaudinn's fluid and a saturated aqueous solution of
mercuric chloride with 5 per cent of glacial acetic acid.

HETEROCINETA PHORONOPSIDIS sp. nov.

•

The body is elongated, asymmetrical, and flattened dorso-ventrally. Twenty
living individuals taken at random ranged in length from 26 ^ to 37 p, in width from
11 ju, to 16 /x, and in thickness from 6.5 ^ to 11 /*, averaging about 29 /A by 14 /* by
8/i. As seen in dorsal view (Fig. 1A) the left side of the ciliate is conspicuously
rounded, while the right side is by comparison very little curved. The body is usu-
ally widest at a point a short distance behind the middle and is rounded posteriorly.
The attenuated anterior end is deflected toward the left, truncate at the tip, and
bent ventrally. The reduced ciliary system, to be described presently, is disposed
in a shallow concavity occupying the anterior four-fifths of the ventral surface
(Fig. IB) ; the dorsal surface and that part of the ventral surface posterior to the
ciliary area are convex.

A contractile suctorial tentacle enables the ciliate to attach itself to epithelial
cells of the tentacles of the host and to feed upon their contents. When fully ex-
tended the suctorial tentacle of Heterocineta phoronopsidis is about 4 /u, in length
and l.S/i, in diameter; it is contracted as soon as the ciliate is dissociated from the
host and is seldom preserved in an extended condition in fixed individuals except
those which have been fixed in a position of attachment to the host.

The internal tubular canal (Fig. 1, c) continuous with the suctorial tentacle is
about 1.5/x in diameter in its anterior portion, which is directed dorsally, and be-
comes abruptly narrower in its posterior portion, which is directed ventrally and
obliquely to the right. In some living specimens and in suitable preparations
stained with iron hematoxylin the canal can be traced along the right side of the body
to a point a short distance posterior to the macronucleus.

The cilia of Heterocineta phoronopsidis are about 5 ^ in length and markedly
thigmotactic. They are disposed in eight longitudinal rows limited to the shallow
concavity on the ventral surface (Fig. 1C). All eight rows originate near the base
of the suctorial tentacle. Each of the first five rows from the right margin is about
three-fifths the length of the body. The fourth and fifth rows are as a rule practi-
cally straight, while the outer three are appreciably curved. The remaining three
rows become progressively longer and inflexed in such a way that they end one
behind the other near the mid-line. The eighth and longest row terminates at a
point about four-fifths the distance from the anterior end of the body to the posterior
end. The cilia of the anterior part of the thigmotactic system move rather actively,
those of the posterior part sluggishly.

The shallow groove-like depression which distinguishes Heterocineta phoronop-
sidis from other species of Heterocineta has its inception on the left side of the body
near the anterior end and is about four-fifths the length of the body (Fig. 1A, g).
As it extends posteriorly it comes to lie on the dorsal surface along the left margin.

182

EUGENE N. KOZLOFF

The groove is visible only in living individuals. There are no traces of ciliature at
any point along its course. Staining with iron hematoxylin and impregnation with
protein silver fail to bring out any basal granules in the region occupied by the
groove.

The cytoplasm is colorless and contains a number of small refractile granules in
addition to food inclusions. The refractile granules (Fig. 1A, eg) are apparently
lipoid droplets, as they are dissolved out by toluol used for clearing following stain-
ing. At least one large food-vacuole and usually several smaller ones are present
near the posterior end of the body (Fig. 1, fv). The contents of the food-vacuoles

\ N

FIGURE 1. Heterocineta phoronopsidis sp. nov.

A. Dorsal aspect, from life; B. lateral aspect from right side, from life; C. ventral aspect.
Schaudinn's fixative-iron hematoxylin. Drawn with aid of camera lucida. X 1940.

c = internal tubular canal, eg = cytoplasmic granule, cv = contractile vacuole, fv = food
vacuole, g = dorso-lateral groove, ma = macronucleus, mi — micronucleus.

are seen to consist mainly of ingested nuclei or fragments of nuclei from the epi-
thelial cells of the tentacles of the host.

The contractile vacuole (Fig. 1, cv) lies near the middle of the body and opens
to the exterior on the ciliated ventral surface. I have not distinguished a perma-
nent opening in the pellicle.

The oval or rod-shaped macronucleus (Fig. 1, ma) is placed dorsally near the
center of the body, its longitudinal axis lying obliquely to the longitudinal axis of
the body. In ten individuals fixed in Schaudinn's fluid and stained by the Feulgen
nuclear reaction on the macronucleus ranged in length from 5.25 ^ to 7.5 /j. and in
width from 3 ^ to 4.5 /x.

The fusiform, rod-shaped, or crescentic micronucleus (Fig. 1C, mi) is situated
anterior to the macronucleus. It is very difficult to distinguish in living specimens
and is stained only weakly by iron hematoxylin and the Feulgen nuclear reaction.
In ten individuals fixed in Schaudinn's fluid and stained by the Feulgen reaction

HETEROCINETA PHORONOPSIDIS 183

the micronucleus ranged in length from 1.5//. to 2.25^ and in width from 0.75 p.
to 1.2/t.

When attached to the tentacles of the host Heterocineta phoronopsidis is almost
immobile, exhibiting only a passive vibratory motion due to the energetic movement
of the epithelial cilia. When dissociated from the host the ciliate swims slowly,
usually rotating on its longitudinal axis and tracing wide arcs with its attenuated
anterior end.

Heterocineta phoronopsidis sp. nov.

Diagnosis: Length 26^-37^, average about 29 /j.; width ll/x-16^, average
about 14 /x,; thickness 6. 5 ^,-11 p., average about 8^. The anterior end is attenu-
ated, bent ventrally, and provided with a contractile suctorial tentacle continuous
with an internal tubular canal. The ciliary rows are eight in number and originate
near the base of the suctorial tentacle. The first five rows from the right are about
three-fifths the length of the body, while the remaining three rows become progres-
sively longer and are inflexed in such a way that they end one behind the other
near the mid-line. A groove-like depression, without any trace of ciliature, extends
from the anterior end of the body posteriorly along the dorsal surface close to the
left margin. Ectoparasitic on the tentacles of Phoronopsis viridis Hilton (Tomales
Bay, California). Syntypes are in the collection of the author.

LITERATURE CITED

CHATTON, E., AND A. LWOFF, 1939. Sur la systematique de la tribu des thigmotriches rhyn-
choides. Les deux families des Hypocomidae Biitschli et des Ancistrocomidae n. fam.
Les deux genres nouveaux Heterocoma et Parhypocoma. C. R. A cad. Sci. Paris, 209 :
429.

HILTON, W. A., 1930. Phoronida from the coast of southern California. Jour. Ent. and Zool.,
22: 33.

JAROCKI, J., 1934. Two new hypocomid ciliates, Heterocineta janickii sp. n. and H. Iwoffi sp. n.,
ectoparasites of Physa fontinalis (L. ) and Viviparus fasciatus Miiller. Mem. Acad.
Cracovie, Cl. Sci. math, nat., B(II), 1934: 167.

JAROCKI, J., 1935. Studies on ciliates from fresh-water molluscs. I. General remarks on proto-
zoan parasites of Pulmonata. Transfer experiments with species of Heterocineta and
Chaetogaster limnaei, their additional host. Some new hypocomid ciliates. Bull. int.
Acad. Cracovie, Cl. Sci. math, nat., £(//), 1935: 201.

ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE
MARINE BIOLOGICAL LABORATORY, SUMMER OF 1945

The role of bacteria in the excystment of the dilate Didinium nasutum. C. D.
Beers.

Resting cysts were obtained by the completion-culture method, viz., by preparing small cul-
tures in spring water with paramecia as food. Most of the didinia in such cultures encysted upon
exhaustion of the food supply. Such cysts never became active spontaneously, nor were they
bacteria-free.

Distilled water, sugars, salts of plant acids, pH changes, and metabolites of Paramecium
were ineffective in inducing excystment.

Timothy and lettuce infusions, and peptone and yeast-extract solutions induced 78-94 per
cent excystment within 9-12 hours at 28° C. The tentative conclusion that these substances were
effective excystment-inducing agents per sc was soon negatived by the observation that at the
time of excystment, bacteria (introduced with the cysts) were always flourishing in the media,
which had been originally sterile. To test more adequately the effect of bacteria, these same
four media were inoculated with wild bacteria from Paramecium cultures and incubated 18-24
hours. The bacterized media when tested on cysts induced 89-95 per cent excystment within
3-4 hours at 28° C. and thus produced a distinct acceleration effect.

The special effectiveness of bacterized peptone suggested an examination of the role of amino
acids in excystment. Nine such acids were tested, singly and in mixtures, in buffered solution,
but none yielded an accelerative effect. Only those acids (e.g., histidine, arginine, proline, methi-
onine) and mixtures which supported bacterial growth induced excystment, and then only when
bacteria were flourishing, i.e., after 9-12 hours. Acid mixtures previously inoculated with bac-
teria produced the usual accelerative effect. Hydrolyzed peptone behaved similarly.

Boiling the bacterized acid mixtures, or peptone solutions or hydrolyzates destroyed their
effectiveness, which, however, could be restored by inoculation with bacteria.

The results indicate that excystment in Didinium is induced through the agency of bacterial
action. Further studies are in progress to identify the effective bacteria, and to ascertain the
chemical nature of the substances responsible for excystment.

Cytological studies in Culex. C. A. Berger and Sister Mary Grell.

Cells in the hind-gut of Culex (2n = 6) are diploid at the beginning of larval life and are
highly polyploid at pupation. This polyploid condition arises by repeated chromosome redupli-
cation within the resting nucleus. During metamorphosis these cells undergo mitotic division.
The first division of a 16-ploid cell is described. Unique cytological features of this division
are as follows. There are six groups of chromosomes each composed of eight sister chromo-
somes. Homologous groups are paired, relationally coiled and apparently have their spindle at-
tachment regions fused. In early prophase the association of sister chromosomes is so close that
the eight appear as one. As prophase contraction proceeds the eight sister chromonemata be-
come evident and are seen to be relationally coiled in two's, in pairs of two's, etc. The spindle
attachment region undergoes successive division in late prophase. At metaphase 48 chromo-
somes can be counted. Anaphase separation is regular and homologous or sister chromosomes
pair as they move to the poles. This work can be interpreted as favoring the first part of
Darlington's hypothesis, that chromosomes are attracted in pairs only, but gives no support to
the second part of the hypothesis, that pairs of pairs repel.

Accelerating metamorphosis in the tunicate, Styela partita. Lloyd M. Bertholf.

C. Grave discovered that metamorphosis in tunicate larvae can be hastened by dozens of
different substances, from complicated extracts of tunicate and vertebrate tissues down to
simple salts of several heavy metals, added to sea-water. He concluded that such acceleration

184

ABSTRACTS OF SCIENTIFIC PAPERS 185

is caused by a poisoning of the larval action-system, so that the adult action-system takes over
sooner than normally, and that the chief agent in this poisoning is copper.

To ascertain how specific the need for copper is, an effort was made to hasten metamorpho-
sis by various substances in which copper is absent. Isotonic solutions of NaCl alone or in
combination with other salts and with lactose and sucrose were first used. All these solutions
brought about metamorphosis much sooner than in the controls, provided the larvae were about
4 hours old or older; if younger, the animals usually died before metamorphosis or shortly
afterward.

It is possible, however, that the salts used contained a threshold amount of copper and
other heavy metals as impurities. Hence distilled water alone was next used. This killed the
animals after a few minutes of continuous exposure, but if larvae of about 2 hours or older were
immersed in distilled water for only % to 2 minutes and then transferred to normal sea-water,
metamorphosis was much hastened, and no deleterious effects resulted.

It seems, then, that the effect of copper is not specific, but that similar effects can be pro-
duced by other means, including the physical shock of a large change in osmotic pressure.

Oxidation-reduction studies on Pcnicilliuin notatiim and other organisms. Matilda
Moldenhauer Brooks.

Redox potential and pH measurements by means of the Coleman electrometer were made of
the media in which Pcnicilliuin notatiim and several other organisms were grown. Daily read-
ings were taken for a period of several weeks. Pcnicillium was grown in corn steep medium.
Aspcrgillus flavus, Mycodcrtna, Torula iitilis and Sacchromyces cervcsiac were grown in modi-
fications of Czapek-Doz media. Sterile conditions were maintained.

It was found that rH values (=2 pH + Eh/.03) for Penicillinm were 8.4 to 8.7. For puri-
fied penicillin (100,000 Oxford units) the rH was 8.7. For other organisms it was either higher
or lower. In the case of Pcnicilliuin, the Eh value became very negative ( — 0.25) and the pH.
alkaline (8.5). No other organism studied had these characteristics.

When flasks were tightly stoppered, the rH values were similar to those obtaining in cul-
tures to which KCN had been added. Growth was hindered when aerobes were used and not
affected in the case of facultative anaerobes.

It is suggested that the therapeutic action of penicillin and related organisms depends upon
the balance between Ei, and pH in the blood, which these organisms produce. This factor makes
it incompatible for such organisms as Staphylococcus aurcus, for example, to exist.

Organisation of the giant nerve fiber system in Neanthes vircns. Theodore H.
Bullock.

The presence of giant nerve fibers in certain polychaete annelids has been known on the
basis of anatomical studies, but their function and functional organization have not been investi-
gated. The group is especially suitable for such studies since its members present a great di-
versity of neural development ; giant fibers are present in varying pattern in many species, absent
in others ; the group is large, and favorable species for laboratory study are common. A sur-
vey of the functional anatomy of the giant system in representative forms has been undertaken to
the end of adding perspective to our picture of the evolution of the nervous system and with
the hope of finding material for special studies of nervous physiology. The electrical signs of
nervous activity were used as a tool for revealing the functional anatomy.

The present report will be confined to Neanthes mrens (Nereis vircns}. When the nerve
cord is directly stimulated by single shocks there is recorded from the nerve cord or from the
mid-ventral line of the intact animal, in any other part of the worm, a pattern of large spikes,
several orders of magnitude higher in voltage than the action currents representing spontaneous
activity of the small fibers of the nerve cord. These large spikes have the properties of single
fiber action currents. The first is the largest, has the lowest threshold, fatigues the slowest, and
arrives at a time representing a minimum conduction rate (assuming no delays) of 5 meters/sec.
Unlike the others it is not all or none, but all or half or none ; two independent units are pres-
ent conducting at just the same rate but separable by threshold and fatigue. The second spike
is intermediate in height, threshold, fatigue, and rate (4.5 m/s) between each half of the first
spike and the later ones. A small third spike at 2.5 m/s may be alone or followed by another

186 ABSTRACTS OF SCIENTIFIC PAPERS

like itself. This pattern is constant from specimen to specimen and may be regarded as char-
acteristic of the species. One can expect anatomically at least four giant fibers or conducting
units : a pair larger than the rest but identical in average diameter ; a single unit, next in size ;
and one or two small but still "giant" units. This corresponds precisely with the known
anatomy, there being a pair of large lateral fibers, a smaller median unpaired fiber, and a pair
of still smaller medially placed fibers. The present technic can assure certain relations difficult
to establish histologically. There is no anastomosis between the lateral fibers such as occurs
in Lumbricus; the fibers are all independent conducting units, none being a necessary efferent
or afferent connection of another ; all the fibers are unpolarized, conducting equally well in both
directions (although segmental macrosynapses like those in Lumbricus have been described).
The sensory connections of each fiber can be inferred from responses to mechanical stimuli.
The giants can each be fired through sense organs by local stimulation of the skin (a light tap
or dropping water) within certain segmental levels; the head is not necessary. The median
fiber (second spike with direct electrical stimulation) is fired by stimuli in the anterior quarter,
approximately ; the smallest, slowest giants by stimuli in the posterior three quarters and a
region of overlap of a few segments occurs. The fast lateral giants can be fired from any level
but require stronger stimuli (water dropping from a few cms. higher for example). The evi-
dence suggests a function as mediators of startle responses to three classes of stimuli — weaker
anterior, weaker posterior, and stronger at any level (differences in threshold in different levels
exist for each fiber within this scheme). The two laterals usually fire together but in certain
cases they can be separated.

The plan in general is very like that in Lumbricus although the two belong to different
classes and many polychaetes with just as close a relation have no or very differently organized
giant systems.

The displacement of terns by gulls at the Weepecket Islands. Sears Crowell.

The changes in population at the colony of Common and Roseate Terns at the Weepecket
Islands are described for a period of twenty years. The colony attained, by 1931, a population
of 3500 adult terns. During the past ten years this colony of terns has gradually been replaced
by breeding Herring Gulls. The terns are probably incapable of successful reproductive ac-
tivity if gulls are near, even though the latter do no direct injury to the terns.

The members of the Weepecket colony have been redistributed among other colonies of
southern Massachusetts as shown by recoveries of banded birds.

Conditions favor a recolonization by the terns if the gulls are evicted or abandon the islands.

The influence of drugs on heat narcosis. A. Froehlich.

When the temperature of the surrounding water is slowly raised aquatic animals, such as
crustaceae, fishes, tadpoles and frogs, show complete loss of voluntary and reflex muscular ac-
tivity at a "critical point" of temperature which is characteristic for each species. This con-
dition is reversible ; transference into cool water causes the animals to recover promptly.
"Heat narcosis" resembles narcosis brought about by drugs (alcohol, ether, etc.) in every way,
except that where the former increases oxygen consumption, the drugs diminish oxygen con-
sumption.

For reasons too numerous to mention here, I decided to investigate the influence of theo-
phylline (as theophylline natrio-aceticum) on the "critical point" of heat narcosis. The experi-
ments were performed on Fundulus hctcroclitus at the M.B.L. in Woods Hole during the sum-
mer months of 1944 and 1945 and at the May Institute for Medical Research, Cincinnati, Ohio,
on field frogs and tadpoles during the winter and spring of 1944-1945.

Theophylline given subcutaneously or intramuscularly in doses which had no visible effect
on the behavior of the experimental animals produced a considerable lowering of the "critical
point" in heat narcosis. The same effect was obtained if the animals were placed in a weak
solution of theophylline.

Theophyllinized animals died much sooner than did controls if access to air was restricted.
The water in which such animals died showed far greater acidity due to accumulation of CO2.

Asphyxiation alone produced a lowering of the "critical point" similar to that obtained with
theophylline.

ABSTRACTS OF SCIENTIFIC PAPERS 187

Methylene blue (intramuscularly to Fundulus) produced effects on the "critical point,"
susceptibility to asphyxiation and acidity of the water which were similar to those obtained with
theophylline.

In the experiments with theophylline as well as in those with methylene blue, previous con-
ditioning in a 1 : 100,000 solution of quinine sulfate counteracted to a greater or a lesser degree
the expected lowering of the "critical point."

It can be concluded that the action of theophylline and methylene blue on these experimental
animals is, in part at least, to increase the demand for oxygen, and that quinine reverses this
action by decreasing respiratory metabolism.

As I had previously found (with E. Zak) that an important part of the action of theophyl-
line consists in increasing tissue permeability, I feel justified now in assuming that this phe-
nomenon is caused by a condition of hypoxemia and acidosis (local asphyxia) in the tissues.

Reactions of oyster (Ostrea virginica) to free chlorine. Paul S. Galtsoff.

By measuring the rate of flow of water through the gills and by recording the shell move-
ments it was possible to demonstrate that both the pumping mechanism of the oyster and its
adductor muscle are very sensitive to free chlorine. In many oysters the first treatment with
the concentrations as low as 0.01 or 0.02 p.p. million causes complete cessation of current and
closure of shells, although there are specimens in which complete cessation of pumping and
closing of shells takes place only in the concentrations approaching 0.5 p.p.m. Repeated treat-
ments develop increased tolerance and pumping may be resumed at the concentrations much
stronger than those which produced strong initial effect. Pumping, however, is not maintained
at the concentrations of one p.p.m. or greater.

Variation in the sensitivity and development of tolerance are apparently associated with
the secretion of mucus which provides protective coating for tentacles, mantle, and gills. Ob-
servations with a strobotac show that lateral cilia of the excised gill filaments continue to beat
even at the concentration of 3 p.p.m. The cessation of pumping activity of an intact organism
is due, therefore, not to the failure of the lateral cilia, but to the reaction of the regulatory
mechanism of the pallium, which prevents the entrance of water to the gills, and to a certain
extent to the disturbance of the rhythm of ciliary motion over the entire ciliated surface of the
demibranches.

The presence of free Cl in water may materially impede the purification of oysters. It is
therefore necessary that water, sterilized by chlorination and used in a process of purification,
contains no residual Cl.

Development of granule-free fractions of Arbacia eggs. Ethel Browne Harvey.

A granule-free fraction of the Arbacia punctulata egg is obtained by breaking the egg with
centrifugal force into two halves, and then breaking the lighter (white) half into two quarters,
one of which contains all the remaining granules ; and the other, the "clear quarter," is free of
all granules visible in the living egg. This clear quarter contains the oil, nucleus, and most of
the matrix or ground substance, but no mitochondria, yolk, or pigment. When fertilized, this
clear quarter in many cases throws off a fertilization membrane, cleaves quite regularly, forms
a perfect blastula and gastrula and pluteus. This pluteus may be quite normal with gut and
skeleton, and later develops pigment spots, but is much clearer and less granular than that from
the white half. It is approximately half the size of the pluteus from the white half and quarter
the size of the pluteus from the whole egg. There is a considerable delay throughout develop-
ment beginning with first cleavage, in spite of the fact that two nuclei (<$ and ?) are present
with a small amount of cytoplasm. The visible granules of the egg are therefore not necessary
for development. The important substance in the cytoplasm is the ground substance or matrix,
which is optically empty in the living state.

In vivo and in vitro glycogen utilisation in the avian nematode Ascaridia galli. W.
Malcolm Reid.

Glycogen constitutes one-third or more of the dry weight of many parasitic nematodes
and flatworms. Extensive in viiro experiments upon glycogen utilization have been carried out
by different investigators chiefly upon mammalian nematodes, cestodes, and trematodes. Von

188 ABSTRACTS OF SCIENTIFIC PAPERS

Brand with Ascaris lumbricoides showed that 45 per cent of the glycogen reserve was utilized
by females during 48 hours. Recent experiments upon fowl nematodes and cestodes have shown
a much higher rate of glycogen utilization when the host had been starved for a short time. In
a typical experiment with Ascaridia galli, 75 per cent of the glycogen reserve was utilized in 48
hours by female worms. With the fowl castode, Raillietina ccsticillus, this reserve was depleted
even more rapidly, 94 per cent of the glycogen being utilized in 24 hours.

Until a study using simultaneously in vivo and in vitro methods upon the same parasite has
been completed, a comparison of the results of such experiments can have little meaning. Fur-
thermore, such a study would serve as a check upon the earlier in vitro experiments which need
re-examination now that improvements in technique have brought some of these results under
question.

Glycogen determinations were made upon three groups of A. galli. Group I were controls
and consisted of worms which were removed from the host after a normal feeding period.
Group II worms were starved within the host for 48 hours before glycogen determinations were
made. Group III consisted of parasites removed from the same hosts used for Group I, but
these parasites were starved anaerobically for 48 hours at 41.5° ± 1° C. in one per cent saline
using the same in vitro methods that were used on mammalian forms. Separate determinations
were made on both males and females since sex differences in glycogen content were known to
exist. The mean glycogen content for approximately ten samples for each group expressed in
per cent of the wet weight of the worms is as follows: Group I females, 4.66; Group II females,
1.16; Group III females, 1.01 ; Group I males, 3.81 ; Group II males, 0.43 ; and Group III males,
0.26. The similarity in the rate of glycogen utilization with both males and females under the
two conditions probably indicates that the in vitro methods used by early investigators reflect
reliable information about normal glycogen metabolism within the host. Comparison between
the glycogen utilization in the avian A. galli with the mammalian A. lumbricoides indicates that
the much higher utilization rate in A. galli is real and not due to differences in technique.

Balanced centerwell solutions for manometric experimentation with cyanide.
W. A. Robbie.

It has been demonstrated, both experimentally and theoretically, that the potassium cyanide-
potassium hydroxide absorption solutions recommended by Krebs (1935, Biochem. Journ., 29:
1620) are not in hydrogen cyanide equilibrium with the experimental fluids for which they were
designed. It is possible, however, to prepare, on the basis of experimental determinations, po-
tassium cyanide-potassium hydroxide mixtures which will absorb carbon dioxide and maintain
hydrogen cyanide equilibria with cyanide solutions of 0.011 M or less. The hydrogen cyanide
tension of calcium cyanide solutions saturated with calcium hydroxide varies only with the con-
centration of the calcium cyanide and the temperature. This type of centerwell mixture will
absorb carbon dioxide effectively and maintain equilibrium with hydrogen cyanide solutions up
to 0.01 M.

Studies of the muscle tiuitch recorded by electronic methods* Alexander Sandow.

Piezoelectric, cathode-ray oscillographic methods have been devised for recording the various
mechanical changes of the isometric twitch of skeletal muscles. To register the latency relaxa-
tion, LR (the minute precontractile elongation of a stimulated muscle during the latter half of
the latent period), the apparatus is used, in effect, as an electronic lever which converts the LR
into a 500,000 X magnified deflection on the cathode-ray screen. The piezoelectric pulse corre-
sponding to the main contraction and relaxation periods is electronically differentiated and thus
at each instant the cathode-ray deflection for this record is proportional to the rate of tension
change in the course of the twitch.

These methods have been used to study the effect of maximal tetani of lengths from ty, to
10 sec. on the mechanical features of the twitch of the frog sartorius. The results prove that
the separate processes that underlie the LR, the use of tension, and the post-contractile relaxa-
tion, are each uniquely affected by the tetanic activity. E.g., a 2 sec. tetanus causes a 10 per

* Supported in part by a grant from the Penrose Fund of the American Philosophical So-
ciety.

ABSTRACTS OF SCIENTIFIC PAPERS 189

cent increase in the maximum rate of tension rise in a twitch, but a 40-60 per cent increase in
the maximum rate of relaxation. The great lability of the relaxation process associated with
the new chemical environment induced by the activity is specially significant in indicating that
relaxation is not passive but is chemically driven.

The LR shows certain temporal features like those of Brown's alpha-process, thus indicat-
ing that it is an external mechanical sign of the alpha-process. Detailed analysis of the effect
of activity and of pH on the LR, especially in reference to the duration of the latent period,
suggests that the latent period is an interval during which myosin-ATPase is splitting ATP,
and leads to the inference, now being subject to further test, that the LR corresponds to the
formation of an enzyme-substrate complex between myosin and ATP which provides a mecha-
nism for directly energizing and activating the myosin for contraction.

Experimentally induced tumors in an insect. Berta Scharrer.

