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
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
VOLUME 91
AUGUST TO DECEMBER, 1946
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
11
THE BIOLOGICAL BULLETIN is issued six times a year at the
Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn-
sylvania.
Subscriptions and similar matter should be addressed to The
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Massachusetts. Agent for Great Britain: Wheldon and Wesley,
Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London,
W. C. 2. Single numbers, $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 September 1, and to the De-
partment 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.
1ANCASTFR PRESS. INC., LANCASTER, PA
CONTENTS
No. 1. AUGUST, 1946
PAGE
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY ... 1
POMERAT, C. M., AND C. M. WEISS
The influence of texture and composition of surface on the attachment
of sedentary marine organisms 57
SCOTT, SISTER FLORENCE MARIE
The developmental history of Amaroecium constellatum. II. Organo-
genesis of the larval action system 66
GIESE, ARTHUR C.
Comparative sensitivity of sperm and eggs to ultraviolet radiations . 81
CARRIKER, MELBOURNE ROMAINE
' Observations on the functioning of the alimentary system of the snail
Lymnaea stagnalis appressa Say . . 88
CHEN, TZE-TUAN
Temporary pair formation in Paramecium bursaria . 112
No. 2. OCTOBER, 1946
WEISZ, PAUL B.
The space-time pattern of segment formation in Artemia salina. . 119
JAKUS, M. A., AND C. E. HALL
Electron microscope observations of the trichocysts and cilia in Para-
mecium 141
PEASE, DANIEL C.
Hydrostatic pressure effects upon the spindle figure and chromosome
movement. II. Experiments on the meiotic divisions of Tradescantia
pollen mother cells 145
BROWN, FRANK A. JR., AND LORRAINE M. SAIGH
The comparative distribution of two chromatophorotropic hormones
(CDH and CBLH) in Crustacean nervous systems 170
MORRISON, PETER R.
Physiological observations on water loss and oxygen consumption in
Peripatus 181
KOZLOFF, EUGENE N.
Studies on ciliates of the family Ancistrocomidae Chatton and Lowff
(order Holotricha, suborder Thigmotricha). III. Ancistrocoma pelse-
neeri Chatton and Lwoff, Ancistrocoma dissimilis sp. nov., and Hypo-
comagalma pholadidis sp. nov .... 189
KOZLOFF, EUGENE N.
Studies on ciliates of the family Ancistrocomidae Chatton and Lwoff
(order Holotricha, suborder Thigmotricha). IV. Heterocineta janickii
Jarocki, Heterocineta goniobasidis sp. nov., Heterocineta fluminicolae
sp. nov., and Enerthecoma properans Jarocki 200
iii
60544
iv CONTENTS
ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL
LABORATORY, SUMMER OF 1946 210
PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GENERAL PHYSI-
OLOGISTS . . 236
No. 3. DECEMBER, 1946
WHITING, P. W.
A strongly intersexual female in Habrobracon . . 243
TOBIAS, J. M., AND J. J. KOLLROS
Loci of action of DDT in the cockroach (Periplaneta americana) . 247
BEERS, C. D.
Tillina magna: Micronuclear number, encystment and vitality in diverse
clones; capabilities of amicronucleate races. . . . 256
SCOTT, ALLAN
The effect of low temperature and of hypotonicity on the morphology
of the cleavage furrow in Arbacia eggs. . . 272
BODENSTEIN, DIETRICH
Developmental relations between genital ducts and gonads in Droso-
phila 288
LEHMAN, H. E.
A histological study of Syndisyrinx franciscanus, gen. et sp. nov., an
endoparasitic rhabclocoel of the sea urchin, Strongylocentrotus francis-
canus 295
SPOOR, W. A.
A quantitative study of the relationship between the activity and oxygen
consumption of the goldfish, and its application to the measurement of
respiratory metabolism in fishes .
Vol. 91, No. 1 August, 1946
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-EIGHTH REPORT, FOR THE YEAR 1945 — FIFTY-EIGHTH YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 14, 1945) .... .1
STANDING COMMITTEES
II. ACT OF INCORPORATION
III. BY-LAWS OF THE CORPORATION
IV. REPORT OF THE TREASURER
V. REPORT OF THE LIBRARIAN
VI. REPORT OF THE DIRECTOR 11
Statement 11
Addenda :
1 . Publications from this Laboratory during the years 1941-1945. . 13
2. The Staff 33
3. Investigators and Students 35
4. Tabular View of Attendance, 1941-1945 41
5. Subscribing and Co-operating Institutions 42
6. Evening Lectures 42
7. Shorter Scientific Papers 43
8. Members of the Corporation 44
I. TRUSTEES
EX OFFICIO
FRANK R. LILLIE, President Emeritus of the Corporation, The University of Chicago
LAWRASON RIGGS, President of the Corporation, 120 Broadway, New York City
E. NEWTON HARVEY, Vice President of the Corporation, Princeton University
CHARLES PACKARD, Director, Marine Biological Laboratory
OTTO C. GLASER, Clerk of the Corporation, Amherst College
DONALD M. BRODIE, Treasurer, 522 Fifth Avenue, New York City
EMERITUS
E. G. CONKLIN, Princeton University
B. M. DUGGAR, University of Wisconsin
W. E. GARREY, Vanderbilt University
R. A. HARPER, Columbia University
Ross G. HARRISON, Yale University
H. S. JENNINGS, University of California
F. P. KNOWLTON, Syracuse University
2 MARINE BIOLOGICAL LABORATORY
R. S. LILLIE, The University of Chicago
*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 1949
W. R. AMBERSON, University of Maryland School of Medicine
P. B. ARMSTRONG, Syracuse University
L. G. BARTH, Columbia University
S. C. BROOKS, University of California
W. C. CURTIS, University of Missouri
H. B. GOODRICH, Wesleyan University
A. C. REDFIELD, Harvard University
C. C. SPEIDEL, University of Virginia
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
A. K. PARPART, Princeton University
FRANZ SCHRADER, Columbia University
B. H. WILLIER, Johns Hopkins University
EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES
LAWRASON RIGGS, Ex officio. Chairman
E. N. HARVEY, Ex officio
* Deceased.
ACT OF INCORPORATION
D. M. BRODIE, Ex officio
CHARLES PACKARD, Ex officio
P. B. ARMSTRONG, to serve until 1947
L. G. EARTH, to serve until 1946
P. S. GALTSOFF, to serve until 1947
WM. RANDOLPH TAYLOR, to serve until 1946
THE LIBRARY COMMITTEE
A. C. REDFIELD, Chairman
E. G. BALL
S. C. BROOKS
M. E. KRAHL
J. W. MAYOR
THE APPARATUS COMMITTEE
E. P. LITTLE, 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
F. M. LANDIS, Chairman
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
II. ACT OF INCORPORATION
No. 3170
COMMONWEALTH OF MASSACHUSETTS
Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T.
Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells,
William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves
with the intention of forming a Corporation under the name of the Marine Biological
Laboratory, for the purpose of establishing and maintaining a laboratory or station for
4 MARINE BIOLOGICAL LABORATORY
scientific study and investigation, and a school for instruction in biology and natural his-
tory, and have complied with the provisions of the statutes of this Commonwealth in such
case made and provided, as appears from the certificate of the President, Treasurer, and
Trustees of said Corporation, duly approved by the Commissioner of Corporations, and
recorded in this office ;
Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu-
setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi-
ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck,
their associates and successors, are legally organized and established as, and are hereby
made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB-
ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties,
and restrictions, which by law appertain thereto.
Witness my official signature hereunto subscribed, and the seal of the Commonwealth
of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord
One Thousand Eight Hundred and Eighty-Eight.
[SEAL]
HENRY B. PIERCE,
Secretary of 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 arid 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 :
REPORT OF THE TREASURER 5
(B) Trustees ex officio, who shall be the President and Vice President of the Cor-
poration, the Director of the Laboratory, the Associate Director, the Treasurer, and
the Clerk;
(C) Trustees 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 1945.
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 de-
tailed reports :
6 MARINE BIOLOGICAL LABORATORY
I. Assets
1. Endowment Assets
As of December 31, 1945, the total book value of all the Endowment Assets,
including the Scholarship Funds, was $966,772.16, a loss for the year of $17,128.41.
The decline was due, as in the last two years, to losses on the mortgage participa-
tions on New York City realty held in the Trust Funds.
At the end of the year $831,993.01 was invested in marketable securities (bonds,
preferred stocks and common stocks) with a market value of $910,162.31. $125,-
753.85 was invested in mortgage participations on New York City real estate.
$9,025.30 was in uninvested principal cash.
The Treasurer's estimate of the actual value of the $125,753.85 in mortgage
notes and participations held on December 31 is $85,750.00. With the market
value of $910,162.31 on marketable securities and the $9,025.30 in cash this makes
a total current valuation of $1,004,937.61 compared with total book value of
$966,772.16.
The increase for the year in market values, $75,454.79, is largely due to the
rise in common stock prices.
t
2. Plant Assets
There were no changes of any consequence in Plant Assets during the year.
The Reserve Fund was increased nearly $10,000.00 to a total of $26,830.71 by the
transfer of the Crane Co. dividends, part of the General Biological Supply House
dividends and other items of current income.
3. Current Assets
The total of current assets increased $10,730.68 during 1945 to a total of
$212,970.35. Current Liabilities at the end of the year were $2,754.70. Current
Surplus increased $12,277.94 to a total of $196,337.90.
II. Income and Expenditures
The return to more normal operations for the Laboratory last summer resulted
in larger totals for both income and expense items. Total income was $182,818.23,
total expense including depreciation reserves of $26,968.12 was $173,044.95, giving
a net surplus for the year of $9,773.28.
EXHIBIT A
MARINE BIOLOGICAL LABORATORY BALACE SHEET, DECEMBER 31, 1945
Assets
Endowment Assets and Equities :
Securities and Cash in Hands of Central Hanover Bank and
Trust Company, New York, Trustee $ 950,130.04
Securities and Cash in Minor Funds 16,642.12
$ 966,772.16
REPORT OF THE TREASURER
Plant Assets :
Land $ 111,425.38
Buildings 1,326,345.54
Equipment 187,837.87
Library 337,266.01
Less Reserve for Depreciation
Reserve Fund, Securities and Cash
Book Fund, Securities and Cash .
$1,962,874.80
677,140.22
Current Assets :
Cash
Accounts Receivable
Inventories :
Supply Department $ 44,441.66
Biological Bulletin 20,1 17.40
$1,285,734.58
26,830.71
18,282.46
$ 30,467.02
20,396.05
64,559.06
$1,330,847.75
Investments :
Devil's Lane Property $ 46,556.99
Gansett Property 1,749.92
Stock in General Biological Supply House,
Inc 12,700.00
Other Investment Stocks 20,095.00
Retirement Fund 11,517.82
Prepaid Insurance
Items in Suspense
92,619.73
4,033.08
895.41
$ 212,970.35
Total Assets $2,510,590.26
Liabilities
Endowment Funds :
Endowment Funds $ 948.646.S2
Reserve for Amortization of Bond Premiums.. 1,483.22
Minor Funds
Plant Funds :
Mortgage Notes Payable
Donations and Gifts $1,172,564.04
Other Investments in Plant from Gifts and
Current Funds 153,283.71
Current Liabilities and Surplus :
Accounts Payable
Items in Suspense
Reserve for Repairs and Replacements
Current Surplus
$ 950,130.04
16,642.12
$ 5,000.00
$1,325,847.75
$ 2,754.70
1,799.63
12,078.12
196,337.90
$ 966,772.16
$1,330,847.75
$ 212,970.35
Total Liabilities $2,510,590.26
MARINE BIOLOGICAL LABORATORY
EXHIBIT B
MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE,
YEAR ENDED DECEMBER 31, 1945
Total Net
Expense Income Expense Income
Income :
General Endowment Fund
$ 32,214.07
$ 32,214.07
Library Fund
9,479.18
9,479.18
Donations
755.00
755.00
Instruction -.
$ 9,554.39
7,220.00
$ 2,334.39
Research
4,550.59
17,434.24
12,883.65
Evening Lectures
86.35
86.35
Biological Bulletin and Membership Dues.
6,393.65
8.775.63
2,381.98
Supply Department
39,255.03
47,812.56
8,557.53
Mess
24,146.52
20,750.36
3,396.16
Dormitories
27,443.23
14,547.91
12,895.32
(Interest and Depreciation charged to above
3 Departments)
(25,574.03)
25,574.03
Dividends, General Biological Supply House,
Inc
14.732.00
14.732.0C
Dividends, Other Investment Stocks
725.00
725.00
Rents :
Bar Neck Property
767.65
6,000.00
5,232.35
Janitor House
30.89
360.00
329.11
Danchakoff Cottages
240.86
275.00
34.14
Sale of Library Duplicates, Micro Film, etc.
344.74
344.74
Microscope and Apparatus Rental
1,372.54
1,372.54
Sundry Income
20.00
20.00
Maintenance of Plant :
Buildings and Grounds
23,642.27
23,642.27
Apparatus Department
4,911.52
4,911.52
Chemical Department
2,265.30
.
2,265.30
Library Expense
6,487.95
/
6,487.95
Workmen's Compensation Insurance ....
526.63
526.63
Truck Expense
238.60
238.60
Bay Shore Property
92.78
92.78
Great Cedar Swamp
21.00
21.00
General Expenses :
Administration Expense 15,168.99 15,168.99
Endowment Fund Trustee and Safe-Keep-
ing 1,028.45 1,028.45
Bad Debts 375.97 375.97
Special Repairs on account of 1944 Hurri-
cane Damage 4,297.24 4,297.24
Interest 125.00 125.00
Reserve for Depreciation 26,968.12 26,968.12
$173,044.95 $182,818.23 $104,862.04 $114,635.32
Excess of Income over Expense carried to
Current Surplus 9,773.28 9,773.28
$182,818.23 $114,635.32
REPORT OF THE LIBRARIAN
EXHIBIT C
MARINE BIOLOGICAL LABORATORY. CURRENT SURPLUS ACCOUNT,
YEAR ENDED DECEMBER 31, 1945
Balance January 1, 1945 $184,059.96
Add:
Excess of Income over Expense for Year as shown, in Exhibit B . . $ 9,773.28
Gain on Gansett Lots Sold 464.18
Bad Debts Recovered 82.23
Mortgage Payable, Transferred to Plant Funds 5,000.00
Reserve for Depreciation Charged to Plant Funds 26.968.J2 42,287.81
$226,347.77
Deduct :
Payments from Current Funds during Year for Plant Assets as
shown in Schedule IV:
Buildings $ 7,402.65
Equipment 4,462.50
Library 7,500.43
$19,365.58
Less Received for Plant Assets Sold 5,600.00
$13,765.58
Pensions Paid $ 3,460.00
Loss on Retirement Fund Securities 847.32
$ 4,307.32
Less Retirement Fund Income 311.79
$ 3,995.53
Transfers to Reserve Fund :
Portion of Dividends from General Biological Sup-
ply House, Inc $ 2,500.00
Dividends from Crane Co 625.00
Income from Operation and Sale of Property 445-
51 W. 23rd and 450-2 W. 24th Sts., N. Y. C. 8,947.72
Gansett Property Profits, 1944 176.04 12,248.76 30,009.87
Balance, December 31, 1945 $196,337.90
Respectfully submitted,
DONALD M. BRODIE,
Treasurer
V. REPORT OF THE LIBRARIAN
The sum $12,262.54 appropriated to the library in 1945 was expended as
follows: books, $469.99; serials, $2,625.79; binding, $577.60; express, $43.22;
supplies, $147.84; salaries, $7,262.54 ($1,150 of this sum was contributed by the
Woods Hole Oceanographic Institution) ; back sets, $1,104.98; insurance, $45.00;
10 MARINE BIOLOGICAL LABORATORY
sundries, $5.00 ; total, $12,281 .96. The cash receipts of the library totalled $344.74 :
for microfilms, $220.34 ($62.17 expenses paid by the library and accounted above
under "supplies") ; sale of duplicates, $122.74; sale of the "Serial List," Biological
Bulletin supplement number, $1.66. This sum, $344.74, reverts to the laboratory
and does not include rent payments for library readers which are collected by the
main office. There were 49 library readers accommodated in the library during
the summer of 1945.
Of the Carnegie of New York Fund, $126.35 was expended for the completion
of one journal and the partial completion of another.
The sum appropriated by the Woods Hole Oceanographic Institution in 1945
for purchases was $800. A balance of $949.39 remaining from 1944 made an
available total of $1,749.39. Of this sum $947.12 was expended, leaving a balance
of $802.27 towards future purchases. In addition to the above, the Woods Hole
Oceanographic Institution contributed $1,150 (see above under salaries).
During 1945 the library received 902 current journals: 279 (8 new) by sub-
scription to the Marine Biological Laboratory; 30 (7 new) to the Woods Hole
Oceanographic Institution; exchanges 352 (6 new; 145 reinstated foreign) and 58
(35 foreign reinstated) with the Woods Hole Oceanographic Institution publica-
tions; 177 as gifts to the former and 6 to the latter. The library acquired 206
books : 77 by purchase of the Marine Biological Laboratory ; 44 by purchase of
the Woods Hole Oceanographic Institution; 10 gifts by the authors; 46 gifts by
the publishers ; 20 by miscellaneous donors and 9 from Miss Jane Strong. There
were 22 back sets of serial publications completed: 14 purchased by the Marine
Biological Laboratory (one with the Carnegie Fund) ; 2 by the Woods Hole
Oceanographic Institution ; 5 by exchange of the "Biological Bulletin" and one by
exchange of duplicate material. Partially completed sets were 54: purchased by
the Marine Biology Laboratory, 27 (1 by the Carnegie Fund) ; purchased by the
Woods Hole Oceanographic Institution, 2 ; by exchange with the "Biological
Bulletin," 2; by gift and exchange of duplicate material, 23.
The reprint additions to the library were 4,620; current of 1944, 604; current
of 1945, 64; and of previous dates, 3,952. A total of 6,390, 2,130 not duplicates
of our holdings, were presented to the library ; 4,295 by Mrs. Meigs ; 67 by Dr.
H. G. Cassidy; 627 by the University of Utah; 26 by Dr. B. M. Davis; and 1,375
by Dr. L. C. Wyman. The large collection of Dr. Carrey's reprints presented last
year have not as yet been counted nor started on the way toward cataloguing.
At the end of the year 1945 the library contained 53,990 bound volumes and
137,674 reprints.
Readers of the library report will be glad to note the number of foreign ex-
changes that have already been reinstated during 1945, both for the "Biological
Bulletin" and for the Woods Hole Oceanographic Institution publications : 145 for
the former and 35 for the latter. Next year's report will probably show the pre-war
number reinstated save only for Germany and perhaps for Russia since we get very
poor response from that country. Nor have we heard anything in regard to the
German journals on order with Otto Harrassowitz which are apparently stalled
if not destroyed in Leipzig. The library committee that is working with the State
Department to get these released has nothing so far to report to this library.
REPORT OF THE DIRECTOR
VI. REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY:
Gentlemen:
I herewith submit a report on the fifty-eighth session of the Marine Biological
Laboratory.
1. Attendance
Since 1943 our total' attendance has increased from the low point reached that
year. In 1944 it was 53 per cent of the pre-war average of 490; in 1945 it was
63 per cent. This increase is found among the independent investigators and the
students; the beginning investigators and research assistants, who, as I explained
in the last report, belong in one group, are still sparsely represented. In 1945
there were only 36, whereas the pre-war average was 130. The advance registra-
tion for 1946 shows an encouraging increase in this group. Many of the appli-
cants are veterans who are taking advantage of Government funds provided under
the G. I. Bill of Rights.
2. Building Repairs
One of the inevitable effects of the war has been the deterioration of our build-
ings. Lack of materials and labor has up until now prevented all but the most
essential repairs from being made. Fortunately, we are now able to begin to put
our house in order, in spite of the shortage of some critical materials. To deter-
mine what work should be done, a Committee on Special Repairs, under the able
leadership of Mr. C. L. Claff, conducted a thorough survey of all of the buildings
and made detailed recommendations. The report, a model of completeness as
drawn up by Mr. Claff, calls for the ultimate expenditure of approximately $145,000
for present repairs and future desirable improvements not only in the buildings but
also in equipment for the Apparatus Department and the Supply Department.
The Executive Committee voted to expend the entire Reserve Fund, amounting
to $25,000, and all but a minimum of the current cash on hand for making the most
urgently needed repairs at once. It also laid plans for securing outside funds with
which to complete the changes called for in the report, and to purchase apparatus.
In addition, funds for a new building and for additional endowment are to be
sought.
Many of the essential repairs have already been made. The Mess kitchen,
never properly restored after the Navy occupation, is now in good condition, and
improvements in the dining room have been made. The Botany Building, unused
for several seasons, has been put to rights with new plumbing, wall tables, shelves,
and other fixtures. Replacements have been made in the Supply Department and
Rockefeller Building; hot water systems are installed in the residences heretofore
not so provided ; and much painting has been done. This work was accomplished
in large measure by our permanent staff, under the direction of Mr. MacNaught.
All the men worked faithfully and energetically, and have completed the assigned
tasks in a most satisfactory manner. It is hoped that waterproofing of the Crane
and Brick Buildings may be completed before the 1946 season begins. As soon as
this has been satisfactorily finished, those Laboratory rooms which have been dam-
aged by water can be made presentable.
12 MARINE BIOLOGICAL LABORATORY
3. The Housing Problem
An unexpected outcome of war-time activities is the housing shortage. Before
the war our residences and the houses in the village could accommodate 450 to 500
persons during the course of a summer. But when the Oceanographic Institution
emharked on extensive defense projects, the number of its workers increased from
comparatively few to upwards of 250, most of whom are year round residents.
They now occupy most of the available houses in the village ; some are forced to
live as far away as North Falmouth and Hyannis. As a result of this crowding
we shall be unable, in the summer of 1946, to take care of more than 375 investi-
gators and students — that is, about 100 less than our pre-war average. Indeed,
we can accommodate this number only because the authorities of the U. S. Fish
and Wild Life Commission have granted us the use of a part of the Fisheries
residence. For their cooperation in this, and in many other ways, the Laboratory
is grateful.
When it is possible once more to build houses, some of this pressure for living
space will be relieved. To encourage investigators to have homes here in Woods
Hole, the Laboratory has opened up the Devil's Lane tract, situated a mile and
more from the center of the village, between the State Road to Falmouth and the
railroad. About 100 lots will presently be available.
In the meantime, the number of applicants for research space will undoubtedly
increase, and we shall be unable to find places for all cmalified investigators who
wish to come. The Administration thus faces the unwelcome prospect of having
to choose between applicants. The Executive Committee has ruled that investi-
gators, instructors, and students should have preference over Library readers in
the residences and at the Mess. But some further method of selection must be
followed until the housing shortage is relieved.
4. Financial Problems
The report of the Treasurer shows that our financial condition is sound; that
is, we are free from debt, and have about $57,000 in the Reserve and Current Cash
accounts. But most of this has already been ear-marked to pay for the most
necessary repairs, and for foreign journals not yet delivered. We shall still need
a larger amount for other needed repairs and replacements. When these have
been made we can say that our regular income from all sources is sufficient to
maintain the Laboratory on its present basis. But in order to expand our research
facilities we must have additional funds. It is estimated that $30,000 each year
should be spent for this purpose.
5. Gifts
Mr. Allen R. Memhard has provided a fund of $1,000, the income of which
may be awarded to a qualified student who has completed the Embryology course.
Mrs. Adele K. Strieker has presented to the Laboratory the sum of $50 in
memory of her son, Capt. George J. Strieker, who worked here during the sum-
mers of 1933 and 1934.
Dr. A. C. Redfield contributed $100 for a hedge and trees to be planted to the
east of the Stone Building.
Donations for current purposes received during the year were as follows :
Mrs. E. B. Meigs, $25.00; Dr. William D. Curtis, $100.00; M. B. L. Associates,
$630.00.
REPORT OF THE DIRECTOR 13
6. Deaths
This year \ve have sustained irreparable losses by death ; Dr. T. H. Morgan,
Trustee since 1897, whose scientific achievements and devotion to this Laboratory
from its earliest days contributed greatly to its growth in usefulness and influence,
and Dr. C. E. McClung, elected Trustee in 1913, active in all Laboratory affairs,
especially in the building up of our great Library.
7. Election 'of Trustees
At the meeting of the Corporation, held August 14, 1945. the following were
elected Trustees Emeriti: Dr. F. P. Knowlton, elected Trustee in 1922; Dr. R. S.
Lillie, elected Trustee in 1921.
The following were elected Trustees: Dr. P. B. Armstrong, Professor of
Anatomy, College of Medicine, Syracuse University ; Dr. A. K. Parpart, Associate
Professor of Biology, Princeton University.
8. Publications
The Executive Committee voted to print in this Report a list of papers, based
wholly or in part on work done at this Laboratory, and published during the years
1941-1945. A similar list, which appeared in the Annual Report of 1908, covered
the years from the beginning of the Laboratory in 1888 to 1907. It is hoped that
eventually a complete compilation of titles to include the intervening years may
be made.
Appended as parts of this Report are :
1. Publications from this Laboratory during the years 1941-1945.
2. The Staff.
3. Investigators and Students.
4. Tabular View of Attendance, 1941-1945.
5. Subscribing and Cooperating Institutions.
6. Evening Lectures.
7. Shorter Scientific Papers.
8. Members of the Corporation.
Respectfully submitted,
CHARLES PACKARD,
Director
(
1. PUBLICATIONS FROM THE MARINE BIOLOGICAL LABORATORY, 1941-1945
Note: An asterisk before a title indicates that the work was done only in part at this Laboratory.
ABELL, R. G. On the comparative permeability of blood capillary sprouts, newly formed capil-
laries, and older capillaries. Anat. Rcc., 82: 1942.
ABELL, R. G. See also Zweifach, Abell, Chambers, and Clowes, 1945.
ABELL, R. G. AND I. H. PAGE. *Behavior of the arterioles in hypertensive rabbits and in normal
rabbits following injections of angiotonin. Biol. Bull., 81: 1941 (abs.).
ABELL, R. G. AND I. H. PAGE. *The reaction of peripheral blood vessels to angiotonin, renin,
and other pressor agents. Jour. E.rp. Med., 75 : 1942.
ADDISON, W. H. F. *The distribution of elastic tissue in the arterial pathway to the carotid
bodies in the adult dog. Bio!. Bull., 81: 1941 (abs.).
ADDISON, W. H. F. *Histologic methods adapted for rat tissues. A Chapter in "The Rat in
Laboratory Investigation," Lippincott, 1942.
ADDISON, W. H. F. The hypophysis of the goose-fish. Anat. Rcc.. 85: 1943 (abs.).
14 MARINE BIOLOGICAL LABORATORY
ADDISON, W. H F. *The extent of the carotid pressoreceptor area in the cat as indicated by
its special elastic-tissue wall. Anat. Rec., 91 : 1945.
ADDISON, W. H. F. *The arterial relations of the glomus caroticum in the rabbit. Anat. Rec.,
88: 1944 (abs.).
ALBAUM, H. G. AND BARRY COMMONER. The relation between the four-carbon acids and the
growth of bat seedlings. Blol. Bull, 80: 1941.
ALLEE, W. C. *Integration of problems concerning protozoan populations with those of gen-
eral biology. Amer. Nat., 75: 1941.
ALLEE, W. C. AND RUTH M. MERWIN. The effect of carbon dioxide on the rate of cleavage
in frog's eggs. Anat. Rec. Suppl, 81: 1941 (abs.).
ALLEE, W. C. AND RUTH M. MERWIN. The effect of low concentration of carbon dioxide on
the cleavage rate in frog's eggs. Ecology, 24: 1943.
ALLEE, W. C, A. J. FINKEL AND H. R. GARNER. Factors affecting rate of cleavage in Arbacia;
the accelerating action of copper. Anat. Rec. SuppL, 81: 1941 (abs.).
ALLEE, W. C., A. J. FINKEL AND H. R. GARNER. Copper and the acceleration of cleavage.
Jour. Cell. Comp. Physiol, 20: 1942.
ALLEE, W. C., A. J. FINKEL, H. R. GARNER, R. M. MERWIN AND G. E. EVANS. Some effects
of homotypic extracts on the rate of cleavage of Arbacia eggs. Biol. Bull., 83 : 1942.
ALLEE, W. C. AND MARJORIE B. DOUGLIS. A dominance order in the hermit crab, Pagurus
longicarpus Say. Ecology, 26 : 1945.
ALSUP, F. W. Photodynamic action in the eggs of Nereis limbata. Jour. Cell. Comp. Physiol.,
17: 1941.
ALSUP, F. W. Photodynamic studies on Arbacia eggs. Biol. Bull., 81 : 1941.
ALSUP, F. W. The effects of light alone and photodynamic action on relative viscosity of
Amoeba protoplasm. Physiol. Zool., 15: 1942.
ANDERSON, T. F. *The study of colloids with the electron microscope. Advances in Colloid
Science, 1 : 1942.
ANDERSON, T. F. *The application of the electron microscope to biology. Collecting Net, 17 :
1942.
ANDERSON, T. F. See also Harvey, E. B. and Anderson, 1943; Luria, Delbruck and Anderson,
1943 ; Richards, Steinbach and Anderson, 1943.
ANDERSON, T. F. AND A. G. RICHARDS. Nature through the electron microscope. Scientific
Month., 55 : 1942.
ANDREW, WARREN. The reticular nature of glia fibers in the cerebrum of the frog and in the
higher vertebrates. Jour. Comp. Neural., 79 : 1943.
ANGERER, C. A. Sec also Hartman, Lewis, Brownell, Sheldon and Angerer. Physiol. Zool.,
17: 1944.
ANGERER, C. A. AND H. ANGERER. *Weight variations of muscles of adrenalectomized frogs in
normal and hypotonic Ringer solutions. Amer. Jour. Physiol., 133: 1941.
ANGERER, C. A. AND K. M. WILBUR. *The action of various types of electric fields on the
relative viscosity of plasmagel of Amoeba proteus. Physiol. Zool., 16 : 1943.
BAILEY, BASIL, P. KORAN AND H. C. BRADLEY. The autolysis of muscle of highly active and
less active fish. Biol. Bull, 83 : 1942.
BAKER, GLADYS E. *Studies in the genus Physalacria. Bull. Torrey Bot. Club, 68 : 1941.
BAKER, H. D. *Notes on Salasiella from Mexico. Nautilus, 54: 1941.
BAKER, H. D. *Zonitid snails from Pacific Islands. Bull. Bishop Museum, Honolulu, 166:
1941.
BAKER, H. D. *Some Haplotrematidae. Nautilus, 54: 1941.
BAKER, H. D. *Puerto Rican Oleacinidae. Nautilus, 55 : 1941.
BAKER, H. D. ^Outline of American Oleacininae and new species from Mexico. Nautilus,
55: 1941.
BAKER, H. D. *A new genus of Mexican helicids. Nautilus, 56: 1942.
BAKER, H. D. *A new genus of Chinese Microcystinae. Nautilus, 56 : 1942.
BAKER, H. D. *Some Antillean helicids. Nautilus, 56: 1943.
BAKER, H. D. *The mainland genera of American Oleacininae. Proc. Acad. Nat. Sci., Phila-
delphia, 95 : 1943.
BAKER, R. F. See Cole and Baker, 1941.
BALL, E. G. A blue chromoprotein found in the eggs of the goose-barnacle. Jour. Biol. Chcm.,
152: 1944.
REPORT OF THE DIRECTOR 15
BALL, ERNEST. The effects of synthetic growth substances on the shoot apex of Tropaeolum
majus. Amer. Jour. Bot., 31 : 1944.
BALLARD, W. W. The mechanism for synchronous spawning in Hydractinia and Pennaria
B iol Bull, 82 : 1942.
BALLENTINE, ROBERT. See Parpart and Ballentine, 1943.
BARNES, W. A. AND O. B. FURTH. Roentgen rays in single and parabiotic mice. Amer. Jour.
Roentgenol. and Radium Therap., 69: 1943.
BARRON, E. S. G. AND J. M. GOLDINGER. Pyruvate metabolism in fertilized and non-fertilized
sea urchin eggs. Biol. Bull., 81 : 1941.
EARTH, L. G. *Neural differentiation without organizer. Jour. Exp. Zool., 87: 1941.
BARTH, L. G. *Oxygen consumption of the amphibian gastrula. Physiol. Zool., 15 : 1942.
BARTH, L. G. *Colloid chemistry of development. A chapter in Alexander's Colloid Chem-
istry. New York, 1944.
BARTH, L. G. The determination of the regenerating hydranth in Tubularia. Physiol. Zool.,
17: 1944.
BARTH, L. G. Sec also Goldin and Earth, 1941.
BARTLETT, J. H. *Transient Anode Phenomena. Trans. Electrochem. Soc., 87 : 1945.
BARTLETT, J. H. *Periodic phenomena at anodes. Phys. Rev., 67 : 1945.
BEAMS, H. W. See Evans, Beams and Smith, 1941.
BEERS, C. D. The role of bacteria in the excystment of the ciliate Didinium nasutum. Biol.
Bull., 89: 1945 (abs.).
BELDA, W. H. *Permeability to water in Pelomyxa carolinensis. 1. Changes in volume of
P. carolinensis in solutions of different osmotic concentration. The Salesianum, 37: 1942.
BELDA, W. H. 2. The contractile vacuoles of P. carolinensis. The Salesianum, 37 : 1942.
BELDA, W. H. 3. The permeability constant for water in P. caroliensis. The Salesianum,
38: 1943.
BERGER, C. A. *Some criteria for judging the degree of polyploidy of cells in the resting stage.
Amer. Nat., 75: 1941.
BERGER, C. A. *Reinvestigation of polysomaty in Spinacia. Bot. Gaz., 102: 1941.
BERGER, C. A. ^Multiple chromosome complexes in animals and polysomaty in plants. Cold
Spring Harbor Symposia, 9: 1941.
BERGER, C. A. *Experimental studies on the cytology of Allium. Torreya (abs.) 44 : 1944.
BERGER, C. A. AND E. R. WITKUS. *A cytological study of c-mitosis in the polysomatic plant
Spinacia oleracea, with comparative observations on Allium cepa. Bull. Torrey. Bot. Club,
70f: 1943.
BERGER, C. A. AND E. R. WITKUS. *Veratrine, a new polyploidy inducing agent. Jour. Hered.,
35: 1944.
BERGER, C. A., E. R. WITKUS AND B. J. SULLIVAN. *The cytological effects of benzene vapor.
Bull. Torrey Bot. Club, 71 : 1944.
BERTHOLF, L. M. Accelerating metamorphosis in the tunicate, Styela partita. Biol. Bull., 89 :
1945 (abs.).
BERTHOLF, L. M. AND S. O. MAST. Metamorphosis in the larva of the tunicate, Styela partita.
Biol. Bull., 87: 1944 (abs.).
BEVELANDER, GERRIT. ^Radioactive phosphate absorption by dentin and enamel. Jour. Dent.
Res., 24: 1945.
BEVELANDER, GERRIT. *The histochemical localization of alkaline phosphatase in the developing
tooth. Jour. Cell. Comp. Physiol., 26 : 1945.
BEVELANDER, GERRIT. *The localization of phosphatase in the cyclic growth of the hair,. Anat.
Rcc., 91 : 1945.
BIRMINGHAM, LLOYD. Regeneration in the early zooid of Amaroucium constellatum. Biol.
Bull, 81: 1941 (abs.).
BLISS, A. F. Derived photosensitive pigments from invertebrate eyes. Jour. Gen. Physiol,
26: 1943.
BOCHE, R. D. AND J. B. BUCK. Studies on the hydrogen ion concentration of insect blood and
their bearing on in vitro cytological technique. Physiol. Zool, 15 : 1942.
BODIAN, DAVID. *Cytological aspects of synaptic function. Physiol. Rev., 22 : 1942.
BODIAN, DAVID. *Poliomyelitic changes in tnultinucleated neurons, with special reference to
the site of action of virus in the cell. Bull. Johns Hopkins Hosp., 77 : 1945.
16 MARINE BIOLOGICAL LABORATORY
BODIAN, DAVID AND R. C. MELLORS. *The regenerative cycle of motor neurons, with special
reference to phosphatase activity. Jour. E.\-p. Mcd., 81 : 1945.
BOELL, E. J. AND L. L. WOODRUFF. Respiratory metabolism and mating types in Paramoecium
calkinsi. Jour. E.vpcr. Zoo!., 87: 1941.
BOTSFORD, E. FRANCES. The effect of physostigmine on the responses of earthworm body wall
preparations to successive stimuli. Biol. Bull., 80: 1941.
BRADLEY, H. C. Sec Bailey, Koran and Bradley, 1942.
BROOKS, M. M. *Infrared spectrophotometric studies on hemoglobin as affected by cyanide,
methylene blue, and carbon monoxide. Amcr. Jour. PhysioL, 132: 1941.
BROOKS, M. M. Effects of CO and methylene blue upon O~ consumption of shark blood. Proc.
Soc. Exp. Biol. Mcd., 46: 1941.
BROOKS, M. M. Interpretations of effects of CO and CN on oxidations in living cells. Col-
lecting Net, 16:' 1941.
BROOKS, M. M. Further interpretations of the effects of CO and CN on oxidations in living
cells. Biol. Bull., 81: 1941.
BROOKS, M. M. The effect of thiamine chloride on the oxygen consumption and the develop-
ment of Arbacia punctulata at different stages. Biol. Bull., 83 : 1942.
BROOKS, M. M. Mechanism of fertilization of eggs. Federation Proc., 2 : 1943.
BROOKS, M. M. Methylene blue, potassium cyanide, and carbon monoxide as indicators for
studying the oxidation-reduction potentials of developing marine eggs. Biol. Bull., 84 :
1943.
BROOKS, M. M. *E1 mecanismo de accion del azul de metileno en las celulas vivas. Adas
Acad. Nacional de Ciensa c.ractas, fisicas y naliialcs de Lima, Peru, 7 : 1944.
BROOKS, M. M. *The effect of methylene blue on performance efficiency at high altitudes.
Jour. Aviation Mcd., 16: 1945.
BROOKS, M. M. *Oxidation-reduction studies on Penicillium notatum and other organisms.
Biol. Bull., 89 : 1945.
BROOKS, M. M. Electrode potential measurements of Penicillium notatum. Federation Proc.,
4: 1945.
BROOKS, M. M. Mechanism of fertilization of eggs. Federation Proc., 5: 1945.
BROOKS, M. M. AND S. C. BROOKS. ^Permeability of Living Cells. Gebriider Borntraeger,
Berlin. Preprinted by Edwards Bros., 1945.
BROOKS, S. C. Intake and loss of phosphate ions by eggs and larvae of Arbacia and Asterias.
Biol. Bull., 83 : 1942.
BROOKS, S. C. Intake and loss of ions by living cells. 1. Eggs and larvae of Arbacia punctu-
lata and Asterias forbesi exposed to phosphate and sodium ions. Biol. Bull., 84: 1943.
BROOKS, S. C. 2. Early changes of phosphate content of Fundulus eggs. Biol. Bull., 84 : 1943.
BROOKS, S. C. *The permeability of cells. Science, 100: 1944.
BROOKS, S. C. Sec also Brooks and Brooks, 1945.
BROWN, D. E. S. Sec Marsland and Brown, 1941, 1942; Marsland, Johnson and Brown, 1941,
1942; Hiatt. Brown, Quinn and MacDuffie, 1945.
BROWN, F. A. AND O. CUNNINGHAM. Upon the presence and distribution of a chromatophoro-
tropic principle in the central nervous system of Limulus. Biol. Bull., 81 : 1941.
BROWN, F. A. AND V. J. WULFF. Chromatophore types in Crago and their endocrine control.
Jour. Cell. Comp. PhysioL, 18: 1941.
BROWNELL, K. A. Sec Hartman, Lewis, Brownell, Shelden and Walther, 1941 ; Hartman,
Lewis, Brownell, Angerer and Shelden, 1944.
BUCHSBAUM, RALPH AND R. WILLIAMSON. The rate of elongation and constriction of dividing
sea urchin eggs as a test of a mathematical theory of cell division. PhysioL ZooL, 16 : 1943.
BUCK, J. B. *Micromanipulation of salivary gland chromosomes. Jour. Hered., 33 : 1942.
BUCK, J. B. Sec also Boche and Buck, 1942.
BUCK, J. B. AND A. M. MELLAND. ^Methods for isolating, collecting, and orienting salivary
gland chromosomes for diffraction analysis. Jour. Hercd., 33: 1942.
BUDINGTON, R. A. The ciliary transport-system of Asterias forbesii. Biol. Bull., 83 : 1942.
BULLOCK, T. H. Neuromuscular facilitation in scyphomedusae. Jour. Cell. Comp. PhysioL,
22: 1943.
BULLOCK, T. H. The giant nerve fiber system in balanoglossids. Jour. Comp. Neural. , 80:
1944.
REPORT OF THE DIRECTOR 17
BULLOCK, T. H. Problems in the comparative study of brain waves. Yale Jour. Biol. Mcd.,
17: 1945.
BULLOCK, T. H. Organization of the giant nerve fiber system in Neanthes virens. Biol. Bull.,
89 : 1945.
'BULLOCK, T. H. Anatomical organization of the nervous system of Enteropneusta. Quart. Jour.
Micr. Set., 86 : 1945.
BULLOCK, T. H. AND D. NACHMANSOHN. Choline esterase in primitive nervous systems.
Jour. Cell. Comp. Physiol, 20: 1942.
CABLE, R. M. Sec also Hunninen and Cable, 1941, 1943.
CABLE, R. M. AND A. V. HUNNINEN. Studies on Deropristis inflata, its life history and affi-
nities to trematodes of the family Acanthocolpidae. Biol. Bull.. 82 : 1942.
CABLE, R. M. AND A. V. HUNNINEN. Studies on the life history of Siphodera vinaledwardsii.
Jour. Parasitol., 28 : 1942.
CAHEN, R. L. *The effects of morphine on the cortical activity of the rat. Yale Jour. Biol.
Mcd., 16: 1944.
CAHEN, R. L. *The influence of morphine on tissue permeability and the spreading effect of
hyaluronidase. Yale Jour. Biol. Mcd., 16 : 1944.
CAHEN, R. L. *Urinary 17 ketosteroids in metabolism. 1. Standardized chemical estimation.
Jour. Biol. Chem., 152: 1944.
CANNAN, R. K. *The hydrogen ion dissociation curve of beta-lactoglobulin. Jour. Biol. Chem.,
142: 1942.
CANNAN, R. K. *The dicarboxylic amino acids in protein hydrolysates. Jour. Biol. Chem.,
152: 1944.
CARSON, H. L. A comparative study of the apical cell of the insect testis. Jour. Morph., 77 :
1945.
CHAMBERS, ROBERT. The intrinsic expansibility of the fertilization membrane of echinoderm
ova. Jour. Cell. Comp. Physiol., 19: 1942.
CHAMBERS, ROBERT. Electrolytic solutions compatible with the maintenance of protoplasmic
structures. Biol. Symposia, 10: 1943.
CHAMBERS, ROBERT. Post war Biology rehabilitation. Science. 100: 1944.
CHAMBERS, ROBERT. Some physical properties of protoplasm. Chapter in Alexander's Colloid
Chemistry, 1944.
CHAMBERS, ROBERT. ^Rehabilitation of the biological sciences in the post-war period. Am.
Nat., 79: 1945.
CHAMBERS, ROBERT, B. W. ZWEIFACH, B. E. LOWENSTEIN AND R. E. LEE. Vaso-excitor and
vaso-depressor substances as "toxic" factors in experimentally induced shock. Proc. Soc.
Exp. Biol. Med., 56: 1944.
CHAMBERS, ROBERT, B. W. ZWEIFACH AND B. E. LOWENSTEIN. ^Circulatory reactions of rats,
traumatized in the Noble-Collip drum. Am. J. Physiol., 139: 1943.
CHAMBERS, ROBERT, B. W. ZWEIFACH AND B. E. LOWENSTEIN. *The peripheral circulation
during the tourniquet syndrome in the rat. Ann. Surg., 120: 1944.
CHASE, A. M. *Effect of azide on Cypridina luciferin. Collecting Net, 16 : 1941.
CHASE, A. M. Observations on luminescence in Mnemiopsis. Biol. Bull. 81: 1941 (abs.).
CHASE, A. M. *The reaction of Cypridina luciferin with azide. Jour. Cell Comp. Physiol., 19 :
1942.
CHASE, A. M. *The absorption spectrum of luciferin and oxidized luciferin. Jour. Biol. Chem.,
150: 1943.
CHASE, A. M. *The visible absorption band of reduced luciferin. Jour. Biol. Chem., 159: 1945.
CHENEY, R. H. Myofibrillar modifications induced by caffeine in cardiac muscle of the frog.
Jour. Comp. Physiol., 18: 1941.
CHENEY, R. H. Caffeine effect in fertilization and development of Arbacia eggs. Biol. Bull.,
83: 1942 (abs.).
CHENEY, R. H. Oxygen consumption of caffeinized Arbacia eggs. Biol. Bull., 83: 1942. (abs.).
CHENEY, R. H. Development of Arbacia eggs in caffeinized sea water. Anat. Rec., 84: 1942
(abs.).
CHENEY, R. H. Inhibitory effect of caffeine on oxygen consumption in Arbacia eggs. Anat.
Rec., 84: 1942 (abs.).
CHENEY, R. H. *Variation in reproductive phenomena by caffeine. Federation Proc., 3 : 1944
(abs.).
18 MARINE BIOLOGICAL LABORATORY
CHENEY, R. H. The effects of caffeine on oxygen consumption and cell division in the fertilized
egg of the sea urchin. Jour. Gen. Physiol., 29: 1945.
CHURNEY, LEON. The osmotic properties of the nucleus. Biol. Bull., 82 : 1942.
CLAFF, C. L. *Glass electrode for determination of hydrogen ion activity of small quantities
of culture media. Science, 94: 1941.
CLAFF, C. L., VIRGINA C. DEWEY AND G. W. KIDDER. *Feeding mechanisms in three species
of Bresslaua. Biol. Bull., 81 : 1941.
CLAFF, C. L. AND O. SWENSON. *Micro glass electrode technique for determination of hydrogen-
ion activity of blood and other biological fluids. Jour. Biol. Chcin., 152: 1944.
CLARK, E. R. AND ELEANOR L. CLARK. ^Microscopic observations on the formation of cartilage
and bone in the living mammal. Amcr. Jour. Anat., 70: 1942.
CLARK, E. R. AND ELEANOR L. CLARK. *Caliber changes in minute blood vessels observed in
living mammal. Amer. Jour. Anat., 73: 1943.
CLARK, E. R. AND ELEANOR L. CLARK *The formation of venae comites. Anat. Rec., 85: 1943.
CLARK, E. R. AND ELEANOR L. CLARK. *Growth and behavior of epidermis as observed mi-
croscopically in the living, in chambers introduced in the rabbit's ear. Anat. Rcc., 88 : 1944.
CLOWES, G. H. A. *Interactions of biologically significant substances in surface films, with
especial reference to two-dimensional solutions and association complexes formed by car-
cinogenic hydrocarbons and sterols. Publication 21. Amer. Assoc. Advance. Sci., 1942.
CLOWES, G. H. A. See also Krahl, Keltch, Neubeck and Clowes, 1941 ; Keltch, Baker, Krahl
and Clowes, 1941; Davis, Baker and Clowes, 1941, 1942; Krahl, Jandorff and Clowes, 1942;
Powell, Krahl and Clowes, 1942; Hutchens, Keltch, Krahl and Clowes, 1942; Zweifach,
Abell, Chambers and Clowes, 1945.
COLE, K. S. Rectification and inductance in the squid giant axon. Jour. Gen. Ph\siol., 25: 1941.
COLE, K. S. See also Guttman and Cole, 1941 ; Curtis and Cole, 1942, 1944.
COLE, K. S. AND R. F. BAKER. Transverse impedance of the squid giant axon during current
flow. Jour. Gen. Physiol., 24: 1941.
COLE, K. S. AND R. F. BAKER. Longitudinal impedance of the squid giant axon. Jour. Gen.
Physiol., 24 : 1941.
COLE, K. S. AND H. J. CURTIS. Membrane potential of the squid giant axon during current flow.
Jour. Gen. Physiol., 24: 1941.
COLE, K. S. AND H. J. CURTIS. *Electrical Physiology : Electrical resistance and impedance of
cells and tissues. Chapter in Medical Physics. Chicago, 1944.
COLE, K. S. AND R. H. COLE. *Dispersion and absorption in dielectrics. 1. Alternating current
characteristics. Jour. Cheni. Phys.,2: 1941.
COLE, K. S. AND R. H. COLE. *2. Direct current characteristics. Jour. Chcm. Phys. 10 : 1942.
COLWIN, LAURA H. Binary fission and conjugation in Urceolaria synapta, with special refer-
ence to the nuclear phenomena. Jour. Morph., 75: 1944.
COMMONER, BARRY. Sec Albaum and Commoner, 1941.
CORNMAN, IVOR. Characteristics of the acceleration of Arbacia cleavage in hypotonic sea-
water. Biol. Bull. 81: 1941 (abs.).
CORNMAN, IVOR. Sperm activation by Arbacia egg extractives, with special reference to
echinoehrome. Biol. Bull. 80: 1941.
CORNMAN, IVOR. Acceleration of cleavage of Arbacia eggs by hypotonic sea water. Biol. Bull.,
84: 1943.
COSTELLO, D. P. ^Advances in Zoology during 1940. hid. Engineer. Chcm., 19: 1941.
COSTELLO, D. P. Segregation of ooplasmic constituents. Jour. Elisha Mitchell Sci. Soc., 61 :
1945.
COSTELLO, D. P. Experimental studies in germinal localization in Nereis. 1. The development
of isolated blastomeres. Jour. E.rp. Zool., 100: 1945.
COSTELLO, D. P. WITH G. I. LAVIN. Ultra violet photomicroscopy of the Nereis and Asterias
egg. Anat. Rcc. Suppl., 87: 1943. (abs.).
CROUSE, HELEN V. *Translocations in Sciara ; their bearing on chromosome behavior and sex
determination. Univ. Missouri Res. Bull., 379: 1943.
CROUSE, HELEN V. See also Ris and Crouse, 1945.
CROWELL, SEARS. A comparison of shells utilized by Hydractinia and Podocoryne. Ecology,
26 : 1945.
CUNNINGHAM, O. Sec Brown and Cunningham, 1941.
REPORT OF THE DIRECTOR 19
CURTIS, H. J. See also Cole and Curtis, 1941.
CURTIS, H. J. AND K. S. COLE. Membrane resting and action potentials' from the squid giant
axon. Jour. Cell. Comfy. Physiol., 19: 1942.
DAVSON-, HUGH. See also Shapiro and Davson, 1941.
DAVSON, HUGH AND J. M. REINER. Ionic permeability; an enzyme-like factor concerned in
the migration of sodium through the cat erythrocyte membrane. Jour. Cell. Comp. Physiol
20: 1942.
DELBRUCK, M. See Luria, Delbruck and Anderson, 1943.
DENT, J. N. *The embryonic development of Plethodon cinereus as correlated with the dif-
ferentiation and functioning of the thyroid gland. Jour. Morph. 71 : 1942.
DENT, J. N. See also Lynn and Dent, 1941.
DEWEY, VIRGINIA C. See also Claff, Dewey and Kidder, 1941.
DEWEY, VIRGINIA C. AND G. W. KIDDER. The possibility of thiamin synthesis by ciliates. Biol.
Bull., 81: 1941. (abs.).
DOUGLIS, MARJORIE B. See Alice and Douglis, 1945.
DZIEMIAN, A. J. *The permeability and the lipid content of immature red cells. Jour. Cell.
Comp. Physiol., 20 : 1942.
EVANS, T. C., H. W. BEAMS, AND M. E. SMITH. Effects of roentgen radiation on the jelly of
the Arbacia egg. Biol. Bull. 80: 1941.
EVANS, T. C. AND J. C. SLAUGHTER. Effect of sea water on the radiosensitivity of Arbacia
sperm. Biol. Bull., 81 : 1941 (abs.).
EVANS, T. C. AND J. C. SLAUGHTER, E. P. LITTLE AND G. FAILLA. Influence of the medium on
radiation injury of sperm. Radiology, 39: 1942.
FAILLA, G. See Evans, Slaughter, Little and Failla, 1942.
FAUST, E. C. *Clinical Parasitology. Co-author with C. F. Craig, Lea and Febiger, 1945.
FINKEL, A. J. See Alice, Finkel and Garner, 1941, 1942; Alice, Finkel, Garner, Merwin and
Evans, 1942
FISHER, K. C. The fractionation of cellular respiration by the use of narcotics. Biol. Bull., 81 :
1941 (abs.).
FREEDMAN, W. B. AND R. WALKER. *Size, development, and innervation of labyrinth sensory
areas in Squalus. Jour. Comp. Neur., 77 : 1942.
FREIS, E. F. B. Some neurohumoral evidence for double innervation of xanthophores in kill-
fish. Biol. Bull., 82 : 1942.
FRISCH, J. A. *The rate of pulsation of the posterior contractile vacuole in Paramoecium
woodruffi and P. calkinsi. Anat. Rec. 89: 1944 (abs.).
FRISCH, J. A. *The rate of adaptation of P. caudatum to sea water. Anat. Rec. 89: 1944 (abs.).
FROEHLICH, ALFRED. The influence of drugs on heat narcosis. Biol. Bull. 89: 1945 (abs.).
FURTH, JACOB. *The teaching of experimental pathology. Arch. Path., 34 : 1942.
FURTH, JACOB. Neoplastic growth. Ann. Rev. Physiol., 6: 1944.
FURTH, JACOB, R. K. COLE AND M. C. BOON. *The effect of maternal influence upon spontane-
ous leukemia of mice. Cancer Research, 2 : 1942.
FURTH, JACOB AND M. C. BOON. ^Enhancement of leukemogenic action of methylcholanthrene
by pre-irradiation with X-rays. Science, 98 : 1943.
FURTH, JACOB, M. C. BOON AND N. KALISS. *On the genetic character of neoplastic cells as
determined in transplantation experiments : with notes on the somatic mutation theory.
Cancer Research, 4 : 1944.
GABRIEL, M. L. The effect of temperature on vertebral variations is Fundulus. Biol. Bull., 83 :
1942 (abs.).
GABRIEL, M. L. Factors affecting the number and form of vertebrae in Fundulus. Jour. Exp.
Zoo!., 95 : 1944.
GALTSOFF, P. S. Storage and distribution of manganese in Ostrea virginica. Collecting Net, 16 :
1941.
GALTSOFF, P. S. Accumulation of manganese and the sexual cycle in Ostrea virginica. Physiol.
Zool., 15 : 1942.
GALTSOFF, P. S. Reaction of the oyster to free chlorine. Biol. Bull., 89: 1945 (abs.).
GARNER, H. R. See Alice, Finkel and Garner, 1941, 1942.
CARREY, W. E. Action of acetylcholine on the heart of Limulus. Amer. Jour. Physiol., 133 :
1941.
20 MARINE BIOLOGICAL LABORATORY
CARREY, W. E. An analysis of the action of acetylcholine on the cardiac ganglion of Limulus.
Amer. Jour. Physiol. 136: 1942.
GATES, R. R. Tests of nuceoli and cytoplasmic granules in marine eggs. Biol. Bull., 81 : 1941.
GATES, R. R. *Nucleoli and phylogeny. Collecting Net, 17 : 1942.
GATES, R. R. *Chromosome numbers in mammals and man. Science, 96 : 1942.
GATES, R. R. *Symbols for human genes. Science, 95 : 1942.t
GATES, R. R. *Nucleoli and related nuclear structures. Bot. Rev., 8 : 1942.
GATES, R. R. *Our ancestors, ourselves, our descendants. Medical Genetics and Eugenics, 2 :
1943.
GATES, R. R. (with G. N. PATHAK). *Variations in the offspring. of tetraploid Oenotheras.
Amer. Naturalist, 78 : 1944.
GIDGE, NATALIE M. -AND S. M. ROSE. The role of larval skin in promoting limb regeneration
in adult Anura. Jour. Exp. Zool., 97 : 1944.
GIESE, A. C. *Studies on nutrition of dim and bright variants of a species of luminous bacteria.
Jour. Bact., 46: 1943.
GIESE, A. C. *The action of azide on luminescence, respiration, and growth of the luminous
bacteria. Jour. Cell. Comp. Physiol., 26 : 1945.
GIESE, A. C. ""Ultraviolet radiations and life. Physiol. Zool., 18 : 1945.
GIESE, A. C. *Effects of ultraviolet radiations on luminescence and respiration of Achromobac-
ter fischeri. Jour. Cell. Comp. Physiol., 17: 1941.
GIESE, A. C. AND E. L. TATUM. *The effect of some vitamins of the B-complex on respiration
of mutants of Neurospora. Biol. Bull., 83: 1942 (abs.).
GILBERT, P. W. *The urogenital system of the male frilled shark, Chlamydoselachus anguineus.
Anat. Rec., 84: 1942 (abs.).
GILBERT, P. W. *The morphology of the male urogenital system of the frilled shark Chlamy-
doselachus anguineus. Jour. Morph., 3: 1943.
GILMAN, L. C. *Mating types in diverse races of Paramoecium caudatum. Biol. Bull., 80:
1941.
GLASER, O. C. Protein metabolism and embryonic growth rate. Biol. Bull., 83: 1942 (abs.).
GOLDIN, A. Factors influencing regeneration and polarity determination in Tubularia crocea.
Biol. Bull, 82 : 1942.
GOLDIN, A. A quantitative study of the interrelationships of oxygen and hydrogen ion concen-
tration in influencing Tubularia regeneration. Biol. Bull., 82 : 1942.
GOLDIN, A. See also Spiegelman and Goldin, 1944.
GOLDIN, A. AND L. G. BARTH. Regeneration of coenosarc fragments removed from the stem
of Tubularia crocea. Biol. Bull, 81 : 1941.
GOLDINGER, J. M. Sec Barren and Goldinger, 1941.
GOODCHILD, CHAUNCEY. *Additional observations on the life history of Gorgodera amplicava.
Jour. Parasitol, 31 : 1945.
GOODRICH, H. B., N. D. JOSEPHSON, J. P. TRINKHAUS AND JEANNE M. SLATE. *The cellular
expression and genetics of two new genes in Lebistes reticulatus. Genetics, 29 : 1944.
GRANICK, SAM. See Michaelis and Granick, 1945.
GRAVE, B. H. The sexual cycle of the shipworm, Teredo navalis. Biol. Bull, 82: 1942 (abs.).
GRAVE, CASWELL. Further studies of metamorphosis of ascidian larvae. Biol. Bull, 81 : 1941
(abs:).
GRAVE, CASWELL. The "eye spot" and light response of the larva of Cynthia partita. Biol Bull,
81: 1941 (abs.).
GRAVE, CASWELL, AND S. O. MAST. The larva of Styela (Cynthia) partita; structure, ac-
tivities, and duration of life. Jour. Morph., 75 : 1944.
GRAY, I. E. *Changes in weight and water content during the life cycle of the wood-eating
beetle, Passalus cornutus. Biol Bull, 86: 1944.
GROSCH, D. S. *The relation of cell size and organ size to mortality in Habrobracon. Growth,
9: 1945.
GUTTMAN, RITA. *Action of potassium and narcotics on rectification in nerve and muscle.
Jour. Gen. Physiol, 28 : 1944.
GUTTMAN, RITA AND K. S. COLE. The rectifying property of the giant axon of the squid.
Collecting Net, 16: 1941.
GUTTMAN, RITA AND K. S. COLE. Electrical rectification in single nerve fibers. Proc. Soc.
Exp. Biol.Afed.,48: 1941.
REPORT OF THE DIRECTOR 21
HAGEK, R. P. *Sex linkage of stubby in Habrobracon. Biol. Bull., 81: 1941 (abs.).
HAMILTON, H. L. *The influence of hormones on the differentiation of melanophores in birds.
Biol. Bull, 81: 1941 (abs.).
HAMILTON, H. L. AND B. H. WILLIER. ^Developmental Physiology. Ann. Rev. Physiol 4-
1942.
HARNLY, M. H. *Wing form and gene function in nine genotypes of Drosophila melanogaster.
Biol. Bull., 82 : 1942.
HARRIS, D. L. The osmotic properties of cytoplasmic granules of the sea urchin egg. Biol.
Bull., 85 : 1943.
HARTMAN, F. A., L. A. LEWIS, K. A. BROWNELL, F. F. SHELDEN AND R. F. WALTHER. Some
blood constituents of the normal skate. Physiol. Zool. 14: 1941.
HARTMAN, F. A., F. F. SHELDEN, AND E. L. GREEN. Weights of interrenal glands of elasmo-
branchs. Anat. Rcc., 87 : 1943.
HARTMAN, F. A., L. A. LEWIS, K. A. BROWNELL, C. A. ANGERER AND F. F. SHELDEN. Effect of
interrenalectomy on some blood constituents in the skate. Physiol. Zool., 17 : 1944.
HARVEY, ETHEL BROWNE. Relation of the size of "halves" of the Arbacia punctulata egg to
centrifugal force. Biol. Bull., 80: 1941.
HARVEY ETHEL BROWNE. The cytology of fertilization and cleavage of Arbacia punctulata.
Turtox Neivs, 19: 1941.
HARVEY ETHEL BROWNE. Cross fertilization of echinoderms. Science, 94 : 1941.
HARVEY ETHEL BROWNE. Vital staining of the centrifuged Arbacia egg. Biol. Bull., 81 : 1941.
HARVEY ETHEL BROWNE. Maternal inheritance in echinoderm hybrids. Biol. Bull., 81 : 1941.
(abs.).
HARVEY ETHEL BROWNE. Rate of breaking and size of the "halves" of the Arbacia egg when
centrigued in hypo- and hypertonic sea water. Biol. Bull., 85 : 1943.
HARVEY, ETHEL BROWNE. *Early biological photomicrographs. Jour. Biol. Photograph. Assoc.,
13: 1945.
HARVEY ETHEL BROWNE. Stratification and breaking of the Arbacia egg when centrifuged in
single salt solutions. Biol. Bull., 89 : 1945.
HARVEY ETHEL BROWNE. Development of granule-free fractions of Arbacia eggs. Biol. Bull.,
89: 1945 (abs.).
HARVEY ETHEL BROWNE AND T. F. ANDERSON. The spermatozoan and fertilization membrane
of Arbacia, as shown by the electron microscope. Biol. Bull., 85: 1943.
HARVEY ETHEL BROWNE AND G. I. LAVIN. The chromatin in the living Arbacia egg; and the
cytoplasm of the centrifuged egg as photographed by ultraviolet light. Biol. Bull., 86 :
1944.
HARVEY, E. N. Stimulation of cells by intense flashes of ultraviolet light. Jour. Gen. Physiol.,
25 : 1942.
HARVEY, E. N. Note on the red luminescence and the red pigment of the "railroad worm."
Jour. Cell. Comp. Physiol., 25 : 1945.
HARVEY, E. N. AND H. SHAPIRO. The recovery period (relaxation) of marine eggs after de-
formation. Jour. Cell. Comp. Physiol., 17 : 1941.
HARVEY, E. N. AND F. J. M. SICHEL. The response of single striated muscle fibers to intense
flashes of ultraviolet light. Jour. Cell. Comp. Physiol., 19: 1942.
HARVEY, E. N. AND F. J. M. SICHEL. A method of recording the dimensions of muscle fiber
striations during contraction. Federation Proc., 1 : 1942.
HARVEY, E. N. AND F. J. M. SICHEL. High speed linear photography. Jour. Cell. Comp.
Physiol., 25 : 1945.
HARVEY, E. N., D. K. BARNES, W. D. MCELROY, A. H. WHLTELY, D. C. PEASE AND K. W.
COOPER. *Bubble formation in animals. I. Physical factors. Jour. Cell. Comp. Physiol.,
24: 1944.
HASSETT, C. C. *Photodynamic action in the flagellate Peranema trichophorum with special
reference to motor response to light. Physiol. Zool., 17 : 1944.
HAYASHI, TERU. Dilution medium and survival of the spermatozoa of Arbacia punctulata.
I. Effect of the medium on fertilizing power. Biol. Bull., 89: 1945.
HAYWOOD, CHARLOTTE. The permeability of the toadfish liver to inulin, with and without
choleretics. Federation Proc., 2: 1943 (abs.).
HAYWOOD, CHARLOTTE, VIRGINIA C. DICKERSON, AND MARGARET C. COLLINS. *The secretion
of dye by the fish liver. Jour. Cell. Comp. Physiol., 25 : 1945.
MARINE BIOLOGICAL LABORATORY
HEILBRUNN, L. V. *An Outline of General Physiology. 2nd Ed. Saunders. 1943.
HEWATT, W. G. A method of narcotizing Holothurians. Science, 97 : 1943.
HIATT, E. P. AND G. P. QUINN. *The distribution of quinine, quinidine, cinchonine, and
cinchonidine in fluids and tissues of dogs. Jour. Pharmacol. Exp. Thcrap., 83 : 1945.
HIATT, E. P., D. E. S. BROWN, G. P. QUINN AND K. MACDUFFIE. *The blocking action of
the cinchona alkaloids and certain related compounds on the cardioinhibitory vagus endings
of the dog. Jour. Pharmacol. Exp. Therap., 85 : 1945.
HIBBARD, HOPE AND G. I. LAVIN. *A study of the Golgi apparatus in chicken gizzard epithelium
by means of the quartz microscope. Biol. Bull., 89: 1945.
HIESTAND, W. A. Oxygen consumption of the sea cucumber as a function of oxygen tension
and hydrogen ion concentration of the surrounding medium. Trans. Wisconsin A cad. Sci-
ences, Arts and Letters, 32 : 1941.
HIESTAND, W. A. Action of certain drugs on the sea star, Asterias forbesii. Proc. Soc. Exp.
Biol. Med., 52 : 1943.
HILL, S. E. The relation between protoplasmic streaming and the action potential in Nitella
and Chara. Biol. Bull., 81: 1941 (abs.).
HOLLINGSWORTH, JOSEPHINE. Activation of Cumingia and Arbacia eggs by bivalent cations.
Biol. Bull, 81: 1941.
HOPKINS, D. L. See Mast and Hopkins, 1941.
HORN, ANNABELLE. *Proof for multiple allelism of sex differentiating factors in Habrobracon.
Amer. Nat., 77: 1943.
HOUCK, C. R. The effects of bichloride of mercury upon the luminescence and respiration of
the luminous bacterium, Achromobacter fischeri. Jour. Cell. Comp. Physiol., 20 : 1942.
HUNNINEN, A. V. See also Cable and Hunninen, 1941, 1942.
HUNNINEN, A. V. AND R. M. CABLE. Life history of Lecithaster confusus. Jour. ParasitoL,
29: 1943.
HUNNINEN,- A. V. AND R. M. CABLE. The life history of Podocotyle atomon. Trans. Amcr.
Microscop. Soc., 62: 1943.
HUTCHENS, J. O. The utilization of ammonia by Chilomonas paramoecium. Biol. Bull., 81 :
1941 (abs.).
HUTCHENS, J. O., A. K. KELTCH, M. E. KRAHL AND G. H. A. CLOWES. Studies on cell metab-
olism and cell division. VI. Observations on the glycogen content, carbohydrate consump-
tion, lactic acid production, and ammonia production of eggs of Arbacia punctulata. Jour.
Gen. Physiol., 25 : 1942.
HUTCHENS, J. O., M. J. KOPAC AND M. E. KRAHL. The cytochrome content of centrifugally
separated fractions of unfertilized Arbacia eggs. Jour. Cell. Comp. Physiol., 20 : 1942.
IRVING, LAURENCE. See Root and Irving, 1941.
JACOBS, M. H. Sec also Netzky and Jacobs, 1941 ; Stewart and Jacobs, 1941.
JACOBS, M. H. AND DOROTHY R. STEWART. Catalysis of ionic exchange by bicarbonates. Biol.
Bull., 81 : 1941.
JACOBS, M. H. AND DOROTHY R. STEWART. The role of carbonic anhydrase in certain exchange
involving the erythrocyte. Jour. Gen. Physiol., 25 : 1942.
JACOBS, M. H. AND DOROTHY R. STEWART. *A biological method for the quantitative estima-
tion of certain organic bases. Amer. Jour. Med. Sci., 206 : 1943.
JACOBS, M. H. AND DOROTHY R. STEWART. *Osmotic equilibria between the erythrocyte and
a complex external solution. Amer. Jour. Med. Sci., 209: 1945.
JACOBS, M. H. AND J. D. HELM. *Some apparent differences between the erythrocytes of white
and negro subjects. Jour. Cell. Comp. Physiol., 22: 1943.
JACOBS, M. H., DOROTHY R. STEWART AND MARY K. BUTLER. *Some effects of tannic acid on
the cell surface. Amer. Jour. Med. Sci., 205 : 1943.
JAEGER, LUCENA. Glycogen utilization by the amphibian gastrula in relation to invagination
and induction. Jour. Cell. Comp. Physiol., 25 : 1945.
JANDORF, B. J. AND M. E. KRAHL. Studies on cell metabolism and division. VIII. The diphos-
phopyridine nucleotide content of eggs of Arbacia punctulata. Jour. Gen. Physiol., 25 :
1942.
JONES, E. R. *The morphology of Enterostomula gram. Jour. Morph., 68: 1941.
JONES, E. R. AND W. J. HAYES, JR. *Microdalyellia gilesi, a new Turbellarian worm from
Mountain Lake, Va. Amer. Midland Naturalist, 26: 1941.
REPORT OF THE DIRECTOR 23
KALISS, NATHAN. Sec Furth, Boon and Kaliss, 1944.
KAWATA, N. See Steinbach, Spiegelman and Kawata, 1944.
KAYLOR, C. T. *Studies on experimental haploidy in salamander larvae. Blol. Bull., 81 : 1941.
KAYLOR, C. T. *Sex differentiation in two androgenetic salamander larvae. Anat Rec 87 •
1943.
KELTCH, ANNA K. See Krahl, Keltch, Neubeck and Clowes, 1941 ; Hutchens, Keltch, Krahl
and Clowes, 1942.
KIDDER, G. W. *Growth studies on ciliates. VII. Comparative growth characteristics of four
species of sterile ciliates. Biol. Bull., 80 : 1941.
KIDDER, G. W. See also Claff, Dewey and Kidder, 1941 ; Burt, Kidder and Gaff, 1941.
KNOWLTON, F. P. Observations on the dual contraction of crustacean muscle. Biol. Bull., 82 :
1942.
KNOWLTON, F. P. An investigation of inhibition by direct stimulation of the turtle's heart.
Amer. Jour. Physiol., 135: 1942.
KNOWLTON, F. P. The action of certain drugs on crustacean muscle. Jour. Exp. Pharmacol.
Exp. Therap., 75: 1942.
KNOWLTON, F. P. """Inhibition of the turtle's atria by single induction shocks applied directly.
Amer. Jour. Physiol., 140: 1943.
KOPAC, M. J. Disintegration of the fertilization membrane of Arbacia by the action of an
"enzyme." Jour. Cell. Comp. Physiol., 18: 1941.
KOPAC, M. J. See also Hutchens, Kopac and Krahl, 1942.
KRAHL, M. E. See also Hutchens, Keltch, Krahl and Clowes, 1942; Jandorf and Krahl, 1942;
Hutchens, Kopac and Krahl, 1942.
KRAHL, M. E., A. K. KELTCH, C. E. NEUBECK AND G. H. A. CLOWES. Studies on cell metab-
olism and cell division. V. Cytochrome oxidase activity in eggs of Arbacia punctulata.
Jour. Gen. Physiol., 24: 1941.
KRAHL, M. E., B. J. JANDORF AND G. H. A. CLOWES. Studies on cell metabolism and cell
division. VII. Observations on the amount and possible function of diphosphothiamine in
eggs of Arbacia punctulata. Jour. Gen. Physiol., 25 : 1942.
KRASNOW, FRANCES. *The physiological significance of phospholipid in human saliva. Jour.
Dental Res., 24: 1945.
LANCEFIELD, REBECCA C. *Studies on the antigenic composition of Group A hemolytic strepto-
cocci. I. Effects of proteolytic enzymes on streptococcal cells. Jour. Exp. Med., 78 : 1943.
LANCEFIELD, REBECCA C. AND W. A. STEWART. *II. The occurrence of strains in a given type
containing M but no T antigen. Jour. Exp. Med., 79: 1944.
LAVIN, G. J. Some observations with a simplified quartz microscope. Biol. Bull., 83: 1942
(abs.).
LAVIN, G. J. *Simplified ultraviolet microscopy. Rec. Scientific Instruments, 14: 1943.
LAVIN, G. J. See also Costello and Lavin, 1943; Harvey and Lavin, 1944; Hibbard and Lavin,
1945.
LAZAROW, ARNOLD. *The chemical organisation of the cytoplasm. Biol. Bull., 87: 1944.
LEE, R. E. The occurrence of female sword-fish in southern New England waters, with a
description of their reproductive condition. Copeia, 1942.
LEE, R. E. The hypophysis of the broad-billed sword-fish, Xiphias gladius. Biol. Bull 82 :
1942.
LEE, R. E. Notes on the color changes of the sea robin, with special reference to the erythro-
phores. Jour. Exp. Zool, 91 : 1942.
LEE, R. E. Pituitary function in the chromatic physiology of Opsanus tau. Biol. Bull 83 •
1942 (abs.).
LEE, R. E. *A quantitative survey of the invertebrate bottom fauna in Menemsha Bight. Biol
Bull. 86: 1944.
•LEE, R. E. See also Chambers, Zweifach, Lowenstein and Lee, 1944; Zweifach, Lee, Hyman
and Chambers, 1944.
LEFEVRE, P. F. Certain chemical factors influencing artificial activation of Nereis eggs Biol
Bull, 89 : 1945.
LEVY, MILTON AND A. H. PALMER. *Amino peptidase. Jour. Biol. Chein., 150 : 1943.
LEWIS, L. A. See Hartman, Lewis, Brownell, Shelden and Walther, 1941 ; Hartman, Lewis,
Brownell, Angerer and Shelden, 1944.
24 MARINE BIOLOGICAL LABORATORY
LEWIS, MARGARET R. *The failure of purified penicillin to retard the growth of sarcoma in
mice. Science, 100: 1944.
LEWIS, MARGARET R. *A study of inducement and transplantability of sarcoma in rats.
Growth, 9 : 1945.
LEWIS, W. H. The superficial gel layer and its role in development. Blol. Bull., 87 : 1944.
LIEBMAN, EMIL. *The coelomocytes of Lumbricidae. Jour. Morph., 71 : 1942.
LILLIE, F. R. Further experiments on artificial parthenogenesis in starfish eggs, with a review.
Physiol. Zool, 14: 1941.
LILLIE, F. R. The Woods Hole Marine Biological Laboratory. Univ. Chicago Press, 1944.
LILLIE, R. S. *The problem of synthesis in Biology. Philosoph. Science, 9: 1942.
LILLIE, R. S. *Living systems and non-living systems. Philosoph. Science, 9 : 1942.
LILLIE, R. S. *The. psychic factor in living organisms. Philosoph. Science, 10: 1943.
LILLIE, R. S. *Vital organization and the psychic factor. Philosoph. Science, 11 : 1944.
LILLIE, R. S. *General Biology and Philosophy of the Organism. Univ. Chicago Press, 1945.
LITTLE, E. P. See Evans, Slaughter, Little and Failla, 1942.
LLOYD, D. P. C. *Activity in neurons of the bulbospinal correlation system. Jour. Neuro-
physiol., 4: 1941.
LOEWI, OTTO. *Chemical transmission of nerve impulses. Science Progress, 4 : 1945.
LOEWI, OTTO. ^Aspects of the transmission of the nervous impulse. Jour. Mt. Sinai Hasp.,
12: 1945.
LUCAS, A. M. AND J. SNEDECOR. Coordination of ciliary movement in the Modiolus gill. Biol.
Bull., 81: 1941 (abs.).
LUCRE, BALDUIN, A. K. PARPART AND R. A. RICCA. Failure of choleic acids in carcinogenic
hydrocarbons to alter permeability of marine eggs and of mammalian erythrocytes. Cancer
Research, 1: 1941.
LURIA, S. E., M. DELBRUCK AND T. F. ANDERSON. Electron microscope studies of bacterial
viruses. Jour. Bad., 46 : 1943.
LYNN, W. G. *The embryology of Eleutherodactylus nubicola, an anuran which has no tadpole
stage. Carnegie Contrib. to Embryol., 30: 1942.
LYNN, W. G. AND J. M. DENT. Notes on Plethodon cinereus and Hemidactylium scutatum on
Cape Cod. Copeia, 1941.
MACLEAN, BERNICE. Sec Shapiro, 1945.
MARSLAND, D. A. *Protoplasmic streaming. Chronica Botan., 6: 1941.
MARSLAND, D. A. ^Protoplasmic streaming. Collecting Net, 16: 1941.
MARSLAND, D. A. *Protoplasmic streaming in relation to gel structure in the cytoplasm.
Chapter in The Structure of Protoplasm, Iowa State College Press, 1942.
MARSLAND, D. A. The contractile mechanism in unicellular chromatophores. Biol. Bull., 83 :
1942 (abs.).
MARSLAND, D. A. ^Quieting Paramoecium for the elementary student. Science, 98 : 1943.
MARSLAND, D. A. Mechanism of pigment displacement in unicellular chromatophores. Biol.
Bull., 87 : 1944.
MARSLAND, D. A. *Principles of Modern Biology. Holt and Co., New York, 1945.
MARSLAND, D. A. AND D. E. S. BROWN. The action of pressure on sol-gel equilibria. Anat.
Rec., 81: Suppl., 1941.
MARSLAND, D. A. AND D. E. S. BROWN. The effects of pressure on sol-gel equilibria, with
special reference to myosin and other protoplasmic gels. Jour. Cell. Comp. Physiol., 20 :
1942.
MARSLAND, D. A. AND R. RUGH. *Effects of pressure on maturation, cleavage, and early de-
velopment of the frog's egg. Anat. Rec., 82 : 1942.
MARSLAND, D. A. AND R. RUGH. *The effect of hydrostatic pressure upon the early develop-
ment of the frog's egg. Proc. Amer. Philosoph. Soc., 86: 1943.
MARTIN, W. E. Cerama solemyae, probably a blood fluke from the marine pelecypod, Solemya
velum. Jour. Parasitol, 30 : 1944.
MARTIN, W. E. Studies on trematodes of Woods Hole. IV. Additional observations upon
Cercaria loossi developing in an Annelid. Trans. Amcr. Micro. Soc., 63: 1944.
MARTIN, W. E. Two new species of marine cercariae. Trans. Amcr. Micro. Soc., 64: 1945.
MAST, S. O. *The hydrogen ion concentration of the content of the food vacuoles and the
cytoplasm in Amoeba and other phenomena concerning the food vacuoles. Biol. Bull.. 83 :
1942.
REPORT OF THE DIRECTOR 25
MAST, S. O. A new peritrich belonging to the genus Ophridium. Trans. Amer. Mic. Soc., 63 :
1944.
MAST, S. O. Sec also Bertholf and Mast, 1944; Grave and Mast, 1944.
MAST, S. O. AND D. L. HOPKINS. *Regulation of the water content of Amoeba mira and
adaptation to changes in the osmotic concentration of the surrounding medium. Jour. Cell.
Comp. Physiol, 17: 1941.
MAST, S. O. AND D. M. PACE. *The effect of phosphorus on metabolism in Chilomonas para-
moecium. Jour. Cell. Comp. Physiol., 20 : 1942.
MAST, S. O. AND W. G. BOWEN. *The food vacuole in the Peritricha, with special reference
to the hydrogen ion concentration of its content and of the cytoplasm. Biol. Bull., 87 : 1944.
MCELROY, W. D. See Harvey, Barnes, McElroy, Whitely, Pease and Cooper, 1944.
MELLAND, A. M. See Buck and Melland, 1942.
MENKIN, VALY. *Studies on the chemical basis of fever. Biol. Bull.. 87 : 1944.
MERWIN, RUTH M. See Alice and Merwin, 1941 ; Alice, Finkel, Garner, Merwin and Evans,
1942; Alice and Merwin, 1943.
METZ, C. B. *The inactivation of fertilizin and its conversion to the "univalent" form by
X-rays and ultraviolet light. Biol. Bull., 82 : 1942.
METZ, C. B. *The agglutination of starfish sperm by fertilizin. Biol. Bull., 89 : 1945.
MEYERHOF, BETTINA. See Nachmansohn and Meyerhof, 1941.
MEYERHOF, OTTO. *Nature, function, and distribution of the phosphagens in the animal king-
dom. Collecting Net, 16: 1941.
MICHAELIS, L. AND SAM GRANiCK. *Metachromasy of basic dye stuffs. Jour. Amer. Chem.
Soc., 67 : 1945.
MILLER, J. A. Some effects of covering the perisarc upon tubularian regeneration. Biol. Bull.,
83: 1942.
MIRSKY, A. E. See Pollister and Mirsky, 1942, 1943.
MOOG, FLORENCE. The influence of temperature on reconstitution in Tubularia. Biol. Bull.,
81: 1941 (abs.).
MOOG, FLORENCE. Some effects of temperature in the regeneration of Tubularia. Biol. Bull.,
83: 1942 (abs.).
MOOG, FLORENCE. See also Spiegelman and Moog, 1944.
MOOG, FLORENCE AND S. SPIEGELMAN. Effects of some respiratory inhibitors on respiration
and reconstitution in Tubularia. Proc. Soc. Exp. Biol. Med., 49: 1942.
MORGAN, T. H. *Further experiments in cross- and self-fertilization of Ciona at Woods Hole
and Corona del Mar. Biol. Bull., 80: 1941.
MORGAN, T. H. Cross- and self-fertilization in the Ascidian, Styela. Biol. Bull., 82 : 1942.
MORGAN, T. H. Cross- and self-fertilization in the Ascidian, Molgula manhattensis. Biol.
Bull., 82 : 1942.
MUIR, R. M. Effect of radiation from radioactive isotopes on the protoplasm of Spirogyra.
Jour. Cell. Comp. Physiol., 19: 1942.
NACHMANSOHN, DAVID. On the mechanism of transmission of nerve impulses. Collecting
Net, 17 : 1942.
NACHMANSOHN, DAVID. On the energy source of the nerve action potential. Biol. Bull., 87:
1944.
NACHMANSOHN, DAVID AND B. MEYERHOF. Relation between electrical changes during nerve
activity and concentration of choline esterase. Jour. Neurophysiol., 4: 1941.
NACHMANSOHN, DAVID AND H. B. STEINBACH. On the localization of enzymes in nerve fibers.
Science, 95: 1942.
NACHMANSOHN, DAVID AND H. B. STEINBACH. Localization of enzymes in nerves. 1. Suc-
cinic dehydrogenase and vitamin B!. Jour. Neurophysiol., 5 : 1942.
NAVEZ, A. E., J. D. CRAWFORD, D. BENEDICT AND A. B. DuBois. On the metabolism of the
heart of Venus mercenaria. Biol. Bull., 81 : 1941.
NETSKY, M. G. AND M. H. JACOBS. Some effects of desoxycorticosterone and related com-
pounds on the mammalian red cell. Biol. Bull., 81 : 1941.
NEUBECK, C. E. See Krahl, Keltch, Neubeck and Clowes, 1941.
O'BRIEN, J. P. ^Studies on the effects of X-rays on regeneration in the fragmenting oligo-
chaete, Nais paraguayensis. Growth, 6 : 1942.
OLSON, MAGNUS. Histology of the radula protractor muscles of Busycon canaliculatum. Biol.
Bull, 82 : 1942.
26 MARINE BIOLOGICAL LABORATORY
OPPENHEIMER, JANE M. The anatomical relationships of abnormally located Mauther's cells
in Fundulus embryos, four. Com p. Neural., 74: 1941.
ORMSBEE, R. A. *The normal growth of Tetrahymena geleii. Biol. Bull., 82 : 1942.
OSBORN, C. M. Studies on the growth of integumentary pigment in the lower vertebrates.
*I. The origin of artificially developed melanophores on the normally unpigmented ventral
surface of the summer flounder. Biol. Bull., 81: 1941.
OSBORN, C. M. II. The role of the hypophysis in melanogenesis in the common catfish. Biol.
Bull., 81: 1941.
OSTERHOUT, W. J. V. Some properties of protoplasmic gels. I. Tension in the chloroplast of
Spirogyra. Jour. Gen. Physiol., 29 : 1945.
PACE, D. M. See Mast and Pace, 1942.
PACKARD, CHARLES. *Roentgen radiation in biological research. Radiology, 45 : 1945.
PACKARD, CHARLES AND F. M. EXNER. *Comparison of physical and biological methods of
depth dose measurement. Radiology, 44 : 1945.
PARK, THOMAS. The laboratory population as a test of a comprehensive ecological system.
Quart. Rev. Biol., 16: 1941.
PARK, THOMAS, ELLA V. GREGG AND CATHERINE Z. LUTHERMAN. *Studies in population
physiology. X. Interspecific competition in populations of granary beetles. Physiol. Zool.,
14: 1941.
PARKER, G. H. Melanophore bands and areas due to nerve cutting, in relation to the protracted
activity of nerves. Jour. Gen. Physiol., 24: 1941.
PARKER, G. H. The methods of excitation of melanophores in the skin of the catfish Ameiurus.
Science, 93: 1941.
PARKER, G. H. Limited responses of melanophores as determined by activating agents. Sci-
ence, 93: 1941.
PARKER, G. H. The responses of catfish melanophores to ergotamine. Biol. Bull., 81 : 1941.
PARKER, G. H. The organization of the melanophore system in bony fishes. Biol. Bull., 81 :
1941.
PARKER, G. H. Hypersensitization of catfish melanophores to adrenalin by denervation. Biol.
Bull., 81: 1941.
PARKER, G. H. The method of activation of melanophores and the limitations of melanophore
responses in the catfish Ameiurus. Proc. Amcr. Philosoph. Soc., 85: 1941.
PARKER, G. H. The activity of peripherally stored neurohumors in catfishes. Jour. Gen.
Physiol., 25: 1941.
PARKER, G. H. *Color changes in Mustelus and other elasmobranch fishes. Jour. Exp. Zool.,
89: 1942.
PARKER, G. H. *Sensitization of melanophores by nerve cutting. Proc. Nat. Acad. Sci., 28:
1942.
PARKER, G. H. Methods of estimating the effects of melanophore changes in animal coloration.
Biol. Bull., 84: 1943.
PARKER, G. H. Color changes in the American eel, Anguilla rostrata. Anat. Rcc., 87: 1943.
PARKER, G. H. *The time factor in chromatophore responses. Proc. Amer. Philosoph. Soc.,
87 : 1944.
PARKER, G. H. Melanophore activators in the common American eel Anguilla rostrata. Jour.
Exp. Zool., 98: 1945.
PARPART, A. K. Lipoprotein complexes in the egg of Arbacia. Biol. Bull., 81 : 1941.
PARPART, A. K. The preparation of red cell membranes. Jour. Cell. Comp. Physiol., 19: 1942.
PARPART, A. K. See also Lucke, Parpart and Ricca, 1941 ; Chase, Lorenz, Parpart and Gregg,
1944.
PARPART, A. K. AND R. BALLENTINE. *Hematocrit determination of red cell volume. Science,
98: 1943.
PEASE, D. C. Sec Harvey, Barnes, McElroy, Whitely, Pease and Cooper, 1944.
PIERCE, MADELENE E. Response of melanophores of the skin to injections of adrenalin, with
special reference to body weight of the animal. Jour. Exp. Zool., 86: 1941.
PLOUGH, H. H. *Spontaneous mutability in Drosophila. Cold Spring Harbor Svmposia, 9 :
1941.
PLOUGH, H. H. ^Temperature and evolution. Biol. Symposia, 6 : 1942.
POLLISTER, A. W. *Mitochondrial orientations and molecular patterns. Physiol. Zool., 14 :
1941.
REPORT OF THE DIRECTOR 27
POLLISTER, A. W. AND A. E. MiRSKY. *Nucleoproteins of cell nuclei. Proc. Nat. Acad. Sci.,
28: 1942.
POLLISTER, A. W. AND A. E. MIRSKY. *Studies on the chemistry of chromatin. Trans. N. Y.
Acad. Sci., 5 : 1943.
POLLISTER, A. W. AND A. E. MIRSKY. *Fibrous nucleoproteins of chromatin. Biol. Symposia,
10: 1943.
POLLISTER, A. W. AND P. F. POLLISTER. *Relation between centriole and centromere in a
typical spermatogenesis of viviparid snails. Ann. N. Y. Acad. Sci., 45 : 1943.
PRICE, DOROTHY. *A comparison of the reactions of male and female rat prostate transplants.
Anat. Rec., 82 : 1942.
PROSSER, C. L. An analysis of the action of acetylcholine on hearts, particularly in Arthopods.
Biol. Bull., 83 : 1942.
PROSSER, C. L. Single unit analysis of the heart ganglion discharge in Limulus polyphemus.
Jour. Cell. Comp. Physiol., 21 : 1943.
PROSSER, C. L. AND G. L. ZIMMERMAN. Comparative pharmacology of myogenic and neuro-
genic hearts. Biol. Bull., 81: 1941 (abs.).
QUINN, GERTRUDE P. Sec Hiatt and Quinn, 1945.
RECKNAGEL, RICHARD. See Wilbur and Recknagel, 1943.
REID, W. M. *The relationship between glycogen depletion in the nematode Ascaridia galli
and the elimination of the parasite by the host. Amcr. Jour. Hyg., 41 : 1945.
REID, W. M. *In vivo and in vitro glycogen utilization in the fowl nematode Ascaridia galli.
Biol. Bull., 89: 1945 (abs.).
REINHARD, E. G. A hermit crab as intermediate host of Polymorphus. Jour. Parasitol., 30 :
1944.
REINHARD, E. G. Paguritherium alatum, an entoniscian parasite of Pagurus longicarpus. Jour.
Parasitol., 31 : 1945.
RICCA, R. A. See Lucke, Parpart and Ricca, 1941.
RICHARDS, A. G. The interfibrillar material in the central nervous system of mosquito larvae.
Biol. Bull,, 83: 1942 (abs.).
RICHARDS, A. G. *Lipid nerve sheaths in insects and their probable relation to insecticide
action. Jour. N. Y. Entomol. Soc., 51 : 1943.
RICHARDS, A. G. *The structure of living insect nerves and nerve sheaths as deduced from the
optical properties. Jour. N. Y. Entomol. Soc., 52 : 1944.
RICHARDS, A. G. AND JANE L. WEYGANT. *The selective penetration of fat solvents into the
nervous system of mosquito larvae. Jour. N. Y. Entomol. Soc., 53 : 1945.
RICHARDS, A. G., H. B. STEINBACH AND T. F. ANDERSON. Electron microscope studies of
squid giant nerve axoplasm. Jour. Cell. Comp. Physiol., 21 : 1943.
Ris, HANS. *A cytological and experimental analysis of the meiotic behavior of the univalent
X-chromosome in the bearberry aphid Tamalia. Jour. Exp. Zool., 90 : 1942.
Ris, HANS. *A quantitative study of anaphase movement in the aphid Tamalia. Biol, Bull.,
85: 1943.
Ris, HANS AND HELEN GROUSE. *Structure of the salivary gland chromosomes of Diptera.
Proc. Nat. Acad. Sci,, 31 : 1945.
ROBBIE, W. A. Balanced centerwell solutions for manometric experimentation. Biol. Bull., 89 :
1945 (abs.).
ROGICK, MARY D. Resistance of fresh water Bryozoa to dessication. Biodynamica, 3: 1941.
ROGICK, MARY D. Supplementary note on the effect of the 1938 hurricane. Ohio J. Sci,, 41 :
1941.
ROGICK, MARY D. ^Studies on fresh-water Bryozoa XV. Hyalinella punctata growth data.
Ohio Jour. Sci., 45 : 1945.
ROGICK, MARY D. Field trips with a long-range purpose. Amcr. Biol. Teacher, 7 : 1945.
ROGICK, MARY D. Studies on marine Bryozoa. I. Aeverrillia setigera. Biol. Bull., 89 : 1945.
ROGICK, MARY D. *Studies on fresh-water Bryozoa. XVI. Fredericella australiensis. Biol.
Bull., 89 : 1945.
ROGICK, MARY D. "Calcining" specimens. Amcr. Biol. Teacher, 8 : 1945.
ROOT, R. W. AND LAURENCE IRVING. The equilibrium between hemoglobin and oxygen in
whole and hemolyzed blood of the tautog, with a theory of the Haldane effect. Biol. Bull.,
81 : 1941.
MARINE BIOLOGICAL LABORATORY
ROSE, S. M. *A method for inducing limb regeneration in adult Anura. Proc. Soc. Exp. Biol.
Med., 49 : 1942.
ROSE, S. M. *Causes for loss of regenerative power in adult Anura. Anat. Rec., 89: 1944.
ROSE, S. M. *Methods for initiating limb regeneration in adult Anura. Jour. Exp. Zool., 95 :
1944.
ROSE, S. M. *The effect of NaCl in simulating regeneration of limbs of frogs. Jour. Morph.,
77: 1945.
ROSE, S. M. AND FLORENCE C. ROSE. The role of a cut surface in Tubularia regeneration.
Physiol. Zool., 14: 1941.
ROSE, S. M. See also Gidge and Rose, 1944.
RUGH, ROBERT. See Marsland and Rugh, 1942.
RUNYON, E. H. Aggregation of separate cells of Dictyostelium to form a multicellular body.
Biol. Bull, 83: 1943 (abs.).
SANDOW, ALEXANDER. Studies of the muscle twitch recorded by electronic methods. Biol.
Bull, 89: 1945 (abs.).
SAYLES, L. P. Regeneration in the polychaete, Clymenella torquata. Biol Rev. of the City
College, N. Y ., 3 : 1941.
SAYLES, L. P. Buds induced in Clymenella torquata by implants of nerve cord and neighboring
tissues derived from the mid-body region of worms of the same species. Biol. Bull, 82 :
1942.
SAYLES, L. P. Implants consisting of young buds, formed in anterior regeneration in Cly-
menella, plus the nerve cord of the adjacent old part. Jour. Exp. Zool, 94: 1943.
SCHAEFFER, MORRIS. *Preparation of influenza virus. Proc. Soc. Exp. Biol Med., 51 : 1942.
SCHALLEK, WILLIAM. The reaction of certain Crustacea to direct and to diffuse light. Biol
Bull, 84: 1943.
SCHALLEK, WILLIAM. Action of potassium on bound acetylcholine in lobster nerve cord.
Jour. Cell Comp. Physiol, 26 : 1945.
SCHARRER, BERTA. *Neurosecretion. II. Neurosecretory cells in the central nervous system of
cockroaches. Jour. Comp. Neural, 74: 1941.
SCHARRER, BERTA. III. The cerebral organ of the nemerteans. Jour. Comp. Neurol, 74: 1941.
SCHARRER, BERTA. IV. Localization of neurosecretory cells in the central nervous system of
Limulus. Biol Bull, 81: 1941.
SCHARRER, BERTA. *Endocrines in Invertebrates. Physiol. Re^'., 21 : 1941.
SCHARRER, BERTA. *Experimental tumors after nerve section in an insect. Proc. Soc. Biol.
Med., 60: 1945.
SCHARRER, BERTA. See also Scharrer and Scharrer, 1945.
SCHARRER, BERTA AND E. SCHARRER. Neurosecretion VI. A comparison between the inter-
cerebralis-cardiacumallatum system of the insects and the hypothalamo-hypophyseal system
of the vertebrates. Biol Bull, 87: 1944.
SCHARRER, ERNST. Neurosecretion I. The nucleus preopticus of Fundulus. Jour. Comp.
Neurol, 74: 1941.
SCHARRER, ERNST. The capillary bed of the central nervous system of certain invertebrates.
Biol Bull, 87 : 1944.
SCHARRER, ERNST. *The blood vessels of the nervous tissue. Quart. Rev. Biol, 19 : 1944.
SCHARRER, ERNST. Capillaries and mitochrondria in neurophil. Jour. Comp. Neurol, 1945.
SCHARRER, ERNST. See also Scharrer and Scharrer, 1944.
SCHARRER, ERNST, S. L. PALAY AND R. G. NILGES. Neurosecretion VIII. The Nissl sub-
stance in secreting nerve cells. Anat. Rec., 92 : 1945.
SCHARRER, ERNST AND BERTA SCHARRER. Neurosecretion. Physiol Rev., 25: 1945.
SCHECHTER, VICTOR. Experimental studies upon the egg cells of the clam, Mactra solidissima,
with special reference to longevity. Jour. Exp. Zool, 86: 1941.
SCHECHTER, VICTOR. *Oxygen as a factor in the polarity of Corymorpha palma. Physiol.
Zool, 14: 1941.
SCHECHTER, VICTOR. *Tolerance of the snail, Thais floridana to waters of low salinity and
the effect of size. Ecology, 24 : 1943.
SCHECHTER, VICTOR. *Two flatworms from the oyster-drilling snail, Thais floridana. Jour.
Parasitol, 29 : 1943.
SCHMITT, F. O. *Structural proteins of cells and tissues. In Advances in Protein Chemistry,
1: 1945.
REPORT OF THE DIRECTOR 29
SCOTT, ALLAN. Reversal of sex production in Micromalthus. Biol. Bull., 83: 1942 (abs.).
SCOTT, SISTER FLORENCE MARIE. The early embryonic development of Amaroecium constel-
latum. Biol. Bull, 83: 1942 (abs.).
SCOTT, SISTER FLORENCE MARIE. The developmental history of Amaroecium constellatum.
1. Early embryonic development. Biol. Bull., 88: 1945.
SEVAG, M. G. *Immuno-catalysis. C. C. Thomas, Springfield, 111., 1945.
SHAEFFER, A. A. A fourteen day rhythm in the left-right spiralling ratio of Flabellula citata.
Biol. Bull. ,83: 1942 (abs.).
SHANES, A. M. Current, voltage, and resistance characteristics of injured nerves. Biol. Bull
87: 1944 (abs.).
SHANES, A. M. Frog nerve as a generator of current and voltage. Jour. Cell. Comp. Ph\sioL,
24: 1944.
SHANES, A. M. Evidence of a metabolic effect by potassium in lowering the injury potential
of invertebrate nerve. Biol. Bull, 89: 1945 (abs.).
SHAPIRO, H. H. AND BERNICE L. MACLEAN. *Transplantation of developing tooth germs in
the mandible of the cat. Jour. Dental Res., 24: 1945.
SHAPIRO, HERBERT. Oxygen utilization by starfish eggs. Amcr. Jour. Physiol, 133: 1941.
SHAPIRO, HERBERT. Metabolism and fertilization in the starfish egg. Collecting Net, 16: 1941.
SHAPIRO, HERBERT. Centrifugal elongation of cells, and some conditions governing the return
to sphericity, and cleavage time. Jour. Cell Comp. Physiol, 18 : 1941
SHAPIRO, HER^ERT. Water permeability of the Chaetopterus egg before and after fertilization.
Jour. Cell Comp. Physiol, 18 : 1941.
SHAPIRO, HERBERT AND HUGH DAVSON. Permeability of the Arbacia egg to potassium. Biol
Bull, 81: 1941.
SHAPIRO, HERBERT. Metabolism and fertilization in the starfish egg. Biol Bull, 81 : 1941.
SHAPIRO, HERBERT. The speed of membrane formation. Anat. Rcc., 81 : 1941.
SHAPIRO, HERBERT. See also Harvey and Shapiro, 1941.
SHAW, MYRTLE. Sec Sickles and Shaw, 1941.
SHELDEN, F. F. See Hartman, Lewis, Brownell and Shelden, 1941 ; Hartman, Shelden and
Green, 1943; Hartman, Lewis, Brownell, Angerer, and Shelden, 1944.
SICHEL, F. J. M. *The relative elasticity of the sarcolemma and of the entire skeletal muscle
fiber. Amer. Jour. Physiol, 133 : 1941.
SICHEL, F. J. M. Sec also Harvey and Sichel, 1942, 1945.
SICKLES, GRACE M. AND MYRTLE SHAW. The production of specific antisera for enzymes that
decompose pneumococcus carbohydrates types 3 and 8. Jour. Bacterial, 42: 1941.
SLAUGHTER, J. C. See Evans, Slaughter, Little, and Failla, 1942.
SLIFER, ELEANOR H. *A mutant stock of Drosophila with extra sex-combs. Jour. Exp. Zool
90: 1942.
SLIFER, ELEANOR H. *The internal genitalia of some previously unstudied species of female
Acrididae. Jour. Morph. 72: 1943.
SLIFER, ELEANOR H. *The internal genitalia of female Tetrigidae, Eumastacidae, and Proscopi-
idae. Jour. Morph., 73 : 1943.
SMELSER, G. K. The oxygen consumption of eye muscles of thyroid-ectomized and thyroxin in-
jected guinea pigs. Amer. Jour. Physiol, 142: 1944.
SMITH, M. E. See Evans, Beams, and Smith, 1941.
SOSA, J. M. *Woods Hole, Acropolis de los biologos. El. Dia., 1943.
SOSA, J. M. *Quince meses en los Estados Unidos de Norte America. Anales Facultad de
Med. Montevideo, 30: 1945.
SPIEGELMAN, S. Mass and time relationships in the regeneration of Tubularia. Biol Bull, 83 :
1942 (abs.).
SPIEGELMAN, S. See also Moog and Spiegelman, 1942 ; Steinbach and Spiegelman, 1943, 1944.
SPIEGELMAN, S. AND A. GOLDIN. A comparison of regeneration and respiration rates of
Tubularia. Proc. Soc. Exp. Biol Med. 55 : 1944.
STEINBACH, H. B. Chloride in the giant axons of the squid. Jour. Cell Comp. Physiol, 17 :
1941.
STEINBACH, H. B. See also Nachmansohn and Steinbach, 1942 ; Richards, Steinbach and
Anderson, 1943.
30 MARINE BIOLOGICAL LABORATORY
STEIXBACH, H. B. AND S. SPIEGELMAN. The sodium and potassium balance in squid nerve axo-
plasm. Jour. Cell. Comp. Physiol, 22 : 1943.
STEINBACH, H. B. AND N. KAWATA. The recovery of the cut surface of the scallop muscle.
Fed. Proc., 3 : 1944.
STEINBACH, H. B., S. SPIEGELMAN AND N. KAWATA. Rectification and injury potential in
squid axons. Fed. Proc., 3 : 1944.
STEINBACH, H. B., S. SPIEGELMAN AND N. KAWATA. The effects of potassium and calcium on
the electrical properties of squid axons. Jour. Cell. Comp. Physiol. 24: 1944.
STERN, K. G. *Oxidases, Peroxidases, and Catalase. Symposium on respiratory enzymes.
Madison, Wis., 1942.
STERN, K. G. Physical-chemical studies on chromosomal nuceloproteins. Biol. Bull., 89 : 1945
(abs.).
STERN, K. G. AND S. F. VELICK. The effect of centrifugation upon the oxygen consumption of
Arbacia eggs. Biol. Bull, 81 : 1941.
STEWART, DOROTHY R. See Jacobs and Stewart, 1941, 1942, 1945 ; Jacobs, Stewart and Butler,
1943.
