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THE
BIOLOGICAL BULLETIN
PUBLISHED BY
THE MARINE BIOLOGICAL LABORATORY
Editorial Board
E. G. CONKLIN, Princeton University
E. N. HARVEY, Princeton University
SELIG HECHT, Columbia University
LEIGH HOADLEY, Harvard University
L. IRVING, Swarthmore College
M. H. JACOBS, University of Pennsylvania
H. S. JENNINGS, Johns Hopkins University
FRANK R. LILLIE, University of Chicago CARL R. MOORE, University of Chicago GEORGE T. MOORE, Missouri Botanical Garden T. H. MORGAN, California Institute of Technology G. H. PARKER, Harvard University A. C. REDFIELD, Harvard University F. SCHRADER, Columbia University
H. B. STEINBACH, Washington University Managing Editor
VOLUME 85
AUGUST TO DECEMBER, 1943
Printed and Issued by LANCASTER PRESS, Inc.
PRINCE 8C 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 October 1, and to the Depart- ment of Zoology, Washington University, St. Louis, .Missouri, during the remainder of the year.
Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912.
LANCASTER PRESS, INC., LANCASTER, PA.
CONTENTS
No. 1. AUGUST, 1943
PAGE
ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1
SONNEBORN, T. M., AND RUTH V. DlPPELL
Sexual Isolation, Mating Types, and Sexual Responses to Diverse Con- ditions in Variety 4, Paramecium Aurelia 36
HOVANITZ, WILLIAM
Hybridization and Seasonal Segregation in Two Races of a Butterfly Occurring Together in Two Localities 44
LEVINE, HARRY P.
Species Differences in Rates of Osmotic Hemolysis Within the Genus Peromyscus 52
LAWSON, CHESTER A.
Germarial Differences and the Production of Aphid Types 60
LOOSANOFF, VICTOR L., AND JAMES B. ENGLE
Polydora in Oysters Suspended in Water 69
WAIT, ROBERT B.
The Action of Acetylcholine on the Isolated Heart of Venus Mercenaria 79
No, 2. OCTOBER, 1943
PYLE, ROBERT W.
The Histogenesis and Cyclic Phenomena of the Sinus Gland and X- Organ in Crustacea 87
BURT, AGNES SANXAY
Neurulation in Mechanically and Chemically Inhibited Amblystoma. . 103
PRATT, DAVID M.
Analysis of Population Development in Daphnia at Different Tempera- tures 116
HARVEY, ETHEL BROWNE
Rate of Breaking and Size of the "Halves" of the Arbacia Punctulata Egg when Centrifuged in Hypo- and Hypertonic Sea Water 141
HARVEY, ETHEL BROWNE, AND THOMAS F. ANDERSON
The Spermatozoon and Fertilization Membrane of Arbacia Punctulata
as Shown by the Electron Microscope 151
BODINE, JOSEPH HALL, AND THEODORE NEWTON TAHMISIAN
The Development of an Enzyme (Tyrosinase) in the Parthenogenetic Egg of the Grasshopper, Melanoplus Differentialis 157
Ris, HANS
A Quantitative Study of Anaphase Movement in the Aphid Tamalia. . 164
iv CONTENTS
PAGE
No. 3. DECEMBER, 1943
HARRIS, DANIEL L.
The Osmotic Properties of Cytoplasmic Granules of the Sea Urchin Egg 179
WILBUR, KARL M., AND RICHARD O. RECKNAGEL
The Radiosensitivity of Eggs of Arbacia Punctulata in Various Salt Solutions 193
CLARKE, GEORGE L., E. LOWE PIERCE AND DEAN F. BUMPUS
The Distribution and Reproduction of Sagitta Elegans on Georges Bank
in Relation to the Hydrographical Conditions 201
STUNKARD, HORACE W.
The Morphology and Life History of the Digenetic Trematode, Zoogo- noides Laevis Linton, 1940 227
WHITING, P. W.
Intersexual Females and Intersexuality in Habrobracon 238
THIVY, FRANCESCA
New Records of Some Marine Chaetophoraceae and Chaetosphaeridia- ceae for North America t. . . . 244
HUGHES-SCHRADER, SALLY
Polarization, Kinetochore Movements, and Bivalent Structure in the Meiosis of Male Mantids. 265
Vol. 85, No. 1 August, 1943
THE
BIOLOGICAL BULLETIN
PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY
THE MARINE BIOLOGICAL LABORATORY
FORTY-FIFTH REPORT, FOR THE YEAR 1942 — FIFTY-FIFTH YEAR
I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 11, 1942) .... 1
STANDING COMMITTEES 2
II. ACT OF INCORPORATION 3
III. BY-LAWS OF THE CORPORATION 4
IV. REPORT OF THE TREASURER 5
V. REPORT OF THE LIBRARIAN 9
VI. REPORT OF THE DIRECTOR 11
Statement 11
Addenda :
1. The Staff, 1942 13
2. Investigators and Students, 1942 16
3. Tabular View of Attendance 21
4. Subscribing and Co-operating Institutions, 1942 22
5. Evening Lectures, 1942 22
6. Shorter Scientific Papers, 1942 23
7. Members of the Corporation 24
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
Ross G. HARRISON, Yale University.
H. S. JENNINGS, University of California.
C. E. McCLUNG, University of Pennsylvania.
S. O. MAST, Johns Hopkins University.
A. P. MATHEWS, University of Cincinnati.
T. H. MORGAN, California Institute of Technology.
W. J. V. OSTERHOUT, Rockefeller Institute.
G. H. PARKER, Harvard University.
W. B. SCOTT, Princeton University.
1
MARINE BIOLOGICAL LABORATORY
TO SERVE UNTIL 1946
DUGALD E. S. BROWN, New York University. E. R. CLARK, University of Pennsylvania. OTTO C. GLASER, Amherst College.
E. N. HARVEY, Princeton University.
M. H. JACOBS, University of Pennsylvania.
F. P. KNOWLTON, Syracuse University. FRANZ SCHRADER, Columbia University.
B. H. WILLIER, Johns Hopkins University.
TO SERVE UNTIL 1945
W. R. AMBERSON, University of Maryland School of Medicine.
S. C. BROOKS, University of California.
W. C. CURTIS, University of Missouri.
H. B. GOODRICH, Wesleyan University.
I. F. LEWIS, University of Virginia.
R. S. LILLIE, The University of Chicago.
A. C. REDFIELD, Harvard University.
C. C. SPEIDEL, University of Virginia.
TO SERVE UNTIL 1944
ERIC G. BALL, Harvard University Medical School.
R. CHAMBERS, Washington Square College, New York University.
EUGENE F. DuBois, Cornell University Medical College.
W. E. CARREY, Vanderbilt University Medical School.
COLUMBUS ISELIN, Woods Hole Oceanographic Institution.
C. W. METZ, University of Pennsylvania. H. H. PLOUGH, Amherst College.
W. R. TAYLOR, University of Michigan.
TO SERVE UNTIL 1943
W. C. ALLEE, The University of Chicago.
G. H. A. CLOWES, Lilly Research Laboratory.
B. M. DUGGAR, University of Wisconsin.
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.
EXECUTIVE COMMITTEE OF THE BOARD OK TRUSTEES
LAWRASON RIGGS, Ex officio, Chairman. E. N. HARVEY, Ex officio.
D. M. BRODIE, Ex officio. CHARLES PACKARD, Ex officio.
D. E. S. BROWN, to serve until 1943.
B. H. WILLIER, to serve until 1943.
C. W. METZ, to serve until 1944. OTTO C. GLASER, to serve until 1944.
ACT OF INCORPORATION THE LIBRARY COMMITTEE
A. C. REDFIELD, Chairman. E. G. BALL. S. C. BROOKS. M. E. KRAHL. J. W. MAYOR.
D. E. S. BROWN, Chairman. C. L. CLAFF. G. FAILLA. S. E. HILL. A. K. PARPART.
THE APPARATUS COMMITTEE
THE SUPPLY DEPARTMENT COMMITTEE
L. G. EARTH, Chairman. E. G. BALL. P. S. GALSOFF. R. T. KEMPTON. D. A. MARSLAND.
THE EVENING LECTURE COMMITTEE
B. H. WILLIER, Chairman. M. H. JACOBS. CHARLES PACKARD.
H. B. GOODRICH, Chairman. W. C. ALLEE. S. C. BROOKS. VIKTOR HAMBURGER. CHARLES PACKARD.
THE INSTRUCTION COMMITTEE
II. ACT OF INCORPORATION No. 3170
COMMONWEALTH OF MASSACHUSETTS
Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, William G. Farlow, Anna D. Phillips and B. H. Van Vleck have associated themselves with the intention of forming a Corporation under the name of the Marine Biological Laboratory, for the purpose of establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural his- tory, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the certificate of the President, Treasurer, and Trustees of said Corporation, duly approved by the Commissioner of Corporations, and recorded in this office ;
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
MARINE BIOLOGICAL LABORATORY
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 Connnomvealtli.
III. BY-LAWS OF THE CORPORATION OF THE MARINE
BIOLOGICAL LABORATORY
I. The annual meeting of the members shall be held on the second Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 11.30 A.M., daylight saving time, in each year, and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve one year, and eight Trustees to serve four years. There shall be thirty-two Trustees thus chosen divided into four classes, each to serve four years, and in addition there shall be two groups of Trustees as follows : (a) Trustees ex officio, who shall be the President of the Corporation, the Director of the Laboratory, the Associate Director, the Treasurer and the Clerk; (b) Trustees Emeritus, who shall be elected from the Trustees by the Cor- poration. 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 rights of the Trustees except that Trustees Emeritus shall not have the right to vote.
The Trustees and officers shall hold their respective offices until their successors are chosen and have qualified in their stead.
II. Special meetings of the members may be called by the Trustees to be held in Boston or in Woods Hole at such time and place as may be designated.
III. Inasmuch as the time and place of the Annual Meeting of Members 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 said meeting, at least fifteen (15) days before such meeting, to each member at his or her address as shown on the records of the Corporation.
IV. Twenty-five members shall constitute a quorum at any meeting.
V. The Trustees shall have the control and management of the affairs of the Corpora- tion; they shall present a report of its condition at every annual meeting; they shall elect one of their number President of the Corporation who shall also be Chairman of the Board of Trustees; they shall appoint a Director of the Laboratory; and they may choose such other officers and agents as they may think best ; they may fix the compensation and define the duties of all the officers and agents ; and may remove them, or any of them, except those chosen by the members, at any time ; they may fill vacancies occurring in any manner in their own number or in any of the offices. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best.
VI. Meetings of the Trustees shall be called by the President, or by any two Trustees, and the Secretary shall give notice thereof by written or printed notice sent to each Trustee by mail, postpaid. Seven Trustees shall constitute a quorum for the transaction of busi- ness. The Board of Trustees shall have power to choose an Executive Committee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient.
REPORT OF THE TREASURER
VII. The accounts of the Treasurer shall be audited annually by a certified public ac- countant.
VIII. The consent of every Trustee shall be necessary to dissolution of the Marine Biological Laboratory. In case of dissolution, the property shall be disposed of in such manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees.
IX. These By-laws may be altered at any meeting of the Trustees, provided that the notice of such meeting shall state that an alteration of the By-laws will be acted upon.
X. Any member in good standing may vote at any meeting, either in person or by proxy duly executed.
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 1942.
The accounts have been audited by Messrs. Seamans, Stetson and Tuttle, certified public accountants. A copy of their report is on file at the Laboratory and is open to inspection by members of the Corporation.
The principal summaries of their report — The Balance Sheet, Statement of Income and Expense, and Current Surplus Account — are appended hereto as Exhibits A, B and C.
The following are some general statements and observations based on the detailed reports :
/. Assets
1. Endowment Assets
At the end of 1942 the total of all the Endowment Assets was $1,071,990.90, a loss of $7,821.17 from the preceding total, due largely to the loss of $8.801.13 incurred in the sale of one of the New York City real estate holdings on which the Laboratory had held a mortgage participation. The market value of the marketable securities increased slightly during the year. Using book values for the mortgage and real estate participations for which there are no market values, the total market value of all Endowment Assets was $1,020,282.41, compared with $999,599.86 at the end of 1941.
2. Plant Assets
The total of Plant Assets (excluding the Gansett and Devil's Lane Tracts) was $1,357.761.97 after deduction of $608,146.02 accumulated Depreciation Reserve. This represents a decrease of $18,407.84. Actual additions to Plant Assets during the year totalled $12,208.30 but this gain was more than offset by depreciation charges on buildings and equipment amounting to $26,935.14.
During the year $108.00 was expended on the construction of the Library addition. This left a balance of $2,762.07 remaining from the gifts totalling $110,400.00 received in 1940 and 1941 from the Rockefeller Foundation for the addition. This unexpended balance of $2.762.07 was returned to the Rockefeller Foundation in December in accordance witb the understanding with the donor.
6 A1ARINE BIOLOGICAL LABORATORY
3. Current Assets
Current Assets, including cash, inventories and investments not in the Endow- ment Funds, amounted to $164,669.02, a decrease of $7,001.09. Current Liabilities (accounts payable) were $4,996.85 as compared with $6,423.47 so that Current Surplus was down only $5,574.47 to a total of $159,672.77.
II. Income and Expenditures
Total Income was $146,069.76, a decrease of $16,676.94 from 1941. Total expenditures including the $26,935.14 added to Depreciation Reserves were $163,- 281.69, a decrease of $8,947.02. The deficit for the year was, therefore, $17,211.93 as compared with the 1941 deficit of $9,482.01.
The decline in income was due to several factors. Income from the General Endowment and Library Funds was down from $38,879.73 in 1941 to $35,883.81. Dividends from the General Biological Supply House, Inc., dropped from $17,780.00 to $10,922.00. "Research" net income declined from $8,606.65 to $4,948.42. "Instruction" resulted in a net loss of $2,063.57 instead of the 1941 net profit of $176.45.
The Laboratory Administration met the problem created by reduced income by reducing operating expenses as shown in the detailed appendices. Maintenance expenses were substantially reduced and the usual deficits in operation of the mess, dormitory and supply departments (deficits caused only by depreciation and rental charges) were lessened. The rentals received from the United States Navy for the Laboratory properties under lease (Mess Hall, Apartment House, etc.) were also of assistance in reducing the deficit. Such rentals actually paid in 1942 amounted to $10.847.47 and were allocated to the respective accounts. In addition as of December 31st there were rental accruals clue from the Navy of $1,677.47.
EXHIBIT A MARINE BIOLOGICAL LABORATORY BALANCE SHEET, DECEMBER 31, 1942
Assets
Endowment Assets and Equities :
Securities and Cash in Hands of Central Hanover Bank and
Trust Company, New York, Trustee $1,062,364.97
Securities and Cash in Minor Funds 9,625.93
$1,071.990.90 Plant Assets :
Land $ 111,425.38
Buildings 1,322,315.51
Equipment 185,313.69
Library 320,069.89
$1,939.124.47 Less Reserve for Depreciation 608,146.02
$1,330,978.45
Cash in Reserve Fund 4,273.51
Cash in Book Fund . 22,510.01
$1,357,761.97
REPORT OF THE TREASURER
Current Assets :
Cash $ 4.965.40
Accounts Receivable 18.537.86
Inventories :
Supply Department $ 31.683.18
Biological Bulletin 12.768.29
$ 44,451.47 Investments :
Devil's Lane Property $ 45,720.27
Gansett Property 100.68
Stock in General Biological Supply House,
Inc 12,700.00
Other Investment Stocks 17,770.00
Retirement Fund 14,137.88
$ 90,428.83
Prepaid Insurance 4,291.72
Items in Suspense 1,994.34
$ 164,669.62
Total Assets $2,594,422.49
Liabilities
Endowment Funds :
Endowment Funds $1,060.069.32
Reserve for Amortization of Bond Premiums.. 2.295.65
$1,062.364.97 Minor Funds 9,625.93
$1,071,990.90 Plant Liabilities and Surplus :
Donations and Gifts $1,172,564.04
Other Investments in Plant from Gifts and Current Funds . 185,197.93
$1,357,761.97 Current Liabilities and Surplus :
Accounts Payable $ 4,996.85
Current Surplus (Exhibit C) 159,672.77
$ 164,669.62 Total Liabilities $2,594,422.49
EXHIBIT B
MARINE BIOLOGICAL LABORATORY INCOME AND EXPENSE, YEAR ENDED DECEMBER 31, 1942
Income :
Total Net
Expense Income Expense Income
General Endowment Fund $ 29,549.85 $ 29,549.85
Library Fund 6,333.96 6,333.96
Instruction $ 7,508.57 5,445.00 $ 2,063.57
Research 5,600.98 10,549.40 4,948.42
8
MARINE BIOLOGICAL LABORATORY
Evening Lectures 8.95
Biological Bulletin and Membership Dues.. 7,575.89
Supply Department 42,306.45
Mess 16,040.39
Dormitories 21,151.71
(Interest and Depreciation charged to above
3 Departments) 24,197.25
Dividends, General Biological Supply House,
Inc
Dividends, Crane Company
Rents :
Bar Neck Property 648.80
Janitor House 24.16
Danchakoff Cottages 270.11
Lecture Hall and Botany Building
Sale of Library Duplicates and Micro Films
Apparatus Rental
Sundry Income
Maintenance of Plant :
Buildings and Grounds 21,419.05
Apparatus Department 5,952.23
Chemical Department 2,789.77
Library Expense 7,883.29
Workmen's Compensation Insurance .... 541.87
Truck Expense 307.04
Bay Shore Property 86.57
Great Cedar Swamp 19.35
General Expenses :
Administration Expense 12,501.53
Endowment Fund Trustee and Safe- keeping 1,014.45
Bad Debts 355.78
Special Repairs, Supply Dep't Stone Build- ing 5,811.86
Payment to former Technical Director .... 725.00
Reserve for Depreciation 26,935.14
8,244.44 40,607.40 14,436.11 12,676.29
10,922.00 500.00
4,338.02 360.00 600.00 666.66
89.82 689.63
60.28
8.95
1,699.05 1,604.28 8,475.42
668.55
21,419.05
5,952.23
2,789.77
7,883.29
541.87
307.04
86.57
19.35
12,501.53
1,014.45 355.78
5,811.86
725.00 26,935.14
24,197.25
10,922.00 500.00
3,690.12 335.84 329.89 666.66
89.82 689.63
60.28
Excess of Expense over Income carried to Current Surplus
$163,281.69 $146.069.76 $100,194.20 $ 82,982.27
$ 17,211.93 $163,281.69
$ 17,211.93 $100,194.20
EXHIBIT C
MARINE BIOLOGICAL LABORATORY, CURRENT SURPLUS ACCOUNT YEAR ENDED DECEMBER 31, 1942
Balance, January 1, 1942 $165,247.24
Add:
Reserve for Depreciation Charged to Plant Funds $26,935.14
Bad Debts Recovered 36.39
Gain on Gansett Lot Sold 47.83
$ 27,019.36 $192,266.60
REPORT OF THE LIBRARIAN
Deduct :
Excess of Expense over Income for Year as shown in Exhibit B. . $17,211.93 Payments from Current Funds during Year for Plant Assets :
Buildings $ 3,192.99
Equipment 1,580.98
Library 7,449.33
$12,223.30 Less Received for Plant Assets Disposed of 15.00
$12,208.30
Pensions Paid $ 3,460.00
Less Retirement Fund Income 286.40
$ 3,173.60
$ 32,593.83
Balance, December 31, 1942 $159,672.77
Respectfully Submitted,
DONALD M. BRODIE,
Treasurer.
V. REPORT OF THE LIBRARIAN
The Library budget for 1942 was greatly reduced by action of the Executive Committee. For the years 193-1 — 41 inclusive it was $18,850 per year, with only slight variations; for 1942 it was $12,200, a decrease of more than $6,000. Since 1940 we have received fewer and fewer European continental journals, until now practically none come in. Our subscriptions, however, are kept up, and the jour- nals which cannot be delivered are being stored for the duration. Meanwhile no payments for these subscriptions have been made. For this reason there was an unexpended balance at the end of 1940. In 1940 the Library Committee requested that the balance, amounting to $3,977.18, be placed in a reserve fund from which to pay for the journals and back sets at such a time as they might be delivered. This request was granted by the Executive Committee. Early in the year 1941 a sum of $2,228.32 wras so spent. A similar request in 1941 was not granted, and the unexpended balance of $2,663.48 for that year reverted to the general fund of the Laboratory. No request for a reserve fund was made in 1942. The Labora- tory is now committed to pay for three years of foreign subscriptions, assuming that the journals can be delivered at some future time. There is now no adequate re- serve fund from which such payments may be made.
This year the $12,200 appropriated was expended as follows: books, $91.06; serials, $1,489.28; binding, $1,084.05; express, $174.63; supplies, $471.15; sal- aries, $7,200; back sets, $1,797.92; sundries, $26.75; and insurance, $45.00; total, $12,379.84. The sales of duplicates brought in this year $26.06 and the income from the microfilm service inaugurated in the summer amounted to $63.76, the expenses for this latter having been charged to "supplies" and "salaries."
From the "Carnegie Fund" $2,239.01 was spent for back sets and journals and $250.98 for valuable books that we term biological "classics"; in all, 15 completed back sets, 24 partially completed and 23 "classics."
10 MARINE BIOLOGICAL LABORATORY
The Woods Hole Oceanographic Institution appropriated $800 to the Library for 1942 and a balance of $154.65 remained from the 1941 budget. An expended sum of $884.54 has been reported to the Director. A balance of $70.11 was carried on to the year 1943.
Since practically no current issues of journals have come to us from Europe since June 1941, it seems best in this report to give the figures for current journals actually received rather than for the subscriptions and exchange orders due us. This explains the sharp drop in this item that follows as compared with that for 1941. In 1942 the Library received 637 current publications (1,297 in 1941) : 227 (11 new) in subscriptions to the Marine Biological Laboratory, 18 (1 new) to the Woods Hole Oceanographic Institution; 209 exchanges, 192 (2 new) with the "Biological Bulletin" and 17 (0 new) with the Woods Hole Oceanographic Institu- tion publications; 178 gifts to the former and 5 to the latter. The Marine Biologi- cal Laboratory acquired 107 books ; 43 by purchase of the Marine Biological Labora- tory (23 "classics" see above), 15 by purchase of the Woods Hole Oceanographic Institution; 16 as gifts from the authors, 30 from publishers and 3 miscellaneous. There were 47 back sets of serial publications completed ; 34 purchased by the Marine Biological Laboratory (1 5 with "Carnegie Fund") ; 2 by the Woods Hole Oceanographic Institution ; 1 secured by exchange of the "Biological Bulletin" ; 5 as gifts to the same and 5 by exchange of duplicate material. Partially completed sets were 164: purchased by the Marine Biological Laboratory, 59 (24 with "Car- negie Fund") ; by the Woods Hole Oceanographic Institution, 3; by exchange of the "Biological Bulletin," 1 ; by gift to the same, 44 ; and by exchange of duplicate material, 57. The reprint additions number 3,097: current of 1941, 436; current of 1942, 23 ; and of previous dates, 2,638. The present holdings of the Library are 50,937 bound volumes and 122,723 reprints.
