HfMlmlHHHHHKHHKStj THE , BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden E. N. HARVEY, Princeton University G. H. PARKER, Harvard University LEIGH HOADLEY, Harvard University A. C. REDFIELD, Harvard University L. IRVING, Swarthmore College F. SCHRADER, Columbia University M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University H. B. STEINBACH, University of Minnesota Managing Editor VOLUME 95 AUGUST TO DECEMBER, 1948 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. H 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 June 15 and September 1, and to the De- partment of Zoology, University of Minnesota, Minneapolis 14, Minnesota, 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, 1948 PAGE ANNUAL REPORT OF THE MARINE BIOLOGICAL LABORATORY 1 HEILBRUNN, L. V. AND W. L. WILSON Protoplasmic viscosity changes during mitosis in the egg of the Chaetop- terus 57 PEQUEGNAT, WILLIS E. Inhibition of fertilization in Arbacia by blood extracts 69 BLACK, VIRGINIA S. Changes in density, weight, chloride, and swimbladder gas in the killifish, Fundulus heteroclitus, in fresh water and sea water 83 BERRILL, N. J. A new method of reproduction in Obelia 94 IFFT, JOHN D. AND DONALD J. ZINN Tooth succession in the smooth dogfish, Mustelus canis 100 MENZIES, ROBERT J. AND RICHARD J. WAIDZUNAS Postembryonic growth changes in the isopod Pentidotea resecata (Stimpson) with remarks on their taxonomic significance 107 HASSETT, CHARLES C. The utilization of sugars and other substances by Drosophila 114 BOREI, HANS Respiration of oocytes, unfertilized eggs and fertilized eggs from Psam- mechinus and Asterias 124 No. 2. OCTOBER, 1948 ADDRESSES AT THE LILLIE MEMORIAL MEETING, WOODS HOLE, AUGUST 11, 1948 151 HSU, W. SlANG Some observations on the Golgi material in the larval epidermal cells of Drosophila melanogaster 163 SMITH, RALPH I. The role of the sinus glands in retinal pigment migration in grapsoid crabs 169 SCHARRER, BERTA The prothoracic glands of Leucophaea maderae (Orthoptera) 186 VON BRAND, THEODOR, M. O. NOLAN, AND ELIZABETH ROGERS MANN Observations on the respiration of Australorbis glabratus and some other aquatic snails 199 ROGERS-TALBERT, R. The fungus Lagenidium callinectes Couch (1942) on eggs of the blue crab in Chesapeake Bay 214 iii iv CONTENTS SCUDAMORE, HAROLD H. Factors influencing molting and the sexual cycles in the crayfish 229 ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1948 238 PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GENERAL PHYSI- OLOGISTS 281 No. 3. DECEMBER, 1948 BERRILL, N. J. The life cycle of Aselomaris michaeli, a new gymnoblastic hydroid .... 289 COLWIN, LAURA HUNTER Note on the spawning of the holothurian, Thyone briareus (Lesueur) .... 296 DAS, S. M. The physiology of excretion in Molgula (Tunicata, Ascidiacea) 307 JOHNSON, MARTIN W. AND J. BENNET OLSON The life history and biology of a marine harpacticoid copepod, Tisbe furcata (Baird) 320 LEFEVRE, PAUL G. Further chemical aspects of the sensitization and activation reactions of Nereis eggs 333 WELSH, JOHN H. AND RAE TAUB The action of choline and related compounds on the heart of Venus mercenaria 346 WHITING, ANNA R. Incidence and origin of androgenetic males in X-rayed Habrobracon eggs 354 PAPERS PRESENTED AT GENERAL SCIENTIFIC MEETINGS, MARINE BIOLOG- ICAL LABORATORY, SUMMER OF 1948: ERRATUM 361 Vol. 95. No. 1 August, 1948 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE MARINE BIOLOGICAL LABORATORY FIFTIETH REPORT, FOR THE YEAR 1947 — SIXTIETH YEAR I. TRUSTEES AMD EXECUTIVE COMMITTEE (AS OF AUGUST 12, 1947) 1 STANDING COMMITTEES II. ACT OF INCORPORATION 4 III. BY-LAWS OF THE CORPORATION 4 IV. REPORT OF THE TREASURER 5 V. REPORT OF THE LIBRARIAN 11 VI. REPORT OF THE DIRECTOR 16 Statement 16 Addenda : 1 . Memorials to Deceased Trustees 20 2. The Staff 30 3. Investigators and Students 32 4. Tabular View of Attendance, 1943-1947 41 5. Subscribing and Co-operating Institutions 41 6. Evening Lectures 42 7. Shorter Scientific Papers (Seminars) 43 8. Members of the Corporation 43 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 EMERITI E. G. CONKLIN, Princeton University W. C. CURTIS, University of Missouri B. M. DUGGAR, University of Wisconsin W. E. GARREY, Vanderbilt University Ross G. HARRISON, Yale University F. P. KNOWLTON, Syracuse University * Deceased. MARINE BIOLOGICAL LABORATORY R. S. LILLIE, The University of Chicago A. P. MATHEWS, University of Cincinnati W. J. V. OSTERHOUT, Rockefeller Institute G. H. PARKER, Harvard University TO SERVE UNTIL 1951 W. C. ALLEE, The University of Chicago C. L. CLAFF, Randolph, Mass. G. H. A. CLOWES, Lilly Research Laboratory K. S. COLE, The University of Chicago P. S. GALTSOFF, U. S. Fish and Wild Life Service L. V. HEILBRUNN, University of Pennsylvania J. H. NORTHROP, Rockefeller Institute A. H. STURTEVANT, California Institute of Technology TO SERVE UNTIL 1950 DUGALD E. S. BROWN, Bermuda Biological Station D. P. COSTELLO, University of North Carolina M. H. JACOBS, University of Pennsylvania D. A. MARSLAND, New York University A. K. PARPART, Princeton University FRANZ SCHRADER, Columbia University H. B. STEINBACH, University of Minnesota B. H. WILLIER, Johns Hopkins University TO SERVE UNTIL 1949 W. R. AMBERSON, University of Maryland School of Medicine P. B. ARMSTRONG, Syracuse University L. G. BARTH, Columbia University S. C. BROOKS, University of California F. A. BROWN, JR., Northwestern University H. B. GOODRICH, Wesleyan University A. C. REDFIELD, Harvard University C. C. SPEIDEL, University of Virginia TO SERVE UNTIL 1948 ERIC G. BALL, Harvard University Medical School R. CHAMBERS, Washington Square College, New York University EUGENE F. DuBois, Cornell University Medical College COLUMBUS ISELIN, Woods Hole Oceanographic Institution C. W. METZ, University of Pennsylvania H. H. PLOUGH, Amherst College E. W. SINNOTT, Yale University W. R. TAYLOR, University of Michigan EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES LAWRASON RIGGS, Ex officio, Chairman E. N. HARVEY, Ex officio D. M. BRODIE, Ex officio CHARLES PACKARD, Ex officio P. B. ARMSTRONG, to serve until 1948 P. S. GALTSOFF, to serve until 1948 TUUSTKKS M. H. JACOBS, to serve until 1<>4(> A. K. PARPART, to serve until 1949 (.'. C. SPEIDEL, to serve until 1950 H. B. STEINBACH, to serve until 1950 THE LIBRARY COMMITTEE \V. R. TAYI.OK, Cliainnaii K. S. COLE E. N. HARVEY M. E. KRAHL A. C. REDFIELD E. P. LITTLE, Chairman C. L. CLAFF G. FAILLA A. K. PARPART THE APPARATUS COMMITTEE THE SUPPLY DEPARTMENT COMMITTEE P. B. ARMSTRONG, Chairman P. S. GALTSOFF R. T. KEMPTON D. A. MARSLAND CHARLES PACKARD THE EVENING LECTURE COMMITTEE CHARLES PACKARD, Chairman L. G. BARTH E. M. LANDIS THE INSTRUCTION COMMITTEE H. B. GOODRICH, Chairman W. C. ALLEE S. C. BROOKS VIKTOR HAMBURGER CHARLES PACKARD, Ex officio THE BUILDINGS AND GROUNDS COMMITTEE C. LLOYD CLAFF, Chairman D. P. COSTELLO ROBERTS RUGH A. C. SCOTT MRS. C. C. SPEIDEL 4 MARINE BIOLOGICAL LABORATORY II. ACT OF INCORPORATION No. 3170 COMMONWEALTH OF MASSACHUSETTS Be It Known, That whereas Alpheus Hyatt, William Sanford Stevens, William T. Sedgwick, Edward G. Gardiner, Susan Minns, Charles Sedgwick Minot, Samuel Wells, William G. Farlow, Anna D. Phillips, and B. H. Van Vleck have associated themselves with the intention of forming a Corporation under the name of the Marine Biological Laboratory, for the purpose of establishing and maintaining a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural his- tory, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the certificate of the President, Treasurer, and Trustees of said Corporation, duly approved by the Commissioner of Corporations, and recorded in this office ; Now, therefore, I, HENRY B. PIERCE, Secretary of the Commonwealth of Massachu- setts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardi- ner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and successors, are legally organized and established as, and are hereby made, an existing Corporation, under the name of the MARINE BIOLOGICAL LAB- ORATORY, with the powers, rights, and privileges, and subject to the limitations, duties, and restrictions, which by law appertain thereto. Witness my official signature hereunto subscribed, and the seal of the Commonwealth of Massachusetts hereunto affixed, this twentieth day of March, in the year of our Lord One Thousand Eight Hundred and Eighty-Eight. [SEAL] HENRY B. PIERCE, Secretary of the Commonwealth. III. BY-LAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY I. The members of the Corporation shall consist of persons elected by the Board of Trustees. II. The officers of the Corporation shall consist of a President, Vice President, Di- rector, Treasurer, and Clerk. III. The Annual Meeting of the members shall be held on the second Tuesday in August in each year, at the Laboratory in Woods Hole, Massachusetts, at 11:30 A.M., and at such meeting the members shall choose by ballot a Treasurer and a Clerk to serve one year, and eight Trustees to serve four years, and shall transact such other business as may properly come before the meeting. Special meetings of the members may be called by the Trustees to be held at such time and place as may be designated. IV. Twenty-five members shall constitute a quorum at any meeting. V. Any member in good standing may vote at any meeting, either in person or by proxy duly executed. VI. Inasmuch as the time and place of the Annual Meeting of members are fixed by these By-laws, no notice of the Annual Meeting need be given. Notice of any special meeting of members, however, shall be given by the Clerk by mailing notice of the time and place and purpose of such meeting, at least fifteen (15) days before such meeting, to each member at his or her address as shown on the records of the Corporation. VII. The Annual Meeting of the Trustees shall be held on the second Tuesday in August in each year, at the Laboratory in Woods Hole, Mass., at 10 A.M. Special REPORT OF THE TREASURER meetings of the Trustees shall be called by the President, or by any seven Trustees, to be held at such time and place as may be designated, and the Secretary shall give notice thereof by written or printed notice, mailed to each Trustee at his address as shown on the records of the Corporation, at least one (1) week before the meeting. At such special meeting only matters stated in the notice shall be considered. Seven Trustees of those eligible to vote shall constitute a quorum for the transaction of business at any meeting. VIII. There shall be three groups of Trustees : (A) Thirty-two Trustees chosen by the Corporation, divided into four classes, each to serve four years ; and in addition there shall be two groups of Trustees as follows: (B) Trustees ex officio, who shall be the President and Vice President of the Cor- poration, the Director of the Laboratory, the Associate Director, the Treasurer, and the Clerk; (C) Trustees Emeriti, who shall be elected from present or former Trustees by the Corporation. Any regular Trustee who has attained the age of seventy years shall con- tinue to serve as Trustee until the next Annual Meeting of the Corporation, whereupon his office as regular Trustee shall become vacant and be filled by election by the Corpora- tion and he shall become eligible for election as Trustee Emeritus for life. The Trustees ex officio and Emeritus shall have all the rights of the Trustees except that Trustees Emeritus shall not have the right to vote. The Trustees and officers shall hold their respective offices until their successors are chosen and have qualified in their stead. IX. The Trustees shall have the control and management of the affairs of the Cor- poration ; they shall elect a President of the Corporation who shall also be Chairman of the Board of Trustees; and shall also elect a Vice President of the Corporation who shall also be the Vice Chairman of the Board of Trustees ; they shall appoint a Director of the Laboratory; and they may choose such other officers and agents as they may think best; they may fix the compensation and define the duties of all the officers and agents; and may remove them, or any of them, except those chosen by the members, at any time ; they may fill vacancies occurring in any manner in their own number or in any of the offices. The Board of Trustees shall have the power to choose an Executive Commit- tee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. They shall from time to time elect members to the Cor- poration upon such terms and conditions as they may think best. X. Any person interested in the Laboratory may be elected by the Trustees to a group to be known as Associates of the Marine Biological Laboratory. XI. The consent of every Trustee shall be necessary to dissolution of the Marine Bi- ological Laboratory. In case of dissolution, the property shall be disposed of in such manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees. XII. The account of the Treasurer shall be audited annually by a certified public accountant. XIII. These By-laws may be altered at any meeting of the Trustees, provided that the notice of such meeting shall state that an alteration of the By-laws will be acted upon. IV. THE REPORT OF THE TREASURER To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY: Gentlemen: The accounts of the Marine Biological Laboratory for the year 1947 have been audited as heretofore by Messrs. Seamans, Stetson and Tuttle, certified public ac- 6 MARINE BIOLOGICAL LABORATORY countants of Boston, and a copy of their audit is available for inspection at any time in the Laboratory office. There were few changes in the Balance Sheet from the preceding year, and it is appended as Exhibit A. As of December 31, 1947, the total book value of all the Endowment Assets, including the Scholarship Funds, was $978,677.67, an increase of $1,478.37. The securities and cash comprising these assets had at the end of the year a market value of $989,814.16. Plant Assets (Land, Buildings and Equip- ment) amounted to $1,311,950.55, a reduction for the year of $1,013.55. Current Assets were increased $10,156.31 to a total of $231,008.19. Current Liabilities (Accounts Payable) were $11,621.59. In recent years the Treasurer's Report has also included the "Exhibit B — In- come and Expense" of the Auditors' Report. This year there is being substituted a summary of "Receipts and Expenditures" prepared by the Treasurer giving the actual cash transactions during the year. The Auditors' statement of "Income and Expense" is a necessary accounting of the books of the Laboratory as they are set up, but does not, in the Treasurer's judgment, give a simple picture of the moneys received and spent during the year. It includes items of a purely bookkeeping character such as depreciation, the value of Library serials received through ex- change, and interdepartmental charges. It does not include expenditures for capi- tal items including some apparatus, books and serials for the Library, etc., even though some of these are normal operating expenses, nor either pensions paid nor funds paid into the Retirement Fund. (All of these are accounted for elsewhere in the Auditors' Report, Schedule IV or Exhibit B.) It does include all gifts in "income" even though some of these gifts are designated for such special purposes as a new boat, and cannot be used for current expenses. Statement I which follows is a summary of the actual financial transactions of the year except for donations for special purposes, special agency accounts, and real estate development accounts which are listed subsequently. Actual receipts in 1947 for current operations were $250,098.72. Current expenditures were $253,822.09 and are listed in detail by departments in "II. Current Expenses." An additional $32,806.56 was spent on repairs and special purchases entered on the books as "Plant Assets" and listed below in "III. Additions to Capital Assets from Current Funds." The total of the year's expenditures is therefore $286,628.65. After the deduction of $2,671.07 transferred from the Carnegie Book Fund and used for the purchase of some of the Library items, the resulting cash deficit for the year is $33,858.86. This was taken care of by using $10,000 of the Reserve Fund (re- ducing it to $6.218.88) and by reducing the cash balances in the checking accounts to a total of $7,949.16 at the end of the year. It should be noted that at the end of the year there was a net increase of the ex- cess of Accounts Receivable over Accounts Payable amounting to $5,776.01, and an increase in the value of Supply Department inventory of $4,848.29. If these two items are taken into account, the actual deficit for the year is reduced to $23,234.56. On the other hand, if the reserve for depreciation is deducted, as it properly is in the Auditors' Report, the deficit would be $25,806.10 greater or $49,040.66. REPORT OF THE TREASURER /. Cash Statement jor Year Ended December 31 , 1947 Expenditures Receipts Additions to Total Current Capital Assets Expenditures Membership Dues $ 2,238.00 Donations for Current Expenses 1 . . 944.95 Income from Endowment 35,616.77 Income other Securities 21,492.00 Real Estate Rentals 6,360.00 $ 1,002.84 $ 1,002.84 Instruction 12,630.00 7,824.94 7,824.94 Research (incl. Apparatus and Chemi- cal Depts.) 25,193.34 17,372.60 $ 2,127.22 19,499.82 Mess 32,029.42 33,965.04 1,880.93 35,845.87 Dormitories and Apt. House 16,600.10 13,570.83 11,013.25 24,584.08 Library2 3,000.00 11,649.19 9,153.70 20.801.89 Buildings and Grounds 46,184.88 5,882.06 52,066.94 Supply Department 3 87,462.76 86,551.69 2,749.50 89,301.19 "Biological Bulletin" 6,044.42 9,812.79 9,812.79 Administration 24,336.70 24,336.70 Miscellaneous 486.96 1,551.59 1,551.59 $250,098.72 $253,822.09 $32,806.56 $286,628.65 Total Expenditures $286,628.65 Total Receipts 250,098.72 36,529.93 Deduct Carnegie Book Fund Payment 2,671.07 Deficit for Year $ 33,858.86 None of the totals in the above Statement include any interdepartment charges, nor any charges for depreciation or interest on investment. 1 Donations were $775 given by the "Associates" of the Laboratory for apparatus, and $169.95 contributed for a Washing Machine. 2 The Library income of $3,000 is the payment from the Oceanographic Institution towards Library expenses. The monetary value of serials received in exchange for the "Bulletin," estimated at $4,937.80, is not included in the above, nor is the $1,350 received from the Oceano- graphic Institution for the purchase of books for their account. 3 The actual sales of the Supply Department were $96,191.85. The values of specimens and supplies furnished Research and Instruction Depts. were $7,132.44 and $4,672.05 respectively. If these values are taken into account and also the gain in inventory of $4,848.29, the increase in accounts receivable of $5,233.85, and a debit charge of $1,800 for administrative and mainte- nance expense, there would be a net profit of $20,997.70 on the operations of the Supply Depart- ment for 1947. This does not take into account the $2,749.50 spent for capital items, or the Auditors' charges of $1,461.67 for depreciation and $2,221.29 for interest on investment. If these had been included, the net profit for the Supply Department would have been $14,565.24. MARINE BIOLOGICAL LABORATORY II. Current Expenses for 1947 by Departments Administration Salaries $ 18,851.55 Central Hanover Bank Trustee Commissions 1,034.87 Falmouth Nat'l Bank Service Charges 142.45 Audit 1,045.83 Treasurer's Office 600.00 Advertising 333.34 Office Supplies 837.74 Sundries (Telephone, Postage, etc.) 1,682.82 Deduct Cash Receipts Instruction Salaries and Travel Allowances Sundries Research (Incl. Apparatus and Chemical Depts.) Salaries Travel Repairs Supplies and Sundries Deduct Cash Receipts Library Salaries Office Supplies Sundries Buildings and Grounds Salaries and Wages Fuel Gas Light and Power ... Water Insurance Repairs Sundries Deduct Cash Receipts Dormitories and Apt. House Salaries and Wages Lighting, Gas and Water Repairs to Bids, and Grounds . . Outside Rentals Laundry Insurance Sundries 24,528.60 191.90 24,336.70 7,273.80 551.14 7,824.94 11,378.87 200.00 798.67 6,763.96 19,141.50 1,768.90 17,372.60 10,881.25 423.29 343.65 11,648.19 22,630.19 2,825.30 1,871.81 2,749.00 623.88 1,666.23 10,605.74 4,190.39 47,162.54 977.66 46,184.88 4,800.50 2,134.87 2,820.28 500.00 1,390.59 734.22 1,190.37 $ 13,570.83 Mess Salaries and Wages $ 8,431.22 Cost of Food 21,326.59 Gas, Water, Light and Power.. 1,650.78 Repairs 231.03 Replacement of Dishes, etc. ... 391.15 Insurance 603.69 Laundry 311.80 Freight and Express 102.07 Sundries 916.71 Supply Department Salaries and Wages Purchase of Specimens Chemicals Containers Boat Expenses Truck Expenses Freight and Express Fuel Light Office Supplies Telephone and Telegraph Insurance Advertising Specimens and Supplies pur- chased for Research Sundries 'Biological Bulletin" Salaries and Wages Printing, etc Real Estate (Rented) Taxes and Insurance on Bar Neck Property (Garage) and Janitor's House Other Expenses Workmen's Compens. Ins Truck Expense Bay Shore and Great Cedar Swamp Expenses Interest on Mortgage Evening Lectures Special Repairs, 1944 Hurricane Damage 33,965.04 30.756.78 38,668.92 2,579.60 3,708.63 1,972.44 825.69 3,313.63 775.37 96.00 511.18 317.10 873.74 344.54 1,058.40 749.66 86,551.69 2,101.00 7,711.79 9,812.79 1,002.84 725.28 222.26 203.72 250.00 128.96 21.37 1,551.59 Total Expenses $253,822.09 REPORT OF THE TREASURER ///. Additions to Capital Assets jroni Current Funds A. Land Improvements Bake House Lot. $ 201.00 201.00 B. Buildings Waterproofing of Dormitory . . . 4,000.00 Waterproofing Apt. House 2,780.00 Dormitories 3,349.32 Brick Laboratory 2,116.75 Wharf 450.00 Other Buildings 1,081.14 $13,777.21 C. Equipment Apparatus Department $ 2,127.22 Dormitories 883.93 Mess 1,880.83 Brick Laboratory 1,273.60 Carpenter Shop 347.62 Old Main Building 411.95 Supply Department 2,749.50 9,674.65 D. Library Back Sets 2,587.72 Books 801.85 Serials 3,901.46 Reprints 10.76 Binding 1,851.91 9,153.70 Total Additions $32,806.56 IV. Gifts for Special Purposes A. Boat Fund Contributions Received $9,335.00 Payments on New Boat 4,032.21 Balance Dec. 31, 1947 5,302.79 In addition securities were received in 1947 from Mrs. W. Murray Crane for the Boat Fund that were subsequently sold for $784.53. B. Dr. Frank R. Lillie Memorial Fund Initial Contribution from Dr. G. H. A. Clowes $1,000.62 V. Real Estate Accounts A. Devil's Lane Property Cash Received in 1947 from Sale of Lots $5,198.00 Disbursements : Taxes $ 286.66 Road Construction 4,161.66 4,448.32 Cost of Devil's Lane Property to Dec. 31, 1947 was $54,296.13. Eighteen lots were sold in 1946 and 1947 for $14,250.00. $6,990.50 was paid in 1946 and 1947 on these purchases. The Devil's Lane Property as of Dec. 31, 1947, was carried on the books at $40,046.13, with the unpaid installments on the lots sold amounting to $7,259.50 carried as Accounts Receivable. B. Gansett Property No cash transactions in 1947 except payment of $87.69 taxes. Gansett Property including Accounts Receivable of $970, and deducting Reserve of $1,950 is now carried on the books at $1,162.36. 10 MARINE BIOLOGICAL LABORATORY [7. Agency Accounts A. Fclloivship Fund Cash Received from the Lalor Foundation $ 5,000.00 Disbursements : » For Fellowships $2,512.50 For Apparatus, Supplies and Laboratory Space .... 1,444.47 3,956.97 Balance, Dec. 31, 1947 1,043.03 B. Cancer Research Account Cash Received from U. S. Public Health Service as grant-in-aid for "The Mechanism of Cell Division and Protoplasmic Growth" (under direction of Dr. Robert Chambers) $25,000.00 Cash paid for Salaries, Laboratory Space, Apparatus and Sup- plies 6,278.81 Balance, Dec. 31, 1947 18,721.19 EXHIBIT A MARINE BIOLOGICAL LABORATORY BALANCE SHEET, DECEMBER 31, 1947 (From Auditors' Report) Assets Endowment Assets and Equities: Securities and Cash in Hands of Central Hanover Bank and Trust Company, New York, Trustee $ 961,036.65 Securities and Cash in Minor Funds 17,641.02$ 978.677.67 Plant Assets: Land $ 1 10,626.38 Buildings 1,337,188.88 Equipment 202,358.59 Library 363,325.72 $2,013,499.57 Less Reserve for Depreciation 722,069.52 $1,291,430.05 Reserve Fund, Cash 6,218.88 Book Fund, Securities and Cash 14,301.62 1,311,950.55 Current Assets: Cash 18,648.18 Mortgage Note Receivable 2,425.00 Accounts Receivable 39,100.94 Inventories : Supply Department $ 43,932.10 "Biological Bulletin" 16,775.67 60,707.77 Investments : Devil's Lane Property 47,305.63 Gansett Property 1,162.36 Stock in General Biological Supply House, Inc. 12,700.00 Other Investment Securities 21,464.00 Retirement Fund 16,388.44 99,020.43 Prepaid Insurance 5,575.99 Items in Suspense (Debits) 5,529.88 231,008.19 $2,521,636.41 REPORT OF THE LIBRARIAN Liabilities Endozi'inait Funds: Endowment Funds $ 959,619.12 Reserve for Amortization 1,417.53 $ 961,036.65 Minor Funds 17,641.02 $ 978,677.67 Plant Funds: Mortgage Note Payable $ 5,000.00 Donations and Gifts $1,172,564.04 Other Investments in Plant from Gifts and Cur- rent Funds 134,386.51 1,306,950.55 1,311,950.55 Current Liabilities and Surplus: Accounts Payable $ 11,621.59 Items in Suspense (Credits) 1,937.38 Reserve for Repairs and Replacements 7,166.71 Current Surplus 210,282.51 231,008.19 $2,521,636.41 Respectfully submitted, DONALD M. BRODIE, Treasurer V. REPORT OF THE LIBRARIAN 1947 The sum of $11,500 appropriated to the library in 1947 was expended as fol- lows: books, $588.11; serials, $3.936.94; binding, $1,851.91; supplies. $444.99; express. $117.52; salaries, $9,475.25 ($3,000 of this sum was contributed by the Woods Hole Oceanographic Institution); back sets, $130.39; insurance, $45.00; sundries, $247.11; total, $16,837.22. The cash receipts of the library totalled: for microfilms, $222.76 (the cost was $531.47) ; sale of duplicates, $264.20. These re- ceipts revert to the Laboratory; so also do the fees from library readers. There were 55 of these readers in the library during the year. Of the Carnegie Corporation of New York Fund, $2.457.33 was expended for the completion of 17 back sets and for the partial completion of 12 back set ; $213.74 was expended for 10 books. The Woods Hole Oceanographic Institution budget was $800 plus $500 for additional purchases made during the year. The total spent was $1,322.28. The Woods Hole Oceanographic Institution also contributed $3.000 (see above under "salaries"). During 1947, the library received 1.201 current journals: 327 (13 new) by sub- scription to the Marine Biological Laboratory; 48 (3 new) to the \Voods Hole Oceanographic Institution; exchanges with the "Biological Bulletin." 512 (12 new; 43 reinstated foreign) and 130 (21 new; 3 reinstated foreign) with the Woods Hole Oceanographic Institution publications; 173 (8 new) as gifts to the former and 11 12 MARINE BIOLOGICAL LABORATORY (3 new) to the latter. The library acquired 178 books: 55 by purchase of the Marine Biological Laboratory ; 18 by purchase of the Woods Hole Oceanographic Institution ; 9 by gift of the authors ; 43 by the publishers ; and 53 by various donors, those of Dr. Oscar W. Richards (8) ; Dr. W. J. V. Osterhout (1) ; Dr. Bradley M. Davis (2) and Dr. Paul S. Galtsoff (2) among the most notable. There were 32 back sets of serials completed ; 19 purchased by the Marine Biological Laboratory (17 with the "Carnegie Fund") ; 5 by the Woods Hole Oceanographic Institution; 6 by exchange with the "Biological Bulletin"; 2 by exchange with the Woods Hole Oceanographic Institution publications. Partially completed sets numbered 56: purchased by the Marine Biological Laboratory, 29 (12 with the "Carnegie Fund") ; purchased by the Woods Hole Oceanographic Institution, 1 1 ; by exchange with the "Biological Bulletin," 1 ; by exchange with the Woods Hole Oceanographic Institu- tion publications, 6; by gift and exchange of duplicate material, 9. The reprint additions to the library wrere 6,926 ; current of 1947, 257 ; current of 1946, 1,041 ; and of previous dates, 5,628. Acknowledgment is made to Drs. F. A. Hartman, Charles Packard, and F. K. Knowlton for valuable contributions to the reprint collection. Also, through Dr. E. G. Butler of Princeton University, the re- print collection of Dr. Ulric Dahlgren was presented to the library. At the end of the year 1947 the library contained 56,594 bound volumes and 149,218 reprints. In the fall 96 titles of the German journals delayed since 1940 were received in scattered volumes and numbers. The majority of these were for the years 1940-42. The above report for 1947 is given in a format similar to preceding years. There follows a summary of the main events in the growth of the library from 1924 to 1947 inclusive. The figures of the varying budgets and acquisitions have been transformed for easy reading into graphs covering these twenty-four years. The data, as well as the running account, have been gathered for the most part from the yearly reports of the librarian. The year 1924 was chosen for the starting point for several reasons. For one, a fair account of the library from its inception in 1888 to 1924 has already been pub- lished in the "Collecting Net" of 1929. But more important, the date marks a change in the administration of the library, in its expansion and in its budget. Previous to 1924 there is no mention of a library committee. The librarian (Dr. H. McE. Knower, followed by Dr. R. P. Bigelow) directed the work with the help of a paid assistant from 1914 to 1923. In 1924 a committee was appointed by the trustees with Dr. C. E. McClung as chairman, and Mrs. T. H. Montgomery, Jr. was made the full-time librarian. Dr. McClung remained as chairman through 1924-1931. Professor E. G. Conklin followed until 1941 and Dr. A. C. Redfield served in this office from 1942 to the end of 1947. In 1924, also, the present li- brary was under construction; and in 1925 the collection was moved from room 217 in the Crane Building to its present position. At this time the library acquired a secretary, Miss Deborah Lawrence, whose invaluable assistance has continued throughout the development of the library into the present. The special significance of the period beginning in 1924 consists in the change that occurred in the library budget. Careful checks having been made to list the journals and books necessary for research in an expanding library, large sums of money were secured to purchase these, especially the back sets of needed journals. The accumulated "library fund" REPORT OF THE LIBRARIAN 13 of $8,000 was made available for 1924-25. In 1925 the General Education Board contributed $50,000 on the condition that it should be spent for back sets during the years 1926-30. In 1929 the General Education Board gave $200,000 for the gen- eral purposes of the library. In addition to these gifts there have been special funds which came to the li- brary later, not a part of the regular annual budget. The Woods Hole Oceano- graphic Institution, established in 1930, in that year contributed $5,000 for back sets and books, and for current journals on oceanography. Subsequently its annual budget for this purpose was $1,000 to $600, increased in 1941 to $800. In addition it has contributed to our library salary budget. The appropriation for this purpose was $1,100 in 1944, $1,700 in 1946 and $3,000 in 1947. The "Carnegie Fund" of $25,000 became available in 1941. Since the gift was not conditioned by a time limit for its expenditure and since the market for rare sets has been limited during and after the war years there remains a balance of this fund amounting to about $10,000. The spacious library, begun in 1924, with five floors of stacks sufficient to hold 100,000 volumes, large reading and cataloguing rooms and librarian's office was thought to be adequate for many years. As early as 1936, however, when the li- brary had acquired half this number of volumes and about 100,000 reprints the librarian reported "it is necessary to explain also how 50,000 volumes (which will be the total in four years if growth occurs at the present rate of 2,000 annually, and and 108,000 reprints if each years adds 3,500) will completely fill space that in 1925 was estimated to be adequate for 100,000 volumes, or 20,000 on each of five floors. The reprint floor at once reduces the available space for volumes to a capacity of 80,000. Besides this the many serial sets and books of quarto size, and over, re- duce the space, and half of the bound serials recorded in our count are in reality two volumes bound together, so that the library will at the end of the year 1940 ac- tually be housing more nearly 75,000 volumes, counted as volumes and not by the accession number, and 108,000 reprints." This prediction was amply fulfilled. By 1940 all available space in the stacks and wall shelves was occupied. An appeal to the Rockefeller Foundation for funds to build an addition met with a generous re- sponse. This gift of $110,400 was used, during the fall and winter of 1940-41, to erect a new wing which more than doubled the capacity of the library. Special pro- vision was made for readers who can now enjoy private, well-lighted tables. Through the period from 1924 until 1941 Dr. Frank R. Lillie was President of the Corporation and Chairman of the Executive Committee of the Laboratory. Any analysis of the steps taken in the development of the library throughout these years points directly to his wisdom, forethought and executive ability. He con- ceived its broad plan and under his guidance the library has grown from a small beginning to its present outstanding position. The cost of building up and maintaining the library is shown in Figure 1. The total expenditures for each year since 1924 are given, and the amounts used for serials and books, and for back sets. Other expenses, such as salaries, binding, supplies, etc., are not shown. Reference has already been made to the great in- crease in overall expenditure from 1924 to 1932. The effects of the depression then began to be felt. The income of the Library Endowment and of the Labora- tory as a whole dropped sharply. The library budgets have been successively re- 14 MARINE BIOLOGICAL LABORATORY duced. This is especially noticeable during the war years when foreign journals, those from Germany in particular, could not be delivered and paid for. The ex- tent of the drop in the number of journals received at that time is seen in Figure 2. Beginning in 1944 the number has risen rapidly until now the total is 1,201, as con- trasted with 1,339 in 1936. Some of the back numbers published during the war are gradually coming in. The yearly additions of volumes of serials and reprints is shown in Figure 3. At present there are 56,594 such volumes and in addition 8,000 books. Attention is called to the reprint receipts from 1924 to 1932, an increase of nearly 7,000 each year. During these years the large collections from Dr. Whitman's library and 35OOO - 3OOOO - asooo- 20 OOO - I5OOO - IOOOO- 5000- 1925 1910 1935 1945 FIGURE 1 those of Sidney I. Smith and Maynard M. Metcalf were catalogued. At this time also about 5,000 reprints previously listed under books were shifted from the count of volumes to that of reprints. In 1938, besides other generous gifts, a collection from Dr. F. R. Lillie was recorded, and in 1943-1947 Dr. Rudolf Hober's large contribution, those of Drs. E. B. Meigs, W. E. Carrey, A. C. Redfield, Ulric Dahl- gren, and others, greatly increased the count. Much of the work of the Library Committee and of the staff has already been indicated. But in a summary of the past twenty-four years it is appropriate to note other services that are not as obvious from a study of the tables. That the purchase of journals and books was preceded by a careful selection is self-evident. In the matter of choice the investigators who use the library have always been the arbiters. To aid them in this the librarian, under the direction of the Library REPORT OF THE LIBRARIAN 15 1000 500 T~T fslumbcr of Journals Received Annualy i Total number of - - Complete Sets of Journals I J I 1950 1935 FIGURE 2 I94O Committee has, each summer since 1927, compiled a list of desirable journals and books. The titles on these lists recommended by the investigators, as well as their own suggestions, were obtained either by purchase or exchange. Policies regard- ing exchange material were decided at the summer meeting of the Library Com- mittee. Another service rendered by the staff was the preparation of the complete list of journal titles, published as a supplement to the "Biological Bulletin." The full titles and holdings of each journal, with cross references of duplication in titles, were arranged in alphabetical order exactly as they appear on the shelves of the 1500001 I r 100,000 50.000 i r 1 r Reprmb j L 1930 j L 1955 FIGURE 3 '94O j L 1945 16 MARINE BIOLOGICAL LABORATORY library. This made a volume of 80 pages in its initial publication in 1943. Addi- tions, in similar form, have been published in each subsequent year. For the benefit of the investigators the "sales room" of duplicate reprints and books has been in operation for many years. The microfilm service, initiated in 1942, has been used extensively by investi- gators to secure literature not available to them elsewhere. This service has re- sulted in a reduction in the number of loans requested by outside libraries during the winter months. In this connection the printed list of journals, mentioned above, has been helpful. The most difficult of the services carried on throughout this period was that of the catalogues. A journal catalogue separated from that of the books and reprints and complete in cross-references proved satisfactory. It seems to present no prob- lem for its future use. On the other hand, the catalogue for books and reprints has always presented difficulties. That the books should be catalogued by subject as well as by author has never been questioned. \Yhether the enormous collections of reprints should be catalogued in the same way has not met the same unanimity of opinion. Finally the librarian established a system of assigning subjects by which the catalogue became less bulky. By this method a bibliography card was placed in front of each author's catalogued works. On this card a list of his sub- jects appears and on a subject card a cross-reference is given to the author's name. In this way many names appear as cross-references from the subject, thus eliminat- ing the making of repetitive subject cards. After this system was installed through- out the catalogue, it was estimated that the card count was 472.500. If the new entries following 1947 can be continued in this manner the catalogue should form a useful guide to those unacquainted with the literature of the reprint collection. This account of the library is not complete without a very special acknowledg- ment of the constant attention given to its development by the Directors (Dr. Mer- kel M. Jacobs and Dr. Charles Packard) and by the Library Committee members. All problems, large or small, in policy or in execution, that were laid before them, received attentive guidance and encouragement. Through their suggestions and moral support the work of the library has maintained its growth and stability. PRISCILLA B. MONTGOMERY, L ibrarian ( retired ) VI. THE REPORT OF THE DIRECTOR To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY Gentlemen: I submit herewith a report of the sixtieth session of the Marine Biological Lab- oratory for the year 1947. The Laboratory has suffered an irreparable loss in the death of Dr. F. R. Lillie. For more than forty years he was its guiding spirit ; under his leadership it grew from a small, struggling institution to its present eminence. His career began and ended in Woods Hole. Here he came as a young student, and here, full of years and honors, he was buried. For fifty-six consecutive seasons he returned, serving as instructor, investigator, and administrator, always with the interests of the Lab- REPORT OF THE DIRECTOR 17 oratory at heart. On becoming Director in 1907 he said, "Our purpose is essen- tially ideal, and its pursuit demands our best efforts and our loyalty." When he resigned in 1926 the trustees wrote, "The Trustees appreciate the conspicuous abil- ity, combined with unselfishness, with which you have guided the Laboratory. To you, in large measure, is due the steady broadening in scope and method that has been so conspicuous a feature of its work in recent years. As its Director you began with an institution already rich in achievement but still poverty stricken in respect to material things. You have left it unsurpassed in equipment and endow- ment, the center of activities that exert an always increasing influence on scientific progress throughout the world." In his reply Dr. Lillie characteristically gave all credit to others — to the Trustees, to Mr. Crane, to the technical staff, and added, "Patience and faith were the only other necessary factors. I have no fear for the future as long as the Woods Hole spirit survives." Genuine cooperation and mutual helpfulness, he said, constitute the basis of this spirit. But elsewhere he added this note of warning, "We must not allow ourselves to forget that the principles for which we stand are never en- tirely won." Those of us who worked with him will not forget his quiet and unhurried ap- proach to current problems, his ability to foresee future needs, his untiring and suc- cessful efforts to meet them ; and his lifelong devotion to the welfare of this Lab- oratory. It is fitting that a tablet to his memory should be placed in the entrance of the building. But more than that, we should build an F. R. Lillie Laboratory which will remind future generations of biologists of our debt to him. 1. Changes in Personnel Mrs. Montgomery, our Librarian since 1924, retired at the end of 1947. For twenty-three years she carried on the business of the Library, beginning at the time when it was a small and insignificant collection, and continuing throughout the en- tire period of its expansion. In addition to her regular Annual Report she has pre- sented a brief history of the Library during her incumbency. Of her own impor- tant and successful work she says little. But all who use the Library are fully aware that she, more than anyone else, is responsible for its completeness, its fine arrangement, and its perfect condition. We owe her a debt of gratitude for her outstanding work in the development of this world famous part of the Laboratory. Miss Deborah Lawrence, who has worked with Mrs. Montgomery since 1925, has been appointed Acting Librarian. Under her supervision the Library con- tinues to be carried on in a most satisfactory way. Mr. Larkin, Superintendent of Buildings and Grounds, also retired this year. Through a long term of service he has been devoted to the interests of the Labora- tory, on hand in emergencies at any time of clay or night, always capably carrying on the work of his department. He has been retained as Consultant because of his intimate knowledge of the complicated installations in our buildings. Mr. Robert Kahler, for many years Mr. Larkin's assistant, is now in charge of the plant, and has demonstrated his competence and resourcefulness in meeting the usual and unusual problems connected with the work. Mr. Homer P. Smith joined the staff in July 1947 as Assistant Business Man- ager. The need of a second man in the office has been felt for some time for the 18 MARINE BIOLOGICAL LABORATORY business of the Laboratory has increased greatly in recent years, and one man could not be expected to carry the entire burden. Under Mr. MacNaught's tutelage he has become familiar with the various phases of work, has adapted himself to the situation, and has proved to be a valuable addition. 2. New Boats The Laboratory is now well provided with boats for all its various needs. New craft were urgently needed to replace the Sagitta, considered unsafe after 40 years of service, to collect the large amount of material required by schools and colleges, and to carry the classes on their field trips. It was, therefore, decided to make an appeal for funds to all members of the Corporation, to all workers at the Laboratory, and to friends living in the vicinity of Woods Hole. The Boat Fund Committee, under the direction of Dr. Heilbrunn, was highly successful in obtaining contribu- tions, the total amount being $10,035. Among the nearly 300 donors were the Lilly Endowment $3,000, Mr. William Proctor $1,000, and Dr. G. H. A. Clowes $1,000. Dr. Redfield, Chairman of the Committee appointed to secure a suitable boat, after visiting a number of boat yards, advised that the new craft should be designed by Mr. Crocker, a naval architect. After the Committee had approved the plans, construction was begun and carried forward without delay. The boat, 33 feet on the water line, has a cruising speed of 12 knots, and is equipped with dredging gear and a two-way radio. It will be in service in the summer of 1948. In the spring of 1948 Mr. Crocker reported that a much larger boat, which he designed, could be bought for $12,000 — about half the original cost. Mr. Walter Kahler inspected it thoroughly and urged that the Laboratory acquire it. The Ex- ecutive Committee approved its purchase. The boat, large enough to accommo- date the entire Zoology class, will be used during the summer of 1948. 3. Repairs and Improvements In the course of the past two years we have made many repairs and improve- ments in several of our buildings. The much needed waterproofing, roof repairs, and outside painting of the Brick Building have been completed. The Apparatus Department is now situated in the well lighted and airy basement of the new wing of the Library. The rooms which were vacated will be now used as laboratories. The Chemical Department now has an air-conditioned room where special meas- urements can be made. The south wing of Old Main has been shored up so that the floors no longer vibrate as freely as in the past. Useful changes have been made in the Botany and Rockefeller Buildings. In the Stone Building a freight elevator is under construction. The top floor can be used for the storage of Supply Depart- ment materials. The Drew House has been put into good shape and painted ; a new apartment was made out of the reception room of the Apartment House. Here also extensive repairs to the balcony supports were imperative. These and other much needed changes and repairs have cost nearly $38,000. We must next make extensive alterations in the Supply Department Building, and put in good condition the wooden residences. Each year at least one of our buildings should be restored to first class shape. In this way they can be prevented from falling into serious disrepair. REPORT OF THE DIRECTOR 19 4. The Housing Problem Each year since the war ended we have experienced great difficulty in providing living quarters for those who want to work here. This is, to a considerable extent, due to the fact that, compared with former years, many more of the young investi- gators and students are married and require apartments, suites, or other special ac- commodations for their growing families. The Laboratory can provide for a rela- tively small proportion of these applicants ; in the village there is a reluctance to rent rooms to families with children. As a result a number of good investigators are forced to withdraw their requests for research space. When the housing situation became critical soon after the Brick Building was finished, Dr. Lillie urged the erection of a building to accommodate a large number of single investigators, and, in addition, ten bungalows for families. Today we can provide for the first group, but for the latter we need more apartments or small houses. To add to our present Apartment House or to erect a new one is too costly a venture at this time. But simple houses could be built on Devil's Lane property at a moderate cost. I believe that if the Laboratory built several, with a view to selling them to our members on easy terms, we could soon dispose of them, together with the lots on which they stand. Without doubt such a housing project would stimulate others to purchase Devil's Lane lots. Thus both the workers and the Laboratory would be benefited. \ 5. Lalor Fellowships Lalor Fellowships, established by the Lalor Foundation, were granted to the fol- lowing investigators : Senior Fellow : Prof. Jean Brachet, University of Brussels, Visiting Professor at the University of Pennsylvania Junior Fellows: Dr. I. M. Klotz — Northwestern University Dr. Arnold Lazarow — Western Reserve University Dr. Benjamin Libet — University of Chicago Dr. Claude Villee — Harvard LIniversity 6. Winter Research The Laboratory of Experimental Cell Research, under the direction of Dr. Rob- ert Chambers, has been engaged since the Fall of 1947 in the study of the mech- anism of cell division and growth, employing micromanipulation methods and tissue cultures. 7. Gifts The Laboratory gratefully acknowledges the following gifts : The Associates of the Marine Biological Laboratory, $775 Dr. G. H. A. Clowes— for a Lillie Memorial, $1,000 Mrs. W. Murray Crane, $700 Contributors to the Boat Fund, $10.035 20 MARINE BIOLOGICAL LABORATORY 8. Election of Trustees At the Meeting of the Corporation on August 12, 1947, the following trustees were elected : Class of 1951 W. C. Allee P. S. Galtsoff C. L. Claff L. V. Heilbrunn G. H. A. Clowes J. H. Northrop K. S. Cole A. H. Sturtevant 9. There are appended as parts of this report: 1. Memorials to Prof. William B. Scott, Prof. Robert A. Harper, Mr. George M. Gray, Professor Herbert S. Jennings, Professor Samuel O. Mast, and Professor L. L. Woodruff 2. The Staff 3. Investigators and Students 4. Tabular View of Attendance 5. Subscribing and Cooperating Institutions 6. Evening Lectures 7. Shorter Scientific Papers (Seminars) 8. Members of the Corporation Respectfully submitted, CHARLES PACKARD, Director 1. MEMORIALS irilliain Hen-yimm Scott, 1858-1947 By E. G. Conklin William Berryman Scott, the oldest member of the Board of Trustees of the Marine Biological Laboratory, was born on the birthday of Charles Darwin and Abraham Lincoln, February 12, but 49 years later than these great predecessors, viz. 1858. He died in his 90th year on March 29, 1947 in Princeton, New Jersey, his life-long home. He and his colleague at Princeton, William Libbey, first visited Woods Hole in 1883 on invitation Spencer F. Baird, head of the U. S. Fish Commission, to con- fer with him on plans for the development of the Fisheries Station at Woods Hole, and as a result, Libbey contributed in the name of Princeton University, $1,000, toward the purchase of the land on which the Station was established. In 1890, two years after the founding of the Marine Biological Laboratory, Prof. Scott became a member of the Corporation and in 1897 he was elected a Trustee and continued in that office and as Trustee Emeritus until his death. During these forty years as Trustee, he made it a point of honor to be present whenever possible at the annual meetings, and in 1897 and 1898, he gave lectures at the Laboratory on the methods and results of his paleontological researches. REPORT OF THE DIRECTOR 21 Although he was for fifty years a member of the staff of the Department of Ge- ology at Princeton, and for forty-six years head of that department, he was pri- marily a zoologist. At the close of his senior year in college, he and two other classmates, Osborn and Speir, organized a scientific expedition to Colorado and Wyoming. The collection of vertebrate fossils made that summer of 1877 was described in their first scientific publication and was instrumental in shaping the future careers of Scott and Osborn. After a year of graduate study at Princeton and a second expedition to the West in the summer of 1878, Scott spent two years in graduate study in Europe; first with Huxley in London, then with Balfour in Cambridge, and finally with Gegenbaur in Heidelberg. His work with these mas- ters was in anatomy and embryology. Under the stimulus of Balfour, he, with Os- born, completed and published a study on 'The Early Development of the Common Newt," the first such study on the embryology of a urodele. In Gegenbaur's lab- oratory he was assigned for study the valuable material which had been collected by Dr. Calberla, deceased, on the embryology of a cyclostome fish and the results of this study were published in Gegenbaur's Morphologisches Jahrbuch in 1880 as his thesis for the Ph.D. degree, with the title, "Beitrage zur Entwicklungsgeschichte der Petromyzonten." Thus at the early age of twenty-two years, he had finished his work for the doc- tor's degree, published three important papers, been a leader in two exploring ex- peditions, and had met on terms of intimate friendship and cooperation, some of the foremost scientists and scholars of Europe and America. On his return from Europe, he was appointed assistant in geology at Princeton and three years later was made full professor in that department, which position he continued to hold until he had completed fifty years of teaching at Princeton Uni- versity. During that time, he made eight additional exploring expeditions to the West and published more than 150 paleontological reports. Perhaps his most mon- umental work was the "Reports of the Princeton Expedition to Patagonia" which was published in nine magnificent volumes, of which he was editor and co-author. His later work, undertaken after he was seventy-six years old, in association with his former student and colleague, Dr. Jepson, was a great monograph of 1,000 pages and 100 plates on the "Mammalian Fauna of the White River Oligocene" ; while his latest work was a similar monograph on the "Mammalian Fauna of the Uinta Formation," upon the final pages of which he was at work until two days before his death. In addition to these research publications, he was the author of a number of im- portant books of a more general nature, among them, an "Introduction to Geology," which ran through three editions. "A History of Land Mammals of the Western Hemisphere," two editions ; "Physiography, the Science of the Abode of Man" ; "The Theory of Evolution" ; and finally, a most interesting and important autobiog- raphy, "Some Memories of a Paleontologist." He was abundantly honored both in Europe and America by universities and learned societies. The Universities of Pennsylvania, Harvard, Princeton and Ox- ford, gave him honorary degrees. He received ten medals and awards from learned societies here and abroad. He was elected a member of the American Philosophical Society when he was twenty-eight years old and at the time of his death, had been a member for more than sixty years. For seven years he was president of that soci- 22 MARINE BIOLOGICAL LABORATORY ety of which his great, great, great-grandfather, Benjamin Franklin, had been founder and first president. He was also president of the Geological Society of America (1925) and of the Paleontological Society (1911), and a member of the National Academy of Sciences, the American and British Associations for Advance- ment of Science, the American Academy of Arts and Sciences, the Academy of Nat- ural Sciences of Philadelphia, the New York Academy of Sciences, the Washington Academy of Sciences, the Geological, Zoological and Linnean Societies of London. Professor Scott was a brilliant lecturer and he often enlivened scientific meetings with his humorous stories and his phenomenal memories of great men and events. Nevertheless, he was a scholar and thinker rather than a popular lecturer or writer. Fortunately, he has recorded in his autobiography many of his memories of some of the greatest men of his generation. The Corporation and Trustees of the Marine Biological Laboratory record their sorrow in the loss of one of their oldest and most distinguished members and trans- mit to the members of his family this expression of their esteem and sympathy. Dr. Robert A. Harper By Edmund W. Sinnott In Dr. Harper's death, plant science has lost a man who for years was one of its greatest leaders. Dr. Harper was born on January 21, 1862 at Le Claire, Iowa. He received his B.A. at Oberlin in 1886 and then for two years taught Latin and Greek at Gates College. From 1889 to 1891 he was instructor in science at Lake Forest Academy. He took an M.A. at Oberlin 1891 and then served for a time as professor of bot- any and geology at Lake Forest College. Dr. Harper's chief interests centered more and more in botany and he soon determined to make this his career. As so many young botanists did in the nineties, he went to Germany for graduate work. At Bonn he came under the influence of Strasburger and other notable teachers, taking his doctorate in 1896. Here began his life-long interest in cytology. Soon after his return to America, he went to the University of Wisconsin where he became professor of botany and head of the de- partment. In 1911 he was called to Columbia University as Torrey Professor of Botany, serving until his retirement in 1930. Here he reorganized the department and greatly widened its scope and activities. He was keenly interested in the New York Botanical Garden, the Boyce Thompson Institute for Plant Research and the Torrey Botanical Club. Dr. Harper was a student of plant cells since his days at Bonn, but he was much more than a mere cytologist> He liked to describe himself as a cellular physiologist and studied many aspects of the activities of all sorts of plant cells. He was for a long time especially concerned with a study of reproduction in the fungi, and his work on nuclear behavior in the ascomycetes is classic. Studies of development and morphogenesis particularly appealed to him and he was a stout supporter of the view that the phenomena of development are best approached through a knowledge of the behavior of cells. He was much interested in some of the simpler algae, no- tably Pediastrum and Hydrodictyon, in which he investigated the problems of cel- lular activities. REPORT OF THE DIRECTOR 23 Unlike so many biologists of today, Dr. Harper had a wide knowledge not only of his own science, but of others and of wider fields of learning. He was a good field botanist, a successful farmer, a skilled experimenter, and a man of wide read- ing and erudition. He encouraged his