STEWART, DOROTHY R. AND M. H. JACOBS. The role of carbonic anhydrase in the catalysis of
ionic exchanges by bicarbonates. Biol. Bull., 81 : 1941 (abs.).
STEWART, W. A. See Lancefield and Stewart, 1944.
STILES, K. A. *Handbook of microscopic characteristics of tissues and organs. Blakiston,
1943.
STILES, K. A. ^Laboratory explorations in general zoology. Macmillan, 1943.
STOREY, ALMA G. *Gametophytes of Marattia sambucina and Macroglossum Smithii. Bot.
Gas., 103 : 1942.
STOKEY, ALMA G. *The gametophyte of Dipteria conjugata. Bot. Gas., 106: 1945.
STUNKARD, H. W. Specificity and host-relations in the trematode genus Zoogonus. Biol. Bull.,
81 : 1941.
STUNKARD, H. W. Pathology and immunity to infection with heterophyid trematodes. Biol.
Bull., 81 : 1941.
STUNKARD, H. W. Studies on pathology and resistance in terms and dogs infected with the
heterophyid trematode, Cryptocotyle lingua. Trans. Amer. Microscop. Soc., 61 : 1942.
STUNKARD, H. W. The morphology and life history of the digenetic trematode, Zoogonoides
laevis. Biol. Bull., 85 : 1943.
TAFT, C. H. The effects of a mixture of high molecular alkyl-dimethyl-benzyl-ammonium
chlorides on the isolated heart of Limulus polyphemus. Proc. and Trans. Texas A cad.
Set., 28 : 1945.
TAFT, C. H. The action of amino acids on color changes in Fundulus. Science, 101 : 1945.
TAFT, C. H. *Action of quitenine on the living Tautog and Toadfish. Biol. Bull., 89: 1945
(abs.).
TAFT, C. H. AND J. A. PLACE. *The comparative effects of the subcutaneous injection of
quitenine on the kidneys of glomerular and aglomerular fish. Texas Rep. on Biol. Mcd. 2 :
1944.
TAYLOR, W. R. Reappearance of rare New England marine algae. Rhodora, 43: 1941.
TAYLOR, W. R. *Notes on the marine algae of Texas. Mich. Acad. Sci. Artes. and Letter, 26 :
1941.
TAYLOR, W. R. Tropical marine algae of the Arthur Schott Herbarium. Field Mus. Nat.
Hist. Bot., 230 : 1942.
TAYLOR, W. R. *Marine algae of the Allan Hancock Expedition to the Caribbean, 1937. Allan
Hancock Atlantic Exped. 2: 1942.
TAYLOR, W. R. *Marine algae from Haiti. Mich. Acad., 28 : 1943.
TAYLOR, W. R. *The collecting of seaweeds and fresh water algae. Instructions to naturalists
in the armed forces for botanical work. 1944 2nd ed. 1945.
TAYLOR, W. R. *William Gilson Farlow, promotor of phycological research in America. Far-
lowia, 2 : 1945.
TAYLOR, W. R. *Pacific marine algae of the Allan Hancock Expeditions to the Galapagos
Islands. Allan Hancock Pacific Exp. 12 : 1945.
TEWINKEL, Lois E. Structures concerned with yolk absorption in Squalus acanthias. Biol.
Bull, 81: 1941 (abs.).
REPORT OF THE DIRECTOR 31
TE\VINKEL, Lois E. Observations on later phases of embryonic nutrition in Squalus acanthias.
Jour. Morph., 73: 1943.
TE\VINKEL, Lois E. Embryonic nourishment in the spiny dogfish. Wards Nat. Hist. Bull.,
19: 1945.
THIVY, FRANCESA. A new species of Ectochaete from Woods Hole. Biol. Bull., 83 : 1942.
THIVY, FRANCESA. New records of some marine Chaetophoraceae and Chaetosphaeridiaceae
for North America. Biol. Bull., 85 : 1943.
TRACER, WILLIAM. *The nutrition of invertebrates. Physiol. Rev., 21 : 1941.
TRACER, WILLIAM. *Studies on conditions affecting the survival in vitro of a malarial para-
site. Biol. Bull., 81: 1941 (abs.).
TRINKHAUS, J. P. See Goodrich, Josephson, Trinkhaus and Slate, 1944.
TROMBETTA, VIVIAN (Mrs. Roland Walker). The cytonuclear ratio. Bot. Rev., 8: 1942.
VON DACH, HERMAN. Respiration of a colorless flagellate, Astasia klebsii. Biol. Bull., 82 :
1942.
VON SALLMAN, L. J. K. *Hydrogen ion concentration of the vitreous in the living eye. Arch.
Ophthalmol., 33 : 1945.
WALKER, ROLAND AND G. C. BENNET. *Size relations in the optic system of telescope-eyed
goldfish. Trans. Connecticut Acad. Art and Sci., 36: 1945.
WALKER, ROLAND. See also Freedman and Walker, 1942.
WARREN, C. O. *The role of bicarbonate in the action of serum in supporting tissue respira-
tion. Jour. Biol. Chem., 156 : 1944.
WARREN, C. O. *The effect of thiouracil on the respiration of bone marrow and leucocytes
in vitro. Amcr. Jour. Physiol., 71 : 1944.
WATERMAN, A. J. The action of drugs on the compound ascidian, Perophora viridis, as in-
dicated by the activity of the intact heart. Physiol. Zool., 15: 1942.
WATERMAN, A. J. Further study of the action of drugs on the heart of the compound ascidian.
Physiol. Zool., 16 : 1943.
WATTERSON, R. L. *Some aspects of pigment deposition in feather germs of chick embryos.
Biol. Bull., 81: 1941 (abs.).
WATTERSON, R. L. Asexual reproduction in the colonial tunicate Botryllus schlosseri, with
special reference to the developmental history of intersiphonal band of pigment cells. Biol.
Bull. 88 : 1945.
WEIDENREICH, FRANZ. *The brachycephalization of recent mankind. Southwestern. Jour.
Anthropol, 1 : 1945.
WENRICH, D. H. *The morphology of some protozoan parasites in relation to microtechnique.
Jour. Parasitol., 27: 1941.
WENRICH, D. H. *Observations on the food habits of Entamoeba muris and Entamoeba ra-
narum. Biol. Bull., 81: 1941.
WENRICH, D. H. Morphology of the intestinal trichomonad flagellates in man and of similar
forms in monkeys, cats, dogs, and rats. Jour. Morph., 74 : 1944.
WENRICH, D. H. Comparative morphology of the trichomonad flagellates of man. Amcr. Jour.
Trap. Med., 24 : 1944.
WENRICH, D. H. Nuclear structure and nuclear division in Dientamoeba fragilis. Jour.
Morph., 74: 1944.
WENRICH, D. H. Studies on Dientamoeba fragilis. IV. Further observations with an outline
of present day knowledge of this species. Jour. Parasitol., 30 : 1944.
WHALEY, W. G. AND C. Y. WHALEY. A developmental analysis of inherited leaf patterns in
Tropaeolum. Amcr. Jour. Botany, 29 : 1942.
WHITING, ANNA R. *X-ray sensitivity of first meiotic prophase and metaphase in Habrobracon
eggs. Genetics, 27 : 1942.
WHITING, ANNA R. *Effects of X-rays on hatchability and on chromosomes of Habrobracon
eggs treated in first meiotic prophase and metaphase. Amcr. Nat., 79 : 1945.
WHITING, P. W. *The cytogenetics of sex determination. Proc. 7th Internat. Genctical Cong.,
1941.
WHITING, P. W. *Sex determination in Habrobracon. Proc. 7th Internat. Genctical Cong.,
1941.
WHITING, P. W. *Multiple alleles in complementary sex determination of Habrobracon.
Genetics, 28: 1943.
32 MARINE BIOLOGICAL LABORATORY
WHITING, P. W. *Intersexual females and intersexuality in Habrobracon. Biol. Bull., 85 :
1943.
WHITING, P. W. *Androgenesis in the parasitic wasp Habrobracon. Jour. Hered., 34: 1944.
WHITING, P. W. *The problem ®f reversal of male haploidy by selection. Biol. Bull., 89:
1945 (abs.).
WHITING, P. W. *The evolution of male haploidy. Quart. Rev. Biol., 20 : 1945.
WICHTERMAN, RALPH. *Pure line mass cultures for demonstrating the mating reaction and
conjugation in Paramoecium. Turtox News, 22 : 1944.
WICHTERMAN, RALPH. *Recent discoveries of nuclear processes and sexual phenomena in
Paramoecium. Turtox News, 22 : 1944.
WICHTERMAN, RALPH. A modified petri dish for continuous temperature observation. Science,
101: 1945.
WTIERCINSKI, F. J. An experimental study of intracellular pH in the Arbacia egg. Biol. Bull.,
81: 1941.
WIERCINSKI, F. J. An experimental study of protoplasmic pH determination. I. Amoebae and
Arbacia punctulata. Biol. Bull., 86 : 1944.
WILBUR, K. M. The stimulating action of citrates and oxalates on the Nereis egg. Physiol.
Zoo/., 14: 1941.
WILBUR, K. M. See also Angerer and Wilbur, 1943.
WILBUR, K. M. AND R. O. RECKNAGEL. The radiosensitivity of eggs of Arbacia punctulata in
various salt solutions. Biol. Bull., 85 : 1943.
WILHELMI, R. W. *The application of the precipitin technique to theories concerning the origin
of vertebrates. Biol. Bull., 82 : 1942.
WILLIAMSON, R. R. See Buchsbaum and Williamson, 1943.
WILLIER, B. H. *Melanophore control of the sexual dimorphism of feather pigmentation in
the Barred Plymouth Rock. Biol. Bull., 87 : 1944.
WILLIER, B. H. AND MARY E. RAWLES. *Genotypic control of feather color pattern as demon-
strated by the effects of a sex-linked gene upon the melanophores. Genetics, 29 : 1944.
WILLIER, B. H. AND MARY E. RAWLES. Melanophore control of the sexual dimorphism of
feather pigmentation pattern in the Barred Plymouth Rock fowl. Yale Jour. Biol. Med.,
17: 1944.
WITKUS, ELEANOR R. *Some hints on smear technique. Turtox News, 23 : 1945.
WITKUS, ELEANOR R. *Endomitotic tapetal cell divisions in Spinacia. Amer. Jour. Botany,
32: 1945.
WITKUS, ELEANOR R. *Endomitosis in plants. Biol. Bull., 89: 1945 (abs.).
WITKUS, ELEANOR R. Sec also Berger and Witkus, 1943 ; Berger, Sullivan and Witkus, 1944.
WOODRUFF, L. L. Sec Boell and Woodruff, 1941.
WOODWARD, ALVALYN E. AND J. M. CONDRIN. *Physiological studies on hibernation in the
chipmunk. Physiol. Zool, 18: 1945.
WRINCH, DOROTHY. *The native protein theory of the structure of protoplasm. Cold Spring
Harbor Symposia, 9: 1941.
WRINCH, DOROTHY. Proteins in action. Collecting Net, 16: 1941.
WRINCH, DOROTHY. Further implication of flexible protein frameworks. Collecting Net, 16 :
1941.
WRINCH, DOROTHY. The structure of biologically active membranes. Biol. Bull., 83: 1942
(abs.).
WRINCH, DOROTHY. *Native proteins, flexible frameworks and cytoplasmic organization.
Nature, 150: 1942.
WRINCH, DOROTHY. *Growth and form. Isis, 34: 1943.
WRINCH, DOROTHY. Native protein crystallography and diffraction patterns. Biol. Bull., 87 :
1944.
WRINCH, DOROTHY. Fourier transforms and structure factors. Phys. Rev., 67 : 1945.
WRINCH, DOROTHY. A tetrahedral framework for native proteins? Biol. Bull., 89: 1945.
WULFF, V. J. Sec Brown and Wulff, 1941.
YNTEMA, C. L. *An experimental study on the origin of the sensory neurones and sheath cells
of the IXth and Xth cranial nerves in Amblystoma punctatum. Jour. Exp. Zool., 92: 1943.
YNTEMA, C. L. ^Experiments on the origin of the sensory ganglia of the facial nerv»e in the
chick. Jour. Comp. Ncur., 81 : 1944.
REPORT OF THE DIRECTOR 33
YNTEMA, C. L. AND W. S. HAMMOND. ""Depletions and abnormalities in the cervical sympa-
thetic system of the chick following extirpation of neural crest. Jour. E.r/>. Zoo/., 100 :
1945.
ZINN, D. J. AND R. W. PERNAK. Mystacocarida, a new order of Crustacea from intcrtidal
beaches in Massachusetts and Connecticut. Smithsonian Miscellaneous Collections, 103:
1943.
ZWEIFACH, B. J., R. E. LEE, C. HYMAN AND R. CHAMBERS. *Omental circulation in morph-
inized dogs subjected to graded hemorrhage. Annals Surg., 120: 1944.
ZWEIFACH, B. J. See also Chambers and Zweifach, 1944.
ZWEIFACH, B. J., R. G. ABELL, R. CHAMBERS AND G. H. A. CLOWES. Role of the decompensa-
tory reactions of peripheral blood vessels in tourniquet shock. Surg. Gyn. Obstct., 80 : 1945.
2. THE STAFF, 1945
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
F. A. BROWN, Associate Professor of Zoology, Northwestern University, in charge of
course.
T. H. BULLOCK, Assistant Professor of Neurology University of Missouri Medical School.
W. D. BURBANCK, Associate Professor of Biology, Drury College.
C. G. GOODCHILD, Associate Professor of Biology, Southwest Missouri State Teachers
College.
JOHN H. LOCH HE AD, 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.
III. LABORATORY ASSISTANT
ANTOIN BACA, Duke University Medical School.
EMBRYOLOGY
I. CONSULTANTS
H. B. GOODRICH, Professor of Biology, Wesleyan University.
34 MARINE BIOLOGICAL LABORATORY
II. INSTRUCTORS
W. W. BALLARD, Professor of Zoology, Dartmouth College.
DONALD P. COSTELLO, Professor of Zoology, University of North Carolina.
VIKTOR HAMBURGER, Professor of Zoology, Washington University, in charge of course.
JANE M. OPPENHEIMER, Assistant Professor in Biology, Bryn Mawr College.
III. RESEARCH ASSISTANT
MARJORIE HOPKINS, University of California.
IV. LABORATORY ASSISTANTS
CATHERINE HENLEY, The Johns Hopkins University.
ELEANOR LERNER, Washington University.
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. CARREY, Professor of Physiology, Vanderbilt University Medical School.
MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania.
II. INSTRUCTORS
ROBERT BALLENTINE, Lecturer in Zoology, Columbia University (absent in 1945).
AURIN CHASE, Assistant Professor of Biology, Princeton University.
ARTHUR C. GIESE, Associate Professor of Biology, Stanford University (absent in 1945 I.
E. S. GUZMAN BARROX, Associate Professor of Biochemistry, The University of Chicago.
RUDOLF T. KEMPTOX, Professor of Zoology, Vassar College (absent in 1945).
ARTHUR K. PARPART, Associate Professor of Biology, Princeton University, in charge
of course.
ROBERT RAMSEY, Associate Professor of Physiology, Medical College of Virginia.
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
REPORT OF THE DIRECTOR
APPARATUS DEPARTMENT
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager
J. D. GRAHAM DOROTHY LEFEVRE
CHEMICAL DEPARTMENT
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager
SUPPLY DEPARTMENT
JAMES Mclxxis, Manager
D. J. Zixx. Naturalist
RUTH CROWELL GRACE M. KENNERSON
M. B. GRAY W. E. KAHLER F. N. WHITMAN
A. M. HILTON G. LEHY
GENERAL OFFICE
F. M. MACNAUGHT, Business Manager
POLLY L. CROWELL MRS. LILA S. MYERS
*
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
3. INVESTIGATORS AND STUDENTS
• Independent Investigators, 1945
ABELL, RICHARD G., Assistant Professor of Anatomy, University of Pennsylvania.
ADDISON, WILLIAM H. F., Professor of Normal Histology and Embryology, University of
Pennsylvania.
ANDERSON, RUBERT S., Assistant Professor of Physiology, University of Maryland.
ANFINSON, CHRISTIAN B., JR., Instructor in Biological Chemistry, Harvard Medical School.
ARMSTRONG, PHILIP B., Professor of Anatomy, Syracuse University.
ARONOFF, SAMUEL, Instructor, University of Chicago.
AXELRAD, ARTHUR A., Investigator, McGill University.
BALL, ERIC G., Associate Professor of Biological Chemistry, Harvard Medical School.
BALLARD, W. W., Professor of Zoology, Dartmouth College.
BALLENTINE, ROBERT, Instructor, Columbia University.
BARRON, E. S. GUZMAN, Associate Professor of Biochemistry, The University of Chicago.
EARTH, L. G., Associate Professor of Zoology, Columbia University.
BEERS, CHARLES DALE, Professor of Zoology, University of North Carolina.
BERGER, CHARLES A., Professor of Cytology, Fordham University.
BERTHOLF, LLOYD M., Professor of Biology, Western Maryland College.
BEVELANDER, GERRIT, Associate Professor of Anatomy, New York University.
BLISS, ALFRED F., Instructor in Physiology and Pharmacology, Union University.
BODIAN, DAVID, Associate in Epidemiology, Johns Hopkins University.
36 MARINE BIOLOGICAL LABORATORY
BRONK, DETLEV W., Professor of Biophysics, Johnson Foundation.
BROOKS, MATILDA M., Research Associate in Biology, University of California.
BROOKS, SUMNER C., Professor of Zoology, University of California.
BROWN, DUGALD E. S., Professor of Physiology, New York University.
BROWN, FRANK A., JR., Associate Professor of Zoology, Northwestern University.
BROWNELL, KATHARINE A., Research Associate, Ohio State University.
BUDINGTON, ROBERT A., Professor of Zoology, Emeritus, Oberlin College.
BULLOCK, THEODORE H., Assistant Professor of Anatomy, University of Missouri.
BURBANCK, WILLIAM D., Associate Professor of Biology, Drury College.
BURKHOLDER, PAUL R., Professor of Botany. Yale University.
CHAMBERS, ROBERT, Research Professor of Biology, New York University.
CHASE, AURIN M., Assistant Professor of Biology, Princeton University.
CHENEY, RALPH H., Chairman Biology Department, Long Island University.
CHIDESTER, F. E., Research Worker, Lee Foundation.
CLAFF, C. LLOYD, Research Fellow in Surgery, Harvard Medical School.
CLARK, ELEANOR L., Voluntary Research Worker, University of Pennsylvania.
CLARK, ELIOT R., Professor of Anatomy, University of Pennsylvania.
CLAUDE, ALBERT, The Rockefeller Institute for Medical Research.
CLEMENT, A. C., Associate Professor in Biology. College of Charleston.
CLOWES, G. H. A., Director of Research, Lilly Research Laboratories.
CONKLIN, EDWIN G., Professor of Zoology, Emeritus, Princeton University.
COPELAND, MANTON, Professor of Biology, Bowdoin College.
COSTELLO, DONALD P., Professor of Zoology, University of North Carolina.
CRAMPTOX, HENRY E., Professor Emeritus. Columbia L'niversity.
CROASDALE, HANNAH T., Technical Assistant, Dartmouth College.
CROUSE, HELEN V., Research Associate, University of Pennsylvania.
CROWELL, SEARS, Assistant Professor of Zoology, Miami University.
FROEHLICH, ALFRED, Associate, May Institute for Medical Research.
FURCHGOTT, ROBERT F., Research Associate, Cornell L^niversity.
FURTH, JACOB, Professor of Pathology, Cornell University.
GAFFRON, HANS, Assistant Professor of Biochemistry, Research Associate, University of Chicago.
GALTSOFF, PAUL S., Senior Biologist, U. S. Fish and Wildlife Service.
CARREY, W. E., Professor of Physiology, Emeritus, Vanderbilt University, School of Medicine.
GLASER, OTTO C., Professor of Biology, Amherst College.
GOODCHILD, DR. C. G., Associate Professor of Biology, State Teachers College.
GORBMAN, AUBREY, Instructor in Biology, Wayne University.
GOULD, HARLEY N., Professor of Biology, H. Sophie Newcomb College.
GRAND, C. G., Research Associate, New York University.
HAMBURGER, VIKTOR, Professor of Zoology, Washington University.
HARTMAN, FRANK A., Professor and Chairman of Department of Physiology, Ohio State
University.
HARVEY, ETHEL BROWNE, Independent Investigator Biology Department, Princeton University.
HARVEY, E. NEWTON, Professor of Physiology, Princeton University.
HAYASHI, TERU, Instructor in Zoology, University of Missouri.
HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College.
HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania.
HIBBARD, HOPE, Professor of Zoology, Oberlin College.
HICKSON, ANNA KELTCH, Research Chemist, Eli Lilly & Company.
HOPKINS, HOYT S., Associate Professor of Physiology, New York University, College of
Dentistry.
HUBER, WOLFGANG, Senior Research Chemist, Winthrop Chemical Company.
JACOBS, M. H., Professor of General Physiology, University of Pennsylvania.
JAEGER, LUCENA, Research Associate, Columbia University.
JENKINS, GEORGE B., Professor of Anatomy, Emeritus, George Washington University.
JOHLIN, J. M., Associate Professor of Biochemistry, Vanderbilt University.
JOHNSON, FRANK H., Assistant Professor of Biology, Princeton University.
KRAHL, M. E., Instructor in Pharmacology, Columbia University.
LANDIS, EUGENE M., Professor of Physiology and Head of Department, Harvard Medical
School.
REPORT OF THE DIRECTOR 37
LAVIN, GEORGE I., In charge of Spectroscopic Laboratory, Rockefeller Institute for Medical
Research.
LEE, RICHARD E., Student, Columbia University.
LIEBEN, FRITZ, Research Fellow, Johns Hopkins University.
LILLIE, RALPH S., Professor of Physiology, Emeritus, University of Chicago.
LOCH HEAD, JOHN H., Assistant Professor of Zoology, University of Vermont.
McCLUNG, C. E., Professor of Zoology, Emeritus, University of Pennsylvania.
MACLEAN, BERNICE L., Assistant Professor, Department Biological Sciences, Hunter College.
MAC.ALHAES, HULDA, Instructor in Zoology, Duke University.
MARKS, MILDRED H., Student, Massachusetts Institute of Technology.
MARSLAND, DOUGLAS A., Associate Professor of Biology, New York University.
MAST, S. O., Professor of Zoology, Emeritus, Johns Hopkins University.
MATHEWS, ALBERT P., Professor of Biochemistry, Emeritus, University of Cincinnati.
MATTHEWS, SAMUEL A., Professor of Biology, Williams College.
MEMHARD, ALLEN R., Crescent Road, Riverside, Connecticut.
MENKIN, VALY, Assistant Professor of Pathology, Duke University.
METZ, CHARLES W., Director Zoological Laboratory, University of Pennsylvania.
MICHAELIS, LEONOR, Member Emeritus, Rockefeller Institute for Medical Research.
NACHMANSOHN, DAVID, Research Associate in Neurology, Columbia University.
NORTHROP, JOHN H., Member of the Institute, Rockefeller Institute for Medical Research.
OPPENHEIMER, JANE M., Assistant Professor of Biology, Bryn Mawr College.
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research.
PARPART, ARTHUR K., Associate Professor of Biology, Princeton University.
PIERCE, MADELEXE E., Associate Professor of Zoology, Vassar College.
RAMSEY, ROBERT W., Associate Professor of Physiology, Medical College of Virginia.
RANKIN, JOHN S., JR., Assistant Professor of Zoology, University of Connecticut.
REID, W. MALCOLM, Assistant Professor of Biology, Monmouth College.
RIKER, WALTER F., JR., Instructor in Medicine and Pharmacology, Cornell University Medical
School.
Ris, HANS, Assistant in Physiology, Rockefeller Institute for Medical Research.
ROBBIE, WILBUR A., Research Associate, State University of Iowa.
ROGICK, MARY DORA, Professor of Biology, College of New Rochelle.
SAMPSON, MYRA M., Professor of Zoology, Smith College.
SANDOW, ALEXANDER, Assistant Professor of Biology, New York University.
SCHAEFFER, A. A., Professor of Biology, Temple University.
SCHARRER, ERNST A., Assistant Professor of Anatomy, Western Reserve University School of
Medicine.
SCOTT, SISTER FLORENCE MARIE, Professor of Zoology, Seton Hill College.
SCOTT, GEORGE T., Instructor, Oberlin College.
SHANES, ABRAHAM M., Assistant Professor of Physiology. New York University College of
Dentistry.
SHAPIRO, HARRY H., Assistant Professor of Anatomy, Columbia University.
SLIFER, ELEANOR H., Assistant Professor of Zoology, State University of Iowa.
SMITH, DIETRICH CONRAD, Associate Professor of Physiology, University of Maryland, School
of Medicine.
STERN, KURT G., Lecturer in Department of Chemistry, Polytechnic Institute of Brooklyn.
STEWART, DOROTHY R., Fellow in Physiology, University of Pennsylvania.
STOKEY, ALMA G., Professor of Plant Science, Emeritus, Mount Holyoke College.
STUNKARD, H. W., Professor of Biology, New York University.
TAFT, CHARLES H., Associate Professor of Pharmacology, Medical Branch, University of Texas.
TAYLOR, WILLIAM RANDOLPH, Professor of Botany, University of Michigan.
TEWINKEL, Lois E., Associate Professor of Zoology, Smith College.
THIVY, FRANCESCA, Professor of Biology, Women's Christian College.
VILLEE, CLAUDE A., Assistant Professor of Zoology, University of North Carolina.
WAINIO, WALTER W., Assistant Professor of Physiology, New York University, College of
Dentistry.
WARREN, CHARLES O., Assistant Professor of Physiology, Cornell University Medical College.
WENRICH, D. H., Professor of Zoology, University of Pennsylvania.
WHITING, ANNA R., Visiting Investigator, University of Pennsylvania.
MARINE BIOLOGICAL LABORATORY
WHITING, P. W., Associate Professor of Zoology. University of Pennsylvania.
WICHTERMAN, RALPH, Assistant Professor of Biology, Temple University.
WILLIER, B. H., Professor of Zoology and Director of the Biological Laboratories, Johns
Hopkins University.
WINSOR, CHARLES P., Research Associate, Princeton University.
WITKUS, ELEANOR R., Instructor in Botany and Bacteriology, Fordham University.
WOODWARD, ALVALYN E., Assistant Professor, University of Michigan.
WOODWARD, ARTHUR A., JR., Research Assistant, University of Pennsylvania.
WOOLEY, D. W., Associate. Rockefeller Institute for Medical Research.
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, 1945
BROWN, ELLEN, Commonwealth Fellow, University of California Medical School.
BROWN, VIRGINIA H., Graduate Student, Ohio State University.
COYNE, CHRISTOPHER J., Student, University of Pennsylvania.
DAVIDSON, MARGARET E., Demonstrator and Assistant to Dr. Berrill, McGill University.
KRUGELIS, EDITH J., Graduate Student, Columbia University.
LERNER, ELEANOR, Fellow in Zoology, Washington University.
LOVELACE, ROBERTA, Teaching Fellow, University of North Carolina.
MILLER, HELMA C., Assistant, Johns Hopkins University.
SCHNEYER, LEON H., Instructor, New York University, College of Dentistry.
WILSON, WALTER, Graduate Student, University of Pennsylvania.
Research Assistants, 1945
ABRAMSKY, TESS, Research Assistant, Rockefeller Institute for Medical Research.
BRUNELLI, ELEANOR L., Research Assistant, New York University, College of Dentistry.
DEFALCO, ROSE H., Research Assistant, University of Pennsylvania.
FISCHL, MATHILDA, Research Assistant in Medicine, Cornell University.
FRANZ, RUTH ESTELLE, Research Assistant, Yale University.
GARZOLI, RAY F., Graduate Student, University of California.
GOULD, DAVID, Research Technician, New York University.
HARLOW, JANET, Technician, Syracuse University.
HELFMAN, MYRNA, Technician, New York University.
HENLEY, CATHERINE, Graduate Teaching Assistant, Johns Hopkins University.
HONEGGER, CAROL, Student, Temple University.
LAWLER, H. CLAIRE, Research Assistant, New York University.
LEVIN, ISAAC, Research Assistant, Princeton University.
LEVY, BETTY, Research Assistant, Rockefeller Institute for Medical Research.
LOOFBOURROW, G. N., Instructor, Rhode Island State College.
MCVEIGH, IDA, Research Assistant in Botany, Yale University.
METZ, DELILAH B., Research Assistant, Eli Lilly & Co.
MINER, KARYL, Research Assistant, New York University.
MITCHELL, CONSTANCE, Research Assistant, University of Pennsylvania.
PETTENGILL, OLIVE S., Student, Temple University.
QUINN, GERTRUDE P., Research Assistant, New York University.
ROTHENBERG, M. A., Research Assistant in Biochemistry, College of Physicians and Surgeons.
UHLMAN, GLORIA E., Research Assistant, Yale University.
WALTERS, C. PATRICIA, Research Assistant, Eli Lilly & Co.
WARNER, CHARLOTTE, Medical Student, University of Pennsylvania.
ZACKS, SUMNER L, Student, Brookline High School.
Library Readers, 1945
AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland.
BECK, LYLE V., Associate Professor of Physiology, Hahnemann Medical College.
REPORT OF THE DIRECTOR 39
BENDICH, AARON, Member War Research Division, Neurological Institute, New York.
BLOCK, RICHARD J., Associate, New York Medical College.
BREHME, KATHERINE S., Lecturer, Cornell University Medical College.
CAHEN, RAYMOND L., Research Assistant, Yale University, Medical School.
CARSON, HAMPTON L., Instructor in Zoology, Washington University.
CASSIDY, HAROLD G., Assistant Professor of Chemistry, Yale University.
COLWIN, LAURA HUNTER, Instructor, Pennsylvania College for Women.
FRIEDEMANN, ULRICH H., Head of Department of Bacteriology, Brooklyn Jewish Hospital.
FRISCH, JOHN A., Professor of Biology, Canisius College.
GATES, R. RUGGLES, Professor Emeritus, University of London.
GUREWICH, VLADIMIR, Assistant Visiting Physician, Bellevue Hospital.
KABAT, ELVIN A., Research Associate in Biochemistry, College of Physicians and Surgeons.
KAYLOR, CORNELIUS T., Assistant Professor of Anatomy, Syracuse University.
KELLER, RUDOLPH, Researcher, Robinson Foundation, New York.
KRASNOW, FRANCES, Head of Department of Research, Guggenheim Dental Foundation.
LANGE, MATHILDE M., Professor of Zoology, Head of Department of Biology, W'heaton College.
LOEWI, OTTO, Research Professor of Pharmacology, New York University, College of Medicine.
MARINELLI, LEONIDAS, Physicist, Memorial Hospital.
MAVOR, JAMES W., Professor of Biology, Union College.
MAYER, MANFRED M., Scientific Staff, War Research Division, Columbia University.
METZ, CHARLES B., Instructor in Biology, Wesleyan University.
MEYERHOF, DR. OTTO, Research Professor of Biochemistry, University of Pennsylvania.
MOLDAVER, JOSEPH. Research Associate in Neurology, Columbia University.
MOORE, JOHN A., Assistant Professor of Zoology, Barnard College.
MOSCHCOWITZ, ELI, Assistant Professor of Chemical Medicine, Columbia University.
OSEASOHN, ROBERT O., Long Island College of Medicine.
PERRY, BARBARA H., Graduate Student and Teaching Fellow in Zoology, Smith College.
PONDER, ERIC, Research, Nassau Hospital.
RAMSDEN, ETHEL J., Instructor in Biology, Montclair Teachers Colle;-ir.
ROBINSON, MILES H., Instructor in Pharmacology, University of Pennsylvania.
RYAN, FRANCIS J., Assistant Professor, Columbia University.
SCOTT, ALLAN, Assistant Professor of Biology, Union College.
STRAUSS, WILLIAM L., JR., Associate Professor of Anatomy, Johns Hopkins University.
VoxDACH, HERMAN, Assistant Professor of Physiology, Georgetown Medical School.
WALLACH, JACQUES B., Long Island College of Medicine.
ZORZOLI, ANITA, Assistant Instructor, New York University.
Students, 1945
BOTANY
BARRACLOUGH, MARY EDITH, Student, Smith College.
DIETZ, ALMA, Assistant in Biology, American International College.
GARDNER, ELIZABETH B., Radcliffe College.
MOUL, EDWIN THEODORE, Botany Assistant, University of Pennsylvania.
SMITH, MATTIE Lot', Student, Radcliffe College.
EMBRYOLOGY
BEACH, JANET, Student, University of Connecticut.
BERNIER, GERMAINE, University of Montreal, Quebec, Canada.
BERRY, BETH SINCLAIR, Student, Rockford College, Illinois.
CARTER, MARJORIE ESTELLE, Teacher, Georgia State Women's College.
CHIRICO, ANNA MARIE, Student. Seton Hill College.
CLARK, CARL CYRUS, Student, Amherst College. •
COPINGER, ANNE STEVENS, Goucher College.
EHRLICH, MIRIAM, Knox College.
Izzo, MARY JANE, University of Rochester.
KELL, AMY, University of Illinois.
LEVIN, ILANE B., Goucher College.
40 MARINE BIOLOGICAL LABORATORY
LODICO, DOROTHY GERALDINE, University of Rochester.
LOVELACE, LOLLIE ROBERTA, Teaching Fellow, University of North Carolina.
MARKER, MURIEL JOSEPHINE, Student, Colby College.
MEZGER, LISELOTTE, Student, Bryn Mawr College.
MILLER, HELMA C., Graduate Assistant, Johns Hopkins University.
PERKINS, BARBARA BURNHAM, University of Connecticut.
RAYMOND, BARBARA, Student, Swarthmore College.
RICE, MARY ESTHER, Assistant in Biology Laboratory, Drew University.
ROBERTS, ELIZABETH S., Assistant in Biology, Wilson College.
RUDERMAN, CLAIRE, Teaching Assistant, University of Rochester.
THORBY, JEAN ADELAIDE, Student, Rockford College.
UPHOFF, DELTA Ev University of Rochester.
PHYSIOLOGY
BRUST, MANFRED, Student, New York University.
COOK, JOHN ALFRED, George Washington University.
FERGUSON, ALICE HOWARD, Graduate Assistant, Louisiana State University.
FLINKER, MARIE-LOUISE M., Assistant in Physiology, Vassar College.
FOGERSON, VIRGINIA LEE, Student, Drury College.
FOSTER, ELIZABETH JANE, Student, University of Illinois.
GOLDSMITH, YVETTE, Perth Amboy, New Jersey.
HAJEK, NORMA MARY, Cornell University.
HECHT, LISELOTTE ISABELLA, Student, University of Michigan.
RESNICK, OSCAR, Resident Scholar, Harvard University.
WEISS, MICHAEL S., Student, Washington Square College.
WOLFF, MARY LYDA, Instructor, Cedar Crest College.
WORKEN, BARNEY, 3400 Wayne Avenue, New York City.
ZOOLOGY
ARONOWITZ, OLGA, New York University.
BATES, MARY FLORENCE, Student, Vassar College.
BAYORS, WINIFRED M., Student Seton Hill College.
BEAL, JUDITH D., Vassar College.
BENJAMIN, MRS. REZSIN C., Undergraduate Student, University of Rochester.
BERNARD, SISTER MARIE, Fordham University.
BERNIER, GERMAINE, Instructor, University of Montreal.
BEZILLA, HELEN, Student, Seton Hill College.
BRADIN, JOHN L., Northwestern University.
CALVERT, JULIE NEIL, Student, Wilson College.
CARLSON, ALICE MARIE, Laboratory Assistant, University of Minnesota.
CHAFFIN, EVELYN L., Student. Drury College.
CLARK, CARL CYRUS, Amherst College.
CUMMINGS, REV. GEORGE W., Graduate Student, Catholic University.
DAILEY, DOROTHY HELEN, Depamv University.
DAWSON, MARY JEAN, Student, Mt. Holyoke College.
DEMPSEY, ELLEN, Oberlin College.
DICKASON, MARY ELIZABETH, Student, Smith College.
FARNHAM, CAROL JEAN, Student, Drury College.
FREITAG, JANET FAITH, Student, University of Connecticut.
GOLDIS, BERNICE RUTH, Graduate Student, University of Pennsylvania.
HANLON, REV. JAMES J., Graduate Student, Fordham University.
HILL, SHIRLEY B., Student, Vassar College.
HINES, EILEEN BARBARA, State University of Iowa.
JONES, DOROTHY B., Student, University of Connecticut.
JOSITA, SISTER M., Student, Fordham University.
JULIER, EDITH VAILLANT, Student, Vassar College.
REPORT OF THE DIRECTOR
41
KREKELER, CARL H., Student, Washington University.
KUHN, ALICE ROBERTS, Western-Maryland College.
LOWENS, MARY DOROTHY, Student, Swarthmore College.
McCLAiN, MARYLOW, Student, Swarthmore College.
MCGREGOR, ELIZABETH, Instructor, Mount Holyoke College.
McVicKER, SISTER MAUREEN, Teacher of Biology, St. Joseph's College for Women
MALLOCH, JEAN, Vassar College.
MEIHACK, HELEN LLOYD, Student, Oberlin College.
MINA, FRANK A., Laboratory Instructor, Fordham University.
OSBORN, JOAN A., Student, Barnard College.
PETERS, REV. JOSEPH J., Graduate Student, Fordham University.
RAYMOND, BARBARA, Student, Swarthmore College.
RIGGS, AUSTIN F., Student, Harvard University.
ROGERS, HENRY CRAMPTON, Deerfield Academy.
SCHAEFER, GERTRUDE, Undergraduate, Temple University.
SEAMAN, ARLENE, Zoology Assistant, Cornell University.
SNIPES, ANNE, Wheaton College.
STEES, NANCY, Teacher, West Chester State Teachers College.
SURRARRER, THOMAS C, Professor of Biology, Baldwin-Wallace College.
THORNTON, DOROTHY GOLDEN, Assistant in Zoology Dept., Wellesley College.
TUPPER, LYLA, Graduate Student, Northwestern University.
UBER, VIRGINIA M., Student, Pennsylvania College for Women.
WAX, FLORENCE SIMA, Student, Oberlin College.
WHYTE, MARJORIE ANN, Assistant, Cornell University.
WILCOX, BARBARA L., Student, Radcliffe College.
WILLIAMS, OLWEN, Teacher of Biology and Chemistry, The Putney School.
WILSON, FAITH EVELYN, Johns Hopkins University.
WILSON, MARIE ELLEN, Student, Western Maryland College.
4. TABULAR VIEW OF ATTENDANCE
1942 1943 1944 1945
INVESTIGATORS — Total 337
Independent 197
Under instruction / 59
Library readers 31
Research assistants 50
STUDENTS — Total 131
Zoology 55
Embryology 37
Physiology 24
Botany 15
TOTAL ATTENDANCE 468
Less persons registered as both students and investigators 7
461
INSTITUTIONS REPRESENTED — Total 144
By investigators 102
By students 72
SCHOOLS AND ACADEMIES REPRESENTED
By investigators 5
By students 2
FOREIGN INSTITUTIONS REPRESENTED
By investigators 3
By students 1
201
132
16
28
25
74
36
24
6
8
275
2
273
126
83
43
160
89
19
35
17
68
47
13
8
228
6
222
116
70
41
2
1
— 2
193
112
11
50
20
75
37
23
10
5
276
1
275
106
74
41
1
2
2
3
212
138
10
38
26
96
55
23
13
5
308
124
100
49
2
2
42
MARINE BIOLOGICAL LABORATORY
5. SUBSCRIBING AND COOPERATING INSTITUTIONS
1945
Albany Medical College
Amherst College
Biological Institute, Philadelphia, Pennsylvania
Bowdoin College
Bryn Mawr College
Cathedral College
The 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
Goucher College
Harvard University
Harvard University Medical School
Industrial and Engineering Chemistry, of the
American Chemical Society
Johns Hopkins University
Johns Hopkins Medical School
Lee Foundation
Eli Lilly & Company
Long Island University
Macy Foundation
Massachusetts Institute of Technology
McGill University
Miami University
Mount Holyoke College
New York University
New York University College of Medicine
New York University School of Dentistry
New York University Washington Square
College
Oberlin College
Ohio State University
Pennsylvania College for Women
Princeton University
Radcliffe College
Rockefeller Institute for Medical Research
St. Joseph College for Women
Smith College
State University of Iowa
Syracuse University
Syracuse University Medical School
Temple University
University of Chicago
University of Connecticut
University of Illinois
University of Maryland Medical School
University of Michigan
University of Missouri
University of Pennsylvania
L'niversity of Pennsylvania School of Medicine
University of Rochester
Vanderbilt University Medical School
Vassar College
Washington University
Wayne University
Wellesley College
Wesleyan University
Western Maryland College
Western Reserve University
Wheaton College
Williams College
Wilson College
Woods Hole Oceanographic Institution
Yale University
6. EVENING LECTURES, 1945
Friday, June 29
PROF. P. W. WHITING "The Development of Hymenopteran Ge-
netics."
Friday, July 6
DR. R. R. GATES "Human Heredity in Relation to Animal
Genetics."
Friday, July 13
DR. I. FANKUCHEN "X-Ray Diffraction and Protein Structure."
Friday, July 20
PROF. S. C. BROOKS "Our Interrelationships with South Ameri-
can Universities, together with Illustrated
Travel Notes."
Friday, July 27
PROF. E. G. BUTLER "Problems of Differentiation and Dediffer-
entiation in Amputated Urodele Limbs."
REPORT OF THE DIRECTOR 43
Friday, August 3
DR. BOSTWICK H. KETCHUM "The Prevention of Ship Bottom Fouling."
Friday, August 10
DR. DANIEL MERRIMAN "A Study in Pure and Applied Marine Bi-
ology. The Life History and Economic
Importance of the Ocean Pout."
Friday, August 17
DR. DETLEV W. BRONK "Biological Research During the War and
Postwar Periods."
Friday, August 24
DR. F. L. HISAVV "Endocrines and the Evolution of Vivi-
parity among the Vertebrates."
Monday, August 27
GEORGE G. LOWER "Local Invertebrates."
•
Wednesday, August 29
DR. PAUL S. GALTSOFF "Impressions of a Biologist at the San
Francisco Conference."
Thursday, August 30
MAJOR A. H. NEUFELD "Medical Research Organization in the Ca-
nadian Army."
Thursday, August 30
CAPT. W. R. DURYEE "Medical Military Training."
7. SHORTER SCIENTIFIC PAPERS, 1945
Tuesday, July 24
DR. M. M. BROOKS "The Redox Potential of Penicillium rota-
turn Medium under Some Different Con-
ditions of Growth."
DR. WILBUR ROBBIE "The Use of Cyanide in Manometric Ex-
perimentation."
DR. SEARS CROWELL "The Displacement of Terns by Gulls at
Weepecket Island."
Tuesday, July 31
DR. P. W. WHITING "The Problem of Reversal of Male Hap-
loidy by Selection."
DR. BERTA SCHARRER "Experimental Tumors after Nerve Section
in an Insect."
DR. P. S. GALTSOFF "Reactions of Oysters to Free Chlorine."
Tuesday, August 7
DR. T. H. BULLOCK "Organization of Giant Nerve Fibers in cer-
tain Polychaetes."
DR. ERNST SCHARRER "The Origin of Neurosecretory Granules
from Basophil Substances in the Nerve
Cells of Fishes."
DR. C. H. TAFT "The Action of Quitenine on the Livers of
Tautog and Toadfish."
DR. A. M. SHANES "Evidence of a Metabolic Effect by Potas-
sium in Lowering the Injury Potential of
Nerve."
44 MARINE BIOLOGICAL LABORATORY
Tuesday, August 14
DR. R. CHAMBERS "Interrelations between Sperm-Nucleus,
Egg-Nucleus and Cytoplasm in Asterias
Egg."
DR. KURT G. STERN "Physical-chemical Studies on Chromosomal
Nucleoproteins."
Tuesday, August 21
DR. DOROTHY WRINCH "Hemoglobin and other Native Proteins."
DR. E. R. WIT.KUS "Endomitosis in Plants."
DR. C. A. BERGER "Recent Cytological Studies in Culex."
Thursday, August 23
DR. ETHEL B. HARVEY "Development of Granule-free Fractions of
Arbacia eggs."
DR. ALEXANDER SANDOW "Studies of the Muscle Twitch by Methods
of Electronic Recording."
DR. C. D. BEERS "The Role of Bacteria in the Excystment of
the Ciliate Didinium."
Monday, August 27
DR. ANNA R. WHITING "Differences in Sensitivity, Hatchability
Curves and Cytological Effects between
Eggs X-rayed in First Meiotic Prophase
and Metaphase."
DR. W. W. WAINIO "Aerobic Oxidation of Simple sugars by
Mammalian Liver."
DR. DUGALD E. S. BROWN "The Role of Myosin and Myosin Triphos-
phatase ;';; Vitro and in Muscle."
Tuesday, August 28
DR. LLOYD M. BERTHOLF "Accelerating Metamorphosis in the Tuni-
cate Styela."
DR. ALFRED FROELICH "The Influence of Drugs on Heat-narcosis."
DR. W. MALCOLM REID "In Vivo and in Vitro Glycogen Utiliza-
tion in the Avial Nematode Ascardia
Galli."
8. MEMBERS OF THE CORPORATION, 1945
1. LIFE MEMBERS
ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France.
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.
JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts.
JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts.
KING, MR. CHAS. A.
REPORT OF THE DIRECTOR 45
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.
STRONG, DR. O. S., Columbia University, New York City, New York.
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.
ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New
York.
ALBAUM, DR. HARRY G., Biology Dept., Brooklyn College, Brooklyn, N. Y.
ALBERT, DR. ALEXANDER, 383 Harvard Street, Cambridge, Mass.
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.
BARTH, DR. L. G., Department of Zoology, Columbia University, New York City,
New York.
46 MARINE BIOLOGICAL LABORATORY
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., Dickinson House, South Haclley, Massachusetts.
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 Medical School, St. Louis, Missouri.
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.
BUCK, DR. JOHN B., Industrial Hygiene Research Lab., National Institute of
Health, Bethesda, Maryland.
BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.
BUDINGTON, PROF. R. A., Winter Park, Florida.
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.
BULLOCK, DR. T. L., University of Missouri, Columbia, Missouri.
BURBANCK, DR. WILLIAM D., Department of Biology, Drury College, Springfield,
Missouri.
BURKENROAD, DR. M. D., Yale University, New Haven, Connecticut.
BUTLER, DR. E. G., Princeton University, Princeton, N. J.
BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Pennsylvania.
CAMERON, DR. J. A., Baylor College of Dentistry, Dallas, Texas.
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.
REPORT OF THE DIRECTOR 47
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, 1621 Connecticut Are., 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, DR. LEON, 155 Powell Lane, Upper Darby, Pennsylvania.
CLAFF, MR. C. LLOYD, Research Fellow in Surgery, Harvard Medical School,
Boston, Mass.
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., Harvard University Biol. Lab., 16 Divinity Ave., Cambridge
38, Mass.
CLELAND, PROF. RALPH E., Indiana University, Bloomington, Indiana.
CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana.
COE, PROF. W. R., Yale University, New Haven, Connecticut.
COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts.
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.
COPELAND, 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.
CROUSE, DR. HELEN V., University of Pennsylvania, Philadelphia, Pennsylvania.
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.
48 MARINE BIOLOGICAL LABORATORY
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., 1175 Centre Street, Newton, 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.
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.
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.
REPORT OF THE DIRECTOR 49
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., 919 20th Street, 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.
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., Duke University, Durham, North Carolina.
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio.
HILL, DR. SAMUEL E., 18 Collins Avenue, Troy, New York.
HINRICHS, DR. MARIE, Department of Physiology and Health Education, Southern
Illinois Normal University, Carbondale, Illinois.
50 MARINE BIOLOGICAL LABORATORY
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. D.WIGHT 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, LT. COL. LAURENCE, Wright Field, Dayton, Ohio.
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts.
JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsylvania, Phila-
delphia, Pennsylvania.
JENKINS, DR. GEORGE B., 1336 Parkwood Place, 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, Williamsburg, 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.
KIDDER, JEROME F., Woods Hole, Massachusetts.
KILLE, DR. FRANK R., Carleton College, Northfield, Minnesota.
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.
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.
LANDIS, DR. E. M., Harvard Medical School, Boston, Massachusetts.
LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.
REPORT OF THE DIRECTOR 51
LAVIN, DR. GEORGE I., 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.
LOCHHEAD, DR. JOHN H., Department of Zoology, University of Vermont, Bur-
lington, Vermont.
LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri.
LOEB, DR. R. F., 180 Ft. Washington Avenue, New York City, New York.
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.
LUCRE, 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, Dept. of Zoology, University of California, Los
Angeles 24, California.
LYNN, DR. WILLIAM G., Department of Biology, The Catholic University of Amer-
ica, Washington, D. C.
MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia.
MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa-
chusetts.
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.. 153 Cortland Avenue, Winter Park, Florida.
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.
MAYOR, 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.
MEM HARD, MR. A. R., Riverside, Connecticut.
52 MARINE BIOLOGICAL LABORATORY
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.
MOORE, DR. J. A., Barnard College, New York City, New York.
MORGAN, DR. ISABEL M., Poliomyelitis Research Center, 1901 E. Madison Street,
Baltimore 5, Maryland.
MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue,
New York City, New York.
MULLER, PROF. H. J., Department of Zoology, Indiana University, Bloomington,
Indiana.
NACHMANSOHN, DR. D., College of Physicians and Surgeons, 630 W. 168th Street,
New York City, New York.
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. SEVERQ, 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., Ohio 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., 5 Acacia Street, Cambridge, Massachusetts.
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.
REPORT OF THE DIRECTOR 53
PIERCE, DR. MADELENE E., Vassaf 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., 53 Alexander Street, Manchester. Connecticut.
PRATT, DR. FREDERICK H., Wellesley Hills 82, Massachusetts.
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.
REID, DR. W. M., Monmouth College, Monmouth, Illinois.
RENN, DR. CHARLES E., Harvard University, Cambridge, Massachusetts.
RENSHAW, DR. 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.
RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma.
RICHARDS, DR. A. GLENN, Entomology Department. University Farm, Univ. of
Minnesota, St. Paul 8, Minnesota.
RICHARDS. DR. O. W.. Research Dept. American Optical Co., 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.
SAMPSON, DR. MYRA M., Smith College, Northampton, Massachusetts.
SASLOW, DR. GEORGE, Washington University Medical School, St. Louis, Missouri.
SAUNDERS, LAWRENCE, W. B. Saunders Publishing Company, Philadelphia, Penn-
sylvania.
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.
54 MARINE BIOLOGICAL LABORATORY
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.
SCHMITT, PROF. F. O., Department of Biology, Massachusetts Institute of Tech-
nology, 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.
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.
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.
STOREY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts.
REPORT OF THE DIRECTOR 55
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, Univ. of California, College of Agriculture,
Davis, 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.
TE\VINKEL, DR. L. E., Department of Zoology, Smith College, Northampton,
Massachusetts.
TURNER, DR. ABBY H., Mt. Holyoke College, South Hadley, Massachusetts.
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.
WAINIO, DR. W. W., New York University, College of Dentistry, New York City.
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.
56
MARINE BIOLOGICAL LABORATORY
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.
YOU-NG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland.
9. ASSOCIATES OF THE MARINE BIOLOGICAL LABORATORY
BARTOVV. MRS. FRANCIS D.
BEHNKE. JOHN.
BROWN, MR. AND MRS. THEODORE.
CALKINS, MRS. GARY N.
COOPER, CHARLES P.
CROSSLEY, MR. AND MRS. ARCHIBALD.
CROWELL, PRINCE.
CURTIS, DR. WILLIAM D.
FAY, MRS. H. H.
FOSTER, RICHARD W.
GARFIELD, IRVIN McD.
GREEN, GEORGE S.
GREEN, Miss GLADYS.
HARRISON, R. G., JR.
HUNT, MRS. REID.
JANNEY, MRS. WALTER.
KNOWER, MRS. H. McE.
LILLIE, MRS. F. R.
McMlTCHELL, MRS. J. McC.
MURPHY, DR. W. J.
NEWTON, Miss HELEN.
NIMS, MRS. E. D.
NORMAN, EDWARD A.
RIGGS, MRS. LAWRASON.
RUDD, MRS. H. W. D.
SAUNDERS, MRS. LAWRENCE.
STOCKARD, MRS. C. R.
SWOPE, MR. AND MRS. GERARD.
TEBBETS, WALTER.
WEBSTER, MR. AND MRS. E. S.
WICK, MRS. MYRON T.
WILSON, MRS. E. B.
^v
C
<*D
LIBRA*\
J*4S*
o •*
*
THE INFLUENCE OF TEXTURE AND COMPOSITION OF
SURFACE ON THE ATTACHMENT OF SEDENTARY
MARINE ORGANISMS*
C. M. POMEKAT AND C. M. WEISS
Medical Branch, University of Tc.vas and the IToods Hole Oceanographic Institution *
Marine installations of various kinds necessitate exposure of construction mate-
rials under sea water. Data dealing with the amount of fouling accumulated by
such materials are not abundant. Information which might be of aid to the scien-
tist seeking the most favorable material upon which to collect sedentary organisms
for study is also scanty. The present study was undertaken to determine the effect
TABLE I
Effect of surface texture of glass on attachment of sedentary organisms. (Numbers of individuals
on each surface of 80 square inches of plate)
Surface number
Plain
0
Sand-
blasted
1
Factrolite
2
Prestlitt-
.?
Ribbed
4
Pentecor
5
Series No. 1, Tahiti Beach1
39 days (8/22/42-9/30/42)
Hydroides sp.
143
265
152
506
349
197
Spirorbis sp.
85 1 88
122
90
163
110
Barnacles
1,948 1.072 1,162
975
1,674
2,140
Total
2,176 1,525
1 ,436
1,571
2,186
2,447
Average pop.
725.3 508.3
478.7
523.7 ' 728.7
815.7
Average/square inch
9.1
6.4
6.0
6.5
9.1
10.2
Series No. 2, Miami Beach-
17 days (8/22/42-9/8/42)
Wet weight (grams)
5 1 .0
45.5
50.0
41.0
50.0
4 1 .0
Drv weight (grams)
8.5
6.4
7.9
5.5
7.5
8.8
Barnacles
308
227
268
213
263
331
Series No. J, Miami Bcach-
30 days (9/15/42-10/15/42)
Wet weigh 1 (grams)
164.5
174.0
149.0
1 50.0
126.0
155.5
1 )ry weight (grams)
51.5
50.0
30.0
24.0
24.0
33.0
Barnacles
642
515
554
778
798
977
1 Subtropical testing service.
2 Beach boat slips.
* The observations described here were made while the authors were engaged by the Woods
Hole Oceanographic Institution in an investigation of fouling, under contract with the Bureau
of Ships, Navy Department, which has given permission for their publication. The opinions
presented here are those of the authors and do not necessarily reflect the official opinion of the
Navy Department or the naval service at large. Contribution No. 349 from the Woods Hole
Oceanographic Institution.
57
58
C. M. POMERAT AND C. M. WEISS
of surface irregularities and of substrate composition on the establishment of sessile
populations. The experiments were conducted in Biscayne Bay at Miami, Florida,
where subtropical conditions favor the attachment of fouling organisms throughout
the year.
Grateful acknowledgment is made to Dr. A. C. Redfield and Dr. F. G. Walton
Smith for many helpful suggestions.
FIGURE 1. Glass surfaces used in testing the relation of surface irregularities to fouling. 0. Plain.
1. Sandblasted. 2. Factrolite. 3. Prestlite. 4. Ribbed. 5. Pentecor.
EFFECT OF SURFACE IRREGULARITY
Commercial glasses, manufactured by the Pittsburgh Glass Company, with
various surface irregularities were used for this study. Six 8 X 10 inch glass plates
were assembled, irregular surface down, in a rack suitable for floating on the surface
of the water. The floats were constructed in such a way that sea water could move
freely on both sides of the exposed surface. The backs of the panels which were
all relatively smooth were placed upward. The fouling on the back surfaces was
ATTACHMENT OF MARINE ORGANISMS 59
not recorded. The surface irregularities of the panels are shown in Figure 1 and
may be described as follows :
Surface Number:
0. Plain Smooth glass, polished.
1 . Sandblasted Glass sandblasted on lower side.
2. Factrolite Surface consisted of pyramidal depressions of which there were
about 144 per square centimeter.
3. Prestlitc Approximately nine pyramidal depressions per square centi-
meter.
4. Ribbed Surface of V-shaped grooves, nine grooves per centimeter of
width.
5. Pcntecor Approximately three V-shaped grooves per centimeter of
width.
Results obtained from three series of experiments in which the glass surfaces
were exposed are shown in Table 1.
The Sessile populations which grew on the glass plates were composed pri-
marily of barnacles and tubeworms, with irregular, perhaps seasonal, appearances
of tunicates and Anoinia sp. Barnacles (B. iinprorisns and B. amphitritc niveus in
order of relative abundance) were numerous in both locations, but those at Tahiti
Beach were always very small compared to those at Miami Beach. Many more
barnacles attached to the lower (shaded) surfaces of the panels than to the upper
surfaces where light was more abundant. This is in agreement with the experience
of Pomerat and Reiner ( 1(M2). who report that larger numbers of barnacles accu-
mulate on dark surfaces than on light surfaces. The shaded undersides of the glass
panels, being darker than the upper sides, appear to attract more cyprids and hence
show a greater barnacle accumulation.
The various surface textures of glass had little influence on the number of at-
tached organisms. In these experiments barnacles were consistently slightly more
numerous on Pentecor than on smooth glass. This behavior was confirmed in the
experiment reported in the following section although conditions of exposure were
not exactly parallel. In the first experiment the glass panels were floated at the
surface in a shaded location, while in the second they hung vertically below low tide
TABLE II
Influence of substrate on fouling, sixty days' exposure at the beach boat slips, September 25, 1942-
November 25, 1942
Weight of fouling on panel area of 264 sq. in., that of wood
panels employed
Substrate Wet weight grams Dry wt. grams
1. Dade County pine 675.1 346.5
2. Gum 1127.6 531.4
3. Magnolia 1165.4 446.4
4. White pine 968.7 446.8
5. Cypress 954.8 392.0
6. Tile 980.1* 487.3
7. Cement 1033.0* 534.1
8. Glass 386.1 167.3
* Corrected to an area, 264 sq. in., equal to that of the wood panels.
60
C. M. POMERAT AND C. M. WElSS
under sun exposure. Counts of tubeworms were made on only one set of expo-
sures. H \droidcs sp. was most abundant on Prestlite and Sf>irorbis sp. was most
abundant on sandblasted gla.ss.
COMPOSITION OF THE SURFACE
Unpainted panels of wood of five species, clay roofing tiles, cement roofing plates,
and a glass panel were exposed for 60 days at the Reach Roat Slips in Miami Reach.
TABLE 1 1 1
Effect of substrate on fouling, exposures of three months at South Dock, Belle Isle, Miami Beach,
Florida, January 9, 1943-April 9, 1943, all materials applied to, or mounted on, wood unless
otherwise noted
Composition of surfaces
\Yet
weight*
(grams)
Dry
weight*
(grams)
Number*
of bar-
nacles
Notes
Plastics
1. Celluloid
3.8,
2.2
11
Thin coat of algae.
2. Plasticel
24.3
12.2
124
Barnacles' bases easily removed.
3. Lucite
5.6
1.7
41
4. Formica
6.9
3.2
11
5. Isobutyl
15.4
7.2
70
Film applied to glass panel.
Methacrylate
Plastic peels intact with barnacles.
Glass
6. Prestlite
57.0
25.2
176
Some barnacles 12 mm. across.
7. Pentecor
46.0
25.0
148
Some barnacles 12 mm. across.
8. Sandblasted
23.6
7.0
46
6 calcareous tubeworms; tunica tes.
9. Smooth
4.5
1.7
16
Green slime may have caused fish to
remove young barnacles.
Paints and ingredients**
-
Coatings applied to steel
panels
10. Ester gum vehicle
36.3
8.1
58
Tunica tes and bryozoa.
1 1 . Rosin vehicle
2.7
0.4
0
Fish spawn both sides.
12. Anticorrosive paint 42-A
2.7
0.5
9
Baracles very small.
13. Vehicle of 15RC
6.1
3.3
43
14. Antifouling paint 7C
0.0
0.0
0
Some slime film.
15. Antifouling paint 8C
0.6
0.3
14
Small barnacles close to edge.
Coatings Applied to Wood
16. Ceraloid
57.6
38.5
183
17. Paraffin
11.3
6.1
59
Lomnoria active in breaking paraffin.
18. Asphalt u m
121.4
34.3
768
Barnacles onlv.
19. Asphaltum varnish
67.8
13.8
256
Some bryozoa.
20. Spar varnish
45.1
7.0
304
2 1 . Navy grev
41.6
5.6
150
Algae.
22. Anti-corrosive 42-A
48.2
10.7
156
* Corrected to an area of 144 square inches.
* Anticorrosive 42A is a standard Navy formula. Vehicle of 15RC is the non-pigmented
portion of a standard Navy antifouling paint. Antifouling paints 7C and 8C are experimental
modifications of a standard Navy antifouling paint of the cold plastic type in which the toxic
pigment is reduced to 50 and 60 percent of the normal value.
ATTACHMENT OF MARINE ORGANISMS
61
TABI.K I II — Continued
Wet
Dry
Number*
Composition of surfaces
weight*
weight*
of bar-
Notes
(grams)
(grams)
nacles
Woods
23. Dade County pine
395.2
120.7
748
Bryozoa.
(soaked 60 days)
24. Gum (soaked 60 days)
452.1
133.4
686
25. Hade County pine
144.3
27.3
125
Hydrozoa, bryozoa.
(unsoaked)
26. Gum (unsoaked)
249.8
43.5
222
27. Soft pine
57.6
11.5
184
28. Teak
143.8
88.7
306
Large barnacles.
29. Maderia
173.7
84.2
358
Manv lish eggs.
30. Greenheart
77.0
40.8
342
31. Balsa
2.9
1.6
5
Wood verv soft.
Metals
32. Steel
224.4
42.8
88
33. Galvanized iron
2.6
0.7
6
Barnacles easily removed.
34. Zinc
1.0
0.2
0
Active corrosion.
35. Lead
30.6
50.9
396
Large barnacles.
36. Monel
1.6
0.5
6
Manv fish eggs.
37. Nickel
43.2
10.7
126
38. Galvanized iron pipe
4.7
3.0
27
Barnacle on rusted threads and dam-
aged edges.
Miscellaneous
39. Linoleum
79.7
23.0
193
40. Deck canvas no. 10
5.1
2.3
7
Sagging-algae eaten by fish.
41 . Sole leather
32.4
12.4
66
42. Masonite, heat tempered
138.6
31.8
594
Brown tunicates.
43. Asbestos
284.2
65.9
980
Bryozoa, Anoniia, hydrozoa, calcar-
eous tubeworm.
All panels were suspended in a vertical position approximately two feet beneath the
mean low water mark. The site was well shaded by a protecting roof. The results
obtained are presented in Table II.
The weights of the populations (barnacles, tubeworms, tunicates, bryozoa, and
algae) which accumulated on the woods, tile, and cement were of the same order of
magnitude, though variations as great as 59 per cent were observed. The weight
of organisms accumulated on glass was approximately 30 per cent of that collected
from other substrate materials.
A much larger number of materials were tested in a second experiment, the re-
sults of which are given in Table III. Exposure was made for three months at
South Dock, Belle Isle, in Miami Beach, where conditions of bright sunlight, active
current movement, and moderate fouling incidence were found. Growth on the
panels consisted primarily of barnacles (B. hnprovisus and B. amphitrite niveus)
with occasional tufts of hydrozoa and patches of colonial tunicates. A blanket of
algae having very short filaments grew on panels of light color or shaded back-
ground. Large sets of fish eggs were found on the rosin vehicle and Monel. Bor-
ings of Liuuwria sp. were everywhere evident in unprotected wood.
62
C. M. POMERAT AND C. M. WEISS
The substrates accumulating heaviest populations were asbestos, asphaltum,
Dade County pine (pre-soaked 60 days), gum wood (pre-soaked 60 days) and
Masonite. Asbestos shingle, commonly used as clapboarding, yielded the richest
harvest as measured by the number of barnacles. A comparison of asbestos and
Masonite, two of the best collectors, is shown in Figure 2. The asphaltum used
was of the type employed as aquarium cement. It accumulated barnacles only.
Panels of gum and Dade County pine, which had been exposed for 60 days in
the earlier test reported in Table II, were included for comparison with unsoaked
specimens of these woods. The unsoaked woods developed much less fouling.
'
'
'-
, Jf B
• j
W&* I
• < , •;,
i } i ' •;' -V i
. * " > i /
". -1' ' •/'- :J^
"
^ ' <'' J;.^"
' '.'.. ."..•<'.:• " v'
' * 1^9" & 3 ~ft- ^B m > ' T . < •
•.V. : '£\ , ' - •
' . ' I :" ' -
:*..•».,, *| • • • •-,«"(- ',*
'•' • :- ' ••'' ' •••"*•*
': . •••"•!*
2. Accumulation oi fouling organisms on masonite and asbestos after 90 days' exposure
at Belle Isle, Miami Beach, Florida.
Intact galvanizing on iron was very resistant to marine life. No barnacles were
obtained on zinc, on the experimental antifouling paint 7C or on rosin vehicle, a
common paint component.
Materials with hard non-porous and non-fibrous surfaces were in general rather
poor collectors of fouling. The best accumulation of sedentary populations was
found on surfaces which were porous and/or fibrous. Surface of paints, paint in-
gredients and linoleum are in general non-porous and non-fibrous. Compared to
the size and strength of the barnacle cyprid they are also smooth and hard. The
histogram (Fig. 3) summarizes the collective efficiency of the substrates.
Some results were undoubtedly spurious, and these should be noted. Fouling
on the antifouling paint SC which occurred along the edges of the panel was probably
ATTACHMENT OF MARINE ORGANISMS
63
Grams Wet Weight of Foulini
lOO
200
400
300
PLASTICS
— — GJ.ASSZS
PA/HTS AND PAINT
BALSA
MSTALS (except Steel) - STEEL
•CAHVAS-LEATHIK.-LIMOLEUM - MASOMIT£ - ASBESTOS
WOODS
FICUKE 3. Relative amounts of foulins; on various classes ot materials used as test panels.
FIGURE 4. Bases of barnacles grown on various substrates. A. Navy grey. B. Antifouling
paint 15RC. C. Ester gum. D. Anticorrosive paint 42A.
64
C. M. POMERAT AND C. M. WEISS
due to imperfections of the paint surface. In contrast, 7C which contained less
copper was not fouled. Deck canvas and smooth glass both supported a culture of
green algae which evidently served as food for fish. Active feeding on these panels
unquestionably disturbed other fouling organisms. Balsa wood was apparently
sloughing its surface and thus loosening attached forms. In spite of these minor
qualifications, the results involve a range of population numbers sufficiently wide
to indicate the relative merits of the substrates used.
One of the most interesting of the results was observed when barnacles were
removed from the various substrates. Some of the substrates bore barnacles with
deeply scalloped margins (Fig. 4) instead of the typical smooth edges. These
margins suggested that localized irregular marginal growth interruptions had taken
place. Such barnacles were collected from :
Spar varnish
Linoleum
Navy grey topside paint (P— 50)
Antifouling paint vehicle 15RC
Ester gum paint vehicle
Anticorrosive paint 42-. \
"'
ft*, c?
FIGURE 5. Bases of barnacles grown on various substrates. A. Isobutyl methacrylate.
B. Plasticel. C. Soft paraffin. D. Ceraloid.
ATTACHMENT OF MARINE ORGANISMS 65
Barnacles growing on soft paraffin had distinctly concave bases. Mosaics of
bases witb angular margins were typical of barnacles attached to lead but were also
found on other overcrowded substrates. It was possible to remove barnacles with
intact bases very easily from several materials, including plasticel, ceraloid, and iso-
butyl methacrylate (Fig. 5). This finding might prove useful in designing experi-
ments in which the minute anatomy of basal structures was to be studied.
SUMMARY
1. Submerged samples of 40 different construction materials were used as sub-
strates for the collection of sedentary populations. The barnacle counts in the popu-
lations ranged from 980 on asbestos shingles to zero on zinc and on two paint coat-
ings, after three months' immersion in Biscayne Bay at Miami Beach, Florida.
2. Various surface textures of glass plates were found to exert no significant
influence on the accumulation and growth of sedentary marine organisms, although
smooth clear glass accumulated smaller populations in the comparatively short expo-
sure periods, 1—3 months .
3. The results suggest that efficiency of a substrate as a fouling collector is in
general correlated with porosity of surface or with fibrous nature of surface.
Smooth, non-porous, non-fibrous surfaces, especially if also hard, seem to be poor
accumulators of sedentary organisms.
4. Further testing of substrates is greatly to be desired in this connection.
REFERENCES
POMERAT, C. M., AND E. R. REINER, 1942. The influence of surface angle and of light on the
attachment of barnacles and other sedentary organisms. Biol Bull., 82 (1) : 14.
THE DEVELOPMENTAL HISTORY OF AMAROECIUM CONSTEL-
LATUM. II. ORGANOGENESIS OF THE LARVAL
ACTION SYSTEM
SISTER FLORENCE MARIE SCOTT
The Marine Biological Laboratory, Woods Hole, Mass.. and the Biology Department, Scion Hill
College. Greensburg, Pennsylvania
INTRODUCTION
The early development of the embryo of A-maroecium constellation has been pre-
sented in a previous paper (Scott, 1945). The accumulation of yolk modifies the
pattern of mosaic development characteristic of Tunicates to the extent that gastru-
lation is accomplished in an atypical manner. Convergence of the cells of the lateral
margins of the posterior blastoporal lip is accomplished to the left of the mid-line.
The neural plate elongates posteriorly at the place where the lateral blastoporal lips
meet and close. The chordal cells are inflected at the anterior lip and lie in the
median axis. The potential muscle cells of the morphological right side lie dorsal
to the notochord as a result of their growth across the mid-dorsal plane, the muscle
cells of the morphological left side lie below the level of the notochord on the curved
left side of the embryo. The two groups of muscle cells are separated by the poste-
rior extension of the neural plate.
MATERIALS AND METHODS
Amaroecium constellation is abundant along the eastern coast of the United
States. The breeding season lasts throughout the summer months. Material for
this study was collected at Woods Hole, Massachusetts. The embryos, squeezed
from adult colonies, were selected and arranged into a progressive series of stages
for study. They were fixed in Bouin's fluid. Some were stained by Conklin's
modification of Delafield's haematoxylin, others with borax-carmine, then cleared
according to the benzyl-benzoate method described in Romeis' "Taschenbuch der
Mikroscopischen Technik." Corresponding stages were sectioned serially, stained
in Mayer's or Gallagher's or iron haematoxylin, and counterstained with eosin or
triosin. All drawings were made with the aid of a camera lucida. The photo-
micrographs were made with a Leitz "Macca" camera using Zeiss apochromat, 20 X ,
and fluorite oil immersion, 100 X, objectives with a Zeiss microscope.
Later embryonic development
It seems advisable to present a descriptive series of developmental stages that
may be used as points of reference for structures differentiating during the organ-
forming period. For convenience the developmental period following gastrulation
is divided into four stages; 1) the tail bud stage, 2) early tadpole stage, 3) pre-
hatching stage, and 4) the free-swimming tadpole stage. The free-swimming larva
66
AMAROECIUM CONSTELLATUM. II
67
or tadpole has been described thoroughly by Grave (1921) and shall be presented
here in brief summary since reference to it is necessary. A short description of the
external appearance of these stages will be given first and referred to in subsequent
treatment of organogenesis as Stages I, II, III, and IV. The terms, larval action
system and adult action system, used by Grave (1935, 1944) will be adopted for
the structures functioning during larval life and those functioning during adult life
respectively.
The tail bud stage
By the end of gastrulation the embryo is approximately spherical except for a
shallow postero-ventral invagination of the ectoderm constricting tail from trunk
region. The furrow appearing first on the right side is deeper there, and less deep
as it extends to the left side. The tail bud is short and rounded, curving immedi-
ately toward the ventral side of the trunk. Through the thin epidermis quadruple
rows of large muscle cells can be seen lying dorsal and ventral to the notochord.
The neural plate is elevated at the periphery to form the neural groove, enclosing;
anteriorly a wide depression, the presumptive brain region, posteriorly a narrow,
trough-like depression lying to the left of the notochord, the presumptive neural
tube (Fig. I A).
h.v
FIGURE 1. A. Stage I, embryo before neural folds close. 160 X. B. Stage II, early tad-
pole; beginning of differentiation of digestive and nervous systems. 160 X. b. v., brain vesicle;
d. ph., dorsal diverticulum of pharynx; m. bd., muscle band; n. t., neural tube; ph., pharynx;
y. m., yolk mass.
A transverse section through the tail bud stage discloses that the embryo is solid.
A single layer of definitive endoderm lies under the concave neural plate (Fig. 5£).
This layer of cells develops from the cells that form the superficial "pseudo-
invagination" cavity of gastrulation. The depression closes by a reversal in change
of shape of the cells involved rather than by approximation of the lips of the blasto-
pore thus producing a solid archenteron (Scott, 1945). The endodermal cells
spread under and anterior to the neural plate. Ventral to them is located the mass
of heavily yolk-laden cells derived from the macromeres. Wedged between the
68 FLORENCE MARIE SCOTT
thin ectoderm and the solid endoderm on either side is a mass of mesenchyme, small,
polygonal cells with prominent nuclei (Fig. 5£).
Posteriorly the definitive endoderm lies adjacent to the chordal cells which are
beginning to interdigitate in the base of the tail bud. The mesenchyme terminates
abruptly in this region against the muscle cells of the tail.
Early tadpole stage
The embryo increases in size and acquires the shape that justifies its being called
"tadpole." The trunk region elongates slightly in the antero-posterior axis remain-
ing curved at the anterior end. The tail encircles the body meridionally as it grows
in length. The embryo is still opaque.
The neural folds are closed, the position of the sensory vesicle being marked by
aggregations of black pigment which show through the surface of the body. The
neural tube is faintly visible along the side of the tail. More conspicuous are the
large muscle cells dorsal and ventral to the prominent notochord which forms the
axis of the tail throughout its length. Dorsally, on either side of the sensory vesicle
there is a slight ectodermal invagination, rudiments of the atrial chambers. The
embryo is confined within a test the cells of which are arranged in a compact layer
(Fig. IB).
Pre-hatching stage
Changes in the external appearance of the later embryo depend on the develop-
ment of siphons and adhesive papillae and the secretion of a tunic. As body growth
continues and organs of the larval action system differentiate, the body becomes
transparent except where the mass of yolk is lodged in the pharynx.
The trunk region continues to elongate antero-posteriorly becoming elliptical in
shape. Posteriorly the body narrows to the base of the tail ; anteriorly it flares in
the dorso-ventral axis in relation to the vertical position of the adhesive papillae.
Laterally the body is compressed. A thickening layer of tunic invests the entire
trunk. It is indented at the junction of trunk and tail and continues over the sur-
face of the tail. The tail encircles the body meridionally being pressed into a groove
in the tunic. The tunic of the tail projects laterally into fins.
The sensory vesicle occupies a dorsal position at the posterior end of the trunk.
Two masses of pigment project into its cavity. Immediately in front of it lies the
elevation of the oral siphon ; behind it and on the posterior curve of the body lies the
atrial siphon. Much of the internal structure is visible through the tunic and
mantle. The incipient adhesive papillae appear as three disc-like projections in
verticle series at the rounded anterior end (Fig. 2).
The free-swimming tadpole stage
The trunk of the tadpole of Amaroecium at its release measures about 600 micra
in length; it measures about 270 micra in depth. The tubular atria with their triple
rows of gill slits are pressed into the dorsal pharynx through half of its length
posteriorly. An obvious structure in the pharynx is the dorsal, heavily ridged endo-
style which seems to rest on the lateral masses of yolk that form the wall of the
pharynx. The transparent pericardium occupies a large space below the yolk ante-
AMAROECIUM CONSTELLATUM. II
69
riorly in front of the loop of alimentary tract. On the right side of the body the
stomach extends along the posterior and ventral curvature of the yolk. On the left
the narrow intestine curves along the side of the stomach up to the left atrium where
it terminates. The root of the tail lies in the posterior third of the length of the
body.
Anteriorly, the adhesive papillae project into the tunic in a vertical row slightly
to the right of the median plane. The test vesicles lie loosely within the tunic or
many of them, even at the time of hatching, retain a slender connection with the
cone or ridge from which they originate. Where the tail is continuous with the
trunk the tunic dips clown into an abrupt pocket. The epidermis secretes a thin
sheath of tunic about the tail. Laterally it expands into wide sail-like fins (Fig. 3).
,5.V.
ad.
ps~
FIGURE 2. Stage III, lateral view of tadpole with incipient adhesive papillae. About 120 X.
ad. p., adhesive papillae; end., endostyle ; ep., epidermis; /. a., left atrium; >i. t., neural tube; oes.,
oesophagus; p. c., pericardia! cavity; ph., pharynx; st., stomach-intestine rudiment; s. v., sensory
vesicle ; tu., tunic ; y. »i., yolk mass.
ORGANOGENESIS OF THE LARVAL ACTION SYSTEM
Digestive system
The pharyngeal cavity develops in Stage II by delamination between the layer
of definitive endoderm and the mass of yolk-laden cells, appearing first below the
brain and spreading from that point (Fig. IB, 5F, 6E}. It extends back to the
base of the notochord as an upwardly directed diverticulum. Ventral to the base
of this projection a second invagination appears, the rudiment of the stomach and
intestine located a little to the right of the median plane on the inner side of the
visceral ganglion (Fig. 2, 6F).
The pharynx deepens in Stage III encroaching upon the mass of yolk cells.
Gradually thin septa of epithelium divide the yolk mass into four compact longi-
tudinal columns, the two on each side being continuous at the bottom. The central
70
FLORENCE MARIE SCOTT
two are lower than the outer two, thus providing greater depth for the limited
pharyngeal cavity (Fig. 6A, F). This supply of nutritive material in the pharynx
remains to be digested during the active life of the larva and throughout the critical
period of metamorphosis. All other tissues lose their meager supply of yolk almost
entirely, leaving their cytoplasm clear.
Along the roof of the pharynx, anterior to the place of origin of the oral siphon,
the epithelium rises up into a double fold enclosing the endostyle, restricted to the
dorsal side above and between the lateral masses of yolk and passing to the anterior
end of the yolk mass (Fig. 2, 6A}. Before the tadpole is released from its test, the
cells in the floor of the groove develop long cilia. The pharynx grows out above
and below the atrial sacs, bringing the mesial atrial and lateral pharyngeal walls into
intimate contact (Fig. 65).
Due to the combined activity of atrial and pharyngeal epithelia, three horizontal
rows of gill slits are formed, each consisting of seven or eight perforations. The
oral Siphon
FIGURE 3. Stage IV, tadpole at hatching. About 120 X. end., endostyle; cp., epidermis:
int., intestine; p. c., pericardia! cavity; st., stomach; s. v., sensory vesicle; te. v., test vesicles:
tit., tunic ; y. m., yolk mass.
bordering cells of each gill develop a heavy brush of cilia, precocious equipment from
the functional point of view. Even though the mouth breaks through to the bran-
chial chamber, the tunic fills up the oral and atrial siphons until metamorphosis is
completed.
The rudiment of the oesophagus grows forward along the curvature of the yolk
and dilates to form the stomach. The diverticulum extends to the midventral region
of yolk where it turns sharply upon itself and continues backward as the slender
intestine. With a gradual slope upward the intestine retraces the course of the
stomach on its left side terminating ventral to the posterior end of the left atrium
(Fig. 3, 6B). Later the anus opens into the atrium here.
There are no cilia evident in the intestine or stomach during this period of de-
velopment. The wall of the stomach is thicker than the wall of the remaining parts
of the digestive tract although the alimentary epithelium, throughout its length, con-
sists of a single layer of cells.
AMAROECIUM CONSTELLATUM. II 71
With rapid general growth of the body, the loop of intestine and stomach in-
creases in length anteriorly, extending through the posterior half of the body cavity
below and behind the yolk-laden pharynx (Fig. 3). The pericardium lies directly
in front of it. Between the arms of the loop posteriorly are lodged the bases of
the axial organs of the tail.
Atrium — During Stage II the atrium or peribranchial sac appears as a
pair of ectodermal invaginations, one on either side of the sensory vesicle (Fig.
6£). At the place of its origin the neck of each depression constricts and separates
from the surface.
In the transition from Stage II to Stage III, the atria, in contact with the lateral
endodermal wall of the pharynx, grow in an anterior direction only, with the result
that the atrial chambers are horizontal capsular cavities located dorsally, one on
either side of the pharynx (Fig. 6B). They extend through the posterior two-
thirds of the trunk, curving gently upward posteriorly where they grow towards
each other and unite behind the pharynx (Fig. 3). The atrial siphon opens
through the dorsal wall of this connecting canal between the two cavities.
The atrial walls are characteristically thin and the cells lose their intercell mem-
branes. Occasional yolk granules are scattered through the cytoplasm. During
Stage III the gill slits perforate the walls in three horizontal rows on the inner side
in direct contact with the wall of the pharynx. The lowermost row develops first,
the atrial and pharyngeal fusing first in these regions. The slits number between
seven and nine in each row. Later in the free-swimming period the cells bordering
the gill aperture produce long cilia. The endoderm has no part in atrial formation
except insofar as the gill slits are the product of joint activity of atrial and pharyn-
geal walls (Caullery, 1895).
Oral and Atrial Siphons — Late in Stage III the dorsal ectoderm in front
of the sensory vesicle thickens and invaginates, pushing the endoderm of the pharynx
before it. The circle of epidermis around the invaginated area becomes elevated,
giving the oral siphon a crater-like appearance (Fig. 68} . The floor of the invagi-
nation thins out in a flat layer against the pharyngeal roof with which it is in con-
tact. The lower part of the cavity projects outward from the center and produces
a ring-shaped extension on the mouth opening. The oral cavity assumes the shape
of a flask with a long neck and a flattened base (Fig. 3). Into this ectodermal
cavity, or stomodaeum, the hypophysial duct opens, just before hatching of the tad-
pole. Although the oral plate breaks through late in the tadpole's development,
the tunic fills up the stomodaeal portion and prevents the passage of both food and
water during larval life.
The atrial siphon, like the oral, is formed by ectodermal invagination. The
thickened mantle is elevated, raising the siphon above the level of the rest of the
mantle in knob-like fashion (Fig. 6C). The floor of the invagination fuses with
the dorsal wall of the connecting arm of the atrium. The atrial siphon is situated
on the downward curve of the dorsal surface just posterior to the sensory vesicle
and anterior to the insertion of the tail (Fig. 3, 6G, H). The epithelial lining of
the oral and atrial siphons projects into each opening at several points forming
small tentacles. The mesenchymatous muscles in the mantle in this region provide
the contractile elements that control the apertures when the siphons begin to function.
FLORENCE MARIE SCOTT
Heart and pericardium
Towards the end of Stage III, the endodermal cells extend completely around
the yolk mass as a definite epithelium. Mid-ventrally it evaginates into the body
space and constricts off from the yolk epithelium. The bladder-like vesicle is the
pericardium which invaginates mid-dorsally into an inner enclosed vesicle, the heart.
The cells lose their inter-cell membranes and the nuclei bulge irregularly in both
cardial and pericardial walls (Fig. 6 A). The heart does not develop beyond this
point at present, the circulatory system not functioning during larval life.
The nervous system
The neural folds of Stage I close in the early phase of Stage II thus forming the
hollow nervous system typical of chordates except in one point, the curving of the
neural tube through 90° to the left of the brain region. The anterior portion of the
nervous system produces the sensory vesicle with its sensory organs, the hypophysis,
definitive ganglion, and the so-called subneural gland. The intermediate part in-
cluding the origin of curvature and a small contribution from the brain region gives
rise to the visceral ganglion and the spinal enlargement, the posterior part becomes
the neural tube.
The cavity of the brain region is slightly dilated and its wall uniformly thick.
The neural tube consists, in section, of four cuboidal cells surrounding a small lumen
(Fig. 4C). Cell membranes in both regions are distinct at this stage, the nuclei
are large and contain heavily staining nucleoli. The cytoplasm is reticular in ap-
pearance and has occasional yolk granules.
During Stage III the brain vesicle differentiates into two structures, the sensory
vesicle in the entire right side and the rudiment of the hypophysis on the left poste-
rior side (Fig. 4A, 5 A}. The vesicle expands, its walls becoming thin; the rudi-
ment of the hypophysis remains small with thick walls. This secondary cavity is
separated completely from the sensory vesicle at the region of evagination but their
walls remain attached throughout subsequent development (Fig. 5B, C).
The sensory vesicle — Two sensory structures develop in the sensory vesi-
cle, the statolith and the eye. The left posterior wall of the vesicle thickens, the
right wall expands dorsally and laterally ; all the cells lose their inter-cell mem-
branes. The left wall of the cavity remains thick and constitutes the sensory
ganglion of the brain. One cell on the ventro-anterior wall projects into the cavity
and large pigment granules are deposited in its cytoplasm ; these coalesce to form
the statolith (Fig. 4B, SB). In Stage IV the statolith is a spherical mass of pig-
ment confined within the cell membrane and attached to the ganglionic wall by a
stout stalk, the remaining part of the cell (Fig. 4D, SC, D}.
A group of cells situated dorso-laterally at the left posterior limit of the vesicle
initiates the development of the eye by the deposition of pigment granules of much
smaller size than those that form the statolith. Absence of cell membranes makes
it difficult to ascertain the number of cells that participate in this activity. The pig-
ment is deposited in the shape of a cup, its concavity facing dorso-laterally and to
the right within the vesicle. Three ganglionic cells which retain their membranes
fill up the concavity in series. They secrete globules of liquid which increase in size
both by the gradual addition of the secretion and by the fusion of globules. The
globules of liquid form the so-called lens cell (Fig. 4A, B, D, 5D). The nuclei
AMAROECIUM CONSTELLATUM. II
73
..ret
FIGURE 4. .4. Transverse section through brain after the neural folds close, Stage II. 750 X.
B. Longitudinal section through brain of same stage. 750 X. C. Cross section through tail of
tadpole, Stage III. 300 X. D. Section through brain of tadpole just before hatching, oblique to
include both sensory organs. 750 X. E. Longitudinal section through tail of tadpole in Stage
III. 300 X. F. Section through epidermis and test of Stage III. 750 X. G. Reconstruction
of brain and related structures of Stage III, viewed from left side. 300 X. b. v., brain vesicle;
con. fib., contractile fibrils; dcf. g., definitive ganglion; cp., epidermis; hyp., hypophysis; /. c.,
lens cell; m. bd., muscle band; n. c., neural canal; nch., notochord ; n. t., neural tube; ret., retinal
cells of eye; s. gn., sensory ganglion; sn. gl., subneural gland; stat., statolith ; .?. c., sensory cell;
s. e., spinal enlargement ; .y. in., smooth muscle cells of mantle ; j. p., sensory pigment ; t. c., test
cells ; tu., tunic ; v. g., visceral ganglion ; y. g., yolk granules.
74 FLORENCE MARIE SCOTT
which at first occupy a central position in the cells are pushed to the periphery as
the lenses, increasing in size, come eventually to monopolize the entire cell.
The pigment granules of the eye always remain discrete, not coalescing as do
those of the otolith. Extending through the concentrated pigment are small rods
of clear cytoplasm. They run from the hase of the cup back towards the ganglion.
Seven or eight of them may be seen in embryos of Stage IV that are mounted in a
mixture of benzyl-benzoate and oil of wintergreen.
The hypophysis — The rudiment of the hypophysis early in Stage III ap-
pears as an extension or small evagination of the brain cavity (Fig. 4A, 5 A). The
cells retain their membranes, their nuclei are smaller than those of the adjoining
part of the brain. Histologically they present the appearance of epithelial tissue.
Upon its separation from the primary cavity during Stage III it elongates antero-
posteriorly along the left side of the sensory ganglion (Fig. 4G). In Stage IV it
ends blindly at the posterior wall of the oral siphon. Later these walls fuse and the
hypophysis communicates with the posterior region of the stomodaeum, extending
along the side of the ganglion with a gentle slope upward as far as the atrial siphon
where it terminates blindly. The floor of the duct, corresponding in position to
the region of the eye, deepens abruptly (Fig. 4D, G). The ventral wall of the
pocket becomes slightly thicker, the indentation with its thickened floor constituting
the subneural gland. Hjort (1896) reviews the opposing views concerning this
structure in the early works on Tunicates.
The definitive ganglion — By a proliferation of cells in the mid-region of
its roof in Stage II the hypophysial duct produces an oval mass containing small
nuclei similar to those in the hypophysial duct itself. The cell membranes dis-
appear and the nuclei wander out toward the periphery where they collect in several
rows with the granular cytoplasm concentrated in the center (Fig. 4D, 4G, SC).
This part of the nervous system, the definitive ganglion, persists through meta-
morphosis and together with the hypophysis gives rise to the permanent nervous
system of the adult.
Visceral ganglion — The visceral ganglion originates in that part of the
neural plate that curves toward the left in Stage I. The lumen is obliterated, the
large nuclei migrate to the periphery leaving the medulla mass of interlacing fibrils
and granules (Fig. 4D). The visceral ganglion lies posterior to and ventral to the
sensory vesicle. Dorsally where it merges with the sensory ganglion, it exceeds the
sensory vesicle in diameter but it gradually diminishes in diameter towards the base
of the tail where it is continuous with the neural tube. At the junction there is a
slight enlargement called the spinal enlargement (Fig. 4G). The neural tube re-
tains its lumen. It runs through the length of the tail to the left of the notochord.
In Stage IV a single nerve emerges from the visceral ganglion on its right side just
below the hypophysis. It runs anteriorly and sends out branches to the smooth
musculature of the mantle.
THE NOTOCHORD
At the end of the gastrulation period the chordal cells lie under the posterior part
of the neural plate. Anteriorly adjacent to them are endodermal cells; dorsally, the
potential muscle cells of the right lateral margin of the blastopore ; ventro-laterally,
the potential muscle cells of the left lateral margin of the blastopore. Posteriorly
the chordal cells extend into the rudiment of the tail.
AMAROECIUM CONSTELLATUM. II
75
. Vi
• ., • "I
*
FIGURE 5. A. Transverse section corresponding to Figure 4 A. 225 X. B. Transverse sec-
tion through brain of Stage III, hypophysis separated from brain vesicle. 650 X. C, D. Sec-
lions through sensory vesicle and definitive ganglion of Stage IV; oblique, thus including both
sensory organs. 650 X. E. Transverse section through Stage I; anterior region. 150 X. F.
Transverse section through early Stage II; neural folds closed. 150 X. G. Longitudinal sec-
tion through adhesive papilla of Stage III. 650 X. d. c., definitive endoderm ; dcf. <;., definitive
ganglion; <//. c., gland cells; //y/1., hypophysis; inch., mesenchyme cells; 11. p., neural plate; stal.,
statolith.
Some of the endodermal cells of the yolk mass lie along the right side of the
chordal cells and when the tail is constricted from the trunk region these cells form
the loose column of caudal endoderm. In Stage IV little of it remains (Fig. 4C,
65).
In Stage I the notochordal cells begin to shift in position. They interdigitate
into a row of disc-shaped cells occupying the central axis of the short tail. The cells
76 FLORENCE MARIE SCOTT
resemble the endodermal cells of the yolk mass in possessing delicate membranes,
nuclei smaller than those of adjoining muscle cells, and yolk granules.
During Stages II and III the notochord elongates as the tail lengthens. The
chordal cells lengthen ; the inter-cell membranes separate from each other converting
them into hour-glass shaped cells with the nucleus resting in the constricted neck
between the peripheral masses of protoplasm (Fig. 4E).
In Stage IV the cell halves separate completely giving the chord the appearance
of a tube with a scalloped lining. The proximal end retains its relationship with
the hinder end of the pear-shaped mass of yolk between the atrial cavities and the
arms of the digestive tract (Fig. 6C, H). Distally it corresponds in length to the
neural tube and tail muscles.
Muscle cells of the tail
Mesoderm differentiates into three structures of the larva, one of which is re-
stricted to the larval action system, two of which function in both the larval and adult
action systems. The former includes the muscles of the tail, the latter the muscles
of the mantle and mesenchymatous connective tissue in the body cavity. The asym-
metry of the posterior lip of the blastopore at the end of gastrulation (Stage I)
places the muscle cells of the right side dorsal to the chordal cells and to the right
of the neural plate at its posterior end, the muscle cells of the left side to the left of
the posterior neural plate but ventral to the notochord (Fig. 4C). Each band is
made up of four cells in fairly regular rows.
In Stage II the myoblasts are the most prominent cells in the body because of
their size and heavy membranes. Each cell contains a large faintly reticular nucleus
with a conspicuous nucleolus. The deeper cytoplasm is grossly reticular and retains
an occasional yolk granule (Fig. 6D).
In Stage III the peripheral cytoplasm elaborates in its cortex, in a slightly spiral
direction, along the longitudinal axis rows of contractile fibrillae composed of minute
granules so distributed that they resemble the individual myofibrillae of striated
muscle of the higher chordates (Fig. 4E, 6D). The myofibrillae are continuous
from one cell to another throughout the length of the muscle bands. Grave (1921)
has described this in the free-swimming tadpole of Amaroecium. The bases of the
muscle bands, like that of the notochord in Stage IV, are located well within the
posterior part of the trunk just behind the mass of yolk (Fig. 6H).
Muscles of the mantle
In the late embryonic period (Stage III) many of the mesenchyme cells located
directly under the ectoderm unite end to end to form the smooth fibres of the mantle
(Fig. 4F). One set of such muscle fibres radiates from each of the siphons. The
other set encircles the trunk obliquely from the dorsal to the ventral side.
Mcscnclivinc of the body cavity
In Stage I two compact lateral masses of mesenchyme cells lie pressed tightly
between the nutritive endoderm and shallow' ectodermal cells. The one on the right
side is disposed more dorsally than the one on the left side (Fig. 5E). They ex-
tend from the posterior muscle cells towards the anterior end of the body.
AMAROECIUM CONSTELLATUM. II
77
w
/^
*}
.---or.S
•
&
Ph *VA KF- ^V
'it 7Kf5 f ^4 i
. -)' fed
,
PC— V-^ / /•*
v.,.. ' ... ^gjf
"1 C
' • r
kj -»
H
FIGURE 6. A, B, C. Transverse sections through tadpoles of Stage IV; A, anterior. B. In
region of oral siphon. C. In region of atrial siphon. About 200 X. D. Section through part
of muscle hand ; middle cell through center of muscle cell, lateral cell through peripheral cyto-
plasm where myonbrillae are formed. About 850 X. E. Transverse section through Stage II to
show atrial invaginations. 300 X. /;. Longitudinal section through Stage III. About 150 X.
G. Tadpole just before hatching, chorion not ruptured. About 250 X. H. Tadpole at hatching.
Note insertion of notochonl at posterior end of trunk. About 250 X. ad. p., adhesive papilla;
at., atrium; at. in., atrial invagination ; at. .?., atrial siphon; end., endostyle ; int., intestine; i)i. c.,
muscle cell; ncli.. notochonl; or. .v.. oral siphon; ph.. pharynx; p. c., pericardial cavity; st..
stomach ; ^. v., sensor}- vesicle ; v, y., visceral ganglion.
78 FLORENCE MARIE SCOTT
In Stage II both masses of cells multiply and spread out under the ectoderm in
all directions except posteriorly. A small amount of mesenchyme is found in the
tail, probably derived from the cells in the mid-region of the posterior lip of the
blastopore. Being crowded together the cells appear angular in section. The nu-
clei are relatively large (Fig. 4F, 5(7).
During the transition from Stage II to Stage III growth of the body and absorp-
tion of the yolk effect a separation between the epithelial cells of the epidermis and
the endodermal cells (Fig. SF, 6F). As the body cavity enlarges the cells round
up, separate from each other, and wander freely about, dividing frequently and even-
tually filling up all available space except in the area around the base of the tail ( Fig.
6A,B,Q.
Other mesenchyme cells assume stellate shape and send out long slender strain IN
of protoplasm by means of which they form a reticulum of mesenchymatous tissue.
This is the nearest approach to a coelomic epithelium that is found in Tunicates with
the possible exception of the perivisceral cavity of Ciona.
The mantle and tunic
The epidermis in Stage I is a layer of thin cells small in surface view dorsally
where they adjoin the neural plate, larger towards the ventral body region. In
Stage II the cells are of uniformly small size and cuboidal in section except where
they invaginate to form the atrium and are columnar in shape. In surface view all
present the characteristic polygonal arrangement of epithelial tissue.
During Stage III the protoplasm becomes vacuolated medially, the nuclei being
pushed to the periphery where the cytoplasm is more granular ( Fig. 5B, 6E). The
epidermal cells grow thinner as development progresses and the inter-cell mem-
branes disappear. \Yhen the epidermis has assumed the characteristic appearance
of the Tunicate mantle in Stage III it secretes a thick layer of structureless tunic.
Occasional cells of the test of the ovum are trapped in the clear tunicin, the greater
number, however, being pushed with the test ahead of the tunic (Fig. 6C). The
tunic is grooved where it is secreted about the tail and when the tail is released, with
the disappearance of the test, the groove remains in evidence marking the embryonic
position of the tail.
Dcrh'ati'i'cs <>j tJie epidermis
Adhesive papillae — A conspicuous feature of the Amaroecium larva is a
vertical row of three adhesive papillae at the anterior end (Fig. 3, 6/;, 6", H). Each
papilla first appears early in Stage III as a local thickening of ectoderm forming a
pad of columnar cells. The cells at the periphery of the thickened pad form a stem
which increases in length as the tunic thickens, the whole organ becoming goblet-
shaped. It retains its connection with the body cavity through its slender hollow
stem (Fig. 6G, H). The papillae extend through the thickness of the tunic and
are exposed at its surface. Cells that constitute the functional portion become vacu-
olated and reticular proximally and toward the center of the cup, where the long
cells converge, they produce secretion granules which lodge in the concavity of the
papilla (Fig. 56"). The bordering epidermis surrounds the disc forming a thin
layer over the cup-shaped depression. During the free-swimming life of the larva
AMAROECIUM CONSTELLATUM. II 79
the secretion granules are converted into a viscid substance by means of which the
tadpole becomes attached. The entire glandular structure is of ectodermal origin.
Grave (1921) from his study of the fully formed tadpole supposed that mesenchyme
cells gave rise to the glandular portion of the papilla. Mesenchyme cells wander
from the body cavity into the hollow stalk but they are not incorporated into its
structure. The tail encircling the body crowds the papillae a little to the right of
the sagittal plane thus adding to the asymmetry of the larva. The three papillae
cannot be homologized with the tactile papillae of Botryllus, which are integral parts
of the peripheral nervous system. Here they serve only as gross organs of
attachment.
Test vesicles — During Stage III, when the adhesive papillae are differ-
entiating, the test vesicles originate as numerous small ectodermal evaginations in
four distinct regions at the anterior end of the trunk. Two groups, separated from
each other by the median papilla, are directed forward. The dorsal group is de-
rived from a short ridge extending in the direction of the oral siphon. The ventral
group, below the ventral papilla, is derived from a long ridge extending posteriorly
through about a third of the length of the trunk (Fig. 5, 6G, H). The vesicles
themselves originate as independent hollow slender projections of the ectoderm.
The attached end of each evagination becomes narrow, finally constricting off and
severing its connection at the base. Frequently this separation is not effected by the
time of hatching of the vesicle still being attached to the epidermis by their stalk-like
bases. When detached the slightly pear-shaped vesicle rounds up and becomes a
sphere consisting of a single layer of cells which lose their definition on the proximal
side where they are extremely thin.
The use of the word "test" in connection with these vesicles is unfortunate. The
chorion of the egg of Tunicates is called the test and the cells that either lie freely
in the enclosed liquid or are resolved into pavement epithelium are called the test
cells. The tunic of the tadpole is a purely ectodermal derivative. The tunic of the
adult colonies being the product of secretory activity of these vesicles, the vesicles
should, with greater accuracy, be called the "tunic vesicles."
SUMMARY
1. The digestive system of Amaroecium lacks an open archenteron at the end of
gastrulation. The pharynx appears as a narrow incision with a thin roof and heavy
floor. An oesophageal evagination differentiates into stomach and intestine.
2. Heart and pericardium originate from the floor of the pharynx.
3. Atrium and siphons are ectodermal structures that become associated with
the digestive system.
4. The nervous system consists of a sensory vesicle enclosing two sensory masses
of pigment, a hypophysis lying beside two sensory ganglion, a visceral ganglion de-
scending laterally to the neural tube which lies to the left of the notochord through-
out the length of the tail.
5. The notocord is derived from chordal cells invaginated at gastrulation. Its
cells become vacuolated. The notochord is confined to the tail and posteriormost
region of the trunk.
6. Muscle cells differentiate from mesodermal cells of the blastoporal margins.
Asymmetry of the blastopore places the cells of its right margin dorsal to the noto-
80 FLORENCE MARIE SCOTT
chord, the cells of the left margin ventral to the notochord. Each band of muscle
cells consists of four longitudinal rows. Cells separate from the two lateral masses
of mesenchyme and move into the body space of the developing tadpole. They give
rise to muscles of the mantle.
LITERATURE CITED
CAULLERY, M., 1895. Contribution 1'fitude des Ascidies Composees. Bull, dc la France ct de la
Belglquc, 27: 1-158.
CONKLIN, E. G., 1905. Organization and cell lineage of the Ascidian egg. Jour. Acad. Nat.
Sci. Phild., 13: 1-119.
GRAVE, C., 1920. The origin, function and fate of the test vesicles of Amaroucium constellatum.
Anat. Rcc., 17 : 350.
GRAVE, C., 1921. Amaroucium constellatum — The structure and organization of the tadpole
larva. Jour. Morfh., 36: 71-101.
GRAVE, C., 1935. Metamorphosis of Ascidian larvae. Papers from the Tortugas Laboratory,
29 : 209-292.
GRAVE, C., 1944. The larva of Styela (Cynthia) partita: Structure, activities and duration of
life. Jour. Morph., 75 : 173-190.
HJORT, J., 1896. Germ layer studies based upon the development of Ascidians. Cliristiania.
MAURICE, C. ET M. SCHULGIN, 1884. Embryogenie de 1'Amaroecium proliferum (Ascidie com-
posee). Ann. Sci. Nat., (6) 17: I^t6.
SCOTT, SISTER FLORENCE M., 1945. The developmental history of Amaroecium constellatum. I.
Early embryonic development. Biol. Bull., 88: 126-138.
COMPARATIVE SENSITIVITY OF SPERM AND EGGS TO
ULTRAVIOLET RADIATIONS
ARTHUR C. GIESE *
Marine Biological Laboratory, Woods Hole, Mass, and Hopkins Marine Station,
Pacific Grove, Calif, and Stanford University, California
The sperm of the sea urchin are more sensitive to ultraviolet radiations than
the eggs when the effectiveness of the rays is compared by the retardation of cleav-
age of unexposed eggs fertilized with irradiated sperm on the one hand and of ir-
radiated eggs fertilized with unexposed sperm on the other (Giese, 1939c). It
would be interesting to know whether sperm are generally more susceptible to these
radiations than eggs ; therefore, the experiments were repeated on a number of
marine forms. It is also desirable to find an explanation for this differential sus-
ceptibility in those cases where it occurs. Insight might be gained by determining
action spectra for the sperm and egg, therefore the relative efficiency of action of
different wave-lengths of ultraviolet light in retarding cleavage of irradiated eggs
and of eggs fertilized with irradiated sperm was determined as described below.