Very few of the current reprints received were catalogued during 1942. From May until November three members of the staff spent the major part of their work- ing hours on the "List of serial holdings" to be published as a "supplement" to the "Biological Bulletin" in the February 1943 issue. The total reprints of date 1941 therefore will be recorded, as well as those of the date 1942, in the 1943 report. The current reprints separated from those of previous dates were first counted in the 1937 report and are summarized as follows: 1936-37, 4,602; 1938, 2,453; 1939, 2,246; and 1940, 1,887. The decline in current reprints in 1940 continues in 1941-42. It would seem that the efforts made so far by the Librarian to impress upon investigators the importance of these current reprints can have had no sus- tained effect. The best results were obtained by personal interviews of the Library with individuals and credit must be given to those, and they are considerable in number, who do conscientiously send their reprints as issued. Perhaps a better method of keeping the collection to date may be devised when the war conditions are over.
During the year seven valuable gifts in non-current reprints were received ; a total of 17,017. Of these 7,759 were new to us and will be filed for use ; 9,258, be- ing duplicate, will be placed in our duplicate files and any third copies will be for sale. The Library is indebted to Dr. Rudolf Hober for the generous gift of his collection of 7,217 reprints in the subjects of physical and physiological chemistry and physiology; to Dr. H. E. Crampton, for the high figure of 5,102 reprints on miscellaneous subjects; from Mrs. H. J. Fry and Dr. Robert Chambers, Dr. Fry's
REPORT OF THE DIRECTOR 11
collection in cytology, 2,660 in all; Dr. D. J. Edwards contributed 606 reprints; Dr. E. J. Herrick, 383; Dr. Libbie H. Hyman, 965; and Dr. B. M. Davis, 84. Miss Mathilda Koch kindly sent to us several sets of journals and four books from the Library of her brother, Dr. Waldemar Koch, with the understanding that the books should be incorporated in the Library, and the journals, which are duplicates to us, should revert to our use for sale or exchange in case the sale of these is not consummated within a given period. The addition of 7,759 reprints to the back files of reprints is the highest number that has ever been added in one year to the Library's collection and Dr. Hober's gift is the largest single collection that this Library has ever received.
VI. THE REPORT OF THE DIRECTOR
To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY : Gentlemen:
I beg to submit herewith a report of the fifty-fifth session of the Marine Biologi- cal Laboratory for the year 1942.
1. Changes in Personnel. At the Trustees' meeting in 1940, Dr. F. R. Lillie presented his resignation as President of the Corporation and Chairman of the Executive Committee. He was persuaded, however, to continue his duties until suitable preparations could be made for naming his successor. The Committee entrusted with this responsibility agreed that the office of President deals largely with the external relations of the Laboratory and that search should be made for some one who would appropriately represent the Laboratory in this field. At the same time they felt that there should be a Vice President who would represent Biology. After clue consideration, Lawrason Riggs, Treasurer of the Corporation since 1924, was nominated for the Presidency, and Dr. E. Newton Harvey was named to fill the newly created position of Vice President. They were formally elected to these < ffires at the Trustees' meeting in 1942. At the Corporation meet- ing, Mr. Donald M. Brodie, formerly manager of Mr. C. R. Crane's New York office, was elected Treasurer in place of Mr. Riggs, and Dr. Otto Glaser was elected Clerk of the Corporation in place of Dr. P. B. Armstrong who resigned because of pressure of war work. The Laboratory is fortunate in securing the services of these men and confidently gives them its whole-hearted support. Dr. Lillie was named President Emeritus of the Corporation. Dr. Packard, the Director, was made Resident Director, and assumed his full time duties at the Laboratory on October 1, 1942.
2. Financial. At the present time the financial condition of the Laboratory is satisfactory, even though our income has fallen during the past few years. In 1942 it was about 16 per cent below the average of the preceding eight years, the decrease being due, in large measure, to a sharp drop in returns from the endow- ment funds and from dividends, from research fees, and from the courses of in- struction. It is a matter of gratification that many of our subscribing institutions have continued their support even though they can send few representatives or none at all. On the other hand, the income from the Supply Department increased, and so also did the item of Rentals, a result of the Navy's occupation of the Apart- ment House, the Mess, and other buildings. At the same time our expenditures have been reduced. The cost of maintaining the buildings, and of administration,
12 MARINE BIOLOGICAL LABORATORY
has fallen somewhat below the average, but the chief reductions are in the appropri- ations for the Library and the Apparatus Department. These economies have, in a sense, been forced upon us. A large proportion of the foreign journals, to which we are still subscribing, can no longer be delivered to us; our payments for them have therefore ceased. Then, also, we can buy little new scientific equipment. Thus far this has not worked any great hardship on the investigators, for with reduced attendance, the call for apparatus has lessened. What we have on hand can be adapted to new needs with the aid of the Apparatus Department staff. Un- der these conditions the Laboratory can continue to operate within its budget.
But these conditions will not long continue. When normal interchange with Europe is re-established we shall presumably receive the journals now held in storage for us, and for them we must pay approximately the amount which was taken from the Library appropriation. So, also, after the war we shall need a sub- stantial sum for the replacement of old apparatus, and more particularly, for the purchase of new tools for research to be used in new fields, such as electronics, which have been so greatly developed within the last few years.
3. Attendance. A comparison of the attendance at the Laboratory in 1941 and 1942 with the average of the preceding five-year period shows how seriously the war is affecting us. The years 1936-1940 marked the highest attendance in our history. In 1937 the total registration was 511, and in 1940, 507. The decline began in 1941 when the number of independent investigators fell off noticeably. The other groups, however, were present in normal numbers. In 1942 the attendance in all groups declined sharply, the change being most marked among the younger mem- bers. Only about one-third of the usual number of assistants was present, and only one-fourth of the beginning investigators. In the classes, attendance dropped to about two-thirds of the average except in Physiology where the falling off was greater. Many of the investigators taught at their colleges throughout the sum- mer, and will continue to do so for the duration. Others are engaged on wartime problems which they are carrying on at their own institutions. In many cases this research is in their chosen field, so their time is by no means lost. Indeed in some instances it has already opened up new fields for future exploration. But the armed services have absorbed a large proportion of the younger generation who normally would take our courses or begin research. For some time to come, few young and vigorous minds will be added to our list of investigators. All the more it is en- cumbent upon those who remain to continue and to extend their peacetime investi- gations.
RECORD OF ATTENDANCE, 1936-1942
1936 1937 1938 1939 1940 Ave. 1941 1942
Independent Investigators 226 256 246 213 253 239 228 16.0
Assistants 57 61 81 79 71 70 50 25
Beginning Investigators 76 74 53 60 62 65 59 16
Students 138 133 132 133 128 133 131 74
Corrected Totals 473 511 496 471 507 489 461 273
4. Losses by Death. In the death of Dr. Calkins the Laboratory loses a de- voted friend. His important services as Clerk of the Corporation, Secretary of the Board of Trustees, as an active member of many committees, and as head of the Protozoology course, will long be remembered.
REPORT OF THE DIRECTOR 13
During the year also occurred the death of one of our Life Members, Dr. A. Law- rence Lowell, who served as Clerk of the Corporation from 1890 to 1894.
5. The Stone Building. During the fall and winter the Stone Building has been completely renovated. The decision to do this was made when the Executive Com- mittee, after a tour of inspection, realized how serious the condition was. Only a part of the basement could be used ; the stairs were no longer safe ; the first and second floors were not strong enough to permit the storage of heavy tanks ; the shingles and trim were in bad shape. These deficiencies have been corrected. The entire basement is now available for storage, there is a new concrete floor, a new heating system, and adequate plumbing and lighting. To provide more head room, the ceiling was raised 18 inches. Many steel columns, both in the basement and in the first floor, support the great carrying beams which are still sound. The front of the first floor is now divided into offices and laboratories. The business of the Sup- ply Department can therefore be carried on in the Stone building, leaving the wooden building to be used primarily for the preparation of material. These changes were planned and carried out by Mr. Larkin and Mr. Hemenway, the latter bearing the larger share of the work. Both the inside and the outside of the building are now in excellent condition.
In summary, the Laboratory during these disturbing times is maintaining its usual services, and by rigid economies, is balancing its budget. This quiescent pe- riod will not long continue ; we must prepare for an expansion of our research facilities soon after the war ends.
6. Election of Trustees. At the meeting of the Corporation held August 11, 1942, the following Trustees were elected Trustees Emeritus :
A. P. Mathews, University of Cincinnati S. O. Mast, The Johns Hopkins University The new Trustees elected at that meeting are :
Eugene F. DuBois, Class of 1944
Eric G. Ball, Class of 1944
7. There are appended as parts of this report :
1. The Staff.
2. Investigators and Students.
3. Tabular View of Attendance, 1938-1942.
4. Subscribing and Co-operating Institutions.
5. Evening Lectures.
6. Shorter Scientific Papers.
7. Members of the Corporation.
Respectfully submitted,
CHARLES PACKARD,
Director
1. THE STAFF, 1942
CHARLES PACKARD, Director, Marine Biological Laboratory, Woods Hole, Massachusetts.
SENIOR STAFF OF INVESTIGATION
GARY N. CALKINS, Professor Emeritus of Protozoology, in residence, Columbia Univer- sity.
14 MARINE BIOLOGICAL LABORATORY
E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University.
CASWELL GRAVE, Professor of Zoology, Emeritus, Washington University.
FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of Chicago.
RALPH S. LILLIE, Professor of General Physiology, The University of Chicago.
C. E. McCLUNG, Professor of Zoology, Emeritus, University of Pennsylvania.
S. O. MAST, Professor of Zoology, Johns Hopkins University.
A. P. MATHEWS, Professor Emeritus, Biochemistry, 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
A. J. WATERMAN, Associate Professor of Biology, Williams College, in charge of course. JOHN B. BUCK, Assistant Professor of Zoology, University of Rochester. M. D. BURKENROAD, Assistant Curator, Bingham Oceanographic Foundation, Yale Uni- versity.
W. G. HEWATT, Professor of Biology, Texas Christian University. W. E. MARTIN, Associate Professor of Zoology. DePauw University. N. T. MATTOX, Assistant Professor of Zoology, Miami University. R. W. WILHELMI, Instructor in Zoology, University of Missouri.
III. LABORATORY ASSISTANT RUTH MERWIN, University of Chicago.
EMBRYOLOGY
I. CONSULTANTS
L. G. BARTH, Assistant Professor of Zoology, Columbia University. H. B. GOODRICH, Professor of Biology, Wesleyan University.
II. INSTRUCTORS
VIKTOR HAMBURGER, Professor of Zoology, Washington University, in charge of course. DONALD P. COSTELLO, Assistant Professor of Zoology, University of North Carolina (ab- sent in 1942).
CHARLES B. METZ, Teaching Fellow, California Institute of Technology. OLIN RULON, Assistant Professor of Biology, Wayne University. RAY L. WATTERSON, Instructor in Embryology, Dartmouth College.
PHYSIOLOGY I. CONSULTANTS
WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of
Medicine.
HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin. WALTER E. GARREY, Professor of Physiology, Vanderbilt University Medical School. MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania.
REPORT OF THE DIRECTOR 15
II. INSTRUCTORS
RUDOLF T. KEMPTON, Professor of Zoology, Vassar College, in charge of course. KENNETH C. FISHER, Assistant Professor of Experimental Biology, University of To- ronto. ARTHUR C. GIESE, Associate Professor of Biology, Stanford University.
F. J. M. SICHEL, Assistant Professor of Physiology, University of Vermont, College of
Medicine.
BOTANY
I. CONSULTANTS
S. C. BROOKS, Professor of Zoology, University of California.
D. R. GODDARD, Assistant Professor of Botany, University of Rochester.
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 MARY A. ROHAN S. MABELL THOMBS
APPARATUS DEPARTMENT
E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager J. D. GRAHAM R. S. LILJESTRAND
CHEMICAL DEPARTMENT
KENNETH C. BALLARD, Lawrence High School, Falmouth, Mass., Manager
SUPPLY DEPARTMENT
JAMES MC!NNIS, Manager
RUTH CROWELL GRACE HARMAN M. B. GRAY W. E. KAHLER G. LEHY
A. M. HILTON A. W. LEATHERS F. N. WHITMAN
GENERAL OFFICE
F. M. MACNAUGHT, Business Manager POLLY L. CROWELL GLADE F. ALLEN
GENERAL MAINTENANCE
T. E. LARKIN, Superintendent
F. A. CANNON T. E. TAWELL
W. C. HEMENWAY R. F. TRAVIS
R. W. KAHLER J. WYNNE
THE GEORGE M. GRAY MUSEUM GEORGE M. GRAY, Curator Emeritus
16 MARINE BIOLOGICAL LABORATORY
2. INVESTIGATORS AND STUDENTS
Independent Investigators, 1942
ADDISON, WILLIAM H. F., Professor of Normal Histology and Embryology, University of Pennsylvania, School of Medicine.
ANDERSON, THOMAS F., RCA Fellow, National Research Council.
ANDREW, WARREN, Assistant-Professor of Histology and Embryology, Baylor University, Col- lege of Medicine.
BAKER, HORACE B., Professor of Zoology, University of Pennsylvania.
BALL, ERIC G., Associate Professor, Department of Biological Chemistry, Harvard Medical School.
BALL, ERNEST, National Research Fellow in Botany, Yale University.
BARTH, L. G., Assistant Professor of Zoology, Columbia University.
BARTLETT, JAMES H., JR., Associate Professor of Theoretical Physics, University of Illinois.
BERGER, CHARLES A., Professor of Cytology and Genetics, Fordham University.
BISSONNETTE, T. H., Professor of Biology, Trinity College.
BLUM, JOHN L., Instructor in Biology, Canisius College.
BODIAN, DAVID, Associate in Epidemiology, Johns Hopkins University.
BOTSFORD, E. FRANCES, Associate Professor of Zoology, Connecticut College.
BROOKS, MATILDA M., Research Associate, University of California.
BROOKS, SUMNER C., Professor of Zoology, University of California.
BUCK, JOHN B., Assistant Professor of Zoology, University of Rochester.
BUDINGTON, R. A., Professor of Zoology, Emeritus, Oberlin College.
BURKENROAD, MARTIN D., Assistant Curator, Peabody Museum, Yale University.
CANNAN, R. KEITH, Professor, New York University College of Medicine.
CHAMBERS, ROBERT, Research Professor of Biology, Washington Square College, New York University
CHENEY, RALPH H., Professor of Biology, Long Island University.
CHILD, RUTH C., Assistant Professor, Wellesley College.
CLARK, ELEANOR L., Department of Anatomy, University of Pennsylvania.
CLARK, ELIOT R., Professor and Director of Department of Anatomy, University of Pennsyl- vania, School of Medicine.
CLOWES, G. H. A., Director of Research, Eli Lilly and Company.
CON KLIN, EDWIN G., Professor of Biology, Emeritus, Princeton University.
COPELAND, MANTON, Professor of Biology, Bowdoin College.
CROASDALE, HANNAH T., Technical Assistant, Dartmouth College.
DELBRUCK, MAX, Instructor in Physics, Vanderbilt University.
DREYER, NICHOLAS B., Associate Professor of Pharmacology, Long Island College of Medicine.
EAKIN, RICHARD M., Assistant Professor of Zoology, University of California.
ELIZABETH, SISTER MIRIAM, Associate Professor of Biology, Chestnut Hill College.
EVANS, TITUS C., Research Assistant Professor of Radiology, State University of Iowa.
FAILLA, G., Physicist, Memorial Hospital.
FISHER, KENNETH C., Assistant Professor of Physiological Zoology, University of Toronto.
FREY, DAVID G., Junior Aquatic Biologist, U. S. Fish and Wildlife Service.
FRISCH, JOHN A., Professor of Biology, Head of Biology Department, Canisius College.
GABRIEL, MORDECAI L., Lecturer in Zoology, Columbia University.
GALTSOFF, PAUL S., Biologist in Charge Shellfish Investigation, U. S. Fish and Wildlife Service.
GARREY, W. E., Professor of Physiology, Vanderbilt University School of Medicine.
GIESE, ARTHUR C., Associate Professor of Biology, Stanford University.
GLASER, OTTO C., Professor of Biology, Amherst College.
GRAND, C. G., Research Associate, Washington Square College, New York University.
GRAVE, CAS WELL, Professor of Zoology, Emeritus, Washington University.
GUREWICH, VLADIMIR, Clinical Assistant and Attending Physician, Cornell Division of the Bellevue Hospital.
HAMBURGER, VIKTOR, Professor of Zoology, Washington University.
REPORT OF THE DIRECTOR 17
HARTMAN, FRANK A., Professor and Chairman Department of Physiology, Ohio State Uni- versity.
HARVEY, ETHEL B., Research Investigator, Princeton University.
HARVEY, E. NEWTON, Professor of Physiology, Princeton University.
HAUGAARD, G., Research Assistant, Harvard University.
HAYWOOD, CHARLOTTE, Professor of Physiology, Mount Holyoke College.
HEILBRUNN, L. V., Associate Professor of Zoology, University of Pennsylvania.
HENRY, RICHARD J., Medical Student, School of Medicine, University of Pennsylvania.
HEWATT, WILLIS G., Professor of Biology, Texas Christian University.
HILL, SAMUEL E., Professor of Biology, Russell Sage College.
HOPKINS, HOYT S., Associate Professor of Physiology, New York University College of Den- tistry.
HOWE, H. E., Editor, Industrial and Engineering Chemistry.
HYMAN, CHESTER, Research Assistant, New York University.
JACOBS, M. H., Professor of General Physiology, University of Pennsylvania Medical School.
JOHLIN, J. M., Associate Professor, Vanderbilt University School of Medicine.
KEMPTON, RUDOLF T., Professor of Zoology, Vassar College.
KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of Medicine.
KOPAC, M. J., Visiting Assistant Professor of Biology, New York University.
KRAHL, M. E., Research Chemist, Eli Lilly and Company.
LILLIE, FRANK R., Professor of Embryology, Emeritus, The University of Chicago.
LILLIE, RALPH S., Professor of Physiology, Emeritus, The University of Chicago.
LITTLE, ELBERT P., Instructor in Science, Phillips Exeter Academy.
LOWENSTEIN, B. E., Research Associate, New York University, Washington Square College.
LURIA, SALVADOR E., Research Assistant in Surgical Bacteriology, Columbia University.
McBRiDE, ARTHUR F., Curator, Marine Studios Inc.
McCLUNG, C. E., Professor of Zoology, Emeritus, University of Pennsylvania.
MARSLAND, DOUGLAS A., Assistant Professor of Biology, Washington Square College, New York University.
MARTIN, WALTER E., Associate Professor of Zoology, DePauw University.
MAST, S. O., Professor of Zoology, Johns Hopkins University.
MATHEWS, A. P., Professor of Biochemistry, Emeritus, University of Cincinnati.
MATTOX, N. T., Assistant Professor of Zoology, Miami University.
MAYOR, JAMES W., Professor of Biology, Union College.
MEMHARD, ALLEN R., Crescent Rd., Riverside, Connecticut.
MENKIN, VALY, Assistant Professor of Pathology, Harvard Medical School.
METZ, CHARLES W., Head, Department of Zoology, University of Pennsylvania.
MOLTER, JOHN A., Graduate Student, University of Pennsylvania.
MOOG, FLORENCE, Graduate Student, Columbia University.
MORGAN, T. H., Professor of Biology, California Institute of Technology.
NABRIT, S. MILTON, Professor of Biology, Atlanta University.
NACHMANSOHN, DAVID, Research Associate, Columbia University.
O'BRIEN, JOHN A., Instructor in Biology, Catholic University of America.
OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research.
PACKARD, CHARLES, Director, Marine Biological Laboratory.
PIERSON, BERNICE F., Instructor in Biology, National Park College.
PLOUGH, HAROLD H., Professor of Biology, Amherst College.
POLLISTER, ARTHUR W., Associate Professor of Zoology, Columbia University.
POMERAT, GERARD R., Instructor in Biology, Harvard University.
RICHARDS, A. GLENN, JR., Instructor in Zoology, University of Pennsylvania.
Ris, HANS, Zoology Department, Columbia University.
RUGH, ROBERTS, Associate Professor, Washington Square College, New York University.
RULON, OLIN, Assistant Professor, Wayne University.
RUNYON, ERNEST H., Associate Professor of Botany, Agnes Scott College.
SCHALLEK, WILLIAM B., Biological Laboratories, Harvard University.
SCHAEFFER, A. A., Professor and Chairman of the Department of Biology, Temple University.
18 MARINE BIOLOGICAL LABORATORY
SCOTT, ALLAN C, Assistant Professor of Biology, Union College.
SCOTT, SISTER FLORENCE M., Professor of Zoology, Seton Hill College.
SHANES, ABRAHAM M., Instructor in Physiology, New York University, College of Dentistry.
SHAW, MYRTLE, Senior Bacteriologist, New York State Department of Health.
SHELDEN, FREDERICK F., Instructor in Physiology, Ohio State University.
SICHEL, ELSA KEIL, Head of the Science Department, Vermont State Normal School.
SICHEL, F. J. M., Assistant Professor of Physiology, University of Vermont, College of Medi- cine.
SIMPSON, JENNIE L. S., Assistant Professor of Botany, Hunter College.
SLIFER, ELEANOR H., Assistant Professor, Department of Zoology, State University of Iowa.
SMELSER, GEORGE K., Assistant Professor of Anatomy, Columbia University College of Physi- cians and Surgeons.
SPRINGER, STEWART, Marine Studios, Inc.
STEINBACH, H. B., Associate Professor of Zoology, Washington University.
STEWART, DOROTHY R., Associate Professor of Biology, Skidmore College.
STOREY, ALMA G., Professor Emeritus, Mount Holyoke College.
STUNKARD, HORACE W., Professor of Biology, New York University.
TAYLOR, WILLIAM R., Professor of Botany, University of Michigan.
TsWiNKEL, Lois E., Assistant Professor of Zoology, Smith College.
THIVY, FRANCESCA, Graduate Student, University of Michigan.
TRINKAUS, J. PHILIP, Graduate Student, Johns Hopkins University.
TURNER, ABBY H., Professor of Physiology, Emeritus, Mount Holyoke College.
VON SALLMANN, LUDWIG J., Assistant Professor in Ophthalmology, College of Physicians and Surgeons, Columbia University.
WATERMAN, ALLYN J., Associate Professor of Biology, Williams College.
WENRICH, D. H., Professor of Zoology, University of Pennsylvania.
WENSTRUP, EDWARD J., Head, Department of Biology, St. Vincent College.
WHITING, P. W., Associate Professor of Zoology, University of Pennsylvania.
WIERCINSKI, FLOYD J., Research Assistant, University of Pennsylvania.
WILBUR, KARL M., Instructor, Ohio State University.
WILHELMI, RAYMOND W., Instructor in Zoology, University of Missouri.
WILLIER, B. H., Professor of Zoology, The Johns Hopkins University.