In Lcucophaca madcrac, a large Orthopteran, the recurrent nerve was cut at various levels.
This nerve, which belongs to the stomatogastric nervous system, innervates the anterior portion
of the alimentary canal as well as the salivary glands and their reservoir. Within ten days to
several months after the operations tumors developed in organs innervated by the recurrent
nerve. Frequent sites of tumorous growth were the anterior portion of the mid-gut and the
salivary reservoir. In the fore-gut and in the salivary glands well developed tumors were rela-
tively rare. Several types of control operations, such as allatectomy and castration in which the
recurrent nerve had remained intact, did not cause the development of tumors. Some of the
tumors obtained after the cutting of the recurrent nerve attained considerable sizes. Histo-
logically they consist of layers of cells which show various degrees of abnormality. In advanced
stages part of the cells break down into a debris of brown color. About 300 specimens, nymphs
as well as male and female adults, with experimental tumors, were studied.

The origin of neurosccretory granules from basophil constituents of the nerve cells
in fishes. Ernest Scharrer.

Neurosecretory granules do not appear to be formed in association with the Golgi apparatus
or the mitochondria, but with the basophil constituents of the secreting nerve cells. Three
modes of origin of the granules have been observed. In the preoptic nucleus of most fishes the
granules originate in association with the peripherally located Nissl bodies. The latter diminish
to the extent to which the acidophil neurosecretory granules increase. In a second type found
in the preoptic nucleus of Ameiurus, Noturus, Centropristes, and others the nuclei of the secret-
ing nerve cells show imaginations. These are filled with basophil cytoplasm which may con-
tain acidophil granules. In a third type which is characteristic of the nucleus lateralis tuberis
of catfishes, the acidophil granules originate within the nuclei of the cells, apparently at the
expense of the nuclear chromatin. All three types may occur in the preoptic nucleus of
Centropristes.

Evidence of a metabolic effect by potassium in lowering the injury potential of in-
vertebrate nerve.* Abraham M. Shanes.

The action of potassium on the injury potential of spider and blue crab nerve has been
studied over a concentration range of one to 530 mM. When the magnitude of these potentials
is plotted against the logarithm of potassium concentration, the relative effectiveness of low
potassium concentrations in lowering the potential is found to be % that of concentrations
above 30 to 40 mM. The data may be replotted on a log-log graph on the assumption that po-
tassium is inactivating an enzyme, the active form of which is proportional to the resting po-
tential. Two straight lines intersecting at 40 mM fit the data very well, the slope at lower
concentrations being about Vz and at higher concentrations about one. This graph is like one
which has been obtained for the effect of urethane on oxygen consumption in yeast and Arbacia ;
in this case the inhibitor is believed to act on two processes. The same interpretation may be
applied to potassium.

* Aided in part by a grant from the American Academy of Arts and Sciences.

190 ABSTRACTS OF SCIENTIFIC PAPERS

The similarity of potassium to an actual inhibitor is even stronger if consideration is given
to the effect on activity. Only at a concentration corresponding to almost complete cessation of
the process affected at low concentration does activity appreciably and suddenly decrease.
Thus, in crab nerve, conduction ceases between 37 and 42 mM. Potassium and excitability is
unaffected up to 37 mM.

The effect of low potassium concentrations is definitely correlated with the simultaneous
inhibition of an aerobic metabolic process which supports the injury potential. In concentra-
tions of 10 to 30 mM potassium eliminates % of this process — values corresponding closely to
those obtained previously in frog nerve.

Physical-chemical studies on chromosomal nucleoproteins.* Kurt G. Stern.

The object of this research is to determine the size and shape of desoxyribosenucleoproteins,
isolated from cell nuclei, with the aid of such quantitative methods as ultracentrifugation, diffu-
sion, electrophoresis, viscosity, x-ray diffraction, and similar techniques. In this cooperative
study, S. Davis, P. Macaluso, S. C. Shen, and I. Fankuchen are collaborating with the writer.

Thus far, the desoxyribosenucleoproteins from the nuclei of chicken red blood cells and from
calf thymus gland have been studied. Measurements in the analytical ultracentrifuge, in the
diffusion apparatus, and in Ostwald viscometers, performed on solutions of these purified nucleo-
proteins in one M. NaCl, indicate a molecular weight of the order of two to three million and
axial ratios varying from 35 : 1 to 100: 1. The discrepancy of the results obtained with inde-
pendent techniques casts considerable doubt on the suitability of this solvent, proposed by Mirsky
and Pollister, with regard to the native state of the nucleoproteins. It appears that these conju-
gated proteins are appreciably dissociated in M.NaCl-solution. According to preliminary ex-
periments, one M.glycine appears to be a solvent better suited for physical-chemical studies on
these macromolecules.

The theory that these desoxyribosenucleoproteins are capable of assuming a more or less
helical shape in solution as a function of the nature and ionic strength of the solvent, is advanced
as a working hypothesis. Thus it is assumed that these molecules reflect in their configuration,
on a molecular scale, the coiling and uncoiling of the chromosomes of which they represent im-
portant constituents. Plastic models, constructed in accordance with this hypothesis, were dem-
onstrated at the Seminar.

Action of quitenine on the livers of tautog and toadfish. Charles H. Taft.

When quinine is treated with potassium permanganate the vinyl group is oxidized to a
carboxyl group yielding quitenine.

It has been shown (Dauber, M., 1920; Zeit. filr E.rpt. Path. u. Therapic, 21: 311) that
quitenine had a damaging action on kidney tubules. Taft and Place (1944; Texas Reports on
Biol. and Med., 2: 61) showed that quitenine was more injurious to the kidneys of a glomerular
fish than to the kidneys of an aglomerular fish.

Quitenine dihydrochloride in a 0.25 molar solution was injected subcutaneously into the
side of the fish. The doses used were 1, 2, and 4 mM/Kg. Fish were killed by a blow on the
head after varying intervals of time. The livers were placed in Bouin solution. Sections were
cut 6/x thick.

On gross examination a few tautog livers were abnormally soft. Gall bladder was a
greenish blue in all cases. In the toadfish the liver was soft in a few cases. Color of gall
bladder ranged from white through pale pink, orange, yellow green to green. Variation in
color is probably due to variation in amount of bile pigment production or to oxidation of bile
pigment. The von Kupffer cells were undamaged as were pancreatic cells of hepatopancreas.

Microscopic examination of toadfish liver shows fatty metamorphosis and some parenchy-
matous degeneration. Microscopic examination of the tautog liver showed fatty metamorphosis,
albuminous degeneration, hydropic degeneration, and parenchymatous degeneration.

Quitenine is more damaging to the liver of the tautog than to the liver of the toadfish.
The damage to the livers is not as severe as it was in the kidneys.

* This work was made possible by a grant of The Carrie S. Scheuer Foundation of New
York.

ABSTRACTS OF SCIENTIFIC PAPERS 191

Differences in sensitivity, hatchability curves, and cytological effects betzveen Habro-
bracon eggs x-rayed in first meiotic prophase and metaphase. Anna R. Whiting.

Unlaid Habrobracon eggs were x-rayed in first meiotic prophase (diplotene) and in late
metaphase and allowed to develop parthenogenetically. Those treated in prophase have SO per
cent hatchability at about 12,000 r (lethal dose about 45,000 r) ; give an exponential hatchability
curve which tends to become linear when dose is fractionated ; may show, after treatment, frag-
ments or bridges or both in division I, in division II or in both. Those treated in late meta-
phase have 50 per cent hatchability at about 400 r (lethal dose about 2,000 r) ; give a linear
hatchability curve which does not change with fractionation of dose ; may show fragments but
no bridges in division I, either or both in division II. All eggs treated in either stage with
lethal dose develop at least to first cleavage (20 per cent continue to blastoderm) ; show bridges
and sometimes fragments in cleavage. A correlation of chromosome form, movement, and
tension at time of treatment with sensitivity and cytological effects exists which suggests that
x-ray injury is due to direct "hits" on chromosomes, and that sensitivity is associated with de-
gree of tension to which chromosomes are exposed during irradiation ; that nature of chromo-
some changes is due to their form and proximity during treatment. Lethal dose is not lethal to
the treated cell (oocyte) but to its descendents (embryo) since chromosome fragmentation is
not lethal, loss of fragments is.

The problem of reversal of male Jiaploidy by selection. P. W. Whiting.

Except for the almost sterile, highly inviable diploid males of the wasp Habrobracon ob-
tained in experimental cultures, diploid males are unknown in the Hymenoptera, as also in
rotifers, thrips, mites except Mesostigmata, aleurodids and iceryine coccids, and in the beetle
Micromalthus. It is probable that all normal males in these groups are haploid and that male
haploidy has been attained in an evolutionary sense not more than six or seven known times.
One of these attainments, taking place in an ancestral hymenopteron probably in the early Juras-
sic, has come to involve the entire order. Three conditions characterize male haploidy: (1)
Production of males from reduced unfertilized eggs. (2) Reduction or omission of meiosis in
spermatogenesis. (3) Complementary sex determination with heterozygous "double dominant"
females. The problem of reversal of male haploidy is not to attempt to re-integrate any Juras-
sic protohymenopteran species, but rather to obtain by methods of genetics a strain of Habro-
bracon juglandis with normal biparentalism of males as well as of females. Inbreeding gives
diploid males homozygous for sex. Selection has increased their viability from one to sixty per
cent as compared with females. Cell size of diploid males is abnormally large, but is reduced
somewhat in strains of high viability. Spermatogenesis of diploid males is of the haploid type,
lacking chromosome synapsis and resulting in diploid sperm. If a strain with chromosome
synapsis can be derived, it is considered that the problem can be solved, since sex determination
should then shift from the complementary to the back-cross type with digametic females.

Endomitosis in plants. E. R. Witkus.

The process called endomitosis was discovered by Geitler in 1939 in insect material. Dur-
ing this process there is a chromosomal reduplication without a nuclear division, no spindle is
present and there is no true anaphase movement of chromosomes. Throughout the whole proc-
ess the nuclear membrane remains intact. Geitler divided the process into four stages, which
he termed endoprophase, endometaphase, endoanaphase, and endotelophase. During endopro-
phase the chromosomes become shorter and thicker. The stage at which the chromosomes have
reached their highest degree of contraction is called endometaphase. The nuclear membrane is
intact and the chromosomes are not aligned on an equatorial plate. The SA-region of the
chromosomes divides and the chromatids or now endoanaphase chromosomes slightly separate.
After this separation the chromosomes undergo reversion to the resting stage. This reversion
process occurs during endotelophase. The resulting cell then is tetraploid.

This process was also found to occur in the tapetal cells of Spinacla oleracca (Spinach)
and apparently this is the first time that endomitosis, as defined by Geitler, has been reported for
plant material.

The tapetal cells of Spinacia undergo two successive divisions during the early prophase
stages of meiosis. The first division is an incomplete mitotic division resulting in binucleate

192 ABSTRACTS OF SCIENTIFIC PAPERS

cells or in cells having dumb-bell shaped nuclei. The second of these divisions is in all cases
endomitotic.

It becomes increasingly apparent that polyploidy brought about by a chromosomal redupli-
cation without a nuclear division is of quite common occurrence in both plant and animal ma-
terial. Endomitosis is only one of three known methods by which this can occur, although it
has often been confused with all three in recent cytological literature. The first method is by a
repeated reduplication in the resting nucleus as illustrated in the multiple complex cells of mos-
quito. The second is simply by a double reduplication in the resting nucleus as shown by cer-
tain cells in the root tips of polysomatic plants such as Spitiacia. The third is by endomitosis.

It is interesting also to note that polyploidy arises by different methods in the root tip and
tapetal cells of Spinacia oleracea.

A tetrahedral framczvork for native proteins? Dorothy Wrinch.

It was suggested in 1936 that a fabric or atomic lamina is an essential element in the struc-
ture of native proteins and the lactim cyclol fabric was formulated as a working hypothesis
(Nature, 137: 411). Today four different types of fabric — all necessarily polypeptide fabrics-
are open for discussion; the lactim and enol cyclol fabrics, the hydrogen-bridged linear poly-
peptide fabrics, and the fabrics in which cyclized polypeptides are interlocked by hydrogen
bridges (Jordan Lloyd and Wrinch, 1936; Nature, 138: 758; Astbury and Wrinch, ibid., 139:
798; Wrinch, 1940; Phil. Mag., 30: 64; 1941, 31: 177). This idea of an atomic lamina or
fabric as characteristic of native proteins has been widely accepted and adopted. It has now
been used to interpret the x-ray intensities in a study of horse methemoglobin in crystalline
form.

We wish now to suggest that these (hoi) intensities (Perutz, 1942; Nature, 150: 324) sug-
gest not only laminae parallel to the c-planes (Boyes- Watson and Perutz, 1943; Nature, 151:
714) but also a second set of laminae at approximately the tetrahedral angle to the first. For
this and a number of other reasons, the hypothesis is put forward that the native protein unit
(of which there may be one, two, or more in the native protein particle) is built on a tetrahedral
framework, the possibility that the enveloping truncated tetrahedron of the framework may be
an octahedron not being excluded. In the case of horse methemoglobin, this suggestion implies
trigonality of the individual frameworks about an axis approximately normal to the c-planes ;
thus it offers an interpretation of the fact that the lattice points and c-face centers in these planes
form a triangular network which is very nearly equilateral. The hypothesis appears to bear
very closely upon the fact that twins, trillings, and other compound crystals are extremely com-
mon in many different hemoglobins (Reichert and Brown, 1909; The Crystallography of the
Hemoglobins, Washington, D. C.). This we propose for discussion the possibility that such
situations as the apposition of tetrahedral frameworks by displacements plus rotations of_(l)
%, % about the (111) axis; or (2) %, %, % about the (111) axis; or (3) % about the (110)
or (112) axes, or %, }£, % about the (100) axes, etc., are here realized. Attention is also di-
rected to the obvious manner in which this postulate lends itself to the interpretation of the
space groups and classes of crystal symmetries found in x-ray (Fankuchen, 1941; Ann. N. Y.
Acad. Sci., 41 : 157) or classical studies of the native proteins.

Vol. 89, No. 3 December, 1945

THE

BIOLOGICAL BULLETIN

PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY

NATURAL HETEROAGGLUTININS IN THE SERUM OF THE SPINY

LOBSTER, PANULIRUS INTERRUPTUS. II. CHEMICAL AND

ANTIGENIC RELATION TO BLOOD PROTEINS1

ALBERT TYLER AND BRADLEY T. SCHEER

JVilliam G. Kcrckhoff Laboratories of the Biological Sciences, California Institute of

Technology, Pasadena

In a previous report (Tyler and Metz, 1945) it has been shown that lobster-
serum contains at least ten heteroagglutinins for sperm or blood cells of various
animals. Each of the heteroagglutinins was found to act on all the species tested
that belong to the same group of animals. Since the group, in most instances,
represents a taxonomic class, the heteroagglutinins are termed class-specific. The
heteroagglutinins were found to be most probably protein, and by means of
electrophoresis they were shown to be distinct from the hemocyanin which Allison
and Cole (1940) and Clark and Burnet (1942) had considered to be the sole
protein present in lobster-serum.

The relatively small amount found to be present accounts for Allison and Cole's
conclusion which was based on approximate identity of the copper to protein nitrogen
ratios of purified hemocyanin and of whole serum. Clark and Burnet's evidence
was actually to the effect that there is no protein present with active antigenic
properties different from that of pure hemocyanin. This is in accord with the
results obtained with antisera prepared against hetefoagglutinin by injecting rabbits
with agglutinin that had been absorbed on rabbit cells. In the present paper a
precipitation method for preparing the heteroagglutinins free of hemocyanin is
described, and results of an electrophoretic examination of the material are pre-
sented. The agglutinating action of fibrinogen preparations from plasma and
further serological tests are also reported.

MATERIAL AND METHODS

Blood is quite easily obtainable from lobsters by means of a syringe inserted,
between cephalothorax and abdomen, into the pericardial chamber. A twelve-inch
lobster yields, in this manner, about 20 to 30 ml. of blood. For serum the blood
was generally defibrinated by shaking with glass beads, filtered, and centrifuged ;
or it was occasionally allowed to clot, forced through a fine mesh wire screen, and
centrifuged. For plasma the blood was drawn into a small amount of sodium

1 This work has been aided by grants from the American Philosophical Society and the
Rockefeller Foundation.

193

194 ALBERT TYLER AND BRADLEY T. SCHEER

citrate solution, then subsequently filtered, centrifuged, and dialyzed against saline.
One volume of 10 per cent citrate suffices to prevent fibrin-clotting in about 30
volumes of blood.

The agglutinative tests were made as previously described (Tyler and Metz,
1945) by mixing equal volumes of the sperm or blood cells (of sea-urchin, sheep,
or other animal) and of serial two-fold dilutions of the test-solution adjusted to the
appropriate salinity. Deviations from these proportions are specified in the tests.

EXPERIMENTAL PART
Separation of heteroagglutinins from licuiocyanin b\ isoelectric precipitation

Hemocyanin was prepared from serum by isoelectric precipitation essentially as
described by Allison and Cole (1940) and by Rawlinson (1940). This consists
in dialysis against distilled water and then against dilute acetate buffer at the pH of
the isoelectric point. Further purification is obtained by repeated solution in dilute
ammonia and reprecipitation, by addition of acetate buffer (0.1 M., pH 4.5).

Rawlinson (1940), in the course of purification of hemocyanin from the plasma
of the Australian spiny lobster, noted the presence of small amounts of protein
which he considered to be fibrinogen. Such a non-hemocyanin protein is obtainable
from the serum of the California spiny lobster, Panulirus interruptus.

When samples of serum or plasma of Panulirus were dialyzed against dilute,
pH 4.5, acetate buffer, there invariably appeared small amounts of a pale precipitate
that separated before the hemocyanin started to come down. The precipitates
ranged in color from white to pink. After centrifugation, washing with distilled
water and solution in dilute ammonia, the material was reprecipitated by slow
addition of 0.01 M., pH 4.5 acetate buffer. The material was regularly found to
start to precipitate at pH 5.0 and reach a maximum at pH 4.8. From the super-
natants of the first precipitates the blue-colored hemocyanin was precipitated by
continuation of the dialysis against the pH 4.5 buffer. The hemocyanin was
obtained in crystalline form from concentrated solutions of it in dilute ammonia
by the slow addition of dilute acetate buffer. Its precipitation was found to begin
at pH 4.6 and to be complete at 4.5 to 4.4.

Samples of the purified hemocyanin and of the pale precipitate were tested for
their ability to agglutinate the sperm or blood cells of various animals. After
adjustment of the solution to appropriate pH and salinity by dialysis, they were
tested on one per cent suspensions of the sperm of the polychaet, Chaetopterus
variopedatus ; the sea cucumber, Sticliopus calif oniicus; the starfish, Pisaster
ochraceus ; the sea-urchin, Strongylocentrotus purpuratus ; the sea-squirt, Ciona
intestinalis; and the grunion (smelt), Leuristhes tennis; and of the erythrocytes
of the sand bass, Paralabra.v maculatojasciatus ; the frog, Rana pipicns; the
chuckwalla, Sauromalus ater; the pigeon, and sheep. The hemocyanin prepara-
tions, containing this material in amounts as great as or greater than normally
present in the serum, were found to be completely inactive. The preparations of
the pale, pH 4.8-5.0, precipitate gave very good agglutination of the cells of all the
above named species.

Titer determinations were made with one of these preparations on sperm of
Strongylocentrotus. In this case 0.2 ml. of serial two-fold dilutions of the solution
were mixed with one drop of 10 per cent sperm-suspension. The protein con-

HETEROAGGLUTININS IN LOBSTER-BLOOD

195

centration (from Kjeldahl nitrogen determination) of the solution was 0.7 per
cent and its titer (minimum dilution giving definite microscopic agglutination) was
128. A sample of serum containing 5 per cent protein gave at the same time a titer
of 256. This preparation showed, then, about 3^/2 times the activity of the whole

serum.

Electrophoretic examination of the pale precipitate -

Another sample of the material freed of hemocyanin was reprecipitated at pH 5,
dissolved in dilute ammonia, and dialyzed for 2 days in the cold against barbiturate
buffer (fji = 0.05) at pH 7.7. It was then examined electrophoretically in the

FIGURE 1. Electrophoretic patterns of pale (pH 5) precipitate from lobster-serum, a, de-

scending (desc.) side; b, ascending (asc.) side; after 59 minutes of electrophoresis at pH 7.7,

ionic strength 0.05 and 14.8 ma. Arrows show direction of migration. See text for further
description.

2 The apparatus employed was that constructed in the Division of Chemistry by Dr. Stanley
M. Swingle to whom we are indebted for the electrophoresis of this material.

196

ALBERT TYLER AND BRADLEY T. SCHEER

Tiselius' (1937) apparatus using the scanning method of Longsworth (1939).
After 59 minutes of electrophoresis with a current of 14.8 ma., the patterns shown
in Figure 1 were obtained. As may be seen from the figure two components,
besides the 8- or e-boundaries, are present in the serum. From the relative areas
covered by the peaks the ratio of amount of slow component to that of fast compo-
nent is approximately 5:1. At the end of the run the fast moving component was
removed from the ascending side and the slow component (plus 8), from the
descending side of the electrophoresis cell. After dialysis against normal saline,
determinations were made of their agglutinative titers for rabbit cells and of the
Kjeldahl nitrogen content. Samples of the original solution of the pale precipitate
(taken from the cell after the run) and of normal lobster-serum were tested at the
same time. The results are given in Table I. The nitrogen content of the
solutions does not represent the relative concentrations of the components present
in the original solution since there was some dilution with buffer upon their
removal from the electrophoresis cell. As may be seen in Table I, the solution
of the fast component showed no agglutinative activity for rabbit-erythrocytes
although its nitrogen content was about one-third that of the slow component. The
slow component proved highly active, giving almost twice the titer (per mg. N.
content of solution) of the original solution and 24 times that of whole serum.
This is approximately the order of magnitude of activity obtained (Tyler and
Metz, 1945) for the components isolated by electrophoresis from whole serum.

TABLE I

Agglutinative titers of components obtained by electrophoresis
of the pale (pH 5) precipitate from lobster-serum

Material

mg. Kjeldahl N.
per ml.

Agglutinative titer on
1% rabbit cells

Titer/mg. N.

Fast component (F of Figure 1)

0.29

Slow component (S of Figure 1)

1.008

128

Original solution (from cell-residue)

3.85

256

66.5

Whole serum

11.97

5.3

The slow component obtained here was also tested on cells of all the animals
listed on page 194, with the exception of Sanromahis and Lcnristhcs. It proved to
be highly active with all of them. In the previous report lobster-serum was shown
to contain at least ten "class-specific" heteroagglutinins. It is evident from the
present results that these are represented by a single electrophoretic component of
the serum, unless there is some active component in the stationary 8- or e-boundary.
The latter is, however, highly unlikely since the original material for the present test
was obtained by precipitation at pH 4.8 to 5.0 and the electrophoresis was run at
pH 7.7. For any material to remain in these stationary boundaries it would have
to be isoelectric at the latter pH.

Preparation of fibrinogcn and tests for heteroagglutinating activity

Lobster-plasma upon being brought to 25 per cent saturation with ammonium
sulfate formed a white to pink precipitate which separated easily upon centrifuga-

HETEROAGGLUTININS IN LOBSTER-BLOOD

197

tion. The precipitate was washed with distilled water and dissolved in sea water.
Addition of fresh lobster-blood-cells to the solution caused it to form a firm clot.
A pH 5.0 precipitate obtained directly from plasma was found to contain fibrinogen,
which could be separated from the remaining protein material by precipitation with
ammonium sulfate. None of the prepartions from serum were found to contain
fibrinogen.

TABLE II

Agglutinative tilers of protein preparations from plasma and serum

Material

mg. Kjeldahl N.
per ml.

Agglutinative titer on
Strongylocentrotus sperm

Titer per mg. N.

Fibrinogen preparation (I)
Pale precipitate (II)
Hemocyanin
Whole serum

1.25

1.25
7.4
8.5

32 to 64
64 to 128
0
128 to 256

26 to 51
51 to 102
0
15 to 30

Plasma

8.5

256 to 512

30 to 60

A fibrinogen preparation (I) was obtained from whole plasma by 25 per cent
saturation with ammonium sulfate. The precipitate was dissolved and reprecipi-
tated by dialysis to pH 5.0. This preparation was tested for agglutinating action
on sperm of Strongyloccntrotus in the same manner as on page 194. The super-
natant from the 25 per cent ammonium sulfate precipitate was dialyzed against tap
water and then brought to approximately pH 5 by dialysis against pH 4.5 buffer.
This gave a pale precipitate (II) which resembled the pale precipitate from serum.
After solution and dialysis against sea water it, too, was tested for agglutinating
activity. The results are given in Table II along with simultaneous tests of whole
serum, plasma, and hemocyanin. The presence of calcium in the sperm suspension
does not interfere with the tests, since clotting of the fibrinogen does not occur
unless fresh lobster-blood-cells are added. As the table shows, plasma has about
twice the agglutinating activity of serum. The fibrinogen preparation proved about
half as active as the pale precipitate.

Another pale precipitate was also obtained directly from plasma by dialysis
against pH 4.5 buffer. When the precipitate was dissolved and brought to 25 per
cent saturation with ammonium sulfate there separated out some material that
proved to be fibrinogen. It appears from the experiments reported above that the
isoelectric point of fibrinogen is not greatly different from that of the heteroag-
glutinin found in serum. This conclusion was verified by Mr. Maurice Rapport,
who repeated some of our experiments, and made an electrophoretic examination
of plasma and of protein preparations separated from plasma. The pH 5.0 pre-
cipitate from plasma showed two electrophoretic components, the patterns being
similar to those of Figure 1 . The smaller, faster component probably corresponded
to the fast component observed in serum preparations. The other component,
containing agglutinating activity, could not be separated further during 100 minutes
of electrophoresis at pH 7.3, 1.2° C. and 20 ma.

Precipitation of the pH 5.0 precipitate from plasma with ammonium sulfate
at 40 per cent of saturation removed nearly all of the agglutinating activity, but
left behind a small amount of protein material. The ammonium sulfate precipitate

198 ALBERT TYLER AND BRADLEY T. SCHEER

contained 3.5 mg. Kjeldahl N./ml, and had a titer of 64 against Strongylocentrotus
sperm. The supernatant contained 1.6 mg. N./ml, and titrated only to 4. Mr.
Rapport showed that this small residue migrated rapidly in the electrophoresis
apparatus at pH 7.3. It probably corresponded to the fast component from serum.
In the absence of more exhaustive chemical and electrophoretic separations it
is not possible to decide with certainty whether the agglutinative activity found in
fibrinogen preparations is associated with fibrinogen itself, or is due to the presence
in these preparations of the heteroagglutinin fraction which is present in serum.