students to train themselves broadly and not to be carried away by the fashions of the time. He was a stimulating teacher, and in a discussion, delighted to take the less popular side and to defend it vigorously. He was highly critical and a foe of slipshod works. His own papers were beauti- fully done and his drawings, in particular, were remarkably fine. Dr. Harper had close contacts with Woods Hole for many years. He was a student here in 1891, along with Bradley Moore Davis, C. P. Sigerfoos and Kath- erine Foote. In 1893, he worked as an investigator at the Laboratory. Fre- quently during ensuing years he came to Woods Hole for part of the summer. He was elected to the Board of Trustees in 1911 and in 1932 became Trustee Emeritus. In recent time he has rarely come to the Laboratory but devoted all his time to his farm at Bedford, Virginia, where he spent his last years happily close to the soil and with plants he loved. Dr. Harper's warm and friendly personality endeared him to his many students and to a host of friends all over the world. He was a great teacher, a friendly op- ponent in debate, and a constant stimulus and inspiration to all who knew him. He will be sorely missed. George M. Gray By W. C. Curtis George Milton Gray was born at Bristol, Rhode Island, November 2, 1856. As a boy he was interested in natural history, particularly in birds and insects. When a young man he worked as a taxidermist, gave lectures on birds at a boys' camp and made collections in the vogue of his day. He was thus a naturalist from his youth up. Later, he was discovered by Dr. H. C. Bumpus and engaged as a tech- nician in the Department of Zoology at Brown University. Mr. Gray first came to the Marine Biological Laboratory in the summer of 1891 as a laboratory assistant. He served in this capacity for six summers (1891-96), as collector for two sum- mers (1897-98), and became Curator of the Supply Department in 1899. Although this department was established in 1891, its effective operation began with Mr. Gray. At first the orders were filled only in the summer months. In the years 1896-97 and 1897-98, a stock was shipped to Williams College, and sales to- taling about $125 per year were made during each of these winter periods. The trustees were elated when it was announced that the sales might exceed $500 for the summer of 1897 and again in 1898. In September 1899, when Mr. Gray be- came Curator and a year-round appointee of the Laboratory, the department began its continuous existence at Woods Hole. It was then located in the basement space between the two wings of the Old Main Building. In winter the supplies were moved to the invertebrate laboratory where a stove was set up to keep Mr. Gray and the specimens from freezing. The Stone Building, to which the department was transferred several years later, was luxury indeed when he was first established there. As a result of his untiring efforts during these early years, the department flourished. In the year 1912 the sales totaled $13,966.35. In 1925 the total was 24 MARINE BIOLOGICAL LABORATORY $57,771.67. The creation of this necessary adjunct to the work of the Laboratory and important source of revenue was largely the work of George M. Gray. Mr. Gray not only established the reputation of the department for reliability and for quality of material, he also established a record of service to classes and investi- gators. Beginning as a one-man organization, it employed an increasing number of assistants, and the role of those who "worked in the Supply Department" for a summer or two and later attained distinction in some field of biological science is an impressive one. Another notable contribution by Mr. Gray was his personal influence upon these youngsters. In later years he was able to devote more time to aspects of the work in which he was particularly interested. After he withdrew from the Supply Department (1931), he became Curator of the Museum, which was named in his honor and was his special pride. He was fairly active even as Curator Emeritus since 1935. He died December 1, 1946 and is buried in the Woods Hole Cemetery. Writing in a laboratory room, the windows of which look out upon the Hole and the islands where I worked as his assistant fifty years ago this summer, I cannot forbear my personal tribute. I have always thought him one of the most honest and kindly men I ever know. He was my close friend always from that summer long ago. About that time I was taken by a remark, made sadly by an elderly clergyman wise in the ways of men, that he had known some individuals to whom he thought the term "Christians" might be applicable although he had never seen any reason for applying it to church members as a group. I thought then and I have thought ever since that George Milton Gray was one of the few among my acquaintances to whom I would apply that term. We of the Marine Biological Laboratory never had a more devoted service nor greater loyalty than he gave us. He well exemplified the dictum of his faith : "He that would be great among you let him be the servant of you all." Dr. Herbert Spencer Jennings By O. C. Glaser With the death of Herbert Spencer Jennings, at Santa Monica, California, on April 14, 1947, the Marine Biological Laboratory lost one of its most distinguished members — a trustee for 33 years and a trustee emeritus since 1938. The son of a physician, Jennings was born in Tonica, Illinois, on April 8, 1868, and educated as he said "in most states of the Union." This migratory life con- tinued with only short interludes until he was 38. Like his namesake, he was pre- cocious and for years hard pressed by economic difficulties and the struggle for scientific opportunity. At 20 he prepared for college, so to speak, as an Assistant Professor of Botany and Horticulture at the Agricultural and Mechanical College of Texas. As an undergraduate at Ann Arbor, he joined the Biological Survey of the Great Lakes conducted by the Michigan State Board of Fisheries and laid the foundations for his first monograph on Rotifers, published in 1894 — a year after receiving his B.S. from the University of Michigan. At Harvard he received the M.S. in 1895 and his Ph.D. in 1896. His thesis on cell-lineage of the rotifer Asplanchna herrickii, related the orientation of the spindles to the general problems of developmental mechanics whose solution he projected into the molecular realm. Travelling fellowships enabled Jennings to spend the following year with Max REPORT OF THE DIRECTOR Verworn in Biedermann's Laboratory at Jena, Mere began his preoccupation with the reactions of unicelluar organisms. Returning in 1897. lie became Professor of Botany and Bacteriology at the Ag- ricultural College of Montana; accepted next instructorships, first at Dartmouth, then at Michigan where he became Assistant Professor of Zoology in 1900 and remained until called in 1903 to an Assistant Professor at the University of Pennsylvania. These frequent translocations must have had serious disadvantages but his tre- mendous drive and unbounded enthusiasm found or created time for both teaching and research. By 1906 when he left Pennsylvania, he had to his credit at least 40 publications. The joint text with Reighard on the Anatomy of the Cat based on his own dissections and illustrated by Mrs. Jennings, belongs to the second Michigan period. Here too, as Director in 1901 of the Biological Survey of the Great Lakes under the U. S. B. F., he gathered material for three additional mono- graphs on the Systematics of Rotifers and in connection with his teaching gave the first expression to a lively and recurrent interest in simulacra. Outstanding among the special contributions were his Psychology of the Protozoa in which Paramecium is shown to have "hardly taken the first step in the evolution of mind," and his analysis of the Biological Significance of Asymmetry. There were further studies on stimulation in Protozoa, followed by more from Pennsylvania. There were also publications on the behavior of the earthworm, the sea-anemone and the star- fish— the latter a veritable storehouse of information on the activities of this animal- appearing in 1907. Many of his results during the Philadelphia period were either summarized in the great monograph on the behavior of lower organisms published by the Carnegie Institution or in the famous Behavior of the Lower Organisms appearing in the Columbia Biological Series in 1906; reprinted in 1915; and trans- lated by Ernst Mangold in 1914 into German. The chief product of all his meticu- lous observations and simple experiments was a general outlook with variability, modifiability, unity and adaptiveness of behavior as the central themes. With his transfer in 1906 to an Associate Professorship of Experimental Zool- ogy at the Johns Hopkins University and his subsequent elevation to the Henry Walters Chair of Zoology and Directorship of the laboratory — both in succession to William Keith Brooks — Dr. Jennings entered upon his only long tour of duty at any one institution. He also changed his field of investigation. Although the- oretical and controversial writings on behavior continued to appear, 1908 marks the beginning of a long series of researches on the life-cycles, heredity, variation, and evolution of Protozoa, notably Paramecium, Arcella, and DifHugia. In 1928, he reverted to his rotifers and, with Ruth Stocking Lynch, published two papers on Age, Mortality, Fertility, and Individual Diversities in Proales sordida. During his genetic phase, Jennings substituted for full verbal description, long tables of measurements and enumerations. He became a biometrician. So highly did he perfect his mathematical techniques and insights that he was called upon to act as a statistician for the Food Administration during the first World War. Among his most important genetic results we must cite the analysis of conjugation in Paramecium whose significance he found in the diversities so created rather than in any rejuvenating effects; his pure lines in the same organism; and finally, the discovery of mating types — at long last the key to Protozoan Genetics. Through- 26 MARINE BIOLOGICAL LABORATORY out this period, Jennings strengthened his position as the apostle of diversity. He also developed further his inborn sensitivities to the more general intellectual and social climate. Contributions on special and general methodology such as genetic method and Radical Experimental Analysis appeared in a stream of critical evalu- ations of Vitalism, Mechanism, Determination and Freedom. In the social area, he wrote on Immigration, Defectives, "Undesirable Aliens," Racial Progress, the Family and Marriage. Apparently unaware of his powers, he invaded more dis- tant territory. In his essay on the advantages of Growing Old, the euphorious state created by the presentation of a portrait of one's self and the pessimistic out- look of a young man trying to lead the life of a productive scholar on a $900 instruc- torship, are contrasted with humor, pathos and artistry sufficient to suggest pure literature. All told, the output of 32 years at Johns Hopkins amounts to about 120 papers, long or short, and seven books. Modest, shy, nervous and frail, Jennings nevertheless accepted many outside lecture engagements. He spoke with great animation and charm to audiences in- variably responsive to his sincere excitement and well ordered presentations. He gave more than one of our evening lectures. Few who heard his mathematical analysis of the data on genie linear array will forget his enraptured delight with the Naperian Case or the suspense he created and maintained until the final unveiling of Morgan's own theory at the very end. He gave the Terry lectures at Yale ; the Vanuxem, at Princeton ; the Leidy, at Pennsylvania, where he received the first award of the Leidy medal. In 1931-32 he was Visiting Professor at Keio University, Tokio, and in '35-'36, Eastman Visiting Professor at Oxford. After retirement, he gave the Patten lec- tures at Indiana ; became Visiting Professor for one year and remained as Research Associate at the University of California in Los Angeles. Dr. Jennings was active on the Editorial Boards of four Journals ; was a mem- ber of the National Academy, the American Philosophical Society and other cov- eted American Academies. He was President of the American Society of Zoolo- gists (1909). His presidential address (1911) to the American Naturalists on Heredity and Personality — one of the most memorable of his gems — exposed, with a gaiety born of many insights, the genetic and environmental odds against the birth of any particular individual. Foreign recognitions included Honorary Fel- lowship in the Royal Microscopical Society, Corresponding Memberships in the Russian Academy of Sciences and in the Societe Biologique de France or de Paris —he was not certain which. His honorary degrees proved quite unmanageable. The British Who's Who for 1944 records eight such degrees ; the American coun- terpart and the American Men of Science both also in 1944 each list six. Agree- ment on totals, however, obscures the true diversities among four D.Sc.'s, three LL.D.'s and an Oxford A.M. ; one D.Sc. and five LL.D.'s ; and four D.Sc.'s with two LL.D.'s. One highly reputable LL.D. he avoided. As long as there were Paramecia to measure, count and keep in order, why bother about honorary degrees ? Whoever recalls him will continue to regret that his work at the Marine Bio- logical Laboratory ended in 1933 and that he was a regular attendant only during the decade of the twenties. We should have liked to share him with many others. His inspiring example and achievement can be fully appreciated only against his background of almost continuous ill health. Yet he was always friendly and cheer- REPORT OF THE DIRECTOR 27 ful ; always excited about something ; and ready to discuss a case, not for the sake of argument, but because of his passions for both sides of every question ; for clarity of mind and for fairness. Impelled as he was by ceaseless cerebration, he neverthe- less lists two related forms of recreation— travel and motoring. As a travelling companion, one can hardly imagine another more delightful; however, his friends who either rode with him or merely observed him spiraling briskly down the main street of Woods Hole were far too apprehensive to benefit from the recreative pow- ers of his driving. Samuel Ottmar Mast By B. H. Willier On October 5. 1871, Samuel Ottmar Mast was born on a farm near Ann Arbor, Michigan. His early schooling, academic training and teaching experience were in his native state. After obtaining a "full diploma" in 1897 from the Michigan State Normal College, he went to the University of Michigan where he received the B.S. degree in 1899. From 1899 to 1908, he was Professor of Botany and Biology at Hope College. Holland, Michigan. In 1906, he received the Ph.D. de- gree in zoology from Harvard University. He then came to the Eastern seaboard where he spent the remainder of his life. For a period of three years (1908-1911) he was a member of the biology staff at Goucher College. In the autumn of 1911, he joined the zoology staff at Johns Hopkins as associate professor which soon cul- minated in a Professorship of Zoology and later upon the retirement of the late Professor H. S. Jennings in the chairmanship of the Department of Zoology (1938-41). Since 1942, he has been professor emeritus of zoology. According to the records, he first attended the Marine Biological Laboratory during the summer session of 1907. In 1908, he was elected a member of the Corporation and later (1936-1942) served as a member of the Board of Trustees and of its executive committee for two years (1938-1940). Since 1942 he has been Trustee Emeritus. He and his family have been regular summer residents of Woods Hole for a period of over twenty years. Over these years, he has been in regular attendance as an investigator and his interest in the laboratory has been con- stant and genuine. In 1908, he married Grace Rebecca Tennent, the sister of the late David Hilt Tennent of Bryn Mawr and of this laboratory. She and three daughters and many grandchildren survive him. He died quite suddenly on Monday evening, February 3, 1947, at the age of 75 at his home in Roland Park in the city of Baltimore. The life long work of Professor Mast was directed toward an understanding of the physiology of the "lower" organisms, especially the Protozoa. His major in- terest was in the mechanisms of behavior of these forms and more specifically in their reactions to light. This is best exemplified in his most significant book, "Light and the Behavior of Organisms" (1911) and in his numerous published papers on the motor responses, factors in the process of orientation, etc. of a variety of unicel- lular animals and other invertebrates. In 1926, as a result of his interest in the behavior of Amoeba proteus he formulated a theory to account for amoeboid move- ment, which has received wide recognition. His wide interest in the physiology of the Protozoa led him later to make a study of the nutrition of the colorless flagellate, Chilomonas. Together with Dr. Donald M. Pace and other students, he showed 28 MARINE BIOLOGICAL LABORATORY that this organism in the total absence of light can synthetize carbohydrates, fats, proteins and protoplasm from a few simple inorganic salts, resembling in this re- spect, the green plants and certain bacteria. During the last few years of his life, Mast turned his attention to an investigation of the processes of feeding and diges- tion in the Protozoa, which culminated in his most significant paper on this subject entitled, "The Food-Vacuole of Paramecium." This work is a fitting and lasting example of the exactness and care which characterized all of his researches and pub- lications. His every publication was marked by the meticulous care with which each word and phrase were weighed to make sure they meant exactly what he had in mind, no more and no less. Professor Mast commanded to a marked degree the loyalty and admiration of graduate students. He had many pupils and has trained a whole generation of zo- ologists who have much to thank him for. His loss is deeply felt by many friends, former colleagues and students, and no less by the community of biologists at Woods Hole. Dr. Lorrandc Loss Woodruff By R. G. Harrison Lorande Loss Woodruff, Colgate Professor of Protozoology at Yale University and Director of the Osborn Zoological Laboratory, died at his home in New Haven after a long illness on June 23, 1947 in his 68th year. With his passing, the Cor- poration of the Marine Biological Laboratory loses a member of more than forty years standing and the Board of Trustees, one who had served faithfully for 24 years. Dr. Woodruff was born in New York on July 14, 1879, and received his educa- tion in his native city, graduating from Columbia University with the A.B. degree in 1901 and the Ph.D. in 1905. Before completing his graduate work, he was ap- pointed Assistant and later Instructor in Biology at Williams College, where he remained until he was called to Yale in 1907. There he served successively as In- structor, Assistant Professor and Professor, until his death. He became Chairman of the department and Director of the Osborn Zoological Laboratory in 1938, but took leave of absence in November 1946 on account of ill health. His connection with the Marine Biological Laboratory began in 1905 when he attended the summer session as Investigator and Instructor in the Invertebrate Course and was elected to membership in the Corporation. Four years later, he joined the Staff of the course in Embryology of which he remained a member un- til 1914. During the absence of Dr. Calkins in the summer of 1927, he was in charge of the course in Protozoology. Elected to the Board of Trustees in 1923, he served with them until his death and during the years 1930-32, he was a member of the Executive Committee. Coming to Yale at a time when a radical reorganization of the instruction in bi- ology was to be undertaken, Woodruff took part from the first in teaching general biology and in 1910 assumed full charge of the general course in Yale College. This he built up into one of the soundest and at the same time, most popular courses in the University. Through the years, thousands of students listened to his mas- terly lectures, later incorporated in his textbook, "The Foundations of Biology," which has been very widely used and has run through six editions. REPORT OF THE DIRECTOR Woodruff's research was exclusively in the field of unicellular organisms. Be- ginning with his doctoral dissertation, which was done under the direction of the late Professor Calkins and published in 1905, he made many contributions to our knowledge of the life history of ciliates, their division rate, nuclear reorganization, the effect of environmental factors on their life cycle. He is perhaps best known for the famous pedigreed race of Paramecium aurelia, which was carried for eight years with daily isolation of the products of division, thus precluding conjugation and showing that these organisms can reproduce asexually indefinitely without dy- ing out. In the first eight years over 5.000 generations were obtained and after- ward the culture was carried in a less rigorous manner though sufficiently carefully to exclude conjugation except possibly between closely related individuals. The culture has now reached more than 24,000 generations without loss of vigor. In the course of this work, Woodruff and Erdmann discovered that, corresponding to the rhythms in division rate, the nuclei of the paramecia undergo a reorganization process which they termed "endomixis" and which they described as a form of nu- clear reorganization with syncaryon formation. This stirred up much discussion and more recently the process has been described by others as autogamy, involving fusion between micronuclei from the same cell. His research naturally attracted graduate students, and throughout the years many have written their dissertations under his direction and carried his methods to new centers, just as his assistants and students in the course in general biology, many of whom have become teachers, have spread his ideas of the teaching of bi- ology throughout the land. Woodruff was intensely interested in the history of science. Early in his career at Yale, he organized a course in the history of biology which he continued through- out his life. He was a collector of scientific books of historical significance. He published a number of essays and addresses in this field and organized two series of lectures on the history of science under the auspices of Gamma Alpha Fraternity, which were later published in book form under his editorship. A paper on "Baker on the Microscope and the Polypa" led to a friendly encounter with a descendant of Trembly, the famous author of the treatise on Hydra published in 1744. Woodruff was Chairman of the Division of Biology and Agriculture of the Na- tional Research Council, 1928-29. He was a member of many scientific societies, including the National Academy of Sciences, the American Society of Zoologists, of which he was Secretary-Treasurer, 1907-09 and President in 1942, the American Physiological Society, the American Society of Naturalists (Vice-President, 1923), the American Association for the Advancement of Science, (Fellow) and others. He was a member of Phi Beta Kappa, Gamma Alpha and Sigma Xi, having been President of the Yale Chapter of the last in 1915. He lectured on Protozoology at four summer sessions of the Mountain Laboratory of the University of Virginia. For two terms of three years each, he was an Associate Editor of the Journal of Morphology. In 1935, lie received the Townsend Harris medal from the College of the City of New York, where he had been as a student before entering Columbia. For one who was closely associated with Woodruff for nearly forty years, it is difficult to realize that this intimate relation has been forever broken. The associa- tion was one of mutual trust throughout and without serious disagreement. He was always on the side of high standards, which he applied to himself as well as to 30 MARINE BIOLOGICAL LABORATORY others. Indeed, this was one of his outstanding qualities, as was his intense loyalty to the institutions he served. He was quiet and reserved, but with a kindliness that meant much to those about him. With all of his reserve, he could be deeply moved, and he never recovered from the shock of Mrs. Woodruff's death, which came with such cruel suddenness in March 1946. The members of the Corporation of the Marine Biological Laboratory desire to record their sorrow over the loss of one of their body, a friend and a fellow servant whom they will miss acutely and whose memory they will always cherish. 2. THE STAFF, 1947 CHARLES PACKARD, Director, Marine Biological Laboratory, Woods Hole, Massachusetts. SENIOR STAFF OF INVESTIGATION E. G. CONKLIN, Professor of Zoology, Emeritus, Princeton University. *FRANK R. LILLIE, Professor of Embryology, Emeritus, The University of Chicago. RALPH S. LILLIE, Professor of General Physiology, Emeritus, The University of Chicago. A. P. MATHEWS, Professor of Biochemistry, Emeritus, University of Cincinnati. G. H. PARKER, Professor of Zoology, Emeritus, Harvard University. ZOOLOGY I. CONSULTANTS T. H. BISSONNETTE, Professor of Biology, Trinity College. LIBBIE H. HYMAN, American Museum of Natural History. A. C. REDFIELD, Woods Hole Oceanographic Institution. II. INSTRUCTORS F. A. BROWN, Associate Professor of Zoology, Northwestern University, in charge of course. W. D. BURBANCK, Professor of Biology, Drury College. C. G. GOODCHILD, Professor of Biology, S.W. Missouri State College. JOHN H. LOCHHEAD, Professor of Zoology, University of Vermont. MADELENE E. PIERCE, Associate Professor of Zoology, Vassar College. W. M. REID, Associate Professor of Biology, Monmouth College. MARY D. ROGICK, Professor of Biology, College of New Rochelle. T. H. WATERMAN, Instructor in Biology, Yale University. III. LABORATORY ASSISTANTS VIRGINIA L. FOGERSON, Vassar College. AMOS L. HOPKINS, Harvard University. MARIE WILSON, Western Maryland College. EMBRYOLOGY I. CONSULTANTS P. B. ARMSTRONG, Professor of Anatomy, College of Medicine, Syracuse University H. B. GOODRICH, Professor of Biology, Wesleyan University. * Deceased. REPORT OF THE DIRECTOR 31 II. INSTRUCTORS DONALD P. COSTELLO, Professor of Zoology, University of North Carolina, in charge of course. HOWARD L. HAMILTON, Assistant Professor of Zoology, Iowa State College. JOHN A. MOORE, Assistant Professor of Zoology, Barnard College. JEAN BRACKET. Professor of Experimental Morphology, University Brussels. III. RESEARCH ASSISTANT MARJORIE HOPKINS Fox, University of California. IV. LABORATORY ASSISTANTS CATHERINE HENLEY, The Johns Hopkins University. ALICE H. FERGUSON. PHYSIOLOGY I. CONSULTANTS WILLIAM R. AMBERSON, Professor of Physiology, University of Maryland, School of Medicine. HAROLD C. BRADLEY, Professor of Physiological Chemistry, University of Wisconsin. WALTER E. CARREY, Professor of Physiology, Vanderbilt University Medical School. MERKEL H. JACOBS, Professor of Physiology, University of Pennsylvania. II. INSTRUCTORS ARTHUR K. PARPART, Associate Professor of Biology, Princeton Universtiy, in charge of course. E. S. GUZMAN BARRON, Associate Professor of Biochemistry, The University of Chicago. RUDOLPH T. KEMPTON, Professor of Zoology, Yasser College (absent in 1946). M. J. KOPAC, Assistant Professor, New York University. GEORGE WALD, Associate Professor of Biology, Harvard University. DOROTHY WRINCH, Lecturer, Smith College. BOTANY I. CONSULTANTS S. C. BROOKS, Professor of Zoology, University of California. P. R. BURKHOLDER, Eaton Professor of Botany, Yale University. W. R. TAYLOR. University of Michigan. II. INSTRUCTORS MAXWELL S. DOTY, Instructor of Botany, Northwestern University. In Charge of Course. HANNAH CROASDALE, Dartmouth College. ISABELLA ABBOTT, University of California. III. RESEARCH ASSISTANTS R. D. NORTHCRAFT, Massachusetts State College. R. D. WOOD, Northwestern University. 32 MARINE BIOLOGICAL LABORATORY IV. LABORATORY ASSISTANT EDWIN T. MOUL, University of Pennsylvania. 9 EXPERIMENTAL RADIOLOGY G. FAILLA, College of Physicians and Surgeons, Columbia University. L. ROBINSON HYDE, Phillips Exeter Academy, Exeter, N. H. LIBRARY PRISCILLA B. MONTGOMERY (MRS. THOMAS H. MONTGOMERY, JR.), Librarian DEBORAH LAWRENCE, Assistant Librarian MARGARET P. MC!NNIS MARY A. ROHAN JEAN GOODFELLOW APPARATUS DEPARTMENT E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager J. D. GRAHAM DOROTHY LEFEVRE CHEMICAL DEPARTMENT E. P. LITTLE, Phillips Exeter Academy, Exeter, N. H., Manager SUPPLY DEPARTMENT JAMES MC!NNIS, Manager JOHN S. RANKIN, Naturalist RUTH CROWELL MARCIA MCLAUGHLIN M. B. GRAY W. E. KAHLER R. E. TONKS A. M. HILTON W. S. LANDERS F. N. WHITMAN G. LEHY GENERAL OFFICE F. M. MACNAUGHT, Business Manager HOMER P. SMITH, Assistant Business Manager POLLY L. CROWELL MRS. LILA S. MYERS GENERAL MAINTENANCE T. E. LARKIN, Superintendent ROBERT ADAMS G. T. NICKELSON, JR. R. W. KAHLER A. J. PIERCE GEO. KAHLER T. E. T A WELL THE GEORGE M. GRAY MUSEUM 3. INVESTIGATORS AND STUDENTS Independent Investigators, 1947 ABBOTT, ISABELLA A., Graduate Student, University of California. ABELSON, PHILIP H., Chairman Bio Physics Section, Carnegie Inst. of Washington. ALLEN, M. JEAN, Instructor in Biology, Mather College, Western Reserve University. REPORT OF THE DIRECTOR ANDERSON, RUBERT S., Assistant Professor of Physiology, University of Maryland Medical School. ARMSTRONG, PHILIP B., Professor of Anatomy, College of Medicine, Syracuse University. ATLAS, M-EYER, Assistant Professor of Biology, Yeshiva University. BAEZ, SILVIO, Research Fellow, Cornell Medical College. BALL, ERIC G., Professor of Biological Chemistry, Harvard Medical School. BARROX, E. S. GUZMAN, Associate Professor of Biochemistry, The University of Chicago. BARTLETT, JAMES H., Professor of Theoretical Physics, University of Illinois. BARTON, ELEANOR, Assistant Professor of Zoology, N. J. College for Women, Rutgers Uni- versity. BERGER, CHARLES A., Director, Biological Laboratory, Fordham University. BLISS. ALFRED F., Assistant Professor of Physiology, Tufts College Medical School. BLUM, HAROLD F., Guggenheim Fellow, National Cancer Institute. BOELL, E. J., Professor of Biology, Yale University. BONXER, JOHN T., Junior Fellow, Harvard University. BRACKET, JEAN L. A., Visiting Professor, University of Pennsylvania. BRIDGMAX, JOSEPHINE, Associate Professor of Biology, Limestone College. BROXK, DETLEV W., Professor of Biophysics and Director, Johnson Foundation, University of Pennsylvania. BROWX, FRAXK A., JR., Professor of Zoology, Northwestern University. BROWXELL, KATHERIXE A., Instructor, Ohio State University. BRUST, MANFRED, Graduate Student, University of Chicago. BUDIXGTON. ROBERT A., Professor of Zoology Emeritus, Oberlin College. Buxo, WASHINGTON, Professor and Director Department of Histology, School of Medicine, Montevideo. BURBANCK, W. D., Professor of Biology, Drury College. BURGER, AXDRE, Visitor at Harvard University. CHAMBERS, FRANCIS W., JR., Lt. Com. U. S. Navy through Columbia University. CHAMBERS, ROBERT, Research Professor of Biology, New York University. CHASE, AURIN M., Assistant Professor of Biology, Princeton University. CHENEY, RALPH H., Associate Professor of Biology, Brooklyn College. CHRYSTALL, FRIEDA L., Teacher of Biology, New York City Public High School. CLAFF, C. LLOYD, Research Fellow in Surgery, Harvard Medical School. CLARK, A. M., Instructor in Biology, University of Delaware. CLARK, ELIOT R., Professor of Anatomy, University of Pennsylvania School of Medicine. CLARK, L. B., Professor of Biology and Chairman of Department, Union College. CLEMENT, A. C., Professor of Biology, College of Charleston. CLOWES, G. H. A., Research Director Emeritus, Eli Lilly & Company. COLE, KENNETH S., Professor of Biophysics, University of Chicago. COLWIN, ARTHUR L., Assistant Professor of Biology, Queens College. CONKLIN, E. G., Professor Emeritus of Biology, Princeton University. COOPER, KENNETH W., Associate Professor of Biology, Princeton University. COPLEY, A. L., Research Associate, N. Y. U. Washington Square College. COPELAND, D. E., Assistant Professor of Zoology, Broxvn University. CORNMAN, IVOR, Research Fellow, Sloan-Kettering Institute. COSTELLO, DONALD P., Professor of Zoology, University of North Carolina. CROADALE, HANNAH T., Associate in Zoology, Dartmouth College. CROWELL, SEARS, Associate Professor of Zoology, Miami University. CURTIS, WINSTON C., Professor Emeritus of Zoology, University of Missouri. DAN, JEAN C., Independent Investigator, Misaki Marine Biological Station, Japan. DOTY, MAXWELL S., Assistant Professor of Botany, Northwestern University. DUMM, MARY E., Instructor in Chemistry, New York University Medical College. DURYEE, WILLIAM R., Guest Investigator, Carnegie Institute of Washington. EDGERLEY, ROBERT H., Assistant Professor of Zoology, University of Alabama. EICHEL, BERTRAM, Teaching Research Fellow, N. Y. U. College of Dentistry. EVANS, TITUS C., Assistant Professor of Radiobiology, College of Physicians and Surgeons. FAILLA, G., Professor of Radiology, College of Physicians and Surgeons. FIGGE, FRANK H. J., Associate Professor of Anatomy, University of Maryland School of Medi- cine. 34 MARINE BIOLOGICAL LABORATORY FISHER, HARVEY F., Western Reserve University, FROEHLICH, ALFRED, Associate, May Institute for Medical Research. GABRIEL, MORDECAI L., Instructor of Biology, Brooklyn College. GILMAN, LAUREN C., Assistant Professor of Zoology, University of South Dakota. GLASER, OTTO, Professor of Biology, Amherst College. GOODCHILD, C. G., Professor of Biology, Missouri State College. GOODRICH, H. B., Professor of Biology, Wesleyan University. GORBMAN, AUBREY, Assistant Professor of Zoology, Columbia University. GOULD, HARLEY N., Professor of Biology, Newcomb College. GROSCH, DANIEL S., Assistant Professor, University of North Carolina. GRUNDFEST, HARRY, Research Associate in Neurology, Columbia University Medical School. HALL, THOMAS S., Associate Professor of Zoology, Washington University. HAMILTON, HOWARD L., Assistant Professor of Zoology, Iowa State College. HARTMAN, FRANK A., Professor of Physiology, Ohio State University. HARVEY, ETHEL BROWNE, Research Biology Department, Princeton University. HARVEY, E. NEWTON, Professor of Physiology, Princeton University. HEIDENTHAL, GERTRUDE, Assistant Professor of Biology, Russell Sage College. HEILBRUNN, L. V., Professor of Zoology, University of Pennsylvania. HIBBARD, HOPE, Professor of Zoology, Oberlin College. HICKSON, ANNA KELTCH, Research Chemist, Eli Lilly & Company. HINTON, TAYLOR, Instructor, Amherst College. HOPKINS, HOYT S., Associate Professor of Physiology, N. Y. U. College of Dentistry. HSIAO, SIDNEY C., Visitor and Seessel Fellow, Yale University. HUTCHINGS, Lois M., Instructor, N. Y. U. Washington Square College. IFFT, JOHN D., Assistant Professor of Biology, Simmons College. JABLONS, BENJAMIN, Associate Clinical Professor, New York Medical College. JACOBS, M. H., Professor of General Physiology, University of Pennsylvania. JENKINS, GEORGE B., Emeritus Professor of Anatomy, George Washington University. JEROME, SISTER FRANCIS, Professor of Biology, Ohio State University. KARUSH, FRED, Research Associate, N. Y. .U. Medical School. KEMP, MARGARET, Associate Professor of Botany, Smith College. KEMPTON, RUDOLPH T., Professor of Zoology, Vassar College. KISCH, BRUNO, Professor at Yeshiva University. KITCHEN, I. C., Research Fellow, Princeton University. KLOTZ, IRVING M., Assistant Professor of Chemistry, Northwestern University. KOPAC, M. J., Associate Professor of Biology, N. Y. U. Washington Square College. KRAHL, M. E., Assistant Professor of Pharmacology, Washington University. KREEZER, GEORGE L., Guggenheim Fellow, Princeton University. KRUGELIS, EDITH J., Research Associate, University of Pennsylvania. KUFFLER, STEPHEN W., Assistant Professor of Physiological Optics, Johns Hopkins Medical School. LAVIN, GEORGE L, in charge of Spectroscopic Laboratory, Rockefeller Institute for Medical Research. LAZAROW, ARNOLD, Assistant Professor of Anatomy, Western Reserve LTniversity. LEFEVRE, PAUL G., Instructor in Physiology, University of Vermont. LIBET. BENJAMIN, Instructor in Physiology, University of Chicago. Liu, CHIEN-KANG, Graduate Student, McGill University. LILLIE, RALPH S., Professor Emeritus of Physiology, University of Chicago. LOCHHEAD, JOHN H., Assistant Professor of Zoology, University of Vermont. LYNN, W. GARDNER, Associate Professor, The Catholic University of America. MAcDouGALL, MARY S., Professor of Zoology, Agnes Scott College. MARMONT, GEORGE, Assistant Professor of Biophysics, University of Chicago. MARINELLI, L., Physicist, Memorial Hospital. MARSHAK, ALFRED, Research Associate, New York University Medical College. MARSLAND, DOUGLAS A., Associate Professor of Biology, N. Y. U. Washington Square College. MAVOR, JAMES W., Research Professor of Biology, Union College. MAZIA, DANIEL, Associate Professor of Zoology, University of Missouri. MCDONALD, SISTER ELIZABETH SETON, Professor of Biology, College of Mt. St. Joseph. REPORT OF THE DIRECTOR 35 MEM HARP, ALLEN R., Riverside, Connecticut. METZ, CHARLES B., Assistant Professor of Zoology, Yale University. METZ, C. W., Chairman, Department of Zoology, University of Pennsylvania. MILLER, JAMES A., Chairman, Department of Anatomy, Emory University. MOORE. JOHN A., Assistant Professor of Zoology, Barnard College. MOUL, EDWIN T., Assistant Instructor of Botany, University of Pennsylvania. NABRIT, S. M., Professor of Biology, Atlanta University and Moreland College. NACHMANSOHN, DAVID, Research Associate in Neurology College of Physicians and Surgeons. NIE, DASHU, Research Fellow, Institute of Zoology, Academia Sinica, China. NORTHROP, JOHN H., Member of the Institute, Rockefeller Institute, Princeton. O'BRIEN, JOHN A., JR., Assistant Professor of Biology, Catholic University of America. OLMSTED, FREDERICK, Member Research Staff, Cleveland Clinic. OSTERHOUT, W. J. V., Member Emeritus, Rockefeller Institute for Medical Research. PAPPENHEIMER, A. M., Associate Professor of Bacteriology, New York University. PARMENTER, CHARLES L., Professor of Zoology, University of Pennsylvania. PARPART, ARTHUR K., Professor of Biology, Princeton University. PATT, DONALD L, Instructor in Biology, Micldlebury College. PEQUEGNAT, WILLIS E., Assistant Professor of Zoology, Pomona College. PIERCE, MADELENE E., Associate Professor, Vassar College. PLOUGH, HAROLD H., Professor of Biology, Amherst College. PROSSER, C. LAD'D, Associate Professor of Zoology, University of Illinois. REID, W. MALCOLM, Associate Professor and Department Head of Biology, Monmouth College. ROBBIE, WILBUR A., Research Assistant Professor, State University of Iowa. ROGICK, MARY D., Professor of Biology, College of New Rochelle. ROOFE, PAUL G., Professor of Anatomy, University of Kansas. ROTH, ALEXANDER, Research Assistant, University of Kansas. RUDZINSKI, ANNA MARIA, Research Worker, Washington Square College. RUGH, ROBERTS, Associate Professor of Biology, N. Y. U. Washington Square College. RULON, OLIN, Assistant Professor of Biology, Wayne University. SCHMIDT, GERHART, Research Fellow, Tufts Medical School. SCH NEVER, LEON H., Instructor, New York University Dental College. SCOTT, ALLAN, Associate Professor of Biology, Union College. SHANES, ABRAHAM M., Assistant Professor of Physiology, New York University College of Dentistry. SHAPIRO, HERBERT, Physiologist, National Institute of Health. SHERMAN, FREDERICK G., Instructor of Biology, Brown University. SICHEL, F., Professor of Physiology, University of Vermont. SLIFER, ELEANOR H., Assistant Professor of Zoology, State University of Iowa. SMITH, SYDNEY, University Lecturer, Cambridge University, England. STEWART, DOROTHY R., Assistant Professor of Anatomy, Western Reserve Medical School. SPEIDEL, CARL C., Professor of Anatomy, University of Virginia. STEINBACH, H. B., Professor of Zoology, University of Minnesota. STOKEY, ALMA G., Professor Emeritus, Mount Holyoke College. STOUDT, HARRY N., Instructor in Biology, Temple University. STRAUS, WILLIAM L., Associate Professor of Anatomy, Johns Hopkins University. STUNKARD, HORACE W., Professor of Biology, New York University. SZENT-GYORGYI, ALBERT, Professor of Biochemistry, Budapest University, Budapest. TAYLOR, WM. RANDOLPH, Professor of Botany, University of Michigan. TOWNSEND, GRACE G., Professor of Biological Science, Cincinnati College of Pharmacy. TRACY, HENRY, Professor of Anatomy, University of Kansas. TREITEL, OTTO, Research^ Associate, Botanical Laboratory, University of Pennsylvania. VILLEE, CLAUDE A., instructor in Biological Chemistry, Harvard Medical School. WAINIO, WALTER W., Assistant Professor, New York University College of Dentistry. WALD, GEORGE, Associate Professor, Harvard University. WARD, HELEN L., Instructor in Zoology, University of Tennessee. WARNER, ROBERT C, Assistant Professor of Chemistry, New York University College of Medi- cine. WATERMAN, TALBOT, Instructor,Yale University. 36 MARINE BIOLOGICAL LABORATORY WATTERSON, RAY L., Assistant Professor of Zoology, University of Chicago. WHITING, ANNA R., Guest Investigator, University of Pennsylvania. WHITING, P. W., Professor of Zoology (Genetics), University of Pennsylvania. WICHTERMAN, RALPH, Associate Professor of Biology, Temple University. WILBER. CHARLES G., Assistant Professor of Physiology, Fordham University. WILDE, C. E., JR., Instructor of Biology, Princeton University. WINSOR, CHARLES P., Assistant Professor, School of Hygiene, Johns Hopkins University. WITKUS, ELEANOR R., Instructor in Botany and Cytology, Fordham University. WRINCH, DOROTHY, Lecturer, Smith College. YNTEMA, CHESTER L., Associate Professor of Anatomy, Syracuse University Medical College. ZINN, DONALD J., Instructor in Zoology, Rhode Island State College. ZORZOLI, ANITA, Instructor in Physiology and Biochemistry, Washington University. ZWEIFACH, BENJAMIN W., Assistant Professor of Physiology, Cornell Medical College. Beginning Investigators, 1947 • ALSCHER, RUTH P., Instructor in Biology, Manhattanville College. ALLEN, MARY BELLE, Research Fellow, Washington University. BERG, GEORGE G., Graduate Student, Columbia University. BLUMENTHAL, GERTRUDE, University of Pennsylvania. BRONFENBRENNER, ALICE, Medical Student, Washington University Medical School. CRANE, ROBERT K., Graduate Student, Harvard University. FAHEY, ELIZABETH M., Boston University. FERGUSON, ALICE, Graduate Assistant, Louisiana State University. FOGERSON, VIRGINIA L., Assistant in Zoology, Vassar College. FONTANELLA, M. A., Instructor in Comparative Anatomy, Fordham University. FOWLE, ANN M. C., Research Assistant, University of Toronto. GOLLUB, SEYMOUR, Student, University of Pennsylvania. GREEN, JAMES W., Graduate Student, Princeton University. HERBRUCK, BRUCE K., Student, Western Reserve University School of Medicine. KUNTZ, ELOISE, Assistant in Biology, Brown University. LERNER, ELEANOR, Fellow in Zoology, Washington University. LOVELACE, ROBERTA, Teaching Fellow, University of North Carolina. LUMB, ETHEL SUE, Graduate Assistant in Zoology, Washington University. MARTIN, BARBARA A., Assistant in Zoology, Barnard College. McLEAN, DOROTHY J., Demonstrator, University of Toronto. NELSON, THOMAS CLIFFORD, Graduate Assistant, Columbia University. NURNBERG, MIRIAM, Graduate Student, New York University. PETTENGILL, OLIVE S., Graduate Assistant in Physiology, Brown University. RANSMEIER, ROBERT E., Graduate Student, University of Chicago. RECKNAGEL, RICHARD O., Student, University of Pennsylvania. SCHLESINGER, ARTHUR H., Research Fellow, Northwestern University. SHAVER, JOHN R., Instructor in Zoology, University of Pennsylvania. SIROT, GUSTAVE, Western Reserve University. TAYLOR, BABETTE, Graduate Student, University of Minnesota. TIETZE, F., Research Fellow, Northwestern University. VAN HOESEN, Drusilla, Graduate Student, University of Pennsylvania. WEINER, MILTON H., Student, Western Reserve University. WEINSTEIN, H. G., Research Assistant, University of Illinois. WILSON, JEAN R., Graduate Student, University of Pennsylvania. WILSON, WALTER L., Assistant Instructor in Zoology, University of Pennsylvania. WITTENBERG, JONATHAN, Student, Columbia University. • Research Assistants, 1947 ABRAMSKY, TESS, Research Assistant, Rockefeller Institute for Medical Research. BENSON, ELEANORE, Research Assistant, University of Pennsylvania. BERMAN, JACK H., Graduate Student, Western Reserve University. BERMAN, RUTH, Student and Research Assistant, University of Pennsylvania. CARLSON, FRANCIS D., Research Assistant, Johnson Foundation, University of Pennsylvania. REPORT OF THE DIRECTOR CLIPPINGER, F. W., Student, Drury College. CONNELLY, C. M., Graduate Student, Johnson Foundation, University of Pennsylvania. COOPER, OCTAVIA, Research Assistant, Harvard Medical School. COOPERSTEIN, SHERWIN, Student, New York University College of Dentistry. DANUFSKY, PHILIP, Research Assistant, University of Pennsylvania. DEY, THOMAS E., Research Technician, Princeton University. DIETZ, ALMA, Student, University of Michigan. ESTABLE, JOSE J., Professor of Pharmacology, University of Montevideo, Uruguay. FERREIRA, Hiss M., Fellow in Biophysics, University of Chicago. FOLEY, MARY T., Research Assistant, Yale University. Fox, MARJORIE HOPKINS, Instructor, San Francisco Junior College. GOLD, MARCIA, Research Assistant, University of Chicago. HENDEE, EDELMIRA D., Research Assistant, New York University Medical School. HENLEY, CATHERINE, Research Assistant, University of North Carolina. HIRSCHHORN, HENRY A., Student, New York University. HOARE, CATHERINE V., Graduate Assistant in Bacteriology, Brown University. HONEGGER, CAROL M., Instructor, Temple University. JAFFE, LIONEL F., Harvard University. KEMP, GRACE, Graduate Assistant, Wesleyan University. METZ, DELILAH B., Research Associate in Medicine, Cornell Medical School. MEZGER, LISELOTTE, Washington University. MITCHELL, CONSTANCE, Research Assistant, University of Pennsylvania. NEFF, ROBERT J., Research Assistant, University of Missouri. NORRIS, KARL H., Electronic Engineer, University of Chicago. NORTHCRAFT, RICHARD D., Instructor, Rutgers University. PORTIS, RICHARD A., Graduate Student, Western Reserve University. RALL, WILFRID, Fellow in Biophysics, University of Chicago. RIESER, PETER, Research Assistant, University of Pennsylvania. ROTHENBERG, M. A., Research Assistant in Neurology, Columbia University. SANDEEN, MURIEL, Teaching Assistant, Northwestern University. SEAMAN, GERALD R., Graduate Assistant, Fordham University. VOLKMAN, ALVIN, Graduate, Union College. WALTERS, C. PATRICIA, Research Assistant, Eli Lilly & Company. WEBB, MARGUERITE, Teaching Assistant, Northwestern University. WEISS, MICHAEL S., Research Assistant in Neurology, College of Physicians and Surgeons. WHEELER, CHARLES B., Research Assistant, Anatomy Department, University of Kansas. WILLIS, MARION, Research Assistant, University of Pennsylvania. WILSON, MARIE, Assistant in Zoology, Northwestern University. WOODWARD, ARTHUR A., JR., Instructor in Zoology, University of Pennsylvania. YOUNG, MARCIA A., Technical Assistant, Ohio State University. Library Readers, 1947 * BERG, WILLIAM E., Research Fellow in Medical Physics, University of California. BERNHEIMER, ALLAN W., Assistant Professor of Bacteriology, New York University College of Medicine. BLOCH, ROBERT, Research Associate in Botany, Yale University. CANTONI, G. L., Assistant Professor in Pharmacology, Long Island College of Medicine. CLIFFORD, SISTER ADELE, Teacher, College of Mount St. Joseph. DEANE, HELEN W., Instructor, Harvard Medical School. FRIES, E. F. B., Assistant Professor of Biology, City College of New York. GATES, R. RUGGLES, Research Fellow in Biology, Harvard University. GRANT, MADELEINE, Member Teaching Faculty, Sarah Lawrence College. GUDERNATSCH, FREDERICK, Visiting Professor, New York University. GUREWICH, VLADIMIR, Assistant Visiting Physician, Bellevue Hospital. HARRISON, JOHN W., Student, Medical School, University of Pennsylvania. HOBSON, LAWRENCE B., Assistant. Resident, New York Hospital. 38 MARINE BIOLOGICAL LABORATORY JOFTES, DAVID L., Assistant in Biology, Tufts College. JONASSEN, HANS B., Assistant Professor of Chemistry, Tulane University. LANGE, MATHILDE M., Professor of Zoology, Wheaton College. LEDERBERG, JOSHUA, Fellow, Jane Coffin Childs Fund, Yale University. LEVITT, LEO C, Graduate Student in Physics, Princeton University. LEVEY, STANLEY, Instructor in Biochemistry, Wayne University College of Medicine. LOEWI, OTTO, Research Professor of Pharmacology, New York University College of Medicine. MEYERSHOF, OTTO, Research Professor of Biochemistry, University of Pennsylvania. MOUTON, ROBERT F., Head of Mission, Belgium. OSTER, ROBERT H., Associate Professor of Physiology, University of Maryland School of Medicine. PICK, JOSEPH, Associate Professor of Anatomy, New York University College of Medicine. PRICE, BRONSON, Analyst, U. S. Public Health Service. ROSE, S. MERYL, Assistant Professor, Smith College. RYAN, FRANCIS J., Assistant Professor of Zoology, Columbia University. SCHNEIDER, LILLIAN K., Research Assistant in Microbiology, Columbia University. SCHWARTZMAN, GREGORY, Head of Department of Bacteriology, Mt. Sinai Hospital. SIEGEL, BENJAMIN, Associate in Laboratory of Electron Microscopy, Polytechnic Institute. SPRATT, NELSON T., JR., Assistant Professor of Biology, Johns Hopkins University. TAUSSKY, HERTHA H., Research Associate, Cornell University Medical College. VONSALLMAN, LuowiG, Associate Professor, College of Physicians and Surgeons. WAGMAN, IRVING -H., Associate in Physiology, Jefferson Medical College. WATTS, NELLIE P., Pharmacologist, Abbott Laboratories. ZAWADZKI, BRONISLAW, Fellow, College of Physicians and Surgeons. Students, 1947 BOTANY BOYLE, E. MARIE, Science Teacher, Baldwin School. CADORET, REMI, Student, Harvard College. COYLE, ELIZABETH E., Assistant Professor of Biology, College of Wooster. DEARDEN, ELIZABETH R., Research Assistant, University of Toronto. DIPPELL, RUTH D., Research Associate, Indiana University. DWORKIN, ZELMAN Z., Instructor, University of Connecticut. ERSKINE, DAVID S.', Acadia University. FERGUSON, EDWARD L., Undergraduate, Wesleyan University. GAGE, MARILYN A., Student, Pennsylvania College for Women. GRIMM, MADELON R., Research Assistant in Bacteriology, Amherst College. HOLMES, ROBERT W., Student, Haverford College. HULBURT, EDWARD M., Graduate Student, University of Michigan. HYDE, BEAL B., Student, Harvard University. LAW SON, DOROTHY L., Wellesley College. SPIEGEL, LEONARD E., Drew University. WOOD, RICHARD D., Northwestern University. WOOD, URDA K., Northwestern University. EMBRYOLOGY BAUER, MARK H., Graduate Student, Princeton University. BLAIR, CHARLES B., Graduate Student, Instructor, University of North Carolina. BOLTON, ELLIS T., Graduate Assistant, Rutgers University. BUCHANAN, DOUGLAS, Assistant in Biochemistry, Dartmouth Medical School. CALVERT, JULIE N., Demonstrator in Biology, Bryn Mawr College. CHMIELOWSKI, ADAM A., Graduate Assistant, Marquette University. CLOUD, PRESTON E., JR., Assistant Professor of Geology, Harvard University. CORTELYOU, REV. J. R., Student, Northwestern University. REPORT OF THE DIRECTOR 39 DICKASON, MARY E., Smith College. EDWARDS. JOHN P., Graduate, Drury College. GOMBERG, CHARLES, Student, McGill University. GREGG, JAMES H., Graduate Student, Princeton University. HILL, HENRIETTA J., Dickinson College. HINCHEY, M. CATHERINE, Instructor in Biology, Temple University. HOLTZER, HOWARD, Student, University of Chicago. HOPKINS, AMOS L., Harvard College. ISAAC, ISAAC B., Wesleyan University. KLAU, HELEXE H., Graduate Assistant, University of Oklahoma. LEONE, CHARLES A., Instructor, Rutgers University. MAGDEBARGER, ALICE E., Student, Goucher College. MAXON, MARION G., Graduate Assistant, Claremont Graduate School. NELSON, BETTY G., Graduate Student, Johns Hopkins University. ODUM, HOWARD T., Graduate Student, University of North Carolina. PADYKULA, HELEN A., Graduate Assistant, Mt. Holyoke College. RIGGS, AUSTIN F., Student, Harvard College. ROBBINS, MARILYN, Student, Stanford University. ROSENBLOOM, LiBBY, Laboratory Assistant, University of Michigan. Russo, EVELYN E., Rosemont College. SHRADER, RUTH E., Graduate Student, Yale University Medical School. STOLACK, RICHARD B., Graduate Assistant, University of North Carolina. WEINSTEIN, HYMAN G., Research Assistant, University of Chicago. WENGER, BYRON S., Graduate Assistant, Washington University. ZUCKERKANDL, EMIL, University of Illinois. PHYSIOLOGY ARDAO, MARIA L, Assistant Professor of Chemistry, Montevideo, Uruguay. BATEMAN, MARGARET M., Graduate Student, Washington University. BICKS, RICHARD O., Long Island College of Medicine. BLOCK, SAMUEL W., Hammond, Louisiana. COSGROVE, WILLIAM B., Graduate Assistant, New York University. DAS, S. M., Government Scholar, Government of India. EDELBERG, ROBERT E., Graduate Student, University of Pennsylvania. FIALA, SILVIO, Charles University, Prague. FRIEDMAN, FLORENCE L., Teaching Assistant, Washington University. GOURLEY, D. R. H., Research Assistant, University of Toronto. HAMILTON, JAMES D., Fellow, Department of Medical Research, University of Western Ontario. HANKE, HARRIETT, Teaching Fellow in Biology, New York University. IRVING, JACK HOWARD, Graduate Student, Princeton University. KAUPE, WALTER, Massachusetts Institute of Technology. KELLOGG, RALPH H., Teaching Fellow in Physiology, Harvard Medical School. LAYTON, LAURENCE L., Assistant Professor, Johns Hopkins University. LOVE, WARNER E., Graduate Student. University of Pennsylvania. McCANN, WILLIAM P., Cornell University Medical College. MOSKOVIC, SAMUEL, Graduate School, New York University. ^. NELSON, LEONARD, Graduate Teaching Assistant, Washington University. PAULSEN, ELIZABETH C, Instructor in Zoology, University of Vermont. PERLMAN, PRESTON L., Research Fellow, Cornell University. RANSMIER, ROBERT E., Graduate Student, University of Chicago. STEELE, ALLOYS C., Research Fellow, University of Toronto. STEKLER, BURTON, Student, New York University Medical School. TAYLOR, ROBERT E., Fellow in Physiology, Strong Memorial Hospital. 