MATERIALS AND METHODS
Arbacia punctulata, Nereis limbata, Chaetoptcrus pcrgamcntaccus, and Mactra
sp. were studied at Woods Hole, Mass. Strongylocentrotus franciscaims and S.
purpuratus, collected at Moss Beach, and Urcchis caiipo collected at Bolinas Bay,
California, were used at Stanford University, and Dendrastcr e.vccntricns and Pa-
tcria miniata were studied at the Hopkins Marine Station, each type of egg being
used during the active breeding season.
The methods for studying the eggs were similar to those previously described
(Giese, 1938). Except for the work on the action spectrum, the mercury argon
discharge tube which emits about 85 per cent of its light at A 2537 A was used and
the light intensity was measured with a Hanovia Ultraviolet Meter (No. 949). The
dishes were kept in running sea water to attain a lower temperature than that of the
room. The work on the action spectrum was done with a mercury arc and a natural
quartz monochromator and the intensity of the light was measured with a thermopile
as in previous studies (Giese, 1938). The eggs were kept in dishes in moist cham-
bers and in a constant temperature room at 15° C.
Sperm were used in dilutions of between 1 : 200 and 1 : 1 .000 of the spawn. Such
dilution is necessary because in denser suspensions ultraviolet light is completely
removed by the sperm first reached. Irradiated sperm lose their fertilizing power
rapidly, therefore they must be used soon after exposure (see Hinrichs, 1927, for
studies on inactivation).
1 This work was supported in part by grants from the Rockefeller Foundation. The writer
is indebted to Dr. C. Packard, Director of the Marine Biological Laboratory, and to Dr. L. R.
Blinks, Director of the Hopkins Marine Station, for making available the facilities and for the
many kindnesses extended to the author during the course of this work.
81
82
ARTHUR C. GIESE
EXPERIMENTAL
Comparison of various eggs
A summary of the general results obtained with all the eggs studied will be found
in Table I. Not all the eggs respond to ultraviolet radiations in the same way.
Thus cleavage of eggs of Arbacia and Strongylocentrotus is merely slowed up but
remains normal after small and medium dosages so that comparisons of the effects
of various dosages and wave-lengths is relatively easy. Abnormalities only appear
after larger dosages. In Urechis, Nereis, and some of the other eggs the threshold
for abnormal development is relatively low. While per cent of abnormal develop-
ment could be used for analysis of effects of radiations, it would be much more
difficult.
It is readily apparent that with regard to ultraviolet susceptibility, there are two
types of sperm : in the Echinoderms, especially Arbacia and Strongylocentrotus, the
TABLE I
Comparative action of ultraviolet radiation2 on eggs and sperm of various marine animals
Species
Effects on eggs
Effects on sperm
Strongylocentrotus
purpuratus
Arbacia punctulata
Dendr aster excen-
tricus
Urechis caupo
Chaetopterus perga-
mentaceus
Nereis limbata
Mactra sp.
Delay just noticeable after about
100 ergs/mm.2; will develop even
after 4,000; after 8,000 ergs/mm.2
become quite abnormal.
Noticeable delay after 200 ergs/
mm.2 but even after 2,000 ergs/
mm.2 plutei, normal but smaller
than controls, develop from eggs.
After 4,000-8,000 ergs/mm.2 eggs
are quite abnormal.
Slight delay after 1,600 ergs/mm.2;
strong after 6,400; quite abnormal
after 25,000 ergs/mm.2
Some delay after 200 ergs/mm.2
Marked injury with abnormal
cleavage after 5,000 ergs/mm.2
Slight delay only after about 4,000
ergs/mm.2; after 16,000 ergs/mm.2
still cleave but much delay and
many cytolize.
Even after 4,000 ergs/mm.2 de-
velop with little delay to the
trochophore stage; after 8,000 ergs
show delayed cleavage.
Very slight delay after 500 ergs/
mm.2; striking after 4,000-8,000.
Noticeable delay3 even after 10-
20 ergs/mm.2 Marked retardation
as dosage above this is used.
Noticeable delay even after less
than 50 ergs/mm.2 After 250 ergs
still develop larvae but after 500
quite abnormal. Even after 4,000
ergs/mm.2 sperm activate eggs.
Slight delay after 200 ergs/mm.2;
abnormal after 800 ergs/mm2.
Marked abnormalities after 200
ergs/mm.2
Slight delay after 2,000 ergs/mm.2;
killed after about 8,000-16,000
ergs/mm2.
Slight delay between 4,000-8,000
ergs/mm.2 8,000 kills most sperm.
Delay after 250 ergs/mm.2 and
progressive delay thereafter.
2 The measurements with the Hanovia meter are accurate to about 10 per cent as checked by
thermopile measurements in one instance.
3 Amounting to 15-30 minutes delay at the third cleavage of eggs fertilized with such irradiated
sperm. Marked delay means a delay of an hour or more.
SENSITIVITY OF SPERM AND EGGS
83
sperm are much more sensitive than the eggs ; in the worms such as Urechis,
Nereis, and Chaetopterus the sperm is slightly if at all more sensitive than the egg,
as judged by cleavage delay.
Such a lack of differences in susceptibility of the gametes might be more ap-
parent than real. It is possible that when there is little or no cleavage delay follow-
ing fertilization of an egg with an irradiated sperm, the sperm may be serving only
to activate the egg to haploid parthenogenesis. Eggs of Arbacia and of Chaetop-
terus were, therefore, fertilized with sperm treated either to a small dosage or to a
medium dosage of radiations and at appropriate intervals samples were fixed in
Benin's fluid and stained with iron hematoxylin. Although the preparations were
k
ACTION SPECTRUM FOR RETARDATION OF CLEAVAGE
100
80
\
I6"
40
20
SPERM^
A
X. S
EGGS D
SETTING ACTION AT 2804
N
\ EQUAL TO 100
\
\
\
\
\
\
\
P
EGGS COMPARED TO SPERM
ON AN ABSOLUTE BASIS
2400
2600 280O
WAVELENGTH IN A
3000
320O
FIGURE 1. Action spectra for retardation of cleavage of eggs fertilized with irradiated sperm
at A and for irradiated eggs at C. At B the data for the eggs are compared on a relative basis
setting the value at \ 2,804 A as 100 per cent efficient. See text below.
not entirely satisfactory, evidence for pronuclear fusion was observed in both cases.
No lagging or disintegrating sperm were observed in the cytoplasm of either egg.
Since neither cytological nor physiological evidence suggests parthenogenesis, it
seems likely that for the dosage ranges tested the delayed cleavage follows fusion
of the gametic nuclei. The difference between the two types of sperm must lie in
some other factor. Possible explanations will be considered in the discussion.
The data in Table I show that the threshold for effects on cleavage is quite dif-
ferent for eggs of different species. Thus Strongylocentrotus, Arbacia, Mactra,
and Urechis eggs are retarded after brief exposures to ultraviolet as compared to
84
ARTHUR C. GIESE
Nereis, Chaetopterus, and Dendraster. Whether this is due to mere physical
screening by some inert materials in the egg or to differences in concentration of
some light sensitive materials is not known.
Action spectra for egg and sperm
If irradiation of the nucleus alone causes retardation of division of the cell, the
same action spectrum should be found for egg and sperm ; that is, there should be
no qualitative difference in effectiveness of different wave-lengths even though the
general susceptibility of the sperm is greater. If elements in the cell other than the
ACTION SPECTRA FOR SPERM AND EGG AND PROTEIN ABSORPTION
100 i-
i
I
I
i
\ ABSORPTION BY
SERUM ALBUMIN
' SPERM
ACTION
SPECTRUM
\
ABSORPTION
BY NUCLEIC ACID
EGG ACT/ON
SPECTRUM
20 -
£300
2500
270O
2900
3100
WAVELENGTH IN ANGSTROM UNITS
FIGURE 2. Comparison of the action spectra of Figure 1 with absorption spectra of nucleic
acid and serum albumin. Data for nuclei acid from Caspersson (1938), for proteins from
Smith (1929). Note that the action spectrum for the egg is quite different from the absorption
spectrum for albumin at both ends.
nucleus are involved the egg may show an action spectrum different from that of
the sperm.
The methods employed for the studies at different wave-lengths are similar to
those already described elsewhere (Giese, 1938, 1939c), therefore, only the briefest
mention need be made of them. The irradiated eggs are fertilized with normal
sperm. The rate of division is then determined by observing for percentage of
cleavage every 15 minutes. The times at which the eggs reach the 2, 4, 8, 16, and
32 cell stages are recorded and the number of cleavages is plotted against the time
after fertilization and compared with the control. The increase in time required to
reach the third cleavage is taken as a measure of the retarding action of the radi-
SENSITIVITY OF SPERM AND EGGS 85
ations. The retardation is then plotted against dosage. From such curves for each
of the wave-lengths the dosage required to bring about a given retardation can be
determined. For Figures 1 and 2 the reciprocals of the relative amounts of energy
at different wave-lengths required to produce a retardation of division by 1.5 hours
were determined. In Figure 1 at A and C the sperm and egg are compared on
this basis and a great difference in susceptibility between the gametes is evident.
In B the data for the eggs are compared amongst themselves on a relative basis
setting the action at A 2,804 A as 100 per cent efficient.
The shape of the curves indicates that different materials are being affected in
the two cases, since the action spectrum is considered to be a measure of the ab-
sorption by the active constituent. To see if the absorbing materials can be identi-
fied the absorption spectra for serum albumin and nucleic acid are given in Figure
2. It is apparent that the action spectrum for sperm matches the absorption spec-
trum for nucleic acid better than the absorption spectrum for albumin ; the reverse
is true for the action spectrum of the egg. Since the simple proteins and nucleo-
proteins are the major structural constituents of the cell and none of the other or-
ganic or inorganic constituents have very specific absorption, the resemblances while
imperfect are indicative of absorption by these two classes of compounds in the
action of ultraviolet radiations on the gametes.
DISCUSSION
The occurrence of a differential susceptibility of gametes with the sperm more
sensitive to ultraviolet light than the egg as first found in the sea urchin, Strongylo-
centrotus pwpuratus, was verified on Arbacia and Dendraster and in preliminary
trials on Pateria and 6\ franciscanus but not on Urechis, Chaetopterus, and Nereis.
In the latter forms the sperm appears to be only slightly more sensitive than the egg
(Table I). The former group of species belongs embryologically to the radially-
cleaving, indeterminate egg type, the latter group to the spiral determinate type.
In addition, the radial eggs used here are mature or nearly so at the time of shedding
whereas the spiral eggs are generally immature. An illustration of the difference
in response to ultraviolet light, depending on this difference in organization is seen
in the local "burns" occurring in the spiral eggs. Thus a Nereis egg given a uni-
lateral dosage of between 8,000-16,000 ergs/mm.2 may develop apparently normally
except on the burned surface which appears blistered. A Strongylocentrotus egg
on the other hand unless given a large dosage of light will show general effects dis-
tributed throughout the retarded egg. However, it is not possible to say which
features of the organization account for the difference in sensitivity of the eggs and
sperm of the two groups.
One might envisage that in eggs the retarding effects of radiation on cleavage are
due to the inactivation, by substances formed during irradiation, of some catalyst
which is necessary for the reactions involved in cleavage. In one group of eggs
perhaps the catalyst is present in excess of that necessary for a characteristic rate
of cleavage, the rate being controlled by some other limiting factor, in the other it is
present in just adequate concentration and itself constitutes the limiting factor.
Even a considerable dosage of radiations will not reduce the concentration of cata-
lyst below the critical level in the first case but will readily do so in the second. In
the first case no cleavage delay would be expected until very large dosages of radi-
86 ARTHUR C. GIESE
ations had been administered, in the second the cleavage should be affected after
very small dosages. One would have to assume that irradiated sperm on penetrat-
ing unirradiated eggs introduce similar cleavage-inhibiting substances acting on the
catalyst as those formed in the irradiated egg. In this case also the effect on cleav-
age should depend upon the amount of catalyst present in the egg — if in excess, the
cleavage should not be easily inhibited, if limiting, the reverse should be true. \Ye
should expect both sperm and egg to be relatively insensitive to the radiations in
the former and this is found in most spirally cleaving eggs.
Against the above postulation is the fact that the action spectrum for sperm re-
sembles nucleoprotein absorption while for the egg it resembles simple protein ab-
sorption indicating two different ultraviolet absorbing materials in the gametes by
which the cleavage-retarding effect is produced. It is possible that absorption by
both of these types of proteins leads to the formation of toxic photoproducts which
inhibit the same catalyst. It is also possible that the toxic substance is much more
rapidly formed by the nucleoproteins, but the necessary assumptions strain the
imagination.
It should be pointed out that the retardation of the early cleavage- is only the
initial effect of the radiation. If the delayed effect could be studied we might find
that the recovery from injury to the egg would resemble absorption by nucleo-
protein, indicating a more lasting injury to the nucleus than to the cytoplasm, as is
the case for division of Paramecium (Giese, 1945a). Because the number of cells
cannot be satisfactorily determined in the later cleavages such experiments were not
attempted with eggs.
The action spectrum obtained for the egg is similar to that observed for "cyto-
plasmic" effects such as increased time of ciliary reversal, retardation of excystment,
immobilization of cilia, and prevention of hatching of eggs. The action spectrum
for the sperm resembles that for "nuclear" effects such as recovery of paramecia
from sublethal effects, bactericidal and fungicidal effects and the production of muta-
tions (see Giese, 1945b, for references). It is interesting to note the difference be-
tween the action spectrum for retardation of cleavage of the egg and for activation
studied by Hollaender (1938). In the latter case no action was found until about
A 2,650 A and the effectiveness of the light increased as the wave-length decreased.
The mechanism of action of the ligbi must be different in these two instances. The
action spectrum data thus lay the foundation for further analysis of the effect of
these radiations upon gametes.
SUMMARY
1. The action spectrum for the retardation of division of eggs fertilized with ir-
radiated sperm resembles the absorption of ultraviolet light by nucleoproteins.
2. The action spectrum for retardation of division of irradiated eggs of the sea
urchin resembles absorption by simple proteins like albumin except that at the short
wave-length end there is no increase in action at A 2,483 A where absorption shows
a definite upswing.
3. The absolute amount of energy required to affect division to the same extent
by affecting the sperm is very much less than that required to affect eggs.
4. Other Echinoderms tested show a similar difference in susceptibility of eggs
and sperm : 6\ jranciscanus, Arbacia punctnlata, Dendraster ex centric us, and Pa-
ter la miniata
SENSITIVITY OF SPERM AND EGGS 87
5. Animals other than Echinoderms tested did not show as striking a difference
between susceptibility of eggs and sperm : Urechis caupo, Mactra sp., Chactoptcrus
pcrgamentaceus, and Nereis limb at a.
6. In the eggs listed in paragraph 5, determinations are made more difficult by
the tendency for the eggs to show irregular cleavage rather than retarded cleavage
as the dosage increases. Such irregular cleavage occurs in Echinoderm eggs as well
but the threshold is higher.
7. If both eggs and sperm of the sea urchin are irradiated the effect on the rate
of division is less than the sum of the effects which would be expected on each of the
gametes alone. However, the percentage of abnormal cleavage greatly increases.
LITERATURE CITED
CASPKRSSON, T., 1936. Uher den chemischen Aufbau des Strukturen des Zellkernes. Skandinav.
arch. f. physiol. Suppl, 8 to Vol. 73, 1-151.
GIESE, A. C., 1938. The effects of ultraviolet radiations of various wavelengths upon cleavage
of sea urchin eggs. Biol. Bull., 65 : 238-247.
GIESE, A. C., 1939a. Retardation of early cleavage of Urechis by ultraviolet light. PhvsioJ.
' Zool., 12 : 319-327.
GIESE, A. C., 1939b. Ultraviolet light and cell division. Effects of x 2654 and 2804A upon
Paramecium caudatum. /. Cell. Conip. Physiol., 13: 139-150.
GIESE, A. C., 1939c. Ultraviolet radiation and cell division. Nuclear sensitivity : effect of ir-
radiation of sea urchin sperm. /. Cell. Conip. Physiol., 14: 371-382.
GIESE, A. C., 1945a. The ultraviolet action spectrum for retardation of division of Paramecium.
/. Cell. Comp. Physiol., 26 : 47-55.
GIESE, A. C., 1945b. Ultraviolet radiations and life. Physiol. Zool., 18 : 223-250.
HINKICHS, M. A., 1927. Ultraviolet radiation and the fertilizing power of Arbacia sperm.
Biol. Bull., 53: 416-437.
HOI.LAENDER, A., 1938. Monochromatic ultraviolet radiation as an activating agent for the eggs
of Arbacia punctulata. Biol. Bull., 75 : 248-257.
SMITH, F. C., 1929. The ultraviolet absorption spectra of certain aromatic ainino acids and of
proteins. Proc. Roy. Soc. London B, 104: 198-205.
OBSERVATIONS ON THE FUNCTIONING OF THE ALIMENTARY
SYSTEM OF THE SNAIL LYMNAEA STAGNALIS
APPRESSA SAY
MELBOURNE ROMAINE CARRIKER
Zoological Laboratory of the University of Wisconsin, Madison
INTRODUCTION
Although records exist of functional studies on the alimentary system of Basom-
matophora as far back as the early eighteen hundreds, the detailed story of the
course and ultimate fate of food in the alimentary tract and the simultaneous move-
ments of the tract is thinly scattered and far from complete. In the more recent
emphasis placed on some gastropods because of their importance as vectors of para-
sites of man, domestic animals, wild game, and fish, it is vitally important that the
normal physiology of the system most frequented by these parasites be better known.
It is the purpose of this paper to integrate the previous work on the physiology
of the alimentary system of Lymnaea st agnails and allied forms (suborder Basom-
matophora, order Pulmonata) with original research on the same system in L. s.
apprcssa Say. The basic morphological (Carriker, 1945) and histological (Car-
riker and Bilstad, 1946) studies on this system in L. s. apprcssa have been com-
pleted and are in press. All terms used in this research have been described in these
two papers.
L. s. apprcssa has been selected for this research because it is a representative
vector and because of its excellent response to laboratory culture, its relatively
large size (maximum shell length, 62.5 mm.) as compared with other fresh water
pulmonates, its short life cycle, and its relatively thin semitransparent shell and semi-
transparent tissues. Snails used in the research were cultured entirely in the labo-
ratory. They were grown through many generations in large battery jars and fed
on lettuce and cooked "cream of wheat" cereal. The water in the jars was aerated
by means of a small Marco air pump (Noland and Carriker, 1946). The original
snails were collected in Fox Lake, Wisconsin, in 1939. Parasite-free cultures
(especially of trematodes) from the original snails were obtained by the isolation
of the egg mass soon after oviposition in separate aquaria. Each new culture was
started in this way rendering transmission of infection very improbable. Detailed
examination of succeeding generations has not disclosed parasites.
This work was carried out at the University of Wisconsin (1939-1943) under
the stimulating guidance of Prof. L. E. Noland, whose advice, encouragement, and
friendly cooperation were much appreciated.
HISTORICAL REVIEW
Scanty observations on the function of the anterior part of the alimentary tract
of Lymnaea were given by Semper (1857), Geddes (1879) and Moquin-Tandon
(1885) ; more detailed information was given by Amaudrut (1898), Pieron (1908)
88
ALIMENTARY SYSTEM OF LYMNAEA 89
and by Baecker (1932). The stomach region was investigated by Gartenauer
(1875), Moquin-Tandon (1885), Colton (1908) and Heidermanns (1924). These
experimental contributions of Colton and of Heidermanns, particularly of the
latter, are noteworthy. The liver has been the object of most of the physiologi-
cal work although the research has usually been incidental to that on the stylom-
matophoran Helix: Barfurth (1880, 1881, 1883a and b), Frenzel (1886), Cuenot
(1892), Enriques (1901, 1902), Faust (1920), Peczenik (1925) and Krijgsman
(1928). Only the investigation of Peczenik is exclusively on L. stagnalis.
EXPERIMENTAL METHODS AND RESULTS
Lymnaea physiological salt solution
The study of the living system has required the development of a physiological
salt solution which will approximate the ionic and osmotic balances of the blood of
Lymnaea more closely than do such commonly used solutions as Ringer's. On the
basis of incomplete data given by Duval (1928) on Lymnaea and by Bernard and
Bonnet (1930) on Helix on the molecular concentration of blood, the following
solution was developed for L. s. appressa:
NaCl 2.0 gms. per liter
NaHCO., 2.0 " " "
KH2PO4 0.1 " " "
MgCl2 0.3 " " "
CaCl2 0.3 " " "
This solution consists of 0.47 per cent salts and gives a pH of approximately 7.8.
After about a week considerable precipitation of CaCO., occurs, although this
seems to have no noticeable effect on the isolated organs. The vas deferens was
used in testing the solution and was found superior to the heart for this purpose.
The vas deferens, terminal preputium and prostate gland were removed under the
physiological salt solution from the cephalic hemocoel without bruising. This por-
tion of the reproductive tract is in part a strong muscular tube which is easily ex-
cised and maintains a continuous squirming motion as long as the tissues are alive.
It continued squirming for about 66 hours in the solution described above. A
Ringer's solution of 0.7 per cent salts keeps it moving for about 12 hours, although
at a much reduced rate.
Hydrogen ion concentration
The first work on the estimation of the pH of the alimentary tract of a fresh
water snail seems to be that done by A. H. Rosenbloom on L. s. appressa in his
bachelor's thesis in 1942 (unpublished) in this laboratory. He has kindly con-
sented to the incorporation of his results in this paper. His method was essentially
the colorimetric one employed by Yonge (1925) : fluids from the various lumina of
the alimentary tract of the snails under variable feeding conditions were pressed
out onto paraffined plates and thoroughly mixed with indicators (brom-thymol
blue, neutral red, and methyl red). The colors were compared with those of indi-
cators freshly prepared in buffered solutions checked on a Coleman pH electrome-
ter. The results are given in Table I :
90
MELBOURNE ROMAINE CARRIKER
TABLE I
pH of the contents of various lumina of the alimentary tract of L. s. appressa
Organ
Average pH
Maximum and
minimum pH
Number of tests
Number of snails
Buccal cavitv
Same as water in
external medium.
Proesophagus
6.9
7.2-6.3
10
10
Postesophagus
7.2
7.6-6.6
10
10
Gizzard and crop .
6.4
7.2-6.3
12
12
Pylorus ....
6.6
7.0-5.8
10
10
Intestine
7.1
7.8-6.2
35
15
Enzymes
Preliminary tests were made for non-purified cathepsin, pepsin, trypsin, and
amylase. The tests for the proteinases were made according to the methods of
Anson (1938), Bradley (1938) and Folin and Ciocalteu (1927) ; those on amylase,
by the iodine test of Hawk and Bergeim (1937). Semi-micro technics were ap-
plied to large numbers of the excised organs.
Maximum catheptic activity (at pH 3) over a ten-day period was found in the
liver. That occurring in the buccal mass and gizzard and other portions of the
alimentary system was not significant as compared to that in the liver. In an effort
to determine to what extent cathepsin might be secreted from the liver, gut fluid
from which the amebocytes had been centrifuged was tested. Under the conditions
of the experiment, at least, no cathepsin was found in the gut juice. In some tests
tryptic activity was found in the salivary glands. A very active amylase, optimum
pH 7, was present in the salivary glands and in the liver.
The only investigation of the hydrolytic enzymes of the alimentary system of the
Basommatophora reported in the literature is that by Heidermanns (1924). He
described a positive test for cellulase present in the digestive juice of the stomach
organs (crop, gizzard, and pylorus) of L. stagnalis.
Ciliatlon
Ciliary currents were studied by the injection of fine carmine suspensions in
Lymnaea physiological salt solution through various portions of the exposed tract,
by application of carmine particles to the epithelium of the opened tract and by
placing small bits of gut wall in a carmine suspension on an uncovered microscopic
slide under high magnification. In some dissections the undisturbed food particles
were seen passing through various portions of the excised gut on the natural ciliary
currents.
No work has been performed previously on the ciliation of the alimentary system
of the Basommatophora. Merton (1923) in his research on the external ciliation
of pulmonates included a brief study of the ciliation of the hepatic ducts of Helix.
The entire alimentary system of L. s. apprcssa, with the exception of the gizzard
and portions of the buccal cavity, is ciliated (see later in this paper), Figures 3, 9,
and 11.
ALIMENTARY SYSTEM OF LYMNAEA 91
Muscular activity
The activity of the alimentary system was observed under binoculars through the
transparent walls of normal living young snails and in adult unanesthetized snails
opened under Lymnaea physiological salt solution. The independent activity of
the radula over the odontophore was clearly observed and conclusively verified by
watching snails under the binocular under the following conditions : snails deprived
of food for a day were placed in a finger bowl of well aerated water to which had
been added strips of lettuce (1-2 mm. wide). A Petri dish was floated over the
lettuce and the water. As the snails crawled upside down under the glass, feeding
on the lettuce, the action of the radula and mouth parts was clearly visible under a
strong beam of light.
Sand in the gizzard
In order to check the experiments of Heidermanns (1924) and to add additional
information on the role of sand in the comminution of food by the gizzard of L. s.
apprcssa, the following experiments were devised.
Sixteen adult snails were placed in each of four aerated aquaria containing a
one-half inch mesh wire platform over the bottom. By means of this contrivance
the feces were removed from the vicinity of the snails soon after defecation. To
three of the aquaria the following foods were added respectively : ( 1 ) cooked "cream
of wheat," (2) filter paper, and (3) lettuce. (4) No food was added to the fourth
tank. (5) A fifth tank was assembled as a control without the wire platform and
with lettuce and sand. One snail from each aquarium was killed daily and opened
immediately. After ten days the following was disclosed : eight of the forty-three
experimental pulmonates contained no sand in the tract, thereby showing that it is
possible to rid completely the tracts of a few of the snails of sand ; however, there
was extensive variation in the ability of the different snails to retain sand. As the
quantity of sand in the gut decreased, the snails consumed less food, until in the
absence of sand in the tract, no food was ingested and the guts became void of food
material and feces. The different diets indicated no significant difference in their
respective values as sand eliminators. Sand was found most abundantly in the giz-
zard lumen, then in decreasing amounts in the crop and retrocurrent passage of the
pylorus '(anatomical terminology has been described elsewhere, Carriker, 1945).
After the quantity of sand in the lumen of the gizzard reached a certain low level,
it was retained with surprising tenacity for many days. The material in the fecal
pellets of the control snails, particularly of the gizzard residues, was markedly
brown and more thoroughly triturated than those of snails with sand-free diets.
In a second set of experiments snails approximately 10 mm. in length were
placed in a one-quarter inch mesh wire basket suspended in a large laboratory snail
stock tank. The feces, propelled by the sluggish circulation of the water in the
tank, passed out of the basket. All lettuce placed in the basket was carefully washed
to remove sand. The experiment was continued for several months. In spite of
precautions, small quantities of fine sand were always present in the tracts of some
of the animals ; however, this did not seem to be enough for proper trituration as
many of the snails died abnormally at an early age and none reached the normal
adult size of the control snails in the tank outside the experimental basket. There
MELBOURNE ROMAINE CARRIKER
is unquestionably a vital need for the presence of at least a limited quantity of sand
in the gizzard of these snails for sufficient breaking down of the food.
These results are in agreement with the findings of Heidermanns (1924) and of
Colton (1908). Heidermanns accidently discovered that the only way to entirely
remove the sand from a live snail was to cause it to hibernate, in which state it
emitted the total contents of the tract. Colton noted that in the presence of sand
the plant food was cut to pieces by L. colnniclla, but that in the absence of sand it
went unmolested.
Digestive cell ingestiou
By the use of a method patterned after that of Peczenik (1925) the ingestion of
participate food by the digestive cells was investigated. White of egg was strained
through cheese cloth. Carbon (lamp black) was ground into the egg albumen and
the mixture was thoroughly beaten. This was steamed to a stiff mass and fed to
snails starved for a few days. After feeding commenced, the snails were opened
every other day. Indigestible residues within vacuoles in the digestive cells as well
as similar residues in the fecal pellets showed the presence of very minute particles
of carbon, particles not present in the control snails. The indigestible residues in
the digestive cells appeared very similar to the albumen passing down the intestine
in the gizzard residues.
Fecal rhythms
Some information was gathered on the rhythms of the liver and of the gizzard
by a study of the rate and extent of passage of the various fecal strings. The fecal
pellets of a 40 mm. snail were observed daily for twenty-four days. The animal
was isolated in a two-liter glass jar over the bottom of which was placed a paraf-
fined one-half inch mesh galvanized metal screen, so that all fecal pellets fell to the
bottom of the jar and could not be reconsumed. The mollusc was fed lettuce on
which was sufficient sand for the needs of the stomach region. Three egg masses
were oviposited by the snail, and it added 2 mm. of shell during the twenty-four
day period. Upon dissection at the end of the experiment the animal appeared nor-
mal in all respects. For the first ten days the pellets were collected and examined
microscopically every few hours during the day ; during the latter part of the ex-
periment they were collected every twelve hours. Numerous examinations were
made of fecal pellets from the stock snail tanks to corroborate the findings on the
experimental snail.
PHYSIOLOGY OF THE ALIMENTARY TRACT
Bitccal mass and esophagus
L. s. appressa is primarily an herbivore. In the laboratory it may complete its
life cycle on lettuce alone and in its natural state feeds on the aquatic vegetation of
its surroundings. Specialization of the alimentary system (Carriker, 1945) has
been in keeping with a plant diet. However, animal food is also consumed as has
been observed by Walter ( 1906) and by seven other authors cited by him. Repeat-
edly in this laboratory L. s. appressa has been observed to eat the bodies out of the
ALIMENTARY SYSTEM OF LYMNAEA 93
shells of dead snails in the aquaria. Biochemical tests disclose the presence of some
tryptic activity in the secretion of the salivary glands.
Pieron (1908) has found in L. auricularia and L. stagnalis that there is a total
absence of food discrimination in the buccal mass and that their feeding is a reflex
which keeps the radula working most of the time. The only portion of the body
showing any discrimination is the anterior surface of the foot which contains faintly
sensitive chemoreceptors. In aquaria in this laboratory L. s. appressa rasps much
of the time, whether on lettuce or over the newly cleaned glass surface of its tank.
However it does also pass through regular "resting" periods in which no rasping
occurs. In the rasping stroke the radula passes first to one side and then to the
other describing a broad feeding track.
Feeding can be followed clearly in normal immature "albino" L. s. appressa (a
strain with very little dark pigment) feeding on a "cream of wheat" food mixture
blackened with lamp black. This can lie seen to pass as far as the stomach region.
On the protractor stroke the radula cups to an elongated spoon-shaped trowel about
one-half the width of the upper mandible, and working against this, cuts out long
narrow bits of food. Each denticle is sharp so the concerted action of the numerous
denticles on the radula, sliding independently over the odontophore, provides an
effective cutting-rasping apparatus. The food bits are pushed back through the
dorsal food channel to the rear of the buccal cavity which dilates to receive them.
The tip of the radula closely appresses to the dorsal wall of the buccal cavity in its
rearward passage, as attested by the jagged pattern of the dorsal chitinous surface.
The buccal aperture constricts strongly and rapidly after the receding radula. Some
bits of food are dropped and remain in the dorsal food channel for the next rear-
ward swing of the food-laden radula. Several food bits clump in the rear of the
buccal cavity prior to being forced down the esophagus. The radula functions
principally in cutting pieces of food of suitably small dimensions for convenient
transport through the anterior portion of the alimentary tract ; it does not triturate
the food to any considerable degree.
Only the posterior third of the buccal cavity is ciliated. These cilia and those
in the densely ciliated esophagus beat strongly posteriorly, bearing food bits from
the rear of the buccal cavity to the crop.
In connection with the functioning of the buccal mass, refer to a previous paper
(Carriker, 1945) for the names, origin, and insertion and relations of the muscles
and parts of the mass. The muscular activity of the buccal mass is divisible into
four major synchronous movements: (1) opening and closing of the oral aperture
and consequent spreading and approximation of the mandibles and lips, as well as
dilation and contraction of the circular muscles about the anterior portion of the
buccal cavity, (2) backward-forward and simultaneous elevator-depressor move-
ments of the odontophore, with some slight turning of the odontophore on its longi-
tudinal axis and some movement to the right and to the left, (3) movement of the
radula and radular sac over the cartilage, and (4) backward -forward and simul-
taneous elevator-depressor movements of the entire buccal mass. Consequently
there exist in the buccal mass three intrinsic focal points about which the ma-
jority of the muscles radiate: (1) the oral aperture, (2) the odontophoral cartilage,
and (3) the radula and the radular sac.
The activity of the odontophore with respect to the remainder of the buccal mass
may be arbitrarily divided into four phases, and described as follows: (1) the quies-
94 MELBOURNE ROMAINE CARRIKER
cent stage in which the odontophore lies at rest in the rear of the huccal cavity with
its longitudinal axis in a dorsoventral position. (2) The protracting stroke in
which the proximal end of the odontophore swings in an arc of about 130° from its
basal position to a point where it lies above the plane of the distal end, which then
is in a position to pass partly out of the buccal cavity, bringing the radula against
the substratum. At the beginning of this stroke the odontophore assumes a hori-
zontal position as a result of the lowering of the distal end by contraction of the
dorsal odontophoral flexor muscle, and a simultaneous raising of the proximal end
by strong contraction of the posterior jugalis muscle. The oral aperture and the
anterior portions of the buccal mass dilate to permit partial protrusion of the odon-
tophore through the mouth ; the labial retractors, suboral dilators and dorsomandibu-
lar dilators spread the mouth. The extrinsic postventral levators and posterior
jugalis further raise the rear of the buccal mass so that the distal tip of the odon-
tophore is directed towards the oral aperture, to which it seems to be guided prin-
cipally by the action of the dorsal odontophoral flexor muscles. The inframedian
radular tensors draw the radula over the distal end of the cartilage to the point
where most of the radula outside the radular sac lies on the under side of the hori-
zontally inclined cartilage, and the collostylar hood lies just behind the distal crest
of the cartilage. The combined action of the radular sac and cartilage tensors holds
the radula tautly drawn over the cartilage in readiness for the rasping stroke. Con-
traction of the intracartilage tensors adds considerably to the rigidity of the cushion
under the radula. As Woodward (1895) points out for Natalina caffra, the fibers
of the cartilage act in much the same way as the intrinsic muscles of the human
tongue and in contraction cause an elongation and consequent slight protrusion of
the radula. The pressure of the blood in the odontophoral sinus probably provides
further turgidity. Contraction of the extrinsic as well as of the intrinsic protractor
muscles brings the odontophore to the substratum. (3) In the rasping stroke the
distal tip of the odontophore is drawn over the substrate in a licking motion. The
radula, independent of the principal motion of the cartilage under it, is itself simul-
taneously slid quickly backward most of its length over the cartilage by the action
of the heavy supralateral and supramedian radular tensor muscles. The odonto-
phore is aided by contraction of the extrinsic preventral levator muscles which pull
the anteroventral floor of the buccal cavity forward and upward. As the mouth
opens during the previous stroke, the cutting distal margin of the dorsal mandible
is turned partly forward by contraction of the dorsomandibular dilators and
possibly the posterior jugals. Thus as the radula rasps forward it makes
connection with and scrapes past the inner side of the dorsal mandible, much
as two jaws would come together, so that the snail when feeding on thin por-
tions of lettuce actually "bites" off pieces with each rasping stroke. It is only when
feeding on thicker foods that true "rasping" comes into play. The dorsal mandible
is governed by the dorsomandibular approximator muscle. The lateral mandibles
afford mechanical protection to the sides of the mouth, and close in medially after
the radula and under and behind the dorsal mandible. (4) The retractor stroke
returns the odontophore to the resting condition, and completes the cycle, by action
of the extrinsic retractor muscles and the supralateral and supramedian radular ten-
sors and relaxation of the protractors. The oral aperture is closed after the reced-
ing odontophore by action of the labial sphincter and the mandibular approximator
muscles ; the buccal cavity, by a contraction of the buccal sphincter and related mus-
ALIMENTARY SYSTEM OF LYMNAEA 95
cles of the walls. In assuming the resting- position, the raclular sac is depressed
behind the cartilage and the radula rests principally behind the vertically arranged
cartilage so that the ventral tip of the sac projects slightly below the level of the
buccal mass. As observed by Amaudrut (1898) for Lyiniiaca, the ventral wall of
the buccal cavity between the esophageal ledge and the collostylar hood is also de-
pressed, forming a slight dilation in front of the esophageal opening. As both the
oral aperture and the proesophagus are closed during the retractive stroke of the
radula, it is likely that this dilation is instrumental in creating a slight vacuum in
front of the esophageal opening which aids in disengaging food particles from the
radula. The dilation is caused principally by depression of the radular sac and
possibly by contraction of the superior suspensor muscle of the radular sac and the
hood tensor muscles.
The proesophagus is limited in its muscular activity to slight peristaltic waves
proceeding towards the postesophagus ; while the latter undergoes pronounced peri-
staltic activity in either a forward or a backward direction, dilating broadly and
contracting its entire length. In dilation it may become so large as to fill much of
the cephalic hemocoel of the expanded mollusc. In expansion it is filled with a
reddish fluid from the stomach region and food particles.
In the buccal cavity the food receives generous quantities of fluid from the buccal
gland cells, a fluid which is probably mostly mucoid in nature, judging from the
positive mucicarmine stain and from negative tests for amylase and trypsin. This
does not however preclude the possibility of the presence of other enzymes which
were not tested for. As food passes under the openings of the salivary ducts it
receives mucus, amylase, trypsin, and possibly other enzymes from the salivary
glands.
The proesophagus adds more secretion from buccal glands and mucous cells.
The postesophagus functions as a temporary reservoir for the retention of food
when the crop is full. Being capable of considerable distension, it may retain
larger quantities of food than the crop. Digestion begins in the postesophagus
because of enzymatic secretions received from the salivary glands.
Stomach region
Comminution of food particles is completed in the crop, gizzard, and anterior
portions of the retrocurrent passage of the pylorus. These three organs act as a
unit comparable to a grist-mill. The kneading motion of the anterior and posterior
gizzard constrictor muscles and the gizzard lobes over the sand in the lumen pro-
vides the grinding action. Food bits forced between the sand are soon crushed to
minute particles upon which the digestive enzymes may act more efficiently. Two
synchronized movements are present in the gizzard. In the first the anterior and
posterior gizzard constrictor muscles alternate smoothly in mild contraction, thus
mixing and forcing the contents of the gizzard slowly back and forth ; in the second,
not as frequent as the first, the bulk of the gizzard compressor muscles contract
suddenly and strongly, bringing pressure to bear on the contents of the gizzard.
The presence of gritty material in the gizzard of the Lymnaeidae has been noted by
many: Cuvier (1817), Wetherby (1879), Whitfield (1882), Moquin-Tandon
(1885), Colton (1908), F. C. Baker (1900, 1911), and Heidermanns (1924).
In the crop, all ciliary currents lead to the anterior margin of the right gizzard
96 MELBOURNE ROMAINE CARRIKER
pad, those on the left side beating ventrad and over to the right (Fig. 3). Thus
fine food material accumulates on the right side of the crop at the anterior edge of
the right gizzard lobe. The crop receives food from the postesophagus and forces
it into the gizzard lumen. When ample sand is accessible to the animal, the crop
and anterior portions of the retrocurrent pyloric passage are both filled with it.
The walls of these organs act as mechanical obstructions to the open ends of the
gizzard lumen and concentrate the pressure of the gizzard musculature upon the
contents. They also cooperate in the muscular activity of the gizzard in a unified
kneading and a slow rotation of the gritty contents. The retrocurrent passage re-
turns to the crop those particles which have been dislodged from the gizzard con-
tents by muscular movements of the stomach region. In this fashion the contents
of the gizzard undergo thorough comminution and partial digestion before the resi-
dues are shunted down the procurrent passage to the prointestine.
The epithelium of the stomach region bears a complicated system of ciliary cur-
rents (Figs. 1, 2, 3, 9). Cilia in the procurrent passage direct fine particles from
the right ventral side of the gizzard cavity to the prointestine. Those in the retro-
current passage are directed anteriad towards the left side of the gizzard cavity.
The dorsal passage bears what in fixed sections appears to be nothing more than a
brush border. Even in carmine suspensions under high magnification no distinct
current could be detected in it. The cilia on the ventral fold are divided into two
distinct functional areas : those on the right half of the fold beat obliquely posteriad
and laterally in the direction of the currents in the procurrent passage ; those on the
left half, obliquely anterolaterad in the direction of the gizzard and the currents in
the retrocurrent passage. The currents on the minor fold whip obliquely antero-
laterad; those on the medial half of the major fold pass obliquely anterolaterad;
while those on the lateral half of the major fold and those on the medial half of the
fold adjacent the hepatic vestibule reach posterolaterad. The ciliary currents in the
retrocurrent passage are noticeably faster than those in the procurrent passage.
Currents on the atrial corrugations run into the incurrent tubule of the cecum.
Thus the pylorus in cross section (Fig. 2) is composed of three channels, each with
distinct ciliary currents and of three folds which almost meet centrally and whose
EXPLANATION OF PLATE I
(All figures concern L. s. appressa)
FIGURE 1. Stereogram of the pylorus, hepatic vestibule, atrium, cecum, anterior portion of
prointestine, and liver lobes. The vascularization is stressed. (Small arrows indicate the flow
of blood in the arteries ; large arrows, the direction of movement of the contents of this part of
the tract.) X 6.
FIGURE 2. Stereogram of cross-section of the pylorus, taken midway between the gizzard
and the hepatic vestibule. The stippled surfaces are heavily ciliated. (The small arrows in-
dicate the direction of the ciliary beat; the large arrows, the direction of passage of material in
the pylorus. The arrows with broken stems designate the direction of ciliary beat on surfaces
behind the folds.) X 25.
ABBREVIATIONS
AT, atrial artery; CC, cecal artery; d.p.p., dorsal pyloric passage; GD, dorsogastric artery;
HN, minor hepatic artery; HP, prohepatic artery; IP, prointestinal artery; »;./>./., major pyloric
fold; n.p.j., minor pyloric fold; pc.p., procurrent pyloric passage; PM, major pyloric artery;
PN, minor pyloric artery; PP, propyloric artery; PV , ventropyloric artery; re. p., retrocurrent
pyloric passage ; v.p.j., ventropyloric fold ; VT, vestibular vascular arborescence.
ALIMENTARY SYSTEM OF LYMNAEA
97
PLATE I
PI/
or /ode of //t/er
typhlosote
-pm/f)fest/ne
ftepot/c vestibule
Aepafic ducts
/ode
c/orsa/ passage
minor-
major fo/d
uentra/ fo/d
ct/iatecf str/a
ci/ia
98 MELBOURNE ROMAINE CARRIKER
ciliary currents pass out of the dorsal into both the procurrent and the retrocurrent
passages. The major fold in addition bears a thin longitudinal strip of long cilia
at its boundary with the dorsal passage. The major and minor folds in the living
animal nearly always touch along their crests, so that the fluid contents of the dorsal
passage may pass into the two ventral passages but coarse material from the ventral
passage may not pass into the dorsal passage. The juxtaposition of the two folds
is continued under the hepatic vestibule, where the folds provide a ventral floor to
this chamber. At this point the cilia on the folds direct a powerful current out and
away from the vestibule, again preventing the entrance of coarse material into the
hepatic ducts and liver.
As discovered for Helix by Merton (1923), the corrugations of the larger proxi-
mal portions of the hepatic ducts of L. s. apprcssa bear two ciliary countercurrents
(Fig. 11) : the cilia on the crests of the corrugations are long and beat into the
liver, those in the grooves are shorter and pass particles in the direction of the he-
patic vestibule and into the incurrent tubule of the cecum. The particles in the
grooves are quickly entrapped in mucus secreted there and formed into delicate
strings. The currents directed into the liver could be traced with certainty only in
the large hepatic ducts, although cilia were observed as far as the peripheral folli-
cles in isolated bits of living liver tissue. Yonge (1936) states that in Mollusca
where food passes into the liver and waste material out, the ducts are ciliated in
such a way that an inward passage exists on one side and an outward one, on the
other. Such counter currents could not be determined in L. s. appressa.
In the cecum the cilia on the cecal folds beat off the folds into the tubules (Fig.
9) ; those in the incurrent tubule pass carmine particles directly to the distal end
and around this into the excurrent tubule. Here the cilia beat circumferentially,
rotating the contents of the tubule along the longitudinal axis. In the continuation
of the excurrent tubule across the pyloric wall the ciliary stream is directed towards
the prointestine.
The crop, pylorus, liver, and hepatic ducts are as active as the postesophagus.
Besides the usual peristaltic movements, they undergo a series of violent alternating
pulsations, here designated pulsatory movements, in which the crop, pylorus, hepatic
ducts, and liver pulsate successively, forcing the fluid contents slowly back and
forth in swirling currents. In the pylorus the pulsations commence at a point be-
tween the typhlosole and the atrium and pass towards the gizzard. They are of
two types : ( 1 ) very strong pulsations in which the entire structure contracts and
(2) minor pulsations running over restricted portions of the pylorus. In the liver
the pulsations pass as far as the terminal follicles. This marked movement is most
vividly observed in bits of living liver tissue under high magnification. Individual
cells are seen to move against each other by contraction of the thin muscular con-
nective sheet enveloping each follicle. The pylorus undergoes the most pronounced
movements and appears to lead the other organs in activity. The incurrent tubule
of the cecum is relatively thin-walled and does not appear to undergo peristaltic
activity. The excurrent tubule is thicker-walled and has definite peristaltic move-
ment in the direction of the outlet.
It follows then that one of the important functions of the pylorus is that of a
filter chamber, separating the digested and the fine, partly digested food particles
from the gross material and sand. This is the conclusion which Heidermanns
(1924) also reached when he stated that most of the time sand and gross material
ALIMENTARY SYSTEM OF LYMNAEA 99
are kept from passing into the liver by the pyloric folds. The major and minor
folds remain in close approximation along their crests, leaving a narrow slit be-
tween the dorsal and the ventral passages which may be called the pyloric filter.
The cilia on the folds are well developed and beat away from the dorsal passage.
During the pulsatory movements of the stomach region only the finest particles and
fluid material are permitted ingress to the liver through this filter. The pulsatory
currents, as these in the gut lumen may be named, are relatively strong and in their
streaming between the sand particles and foot bits in the gizzard cavity dislodge
large particles of food. Those which are carried into the pro- and retrocurrent
passages and which are too large to pass through the pyloric filter, become entangled
in the ciliary currents of the folds and are carried quickly back to the left side of
the gizzard lumen by way of the retrocurrent passage. The particles carried into
the crop on the forward streaming of the contents are soon entangled in the ciliary
currents of the crop and conveyed to the right side of the gizzard lumen. Here,
then, is a delicate adjustment by which the larger particles dislodged from the giz-
zard contents are equally redistributed for further grinding within the gizzard.
At certain intervals during the day the pulsatory movements appear to cease and
a portion of the residual material and sand in the gizzard pass out through the pro-
current passage to the prointestine. The propulsion of gizzard strings (Fig. 10),
as these residues may be named, through the procurrent passage is very slow and
mostly by cilia supplemented by slight peristalsis. Cilia were found active through-
out all portions of the alimentary tract whenever opened ; no cessation of ciliary
activity (as occurs in some lamellibranchs during increase of CO2 concentration)
or reversal of beating was observed. During emission of the gizzard string, the
large portion of the ventral pyloric fold which partly occludes the gizzard lumen
flattens to enlarge the opening. As suggested by Howells (1942) for Aplysia, it
appears that the shape and position of the pyloric folds in L. s. appressa are partly
maintained by blood pressure in the sinuses.
To what extent digestion does occur in the postesophagus, crop, gizzard, and
pylorus is questionable. As amylase from the liver and from the salivary glands,
trypsin from the salivary glands and cellulase, at least, are present in the gut con-
tents, some food may be partly hydrolized. Part of the remaining available food
is reduced mechanically to particles small enough for ingestion by the digestive
cells of the liver. The amebocytes of the gut also appear to aid in digestion. Ac-
cording to Heidermanns (1924) fats and carbohydrates are absorbed in the pylorus
by the ciliated cells.
The pyloric filter permits only minute food particles to pass into the liver. Most
of the radular teeth which are discarded continuously from the radula throughout the
life of the snail (Carriker, 1943a) and grains of sand as large as 90^., by reason of
the fact that they are considerably heavier than the lighter food particles of the
same dimensions, are carried past the cilia by the force of the pulsatory currents.
The larger free food particles, especially of lettuce, are very light and are readily
barred by the cilia of the filter. In the proximal portions of the hepatic ducts, be-
cause of counter ciliary currents, only the finer particles that fall into the grooves of
the corrugations can be carried towards the cecum ; thus teeth and larger sand
grains are held at this point by the ciliary currents of the crests of the corrugations
until sufficient fecal material passes out of the liver to carry them with it.
Ciliation of the crests of the corrugations may play a small role in the conduction
100 MELBOURNE ROMAINE CARRIKER
of food material into the follicles of the liver, but probably the principal conveyers
are the pulsatory currents. Food in solution and in suspension is thus brought to
all the internal surfaces of the liver follicles. Larger particles finding entrance
through the filter and too large to remain readily in suspension appear to fall to
the ductal epithelium. The smaller of these are soon propelled into the grooves of
the corrugations. Liberal quantities of mucus are secreted there, trapping the par-
ticles in mucous strings which pass towards the cecum, coalescing as they advance
into the larger grooves (Fig. 11). From the incurrent cecal tubule the mucoid
strings pass around the distal end of the cecum into the excurrent tubule. There
the material receives a further transparent layer of mucoid and cementing material
and is rotated into a smooth cylindrical continuous string, here designated the cccal
string (Fig. 10). This, partly by ciliary action and partly by peristalsis, then passes
on into the prointestine across the atrium. In snails feeding on green lettuce the
strings are a vivid green because of a heavy accumulation of bits of chlorophyll
bearing bodies which become entangled in mucous strings in the hepatic ducts.
In gastropods fed on a food containing carbon, the cecal strings are a dense black.
In animals on a starvation diet, the cecum continues to pass out cecal strings, just
as in the feeding animal, but the strings are a mucoid, transparent, milky-white color
and much reduced in diameter. It thus would seem that the function of the grooves
in the hepatic corrugations and of the cecum is to collect and eliminate those fine
particles which pass through the pyloric filter but which are too large to be engulfed
by the digestive cells and which are thus mechanically eliminated by a "supple-
mentary filter." Cecal strings pass out continuously, apparently at the same uni-
form rate and without apparent interruption. They provide a kind of "time clock"
by which the rate of passage of the gizzard strings and the residues from the liver
can be compared (Fig. 10).
EXPLANATION 01- PLATE 11
(All figures concern L. s. appressa )
FIGURE 3. Ciliation currents of the postesophagus, crop, gizzard, pylorus, hepatic vestibule,
atrium, and anterior portion of prointestine. The tract has been slit ventrally and spread. X 6.
FIGURE 4. Irregular blue-green excretion bodies (in vacuoles) taken from the liver string.
X500.
FIGURE 5. Smooth blue-green, or brown, excretion bodies (in vacuoles) taken from the
liver string. X 500.
FIGURE 6. "Signet" excretion body (in vacuole) appearing in the liver strings. X 500.
FIGURE 7. Clear nodules found in the liver strings which when pressed out under the cover
slip display their crystalline nature. They dissolve in dilute HC1 and seem very similar to the
calciferous concretions of the vesicular cells of the connective tissue. X 500.
FIGURE 8. Indigestible residues from digestive cell (in vacuole), found abundantly in liver
strings. X 500.
FIGURE 9. Ciliation currents of the cecum, which has been opened along the incurrent cecal
tubule and spread flat. X 6.
FIGURE 10. Typical fecal pellet, showing the gizzard, liver and cecal strings, and the im-
pression of the typhlosole in the pellet. X 6.
FIGURE 11. Portion of the corrugated epithelium of the hepatic duct, taken at the opening of
the duct into the hepatic vestibule. (Large arrows indicate the direction of the ciliary currents
in the grooves ; the small arrows, that on the crests of the corrugations. ) X 50.
ABBREVIATIONS
c.s., cecal string; cxcur. tubule, excurrent tubule; g.s., gizzard string; inc. tubule, incurrent
tubule; l.s., liver string; s., sand; t.i., impression of typhlosole in fecal pellet.
PLATE II
f-v, ::> !. ---\ ••:-!•
^itfY
wnm^M'm
procarren t
pa-ssoge
^wm/'-m^r^----
:• 0:py' -mr< -*-f -•/-•
Hl^f^Hii -x// ••. /.»
passage
'or fo/d
/ni/jor fo/d
. fa date
ceco/ fo/o?
ezcur. fa bate
w
1 «^. ;V
H^- &-':.'• '. ' • . .^
•'-•> sJ^'r.iX'.. •:• • .
102 MELBOURNE ROMAINE CARRIKER
The excretory bodies and indigestible residues in the liver are voided periodi-
cally. These are passed simultaneously in minute mucous strings from all parts of
the liver towards the central hepatic ducts, there converging into larger strings
which pass in the direction of the hepatic vestibule. At the proximal end of the
hepatic ducts this material fills most of the main duct. The combined currents in
the grooves of the corrugations appear to exert a stronger force than those on the
crests, so forcing the waste material directly into the hepatic vestibule (Fig. 11).
There it is caught by the outward flowing ciliary currents on the major and minor
pyloric folds and passed rapidly into the prointestine. The excretory bodies and
indigestible residues passing from both lobes of the liver are compressed in the
hepatic vestibule into one bulky string which is distinct from the cecal and from
the gizzard string and may be called the liver string (Fig. 10). It is drawn out
of the liver at the same rate as the cecal string passes out of the ceum. Both strings
are usually found parallel to each other and uncoiled in the fecal pellets. The giz-
zard string, on the other hand, passes out much more slowly so that the cecal string
occurs loosely and abundantly coiled therein (Fig. 10). A lapse of time seems to
occur between the exit of the gizzard string and that of the liver string, as indicated
by a conspicuous coiling of the cecal string between the last portion of the gizzard
string and the forward end of the liver string. The gizzard string follows the liver
string immediately, as indicated by no noticeable coiling of the cecal string between
the two. There is also some evidence that, as the liver string is drawn from the
liver, the pulsations of the stomach region cease. In animals opened for physiologi-
cal observation of the tract, the stomach region was never pulsating when the liver
strings were passing out of the liver. This is desirable to prevent the dismember-
ment of the strings and their mixing with food material brought into the liver by
the pulsatory currents. The merger of the strings in the prointestine produces the
fecal pellets.
The pylorus is composed of a complicated system of folds and passages, it is in-
nervated by a pair of complex nerve plexuses and a nerve net, and all of the parts
are exceptionally well vascularized. Functionally there is present in this portion
of the tract an intricate system of counter ciliary currents and synchronized mus-
cular movements, as well as partial vascular control of the folds. The pylorus is
thus well equipped to convey digestive fluids from the liver to the gizzard and crop,
to bear digested and semi-digested particles into the liver from the gizzard, to ex-
clude large sand and other large particulate matter from the liver and transfer such
residues to the prointestine, to receive waste material from the liver and transport
it to the intestine, to act in conjunction with the cecum, liver, and hepatic ducts in
shunting a continuous string of residual particles from the walls of these organs
into the prointestine, to secrete fluids (of unknown nature) and finally to absorb
fats and carbohydrates.
Liver
The liver is probably the most important organ of digestion in the alimentary
system of the gastropods. Peczenik (1925) shows, as has been indicated in this
work also in feeding experiments, that such proteins as egg albumen are engulfed
and digested intracellularly in the digestive cells, and the indigestible residues are
cast out in vacuoles. Krijgsman (1928) believes that digestive cells in Lymnaea
ALIMENTARY SYSTEM OF LYMNAEA 103
are also secretory as well as absorptive, as he has often observed numerous typical
secretion granules in the liver cells of starved snails. Biochemical tests indicate
that the greatest catheptic activity of the snail body is localized in the liver, yet none
of this activity has been found in the fluid of the gut. This is in keeping with cath-
eptic systems in other animals in which the enzyme has been shown to exist entirely
as an intracellular protease. Hurst (1927) writes that in PJiysa fat and glycogen
are stored in the digestive cells. Fat was also found in the lime cells of Helix by
Griinbauin (1913). The problem of what size of food particle is engulfed through
the distal membrane of the digestive cells is still an open question. It is likely, as
indicated by the work of Krijgsman (1925. 1928) on Hcli.r. that the lime cells
function in storing and in periodically secreting a buffering agent which adjusts
the pH of the gut juice ; this point has not been investigated in L. s. appressa. The
mucous cells of the liver provide the mucus utilized in the binding of the indi-
gestible residues and the excretory bodies into the liver strings.
Amebocytes were found in varying numbers in the contents of the lumina of the
liver, postesophagus, gizzard, and pylorus. These were similar to those seen in
the blood. In some instances those in the gut contained fecal vacuoles so large as
to force the cell into a peripheral lobate ring.
Rhythmic activity of the liver is suggested by inspection of sectioned liver tissue,
of fecal pellets and of the living organ in various phases of its activity. Pulsatory
movements of the stomach region are apparently interrupted only during the pas-
sage of liver strings and of gizzard strings. This may explain why smaller hepatic
excretory bodies occur in the upper pylorus, gizzard, crop, and postesophagus in
such insignificant numbers. If the pulsatory currents persisted during the elimina-
tion of the liver residues one would expect to find liver string detritus scattered
over the gut in as great profusion as in the liver, along with the reddish colored se-
cretions from the liver.
The inclusion bodies of the digestive cells of L. s. appressa have been studied
in detail in the living cells of normally feeding snails, starved snails, snails fed on
special diets and in preserved tissue sections. The egested bodies have been fol-
lowed in the fecal pellets over a period of weeks. The results of the study clearly
indicate the presence in the digestive cells of excretion bodies, of indigestible resi-
dues and of secretion in separate vacuoles.
Figure 8 illustrates a vacuole from the digestive cells which is filled with indi-
gestible particles. These vacuoles measure 12 to 25 /j. in diameter. In snails feed-
ing on lettuce the contents are colored a greenish brown to dark brown and are
composed of minute irregular particles, some of the larger ones of which measure
about 3 ju, in diameter. In the digestive cells they occur one per cell and in varying
stages of particulate concentration. These constitute the bulk of the liver strings
and retain their identity in fecal pellets which have been voided for several days.
The secretion granules are clearly evident in preserved histological sections
stained with iron hematoxylin, especially grouped towards the distal area of the cell.
Larger granules measure as much as 4 ^ in diameter.
The excretion vacuoles (Figs. 4, 5, 6) when in the cells may measure as much
as 25 p. in diameter, but in the fecal pellets have shrunk somewhat. In the living
cells excretion bodies are found in variable form and color and are best observed
when the cells are slowly pressed out under a cover slip as the fluids evaporate.
The cell contents then pass rolling and turning from the ruptured cells, exposing the
104 MELBOURNE ROMAINE CARRIKER
different surfaces of the inclusions. There is one series in which the vacuoles range
from small to large vacuoles containing variable numbers and sizes of minute blue-
green, translucent, many-angled particles. The smaller particles are in constant
Brownian movement, dancing around like a swarm of bees, and indicating the low
viscosity of the fluids in the vacuoles (Fig. 4). In a second series the same vari-
ation in size of the vacuole is encountered but the blue-green bodies are present in
groups of only one to four per vacuole and are spherical and smooth ( Fig. 5 ) . In
a third series the vacuoles and bodies are identical in form to the second series, but
the color of the bodies varies from a light brown to a dark solid brown. The largest
of these bodies are sometimes found free of the vacuoles. When compressed under
a cover slip they spread with a flowing viscous movement, much as a drop of heavy
molasses spreads when pressed between two smooth surfaces. In the fecal pellets
these vacuoles are usually found varying in diameter from 3 to 15 ju, and the vacuole
membrane presses closely around the excretion body. A fourth type of excretion
body is found which varies in diameter from 12 to 18 /*, is colored a dark brown
with a smooth center and possesses a periphery of irregular markings, such that the
body resembles a signet ring (Fig. 6). The excretion bodies described above are
present principally in the liver strings, and only in negligible numbers in the cecal
strings. The "browns" and "signets," particularly, stain with methylene blue and
neutral red and do not dissolve in strong HC1. The different types described are
not all present in any liver string in equal abundance at any one time, but vary
independently, in a sequence which did not seem significant. Because of the transi-
tional stages between some of these excretion bodies it is probable that they are all
different phases of the same type of metabolic excretion ; but the method of their
formation is still a puzzle.
Intestine and rectum
Cilia on the typhlosole beat towards the lateral sides of the typhlosole (Fig. 3) ;
those over the prointestine around the typhlosole beat circumferentially and some-
what obliquely from the dorsal to the ventral sides in a symmetrical pattern. The
division of the currents occurs along the dorsal line of the prointestine. Over the
pellet-compressor the cilia beat transversely across the intestine. The raphe bears
a strong current which streams directly posteriad. Thus in the pellet-forming re-
gion, through ciliation and muscular movement, loose particles are gathered, rolled
inward about the typhlosole and folded into a compact pellet. Strong ciliary cur-
rents in the remainder of the intestine and rectum are limited almost entirely to
the costae, raphe, and pseudoraphe ; cilia of the intercostal surfaces are relatively
short and weak. Peristaltic activity is evident throughout the intestine and rectum,
being noticeably strongest in the early portions of the prointestine, just behind the
pellet-compressor.
Abundant vascularization of the prointestine, in contrast to the relatively poor
vascularization of the esophagus, suggests that this region of the intestine may also
function in the absorption of food and water.
Consolidation of the cecal and liver strings occurs at the hepatic vestibule ; of the
gizzard residues and cecal string, in the pellet-forming region. The cecal string as
it is moulded in the cecum is already a smooth well cemented string and undergoes
no further change as it is forced continuously across the outer margin of the atrium.
ALIMENTARY SYSTEM OF LYMNAEA
105
The liver string, characterized hy a fine dark brown mottling and almost as well
concentrated as the cecal string, receives a final transparent envelope of cementing
fluid which binds the cecal string to it (Fig. 10).
The chief function of the pellet-forming region is that of consolidating and ce-
menting the loose straggling gizzard residues which constitute by far the greatest
bulk of the fecal pellet. The large numbers of mucous cells, basophilic flask cells
and basal secreting cells about the pellet-forming region are indicative of the large
quantities of cementing substance secreted during the moulding of the pellets. By
means of ciliary streams and constriction of the tube at the pellet-forming region
the gizzard residues are pressed into pellets, and the cecal strings, lying loosely
40
30
20
10
length
/engfn fiver •str/fryi
number t/cer strings
10
15
20
25
Days
FIGURE 1. Length in millimeters of the liver and gizzard strings and number of liver strings
of the fecal pellets, calculated on a twenty-four hour basis. These were voided in a period of
twenty-four days by a forty millimeter L. s. afiprcssa. The vertical arrows indicate the time
at which egg masses were oviposited.
coiled in these residues, are simultaneously incorporated in the pellets. These are
then forced out of the pellet-forming region by ciliary activity and by strong peri-
staltic movements which are noticeably stronger immediately behind the pellet-
compressor. Peristaltic activity gradually diminishes in the direction of the anus.
The conspicuous impression of the typhlosole remains in the fecal pellet, particu-
larly in the gizzard string portion, as long after defecation as the pellet retains its
form. Moore (1931) has found variable patterns in the fecal pellets of different
Gastropoda and points out the importance of identification of animals by means of
their pellets. A most striking fact about fecal pellet formation is the extreme com-
106 MELBOURNE ROMAINE CARRIKER
pleteness with which fecal material is compressed and cemented. This presumably
prevents fouling of any portion of the tract.
For any given snail the diameter of the gizzard string portion of the fecal pellet
is constant, varying principally with the size of the snail. The liver string varies
in diameter from that of the gizzard string to that of the fecal string. Figure 1
indicates for a forty millimeter snail over a period of twenty-four days the rate and
extent of voidance of fecal pellets. For the tabulation of this data the fecal pellets
were collected daily and arranged end to end under the binoculars and measured to
the nearest millimeter. The measurements given indicate only the lengths of the
gizzard and liver strings, as the cecal string generally occurs embedded in the first
two strings. The diameter of the gizzard string is reliably constant ; that of the
liver, less so.
Most conspicuous is the fact that the quantity of fecal pellets voided daily is
quite variable from day to day. The quantity of gizzard strings fluctuates far more
erratically than does that of the liver strings, indicating that the volume of material
utilized by the liver is more constant than that which may pass through the gizzard.
The number of liver strings is a more conservative indicator than the length of
strings, and is probably not as accurate. Passage of food through the gizzard, and
thus food consumption, seems to diminish during oviposition.
As indicated by the following data, feces were voided in about equal quantity
day and night, with just a slight daily increase, over a period of twenty days (9 P.M.
to 9 A.M., and 9 A.M. to 9 P.M., respectively) :
Pellets Night Day
Total length of pellets, mm 1,987 2,134
Total length of liver strings, mm 530 588
Total number of liver strings 110 113
The total length of fecal pellets passed in the twenty-four days was 5,645 mm. ;
and the total length of liver strings, 1,491 mm., was passed in 289 liver strings, giv-
ing an average length of 5.1 mm. per liver string. Actually the liver strings varied
in length from one to 10 mm. The average calculated length of fecal pellets passed
in twenty-four hours was 235 mm. ; of liver strings, 62 mm. In a normally feeding
snail the sequence of the liver strings with the gizzard strings was always one of
alternation. Liver strings do not generally mix with the gizzard strings. Gizzard
strings as long as 52 mm. were found connecting liver strings. Three typical series
of fecal pellets taken from days one, two, and three on Figure 1 are given below.
The liver and gizzard strings are represented by the lengths in millimeters of the
strings in the order of their elimination ; the figures for lengths of the gizzard strings
are italicized. The total time for elimination of the pellets is given to the right in
parenthesis :
(1) 640 7 33 7 11 644 5 (5 hrs. 15 rnins.)
(2) 48 7 52 8 13 4 50 5 38 6 (10 hrs. 15 mins.)
(3) 207 87226299243 (10 hrs. 30 mins.)
As indicated by the curve for total fecal pellets in Figure 1 and by the lengths of
the gizzard strings in the series above, consumption of food appeared to follow an
alternating heavy and light cycle.
ALIMENTARY SYSTEM OF LYMNAEA 107
In snails deprived of food the elimination of the gizzard strings ceased and liver
strings then became connected only by slender lengths of cecal strings. When
starvation had continued for ten or more days nothing but delicate white cecal
strings and a few much reduced liver strings containing metabolic excretion bodies
were found in the intestine.
A. H. Rosenbloom (unpublished bachelor's thesis, 1942) by feeding colored
food to L. s. appressa at different times through a period of a month found that in
normally feeding snails of approximately forty millimeters shell length the minimum
time for the passage of food from the mouth to the anus was two hours and twenty
minutes ; in snails previously starved for a week, five hours and fifty minutes. He
found also that previously starved snails feed for a longer consecutive time than
do normally feeding snails. The present investigation shows clearly that the ali-
mentary system becomes completely emptied of food a few days after starvation
commences. Considerably more food and a longer time are required for a starved
animal to fill the alimentary tract with food to the point where fecal material is
voided than for a normally feeding snail.
The rhythm of passage of liver strings is in keeping with the rhythm of the liver
itself in which all digestive cells appear to assimilate food together and discharge
indigestible residues simultaneously. This cycle, as indicated by the passage of
liver strings, is not completely unvarying, because the number of liver strings dis-
charged daily varied approximately from eight to nineteen. Thus the interval be-
tween the discharge of liver residues, probably the time during which the liver was
digesting food, varied in this experiment from seventy-five minutes to three hours.
It is possible that oviposition ( Fig. 1 ) may account for some of the variability.
There seems to be nothing in the literature concerning fecal cycles in the Gas-
tropoda. Some few scattered observations are reported on the length of the fecal
pellets. For example, Heidermanns (1924) writes that a 48 mm. L. stagnalis
with a 90 mm. intestine, eliminated 120 mm. of feces in 24 hours.
The long intestine is characteristic of the herbivorous snail nutrition of L. s.
appressa. One of the most striking facts about the functioning of the alimentary
system is the meticulous care with which all loose particles are collected and properly
disposed of, in this way serving as a highly efficient sanitation system. The fecal
pellets receive additional external layers of cementing material as they pass down
the length of the intestine and rectum. The pH of the intestine is slightly more
alkaline than that in the stomach region. As pointed out by Yonge (1935) mucus
is an amphoteric protein whose viscosity is augmented by higher pH, thus more
efficient consolidation of the feces occurs. Elimination of the fecal pellets through
the anus is a fairly rapid and uniform process. The strong anal sphincter muscle
remains tightly contracted except during defecation. Fecal pellets, being slightly
heavier than water, settle slowly to the bottom of the aquaria. The marked effi-
ciency of the mucoid coating over the feces is indicated by the extended period after
defecation that pellets retain their identity. Thus it would seem that the alimentary
system has not only become specialized in the maintenance of hygienic conditions
within the system, but also in furthering a healthy external environment.
Fecal pellets are ingested by snails even in the presence of fresh food and the
animals appear to derive some nourishment from them. It is to be recalled that
the gizzard is not a thoroughly efficient grinding mechanism and in many cases,
108 MELBOURNE ROMAINE CARRIKER
particularly in the absence of sufficient fine sand, considerable unused available
food passes out in the gizzard strings.
DISCUSSION
The question as to whether the radula slides over the cartilage independent of
cartilage activity has been a favorite point of academic controversy with certain
malacologists for some time (in Lymnaeidae see Geddes, 1879; Amaudrut, 1898;
and Pelseneer, 1935; in the Stenoglossa, a review: Carriker, 1943b). In L. s.
appressa (and possibly in the majority of snails carefully investigated) there is no
question but that the principal activity of the radula is that effected by the action
of the cartilage and muscles under it, and a sliding of the total radula over the
cartilage independent of the movement of the cartilage.
A study of the movements of the gut in L. s. apprcssa suggests that rather than
the presence of different pH in the different portions of the gut, the pH may vary
with the rhythms and secretions of the liver, the secretions of the salivary glands,
the secretions of the unicellular glands of the gut wall and with feeding. It is quite
unlikely that with the constant mixing of the gut contents as a result of the pulsatory
movements at certain periods, the pH would vary markedly in the different lumina
of the tract at any time. The wide range obtained between the maximum and the
minimum pH's and the insignificant variation of the maximum and of the minimum
pH's is in keeping with this suggestion. The partial isolation of the intestine from
the movements of the stomach region is in keeping with the slightly higher pH found
in the intestinal lumen.
The complexity and abundance of nervous tissues about the stomach region sug-
gests a possible nervous control of the movements of the stomach region and of the
liver. In its muscular structure there is no doubt that the buccal mass is the most
complex organ in the alimentary system ; functionally it appears that the region in
and about the pylorus is the most intricate. The dense ramifications of blood ves-
sels, the presence of two nerve plexuses, the intricate series of folds and the compli-
cated ciliary streams in this region lend credence to this postulation.
Heidermanns (1924) has opened the question of the function of sand in the
basommatophoran gizzard in his comparative study of Ancylus, Planorbis, Physa,
Lymnaea and certain stylommatophorans. He points out that in land pulmonates
the flaring portion of the esophagus is called the stomach, whereas in the aquatic
pulmonates the esophagus is normal and the stomach has become differentiated into
the crop, gizzard and pylorus. Thus the Stylommatophora have no organs that
could properly be homologized with the stomach of the Basommatophora. The giz-
zard and, with few exceptions, sand in the tract are absent from the land pulmo-
nates. The gizzard, he states, reaches its peak of specialization in L. stagnalis and
probably rose by reason of the ingestion of sand with food. He observed that in all
Basommatophora the gizzard originates in front of the first flexure of the gut, appar-
ently as a muscular band whose primitive function was to dispose of sand masses
tending to congest there. This primitive type of. gizzard is exemplified by that of
Ancylus and the intermediate type by that of Planorbis. Heidermanns in support
of his theory of the origin of the gizzard through a specialization of a primordial
portion of undifferentiated gut, attempted to show modification of the gizzard in
one snail generation by the use of various diets. As might be anticipated, he got
no significant structural changes.
ALIMENTARY SYSTEM OF LYMNAEA 109
The fact that Lymnaca possesses the gizzard grinding mill may explain the ob-
servation stressed by Heidermanns that the cellulase of this snail is less active than
that of Helix which has not developed a gizzard and consequently needs a strong
cellulase for the hydrolysis of the cell walls of plant food consumed.
There is striking similarity in the functioning of the alimentary tract of the
herbivore Onchidella ccltica, ably presented by Fretter (1943) in a recent paper,
and that of L. s. apprcssa. Perhaps this similarity is not to be wondered at when,
as Fretter writes, "Many of the features which the Onchidiidae share with the pul-
monates may be attributed to the close origin of the two groups, the similarity of
their diet and their air-breathing habit."
SUMMARY
1. A balanced physiological salt solution was developed which maintains con-
tractions of the vas deferens for approximately 66 hours.
2. Cathepsin was found in greatest concentration in the liver and no activity
could be ascertained in the gut fluids. Some trypsin was indicated in the salivary
glands. Amylase showed greatest activity in the salivary glands and the liver.
3. Muscular activity of the alimentary system involves the manipulation of the
mouth parts in the buccal mass, peristalsis in the remainder of the tract, marked
pulsatory movements of the postesophagus, crop, pylorus and liver, and a kneading
motion of the gizzard. The radula is moved principally by the action of the odonto-
phore but also operates independently of it.
4. The entire alimentary system, with the exception of the gizzard and parts of
the buccal cavity, is ciliated. The cilia show definite directional streams which
function in propelling food particles, in sorting food and in consolidating fine refuse
particles with the aid of mucoid substances.
5. Sand is consumed normally by the snail and is necessary for the proper
functioning of the gizzard in the crushing of food particles. Very little trituration
is performed by the mouth parts.
6. The pylorus is composed of a complicated system of folds and passages and
counter ciliary currents and functions as a filter which permits only the soluble and
the finer food particles to pass into the liver. It shunts the undigested residues
from the gizzard into the prointestine.
7. In the liver the digestive cells function in secretion, assimilation-, intracellular
digestion and excretion. The indigestible foods and the excretory products, as vari-
ably shaped and colored inclusion bodies, are eliminated in vacuoles.
8. The cecum functions in collecting the finer residues from the liver and forces
them in a continuous string into the prointestine.
9. The residual material coming from the gizzard, liver and cecum is charac-
teristic for each organ and is readily identified as distinct in the fecal pellet.
10. The prointestine is specialized in the final consolidation of gizzard, liver and
cecal strings with the aid of cementing substances secreted by the basophilic flask
cells and the basal cells.
11. The rhythmic nature of the liver is disclosed principally by a study of the
fecal pellets.
12. L. s. apprcssa is an herbivore. Food bits are cut away by the radula and
swallowed. In the buccal cavity the food receives mucus from the buccal gland
110 MELBOURNE ROMAINE CARRIKER
cells, mucous cells and the salivaries and enzymes from the latter. Temporary stor-
age and initial digestion occur in the postesophagus. Digestive fluids pass up from
the liver in the pulsatory movements of the stomach region which keep the fluid gut
contents in constant circulation. The crop, gizzard and anterior portion of the
retrocurrent passage of the pylorus comminute the food. Amebocytes present in the
gut contents appear to aid in digestion. Soluble and fine particles of food pass
through the pyloric filter into the liver where it is assimilated by the digestive cells.
Assimilation also occurs in the pylorus and absorption possibly in the intestine.
There is some evidence that the pulsatory movements of the stomach region cease
during the passage of the gizzard and the liver strings.
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TEMPORARY PAIR FORMATION IN PARAMECIUM
BURSARIA 1
TZE-TUAN CHEN
Department of Zoology, University of California, Los Angeles
In Parame'cium bursaria, the two members of a conjugating pair normally re-
main united twenty to thirty-eight or more hours. During this time various nu-
clear processes take place, including three pregamic divisions, exchange and fusion
of pronuclei, and three post-zygotic divisions. Clones that are capable of under-
going normal conjugation as described above are called "normal clones."
But there are some clones of this species which are abnormal - in that when they
are mixed with normal clones the pairs formed are not lasting but separate within
a few hours. An examination was made of such temporary pairs in order to dis-
cover what nuclear or other changes occur in them.
Fourteen clones of Parameciion bursaria, all belonging to Variety I, have been
used in the present study. These clones, all of which were collected in nature, are
listed in Table I with data on each clone including (1) the mating type to which
it belongs and (2) the locality where it was collected.
TABLE I
Clones of Paramecium bursaria employed in study of temporary pair formation
Clone number
Original designation
of clone
Mating type
Locality collected
1
SAaS
A
Santa Ana River, Cal.
2
Or3
A
Vicinity of Capistrano, Cal.
3
SGa3
A
San Gabriel River, Cal.
4
BG35
A
Los Angeles, Cal.
5
La3
B
Laguna Canyon, Cal.
6
SAa7
B
Santa Ana River, Cal.
7
UC13
B
Los Angeles, Cal.
8
BH2
B
Beverly Hills, Cal.
9
SAa4
D
Santa Ana River, Cal.
10
LP10
D
Lone Pine, Cal.
11
UC14
D
Los Angeles, Cal.
12
SAal
C
Santa Ana River, Cal.
13
BH7
C
Beverly Hills, Cal.
14
BH101
C
Beverly Hills, Cal.
Clones 1-11 are normal clones in that they are capable of undergoing normal conjugation.
Clones 12-14 are abnormal clones in that when they are mixed with normal clones the pairs formed
are not lasting but separate within a few hours.
1 This work was aided by grants from the Committee for Research in Problems of Sex,
National Research Council ; and from the Joseph Henry Fund of the National Academy of
Sciences.
- These clones are considered abnormal here only because they are incapable of taking part
in the formation of lasting pairs when they are mixed with other, normal clones.
112
TEMPORARY PAIR FORMATION IN PARAMECIUM 113
The animals were cultured in essentially the manner described by Jennings
(1939). For cytological study the animals were fixed in Schaudinn's fluid con-
taining glacial acetic acid, stained in iron hematoxylin, and destained in saturated
aqueous solution of picric acid, following the technique the writer has described
(Chen, 1944).
EXPERIMENTAL STUDIES
Most of the present work was done with the two abnormal clones (BH7, SAal),
although some study was also made on a third abnormal clone (BH101). All of
these three abnormal clones belong to mating type C of Variety I. These abnormal
clones were mixed with normal clones. Eleven such normal clones (all belonging
to Variety I) were used. Four of these normal clones belong to mating type A;
four to type B ; and three to type D (see Table I).
As an example of the phenomenon of temporary pair formation, the reaction be-
tween the abnormal clone SAal and the normal clone UC13 will be described. On
November 27. 1940, a large number of animals belonging to each of these two clones
were mixed at about eleven o'clock in the morning. Strong agglutinative mating
reaction occurred almost immediately. Half an hour after mixture, pairs were
being formed. An hour after mixture (about noon) many pairs were formed.
But in the early afternoon the pairs broke apart into single animals. By five o'clock
all but a few pairs had separated. By evening all had separated.
Such temporary pair formation was also observed when the abnormal clone
SAal was mixed with the following normal clones: Or3, SGa3, SAaS, La3, SAa7,
BH2, SAa4, LP10. and UC14; or when the abnormal clone BH7 was mixed with
the normal clone LP10; or when the abnormal clone BH101 was mixed with the
normal clone BG35.
If such a mixture was placed in a moist chamber and kept from drying (with
occasional replacement of the fluid that evaporated), the typical agglutinative mating
reaction and temporary pair formation recurred the following day and almost daily
over a period of many days. Some such mixtures were kept under daily observa-
tion over a period of nineteen days. The following is the characteristic daily be-
havior of the animals in such a mixture. The agglutinative mating reaction occurs
in the late morning. By noon, many pairs are formed. These pairs persist for a
few hours. Between four and six o'clock in the afternoon only a few pairs are
found. In the early evening one or two pairs may remain ; none can be found after
nine o'clock in the evening.
CYTOLOGICAL STUDIES
Nuclear conditions in the clones that jorm only temporary pairs
The writer has made a cytological study of twenty-one abnormal clones, includ-
ing the two clones BH7 and SAal, and nineteen of the twenty-two such clones de-
scribed by Jennings (1944). It was found that fifteen of these clones possess
micronuclei, while six appear to be amicronucleate.
Thus the amicronucleate condition is not the general cause of the peculiar be-
havior of these abnormal clones. It is probable that the persistence of the amicro-
nucleate condition is a result of the inability to conjugate and acquire a micronucleus,
rather than the cause of it. Apparently there are conjugating and non-conjugating
114 TZE-TUAN CHEN
races of amicronucleate ciliates. In nature those that can conjugate do so and ac-
quire a micronucleus, leaving in the amicronucleate condition only those incapable
of conjugation. In my experience with P. bursaria, which includes a study of the
nuclei and chromosomes of many clones (collected from different parts of the United
States. Canada, Russia, England, Ireland, and Czechoslovakia), the only amicro-
nucleate animals found in nature are those which cannot conjugate. Since they can-
not conjugate, it is likely that such clones will be permanently amicronucleate. In
nature any amicronucleate animal that can conjugate would not remain amicro-
nucleate for long, since it would become micronucleate after mating with a normal
animal from whom it receives a pronucleus as a result of conjugation (Chen, 1940).
Amicronucleate animals that can conjugate have been found in P. bnrsaria (Chen,
1940) 3 and in Euplotcs patella (Kimball, 1941). They arose spontaneously in labo-
ratory cultures.
Nuclear changes in temporary pair jormation
To determine whether nuclear changes occur in temporary pair formation, a series
of preparations were made, in December, 1940, of temporary pairs (abnormal clone
SAal X normal clone UC13) 4 and a number of separated animals belonging to the
latter clone. The material included pairs 5 to 6 hours after onset of temporary
mating, separated animals a few hours after separation, 13 to 17 hours after separa-
tion, and 21 hours after separation. The micronuclei in these temporary pairs and
separated animals were compared with the micronuclei of vegetative animals of
clone UC13 (not mixed with any other clone). It was found that micronuclei in
the majority of the temporary pairs and of the separated animals were slightly
swollen. In some, no nuclear changes were apparent.
In June, 1943, a series of preparations were made of temporary pairs (abnormal
clone BH101 X normal clone BG35) 5 and a number of separated animals belonging
3 The writer has recently found some additional cases of conjugation between amicronucleate
and normal animals in Pafaincciinn bnrsaria, in Variety III. (Normal nuclear changes occur
in the conjugants having the micronuclei.) These amicronucleate animals arose spontaneously
in laboratory cultures.
Schwartz (1939) in a brief preliminary paper reported "conjugation" in Paramecium
bursaria between amicronucleate and normal animals and between two amicronucleate animals.
In view of the lack of details in this report, it is impossible to tell whether temporary or lasting
pair formation took place.
4 Clone SAal appears to be amicronucleate; clone UC13 has a deeply staining micronucleus.
5 Clone BH101 has a small, lightly staining micronucleus ; clone BG35 has a relatively large,
deeply staining micronucleus.
EXPLANATION OF FIGURES
FIGURES 1-34. Micronuclei of animals belonging to clone UC13 before, during, and after
temporary pairing with animals of clone BH7 (drawn by Mr. Earl Nielsen). All drawings
were made with a camera lucida. X 3,300.
FIGURES 1-5. Resting micronuclei of vegetative animals.
FIGURES 6-10. Micronuclei in the members of temporary pairs 4 hours after onset of pair-
ing.
FIGURES 11-16. Micronuclei in the separated animals 18 hours after separation.
FIGURES 17-22. Micronuclei in the separated animals 30 hours after separation.
FIGURES 23-28. Micronuclei in the separated animals 42 hours after separation.
FIGURES 29-34. Micronuclei in the separated animals 51 hours after separation.
TEMPORARY PAIR FORMATION IN PARAMECIUM
115
6
1 • 29
17
c
&
v£-
II
12
,' f
'• r
1
25
31
FIGURES 1-34.
13
J'i
14
116 TZE-TUAN CHEN
to the latter clone. The material included pairs 3 to 4 hours after onset of temporary
pairing, and separated animals 2 hours after separation, and a day after separation.
It was found that the micronuclei in the majority or most of the temporary pairs
and separated animals were slightly swollen. In others no nuclear changes were
apparent.
In October, 1944, a series of preparations were made of the temporary pairs (ab-
normal clone BH7 X normal clone UC13) 6 and separated animals belonging to the
latter clone. The material included pairs 4 hours after onset of temporary mating,
and separated animals 7 hours after separation, 18 hours after separation, 30 hours
after separation; 42 hours after separation, 51 hours after separation. A series of
preparations of vegetative animals of clone UC13 (not mixed with any other clone)
were used as controls (Figs. 1-5). It was found that the micronuclei in nearly all
of the temporary pairs and separated animals were slightly but noticeably swollen
(Figs. 6-34). This is true even of the separated animals 51 hours after separation
(Figs. 29-34), indicating that the physiological effect of the contact between the
animals in temporary pairing (as shown by the swelling of the micronucleus) is of
long duration.
GENERAL RELATIONS
The temporary pair formation described in the present paper is similar to that
reported by Sonneborn (1942) in P. aurclia and by Jennings (1944) in P. bursaria.
Sonneborn (1942) concluded from his data that cell adhesion occurring in the initial
stage of the mating reaction and cell fusion occurring during subsequent conjugation
are due to two different mechanisms.
SUMMARY AND CONCLUSIONS
1. In normal conjugation of Paramecium bursaria, the two members of each pair
remain united for 20 to 38 or more hours, during which time various nuclear proc-
esses take place including three pregamic divisions, exchange and fusion of pro-
nuclei, and three post-zygotic divisions. Clones that are capable of undergoing nor-
mal conjugation as described above are called "normal clones."
2. Some clones of this species are abnormal in that when they are mixed with
normal clones the pairs formed are not lasting but separate within a few hours.
3. In temporary pair formation, the animals of diverse mating types when mixed
exhibit the typical agglutinative mating reaction. Within an hour many pairs are
formed but in a few hours these pairs break apart into single animals.
4. If such a mixture is placed in a moist chamber and kept from drying (with
occasional replacement of fluid that evaporates) such agglutinative mating reaction
and temporary pair formation will recur daily over a period of many days.
5. Cytological study of 21 such abnormal clones shows that most of these clones
have micronuclei ; some appear to be amicronucleate. Therefore, amicronucleate
condition cannot explain the incapacity for taking part in the formation of lasting
pairs. It is probable that the persistence of the amicronucleate condition is a result
of the inability to conjugate and acquire a micronucleus rather than the cause of it.
6. In temporary pair formation, there are no conspicuous nuclear changes either
in the pairs or in the animals after their separation. In the majority of the tempo-
G Clofie BH7 appears to be amicronucleate.
TEMPORARY PAIR FORMATION IN PARAMECIUM 117
rary pairs and separated animals, there is, however, a slight swelling of the micro-
nuclei. This swelling persists for a considerable length of time after the separation
of the animals, indicating that the physiological effect of the contact between the ani-
mals in temporary mating (as shown by the swelling of the micronuclei) is of long
duration.
LITERATURE CITED
CHEN, T. T., 1940. Conjugation in Paramecium bursaria between animals with diverse nuclear
constitutions. Jour. Hercd., 31 : 185-196.
CHEN, T. T., 1944. Staining nuclei and chromosomes in Protozoa. Stain Tcchn., 19 : 83-90.
JENNINGS, H. S., 1939. Genetics of Paramecium bursaria. I. Mating types and groups, their
interrelations and distribution ; mating behavior and self-sterility. Genetics, 24 : 202-
233.
JENNINGS, H. S., 1944. Paramecium bursaria: life history. I. Immaturity, maturity and age.
Bio!. Bull., 86: 131-145.
KIMBALL, R. F., 1941. Double animals and amicronucleate animals in Euplotes patella with
particular reference to their conjugation. Jour. Exp. Zool., 86 : 1-32.
SCHWARTZ, V., 1939. Konjugation micronucleusloser Paramaecien. Natumnss., 27: 724.
SONNEBORN, T. M., 1942. Evidence for two distinct mechanisms in the mating reaction of
Paramecium aurelia. Anat. Rec., 84 : 542-543.
Vol. 91, No. 2 October, 1946
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE SPACE-TIME PATTERN OF SEGMENT FORMATION
IN ARTEMIA SALINA
PAUL B. WEISZ
Department of Zoology, McGill University, Montreal, Canada
i
INTRODUCTION
The present work was carried out in an attempt to arrive at a primary under-
standing of the regularities and laws in the phenomenon of metameric segmentation,
as related to the shape and size of animals. To date this phenomenon, although of
widespread occurrence amongst the higher animal phyla and thus probably an in-
tegral part in the more complex patterns of evolutionary organization, was never-
theless surprisingly rarely, if at all, subjected to analytical inquiry. The reason
for this can probably be found in an essential lack in the past of well-defined con-
cepts about the interrelations between mass, shape, growth, and degree of develop-
ment of living organisms. The problem of segment formation in relation to size
and shape is primarily one involving a clear appreciation of the dynamic geometry
of living matter, and initial insight into the problem can therefore only emerge from
rigorous observation on a quantitative level, followed preferably by geometrical
and mathematical analysis. Such a method has been employed in the present
work, and the results gained are conclusive enough not only to point the way for
further study of the problem at hand, but also to promise reasonable success in the
application of the quantitative, geometrical method to questions of biological space-
time pattern in general.
The choice of Artemia has proven particularly fortunate for a study of meta-
meric segmentation. The animal, held to be amongst the most primitive of living
Crustaceans (Lockhead, 1941), develops few, if any, specialized structural features
which would ordinarily tend to obscure the fundamental processes of morphogenesis.
Moreover, the development of as many as nineteen body segments, a further primi-
tive trait, is of obvious advantage in the investigation of the underlying principles
of formation. Also, Artemia is easily obtained and can be reared in the laboratory
without difficulty.
METHODS AND MATERIALS
Larvae of Artemia salina were obtained from commercial, air-dried egg cysts.
Since excystment is retarded or inhibited in water above a certain salinity (Jennings
and Whitaker, 1941), water of a specific gravity of 1.020 was used throughout as
the initial medium. The egg shells cracked open usually 12 to 18 hours after
contact with the water, and emergence of the larvae (Whitaker, 1940) took place
119
120 PAUL B. WEISZ
between 18 and 24 hours. Portions of five stock solutions of brines with different
salt concentration were employed as further media. The solutions were obtained
from the original sea water by either diluting with doubly glass distilled water or
concentrating with NaCl to specific gravities of 1.022, 1.033, 1.047, 1.066, and
1.085, respectively. All solutions were vigorously aerated daily, and possible
deviations from the proper specific gravity were adjusted in weekly intervals. All
work was carried out at room temperature, corresponding to an average water
temperature of 21-22° C. As soon as the embryos had emerged, still enclosed
within the fine hatching membrane, they were transferred to water from either of
the five stock solutions. The moment of hatching, occurring within 24 to 30 hours
after the cysts had first made water contact, was taken as zero time for all further
determinations.
For observation the larvae were reared singly, in heavy crystal watch glasses.
In the course of several observational series, a total of up to 100 individuals were
observed, at least for certain periods of their development; of the 100, about 25
individuals, evenly distributed among the solutions, were reared from hatching to
the adult stage. The presence or absence of a molted shell, the time, the tempera-
ture, the stage of development reached, and a series of measurements on bodily
proportion were recorded for each individual twice daily in the earlier stages and
daily for later stages. The animals were fed once every two days on a sea-water-
yeast suspension. Each watch glass containing an animal was covered so that
evaporation was nearly abolished, but a minimum of air circulation was always
allowed for to equilibrize the CO2 released by the yeast and the animal. The water
was changed at two-day intervals for the younger stages and daily for older ones.
Larval body measurements were taken under the microscope with the help -of
a hemocytometer slide whose grid allows the direct reading off of lengths of 50
micra and consistent estimations of lengths of 10, 20, and 30 micra. If the larva
is placed on a coverslip with a minimum of water, the whole can be adjusted in
relation to the grid ; evaporation is sufficiently slow to allow five or six measure-
ments at a time. The error inherent in this method, viz., the parallax due to the
thickness of the coverslip, is small enough to be negligible ; also, since all measure-
ments were taken in this way, the relative values are consistent.
PRELIMINARY OBSERVATIONS
Barigozzi (1939) and Rugh (1941) observed the total developmental time from
hatching to the adult stage of Artemia to be 3 to 4 weeks. This is true as a broad
generalization, but with the egg cysts in the various salinity media here employed,
certain statistically preferred tendencies become apparent, expressed empirically by
r)_r). o
*-> x — •k'O ^
-e.s
where Dx is the time, in days, for complete development from hatching in a solution
of salt concentration x\ D0, the (hypothetical) time, similarly, for development in
distilled water (the algebraic value turned out to be 36.55) ; and Sx, the specific
gravity of the solution of concentration x. This relation holds good only in a
statistical sense, within a specific gravity range of 1.020 and 1.1 ; it indicates that
with higher salt concentrations the rate of development tends to be greater (Fig. 1).
Irrespective of concentration a sigmoid curve of growth is always obtained.
ARTEMIA SEGMENTATION PATTERN
121
Morphologically, different salinities have no differential effect on relative body
proportions, a result to be expected in view of the conclusions of Bond (1932).
An inverse relation between total size and salinity, observed by Bond, Heath
(1924), and Warren (1938) for larvae from non-excysted eggs in the natural
habitat, however, could not be observed for the excysted larvae here used; in the
latter, total sizes are identical at equivalent stages of development, irrespective of
salinity.
The number of molts between hatching and sexual maturity is not constant.
Even when reared in the same medium, slight differences in molting frequency
between several larvae may occur. Moreover, there exists a rough statistical
relation between salinity and the total number of molts, approximating closely the
0 I/ S4 5 fi 7 8 9 10 II 12 13 14 15 IS 17
05
FIGURE 1. The effect of salinity on developmental time, from hatching to sexual maturity;
absolute growth curves. Abscissa: total larval length; Ordinate: time in days. A-E, media
of salt water, specific gravities from 1.022-1.085 respectively; numbers 1-19 above abscissa refer
to number of body segments present.
above relation between salinity and the time required for complete development;
in general, however, the number of molts for a given salinity is somewhat lower than
the number of days required for development. In larvae from excysted eggs and
under artificial food conditions, a range of 12 to 16 molts was observed between
hatching and maturity, at a specific gravity of 1 .085 ; this compares with 25 to 29
molts at a specific gravity of 1.022, and gradually decreasing molting frequencies
for the intermediate salinity ranges. A staging of larval development according
to molts, as Heath has done for non-excysted individuals, would therefore not be
possible in the present case. Heath's 13 stages would hold for excysted larvae
only when reared in brine of a specific gravity of 1 .085 ; even then certain definite
differences in the degree of development of equivalent molting stages can be
122 PAUL B. WEISZ
observed, as comparison of Heath's descriptions with those below makes apparent.
With increasing developmental age the duration of instars increases; a 12 to
24 hour interval between molts in the younger stages compares with 24 to 30
hour intervals in older ones. The two factors of salinity and developmental age
also determine the size increase between molts ; for higher salinities, as well as for
older larvae, the size increase is greater. There is no observable relation however
between the time at which a molt occurs and the size or the developmental stage
attained, irrespective of whether test larvae are reared in the same or at different
salinities. Molting is also greatly influenced by the food supply. Starving animals
do not molt ; after 3-5 days an abortive attempt at molting is made which usually
results in the death of the animal. Conversely, overfed larvae may molt twice in
rapid succession without undue increase in size.
ANALYSIS OF SEGMENT FORMATION
Observations and definitions
The larval development of Artemia can best be dealt with in terms of the
number of body segments present. The first three segments become visible almost
simultaneously at a total larval size of 0.745 mm. (stage 3), after the embryonic
yolk has been digested away, and the termination of the hatching, nauplius, and
metanauplius stages can therefore be represented as the termination of stages 0, 1,
and 2, respectively ; at the end of any following stage the stage number will thus indi-
cate directly the number of body segments present. It will be convenient to distin-
guish between a thoracic period of development, comprising the first 11 stages, and
an abdominal period, including stages 12 to 19; the latter can again be divided into
a genital period (stages 12 and 13) and a post-abdominal 'one (stages 14 to 19).
Except for the first three, each individual segment is initially recognizable as a
transverse ring of thickened mesoderm, the segment rudiment, immediately under-
neath the otherwise smooth epidermal layers (segmental stage a). Later, partial
transverse constrictions appear externally in the epidermis and the chitin, in a
plane just posterior to that of the segment rudiment (segmental stage b}. Even-
tually, the constrictions become complete and deepen, with a concomitant bulging
out of the body wall in the region of the segment rudiment (segmental stage c).
At this stage, the segment can be considered "laid down," its shape resembling
more or less a short cylinder. In thoracic segments, appendage buds appear in
stage c ventro-laterally, on either side. The segments are considered mature when
their pairs of swimming appendages first become independently motile. Stages a
to c of the first and second, and stages a and b of the third segment can never be
clearly seen; the first stages of these segments are attained prior to hatching and
during the nauplius and metanauplius phases, when the presence of dense yolk
conceals details of structure. As these segments become plainly visible in the
third stage of the thoracic period, segment 3 is in stage c, but segments 1 and 2
are already correspondingly ahead, both in size and the degree of their development.
At the end of the thoracic period the llth segment has reached stage c and the
first five segments have become mature. The llth segment attains maturity at
the end of the abdominal phase of development (stage 19). Appendage buds
similar to those on more anterior segments also develop on segments 12 and 13.
But instead of developing into swimming appendages the buds on either side of
ARTEMIA SEGMENTATION PATTERN 123
both segments enlarge, and in the female fuse into a sac in stage 18, forming the
left and right brood pouch; no male larvae were investigated. The remaining six
segments develop similarly from segment rudiments, but no appendage buds are
ever formed and stage c represents the first stage of maturity. At the end of the
abdominal period the 19th and last segment has become mature. In the head, the
ocellus becomes pigmented in stage 2 and the compound eyes in stage 4. The
maxillae and maxillulae also form in stage 2. The end of stage 19 marks the time
when the gnathobase and the setae have been lost entirely from the second antenna.
After stage 19 an arbitrary number of non-segmental stages ensue before sexual
maturity is reached.
When individuals in identical stages of development from the same or from
different salinity media are compared, it is strikingly apparent that total lengths
and body proportions in general fall within well-defined size-classes ; the deviations
from the underlying averages in no case exceed ± 3 per cent. In Table I the
averages of a variety of body measurements are shown, from the 25 individuals
watched throughout development, with the stage number as the basis of calculation ;
these values, within ± 3 per cent, are true for individuals from any of the salinities
here examined. A schematic diagram of an Artemia larva indicates, in Figure 2,
how the various entities have been defined. Head length is understood to include
maxillar and maxillular segments. The length of a segment refers to axial and
the width to its lateral extent. Total abdominal length is the length of the seg-
mental portion, whether actually cut up into segments or not, plus the length of a
terminating anal piece; the segmental portion is the pygidium of annelid forms,
and in Artemia is readily distinguished from the anal piece, or urosome, by a con-
striction. During the abdominal period of development, the segmental abdomen
contains a genital region composed of segments 12 and 13, as well as a post-
abdomen (presumptive segments 14 to 19) with segmented and non-segmented
portions.
From observation and from examination of the data in Table I the following
facts concerning the formation of segments in relation to larval shape and size are
consistently found to occur :
1. Every thoracic segment when newly formed (stage c) has a fixed length
of 0.03 mm. and a fixed width of 0.144 mm.
2. Every time a new thoracic segment is laid down in stage c, preceding seg-
ments increase in length and in width.
3. Throughout the thoracic period, the segmental part of the abdomen has a
constant average length of 0.249 mm.; its anterior width, being slightly smaller
than the width of the newest segment, is also constant (C = 0.142 mm.).
4. During the thoracic period, the lateral contour-lines of the thorax are straight
lines converging posteriorly; the lateral abdominal contour-lines are also straight,
but generally they converge with a greater degree of taper than the thoracic contours.
5. Appendages are longer the more anterior they are; the line joining the tips
of the appendages on one side of the body is more or less a straight line.
6. As the llth segment appears in stage c, the 5th segment has matured and
the 19th segment has appeared in stage a.
7. Between stage a and stage c of the thoracic segments, 4 stages of larval
development intervene ; an interval of more than 4 developmental stages is neces-
sary for an abdominal segment to reach stage c from stage a.
124
PAUL B. WEISZ
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ARTEMIA SEGMENTATION PATTERN
125
8. Between stage c and maturity in the first 5 segments, 6 stages of develop-
ment intervene; 8 stages intervene before maturity of segments 6 to 11.
9. The anterior width of the non-segmented portion of the abdomen during the
abdominal phase is of a fixed and constant magnitude and identical to the constant
anterior width of the abdomen during the thoracic phase ; thus at the end of stage
wu
FIGURE 2. Schematic diagram of a larva of Artcmia salina. A, larva in the thoracic
period ; B, larva in the post-abdominal period of development. A, length of segmental abdomen
(pygidium) ; a, angle of thoracic taper; /3, angle of abdominal taper; C, Wo, WA0, constant
anterior width of segmental abdomen; GL, length of genital region; H, head length; NSPA,
non-segmented post-abdomen ; P, length of post-abdomen ; PS, PSm, length of a post-abdominal
segment; SPAL, length of segmented post-abdomen; SR, segment rudiment; T, Tn, Tm, length
of thorax ; TAL, total abdominal length ; TS, TSn, length of first thoracic segment ; TSL, Ti,
length of last (newest) thoracic segment: Tot.L, total larval length; U, urosomal length; W,
Wn, width of first thoracic segment; WTSL, Wi, width of last thoracic segment; WA, WAr,
width of first abdominal (12th) segment; WASL, width of last (newest) abdominal segment;
WU, anterior width of urosome.
126 PAUL B. WEISZ
19, the width of the urosome is the same as the posterior width of the head in
stage 0 when segmental development started. A constant width has seemingly
travelled down the larva.
10. At the beginning of stage 12, the segmental abdomen starts to grow in
length at a fast rate, having retained a constant length in the thoracic period.
.11. Segments 12 and 13 are of equal length at any time after their formation;
they are individually always somewhat longer than the llth thoracic segment and
become progressively shorter, relatively, than the 14th segment. At stage 18,
6 developmental stages after segment 12 has reached stage c, segment 12 and 13
fuse to form the brood pouch in the female and can then be considered matured.
The interval for attainment of maturity is thus equal to the similar interval in the
first 5 thoracic segments.
12. Segments 14 to 19 are not of equal length when formed ; more posterior
segments, when formed, are longer than more anterior ones when formed. Also,
any one of these segments has always one-sixth of the length of the post abdomen,
and at a given time post-abdominal segments are of equal length.