Antigenic relationship of the blood proteins

Two rabbits that were each given two courses of intravenous and intra-abdominal
injections with a total of 375 mg. of purified hemocyanin produced very good
precipitating antisera. The titers (end point of precipitation on mixing equal
volumes of antiserum and serial dilutions of a 10 per cent hemocyanin solution)
ranged from 10,000 to 20,000 in terms of antigen dilution and optimal proportions
(second optimum, see below) were obtained at approximately one volume of 10
per cent hemocyanin to 10 to 20 volumes of antiserum. The antisera also reacted
very well with whole lobster-serum, the optimal proportions point being about 9
volumes of antiserum to one volume of the lobster-serum.

Tests were then made of the ability of antiserum vs. hemocyanin to remove
natural heteroagglutinin from whole lobster-serum. One volume of lobster-serum
was absorbed with 9 volumes of the rabbit antiserum and the supernatant tested
for ability to agglutinate rabbit-erythrocytes and Strongylocentrotus sperm. The
absorbed serum gave no reaction with these cells, while control lobster-serum gave
good agglutination out to dilutions of 1/90 (+ + + reaction) with the rabbit cells
and 1/80 (+ reaction) with the Strongylocentrotus cells respectively.

It appears, then, that antibodies prepared against hemocyanin also react with
the natural heteroagglutinins present in lobster-serum.

One of the antihemocyanin rabbit sera was also titrated with the solution of
electrophoretically purified heteroagglutinin (slow component). A titer (dilution
of antigen) of 128 was obtained for this solution which contained one mg. Kjeldahl
N. per ml. A control hemocyanin solution containing 8 mg. N. gave a minimum
titer (end point not reached) of 4096, or 512 per mg. N.

Another antiserum against hemocyanin was also titrated with various protein
fractions separated from lobster-blood. The titer (dilution of antigen) of reprecipi-
tated hemocyanin was 20,000 for a solution containing 6.6 mg. Kjeldahl N. per
ml. or 3000 per mg. N. For the heteroagglutinin (pH 5 precipitate from serum,
reprecipitated), the titer was 200 for a solution containing 1.6 mg. N. per ml. or
125 per mg. N. For the fibrinogen (ammonium sulfate precipitate from plasma),
the titer was 200 for 3.4 mg. N. per ml. or 60 per mg. N.

In these titrations, it was sometimes noted that precipitation occurred in the first
few tubes, containing concentrated antigen solutions. In intermediate dilutions,
no precipitation occurred, but a second zone of precipitation appeared in the higher
dilutions. This \vas noted both with hemocyanin and fibrinogen, but not with the
agglutinin preparation (pale precipitate from serum) used. Boyden and deFalco
(1943) reported a similar double zone phenomenon with Homarns serum titrated
against anti-//o;nan<^-hemocyanin. They pointed out that this is indicative of the

HETEROAGGLUTININS IN LOBSTER-BLOOD 199

presence of two kinds of antibodies in the antisera. However, this does not seem
to be the entire explanation, since we find that absorption of a sample of antiserum
with an amount of hemocyanin which corresponds to the lower of the two optima
removes all antibody for the homologous antigen, as well as for fibrinogen and
pale precipitate.

Two rabbits were also immunized with whole lobster-serum, each receiving a
total of 5.5 ml. of serum in two courses of three injections each, with three weeks
rest between courses. The antisera obtained one week after the last injection gave
very good precipitation with the homologous antigen, optimal proportions (second
optimum) being obtained with one volume of lobster-serum to approximately 16
volumes of antiserum. A sample was absorbed with purified hemocyanin and
tested on whole serum, a concentrated solution of the pale (pH 5) precipitate, and
a fibrinogen preparation. It failed to give precipitation with any of these antigens.
This confirms the findings of Clark and Burnet (1942) and indicates that the other
blood proteins have no active antigenic groups other than those present in the
hemocyanin. Alternatively, the results might be explained on the basis of com-
petition of antigens (see Sachs, 1929), such that the rabbit does not form anti-
bodies against other antigens when one powerful antigen (the hemocyanin) is
present in excess in the material (whole lobster-serum) used for immunization.
However, in view of the analogous results obtained (Tyler and Metz, 1945) with
antisera prepared against heteroagglutinin, and with antihemocyanin sera (above),
the alternate explanation seems highly unlikely.

SUMMARY

1. Lobster-serum contains small amounts of other protein constituents besides
hemocyanin.

2. The "class-specific" heteroagglutinins of lobster-serum are found to reside in
a component that is obtained free of hemocyanin by isoelectric precipitation at
pH 4.8 to 5.0.

3. Electrophoresis of this "pale precipitate" reveals the presence of two com-
ponents, of which the more slowrly migrating one bears the heteroagglutinating
activity. The ten separate "class-specific" heteroagglutinins are thus evidently
represented by a single electrophoretic component.

4. There is some indication that fibrinogen obtained from the lobster plasma
may also act as heteroagglutinin.

5. Antibodies produced in rabbits against purified hemocyanin also react with
the slow electrophoretic component (heteroagglutinin) of the pale precipitate and
with fibrinogen. Absorption tests with antisera vs. whole lobster-serum fail to
reveal the presence of any specific antigenic groups other than those of the hemo-
cyanin. The other blood proteins are, then, evidently serologically equivalent to
hemocyanin.

LITERATURE CITED

ALLISON, J. B., AND W. H. COLE, 1940. The nitrogen, copper and hemocyanin content of the

sera of several arthropods. Jour. Biol. Chem., 135 : 259-265.
CLARK, ELLEN AND F. M. BURNET, 1942. The application of the serological methods to the

study of Crustacea. Austral. Jour. E.vp. Bio!, and Mcd. Sci., 20 : 89-95.

200 ALBERT TYLER AND BRADLEY T. SCHEER

BOYDEN, A., AND R. J. DEFALCO, 1943. Report on the use of the photronreflectometer in sero-

logical comparisons. Physiol. Zool., 16: 229-241.
LONGSWORTH, L. G., 1939. A modification of the Schlieren method for use in electrophoretic

analysis. Jour. Amcr. Chcni. Soc., 61 : 529-530.
RAWLINSON, W. A., 1940. Crystalline haemocyanin : some physical and chemical constants.

Austral. Jam: Exp. Biol. and Med. Sci., 18: 131-140.
SACHS, H., 1929. Antigene und Antikorper (c) die Konkurrenz der Antigene. Handbuch dcr

Norm, und Path. Physiol., 13 : 444-446.
TISELIUS, A., 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans.

Faraday Soc., 33: 524-531.
TYLER, A., AND C. B. METZ, 1945. Natural heteroagglutinins in the serum of the spiny lobster,

Panulirus interruptus. I. Taxonomic range of activity, electrophoretic and immunizing

properties. Jour. E.rper. Zool., in press.

STUDIES ON MARINE BRYOZOA. I. AEVERRILLIA
SETIGERA (HINCKS) 1887

. MARY DORA ROGICK

Marine Biological Laboratory and College of Nczv Rochclle

TABLE OF CONTENTS

PAGE

Introduction 201

Distribution 201

Ecology 202

Description of species 203

Table I 205

Discussion 212

Summary 213

Literature cited 213

Explanation of Plate I 206

Explanation of Plate II 208

INTRODUCTION

During the summer of 1944 collections of Aevcrrillia setigera were made at
New Bedford and Woods Hole, Massachusetts. Perusal of literature pertaining
to this species showed that a more complete account of this form would not be amiss.
This article brings together all available distribution and anatomical data previously
given for this form and adds to it some new distribution data, more complete
illustrations than were heretofore available and a considerable amount of anatomical
and some ecological data.

The writer wishes to acknowledge, with sincere appreciation, the kindness of
Dr. Hannah Croasdale of Dartmouth College and of the Marine Biological Labora-
tory of Woods Hole, Mass., who collected the first specimens of A. setigera from
New Bedford, Mass., and turned them over to the writer for study, and to Dr.
Raymond C. Osburn of the University of Southern California who so kindly
checked the specimens, confirming the identification and who offered many helpful
suggestions.

DISTRIBUTION

The species Buskia setigera has been reported previously by the following
authorities from the localities listed below :

Hincks, 1887 (pp. 121, 127-128; PI. XII, Figs. 9-13), from the Gulf of Bengal.

around the Mergui Archipelago.
Kirkpatrick, 1890a (pp. 603, 612), between Australia and New Guinea in the

Torres Straits, 20 miles off Warrior Island.

Kirkpatrick, 1890b (p. 17), off Tizard Banks in the China Sea.
Thornely, 1905 (p. 128), from Ceylon.

201

202 MARY DORA ROGICK

Harmer, 1915 (pp. 87-88; PI. 5, Figs. 8-10), from the Bay of Bima (India), Bay
of Badjo, west coast of Flores (Malay Archipelago), Makassar, Borneo Bank,
off Pulu Jedan, east coast of Aru Islands, and also in the following unnamed
locations: Station 164, at 1°42'.5 S. ; 130°47'.5 E. ; Station 166, at 2°28'.5 S.;
131°3'.3 E.

Thornely, 1916, off Poshetra Head, Kattiawar, and Ceylon.

Hastings, 1927 (p. 351), at Menzaleh Lock and other stations at the Suez Canal.

Livingstone, 1927 (p. 67), from Queensland, Australia.

Hastings, 1932 (p. 407), from Penguin Channel and N. E. Low Island, Great
Barrier Reef, Australia.

Osburn, 1933 (p. 64), from Porto Rico.

Marcus, 1937 (p. 143 ; PL 29, Fig. 76), from Bay of Santos, Brazil, South America.

Osburn, 1940 (p. 343), from Porto Rico.

Hutchins, 1945 (p. 539), Pine Orchard, Long Island Sound, Connecticut, U.S.A.

Additional discussion of the species occurs in the following articles:

Osburn and Veth, 1922; (p. 159).

Marcus, 1938; (p. 61).

Marcus, 1939; (pp. 168, 171).

Marcus, 1941; (pp. 74-77, 147; PL X, Fig. 45).

The above reports indicate that the species is distributed near several continents,

—Africa (Suez Canal), Asia, Australia, South America, and North America, and

also around several islands, including Porto Rico. The present article reports

its occurrence around the State of Massachusetts, extending the northerly range

of this species to 41°38' N. Latitude.

Averrillia setigera was found in two Massachusetts localities. The first collec-
tion was made by Dr. Hannah Croasdale on July 29, 1944, at Black Rock in the
Harbor of New Bedford, Mass. The next collections were made by the author
on August 4, 13, and 14, 1944, at Stony Beach, Woods Hole, Mass. Further
details of the nature of the collecting site and the associated biota will be given in
the ECOLOGY section.

ECOLOGY

The New Bedford Harbor specimens were collected by Dr. Croasdale at the
time of low tide, from the littorine region around Black Rock, along with red
algae, at a depth of less than 2 feet below the surface of the water. The Woods
Hole specimens came from a large, partially submerged boulder located approxi-
mately 50 yards from shore. The sea bottom around the boulder is largely sand
although there are some rocks a short distance away on each side of the boulder.
The general locality is not subjected to strong wave action. The boulder is almost
completely submerged at high tide but is about half exposed at low tide. Its sides
are well covered with algae of various kinds as well as with a luxuriant fauna.
The A. setigera colonies were collected at low tide, a foot or two below water level,
by gathering likely looking Chondrus and Ascophyllum algae off the boulder.

The Woods Hole A. setigera specimens were found growing in close associa-
tion with the following animal forms : Folliculina, Vorticella, Sycon, Obelia,
Sertularia, other hydroids, Bowerbankia gracilis, Bugula flabellata, Crisia eburnea,

MARINE BRYOZOA. I 203

Hippothoa hyalina, Pedicellina ccrnua, and Stephanosella biapcrta. The Aever-
rillia autozoids, and in some instances stolons, had a few Folliculina, Vorticella, or
Pedicellina, growing on them. The A. set ig era colonies grew on hydroid stems and
on the same algal thalli (Chondnis and Ascophyllum) as Bugula flabellata,
Hippothoa hyalina, Crisia, and the other Bryozoa.

Aeverrillia setigera has been collected from varying depths, from one or two
feet below tide mark (present author) to much greater depths (other writers).
Kirkpatrick found specimens at depths of 5l/2 and 27 fathoms; Thornely (1916),
at 7 fathoms; Hastings (1932), at 8 to 15V2 fathoms; Marcus (1937), at 17
meters ; while Harmer found specimens at greater depths : 0 to 40 meters, 55
meters, 59 meters and 118 meters.

This Bryozoan grows on the following types of substratum : 1 , on broken shells
(Kirkpatrick, 1890a) ; 2, on stems of Idia pristis (Thornely, 1916) ; 3, on stems
of hydroids and Bryozoa (Osburn, 1940) ; 4, on stems of Nellia oculata Busk
(Hincks, 1887); 5, on hydroids and the following Bryozoa: Bugula, Catenicella
and Valkeria atlantica, which were dredged from areas whose bottom consisted of
such materials as mud, sand, hard coarse sand, coral, shells, and stones (Harmer,
1915) ; and 6, on hydroids like Obelia and algae like Chondrus and Ascophyllum,
in close association with many other already mentioned animal forms (present
paper).

DESCRIPTION OF SPECIES

The status of Bryozoa as an entire group is still an unsettled problem. It has
been considered a Phylum, a Sub-phylum and a Class. Each category has its
earnest and qualified supporters. With this in mind the following taxonomy of
the Aeverrillia species, patterned after the work of Dr. Marcus, is given :

BRYOZOA Ehrenberg 1831

Class ECTOPROCTA Nitsche 1869
Order GYMNOLAEMATA Allman 1856
Sub-order CTENOSTOMATA Busk 1852
Group STOLONIFERA Ehlers 1876
Family Valkeriidae or Mimosellidae ?
Genus Aeverrillia Marcus 1941
Species setigera Hincks 1887

The classification of Aeverrillia setigera has undergone a few changes since its
original description by Hincks in 1887. Its generic names were Buskia, Hippur-
aria, and now Aeverrillia. The latter genus was erected in 1941 by Dr. Marcus
in honor of A. E. Verrill.

The question regarding the family into which it should be placed is set forth by
Marcus (1941, p. 147) thus: "Aeverrillia does not need a new family; the genus
can be placed in the Valkeriidae or perhaps in the Mimosellidae as now enlarged
by Bassler (1935, p. 8)." Earlier the species had been placed among the Triticel-
lidae, the Buskiidae, and eventually into the Valkeriidae.

The colonies' are delicate yellowish or very pale amber colored transparent
traceries closely adherent to various living and non-living submerged objects. They
are barely big enough to be seen with the unaided eye. They consist of paired

204 MARY DORA ROGICK

individuals connected by slender stolons. The stolons and individuals are chitinized
and firm-walled. The stolons especially have a well thickened wall.

Bryozoa exhibit polymorphism. The Aeverrillia colony consists of three types
of structures or possibly individuals, namely stolons, peduncles, and autozoids.

In the colony there is a main or primary axis or stolon and lesser (secondary and
sometimes tertiary) stolons (Fig. 7). The lesser stolons are more apparent in older
colonies than in young ones.

These stolons, according to Dr. Marcus, are composed of kenozooecia. The
long slender tubular kenozooecia of each stolon grow longitudinally and are
attached end to end. Those of the secondary stolons have their origin at the sides
of the primary stolon usually with a peduncle intervening between the primary and
secondary stolons. The tertiaries have their origin at the sides of the secondaries,
likewise usually with an intervening peduncle. Some stolons appear to arise directly
from other stolons without an intervening peduncle (Fig. 8). Also, one of a pair
of opposite stolons may arise from a stolon without the intervention of a peduncle
while its partner may have a peduncle between it and its parent stolon (Fig. 8).
Whether this barrenness of one stolon may be a temporary or a permanent condition
is uncertain.

The primary and secondary stolons are at right angles, approximately, to each
other but it is difficult to say the same about the tertiaries because the latter are
sometimes twisted, gnarled, and not often found running in a straight line because
of the limited area of the substratum on which the specimens grow. The secondary
stolons usually originate in pairs, one stolon on each side of the primary stolon
and directly opposite the other, growing away from each other.

The primary stolons, possibly because they are older, have thicker walls than
the secondary stolons. The primaries are also somewhat straighter than the
secondary and tertiary stolons but that again may be due to the limited substratum.
Anastomoses occur occasionally, especially where there are tertiary and secondary
stolons over a crowded or limited substratum. Hincks suggested the possibility of
anastomosis of branches.

Primary stolons are very closely and entirely adherent to the substratum which
in many cases proves to be a hydroid stem or Chondrus or Ascophyllum thallus.
The primaries follow the stems or thalli in a fairly straight line for some distance.
The secondaries and tertiaries must find what surface they can. Some of the
lesser stolons look as if they are not necessarily attached along their entire length.

Generally the kenozooecia of the stolons are slightly enlarged distally at the
point of origin of the lateral kenozooecia or peduncles. Transverse uniporous
septa mark the proximal and distal limits of the kenozooecia along the stolons (Figs.
2 and 10). There are septa also at the points of origin of the lateral branches on
the main stolons (Figs. 16 and 18). The region of the septum is sometimes
referred to as the node and the stretch of stolon between two transverse septa, as
the internode.

Stolon length is variable (Figs. 7 and 9, Table I). Some secondary stolons
are short, some long. Some tertiary stolons are considerably longer than the
primaries or than some of the secondaries. Stolon diameter is given in Table I.

The stolons under low power observation (75 X magnification) appear empty or
tubular but under higher magnification (430 X ) a cellular lining membrane is
evident within them.

MARINE BRYOZOA, I

205

TABLE I

Measurements of Massachusetts specimens of Aeverrillia setigera

Part

Number of
readings

Maximum

Minimum

Average

Refer to
Figs.

A. Length or height of furled setig-
erous collar

0.602 mm.

0.440 mm.

0.531 mm.

6, 17

B. Diameter at distal end of un-
furled setigerous collar

0.537 mm.

0.370 mm.

0.440 mm.

C. Diameter at the basal, proxi-
mal end of the setigerous collar

0.110 mm.

0.059 mm.

0.083 mm.

6, 17

D. Length of orificial spine

0.259 mm.

0.141 mm.

0.204 mm.

E. Diameter of extruded vestibu-
lar membrane

0.111 mm.

F. Length of extruded vestibular
membrane

0.321 mm.

G. Length of tentacular sheath

0.237 mm.

H. Diameter of tentacular sheath

0.096 mm.

0.074 mm.

0.085 mm.

6, 11

I. Autozoid width at widest part

0.212 mm.

0.170 mm.

0.185 mm.

J. Autozoid length from base of
zoid to base of orificial spines

0.592 mm.

0.481 mm.

0.552 mm.

K. Stolon diameter, at the normal
thickness, not the swollen area
of the stolon

0.049 mm.

0.015 mm.

0.027 mm.

La. Length of shorter lateral surface
of the clasping processes

0.179 mm.

0.043 mm.

0.110 mm.

Lb. Length of longer lateral surface
of the clasping processes

0.182 mm.

0.077 mm.

0.119 mm.

M. Width of stolon at most swollen
part, near node

0.051 mm.

0.034 mm.

0.040 mm.

N. Length of peduncle

0.170 mm.

0.068 mm.

0.114 mm.

O. Diameter of peduncle

0.071 mm.

0.039 mm.

0.058 mm.

P. Length of internode

1.013 mm.

0.294 mm.

0.658 mm.

S. Number of tentacles

6, 13, 14

T. Number of setae in setigerous
collar

206 MARY DORA ROGICK

PLATE I

All figures except Figures 2 and 6 have been drawn with the aid of a camera lucida. All
are of Aevcrrillia setigera.

FIGURE 1. A chitinized sconce of the proventriculus, seen from the lumen side. Note the
converging rows of teeth. Drawn to the same scale as Figure 10. This and Figures 3, 4, and 5
are from gizzard remains found in empty, degenerated autozooecia.

FIGURE 2. Detail of the uniporous septum which occurs along the stolons.

FIGURE 3. Latero-basal view, from the concave side of the chitinized gizzard sconce. All
the softer parts of the gizzard have disintegrated, leaving only the hardened plate or sconce.
Drawn to the same scale as Figure 10.

FIGURE 4. Side view of a somewhat flattened chitinized gizzard sconce. Drawn to the
same scale as Figure 10.

FIGURE 5. Side view of a chitinized gizzard sconce of the more usual shape. Some of the
teeth are darker than others. Drawn to the same scale as Figure 10.

FIGURE 6. A diagram showing several things : the relation between the open or unfurled
setigerous collar, the eight tentacles, three of the four zooecial spines around the orifice and the
lettered areas A through G along which measurements for Table I have been made. The same
letters are found listed in the first column of the Table.

Line A stands for the length or height of the setigerous collar. It was measured only when
furled or very slightly unfurled.

Line B represents the diameter of an unfurled setigerous collar, at its distal, expanded end.

Line C represents the diameter at the basal or proximal end of the setigerous collar.

Line D represents the length of the orificial spine.

Line E represents the diameter of the vestibular membrane and the area it encloses.

Line F represents the length of the vestibular membrane.

Line G represents the length of the tentacular sheath or the distance between the lophophore
and the base of the setigerous collar, C- — C.

FIGURE 7. Part of an old zoarium or colony showing the growth habit, anastomosis of
branches (AN), primary stolon (P. ST.) and secondary stolon (S.ST.). All the autozooecia
are empty of polypides. One (Z) has the setigerous collar in place yet but has no polypide.
Some of the zooecia have two or three acuminate processes (B.P.). Measurements of these
acuminate processes were made along two surfaces, the shorter (La) and the longer (Lb).
These figures are to be found in Table I. The "membranous" area mentioned by Hincks is not
very plain on most specimens. However, there is a hint of it in the second and fourth zooecia
from the top. Drawn from freshly collected material on Aug. 14, 1944, and to the scale shown
at its base.

FIGURE 8. An autozooecium attached to a very long secondary stolon. The location of
certain measurements mentioned in Table I is indicated on the drawing.

Line I represents the diameter of the autozoid.

Line J represents the length or height of the autozoid exclusive of spines.

Line K represents the diameter of the stolon along most of its length and not at the swollen
areas.

Line M represents the diameter of the stolon at the slightly swollen node region.

Line O represents the height or thickness of the peduncle.

The faintly curving line along the autozooecium suggests the location of the "membranous"
area. The scale below belongs with this sketch.

FIGURE 9. Part of a colony showing four autozoids growing quite regularly in pairs, on
peduncles at opposite sides of the primary stolon. Two are empty, the third has a setigerous
collar and the fourth has a living polypide within. A darker gizzard is evident within the last.
The scale directly below belongs to this colony.

FIGURE 10. The section of the stolon showing a septum and the swollen part represented
by M in Figure 8. This is the region of the node. Drawn to the scale directly below. Figures
1, 3, 4, 5, and 11 also are drawn to this scale.

FIGURE 11. Part of an autozoid showing the basal region of the setigerous collar through
which is lightly indicated the tentacle-bearing lophophore and tentacular sheath. At the base of
the setigerous collar are placed two small letters H which represent the width of the tentacular
sheath. The measurements are found in Table I. Below the setigerous collar is the vestibular
membrane through which are visible muscle fibers. Drawn to the same scale as Figure 10.

MARINE BRYOZOA, I

207

PLATE I

208 MARY DORA ROGICK

The second type of structure or possibly individual (?) in A. se tig era is the
peduncle, so designated by Marcus (1937, p. 142). This is a short much swollen
segment generally placed between stolons which are at right angles to each other
and found at the base of the autozoids (Figs. 8 and 16). It originates from a
stolon and gives rise to a stolon and an autozoid. It is cut off from the stolons and
autozoid by a uniporous septum. A peduncle is more swollen and of shorter length
than the stolon kenozooecium and has a lining membrane. In one instance there
appeared a few transverse fibers inside a peduncle.

The third type of individual in an A. sctigera colony is the autozoid. It arises
from the peduncle. The autozoids are just big enough to see with the unaided eye.
Harmer (1915, p. 87) gave their length as 0.48-0.55 mm. and Osburn (1940, p.
343) as 0.50-0.60 mm. Their width was given as 0.18 mm. (Harmer, 1915 and
Osburn, 1940). Measurements of the Massachusetts specimens are given in
Table I.

The autozoids occur in pairs bilaterally placed with respect to the primary stolon
(Fig. 9). Where secondary stolons are well developed the autozoids occur in the
same manner along the secondary stolon. Occasionally one of the paired autozoids
is missing but a stub of its peduncle or a stolon may be present in its place (Fig. 8).
These paired autozoids are not truly parallel but converge slightly basally as shown

PLATE II

All figures are drawn with the aid of a camera lucida and are of Aeverrillia sctigera.

FIGURE 12. A part of the unfurled setigerous collar, showing the delicate transparent mem-
brane which folds like a fan. Its stiff supporting ribs or setae are transparent also. Drawn
to the scale at left.

FIGURE 13. An autozoid in which a very young polypide is growing. A characteristic
setigerous collar is not yet present although its Anlage (SC) is visible. Eight tentacles can be
counted. The digestive tract is small. A gizzard or proventriculus is present in it. Drawn
from living material on August 13, 1944, to the scale shown directly below.

FIGURE 14. Another young autozoid but slightly older than that of the preceding figure.
Drawn to the same scale.

FIGURE 15. View of a mature autozoid showing an almost completely retracted polypide,
a very long folded setigerous collar partially withdrawn, the U-shaped digestive tract twisted
around in the lower half of the zooecium. The gizzard (GZ) is oriented in such a manner that
one is looking along its vertical axis. Some of the body wall and polypide musculature is shown,
particularly the circularly arranged parietal muscles (PM). The acuminate process is barely
visible. Drawn to the same scale as Figure 12.

FIGURE 16. A partly retracted autozoid. The tentacle tips are just barely visible in the
dark mass at the base of the spine-bearing processes. The somewhat indistinctly depicted diges-
tive tract is in the basal part of the zooecium. Only a part of the autozoid at left is shown.
The scale directly above the setigerous collar applies to this figure.

FIGURE 17. A folded setigerous collar showing typical twisting of the supporting ribs or
setae. The transparent membrane is faintly indicated at the distal end. Drawn to the scale
directly below.

FIGURE 18. A young autozoid is shown at left. Only a part of the right one is included.
The young polypide has eight tentacles and a U-shaped digestive tract. The setigerous collar
is not visible but its Anlage (SC) is present. The vestibule (V) and the parieto-vaginal muscles
(PVM) are plain. Line N represents the length of the peduncle which bears the autozoid.
Measurements of it are given in Table I. Drawn to the same scale as Figure 13, on Aug. 13,
1944, from fresh material.

FIGURE 19. Three of the four chitinized gizzard sconces. The teeth are darker than the
rest of the disc in this particular case. Muscle fibers encircle the cluster of four sconces and
are here indicated by horizontal or parallel lines. Drawn to the scale at left.