40 MARINE BIOLOGICAL LABORATORY INVERTEBRATE ZOOLOGY ALLEN, JOHN M., Drury College. ALSCHER, RUTH P., Instructor in Biology, Manhattanville College. ANDERTON, LAURA G., Laboratory Assistant, Brown University. BAUER, EDWARD CHARLES, Undergraduate Assistant, University of Connecticut. Boss, WILLIS R., University of Iowa. BOYER, GEORGE F., Graduate Student, University of Illinois. BRAGG, NANA I., Student, Oberlin College. BUCKLIN, DONALD H., Graduate Assistant, Amherst College. BULL, ALICE L., Laboratory Assistant, Alt. Holyoke College. BURGER, ANDRE, Harvard University. CHAMBERLIN, J. LOCKWOOD, Tufts College. CLOUD, PRESTON E., Assistant Professor, Harvard University. COLE, GERALD A., Laboratory Assistant, University of Minnesota. CORLISS, CLARK E., Graduate Student, University of Massachusetts. DANIEL, EDWIN E., Student, Johns Hopkins University. EHRLICH, MIRIAM, Graduate Student, Yale University. EPSTEIN, HOWARD E., Assistant, Department of Comparative Anatomy, Cornell University. EVANS, JEANNE F., Student, University of Pennsylvania. FLYNN, JOYCE M., Newton, Massachusetts. FORD, BENJAMIN P., Princeton University. FORD, DONALD H., Laboratory Assistant, Wesleyan University. FORD, PRISCILLA W., Wellesley College. FULLER, FORST D., Instructor in Zoology, Purdue University. GLOCKLER, ANNABEL, Western Maryland College. GOODSMITH, WILLOUGHBY, Teaching Assistant and Student, Amherst College. HAY, ELIZABETH D., Undergraduate, Smith College. HENOCH, STEPHANIE D., Graduate Assistant, Indiana University. HOWARD, ROBERT S., University of Chicago. HUTCHINGS, Lois M., Instructor, N. Y. U. Washington Square College. KAMNER, SANDRA L., Goucher College. KELLER, MILDRED E., Randolph-Macon Woman's College. MANDLOWITZ, SAMUEL, Tulane University. McCuLLOUGH, KIRK W., Instructor, Washington and Jefferson College. McELLiGOTT, JANE K., Graduate Student, Fordham University. McGyiRE, IRENE E., Fordham University. McWniNNiE, MARY A., Instructor in Zoology, DePaul University. MELTZER, JAY, Student, Princeton University. ODUM, HOWARD T., Graduate Student, University of North Carolina. PERNA, VINCENT, Student, Wesleyan University. PRONKO, ROBERT C., Student, Drury College. RADFORD, BETTY J., Assistant in Biology Department, Agnes Scott College. SAUSE, GLADYS E., Western Maryland College. SPRINGER, ELEANOR V., Vassar College. STAY, BARBARA A., Student, Vassar College. STEEVES, TAYLOR A., East Weymouth, University of Massachusetts. STOLACK, RICHARD B., Graduate Student, University of North Carolina. SUSCA, Louis, Graduate Student, Fordham University. THAYER, PHILIP S., Amherst College. TRENT, JANE, Assistant, Wesleyan University. WENGER, BYRON S., Graduate Assistant, Washington University. WINN, HUDSON S., Graduate Teaching Assistant, Northwestern University. WINSTON, PAUL W., Student, University of Massachusetts. WITHROW, JOANNA E. F., Student, Oberlin College. WOLF, DORIS E., Graduate Student, University of Minnesota. WONG, AN CHI, Wellesley College. REPORT OF THE DIRKcTOR 41 4. TABULAR VIEW OF ATTENDANCE 1943 1944 1945 1946 1947 I NVESTICATORS— Total 160 193 212 267 299 Independent 89 112 138 175 182 Under instruction 19 11 10 29 36 Library readers 35 50 38 38 36 Research assistants 17 20 26 25 45 STUDENTS— Total 68 75 96 122 131 Zoology 47 37 55 57 55 Embryology 13 23 23 30 33 Physiology 8 10 13 26 26 Botany 5 5 9 17 TOTAL ATTENDANCE 228 276 308 389 430 Less persons registered as both students and investigators 61 2 222 275 428 INSTITUTIONS REPRESENTED — TOTAL 116 106 124 141 By investigators 70 74 100 102 114 By students 41 41 49 56 56 SCHOOLS AND ACADEMIES REPRESENTED By investigators 2 1 2 2 1 By students 1 2 2 1 FOREIGN INSTITUTIONS REPRESENTED By investigators 2 2 1 7 7 By students 3 5 3 5. SUBSCRIBING AND COOPERATING INSTITUTIONS, 1947 Cooperating Institutions American Philosophical Society (Penrose Fund) Amherst College Brooklyn College Brown University Bryn Mawr College The Catholic University of America College of Mt. St. Joseph on the Ohio Columbia University Cornell University Duke University Fordham University Goucher College Harvard University Harvard University Medical School Johns Hopkins Medical School Johns Hopkins University Johnson Foundation Eli Lilly & Company Macy Foundation Massachusetts Institute of Technology Memorial Hospital Miami University New York University New York University College of Medicine New York University School of Dentistry New York University Washington Square College Oberlin College Ohio State University Pomona College Princeton University Rockefeller Institute of Medical Research State University of Iowa Syracuse University Medical School Temple University Tufts College Union College University of Chicago University of Illinois University of Kansas University of Maryland Medical School University of Missouri University of Pennsylvania University of Pennsylvania School of Medi- cine University of Rochester University of Toronto University of Vermont University of Virginia Vassar College Washington University Wayne University Wellesley College Wesleyan University Western Reserve Medical School Wilson College Woods Hole Oceanographic Institution Yale University 42 MARINE BIOLOGICAL LABORATORY Subscribing Institutions Atlanta University, Moreland College Rockefeller Foundation Boston Dispensary Russell Sage College Carnegie Inst. of Washington Rutgers University College of Charleston Simmons College College of Physicians and Surgeons Smith College Indiana University St. John's College Mount Sinai Hospital University of Delaware National Cancer Institute University of Massachusetts North Carolina State College of Agriculture University of Michigan & Engineering University of Minnesota Pennsylvania College for Women University of Western Ontario 6. THE FRIDAY EVENING LECTURES, 1947 June 27 DR. GOTTFRIED FRAENKEL "Nutritional Research with Insects." July 3 DR. JEAN BRACKET "Metabolism of Nucleic Acids in Embryonic Development." July 11 PROF. E. L. TATUM "Mutation in Microorganisms." July 25 DR. F. O. SCHMITT "Studies of the Ultra Structure of Connec- tive Tissue Constituents." August 1 DR. PHILIP H. ABELSON "Tracer Isotopes in Biology." August 8 PROF. ALFRED S. ROMER In celebration of the 74th Anniversary of the founding of the Agassiz Laboratory at Penikese. August 15 DR. JOHN A. MOORE "Early Development of Amphibian Hy- brids." August 22 DR. DANIEL MAZIA . "The Molecular Structure of Chromo- somes." OTHER LECTURES July 24 DR. PAUL S. GALTSOFF "The Bikini Atom Bomb Test." July 30 DR. K. J. HEINICKE "Recent Developments in Microscopy." August 13 DR. OSCAR W. RICHARDS "Phase Microscopy, with Special Reference to Biology." August 18 DR. H. J. ABRAHAM U. S. State Dept. Associate Director of Re- lations with UNESCO. August 27 DR. ALBERT SZENT-GYORGYI "Muscular Contraction." August 28 PROF. Louis VAN DEN BERGHE "National Parks and Scientific Research in the Congo." REPORT OF THE DIRECTOR 43 7. SEMINARS, 1947 July 15 ANNA R. WHITING "Androgenetic Males from Eggs X-rayed with Dose Many Times Lethal." T. S. HALL AND FLORENCE MOOG ''Effects of Sodium Azide in Solution upon the Rate of Amphibian Development." C. A. YILLEE AND- H. B. BissELL "Nucleic Acids and Nucleotides as Growth Factors in Drosophila." July 22 IVOR CORNMAN "The Effects of Podophyllin on the Matura- tion and Cleavage of the Starfish Egg." C. G. WILBER "The Synthesis of Lipids from Protein in Colpidium Catnpylum." W. W. WAINIO, S. COOPERSTEIN, S. KOLLEN AND B. EiCHEL "The Preparation of a Soluble Cytochrome Oxidase." July 29 OTTO MYERHOFF AND JEAN R. WILSON . . "Glycolysis of Glucose, Fructose, and Hex- osephosphates in Tumor and Brain Ex- tracts." B. LIBET "Relatively Steady Potentials and Brain Ac- tivity." f . T. BONNER "Morphogenetic Movement in the Slime Molds." August 5 EDITH J. KRUGELIS "Alkalin Phosphatase Localization in Early Embryonic Development." J. R. SHAVER "Experimental Study of the 'Second Fac- tor' in Artificial Parthenogenesis in Frog Eggs." ROBERT BLOCH "Irreversible Differentiation in Certain Plant Cell Lineages." August 12 DOROTHY WRINCH "Biological Specificity and Biological Mor- phology." TAYLOR HINTON "Factors Influencing the Expression of 'Po- sition Effects.' ' D. E. COPELAND "The Cytological Basis of Salt Excretion from the Gills of Fundulus heteroclitus." August 19 P. W. WHITING "Spermatogenesis in Sphecoid Wasps." ETHEL BROWNE HARVEY "Bermuda Sea Urchins and Their Eggs." PAUL S. GALTSOFF "The Red Tide along the Gulf Coast and Florida." 8. MEMBERS OF THE CORPORATION, 1947 1. LIFE MEMBERS *ALLIS, MR. E. J., JR., Palais Carnoles, Menton, France. BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, New York. BILLINGS, MR. R. C., 66 Franklin Street, Boston, Massachusetts. CALVERT, DR. PHILIP P., University of Pennsylvania, Philadelphia, Pennsylvania. COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin. * Deceased. 44 MARINE BIOLOGICAL LABORATORY CONKLIN, PROF. EDWIN G., Princeton University, Princeton, New Jersey. COWDRY, DR. E. V., Washington University, St. Louis, Missouri. JACKSON, MR. CHAS. C, 24 Congress Street, Boston, Massachusetts. JACKSON, Miss M. C., 88 Marlboro Street, Boston, Massachusetts. KING, MR. CHAS. A. KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York. LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Maryland. MEANS, DR. J. H., 15 Chestnut Street, Boston, Massachusetts. MOORE, DR. GEORGE T., Missouri Botanical Gardens, St. Louis, Missouri. MOORE, DR. J. PERCY, University of Pennsylvania, Philadelphia, Pa. MORGAN, MRS. T. H., Pasadena, California. No YES, Miss EVA J. PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pennsylvania. SCOTT, DR. ERNEST L., Columbia University, New York City, New York. SEARS, DR. HENRY F., 86 Beacon Street, Boston, Massachusetts. SHEDD, MR. E. A. STRONG, DR. O. S., Columbia University, New York City, New York. WAITE, PROF. F. C., 144 Locust Street, Dover, New Hampshire. WALLACE, LOUISE B., 359 Lytton Avenue, Palo Alto, California. 2. REGULAR MEMBERS ADAMS, DR. A. ELIZABETH, Mount Holyoke College, South Hadley, Massachusetts. ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia, Pennsylvania. ADOLPH, DR. EDWARD F., University of Rochester Medical School, Rochester, New York. ALBAUM, DR. HARRY G., Biology Dept., Brooklyn College, Brooklyn, N. Y. ALBERT, DR. ALEXANDER, Mayo Clinic, Rochester, Minnesota. ALLEE, DR. W. C., The University of Chicago, Chicago, Illinois. AMBERSON, DR. WILLIAM R., Department of Physiology, University of Maryland, School of Medicine. ANDERSON, DR. RUBERT S., University of Maryland School of Medicine, Depart- ment of Physiology, Baltimore, Maryland. ANDERSON, DR. T. F., University of Pennsylvania, Philadelphia, Pennsylvania. ANGERER, PROF. C. A., Department of Physiology, Ohio State College, Columbus, 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. REPORT OF THE DIRECTOR 45 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. BARTLETT, DR. JAMES H., Department of Physics, University of Illinois, Urbana, Illinois. BEADLE, DR. G. W., School of Biological Sciences, Stanford University, California. BEAMS, DR. HAROLD W., Department of Zoology, State University of Iowa, Iowa City, Iowa. BECK, DR. L. V., Edgey Road and Beech Avenue, Bethesda, Maryland. BEERS, C. D., University of North Carolina, Chapel Hill, North Carolina. BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, Louisiana. BERTHOLF, DR. LLOYD M., Western Maryland College, Westminster, Maryland. BEVELANDER, DR. GERRIT, New York University School of Medicine, New York City, New York. BIGELOW, DR. H. B., Museum of Comparative Zoology, Cambridge, Massachusetts. BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Massa- chusetts. BISSONNETTE, DR. T. HUME, Trinity College, Hartford, Connecticut. BLANCHARD, PROF. K. C., Johns Hopkins Medical School, Baltimore, Maryland. BLUM, DR. HAROLD F.. Department of Biology, Princeton University, Princeton, New Jersey. BODINE, DR. J. H., Department of Zoology, State University of Iowa, Iowa City, Iowa. BOELL, DR. EDGAR J., Yale University, New Haven, Connecticut. BORING, DR. ALICE M., Yenching University, Peiping, China. BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wisconsin. BRODIE, MR. DONALD M., 522 Fifth Avenue, New York City, New York. BRONFENBRENNER, DR. JACQUES J., Department of Bacteriology, Washington Uni- versity Medical School, St. Louis, Missouri. BRONK, DR. DETLEV W., Johnson Foundation, University of Pennsylvania, Phila- delphia, Pennsylvania. BROOKS, DR. MATILDA M., University of California, Department of Zoology, Berke- ley, California. BROOKS, DR. S. C., University of California, Berkeley, California. BROWN, DR. DUGALD E. S.. Bermuda Biological Station, Bermuda. BROWN, DR. FRANK A., JR., Department of Zoology, Northwestern University, Evanston, Illinois. BROWNELL, DR. KATHERINE A., Ohio State University, Columbus, Ohio. BUCK, DR. JOHN B., Industrial Hygiene Research Lab., National Institute of Health, Bethesda, Maryland. BUCKINGHAM, Miss EDITH N., Sudbury, Massachusetts. BUDINGTON, PROF. R. A., Winter Park, Florida. BULLINGTON, DR. W. E., Randolph-Macon College, Ashland, Virginia. BULLOCK, DR. T. H., University of California, Los Angeles 24, California. BURBANCK, DR. WILLIAM D., Department of Biology, Drury College, Springfield, Missouri. 46 MARINE BIOLOGICAL LABORATORY BURKENROAD, DR. M. D., Yale University, New Haven, Connecticut. BURKHOLDER, DR. PAUL R., Yale University, New Haven, Connecticut. BUTLER, DR. E. G., Princeton University, Princeton, N. J. CAMERON, DR. J. A., Baylor College of Dentistry, Dallas. Texas. CANNAN, PROF. R. K., New York University College of Medicine, 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, New York City, New York. CATTELL, MR. WARE, Cosmos Club, Washington, D. C. CHAMBERS, DR. ROBERT, Woods Hole, Massachusetts. CHASE, DR. AURIN M., Princeton University, Princeton, New Jersey. CHEYNEY, DR. RALPH H., Biology Department, Brooklyn College, Brooklyn 10, New York. CHILD, PROF. C. M., Jordan Hall, Stanford University, California. CHURNEY, DR. LEON, Dept. of Physiology, Louisiana State University School of Medicine, New Orleans 13, Louisiana. CLAFF, MR. C. LLOYD, 31 West Street, Randolph, Massachusetts. CLARK, PROF. E. R., Wistar Institute, Woodland Avenue and 36th Street, Philadel- phia 4, Pennsylvania. CLARK, DR. LEONARD B.( Department of Biology, Union College, Schenectady, New York. CLARKE, DR. G. L., Department of Biology, Harvard University, Cambridge 38, Mass. CLELAND, PROF. RALPH E., Indiana University, Bloomington, Indiana. CLOWES, DR. G. H. A., Eli Lilly and Company, Indianapolis, Indiana. COE, PROF. W. R., Yale University, New Haven, Connecticut. COHN, DR. EDWIN J., 183 Brattle Street, Cambridge, Massachusetts. COLE, DR. ELBERT C., Department of Biology, Williams College, Williamstown, Massachusetts. COLE, DR. KENNETH S., University of Chicago, Chicago, Illinois. COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio. COLTON, PROF. H. S., Box 601, Flagstaff, Arizona. COLWIN, DR. ARTHUR L., Queens College, Flushing, Long Island, New York. COOPER, DR. KENNETH W., Department of Biology, Princeton University, Prince- ton, New Jersey. COPELAND, DR. D. E., Department of Zoology, Brown University, Providence, Rhode Island. COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine. COSTELLO, DR. DONALD P., Department of Zoology, University of North Carolina, Chapel Hill, North Carolina. REPORT OF THE DIRECTOR 47 COSTELLO, DR. HELEN MILLER, Department of Zoology, University of North Caro- lina, Chapel Hill, North Carolina. CRAMPTON, PROF. H. E., American Museum of Natural History, New York City, New York. CRANE, JOHN O., Woods Hole, Massachusetts. CRANE, MRS. W. MURRAY, Woods Hole, Massachusetts. CROASDALE, HANNAH T., Dartmouth College, Hanover, New Hampshire. GROUSE, DR. HELEN V., University of Pennsylvania, Philadelphia, Pennsylvania. CROWELL, DR. P. S., JR., Department of Zoology, Miami University, Oxford, Ohio. CURTIS, DR. MAYNIE R., 377 Dexter Trail, Mason, Michigan. CURTIS, PROF. W. C., University of Missouri, Columbia, Missouri. DAN, DR. KATSUMA, Misaki Biological Station, Misaki, Japan. DAVIS, DR. DONALD W., College of William and Mary, Williamsburg, Virginia. DAWSON, DR. A. B., Harvard University, Cambridge, Massachusetts. DAVVSON, 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. DOTY, DR. MAXWELL S., Northwestern University, Department of Botany, Evans- ton, Illinois. DuBois, DR. EUGENE F., Cornell University Medical College, New York City, New York. DUGGAR, DR. BENJAMIN M., c/o Lederle Laboratories Inc., Pearl River, New York. DUNGAY, DR. NEIL S., Carleton College, Northfield, Minnesota. DURYEE, DR. WILLIAM R., Dept. of Terrestrial Magnetism, Washington 15, D. C. ELLIS, DR. F. W., 1175 Centre Street, Newton, Massachusetts. EVANS, DR. TITUS C., Radiation Research Laboratory, College of Medicine, Iowa City, Iowa. FAILLA, DR. G., College of Physicians and Surgeons, New York City, New York. FAURE-FREMIET, PROF. EMMANUEL, College de France, Paris, France. FAUST, DR. ERNEST C., Tulane University of Louisiana, New Orleans, Louisiana. FERGUSON, DR. JAMES K. W., Department of Pharmacology, University of Toronto, Ontario, Canada. FIGGE, DR. F. H. J., Lombard and Greene Streets, Baltimore, Maryland. FISCHER, DR. ERNST, Baruch Centre of Physical Medicine, Medical College of Vir- ginia, Richmond 19, Virginia. FISHER, DR. JEANNE M., Department of Biochemistry, University of Toronto, To- ronto, Canada. 48 MARINE BIOLOGICAL LABORATORY FISHER, DR. KENNETH C., Department of Biology, University of Toronto, Toronto, Canada. FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Massachu- setts. FRISCH, DR. JOHN A., Canisius College, Buffalo, New York. FURTH, DR. JACOB, V. A. Hospital (Lisbon) Laboratories, Dallas, Texas. GALTSOFF, DR. PAUL S., 420 Cumberland Avenue, Somerset, Chevy Chase, Mary- land. CARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee. GASSER, DR. HERBERT, Director, Rockefeller Institute, New York City, New York. GATES, DR. REGINALD R., Woods Hole, Massachusetts. GEISER, DR. S. W., Southern Methodist University, Dallas, Texas. GERARD, PROF. R. W., The University of Chicago, Chicago, Illinois. GLASER, PROF. O. C., Amherst College, Amherst, Massachusetts. GOLDFORB, PROF. A. J., College of the City of New York, New York City, New York. GOODCHILD, DR. CHAUNCEY G., State Teachers College, Springfield, Missouri. GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut. GOTTSCHALL, DR. GERTRUDE Y., 315 East 68th Street, New York 21, New York. GOULD, DR. H. N., Newcomb College, New Orleans 18, Louisiana. GRAHAM, DR. J. Y., Roberts, Wisconsin. GRAND, CONSTANTINE G., Biology Department, Washington Square College, New York University, Washington Square, New York City, New York. GRANT, DR. MADELEINE P., Woods Hole, Massachusetts. GRAVE, PROF. B. H., DePauw University, Greencastle, Indiana. GRAY, PROF. IRVING E., Duke University, Durham, North Carolina. GREGG, DR. J. R., Department of Zoology, Columbia University, New York 27, New York. GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City, New York. GRUNDFEST, DR. HARRY, Columbia University College of Physicians and Surgeons, New York City, New York. GUDERNATSCH, DR. J. FREDERICK, New York University, 100 Washington Square, New York City, New York. GUTHRIE, DR. MARY J., University of Missouri, Columbia, Missouri. GUYER, PROF. M. F., University of Wisconsin, Madison, Wisconsin. HAGUE, DR. FLORENCE, Sweet Briar College, Sweet Briar, Virginia. HALL, PROF. FRANK G., Duke University, Durham, North Carolina. HAMBURGER, DR. VIKTOR, Department of Zoology, Washington University, St. Louis, Missouri. HAMILTON, DR. HOWARD L., Iowa State College, Ames, Iowa. HANCE, DR. ROBERT T., The Cincinnati Milling Machine Co., Cincinnati 9, Ohio. 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. HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut. HARTLINE, DR.'H. KEFFER, University of Pennsylvania, Philadelphia, Pennsylvania. REPORT OF THE DIRECTOR 49 HARTMAN, DR. FRANK A., Hamilton Hall, Ohio State University, Columbus, Ohio. HARVEY, DR. E. NEWTON, Guyot Hall, Princeton University, Princeton, New Jer- sey. HARVEY, DR. ETHEL BROWNE, 48 Cleveland Lane, Princeton, New Jersey. HAUSCHKA, DR. T. S., Institute for Cancer Research, Philadelphia 30, Pennsyl- vania. HAYDEN, DR. MARGARET A., Wellesley College, Wellesley, Massachusetts. HAYES, DR. FREDERICK R., Zoological Laboratory, Dalhousie University, Halifax, Nova Scotia. HAYWOOD, DR. CHARLOTTE, Mount Holyoke College, South Hadley, Massachusetts. HECHT, DR. SELIG, Columbia University, New York City, New York. HEILBRUNN, DR. L. V., Department of Zoology, University of Pennsylvania, Phila- delphia, Pennsylvania. 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., 18 Collins Avenue, Troy, New York. HINRICHS, DR. MARIE, Department of Physiology and Health Education, Southern Illinois Normal University, Carbondale, Illinois. HISAW, DR. F. L., Harvard University, Cambridge, Massachusetts. HOADLEY, DR. LEIGH, Biological Laboratories, 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, P.O. Box W., Clinton Laboratories, Oak Ridge, Tennessee. HOPKINS, DR. DWIGHT L., Mundelein College, Chicago, Illinois. HOPKINS, DR. HOYT S., New York University, College of Dentistry, New York City, New York. HYMAN, DR. LIBBIE H., American Museum of Natural History, New York City, New York. IRVING, LT. COL. LAURENCE, Swarthmore College, Department of Zoology, Swarth- more, 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., 1336 Parkwood Place, N.W., Washington, D. C. JOHLIN, DR. J. M., Vanderbilt University Medical School, Nashville, Tennessee. JONES. DR. E. RUFFIN, University of Florida, Gainesville, Florida. KAAN, DR. HELEN W., National Research Council, 2101 Constitution Avenue, Washington, D. C. KAUFMANN, PROF. B. P., Carnegie Institution, Cold Spring Harbor, Long Island, New York. KEMPTON, PROF. RUDOLF T., Vassar College, Poughkeepsie, New York. 50 MARINE BIOLOGICAL LABORATORY KIDDER, DR. GEORGE W., Amherst College, Amherst, Massachusetts. KIDDER, JEROME F., Woods Hole, Massachusetts. KILLE, DR. FRANK R., Carleton College, Northfield, Minnesota. KINDRED, DR. J. E., University of Virginia, Charlottesville, Virginia. KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania. KING, DR. ROBERT L., State University of Iowa, Iowa City, Iowa. KLOTZ, DR. I. M., Department of Chemistry, Northwestern University, Evanston, Illinois. KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York. KOPAC, DR. M. J., Washington Square College, New York University, New York City, New York. KRAHL, DR. M. E., Washington University School of Medicine, Department of Pharmacology, St. Louis, Missouri. KRIEG, DR. WENDELL J. S., 303 East Chicago Ave., Chicago, Illinois. LANCEFIELD, DR. D. E., Queens College, Flushing, New York. LANCEFIELD, DR. REBECCA C., Rockefeller Institute, New York City, New York. LANDIS, DR. E. M., Harvard Medical School, Boston, Massachusetts. LANGE, DR. MATHILDE M., Wheaton College, Norton, Massachusetts. LAVIN, DR. GEORGE I., Rockefeller Institute, New York City, New York. LAZAROW, DR. ARNOLD, Western Reserve University School of Medicine, Cleveland 6, Ohio. LEWIS, PROF. I. F., University of Virginia, Charlottesville, Virginia. *LILLIE, PROF. FRANK R., The University of Chicago, Chicago, Illinois. LILLIE, PROF. RALPH S., The University of Chicago, Chicago, Illinois. LITTLE, DR. E. P., Phillips Exeter Academy, Exeter, New Hampshire. LOCH HEAD, DR. JOHN H., Department of Zoology, University of Vermont, Bur- lington, Vermont. LOEB, PROF. LEO, 40 Crestwood Drive, St. Louis, Missouri. LOEB, DR. R. F., Department of Medicine, College of Physicians and Surgeons, New York City, New York. LOEWI, PROF. OTTO, 155 East 93d Street, New York City, New York. LOWT.HER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York City, New York. LUCAS, DR. ALFRED M., Regional Poultry Research Laboratory, East Lansing, Michigan. LUCRE, PROF. BALDUIN, University of Pennsylvania, Philadelphia, Pennsylvania. LYNCH, DR. CLARA J., Rockefeller Institute, New York City, New York. LYNCH, DR. RUTH STOCKING, Dept. of Zoology, University of California, Los Angeles 24, California. LYNN, DR. WILLIAM G., Department of Biology, The Catholic University of Amer- ica, Washington, D. C. MACDOUGALL, DR. MARY S., Agnes Scott College, Decatur, Georgia. MACNAUGHT, MR. FRANK M., Marine Biological Laboratory, Woods Hole, Massa- chusetts. McCoucH, DR. MARGARET SUMWALT, University of Pennsylvania Medical School, Philadelphia, Pa. * Deceased. REPORT OF THE DIRECTOR 51 MCGREGOR, DR. J. H., Columbia University, New York City, New York. MACKLIN, DR. CHARLES C, School of Medicine, University of Western Ontario, London, Canada. MAGRUDER, DR. SAMUEL R., Department of Anatomy, Tufts Medical School, Bos- ton, Massachusetts. MALONE, PROF. E. F., 153 Cortland Avenue, Winter Park, Florida. MANWELL, DR. REGINALD D., Syracuse University, Syracuse, New York. MARSLAND, DR. DOUGLAS A., Washington Square College, New York University, New York City, New York. MARTIN, PROF. E. A., Department of Biology, Brooklyn College, Brooklyn, New York. MATHEWS, PROF. A. P., Woods Hole, Massachusetts. MATTHEWS, DR. SAMUEL A., Thompson Biological Laboratory, Williams College, Williamstown, Massachusetts. MAYOR, PROF. JAMES W., 24 Edward Street, Belmont, Massachusetts. MAZIA, DR. DANIEL, University of Missouri, Department of Zoology, Columbia, Missouri. MEDES, DR. GRACE, Lankenau Research Institute, Philadelphia, Pennsylvania. MEIGS, MRS. E. B., 1736 M Street, N.W., Washington, D. C. MF.MHARD, MR. A. R., Riverside, Connecticut. MENKIN, DR. VALY, Department of Surgical Research, Temple University Medical School, Philadelphia, Pennsylvania. METZ, DR. C. B., Osborn Zoological Laboratory, Yale University, New Haven, Connecticut. METZ, PROF. CHARLES W., University of Pennsylvania, Philadelphia, Pennsylvania. MICHAELIS, DR. LEONOR, Rockefeller Institute, New York City, New York. MILLER, DR. J. A., Emory University, Atlanta 3, Georgia. MILNE, DR. LORUS J., Zoology Department, University of Vermont, Burlington, Vermont. 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. MOORE, DR. J. A., Barnard College, New York City, New York. MORGAN, DR. ISABEL M., Poliomyelitis Research Center, Baltimore 5, Maryland. MORRILL, PROF. C. V., Cornell University Medical College, New York City, New York. MULLER, PROF. H. J., Department of Zoology, Indiana University, Bloomington, Indiana. NABRIT, DR. S. M., Atlanta University, Morehouse College, Atlanta, Georgia. NACHMANSOHN, DR. D., College of Physicians and Surgeons, New York City, New York. NAVEZ, DR. ALBERT E., Department of Biology, Milton Academy, Milton, Massa- chusetts. NEWMAN, PROF. H. H., 173 Devon Drive, Clearwater, Florida. NICHOLS, DR. M. LOUISE, Rosemont, Pennsylvania. 52 MARINE BIOLOGICAL LABORATORY *NONIDEZ, DR. JOSE F., Cornell University Medical College, New York City, New York. NORTHROP, DR. JOHN H., The Rockefeller Institute, Princeton, New Jersey. OCHOA, DR. SEVERO, New York University, College of Medicine, New York 16, New York. OPPENHEIMER, DR. JANE M., Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania. .OSBURN, PROF. R. C., Ohio State University, Columbus, Ohio. OSTER, DR. ROBERT H., University of Maryland, School of Medicine, Baltimore, Maryland. OSTERHOUT, PROF. W. J. V., Rockefeller Institute, New York City, New York. OSTERHOUT, MRS. MARIAN IRWIN, Rockefeller Institute, New York City, New York. PACKARD, DR. CHARLES, Marine Biological Laboratory, Woods Hole, Massachu- setts. PAGE, DR. IRVINE H., Cleveland Clinic, Cleveland, Ohio. PAPPENHEIMER, DR. A. M., 5 Acacia Street, Cambridge, Massachusetts. PARKER, PROF. G. H., Harvard University, Cambridge, Massachusetts. PARMENTER, DR. C. L., Department of Zoology, University of Pennsylvania, Phila- delphia, Pennsylvania. PARPART, DR. ARTHUR K., Princeton University, Princeton, New Jersey. PATTEN, DR. BRADLEY M., University of Michigan Medical School, Ann Arbor, Michigan. PAYNE, PROF. F., University of Indiana, Bloomington, Indiana. PEEBLES, PROF. FLORENCE, 380 Rosemont Avenue, Pasadena, California. PIERCE, DR. MADELENE E., Vassar College, Poughkeepsie, New York. PINNEY, DR. MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin. PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Massachusetts. POLLISTER, DR. A. W., Columbia University, New York City, New York. POND, DR. SAMUEL E., 53 Alexander Street, Manchester, Connecticut. PRATT, DR. FREDERICK H., Wellesley Hills 82, Massachusetts. PROSSER, DR. C. LADD, University of Illinois, Urbana, Illinois. RAMSEY, DR. ROBERT W., University of Virginia Medical School, Richmond, Vir- ginia. RAND, DR. HERBERT W., Harvard University, Cambridge, Massachusetts. RANKIN, DR. JOHN S., Zoology Department, University of Connecticut, Storrs, Connecticut. REDFIELD, DR. ALFRED C., Harvard University, Cambridge, Massachusetts. REID, DR. W. M., Monmouth College, Monmouth, Illinois. RENN, DR. CHARLES E., Sanitary Laboratories, The Johns Hopkins University, Baltimore, Maryland. RENSHAW, DR. BIRDSEY, Department of Physiology, University of Oregon Medical School, Portland, Oregon. REZNIKOFF, DR. PAUL, Cornell University Medical College, New York City, New York. RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio. * Deceased. REPORT OF THE DIRECTOR 53 RICHARDS, PROF. A., University of Oklahoma, Norman, Oklahoma. RICHARDS, DR. A. GLENN, Entomology Department, University Farm, Univ. of Minnesota, St. Paul 8, Minnesota. RICHARDS, DR. O. W., Research Department, American Optical Co., Buffalo, New York. RIGGS, LAWRASON, JR., 120 Broadway, New York City, New York. ROBBIE, DR. W. A., University Hospital, Iowa City, Iowa. ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio. ROGICK, DR. MARY D., College of New Rochelle, New Rochelle, New York. ROMER, DR. ALFRED S., Harvard University, Cambridge, Massachusetts. ROOT, DR. R. W., Department of Biology, College of the City of New York, New York City, New York. ROOT, DR. W. S., College of Physicians and Surgeons, Department of Physiology, New York City, New York. RUEBUSH, DR. T. K., Dayton, Virginia. RUGH, DR. ROBERTS, Department of Biology, Washington Square College, New York University, New York City, New York. RYAN, DR. FRANCIS J., Columbia University, New York City, N. Y. SAMPSON, DR. MYRA M., Smith College, Northampton, Massachusetts. SASLOW, DR. GEORGE, Washington University Medical School, St. Louis, Missouri SAUNDERS, LAWRENCE, W. B. Saunders Publishing Company, Philadelphia, Penn- sylvania. SCHAEFFER, DR. ASA A., Biology Department, Temple University, Philadelphia, Pennsylvania. SCHARRER, DR. ERNST A., Department of Anatomy, University of Colorado School of Medicine and Hospitals, Denver, Colorado. SCHECHTER, DR. VICTOR, College of the City of New York, New York City, New York. SCHMIDT, DR. L. H., Christ Hospital, Cincinnati, Ohio. SCHMITT, PROF. F. O., Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts. SCHOTTE, DR. OSCAR E., Department of Biology, Amherst College, Amherst, Massa- chusetts. SCHRADER, DR. FRANZ, Department of Zoology, Columbia University, New York City, New York. SCHRADER, DR. SALLY HUGHES, Department of Zoology, Columbia University, New York City, New York. SCHRAMM, PROF. J. R., University of Pennsylvania, Philadelphia, Pennsylvania. SCOTT, DR. ALLAN C, Union College, Schenectady, New York. SCOTT, SISTER FLORENCE MARIE, Professor of Biology, Seton Hill College, Greens- burg, Pennsylvania. SCOTT, DR. GEORGE T., Oberlin College, Oberlin, Ohio. SEMPLE, MRS. R. BOWLING, 140 Columbia Heights, Brooklyn, New York. SEVERINGHAUS, DR. AURA E., Department of Anatomy, College of Physicians and Surgeons, New York City, New York. SHANES, DR. ABRAHAM M., Department of Physiology and Biophysics, George- town University, School of Medicine, Washington, D. C. 54 MARINE BIOLOGICAL LABORATORY SHAPIRO, DR. HERBERT, National Institute of Health, Bethesda, Maryland. SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan. SHUMWAY, DR. WALDO, Stevens Institute of Technology, Hoboken, New Jersey. 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, Baltimore, Maryland. SNYDER, PROF. L. H., University of Oklahoma, Norman, Oklahoma. SONNEBORN, DR. T. M., Department of Zoology, Indiana University, Bloomington, Indiana. SPEIDEL, DR. CARL C, University of Virginia, University, Virginia. STEINBACH, DR. HENRY BURR, University of Minnesota, Minneapolis, Minnesota. STERN, DR. CURT, Department of Zoology, University of Rochester, Rochester, New York. STERN, DR. KURT G., Polytechnic Institute, Department of Chemistry, Brooklyn New York. STEWART, DR. DOROTHY, Rockford College, Rockford, Illinois. STOREY, DR. ALMA G., Department of Botany, Mount Holyoke College, South Hadley, Massachusetts. STRAUS, DR. W. L., Johns Hopkins Medical School, Baltimore, Maryland. STUNKARD, DR. HORACE W., New York University, University Heights, New York. STURTEVANT, DR. ALFRED H., California Institute of Technology, Pasadena, California. TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio. TAYLOR, DR. WILLIAM R., University of Michigan, Ann Arbor, Michigan. TEWINKEL, DR. L. E., Department of Zoology, Smith College, Northampton, Massachusetts. TRACY, DR. HENRY C., University of Kansas, Lawrence, Kansas. TURNER, DR. ABBY H., Mt. Holyoke College, South Hadley, Massachusetts. TURNER, PROF. C. L., Northwestern University, Evanston, Illinois. TYLER, DR. ALBERT, California Institute of Technology, Pasadena, California. UHLENHUTH, DR. EDUARD, University of Maryland, School of Medicine, Balti- more, Maryland. VILLEE, DR. CLAUDE A., JR., Harvard Medical School, Boston, Massachusetts. VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, Ohio. WAINIO, DR. W. W., New York University, College of Dentistry, New York City. WALD, DR. GEORGE, Biological Laboratories, Harvard University, Cambridge, Massachusetts. WARBASSE, DR. JAMES P., Woods Hole, Massachusetts. WARREN, DR. HERBERT S., 1405 Greywall Lane, Overbrook Hills, Philadelphia 31, Pennsylvania. WATERMAN, DR. ALLYN J., Department of Biology, Williams College, Williams- town, Massachusetts. REPORT OF THE DIRECTOR 55 WEISS, DR. PAUL A., Department of Zoology, The University of Chicago, Chicago, Illinois. WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pennsylvania. WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, North Dakota. WHITAKER, DR. DOUGLAS M., Stanford University, California. WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pennsylvania. WHITING, DR. ANNA R., University of Pennsylvania, Philadelphia, Pennsylvania. WHITING, DR. PHINEAS W., Zoological Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania. 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. WILBER, DR. C. G., Fordham University, Biological Laboratory, New York, New York. 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. WOODWARD, DR. ALVALYN E., Zoology Department, University of Michigan, Ann Arbor, Michigan. WRINCH, DR. DOROTHY, Smith College, Northampton, Massachusetts. YNTEMA, DR. C. L., Department of Anatomy, Syracuse University Medical College, Syracuse, New York. YOUNG, DR. B. P., Cornell University, Ithaca, New York. YOUNG, DR. D. B., 7128 Hampden Lane, Bethesda, Maryland. ZINN, DR. DONALD J., Rhode Island State College, Kingston, Rhode Island. 9. ASSOCIATES OF THE MARINE BIOLOGICAL LABORATORY ADLER, MRS. CYRUS ALLEN, MR. AND MRS. EUGENE BARTOW, MRS. FRANCIS D. BEHNKE, JOHN A. BROWN, MRS. THEODORE E. CALKINS, MRS. GARY N. CLOWES, MRS. G. H. A. COOPER, MRS. CHARLES F. CROSSLEY, MR. AND MRS. ARCHIBALD CROWELL, PRINCE S. CURTIS, W. D. DRAPER, MRS. MARY C. ELSMITH, MRS. DOROTHY FISHER, MRS. BRUCE CRANE FOSTER, RICHARD W. GARFIELD, I. McD. GREENE, GEORGE GREENE, Miss GLADYS M. HARRISON, Ross G., JR. HUNT, MRS. REID JANNEY, MRS. WALTER C. KEITH, MR. AND MRS. HAROLD C. KlDDER, MR. AND MRS. HENRY M. KIDDER, MRS. JEROME KNOWER, MRS. HENRY LILLIE, MRS. FRANK R. MARVIN, MRS. A. H. MITCHELL, MRS. JAMES McC. MIXTER, MRS. JASON MOORE, MRS. WILLIAM A. 56 MARINE BIOLOGICAL LABORATORY MOTLEY, MRS. THOMAS MURPHY, DR. WALTER J. NEWTON, Miss HELEN NIMS, MRS. E. D. NORMAN; MR. AND MRS. EDWARD OPPENHEIM-ERRER, DR. AND MRS. PAUL PARK, MALCOLM RENTSCHLER, MR. AND MRS. GEORGE RIGGS, MRS. LAWRASON RUDD, MRS. H. W. DWIGHT SAUNDERS, MRS. LAWRENCE SPACKMAN, Miss EMILY S. STEEL, RICHARD STOCKARD, MRS. CHARLES R. STRECHER, MRS. SWOPE, GERARD TEBBETTS, MR. AND MRS. WALTER TRUSLOW, MR. AND MRS. ARTHUR WARD, MR. AND MRS. FRANCIS T. WEBSTER, MRS. EDWIN S. WICK, MRS. MYRON T. WlCKERSHAM, MRS. BERTHA T. WILSON, MRS. EDMUND B. WOLFINSOHN, MRS. WOLFE PROTOPLASMIC VISCOSITY CHANGES DURING MITOSIS IN THE EGG OF CHAETOPTERUS L. V. HEILBRUNN AND W. L. WILSON Department of Zoology, University of Pennsylvania, Philadelphia, and the Marine Biological Laboratory, Woods Hole, Massachusetts x In spite of the enormous effort that has been spent in order to discover the basic cause of cancer, a disease primarily due to the initiation of cell division in cells which normally do not divide, there has been but little advance in the past twenty years in our understanding of the basic physiology of cell division. Of the three main theories which have been proposed to account for the initiation of cell division, only one survives and that has had but little test. The idea that cell divi- sion is caused by an increase in cell permeability can scarcely be held at the present time, for in marine egg cells the calcium ion is a potent agent for promoting mitosis (See Pasteels, 1941 ; Hollingsworth. 1941) and the calcium ion is well known for its effect in decreasing rather than increasing cell permeability. Secondly, at the present time it can hardly be maintained that an increase in respiration is the pri- mary cause of initiation of cell division. When certain types of cells are incited to divide, the respiration does increase, but other types of cells show no such effect, and in still other cells the respiration decreases (Whitaker, 1931a, b, c; 1933a, b; compare also Holter and Zeuthen, 1944). Nor, on the basis of present evidence, can it be held that a particular respiratory system is involved ; at any rate the attempt to argue that in the sea-urchin egg the cytochrome oxidase system is acti- vated when the cell is incited to divide is rather an expression of wishful thinking than of careful experimentation (See Robbie, 1946). We are left then with the third of the three major theories, the view that the primary impetus to cell division is a mitotic gelation akin to the gelation which occurs generally in cells when they respond to stimulation. This colloidal theory of cell division is discussed at some length in the second edition of Heilbrunn's Outline of General Physiology (1943, see Chapt. 42). One of the reasons for believing in the colloidal theory is that in those cells in which viscosity studies have been made, the appearance of the mitotic spindle appears to be preceded by a very definite gelation of the cytoplasm. Actually, how- ever, only a few types of cells have been studied, and if this point is to be firmly established, we should have additional information for other types of cells. We were led to a study of the egg of the annelid Chaetopterus pergamentaceus Cuvier, because this worm is found in suitable numbers at Woods Hole and can be obtained for study, and also because the egg represents a type similar to Cumingia in that fertilization occurs at the time of the first maturation spindle. Unfor- tunately, at the present time Cumingia is very rare at Woods Hole and therefore can not be used for experimental work. 1 The research on which this paper is based was aided by a grant from the United States Public Health Service. 57 58 L. V. HEILBRUNN AND W. L. WILSON Chaetopterus eggs can be obtained throughout the summer (Compare Mead, 1898). One female worm provides enough eggs for several experiments. When the eggs are shed into the sea water, they are in the germinal vesicle stage, but as soon as they enter the sea water, maturation begins and proceeds until the meta- phase stage of the first maturation division is reached. Then, if the egg is fertilized, the maturation divisions continue and cleavage follows. Obviously, the Chaetopterus egg is convenient for study. We plan to use it in various types of experimental work. We were interested therefore in knowing the normal cycle of viscosity change and how this cycle was related to the mitotic phenomena occurring between the time of fertilization and the first cleavage. Strangely enough, in spite of the great amount of cytological work on mitosis, there is no complete minute by minute time record of what can be seen in fixed and sectioned material during the progressive stages of mitosis in marine eggs. METHODS The sexes of Chaetopterus can be determined from the color of the gametes contained in the transparent parapodia. The eggs are yellow and the sperm are white. A few posterior segments of a worm were cut off and placed in a small stender dish containing about 20 ml. of sea water. The parapodia of these excised segments were cut open and as a result the eggs or the sperm, as the case might be, exuded into the sea water. Eggs were filtered through cheesecloth to remove the excess jelly and extraneous tissue and then were washed by decantation. The eggs are usually so abundant that only a few segments of the worm provide enough eggs for a single experiment. A sperm suspension was prepared by removing the parapodia and segments from the stender dish and then adding to the original 20 ml. another 10 ml. of sea water. Two or three drops of this suspension were used to fertilize one batch of eggs. A worm does not die following the removal of several segments and indeed the same worm may be used several times. In all of our work, the eggs of only one female were used in any given series of experiments. The eggs were kept in a water bath maintained at a constant tem- perature of 21° C. In some experiments, the temperature varied slightly, but ordinarily the variation was not greater than two or three tenths of a degree. As soon as the eggs began to show indications of cleavage, counts were made as rapidly as possible in order to determine with reasonable precision the exact time at which 50 per cent of the eggs had divided. In these counts, all eggs in which a cleavage plane had begun to travel across the egg were regarded as cleaved. Actually, the passage of the cleavage plane through the egg takes an appreciable time, perhaps a minute or more, so that the cleavage times we recorded are somewhat less than the times would be if we considered as cleavage time the time at which the egg is completely divided. In making rapid counts on living eggs, it would scarcely be possible to use as a criterion of cleavage the complete division of the egg. At a tem- perature of 21° C. we found the average cleavage time to be 56 minutes. Protoplasmic viscosity tests were made with an Emerson hand centrifuge. The handle of the centrifuge was turned once every two seconds. This represents a speed of 85 revolutions per second. The radius of turn was 8 cm. The cen- trifugal force was calculated to be 2325 times gravity. A few preliminary tests of protoplasmic viscosity during mitosis showed that the viscosity changes in the VISCOSITY CHANGES DURING MITOSIS 59 dividing Chaetopterus egg are not as pronounced as they are in the egg of Cumingia or in the egg of Arbacia. If tests are to be made at frequent intervals, observations must be made Tapidly. This can introduce uncertainty. We were worried over the possibility that subjective impressions might creep in. Accordingly, we decided on the following procedure. For any given test, the centrifuge was turned by Mrs. Jean Wilson. As soon as the turning was completed, she passed the centrifuge tube to one of us as quickly as possible. The eggs were then removed from the tube to two microscope slides. Each of us had a microscope, and we observed the centrifuged eggs independently. In this way, we were able to make tests at one minute intervals and although the observations were necessarily very rapid, when we compared our results at the end of a series of tests, we found remarkably good agreement. When a. Chaetopterus egg is centrifuged, lighter (presumably fat) granules move to the centripetal pole, and heavier yolk granules move to the centrifugal pole. There is a cortical layer of granules which does not move at all. Details of the appearance of centrifuged eggs are given by Lillie (1906). When we observed the centrifuged eggs, we recorded everything that we could see. If the viscosity is relatively low, the yolk granules move more readily through the egg ; the fat granules also move more readily. It is possible to observe a shift of the yolk granules before any movement of the fat granules can be noted. One can follow viscosity change either by considering the movement of heavy or light granules. In order to obtain definite quantitative values for viscosity, we chose as an endpoint the appearance of a definite accumulation of fat granules at the centripetal pole of the egg. The number of seconds required to give this accumulation was used in plotting a viscosity curve. The eggs of Chaetopterus are not very transparent. We wanted to know exactly what was happening in the mitotic process during every minute between fertilization and cleavage. This necessitated preparation of sections. Eggs were fixed at one minute intervals either in Benin's fluid or in Meves' fluid. We fixed four complete series of eggs, and all four series were imbedded in paraffin. Sec- tions were made from only one series (Bouin), the others being kept in reserve. Sectioning and staining were done by Miss Drusilla Van Hoesen. Our sections are eight microns thick and they are stained with Heidenhain's hematoxylin. RESULTS The viscosity changes in the protoplasm of the Chaetopterus egg during the period between fertilization and cleavage are shown in Figure 1. The viscosity figures represent relative values and they have no absolute basis other than that they represent the number of seconds of centrifugation necessary to arrive at the endpoint described in the previous section. In order to obtain the viscosity values, after some preliminary tests, we ran through 12 complete series, in each of which the eggs were centrifuged at minute intervals for a given length of time. The times of centrifuging in the various tests were 4, 5, 7,9, 10, 11, 12, 13, 14, 15, 17. and 18 seconds. Fertilization is followed by a drop in viscosity ; then while the maturation divi- sions are proceeding, the viscosity remains constant. In one or two of our series, we did get some indication that during the course of the maturation divisions, there 60 L. V. HEILBRUNN AND W. L. WILSON might be minor fluctuations in viscosity, but the weight of evidence is against any change whatsoever. This may seem strange, for in the egg of the clam Cumingia one of us had noted marked changes in viscosity during the maturation divisions (Heilbrunn, 1921). The difference between the egg of Chaetopterus and that of Cumingia is, however, easy to understand. In the relatively small egg of Cumingia, the maturation spindles occupy a rather large fraction of the egg volume. Thus Morris (1917) states that the first polar spindle "is large, and lies near the center of the egg. It might, in fact, be mistaken for a cleavage spindle in the metaphase, if it were not for the form of the chromosomes." Similarly, Jordan (1910) shows the first maturation spindle of Cumingia as a large structure extending through most of the egg; the distance between the centers of the centrospheres of this spindle is approximately half the diameter of the egg. On the other hand, the 15 >- 12 S9 to >' 6 I I I I I I 5 10 15 20 25 30 35 40 45 50 55 MINUTES AFTER FERTILIZATION FIGURE 1. Protoplasmic viscosity changes in the egg of Chaetopterus during the time between fertilization and first cleavage. maturation spindles of the Chaetopterus egg are relatively small. Thus, Lillie (1906) shows the fully formed maturation spindle of the Chaetopterus egg as small. In his Figures 3 and 4, the. distance between the centers of its two centrospheres is only one-sixth of the diameter of the egg (Compare also Mead, 1898). Lillie (1906) showed that the cortex of the Chaetopterus egg was relatively viscous, con- taining granules which did not move when the egg was subjected to reasonably strong centrifugal force. We conceive, therefore, of the maturation spindles of the Chaetopterus egg as being relatively small bodies only several times as long as the cortical layer is thick and extending only a relatively short distance into the fluid region of the egg. Following the maturation divisions, the protoplasm undergoes a sharp increase in viscosity. Our curve shows it to be approximately a two-fold increase. Some of our data indicated a somewhat greater change, but we preferred a conservative estimate. The viscosity increase is followed by a decrease in viscosity, and then VISCOSITY CHANGES DURING MITOSIS 61 just before cleavage, the viscosity increases sharply again. These major changes in protoplasmic viscosity are related to the process of mitosis. In earlier work on the eggs of Cumingia and Arbacia, it was found that "the appearance of a spindle is preceded by an increase in viscosity and followed by a decrease in viscosity." It is of interest now to inquire as to whether the same correlations exist for the Chaetopterus egg. Because of the fact that the Chaetopterus egg is one of the few invertebrate eggs that can conveniently be studied at Woods Hole, and because also of the present great interest in cell division, it was thought worth while to establish a complete time record of the mitotic changes in this egg as they occur during the interval between fertilization and first cleavage. One difficulty in presenting such a time record is the uncertainty of terminology. Mitosis is a continuous process and the various stages of this process can not be sharply delimited from each other. Moreover, not all authorities on mitosis agree in the way they define the stages. And even if a definition is rather uniformly followed, it is not always easy to apply it in such a way as to give a clear-cut decision as to when one stage ends and an- other begins. Thus, the telophase may be defined as the stage in which the chro- mosomes reach the poles of the spindle and begin to transform into vesicles or other structures characteristic of the resting stage. Now on a time basis, these two processes may not be simultaneous, and if one seeks to make sharp time distinc- tions, one must choose either the one or the other. Furthermore, the situation is complicated by the rapid succession of mitoses in an egg cell. Thus there may be no resting stage at all between two successive divisions, and there may not even be a complete telophase between the first and second maturation divisions. For us it seemed wisest, if we were to present our results in tabular form, to make arbitrary criteria and distinctions. For our purposes, we shall define the metaphase as the stage in which the chromosomes are arranged along the equatorial plate of the spindle. We regard the anaphase as beginning as soon as the chro- mosomes have divided sufficiently so that we can see a space between the two groups of chromosomes. During anaphase, the two sets of chromosomes migrate toward the poles. It is hard to tell exactly when they have reached the poles. Accord- ingly, for this material, we chose as the distinction between anaphase and telophase, the moment when at least some of the chromosomes show signs of vesiculation. Actually, this distinction may depend to a slight extent on the depth to which the sections have been stained, but the difference between lightly and darkly stained sections does not appear to be great. On the basis of these distinctions, it was usually not too hard for us to tell when the egg cells were in metaphase, anaphase, or telophase. Prophases were more difficult. At the end of the first maturation division, after the first polar body has been separated off, there is an intermediate series of stages which are hard to classify. The polar body is given off 9 minutes after fertilization. At 10, 11, and 12 minutes after fertilization, one typically sees remnants of the first maturation spindle. At these times, the chromosomes are not vesiculated, so that according to our previous definition, it is not proper to call this stage a telophase. We might refer to it as an interphase, but we prefer to con- sider it as a late anaphase. At 13 minutes after fertilization, many half spindles appear in the sections. Following this is a stage in which the second maturation spindle appears ; typically it lies in a plane perpendicular to the radius of the egg. 62 L. V. HEILBRUNN AND W. L. WILSON The chromosomes are frequently scattered along this second maturation spindle. This stage we refer to as the prophase of the second maturation spindle. The spindle then turns so that at metaphase it is perpendicular to the surface of the egg. The second polar body is given off at 23 minutes after fertilization. The egg chromosomes now go into a very definite telophase stage and form discrete vacuoles. The egg nucleus is irregular in shape and may look like a bunch of grapes. At this stage, the male pronucleus, in sympathy as it were, may also become lobulated. There is thus a very definite telophase stage (at least in so far as the egg pro- nucleus is concerned). But between this telophase stage and the late prophase stage of the cleavage mitosis, it is hard to find distinctions which can be used for the purposes of our time scale. The male and the female pronuclei approach each other. There is thus a stage in which the pronuclei are separate and a stage when they are apposed. Usually, by the time they are apposed, the vesicular lobate appearance of the female pronucleus has disappeared so that this nucleus is now a smoothly spherical body with its chromatin either in a resting stage or in a condi- tion indicative of an early prophase. There are exceptional cases in which the female pronucleus preserves its telophase appearance even though it is close to the male pronucleus. Then comes a stage in which the two pronuclei are fused together, or at least are apparently fused together. Mead (1898) says that an actual fusion of the pronuclei does not occur, but in many instances we were not able to detect a line of demarcation between the two pronuclei ; and indeed in Mead's Figure 40, such a demarcation line is questionable. Accordingly, we dis- tinguished a fusion nucleus stage. During this stage, the condition of the nucleus is almost certainly what most authors would call early prophase. There then comes a stage in which the nuclear membrane gradually breaks down and the chromosomes are arranged along the developing mitotic spindle ; this stage is called late prophase. In our time schedule, therefore, we distinguished the following stages: 1st maturation metaphase, 1st maturation anaphase, 2nd maturation prophase, 2nd maturation metaphase, 2nd maturation anaphase, 2nd maturation telophase, pro- nuclei separate, pronuclei apposed, fusion nucleus, late prophase of cleavage mitosis, metaphase, anaphase, and telophase (of cleavage mitosis). These stages are ab- breviated in the headings of the table which gives a record of our findings. The data for the table were collected from a study of the slides prepared as described previously. For each minute of the time between fertilization and cleavage, we counted 20 cases in which the condition seemed clear in terms of one of the above- mentioned categories. The work was shared, and each of us made ten counts for each minute. On comparing our results, we found essential agreement. The stages listed in our table do not give information on one point of consid- erable importance. They do not indicate at what moment the cleavage spindle first makes its appearance. After careful study of the sectioned material we have decided that the following series of events occurs. At 30 and 31 minutes after fertilization, the two pronuclei begin to come close to each other. There is at this time a large sperm aster with a large centrosphere. As the two pronuclei come still closer to each other (at 32 and 33 minutes after fertilization), between them they squeeze the centrosphere of the sperm aster into an elongated shape, so that it may form a narrow band between the two. This is the stage illustrated in Figure 39 of Mead (1898). This elongated centrosphere, with its astral rays VISCOSITY CHANGES DURING MITOSIS 63 divided into two groups stretching well out into the cytoplasm, is not the definitive mitotic spindle, as subsequent stages indicate. Nevertheless, the line connecting the two sets of astral rays and the line along which the pronuclei fuse is almost always in the direction of the future spindle, for this line is typically perpendicular TABLE I Mitotic stages in the Chaetopterns egg as a function of time (minutes) at 21° C. Further explanation in text Time 1st M 1st A 2nd P 2nd M 2nd A 2nd T PNS PNA FN LP M A T 0 20 1 20 2 20 3 20 4 20 5 20 6 19 1 7 12 8 8 3 17 9 20* 10 20 11 20 12 19 1 13 15 5 14 8 8 4 15 9 11 16 3 17 17 20 18 19 1 19 16 4 20 13 7 21 6 14 22 1 19 23 20* 24 18 2 25 8 12 26 20 27 18 2 28 15 5 29 14 4 2 * Indicates time of appearance of 1st and 2nd polar bodies. 64 L. V. HEILBRUNN AND W. L. WILSON TABLE I — Continued Time 1st M 1st A 2nd P 2nd M 2nd A 2nd T PNS PNA FN LP M A T 30 8 9 3 31 2 7 11 32 1 5 14 33 1 19 34 20 35 20 36 9 11 37 8 12 38 3 17 39 4 16 40 13 7 41 5 15 42 4 15 1 43 16 4 44 15 5 45 3 17 46 19 1 47 20 48 18 2 49 16 4 50 3 17 51 20 52 20 53 16 4 54 6 14 55 1 19 56 20 to the egg axis as indicated by the position of the polar bodies. This is also shown in Mead's Figure 39. Although at 32 and 33 minutes after fertilization, the astral rays are well developed, subsequently they seem to fade, so that 35 minutes after fertilization the asters either do not appear at all, or if present, they are faint. At this time there is no spindle. In the next three or four minutes, one occasionally sees instances of a double aster at one side of the fusion nucleus with what is apparently an embryonic spindle being stretched out between the two asters. Whether this is a general condition or not, only further study can decide. On one point we are certain, the definitive mitotic spindle does not appear until approximately 40 minutes after fertilization. In our study of eggs fixed at 40 VISCOSITY CHANGES DURING MITOSIS 65 minutes after fertilization, we found 15 out of 20 with the fusion nucleus elongated and pointed at its ends. In the pointed ends of these nuclei, spindle fibers show in 7 out of the 15 cases. Thus 7 out of 20 cells showed a true spindle. Probably this is somewhat less than the true proportion, for a nucleus might well be cut so that spindle fibers though present would not be visible. As far as our observations go, they indicate rather clearly that the moment at which the definitive mitotic spindle appears is 40 minutes after fertilization. Let us now attempt to correlate our viscosity curve with the mitotic changes as shown by our observations of the fixed material. The viscosity curve shows a minimum of viscosity from 44 minutes after fertilization to 50 minutes after fertili- zation. This is almost exactly the time during which the cell is in metaphase, for the table shows the metaphase period to extend from 45 to 49 minutes after fertili- zation. We have chosen as the moment at which the definitive mitotic spindle appears as 40 minutes after fertilization. This is essentially simultaneous with the moment at which the viscosity of the protoplasm begins to drop. We conclude therefore that the appearance of the cleavage spindle in the Chaetopterus egg is preceded by a period in which the protoplasm is relatively viscous. As soon as the spindle is formed, the viscosity drops. The metaphase is the stage at which the viscosity of the protoplasm is at a minimum. Finally, we should like to express our admiration of the cytological study made by Mead in 1898. In general we confirm his findings. There are one or two minor points in which we might differ. In his Figure 46, which represents what we would call an anaphase, he shows some bodies in the equatorial plane of the spindle; these he calls nucleoli. Lillie (1906) in his Figure 25 illlustrates similar bodies which he labels as "chromatin masses cut off from the chromosomes." We have frequently seen these bodies in the equatorial plane of the spindle during the anaphase. However, our sections seem to indicate that they are neither nucleoli or chromosome fragments, but rather cytoplasmic granules which have pushed their way into the equatorial plane of the spindle. If this is correct, it is an observation which may have some importance in the interpretation of the mitotic spindle, but we made no careful study of the phenomenon. We should merely like to call the attention of the cytologists to it. DISCUSSION Our results provide a suitable basis for further work on the protoplasm of the dividing Chaetopterus egg, and we hope in the future to study the action of radia- tion and other agents in terms of their effect on the protoplasmic viscosity. The viscosity curve that we have plotted for the Chaetopterus egg is essentially the same as that reported earlier for Arbacia and Cumingia. Heilbrunn (1921) stated that "The viscosity changes in Arbacia and Cumingia are absolutely parallel. In each case the appearance of a spindle is preceded by an increase in viscosity and followed by a decrease 'in viscosity. And in both eggs division of the cell is immediately preceded by a viscosity increase." As a matter of fact, all authors who have made objective measurements of protoplasmic viscosity during mitosis are in substantial agreement. A survey of much of the literature is given by Carlson (1946). 