WOLF, E. ALFRED, Associate Professor of Biology, University of Pittsburgh.
WOODRUFF, LORANDE L., Professor of Protozoology and Director of the Osborn Zoological Laboratory, Yale University.
WRINCH, DOROTHY, Visiting Professor, Smith, Amherst and Mt. Holyoke Colleges.
ZWEIFACH, BENJAMIN W., Research Associate in Biology, New York University.
Beginning Investigators
BRUMMER, DONALD L., Student, New York University, College of Medicine.
CLARK, ARNOLD M., Graduate Student, University of Pennsylvania.
COLE, EDITH, Undergraduate Assistant, Pennsylvania College for Women.
DANIEL, SISTER PAUL, Instructor, Chestnut Hill College.
FERGUSON, FREDERICK P., Teaching Assistant, University of Minnesota.
GROSCH, DANIEL S., Assistant Instructor, University of Pennsylvania.
HINCHEY, M. CATHERINE, Instructor in Biology, Temple University.
JAEGER, LUCENA, Graduate Student, Columbia University.
KELTCH, ANNA K., Research Chemist, Eli Lilly and Co.
LEFEVRE, PAUL G., Research Assistant, University of Pennsylvania.
METZ, CHARLES B., Teaching Fellow, California Institute of Technology.
NELSON, LEONARD, Student, University of Pennsylvania.
SOUTHWICK, MILDRED D., Instructor of Plant Science, Vassar College.
TAYLOR, HARRIETT E., Graduate Assistant, Columbia University.
WATTERSON, RAY L., Instructor. Dartmouth College.
WILSON, WALTER L., Graduate Student, University of Pennsylvania.
REPORT OF THE DIRECTOR 19
Research Assistants
ATKINSON, LENETTE R., Research Assistant, Amherst College.
BARBER, AVA J., Senior Student, University of California.
BOND, CHRISTIANA, Secretary, University of Maryland Medical School.
BROWNELL, KATHARINE A., Research Associate, Ohio State University.
BUTLER, MARY K., Research Assistant, University of Pennsylvania.
COOK, ELIZABETH J., Research Assistant, Harvard University.
DYTCHE, MARYON M., Graduate Assistant, University of Pittsburgh.
EHRENFELD, KLARA, Research Assistant, Amherst College.
GARZOLI, RAY F., Graduate Student, University of California.
HEIDENTHAL, GERTRUDE, Research Assistant, University of Pennsylvania.
HOHWIELER, HAROLD J., Graduate Assistant, Washington University.
JACOBS, JOYE E., Research Assistant, University of Maryland Medical School.
KIBRICK, ANDRE C, Teaching Assistant, New York University Medical College.
KIELICH, E. RANDOLPH, Graduate Assistant, Canisius College.
KRUGEI.IS, EDITH J., Research Assistant, Columbia University.
LONG, M. JEANNE, Research Assistant, New York University.
MACHADO, ANGELO L., Research Fellow, Yale University Medical School.
MERRITT, FRANCES A., Laboratory Assistant, Eli Lilly & Co.
PHILLIPS, CLYDE, Assistant in Anatomy, Morehouse College.
SMITH, DOUGLAS F., Research Assistant, Ohio State University.
SPIEGELMAN, S., Research Assistant, Washington University.
STEVENS, HAZEL A., Laboratory Assistant, Eli Lilly and Co.
STEVENS, KATHARINE, Student, Vassar College.
WOODWARD, ARTHUR A., JR., Research Assistant, Wesleyan University.
WURTZ, CHARLES B., Graduate Student Assistant, University of Pittsburgh.
Library Readers, 1942
AMBERSON, WILLIAM R., Professor of Physiology, University of Maryland Medical School.
BECK, L. V., Instructor in Physiology, Hahnemann Medical College.
BELDA, WALTER H., Assistant Professor, Fordham University.
BLOCK, ROBERT, Research Assistant, Yale University.
CASSIDY, HAROLD G., Yale University.
CLARK, HELEN, Instructor in Zoology, Hunter College of the City of New York.
DIAMOND, Louis K., Associate in Pediatrics, Harvard Medical School.
DIAMOND, MOSES, Associate Professor, Columbia University Dental School.
EVERETT, GUY M., Weaver Research Fellow, University of Maryland Medical School.
FOWLER, COLEEN, Johns Hopkins University.
GATES, R. R., Professor, University of London.
HUTCHINGS, Lois M., Teacher of Biology, Weequahic High School.
JONES, ARTHUR W., Research Fellow in Zoology, University of Virginia.
KREEZER, GEORGE L., Assistant Professor of Psychology, Cornell University.
LAVIN, GEORGE, Rockefeller Institute for Medical Research.
LEVINE, PHILIP, Bacteriologist and Serologist, Beth Israel Hospital.
LOEWI, OTTO, Research Professor, New York University College of Medicine.
LUDWIG, FRANCIS W., Instructor, Villanova College.
MEYERHOF, N. OTTO, Research Professor of Biochemistry, University of Pennsylvania.
MITCHELL, PHILIP H., Professor of Biology, Brown University.
NEWELL, JAMES W., Student, Cornell University Medical College.
OSTER, ROBERT H., Assistant Professor of Physiology, University of Maryland Medical School.
RENSHAW, BIRDSEY, Assistant Professor, Oberlin College.
ROBERTS, EDITH, Chairman, Department of Botany, Vassar College.
SEVAG, M. G., Assistant Professor of Biochemistry, University of Pennsylvania School of
Medicine.
SHAPIRO, HERBERT, Instructor in Physiology, Hahnemann Medical College. SHWARTZMAN, GREGORY, Head of Department of Bacteriology, The Mount Sinai Hospital. STILES, KARL A., Professor of Biology, Coe College.
20 MARINE BIOLOGICAL LABORATORY
Students, 1942 BOTANY
ARROWSMITH, HAROLD N.. JR., Student, Johns Hopkins University. BEHNKE, JANE, Student, Wellesley College. BOOTH, MARY L., Student, Smith College. HITCHCOCK, MARGARET V., Goucher College.
KINGSLEY, EUNICE L., Assistant Prof, of Botany, Kansas State College. PAULL, JOHN J., Student, Washington and Jefferson College. RICHARDSON, EDWARD A., Graduate Assistant, Rutgers University. YOUNG, MARGARET E., Assistant in Botany, Wellesley College.
EMBRYOLOGY
BEARDSLEY, MARGARET, Smith College.
Boss, MARY B., Goucher College.
BUGGS, CHARLES W., Prof, of Biology and Head, Division of the Sciences, Dillard University.
CARPENTER, ELIZABETH, Graduate Assistant, Mount Holyoke College.
CHURCHILL, WARREN S., Assistant in Zoology, University of Illinois.
COLE, EDITH, Undergraduate Assistant, Pennsylvania College for Women.
DODD, SAMUEL G., Wesleyan University.
DUNN, BARBARA, Graduate Assistant, Wellesley College.
ELIAS, CATHERINE, Volunteer Laboratory Assistant, Connecticut College.
FOSTER, JAMES J., Graduate Assistant, Amherst College.
GAJDUSEK, D. CARLETON, Student, University of Rochester.
GEISLER, SISTER FRANCIS S., S.S.J., Student, Catholic University.
LITTRELL, JUNE L., Assistant, University of Illinois.
MEMHARD, ALLEN R., Crescent Road, Riverside, Conn.
NEWFANG, DOROTHY, Mount Holyoke College.
NICKERSON, MARK, Graduate Assistant, Johns Hopkins University.
PHILBRICK, MADELINE G., Russell Sage College.
POINDEXTER, JOAN, Smith College.
PRODELL, JOHN H., Brothers College of Drew University.
REYER, RANDALL W., Cornell University.
SEITNER, MARGARET M., Hunter College of the City of New York.
SENYARD, JUANITA, Graduate Assistant, Mount Holyoke College.
SHEA, SAMUEL E., JR., Student Laboratory Instructor, Canisius College.
WOOD, MARCIA, Student, Russell Sage College.
PHYSIOLOGY
CHRISTIANSEN, GERTRUDE M., Assistant, Wellesley College.
HARDENBERGH, ESTHER, Student, Mount Holyoke College.
LARSON, VIRGINIA P., Assistant in Physiology, Vassar College.
Low, EVA M., Student, Radcliffe College.
OSTERMAN, GEORGE B., Instructor, Washington and Jefferson College.
POKER, NATHAN, Brooklyn College.
ZOOLOGY
AVILA, ENRIQUE, Compania Administradora del Guano, Lima, Peru.
BENSON, JOHN A., Undergraduate Assistant, Wesleyan University.
BREARLEY, MARGERY, Graduate Student, Mount Holyoke College.
CHRONIAK, WALTER, Massachusetts State College.
COLE, ELSIE L., Heidelberg College.
COLE, M. ETHEL, Teacher, Frick Educational Commission.
COLLARD, LAVERNE E., Oberlin College.
COSBY, EVELYN L., Laboratory Instructor in Botany, University of Richmond.
REPORT OF THE DIRECTOR 21
CREGAR, MARY, Wilson College.
DAUGHADAY, ELEANOR F., Vassar College.
DINTIMAN, SARA MAE, Rutgers University.
DONALDSON, SARA L., Graduate Assistant, Syracuse University.
DOOCHIN, HERMAN D., Student, University of Miami.
FOGG, N. W., Student, American International College.
FOSTER, JAMES J., Graduate Assistant, Amherst College.
FRANKLIN, REV. ROGER G., Prof, of Biology, St. Joseph's Seminary.
HAAS, ELIZABETH, Bennington College.
HUFFORD, VIRGINIA, Oberlin College.
HYDE, JANE E., Student, Radcliffe College.
JOHNSON, VIENO T., 44 Francis Ave., Cambridge, Mass.
KEISTER, MARGARET L., Instructor, Wheaton College.
LESAGE, MAURICE C., Teacher, Society of Divine Word.
LORENTZ, JOHN J., Graduate Student, Fordham University.
MANNY, ELLA T., Sarah Lawrence College.
NEWCOMER, STANLEY, Assistant, Cornell University.
O'RouRK, ANN E., Duke University.
PETERSON, HAROLD L., Student Assistant, Drew University.
PHILBRICK, MADELINE G., Student, Russell Sage College.
RAYNER, HARRIET A., Massachusetts State College.
SAUNDERS, JOHN W., Graduate Assistant, Johns Hopkins University.
SCHMEISSER, ELIZABETH F., Student, Sweet Briar College.
TAFT, EDITH D., Wheaton College.
WATERMAN, GEORGE E., Professor of Biology, Assumption College.
WECKSTEIN, ABRAHAM M., Instructor of Biology, New York University.
WHITE, MARCIA R., Student, Cornell University.
WOOD, MARCIA, Student, Russell Sage College.
3. TABULAR VIEW OF ATTENDANCE
193S 1939 1940 1941 1942
INVESTIGATORS— Total 380 352 386 337 201
Independent 246 213 253 197 132
Under instruction 53 60 62 59 16
Research assistants 81 79 71 50 25
Library readers 31 28
STUDENTS— Total 132 133 128 131 74
Zoology 54 55 55 36
Protozoology (not given after 1940 ) .' 10 12 7
Embryology 34 36 34 37 24
Physiology 22 21 22 24 6
Botany 12 9 10 15 8
TOTAL ATTENDANCE 512 485 514 468 275
Less persons registered as both students and investi- gators 16 14 7 2
496 471 507 461 273
INSTITUTIONS REPRESENTED— Total 151 162 148 144 126
By investigators 125 132 112 102 83
By students 67 79 72 43
SCHOOLS AND ACADEMIES REPRESENTED
By investigators 4 2 1 5 2
By students 1 2 2 2 0
FOREIGN INSTITUTIONS REPRESENTED
By investigators 14 8 2 3 0
Bv students . 31110
22
MARINE BIOLOGICAL LABORATORY
4. SUBSCRIBING AND CO-OPERATING INSTITUTIONS
1942
Amherst College
Atlanta University
Beth Israel Hospital
Biological Institute, Philadelphia, Pennsyl- vania
Bowdoin College
Brooklyn College
Brown University
Bryn Mawr College
Canisius College
College of Physicians and Surgeons
Columbia University
Cornell University
Cornell University Medical College
Drew University
Duke University
Fordham University
Frick Educational Commission
Goucher College
Harvard University
Harvard University Medical School
Heidelburg College
Hunter College
Industrial and Engineering Chemistry, of the American Chemical Society
John and Mary Markle Foundation
Johns Hopkins University
Julius Rosenwald Fund
Eli Lilly and Co.
Long Island University
Marine Studios, Inc.
Massachusetts State College
Morehouse College
Mount Sinai Hospital, New York City
National Research Council
New York State Department of Health
New York University
New York University College of Medicine New York University Washington Square
College
Oberlin College Ohio State University Pennsylvania College for Women Princeton University Radcliffe College
Rockefeller Institute for Medical Research Russell Sage College Rutgers University
St. Joseph's Seminary, Dunwoodie, New York Smith College State University of Iowa Sweet Briar College Syracuse University Tufts College Union College University of Cincinnati University of Illinois University of Maryland Medical School University of Missouri L^niversity of Pennsylvania University of Pennsylvania School of Medicine University of Pittsburgh University of Rochester Vanderbilt University Vanderbilt University Medical School Vassar College Villanova College Washington University Wellesley College Wesleyan University Wheaton College
Woods Hole Oceanographic Institution Yale University
5. EVENING LECTURES, 1942
Friday, June 26
DR. MICHAEL HEIDELBERGER ''Biological Aspects of Immunity and Com- plement Action." Friday, July 3
DR. DONALD R. GRIFFIN "Echo Sounding by Flying Bats."
Friday, July 10
DR. R. RUGGLES GATES "The Nucleolus and Phylogeny."
Friday, July 17
DR. E. NEWTON HARVEY "Animal Luminescence."
Friday, July 24
MR. PER HOST "Norway Fights On."
Friday, July 31
REPORT OF THE DIRECTOR
DR. DAVID NACHMANSOHN ''On the Mechanism of Transmission of
Nerve Impulses." Friday, August 7
PROF. SELMAN A. WAKSMAN "Science in Soviet Russia on the Eve of the
World War." Friday, August 14
DR. A. GLENN RICHARDS, JR "Electron Microscope Studies of Insect
Structures and Tissues." Friday, August 21
DR. ROBERT F. GRIGGS "Timber Lines as Indices of Climatic
Change." Thursday, August 27
DR. OSCAR W. RICHARDS "The Precision of Sectioning with a Micro- tome." Friday, August 28
DR. C. W. METZ "Evolutionary Chromosome Changes in Sci-
ara as Shown by the Giant Salivary Gland Chromosomes."
6. SHORTER SCIENTIFIC PAPERS, 1942
Tuesday, July 21
DR. K. C. FISHER AND
GRACE W. SCOTT "The physiological basis of temperature 'se- lection' by fish."
DR. J. R. STERN AND
K. C. FISHER "The action of narcotics on oxygen con- sumption of resting and caffeinized frog muscle."
DR. A. C. GIESE AND
E. L. TATUM "Effects of vitamins of the B-complex on
respiration of Neurospora mutants." Tuesday, August 4
MR. SOL SPIEGELMAN "Differential effects on the mass and time
of appearance of regenerants in Tubu- laria."
Miss FLORENCE MOOG "Some effects of temperature in the regen- eration of Tubularia."
DR. MORDECAI GABRIEL "The effect of temperature on vertebral vari- ations in Fundulus heteroclitus." Tuesday, August 18
DR. DOROTHY WRINCH "The structure of biologically active mem- branes."
DR. DOUGLAS MARSLAND "The contractile mechanism in unicellular
melanophores."
DR. E. H. RUNYON "The aggregation of separate cells of Dicty-
ostelium to form a multicellular body." Tuesday, August 25
DR. G. M. EVERETT "Vitamin B, deficiency in the cat." Motion
pictures in color.
DR. T. H. BISSONNETTE "Experimental modification of molts, and
color-changes by controlled lighting of the Bonaparte weasel."
24 MARINE BIOLOGICAL LABORATORY
7. MEMBERS OF THE CORPORATION, 1942
1. LIFE MEMBERS
ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France.
ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Maryland.
BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York.
BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts.
CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania.
COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin.
CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey.
COWDRY, DR. E. V., Washington University, St. Louis, Missouri.
EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Massachusetts.
FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France.
GARDINER, MRS. E. G., Woods Hole, Massachusetts.
JACKSON, MR. CHAS. C., 24 Congress Street, Boston, Massachusetts.
JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts.
KING, MR. CHAS. A.
KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York.
LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland.
LOWELL, MR. A. L., 17 Quincy Street, Cambridge, Massachusetts.
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, MR. J. PIERPONT, JR., Wall and Broad Streets. New York City, New
York.
MORGAN, MRS. T. H., Pasadena, California. MORGAN, PROF. T. H., Director of Biological Laboratory, California Institute of
Technology, Pasadena, California.
MORRILL, DR. A. D., Hamilton College, Clinton, New York. NOYES, Miss EVA J.
PORTER, DR. H. C., University of Pennsylvania. Philadelphia, Pennsylvania. SCOTT, DR. ERNEST L., Columbia University, New York City, New York. SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts. SHEDD, MR. E. A. THORNDIKE, DR. EDWARD L., Teachers College, Columbia University, New York
City, New York.
TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, New York. TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, Illinois. WAITE, PROF. F. C., 144 Locust Street, Dover, New Hampshire. WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California.
2. REGULAR MEMBERS
ABRAMOWITZ, DR. ALEXANDER A., Biological Laboratories, Harvard University,
Cambridge, Massachusetts.
ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts. ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia.
Pennsylvania.
REPORT OF THE DIRECTOR
ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New York.
ALBAUM, DR. HARRY G., 3115 Avenue I, Brooklyn, New York.
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., Memorial Hospital, 444 East 68th Street, New York City, New York.
ANGERER, DR. CLIFFORD A., Department of Physiology, Ohio State University, Co- lumbus, Ohio.
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.
BEADLE, DR. G. \V., School of Biological Sciences, Stanford University, California.
BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, low^a City, Iowa.
BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana.
BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts.
BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Massa- chusetts.
BINFORD, PROF. RAYMOND, Buck Creek Camp, Marion, North Carolina.
BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut.
BLANCHARD, PROF. KENNETH C., Washington Square College, New York Univer- sity, New York City, New York.
BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City, Iowa.
BORING, DR. ALICE M., Yenching University, Peking, China.
BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wisconsin.
BRODIE, MR. DONALD M., 522 Fifth Avenue, New York City, New York.
BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, \Vashington 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.
26 MARINE BIOLOGICAL LABORATORY
BROWN, DR. DUGALD E. S., New York University, College of Dentistry, 209 East 23d Street, New York City, New York.
BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern University, Evanston, Illinois.
BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts.
BUCK, DR. JOHN B., Department of Zoology, University of Rochester, Rochester, New York.
BUDINGTON, PROF. R. A., Winter Park, Florida.
BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia.
BUMPUS, PROF. H. C., Duxbury, Massachusetts.
BYRNES, DR. ESTHER F., 1803 North Camac Street, Philadelphia, Pennsylvania.
CALKINS, PROF. GARY N., Columbia University, New York City, New York.
CANNAN, PROF. R. K., New York University College of Medicine, 477 First Ave- nue, New York City, New York.
CARLSON, PROF. A. J., Department of Physiology, The University of Chicago, Chi- cago, Illinois.
CAROTHERS, DR. E. ELEANOR, 134 Avenue C. East, Kingman, Kansas.
CARPENTER, DR. RUSSELL L., Tufts College, Tufts College, Massachusetts.
CARROLL, PROF. MITCHELL, Franklin and Marshall College, Lancaster, Pennsyl- vania.
CARVER, PROF. GAIL L., Mercer University, Macon, Georgia.
CATTELL, DR. McKEEN, Cornell University Medical College. 1300 York Avenue, New York City, New York.
CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, New York.
CATTELL, MR. WARE, Smithsonian Institution Building, 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, Department of Biology, Brown University, Providence, Rhode Island.
CLARK, PROF. E. R., University of Pennsylvania Medical School, Philadelphia, Pennsylvania.
CLARK, DR. LEONARD B., Department of Biology, Union College, Schenectady, New York.
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., College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York City, New York.
COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio.
REPORT OF THE DIRECTOR 27
COLTON, PROF. N. 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., Barnard College, Columbia University, New York City, New York.
CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio.
CURTIS, DR. MAYNIE R., 377 Dexter Trail, Mason, Michigan.
CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri.
DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan.
DAVIS, DR. DONALD W., College of William and Mary, Williamsburg, Virginia.
DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts.
DAWSON, DR. J. A., The College of the City of New York, New York City, New York.
DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut.
DEMEREC, DR. M., Carnegie Institution of Washington, Cold Spring Harbor, Long Island, New York.
DILLER, DR. WILLIAM F., 1016 South 45th Street, Philadelphia, Pennsylvania.
DODDS, PROF. G. S., Medical School, University of West Virginia, Morgantown, West Virginia.
DOLLEY, PROF. WILLIAM L., University of Buffalo, Buffalo, New York.
DONALDSON, DR. JOHN C., University of Pittsburgh, School of Medicine, Pitts- burgh, Pennsylvania.
DuBois, DR. EUGENE F., Cornell University Medical College, 1300 York Avenue, New York City, New York.
DUGGAR, DR. BENJAMIN M., University of Wisconsin, Madison, Wisconsin.
DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota.
DURYEE, DR. WILLIAM R., Department of Biology, Washington Square College, New York University, New York City, New York.
EDWARDS, DR. D. J., Cornell University Medical College, 1300 York Avenue, New York City, New York.
ELLIS, DR. F. W., Monson Massachusetts.
EVANS, DR. TITUS C., 723 Kirkwood, Iowa City, Iowa.
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.
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.
MARINE BIOLOGICAL LABORATORY
FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto,
Canada.
FLEISHER, DR. MOVER S., 20 North Kingshighway, St. Louis, Missouri. FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachu- setts.
FRISCH, DR. JOHN A., Canisius College, Buffalo, New York. FURTH, DR. JACOB, Cornell University Medical College, 1300 York Avenue, New
York City, New York.
GAGE, PROF. S. H., Cornell University, Ithaca, New York.
GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Mary- land.
GARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee. GEISER, DR. S. W., Southern Methodist University, Dallas, Texas. GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois. GLASER, PROF. O. C., Amherst College, Amherst, Massachusetts. GOLDFORB, PROF. A. J., College of the City of New York, Convent Avenue and 139th
Street, New York City, New York.
GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut. GOTTSCHALL, DR. GERTRUDE Y., 1630 Rhode Island Avenue, N.W., Washington,
D. C.
GRAHAM, DR. J. Y., University of Alabama, University, Alabama. 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. GRAVE, PROF. CASWELL, Washington University, St. Louis, Missouri. GRAY, PROF. IRVING E., Duke University, Durham, North Carolina. GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City,
New York. GUDERNATSCH, J. FREDRICK, 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., Department of Biology, Duquesne University, Pittsburgh,
Pennsylvania. 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.