MARINE BRYOZOA, I

209

210 MARY DORA ROGICK

in Figure 9. They are not upright but are recumbent at an angle close to the
substratum. The basal part rests directly on the substratum, or is very close to it,
while the distal part is free. The autozoids are somewhat elongate ovate with the
broad end attached. The side nearest the substratum and the inter-autozoidal
stolon is slightly flatter than the opposite side. At its point of origin the autozoid
may be globose as in Figure 14 or slightly "stemmed" as in the right-hand individual
of Figure 18.

The lower half of the autozoid is swollen slightly. From it arise from one to
four, usually two, acuminate clasping processes (Figs. 7 and 16), which were called
"tubular adherent processes" by Hincks (1887, p. 128) and "spines" by Harmer
(1915, p. 87). They are placed obliquely on the zoid. They may touch the
stolons or the neighboring autozoid or else cling to the substratum without touching
either the adjacent autozoid or the stolon. Colonies in place on hydroids show some
of these clasping processes curling around the hydroid stems, closely adherent.
These clasping processes are hollow and not separated by any sort of septum from
the rest of the zoid.

Hincks (1887, p. 127) described a large aperture closed by a membranous wall
on the greater part of the ventral side of the autozoid. It is difficult to see in the
Massachusetts specimens although indications of it are present in Figures 7 and 8.
In Figure 7, it is evident on the second and fourth autozoids from the top. More-
over, it appears chitinized rather than membranous.

The distal tapering end of the autozoid has four spine-bearing processes (basal
segments or flaps). Occasionally more than four flaps may occur. Harmer
(1915, p. 88) reported a specimen with eight. This condition however is very
infrequent. These flaps are arranged around the zooecial orifice through which
the setigerous collar may be protruded.

The position of these distal triangular flaps is not rigidly, immovably fixed.
The line of bending is at the base of the triangle. Sometimes the flaps may be
flexed inward so that their spines may cross each other above the orifice as in the
top left-hand zoid of Figure 9, or in Figure 16. This is the usual position when
the setigerous collar is withdrawn into the autozoid. When the setigerous collar
is out the flaps are bent outward as in Figure 6. This is the condition also in many
empty zooecia. Whether there are any muscle fibers controlling the movement of
these flaps was impossible to determine from the material at hand.

The flaps are more heavily chitinized than the surrounding zooecial wall. The
difference is quite noticeable.

The apex of a triangular flap is rounded in all views. A sharply tapering,
slightly irregular orificial spine is set shallowly into this rounded area. The spine
is hollow, but so far as it is possible to determine its cavity is not continuous with
the cavity of the flap but is cut off by a septum. In Porto Rican specimens the
spines measured 0.20-0.30 mm. (Osburn, 1940, p. 343). Measurements of Massa-
chusetts specimens are given in Table I.

The setigerous collar is long and very slender when furled. Harmer (1915,
p. 88) gives its length as 0.46 mm. and its breadth at the distal end as 0.130 mm.
This last figure is undoubtedly of a partly furled individual. The dimensions of
the Massachusetts specimens are included in Table I.

The setigerous collar can be protruded clear out of the autozoid (Fig. 6). On
the other hand, it also can be completely withdrawn into the autozoid. In fact it

MARINE BRYOZOA. I 211

can be pulled in so far that its uppermost or distal tip is halfway down inside the
zoid. There are muscular fibers attached to its base (Fig. 11). When it is com-
pletely withdrawn the tentacles are below it. When it is protruded and expanded
die tentacles are within its circle of setae (Fig. 6).

Hincks (PI. XII, Fig. 13), Harmer (PI. V, Fig. 9), Marcus (1937, PI. XXIX,
Fig. 76) and the present writer (Figs. 6, 11, 15, and 17) have pictured the peculiar
spiral twisting of the setae of the collar. The setae reinforce a delicate, colorless,
transparent membrane which folds neatly like a fan along scarcely discernible creases
between adjacent setae, when the collar is being withdrawn (Figs. 6, 12, and 17).
The setae or ribs supporting the collar are extremely regular in diameter from base
almost to the very tip.

The collar is often found in excellent condition even when all the zoid contents
except the zooecial wall have disintegrated.

In young zoids as represented in Figures 13, 14, and 18 the setigerous collar is
not yet completed but is represented by a mass of germinative tissue, SC, which
temporarily forms a flexible canopy above the tentacles, at the bottom of the
vestibule.

The vestibule is the cavity down which the setigerous collar travels when being
withdrawn. Its wall is formed by a soft vestibular membrane, to which are
attached a number of fibers which constitute the parieto- vaginal muscles. The
vestibular membrane is shown withdrawn or introverted in Figure 18 and extruded
in Figure 6.

The circular lophophore bears eight tentacles (Figs. 6, 13, and 14). This
number is in agreement with the statements of Harmer and Marcus.

The tentacles, when retracted, are pulled into the introverted tentacular sheath
in a manner characteristic of the Bryozoa (please compare Figs. 6 and 18).

They surround the entrance to the digestive system which is a U-shaped tract
consisting of pharynx, esophagus, proventriculus, stomach and intestine. The
most interesting features about the tract are the great length of the esophagus and
the presence of a muscular and chitinized proventriculus or gizzard between the
stomach and esophagus.

The proventriculi of various species of Biiskia or Aeverrillia are illustrated in
papers by Osburn and Veth (1922, Plate I) and Marcus (1941, Plate X, Figs. 44B
and 45). Marcus figures the gizzard of both A. arinata and A. setigera. How-
ever, the proventriculus of the Massachusetts specimens of A. setigera resembles
that of his A. arinata as much as it does that of his A. setigera.

The proventriculus of the Massachusetts A. setigera is a compact, rounded organ
consisting of four conical chitinous sconces capping the internal epithelium. A
wide band of circular muscle fibers surrounds these four sconces (Fig. 19). An
end view of the proventriculus showing the relation of the four sconces to each other
is pictured clearly in Figure 15 and suggested in Figures 9 and 18. A side view,
showing the relation of the circular musculature to the sconces and the relative
position of the proventriculus in the polypide, is depicted in Figures 13, 14, and 16.
A detailed picture of the arrangement of the chitinous and sometimes brown-colored
denticles on the sconces appears in Figures 1, 3, 4, 5, and 19. The denticles seem
to have a definite arrangement in several roughly V-shaped rows. They are of
various sizes. Their color ranges from pale yellow to brown. The shape of each
sconce at the base ranges from a broad ellipse to a circle. In side view the sconce

212 MARY DORA ROGICK

may appear globose, conical, or even slightly flattened, except for the projecting
teeth. Careful inspection of an old or empty colony may occasionally reveal
sconces of degenerated polypides still within the otherwise empty zooecia. Because
the sconces are usually transparent, pale yellow, and small it is easy to overlook
them. In degenerating polypides the gizzard can usually be distinguished as the
central part of a dark mass of degenerating material.

The relations of the stomach and intestine to the gizzard and to the lophophore
can be seen in Figures 13, 14, and 18. In these three instances the digestive tract
is empty. In a mature feeding individual the digestive tract is considerably longer,
as a study of Figure 15 will show. The intestine opens outside the circle of tenta-
cles— a characteristic of the Ectoprocta.

The musculature of the lower half of the autozoid was difficult to study partly
for lack of sufficient living material and partly because in a mature zoid the digestive
tract occupies so much of the interior. However Figure 13 does show a sugges-
tion of a band of retractor muscle fibers attached to the base of the tentaculer crown
or the upper part of the digestive tract.

The other major muscles attaching to the body wall are the horizontally or
circularly arranged parietal muscles. Harmer (1915 p. 88) states that three
groups of parietal muscles are visible in his specimens. In the Massachusetts
specimens it appears as if there are four groups (Fig. 15).

In a few near-empty zooecia, from which the musculature, tentacles, setigerous
collar, and digestive tract were missing but which had a brown body (a mass of
dedifferentiating or degenerating tissue) in the upper half of the zooecium, was
noticed a rather peculiar globular membranous sac attached to the base of the inte-
rior of the autozoid, in the vicinity of the septum which separates the autozoid from
the peduncle. This globose mass was hollow. Its wall was soft membranous, and
turgid. It is not figured here. Its appearance and position suggest one of two
possibilities: 1, it may be a regenerating mass which would give rise to a new poly-
pide within the old zooecium ; or 2, it may represent the remains of a degenerating
polypide, exclusive of the brown body which was already evident in the upper half
of the zooeckim. In the fresh water Bryozoa, when polypides of a colony degen-
erate, sometimes the wall of the colony forms a hollow membranous sac which
may either degenerate completely or give rise to a new colony (Rogick, 1938; p.
197).

In studying any form, measurements are extremely helpful. Therefore, as com-
plete a set of measurements of A. setigera as was possible was made and is arranged
in Table I. The letters and parts A to P are clearly indicated in the drawings of
Plates I or II.

DISCUSSION

Aeverrillia setigera seems very widely distributed circumtropically. It has
been reported previously from such widely scattered localities as north and east of
Australia, China Sea, Gulf of Bengal, Malay Archipelago, Suez Canal, Porto Rico,
Brazil's Bay of Santos, etc., whose latitudes range from approximately 24° S to
31° N. The present report extends its range to 41°38' N. Latitude. A recent
report (Hutchins, 1945; from Long Island Sound) cites its occurrence slightly
south of the present paper. In spite of this extensive range the number of reports

MARINE BRYOZOA, I 213

on the occurrence of this species have not been too numerous : Harmer, Hastings,
Hincks, Hutchins, Kirkpatrick, Livingstone, Marcus, Osburn, Thornely, and the
present writer.

The Massachusetts specimens agree essentially in measurements and appearance
with those found in more southerly waters (Gulf of Bengal, South America, and
Porto Rico) by previous workers.

Because of their small size and inconspicuous appearance they are easily over-
looked when collecting. Very little is known of their behavior, embryology, life
history, and physiology. A study should be made of these as well as of colony
degeneration, regeneration, rate of growth, development of the proventriculus and
setigerous collar, the location and development of the reproductive system, and the
nature of the larva. All the work done so far on this form has been of taxonomic
nature. The present paper has added a more complete account of the anatomy,
included measurements of a number of parts hitherto unmeasured and added a
more complete series of diagrams than have existed previously for this species.

SUMMARY

1. Aeverrillia sctigera was found at Woods Hole and at New Bedford, Mass.
This extends its northerly range to 41°38' N. Latitude.

2. The Massachusetts specimens agree closely in appearance and measurements
with specimens from more southerly waters of the Gulf of Bengal, Malay Archi-
pelago, South America, and Porto Rico.

3. Measurements of many structures or parts not measured by other workers
are here included.

4. The species has been more fully illustrated.

5. The species did not seem to be abundant in the localities from which it has
just been reported.

LITERATURE CITED

BASSLER, R. S., 1935. Fossilium Catalogus. I. Animalia, pars 67 : Bryozoa. 's-Gravcnhage.

p. 1-229.
HARMER, S. F., 1915. The Polyzoa of the Siboga Expedition, Part 1. The Entoprocta, Cteno-

stomata and Cyclostomata. Siboga-Expcditie, Mongr. 28a, Livr. 75: 1-180; PI. 1-12.
HASTINGS, A. B., 1927. Report on the Polyzoa of the Suez Canal. Trans. Zool. Soc. London,

22 (pt. 3, no. 8) : 331-354.
HASTINGS, A. B., 1932. The Polyzoa, with a note on an associated hydroid. Great Barrier Reef

E.rped. 1928-29, Sci. Reports, 4 (12) : 399-458, PI. 1. Brit. Mits. Nat. Hist.
HINCKS, T., 1887. On the Polyzoa and Hydroida of the Mergui Archipelago collected ... by

Dr. J. Anderson. Jour. Linn. Soc. Zool, 21: 121-135; PI. 12.
HUTCHINS, L. W., 1945. An annotated check-list of the salt-water Bryozoa of Long Island

Sound. Trans. Conn. Acad. Arts and Sci., 36: 533-551.
KIRKPATRICK, R., 1890a. Hydroida and Polyzoa. Reports on the Collection made in Torres

Straits by Prof. A. C. Haddon 1888-1889. Sci. Proc. R. Dublin Soc.. n.s., 6: 603-625;

PI. 14-17.
KIRKPATRICK, R., 1890b. Report upon the Hydrozoa and Polyzoa collected by P. W. Bassett-

Smith . . . during the survey of the Tizard and Macclesfield Banks in the China Sea,

by H.M.S. "Rambler." Ann. Mag. Nat. Hist., ser. 6, 5: 11-24; PI. 3-5.
LIVINGSTONE, A., 1927. Studies on Australian Bryozoa, No. 5. A checklist of the marine

Bryozoa of Queensland. Rcc. Austral. Mus., 16 (1) : 50-69.
MARCUS, E., 1937. Bryozoarios marinhos brasileiros, I. Vn'w. Sao Paulo, Bol. Faculd. Filos.,

Cienc. e Letr., I. Zool., 1 : 3-224 ; PI. 1-29.

214 MARY DORA ROGICK

MARCUS, E., 1938. Bryozoarios marinhos brasileiros, II. Univ. Sao Paulo, Bol. Faculd. Filos.,

Cienc. e Lctr., IV. Zool, 2: 1-196; PI. 1-29.
MARCUS, E., 1939. Briozoarios marinhos Brasileiros. III. Unii>. Sao Paulo, Bol. Faciild.

Filos., Cienc. e Letr., XIII. Zool., 3: 111-354; PI. 5-31.
MARCUS, E., 1941. Sobre os Briozoa do Brasil. Univ. Sao Paulo, Bol. Faculd. Filos., Cienc. c

Letr., XXII. Zool., 5 : 3-208 ; PI. 1-18.
OSBURN, R. C., 1933. Bryozoa of the Mount Desert Region. Biol. Surv. Mt. Desert Region,

Part 5: 291-385; PI. 1-15.
OSBURN, R. C., 1940. Bryozoa of Porto Rico with a resume of the West Indian Bryozoan

Fauna. Sci. Surv. Porto Rico and the Virgin Islands, 16 (3) : 321^86; PI. 1-9. N. Y.

A cad. Sci.
OSBURN, R. C, AND R. M. VETH, 1922. A new type of Bryozoan gizzard, with remarks on the

Genus Buskia. Ohio Jour. Sci, 22 (6) : 158-163.
ROGICK, M. D., 1938. Studies on Fresh Water Bryozoa. VII. On the viability of dried stato-

blasts of Lophopodclla carteri var. typica. Trans. Amer. Micr. Soc., 57 (2) : 178-199.
THORNELY, L. R., 1905. Report on the Polyzoa ... at Ceylon, in Herdman's Kept. Ceylon

Pearl Oyster Fish., vol. 4, Supplement. Report, 26: 107-130.
THORNELY, L. R., 1916. Report on the Polyzoa collected at Okhamandal in Kattiawar in 1905-

1906. Hornell, Kept. Gov. Baroda Mar. Zool. Okhamandal, Part 2: 157-165.

STUDIES ON FRESH-WATER BRYOZOA. XVI. FREDERICELLA
AUSTRALIENSIS VAR. BROWNI, N. VAR.

MARY DORA ROGICK

Marine Biological Laboratory and College of Nczv Rochelle

TABLE OF CONTENTS

PAGE

Introduction 215

Fredericella australiensis (emend.)

Description 216

Discussion 217

Growth habit 217

Dissepiments or septa % 217

Keel .' 217

Zooecial tube 217

Ectocyst 218

Polypide 218

Tentacular crown 218

Sessoblasts 224

Distribution 225

Key to varieties 226

Table I 219

Fredericella australiensis var. broivni

, Description and discussion 226

Table II 225

Summary 227

Literature cited 228

Plate I, Explanation 220

Plate II, Explanation 222

INTRODUCTION

This study deals with a Fredericella, F. australiensis Goddard 1909, which was
reduced to variety rank and to which were added two other varieties, one of them
new. The new variety is here named F. australiensis var. browni, in honor of Dr.
Claudeous J. D. Brown of the Michigan Department of Conservation, Ann Arbor,
Michigan, who most generously turned over the material to the author for further
study.

The specimens were collected in fair abundance on August 3, 1942, from rocks
in an alkali pond about three miles northeast of Church Butte, Uinta County,
Wyoming, U.S.A., by Dr. Henry van der Schalie of the University of Michigan, at
Ann Arbor.

The writer wishes to express her deep appreciation to both Dr. van der Schalie
and Dr. Brown for the opportunity to examine the specimens and to make the
present study.

215

216 MARY DORA ROGICK

Observations were made on preserved material which was dissected and on
preserved material which had to be imbedded and sectioned. No living specimens
were available. Dissection and sectioning were necessary to determine tentacle
number, diameter of various parts, and internal structure since the zooecial wall
was too opaque to permit ready observation of internal structures.

It was necessary to create a new variety, var. broivni, for the Wyoming form
because it resembled very closely in some respects and differed somewhat in other
respects from two other forms known heretofore as Fredericella sultana subsp.
transcaucasica AbricossofT 1927 and Fredericetla australiensis Goddard 1909.

It was necessary to reduce the original F. australiensis of Goddard to variety
rank, making it F. australiensis var. australiensis and to add to it two other varieties
because the three forms so closely resembled each other and differed noticeably
from the long established species of Fredericella sultana. Consequently, the former
F. australiensis Goddard and the F. sultana subsp. transcaucasica Abricossoff become
varieties under the emended F. australiensis, namely, F. australiensis var. australi-
ensis and F. australiensis var. transcaucasica. The finding of the Wyoming speci-
mens adds a third variety, browni to this emended species.

FREDERICELLA AUSTRALIENSIS, EMENDED
Description

The colony is attached along the bases of a number of zooecia whose tips become
erect at the distal end and eventually give rise to upright branches which usually do
not fuse into a solid mass but which form rather openly branched tufts (Fig. 4).
Branching is antler-like or very roughly dichotomous. Septa or dissepiments are
absent. Zooecial tubes are slightly wider than those of F. sultana. The degree of
incrustation of the ectocyst varies from almost none in var. transcaucasica to a
considerable amount in var. brozvni and var. australiensis. Floatoblasts are absent.
Sessoblasts are rounded or very broadly elliptical, not reniform or very elongate
as those of F. sultana. They are shorter and broader than those of F. sultana.
More exact data or measurements will be given in the "Discussion" section. The
terms sessoblasts and floatoblasts have been defined in the author's Study XIV. The
F. australiensis polypides are shorter and stubbier than those of F. sultana and are
restricted to the zooecial tips whereas those of the latter species are longer and
extended further down into the zooecial tubes. The tentacle number is larger in
F. australiensis than in F. sultana. The former has approximately 24 to 30 ten-
tacles while the latter has about 17 to 24 tentacles. The lophophore is decidedly
elliptical in var. australiensis. In the other two varieties it is uncertain whether the
lophophore is nearly circular or definitely elliptical. Living specimens are necessary
to determine this point. However, the lophophore is not horseshoe-shaped, except
only in the retracted condition. An epistome is present.

Fredericella australienisis is characterized by the rounded, broadly elliptical
shape of the sessoblasts, the larger number of tentacles and greater zooecial tube
diameter, all admittedly somewhat variable characters but unfortunately almost the
only ones, barring nature of colony growth and degree of incrustation which in them-
selves are variable, on which one can make a distinction in this genus.

FRESH-WATER BRYOZOA, XVI 217

Discussion

Groii'th habit

Fredericella australiensis and F. sultana have a similar growth habit and colonial
appearance. The mode of branching is similar. Zoids are adherent for a distance
then give off upright branches. Branching is antler-like or very roughly dichoto-
mous in both.

Dissepiments or septa

Allrnan (1856, p. 112) says of F. sultana, "At the origin of the branches there is
frequently found a more or less perfect septum." His Plate IX, Figure 3, shows
an imperfect or partial septum, i.e., a septum with a hole in it. This chitinous
septum is located at the commencement of a branch. Kraepelin (1887) calls the
dissepiments rudimentary. In F. australiensis there seem to be no septa at the
start of the branches. Goddard (1909, p. 490) finds none in var. australiensis.
Abricossoff (1927b, p. 88) shows none in his Figure 2 of transcaucasica, and
there appear to be none in var. browni (present study).

Keel

There seems to be relatively little difference between F. sultana and F. australi-
ensis in this character. The zooecial tubes are cylindrical or nearly so in younger
F. sultana zooecia and keeled in older specimens, so there occur specimens with
and without a keel. This is true also of F. australiensis — some individuals may
have and others may lack a keel.

Zooecial tube

The two species differ very slightly in the shape of the zooecial tubes, when
viewed in cross section. The F. sultana tubes vary in cross section from cylindrical
in unkeeled specimens to somewhat pear-shaped in keeled ones. In F. australiensis
the tube cross section ranges from an ellipse (in var. brozvni, Figs. 1 and 10) to a
rough triangle (var. australiensis').

There is a greater difference between the two species in width of zooecial tubes.
Those of F. sultana are more slender. The diameter of F. sultana zooecial tubes
of New Rochelle and Lake Erie specimens as given in Study IX (Rogick, 1940,
p. 195) ranged from 0.16 to 0.35 mm. and averaged 0.24 mm. for 44 readings.
Abricossoff (1927b, p. 91) said that in the U.S.S.R. Fredericella sultana the
zooecial tube was not more than 0.4 mm. wide. He placed that as the upper limit
but did not give the minimum nor average measurements for the point in question.
The zooecial tubes of F. australiensis are greater in diameter than those of F. sultana.
Abricossoff (1927b, p. 91) gives the average diameter in transcaucasica as 0.5 mm.
while the present writer gives a range of 0.259 to 0.576 mm. or an average of 0.391
mm. for the most typical region of a var. browni zooecial tube. Thus it would
seem that as regards this particular character, var. browni is somewhat closer to
F. sultana than is var. transcaucasica.

218 MARY DORA ROGICK

Ectocyst

There is little difference in appearance between the two species so far as
chitinized ectocyst is concerned. In F. sultana the degree of incrustation of the
ectocyst may vary to such an extent that the zooecial tubes may be translucent to
opaque, generally favoring the latter. Debris, stone particles and even algae may
attach to it. In F. australiensis the degree of incrustation varies also from ex-
tremely little in var. transcaucasica to the usual "opaque," reasonably well incrusted
amount in the other two varieties. Sand grains and debris form part of the
incrustation. The color of the ectocyst varies from tan to light brown, in F.
australiensis.

Polypide

Kraepelin (1887, p. .99) says that polypides of F. sultana are very long. Allman
(1856, PL IX, Fig. 7) shows such a specimen. In samples observed by various
workers, including the present one, the polypides of this species seemed long and
slender. On the other hand, in F. australiensis, the polypides appear distinctly
shorter and stubbier, and are restricted to the zooecial tips (see Goddard, 1909,
Fig. 12). Since no digestive tract measurements exist for F. sultana it is necessary
to judge the relative length of its tract by studying Allman's and other workers'
drawings. These measurements would vary with the age and condition of nourish-
ment of the polypides.

Tentacular crown

In F. sultana the tentacles are long and slender but no measurements exist for
them so far as can be determined. In F. australiensis the tentacles are generally
shorter and stubbier with the possible exception of var. australiensis. In the latter
variety they measure about one mm. in length and 0.01 mm. in diameter. In var.
browni the tentacles are shorter and thicker. Unfortunately not too many were in
a position to be measured accurately so that one had to depend on the general
appearance of those dissected out of the colonies and on a few which were sectioned
in the proper plane. These ranged from 0.383 to 0.514 mm. in length and from
0.019 to 0.029 mm. in width (Table II). This is shorter and wider than in var.
australiensis. No measurements are available for var. transcaucasica tentacles.
One has to judge them from AbricossofFs (1927b, p. 88, Fig. 2) figure in which
they appear shorter and stubbier than tentacles of his F. sultana (ibid., Fig. 1).

The number of tentacles does not seem to vary as much in Fredericella individ-
uals as it does in those of Pluinatella and Hyalinella. In Hyalinella punctata, the
author (1945, Study XV. p. 69) found that the ancestrula or first polypide of a
colony could be distinguished from successive polypides on the basis of the number
of tentacles. It had about 10± less than successive polypides did. Whether the
same general principle holds for Fredericella and other fresh-water forms could
easily enough be determined by germinating statoblasts of the various forms and
keeping accurate counts of the number of tentacles developed in each zoid.

The tentacle number of the two species of Fredericella is different. In F.
sultana it ranges from 17 to 24, with 20 to 22 being the most common number.
In F. australiensis the number ranges from 24 to 30.

FRESH-WATER BRYOZOA, XVI

219

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220 MARY DORA ROGICK

Previous authors have given ample data on the tentacle number of F. sultana.
Allman (1856, p. 112) states that this species has about 24 tentacles. His Plate
IX, Figure 2, shows 20 to 24 tentacles on various polypicles while his Figure 7
(same Plate) shows 25. Nowhere does he call attention to this large number
however. Hyatt's (1868, p. 220) F. rcg'ma, now a synonym for F. sultana, had
18 to 22. Kraepelin's (1887, pp. 92, 103) specimens had 20 to 22 as a rule but
could also range from 18 to 24. Braem's (1890, p. 11) ranged from 20 to 22, with
one specimen being found which had only 17. Toriumi's (1941, pp. 196-197) had
17 to 23. The present writer has found New Rochelle specimens with 24 (1940,
Study IX, p. 195), Lake Erie specimens showing the full range of 18 to 24, but
usually with 20 to 22 tentacles (1935, Study II, p. 250).

Borg (1937, pp. 272-275) reported the collection of a F. sultana, from the
Sahara region of Africa, which had 24 to 28 tentacles, wider zooecial tubes than the

EXPLANATION OF PLATE I

All figures are of Fredcricclla australiensis var. browni from the Wyoming collection and
have been drawn with the aid of a camera lucida.

FIGURE 1. Cross section through a sand and debris incrusted zooecial tube near the tip,
which at this level contains the retracted tentacular crowns of two polypides. The tentacular
crown at left has been sectioned through the lophophore region at the bases (TB) of the tenta-
cles and through the epistome (EP). The lophophore bears 25 tentacles in this specimen and
their bases (TB) at this level are somewhat triangular. The heavy staining of the nuclei ac-
counts for the darkest wavy "stratum" of the tentacles. The lightly stippled material immedi-
ately on either side of this dark nuclear "line" or "band" is cytoplasmic material. At this level
the surface of the tentacles facing the epistome is ciliated but that is not shown on the drawing.
The tentacular crown at left appears to be horseshoe-shaped but that is because it is in the re-
tracted condition. Such a condition also occurs in a retracted F. sultana polypide (see Braem,
1890, PI. V, Fig. 68). The group of 27 tentacles (T) at right belongs to a second polypide.
The tentacular cell nuclei are more conspicuous on the inner border of each tentacle where the
cells are taller and closer together than on the outer border where the cells are flatter. By inner
border is meant the surface facing the epistome and by outer border is meant the surface at the
periphery of the tentacular crown. That orientation is best noted in the outermost circle of
tentacles. Those within the circle are less regularly oriented. Here again, the cilia have been
omitted from the drawing. The zooecial tube is a somewhat longer ellipse here near the zooecial
tip than at a level lower down along the tube, as shown in Figure 10. The wall of the tube
varies in thickness because of the incrustation. Drawn to Scale A which is 0.073 mm. long.