66 L. V. HEILBRUNN AND W. L. WILSON If one excludes the work done by subjective and non-quantitative methods, there is only one discordant paper. Fry and Parks in 1934 published what we believe to be a masterpiece of distortion. They made a few centrifuge measure- ments and then used Heilbrunn's data in plotting their curves, stating that Heil- brunn's measurements were more complete and accurate than their own. After doing this, they insist in a final discussion that Heilbrunn is wrong. They reach this strange conclusion by misquoting and distorting the views not only of Heil- brunn but also of almost every other worker in the field. Actually, though Fry and Parks claimed to have copied Heilbrunn's curves, this is not exactly true. The rise in viscosity which Heilbrunn found to occur in the Arbacia egg ten minutes after fertilization (for a cleavage time of 50 minutes) is shifted by Fry and Parks so that it occurs approximately seven or eight minutes after fertilization (for a cleavage time of 67 minutes). Thus in Heilbrunn's curve, viscosity rises only after one-fifth of the time between fertilization and cleavage has elapsed, whereas in the curve stated by Fry and Parks to be a copy of Heilbrunn's curve, the rise occurs when about one-ninth of the time between cleavage and fertilization has elapsed. Needless to say, this shift favors the interpretation Fry and Parks endorse. Moreover, the final upsweep of the 'Arbacia curve is shifted so as to make the metaphase of division come in a period of high rather than low viscosity. Fry and Parks claim to find agreement between their curves, which they state to be Heilbrunn's curves, and Chambers' opinions on viscosity change during mitosis, opinions based on subjective microdissection studies of various species of eggs at uncertain times. This they do in order to make Heilbrunn's curves fit what Fry and Parks regard as Chambers' opinions. In their Chart 5, Fry and Parks credit Chambers with maintaining that the metaphase is a stage during which the proto- plasm is fluid. But this is the exact opposite of what Chambers says. Thus Chambers (1919) states: "The time of appearance of the amphiaster until comple- tion of cleavage lasts from 10 to 15 minutes. The increased viscosity of the egg during the amphiaster stage could be more easily demonstrated by the needle in the eggs of Echinarachnius and Cerebratulus than in those of Arbacia." The facts of the case are as we have stated them, and no amount of distortion can hide the fact that the appearance of the mitotic spindle is preceded by a stage of high viscosity and followed by a stage of low viscosity. Heilbrunn (1921) suggested that "it is as though the spindle were coagulated out of the protoplasm." Recent work has indicated that in some types of proteins, gelation may result in the formation of a spindle-shaped structure called a tactoid (Bernal and Fankuchen, 1941). Bernal (1940) believes that the spindle is actually a tactoid. Perhaps there are other correlations that may be made between changes in the protoplasm and the series of viscosity changes that we have described. For one thing, the stage of increasing viscosity occurs at a time when water is being taken from the cytoplasm by the enlarging pronuclei. Then, when the spindle appears, the nuclear membrane breaks down and this might involve an increase in the water content of the cytoplasm. Carlson (1946) suggests that changes in the viscosity of protoplasm during mitosis may be due to changes in the nucleic acid content of the cytoplasm. He thinks that a high content of nucleic acid in the cytoplasm would tend to produce a high viscosity. Carlson states that Brachet and also Painter found the cytoplasmic nucleic acids abundant in early prophase, less abun- dant or entirely absent from late prophase through anaphase, and increasing in VISCOSITY CHANGES DURING MITOSIS 67 amount following division ; but in the papers cited by Carlson it is not possible for us to find any data on the changes in nucleic acid content of the cytoplasm during various stages of mitosis. That there is an exact correlation between the amount of cytoplasmic nucleic acid and the protoplasmic viscosity is very doubtful, and certainly it has not in any sense been established. For one thing, the unfertilized sea-urchin egg is apparently rich in cytoplasmic nucleic acids (see, for example, Caspersson and Schultz, 1940), and yet this unfertilized egg has a low protoplasmic viscosity (Heilbrunn, 1920). SUMMARY 1. The viscosity of Chaetopterus egg protoplasm was determined at one minute intervals during the period between fertilization and cleavage. 2. By studying fixed, sectioned and stained material, the course of the mitotic processes in the Chaetopterus egg was followed minute by minute. 3. During the cleavage mitosis, marked changes in protoplasmic viscosity occur, and these are similar to the changes already described for the eggs of Arbacia and Cumingia. 4. The appearance of the mitotic spindle is preceded by an increase in proto- plasmic viscosity and is followed by a decrease in protoplasmic viscosity. 5. During the metaphase, the protoplasmic viscosity is low. 6. Just before the cell divides, the protoplasmic viscosity increases markedly. LITERATURE CITED BERNAL, J. D., 1940. Structural units in cellular physiology. The cell and protoplasm. Pub- lication of the American Association for the Advancement of Science, No. 14, pp. 199-205. BERNAL, J. D., AND I. FANKUCHEN, 1941. X-ray and crystallographic studies of plant virus preparations. Jour. Gen. PhysioL, 25: 111-146, 147-165. CARLSON, J. GORDON, 1946. Protoplasmic viscosity changes in different regions of the grass- hopper neuroblast during mitosis. Biol. Bull., 90: 109-121. CASPERSSON, T., AND JACK SCHULTZ, 1940. Ribonucleic acids in both nucleus and cytoplasm. and the function of the nucleolus. Proc. Nat. Acad. Sd., 26: 507-515. CHAMBERS, R., 1919. Changes in protoplasmic consistency and their relation to cell division. Jour. Gen. PhysioL, 2 : 49-68. FRY, HENRY J., AND MARK E. PARKS, 1934. Studies of the mitotic figure. IV. Mitotic changes and viscosity changes in eggs of Arbacia, Cumingia, and Nereis. Protoplasma, 21 : 473-499. HEILBRUNN, L. V., 1920. An experimental study of cell division. I. The physical conditions which determine the appearance of the spindle in sea-urchin eggs. Jour. E.rp. Zool., 30: 211-237. HEILBRUNN, L. V., 1921. Protoplasmic viscosity changes during mitosis. Jour. Exp. Zool., 34: 417-447. HEILBRUNN, L. V., 1943. An outline of general physiology. 2nd Ed., W. B. Saunders Co., Phila. HOLLINGSWORTH, J., 1941. Activation of Cumingia and Arbacia eggs by bivalent cations. Biol. Bull.. 81 : 261-276. HOLTER, H., AND E. ZEUTHEN, 1944. The respiration of the egg and embryo of the ascidian, Ciona intestinalis L. Compt.-rend. Lab. Carlsberg, ser. chim., 25 : 33-65. JORDAN, H. E., 1910. A cytological study of the egg of Cumingia with special reference to the history of the chromosomes and the centrosome. Arch. f. Zellf., 4 : 243-253. LILLIE, F. R., 1906. Observations and experiments concerning the elementary phenomena of embryonic development in Chaetopterus. Jour. E.rp. Zool., 3: 153-268. 68 L. V. HEILBRUNN AND W. L. WILSON MEAD, A. D., 1898. The origin and behavior of the centrosomes in the annelid egg. J 'our. of Morph., 14: 181-218. MORRIS, M., 1917. A cytological study of artificial parthenogenesis in Cumingia. Jour. Exp. Zool, 22 : 1-52. PASTEELS, J., 1941. Sur quelques particularites de 1'activation de 1'oeuf d'oursin (Psammechinus miliaris). Bull. Cl. Sc. Acad. roy. Belg. 5e serie, 27: 123-129. ROBBIE, W. A., 1946. The effect of cyanide on the oxygen consumption and cleavage of the sea urchin egg. Jour. Cell, and Comp. Physiol., 28 : 305-324. WHITAKER, D. M., 1931a. On the rate of oxygen consumption by fertilized and unfertilized eggs. I. Fucus vesiculosus. Jour. Gen. Physiol., 15 : 167-182. WHITAKER, D. M., 1931b. On the rate of oxygen consumption by fertilized and unfertilized eggs. II. Cumingia tellinoides. Jour. Gen. Physiol., 15 : 183-190. WHITAKER, D. M., 1931c. On the rate of oxygen consumption by fertilized and unfertilized eggs. III. Nereis limbata. Jour. Gen. Physiol, 15: 191-200. WHITAKER, D. M., 1933a. On the rate of oxygen consumption by fertilized and unfertilized eggs. IV. Chaetopterus, and Arbacia punctulata. Jour. Gen. Physiol., 16 : 475-495. WHITAKER, D. M., 1933b. On the rate of oxygen consumption by fertilized and unfertilized eggs. V. Comparisons and interpretations. Jour. Gen. Physiol., 16 : 497-528. INHIBITION OF FERTILIZATION IN ARBACIA BY BLOOD EXTRACTS WILLIS E. PEQUEGNAT Marine Biological Laboratory, Woods Hole, Massachusetts, and Department of Zoology, Pomona College, Claremont, California From the time of publication of F. R. Lillie's paper (1914) on fertilization in Arbacia, some embryologists have maintained that the serum of Arbacia blood * provided an effective block to fertilization in this and a few other marine inverte- brates. Lillie formulated the hypothesis that fertilization in Arbacia was actuated through the conjoining of certain constituents of egg and sperm by a substance called fertilizin, the presence of which in solution could be detected by the agglu- tinating action which it exerted upon sperm in aqueous suspension (see Tyler, 1948. for recent review of the subject). Asserting that filtered blood of Arbacia was capable of inhibiting fertilization while it did not prevent fertilizin from agglu- tinating sperm, Lillie linked this inhibitory action into his conception of the mecha- nism of fertilization by postulating that the serum-inhibitor prevented the uniting of fertilizin with the necessary constituent of the egg. Oshima (1921) published the results of a few experiments which had motivated him to suggest that an external ("dermal") secretion was responsible for the inhi- bition observed by Lillie. That Oshima was not prepared to enter a complete denial of Lillie's conclusions is evidenced by his admitting that filtered blood was capable of exhibiting a weak though unpredictable inhibitory influence upon the fertilizabil- ity of the egg. Interestingly enough the degree of inhibitory action considered weak by Oshima fell within the range certainly considered significant by Lillie. Furthermore, it is worthy of note that Lillie was not able to offer a satisfactory explanation of the fact that the potency of undiluted blood samples displayed de- grees of inhibitory effectiveness varying from zero to one hundred per cent. None- theless, largely through the influence of E. E. Just, little or no attention was paid to Oshima's suggestions by the majority of interested embryologists, except, per- haps, for Harvey (1939). Apparently critical data confirming Lillie's conclusions were brought forth by Just (1922), who, at the same time, brushed aside Oshima's contraindications without any statement that he had attempted to repeat the latter's experiments. Also. Just stated that the most plausible explanation of Oshima's results would depend upon the presence of excretory or defecatory wastes in his solutions. The matter rested at this point until the summer of 1946 when, at the sugges- tion of Dr. Albert Tyler, Richard L. Murtland, Albert H. Banner, and the present 1 It has been convenient to use the word blood as a synonym of the term perivisceral fluid, even though strict interpretation may not warrant the practice. For present purposes the words serum and plasma are considered as literal equivalents when applied to Arbacia, since in this organism the clot is believed to be purely of cellular composition. Since Lillie and Just had previously used serum to denote the material obtained from whole blood by clotting, filtering, or centrifugation, I have followed this choice entirely for the virtue of consistency. 69 70 WILLIS PEQUEGNAT author - collaborated briefly in repeating a few of Lillie's experiments. Becoming interested in the mechanism of this inhibitory action, I carried on alone during the latter part of the summer of 1946 and returned to Woods Hole in the summer of 1947 to proceed with the same problem. Although at the time I was unaware of Oshima's publication, I undertook to verify Lillie's observations before proceeding to a study of the modus operandi of the inhibitor. Following Lillie's techniques as closely as possible, I obtained results which corroborated his. Thus, I was convinced that his conclusion to the effect that Arbacia serum contained a factor capable of inhibiting fertilization was valid. But when, during the summer of 1947, I introduced techniques of collecting blood designed to yield uncontaminated samples, I obtained results which revealed that Lillie's original description of the source of the inhibitor must be modified. During the course of this study, it was found that serum samples removed by syringe so that they were uncontaminated with drainage from the exterior of the test did not possess inhibitory activity. Furthermore, it was found that sea water extracts from the tests of intact Arbacia were not capable of inhibiting fertilization. Thus, it became evident that some step in Lillie's technique, which I had followed pre- viously in obtaining corroboratory results, was responsible for the appearance of the inhibitor in the serum samples. Chosen as the most likely cause was the fact that prior to opening the perivisceral cavity both Lillie and Just rinsed their urchins in tap water, presumably to kill any sperm present on the test which would other- wise fertilize samples of eggs. I had noticed that the application of tap water, even when followed by a sea water rinse within a few seconds, caused a yellow substance to appear in the excess water draining from the test of the as yet intact animal. A detailed study of this phenomenon revealed that this yellow exudate was capable of inhibiting fertilization. Additional experiments revealed that the immediate source of this inhibitor was to be found in certain granules or cells located in the tube feet and a few other organs. And, contrary to the findings of Lillie and Just, the ultimate source of the inhibitor was found to be some of the blood cells found in the perivisceral fluid. The present paper gives the details of these experiments. METHOD The sea-urchin Arbacia punctulata was the principal animal used in these experiments. Perivisceral fluid was removed from Arbacia by methods designed both to permit contamination from the outside and by methods devised to prevent such contamination. In addition, various techniques were devised which might supply information relative to the ultimate source of the inhibitor. Also, one significant step not used by previous workers was added to the routine handling of all samples. Having noted that the pH of sea-urchin blood was lower than that of sea water, and since this in itself may interfere with fertilization (Tyler and Scheer, 1937), it was decided that the pH of all samples should be adjusted to that of sea water. Moreover, in order to obviate any modification of results arising from undue concentrations of egg or sperm secretions, all samples containing gametes were discarded. In most experiments one drop each of eggs and sperms were introduced into 2 cc. of fluid, be it sea water or extract, in Syracuse dishes. In each instance the - Three members of the 1946 Embryology Class of the Marine Biological Laboratory. INHIBITION OF FERTILIZATION 71 eggs from a single female were used for each series of experiments. The average concentration of eggs in suspension was found by actual count to be between 2200 and 2500 eggs per drop. Fresh sperm suspensions were used for fertilizations and were made by diluting one drop of dry sperm with the equivalent of 99 drops of sea water. The various fluids were tested in serial two-fold dilutions of 2 cc. down to 64-fold. Determinations of the percentage of fertilization were made by actual counts under low magnification from three to five hours after insemination. The practice of first scanning the dish and then counting between four and five hundred eggs along two diameters was followed consistently. Most of the experiments with differently prepared fluids were run simultane- ously, as is indicated by similar dates in the tables. EXPERIMENTS Series I. This experiment was carried out essentially as outlined by Lillie and Just, as follows: (1) Arbacia rinsed in tap water for a few seconds, shaken and rinsed in filtered sea water; (2) animals permitted to drain, cut made with scissors around peristome, and fluid drained into Syracuse dish; (3) clot permitted to form, checked for presence of gametes, then filtered; and finally (4) samples were centrifuged lightly and the pH adjusted to that of sea water. The fluid was then used undiluted, or diluted as described above. In each instance the fluid obtained in this way had a yellowish tinge. This series of experiments, involving a total of 42 animals, was repeated seven times between July 8 and August 2, 1947. From the results tabulated in the left half of Table I it can be observed that this solution was effective in blocking fertili- zation when used undiluted. An average of approximately 3 per cent fertilizations was obtained in undiluted fluids as compared to nearly 100 per cent fertilizations of eggs in sea water controls. A summary of part of Lillie's work (1914), in which undiluted fluid collected from 50 Arbacia in the same manner and within the above dates was used, gives an average of 50 per cent fertilizations, as compared to 97 per cent in the sea water controls. The apartness of our results can be explained in part by the fact that his samples were used individually, while in my experiments fluid from all individuals was pooled before being tested. For exam- ple, Lillie's data show that the serum obtained by him from one individual con- tained no inhibitor, while the serum from another contained enough to inhibit all eggs tested. This would yield an average of 50 per cent inhibition. On the other hand, if these two samples had been pooled before being tested, it is possible that sufficient inhibitor would be present in the mixture to give complete inhibition. A comparison of the effects of diluting the serum show this conjecture to be valid. Thus, the percentage of fertilization increased when the fluid collected on August 2 was diluted, as follows : Percentage serum (in sea water) 100 50 12.5 6.2 Percentage fertilization 0 16 68 99 100 In this experiment I obtained an average of 32 per cent inhibition with a 25 per cent solution of serum, and Lillie's data show that he obtained 30 per cent inhibi- tion when using a 20 per cent solution of serum obtained in the same manner. 72 WILLIS PEQUEGNAT Series II. The tap water rinse was eliminated in this experiment; otherwise all procedures were the same as those outlined in Series I. Again, fluid from a total of 42 animals was tested on seven occasions between July 10 and August 3, 1947. The fluid in each case was clear, not yellow. The results, as tabulated in the right half of Table I, offer a marked contrast to those of Series I. There is no significant difference between the percentages of fertilizations obtained from eggs inseminated in undiluted serum and those in- seminated in sea water (both yielding approximately 99 per cent fertilizations). Hence it became apparent that the application of tap water was linked in some manner with the appearance of the inhibitor. Additional experiments were per- formed in order to determine whether it was being liberated into the serum from within the animal or from the outside. TABLE I Serum obtained by cutting peristome With tap water rinse (Series I) Without tap water rinse (Series II) Percentage fertilizations Percentage fertilizations July pH Adjusted pH July pH Adjusted PH Serum (100%) Sea water Serum (100%) Sea water 8 7.5 8.0 0 100 10 7.6 8.0 100 100 10 7.4 8.0 0.5 100 12 7.6 7.9 99 99 12 7.6 8.0 0 99 14 7.7 8.0 100 100 14 7.6 8.0 0 100 21 7.5 7.9 100 100 26 7.2 8.0 10 100 26 7.5 8.0 95 100 27 7.5 8.0 5 99 Aug. Aug. 2 7.6 8.0 100 100 2 7.5 8.0 0 100 3 7.0 8.0 100 100 Scries III. In order to circumvent contamination of serum with external drainage, the fluid was withdrawn by means of a 10 cc. syringe equipped with a No. 22 gauge hypodermic needle. The needle was introduced into the perivisceral cavity through the peristome about 3 mm. from Aristotle's lantern. Care had to be exercised to keep the needle from penetrating the gonads and to prevent the plunger from crushing blood cells when fluid was expelled from the syringe. When the proper depth of penetration had been determined, the needle was ensheathed with rubber tubing long enough to stop it at the desired level. All animals used in this experiment were rinsed momentarily in tap water and then in sea water prior to withdrawing the fluid. Subsequently, the fluid was prepared and used exactly as described previously. The fluid in all cases was clear, not yellow. Eight experiments were performed with serum collected in this manner be- tween July 8 and August 3. The data from this series are tabulated in Table II. An average of 99.1 per cent fertilizations was obtained from eggs fertilized in all undiluted samples of this serum. This evidence, when coupled with the results of Series I, indicated that the inhibitor evoked by tap water drained into the peri- visceral fluid from the outside when the latter was collected by cutting the peri- stome. Moreover, there seems little reason for doubting that these facts explain INHIBITION OF FERTILIZATION 73 the large range of variation in potency of inhibitor recorded both by Lillie and Just, because the amount of drainage in their samples would have varied inversely with the time elapsing between rinsing the animals and withdrawing the fluid, and directly with the time required to drain each animal. Thus far the following facts have been ascertained : ( 1 ) that the inhibitor of fertilization is not found in the serum of the intact Arbacia; (2) that tap water causes the inhibitor to appear in samples of serum collected by the method of Lillie and Just (Series I) ; and (3) that the inhibitor so evoked comes from the outside of the animal. It is important to note that when inhibition has been observed up to this point perivisceral fluid plus external drainage have been in solution together. Further experiments were performed to reveal whether this complex was necessary for TABLE II Serum withdrawn by syringe after tap water rinse (SERIES III) Percentage of fertilizations July pH Adjusted pH Serum (100%) Sea water 8 7.5 7.9 100 100 10 7.6 8.0 100 100 12 7.6 7.9 98 99 14 7.7 8.0 99.5 100 17 7.9 7.9 100 100 26 7.5 7.9 95 100 Aug. 2 7.5 8.0 100 100 3 7.0 8.0 100 • 100 inhibition, or whether the yellow drainage alone was sufficient to cause inhibition of fertilization. The fourth series of experiments was devoted to this problem. Series IV. Several Arbacia were washed in filtered sea water to remove debris and wastes, then rinsed under the tap for fifteen seconds, and finally submerged briefly in filtered sea water to correct hypotonicity. After being shaken the animals were placed edgewise in glass funnels fitted with moistened filter paper, and the yellow drainage collected in centrifuge tubes. An equal number of control animals was subjected to the same treatment except that no tap water rinse was administered. Material from any animals that proceeded to defecate or shed gametes was discarded. The animals were left in the funnels a maximum of 20 minutes, or until draining ceased. The filtrate was centrifuged, although no visible separation occurred, and the pH adjusted to that of sea water. The clear, yellow fluid was then used as before. This experiment was repeated seven times between July 10 and August 2, using a total of 68 animals. The results shown in Table III indicate that this material alone is sufficient to bring about inhibition of fertilization. In only two instances was fertilization obtained in this material when undiluted, and then only an average of 2 per cent of the eggs were activated. These data show also that 74 WILLIS PEQUEGNAT TABLE III (SERIES IV) Yellow fluid obtained from the test by draining after tap water rinse Percentage of fertilizations July PH Adjusted pH Fluid dilutions (per cent) Rinse Sea water control control 100 100 100 50 25 12 6 3 10 7.6 8.1 1 — — — — . ' 97 99 12 7.8 8.0 0 — — — — — 100 100 14 7.9 8.0 0 — . — — — . — 100 100 17 7.8 8.1 0 0 0 0 17 100 96 97 19 7.6 7.9 0 0 0 0 0 — - 95 98 *27 7.5 7.0 0 0 0 0 0 0 27 7.5 8.0 3 2 3 3 3 99 100 100 27 7.5 9.0 21 29 10 10 21 84 Aug. 2 7.6 8.0 0 0 0 0 1 5 100 100 * The experiment of July 27 was carried out at three pH's. The inhibitor's effectiveness is apparently reduced at pH 9. Note also an increase of effectiveness with moderate dilution; this occurred at other pH's as well. these solutions effectively blocked fertilization in dilutions ranging from 50 to 3 per cent by volume. As a check some of this material was mixed in varying amounts with perivisceral fluid which alone had no effect on fertilization. As was anticipated, the previously impotent serum now became inhibitory to fertilization in proportion to the amount of yellow drainage added (Table IV). No appreci- able difference in the inhibitor's activity could be detected between that diluted with sea water and that diluted in serum. Since the liquid draining from the control animals (those not rinsed in tap water) was found to be ineffective in blocking fertilization, it appears unlikely that this inhibition is due to the presence of soluble wastes, at least as proposed by Just in answer to Oshima's report. TABLE IV (SERIES IV) Inhibitor mixed with impotent serum Percentage of components in mixture Percentage fertilizations Serum Inhibitor Mixture Sea water 95 5 98 100 90 10 78 — 85 15 24 — 80 20 2 — 75 25 0 — 70 30 0 — 50 50 0 100 INHIBITION 01" FERTILIZATION 75 Scries V. Much more potent solutions of inhibitor were obtained by (1) placing urchins directly into distilled water to depths not exceeding the greatest circumference of the shell and permitting them to remain 15 minutes, and (2) adjusting the osmotic value of the solution with sea water concentrated by evapora- tion. Control animals were placed in sea water to soak for the same period of time as the test animals. The osmotic values of these test solutions were checked by comparing the diameters of test eggs with those of the controls. No significant variations were observed. In addition one control was composed of equal volumes of distilled water and sea water concentrated to half its original volume by evaporation. Although this experiment was run on several occasions, only one will be de- scribed in detail since all were essentially the same. On July 31 half a dozen Arbacia were placed in succession into 40 cc. of distilled water and permitted to remain approximately 5 minutes each. Six control animals were placed into the same volume of sea water in the same manner. The distilled water was imme- diately colored yellow, while the sea water remained clear and colorless through- out. After filtration, centrifugation, and adjustment of osmotic value. 62 cc. of yellow fluid were obtained. This obviously represents a much greater dilution per animal than in previous experiments. But despite this fact this solution prevented fertilization completely in serial dilutions down to 1 per cent. The sea water in which control animals had stood gave 100 per cent fertilizations, as did the other control solution. Series VI. In order to narrow down the locus of origin of the inhibitor, five animals were cut into halves along the oral-aboral axis. All internal organs were removed and the inside of the tests scrubbed in sea water with a brush. Following this the sectioned tests were soaked for one hour in sea water, which was not dis- colored in the process. Then the tests were rinsed for a few seconds in tap water and sea water, and permitted to drain into a clean finger bowl. The drainage was yellow. The spines on the tests were still moving when the fluid was removed after one hour. The pH was adjusted from 7.6 to 8.0, and the material tested. • The following results were obtained : Percentage of extract 100 50 25 12 6 Percentage of fertilizations 3 6 3 3 92 100 When the same tests were again rinsed in tap water and sea water and permitted to drain, only 2 cc. of fluid were obtained. When used undiluted this second drainage gave 10 per cent aberrant cleavages. Controls gave 98 and 99 per cent fertilizations, respectively. These results provided additional evidence that some external structure was the source of the inhibitor. Scries VII. A study of individual tube feet under the microscope revealed a layer of closely packed, yellow granules or cells just beneath the outer epithelium. These granules maintained their integrity while immersed in sea water. But when the sea water was replaced with tap water, all traces of yellow material disappeared from within the feet. Simultaneously with this disappearance, the water around the feet was colored bright yellow. It is important perhaps to note that this material diffused through the outer epithelium and did not pass into the lumen of the foot. Tests run on this yellow material proved that if possessed the property 76 WILLIS PEQUEGNAT of inhibiting fertilization in a manner similar to that observed previously. One experiment will be described in greater detail. Part of the tube feet from the oral hemispheres of four Arbacia were removed. This was done by letting the animal attach to a glass plate and then pulling the plate away. The tube feet were soaked in several rinses of sea water, covered momentarily with tap water and then placed into 4 cc. of clean sea water. The latter water was immediately colored yellow. After five minutes the supernatant fluid was decanted and its volume noted to be 5 cc. It was then filtered and the pH raised from 7.6 to 8.0. It is worthy of note that this extract was brighter yellow than that obtained by placing the whole animal in distilled water. The color of the extract deepened to a certain extent when the pH was elevated. Interestingly enough I found subsequently that it became colorless at pH 4 and below, and a darker yellow at pH 8 and above. This tube-foot extract proved to be very effective at blocking fertilization. In concentrations running from 100 to 12 per cent no fertilizations occurred, and only 1 per cent of the eggs was fertilized in dishes containing as little as 3 per cent extract in sea water. This degree of effectiveness is made more remarkable by the fact that because of the dilution intrinsic to the method of extraction the extract represented only 20 per cent by volume of the inhibitor solution that exuded from the tube feet. Hence, the 12 per cent solution in the series of 2 cc. dilutions would actually contain a maximum of 0.025 cc. of inhibitor, or approximately 1.25 X 10'5 cc. per egg. Because the color of this extract was not the same shade as that obtained when intact animals were used, I searched for other sources. It was found that spines gave forth a small amount of inhibitor, but only from their bases where epithelium was to be found. The bodies and tips of the spines, which in many instances had no fleshy covering, gave up a purplish substance which had no significant effect on fertilization. When this substance was mixed in small amounts with the ex- tract from tube feet, however, the latter assumed the color of the extract from the intact animals. Series VIII. Further work revealed that the inhibitor was carried by at least one type of amoebocyte found in the perivisceral fluid. Blood was removed by cutting the peristome, but attention is called to the fact that the animals were not rinsed in tap water. And, instead of filtering the blood as before, the plasma or serum was separated from the cells by light centrifugation and then decanted into clean flasks. At this time, an equal volume of sea water was added to the clot in the tube and the two mixed by shaking and rapid centrifugation. Whereas the plasma was colorless, the supernatant sea water solution was the same bright yellow as the extract from the tube feet. The pH of the two solutions offered an addi- tional point of contrast. Whereas the pH of the plasma was 7, that of the yellow extract was 6.3, despite the fact that the sea water was pH 7.9 at the time of its addition to the clot. It is suggested that this depression of pH was caused by the liberation of the acid contents of the colorless amoebocytes. Before being tested, both solutions were brought up to pH 8. A further contrast of properties of these two solutions was observed when they were tested : the percentage of fertilization in solutions of plasma equalled that of the controls, in this case 99 per cent ; the yellow extract, however, permitted no INHIBITION OF FERTILIZATION 77 fertilizations when used undiluted. A two-fold dilution of the extract permitted only 10 per cent of the eggs to be fertilized. These results showed clearly that the inhibitor was carried by certain blood cells, and in such a manner that it did not normally pass from them into the plasma. Attempts to isolate the specific type or types of blood cells that carried the inhibitor were nullified by the fact that no practical method was devised for preventing the blood from clotting. The methods usually employed to prevent clotting of vertebrate blood were found to be of no value. Nonetheless, it was possible to observe microscopically that upon cytolysis the amoebocytes with yellow spherules (for classification of blood cells, see Kindred, 1926) gave up a yellow substance which upon addition of acid became decolorized as does a solution of the inhibitor. Two additional observations also serve to link the inhibitor obtained by methods described previously with that obtained directly from the blood cells. It was possible to demonstrate that the potency of inhibitor extracts obtained from blood clots of animals that had been soaked previously in distilled water was less than that obtained from untreated animals. For example, blood was removed by syringe from six animals which had been used just previously for obtaining inhibitor by soaking in distilled water (after the method of Series V). The blood was then centrifuged, the plasma decanted and replaced by sea water, and the mixture shaken and centrifuged rapidly. These solutions were tested with the result that no fertilizations were permitted in the extract obtained by soaking in distilled water; 50 per cent fertilizations were obtained from the undiluted sea water extract of the clot; and 100 per cent fertilizations were obtained in the plasma and sea water controls. The reciprocal of the above was also found to be true, viz., that animals from which all possible perivisceral fluid had been removed by syringe produced weaker solutions of inhibitor obtained by application of tap water (after the method of Series IV). I took six animals from which pervisceral^ fluid had just been removed by syringe, and rinsed them briefly in tap water and*£ea water, and then placed them into funnels from which the drainage was collected. This drainage permitted an average of 32 per cent fertilizations when used undiluted. All of the observations made in this series of experiments lend some support to the opinion that the yellow granules observed in the tube feet may actually be yellow amoebocytes that are free to move between tube feet and the perivisceral cavity. Series IX. The following experiment was devised to show whether or not the inhibitory effect of blood extracts upon eggs was reversible. One drop of eggs was placed into 2 cc. of undiluted inhibitor solution contained in each of six Syra- cuse dishes. After insemination the dishes were placed in running sea water on the water table. No fertilizations resulted in any of the dishes. At the end of two hours, the inhibitor solution was pipetted from one of the dishes and the eggs washed twice in fresh sea water and then reinseminated. Eggs in the remaining dishes were handled in the same manner 4, 8, 12, 14 and 24 hours after being intro- duced into the inhibitor solution. A series of six control dishes contained approxi- mately the same number of eggs in 2 cc. of sea water ; these were fertilized in series after the same intervals of time. The results are tabulated in Table V. Although there are some indications that some eggs were damaged by standing in the inhibitor solution, there is definite evidence that this blocking of fertilization is reversible. 78 WILLIS PEQUEGNAT TABLE V (SERIES IX) Fertilization of blocked eggs after washing Percentage fertilizations Exposure to inhibitor (hours) Before washing After washing Controls 2 0 97 100 4 0 92 98 8 0 95 96 12 0 *90 94 14 0 f99 98 24 0 90 93 * About 30 per cent exhibited polyspermy. t About 10 per cent aberrant cleavages. Scries X. Eggs that were observed to develop in various dilutions of inhibitor had indicated that post-fertilization developmental processes were not appreciably affected, but one experiment was run to test this point more effectively. Eggs were inseminated in sea water and transferred as quickly as possible into dishes containing undiluted inhibitor. Subsequent examinations revealed no significant differences between those samples of eggs that were placed in inhibitor and those that remained in the sea water. The zygotes were kept in the original inhibitor solution until they had reached the swimming stage. At this time they were transferred to sea water and carried on to the pluteus stage. Two observations were made during this time : ( 1 ) there wrere some indications that the rate of development was retarded slightly by the inhibitor, and (2) that the plutei devel- oping from inhibitor-treated zygotes were smaller than the controls. These obser- vations were not investigajld further. DISCUSSION The results obtained from Series II and III of experiments refute the validity of the conclusion of Lillie and Just that the serum of Arbacia blood normally con- tains an inhibitor to fertilization. Although Series. VIII showed that the inhibitor was carried by certain blood cells, the results of Series II indicated that the inhibi- tor did not leave the blood cells to enter the serum. In fact, the inhibitor was obtained from the cells in Series VIII only after vigorous shaking and rapid centrifugation. Since it has been shown that the inhibitor can be evoked by the application of tap water to the outside of the animal, it may be concluded that the inhibitor observed by them entered their samples from the outside of their urchins as a result of the application of tap water. Furthermore, the supposed variations in potency of the inhibitor reported by Lillie and Just were shown to be illusory by the results of Series I and III. Certainly the property of inhibition is not of itself particularly interesting for no doubt a large number of substances could be used to inhibit fertilization in Arbacia, but most of them would very likely be inimical in one way or another to the gametes. Therefore, the fact that eggs appear to be fundamentally unharmed INHIBITION OF KKKTIUZATION 79 by exposures to this natural inhibitor ranging from a few seconds to many hours serves to heighten one's interest. It has been shown that eggs which have remained blocked up to 24 hours in this inhibitor can be fertilized, provided they are washed thoroughly in sea water and reinseminated. Moreover, Just (1922) reported that he obtained development in blocked eggs (inseminated in inhibitor solution) with- out reinsemination, so long as the eggs were washed within two hours after fertilization. Except for a slight depression of the rate of development, this inhibitor exerted no appreciable influence upon post-fertilization changes in the egg. Eggs that were fertilized at one instant and transferred immediately to inhibitor proceeded to develop into normal blastulae ; yet when eggs were introduced into potent inhibi- tor and sperm added as quickly as possible blocking was complete. This latter observation supports the hypothesis that the inhibitor acts at the surface of the egg. Other evidence may be brought to bear on this point. The inhibitor appeared to remove part of the egg's jelly layer, in proportion to concentration or to the duration of exposure. Inferred at first from the observa- tion that eggs tended during the period of contact with inhibitor to aggregate more compactly than eggs in sea water, this conclusion was strengthened by the addition of dilute solutions of Janus Green B. Furthermore, the jelly layer of Chaetopterus eggs exhibited a marked affinity for the Arbacia inhibitor by staining a deep yellow during exposure, but the jelly layer was not removed by it. It is interesting to note also that this yellow cast was not removed by subsequent washings. Because the inhibitor obtained from Arbacia was observed to prevent fertilization of Chae- topterus eggs, it is unfortunate that no attempt was made to determine their fer- tilizability after washing. This might well have revealed whether the inhibitor itself is yellow or is only associated with the pigment in solution. While referring to associated species, it is appropriate to record that the Arbacia inhibitor does suppress fertilization in the sand-dollar, Echinarachnius parma. This fact was reported by all previous workers. In addition. Just (f923) stated that the blood of this sand-dollar blocked the fertilization of its eggs. It is possible, however, that this observation is subject to the same criticism herein advanced against his interpretation of the Arbacia inhibitor, because I observed that tap water evoked a similar response from Echinarachnius. Unfortunately. I could find no complete description of the method he used in obtaining this fluid. Normal fertilization membranes were seldom observed on eggs that were ferti- lized in fresh sea water after prolonged exposures to the inhibitor. It is possible that this condition resulted from simple aging of the eggs. But in some instances no membranes could be observed even after the eggs began to cleave. That this was not tight membrane development is attested to by those extreme cases in which the blastomeres rounded up and were as easily separable as those of eggs treated with Ca-free sea water. It was more difficult to observe definitive effects of the inhibitor on sperm. Little positive evidence as yet obtained rules out the possibility that the inhibitor acts directly upon the sperm. But this position could be rendered less tenable by several observations. In the first place, sperms appeared to be stimulated to greater activity when in the presence of inhibitor ; and, secondly, they continued to move about inhibited eggs long after all evidence of motility of sperm had dis- 80 WILLIS PEQUEGNAT appeared in the controls. One point in this connection that might be of value in future work is the fact that the sperms which persisted in activity longest appeared to have lost their ability to attach to the egg. Thus, they moved about aimlessly among the eggs without attempting to penetrate. Finally, Just (1922) reported the actual penetration of sperms into the cortex and cytoplasm of blocked eggs. Presumably these were the sperms that were able to consummate fertilization when such blocked eggs were washed within the two-hour limit but not reinseminated. Differences of opinion have arisen concerning the interaction, if any, between inhibitor and fertilizin. Lillie (1914) concluded that the effects of the inhibitor could be nullified by mixing it with fertilizin. One cannot question the data from which he drew this conclusion, but it appears to the present author that the method that he used to obtain neutralization of the inhibitor permits another interpretation of his data. In order to combine fertilizin with inhibitor. Lillie mixed serum and whole eggs in the ratio of two parts serum to one part eggs. Time intervening, the mixture was filtered and the filtrate tested for inhibitory activity. His data show that the untreated serum permitted only 0.5 per cent of the eggs tested to be fertilized, while the treated serum permitted 99.0 per cent of the eggs tested to be fertilized. Lillie concluded that the fertilizin had neutralized the inhibitor in the serum. But it is possible that little or no inhibitor was left in the filtrate. The basis for this interpretation is supplied by an experiment not previously described. I mixed 4 cc. of inhibitor solution, obtained in the manner of Lillie, with 2 cc. of a suspension of eggs computed to contain approximately 0.75 cc. of sea water (Solution 1). Another 4 cc. of the same inhibitor were mixed with 0.75 cc. of sea water (Solution 2). A third solution was prepared by adding 2 cc. of strong egg-water (known to agglutinate sperm) with another 2 cc. of the inhibitor solu- tion (Solution 3). After an interval of twenty minutes, all tubes were centrifuged lightly and 2 cc. samples were removed carefully from the top of each tube, and tested in the usual manner along with sea water controls. The following results were obtained : Solution 1 «gave 75 per cent fertilization ; Solutions 2 and 3 gave 0.0 per cent fertilizations ; and 99 per cent of the eggs in the sea water controls were fertilized. These data suggested that the inhibitor combined in some manner with the eggs. Also, the fact that no fertilization occurred in the mixture of inhibitor and egg- water (Solution 3) supports the contention that the fertilizin does not neutralize the inhibitor, at least in the same sense of the word as used by Lillie. The results of this experiment (particularly from Solution 1) provide a tenta- tive explanation of the retarded activity of eggs that have stood in dilutions of inhibitor for some time after insemination. It is obvious that individual eggs in any sample are affected differentially by the inhibitor ; otherwise there could be no explanation of the interesting fact that in dilutions of inhibitor some eggs are fertilized while others are not. Furthermore, one should recall that blocked eggs may be reversed to a state of fertilizability by soaking them in sea water. There- fore, it is possible that eggs which at first have the minimum of inhibitor necessary to prevent fertilization give this up slowly when the diffusion gradient has reversed, as the result of the greater affinity of other eggs for inhibitor. Just what condi- tions in or on the egg account for this differential reaction to inhibitor, I cannot say. Although it appeared that fertilizin exerted no appreciable influence upon the INHIBITION OF FERTILIZATION 81 activity of the inhibitor, some evidence was obtained which indicated that a reverse influence did exist. On the one hand, as indicated in the introductory paragraph of this report, Lillie asserted that the inhibitor did not reduce the ability of fertilizin to agglutinate sperms. On the other hand, I found that concentrated solutions of inhibitor, when mixed in the ratio of 1 : 3 with egg-water capable of agglutinating sperm, would reduce the time required for reversal of agglutination. When these substances were mixed in equal parts, the egg-water lost its ability to bring about agglutination. An interesting aspect of this problem is revealed by the close parallel between certain properties shared by the inhibitor and material extracted by Tyler (1940) from Arbacia eggs. Both of these materials are yellow ; both appear to negate the sperm-agglutinating power of filtered egg suspension ; both exhibit tendencies to cause clumping of eggs ; and, under certain conditions, both reduce the fertilizability of eggs. The material extracted from eggs, however, produces a visible precipita- tion membrane on the egg's jelly layer ; no membrane of this type has as yet been observed upon applying the blood inhibitor. Nonetheless, this parallelism between the properties of these two extracts is such that further study is indicated. SUMMARY 1. Whole perivisceral fluid (blood) of Arbacia contains a substance capable of inhibiting fertilization. 2. Contrary to the conclusions of previous investigators, this inhibitor is not normally present in the serum. Rather certain blood cells, particularly the amoebo- cytes with yellow spherules, are the ultimate source of the inhibitor. 3. The inhibitor believed by Lillie and Just to be found in the serum of Arbacia blood actually entered their samples as a contaminant from the outside of their animals. 4. The external application of tap water causes the inhibitor to appear in the drainage from the test. Under these conditions the inhibitor emanates from yellow bodies found in the hypodermis of the tube feet. 5. The supposedly variable potency of inhibitor reported by previous workers can be explained by the technique used in obtaining samples, and the methods used in testing its strength. In reality former workers were testing inhibitor in varying dilutions rather than testing the potency of a standard amount of inhibitor. 6. This inhibitor does react with the egg's jelly layer and can modify the fertili- zation membrane in proportion to concentration and duration of exposure. Eggs that are inhibited for short intervals of time (1-4 hours) can be fertilized and will develop- normally (i.e. with membranes, etc.), provided they are rinsed thoroughly in fresh sea water. 7. Fertilizin is believed to have little influence on the activity of the inhibitor beyond a simple dilution effect. On the other hand, the sperm-agglutinating power of fertilizin-bearing solutions can be reduced or nullified by the addition of sufficient inhibitor. 8. It is suggested that this blood inhibitor may be related to an egg-agglutinin extracted from the egg itself. 82 WILLIS PEQUEGNAT LITERATURE CITED HARVEY, E. B., 1939. Arbacia. The Collecting Net, 14, No. 8. JUST, E. E., 1922. The effect of Arbacia blood on the fertilization-reaction. Biol. Bull., 43: 411-422. JUST, E. E., 1923. The fertilization-reaction in Echinarachnius parma. VII. Biol. Bull., 44, 10-16. KINDRED, J. E., 1926. A study of the genetic relationships of the amoebocytes with spherules in Arbacia. Biol. Bull., 50 : 147-154. LILLIE, F. R., 1914. Studies of fertilization. VI. The mechanism of fertilization in Arbacia. Jour. E.vp. Zool., 16 : 523-588. OSHIMA, H., 1921. Inhibitory effect of dermal secretion of the sea-urchin upon the fertilizability of the egg. Science, 54 : 578-580. TYLER, A., AND B. T. SCHEER, 1937. Inhibition of fertilization in eggs of marine animals by means of acid. Jour. E.vp. Zool., 75 : 179-197. TYLER, A., 1940. Agglutination of sea-urchin eggs by means of a substance extracted from the eggs. Proc. Nat. Acad. Sci., 26: 249-256. TYLER, A., 1948. Fertilization and immunity. Physiol. Rev., 28 : 180-219. CHANGES IN DENSITY, WEIGHT, CHLORIDE, AND SWIMBLADDER GAS IN THE KILLIFISH, FUNDULUS HETEROCLITUS, IN FRESH WATER AND SEA WATER VIRGINIA S. BLACK Department of Biology, Dallwusic University, Halifax INTRODUCTION The field of osmotic regulation in aquatic animals has received much attention during the last fifty years. In fish this work has been largely directed toward a study of the euryhaline species such as the eel and salmon (Krogh, 1939). The theories currently accepted for the maintenance of water and salt balance by normal fish in sea water and fresh water were first thoroughly reviewed by Smith (1932), and have been well summarized by Krogh (1939), Baldwin (1940), and Scheer (1948). The killifish, Fundulus, has been used by many investigators, probably because it is one of the few small euryhaline genera which are available in quantity and adapt readily to aquarium life. The most extensive work dealing with the effect of density changes on Fundulus and other fish was carried out by Sumner (1905). His experiments are based mainly on viability of groups of fish in various salini- ties and fresh water. He also measured weight changes and changes in chloride in the water and in the tissues of Fundulus resulting from removal from sea water to fresh water and vice versa. The present investigation was designed to obtain serial quantitative measure- ments of rapid adjustments of a marine fish to fresh water, and so construct a more complete picture of a reaction whose qualitative aspects are already known. The author is deeply indebted to Dr. F. R. Hayes for invaluable assistance in formulating the problem and also for information and helpful suggestions regarding the measurements of density of fishes. MATERIAL AND GENERAL PROCEDURE • Live specimens of Fundulus hcteroclitus, commonly known as killifish, mummi- chog, or salt water "minnow," were obtained from salt water flats northeast of Halifax, Nova Scotia. This species is normally found in the sea, in estuaries, and in brackish waters. In the laboratory the fish were kept in large glass aquaria (10 inches by 17.5 inches) having a depth of water of 6.5 inches. Stock sea water was obtained from the Northwest Arm, Halifax. The tap water was derived from the Halifax civic water supply. An analysis of the water made in 1940 '(Leverin, 1942) from samples at the pumping station is given in Table I. Experiments were not begun until the fish had been in the laboratory for at least two days. Fish which had been in the laboratory more than a week were not used to begin a series of experiments. The fish were not fed during the period of the experiment, but stock fish were fed every two days on Aylmer's canned beef 83 84 VIRGINIA S. BLACK TABLE I A nalysis of Halifax water supply, July 1940 (Lever in, 1942) Parts per million Parts per million Color 30.0 Bicarbonate (HCO3) None Alkalinity as CaCO3 None Sulphate (SO4) 6.6 Residue on evaporation dried at 30.0 Chloride (Cl) 2.5 Silica (SiO») 4.0 Nitrate (NO3) 0.35 Iron (Fe) 0.05 Total hardness as CaCOs 18.8 Calcium (Ca) 5.7 Calcium hardness 14.3 Magnesium (Mg) 1.1 Magnesium hardness 4.5 TABLE II Density, weight, chloride and swimbladder gases of F. heteroclitus in sea water Date 1947 Sex Weight grams Density (see text) Chloride m.eq./kilo wet tissue Swimbladder C02 % Oz % Vol. swimbladder Wt. of fish Series I • June 2 rf1 8.28 42 6.2 11.9 0.060 9 6.34 6.0 10.1 0.040 9 4.50 58 2.3 13.1 0.060 4 9 6.98 55 3.1 10.5 0.063 tf 7.87 58 1.3 14.2 0.051 7 9 6.86 66 0 12.6 0.044 9 7.15 60 2.7 10.2 0.050 9 9 3.40 1.017 50 1.2 15.5 0.048 10 9 6.90 1.027 55 0.6 12.1 0.061 A verage 6.5 56 2.6 12.2 0.053 Series II July 21 9 2.09 50 1.2 15.1 0.033 9 1.51 50 rf 4.43 3.6 17.3 0.056 9 10.18 3.2 14.0 0.054 rf1 1.87 2.3 21.4 0.029 22 9 1.47 50 3.0 20.0 0.029 23 9 1.67] 55 1.1 15.1 0.031 9 1.26} 1.026 52 0.8 12.5 0.032 cf, 9 1.74 j 46 24 c? 1.71 51 1.3 17.5 0.039 Aug. 1 9 1.54] 58 0.5 11.8 0.045 9 4.72} 1.024 60 0.8 10.7 0.044 ^ PANTIN, C. F. A., 1931. The adaptation of Gnnda ulvac to salinity. III. The electrolyte exchange. Jour. E.\-p. Biol., 8 : 82-94. PETERS, J. P., AND D. D. VAN SLYKE, 1932. Quantitative clinical chemistry. Volume II, Methods. Baltimore, Williams & Wilkins Co. RAUTHER, M., 1937. Die Schwimmblase. Handbuch dcr vergleichenden Anatomic der Wirbel- tiere, 3 : 883-908. SCHEER, B. T., 1948. Comparative physiology. New York, Wiley & Sons. 563 pp. SCOTT, G. G., 1910. Effects of changes in the density of water upon the blood of fishes. Bull. U. S. Bur. Fish., 28: 1143-1150. SMITH, H. W., 1932. Water regulation and its evolution in the fishes. Quart. Rev. Biol., 7 : 1-26. SUMNER, F. B., 1905. The physiological effects upon fishes of changes in the density and salinity of the water. Bull. U. S. Bur. Fish., 25 : 53-108. WEIL, E., AND C. F. A. PANTIN, 1931. The adaptation of Gunda ulvac to salinity. II. The water exchange. Jour. Exp. Biol., 8: 73-81. A NEW METHOD OF REPRODUCTION IN OBELIA N. J. BERRILL McGill University, Montreal In view of the eminence of Obelia as a zoological type enthroned in all text books, and the consequent widespread study of innumerable specimens, it is sur- prising that there could be an important method of reproduction of this genus so far unreported. The observations recorded here were made in the course of extensive investi- gation of growth and form in Obelia, involving day by day study of specific colony sites through the summer of 1947 at Boothbay Harbor. Temperature changes were followed closely since colonies of Obelia and other hydroids fluctuated greatly, disappearing and reappearing as temperatures rose and fell markedly above and below 20° C. Three species were studied, all associated- with one float, Obelia articulata, O. genieulata and O. longissimus. For nearly two months of excessively high temperatures during July and August, no colonies could be found. With the onset of offshore winds, the warm surface water blew out of the bay, to be replaced by bottom water 8 to 10 degrees colder. With this lowering of the temperature, small Obelia colonies appeared in relatively large numbers. Calm weather with no wind except the daily inshore breeze allowed the surface bay waters to warm up again to about 21° C. for a few days, followed by a slow fall to lower temperatures. The growth or reproductive procedures described here were responses to these changes. OBELIA ARTICULATA This species grew attached to laminaria. Colonies are relatively small but well branched, and in general are intermediate in character between the single un- branched stems of O. genieulata and the enormously long and branching colonies of 0. longissimus. The intermediate character is again shown in the distribution of the gonangia. In 0. genieulata they grow out from the angles made by the hydranths and the stem. In 0. longissimus they appear at the angles made by hydranths with lateral branches but only at the basal region of a colony after it has already become massive. In O. articulata they appear when the colony is small, but at angles between hydranths and secondary branches, not in connection with the main stem. Similarly the growing tip of the main stem in colonies of all three species varies in series. It is essentially a stolonic type of growth like that of the creeping stolons. In O. longissimus it grows rapidly and vigorously, giving off secondary stolonic outgrowths regularly at a certain distance from the tip, and these behave in much the same way. Hydranths are mainly tertiary outgrowths, at least. In O. genieulata terminal stolonic growth is very limited, lateral branches are not formed, and the tip itself usually differentiates into a hydranth. 0. articu- lata lies between. 