13. When segment 19 reaches stage c, the llth segment attains maturity.
14. During the abdominal phase, the thorax changes shape in the following
way: the 2nd segment becomes longer and wider than the 1st, then the 3rd larger
than the 2nd, etc., and the 5th becomes the largest, coincident with the end of
stage 19. As a result, the lateral thoracic contours become curved, the widest part
of the thorax being at segment 2 in stage 16, at segment 3 in stage 17, etc., and at
segment 5 in stage 19.
15. Similar differential increases take place in the appendages; at stage 19,
the 5th pair of swimming appendages is longest and appendageal length regularly
decreases towards the 1st and the llth pair. The line joining the appendage tips
on one side of the thorax is now also curved.
16. During the abdominal phase, and paralleling the differential increases in
the thoracic segments, a progressive dorsal thoracic curvature develops, with an
analogous shift backwards of the maximal flexure ; the latter arrives similarly at
segment 5 at the end of stage 19. Due to this flexure the head now appears bent
ventrad.
17. The lateral contours of the abdomen remain straight lines throughout the
abdominal phase, with a definite taper directed backwards.
18. At the end of stage 19 segmental development is completed ; further devel-
opment is still to take place in the head. The essential overall shape of the animal
as now established, i.e., the possession of a barrel-shaped thorax and a straight
tapering abdomen, is carried through to sexual maturity, although changes of detail
do still occur.
These observations are now to be interpreted and integrated analytically.
The thoracic phase of development
The lengths of the first thoracic segment, in successive stages, are 0.03, 0.04,
0.05, 0.058, 0.065, 0.076 mm., etc. (Table I). The differences between these
values, taken for all 11 thoracic stages, are very close to an average difference of
0.0097 mm. The length of the first thoracic segment in successive stages can
ARTEMIA SEGMENTATION PATTERN 127
therefore be expressed as an arithmetical series
TSn = (TSi -- TS0) + (n-- l)-Aj (1)
where TSn refers to the length of the first thoracic segment at stage n; n, to the
successive stage numbers from 1 to 11 (and thus to the number of segments
present at the time) ; (TS\ — TSo), to the initial length of the first segment at
the end of stage 1 ; and As, to the increase of segmental length per stage (0.0097
mm.). The expression would mean that the first segment grows in length by a
constant amount As during each stage.
Since every other thoracic segment is known to start off with an identical value
for (TSi — TSo), viz., 0.03 mm., it could be possible that other thoracic segments
also increase a constant amount As during each stage. If that were true, then the
newest segment, at any given stage, would have a length of (TSi -• TSQ}, the
segment immediately preceding it a length of (TSi -- TSo) + As, the third but
last a length of (TSi -• TS0) + 2As, . . . etc., and the first segment again a
length of [(7\S"i -- TSo) + (n -- l)-As]. In other words the length of the entire
thorax, being the sum of individual segments, should be the sum of an arithmetical
series whose first term is (TSi -• TS0) and whose last term is [(TSi — T5"0)
+ (n -- l)-As], This can be put as
Tn = n- (TS, - TS0) + -As (2)
where Tn is the total thoracic length at stage n; and (TSi -• TSQ), the constant
length of the newest segment (or the length of the first segment when in stage c).
Taking As as 0.0097 mm. and (TSi -• TS0) as 0.03 mm., Tn for successive values
of n can be calculated. These calculated values are compared with the observed
values for thoracic length in Table II ; the largest discrepancy is only approximately
5 per cent, and the original suggestion is thus shown to be fact, i.e., every thoracic
segment grows in length for a constant amount As, in each stage of the thoracic
period.
A similar approach can be employed to analyze thoracic changes in width.
While in stage 0 thoracic length Tn is also 0, the anterior width of the presumptive
thorax is already 0.142 mm. (C). In stage 1, the width of the first segment is
0.144 mm. (Table I), and the initial increase (W\--C), analogous to (TS\
• TSo) in equations 1 and 2, is therefore 0.002 mm. ; the anterior width of the
presumptive second segment is again C = 0.142 mm. (each segment after stage c
being regarded as a short cylinder). In succeeding stages, the width of the first
segment increases 0.002, 0.004, 0.005 mm. . . . etc. (Table I). The increments
per stage are then not constant, as they were for segmental length, but the figures
suggest that the increases of the increments per stage might be constant. If the
increment in stage 2 were 0.003 instead of 0.002 mm., the increase Azy over the
initial increment (W\ — C) would be 0.001, and (W\ -- C) + Aw would repre-
sent the increase in width during stage 2. Similarly (W\ — C) + 2Aw and
(W\ — C) + 3 Aw would indicate the increases during stages 3 and 4 respectively.
In general,
(Wn - Wn^ = (Wi - C) + (n - 1) -Aw (3)
128
PAUL B. WEISZ
would be true, where (Wn~ Wn-i) represents the increase in width of the first
segment during stage n. The total width increase of the first segment during the
first n stages would then be the sum of an arithmetical series whose first term is
(Wi — C) and whose last term is [(Wi -- C) + (n -- 1) -Aw], for similar reasons
as in thoracic length ; or
and
n(n -- 1)
n(n -• 1)
Aw
(4)
(5)
TABLE II
Calculated and observed magnitudes of certain larval body regions, in millimeters
Thoracic length
Width of 1st thoracic segment
Observed
Calculated
Observed
Calculated
. 1
0.030
0.030
0.144
0.144
2
0.070
0.069
0.146
0.147
3
0.122
0.119
0.150
0.151
4
0.185
0.178
0.155
, 0.156
5
0.242
0.247
0.162
0.162
6
0.311
0.325
0.167
0.169
7
0.390
0.413
0.179
0.177
8
0.480
0.511
0.191
0.186
9
0.600
0.619
0.209
0.196
10
0.733
0.736
0.225
0.207
11
0.861
0.863
0.245
0.220
Length of post-abdomen
Length of a post-abdominal
segment
Width of 12th segment
Observed
Calculated
Observed
Calculated
Observed
Calculated
12
(0.29)
(0.28)
(0.041)
(0.04)
0.150
0.147
13
(0.325)
(0.30)
(0.054)
(0.05)
0.160
0.154
14
0.38
0.36
0.06
0.06
0.170
0.163
15
0.45
0.45
0.08
0.075
0.185
0.174
16
0.58
0.57
0.09
0.095
0.190
0.187
17
0.73
0.72
0.12
0.12
0.200
0.202
18
0.91
0.90
0.15
0.15
0.210
0.219
19
1.13
1.1-1-
0.18
0.185
0.230
0.238
Taking for (Wi — C) and Aw the values 0.002 and 0.001 mm. respectively, Wn
has been calculated for successive values of n, and the comparison with the observed
values is shown in Table II. The percentage discrepancies are greater than those
observed for thoracic length, but nevertheless insignificant in view of the greater
difficulty of taking accurate measurements of entities of so much smaller magni-
tude. It is to be concluded that the width of the first thoracic segment grows
similarly as the length of the thorax, i.e., by adding, in each stage, another term
ARTEMIA SEGMENTATION PATTERN 129
of an arithmetical series in which consecutive terms differ by a constant amount Aw.
It must now be shown that other thoracic segments also increase in width
according to equations 4 and 5 ; actual measurements for these segments have not
been taken, but the proof can be arrived at indirectly. It is known from observa-
tion that the lateral thoracic contours are straight lines converging posteriorly. The
angle of taper a (Fig. 2) is always expressed by
Wn-C
tan a = ?T (6)
LL „
and this angle, on calculation, is seen to be very nearly constant for successive
values of n. For n—\ and n — 11, tan a equals 0.033 and 0.045 respectively;
the average from all eleven values is 0.039, corresponding to an angle of 2° 18',
± 15'. Since the contours are then straight lines, with a constant taper in all
thoracic stages, the taper of individual segments must also be constant and identical,
i.e., (IV n -- Wn-\)/2TSn .', as the length TSV of a given segment in a given stage
can be shown to be equal to the length, in the preceding stage, of the segment
immediately anterior to it, an analogous equality must obtain for the width of a
segment, for the taper in each case must be identical. In other words, when the
width of the first segment is Wn, the width of the succeeding segment is Wn_\,
in the same stage; this proves however, by extension, that all thoracic segments
must increase in a manner identical to the first, since Wn and Wn-\ represent sums
of the same arithmetical series as that in equation 5, Wn containing one term more
than Wn-i-
The segmental abdomen during the thoracic phase maintains a constant length
(A = 0.249 mm.) and a constant anterior width (C = 0.142 mm.). The posterior
width W U, identical to the "width of the urosome," however increases (Table I).
The angle of taper ft, therefore, expressed by
C -
~~
2A '-
decreases. Stated in other words, the convergence of the abdominal contour-lines
gradually diminishes. A stage will eventually be reached at which the thoracic
and abdominal contours will form continuous straight lines, the thoracic contours
having a constant taper (equation 6) ; at this time
tan a = tan ft
and
Wn-C C - WUn
(8)
2Tn 2A
from which WUn can be calculated, all other terms being known. WUn from
equation (8) is 0.123 mm.; the value of WUn closest to this in Table I is 0.125
mm. in stage 11. It follows therefore that the thoracic and abdominal contours
become continuous straight lines as the end of the thoracic period of development
is reached.
For analytical purposes thoracic shape during the thoracic period can be re-
garded as a regular cone from which the tip was cut off (frustrum of a cone).
Dorso-ventral extent at any level would be very nearly equal to the lateral width
130 PAUL B. WEISZ
at that level. The diameters of the end faces of the frustrum can thus be assumed
to be Wn and W-L respectively, and since the length of the frustrum is always given
by Tn, the volume and the surface area of the thorax can be approximated by the
use of known geometrical formulae. If the volume V\ of the first segment is
known the total thoracic volume Vn at any stage can also be calculated from a
sum-of-a-series equation, of the general form
which must obtain, since both length and width changes are governed by such
equations. Furthermore, As and Aw are obviously related mathematically to
Av. In sum, if the initial size and shape of the thorax (n— 1), and the .values
As and Aw are known, the size and shape of the thorax at any further thoracic
stage can be predicted.
The abdominal phase of development
Abdominal growth. — At the beginning of the abdominal period, the segmental
abdomen starts to grow in length, having been constant before. During stages 12
and 13 the abdominal increases are 0.08 mm. per stage, or almost exactly 8 X As
(Table I) ; since the initial abdominal length at the beginning of stage 12 (or at
the end of stage 11) is 0.249 mm. or approximately 8 X 0.03 m., it follows that during
the genital period each 0.03 mm. portion of the segmental abdomen grows an
amount As per stage. In other words, the segmental abdomen behaves as though
it were already cut up into its eight segments, and each of these hypothetical seg-
ments has the same antero-posterior growth potential as thoracic segments when
first laid down, viz., increasing As per stage after having a length of 0.03 mm. If
the 12th segment were laid down in the manner in which thoracic segments are
formed, it would reach stage c at a length of 0.03 mm. But after stage 11 the
entire segmental abdomen has already started to grow, at a rate of As per stage
per 0.03 mm. Thus at the end of stage 12 when the 12th segment reaches stage c,
it will be 0.03 + As, or 0.04 mm. instead of 0.03 mm. long ; the entire segmental
abdomen should then be eight times 0.04, or 0.32 mm., and the post-abdomen 0.28
mm. long. Analogously during stage 13, each 0.04 mm. portion of the segmental
abdomen will now add an amount As, so that segment 13 when in stage c will be
0.05 mm. and the entire segmental abdomen eight times 0.05, or 0.40 mm. long.
At this point the genital region should be 0.10 (2 X 0.05) mm. and the post-abdomen
0.30 mm. long. Actual figures in Table I support such an interpretation rather
well, and the conclusion is justified that during the genital period the segmental
tissue of the abdomen acquires the same growth potential in length as that of
equivalent amounts of thoracic tissue during the thoracic period.
The genital region continues to grow in length at the indicated rate, as the data
in Table I tend to show. The post-abdomen would similarly do so, were it not
for the fact that another change in the mode of growth occurred at the end of stage
13. Successive post-abdominal lengths Pm from stage 13 on are 0.325, 0.38, 0.45,
0.58 mm. etc., in other words the increments are increasing. A sum-of-a-series
expression, similar to that for thoracic width changes, fits these figures very
ARTEMIA SEGMENTATION PATTERN 131
closely, i.e.,
Pm - Fo = (Pi - Fo) -m + m(m~ 1} • A/> ( (10)
and
where Pm represents the total post-abdominal length for stages 14 to 19 ; PQ, the
initial length at the end of stage 13; PI, the length at the end of stage 14; m, the
successive integers from 1 to 6; and A/>, the increments per stage over the initial
increase (Pi — P0). The theoretical value for P0 was previously seen to be 0.30
mm., and with 0.06 and 0.03 for (Px — P0) and A/? respectively, the calculated
values for Pm compare well with the observed ones (Table II).
If equation (11) is divided by six, the growth formula for individual segments
is obtained, since each of these segments is one-sixth of the entire post-abdomen;
+ (PS, - PS0) -m + • A(/>) (12)
PSo, PSi, and A(/>) are 0.05, 0.06 and 0.005 mm. respectively, and (PSl - PS0)
is therefore 0.01, or very closely As; thus the initial increase of the presumptive
segments 14 to 19, at the beginning of the post-abdominal period, is identical to
the increase of these tissues during stages 12 and 13, and this increment is then
augmented by a constant amount A(/>) in each subsequent stage. What is re-
sponsible for this change in the mode of growth of post-abdominal segments? It
is more than likely that non-formation of appendages is related to this, inasmuch
as newly formed tissue will not be diverted for the establishment and subsequent
growth of appendage buds ; augmented growth of the segments would therefore
be facilitated. It can now be stated in general, that while body segments are
formed, length increments per stage for all segments are constant, but the incre-
ments may be added to an initial length as in thoracic and genital segments, or to
an initial increase of length, as in post-abdominal segments.
As in thoracic segmentation, the anterior width of the segmental abdomen has
the constant value C = 0.142 mm., during the abdominal period. This value is
the anterior abdominal width at the end of stage 11, and the anterior width of the
presumptive 13th segment at the end of stage 12. The 12th segment, by this time,
has attained a width of 0.15 mm. (Table I), and in succeeding stages this width
increases to 0.16, 0.17, 0.185 mm. . . . etc. As for thoracic width the increases
are found not to be uniformly constant, and a sum-of-a-series expression again
approaches the data best, i.e.,
WAr-C --r-(WAi-C} +2 "Awa (13)
and
J^r = C + r(0Mi - C) + r(r7 n-A7C'a (14)
where WAr represents the width of the 12th segment at a stage r of the abdominal
period; (WA\ — C), the initial increase in width during stage 12; Awa, the in-
crease in width, per stage, over the increment during the preceding stage; and r,
132 PAUL B. WEISZ
the successive integers from 1 to 8. If for (IVAi — C) and Awa 0.005 and
0.002 mm. respectively are taken, the calculated values for WAr compare well
with the observed ones (Table II).
Other abdominal segments can be shown to follow a similar mode of growth
in width. The lateral contours being straight lines, the angle of taper /? is
expressed by
WAr - WUr
where Ar is the length of the entire segmental abdomen, i.e., genital plus post-
abdominal lengths, and other values as before. Tan /?, when calculated from
Table I for successive values of r, centers about the average of 0.036 ± 0.004 ; in
other words, the abdominal taper does not only remain constant during the ab-
dominal period, but this taper is also practically identical with that reached by the
segmental abdomen at the end of stage 11 (cf. above, equation 8).
Unlike thoracic segments, which start development at stage c with the same
length as that of more anterior segments at stage c, the abdominal segments begin
development at a length identical with that of more anterior segments at the same
time. In maintaining a constant taper, the initial increase of any presumptive
thoracic segment over the width C is always expressed by the first term of the
series applying to thoracic width (equations 3, 4, and 5), and the later a segment
arises the fewer terms of the series can it add to its width during the thoracic period.
Since abdominal segments have now also been shown to maintain a constant taper,
and since their lengths at stage c are equal to those of more anterior segments
already beyond stage c, an analogous relation must similarly exist for segmental
width ; namely, the initial increase of a presumptive abdominal segment over the
width C must be identical to the width increase experienced by other abdominal
segments at the same time. If (WA]_ -- C) in equation (13) represents the initial
increase of segment 12, then (WA2 — WA\) would do similarly for segment 13.
In other words, the width of both segments follow the same series, but the second
term for segment 12 becomes the first term for segment 13; the third term for
segment 12, similarly, becomes the first term for segment 14, etc., and the eighth
and last term for segment 12 is the first and last term for segment 19. Thus as
with thoracic segments, the later an abdominal segment arises the fewer terms are
added to its width, but while the width increases of thoracic segments start with
the same and end with consecutive terms, those of abdominal segments start with
consecutive and end with the same terms of the series.
It should be observed parenthetically that equation (13) may have a slightly
different constant Awa for the genital and post-abdominal segments respectively,
reflecting the different modes of growth in length of these two groups of segments ;
or, if the constant is actually identical the lateral abdominal contour would theo-
retically not be an exact continuous straight line, but rather two straight lines with
slightly different taper, joined between segments 13 and 14. In either case, the
difference would be so small as to be unnoticeable in practice ; with the present
techniques of observation and measurement, a single series relation holds for both
groups of segments, and even if two separate series could be established with finer
means, the principle of growth in width as outlined above would nevertheless hold.
As for segmental growth in length, a general conclusion can now be stated for
ARTEMIA SEGMENTATION PATTERN 133
growth in width, viz., width increments per stage for all body segments are con-
stant, and the increments are always added to an initial increase in width. The
combined generalization is also true, that total segmental mass increments per stage
are constant, and the increments are added either to initial masses or to initial
increases of mass.
Thoracic growth. — One of four possible reasons could a priori be advanced
in an attempt to account for the differential size changes in the thorax, such that
the 5th segment ultimately becomes largest, during the abdominal period: i.e.,
either the segment rudiments in stage a differ in initial size but follow the same
growth curves ; or the analogous converse ; or either the rudiments have both equal
initial size and identical growth curves ; or the analogous negative. Since for all
thoracic segments four stage-intervals elapse between stage a and stage c, length
and width magnitudes at stage c are identical, and the increments per stage, no
matter at which segmental stage, are identical (i.e., As), only the conclusion is
admissible that thoracic segment rudiments have equal initial sizes and follow
growth curves of the same shape. Under such conditions there are two factors
which must be held responsible for the observed growth of thoracic segments, i.e.,
the time lag in the formation of consecutive segments, and segmental age. The
time lag fully accounts for the regular gradation of segmental sizes at the end of
the thoracic period and for the constant taper of the thorax ; as will be demon-
strated below, the influence of this original time lag carries over importantly into
the abdominal phase, and this, together with the factor of segmental age, can indeed
be made the basis for a consistent interpretation of the manner of thoracic growth.
Data in Table I show that thoracic length remains constant during the genital
phase. Hereafter the values for length fit the equation
r/-r-i / T-> T \ 1% \ 1H 1 / i f 1 S- \
m-: Tm_! + (7\ - • T0)m - -^- - As (16)
where T0 and 7\ represent thoracic length at the end of stage 11 and stage 14
respectively, and m, as before, the integers from 1 to 6. With 0.86 and 0.92 mm.
for TO and 7\, and As as before, successive calculated values for T,,, are 0.92, 1.03,
1.18, 1.36, 1.56, and 1.77 mm., significantly close to the observed data; the in-
creases per stage are therefore 0.06, 0.11, 0.15, 0.18, 0.20, and 0.21 mm., and the
differences between the increases are seen to diminish in a regular manner.
The scheme in Table III will account for such a series of increases. The
figures in this table represent multiples of As and they show the length increase
of the indicated segment during the indicated stage. Sums of figures in vertical
rows, multiplied by As, indicate the increases of the entire thorax during the given
stages, and successive sums are seen to be equal to the values for the increases per
stage as calculated from equation (16). Horizontal sums, multiplied by As, give
the total increments of any thoracic segment during the post-abdominal period.
This scheme is reproduced somewhat differently in Table IV, in which the figures,
multiplied by As, indicate directly the size of any of the 19 body segments at any
of the 19 developmental stages ; vertical sums have meanings analogous to equiva •
lent sums in Table III.
It will be observed that all formulae previously deduced in connection with
length increases are inherent in the figures in Table IV ; observational data are
134
PAUL B. WEISZ
also incorporated. For example, the first segment when reaching maturity in
stage 7 has a length of 0.09 mm. (cf. Tahle I). Succeeding thoracic segments
must also mature at this size, in consequence to the equality of their growth curves ;
thus segment 5 is shown to mature in stage 11 when the 19th segment appears in
stage a, and segment 11 in stage 19, in conformity to the observational data in
Table I. The scheme in Table IV also shows well the successive segmental pro-
portions in the thorax during the abdominal phase. In stage 16, segments 1 and 2
are longest, in stage 17 similarly segments 2 and 3, etc. ; maximal segmental length
thus shifts caudad, fully corroborating observation.
Segmental -growth of the thorax as indicated in the table can be interpreted
provided two assumptions are postulated, i.e., (a) a segment can no longer grow
by regularly increasing amounts after having passed through 14 segmental stages,
TABLE III
Scheme of segmental increments, in multiples of As, in the thorax during the post-abdominal
phase of development
Stage
Total
14
15
16
17
18
19
increases
Segment
1
0
1
1
1
1
1
5
2
0
1
2
2
2
2
9
3
0
1
2
3
3
3
12
4
0
1
2
3
4
4
14
5
0
1
2
3
4
5
15
6
1
1
1
1
1
1
6
7
1
1
1
1
1
1
6
8
1
1
1
1
1
1
6
9
1
1
1
1
1
1
6
10
1
1
1
1
1
1
6
11
1
1
1
1
1
1
6
Total
thoracic
increases
6
11
15
18
20
21
counted from stage c, and (b) a segment, in order to grow by increasing amounts
at all, must have matured within the first 6 segmental stages of its existence,
counted from stage r. These two provisions constitute the limiting conditions of
segmental age.
Table IV reveals that only the first 5 segments fulfill the second condition ;
segments 6 to 1 1 would also have matured in 6 stages of their individual existence,
were it not for the fact that no thoracic growth takes place during the genital period,
and maturation of the posterior thoracic segments is therefore delayed by two
stages. Thus only the first five segments would be able to grow by increasing
amounts, whenever such growth was made possible. It has been shown previously
that at the beginning of the post-abdominal phase, the post-abdomen ceases to grow
by constant increments and begins growth by increasing increments, with an initial
ARTEMIA SEGMENTATION PATTERN
135
increase during stage 14 equal to that of stage 13. Apparently the phenomenon of
increasing increments at this time is not confined to the post-abdomen but also
affects thoracic segments, subject to the limiting provisions stated above. Thus
the first five segments have an initial increase equal to the increment during stage
13, viz., 0; segments 6 to 11, not fulfilling condition (b), simply continue at their
former constant rates, viz., As per stage (cf. data in Table III, under increases
during stage 14). From here on, the first five segments augment their increases
by As in every stage, until their 14th segmental stage is passed ; then, by assump-
tion, the increment of the 14th segmental stage can no longer be augmented, but
TABLE IV
Scheme of growth of body segments, in multiples of A.v
(a refers to segmental stage a of any given segment)
Stage
•
Thoracic phase
Genital
phase
Post-abdominal
phase
1234567
8
9
10
11 12 13
14
15
16
17
18
19
Seg-
ment
1
3456789
10
11
12
13
13
14
15
16
17
18
2
345678
9
10
11
12
12
13
15
17
19
21
3
34567
8
9
10
11
11
12
14
17
20
23
4
3456
7
8
9
10
10
11
13
16
20
24
5
a 345
6
7
8
9
9
10
12
15
19
24
6
a 34
5
6
7
8
9
10
11
12
13
14
7
a 3
4
5
6
7
8
9
10
11
12
13
8
a
3
4
5
6
7
8
9
10
11
12
9
a
3
4. 5
6
7
8
9
10
11
10
a
3
4
5
6
7
8
9
10
11
a
3
4
5
6
7
8
9
12
(a)
4 5
6
7
8
9
10
11
13
a
5
6
7
8
9
10
11
14
(a)
6
7.5
9.5
12
15
18.5
15
a
7.5
9.5
12
15
18.5
16
(a)
9.5
12
15
18.5
17
a
12
15
18.5
18
(a)
15
18.5
19
a
18.5
is retained as a constant increment till growth stops altogether. Thus segment
one has increased its increment of zero by As at the end of stage 15 (Tables III
and IV) ; but at this point its 14th segmental stage has already been passed and
hereafter only a-constant increment of As per stage is possible. Segment 2 on the
other hand is younger than segment one, being laid down in stage c with a time
lag of one developmental stage. By the end of stage 15, therefore, when segment
one has just passed its 14th segmental stage, segment 2 has only passed its 13th
segmental stage and its increment of As during stage 15 can be augmented once
more by As; when the 2nd segment has passed its 14th segmental stage, its in-
136 PAUL B. WEISZ
creases in subsequent stages will therefore be 2 As per stage. Similarly, segments
3, 4, and 5, each being one stage younger than the preceding segment, are able to
augment their increments by As 3, 4, and 5 times respectively, before they com-
plete the 14th segmental stage. Segment 5 in consequence is as long as segment
4 at the end of stage 19, but the former will continue to grow at a rate of 5 As
per stage, while the rate of the latter can only be 4 As per stage ; at any time after
stage 19 therefore the fifth segment will be longest.
Analogous changes occur with regard to thoracic growth in width, and the
thoracic cone-frustrum of stage 11 gradually assumes the shape of a barrel, with
the "waist" at segment 5 after stage 19. The dorsal thoracic curvature of the
animal, arising similarly after stage 11, can also be interpreted as a result of
differential segmental increases in a dorsal direction, according to a scheme resem-
bling that in Table III.
The genital segments have been noted to mature, i.e., to form a broodpouch, in
stage 18. Table IV reveals that at the end of this stage, segment 12 has just com-
pleted its 6th, and segment 13, its 5th stage of segmental development, counted
from stage c. Thus both segments fulfill one of the two age conditions assumed
for thoracic segments ; the fulfillment of the other might be expected. Observa-
tion proves that this is actually so. Genital segments of older larvae are known
to bulge considerably beyond the general abdominal contour, giving them a knobby
appearance. This could not be possible if the constant increases observed up to
stage 18 were maintained any further; rather, after stage 18 the initial increment
of an increasing rate will again be equal to the increase during the stage just
passed, viz., As, and during a 20th stage this increment will be augmented by a
given amount, during a 21st stage by twice this amount, etc., till the 14th seg-
mental stage is passed.
The post-abdominal segments have previously been shown to grow by regu-
larly augmented increases as soon as they afe laid down. But since these segments
bear no appendages, stage c for them is equivalent to attainment of maturity, as
already observed above. Maturity thus proves to be an important temporal
threshold for all body segments, and the statement that augmented growth will
occur in any mature segment, provided maturity was reached in a definite time,
has general application ; the concept of segmental maturity is apparently not only
a working hypothesis, as has been assumed at the start, but seems to have real
biological meaning.
There is no doubt that the scheme of growth here presented describes correctly
the actual events of later thoracic development ; but the assumptions, while justified
by the interpretations they allow, still remain to be explained. Only tissue culture
studies will be able to reveal why segments not matured in the first 6 stages of
existence are at too early a stage of development, and why segments after 14 stages
of existence are at too advanced a stage to do more than keep up a constant rate.
Growth of appendages; integration oj segmental development
In the preceding section it has been reasoned that segment rudiments in the
thorax have equal initial size and identical growth curves; observation tends to
confirm not only this but also that equal-sized rudiments develop for all body
segments. It can be assumed that in these rudiments certain tissue masses (ap-
ARTEMIA SEGMENTATION PATTERN 137
pendage rudiments), initially also of equal size and of equal growth capacity in
equal times, differentiate independently towards the establishment of appendage
buds. Such buds however never appear in the post-abdomen, and when they
appear in other regions they may develop into swimming appendages or into a
broodpouch. In the evidence presented in Tables I and IV, an important clue
can be found to at least one of the factors preventing serial analogy despite the
observed serial homology in appendageal development.
Every thoracic appendage rudiment reaches the bud stage after an interval of
four developmental stages. The segment as a whole is at stage c at this point,
and the appendage buds of any thoracic segment must be of identical size, due to
the identity of initial size and of growth capacity for all appendage rudiments, and
of identical shape, since every thoracic segment at stage c has identical propor-
tions. Enough appendageal tissue has apparently been manufactured, during the
four preceding stages, to initiate the development of a swimming appendage.
When a genital segment reaches stage c, 4 + and 5 developmental stages have
elapsed since stage a. The appendage rudiments therefore have time to manu-
facture proportionately more appendageal tissue, at the same intensity as that of
thoracic rudiments. If the genital segments in stage c had larger sizes, propor-
tionate to the longer time interval available, the appendage buds of genital segments
would have the same size and shape as those of thoracic segments. However, both
the length and the width of genital segments are greater in stage c than the size
which would be proportionate to the longer time of formation. The length of
any thoracic segment when laid down is 0.03 mm., for example, and four develop-
mental stages have elapsed since stage a ; the length/time ratio is thus 0.03/4. In
genital segments this ratio is larger, viz., 0.04/4 + and 0.05/5, and analogously for
width. Appendageal tissue in genital segments can therefore not be developed in
sufficient quantity, in proportion to segmental size, to produce appendage buds of
dimensions equal to those of thoracic buds, even though more time is available.
Genital buds will thus be relatively smaller and flatter, and the amount of appenda-
geal tissue manufactured will be spread more thinly over the presumptive appen-
dage region ; the quantity of tissue present per unit area is apparently already
below the threshold necessary for the formation of comparatively specialized
swimming appendages, and only enough tissue is available to initiate the formation
of a relatively simple sac.
In post-abdominal segments at stage c the size/time ratio becomes progressively
larger still, and appendageal tissue consequently cannot even accumulate in quanti-
ties sufficient to form a bud.
After the appendage buds are laid down, an appendage retains a definite size-
proportionality to the segment bearing it. When, for example, the thoracic con-
tour is a straight line, during the thoracic phase of development, the line joining
the tips of the appendages on one side is also a straight line, and, as with the
segments themselves, the time lag in bud formation accounts for the taper. Simi-
larly, as the thorax gradually becomes barrel-shaped in the abdominal phase, the
transformation is reflected in differential length increases in the appendages, and
when the appendageal tips on one side of the body are joined by a line, the result
is an analogously barrel-shaped contour.
From the above analyses, the following integrated sequence of events becomes
apparent with regard to segmental development.
138 PAUL B. WEISZ
Shortly before hatching segmental rudiments of equal size begin to be formed,
at a rate of one per developmental stage ; with a time lag of four stages, segments
are constricted off in posterior succession, all with constant initial sizes and in-
creasing by a constant amount during each stage. As the first segment reaches
maturity, the rate of segment rudiment formation increases to two per stage.
Rudiments are laid down at this rate till the newest rudiment appears at the
posterior end of the segmental abdomen which latter had so 'far maintained a
constant length. The last formed rudiment happens to be the 19th and by this
time, 11 segments have been constricted off, five of which have already matured.
The process of rudiment deposition and segment constriction could be assumed
to go on at length, were it not for the fact that the "end" of the animal has been
reached. This is apparently the cue for a general change in the mode of growth.
The entire segmental abdomen begins growth, increasing as yet equal amounts
per stage, and the thorax ceases to grow. After two genital segments of equal
size are formed another general change occurs* to the effect that hereafter any
segment maturing within a definite time may grow by augmented increases, as
described in detail above. This type of growth is maintained, in each segment in
which it takes place until the 14th segmental stage is passed, whereupon the total
increment of the 14th stage is reproduced without further increase in each suc-
ceeding stage. Segments not matured within the required time continue to grow
by constant increments. The eventual result of this varied manner of growth,
maintained up to sexual maturity, is the barrel-shape of the thorax, the presence
of a dorsal thoracic curvature, the knobby appearance of the broodpouch seg-
ments, etc.
DISCUSSION
Throughout the present analysis of metamerism in Artemia salina, the time
scale employed was that of developmental stages, defined as the number of body
segments present. It must be eminently realized that this is a scale of relative,
biological time. Events in nature take place in a space-time continuum, and to
Artemia equivalent happenings in space, i.e., the establishment of segments, must
be correlated to the passage of equivalent units of (relative) time, i.e., what here
had been called "stages." In hours and minutes, segment formation occurs of
course not in equivalent times, since the phenomenon is dependent on the environ-
ment on the one hand, and on changes in growth rates with age on the other.
Artemia and other similarly primitive forms are particularly suited for a ready
identification of relative time, but in segmented animals of greater complexity, as
well as in non-segmented groups, "equivalent happenings in space" cannot be picked
out with comparative ease, and it will be more difficult to tell what the relative time
scale actually is ; but that it is intrinsically present in biological phenomena has
already been acknowledged by others. Thus Needham (1942), after briefly re-
viewing the pertinent literature, states :
"Mouse time must bear the same, or a similar, relation to elephant time as
mouse spatial magnitudes to elephant spatial magnitudes. Indeed, unless the
time factor is brought into account, we may understand morphological similarity,
but we can never hope to understand physiological, still less embryological,
similarity."
ARTEMIA SEGMENTATION PATTERN 139
t
Measurements on Artemia in absolute time would never have brought to light
the truly amazing simplicity of the laws of segment formation, as given by the series
and the sum-of-series formulae, and in terms of relative time these formulae as-
sume a simple biological meaning, viz., (a) that equivalent spatial events take
place during equivalent relative times, and also (b) that equivalent spatial events
take place in tissues of equivalent relative age. For illustration, the thorax during
the thoracic period of development may be considered, where the increments per
stage of (TSi -- TS0) and (W\ -- C) (equations 1 and 3) are indeed equivalent
and constant, and where every other segment grows similarly in this same manner ;
summation of the increments must then result in the sum-of-series expression.
Analogous interpretations, based on the idea of spatial and temporal equivalence
can be adduced in every other case in which the formulae hold, i.e., virtually for
the entire period of segmental development. Before and after this period, relative
time is of course still operative, but its expression is latent, inasmuch as its passage
is not paralleled by morphological events clearly identified as equivalent. The
same would be true for the majority of living organisms, but it can be asserted
with a fair amount of logical conviction, that if and when it will be possible to
make explicit the relative time scales of living organisms as a whole, size incre-
ments in relative time units will be found to be equivalent, and series formulae of
linear, quadratic, and perhaps even of higher degree will be found to hold.
It should in general be useful to have a specific term to distinguish relative
biological time from absolute duration ; the concept as a whole might be called
"biochronism," and the relative time scale could be said to have one "biochron"
as its unit. Also, in order to transcend the usual connotations of "growth rate,"
"biochronal rate" could be substituted. Whenever in the text above "increase per
developmental stage" was mentioned, "increase per biochron" was really implied.
In this connection, the type of analysis in the present report is clearly different
from "allometric," "heterauxetic," or "heterogonic" inquiries. The term "mor-
phometry" is suggested to indicate generally any quantitative appreciation of or-
ganic size, shape, and time as an integrated dynamic pattern. While it is realized
that apologies are in order, more or less categorically, for the introduction of any
new term into present-day biology, it should be kept in mind that new terms become
unavoidable as different methods of inquiry and fresh fields of study appear.
Two immediate issues have not been touched on at all in the present analysis.
First, what determines the changes in the mode of segmental growth at the end
of both the thoracic and the genital periods? That the changes occur is fairly
definitely established, and this would support the view that division of segmental
development into periods is real, i.e., physiological as well as morphological. But
beyond that, speculation into the nature and history of the changes would be futile,
for lack of direct evidence. Secondly, and this is the fundamental question in the
study of metamerism, why are segments formed at all? It will readily be ad-
mitted that even an attempt to answer this problem can only be made after a great
deal more is known about segmentation phenomena as a whole.
Excepting these two questions however, the final size and shape of Artemia
nevertheless has here been accounted for in terms of initial body proportions much
as Berrill (1941) has done for the ascidian, Botryllus. When copepods, crayfish,
and other diverse crustacean forms oE higher evolutionary rank are considered,
similarly in possession of a barrel-shaped thorax and a straight tapering abdomen,
140 PAUL B. WEISZ
it is perhaps justifiable to reflect that Crustacea as a group might have evolved
with a single and basic geometrical pattern of growth.
SUMMARY
1. Growth and the dynamic pattern of segment formation in excysted larvae of
Artemia salina have been quantitatively studied. The final shape of Artemia at
sexual maturity can be accounted for in terms of initial shape at hatching.
2. In analyzing the pattern of metamerism, the stages of development are
gauged by the number of body segments present. Growth during the entire period
of segment formation is found to be governed by arithmetical series and sum-of-
series relations, implying that growth increments per stage over either initial sizes
or initial increases are constant and identical for thoracic, genital, and abdominal
segments, respectively. Later transformations of larval shape, resulting in the
barrel-shape of the thorax, the presence of a dorsal thoracic curvature, the knobby
appearance of the genital segments, and the presence of a straight tapering ab-
domen, are accounted for analytically on the basis of concepts concerning the age
of segments and the time lag involved in segment formation.
3. The presence, absence, and the difference of structure of appendages are
shown to be determined, at least in part, by the size of segments when first laid
down, and by the time available for appendage rudiments to form appendageal
tissues..
4. The time scale employed in the analysis of the segmentation pattern in
Artemia is interpreted to be a relative, biological one, and the meaning of the series
formulae with regard to this relative scale is illustrated. The notion of "bio-
chronism" is introduced, as a general concept applying to biological events in
relative time.
LITERATURE CITED
BARIGOZZI, C, 1939. La biologia di Artemia salina Leach studiata aquario. Atti Soc. Ital.
Sci. Nat., 78 : 137-160.
BERRILL, N. J., 1941. Size and morphogenesis in the bud of Botryllus. Biol. Bull, 80: 185-193.
BOND, R. M., 1932. Observations on Artemia "franciscana" Kellogg, especially on the relation
of environment to morphology. Int. Rev. dcr gcs. Hydrobiol. u. Hydrogr., 28: 117-125.
HEATH, H., 1924. The external development of certain phyllopods. Jour. Morph., 38 : 453-483.
JENNINGS, R. H. AND D. M. WHITAKER, 1941. The effect of salinity upon the rate of excyst-
ment of Artemia. Biol. Bull, 80: 194-201.
LOCKHEAD, J. H., 1941. Artemia, the brine shrimp. Turtox 'News, 19: 41—45.
NEEDHAM, J., 1942. Biochemistry and morphogenesis. Cambr. Univ. Press, Cambridge, 1942
(p. 561).
RUGH, R., 1941. Experimental embryology. New York Univ. Press, N. Y., 1941 (p. 206).
WARREN, H. S., 1938. The segmental excretory glands of Artemia salina Linn. var. principalis
Simon (the brine shrimp). Jour. Morph., 62: 263-289.
WHITAKER, D. M., 1940. The tolerance of Artemia cysts for cold and high vacuum. Jour.
E.rp. Zoo/. 83 : 391-399.
ELECTRON MICROSCOPE OBSERVATIONS OF THE
TRICHOCYSTS AND CILIA OF PARAMECIUM
M. A. JAKUS AND C. E. HALL
/ V/></r///;<'»/ /if Binliiiiy. Massachusetts Institute of Tcclui/>!o//y, Cambridge, .Massachusetts
In previous publications, electron micrographs have been shown of trichocysts
(Jakus, 1945) and of cilia (Schmitt, Hall, and Jakus, 1943). Recently we have
re-examined both these organelles using the shadow-casting technique of Williams
and Wyckoff (1945). The new technique shows structural detail with improved
clarity and reveals some features not previously visible in specimens prepared in
the conventional manner.
TUNGSTEN FILAMENT
WITH METAL
METAL DEPOSIT
^ COLLODION FILM
"SHADOW"
FIGUKE 1. Diagram uf shadow-casting technique.
The shadow-casting technique is illustrated diagrammatically in Figure 1. A
specimen is placed in a vacuum bell- jar containing a conical tungsten filament in
which are placed some small pieces of a suitable metal such as chromium. When
the filament is raised to a high temperature by the passage of an electric current
the metal evaporates, travelling in straight lines and depositing on the specimen as
indicated. Structures projecting above the surface of the supporting film cast per-
manent shadows to the "leeward" and intercept metal to the "windward." Speci-
mens are then examined in the electron microscope in the usual manner. In posi-
tive prints the shadows appear bright because they represent relatively transparent
regions in the object. It is customary, therefore, to prepare micrographs as nega-
tive prints so that the shadows will appear darker than the background.
TRICHOCYSTS
The structure and properties of the trichocysts of Paramecium have been de-
scribed in a previous paper (Jakus, 1945). In electron micrographs, the discharged
trichocyst consists of a sharply-pointed tip and an elongated, cross-striated shaft
with a periodicity of about 550 A. The cross-striated structure appears to be a
141
142 M. A. JAKUS AND C. E. HALL
thin membrane formed by the lateral aggregation of fine fibrils. The tip, in con-
trast to the shaft, is quite opaque. The reason for this opacity was not obvious.
Further information about the morphology of the dried extruded trichocyst is
obtained from electron micrographs of shadowed specimens (Fig. 2). The tip is seen
to be a compact structure which stands up from the film and is not flattened to any
great extent as a result of dehydration. The contour of its shadow indicates that
it is shaped somewhat like a golf tee. In contrast to the tip, the dried shaft is very
flat, as is evident from the short shadow it casts. The cross striations previously
observed are enhanced by the metal, indicating that the surface has a regularly cor-
rugated contour. The elevated regions correspond to the darker bands in both un-
treated trichocysts and those stained with phosphotungstic acid. Other details of
structure observed previously may also be found in some shadowed trichocysts.
These are the fine longitudinal striations of the shaft membrane and the larger
periodicity (2.200 A) frequently noted along the shaft. The latter may appear
simply as a slight further intensification of every fourth dark band, suggesting that
these ridges have a somewhat higher elevation than do the others.
In some specimens the pointed tip appears regularly cross-striated, if the amount
of metal deposited has not been excessive and the orientation of the tip is approxi-
mately parallel to the direction of deposition. This banding has not been seen in
either stained or unstained specimens and, while it is readily visible in the original
micrographs of shadowed tips, it is not considered to be of sufficient clarity for re-
production. Although relatively constant in any one tip, the spacing varied from
280 to 365 A in the different tips measured and had an average value of about 300 A.
This is to be compared with the average period of about 550 A in the trichocyst
shaft.
>-
CILIA
The cilia of Paramecium are shed quite readily if the cell is injured and both
intact cilia and fragments are observed frequently in preparations of trichocysts.
Each cilium consists of a bundle of fibrils (about eleven in number), extending the
full length of the cilium (Fig. 3). The diameter of the dried fibrils lies between
300 and 500 A. It may be of significance that both the number of fibrils and their
diameter are in close agreement with the corresponding values observed in the sperm
tails of numerous animal forms ( Schmitt, Hall, and Jakus. 1943).
In fixed preparations (for example, with OsO4), the component fibrils usually
adhere to form a compact bundle, while in unfixed cilia they separate to a greater
or lesser extent. They are clearly defined in shadowed specimens. Usually the
separation of fibrils is not complete and they remain in close contact near the end of
the cilium which was attached to the cell. Here they appear sometimes to be joined
into two closely adjacent bundles.
It is not evident what holds the fibrils together in the living cilium. No spiral
sheath similar to that observed in mammalian sperm tails (Schmitt. Hall, and Jakus,
1943) or in Euglena flagella (Brown. 1945) has been seen. If a sheath does exist,
it must be very fragile and easily ruptured. In some cilia, a rather poorly-defined
cross-striation has been noted, particularly in two or more adjacent fibrils. This
striation appears to be unlike that of clearly cross-striated proteins and, if it is not
an inherent periodicity in the fibril, it may represent the remnants of some binding
or enveloping structure.
TRICHOCYSTS AND CILIA
143
FIGURE 2. Trichocysts from Paramecium, shadow-cast with chromium. X 16,000.
FIGURE 3. Ciliuni from Paramecium, shadow-cast with chromium. X 11,000.
144 M. A. JAKUS AND C. E. tJALL
SUMMARY
Electron micrographs of shadow-cast trichocysts of Paramecium show that the
dried trichocyst shaft is flattened on the supporting film, while the pointed tip is
apparently more resistant to collapse on dehydration. Accentuation, by the metal,
of the cross striation previously observed in the shaft indicates that the periodicity
is accompanied by corrugation of the dried surface. A cross striation in the tip is
also visible in some micrographs of shadow-cast specimens. In the few cases where
the periodicity could be measured, the average spacing was about 300 A, as com-
pared to about 550 A for the well-defined shaft striation.
In electron micrographs of shadow-cast specimens of Paramecium cilia, the
component fibrils are seen with greatly increased clarity.
LITERATURE CITED
BROWN, H. P., 1945. On the structure and mechanics of the protozoan flagellum. Ohio Jour.
Science. 45: 247-301.
JAKUS, M. A., 1945. The structure and properties of the trichocysts of Paramecium. Jour.
E.r/>. ZooL. 100: 457-485.
SCHMITT, F. O., C. E. HALL, AND M. A. JAKUS, 1943. The ultrastructure of protoplasmic
fibrils. Biol. Symp.. 10: 261-276.
WILLIAMS, R. C., AND R. W. G. WYCKOFF, 1945. Electron shadow-micrography of virus par-
ticles. Proc. Soc. E.\-p. Hiol. Mcd., 58: 265-270.
HYDROSTATIC PRESSURE EFFECTS UPON THE SPINDLE FIGURE
AND CHROMOSOME MOVEMENT. 11. EXPERIMENTS ON
THE MEIOTIC DIVISIONS OF TRADESCANTIA
POLLEN MOTHER CELLS
DANIEL C. PEASE
Department of Anatomy, the Medical School, the University of Southern California,
Los Angeles, California
INTRODUCTION
Hydrostatic pressure increments are known to reduce progressively the rigidity
of plasmagels and the viscosity of plasmasols. Eventually complete solation results.
Marsland (1939 and 1942) has been able to formulate what appears to be a gen-
eral quantitative law on the basis of a considerable volume of work with very di-
verse material. He has found that with each increment of 1,000 lbs./in.2 hydro-
static pressure, the relative rigidity or viscosity decreased to 76 per cent of the
initial value. This applied no matter whether the cytoplasm of amoebae, Arbacia
eggs, or Elodca was being studied. Furthermore, these direct effects have always
proved very rapidly reversible when the pressure was released. The subsequent
pattern of cell events, however, has sometimes been found to have been changed by
new reorganization patterns (cf., Pease, 1940, 1941).
In the first paper of this series (Pease, 1941), experiments w7ere reported in
which advantage was taken of these known effects of hydrostatic pressure to study
the first cleavage division spindle apparatus in Urechis eggs. The material was not
well suited for this sort of work, and some interpretations were open to question.
However, the following facts were clear and significant. 1 ) Pressure could so
affect the cell that no trace of the spindle figure appeared in the fixed preparations,
and presumably the spindle had been completely liquified. 2) The pressures de-
stroying the spindle blocked all anaphase movement. 3) The chromosomes ag-
gregated in clumps (originally thought to be vesicles) under lower pressures than
were required to block anaphase movement. 4) Numerous cytasters appeared in
material given a brief recovery period before fixation. 5) Peculiar "half-spindles"
developed de novo within cytasters whenever the latter came in contact with nuclear
material. 6) By their very nature, the half-spindles lacked "continuous fibers"
since only one pole was involved, and also there were no "interzonal fibers." 7)
Yet there was ample evidence that such half-spindles were functional in moving
chromosomes, and even recently-formed nuclei with membranes were at least de-
formed, and* probably moved, by them. The role of cytoplasmic components in the
spindle was stressed (perhaps unduly), and the role of the "traction fibers" mini-
mized (perhaps incorrectly as will be seen later).
To find out whether or not nuclear gels behaved in the same manner as cyto-
plasmic gels when hydrostatic pressures were applied, the extraordinary equational
meiotic division in Stcatococcus spermatocytes has been studied in unpublished work
by the author. In these cells the spindle is formed inside the nuclear membrane,
145
146 DANIEL C. PEASE
and the anaphase movement nearly completed, before the nuclear membrane dis-
integrates. In this case, there can be no question but that the whole spindle appa-
ratus is of nuclear derivation. It was found that sufficiently high pressures de-
stroyed it by liquefaction, and anaphase movement was blocked. The spindle
re-formed once more when the pressure was removed and the cells allowed a short
recovery period. Thus the physiological action of hydrostatic pressure appears to
be qualitatively identical in gels of nuclear and cytoplasmic origin.
For the present work, Tradcscentia pollen mother cells (PMC) were selected
as material for several reasons. The spindle is characterized by relatively enor-
mous "traction fibers" going to the poles from comparatively large and easily visible
kinetochores. The cells have the advantage of a small number of chromosomes
which are relatively large. The only important disadvantages are the impossibility
of getting controls which necessarily divide at the same time as the experimental
material, and the extreme difficulties (which proved insuperable with pressure tech-
niques) of actually observing the divisions in vivo (cf., Shimakura, 1934).
The material was collected and prepared at Stanford University, and the author
is indebted to Dr. Reed Rollins of that institution's botany department for technical
advice on handling procedures and for the plants which were used. The material
was studied mostly at Columbia University before the war interrupted the work.
Dr. F. Schrader, Dr. S. Hughes-Schrader, and Dr. H. Ris followed its course with
interest, enthusiasm, and valuable suggestions. Dr. C. W. Metz of the University
of Pennsylvania also contributed excellent comments on an early draft of the
manuscript.
MATERIAL AND METHODS
The half dozen Tradcscantia paludosa plants used in these experiments pos-
sessed six pairs of chromosomes. They were derived from a common stock. The
anthers were prepared by separating the connective which joins the two lobes. One
lobe was then fixed as a control just at the time of pressure application to the other
lobe. The bisection of the anthers with a small lance could be accomplished easily
without rupturing the anther lobe walls. The lobes were handled and finally
mounted in a 7.4 gm./lOO ml. saccharose (Merck, C. P.) solution which Shimakura
(1934) has found to be isotonic with Tradcscantia pollen mother cells.
The pressure bomb used in these experiments held a half dram homeopathic vial,
and was so designed that it could be opened very rapidly. After filling with sugar
solution and a few anther lobes, the vial was sealed with "Parafilm" wax sheet held
in place with a rubber band. The experimental material was always kept under the
desired hydrostatic pressure for a one hour period. In a few experiments the mate-
rial was fixed 30 minutes after the release of pressure which allowed time for some
recovery. But in most of the experiments, the pressure was released, the bomb
opened, and the fixative added within one minute. Preliminary experiments had
shown that there was no appreciable reorganization within that short time limit.
Experiments were performed using 1,000 Ib. pressure increments from 1,000 to
6,000 lbs./in.2, and with 8,000, 10,000, and 15,000 lbs./in.2 Control experiments
were performed giving identical treatment, but at atmospheric pressure, and at the
relatively low pressure of 150 lbs./in.2
Bouin's fixative, to which 3 per cent urea was added, was used throughout.
For study, eight micra sections were prepared, and stained by Heidenhain's hema-
ANAPHASE MOVEMENT UNDER PRESSURE. II 147
toxylin method. Both mordanting and staining were prolonged (never less than
5 hours each), and the sections were destained in saturated picric acid in such a
fashion that considerable stain remained in the cytoplasm. There was a good deal
of shrinkage, but the cytoplasmic differentiation (particularly of the spindle) was
good.
RESULTS
Effects upon the first division spindle
The first division spindle was particularly sensitive to a critical hydrostatic pres-
sure that was found to be between 4,000 and 5,000 lbs./in.2 Even after 4,000 Ibs.
had been applied, the spindle figures looked essentially normal. There was no re-
duction in the length or diameter of "traction fibers" (compare Fig. 28 with Figs.
25 and 26). However, many of the "continuous fibers" had apparently been lost
for the net effect was a more diffuse looking spindle mass with fewer and less con-
spicuous continuous fibers. The abnormalities of chromosome movement under
even the lower pressures prevented any adequate study of "interzonal connections,"
but occasional examples that looked normal have been found after 4,000 lbs./in.2
In striking contrast were the results after 5,000 Ibs. had been applied. The
traction fibers were then reduced in length and in diameter so that they appeared
as delicate structures (Fig. 30). Small numbers of faint and very thin continuous
fibers were usually visible, although not always. Ordinarily 6,000 Ibs. pressure
obliterated the spindle completely, but in a small fraction of the cells a fine residual
fiber structure remained visible. Figure 31 is a photograph of the heaviest and
most extensive fibers which have been observed in material fixed after an exposure
to this pressure. It must be emphasized that this is an entirely atypical cell. No
sign of continuous fibers has been seen after exposures to 8,000 Ibs., and it was the
very rare cell which showed indications of traction fibers. When visible, as in
Figure 33 (arrows), they were thin and short. No oriented fiber structure of any
sort was ever observed after exposures to 10,000 or 15,000 lbs./in.2
In summary, it can be said that the first division spindle looked essentially nor-
mal after treatments with 4,000 lbs./in.2 pressure, but was profoundly affected by
5,000 Ibs. This demarkation was really very sharp !
Effects upon the second division spindle
The spindle of the second meiotic division was considerably more resistant to
hydrostatic pressure than that of the first division. The spindles appeared nearly
normal after 4,000 lbs./in.2 pressure, and after 6,000 Ibs. the spindles of some cells did
not seem to be greatly affected. After 6,000 Ibs. pressure there was a considerable
individual variability in different cells, even within the same anther lobe. The best
spindles were somewhat fainter than normal, and the fibers seemed generally thin-
ner, but they sometimes extended from one pole to the other. After 8,000 Ibs. pres-
sure there were occasionally evidences of traction and continuous fibers, although
they were always thin and faint if present. No fiber structure was ever visible
after pressures of 10,000 Ibs. or more.
It thus appears that the second division spindle withstood nearly 2,000 lbs./in.2
more pressure than the spindle of the first division. It will appear later that the
pressure required to block anaphase movement was similarly proportional.
148 DANIEL C. PEASE
It may also be noted here that there was a little evidence that the spindles of the
somatic cells in the connective were even more resistant to pressure, and were not
entirely destroyed unless pressures in excess of 8,000 Ibs. were applied.
i
Effects upon the chromosomes — fusion
Increasing hydrostatic pressures made the chromosomes progressively more
"sticky" and "soft." Chromosomes tended to aggregate in fused masses. In Fig-
ure 27 a metaphase plate is shown, fixed just after the release of 2,000 Ibs. pressure.
It will be noted that there are stained "bridges" connecting all of the chromosomes.
At this low pressure, the bridges were, on the average, only slightly heavier than
comparable bridges which could be found in controls of the proper stage. However,
they persisted much longer than normally, well into the anaphase stages.
When pressures of 3,000 Ibs. or more were applied, the inter-chromosomal
bridges tended to become much thicker, and entirely out of the range of normal
variation. Figure 32 shows such connections in a cell fixed just after the release of
6,000 Ibs. pressure. With progressively higher pressures, there was an increasing
tendency for the fusion of chromosomes into a single mass. This can be seen in
Figures 33 and 34. The extreme condition was reached at 15,000 Ibs. /in.2 when
it was nearly always quite impossible to recognize individual chromosomes. This
is well shown in Figure 36.
It must be emphasized that the preceding description and the photographs are
typical of cells to which the pressure was applied in late metaphase stages. When
the pressure was applied to early metaphases, the chromosomes showed a much
greater degree of fusion for corresponding pressures. Of considerable importance
must have been the proximity of chromosomes, and probably also the initial pres-
ence of thin connections. The existence of some movement in the low pressure
range may have aided the process.
Not only were metaphase chromosomes fused together by treatment with hydro-
static pressures, but a comparable effect was observed with late diakinesis chro-
mosomes before the nuclear membrane broke down. Here the chromosomes are
apparently normally kept separate from one another by gel structure within the nu-
cleus, for nucleoplasm strands showed clearly enough in fixed preparations. These
strands continued to be visible until pressures of 6,000 or 8,000 Ibs./in.2 were ap-
plied. As long as they were present the chromosomes kept apart and did not fuse.
After the higher pressures the strands were no longer visible, and the chromosomes
were all in a single clump together. But, as with the metaphase chromosomes, the
individual chromosomes did not lose their visible identity until pressures of 15.000
Ibs. were applied.
At metaphase, the chromosomes were not only found fused laterally in the plane
of the equatorial plate, but the homologous chromosomes were also fused together
so that their separation was greatly complicated. This was very obvious when first
diyision anaphases fixed just after the release of 3,000 or 4,000 Ibs. pressure were
studied. Practically every cell showed evidences of fusion with bridges that were
often long and massive (cf.. Figs. 1-12). Such bridges always stained just as the
chromosome proper with hematoxylin (Fig. 39), and the larger ones, at least, were
stained by the Feulgen reaction. These bridges were frequently between homolo-
gous chromosomes, but also commonly involved lateral fusion with non-homologous
chromosomes.
ANAPHASE MOVEMENT UNDER PRESSURE. II 149
Even more massive bridges were found in the second meiotic division material
subjected to the higher pressures which still allowed a good spindle to exist. Then,
after 6,000 lbs./in.2 pressure, most or all of the chromosomes were frequently so
fused together that they nearly lost their visible identity. However, the mass of
chromosomes often would be strung out from one end of the cell to the other (Fig.
15).
It should be noted that the chromosomes of somatic cells showed the same type
of fusion. These have occasionally been seen in the tissue of the connective, and
Figure 41 shows one bridge out of a total of three present in such a cell fixed just
after the release of 4,000 Ibs. pressure.
Effects upon the chromosomes — rounding
It should be emphasized that all of the fusion bridges between chromosomes had
rounded outlines. This shows well in Figures 27 and 32, and suggests a consider-
able plasticity.
In addition, the chromosomes as a whole tended to round up under the higher
pressures. This was most obvious in the second division chromatids which were
V-shaped with relatively long and thin arms. After 3,000 Ibs. pressure there was
very little noticeable change in shape even though there might be some fusion (Fig.
13). However, after 4,000 Ibs. there was a striking alteration. The chromatids
were then decidedly thickened and shortened (Fig. 14). This tendency became
more pronounced with increased pressures (Fig. 15, 6,000 Ibs.).
The short and thick chromosomes of the first meiotic division were not as suited
for study, but the same tendency was obviously present. Particularly after 10,000
Ibs., when the identity of individual chromosomes could still be seen, they were de-
cidedly shortened and rounded except at the kinetochore region (Fig. 34).
Effects upon the chromosomes — the spindle attachment region
The first meiotic division material gave the impression that 1,000-3,000 lbs./in.2
pressure allowed a greater extension of the attachment region of the chromosomes
than was normal (compare Fig. 26 with 25). More particularly, this region of
some chromosomes was extended far beyond what could be found in the controls.
The attachment region gave the impression of being unduly short in the material
exposed to 4,000 Ibs. pressure. An attempt to measure statistical samples was de-
cided upon.
In Table I the mean extensions of the attachment regions of first division chro-
mosomes are given for pressures up to 4,000 lbs./in.2 There were, of course, real
difficulties in measuring such small distances, but errors should have cancelled out
in the averages. While no great reliance should be placed on the absolute values,
they certainly indicate the general trend.
The measurements were made with a filar micrometer. In each group, 50 meas-
urements were made at random, excepting that only cells in anaphase were selected,
and individual chromosomes that had not yet separated and left the metaphase plate
were measured. The micrometer hair was moved up to a chromosome until it just
touched the distal tip of the kinetochore (indicated by the arrows in Figs. 25 and
26), and a reading made. Then the hair was swung across the field, and moved
back in the other direction until the hair just touched the base of the attachment
150
DANIEL C. PEASE
stalk which was ordinarily rather well defined from the body of the chromosome by
its relative translucency. Then a second reading was made. The difference meas-
ured the length of the stalk plus the width of the hair in the micrometer. The hair
width was measured in the same way in relation to a fixed point, and this value was
subtracted from all of the measurements. The figures were then converted to micra.
The control measurements actually used for comparison were combined from data
upon the control anther lobes of the 1,000 and 3,000 Ib. experimental material, and
a control anther which was left mounted in the bomb for one hour before fixation,
but without pressure.
It is to be- concluded that the mean length of the attachment stalk was definitely
increased by pressures from 1,000 to 3,000 lbs./in.2, and it has also been found that
there is no overlap in the extreme extensions between control cells and experimental
cells exposed to this pressure range. With 4,000 Ibs. pressure the mean extension
was significantly less than in the controls, and the greatest extensions found after
this treatment did not even approach the maxima found in the controls.
TABLE I
Pounds pressure
Mean extension
in micra
Percentage increase
in length
Percentage overlap with
control mean
control
0.85
1,000
1.4
59.4
2
2,000
1.2
38.6
6
3,000
1.2
42.9
4
4,000
0.68
-19.9
22
The distance between the tip of the kinetochore and the base of its stalk is given in the
second column. In the experimental series, 50 measurements were made at random, excepting
only that early anaphase cells were selected. The control average, however, is a combined average
of three sets of measurements upon different material. The mean percentage increases in length
are based upon figures carried to the third decimal place. The last column gives the percentage
of measurements which overlapped the mean of the control.
Effects upon the chromosomes — chromoneinata
We have already seen that late prophase and metaphase chromosomes fused to-
gether and rounded up under the influence of hydrostatic pressure. This, however,
only applied to condensed chromosomes. Uncondensed early prophase chromo-
somes did not seem to be affected by even the highest pressures employed. This
agrees with the findings of Pease and Regnery (1941) who were unable to detect
any effect of 15,000 lbs./in.2 pressure upon Drosophila salivary chromosomes which
are similarly uncondensed. It must be admitted that no detailed study has been
made of the early prophase chromosomes. While there was certainly no general
clumping, it is possible that very local fusions could have been overlooked, but there
was no indication of shortening or thickening.
An "accidental experiment" gave further information, and additional reason for
believing that the chromonemata were not affected by hydrostatic pressure. An
anther lobe which had been pricked was exposed to 15,000 lbs./in.2 pressure for one
hour and was then rapidly fixed in the usual fashion. The surrounding sugar solu-
ANAPHASE MOVEMENT UNDER PRESSURE. II
151
tion had entered the anther, and apparently was somewhat hypertonic. All of the
cells were slightly plasmolized and had more or less swollen chromosomes. In one
small section of the anther, conditions were such that the spiral structure was visible.
Figures 37a and b are photographs of one of these early anaphase cells, and it is
obvious that the spiral structure was unaffected. Oddly enough there was no tend-
ency for the chromosomes to fuse under these circumstances.
First division cells. Figures 1-10 are of sections from material which was fixed just after
the release of 4,000 lbs./in.2 pressure. Figures 11 and 12 are of sections fixed just after the re-
lease of 3,000 Ihs. pressure. The broken lines represent traction fibers except in Figure 7 where
they represent the pathways of "continuous fibers." All of the chromosomes visible were not
necessarily included.
Abnormalities of chromosome movement under pressure
Because of the fusion of metaphase chromosomes, even by relatively low pres-
sures, their ultimate distribution to the two spindle poles was usually very abnormal
whenever anaphase movement took place during the pressure treatment. The par-
ticular pattern which resulted apparently depended upon the balance between ana-
152
DANIEL C. PEASE
phase forces and the local resistances of whatever fused bridges happened to be pres-
ent. Greater or lesser fusions might occur between homologous chromosomes and,
laterally, between non-homologous chromosomes. Almost any conceivable vari-
ation in the resulting pattern could be found in all degrees. Some of the more
interesting variations which have been seen are included in Figures 1-15, which
are also perfectly typical of material exposed to 3,000 or 4,000 Ibs. pressure.
Homologous chromosomes might be so extensively fused that separation could
not occur. Such pairs of chromosomes, fused as in Figure 2 in the metaphase plate
Second division cells. Figure 13 is from material fixed just after the release of 3,000
lbs./in.2 pressure; Figure 14, after 4,000 Ibs. pressure; and Figure 15, after 6,000 Ibs. pressure.
Figure 16 is from recovery material, fixed 30 minutes after the release of 10,000 Ibs. pressure.
The broken lines indicate traction fibers except in the upper cell of Figure 15 in which they
indicate the pathways of the "continuous fibers." Not all visible chromatids were necessarily
included.
Figures 17-24 are all from first division recovery material which was fixed 30 minutes after
the release of 10,000 Ibs. /in.2 pressure. The broken lines indicate traction fibers. Not all visible
chromosomes were included except in the last three figures.
ANAPHASE MOVEMENT UNDER PRESSURE. II
153
27
28
Figure 25 is a first division early anaphase control exposed in the bomb for an hour (but
without pressure) before fixing. Figure 26 is of a cell fixed just after the release of 2,000
lbs./in.2 pressure. Figure 27 is a metaphase plate of the same material. Figure 28 is of a cell
fixed just after the release of 4,000 Ibs. pressure, and note the anaphase separation of the homolo-
gous chromosomes a' and a". The small arrows indicate the distal ends of the kinetochores.
The magnification of these and the following photographs is approximately X 3,000.
154 DANIEL C. PEASE
region, would presumably have remained there, and eventually formed micronuclei
(Figs. 9 and 10).
Even though there was no lateral fusion with other chromosomes, there might
be slight differences in the forces directed towards the two poles, or possibly in the
strength of the traction fibers going to opposite poles. An extensively fused pair
of chromosomes might then go as a unit to one pole (Fig. 5). Then there would
always be an abnormally long, but otherwise normal looking traction fiber (with
full thickness) going most of the way across the cell to the other pole.
Figures 4 and 5 show very extensive lateral fusion between non-homologous
chromosomes. -Such anaphase cells would probably have given rise to extensive
bridges in telophase, and between daughter nuclei, such as are shown in Figures 8,
10, and 12.
In Figure 6 the lower member of a pair of homologous chromosomes, indicated
by an arow, was laterally fused with a non-homologous chromosome going to the
upper pole. Seemingly it was being carried to that pole in spite of its traction fiber
to the other pole.
We have already spoken of the massive bridges which characterized the second
meiotic division material exposed to 6,000 Ibs. pressure, and which often involved
all of the chromatids (Fig. 15). There was less fusion with lower pressures, and
the abnormalities more nearly resembled what has just been described for the first
division.
The critical pressure blocking anapliasc movement
The best evidence for chromosome movement under pressure is certainly the
presence of extensive bridging. The author sees no rational way of accounting
for the bridges other than to suppose that anaphase movement occurred after the
chromosomes established fusions in the metaphase plate and then pulled out the
bridging connections.
With this as a criterion of movement, it is possible to state that anaphase move-
ment continued at 4,000 Ibs. /in.2 hydrostatic pressure in the first meiotic division,
but was blocked by 5.000 Ibs. pressure. No extended bridge has been seen in any
cell of this division exposed to 5,000 or more pounds pressure. Nor were there
ever signs of asynchrony, or of directionally atypical movements.
It must also be emphasized that abnormal division resulting from fusion charac-
terized practically ez>cry anaphase cell exposed to 4,000 Ibs. pressure. It was also
extremely common after 3,000 Ib. treatments. Similar abnormalities appeared on
a lesser scale after 1,000 or 2,000 Ibs., but then the separation was more frequently
fairly normal, and characterized only by loss of division synchrony.
In the second meiotic division very abnormal anaphase movement involving
massive fusions took place in some cells exposed to 6,000 Ibs. /in.2 pressure (Fig.
15), but none wras possible at 8,000 Ibs.
Bridging has been found even after 8,000 Ibs. pressure in the somatic cells of
the connective. Figure 42 is from a somatic cell forming daughter nuclei at this
pressure, and two out of a total of five bridges are visible in the plane of the
photograph.
In the meiotic divisions, at least, the presence of a good visible spindle was corre-
lated with anaphase movement. When the spindle was obviously considerably af-
ANAPHASE MOVEMENT UNDER PRESSURE. II
155
.
30.
31
32.
Figure 29 is a late anaphase cell from the same material as Figure 28 (exposed to 4,000
lbs./in.2 pressure). Figure 30 is a cell fixed just after the release of 5,000 Ibs. pressure. Fig-
ures 31 and 32 are from material fixed just after the release of 6,000 Ibs. pressure.
156 DANIEL C. PEASE
fected there were no longer evidences of anaphase movement. This was also prob-
ably true of the somatic cells, but they have not been carefully studied. It is clear
that movement is most sensitive to hydrostatic pressure during the first meiotic divi-
sion, withstands nearly 2,000 Ibs. more pressure in the second division, and seem-
ingly about 2,000 Ibs. more in the somatic cells. This, in turn, appears due to dif-
ferent characteristics of the spindle gels, rather than being due to differential pres-
sure effects upon the chromosomes. For in the first and second meiotic divisions,
and probably also in the somatic divisions, the chromosomes seemed affected equally
by equal pressures.
Spindle recovery after pressure release
At the time of making these experiments the importance of the recovery stages
was largely unsuspected, and relatively little material was gathered. But after one
hour exposures to 10,000 and 15,000 Ibs. /in.- pressures, some experimental material
was removed from the bomb and given a 30 minute recovery period before fixing.
Many of these cells showed excellent spindles with massive traction fibers (Fig. 38).
Of particular interest is the fact that the traction fibers of these recovery spindles
were de novo formations. Conclusive evidence of this was afforded by paired ho-
mologous chromosomes (still fused as a result of the pressure treatment) which
formed traction fibers from both kinetochores that went to the same pole. Figure
39 is a photograph of such a condition. Figure 40a is a drawing of another ex-
ample. Figure 40fr seems further complicated for apparently one traction fiber had
to curve around a blocking chromosome before its direction to the "wrong" pole
could become definitive. In Figure 40r each traction fiber can probably be con-
sidered as having gone to the "wrong" pole so that the original polarity of each
chromosome was entirely reversed.
Figures such as those described in the last paragraph were not rare, although
out of the ordinary. They were never seen in the controls, nor is the author aware
of similar accounts in the literature.
Most commonly the spindle appeared to re-form nearly along its original axis
if it is assumed that the metaphase plate was not displaced, and remained as an
index of that polarity. The pattern thus usually seemed very nearly normal.
However, the long axis of the new spindle was sometimes very oblique to the plate,
and presumably to the original spindle axis. In extreme cases a 90° shift was
indicated.
Also, not infrequently multipolar spindles were found which were very rare in
the control material. Three-pole spindles such as Figure 24 were fairly common,
and a few four-pole spindles have been seen. All possible variants were seen with
equal or very unequal poles, spaced equidistant from one another, or barely
separated.
These several lines of evidence all imply that the spindle was re-formed de novo,
and was not rebuilt upon residual structure which had survived the pressure treat-
ment and persisted to give a framework. New patterns appeared, and whatever
molecules were involved, they were at least rearranged.
The development of the recovery spindle
One can select a series of cells which apparently show the different steps of
spindle re-formation after the release of pressure. In some cells fiber structure con-
ANAPHASE MOVEMENT UNDER PRESSURE. II
157
J
-
35.
36.
Figure 33 is of a cell fixed just after the release of 8,000 lbs./in.: pressure (the arrows
indicate very faintly visible traction fibers), and Figure 34 after 10,000 Ibs. pressure. Figure 35
is of a cell fixed just after the release of 15,000 Ibs. pressure, and the orientation is thought to
be in the plane of the original spindle axis. Figure 36 is from the same material, but sectioned
in the plane of the metaphase plate.
158 DANIEL C. PEASE
sisted of thin fibrils tangled around the clumped chromosomes of the equatorial
plate, and without any polar orientation. The fiber direction was roughly circum-
ferential to the enclosed mass of chromosomes (as in a cocoon. Fig. 44). This
could be regarded as the first recovery stage.
Many cells showed polarized fibers as in Figure 45a. The section of Figure 45
is oblique to the spindle axis. The focus of Figure 45a is tangent to the slant height
of the cone which makes up one-half of the entire spindle (the "surface" of the
spindle, so to say). The visible fibers are the continuous fibers of the new spindle.
Figure 45b is a lower focus of the same cell. It should be observed that there are
no continuous fibers in the center of the cone. Instead, there are only slight indi-
cations of traction fibers. The continuous fibers were thus largely peripheral, but
the extensive lateral fusion of the chromosomes to make a practically solid meta-
phase plate probably had much to do with this morphological pattern which was
typical of recovery material.
Traction fibers were not seen in cells without polarized continuous fibers. But
when the latter had formed, traction fibers could usually be found. In some cells
they would be thin and short, in others longer and more massive. Thus the trac-
tion fibers appeared to "grow" outward directly away from the kinetochore region,
and full thickness was not achieved until they practically reached the poles.
It was possible to find many minor irregularities in the developmental pattern
of traction fibers. These resulted whenever the kinetochore pointed in some other
direction than directly towards a pole. A graded series could be found, the ex-
treme examples being when kinetochores pointed more or less to "wrong" poles.
Invariably the base of the traction fiber extended directly away from the kinetochore,
and it did not bend towards a pole until it became associated with continuous fibers.
The bend would then be towards the pole less than 90° away from the initial growth
direction even if this happened to be the "wrong" pole. It thus looked as though
the growth direction was unimpeded until the traction fiber became associated with
continuous fibers, and then the further extension of the traction fiber followed the
path of least" resistance in the pattern expressed by the continuous fibers. Thus the
traction fiber' even developed around obstructions as in Figure 40b.
The fusion of traction fibers
A very rare situation casts further light on the formation of traction fibers if
the interpretation is correct. It was possible to find non-homologous chromosomes
in the recovery material which appeared to be bridged across the kinetochore re-
gions. A photograph of such a bridge is shown in Figure 43. These bridges dif-
fered from all the other ordinary bridges which have been seen in that they were
achromatic. Although they were short, they had exactly the appearance in the
fixed and stained preparations that traction fibers had. They certainly gave the
impression that they represented fused traction fibers, traction fibers which started
to develop from each separate kinetochore in opposite directions, and which grew
terminally into each other to fuse end to end.
The author hesitates to emphasize these structures. The material has been
thoroughly searched and only two good examples have been seen, plus another which
was more questionable because overlying material partially obscured it. There
may be good reason for their rarity, for it is obviously an exceptional situation to
have two kinetochores pointed directly towards each other. If we accept their
ANAPHASE MOVEMENT UNDER PRESSURE. II
159
37
a.
b. 38.
39.
A 40.
Figure 37 is from a slightly plasmolized cell fixed just after the release of 15,000 lbs./in.2
pressure (a and b are different focal levels). Figures 38-40 are from recovery material fixed
30 minutes after the release of 10,000 Ibs. pressure. In Figure 38 note the bridge, br. In Fig-
ures 39 and 40 de novo recovery traction fibers of fused homologous chromosomes go to the
"wrong" pole. The direction of a pole is indicated by arrows in Figure 40.
160
DANIEL C. PEASE
42.
ijte
a.
.
44.
b.
Figure 41 is of a somatic anaphase cell fixed just after the release of 4,000 Ibs. /in.2 pressure.
Figure 42 is of a somatic cell forming daughter nuclei, fixed just after the release of 8,000 Ibs.
pressure. Figure 43 is from recovery material fixed 30 minutes after the release of 10,000 Ibs.
pressure, and shows achromatic bridging between non-homologous chromosomes (fused trac-
tion fibers?). Figure 44 shows an early stage of spindle recovery in material fixed 30 minutes
after the release of 10,000 Ibs. pressure. Figure 45 is from the same material, but spindle re-
covery is more advanced (a and /> are different focal levels of the same cell).
ANAPHASE MOVEMENT UNDER PRESSURE. II 161
reality and the above interpretation, however, the implications are of considerable
interest, for it means that developing fibers can mutually terminalize each other.
Yet there is no effect as far as lateral growth is concerned, and the fibers thicken
as normally. There is simply no growing end left. We can say that fibers extend
by terminal additions rather than from the kinetochore, or by elongation from
within their length.
Having gone this far, we can make another deduction as to the role of the
kinetochore in traction fiber formation. We can regard it as an "organizing center"
which initiates linear extension and controls fiber diameter. The linear gowth is
self perpetuating once started until the fiber reaches a pole, or is terminalized as
above. The fiber thickens by further organization at the kinetochore, and additional
linear growth parallel to the initially thin fiber, thus adding enclosing layers. The
final fiber has a thickness equal to the diameter of the organizing center. The au-
thor reiterates that this hypothesis has a slender experimental basis, and depends
upon a correct interpretation of three figures.
Chromosome movement in recovery material
There were obvious indications of chromosome movement in recovery material
after the spindles re-formed. The movement was abnormal because of strong and
persistent fusion bridges,, and in many 'ways resembled the anaphase movement
which occurred under low pressures (3,000 and 4,000 Ibs.).
Frequently fused pairs of homologous chromosomes were found going to, or
after they had reached, a single pole as in Figures 38 and 17. In such cases one
traction fiber extended all the way across the cell to the other pole but seemed to
be of normal thickness. This type of movement often seemed to be aided by lateral
fusion with non-homologous chromosomes as in Figures 18 and 21. Less fre-
quently the fusion between homologous chromosomes was relatively slight, and there
would be a partial separation with the formation of more or less long and thin bridges
(Figs. 19, 20, and 38, /»-.). Quite frequently very massive bridges were formed
involving most if not all of the chromosomes which would be fused together (Figs.
22 and 23). There were no important differences between first and second meiotic
division cells (note Fig. 16).
None of the material was allowed a sufficient recovery period so that daughter
nuclei formed in cells which began their anaphase movement after the application
of pressure. It can be presumed, however, that many of the cells would form only
a single nucleus because of an inability on the part of the chromosomes to separate.
Other cells would be expected to form bridged nuclei, and probably multiple
micronuclei.1
Chromosome structure in recovery material
The persistence of chromosome fusion in the recovery material would seem to
suggest just one possibility — that the initial fusion under high pressure must have
been due to at least a partial liquefaction of some chromosomal element, and that
the fusion bridges then gelled when the pressure was released. In the recovery
material the chromosomes were thus stuck together by very viscous bridges.' After
examining a great deal of material, the author is of the opinion that it is very doubt-
1 Pease (1941) definitely found this to be the case in Urechis eggs.
162 DANIEL C. PEASE
ful that fused chromosomes were ever able to separate completely before the forma-
tion of daughter nuclei. Most commonly there were few signs of any separation,
but even in extreme cases, thin and very long bridges persisted as in Figures 19
and 20. The moderately thick bridges, at least, stained with Feulgen.
There is another, and much more puzzling, aspect of chromosome structure
which is brought to light by a study of the recovery material. Even after the re-
lease of 15,000 lbs./in.2 pressure (which resulted in the very complete fusion of the
chromosomes as in Figure 36) the chromosomes regained their visible identity and
their approximately normal shape. This tendency can be seen (in 10,000 Ibs. mate-
rial) by comparing Figure 38 with Figure 34, but it is best seen by comparing the
long chromatids of the second meiotic division (compare Fig. 16 with Figs. 14 and
15). In regaining the normal shape, the fusion areas must necessarily have been
reduced in cross-section, and it is likely that some fusion bridges were lost entirely
during this change. The effects of this change were best demonstrated by the sepa-
ration of the second division chromatids in material recovering from 10,000 Ibs.
pressure. Extensive separation sometimes occurred, thus differing in degree from
the first division. Figure 16 gives an indication of typical difficulties which were
essentially the same as in the first division.
Absolute pressure and recovery rate
In Urechis egg material Pease (1941) found that the rate of recovery was
roughly proportional to the absolute pressure which had been applied. In the
Tradescantia PMC material we can only compare the effects of 10,000 and 15,000
lbs./in.2 pressures. Comparison is subjective, but there was not the slightest doubt
but that the cells subjected to 10,000 Ibs. pressure showed a much greater amount
of recovery of the spindle elements in 30 minutes than the cells exposed to 15,000
Ibs. showed in the same length of time. Fully developed new spindles were only
rarely found in the 15,000 Ib. material, but were common in the 10,000 Ib. mate-
rial. In both, however, the chromosomes had regained their visible identity and
approximately normal shapes.
CONCLUSIONS
A single hypothesis readily accounts for most of the manifold effects of hydro-
static pressure upon spindle, chromosomes, and anaphase movement. This sup-
poses that increasing hydrostatic pressures progressively reduce gel rigidity, with
liquefaction as the end result. Conversely, after the release of pressure, conditions
return to a state such that gel structures can be re-formed once more. There is,
of course, an excellent experimental background for this thesis, particularly in so
far as it applies to cytoplasmic systems. This has been indicated in the introduction,
and has been outlined at greater length in the first paper of this series (Pease, 1941 ).
It is, however, unfortunate that this work depends upon an interpretation of
fixed material. However, we have every reason for believing that the presence of
good fiber structures in such material is a good index of oriented gel structure in
life. It is only on that assumption that a comprehensive pattern appears, consistent
throughout its details. It is true that whenever we have contributory evidence of
liquefaction (such as a block of anaphase movement), we do not find fiber struc-
tures in the cytological material. Apparently extensive fiber structures are only
ANAPHASE MOVEMENT UNDER PRESSURE. II 163
precipitated by fixation agents when molecules are at least organized into an oriented
pattern and probably also concentrated in a gel.
Spindle structure and formation
In view of the above considerations, it is not surprising to find that the spindle
no longer appears in cytological preparations after a critical pressure has been ap-
plied before fixation. This is to be interpreted as indicating a liquefaction of pre-
existing gel structures, with a consequent loss of molecular organization.
It has been demonstrated that the pattern of the recovery spindle can be very
different from that of the original spindle. High hydrostatic pressure seems able
to break down the oriented structure of the original spindle so completely that it
re-forms de novo, and sometimes with a new polarity. In the re-formation of the
spindle much the same protoplasmic material may well be used, but the unit mole-
cules or micells are rearranged in a different manner, just as a pile of second-hand
bricks might be rearranged to build a new house. This conclusion can probably be
accepted as a generalization for it agrees with the findings in Urechis eggs which,
in their formation of "half spindles," were even more striking (Pease, 1941), with
certain other observations on cytoplasmic systems (cf., Pease, 1940), and with gen-
eral theory.
It is not clear just what does orient the new spindle axis in Tradcscantia PMC.
Cytasters accomplished this end in Urechis eggs, and obviously played the impor-
tant role. These were never observed in the PMC material. Instead, we find a
strong tendency for the new axis to coincide more or less with the original. The
recovery spindle encountered one unusual difficulty in its organization in that the
chromosomes were no longer completely separate entities. After the higher pres-
sures there was usually a continuous plate of fused chromosomes in the equatorial
region. Continuous fibers did not, indeed could not, penetrate this obstruction.
However, note that homologues were not even found as half spindle components.
Continuous fibers were only found sweeping around the blocking mass leaving the
core of the spindle devoid of visible oriented structure except for traction fibers.
Apparently, therefore, the continuous fibers are entirely a product of the cytoplasm,
and are not directly related to the chromosomes. The latter, in fact, are obstacles
to be by-passed. This does not, however, preclude the possibility of a generalized
interaction between chromosomes and cytoplasm in that the former may "activate"
the latter to form gel structures. Such an "activation" was quite definitely shown
by Urechis eggs recovering from the effects of hydrostatic pressure (Pease, 1941).
A more accurate interpretation might be not to stress the continuous fibers as such,
but to consider them simply as an index of a more fundamental structural organiza-
tion of molecules. They thus may signify nothing more than the basic pattern of
an extensive gel framework.
On the other hand, the kinetochore apparently quite specifically "organizes" the
protoplasm to form the attached traction fiber. This process is partially separable
from the development of continuous fibers. We have good reason for believing
that developing traction fibers simply follow the path of least resistance in the struc-
tural pattern of the bulk of the spindle, which, in turn, is expressed by the distribu-
tion of the continuous fibers. Thus the structural pattern of the body of the spindle
limits the course taken by the traction fibers as they develop outwards away from
the kinetochores. It seems likely that this is a progressive wave of molecular or-
164 DANIEL C. PEASE
ganization. This view is quite similar to that of Schrader (1932), although based
upon different evidence. However, it is fundamentally distinct from that of Belar
(1929) who supposed a very different relationship between traction and continuous
fiber. Further tentative conclusions on the growth of traction fibers have already
been given in describing the experimental results.
The extension of the attachment region in chromosomes subjected to relatively
low pressures indicates a real pull by or through the traction fibers. It is almost
impossible to imagine that it could be due to "repulsive forces" between the kineto-
chores for, if that was so, the extension should continue to increase with progres-
sively higher, pressures which further soften the chromosomes. Instead, we find the
extension to be subnormal while we still have evidence of traction fibers and ana-
phase movement (at 4,000 Ibs. in the first meiotic division). Our conclusion, then,
is that the traction fiber is a reasonably stiff gel. No doubt it progressively loses
rigidity with increasing pressure, but it has a margin of strength, and there is no
important weakness until a pressure threshold is passed. The extension of the
attachment stalk is therefore thought due to a pressure effect upon the chromosome
itself so that it is softened, and can be unduly pulled out. The subnormal exten-
sion at 4,000 Ibs. indicates a significant weakness of either the traction fiber or
available force. It is interesting for comparison that the centrifuging experiments
of Shimamura (1940) with comparable material (Liliwn PMC) also lead to the
conclusion that the traction fiber is a fairly stiff gelled structure. The latter's
work seems to the author to be quite conclusive.
Chromosome structure
It seems obvious that some portion of the condensed chromosome tends to be
softened, and finally liquefied, by hydrostatic pressures. Since there was no ap-
parent effect upon uncondensed chromosomes, or upon the spirally coiled chro-
monemata, the portion affected would seem to be the "matrix" (no morphologically
separable "sheath" is visible, and presumably more than a sheath would be involved
when the attachment region is extended).2
A critical analysis of the data, however, discloses some relationships that cannot
yet be interpreted with any assurance of certainty. The normal presence of an at-
tachment stalk, and its further extension under relatively low pressures, suggests
that the rigidity of the matrix is normally low, but is further reduced by pressure.
One might suppose it to be viscous rather than a stiff gel. While the spindle gels
are liquefied by moderate pressures, the matrix is not entirely liquefied until pres-
sures of about 15,000 Ibs. /in.2 are applied when the chromosomes so fuse that they
lose their visible identity. Thus a structural viscosity appears to persist and with-
stand very considerable pressures.
It is a fair assumption that the spindle gels obey Marsland's (1939) law, so that
their rigidity is reduced 24 per cent by each pressure increment of 1,000 lbs./in.-
Liquefaction then occurs at a critical pressure, when gel linkages tend to break more
2 In the first paper of this series (Pease, 1941) chromosome aggregation was described in
Urechis eggs subjected to hydrostatic pressure. The cytological appearance suggested that a
"sheath" was involved in this fusion rather than the matrix. The Urechis chromosomes were
so small, though; that the details were not visible. In view of the present work it seems more
likely that the matrix as a whole was involved.
ANAPHASE MOVEMENT UNDER PRESSURE. II 165
rapidly than they can be formed. Whereas we can probably apply Mainland's law
to the spindle gels, it does not seem applicable to the chromosome matrix, unless we
assume that the matrix material has a much lower pressure/rigidity constant than
cytoplasmic or spindle gels, i.e., much less than 24 per cent per 1,000 lbs./in.2
That other different gels in vitro do, in fact, have different constants has been dem-
onstrated by Marsland and Brown (1942).
There is yet another aspect of chromosome structure to be considered. Why is
it that with increasing pressures we find chromosomes rounding up and tending to
fuse into a single mass? This looks like an interfacial phenomenon to be exp' lined
on the basis of surface tension laws. We do not observe this with uncondensed
chromosomes. The author does not see how these and related observations can
be explained except by the assumption that a true interface does exist between con-
densed chromosome and surrounding protoplasm (cf., Hirschler, 1942). Many
workers do not believe that there is an osmotically active membrane separating
chromosome from protoplasm, although this could explain many of the observa-
tions of chromosome swelling. However, a real interfacial boundary would not
necessarily imply an osmotically active system.
In any case, it can be presumed safely that any intracellular interface would exert
only a very low tension, certainly not more than a fraction of a dyne, or the very
few dynes, that have invariably been recorded for water/cell interfaces, or intra-
cellular oil/protoplasm interfaces (cf., Harvey and Shapiro, 1934 and Harvey and
Schoepfle, 1939). The presence and properties of dissolved proteins would always
prevent high values. Thus any interfacial tension at the surface of a chromosome
would be so low that complete rounding of the aspherical shape would occur only
when both chromosome and surrounding protoplasm were essentially fluid, and
practically without structural viscosity. It is only at a pressure of about 15,000
Ibs./in.2 that the observed effect indicates these conditions as being nearly fulfilled.
The spindle in chromosome •movement
It has already been pointed out that there is a direct and definite correlation be-
tween anaphase movement and the presence of a good visible spindle. Hence, our
outstanding conclusion is that the presence of gel structure in a spindle is essential
for anaphase movement. When the gel rigidity is sufficiently reduced, the move-
ment necessarily ceases. Other types of experimentation have less directly led to
the same conclusion (cf., particularly the work of von Mollendorff, 1938 and 1939,
on the specific effects of chemical agents). On the other hand, hypotheses involving
attractive or repulsive forces are well nigh incompatible with the results. It is
hard to imagine hydrostatic pressure affecting such forces, particularly in the low
pressure range. Under pressure, with conditions of reduced viscosity, the chro-
mosomes should move apart all the more rapidly and easily if such forces were in-
volved. Furthermore, since Marsland's law relating pressure and viscosity ex-
presses a logarithmic relationship, the effect should be most noticeable in the low
pressure range. Obviously this is in direct disagreement with the present findings.
But what is the role of gel structure in anaphase movement? Certainly there
are at least two separable structures to be considered — the traction fibers and the
spindle mass.
Considering the traction fibers first, Cornman (1944) in a thought-provoking
review comes to the conclusion that they are contractile structures and supply the
166 DANIEL C. PEASE
force for movement. However. Cornman ignores one major difficulty in his other-
wise excellent analysis. No one has yet been able to demonstrate that traction
fibers thicken as they shorten, although this would be expected if we were dealing
with contractile bodies. The author has certainly seen no evidence of this in his
own preparations, nor has he been able to observe the converse of any visible thin-
ning when a traction fiber was extended all the way across the cell from one pole
to the other. We, therefore, seem to require a different explanation.
It is the author's thought that Schrader (1932) was correct in regarding trac-
tion fibers as being no more than passively semi-elastic structures. . This has been
given excellent experimental foundation by Ris (1943) who has been able to meas-
ure directly anaphase movement in living cells (insect spermatogonia and spermato-
cytes). In some cases he has demonstrated that anaphase movement is very defi-
nitely a two step process. The first, relatively rapid movement can be explained
as due to the release of elastic tension so that the traction fibers do actually shorten.
The remaining movement is then due to the spindle mass, with the traction fibers
serving simply as passive connections to the chromosomes. Lewis '(1939) pro-
duced an accelerated motion picture of dividing fibroblasts in vitro which beauti-
fully showed the same phenomenon, although he has not commented upon it.
A general hypothesis of anaphase movement can be advanced on the assumption
that the traction fiber is anchored at one end to the chromosome, and along some of
its length to the larger gelled mass of the spindle which, in turn, is in motion. Thus
it is simply a more or less elastic connection from the spindle body to the chromo-
some— a rope, so to say, between the machine and the load. This interpretation
forces our attention to the body of the spindle.
The analysis of anaphase movement by Belar (1929) does much to delimit the
problem, even though we cannot accept his general hypothesis. He demonstrated
that it was impossible to account for the total movement on the basis of simple
swelling or elongation of the main spindle mass (or, more specifically, the Stemm-
korper). There is, however, an obvious way to avoid the difficulties outlined by
Belar (other than his own solution), and still be consistent with his findings and
other knowledge.
It is proposed that motion and force may be imparted to the spindle mass by
means of two phase transformations. The postulate supposes that gel material is
added either in the interzonal region 3 or along the greater part of the spindle,
while a proportional solation occurs at the poles. Thus a material circulation is
established, but a circulation by means of sol-gel-sol transformations rather than
within a single phase. Actually a somewhat comparable idea has been proposed
by Wassermann (1929 and 1939). Such an idea would be regarded by many as
entirely too speculative, and not subject to either proof or disproof. The author,
however, wishes to point out some comparable effects which are not likely to be
known to most cytologists.
Dan ct al. (1938 and 1940) discovered a remarkable phenomenon in dividing
sea urchin eggs. After the furrow completes its intrusion, an entirely new region
of gelled cortex is added in the center of the furrow region as the original cortical
3 Note that Schmidt (1939) did not find birefringence with polarized light in the mid-region
of sea urchin egg spindles, and that Shimamura (1940) found this to be the "weak" region in
centrifuging experiments upon Lilium PMC.
ANAPHASE MOVEMENT UNDER PRESSURE. II 167
material backs out. Pease (1943) calculated that this de novo cortex came to cover
about 11 per cent of the cell surface. This gel growth is obviously analogous to a
system that could very well work within a spindle.
Since the advent of hydrostatic pressure techniques, it has also become clear
that all sorts of other cell processes involving movement are dependent upon gel
structure. Thus amoeboid movement, cyclosis, streaming, cytoplasmic division,
the movement of pigment granules, and the pole cell nuclei of Drosophila eggs, and
even sperm penetration both through the egg surface and also to their final central
position all cease (reversibly) when the gel is liquefied. All of these movements
depend upon the rather unexpected, and admittedly little understood, properties of
protoplasmic gels. Obviously the gel rearranges itself, and is itself in motion (cf.,
the review of Marsland, 1942). No doubt gel-sol transformations are usually if
not always involved along with the rearrangement. Thus we do find empirically
a common denominator for all movements other than such specialized activities as
muscle contraction and ciliary motion. The author believes that a general theory
of anaphase movement is in sight, and that it will come from a better physico-
chemical understanding of protoplasmic gel-sol systems.
SUMMARY
Hydrostatic pressures have been applied to Tradescantia pollen mother cells as
a technique for studying the structure of division spindles and chromosomes and
the mechanics of anaphase movement. The procedure has given pertinent informa-
tion by virtue of the fact that increasing pressures progressively reduce gel rigidity.
Sufficiently high pressure results in liquefaction. Yet the effects are reversible.
The spindle of the first meiotic division was but slightly affected by 4,000
lbs./in.2 pressure, yet was mostly liquefied by 5,000 Ibs. The spindle of the second
meiotic division withstood about 2,000 Ibs. more pressure. The somatic cells were
even more resistant.
Condensed chromosomes were significantly softened by even 1,000 lbs./in.2
pressure as indicated by an undue elongation of the kinetochore stalk. Fusion
bridges became particularly obvious when 3,000 Ibs. was applied. Significant short-
ening and rounding occurred at 4,000 Ibs. Total fusion and rounding, indicating
complete liquefaction of the matrix, did not occur until pressures of 15,000 lbs./in.2
were applied. The fusion and rounding appeared to be a surface tension effect, and
suggested the existence of a true interfacial membrane between condensed chromo-
some and cytoplasm. Not even these highest pressures, however, affected the un-
condensed prophase chromosomes so that the effect of pressure was thought to be
only upon the matrix material.
Chromosome movement was limited to those pressures which did not liquefy
the spindle. The presence of fusion bridges, however, resulted in very abnormal
movement.
After the release of high pressures, spindles re-formed. That these were de
novo structures was indicated by their sometimes abnormal orientation, by the fre-
quency of multipolar spindles, and by abnormalities in the course of traction fibers.
Thus, the traction fibers of two homologous chromosomes might go to a single
pole. Abnormalities made it seem likely that the growth of traction fibers was in
a large measure independent of the growth of the body of the spindle. The direc-
168 DANIEL C. PEASE
tion of growth of the traction fiber was not specifically oriented until it reached the
oriented bulk of the spindle.
Chromosome movement in recovery material was abnormal in that the fusion
bridges persisted. Thus the chromosome matrix which had been liquefied, had
become highly viscous once more. Under such circumstances, homologous chromo-
somes frequently went to a single pole, and the traction fiber to the other pole ex-
tended all the way across the cell. However, such traction fibers were not thinner
than normal.
The outstanding conclusion is that a gel structure in the spindle is essential for
anaphase movement. The traction fiber apparently serves as nothing more than
a semi-elastic connection between the chromosome and the main mass of the spindle
which, in turn, is in motion. It is suggested that motion and force is imparted by
means of sol-gel-sol transformations, with gel being added to the central bulk of the
spindle while a proportional solation goes on at the poles.
LITERATURE CITED
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tocyten von Chorthippus (Stenobothrus) lineatus Panz. Arch. f. Entivickmcch., 118:
359-484.
CORNMAN, I., 1944. A summary of evidence in favor of the traction fiber in mitosis. Amer.
Nat., 78 : 41(M22.
DAN, K., AND J. C. DAN, 1940. Behavior of the cell surface during cleavage. III. On the
formation of new surface in the eggs of Strongylocentrotus pulcherrimus. Biol. Bull.,
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DAN, K., J. C. DAN, AND T. YANAGITA, 1938. Behavior of the cell surface during cleavage. II.
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HARVEY, E. N., AND G. SCHOEPFLE, 1939. The interfacial tension of intracellular oil drops in
the eggs of Daphnia pulex and in Amoeba proteus. Jour. Cell. Comp. Physiol., 13:
383-389.
HARVEY, E. N., AND H. SHAPIRO, 1934. The interfacial tension between oil and protoplasm
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cyten der Rhynchoten-Art Palomena viridissima Poder. Naturw., 30: 105.
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Pa.) Anat. Rcc., 80: 396, 1941 (review).
MARSLAND, D. A., 1939. The effects of high hydrostatic pressure upon the mechanics of cell
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with special reference to myosin and other protoplasmic gels. Jour. Cell. Cornp.
Physiol., 20 : 295-305.
VON MOLLENDORF, W. V., 1938. Zur Kenntis der Mitose. I. Arch. f. c.rp. Zellforsch., 12 : 1-66.
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29 : 706.
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tern in the Chaetopterus egg. Biol. Bull., 78: 103-110.
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movement. I. Experiments on the first mitotic division of Urechis eggs. Jour.
Morph., 69: 405-441.
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ANAPHASE MOVEMENT UNDER PRESSURE. II 169
PEASE, D. C. Hydrostatic pressure effects upon the spindle figure and chromosome movement.
III. Experiments on the intranuclear meiotic division of Steatococcus spermatocytes
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hydrostatic pressure. Jour. Cell. Comp. Physiol., 17 : 397-398.
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Fujii et al. On the mechanism of nuclear division and chromosome arrangement. IV.)
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der mikroskopischen Anatomic des Menschen. Springer, Berlin.
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THE COMPARATIVE DISTRIBUTION OF TWO CHROMA-
TOPHOROTROPIC HORMONES (CDH AND CBLH)
IN CRUSTACEAN NERVOUS SYSTEMS
FRANK A. BROWN, JR., AND LORRAINE M. SAIGH
Department of Zoology, N or thivc stern University, Evanston, 111., and
Marine Biological Laboratory, Woods Hole, Mass.
INTRODUCTION
It was demonstrated by Brown (1933) that sea-water extracts of the crustacean
central nervous organs contained material having a definite and characteristic effect
upon certain chromatophores of the body. The nervous organs were the only tis-
sues of the body other than the eyestalks, with their included sinus glands, that
yielded such a chromatophorotropically active substance, thus suggesting that the
former possibly contained a source or sources of normal, color-changing hormonal
material. In the shrimp, Palaeinonetes, injection of extracts of the nervous system
were shown to bring about a rapid blanching of dark-colored specimens through
concentration of the red and yellow pigments within the chromatophores, an action
similar to that which could be induced by extracts of the sinus gland of the eyestalk.
Similar activity of the nervous system was described by Hosoi (1934) for
Penacus japonicus and by Hanstrom (1937) for Penacus brasilicnsis. Knowles
(1939) found that extracts of the central nervous system of Lcander adspersus
caused concentration of the white pigment within that species. Concentration of
white pigment by extracts of central nervous system was also reported for Cambarus
by Brown and Meglitsch (1940) who worked with the chromatophores in isolated
pieces of integument. Sinus gland extracts had an antagonistic action upon this
pigment, thus proving that the sinus glands and nervous system did not yield exclu-
sively identical chromatophorotropic substances.
Evidence that the central nervous organs contained sources of hormones nor-
mally involved in the adaptive color-changes of Palacmonetcs was presented by
Brown (1935) who found that any vigorous stimulation of the cut ends of the optic
nerves in darkened eyestalkless specimens would induce a blanching characteristic
of that following injection of extracts of central nervous organs. Roller (1930)
had also observed comparable responses of eyestalkless Crago but did not at that
time consider the central nervous organs to be a source of the active material.
More convincing evidence for the production of a normal chromatophorotropic
hormone in the crustacean nervous system was presented by Brown and Ederstrom
(1940). Their observations concerned the reactions of the particularly sensitive
melanophores in the telson and uropods of the shrimp, Crago. Amputation of the
eyestalks of a white-adapted animal brought about, within 3-6 minutes, a complete
dispersion of black pigment in the melanophores giving the animal a "black-tailed"
appearance. The condition persisted for about an hour whereupon the pigment re-
turned to its former concentrated state, the latter condition typically lasting for
several days. Brown and Ederstrom found that the black pigment could be caused
170
HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 171
to disperse again by stimulation of the eyestubs or by the injection of extracts of
the circumoesophageal connectives. Upon more extensive experimentation they
concluded that the mid-region of the connectives, including the connective ganglia,
contained the origin of the Crago tail-darkening hormone (CDH) involved here.
The results of these investigators were confirmed and extended when Brown and
Wulff (1941) gave evidence for a second chromatophorotropic principle within the
central nervous system, namely a Crago body-lightening hormone (CBLH) by de-
scribing that strong stimulation of the eyestubs simultaneously darkened the telson
and uropods and lightened the remainder of the body, an action duplicated by in-
jection of extracts of the central nervous system as a whole. It was shown that
these two actions were due to two separable principles in that injection of ethyl -
alcohol extracts of the nervous system gave only body-lightening action, the tail-
darkening principle remaining in the alcohol-insoluble residue, and, that mild stimu-
lation of the eyestubs of eyestalkless animals produced both tail-darkening and
body-darkening. Brown and Wulff speculated that CDH was. in the absence of
CBLH, a general body-darkening principle. This hypothesis was more specifically
set forth and given experimental support by Brown (1946) who clearly demon-
strated the source of this darkening principle to lie, not in the circumoesophageal
connectives proper, but in the minute tritocerebral commissure interconnecting the
connectives immediately posterior to the oesophagus. Injection of sea-water ex-
tract of this commissure in various experiments produced in every case tail-
darkening but various degrees of either body-lightening or body-darkening. The
variable effects upon the body seemed reasonably explained in terms of varying
concentrations of an antagonistic body-lightening principle.
In the following experiments a survey was made of the effects of sea- water ex-
tracts of the central nervous systems of thirteen species of higher crustaceans repre-
senting the Isopoda, Natantia, Ashicura, Anomura, and Brachyura upon Crago
color-change. The distribution of both the Crago tail-darkening hormone, CDH,
and the Crago body-lightening hormone, CBLH, was considered. We have con-
cerned ourselves primarily with the presence or absence of each substance within
the centra] nervous systems and, when the hormones are present in a particular
species, with a survey of the relative concentrations of the principles within the
parts containing the hormone in question.
EXPERIMENTS AND RESULTS
The experiments to determine the distribution of CDH and CBLH were con-
ducted in the following manner. Animals for use in assaying the concentration
of active principles in extracts of nervous tissue were first prepared. The eyestalks
of a number of Crago septevnspinosus, ranging from 3-6 cm. in length, were ampu-
tated by means of a sharp scalpel and the eyestubs cauterized with an electric cautery
needle. No animals were used for assay purposes until at least twelve hours fol-
lowing this operation, at which time they could best be described as possessing
mottled black and white bodies and light telson and uropods (see Fig. \A, control).