FIGURE 2. A branch from a colony, showing the zooecial tubes (Z) closely adherent to the
substratum (SM), which in this instance is blacked in. The tips of the zooecia are not generally
attached to the substratum but are free and directed upward. The condition of the tips indicates
. that all the polypides are retracted. Drawn to Scale C.

FIGURE 3. Six sessoblasts (SS) shown inside the thin, translucent, tubular, cellular or mem-
branous endocyst (EN). The ectocyst has been removed from the specimen. The cement ring
is the darkest part of the sessoblast here. Three of the sessoblasts are turned a little so that
one edge shows, but the other does not. The endocyst was torn at the right during dissection
and the right-hand statoblast is partly out of it. Drawn to Scale B, which is 0.3 mm. long.

FIGURE 4. Habit sketch of a part of a colony or zoarium showing the adherent base, the
upright branches and the mode of dichotomous branching. The substratum is shown in black.
When the zooecial tips appear as in this figure their tentacles are either generally retracted or
else the tips may be empty. It is sometimes hard to tell if the colony has polypides within it or
not because the ectocyst is fairly opaque, so that only very dark structures like the sessoblasts
are perceptible with any ease. Since polypide parts are light in color they usually do not show
through the ectocyst but have to be dissected out for study. If a colony has been empty a long
time the zooecial tips may be broken off and then their emptiness, of course, is evident. Drawn
to Scale C which is equivalent to one mm.

FRESH-WATER BRYOZOA, XVI

221

EP TB

I MM

PLATE,!

MARY DORA ROGICK

ordinary F. sultana, and statoblasts which were extremely variable (Table I) and
in many cases rounded or oval. Some of his specimens (Borg, 1937, PI. XVII,
Figs. 2 and 3) look very much like F. sultana and probably are but his Figure 1
(same plate) appears definitely to belong to F. australiensis. Judging by tentacle
number, zooecial tube diameter, and appearance of the pictured statoblast inside
its tubes, it seems to agree favorably with var. browni.

Borg (1937, p. 275) also mentions very incidentally another interesting form
of Fredericella, F. sultana forma major, from the north of Sweden, which has 28
to 32 tentacles and is generally of a greater width (presumably zooecial tube
width). This would be in conformity with F. australiensis. Unfortunately how-
ever, he gives no description, pictures, or dimensions of it so that its status is quite
uncertain. It may either prove a new species of Fredericella or a new variety of
F. australiensis. At any rate it would be worth a fuller investigation.

EXPLANATION OF PLATE II

These are all figures of F. australiensis var. brozvui (from the Wyoming locality) and were
drawn with the aid of a camera lucida.

FIGURE 5. Surface view of the greater jjart of one fairly young completed sessoblast. The
chitinous substance of the valve gradually thins out toward the center which part is the last to
be closed over by the chitin in development. In this specimen the central region was thinnest
and palest in color. Drawn to Scale H.

FIGURE 6. An abnormally shaped sessoblast. There were relatively few mis-shapen sesso-
blasts found in the collection and this was one of them. Its drawing is included as a contrast to
the typical sessoblasts shown in Figures 9 and 11. The sessoblast valves are joined together at
the border in what is sometimes called a cement ring (CR). The sessoblast contains opaque
germinative material (GM) occupying almost all the space between the two capsule valves. The
cement ring is dark amber color while the valves are a paler amber.

FIGURE 7. A tentacular crown dissected from a zooecial tube, from preserved material. It
shows the relative length of the tentacles. The tentacular mass was slightly disarranged during
dissection. Drawn to Scale D whose length is given below the figure.

FIGURE 8. A side or edge view of a sessoblast. The two irregular dark patches (CH) on
one valve are chitinous material which grows on some of the sessoblasts, attaching them to the
substratum, or to the wall of the colony. A face view of a similar growth is shown in Figure 9.
Drawn to the same scale as Figures 9 and 11.

FIGURE 9. A portion of the cellular endocyst tube (EN) enclosing a sessoblast (SS) on
which are growing several irregular or crescent-shaped patches of chitin (CH). The sessoblast
is typical, normal. Drawn to Scale E.

FIGURE 10. A cross section of a zooecial tube taken about midway between the tip and the
base, shown in silhouette. This section is more typical of the elliptical shape of the ectocyst tube
than is Figure 1, which was taken near the tip which housed the broadest part of the polypides.
The irregularity of the zooecial wall is due to the material incrusting it (see Figs. 1 and 14).
Drawn to Scale F.

FIGURE 11. A sessoblast showing the internal germinal mass (GM) shining through the
deep amber-colored translucent capsule. The colors of the rest of the sessoblast at the line of
junction of the two valves are as follows. The outermost stippled ring is dark reddish amber
while the ring shown in black is a very dark brown. These two dark outer bands represent the
cement ring area. The shape of the sessoblast is typical for this variety and species. Drawn to
Scale E.

FIGURE 12. Surface view of a portion of a sessoblast valve which is older than that por-
trayed in Figure 5. A delicate raised chitinous tracery, here shown in black, covers it. Drawn
to Scale G.

FIGURE 13. Surface view of a portion of still older sessoblast valve than shown in Figure 12.
The raised tracery is coarser, darker, and more prominent. Drawn to Scale G.

FIGURE 14. Surface view of ectocyst showing the minute sand grains and other debris im-
bedded in it. Drawn to Scale H.

FRESH-WATER BRYOZOA, XVI

223

• '

.072
MM.

0734 MM.-

.3232 MM.

L.05I MM.

O.I MM.

PLATE II

224 MARY DORA ROGICK

Borg mentions that Kraepelin (1914, reference not available to present author)
has collected specimens of Fredericella from Rhodesia, Africa, which have stato-
blasts which are about one third smaller than ordinary German F. sultana speci-
mens. Nothing is said about the number of tentacles in the Rhodesian form.

The shape of the expanded tentacular crown in F. sultana is nearly circular.
In F. australiensis var. australiensis the lophophore is very definitely elliptical in
shape, measuring 0.23x0.38 mm. In var. brozvni, it can not be said for certain
what the shape is in expanded lophophores since all were retracted in the material
studied. Abricossoff makes no mention of this point in var. transcaucasica. Cross
sections of retracted F. sultana and F. australiensis look similar except that the
latter species has a greater number of tentacles. When the polypides of both
species are withdrawn, their lophophores assume a crescent or horseshoe shape
(Fig. 1 of present study; Braem, 1890, PI. V, Fig. 68; Goddard, 1909, p. 491 and
PI. XLVII, Fig. 5).

Scssoblasts

Statoblasts are extremely important in identification of fresh-water Bryozoa,
but those of Fredericella, Pluniatella, and Hyalinclla often are not entirely adequate
in themselves, especially when present in very small numbers, to determine the
exact variety or sometimes even the species to which they belong. It is necessary
that sufficient specimens be available so that the normal type of statoblast can be
observed, for there is so much variation in shape and size that one can readily be
misled by examination of just one or two lone statoblasts. There is a great amount
of intergradation between statoblasts of different varieties and species. Almost
every worker has rather helplessly commented on the fact, yet has been unable to
find a criterion that is invariable by which to identify the species and varieties.
Statoblasts alone of the above forms are often insufficient for absolute identification.
One should also have the colonies and polypides, living and preserved, in sufficient
quantity to really make accurate identifications.

In Fredericella there is apparently a complete series of intergrading sessoblasts
between F. sultana and F. australiensis. However, the vast majority of the F.
sultana statoblasts are reniform or quite elongated while the majority of the F.
australiensis sessoblasts are more rounded or broadly elliptical in outline.

The extreme dimensions for F. sultana sessoblasts are : length range from 0.27
to 0.57 mm. and width range from 0.139 to 0.37 mm. The minimal figures above
are from some Lake Erie specimens (Rogick, 1935, Study II, p. 250) and the
maximal figures are for some European specimens (Kraepelin, 1887, p. 104). As
a rule, the average length and width figures show that F. sultana sessoblasts are
considerably longer than wide, a fact that can not always be fully appreciated from
lone maximum and minimum figures. The extreme dimensions, so far determined
for F. australiensis sessoblasts, are: length range from 0.331 to 0.470 mm., width
range from 0.267 to 0.367 mm. if var. brozvni and var. transcaucasica (Tables I and
II) are considered, or 0.22? to 0.367 if Dr. Borg's African specimens are included in
these computations and if the African forms should all prove to belong to F. australi-
ensis and not to F. sultana. The reason for the question mark after 0.22 in the pre-
ceding sentence is that this particular measurement may or may not have been of this
species or variety. The average length and width of F. australiensis statoblasts,

FRESH-WATER BRYOZOA, XVI

225

TABLE II

Measurements of Fredericella australiensis var. brownifrom Wyoming

Part or structure

Maximum

Minimum

Average

Number of
readings

A. Sessoblast

1. Total length
2. Total width

0.461 mm.
0.367 mm.

0.331 mm.
0.266 mm.

0.382 mm.
0.316 mm.

69
69

3. Thickness in middle

0.101 mm.

. 4. Cement ring diameter

0.014 mm.

B. Zooecial tube diameter along the longer
of the two transverse axes

0.576 mm.

0.259 mm.

0.391 mm.

C. Tentacles

1. Number

26-27

2. Length
3. Broadest part of the shorter trans-
verse diameter

0.514 mm.
0.029 mm.

0.383 mm.
0.020 mm.

0.451 mm.
0.025 mm.

3
10

4. Longer transverse diameter (at right
angles to preceding measurement)

0.027 mm.

0.019 mm.

0.024 mm.

D. Lophophore retracted within zooecial
tube:

1. Antero-posterior diameter
2. Lateral diameter

0.308 mm.
0.170 mm.

0.147 mm.
0.111 mm.

0.182 mm.
0.133 mm.

8
8

E. Epistome
1. Antero-posterior diameter
2. Lateral diameter

0.019 mm.
0.056 mm.

F. Esophagus
1. Length
2. Width

0.060 mm.

0.051 mm.

0.193 mm.
0.054 mm.

1
3

G. Stomach

1. Length
2. Width

0.653 mm.
0.070 mm.

0.634 mm.
0.066 mm.

0.644 mm.
0.068 mm.

2
2

at least of the broivni variety, show that the statoblasts are more nearly a broad
ellipse than are those of F. sultana. The F. australiensis sessoblasts are generally
slightly flattened on one side and very probably roughened by various markings on
the other, when mature (Figs. 12 and 13). Neither Goddard nor Abricossoff
mention the nature or pattern of the surface markings on their specimens' sesso-
blasts. Variety broivni however had some sessoblasts with markings (Figs. 12 and
13) ; so does F. sultana (Rogick, 1937, p. 102, Fig. 1).

Distribution

Fredericella australiensis has a widely scattered distribution although it has
been reported relatively few times. Its three varieties are distributed as follows.
Variety australiensis occurs in the water supply system at Pott's Hill in New South
Wales, Australia (Goddard 1909, pp. 487-489). Goddard reported that the F.

226 MARY DORA ROGICK

sultana recorded earlier from Australia by Whitelegge is probably his own F.
australiensis. Variety transcaucasica occurs in Lake Madatapeen, Tiflis District,
the Transcaucasus, in the U.S.S.R. (Abricossoff 1927a, p. 308 and 1927b, p. 91).
This variety was collected by B. S. Winograd on July 1, 1915 and later identified
by Dr. Abricossoff. Variety browni occurs in Uinta County, Wyoming, U.S.A.
Some of Dr. Borg's material from rivers in the Sahara region of North Africa is
very likely F. australiensis var. browni. This widens the distribution of F. australi-
ensis to 4 ? continents : Africa ?, Australia, Eurasia, and North America.

Key to Varieties of Frcdericella australiensis

1 (2) Chitinous ectocyst well incrusted with sand grains and debris ; rather opaque 3

2 (1) Chitinous ectocyst very little incrusted; very transparent; zooecia about 0.5 mm. wide;

sessoblasts average 0.315 X 0.47 mm var. transcaucasica

3 (4) Tentacle number 24-28; sessoblast average 0.316x0.382 mm.; zooecial tubes elliptical in

cross section var. browni

4 (3) Tentacle number 28-30; zooecial tubes roughly triangular in cross section

var. australiensis

FREDERICELLA AUSTRALIENSIS VAR. BROWNI, NEW VARIETY
Description and Discussion

This variety is illustrated in Figures 1 through 14. Its measurements are given
in Table II. Its points of difference and resemblance as compared with the other
two varieties are briefly summed up in Table I. Some gaps exist in the information
about this variety and they are : 1, the shape and dimensions of the expanded lopho-
phore and 2, the unavailability of living specimens for a more complete study of
tentacle and polypide size and variation. However, on the basis of the preserved
material available, the following description of the variety can be made.

Variety brozvni has a thin chitinous ectocyst well incrusted with sand grains and
debris (Figs. 1 and 14). It is of light tan color and rather opaque. The opacity
of the zooecia is such that it is possible to see whether the much darker colored
sessoblasts are present, but not whether polypides are present because the light
color of the polypides blends in so well with the color of the incrusted ectocyst.
To determine if tubes contain polypides it is frequently necessary to tear them apart.
Only then are the polypides visible.

Basal zooecia are recumbent or adherent in their more proximal part, with the
tips directed upwards (Figs. 2 and 4). From these arise erect branches (Fig. 4).
The zooecia are generally elliptical in outline (Figs. 1 and 10). Occasionally a
faint keel may be present (Fig. 2) but usually it is not noticeable. The colony
appears upon rocks as a coarse tracery or tufted mass, depending upon the number
of polypides in it. If the number of polypides is small or if the periphery of the
colony is examined there will be located the more adherent members. If the colony
is luxuriantly branched and on a rather limited substratum then it has many more
upright branches. These are not fused together but retain their individuality and
open mode of branching. The zooecia are usually very long (Fig. 4). The
ectocyst has considerable rigidity and firmness. The zooecia are somewhat wider
than in F. sultana. Those of var. browni are not as wide apparently as those of
var. transcaucasica (Table I). The ectocyst is too opaque to be able to see
dissepiments or incomplete septa at the commencement of the zoids even if they

FRESH-WATER BRYOZOA, XVI 227

were present in this variety. Such dissepiments occur in F. sultana. A diligent
search was made through sectioned and dissected F. australiensis var. browni
material but no dissepiments could be found.

The ectocyst is lined with a soft thin transparent membranous endocyst. The
endocyst encloses the polypides and sessoblasts (Figs. 3 and 9).

The polypides of var. browni appear short and stubby. The tentacles, especially,
seem so, perhaps because of their considerable number, 24—28 (Fig. 7). The
tentacles ranged in number from 24 to 28 but the usual number was 26 or 27, just
as Borg had found in his African specimens. Of course, the condition of the colony,
the length of the polypides 'and tentacles are greatly influenced by the state of
nutrition of the colony. The better fed the colony, the longer the polypides and
tentacles. However, the var. browni specimens seemed well enough nourished.
Their digestive tracts were well filled with algal food.

The parts of the digestive tract are the same as for F. sultana and Plumatclla
repens — ciliated mouth guarded by the epistome, ciliated pharynx, esophagus,
stomach, and intestine.

The reproductive organs were not observed.

The sessoblasts of var. browni are generally smooth on one side (Fig. 5) and
roughened on the other (Figs. 12 and 13). However, some older sessoblasts may
show roughening or markings on both sides, and in addition, chitinous material
may begin to grow on the valve of the statoblast (Fig. 8), attaching it to the
endocyst (Fig. 9) or to the body wall and possibly eventually to the substratum.

Variety browni' s sessoblast shape is best shown in Figures 9 and 11, which are
typical. Abnormal specimens occasionally occur and one such is shown for con-
trast in Figure 6.

The colors of the sessoblasts range from reddish yellow to brown, depending
upon the age ; the older, the darker.

There were quite a number of sessoblasts present in the zooecial tubes of the
Wyoming specimens at the time of collection (August).

The sessoblasts were so distinctive in shape and general proportions that it was
immediately evident that one was not dealing with F. sultana but with a form
related to AbricossofFs and .Goddard's specimens — a distinct species — F.
australiensis.

The decision to make each of these forms (F. australiensis, F. sultana trans-
caucasica, and the Wyoming specimens) a separate variety of F. australiensis was
based on the great similarity to each other so far as the shape of their statoblasts
was concerned and their slight but distinct differences as regards the nature of the
ectocyst and the number of tentacles (refer to Key to Varieties and Tables I and II).

SUMMARY

1. The species Frederic clla australiensis has been emended to include three
varieties.

2. A new variety, F. australiensis var. browni, has been erected.

3. Two other previously recorded forms, F. australiensis Goddard 1909 and
F. sultana subsp. transcaucasica Abricossoff 1927 have been reduced to the status
of varieties under the emended F. australiensis.

4. The emended F. australiensis is characterized by its rounded or broadly

228 , MARY DORA ROGICK

elliptical sessoblasts, its wider zooecial tubes, its greater tentacle number, its lack
of dissepiments and the shorter stubbier tentacles and polypides which are generally
confined to the tips of the tubes. These features distinguish it from F. sultana.

5. The varieties australiensis, browni, and transcaucasica are placed in F.
australiensis because they possess the above characteristics.

6. The three varieties are distinguished from each other on the basis of degree
of incrustation of their ectocyst, the difference in number of tentacles, appearance
of the zooecial tubes in cross section and miscellaneous measurements.

7. Fredericella australiensis has a wide but scattered distribution. It is repre-
sented in Australia by var. australiensis; in Eurasia (the U.S.S.R.), by var trans-
caucasica, in Africa?; and in North America, by var. browni.

8. The specimens which were immediately responsible for the erection of the
new variety, F. australiensis var. browni, were obtained through the kindness of Dr.
C. J. D. Brown and Dr. H. van der Schalie of Ann Arbor, Michigan, who turned
the collection over to the author for study. The specimens were collected by Dr.
van der Schalie on August 3, 1942, from rocks in an alkali pond about three miles
northeast of Church Butte, Uinta County, Wyoming, U.S.A.

9. The study includes 14 illustrations and one table of measurements dealing
with var. browni and one table of comparison between the three varieties.

10. A brief summary of available measurements and other data on F. sultana
is given.

LITERATURE CITED

ABRICOSSOFF, G., 1927a. Uber die Susswasser-Bryozoen der USSR. Compt. Rend, d I'Acad.

Sci. de I'URSS., 1927 : 307-312.
ABRICOSSOFF, G., 19275. To the knowledge of the fauna of the Bryozoa of the Caucasus. Russ.

Hydrobiol. Zeitschrift, Saratow, USSR, 6 (3/5) : 84-92.
ALLMAN, G., 1856. A monograph of the fresh-water Polyzoa, including all the known species,

both British and foreign. Ray. Soc., London, 120 pp., 11 PI.
BORG, F., 1937. Sur quelques Bryozoaires d'eau douce Nord-Africains. Bull. Soc. d'Hist. Nat.

de I'Ajrique du Nord, 27 (7) : 271-283.
BRAEM, F., 1890. Untersuchungen iiber die Bryozoen des siissen Wassers. Bibliotheca Zo-

ologica, Heft 6. 154 pp., 15 PI.
GODDARD, E. J., 1909. Australian freshwater Polyzoa. Part 1. Proc. Linn. Soc. N. S. W ., 34:

487-496. 1 PI.

HARMER, S. F., 1913. The Polyzoa of Waterworks. Proc. Zool. Soc. London, 1913 : 426-457.
HYATT, A., 1868. Article X. Observations on Polyzoa, Suborder Phylactolaemata. Connn.

Essc.r hist., 5 : 193-232.
KRAEPELIN, K., 1887. Die deutschen Siisswasserbryozoen. Eine Monographic. I. Anat.-Syst

Teil. Abhandl. d. natiirw. Vere'ms Hamburg, 10: 1-168.
KRAEPELIN, K., 1914. Bryozoa. Bcitriigc s. Kcunt. d. Land u. Siissivasscrfauna Dcutsch-

Sudwcst-Afrikas. Ergebn. d. Hamburger Dcutch-Siidwcst-Ajr. Studicnreise, 1911.

(Ref. not available to present author.)
ROGICK, M. D., 1935. Studies on fresh-water Bryozoa, II. Trans. Amcr. Micr. Soc., 54 (3) :

245-263.

ROGICK, M. D., 1937. Studies on fresh-water Bryozoa, V. Ohio Jour. Sci., 37 (2) : 99-104.
ROGICK, M. D., 1940. Studies on fresh-water Bryozoa, IX. Trans. Amer. Micr. Soc., 59 (2) :

187-204.
ROGICK, M. D., 1943. Studies on fresh-water Bryozoa, XIV. Annals N. Y. Acad. Sci., 45

(4) : 163-178. 3 PI.

ROGICK, M. D., 1945. Studies on fresh-water Bryozoa, XV. Ohio Acad. Sci., 45 (2) : 55-79.
TORIUMI, M., 1941. Studies on fresh-water Bryozoa of Tapan, I. Sci. Repts. Tohoku Imper.

Univ. (4: Biol.), 16: 193-215.

STUDIES ON THE BIOCHEMISTRY OF TETRAHYMENA. VII.
RIBOFLAVIN, PANTOTHEN, BIOTIN, NIACIN AND
PYRIDOXINE IN THE GROWTH OF T. GELEII W

GEORGE W. KIDDER AXD VIRGINIA C. DEWEY1

Arnold Biological Laboratory, Broitm University, Providence, R. I.

With the substitution of chemically known materials for all but one fraction in
the medium for the growth of Tetrahymena it has been possible to determine with
some degree of exactness the specific vitamin requirements of this important ciliate.
When proteins, such as casein or gelatin, or peptones are used as the base medium it
has been impossible to determine the importance of those vitamins which were
stable to treatments which would not also destroy other essential materials. Using
these types of media, claims have been made for the essential nature of thiamine and
of riboflavin for Teirahyuiena geleii (Hall and Cosgrove, 1944; Hall, 1944). It
was earlier indicated (Kidder and Dewey, 1942) and later conclusively proven
(Kidder and Dewey, 1944; 1945a; 1945b) that at least eight strains of Tetrahy-
mena could grow in a medium in which the thiamine had been destroyed.

When it was found that T. geleii could be grown successfully in a mixture
of amino acids (Kidder and Dewey, 1945c) and that two of the three "unknown
growth factors" could be replaced with nucleic acid derivatives (Kidder and Dewey,
1945d) and that the remaining "unknown growth factor" (Factor II) was relatively
stable and was not adsorbed readily on activated charcoal, it became possible to
examine the effects of the omission of a number of the vitamins. Hitherto these
vitamins had been added routinely to guard against the possibility of any one of
them proving to be a limiting factor. It was found (Kidder, 1945) that folic acid
is an essential growth factor for T. geleii W, this fact being obscured previously by
the necessary inclusion of Factor I (containing folic acid) as the lead acetate precipi-
tate fractions of raw materials, the Factor I activity being readily absorbable on
activated charcoal.

The present work has been made possible by the utilization of a number of
different treatments of the Factor II preparations and the inclusion of all other
constituents of the medium as chemically pure materials. Furthermore, this work
would not have been possible without the employment of a microbiological method
for the detection of traces of the growth factors under consideration. We have
utilized Lactobacillus casei as a tool in this study, and while we have made no
attempts to assay various preparations quantitatively, we have used the bacterial
method for determining the total lack of the vitamin under immediate consideration.
It has been possible also, to show that the ciliate possesses the ability to synthesize
certain of the B vitamins, by determining the increase of the vitamin by the L. casei
test after the growth of the ciliate.

1 Aided by grants from the Morgan Edwards Fellowship Fund and the Manufacturers Re-
search Fund for Bacteriology and Protozoology of Brown University. Present address Bio-
logical Laboratory, Stanford University.

229

230 GEORGE W. KIDDER AND VIRGINIA C. DEWEY

MATERIALS AND METHODS
Organisms

The ciliate used in this study was Tetrahymena geleii W, which has been main-
tained in pure (bacteria-free) culture in this laboratory for a number of years and
which has been used in numerous previous studies (Kidder and Dewey, 1942—1945).
The organism has been grown in amino acid media for the past one and one-half
years and all inocula for the present series were taken from these stocks.

Lactobacillus easei 912 was used for the microbiological testing of experimental
media. This organism was obtained from the Squibb Institute for Medical Research
through the courtesy of Dr. Vincent Groupe. Stocks were carried in yeast extract-
dextrose-agar stab cultures, transplants being made at monthly intervals, incubated
at 37° C. for 24 hours and then placed in the refrigerator.

Ciliate base medium

One type of base medium was used routinely. This appears in Table I with
the complete supplements. Each vitamin under investigation was omitted from
the medium separately.

Preparation of Factor II

It has been necessary to treat the Factor II preparations in various appropriate
ways in order to eliminate the different vitamins studied. In the earlier work
(Kidder and Dewey, 1945d) the prime consideration in the Factor II preparation
was the elimination of Factors I and III activity, and the methods used did not
necessarily render the preparation vitamin free. In this study the inclusion of
Factor I and Factor III activity was of no particular importance, and so attempts
were made to eliminate the vitamin under consideration and still retain maximum
Factor II activity. This latter was not always possible as some of the treatments
used not only removed or destroyed the vitamin but also lowered the Factor II
activity. Nevertheless preparations which were satisfactory for this study were
obtained, and these will be described under the heading of each vitamin.

Riboflavin-frcc preparation (SL531).

Liver Fraction L2 (50 grams) was dissolved in one liter of distilled water and
a 40 per cent solution of normal lead acetate was added until no more precipitate
formed. The precipitate was removed by filtration with the aid of Celite and
discarded. The filtrate was neutralized with NaOH and treated with an excess of
basic lead acetate. The second precipitate was removed and discarded, the excess
lead removed with 9 per cent oxalic acid and the excess oxalic removed as the
oxalate with Ca(OH)L,. Tests at this stage showed the presence of large amounts
of riboflavin, but after adsorption with 10 grams of Norit A for one hour at room
temperature at pH 3.5 the riboflavin had been quantitatively removed. This
preparation was used in a concentration of one part in ten parts of final medium.

- Furnished through the courtesy of Dr. David Klein and the Wilson Laboratories.