94 REPRODUCTION IN OBELI. \ 95 In any species a rise in temperature, especially when in excess of 20° C, tends to maintain or promote stolonic growth at the end of a stem or branch of any order and conversely to inhibit hydranth differentiation. The outgrowths capable of responding in one way or another to different temperature conditions may be of varying origin. They may be the terminal tips of the main stem and secondary branches, tips of branches of a more subsidiary order, or the tips of presumptive gonangia at stem or branch angles. A marked rise in temperature results in prolonged growth of a stolonic char- acter in the first two cases, the long slender branches thus formed remaining an integral part of the colony, even though they may in no way contribute to its wel- fare. In the third case, those normally destined to become gonangia, the reaction is different. A gonangium in its earliest recognizable stage is shown in Figure 1A, growing from the non-annulated region immediately below a hydranth. It consists of an outgrowth with several annulations, terminating in a relatively large bulb with a short central cone and wide shoulders. This persists and grows as the distal cap of the gonangium. In the same figure on the same scale are shown outgrowths from homologous locations, but from colonies subject to higher temperatures. Annulated growth, instead of stopping after t\vo or three annulations and forming the wide gonangium rudiment, continues until ten or a dozen shallow annulations have occurred. The final surge corresponding to the establishment of the gonan- gium leads instead to the formation of a massive elongate stolonic structure with no further trace of annulations. It is similar to the terminal stolonic growth at the ends of branches, but with two differences, it is much more massive and of greater girth, and is so vigorous that the stem uniting it to its point of origin becomes attenuated to the point of rupture (Fig. IB). Distally each such mass grows rapidly, while it resorbs correspondingly at the proximal end. The separation usually occurs at the region where the annulated growth transforms into a steady surge (Fig. 1C), the part left attached to the colony retracting proximally as the tension is relaxed, while the congested terminal units slide out of the thin but wide perisarcal tube to float freely in the surrounding water. The question that arises at once is whether this is a normal process or a response to the disturbance of collection and subsequent examination. Obelia and similar hydroids are notoriously susceptible and it is a common experience to find hydranths and other terminals in process of regression with distal parts of the coenosarc often isolated within the perisarc from the main body. This possibility was considered immediately, and the following is the evidence that the process is a normal one for the sea temperature prevailing at the time. Colonies picked off the laminaria and dropped into formalin within a few seconds of emergence from the water exhibited the phenomenon to as great a degree as any. The colonies under live examination were fresh, had hydranths with active tentacles and manubrium, possessed hydranth buds that progressed normally to complete development, and showed no trace of resorption at any of the terminals. Small colonies left standing in finger bowls liberated literally hundreds of gonangial terminals overnight and wrere still in active process the following day. Lastly, there is the evidence that they possess a useful function. In the first place, fragments of ordinary terminals of equivalent length but smaller diameter can reattach and survive for a week or two. They do not develop hydranths unless B A C FIGURE 1. Production of free gonangial buds in Obelia articulata. A Hydranth stalk with young gonangium. B. Hydranth stalk with gonangial buds in process of formation and liberation. C. Gonangial bud showing constriction at junction of annulated and non-annulated regions, fb, free buds; g, young gonangium; h, hydranth; rb, retracted stalk after liberation of bud. 96 REPRODUCTION IN OBELIA 97 several times as long. The isolated gonangial terminals on the other hand become attached to a solid substratum immediately upon contact. After about 12 hours, each fragment is about twice its original length and half its girth. The original perisarc, however, indicates that most of the tissue is now the result of new distal growth and proximal resorption (Fig. 2B). At the same time an annulated up- B A FIGURE 2. Development of free gonangial bud of Obclia articuhta. A. Bud at time of attachment. B. Twelve hours later with hydranth buds growing verti- cally and empty perisarc indicating extent of proximal resorption and distal growth. C. Twenty-four hours after attachment, with hydranth bud at tentacle rudiment stage, and with secondary distal outgrowth, d, distal growing region; lib, hydranth bud. growth from the middle of the fragment indicates a developing hydranth. In Figure 2C a fragment is shown typical of the condition 24 hours after liberation. A lateral creeping stolonic terminal has started, while the hydranth has progressed to the tentacle rudiment stage. In the great majority of the liberated fragments, functional hydranths were present on the second day and new colonies thus started. 98 N. J. BERRILL fb C FIGURE 3. Production of gonangial buds in Obclia garicitlata. A. Complete sprig with two advanced gonangial buds. B. Part of stem showing young gonangium and two stages in production of gonangial buds. C. Bud showing sharp demarkation between massive presumptive free bud and attenuated proximal stalk. //;, presumptive free bud ; g, gonangium ; //, hydranth : />, perisarc. REPRODUCTION IN OBELIA The immediate developmental capacities are accordingly somewhat superior to those of the average planula. OBELIA GENICULATA Colonies of Obclia (/cnicitlntii were collected a day later than those of O. articii- lata, when the water temperature was already failing. The great majority of stems had the appearance shown in Figure 3A. Xo gonangia were present, but in their place were large congested terminals similar to those of 0. articnlata. Detail as seen in Figure 3C indicates that the process is essentially the same, the distal part of the massive stolonic outgrowth growing rapidly at the expense of proximal tissue. In fact the proximal half of the outgrowth becomes so attenuated that the lumen is obliterated. \Yhile actual separation was not observed in this species, continued distal growth after the occlusion of the lumen must inevitably result in a break in the attenuated proximal part. In Figure 3B two stages are shown, one with an attenuated stalk and a younger stage with wide lumen through- out. A third, the most anterior, is a younger outgrowth and is developing into a typical gonanginm. suggesting that the external temperature had already dropped below the critical value at the time of its initiation. At the same time it indicates the relative scale of the two forms of growth from the hydranth-stem angles, and the comparative massiveness of the high-temperatures' outgrowth. OBELIA LONGISSIMA This species is included merely as a basis for comparison. It is typified by the very extensive growth of the primary and secondary terminals, leading to the formation of relatively enormous colonies. Gonangia appear in secondary and other angles at the base of the colony only after it has attained a fairly large size. During the warmer summer months growth is directed mainly into the vigorous terminals, and there appears to be little tendency to form gonangial outgrowths at all. They are most abundant during late winter and early spring. Consequently the type of asexual reproduction just described for 0. articnlata and 0. geniculata is here probably of very rare occurrence^, if it occurs at all, for without the initiation of gonangial outgrowths of any kind, no response in either direction is possible. SUMMARY A method of asexual reproduction previously unrecorded is described for Obclia articitlata and Obclia geniculata. When water temperatures markedly exceed about 20° C. presumptive gonangial outgrowths continue growth as massive stolonic terminals that rapidly constrict off, leave the colony and settle elsewhere to establish new colonies in large numbers. In size and potentiality these reproductive units somewhat exceed those of typical campanulid planulae. TOOTH SUCCESSION IN THE SMOOTH DOGFISH, MUSTELUS CANIS JOHN D. IFFT Simmons College, Boston AND DONALD J. ZINN Rhode Island State College The arrangement of the teeth of sharks in a series of rows is well known. In some species, such as tiger sharks and sand sharks, with large conical teeth, newly formed teeth appear to be formed in the hack rows while older teeth are in front. This impression led Owen in 1866 to state, "... the whole phalanx of their numerous teeth is ever marching slowly forwards in rotary progress over the alveolar border of the jaw, the teeth being successively cast oft" as they reach the outer margin, and new teeth rising from the mucous membrane behind the rear rank of the phalanx." Owen's theory of tooth replacement in sharks is the com- monly accepted one today and is found in most comparative anatomy texts. This theory apparently was based only on morphological evidence without experimental proof ; a search of the literature has failed to reveal reports of any experiments testing the theory. However, the morphological evidence is quite convincing and accounts for the general acceptance of the theory. Within recent years Owen's hypothesis has been challenged by Cawston in a series of papers (1939; 1940a, b, "c ; 1941a, b, c; 1944; 1945). He has doubted that sharks shed their teeth but if they do he denies the possibility of replacement occurring by the forward movement of teeth from the rear. That sharks shed their teeth is confirmed by Breder (1942) who noticed the sloughing of teeth by sand sharks (Carcharius littoralis) in the tanks at the New York Aquarium. Whether the lost teeth are replaced and the manner of this replacement if it occurs apparently has not been observed. It is the purpose of this investigation to inquire experi- mentally into the question of polyphyodonty in selachians. MATERIALS AND METHODS It was thought at the beginning of this work at Woods Hole, Massachusetts, that both the spiny dogfish (Squalits acantJiias), and the smooth dogfish (Miistcliis canis} could be used. However, the spiny dogfish would not live in the aquaria. Perhaps this may be caused by normal summer salt water temperature in Woods Hole being lethal for the spiny dogfish but not for the smooth dogfish. This was suggested by William Schroeder, Jr., of the Woods Hole Oceanographic Institute who in conversation with the authors pointed out the coastwise migrations of the spiny dogfish paralleling temperature isotherms. Since Sqiialiis proved unsatisfactory, Alitstelits canis, collected at Woods Hole, Massachusetts, wrere used in these experiments. A total of 23 adult animals were 100 TOOTH SUCCESSION IN MUSTELUS CANIS 101 used, one group of 12 in the summer of 1946 and a second group of 11 in the summer of 1947. The animals ranged in size from H1/^" to 39" with the majority being over 24" in length; 11 were males, 12 females. They were kept in a large paraffin-lined cement tank supplied with running sea water and were fed every other day on chopped fish. The dogfish were anesthetized by cooling in ice water according to the method of Parker (1937) and a varying number of teeth, as described below, were removed with forceps from the lower jaws. In order to follow the movements of the remain- ing teeth they were marked with silver nitrate solution precipitated with stannous chloride. While the stain subsequently was worn away from the surface of the teeth, sufficient amounts remained on the sides of the teeth to mark them adequately. This species has pavement teeth, somewhat diamond-shaped and arranged in com- pact rows (see Fig. 3). Sections were made of the jaws using both paraffin and celloidin technics following decalcification. Mallory's stain as well as haemo- toxylin and borax-carmine was used. We wish to thank the Woods Hole Oceanographic Institute and the Marine Biological Laboratory for the use of their facilities. EXPERIMENTS AND OBSERVATIONS The preliminary experiments were designed to determine if tooth replacement occurs in Mustchts. For this purpose 12 animals were divided into four groups. In the first group of three animals, six teeth of the first row in the mid-line of the lower jaw were removed. These animals died six, eight, and 11 days respectively after the operation. The cause of death was not ascertained although it probably was not the result of the operation since one of the unoperated controls died during the same period. The teeth were not replaced in this period. Serial sagittal sec- tions at 10^ revealed no change had taken place and the jaws presented the usual appearance with tooth buds in successive stages of development posterior to the area of the erupted teeth. The second group contained four animals from each of which 22 teeth were extracted from a triangular area, five rows deep; the apex of the triangle pointed posteriorly. Figure 1 is a photograph of a jaw of this group. Two of the fish died before replacement occurred, after eight and 12 days respectively. The re- maining two replaced the teeth within 50 days. Figures 2 and 3 are photographs of the jaw of one of these latter fish. It can be seen that the replaced teeth are arranged in the normal pattern. Sections of these jaws also were normal in appearance (Fig. 4). The third group of three animals had the first row of teeth removed. Two died on the following day but the third had replaced the teeth when examined 93 days later. The rate of replacement was not obtained for this animal. The fourth group consisted of the two control animals. Both were anesthetized by cooling but were not operated upon. One died the following day, the other in 18 days. The cause of death was not determined although the method of anes- thetizing might have been a contributory cause. The second series of experiments were designed to discover the manner in which the tooth replacement occurred. The 11 dogfish of this series were divided into three groups. In the first group of four, each of the fish had 12 teeth in all ex- 102 JOHN D. IFFT AND DONALD J. ZINN PLATE I TOOTH SUCCESSION IN MUSTELUS CANIS 103 tracted from the anterior first two rows in the center section of the lower jaw. The remainder of the teeth with the exception of the two first rows lateral to the extracted area were marked with silver nitrate. One animal died on the ninth day and no change in the teeth was found. The other three were examined 25 days later and all had replaced the extracted teeth with teeth bearing silver nitrate marks. In addition, the teeth lateral to the extracted area, previously unmarked, now were replaced by teeth bearing silver nitrate markings. This would seem to indicate, therefore, that within the 25-day period, two rows of teeth moved forward and replaced the former first two rows. The second group of this series consisted of five animals in which either two, three, or four rows in the center section were removed, and the tooth-bud area back of the region from which the teeth had been extracted, was cauterized with an electric cautery. Four of these animals died in three, five, 12 and 13 days respec- tively. The remaining animal of the group lived and was killed 25 days later. In the three cauterized dogfish living 12, 13, and 25 days the tooth area in front of the region cauterized was disorganized: many teeth in addition to those extracted had fallen out and only a few scattered teeth remained in the center area. Figure 6 is a photograph of the jaw of one of these fish. No replacement of teeth had occurred in any of this group including the animal killed after 25 days. A section (Fig. 7) from this latter dogfish taken through the cauterized area and the region anterior to it shows the drastic disorganization resulting from the cauterization. The tooth buds were destroyed and parts of the jaw cartilage degenerated. The oral epithelium and underlying connective tissue appeared to be sloughing off. The third group contained two animals in which all but the first two rows of teeth were marked with silver nitrate but no teeth were extracted. Both of these fish died six days later ; there were no observable changes in the teeth. Certain general observations of the teeth were made. It was found that the first or outermost row of teeth was irregular while the preceding rows are quite regular. This would seem to indicate that the teeth are normally lost singly from the first row as has been observed in other species. Great regularity was observed in the posterior rows and in the animals examined there were no indications of tooth-loss except in the first row. The number of exposed rows of teeth varied from eight to 11. No sexual differences in the teeth were seen. The arrangement of the teeth in the upper jaws appeared to be similar to that of the lower jaws. PLATE I FIGURE 1. View of jaws of dogfish showing triangular area in center of lower jaw from which teeth have been extracted. About one-third natural size. FIGURE 2. Dorsal view of jaw of animal in Figure 1 fifty days after removal of teeth show- ing the complete replacement of the teeth. About one-third natural size. FIGURE 3. Ventral view of jaw in Figure 2. About one-third natural size. FIGURE 4. A sagittal section at 10 microns of the jaw seen in Figure 2. Tooth buds can be seen back of the erupted teeth. About X 10. FIGURE 5. A view of the tooth bud area from Figure 4. About X 33. FIGURE 6. A dorsal view of a jaw in which 4 rows of teeth were removed in the center section and the tooth buds back of this region were cauterized. No replacement had occurred after 25 days. About one-third natural size. FIGURE 7. A sagittal section at 10 microns of the jaw seen in Figure 6, showing the dis- organization resulting from the cauterization. About X 10. 104 JOHN D. IFFT AND DONALD J. ZINN Tooth-bud areas were never found except behind the tooth-bearing region. Figure 5 is a photograph of the tooth-bud area. The tooth buds can be seen to be progressively larger and more mature in a postero-anterior direction. Particular care was taken to search for buds underlying the outermost rows but none were found. It would appear, therefore, that the only source of new teeth are these buds back of the erupted tooth area. CONCLUSIONS AND DISCUSSIONS From the experiments described above it seems apparent that in Mustelus canis teeth can be replaced and that this replacement occurs in the manner hypothecated by Owen ; that is, by the moving forward of the teeth from the rear. The fact that marked teeth from posterior areas were seen later to occupy areas where teeth had been removed seems conclusive evidence in favor of Owen's view. It is not certain from the experiments what the normal rate of replacement is since the animals which were to have been used to test this point died before such information could be obtained. However, the rate of replacement in the operated animals was quite rapid, being approximately of the order of one row replaced in ten to twelve days. The experiment in which the tooth buds back of the center area of the jaw were removed by cautery was done to determine whether replacement occurred in the absence of the posterior tooth buds. In the one surviving animal replacement had not taken place although in the same length of time non-cauterized dogfish did replace teeth. While the experiment apparently bears out the role of the posterior tooth buds in replacement it may be criticized on the ground that the unexpected general disorganization and degeneration resulting from the cauterization would prevent replacement from any source. However, even if this experiment is omitted from consideration, there is sufficient evidence from the other experiments to support the contention that Owen's hypothesis -is correct. From a study of Cawston's papers it would appear that his views are based on gross examination only and without a study of histological sections. Otherwise it is difficult to account for his statement (1941a) : "New tooth formation behind the normal number of rows of teeth in species of shark has never been observed, though dental germs should be present if the alleged replacement of teeth by revolv- ing of the gum forwards ever occurred in adult specimens." In the same paper he also states: "At the anterior border of the teeth of Mustelus canis (Mitch.) one sees round or oval dental germs in process of development into the flattened closely set teeth of the adult, which reveal the characteristic wrinkled surface very early." As we have noted earlier, and as can be seen from the photographs of the sections (Figs. 4, 5), tooth buds are found back of the erupted teeth and are not found in the front region of the jaw. There is no evidence that new teeth are being formed in the front row of Mustelus. In a later paper (1944), Cawston states that there is no provision for replace- ment of lost teeth in selachians and that growth may continue throughout life. In earlier papers (1939, 1941a) he considers that a tooth is renewed at the site where one is lost. He considers that this replacement obtains by vertical succession (1941b). Unless we are misinterpreting the statements it would appear that Cawston's viewpoint has changed from a possibility of vertical succession in tooth replacement to the hypothesis that no replacement of any type occurs. TOOTH SUCCESSION IN MUSTELUS CANIS 105 Other observers besides Owen have concluded by studying the morphology of the jaw that replacement occurs by the forward movement of the back teeth. For example, Budker (1938) states: "Lorsque la dent est tombee, une autre, dite 'dent de remplacement' et provenant des rangees de remplacement disposees derriere les rangees fonctionelles, vient prendre sa place." This author also observed that tooth buds did not develop at the site of the lost tooth. The cause of the falling-out of the teeth was also studied by Budker in various species such as Scyliorhinus canicula. He accounted for this loss by the destruc- tion of the dentinal basal plates which anchor the tooth in the underlying connective tissue by specialized cells similar to osteoclasts which cells also reduce the dentine of the older tooth as a whole. Benzer (1944), on the other hand, reports that the dentine of Mustelus grows progressively thicker in older teeth. He did not note that the dentine was later destroyed. The jaws of ten other species of sharks were examined by the authors through the courtesy of Mr. Schroeder at the Museum of Comparative Zoology at Harvard University. Included in the group were three species of the Port Jackson shark (Cestracion or Heterodontus} which have pointed biting teeth in front and flat crushing teeth in the remainder of the jaw. It was observed, however, that the teeth in any section of the jaw are the same in an antero-posterior direction and consequently could be replaced in the manner described for Mustelus. No mor- phological indications were found in any of the other species examined contradicting Owen's hypothesis. SUMMARY 1. Twenty-two teeth extracted in a triangular area five rows deep from the front of the tooth-bearing region of the lower jaw of Mustelus canis were replaced within 50 days. 2. Marking of the posterior teeth with silver nitrate indicated that extracted teeth were replaced from behind by these marked teeth. The replacement rate was approximately one row in 10 to 12 days. 3. Tooth buds were found only back of the erupted teeth and never elsewhere. 4. Destruction of the tooth buds by cautery prevented replacement. 5. It is concluded that Owen's hypothesis of the replacement of sharks' teeth by the forward movement of the posterior teeth is correct and that Cawston's objections to the theory are not tenable. LITERATURE CITED BENZER, PAUL, 1944. Morphology of calcification in Squalus acanthias. Copcia, 217-224. BREDER, C. M., JR., 1942. The shedding of teeth by Carcharias littoralis (Mitchill). Copeia, 42-44. BUDKER, P., 1938. Les cryptes sensorielles et les denticules cutanes des Plagiostomes. Ann. Inst. Occanogr., 18 : 207-288. CAWSTON, F. G., 1939. Succession of teeth in sharks, Selachii. Jour, of Trop. Med. (London}, 42: 7. CAWSTON, F. G., 1940a. A consideration of the replacement of teeth in sharks and fangs in snakes. Dental Record (London}, 60: 435-439. CAWSTON, F. G., 1940b. A consideration of the alleged succession of teeth by revolving of the tooth-bearing area. So. Ajric. Dental Jour., 14 : 412-413. 106 JOHN D. IFFT AND DONALD J. ZINN CAWSTON, F. G., 1940c. The dentition of fishes and reptiles with special reference to the re- placement of teeth. Indian Jour. Vet. Sci., 10: 239-300. CAWSTON, F. G., 1941a. A consideration of the teeth of embryonic and immature skates and rays in relation to the successional theory of teeth. So. Afric. Dental Jour., 15 : 95-98. CAWSTON, F. G., 1941b. A note on the development and survival of teeth, especially Selachian. Dental Record (London), 61 : 291-293. CAWSTON, F. G., 1941c. Further observations on the dentition of Batoidei. Dental Record (London), 61: 327-328. CAWSTON, F. G., 1944. The shedding of selachian teeth and its relation to tooth replacement in fishes and reptiles. Copcia, 184-185. CAWSTON, F. G., 1945. Consideration of the successional theory as applied to the dentition of Pagrus nasutus (the Mussel-Crusher) and some reptiles. Trans. Roy. Soc. So. Africa, 30 : 267-270. OWEN, RICHARD, 1866. Anatomy of Vertebrates. London. PARKER, G. H., 1937. Integumentary color changes of Elasmobranch fishes especially of Mus- telus. Proc. Amcr. Phil. Soc., 77: 223-247. POSTEMBRYONIC GROWTH CHANGES IN THE ISOPOD PENTI- DOTEA RESECATA (STIMPSON) WITH REMARKS ON THEIR TAXONOMIC SIGNIFICANCE ROBERT J. MENZIES AND RICHARD J. WAIDZUNAS Pacific Marine Station, College of The Pacific, Dillon Beach, California INTRODUCTION This study was the result of the observation that the number of setae of the seventh peraeopod of Pentidotea resccata (Stimpson) (Valvifera: Idotheidae) was markedly variable. It was decided to conduct an investigation including features other than peraeopod setal number, in order to determine which features remained relatively stable and were thus of specific significance in the classification of the marine idotheids. The nature of the variations in certain features was found to be directly related to the size of the specimens, and thus to growth ; and it is believed that these variations are of basic significance to isopod taxonomy. The features at present used to distinguish marine isopods of North America of the family Idotheidae include the number of segments of the flagellum of the second antennae, the number of segments of the palp of the maxilliped. and the shape of the posterior margin of the telson (Richardson, 1905 and references, pp. 346-408). It is most significant that, in the species investigated, it was these features that demonstrated the greatest degree of growth variation. The material consisted of ten adult and seven juvenile specimens which ranged in length from 5.2 mm. to 43.0 mm. and of ten far advanced embryos of 2.2 mm. length, removed from the marsupium of an adult female of the species. The speci- mens were collected by the writers during the summer of 1947 from eelgrass, Zostera sp., located on the sand flats of Tomales Bay. Marin County, California, where the species is fairly abundant. The head. The most stable features of the head during growth included the shape and location of the eyes (Fig. 8) and also the relationship of the frontal laminae to one another and to the anterior-dorsal border of the head. In embryos, however, these features were not developed. The number of segments to the flagellum of the second antennae was found to increase in direct proportion to the size of the specimen at least until adult status was reached. This phenomenon was observed in part by Hale (1946, Fig. 19, p. 193) in his description of Antarcturns horrid-its Tattersall (Arcturidae). Em- bryos 2.2 mm. long had two segments to the flagellum of the second antenna. In a specimen of 5.2 mm. and one of 6.0 mm. length the number of segments was four. In two specimens of 8 mm. length, one of 9 mm. length and in one specimen of 10 mm. length the number of segments was seven ; while in one specimen of 9.5 mm. length the number of segments was eight. Even among the larger specimens the number of segments of the flagellum was observed to vary considerably. The length of the flagellum appears to be proportional to the body length in the juvenile 107 108 ROBERT J. MENZIES AND RICHARD J. WAIDZUNAS PLATE I Pentidotea resccata FIGURE 1. Abdomen, dorsal view, embryo, 2.2 mm. length. Magnification as indicated. FIGURE 2. Abdomen, dorsal view, juvenile, 5.2 mm. length. Magnification as indicated. FIGURE 3. Abdomen, dorsal view, small adult male, 9.5 mm. length. Magnification as per Figure 2. FIGURE 4. Flagellum second antenna, left, embryo, 2.2 mm. length. Magnification as per Figure 1. FIGURE 5. Flagellum second antenna, left, juvenile, 5.2 mm. length. Magnification as per Figure 1. FIGURE 6. Flagellum second antenna, left, small adult male, 9.5 mm. length. Magnifica- tion as per Figure 1. GROWTH CHANGES IN THE ISOPOD 109 specimens of P. rcsccafo, while in the adult specimens the length of the flagellum varied. In specimens of 6.5 mm. and below, the palp of the maxilliped was four jointed. More developed specimens had a maxilliped palp of five segments. At present the only character used to distinguish the genus Pentidotca (Richardson, 1905, p. 368) from Idothea' (ibid., p. 356) is the presence of a five-jointed palp in the former and of a four-jointed palp in the latter (see also Light, 1941, p. 87). It seems evident to the writers that either Pentidotca must be considered a synonym of Idothea or that the generic differences must be redefined. The increase in setae on the tip of the endopodite of the maxilliped was found to be correlated directly with the size of the specimen (compare Fig. 17 and Fig. 19). The same was true of the "hairiness" of the median border of the palp of the maxilliped. Only the presence of a single coupling-hook on the median border of each maxilliped was found to be constant (Figs. 17 and 19, "x"). The perion. The relationship in length of the lateral border of the epimeral segments of the perion to the length of the lateral border of the perion segments themselves appeared to be constant, yet measurements made at the second and third perion segments showed considerable variation. This variation did not cor- relate directly with a size increase of the specimens and the writers believe that the difficulty in obtaining accurate measurements of these structures accounts for the irregularity. Observations indicate that the seventh perion segment remained in an undeveloped state in juvenile animals as large as 5.2 mm. Its retarded devel- opment was best indicated on embryo specimens. The general narrow shape of the animal was maintained in animals of all sizes. Ovigerous specimens showed a distinct lateral widening of the segments of the perion concerned with the marsu- pium development. The peraeopod. The number of setae on the seventh peraeopod was examined carefully in the hope that a very definite non-variable structural feature could be found. The number of setae on the ventral surface of the propodus was observed to be directly proportional to the size of the animal. Three distinct types of setae are discernible; a "saw-toothed" seta (Fig. 11), a "file-toothed" seta (Fig. 10). and a "simple" seta (Fig. 12). A once specialized seta observed on the propodus of the seventh peraeopod of a small animal was always found without modification, other than increase in size, on the propodus of the seventh peraeopod of a larger animal. The "saw-toothed" type seta was constant in number and location. The "file-toothed" type seta was more numerous on the propodus of the seventh peraeopod of larger specimens and the same is true of the "simple" type setae which most frequently surrounded the "file-toothed" seta. Evidence indicates that the "file-toothed" seta is developed, in part at least, from one of the "simple" type. It was found that the seventh peraeopod was weakly developed in very small specimens (below 6.0 mm. length) ; whereas the first to the sixth peraeopods were FIGURE 7. Flagellum second antenna, left, adult male, 20.5 mm. length. Magnification as per Figure 2. FIGURE 8. Eye, left, lateral view, adult male, 20.5 mm. length ; "P" posterior, "A" anterior, "D" dorsal, "V" ventral. Magnification as per Figure 2. FIGURE 9. Anterior-dorsal border and first two frontal laminae, adult male, 20.5 mm. length. Magnification as per Figure 2. 110 ROBERT J. MENZIES AND RICHARD J. WAIDZUNAS PLATE II Pentidotea rcsecata FIGURE 10. File-toothed seta of ventral surface of propodus, see "b" of Figure 15. Mag- nification as indicated. FIGURE 11. Saw-toothed seta of ventral surface of propodus, see "a" of Figure 15. Mag- nification as per Figure 10. FIGURE 12. Simple seta of ventral surface of propodus, see "c" of Figure 15. Magnifica- tion as per Figure 10. GROWTH CHANGES IN THE ISOPOD 111 well developed. The retardation in the development of the seventh peraeopod has been observed in other isopods (So'mme, 1940, Limnoriidae, p. 158; Faxon, 1882, Asellidae, pi. vi, Fig. 19; Hult. 1941, Parasellidae, p. 39). The telson. The concavity of the posterior margin of the telson has been regarded as the most diagnostic and key feature of this species. Actually, how- ever, the margin was found by the writers to change very gradually from one with a convex posterior border in the embryo (Fig. 1) to a slightly concave margin in specimens of 6.5 mm. in length (Fig. 2) ; until, when the adult condition is reached (Fig. 20), the concavity is most developed. The uropod conforms to the shape of the telson and therefore varies in ac- cordance with its size and shape. The number of movable setae at the lateral articular distal border of the penultimate uropod segment varied in number from one to two regardless of size or sex of the specimen. It is evident from the above observations that very young specimens of Penti- dotea resecata might very well be placed in the genus Idothea and considered new to science by an investigator unaware of the developmental nature of the maxil- liped palp. Such would be true at least as long as the two genera Idothea and Pentidotea remain so briefly designated. Indeed one writer, Fee ( 1926, p. 18, Fig. 12) did just that in describing Idothea rnjescens from specimens which appar- ently are juvenile specimens of Pentidotea resecata (Stimpson). The suggestion of course from the above is that authors of new species of Idotheid genera (as well as isopods in general) not only indicate the measurements of the types but also give measurements of all specimens figured or described in the text. To date such a procedure has been followed by only a very limited num- ber of workers and even then without any marked degree of consistency. SUMMARY In an attempt to find constant characteristics which may be relied upon as specifically diagnostic in the marine isopod Pentidotea resecata, the following fea- tures proved to be especially significant constant features regardless of the size of the specimen: (1) structural interrelationships of the frontal laminae, (2) epi- meral plate length in relation to the length of the lateral border of the corresponding perion segment, (3) the character of the setation and certain features of peraeopod morphology, (4) general body shape. Features showing numerical increase which was found to be directly propor- tional to the size of the animal and thus believed to be of very limited taxonomic utility include : ( 1 ) number of segments to the flagellum of the second antennae, (2) number of segments to the palp of the maxilliped, (3) number of setae of the maxilliped and of the peraeopods. FIGURE. 13. Seventh peraeopod, right, juvenile, below 6.0 mm. length. Magnification as per Figure 1. FIGURE 14. Seventh peraeopod, left, juvenile, above 6.0 mm. length. Magnification as per Figure 1. FIGURE 15. Propodus and dactylus of seventh peraeopod, small adult male, 9-.S mm. length. Magnification as per Figure 1. FIGURE 16. Ventral border of propodus of seventh peraeopod of large adult male, 20.5 mm. length. Magnification as per Figure 1. 112 ROBERT J. MENZIES AND RICHARD J. WAIDZUNAS PLATE III 20 Pentidotea rcsccata Magnification as indicated FIGURE 17. Maxilliped, left, small adult male, 9.5 mm. length ; "x" is the coupling-hook. FIGURE 18. Uropod, left, small adult male, 9.5 mm. length. FIGURE 19. Maxilliped, left, juvenile, 5.2 mm. length; "x" is the coupling-hook. FIGURE 20. Dorsal view, adult male, 20.5 mm. length. GROWTH CHANGES IN THE ISOPOD 113 It would seem to be necessary, in view of the findings, to reexamine the status of species and genera which owe their existence exclusively or in part to charac- teristics here shown, in this species at least, to be variable in different age groups. One species IdotJica rnjcsccns Fee, apparently based on immature specimens, is considered a synonym of Pcntidotca rcsccata (Stimpson). LITERATURE CITED FAXON, WALTER, 1882. Selections from embryological monographs, No. 1, Crustacea. Man. Mns. Coin p. Zool. Harvard, Vol. IX, No. 1. FEE, A. R., 1926. The Isopoda of Departure Bay and vicinity' with descriptions of new species, variations and color notes. Contr. Canadian Biol. and Fish., 3: 13-35. HALE, HERBERT M., 1946. Isopoda-Valvifera, B.A.N.Z. Antarctic Research Expedition 1929- 1931, Reports— Scries B (Zoology and Botany), V: 161-212. HULT. JORAN, 1941. On the soft-bottom isopods of the Skager Rak. Zoologiska Bidrag Fran Uppsala, 21 : 1-234. LIGHT, S. F., 1941. Laboratory and field tc.rt in invertebrate zoology, pp. 1-232. Stanford University Press. RICHARDSON, H., 1905. Monograph on the isopods of North America. Bull. U. S. Nat. Mus. No. 54, pp. 1-727. S0MME, OLAUG M., 1940. A study of the life history of the gribble Limnoria lignorum (Rathke) in Norway. Sacrfrvkk ar Nvtt Maqasin for Natnrvidcnskapcnc, 81: 145- 205. THE UTILIZATION OF SUGARS AND OTHER SUBSTANCES BY DROSOPHILA CHARLES C. HASSETT From the Medical Division, Army Chemical Corps, Army Chemical Center, Maryland Studies have been made of the use of carbohydrates and other food material by several insects, e.g. the honey-bee (Bertholf, 1927; Phillips, 1927; Vogel, 1931), the blow-fly (Fraenkel, 1936, 1940), the Mexican fruit fly, Anastrcpha ludcns (Baker et al., 1944), and a number of others. The reviews of Trager, 1941 and 1947, and Uvarov, 1928, furnish extensive references. Drosophila melanogastcr seems, however, to have escaped attention in this connection heretofore. Experi- ments have now been made on the ability of this fly to utilize a large number of carbohydrates and related compounds, as well as some substances of other classes. In addition, an estimate of the relative nutritional efficiency of these substances has been made. MATERIAL AND METHODS Adults. To rear flies for these tests, the standard corn meal, agar, and sugar medium, in half-pint milk bottles, with an inoculation of fresh yeast, was used. As soon as the larvae reached full size and began to leave the medium, a layer of sawdust was added. This prevented the adults from obtaining any food until they were transferred to test bottles. The flies were used as soon as possible, never more than 24 hours after emergence. Test bottles were set up as follows : solutions to be tested were put into 10 ml. vials stoppered with a roll of filter paper which served as a wick. About 50 ml. of 1 .5 per cent agar was poured into a half-pint milk bottle : this maintained moisture and facilitated counting dead flies. For non-fermentable substances the vials were simply embedded in the agar base, otherwise they were wrapped in strips of paper toweling to form a plug for the milk bottle. This stopper could be changed readily and fresh solutions offered the flies, eliminating the complications of bacterial growth. It was found desirable to transfer the flies to fresh bottles after about two weeks if they survived, since otherwise dead flies were eaten by larvae and counting became difficult. One hundred flies were used for each test. They were divided among three bottles for convenience in counting. The dead flies in the bottles were counted each day. Initially the number of days required for 50 per cent of the flies to die was used as a means of evaluating the degree of utilization of a substance, but it was found that many of the materials having low values could not be differentiated without making counts at shorter intervals, which was impractical. A better index was achieved by totalling the daily survival percentages and using the result- ing number as an index of nutritive value. For example, when formic acid was fed to flies, all survived the first day, 43 per cent the second, none the third. The "score" was, therefore, 143. 114 UTILIZATION OF SUBSTANCES BY DROSOPHILA 115 Larvae. Three of the common sugars were tested on sterile larvae. Eggs were obtained by allowing flies to deposit them on small dishes of agar for about two hours ; the eggs were then collected and sterilized by immersion in 85 per cent alcohol for 10 minutes and transferred to shell vials containing 10 ml. of sterile culture medium. Each vial contained the following: powdered agar, 150 mg. ; dried brewer's yeast, 50 mg. ; sugar, 50 mg. ; distilled wrater, 10 ml. The same medium, minus sugar, is the "starvation diet" of Beadle et al. (1938), and this, together with their "adequate" diet of 2 per cent yeast, was used for comparison with the sugar supplemented media. Each vial was seeded with 40 eggs and maintained at 25° C. After the forma- tion of pupae, the vials were examined daily and when all the adults had emerged, counts were made to ascertain: (a) number of adults; (b) number of pupae not completing metamorphosis; (c) number of unhatched eggs. The larvae some- times churned the medium so that unhatched eggs were lost, but a large number of vials were found with eggs and egg cases undisturbed ; from these it was calcu- lated that an average of 4 eggs per vial failed to hatch. The numbers of eggs given in Table IV represent, therefore, 36 eggs per vial. RESULTS If flies are put into dry bottles, they are all dead within 48 hours : their score is 65. If a layer of agar is put into the bottles, the score is 110; if, in addition, a vial of distilled water is supplied, the score rises to 120. On standard corn meal, agar, and sugar medium, they live a long time: the score for that is 4418. Table I shows the scores calculated as described above, and the day on which 50 per cent of the flies in each test were left alive. From the data it can be seen that adults of Drosophila uiclanogaster can live on a large number of substances in several classes of chemical compounds, but that the sugars and their close deriva- tives are best for maintaining these insects. Even in the sugars, each subgroup is found to contain substances which cannot be utilized. If flies are supplied with pure sugar solutions, they survive for periods de- pendent upon the degree of utilization of the sugar and its concentration. Poorly utilized sugars like xylose sustain life only for short periods, even in concentrated solutions, while well utilized sugars like sucrose maintain life for longer and longer periods as the concentration increases. The limit in this direction seems to be reached between M/10 and M/5 for sucrose, for further increases in the concen- tration fail to increase survival. Groups of flies tested with concentrations of sucrose as follows: M/5, M/2. M, and 2M gave results no better than M/10, and indeed, the higher concentrations showed a tendency to decrease the life span slightly, but other factors such as osmotic pressure might enter to account for this. The substances which were tested gave scores ranging from that of rafrlnose. 2600, to guanine, 13. as shown in Table I. Three groups of substances can be distinguished : Group 1. Substances which appear to be inert, with scores close to that of water. Because of the natural variability of different batches of flies, and tem- perature conditions as noted previously, one could not expect sharply demarcated groups, and in fact there is a continuous gradation of scores. Probably all sub- stances with scores between 100 and 150 should be called inert. This group would 116 CHARLES C. HASSETT TABLE I The survival of adult Drosophila melanogaster on various substances, given as summations of daily survival percentages (A), and as days required for 50 per cent mortality (B). Except where noted, solutions are M/10. Each test represents 100 flies. A B A B A B Controls Trisaccharides Carboxylic acids Dry bottle 65 1 Ramnose 2600 28 Butyric 205 3 Bottle with agar 110 2 Melezitose 2432 26 Acetic 202 3 Water (442 flies) 120 2 Raffinose, M/20 1460 15 Formic 143 2 Standard medium 4418 45 Melezitose, M/20 909 14 Valeric 133 2 Propionic 113 2 Pen loses Polysaccharides Lactic, M/2 377 5 M/5 327 4 D-Xylose, M/2 680 tj Dextrin, 1% 778 8 M/10 208 3 Ribose 340 4 Starch, 1% 334 4 M/20 153 2 D-Xylose 211 3 Glycogen, 1% 298 4 Pvruvic, M/5 100 2 L-Fucose 169 3 Inulin, sat. sol. 160 7 M/10 90 2 D-Arabinose 166 3 M/20 75 . 7 D-Xylose, M/20 131 2 Alcohols Glycolic 107 2 L-Arabinose, M/2 101 2 Levulinic 97 2 i.-Rhamnose, M 80 2 Ethyl, M/5 172 3 Succinic 367 4 D-Arabinose, M/2 69 2 Ethyl, M/2 99 2 Pimelic 160 3 L-Rhamnose 68 2 Ethyl, M/10 93 2 Glutaric 124 2 L-Arabinose 64 2 w-Butyl 102 2 Malonic 88 2 tert-Amyl 100 2 Azelaic 80 2 Hexoses w-Amyl 99 2 Adipic 70 2 iso-Butyl 96 2 Oxalic 20 1 D-Fructose 1855 18 sec-Butyl 95 2 Malic 234 3 Glucose 1521 16 tert-Butvl 50 Aconitic 162 3 D-Mannose 1415 14 I laconic 158 3 Polyhydric alcohols Fumaric 151 2 D-Fructose, M/20 1033 11 Maleic 120 2 D-Galactose 945 9 Glycerol 1369 14 m-Tartaric 97 2 Gluctose, M/20 663 7 Mannitol 729 6 Citric 413 4 D-Galactose, M/20 235 3 Inositol 572 6 L-Sorbose 191 3 Sorbitol 358 5 Salts L-Sorbose, M/2 68 2 Adonitol 308 4 w-Erythritol 170 3 Sodium succinate 115 2 Disaccharides Dulcitol, M/5 119 2 Sodium citrate 105 2 Dulcitol 108 2 Sodium lactate 115 2 Sucrose 2218 24 Arabitol 107 2 Sodium malonate 99 2 Sucrose, M/5 2141 23 w-Erythritol, M/2 86 2 Maltose 2040 17 £e»ta-Erythritol, Sucrose, M 2010 22 M/2 51 1 Trehalose 1864 21 M/10 40 1 Maltose, M/20 1668 16 Sucrose, M/2 1624 20 Glycols Sucrose, 2M 1516 16 Sucrose, M/20 1506 14 Propylene 172 3 Melibiose 1237 12 Diethylene 160 ? Sucrose, M/40 382 4 Ethylene 124 2 Lactose 179 3 Dipropylene 60 2 Lactose, M/2 153 2 Lactose, M/20 100 2 Cellobiose 84 2 Cellobiose, M/2 40 1 UTILIZATION OF SUBSTANCES BY DROSOPHILA TABLE I — Continued 117 A B A B Amino acids Miscellaneous Glycine 202 3 Yeast-sucrose, equal parts, dry 2074 24 DL-Methionine 195 3 alpha-Methylglucoside 639 6 L-Glutamic acid 124 2 Yeast, fresh 2% suspension 165 3 DL-Aspartic acid 122 2 Parenamine, 1% (proprietary casein 147 2 DL-Alanine 108 2 hydrolysate) Beta alanine 108 2 Amygdalin 139 2 L-Cystine (sat. sol.) 102 2 Yeast, fresh dry 128 2 L-Cysteine 101 2 Catechol 126 2 DL-Glutamic acid 101 2 Albumin, 1% 117 2 DL-Threonine 101 2 Lecithin, 1% 116 2 L-Arginine 95 2 Charcoal, dry 107 2 DL-Phenylalanine 93 2 Glucosamine 106 2 i.-Histidine 89 2 Casein, dry 106 2 DL-Isoleucine 72 2 Gulonic lactone, 4% 105 2 L-Lysine 71 2 Magnesium hexosediphosphate 104 ? L-Proline 70 2 Glucoheptonic lactone, 4% 100 2 L-Leucine (sat. sol.) 67 2 D-Galacturonic acid 98 2 L-Hydroxyproline 66 2 Xylan (sat. sol.) 94 2 DL-Tryptophane (sat. sol.) 63 2 Sucrose acetate 93 2 L-Tryptophane (sat. sol.) 62 2 Mucic acid 90 2 L-Tryosine (sat. sol.) 57 2 Calcium glucoheptonate, 4% 84 2 DL-Leucine (sat. sol.) 55 2 Nucleic acid (sat. sol.) 83 2 DL-Norleucine 55 2 Sodium nucleate, 1% 82 2 DL-Serine 51 1 Yeast, dried, suspension 80 2 DL-Valine 51 1 Milk, powdered 78 2 Yeast, dried 64 2 Starch, Lintner, dry 45 1 Xanthine (sat. sol.) 15 1 Guanine 13 1 Uracil 13 1 include not only substances not utilized when ingested, but those which might be utilized somewhat, were they not also slightly repellent so that the flies do not drink the solutions. Group 2. Substances which are utilized by Drosophila, shown by scores higher than that of water. This group includes anything \vhich prolonged the life of the flies in any degree, from such poor nutrients as xylose to the best of the higher sugars. Sugars, particularly the mono-, di-, and trisaccharides, lead in this group, but moderately good results were obtained with dextrin, glycerol, mannitol, inositol, and alpha-methylglucoside. Some prolongation of life was obtained with starch, glycogen. sorbitol, adonitol, and with butyric, acetic, lactic, succinic, malic, and citric acids. The only amino acids showing any usefulness were methionine and glycine. A few other substances, such as ethyl alcohol, propylene and diethylene glycol, aconitic and itaconic acids, were doubtful. Proteins alone, e.g. albumin, were of no value, nor were such products as casein, yeast, or milk. The low values obtained with dry yeast (64) and starch (45) prompted a test with an inert powder. Charcoal was selected, and the relatively high score (107) suggests that there is something definitely harmful in dry starch and yeast, but whether its nature is 118 CHARLES C. HASSETT physical or chemical has not yet been ascertained. Dry yeast mixed with an equal amount of powdered sugar, on the other hand, makes an excellent food, giving a score of 2074. In order to obtain a more exact comparison of nutritive value among some of the commoner sugars, seven were tested under identical conditions. The molarities of the solutions were chosen to equate the mono- and disaccharides with respect to weight per unit volume. Lactose, M/20, and xylose, M/10, showed no nutritive value, and galactose, M/10, very little. The other sugars were, in order of increas- ing nutritive value: glucose, M/10, 1375; sucrose, M/20, 1440; maltose, M/20, 1720; fructose, M/10, 1833. These scores and the curves of Figure 1 show there was little variation in this group, also that the results were nearly the same as those shown in Table I for the larger series of experiments. The longevity of flies fed on di- and trisaccharides was compared, under identical conditions, with that of flies fed on the constituent monosaccharides. Table II TABLE II A comparison of sonic di- and trisaccharides with their hc.rosc constituents. Each pair was run with flies from the same batch, under identical temperature conditions. Substance Cone. Score Substance Cone. Score Sucrose Fructose Glucose M/20 M/20 M/20 1455 1421 Raffinose Fructose Glucose Galactose M/20 M/20 M/20 M/20 1460 1492 Maltose Glucose M/20 M/10 1466 1363 Melezitose Glucose Fructose M/20 M/10 M/20 1257 1285 Trehalose Glucose M/20 M/10 1064 1285 shows that there was little difference in the results, a mixture of fructose and glucose being as good as an equivalent amount of sucrose, etc. Larvae. The results obtained in rearing sterile larvae on yeast and on yeast- sugar mixtures are given in Table IV. No significant difference was found in the number of flies produced by the three sugar media. A significant difference was found when adequate amounts of yeast were supplied, and an increase in the amount of sugar might have increased the yield. Since the object of the experi- ment was to differentiate among the sugars, if possible, by putting the larvae into somewhat unfavorable conditions, this was not done. Flies consuming fructose developed more rapidly than those on sucrose and glucose, though less rapidly than those having a full yeast diet. Group 3. Substances which have low scores, and are therefore toxic or repel- lent. Flies in a bottle having a layer of agar live almost as long as if they are supplied with drinking water. Substances which are merely repellent will, there- fore, be difficult to separate from those which are nutritionally inert. Toxic sub- UTILIZATION OF SUBSTANCES BY DROSOPHILA 110 stances should give much lower scores and be accordingly easier to single out. Guanine, for example, is clearly toxic. Variations in toxicity and in the flies themselves naturally militate against any sharp distinction, so that further experi- ments were performed to bring out hidden differences. The difference between toxic and repellent substances can sometimes be demonstrated readily by offering a questionable solution alone and in combination with a separate vial of water. Rhamnose alone, for example, gave a score of 68, but when the flies were offered 10 15 20 DAYS OF SURVIVAL 30 FIGURE 1. The duration of life of adult fruit flies fed solutions of various sugars. Lactose, M/20, ; water, 3; xylose, M/10, X; galactose, M/10, O; glucose, M/10, o; sucrose, M/20, •; maltose, M/20, + ; fructose, M/10, O. an additional vial of water, the score rose to 100. No discrimination was evidenced, and presumably the flies lived longer because they drank less of the rhamnose solu- tion. When repellency is suspected, however, something must be used to insure the ingestion of the solution. Vogel (1931) used sucrose solution, and a M/40 solution of sucrose was found useful in these experiments. Testing a large number of flies with this solution alone gave a score of 382. Table III shows how the results differed when various substances were added to it. Dulcitol alone is seem- ingly inert in M/10 solution, but when M/40 sucrose is added, the flies live longer than in sugar alone (score 508). Isoleucine is inert either way. D-Arabinose. on the other hand, prolongs life slightly when alone but shortens it when added to the sucrose solution, a puzzling result, to be sure. Sorbose would seem to be toxic either alone or in sucrose solutions, as do tartaric acid, norleucine and histidine, while valine, which is toxic when alone, can probably be detoxified when sucrose is present. 120 CHARLES C HASSETT TABLE III The effect of certain substances an Drosophila when dissolved in water and in M/40 sucrose. Each pair run under identical conditions. Score Substance Cone. In water In M/40 sucrose Cellobiose M/10 84 396 Dulcitol M/5 119 508 D-Arabinose M/5 170 162 L-Sorbose M/2 68 285 M-Tartaric acid M/5 102 124 D-Tartaric acid M/5 80 115 DL-Norleucine M/10 20 83 DL-Valine M/10 24 353 DL-Isoleucine M/10 11 1 360 L-Histidine M/10 93 203 DISCUSSION As noted above, the question of what sugars can be utilized by insects has been investigated for several species. The results in hand for the adult and larval bee, the adult blowfly, and for the adult fruit flies Anastrepha and Drosophila, indicate almost identical abilities to utilize sugars, as nearly as the data are comparable. The really clear cut differences reported are as follows : mannose is used by Calliphora, Anastrepha and Drosophila, but not by the bee. Indeed von Frisch (1934) and Staudenmayer (1939) have reported a specific toxicity of mannose for the bee. Melibiose, dextrin, starch, and glycerol are not used by adult bees, but TABLE IV The development of sterile Drosophila larvae on low yeast, /o?cr yeast plus sugars, and ade- quate yeast diets. Medium Number of eggs (36/vial) Number of pupae Mean number of pupae per vial Difference divided by prob. error of difference Number of adults Mean number of adults per vial Difference divided by prob. error of difference Mean number of days for emergence of all flies 0.5% yeast 252 73 10.3±2.9* 2.0 67 9.6±3.3* 2.0 20.5±3.4* 0.5% yeast 0.5% glucose 180 106 21.2±4.1 102 20.4±4.3 21.0±1.7 0.8 0.6 0.5% yeast 0.5% sucrose 540 371 28.5±2.3 353 27.1±2.3 21.5±1.3 2.0 1.4 0.5% yeast 0.5% fructose 216 183 30. 5 ±1.0 178 29.7±1.8 16.2±3.7 3.0 4.5 2.0% yeast 72 70 35.0±0.9 70 35.0±0.9 12.0±0.0 * Probable error. UTILIZATION OF SUBSTANCES BY DROSOPHILA 121 are by Callipliora and Drosophila. Inositol is utilized by Drosophila, but not by the others, and arabinose is used by Apis alone. There are other differences re- ported, such as the use of fucose by Drosophila and not by other forms, but the degree of utilization is so small that the difference is unimportant. The present experiments do show, however, that no substance should be judged inert until it has been tested in several concentrations, e.g. xylose is very poor in M/10 or less, but definitely useful in M/2. Also, substances should not be finally classified as useless or toxic unless they are offered in such form that ingestion is certain. Dul- citol, for example, is apparently inert for Drosophila when given alone, even up to M/5, yet when it is dissolved in M/40 sucrose, the flies live longer. The comments of Yogel (1931), Haslinger (1935) and Fraenkel (1940) are also pertinent to this point. The ability of Calliphora and Drosophila to utilize glycogen and starch is clear, although it is much less than the ability to utilize sugars. The danger of using a partially hydrolyzed starch should be noted. DrosopJiila fed Lintner's soluble starch, one per cent, gave a score of 625, whereas sugar-free corn starch scored only 334. Reducing sugar was readily demonstrated in the soluble starch, which may account for the partial development of Acdes larvae reported by Hinman (1933). The question of which sugar is best, which was raised by Bertholf (1927), is. perhaps, one applicable only to the individual species. It is further complicated by the variety of standards adopted by various investigators. Yet it is interesting to note that the "physiological sugar," glucose, is consistently poorer than others, being rated second by Phillips, third by Baker and Fraenkel, and fourth by Bertholf and in the present experiments, when only sucrose, maltose, glucose, and fructose are considered. Fructose, on the other hand, is rated first by Phillips, equal to sucrose by Fraenkel, second to sucrose by Bertholf, and in the present experiments it was superior to the others. Indeed, a comparison of scores for M/10 fructose and M/20 raffinose indicates that fructose is superior to the trisaccharides also. Sucrose is at or near the top in all. The curve for galactose in Figure 1 is also of some interest. The initial mor- tality was so heavy that it suggested reduced powers for utilization of galactose. or greater power of mobilizing enzymes, on the part of one of the two portions of the population. A repetition of the experiment yielded similar results. The basis of the variability is not known but it will be investigated. Partial successes were obtained with the substances regarded as intermediate products of carbohydrate metabolism. None of these was utilized by Calliphora (Fraenkel) ; DrosopJiila, however, survives a short time on citric, malic, succinic. lactic, butyric, and acetic acids, and possibly also on aconitic, itaconic, fumaric, and pimelic acids, although these are on the borderline. Since there is such close agreement in other respects, these data suggest that the blowfly might be able to metabolize the compounds in question, a possibility which Fraenkel has pointed out. In an experiment in which the present technique was used with Lucilia sericata, the flies died about as rapidly when offered M/10 citric acid or dry citric acid as they did when offered water alone. Calliphora was not available for this test, but the results with Lucilia suggest that if blowflies are able to metabolize any of the intermediates, some other means must be employed for introduction of the material. According to Weidenhagen (1931), and the somewhat modified point of view 122 CHARLES C. HASSETT of Pigman (1944), all carbohydrates can be split by a small number of enzymes. With Weidenhagen's work in mind, Fraenkel concludes that only two enzymes, an alpha-glucosidase and an alpha-galactosidase, need exist in Calliphora to split all the carbohydrates that the blowfly utilizes. Drosopliila evidently depends largely on the same two, but may have in addition a fructofuranosidase, which would be needed to utilize inulin, and could also act on sucrose. An amylase, too, must be present to split starch and glycogen. While the longevity of the fruit fly on sugar alone may seem remarkable (50 per cent survival up to four weeks), the much greater longevity on the standard culture medium which furnishes carbohydrate directly and protein and accessory factors from the yeasts growing on the medium suggests that the addition of traces of other substances to the sugar solution might increase survival greatly. A fur- ther point on longevity is that the present method is not calculated to produce the longest lived flies. According to Pearl, Miner and Parker (1927), the maximum longevity of Drosophila is found in relatively crowded populations, about 50 flies in a 30 ml. vial having given best results in their experiments. SUMMARY 1. Drosophila niclanoyaster can survive for varying periods on pure solutions of many compounds, including sugars, polysaccharides, polyhydric alcohols, aliphatic acids, etc. 2. In equivalent solutions, the order of usefulness of some common sugars was found to be : fructose > maltose > sucrose > glucose > galactose > xylose > lac- tose. 3. There is no significant difference in life span between flies fed on disac- charides and their constituent monosaccharides. 