REPORT OF THE DIRECTOR
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.
HAZEN, DR. T. E., Barnard College, Columbia University, New York City, New York.
HECHT, DR. SELIG, Columbia University, New York City, Ne\v 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.
HIBBARD, DR. HOPE, Department of Zoology, Oberlin College, Oberlin, Ohio.
HILL, DR. SAMUEL E., Department of Biology. Russell Sage College, Troy, New York.
HINRICHS, DR. MARIE, Department of Physiology and Health Education, South Illinois Normal University, Carbondale, Illinois.
HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts.
HOADLEY, DR. LEIGH, Harvard University, Cambridge, Massachusetts.
HOBER, DR. RUDOLF, University of Pennsylvania, Philadelphia, Pennsylvania.
HODGE, DR. CHARLES, IV, Temple University, Department of Zoology, Philadelphia, Pennsylvania.
HOGUE, DR. MARY J., University of Pennsylvania Medical School, Philadelphia, Pennsylvania.
HOLLAENDER, DR. ALEXANDER, c/o National Institute of Health, Laboratory of In- dustrial Hygiene, Bethesda, Maryland.
HOOKER, PROF. DAVENPORT, University of Pittsburgh, School of Medicine, Depart- ment of Anatomy, Pittsburgh, Pennsylvania.
HOPKINS, DR. DWIGHT L., Mundelein College, 6363 Sheridan Road, Chicago, Illi- nois.
HOPKINS, DR. HOYT S., New York University, College of Dentistry, New York- City, New York.
HOWE,' DR. H. E., 1155 16th St., N.W., American Chemical Society Bldg., Wash- ington, D. C.
HOWLAND, DR. RUTH B., Washington Square College, New York University. Washington Square East, New York City, New York.
HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia.
HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City. New York.
IRVING, PROF. LAURENCE, Swarthmore College, Swarthmore, Pennsylvania.
ISELIN, MR. COLUMBUS O'D., Woods Hole, Massachusetts.
JACOBS, PROF. MERKEL H., School of Medicine, University of Pennsylvania, Phila- delphia, Pennsylvania.
JENKINS, DR. GEORGE B., 30 Gallatin Street, N.W., Washington, D. C.
30 MARINE BIOLOGICAL LABORATORY
JENNINGS, PROF. H. S., Department of Zoology, University of California, Los An- geles, California.
JEWETT, PROF. J. R., 44 Francis Avenue, Cambridge, Massachusetts.
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.
KILLE, DR. FRANK R., Swarthmore College. Swarthmore, Pennsylvania.
KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia.
KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, 36th Street and Woodland Avenue, Philadelphia, Pennsylvania.
KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa.
KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York.
KOPAC, DR. M. J., Washington Square College, New York University, New York- City, New York.
KORR, DR. I. M., Department of Physiology, New York University, College of Medi- cine, 477 First Avenue, New York City, New York.
KRAHL, DR. M. E., Lilly Research Laboratories, Indianapolis, Indiana.
KRIEG, D*. WENDELL J. S., New York University, College of Medicine, 477 First Avenue, New York City, New York.
LANCEFIELD, DR. D. E., Queens College, Flushing, New York.
LANCEFIELD, DR. REBECCA C, Rockefeller Institute, 66th Street and York Avenue, Newr York City, New York.
LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts.
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.
LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri.
LOEWI, PROF. OTTO, 155 East 93d Street, New York City, New York.
LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York City, New York.
LUCAS, DR. ALFRED M., Zoological Laboratory, Iowa State College, Ames, Iowa.
LUCAS, DR. MIRIAM SCOTT, Department of Zoology, Iowa State College, Ames, Iowa.
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, Maryland State Teachers College, Towson, Mary- land.
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.
MACLENNAN, DR. RONALD F., 174 Forest Street, Oberlin, Ohio.
MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa- chusetts.
REPORT OF THE DIRECTOR 31
McCLUNG, PROF. C. E., 417 Harvard Avenue, Swarthmore, Pennsylvania.
McCoucH, DR. MARGARET SUM WALT, University of Pennsylvania Medical School, Philadelphia, Pa.
MCGREGOR, DR. J. H., Columbia University, New York City, New York.
MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario, London, Canada.
MAGRUDER. DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Bos- ton, Massachusetts.
MALONE, PROF. E. F., College of Medicine, University of Cincinnati, Department of Anatomy, Cincinnati, Ohio.
MAN WELL, DR. REGINALD D., Syracuse University, Syracuse, New York.
MARSLAND, DR. DOUGLAS A., Washington Square College, New York University, Xew 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., University of Cincinnati, Cincinnati, Ohio.
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, University of Missouri, Columbia, Missouri.
MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania.
MEIGS, MRS. E. B., 1736 M Street, N.W., Washington. D. C.
MENKIN. DR. VALY, Harvard Medical School, Boston, Massachusetts.
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. F., Department of Zoology, University of Minnesota, Minne- apolis, Minnesota.
MITCHELL, DR.' PHILIP H., Brown University, Providence, Rhode Island.
MOORE, DR. CARL R., The University of Chicago, Chicago, Illinois.
MORGAN, DR. ISABEL M., Rockefeller Institute, York Avenue at 66th Street, New York City, New York.
MORGULIS, DR. SERGIUS, University of Nebraska, Omaha, Nebraska.
MORRILL, PROF. C. V., Cornell University Medical College, 1300 York Avenue, New York City, New York.
MOSER, DR. FLOYD, Department of Biology, University of Alabama, University, Alabama.
MULLER, PROF. H. J., Amherst College, Amherst, Massachusetts.
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.
32 MARINE BIOLOGICAL LABORATORY
NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey.
OKKELBERG, DR. PETER, Department of Zoology, University of Michigan, Ann Arbor, Michigan.
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., Lilly Laboratory Clinical Research, Indianapolis City Hos- pital, Indianapolis, Indiana.
PAPPENHEIMER, DR. A. M., Columbia University, New York City, New York.
PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts.
PARMENTER, DR. C. L., Department of Zoology, University of Pennsylvania, Phila- delphia, Pennsylvania.
PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jersey.
PATTEN, DR. BRADLEY M., University of Michigan Medical School. Ann Arbor. Michigan.
PAYNE, PROF. F., University of Indiana, Bloomington, Indiana.
PEEBLES, PROF. FLORENCE, Lewis and Clark College, Portland, Oregon.
PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin.
PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts.
POLLISTER, DR. A. W., Columbia University, New York City, New York.
POND, DR. SAMUEL E., 1203 Enfield Street, Thompsonville, Connecticut.
PRATT, DR. FREDERICK H., Boston University, School of Medicine, Boston, Massa- chusetts.
PROSSER, DR. C. LADD, University of Illinois, Urbana, Illinois.
RAND, DR. HERBERT W., Harvard University, Cambridge, Massachusetts.
RANKIN, DR. JOHN S., Zoology Department, University of Washington, Seattle, Washington.
REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massachusetts.
RENSHAW, PROF. BIRDSEY, 4600 Harling Lane, Bethesda, Maryland.
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, PROF. A. G., Department of Zoology, University of Pennsylvania, Phila- delphia, Pennsylvania.
RICHARDS, DR. O. W., Research Department, Spencer Lens Company, 19 Doat Street, Buffalo, New York.
RIGGS, LAWRASON, JR., 120 Broadway, New York City, New York.
ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio.
ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts.
REPORT OF THE DIRECTOR
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., Naval Medical School, National Naval Medical Center, Beth- esda, Maryland.
RUGH, DR. ROBERTS, Department of Biology, Washington Square College, New York University, New York City, New York.
SASLOW, DR. GEORGE, 72 Grozier Road, Cambridge, Massachusetts.
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.
SCHECHTER, DR. VICTOR, College of the City of New York, 139th Street and Con- vent Avenue, New York City, New York.
SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio.
SCHMITT, PROF. F. O., Department of Biology and Public Health, Massachusetts Institute of Technology, Cambridge, Massachusetts.
SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massa- chusetts.
SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York City, New York.
SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia University, New York City, New York.
SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Pennsylvania.
SCOTT, DR. ALLAN C.. Union College, Schenectady, New York.
SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, New Jersey.
SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College, Greens- burg, Pennsylvania.
SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York.
SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, 630 West 168th Street, New York City, New York.
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.
SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio.
34 MARINE BIOLOGICAL LABORATORY
SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington,
Indiana.
SPEIDEL, DR. CARL C., University of Virginia. University, Virginia. STABLER, DR. ROBERT M., Department of Zoology, University of Pennsylvania,
Philadelphia, Pennsylvania.
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., Overly Biochemical Research Foundation, 254 W. 31st
Street, New York City, New York.
STEWART, DR. DOROTHY R., Skidmore College, Saratoga Springs, New York. STOKEY, DR. ALMA G., Department of Botany, Mount Holyoke College, South
Hadley, Massachusetts. STRONG, PROF. O. S., College of Physicians and Surgeons, Columbia University,
New York City, New York. STUNKARD, DR. HORACE W., New York University, University Heights, New
York. STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena,
California. SUMMERS, DR. FRANCIS MARION. Department of Biology, College of the City of
New York. New York City, New York. SWETT, DR. FRANCIS H., Duke University Medical School, Durham, North
Carolina.
TAFT, DR. CHARLES H., JR., University of Texas Medical School, Galveston, Texas. TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio. TAYLOR, DR. C. V., Leland Stanford University, Leland Stanford, California. TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michigan. TEWINKEL, DR. L. E.. Department of Zoology, Smith College, Northampton,
Massachusetts. TURNER, DR. ABBY H., Department of Physiology, Mount 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.
UNGER, DR. W. BYERS, Dartmouth College, Hanover, New Hampshire. VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, Ohio. WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge,
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.
REPORT OF THE DIRECTOR
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. YNTEMA, DR. C. L., Department of Anatomy, Cornell University Medical College,
1300 York Avenue, New York City, New York. YOUNG, DR. B. P., Cornell University, Ithaca, New York. YOUNG, DR. D. B.. 7128 Hampden Lane, Bethesda, Maryland.
SEXUAL ISOLATION, MATING TYPES, AND SEXUAL RESPONSES TO DIVERSE CONDITIONS IN VARIETY 4, PARAMECIUM AURELIA l
T. M. SONNEBORN 2 AND RUTH V. DIPPELL
(Department of Zoology, Indiana University, Bloomingtori)
In previous publications (Sonneborn, 1938; 1939; 1943) the species Para- rnecium aurelia has been shown to consist of a number of sexually isolated and physiologically distinct groups of races. Their sexual isolation is perhaps sufficient ground for assigning these groups to different species; but as all are morphologically similar and conform to the description of the species Paramecium aurelia, it seems more practical for the present at least to designate them as varieties of this species. Each of these varieties consists of two classes of in- dividuals that are morphologically identical but physiologically different. These two classes of individuals mate with each other, but neither class mates with other individuals of the same class or with either of the two classes that occur in any other variety of the species. The two classes of individuals within each variety are known as mating types and, in P. aurelia, they are designated by Roman numerals. The diverse varieties are designated by Arabic numerals.
The present paper is the first of a series dealing with the general biology and genetics of variety 4, containing the mating types VII and VIII. Each variety thus far studied has proven to be specially favorable for the study of certain problems of protozoan biology and genetics not so readily investigated in other varieties. As will appear in the course of this series of papers, investigations on variety 4 have yielded information on a number of important problems. In this first paper of the series we set forth the foundation on which the work of the later papers is based : demonstration of the existence of variety 4, and an account of its mating types and the conditions under which they mate.
MATERIAL
Among the 53 races of P. aurelia collected from different sources in nature and studied in this laboratory, only the following four belong to variety 4: Race 29 collected by Dr. R. F. Kimball from Ben's Run, Hebbville, Maryland, in
1938.
Race 32 collected by Dr. Kimball from a pond in Towson, Maryland, in 1938. Race 47 collected by Dr. A. C. Giese from a pool across the Bay from Berkeley, California, and sent to me in February 1939. Race 51 collected by Mrs. Aner Laubscher at Spencer, Indiana, in August 1939.
Before intensive study of these races began in the spring of 1942, they were maintained in quart jars of hay infusion to which boiled hay strips were added every month or two. In the course of this period, race 47 either changed one
1 Contribution No. 318 from the Department of Zoology, Indiana University.
2 Aided by a grant from the Rockefeller Foundation.
36
MATING TYPES IN PARAMECIUM AURELIA 37
of its characters or was mislabelled, for in 1939 it produced a unique type of lethal action on other races and no trace of this action has appeared in our recent work. In the following studies, these four races were cultivated in desiccated lettuce infusion to which a pure culture of the bacterium Aerobacter aerogenes was added.
OCCURRENCE, SEXUAL ISOLATION AND MATING TYPES OF VARIETY 4
In order to discover whether a race or group of races constitutes a new variety (in the sense in which this term is employed here, i.e., a sexually isolated group of races), it is required to demonstrate that it contains mating types which inter- breed with each other but not with those in any other known variety. This is made possible by the fact that all the mating types so far found in P. aurelia ordinarily reproduce true to type during vegetative reproduction and so yield from a single individual a clone containing one mating type only. Samples of clones of unidentified races may then be mixed with samples of sexually reactive clones of each of the known mating types. If no mating occurs in any of these mixtures, this is evidence that the new races do not contain any of the known mating types; but the evidence is not convincing unless it is certain that the clones of the new races, as well as those of the known mating types, are in sexually reactive condition at the time the tests are carried out. This can be achieved only when the clones of the new races mate with each other in appropriate combinations.
Such an analysis was carried out on the four races discusssed in this paper. Clones of each of these races were mixed with sexually reactive cultures of each of the six known mating types (I, II, III, IV, V, and VI) and no mating resulted. As repeated trials gave the same result, the six known mating types seemed not to occur among the four new races. However, at that time mating also failed to occur in mixtures of different clones and different races of the four new races. Under such conditions, conclusive proof that they constituted a new variety could not be given; they might simply have been immature. In April 1942 this diffi- culty disappeared when mating was observed for the first time in race 32. As some of the individuals were coming together in preparation for conjugation, they were separated before they had time to unite firmly and cultures were grown from the isolated members of the split pairs. The resulting clones proved to be of unlike mating types for no mating occurred within either clone alone, but the characteristic clumping reaction and conjugation took place when samples of the two clones from a split pair were mixed together. The same clones, while in this reactive condition, failed to clump or conjugate with any of the six pre- viously known mating types, although all of these were at the time in highly reactive sexual condition. Hence, there occur in race 32 two mating types unlike any of those previously known. They were therefore called mating types VII and VIII. All clones of race 32 available at that time, and subsequently, have been found to belong to either one or the other of these two mating types. When these two types were mixed with samples of clones of the remaining three races (29, 47 and 51), clumping and conjugation occurred in the mixtures with type: VIII, but not in the mixtures with type VII. These three races therefore con- tained type VII only and all clones examined at that time in these three races
38 SONNEBORN AND DIPPELL
were found to be of type VII. The four races 29, 32, 47 and 51 thus constitute a fourth variety with two new mating types VII and VIII.
Subsequently, and at a definitely known time, type VIII arose independently in race 51, but it has still not been found in races 29 or 47 in spite of a prolonged and intensive search for it. However, type VIII might well arise eventually in these races also as it has already done in the other two races.
SEXUAL RESPONSES TO DIVERSE CONDITIONS IN VARIETY 4
The nutritive conditions for conjugation appear to be the same in variety 4 as in the three previously described varieties: the animals must be neither very well fed nor completely starved, but in a declining nutritive condition. The strongest mating reactions take place when there are in progress, in the cultures to be mixed, the last fissions before the food supply is exhausted.
As diurnal periodicities in the occurrence of the mating reaction exist in two of the three previously described varieties of P. aurelia (Sonneborn, 1938; 1939), the possibility of its occurrence was examined in variety 4. For this purpose, cultures of the races 29 and 47 and cultures of each mating type in the races 32 and 51 were prepared by growing them for 6 days exposed to the light of a north window during the daylight hours. The plan was to mix samples of each of the type VIII cultures (from races 32 and 51) with each of the type VII cultures (from all four of the races) at four-hour intervals through at least one complete cycle of 24 hours. In order to be sure to have cultures in the proper nutritive condi- tion at all times, the six original cultures were subcultured in triplicate the evening before the tests were to be made and the three subcultures of each original were fed in the ratio of 1 : 2 : 4 volumes of culture fluid. During the daylight hours there was no difficulty in making the required mixtures, but at night precautions had to be taken to avoid exposing the cultures to light in so far as possible. This was accomplished as follows. Samples of all the cultures to be mixed at night were put into depression slides before dark. The two depressions of each slide contained two cultures that were later to be mixed. There was a separate slide for each combination and each time of mixture, with ample duplicates for emer- gencies. All of these slides were placed in moist chambers and were covered at night with black cloth. At the time for mixture, a very dim flashlight was di- rected away from the culture dishes, the appropriate slides were removed from the moist chambers, and the fluid from one depression on each slide was pipetted into the other depression of the same slide. Two or three minutes later the mix- ture was examined under the microscope with the faint light from the flashlight. The mixtures were then returned to the cloth-covered moist chambers.
A complete set of eight mixtures was made every four hours beginning at 5:15 P.M. on February 13 and continuing until 9:15 P.M. on February 14 Additional sets were made on other days at various times from 8 A.M. to 10 P.M. The agglutinative mating reaction occurred at once in mixtures made at every one of the different hours tested. There was thus no indication of any diurnal periodicity in the mating reaction. In this respect variety 4 is like variety 1 and unlike varieties 2 and 3 (Sonneborn, 1938; 1939).
The relation of temperature to the occurrence of conjugation was studied in five series of experiments. In each series, the same eight combinations of cul-
MATING TYPES IN PARAMECIUM AURELIA 39
tures were brought together as in the preceding experiments on diurnal periodicity. In series 1, each of the six cultures was grown for 6 days at 9°, 16.5°, 20° and 25° C. ; then a set of eight mixtures wras made and retained at each temperature and duplicate sets from 9° and 16.5° were immediately placed at 25°. In series 2, the same cultures were grown for one day at 9°, 15.5°, 21° and 25.5°; mixtures were made as in series 1, duplicate sets of mixtures from the two lower tempera- tures again being placed at once at the highest temperature. In series 3, the same six cultures were grown for 13 days at 9°, 15.5° and 26°; then mixtures were made and retained at the same temperatures and duplicate sets of mixtures from the two lower temperatures were again placed at the highest temperature; in addi- tion two extra sets of mixtures were made from the 26° cultures: one was im- mediately placed at 9° and the other at 15.5°. In series 4, cultures were grown for one day at 22°, 30° and 36°; one set of mixtures was made and retained at each temperature, one set from 30° and one from 36° was placed at 22° and two sets from 22° were placed at 30° and 36° respectively. In series 5 the six cultures were grown for several days at 21°, then five sets of mixtures were placed at 10°, 19°, 24.5°, 29° and 39°, respectively. We report first the results on mixtures retained at the temperatures at which the cultures were grown, then the results of changing the temperature at the time the mixtures were made.
Cultures Grown and Tested at 9° C. Three sets of eight mixtures between types VII and VIII (series 1, 2, and 3) were grown and tested at 9°. In 20 of these mixtures no conjugation occurred at all; in the other four mixtures (all from series 1) less than 3 per cent of the animals conjugated. The mixtures of series 1 were observed 8^ hours; series 2, 31 hours; and series 3, 23 days. Thus at 9° conjugation occurs in but a small proportion of mixtures and among only small proportions of the animals in these.
Cultures Grown and Tested at 15.5° to 16.5° C. Three sets (series 1,2, and 3) of eight mixtures each were grown and tested at this temperature. The first two sets reacted poorly: half of the 16 mixtures gave no conjugation at all and the other half gave only 1 to 3 per cent conjugation. In the third set, one mixture gave 50 per cent conjugation and the other seven gave 15 to 25 per cent. Thus conjugation occurs in more of the cultures and may occur in a much higher pro- portion of the animals of a culture at this temperature than at 9°.
Cultures Grown and Tested at 20° to 22° The 24 mixtures (series 1, 2 and 4) grown and tested at this temperature all gave large proportions of conjugants — 30 per cent to 90 per cent — and most of them gave immediate strong agglutinative reactions at the time of mixture. The latter did not occur at all at the lower temperatures.
Cultures Grown and Tested at 25° to 26°. Of the 24 mixtures made at this temperature, four proved unsuitable for study. The remaining 20 gave 40 per cent to 90 per cent conjugation and most gave strong immediate agglutinative mating reactions at the time of mixture.
Cultures Grown and Tested at 30°. The eight mixtures (series 4) at this temperature all gave immediate strong mating reactions and high percentages of conjugants.
Cultures Grown and Tested at 36°. The eight mixtures at this temperature (series 4) gave from 2 to 20 per cent conjugation.
At 39° cultures could not be grown, but the effects of this temperature, as set
40 SONNEBORN AND DIPPELL
forth below, were studied in cultures grown at lower temperatures and placed at 39° immediately after mixture.
From the preceding, it appears that the optimal temperatures for conjuga- tion in variety 4 extend from 20° to 30°; that the amount of conjugation obtained is approximately the same throughout this range of temperature; that the amount decreases both as temperature rises and falls away from this range; and that it occurs but rarely at 9°.
In the following paragraphs are presented the results of changing temperature at the time cultures of types VII and VIII are mixed together. The changes of temperature investigated were: (a) changes within the optimal range (20° to 30°); (6) changes from optimal to non-optimal temperatures; and (c) changes from non-optimal to optimal temperatures. The results, which are presented in this order, confirm and extend the conclusions in the preceding paragraph concerning the relation of temperature to the occurrence of conjugation in variety 4.
Changes of Temperature within the Optimal Range (20° to 30°). The following changes of temperature within the optimal range were investigated: cultures grown at 21°-22° were placed at the time of mixture at 24.5° (series 5), at 29° (series 5), and at 30° (series 4) ; and cultures grown at 30° were placed at the time of mixture at 22° (series 4). In each experiment, as in all of those that follow, a complete set of eight mixtures was again made in the way set forth in the pre- ceding section. After all of these changes of temperature, the proportions of conjugants obtained in the mixtures were not significantly different from those obtained in other mixtures of the same cultures kept at the original temperatures. Hence, change of temperature within the optimal range has no effect on the pro- portion of conjugants obtained.
Changes from Optimal to Non-optimal Temperatures. When cultures of the two mating types were grown at a temperature within the range 20° to 30°, were mixed together and placed immediately at a temperature well outside this range, the proportions of animals that conjugated were always less than in corresponding controls retained after mixture at the original temperature.
In two experiments the temperature was raised from 21° or 22° to well over 30°. In one experiment, increase of temperature from 22° to 36° (series 4) re- sulted in no conjugation at all in two of the mixtures and in less than 12 per cent conjugation in the other six mixtures. The corresponding control mixtures retained at 22° gave in each of the eight mixtures from 30 to 90 per cent conjuga- tion, or seven to eight times as much as in those placed at 36°. In the other experiment, increase of temperature from 21° to 39° resulted in no conjugation at all in any of the eight mixtures; but the corresponding eight control mixtures re- tained at 21° all conjugated in high proportions. Hence the upper limit of temperature for the occurrence of conjugation in variety 4 lies between 36° and 39°.