BIOCHEMISTRY OF TETRAHYMENA

231

TABLE I

Base Medium

micrograms/ml.

biotin methyl ester3 0.00005

calcium pantothenate3 0.10

thiamine hydrochloride 0.10

nicotinamide3 0.10

riboflavin3 0.10

pyridoxine hydrochloride3 0.10

/>-aminobenzoic acid 0.10

z-inositol 1.00

choline chloride 1.00

folic acid concentrate4 1.00

mg./ml.

hydrolyzed yeast nucleic acid5 0.05

Factor II preparation (see text)

mg./ml

)-arginine mono-hydrochloride . 0.82
l(— )-histidine mono-hydrochloride. 0.10

rf/-isoleucine 0.35

dMeucine 0.35

dMysine 0.60

J/-methionine 0.34

d/-phenylalanine 0.14

dl-ser'me 0.04

<i/-threonine 0.20

/( — )-tryptophane 0.10

dl-va\'me 0.20

dextrose 2.00

MgSO4.7H2O 0.10

K2HPO4 0.10

CaCl2.2 H2O 0.05

FeCl3.6H2O 0.00125

MnCl2.4 H2O 0.00005

ZnCl2 0.00005

Pantothen-jree preparation (8L531H}.

Although pantothenic acid is adsorbed on activated charcoal the time and tem-
perature allowed in preparing the riboflavin-free medium is insufficient for the
complete removal of pantothen. Raising the temperature, increasing the time, or
increasing the amount of Norit used was not practical as the Factor II activity was
greatly reduced (Kidder and Dewey, 1945d). Therefore advantage was taken
of the sensitivity of pantothenic acid to alkali and heat and the riboflavin-free prepa-
ration was adjusted to pH 10.0 with NaOH and autoclaved for two hours. The
Factor II activity was somewhat reduced by this treatment, but the preparation
was entirely satisfactory for use. L. easel tests showed that the pantothenic acid
content had been lowered to an insignificant amount. This preparation was used
in concentrations of one part in ten parts of final medium.

Biotin-jree preparation (8L5C1}

The most active biotin-free preparation, and therefore the one used in this study,
was made in the following manner. Ten grams of Liver Fraction L was dissolved
in 200 ml. of distilled water and brought to boiling. To this boiling mixture were
added 10 ml. of a 10 per cent solution of NaHSO3 and 10 ml. of a 10 per cent
solution of CuSO4, and boiling was continued for 3-5 minutes. The precipitate was
removed on a fluted filter and the process repeated once. The copper was removed
as CuS after treating with 15 per cent Na2S and the sulfate and sulfite removed
as the barium salts after treatment with Ba(OH)2. The volume of the filtrate
was reduced to 200 ml. and a 100 ml. aliquot was adjusted to pH 3.5. Two grams

3 Omitted singly in the appropriate series of experiments.

4 The folic acid concentrate used had a potency of 5000 and was furnished through the
courtesy of Dr. R. J. Williams.

5 Assays of the hydrolyzed yeast nucleic acid with L. easel showed it to be free of riboflavin,
pantothen, biotin, niacin, and pyridoxine but appreciable amounts of folic acid were present.

232 GEORGE W. KIDDER AND VIRGINIA C. DEWEY

of Norit A was added and adsorption allowed to continue for one hour at room
temperature with constant stirring. This preparation was used in a concentration
of one part in twenty parts of final medium.

Niacin-free preparation (8L5C2)

The use of copper precipitation, described above, was designed for the removal
of nicotinic acid. While most of the niacin activity was removed by this method, as
shown by the L. casci test, enough remained to warrant further treatment. Accord-
ingly the nitrate from the copper precipitation was extracted with w-butanol for 96
hours in a continuous extraction apparatus (Wilson, Grauer, and Saier, 1940).
It is known that nicotinamide is readily extracted with butanol, and after this
treatment the extract was found to be entirely devoid of niacin activity, even when
tested with L. casci in amounts four times greater than those used as a supplement
for the ciliate. This preparation was used in a concentration of one part in twenty
parts of final medium.

Pyrido.vine-free preparation (8L531L)

This preparation was the least successful of any used. While it was possible to
treat crude extracts and various filtrates in ways which would remove all pyridoxine
activity for L. easel, it was usually found that the Factor II activity was also lowered
to a point where the preparation was very inferior as a ciliate supplement. There-
fore, the most satisfactory preparation, and the one finally used, was very low in
Factor II activity, and the results obtained cannot be compared directly with those
of the other vitamins tested. This preparation was made by exposing an alkaline
lead acetate filtrate fraction (8L531), to direct illumination from a 300 watt electric
bulb at a distance of 8 inches for a period of 72 hours. This method was used by
Hochberg et al (1943) for pyridoxine destruction. Besides the destruction of
appreciable amounts of the Factor II, another disadvantage of the technique was the
excessive evaporation which took place during the treatment. It was necessary to
add distilled water at frequent intervals to prevent the preparation from drying
down. This preparation was used in a concentration of one part in ten parts of
final medium.

Assay procedure

The base medium employed for the testing of the various preparations was the
16 amino acid mixture suggested by Hutchings and Peterson (1943). This was
chosen in preference to the casein hydrolysate medium of Landy and Dicken (1942)
because of the known composition of the former and the fact that lower blanks can
be obtained. While the amino acid medium does not permit the production of as
much acid by the bacteria it is very satisfactory for determining the presence or
absence of a known vitamin.

Because of the scarcity of amino acids we have modified the usual procedure.
The amino acid medium is made up for stock in double strength and the sugar,
acetate, salts, purines and pyrimidine are added in double strength. For a test,
this complete base medium is measured into 125 X 7 mm. Pyrex tubes in one ml.
volumes. The material to be tested is added in appropriate amounts and a mixture

BIOCHEMISTRY OF TETRAHYMENA 233

of the vitamins, minus the one for which the preparation is being tested, is added.
The volume is then made up to 2 ml. with distilled water. Two controls were run
with each test, one containing base medium and a complete set of supplements except
for the vitamin under test. The second control contained the base medium plus the
complete supplement and plus the Factor II preparation. The first served as a
control on carry-over growth. The second was a control on the possible toxicity
of the Factor II preparation. When titrations were made the figure from the first
control was subtracted from the figure from the unknown preparation. Inasmuch
as a small volume of medium was used it was found advantageous and more accurate
to reduce the standard hydroxide to 0.05 N. The NaOH was standardized with
0.05 N oxalic acid, and the amount of acid produced after 96 hours of growth at
37° C. was titrated directly, using brom thymol blue as an indicator. The longer
incubation period was used for maximum acid production, for in this way the test
becomes more sensitive for traces of vitamins.

After many trials, the usual drop method of inoculation of L. easel was aban-
doned in favor of inoculating with a straight needle. This eliminates the necessity
for washing the bacteria and blanks are just as low. The inocula were always
'made from yeast extract cultures which had incubated for 18—24 hours at 37° C.

While standard curves, using this method, have been made for all the vitamins
studied the results obtained with our preparations do not permit quantitative state-
ments as to amounts inasmuch as the tests were always made at very high levels
and stimulatory materials in the Factor II preparations were invariably present.
We were interested, moreover, first in the determination of the vitamin-free con-
dition of our media, and second, in the biosynthesis of the vitamins by the ciliates.
In the latter case, assays were employed on the medium before and after ciliate
growth and the difference of acid production between the two compared directly.

It has been pointed out (see Cheldelin et al, 1942) that many of the B vitamins
occur in a bound form in tissues and must be liberated by some means for satisfac-
tory tests. There was the possibility that bound vitamins in the Factor II prepara-
tions might be available for the ciliate but not for L. easel, and these would invalidate
any conclusions which were based on the vitamin-free nature of the preparation by
the L. easel test. Enzymatic digestion was carried out on all the preparations,
therefore, in order to test for the total vitamin content. Accordingly takadiastase
and pepsin in quantities of one per cent each of the total solids of the preparation
to be tested were used. The preparation was allowed to digest under toluene for
24 hours at 37° C. at pH 3.5. After steaming, the digest was added to the assay
tubes, as described above, and a control of equivalent amounts of the enzymes
added to parallel tubes. This latter control is obviously necessary as the enzymes
are not vitamin-free. Data on the results of assays of the Factor II preparations
used are presented in Table II.

Ciliate cultures

It was the usual practice, when testing for the effects of one of the known
vitamins, to grow the ciliate through at least three serial tube transplants in the
medium containing the vitamin being investigated, paralleled with the same medium
minus the vitamin. Transplants were made at 72 hour intervals with a bacterio-
logical loop delivering approximately 0.005 ml. of fluid. All incubation was at

234

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

25° C. Growth rate was followed by inoculating appropriate amounts of third
transplant ciliates (36 hours old) into like media in culture flasks (Kidder, 1941).
After inoculation of the flasks (as near 100/ml. as possible) samples were drawn
and the initial inoculum determined. Growth thereafter was ascertained by samp-
ling at intervals until the termination of the experiment. In all cases the flask
series were repeated at least once and the figures averaged.

TABLE II

Assay of Factor II Preparations with Lactobacillus casei 912

Vitamin omitted from base medium

No.

Additions

Riboflavin

Pantothen

Biotin

Nicotinamide

Pyridoxine

Folic acid

None

0.05

0.00

0.05

0.49

0.12

0.62

Enzyme

0.13

. 0.38

0.17

0.75

0.10

0.90

preparation

8L531

0.05

3.79

2.88

4.72

3.86

0.00

8L531H

0.03

0.12

2.79

4.80

2.60

0.00

8L5C1

0.00

1.53

0.07

0.22

3.10

4.36

8L5C2

3.42

0.87

2.93

0.00

3.65

4.65

8L531L

0.00

3.74

3.00

4.51

0.09

0.00

Figures represent ml. of 0.05 N acid per culture (2 ml.). All figures corrected for uninocu-
lated blanks. Line 2 corrected for carry-over growth (Line 1). Lines 3-7 corrected for vitamin
content of enzyme preparation (Line 2).

One obvious objection to the flask technique is the possibility of introducing the
vitamin being investigated from the rubber vaccine tip used in the sampling port.
This possibility was diminished by boiling the vaccine tips for one hour previous
to setting up the flasks. As a check on the tips uninoculated flasks were manipu-
lated in the same manner as the experimental cultures and the samples tested with
L. casei for the vitamin being studied. In no case were these detectable amounts
of the vitamins present. Sampling needles were made chemically clean as well as
sterile before use.

Growth rate during the logarithmic phase was calculated by the formula

•I. 1 O

a = : — —. where t = the time in hours during which the population has been

log b — log a

increasing exponentially, a =the number of cells per unit volume at the beginning,
and b — the number of cells at the end of time, t.

RESULTS

After obtaining Factor II preparations which were free of the vitamins to be
studied, preliminary experiments were set up to determine which vitamins, if any,
were essential growth factors for Tetrahymena geleii W. Accordingly serial trans-
plants were made in the appropriate media, one set with the vitamin present, and
the other with the vitamin omitted. It was immediately apparent that the ciliate
lacked all ability to synthesize folic acid (Kidder, 1945) but the absence of none of
the other vitamins did more than lower the growth rate and the yield. Growth in

BIOCHEMISTRY OF TETRAHYMENA

235

o
6

• CONTROL G = 4.37 MRS.

O MINUS RIBOFLAVIN G = 5.2l MRS.

HOURS

FIGURE 1. Effect of the omission of riboflavin. Factor II preparation used was 8LS31.

Average of two separate experiments.

TABLE III

Summary of Growth Data

Medium

Generation time in hours

Population per ml. at end
of log. phase

Population per ml. at
96 hours

Control
Minus riboflavin

4.37
5.21

58,000
19,000

164,000
67,000

Control
Minus pantothen

4.57
4.60

32,000
34,500

90,000
41,000

Control
Minus biotin

4.32
5.01

45,500
15,000

152,000
96,000

Control
Minus nicotinamide

4.17
8.40

49,000
7,500

170,000
79,000

the sixth serial transplant was possible for all series except that lacking exogenous
folic acid.

In order to gain quantitative information regarding the stimulatory effect that
was apparent in the tube cultures, growth flasks were inoculated from third trans-
plant tubes and the growth followed by frequent sampling. In the case of pyridox-
ine, however, the flask cultures were omitted, as the Factor II preparation necessarily
used was relatively inactive and the growth was erratic, even when pyridoxine was

236

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

• CONTROL G=4.57 MRS.

O MINUS PANTOTHEN G=4.60

HOURS

FIGURE 2. Effect of the omission of pantothenic acid. Factor II preparation used with 8L531L.

Average of two experiments.

• CONTROL G = 432HRS.

O MINUS BIOTIN G = 5.0I MRS

HOU RS

FIGURE 3. Effect of the omission of biotin. Factor II preparation used was 8L5C1.

Average of two separate experiments.

BIOCHEMISTRY OF TETRAHYMENA

237

• CONTROL G = 4.I7 MRS.

O MINUS NICOTINAMIDE G= 8. 40 MRS

H OU RS

FIGURE 4. Effect of the omission of nicotinamide. Factor II preparation used was 8L5C2.

Average of three separate experiments.

present. While qualitative data are lacking for the pyridixine series, nevertheless
we can say from the serial tube transplants that this vitamin appears to be only
stimulatory for T. geleii W.

The omission of riboflavin from the medium resulted in slower growth during
the exponential period. Thus the generation time was raised from 4.47 hours in
the control flasks to 5.21 hours. The maximum yields were reduced to less than
half of those in the control flasks (Fig. 1 ; Table III).

The ciliates appear to synthesize pantothen at a rate which equals the demands
for rapid growth, as judged by the almost identical growth rates in the pantothen-
containing and the pantothen-free media (Fig. 2; Table III). In all cases, how-
ever, the maximum yield was significantly lower in the pantothen-free cultures.

A comparison of the growth curves, generation times and yields for biotin-free
and riboflavin-free media (Figs. 1, 3; Table III) shows remarkable similarity.
The rate of synthesis of biotin by the ciliates appears to be low, indicating the
stimulatory status of this vitamin. We possess added data on biotin substantiating
its non-essential nature for T. geleii W. Early in this series of investigations the
effect of raw egg white and avidin concentrates were studied as a means of determin-
ing whether or not the ciliate required biotin. Egg white was taken aseptically and
added to tubes containing 5 ml. of one per cent proteose-peptone, each tube receiving
0.1 ml. According to Eakin, Snell, and Williams (1941), this amount of egg
white is enough to inactivate 0.05 micrograms of biotin. The analysis of proteose-
peptone made by Stokes, Gunness, and Foster (1944) shows that one gram contains

238

GEORGE W. KIDDER AND VIRGINIA C. DEWEY

0.2 micrograms of biotin, hence our tubes each contained 0.01 micrograms of the
vitamin. The amount of raw egg white used, therefore, was enough to inactivate
five times more biotin than was present. Indefinitely transplantable growth
occurred in the proteose-peptone plus egg white. Likewise, the use of avidin
concentrates in quantities far in excess of that needed to inactivate all of the biotin
present, produced similar results. In this case the avidin was allowed to act on the
proteose-peptone, the peptone removed as the diffusate in dialysis, the peptone
being used as the medium. Similar results were obtained with proteose-peptone
treated with H2O2 in a manner similar to that described by Garnjobst, Tatum,
and Taylor (1943). While it is clear that biotin is not required by T. geleii W
this vitamin is stimulatory.

TABLE IV

Assay Data (L. casei) Before and After the Growth of T. Geleii W

Factor II preparation

Additions for assay (1 : 10)

8L531

8L531H

8L5C1

8L5C2

8LS31L

Plus
ribo-
flavin

Minus
ribo-
flavin

Plus
panto-
then

Minus
panto-
then

Plus
biotin

Minus
biotin

Plus
nicotin-
amide

Minus
nicotin-
amide

Plus
pyri-
doxine

Minus
pyri-
doxine

Before inoculation

4.78

0.07

3.90

0.17

3.92

0.08

3.88

0.00

3.61

0.16

After 72 hr. cilia te growth.

4.60

1.18

3.94

1.71

3.90

1.56

3.80

0.21

3.48

0.22

Medium plus cells

Supernatant of 72 hr. cili-

4.82

0.06

4.13

0.10

3.86

0.10

3.71

0.16

3.52

0.11

ate culture

Washed ciliates from 72 hr.

4.80

1.07

3.94

1.64

3.91

1.05

3.75

0.25

3.61

0.15

culture

Figures represent ml. of 0.05 N acid per culture (2 ml.). All figures corrected for uninocu-
lated blanks and for carry-over growth.

While T. geleii W can be transplanted indefinitely in the absence of exogenous
nictotinamide this vitamin (or nicotinic acid) is an active stimulant. The genera-
tion time is doubled when the ciliate is grown in niacin-free medium as compared
to that in the nicotinamide-containing control (Fig. 4; Table III). While the
population density at 96 hours is less than one-half that of the control (which is
similar to the cases of riboflavin, biotin, and pantothen), the population at the end
of the logarithmic phase is extremely low (approximately 7000/ml.).

It was of interest to determine whether or not T. geleii W would synthesize
amounts of the vitamins which could be detected with the assay methods used.
Accordingly the five types of media used above were set up for serial transplants
and an aliquot of each was assayed with L. casei. After the ciliates had grown
for 72 hours in the third transplants, assays were again made for the various
vitamins. These assays were of three different types. One was on the whole
medium (medium plus cells) ; one, on the supernatant fluid following centrifugation
after chilling (Kidder, Stuart, McGann and Dewey, 1945), and the third was on
washed cells equivalent to the concentrations found in the whole medium. The
samples to be tested were added to the L. casei base medium and sterilized by auto-

BIOCHEMISTRY OF TETRAHYMENA 239

claving. The results of these experiments are given in Table IV. Appreciable
amounts of riboflavin, pantothen, and biotin are synthesized by the ciliates. In-
creases in amounts of niacin are so small that they probably lack significance and
there appears to be no increase in pyridoxine. It must be remembered, however,
that the growth in the niacin-free medium is less at 72 hours than in the ribo-
flavin-, pantothen-, or biotin-free media, while the maximum population reached in
the pyridoxine-free medium never exceeded 20,000 ciliates per ml. The amounts of
the vitamins detected represent minimums, as no attempt was made to release any
which may have been bound (except by autoclaving). It is to be noted that all
vitamins which were synthesized remained in the cells. This was also found to be
true in the case of the biosynthesis of thiamine by T. geleii W (Kidder and Dewey,
1942).

DISCUSSION

Due to the various treatments necessary for the removal of vitamins none of the
Factor II preparations used in this study produced as high yields as had been
previously obtained (Kidder and Dewey, 1945d ; Kidder, 1945). While the ribo-
flavin-free preparation was essentially the same as had been used for the study of
purines and pyrimidines and of folic acid, variations in potency of Factor II activity
were evident. This is due almost entirely to the degree of adsorption on the
activated charcoal. Slight variations of temperature appear to effect the degree to
which Factor II is lost, so that a critical balance is found between the complete
removal of the vitamins and the loss of Factor II activity. In this study the
emphasis was placed on the vitamin removal at a sacrifice of yield.

The findings of Hall and Cosgrove (1944) on the importance of riboflavin for
their strain of Tetrahymena geleii does not seem inconsistent with the present
observations. They state that heat — and alkali-treated casein did not support
growth unless supplemented with thiamine, and even then poorly. The addition of
riboflavin together with the thiamine, however, permitted as good growth as did
the casein medium before heating. There can be no doubt as to the stimulatory
effect of riboflavin, and it is altogether possible that it may function as a detoxifying
agent as well. The detoxifying action of thiamine has been suggested previously
in this connection (Kidder and Dewey, 1944).

In addition to the vitamins which have already been investigated for T. geleii
W there remain at least three of the commonly recognized ones about which little
is known. These are />-aminobenzoic acid, inositol and choline. As yet we have
not had the opportunity to test for the last two, but preliminary work has been
started on the first. The commonly employed technique of adding sulfonamides
to the medium has indicated that this ciliate requires excessive amounts of the
inhibitor to effect growth. The inhibition to growth at these high levels is not
completely reversed with />-aminobenzoic acid, and the evidence indicates that
purines are also involved. This study awaits completion and will be reported at a
later date, but it appears that T. geleii W may be independent of an exogenous
supply of />-aminobenzoic acid.

The only other protozoan of animal nature about which there appears to be critical
data regarding the requirements of the vitamins studied here is Colpoda duodenaria
(Tatum, Garnjobst, and Taylor, 1942; Garnjobst, Tatum and Taylor, 1943).

240 GEORGE W. KIDDER AND VIRGINIA C. DEWEY

Colpoda requires large amounts of thiamine, pantothen, riboflavin, nicotinamide,
and pyridoxine. It does not require />-aminobenzoic acid, biotin, or inositol, while
the status of choline and folic acid is still unknown. Moreover, Colpoda was
shown (Garnjobst, Tatum, and Taylor, 1943) by the Nenrospora test of Tatum and
Beadle (1942) to either release bound biotin from the bacterial "plasmoptyzate"
used or to synthesize this vitamin. This biotin appeared in the medium, however,
and in this way differs from the condition found with T. geleii W where all of the
vitamins arising by biosynthesis appear to bound in the cell protoplasm.

The biochemical investigations of Tetrahymena gelcii W which have so far
been completed permit a fairly complete view of its synthetic abilities. Added
carbon sources appear to be unnecessary except as they may perform a sparing
action on the amino acids. Inorganic salts certainly are essential (Hall and
Cosgrove, 1944; Kidder and Dewey, 1944) although the question of which elements
need to be included is yet to be determined. The commonly employed inorganic
salts usually accepted as being physiologically important satisfy the ciliate require-
ments. Nine amino acids are to be classed as indispensable for this strain (histi-
dine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane,
and valine) while arginine is synthesized at so low a rate that its inclusion becomes
obligator)-. Serine is extremely stimulatory, but its place ma}' be taken by others
of the dispensable amino acids (Kidder and Dewey, 1945c). The list of essential
growth factors for this strain is not long. Purines (most effectively guanine) and
pyrimidines (cytidylic acid and/or uracil) must be supplied in rather large amounts
(Kidder and Dewey, 1945d), and folic acid must be present in amounts in excess
of that required for most of the folic acid-requiring bacteria (Kidder, 1945).
Factor II must be supplied. This substance (or substances) is still chemically
undefined, but it possesses similarities to the "streptogenin" of Woolley (1941) and
Sprince and Woolley (1944).

Biosynthesis of riboflavin, pantothen, and biotin can be accomplished by T.
geleii W. Indefinitely transplantable growth results without exogenous thiamine
(Kidder and Dewey, 1942; 1944; 1945b), riboflavin, pantothen, biotin, niacin, or
pyridoxine. There is some evidence to indicate that /'-aminobenzoic acid may not
be essential, and the status of inositol and choline is still unknown.

For practical purposes it is always of advantage to include any substances of a
stimulatory nature. The absence of any one of the stimulatory substances (thiam-
ine, riboflavin, pantothen, biotin, niacin, pyridoxine) will become a limiting factor,
decreasing the growth rate or the maximum yield or the longevity of the culture
(Johnson and Baker, 1942; Hall, 1944). The stimulatory vitamins should be
included in the culture medium of this ciliate when maximum growth is desired.

SUMMARY

1. It has been possible to prepare media for the growth of Tetrahymena geleii
W which are free of riboflavin, pantothen, biotin, niacin. and pyridoxine, as
determined by the Lactobacillus easel test.

2. T. geleii W is not dependent on an exogenous source of any one of the above
vitamins. Omission of any one, however, reduces the maximum yield and, with
the single exception of pantothen, the growth rate.

BIOCHEMISTRY OF TETRAHYMENA 241

3. Biosynthesis of appreciable amounts of riboflavin, pantothen, and biotin
occurs. These vitamins are found bound in the cell protoplasm. No significant
increases of pyridoxine by biosynthesis were found.

4. The five vitamins listed are not essential growth factors for T. gelcii W but are
stimulatory factors, and as such should be included in the medium for optimum
growth.

LITERATURE CITED

CHELDELIN, V. H., M. A. EPPRIGHT, E. E. SNELL, AND B. M. GUIRARD, 1942. Enzymatic libera-
tion of B vitamins from plant and animal tissues. Univ. Texas Publ. No. 4237 : 15-36.

EAKIN, R. E., E. E. SNELL, AND R. J. WILLIAMS, 1941. The concentration and assay of avidin,
the injury producing protein in raw egg white. Jour. Biol. Chem., 140: 535-543.

GARNJOBST, L., E. L. TATUM, AND C. V. TAYLOR, 1943. Further studies on the nutritional re-
quirements of Colpoda duodenaria. Jour. Cell. Comp. Physiol., 21 : 199-212.

HALL, R. P., 1944. Comparative effects of certain vitamins on populations of Glaucoma piri-
formis. Physiol. Zool, 17 : 200-209.

HALL, R. P., AND W. B. COSGROVE, 1944. The question of the synthesis of thiamine by the ciliate,
Glaucoma piriformis. Biol. Bull., 86: 31-40.

HOCHBERG, M., D. MELNICK, L. SIEGEL, AND B. L. OSER, 1943. Destruction of vitamin B6 (pyri-
doxine) by light. Jour. Biol. Chem., 148: 253-254.

HUTCHINGS, B. L., AND W. H. PETERSON, 1943. Amino acid requirement of Lactobacillus casei.
Proc. Soc. E.vp. Biol. Med., 52 : 36-38.

KIDDER, G. W., 1941. Growth studies on ciliates. V. The acceleration and inhibition of ciliate
growth in biologically conditioned medium. Physiol. Zool., 14 : 209-226.

KIDDER, G. W., 1945. Studies on the biochemistry of Tetrahymena. VI. Folic acid as a growth
factor for T. geleii W. Arch Biochem. (in press).

KIDDER, G. W., AND V. C. DEWEY, 1942. The biosynthesis of thiamine by normally athiamino-
genic microorganisms. Growth, 6 : 405-418.

KIDDER, G. W., AND V. C. DEWEY, 1944. Thiamine and Tetrahymena. Biol. Bull.. 87 : 121-133.

KIDDER, G. W., AND V. C. DEWEY, 1945a. Studies on the biochemistry of Tetrahymena. III.
Strain differences. Physiol. Zool., 18: 136-157.

KIDDER, G. W., AND V. C. DEWEY, 1945b. Studies on the biochemistry of Tetrahymena. IV.
Amino acids and their relation to the biosynthesis of thiamine. Biol. Bull., 89: 131-143.

KIDDER, G. W., AND V. C. DEWEY, 1945c. Studies on the biochemistry of Tetrahymena. I.
Amino acid requirements. Arch. Biochem., 6: 425-432.

KIDDER, G. W., AND V. C. DEWEY, 1945d. Studies on the biochemistry of Tetramymena. V.
The chemical nature of Factors I and III. Arch. Biochem. (in press).

KIDDER, G. W., C. A. STUART, V. G. McGANN, AND V. C. DEWEY, 1945. Antigenic relation-
ships in the genus Tetrahymena. Physiol. Zool., 18: 415.