4. Doubtful sugars can usually be resolved into toxic, repellent, or slightly useful substances by offering them in dilute sucrose solutions. 5. On a sterile, "starvation" diet, larvae develop better on fructose than on glucose. 6. On the basis of survival when fed pure substances, Drosophila seems to possess alpha-glucosidase, alpha-galactosidase, beta-fructofuranosidase and amylase. LITERATURE CITED BAKER, A. C., W. E. STONE, C. C. PLUMMER AND M. McPnAiL, 1944. A review of studies on the Mexican fruit fly and related Mexican species. U. S. D. A. Misc. Pitbl. 531. BEADLE, G. W., E. L. TATUM AND C. W. CLANCY, 1938. Food level in relation to rate of development and eye pigmentation in Drosophila melanogaster. Blol. Bull., 75 : 447. BERTHOLF, L. M.. 1927. The utilization of carbohydrates as food by honeybee larvae. Jour. Agrlc. Res., 35: 429. FRAENKEL, G., 1936. Utilization of sugars by Calliphora, Dipt. Nature, 137 : 237. FRAENKEL, G., 1940. Utilization and digestion of carbohydrates by the adult blowfly. Brit. Jour. Exp. Biol, 17 : 18. FRISCH, K. VON, 1934. Uber den Geschmacksinn der Biene. Ein Beitrag zur vergleichenden Physiologic des Geschmacks. Zclt. f. I'crgl. Phys'wl., 21: 1. HASLINGER, F., 1935. Uber den Geschmacksinn von Calliphora Erythrocephala Meigen und iiber die Verwertung von Zuckern und Zuckeralkoholen durch diese Fliege. Zeit. f. vergl. Physiol, 22 : 614. HINMAN, E. H., 1933. Enzymes in the digestive tract of mos.quitoes. Ann. Ent. Soc.. Amcr., 26 (1) : 45. UTILIZATION OF SUBSTANCES BY DROSOPHILA 123 PEARL, R., J. R. MINER AND S. L. PARKER, 1927. Experimental studies on the duration of life. IV. Data on the influence of density of population on the duration of life in Drosophila. Amcr. Nat., 289. PHILLIPS, E. F., 1927. The utilization of carbohydrates by honeybees. Jour. Agric. Res., 35: 385. PIGMAN, W. W., 1944. In Advances in cnzymology, v. 4, ed. by Nord and Werkman. Inter- science Publ., Inc., New York. STAUDENMAYER, T., 1939. Die Giftigkeit der Mannose fiir Bienen und andere Insekten. Zcit. j. vergl. Physio!., 26 : 644. TRACER, W., 1941. The nutrition of invertebrates. Physiol. Rev., 21 : 1. TRACER, W., 1947. Insect nutrition. Biol. Rev., 22: 148. UVAROV, B. P., 1928. Insect nutrition and metabolism. Trans. Ent. Soc. Loud., 76: 255. VOGEL, B., 1931. Uber die Beziehungen zwischen Siissgeschmack und Nahrwert von Zuckern und Zuckeralkoholen bei der Honigbiene. Zcit. f. vcryl. Physiol., 14: 273. WEIDENHAGEN, R., 1931. Spezifitat und Wirkungsmechanismus der Carbohydrasen. Ergeb. Ensymforsch., 1 : 168. RESPIRATION OF OOCYTES, UNFERTILIZED EGGS AND FERTILIZED EGGS FROM PSAMMECHINUS AND ASTERIAS HANS BOREI W enner-Greri s Institute for Experimental Biology, University of Stockholm 1. Introduction 124 2. General remarks on material and methods 125 3. Experiments and interpretations 3.1 Respiration of oocytes and unfertilized eggs 3.11 Psammechinus 129 3.12 Asterias 134 3.2 Respiration before and after fertilization 3.21 Psammechinus 137 3.22 Asterias 141 3.3 Cleavage rate 141 4. General discussion 143 5. Summary 148 6. Literature cited 149 1. INTRODUCTION Using Cartesian diver micro-respiration technique Lindahl and Holter (1941) measured the oxygen consumption rate of primary oocytes, mature unfertilized eggs and fertilized eggs of the sea-urchin Paracentrotus lividus. They found that the oocyte respiration is markedly higher than that of the unfertilized egg and that it probably exceeds that of the newly fertilized egg. On the other hand Boell, Chambers, Glancy and Stern (1940) stated, in an earlier brief note, that in similar diver measurements the mature unfertilized Arbacia egg reveals a higher oxygen consumption and a higher capacity to oxidize p-phenylenediamine than does the immature egg (oocyte). Because of these two quite divergent results it was considered to be of interest to investigate, and eventually settle the matter, by using a third sea-urchin species. Psammechinus miliaris from the Swedish West Coast was chosen. This species offers some special advantages : its spawning period is relatively long ; its oocytes can be obtained regularly during practically the whole of the spawning period ; and, there are three distinct cytoplasmic maturity stages of the egg, viz., under-ripeness, ripeness and over-ripeness. These maturity stages are characterized by differences in fertilizability, in fertilization membrane appearance and in reaction to hypertonic medium (cf. Runnstrom and Monne, 1945). Thus it might be possible to follow in detail any gradual alteration in oxygen consumption during the entire egg matura- tion process. Since it is necessary selectively to pick out the desired cells from the cell mixture, which is extruded from the ovary, the diver technique, which permits the measurement of the oxygen consumption of as few as about a hundred oocytes or resting eggs, will be very suitable. As only a small number of cells are 124 ECHINODERM FGG AND OOCYTE RESPIRATION 125 necessary for each experiment, it is possible to charge any desired parallel diver unit with cells from the same ovary. Furthermore, it was thought that a comparison between the sea-urchin oocyte or egg respiration and that of the starfish oocyte or egg might throw some light on the general laws of echinoderm egg metabolism. Tang (1931), using a Warburg technique, found that mature and immature (oocytes) Asterias eggs used up the same oxygen amount per time unit. Boell, Chambers, Clancy and Stern (1940) arrived at the same result using diver technique, but Brooks (1943) in Warburg experiments found a lower consumption in immature eggs than in mature. In the present paper diver measurements on Asterias glacialis oocytes and eggs will be reported. In addition, diver measurement data concerning the oxygen consumption of the fertilized sea-urchin and starfish egg are given and interpreted. In Asterias the respiration is only followed over the first mitosis, but in Psammechinus it is measured until some hours after hatching. Abbreviations used in the text : Ps.: Psammcchinns iniliaris Ast.: Asterias glacialis Par.: Paraccntrotus livid us 2. GENERAL REMARKS ON MATERIAL AND METHODS 2.1. Animals Ps. occurs in two phenotypic varieties : one, called the Z-form, is the trivial littoral form ; the other, called the S-form, is found at greater depths. They differ in size and morphological appearance and, of special interest in this investigation, in the spawning period. The Z-fonn has fertilizable eggs from the middle of June until the middle of July, the S-form during July and August. Concerning living conditions, distribution and biology, cf. Lindahl and Runnstrom (1929) and Borei and Wernstedt (1935). The Ps. Z-form animals were dredged from about 6 m. depth. They were kept in a wire mesh cage immersed in the surface water off the station pier, where temperature and salinity conditions were approximately the same as those at the dredging-locality. The sea-urchins were used for experiments within a few days of being caught. The Ps. S-form sea-urchins were caught at about 20-30 m. depth. They were brought to the station immersed in 32-33 °/oo salinity water in a big Dewar vessel to keep the temperature low. At the station they were transferred to aquaria with running sea water, where the salinity was about 32-33 °/«o and the temperature between 15-17° C. The conditions at the dredging- localities were about the same with regard to salinity, but somewhat lower as regards tem- perature. The animals were mostly used for experiments on the day of capture ; in some rare cases they were not used until the next day. Ast. was dredged from 30-40 m. depth. The animals were brought to the station and kept there in the manner described above for the Ps. S-form. They were invariably used for ex- periments on the day of capture. Their spawning period falls mainly in May and June. 2.2. Diver technique The technique of Cartesian diver measurements has been described in detail by Holter (1943). Only points of special interest will be mentioned here. The divers used were of standard type (volume 8-10 Ml.) made of Jena Cerate glass (0 = 2.412). They were charged as follows, according to the "Diver charge Type I" of Borei (1948) : 126 HANS BOREI Mouth seal: Holler's medium (.\t = 1.325) Neck seal IrO.5 Ml. paraffin oil Oo, =0.87) Neck seal II: 0.5 Ml. isotonic sodium hydroxide"! , ^ . ... Bottom drop : 0.8 Ml. sea water cell suspension/ (cf. Borei (1948), Figure 1:1). The cells were extruded from the ovary after this had been removed from the body, sifted through bolting-cloth and washed three times in sea water before being picked up in a braking pipette. The salinity of the sea water of the cell suspension varied according to the material. For the Ps. Z-form water of 24.6 %„ S was mostly used. This salinity figure approximately equals the medium salinity during the summer months of the surface water off the station pier where the animals were kept. Occasionally higher salinities occur. Thus it was sometimes found more correct to apply water of 27 or 29 °/00 S. For the Ps. S-fonn which lives in water oi higher salinity and was therefore kept in the station aquarium sea water; 32-33 %,, S sea water was used. In the Ast. experiments the salinity was 29 °/oo throughout. This salinity is somewhat lower than that on the dredging-localities, but had to be used owing to some tem- porary trouble with the station sea water pipe-line. All salinities were checked by titrimetric estimations according to Borei (1947). For pH control potentiometric measurements (glass electrode) were employed. Isotonic NaOH solutions for the diver neck seal II were prepared from a stock solution. (From the sea water freezing points tabulated by Knudsen (1903) it can be calculated that 0.365 N NaOH is isotonic to 25 %,„ S sea water.) The temperature in the experiments was mainly 18° C. For some Ps. experiments tem- peratures between 15-21° C. were employed, owing to the requirements of simultaneous measure- ments fof other investigations. The maximum temperature for normal larval development of Ps. was studied by Runnstrum (1927) and found to be 22° C. Ast. belongs to the same species group, the mediterranean-boreal, and is likely to have about the same upper temperature limit for normal development. The temperatures in the experiments are thus well below the critical level. The number of eggs per diver was 40-50 for Ast., 70-120 for Ps. when unfertilized, and 40-50 for Ps. when fertilized. These numbers give approximately 12.5, 8 and 9 X 10'^ Ml. oxygen consumed per hour respectively in the most crowded divers, i.e. 8p lies between 1-2 cm. per hour. This rate is best suited to keep the errors of the diver apparatus low ( cf . Holter, 1943) and lies, moreover, within the range 3-18 X 10~:| Ml. per hour, which Lindahl and Holter (1940) in diver experiments on Par. found to be characterized by direct proportionality between number of cells and oxygen consumption. Lindahl and Holter (1940) further found that diver and Warburg experiments which were performed simultaneously gave very consistent results. The same applies to the present diver experiments on unfertilized Ps. eggs compared with the Warburg experiments of Borei (1934) (cf. 3.112.2). Thus the oxygen supply is apparently not the limiting factor in these diver experiments. This view is further supported by the fact that in the course of the experiments the oxygen pressure within the diver does not decrease more than 2 mm. Hg at the most, whereas it is generally agreed (cf. Tang, 1941) that the sea- urchin egg respiration is unaffected by a decrease in oxygen pressure from 160 mm. down to 40 mm. The number of cells per volume of cell suspension is about the same in diver experi- ments as in Warburg ones, or slightly lower. After completed diver measurements the cells were washed out of the divers with sea water, re-counted and then microscopically observed as to condition and development. In applicable cases even fertilizability controls were undertaken. Only those experiments were accepted in which the cells passed these post-diver-measurement controls. 2.3. Evaluation of results The oxygen used up during the experiment, 5r. is calculated from the read pressure differ- ence, 5p, according to the formula ECHINODFRM K(i(, AND OOCYTK RESPIRATION 127 Using this formula 8v will be given in Ml. measured at 0° C, and normal barometric pressure, provided Sp is stated in cm. Brodie, p» is the normal barometric pressure in cm. Brodie (=1000), T is the temperature of the experiment in °K., 7,, stands for 273° K., and if r stands for the total gas space in Ml. of the charged diver at equilibrium pressure. It will be noted that this formula is similar to that used by Holter ( 1943 ) p. 466 in cases where the solubility of the measured gas is low, and where formed CO, is absorbed away, but a tem- perature correction has been added in order to render a comparison between the measurements possible even if the latter have been taken at different temperatures (see, however, below). In the formula the absorption of oxygen in the liquid phases of the charge has been disregarded as it is a very small quantity. From the diver equation given by Linderstrom-Lang (1943) p. 363 the following expression for r may be derived: (2) V = eD I where x and G i = densities of the aqueous charge, the paraffin oil, the medium and the diver glass. The formula may be shortened to : = cjD-A + B + C (3) where go- A may be defined as the total gas space in M!. of the uncharged diver at equilibrium pressure. This is a constant characteristic of the individual diver. For calculating A the graph in Figure la may be of help. For a given medium (in this investigation Holter's medium, = 1.325; cf. Holter (1943) p. 412, has been used) A is solely a function of 4>Gi. 0.4 O.3 0.2 I 325 u to 2.O 2.5 3.O DENSITY OF GLASS O -O.I 00 1 S ^ ^ N^ ^ ( b X -N, N^ k. S ^ ^ X, -0.3 Oc \ \ s, 's v \. s ^ s X ^^ (OIL) k ^, (AQ UE ^E OUS) ^ /: i:c .325 O ).87 ^ Oe ^ -0.7 -0.8C ) O.5 I.O 1.5 2.O VOLUME OF CHARGE (pi.) FIGURE 1. Graphs for calculating the values of the constants A, B and C in the formula for V , the total gas space of the charged diver at equilibrium pressure. The constants B and C are governed by the adopted type of charge, but are independent of the characteristics of the diver. They may be found from the graphs in Figure Ib. The value of A ought to be stated to the third figure ; B and C will be sufficiently correct if stated to the second. As stated in 2.2., measurements were usually performe'd at 18° C., but in some experiments on Ps. cells the temperature was varied within the limits 15-21° C. Measurements at different 128 HANS BORE1 temperatures are not comparable if merely calculated according to formula (1), as the cellular oxygen consumption rate is itself a function of temperature. As will be shown in a later paper (jointly with S. Lybing) this function in the unfertilized Ps. egg is characterized by a Q,0 ^22.25. Adopting this value, the consumption rates at any temperature within the range may be converted to consumption rates at 18° C. The same applies when the results of other investigators are compared with those given in this paper. The same Q,0 value may, without significant errors, be used even for fertilized eggs. If all corrections and constants are now put together the consumption formula (1) is simplified to : 8v= V-Sp-K. (4) The value of K for different experimental temperatures when adopting <2,0 = 2.25 will be found from Table I. The technical errors of the method will no doubt remain below 5 per cent (cf. Holter, 1943). The biological scattering of the gained figures is, however, much greater. (This scat- tering is frequently met with by investigators who study oxygen consumption in marine inverte- brates. Thus, using apparently identical objects one frequently comes across biological scat- tering amounting to the horrifying value of over 100 per cent.) In order to get a measure on the biological scattering in this investigation the standard deviation, -12 HOURS AFTER FERTILIZATION 456789 HOURS AFTER FERTILIZATION 10 12 13 O I 234567 HOURS AFTER REMOVAL FROM OVARY FIGURE 3. Oxygen consumption of Psammechinus eggs before and after fertilization. The values are obtained from parallel divers, one with unfertilized, the other with fertilized eggs. For comparison the results of Gray (1926) on Psammechinus millarls (17° C.) and of Lindahl (1939) on Paraccntrotus Ihidus (22° C.) together with those of this investigation (18° C.) are plotted on a relative scale (extrapolated values at 30 minutes after fertilization — 100) in the small right hand bottom graph. In this graph the times of hatching are indicated by arrows. In the main graph the position of the telophase (appearance of cleavage furrow) of the earlier mitoses is indicated. ECHINODERM EGG AND OOCYTE RESPIRATION 141 the results of Lindaftk (1939) on fertilized eggs of Par. From the results on Ps. oocytes and unfertilized Ps. eggs presented in this paper (cf. 3.112 and 3.113) it is apparent that the respiration of the Ps. and Par. cells differs. Thus results from the two species must not be represented as a continuation in one and the same curve. As has been previously mentioned it has not been possible to use the diver technique to measure the respiration until 40 minutes after the fertilization. Before this time there is said, however, to be a higher oxygen consumption than at any time during the next few hours. This was first indicated in measurements of Shearer (1922a) on Psammechinus inicrotubcrculatns and later studied in detail by Runnstrom (1933) and by Laser and Rothschild (1939) working on Par. and Ps., respectively. Nevertheless the exact shape of this part of the curve is still uncertain. (The slight temporary rise over the unfertilized egg value immediately after fertili- zation, which is indicated in Zeuthen (1947b) in his main graph, is not drawn in accordance with the findings of the mentioned investigators.) The period between fertilization and the first diver measurements has been left empty in Figure 3, but one must, however, keep in mind that during this period oxygen consumption rates, higher than that at 40 minutes, may have occurred. 3.22. Astcrias Comparing the respiration rates of unfertilized and fertilized Astenas eggs Loeb and Wasteneys (1912) (Winkler measurements) and Tang (1931) (War- burg technique) found no differences. In Cartesian diver experiments Boell and co-workers (1940) confirmed this. The results in the present paper in no way differ from these results. Here the respiration has been followed, however, for a longer space of time than in the earlier investigations, namely over a period of more than 200 minutes after fertilization, i.e. over the first mitosis. A gradual increase in oxygen consumption rate is to be noted during this time, as will be seen from Figure 4 (concerning the cell material, cf. 3.121). z U.O I4O %3> 120 ccz FERTILI- ZATION 1st MITOSIS O 6O I2O ISO MINUTES AFTER FERTILIZATION FIGURE 4. Oxygen consumption of Asterias eggs before and after fertilization. The exact position of the first mitosis telophase in relation to the time of fertilization de- pends on the state of the egg when fertilized (secondary oocyte or egg). In a batch of cells mitosis may thus not occur synchronously. 3.3. Cleavage rate In previous chapters reference has been made to the succession of mitoses and the time of their occurrence after fertilization. Though this cleavage rate in Ps. has repeatedly been studied by various authors, it has, for the sake of control, been checked even in this investiga- 142 HANS BOREI TABLE IX Cleavage rate of fertilized Psammechinus eggs Concerning right time values in Zeuthen's experiments cf. 3.21 Author Gray (1926) Zeuthen (1947b) Borei (this investig.) Temperature. . 17° C 16° C 18° C. Salinity 34-35%o 32»/oo 33.2o/oo Time after fertilization: 1st mitosis 67 mins. 56 mins. 2nd 100 — 83 3rd 132 — 127 4th 168 165 mins. 160 5th 203 200 ~200 6th 238 240 ~240 7th 271 (290) — Hatching 9 hours 9 hours 30 mins. 8 hours 35 mins. tion. Found discrepancies were, however, with exception of the first cleavage time, of minor importance as is shown in Table IX. It should be noted that in this table the values of Borei and Gray were obtained from cultures, whereas Zeuthen's are from observations on dividing eggs inside a diver. In this latter case the hatching time is somewhat delayed. As has been mentioned before (3.21) even in the present investigation a minor delay has been observed in cells when inside a diver. In Figure 5 the cleavage sequence for Ps. and Ast. has been represented graphically. For comparison the cleavage rate for the irregular sea-urchin Echinocardhim cordatum has been plotted in the same figure. All values were obtained during this investigation. HATCHING PS. AST. ECD AST. PS. : PSAMMECHINUS MILIARIS ECD. : ECHINOCARDIUM CORDATUM AST. : ASTERIAS GLACIALIS 17-18 °C. _L _L IO 15 2O HOURS AFTER FERTILIZATION 25 FIGURE 5. Cleavage rate in Psammechinus, Asterias and Echinocardium. Values obtained from observations on sparse cultures (200 eggs in 10 ml. sea water). The mitoses are represented from the appearance of the cleavage furrow (teleophase). The position of the first cleavage in Ast. is dependent on the state of the egg when fertilized ; in the repre- sented sequence the germinal vesicle had just broken down when the sperms were added. ECHINODERM EGG AND OOCYTE RESPIRATION 143 4. GENERAL DISCUSSION Many investigators consider that they have reason to look upon the mature sea-urchin egg as a resting cell between the growing oocyte and the developing fertilized egg. (For literature see Lindahl and Holter, 1941.) The oxygen con- sumption measurements with diver technique performed by Lindahl and Holter (1941) on Par. oocytes, mature unfertilized eggs and fertilized eggs seem to give excellent support to this view. They found that the primary oocytes apparently have a respiration even slightly higher than that of the newly fertilized egg, whereas the unfertilized eggs have a considerably lower oxygen consumption. The authors compare the state of the unfertilized egg with the diapause of certain insect eggs. The investigations of Whitaker (1933) indicate that the low unfer- tilized egg respiration in comparison with that of the fertilized egg is, among marine invertebrates, peculiar to the sea-urchin group. The experiments in this paper show, however, that the sea-urchin group is not quite homogeneous in respect to oocyte respiration, as may be seen from Fig- ure 6. Though the oocyte oxygen consumption was measured in principally the same way as in Lindahl and Holter's investigation it was found that Ps. oocytes, in contradistinction to those of Par., consumed oxygen at a rather low rate, not differing very much from that of the unfertilized egg. A new fact, however, is revealed in this investigation concerning the sea-urchin egg respiration, viz. the rapidly decreasing rate of oxygen consumption after the cell has been removed from the ovary (cf. Fig. 2). This declining respiration rapidly and asymptotically approaches a considerably lower value than the initial one (cf. parallels in the endogenous respiration of baker's yeast, kinetically studied by Borei, 1942, and briefly reviewed in 3.112.1). According to these facts the initial rate of oxygen consumption upon egg removal may even be higher than the rate of the fertilized egg during the first hours after activation (cf. Fig. 3). Such a rapidly declining respiration has also been found to be characteristic of the Ps. oocytes (3.113). In all previous investigations the cell material must undoubtedly, though never stated, have been at least some hours old (reckoned from the time of removal from the ovary) when used in consumption measurements. Thus in the present investigation only such Ps. and Ast. meas- urements as were obtained from material of that age may be compared with the values of the previous authors. This means that only the measurements on the low level stage which are given in 3.112.2, 3.113 and 3.122 and summarized in Figure 6 may be used for comparison. If differences now exist, as they probably do, both between the species and between the different kinds of cells within the species, as regards the rapidity with which the low level state is reached, i.e., in steepness of the declining curve, it might in the Par. case be that one has hit upon oocytes which show a very slow decrease, whereas in the Ps. case the oocytes show a rapid decrease. Under such circumstances the apparent contradictions between Ps. and Par. oocyte respiration can be understood. In the case of Boell and co-workers' (1940) findings in Arbacia of a lower respiration in the oocytes than in the eggs, it might be either that the oocyte respiration decrease is especially rapid or that the respiration decrease of the eggs is especially slow in that species, or it might be that the time which has elapsed since the removal from the ovary is longer for the oocytes. As no technical points 144 HANS BOREl whatsoever are stated in Boell and co-workers' paper it is impossible to draw any conclusions. It is very interesting to discover that there is general agreement within the sea-urchin group as to a low respiration in the unfertilized egg (when measured some hours after removal from the ovary) in comparison with the respiration of the fertilized cell. (The reviews of Whitaker (1933) and Ballantine (1940) and the results given in 3.21 might be consulted.) Now Loeb and Wasteneys (1912) have found that unfertilized Asterias eggs which respire at the same rate as the O.3 a: O 08 a: u O.2 u U owo.i PARACENTROTUS PSAMMECHINUS PS PAR. AST. ASTERIAS i/> O O 8 LJ o. or u a z o O Q- § S z O I- N U UJ g 39V1S U U I C\J FIGURE 6. Comparison on a cell volume basis between the oxygen consumption of different female reproduction cells from Psamtnechinus, Paracentrotus and Asterias. The results on Ps. and Ast. are from the present investigation ; those on Par. from Lindahl and Holter's (1941). In the unfertilized cells the oxygen consumption was always measured several hours after cell removal from the ovary. All rates are uniformly calculated for 18° C. and expressed as if measured at 0° C. and at normal barometric pressure. Average cell volume values used: Ps. 5.56 X 1(T4 /*!., Par. 5.75 X 1(T4 /*!. and Ast. 25.2 X 1(T4 /*!. ECHINODERM EGG AND OOCYTE RESPIRATION 145 fertilized ones, die very rapidly if not kept anaerobically (i.e., if the oxidative proc- esses within the eggs are allowed to continue at an unlimited high level). In view of this and the considerations of other authors one is inclined to look upon the low respiration of the sea-urchin egg as a natural precaution in order to facilitate long life for the shed egg. In this connection one must consider the actual conditions of the localities at time of spawning. The animals there live rather close together and spawning probably sweeps epidemically and simultaneously over the specimens, both male and female, of a given species in a community. Thus a rapid fertiliza- tion may be secured, which would mean that the egg respiration would only have time to enter the very first part of the declining curve. It is possible that eggs, which accidentally are not fertilized at once, are preserved for future activation through this low-level mechanism, which thus promotes the reproduction possibili- ties of the group. On the other hand, however, the similar declining curve of the oocyte respiration will probably have no biological significance whatsoever because, as is generally agreed, the sea-urchin oocytes are never shed. The biochemical mechanism that causes the differences in respiration between fertilized and unfertilized sea-urchin eggs, as studied by Runnstrom (1930, 1933, 1935), Orstrom (1932), Korr (1937) and others, is likely to be characterized in the unfertilized egg by a block in the chain of carriers which, in the fertilized egg, mediates the oxidation of the substrates. This chain of carriers is supposed to include the cytochrome-system. -Runnstrom considered that the oxidase was unsaturated with its substrate, and later investigations have furthered the view that substrates, dehydrogenases and oxidase are kept apart in the unfertilized egg. It is remarkable that the respiration of the unfertilized egg is insensitive to inhibitors affecting the cytochrome-system, whereas the respiration of the fertilized egg is very sensitive. In all these investigations it has been pointed out that the respira- tion of the unfertilized egg is low in comparison with that of the fertilized egg. This means that the studies have been performed on unfertilized eggs, in which the declining respiration part has already reached a low level. As the initial respira- tion of the unfertilized egg (when just removed from the ovary) can be even higher than that of the fertilized egg, the question therefore arises as to the relation between the rapidly declining (monomolecular) respiration part of the unfertilized egg and the respiration of the fertilized egg. Whether or not the oxidase is as important for this part of the respiration of the unfertilized egg as it is for the fertilized egg can probably be settled if inhibition experiments are performed as early as possible after the eggs have been removed from the ovary, i.e. when the declining respiration is still prominent. The oxygen consumption figures in the three stages of ripeness of the unfer- tilized Ps. egg reveal that the respiration value slightly decreases with growing ripeness (cf. 3.113 and Fig. 6). It has not been possible to establish any differ- ence in the early declining respiration of these stages. The material is, however, too small for convincing interpretations. (It should be kept in mind that the respiration of unfertilized sea-urchin eggs when kept for a long time in sea water undergoes a change, thus becoming more like that of the fertilized egg.) In the case of the Ast. egg it has been found that the respiration is lower in the primary oocyte than in either the secondary oocyte or the unfertilized egg (cf. 3.122 and Fig. 6), but this finding is in contradiction to earlier ones (Tang, 1931, and Boell and co-workers. 1940; cf., however, Brooks, 1943). As the eggs on natural 146 HANS BOREI spawning are shed with broken clown germinal vesicle, i.e. as secondary oocytes or as eggs (cf. Runnstrom, 1944), the jump in respiration rate between primary and secondary oocytes does not occur outside the ovary. The respiration after fertili- zation proceeds (as has been reported by several previous investigators) at much the same level as before. As the decreasing part of the respiration of the cells after their removal from the ovary is not very marked in this material, the rate of respiration of the fertilized eggs may not, under natural conditions, differ very much from that found in this investigation (3.22), even if the fertilization takes place very soon after spawning. Thus if the rates of respiration of the fertilized sea-urchin eggs and the fertilized starfish eggs are compared on a cell volume basis (see Fig. 6), a very much lower respiration in the case of the starfish will be found. Whitaker (1933) has argued that the oxygen consumption of fertilized marine animal eggs from several invertebrate phyla and even of other developing cells, wrhen compared on a cell volume basis, will show a remarkable consistency, whereas the respiration values of the unfertilized cells are widely scattered. It may be read from Whitaker's discussion, though not stated in these terms, that the respiration of the fertilized eggs is thought to be intimately connected with the work of morpho- genesis and with biochemical activities connected with growth, and that in develop- ing cells the amount of oxygen required for these purposes is about equal per volume of cytoplasmic matter. It is stated, however, that big cells, especially yolky eggs respire at a much lower rate. Other factors to be considered will, no doubt, be different degrees of cytoplasmic hydration, inert inclusions in vacuoles, etc., dead protecting or otherwise supporting structures, etc. The measurements by Tang (1931) on Ast. egg respiration before and after fertilization gave too low values to fit in Whitaker's scheme. The latter author severely criticizes technical weak- nesses in Tang's measurements and leaves them out of his survey. The present investigation has certainly found Tang's values to be notably low (cf. 3.122), but still the fertilized Ast. egg respiration is remarkably low in comparison with that of the sea-urchin egg (cf. Fig. 6). It should be borne in mind that the Ast. egg has a volume that is about 5 times greater than that of the Ps. or Par. egg. This fact may probably, in the light of the discussion on the connection between body size and metabolic rate recently put forward by Zeuthen (1947a), be of more impor- tance than considerations concerning supposed morphogenetic work. To summarize the egg respiratory conditions found in this investigation to- gether with those previously known, the graphical schemes in Figure 7 may serve. The Ps. ripe egg (Fig. 7a) (cf. 3.112.1) has very probably a high respiration level in the ovary ; at least it starts with high respiration velocity when brought into sea water. The oxygen consumption rate rapidly decreases and within some hours reaches a low and fairly constant level. The oocyte behaves similarly. At fertilization the rate immediately rises to around the level of the just removed egg, drops slightly and thereafter proceeds after an exponentially increasing curve (cf. 3.21). After natural spawning, fertilization probably occurs very soon, thus leav- ing the decreasing curve without much importance. Only ripe eggs are fertilizable. The Ast. primary oocyte oxygen consumption rate (Fig. 7b) (cf. 3.122) de- creases comparatively slowly in sea water. If the oocyte is ripe it soon starts the first meiosis, thereby increasing its respiration considerably. The second meiosis soon follows and a slow decrease in oxygen consumption continues until fertiliza- ECHINODERM EGG AND OOCYTE RESPIRATION 147 tion occurs. After fertilization the consumption proceeds as an exponentially increasing curve (cf. 3.22). On fertilization no sudden jump in consumption rate occurs as in Ps. When at natural spawning the cell leaves the ovary its nuclear membrane has disappeared. Thus the jump in the rate of respiration on transformation from primary to secondary oocyte has already occurred. Even oocytes may be fertilized, but then the time which elapses before the first mitosis will be longer thus allowing meioses to occur and thereafter the resting sperm nucleus to unite with the egg nucleus. o: UJ O U. O Ld _ O ££ UJ Q. Q. 2 CO 6 O z LU O X O EMBRYO EGG PSAMMECHINUS TIME OOCYTE EGG ASTERIAS OOCYTE TIME FIGURE 7. Generalized schemes of oxygen consumption per cell volume of reproduction cells of Psammechinus and Asterias. Time span. of evaluated diver measurements as well as location of comparison value on respiration curve indicated for each cell type. 148 HANS BOREI It is interesting to note that in fertilized eggs of both Ps. and Ast. the increase in oxygen consumption, which takes place during the first hours of development after fertilization, seems to be of the same exponential type in both species. This will be seen from Figure 8, where values from the first 4 hours' development in both species have been plotted on a relative scale putting the values at 40 minutes after fertilization = 100. u.g'40 Of= £L u§ 120 ,,0 IOO LJ 8O O PSAMMECHINUS • ASTERIAS O 60 I2O ISO 24O MINUTES AFTER FERTILIZATION FIGURE 8. Relative oxygen consumption of fertilized Psammechinus and Astcrias eggs. Values at 40 minutes after fertilization put = 100. 5. SUMMARY In Cartesian diver experiments on the oxygen consumption of oocytes, unfer- tilized eggs and fertilized eggs from the sea-urchin Psammechinus miliaris and the starfish Asterias glacialis it was found : 1 . The respiration of ripe Ps. eggs declines rapidly after they have been re- moved from the ovary into sea water. Starting at a rate that may exceed that of newly fertilized eggs it has thus, after some hours, attained a comparatively low and fairly constant level. The declining curve on kinetical analysis proves to be composed of a monomolecular and a constant part. The respiration curve of Ps. oocytes is of a similar type. In Ast. oocytes and eggs the respiratory decrease, though present, is not so prominent as in Ps. cells (3.112.1, 3.113, 3.122, Fig. 2). 2. Though there is a real difference in size between the eggs of the two Ps. phenotypes (the littoral Z-fonu and the S-fonn of the depths) no difference is found in the rate of respiration (3.112.2, 3.114). 3. Measurements on Ps. oocytes and eggs some hours after removal from the ovary show that the oocytes have only a slightly higher respiration than the eggs. The earlier investigations (Lindahl and Holter, 1941) on Paraccntrotus lividus eggs showed that these oocytes maintain a rate of respiration even higher than that of the newly fertilized egg. The findings in Par. might be ascribed to a slow respiration decrease in the oocytes, whereas the decrease is more rapid in the eggs. In Ps. the decrease is about equal in oocytes and eggs (3.112.2, 3.113, 4, Fig. 6). 4. In Ast. the primary oocytes respire at a much lower rate than do the sec- ondary ones or the eggs (3.122, 4, Fig. 6). 5. In Ps. there is a gradual slight decrease in egg respiration with advancing cytoplasmic maturity (3.113). 6. In both Ps. and Ast. the respiration of oocytes in ovarial fluid seems to be of the same order of magnitude as that of oocytes in sea water (3.113, 3.122). ECHINODERM EGG AND OOCYTE RESPIRATION 149 7. The shape of the respiration curve in Ps. after fertilization is in full con- cordance with earlier results obtained with different techniques by Gray (1926) and Lindahl (1939) (3.21, Fig. 3). 8. The value of the rise in respiration, that occurs in sea-urchin eggs on fertili- zation, may entirely depend on where on the slope of the decreasing egg respiration curve fertilization occurs. (This rise is characteristic for sea-urchin eggs and has repeatedly been found by earlier investigators.) It is thought that on natural spawning the rise is rather feebly marked because of early fertilization, and that correspondingly the low level respiration of the unfertilized egg may not be reached (3.21, 4, Figs. 3 and 7). 9. In Ast. there is no immediate rise in respiration after fertilization, but there is a gradual rise which exactly resembles the exponential increase in newly fertilized sea-urchin eggs (after the first sudden increase has passed). The rise from the oocyte respiration level to that of the egg will, under natural conditions, not occur outside the ovarv. as the cells are shed with broken down nuclear membranes (3.22, 4, Figs. 4, 7 and 8). Cleavage rates are given up to the sixth mitosis for Ps., Ast. and Echinocardium cordatum; hatching time is noted (3.3, Fig. 5). It is discussed whether the decrease in respiration of the unfertilized sea-urchin egg after its removal from the ovary has any possible significance for the biochemi- cal aspects of the sea-urchin egg respiration (4). If the respiration rates found in this investigation are compared on a cell volume basis it is found that the Ast. egg will not fit into the generalized scheme of Whitaker (1933) for marine invertebrate eggs; it is discussed why the Ast. egg respiration is so comparatively low (4, Fig. 6). Acknowledgments: This investigation was carried out during 1946 and 1947 on the Swedish West Coast at the Kristineberg Zoological Station of the Royal Swedish Academy of Science. The author wishes to express his deep gratitude for laboratory facilities and for the great pains taken by the Station Staff in supplying materials. He is much indebted to Professor J. Runn- strom for encouragement and for keen interest in the subject. LITERATURE CITED BALLENTINE, R., 1940. Jour. Cell. Comfy. Physiol., 15: 217. BOELL, E. J., R. CHAMBERS, E. A. CLANCY, AND K. G. STERN, 1940. B'wl. Bull., 79: 352. BOREI, H., 1940. Zeit. v. Physiol.. 20 : 258. BOREI, H., 1942. Biochem. Zeit., 312 : 160. BOREI, H., 1947. Arkiv. Kemi. Mineral. Gcol, 25B, No. 7. BOREI, H., 1948. Arkiv. Zool, 40A, No. 13. BOREI, H., AND C. WERNSTEDT, 1935. Arkiv. Zool., 28A, No. 14. BROOKS, M. M., 1943. B'wl. Bull., 84: 164. CHAMBERS, R., AND E. L. CHAMBERS, 1940. B'wl. Bull., 79: 340. COSTELLO, D. P., 1935. Physiol. Zool, 8: 65. GRAY, J., 1926. Brit. Jour. Exp. Biol, 4: 313. HOBSON, A. D., 1932. Jour. Exp. Biol., 9: 69. HOLTER, H., 1943. Compt.-rcnd. Carlsbcrg, Ser. chim., 24 : 399. HOLIER, H., AND E. ZEUTHEN, 1944. Compt.-rend. Carlsberg, Ser. chim., 25 : 33. HORSTADIUS, S., 1939. Publ. stas. zool. Napoli, 17: 221. KNUDSEN, M., 1903. Publ. circ., No. 5. KORR, I. M., 1937. Jour. Cell. Comp. Physiol., 10: 461. LASER, H., AND LORD ROTHSCHILD, 1939. Proc. Roy. Soc. (London], B126: 539. 150 HANS BOREI LINDAHL, P. E., 1939. Zcit. v. PhysioL, 27: 233. LINDAHL, P. E., AND H. HoLTER, 1940. C 'o m pt. -rend. Carlsbcrg, Scr. chiin., 23 : 257. LINDAHL, P. E., AND H. HOLTER, 1941. Compt.-rcnd. Carlsbcrg, Ser. chim., 24: 49. LINDAHL, P. E., AND L. O. OHMAN, 1938. Biol. Zentr., 58: 179. LINDAHL, P. E., AND J. RUNNSTROM, 1929. Acta Zool. (Stockholm), 10: 401. LINDERSTROM-LANG, K., 1943. Compt.-rend. Carlsberg, Ser. chim.. 24: 333. LOEB, J., AND H. WASTENEYS, 1912. Arch. Entitnck. Organ., 35: 555. ORSTROM, A., 1932. Protoplasma, 15: 566. RUNNSTROM, J., 1930. Protoplasma, 10: 106. RUNNSTROM, J., 1933. Protoplasma, 20: 1. RUNNSTROM, J., 1935. Biol. Bull., 68: 327. RUNNSTROM, J., 1944. Acta Zool. (Stockholm], 25: 159. RUNNSTROM, J., AND L. MONNE, 1945. Arkiv. Zool., 36A, No. 20. RUNNSTROM, S., 1927. Bergcns Museums Arbok., Naturr. Rckke., No. 2. SHEARER, C, 1922a. Proc. Roy. Soc. (London), B93 : 213. SHEARER, C., 1922b. Proc. Ro\>. Soc. (London), B93 : 410. TANG, P. S., 1931. Biol. Bull., 61 : 468. TANG, P. S., 1941. Quart. Rev. Biol. 16: 173. WHITAKER, D. M., 1933. Jour. Gen. PhysioL, 16 : 497. WICKLUND, E., 1947. Arkiv. Zool., 40A, No. 5. ZEUTHEN, E., 1947a. Compt.-rcnd. Carlsbcrg, Scr. chim., 26: 17. ZEUTHEN, E., 1947b. Nature, 160: 577. FRANK RATTRAY LILLIE Vol. 95, No. 2 October, 1948 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY ADDRESSES AT THE LILLIE MEMORIAL MEETING WOODS HOLE, AUGUST 11, 1948 B. H. WILLIER, R. G. HARRISON, H. B. BIGELOW, E. G. CONKLIN FRANK RATTRAY LILLIE 1870-1947 The Work mid Accomplishments of Frank R. Lillie at Chicago It is my privilege and honor tonight to review and appraise the work and ac- complishments of Frank Rattray Lillie at the University of Chicago. The year fol- lowing his graduation in 1891 from the University of Toronto he was fellow in zoology at Clark University, where he began graduate studies under the direction of C. O. Whitman, one of the early leaders in American zoology and the first direc- tor and one of the guiding spirits in the early days of the Marine Biological Labora- tory. In 1892 the dynamic President Harper lured Whitman, among other prominent scientists, away from Clark University to the newly founded University of Chicago. With affectionate admiration and devotion every student went with Whitman. There two years later (in 1894) Lillie received, at the age of 24, the degree Doctor of Philosophy in Zoology. In accord with the trend in embryology of those days, his thesis dealt with the phenomena of cell lineage as it occurs not in a marine species, but in the fresh water clam, Unio. Worthy of special mention here is an evaluation of this work in his own words, written only four years ago : "A feature of special interest in the first publication was a discovery I made in studying the cell-lineage of Unio, that the behavior of the individual cells was adaptive and that varying sizes, rates and direction of division of cells were directly related to the subsequent events." To him development at any particular time and place is always a special feature directly related to functional need. Herein lies the key to his philosophy of embryology and of biology in general. Except for a period of six years following the doctorate, his professional life was intimately associated with the University of Chicago from the time of its founding. During this period he was for 5 years (1894-99) instructor of zoology at the Uni- versity of Michigan and for one year (1899-1900) Professor of Biology at Vassar College. In the fall of 1900 he was called back to Chicago as Assistant Professor of Zoology, which in 1906 culminated in a professorship of Embryology at the age of thirty-six years. Upon the death of Whitman in 1910, Lillie succeeded him as chairman of the Department of Zoology and continued in that position until 1931, a span of 21 years. From 1931 to 1935 he was Dean of the Division of the Biological 151 152 WILLIER, HARRISON, BIGELOW, AND CONKLIN Sciences. Concurrently in recognition of his outstanding achievement in research and service to the university and to the science of biology at large he was the Andrew MacLeish Distinguished Service Professor of Embryology, and there- after held this title emeritus until his death. Outstanding among his accomplishments as chairman was an effective depart- mental organization which was characterized by a lack of elaborate administrative machinery and by a policy which encouraged both freedom and initiative for the individual, whether staff or student. In dignity and simplicity he administered without seeming to do so. His was a leadership in which everyone knew his posi- tion, responsibility, and opportunity. With the growth of the department, the need for additional research facilities became increasingly great. This need was met to a large extent in 1936 when he and Mrs. Lillie very generously presented to the university The Whitman Laboratory of Experimental Zoology. Named in honor of Professor Whitman, the first head of the department, it stands today as a monu- ment to the research ideals of both teacher and student. The four-year term as Dean of the Division of Biological Sciences began only one year after the clinical departments of a new medical school were set up on the Midway campus. In Dr. Lillie's own words his "special task as dean was to amalgamate the old established pre-clinical departments with the newly established clinical departments and hospitals into a coherent medical school." Through much patience, vision, and understanding he succeeded in uniting the departments of basic sciences and clinical biology into a cooperating group which is unrivalled elsewhere. Today this working union stands as a model of what can be accomplished along these lines. Of this achievement Dr. Lillie was justly proud. Professor Lillie's influence as a teacher had a far-reaching effect on students. His lectures were always characterized by a masterful precision of organization and conciseness of statement, the force of which was perhaps sometimes not fully ef- fective owing to his soft-spoken and undramatic manner of delivery. The graduate students were not taught in the conventional way. "The student was trained to think by one who directs without seeming to do so, and was attracted first of all to the organization of the seminar and graduate courses in which the results of research, interpretations, and theories were ingeniously knit together around a central theme. Thus, the alert student was able to see how an apparently insignifi- cant detail was concisely and cleverly woven into a concept with significant implica- tions. The student soon learned to judge and evaluate his own performance in the seminars. The example somehow led him to strive for perfection in organization and clear thinking. The young student when he began research was to a large extent thrown upon his own resources. He found out for himself whether he was fitted to be an independent investigator. Once the problem was suggested and the way of approach briefly sketched, the student knew that results were expected. Only when a preliminary result was obtained did the student report to Dr. Lillie, and even then only when he was prepared to ma'ke a possible interpretation." (Anat. Rcc., 100: 409.) Although his scientific life was shared almost co-equally between the Marine Biological Laboratory and the University of Chicago and these interests are con- sequently difficult to separate, I shall confine my remarks only to those discoveries which were made solely at the latter institution. Lillie's studies on the action of ADDRESSES AT THE LILLIE MEMORIAL MEETING 153 sex hormones in foetal life of cattle grew out of attempts to find an explanation of the sterility and partial inversion of the female of two-sexed cattle twins, popularly known for a century or more among cattle breeders as the free-martin. Apparently his attention was first drawn to this peculiar condition in a herd of cattle on the family farm northwest of Chicago near the village of Wheeling, Illinois. The emhryological phases as worked out were made possible by the close proximity of the university to the stockyards, in which large numbers of cattle are slaughtered daily. As a student, I remember seeing him garbed immaculately in a white gown and wearing rubber gloves, examining and dissecting pregnant uteri containing young twins which the collector had rushed to his laboratory table in a breathless manner. He found that when the foetal membranes of male and female embryos are fused so that the blood vessels (especially the arteries) of the two are continuous, the female embryo is modified in the male direction. Furthermore, if no blood connection occurs both partners are normal. Without goin.. !•.'• •• . • • <¥ 8 DROSOPHILA LARVAL EPIDERMAL CELLS 167 two cells, a Golgi-material origin of the spherical globules is unmistakable. Figure 3 shows practically all the stages of the development of a secretion globule in relation to Golgi material, from apparently homogeneous bits of Golgi material to mature globules lying free in the cytoplasm. Thus, regarding the origin, growth and releasing of the secretion globules in relation to Golgi bodies, the situation as found in the epidermal cells may be summed up as follows : Inactive pieces of Golgi material appear homogeneous ; but when secretory synthesis has proceeded sufficiently far, there becomes visible one light area in each Golgi body. This area has been interpreted, on evidences which have been reported in a previous paper (1947), to be a globule of elaboration product viewed through a layer of Golgi material. As the globule increases in size, the light area in most of the Golgi bodies becomes less and less colored as a result of the continual thinning of the overlying layer of Golgi material on the surface of the globule. A time will eventually be reached when the Golgi shell will no longer be able to contain the enlarging globule within it and will mechanically break into small irregular pieces, releasing its contents into the cytoplasm. This series of changes almost exactly duplicates what has been seen in the other larval tissues of Drosophila definitely known to have a glandular function (1947, 1948). What I consider as most instructive and significant, however, are strips of cells, usually five to six in number, which have often been observed with their cell mem- brane broken but with their nuclei and part of their cytoplasm still intact (Fig. 8). These cells remind one most vividly of the method of discharging their secretory product seen in the larval mid-gut epithelium and salivary gland cells of this same fly (1947, 1948). When one adds to this situation the relation observed to be exist- ing between the Golgi bodies and the globules which they elaborate, as pointed out in the preceding paragraph, the suggestion becomes more than probable that in the Drosophila larvae, the epidermal cells may also serve as internal secretion glands. Thus, Figures 1 to 7 may be taken as showing graphically the various stages of secretory synthesis which an epidermal cell passes through — from a stage wherein the cytoplasm contains no free secretory globules but numerous Golgi bodies (some are visibly homogeneous while others show a light center) to a stage in which the cell is extremely vacuolated due to the confluence of a large number of secretory globules elaborated and set free in the cytoplasm by the Golgi bodies. Figure 8, finally, illustrates the merocrine method of discharging secretion by the cell. Hav- ing failed to see any replacement cells in the epidermis, I assume that after having PLATE II FIGURE 5. A cell showing the vacuolated condition of the cytoplasm near the basement membrane. A few unfused globules are still visible within the vacuolated area at a ; a group of Golgi bodies all at about the same stage of secretory synthesis are seen at b. Note the two large globules not yet freed from their respective Golgi shell and also the bits of apparently homogeneous Golgi material in the cytoplasm near the cuticula end of the cell. FIGURE 6. A cell showing the Golgi shells around the globules already or about to be broken into small pieces which have the rugged surface and irregular shapes characteristic of apparently homogeneous and inactive bits of Golgi material. FIGURE 7. A cell showing its cytoplasm extremely vacuolated with apparently homogeneous bits of Golgi material embedded on strands of cytoplasm; at the right end of the cell may yet be seen some globules in the process of fusing. FIGURE 8. A cell with its cell membrane broken discharging its secretion and also a portion of its Golgi material into the body cavity of the larva. 168 W. SIANG HSU discharged its store of secretion, each cell is capable of repairing itself and starting another cycle of secretion when proper conditions are again present. This is the situation which prevails in both the mid-gut epithelium and the salivary gland cells. It is interesting to record that it was after I had reached the conclusion that the cells of Drosophila larvae may serve as internal secretion glands that my search into the literature for some supporting opinion led me to a paragraph by Wiggles- worth (1934) in his study on ecdysis in Rhodnius, which I quote: "The histo- logical evidence therefore favours the idea that the corpus allatum is responsible for secreting the moulting hormones which must be derived from the growing cells themselves, and this raises the question whether the general epidermal cells may not be responsible for the initial moulting hormone. This possibility cannot be entirely excluded ; but the epidermal cells are not innervated, and it is therefore probable that any hormones they secrete appear only when their own growth has been stimulated by the hormone from the head." This is the first reference to the epidermal cells as internal secretion organs which has come to my knowledge, al- though it must be admitted that my search in the literature in that regard is not an exhaustive one. However, in quoting Wigglesworth, I do not claim that my obser- vations prove that the epidermal cells are "responsible for the initial moulting hor- mone" in Drosophila larvae. Needless to say, this question merits more particular examination. I only claim that according to my material it is difficult to dismiss the idea that the epidermal cells of a Drosophila larva are capable of secreting some substance and that the secretion is discharged directly into the body cavity of the larva. SUMMARY 1. On the strength of observations set forth in the following paragraphs, it has been concluded that the epidermal cells of Drosophila larvae seem to act as internal secretion organs, at least at the age when the larvae are about one day before pupation. 