A relatively simple but effective method was used in the preparation of central-
nervous-system extracts. The donor of the nervous tissue first had eyestalks re-
moved and stubs cauterized in the same manner as described above for Crago. The
dorsal portion of the exoskeleton was then cut away. After removing surrounding
172
FRANK A. BROWN, JR., AND LORRAINE M. SAIGH
viscera and muscles the nervous organs were removed under a dissecting micro-
scope by carefully severing the nerves about the brain, thoracic and abdominal cords
and gently lifting the entire system out of the animal. Particular caution was ob-
served in the removal of the circumoesophageal connectives so as to prevent any
damage to the tritocerebral commissure. The nervous system was then placed in
a watchglass containing a small amount of sea-water and divided by means of a
sharp scalpel into the desired portions which usually comprised brain, connectives,
thoracic cord, and abdominal cord.
A
B
FIGURE 1. ^.Darkening of eyestalkless Crago following injection of a sea-water extract
of the abdominal nerve cord of Homarns (cone. = 1 cord/0.5 ml. sea-water). The two speci-
mens on the left are two uninjected ones used for a control. The injections for the animals on
the right were made 15 min. before the photographs were made. B. Lightening of eyestalkless
Crago following injection of a sea-water extract of the circumoesophageal connectives of fY<;
(cone. = 3 pr. conn, to 0.2 ml. sea-water). The two specimens on the right were injected 8
minutes before the photographs were made.
HORMONES IN CRUSTACEAN NERVOUS SYSTEMS
173
Following this procedure the organs were transferred to individual glass mortars
where excess sea-water was removed and the tissues allowed to dry partially. The
tissue was then triturated with a measured amount of sea-water varying in quantity
with the different species from 0.1-0.5 cc. per portion depending upon the size of
the nervous system as a whole. In some cases, such as that of Idothca, it was
necessary to use the parts of several nervous systems in the preparation of each
extract in order to obtain adequate concentration and amount for assay. All ex-
tracts were centrifuged for three minutes at approximately 3,500 R.P.M. and the
supernatant liquid of each injected into the dorsal musculature of the abdomen of
at least two test-animals prepared as described above. The amount of extract in-
jected into each varied with the size of the test-animal, but was normally between
TABLE I
Responses of eyestalkless Crago to injection of extracts of various portions of the central nervous system
of other crustaceans. No. of cases signifies the number of donors
Body-lightening © or
Tail-darkening darkening ©
Time (min.) Time (min.)
Species
Organ
No.
cases
0
5
10
15
30
45
60
0
5
10
15
30
45
60
Homarus
Brain
Connectives
Thoracic cord
Abdominal cord
7
8
8
2
0.0
0.0
0.0
0.0
3.3
1.9
1.6
1.0
3.6
2.3
2.1
1.5
3.9
2.3
2.2
2.5
3.7
2.3
2.8
3.0
3.7
2.1
2.7
2.5
1.6
1.4
2.5
1.5
0.0
0.0
0.0
0.0
0.0
-1.4
-0.8
+3.0
+0.7
-1.0
-0.3
+3.5
+2.0
- 0.0
+0.4
+4.0
+2.6
+0.6
+2.8
+4.0
+ 1.9
+0.4
+2.8
+4.0
+0.5
0.0
+4.0
Cambarus
Brain
Connectives
Thoracic cord
Abdominal cord
10
10
10
10
0.0
0.0
0.0
0.0
3.4
2.5
2.7
2.6
3.4
2.9
3.1
2.8
3.4
3.2
3.4
2.8
2.6
2.2
2.9
2.5
1.3
0.9
2.1
1.4
0.4
0.3
1.3
1.0
0.0
0.0
0.0
0.0
+0.8
-1.7
-1.9
+0.8
+ 1.2
-0.9
-1.7
+ 1.2
+ 1.7
-0.6
-0.9
+ 1.4
+0.9
-0.1
+0.1
+ 1.1
+0.4
+0.1
+0.3
+0.6
+0.2
+0.3
0.0
+0.2
Upogebia
Brain
Connectives
Thoracic cord
Abdominal cord
7
6
7
7
().()
0.0
0.0
0.0
1.3
0.2
1.5
1.0
2.3
0.6
2.1
1.4
2.3
0.0
2.7
1.4
2.2
0.0
2.5
1.2
0.8
0.0
2.0
0.9
0.0
0.0
1.4
0.4
0.0
0.0
0.0
0.0
-2.0
-2.3
-0.2
-0.6
-1.7
-2.6
+0.7
-0.6
-1.5
-2.0
+ 1.2
-0.3
-0.4
-1.7
+0.8
0.0
-0.2
-0.4
+0.5
0.0
0.0
-0.2
+0.2
0.0
Pagurus
Brain
Connectives
Thoracic cord
8
8
8
0.0
0.0
0.0
0.4
0.0
1.9
0.5
0.0
2.9
0.4
0.0
3.1
0.2
0.0
2.4
0.2
0.0
1.4
0.0
0.0
0.2
0.0
0.0
0.0
- .6
- .8
- .3
-1.6
-1.6
-1.3
-1.1
-1.0
-1.3
-0.5
0.0
-0.5
-0.1
+0.1
+0.2
0.0
+0.1
0.0
Emerita
Brain
Connectives
Thoracic cord
8
8
8
0.0
0.0
0.0
0.0
0.0
1.0
0.0
0.0
1.6
0.0
0.0
1.6
0.0
0.0
1.0
0.0
0.0
0.5
0.0
0.0
0.1
0.0
0.0
0.0
- .5
- .4
-0.9
-0.6
-0.6
-0.6
-0.5
-0.2
-0.1
-0.2
0.0
-0.1
0.0
0.0
0.0
0.0
0.0
0.0
Libinia
Brain
Connectives
Thoracic cord
7
7
7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
-2.6
-1.7
-1.7
-2.8
-1.8
-1.7
-2.0
-1.7
-1.8
-1.0
-0.4
-0.9
-0.3
-0.1
-0.4
0.0
0.0
0.0
0.025 and 0.04 cc. Sea-water injected or uninjected controls were observed simul-
taneously with all test-animals.
Observations of the color changes in both body and tail were taken at five-
minute intervals up to fifteen minutes and at fifteen-minute intervals thereafter.
The degree of darkness of the tail or body was described within the range, + 1 to
+ 4, the number + 4 representing the maximum extent of darkening and the num-
ber + 1, the minimum observable one. In a similar manner body-lightening was
indicated by the range, - - 1 to -- 4, with -- 4 denoting the greatest extent of body-
lightening. Final results for a number of experiments were averaged and are pre-
sented in tabular -form in Table I. These results have been further analyzed so as
to present the distribution of CDH and CBLH within the central nervous system
174
FRANK A. BROWN, JR., AND LORRAINE M. SAIGH
of each of the species considered (see Tables II and III). In these tables, the
relative distribution of activity of the hormones is calculated for the various portions
of the nervous system for each species.
This was done as follows. The average values of the chromatophores at 5, 10,
15, and 30 minutes following extract-injection were of themselves averaged. Then
for Table II the portion of the nervous system producing maximum darkening was
TABLE II
The quantitative distribution of CDH activity within the central nervous systems of a number of
crustaceans. The region of maximum activity is arbitrarily given the value 1.00. It is important to
note that each portion of the nervous system, regardless of size, is extracted in an equal volume of sea-
water, and the relative concentrations of the principles investigated are expressed solely in terms of their
activities. This note applies equally to Table III.
Classification
Species or genus
Brain
Connec-
tives
Thoracic
cord
Abdominal
cord
Isopoda
Decapoda
Natantia
Reptantia
Astacura
Anomura
Brachyura
Oxyrhyncha
Brachyrhyncha
Idothea baltica
Crago scptemspinosus
Palaemonetes vulgaris
Homarus americanus
Cambarus virilis
Upogebia affinis
Pagurus sp.
Emerita talpoidea
Libinia sp.
Cancer irroratus
Carcinides maenas
Ovalipes ocellatus
Uca pugilator
1.00
1.00
1.00
1.00
some
0.06
1.00
0.22
0.21
0.85
1.00
0.97
0.98
1.00
0.61
0.61
0.53
1.00
0.84
0.94
0.84
1.00
0.10
0.80
0.65
0.17
0
1.00
—
0
0
1.00
—
0
0
0
—
0
0
0
—
0
0
0
—
0
0
0
—
0
0
0
—
arbitrarily given the value 1.00, the activity of the other parts being expressed in
terms of simple proportions of this. For Table III the part showing maximum
lightening was given the value -- 1.00 with the activity of other parts similarly ex-
pressed proportionately. The positive values in the latter table obviously indicate
darkening rather than lightening.
Within the single species of Isopoda investigated, Idothea baltica, there appears
to be roughly a uniform distribution of CDH throughout the central nervous sys-
tem, all organs darkening the telson and uropods of Crago to approximately the
HORMONES IN CRUSTACEAN NERVOUS SYSTEMS
175
same degree. Great variations in distribution of the hormones occur among the
decapods. The Natantian, Crago apparently possesses significant CDH activity only
in the regions of the circumoesophageal connectives. CDH is differentially distrib-
uted throughout the central nervous system of the anomurans with highest quantity
usually in the posterior region of the thoracic cord, is relatively uniformly distributed
within the central nervous system of the astacurans and Palaemonetes, and is en-
tirely absent within that of the brachyurans.
The quantitative distribution of CBLH was considered here solely within the
reptantian nervous system, although it is known to be present throughout the cen-
tral nervous system of the natantians (Brown and Wulff, 1941). Both the anomu-
rans and brachyurans show wide distribution of this principle throughout brain.
TABLE III
The quantitative distribution of CBLH activity within the central nervous systems of a number
of crustaceans. The region of maximum body -lightening is arbitrarily assigned the value — 1.00.
The + values indicate body-darkening.
Classification
Species or genus
Brain
Connec-
tives
Thoracic
cord
Abdominal
cord
Isopoda
Idothea baltica
pres.
pres.
pres.
pres.
Decapoda
Natantia
Crago septemspinosus
pres.
pres.
pres.
pres.
Palaemonetes vulgaris
pres.
pres.
pres.
pres.
Reptantia
Astacura
Homarus americanus
+ 2.40
-1.00
+ 2.20
+ 7.20
Cambarus virilis
+ 1.09
-0.73
-1.00
+ 1.00
Anomura
Upogebia affinis
-0.64
-1.00
+0.27
-0.18
Pagurus sp.
-0.92
-0.85
-1.00
Emerita talpoidea
-1.00
-0.86
-0.57
Brachyura
Oxyrhyncha
Libinia sp.
-1.00
-0.67
-0.71
Brachyrhyncha
Cancer irroratus
pres.
pres.
pres.
Carcinides maenas
pres.
pres.
pres.
Ovalipes ocellatus
pres.
pres.
pres.
Uca pugilator
pres.
pres.
pres.
connectives, and thoracic cord. However, a striking feature is noted in the asta-
curans and the natantian, Palaemonetes, in which a darkening (see Fig. I A), as well
as a lightening, of the body occurs.
The two species of astacurans with which we have concerned ourselves more or
less parallel one another with respect to the distribution of CDH. In Homarus and
Cambarus the region of greatest quantity of this principle is the brain, and is fol-
lowed by an apparent gradual diminution of the substance from anterior to posterior
within the nervous system. The problem of CBLH distribution seems somewhat
more complex since, as has been previously mentioned, certain of these nervous-
system extracts appear to produce body-darkening preceded by a body-lightening.
The abdominal-cord extract is particularly active in body-darkening and only the
176
FRANK A. BROWN, JR., AND LORRAINE M. SAIGH
connectives and thoracic cords of Homarus and Cambarus show any body-lightening
activity at all. In these cases where body-lightening is indicated, the lightening
persists for only a short time and is followed by a definite darkening. These ob-
servations suggest that the body-darkening activity observable for extracts of the
astacuran central nervous system is explainable in terms of CDH. It is significant
that in no case is body-darkening ever obtained from a portion of the nervous
system lacking tail-darkening activity. However, since there is no essential direct
correlation between the degree of tail-darkening and the degree of body-darkening
even within a single species, the observed results must be the consequences of vary-
ing proportions of the two principles within the extracts, with the degree of influ-
ence of either one being a function of its relative concentration at any given instant.
There are significant differences in the distribution of CDH within the group
of anomurans. Pagurus and Emcrita exhibit similar tail-darkening activities and
these are shown chiefly by thoracic cord extracts. On the other hand, extracts of
TABLE IV
The responses of eyestalkless Crago to injections of extracts of parts of the thoracic cord of some
anomurans, showing the differing distributions of CBLH and CDH activity. No. of cases signifies
number of donors.
Body-lightening 0 or
Tail-darkening darkening ©
Time (min.) Time (min.)
Species
Part of thor.
No.
0
5
10
15
30
45
60
0
5
10
15
30
45
60
cases
Pagurus pollicaris
Anterior J£
8
0.0
0.3
0.3
0.4
0.3
0.0
0.0
0.0
-2.3
-2.2
-2.0
-0.6
-0.3
0.0
Second J4
8
0.0
0.6
0.7
0.7
0.2
0.0
0.0
0.0
-0.7
-0.6
-0.3
-0.3
0.0
0.0
Third y±
8
0.0
1.5
1.6
1.6
0.5
0.5
0.4
0.0
-0.4
-0.4
-0.3
0.0
0.0
0.0
Posterior J£
8
0.0
2.6
2.6
2.4
0.9
0.5
0.0
0.0
-0.5
-0.5
-0.3
-0.1
0.0
0.0
Pagurus
Anterior J£
6
0.0
0.5
0.5
0.5
0.0
0.0
0.0
0.0
-1.7
-1.6
-1.2
-0.4
-0.2
0.0
longicarpus
Second J4
6
0.0
0.2
0.2
0.2
0.0
0.0
0.0
0.0
-1.0
-1.0
-0.3
0.0
0.0
0.0
Third M
6
0.0
1.3
1.4
1.5
0.8
0.5
0.2
0.0
-1.2
-0.8
-0.2
0.0
+0.2
+0.2
Posterior J£
6
0.0
2.0
2.0
2.0
1.2
0.5
0.0
0.0
-0.5
-0.4
-0.2
-0.2
0.0
0.0
Emerila talpoidea
Anterior J^
4
0.0
0.8
0.8
0.8
0.3
0.0
0.0
0.0
-2.3
-2.3
-1.8
-0.5
0.0
0.0
Second }-£
4
0.0
0.0
0.0
0.0
0.0
().()
0.0
().()
0.0
0.0
0.0
0.0
0.0
0.0
Third H
4
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
-0.3
-0.3
0.0
0.0
0.0
0.0
Fourth Ji
4
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
-0.5
-0.5
0.0
0.0
0.0
0.0
Posterior J£
4
0.0
0.3
1.8
1.8
0.3
0.1
0.0
0.0
-0.3
+0.8
+0.8
0.0
0.0
0.0
brain, thoracic and abdominal cords of Upogebia all contain notable amounts of
CDH. Another similarity between extracts from Pagurus and Emerita is seen in
the distribution of CBLH. CBLH is found in considerable amounts in brain, con-
nectives, and thoracic cord of both genera. However, the thoracic cord extracts
of Upogebia show almost a complete absence of CBLH activity while the extracts
of the remaining parts of the central nervous system produce a definite body-
lightening, the connectives being most active in this respect.
The absence of CDH within the brachyurans investigated as well as the restric-
tion of this principle to the connective region of the natantians studied confirms the
results of Brown and Ederstrom (1940). Experimental data show that moderate
amounts of CBLH are found in brain, thoracic cord, and connectives. Although
results for CBLH distribution for brachyurans are shown only for Libinia, it has
been found that they are qualitatively the same for Uca, Cancer, Carcinides, and
HORMONES IN CRUSTACEAN NERVOUS SYSTEMS 177
Oval i pcs. The striking body -lightening effect of a strong extract of Uca connec-
tives and commissures is illustrated in Figure IB.
An attempt was made to analyze further the localization of CDH and CBLH
within the thoracic cords of Emcrita and two species of Pagnnis: pollicaris and
longicarpns (Table IV). The procedure consisted of dividing the thoracic cords
into a number of approximately equal portions, four in the case of Pagunis and
five in that of Emerita. It was observed that the concentration of CDH within
the thoracic cord of both P. pollicaris and P. longicarpus is greatest in the posterior
fourth of the cord and decreases gradually along the cord as one proceeds anteriorly.
In Emcrita the highest region of CDH concentration is also the posterior portion
of the thoracic cord. However, there is a lack of CDH in any of the central por-
tions of the thoracic cord in Emcrita. It would seem then that the distribution of
CDH in the thoracic cord of Emcrita is more restricted than in Pagunis.
The distribution of CBLH in the thoracic cord of P. pollicaris and P. longicarpus
is similar. The most intense body-lightening effect is brought about by extracts
of the anterior fourth of the cord while less intense reactions are produced by ex-
tracts of the remaining portions. Experiments with extracts of Emcrita thoracic
cord indicate a higher concentration of CBLH in the anterior portion of the cord,
and apparent absence of CBLH in the second portion and only slight amounts of
the principle in the third, fourth and fifth divisions of the cord. In summarizing
the distribution of CDH and CBLH within the thoracic cords of Pagnnis and
Emcrita we can say that CDH is relatively more concentrated posteriorly in the
thoracic cord while CBLH appears more concentrated anteriorly.
DISCUSSION OF RESULTS
The effect of the extracts of the central nervous system upon the dark pigments
of the telson and uropods of Crago possesses a characteristic pattern in each of the
major groups of the order Decapoda. In the Xatantian, Crago, we have observed
the restriction of CDH activity to the circumoesophageal connectives, whereas the
Astacnra and Palacmonctcs exhibit a more generalized occurrence of the hormone
within the organs of the central nervous system. However, as one proceeds to the
Anoinnra. these contain changes from the widespread condition in the astacurans to
a more specialized one as evidenced by the restriction of CDH in the thoracic cord of
two of the three genera examined. Finallv there is an entire lack of CDH among the
O J
brachyurans.
Experimental data concerning the distribution of CBLH in the reptantians pre-
sent an interesting problem. Although both the anomurans and brachyurans pos-
sess the body-lightening hormone in varying amounts throughout the entire central
nervous system, the astacurans appear to limit the hormone to connectives and
thoracic cord. The simplest explanation for the body-darkening activity of the
astacuran central-nervous-system extracts involves action of the tail-darkening prin-
ciple. It is thought that CDH produces body-darkening after CBLH has been ex-
hausted or in the absence of CBLH. This is indicated in Figure 2 in which selected
portions of the central nervous system of Libinia, Cainbanis, and Homarus are
shown to produce a graded series of differential effects upon the coloration of the
body of eyestalkless Crago. These range all the \vay from maximum body-lightening
and no trace of darkening (Libinia brain) through initial lightening followed by
178
FRANK A. BROWN, JR., AND LORRA1NK M. SAIGH
darkening, to immediate and extensive body-darkening (Hoinanis abdominal cord).
These results are believed to be explained in terms of different relative amounts of
CDH and CBLH. The former is known to be absent in the case of Libinia, and
it is assumed that the latter is absent or nearly so in the case of Honianis abdominal
cord. In the case of the extracts of Honuinis thoracic cord and connectives and
those of Cainbarus thoracic cord, CBLH is present in small amounts and lightens
the body for a short time, thereby delaying the darkening influence of CDH on the
body.
A comparison of tail-darkening and body-darkening within Craf/o injected with
nervous system extracts from numerous sources suggests a rough positive correla-
tion between the two (Fig. 3). Generally speaking, we may infer from these data
that the tendency towards body-darkening is greater in those animals showing a
high degree of tail-darkening. This gives further support for an active role of
CDH in body-darkening.
Unlike the Dccapoda the Isof>oda apparently exhibit a uniform distribution of
CDH within the central nervous system. However, since only a single species was
considered, further experimentation is deemed necessary before any decisive state-
ment is made concerning CDH distribution within this group.
10
20
TIME
30
IN MINUTES
FIGURE 2. The influences of extracts of selected portions of the central nervous system of
some crustaceans upon the body coloration of eyestalkless Crago.
From most positive to most negative at the end of 10 minutes are shown, respectively, Huniants
abdominal cord, Homarus brain, Hoiuanis thoracic cord, Huniants circumoesophageal connectives,
Cainbarus thoracic cord, and J.ibinia brain. Concentration in each experiment was: organs of
one specimen/0.5 ml. sea-water.
HORMONES IN CRUSTACEAN NERVOUS SYSTEMS
179
20
15
O
10
o-
o o
0°°
o
o
o
00
o o o oo
-IO -5 O +5
BODY LIGHTENING (-) OR DARKENING
•HO
FIGURE 3. The general relationship between the degree of darkening or lightening of the
body proper of eyestalkless Crago and the degree of darkening of the telson and uropods.
Darkening of the tail is expressed as the algebraic sum of the intensities of the reactions at 5,
10, 15, 30, 45, and 60 min. following extract injection, thereby including a measure of both in-
tensity and duration of the effect. Body-lightening, being more rapidly transitory, is expressed
as the algebraic sum of the values at 5, 10, 15, and 30 min.
SUMMARY
1. A survey was made of the effects upon Crago color-change of sea-water ex-
tracts of various parts of the central nervous system of 'thirteen species of higher
crustaceans. The crustaceans represented the groups Isopoda, Natantia, Astacura,
Anomura, and Brachyura.
2. Extracts of various portions of the nervous system among the various groups
showed wide differences in their total chromatophorotropic activities, producing
various degrees of telson and uropod darkening and of body-lightening and
darkening.
3. An analysis of the results gave support to the hypothesis that most crustacean
nervous systems possess at least two principles, a) a Crago body-lightening prin-
ciple, CBLH, lightening all portions of the body except telson and uropods, and b}
a CVa<70-darkening hormone, CDH, darkening the telson and uropods, and, in the
absence of CBLH, the body as well.
180 FRANK A. BROWN, JR., AND LORRAINE M. SAIGtt
4. CBLH is more or less uniformly distributed throughout the nervous systems
of all the species examined except the astacurans in which it is demonstrated only
for the circumoesophageal connectives and thoracic cord.
5. CDH is restricted to the circumoesophageal connective region of the Natantia,
is differentially distributed throughout the nervous systems of anomurans, with
highest concentration in the posterior region of the thoracic cord, and is distributed
throughout the nervous systems of the other species except the brachyurans in
wrhich it is absent.
LITERATURE CITED
BROWN, F. A., JR., 1933. The controlling mechanism of chromatophores in Palaemonetes.
Proc. Nat. Acad. Sci, Washington, 19: 327-329.
BROWN, F. A., JR., 1935. Control of pigment migration within the chromatophores of Palaemo-
netes vulgaris. Jour. Exp. Zool., 71 : 1-15.
BROWN, F. A., JR., 1946. The source and activity of Crago-darkening hormone (CDH).
Physiol. Zool., 19: 215-223.
BROWN, F. A., JR., AND H. E. EDERSTROM, 1940. Dual control of certain black chromatophores
of Crago. Jour. Exp. Zool., 85 : 53-69.
BROWN, F. A., JR., AND A. MEGLITSCH, 1940. Comparison of the chromatophorotropic activity
of insect corpora cardiaca with that of crustacean sinus glands. Biol. Bull., 79 : 409-
418.
BROWN, F. A., JR., AND V. J. WULFF, 1941. Chromatophore types in Crago and their endo-
crine control. Jour. Cell. Comp. Physiol., 18 : 339-353.
HANSTROM, B., 1937. Die Sinusdruse und der hormonal bedingte Farbwechsel der Crustaceen.
Kungl. Svenska Vetenskap. Handl, 16: Nr. 3, 1-99.
Hosoi, T., 1934. Chromatophore activating substance in the shrimps. Jour. Fac. Sci. Imp.
Univ. Tokyo, 3 : 265-270.
KNOWLES, F. G. W., 1939. The control of white-reflecting chromatophores in Crustacea. Pub-
bli. Stas. Napoli, 17: 174-182.
ROLLER, G., 1930. Weitere Untersuchungen iiber FaYw'echsel und Farbwechsel-hormonen.
Biol. Centralbl, 50: 759-768.
PHYSIOLOGICAL OBSERVATIONS ON WATER LOSS AND
OXYGEN CONSUMPTION IN PERIPATUS x
PETER R. MORRISON
Biological Laboratories, Harvard University, Cambridge, Massachusetts
The small group of species which comprises the Onychophora have long been
of interest because of their unique combination of arthropod and annelid characters
which places them in a phylogenetic position intermediate to those two extensive
groups (Snodgrass, 1938). They are further of interest because of their close
homogeneity despite a sporadic distribution that encompasses a large portion of the
world and points to an ancient separation of some of the genera (Clark, 1915 ; Brues,
1923). This homogeneity appears to be physiological and ecological as well as
morphological,2 since Peripatus is restricted everywhere to a moist but terres'rial
environment. Further, their sporadic and fluctuating local distribution suggests
that environmental variation, presumably in moisture, is actively limiting their oc-
currence even in the regions where they are found.
Physiological observations on members of this group, then, are of interest, and
it is of particular interest to examine the process of water loss and to contrast Perip-
atus in this respect to comparable annelids and arthropods. Manton and Ramsay
(1937) have reported a value for water loss in Peripatus (Pcripatopsis) at 30°
and with wind velocity of 7.0 m./sec. (16 m.p.h.). These conditions, however,
seem rather severe for a species which is uncomfortable at temperatures above 20°
(Manton, 1938) and which, living in crevices, must have little exposure to wind.
The experiments reported here were made under conditions which more nearly ap-
proximate those encountered by the animal in nature.
In this connection reports in the literature suggest that there may have been
some temperature adaptation in the Onychophora. Thus the two species studied
here, both from Panama (lat. 9° N.), stayed in good condition at a temperature of
25° =t. In contrast as already noted, Peripatus from near Cape Town (lat. 34° S.)
became uneasy at temperatures above 20° although low temperatures, even down to
freezing, did not bother them. They survived very well in England (Manton,
1938; Sedgwick, 1885) as have specimens from New Zealand (lat. 40° S.). The
latter were only successfully transported through the intervening tropical regions
with the aid of refrigeration (Sedgwick, 1887). Peripatus from New Zealand
(Hutton, 1876) and from Australia (Steel, 1896) are reported to become torpid
during the winter but with no subsequent ill effects. On the other hand Sclater
(1887) reported that his specimens from British Guiana (lat. 7° N.) successfully
1 These observations are by no means complete, but because the literature contains little data on
living Onychophora, particularly on New World species, and because there was no immediate
prospect of obtaining a further supply of these unusual animals, it seemed advisable to present
them at this time.
2 It should be noted, however, that for such a small group of Onychophora show remarkable
diversity in their embryological development and their mode of reproduction.
181
182 PETER R. MORRISON
survived the trip to England, "but unfortunately were much affected by the cold,
and were therefore killed."
MATERIAL
These Peripatus were secured on Barro Colorado Island, Canal Zone, through
the great kindness of Mr. James Zetek. Two species (note Clark and Zetek, 1946)
were obtained, the larger of which (Epipcripatus brasiliensis varians) had a con-
tracted length of 50 mm. and was uniformly colored a rich red-brown. The smaller
species (Oroperipatus corradi) had a contracted length of 25 mm. and was a choco-
late color with lighter underside and with darker legs and a dark, median, dorsal
stripe 0.3 mm. in width. The animals were taken in -early September and these
observations were made in Cambridge about a month later. During the interim
they were kept in moist forest debris but were not given suitable food other than the
supply of termites initially in the debris. The animals survived the trip well and
apparently stayed in good health until just before death which presumably occurred
through starvation.
The general behavior of these individuals corresponded to that described for
other species (Manton, 1938; Holliday, 1942; Andrews, 1933; Steel, 1896; Sedg-
wick, 1885; etc.). They were retiring and preferred to remain inactive in some
dark crevice. They are sensitive to light but react even more sharply to dryness
which stimulates them to constant activity. The smaller species were definitely
more sensitive in this respect and could not be held still even for a moment.
An occurrence involving an individual of the larger species may be of particular
interest. On the occasion of mechanical injury to one of its antennae that member
was placed in the mouth and the injured portion, about half the length, was removed.
The stump healed and the individual did not appear to be inconvenienced by the
loss. Parturition as observed in these specimens has been described elsewhere
(see Morrison, 1946).
The rate of oxygen consumption and water loss in Peripatus was compared
to several arthropods and annelids of fairly similar size, habitat and body form :
centipeds (Lithobius) ; millipeds (Julits} ; sow bugs (Onlscus) ; and earthworms.
These were all collected locally with the exception of one small tropical earthworm
found among the debris.
OBSERVATIONS
Sensory responses
With the exception of the antennae the animals showed equal tactile sensitivity
all over the body, on the dorsal and ventral surfaces and on the legs. A very light
stimulation could be applied with no response, a light one produced a local with-
drawal of a leg or small section of the body, while a strong stimulus led to a general
withdrawal. Holliday (1942) noted that fairly large wood lice and centipeds could
crawl over the body of a Peripatus without evoking any response. The antennae
are much more sensitive and the lightest touch here results in the retraction of one
or both. With stronger or repeated stimulation the animal will completely contract
and change its direction of progression ; further irritation provoked the well known
ejection from the slime glands. These responses are in accord with the histological
findings of Manton (1937) that while a single well ensconced sense capsule was
OBSERVATIONS ON PERIPATUS 183
found in each primary body papilla, each antennal papilla bore at least three much
more exposed capsules with much heavier innervation.
The animals usually walked forwards but when startled would often reverse
their direction, apparently walking backwards with equal ease. Occasionally they
would half turn backwards and then move in the form of a "U" with the legs of
the anterior half walking forward and those of the posterior half walking backwards.
This mode of progression must impose an interesting problem in coordination.
The response of the animals to a single point source of light (a two-cell flashlight
with reflector and glass removed, at a distance of 0.5 to 1 m.) was recorded by trac-
ing their path on a large underlying sheet of paper. A number of records were
made both with the light fixed and with it moved through 90 or 180° halfway
through the record. Examination of the records showed no oriented negative
phototropism ; indeed, the animals actually travelled towards the light more often
than away from it. Thus these animals would appear to be unable to localize light
but only to be aware of it. This corresponds to the observations of Manton (1938)
that the movement of objects near Peripatus elicited a response only when accom-
panied by air movement. These experiments were not carried out in a saturated
environment, however, and it is possible that with the very strong stimulus of dry-
ness removed, some phototropic pattern might be observed.
Water balance
In measuring water loss the animals were placed in large (D = 5 cm.) flat,
weighing bottles containing a layer of calcium chloride covered by a floor of brass
gauze. Measurements were made at 24° which is within the range normally en-
countered by these species (Kenoyer, 1929), and for periods of 30 minutes. No
circulation was supplied, the movement of the animals themselves providing for
convection. The Peripatus were particularly uneasy in this very dry atmosphere
and kept in constant and vigorous motion.
The values obtained for the two species of Peripatus and for several other ani-
mals are summarized in Table I. Water loss has been computed on the basis of
both body weight, and the two-thirds power of the body weight.3 The latter is
perhaps a more reasonable basis for comparing animals of different size. The two
values for Peripatus agree well and lie between those found for the annelids and
arthropods. They indicate that Peripatus has a twofold advantage over the earth-
worm 4 in the conservation of water ; and that it is at a twofold disadvantage as
compared to the centiped, the most xerosensitive arthropod studied. Other arthro-
pods showed values ranging down to one-twentieth that observed in Peripatus.
These data are presented graphically in Figure 1.
Manton and Ramsay (1937) reported on water loss in Peripatus under the
much more rigorous conditions of 30° with a 7 m./sec. (16 m.p.h.) wind and a rela-
3 This quantity is proportional to the surface area in animals of similar body form. In
Peripatus and the arthropods where the actual body surface is increased by appendages and
papillae, loss of water very probably takes place largely through the trachae (note Mellanby,
1935). Water loss will therefore be related to respiration which is also roughly proportional
to the two-thirds power of the body weight in animals of different size (Krogh, 1916).
4 This will be a minimum figure since the body weight of the earthworm includes a con-
siderable amount of dirt in the gut. These earthworms were kept in clean wet containers for
l1/^ days before use, during which time they evacuated up to 15 per cent of their weight, but
more undoubtedly remained.
184
PETER R. MORRISON
tive humidity of 27.5 per cent. They found a value of 13.0 mg./g. min. or 2 to 3
times our value. A similarly measured value for an earthworm was about half as
large on a weight basis or of equal magnitude on the basis of surface area. How-
ever, the advisability of making measurements under physiological conditions should
be stressed since under abnormal circumstances quite different relations may hold.
Thus, for example, Ramsay (1935) showed that in the cockroach water was lost
O
UJ
Q_
i
25
20
* 15
c/)
O
.J
CL
UJ
10
0.2
0.4
0.6
0.8
BODY WEIGHT IN GRAMS
FIGURE 1. Water loss in Peripatus and other animals at 24° over calcium chloride as a
function of the body weight. Open circles, earthworms ; crossed circles, Peripatus ; half-closed
circles, centipeds ; lined circles, sow bugs ; closed circles, millipeds. The curves represent
}" -= K (X)2/3, where the values for K are the average values given in Table I.
much more rapidly at temperatures above 30° with an apparent breakdown of the
hydrophobic character of the body surface.
In considering this function it is of interest to note that Clark (1915) concluded
on the basis of distributional and taxonomic considerations that the Onychophora
had originally evolved in a cooler rather than a warmer environment. Thus, the
more primitive groups are found on mountains or in the "temperate" regions while
OBSERVATIONS ON PERIPATUS
185
the more recent forms are tropical. This is, of course, entirely in accord with the
physiological considerations since the xerotic stress would be reduced at a lower
temperature and such an environment would be more favorable for evolution from
an aquatic to a terrestrial mode of life.
TABLE I
Water loss in Peripatus and other animals at 24° over calcium chloride
Water loss
. . .
Number and weight
Duration of experi-
in mg.
ment in min.
mg./g.min.
mg./g.2/3min.
Earthworm
884
15
7.4
7.1
703
15
12.0
10.4
360
15
13.4
9.6
208
10
17.3
; 10.4
105
8
22.6
10.7
Peripatus
Epiperipatus
788
30
5.2
4.7
Oroperipatiis
423
30
6.6
5.0
Centiped
150
60
5.6
3.0
135
20
5.9
3.1
4X95
60
4.1
1.9
4X63
30
7.2
2.8
Sow bug
158
25
3.0
1.6
97
40
2.5
1.2
6X49
60
3.0
1.1
48
20
4.5
1.6
Milliped
3X76
120
0.56
0.24
3X98
600
0.44
0.21
A verages
Earthworm
15 Experiments
9.9
Peripatus
2 Experiments
4.9
Centiped
4 Experiments
2.5
Sow bug
5 Experiments
1.3
Milliped
2 Experiments
0.22
Respiration
The oxygen consumption of the larger species of Peripatus and of various other
animals was measured in a Warburg apparatus.5 Carbon dioxide was absorbed in
sodium hydroxide in a small cup fused to the bottom of the chamber. The animals
were placed directly in the chamber and were kept from the lye by a small screen
shield. Measurements were made at 25.0° C. over a period of 60 minutes.
The results on Peripatus are shown in Figure 2. After a restless initial period
(10 minutes) it settled down to a very uniform rate of oxygen consumption. The
centipeds, also shown in Figure 2, were less regular. The results for the various
5 I am indebted to Dr. William Carroll for the use of his calibrated Warburg assembly.
186
PETER R. MORRISON
animals are summarized in Table II. The exact significance of the "resting" or
"basal" oxygen consumption is not known but some correlation between it and the
"intensity" of the organism has been observed. Compared on a weight basis Perip-
atus consumes oxygen at the same rate as the earthworm and at about half the rate
of the arthropods. It has been observed, however, that within a given group, the
metabolism per unit of weight varies with the size of the animal (note Edwards,
1946, for example), and that the metabolism is more nearly proportional to some
16
ID
O
Q.
2
ID
(/)
Z
O
o
LL)
O
X
o
8
20
40
60
TIME IN MINUTES
FIGURE 2. Oxygen consumption in Peripatus and the centiped as a function of time. Open
circles, Peripatus (Epiperipatus), 0.68 g. ; closed circles, 3 centipeds, total weight 0.33 g. ;
temperature, 25°.
lower power of the weight. As a first approximation this may be taken as the
two-thirds power (Krogh, 1916). When the oxygen consumption is compared on
this basis, Peripatus agrees more closely with the arthropods and has a higher value
than the earthworm.
The hydrophobic character of the body surface has been noted by many ob-
servers. It is particularly evident when the animal is submerged since the body
papillae hold the water away from the body surface and leave the animal entirely
OBSERVATIONS ON PERIPATUS
187
surrounded by a sheath of air. It would seem entirely possible that this air sheath
may function as a respiratory surface under water. Such a mechanism has been
demonstrated in certain aquatic insects which carry down an air supply by means
of hydrophobic hairs and which, by this means, greatly extend their periods under
water (Krogh, 1941 ; Wigglesworth, 1931). Since Peripatus must be often covered
by water in rainstorms, particularly as its lack of resistance to dessication forces it
to frequent wet places, this mechanism could be of real utility and have a consider-
able survival value. This would provide a functional explanation for the papilla-
covered body surface which is characteristic and unique in the Onychophora.
TABLE II
Oxygen consumption in Peripatus and other animals at 25° C.
Oxygen consumption
Animal
Weight in mg.
cc./g.hr.
cc./g.2«hr.
Earthworm
96
0.22
0.10 '
Peripatus
(Epiperipatus)
680
0.23
0.20
Millipeds
3X111
0.46
0.22
Centipeds
2X69
0.56
0.22
Pill bugs
5X61
0.35 2
0.14
1 Lesser (1908) reported values of 0.4 cc. per g.2/3hr. at 19° at which temperature the oxygen
consumption should be about half that measured at 25° (Vernon, 1897).
2 Edwards (1946) reports a similar value but at a temperature of 17°.
SUMMARY
The Onychophora represent a morphological transition between the annelids and
the arthropods. They also represent a physiological transition between the aquatic
and the terrestrial environment. In the latter transition the most important adapta-
tions are those involving the functions of water conservation and respiration.
The ability of Peripatus to conserve water has been compared to that of com-
parable annelids and arthropods. Peripatus is shown to be intermediate to those
two groups in this function, losing twice as much water as the centiped, but only
one-half as much as the earthworm. This corresponds to its taxonomic and ecologi-
cal positions.
The "resting" rate of oxygen consumption has also been compared to other ani-
mals. The rate in Peripatus is comparable to that in the arthropods and larger than
that in the earthworm.
It is suggested that the unique papilla-covered body surface may represent an
adaptation for underwater respiration to meet the environmental restriction imposed
by the inadequate regulation of water loss.
LITERATURE CITED
ANDREWS, E. A., 1933. Peripatus in Jamaica. Quart. Rev. Biol., 8 : 155.
BRUES, C. T., 1923. The geographical distribution of the Onychophora. Amcr. Nat., 57: 210.
CLARK, A. H., 1915. The present distribution of the Onychophora, a group of terrestrial in-
vertebrates. Smithsonian Misc. Coll., 65 : 1-25.
188 PETER R. MORRISON
CLARK, A. H., AND J. ZETEK, 1946. The Onychophores of Panama and the Canal Zone. Proc.
U. S. Nat. Mus., 96 : 205.
EDWARDS, G. A., 1946. The influence of the temperature upon the oxygen consumption of sev-
eral arthropods. Jour. Cell. Comp. Physiol., 27 : 53.
HOLLIDAY, R. A., 1942. Some observations on Natal Onychophora. Ann. Natal. Mus., 10: 237.
HUTTON, F. W., 1876. On Peripatus Novae-Zealandiae. Ann. Mag. Nat. Hist. (4) 18: 361.
KENOYER, L. A., 1929. General and successional ecology of the lower tropical rain forest at
Barro Colorado Island, Panama. Ecology, 10: 210.
KROGH, A., 1916. The respiratory exchange of animals and man. London, Longmans, Green
and Co., 173 pp.
KROGH, A., 1941. The comparative physiology of respiratory mechanisms. Philadelphia, The
University of Pennsylvania Press, 172 pp.
LESSER, E. J., 1908. Chemische Prozesse bei Regenwiirmern. Zeit, f. Biol., 50: 421.
MANTON, S. M., 1937. Studies on the Onychophora. II. The feeding, digestion, excretion and
food storage of Peripatopsis. Proc. Roy. Soc. London, B, 227 : 411.
MANTON, S. M., 1938. Studies on the Onychophora. VI. The life history of Peripatopsis.
Ann. Mag. Nat. Hist. (11), 1 : 515.
MANTON, S. M., AND J. A. RAMSAY, 1937. Studies on the Onychophora. III. The control of
water loss in Peripatopsis. Jour. Exp. Bio!., 14: 470.
MELLANBY, K., 1935. The evaporation of water from insects. Biol. Rev., 10: 317.
MORRISON, P. R., 1946. Parturition in Peripatus. Psyche (In press).
RAMSAY, J. A., 1935. The evaporation of water from the cockroach. /. Exp. Biol., 12 : 373.
SCLATER, W. L., 1887. Notes on the Peripatus of British Guiana. Proc. Zool. Soc. London,
130.
SEDGWICK, A., 1885. A monograph on the development of Peripatus capensis. Quart. Jour.
Micr. Sci., 25 : 449.
SEDGWICK, A., 1887. A monograph of the genus Peripatus. Quart. Jour. Micr. Sci., 28: 431.
SNODGRASS, R. E., 1938. Evolution of the Annelida, Onychophora, and Arthropoda. Smith-
sonian Misc. Coll., 97 : 6.
STEEL, T., 1896. Observations on Peripatus. Proc. Linn can Soc. New South Wales, 21 : 94.
VERNON, H. M., 1897. The relation of the respiratory exchange of cold blooded animals to
temperature. Jour. Physiol., 21 : 443.
WIGGLESWORTH, V. B., 1931. The respiration of insects. Biol. Rev., 6: 181.
J
STUDIES ON CILIATES OF THE FAMILY ANCISTROCOMIDAE
CHATTON AND LWOFF (ORDER HOLOTRICHA,
SUBORDER THIGMOTRICHA)
III. ANCISTROCOMA PELSENEERI CHATTON AND LWOFF,
ANCISTROCOMA DISSIMILIS SP. NOV., AND
HYPOCOMAGALMA PHOLADIDIS SP. NOV.
EUGENE N. KOZLOFF
Lewis and Clark College, Portland, Oregon
INTRODUCTION
Chatton and Lwoff described in 1926 two ciliates for which they created the
genus Ancistrocorna: A. pelseneeri, from the gills and palps of Macoma balthica
(L.) ; and A. pholadis, from Barnea (Pholas) Candida (L.). Their descriptions of
these two species are of a preliminary nature and are not accompanied by illustra-
tions. More detailed descriptions of A. pelseneeri, together with illustrations, are
given in two papers of Raabe (1934, 1938).
Kofoid and Bush (1936) described as Parachaenia myae a ciliate from the peri-
cardial cavity and excurrent siphon of My a arenaria L. which Kirby (1941) noted
was in several respects apparently identical with A. pelseneeri. Kudo (1946),
however, listed Parachaenia myae as a valid species in the suborder Gymnostomata.
Kofoid and Bush stated that they did not find P. myae in any other molluscs which
were present in the same localities as the host species. I have studied the ciliate
associated with Mya arenaria in San Francisco Bay and have compared it with
similar forms from Cryptomya calif ornica (Conrad), Macoma inconspicua Broderip
and Sowerby,1 Macoma nasuta (Conrad), and Macoma ims (Hanley) from San
Francisco Bay, and from Macoma sect a (Conrad) from Tomales Bay, California.
I have concluded that the ciliate described by Kofoid and Bush as Parachaenia myae
is not specific in Mya arenaria and that P. myae is identical with Ancistrbcoma
pelseneeri Chatton and Lwoff.
On the gills and palps of the rock-boring piddock Pholadidea penita (Conrad)
there occurs a species of Ancistrocoma which is clearly distinct from A. pelseneeri
and which I will describe in this paper as Ancistrocoma dissimilis sp. nov. Another
ciliate I have studied from P. penita is referable to the genus Hypocomagalma, cre-
ated by Jarocki and Raabe (1932) for H. dreissenae, from the fresh water mussel
Drcissena polyrnorpha (Pall.). It will be described herein as Hypocomagalma
pholadidis sp. nov.
1 By some malacologists the small species of Macoma referred to in this paper as M. incon-
spicua is considered to be conspecific with M. balthica; by others it is considered to be a sub-
species of M. balthica. No conclusive evidence has been presented in the literature in recent
years either to support or refute these contentions.
189
190
EUGENE N. KOZLOFF
ANCISTROCOMA PELSENEERI CHATTON AND LWOFF
(Figure 1 ; Plate I, Figs. 1, 2)
The body is elongated and somewhat flattened dorso-ventrally.2 As seen in
lateral view, the ciliate is banana-shaped, the ventral surface being incurved. The
anterior end is more or less attenuated. The body is usually wjdest and thickest
in its posterior third. Forty living individuals taken at random from Mya arenaria
ranged in length from 50^ to 83 p., in width from 14 /A to 20 /*, and in thickness
from 11 /A to 16^,, averaging about 62 ,11 by 16 /x by 12.5 //. Twenty individuals
from Macoma inconspiaia ranged in length from 52 /u. to 78 /*, in width from 14 ju,
to 19 p., and in thickness from 11 p. to 15 /JL.
FIGURE 1. Ancistrocoma pelsenceri Chatton and Lwoff. Distribution of ciliary rows, somewhat
diagrammatic.3 A, dorsal aspect ; B, ventral aspect.
The anterior end is provided with a contractile suctorial tentacle which enables
the ciliate to attach itself to the epithelial cells of the gills and palps of the host and
to suck out their contents. The internal tubular canal continuous with the tentacle
is directed at first dorsally and then ventrally and obliquely toward the right side
of the body. It can usually be traced in fixed individuals stained with iron hema-
toxylin for about two-thirds the length of the body. Kofoid and Bush suggested
only in the title of their paper that the form which they named Parachaenia myae
was parasitic in Mya arenaria, but did not describe attachment of the ciliate to the
epithelium. They found the ciliate in the pericardial cavity and excurrent siphon
2 Kofoid and Bush stated in their description of "Parachaenia myae" that the body of this
ciliate is bilaterally compressed, the transverse diameter being about two-thirds the dorso-ventral
diameter. Obviously their orientation of the form in question is not in agreement with the
orientation assigned to it by Chatton and Lwoff, Raabe, and myself.
3 All text-figures illustrating this paper are based on camera lucida drawings of individuals
fixed in Schaudinn's fluid and impregnated with activated silver albumose (protargol).
CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 191
oi the clams and apparently believed it to be unattached and to feed as a gymno-
stome, by producing a current in the medium by means of vigorous ciliary activity
which carries food particles to the mouth. They stated that they observed a few
instances of food taking, in which "debris containing bacteria enters the mouth and
moves along the cytopharynx, forming little globules which continue back and aggre-
gate in the large food vacuoles which distend the posterior part of the body." They
stated further that "stained specimens show some vacuoles containing broken-up
nuclear material similar to that of the epithelial cells which are removed when the
fluid is taken from the clam." I have not observed any instances of ingestion of
food such as that described by Kofoid and Bush, and although I admit it is perhaps
possible for the ciliates to ingest food in this manner, I believe that they are pri-
marily branchial parasites which feed by means of the suctorial tentacle.
The cilia of A. pelseneeri are disposed on the ventral, lateral, and dorso-lateral
surfaces of the body in longitudinal rows originating at the anterior end. In all
individuals which I examined carefully the number of ciliary rows was fourteen,
but Raabe stated that in some specimens there are but thirteen rows. According to
Raabe the ciliary system is composed of three separate complexes, the first consist-
ing of eight or nine rows spiralling from the left side of the body toward the right
and terminating progressively more posteriorly on the ventral surface, the second
consisting of two approximately meridional rows situated on the central part of the
ventral surface, and the third consisting of three rows spiralling from the right side
of the body toward the left and terminating on the ventral surface. After studying
a large number of the ciliates from Mya arenaria and Macoma inconspicua I cannot
agree with Raabe on this matter. The ciliary rows appear collectively to form a
single complex. There are usually five approximately equal rowrs about two-thirds
the length of the body occupying the central portion of the ventral surface ; these are
bounded on the right by three progressively longer and more widely-spaced rows
and on the left by six progressively longer and more widely-spaced rows. In some
specimens the number of longer rows on the left side is greater than six, in which
case the number of approximately equal and more or less meridional rows is pro-
portionately decreased. Some of the outer rows on either side of the body, which
originate on the lateral margins or on the dorsal surface, curve ventrally as they ex-
tend posteriorly, but the last two rows on the left side and the last row on the right
side are typically dorso-lateral in position over their entire length. The outermost
row on either side extends almost to the posterior tip of the body. Kofoid and
Bush stated that the ciliary rows of "Parachacnia myac" may unite with one an-
other, but I have never observed this to be the case, although in some seriously
shrunken fixed individuals a few of the rows converge in such a way that they ap-
pear to be united.
In one of the illustrations accompanying the first of Raabe's papers in which
there is a detailed discussion of A. pelseneeri (1934) the, outermost ciliary row on
the right side of the body is shown to originate as far anteriorly as the more cen-
tral rows, while the outer three or four rows on the left side are shown to originate
progressively more posteriorly. According to my own observations, however, the
outermost row on the right side originates at about the same level as the last row
on the left side. In all suitably impregnated individuals which I have studied the
eighth row from the right side originates a little posterior to the level of origin of
the adjacent ventral rows.
192 EUGENE N. KOZLOFF
The cilia of A. pelseneeri are 8 ^ to 10 p. in length. Those at the anterior end
of the body are usually the more active and may be employed for thigmotactic at-
tachment. Kofoid and Bush stated that the cilia of the "dorso-bilateral region" of
"Parachaenia myae" are about 20 /x long near the anterior end, becoming somewhat
shorter posteriorly ; the cilia of the ventral surface, on the other hand, were said
by them to be about one-half the length of those of the dorso-bilateral area. I have
noted, however, no significant disparity between the lengths of the cilia of various
parts of the ciliary system. When dissociated from the host the ciliate swims ener-
getically, rotating on its longitudinal axis or swaying from side to side.
In the original description of A. pelseneeri given by Chatton and Lwoff refer-
ence is made to a "frange peristomienne" which they supposed corresponded to the
peristomal fringe of cilia in species of Ancistrmna. In his paper of 1934, Raabe
described a short (approximately 13 /x long) row of basal granules lying in a dorsal
anterior depression just above the anterior part of the internal tubular canal which
he thought may represent the "frange peristomienne" described by Chatton and
Lwoff. In his paper of 1938, however, Raabe stated that on certain of his prepara-
tions of this ciliate he could distinguish a row of basal granules such as he described
in 1934, but did not refer to it as the peristomal fringe, and suggested that Chatton
and Lwoff may have mistaken the stained outline of the internal tubular canal for
a row of basal granules homologous with those of the peristomal fringe of ancis-
trumid ciliates. In my study of living, stained, and impregnated individuals of the
ciliate I believe to be A. pelseneeri I have found no evidence whatever of a dorsal
anterior depression or a row of basal granules such as that described by Raabe.
Kofoid and Bush described internal fibrillar structures, wrhich they believed to
represent elements of the neuromotor system, extending for a short distance poste-
riorly from an annular commissure ("cytostomal ring") around the "cytostome."
One of the fibrils was said by them to pass along the internal tubular canal ("cyto-
pharynx") to a slight thickening on the surface of the canal, then "towards the
dorsal surface where it joins a relatively large granule which is closely associated
with the mid-dorsal ciliary fibril." They stated further that "from points of the
cytostomal ring on the ventral side, two fibrils are given off which soon unite and
continue as a slender thread along the ventral surface of the cytopharynx." I have
been unable to detect any structures in A. pelseneeri which might be construed as
elements of a neuromotor system, but perhaps it is a siderophilic fibril-like structure
of the type that Kofoid and Bush described that Raabe may have thought to repre-
sent a series of basal granules. The "cytostomal ring" around the "cytopharynx"
was stated by Kofoid and Bush to be connected with the longitudinal ciliary rows,
EXPLANATION OF PLATE I
FIGURE 1. Ancistrocoma pelseneeri Chatton and Lwoff (from Mya arenaria). Ventral
aspect. Heidenhain's "susa" fixative-iron hematoxylin. X 1,680.
FIGURE 2. Ancistrocoma pelseneeri Chatton and Lwoff (from Macoma inconspicua) .
Lateral aspect from left side, from life.
FIGURE 3. Ancistrocoma dissimilis sp. nov. Ventral aspect. Schaudinn's fixative-iron
hematoxylin. X 1,680.
FIGURE 4. Hypocomagalma pholadidis sp. nov. Dorsal aspect. Schaudinn's fixative-iron
hematoxylin. X 1,260.
FIGURE 5. Hypocomagalma pholadidis sp. nov. Ventral aspect. Schaudinn's fixative-iron
hematoxylin. X 1,260.
CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill
193
ii\\
-
ii
.
r
JK;
PLATE I
194 EUGENE N. KOZLOFF
but I have not observed this to be the case in A. pclscnccri. As has been pointed
out above, some of the rows do not originate as close to the base of the suctorial
tentacle as others. It is possible that the structure referred to by Kofoid and Bush
as the "cytostomal ring" represents the siderophilic anterior edge of the contracted
suctorial tentacle.
The cytoplasm is colorless and contains numerous small refractile granules of a
lipoid substance. In the posterior part of the body there are in addition to typical
food vacuoles containing ingested fragments of epithelial cells one or more large
vacuoles containing globular masses usually of a dense, homogeneous character.
Raabe referred to this type of vacuole as "Konkrementenvacuole" and suggested
that since he observed the internal tubular canal to terminate very near the
"Konkrementenvacuole" the material within the vacuole may represent an accumula-
tion of waste material which was not digested and absorbed as the ingested food
material passed backward down the canal. It is quite true that these concrement
vacuoles do not resemble the typical food vacuoles of most other ancistrocomid
ciliates which I have studied. It would be interesting to determine whether or not
digestion and absorption take place in the internal tubular canal, and how the mate-
rial in the concrement vacuole, if it represents undigested wastes, is gotten rid of
by the ciliate.
The macronucleus is usually sausage-shaped, rarely ovoid, and typically is situ-
ated dorsally near the middle of the body. In some fixed specimens stained with
iron hematoxylin the chromatin appears to be distributed in irregular masses scat-
tered through the macronuclear material ; in other iron hematoxylin preparations
and in most specimens stained by the Feulgen reaction the chromatin is aggregated
into a dense reticulum enclosing vacuole-like clear spaces. In twenty individuals
from Mya arcnaria fixed in Schaudinn's fluid and stained by the Feulgen reaction
the macronucleus ranged in length from 11 ^ to 16 p. and in width from 4 p. to 7 p.
The micronucleus is ovoid, fusiform, or sausage-shaped, and usually is seen to
lie to the right of the macronucleus. In fixed and stained specimens the chromatin
is ordinarily aggregated into granules. In twenty individuals from Mya arcnaria
fixed in Schaudinn's fluid and stained by the Feulgen reaction the micronucleus
ranged in size from 1.2 /x by 3 ^ to 2.1 /j, by 3.2 ju.
Ancistrocoina pclscnccri is very common in M\a arcnaria in all localities in San
Francisco Bay where I have collected this mollusc. I have found it to be present,
although usually in smaller numbers, also in Cryptoniya calljornlca, Maconia incon-
splcua, M. nasuta, and M. irus from several localities in San Francisco Bay, and in
Macoma sccta from Tomales Bay. It is peculiar that this ciliate was not recorded
by Raabe from Mya arenaria at the marine biological station at Hel. Raabe listed
Sphcnophyra doslniac Chatton and LwofF. Hypocomidium grainun Raabe, and a
species of Ancistruma which he provisionally referred to A. cyclidioides (Issel),
from M. arcnaria. I have found 6". doslniac in a small percentage of M. arcnaria
and in a fairly large percentage of Cryptomya calijornica from San Francisco Bay.
I have also found in M. arcnaria the ciliate thought by Raabe to be A. cyclidioides,
but not Hypocomidium graimin.
Ancistrocoina pclscnccri Chatton and Lzvoff (= Parachacnia myac Kofoid and
Bush} •
Diagnosis: Length 50 /*-83 /JL (according to Kofoid and Bush 40/x-100/i), aver-
age about 62 p.; width 14//.-20/*, average about 16^; thickness 11 ^-16 /x, average
CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 195
about 12.5 JJL. The ciliary rows are fourteen (according to Raabe thirteen or four-
teen) in number and are distributed on the ventral, lateral, and dorso-lateral sur-
faces of the body. There are usually five approximately equal rows about two-
thirds the length of the body on the ventral surface, bounded on the right by three
progressively longer and more widely-spaced rows and on the left by six progres-
sively longer and more widely-spaced rowrs. The outermost row on either side
extends almost to the posterior tip of the body. The more central rows originate
close to the base of the suctorial tentacle, while the several outer rows on either side
originate progressively more posteriorly on the lateral margins and the dorsal sur-
face. Some of these rows curve ventrally as they extend posteriorly, but the two
outer rows on the left side and the outermost row on the right side are typically
dorso-lateral in position over their entire length. The macronucleus is usually
sausage-shaped. The micronucleus is ovoid, fusiform, or sausage-shaped. Para-
sitic on the epithelium of the gills and palps of Maconia balthica (L.) (Wimereux
[Chatton and Lwroff] ; Hel [Raabe] ) ; J\Iacoina inconspicua Broderip and Sowerby,
Macoma uasuta (Conrad), Maconia inis (Hanley), Cryptomya calijornica (Con-
rad) (San Francisco Bay, California) ; Maconia sccta (Conrad) (Tomales Bay,
California) ; My a arcnaria L. (Tomales Bay [Kofoid and Bush] ; San Francisco
Bay).
ANCISTROCOMA DISSIMILIS SP. NOV.
(Figure 2; Plate I, Fig. 3)
The body is elongated, attenuated anteriorly, and somewhat flattened dorso-
ventrally. The ciliary system, to be described presently, is disposed for the most
part on the incurved and slightly concave ventral surface. The body is widest and
thickest in its posterior third and rounded posteriorly. Twenty living individuals
taken at random ranged in length from 33 /x to 51 /x, in width from 10 ^ to 14.5 /x,
and in thickness from 8 /x to 12 /x, averaging about 44 ^ by 13 ^ by 10 /x.
The anterior end is provided with a contractile suctorial tentacle continuous
with an internal tubular canal. The canal is directed at first dorsally and then
ventrally and obliquely toward the right side of the body. In fixed specimens
stained with iron hematoxylin it can usually be traced posteriorly for about one-half
the length of the body.
The cilia of A. disshnilis are 7 it to 8 /x in length and are disposed in longitudinal
rows originating at the anterior end. The typical number of ciliary rows is eleven,
but specimens with twelve rows are not uncommon, and I have seen some with four-
teen rows. There are usually five approximately equal rows about three-fifths the
length of the body occupying the central portion of the ventral surface ; these are
bounded on either side by three progressively longer rows, the outermost rows being
three-fourths to four-fifths the length of the body. In specimens having twelve
ciliary rows there are four longer ro\vs on the left side instead of three ; in speci-
mens having fourteen rows there are four longer rows on the right side and five
longer rows on the left. In some cases, particularly if the number of ciliary rows
exceeds eleven, the five central rows are of unequal length, becoming progressively
longer from right to left. One or two of the outer rows on either side originate
on the lateral margin or the dorsal surface, usually a short distance posterior to the
level of origin of the other rows. These rows curve ventrally and inward as they
extend posteriorly, so that at least their distal portions are visible in ventral view.
196
EUGENE N. KOZLOFF
The cytoplasm is colorless and contains numerous small refractile granules of a
lipoid substance in addition to food inclusions. One or more larger food vacuoles
are usually present in the posterior part of the body. The contractile vacuole lies
near the middle of the body and opens to the exterior on the ventral surface.
The macronucleus is ovoid and situated dorsally near the middle of the body.
In fixed and stained preparations the outline of the macronucleus is nearly always
very irregular and the chromatin appears to be aggregated into a dense reticulum
enclosing vacuole-like clear spaces of varying size. In twenty individuals fixed in
Schaudinn's fluid and stained with iron hematoxylin the macronucleus ranged in
length from 6.8 ^ to 13.7 /n and in width from 5.4 ^ to 7.2 /A.
The micronucleus is typically ovoid, rarely spherical, and commonly is situated
a short distance anterior to or to one side of the macronucleus. In fixed and stained
FIGURE 2. Ancistrocoma dissimilis sp. nov. Distribution of ciliary rows, somewhat
diagrammatic. A, dorsal aspect ; B, ventral aspect.
preparations the chromatin appears to be dispersed in granules of varying size. In
twenty individuals fixed in Schaudinn's fluid and stained with iron hematoxylin
the micronucleus ranged in size from 2.2 ^ by 2.4 ,u to 2.2 ^ by 3.2 p..
I found Ancistrocoma dissimilis to be present on the gills and palps of twenty-
one of thirty-six specimens of Pholadidca pcnita which I examined from localities
near Moss Beach, California. It is sometimes found in association with Hypo-
comagalma pholadidis. In some individuals of P. penita I have encountered a ciliate
of the genus Sphenophrya which I hope to describe in a later paper and a species
of Boveria which may also be new.
Ancistrocoma disshnilis sp. nov.
Diagnosis: Length 33^-51^, average about 44^; width lO/x-14.5^, average
about 13 ju,; thickness 8 fi-12 /JL, average about 10 /x. The ciliary rows are eleven to
fourteen (typically eleven) in number and are distributed for the most part on the
CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill • 197
ventral surface and lateral margins of the body. Most of the rows originate on the
ventral surface close to the base of the suctorial tentacle, while one or two outer
rows on either side originate on the lateral margin or the dorsal surface and curve
ventrally and inward as they extend posteriorly. There are usually five approxi-
mately equal rows about three-fifths the length of the body bounded on the right
by three progressively longer rows and on the left by four progressively longer
rows. The outermost row on either side is three-fourths to four-fifths the length of
the body. The macronucleus is ovoid. The micronucleus is typically ovoid. Para-
sitic on the gills and palps of Pholadidea penita (Conrad) (Moss Beach, California).
Syntypes are in the collection of the author.
HYPOCOMAGALMA PHOLADIDIS SP. NOV.
(Figure 3; Plate I, Figs. 4, 5)
The body is elongated, strongly attenuated anteriorly, and markedly asymmetri-
cal. The anterior end is deflected toward the left and bent ventrally. The dorso-
ventral flattening characteristic of most ancistrocomid ciliates is not conspicuous in
this species. As viewed from the posterior end the body appears in its middle and
posterior portions to be almost as thick as wide. In its anterior third the body is
nearly round in cross section. Most fixed specimens are considerably distorted and
compressed in such a way that they appear to be widest near the middle. Twenty
living individuals taken at random ranged in length from 63 /* to 89 ju, in width from
18 ju to 25 JJL, and in thickness from 16 /z to 21 /*, averaging about 76 ^, by 22 /x by 19 //..
The anterior end is provided with a contractile suctorial tentacle continuous
with an internal tubular canal. The canal can usually be traced in fixed specimens
stained with iron hematoxylin down the middle of the attenuated anterior part of
the body and then obliquely toward the right side. I have not succeeded in demon-
strating the course of the canal beyond the anterior one-third of the body.
The cilia of Hypocomagalma pJwladidis are approximately 9/x to 10 ^ in length.
The ciliary system consists of twenty-four or twenty-five longitudinal rows. The
body is almost completely invested by cilia except for a cilia-free "cap" at the pos-
terior end. Two rows on the right side of the body usually appear to be set apart
from the others, but in some specimens the spacing between these rows and the adja-
cent rows on either side is not significantly wider than the spacing between some
of the other rows. Perhaps these two rows are homologous with the one or two
rows constituting the right ciliary complex of Crebricoma carinata (Raabe), Insig-
nicoma venusta Kozloff, and species of Hypocomides. They originate near the base
of the suctorial tentacle on the right margin or the dorsal surface close to the right
margin and curve ventrally and to the left as they extend backward. The outer
row, as seen in ventral view, is the longer and extends almost to the posterior end
of the body. The inner row terminates a short distance more anteriorly than the
outer row, but is conspicuously longer than the first of the next series of rows,
which usually is about two-thirds the length of the body. The first eight to ten
rows to the left of the two longer rows all originate at about the same level on the
ventral surface close to the base of the suctorial tentacle. The remaining rows,
which are disposed along the left margin of the body and on the dorsal surface,
originate progressively more posteriorly. The tenth or eleventh row of this com-
plex is usually the longest, although some of the shorter rows on the dorsal surface
198
EUGENE N. KOZLOFF
may terminate more posteriorly. The last ciliary row on the right side of the
dorsal surface is always the shortest row, originating at a point about one-third the
distance from the anterior end of the body to the posterior end and terminating at
a point about three-fourths or four-fifths the distance from the anterior end to the
posterior end.
The cytoplasm is colorless and contains numerous small refractile granules of a
lipoid substance in addition to food inclusions. One or more larger food vacuoles
containing fragments of cells from the epithelial tissues of the gills or palps of the
host are usually evident in the posterior part of the body. The contractile vacuole,
when single, is located near the middle of the body and opens to the exterior on
the ventral surface. In a larger percentage of the living specimens of H. pholadidis
which I examined there were two or more contractile vacuoles scattered through
FIGURE 3. Hypocomagaluia pholadidis sp. nov. Distribution of ciliary rows, somewhat
diagrammatic. A, dorsal aspect; B, ventral aspect.
the body which emptied their contents to the exterior on the ventral surface. In a
large percentage of the living specimens of H. pholadidis which I examined there
were two or more contractile vacuoles scattered through the body which emptied
their contents to the exterior independently of one another. Jarocki and Raabe
(1932) reported that in H. dreissenae the contractile vacuole was sometimes single,
but that in some specimens there were several smaller ones.
The macronucleus typically is sausage-shaped and lies in the posterior third of
the body, its longitudinal axis placed obliquely to the longitudinal axis of the body.
In light iron hematoxylin preparations and in specimens stained by the Feulgen
reaction the chromatin of the macronucleus appears to be aggregated into a dense
reticulum enclosing vacuole-like spaces which frequently contain globular masses
of deeply-staining material. In ten . individuals fixed in Schaudinn's fluid and
CILIATES OF THE FAMILY ANCISTROCOMIDAE. Ill 199
stained by the Feulgen reaction the macronucleus ranged in length from 12.5 ju, to
20 p. and in width from 5 /* to 8.9 /*.
The micronucleus is spherical and usually is situated a short distance anterior
to or to one side of the macronucleus. In most fixed and stained preparations the
chromatin appears to be homogeneous, although in some the chromatin appears to
be in part aggregated into granules or peripheral strands. In ten individuals fixed
in Schaudinn's fluid and stained by the Feulgen reaction the diameter of the micro-
nucleus ranged from 2.4 p. to 3.3 p..
I found Hypocomagalma pholadidis to be present on the gills and palps of
twenty-eight of thirty-six specimens of Pholadidea penita which I examined from
localities near Moss Beach, California. When the ciliate is dissociated from the
host it swims erratically, usually rotating on its longitudinal axis and tracing wide
arcs with its attenuated anterior end. The cilia of the anterior half of the body are
more active than those of the posterior half and are sometimes observed to beat
metachronously. The ventral cilia near the base of the suctorial tentacle are mark-
edly thigmotactic.
Hypocomagalma pholadidis sp. nov.
Diagnosis: Length 63 /*-S9 /u, average about 76 /*; width 18^-25^, average
about 22 p; thickness 16,u-21 /x, average about 19ft. The anterior end of the body
is attenuated, conspicuously deflected toward the left, and bent ventrally. The
ciliary system consists of twenty-four or twenty-five rows. Two long rows on the
right side of the body appear in most specimens to be set apart from the remaining
rows ; these two rows originate near the base of the suctorial tentacle and extend
almost to the posterior end of the body. The first eight to ten rows to the left of
these two longer rows originate at about the same level on the ventral surface, while
the remaining rows, disposed along the left lateral margin and the dorsal surface,
originate progressively more posteriorly. The contractile vacuole may be single or
represented by several independent vacuoles opening to the exterior on the ventral
surface. The macronucleus is sausage-shaped. The micronucleus is spherical.
Parasitic on the epithelium of the gills and palps of Pholadidea penita (Conrad)
(Moss Beach, California). Syntypes are in the collection of the author.
LITERATURE CITED
CHATTON, E., AND A. LWOFF, 1926. Diagnoses de cilies thigmotriches nouveaux. Bull. Soc.
Zool. France, 51 : 345.
JAROCKI, J., AND Z. RAABE, 1932. t)ber drei neue Infusorien-Genera der Familie Hypocomidae
(Ciliata Thigmotricha), Parasiten in Susswassernmuscheln. Bull. int. Acad. Cracovie,
Cl. Sci. math, not., B(II), 1932: 29.
KIRBY, H., 1941. Relationships between certain Protozoa and other animals. In Calkins, G.,
and F. Summers (editors) : Protozoa in Biological Research. Columbia University
Press, New York.
KOFOID, C., AND M. BUSH, 1936. The life cycle of Parachaenia myae gen. nov., sp. nov., a ciliate
parasitic in Mya arenaria Linn, from San Francisco Bay, California. Bull. Mus. Hist.
not. Belgique, 12, No. 22.
KUDO, R., 1946. Protozoology. 3rd ed. Charles C. Thomas, Springfield.
RAABE, Z., 1945. tiber einige an den Kiemen von Mytilus edulis L. und Macoma balthica (L.)
parasitierende Ciliaten-Arten. Ann. Mus. sool. polon., 10: 289.
RAABE, Z., 1938. Weitere Untersuchungen an parasitischen Ciliaten aus dem polnischen Teil
der Ostsee. II. Ciliata Thigmotricha aus den Familien: Hypocomidae Biitschli und
Sphaenophryidae Ch. & Lw. Ann. Mus. zo.ol. polon., 13: 41.
STUDIES ON CILIATES OF THE FAMILY ANCISTROCOMIDAE
CHATTON AND LWOFF (ORDER HOLOTRICHA,
SUBORDER THIGMOTRICHA)
IV. HETEROCINETA JANICKII JAROCKI, HETEROCINETA
GONIOBASIDIS SP. NOV., HETEROCINETA FLUMINI-
COLAE SP. NOV., AND ENERTHECOMA
PROPERANS JAROCKI
EUGENE N. KOZLOFF
Lewis and Clark College, Portland, Oregon
INTRODUCTION
The genus Heterocineta was established by Mavrodiadi (1923) for a ciliate
which he named Heterocincta anodontae, and which he had formerly believed to
represent a gregariniform stage in the development of Conchaphthirus anodontae
(Ehrenberg). Unaware of the fact that Mavrodiadi had abandoned his earlier con-
ception and applied the name Heterocincta anodontae to this ciliate, Jarocki and
Raabe (1932) described the same species, from Anodonta cygnca (L.) and Unio
pictorum L., as Hypocomatophora unionidarum. Jarocki later (1934) pointed out
that Hypocomatophora unionidarum was a synonym of Hcterocineta anodontae.
In his papers of 1934 and 1935 Jarocki described seven additional species of
the genus Heterocincta ectoparasitic on fresh water gastropods : H. janickii, from
Physa fontinalis (L.) ; H. Iwoffi, from Viviparus jasciatus Miiller ; H. chattoni, from
Radix ovata (Drap.) ; H. krsysiki, from Bithynia tcntaculata (L.) ; H. maziarskii,
from Coretus corncus (L.) ; H. turi, from Tropidiscus planorbis (L.) and Spiralina
vortex (L.) ; and H. siedlcckii, from Acroloxus lacustris (L. ). In 1945 I de-
scribed as Heterocineta phoronopsidis a ciliate from the tentacles of Phoronopsis
viridis Hilton. This species is the only representative of the genus thus far de-
scribed which is not a parasite of fresh water molluscs or of the annelid commensal
Chaetogastcr limnaci von Baer when the latter is associated with infected snails.
On the fresh water prosobranch snails Goniobasis plicijera silicula (Gould) and
Fluminicola virens (Lea) I have found two new species of Heterocincta which will
be described herein as H. goniobasidis sp. nov. and H. fluminicolae sp. nov. I have
also studied a species of Heterocineta from Physa cooperi Tryon which agrees with
the original description of H. janickii. It seems advisable, for comparative pur-
poses, and in view of the fact that Jarocki's description of H. janickii is not accom-
panied by illustrations, to include an account of the morphology of this form in the
present paper.
The genus Encrthccoina was proposed by Jarocki (1935) for a single species,
E. propcrans, parasitic on the gills of Viviparus jasciatus. Although the original
description of this species is quite adequate, it is not supplemented by illustrations,
and the second installment of Jarocki's "Studies on ciliates from fresh-water mol-
luscs," in which figures of E. properans and several other ciliates were to be pub-
200
CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV
201
lished, has not come to my attention. A ciliate which I have found to infest Vivi-
parus malleatus (Reeve) apparently is identical with E. properans. This ciliate
will be described and illustrated here.
HETEROCINETA JANICKII JAROCKI
(Figure 1; Plate I, Fig. 1)
The body is elongated and flattened dorso-ventrally. The anterior end is attenu-
ated, bent ventrally, and deflected slightly toward the left. The anterior one-half
of the left margin is not quite so rounded as the right margin and typically is nearly
straight or weakly indented. The body is widest a short distance behind the mid-
dle and rounded posteriorly. The ciliary system, to be described presently, is dis-
posed on a shallow concavity occupying the anterior two-thirds of the ventral sur-
face ; the dorsal surface and that part of the ventral surface posterior to the ciliary
area are convex. Twenty living individuals from Pliysa cooperi ranged in length
from 25 fj. to 32 //., in width from 12 /x to 15^,, and in thickness from 10 /x to 12 /j.,
FIGURE 1.
^> X-
Hcterocineta janickii Jarocki. Distribution of ciliary rows, somewhat diagram-
matic.1 Ventral aspect.
averaging about 30^ by 14 ^ by 11 /x. The specimens of H. janickii from Pliysa
joiitinalis which were studied by Jarocki ranged in length from 23^ to 32 (*,, in
width from 12^ to 17 /*, and in thickness from 10^, to 13^.
The anterior end of the body is provided with a short contractile suctorial ten-
tacle which enables the ciliate to attach itself to the epithelial cells of the host and
to feed upon their contents. When fully extended the tentacle is about 3 /* to 4 ju,
(according to Jarocki about 4.5 p.) in length. The internal tubular canal continuous
with the tentacle is directed at first dorsally and then ventrally and obliquely toward
the right side, and in specimens stained with iron hematoxylin can usually be traced
for about one-half the length of the body.
The ciliary system consists of eight longitudinal rows originating close to the
base of the suctorial tentacle. The first four rows from the right side are approxi-
mately one-half the length of the body. The remaining four rows become increas-
ingly longer and terminate one behind the other a little to the left of the midline.
1 The text figures illustrating this paper are based on camera lucida drawings of specimens
impregnated with silver nitrate by Klein's method.
202 EUGENE N. KOZLOFF
The longest row is about two-thirds the length of the body. The cilia are about
6 p. to 7 fji (according to Jarocki about 5 p. to 7 /x) in length. While attached to the
skin of the host the parasites are as a rule almost immobile, their cilia exhibiting
only a feeble motion. When dissociated from the host Hetcrocineta janickii swims
sluggishly, usually rotating on its longitudinal axis and tracing wide arcs with its
attenuated anterior end.
The cytoplasm is colorless and contains numerous small refractile granules in
addition to food inclusions. One or more large food vacuoles are present in the
posteriorpart of the body behind the macronucleus. The contractile vacuole is situ-
ated near the middle of the body and opens to the exterior on the ventral surface.
I have observed no permanent opening in the pellicle.
The macronucleus is typically sausage-shaped and is located near the middle of
the body or somewhat posterior to the middle. As seen in dorsal or ventral view
the longitudinal axis of the macronucleus is placed obliquely to the longitudinal axis
of the body. As seen in lateral view, the anterior end of the macronucleus is di-
rected dorsally, while the posterior end is directed ventrally. In fixed and stained
preparations the chrbmatin appears to be more or less homogeneous. In ten indi-
viduals fixed in Schaudinn's fluid and stained by the Feulgen reaction the macro-
nucleus ranged in length from 7 //. to 1 1 ju, and in width from 4 /* to 5 /JL.
The micronucleus is ovoid or spherical and is situated near the dorsal surface
anterior to or to one side of the macronucleus. In most fixed and stained specimens
the chromatin is homogeneous, although in some it appears to be concentrated in
peripheral granules. In ten individuals fixed in Schaudinn's fluid and stained with
iron hematoxylin the size of the micronucleus ranged from 1 .4 p. by 1 .4 ^ to A .6 /A
by 2 p..
Hetcrocineta janickii was present in very small numbers on the tentacles, mantle,
and margins of the foot of most of the specimens of Physa coopcri which I col-
lected in a stream near Mt. Eden, California. The degree of infestation increased
rapidly on snails kept in laboratory aquaria for a period of six weeks.
Heterocineta janickii Jarocki
Diagnosis: Length 25 ju-32 p. (according to Jarocki 23/x.-32/x), average about
30 p; width 12/^-15 p. (according to Jarocki 12/x-17/t), average about 14/x; thick-
ness 10/x-12/x (according to Jarocki 10/x-13/x), average about 11 p.. The ciliary
system consists of eight rows originating close to the base of the suctorial tentacle.
The first four rows from the right are about one-half the length of the body, while
the remaining four rows become progressively longer and terminate one behind the
other a little to the left of the midline. The longest row is about two-thirds the
length of the body. Parasitic on the epithelium of the tentacles, mantle, and foot
of Physa jontinalis (L.) (Warsaw [Jarocki])' and Physa coopcri Tryon (Mt.
Eden, California).
HETEROCINETA GONIOBASIDIS SP. NOV.
(Figure 2; Plate I, Figs. 2, 3)
The body is elongated and flattened dorso-ventrally. The anterior end is at-
tenuated, bent ventrally, and deflected slightly toward the left. The anterior one-
half of the left margin is not so rounded as the right margin and typically is nearly
CILIATES OF THE FAMILY ANC1STROCOMIDAE. IV
203
straight or weakly indented. The body is widest at the middle or a short distance
anterior to the middle. The ciliary system is disposed on a shallow concavity oc-
cupying the anterior two-thirds of the ventral surface ; the dorsal surface and that
part of the ventral surface posterior to the ciliary area are convex. Twenty-five
living specimens taken at random ranged in length from 36 /j. to 48 /JL, in width from
15,u, to 20 fj., and in thickness from 11 /A to 14 /JL, averaging about 43 /x by 18 /A
by 13 fjL.
The anterior end is provided with a contractile suctorial tentacle continuous with
an internal tubular canal. The nature of the canal is very similar to that of other
members of the genus. It is directed at first dorsally and then ventrally and ob-
liquely toward the right side of the body. It can be traced in most fixed specimens
stained with iron hematoxylin for about one-half to two-thirds of the length of the
body.
FIGURE 2.
Hetcrocineta goniobasidis sp. nov. Distribution of ciliary rows, somewhat
diagrammatic. Ventral aspect.
The cilia of H. goniobasidis are about 9 /A long. Those of the anterior part of
the ciliary system are markedly thigmotactic. The ciliary system consists of ten
longitudinal rows. The first six rows are approximately the same length, being
about one-half the length of the body, although on careful examination the first
row is seen to originate some distance posterior to the level of origin of the other
five rows. The seventh, eighth, ninth, and tenth rows originate progressively more
posteriorly and become increasingly longer, terminating one behind the other a little
to the left of the midline. The longest row is two-thirds to three-fourths the length
of the body. The last one or two rows usually originate on the left margin and
curve ventrally as they extend backward. The cilia of the distal portions of the
longer rows are nearly always practically motionless and directed posteriorly. When
dissociated from the host the ciliate swims sluggishly and erratically, rotating on its
longitudinal axis.
The cytoplasm is colorless and contains numerous refractile granules of a lipoid
substance in addition to food inclusions. There are usually one or two large food
vacuoles in the posterior part of the body behind the macronucleus. The contractile
vacuole is central and opens to the exterior on the ventral surface.
204 EUGENE N. KOZLOFF
The macronucleus is situated in the middle portion of the body. It is elongated
and typically somewhat narrower at its anterior end than at its posterior end. As
seen in dorsal or ventral aspect, the longitudinal axis of the macronucleus is placed
obliquely to the longitudinal axis of the body. As seen in lateral view, the anterior
end of the macronucleus is directed dorsally, while the posterior end is directed
ventrally. In ten individuals fixed in Schaudinn's fluid and stained with iron
hematoxylin the macronucleus ranged in length from 10^ to 13. 5 /A and in width
from 4 ^ to 5.5 /x.
The spherical or ovoid micronucleus is very difficult to distinguish in the living
ciliates. It is usually situated near the dorsal surface a short distance anterior to
the macronucleus. In fixed and stained preparations the micronucleus is vesicular,
the chromatin being concentrated along the periphery. In ten individuals fixed in
Schaudinn's fluid and stained with iron hematoxylin the micronucleus ranged in
size from 1 .2 ^ by 1 .5 ;u, to 1 .5 p, by 1.7 /i.
Heterocineta goniobasidis was found to be present on the epithelium of the gills
and mantle of a small percentage of the specimens of Goniobasis plicifcra silicula
which I collected in Crystal Springs Creek, in Portland, Oregon. The degree of
infestation on freshly collected snails was very low, but increased during the four
weeks the specimens were kept in laboratory aquaria.
Heterocineta goniobasidis sp. nov.
Diagnosis: Length 36^ — 1-8//., average about 43 yu.; width 15^-20/x, average
about 18/x; thickness ll/x-14/x, average about 13/x. The ciliary system h com-
posed of ten rows. The first six rows from the right side are about one-half the
length of the body and, with the exception of the first row, originate close to the
base of the suctorial tentacle. The remaining rows originate progressively more
posteriorly and become increasingly longer, terminating one behind the other a little
to the left of the midline. The longest row is two-thirds to three-fourths the length
of the body. Parasitic on the gills and mantle of Goniobasis plicifera silicula
(Gould) (Portland, Oregon). Syntypes are in the collection of the author.
HETEROCINETA FLUMINICOLAE SP. NOV.
(Figure 3; Plate I, Fig. 4)
The body is elongated and flattened dorso-ventrally. The anterior end is at-
tenuated, bent ventrally, and deflected slightly toward the left. The anterior part
•
EXPLANATION OF PLATE I
All figures except Figure 2 have been prepared with the aid of a camera lucida.
FIGURE 1. Heterocineta janickii Jarocki. Ventral aspect. Schaudinn's fixative-iron hema-
toxylin. X 1,720.
FIGURE 2. 'Heterocineta goniobasidis sp. nov. Lateral aspect from left side, from life.
FIGURE 3. Heterocineta goniobasidis sp. nov. Ventral aspect. Schaudinn's fixative-iron
hematoxylin. X 1,720.
FIGURE 4. Heterocineta fluniinicolae sp. nov. Ventral aspect. Schaudinn's fixative-iron
hematoxylin. X 1,720.
FIGURE 5. Encrthccoma properans Jarocki. Macro- and micronuclei from three specimens.
Schaudinn's fixative-Feulgen reaction. X 1,720.
FIGURE 6. Enerthecoma properans Jarocki. Ventral aspect. Schaudinn's fixative-iron
hematoxylin. X 1,720.
CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV
205
ff.
PLATE I
206
EUGENE N. KOZLOFF
of the left margin is not so rounded as the right margin and typically is weakly
indented. The body is widest a short distance behind the middle and rounded
posteriorly. The ciliary system is disposed on a shallow concavity occupying the
major portion of the ventral surface ; the dorsal surface and that part of the ventral
surface posterior to the ciliary area are convex. Twenty-five living individuals
taken at random ranged in length from 30^ to 36 /x, in width from 13 /x to 17 /M,
and in thickness from 10 /A to 12 //,, averaging about 33 /A by 15/u by 11 /*.
The anterior end is provided with a contractile suctorial tentacle continuous
with an 'internal tubular canal. The canal is directed at first ventrally and then
obliquely toward the right side of the body. It can be traced in most fixed speci-
mens stained with iron hematoxylin for about one-half the length of the body.
The cilia of H. fluminicolae are about 6/x or 7 p. long. Those of the anterior
part of the ciliary system are strongly thigmotactic. The ciliary system consists of
ten longitudinal rows. The first row on the right side of the ciliary complex origi-
FIGURE 3. Hctcrochicla flnininicolac sp. nov. Distribution of ciliary rows, somewhat
diagrammatic. Ventral aspect.
nates close to the base of the suctorial tentacle ; each of the remaining rows origi-
nates progressively more posteriorly. The first six rows from the right side are
approximately the same length, being about two-thirds the length of the body. The
last four rows become increasingly longer and incurved in such a \vay that they
terminate one behind the other not far to the left of the midline. The longest row
usually extends almost to the posterior end of the body. The cilia of the distal
portions of these longer rows are usually directed posteriorly. When the ciliate is
dissociated from the host it swims erratically, rotating on its longitudinal axis and
tracing wide arcs with its anterior end.
The cytoplasm is colorless and contains numerous small refractile granules of
a lipoid substance in addition to food inclusions. One or more large food vacuoles
are usually present in the posterior part of the body behind the macronucleus. The
contractile vacuole is central and opens to the exterior on the ventral surface. I
have not observed a permanent opening in the pellicle.
The sausage-shaped macronucleus is situated dorsally a short distance behind
the middle of the body with its longitudinal axis placed obliquely to the longitudinal
CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 207
axis of the body. In fixed and stained preparations the chromatin appears to be
more or less homogeneous. In ten individuals fixed in Schaudinn's fluid and
stained with iron hematoxylin the macronucleus ranged in length from 7.4 p, to 10 p.
and in width from 3.9 p, to 4.4 /JL.
The micronucleus is round, fusiform, or ovoid, and .is usually placed dorsally
near the middle of the body anterior to or to one side of the macronucleus. In
fixed and stained specimens the chromatin is seen to be concentrated primarily along
the periphery. In ten individuals fixed in Schaudinn's fluid and stained with iron
hematoxylin the micronucleus ranged in size from 1.5^ by 1.2 /JL to 1.7 p, by 1.5 /A.
Heterocineta fluminicolae was present in small numbers on the epithelium of the
gills and the edge of the mantle of nearly all specimens of Fluminicola virens which
I collected in Crystal Springs Creek in Portland, Oregon.
Heterocineta fluminicolae sp. nov.
Diagnosis : Length 30 /A-36 /JL, average about 33 p. ; width 13 p.-\7 p., average about
15 p.; thickness lO/t-12//,, average about 11 p. The ciliary system is composed of
ten rows originating progressively more posteriorly from the right side to the left.
The first six rows from the right side are about two-thirds the length of the body.
The remaining four rows become increasingly longer and terminate one behind the
other a little to the left of the midline. The longest row extends almost to the pos-
terior end of the body. Parasitic on the gills and mantle of Fluminicola virens
(Lea) (Portland, Oregon). Syntypes are in the collection of the author.
ENERTHECOMA PROPERANS JAROCKI
(Figure 4; Plate I, Figs. 5, 6)
The body is elongated, nearly symmetrical as seen in dorsal or ventral view, at-
tenuated anteriorly, and flattened dorso-ventrally. The anterior end is bent ven-
trally and deflected inconspicuously toward the left. The ciliary system is disposed
on a narrow, relatively flat area occupying the anterior two-thirds of the ventral
surface ; the dorsal surface and that part of the ventral surface posterior to the
ciliary area are convex. The body is widest at a point about two-thirds the dis-
tance from the anterior end to the posterior end. Twenty-five living individuals
taken at random from Viviparus malleatus ranged in length from 32 p, to 56 p, in
width from 13 ^ to 21 p., aiid in thickness from 10 p. to 13 /x, averaging about 44 p.
by 18 p. by 11.5^. Specimens from Viviparus fasciatus which were measured by
Jarocki ranged in length from 33 p. to 60 p., in \vidth from 15 p. to 22 p, and in thick-
ness from 10 p. to 13 p..
The contractile suctorial tentacle is continuous with an internal tubular canal
which is directed at first dorsally and then ventrally and obliquely toward the right
side of the body. In specimens stained with iron hematoxylin the canal can usually
be traced for about two-thirds or three-fourths the length of the body.
The ciliary system is composed of eight approximately equal rows about two-
thirds the length of the body. These rows originate close to the base of the suc-
torial tentacle. The first five rows from the right side are usually a little more
widely spaced than the last three rows. This was noted also by Jarocki. who stated
that the ciliary system was separated into two complexes by an "inconsiderable
eminence stretching from the base of the tentacle to the end of the system," which
208
EUGENE N. KOZLOFF
segregated the five rows on the right from the three rows on the left. This eminence
was evident on many of the living specimens which I examined but is never con-
spicuous. The cilia of E. proper ans are about 9 ^ in length and exhibit a feeble
undulatory motion while the parasites are attached to the epithelium of the gills of
the host. When dissociated from the host the ciliates swim slow and erratically,
usually rotating on their longitudinal axes.
The cytoplasm is colorless and contains numerous small refractile granules ot
a lipoid substance in addition to food inclusions. One or more larger food vacuoles
are usually present in the posterior part of the body. The contractile vacuole is
situated a short distance behind the middle of the body and opens to the exterior
on the ventral surface. I have not detected a permanent opening in the pellicle.
The macronucleus is typically sausage-shaped and is situated in the posterior
half of the body with its longitudinal axis placed obliquely to the longitudinal axis
FIGURE 4. Enerthccoma propcrans Jarocki. Distribution of ciliary rows, somewhat
diagrammatic. Ventral aspect.
of the body. In specimens stained with iron hematoxylin the chromatin appears
to be more or less homogeneous, but in preparations stained by the Feulgen re-
action it appears to be organized into a dense reticulum enclosing vacuole-like clear
spaces of varying size. In ten individuals fixed in Schaudinn's fluid and stained
by the Feulgen reaction the macronucleus ranged in length from 10 /A to 19 ^ and
in width from 4 ^ to 7 p.
The micronucleus is situated anterior to or to one side of the macronucleus.
In most of the individuals of E. properans which I examined, the micronucleus is
elongated and more or less fusiform. I have observed very few specimens to have
a round micronucleus such as that described by Jarocki. The micronucleus does
not stain readily with iron hematoxylin and it is possible that Jarocki may have
mistaken food inclusions for micronuclei. In specimens stained by the Feulgen
reaction the chromatin of the micronucleus appears to be concentrated in peripheral
granules or strands. In ten individuals fixed in Schaudinn's solution and stained
CILIATES OF THE FAMILY ANCISTROCOMIDAE. IV 209
by the Feulgen reaction the micronucleus ranged in size from 0.8 p. by 2.3 /* to 1 /*
by 3.8 n.
Enerthecoma properans was abundant on the gills of nearly all specimens of
Viviparus mallcatus which I collected in Stow Lake, San Francisco, California,
and in Evans Lake, Riverside, California. It is undoubtedly a common parasite
of this introduced snail wherever the latter has become established.
Enerthecoma properans Jarocki
Diagnosis: Length 32/^-56^ (according to Jarocki 33^-60^), average about
44/.I. ; width 13 /x-21 p. (according to Jarocki 15/x-22/x), average about 18 /x; thick-
ness 10fi-13/x, average about 11.5/x. The ciliary system is composed of eight ap-
proximately equal rows about two-thirds the length of the body which originate
close to the base of the suctorial tentacle and occupy a narrow, relatively flat area
on the ventral surface. The first five row's from the right are more widely-spaced
than the remaining three rows, and in living specimens appear to be segregated
from the latter by an inconspicuous longitudinal eminence. The macronucleus is
elongated ; the micronucleus is typically elongated and more or less fusiform (ac-
cording to Jarocki, spherical). Parasitic on the gills of Viviparus jasciatus Miiller
(Warsaw [Jarocki]) and Viviparus mallcatus (Reeve) (San Francisco, California;
Riverside, California).
LITERATURE CITED
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. Cracovic, Cl. Sci. math, not., B, 1934: 167.
JAROCKI, J., 1935. Studies on ciliates from fresh-water molluscs. I. General remarks on pro-
tozoan parasites of Pulmonata. Transfer experiments with species of Heterocineta
and Chaetogaster limnaei, their additional host. Some new hypocomid ciliates. Bull,
int. Acad. Cracovic, Cl. Sci. math, nat., B (II), 1935: 201.
JAROCKI, J., AND Z. RAABE, 1932. Uber drei neue Infusorien-Genera der Familie Hypocomidae
(Ciliata Thigmotricha), Parasiten in Siisswassermuscheln. Bull. int. Acad. Cracovie,
Cl. Sci. math, nat., B (II), 1932: 29.
KOZLOFF, E., 1945. Heterocineta phoronopsidis sp. nov., a ciliate from the tentacles of Phoro-
nopsis viridis Hilton. Biol. Bull., 89 : 180.
MAVRODIADI, P., 1923. "Kosoe" delenie u infuzoril. Pratsy Bclaruskaga dziarshafinaga univer-
sytctu H Mcuskn, 4-5 : 166.
PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED
AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1946
JULY 9
DR. J. E. KINDRED. No abstract submitted.
The cyanide sensitivity of the unfertilized sea urchin egg. W. A. ROBBIE.
Reinvestigation of the cyanide sensitivity of unfertilized eggs of Arbacia punctulata, using
recently devised methods for the control of HCN concentration in manometric experiments,
showed that there was a definite inhibition of respiration. The respiration is depressed by
concentrations of HCN as low as 10~5 M., and for a four-hour period in KT4 M. it is only 40
per cent of the control value. There is complete inhibition for the first hour or more. In 4 per
cent CX-96 per cent Nj mixture there is no depression of the respiration of the control egg, but
on the addition of 10~l M. HCN the oxygen consumption is reduced, for a four-hour exposure,
to 20 per cent of the control level.
At concentrations of cyanide higher than 10 4 M. there is apparently a stimulation in oxygen
uptake. This is increased with high and reduced with low oxygen tensions. It is possibly
associated with oxidations proceeding through a cyanide-hemin system, or with the metabolism
of a carbohydrate intermediate catalyzed by HCN.
Inhibition of fertilisation in sea urchins by means of univalent antibodies vs.
antifertilisin. ALBERT TYLER.
In order to obtain further information as to the role of the specific interacting substance
of eggs and sperm in fertilization, antisera were prepared against them by immunization of
rabbits, and the antibodies tested for their ability to interfere with fertilization. The present
report concerns tests with antibodies prepared against purified anifertilizin derived from sperm
of the sea urchin Lytcchinus pictus and the gephyrean worm Urcchis caupo. The antisera ag-
glutinate the species sperm to high titer, but cannot be used directly to test for specific action on
fertilization, since the mechanical effect of tying up the sperm would itself constitute a block to
fertilization. However, by a previously described method, namely photo-oxidation, antibodies
can be converted into a non-agglutinating form, termed "univalent." This treatment was,
therefore, applied to the anti-antifertilizin sera and the "univalent" antibodies thus obtained
were tested for possible action on the ability of the sperm to fertilize eggs of the same species.
The results showed a considerable reduction in fertilizing power of the sperm, ranging in dif-
ferent tests from 32-fold to greater than 128-fold. At the same time, the motility of the treated
sperm was found to be quite as high as the controls.
JULY 16
Intermediate steps in the visual cycle. A. F. BLISS.
The primary functions of a visual pigment are absorption of radiant energy and its transfer
to the stimulatory mechanism of the visual cell. At present four such pigments are known :
rhodopsin and porphyropsin, the photosensitive pigments of vertebrate night vision; iodopsin,
the corresponding pigment of daylight vision; and cephalopsin, the photostable red pigment of
cephalopods and probably other invertebrates (/. Gen. Physiol, 1943). Instability in the light
has generally been accepted as a diagnostic test for a visual pigment. The existence of a light-
stable visual pigment in the squid however throws doubt on the validity of this assumption.
The bleaching by light of vertebrate visual pigments is nevertheless an interesting and
complex process which has not been adequately analyzed into its component steps. The first
known product of bleaching visual purple is a thermally unstable complex lipid, called Tran-
210
PRESENTED AT MARINE BIOLOGICAL LABORATORY 211
sient Orange by Lythgoe and Provisual Red by Krause. Its sluggish reaction to base and tem-
perature near 0° C. suggest that it is the acid tautomer of the first relatively stable product of
bleaching, appropriately named Indicator Yellow by Lythgoe. Acid Indicator Yellow is a red
lipid, becoming reversibly decolorized in base, and irreversibly converted in chloroform to the
greenish yellow carotenoid retinene, extracted by Wald from freshly bleached frog retinas. If
bleached retinas are allowed to stand an hour before extraction, retinene is no longer found.
Instead an equivalent amount of vitamin A is extracted. Retinene, however, is not the precursor
to vitamin A, but is due to the irreversible side reaction described above. In the normal retina
and in fresh neutral solution Indicator Yellow forms vitamin A under the influence of a labile
protein, the reaction being presumbaly enzymatic in nature. In the dark, dissolved rhodopsin
is reformed in part from Indicator Yellow. In the living animal the vitamin A released by
bleaching is reincorporated into rhodopsin by unknown means.
MR. W. H. PRICE. No abstract submitted.
The dependence of the resting potential of nerve on potassium, calcium, and
hydrogen ions.* ABRAHAM M. SHANES.
On the basis of new as well as recent -experimental results it now appears possible to de-
scribe a specific mechanism necessary and sufficient to account for the relationships between the
resting potential and metabolic processes in frog nerve. Cellular hydrogen ion production ac-
companied by an exchange with extracellular potassium ions is apparently involved ; under the
conditions of study this process contributes about 50 per cent of the total resting potential.
Calcium reduces the rate of ionic exchange, an effect of possible importance in the energy ex-
penditure necessary to maintain concentration gradients and associated potentials.
The evidence consists of demonstrating first that hydrogen ions from (1) CO, produced
by nerve, (2) CO2 applied to nerve, and (3) lactic acid and possibly other sources of acid within
the fibers are directly concerned with the production and maintenance of the potentials. This
has been possible chiefly with the aid of inhibitors of carbonic anhydrase — sulfanilamide and
thiophene-2-sulfonamide — by means of which the role of hydrogen ions can be followed during
anoxia, upon return to oxygen following anoxia, and to some extent during relatively normal
aerobic conditions. The changes of potential associated with the application of CQ..-O2 mix-
tures and related experiments show that the effectiveness of the hydrogen ions is dependent on
the ionic gradients established.
The involvement of extracellular potassium is demonstrated by the suppression in its ab-
sence of the changes in potential normally produced by CO2. This effect is used to show, fur-
ther, that the potassium in the extracellular spaces is reduced by the rapid large increase in
potential induced by the return of the anoxic nerves to oxygen. A small secondary decline of
potential which follows the rise in CO2 and which is independent of extracellular buffering is
also dependent on extracellular potassium, which suggests that the dense connective tissue or
the other sheathing materials of nerve are interfering with the potassium exchange between the
extracellular space immediately adjacent to the fibers and that more remote.
Calcium slows the rate of potential rise upon application of CO2 and this effect is directly
related to calcium concentration ; in view of the above results and available evidence, this is
interpreted as an effect on ionic exchange. At lower concentrations calcium depresses to almost
the same degree the potential changes in response to oxygen following anoxia and to CO2 ;
higher calcium concentrations, known to suppress the metabolic processes, exert a more marked
effect on the former.
These results therefore focus attention on factors important in the production and modifica-
tion of the resting potential. The action of any agent on the potential must be considered from
several possible standpoints: (1) inhibition or activation of metabolism or of carbonic anhy-
drase, (2) production of hydrogen ions, (3) production of a membrane diffusion potential, (4)
modification of equilibrium or membrane diffusion potentials. The experimental procedures
which have been applied provide means of distinguishing these possibilities. In view of the
conclusions reached, these methods should also prove useful in studies of the "potassium pump"
and of the biochemical processes concerned with CO2 production and fixation in relatively intact
cells, both are problems of considerable interest at the present time.
* Aided by a grant from the Penrose Fund of the American Philosophical Society.
212 PRESENTED AT MARINE BIOLOGICAL LABORATORY
DR. J T. BONNER. No abstract submitted.
JULY 23
Oxidation-reduction studies as a clue to the mechanism of fertilisation of marine
eggs. MATILDA M. BROOKS.
Eggs, sperm, and larvae at stages up to pluteus of three marine animals (Arbacia punctu-
lata, Asterias Forbesii and Chactopterus pcrgcmentaceus) were measured for Eh and Ph. The
egg, sperm or larvae were centrifuged and 1 cc. of the mass used for measurement in a glass
vessel in the Coleman electrometer. It was found that there is a definite correlation between
the rate of Q.. consumption and the redox potential of these cells. It was also found that the
redox potential of sea water, as diluted by hypertonic NaCl, CaCU, MgCl,., butyric acid or su-
crose, became more negative than that of sea water alone. These facts were used as a basis
for the hypothesis that a proper redox potential or ratio of oxidants to reductants of the respira-
tory enzymes was necessary for producing fertilization of the egg. The hypothesis as presented
states that the redox potential of the external solution or sperm as compared with that of the
exterior of the egg itself is an important factor in producing fertilization of the egg.
From the results with KCN in sea water which produced fertilization membranes but not
cleavage, it was concluded that the formation of the fertilization membrane is not associated
with oxidations, and appears rather to be due to change in the physical aggregation of some
proteins at the surface of the egg or to a denaturation process occurring as the redox potentials
is changing.
DR. C. L. YNTEMA. No abstract submitted.
JULY 30
The action oj napthoquinone antimalarials on respiratory systems. CHRISTIAN
B. ANFISEN AND ERIC G. BALL.
In confirmation of the findings of Wendel (unpublished reports) a series of 2-hydroxy-3-
alkyl-naphthoquinones have been found to exert a powerful inhibitory effect on the respiratory
metabolism of the malarial parasite. The most powerful tested to date is the compound 2-
hydroxy-3(2-methyl-octyl)-naphthoquinone-l,4 (M-285) which, at a level of 1 mg./liter in-
hibits Plasmodium kiwzvlcsi respiration 60 per cent. In experiments to localize the site of ac-
tion of M-285 in the main respiratory chain of enzymes it was found that p-phenylene-diamine
oxidation, requiring only the cytochrome system, was not inhibited by the drug, while the oxida-
tion of succinate to fumarate by succinic oxidase prepared from beef heart was completely in-
hibited at about 1 mg./liter. The drug, therefore, appears to inhibit at an oxidation-reduction
potention level below that of cytochrome C. Succinic dehydrogenase activity, as measured by the
Thunberg methylene blue technique, was only very slightly diminished even at high drug con-
centrations. Similarly, the respiration of both fertilized and unfertilized eggs of Arbacia punc-
tulata, neither presumably containing succinic dehydrogenase, was inhibited strongly at levels
as low as 0.1 mg./liter (2 X 10"7 M.).This enzyme, therefore, does not seem to be the inhibited
system. A number of flavoproteins including d-amino acid oxidase and xanthine oxidase, as
well as several systems involving the mediation of the pyridine nucleotides, showed no decrease
in activity in the presence of the drug.
It appears possible that the naphthoquinones under study are inhibiting a hitherto un-
detected enzyme or enzyme group in the main chain of oxidative metabolism having an E0 below
that of cytochrome C and above that of the flavoproteins.