LANDY, M., AND D. M. DICKEN, 1942. A microbiological assay method for six B vitamins using
Lactobacillus casei and a medium of essentially known composition. Jour. Lab. Cliu.
Med., 27 : 1086-1092.

SPRINGE, H., AND D. W. WOOLLEY, 1944. Relationship of a new growth factor required by
certain hemolytic streptococci to growth phenomena in other bacteria. Jour. E.rp. Med.,
80: 213-217. '

STOKES, J. L., M. GUNNESS, AND J. W. FOSTER, 1944. Vitamin content of ingredients of micro-
biological culture media. Jour. Bact., 47 : 293-299.

TATUM, E. L., AND G. \V. BEADLE, 1942. The relation of genetics to growth factors and hor-
mones. Grozt'th, 6: 27-35.

TATUM, E. L., L. GARNJOBST, AND C. V. TAYLOR, 1942. Vitamin requirements of Colpoda
duodenaria. Jour. Cell. Comp. Physiol., 20: 211-224.

WILSON, D., R. C. GRAUER, AND E. SAIER, 1940. A simplified continuous extractor for estrogens
and anclrogens. Jour. Lab. Clin. Med., 26: 581-585.

WOOLLEY, D. W., 1941. A new growth factor required by certain hemolytic streptococci. Jour.
Exp. Med., 73 : 487-492.

THE STRUCTURE OF MEIOTIC CHROMOSOMES IN THE

GRASSHOPPER AND ITS BEARING ON THE

NATURE OF "CHROMOMERES" AND

"LAMP-BRUSH CHROMOSOMES"

HANS RIS
Rockefeller Institute jor Medical Research, New York *

The nature of the gene is one of the fundamental problems in modern biology.
Since the genes are located in the chromosomes, the structure, chemistry, and
metabolism of the chromosomes are of special significance for the understanding of
the gene and gene action. The prevalent interpretation of chromosome structure
has developed as a kind of compromise between two originally opposed views,
the "chromomere hypothesis" of Balbiani, Pfitzner, and Strasburger and the
"chromonema hypothesis" of Baranetzky, Bonnevie, and Vejdovsky.2 According
to the "chromomere hypothesis," the chromosome consists of a series of small beads
or discs strung together. During prophase they approach each other, fuse into
larger complexes, and finally disappear in the thick rod-shaped metaphase chromo-
somes. For the "chromonema hypothesis" on the other hand, the fundamental
unit of the chromosome is a coiled thread, tightly wound in a helix at metaphase
and more or less uncoiled during interphase. Both chromomeres and spirals were
discovered about the same time (Balibiani, 1876; Pfitzner, 1882; Baranetzky,
1880). Yet more and more structures first described as "chromomeres" have
turned out to be coils and today the "chromomere" is in full retreat into the sub-
microscopic level. Strasburger's "chromomeres" in Tradescantia pollen mother
cells had been clearly shown to be spirals by Baranetzky (1880) ; Pfitzner's
"granules" in somatic prophases of the salamander were resolved into coils by
Schneider (1910) and by Lee (1921), who concluded that all "chromomeres" are
in reality turns in the helix. The modern view which is accepted by most cytolo-
gists today and is based mainly on Heitz (1935), holds that the true "chromo-
meres" (Belling's ultimate chromomeres) can only be seen in the prophase of
meiosis (leptotene) and in the curious giant chromosomes of dipteran larvae, where
the chromonemata are assumed to be completely uncoiled. According to this view
(Reuter, 1930; Heitz, 1935; Darlington, 1937; White, 1937; Geitler, 1938;
Koltzoff, 1938; Kuwada, 1939; Nebel, 1939; Huskins, 1941, 1942; Straub, 1943)
the chromonema consists of chromomeres of different but constant size, rich in
nucleic acid, connected by protein fibrils. The chromomeres bear the genes, they
reproduce as specific units and they synapse in meiotic prophase. They are the
visible expression of the linear arrangement of the genes.

1 Part of the work for this paper was done in the Department of Biology, Johns Hopkins
University.

2 The "vacuolization hypothesis" of Gregoire and his school, denying both chromomeres and
chromonemata, has been thoroughly disproved by the work of the last twenty years and need
not be discussed here.

242

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 243

Yet even in leptotene chromosomes the "chromomeres" were found to be coils
by several authors. They were first described as such in Tradescantia by Kauf-
mann (1931), who nevertheless accepted the "chromomere" interpretation for other
plants and animals (Kaufmann, 1936). Koshy (1934, 1937) found the leptotene
chromosome to be coiled in Allium and Aloe, Naithani (1937) in Hyacinthus.
Smith (1932) suggested that the beadlike appearance of the leptotene in Galtonia
might be due to twists in the chromonema and Hoare (1934) noted that the
zygotene threads give the impression of two tightly coiled chromonemata. Kuwada
(1939) pointed out that sharp turns in the coils might easily be mistaken for
"chromomeres." In Tradescantia, Swanson (1943) found no "chromomeres"
which could not be resolved into coils, and he suggested that a chromomere pattern
such as that in maize might be due to differential spiralization.

Yet most recent discussions on the gene and chromosome structure cling
tenaciously to the belief that "chromomeres" are -real (e.g., Schultz, 1944). The
main evidence usually presented, besides the salivary chromosomes of dipteran
larvae, is the observations of Wenrich (1916), Lewis and Robertson (1916), and
Chambers (1924) on the large chromosomes in grasshopper spermatocytes. To
re-examine this evidence is the purpose of the present investigation.

MATERIAL AND METHODS

Spermatocytes of Clwrthippus curtipennis, Chorthophaga viridifasciata, Disso-
steira Carolina, Melanoplus femur-rubrum, Arphia sp., Hippiscus sp., and Orphulella
sp. were studied in sections (fixation: B 15 and Sanfelice, stain: Feulgen), and
aceto-orcein smears. For the detailed study of leptotene chromosomes sections
stained with Feulgen were found to be more reliable than smears. To uncoil
chromosomes, testes were submersed for one-two hours in 2-10~3 M KCN in
Belar solution (Belaf, 1929) before smearing (Oura, 1936). The optics used
consisted of a Zeiss aplantic condenser N.A. 1.4, Zeiss 2 mm. objective N.A. 1.4
and 15 X ocular. The photographs (except Figure 12) were taken with the same
optics and a Bausch and Lomb photomicrographic camera type H. The stereo-
scopic photographs were made by shifting the substage diaphragm maximally to
the left and right respectively for the two exposures. 3

THE STRUCTURE OF LEPTOTENE CHROMOSOMES

On casual examination the slender, irregularly twisted chromosomes at lepto-
tene have a beaded appearance as has been so often described in the literature
(for a review see Renter, 1930). A detailed study with the best optics and a
delicate use of the fine adjustment screw of the microscope, however, resolves the
beads or "chromomeres" into turns of a narrowly pitched coil 4 (Figures 1, 6a, and
13). With Feulgen the chromosome stains evenly throughout its length and there
are no Feulgen-negative "interchromomeric fibrils." This uniform nature of the

3 1 wish to thank Mr. John Spurbeck, Dept. of Biology, Johns Hopkins University, for help
with the photomicrographs.

4 Mr. L. Vanderlyn, Dept. of Zoology, University of Pennsylvania, informs me that he has
come independently to the conclusion that the "chromomeres" are in reality gyres in the chro-
monemata. In a forthcoming paper he will trace the origin of these from the unpacking coils
of the preleptotene in Podisina alpina.

244 HANS RIS

leptotene chromosomes can best be seen in well fixed sections. A chromosome,
followed with the fine adjustment as it winds itself through the nucleus, is seen
to be a thread of uniform thickness thrown into a tight, irregular helix. The
narrow turns of this coil where the chromosome overlaps itself, appear as "chromo
meres." The gyres can vary in wridth and may be unevenly spaced (see Figure 13).
This can give the impression of different sized chromomeres. The width of the
thread and the tightness of the helix are characteristic for each species of grass-
hopper studied. In aceto-orcein smears, when the chromosome has been under
shear or pressure, an apparent chromomeric structure is more pronounced. This
is due to the wax-like consistency of the chromosome which causes its gyres to fuse
or be pulled out and otherwise distorted. Chromosomes, in which the coils can be
clearly seen, can easily be transformed into the classical string of beads simply by
exerting pressure on the coverslip and smearing them out. It is interesting to
note in this connection that Belling (1931) emphasized that chromomeres are not
clear in sections and that one has to use smears to make them visible.

When does that tight irregular coil of the leptotene chromosome originate?
Is there any stage when the chromonemata are completely stretched out and without
any signs of coiling? In all the grasshoppers studied no chromosome was found
that did not show some degree of coiling. Furthermore, the characteristic coil
of the leptotene chromosome is already present in the interphase and unravelling
stage of preleptotene. We must assume that the leptotene spiral originates in the
interphase or telophase of the preceding division. This origin of a prophase helix
in the preceding telophase has been demonstrated by Sparrow (1942) in the
microspore division in Tradescantia. The chromosome of the unravelling stage is
thus doubly coiled (Figure 7). It shows the wide gyres of the previous metaphase
relaxing into the relic coils of leptotene and the small tight helix which is destined
to enlarge during pachytene and become the major coil of the first meiotic meta-
phase chromosome. This structure of the preleptotene chromosome was indicated
clearly in McClung's figures for Mecostethus lineatus (esp. Figure 43, McClung,
1927). The heteropycnotic X chromosome in the prophase of grasshopper sperm-
atocytes, which does not unwind in preleptotene and is thus comparable to the
preleptotene autosomes in structure, similarly discloses a small tight helix and a
wide irregular coil as Coleman (1943) has demonstrated.

Since the preleptotene chromosome consists of at least two chromonemata the
leptotene chromosome also must be double (Robertson, 1931). The split between
the chromatids can sometimes be discerned, especially in the turns of the coil, but
usually the sister strands are closely appressed. They seem to form a plectonemic
spiral, though this could not be determined with certainty.

THE STRUCTURE OF ZYGOTENE CHROMOSOMES

The pairing of homologous chromosomes at zygotene thus takes place between
two coiled structures. The gyres of the two chromosomes fit into each other and
become more or less closely appressed (Figures 2 and 6b). The bivalent now
forms a paranemic coil. Just as the gyres in leptotene were mistaken for "chromo-
meres," so the gyres of the parallel coil in the bivalent were thought to be paired
"chromomeres."

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER

245

FIGURES 1-5. Diagrammatic representation of chromosome structure during meiotic pro-
phase of the grasshopper.

FIGURE 1. Leptotene.

FIGURE 2. Zygotene.

FIGURE 3. Pachytene. The hoinologues can be either slightly separated or closely ap-
pressed.

FIGURE 4. Later pachytene. Appearance of the minor coil.

FIGURE 5. Diplotene. The chromonemata have separated laterally. This represents in
essence also the structure of "lamp-brush chromosomes."

246 HANS RIS

THE STRUCTURE OF PACHYTENE CHROMOSOMES

During pachytene the helices of the paired chromosomes increase in width and
the number of gyres decreases. This process is identical to that described by
Swanson (1942a) for Tradescantia (despiralization cycle). If the chromosomes
are closely appressecl only one helix is visible. When the coils separate slightly
a reticular or vacuolated appearance is produced, though often two parallel helices
can be clearly discerned (Figures 3 and 8). In late pachytene an irregular waviness
appears on the gyres of the pachytene coil ; this sometimes looks like a very fine
spiral of narrow pitch. It most likely corresponds to the minor spiral described in
plant chromosomes (Figures 4 and 9).

THE STRUCTURE OF THE CHROMOSOMES DURING DIPLOTENE AND DIAKINESIS

In this stage the chromosomes are most difficult to analyze. They are usually
described in the literature as diffuse, having fuzzy or woolly fringes (see for instance
Nebel and Ruttle, 1937). The better the general fixation seems to be, the less
distinct or sharp the chromosomes appear. However, after submersing the cells
for one to two hours in 2 • 10~3 M KCN in Belar solution and staining in aceto-
orcein, the structure of the diakinesis chromosome and the reason for its woolly
appearance becomes quite clear. The lateral separation of the chromonemata which
had already begun in pachytene has progressed much further, so that their gyres now
overlap only within a narrow central region. This region appears as a beaded
darker core of the chromosome. The gyres of the major coil of the chromonemata
form loops projecting beyond this central core (Figures 5 and 14). It is these
loops of the individual chromonemata which give the chromosome its hairy appear-
ance. If the separation of the coiled threads is great the chromosome looks like a
dark, beaded rod with loops or hairs at regular intervals (Figure 14a). When
the lateral shifting is less the chromosome gives the impression of a double beaded
rod, the loops or hairs now of course being shorter (Figure 14b). These appear-
ances can easily be explained on a model of four simultaneously coiled wires.
Sometimes one or more irregular turns of the minor coil can be seen on the loops.

In this stage there is further evidence against the reality of "chromomeres."
If the apparent thickenings in the leptotene chromosome were constant units of
definite size, they should be visible also in the loops of the diplotene chromatids.

PLATE I

FIGURE 6. Chorthophaga, zygotene. Pretreated with ammonia vapor. Aceto-orcein smear.
Note the coil of the univalent at a and the paranemic helix of the bivalent at b.

FIGURE 7. Chorthophaga, preleptotene. Aceto-orcein smear. Irregular "major coil" in
the process of unravelling. The narrowly pitched helix ("minor coil") corresponds to the lepto-
tene spiral (arrows).

FIGURE 8. Chorthippus, early pachytene. Section. Fixed with Sanfelice and stained with
Feulgen.

FIGURE 9. Hippiscus, late pachytene. Section. Fixed with Sanfelice and stained with
Feulgen.

FIGURES 10 AND 11. Orphulella, pachytene. Pretreated for 2 hours in KCN. Aceto-orcein
smear. The heterochromatic knobs have been resolved into coils (arrows).

FIGURE 12. Fragment of a "lamp-brush chromosome" from a frog oocyte. Aceto-orcein
smear. Note the loops of the major coil and the minor coil (arrows). Zeiss 3 mm. objective,
15 X ocular.

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 247

R • ^

A *v« %-
7 ..8 rf ' ' *

• •*»- *

" ' '.,*'

t ,. •*' x ;

* -v'

10 n" . 11

PLATE I

248 HANS RIS

These chromatids, however, never show any beaded structure. The despiraliza-
tion already noted in pachytene has continued and has resulted in an increase in
width and decrease in the number of gyres with a consequent shortening and
thickening of the chromosome.

THE STRUCTURE OF METAPHASE CHROMOSOMES

At the end of diakinesis the gyres of the chromatids become more closely spaced
along the chromosome axis, leading to a further shortening of the chromosome and
a fusion of the "chromatic coating" ( Ris, 1942) of the individual chromatids, so
that a uniformly staining body results. The chromatids retain their lateral separa-
tion, causing what is sometimes observed as a reticulate or vacuolated appearance
of the metaphase chromosomes.

THE NATURE OF THE HETEROPYCNOTIC REGIONS IN ORPHULELLA

During meiotic prophase the chromosomes of Orphulella carry small, knob-like,
darkly staining bodies, especially at their ends. These structures resemble the large
"chromomeres" described by Wenrich (1916) in Phrynotettix. Treatment with
KCN for 3 hours causes a loosening of the chromosome helix and shows that these
knobs are tightly coiled regions of the chromosome (Figures 10 and 11). It is
evident that the different appearance of such heteropycnotic regions in meiotic
chromosomes is mainly due to differential coiling of the chromonemata as has been
shown for the X chromosome by Coleman (1943). Similarly Wilson and Booth-
royd (1944) have demonstrated that heterochromatic differentiations after cold
treatment are the result of differential coiling.

DISCUSSION

Chromomeres

The synthesis of cytology and genetics in the chromosome theory of inheritance
has had a stimulating effect on the investigation of chromosomes. Yet the knowl-
edge of the intimate structure of the chromosome has been retarded rather than
furthered by the influence of genetics. The constant desire to find visual expres-
sion of the linear order of genes has led to the perpetuation of misinterpretations
of the microscopic image. Indeed cytogenetics has established beyond doubt the
longitudinal differentiation of chromosomes, but it is not justifiable to conclude
that the units of this differentiation are microscopically visible particles. Thus
observations which did not agree with the "chroinomere" hypothesis tended to be
ignored. The extensive literature on the subject (see Renter, 1930) shows the
widespread acceptance as well as the great versatility of the chroinomere concept.
Almost any expression of unevenness along the chromosome was at one time or
other called "chroinomere." The first pictures of "chromomeres" were published
by Balbiani (1876) and Pfitzner (1882). Botli described prophase and metaphase
chromosomes in somatic cells. Today there can be no doubt that they saw the
gyres of the somatic helix (Schneider, 1910; Lee, 1921; Creighton, 1938).
Strasburger (1882) and Farmer and Shove (1905) described disc-like "chromo-
meres" in meiotic metaphase chromosomes of Tradescantia. We know now that
they mistook the gyres of the major coil for discs. Quite often chromocenters in

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 249

•

, "•'

•

'jt' ***'

i » fV-' - > -ia !.V-;^.: ;

13 _ .

.m <M» »JL1- L*t^i

'.. '

^p *

PLATE II

FIGURE 13. Stereophotomicrograph, Chorthippus leptotene. Section. Fixed with Sanfelice
and stained with Feulgen and Iron hematoxylin. Note the coiled leptotene chromosomes
(arrow).

FIGURE 14. Stereophotomicrograph, Hippiscus diakinesis. Pretreated with KCN. Aceto-
orcein smear. Note the loops of the major coil which give the chromosomes at this stage the
fuzzy appearance.

250 HANS RIS

interphase nuclei and heteropycnotic regions on the chromosome, such as found in
the X chromosome of Notonecta indica (Browne, 1916), were called "chromo-
meres" (cf. Heitz, 1929). Shinke (1937) and Coleman (1940, 1941) have shown
that such heteropycnotic regions are parts of the chromonema which remain
tightly coiled or become precociously coiled. This could be confirmed in the
present paper for the "knobs" of the meiotic chromosomes of Orphulella. Thus,
one more "chromomere" was reduced to chromonematic coiling. There remained
the "ultimate chromomere" of Belling (1928), the only bona fide "chromomere"
according to most modern cytologists. This "chromomere" can only be seen in
meiotic prophase and in salivary chromosomes of clipteran larvae, where the
chromonemata are assumed to be maximally stretched. Let us examine point for
point the evidence which is given for the reality of these "chromomeres" (see
reviews cited in introduction).

(a) "The chromomeres are seen in living cells and cannot be artefacts."
Belaf (1928) described "chromomeres" in living spermatocytes of the grasshopper.
An analysis of his figure shows that he did not see chromomeres but the coils of
diakinesis chromosomes. Lewis and Robertson (1916) and Chambers (1924)
found "chromomeres" in the leptotene of living grasshopper spermatocytes. This
may show that the structures observed are not fixation artefacts, but it certainly
is easier to misinterpret narrow coils as granules in unstained cells where the
chromosomes are hardly visible, than in well stained preparations. Yet there is a
very interesting observation by Chambers (1924, page 270) which seems to have
been overlooked by himself as well as most reviewers of chromosome structure.
He writes : "If one of the early prophase chromosomes with ragged granular outlines
be seized with a needle and rapidly pulled across the field so as to stretch it, the
granules disappear and the whole substance becomes homogeneous." So Chamber's
microdissection study does not support the "chromomere" hypothesis, but rather the
assumption of a uniform but coiled leptotene chromosome.

(b) "The chromomeres have specific and constant sizes and form a definite
pattern." The classical examples are Dendrocoelum (Gelei, 1921) and Phryno-
tettix (Wenrich, 1916). The observed patterns in these and other forms are an
expression of the longitudinal differentiation of the chromosome. This differentia-
tion is real. But the nature of this differentiation now turns out to be differential
coiling and not a sequence of discrete bodies of different sizes. The large
"chromomeres" in Phrynotettix are heterochromatic regions along the chromosome
similar to those found in certain plant chromosomes and those described for Orphu-
lella in this paper. In J'eltlieiinia viridijolia Coleman (1940) could show that such
heterochromatic regions are closely coiled sections of the chromonema. They
correspond in structure to the differential segment in Rhoeo (Coleman, 1941) and
the chromocenters in various animals and plants (Shinke, 1937). The knobs
in maize are most probably of a similar nature.

(c) "The chromomeres of homologous chromosomes pair specifically at zygo-
tene." Just as the turns in the spiral give the impression of "chromomeres" at
leptotene, the paranemic spiral of the paired bivalent simulates a row of paired
granules. Since homologous regions of the chromosomes pair, it is evident that
heterochromatic sections will come to lie side by side in the pachytene chromosomes.

(d) "The number of chromomeres in leptotene corresponds approximately to
the number of genes in Lilinin (Belling, 1928). In salivary chromosomes the

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 251

bands, which correspond to the leptotene chromomeres, ^verc shown to be closely
associated with certain genes (Muller and Prokofyeva, 1935)."

Balling's estimate of the number of genes in Lilium was entirely arbitrary and
he had no direct evidence for a correlation of "ultimate chromomeres" and genes.
In salivary chromosomes of Drosophila, however, a great number of workers have
proven beyond doubt that the visible "bands" are correlated with certain genes.
A recent analysis of the salivary chromosomes of Sciara in collaboration with Dr.
Helen Grouse (in press) has shown that the "granules" and "bands" are misinter-
pretations of a very complicated spiralization of a bundle of chromonemata. What
has been described as a "chromomere" corresponding to a gene represents in reality
a region of relatively considerable length along the chromonema. The cytogenetic
work on Drosophila salivary chromosomes is not evidence for a "chromomeric"
structure of the chromonema, but shows that certain sections of the uniform
chromonematic thread correspond to definite genes and that the detailed nature
of the coiling in these interphase chromosomes is closely correlated with a genetic
specificity on a submicroscopic level.

In summary this is the evidence against the existence of "chromomeres" : (a)
In living cells the microdissection experiment of Chambers (1924) shows that the
leptotene chromosome can be stretched into a uniform thread, (b) In several
plants such as Tradescantia (Kaufmann, 1931; Swanson, 1943), Alliuin and Aloe
(Koshy, 1934, 1937), Hyacinthus (Naithani, 1937), and in the grasshopper the
leptotene chromosome consists of a uniform, coiled thread, Feulgen-positive through-
out its length. No evidence of interchromomeric fibrils can be found. The lepto-
tene coils can be followed into the pachytene where they increase in width and
decrease in number. This explains the observation of many authors (e.g., Belling,
1931) that the "chromomeres" increase in size and decrease in number during the
course of prophase. (c) In the diplotene chromosomes of the grasshopper no
"chromomeres" can be seen in the large loops of the chromatids. If specific "chro-
momeric" granules were present at leptotene they should be visible also in the chro-
monema of diplotene. (d) McClintock (1944) has shown in maize that at least
one gene is located in the interchromomeric thread between the terminal knob and
the first "chromomere" on chromosome nine. This disproves definitely the idea, at
least for maize, that the genes are necessarily located in the "chromomeres" which
are connected by non-genie fibrils.

Diplotene chromosomes and "lauip-bmsli chromosomes"

The coiling cycle in the grasshopper appears to be identical with that described
by Swanson (1942, 1943) for Tradescantia. The leptotene coil develops into the
major coil of diakinesis and metaphase through despiralization. There is no
definite minor coil, but from late pachytene on, an irregular waviness appears on
the loops of the chromatids, resembling an incipient helix. A minor coil was seen
in spermatocytes of another orthopteran, Podisma, by Makino (1936). In
Trillium (Huskins, 1941) there seems to be a similar waviness instead of a definite
helix as was demonstrated for Tradescantia. This difference in the appearance of
the minor coil seems to be mainly one of timing of the spiralization cycle as Kuwada
(1938) has suggested. In the grasshopper the chromatids have never been seen
completely separated in diakinesis or metaphase. Their coils sometimes appear

252 HANS RIS

interlocked as Kuwada (1938) found in Tradescantia, but this could not be definitely
determined. Swanson (1942b) has shown that the terminalization of chiasmata
is correlated with the despiralization of the major coil in Tradescantia. The same
process takes place in the grasshopper and it is most likely that here, too. term-
inalization of chiasmata is the consequence of despiralization of the major coil.

The diffuse appearance of orthopteran as well as most other animal chromo-
somes in diplotene has made their analysis rather difficult. The chromonema is
generally of smaller diameter than in plant chromosomes and therefore the delicate
loops of the major coils escaped observation. This diffuse structure is due to a
lateral separation of the chromatids in contrast to the usual appression of the
chromatids in plant chromosomes. Under certain conditions, and especially in
diakinesis, plant chromosomes also show a separation of chromatids. They then
give the same pictures as diplotene chromosomes of animals (see the anaphase
chromosome of desynaptic Trillium in Figure 9 of Sparrow, Huskins and Wilson,
1941 ; Swanson, 1942a, 1943, and Kuwada and Nakamura, 1938 for Tradescantia).
Plant and animal chromosomes have often been described as reticulate or vacuo-
lated. Gregoire and his school based on this their "vacuolization hypothesis" of
chromosome structure. All their pictures can today be explained on the simple
assumption of a multiple stranded helix with the chromonemata more or less
appressed or opened up.

When the lateral separation of the chromonemata is great and the loops only
faintly stained, the chromosome may appear covered with a layer of achromatic
material (often described as "matrix" or "sheath"; see for instance Lee, 1921 and
McClung, 1941, Figure 7). Probably many a "matrix" in the literature is nothing
but the apparant connection between faintly staining outer loops, running at an even
distance from the darker core of the chromosome where the chromonemata over-
lap. Makino (1936) published some photographs of diakinesis and metaphase
chromosomes of Podisma which at first seem to contradict my interpretation of
these stages. He shows a dark inner coil sometimes appearing double, surrounded
by a light "matrix." Faint strands are sometimes seen to connect the central
spiral with the border of the "matrix." Yet it is very easy to understand these
figures with the help of a model of four wires coiled together. When two are
maximally separated laterally and two stay appressed in the center, Makino's coil
and matrix become explainable. The outer coils are not at all or only faintly
stained in his gentian violet preparations and their outer boundary suggests the
presence of a "matrix."

The previous studies of diplotene chromosomes of Orthoptera have completely
ignored these outer gyres of the chromonemata. They were described as woolly
threads or brushlike projections on the surface of the chromosome, but not as an
essential part of it. Thus the pictures of Hearne and Huskins (1934), Nebel and
Ruttle (1937), Darlington (1936), and the McClung school are based on optical
• illusions or too light staining. What were described as "chromomeres" in this
stage are the points of overlap of the chromonemata. Darlington (1936) has
studied relational coiling of chromatids and chromosomes in pachytene and diplo-
tene. What he pictured as one single chromatid, however, is not a continuous
structure, but a series of nodes of separate overlapping major coils. His relational
coil of chromatids is therefore an optical illusion. Only a complete stretching of

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 253

the major coil could reveal whether the chromatids are wound around each other
(see Kuwada, 1938).