The temperature was lowered from 21° or 26° to well below 20° in three experi- ments. In one (series 5) the temperature was reduced from 21° to 10°. After 2 hours, the eight mixtures at 10° had less than half as many pairs of "conjugants" as the eight control mixtures retained at 21°. Moreover, while the pairs in the 21° mixtures were tightly united, those in the 10° mixtures were not. As will appear immediately, there is reason to believe that all of the latter pairs would have separated without having conjugated. Evidence for this was obtained in
MATING TYPES IN PARAMECIUM AURELIA 41
the second experiment (series 3) in which the temperature was reduced from 26° to 9°. Each of the eight control mixtures retained at 26° yielded more than 50 per cent of the animals tightly united in conjugation within 4 hours; but the eight mixtures at 9° contained at this time less than 10 per cent of the animals in pairs and these pairs were still loosely united. Soon thereafter all these pairs broke apart without having united in true conjugation and no other pairs formed, even loosely, within the next four days (compare with variety 1, Sonneborn, 1941). Reduction of temperature from over 20° to 10° or less thus suppresses conjuga- tion just as does an increase of temperature to 39°. The third 'experiment (series 3) involved reduction of temperature from 26° to 15.5°. These eight mixtures each gave from 15 to 20 per cent conjugation, while each of the corresponding control mixtures at 26° gave more than 50 per cent conjugation within four hours.
All five of these experiments agree in showing that change from a temperature of 21° to 26° to one well below 20° or well above 30° results invariably in consider- able reduction in the proportion of animals that conjugate. When the new tem- perature is as low as 10° or as high as 39°, conjugation is completely suppressed.
Changes from Non-optimal to Optimal Temperatures. Such changes include both reductions from very high to moderate temperatures and increases from very low to moderate temperatures. Both types of changes resulted in increases in the amount of conjugation. Thus, eight mixtures of cultures grown at 36° and placed immediately at 22° gave 10 to 70 per cent conjugation in 6£ hours, while corresponding control mixtures retained at 36° gave only 2 to 20 per cent con- jugation in the same time. Further, three sets of cultures grown at 9° were mixed and placed at 25°-26°. All 24 of these mixtures yielded conjugants in proportions varying from 10 to 90 per cent; but 20 of the 24 control mixtures retained at 9° yielded no conjugants at all and the other four gave less than 3 per cent conjuga- tion. Finally, three sets of cultures grown at 15.5°-16.5° were mixed and placed at 25°-26°. All 24 of these mixtures conjugated and gave higher proportions of conjugants than the corresponding controls kept at 15.5°-16.5°. For example, in one set, seven of the mixtures yielded 40 to 65 per cent conjugants while the corresponding controls yielded only 15 to 25 per cent; and the eighth mixture gave 75 per cent conjugation, its control only 50 per cent.
In general, when the temperature is changed at the time cultures of diverse mating type are mixed, the percentage of conjugation that results is unaffected if both the original and final temperatures are moderate (20° to 30°) ; it is greatly increased if the original temperature is extreme (36° and above, or 16° and below) and the final temperature moderate; and it is greatly decreased if the original temperature is moderate and the final temperature extreme. The optimal tem- peratures for the occurrence of conjugation in variety 4 are thus moderate (be- tween 20° and 30°), regardless of whether mixtures are made from cultures grown at these or other temperatures. Conversely, as the temperature at which the mixtures are placed diverges from this optimum range (either above it or below) , the percentage of conjugation decreases.
DISCUSSION
The conditions for conjugation in variety 4 differ markedly from those for varieties 2 and 3 in the same ways that the conditions for conjugation in variety 1
42 SONNEBORN AND DIPPELL
do (Sonneborn, 1938; 1939). Both varieties 1 and 4 lack a diurnal periodicity in sexual reactivity. Both are able to conjugate over a wide range of tempera- tures. Both give smaller proportions of conjugants as temperature decreases below 20°. Both react to a sudden reduction of the temperature to 10° by dis- continuing a mating reaction previously begun. Both are occasionally able to conjugate at this low temperature, if cultures of opposite types are grown at the same temperature some time before mixture. Nevertheless, varieties 1 and 4 do differ slightly in the conditions for conjugation; but the differences appear only at higher temperatures. Variety 4 gives maximum mating reactions between 20° and 30°, weak ones at 36° and fails to conjugate at 39°. Variety 1 gives maxi- mum reactions between 20° and 38° and then suddenly fails to conjugate as the temperature rises to 40°. Thus, although conjugation occurs over practically the same range of temperature in the two varieties, the range of temperature for maximum sexual reactivity and the rate at which sexual reactivity decreases as the temperature rises above the optimum differ in the two varieties. At 36° the difference appears clearly: variety 1 gives a maximum reaction, while variety 4 conjugates but poorly. Thus it is possible to distinguish these four varieties of P. aurelia not only by their mating types, but also by the sexual responses to diverse conditions. Whether the latter will hold for all varieties of P. aurelia remains to be discovered. Four more varieties are under cultivation (reported in part in Sonneborn, 1943) in our laboratory and many more must exist in nature; but the sexual responses of these to diverse conditions have not yet been investigated.
SUMMARY
Among the 53 races of P. aurelia that have been investigated, four races (29, 32, 47 and 51) do not conjugate with any of the three previously described varieties. They constitute a fourth variety with two new interbreeding mating types, VII and VIII. Mating type VII occurs in all four of these races, but mat- ing type VIII has appeared only in the two races 32 and 51.
The mating types VII and VIII give with each other the agglutinative mating reaction characteristic of Paramecium and proceed to conjugate. As in the other three varieties, agglutination and conjugation occur only when mixture is made between cultures of the two types that are neither well-fed nor starved, but are nearing the stage of nutritive exhaustion. Like variety 1, but unlike varieties 2 and 3, variety 4 shows no diurnal periodicity in sexual reactivity: cultures ex- posed to the natural alternation of daylight and night are capable of reacting sexually at any hour. Further, again like variety 1 and unlike varieties 2 and 3, variety 4 can react sexually throughout the range of temperatures from 9° to 36°, but not at 39°. At 16°, the sexual reactions are weak, leading to but a small proportion of conjugants. In mixtures made at higher temperatures and trans- ferred at once to 9°, pairs begin to form but break apart without conjugating; however, if cultures are first adapted to 9° before they are mixed, a small propor- tion of true conjugation may occur at this temperature. In all these details, varieties 1 and 4 are alike; but they differ in behavior at the higher temperatures. The maximum optimum temperature for conjugation lies between 30° and 36° in variety 4, between 38° and 40° in variety 1. Thus at 36°, variety 1 gives a maximum sexual reaction, while variety 4 gives only 12 to 25 per cent of the
MATING TYPES IN PARAMECIUM AURELIA 43
optimum. Variety 4 shows a gradual falling off in sexual reactivity as tempera- ture increases above the optimum, while variety 1 shows a sudden cessation of sexual reactivity at a temperature only 2° above the optimum.
It is thus possible to distinguish these four varieties of P. aurelia not only by their mating types, but also by the sexual responses to diverse conditions.
LITERATURE CITED
SONNEBORN, T. M., 1938. Mating types in Paramecium aurelia: diverse conditions for mating in
different stocks; occurrence, number and interrelations of the types. Proc. Amer. Phil.
Soc., 79: 411-434. SONNEBORN, T. M., 1939. Paramecium aurelia: mating types and groups; lethal interactions;
determination and inheritance. Amer. Nat., 73: 390-413. SONNEBORN, T. M., 1941. The effect of temperature on mating reactivity in Paramecium aurelia,
variety 1. Anat. Rec. 81 (suppl): 131. SONNEBORN, T. M., 1943. More mating types and varieties in Paramecium aurelia. Anat. Rec.,
84(4): 92.
HYBRIDIZATION AND SEASONAL SEGREGATION IN
TWO RACES OF A BUTTERFLY OCCURRING
TOGETHER IN TWO LOCALITIES
WILLIAM HOVANITZ
(California Institute of Technology, Pasadena) %
The yellow and orange butterfly, Colias chrysotheme, exists in the form of two complexes known as the orange-race and the yellow-race (Hovanitz, 1943a; 1943b). These races have different geographical distributions but overlap over a tremendous territory from the Sierra-Cascade divide in western North America to the Atlantic ocean in the east and from southern Canada in the north through Mexico in the south (Hovanitz, 1943c). Each race usually occupies a different ecologic niche so that nearly pure populations of each may be found in this area as well as outside the zone of overlap. In certain localities, however, the same ecologic niche is partly occupied by both races, resulting in considerable hy- bridization between them.
Two localities where the races occupy the same niche for the most part were analyzed from 1941 to 1943 in order to study the behavior of each in relation to its environment, and to get an indication of the extent of hybridization between them. These places were at Mono Lake Valley, Mono County, California, and at Round Valley (near Bishop), Inyo County, California. Their positions are indicated on a map (Hovanitz, 1943d) ; they are just east of the Sierra Nevada in the western Great Basin.
The Seasonal Distribution of Adults
Orange butterflies are present throughout the entire warm season of the year at both Round Valley and Mono Lake. It is easier, however, to get a good sample in midsummer as compared with early spring or autumn. The abundance of orange adults apparently is at a minimum at each end of the growing season and at a maximum in midsummer.
The yellow butterflies at Mono Lake are more irregular in seasonal distribu- tion than the orange (Fig. 1). The 1941 samples (Table I) show a high relative frequency of yellow to orange in May, and then a complete drop to none present at all in June. A rise to a second maximum in late July is apparent with a gradual drop again to none at all in September. Early in October there is a third maximum. This suggests three distinct broods per year at Mono Lake with an elapsed egg, larval and pupal development time of two months between each. This time compares with a development rate of three to four weeks at a constant laboratory temperature of 25° C. Mono Lake has a rather low air temperature, especially at night; in the day time, the direct radiation from the sun is the primary source of heat.
The 1942 samples at Mono Lake show much the same seasonal distribution.
44
HYBRIDIZATION IN BUTTERFLIES 45
The first adult flight was apparently not observed; it is probably very short in duration. The 1942 samples were obtained at monthly intervals rather than semi- monthly as in 1941; therefore, the chance of missing a short adult flight is in- creased. The second and third broods of 1942 are to be found indicated in the figure a few weeks earlier than in the preceding year. As 1942 was a warmer year for Mono Lake than was 1941, an earlier start in larval development in the spring, with a consequent shift forward in the successive broods, would thus be expected.
The two 1940 samples at Mono Lake show no yellow butterflies present at all. Therefore, it would appear that they were obtained in a yellow interbrood period (Fig. 1).
The frequency at Round Valley does not follow this sequence of events (Fig. 1). Neither the 1941 nor the 1942 samples show any correlation with those
%
80 70 60 50 40- 30 20 •
\ A ,
|Q. M0t*0 LAKE ------- l( ' / \'o
ROUND VALLEY - \ / \ \' ,''
\ / / \ V" /
o ...... - v — •«..-.
APRIL MAY JUNE JULY AUG. SEPT. OCT
FIGURE 1. Frequency of yellow to orange butterflies at Mono Lake and Round Valley, California, throughout the season. Note the complete absence of yellow at certain times at Mono Lake as compared with Round Valley.
of Mono Lake (Table I). This shows the complete lack of intermixing between the two places though they are only fifty miles apart. The 1941 Round Valley curve is high in late June (60 per cent yellow) and drops to a low in late July (25 per cent yellow). A rise occurs in mid-August (53 per cent), with a subsequent drop again the first of September (34 per cent), and then a last rise in early October (55 per cent). If these fluctuations represent successive broods not completely separated one from the other, then there are many more generations present per year at Round Valley than at Mono Lake. This would be expected considering the warmer climate at the former place (Round Valley is at an elevation of 4500 feet and Mono Lake at 6500 feet). Latitudinal differences in brood number per year parallel these altitudinal ones. There are three generations per year near Washington, D. C. (and Mono Lake), two generations near Hanover, N. H. (and
46
WILLIAM HOVANITZ
central British Columbia) and one generation in Alaska and Yukon Territory. At Round Valley there are probably four or more.
The 1942 samples are more extensive at Round Valley than are those of 1941 (Table I). There is a low of yellow (40 per cent) in early spring, rising to a high within the month of 75 per cent and later 79 per cent, with a rather constant frequency of 65 per cent yellow the remainder of the year. This curve shows little evidence of a series of broods or generations during the year. At the rather high temperatures prevailing in the valley during the summer (around 40° C.
TABLE I
The frequency of the yellow-race as compared with the orange-race butterflies at Mono Lake and Round
Valley, Calif. Standard errors are used in this and the other tables. The "many"
is not included in the figures of totals but indicates the presence of yellow alone.
|
Round Valley |
Mono Lake |
|||
|
% yellow |
N |
% yellow |
N |
|
|
1940 |
— |
|||
|
Aug. 11 |
— |
— |
0.0 |
105 |
|
Oct. 20 |
— |
— |
0.0 |
46 |
|
1941 |
50.50 ± 2.89 |
299 |
5.84 ± 0.63 |
1,387 |
|
May 4 |
many |
many |
— |
— |
|
May 19 |
— |
— |
46.27 |
75 |
|
June 8 |
— |
— |
0.0 |
91 |
|
June 24 |
61.19 |
134 |
0.0 |
70 |
|
JulyS |
50.82 |
61 |
0.89 |
678 |
|
July 26 |
22.92 |
48 |
12.66 |
237 |
|
Aug. 15 |
52.63 |
19 |
4.21 |
95 |
|
Sept. 2 |
33.33 |
15 |
0.0 |
20 |
|
Oct. 4 |
54.55 |
22 |
9.01 |
121 |
|
1942 |
65.96 ± 1.50 |
987 |
10.68 ± 0.88 |
1,236 |
|
April 1 |
40.82 |
49 |
— |
— |
|
April 25 |
74.55 |
110 |
— |
— |
|
June 12 |
77.14 |
140 |
0.0 |
many |
|
July 7, 8 |
63.70 |
540 |
16.16 |
396 |
|
Aug. 6, 7 |
65.22 |
69 |
0.0 |
434 |
|
Sept. 16 |
65.82 |
79 |
10.67 |
406 |
|
1940-41-42 |
62.36 ± 1.35 |
1,286 |
7.68 ±0.51 |
2,774 |
during the day, and fluctuating but not very cool at night), the succession of generations would be at about one month intervals. The samples were made at this interval of time, so it is quite possible that the sampling periods coincided with the periods of adult emergence. Were this the case, the results would show a rather constant seasonal frequency. On the other hand, it is possible that the variations in development rate between individuals owing to micro-temperature differences in the locality have completely eliminated the inter-brood population minima. This has been shown to be partially true for the second and third broods in the vicinity of Washington, D. C., as well as for New York state. In these
HYBRIDIZATION IN BUTTERFLIES
47
places, only the breaks between broods one and two are clearly defined by the absence of adults.
A higher frequency of yellow at Round Valley than at Mono Lake, at all times, is apparent (Fig. 1). Several factors combine to create this difference: (1) more larval food is present at Round Valley (Trifolium), (2) Round Valley is farther ecologically from the source of the migrant orange-race individuals (San Joaquin Valley), for these are more likely to stop in the mountain meadows than to proceed through the desert to Round Valley. The frequency of yellow is given as compared with orange. When the orange frequency goes down, the yellow will appear to rise in the curve. (3) The longer and warmer growing season at Round Valley gives more time for the resident population size to be built up. This has been shown elsewhere by the increased numbers of individuals in the second and third broods at Washington, D. C., and New York as compared with the first spring brood.
TABLE II
The frequency of intermediates in the mixed population of orange and yellow races of Colias chryso-
theme at Round Valley, California. (The total given in Table I does not
include intermediates; hence, it is smaller than that given here.)
|
1941 |
1942 |
||||
|
% intermediates |
% intermediates |
||||
|
May 4 |
— . |
many |
April 1 |
10.91 |
55 |
|
June 24 |
14.65 |
157 |
April 25 |
7.56 |
119 |
|
July 5 |
11.59 |
69 |
June 12 |
14.15 |
163 |
|
July 26 |
9.43 |
53 |
July?, 8 |
6.08 |
575 |
|
Aug. 15 |
32.14 |
28 |
Aug. 6, 7 |
6.76 |
74 |
|
Sept. 2 |
0.0 |
15 |
Sept. 16 |
12.22 |
90 |
|
Oct. 4 |
0.0 |
22 |
1942 |
8.27 ±0.84 |
1,076 |
|
1941 |
13.08 ± 1.82 |
344 |
1941-42 |
9.44 ± 0.78 |
1,420 |
A higher frequency of yellows at Round Valley in 1942 as compared with the 1941 samples is also indicated. The latter samples were obtained in a mixed alfalfa-red clover field at the periphery of the large meadow which constitutes the primary ecologic niche for the yellow-race. The 1942 samples were made at a different field one mile from the latter (containing alfalfa, red-clover, white clover and native perennial clovers) in the center of the meadow. This field would be in the midst of the population for the yellow-race whereas the former field is on the periphery. For the migratory orange-race (Hovanitz, 1943d), no part of the meadow would constitute a population center. The higher frequency of yellow in 1942, therefore, can be accounted for by this change in position of the place sampled.
Hybridization Between the Races
Genetic data on crosses involving the races and on progeny from wild inter- mediates between the races indicate that crossing is easily possible and occurs
48 WILLIAM HOVANITZ
frequently (Hovanitz, 1943b). Also, the indications are that there is no genetic sterility between the races. The FI is an exact intermediate of a light orange color; F2 and backcrosses give the range of intermediates expected on a multiple factor distribution of genes.
The range of colors from the parental types through the intermediates is given in a range from yellow to orange of 1 to 10. From genetic results, it is known that grades 1 and 2 are pure parental types, breeding true for the yellow race. In the pure populations of orange race, there is a range of yellow to orange from 1 to 10 but from about 1 to 7 or 8 these are exceedingly rare (Hovanitz, 1943e). Therefore, grades 8 to 10 in the males and 7 to 10 in the females are considered as "parental types" for the Round Valley population. It is under- stood that grades 7 or 8 may be intermediates or that some lower grades may be parental types but that these will be insignificantly small.
40' ••.....
1941
1942
30-
20-
10-
APRIL MAY JUNE JULY AUG. SEPT. OCT.
FIGURE 2. Frequency of intermediates between yellow and orange in the population at Round Valley, California, during the two years 1941-42.
On the basis of grades 3 through 7 in the males and 3 through 6 in the females, the frequency of intermediates in the Round Valley populations have been calcu- lated (Table II). It is seen that there is but little seasonal change in the abun- dance of intermediates (Fig. 2). A high of 30 per cent in August 1941 is possibly a result of the small sample size. An average of about 10 per cent intermediates is usual.
Range of Wild Intermediates
The statistical consequences of continued interbreeding between the orange and yellow races should be a single race combining the characteristics of each parental type. But the two races have maintained their primary discreteness after more than 70 years of such interbreeding, and probably for many centuries (Hovanitz, 1943b; 1943c). Were the interbreeding only of very recent origin, the hybrid range would show a very high frequency of FI intermediates (grades 5 or 6) and a lower range of F2, F3 and backcross intermediates (grades 3-4, 7-8). The data on wild individuals (Fig. 3) do not show this higher frequency of FI to
HYBRIDIZATION IN BUTTERFLIES
49
any great extent. The female curve may be masked by the normally low orange female grades. The male range shows a somewhat higher frequency of grade 5 than the other intermediates. The lack of the FI intermediates compared with
123456789 10
1234 56 7 8 9 10
FIGURE 3. Histograms showing the range of variation in the intermediates between the orange and yellow races at Round Valley, California, 1941-42. Male on left and female on right. The smaller numbers represent the numbers of individuals in a given class and the larger represent the grade or class of intermediates.
Fn intermediates may be due to many factors of which a general lower viability seems to be the most likely (Hovanitz, 1943b).
DISCUSSION
The data on the existence of the two races of Colias living in the same locality suggest how ecologic and physiological differences can be maintained in units
50 WILLIAM HOVANITZ
which may be called species. The races are not here called species for some genes are easily and often interexchanged (Hovanitz, 1943b). However, other genes are not effectively segregated in this way. This suggests that the significant gene complex characterizing each race and giving it individuality is not broken down in hybrid crosses.
Since the color difference separating the races is a multiple factor one and these factors are segregated independently of the basic complex, it might still be ex- pected that a complete intermediate population would be produced, separated only by the non-visible basic complex. The reason for this lack of complete blending of characters probably lies in a combination of the following conditions:
(a) Sexual selection (Hovanitz, 1943b) may prevent sufficient intercrossing to be effective.
(&) Eggs genetically determined to be yellow-complex laid on alfalfa will later result in sterile adults or the subsequent larvae may die; also the reciprocal on red clover (Hovanitz, 1943; 1943b).
(c) The intermediates of all types are probably less viable than the parental types and many of them will be sterile on the food plant upon which they feed (Hovanitz, 1942; 1943b).
(d~) The diapause associated with the one complex (Hovanitz, 1942, 1943b) tends to keep the races ecologically separated.
(e) The supplementary color genes of each normal type probably act better in unison with the basic complex than any intermediate segregation of genes.
(/) The different ecological niche occupied by the food plants necessary for each complex aids in preventing hybridization (Hovanitz, 1943c).
Summary
1. Two localities where the two races of Colias chrysotheme occur together are described (Mono Lake and Round Valley, Calif.).
2. In these places, the yellow-race has definite broods during the season. The orange race apparently does not.
3. The yellow-race has more seasonal generations when a population is at a lower elevation (Round Valley) than at a higher elevation (Mono Lake). This compares with latitudinal differences of the same type.
4. The two localities are 50 miles apart, but show no correlation in seasonal generations.
5. The yellow-race generations at the higher elevation are separated by inter- brood periods with no adults. At the lower elevation, the generations merge one into the other.
6. Hybrid intermediates are present at one locality rather constantly at a frequency of about 10 per cent.
7. The range of color intermediates is not trimodal, but a U-shaped curve. This is probably due to a low viability of the FI. A trimodal curve is expected under conditions of very recent hybridization and all intermediates with long- time hybridization.
8. Several reasons are given to account for the lack of complete blending be- tween the races after years of hybridization.
HYBRIDIZATION IN BUTTERFLIES 51
A cknowledgments
This work was carried on through the encouragement of Professors T. H. Morgan and A. H. Sturtevant to whom the author is grateful.
LITERATURE CITED
HOVANITZ, W., 1942. The biology of racial or species differences in Colias, Bull. Ecol. Soc. Amer.,
23: 68 (Abstract). HOVANITZ, W., 1943a. The nomenclature of the Colias chrysotheme complex in North America.