Chemical sense and taste in the Sea Robin, Prionotus. ERNST SCHARRER.
The differentiation of taste and chemical sense is partly based on the concept that chemical
sensitivity can evoke only negative or defensive reactions ; positive reactions to food are medi-
ated by the sense of taste (Kappers, Huber, Crosby, 1936, p. 347). Observations in the sea
robin, Prionotus, do not support this conclusion. Prionotus possesses three free fin rays. Their
PRESENTED AT MARINE BIOLOGICAL LABORATORY 213
epithelium is innervated by spinal nerves ; taste buds are absent. The afferent fibers end in
accessory lobes on the dorsal surface of the cephalic end of the spinal cord. Secondary fibers
from these lobes which represent the greatly enlarged dorsal horns, ascend cephalad to the
funicular nucleus from which fibers pass ventromedially, crossing in the ventral commissure,
and ending in the contralateral ventral horn. When the free fin rays of blinded and sufficiently
hungry sea robins are stimulated with extracts of clams or crabs the animals react positively by
turning to and snapping in the direction from where the juice comes. Positive reactions to
chemical stimuli are mediated in this case by spinal nerves and in the absence of taste buds.
The differentiation between chemical sense and taste can, therefore, be based only on the inner-
vation and the presence or absence of taste buds. The reaction of the animal cannot be used
as a criterion.
Studies o\ the respiration of the iniaginal discs of Drosophila using the Cartesian
diver ultramicrorespiromctcr. CLAUDE A. VILLEE.
A determination of the effects of a mutant gene on the metabolic activity of a particular
group of cells provides a basic approach to the analysis of gene action in development. In
most animals it is impossible to locate exactly the cells which will give rise to a particular
structure, but in Drosophila each organ develops from a discrete group of cells, an imaginal
disc, which can be dissected out of the larva. The rate of respiration of wing and leg
discs from wild, "miniature" wing, and "vestigial" wing stocks were determined by the
Cartesian diver ultramicrorespirometer and their weights measured by the quartz fiber balance
of Lowry. The legs of the adults of all three stocks are normal, the wings of adult "miniature"
flies are about two-thirds normal size but of normal shape, and the wings of adult "vestigial"
flies are small misshapen stumps, less than one-quarter the size of the normal wing. At each
of several stages studied, before, at and after pupation, the Qo2 of wild type discs and the leg
discs of all stocks used varied only slightly from 20 cu. mm. O2 per hour per milligram of
tissue. The Qo2 of "miniature" wing discs was 18 and of "vestigial" wing discs 9 cu. mm. O,.
per hour per milligram of tissue. The weights of "vestigial," "miniature" and wild type wing
discs are the same at corresponding developmental stages in the larvae and early (1-2 hour)
pupae. The discs contain considerable reserves of substrate and will respire in the divers
twelve hours or more. The mutant genes "vestigial" and "miniature" produce their effects
by altering the rate of some chemical reaction in the wing disc of the larva which is reflected
by a lowered rate of oxygen consumption. These results do not mean that the "miniature"
and "vestigial" genes affect the same chemical reaction in development to a different extent
but rather that in affecting different processes they each lower the overall metabolic rate of the
disc. The metabolism of the leg discs, and probably of the other discs as well, is not changed,
although the cells contain the mutant gene. The "vestigial" and "miniature" genes therefore
produce their physiological as well as their morphological effects only in certain cells of the
body, presumably due to the interaction of the gene or gene products with specific components
of the cytoplasm of those cells.
AUGUST 6
The specificity of chlorine est erase. PHILIP B. ARMSTRONG.
The relative rates of hydrolysis of choline esters acting at the nerve terminations in the
sphincter pupillae of the turtle could be inferred by determining the relative potentiations by
eserine of threshold concentrations for pupillary constriction of the choline esters. A com-
parison of the potentiations in vivo with the relative hydrolysis rates of the choline esters
by the purified specific choline esterase in vitro indicates that the enzyme as it functions in vivo
is as specific if not more so than in vitro. The choline ester substrate concentrations in vivo
at which eserine was effective were much lower than those for effective substrate hydrolysis
in vitro.
DR. T. H. BULLOCK. No abstract submitted.
214 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The endocrine role of the corpora allata in insects. BERTA SCHARRER.
In Lcncophaca wadcrac (Orthoptera) extirpation of the corpora allata at nymphal stages
earlier than the last causes an abbreviation of development (suppression of molts) which results
in animals with adult-like characters ("adultoids"). In operated seventh instars the following
nymphal molt is suppressed, and the animals emerge as adultoids, resembling normal adults
except for their smaller size and comparatively shorter wings. Allatectomized sixth or fifth
instars result in "pre-dultoid" stages which show less adultoid differentiation and require an
additional molt before becoming adultoids. In the adult insect the corpora allata are necessary
for the development of the eggs. In females allatectomized shortly after the beginning of a
reproductive cycle the eggs do not develop appreciably beyond the stage typical of the ovary
at the time of operation. The accessory sex glands in these operated females show little or no
sign of secretion in contrast to normal control glands. Reimplantation of the corpora allata
into allatectomized females causes the eggs and the nymphs hatching from them to develop
as normally as those of unoperated animals. In a series of experiments in which the time of
allatectomy is varied it can be demonstrated that the corpora allata are necessary throughout
the period of growth and yolk deposition which constitutes about the first third of the total
period required for the development of the eggs. The corpora allata are apparently not essential
for the reproductive activity of male Leucophaea. Allatectomized males when mated with
normal virgin females are capable of fertilizing the eggs.
Contrasts between visible and dominant lethal mutation rates in x-rayed Habro-
bracon eggs. ANNA R. WHITING AND H. C. GEORGE.
Senior author has previously reported that eggs x-rayed in late metaphase I have lethal dose
about 2,000 r and one-hit dose-hatchability curve. Death appears to be due to terminal deletions.
Eggs x-rayed in prophase I have lethal dose about 45,000 r and complex dose-hatchability curve.
Death appears to be due to several factors, including translocations and inversions. Majority of
lethal effects in both stages are dominant. Recently, two groups of females were treated with
doses giving about 90 per cent mortality, one with 1,120 r for metaphase I and the other with
28,000 r for prophase I. They were then crossed with untreated males and their daughters were
F, 5? heterozygous
tested for heterozygosis for visible mutations. -p — ~T— - was 2.11 per cent lor eggs
.b, Y¥ tested
treated in metaphase I, 12.69 per cent for eggs treated in prophase I. By x2 test there is less
than one chance in one hundred that these stages belong to same class in respect to visible muta-
tion rate although they have same dominant lethality rate. This strengthens theory of terminal
deletions (which would not produce visibles) as most common response to x-rays of metaphase
I. Visibles produced in metaphase I are probably genie and their low percentage is what
would be expected at low doses tolerated by this stage. Most visibles from treated prophase
I are probably also genie although a few may be due to position effects of translocations or
inversions. Their high percentage is possible because of high doses tolerated.
AUGUST 13
A new factor from the adrenal influencing fat deposition in the liver. KATHERINE
A. BROW NELL.
Starvation in the normal mouse leads to a large deposition of fat in the liver. This fails
to occur after adrenalectomy. With these facts as a basis we have developed a test for a fat
factor in various fractions prepared from ox adrenals.
The method is briefly as follows : Adrenalectomized mice are fed for 24 hours then fasted
for 24 hours. During this 48-hour period they are injected every 6 hours with 0.2 cc. of the
preparation to be tested. Two to 3 hours after the final injection the livers are removed and
the total lipid determined gravimetrically.
Over 30 fractions from the adrenal gland including crystalline compounds have been tested
by this method. The table shows results on adrenalectomized untreated animals; two fractions,
a whole extract from which these fractions were taken and three crystalline compounds already
PRESENTED AT MARINE BIOLOGICAL LABORATORY
215
proven to have glyconeogenic potency. Both fractions are crude, being specific in only one
respect — namely, that the carbohydrate factor fraction has no electrolyte potency and the sodium
factor fraction no glyconeogenic potency. The only fraction that gave a highly significant
response was that containing the carbohydrate factor. The low response given by whole
extract, we attribute to inhibiting substances, three of which have been tested.
Since the liver fat response was given almost exclusively by the carbohydrate factor
fraction, some of the crystalline compounds having glyconeogenic properties were tried to de-
termine whether or not they were responsible. The table shows that the only one used which
gave a significant response was dehydrocorticosterone ; a 25 per cent increase over the control
level and in order to obtain this response two and one half times as much pure substance (0.96
mgm.) was used as that estimated to be present in our carbohydrate factor fraction (0.35
mgin.). The other two compounds, corticosterone and 17-hydroxy- 11 -dehydrocorticosterone,
gave liver fat responses only on the borderline of significance and to obtain even these small
responses two to two and one half times as much material was used as that estimated to be
present in the carbohydrate factor fraction. The fourth known glyconeogenic compound,
hydroxycorticosterone, we were unable to test on account of lack of material.
There remain two possibilties : (1) that hydroxycorticosterone is the fat factor. If so,
the effect on fat metabolism is a new property. (2) There is in the carbohydrate factor fraction
a new factor regulating fat deposition in the liver.
Effect of adrenal fractions on deposition of fat in the liver
Treatment
No. of animals
Total lipid per cent
Increase per cent
Adect. untreated
29
6.31
—
Carbo. factor fraction *
15
8.42
33
No factor fraction *
7
6.74
9
Whole extract *
7
7.13
13
Dehydrocorticosterone f
8
7.87
25
Corticosterone f
8
7.11
13
1 7-hydroxy- 1 1 -dehydrocorticosterone t
7
6.87
9
* The extracts represent 300 gm. of tissue per cc.
t The solutions of crystals represent 0.6 mgm. solid per cc.
Hyper activity of the adrenal cortex. FRANK A. HARTMAN.
•
At rest or under conditions of minimal activity there is a basal secretion of adrenal cortical
hormones. In response to various stresses such as exercise, exposure to cold, trauma, anoxia,
and poisons, there is an increase in output of the hormones which subsides after the stimulus dis-
appears. After removal of a large proportion of both adrenals by enucleation, in the mouse, a
considerable rise in the basal secretion occurs. This higher level of secretion is maintained for
months. The following table illustrates these changes. Fat and glycogen (as sugar) in the
liver were determined after 24 hours' starvation.
Values indicating changes in hormone production after
enucleation of both adrenals
Normal
Adrenalectomized
Enucleated 2 days
Enucleated 7 days
Enucleated 15 days
Enucleated 29 days
Enucleated 99 days
Total lipid
per cent
8.5
6.3
6.6
11.8
10.0
10.0
Glycogen
per cent
0.12
0.04
0.24
0.58
216 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The wide difference in time at which the peaks for the production of the fat factor and car-
bohydrate factor occur, is evidence that the two factors are not identical.
By enucleation we removed an average of 75 per cent of the adrenal tissue. Less than 25
per cent of the active tissue remained since the circulation was disturbed and this 25 included the
capsule. Thirteen days after enucleation the adrenals averaged 0.69 per cent of the body weight
which is one-half the normal weight. Removal of cortical tissue probably reduces the in-
hibitory effect on the adrenotrophic hormone production by the pituitary so that after a lag
of three or four days there is sufficient recovery of the remaining cortices to respond to the in-
creased output of adrenotrophic hormone. However, the new level of cortical hormone pro-
duction .does not return the adrenotrophic output to the old level. Thus a higher basal level is
established. The performance of a relatively small number of cortical cells indicates a large fac-
tor of safety. This capacity of cortical cells for sustained activity in disease where a large
proportion of cortical tissue is destroyed is important in prolonging life.
There is now evidence for three mother hormones secreted by the adrenal cortex ; the fat
factor, the carbohydrate factor, and the sodium factor.
Studies on the mechanism of allo.nin action. ARNOLD LAZAROW AND STANLEY
LEVEY.
A number of compounds related to alloxan were synthesized and tested for their diabetogenic
effect. These compounds were injected intraperitoneally into rats in high doses and the blood
sugar was determined at 0. 1. 3, 8, 24, 48, and 72 hours after injection. Alloxan, N-methyl
alloxan, and alloxantin which dissociates into alloxan all produced diabetes. N-N-dimethyl
alloxan was toxic and, therefore, could not be injected in doses equivalent to that required for
the production of diabetes with alloxan. Since alloxan is a ureid of mesoxalic acid, some deriva-
tives were prepared in which the urea or mesoxalic acid portions of the molecule were intact.
None of these (mesoxalamide, mesoxalic acid, dimethyl mesoxylate, or diacetyl urea) produced
diabetes in the doses used. Freshly prepared dialuric acid, alloxanic acid, and barbituric acid
did not produce diabetes ; whereas, dialuric acid which was allowed to stand overnight was di-
abetogenic. (This is interpreted as oxidation of dialuric acid to alloxan by molecular oxygen.)
Slight alterations in the structure of alloxan abolish its diabetogenic effect.
It has been reported by other investigators that alloxan combines with sulfhydryl groups of
proteins and that on injection it produces a rapid drop in the blood and tissue glutathione. Since
one of us has shown that injection of glutathione or cysteine immediately preceeding a diabetogenic
dose of alloxan completely protected the animals from diabetes ; and since others have shown that
pancreas contains less glutathione than do other tissues ; it was suggested that variations in
tissue glutathione may determine the selectivity of alloxan. Studies are now being carried out
to determine the glutathione content of the beta cells of the pancreas which are selectively de-
stroyed by alloxan.
Biological specificity and the synthesis of native proteins. DOROTHY WRINCH.
A common starting point for the discussion of biological specificity today is the assumption
that biological function is an outward and visible sign of atomic pattern. Furthermore indications
from many fields reinforce the old assumption that the native protein is the dominant structure
type in all living systems. A vast number of physiological problems turn upon questions of
atomic pattern, particularly such matters as (1) local stereochemical features and (2) the pres-
ence of internal OH...O, NH...O and NH...N bridges and of linkages dependent upon the
presence of a foreign ion.
Of all these problems, the most fundamental is the synthesis of native proteins. We must
presume that the power of native proteins to produce replicas of themselves depends in some
basic way upon their structure, and that it is intimately related to the presence on native protein
surfaces of 'active patches' to use Warburg's term, each of which functioning as a template or
mold permits the laying down on itself of a complementary constellation.
It is useful to notice that the associations of simple molecules within crystals offer many ex-
amples of such complementary constellations, e.g., (1) hexamethylene tetramine, with pairs
which are not identical associated about tetrahedrally related planes with a common three-fold
axis and (2) the phosphotungstic acid 29-hydrate with identical (i.e., self complementary) con-
PRESENTED AT MARINE BIOLOGICAL LABORATORY 217
stellations associated about such planes with a common three-fold axis and, in addition, self-com-
plementary constellations associated about cube planes with a common two-fold axis.
Visualizing the formation of new 'active patches' on the surfaces of an already existing
species of native protein molecules, we see that such new constellations comprise the material re-
quired for the formation of a new and identical molecule if (1) the species carries complementary
patches (which may be but need not be individually self-complementary) and (2) the molecule
is wholly made up of such patches, i.e., is a surface structure.
In order to have a mechanism whereby these isolated constellations on several different
molecules may be integrated so as to interlock in the same spatial pattern as in the original mole-
cule, something has to be postulated as to the capacity of the original molecules to form a crystal.
Thus for example, let us visualize a body-centered cubic lattice with molecules placed a: the 8
body-centers and the 6 cube corners nearest the origin, with the molecule at the origin missing.
With the complementary constellations in position on each of the 8+6 faces of the molecules
turned to the origin, we have a situation in which interlocking, possibly in a number of distinct
steps, could take place, the resulting molecule being a replica of the original molecule. This is
but one example of a number of such possibilities, with the original molecules characterized by
antipodal pairs of complementary patches. All, however, have in common the dependence upon
the capacity of the original molecules to crystallize, an outstanding characteristic and most re-
markable property of unnumbered native proteins. Similarly, all theories as to the formation
of new native protein molecules by autocatalysis must, it would seem, have in common the picture
of such molecules as surface structures, i.e., atomic fabric cages.
AUGUST 20
Naturally occurring polyploidy in the development of Allium cepa L. Dr. C. A.
BERGER.
One of the factors in the developmental pattern of Allium cepa is the formation of some
tetraploid cells and their division as tetraploids. These cells are found throughout the cortex of
the cotyledon and of the intermediate region between root and shoot. They are found in seed-
lings between 20 and 40 mm. in length. They are never found in the root. During prophase of
mitosis in tetraploid cells the chromosomes are closely paired and relationally coiled. The two
members of each pair are united at a single undivided SA-region. These cytological details
show that the chromosomes have not separated since the time ofxtheir formation. Since the pair-
ing and relational coiling is present from earliest prophase the double chromosome reduplica-
tion must have taken place during the resting stage immediately preceding the 4n division. At
metaphase the tetrachromosomes undergo two successive divisions of the SA-region and ana-
phase is normal. Since no tetraploid division figures were found with unpaired chromosomes it
was concluded that only one division of these tetraploid cells occurs.
Chick embryology at the medical schools of Ancient Greece. TAGE U. H. EL-
LINGER.
Of the seventy titles comprising the Hippocratic Corpus, the most significant work, from
a biological standpoint, is the lecture on embryology represented by the two texts On Semen and
On the Development of the Child. It deals with human embryology from the formation of the
semen to the birth of the child. The author is unknown, but he was not Hippocrates nor one
of his followers. His work reflects the teachings of the medical school at Cnidos and of that
of Empedocles whose influence is evident in doctrine as well as in scientific method and in the
choice of vocabulary. This pre-Aristotelian author, who wrote in the last quarter of the fifth
century B.C., at the time of Socrates, was indeed a very great scientist and a great teacher as well.
To the modern reader perhaps the most amazing revelation is the use made of observations
on chick embryology in explaining to the students the development of the human embryo. The
following quotations are in the author's translation.
In chapter 13, the Greek physician after describing a "semen which had stayed six days in
the womb and which fell out," adds "A little later I will describe another test in addition to this
one, that will enable anyone who seeks knowledge to see this for himself, as well as a proof that
my whole discourse is correct, as far as that is possible for a mortal discussing such a matter."
218 PRESENTED AT MARINE BIOLOGICAL LABORATORY
He returns to this topic in chapter 29 : "Now I shall recount the crucial test, that I promised
a little while ago to make known, which is as clear as possible to a human intelligence and makes
plain to anyone who wants to be informed about it, that the semen is in a membrane and that the
navel is in the middle of it, and that it first draws air in and expels it outward," (according to
the Empedocles* pneuma theory of differentiation) "and that there are membranes from the navel.
You will also find the further growth of the child, as I have described it, to be from beginning
to end, such as it is in my account, if you will apply the method of inquiry that I am about to
describe. Take twenty eggs or more and give them to hatch to two hens or more ; then on every
day from the second to the last, that of hatching, remove an egg, break it and examine it. You
will find that everything in it conforms with my statements, in so far as one can compare the
growth of a bird with that of man. That there are membranes extending from the navel, and
all my other statements about the child, you will find illustrated from beginning to end in the
hen's egg ; and he who has not yet made these observations will be surprised that there is a
navel in a hen's egg. Such are the facts, and such is my account of them."
Again in chapter 30, the Greek author advances chick observations to illustrate and explain
conditions in man. He states : "Now in proof of my theory, that it is the lack of nourishment
that causes the child to come forth, provided it suffers no violence, I offer the following evidence.
The bird develops from the yolk of the egg in the following way. Under the brooding mother
the egg is heated and the content of matter inside receives the impulse to development from the
mother. When the content of the egg is heated, it forms air and attracts other cold air from
the atmosphere through the egg; for the egg is porous enough to admit the attracted air in
sufficient quantity to the matter inside. The bird grows in the egg and is differentiated in the
same or in a similar way to the child, as I have already said above. It develops from the yolk,
but it receives its nourishment and material for growth from the white that is in the egg. This
was at once apparent to all those who have given attention to it. Whenever nourishment from the
egg is insufficient for the chick, then, not having sufficient nourishment to live on, it moves vio-
lently in the egg seeking more nourishment, and the membranes about it burst. When the mother
notices that the chick has moved violently, she pecks and removes the shell. And this happens
in twenty days. And it is evident that this is so, for, when the mother pecks the shell of the egg,
there remains in it no liquid worth mentioning, since it has been expended on the chick."
Reproductive economy in closccrossed species zvith haploid males. P. W. WHIT-
ING AND RUDOLPH G. SCHMEIDER.
According to the multiple-allele theory of sex determination, proved true for the wasp
Habrobracon, every mating must involve either three or two sex alleles. The three-allele
matings produce only females (sex heterozygotes) and normal (haploid) males (azygotes) ; but
the two-allele matings produce also sex-homozygotes which either develop into sterile (diploid)
males or are inviable. Outcrossing reduces the chance for two-allele crosses with their attend-
ant reproductive wastage. The Habrobracon theory has been tentatively applied to the six or
seven invertebrate groups characterized by male haploidy. Since many species, however, re-
produce with much inbreeding, this theory would imply loss approximating half of the fertilized
eggs. It has now been shown that in the wasp Melittobia over 90 per cent of the eggs from close-
crosses, including selfcrosses (mother X haploid son), may develop into females. If Melittobia
females are sex-heterozygotes, some method must therefore have been evolved other than multiple
alleles for avoiding production of sex-homozygotes equal in number to the females. Although
the method of sex determination in Melittobia is not yet understood, it has now for the first time
been shown that reproductive economy is high in a closecrossed species with haploid males.
A comparative study of the lipids in some marine annelidcs. CHARLES G. WILBER.
Studies on the metabolism of lipids have been in the past confined to observations made on
vertebrates. Very few studies have been made on the lipids in the invertebrates; consequently
a detailed investigation seems justified.
The following marine annelides were studied: Nereis pelagica, Amphitrite ornata, Arcnicola
marina, Phascolosoma gouldii, Lepidonotus squamatus, Glycera americana, and Chactoptcrns
variopedatus.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 219
Whole worms or individual tissues were prepared by grinding or in the Waring-blendor.
Lipids were extracted with boiling alcohol. Phospholipids were precipitated with acetone and
magnesium chloride and estimated by oxidation-titration method of Bloor. Fatty acids were
estimated by oxidation-titration and cholesterol colorimetrically using the acetic anhydride-sul-
furic acid reagent. The ratios, cholesterol/fatty acid (lipocytic index) and cholesterol/phos-
pholipid, were calculated.
It was found that the absolute values of the various lipids in the same species and in different
species were not always the same. On the other hand, the lipocytic index and the relation,
cholesterol/phospholipid, were constant for a given species and tissue. If the lipocytic index of
each worm were plotted against the phospholipid of the same worm the points representing the
various species fell along a straight line ; a similar straight line was obtained when the cholesterol
was plotted against phospholipid.
There is, therefore, an apparent relationship between cholesterol and phospholipid and be-
tween phospholipid and lipocytic index in marine annelides. Tissues with a high lipocytic index
or high cholesterol content have a high phospholipid content. These results indicate that in
the marine annelides, just as Bloor found in the vertebrates, since cholesterol is associated with
and in constant relation to phospholipids, it is probably a normal protoplasmic constituent. These
results confirm in part the results of analyses made on vertebrate tissues and agree with the con-
tention of Mayer and Schaeffer that the lipocytic index is characteristic of the organ of an animal
in a given species.
GENERAL SCIENTIFIC MEETINGS
AUGUST 23
Vascular reactions to ergonovine maleate * as seen directly with the microscope
in the living mammal.1 RICHARD G. ABELL.
Erognovine was injected intravenously in amounts varying from 0.005 mgm. to 0.2 mgm.,
and its effect upon the arterioles, capillaries and venules observed directly with the microscope
in transparent 'moat' chambers (Abell and Clark, '32) in rabbits' ears. The clinical intravenous
dose of ergonovine is 0.1 mgm. The equivalent dose in the rabbit is approximately 0.005 mgm.
Injections of 0.005 mgra. caused constriction of arterioles to approximately 0.7 to 0.9 of their con-
trol diameters, and a slight reduction in velocity of flow. The arterioles returned to their con-
trol diameters and the flow to its control rate within 3 to 5 minutes. Daily injections of 0.005
mgm. made for a period of 2 weeks caused similar results. One hundredth mgm. (twice the
clinical dose) caused constriction of the arterioles to approximately 0.6 to 0.8 of their control
diameters, and a slightly greater reduction in rate of flow than 0.005 mgm. One tenth mgm.
(20 times the clinical dose) caused arterioles 15 to 30 microns in diameter to constrict to the point
of obliterating their lumens and stopping the blood flow for approximately 30 seconds to one
minute. The vessels relaxed to their control diameters within approximately 12 minutes. Two
tenths mgm. (40 times the clinical dose) caused more vigorous and prolonged arteriolar con-
striction, which lasted for from 1 to 1% minutes, and stopped all of the blood flow within the
chamber. The venules constricted to approximately 0.6 to 0.7 of their control diameters. The
arterioles returned to their control diameters in approximately 15 to 20 minutes. Four injections
of 0.2 mgm. at 15 minute intervals made the small arterioles (15 to 30 microns in diameter) un-
responsive to further injections, but not the larger arterioles (80 to 90 microns). Intravenous
injections of 0.025 mgm. of epinephrine while the small arterioles were still unresponsive to
ergonovine, caused them to constrict to the point of obliterating their lumens, which is the typical
response to this amount of epinephrine.
None of the above injections caused any sign of injury to the blood vessels, or any abnormali-
ties in appearance and distribution of the red blood cells, the white blood cells, or the platelets.
Thus it is clear that ergonovine maleate, which is used widely to prevent post partum hemorrhage
and to give symptomatic relief of migraine headache, does not cause any observable injury to the
blood vessels and associated structures even when given in amounts of 40 times the clinical dose.
*"Ergotrate" (Ergonovine Maleate, U.S. P., Lilly).
1 This work was aided by a grant made by Eli Lilly and Company to the Department of
Anatomy of the University of Pennsylvania Medical School.
220 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The effect of halogcnated alkyl amines on the respiration of Arbacia eggs and
sperm. E. S. GUZMAN BARRON, E. G. MENDES AND H. T. NARAHARA.
Halogenated alkyl amines at 0.001 M concentration produce an inhibition of the respiration
of animal tissues, and complete inhibition of pyruvate and choline oxidation (Barron et al.1). In
smaller concentrations the early cleavage of the fertilized sea urchin egg is inhibited or retarded
(Cannan et al.1). There is also inhibition of mitosis in the corneal epithelium of mammals
(Friedenwald and Scholz 1) and a high incidence of sex-linked lethals as well as a significant
number of translocations and inversions in the chromosomes of Drosophila inelanogastcr ( Auer-
bach et al.1).
Dichloroethylmethylamine HC1, and trichloroethylamine HC1 at a concentration of 0.001 M.
and dissolved in sea water, produced a definite increase in the respiration of sea urchin sperm
(from 170 to 50 per cent). The increase of respiration could be noticed even with 1 X 10"5 M.
The respiration of sea urchin eggs, fertilized or unfertilized, was slightly inhibited by this con-
centration of alkyl amine (14 to 17 per cent). Higher concentrations produced inhibition of
respiration probably due to a decrease in pH as a result of the hydrolysis of these compounds.
When the alkyl amines were previously neutralized and the sperm and eggs suspended in 0.05 M
citrate buffer, pH 6.8, the effect of the alkyl amines was erratic. It is quite possible that
penetration of the alkyl amines into the cell occurs only in an acid milieu.
The experiments of Cannan et al.1 on retardation of the rate of cleavage of fertilized Ar-
bacia eggs were confirmed. Eggs treated with 0.001 M dichloroethylmethylamine HCL (dis-
solved in sea water) for 15 minutes prior to insemination, and fertilized eggs treated at the time
of the first cleavage showed a definite retardation in the rate of cleavage. Furthermore none
of the treated eggs reached the pluteus stage.
The effect of uranyl nitrate on the respiration of Arbacia- sperm. D. BENEDICT
AND E. S. G. BARRON.
Uranium, like other heavy metals, is quite toxic and it has been extensively used for the
production and study of experimental nephritis. Uranyl nitrate in concentrations varying from
10~2 to 5 X 10"5 M. inhibited the respiration of Arbacia sperm. The inhibition was complete at
5 X 10~4 M. (92 per cent inhibition). W4 M. UO2(NOS)2 produced partial inhibition (from 53
to 15 per cent), 5 X 10"5 M. inhibited 15 per cent, and 10"5 M. had no effect at all. This inhibi-
tion must be due to combination of respiratory enzymes with uranium, a combination which can be
reversed completely on addition of a citrate at a ratio of U : citrate of 1 : 2. Addition of phosphate
at a ratio of 1 :100 brought only partial release (25 per cent). The experiments were performed
in acetate-sea water buffer at pH 6.4 to avoid precipitation of the uranyl salt. Dry weights of
sperm were obtained after centrifugation of the sperm at 16,000 g. There was in the control
experiments a rise in the pH value of about 0.6 units at the end of one hour, probably due to the
formation of NH:!.
Some properties of purified squid visual pigment. ALFRED F. BLISS.
The photostable red visual pigment of the squid (Bliss, 1943, Jour. Gen. Pliysiol.) was
found to become reversibly light sensitive in the presence of formalin. A method was devised
for the extraction of this pigment in a state of purity approximating that of the best prepara-
tions of vertebrate rhodopsin. The principal impurity of previous extracts, melanoprotein, was
rendered insoluble by the following procedure. Retinas were rinsed in distilled water and kept
frozen until use. They were then homogenizd with 0.2 M NaL. HPO4 and centrifuged. The resi-
due was washed with pH 4.5 buffer and distilled water. The visual pigment was extracted with
3 per cent digitonin at 6° C. for 2 minutes and centrifuged 5 minutes. The absorption spectrum
of the extracted pigment did not differ significantly from that of rhodopsin. In its chemical prop-
erties it differed significantly from rhodopsin, since it was rapidly destroyed by digitonin even at
6° C. The primary breakdown product in cold acetone was, like that of rhodopsin (Bliss, 1946,
Biol. Bull.), the acid tautomer of the lipid "Indicator Yellow." Because of the distinctive prop-
erties of the squid rhodopsin, a differentiating name, cephalopsin, is suggested.
1 All quoted from Gilman, A., and Philips, F., Science 103: 409 (1946).
PRESENTED AT MARINE BIOLOGICAL LABORATORY 221
Studies on the viscosity and elasticity of striated muscle. MANFRED BRUST.
By the use of a spring vibrating against the resistance of frogs' (Rana pipicns) sartorius
muscles — as described by Gasser and Hill (Proc. Roy. Soc. B. 96: 398, 1924) — the effects of
urea and iodoacetic acid (IAA) on the viscosity and elasticity of these muscles were studied.
All initial slack was removed from the system by stretching the muscles 17.5 per cent beyond
their resting length and putting them under 3.5 gm. tension.
Thirty minutes immersion in solutions of 2.5 M urea in Ringer's shortens the muscles on
the average by 26.1 per cent. When extended to their original length they still exert the ten-
sion originally exerted at that length. They will not return to their urea induced length when re-
leased from stretch. Their viscosity is reduced on the average of 53.6 + 13.0 per cent of that
in the untreated muscles, while the elasticity is similarly reduced to 60.4 + 18.6 per cent.
Sixty minutes immersion of Ringer's equilibrated muscles in 1-80000 IAA (6.72 X 10"5 M)
in Ringer's sometimes causes a rise in viscosity and elasticity even without activity by the poisoned
muscles. Summer frogs show this response less often than winter frogs. Measurements made
during 30 second rest periods between 5 second isometric tetani show a short initial decrease fol-
lowed by a gradual increase in both viscosity and elasticity. Average maximum rigor values of
181 per cent and 258 per cent respectively of the untreated muscle values are attained.
The urea results would agree with the findings by other authors that this agent disrupts myosin
and other protein molecules thus transforming them into disconnected less asymmetric entities.
Collagen is not believed to be markedly affected since muscle shape is maintained while tension
remains the same as before treatment at the same lengths. The IAA results would agree with
the progressively diminishing solubility and increase in hardness of actomyosin in gradually de-
creasing concentrations of adenosine triphosphate reported by the Szent-Gyorgyi group (Acta
I'liysiol. Scand. 9: Snppl. xxv, 1945).
Arterial anastomoses. ELIOT R. CLARK AND ELEANOR LINTON CLARK.
This study represents an attempt to discover factors responsible for the presence or absence
of arterial anastomoses, which vary so greatly in different organs.
The governing factor appears to be the histo-mechanical principle established by R. Thoma
in 1892, corroborated by E. R. Clark in 1918 in studies on living vessels in the tadpole's tail, that
the size of the lumen of an artery is regulated by the amount of blood flow. In the absence of
flow, the lumen is reduced to zero and the artery obliterated. In order, then, for arterial an^to-
moses to survive, conditions must be such as to provide a fldw of blood through the .terminal
connecting portion.
In most cases this requires the presence of factors which force the blood to flow part of the
time in one and part of the time in the reverse direction. Such factors are present in the periph-
eral parts of the body in the form of varying outside pressures that are exerted irregularly upon
large supplying arteries or small distributing arterioles.
A study, with the aid of artificial chambers, of the living circulation in the rabbit's ear, where
anastomeses are abundant, reveals frequent reversals of flow in connecting portions of anasto-
moses, but controlled by an unsuspected factor, namely, the irregular contraction of the arteries or
arterioles themselves, described in an earlier paper.
In types of artificial chambers, installed in rabbits' ears, which are invaded by new tissue,
there are often arterioles that, for weeks, are unprovided with nerves and hence contract little,
if at all. In many such chambers no arterial anastomoses survive. However, in this type of
chamber, occasionally arterioles receive a nerve supply, and in such cases arterial anastomoses
may survive. In every case in which such anastomoses have persisted in newly-formed tissue,
there have been frequent reversals of flow in the connecting portion.
The effects of the ultra-violet radiations on Styela eggs. A. M. DALCQ.
The M.D.L. installation for microphotography with U.V. rays (2537 A) may be used for
irradiating part of the Ascidian egg or certain of the various blastomeres up to the Vlll-cell
stage. The method was worked out with the aid of Dr. G. I. Lavin. The egg is placed in a
drop of sea water near the edge of a thin quartz coverslip, which is itself put on the transverse
arm of the mechanical stage. The coverslip is adjusted under the microscope in such a way
that the part of the egg to be irradiated protrudes over the edge of the metallic stage arm which
PRESENTED AT MARINE BIOLOGICAL LABORATORY
acts as a protection screen for the rest of the egg. Attention should be paid to two sources of
error: (1) the effect of hypertony due to evaporation of the drop and (2) to the reflexion of
the rays by the objective lens of the microscope, which is easily eliminated by interposing some
black paper during the irradiation. In exploring a considerable range of exposure no favorable
effect of the irradiation could be found. If feeble, it produces a delayed disorganization of the
embryonic layers. If stronger, it stops the cleavage with rapidity varying according to the
dosage. In order to obtain stopping of the next cleavage, exposures of at least 10 minutes are
necessary. When a division is suppressed, the cell may manifest a delayed attempt at cleavage,
but this is always abortive. After exposure of the unsegmented eggs, deviations of the first
cleavage plane may be observed. Observations of the movements of yolk and yellow pigment
and the elongation of the cell-body indicate that the effect of the radiation is not primarily on
the nuclear activity. That the influence of the rays is exerted on the surface protoplasm is
shown by the transitory appearance of alterations of the surface film (small protuberances,
"blisters") in coincidence with attempts at cleavage.
By means of this method, the division of one or more blastomeres of the II, IV, and VIII
cell stages has been inhibited. The non-irradiated cells exhibit normal development with respect
to mitotic rhythm and arrangement. Their capacity for differentiation, which seems rather poor
when large blastomeres remain undivided in the germ, must still be studied in sections.
A correlation betzveen gill surface and activity in marine fishes. I. E. GRAY.
The units of respiration in the gills of fishes are the numerous microscopic secondary lamel-
lae which appear as thin, leaf-like plates set at right angles to the main axes of the primary
lamellae. Within each plate lies a capillary network through which the interchange of gases
takes place. Among fishes there are species differences, not only in the number of gills, but
also in the number and length of the gill filaments (primary lamellae) and in the number of
respiratory units (secondary lamellae). By determining the number of respiratory units per
gram of body weight it is possible to obtain an estimate of the relative respiratory ability of
different fishes.
There is a marked contrast in the number of respiratory units per gram of body weight
between the active, surface, migratory fishes (mackerel, 2550; butterfish, 1725; menhaden,
1685) and the sluggish bottom fishes (flounder, 265; toadfish, 135; goosefish, 50). The number
of respiratory units of fishes of medium activity fall between these two extremes (scup, 1325;
sea 'trout, 1250; sea bass, 1110; eel, 900; sea robin, 800: puffer, 505; tautog, 440). A four
hundred gram mackerel has a total of nearly three-fourths million respiratory units while a
toadfish of the same weight has only fifty thousand. The number of respiratory units is also
directly correlated with the amounts of sugar and hemoglobin in the blood.
The distribution of lipid between the light and heavy halves of the Arbacia egg.
F. R. HUNTER AND A. K. PARPART.
Unfertilized Arbacia eggs were centrifuged for 10-20 minutes in an air turbine at ap-
proximately 16,000 X G. in a medium of graded density obtained by mixing sea water and 0.95
1 molal sucrose. The light and heavy halves which resulted were collected, packed in an air
turbine, frozen, dried in a vacuum desiccator and weighed. This dried material was then ex-
tracted with ether, dried and again weighed. The loss in weight was taken as a measure of
the amount of free fats and sterols. This material was then extracted with alcohol-ether and
again dried and weighed. This was considered to give a value for the bound lipid. In order
to relate the amount of lipid to the number of halves, counts were made on suspensions of halves
prior to drying. The following values expressed as nigs, of lipid per million halves were' ob-
tained: heavy halves — 6.6 (ether fraction), 12.2 (alcohol-ether fraction) ; light halves — 2.2
(ether fraction), 9.6 (alcohol-ether fraction). Thus, 75.0', per cent of the free fats and sterols,
56.0 of the bound lifids and 61.6 per cent of the total lipids are in the heavy halves. The sum
of the total lipids in the two halves is equal to 30.6 mgs. per 10"' cells which compares favorably
with the value 34.1 mgs. per 10" cells calculated from the data given by Parpart (Biol. Bull.,
81: 296, 1941) for unfertilized, whole eggs. Similarly a comparison can be made between the
sum of the bound lipids of the two halves and of the whole egg. Their values are 71.3 per
cent and 77 per cent, respectively.
PRESENTED AT MARINE BIOLOGICAL LABORATORY
Evidence for enzymatic participation in the penetration of the human erythrocyte
by glyccrol. PAUL G. LEFEVRE.
Jacobs and his associates have reported that an amount of copper sufficient to cover only
a very small fraction of the surface of the cells involved markedly inhibits hemolysis of human
red cells in isotonic glycerol. This report concerns the extension of this finding to the effects
of other substances which inhibit the same types of enzymes affected by traces of copper.
Following the pattern prescribed by Barren and Singer for identification of sulfhydryl
activity, iodine, mercuric ion, the arsenical Mapharsen, and p-chloromercuribenzoate were shown
to inhibit hemolysis by glycerol, buffered at pH 7.1. This inhibition failed in the presence of
cysteine, glutathione, or thioglycolic acid ; and could be reversed by later addition of these sub-
stances at 2-3 times the concentration of the inhibitor, except with Mapharsen. These relations
indicate strongly that active •- SH groups are involved in carrying glycerol into the cell.
Though sensitive to the inhibitors mentioned, the hemolytic process was not affected by iodoace-
tate ; this indicates that the sulfhydryl groups involved are of the difficultly available type, not
inactivated by the alkylating agents.
Since phosphorylation is apparently essential in transfer of sugars and other substances
across the membranes of the kidney tubule and the intestinal cell, it is proposed tentatively that
the enzymatic step involved in the present studies is the phosphorylation of glycerol. Adenosine
triphosphatase, capable of this step, is present in the erythrocyte, and shows the same pattern
of sensitivity to inhibitors as found in the present instance, as well as similar relations of ac-
tivity to pH. Further, more decisive tests of the proposed identity of the enzymatic factor are
planned.
"Accommodation" and opening excitation in nerve and muscle. PAUL G. LE-
FEVRE.
In his mathematical analysis of electrical excitation in 1936, Hill pointed out that the accom-
modative process (recession of threshold under the influence of a stimulus) itself accounted for
the phenomenon of excitation at the anode at the "break" of a constant current. There seems
to have been no attempt to test this neglected implication of Hill's theory: that "accommodation"
is an essential prerequisite for "opening excitation" at the anode. This report concerns the
occurrence of opening excitation in tissues showing no accommodation.
Following Solandt's practise, frog sciatic nerves were treated with citrate until they no
longer showed any accommodation : their threshold was independent of the rate of increase of
the excitatory current (delivered with Solandt's condenser-charge arrangement). In such
preparations, in spite of the absence of accommodation, there was no difficulty in eliciting an
anodal response at the cessation of a steady current. The same result was readily obtained with
exposed sciatic nerves of anesthetized rats treated with citrate.
Frog sartorii, or the pharyngeal retractors of Thyone, if stimulated in the nerve-free re-
gions, or following neural degeneration, also showed no accommodation. But all attempts to
demonstrate any response at "break" in these muscles failed ; this is in accordance with the
predictions of Hill's theory. Only in the case of citrated nerves was seen the troublesome occur-
rence of opening excitation in the absence of any accommodation.
A further analysis of this matter is planned, to determine whether the results may be ex-
plained on the basis of a postulated fundamental difference between accommodation at the
cathode and that at the anode ; the latter persisting in the absence of the calcium ion required
by the former.
A photometric study of the kinetics of fibrin formation. JOSEPH LEIN.
The clotting of fibrinogen solutions by thrombin was studied by measuring the optical
density and light scatter as the process occurred. The results can be analyzed kinetically only
when purified preparations were used. If other plasma proteins are present the degree of light
scatter also depends on their concentrations. This is believed due to a trapping effect of non-
clottable proteins by the fibrin as it is formed. The light scatter studies were particularly useful
in the kinetic analysis of the clotting process.
224 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The reaction was considered from a polymerization viewpoint, the fibrin representing the
polymer formed through the action of thrombin on the monomer fibrinogen. First order re-
action kinetics were employed. The following assumptions were made. (1) With constant
thrombin concentrations the rate of increase of the polymer size is proportional to the fibrinogen
concentration (dN/dT — K,F~). (2) The rate of decrease of the fibrinogen is proportional to
the fibrinogen concentration (— dF/dT -= K~F). (3) The light scatter under the conditions
of the experiment is proportional to the increase in particle size once it reaches the critical size
(N0) which first scatters light. (LS — KS(N — N0). From these formulas a relationship
was derived that included light scatter (LS}, the initial concentration of fibrinogen (Fn), time
(T), and time (Ta) for the polymer to reach a size (AM that would scatter light. The for-
mula may be expressed as :
log (K.KJK, F.e-K^T, - LS) = - 0.434 K,T + log KtKJK, Fn
The relationship was tested on a series of experiments in which the initial concentration
of fibrinogen was altered, the thrombin being kept constant. The calculated values of the con-
stants agreed with the experimental values within a 6 per cent average deviation. It thus ap-
pears that the course of the reaction may be considered to be molecular and that thrombin acts
as a true catalyst, not forming part of the final fibrin product.
The effect of iodacetate on the changes in muscular latency induced by activity.
A. SANDOW. No abstract submitted.
Formation of the nuclear membrane and other mitotic events in Chaos chaos Linn
and Chaos ncos (nczv species). A. A. SCHAEFFER.
The mitotic stages of the amebas mentioned are easily followed in the living animal. The
principal stages are the following: 1. the nucleus about to divide swells up to about 6 times its
former volume. 2. The chromatin grains (300 to 600 in number) gradually disappear, as if
going into solution. Some of these grains coalesce before going into solution. 3. A new mass
of small grains (about 2500) appear, before all the larger grains of the so-called resting nucleus
have disappeared. These small grains arrange themselves first as a lens-shaped cloud, then as
a plate of about 2 grains thickness. At this stage the plate of grains is occasionally seen to be
indistinctly divided into at least 4, possibly as many as 8 or 12, smaller groups of equal size.
4. This plate of grains then separates into 2 plates which rapidly move apart. 5. Fibers analo-
gous to, if not identical with, spindle fibers, appear between the plates, as the plates separate.
Fibers also appear on the other face of the plates. All fibers are at first horizontal and parallel.
6. The plates separate and the inter-plate fibers lengthen until the plates are separated to about
2 or 3 times their diameter when, because of the streaming of the protoplasm, the plates are
torn apart. During this time the nuclear membrane breaks into pieces which eventually com-
pletely disappear. 7. The separated plates, still granular, become bent like a concavo-convex
lens, with polar fibers still attached. The granules soon disappear, leaving a very thin, per-
fectly homogeneous flat disk that shows a brilliant blue green color when seen on edge. No
refractory edge can be made out. This stage is difficult to see. 8. After a few minutes the
disk shrinks in diameter and is thrown into rope-like folds around the periphery. 9. Very soon
thereafter a refractory edge begins to appear as the folds disappear. 10. Very fine grains pres-
ently begin to appear until about 1,400 are formed. Many of these coalesce to form larger
grains until only about 600 to 700 remain in the newly formed daughter nucleus. (Further
reduction in number may occur during the next few hours.) While the small grains are ap-
pearing, the edge of the nucleus becomes more and more refractory until in the new nucleus it
is seen as the new nuclear membrane. The steps outlined here require about 28 minutes.
The mitotic events of Chaos difflucns, as far as they have been observed, are practically
identical with those of the above-mentioned species, except for size.
Correlated histories of individual sense organs and their nerves, as seen in living
frog tadpoles. CARL CASKEY SPEIDEL.
In the living frog tadpole it is possible to make daily observations on the same individual
nerves and sense organs of the lateral-line for many weeks or months. By suitable operations
PRESENTED AT MARINE BIOLOGICAL LABORATORY 225
some sense organs may be deprived of their nerve supply (nerveless organs), and conversely,
some aberrant lateral-line branches may be induced to grow without reaching any sense organ
(organless nerves).
Prolonged observations of nerveless organs (1 to 21 months) reveal the following: (1)
During the first two months in regenerating or growing zones the sense organs are largely
independent of their nerve supply. They grow and divide readily. (2) During later months,
however, regressive changes of atrophy and degeneration take place. The organs become
smaller. Some degenerate and disappear. Occasionally, however, a nerveless organ may per-
sist for more than a year.
Prolonged observations of organless nerves reveal the following: (1) During the first two
months they are largely independent of the sense organs. They grow and become provided
with both neurilemma and myelin sheaths. (2) During later months, however, regressive
changes ensue. The myelin sheath is not maintained on any functionless fiber. It becomes
thinner and ultimately disappears. The untnyelinated fiber resulting may then itself degenerate,
leaving only a collapsed neurilemma tube.
Thus, the structural integrity of both lateral-line sense organ and nerve fiber is definitely
correlated with the successful establishment of a functional relationship between the two.
Sense hairs and orange granules are specialized structures of lateral-line organs. The be-
havior of both of these under various experimental conditions indicates their relative inde-
pendence of nerve influence.
Many other histories involving nerve and sense organ relations in wound zones have also
been recorded. Illustrative cine-photomicrographs have been made.
The effect of prolonged starvation on the lipids in Phascolosoma gonldii. CHARLES
G. WILBER.
It is known that the muscle in vertebrates serves as a storehouse of fats and that during
starvation the fats in muscle decrease whereas the fat in various internal organs is not changed.
Whether this is true for invertebrates is not known.
In order to throw light on the problem, worms (Phascolosoma c/onldii) were starved for
one month and then the whole worm, the muscle, and the perivisceral fluid respectively were
analyzed for phospholipid, for cholesterol, and for fatty acid. These results were compared
with the results of similar analyses made on control worms.
It was found that in the whole worm there was a loss of all lipid constituents. In the peri-
visceral fluid, phospholipid and fatty acid decreased greatly, but cholesterol did not decrease.
In the muscle there was an apparent increase in lipid material which can be explained on the
basis of absorption of some of the tissue. In muscle the fatty acid is decreased, as is clear from
the larger lipocytic coefficient of the muscle of starved worms.
It is concluded that the perivisceral fluid serves as a storehouse of lipid in Phascolosoma
and that the muscle does not. Moreover, it seems that phospholipid and fatty acid are used
during starvation. Whether cholesterol is also used is not certain. Phascolosoma differs,
therefore, from the vertebrates in the use of phospholipid during starvation and in the fact that
muscle is not the important storehouse of fat.
Protoplasmic clotting in isolated muscle fibers. ARTHUR A. WOODWARD.
Isolated muscle fibers provide a material favorably adapted to the quantitative study of
protoplasmic clotting. The cut ends form clots which pass in waves over the length of the fiber.
The rate of the clotting reaction can be measured and is expressed as mm. of fiber converted into
clot per minute. Single fibers are teased from the adductor magnus of Rana pipicus; all solutions
used are kept at pH 7.1 — 7.4 with glycine buffer.
In Ringer's solution, used as a standard for comparison, the rate of clot formation is constant
for a given fiber and varies only moderately from fiber to fiber within a muscle. The normal
rate is about 0.050 mm./min.
Ca ion causes a very rapid clot formation ; in this case it is shown that the rate is largely
a function of the speed with which Ca diffuses into the end of the fiber, the protoplasm clotting
with great rapidity once it is exposed to free Ca ion.
226 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The clotting process is relatively insensitive to pH changes in the region from pH 5 to pH 9 ;
above and below this the rate increases very rapidly. Near the regions in which thrombin is
inactivated liquefaction has been observed under certain conditions.
Solutions of crystalline trypsin cause clot formation at a rate averaging about 50 times that
of the control. In the absence of Ca, trypsin produces only a slight increase over the control.
Crystalline chymotrypsin is much less active than trypsin and also has very little effect in the
absence of Ca. Preparations of crude papain cause clot formation at a moderately high rate ;
addition of glutathione increases the effect to the magnitude of that produced by trypsin. Ab-
sence of Ca has no effect on the action of papain.
AUGUST 24
Some aspects of the histology and physiology of luminescence in "railroad worms."
JOHN B. BUCK.
In the Uruguayan "railroad worm," Phrixothrix, the lateral photogenic organs are small
compact ovoid masses of small dense cells near the posterior edges of the segments somewhat
above the spiracular level. The organ is apparently supplied by one trachea ramifying profusely
between the cells. There are no end-cells. Large oenocyte-like cells are present near some of
the lateral photogenic organs and elsewhere.
In Phengodcs, a close American relative of Phrixothrix, the lateral organs are in the pos-
terior ends of horizontal rolls of tissue which extend along the segments ventral to the spiracles.
Light is also emitted along the dorsal posterior edges of most of the segments. Both lateral
and dorsal organs apparently consist of loose aggregations of very large oenocyte-like cells with-
out end-cells or special tracheal supply. Similar cells are present in small numbers in parts of
the body not regarded as luminous but not in the lateral tissue roll except in the region which
emits light. Further evidence is furnished by the observation that the light of Phcnyodcs can be
seen microscopically to come from clusters of round or oval spots corresponding in shape, posi-
tion, number, and size to the oencyte-like cells.
The photogenic organ of Phyri.vothrix is very similar to that in the larval firefly and agrees
with the generalization that luminous beetles which produce a lingering glow rather than a short
flash, have organs of relatively simple structure without end-cells.
The photogenic organs of Phcngodes are the simplest yet known in insects and represent
the first time, that bioluminescence has been ascribed to eonocytes. A corresponding physiological
simplicity may be the fact that the light is continuous.
Phengodcs dims in the vapor of KT2 and 1CT3 M. HCN at about the same rate as luminous
bacteria, and faster than fireflies.
Effect of caffeine concentration upon retardation of Arbacia development. RALPH
HOLT CHENEY.
Sea urchin eggs and sperm were subjected to eight different concentrations of caffeine-in-
sea-water for 15 minutes, then mixed for fertilization and the developmental rates in S.W. and
S.W.C. were compared with the normal rate of untreated ova and sperm. Observations were
made at intervals during a three-day 'period. Normal time rates were accepted as stated by E.B.
Harvey, 1940 (Biol. Bull, 79, (1) "Plate II, photographs 16-32 inclusive).
All eggs utilized in a single experiment were obtained from the same female and all sperm
from one male. Eggs and sperm were shed directly into S.W. or S.W.C. prior to mixing. The
series of six combinations presented in 1942 (Biol. Bull., 83) were repeated and extended to a
full three day period as follows :— N? X Nrf,' N? X Crf, C? X Nrf, C? X Cd, all developed in
normal sea water after the original immersion of fifteen minutes after shedding as indicated
into S.W. or S.W.C. In the cases of C? X NJ1 and C$ X Cd1, each was developed also in S.W.C.
Results indicated that the period of immersion (15 min.) in the caffeine concentrations em-
ployed prior to mixing the gametes did not render the ova non-fertilizable subsequently nor
destroy the ability of the sperm to fertilize. Eggs and/or sperm, however, were not unaffected
at least by higher concentrations, since C? X Cd cultures, although they did form the fertilization
membrane when mixed and developed in uncaffeinized S.W., the fertilized ova never survived
longer than the early cleavage stages.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 227
Plutei developed in normal time and form in all of the six combinations of 0.002 per cent and
0.004 per cent S.W.C. Gametes shed into two per centum S.W.C., in each of the four combina-
tions developed in S.W. formed the P.M. but showed retarded development and in no case
reached the pluteus stage before death. In the two combinations developed in S.W.C., the P.M.
was not formed. Intermediate percentages used between these extremes of concentration gave
intermediate effects indicating that in general, the effects were directly proportional to the con-
centration of the caffeine.
Other experimental series subjected normally fertilized ova (N? X N^1 in S.W.) which had
developed to a desired stage of development, to the different concentrations of caffeine-in-S.W.
Results here indicated similarly that the retardation effect upon the development time was pro-
portional to the concentration.
Shape changes in flic denuded Nereis egg preceding first cleavage. ALBERTA T.
JONES.
•It is known from Hoadley (1934) that Nereis eggs undergo a series of amoeboid changes
prior to first cleavage. Neither the reason for this phenomenon nor the exact pattern followed
has been completely described. It is the purpose of this paper to report the principal findings on
the pattern of shape changes up to first cleavage in the egg of Nereis limbata. The denuded egg
was used to eliminate the complications of membrane and external jelly.
Gametes were taken from animals caught the previous night in Eel Pond (Woods Hole) and
artificial insemination was carried out. The fertilized eggs were denuded by treatment with
alkaline 0.53 molar NaCl solution brought to pH 10.5 by addition of NaL,CO3. This is the method
used by Costello (1939 and 1945). Observations began when the denuded eggs were rinsed free
of alkali and the first polar body had formed. Outline drawings of the eggs were made with a
camera lucida at three minute intervals. To serve as reference points, the position of polar
bodies and oil droplets was indicated.
Hoadley states: — "pulsations of the (Nereis) egg are of two sorts, one of which is quite
extensive and results in general distortion of the sphere, and the other of which results in surface
irregularities which appear more or less localized." The shape changes discernible in denuded
eggs seem to correspond to Hoadley's first category. A consistent pattern of sequences, dif-
ferent from those described by Hoadley for the intact egg, has been found. The sequences in-
clude such general distortions as : ( 1 ) polar flattening followed by rerounding ; (2 ) elongation
in the polar axis followed by rerounding; and, (3) elongation in the equatorial axis followed by
formation of the first cleavage plane. The magnitude of these changes, in comparison with the
pulsations observed by Hoadley, may be attributed to the absence of jelly mass and membrane.
It may be concluded that shape changes in the denuded Nereis egg prior to first cleavage
proceed (1) according to a definite pattern; and (2) always with a particular relation to the
polar axis of the egg.
'Hormone control of dchydrogenasc activity of Crustacean tissues. ELOISE KUNTZ.
Sea water extracts of the sinus glands of Libinia cmarginata, Honiants americanus, Uca
pugilator and U. pugnax and similarly prepared extracts of the central nervous system of Libinia,
Homarus and the arachnoid, Limulus polyphcmus, were made. These were boiled and centri-
fuged and the supernatant fluid was used. The extracts were tested for their effect upon de-
hydrogenase activity of gastric gland and muscle of Libinia, Honiants and Limulus, which were
measured in Thunberg tubes with methylene blue as the hydrogen acceptor.
The effect of sinus gland extract was dependent upon the concentration. Half of a
Libinia sinus gland doubled the rate of methylene blue reduction, but the reduction rate rapidly
fell to slightly above that of the controls with increasing concentrations. Half of a Uca pugilator
sinus gland also doubled the reduction rate, but activity remained high for concentrations of I
sinus glands, falling to the level of the controls at 6 sinus glands. Here it remained. Uca
pugnax showed strong inhibitory action in concentrations of 6 to 12 sinus glands. The character
of the curves suggests the possibility of two active substances which vary in relative proportions
in different species.
PRESENTED AT MARINE BIOLOGICAL LABORATORY
Central nervous system extracts of Homarus, Libinia and Limuhts in concentrations of 0.7
mg. tissue per cc. strongly stimulated dehydrogenase activity. Extracts of other tissues were
ineffective at several times this concentration.
Localization of hormone production within the nervous system was demonstrated. In
Homarus the circumoesophageal ganglia and second ventral ganglion were most effective. The
brain and suboesophageal ganglion were ineffective. The remainder of the ventral cord had a
relatively weak action. All parts of the circumoesophageal ring of Limulus were effective.
An antagonistic action of sinus gland and nervous system extract was demonstrated. The ad-
dition of sinus gland hormone to a system stimulated by central nervous system extracts de-
pressed dehydrogenase activity to that of the controls.
A comparative study of cholinest erase activity in normal and gcneticallv deficient
strains of Drosop/iila melanogaster. DR. F. POULSON AND E. J. ROELL.
Cholinesterase activity has been determined in late embryos of several strains of Drosophila
melanogaster by means of the cartesian diver technique which measures the evolution of CO^
from Ringer-bicarbonate solution following hydrolysis of acetylcholine in the presence of an
atmosphere of 95 per cent N2 and 5 per cent CO2. Timed eggs from a stock of the deficiency
known as Notch8 crossed to Canton-S wild strain (Ns/+) were dechorionated by Slifer's hypo-
chlorite method and classified as normal or Notch-deficient. At 24 hours normals are larvae
ready to hatch, while the deficient male embryos are strikingly abnormal and possess a nervous
system about three times normal size. The ratio of types is 3 normal : 1 abnormal. Embryos
were cut up and placed in divers containing 1 mm.:f Ringer-bicarbonate and 0.5 mm.3 of 1.5 per
cent acetylcholine. Although it is possible to carry out measurements on single embryos, two
to five embryos per diver were used in most experiments. Readings were made at ten minute
intervals for one hour after the divers had reached thermal equilibrium.
A series of four determinations on 24-hour normals gave an average of 12.8 m ju.l. CO2/em-
bryo/hour. A series of five determinations on Notch 8 deficient male embryos gave an average of
34.0 m./z.l. COo/embryo/hour. The cholinesterase activity of Notch embryos is 2.7 times that of
normal, which is nearly the same as the volume ratio of Notch/normal nervous systems, 3.3 as
determined by planimeter from camera lucida outlines of sections. Thus cholinesterase activity
is proportional to volume of nervous tissue. Notch-deficient embryos of other strains have given
similar results. Thus the Notch male nervous system while abnormal in size and morphology
is biochemically normal with respect to cholinesterase. A first step has been made in studying
the rate of increase with development of cholinesterase activity in both normal and deficient em-
bryos. At 18.5 hours the activity of the Notch embryo is 8.3 m.ju.l. CO2/hour, that of normal 3.8
m.ju.l. COo/hour. In the unhatched Notch embryo at 48 hours the activity increases to 51.0
m.fji.l. COn/hour.
As checks, cholinesterase activity of unfertilized eggs and methyl butyrase activity of normal
and Notch embryos were measured and found to be negligible.
To determine the location of cholinesterase in normal embryos, central nervous systems were
dissected out and their cholinesterase activity measured separately from the remaining portion of
the embryos. One determination has given a value of 17.0 m. .l./N.S./hour. The value for the
remnant is 2.6 m./i.l./hour. Since the central nervous system makes up not more than one-
sixth the embryonic volume the cholinesterase activity there is roughly forty times that in the
remnant.
Possible metabolic and pliysical chemical factors in the production of the injury
potential in spider crab nerve* A. M. SHANES.
In contrast to frog sciatic nerve, spider crab nerve is permeable to both potassium and
chloride ions and to a lesser extent to sodium. The relationship between metabolism and the
potentials therefore cannot be the same as in frog nerve which is highly impermeable to chlo-
ride and other small anions as well as to sodium. This is confirmed by the following observa-
tions: (1) Although 0.002 to 0.0006 M iodoacetate (IAA) produces a continuous slow fall in
* Aided by grants from the Penrose Fund of the American Philosophical Society and from
the American Academy of Arts and Sciences.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 229
potential in oxygen, 0.02 M pyruvate is unable to counteract this inhibition; (2) pyruvate
accentuates IAA inhibition of the post-anoxic recovery of potential ; (3) the decline of potential
in nitrogen is more rapid than in frog nerve and is not hastened by IAA ; (4) glucose does not
retard the fall in potential during anoxia and inhibits a recovery in oxygen. The inhibitory
effects of pyruvate and glucose may be the result of acid production, for 5 per cent CO2 lowers
the potential in this system in contrast to its effect in frog nerve.
These results provide a basis for understanding some peculiarities of carbohydrate metabo-
lism in spider crabs. For example, blood sugar levels average only 1 mg. per cent, nerve gly-
cogen ranges from 500 to 2,000 mg. per cent wet weight (Kleinholtz, unpublished), and the
breakdown of glycogen is reported to be largely to simple sugars as well as to lactic acid.
The potassium content of these fibers is known to be high and the potential is inversely
related to the extracellular potassium concentration. Consequently the injury potential is
probably a potassium concentration potential as in frog nerve. In yeast (Rothstein and Haege,
1943) potassium retention is stoichiometrically related to hydrogen ions lost to the medium when
glucose is assimilated to form reserve carbohydrate. This mechanism may explain the high
levels of both glycogen and potassium, and hence the metabolism-potential relationship, in spider
crab nerve.
Some effects of tannic acid on osmotic hemolysis. T. H. WILSON AND M. H.
JACOBS.
Human erythrocytes are less easily hemolyzed in hypotonic solutions of NaCl in the pres-
ence of tannic acid than in its absence. The salt solution employed in the present experiments
ranged from 0.091 to 0.069 M and those of tannic acid from 1/800 to 1/51,200 per cent. Even
at the lowest of these concentrations of tannic acid there was a marked protective effect. In
hypotonic solutions of Na2SO4 ranging from 0.045 to 0.031 M and with the same concentrations
of tannic acid as before, the effect was the exact opposite, hemolysis invariably being increased
except at the lowest concentrations of tannic acid. Very similar effects, somewhat complicated
by the permeability of the erythrocyte to ammonium salts, were obtained with NH4C1 and
(N,H4)2SO4, respectively.
In the light of results obtained with molecular films of proteins by Schulman and others,
the action of the tannic acid in the chloride solutions might be explained either by a strengthen-
ing of the cell surface or by a decrease in its permeability to hemoglobin. Such an action in the
case of the sulfate solutions is not necessarily excluded, but it seems to be overshadowed by an-
other effect of a different nature, namely, the decreased permeability to anions produced by
tannic acid, described elsewhere by Jacobs, Stewart and Butler. Since swelling of the erythro-
cyte is known to be opposed by the exchange of bivalent sulfate ions from the outside for uni-
valent anions from the inside, tannic acid, by hindering this exchange, might in this particular
case indirectly favor hemolysis, despite its more direct protective effect on the cell surface.
The effect of roentgen radiation on protoplasmic viscosity changes during mitosis.
WALTER L. WILSON.
Roentgen radiation has a marked effect on the protoplasmic viscosity of the dividing sea-
urchin egg. If the eggs, or sperm, or both the eggs and sperm of Arbacia punctulata are ir-
radiated before fertilization, then the normal pattern of viscosity change is altered. In the
control the viscosity was low shortly after fertilization, then increased to a peak at 15 minutes
(23° C.). It remained high for 6-10 minutes and then decreased. This decrease was markedly
retarded by irradiation of the sperm or eggs (11,300 r at 6400 r/m), or both the sperm and
eggs (5,000 r at 6400 r/m) before fertilization. In these experiments the viscosity remained
high two or three times longer than in the controls. In three experiments out of ten in which
irradiated eggs were fertilized with normal sperm, the viscosity increased to a value almost
twice that of the controls.
Biological specificity and the synthesis, oj native proteins. D. WRINCH. No ab-
stract submitted.
230 PRESENTED AT MARINE BIOLOGICAL LABORATORY
PAPERS READ BY TITLE
The effects of massive doses of ergonovine male ate * upon the smaller blood
vessels as seen directly with the microscope in the living mammal.1 RICHARD
G. ABELL.
In these experiments, as in those described above, the blood vessels were studied in trans-
parent 'moat' chambers in rabbits' ears. All injections were of 3.0 mgm., and all were made
intravenously. Three mgm. in a rabbit corresponds to 60 mgm. in a man, which is 600 times
the clinical intravenous dose. In these experiments the injection of 3.0 mgm. caused complete
arteriolar constriction for 3 to 4 minutes. The larger arterioles (80 to 90 microns in diameter)
remained narrowed to approximately one-half of their control diameters for 3 to 4 hours. A
temporary constriction of the venules to from 0.5 to 0.8 of their control diameters occurred.
In addition, this amount of ergonovine caused thicking of leukocytes to the walls of the arte-
rioles, capillaries and venules. The degree of sticking varied widely in different rabbits ; in
some cases it was slight ; in others large numbers of leukocytes stuck to the walls of the capil-
laries and venules, and emigrated into the surrounding tissue. In one rabbit injections of 3.0
mgm. were followed by the formation of leukocytic emboli, which blocked many of the capil-
laries and venules and formed thrombi. The reaction was reversible and the thrombi usually
disappeared within approximately 4 hours following the injections. This is in accord with the
flow toxicity of ergonovine, and its failure to produce gangrene on repeated injections.
As shown by the work of numerous investigators, two other ergot alkaloids, ergotoxine and
ergotamine, do produce gangrene on .repeated injection. Such gangrene also occurs in ergot
poisoning and is due to obliterative endarteritis and thrombosis. The formation of these
thrombi is usually attributed to prolonged constriction of the small arteries, and interruption of
the blood flow, but this is entirely hypothetical.
In the present experiments thrombi were formed due to the increase in stickiness of the
endothelium toward leukocytes and of the leukocytes toward each other.
Perhaps gangrene produced by ergot and its more toxic alkaloids may be caused by a more
severe and prolonged reaction of the type described above.
Secretory cells in the branchial epithelium of fislics. GERRIT BEVELANDER.
It was shown by Smith (1930, 1931, 1932) that the osmotic regulation of the body fluids in
fresh and salt water teleosts and in elasmobranchs is effected considerably by the extrarenal
excretion of salt (NaCl and KC1) under conditions that probably involve considerable osmotic
work. It was further inferred that this exchange occurred in the gills. A previous study of
the branchial epithelium in an extensive and widely divergent group of fishes (Bevelander
(1935), led this writer to conclude that the only specialization occurring in the branchial epi-
thelium of fishes consists in a thicker epithelium in the elasmobranchs than in teleosts and the
presence of numerous mucous cells in all species examined. The cells which we described as
mucous, were alleged to be "chloride secreting" cells in Anguilla and in some fresh water tele-
osts, but not in elasmobranchs by Keyes and Willmer (1932).
A re-examination of this problem included the experimental stimulation of secretion of the
cells in dispute in representative teleosts and elasmobranchs. These cells were then subjected
to a number of histochemical tests and were shown to be positive for mucin. Further, the oral
and opercular membranes were also examined and it appears unlikely on the basis of structure
that they are concerned with osmotic regulation.
In order to comply with the observed physiological data, the cells which are responsible
for extrarenal excretion must be in intimate relation with the blood supply and the external
milieu, they must be very extensive to account for the considerable work performed, and finally
they must be present in teleosts and elasmobranchs. Our observations reaffirm the absence of
any specialized structures ; the only cells which comply with the three criteria required are the
* 'Ergotrate' (Ergonovine Maleate, U.S. P., Lilly).
1 This work was aided by a grant made by Eli Lilly and Company to the Department of
Anatomy of the University of Pennsylvania Medical School.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 231
respiratory epithelial themselves. It is further suggested that the observed conservation of
urea in the elasmobranch gills may be affected by the relatively thick respiratory epithelium
which covers the gill filaments.
A modified Crompton formula for the latent heat of vaporisation. ALBERT P.
MATHEWS.
The general formula for the latent heat, L, which I have found is
(1) L = CR'Thie(d/D)
R' is the actual value of the gas constant in the liquid phase, constantly falling as molecular co-
aggregation increases with falling temperature, d is the liquid density and D that of the vapor,
C is a constant peculiar to the substance, often not far from 2.
The values of C and R' are obtained from the following general formulas which apply to all
non-associating substances with the possible exceptions of hydrogen and helium.
(2) C =C(R/R'e)(L/(L-E)-)a
)
(3) C - 1.1292- (3/8)5-+ (9/64 ) S2 = 0.8792 + ( (3/8)5 - 0.5)2
5 in (3) is the critical coefficient: RTc/pcVc in which R has its ideal value. 5 may be com-
puted by (4) :
(4) S = [(dmRTc/Mpc) + 16((7c-D/ro) -12((TC-T)/TCY]
/[I + 5.158((7\, - T)/Te) - 3.158 ((r. - T)/Tcy]
dm is the mean density of saturated vapor and liquid at temperature, T ; pc and Tc the critical
pressure and temperature.
(5) (L/(L-£))«=
L-E is the internal latent heat of vaporization. (5) is obtained from (6).
(6) ((T/p)(dp/dT))e = (L/£)c=l + (27S2tf'c)/64 R")
(7) R'c/R = (512 - 64S + 216S2 - 27S3)/512.S
R'T in (1) is obtained from (8).
(8) (L/(L-E) )r = R'T/R'. = 1 + ( (L/(L-E) )„ - 1) ( (9/16) (T/TC) + 7/16) (7YTC)2
(9) R', = R
At absolute zero L/ (L-E) is 1 and it advances with temperature as co-aggregation diminishes.
In the ideal state C, 5 and (L/£)c are 1. When 5 has its highest value of 4 in a normal sub-
stance (L/E)C will be 7.3346 and, when 5" has its lowest value of 3/8, (L/E)C will be 4. 5 may
be calculated also from the latent heat of vaporization at any temperature, C' being equal to
(L-E)/RTln.(d/D) by (10):
(10) 5= (8/3) (0.5 + V (C- 0.8792))
Obtained from (3) above; E being taken, with small error usually, as equal to p(V-v).
Formula (1) above is an easier and more accurate way of computing the latent heat of
vaporization than by the thermodynamic equation: L— (Tdp/dT) (V-v). The results by (1)
agree usually within 1 per cent with the experimental determinations at the normal boiling point
as made by J. H. Mathews and others. The derivation of all the foregoing formulas will be
given in the full papers together with examples of application to specific cases and also the general
formula for the Cailletet and Mathias law (11) :
(11) rfm = rf.[l + (5.158- 16/5) ((T,-T)/Tc) + (12/5 - 3.158) ((7. -
232 PRESENTED AT MARINE BIOLOGICAL LABORATORY
Heat Death. PAUL R. ORR.
I. Time-temperature relationships in marine animals.
Temperature as an intrinsic ecological factor which determines, to a great extent, the abun-
dance, life cycle, and distribution of marine organisms, is a well-established fact. However, the
duration of exposure required to produce death at each temperature in the effective series has
not been taken into consideration. Thus, in order to state accurately the conditions of heat
death, it is necessary to plot a curve in which both variables, temperature and time, are
represented.
Heat death curves have been plotted for Uca pugilator, Asterias forbesi, Ophiodenna
brevispuium, Arbacia punctulata, Nassa obsolcta, Fiindulus hctcrnclitus.
All of the curves have approximately the same shape. For a relatively slight rise in tem-
perature there is a marked drop in the length of exposure necessary to cause death. This rela-
tionship is not one of direct proportionality.
II. Differential response of the entire animal (Rana pificns) and several of its organ systems.
Whether we are dealing with cells or multicellular organs and tissues, or the organism as
a whole, we are confronted with the fact that not all of the cells, organs, etc., have the same
sensitivity to heat. An animal exposed to excessive heat for a length of time to cause complete
loss of excitability might well be pronounced dead, for it never again will show any signs of
life as a complete organism. Yet there are parts of the complex animal that are "alive."
The animal as a whole, the tadpole, sciatic nerve, sartorius and gastrocnemius muscles, and
heart were separately studied, and curves were plotted for the heat death points of each. The
data show that in the adult animal the order of death is: (1) the organism as a whole; (2) the
muscular system; (3) heart, and (4) nervous tissue.
All heat death curves plotted are of the same shape, showing a sudden drop followed by a
gradual approach to a constant level.
III. The effect of high temperatures on heart rate in Venus mercenaria.
In the clam heart (Venus mercenaria} we have an automatic mechanism by which the effect
of heat can be studied. By subjecting excised hearts to a series of high temperatures and noting
the heart rate it was possible to determine the lethal point for each temperature and thus plot
a curv.e showing time/temperature relationship.
For the clam heart the same general type of curve was found as shown in previous studies
on marine animals and frogs. That is, there is a point at which the hearts will beat for a rela-
tively long period of time; then as they are subjected to higher temperatures there is a rapid
decrease in heart rate, followed by a leveling off to a constant rate.
Penetration glands in tapeworm Onchosphcres. \Y. MALCOLM REID.
Although various types of cystogenous and penetration glands have long been figured and
studied as a part of the internal structure of trematode cercariae and miracidia, they have not
been recognized in the onchosphere stage of cestodes. A pair of such glands has been found in
the fowl cestodes Raillietina cesticillus (Molin), and Choanotaenia infundibulum (Bloch) and
in a herring gull cestode Hymcnolepis sp. Although these glands may be seen under favorable
conditions without special stains, they respond in the same manner to vital stains as do trema-
tode glands, showing up best with Nile blue sulfate and neutral red. The gland stretches to the
posterior end of the larva, where it appears to be anchored. The secretion pores are located
near the anterior and slightly to the side and above the middle pair of hooks when these hooks
are oriented with the points directed anteriorly and downward. The granular contents may be
seen to move about as the general contour of the glands is changed by the violent contractions
associated with hook movements and at times some of the secretion may be seen exuding from
the pores. A single nucleus is located near the middle of each gland, and the two glands are
connected by a narrow isthmus near the posterior end.
The nature of the secretion has not been determined but it is possible that it assists the
larva in penetration since this granular substance is given off at a time in the life cycle when
the six-hooked embryo must break out of the covering membranes of the egg and penetrate the
gut of an arthropod intermediate host.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 233
Intensity-duration relation in stimulation by light. F. J. M. SICHEL AND P. B.
ARMSTRONG.
The excised sphincter pupillae of many vertebrates will respond by constriction to stimula-
tion by visible light of suitable wave-lengths. In these experiments the sphincter pupilla of the
eel, Anguilla rostrata, was used. The sphincter was excised from small adults, 15 to 18 inches
in length.
The sphincter was pinned out, anterior surface uppermost, on white beeswax. It was
illuminated for observation by transmitted red light, to which the preparation is insensitive.
The source of light for stimulation was a tungsten filament lamp maintained at constant voltage.
This was focussed on the preparation obliquely from above. The intensity of the stimulating
light was varied by Wratten neutral niters and a neutral wedge. The duration of the stimu-
lating flash was controlled by a shutter manually operated and timed by a stop watch. The
criterion of threshold was the smallest contraction visible through a low-power microscope.
An eyepiece filar micrometer was used to advantage in determining the threshold stimulus.
The preparation was bathed in a Ringert's fluid and permitted to become dark-adapted before
each experiment.
The threshold was found to be a function of the duration and of the intensity of the stimu-
lating flash. The intensity-duration relation conforms with Hill's theory of excitation for rec-
tangular stimuli. The chronaxies averaged about 12 seconds, the range being from about 6
seconds to 20 seconds. In terms of the reciprocity law this would mean that the law holds
reasonably well for flashes shorter than, say, 10 seconds. At longer durations the deviation is,
in direction and amount, what would be expected on the basis of Hill's equation for excitation.
There is a definite rheobase, or minimal intensity of the stimulating flash below which excitation
is never produced, even for very long exposure times.
The pattern of flic intrinsic palmar musculature. WILLIAM L. STRAUS, JR.
The intrinsic palmar musculature of tetrapod vertebrates comprises two fundamental series :
(1) a superficial, arising from fascia or tendon, and showing variable tendency toward strati-
fication, and (2) a deep, arising from bone and always arranged in two layers separated by the
deep palmar nerves and vessels. Between the two series lies the mid-palmar space.
In urodeles (Nectunts inaculosus, Cryptobranchus alleghaniensis), the superficial series is
a single layer (flexores breves superficiales) arising from the dor sum of the long flexor tendon.
The deep series is composed of a superficial (contrahentes or adductores) and a deep (flexores
breves profundi, intermetacarpales, interphalangeus III?; in Cryptobranchus also flexores breves
minimi) layer.
In lizards (Sceloporus spinosus, Ctenosaura siinilis}, the superficial series tends to form
two layers — a superficial (flexores breves superficiales, marginal abductors), arising from the
transverse carpal ligament, and a deep (lumbricales), arising from the long flexor tendon; in
Sccloporus, however, such lamination is incomplete, for fibers of the superficial layer also arise
from the long flexor tendon. The deep series again exhibits superficial (contrahentes) and
deep (flexores breves profundi) layers.
In mammals (Didclphis virginiana, Macaco mulatto, Homo}, the superficial series forms
two distinct layers — a superficial (abductor pollicis brevis, flexor pollicis brevis, opponens pol-
licis?, palmaris brevis, flexor V brevis, abductor V; in Didclphis also a flexor brevis manus),
largely from palmar aponeurosis and transverse carpal ligament, and a deep (lumbricales), from
the deep long flexor tendon — separated by the superficial palmar vessels and nerves. The deep
series again has superficial (contrahentes ; only adductor pollicis in man) and deep (interossei,
opponens V) layers.
Muscular homologies, at least between vertebrate classes, cannot be reasonably extended
beyond comparison of entire palmar layers. Direct homology of individual muscle units is
profitless and probably invalid.
234 PRESENTED AT MARINE BIOLOGICAL LABORATORY
The toxicity of a mixture of high molecular alkyl-dimethyl-benzyl ammonium
chlorides to Fundulus. CHARLES H. TAFT.
The mixtures of high molecular alkyl-dimethyl-benzyl ammonium chlorides used is sold by
the Winthrop Chemical Company under the trade name Zephiran Chloride * for use as an anti-
septic or disinfectant.
Taft and Strandtmann (1945. Fed. Proc., 4: 136) showed that under laboratory conditions
this material is an efficient larvicide for the mosquito Culex quinquefasciatus and Aedes aegypti
in dilutions up to 1 : 250,000. It seems desirable to determine its toxicity to some of the animals
it might be brought in contact with if used for this purpose. Taft (1946. Texas Rpts. on Biol.
and Mcd., 4: 25) has reported its toxicity for various invertebrates.
To determine the toxicity by injection fundulus were injected intraperitoneally with different
doses of one per cent solution; 0.25 cc. killed 17 out of 22, 0.05 cc. killed 15 out of 17 while 0.1
cc. killed 24 out of 24 fundulus. When these fish died they were darker than the controls and
in many of them the abdomen was red about the site of injection. When the abdomen was
opened there was frequently a greenish fluid present and the viscera had the appearance of
having been cooked. The liver, gall bladder, heart, kidneys, and gills appeared normal.
Other fundulus were placed in finger bowls containing 225 cc. aerated sea water with dif-
ferent concentrations of the drug. When the fish were placed in dilutions of from approxi-
mately 1 : 2,500 to 1 : 100,000 all the fish died in from 35 to 105 minutes. On autopsy there
were no significant gross changes. A dilution of 1 : 225,000 killed 25 per cent of the fish while
1 : 500,000 did not kill any of the fish exposed to it.
To determine the effects of longer exposure to the drug several fundulus were placed in
battery jars in aerated sea water solution of from 1 : 100,000 to 1 : 400,000 and observed at the
end of 24 hours. All the fish exposed to 1 : 100,000 and 1 : 200,000 were found dead. Twenty-
five per cent of those exposed to 1 : 300,000 died while the 1 : 400,000 solution failed to kill any
fish. It is evident that the effective range of this drug when employed as mosquito larvicide
might be deleterious to fundulus.
Further evidence of polypoidy in the conjugation of green and colorless Paramecium
bnrsaria. RALPH WICHTERMAN.
In a study of the time-relations of the nuclear events in living and Feulgen-stained prepara-
tions through conjugation, instances of polyploidy were encountered. Polyploidy was first re-
corded in Paramecium by Chen (1940, Proc. Nat. Acad. Sci. V: 26) for P. bursaria and this
represents the second report of the phenomenon. Pure-line races of the colorless (255) and
green (B9) paramecia were mated. The individuals of each race have well-defined micronuclei
of approximately equal size.
The three pregamic divisions were found to be remarkably constant in respect to time and
micronuclear behavior at a given temperature. However, in the cytological examination of
many hundreds of joined pairs, approximately 2 per cent were observed in which the micro-
nuclear behavior resulted in the polyploid conditions only after the pregamic divisions. The
crucial stage where polyploidy occurs is found during the period of pronuclear transfer, ap-
proximately 16-18 hours after the animals have been mated. It follows the third suggestion
made by Chen in accounting for polyploidy ; namely, the failure of a migratory pronucleus in
one of the conjugants to migrate to the other conjugant. The result is an individual with one
small pronucleus (the "stationary") which is haploid, and the conjugant with three pronuclei
(two "migratory" and one "stationary") which fuse and form a larger triploid synkaryon.
What is the fate of each nuclear body that is now comparable to the normal synkaryon ? The
subsequent micronuclear stages show a conspicuous and persisting size difference in all later
stages and hence are recognized easily. In the haploid conjugant, late anaphase stages (com-
parable to postgamic ones) measure 10.8 /z in length and are very narrow; similar stages in
the triploid co-conjugant measure 27 /JL in length and are proportionately wider. Their division
products measure 8 (j. in the haploid and 15.5 fj. in the triploid individuals respectively.
While polyploidy occurs in only 2 per cent of the cases in this material, it nevertheless cre-
ates variation in micronuclear composition and is therefore of evolutionary significance.
* Kindly furnished by the Winthrop Chemical Company.
PRESENTED AT MARINE BIOLOGICAL LABORATORY 235
The Lipids in Pelomyxa carolinensis. CHARLES G. WILBER.
In 1942 the author demonstrated that the cytoplasm of Pelomyxa carolinensis contains lipid
material, that this lipid comes from digested food, and that it is composed of a high proportion
of fatty acid. In the latter respect the stored fat differs from that in Amoeba proteus in which
fat is stored in the neutral form. In the previous work, Nile blue sulfate was used to distinguish
neutral fat from fatty acid. This dye has been criticized as a reagent for fat tests. Conse-
quently it seemed desirable to use specific chemical procedures to ascertain the nature of the
lipid material in Pelomyxa.
Ninety mg. (wet weight) of pelomyxae were thoroughly washed in boiled culture fluid.
By repeated centrifugation the cells were broken up and then the lipids were extracted in hot
alcohol. The quantities of phospholipid, cholesterol, and fatty acid were ascertained by the
Bloor method. It was found that in the amount of cellular material used there was no meas-
urable phospholipid or cholesterol. The total weight of fatty acid in 54 mg. of cells was 2.05 mg.
or 3.8 per cent fatty acid.
These results are in agreement with the results previously obtained using Nile blue sulfate.
It seems that in Pelomy.ra carolinensis the lipid material occurs chiefly as fatty acid and that
the amount of other lipids is very small.
The presence of lip as c in Pdomyxa carolinensis. CHARLES G. WILBER.
The digestion of fat in rhizopods has been demonstrated by several investigators. More-
over, it has been shown that the fats digested are incorporated into the cytoplasm. However,
none of the investigations so far has given direct evidence for the presence of lipase in the cyto-
plasm of rhizopods.
Pelomyxae were starved and then ground up in a drop of water. A drop of this solution
was added to a drop of 0.2 per cent emulsion of castor or olive oil and a drop of pure water
was added to another drop of the emulsion as a control. After 30 minutes both drops were
treated with hydroxylamine hydrochloricle and potassium hydroxide. Then after acidification
each drop was treated with 1 per cent ferric chloride solution. In each case a violent brown
color was produced in the control, whereas no color was produced in the drop containing the
ground up pelomyxae.
The above reaction is a test for esters. Lipases are known to be ester ferments. Since the
oil emulsions mixed with ground pelomyxae did not give the characteristic ester reaction, it can
be concluded that the esters were broken down by something in the cytoplasm. We therefore
have direct evidence for the presence of lipase in Pelomyxae carolinensis.
PAPERS PRESENTED AT THE MEETING OF THE SOCIETY
OF GENERAL PHYSIOLOGISTS
SEPTEMBER 5 AND SEPTEMBER 6
FIRST SESSION — S. C. BROOKS, CHAIRMAN
The effect of cold on capillary permeability and fluid movement in the frog.
ELLEN BROWN, M.D.* AND EUGENE M. LANDIS, M.D.
Micro-manipulative methods were used to study the relationship between capillary blood
pressure and the rate of fluid movement through the walls of single capillaries in the frog's
mesentery (a) at ordinary room temperatures of 22.5° to 25.5° C. and (b) when the mesentery
was cooled to between — 2° and + 2° C. Cooling the mesentery decreased capillary perme-
ability, reduced the observed rates of nitration and increased the observed rates of absorption.
The nitration constant of the capillary wall was reduced from the control value of 0.0070
i"-r'/i".2/sec./cm. water pressure to 0.0019, a decrease of 73 per cent. The effective osmotic pres-
sure of the blood within the capillaries was elevated from 10.5 to 13.8 cm. water, an increase
of 31 per cent.
Four possible causes for the increase in apparent or effective osmotic pressure were con-
sidered. (1) An increase of absolute colloid osmotic pressure was excluded because plasma
protein concentrations, calculated from specific gravities of plasma samples, were the same in
the two series of frogs. (2) An increase in effective colloid osmotic pressure due to greater
retention of plasma protein during cooling could also be excluded because the control experi-
ments showed that plasma proteins were already retained completely, or almost completely, even
at room temperature. (3) It is possible, however, that the effective non-protein osmotic pres-
sure might rise if the passage of smaller molecules, e.g., glucose, amino acids, urea or certain
electrolytes, was impeded more than that of water as the permeability of the capillary wall
decreased during chilling. (4) Tliermosmosis might also be responsible because relatively warm
blood was circulating through capillaries surrounded by cooler tissues. Studies are in progress
to determine whether or not this factor modifies the movement of fluid through membranes in
•vitro using a schema which simulates the conditions existing in vivo.
The effects of cold on the capillaries of the frog differ from those observed in mammalian
capillaries because the former become less permeable at 2° C., whereas the latter become more
permeable as temperature falls below 10° C. However, actual freezing of the frog's capillaries
at temperatures of — 5° to — 10° C. increased capillary permeability conspicuously, as shown
by the appearance of stasis during thawing. If the duration of actual freezing was brief this
stasis usually disappeared within a few minutes as the capillary wall regained its normal relative
impermeability to protein.
Bubble formation within single cells. ~f E. NEWTON HARVEY, K. W. COOPER, A.
H. WHITELEY, D. C. PEASE, AND W. D. MCELROY.
Normal living isolated cells (Amoeba sp., Chaos chaos, Paramoecium, Arbacia and Aste-
rias eggs and Nitella) do not form internal gas bubbles if saturated with nitrogen gas at 80 to
120 atmospheres pressure and then suddenly decompressed. Bubbles may form on the outside
of the cells due to contamination with gas nuclei (minute gas phases sticking to hydrophobic
spots). Cells which have been killed by chloroform or formalin likewise form no bubbles within
but sometimes spontaneously dead cells or those previously injured by twisting before subjec-
* Research Fellow in Physiology, Commonwealth Fund.
t Part of the work described in this Abstract was done under a contract recommended by
the Committee on Medical Research between the Office of Research and Development and
Princeton University.
236
PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 237
tion to the high gas pressures do form bubbles within after decompression. A living Nitella
cell just after decompression from high gas pressures and still free of bubbles, will immediately
form a bubble inside if the cell wall is gently pinched (not enough to penetrate the wall) or
twisted. Such bubbles are believed to result from local decreased tensions that tear the liquid,
forming a space or cavity (a vapor phase) into which gas diffuses, forming a gas nucleus
that immediately grows to a bubble. Such cavities can form inside or outside of cells even at
atmospheric pressures. They are believed to be formed during muscular exercise in man,
when the incidence of aviator's bends is greatly increased.
The action of various cations of muscle protoplasm. L. V. HEILBRUNN AND F. J.
WIERCINSKI.
There are two ways to study the colloidal behavior of muscle protoplasm. One way is to
isolate pure proteins and follow their reactions in test tubes; the other way is to subject the
protoplasm itself to reagents and observe the results. In our studies, we injected solutions of
various salts into the interior of isolated muscle fibers of the frog. We then noted the degree
of shortening of the constituents of the muscle. With the aid of a micrometer eyepiece, we were
able to determine the effect of the injections on the length of the fiber. In numerous experiments
we found that rather dilute calcium chloride solutions invariably caused an immediate and pro-
nounced shortening of the protoplasmic constituents of the muscle. On the other hand, potassium
and sodium chloride had very little effect. Even when injected in concentrations isotonic with
the muscle, they ordinarily caused no shortening whatsoever. Rarely, a shortening did follow
injection of isotonic sodium or potassium chloride. This we believe was due to the release of
calcium ion. Magnesium ion likewise causes no shortening of the protoplasmic constituents.
Barium acts like calcium. The results support the calcium ion theory of stimulation and they are
opposed to Szent-Gyorgyi's opinion that potassium is the ion primarily responsible for the con-
traction of muscle.
Further observations on an oligodynamic action of copper and mercury on eryth-
rocytes. M. H. JACOBS AND DOROTHY R. STEWART.
The specific effect of copper in decreasing the permeability of erythrocytes to glycerol seems
to be absent in all species whose erythrocytes show a low degree of permeability to this solute.
It is also lacking in a number which show a very high permeability both to glycerol and to other
hydrophilic solutes of comparable molecular volume. It has so far been found only in those
species whose erythrocytes show a disproportionately great permeability to glycerol, thus sug-
gesting that some special mechanism of penetration may be involved, which is reversibly in-
activated by copper. This generalization is supported by the behavior of the erythrocytes of a
number of birds in which the specific permeability to glycerol is particularly great.
The effects of HgCL in some ways resemble and in others differ from those of CuCL. One
of the most important differences is that HgCU forms a double salt with NaCl, and its activity
is therefore greatly reduced by the presence of any considerable quantities of the latter salt. A
second difference is that HgCL readily enters the erythrocyte, while CuCU does not. These two
fundamental differences are responsible for a number of secondary ones.
That copper may hinder the escape of glycerol from human erythrocytes, as well as its en-
trance into them, is suggested by the following experiment. To a suspension of erythrocytes in
an isotonic salt solution, small amounts of copper and of concentrated glycerol are added, and
the resulting mixture is then diluted with a properly chosen hypotonic salt solution. If the copper
is added before the glycerol, it decreases hemolysis by preventing the entrance of glycerol into the
cells. If the copper is added 30 seconds or more after the glycerol, it increases hemolysis by
preventing the escape of the glycerol that has entered the cells.
The prolytic loss of K from red cells. ERIC PONDER.
The prolytic loss of K, i.e., the loss of K which takes place from red cells exposed to hypolytic
concentrations of lysin, has been measured by means of the flame photometer in systems containing
distearyl lecithin, sodium taurocholate, sodium tetradecyl sulfate, saponin, and digitonin. The
PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS
lysins are added in various concentrations to washed red cells from heparinised human blood,
and the K in the supernatant fluids is determined after various intervals of time at various tem-
peratures. This prolytic loss of K, KP, is compared in every experiment with the loss Ks into
standard systems containing one per cent NaCl alone, without lysin.
The losses K,, and Ks increase with time, so that new steady states are approached logarith-
mically. The values of Kp which correspond to the new steady state depends on the lysin used,
being greatest with taurocholate and smallest with powerful lysins such as digitonin (confirming
an observation of Davson and Danielli). The extent and course of the K losses seem to have no
simple relation to the prolytic phenomenon of the disk-sphere transformation.
Just as the prolytic loss of K occurs without the loss of any Hb, so in concentrations of lysin
sufficient to produce hemolysis the loss of K, expressed as a percentage of the total red cell K,
increases much more rapidly with lysin concentration than does the loss of Hb, expressed as a
percentage of the total Hb. The explanation of these relations depends on whether the loss of K
is treated as being all-or-none in the case of the individual cell, or as being the result of the loss
of part of the K from all the cells. This point has yet to be decided.
SECOND SESSION — L. MICHAELIS, CHAIRMAN
Effect of fluoroacetate on the metabolism of baker's yeast. E. S. GUZMAN BARRON
AND GEORGE KALNITSKY.
•
Among the organic halogen compounds, those containing fluorine occupy a special position
regarding their chemical and physiological properties. Because of the high value of the energy
of the C-F bond and of the electro-negativity of F, the introduction of F into the C atom pro-
duces a greater stability, specially in alyphatic compounds. This is shown on measuring the rate
of combination of cysteine with halogen acetates. At 23°, half-reaction with iodoacetate took
place in 4.4 minutes ; with bromoacetate in 6.2 minutes ; with chloroacetate, in 125 minutes.
With fluoroacetate it did not react at all. There is a certain relationship between the rates of
reaction and the bond-energy values of the C-halogen bonds as well as the electronegativity
values of the halogens. On studying the effect of these halogen acids on the rate of oxidation
of acetate by baker's yeast it was found that 0.001 M of fluoroacetate inhibited it 90 per cent;
bromoacetate, 17 per cent; iodoacetate, chloroacetate, and trifluoroacetate, none at all. On com-
paring the interatomic distances between C and the halogen it can be seen that the C-F bond
with a distance of 1.41 A approaches most closely the distance of the C-H bond, 1.09 A. By in-
creasing the size of the fluoroacetate molecule through the replacement of the other two hydrogens
with fluorine (trifluoroacetate) the inhibiting effect was destroyed. This inhibition is a sub-
strate competitive inhibition, the fluoroacetate occupyng the place of acetate in the protein moiety
of the acetate metabolism enzyme. Increase of the length of the molecule as in fluoropropionate,
fluorobutyrate, and fluorocrotonate destroyed the inhibition. Inhibition was partially reversed
on addition of large amounts of acetate (0.08 M). Inhibition occurs in the first step of acetate
metabolism, namely, condensation with oxaloacetate to give citrate. The formation of citrate
from acetate was completely inhibited with 0.005 M fluoroacetate. In the presence of ethanol,
the rate of O2 uptake was not affected by fluoroacetate up to 42 per cent of the total O? uptake,
when the inhibitory effect appeared. This is indication that ethanol oxidation occurs in three suc-
cessive steps : -oxidation of ethanol to aldehyde ; and of aldehyde to acetate, both unaffected by
fluoroacetate ; and oxidation of acetate, inhibited by it. At the end of the experiment there were
in the control 1572 cmm. O2 used, and 150 cmm. of acetate formed from 940 cmm. of ethanol; in
the presence of fluoroacetate there were 975 cmm. O... used and 890 cmm. of acetate formed.
The effect of sodium aside on Parameciuwi calkinski. E. J. BOELL.
Sodium azide is generally regarded as an inhibitor of respiration by virtue of its inactivation
of cytochrome oxidase. In Parameciiim calkinsi, experiments have shown that this compound, in
a concentration of 0.001 to 0.01 molar, reversibly depresses respiratory activity by 50 to 60 per
cent. Under certain circumstances, however, azide instead of inhibiting respiration serves as a
powerful respiratory stimulant. The stimulating effect of azide seems to depend primarily upon
the pH of the medium. For example, a 0.01 molar solution of NaN3 at pH 6.02 will depress
respiration to a value about 30 per cent of normal ; at pH 6.24 respiration is 70 per cent of nor-
PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 239
mal, while at pH 6.59 the same concentration of azide stimulates respiration of 238 per cent of
normal. Calculation of the hydrazoic acid concentration at these pH values shows that the ef-
fect produced depends, within certain limits, upon the concentration of undissociated HN3.
A study has been made of the mechanism of azide stimulation. It has been found that the
respiratory quotient of normal animals averages 0.99 ; that of animals in the presence of a
stimulating dose of azide averages 1.05. The increased oxidation thus involves the metabolism
of organic substrate. It is also apparently mediated by the normal enzymic mechanisms for it
is sensitive to cyanide. Carbon monoxide, however, exerts only a slightly depressing effect.
The metabolism of Paramecia under normal circumstances is accompanied by the production
of large quantities of ammonia nitrogen. On the assumption that such ammonia production
represents protein breakdown, approximately 75 per cent of the total oxygen consumption of
control animals can be accounted for in this way. Although Paramecia treated with azide show
increased ammonia production, only 22 per cent of the extra oxygen uptake induced by azide can
be accounted for as protein breakdown with ammonia as the end product.
In addition to the effects already noted, azide interferes with the ability of Paramecium
calkinsi to maintain normal water balance. The activity of the contractile vacuoles is greatly
reduced and supernumerary vacuoles are frequently formed.
The oxygen consumption concerned with growth in bacterium coli. KENNETH
FISHER. No abstract submitted.
Enzymatic acetylation and the co enzyme of acetylation. FRITZ LIPMANN.
The mechanism of enzymatic acetylation of aromatic amines has been studied in pigeon liver
homogenates and extracts (Lipmann, F., 1945. /. Biol. Chcm., 160: 173). In this enzymatic
system the condensation of an aromatic amine, like sulfanilamide, with acetate, is effected
through a transfer of phosphate bond energy from adenylprophosphate. (Cf., Nachmansohn,
D. and Machado, A. L., 1943. J. Neurophysiology, 6: 397 for a similar system of choline acety-
lation in brain).
A heat stable and dialysable coenzyme was recently found necessary in this reaction, besides
the energy donor adenylpyrophosphate. The characterization of this new coenzyme is now in
progress in this laboratory in collaboration with Dr. Nathan O. Kaplan. We find the same
coenzyme necessary to complement dialyzed brain extracts for acetylation of choline, although
the brain enzyme is specific for choline and the liver enzyme specific for amines. The coenzyme
is present in largest amounts in brain, liver, and kidney. Appreciable amounts are present in
all tissues tested, including carcinoma. Therefore its action must be a very general one and
probably not merely restricted to acetylation.
The coenzyme is destroyed by intestinal phosphatase with liberation of phosphate. It is
inactivated by a rather general tissue enzyme without liberation of phosphate. The link at-
tacked by the latter enzyme is unknown. The compound follows the general pattern of nucleo-
tide precipitation. Our most active preparations showed sporadic crystals on microscopic ex-
amination. This quite uniform fraction contained adenine, ribose, and phosphate in the propor-
tion 1 to 1 to 2. Acid hydrolysis showed the second phosphate not to be in pyrophosphate
linkage. If we assume the presence of some crystals to indicate near purity, which it not neces-
sarily does, then the content of approximately 50 per cent of adenylic acid in our best prepara-
tions should mean the coenzyme to be of dinucleotide structure. Therein the adenylic acid
should be linked through the second phosphate to an as yet unidentified part. Electrotitration
and cleavage experiments seem to support the outlined constitution.
Penetration and action of cholincsterasc inhibitors. DAVID NACHANSOHN. No
abstract submitted.
The metamorphosis of visual systems in amphibia. GEORGE WALD.
In the rods of the vertebrate retina two visual systems are found. One is based upon the
red photosensitive pigment rhodopsin, engaged in a cycle with vitamin A, ; the other involves
the purple photopigment, porphyropsin, bound in a similar cycle with vitamin A2.
240 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS
The porphyropsin system appears to be the more primitive in vertebrate evolution. The
cyclostome, Petromyzon marinus, possesses only this system. The same is true of all types of
freshwater fish so far examined.
Vertebrates have followed two pathways out of fresh water, one into the sea, the other
to land. Both have led them to the use of vitamin A, in vision. Thus all marine fishes which
have been examined, with the single exception of certain Labridae, have the rhodopsin system
alone ; so also do all the birds and mammals investigated.
Interpolated between freshwater and marine fishes are euryhaline forms, which can exist
as adults in either environment. Among them, the salmons and the "freshwater" eel have mix-
tures o'f the rhodopsin and porphyropsin systems ; while the alewife and white perch have only
the latter. In all these forms the visual system is predominantly or exclusively that normally
associated with the environment in which the fish develops embryonically, and is relatively inde-
pendent of the environment in which it is found as an adult.
Interpolated between freshwater fishes and true land vertebrates are the amphibia. Their
life histories for the most part are closely analogous with those of euryhaline fishes, amphibian
migrations to land replacing fish migrations into the sea.
Adult frogs possess the rhodopsin system and vitamin A, alone. The tadpole of the common
bullfrog, Rana catesbiana, however, has exclusively the porphyropsin-vitamin An system just.
prior to metamorphosis. During metamorphosis it transfers completely to the rhodopsin sys-
tem, which is found alone in the new emerged frog. Partly metamorphosed animals have mix-
tures of both systems, such as have been found otherwise only in euryhaline fishes.
The common newt, Triturus viridescens, begins its life as a gilled larva in fresh water.
After several months it metamorphoses to the land-living red eft; then after 1-2 years of growth
it undergoes a second metamorphosis to the sexually mature newt, returning to the water for
the remainder of its life. The eye of the red eft contains a mixture of vitamins Aj and A2,
predominantly the former; while that of the water-phase adult presents just the reverse propor-
tions of both vitamins. This is a change opposite in direction to that in the frog, but associ-
ated in the same way with the chemical metamorphosis of visual systems.
Amphibia, therefore, like euryhaline fishes possess as a group both the rhodopsin and
porphyropsin systems ; but in amphibia these systems succeed one another as the animal goes
through its basic metamorphoses.
THIRD SESSION — J. H. BODINE, CHAIRMAN
X-ray effects in mixtures of compounds. RUBERT S. ANDERSON.
It has been reported previously that ascorbic acid, as shown by experiments in plasma, has
a preferential ability to react with the materials produced in water by x-rays. Much of the
ascorbic acid reaction is not observed in irradiated muscle. Non-uniform distribution of the
ascorbic acid in muscle would tend to make the observed result too low. Another possibility
is that other compounds are present in muscle which take some of the reactive material away
from the ascorbic acid.
Evidence has been obtained that the ascorbic acid reaction consists in part of a reversible
oxidation, presumably to dehydroascorbic acid. When present during the irradiation, glutathi-
one and cysteine gave substantial, although variable, protection of ascorbic acid against x-rays.
Alanine was much less effective, suggesting that the sulfhydryl grouping is largely responsible,
whether it is a true competitive protection or a reversal of oxidation. Glutathione and cysteine
and possibly protein sulfhydryl groups could thus account for a part of the protective effect of
muscle on ascorbic acid.
There is no evidence that the destruction of a small amount of these compounds woulc
damage a cell. However, the work shows that, in principle, a compound through which the
water reaction might damage the cell could exist.