Many oocytes and spermatocytes in diplotene undergo a so-called "diffuse
stage," which is correlated with the growth of the cell. The chromosomes stain
only faintly and lose their definite shapes ; they may even disappear into a reticular
structure. In the grasshopper the diffuse nature of the chromosomes is due to the
loosening and separation of the individual chromonemata of the major coil. This
more or less pronounced loosening up of the gyres, comhined possibly with some
chemical changes in the composition of the chromatin, can explain the appearance
of diplotene chromosomes during this stage in spermatocytes and oocytes.

The diplotene chromosomes in the large oocytes of some vertebrates have par-
ticularly interested the cytologist ever since their discovery by Riickert in 1892, be-
cause of their tremendous size. Their fuzzy and brush-like appearance warranted
the name "lamp-brush chromosomes." Duryee (1937, 1938, 1939, 1941) has re-
cently studied these chromosomes in great detail in the frog and salamander, and
concludes that (1) they represent paired gelatinous cylinders in which the chromo-
meres are embedded. (2) From these chromomeres lateral loops grow out. He
likens this growth to that of a crystal or the reproduction of a virus. (3) In a
later stage, before the maturation divisions take place, these lateral loops are thrown
off into the cytoplasm as genie products essential for the early embryo.

Painter (1940) came to somewhat different conclusions. He considers "lamp-
brush chromosomes" to be chromosome aggregates, which originated through endo-
mitosis and the loops to correspond to whole chromosomes. Material from thous-
ands of such chromosomes, he maintains, is thrown into the cytoplasm as substrate
for the synthesis of cleavage chromosomes. Koltzoff (1938) thinks that the lateral
projections are side branches of the chromomeres which then are given off into the
cytoplasm.

In contrast to Duryee, Koltzoff, and Painter, it is here suggested that "lamp-
brush chromosomes" are typical diplotene chromosomes which differ from other
diplotene chromosomes only in the tremendous longitudinal growth of the chro-
monemata. The loops are then the major coils of the laterally separated chromone-
mata, the "chromomeres" are simply overlaps of the strands just as in diplotene
chromosomes of the grasshopper. Figure 12 shows a fragment of a "lamp-brush
chromosome" of a frog oocyte, smeared in aceto-orcein. The somewhat distorted
large loops of the major coil and the minor coil are easily visible.

The evidence for this interpretation may be summarized as follows: (a) The
loops are continuous as Riickert (1892) has already observed. He followed the
chromonema for several turns. He also pointed out that the granules ("chromo-
meres") are not real, but optical sections of the overlapping threads. The denser
inner region of the chromosome he described as due to the radial arrangement of
the threads, (b) "Lamp-brush chromosomes" are diplotene chromosomes and ex-
cept for their greater size have the same appearance as the diplotene chromosome
of the grasshopper. Since it has been shown here that the loops are simply the
gyres of the major coil of the separate chromonemata, one can conclude that the
corresponding appearance of the "lamp-brush chromosome" is the result of a similar
structure, (c) Koltzoff (1938) has published drawings of cross sections of "lamp-
brush chromosomes" (his Figure 10, b and c). These cross sections look like a
star with characteristically eight rays. These eight rays are most likely the eight

254 HANS RIS

half-chromatids which form independent loops, though Koltzoff saw them as brush-
like projections.

The reduction in chromosome size just before the meiotic divisions is accom-
plished then not by throwing off parts of the chromosome or entire chromosomes,
but by elimination of material on a submicroscopic level.

The microscopic organization of chromosomes

Kuwada (1939) in his review of chromosome structure predicted that the spiral
theory might well prove capable of harmonizing the various hypotheses of chromo-
some structure. Such a uniform interpretation of the structure of all types of
chromosomes is now possible. The unit of the chromosome is the chromonema, a
microscopically uniform thread. This chromonema is never completely straight-
ened out, but always shows some degree of spiralization. This coiling is not at
random, but, as the salivary chromosomes and heterochromatic regions show, is an
expression of the longitudinal differentiation of the chromonema and closely corre-
lated with the genes. It is, in other words, an expression of submicroscopic struc-
ture and possibly the functional state of the gene (cf. heterochromatin). The mi-
croscopic uniformity of course does not exclude a great variability of submicroscopic
structure and chemical composition along the chromonema. During the mitotic
cycle there develops a condensed chromosome through despiralization of the in-
cipient coil of early prophase. The differentiation of mitotic chromosomes, primary
and secondary constrictions, satellites, and heterochromatic regions are expressions
of the differential coiling of the chromonemata. In the resting nucleus of different
tissues we often find different patterns of heterochromatin. It may be that differ-
ential spiralization of the chromonemata in resting cells is correlated with cell dif-
ferentiation. The chromonema is not uniform in length, but it can vary greatly
from cell to cell in the same organism, as well as in the same cell in different meta-
bolic states. In many synthetically very active cells as for instance some oocytes,
nurse cells, gland cells (dipteran salivary glands), the total amount of chromatin is
greatly increased. This is accomplished by an increase in the number of chromo-
somes (endomitosis, cf. Geitler, 1941), by a growth in length of the chromonemata
(as in "lamp-brush chromosomes") or by both simultaneously (salivary chromo-
somes). In "lamp-brush" and salivary chromosomes the increase in length is
tremendous and would be difficult to understand if only inert "genoplasm" or
"matrix" (Koltzoff, 1938) had increased. More likely it is an increase in the
volume of the gene complex, related to the greater metabolic activity. We have
to look at the gene, therefore, not as a unit of constant and specific size as expressed
in the "chromomere" hypothesis, but as a complex that is greatly variable in mass,
depending on the metabolic activity of the nucleus.

SUMMARY

1. "Chromomeres" do not exist as definite structures. What has been de-
scribed as "chromomeres" are (a) misinterpretations of gyres of the chromonematic
helix (leptotene, somatic prophase) ; (b) points of overlap of chromonemata (diplo-
tene) ; (c) heterochromatic sections consisting of more tightly coiled regions of
the chromonema. The fundamental unit of the chromosome is a microscopically

MEIOTIC CHROMOSOMES IN THE GRASSHOPPER 255

uniform thread. The longitudinal differentiation of the chromosome is clue to dif-
ferential coiling of this chromonema.

2. "Lamp-brush chromosomes" are typical diplotene chromosomes, but with
tremendously enlongated chromonemata. The side branches are the gyres of the
major coils of the individual chromonemata, which have laterally separated from
each other.

I am greatly indebted to Prof. F. Schrader and Dr. S. Hughes-Schrader, Co-
lumbia University, for critically reading the manuscript.

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INDEX

A BSTRACTS of scientific papers presented
at the Marine Biological Laboratory, Sum-
mer of 1945, 184.

Agglutination of starfish sperm by fertilizin, 84.

Annual report of the Marine Biological Labora-
tory, 1.

Arbacia punctulata, dilution medium and sur-
vival of the spermatozoa, 162.

Arbacia punctulata egg when centrifuged in
single salt solutions, stratification and
breaking of the, 72.

Azide and cyanide, the effect of, on the develop-
ment of frogs' eggs, 122.

BIOCHEMISTRY of Tetrahymena, IV, 131.

Biochemistry of Tetrahymena, VII, 229.
Bryozoa, studies on fresh-water. XVI. Fre-

dericella australiensis var. browni, n. var.,

215.
Bryozoa, studies on marine. I. Aeverrillia

setigera (Hincks) 1887, 201.

CHEMICAL factors influencing artificial ac-
tivation of Nereis eggs, 144.

Ciliates from Phytia setifer, 95.

Cochliophilus depressus gen. nov., sp. nov. and
Cochliophilus minor sp. nov., holotrichous
ciliates from the mantle cavity of Phytia
setifer (Cooper), 95.

Cyanide and azide, the effects of, on the de-
velopment of frogs' eggs, 122.

Cyanide, effect of, on respiration of Paramecium
caudatum and Paramecium aurelia, 76.

DEVELOPMENT of marine fouling com-
munities, 103.

DEWEY, VIRGINIA C. See GEORGE W. KIDDER

Dilution medium and survival of the sperma-
tozoa of Arbacia punctulata. I. Effect of
the medium on fertilizing power, 162.

Dominant lethality and correlated chromosome
effects in Habrobracon eggs x-rayed in
diplotene and in late metaphase I, 61.

T^FFECT of cyanide on respiration in Para-
mecium caudatum and Paramecium au-
relia, 76.

pRESH-WATER Bryozoa, XVI, 215.

Frogs' eggs, the effects of cyanide and azide
on the development of, 122.

apparatus, in chicken gizzard epi-
thelium by means of the quartz micro-
scope, a study of the, 157.

Grasshopper, structure of meiotic chromosomes
in the, 242.

•LJABROBRACON eggs x-rayed in diplotene
and in late metaphase I, dominant lethality
and correlated chromosome effects in, 61.

HARVEY, ETHEL BROWNE. Stratification and
breaking of the Arbacia punctulata egg
when centrifuged in single salt solutions,
72.

HAYASHI, TERU. Dilution medium and sur-
vival of the spermatozoa of Arbacia punc-
tulata. I. Effect of the medium on ferti-
lizing power, 162.

Heteroagglutinins in lobster-blood, 193.

Heterocineta phoronopsidis sp. nov., a ciliate
from the tentacles of Phoronopsis viridis
Hilton, 180.

HlBBARD, HOPE AND GEORGE I. LAVIN. A

study of the Golgi apparatus in chicken
gizzard epithelium by means of the quartz
microscope, 157.

Holotrichous ciliates from the mantle cavity of
Phytia setifer (Cooper), 95.

BIDDER, GEORGE W. AND VIRGINIA C.
DEWEY. Studies on the biochemistry of
Tetrahymena. IV. Amino acids and their
relation to the biosynthesis of thiamine,
131.

KIDDER, GEORGE W. AND VIRGINIA C. DEWEY.
Studies on the biochemistry of Tetra-
hymena. VII. Riboflavin, pantothen, bi-
otin, niacin, and pyridoxine in the growth
of T. geleii W, 229^

KOZLOFF, EUGENE N. Cochliophilus depressus
gen. nov., sp. nov. and Cochliophilus
minor sp. nov., holotrichous ciliates from
the mantle cavity of Phytia setifer
(Cooper), 95.

KOZLOFF, EUGENE N. Heterocineta phoronop-
sidis sp. nov., a ciliate from the tentacles
of Phoronopsis viridis Hilton, 180.

T AVIN, GEORGE I. See HOPE HIBBARD,

^ 157.

LEFEVRE, PAUL G. Certain chemical factors

influencing artificial activation of Nereis

eggs, 144.

258

INDEX

259

ARINE Biological Laboratory, Annual re-

port of the, 1.
Marine Bryozoa, I, 201.
Marine fouling communities, the development

of, 103.

Meiotic chromosomes in the grasshopper, 242.
METZ, CHARLES B. The agglutination of star-

fish sperm by fertilizin, 84.
MOOG, FLORENCE. See S. SPIEGELMAN, 122.

eggs, certain chemical factors in-
fluencing artificial activation of, 144.

DACE, D. M. The effect of cyanide on
respiration in Paramecium caudatum and
Paramecium aurelia, 76.

Paramecium caudatum and Paramecium au-
relia, effect of cyanide on respiration of, 76.

D IS, HANS. The structure of meiotic chro-
mosomes in the grasshopper and its bearing
on the nature of "chromomeres" and
"lamp-brush chromosomes," 242.

ROGICK, MARY DORA. Studies on fresh-water
Bryozoa. XVI. Fredericella australiensis
var. browni, n. var., 215.

ROGICK, MARY DORA. Studies on marine
Bryozoa. I. Aeverrillia setigera (Hincks)
1887, 201.

CCHEER, BRADLEY T. The development of

marine fouling communities, 103.
SCHEER, BRADLEY T. See ALBERT TYLER, 193.

Scientific papers presented at the Marine Bio-
logical Laboratory, Summer of 1945, 184.

SPIEGELMAN, SOL AND FLORENCE MOOG. A
comparison of the effects of cyanide and
azide on the development of frogs' eggs,
122.

Starfish sperm, agglutination of, by fertilizin,
84.

Stratification and breaking of the Arbacia
punctulata egg when centrifuged in single
salt solutions, 72.

Structure of meiotic chromosomes in the grass-
hopper and its bearing on the nature of
"chromomeres" and "lamp-brush chromo-
somes," 242.

Studies on the biochemistry of Tetrahymena.
IV. Amino acids and their relation to the
biosynthesis of thiamine, 131.

rpETRAHYMENA, studies on the biochem-

istry of, 131.
TYLER, ALBERT AND BRADLEY T. SCHEER.

Natural hetercagglutinins in the serum of

the spiny lobster, Panulirus interruptus.

II. Chemical and antigenic relation to

blood proteins, 193.

ANNA. Dominant lethality and
correlated chromosome effects in Habro-
bracon eggs x-rayed in diplotene and in
late metaphase I, 61.

V-RAYS and dominant lethality, 61.

Volume 89 Number 1

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago

E. N. HARVEY, Princeton University GEORGE T. MOORE, Missouri Botanical Garden

SELIG HECHT, Columbia University -p. H. MORGAN, California Institute of Technology

LEIGH HOADLEY, Harvard University Q H pARKERt Harvard University

L. IRVING, Swarthmore College RKnprELD Harvard Universitv

M. H. JACOBS, University of Pennsylvania *• C' KEDFIELD. Harvard University

H. S. JENNINGS, Johns Hopkins University F- SCHRADER, Columbia University

FRANK R. LILLIE, University of Chicago DOUGLAS WmTAKER, Stanford University

H. B. STEINBACH, Washington University
Managing Editor

AUGUST, 1945

Printed and Issued by

LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.

SERIAL LIST

A. SERIAL list of the holdings of The Marine Biological Labora-
tory has been published as a separately bound supplement to The
Biological Bulletin. This supplement lists with cross references the
titles of journals in the Library; additional titles and changes are
published annually. A few extra copies of the original list are
still available. Orders may be directed to The Marine Biological
Laboratory.

MICROFILM SERVICE

1 HE Library of The Marine Biological Laboratory is now pre-
pared to supply microfilms of material from periodicals included in
its extensive list. Through the generosity of Dr. Athertone Seidell,
the essential equipment has been set up and put into operation.
The Staff of The Marine Biological Laboratory Library is anxious to
extend the Microfilm Service, particularly at this time when dis-
tance makes the Library somewhat inaccessible to many who nor-
mally use it. Investigators who wish films should send to the Li-
brarian the name of the author of the paper, its title, and the name
of the periodical in which it is printed, together with the volume
and year of publication. The rates are as follows: $.30 for papers
up to 25 pages, and $.10 for each additional 10 pages or fraction
thereof. It is hoped that many investigators will avail themselves
of this service.

Your Biological News

You would not go to the library to read the daily newspaper — probably
you have it delivered at your home to be read at your leisure. Why, then,
depend upon your library for your biological news ?

Biological Abstracts is news nowadays. Abridgments of all the im-
portant biological literature are published promptly — in many cases before
the original articles are available in this country. Only by having your
own copy of Biological Abstracts to read regularly can you be sure that
you are missing none of the literature of particular interest to you. An
abstract of one article alone, which otherwise you would not have seen,
might far more than compensate you for the subscription price.

Biological Abstracts is now published in seven low priced sections, as
well as the complete edition, so that the biological literature may be avail-
able to all individual biologists. Write for full information and ask for a
copy of the section covering your field.

BIOLOGICAL ABSTRACTS

University of Pennsylvania

Philadelphia, Pa.

LANCASTER PRESS, Inc.

LANCASTER, PA.

THE EXPERIENCE we have
gained from printing some
sixty educational publica-
tions has fitted us to meet
the standards of customers
who demand the best.

We shall be happy to have workers at
the MARINE BIOLOGICAL LABORATORY
write for estimates on journals or
monographs. Our prices are moderate.

INSTRUCTIONS TO AUTHORS

The Biological Bulletin accepts papers on a variety of subjects of biologi-
cal interest. In general, a paper will appear within three months of the date of
its acceptance. The Editorial Board requests that manuscripts conform to the
requirements set below.

Manuscripts. Manuscripts should be typed in double or triple spacing on
one side of paper, 8l/2 by 11 inches.

Tables should be typewritten on separate sheets and placed in correct
sequence in the text. Explanations of figures should be typed on a separate
sheet and placed at the end of the text. Footnotes, numbered consecutively,
may be placed on a separate sheet at the end of the paper.

A condensed title or running page head of not more than thirty-five letters
should be included.

Figures. The dimensions of the printed page, 5 by 7% inches, should be
kept in mind in preparing figures for publication. Illustrations should be large
enough so that all details will be clear after appropriate reduction. Explana-
tory matter should be included in legends as far as possible, not lettered on the
illustrations. Figures should be prepared for reproduction as line cuts or half-
tones; other methods will be used only at the author's expense. Figures to be
reproduced as line cuts should be drawn in black ink on white paper or blue-
lined co-ordinate paper; those to be reproduced as halftones should be mounted
on Bristol board and any designating letters or numbers should be made di-
rectly on the figures. The author's name should appear on the reverse side of
all figures. The desired reduction should be specified on each figure.

Literature cited. The list of literature cited should conform to the style set
in this issue of The Biological Bulletin. Papers referred to in the manuscript
should be listed on separate pages headed "Literature Cited."

Mailing. Manuscripts should be packed flat. Large illustrations may be
rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must
be accompanied by photographic reproductions or tracings that may be folded
to page size.

Reprints. Authors will be furnished, free of charge, one hundred reprints
without covers. Additional copies may be obtained at cost; approximate
figures will be furnished upon request.

THE BIOLOGICAL BULLETIN

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

Subscriptions and similar matter should be addressed to The Biologi-
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts.
Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and '4
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers,
$1.75. Subscription per volume (three issues), $4.50.

Communications relative to manuscripts should be sent to the Manag-
ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts,
between July 1 and October 1 , and to the Department of Zoology, Wash-
ington University, St. Louis, Missouri, during the remainder of the year.

Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa.,

under the Act of August 24, 1912.

BIOLOGY MATERIALS

The Supply Department of the Marine Biological Labora-
tory has a complete stock of excellent plain preserved and
injected materials, and would be pleased to quote prices on
school needs.

PRESERVED SPECIMENS

for

Zoology, Botany, Embryology,
and Comparative Anatomy

LIVING SPECIMENS

for
Zoology and Botany

including Protozoan and
Drosophila Cultures, and
Animals for Experimental and
Laboratory Use.

MICROSCOPE SLIDES

for

Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.

CATALOGUES SENT ON REQUEST

Supply Department

MARINE
BIOLOGICAL LABORATORY

Woods Hole, Massachusetts

CONTENTS

Page
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY . . i

WHITING, ANNA

Dominant lethality and correlated chromosome effects in
Habrobracon eggs x-rayed in diplotene and in late meta-
phase 1 61

HARVEY, ETHEL BROWNE

Stratification and breaking of the Arbacia punctulata egg
when centrif uged in single salt solutions 72

PACE, D. M.

The effect of cyanide on respiration in Paramecium caudatum

and Paramecium aurelia 76

METZ, CHARLES B.

The agglutination of starfish sperm by f ertilizin 84

KOZLOFF, EUGENE N.

Cochliophilus depressus gen. nov., sp. nov. and Cochliophilus
minor sp. nov., holotrichous ciliates from the mantle cavity
of Phytia setif er (Cooper) - 95

SCHEER, BRADLEY T.

The development of marine fouling communities 103

SPIEGELMAN, S., AND FLORENCE MOOG

A comparison of the effects of cyanide and azide on the de-
velopment of frogs' eggs 122

Volume 89

Number 2

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania
H. S. JENNINGS, Johns Hopkins University
FRANK R. LILLIE, University of Chicago

CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University
F. SCHRADER, Columbia University
DOUGLAS WHITAKER, Stanford University

H. B. STEINBACH, Washington University
Managing Editor

OCTOBER, 1945

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE & LEMON STS.

LANCASTER, PA.

SERIAL LIST

SERIAL list of the holdings of The Marine Biological Labora-
tory has been published as a separately bound supplement to The
Biological Bulletin. This supplement lists with cross references the
titles of journals in the Library; additional titles and changes are
published annually. A few extra copies of the original list are
still available. Orders may be directed to The Marine Biological
Laboratory.

MICROFILM SERVICE

Your Biological News

You would not go to the library to read the daily newspaper — probably
you have it delivered at your home to be read at your leisure. Why, then,
depend upon your library for your biological news ?

BIOLOGICAL ABSTRACTS

University of Pennsylvania

Philadelphia, Pa.

LANCASTER PRESS, Inc.

LANCASTER, PA.

THE EXPERIENCE we have
gained from printing some
sixty educational publica-
tions has fitted us to meet
the standards of customers
who demand the best.

We shall be happy to have workers at

the MARINE BIOLOGICAL LABORATORY

write for estimates on journals or
monographs. Our prices are moderate.

INSTRUCTIONS TO AUTHORS

Manuscripts. Manuscripts should be typed in double or triple spacing on
one side of paper, SVz by 11 inches.

A condensed title or running page head of not more than thirty-five letters
should be included.

Reprints. Authors will be furnished, free of charge, one hundred reprints
without covers. Additional copies may be obtained at cost; approximate
figures will be furnished upon request.

THE BIOLOGICAL BULLETIN

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

Subscriptions and similar matter should be addressed to The Biologi-
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts.
Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers,
$1.75. Subscription per volume (three issues), $4.50.

Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa.,

under the Act of August 24, 1912.

BIOLOGY MATERIALS

The Supply Department of the Marine Biological Labora-
tory has a complete stock of excellent plain preserved and
injected materials, and would be pleased to quote prices on
school needs.

PRESERVED SPECIMENS

for

Zoology, Botany, Embryology,
and Comparative Anatomy

LIVING SPECIMENS

for
Zoology and Botany

including Protozoan and
Drosophila Cultures, and
Animals for Experimental and
Laboratory Use.

MICROSCOPE SLIDES

for

Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.

CATALOGUES SENT ON REQUEST

Supply Department

MARINE
BIOLOGICAL LABORATORY

Woods Hole, Massachusetts

CONTENTS

Page
KIDDER, GEORGE W., AND VIRGINIA C. DEWEY

Studies on the biochemistry of Tetrahymena. IV. Ammo
acids and their relation to the biosynthesis of thiamine 131

LEFEVRE, PAUL G.

Certain chemical factors influencing artificial activation of
Nereis eggs 144

HIBBARD, HOPE AND GEORGE I. LAVIN

A study of the Golgi apparatus in chicken gizzard epithelium
by means of the quartz microscope 157

HAYASHI, TERU

Dilution medium and survival of the spermatozoa of Arbacia
punctulata. I. Effect of the medium on fertilizing power. . 162

KOZLOFF, EUGENE N.

Heterocineta phoronopsidis sp. nov., a ciliate from the ten-
tacles of Phoronopsis viridis Hilton 180

ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE

BIOLOGICAL LABORATORY, SUMMER OF 1945 184

Volume 89

Number 3

THE

BIOLOGICAL BULLETIN

PUBLISHED BY

THE MARINE BIOLOGICAL LABORATORY

Editorial Board

CARL R. MOORE, University of Chicago
GEORGE T. MOORE, Missouri Botanical Garden
T. H. MORGAN, California Institute of Technology
G. H. PARKER, Harvard University
A. C. REDFEELD, Harvard University
F. SCHRADER, Columbia University
DOUGLAS WHITAKER, Stanford University

H. B. STEINBACH, Washington University
Managing Editor

Marine Biological Laborato

UI BR -A. I* "V

JAN 1 5 1946

WOODS HOLE, MASS.

DECEMBER, 1945

Printed and Issued by

LANCASTER PRESS, Inc.

PRINCE &, LEMON STS.

LANCASTER, PA.

SERIAL LIST

MICROFILM SERVICE

Your Biological News

You would not go to the library to read the daily newspaper — probably
you have it delivered at your home to be read at your leisure. Why, then,
depend upon your library for your biological news?

BIOLOGICAL ABSTRACTS

University of Pennsylvania
Philadelphia, Pa.

LANCASTER PRESS, Inc.

LANCASTER, PA.

THE EXPERIENCE we have
gained from printing some
sixty educational publica-
tions has fitted us to meet
the standards of customers
who demand the best.

We shall be happy to have workers at

the MARINE BIOLOGICAL LABORATORY

write for estimates on journals or
monographs. Our prices are moderate.

INSTRUCTIONS TO AUTHORS

Manuscripts. Manuscripts should be typed in double or triple spacing on
one side of paper, 8Vz by 11 inches.

A condensed title or running page head of not more than thirty-five letters
should be included.

Reprints. Authors will be furnished, free of charge, one hundred reprints
without covers. Additional copies may be obtained at cost; approximate
figures will be furnished upon request.

THE BIOLOGICAL BULLETIN

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

Subscriptions and similar matter should be addressed to The Biologi-
cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts.
Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4
Arthur Street, New Oxford Street, London, W. C. 2. Single numbers,
$1.75. Subscription per volume (three issues), $4.50.

Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa.,

under the Act of August 24, 1912.

BIOLOGY MATERIALS

The Supply Department of the Marine Biological Labora-
tory has a complete stock of excellent plain preserved and
injected materials, and would be pleased to quote prices on
school needs.

PRESERVED SPECIMENS

for

Zoology, Botany, Embryology,
and Comparative Anatomy

LIVING SPECIMENS

for
Zoology and Botany

including Protozoan and
Drosophila Cultures, and
Animals for Experimental and
Laboratory Use.

MICROSCOPE SLIDES

for

Zoology, Botany, Embryology,
Histology, Bacteriology, and
Parasitology.

CATALOGUES SENT ON REQUEST

Supply Department

MARINE
BIOLOGICAL LABORATORY

Woods Hole, Massachusetts

CONTENTS

Page

TYLER, ALBERT AND BRADLEY T. SCHEER

Natural heteroagglutinins in the serum of the spiny lobster,
Panulirus interruptus. II. Chemical and antigenic relation
to blood proteins 193

ROGICK, MARY DORA

Studies on marine Bryozoa. I. Aeverrillia setigera (Hincks)
1887 201

ROGICK, MARY DORA

Studies on fresh-water Bryozoa. XVI. Fredericella austra-
liensis var. browni, n. var 215

KIDDER, GEORGE W. AND VIRGINIA C. DEWEY

Studies on the biochemistry of Tetrahymena. VII. Ribo-
flavin, pantothen, biotin, niacin and pyridoxine in the growth
of T. geleii W 229

Ris, HANS

The structure of meiotic chromosomes in the grasshopper
and its bearing on the nature of "chromomeres" and "lamp-
brush chromosomes" . 242

MBL/WHOI LIBRARY

I I I I I III! • II I • •• ••

lilH 17J7 /

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