American Museum Novitates (in press). HOVANITZ, W., 1943b. Genetic data on the two races of Colias chrysotheme in North America
and on a white form occurring in each. (Awaking publication.) HOVANITZ, W., 1943c. The ecological significance of the color phases of Colias chrysotheme in
North America. Ecology (in press). HOVANITZ, W., 1943d. The distribution of gene frequencies in wild populations of Colias. Genetics
(in press). HOVANITZ, W., 1943e. The pattern elements of the North American Colias of the chrysotheme
group. (Awaiting publication.)
SPECIES DIFFERENCES IN RATES OF OSMOTIC HEMOLYSIS WITHIN THE GENUS PEROMYSCUS *
HARRY P. LEVINE
(Department of Zoology, University of Vermont, Burlington)
INTRODUCTION
That definite species differences exist in the properties of the red cell membrane has been recognized at least since the studies of Rywosch in 1907. The possible significance of such specific differences in regard to zoological classification and animal identification has been pointed out by Jacobs (1931). Investigation of the rates of osmotic hemolysis in approximately 50 species of vertebrates led to the conclusion that "not only may individual species be identified but frequently unmistakable evidences of zoological relationship may be traced throughout a group of similar forms." In 1938 Jacobs and collaborators demonstrated striking differences in the permeability properties of the erythrocytes of the rat and mouse representing closely related genera. The purpose of the present investigation was to demonstrate measurable and consistent differences in the rates of hemolysis among a number of species within the genus Peromyscus.
MATERIALS AND METHODS
The mice used in this investigation consisted of four species representing different degrees of taxonomic relationship (Miller, 1923) from widely separated geographical regions as follows:
Subgenus Haplomylomys Osgood
P. eremicus fraterculus — La Jolla, California Subgenus Peromyscus Gloger Species group — leucopus
P. leucopus noveboracensis — Vermont; Moville, Iowa P. gossypinus palmarius — Sebring, Florida Species group — truei
P. truei truei — Deadman Flat, Arizona
In addition, the guinea pig (Cavia cobaya) representing a distantly related rodent species was used for purposes of contrast.
Blood was obtained from each mouse under light ether anesthesia by cardiac puncture after the method of Hicks and Little (1931). About 0.5 cc. could be removed from a mouse without fatality. The blood was immediately expressed into a small beaker containing about 10 cc. of 0.9 per cent saline and defibrinated by stirring. The suspension was then washed down by centrifuge and the cells restored to the original blood volume with saline.
* Preliminary report presented at the 24th annual meeting of the American Society of Mam- malogists in New York City, April 2, 1942.
52
SPECIES DIFFERENCES IN HEMOLYSIS RATES
53
The substances employed in the hemolysis studies were 0.3 molar solutions in distilled water of non-electrolytes including ethylene glycol, glycerol and ery- thritol representing progressively larger polyhydric alcohol molecules, and thiourea.
The method of determining rates of hemolysis was essentially that described by Jacobs (1930). To 5 cc. of one of the above solutions in a test tube in a water bath maintained at 20° C. was quickly added one drop of blood on a specially prepared plunger which simultaneously stirred the cells, producing an even suspension. With the aid of a stop-watch the time for 75 per cent hemolysis of the cells was determined by comparison with a standard suspension (one drop of the same blood in 20 cc. of saline) in a test tube adjacent to that containing the hemolysing suspension. This comparison was effected by means of a thin band of light viewed through the test tubes. Approximately 75 per cent hemolysis was attained when the band of light was visible in the hemolysing suspension to the same degree as in the standard. In practice the blood to be tested was so
TABLE I
Species differences in rates of osmotic hemolysis
|
Time in seconds for 75 per cent hemolysis at 20° C. in 0.3M |
|||||||||||||
|
Ethylene glycol |
Glycerol |
Erythritol |
Thiourea |
||||||||||
|
Species |
No. |
Low |
High |
Ave. |
Low |
High |
Ave. |
Low |
High |
Ave. |
Low |
High |
Ave. |
|
P. lencopus |
17 |
4.7 |
6.4 |
5.6 |
7.0 |
12.3 |
9.5 |
20.6 |
49.0 |
31.8 |
10.7 |
15.3 |
13.3 |
|
P. gossypinus |
14 |
5.6 |
7.8 |
7.1 |
15.0 |
28.5 |
22.1 |
47.0 |
195.0 |
110.0 |
13.3 |
21.7 |
19.4 |
|
P. truei |
6 |
6.8 |
8.0 |
7.3 |
33.6 |
44.3 |
39.2 |
150.0 |
250.0 |
193.0 |
28.7 |
36.0 |
32.1 |
|
P. eremicus |
15 |
5.6 |
6.7 |
6.0 |
31.0 |
58.5 |
44.3 |
150.0 |
465.0 |
259.0 |
16.8 |
28.5 |
23.3 |
|
Cavia cobaya |
3 |
10.6 |
15.4 |
13.6 |
130.0 |
223.0 |
178.0 |
>30 hrs. <42 hrs. |
116.0 |
143.0 |
126.0 |
adjusted with saline that the band of light was just barely visible through the standard suspension since this offered the most easily recognized end point.
In performing the experiments test tubes were carefully chosen for uniformity, standard suspensions were prepared as soon as the blood samples were obtained, and hemolysis rates were determined immediately. All tests were performed in duplicate whenever possible. Remaining portions of blood samples were kept in refrigeration storage at approximately 4° C. Except for certain storage experi- ments where pooled blood was used, hemolysis rates were obtained with eryth- rocytes from individual animals.
EXPERIMENTAL RESULTS
The method of determining rates of hemolysis as described above was very simple and apparently crude, but with proper care the results of tests performed in duplicate proved to be markedly consistent. Variation in duplicate measure- ments of the time for 75 per cent hemolysis of the red cells in any one of the solutions rarely exceeded 10 per cent and most often was less than 5 per cent. With practice, especially in preparing suitable standard suspensions, duplication
54
HARRY P. LEVINE
was brought to within 2 per cent. It was reasonable to assume, therefore, that the differences in hemolysis times obtained here between one species and another represented true specific differences.
The times for 75 per cent hemolysis of the erythrocytes of the species investi- gated are summarized in Table I. Evidence of zoological relationship is readily apparent. When compared with the rate of hemolysis of guinea pig (Cavia) erythrocytes, the hemolysis rates of all the Peromyscus erythrocytes appear to be of the same order of magnitude. With erythritol, for example, the difference
TABLE II
Comparison of glycerol and thiourea times and G/T ratios of four species in the genus Peromycus
(temperature 20° C.)
hemolysis time in glycerol
G/T ratio =
hemolysis time in thiourea
|
Time in seconds for 75 per cent hemolysis |
G/T Ratio |
||
|
0.3M Glycerol |
0.3M Thiourea |
||
|
P. leucopus |
12.2 |
13.8 |
0.88 |
|
8.1 |
13.7 |
0.59 |
|
|
12.3 |
15.3 |
0.80 |
|
|
7.0 |
10.7 |
0.65 |
|
|
Ave. 9.5 |
13.3 |
0.71 |
|
|
P. gossypinus |
28.5 |
21.4 |
1.33 |
|
22.5 |
20.8 |
1.08 |
|
|
24.4 |
21.7 |
1.12 |
|
|
15.0 |
13.3 |
1.13 |
|
|
Ave. 22.1 |
19.4 |
1.14 |
|
|
P. truei |
44.3 |
33.2 |
1.33 |
|
33.6 |
29.8 |
1.13 |
|
|
43.4 |
36.0 |
1.21 |
|
|
35.6 |
28.7 |
1.24 |
|
|
Ave. 39.2 |
32.1 |
1.22 |
|
|
P. eremicus |
58.5 |
27.3 |
2.11 |
|
40.2 |
22.9 |
1.76 |
|
|
54.6 |
27.5 |
1.98 |
|
|
31.0 |
16.8 |
1.85 |
|
|
Ave. 44.4 |
23.3 |
1.90 |
in hemolysis time between leucopus cells and eremicus cells (of the order 1 : 8) is small when compared with the difference between eremicus cells and guinea pig cells (1 : 540). On the other hand, consistent differences in hemolysis rates among the species within the genus are demonstrable. Leucopus cells are most readily hemolysed by each of the permeating substances; gossypinus cells are hemolysed at a somewhat slower rate. Generally truei and eremicus cells are hemolysed less rapidly than either leucopus or gossypinus cells. It is interesting to note in this regard that leucopus and gossypinus are placed taxonomically within the same species group.
SPECIES DIFFERENCES IN HEMOLYSIS RATES
55
The rates of osmotic hemolysis in glycerol especially often reveal striking specific differences and sometimes offer evidence of relationship (Jacobs, 1931; 1938). From Table I it can be seen that all the Peromyscus red cells attain the condition of 75 per cent hemolysis in less than one minute. Yet the hemolysis times for the red cells of each species are apparently confined to definite limits within this time.
According to Jacobs and associates (1938), comparison of the rates of osmotic hemolysis in glycerol and thiourea within a species may provide an index for species identification. Table II records in the first two columns the hemolysis times in glycerol and in thiourea respectively for each species of mouse investi- gated. The figures in the third column (G/T ratio) are obtained by dividing the glycerol hemolysis time by the thiourea hemolysis time. The data have been selected to show the extent of variation found in each species. The average figure for each species is the arithmetic mean of the results for all individuals
o <
20 30
TIME IN SECONDS
40
5O
60
FIGURE 1. Species differentiation by osmotic hemolysis. Each dot represents an individual plotted along the abscissa in terms of the time for 75 per cent hemolysis in 0.3 molar glycerol at 20° C. and along the ordinate in terms of the G/T ratio. The hollow rectangles represent different species:
A = P. leucopus D = P. eremicus
B = P. gossypinus
C = P. truei
studied within each species. It is evident from the table that the glycerol/ thiourea ratio is constant for each species within fairly narrow limits. Leucopus which has the lowest ratio and eremicus which has the highest ratio are readily separated from gossypinus and truei. Although the latter two species exhibit similar ratios, examination of the first two columns in Table II reveals that in the absolute times for hemolysis in glycerol and in thiourea they are readily differentiated.
Figure 1 records graphically the results which have been summarized in Table II. Each mouse investigated in the present study has been plotted with regard to erythrocyte hemolysis in glycerol (along the abscissa) and with regard to the glycerol/thiourea ratio (along the ordinate). The hollow rectangles enclose all the individuals within a species. This figure shows in a striking way that it may be possible to determine the species to which an individual belongs by the ap- propriate hemolysis tests. For example, at one stage in the course of these
56 HARRY P. LEVINE
experiments a colleague kindly provided two blood samples without revealing the species from which they had been obtained. Hemolysis tests provided the following results:
|
Time in seconds for 75 per cent hemolysis at 20° C. |
|||
|
0.3M glycerol |
0.3M thiourea |
G/T ratio |
|
|
Mouse No. 1 Mouse No. 2 |
12.1 8.5 |
15.0 12.8 |
0.80 0.67 |
Both mice were correctly identified as leucopus.
Some evidence of zoological relationship is apparent in the glycerol/thiourea ratios obtained in this study. As can be noted in Table II, the ratios for leu- copus, gossypinus and truei which are placed in the same taxonomic subgenus are all near one as a constant, while the ratio for eremicus which is placed in another subgenus is near two.
At the inception of this investigation some disconcerting variations in he- molysis times occurred within each species of Peromyscus. This led to an in- vestigation of the effect of storage upon the rate of hemolysis of the red cells. In order to obtain a sufficient quantity for this purpose, it was necessary to use pooled blood of each Peromyscus species, whereas blood from individual guinea pigs was employed. Otherwise all blood samples were treated identically. Fig- ure 2 shows the typical effect of storage upon the hemolysis rates of the Peromyscus and guinea pig red cells. Days in storage are plotted against the hemolysis time in glycerol. The red cells of eachTof the species within the genus Peromyscus show a marked and continued increase in hemolysis time upon storage while the red cells of the guinea pig show very little change during the same period of storage. The reason for this interesting storage effect has not yet been determined.
DISCUSSION
Physiological and biochemical studies of blood have produced results both of broad evolutionary interest and also of value in the field of animal classification and identification. The evolutionary significance of results obtained from the studies of the osmotic pressures of blood (Scott, 1916) is well recognized. The extensive work of Reichert and Brown (1909) on the crystallography of hemo- globin among different species has provided convincing evidence of biochemical relationships among animals in general accord with the accepted taxonomic classi- fication. The versatile and rapidly expanding field of systematic serology (see Boyden, 1942) has been employed on the one hand in the study of the possible origin of vertebrates (Wilhelmi, 1942), and on the other hand, in the investigation of the genetic basis for biochemical differences in the serum and blood cells of species and species-hybrids (Irwin and Cole, 1936; Irwin and Cumley, 1942).
The present investigation has revealed that consistent and measurable differ- ences in the rates of hemolysis of the erythrocytes among very closely related species can be employed successfully to differentiate one species from another. Especially with regard to glycerol penetration, confirming observations by Jacobs,
SPECIES DIFFERENCES IN HEMOLYSIS RATES
57
and with regard to the glycerol/thiourea ratio the results indicate zoological rela- tionship in general agreement with the existing system of classification. Whether such agreement between morphological classification and rate of osmotic he- molysis will always hold among closely related species can be determined only by further investigation.
170
160
iP LEUCOPUS • P GOSSYPINUS »P EREMICUS CAVIA COBAYA
DAYS I N STORAGE
FIGURE 2. The effect of storage upon the rate of osmotic hemolysis (75 per cent) in 0.3 molar glycerol at 20° C. (Blood cells stored in 0.9 per cent NaCl at approximately 4° C.)
Preliminary studies on four offspring of a species-cross between leucopus and gossypinus indicate that these differences may be subject to genetic analysis although as yet the data are not sufficient for definite conclusions. Table III shows that in their hemolysis times in different substances the hybrid red cells are very similar to those of the leucopus parent stock while the values for the glycerol/thiourea ratio lie between those of the two parent stocks.
Specific differences in the properties of the cell membrane have introduced a
58
HARRY P. LEVINE
complicating feature to the problem of cell permeability, yet an understanding of the nature of such specific differences may go far towards a better under- standing of the factors determining the permeability of the cell membrane in general. In the meantime collection of further data on species differences in erythrocyte permeability will serve the useful purpose of developing a physiological means of animal identification.
The author is deeply indebted to Dr. Paul A. Moody who gave unstintedly of his mice and of his time when requested ; to Dr. Lee R. Dice of the University of Michigan who provided some of the mice from which the present stock was originated; and especially to Dr. Merkel H. Jacobs of the University of Pennsyl- vania, under whose guidance the author became acquainted with the described hemolysis techniques at the Marine Biological Laboratory, at Woods Hole, Massachusetts. The author is further indebted to Dr. Jacobs for his kindness in reading the manuscript and for his valuable suggestions.
TABLE III
Comparison of hemolysis times and G/T ratios of a species hybrid and its parent stocks
|
Species |
Time in seconds for 75 per cent hemolysis at 20° C. in 0.3M |
G/T ratio |
|||
|
Ethylene glycol |
Glycerol |
Erythritol |
Thiourea |
||
|
*P. leucopus noveboracensis *P. gossypinns pal mar ins leucopus-gossypinits hybrids |
5.6 7.1 4.8 5.1 |
9.5 22.1 8.0 10.4 |
31.8 110.0 28.0 39.0 |
13.3 19.4 8.6 10.1 |
0.71 1.14 0.93 0.97 |
|
5.2 |
10.2 |
34.0 |
10.5 |
1.03 |
|
|
4.9 |
7.8 |
20.0 |
8.7 |
1.12 |
Average of the species.
SUMMARY
The erythrocytes of four species of mice within the genus Peromyscus were studied with regard to their rates of osmotic hemolysis in ethylene glycol, glycerol, erythritol and thiourea. Consistent species differences in hemolysis times were demonstrated by which it was possible in the case of the individuals studied to identify each species with certainty. Evidence of zoological relationship was apparent in the results.
Refrigeration storage of Peromyscus erythrocytes resulted in progressively decreased rates of hemolysis. Storage of Cavia (guinea pig) erythrocytes had very little effect upon their rates of hemolysis.
LITERATURE CITED
BOYDEN, A., 1942. Systematic serology: A critical appreciation. Physiol. Zool., 15: 109-145. HICKS, R. A., AND C. C. LITTLE, 1931. The blood relationships of four strains of mice. Genetics,
16: 397-421. IRWIN, M. R., AND L. J. COLE, 1936. Immunogenetic studies of species and species hybrids in
doves, and the separation of species — specific substances in the backcross. Jour. Exp.
Zool., 73: 85-108.
SPECIES DIFFERENCES IN HEMOLYSIS RATES 59
IRWIN, M. R., AND R. \V. CUMLEY, 1942. Immunogenetic studies of species; qualitative differ- ences in the serum of backcross progeny following a generic cross in birds. Genetics, 27: 228-237.
JACOBS, M. H., 1930. Osmotic properties of the erythrocyte. I. A simple method for studying the rate of hemolysis. Biol. Bull, 58: 104-122.
JACOBS, M. H., 1931. Osmotic hemolysis and zoological classification. Proc. Amer. Phil. Soc., 70: 363-370.
JACOBS, M. H., H. N. CLASSMAN AND A. K. PARPART, 1938. Osmotic properties of the erythro- cyte. IX. Differences in the permeability of the erythrocytes of two closely related species. Jour. Cell, and Comp. Physiol., 11: 479-494.
MILLER, G. S., JR., 1923. List of North American recent mammals. U. S. Nat. Mus. Bull. 128.
REICHERT, E. T., AND A. P. BROWN, 1909. The crystallography of hemoglobins. Carnegie Inst. of Wash. Pub. No. 116.
RYWOSCH, O., 1907. Vergleichende Untersuchungen iiber die Resistenz der Erythrocyten einiger Saugethiere gegen hamolytische Agentien. Pfliiger Archiv., 116: 229-251.
SCOTT, G. G., 1916. The evolutionary significance of the osmotic pressure of the blood. Amer. Nat., 50: 641-663.
WlLHELMl, R. W., 1942. The application of the precipitin technique to theories concerning the origin of the vertebrates. Biol. Bull., 82: 179-189.
GERMARIAL DIFFERENCES AND THE PRODUCTION
OF APHID TYPES*
CHESTER A. LAWSON
(Department of Zoology, Michigan State College, East Lansing, Michigan)
INTRODUCTION
If germaria exercise any control over the development of differential characters in female aphids (Lawson, 1939; 1940) it is possible that they would give evidence of this control by exhibiting structural peculiarities correlated with the production of specific aphid types. To investigate this possibility the germaria of partheno- genetic females producing different aphid types were compared.
THE GERMARIA
Each adult germarium contains two types of cells, nurse cells and germ cells. The nurse cells are larger than the germ cells and make up the bulk of the ger- marium, so if the germarium controls development it is possible that this control stems from the nurse cells. Their prominence in the germarium at least gives them first choice of the parts to be tested, so in this study the nurse cells only are compared.
The nurse cells of all parthenogenetic germaria are essentially alike (Figs. 1, 2, 3, 4). Each nurse cell is roughly pyramidal in shape (triangular in section) with the base at the periphery of the germarium and the apex in the center. The nucleus lies near the base of the pyramid and is covered on its outer edge and sides by a thin layer of cytoplasm. On the inner border of the nucleus the cytoplasm is thicker and extends inward toward the center of the germarium forming the apex of the pyramid. The cytoplasm seldom forms a sharp point in the center, for here it blends with the secreted substance found in the center of all germaria. The exact line of demarcation between cytoplasm and secreted material is difficult to see. The nuclei of all nurse cells are relatively large and each contains a large elliptical nuceolus and chromatin in the form of thin rods or prophase strands that are interconnected by a fine threadlike network.
In comparing the germaria of different aphid types, structural differences were sought that would serve to differentiate among them. Of several possible struc- tural differences only one stands out with any consistency. This is a size differ- ence. To test the reality of this apparent difference measurements were made and compared of the entire germarium and of individual nuclei within the nurse cells.
* Thanks are due to Professor W. D. Baten of the Mathematics Department who assisted with the calculations and to Professor C. P. Swanson of the Botany Department who made the photomicrographs. Part of this work was done at the Franz Theodore Stone Laboratory, Put- In-Bay, Ohio.
60
PRODUCTION OF APHID TYPES
61
•
r/
'
• * 1
• *
* ** y
v«
• ^
• V,
•. •
0>
FIGURES 1-5. Cross-sections of adult aphid germaria. Figure 1. Winged parthenogenetic female producing gamic embryos (1455 X). Figure 2. Winged parthenogenetic female producing parthenogenetic female embryos (1455 X). Figure 3. Wingless parthenogenetic female produc- ing parthenogenetic female embryos (1455 X). Figure 4. Wingless parthenogenetic female producing male embryos (1455 X). Figure 5. Adult^gamic female (675 X).
62
CHESTER A. LAWSON
As each germarium is approximately spherical in shape its center cross section is circular or elliptical. The diameters of this cross-section were measured in micra and the area computed and this figure used to represent the size of the germarium. A better method of comparing the germaria would be to compare volumes. In order to calculate the volume of any one germarium it is necessary to have three diameters because very few of the germaria are perfect spheres. Two of these are easily measured on the center cross section. The third can be gotten by counting the number of cross sections of the germarium. However, no great reliance can be placed on a measurement arrived at in this manner. Each cross section is ten micra in thickness except the first and the last. These two vary from a fraction of one to ten micra, and as the actual thickness cannot be determined the third diameter has a possible error of twenty micra. Because of this error no confidence can be placed in the calculated volumes and it seems best to restrict the comparisons to the more accurately measureable center areas of the germaria. An occasional irregularity in the circumference of the cross sec- tions introduces a source of error which is probably not great enough to dis- count major size differences, but may affect the results in comparison of minor differences.
Each aphid has nine or ten germaria and all of these that could be measured accurately were measured and all measurements from one type of aphid were grouped and treated statistical ly.
The means and standard deviations of the area of the center cross section of the adult germaria are given in Table I. The germaria of the male-producing
rr\ T
FABLE I
.1 comparison of the areas in square micra of germarial center section
|
Type of female |
Contained embryos |
n |
Mean |
Standard deviation |
|
|
1. |
wingless parth. |
males |
103 |
1474±42 |
422±29 |
|
2. |
wingless parth. |
parth. females |
172 |
731 ±14 |
190±10 |
|
3. |
\\inged parth. |
parth. females |
126 |
599±16 |
178±11 |
|
4. |
winged parth. |
gamic females |
127 |
567±10 |
115±7 |
wingless parthenogenetic females (Fig. 4) are larger than those of the wingless females producing parthenogenetic females (Fig. 3) and these in turn are larger than the germaria of winged females (Figs. 1 and 3). The differences between the means are statistically significant for all except the two winged types.
A difference between two means is considered significant when it is at least twice the standard error of the difference between means.
The Nurse Cell Nuclei
The nuclei of the nurse cells also were measured and compared. All the nuclei in any one germarium were not measured, but only those that were spherical. Many of the nuclei formed long ellipses or varied from the spherical unevenly. These nuclei were rejected in order to reduce the error of measurement and also to reduce the labor. If spherical nuclei only are used, one measurement, the
PRODUCTION OF APHID TYPES
63
diameter, is sufficient; and from this the volume can be calculated. This selection may introduce an error in the results if the shape of the fixed nucleus is correlated with its size, which is unlikely; or if an insufficient number of nuclei are measured in any one aphid type. It is believed that the number measured is sufficiently large to evade this source of error.