Ascorbic acid, glutathione and cysteine partially protect pepsin from the inactivating effect
of x-rays. Alanine is much less active.
If at least a part of the reaction in water is distributed randomly throughout the proteins
of the cell and nucleus, the occasional loss of a molecule or two from compounds represented
by hundreds of molecules need have little effect on the cell although the products formed, such
as denatured proteins, might secondarily be harmful to the cell. However, if there are in the
PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 241
cell or nucleus some thousands of different protein compounds or structures each form of which
is essential to the cell and each one of which is represented by but one or two molecules or
particles, then randomly distributed products of irradiated water might destroy one of these
entities and so damage or kill the cell. This is essentially the argument used by Lea in reaching
the conclusion that his theory led to the expectation of gene and chromosomal effects from
irradiation.
Electrical studies of acetylcholine and choline estcrase. T. C. BARNES.
Acetylcholine passes through the thin oil layer of a bubble of guaiacol-resin-cholesterol so
rapidly that the spike potential must be recorded by an oscillograph. First the acetylcholine
produces a negative phase boundary potential on one side of the oil layer but on reaching the
opposite side of the oil a new potential is established which produces the descending part of the
spike (no esterase required). At the suggestion of Osterhout, solutions were shaken 5 hrs.
with guaiacol with these results : oil with saline 5 X 1(T7 mhos ; same with 0.002 M acetylcholine
35 X 10"7 mhos (conductivity was determined of oil separated from aqueous solution). Apply-
ing the Nernst equation, 0.058 times log conductivity difference (6) gives 40 mv. (observed
phase boundary potential). At the suggestion of Loewi, tetramethylammonium iodide was
found to give no potential on nitrobenzene but 0.05 per cent gave 25 mv. negative on guaiacol.
The type of oil and not the tertiary or quarternary nature of the compound determines electro-
genie effects. Thus prostigmine produces 85 mv. negative on guaiacol compared with 35 mv.
generated by acetylcholine (both 0.002 M). Prostigmine inhibits the cord in spasticity by
flooding with high negative potential which may also act as a stimulus on muscle in myasthenia.
Dialantin and phenobarbital produce positive potential (20 mv. at concentration of 0.05 per cent)
which probably neutralizes the excess negativity of acetylcholine in the brain in epilepsy.
Lyovac plasma reduces the phase boundary potential of 0.05 per cent acetylcholine from 35 to
15 mv. (residual potential is produced by choline). Potential of benzyol choline is destroyed
by serum and part of the mecholyl potential by one per cent ground cat brain. Eserine and
DFP preserve the potential of acetylcholine in the oil-cell in the same manner as in the nerve.
One per cent DFP increases the specific conductance of guaiacol 100 per cent which explains
part of its blocking action on nerve and muscle.
The action-current in cholinergic nerve is probably a phase-boundary potential of acetyl-
choline (sympathin is the electrogenic amine in adrenergic nerve).
Two schools of thought in electrophysiological theory. R. BEUTNER AND T. C.
BARNES.
The older school, entrenched as the hypothesis of sieve membranes retaining negative but
not positive ions, explains everything but solves no problems. The newer school omits hypotheses
and proposes searches for electrogenic materials in tissues by setting up artificial battery systems
composed of lipoid layers (oils) inserted between aqueous salt solutions. Some of these resemble
analogous battery systems containing a tissue in place of the lipoid. One type of system studied
is : — concentrated saline/tissue or lipoid (oil) /diluted saline +. Tissue, in such a set-up, may pro-
duce the maximum e.m.f. of 58 millivolts if the concentrated solution is 1/10 mol. ; the diluted one,
1/100 mol. Only few oils show such an effect, as e.g., fatty acid dissolved in a phenol-derivative,
but not neutral fats, gelatin, etc.
The production of bio-electricity does not depend on such aqueous salt concentrations but on
metabolic processes in tissues, chiefly oxidation. A search for suitable electrogenic systems has
led to the following one (Beutner, Loznerea, 1930) : — saline/reduced substance e.g., a higher
alcohol or lower fatty acid as in dying tissue/oxidized substance e.g., corresponding acid or cor-
responding higher fatty acid as in respiring tissue saline +. For the action current one possible
electrogenic substance is acetylcholine since even dilute solutions produce an e.m.f. in contact
with oils in a system such as : + saline without addition/oil/saline with acetylcholine added
1 : 100,000 to 1 : several million— (Beutner and Barnes, 1941).
'Tissue extract can be used in the place of the oil, also frog's nerve by the Netter technique.
Adrenergic amines produce similar negative potentials but on different oils which are inactive in
contact with choline esters. A difference in chemical composition may therefore be responsible
for the specific function of cholinergic and adrenergic fibers. The rapid disappearance of the
242 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS
negative potential, which occurs even in the absence of choline esterase, may be explained by a
penetration of acetylcholine through a thin lipoid layer (membrane) creating a potential dif-
ference in the opposite direction on the other side. Physico-chemical studies are not needed for
the search for electrogenic substances, but when performed on oil cells, they show the existence
of phase boundary potentials depending on electrolyte distribution ; the charged pore theory fails
to explain the phenomena and is contradictory.
The frequency of x-ray-induced chromated breaks in Tradescantia as modified by
near infrared radiation. C. P. SWANSON AND ALEXANDER HOLLAENDER.
The frequency of x-ray-induced chromatid breaks in Tradescantia can be significantly in-
creased by treatment of the inflorescences with near infrared radiation. Pretreatment with near
infrared radiation for seven hours prior to x-radiation inceased the frequency of single deletions,
double (isochromatid) deletions, and translocations between and within chromosomes; post-treat-
ment increased only single deletions and translocations. A delay of 21 hours between treatment
with infrared and x-rays did not appreciably decrease the effectiveness of the infrared, suggesting
that the changes within the cell induced by the infrared were of a relatively permanent nature.
At the present time, the nature of the effect of infrared is not clearly understood.
Vol. 91, No. 3 December, 1946
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
A STRONGLY INTERSEXUAL FEMALE IX HABROBRACON
P. W. WHITING
University of Pcmisyk-ania, Philadelphia, and the Marine Biological Laboratory, Woods Hole
In the parasitic wasp Habrobracon fuglandis (Ashmead), diploid males have
never shown any tendency toward intersexuality ; they are as definitely male as
their normal haploid brothers. When a "diploid male with female genitalia" was-
found, it was therefore regarded with especial interest. The specimen, designated
freak 994, developing from a heavily x-rayed ( 29,300 r) egg, occurred among the
offspring of a treated wild type (stock 33) female crossed with an untreated lemon
honey male (Experiment by A. R. Whiting. 1945).
Freak 994 shows the heterozygous condition of the semidominant body color
gene lemon inherited from its father. (Note light base of antennae in Figure 1.)
The number of its antennal segments and its large ocelli are male characteristics.
It was to be expected, therefore, that male reproductive reactions would occur.
Several tests at different times failed to evince any response toward females although
the specimen appeared healthy, drank honey water and lived for several days until
fixed in Carnoy fluid. Since- it likewise failed to give any response (female) to
caterpillars, its indifference was probably not due to its sex type but to some un-
known factor.
Because of the small "feminized" genitalia on the "male" body, freak 994 was
at first recorded as a "diploid gynandroid male." ( 1\ nandroids, however, have
always been haploids. They are mosaic males in which the two sexually different
types of male tissue react in a complementary way to feminize the external genitalia
(Whiting, Greb, and Speicher, 1934). Their mosaicism is shown by their asym-
metry, not only in body color, in number of antennal segments, in mutant traits,
and often in wing length, but especially in the external genitalia which are a mixture
of normal male and feminized male structures with much reduplication and irregu-
larity. In freak 994 there are no male genital structures and the female genitalia,
consisting of a pair of sensory gonapophyses with no visible sting, are symmetrical
and larger than in gynandroids. They are much smaller, however, than the female
genitalia found in gynanders which are male-female mosaics with clearly separated
male and female regions. That freak 994 is not a sex mosaic is shown by its
symmetry in body coloration, in antennal flagella with nineteen segments in each
and in length of wings and legs.
Two types of intersexes have hitherto been reported in Habrobracon. (1)
Gynoid, dependent upon a single mutant gene, is a weakly intersexual male, func-
tioning normally as a male, but having certain external traits, including antennae,
feminized. (2) Nine intersexual females were reported (Whiting. 1943) occurring
in a single fraternity. "Superficially, these appear to be the reverse of the gynoid
243
244
P. W. WHITING
males, being more masculine anteriorly, feminine posteriorly." They resemble
freak 994 in head and thorax and in the anterior part of the abdomen which are
altogether like those of the male. In the posterior region, however, the sclerites are
thickened, there is a normal sting and the sensory reproductive appendages are of
full leneth characteristic of the female. "The nine intersexual females must be
FIGURE 1
regarded as UK ire strongly intersexual than gynoid males since antennae, ocelli and
instincts are completely sex reversed." Freak 994 is an intersexual female, com-
parable to these nine but still more strongly intersexual because of greater restric-
tion of the "female" region and reduction of the genitalia.
In Habrobracon, normal haploid males have cells almost as large as the corre-
sponding cells of diploid females and in some stocks they are actually larger (Grosch,
INTERSEX IN HABROBRAO )\ 245
1945). Cells of diploicl males are much larger than are those of females or of
haploicl males. These relationships have heen determined by counts of micro-
chaetae within a given area on the upper surface of the wings, each microchaeta
corresponding to a single cell. Study of the dispersion of microchaetae in freak
994 showed its cell size to be within the range for the female or haploid male and
therefore much smaller than that characteristic of the diploid male. The marked
shift of the intersex in the male direction does not then affect the size of its cells.
It may be fundamentally female, heterozygous for the sex factor. This condition
perhaps prevents the abnormal expansion of cell size while permitting development
of antennae and ocelli of normal male type.
The nine intersexual females previously reported had internal abdominal struc-
tures as in the female with normal poison sac and glands and seminal receptacle.
Each ovary, however, appeared to be a pair of sacs of oogonia showing no differen-
tiation of nurse cells and ova. Serial sections were made of the abdomen of freak
994 and the internal structures were studied. The digestive tract is entirely normal
with the crop greatly distended from honey water feeding. A poison apparatus is
present but imperfectly developed and situated near the median plane, directly
dorsal to the compound posterior nerve ganglion instead of being shifted laterad to
the digestive tract. The poison glands are normal although of somewhat small size.
Their ducts converge to a common duct connecting distally with an imperfect poison
"sac" and proxiinally traversing the very short distance to the region where nor-
mally lies the root of the sting. The poison ''sac," of approximately normal length,
is reduced in diameter to an irregularly scleroti/.ed strand. It is surrounded In-
longitudinal muscles as in a normal female. Nothing corresponding to a seminal
receptacle could be located, nor were any gonads to br found. The tat body appears
normal, surrounding the digestive tract and the poison apparatus dorsally and
laterally.
DISCUSSION
In the report on the nine intersexual females, it was suggested that they might
be accounted for by a dominant mutation in a sex allele changing .vb to xbm. The
intersexes would then be modified females, .ni .vb"1. A similar hypothesis would
cover freak 994. but here the mutation may have been x-ray induced and more
potent than in the previous case so that the intersexuality would be more extreme
with turning-point earlier in development.
Failure to find gonads in freak 994 does not necessarily mean that they were
lacking from the beginning for they may have begun development and then disin-
tegrated.
Comparison may be made between freak 994 and certain types of "deficient"
individuals previously reported in Habrobracon (Whiting, 1926). Some of the
"deficient" had external genitalia lacking but gonads present. Others had testes
of reduced size, or present on one side, lacking on the other. Some of the "de-
ficient" females with no trace of poison apparatus had well differentiated ovaries
with eggs and nurse cells. This is just opposite to the condition found in the
intersexual female, freak 994. There was no intersexuality among the "deficient."
24f) P. W. WHITING
SUMMARY
An inter sexual female developed from a heavily x-rayed egg fertilized by an
untreated sperm. The specimen is more strongly intersexual than a group of nine
previously reported, for its external female genitalia are much reduced, its poison
apparatus defective and its ovaries altogether lacking. Externally, it appears like
a diploid male with small female genitalia.
It is suggested that the x-radiation may have caused a change within a sex-
differentiating allele, so that the heterozygote would develop into an intersex rather
than a normal female.
LITERATURE CITED
GROSCH, D. S., 1945. The relation of cell size and organ size to mortality in Habrobracon.
Growth, 9: 1-17.
WHITING, P. W., 1926. Influence of age of mother on appearance of an hereditary variation
in Habrobracon. Biol. Bull.. 51 : 371-385.
WHITING, P. W., 1943. Tntersexual females and inUTst-xuality in Habrobracon. Biol. Bull,.
85: 238-243.
VYnnrxr,, P. W.. RAYMOND J. GREB AND B. I\. Sri K HKR, 1934. A nr\v type of sex-intergrade.
Biol Bull.. 66: 152-165.
LOCI OF ACTION OF DDT IN THE COCKROACH
(PERIPLANETA AMERICANA)
J. M. TOBIAS AND J. J. KOLLROS *
University of CJiicago To.ricity Laboratory^ and the Department of Physiology
In the cockroach. DDT produces symptoms which clearly reflect involvement
of the neuromuscular apparatus. These are qualitatively much the same in all
arthropods which have been studied, though there are important quantitative dif-
ferences. Thus, in any given animal the time course of the poisoning is a function
of dose, and for a dose of comparable toxicity in terms of final mortality, the symp-
toms unfold and death occurs much more rapidly in some insects (the fly) than in
others (the roach) (Tobias, Kollros, and Savit, 1946a). In the roach, the sequence
of symptoms is initiated by hyperextension of the legs, elevation of the center of
gravity and development of postural instability. The hyperextension then decreases
and is superseded by increasing and generalized tremulousness, involving the head,
body, and all appendages ; the gait becomes ataxic, and minor stimuli of sound or
touch result in great hyperactivity, exhibited mainly in running and climbing. The
animal falls on its back time after time until finally it can no longer right itself. Leg
movements continue in the supine insect with two components, a high frequency
intermittent tremulousness and a slower incoordinated flexion and extension. These
two types of activity possibly reflect the double innervation which has been described
for cockroach muscle (Pringle, 1939), one fiber type producing relatively slow tonic
contractions, the other producing relatively fast twitches. It will be seen later that
after poisoning these two types of movement can be independently altered. Activity
finally diminishes progressively. The fast tremors disappear first and finally there
remain only occasional isolated movements of body wall, tarsi, palpi, cerci, or an-
tennae. When no further somatic movement can be detected, the heart usually
continues to beat for some time, and electrical stimulation of the nerve cord may still
evoke muscle responses. The animal may live in this condition for a day or so and
finally die.
Mammals exhibit a similar symptomatology up to a point. In the rat and dog,
given DDT intravenously or orally, muscular fibrillations and excessive blinking are
followed by tremulousness, ataxia, falling and gross convulsive seizures. The ani-
mal may have a number of convulsions and die in the tonic phase of one or recover
after gradual subsidence of symptoms. There is no period of prostration and nearly
complete immobility as in the insect, because death occurs when systematic respira-
tory movements cease. In the insect, the small amount of body movement and
twitching sufficiently augment diffusion for respiratory exchange. Then too, the
insect is far more resistant to anoxia than is the mammal (Wigglesworth. 1939).
* Department of Zoology and The College. Present address. Zoology Department, Uni-
versity of Iowa.
t This work was carried out under contract with the Medical Division of the Chemical
Warfare Service.
247
248 J. M. TOBIAS AND J. J. KOLLROS
The frog, as might be expected, responds more like the insect than the mammal
(Tobias, Kollros, and Savit, 1946b). Respiratory exchange through the skin can
sustain life, and, after a period of hyperirritability, the animal lies prostrate and more
or less immobile. Such symptomatology has prompted a number of investigations
designed to discover a locus of action of DDT. As will be seen, there probably are
a number of sites of action depending largely on dosage, but this point of view was
only gradually attained.
In mammals (Crescitelli and Oilman, 1946), DDT apparently does not act di-
rectly on either muscle, myoneural junction or spinal cord. Since tremors persist
after decerebration and mesencephalic transection, and since abnormal cerebral and
cerebellar electrical activity persists after atlanto-occipital transection, neither cere-
bral cortex nor basal ganglia can be a critical site of action, and intact spinal afferents
are obviously not necessary lor the central effect. The cerebellum is considered, by
these authors to be the most likely critical site of action in the mammal. Locus of
action has also been investigated in insects. In Drosophila (Bodenstein, 1946).
DDT seems not to act on muscle or myoneural junction, but does act on peripheral
nerve and may act on the central nervous system. In the cockroach (Periplaneta
americana}, DDT has been found to act on nerve in high concentrations (Yeager
and Munson, 1945), and, in low concentrations, on peripheral receptors (probably
proprioceptors) (Roeder and Weiant, 1946). The latter workers also have evi-
dence which they interpret to mean that high concentrations may act directly on
either the myoneural junction or muscle itself. In the crab (Cancer irroratus) there
is evidence for action on motor nerves (Welsh, 1946).
It was the purpose of this study to further investigate loci of action of DDT in
an insect. Because of its large size and ready availability, the cockroach (Peri-
planeta americana} was used throughout.
METHODS
Cockroaches were immobilized by exposure to 100 per cent CO2 for 20-60 sec-
onds or by etherization. After CO2, anesthesia seldom lasted over a minute. Once
anesthetized, the roach was fastened to a bit of cardboard by pins passed through
either side of the pronotum. Appendages were held in any desired position by
pins crossed over the body.
Decapitation was easily achieved by simply cutting the neck with a small scissors.
The exposed stump was sealed with low melting-point paraffin. Ligation of the
neck prior to decapitation to prevent loss of hemolymph did not prolong survival
time. Such animals live about 60 hours (Table I).
To expose a thoracic ganglion, the spinasternum just caudal to the ganglion
was cut through, and the incision extended along the sides of the sternal plate. After
the plate was reflected forward, removal of superficial fatty tissue and tracheal tubes
fully exposed the ganglion. The connectives anterior to the ganglion were held in
a jeweler's forceps and cut with iridectomy scissors. Traction on the connectives
exposed the lateral nerves, which were sectioned. Finally, the posterior connectives
were cut and the ganglion removed. Simple isolation of a ganglion from the rest
of the nerve cord can be achieved without excising it by cutting the connectives
through slits in the cuticle. Complete transection of the entire roach between sets
LOCI OF ACTION OF DDT
249
TABLE J
Effect of lesions of the central nervous system on symptoms of DDT poisoning in the cockroach
Operation
No. roaches
Aver-
age
sur-
vival,
hours
General results
Legs which showed
DDT
effects
Con-
trols,
no
DDT
Operated
Nature
Site
(See Fig. 1 )
Before
DDT
After
DDT
Hyperactivity, tremors
and convulsions
Decapitation
At A
14
14
14
61
56
56
Rare tremors in 3 ani-
mals — no convulsive
activity
Typical DDT effects
in all animals
Typical DDT effects
in all animals
None
All
All
Transaction of
ventral nerve
cord
At C and D (both
anterior and pos-
terior to thoracic
ganglion no. 2)
16
15
—
104
60
None in any animals
Typical DDT effects
in all animals
None
All in 13 animals; 2nd
and 3rd pairs in two
animals
Destruction of
ganglion
Th. 2 (thoracic
ganglion no. 2)
15
IS
—
77
71
None in any animals
Typical DDT effects
in all animals
Leg 2 paralyzed in all
1 and 3 in 13 animals.
1, 2 and 3 in two ani-
mals
of legs results in an isolated segment containing a ganglion, nerves and the attached
legs. Such a preparation, if kept moist, is viable for at least 6 to 8 hours.
Excision of the heart largely prevents circulatory removal of substances applied
to structures to elicit a local effect. Longitudinal incisions through the cuticle, on
either side of the heart tube along its entire length, isolate a strip whose removal
carries the heart with it. The heart may be cauterized with equal ease (Yeager and
Munson, 1945).
Methods for the administration of measured doses of DDT to insects have been
described elsewhere (Tobias, Kollros, and Savit, 1946a).
RESULTS
Localisation experiments with uncontrolled DDT doses
Except where otherwise specified, contact poisoning was carried out by confining
the roach for 5-15 minutes within a glass cylinder coated with DDT previously pre-
cipitated from acetone solution.
Roaches decapitated before or after such contact with DDT behaved like intact
poisoned animals (Table I). Therefore, neither the supra- nor the sub-oesophageal
ganglia are essential for the development or maintenance of DDT-induced motor
activity in the legs or body. Ventral nerve cord connectives were transected both
anterior and posterior to the mesothoracic ganglion (Fig. 1, levels C and D). Ani-
mals so prepared but given no DDT showed incoordination of the mesothoracic legs
when walking, but there were no symptoms which could be confused with those
of DDT poisoning. When such animals were subsequently poisoned, however, the
mesothoracic as well as the other legs exhibited typical abnormal activity (Table I).
After complete transection of the whole body of the poisoned roach, at both these
levels (excised segment Fig. 1), leg tremulousness and hyperactivity continued un-
250.
J. M. TOBIAS AND J. J. KOLLROS
abated in the isolated segment. The application of DDT emulsion or DDT in ace-
tone to the cut surface of such segments obtained from normal roaches evoked typical
DDT effects in the attached legs within a few minutes. The same was true of DDT
applied directly to the exposed ganglion in the otherwise intact animal. Emulsion
or acetone without DDT had no such effect.
The cells of origin of the leg nerves lie within the lateral halves of the thoracic
ganglia, each ganglion in the adult being formed by the midline fusion of two em-
bryonic ganglion masses. Median sagittal section of the ganglion in a poisoned
roach (Fig. 1. level F) did not stop hyperactivity in either of the legs innervated
FIGURE 1. Levels of section in cockroach nervous system.
from the resulting ganglion halves. Therefore, even half a segment contains all the
structures necessary for the maintenance of DDT symptoms in a leg.
If, however, the entire ganglion was removed the results were generally quite
different. Mesothoracic ganglia were removed from thirty normal roaches. The
corresponding legs of all were paralyzed and failed to respond to touch or pressure.
Shortly after the operation, fifteen of the animals were contact poisoned. All
showed typical DDT effects in the pro- and metathoracic legs, but the ganglionecto-
mized mesothoracic legs remained entirely quiet in thirteen and showed only occa-
sional tarsal twitching and some slight movement of the other joints in two. Simi-
larly, ganglionectomy after the development of hyperactivity, rather than before
LOCI OF ACTION OF DDT
251
TABLE II
Experiments on isolated roach segments containing local ganglion, nerves, and legs
No. of
segments
6
3
7
4
Material applied
Nothing
Emulsion* without DDT
Acetone without DDT
1 Per cent DDT emulsion*
10 Per cent DDT in acetone
DDT powder
Route
On cut surface
Injected
On ganglion
Injected into vicinity
of ganglion
On ganglion
Number of segments in which
there was persistent DDT
leg activity
None
None
None
Occurred in all
Occurred in all
Occurred questionably in
one
* Emulsion — 1 per cent DDT, 10 per cent peanut oil, 1 per cent lecithin and 88 per cent
0.90 percent NaCI (5).
poisoning, either stopped or markedly reduced symptoms in the corresponding legs
(Table IV). As was to be expected from these experiments, section of leg nerves
lateral to the ganglion stopped or markedly reduced activity in many (65 per cent)
of the legs (Table III).
These experiments tentatively suggested that the ventral cord ganglion was
critically involved in the motor action of DDT and might itself be a site of action.
Conflicting data, however, were also obtained. It was possible, as also reported by
others (Yeager and Munson, 1945; Roeder and Weiant. 1946). to produce motor
TABLE III
Visible effect of DDT on amputated legs
(Dose not controlled)
No. of
legs
30
58
12
22
35
Source of legs
Normal roaches
DDT poisoned
roaches tremulous
and hyperactive
Normal roaches
Normal roaches
Normal roaches
Treatment after amputation
Normal controls
Emulsion without DDT in-
jected into cut end
Emulsion with 1 per cent
DDT applied to cut end
Emulsion with 1 per cent
DDT injected into cut end
Results after amputation
No spontaneous movement
Continued activity in 20. No
movement in 38
No movement in any
Movement in 1, others all quiet
Movement in 25, other 10 quiet
9
13
Normal roaches
Normal roaches
Acetone without DDT in-
jected into cut end
Acetone with DDT injected
into cut end
Movement in 1, other 8 quiet
Movement in 3, other 10 quiet
252
J. M. TOBIAS AND J. J. KOLLROS
activity in a large percentage of amputated legs by the injection of DDT emulsion
(1 per cent DDT, 1 per cent lecithin, 10 per cent peanut oil, and 88 per cent 0.9
per cent NaC! solution). It will also he recalled that ganglionectomy failed to en-
tirely quiet the legs in two of fifteen experiments (Table T).
Such conflicting data were difficult to interpret. Ganglionectomy or denerva-
tion usually stopped leg activity, but this was not invariably the case, and it was
possible to produce activity in the amputated legs by injection of DDT. It was
suspected that such results might be resolved in terms of DDT dose. Further
experiments were then done with measured doses of DDT.
Localization experiments with controlled doses of DDT
It was immediately found that the effectiveness of ganglionectomy in abolishing
motor effects was inversely related to dose (Table IV). That is, as the dose of
DDT was increased ganglionectomy stopped movement in progressively fewer cases.
TABLI-: IV
Effect of ganglionectomy on symptoms after various doses of DDT
Results of ganglionectomy
No.
experi-
DDT*
ments
Number resulting in complete
Number resulting in a
cessation of activity
reduction of activity
10
Usual moderate contact dose (5-10 mins.
70%
30%
in DDT coated tube)
5
Excessive contact dose (approximately
20%
80%
2 hours in DDT coated tube)
25
5-30 mg. DDT per kg. injected intra-
68%
32%
abdominally in emulsion*"
12
60-70 mg. DDT per kg. injected intra-
50%
50%
abdominally in emulsion**
15
130 mg. DDT per kg. injected intra-
7%
93%
abdominally in emulsion**
* LD-50 for DDT injected intra-abdominally in emulsion is 20 mg. per kg. (Tobias, Kollros,
and Savit, 1946a).
** Emulsion — 1 per cent DDT, 1 per cent lecithin, 10 per cent peanut oil, 88 per cent of
0.9 per cent NaCl.
In all cases, however, even when movements were not entirely stopped they were
both qualitatively and quantitatively changed. The high frequency tremulousness
was always markedly reduced or entirely abolished and the slower movements
were much diminished.
Nicotine, in low concentrations, is known to block synaptic transmission cen-
trally as well as peripherally (Libet and Gerard, 1938; Pringle, 1939) but not
axonal transmission. When applied to the cockroach ganglion there is an initial
burst of electrical hyperactivity (100-800 impulses per sec.) followed by electrical
silence (Pringle, 1939). As would be expected, such application of nicotine to a
ganglion also produces great motor hyperactivity in the attached leg which can be
abolished by amputating the leg (Yeager and Munson, 1945).
Now then, if nicotine applied to a ganglion in a concentration which did not
LOCI OF ACTION OF DDT
253
affect peripheral nerve were to stop DDT symptoms, this would be added evidence
for the importance of the ganglionic cell bodies or synapses in the development and
maintenance of such symptoms. After poisoned roaches became hyperactive the
heart was excised. This did not decrease activity, but served to greatly diminish
circulatory transport of solutions applied for local effects. Solutions were then
applied as small droplets to the ganglion or a region of leg nerve exposed by cuticle
excision.
Dilute nicotine solutions (0.01 per cent in insect Ringer) applied to the leg
nerves of the normal or poisoned roach did not paralyze the leg. Typical DDT
induced activity could not be stopped in this fashion. This was almost surely not
due to failure of nicotine to reach the nerve since spontaneous movement as well
as that following electrical stimulation of the ganglion could be stopped by a similar
TABLE V
Effect of locally* applied nicotine and novocaine on motor symptoms of
DDT poisoning after various doses of DDT
No.
experi-
ments
DDT**
Number of experiments in which activity was modified
1.0% novocaine
0.01% nicotine
Injected into tibia
Injected into tibia
Applied to ganglion
Complete inactivity
Complete inactivity
or reduced activity
Activity
stopped
Activity
reduced
4
10-30 mg. per kg. applied
to body surface 18 hours
before
100%
o%
100%
o%
9
100 mg. per kg. applied to
body surface 18 hours be-
fore
100%
0%
77%
22%
13
4
500 mg. per kg. applied to
body surface
1000 mg. per kg. applied to
body surface
23%
0%
77%
100%
* All experiments on cardiectomized roaches to prevent circulatory removal of substances
applied for local effect.
** DDT applied to surface in acetone. LD-50 for DDT so applied is 10 mg. per kg. (Tobias,
Kollros, and Savit, 1946a).
administration of 1 per cent novocaine. When, however, this nicotine solution was
applied to a ganglion (in the same normal or poisoned animal in which it was in-
effective on peripheral nerve) there was a short-lived burst of great activity in the
legs of the segment, followed by complete immobility or markedly decreased activity.
Since the nicotine was effective in concentrations which did not block peripheral
nerve, it was concluded that it was acting by blocking ganglionic synapses and not
by spill-over to the emerging nerve roots. It is clear (Table V) that, as in the
case of ganglionectomy, the immobilizing effect of nicotine decreased as the dose
of DDT increased, and, as was also true after ganglionectomy, if nicotine did not
stop activity it considerably decreased and modified it.
254 J. M. TOBIAS AND J. J. KOLLROS
DISCUSSION
It is clear that DDT can produce motor symptoms l>y effects peripheral to the
ganglion. It is equally clear, however, that the ganglion plays a role in the
initiation and maintenance of symptoms and that this role is to some extent
dependent upon DDT dose.
Roecler and Weiant (1946) found that, in the cockroach, very low concentra-
tions of DDT can initiate centripetally directed, high frequency (300-400 per sec.),
temporally irregular bursts of nerve impulses, presumably excited by action of
DDT on the campaniform sensilla (presumptive proprioceptors). There was no
evidence of any muscle movement which might have initiated such centrally directed
impulses. Welsh (1946) has demonstrated that DDT in very low concentrations
can also favor repetitive response of motor fibers (Cancer irroratus] to a stimulus
normally evoking single responses, and Yeager and M tin son (1945) have concluded
that high concentrations can produce similar changes in the cockroach.
The results of ganglionectomy, in the cockroach (surgical or nicotine inacti-
vated), after various doses of DDT, support the view that the initiation and con-
tinuation of the hypermotor symptoms of DDT poisoning after low doses of DDT
require an intact sensori-motor reflex arc, and that random afferent impulses in
sensory nerves may indeed, as suggested by Roeder and Weiant, excite motor
neurones in the ganglion to initiate incoordinated muscular activity. From the
experiments here reported, this would appear to be a part of the common sequence
of changes in the roach poisoned by uncontrolled contact doses. The fact that
ganglionectomy becomes less and less effective as the dose of DDT is progressively
increased would suggest that larger amounts of DDT may act directly on motor
nerves. Obviously, these data do not rule out a possible direct action on muscle.
Within the dose ranges which have been used there is, however, no conclusive evi-
dence for a direct action on muscle. This is not to say that such action could not
occur at some sufficient dosage level. In addition, ganglionectomy is seen to have
stopped the rapid tremulousness after any dose which was tried, suggesting that
the high frequency movements may be initiated reflexly rather than by direct action
on motor fibers at large as well as at low doses of DDT. This general picture is
compatible with the subsidence of high frequency tremulousness before subsidence
of slower muscular activity. If the former is reflex and the latter due to direct
nerve action one might expect this order of dropping out on the basis of much
greater fatigability for the reflex arc than for the nerve trunk.
Pattern development of symptoms
• It has been claimed (Laiiger, Martin, and Miiller, 1944) that if DDT be put
on one leg of a fly the development and progression of symptoms follow a definite,
orderly and reproducible path from leg to leg. Such a phenomenon might be very
important indeed for an understanding of the mechanism of DDT action. The
authors have not been able to confirm this finding.
CONCLUSIONS
1. Neither decapitation, section of one or several nerve cord connectives nor
complete transection of the entire insect body at one or several levels between nerve
cord ganglia prevents the development of the typical motor effects of DDT in any
LOCI OF ACTION OF DDT 255
of the legs of the cockroach. After combined antero-posterior isolation of a nerve
cord ganglion, even median sagittal section of the ganglion does not prevent motor
symptoms in the legs still attached to the lateral ganglionic cell masses. There-
fore, the anatomical elements necessary for development of the motor symptoms of
DDT are contained within the lateral half of a body segment which contains the
lateral half of a ganglion, leg nerves and peripheral structures.
2. Since the motor symptoms of DDT poisoning can occur in amputated legs,
in legs whose nerves have been cut, and in legs whose segmental ganglia have been
destroyed, it is possible for DDT to produce its motor effects by action on some
structure or structures peripheral to the segmental ganglion.
3. The motor symptoms of DDT poisoning can be stopped or diminished in a
leg by ganglionectomy, leg nerve section, or ganglion synaptic block with nicotine.
The effectiveness of these procedures is in inverse relation to the dose of DDT
administered. These findings suggest that, in the cockroach, low doses of DDT
may excite motor fibers reflexly by impulses fired into the ganglion over afferent
nerve fibers, whereas high doses may act on elements on the motor side of the
ganglion and thus not require an intact reflex arc. Since ganglionectomy stops the
fast component of the hypermotor activity, however, equally well after large or
small doses of DDT, this component may be reflexly initiated and maintained
after all doses of DDT.
LITERATURE CITED
BODENSTEIN, D., 1946. Investigation on the locus of action of DDT in flies (Drosophila).
Biol. Bull, 90: 148.
CRESCITELLI, F. AND A. OILMAN, 1946. Electrical manifestations of the cerebellum and cerebral
cortex following DDT administration in cats and monkeys. Amer. Jour. Physiol.,
147: 127.
LAUGER, P., H. MARTIN AND P. MULLER, 1944. The constitution and toxic effect of botanicals
and new synthetic insecticides. Hclv. Chim. Acta, 27.
LIBET, B. AND R. W. GERARD, 1938. Automaticity of central neurones after nicotine block of
synapses. Proc. Soc. Exp. Biol. and Mcd., 38 : 886.
PRINGLE, J. W. A., 1939. The motor mechanism of the insect leg. Jour. Exp. Biol., 16 : 220.
ROEDER, K. D. AND E. A. WEIANT, 1946. The site of action of DDT in the cockroach. Sci-
ence, 103 : 304.
SAVIT, J., J. J. KOLLROS AND J. M. TOBIAS, 1946. The measured dose of gamma hexachloro-
cyclohexane (y 666) required to kill flies and cockroaches and a comparison with DDT.
Proc. Soc. Exp. Biol. and Mcd., 62 : 44.
TOBIAS, J. M., J. J. KOLLROS AND J. SAVIT, 1946a. Relation of absorbability to the comparative
toxicity of DDT for insects and mammals. Jour. Pharm. and Exp. Ther., 86 : 287.
TOBIAS, J. M., J. J. KOLLROS AND J. SAVIT, 1946b. Acetylcholine and related substances in
the cockroach, fly and crayfish and the effect of DDT. Jour. Cell. Comp. Physiol.
(In press.)
WELSH, J. H., 1946. Personal communication.
WIGGLESWORTH, V. B., 1939. The principles of insect physiology. Dutton and Co., Inc.. New
York.
YEAGER, J. F. AND S. C. MUNSON, 1945. Physiological evidence of a site of action of DDT
in an insect. Science, 102 : 305.
TILLINA MAGNA: MICRONUCLEAR NUMBER, ENCYSTMENT
AND VITALITY IN DIVERSE CLONES; CAPABILITIES
OF AMICRONUCLEATE RACES
C. D. BEERS
Department of Zoology, University oj North Carolina, Chapel Hill
It is well established that the number of micronuclei in Tillina magna is highly
variable. For example, Gregory (1909) found 6-10, and Ilowaisky (1921), in a
ciliate which he called Pseudocolpoda cochlearis cicnkoivskii, reported 2-6. An ex-
amination of Ilowaisky's text and figures shows conclusively that his ciliate was T.
magna Gruber (1879) as Kahl (1931, p. 282) pointed out. Kahl apparently re-
garded six as the typical number. Bresslau (1922) observed the nuclei in sufficient
detail to note the extrusion of macronuclear material at division and the presence of
several micronuclei, though he reported no counts of their actual number. The
writer (1946), in a study dealing chiefly with the history of the nuclei during divi-
sion and encystment, counted the number of micronuclei in 100 individuals (50 active
and 50 encysted) of each of three clones, and found that it varied from 6 to 11 in one
clone and from 4 to 6 in the other two. Thus the number was found to vary in dif-
ferent individuals of the same clone, and the mean number was found to vary in dif-
ferent clones. Active specimens and resting cysts of any particular clone had on the
average like numbers of micronuclei. Contrary to statements in the literature, it
was shown that the micronuclei divide at the time of cell division, and not indiscrimi-
nately or without regard to cell division. The mechanism by which two daughter
cells may receive unlike numbers of micronuclei at division, thus accounting for vari-
ations in number within a clone, was described.
The significance of the wide variation in micronuclear number is unexplained.
Structurally and physiologically an individual having only 4 micronuclei does not
appear to be fundamentally unlike one having 11 micronuclei. The same condition
prevails in the closely related species, T. canalijcra, which I was formerly disposed
to regard (1945) as identical with T. magna. However, on the basis of informa-
tion furnished me by Dr. George W. Kidder, of Amherst College, it appears that T.
canalifera merits recognition as a valid species, chiefly because of the very conspicu-
ous nature of its canal system. In T. canalifera, Turner (1937) reported 4—14
micronuclei, though Burt, Kidder, and Gaff (1941), in specimens obtained from the
late Dr. Turner, found only one. Hence, it is clear that the micronuclear number
may vary from 1 to 14, yet the evidence indicates that the uninucleate and multi-
nucleate races were equally cultivable, vigorous, and capable of producing normal
resting cysts.
The present study of T. magna was undertaken in order to obtain additional in-
formation concerning two points : ( 1 ) the normal variation in micronuclear number
in various natural races and (2) the significance of such variation. The investiga-
tion of the first point is readily feasible, in that the micronuclei may be counted with
absolute certainty in Feulgen preparations of favorably oriented resting cysts or
256
MICRONUCLEI OF TILL1NA MAGNA 257
medium-sized trophic specimens. The investigation of the second point, though less
suited to direct approach, is not impracticable. A number of questions arise, some
of which submit to experimental analysis. For example, is the number of micro-
nuclei related in any way to size, whether of trophic specimens, division cysts or
resting cysts ; to division rate ; to vitality, meaning capacity to endure with undimin-
ished vigor as generations pass ; to ability to produce resting cysts ; to the viability
of such cysts ; or to ability to excyst ? Of the foregoing measurable characters, the
following were selected as being most readily amenable to experimental investiga-
tion : ability to produce resting cysts, size and viability of such cysts, capacity to
excyst, division rate and vitality. These then, will be considered in relation to
micronuclear number, though not all of them will receive equal consideration. The
study assumed unlooked-for interest when it became evident that three of the races
were amicronucleate. Thus a comparison of the potentialities of micronucleate and
amicronucleate clones became possibile.
MATERIALS AND METHODS
Twenty clones of T. inagna, to be designated numerically, were used in the study.
The progenitors of these respective clones were collected in a meadow, known locally
as Sparrow's Pasture, in the vicinity of Chapel Hill, North Carolina. Comparisons
with clones from other sources were desirable, but unfortunately attempts to collect
Tillina elsewhere in the Chapel Hill region, and in the vicinity of Stanford Univer-
sity, California, and Woods Hole, Massachusetts, were unavailing. In this study
a clone refers to all the progeny which were derived asexually from a single resting
cyst or trophic specimen. The intervention of encystment and subsequent excyst-
ment is not considered to be a valid reason for changing the clonal designation, since
there is no evidence that encystment in Tillina involves a sexual process which
might change the genetic constitution of the clone. (It should perhaps be recalled
that Tillina, like its near relative Colpoda, reproduces within a thin-walled tempo-
rary cyst, from which usually four progeny emerge shortly as a result of two succes-
sive divisions. The term encystment, as used in this study, does not refer to these
temporary division cysts, but to the protective or resting cysts.) It is not defi-
nitely established that all of the twenty clones were genetically different, since their
histories prior to their period of laboratory life were unknown.
The progenitors of clones 1, 2, 6, 8, 9, 11, 12, 13, 15, 17, 18, and 19 were taken
as active specimens on Sept. 10, 1945, and these clones were therefore cultured
simultaneously in the early part of the study. Eight of the foregoing clones, namely,
6, 11, 13, 18, 15, 19, 1, and 17, have already been reported on briefly under the
numerical designations 1 to 8, respectively (Beers, 1946a). In the present paper
my original numerical designations of all clones have been changed for the con-
venience of the reader in using the accompanying tables. The progenitors of clones
3, 14, 16, and 20 were isolated on Feb. 4, 1946, when dried leaves and debris, after
8 months of storage at 19° C., were immersed in weak hay infusion; these clones
were therefore cultured simultaneously. It is evident that they were derived from
dried cysts. The progenitors of clones 4, 5, 7, and 10 were isolated on April 8,
1946, when moist leaves and debris, which had recently washed against the bases of
willow saplings in the meadow, were immersed in hay infusion; these clones were
258 C. D. BEERS
maintained in culture simultaneously toward the end of the study. They were un-
doubtedly derived from wet cysts.
An attempt was made to maintain each of the clones in pure-line culture for a
period of 60 days. Sixteen of the clones were readily cultivable and continued with
undiminished vigor throughout the period ; four were intractable in that their divi-
sion rates declined and the lines encysted well before the end of the period. Thus
the laboratory histories of the clones varied, although the conditions of culture -were
uniform. The details, in relation to micronuclear number, will follow.
Each clone was cultured in depression slides in the form of four sub-lines.
These were maintained at 23° C. in 0.05 per cent lettuce infusion to which suitable
quantities of Psciidomonas fluorescent, grown on nutrient agar, were added as food.
Previous experience has shown that this general procedure, combined with daily
isolations and transfers to fresh environments, meets adequately the cultural needs
of Tillina (Beers, 1944, 1945). Records were made daily of fission rates and other
points of interest.
Surplus animals from the lines were stained on cover glasses by the Feulgen
method in order to make micronuclear counts of active specimens. Small stock
cultures of each clone furnished precystic specimens when the food supply neared
depletion. These specimens were removed and allowed to encyst on cover glasses
in the manner described by Beers (1946). Thus convenient preparations were
available, first for making measurements of living cysts, and then for Feulgen stain-
ing. All measurements and micronuclear counts of cysts were made on single
resting cysts. These are the common type. They are practically spherical and
therefore well suited for making accurate measurements.
NORMAL VARIATION IN NUMBER OF MICRONUCLEI
The data bearing on diversity in micronuclear number in the twenty clones are
summarized in Table I, in which the clones are arranged and numbered in the order
of decreasing mean numbers of micronuclei. The data, ignoring for the present
the mean diameters of resting cysts, are largely self-explanatory. A few points
deserve special mention.
In any particular clone both active specimens and resting cysts showed practi-
cally the same extremes of variation (range) in micronuclear number and had essen-
tially the same mean number of micronuclei.
In different clones the mean numbers of micronuclei were extremely variable.
Some clones (e.g., 1 and 2) had consistently high mean numbers; others (e.g., 16
and 17), consistently low numbers, with many intergrades between these extremes.
Clones 18, 19, and 20 were amicronucleate. This statement is not based on
casual observation, but on an intensive study of these clones. In trophic specimens
and resting cysts the micronuclei of Tillina are not disposed toward secretiveness.
They are never imbedded in the macronucleus. Each has an endosome which stains
intensely and conspicuously by the Feulgen method. In mature resting cysts only
the rod-shaped or ellipsoid macronucleus and the micronuclei stain to any appre-
ciable extent; there is nothing in the cytoplasm to conceal the micronuclei. In
trophic specimens it is true that the food vacuoles also stain, but the micronuclei
always lie in the clear peri-macronuclear space and are not in a position to be con-
cealed by the vacuoles. Moreover, considerable numbers of individuals of clones
MICRONUCLEI OF TILLINA MAGNA
259
18, 19, and 20 were stained. These included not only the usual resting cysts and
medium-sized trophic specimens, but also young cysts, cysts in the process of excyst-
ment, and individuals just excysted. None showed a micronucleus, whereas indi-
viduals of the remaining seventeen clones, stained at the same time by the same
method, invariably showed micronuclei.
The individuals of some clones (e.g., 1, 3, 5, 12) showed great diversity in
micronuclear number within the clone. This fact is brought out clearly by the
range which is cited for these clones, and it is further emphasized by the high
standard deviations in the clones. Clone 12 showed the greatest degree of hetero-
geneity in that the range in micronuclear number extended from 2 to 11, with all
intervening numbers being represented. On the other hand, some clones (e.g.,
2, 4, 8; 13, 15, 16, 17) were relatively homogeneous, with narrow ranges and low
standard deviations. Other clones lay between these extremes. Only the amicro-
nucleate clones showed complete homogeneity.
Thus, it is seen that individuals of a clone exhibit varying numbers of micro-
nuclei, that clones differ with respect to their mean number, and that amicronncleate
clones exist in nature.
The mean number of micronuclei in the 850 micronucleate active specimens of
Table I (representing 17 clones) was 7.06; the mean number in the 850 micro-
nucleate cysts was 7.08. Unfortunately, the number in the progenitor of each clone
TABLE 1
Tillina magna. Variation in number of micronuclei in twenty clones; relation of micronuclear
number to size of cysts. The clones are numbered and arranged as the mean number of micronuclei
(average of means for fifty active specimens and fifty resting cysts} decreases.
Numerical
designation
of clone
Range in number of micronuclei
Mean number of micronuclei
± standard deviation
Mean diameter of
50 resting cysts
in microns
± standard
deviation
50 active
specimens
50 resting
cysts
50 active
specimens
50 resting
cysts
1
10-16
9-16
12.90±1.87
12.32±1.93
85.76± 9.65
2
8-12
9-12
10.48±0.94
10.96±0.87
82.94± 7.65
3
7-14
6-13
9.62±2.30
9.24±2.08
79.68± 8.72
4
7-10
7-10
8.52 ±0.82
8.60±0.95
88.62± 7.25
5
6-14
6-12
8.17±1.95
8.28±1.66
88.36± 9.61
6
6-10
6-10
7.56±1.28
7.78±1.24
93.60± 6.21
7
5-10
5-10
7.25±1.28
7.38±1.36
92.44± 7.08
8
6- 9
6- 9
7.16±0.85
7.06±0.92
78.50± 7.82
9
5- 9
4- 9
6.52±1.15
6.74±1.21
84.16± 8.62
10
4- 8
4- 8
5.98±1.52
5.94±1.46
85.42± 5.28
11
5- 8
5- 9
5.90±1.03
5.76±0.96
86.14± 5.34
12
2-10
2-11
5.74±2.41
5.90±1.97
85.64± 5.32
13
4- 6
4- 6
5.12±0.42
5.10±0.45
80.64± 8.92
14
4- 8
4- 8
5.12±1.51
4.98±1.42
84.18± 4.76
15
4- 6
4- 6
4.86±0.53
5.20±0.57
81.60± 8.41
16
4- 6
4- 6
4.92 ±0.63
4.98±0.75
80.92± 7.37
17
3- 5
3- 5
4.28±0.75
4.14±0.74
91.42± 6.87 '
18
—
—
0
0
86.28± 5.63
19
—
—
0
0
84.32 ±10.36
20
—
—
0
0
81.52± 5.34
260 C. D. BEERS
is unknown, since the micronuclei cannot be identified in living specimens. It
seems reasonable to assume that the progenitor of each had a number approximately
equivalent to the mean which was determined for the clone, and that it produced
some offspring having fewer, and some having more, than its own number.
It is well known that the number of micronuclei is variable in many species of
ciliates. Thus, Paramecium multimicronucleatwn has 2 to 7 (Powers and Mitchell,
1910), though usually 4 (Wenrich, 1928) ; Spathidium spathula, 6 to 9 (Maupas,
1888, p. 247) ; Urostyla grandis, 10 to more than 40 (Tittler, 1935) ; and S tent or
coeruleus, 10 to 42 within a single clone (Schwartz, 1935). On the whole, such
variations within a species appear to have little effect on the structure or behavior
of the individuals and to be without functional significance. This general conclu-
sion is supported by the observations on T. umgiia which follow immediately.
NUMBER OF MICRONUCLEI IN RELATION TO VARIOUS ASPECTS OF CYSTMENT
All the clones produced normal resting cysts upon the depletion of the food
supply in small stock cultures prepared with surplus animals from the lines. Fur-
thermore, all the specimens in such cultures encysted ; none persisted in prolonged
swimming, thereby to perish of starvation. Hence, it is clear that the ability to
encyst is not dependent on the presence of the micronucleus, since amicronucieate
as well as micronucleate clones were able to encyst. Moreover, the cysts of all
the clones remained viable for many months. They could be activated at any time
after the fourth day by immersion in distilled water or 0.05 per cent lettuce infusion.
From 2 to 2.5 hours were required for emergence at 23° C., and practically 100 per
cent of the specimens excysted. No precise figures are given here, since the per-
centage of excystment under various conditions has been dealt with in a previous
paper (Beers, 1945) and the present study contributes nothing new on this point.
'However, the present results show clearly that the viability of resting cysts and
their capacity to excyst are in no wise related to the presence of a micronucleus, or
to the number of micronuclei. Well over 90 per cent of the cysts produced in the
various clones were single ones ; double cysts appeared sporadically, some in ami-
cronucieate clones, some in micronucleate. Amicronucieate cysts undergo the usual
colpodid type of macronuclear reorganization, involving the extrusion of a portion
of the macronuclear substance into the cytoplasm (Taylor and Garnjobst, 1941 ;
Burt, Kidder and Claff, 1941; Beers, 1946).
The size of the cysts in different clones was made the subject of special study,
for it was thought that the number of micronuclei might affect the size of the cysts.
The diameters of fifty living single cysts of each clone were measured, each measure-
ment extending from the outer surface of the ectocyst of one side to the correspond-
ing surface of the other. The results of these measurements are included in Table
I. An inspection of the table shows at once that the calculation of coefficients of
correlation between micronuclear number and cyst size would be of little value,
since cyst size is independent of micronuclear number. For example, if we con-
sider certain extremes in micronuclear number, it is seen that the cysts of clone 1
had a mean diameter of about 85 /A, and those of clone 19 a diameter of 84 p, with
approximately equivalent standard deviations. Clones 2 and 20 and clones 4 and
18 constitute other examples of extreme disparity in micronuclear number with
close agreement in cyst size. Among the micronucleate clones, other examples of
MICRONUCLEI OF TILLINA MAGNA 261
wide divergence in micronuclear number, yet with general uniformity in cyst size,
are furnished by clones 3 and 16 and by clones 6 and 17. On the other hand, some
clones having widely divergent micronuclear numbers produced cysts of dissimilar
sizes, e.g., clones 3 and 17 and clones 6 and 13. Thus it is seen that clones having
widely different micronuclear numbers may produce cysts of equivalent mean sizes
or of dissimilar mean sizes. It must be concluded that there is no relation between
the number of micronuclei and the size of the cysts.
The same conclusion is reached if we adopt another approach and consider cyst
size in clones which had similar, or only slightly different, micronuclear numbers.
For example, clones 6 and 7 had similar mean numbers of micronuclei and they
produced cysts of equivalent mean sizes; clones 15 and 16 constitute a second ex-
ample. On the other hand, clones 7 and 8 had similar micronuclear numbers, but
they produced cysts which differed significantly in mean size; clones 16 and 17
furnish another example. Hence, clones having similar mean micronuclear num-
bers may produce cysts of equivalent mean sizes or of different mean sizes. Again,
it is evident that there is no relation between number of micronuclei and size of
cysts. The mean diameter of the 850 micronucleate cysts of Table I was 85.23 p.;
that of the 150 amicronucleate cysts was 84.04 p..
A word concerning the size of individual cysts may be of interest. In any
particular clone of T. mayna, whether micronucleate or amicronucleate, there is
usually wide variation in cyst size, even though the cysts under immediate consid-
eration are all produced in the same small stock culture — meaning a Columbia
culture dish containing 1 cc. of fluid. Cysts in such a culture often vary in size
from 75 ^ to 95 p. ; extremes of 64 /*, and 104 /A have been noted. The factors which
affect cyst size appear to be of a complex physiological nature and are therefore not
readily identifiable.
The point of greatest interest in this consideration of various aspects of cystment
is the fact that amicronucleate and micronucleate clones behaved alike ; clearly the
micronucleus of T. magna plays a negligible role, if any, with reference to encyst-
ment, viability of cysts, excystment, macronuclear reorganization within the cysts,
and cyst size.
NUMBER OF MICRONUCLEI IN RELATION TO DIVISION RATE AND VITALITY
The cultural histories of the twenty clones are presented in Table II. Clones
1, 2, 3, and 5 could not be maintained in culture for the arbitrary period of 60 days.
The remaining clones were maintained with undiminished vigor and were discon-
tinued at the end of the period.
First, the division rates of the sixteen vigorous clones will be considered in
relation to micronuclear number. Clones 4, 6, 7, and 8, as has been seen, had rela-
tively high micronuclear numbers (means, about 7 to 8.5). The total average
number of generations produced by the four sub-lines of these respective clones
varied from 149 to 174. Thus the clones themselves varied with respect to mean
division rate. Clones 9-12 had intermediate micronuclear numbers (means, 6 to
6.5) ; these clones produced from 149 to 167 generations. Clones 13-17 had low
micronuclear numbers (means, 4 to 5) ; they produced from 160 to 176 generations.
Thus clones having intermediate or low micronuclear numbers produced in general
as many generations, and had therefore the same division rates, as clones having
relatively high micronuclear numbers.
262 C. D. BEERS
Amicronucleate clones 18-20 produced from 154 to 172 generations. Thus the
amicronucleate clones had approximately the same division rates as the micronu-
cleate clones; e.g., clone 19 produced almost as many generations as clone 4; clone
18 produced more than clone 10 ; clone 20 produced about as many as clone 9 or
13. Many kinds of comparisons are possible, and the reader may choose to make
other comparisons between amicronucleate and micronucleate clones. For example,
clones 18-20 had higher division rates (i.e., produced more generations) than clones
8, 10, and 14; but clones 18—20 had lower division rates than clones 4, 7, and 16.
Thus the general conclusion that amicronucleate clones have the same division rates
as micronucleate clones is not invalidated. Sections of amicronucleate division cysts
showed that the macronucleus undergoes the usual reorganization after each of its
divisions, as in normal micronucleate cysts (Burt, Kidder and Gaff, 1941; Beers,,
1946).
Next, the vitality of the sixteen vigorous clones must be considered. An ex-
amination of the number of divisions produced in the successive 5-day periods in
any particular clone shows that the clone was dividing as rapidly at the end of the
experiment as at the beginning. Within the time limits of the experiment, the
clones showed no decrease in vitality as measured by the division rate. How long
the sixteen clones would have continued without diminution in vitality is a question
that cannot be answered on the basis of the available data. The important findings
are these : Some clones which have relatively high micronuclear numbers are as
vigorous as those which have low numbers ; amicronucleate clones are fully as
vigorous as many micronucleate clones.
Clones 1, 2, 3, and 5 must receive special consideration. As has been said, these
clones could not be maintained in culture for the duration of the 60-day experi-
mental period. Clone 1 showed a rapid decrease in fission rate and encysted on
the fourth clay. Some of the cysts were activated and new lines were established.
These in turn declined shortly and encysted. Three additional attempts were made
to culture clone 1 ; each time the lines encysted after 3-5 days. Indeed, clone 1
was so refractory that without these repetitions it would have been impossible to
obtain sufficient specimens for the usual number of micronuclear counts. Clones
2, 3, and 5 likewise could not be maintained in culture for 60 days. Their histories
are presented in sufficient detail in Table II. Following the encystment of the
original lines of these clones, new lines were established with excysted specimens,
but they also declined and encysted after 3-4 weeks of culture. It may be main-
tained that the decline and encystment of these four clones resulted from a failure
to meet their cultural needs. However, the conditions of culture were adequate for
a total of sixteen clones, and it seems not unreasonable to assume that they were
likewise adequate for clones 1, 2, 3, and 5 and to conclude that these clones declined
as a result of intrinsic factors.
It has been shown that clones 1, 2, and 3 had higher micronuclear numbers than
any of the other clones. Clone 5 likewise had a high number, though slightly lower
than clone 4, which was cultivable. The evidence indicates that a large number of
micronuclei may be detrimental to the welfare of the organism and incompatible
with high vitality, but the number of such clones studied was too small to justify
a general conclusion. On the whole, the results show that in T. inagna the rate
of division and the vitalitv of the race are in no wise related to the number of micro-
MICRONUCLEI OF TILLINA MAGNA
263
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264 C. D. BEERS
nuclei, or even to the presence of a micronucleus, since amicronucleate clones proved
to be as vigorous as micronucleate clones.
The origin of the amicronucleate races cannot be accounted for with certainty.
It is agreed that such races may arise at any of four periods in the life of ciliates
generally : at endomixis, by the transformation of all the reconstruction micronuclei
into macronuclei ; at autogamy or conjugation, by an analogous transformation of
all the derivatives of the synkaryon or amphinucleus into macronuclei ; or at division,
by an unequal distribution of the micronuclei to the daughter cells. By studying
dividing individuals of an unusual race of Paramecium caudatum, Wichterman
(1946) observed the simultaneous production of bimicronucleate and amicronu-
cleate daughters. In T. inagna conjugation is of rare occurrence, ahd endomixis
and autogamy are unknown. Therefore, it is likely that amicronucleate races usu-
ally take their origin in an unequal distribution of the products of division. How-
ever, it should not be concluded from the present results that 15 per cent of all
Tillina clones are amicronucleate ; actually such clones appear to be very exceptional.
I have stained many specimens during a period of 8 years; all these specimens had
micronuclei, except for the members of clones 18-20.
DISCUSSION
The functional significance of the nuclear dimorphism of the Euciliata has long
held the attention of protozoologists. In general, the dimorphic condition has been
viewed as representing a segregation of idiochromatin and trophochromatin, the
former in the micronucleus. the latter in the macronucleus. It was originally as-
sumed, since ciliates normally possess both types of nuclei, that both are necessary
for the continued survival of the individual. Although the precise functions of the
respective nuclei are difficult to determine, two lines of investigation have supplied
pertinent findings, namely, a study of the capabilities of amicronucleate ciliates of
natural occurrence and a study of regenerative capacity, survival, and reproduction
in ciliates which have been deprived experimentally of either nucleus, or of both,
whether by merotomy or other operative procedures.
The existence of naturally occurring amicronucleate races has long been conceded
by such authorities as Woodruff (1921), Calkins (1930), and Reichenow (1929,
p. 29) and is now accepted as a fact. The potentialities of these races, as revealed
by intensive laboratory study, have demonstrated that the micronucleus is not at all
necessary for the maintenance of the essential vital processes of the individual,
whereas the macronucleus is indispensable. Aside from the absence of a micro-
nucleus and the manifest inability to carry to completion such processes as endo-
mixis, autogamy and conjugation, amicronucleate races of many ciliates do not differ
structurally or physiologically from micronucleate ones.
Thus, Dawson (1919; 1920) maintained an amicronucleate race of Oxytricha
liyinenostonia in pure-line culture for 289 generations (4 months) and in small mass
cultures for 5 months longer. The absence of micronuclei did not prevent the ani-
mals from attempting to conjugate, but these attempts were abortive. Woodruff
(1921) cultured amicronucleate races of Oxytricha jallax and Urostyla grandis for
246 and 128 generations, respectively, and maintained a race of Paramecium can-
datum in pure-line culture long enough to determine that it was definitely amicro-
MICRONUCLEI OF TILLINA MAGNA 265
nucleate. A few pairs of conjugants occurred in mass cultures of O. falla.v, but
they failed to live when isolated.
Amicronucleate races of other ciliates have likewise been isolated a,nd cultured
long enough to demonstrate not only their viability but their sustained vigor and
good health. Among these are the following: (1) Spathidmm spathula. Moody
(1912) was unable to find micronuclei in her specimens, though she was able to
culture them for 218 generations. It is evident that they were amicronucleate, since
the micronuclei of Spathidium were observed and counted by Maupas and were
found regularly by Woodruff and Spencer (1922). (2) Didimuin nasutum. Pat-
ten (1921) cultured an amicronucleate race, which was derived from an exconjugant
of a normal micronucleate race, for 652 generations. Conjugation occurred in the
amicronucleate race, but the exconjugants invariably died. The resting cysts were
likewise inviable. It is evident that the race arose through the faulty reorganiza-
tion of the exconjugant. (3) Paramecium bursaria. Woodruff (1931) cultured
a race characterized by micronuclear instability for 7 years. Neither endomixis nor
conjugation was observed. The race was originally bimicronucleate ; later it was
variable, exhibiting from 1 to 4 micronuclei ; then for about 4 years it was unimicro-
nucleate ; finally, a derived race showed no micronucleus, although this race was
apparently as healthy and vigorous as its bimicronucleate ancestors which were iso-
lated 7 years earlier. Woodruff (p. 543) aptly points out that "whatever function
the micronuclear apparatus plays, the somatic life of the animals is not obviously
influenced by profound variations in volume or in distribution of micronuclear
material." (4) Urostyla grandis. Tittler (1935) found amicronucleate individuals
in stock cultures which previously contained only micronucleate specimens. They
were indistinguishable externally from their micronucleate progenitors, and they
flourished in mass cultures for 2 years. Their macronuclear divisions followed the
usual complex pattern characteristic of the species. The race produced resting
cysts, some of which could be excysted, although endomixis, which usually occurs
in the precystic forms, was absent. Evidently some of the cysts \vere not entirely
normal, since they showed a tendency to disintegrate, perhaps because of the omis-
sion of endomixis. (5) Colpoda steini. Piekarski (1939) studied comparatively
the structure and reproduction of a micronucleate and an amicronucleate race and
was able to culture the latter for approximately 6 years. Both races were equally
cultivable and vigorous. They reproduced within division cysts from which four
progeny regularly emerged and they produced normal resting cysts. They showed
the same Sequence of events in the division of the macronucleus. These events are
of special interest, in that eight chromatic (Feulgen-positive) bodies appear in the
macronucleus of the young division cyst. Ultimately each of the daughters receives
two of them, and thus the behavior of these bodies suggests an equational distribu-
tion of chromosomes. Piekarski concludes that the absence of a micronucleus had
no recognizable effect on the activities of the animals.
The present study of amicronucleate races of Tilliim inagna likewise demon-
strates the adequacy of the macronucleus, not only for long-continued reproduction
accompanied by sustained vigor, but also for encystment and excystment. Thus
endowed with the ability to produce viable resting cysts, these races would seem to
be capable of indefinite survival, even under the changeable conditions of a natural
environment.
266 C. D. BEERS
Still more remarkable, in connection with the capabilities of amicronucleate races,
are the observations of Sonneborn (1940) and Kimball (1941) on mating types.
In certain races of Paramecmm aurelia Sonneborn found a small percentage of ani-
mals which, upon undergoing autogamy or conjugation, developed a new macro-
nucleus from a fragment of the old macronucleus. Since the micronuclei commonly
disappeared in clones produced by these animals and since the mating type never
changed at macronuclear reorganization, Sonneborn concludes that "hereditary
characters (including mating type) of clones from macronuclear regenerates cannot
be directly determined by micronuclei, for they persist in the absence of micronuclei.
Mating type must be determined by the macronucleus. . . ." Kimball was able to
assign amicronucleate specimens of Euplotcs patella to definite mating types, since
they paired readily with individuals of known mating type. He concludes that "the
micronucleus is thus unnecessary for an animal to be of a definite mating type."
With reference to the role of the nuclei in the regeneration of ciliates following
merotomy, the results obtained with different species are not always in complete
accord. The physical properties of the cytoplasm constitute an experimental vari-
able ; a fluid cytoplasm or a rigid pellicle may interfere with the closure of an injury
and thus affect regeneration adversely. Balamuth (1940) has presented an ex-
cellent review of the extensive literature on this subject. For the present only a
few representative examples of regeneration in ciliates will be considered, with spe-
cial reference to the nuclear components of the merozoa (cell fragments).
It is well known that enucleate fragments of ciliates can neither regenerate nor
continue to live, whereas nucleate fragments regenerate successfully and pursue
normal lives. These conclusions are particularly evident in Balamuth's six-page
tabular summary of the findings on regeneration in the ciliates. As a rule the
macronucleus and micronucleus cannot be separated, and a nucleate fragment, as in
Dembowska's work (1925) on Stylonychia mytilus, usually means one having both
macronuclear and micronuclear material.
However, some investigators have succeeded in obtaining nucleate merozoa of
the two types desirable for an experimental analysis of the role of the individual
nuclei in regeneration; namely, macronucleate (without micronuclei) and micro-
nucleate (without any part of the macronucleus). Reynolds (1932), for example,
in work on an amicronucleate Oxytricha jallax, found that various types of macro-
nucleate merozoa could regenerate their missing parts and resume their normal
physiological activities. Schwartz, using microdissection techniques, was able to
remove the entire macronucleus from Stcntor and yet leave a number of micro-
nuclei in the specimens. These micronucleate individuals never survived. By
means of successive operations, he was also able to remove all the micronuclei from
a few specimens, leaving a portion of the beaded macronucleus in place. These
macronucleate individuals regenerated and could be cultured as pure lines. Thus
he produced experimentally an amicronucleate race, which as regards size and divi-
sion rate was not different from the normal controls. Bishop (1943), employing
the ultra-centrifuge as a means of obtaining merozoa of Oxytricha jallax, obtained
sixty-seven macronucleate fragments, all of which regenerated, and seven micro-
nucleate fragments, none of which regenerated. Twelve of the regenerated macro-
nucleate individuals were cultured as amicronucleate pure lines. Bishop concluded
(p. 451) that "the lack of micronuclear material makes no difference in the regen-
erative capacity, division rate, motility or morphology of Oxytricha jallax"
MICRONUCLEI OF TILLINA MAGNA 267
On the other hand, there is evidence that in some forms the micronucleus, as
well as the macronucleus, is necesesary for regeneration and survival. Thus Rey-
nolds found that both nuclei are necessary for the regeneration of merozoa of Eu-
plotcs patella. This observation is in accord with the results of Taylor and Farber
(1924), who, by means of a micro-pipette, removed the micronucleus from fifty
specimens of E. patella, all of which died within a few days. None produced more
than four progeny. Hence, these authors conclude that "the micronucleus plays
more than a purely germinal role in the life history of Eiiplotes patella." However,
the situation in E. patella is confused, for some of Kimball's unimicronucleate double
animals produced viable amicronucleate individuals at division. Some of them sur-
vived as clones, though with a low division rate ; one such amicronucleate clone sur-
vived for 341 days. The results show, according to Kimball (p. 30), "that the
micronucleus is not essential for continued life in at least some clones of Euplotes
patella, though its absence results in a marked decrease in vigor." In various spe-
cies of Uronycliia (Young, 1922), and in Uroleplus mobilis (Tittler, 1938), Spa-
thidium spathula and Blepharisma undulans (Moore, 1924) both types of nuclei ap-
pear to be necessary for the complete regeneration, growth and division of merozoa.
Thus the evidence afforded by the long-continued culture of a number of natu-
rally occurring amicronucleate races demonstrates conclusively that the macro-
nucleus alone suffices for the maintenance of the vegetative life of the organism-
meaning by vegetative life such diverse activities as locomotion, food capture, diges-
tion, assimilation, growth, excretion, respiration, reproduction, and maintenance of
cell proportions and form. On the basis of this evidence the normal role of the
micronucleus in vegetative life appears to be one of relative passivity. The evidence
from operative procedures shows that in many ciliates the macronucleus alone is
adequate for complete regeneration, as well as for subsequent growth and division,
whereas in other ciliates the micronucleus also is necessary. There is no authenti-
cated case on record in which the micronucleus alone is adequate for the maintenance
of vegetative functions or for regeneration. Balamuth's thorough survey of the
literature leads him to make the following comment in his summary : "Of the dual
nuclear apparatus, only the macronucleus can be shown to function in the actual
regenerative process. The role of the micronucleus in this connection is as yet
unclear ; apparently it is more important in the viability of some forms than in oth-
ers." On the whole, the evidence tends only to emphasize the importance of the
macronucleus and to attest to the validity of Calkins' comment (1930, p. 161) on
this organelle : "Far from being negligible it is on the contrary probably the most
important element of the cell in matters of metabolism, reorganization, and continued
cell life."
The tendency to underestimate the importance of the macronucleus in the life
of the organism results probably from its apparent monotony of structure. Lacking
chromosomes, its division is usually unspectacular. Nevertheless, its mode of origin
is not an incidental phenomenon in the life of the ciliate, for both macro- and micro-
nucleus almost invariably have a common and simultaneous origin. Usually they
develop from the synkaryon of the conjugant, by divisions which appear to be equa-
tional. Again, they develop from the synkaryon of autogamy or from the endo-
mictic micronucleus. Thus they inherit equally from a common nucleus of origin,
and each receives equivalent chromatin elements, chief among which are presumably
the genes. In the definitive micronucleus these elements retain the ability to or-
268 C. D. BEERS
ganize periodically as chromosomes, and thereby to arrest the attention of the ob-
server. Once in the definitive macronucleus, on the contrary, they never again
assemble in the form of chromosomes. However, it is possible that they are dis-
tributed at macronuclear division by a mechanism which is fully as effective as the
mitotic distribution of chromosomes, though less conspicuous. For example, it is
not impossible that they are represented in multiplicate in the macronucleus and
distributed at random throughout its substance. Thus each daughter at division
would be reasonably assured of receiving representatives of every type of chromatin
element. The behavior and the potencies of the macronuclear fragments of Sonne-
born's unusual specimens of Paranieciuin aurelia indicate a multiplicate representa-
tion of the chromatin elements. In these specimens the forty or more macronuclear
fragments grew and segregated during subsequent cell divisions, until there was only
one in each cell. Thus each fragment was adequate for the regeneration of a com-
plete macronucleus and for the continued life of the organism, even in the absence of
micronuclei. Hence, Sonneborn concludes that "the normal macronucleus must con-
tain at least forty complete and discrete genomes." A more precise mechanism for
the distribution of the chromatin elements, involving, for example, a differential
streaming of genetically equivalent elements toward opposite ends of a polarized
macronucleus, may be postulated. Regardless of the type of mechanism involved
in the distribution of the chromatin elements of the macronucleus at division, the
fact remains that inheritance in an amicronucleate ciliate is no less precise, to judge
by the structure and physiological activities of the offspring, than in a micronucleate
ciliate. The fact that the behavior of the macronucleus does not conform to the
chromosome theory of heredity in sensu strict o, in that chromosomes are absent,
may mean simply that a different mechanism for the distribution of the genes is
involved.
Whether the macronucleus of amicronucleate ciliates may justifiably be regarded
as an amphinucleus — one containing idiochromatin as well as trophochromatin, as
Woodruff (1921), Moore and others have suggested — seems doubtful in the light
of recent investigations. Thus it has been shown by Schwartz and by Bishop that
viable amicronucleate races of S tent or and Oxytricha may be derived by experi-
mental means from normal individuals in which the idiochromatin and trophochro-
matin were presumably segregated in the two types of nuclei. The macronucleus
of these individuals, following removal of the micronuclei, was adequate to maintain
all the usual vegetative activities in the derived amicronucleate races.
In conclusion, the evidence shows that the macronucleus is the essential nuclear
element in the vegetative life of ciliates. The micronucleus functions largely, if not
solely, in the periodic replacement of the macronucleus and in the production of
new genetic combinations, some of which undoubtedly render the species better
adapted to survival. The nature of the physiological conditions which call for a
renewal of the macronucleus is not clear; that such renewal meets an imperative
physiological need is shown by the widespread occurrence of the phenomenon in
the Euciliata.
SUMMARY
The number of micronuclei was examined in 50 trophic specimens and 50 resting
cysts of each of 20 clones of Tillina magna, three of which were amicronucleate.
MICRONUCLEI OF TILLINA MAGNA 269
In any particular clone trophic specimens and resting cysts contained approxi-
mately equivalent mean numbers of micronuclei. In different micronucleate clones
the mean number varied from 4.21 to 12.61. The mean for 1,700 specimens of 17
clones was 7.07.
The number in the individuals of any particular micronucleate clone was vari-
able ; some clones showed relatively little variation, e.g., 3 to 5 micronuclei ; others,
considerable variation, e.g., 2 to 11 micronuclei. The smallest number observed in
any micronucleate individual was 2; the largest, 16.
All the clones produced normal resting cysts upon depletion of the food supply
(Pseudomonas fluorescent} . The cysts of different clones were equally viable and
capable of excystment. Their size was unaffected by the number of micronuclei.
Amicronucleate cysts showed the usual macronuclear reorganization. Hence,
neither the number of micronuclei nor the absence of micronuclei affected encyst-
ment, viability and size of cysts, excystment or macronuclear reorganization.
An attempt was made to maintain each clone in pure-line culture for 60 days
and thereby to examine the division rate and vitality. Four clones were refractory
and encysted before 60 days expired. The remaining 16 clones, including the three
amicronucleate ones, survived with undiminished vigor and were discontinued. The
13 micronucleate clones produced from 149 to 176 generations during the 60-day
period; the three amicronucleate clones produced 154, 164, and 172 generations,
respectively. Hence, the 16 surviving clones showed slight differences in their
average daily division rates, but neither the divison rate nor the vitality of these
clones was correlated with variations in micronuclear number or with the absence
of micronuclei. Division cysts of amicronucleate clones showed the usual macro-
nuclear reorganization after each division of the macronucleus. The four refractory
clones had high micronuclear numbers.
Since conjugation is rare and endomixis and autogamy are unknown in Tillina,
it is probable that amicronucleate races arise at division by an unequal distribution
of the daughter micronuclei.
Some of the literature on amicronucleate ciliates and on the regeneration of vari-
ous types of nucleate merozoa is reviewed. The evidence shows that the macro-
nucleus is the indispensable nuclear element in the so-called vegetative life of the
organism, whereas the micronucleus during this period appears to be a relatively
passive organelle. Its chief function concerns the periodic replacement of the macro-
nucleus and the production of new hereditary combinations. Special attention is
directed to the fact that inheritance in an amicronucleate race is no less precise than
in a typical micronucleate race, although the division of the macronucleus is amitotic
and usually reveals no suggestion of true chromosomes. It is evident that the
hereditary mechanism of amicronucleate races, and perhaps of ciliates generally,
differs radically from the conventional chromosomal mechanism of metazoa.
LITERATURE CITED
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Biol, 15: 290-337.
BEERS, C. D., 1944. The maintenance of vitality in pure lines of the ciliate Tillina magna.
Amer. Nat., 78: 68-76.
BEERS, C. D., 1945. Some factors affecting excystment in the ciliate Tillina magna. Physiol.
ZooL, 18 : 80-99.
270 C. D. BEERS
BEERS, C. D., 1946. History of the nuclei of Tillina magna during division and encystment.
Jour. Morph., 78: 181-200.
BEERS, C. D., 1946a. Micronucleate and amicronucleate races of Tillina magna. Anat. Rec.,
94 (3) : 92-93.
BISHOP, E. L., JR., 1943. Studies on the cytology of the hypotrichous infusoria. I. The rela-
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BRESSLAU, E., 1922. tiber Protozoen aus Rasenaufgiissen. Verh. Deutschen Zool. Gesell,
27 : 88-90.
BURT, R. L., G. W. KIDDER, AND C. L. CLAFF, 1941. Nuclear reorganization in the family
Colpodidae. Jour. Morph., 69: 537-561.
CALKINS, G. N., 1930. Uroleptus halseyi Calkins. II. The origin and fate of the macronuclear
chromatin. Arch. f. Protistenk., 69: 151-174.
DAWSON, J. A., 1919. An experimental study of an amicronucleate Oxytricha. I. Study of
the normal animal, with an account of cannibalism. Jour. Exp. Zool., 29 : 473-513.
DAWSON, J. A., 1920. II. The formation of double animals or "twins." Ibid., 30: 129-157.
DEMBOWSKA, W. S., 1925. Studien iiber die Regeneration von Stylonychia mytilus. Arch. f.
• Mikr. Anat. u. Entwmech., 104: 185-209.
GREGORY, L. H., 1909. Observations on the life history of Tillina magna. Jour. Exp. Zool.,
6: 383-431.
GRUBER, A., 1879. Neue Infusorien. Zeit. f. Wiss. Zool, 33 : 439-466.
ILOWAISKY, S. A., 1921. Zwei neue Arten und Gattungen von Infusorien aus dem Wolgabassin.
Arb. d. Biol. Wolga-Station, 6: 95-106.
KAHL, A., 1931. Die Tierwelt Deutschlands, Part 21, 181-398. Jena: Verlag von Gustav
Fischer.
KIMBALL, R. F., 1941. Double animals and amicronucleate animals in Euplotes patella with
particular reference to their conjugation. Jour. Exp. Zool., 86: 1-33.
MAUPAS, E., 1888. Recherches experimentales sur la multiplication des infusoires cilies.
Arch, de Zool. Exp. et Gen., Ser. 2, 6 : 165-277.
MOODY, J. E., 1912. Observations on the life-history of two rare ciliates, Spathidium spathula
and Actinobolus radians. Jour. Morph., 23 : 349-407.
MOORE, E. L., 1924. Regeneration at various phases in the life-history of the infusorians
Spathidium spathula and Blepharisma undulans. Jour. Exp. Zool., 39: 249-316.
PATTEN, M. W., 1921. The life history of an amicronucleate race of Didinium nasutum. Proc.
Soc. Exp. Biol. and Med., 18 : 188-189.
PIEKARSKI, G., 1939. Cytologische Untersuchungen an einem normalen und einem Micro-
nucleus-losen Stamm von Colpoda steini Maupas. Arch. f. Protistenk., 92: 117-130.
POWERS, J. H., AND C. MITCHELL, 1910. A new species of Paramecium (P. multimicro-
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REYNOLDS, M. E. C., 1932. Regeneration in an amicronucleate infusorian. Jour. Exp. Zool.,
62: 327-361.
SCHWARTZ, V., 1935. Versuche iiber Regeneration und Kerndimorphismus bei Stentor coeruleus
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to macronuclear structure, amitosis and genetic determination. Anat. Rcc., 78 (Suppl.) :
53-54.
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Euplotes. Univ. Calif. Publ. Zool, 26: 131-144.
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MICRONUCLEI OF TILLINA MAGNA 271
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THE EFFECT OF LOW TEMPERATURE AND OF HYPOTONICITY
ON THE MORPHOLOGY OF THE CLEAVAGE FURROW
IN ARBACIA EGGS *
ALLAN SCOTT
Department of Biology, Union College, Schenectady, N. Y. and The Marine Biological
Laboratory, Woods Hole, Massachusetts
When Arbacia punctulata eggs are exposed to low temperature during the first
cleavage, a pronounced stalk develops between the daughter blastomeres. A stalk-
also develops at room temperature if the eggs are made to divide in hypotonic sea
water or in sea water lacking calcium ion. The development of a conspicuous
cleavage stalk is not a normal feature of the first cleavage in Arbacia, although it
does occur regularly in some cells ; for example, when fibroblasts divide. The
object of the work reported here was to examine the conditions under which the
stalk is formed in Arbacia and to relate these facts to current theories of the mech-
anism of cleavage. These particular experimental treatments were used because
they were found to affect the appearance of the cleavage stalk.
METHODS
Eggs of Arbacia punctulata in the first cleavage served as experimental material.
Ovulation was induced by the removal of the oral half of the test; eggs emerging
from the genital pores were collected in a dish of sea water. The eggs were al-
lowed to settle and the sea water was decanted after which fresh sea water was
added. Two such washings were carried out to minimize contamination by
coelomic fluid. Fertilization was effected by the use of diluted "dry" sperm, and
the sperm were never more than one hour old.
The fertilization membranes were removed by shaking. A heavy suspension
of eggs was placed in a five-inch test tube, one-half full of the suspension, and
shaken rapidly thirty times. Eggs so treated cleave in time with the controls.
The best time for treatment is at 2% minutes after fertilization, for if shaken earlier,
many exovates are formed, and if shaken later, many eggs retain the fertilization
membrane. The alternative method of removing the fertilization membrane by
treatment with the hatching enzyme (Ishida, 1936) was not attempted.
The hyaline layer was removed in a few experiments by washing the eggs in
calcium-free artificial sea water. This was accomplished by several decantations
and additions of the calcium-free mixture. It was found that the hyaline layer
regenerates somewhat if the eggs are returned to a solution possessing calcium
ions ; hence if eggs are to lack the hyaline layer, they must be allowed to cleave in
the calcium-free mixture.
This study was largely accomplished by photographic means. Photomicro-
1 This investigation was aided by a Grant-in-Aid from the Sigma Xi Alumni Research
Fund.
272
CLEAVAGE FURROW IN ARBACIA EGGS 273
graphs taken at intervals with Leica-Ibso apparatus, were projected as negatives
(1,000 X) and measurements made with dividers.
Temperature was maintained by means of a thermostatically controlled, water
jacketed, glass well, mounted on the microscope stage and connected through a
centrifugal pump to a water bath. By this means temperature could be maintained
within ± 0.2° C. at or about 20° and within ± 0.4° C. at or about 10° C.
Artificial sea water lacking calcium ions was compounded according to the
method of Shapiro (1941). This solution has an osmotic pressure and pH closely
similar to that of normal sea water.
The hypotonic solutions were prepared either by the dilution of normal sea
water or of the calcium-free mixture.
A few observations are presented on polyspermic eggs cleaving to three or to
four cells in one division. Polyspermic development was induced by the method
of Smith and Clowes (1924) which involves fertilization in pH 7.2 sea water and
the return of the eggs to the normal pH of 8.4 within two or three minutes.
RESULTS
Morphology of the cleavage furrow
The shape of the deepening furrow is markedly different under different con-
ditions; it is influenced by temperature, concentration of calcium ion, tonicity and
by presence or absence of the fertilization membrane.
Temperature
At temperatures between 20° C. and 30° C. there is normally no stalk in
cleaving eggs whose fertilization membrane has been removed (free cleavage).
The furrow is peaked at the apex (Figs. 1 and 2). At low temperatures, 6° to
12° C., a real stalk is formed during the latter part of the furrowing. This occurs
whether the egg is enclosed in the fertilization membrane or not. At these low
temperatures eggs undergoing membrane-free cleavage, come to resemble a dumb-
bell with a handle (Figs. 3 and 4).
Calcium ion or urea
Chambers (1938) described the short cleavage stalk which develops when Ar-
bacia punctulata eggs divide in calcium-free solutions at room temperature.
He used isotonic mixtures of sodium chloride and potassium chloride. In the pres-
ent study also a short stalk occurred when the eggs were exposed to calcium-free
sea water. Similarly a short stalk was figured by Moore (1930a and b) and by
Motomura (1934), after treatment with urea solutions.
Fertilization membrane
It is a common practice to remove the fertilization membrane either by dis-
solving it in urea solutions or by shaking an egg suspension rather violently. These
techniques allow the mitotic axis to become much longer and the furrowing is thus
more readily followed. If the eggs are confined in the fertilization membrane at
10° C., the blastomeres tend to stay apart and the walls of the furrow are almost
vertical (Figs. 11 and 12). At the end of the cleavage a stalk connects the two
blastomeres. If this same experiment is varied so that the eggs divide within their
274 ALLAN SCOTT
fertilization membranes at 10° C. and in calcium-free sea water, a cleavage stalk like-
wise develops. In this case, however, the stalk moves eccentrically until it is close
to the fertilization membrane (Figs. 30 through 34). The difference is presumably
due to the fact that the hyaline layer is dissolved in solutions lacking calcium ion
and when the hyaline layer is missing the egg is able to slide around inside the
fertilization membrane.
Membrane-free cleavage in polyspermic eggs
Polyspermic eggs may undergo free cleavage to form four cells in the first divi-
sion. In such cases they frequently divide so that a symmetrical figure is seen from
above. In this circumstance the four blastomeres each rest upon the bottom of the
glass container (Figs. 15, 16, 17, and 18). Frequently one blastomere rests upon
the other three at the end of the cleavage (Fig. 21). The former, more symmetrical
type of cleavage is more readily followed. When such an egg begins to cleave it
first flattens like a biscuit; at this stage it resembles a balloon around which two
rubber bands have been placed at right angles. Such a balloon is flattened on the
two surfaces where the rubber bands cross. Perhaps the egg, like the balloon, is
subject to greater elastic tension in the region where the incipient furrows cross,
and therefore flattens on these surfaces.
As seen from above, the egg periphery is roughly square, with corners rounded
(Fig. 15) ; the wide furrows (at 10° C.) gradually sink towards the center with the
apices of the furrows approaching one another. The upper and lower surfaces
meanwhile remain relatively flat although the two flat surfaces slowly come together.
The whole figure at the stage illustrated in Figure 16 resembles a balloon stretched
closely over four tennis balls with two rubber bands placed at right angles. Finally
a definitive stalk is formed (Fig. 18).
When the furrows first appear, the egg is to be considered as having two equa-
torial furrows; that is, two constricting rings (Fig. 29a), which cross each other.
The quasi-independence of the furrows is demonstrated by some eggs which cleave
in a similar way but in zvhich the furrows incise at different rates (Figs. 19 and 20).
In Figure 29b the furrow separating ab from cd is well in advance of the furrow
separating ad from be. This type of cleavage leads to a figure like Figure 20.
It appears that this curious type of cleavage is brought about by the develop-
ment of two new ring-like tensions which develop around the necks of the indi-
vidual blastomeres after the deeper furrow is well established. As a result of the
deep primary furrow, four new isthmuses are established about the necks of the
four incipient blastomeres (cf., Fig. 29b). Perhaps the most significant feature of
PLATE I
FIGURES 1 AND 2. Egg cleaving at 20° C. in sea water, fertilization membrane removed.
FIGURES 3 AND 4. Egg cleaving at 10° C. in sea water, fertilization membrane removed.
FIGURES 5 AND 6. Egg cleaving at 20° C. in 65 per cent sea water, fertilization membrane
removed.
FIGURE 7. Egg cleaving within the fertilization membrane at 20° C. in sea water.
FIGURE 8. Late cleavage at 20° C. in 65 per cent sea water, fertilization membrane removed.
FIGURES 9 AND 10. Polyspermic egg fertilized in sea water at pH 7.2, transferred to nor-
mal sea water at room temperature until cleavage began. Cleaving at 10° C. in sea water.
FIGURES 11 AND 12. Eggs cleaving within fertilization membrane at 10° C.
CLEAVAGE FURROW IN ARBACIA EGGS
275
I
276
ALLAN SCOT I
this type of cleavage is the bridge-like stalk which results (Figs. 23 and 24). In
these latter figures note that one circumferential furrow deepened symmetrically
and more rapidly than the other. The furrow which started later is very asym-
metrical, being much deeper on one side (cf., at the arrow) than the other. Egg^
cleaving to three cells show a similar behavior (Fig. 22) and when cleavage is com-
FIGURE 13.
in sea water.
Series showing late cleavage and development of the cleavage stalk at 10° C.
plete they may have a Y-shaped stalk, or if one furrow deepens more rapidly than
the others, two stalks may connect to one blastomere (Figs. 9 and 10).
The speed of furrowing in polyspermic eggs cleaving to four cells may be as rapid
as when two cells are being formed, yet it should be remembered that the amount
of new surface formed is much greater when a sphere divides into four equal smaller
spheres. The surface of a sphere divided into two spheres increases 26 per cent,
CLEAVAGE FURROW IN ARBACIA EGGS
277
f
l/V
FIGURE 14. Series showing late cleavage and continued activity of cleavage stalk, during
fourteen minutes in calcium-free sea water at 11° C.
ALLAN SCOTT
if divided to four spheres the surface increases 58 per cent. A polyspermic egg
cleaving to four cells forms about 26 per cent more surface than the normal first
cleavage but it may do so in the same amount of time.
fifiembrane-jree cleavage in hyputonic sea water and in hypotonic calcium-free sea
water
Dilution of the sea water causes a swelling of the egg ; it also causes an unusually
wide furrow to develop during the cleavage and leads to the formation of a stalk
at the end (Figs. 5. 6, and 8). This effect occurs at room temperature (20° C.).
The stalk may become very long if the sea water has been diluted sufficiently. In
mixtures of 65 parts sea water and 35 parts distilled water, for example, the stalk
may finally be 30 micra long. This effect occurs either in the presence or absence
of calcium ion. The stalk region is certainly a relatively rigid gel, for it has suffi-
cient rigidity to push the daughter blastomeres far apart. Figure 8 and Figures 40
through 42 show the process of elongation in these extreme cases. Enlarged photo-
graphs of the stalk at these stages are shown in Figures 43, 44, and 45 with dimen-
sions noted. In Figure 43 the stalk is only 4.4 micra in diameter at one point. In
Figure 44 its minimum width is about 2 micra and it is over 22 micra long. In
Figure 45 the constriction is completed. The stalk is still 5 micra wide at some
points, but it is less than 3 micra in diameter for a third of its length. Chambers
(ibid.) relates that a spherical oil drop lying within the egg in the furrow region is
not deformed until the "external surface of the advancing furrow is 4 to 5 \n from
the surface of the oil." If the egg pictured in Figure 43 has a cortex comparable
in thickness, then the stalk must certainly be all gel by the time its diameter is re-
duced to 7 or 8 fj.. One blastomere sometimes ruptures when eggs cleave in 65
per cent sea water. Xo endoplasm escapes if the stalk has closed. One such closed
stalk is shown in Figure 28; it is 5 micra in diameter. The conclusion that the stalk
is all gel (and yet it continues to constrict) is a most important conclusion for it
strongly supports the contraction theory of cleavage of W. H. Lewis. Close in-
spection at high magnification fails to show any movement of granules located in the
stalk. The active constriction of a 5 /A stalk is recorded in Figures 26 and 27.
PLATE II
FIGURES 15, 16, 17, AXI> 18. Cleavage of a dispermic egg, cleaving in calcium-free sea
water at 10° C. Egg fertilized in pH 7.2 sea water, transferred to sea water at room tem-
perature until beginning of cleavage. Time after fertilization: Figure 15, 72 min. ; Figure 16,
74 min.; Figure 17, 88 min.; Figure 18, 190 min.
FIGURES 19 AND 20. Egg snowing dispermic cleavage. Treatment as in Figures 15-18.
Time after fertilization: Figure 19, 86 min.; Figure 20, 88 min.
FIGURE 21. Dispermic egg. Treatment as in Figures 15-18. One blastomere out of the
horizontal plane.
FIGURE 22. Diagram illustrating two types of cleavage to three cells.
FIGURES 23 AND 24. Egg in 70 per cent sea water at 25° C., after accidental polyspermy.
Time after fertilization: Figure 23, 50 min.; Figure 24, 52 min.
FIGURE 25. Dispermic egg cleaving in sea water at 11° C., following fertilization in pH
7.2 sea water.
FIGURES 26 AND 27. Late cleavage of egg in 65 per cent sea water. Room temperature.
Time after fertilization : Figure 26, 83 min. ; Figure 27, 85 min.
FIGURE 28. Closed stalk following rupture of one blastomere ; 65 per cent calcium-free
sea water.
FIGURE 29. See text.
CLEAVAGE FURROW IN ARBACIA EGGS
279
DIAM. if
b
29
PLATE II
280 ALLAN SCOTT
The stalk
The mitotic axis (greatest length) of eggs undergoing free cleavage becomes
progressively longer at 10° than at 20° C. (compare Figs. 1 and 2 with 3 and 4) ;
moreover the early furrow at 10° C. is much more blunt in contour than is the
furrow of eggs at higher temperatures. A study of the final phase of cleavage under
high power (Fig. 13) shows how the wide furrow is transformed into a stalk.
In Figure 13a the deepening furrow is still blunt with a diameter of about 14
micra. In Figure 13/>, however, the stalk is beginning to square off. The arrows
(Figs. 13d and r) indicate the region where the constriction is most active. The
details are similar and are very clear in eggs cleaving in calcium-free sea water at
10° C. The series of diagrams shown in Figure 14, a to /. again show that the
broad furrow first deepens until the diameter of the waist is about 7 or 8 micra (a
and b), then the stalk is elongated by the constriction of the subequatorial cortex
(c and d, see arrows) ; meanwhile the entire stalk is diminishing in diameter. The
minimum diameter of the stalk is about 4 micra at 10° C. and in calcium-free sea
water ; in hypotonic solutions the diameter is often less. When the diameter of the
stalk diminishes below 4 micra, it does so in local areas only (cf., Fig. 14<y, h, /).
Amoeboid activity and cleavage activity
Many workers have noted that the polar surface of the cell bubbles actively dur-
ing cytokinesis (Bowen, 1920 — in Eucliistits spcnnatocytcs; and Lewis 1942 — in
tissue culture fibroblasts). This is not the case with the egg of the sea urchin dur-
ing the first cleavage, instead the polar surface remains smooth and inactive. How-
ever, a variety of agents, will cause the formation of blebs in the sub-furrow region.
One such agent is hypotonic calcium-free sea water. The blebs usually begin to
form after the completion of the major furrowing and they give rise to sizable
spherules which are cut off by a process very much like cleavage (Figs. 46a, b, c}.
The inactivity of the polar surface may indicate that the cortex there is different
from the equatorial cortex in Arbacia."
Eggs that have been in the hypotonic medium for some time may show a sud-
den rush of endoplasm from one blastomere to the other, often causing the blasto-
meres to become very unequal in size (Fig. 47 and Figs. 35 to 39). 3 This endo-
plasmic flow is a very rapid one, usually lasting only two or three seconds. It is
remarkable, however, that the flow is accompanied toy a rapid deepening of the fur-
row, appearing as though a tension has been suddenly overcome, allowing the fur-
row to constrict much more rapidly than usual. The sub-cortical flow in such eggs
may be down one side of the furrow, through the constricted stalk and up the other
side of the furrow, yet the furrowing continues normally to completion (Fig. 47).
- The view that there is a special substance (a special type of plasmagel) located around
the equator has been espoused by a number of workers. Marsland (1942) and Lewis (1942)
among others. Beams and King (1937) are of the opinion that they have removed the "surface
active material" of Ascaris eggs by centrifugation at 150,000 X gravity.
3 The rush of endoplasm described is in this case related to cleavage. It resembles the
amoeboid activity described by Moser (1940) after urea treatment. Moser, p. 77, cites other
cases from the literature.
CLEAVAGE FURROW IN ARBACIA EGGS
281
lit
HO
PLATE III
FIGURES 30, 31. 32, 33, AND 34. Eccentrically placed cleavage stalk. Eggs in fertilization
membrane at 10° C, in calcium-free sea water.
FIGURES 35, 36, 37, 38, AND 39. Volume changes of individual blastomeres. Calcium-free
sea water.
FIGURES 40, 41, AND 42. Elongation of the cleavage stalk; in 65 per cent sea water at
room temperature. Time intervals: Figures 40-41, 1 min. and 40 sec.; Figures 41-42, 1 min.
and 35 sec.
FIGURES 43, 44, AND 45. Enlargements of Figures 40, 41, and 42. The edges of the nearly
transparent stalk have been, retouched in Figures 4, 8, 26, 27, 28, 43, 44, and 45.
282
ALLAN SCOTT
DISCUSSION
The stalk
The occurrence of a stalk during the cleavage of the Arhacia egg is correlated
.with the degree of gelation of the furrow cortex. Both the observations made in
this paper and those of other workers who have concerned themselves with the de-
gree of gelation of the egg cortex confirm this. The results of several workers are
summarized below :
Brown (1934) : Cortical pigment granules are especially resistant to displace-
ment by centrifugation during the division phase.
Chambers (1938) : The furrow cortex resists disintegration after the two in-
cipient blastomeres have been punctured at the poles.
FIGURE 46
Brown and Marsland (1936) : There is a quantitative decrease in the gel value
of dividing eggs as the hydrostatic pressure is increased. Under high pressures the
furrow regresses.
No one has yet recorded the effect of temperature, hypotonicity and lack of
calcium ion upon the cortex of the dividing Arbacia egg, although these observations
have been made upon the unfertilized egg. A brief summary of this work follows:
Costello (1938) : It takes progressively longer to fragment the eggs as the tem-
perature is lowered.
Cole (1932) and Harvey (1943) : It takes longer to fragment eggs in hypotonic
than in isotonic solutions.
Harvey (1945) : Arbacia eggs break less readily in solutions possessing calcium
ions than in solutions lacking calcium ions.
These treatments (low temperature, hypotonicity and calcium ion) are pre-
cisely the ones which favor the development of a cleavage stalk. It is possible that
CLEAVAGE FURROW IN ARBACIA EGGS
283
these treatments may increase the elastic strength of the egg surface by toughening
the extra cortical structures, but it is probable that they favor cortical gelation as
well.
Hypotheses concerning the mechanism of cleavage; surface tension
Chambers and Kopac (1937) found that clean oil drops of the proper inter-
facial tension with sea water, will coalesce spontaneously with a naked egg (Arbacia,
Lytechinus, and Echinometra). They state: "The tendency to coalescence in the
furrow and polar zones of cleaving eggs (late amphiaster and later) was investi-
gated and no difference was found." They used oils whose approximate tensions
in contact with sea water were 30, 10, and 3 dynes per cm. The fact that coalescence
occurs at all indicates a fluid layer around the egg periphery. Spontaneous coales-
FlGURE 47
cence does not occur in Amoeba protcus (Kopac and Chambers, 1937), which in-
dicates a non-fluid surface. In view of these observations any surface tension
hypothesis is untenable.
Subcortical currents
Chambers (1938) has hypothesized that cleavage results from the activity of
"the sub-cortical currents (which) sweep around the two asters and add gelating
material to the inwardly growing cortex." In this hypothesis he combines his own
observations with those of Schechtman (1937) on localized cortical growth during
the cleavage of the amphibian egg. It was shown in the present paper (page 280)
that normal furrowing may be associated with abnormal currents, which argues
against the importance of such currents for division; moreover Lewis (1942) found
no currents in the dividing fibroblast.
284 ALLAN SCOTT
Astral cleavage
An astral theory of cleavage, much modified from Gray (1931), has been elabor-
ated by Katsuma Dan (1943). He believes that the asters are composed of radiate
fibers with intrinsic rigidity ; he considers them to be anchored to the cortex ; he
believes that the rays cross at the equator ; and he believes that the spindle elongates
autonomously. The following quotation (Dan 1943) summarizes his theory of
cytokinesis : ". . . it was also shown that this concept of the mechanism of cell
division is adequate to explain the stretching phase of the furrow surface. That is,
when two such radiate asters are pushed apart, they can in turn, push the cell mem-
brane of the polar region somewhat as a paint brush would push some object. As
they travel away, howrever, since they enclose the fluid endoplasm within the inter-
spaces of their rays, the fluid endoplasm is carried away from the equatorial
region and the cortex there is sucked in, giving rise to a furrow. The cortex is
stretched as it is pulled in by the suction."
The strength of Dan's hypothesis lies in its ability to explain the differential
stretching and shrinkage of the surface which he and his coworkers observed (Dan,
Yanagita, and Sugiyama, 1937; Dan and Yanagita, 1938; Dan, 1943) and for which
no other explanation has been forthcoming. It appears that Dan's hypothesis will
explain such unusual cleavages as are pictured in Figures 9 and 10 of the present
paper. It could be assumed that one element of the tripolar spindle elongated be-
fore the others causing the asters to move apart, and by the suction mechanism, caus-
ing the development of the initial furrow (Fig. 9 at a). On this assumption the de-
velopment of the other furrows (Figs. 9b and c ) begins later, presumably because
the other two spindles begin their elongation later. The secondary furrow (Fig. 10
at a') is presumably caused by the suction resulting from the separation of the lower
two asters. Similar explanations would doubtless serve for the tetra-astral cleav-
ages shown in Figures 23 and 24 of the present study. One can imagine also that
the crossing rays from all four asters, if they became attached to the cortex, would
explain the flattening of the upper and lower surfaces of the egg observed in Figure
15.
Dan's hypothesis is not in accord with the observations presented here concerning
the continued elongation of the cleavage stalk in hypotonic sea water for it is impos-
sible to see how the astral suction mechanism could explain the further constriction
of a long, completely gelated stalk.
The main weakness of the astral suction hypothesis lies in its limited scope. It
fails to explain undoubted cases of anastral cleavage (tissue culture, for example)
frequently noted in the literature. Dan's easy conclusion that all of these anastral
cases are explainable by his astral suction hypothesis (". . . it is possible to imagine
that in cells of the anastral type, similar gelation systems may be existing although
they cannot be discerned morphologically") is unconvincing.
One of Wilson's observations is discordant with Dan's hypothesis. Wilson ob-
served, in a form which normally has asters, that a spindle need not be present for
complete cleavage to occur. He found that a cleavage furrow may cut in around
the base of an isolated aster and result in a complete cleavage. Compare Wilson
(1901), page 376 and Figure 11.
In one of Chamber's microdissection experiments he bisected the partially cleaved
egg in a plane at 45° to the plane of the furrow (1924, Fig. 36). The cut resulted
CLEAVAGE FURROW IN ARBACIA EGGS
immediately in two cells. However, the original furrow remained on each artificially
produced blastomere and, on each, the furrow gradually cut through forming two
small "cells" as well as two large ones. This continued cleavage seems to be quite
unexplainable by Dan's hypothesis which requires crossed astral rays, an elongat-
ing spindle and a suction produced by the separation of the asters.
Cortical grozt'th or cortical contraction?
Schechtman has proposed another theory of the mechanism of cytokinesis. He
suggested (1937) that the furrow cortex grows by the "intussusception of clear
cytoplasm," but simple growth of the equatorial cortex would not be expected to cut
the egg in half. Other factors must account for the inwardly directed furrow and
its narrowing. It seems clear that there is a stretching of the egg cortex at the time
of furrowing as concluded by Dan et al. (1937, 1938), by Schechtman (1937) and
by Motomura (1940), but whether the stretching is active (the result of growth)
or whether it is passive and due rather to a contracting ring at the head of the fur-
row (Lewis, 1942), is not easy to decide. Schechtman is of the opinion that
"Cleavage is initiated by a contraction of the egg cortex at the site of the future
furrow." And he notes that the "'Cortex becomes thicker and bulges toward the
egg interior." He therefore uses both contraction and cortical growth in his com-
plete hypothesis. The observations made in this paper on the continued constric-
tion of small stalks after they consist entirely of gelated material are taken as strongly
favoring the constricting ring theory of Lewis. For if the gelated stalk is able to
contract at that late stage of cleavage, it seems reasonable to suppose that it possesses
contractile power earlier. The direction of contraction is ringwise about the equa-
tor (Fig. 29c) and it is to be expected that such contraction would draw stained
areas out into fine lines as Schechtman observed, if such areas are located in the fur-
row or subfurrow region.
It would be illuminating to know whether or not kaolin particles placed around
the equator would be brought closer together during the furrowing but no one has
made these observations.
Amoeboid activity and bleb formation
One can scarcely observe the amoeboid behavior of eggs in hypotonic media and
particularly the "normal" false cleavages which occur during the amoeboid phase
preceding pronuclear fusion in the nematode egg (Spek, 1918), without being con-
vinced that a fundamental similarity exists between amoeboid motion and cleavage.