2. The relation of the Golgi bodies to the globules, both inside and outside of the Golgi bodies as observed in the epidermal cells, has been found to be the same as what has been established in cells definitely known to be of a glandular nature in the larvae of this fly. In each piece of Golgi material, a single droplet is seen to make its first appearance and gradually to increase in size, eventually breaking free from the confining Golgi shell. It seems to be the normal procedure for the free separate secretory droplets to coalesce to form big vacuoles; and their further confluence gives to the cells in advanced secretory synthesis an extremely vacuolated appearance. 3. Many epidermal cells have been found with their cell membrane broken, thus releasing their secretion product into the body cavity of the larva. The apparent healthy condition of the nuclei of such cells and the absence of replacement cells in the epidermis would point to a merocrine mechanism of secretion in this case. LITERATURE CITED Hsu, W. S., 1947. On the cytoplasmic elements in the mid-gut epithelium of the larvae of Drosophila melanogaster Meigen. Jour. Morph., 80: 161-194. Hsu, W. S., 1948. The Golgi material and mitochondria in the salivary glands of the larva of Drosophila melanogaster. Quart. Jour. Micr. Sci., 88 (in press). WIGGLESWORTH, V. B., 1934. The physiology of ecdysis in Rhodnius prolixus (Hemiptera). II. Factors controlling moulting and "metamorphosis." Quart. Jour. Micr. Sci., 77 : 191-222. THE ROLE OF THE SINUS GLANDS IN RETINAL PIGMENT MIGRATION IN GRAPSOID CRABS RALPH I. SMITH Department of Zoology, Univ. of California, Berkeley 4 INTRODUCTION Among the several functions ascribed to a hormone or hormones produced by the sinus gland of the crustacean eyestalk is that of controlling the migration of the retinal pigments. Evidence for this function has been presented chiefly by Bennitt (1932a, b), Kleinholz (1934, 1936), and Welsh (1939, 1941) and is well sum- marized in the reviews of Kleinholz (1942), Brown (1944), and Panouse (1947). Briefly stated, the compound eyes of decapod crustaceans typically possess three sets of pigment cells : distal melanophores forming a sleeve about each ommatidium, proximal melanin contained within the retinular cells (photoreceptive cells, proximal pigment cells), and reflecting pigment cells located among and beneath the retinular cells. In day-adaptation the proximal and distal melanins approach each other so as to surround the sensitive rhabdomes, while the reflecting pigment may be shifted proximally beneath the basement membrane of the retina. In night adaptation the proximal and distal melanins move apart in such a way as to leave the rhabdomes exposed to light from all sides, while the reflecting pigment is exposed to form a reflecting layer at the bases of the rhabdomes, visible as a reddish area of "glow" in the dark-adapted eye. The relative extent of movement of the three types of pig- ment differs in detail in various species of crustaceans. Attempts to demonstrate nervous control of retinal pigments in crustaceans have been unsuccessful, while the possibility that these cells are independent effectors is not supported by the available evidence. In particular, the marked diurnal movements of retinal pigments ex- hibited in the eyes of many crustaceans under conditions of constant darkness or of constant illumination would seem to rule out independent response to illumination as anything but an incidental factor in the normal process of pigment migration. Positive evidence for a hormonal control is based upon the discovery by Kleinholz (1934, 1936) that extracts of light-adapted Palaemonetes eyestalks injected into dark-adapted animals in darkness caused the distal and the reflecting pigments to move to the light-adapted position. This work was confirmed on Cambarus by Welsh (1939), who found that the proximal pigment was likewise induced to mi- grate to the light-adapted position if sufficiently strong injections of eyestalk extract were used. It has generally been assumed that the sinus gland is the source of the retinal pigment activator, and Welsh (1941) has shown that aqueous extracts of isolated sinus glands are capable of causing typical pigment migration when injected into dark-adapted crayfish. As will be reported below, similar results may be ob- tained in grapsoid crabs. While the evidence for a hormonal control of crustacean retinal pigments is generally convincing, this concept has been based upon procedures which have not 169 170 RALPH I. SMITH included one of the classical tests of endocrine function, namely, the removal of a suspected organ to produce characteristic symptoms, followed by the injection of extracts or the implantation of the organ to restore the normal conditions. Further- more, most experimental work has been carried out with macrurans. Accordingly, it was felt that a further study of brachyurans would be of value, and that sinus gland extirpation should be made the main line of approach to the problem. If the sinus gland is the source of retinal pigment activating hormone, its removal would be expected to stop diurnal pigment migrations and to maintain the eye in a dark-adapted state. Continued display of retinal pigment migration after sinus gland removal would, on the other hand, indicate that this organ is not the con- trolling factor or at least not an indispensable link in the process. Welsh (1941) has given evidence that the control of diurnal changes in retinal pigments involves central nervous activity, possibly affecting the release of hormone by the sinus gland, but the possibility that this effect can be mediated in the absence of the glands has not been investigated. The present paper reports attempts to clarify the role of the sinus glands in retinal pigment migration by means of operative sinus gland removal. MATERIAL AND METHODS A. Animals Three species of grapsoid crabs common in the San Francisco Bay area have been employed : Hcmigrapsus oregonensis, H. nitdus, and Pachygrapsus crassipes. These animals are hardy, possess well-defined sinus glands, and show a marked "glow" in the eyes in darkness at night. With the exception of the work of Bowman (M.A. Thesis, U. C., Berkeley, Calif., 1948) no previous study of the pigmentary changes and endocrine functions of this group of crabs has been made. In order to reduce possible complications associated with egg-bearing, chiefly males have been used in the work, although enough females have been observed to indicate that their pigmentary responses are the same as displayed by the males. Animals were selected for size, since those less than 17 mm. in carapace width were too small for convenient sinus gland removal, while those exceeding 30 mm. in width were not only too large and active for easy handling under the dissecting microscope, but also possessed such thick and pigmented exoskeletons as to render difficult the observa- tions on body chromatophores sometimes carried on concurrently with studies of retinal pigments. All animals were kept in shallow water in individual covered glass dishes, were fed fresh liver, clam, or crab meat and had the water changed every 3 or 4 days. B. The preparation of active extracts Sinus glands and other organs to be tested for retinal pigment activating effect were taken from animals of approximately the same size as the recipients, so that dosage could be estimated in terms of a fraction of the organ in question. Recipient crabs were measured or weighed before an experiment, and a group of donor animals selected so as to have a slightly greater average width or weight than the recipients. Eyestalks were removed, split open in crab perfusion fluid, and the chain of optic ganglia with the attached sinus gland teased free with fine forceps. The sinus gland was detached and removed to a drop of distilled water in a covered one-inch Syra- cuse dish. The medulla terminalis was similarly placed in a second dish, and the RETINAL PIGMENT MIGRATION IN CRABS 171 group of three more distal ganglia (medulla interna, externa, and lamina gangli- onaris) in a third dish. Other tissues extracted included brain, which in these crabs has about twice the mass of the medulla terminalis, thoracic ganglionic mass, and claw muscle. The respective tissues, when a sufficient amount had been collected, were ground with rounded glass rods, although the small sinus glands ordinarily escaped thorough crushing. Crab perfusion fluid was added to the dishes with a 1 cc. hypodermic syringe, stirred, and then taken back into a marked syringe for transfer to a small test tube. The volume of fluid used being known, a further quantity of perfusion fluid sufficient to dilute the suspension to the desired degree was added to the Syracuse dish, and likewise transferred with the marked syringe to the test tube. Ordinarily, 10 or 20 sinus glands, medullae terminalis, and distal ganglia were used, and made up to give extracts containing the equivalent of 10 organs in one cc. Brains were diluted to 5 per cc., while thoracic nerve mass and claw muscle were dissected out in quantity judged to be a little greater than the mass of brain tissue used. After transfer to test tubes, all extracts were heated in boiling water for 10 minutes, cooled, and used unfiltered. Injections were made at the base of a walking leg, the quantity injected being 0.05 cc., using for each extract the same syringe used in handling that material in preparation. Extracts stored in a refrigerator retained their potency for several days. C. Determination of the relative size of extracted organs In order to arrive at a dilution factor, so that extracts could be diluted to contain a bulk of tissue comparable to that present in an extract of sinus glands, there were dissected out in turn from each eyestalk of four crabs the sinus gland, the medulla terminalis, and the group of three distal optic ganglia. These organs from each eye were placed on a haemocytometer slide in crab perfusion fluid and compressed under the cover glass (0.1 mm. clearance). Camera lucida tracings on good quality graph paper were made, the outlined areas being cut out and weighed in groups. A test made by cutting out squares of known area showed that the combined errors in cutting and in thickness of the paper did not exceed 1.5 per cent, whereas variations in dissection were large. In Table I the relative weights of the separately dissected organs are tabulated, showing the wide variation that may be expected in removing TABLE I Relative sizes of sinus gland and optic ganglia Animals Sinus gland Med. terminalis 3 distal ganglia Width Sex Right Left Right Left Right Left 16.5 mm. 17 mm. 17 mm. 19 mm. 9 9 r~U PROTHORACIC GANGLION FIGURE 1. Ventral aspect of anterior thorax and head (tilted back) of Leucophaea nymph showing position of prothoracic glands. crossing the organs are connected by a narrow tissue bridge. The anterior ends of the tissue bands taper off in the neck region. At the posterior end each band divides into two short branches one of which establishes nervous connection with the prothoracic ganglion. Figure 2, based on methylene blue preparations, shows a branch of a thin nerve entering the prothoracic gland shortly after its emergence from the lateral surface of the ganglion. No ducts are found in connection with - 1 am indebted to Miss Kate Gruen for valuable technical assistance. 188 BERTA SCHARRER these glands ; they are surrounded by blood spaces. It may be assumed, therefore, that the secretory products are given off into the blood. The prothoracic glands are present in both nymphs and adults, but in the latter they become considerably reduced in size and structure soon after emergence. PROTHORACIC GANGLION LEFT.--' PROTHORACIC GLAND FIGURE 2. Innervation of prothoracic gland of Leucopliaca by a lateral branch from the pro- thoracic ganglion, as shown for left side. Diagram based on methylene blue preparations. A. The nyniphal type In the prothoracic gland of the nymph and of the freshly emerged adult the cells are arranged in densely packed layers around the longitudinal axis of the tissue band. The center is occupied by a trachea, a nerve, and several parallel fibers of striated muscle all of which extend throughout the length of the organ (Figs. 3, 4). The nerve is evidently derived from the prothoracic ganglion, since no other nervous NERVE 8ffj •'.^J^ TRACHEA FIGURE 3. Prothoracic gland of nymph of Leucophaea stained supravitally with methylene blue. branch than the one mentioned above (Fig. 2) has been observed to enter the gland. The central nerve, in addition to innervating the musculature of the organ, probably also supplies the glandular tissue ; the delicate branches of the nerve could be traced only for short distances in methylene blue preparations. Due to the contraction of the axial muscle the glandular tissue in fresh and in fixed preparations is more or PROTHORACIC GLANDS OF LEUCOPHAEA 189 less folded, with the result that the width of the organ varies. In the wider por- tions 8 to 12 nuclei may be counted across the width of the cellular layer of one side (Fig. 3). The deeply staining nuclei are the most prominent feature of the gland. They are ovoid and, for the most part, approximately uniform in size and appearance. In regard to their dense arrangement and general morphology they resemble the nuclei of the corpora allata of the same species. In some nymphal glands a number of considerably larger nuclei may be observed. The majority of specimens studied showed some pycnotic nuclei in the prothoracic glands. f .'i -*-. *v»~ C O NNECT *.*v'iS$<*rf£:3f TISSUE " "1". . , ... SHEATH %fs*' ^,"^"'° ^ rt"O»^ ,,-tv & »-~ ' <-•» , .**"*- ,-/-"/ V, fe>-- *rtr ,-.•• V: s?*i FIGURE 4. Prothoracic gland of last instar female nymph of Leucophaea in longitudinal section. Paraffin, 4 micra, Mallory azan. The cytoplasm is not abundant and the cell boundaries are difficult to discern, except under favorable conditions as, for instance, in the periphery of the sectioned organ, where the nuclei are less densely packed. With the methods employed for permanent preparations no appreciable amount of cytoplasmic inclusions indicating a secretory activity of the prothoracic gland cells was observed. In two specimens a few acidophilic granules were seen, in others the cytoplasm showed vacuolization. These observations suggest the possibility that certain cytoplasmic inclusions may be dissolved during the ordinary embedding and staining procedure. This view is further supported by the results of supravital staining. The cytoplasm of fresh tissue subjected to supravital dyes for a suitable period of time contains inclusions, as indicated in Figure 5. Round bodies varying in diameter stain a distinct blue with methylene blue. The staining is not always uniform throughout the particle ; more deeply stained areas may be differentiated from a lighter background. The granular inclusions found to stain red with neutral 190 BERTA SCHARRER red are the same as those taking up methylene blue. This can be demonstrated by placing a methylene blue treated organ in a drop of weak neutral red solution on a slide. When a cover slip is used, the blue stain soon disappears from the granules and is replaced by a red tint. The same individual granules can be observed as they change color. After incubation with osmic acid the cells of the prothoracic glands show black- ened inclusions which, on account of their size and distribution, seem to be identical with the granules appearing in supravitally stained organs. These observations sug- gest that the granular inclusions are lipid in nature. Further than that no definite conclusions can be drawn. On the one hand, these bodies may be interpreted as °0 *o.-o FIGURE 5. Glandular portion of prothoracic gland of last instar nymph of Leucophaca, supra- vitally stained, showing granular inclusions (solid black) and nuclei (in outline). cytological manifestations of a secretory activity of the prothoracic glands, since it is known that certain types of secretion granules stain with neutral red. On the other hand the inclusion bodies may be identified as Golgi material on the basis of their similarity, both as to appearance and stainability, with the Golgi elements described in other invertebrate material (Worley, 1944). The prothoracic glands are ensheathed by a thin connective tissue membrane from which tenuous branches may be seen to enter the glandular tissue. These branches continue into the fibrous network surrounding the muscle elements in the center (Figs. 4, 6). These relationships are best observed in azan preparations where the fibrous elements stain a bright blue. The cells of the prothoracic glands undergo mitotic divisions. Not all specimens studied showed mitoses. In a group of 44 dated nymphs, ranging from instar four to eight, 4 micra serial sections in the horizontal plane through the prothoracic glands were checked for mitotic figures. Specimens fixed immediately after molt- ing, or at intervals up to six days following a molt, had no mitoses ; neither did nymphs preparing for a new molt as indicated by the separation of the old cuticle from the epidermis. However, a certain number of the prothoracic glands from ani- mals fixed during the remainder of the intermolt period, showed mitotic figures. While some of these specimens had low values (approximately one mitosis in ten sections), others had an average of up to eight mitoses per section. Since the intervals between two molts in Leucophaca are subject to considerable variation, PROTHORACIC GLANDS OF LEUCOPHAEA 191 no more precise conclusion can be drawn than that at some time during the intermolt period a probably short, but considerable spurt of mitotic activity takes place in the prothoracic glands. B. Adult type In the male and female imago of more than eight days of "adult age" the pro- thoracic glands are considerably changed in appearance (Fig. 7). The tissue bands have become thin and the microscopic examination shows that they consist almost exclusively of the muscular core. Most, if not all, of the glandular cells have dis- CONNECTIVE TISSUE SHEATH FIGURE 6. Longitudinal section of prothoracic gland of male adult Lciicophaea four days after emergence, showing involutionary process (nuclear breakdown, decrease in width of glandular component). Paraffin, 4 micra, Mallory azan. appeared. The connective tissue elements are more conspicuous than in the nymphal gland. This change of morphological appearance of the prothoracic glands within the short period of transition from the nymphal to the adult life may be interpreted as an indication that in the imago these organs are no longer function- ally active. The steps of this striking involutionary process were traced in a series of adult male and female specimens, fixed at daily intervals after emergence. While the prothoracic glands of the freshly emerged adult resemble the nymphal organ, the first signs of regression may be noticed after 24 hours of adult life. At this stage a small number of the nuclei of the glandular component are pycnotic. Within the next three or four days the cellular breakdown becomes very conspicuous in that a large proportion of pycnotic nuclei or cellular remnants are interspersed with a gradually decreasing number of seemingly still normal nuclei (Fig. 6). The glandular tissue decreases in width, while no apparent change takes place with re- 192 BERTA SCHARRER spect to the muscular component. Animals fixed six, seven, or eight days after emergence show the involution of the prothoracic glands almost completed, practi- cally all of the glandular tissue having disappeared by this time. From this stage on throughout adult life the organs show the same picture of involution (Fig. 7). CONNECTIVE: TISSUE SHEATH FIGURE 7. Longitudinal section (at point of crossing) of remnant of prothoracic glands of male adult Leucophaea one month after emergence. Note disappearance of glandular component, thickening of connective tissue sheath. Paraffin, 4 micra, Mallory azan. DISCUSSION The description given in the preceding paragraphs requires a few words of dis- cussion of the place the organ in question occupies with reference to corresponding (homologous and analogous) structures. There can be little doubt that the pro- thoracic glands of Lcncophaca are homologous with those of the Lepidoptcra. Both are located in the anterior thorax. As in Leucophaea the prothoracic glands of cer- tain Lepidoptcra are more or less branched, band-like structures without ducts (Toyama, 1902/03 ; Lee, 1948). In both, the paired glands are composed of epithe- lioid cell elements with densely packed nuclei and a small amount of cytoplasm. The innervation of the prothoracic glands does not represent a pertinent criterion for their identification, since a considerable variation seems to exist even within the Lepidoptera. In. this group of insects the prothoracic glands may receive fibers PROTHORACIC GLANDS OF LEUCOPHAEA 193 from the subesophageal ganglion, from the prothoracic ganglion, from the meso- thoracic ganglion, and from the connectives between these ganglia (Lee, 1948). The glands of Leucophaea are innervated by fibers from the prothoracic ganglion, an observation which is not at variance with Lee's observations in Lcpldoptera. The prothoracic glands of Lcpidoptera are morphologically well developed and are active as endocrine organs only in the immature insect ; their presence has not been observed in the adult moth (Williams, 1948). In Leucophaea likewise the glandular component of the organ regresses after the emergence of the adult. A feature characteristic of the prothoracic glands of Leucophaea is the presence of striated muscular tissue in the center of the organ. No such elements have been described in the corresponding organs of other insects with the possible exception of the "parenchymatous tracheal organs" of Nepa (p. 195). The physiological sig- nificance of this muscle is unknown. It is possible that it is instrumental in dis- charging the secretory product from the organ. This interpretation is suggested by the occurrence of muscular elements in exocrine glands, such as the Malpighian vessels of certain insects, likewise long, thin structures whose content is propelled towards the alimentary canal by a longitudinal muscle (Palm, 1946). Among ver- tebrates, contractile elements in the cytoplasm of the myoepithelial (basket) cells of certain glands (salivary, mammary, sweat, oral glands, etc.) also are thought to facilitate the expulsion of secretory products. Another parallel exists in the occurrence of myoid cells (reticulum cells with stri- ated fibrous elements) in the thymus of a number of vertebrates (see Bargmann, 1943). Their morphological similarity with the striated components of the pro- thoracic glands of Leucophaea is of interest in view of other features the prothoracic glands of insects and the vertebrate thymus appear to have in common (p. 195). Whereas the homology between the prothoracic glands of Leucophaea and of Lepidoptcra is reasonably well established, it is not always easy to recognize from the descriptions in the literature which of various organs in the head and thorax of different insects correspond to the prothoracic glands. Williams postulated that the "ventral glands" ( Pflugfelder, 1938) of Dixippus (Orthoptera) are homologous with the prothoracic glands of Lepidoptera. In this connection it is highly inter- esting that Pflugfelder (1947) in a more recent publication extended his study of the ventral glands to include a number of insect orders, i.e., Odonata, Epheinerida, Plecoptcra, Sanatoria, Phasmida, Dcrmaptcra, Blattaria, Mantodea, and Isoptera. According to these data there exist in almost all lower Pterygota glandular organs of an endocrine character which, in their topography and histology, show many similarities. These phylogenetically ancient ventral glands are assumed to be de- rived from originally segmental organs whose function was excretory. In some cases the connection with the place of origin, i.e., the ventral epidermis, is still evi- dent. More specifically, the ventral glands developed from the ectodermal canal of their precursor organs in the respective segment. During this transformation the lumen of the excretory duct gradually disappeared, a process which is indicated by the presence of a vestigial lumen in the ventral glands of Plecoptera and Dcrinaptera. The wall of the duct became the endocrine tissue. It is of interest that a similar transformation from nephridial organs to glands of internal secretion occurred in certain crustaceans. The rudimentary antennal gland of the isopod Asclliis, a serial homologue of the ventral glands of insects, is endocrine in nature (Pflugfelder, 1947). 194 BERTA SCHARRER Another recent publication must be considered in a discussion of the problem of homology of the prothoracic glands. Cazal (1947) described organs in Aeschna (Odonata) as "massifs ectodermiques intersegmentaires" which, at least with re- spect to their component situated in the posterior head region, seem to correspond to Pflugfelder's ventral glands. With the prothoracic glands of Lepidoptera and Orthoptera (Leucophaca), Cazal's organs have in common their occurrence in the thorax, their paired, elongated, irregularly lobated structure, and their histological appearance. Like the prothoracic glands of Lencophaea, the "intersegmental or- gans" of Aeschna consist of modified epithelial cells with scanty cytoplasm and closely packed nuclei and exhibit a typical cyclic behavior. A quiescent phase (after each molt) is followed by an active phase (preceding each molt), during which the nuclei show mitotic and pycnotic pictures. A "crisis" (crise cinetique) occurs in the adult which leads to the involution of the organ, comparable to that of the prothoracic glands of other insects, especially of Lcucophaea. An additional point of agreement exists between the observations in Aeschna and those reported in the present paper. The structure of the "intersegmental or- TABLE I Antennal glands 1st maxillary glands 2nd maxillary glands Thoracic nephridia 1 2 3 Crustaceans Present, rudimen- tary, or ab- sent (endo- crine in Rudimen- tary, or ab- sent Present, rudimen- tary, or ab- sent Absent or present (Branchi- ura); with- out canal in Absent or rudimen- tary (Ostra- coda) Absent or rudimen- tary (Ostra- coda) Asellus) Ostracoda Onychophora Rudimen- tary Salivary glands Present Present Present Present Diplopoda Absent Absent Tubular glands Absent Absent Absent Chilopoda Absent Absent Salivary glands? Absent Absent Absent Insects: A pterygota Absent Absent Cephalic nephridia Absent Absent Absent "Lower" Pterygota Absent Corpora allata? Ventral glands Absent Absent Absent "Higher" Pterygota Absent Corpora allata? Salivary glands, spin- ning glands? prothoracic glands (Bombyx) Prothoracic glands? Absent Absent PROTHORACIC GLANDS OF LEUCOPHAEA 195 gans" according to Cazal is identical with that of the corpora allata of the same species. The striking similarity between these endocrine organs and the prothoracic glands in Leucophaea has been discussed (p. 189). Comparable observations in his material and a study of the literature led Pflugfelder (1947) to suggest that the corpora allata, like the ventral glands, may be derived from nephridial organs. If we accept this derivation as correct, the ventral glands of the lower Ptcrygota (like the cephalic nephridia of the Apterygota and the second maxillary glands of crustaceans) can be considered as serial homologues of the corpora allata of ptery- gote insects, as well as of the antennal glands of crustaceans. No derivatives of thoracic nephridial organs are known to exist in the insect orders possessing ventral glands. In all holometabolous insects, in Hciniptcroidea, in Mantis, and perhaps in Blatta, organs corresponding to the ventral glands of other insects studied by Pflugfelder are said to be absent. However, in some of these forms (Lepidoptera, Hymenoptera, Blattaria} organs situated in the thorax, the prothoracic glands ("hypostigmatic glands" of Toyama, "intersegmental organs" of Cazal) appear to take the place of the ventral glands. Furthermore, it is possible that a reinvestiga- tion of the enigmatic "parenchymatous tracheal organs" in the thorax of the Hetnip- teran Nepa (Hamilton, 1931) may link these organs with the prothoracic glands of other insects. The possible homologies of the prothoracic glands are summarized in Table I, which is based largely on Pflugfelder's data concerning the developmental history of nephridial derivatives in the Articulata. With respect to Bombyx, Toyama (1902/03) showed that in the embryo the prothoracic glands develop from the second maxillary segment and, due to a short- ening of this segment in the course of development, subsequently become located in the anterior thoracic region. If a corresponding derivation can be demonstrated in other forms possessing prothoracic glands these glands can be considered homolo- gous with the ventral glands. However, it is quite possible that in certain insects the prothoracic glands may be shown to be derivatives of nephridia of the first thoracic segment, in which case they would be serial homologues of the ventral glands. The variability in the innervation of the prothoracic glands which may be supplied from ganglia of several segments (Lee, 1948; see also Pflugfelder, 1947) would thus be better understood. The striking similarities in the development, structure, and life history of the ventral glands, the intersegmental organs, and the prothoracic glands suggest a correspondence in function (see Table II). It seems justified, therefore, to con- sider them as homologous organs whose endocrine function appears to be concerned with the control of developmental processes, both embryonic and postembryonic. It has been mentioned before (p. 193) that the prothoracic glands of Leucophaea and their homologues also have some features in common with the thymus of the vertebrates ; they are listed in Table III. The most interesting of these parallelisms are the involutionary process at the onset of sexual maturity, and the hastening of this regression by allatectomy 3 and hypophysectomy respectively. The significance of such comparable traits is admittedly conjectural. However, it may be pointed out that there exist other structural and functional correlations between certain organs and organ systems of invertebrates and vertebrates, which 3 These observations will be reported in greater detail in a later publication. 196 BERTA SCHARRER , CU X Function lilii U 3 Q, O — "2 ^-2 S n 2 'C <*« i i £ c ^ Q OjO |-J -4-) QJT> •x'-S c ^ c3 E 2 S.c S.S a o o dj Cd CO '— . *_s «E k*— •§ ^ ca "§ o a ^ o » *cD c cc ^ 5 m OJ 3 "3 •g II •*-> •*-> co n) O Jr; --^ 3 '£ « - S £ c S Kg 0 o .Q C § « S o 2 .y.-y £"§ C/3 .S co .Q ca E = § 1 '£ 2 S c E a E >»t; C i- "g y , i "> t~~ c ^ . 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"53 ° o "3 T. .S -g ."2 •"^ /•*! 7 ^~^ T3 ^ co , QJ w ^D (U L " ca o o "D m T3 ^ o ^ -w o s ^ 12 o£ » O "o CB ||S5^ ||| If •cx,O-,,_rScu ^S^l! 5s ^r,CUC^cU «*$ !^^> >-J C- W r*1 us i— 3 >l! 0 !— • O*-J l«1 S ^ OJ ^J V HH njfei-S CO ca CO -o 15 it: to -M tn 1> , •a 1 « C C w 3 c ^ ca ^ hn ca Sj 2 f OJ .rt t Q/j p ^- "M 5,3's E O £ ~~ X ° ?c j. _t S? £ M u ' — ' *^ ca lf~] ^— • ^"^ 2 2 to ca E d ^ fli rd ^ *^ ^* -*-*,-• c :" dJ -£ O bjo *^ cu C >-. O f« '3 ^- JS '^3 ,9 03 g ^ H- 1 O CU .^H 4-> CU oc co M H-l -4-> PROTHORACIC GLANDS OF LEUCOPHAEA 197 TABLE III Prothoracic glands (and equivalents) Location in posterior head and thorax Derived from bilateral invaginations of ecto- derm Ducts originally present, later rudimentary or absent Segmental development Syncytial structure (Platysamia} Lymphocyte-like cell components (Bombyx) Muscular components (Leucophaea) Maximum size before emergence of adult, in- volution in imago Influenced by endocrine disturbances (involu- tion hastened by allatectomy, Leucophaea) Endocrine function established in Lepidoptera (control of developmental processes) Thymus Location in neck and thorax Derived from bilateral epithelial ingrowths Ducts reduced to rudiments during develop- ment Segmental derivation (from several pharyngeal pouches) Syncytial reticulum So-called thymocytes of lymphocyte type Myoid cells Maximum relative size before puberty, involu- tion after puberty (higher vertebrates) Sensitive toward endocrine imbalance, for in- stance hypophysectomy Doubtful endocrine function related to growth and development cannot be dismissed as superficial and accidental similarities, but which indicate similar principles of functional organization. Thus the intercerebralis-cardiacum- allatum system of insects is analogous to the hypothalamo-hypophyseal system of vertebrates (Hanstrom, 1941; Scharrer and Scharrer, 1944), the internephridial organs of the worm Physcosoma to the interrenal body (Harms, 1921), the x-organ of crustaceans to the thyroid, and the sinus gland of crustaceans to the paraphysis (Hanstrom, 1941). From these examples as from many others the concept evolves ever more clearly that the gap between invertebrates and vertebrates has in the past been magnified out of its true proportions. SUMMARY 1. The prothoracic glands of Leucophaea inadcrac are paired band-like structures located in close proximity to and innervated by the prothoracic ganglion. The longitudinal axis contains striated musculature, a nerve and a trachea. The glands are well developed in nymphal stages, but regress in the imago. 2. In the nymph the glandular tissue consists of layers of dense nuclei sur- rounded by scanty cytoplasm. In their histological appearance the prothoracic glands strikingly resemble the corpora allata of the same species. The nymphal prothoracic glands exhibit a cyclic nuclear activity, characterized by a spurt of mitotic divisions during the intermolt period and by quiescent phases preceding and following each molt. 3. In freshly emerged male and female adults the prothoracic glands are still nymphal in appearance. Involution takes place during the first week of the adult stage. It manifests itself by a breakdown of nuclei and results in a reduction of the tissue bands to practically nothing except the muscular core. 4. The prothoracic glands of Leucophaea are considered to be homologous with the prothoracic glands of Lepidoptera and Hyincnoptcra, with the "ventral glands" of lower Pterygota, with the "intersegmental organs" of Odonata, and possibly with the "parenchymatous tracheal organs" of Hcmiptera. .They have certain features in common with the thymus of the vertebrates. 198 BERTA SCHARRER LITERATURE CITED BARGMANN, W., 1943. Der Thymus. Handb. mikr. Anat. Mensch., edited by W. v. Moellen- dorff, 6 : part 4, pp. 1-172. CAZAL, P., 1947. Recherches sur les glandes endocrines retrocerebrales des insectes. II. Odonates. Arch. zool. e.vper. gen., 85 : 55-82. FUKUDA, S., 1940a. Induction of pupation in silkworm by transplanting the prothoracic gland. Proc. Imp. Acad. Tokyo, 16: 414-416. FUKUDA, S., 1940b. Hormonal control of molting and pupation in the silkworm. Proc. Imp. Acad. Tokyo, 16: 417^20. FUKUDA, S., 1941. Role of the prothoracic gland in differentiation of the imaginal characters in the silkworm pupa. Annot. Zoo/. Japan., 20: 9-13. HAMILTON, M. A., 1931. The morphology of the water-scorpion, Nepa cinerea Linn. (Rhyn- chota, Heteroptera). Proc. Zoo/. Soc. London, 1931 : 3 and 4, pp. 1067-1136. HANSTROM, B., 1941. Einige Parallelen im Bau und in der Herkunft der inkretorischen Organe der Arthropoden und der Vertebraten. Lunds Univ. Arsskr. N. F. Avd. 2, 37 : no. 4, 1-19. HARMS, W., 1921. Morphologische und kausalanalytische Untersuchungen ueber das Interneph- ridialorgan von Physcosoma lanzarotae nov. spec. Arch. Entwickl. Mech., 47 : 307-374. KE, O., 1930. Morphological variation of the prothoracic gland in the domestic and the wild silkworms (In Japanese with English summary). Bultcno Scicnca Fakult. Terkult. Kyushu Imp. Univ. Fukuoka, 4: 12-21. LEE, T. Y., 1948. A comparative morphological study of the prothoracic glandular bands of some lepidopterous larvae with special reference to their innervation. Ann. Ent. Soc. Amer., 41 (in press). PALM, N. B., 1946. Studies on the peristalsis of the malpighian tubes in insects. Lunds Univ. Arsskr. N. F. Avd. 2, 42 : no. 11, 1-39. PFLUGFELDER, O., 1938. Weitere experimentelle Untersuchungen ueber die Funktion der Cor- pora allata von Dixippus morosus Br. Zcit. iviss. Zoo/., 151 : 149-191. PFLUGFELDER, O., 1939. Wechselwirkungen von Druesen innerer Sekretion bei Dixippus moro- sus Br. Zeit. zviss. Zoo/., 152 : 384-408. PFLUGFELDER, O., 1947. Ueber die Ventraldruesen und einige andere inkretorische Organe des Insektenkopfes. Biol. Zcntralbl., 66: 211-235. SCHARRER, B. Hormones in insects. Hormones, chemistry, physiology, and clinical applica- tions, edited by G. Pincus and K. V. Thimann, vol. I (in press). SCHARRER, B., AND E. SCHARRER, 1944. Neurosecretion VI. A comparison between the intercerebralis-cardiacum-allatum system of the insects and the hypothalamo-hypophyseal system of the vertebrates. Biol. Bull., 87 : 242-251. TOYAMA> K., 1902/03. Contributions to the study of silk-worms. I. On the embryology of the silk-worm. Bull. Coll. Agric. Tokyo Imp. Univ., 5: 73-118. WILLIAMS, C. M., 1947. Physiology of insect diapause. II. Interaction between the pupal brain and prothoracic glands in the metamorphosis of the giant silkworm, Platysamia cecropia. Biol. Bull, 93 : 89-98. WILLIAMS, C. M., 1948. Physiology of insect diapause. III. The prothoracic glands in the Cecropia silkworm, with special reference to their significance in embryonic and post- embryonic development. Biol. Bull., 94: 60-65. WORLEY, L. G., 1944. Studies of the vitally stained Golgi apparatus. III. The methylene blue technique and some of its implications. /. Morph., 75: 261-289. OBSERVATIONS ON THE RESPIRATION OF AUSTRALORBIS GLABRATUS AND SOME OTHER AQUATIC SNAILS THEODOR VON BRAND, M. O. NOLAN, AND ELIZABETH ROGERS MANN 1 Division of Tropical Diseases, National Institute of Hcaltli, United States Public Health Service, Bethesda, Maryland Trematode diseases are best eradicated, or at least reduced in their incidence, by interrupting the life cycle of the parasites in the intermediate hosts, that is, in the case of human infections, by instituting campaigns against the snails harboring the juvenile worms. This has been attempted to date by methods founded purely on empirical findings. It seems probable that a study of the physiology of the snails might yield important clues for the development of chemical means of control. From a theoretical standpoint it appears likely that snails may be killed by the use of compounds interfering with the cellular respiratory mechanisms. As a pre- liminary to such work, a study was initiated of the respiration of some fresh water snails. We report in the present paper the results of our experiments on the normal aerobic respiratory physiology of Australorbis glabratus, the intermediate host of Schistosoma mansoni in the West Indies and South America. Included also are some data on the respiration of other snail species, most of them belonging to genera transmitting trematodes of man or lower animals. MATERIAL AND METHODS The following species of snails were employed: 1. Pulmonates Planorbldae : Australorbis glabratus, laboratory reared from Venezuelan speci- mens ; Helisoma duryi, in part specimens freshly collected at Kenilworth Gardens, Md., in part laboratory reared specimens derived from this stock; Tropicorbis obstructiis and T. donbilli, both laboratory reared from speci- mens collected in Texas. Lymnaeidae : Lymnaea stagnalis, collected from Mullet Lake, Michigan; L. palustris from Stemple Creek, Marin County, California; L. obmssa from a canal in the vicinity of Washington, D. C. These snails were used shortly after their arrival in Bethesda. Pliysidae: Pliysa gyrina, in part specimens freshly collected at Kenilworth Gar- dens, Md., in part laboratory reared specimens derived from this stock; Physa sp. specimens freshly collected in a creek near Bethesda, Md., and laboratory reared specimens derived therefrom. 1 The authors are indebted to Dr. E. G. Berry for the determination of many of the snails and for advice concerning their care, to Miss Ruth Rue and Dr. L. Olivier for the contribution of a number of specimens, and to Mr. Benjamin Mehlman for technical assistance during the respiration experiments. 199 200 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN 2. Operculates Amnicolidae: Amnicola limosa. collected in a canal near Washington, D. C. ; Oncomelania quadrasi, laboratory reared specimens derived from snails col- lected in the Philippines; O. nosophora, used some weeks after having been received from Japan. Vhnparidae: Cainpeloma sp., used shortly after having been shipped from Doug- las Lake, Michigan. Melaniidae: Semisulcospira sp., laboratory reared specimens, derived from snails collected in the Philippines. Pomatiopsidae: Pomatiopsis lapidaria, freshly collected specimens from Fairfax County, Virginia. Plenroceridac: Pachychilus sp., specimens collected in Guatemala and kept prior to the determinations for months in an aquarium. Littorinidae: Littorma irrorata, recently collected from Wicomico River, Mary- land. All species, with the exception of Littorma, are fresh water species ; Littorma occurs in brackish water. With the exception of the very few cases in which entirely freshly collected specimens were used, the truly aquatic snails were kept in balanced aquaria in a room with a minimum temperature of 21° C. During the summer months the tempera- ture rose to nearly 30° C. The snails were abundantly fed with lettuce leaves and fish food. From time to time some powdered calcium carbonate was added to the water. Those species (Oncomelania, Pomatiopsis) that lead in nature a semi- aquatic life were kept in aquaria simulating as best as possible their normal habitat (see Ward, Travis, and Rue, 1947, for details) ; their chief food consisted of leaf mold. Before the snails w^ere used for the actual determinations, the water adhering to the specimens was removed with filter paper ; they were then weighed on an ana- lytical balance to the nearest mg. The standard temperature used in all experi- ments, with the exception of those in which the temperature influence was studied, was 30° C. This temperature was slightly higher than that reached by the aquarium water during the summer months and was chosen in order to allow an adequate control of the water bath. This temperature was .well tolerated by all species employed. The respiratory medium was, during the first months of the study, filtered river water. Later on tap water was employed ; this was allowed to stand for a minimum of 24 hours in the laboratory to permit the chlorine to evaporate. No difference in respiratory rate was noticed in either type of water. The respiratory exchange was studied by means of Warburg manometers. The number of snails used for a single determination, the amount of water introduced into the flasks, the size of flasks employed, and the interval between readings varied, depending upon the size of the snails. Up to 16 specimens of the small species, or the juveniles of larger species, were used for each flask. The total capacity of these flasks was about 17 cc., and 2 cc. of water were used as respiratory medium. Of the largest species, single specimens were studied in flasks having a total capacity of about 120 cc. ; in these cases 6 cc. of water served as medium. In this latter RESPIRATION OF SNAILS 201 case an equilibration period of one hour was necessary before the manometers could be closed while 20 minutes were sufficient when the smaller flasks were employed Depending upon the respiratory rate and the size of the flasks, readings were taken every 15 or 30 minutes for periods of two to four hours. The manometers were shaken with an amplitude of 4 cm. 100 times per minute. Most of the snails re- mained very quiet in the vessels ; only the Physa species had a tendency to leave the water and to affix themselves to the sides of the flask. In most cases the oxygen uptake alone was studied, the carbon dioxide being absorbed in the customary manner by means of 10 per cent KOH. In most series atmospheric air served as gas phase. In these series we were interested in the oxygen consumption of the snails in air saturated water, or, what amounts nearly to the same, that taken by pulmonates from the air itself. The question of the influ- ence of varying oxygen tensions was studied only with Aiistralorbis glabratits. In these experiments the gaseous atmosphere was changed by passing gases of known composition for a period of 20 minutes through the manometers. Details will be given in a later section. The respiratory quotient was studied in some series of Aiistralorbis glabratus. Since experience in other experiments had shown that the respiration of these snails stays fairly constant over long periods of time, a modification of Warburg's direct method was employed. The snails were first introduced into flasks containing no KOH and the change in manometer reading was followed for one hour or an hour and a half. The flasks were then removed from the manometers, KOH and filter paper were introduced into the center well and, after reequilibration to the tempera- ture of the water bath, a second set of readings was taken for an equal period as above. The error due to possible changes in the respiratory rates is small since only such experiments were used in which the two sets of readings gave steady values. A few experiments in which either the first or the second set gave incon- sistent readings were rejected. The snails used in these series were handled as little as possible; they were not dried or weighed in order to avoid injuries to the shell. This is a very important point since a pronounced carbon dioxide retention sets in very rapidly after the shell has been cracked. The readings taken in these series were calculated as mm3, oxygen or carbon dioxide per one snail instead of being related to weight or surface area and the RQ was calculated from these values. RESULTS 1. Relations betzueen size and respiratory rate The snails varied considerably in size due both to species differences and to dif- ferences in age of specimens of one species. This gave an opportunity to study the relation between size and rate of oxygen consumption insofar as both inter- and intraspecific comparisons are concerned. The experiments summarized in this sec- tion were carried out from May to August ; there wyas no indication that during this time seasonal variations in oxygen consumption occurred. The determinations were in a few cases carried out with freshly collected specimens ; in most cases well fed aquarium snails were employed. This point is important. It will be shown in a later section that the respiratory rate of snails declines very rapidly during starva- tion. It was even repeatedly noted that determinations carried out on Mondays yielded somewhat lower values than during the remaining days of the week, the 202 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN Z 0.4 t£ 1 1 o.o it O Z N3 0.6-1 -1 0.2-1 Zl- or 1-9 0.2-1 Q. UJ H in O o O 2 II. 4 25 5| \ *»* 12 13 14 13 3 4 3 3°27 S43 . 4 PULMONATES * OPERCULATES i i7i« 15 0.0-21 0.40 0.80 I.6O 2.00 2.40 2.80 LOG OF BODYWEICHT (MC.) AND RELATIVE SURFACE 3.20 4.00 FIGURE 1. Relation between size and rate of oxygen consumption in various species of aquatic snails. Pulmonates : 1. Australorbis glabratus, 2. Physa sp., 3. Physa gyrina, 4. Lymnaea palustris, 5. Hclisoma duryi, 6. Lymnaea obrussa, 7. Tropicorbis donbilli, 8. Tropicorbis ob- structus, 9. Lyinnaca stagnalis. Operculates : 10. Ammcola limosa, 11. Oncomelania quadrasi, 12. Pomatiopsis lapidaria, 13. Scmisulcospira sp., 14. Oncomelania nosophora, 15. Campeloma sp., 16. Pachychilns sp., 17. Littorina irrorata. TABLE 1 Respiration of Australorbis glabratus of various sizes at 30° C. Mean values and, in parentheses, extremes Number of experiments Number of snails Weight of single snail mg. Mm.3 oxygen consumed by 1 snail in 1 hour Relative surface Ratios of Weight Oxygen consumption Surface 6 40 14 5.9 5.8 1 1 1 (12, 16) (5.4, 6.2) 6 24 66 16.8 16.3 4.7 2.8 2.8 (55, 76) (14.0, 20.2) 6 12 153 30.3 28.6 11 5.1 4.9 (122, 201) (25.1, 38.4) 6 24 564 81 68.3 40 14 12 (525, 601) (60, 95) 6 6 1137 110 109 81 19 17 (1000, 1288) (84, 156) 6 6 1903 164 154 136 28 27 (1646, 2220) (105, 284) RESPIRATION OF SNAILS 203 reason being that the snails had more or less exhausted their food supply over the week end. The data summarized in Figure 1 are average figures from 4 to 12 determinations each, in most cases from 6 determinations. The sizes of the snails are also average values. The data for Aiistralorbis glabratus (Table 1) may serve to illustrate these points and the variability introduced by them. The data, as shown in Figure 1, prove three facts. First, the respiratory rate of pulmonates was without exception higher than that of operculates when snails of equal weight were compared. Second, the respiratory rate of both pulmonates and operculates was inversely correlated to the size of the specimens when calculated on the basis of weight. The slope of the straight lines around which the values of the two groups fluctuated was almost identical, indicating that about the same per- centage decline in respiratory rate with increasing weight occurred in both groups. Third, it is quite apparent that straight horizontal lines resulted if the respiratory rates were calculated on the basis of relative surface (Weight2/3) rather than weight. Several reasons may be responsible for the deviations from the average straight lines which in some cases were rather marked. Although we tried to use for the experiments summarized in this section only well fed snails, it is entirely possible that not all species were equally near an optimum diet. Another point is that only the soft parts of the snails are actively metabolizing while our data are based on the complex soft parts plus shell. It was for various reasons not possible to determine in each experiment the shell weight and therefore no attempt was made to correlate the oxygen figures to the weight of the soft parts. Sixteen determinations of the shell weight were carried out at various times during the present experiments on various species; it was found that it varied between 11.2 and 25.4 per cent. It is, of course, also possible that the metabolic rate of various species is inherently some- what different, even if nutrition and all other factors were exactly equal. Despite these deviations, the trend of the curves is convincing. It should be mentioned as especially noteworthy that small and large specimens of a given species followed rather closely the surface law, as shown in Table 1 on the example of Aiistralorbis glabratus. This is remarkable since in many other or- ganisms juvenile specimens show a higher rate of metabolism than would be expected from this relationship. It is pertinent to mention that the australorbids used for these experiments were all taken from a single aquarium and that the experiments with them were all carried out within two weeks. We were therefore dealing with an exceptionally uniform material insofar as food and physical environmental factors were concerned. 2. Influence of oxygen tension The experiments on the influence of oxygen tension on the oxygen consumption were carried out with Aiistralorbis glabratus specimens of two sizes, small snails weighing 30 to 40 mg. each and medium sized snails weighing 300 to 400 mg. each. Fully grown specimens could not be used because the gaseous atmosphere could not be changed conveniently in the large flasks that alone would accommodate them. The rate of oxygen consumption of the snails was first established at the oxygen tension of atmospheric air for a period of 1% to 2 hours with readings taken at 15 minute intervals. A gas mixture of known oxygen tension was then passed for 20 204 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN minutes through the flasks. After the manometers had been closed, the rate of oxygen consumption at the experimental tension was followed for two hours. The temperature was in all experiments 30° C. Twelve experiments were carried out for each of the two groups at each of the four experimental oxygen tensions, and each time new snails were employed. The summary of all experiments (Table 2) indicates that the reactions of both size groups to changes in oxygen tension were very nearly identical. It is probably justifiable to assume that fully grown specimens would have reacted in an essentially TABLE 2 Influence of oxygen tension on the oxygen consumption of Australorbis glabratus at 30° C. Mean values and, in parentheses, extremes Series O? consumption at 160 mm. Hg. tension mm.3/l grn./l hr. Experimental Oz tension mm. Hg. Oa consumption at experimental tension Mm.3/l gm./l hr. Per cent of 160 mm. value A 296 760 288 98±8.0 (254, 318) (250, 334) B 140 760 144 104±3.1 (101, 177) (97, 190) A 255 38 205 81±2.0 (226, 270) (186, 221) B 161 38 138 86±2.3 (122, 187) (99, 178) A 311 13 260 85±3.2 (249, 416) (234, 279) B 163 13 156 95±4.1 (129, 183) (110, 204) A 246 5 29.6 12±0.4 (187, 310) (8.2, 49.2) B 177 5 12.5 7±0.7 (147, 262) (3.8, 17.3) Series A: Snails weighing 30 to 40 mg. each. Series B: Snails weighing 300 to 400 mg. each. identical manner. On the whole, there was little indication that the oxygen con- sumption was markedly influenced by changes in tension between 760 and 13 mm. Somewhere below this latter tension the consumption began to fall off rapidly ; at a tension of 5 mm. only a small fraction of the normal amount was consumed. The data obtained prove conclusively that Australorbis glabratus belongs to the group of invertebrates capable of maintaining a more or less uniform rate of oxygen consumption over a wide range of tensions. RESPIRATION OF SNAILS 205 3. Influence of temperature The influence of temperature on the rate of oxygen consumption of Aitstralorbis glabratits was investigated in the range of 0.3 to 41° C. The experiments were car- ried out with fairly small snails, the individual specimen weighing from 40 to 90 mg. Six snails were introduced into each flask and 12 experiments were conducted for each experimental temperature. New snails were used for each experiment. The respiratory rate of every lot was first established at our standard temperature of 30° C., four readings being taken at 15 minute intervals. The manometers were then transferred to a second water bath regulated to the intended experimental tem- perature. After equilibration the respiration was followed for a two hour period with readings taken at 15 or 30 minute intervals at the higher and lower tempera- tures respectively. The manometers were then transferred back to the original 30° C. water bath and a new set of readings was taken at 15 minute intervals in order to test whether during this recovery period the original level of oxygen consumption would again be reached. The absolute values obtained in the various series during the initial 30° C. period varied somewhat, due probably to the differences in size between snail-s of the TABLE 3 Influence of temperature on the oxygen consumption of Australorbis glabratus, averages and, in parentheses, extremes Initial oxygen consumption at 30° C. mm.3 C>2/1 gm./l hr. Experi- mental temper- ature °C. Oxygen consumption at experimental temperature Oxygen consumption at 30° C. following experimental temperature Mm.3 O2/l gm./l hr. Per cent of initial value First hour Second hour Mm.3 Oz/1 gm./l hr. Per cent of initial value Mm.3 Oz/1 gm./l hr. Per cent of initial value 151 0.3 6.6 4.3±0.25 58 39±2.9 Ill 75±6.9 (119, 186) (3.7, 10.0) (39, 74) (73, 151) 222 5.0 15.2 6.9±0.34 131 60±4.0 225 102±4.7 (169, 260) (11.8,17.4) (101, 162) (185, 290) 208 10.0 21.3 10.2±0.16 204 98±1.9 (155, 256) (16.7,27.9) (166, 253) 184 14.8 48.3 27±1.3 178 99±5.0 175 97±4.9 (136, 224) (40.0, 54.3) (167, 196) (150, 222) 179 19.7 88 50±2.2 185 105±3.9 (141, 234) (76, 96) (158, 214) 175 24.7 115 67±3.5 168 97±2.9 (134, 200) (94, 140) (145, 188) 193 37.0 281 148±7.0 193 99±3.8 (139, 228) (226, 358) (138, 260) 225 41.0 158 71±3.7 152 68±7.2 (190, 282) (111,221) (86, 248) 206 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN various batches and to small differences in their nutritional state. These differences do not, however, interfere with an evaluation of the experiments since the figures found during exposure to the experimental temperatures and those obtained during the recovery period could be expressed in per cent of the initial value, thus elimi- nating any influence of these variations. The oxygen consumption of the snails (Table 3) increased in the range 0.3° to 37° C. At 41° C., however, the animals were definitely damaged. Their respira- tory rate decreased, and it did not come back to the original level during the recovery period. After the end of the recovery period, the snails were kept in beakers over night at room temperature and it was found that all were dead the following morn- ing. The lowest temperature employed, 0.3° C., was also damaging. The respira- tory rate increased only slowly after the snails had been transferred back to 30° C., and, after being kept over night at room temperature, about half the snails were dead. All other temperatures were well tolerated and the respiration returned during the recovery period to the pre-experimental value. 