The means and standard deviations of nurse cell nuclear volumes in cubic micra are given in Tables II and III.
TABLK II
-1 comparison of nurse cell nuclear volume measured in cubic micra
|
Type of female |
Embryos |
n |
Mean |
Standard deviation |
|
|
1. |
gamic |
100 |
5180±314 |
3140±222 |
|
|
2. |
wingless parth. |
males |
205 |
953±28 |
402 ±20 |
|
3. |
wingless parth. |
parth. females |
408 |
326±7 |
140±4 |
|
4. |
winged parth. |
parth. females |
351 |
283 ±7 |
130±5 |
|
5. |
winged parth. |
gamic females |
200 |
151±5 |
64±3 |
TABLE III
Means and standard deviations in cubic micra of nurse cell nuclear volume of parthenogenetic females
producing different types of parthenogenetic embryos
|
Type of aphid |
Embryos |
n |
Mean |
Standard deviation |
|
1 . wingless |
winged and wingless |
200 |
302 ±9 |
125±6 |
|
2. wingless |
winged |
208 |
348±10 |
148 ±7 |
|
3. winged |
winged and wingle^ |
351 |
283±7 |
130±5 |
|
4. winged |
winged |
198 |
279±8 |
129±6 |
|
5. winged |
wingless |
134 |
301±11 |
130±8 |
In Table II are listed the means and standard deviations of the five major aphid types. The differences in the mean nuclear volumes are all statistically significant. Thus the nurse cell nuclei of gamic female germaria (Fig. 5) are larger than any of the others, those of wingless females producing males (Fig. 4) are smaller than the gamic nuclei, but larger than any other parthenogenetic nurse cell nuclei. The wingless females producing parthenogenetic females (Fig. 3) have nurse cell nuclei that are smaller than the male-producing type but larger than those in winged females, while the winged female nurse cell nuclei are smaller than any of the others. There is also a nuclear size difference between the two types of winged females. The winged females producing gamic females (Fig. 1) have smaller nuclei than those producing parthenogenetic females (Fig. 2). In comparing Figures 1 to 5 it should be noted that the magnification of Figure 5 is approximately one-half that of Figures 1, 2, 3, 4.
The size differences shown by the nuclear measurements are in the same direc- tion as those shown by the germarial measurements which suggest that the size of the entire germarium is clue to the size of the nurse cells. One exception to this is seen in the two sets of measurements of the winged parthenogenetic females.
64 CHESTER A. LAWSON
In comparing measurements of germarial center areas (Table I) the winged females producing parthenogenetic females and those producing gamic females are not significantly different. The means are different and direction of difference is the same as that of the nuclear size differences, but the difference is not statistically significant. In comparing the nuclear measurements of these same winged female types (Table II) a very large and significant difference appears.
One of the possible explanations is that no correlation exists between nuclear size and germarial size but rather between germarial size and nuclear (cell) number. If this is true the germaria of winged females producing gamic females should have almost twice as many nuclei as the germaria of winged females pro- ducing parthenogenetic females. A count revealed the same number in both (average 20 to 22). Another possibility is that there might be twice as much cyto- plasm in each nurse cell within the gamic producing germaria, or that the material secreted by the nurse cells is excessive. These possible differences are not ap- parent on comparing the two types of germaria (Figs. 1 and 2) hence it is likely that there is some other explanation at present unknown. Also there remains the possibility that a difference may exist between the germarial areas of the two types of winged females (Table I) that is not shown in these calculations. The number of individuals used for computing the means of the germarial areas are one-half as many as are used in computing the means for nuclear volume of the same individuals (Table II). If n were doubled for the germarial areas a sig- nificant difference might appear.
In Table III are listed the means and standard deviations of parthenogenetic females that arc producing parthenogenetic female offspring. The winged and wingless adults are classified according to whether they are producing either winged or wingless parthenogenetic female offspring or both.
The means are all about the same and none of the differences is statistically significant except for number 2 (wingless females producing winged embryos). This mean is significantly different from all in the table except number 5 (winged females producing wingless offspring). Thus except for one case no size difference is correlated with the production of parthenogenetic types and in this one case the difference is not great so it is possible that some factor other than type of offspring produced the difference.
If this interpretation is correct and there is no real size difference among the nurse cell nuclei in Table III a change must be made in the interpretation of Table II. In this table the mean nuclear sizes of number 3 (wingless partheno- genetic females producing parthenogenetic female embryos) and number 4 (winged parthenogenetic females producing parthenogenetic female embryos) are sig- nificantly different. However, the calculation of the mean of 326 ± 7 of number 3 of Table II includes the data under number 2 of Table III. If these data are eliminated from the calculations the mean becomes 302 ± 9 (a = 125 ± 6) and the difference disappears between this mean and that of the number 4, Table II (winged parthenogenetic females producing parthenogenetic female embryos). Thus the group of data in Table III that shows a questionable difference causes the difference between the winged and wingless parthenogenetic-producing females in Table II. Hence 3 and 4 in Table II probably are not different. There re- main, however, the differences among the other types which are so large that their reality seems beyond doubt.
PRODUCTION OF APHID TYPES 65
Germaria and Embryos of Winged-wingless Intermediates
A study of winged-wingless intermediates offers further evidence that the nurse cell nuclear volume is correlated with the type of offspring produced. In Table IV is presented a comparison of the mean nuclear volume of germarial
TABLE IV
A comparison of volumes in cubic micra of nurse cell nuclei in gerniaria of winged -wingless
parthenogenetic female intermediates with the type of embryos contained
in the vitellaria to which these gerniaria are attached
Mean nuclear volumes Types of embryos
1. 57±9 Gamic female
2. 63 ±9 Gamic female
3. 132±10 Parthenogenetic female
4. 140±9 Parthenogenetic female
5. 157 ±8 Gamic female
6. 194±11 Parthenogenetic female
7. 235 ±14 Gamic and parthenogenetic female
8. 272 ±15 Parthenogenetic female
9. 353 ±31 Parthenogenetic female and male
10. 381 ±37 Parthenogenetic female and male
11. 383 ±23 Parthenogenetic female and male
12. 383±23 Parthenogenetic female
13. 486±58 Parthenogenetic female and male
14. 559±61 Parthenogenetic female and male
15. 732±93 Male
16. 804 ±73 Male and gamic egg
17. 930 ±82 Male
nurse cells in individual winged-wingless intermediates and the type of embryos contained within the ovarioles of the intermediates. From this comparison it is evident that the intermediates having the smallest nuclear volume contain gamic female embryos within their ovarioles, and that as the nuclear volume becomes greater the embryos become parthenogenetic, then both parthenogenetic and male (in which the older embryos are parthenogenetic) then all male embryos and finally the intermediates having the largest nurse cell nuclear volume contain both male embryos and gamic eggs.
This correlation is not exact for intermediates 3 and 4, Table IV, contain parthenogenetic embryos while intermediate 5 has gamic embryos and also has a larger mean nuclear volume than either 3 or 4. Also intermediates 11 and 12 have the same nuclear volume, even though number 11 has both male and par- thenogenetic female embryos, while number 12 has parthenogenetic embryos only. This irregularity may be due to the fact that all of the nuclei in any one inter- mediate could not be measured accurately, or it may be due to the effect of some unknown factor. In any case it seems reasonable to conclude that in winged- wingless intermediates the size of the nurse cell nuclei is correlated in general with the production of specific aphid types.
In one intermediate (17) there is an unusual germarium (Fig. 6) in which the nuclei are of two distinct sizes. The germarium is partly divided in half; one-half containing large nuclei (M = 2264 ± 390) the other half containing small nuclei (M = 445 ± 53). The appearance of two distinct sizes of nuclei within one germarium suggests that the size of any one nurse cell nucleus is determined by
66
CHESTER A. LAWSON
some factor within the germarium and perhaps within the individual nucleus itself. What this factor might be is entirely hypothetical; however, the nuclear size variation suggests polyploidy. No chromosome counts have been made as yet, but as the nuclei of gamic female germaria are filled with small rod-shaped chromosomes and are so much larger than any of the other types of nuclei it is probable that there is more chromatin in the gamic nuclei than in the others.
* *
FIGURES 6-7. Figure 6. Abnormal germarium of a winged-wingless intermediate showing nuclei of two sizes (675 X). Figure 7. Degenerate body (embryo?) found in ovariole of wingless parthenogenetic female producing males (675 X).
PRODUCTION OF APHID TYPES 67
Are all intermediates physiologically wingless?
Shull (1940) has suggested that adult winged-wingless intermediates are not physiologically intermediate but, rather, that they are wingless having progressed during development from a winged to a wingless condition. The structural characters become fixed in an intermediate condition during the transition and remain so during the life of the aphid, but the physiological nature of the individual continues changing until it is completely wingless. As a typical winged individual produces gamic females during the gamic phase of the cycle while a wingless female produces males, the physiological nature of the intermediates was determined by examining the type of offspring produced by them. Thus, if an intermediate produced males it was judged to be physiologically wingless. If it produced gamic females it was winged. Shull concluded that all winged-wingless inter- mediates are physiologically wingless.
An opposite conclusion is indicated by the evidence derived from the inter- mediates used in this study. These intermediates produced both male and gamic female embryos. Consequently some of them were physiologically winged and some wingless.
Degeneration in male-producing wingless females
Wingless females that are producing males not only have distinctive germaria but they also have degenerating cell masses within their ovarioles. The cell masses (Fig. 7) occur in the ovarioles at any position though they were observed most frequently at the end nearest the germarium. They are elliptical in longi- tudinal section and circular in cross section. A vacuolated center area is usually surrounded with a rim of densely staining pycnotic cells. What the degenerating bodies are is questionable but their elliptical shape is similar to young embryos, and furthermore, the rim of cells surrounding a vacuolated non-cellular center area is typical of young male blastulae. Therefore, it is tentatively concluded that the degenerating bodies are embryos that failed to continue development and are being resorbecl. Why degenerating embryos should be characteristic of male- producing wingless females remains an open question.
CONCLUSION
A correlation between the size of the germaria and their nurse cell nuclei and the type of embryos produced seems established. Whether the germaria actually control production of aphid types is still unknown.
SUMMARY
The areas of the center cross-section of adult germaria of parthenogenetic female aphids producing different types of offspring were measured and compared. From this comparison it is evident that the center cross-sections of the germaria of male-producing wingless parthenogenetic females are larger than those of wing- less females producing parthenogenetic females, and these in turn are larger than the cross-section of winged female germaria. All winged females have the same cross-sectional area whether they are producing parthenogenetic or gamic females.
68 CHESTER A. LAWSON
A comparison of the volume of the nurse cell nuclei shows that the nuclei of gamic female germaria are larger than any of the others; those of wingless females producing males are smaller than the gamic nuclei, but larger than any other parthenogenetic nurse cell nuclei. The wingless and winged females producing parthenogenetic females have nurse cell nuclei of the same size, while the nurse cell nuclei of winged females producing gamic females are the smallest of all.
A correlation of the nurse cell nuclear volume of winged-wingless intermediates with the embryos contained in the ovarioles supports the thesis that size of nuclei and type of young produced are interdependent. Those intermediates that con- tained gamic embryos have the smallest nuclei; those with the next in nuclear size have both parthenogenetic and male embryos. The largest contain males only or males and gamic eggs.
LITERATURE CITED
LAWSON, C. A., 1939. The significance of germaria in differentiation of ovarioles of female aphids. Biol. Bull., 77: 135-145.
LAWSON, C. A., 1940. The developmental history of germaria in parthenogenetic female aphids. Ohio. Jour. Sci., 40: 74-81.
SHULL, A. F., 1940. Adult intermediate-winged aphids not physiologically intermediate. Genet- ics, 25: 287-298.
POLYDORA IN OYSTERS SUSPENDED IN THE WATER
VICTOR L. LOOSANOFF AND JAMES B. EXGLE (Fish and Wildlife Service, Fishery Biological Laboratory, Milford, Connecticut)
INTRODUCTION
Among the numerous enemies of oysters the small Polychaete worms of the genus Polydora have long been considered as very destructive. It has been re- ported that sometimes these worms may be responsible for the complete dis- appearance of extensive oyster beds. Such depredations were described by Whitelegge (1890) and Roughley (1922, 1925) who were working in Australian waters, where Polydora caused a heavy mortality among the native oysters. Both authors identified the worm as P. ciliata. It is possible, however, that Whitelegge was mistaken in his identification of the species. According to Wilson (1928) "Whitelegge found the ova and larvae of a species of Polydora attached alongside the adults to the walls of their burrows in oyster shells at Newcastle, in New South Wales. He believed the species to be Polydora ciliata Johns, but his figure of the egg-sacs resembles more closely that given by Soderstrom for Polydora ligni Webster." If Whitelegge was actually mistaken then the destruction of the oysters in Australian waters should be attributed to at least two species of Polydora, namely, P. ciliata and P. ligni.
Several species of Polydora are common along our Atlantic Coast. Lunz (1940, 1941) found that approximately 40 per cent of the oysters of South Caro- lina waters are infested with P. ciliata. This author states in his latest paper that he now has evidence or reports of infestation throughout the entire range of distribution of the American oyster, 0. virginica, in North America. Nelson and Stauber (1940) stated in a brief abstract that many oysters of New Jersey harbored P. ligni Webster. This appears to be the same species which, in the opinion of Wilson, Whitelegge was dealing with in Australia. Kavanagh (1940) found that the Japanese oyster, 0. gigas, planted in Louisiana waters became infested with P. ciliata. Takahashi (1937) reported that P. pacifica was quite commonly present in the shells of the pearl oyster, Pinctada margaritifera.
Polydora or, as it is usually called, mud worm, is also known to infest shells of mollusks other than oysters. Lebour (1907) found that the mussels of the Northumberland beds of England were heavily infested with P. ciliata, and Field (1922) stated that the same species occurs in shells of the mussel, M. edulis, living in American waters.
Polydora usually gains entrance into the oyster while the worm is still in the larval stage, or when very young (W7ilson, 1928; Roughley, 1925). Soon after entering the oyster the worm builds two mud tubes at right angles to the edge of the shell. The accumulated mud irritates the oyster tissue and the mollusk, in self protection, secretes a layer of shell material over the mud tubes. A descrip- tion of the formation of mud blisters has already been given by Whitelegge (1890) and Lunz (1941) and need not be repeated here.
69
70 LOOSANOFF AND ENGLE
It has been the opinion of many investigators that the oysters infested with Polydora are usually very poor. If the infestation persists, they gradually begin to weaken and eventually succumb (Roughley, 1922; 1925). In some instances, as for example in Australia, it has been considered advisable to grow these mollusks on stones, logs or on specially constructed platforms, away from the bottom. Roughley (1922, 1925) believes that the method of keeping the oysters above the bottom mud is an effective means of preventing the infestation. It appears that Roughley 's observations and data fully justify his conclusions in regard to the conditions existing in Australian waters. However, recent work of the authors carried on in Milford Harbor on the Connecticut side of Long Island Sound, showed that some of the habits of our species of Polydora and its effects on American oysters are somewhat different from those described for the Austra- lian species, or previously ascribed to the mud worms common in American waters.
Description of P. Websteri Hartman
The mud worm found in the oysters of Milford Harbor was identified by Dr. Olga Hartman of Allan Hancock Foundation, The University of Southern California, as Polydora websteri Hartman, new name. In personal correspondence with the authors Dr. Hartman states that the original description of the worm, as P. caeca, was published by Webster, 1879. Since the description is faulty and misleading in all essential respects, it has little value for systematists. Dr. Hartman expresses an opinion that, unless caution is taken, the next reviser or systematist is almost certain to refer to our species as the European P. ciliata, since its morphological characters are closely akin to those of the latter. To avoid constant confusion of Polydora websteri, which at present is a systematically unknown species, with P. ciliata and some other species of Polydora that are known to be very numerous in eastern America, Dr. Hartman suggested that a description and the illustrations clearly indicating the characters of the worm should be given in this article. In accordance with the suggestion a description of P. websteri and the illustrations showing some of its morphological characters are offered here. Both the description and illustrations were prepared by Dr. Hartman.
" Polydora websteri Hartman
Polydora caeca Webster, 1879, Trans. Albany Inst., vol. 9, pp. 252-253, Figures
119-122 (not Oersted, 1843). Polydora websteri Hartman (1942 MS on Beaufort Annelids).
The total length consists of about 105 segments and measures (preserved) 20 mm. long or shorter, but the body is usually much contracted and coiled up. The prostomium is clearly bifid at its anterior margin; it may lack eyes or there may be 3 or 4 weakly developed ones in trapezoidal arrangement; the prostomial parts, palpi omitted, are shown in dorsal (Figure 1, a) and ventral (Fig. 1, b) views. The first segment has a notopodial lobe but no notosetae, and the neuro- podium is provided with a fascicle of slender setae. The second to fourth seg- ments are biramous and have larger fascicles of notosetae and neurosetae with posterior lamellae. The fifth or modified segment is longer than the others and has, on either side, a dorsal fascicle of heavy yellow hooks with companion
POLYDORA IN OYSTERS SUSPENDED IN WATER
71
FIGURE 1. Showing certain morphological characters of P. websteri. Explanation in the text.
(Courtesy of Dr. Olga Hartman.)
72 LOOSANOFF AND ENGLE
pennoned setae, and a ventral fascicle of 5 or 6 pointed setae. The seventh setiger has pointed setae in both fascicles. Hooded hooks are present from the neuropodium of the eighth setiger and continued posteriorly to the end. There are no specialized hooks in the last segments. The posterior end terminates in a flattened collarlike disk with a dorsal notch (Fig. 1, c in posterior view) con- siderably wider than the last few segments (Fig. 1, d in lateral view).
Branchiae, first present from the seventh setiger, are at first small but gradually enlarge to their full size in about 5 segments; they are continued through most of the body length but gradually decrease in size in the posterior fourth and are absent from the last 15 or 16 segments.
The heavy hooks of the fifth setiger number about 6 projecting ones in a fas- cicle; they are unique in that the falcate distal end has a hard, chitinous sheath around one side; various views are shown for projecting (Figs. 1, f, g) and em- bedded (Fig. 1, e) ones. The companion pennoned setae (Fig. 1, g) when perfect terminate in an acute point but some may be broken off and appear frayed at the distal ends. The hooded hooks number about 6 in a series in the middle of the body; they have 2 well developed teeth, the major one at a right angle to the shaft (Fig. 1, h). Tubes are fragile, constructed of silt and debris incorporated with mucus, and occur in calcareous shells.
The original description as P. caeca Webster is incomplete in some important details and erroneous in some others. The first segment has neurosetae, not notosetae; the pygidium is interrupted above, not below; the companion setae of the modified segment are pennoned, not capillary; the modified hooks of this seg- ment are not merely falcate but have a sheath that extends some distance around it. There may be weakly developed eyespots.
P. websteri resembles P. ciliata (Johnston) (Fauvel, 1927, Faune de France, Vol. 16, p. 49) in some respects but the two differ in that the first has a prostomial caruncle that extends posteriorly to the end of the third setiger and the modified spines of the fifth setiger have a sheath around one side; in the second the pros- tomial caruncle extends posteriorly to the second setiger and the modified spines have an acute tooth in the concave part of the spine.
The single individual on which Webster's description was based is not known to exist. The collection on which the present description is based is deposited in the Allan Hancock Foundation of the University of Southern California. It was collected from vesicles on empty oyster shells, in the mouth of the Milford River, by Mr. J. B. Engle of the Milford Wildlife Laboratory. Since 1937 I have obtained this species in considerable number from Beaufort, North Carolina, Lemon Bay in southwestern Florida, and Virginia north to Connecticut. It may be widely distributed in intertidal zones of temperate North America.
(On the plate, the small scale near the label indicates^! mm. for prostomium and pygidium and 0.1 mm. for setal structures.)"
The authors wish to express their appreciation to Dr. Olga Hartman for the identification of our species of the mud worm and for preparation of the description and the illustrations of the morphological characters of P. websteri.
OBSERVATIONS
These studies were begun in April, 1940, when five large groups of oysters, ranging from one to 5 years of age, were placed under observation in Milford
POLYDORA IN OYSTERS SUSPENDED IN WATER
73
Harbor. In the summer of the same year another group, composed of individuals of the 1940 set, and thus being only a few weeks old, was added. Altogether over 1000 animals were used in the experiment. All these oysters were brought from the deep water beds of Long Island Sound, where Polydora is very uncommon. Examination of the oysters showed that only about 2 per cent of them had mud vesicles.
Oysters of each year-class were placed on separate, large, wire trays, suspended in the water from a float, which rose and fell with the tide. Even at low tide the trays were at least four feet above the bottom. The oysters remained suspended in the water until November 1, 1942. Thus, the experiment lasted 1\ years, and
FIGURE 2. Shells of an oyster infested with P. websteri. A. Cup valve. B. Flat valve.
covered two winter and three summer periods. At the end of the experiment a random sample consisting of 20 oysters was taken for examination from each year-class group. All the oysters were opened and the condition of their shells and meats noted.
Examination of the shells showed that the oysters of all year-classes were heavily infested with Polydora websteri (Fig. 2). This was true even for those of the 1940 class which were but several weeks old when placed on the trays. The infestation was so heavy that in main' instances separate mud vesicles could not be distinguished. Usually the combination of several vesicles formed large mud blisters. All the shells, with exception of one flat valve belonging to an oyster of the 1940 class, were infested. The class of 1935, comprised of the oldest oysters, had the greatest number of vesicles and blisters, while the youngest class had the
74
LOOSANOFF AND ENGLE
least (Table I). However, since the shells of the older oysters offered much larger areas for infestation than those of the younger class, no direct relationship be- tween the age of the animals and the degree of infestation could be assumed. Such a conception was further sustained by the lack of correlation between the age of the oysters and the degree of infestation in the other four year-classes (Table I). In general, the cup valves of the oysters contained more vesicles and blisters than the flat valves. This again cannot be regarded as significant because the surface of a cup valve is considerably larger in area than that of a flat one. Careful examination of the character and positions of the mud vesicles, and the location of the characteristic double holes on the exterior of the shells through
TABLE I
Number of mud vesicles and blisters found in shells of oysters of different ages grown on the suspended trays and on the bottom. Each sample consisted of 20 oysters.
|
TRAY OYSTERS |
BOTTOM OYSTERS |
|||||||
|
YEAR CLASS |
Cup valve |
Flat valve |
Cup valve |
Flat valve |
||||
|
vesicles |
blisters |
vesicles |
blisters |
vesicles |
blisters |
vesicles |
blisters |
|
|
1935 |
208 |
31 |
177 |
27 |
13 |
1 |
17 |
3 |
|
1936 |
136 |
3 |
103 |
2 |
||||
|
1937 |
188 |
20 |
123 |
17 |
||||
|
1938 |
208 |
39 |
111 |
24 |
||||
|
1939 |
189 |
33 |
156 |
28 |
||||
|
1940 |
126 |
20 |
81 |
8 |
3 |
0 |
4 |
0 |
which the worms communicate with the outside, as well as studies of the cross- sections of the shells clearly indicated that the infestation was not confined ex- clusively to any one year within the experimental period. It was found, as the result of such examination, that the infestation with Polydora began during the summer of 1940 and continued until the end of the experiment.