Moreover the abscission of blebs is strikingly similar to cleavage.4 It is suggested
that any deforming force which establishes an isthmus about the cell or a part of
the cell will result in the development of a contracting ring disposed around the
isthmus, provided that the egg is in the cleavage phase. This view would explain
why the normal egg, deformed by the elongating spindle, cleaves at the equator. It
would explain why cleavage planes cut in around the base of cytasters which are
unconnected to a spindle (Wilson, 1901) and it would explain why blebs formed in
4 Very recently Holtfreuter (1946) has suggested "that in normal cytoplasmic division the
activity of the nucleus and of the endoplasm are of a mere secondary importance." He observed
that isolated, embryonic amphibian cells may develop annular constrictions which lead to the
fragmentation of the cell. He considers, however, that the contraction occurs in the membrane
rather than in the plasmagel layer.
286 ALLAN SCOTT
the sub-furrow region may cut off from the remainder of the egg as reported above.
This hypothesis also agrees with the idea that the enlarging gelated asters play a
mechanical role in localizing the furrow.
SUMMARY
1. Under certain conditions the eggs of Arbacia punctulata develop a cleavage
stalk between the first two hlastomeres. No stalk forms in sea water if the tempera-
ture is in the 20° C. to 30° C. range; low temperature (10° C.) causes the develop-
ment of a stalk in sea wrater; a short stalk develops in isotonic calcium-free sea
water at 20° C. ; a very long stalk develops if eggs are cleaving in hypotonic sea
water (65 per cent).
2. The effect of the above treatments on the appearance of cleaving dispermic
eggs is described.
3. Evidence indicates that stalks of 8 micra diameter are all gel, yet in hypotonic
sea water they continue to constrict and elongate. This is good evidence that the
cortical gel has inherent contractile properties.
4. It is hypothesized that any event which deforms the Arbacia egg (if it is in the
"cleavage phase") leads in some way to an orientation of contraction around the
isthmus. The deforming force may be an enlarging aster, an elongating spindle, or
an endoplasmic flow.
LITERATURE CITED
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BROWN, D. S., 1934. The pressure coefficient of "viscosity" in eggs of Arbacia punctiilata.
Jour. Cell. Comp. Physiol., 5 : 335-346.
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pressure. Jour. Cell. Comp. Physiol., 8: 159-165.
CHAMBERS, R., 1924. The physical structure of protoplasm as determined by micro-dissection
and injection. General cytology, pp. 237-309. The University of Chicago Press.
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I. Protopl., 28 : 66-81.
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when centrifuged in hypo- and hyper-tonic sea water. Biol. Bull., 85: 141-150.
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CLEAVAGE FURROW IN ARBACIA EGGS 287
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the sea urchin Strongylocentrotus purpuratus. Protopl., 9: 9-17.
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DEVELOPMENTAL RELATIONS BETWEEN GENITAL DUCTS
AND GONADS IN DROSOPHILA
DIETRICH BODENSTEIN
Medical Division, Edge-wood Arsenal, Maryland
While studying the reproductive system of Drosophila sinmlans gynandromorphs
Dobzhansky (1931) made the following interesting observation: If female genital
ducts and testes were present in the same individual and if the female ducts were
attached to the testes, the latter underwent extreme degeneration. Yet, the attach-
ment of male genital ducts to ovaries did not affect the development of these organs.
Now we know that in normal development the attachment of the ducts to the
gonads takes place during the early period of pupal life. We know further that by
transplantation of gonads from one individual to another it is possible to obtain at-
tachment of the transplanted organ to the host ducts. This knowledge makes it
possible to attack experimentally the question whether the degeneration of testes
when attached to the female ducts, as observed in Drosophila sinmlans gynandro-
morphs, is a peculiarity of this special case, or whether the phenomenon is a general
one and always occurs when female ducts and testes are brought into contact with
each other.
The problem can be approached experimentally in two ways : the larval testes
can be transplanted into female host larvae or the female genital disc from which
the ducts originate can be transplanted into male larvae. By transplanting two or
three testes into one host, the chance that one transplantal will attach itself to the
host duct is quite good. The chances for attachment of the testes to the female duct
are even better when the six oviducts which arise by outgrowths from the three
transplanted imaginal discs compete with the two host ducts for attachment. In
the following studies both these methods were used.
EXPERIMENTAL
Transplantation of testes into female ducts
Two or three testes of mature virilis larvae were transplanted together into the
abdominal cavity of female host larvae of the same age. After the hosts had
emerged, the condition of the transplants and their relationship to the female genital
system was studied in careful dissections. This series consisted of ten cases. The
following was found. All transplants had failed to assume their characteristic spiral
shape. This was to be expected, since the work of Dobzhansky (1931) and Stern
(1941a and b) had shown that the testes have to be attached to the vas in order to
accomplish their spiral growth. In six out of ten cases one of the transplanted
testes had attached itself to one of the oviducts of the hosts. The attached testis was
always greatly reduced in size and appeared degenerate. Figure 1 shows camera
lucida drawings of five representative cases of this series. It will be noted that the
degenerative reduction occurs only when the testis is attached to the oviduct of
288
DEVELOPMENT IN DROSOPHILA
289
the host (Fig. I A, B, 1) and E). Testes that lie free in the body cavity (Fig. IA
and B) or testes that have been attached to the ovary (Fig. 1C and B) are un-
affected. Thus the principle which produces degeneration is apparently given off
only by the oviducts and depends for its action on a close cellular contact with the
testes. This principle, moreover, seems unable to penetrate larger cell barriers, for
testes which were connected to ovaries which in turn had their normal oviduct con-
nection remained unaffected (Fig. 1C). Yet two testes which had established close
FIGURE 1. The spatial and developmental relations of transplanted testes to the reproductive
system of their female hosts. O, ovary ; OD, oviduct : 7\ to Tx, transplanted testes.
contact with each other had both suffered degenerative reduction, although only one
of these fused organs has actually established contact with the oviduct (Fig. IA
and E).
Transplantation of female genital discs into male hosts
In a second series of experiments, two or three female genital discs from mature
virilis larvae were transplanted together into the body cavity of hosts of the same
age. The condition of the host testes and their relationship to the transplanted
female structures was again studied by dissection. Several of the affected testes
290
DIETRICH BODENSTFIN
were also sectioned and studied histologically. Thirty successful cases were avail-
able for investigation. In seven of these cases, the host testes were not connected
to the transplanted ducts, although the latter had developed well and were found
in the immediate neighborhood of the male gonads. The testes of these seven hosts
were normal in size, shape, and histology. One testis in each additional animal was
not connected with the vas of the host, nor to any of the transplanted ducts. These
testes were not coiled but were otherwise normal. The other testis in two of these
individuals was connected to the vas of the host and was normal, while the other
testis of the third individual, although connected to the vas, was not coiled. The
non-coiling of an attached testis is rare, but has been observed at times in otherwise
normal animals. Whether the inabilitv of attached testes to coil is a result of faultv
*• *
connections with the vas or whether the vas in these cases has lost its growth in-
ducing capacity is not known.
TABI.K I
Transplantation of female genital ducts into mule hosts
Number of
discs trans-
planted
Testis free (round)
Testis (spiral).
Normal attached
Testis attached
to 9 duct
State of degeneration of testis
when attached to 9 duct
One side
Both sides
One side
Both sides
One side
Both sides
One side
Other side
3
Yes*
Yes
+
3
Yes
Yes
+ + + +
3
Yes
Yes
+ + + +
3
Yes
Yes
+ + + +
3
Yes
3
Yes
3
Yes
Yes
+ + + +
2
Yes
Yes
+ + +
2
Yes
Yes
+ + + +
2
Yc-s
+ + + + +
+ + + + +
2
Yes
Yes
+
3
Yes
Yes
+ +
3
.
Yes
3
Yes
Yes
+ + + +
3
Yes
Yes
+ + + + +.+
3
Yes
Yes
+ + + + +
2
Yes
2
Yes
Yes
+ + +
2
Yes
2
Yes
2
Yes
2
Yes
2
Yes
2
Yes
+ + + + +
+ + + + +
2
Yes
Yes
+ + + +
2
Yes
+ + +
+ + + + +
2 h.
Yes
2 h.
Yes
Yes
+ + + +
2 h.
Yes
Yes
+ + + +
2 hv.
Yes
Yes
+ + + +
h. = hydei discs into hydei hosts,
vas, but not spiral.
hv. = hydei discs into virilis host. * = testis attached to
DEVELOPMENT IN DROSOPHILA
291
IT-
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DIETRICH BODENSTEIN
In twenty individuals one or both totes were connected to the oviduct of the
transplant. In some cases one testis was even found to have connection with the
oviducts of two discs. All testes that were attached to oviducts showed a more or
less pronounced degree of reduction and appeared degenerate. Table I summarizes
'the results of this experimental series. The state of degenerative reduction of the
testes in this table is indicated by crosses. One cross signifies slight ; six crosses,
extreme size reduction. Figure 2 shows camera lucida drawings of four representa-
tive cases of this series. Figure 2A is a case listed in Table I having one cross.
Figure 2C is listed by having six crosses and Figure 2D and B are listed by having
five crosses each. Figure 3 illustrates by microphotography an extremely reduced
testis.
>
t
B
FIGURE 3. A, normal adult testes. B, (arrow) testis of the same individual degenerated under
the influence of a transplanted and attached oviduct.
It will be noted from Table I that the reduction of the testes is in most cases
very pronounced. Only six of the testes attached to oviducts were reduced to state
"3" or less, while sixteen testes were degenerated to state "4" or more.
This experimental series thus confirms strikingly the original observation that
testes attached to female oviducts suffer degenerative changes and that it is the
oviduct that elicits the principle causing degeneration. The observation from the
previous experiments that the presence of oviducts f>cr sc has no effect on testis
development is also confirmed, for testes in the presence of as many as three pairs
of oviducts in the immediate organic environment remained normal if they were not
attached to the oviduct. In comparing the attached testes of the two series with
each other, a difference in their general shape was noted. While the transplanted
DEVELOPMENT IN DROSOPHILA
293
testes in the first experimental group were small, roundish bodies, the testes in the
second experimental group were in most cases thin and elongated in shape (com-
pare Fig. 1 with Fig. 2). Now it was found in the second group that in all cases
when the attached testis was thin and elongated it was attached not only to the trans-
planted oviduct but also to the vas efferens of its host (Fig. 2). In those cases,
however, where the testis attached only to the transplanted oviduct, had not estab-
lished connection with the vas, it was roundish. This situation is well illustrated
in Figure 2C. The left testis in this case, a small roundish degenerated organ, is
attached only to the oviduct while the right testis. which is attached to the vas ef-
ferens of the host and to one transplanted oviduct, has become an irregularly elon-
gated structure. The elongated shape of such a degenerate testis is thus due to the
stimulating influence of the vas on the growth of the testis, which in normal develop-
ment leads to the coiling of this organ, while the observed degeneration is caused
by the influence of the oviduct.
FH.IUE 4. Sections through two extremely reduced testes. </, degenerating cells.
Not only virilis but also hydei oviducts cause degeneration of hydei testes at-
tached to them. This was shown by three cases in which larval hydei female discs
were transplanted into hydei male larvae (see Table I).
The factor in the oviduct causing degeneration of the testis by contact is not
species specific, for hydei oviducts will cause virilis testes to degenerate (see Ta-
ble I).
Sections of reduced testes were made and their histology studied. It was found
that, depending upon the degree of reduction of size, the testes contained various
amounts of spermatogonia and spermatocytes in all stages of degeneration. The
remnants of disintegrated cells in the form of granular picnotic masses together with
quite normal appearing cells were observed. Figure 4 shows the condition found
in an extremely reduced testis.
CONCLUSION AND SUMMARY
By transplanting female genital discs into male hosts, attachment to the host
testes of oviducts developed from transplanted genital discs is obtained. In these
cases the attached testes suffer extensive degeneration. Only cellular contact of
294 DIETRICH BODENSTEIN
the oviducts to the testes brings about this phenomenon. Unattached female ducts
do not affect the development of the testis. The principle causing degeneration is
not species specific. The findings indicate that the phenomenon encountered is no
unique instance, but representative when oviduct and testis establish cellular contact
during pupal development.
LITERATURE CITED
BODENSTEIN, D., 1946. The post-embryonic development of Drosophila. In Biology of Dro-
sophila. Pub!. Carnegie lust. IT ash. (in press).
DOBZHANSKY, T., 1931. Interaction between female and male parts in gynandromorphs of
Drosophila simulans. Roux' Arch. Entzv. Mech., 123: 719-746.
STERN, C, 1941a. The growth of testes in Drosophila. I. The relation between vas deferens
and testis within various species. Jour. E.\-p. ZooL, 87: 113-158.
STERN, C., 1941b. The growth of testes in Drosophila. II. The nature of interspecific-
differences. Jour. Exp. ZooL, 87: 159-180.
cV1
r«^Xo*s *;
£ ( L I B ? ( A R V
J*4S'
A HISTOLOGICAL STUDY OF SYNDISYRINX FRANCISCANUS.
GEN. ET SP. NOV., AN ENDOPARASITIC RHABDOCOEL
OF THE SEA URCHIN, STRONGYLOCENTROTUS
FRANCISCANUS 1
H. E. LEHMAN
Department of Zoology of the I'nhrrsity of Xorth Carolina -
I NTRODUCTION
Up to the present time eight genera of worms endoparasitic in echinoderms and
sipunculids have been described that belong to the rhabdocoel family Umagillidae
Wahl, 19101). Schneider described the first species, Anoplodinm parasita, in 1858.
Since then six questionable and three valid species of this genus have been re-
ported from widely separated localities as parasites of holothurians. Their distribu-
tion extends from the Mediterranean, Ionian, and North Seas to Japan and the
Philippines (Bock, 1926). Syndesinis ccliinoniin Francois, 1886, the only spe-
cies of the genus, is found in echinoids. It has been collected in the Mediterranean
(Russo, 1895), Norway (Westblad. 1926), and the English Channel (Braun. 1889).
Three species of the genus C'olUishuna are found in sipunculids at Roscoff (Dorler,
1900), the Gulf of Kola (Beklemischev, 1916), and the Bay of Naples (Wahl,
1910a). The genus Dcsiuotc is represented by one species, D. vora.v, discovered in
a crinoid collected in the Gulf of Kola (Beklemischev, 1916). A single species
parasitic in holothurians has been described in each of four genera, i.e.. a Japanese
form, Xcnoiuctra arbor a Ozaki, 1932, and three reported from the'coa.st of Nor-
way, Wahlia tiiacrostylijera Westblad. 1930, Anoplodlcra valuta \Yesthlad. 1930.
and type genus Uiiiagilla forskalensis Wahl, 1909.
The only reference to a member of the Umagillidae from the Western Hemis-
phere was made by Powers in 1936. He reported the presence of a Syndesmis-
like worm in the coelomic cavity of the echinoid, Ccntrccliinns antillantin. at Tortu-
gas. A complete description was not given; however, as compared with Syndesinis.
noticeable differences were observed in details of the copulatory apparatus and the
arrangement of the shell glands. While the endoparasitic rhabdocoel of Sirongylo-
centrotiis jranciscanns. the large common sea urchin of the California coast, is well
known to some investigators who have worked at Pacific Grove, a description of
tli is worm has not been recorded in the literature prior to the present account.
1 This work was done at the Wilson Zoological Laboratory of the University of North
Carolina in partial fulfillment of the requirements for the degree of Master of Arts. The
author is indebted to Professor D. P. Costello for suggesting the problem, for the slide prep-
arations upon which this study was essentially based, and for the invaluable suggestions and
criticisms rendered during the preparation of this paper. The author wishes to acknowledge his
appreciation to Dr. L. H. Hyman for many valuable recommendations and for permission to
introduce her revised and hitherto unpublished terminology relating to this group. To Miss
Catherine Henley the .author expresses his gratitude' for the translation of a number of the
references cited herein.
- Now in the School of Biological Sciences of Stanford University.
295
296 H. E. LEHMAN
Systematic position :;
Order Rhalxlocoela
Suborder Lecithophora
Section Dalyellioida
Family Umagillidae Wahl, 1910
Subfamily Umagillinae Wahl. 1910
Genus Syndisyrinx. gen. nov.
Genotype Syndisyrinx franciscanus, sp. nov.
Holotypc. A whole mount in the United States National Museum, Washington
D. C.
Repositories oj type material. In each of the following repositories a whole
mount, a transversely sectioned, and a sagittally sectioned preparation selected from
the type material have been deposited : U. S. National Museum, Washington. D. C. ;
American Museum of Natural History, New York City ; British Museum. London ;
California Academy of Science, San Francisco ; Wilson Zoological Laboratory of
the University of North Carolina ; and Museum of Natural History, Stanford Uni-
versity. Additional preserved material may be obtained from the author or from any
of these institutions.
Type locality. Mussel Point. Monterey Peninsula. California, Lat. 36°. 37',
20" N., Long. 121°, 54', 15" W.
Collectors. D. P. Costello, 1937 and H. E. Lehman, 1945.
Distinguishing characteristics. Umagillinae with a single intestine, paired and
branched ovaries, cuticular penis, and a bursa seminalis connected by cuticular ducts
to the seminal receptacle and bursal canal.
MATERIALS AND METHODS
Fifty-four rhabdocoel parasites were obtained from two specimens of the sea
urchin Strongylocentrotus franciscanus (A. Agassiz) by Dr. D. P. Costello in Au-
gust 1937 at Pacific Grove, California. These specimens were fixed in Heath's,
Boveri's, Lillie's and Worcester's solutions. Five of the individuals were sectioned
serially at 10/i and stained with Heidenhain's iron hematoxylin and orange G.
One of these preparations was exceptionally fine and the majority of the accompany-
ing figures \vere made from it. Unfortunately this preparation, which the author
intended to designate as the holotype, was lost when a microscope was stolen.
This material, including the slide preparations, was turned over to me by Dr. Cos-
tello. The morphological study was based on this material.
In the summer of 1945 during June, July, and August, the author collected sev-
eral hundred additional specimens from the same locality. Over sixty urchins were
examined and all were found to be infested ; frequently three dozen or more parasites
were obtained from the intestine of a single host. These worms were fixed in
Heath's and Beauchamp's solutions. Seventy were sectioned serially at 10 ,u and
stained with Mayer's acid hemalum and triosin. Thirty whole mounts stained
with paracarmine were also made. The type material was selected from these prep-
3 Classification according to Bresslau (1933), with the exception of "Family Anoplodiidae
Graff, 1913," which has been rejected in favor of "Family Umagillidae Wahl, 1910b," inasmuch
as no reason is given by Graff for discarding the older name or for selecting Anoplodium as
type genus. The subfamily Umagillinae has been retained as designated by Wahl, 1910b.
A PARASITIC RHABDOCOEL 297
parations. At this time another parasite of Str. jranciscanus was discovered which
differed from Syndlsyrin.v in shape, manner of locomotion, and color. A description
of this worm is being prepared and preliminary examination of sectioned material
indicates a close relationship to Syndesmis cchinorum. Upon the suggestion of
Prof. A. R. Moore, who had occasionally observed parasitic worms in Str. purpur-
atus (Stimpson), forty-seven of these urchins were examined. In twenty-nine of
them, worms that are very similar to, and may be identical with Syndisyrin.r fran-
ciscanits were present in small numbers.
GENERAL MORPHOLOGY
The living animals are bright red with a dark brown or yellow median longi-
tudinal line which marks the extent of the intestine. The worms are flattened
dorsoventrally and have a leaf-like appearance, being rounded at the anterior end
and slightly pointed posterad. Individuals vary in size from 2 to 3 mm. long and
1.6 to 2.5 mm. wide. The body is thickest at approximately one-fourth of the dis-
tance from the anterior end and at this level measures about 0.5 mm. in the dorso-
ventral axis. Laterally and posteriorly the thickness of the body diminishes gradu-
ally to about 0.2 mm. at the periphery. A ciliated epithelium covers the entire
surface ; rhabdites and cuticle are lacking.
The mouth is situated on the ventral surface about one-fourth of the distance
from the anterior end and a common genital pore opens ventrally at the posterior
extremity of the body. The musculature and parenchyma are typical of other
Umagillidae. No excretory system was observed. The strongly muscular pharynx
is typically doliiform and possesses pharyngeal glands ; it communicates by a short
oesophagus with the gut. The intestine, possessing a number of small lateral diver-
ticula, extends posterad under the dorsal epidermis along the mid-line and termi-
nates one-quarter of the distance from the posterior end of the body. The gut con-
tains no permanent lumen and food masses lie in temporary cavities surrounded by
large digestive cells. The brain, composed of two cerebral ganglia connected by a
wide commissure, lies anterior to the pharynx and gives off paired anterior, lateral,
and posterior nerves.
Lobed testes lie lateral to the mid-line in the anterior half of the body. Acces-
sory glands empty into the sperm duct that arises from each testis and passes
anteracl. These paired tubes unite mesially and enter a small spermiducal vesicle
that is continued posterad as a muscular common sperm duct which lies dorsal to
the uterus along the mid-line. This tube terminates in an elongated cuticular stylet,
the penis, which is enlarged and funnel-like at the base. The penis stylet enclosed
in the male antrum extends through the posterior third of the body to the common
genital antrum and over most of its length does not exceed 3 p. in diameter.
Paired vitellaria are found immediately posterior to the testes ; they are greatly
ramified and fill most of the ventrolateral spaces in the middle third of the body.
Posterior to the vitellaria a pair of ovaries is located, one on each side of the
mid-line. Laterally each branches into five or more finger-like lobes. Three or
four collecting ducts from the vitellaria empty with the ovaries and seminal recep-
tacle into the anterior end of the ovovitelline duct. The seminal receptacle is oval
and filled with sperm. Located posterodorsad to this organ is a vesicular, sperm-
filled bursa seminalis connected to the seminal receptacle by a fine cuticular insemi-
298
H. E. LEHMAN
ph.g.
.5
bur. c.
FIGURE 1. Semidiagrammatic median sagittal section.
A PARASITIC RHABDOCOEL 299
nation canal. Arising in close association with this tubule is a similar duct, the
cuticular proximal part of the bursal canal that passes posterad from the bursa
seminalis approximately 60 p, before widening into the posterior muscular portion
of the bursal canal (vagina). A cuticular sheath surrounds the openings of these
two ducts into the bursa seminalis. The composite structure, consisting of this
sheath and the canals passing through it, makes up the bursal valve.
An ovovitelline duct, into which accessory glands empty, arises ventrally at the
anterior end of the seminal receptacle. It passes posterad and unites with the female
antrum. The uterus, lying close to the ventral epidermis, extends anteriorly from
the female antrum almost to the pharynx. At the anterior end of the uterus an egg
capsule containing from one to five ova and numerous yolk cells is generally found.
The capsule is continued posterad as a long coiled whip similar to those found in
related forms. Most of the ventrolateral spaces of the posterior third of the body
are filled by cement glands ; they communicate by many small ducts with the female
antrum. The common genital antrum is an elongated cavity at the posterior end
of the body into which the female antrum enters ventrally, the male antrum and penis
open mesially and the bursal canal is given off dorsally. At its posterior end is the
common genital pore which opens ventrally to the exterior.
HlSTOLOGICAL STRUCTURE
Epidermis
A ciliated epithelium covers both dorsal and ventral surfaces of the body. No
pigment or special gland cells were observed in this layer and a cuticle and rhabdites
are lacking. The cytoplasm of the cells in the epidermal layer is granular and cell
boundaries, though faintly stained, are distinct. The cells covering the dorsal sur-
face are cuboidal and measure 10 /A from basement membrane to external surface.
The cytoplasm of these cells stains moderately with hematoxylin. On the ventral
surface the cells are flattened and are about 7 /A thick and from 12 to 35 ^ wide;
they have little affinity for hematoxylin. Cilia of the ventral epidermis are about
6.5 p. long and are almost twice the length of those found on the dorsal surface.
Cells possessing the staining properties and short cilia characteristic of the dorsal
layer extend for a short distance ventrally around the lateral edges. A zone 4 to 6
cells wide of intermediate nature accomplishes the transition between typical dorsal
and ventral epithelium.
Musculature and parenchyma
The arrangement of the musculature is essentially the same as that described for
other Umagillidae. Under the basement membrane of the surface epithelium is
Abbreviations for Figures 1 and 2.
a.o.d. — accessory glands of ovovitelline duct, a.s.d. — accessory glands of sperm duct, br. —
brain, b.c. — buccal cavity, bur. c. — bursal canal, bur. c'. — cuticular end of bursal canal, b.s. —
bursa seminalis, b.v. — bursal valve, e.g. — cement glands, c.s.d. — common sperm duct, e.c. — egg
capsule, f.a. — female antrum, g.a. — common genital antrum, g.p.— genital pore, int. — intestine,
i.e. — insemination canal, 1. int. — lumen of intestine, m.a. — male antrum, oe. — oesophagus, ov.—
ovary, ov'. — ovum, o.d. — ovovitelline duct, p. — penis, p'. — base of penis, ph. — pharynx, ph. g.
— pharyngeal glands, s.d. — sperm duct, s.r. — seminal receptacle, s.v. — spermiducal vesicle, te.—
testis, u. — uterus, vit. — vitellaria, vit. d. — vitelline ducts, w. — whip of egg capsule, y. — yolk cells.
300
H. E. LEHMAN
c.s.d.
a.s.d.
vit. d.
(7. O. d.
g.a
m
-9-P- 2
FIGURE 2. Semidiagrammatic median frontal section, intestine omitted.
A PARASITIC RHABDOCOEL 301
found a thin layer of suhepidermal muscles (Figs. 3-5, 7, 8). The superficial mus-
cles are circular ; these overlie a longitudinal sheet, and interposed at intervals be-
tween these layers are well-developed oblique fibers. In addition to these, bundles
of fibers attached to the internal organs or the basement membrane of the epidermis
pass dorsoventrally through the parenchyma (Figs. 1-3). The special muscles of
the reproductive and digestive systems will be described in connection with the
organs with which they are associated.
A parenchyma, composed of large, irregularly shaped cells with coarsely granular
or vacuolated cytoplasm, fills most of the spaces between the internal organs and
epidermis. A histologically distinct parenchymatous mass of cells enclosed in a
fibrous capsule extends posterad along the mid-ventral line from the posterior level
of the pharynx to the region in which the female antrum enters the common genital
antrum. The flattened, nonvacuolated cells of this tissue possess finely granular
cytoplasm and are arranged in concentric layers around the reproductive ducts, most
of which pass through the mid-ventral parenchyma (Figs. 3-5, 7). Nowhere within
the parenchyma were flame cells or collecting ducts of an excretory system observed.
Nervous system
The brain is similar in all respects to those described in other members of the
family. It is located just anterior to the pharynx and consists of two ganglia con-
nected bv a wide commissure. Around the central fibrous mass of the brain are
j
numerous ganglionic cells that stain quite evenly with hematoxylin. Poorly devel-
oped anterior, lateral and posterior pairs of nerves leave the brain and can be traced
for short distances into the parenchyma. No theca separates the brain or nerves
from the parenchyma and no special sensory organs were found.
Digestive system
The mouth lies on the ventral surface about one-fourth of the distance from the
anterior end of the body. It opens into a very small buccal cavity lined by flattened
ciliated cells that are continuous externally with the ventral epithelium (Figs. 1, 8).
A sphincter underlying the epithelium regulates the size of the oral opening. Lying
immediately dorsal to the mouth and opening into the buccal cavity is the doliiform
pharynx which has the appearance of a dorsally compressed sphere. Its dorso-
ventral axis is about 0.1 mm. long and its greatest diameter is about 0.17 mm.
Passing dorsoventrally through the pharynx is a funnel-shaped lumen that is nar-
rowest at the oral or ventral end. The musculature of the pharynx is similar in
most details of its organization to that found in Syndcsuiis as described by Russo
(1895). A thin superficial layer of vertical fibers overlies the well-defined muscles
encircling the lumen of the pharynx. In addition to the circular and vertical mus-
cles, radial fibers pass from the lumen to the peripheral surface of the pharynx.
Nonmuscular cells with heavily staining reticular cytoplasm fill the spaces between
the radial fibers (Fig. 8). Surrounding the pharynx is a sharply defined basement
membrane to which are attached numerous short, radially arranged, protractor mus-
cles that extend to the basement membrane of the ventral epidermis. The more
oblique of these fibers serve also as dilators of the pharynx. Poorly developed re-
tractors are attached to the equator of the pharynx and pass to the dorsal surface.
Pharyngeal glands are present encircling the dorsal end of the pharynx. The
302 H. E. LEHMAN
peripheral contours of these glands are lobular and a thin basement membrane sepa-
rates them from the parenchyma. The cells which make up these glands have in-
distinct cell boundaries and dense cytoplasm containing numerous granules that
stain darkly with hematoxylin. Cytoplasmic continuations of the cells extend ven-
trally and line the lumen of the pharynx (Fig. 8). Leading dorsad from the
pharynx is a short oesophagus which passes through the pharyngeal glands and
opens into the anterior end of the intestine.
The intestine lies along the mid-line under the dorsal epidermis and extends pos-
terad from the level of the brain to about one-fourth of the distance from the pos-
terior end of the body (Fig. 1). The width of the gut varies from 0.1 to 0.2 mm.
at the anterior end and diminishes gradually posteriorly. Short diverticula ex-
tend laterally on each side. The epithelium of the intestine is made up of large ir-
regularly shaped cells containing moderately granular cytoplasm. The basal end
of most cells reaches the fibromuscular investing sheath of the intestine that sepa-
rates it from the parenchyma. The lumen of the intestine can only be observed when
ingested material is present ; this condition is similar to that found in some alloeo-
coels. In an animal that has been feeding, food masses often lie in cavities that have
lost all direct communication with the oesophagus (Fig. 1). Food vacuoles of
varying sizes are generally present in the cells surrounding the ingested material
and digestive cells were occasionally observed that had apparently migrated into
the food masses by amoeboid movement.
Male reproductive system
The paired testes lie lateral to the mid-line in the anterior half of the body.
They are approximately 0.5 mm. long and from 0.3 to 0.5 mm. wide.. Each is made
up of four to six vesicular lobes, the lumina of which are in direct communication
with one another (Fig. 2). Separating the testes from the parenchyma is a fibrous
sheath that pentrates and partially subdivides the lobes. The chambers so formed
are filled with developing germ cells and tangled masses of mature spermatozoa
(Fig. 3). Mature sperm are present in all lobes but are more numerous midway
between the anterior and posterior ends of the testes near the wide openings of the
sperm ducts. These ducts run mesially from the testes and enter the mid-ventral
parenchyma, whereupon they diminish to about 10 //, in diameter and generally con-
tinue their course anterad, dorsolateral to the uterus (Figs. 1-3). A thin epithelium
surrounded by loose fibromuscular elements makes up the walls of the sperm ducts.
Near the origin of these ducts from the testes, glandular cells that probably possess
some accessory function are found in the mid-ventral parenchyma adjacent to the
ventral walls of the tubes (Fig. 2).
At varying distances posterior to the pharynx the sperm ducts unite mesially
and enter the anterior end of a common sperm duct which at this point is somewhat
enlarged to form a small spermiducal vesicle (Figs. 1-3). The slightly coiled com-
mon sperm duct continues posterad from the vesicle through the mid-ventral paren-
chyma. It gradually diminishes in diameter from 45 ^ to 12 (j.. Its walls are com-
posed of connective tissue cells surrounded by a sheath of circular, oblique, and
longitudinal muscle fibers. The lumen of the tube is lined by a thin squamous
epithelium that is separated from the theca by a thick basement membrane. Pos-
teriorly, the common sperm duct unites with the enlarged base of the penis at
A PARASITIC RHABDOCOEL 303
about one-third of the distance from the posterior end of the body (Figs. 1, 2, 4).
The penis lies in a muscular sheath, the male antrum, which is a diverticulum of the
genital antrum. Histologically this sheath is similar in most details of its structure
to the common sperm duct ; however, the lining epithelium of the male antrum is
thicker, in some regions almost occluding the lumen, and a thick basement mem-
brane is lacking (Fig. 5). The copulatory organ is a cuticular tubule that extends
through the posterior third of the body and is about 3 ^ in thickness over most of
its length. The lumen of the stylet does not exceed 2 /* in diameter except at the
anterior end of the penis which is enlarged to 12 p, at its union with the posterior end
of the common sperm duct (Figs. 1—3). The rim of the funnel-like base of the
penis is thickened to form a collar ; longitudinal muscles in the walls of the male
antrum and common sperm duct attach to this collar and function as protractors
and retractors of the penis.
Female reproductive system
The paired ovaries lie in the posterior third of the body. Each is made up of
from five to ten lobes that branch dichotomously from common trunks arising
near the anterior end of the seminal receptacle. The lobes of the ovaries are di-
rected posterolaterad and are separated from the parenchyma by a very poorly de-
veloped theca. The branches are made up of dovetailed chains or rouleaux of com-
pressed ova that are proliferated from primordial cells at the distal ends of the lobes
(Fig. 2). Mature ova are approximately 75 /A in diameter and vary in thickness
from 20 to 60 \n. The cytoplasm of immature eggs is at first homogeneous, 'but as
development continues many small peripherally distributed granules appear that
are probably stored nutrient materials. During the period of growth the nuclei of
the ova increase from 7 to 25 p. in diameter and the chromatin granules gradually
lose their affinity for basic dyes. In mature ova only the spherical or oval nu-
cleolus stains deeply with hematoxylin (Fig. 4).
A pair of greatly branched vitellaria lie anterior to the ovaries and fill most of
the ventrolateral spaces in the middle third of the body (Figs. 1, 2). Many of the
dorsoventral muscles of the parenchyma contribute fibers to the diffuse sheath that
encloses these ducts. Primordial cells at the distal ends of the branches give rise to
yolk cells. As the cells increase in size, the cytoplasm which at first is homogeneous,
becomes filled with refractile granules that coalesce to form amber-colored droplets
(Figs. 3, 4). From each side three or four collecting ducts packed with mature
yolk cells pass posterad from the vitellaria and unite near the mid-line shortly be-
fore emptying into the anterior end of the ovovitelline duct (Fig. 2).
The seminal receptacle is somewhat oval and lies ventral to the intestine within
the sheath that surrounds the gut. Its anterior extremity is about one-third of the
distance from the posterior end of the body. The posterior part of this organ is
thin walled and masses of mature spermatozoa are observable in its extensive lumen.
Anteriorly the seminal receptacle opens with the paired ducts of the vitellaria and
ovaries into the ovovitelline duct which arises ventrally in this region (Figs. 1, 2).
The wall of the anterior third of the seminal receptacle is lined by large gland-like
cells that restrict the lumen to a narrow channel 6 to 10 /A wide which connects the
posterior vesicular portion to the ovovitelline duct (Fig. 4).
The bursa seminalis lies dorsal to the vesicular portion of the seminal receptacle.
It is enclosed in the same sheath that surrounds the seminal receptacle and the pos-
304 H. E. LEHMAN
terior end of the intestine (Figs. 1, 2). The large lumen of the bursa seminalis is
lined by an epithelial layer very similar to that lining the posterior part of the seminal
receptacle. In every specimen examined spermatozoa were found in the bursa ;
frequently they were aggregated into roughly spindle-shaped masses in which degener-
ating sperm were observable (Figs. 5, 6). Arising ventrally, or in some cases later-
ally, from the wall of the posterior half of the bursa seminalis is the insemination
canal, a fine cuticular tubule about 4 ^ in diameter connecting the lumina of the
bursa seminalis and seminal receptacle. In close association with the insemination
canal, a second cuticular tube of the same dimensions arises from the wall of the
bursa seminalis and connects the bursa posteriorly to the bursal canal (Figs. 1, 2,
5, 6). Surrounding the ends of the ducts as they penetrate the lining epithelium of
the bursa is a cuticular sheath, 7 /JL in diameter and 10 /x long. The inner end of
this sheath is involuted and fused to the ends of the two ducts (Fig. 6). To desig-
nate this composite cuticular structure made up of the insemination canal, the proxi-
mal end of the bursal canal and the sheath surrounding the ends of these ducts, the
term, "bursal valve," is suggested.
The bursal canal (vagina) is a tubular structure about 0.1 mm. long and 20 ^
in diameter that arises as an anterodorsal continuation of the common genital an-
trum. Its wall is composed of an inner epithelial layer surrounded by a strong
fibromuscular sheath. At the posterior end of the canal the epithelium possesses
cilia-like projections characteristic of the lining of the common genital antrum.
Anteriorly the lumen of the canal is reduced and the thin basement membrane un-
derlying the epithelium becomes continuous with the cuticular wall of the tubule
leading into the bursa seminalis.
A flattened muscular ovovitelline duct (ductus communis) arises ventrally near
the anterior end of the seminal receptacle and receives the ducts of the ovaries and
vitellaria. It passes posterad through the mid-ventral parenchyma to about the level
of the posterior end of the bursa seminalis and here enters the anterior end of the
female antrum (Figs. 1, 2, 5). The ovovitelline duct is approximately 35 ^ wide
but is capable of considerable expansion to allow ova and yolk cells to pass into the
uterus. Circular, oblique and longitudinal muscles are observable in contact with
the thin basement membrane that underlies the lining epithelium ; no fibrous sheath
separates this duct from the cells of the mid-ventral parenchyma. Running parallel
to the ovovitelline duct in the lateral parenchyma are paired accessory glands which
enter the posterior part of the duct prior to its union with the female antrum (Figs.
1, 2, 5). Generally the cytoplasm of these gland cells stains evenly; however, in
some cases the cells were observed to be filled with eosinophil granules.
The uterus arises ventrally from the anterior end of the female antrum. It ex-
tends anterad almost to the pharynx through the mid-ventral parenchyma and
FIGURE 3. Transverse section through egg capsule and spermiducal vesicle (X350).
FIGURE 4. Transverse section through entrance of ovary into seminal receptacle (X500).
Abbreviations for Figures 3 and 4.
a.o.d. — accessory glands of ovovitelline duct, e.g. — cement glands, e.c. — egg capsule, int.—
intestine, mu. — muscle sheath, mu'. — subepidermal muscles, mu".— dorsoventral muscles of
parenchyma, ov. — ovary, ov'. — ovum, p'. — base of penis, pa. — mid-ventral parenchyma, s.d.—
sperm duct, s.r. — seminal receptacle, s.v. — spermiducal vesicle, te. — testis, u. — uterus, \v. — whip
of egg capsule, y. — yolk cells.
A PARASITIC RHABDOCOEL
305
•.?s -ii/**' 4 -'.. . - ••
FIGURES 3-4.
306 H. E. LEHMAN
through its entire course lies very close to the ventral surface of the body (Figs. 1,
2, 4). The anterior end of the uterus is enlarged and encloses an amber-colored,
oval egg capsule containing numerous yolk cells and from one to five spherical eggs
.(Figs. 1, 2, 3). The egg capsule is cuticular and possesses a whip-like prolongation
that extends posterad through the entire length of the uterus and female antrum.
Over most of its length the whip is about 10 /A thick. In the middle portion of the
uterus the whip is often coiled back upon itself a number of times so that its total
length may greatly exceed that of the uterus (Figs. 1, 2). The uterine wall is very
similar in structure to the ovovitelline duct and is able to enlarge greatly to accom-
modate the egg capsule and the folded part of the egg whip (Figs. 3, 4).
The female antrum extends from the posterior ends of the uterus and ovovitelline
duct to the common genital antrum (Figs. 1, 2). The walls are lined by columnar
epithelial cells surrounded by a thin basement membrane and a muscular layer that
is continuous with the fibers enclosing the uterus and ovovitelline duct. The lu-
men is about 12 /A in diameter and the posterior end of the egg whip, when present,
almost completely fills this space (Figs. 5, 7). The ventrolateral spaces of the pos-
terior third of the body contain numerous unicellular cement glands. The cyto-
plasm of these cells is generally uniformly filled with small granules that have a strong
affinity for hematoxylin. Throughout the entire length of the female antrum many
ducts from these glands enter the lateral walls (Figs. 1, 2, 5, 7). The secretions of
the cement glands are believed to be associated with the attachment of the egg cap-
sules to the substrate when expelled. Living animals compressed under a cover
glass were occasionally observed at low magnification to undergo a series of rapid
contractions which resulted in the extrusion of the egg capsule and whip. How-
ever, nothing is known about the normal deposition and attachment of the capsules,
nor are other details of the life cycle understood.
The common genital antrum lies at the posterior end of the body. It is an
elongated tube lined by flattened cells that appear to have cilia about 20 // long which
extend into the lumen (Figs. 1, 2, 7). A diffuse fibrous sheath separates this or-
gan from the parenchyma. The common genital antrum receives the terminal
ducts of both male and female reproductive systems : the bursal canal arises from it
as a dorsal diverticulum ; the male antrum enclosing the penis stylet is given off as
a mesial evagination ; and the female antrum enters it ventrally. The common geni-
tal pore opens on the ventral surface at the posterior end of the body. At this point
FIGURE 5. Transverse section through bursa seminalis and bursal valve (X350).
FIGURE 6. Bursal valve (X 1,050).
FIGURE 7. Transverse section through the entrance of female antrum and male antrum
into the common genital antrum (X 350).
FIGURE 8. Transverse section through pharynx (X 200).
Abbreviations for Figures 5 through 8.
a.o.d. — accessory glands of ovovitelline duct, a.o.d'. — ducts of accessory glands of ovovitel-
line duct, b.c. — buccal cavity, bur. c. — bursal canal, bur. c'. — cuticular end of bursal canal, b.s.—
bursa seminalis, b.v. — bursal valve, cil. — cilia, e.g. — cement glands, d.c.g. — ducts of cement
glands, f.a.— female antrum, g.a. — common genital antrum, int. — intestine, i.e. — insemination
canal, 1. int. — lumen of intestine, m.a.— male antrum, mu. — muscle sheath, mil'. — subepidermal
muscles, mu". — pharyngeal protractor muscles, n. — nerves, oe. — oesophagus, o.d. — ovovitelline
duct, pa. — mid-ventral parenchyma, p. — penis, ph. — pharynx, ph. g. — pharyngeal glands, s. — sper-
matozoa, s'. — degenerating spermatozoa, u. — uterus, w. — whip of egg capsule.
A PARASITIC RHABDOCOEL
307
• CIS&-V
r 1 ^ ' 7 '-• A v •',
J. . .. > • : .-'•-,! ' : ,', :
6
"
7
FIGURES 5-8.
308 H. E. LEHMAN
the ciliated ventral epithelium is invaginated and forms a short bulb-like canal which
meets an outpocketing of the common genital antrum. Sphincters encircle both ends
of this canal and regulate the size of the pore.
DISCUSSION
Comparison of genera
Although the parasite described here is similar in many respects to all genera in
the family Umagillidae, there are certain structural characteristics that do not cor-
respond to those of any previously reported genus of this family. Therefore, it is
considered necessary to establish a new genus to be designated by the name Syndi-
syrinx. This name is intended to describe the complex bursal valve which is not
present in any other genus of the family. The specific name, Syn. franciscanus, is
given to designate the host, Strongylocentrotus franciscanus, in which it was first
found.
For the sake of uniformity in the following comparison of genera of the family
Umagillidae, the morphological nomenclature used by the various authors in their
original descriptions of genera and species has been altered to conform with the
terminology employed in the preceding analysis of Syndisyrinx.
In addition to the fact that both Syndisyrinx and Syndcsmis are found in the in-
testine of echinoids, the morphological characteristics of Syndisyrinx indicate a
closer relationship to Syndcsmis than to the other genera of the family. The loca-
tion and appearance of important organs, viz., muscular pharynx, lobed testes, small
spermiducal vesicle, muscular common sperm duct, ramified vitellaria, dichotomously
branched ovaries, elongated uterus and egg capsule with whip, are very similar in
Syndcsmis and Syndisyrinx and strongly suggest a close relationship between these
two genera. Syndisyrinx differs from Syndcsmis chiefly in the structure and re-
lationships of the bursa seminalis and seminal receptacle. In Syndcsmis a single
vesicle is present for the reception of sperm and cuticular structures such as the
parts which make up the bursal valve of Syndisyrin.\- are lacking. In addition to
these differences, the stucture of -the penis is markedly dissimilar in these twro forms.
The penis of Syndisyrinx is a cuticular hollow stylet attached only at the base,
whereas the copulatory organ of Syndcsmis is a muscular eversible tube with a cu-
ticular lining (Russo, 1895; Fig. 16).
Structures corresponding to the cuticular canals in the bursal valve of Syndi-
syrinx are found in Anoplodicra valuta, Wahlia macrostylifcra, and Dcsmote vorax.
In A. valuta the relationships of the two cuticular canals to the bursa seminalis, as
described by Westblad (1930), are very similar to the arrangement of these struc-
tures in Syndisyrinx. However, the cuticular sheath that surrounds the entrance
of these ducts into the bursa is lacking in A. valuta. There do not appear to be
grounds for concluding that Syndisyrinx and Anoplodiera are closely related since
the appearance and location of the testes and vitellaria, the presence of a single ovary,
and the absence of a female antrum connecting the ovovitelline duct and uterus to
the common genital antrum in A. valuta differ strikingly from the arrangement
found in Syndisyrinx,
In W. macrostylifcra, described by Westblad (1930), and D. vorax, according
to Beklemischev (1916), the proximal end of the bursal canal is cuticular but an in-
semination canal is lacking. In other respects W. macrostylifcra differs from
A PARASITIC RHABDOCOEL 309
Syndisyrinx chiefly in regard to the morphology of the male reproductive system.
The penis stylet is greatly elongated, and paired sperm ducts arising from compact
testes unite and communicate by means of a single duct with the large spermiducal
vesicle situated anterior to the pharynx. Many points of difference are likewise
found by comparing the morphology of Syndisyrinx and Dcsmotc. The most
evident of these are the bipartite gut and the presence of two genital pores, the an-
terior pore by which the uterus opens to the exterior and the posterior pore which
serves for copulation in D. vorax.
The other genera of the family lack cuticular parts in the copulatory complex
comparable to those in the bursal valve of Syndisyrinx and to a greater or less de-
gree exhibit dissimilarities in the location, distribution, number, arrangement and
relationships of organs in the body. In these genera the most conspicuous differ-
ences with respect to Syndisyrinx are : the single ovary and absence of a cuticular
copulatory stylet in the genus Anoplodiuin ; the unbranched ovaries and double-
walled cuticular penis stylet in the genus Umagilla; the absence of a cuticular penis
and the general arrangement of testes and vitellaria in the genus Xcnoinctra; and
the single testis in the genus Collastoma. A manuscript is in preparation which will
deal at greater length with the structural relationships of these forms.
Bursal valve
There is a superficial similarity between the bursal valve of Syndisyrinx and the
cuticular nozzle-like mouthpieces of acoels. In the acoel, Amphichoerus, described
by Graff (1891), and many allied forms, one end of the mouthpiece is generally con-
nected to a vesicular sac or bursa filled with sperm ; the other end is directed toward
the ovary. L. H. Hyman (1937) points out that the function of these mouthpieces
is apparently to direct sperm toward the ova to help insure fertilization. This func-
tion can hardly be ascribed to the insemination canals of Anoplodiera and Syndi-
syrinx which conduct sperm from the bursa seminalis to the seminal receptacle and
not directly to the ova; nor does it seem probable that the insemination canals of
Umagillidae are homologous to these mouthpieces. Noncuticular ducts connect the
bursa seminalis to the seminal receptacle and bursal canal in most genera of Umagil-
lidae, which suggests that cuticular structures are probably of relatively recent
rather than primitive origin. In an analysis of the existing genera, Wahl (1910b)
presents evidence which leads him to conclude that Umagilla is the most primitive
and least modified genus of the family. If one accepts this view, it lends support
to the opinion expressed above, inasmuch as Umagilla lacks any cuticular structures
that might be considered homologous to the bursal valve. It is possible that the
absence of cuticular parts in some of the species is due to a greater degree of simpli-
fication associated with a parasitic existence. However, there is no direct evidence
for this supposition, since in the most closely related free-living families, Grarfillidae
and Dalyelliidae, cuticular structures such as these are not found. This suggests
that these tubules have arisen independently, and until additional information is
available, the insemination canals and bursal canals of Umagillidae should not be
considered as mouthpieces in a true sense.
Although copulation has not been observed in Syndisyrinx, it is believed that the
sperm of one animal are injected by means of the protrusible penis into the bursal
canal of another. Before fertilization can take place, sperm must migrate from the
310 H. E. LEHMAN
bursal canal through its narrow proximal end into the bursa seminalis, there re-
maining until able to find their way through the insemination canal into the seminal
receptacle. Evidently many sperm are unable to accomplish this migration and de-
generate in the lumen of the bursa seminalis. Sperm that do reach the seminal re-
ceptacle must then pass through the constricted anterior part of this organ to fer-
tilize the mature ova that enter the ovovitelline duct at the anterior end of the
seminal receptacle.
It is difficult to explain any selective advantage for the presence of the fine canals
that make up the bursal valve of Syndisyrinx. It was thought at first to be a mech-
anism for the prevention of polyspermy. However, this explanation is negated by
the presence of large masses of spermatozoa in the seminal receptacle. The simplest
explanation for the presence of these ducts is that they act as valves which regulate
the number of spermatozoa entering the bursa seminalis and seminal receptacle. If
this interpretation is correct, it is probable that the function of the bursal valve is
to insure a necessary aging of the sperm in the bursa before fertilization. The cu-
ticular walls are necessary to prevent the collapse of these narrow tubes. It is
evident that the bursal valve restricts the free passage of sperm from the bursa sem-
inalis and therefore as the result of a single copulation, a continuous supply of
sperm may be maintained over a long period of time.
SUMMARY
After completing a histological study of an endoparasitic rhabdocoel from the
Pacific Coast sea urchin, Strongylocentrotus jranciscanus, the following conclusions
have been reached :
1. This parasite belongs to the rhabdocoel family Umagillidae but differs in cer-
tain characteristics from the eight known genera of the family.
2. The distinguishing characteristics are a single intestine, paired and lobed
ovaries and testes, a tubular single-walled ctiticular penis stylet, and cuticular ducts
connecting the bursa seminalis to the bursal canal and seminal receptacle.
3. A characteristic structure typical of this parasite and not present in other
genera of the family is the bursal valve composed of two cuticular tubes, the in-
semination canal and proximal end of the bursal canal, which enter the bursa sem-
inalis through a cuticular cup-like sheath.
4. The parasite here described is given the name Syndisyrinx frandscanus, gen.
et sp. nov.
LITERATURE CITED
BEKLEMISCHEV, W., 1916. Sur les Turbellaries parasites de la cote Mourmanne, II. Rhabdo-
coela. Trav. Soc. Imp. Nat. Petrograd, Zool. et Physiol., Sect. 4, 45: 1-59 (Resume,
60-79).
BOCK, S., 1926. Anoplodium stichopi, ein neuer Parasit von der Westkiiste Skandinaviens.
Zool. Bidrag, Uppsala, 10: 1-30.
BRAUN, M., 1889. Uber parasitische Strudehvurmer in Rostok. Ccntralbl. Bakt. Parasit.,
Abt. 1,5: 41-44.
BRESSLAU, E., 1933. Turbellaria. Kiikenthal und Kntmbach, Handbnch der Zoologic, 2:
264-269.
DORLER, A., 1900. Neue und wenig bekannte rhabdocole Turbellarien. Zcitschr. zviss. Zool.,
68 : 1-42.
A PARASITIC RHABDOCOEL 311
FRANCOIS, P. H., 1886. Stir le Syndesmis, nouveau type de Turbellaries decrit par, W. A.
Silliman. C. R. Acad. Sci. Paris, 103 : 752-754.
GRAFF, L. v., 1891. Die Organisation der Turbellaria acocla, Leipzig (Engelmann), p. 73.
GRAFF, L. v., 1913. Turbellaria, II. Rhabdocoelida. Das Tierreich, Berlin (F. E. Schulze),
35: 152-163.
HYMAN, L. H., 1937. Reproductive system and copulation in Amphiscolops langerhansi (Tur-
bellaria acoela). Biol. Bull., 72: 319-326.
OZAKI, Y., 1932. On a new genus of parasitic Turbellaria, Xenometra, and a new species of
Anoplodium. Jour. Sci. Hiroshima Univ. (Series B, Zoo/.), 1: 81-89.
POWERS, P. B. A., 1936. Studies on the ciliates of sea urchins. A general survey of the
infestations occurring in Tortugas echinoids. Pap. Tortugas Lab. Carnegie hist.
Washington, 29: 319-320.
Russo, A., 1895. Sulla morfologia del Syndesmis echinorum Francois. Ricerche Lab. Anat.,
Roma, Fasc. 1,5: 43-68.
SCHNEIDER, A., 1858. Uber einige Parasiten der Holothuria tubulosa. I. Anoplodium parasita.
Miillcr's Arch. f. Anat. Phys. und u-iss. Mcd., Berlin: 324-329.
WAUL, B., 1909. Untersuchungen liber den Bau der parasitischen Turbellarien aus der Familie
der Dalyelliiden (Vorticiden). II. Teil, Die Genera Umagilla und Syndesmis. Wien,
Sitz.-Bcr. kais. Akad. u'iss. Math.-nat., Abt. 1, 118: 943-965.
WAHL, B., 1910a. Untersuchungen iiber den Bau der parasitischen Turbellarien aus der
Familie der Dalyelliiden (Vorticiden). III. Teil, Das Genus Collastoma. Wien,
Sitz.-Bcr. kais. Akad. iviss. Math.-nat., Abt. 1, 119: 363-391.
WAHL, B., 1910b. Beitrage zur Kenntnis der Dalyelliiden und Umagilliden. Fcstschr. f. R.
Hertivig, Jena (G. Fischer), 2: 41-60.
WESTBLAD, E., 1926. Das Protonephridium der parasitischen Turbellarien. Zoo!. Anz., 67 :
323-333.
WESTBLAD, E., 1930. Anoplodiera voluta und Wahlia macrostylifera, zwei neue parasitische
Turbellarien aus Stichopus tremulus. Zcitschr. f. Morph. u. Okol. Tierc, 19 : 397-426.
A QUANTITATIVE STUDY OF THE RELATIONSHIP BETWEEN
THE ACTIVITY AND OXYGEN CONSUMPTION OF THE
GOLDFISH, AND ITS APPLICATION TO THE MEAS-
UREMENT OF RESPIRATORY METABOLISM
IN FISHES
W. A. SPOOR
Department of Zoology, University of Cincinnati
INTRODUCTION
The fact that fish consume more oxygen when active than when quiescent has
been observed by many investigators (Krogh, 1916; Bowen, 1932; Clausen, 1933,
1936; Wells, 1935; Schlaifer, 1938; Smith and Matthews, 1942), but apparently
no attempt has been made to determine the exact relationship between oxygen con-
sumption and activity in fishes. It is the purpose of this paper to present data
which are believed to provide an objective and quantitative basis for the relationship
between activity and oxygen consumption in the goldfish, and to describe a method
for making the necessary measurements. The method is based on the use of a re-
cording activity detector (Spoor, 1941) combined with a continuous flow system for
measuring oxygen consumption.
The lack of definite information on the activity of fish under experimental condi-
tions has been one of the chief sources of difficulty in work on the respiratory metab-
olism of fishes, and attention has been called to the need for an experimental method
which would make it possible to distinguish between "standard metabolism" and
the increased metabolism due to muscular movements (Wells, 1935). In view of
the fact that the oxygen consumption is affected by changes in the basal metabolic
rate as well as by changes in activity, the importance of such a method is apparent.
The method employed in the present work seems to meet this need, inasmuch as
the state of activity is recorded continuously and periods of inactivity can be selected
for measuring basal oxygen consumption.
Szymanski (1914) and Spencer (1939), using other types of activity detectors,
have reported that goldfish show considerable individual variation in activity and
that the activity pattern is affected by light. Spencer (1939) also found- activity
to be influenced by food. Knowledge of the behavior of the fish under the experi-
mental conditions is of importance in the collection of data on oxygen consumption
in the method to be described, as well as in the interpretation of these data. For
this reason further observations on the patterns and rates of activity and on the ef-
fects of food, light and disturbances are included in the present paper.
THE ACTIVITY OF THE GOLDFISH UNDER EXPERIMENTAL CONDITIONS
Method
Several dozen goldfish (Carassius auratus) ranging between 24 and 96 grams
in weight were selected at random from a stock obtained from a local goldfish farm
312
ACTIVITY AND OXYGEN CONSUMPTION
313
and studied individually in experimental chambers, each of which was equipped with
a recording activity detector. The experimental chambers were set up in a ground
floor aquarium room which was seldom entered except for the purposes of this
study, so that the fish could be left for long periods with relatively little disturbance.
The recording apparatus was kept in another room. Records of the activity of each
fish were started shortly after its introduction into a chamber and continued for pe-
riods ranging from a few days to many months in length, during which the patterns
and rates of activity and the effects of food, light and disturbances upon them were
studied. With a few exceptions, oxygen consumption was not measured in this
series of observations.
The experimental chamber (Fig. 1) consisted of a one-gallon brown glazed
FIGURE 1. Diagram of apparatus for measuring oxygen consumption and activity. (1)
paraffin oil (this was omitted when activity alone was being measured), (2) glass plates, (3)
No. 44 copper wire, (4) to sensitive relay, (5) resistor, (6) wire screen, (7) glass tube, (8)
wire frame protecting paddle. Explanation in text.
crock fitted with a galvanized iron wire screen of % mcn mesh to prevent the fish
from reaching the surface of the water. A glass tube about 3 cm. in diameter was
fitted into an opening in the center of the screen so that it extended 3.5 cm. above
and 3 cm. below the screen ; its purpose will be considered in a later section. The
surface of the water stood about 3 mm. above the screen, the total volume to this
level being 2,600 cc. Water entered the chamber from a constant level reservoir
through 8 mm. glass tubing and left by way of a siphon of 8 mm. glass tubing which
dipped into a constant level drain, the rate of flow (between 70 and 100 cc. a minute)
being regulated by means of a glass stopcock in the inlet. The intake of the siphon
was placed about 5 cm. above the bottom of the chamber, so that feces and other
debris that fell to the bottom did not enter the siphon until they had been broken
into small pieces in the course of their passage upward to the intake. The chamber
was practically self cleaning under these conditions, the flow of the water and the
movements of the fish being sufficient to move debris into the siphon. The fish
314 W. A. SPOOR
could therefore be maintained in the chamber for months without cleaning. A
thistle tube entering the inlet provided for the introduction of food, being closed off
at all other times. The water supply consisted of tap water passed through an ac-
tivated charcoal filter, brought to the desired temperature and aerated until it ap-
proached equilibrium with the atmosphere. Most of the observations were made at
temperatures between 20 and 24° C. The fish seldom extracted more than one-
third of the oxygen from the w^ater at the rates of flow employed, and they usually
took less than this. In view of the findings of Crozier and Stier (1925), Toryu
(1927), and Schlaifer (1938), it seems unlikely that behavior was influenced by
the oxygen tension of the water.
The chamber was enclosed in a wooden case to minimize disturbances and to
make it possible to control the light. The top of the case was fitted with a pane of
glass for natural illumination, and with a wooden cover when either complete dark-
ness or constant light was desired. A ventilator in the side of the case, with baffles
to prevent light from entering, permitted some circulation of the air. The water
inlet, outlet siphon, and a tube leading to a U tube indicating the water level in the
chamber passed through the wall ; a coat of black paint over each tube prevented
light from entering the chamber through these openings.
The detector consisted of a light-weight aluminum paddle suspended in the water
in the experimental chamber by a fine copper wire in such a way that a silver rod
at the top of the paddle shaft passed through a small hole in a fixed silver plate.
Water currents set up by the movements of the fish moved the paddle, causing the
rod to make and break contact with the sides of the hole and thus to activate a sen-
sitive relay. This relay operated the recording apparatus. The blade of the paddle
consisted of aluminum foil (5 cm. long and 2.5 cm. wide) with the corners bent in
at right angles so that the water currents struck a flat surface regardless of their
points of origin. The shaft (10 cm. long) consisted of no. 22 aluminum wire ce-
mented to the blade and imbedded at its upper end (7 cm. above the blade) in a
bakelite insulating rod (2 cm. long and 0.2 cm. in diameter) in the upper end of
which the silver rod (1 cm. long and about 0.04 cm. in diameter) was imbedded.
This silver rod was soldered to a 14.5 cm. length of no. 44 enameled copper wire
held in an insulated binding post attached to a wooden supporting shaft. A
wooden bracket rising from the case supported this shaft in a vertical position
so that the paddle hung in the water through the glass tube in the center of the
screen. A cylindrical frame of galvanized iron wire protected the paddle from
contact with the fish. A small lead weight (about 0.1 gm.) clamped to the
paddle shaft below the bakelite helped to bring the paddle back to the resting
position after displacement by the water currents. The silver plate (about 0.5
cm. square) was attached to the supporting shaft and held in a horizontal posi-
tion about 6 cm. above the screen. The hole in the plate was between 0.08 and 0.1
cm. in diameter. The current to operate the sensitive relay was supplied by a 6 volt
storage battery; the coil of the relay had a resistance of 1,000 ohms. A 5,000 ohm
resistor across the detector contacts prevented sparking and welding without caus-
ing an observable reduction in the sensitivity of the detector. The plate was kept
warm by means of a small insulated heating coil in order to prevent water from
condensing upon it from the humid atmosphere above the chamber.
The sensitivity of the detector could be controlled somewhat by adjusting the
position of the silver rod with respect to the sides of the hole in the plate. The nor-
ACTIVITY AND OXYGEN CONSUMPTION 315
mal movements of the operculum and the position-maintaining fin movements of a
quiescent 25- to 30-gram goldfish were usually sufficient to move the paddle slightly,
and when the rod was close to the plate these movements were recorded. For the
observations to be described, however, the rod was centered in the hole so that the
ordinary respiratory movements did not move the paddle enough to make contact,
these movements being considered as among the basal functions of the fish. Vigor-
ous respiratory movements and any movement that resulted in a change in the posi-
tion of the fish moved the paddle enough to make and break contact, slow swimming
movements causing few, and vigorous activity causing many impulses to be recorded.
At the flow rates used in these experiments the flow of water through the chamber
did not move the paddle.
The sensitive relay activated a counter which in turn caused signal magnets to
record every tenth and hundredth impulse on a long paper kymograph moving about
30 mm. an hour. The frequency of the impulses was such (ranging up to 6,000 an
hour) that they usually could not be counted when recorded individually. The
counter was capable of following and recording at least 10 impulses a second. Time
was recorded in hours beneath the activity record.
Patterns and rates of activity
In agreement with the results of Szymanski (1914) and Spencer (1939), the
goldfish used in this study proved to be quite variable in their patterns and rates of
activity, even when they were maintained under almost identical conditions of light,
feeding, temperature, water supply and disturbance. Three general types of be-
havior appeared when the fish were kept under natural conditions of light: (1)
arhythmic activity, in which no relation to clay or night could be detected; (2)
rhythmic activity, in which the fish were active by day and quiescent at night; (3)
rhythmic activity, in which they were quiescent by day and active at night.
Fish showing the first, arhythmic, type of behavior were extremely variable.
A few were vigorously active da)' and night for periods as long as ten days, others
were moderately active throughout the 24-hour period for weeks at a time, and still
others remained practically inert for similar periods. Some of these arhythmic fish,
particularly those in the last group, showed irregular bursts of activity now and then,
with no apparent relation to the time of day, feeding, or disturbance.
An example of the second type of behavior, diurnal activity and nocturnal quies-
cence, is shown in Figure 2, which is based on the number of impulses recorded by
a 35-gram male goldfish during each hour between 1 P.M. January 11 and 1 P.M.
January 13, 1946. The fish was fed at 11:05 A.M. on January 12, otherwise the
room was not entered between 2:45 P.M. January 11 and 7:15 P.M. January 13.
Aside from the feeding, the effects of which are discussed below, light was the only
known variable, the temperature, rate of flow and aeration of the water being the
same at the end as at the beginning of the period. Most of the fish showing rhythmic
changes in activity 'followed patterns of this type, although the active phase varied
considerably, sometimes being interrupted by several hours of quiescence during
the day, sometimes beginning later in the day, and occasionally continuing well into
the night. The most constant period of quiescence occurred between midnight and
4 A.M., which is in agreement with Spencer's (1939) observations.
The third type of behavior, diurnal quiescence and nocturnal activity, was found
less frequently than the second, although it was not uncommon. An example is
316
W. A. SPOOR
shown in Figure 3, which is based on records of the activity of a 32-gram male gold-
fish between 1 P.M. July 3 and 1 P.M. July 5, 1945. The room was not entered be-
'tween 4 P.M. July 3 and 8 A.M. July 5, and aside from the daily changes in light the
environmental conditions apparently remained constant throughout the period.
The patterns of rhythmic fish did not seem to be fixed, however, even when the
environmental conditions remained unchanged. After several weeks of rhythmic
behavior the fish frequently became arhythmic for several weeks or months, occa-
sionally becoming rhythmic again in the course of extended periods of observation.
This suggests that those fish which did not show daily activity rhythms under the
3000-
2500
2000-
P 1500-
o
1000-
500-
6RM.
I/ /46
2M.
6A.M. I2N.
I/I2/46
6P.M. I2M.
6 AW.
I/I3/46
!2N.
FIGURE 2. Activity pattern of 35-gram male goldfish between 1 P.M. January 11 and 1
P.M. January 13, 1946. Activity is expressed as number of impulses recorded each hour. Tem-
perature 21.5° C. Fed at 11 :05 A.M. January 12.
experimental conditions may have done so eventually had they been studied for lon-
ger periods, and that by chance the observations were made during arhythmic
periods.
Activity and food
The fish were fed rolled oats, commercial fish foods, shredded shrimp, ground
liver or chopped earthworms about three times a week, usually 0.5 to 1 gram at each
feeding. The effects of daily feeding, larger amounts of food and starvation were
also studied. Under the conditions of the experiments the type of food given had
no consistent effects upon activity, but the quantity of food had pronounced effects,
particularly on the total amount of activity. A well fed fish was usually sufficiently
active that the number of impulses recorded in the course of a 24-hour period aver-
aged between 500 and 1,500 an hour, and averages in excess of 2,500 impulses an
ACTIVITY AND OXYGEN CONSUMPTION
317
hour were not uncommon. Starvation caused this rate to decrease markedly, some-
times to fewer than 100 impulses an hour, although as a rule the lowest rates did not
appear until the fish had been starved for a week or so. No fish was observed to
become completely inactive for periods of more than an hour or two, however, even
when starved for two weeks. The effects of feeding after a period of starvation
were striking, activity increasing to normal "well fed" rates within a few minutes.
Doubtless the swimming movements associated with feeding accounted for some of
the activity recorded following the introduction of food, but it seems that the nu-
tritional state also affected the amount of activity. Food given in amounts of one
3000-
2500-
^2000-
H
p I 500-
o
1000-
500-
TJ
J"
6RM. I2M. 6A.M. I2N.
7/3/45 7/4/45
6 P.M.
I2M.
I
6A.M.
7/5/45
2N.
FIGURE 3. Activity pattern of 32-gram male goldfish between 1 P.M. July 3 and 1 P.M. July 5,
1945. Units as in Figure 2. Temperature 23.5° C.
gram or less was usually consumed within three to six hours, but the fish remained
active (in accordance with their activity patterns) for from several days to a week
after they had been fed. Similarly, Spencer ( 1939) found the goldfish to maintain
a'high rate of activity for several hours after feeding, although in his experiments
the food was usually consumed within 15 minutes or less.
The effects of feeding upon activity rhythms were not studied in detail, but the
available data bearing on this question indicate that although the rhythms appearing
under natural conditions of light were frequently modified by the quantity of food
and the time of feeding, they were not causally related to food. Feeding modified
the activity patterns of some fish for part or all of the subsequent 24-hour period,
usually by prolonging the active phase. A response of this type may be seen in
Figure 2. The fish was fed 0.5 gm. of rolled oats at 11 :05 A.M. on January 12 (the
previous feeding being on January 9) ; it will be noted that the activity level re-
mained relatively high for a much longer period on the night of January 12 than on
318 W. A. SPOOR
the preceding night. On the other hand some fish showed no change in activity in
response to feeding, provided of course that they had not been starved. Variations
in the quantity of food and in the time and frequency of feeding did not seem to have
•permanent effects on the activity rhythms, and feeding at the same time each day
did not cause arhythmic fish to become rhythmic.
Activity and light
The goldfish did not seem to be much affected by changes in light intensity while
they were not following daily activity rhythms, but they were usually quite responsive
to light during their periods of rhythmic behavior. In fact, when the fish were well
fed and undisturbed the activity rhythms seemed to be closely related to the daily
changes in natural light, as Szymanski (1914) has reported previously. This view
is supported by several observations in addition to the fact that the active and quies-
cent phases of the cycles usually coincided with day and night. Periods of nocturnal
activity and diurnal quiescence were shown by the 32-gram male goldfish mentioned
above in July and December of 1945. Although the water temperature and other
factors except light were the same during both periods, the nocturnal phase of ac-
tivity usually began earlier in the evening (between 5 and 6 P.M.) and ended later
in the morning (between 7 and 8 A.M.) in December than in July, when it usually
began between 7:30 and 8:30 P.M. and ended between 5 and 6 A.M. This suggests
of course that the nocturnal phase of activity was limited by the setting and rising
of the sun. This fish also responded readily to experimental changes in light in-
tensity, particularly during the day, when darkening the chamber caused its ac-
tivity to increase to levels usually reached only at night. Records were also ob-
tained in which diurnally active and nocturnally quiescent fish remained active on
nights when bright moonlight entered the room in which they were kept. Spencer
(1939) found that the regular diurnal rhythm of the goldfish could be obliterated by
covering the tank by day and lighting artificially at night. This procedure was ac-
companied by night feeding, however, so that the change in activity may not be at-
tributed solely to the reversed lighting.
On the basis of these observations attempts were made to maintain goldfish at
definite rates of activity by exposing them to continuous dim light and to continuous
darkness for periods lasting as long as three weeks, but without success. The fish
did not maintain constant rates of activity under either condition, but continued to
alternate periods of increased activity with periods of relative quiescence. In order
to maintain a low rate of activity it was necessary to starve the fish for about a week,
the relationship between nutritional state and amount of activity being similar to
that described in the preceding section.
Activity and disturbance
The goldfish proved to be extremely sensitive to disturbances. Noise, slight,
changes in the water level, sudden lights, the mere presence of the observer in the
room, or such minor disturbances as the quiet opening and closing of the door to
the room usually caused a change in the rate of activity. Fish that had been active
before the disturbance almost invariably became less active, sometimes practically
motionless, while quiescent fish frequently, although less consistently, became ac-
tive when disturbed. Whichever the response, the original state of activity was
ACTIVITY AND OXYGEN CONSUMPTION 319
usually resumed within a few minutes after the disturbance had ceased. The degree
of response seemed to be related to the amount of disturbance, for when the observer
moved slowly and quietly the change in activity was usually less pronounced, and
recovery more rapid, than after ordinary passage through the room or adjustment
of the apparatus. The effects of disturbances upon the activity of an otherwise
quiescent fish may be seen in Figure 3. The room was entered several times in the
course of the afternoon of July 3 and on July 5, although the experimental chamber
was not approached and the fish could not see the cause of the disturbance. It is
obvious that the rates of activity were higher than at corresponding hours on July 4,
when the room was not entered. Such sensitivity has been observed in other species
of fish by Clausen (1934), who found that a shadow passing over the aquarium
caused increases in the body temperatures of perch and members of the sunfish
group.
THE RELATIONSHIP BETWEEN ACTIVITY AND OXYGEN CONSUMPTION
Method
The activity and corresponding oxygen consumption of individual goldfish were
measured in observation periods ranging in length from 11 to 210 minutes. Ac-
tivity was measured in terms of the number of impulses recorded in a given period,
and the "amount of oxygen consumed by the fish in that period was determined by
means of a continuous flow system. A control chamber similar to the experimental
and housed in the same case was supplied with a continuous stream of water from
the reservoir supplying the fish. The water in each chamber was covered with a
layer of heavy paraffin oil 2.5 cm. thick to retard the diffusion of oxygen from the
air, and a sample of the effluent from each chamber was analyzed for oxygen by the
Winkler method at the beginning and end of each period. The samples were col-
lected in narrow necked glass stoppered bottles of about 270 cc. capacity arranged
to serve as constant level drains (Fig. 1). Each line was arranged so that the
water passed through the outlet siphon to the bottom of the sampling bottle and
overflowed into a funnel so that it could be collected for flow rate determinations.
Although the rates of flow ranged from 70 to 100 cc. a minute in the course of the
study, the rate for any one day's series of samples was held practically constant.
Due care was taken to prevent the diffusion of oxygen into the samples and to ob-
tain representative samples from experimental and control lines. Samples that
were contaminated by participate matter were discarded. The permanganate modi-
fication was used in most of the analyses, but was omitted during some of the
shorter periods. The results obtained with and without the modification were
quite similar, however, which was not unexpected in view of the fact that from four
to six liters of water passed over the fish each hour.
The volume of water flowing through the system in the course of an observation
period being known, together with the oxygen content of the water leaving the
control and experimental chambers at the beginning and end of that period, the
oxygen consumed by the fish could be calculated. The calculations took into ac-
count the change in the amount of oxygen in the constant volume of water in the
chamber. The volume of water displaced by the fish was too small to affect the
calculations.
The samples were collected wTith the foregoing observations on activity patterns
320 W. A. SPOOR
and modifying factors in mind, the periods being timed to yield data at the activity
rates desired, and the method of sampling being modified as necessary to minimize
disturbance of the fish. In the latter connection the outlet tubes were lengthened so
that samples were collected about 10 feet away from the chambers, and the room was
not entered except for sampling and rate of flow determinations. Precautions were
taken to prevent changes in the water level in the experimental chamber as there
were indications that small changes in the level stimulated the fish. These pre-
cautions were necessary also because the volume of water in the chamber, as well as
that flowing through it, entered into the calculations of oxygen consumption.
Samples were discarded if subsequent examinations of the activity records showed
that they had been collected while the fish was undergoing marked changes in ac-
tivity as a result of disturbance or in accordance with an activity rhythm. This was
necessary because although the activity record was instantaneous the change in the
oxygen concentration of the samples tended to lag somewhat behind that in the
chamber, the sample drawn at any instant representing the average of the water
flowing into the bottle in the few minutes preceding its removal. The temperature
of the water was recorded for each observation period in order to avoid discrepancies
attributable to the effect of temperature on metabolic rate (Ege and Krogh, 1914).
Owing to its viscosity and the accumulation of emulsified oil and water at the
oil-water interface, the layer of oil interfered with the movements of the paddle
shaft. Its thickness was therefore reduced to 1 cm. within the central glass tube,
thus permitting the paddle to move about as freely as with a water surface. This
tube extended below the interface far enough to prevent the emulsion from accumu-
lating around the paddle shaft. The oil within the tube had to be changed now and
then, however, to remove the small amount of debris that entered it from beneath.
The detector contacts and bakelite rod were cleaned every few days as a precaution
against their becoming coated with oil, which seemed to spread slo\vly up the paddle
shaft.
It was established by appropriate tests that the layer of paraffin oil was effective
in preventing the diffusion of significant amounts of oxygen into the water from the
atmosphere. In no test did the apparent leakage exceed the limits of error of the
Winkler method itself (Alice and Oesting, 1934), and it was usually considerably
less. The average apparent rate of change for the contents of the experimental
chamber was 0.0015 cc. of oxygen a minute, which was so much smaller than the
rate at wrhich the fish consumed oxygen that even had the apparent change been
real it would have had but little effect on the results. It should be mentioned in this
connection that the oil layer was disturbed relatively little by the movements of the
fish, inasmuch as the wire screen kept the fish out of the oil and glass plates resting
on this screen lessened the churning effects of the water beneath it.
Results
Three goldfish were studied at several temperatures in a total of 104 observation
periods. As the results on all three were much alike, data on but one of the fish,
a 32-gram male on which over two-thirds of the measurements were made, are
presented here.
The relationship between activity and oxygen consumption at temperatures be-
tween 23 and 25° C. is shown in Figure 4, in which oxygen consumption in cubic
ACTIVITY AND OXYGEN CONSUMPTION
321
centimeters per minute is plotted against activity in impulses per minute. Fifty-nine
observation periods are represented, each point corresponding to one period. The
line merely indicates the trend, and has not been fitted to the data mathematically.
As was to be expected, oxygen consumption and activity proved to be closely related,
the relationship apparently being linear above the basal level of oxygen consumption.
Although the values for oxygen consumption at any one rate of activity are seen to
vary somewhat, the trend is clear cut : at high rates of activity the rate of oxygen
.18
.16-
.15-
.13-
z
o
P .12-
o.
01 .1 I '
o
0
.10-
.09-
.08-
.07-
06-
.05-
.04
.03
10
20
30
40 50 60
ACTIVITY
70
80
90 100
FIGURE 4. Activity and oxygen consumption of 32-gram male goldfish. Activity in impulses/
minute; oxygen consumption in cubic centimeters/minute. Temperature 23 to 25° C.
consumption is correspondingly high ; at low activity rates less oxygen is consumed.
The discrepancies that do occur may well have been due to errors in measurement,
rather than to a lack of correspondence between activity and oxygen consumption.
In this connection the data on oxygen consumption follow those on activity quite
closely when the comparison is restricted to one day's series of measurements, thus
ruling out discrepancies attributable to slight differences in the adjustment of the
detector contacts. Such a series is shown in Figure 5, which is based on data ob-
tained with the same fish in a series of thirteen consecutive 15- to 25-minute pe-
riods at 22° C.
According to the slope of the data shown in Figure 4, the basal oxygen con-
322
W. A. SPOOR
sumption of this fish was in the vicinity of 0.040 cc. a minute, or 0.075 cc. per gram
per hour.
DISCUSSION
The results of the present study have a bearing on the collection and interpreta-
tion of data on the respiratory metabolism of fishes, and in the light of these results
the method described seems to offer a number of advantages not found in previous
methods which have been employed for this purpose.
The advantages of the continuous flow method for measuring respiratory metab-
olism in fishes have been discussed by Keys ( 1930) and need not be reviewed here.
In view of the relationship between oxygen consumption and activity, however, the
•100
.03
150
215 230 245 300 315
5OO
30
TIME
FIGURE 5. Activity and oxygen consumption of 32-gram male goldfish in each of thirteen
consecutive observation periods between 1 :35 P.M. and 5 P.M. November 13, 1945. Units as in
Figure 4. Each point on the upper line represents the average rate of oxygen consumption for
the 15- or 25-minute period preceding it. Each point on the lower line represents the average
rate of activity for the corresponding period.
observations on the effects of disturbances may be applied to the use of this method,
inasmuch as the process of sampling may disturb the fish. Should a change in the
rate of activity (and consequently of oxygen consumption) occur at the time of
sampling, the sample would not be representative of the volume of water and unit
of time to which it is related in the calculations. The resulting error could be of
considerable importance, particularly in investigations in which the samples con-
sisted of water flowing directly from the experimental chamber and overflowing
through a sampling bottle. This source of error has been recognized of course, and
in some investigations the experimental chamber has been covered in attempts to
minimize stimulation of the fish. It seems very doubtful, however, whether cover-
ing a goldfish so that it cannot see the investigator is an adequate safeguard against
disturbance. One advantage of using an activity detector in the continuous flow
method then lies in the fact that any sudden change in activity occurring at the
time of sampling can be detected, so that the reliability of the sample may be judged.
Furthermore, the activity record can be used to test the effectiveness of the steps
taken to avoid disturbance.
ACTIVITY AND OXYGEN CONSUMPTION 323
It is of course well known that fluctuations in activity during the test periods
constitute a major obstacle to the correct interpretation of measurements of oxygen
consumption, and numerous attempts to overcome this difficulty have been described
(Ege and Krogh, 1914; Hall, 1929; Adkins, 1930; Keys, 1930; Wells, 1932, 1935;
Clausen, 1933; Smith and Matthews, 1942). These measures include the use of
narcotics, observing that the fish remains quiet, maintaining constant conditions of
light, sampling at the same time each day, restricting the movements of the fish, and
maintaining the fish in an experimental chamber until it appears to have come to
rest or at any rate to have reached a steady state. Although such measures may
permit the establishment of the reality of a change in oxygen consumption in con-
nection with an experimental procedure, they do not appear to give a completely
satisfactory basis for the interpretation of that change. The interpretation must be
based on knowledge of the activity of the fish, inasmuch as oxygen consumption is
affected by changes in the basal metabolic rate as well as by activity. The method
employed must therefore be capable of supplying information on activity and oxygen
consumption at the same time, so that the fraction of the respiratory exchange as-
sociated with basal metabolism may be distinguished from that due to muscular ac-
tivity (Wells, 1935). None of the above methods seems to be adequate for this
purpose.
Narcotics are of doubtful value in studies of this type, even for measuring basal
metabolic rate alone (Adkins, 1930). Among other objections are indications that
an important fraction of the metabolic functions of the fish may be suppressed to
such an extent that the oxygen consumption falls below the basal level as it is gen-
erally understood (Keys and Wells, 1930). In fact, Ege and Krogh (1914) con-
sidered it necessary to use artificial respiration to insure the survival of their gold-
fish, the narcotic having interfered with normal respiratory movements. The other
methods are open to criticism because they are based on the assumption, rather than
the knowledge, that the fish is quiescent or at a constant level of activity under the
conditions of the experiment. The results of the present work suggest that for the
goldfish at any rate this assumption may be unwarranted. The fact that a goldfish
is quiescent while it can be seen should not be taken as proof that it remains so
while unobserved, and it does not seem justifiable to assume that constant environ-
mental conditions mean constant rates of activity. So far as the goldfish is con-
cerned, the individual variations in activity open to question the reliability of methods
based on sampling at the same time each day, particularly if several fish are being
compared. Confining the fish to a small respiration chamber to restrict its move-
ments gives no assurance that it will remain quiescent or even at a constant rate of
activity, and the fact that the oxygen consumption varies over a wide range in such
chambers supports this objection. This method would seem to have a further dis-
advantage for measuring the basal metabolic rate in that a fish confined to a small
tube must swim continuously, however slowly, in order to maintain its position in
the current. The practice of leaving the fish in the respiratory chamber until its
oxygen consumption has reached a relatively low and constant rate (Keys, 1930;
Wells, 1935) is far superior to the earlier techniques, but it is limited in its applica-
tion by the fact that it gives no information as to the amount of activity associated
with the steady state.
The requirements of a satisfactory method appear to be met by combining an
activity detector with the continuous flow system. The rate of activity can then be
324 W. A. SPOOR
measured at the same time that oxygen consumption is determined, and the results
interpreted accordingly. As the activity record is continuous, periods of quiescence
can be selected for measuring basal oxygen consumption, so that it is not necessary
to employ special techniques designed to control activity. In this connection, how-
ever, starvation may be used as a means of prolonging the quiescent state. A fur-
ther advantage of the present method lies in the fact that the fish can be maintained
in good health in the experimental chamber for months, so that measurements of
its respiratory metabolism need not be obscured by the excitement and other effects
of handling.
SUMMARY
1. Apparatus for making continuous records of the activity of isolated and un-
disturbed goldfish is described, together with a method for measuring oxygen con-
sumption and activity simultaneously.
2. The goldfish were quite variable in their patterns and rates of activity under
the experimental conditions. Some fish were diurnally active and nocturnally
quiescent, others followed the opposite pattern and still others were arhythmic
throughout the periods during which they were observed. Moreover, some fish
showed both rhythmic and arhythmic states of activity when studied for periods
extending over several weeks or months.
3. Food, light and minor disturbances had pronounced effects on the activity of
the goldfish.
4. Simultaneous measurements of oxygen consumption and activity are pre-
sented which indicate that the two are closely related above the basal level of oxygen
consumption.
5. The bearing of these observations on the collection and interpretation of data
on the oxygen consumption of the goldfish and on the measurement of its basal
metabolic rate is discussed, and certain advantages of the method are described.
LITERATURE CITED
ADKINS, M., 1930. A method for determining basal metabolism of fishes. Proc. Soc. Exp.
Biol. Med., 28 : 259-263.
ALLEE, W. C. AND R. OESTING, 1934. A critical examination of Winkler's method for deter-
mining dissolved oxygen in respiration studies with aquatic animals. Physiol. Zool.,
7: 509-541.
BOWEN, E. S., 1932. Further studies of the aggregating behavior of Ameiurus melas. Biol.
Bull., 63 : 258-270.
CLAUSEN, R. G., 1933. Fish metabolism under increasing temperature. Trans. Amcr. Fish.
Soc., 63 : 215-219.
CLAUSEN, R. G., 1934. Body temperature of fresh water fishes. Ecology, 15: 139-144.
CLAUSEN, R. G., 1936. Oxygen consumption in fresh water fishes. Ecology, 17 : 216-226.
CROZIER, W. J. AND T. B. STIER, 1925. Critical increment for opercular breathing rhythm of
the goldfish. Jour. Gen. Physiol.. 1 : 699-704.
EGE, R. AND A. KROGH, 1914. On the relation between the temperature and the respiratory
exchange in fishes. Internal. Revue d. ges. Hydrobiol. u. Hydrog., 7 : 48-55.
HALL, F. G., 1929. The influence of varying oxygen tensions upon the rate of oxygen con-
sumption in marine fishes. Amer. Jour. Physiol., 88 : 212-218.
KEYS, A. B., 1930. The measurement of the respiratory exchange of aquatic animals. Biol.
Bull, 59 : 187-198.
KEYS, A. B. AND N. A. WELLS, 1930. Amytal anesthesia in fishes. Jour. Pharm. Exp. Thcrap.,
40: 115-128.
ACTIVITY AND OXYGEN CONSUMPTION 325
KROGH, A., 1916. The respiratory exchange of animals and man. Monographs on Biochemistry.
Longmans, Green and Co., London.
SCHLAIFER, A., 1938. Studies in mass physiology : effect of numbers upon the oxygen consump-
tion and locomotor activity of Carassius auratus. Physiol. Zoo/., 11: 408-424.
SMITH, D. C. AND S. A. MATTHEWS, 1942. The effect of adrenalin on the oxygen consumption
of the fish, Girella nigricans. Aincr. Jour. Physiol., 137 : 533-538.
SPENCER, W. P., 1939. Diurnal activity rhythms in fresh-water fishes. Ohio Jour. Sci., 39:
119-132.
SPOOR, W. A., 1941. A method for measuring the activity of fishes. Ecology, 22: 329-331.
SZYMANSKI, J. S., 1914. Eine Methode zur Untersuchung der Ruhe- und Aktivitatsperioden
bei Tieren. Pfliigcr's Arch. gcs. Physiol., 158: 343-385.
TORYU, Y., 1927. The respiratory exchange in Carassius auratus and the gaseous exchange of
the air bladder. Sci. Kept. Tohoku Imp. Univ., 4 Ser. (Biology), 3: 87-96.
WELLS, N. A., 1932. The importance of the time element in the determination of the respira-
tory metabolism of fishes. Proc. Nat. Acad. Sci., 18: 580-585.
WELLS, N. A., 1935. The influence of temperature upon the respiratory metabolism of the
Pacific killifish, Fundulus parvipinnis. Physiol. Zoo/., 8 : 196-227.
INDEX
A BSTRACTS of scientific papers presented
at the Marine Biological Laboratory, sum-
mer of 1946, 210.
Activity and oxygen consumption of the gold-
fish and its application to the measurement
of respiratory metabolism in fishes, 312.
Amaroecium constellatum, II, 66.
Annual report of the Marine Biological Labora-
tory, 1.
Arbacia eggs, the effect of low temperature and
of hypotonicity on the morphology of the
cleavage furrow, 272.
Artemia salina, the space-time pattern of seg-
ment formation in, 119.
gEERS, C. D. Tillina magna: Micronuclear
number, encystment and vitality in di-
verse clones; capabilities of amicronucleate
races, 256.
BODENSTEIN, DIETRICH. Developmental rela-
tions between genital ducts and gonads in
Drosophila, 288.
BROWN, FRANK A., JR., AND LORRAINE M.
SAIGH. The comparative distribution of
two chromatophorotropic hormones (CDH
and CBLH) in Crustacean nervous sys-
tems, 170.
("^ARRIKER, MELBOURNE ROMAINE. Ob-
servations on the functioning of the ali-
mentary system of the snail Lymnaea
stagnalis appressa Say, 88.
CHEN, TZE-TUAN. Temporary pair formation
in Paramecium bursaria, 112.
Chromosome movement, hydrostatic pressure
effects upon the spindle figure and, 145.
Ciliates of the family Ancistrocomidae Chatton
and Lwoff, III, 189.
Ciliates of the family Ancistrocomidae Chatton
and Lwoff, IV, 200.
Cleavage furrow in Arbacia eggs, 272.
T~)DT, loci of action in cockroach (Periplaneta
americana), 247.
Drosophila, developmental relations between
genital ducts and gonads in, 288.
"pLECTRON microscope observations of the
trichocysts and cilia of Paramecium, 141.
("MESE, ARTHUR C. Comparative sensitivity of
sperm and eggs to ultraviolet radiations, 81.
JLJJABROBRACON, a strongly intersexual
female in, 243.
HALL, C. E. See M. A. JAKUS, 141.
Histological study of Syndisyrinx franciscanus,
~-an endoparasitic rhabdocoel of the sea
urchin, 295
Hormones (CDH and CBLH) in Crustacean
nervous systems, the comparative distribu-
tion of two chromatophorotropic, 170.
INFLUENCE of texture and composition of
surface on the attachment of sedentary
marine organisms, 57.
JAKUS, M. A., AND C. E. HALL. Electron
microscope observations of trichocysts and
cilia of Paramecium, 141.
L£ OLLROS, J. J. See]. M. TOBIAS, 247.
KOZLOFF, EUGENE N. Studies on ciliates
of the family Ancistrocomidae Chatton and
Lwoff (order Holotricha, suborder Thig-
motricha). III. Ancistrocoma pelseneeri
Chatton and Lwoff, Ancistrocoma dissimi-
lis sp. nov., and Hypocomagalma phola-
didis sp. nov., 189.
KOZLOFF, EUGENE N. Studies on ciliates of
the family Ancistrocomidae Chatton and
Lwoff (order Holotricha, suborder Thig-
motricha). IV. Heterocineta janickii Ja-
rocki, Heterocineta goniobasidis sp. nov.,
Heterocineta fluminicolae sp. nov., and
Enerthecoma properans Jarocki, 200.
[EHMAN, H. E. A histological study of
Syndisyrinx franciscanus, gen. et sp. nov.,
an endoparasitic rhabdocoel of the sea
urchin, Strongylocentrotus franciscanus,
295.
Lymnaea stagnalis appressa Say, observations
on the functioning of the alimentary sys-
tem of the snail, 88.
IV/f ARINE Biological Laboratory, annual re-
port, 1.
Marine Biological Laboratory, program and
abstracts of scientific papers presented,
summer of 1946, 210.
MORRISON, PETER R. Physiological observa-
tions on water loss and oxygen consump-
tion in Peripatus, 181.
327
328
INDEX
/~\XYGEN consumption and water loss in
Peripatus, 181.
Oxygen consumption and activity of the gold-
fish, its application to the measurement of
respiratory metabolism in fishes, 312.
DARAMECIUM bursaria, temporary pair
formation in, 112.
Paramecium, electron microscope observations
of the trichocysts and cilia of, 141.
PEASE, DANIEL C. Hydrostatic pressure ef-
fects upon the spindle figure and chromo-
some movement. II. Experiments on the
meiotic divisions of Tradescantia pollen
mother cells, 145.
POMERAT, C. M., AND C. M. WEISS. The in-
fluence of texture and composition of sur-
face on the attachment of sedentary
marine organisms, 57.
CAIGH, LORRAINE M. .See FRANK A. BROWN,
JR., 170.
SCOTT, ALLAN. The effect of low temperature
and of hypotonicity on the morphology of
the cleavage furrow in Arbacia eggs, 272.
SCOTT, SISTER FLORENCE MARIE. The de-
velopmental history of Amaroecium con-
stellatum. II. Organogenesis of the larval
action system, 66.
Society of General Physiologists, papers pre-
sented at the meeting of, 236.
Spindle figure and chromosome movement,
hydrostatic pressure effects upon, 145.
SPOOR, W. A. A quantitative study of the
relationship between the activity and
oxygen consumption of the goldfish, and
its application to the measurement of
respiratory metabolism in fishes, 312.
Studies on ciliates of the family Ancistro-
comidae Chatton and Lwoff (order Holo-
tricha, suborder Thigmotricha). III. An-
cistrocoma pelseneeri Chatton and Lwoff,
Ancistrocoma dissimilis sp. nov., and Hy-
pocomagalma pholadidis sp. nov., 189.
Studies on ciliates of the family Ancistro-
comidae Chatton and Lwoff (order Holo-
tricha, suborder Thigmotricha). IV.
Heterocineta janickii Jarocki, Heterocineta
goniobasidis sp. nov., Heterocineta flumini-
colae sp. nov., and Enerthecoma properans
Jarocki, 200.
nPILLINA magna: Micronuclear number,
encystment and vitality in diverse clones;
capabilities of amicronucleate races, 256.
TOBIAS, J. M., AND J. J. KOLLROS. Loci of
action of DDT in cockroach (Periplaneta
americana), 247.
u
LTRAVIOLET radiations, comparative
sensitivity of sperm and eggs to, 81.
EISS, C. M. See C. M. POMERAT, 57.
WEISZ, PAUL B. The space- time pattern
of segment formation in Artemiasalina, 119.
WHITING, P. W. A strongly intersexual female
in Habrobracon, 243.
Volume 91
<LS (J
Number 1
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
G. H. PARKER, Harvard University
A. C. REDFIELD, Harvard University
F. SCHRADER, Columbia University
DOUGLAS WmTAKER, Stanford University
H. B. STEINBACH, Washington University
Managing Editor
AUGUST, 1946
Marine Biological lal>\>;
LIB S*
SEP 1
WOODS HOIE, MASS.
Printed and Issued by
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D
'URING THE WAR, the libraries of half the world were destroyed in the
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physical damage, they were impoverished by isolation. There is an urgent need
—now — for the printed materials which are basic to the reconstruction of dev-
astated areas and which can help to remove the intellectual blackout of Europe
and the Orient.
There is need for a pooling of resources, for coordinated action in order that
the devastated libraries of the world may be restocked as far as possible with
needed American publications. The American Book Center for War Devastated
Libraries, Inc., has come into being to meet this need. It is a program that is
born of the combined interests of library and educational organizations, of gov-
ernment agencies, and of many other official and non-official bodies in the United
States.
The American Book Center is collecting and is shipping abroad scholarly
books and periodicals which will be useful in research and necessary in the
physical, economic, social and industrial rehabilitation and reconstruction of
Europe and the Far East.
The Center cannot purchase books and periodicals ; it must depend upon gifts
from individuals, institutions, and organizations. Each state will be organized
to participate in the program through the leadership of a state chairman. Other
chairmen will organize interest in the principal subject fields. Cooperation with
these leaders or direct individual contributions are welcomed.
WHAT IS NEEDED: Shipping facilities are precious and demand that
all materials be carefully selected. Emphasis is placed upon publications issued
during the past decade, upon scholarly books which are important contributions
to their fields, upon periodicals (even incomplete volumes) of significance, upon
fiction and non-fiction of distinction. All subjects — history, the social sciences,
music, fine arts, literature, and especially the sciences and technologies — are
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tional reading, books for children and young people, light fiction, materials of
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directly to the Center with regard to specific documents.
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CANNOT ACCEPT MATERIAL WHICH IS SENT COLLECT. Reim-
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tation. When possible, periodicals should be tied together by volume. It will
be helpful if missing issues are noted on incomplete volumes.
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.
MICROFILM SERVICE
•
The Library of The Marine
Biological Laboratory can
supply microfilms of ma-
terial from periodicals in-
cluded in its list. Requests
should include the title of
the paper, the author, peri-
odical, volume and date of
publication.
Rates are as follows: $.30 for
papers up to 25 pages, and $.10
for each additional 10 pages or
fraction thereof.
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
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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, 8Vz 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 September 1, and to the Department of Zoology,
Washington University, St. Louis, Missouri, during the remainder of
the vear.
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
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including Protozoan and
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MARINE
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CONTENTS
Page
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY. ... i
POMERAT, C. M. AND C. M. WEISS
The influence of texture and composition of surface on the
attachment of sedentary marine organisms 57
SCOTT, SISTER FLORENCE MARIE
The developmental history of Amaroecium constellatum. II.
Organogenesis of the larval action system 66
GIESE, ARTHUR C.
Comparative sensitivity of sperm and eggs to ultraviolet
radiations 81
CARRIKER, MELBOURNE ROMAINE
Observations on the functioning of the alimentary system of
the snail Lymnaea stagnalis appressa Say 88
CHEN, TZE-TUAN
Temporary pair formation in Paramecium bursaria 112
Volume 91
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
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
Marine Biological Labofototy
L.I Ell A. R Y
NOV 181946
WOODS HOLE, MASS.
OCTOBER, 1946
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
LANCASTER, PA.
BOOKS-WAR VICTIMS
'URING THE WAR, the libraries of half the world were destroyed in the
fires of battle and in the fires of hate and fanaticism. Where they were spared
physical damage, they were impoverished by isolation. There is an urgent need
—now — for the printed materials which are basic to the reconstruction of dev-
astated areas and which can help to remove the intellectual blackout of Europe
and the Orient.
There is need for a pooling of resources, for coordinated action in order that
the devastated libraries of the world may be restocked as far as possible with
needed American publications. The American Book Center for War Devastated
Libraries, Inc., has come into being to meet this need. It is a program that is
born of the combined interests of library and educational organizations, of gov-
ernment agencies, and of many other official and non-official bodies in the United
States.
The American Book Center is collecting and is shipping abroad scholarly
books and periodicals which will be useful in research and necessary in the
physical, economic, social and industrial rehabilitation and reconstruction of
Europe and the Far East.
The Center cannot purchase books and periodicals ; it must depend upon gifts
from individuals, institutions, and organizations. Each state will be organized
to participate in the program through the leadership of a state chairman. Other
chairmen will organize interest in the principal subject fields. Cooperation with
these leaders or direct individual contributions are welcomed.
WHAT IS NEEDED: Shipping facilities are precious and demand that
all materials be carefully selected. Emphasis is placed upon publications issued
during the past decade, upon scholarly books which are important contributions
to their fields, upon periodicals (even incomplete volumes) of significance, upon
fiction and non-fiction of distinction. All subjects — history, the social sciences,
music, fine arts, literature, and especially the sciences and technologies — are
wanted.
WHAT IS NOT NEEDED: Textbooks, out-dated monographs, recrea-
tional reading, books for children and young people, light fiction, materials of
purely local interest, popular magazines such as Time, Life, National Geographic,
etc., popular non-fiction of little enduring significance such as Gunther's Inside
Europe, Haliburton's Royal Road to Romance, etc. Only carefully selected
federal and local documents are needed, and donors are requested to write
directly to the Center with regard to specific documents.
HOW TO SHIP: All shipments should be sent PREPAID via the
cheapest means of transportation to THE AMERICAN BOOK CENTER,
C/O THE LIBRARY OF CONGRESS, WASHINGTON 25, D. C. Al-
though the Center hopes that donors will assume the costs of transportation of
their materials to Washington, when this is not possible reimbursement will be
made upon notification by card or letter of the amount due. THE CENTER
CANNOT ACCEPT MATERIAL WHICH IS SENT COLLECT. Reim-
bursement cannot be made for packing or other charges beyond actual transpor-
tation. When possible, periodicals should be tied together by volume. It will
be helpful if missing issues are noted on incomplete volumes.
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.
MICROFILM SERVICE
*
The Library of The Marine
Biological Laboratory can
supply microfilms of ma-
terial from periodicals in-
cluded in its list. Requests
should include the title of
the paper, the author, peri-
odical, volume and date of
publication.
Rates are as follows: $.30 for
papers up to 25 pages, and $.10
for each additional 10 pages or
fraction thereof.
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, SVz 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 September 1, and to the Department 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.
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
WEISZ, P/vUL B.
The space-time pattern of segment formation in Artemia
salina 1 19
JAKUS, M. A., AND C. E. HALL
Electron microscope observations of the trichocysts and cilia
of Paramecium 141
PEASE, DANIEL C.
Hydrostatic pressure effects upon the spindle figure and
chromosome movement. II. Experiments on the meiotic
divisions of Tradescantia pollen mother cells 145
BROWN, FRANK A. JR., AND LORRAINE M. SAIGH
The comparative distribution of two chromatophorotropic
hormones (CDH and CBLH) in Crustacean nervous systems 170
MORRISON, PETER R.
Physiological observations on water loss and oxygen con-
sumption in Peripatus 181
KOZLOFF, EUGENE N.
Studies on ciliates of the family Ancistrocomidae Chatton
and Lwoff (order Holotricha, suborder Thigmotricha). III.
Ancistrocoma pelseneeri Chatton and Lwoff, Ancistrocoma
dissimilis sp. nov., and Hypocomagalma pholadidis sp. nov. . 189
KOZLOFF, EUGENE N.
Studies on ciliates of the family Ancistrocomidae Chatton
and Lwoff (order Holotricha, suborder Thigmotricha). IV.
Heterocineta janickii Jarocki, Heterocineta goniobasidis sp.
nov., Heterocineta fluminicolae sp. nov., and Enerthecoma
properans Jarocki 200
ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE
BIOLOGICAL LABORATORY, SUMMER OF 1946 210
PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GEN-
ERAL PHYSIOLOGISTS. 236
Volume 91
Number 3
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
G. H. PARKER, Harvard University
A. C. REDFffiLD, Harvard University
F. SCHRADER, Columbia University
DOUGLAS WHITAKER, Stanford University
H. B. STEINBACH, Washington University
Managing Editor
DECEMBER, 1946
JAN -7 -19-47
WOODS HOLE,
Printed and Issued by
LANCASTER PRESS, Inc.
PRINCE & LEMON STS.
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BOOKS-WAR VICTIMS
D,
'URING THE WAR, the libraries of half the world were destroyed in the
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Your Biological News
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Biological Abstracts is news nowadays. Abridgments of all the im-
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BIOLOGICAL ABSTRACTS
University of Pennsylvania
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MICROFILM SERVICE
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•
CONTENTS
' • '
Page
WHITING, P. W.
A strongly intersexual female in Habrobracon ............. 243
TOBIAS, J. M., AND J. J. KOLLROS
Loci of action of DDT in the cockroach (Periplaneta ameri-
cana) ............................................. 247
BEERS, C. D.
Tillina magna: Micronuclear number, encystment and vitality
in diverse clones; capabilities of amicronucleate races ...... 256
SCOTT, ALLAN
The effect of low temperature and of hypotonicity on the
morphology of the cleavage furrow in Arb'acia eggs ........ 272
BODENSTEIN, DIETRICH
Developmental relations between genital ducts and gonads
in Drosophila ........ ^ ................................. 288
LEHMAN, H. E.
A histological study of Syndisyrinx franciscanus, gen. et sp.
nov., an endoparasitic rhabdocoel of the sea urchin, Strongylo-
centrotus franciscanus ................................. 295
SPOOR, W. A.
A quantitative study of the relationship between the activity
and oxygen consumption of the goldfish, and its application
to the measurement of respiratory metabolism in fishes. ... 312
J
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. VVHOI LIBRARY
UH 17JA D