While examining the shells of the oysters it was noted that in many instances of severe infestation as many as six or seven layers of blisters, superimposing one over the other, could be found over the same shell area. The worms occupying the lowest, and therefore the oldest, blisters were of a larger size than those of the upper ones. The occupants of the upper blisters were, as a rule, very small, indicating that they entered the shell only a short time before examination. Even under such apparently overcrowded conditions the majority of the worms were alive and, judging by the quantities of accumulated mud, very active.
Discovering an unusually heavy infestation of the tray oysters, it was decided to compare the degree of infestation of these animals with that of the mollusks living on the muddy bottom. For this, samples of 20 oysters of the 1935 and 1940 year-classes were taken from the bottom of AJilford Harbor, in the area where the float with the suspended oysters was stationed during the experiment. Examination of the shells of the bottom oysters revealed that they were much less infested than those kept suspended in the trays. Many bottom oysters of the two year-classes were entirely free of mud worms. In the 1935 class, nine
POLYDORA IN OYSTERS SUSPENDED IN WATER
75
cup valves and seven flat ones bore no signs of infestation. The class of 1940 was in even better condition, because 17 cup and 16 flat valves were entirely free of vesicles or blisters (Table I).
In examining the condition of the oysters removed from the trays it was noted that, regardless of the very large number of mud worms infesting their shells, the oyster meats were in an excellent condition. They were unusually "fat," and large in size. They appeared much superior to those of the oysters usually grown in Milford Harbor. To verify this, a comparison was made of the experimental oysters and the animals taken from the bottom of Milford Harbor. It consisted in comparing the weight of the oyster meats in relation to their total weight. Each sample consisted of 20 oysters. The results obtained indicated that the animals suspended on the trays were much better than those collected from the bottom (Table II). This was especially true of the oysters of the 1935 year-class,
TABLE II
Average total weight and weight of meat, and per cent of meat of oysters of different ages grown on the suspended trays and on the bottom.
|
YEAR CLASS |
TRAY OYSTERS |
BOTTOM OYSTERS |
||||
|
Total weight |
Weight of meat |
Per cent of meat |
Total weight |
Weight of meat |
Per cent of meat |
|
|
1935 |
280.4 |
28.3 |
10.1 |
232.5 |
13.2 |
5.7 |
|
1936 |
216.2 |
22.1 |
10.2 |
|||
|
1937 |
202.0 |
21.9 |
10.8 |
|||
|
1938 |
154.2 |
17.2 |
11.1 |
|||
|
1939 |
122.1 |
15.0 |
12.3 |
|||
|
1940 |
73.1 |
10.1 |
13.7 |
21.8 |
2.4 |
11.0 |
where the bottom animals were found to be rather poor. The condition of the bottom oysters of this age-group was further substantiated by the observations made in connection with another series of experiments, dealing with seasonal changes in oysters in Milford Harbor. Samples of these oysters examined on November 15 and December 15, 1942, showed that on those dates the weight of their meats constituted 6.5 and 5.9 per cent of their total weight.
On the basis of the above described observations the conclusion may be formed that a heavy infestation with P. websteri does not necessarily render the oysters poor. As was mentioned previously, the meats of heavily infested tray oysters were in an unusually good condition. Such a condition, of course, cannot be ascribed to commensalism with P. websteri. It indicates, nevertheless, that a heavy infestation of their shells does not prevent oysters from becoming "fat," provided other environmental conditions are favorable for the existence of the mollusks.
Regardless of the fact that the experimental oysters were suspended on the trays, away from the bottom, they were, nevertheless, covered with a very heavy- layer of the deposit consisting of silt, mud and various dead and alive plankton forms. The thickness of this layer usually varied between 1/8 and 1/4 of an inch. Such accumulation of muddy substance was more than sufficient to supply the worms with all the mud needed for their activities. Therefore, no question could
76 LOOSANOFF AND ENGLE
be raised whether or not there was enough mud to be carried by the worms for deposition between the shells of the oysters.
Indirectly, the experiments also provided an answer to the question of whether or not a severe infestation with P. websteri always causes a heavy mortality among the oysters affected. This answer is negative. For example, the most heavily infested year-class was that of 1935. In November 1941, this group consisted of 94 oysters. At the end of the experiment, in November 1942, 90 of these animals were still alive. Therefore, during the last year of the experiment, when infestation with the mud worms was presumably the heaviest, only four animals of the total number of 94 died. Thus, the mortality for the entire year amounted to only 4.3 per cent. This figure is considerably below that of the mortality of oysters of the same age but living under natural conditions, where a death-rate from 8 to 10 per cent is considered as normal.
It was also observed that a heavy infestation with mud worms did not inter- fere with the rapid growth of the oysters. All year-classes of suspended oysters, although heavily infested, showed a considerable increase in growth. The rate of growth greatly exceeded that of the less infested oysters living under natural conditions. The most noticeable difference was recorded in the case of the 1940 year-class, where at the end of two years, the average length of the suspended oysters was 79.2 mm. as compared with 63 mm. for the bottom oysters. Inci- dentally, our observations that the oysters kept off the bottom showed better growth are contradictory to those of Nelson (1921) who, on the basis of his experiments in which he also used wire trays, stated that "There was no appreci- able difference in the rate of growth of oysters on the bottom from that of oysters on the platform above."
DISCUSSION AND SUMMARY
It has been generally assumed that several species of Polychaete worms, such as P. ciliata and P. ligni, are very dangerous enemies of oysters interfering with their fattening and growth, and often causing a heavy mortality among them. It has also been stated that a heavy infestation with Polydora can be avoided if the oysters are grown away from the bottom mud. The method of growing oysters off the bottom is widely used in Australia.
Results of the experiments conducted for a period of 1\ years in Milford Har- bor, Connecticut, indicate that in this body of water certain aspects of the be- havior of at least one species of Polydora and its effects upon infested oysters are different from those observed in Australian waters, or ascribed to the mud worms of certain sections of our Atlantic Coast.
The Milford experiments have shown that mud worms, Polydora websteri, were found in much larger numbers in the shells of the oysters suspended in the water for a period of 1\ years than in those living on the muddy bottom. This indicates that in some areas along the Atlantic Coast of North America the suspen- sion of oysters away from the bottom does not prevent, or eliminate, their infes- tation with the mud worms, P. websteri. Results of the experiments also point to the conclusion that the method of suspension may be regarded as providing sometime more favorable conditions for the mud worms to infest the oysters.
A complete explanation as to why the mud worms preferred the tray oysters to those on the bottom is still lacking. It may be suggested at this time, never-
POLYDORA IN OYSTERS SUSPENDED IN WATER 77
theless, that the difference in salinity at the bottom, and in the zone where the oysters were suspended might have played an important part in the degree of infestation of the two groups. In Milford Harbor, which is a body of water affected by the river discharge and by inflow of salt water from the Sound, the salinity of the upper layers of the water is usually lower than that observed near the bottom. At times such differences are of considerable magnitude. For example, during the rainy period of 1942 occurring in August, the salinity of the surface layer varied between one and five parts per thousand, whereas at the bottom the salinity remained quite steadily above 25 parts per thousand. The fact that the heavily infested tray oysters were living in less salty water than those existing on the bottom may indicate that P. websteri prefers the water of considerably reduced salinity. Lunz (1941), on the basis of his observations in South Carolina, is also of the opinion that P. ciliata is more prevalent in water of low salinity.
The suggestion that P. websteri does not readily infest the oysters living in water of comparatively high salinity is substantiated by the authors' examination of oysters collected from Long Island Sound proper. During the summer of 1942 several thousand oysters of all ages were opened and examined. They were collected from many sections of the oyster-producing area of Connecticut. Very few oysters were found infested with Polydora. The salinity of the water of the area from which the samples were collected is usually above 26 parts per thousand (Loosanoff and Engle, 1940).
If certain species of Polydora, such as P. websteri, prefer water of low salinity, it is quite possible that several outbreaks of infestation of oysters with mud worms may be the result of prolonged rainy periods. In such cases large quantities of fresh water entering inshore shallow areas may considerably reduce the salinity of the water in which oyster beds are located, thus providing favorable conditions for the spreading of Polydora infestation. Experiments on the effects of various salinities upon the activities of Polydora, which are now being conducted by the authors, may throw additional light upon this very interesting and important subject.
Regardless of the heavy infestation with mud worms the meats of the tray oysters were in a far better condition than those of the mollusks living on the bottom. Their growth was also more rapid than that of the less infested animals of the same ages, but living under natural conditions. These two observations indicate that a heavy infestation with P. websteri does not necessarily interfere with the feeding and fattening of oysters, nor impair their growth. The apparent lack of ill effects upon the growth and fattening of oysters can be easily under- stood, if it is remembered that Polydora is not a parasite. Each worm remains in contact with the fleshy tissues of the oysters for a comparatively brief period. As soon as the mollusk covers the intruder and its mud tubes with a layer of shell material, the worm becomes isolated and cannot exert toxic effects upon the tissues of the oyster. It is probable, nevertheless, as Lunz (1941) indicated, that a large number of mud blisters within the shell may restrict the living space of the oyster, and that the animal may be forced to spend considerable energy in secreting the shell material for covering the mud worms. It is also possible that large quantities of mud accumulated by the worms on the bottom may render
LOOSANOFF AND ENGLE
the environmental conditions unfavorable for- the existence of the oysters and may even cause a heavy mortality among those mollusks (Roughley, 1922).
Milford experiments have also shown that a severe infestation with P. websteri did not cause a heavy mortality of the oysters. Our observations coincided with those of Lunz (1941) on P. ciliata who found that "In the five year period during which these pests have been under observation in South Carolina and other south- ern states, no high mortality has been found on oyster beds which could be attributed to the activities of Polydora."
LITERATURE CITED
FIELD, IRVING A., 1922. Biology and economic value of the sea mussel, Mytilus edulis. Bull.
U. S. Bur. Fish., 38: 128-259. KAVANAGH, L. D., 1940. Mud blisters in Japanese oysters imported to Louisiana. Louisiana
Conservation Review for Autumn, 1940: 31-34. LEBOUR, M. V., 1907. The mussel-beds of Northumberland. Northumberland Sea Fisheries
Committee. Report on the Scientific Investigations for the year 1906: 28-46. New Castle-
on-Tyne. LOOSANOFF, VICTOR L., AND JAMES B. ENGLE, 1940. Spawning and setting of oysters in Long
Island Sound in 1937, and discussion of the method for predicting the intensity and time
of oyster setting. Bull. U. S. Bur. Fish., 74: 217-255.
LUNZ, G. R., JR., 1940. The Annelid worm, Polydora, as an oyster pest. Science, 92: 310. LUNZ, G. R., JR., 1941. Polydora, a pest in South Carolina oysters. Journ. of the Elisha Mitchell
Scientific Society, 57: 273-283. NELSON, T. C., 1921. Report of the department of biology of New Jersey Agricultural College
Experiment Station for the year ending June 30, 1920. New Jersey Agricultural Ex- periment Station, 1919-1920: 317-349. NELSON, THURLOW C., AND LESLIE A. STAUBER, 1940. Observations of some common Polychaetes
on New Jersey oyster beds with special reference to Polydora. Anat. Rec., 78: 102. ROUGHLEY, T. C., 1922. Oyster culture on the George's River, New South Wales. Technical
Education Series, No. 25, Technological Museum, Sydney, 1-69.
ROUGHLEY, T. C., 1925. The story of the oyster. Australian Museum Magazine, 2: 1-32. TAKAHASHI, KEIZO, 1937. Notes on the polychaetous annelid Polydora pacifica n. sp. which bores
holes in Pinctada margaritifera (Linne). Palao Trop. Biol. Stat. Studies, 1: 155-167. WHITELEGGE, T., 1890. Report on the worm disease affecting the oysters on the coast of New
South Wales. Records of the Australian Museum, 1: 41. WILSON, DOUGLAS, P., 1928. The larvae of Polydora ciliata Johnston and Polydora hoplura
Claparede. Jour. Mar. Biol. Ass'n N. S., 15: 567-603.
THE ACTION OF ACETYLCHOLINE ON THE ISOLATED HEART OF VENUS MERCENARIA
ROBERT B. WAIT (Biological Laboratories, Harvard University, Cambridge)
INTRODUCTION
The extraordinary sensitivity of the heart of the lamellibranch mollusc, Venus mercenaria, to acetylcholine was first reported by Prosser and Prosser (1937). Smith and Levin (1938) suggested the use of the isolated heart as a test object for acetylcholine and indicated its very much greater sensitivity to acetyl- choline than to choline. The first detailed account of the responses of the Venus heart to acetylcholine and to nerve stimulation was by Prosser (1940). Prosser presented evidence that nervous inhibition of the heart is probably due to the release of acetylcholine at the terminations of nerve fibers from the visceral ganglion.
With the idea of using the isolated Venus heart for determining the acetyl- choline content of tissues, when only small amounts are available, further experi- ments were carried out to ascertain the nature of the concentration-action curve and the imporatance of temperature control. The results will be reported briefly.
METHODS
Supplies of animals were obtained from a local market and stored dry at 5° C. until used. They ordinarily remained in a satisfactory condition for one to two weeks.
Certain minor changes in the method suggested by Prosser (1940) for isolating and perfusing the heart were made, hence the general procedure will be outlined. The soft parts were exposed dorsally by breaking and removing the umbos and hinge of the valves. The mantle and pericardium overlying the heart were cut away exposing the single, median ventricle and the laterally-disposed, thin- walled auricles. A thread was passed under each auricle and tied at the junction with the ventricle. The auricles were cut distal to the threads, also the anterior and posterior blood vessels and the intestine which passes through the heart. The isolated ventricle (which will be spoken of as the "heart") was placed in a bath with a capacity of 10 or 20 cc. when filled to the overflow arm. This was supplied with a common inlet-outlet tube at the bottom for perfusion fluid and an additional inlet for air, needed mainly for stirring since the oxygen requirements of the heart are low. This arrangement is shown in Figure 1. When temperature regulation was desired this chamber was submerged to the overflow arm in a water bath, the temperature of which could be kept constant or varied as desired. The heart was attached to a light heart lever counterweighted to 250 mg. and the beat recorded on a slow kymograph. The advantage of suspending the heart by the auricles is the avoidance of interference by the short length of intestine which
79
80
ROBERT B. WAIT
passes through the longitudinal axis of the ventricle, the amplitude of beat being greater and more constant than when the heart is suspended by the anterior and posterior ends.
| ^ Since the blood of Venus mercenaria is very similar in composition to sea water (Cole, 1940) the latter was used as a perfusion fluid with quite satisfactory results. Glucose was added to the sea water in the proportion of 0.25 grams/liter. Isolated hearts have been kept beating for as long as three days at 15° C. During periods
PERFUSATE
OUTLET
B
FIGURE 1. Perfusion chamber showing arrangement of air inlet and common inlet-outlet for perfusion fluid. Spring clamps at A and B, when opened and closed alternately, allow washing without complete removal of fluid from the chamber.
of washing the level of fluid in the bath was not allowed to drop below the level of the heart as such mechanical disturbance often causes temporary cessation of beat.
The acetylcholine used was in the form of the chloride and a stock solution was made up of 10~3 by weight of the alkaloid in 5 per cent NaH2PO4. This has a pH of approximately 4.0, at which acetylcholine is quite stable. The solution was sealed in small ampoules which were heated at 100° C. in a water bath for five minutes and then stored in a refrigerator until needed. Just before using, the stock acetylcholine was diluted with sea water so that a series of dilutions were at hand from which known amounts, up to one cc., when added to the bath, gave the desired concentration. The acetylcholine was added at the bottom of the bath by means of a hypodermic syringe with a long, small bore, glass tube bent at a right angle, in place of a needle. When the acetylcholine was added a corresponding volume of sea water was automatically displaced from the top of the bath before any appreciable mixing by the stream of air bubbles occurred.
ACETYLCHOLINE ACTION ON VENUS HEART 81
In a given test the acetylcholine was left on the heart for one minute during which time the amplitude reached a new and nearly constant level. The heart was then washed with several changes of sea water and allowed a period of five minutes to recover its original amplitude before a second test was made. - Meas- urement of the amplitude before and near the end of a given test allowed calcula- tion of the amount of inhibition resulting from the action of the drug on the heart, and this was taken as a measure of effect.
Although eserine increases the sensitivity of the Venus heart to acetylcholine it was not used due to the fact that recovery between tests is more rapid in the non-eserinized heart.
RESULTS
1. The concentration-action curve for acetylcholine inhibition.
Besides any theoretical significance in the quantitative relation between the concentration of a drug and its effect on a given biological system it is of practical importance in bioassay to know the nature of the concentration-action curve for the particular drug and the preparation being used. If it is not a straight line one can select that range where the response shows the greatest change with small differences in the concentration of the substance under investigation. Clark (1933) has pointed out that for most potent drugs such as acetylcholine, adrenaline, histamine and nicotine the concentration-action curves follow a hyperbola and that, depending on the kind of preparation and the recording sys- tem, responses are sometimes less accurately measured near the threshold and in other cases as they approach 100 per cent. It will be seen in the case of the Venus heart that the responses are most accurately determined in the vicinity of 50 per cent inhibition.
Concentration-action curves were obtained for 15 isolated Venus hearts. A series of sample records of the responses of one of these hearts is shown in Figure 2. From such records the per cent inhibition for each concentration could be meas- ured and, in each case, when the results were plotted the curve was a hyperbola. Due allowance had to be made in some instances for the apparent complete in- hibition of the heart before the flat portion of the curve was attained. This was due to the inertia in the recording system and the consequent failure of the record to show small residual movement.
In most preparations commonly used in the assay of acetylcholine, such as the isolated frog heart, the frog rectus abdominis and the dorsal muscle of the leech, the range over which graded action is obtained is from 1000 to 10,000 fold (Clark, 1933). In the case of the isolated lobster heart graded effects may be obtained over a 1,000,000 fold range (Welsh, 1942). In such cases it is customary to plot a measure of the effect against the logarithm of the concentration. Such curves are always S-shaped. When the amount of inhibition of the Venus hearts was plotted against the logarithm of the concentration most of the curves were such as is seen in Figure 3, which is that of a typical heart. This curve which was drawn to fit the points emphasizes the difficulty of making accurate measurements as the responses approach maximum. Since it is also difficult to determine accurately small amounts of inhibition it is obvious that in using the Venus heart for bioassay it is better to choose such concentrations of knowns and unknowns that the amount of inhibition produced is between 20 per cent and 80 per cent.
82
ROBERT B. WAIT
ACH 2X10'
ACH 5X 10"
ACH 9XIO
"'2
ACH 10
ACH 2X10"
ACH 7X10
-ii
ACH 9X10"
ACH I 0
"'°
ACH 2X 10
.-10
ACH 2.5 X 10
FIGURE 2. Sample kymograph records from a series on one heart showing graded action of acetylcholine over a range from threshold to complete inhibition.
ACETYLCHOLINE ACTION ON VENUS HEART
83
2. Effect of temperature on the response to acetylcholine.
Some of the concentration-action curves were obtained in February, others as late as May. During February the concentrations of acetylcholine which produced a just measurable inhibition on different hearts were between 5 X 10~12 (1 : 5,000,000,000,000) and 5 X lO"11. During May thresholds were found as high as 5 X 10~10. This was at first thought to be evidence of a seasonal varia- tion in sensitivity, although Prosser (1940) reported the highest sensitivity to
100 -
CD
X
z
LJ
o ac ui a.
20 -
5X10
10
5X10 CONC. ACH.
10
5X10
FIGURE 3. Data from a typical heart showing the decrease in amplitude (per cent inhibition) as a function of the concentration of acetylcholine (ACh).
occur in the spring. The experiments done thus far has been at room tempera- ture and there was some evidence that the sensitivity of a heart was lower when the room temperature was abnormally high. Therefore a few experiments were performed to determine the effect of temperature on the response of the heart to acetylcholine. By means of a bath, with temperature control, the chamber containing the heart, and the perfusion fluid, could be maintained at any tem- perature between 5° and 35° C. The range over which hearts were observed to beat satisfactorily was somewhat less than this. Beginning in some cases at a low temperature and in others at a high, the concentration of acetylcholine was found which would produce a 50 per cent decrease in amplitude. The tempera- ture was then increased or decreased and after a period of adaptation the con- centration of acetylcholine necessary for 50 per cent inhibition was again de-
84
ROBERT B. WAIT
termined. The results on three hearts, which had approximately the same thresh- old sensitivity at a given temperature, are shown in Figure 4. That there is a marked effect of temperature on the response of a given heart to acetylcholine is apparent. Approximately 100 times as much acetylcholine is required to produce 50 per cent inhibition at 30° C. as is required at 10° C. For this reason, and also from a consideration of the average environmental temperature of Venus mer- cenaria, it may be concluded that 15° C. is a satisfactory temperature at which to
30
a.
2
LU
20
10
Id"
10'°
l<59
CONC. ACH.
id*
I07
FIGURE 4. Data from three hearts, each represented by a different symbol, showing the concentration of acetylcholine (ACh) necessary to produce 50 per cent inhibition at different temperatures.
maintain the isolated venus heart for use in bioassay. If temperature control is not employed it is obviously necessary to perform a given set of assays at a nearly constant temperature.
It is probable that the increase in the amount of acetylcholine required to produce a given amount of inhibition, as the temperature is raised, is due to the activation of the enzyme cholinesterase which destroys acetylcholine, and which is present in the Venus heart in small amounts (Jullien, et al, 1938; Smith and Click, 1939).
CONCLUSIONS
This further study of the response of the isolated heart (ventricle preparation) of Venus mercenaria to acetylcholine provides information which confirms and extends that of Prosser (1940). Since the work was done with the practical view- point of eventual use of the preparation in assaying for acetylcholine in tissue extracts, little attention has been directed toward certain interesting theoretical
ACETYLCHOLINE ACTION ON VENUS HEART
problems. The demonstration that the concentration-action curve is a hyper- bola, and recognition of the difficulty of recording beats of small amplitude, indicates that determination of acetylcholine. values can most accurately be made when the concentrations are such as to produce between 20 and 80 per cent de- crease in amplitude.
The importance of temperature control is evident. A heart which is rela- tively insensitive to acetylcholine at 25° to 30° C. becomes 100 times more re- sponsive at 5° to 10° C. A temperature midway in this range has been found to preserve a beat of satisfactory amplitude and frequency for a convenient length of time (12 to 24 hours).
LITERATURE CITED
CLARK, A. J., 1933. The mode of action of drugs on cells. Edward Arnold and Co., London. COLE, W. H., 1940. The composition of fluids and sera of some marine animals and of the sea
water in