2.5 2.0 Z O I- Q- 5 D CO Z O u ^1.5 UJ O UJ U o; LJ Q_ O O 1.0 0.5 _L 32 33 34 35 36 X I04 37 38 FIGURE 2. Temperature relationships of the oxygen consumption of Australorbis glabratus in the range 0.3° to 37° C. expressed according to Arrhenius' equation. Using the percentage oxygen figures, the temperature relationship was then cal- culated according to Arrhenius' equation (Fig. 2) for the range 0.3° to 37° C. A single straight line was obtained and the /A value of 17,400 is entirely within the normal range. Upon projection of the percentage figures on Krogh's (1914) normal curve a very satisfactory agreement to this curve was obtained (Fig. 3). Krogh's curve has been established only for the range 0° to 29° C. The points obtained in the present investigation beyond this range show an excellent fit to an extension of this curve. It would seem possible that this extension may have the same general applicability as the original curve. RESPIRATION OF SNAILS 207 10 15 20 25 30 35 33 FIGURE 3. Projection of the percentage oxygen figures of Australorbis glabratus, taken from Table 3, on Krogh's normal curve and extension of this curve to 37° C. 4. Influence of starvation The influence of starvation on the rate of oxygen consumption was studied with Australorbis glabratus, Hclisoina duryi, Physa sp., and Physa gyrina. Of the three former species six groups of two snails each were employed while six groups of four snails each were used of P. gyrina. The snails of each species were selected as to uniformity in size. Each group was kept in a beaker with about 200 cc. water which was changed at frequent intervals and which was aerated by a slow stream of air bubbling through the water. The experiments were carried out during the summer months, the beakers being kept in a room without temperature control. The water temperature was, however, checked daily. It varied between 21.0° and 29.5° C. ; the average temperature was about 27° C. The rate. of oxygen consump- tion was determined for each group at the start of the starvation period, and after designated periods of starvation ; the temperature during the actual determinations was 30° C. The snails \vere weighed before each determination. The average loss in weight towards the end of the experiments was 18 per cent of the original weight in the case of Physa sp., 13 per cent both in Australorbis glabratus and Physa gyrina, and only 3 per cent in Helisoma duryi. Why this latter species lost relatively so little weight is not clear; it was the species that resisted starvation longest. Because this loss in weight would mask to a certain extent the decrease in metabolic level, if the oxygen values would have been calculated on the basis of the weight the organisms had on a specified day, the values are expressed in mm3, oxygen/1 snail/1 hour. 208 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN Figure 4 shows that the various series ended after three to seven weeks starva- tion. When one snail out of a group died, the group was discarded and the whole series was completed when one of the snails of the last remaining group had died. Obviously then, our figures do not indicate the upper limit of starvation that the O - AUSTRALORBIS GLABRATUS • = HELISOMA DURYI x = PHYSA SP. PHYSA GYRINA 20 24 28 32 DAYS OF STARVATION FIGURE 4. Influence of starvation on the rate of oxygen consumption of four species of pulmonate snails, absolute figures. 100 O =AUSTRALORBIS GLABRATUS • =HELISOMA DURYI x = PHYSA SP. = PHYSA GYRINA 20 24 28 32 DAYS OF STARVATION FIGURE 5. Influence of starvation on the rate of oxygen consumption of four species of pulmonate snails, percentage figures. various species can endure at the temperature prevailing in our experiments. We are even hesitant to assume that all our snails died of actual starvation ; the unavoid- able repeated handling may well have hastened the end of one or the other specimen. It is obvious (Fig. 4) that in all four species the rate of oxygen consumption declined sharply during the initial stages of starvation. Later the decline was much RESPIRATION OF SNAILS 209 less marked, but no completely steady level was reached. In order to test whether the influence of starvation was noticeably different in the various species, the starva- tion values were then calculated in per cent of the initial values. This eliminated the differences in the absolute amounts due to the various sizes of the snails belong- ing to the different species. Figure 5 shows that the values thus obtained fit fairly well to a single curve ; really large deviations occurred only during the first days of starvation. On the whole it is evident that the effect of progressive starvation was quite similar in the four species studied. The respiratory quotient was studied only in the case of Australorbis glabratus. The snails were kept in these experiments in a room with an average temperature of approximately 25° C. but the actual RQ determinations were carried out at our standard temperature of 30° C. The points shown on Figure 6 are mean values of from five to 14 determinations each. BEGINNINING OF STARVATION 0.5 FIGURE 6. Respiratory quotient of Australorbis glabratus under the influence of starvation and subsequent feeding. The respiratory quotient of well fed snails was around 0.85 ; the values found during the progressive stages of starvation fluctuated around the curve shown in Figure 6. A progressive lowering is evident and after three to four weeks starva- tion the surprisingly low value of 0.6 was reached. After 27 days inanition the snails were again fed and the respiratory quotient determined after one and two weeks feeding. It did rise during this period markedly but failed to reach the pre-starvation level. DISCUSSION The present investigation has shown that aquatic pulmonate snails have con- sistently a higher rate of oxygen consumption than operculates of the same size. A definite reason for this difference could not be adduced; it is an illustration of the well known fact that animals with different organization frequently have dif- ferent metabolic levels. In both groups the respiratory rate was inversely correlated to the weight of the specimens, but remained more or less constant if referred to relative surface. This relationship held true both in intraspecific and interspecific comparisons and was especially close in the former case. The question of the relationships between body size and metabolic rate has recently been reviewed by von Buddenbrock ( 1939) , Kleiber ( 1947) , and Zeuthen ( 1947) . While the surface law applies rather 210 TH. VON BRAND, M. O. NOLAN, AND E. R. MANN closely in the case of most vertebrates, the matter is more complex in invertebrates, or when the animal kingdom as a whole is considered. Large deviations are espe- cially apparent in the largest and smallest organisms for which data are available. Insofar as molluscs specifically are concerned, Weinla.nd (1918) found a positive correlation between surface and respiratory rate in Anodonta, while Liebsch (1928) denies such a relationship in terrestrial snails ; he found a positive correlation be- tween weight and respiratory rate. It should be pointed out, however, that the nutritional state of his snails was not uniform and that the size range of his speci- mens was appreciably smaller than that employed by us. An inspection of our data (Fig. 1) shows that within limited size^ ranges lines apparently showing a constancy of weight/O2 relationship could be drawn but that the over all picture is distinctly against the validity of such a procedure. In view of the present results, it would be interesting to reinvestigate the terrestrial snails on a broader basis ; it would be rather remarkable if they differed so fundamentally from the aquatic species. It is true, however, that differences between terrestrial and aquatic snails have been reported also in other respects. It is thus a well established fact that the former show a pronounced dependency of their oxygen consumption on the oxygen tension (Thunberg, 1905; Dahr, 1927; Fischer, 1931 ; Harnisch, 1932) while many marine snails do not (Moore, Edie, Whitley, and Dakin, 1912; Raffy, 1933). Australorbis glabratus belongs, according to the present investigation, to this latter group. This, unquestionably, is due to the haemoglobin in its blood. Although the oxygen dissociation curve of the Australorbis haemoglobin has not yet been studied, it can be presumed to resemble that of the closely related Planorbis. The oxygen affinities of the latter's haemoglobin make it especially suited to procure oxygen at low tensions (Leitch, 1916; Borden, 1931). The fact that Australorbis can hold its oxygen consumption at a normal level even at relatively low tensions may have a bearing on control measures. The application of some chemicals to snail infested waters results in the snails' burrowing into the mud and so escaping the direct action of the poison (W. H. Wright, per- sonal communication). Although it is not known whether Australorbis glabratus specifically reacts in this way, it must be expected that snails with similar respiratory characteristics would not be easily killed by asphyxiation simply by being driven into oxygen-poor surroundings. Under very low oxygen tensions, it is true, the oxygen consumption is markedly lowered. It must be remembered, however, that snails in general are endowed with fairly well developed anaerobic functions (sum- mary of the literature in von Brand, 1946). The temperature relationships of Australorbis glabratus correspond to those commonly found in other invertebrates. The range of temperatures tolerated at least for the short periods employed in the experiments was rather wide. The vari- ations in metabolic level encountered in this range may well have to be taken into consideration in control measures directed against this schistosomiasis-carrying species. Due to the lower metabolic level the snails will consume less food at lower temperatures than at higher ones. A poison, therefore, that acts via the alimentary canal may have to be kept longer at a given concentration in the cooler headwaters of a stream than in the warmer lower regions in order to insure that it reaches adequate concentrations within the tissues of the snail. It should be noted in this connection that according to Luttermoser (1947) in Venezuela at least "the only RESPIRATION OF SNAILS 211 workable method for eradicating the snails was to eliminate them in the headwaters and to destroy them progressively downstream." Very likely, however, the tem- perature range in schistosomiasis-endangered river systems will not be quite so broad as the extremes employed in our laboratory experiments. The influence of starvation on the oxygen consumption of Anstralorbis gla- bratus, Hclisoma duryi, Physa gyrina, and Physa sp. was pronounced and quite similar in the four species. A progressive lowering in metabolic level occurred until the snails finally died. No steady rate of oxygen consumption was reached, the snails resembling in this respect starving warm-blooded animals (Krogh, 1916). The respiratory quotient of Anstralorbis glabratiis sank during starvation from an initial value of 0.85 to the surprisingly low level of around 0.6. Even lower values have been observed by Bellion (1909) in Helix and by Liebsch (1928) in several species of terrestrial pulmonates. Helix was studied towards the end of hibernation, that is, after having starved'for a long time; Liebsch's specimens were probably at least semi-starving. The interpretation of the respiratory quotients of animals having calcareous shells is notoriously difficult ; the following interpretation can therefore be only tentative. In view of the fact that the occurrence of glycogen has been demonstrated in Anstralorbis glabratiis (von Brand and Files, 1947), it does appear probable that the relatively high RQ of well fed snails is due to the utilization of this polysaccharide. The glycogen reserve does not seem to last for a long time ; soon values typical for fat and protein consumption are reached. The very low values found in the last stages may indicate either the new formation of carbohydrate from protein or, possibly, fat, or they may be due to carbon dioxide retention. The data at hand do not permit a decision between these possibilities. The respiratory quotient of australorbids fed again after a starvation period of four weeks rose but failed to reach in two weeks the original level. Before starva- tion, the snails had been kept in a balanced aquarium ; during and after the inanition period they were kept isolated in pairs in beakers. It is possible that they had in the aquarium some accessory food material at their disposal that was lacking in the beakers. But it is equally possible that during the recovery period a certain amount of carbon dioxide retention took place in connection with restitution processes on the shell. We gained at least in some cases the impression that the shells became brittle during protracted periods of starvation but we do not have quantitative data proving this point and emphasize that we do not consider it as more than a possi- bility. It may be mentioned that during hibernation, which of course corresponds to a starvation period, movements of inorganic substances between soft parts and shell likely occur in the case of Helix (von Brand, 1931 ). SUMMARY 1. A study of the rate of oxygen consumption of nine species of pulmonate snails and eight species of operculate snails showed that the pulmonates had consistently a higher metabolic level than the operculates if specimens of equal weight were compared. 2. In both groups, the intensity of oxygen consumption decreased with increas- ing size of the specimens if referred to unit weight, but remained about constant if referred to relative surface. The oxygen/surface relationship held true both in inter- and intra- specific comparisons and was especially close in the latter case. 212 TH. VON BRAND, M O. NOLAN, AND E. R. MANN 3. Aiistralorbis glabratus was able to maintain an approximately steady rate of oxygen consumption over a wide range of oxygen tensions. 4. The oxygen consumption of Aiistralorbis glabratus increased with rising tem- perature in the range of 0.3 to 37° C., but 41° G. was lethal. The temperature relationship calculated according to Arrhenius' equation gave within the tolerated temperature range a straight line. A good fit to Krogh's normal curve was also obtained and an extension of this curve to a higher temperature range than used by Krogh is presented. 5. The intensity of the oxygen consumption of four species of pulmonate snails sank during protracted starvation first rapidly and later on slowly without reaching a steady level. The respiratory quotient of Aiistralorbis glabratus sank during in- anition to very low levels and rose only slowly after feeding was begun again. 6. The possible implications of some of the studied factors on snail control meas- ures are briefly discussed. LITERATURE CITED BELLION, M., 1909. Contribution a 1'etude de 1'hibernation chez les invertebres. Recherches experimentales sur 1'hibernation de 1'escargot {Helix pomatia L.). Ann. Univ. Lyon, N. S. 1, Ease. 27 : 1-139. BORDEN, M. A., 1931. A study of the respiration and of the function of haemoglobin in Planorbis corneiis and Arenicola marina. J. Marine Assoc. Un. Kingdom, N. S. 17: 709-738. DAHR, E., 1927. Studien ueber die Respiration der Landpulmonaten. Lunds Univ. Aarsskrift, N. F. Avd. 2, 23 : 1-120. FISCHER, P. H., 1931. Recherches sur la vie ralentie de 1'escargot (Helix pomatia L.). /. Conchyliologic, 75 : 1-200. HARNISCH, O., 1932. Studien zur Physiologic des Gaswechsels von Tieren ohne Regulierung der Sauerstoffaufnahme bei wechselndem O,-Partialdruck. I. Die Sauerstoffaufnahme. Ztschr. Vcrgl. Physiol., 16 : 335-344. KLEIBER, M., 1947. Body size and metabolic rate. Physiol. Rev., 27: 511-541. KROGH, A., 1914. The quantitative relation between temperature and standard metabolism in animals. Ztschr. Physik.-chcm. Biol., 1 : 491-508. KROGH, A., 1916. The respiratory exchange of animals and man. London. LEITCH, I., 1916. The function of haemoglobin in invertebrates with special reference to Planorbis and Chironomns larvae. /. Physiol., 50 : 370-379. LIEBSCH, W., 1928. Ueber die Atmung einiger Heliciden Eine Untersuchung zum Oberflaech- engesetz. Zoo!. Jahrb. Abt. Allg. Zoo/, und Physiol., 46: 161-208. LUTTERMOSER, G. W., 1947. The control of the blood-fluke disease (schistosomiasis) in Vene- zuela. Inst. Inter-Amer. Affairs. Health and Sanitation Div. Newsletter, Oct. 1947. MOORE, B., EDIE, E. S., WHITLEY, E., AND DAKIN, W. J., 1912. The nutrition and metabolism of marine animals in relationship to (a) dissolved organic matter and (b) particulate organic matter of sea-water. Biochcni. J., 6: 255-296. RAFFY, A., 1933. Recherches sur le metabolisme respiratoire des poikilothermes aquatiques. Ann. Inst. Occanogr. Paris, Ser. IV, 13: 263-393. THUNBERG, T., 1905. Der Gasaustausch einiger niederer Thiere in seiner Abhaengigkeit vom Sauerstoffpartiardruck. Skand. Arch. Physiol., 17: 133-195. VON BRAND, T., 1931. Der Jahreszyklus.ini Stoffbestand der Weinbergschnecke Helix pomatia. Ztschr. Vcrgl, Physiol., 14 : 200-264. VON BRAND, T., 1946. Anaerobiosis in invertebrates. Biodynamica Monogr. No. 4. Normandy, Missouri. VON BRAND, T., AND FILES, V. S., 1947. Chemical and histological observations on the influ- ence of Schistosoma mansoni infection on Aiistralorbis glabratus. J. Parasitol. 33: 476-482. RESPIRATION OF SNAILS 213 VON BCDDENBROCK, W., 1939. Grundriss der vergleichcndcn Physiologic. 2nd. ed. Vol. 2. Berlin. WARD, P. A., TRAVIS, D., AND RUE, R. E., 1947. Methods of establishing and maintaining snails in the laboratory. National hist. Health Bull. No. 189 : 70-80. WEIXLAND, E., 1918. Beobachtungen ueber den Gaswechsel von Anodonta cyidns Rathbun, occurs abundantly in Chesapeake Bay and provides the source for a major seafood industry in the Tidewater section 1 Joint contribution from the Virginia Fisheries Laboratory of the College of William and Mary and Commission of Fisheries of Virginia (Number 28), and from the Department of Biology of the College of William and Mary. 2 Present address : Department of Zoology, University of Wisconsin, Madison, Wisconsin. This work was done in partial fulfillment of requirements for the degree of Master of Arts at the College of William and Mary. 214 FUNGUS INFECTION OF THE BLUE CRAB 215 of Maryland and Virginia. The annual production of the raw product of these crabs is valued at more than a million dollars.3 Churchill (1919) made studies on the life history of this species in Chesapeake Bay. He found that the average blue crab lives two to three years during which time definite migrations take place throughout the bay. In the spawning season, which lasts from May to September of each year, large numbers of egg-bearing females are found in the waters at the mouth of the bay — in the vicinity of Cape Charles and in Hampton Roads (Fig. 1). The gravid female carries her eggs on four pairs of small abdominal appendages (pleopods). These appendages are pro- vided with many hair-like filaments to which the eggs become attached by a glandu- lar secretion when they are extruded from the oviduct (Fig. 2). Incubation is com- pleted in about two weeks in Chesapeake Bay. An egg mass or sponge is estimated to contain about 2,000,000 eggs. The writer has observed that sponges vary a great deal in size, averaging 75 mm. wide, 50 mm. long, and 40 mm. deep. The bulk of so many eggs forces the folded abdomen (apron) away from the ventral side of the cephalothorax until it extends almost posteriorly. Observations have indicated that there is uniform development within a blue crab sponge (Lockhead and Newcombe, 1942), only 1 to 4 per cent of the eggs showing a retarded or un- developed condition. The approximate age of crab embryos can be determined by the color of the sponge. A new sponge is bright orange or yellow due to the large amount of yolk material in the egg. With development, the color of the sponge darkens to brown and finally black as the nutrient yolk is used up and pigment spots appear. Thus, age may be designated by three colors : Yellow, representing the first to the fifth day after eggs are deposited ; brown, the sixth to the eleventh day ; black, the twelfth to the fifteenth day. Hatching releases zoeal larvae which are abundant in the plankton of the lower bay waters. After passing through at least five zoeal instars (Hopkins, 1944), a second larval stage, the megalops, is attained. There is a single megalops instar 4 which molts directly into the first crab stage. The young crabs begin to migrate up-bay or into the near-by rivers. Such crabs hibernate in these waters during the winter, then complete their development and mate the following summer. After mating, if not before, the females begin a migration to the natural spawning area in the vicinity of the Capes. Many arrive in the lower bay at the end of their second summer. They winter here and produce their eggs when conditions become favor- able the following year. A large proportion of the females which mate late in their second summer may winter en route to the capes. Many of them produce their sponges the next summer before they reach the spawning grounds. This partially accounts for the large number of females with yellow sponges and the very few with dark sponges contained in commercial catches of the Egg Island-York Spit area (Fig. 1). Adult male crabs do not make an extensive southward migration as do the 3 For the period 1939-1943 the average annual production of raw product in Chesapeake Bay was 42,807,050 Ibs., averaging an annual value of $1,327,882. Fishery Statistics of the U. S., Fish and Wildlife Service, U. S. Dept. of Interior. 4 M. D. Sandoz, in unpublished data on development of the blue crab, Virginia Fisheries Laboratory. 216 R. ROGERS-TALBERT FIGURE 1. Lower Chesapeake Bay, showing the blue crab sanctuary and areas where blue crabs were collected. Average surface-bottom salinity records are given. (After Wells, Bailey, and Henderson, 1929.) Drawn by G. M. Moore. FUNGUS INFECTION OF THF BLUE CRAB 217 females but remain in the rivers and bay waters along the entire length of the bay where they have matured. The migratory habit of the blue crab endows Maryland seafood dealers with a greater proportion of the soft crab industry because the waters of Maryland and adjoining sections of Virginia are more heavily populated with immature crabs which undergo periodic meltings during their growth. The large population of mature hard crabs in the lower bay is responsible for the crab meat canning industry being located primarily at Hampton, Virginia. Fi<;rRE 2. A segment from the abdomen of a female blue crab. Eggs are borne attached to the longer filaments of the endopodite. (Drawn by R. E. Allen.) To protect the brood stock of blue crabs, the Commission of Fisheries of Vir- ginia maintains a crab sanctuary (Fig. 1) at the mouth of Chesapeake Bay. Opti- mum conditions exist here for the development of blue crab eggs and crab fishing is prohibited in these waters during the spawning season. Examinations of egg- bearing crabs from the sanctuary in 1942 indicated that the fungus parasite, Lagenidium callincctcs Couch, occurred there. This discovery aroused a wide in- terest among fishermen and conservationists and raised a question as to the value of protecting sponge crabs in the area. Furthermore, it pointed to a need for locating the waters where infection exists in order to determine whether the fungus is a general or localized parasite. CHARACTERISTICS OF THE FUNGUS The description of the life history of Lagenidhiiii callincctcs Couch (1942) has been a valuable aid in this study. In his observations of the organism Couch found that when germination of the zoospore begins, a delicate germ tube is sent through the egg membranes. This tube grows rapidly into a network of branched mycelium that soon fills the entire egg (Fig. 3). From the mycelium, stumpy, thumb-like projections, or hyphae, pass through the egg membranes to the outside (Fig. 4). These hyphae quickly mature into sporangia which rupture and discharge new spores to continue the cycle of infection. When the nutrient material of the egg has been 218 R. ROGERS-TALBERT exhausted by the fungus, the mycelium appears to break up into heavy walled, rest- ing cells that seem to be resistant to adverse conditions. However, neither germi- nation of these cells nor a sexual phase of reproduction has yet been observed. In- fected eggs soon give definite indication of being abnormal ; they are opaque and dwarfed, the diameter becoming reduced from about 290 micra to approximately 231 micra (Fig. 4) (Couch, 1942). FIGURE 3. Cross section of a blue crab egg parasitized by Lagcnidium callinectes, showing extensive internal mycelium (400 X). FIGURE 4. Two blue crab eggs from a single pleopod filament (200 X). The parasitized egg (left) demonstrates 8 external hyphae and 3 empty exit tubes. Internal mycelium is seen through the transparent egg membranes. Parasitized egg shows reduction in size. FUNGUS INFECTION OF THE BLUE CRAB 219 The several developmental stages as described by Couch were observed, and it has been possible to maintain the organism under laboratory conditions, thus pro- viding a better understanding of how the parasite destroys the host egg. Among infected eggs collected from Chesapeake Bay and eggs infected under laboratory conditions, the number of external hyphae varies greatly ; usually there are one or two on an egg, but frequently nine or more projections are observed at once. METHODS Studies of this parasitic fungus were carried out by random sampling of sponge crabs and by microscopic examination of the eggs. Lactophenol was used to clear the eggs and expose the mycelium. In this work the age of the sponge was desig- nated by color: yellow, brown, or black. Preliminary sampling up to and including 1943 indicated the waters of heaviest infection. Early in 1944 weekly sampling of various crabbing areas was begun. The samples, consisting of 20 to 25 sponges each, were preserved in 10 per cent formalin as soon as the commercial boats docked, which was only a few hours at most after the crabs were removed from the water. Collections were made in the Hampton Roads-Lynnhaven and Egg Island- York Spit areas. Relatively few sponge crabs are found north of York Spit. To determine the extent of sponge infection, several methods were attempted before a satisfactory one was found. At first, eggs were taken at random from the outside of the mass and examined microscopically. A count totaling 500 eggs was made to estimate the percentage of exterior infection. Then about half the sponge was cut away and the procedure repeated, using eggs from the interior. It was found that infection did not penetrate to the interior, so observations were continued only on the peripheral portion of the sponge. Where infection was observed, several filaments were detached at the base and examined for the progression of fungus along the strand. These methods of computing degrees of infection involved a high probability of error in view of the enormous number of eggs per sponge. It was necessary, therefore, to abandon this plan of estimating the percentage of diseased eggs since it was impossible to count enough in every sponge to determine an accu- rate percentage. Satisfactory results were obtained by setting up a standard based on visible areas of infected eggs. When the fungus has spread through many eggs in a given area of a sponge, the diseased portion in contrast to normal eggs assumes a brown color on yellow sponges and a grayish color on brown and black sponges. This is due to opacity of the eggs caused by the parasite. The following classification was adopted for differentiating the infected sponges in routine collections : Slight — fungus present in microscopic examinations but no areas of infection visible to the naked eye. Moderate — presence of visible areas of infection (which may be one or more) but less than half the sponge visibly infected. Heavy — more than half of sponge periphery visibly infected, but with one or two small areas where infection has not become heavy enough to be seen. Very heavy — a complete peripheral infection with no areas of healthy eggs visible. 220 R. ROGERS-TALBERT From all the samples collected, four sponges were selected which demonstrated the different degrees of infection. From each pleopod of these sponges, 25 filaments were detached at the base and examined microscopically. Observations were made on the depth of fungus penetration, the general condition of interior eggs, and the posibilities of an appreciable hatch of larvae despite the exterior coat of infection. Information on transmission of the fungus was obtained using the following procedures : 1. Several infected and uninfected egg-bearing crabs, selected from commercial catches at Seaford and Hampton, were placed together in aquaria. 2. Healthy and infected eggs from two different sponges were placed at opposite ends of porcelain pans and on opposite sides of large finger bowls. Running water from aquaria containing infected crabs was collected in pans into which normal eggs were then introduced. For controls, healthy eggs were placed in pans of water and females with sponges were placed in aquaria. 3. Infected and uninfected sponges in various stages of development were sus- pended in the York River near shore (Sandoz, Rogers, and Newcombe, 1944). A small cage (30 X 13 X 25 cm.) constructed of window screening on a wooden frame was used to protect the sponges and keep them afloat. Individual pleopods, detached from the sponge, were threaded with string near the base of the protopodite and fastened to hooks inside the cage. To indicate how the salinity factor affects the fungus, a series of salinities ranging from pond water up to the approximate concentration of sea water was prepared, using pond water and salt extracted from York River water. In filaments selected for these salinity tests, the fungus had attacked all eggs within 2 or 3 mm. of the distal end ; below this point eggs were developing normally. One filament was placed in each Petri plate of 50 cc. of water. No other species of crabs has been observed with this infection, so studies were carried out to determine whether or not this parasite has a specific affinity for eggs of Callincctcs sapid-its. Strands of infected blue crab eggs were placed with the healthy eggs of several other species in Syracuse watch glasses containing York River water. The other forms of crabs included the oyster crab (Pinnotheres ostrcnin Say, 1817), the wood crab (Scsanna cincrenm Bosc, 1801), the mud crab (Ncopanopc tcxiana Rathbun, 1900), and the spider crab (Libinia cinarginata Leach, 1815), all of which are found in the area where infected blue crabs occur. RESULTS AND DISCUSSION Lagenidium callincctcs Couch, a peripheral parasite. Microscopic examinations have shown that infection by L. callincctcs is restricted principally to the periphery of a sponge (Fig. 5). In fungus infected crabs, all eggs from the distal end of the strand down about 3 mm. may become infected, but below this eggs are found to be normal. Eggs lying in the interior of a sponge are packed closely together. The fila- ments, which are found only on the posterior side of the pleopods, vary in length from approximately 3 to 22 mm. (Fig. 2). The longer ones extend from the base of the pleopod while the short ones are at the tip. After eggs have been extruded, this variation permits none of the filaments to be buried within the mass. The volume of eggs is so great that the abdomen is pushed away from the cephalothorax FUNGUS INFECTION OF THE BLUE CRAB 221 and water flows freely around the outside of the sponge. The outer eggs of the mass serve as buffers for those on the interior, since commensals and parasites come in contact with these outer eggs first. Also, interior eggs lie closer together and do not seem to permit a rapid flow of water within the sponge, the interspaces only being large enough for water to seep slowly around the eggs. This movement of water is further aided by activities of the mother crab, such as vigorous jerking of her abdomen and frequent stirring of the eggs with her walking legs. L. callincctcs gains a foothold rather quickly, but. never seems able to penetrate to great depths ;.. FIGURE 5. Egg masses of Callincctcs sapidus Rathbun in longitudinal sections, showing a normal (above) and a diseased (below) condition with peripheral infection. within the mass. The female's habit of stirring the eggs with her walking legs may provide some opportunity for fungus spores to get into the interior, for in a few cases infection was found at a distance of 5 or 6 mm. down the filament. In only one sponge were infected eggs ever observed at the base of a filament and this fila- ment measured but 13 mm. in length and was located at the outer end of the pleopod. Occurrence of infection inside the sponge, although uncommon, nevertheless pro- vides positive 'evidence that conditions below the surface of a sponge are suitable for fungus growth. Hence, it seems that the outer eggs must act as a buffer pro- viding a surface for attachment of organisms and a filter for the water that pene- trates the sponge. An infected area of a sponge increases rapidly in diameter while its penetration is much slower. A filament in such an area usually has all the eggs diseased for a length of from one to three millimeters at the distal end. Under the microscope the infection can be seen in various stages. In very heavy infections, the most distal eggs have had their nutrient material exhausted by the mycelium and resting cells have formed while the egg membranes may have started to disintegrate. Adjacent eggs to these have become very opaque and dwarfed and possess external hyphae and sporangia. The diseased eggs which are lowest on the strand are in the earlier R. ROGERS-TALBERT stages of infection with only one or two empty spore cells on the outside ; the internal mycelium is still developing, and very few or no external hyphae are visihle. In this study, examinations were made on disintegrating eggs at the distal ends of the filaments. If the disease destroys eggs rapidly it seems that many of the fila- ments would lose the cuticular covering which supports the eggs. However, no filaments were found where infection had progressed this far. A small percentage of eggs destroyed by infection. In laboratory hatching ex- periments, uninfected egg-bearing filaments yield about a 90 per cent hatch (Sandoz and Rogers, 1944). Numerous empty egg cases observed on sponges removed from spawning grounds also indicate a high hatching percentage of uninfected egg masses in their natural environment. In the case of infected sponges, diseased eggs do not hatch but among the uninfected eggs on the same sponge it was found that the hatching percentage seems to remain high. Several sponges were examined which showed a very heavy peripheral infection beneath which the interior eggs were nearly all hatched out. j On an average-sized sponge of 2,000,000 eggs, about 10 eggs are distributed per millimeter of filament. The average length of all the filaments of a sponge is ap- proximately 12 mm. In very heavy degrees of infection, if the distal 3 mm. of all the filaments were infected, there would be about a 25 per cent infection of the total number of eggs in the sponge. Of the total 2,000,000 eggs, 75 per cent or 1,500,000 eggs are in the interior and do not become infected but complete embryonic develop- ment and hatch normally. Moreover, a very heavy degree of infection occurs in slightly less than 25 per cent of the sponges; therefore it seems unlikely that L. callinectcs can be regarded as a factor in the fluctuations of crab populations. Older sponges more heavily injected. Samples of sponges in any age group show all degrees of infection ; in view of which eggs must be susceptible to fungus spores throughout their developmental period. A large number of moderately in- fected egg masses with diseased patches on opposite sides, or on separate pleopods, indicate that a mass of eggs may be attacked by many spores simultaneously. In most of the sponges where slight infection was present, diseased eggs were found widely distributed over the periphery. Figure 6 shows the incidence of infection by L. callinectcs among sponges of dif- ferent age groups throughout the summer of 1944. Less than 50 per cent of the yellow sponges showed infection while both brown and black sponges had a higher percentage of infection. This increase of infection in brown and black sponges is believed to be due to the eggs being older and, hence, exposed to infection for a longer time. In examining the samples, consideration was given to the amount of infection present on each sponge. The various degrees of infection remained in almost equal proportions throughout the summer, thus indicating a fairly regular cycle for the parasite as regards the continuous infection of sponge crabs during the spawning season. Zocal larvae may become injected. In the laboratory, when infected eggs were present in the filaments, zoeae which had hatched normally often showed evidence of the fungus. It is believed that infection could not have occurred before hatching because the mycelium within 48 hours is able to fill an entire egg (Couch, 1942), and in all probability embryonic development would become disturbed within a few FUNGUS INFECTION OF THK BLUE CRAB 223 hours at most after penetration of the spore. The zoeae possess a very thin exo- skeleton quite similar in appearance and thickness to the egg membranes which spores penetrate easily. It is more probable that zoeal infection occurs following a normal hatching of the egg. None of the larvae taken from plankton have ever been observed with fungus infection. Infected zoeae have been seen only in labora- tory hatching pans, where the larvae must swim about in spore infested water. Under natural conditions larvae hatch from the sponge of the mother crab as she rests on the bottom in warm shallow water. They are positively phototropic and I FIGURE 6. Percentage of yellow, brown, and black sponges infected by Lagcnidium callinectcs. Hampton Roads-Lynnhaven area. 1944. begin to move toward the surface. In this way the young swim away from the old sponge where infection may have contaminated the surrounding water with many motile spores. When the fungus invades the zoeae, the larvae soon weaken and become unable to swim. If such infection does occur in nature, this would explain the absence of diseased individuals from the plankton samples that we have studied. Transmission of infection wider experimental conditions. Laboratory cultures showed that transmission of infection from one egg to another is extremely rapid. Often an entire pan of eggs was destroyed by disease in three or four days, even when the first day showed very few infected eggs. In aquaria, healthy egg-bearing crabs quickly became infected when diseased crabs were introduced. In one case, water from an aquarium inhabited by a single infected female was used in a hatching pan which contained only normal eggs. Within two or three days L. callinectcs was seen and a majority of the eggs soon became infected. In other experiments in which diseased and normal eggs were placed at opposite ends of a pan, the fungus was observed to infect the normal eggs after about two days. 224 R. ROGERS-TALBERT Infected sponges which were suspended in the York River failed to hatch. During the experiment the number of infected eggs increased while the uninfected ones under the same conditions hatched normally, the zoeal larvae escaped and left behind their empty, transparent egg cases. For experimental purposes normal eggs were usually selected from the Seaford catches where diseased crabs were seldom observed. There is no record of infec- tion in the York River ; consequently the chances of fungus having been introduced from the Seaford or York River waters are slight. Examination of controls never showed fungus growth. Factors affecting the fungus. Laboratory experiments have demonstrated a wide salinity tolerance for this fungus. In all salinities, from 5 to 30 p.p.t., hyphal growth and spore formation proceeded rapidly. In fresh water during a two day period there was some development of external hyphae and a few small abnormal sporangia. During a two day period in salinities of approximately 15, 20, 25, and 30 p.p.t. there was such heavy growth that the eggs appeared to be enveloped in a fine white down. New eggs also became infected. In a salinity of 20 p.p.t. where the parasitic growth was extremely heavy, a typically infected crab egg was observed with seven sporangia, four exit tubes, and four hyphae, all visible from one side. TABLE I Percentage of sponges from Chesapeake Bay infected by Lagenidium callinectes Couch during the period 1942-1944 Location Year Number of sponges examined Distribution of infection Percentage of sponge infection Yellow Brown Black Lower Bay 1942 82 1 13 19 40 Lynnhaven Roads 1943 30 3 9 4 53 Lynnhaven Roads 1944 393 78 104 60 62 Lynnhaven River 1943 12 6 1 0 58 Lynnhaven River 1944 37 13 8 0 57 Hampton Roads 1943 15 1 8 4 87 Hampton Roads 1944 136 20 19 16 40 Ballards Marsh 1944 11 0 0 0 0 Seaford 1943 76 0 0 1 1 Seaford 1944 254 6 4 4 5.5 York River (at York- 1944 63 0 0 0 0 town) Rappahannock River 1943 6 0 0 0 0 L. callinectes can withstand sudden changes in salinity. The sponge crab used in this experiment was taken from Lynnhaven where the salinity is about 27 p.p.t. She was carried in a moist basket to the laboratory ; eggs were cut from the sponge and placed in York River water (salinity 20 p.p.t.) for about an hour. When the salinity series was set up, the sponge filaments were transferred directly to pond water and salinities of 5, 10, 15, 20, 25, and 30 p.p.t. In no case except pond water was there apparent retardation in fungus growth. Development in salinity as low as 5 p.p.t. suggests that it may be possible for L. callinectes to become conditioned to very brackish water. FUNGUS INFECTION OF THE BLUE CRAB 225 Low temperatures were observed to retard fungus development somewhat. This was first noticed in hatching experiments in 1942. When diseased eggs were placed in the refrigerator (15 to 16 degrees C.) fungus development and spore for- mation were delayed. This temperature, however, did not prevent sporulation and the spores continued to swim about, but their movement was sluggish. Distribution of Lagcnidium collincctcs in Chesapeake Bay. Extensive samples of sponges collected during 1943—44 have indicated that L. callinectcs is quite com- mon in waters extending from Hampton Roads to Cape Henry (Fig. 1). How- ever, the disease is not confined to these open areas. Samples from neighboring places also revealed the existence of fungus in inlets of the region. Samples from several miles up the Lynnhaven River showed a high percentage of fungus occur- rence. In August, 1943, a sample from this river showed a 58 per cent infection ; in July, 1944, another sample showed a 57 per cent infection (Table 1). In 1942, infected sponges were found in Pagan's Creek, a tributary of the James River. However, in August, 1944, a sample from Ballard's Marsh at the James River Bridge was not infected. In July, 1941, fungus was observed in a sample from Buckroe Beach, which represents the northerly limit of heavily infested waters. — eo — eo — 70 — eo —so — 40 — 30 — 20 — 10 i— O I I I I I LAGENIDIUM CALLINECTES — ~ CARCINONEMERTES CARCINOPHILA i I I I L I l l I I I I L MAY 7 MAY 14 MAY 21 MAY 28 JUNE JUNE JUNE JULY II 16 25 2 JULY 9 JULY JULY JULY AUG AUG AUG 16 23 30 6 13 20 AUG 27 FIGURE 7. Percentage of blue crab sponges infected by the fungus Lagcnidiuni callincctes Couch and by the nemertine Carcinonemertes carcinophila Kolliker during the summer 1944. Hampton Roads-Lynnhaven area. (Dates indicate first day of the week during which collections were made.) 226 R. ROGERS-TALBERT In 1944, sponge crabs first appeared in commercial catches during the second week in May. The fungus was not present until a month later, the first record being taken from a sample collected on June 11 in which 13 out of 20 sponges were infected (Fig. 7). There seems to have been a simultaneous appearance of the fungus in both the Hampton Roads and Lynnhaven areas. This would indicate that the organism is well distributed throughout the region, spends a quiescent winter, and becomes active as soon as favorable conditions return. Egg-bearing crabs dis- appeared soon after August 31, 1944, until which time the fungus was present in more than 50 per cent of the specimens with a small increase during the last of' August. Samples from other regions of Chesapeake Bay have been examined. Through- out the Seaford area infection is uncommon. Several samples taken during June and August showed a 2 to 3 per cent infection. In one sample taken off Egg Island Bar at the mouth of Back River infection occurred in 45 per cent of the sponges. For this region the figure is high ; however, Egg Island Bar is located in waters not far distant from Hampton Roads and the sanctuary where there is infection. In the York River at Yorktown, L. callinecics has not been found. One or two in- fected sponges have been taken from Mobjack Bay, Poquoson River, and the mouths of the York River and Back Creek. The degree of infection in most cases was slight. In these waters however, the majority of spawning crabs are yellow in color and are migrating toward the lower bay where hatching takes place. It is con- cluded that the general migration to the capes of spawning females is responsible for retaining the infection in this locality. When a female has completed spawning, the fungus probably ceases to live on that individual because hatching has depleted the food supply of the parasite. When the young crabs begin their northward mi- gration, it is believed that the parasite remains behind since there is no evidence of an immature crab harboring the organism. The adult females probably die very soon after the completion of spawning so it is doubtful that spreading up the bay from the Lynnhaven area could occur by migration of infected females. Available information suggests that the fungus is localized in waters where female blue crabs hatch their eggs. Occurrence of Lagcnidium callincctes in other species. Laboratory experiments were carried out in an effort to infect eggs of Pinnotheres ostrcuin, Neopanope tc.rana, Libinia einarginata, and Sesanna cinercmn. The crabs used were all col- lected in the Seaford-York River region. Within two to five days, fungus was transmitted to eggs of the oyster crab (Pinnotheres ostreum*) and the mud crab (Neopanope tc.vicuia). Attempts to transmit fungus to eggs of Libinia emarginata and Sesannas cinc- reum were unsuccessful, even though the latter remained alive for more than a week in the laboratory. However, previous hatching experiments with Libinia have never been successful. Other organisms on the crab sponge. In addition to fungus, other organisms, either parasitic or commensal, are frequently found living on the sponge. These organisms, though quite common, seem to do very little damage to the eggs. Pro- tozoan forms of Carchesinni and Eplielota are often attached to the eggs in the peripheral portion of the sponge. When fungus was first observed on the eggs of Callincctes sapidus, a hair-like growth longer than the diameter of a crab egg was noticed. Some eggs showed a FUNGUS INFECTION OF THE BLUE CRAB 227 profuse development of such filaments which at first were confused with the para- sitic fungus. However, the filamentous growth later was recognized to he a Chlamydobacterium (sp.) . In 1944, while conducting crab studies at this laboratory, Dr. Sewell H. Hopkins found the parasitic nemertine Carcinoncincrtes carcinophila Kolliker (compare Humes, 1942) to be very abundant on the gills of the blue crab. This worm was likewise observed embedded in the sponge where it deposited its own eggs in a case entwined around the filaments. In these observations it was noticed that the nemertine and the fungus frequently occurred together (Fig. 7). The factors gov- erning infection by L. callincctes and C. carcinophila appear to be quite similar, since the results show a corresponding periodic fluctuation of the two. Significance of Lagcnidiwn callincctes. From this discussion, Lagenidiinn cal- lincctes has been found to be a peripheral parasite of the egg mass of the blue crab and the data obtained show that it is present in a large percentage of sponges (Table 1 ) . When present, although it spreads rapidly among the peripheral eggs, penetra- tion into the sponge is slow and rarely deep. Meanwhile, the healthy eggs of the interior, which in all cases represent at least three-fourths of the mass, continue their development and hatch normally. This parasite, now evidently established within the spawning area, may possess the potential ability to destroy a great number of blue crab eggs. However, in the light of these observations, prevailing natural conditions seem to hold the fungus in check. It is known that the parasite has a fairly wide temperature and salinity tolerance, but the incubation period of the blue crab lasts only about two weeks which appears to be too brief a time for the fungus to work deeply into the center of an egg mass. SUMMARY 1. The fungus parasite Lagcnidiitni callincctes Couch has been observed to be a peripheral parasite of egg masses of the blue crabs of Chesapeake Bay. 2. Blue crab eggs are susceptible to infection in all stages of their development. Infected areas of a sponge are brown or gray in appearance, depending on the age of the eggs. 3. While the fungus spreads rapidly over the surface of the sponge, it penetrates the egg mass very slowly. Usually the depth of infection is not over 3 mm. 4. Infection is heavier in older sponges which are brown and black than in younger yellow ones, probably due to the longer exposure of older sponges. 5. Peripheral infection does not seem to retard the development of crab eggs in the interior of the sponge, which far outnumber the peripheral eggs. Not over 25 per cent of the eggs of a heavily diseased sponge are infected and only about 14 per cent of the crabs were found to be heavily infected. However, it was not unusual to find 80 or 90 per cent of the crabs in a sample to have some degree of infection. 6. Under laboratory conditions, transmission of infection from egg to egg of the same and different blue crabs is unexpectedly rapid. 7. Development of the fungus was observed to be abnormal in fresh pond water. In salinities from 5 to 30 p.p.t. development proceeded rapidly and indicated a strong tolerance of changes in salt concentration. 228 R. ROGERS-TALBERT 8. Frequently occurring on the peripheral eggs with Lagcnidimn callincctcs are Carchesiwn sp., Ephelota sp., and Chlamydobacterium sp. Carcinonemertes carci- nophila Kolliker is present and shows periodic fluctuations similar to the fungus. 9. Eggs of the oyster crab and the mud crab became infected with L. callincctcs under laboratory conditions. 10. The Hampton Roads-Lynnhaven waters is the area in Chesapeake Bay where L. callincctcs is most common. Only slight infection was observed north of Buckroe Beach. LITERATURE CITED CHURCHILL, E. P., 1919. Life history of the blue crab. Bull. U. S. Bureau of Fisheries, 36, Document No. 870, 1919, pp. 96-123, Washington, D. C. COUCH, J. N., 1942. A new fungus on crab eggs. /. Elisha Mitchell Scientific Society, 58 (2) : 158-162. HOPKINS, S. H., 1944. The external morphology of the third and fourth zoeal stages of the blue crab, Callincctcs sapidns Rathbun. Biological Bit!!., 87 (2) : 145-152. HUMES, A. G., 1942. Morphology, taxonomy, bionomics of the Nemertine genus Carcinone- mertes. 111. Biol. Monographs, 18, No. 4, 105 pages. LOCH HEAD, M. S., AND C. L. NEwcoMBE, 1942. Methods of hatching eggs of the blue crab. Va. Jour, of Sci., 3 : 76-86. SANDOZ, M. D., AND R. ROGERS, 1944. The effect of environmental factors on hatching, molting, and survival of zoeal larvae of the blue crab, Callincctcs safiidiis Rathbun. Ecology, 25 : 216-228. SANDOZ, M. D., R. ROGERS, AND C. L. NEWCOMBE, 1944. Fungus infection of eggs of the blue crao, Callincctcs sapidus Rathbun. Science, 99 : 124. FACTORS INFLUENCING MOLTING AND THE SEXUAL CYCLES IN THE CRAYFISH1 HAROLD H. SCUDAMORE Department of Zoology, Northwestern University INTRODUCTION There are two interesting phenomena associated with the periodic molting and the cycles of sexual functioning in crayfishes that have not been studied in detail. One of these interesting events is the delay in the spring molt of egg-carrying fe- males until after the eggs hatch and the young crayfish leave the female ; the other concerns the changes in the secondary sex characters in the male crayfish at the time of molt (Scudamore, 1942b). This investigation describes some of the factors in- fluencing these two phenomena in the life cycle of the crayfish. The delay in the spring molt of egg-carrying females has been observed by Van Deventer (1937), Tack (1941) and others. According to Tack (1941) the spring molt of males and non-reproducing females of the crayfish, Cambarus immunis, occurs about the middle of April in south central New York with most of the cray- fish molting within the period of a few weeks. However, the females, which are carrying eggs at this time, do not molt until five or six weeks later. The repro- ducing females deposit their eggs in the fall shortly after mating and carry their eggs, attached to their abdominal pleopods, all winter. The eggs hatch about mid- May and the young remain dependent upon the female for a week or longer, while undergoing their first two molts. The egg-bearing female molts a few days after the young leave her pleopods. The spring-molting period of males and non-reproducing females of the crayfish, Cambarus propinqnns, also begins about the middle of April in central Illinois and lasts about three weeks (Van Deventer, 1937). However, the reproducing females do not deposit their eggs until early April. The eggs hatch about the middle of May and the young remain dependent for approximately another week. The egg- bearing females do not molt until late May or early June which is at least three weeks after the male spring-molting period. This delay in molt of egg-bearing females is a protective adaptation, because molting earlier would result in the death of all the embryos. The mechanism pro- ducing this lag in the spring molt of ovigerous females has not been explained ade- quately. However, Hess (1941) reported a delay in molting of the seeded female shrimp, Crangon armillatus, as compared to non-seeded females. He also observed that removal of the embryos from seeded females shortened the period between molts and concluded that the factor, which inhibited molting, was apparently dependent upon attachment of the embryos to the female. 1 The author wishes to acknowledge the constructive criticisms and encouragement offered by Dr. Frank A. Brown, Jr., during the course of this investigation and to express his sincere appreciation to Dr. C. L. Turner for the use of unpublished field data. 229 230 HAROLD H. SCUDAMORE The male secondary sex characters studied were the first pair of abdominal pleopods which are modified as gonopods for. the transfer of spermatozoa to the annulus ventralis of the female during copulation. These gonopods have been de- scribed sufficiently by Turner (1926), Van Deventer (1937), Tack (1941) and others. Male crayfish are classified as Form I, II or "juvenile" on the basis of the morphology and function of the gonopods. Mature males with sexually-functioning gonopods are designated as Form I, and those with non-functioning gonopods, as Form II ; immature males with non-functioning gonopods are classified as "juve- nile." Most mature males change from Form I to Form II at the spring molt and revert to Form I during the summer molt in time to function during the fall mating season, remaining in Form I until the following spring. No satisfactory explanation of these changes in sexual form has been noted in the literature. It is possible that sex hormones are involved in the delay of the spring molt of egg-carrying females and in the changes of sexual form of the male gonopods at the time of molting. However, there is no conclusive evidence for the presence of sex hormones in the crustaceans. As pointed out by Brown (1944) most of the proof for the presence of sex hormones is based on indirect results such as parasitic castra- tion, radiation and regeneration experiments. MATERIALS AND METHODS Most of the laboratory experiments and some field observations were made on the crayfish, Cambarus immunis Hagen ; but a few observations were made on C. propinquus Girard. The stock animals were kept in lead-lined tanks supplied with running tap water and were offered chopped earthworm or liver as food. The experimental crayfish were placed in individual finger bowls which were frequently refilled with fresh tap water and maintained at room temperature. The eyestalks were removed by excising through the basal membrane with a sharp, pointed scalpel and coagulating the open wound with an electric cautery to control hemorrhage. Evidence of an approaching molt was obtained by sacrificing an animal and examining the anterior wall of the stomach for the presence of gastro- liths. The pair of gastroliths were dried in an oven at 100° C. for 24 hours and weighed to determine gastrolith size. The carapace lengths were measured from the posterior margin of the cephalothorax to the tip of the rostrum. The technique of inducing gastrolith formation and molting by removal of both eyestalks, developed by Brown and Cunningham (1939), Kyer (1942), Scudamore (1942a, 1947) and others, permitted (1) a study of secondary sex changes in male crayfish during winter molts induced by eyestalk removal as well as during normal spring and summer molts and (2) an investigation of the role of the eyestalks (sinus glands) upon molting in egg-carrying female crayfish. FIELD OBSERVATIONS Collections of C. propinquus,2 made from a single locality during the spring and summer, illustrate the phenomena of the delay in the spring molt of egg-bearing females and the changes in sexual form of males (Table 1). On March 27th an ice jam had flooded a stream flat and, when the water receded, great numbers of 2 From the unpublished records of collections made by Dr. C. L. Turner in 1921 from Turtle Creek, Rock County, Wisconsin. MOLTING AND SEXUAL CYCLES IN THE CRAYFISH 231 crayfishes remained on the flat, either dead or in a dormant condition. All the males were Form I and had hard, calcareous exoskeletons. None of the females were bearing eggs. In this particular collection there were more females than males. According to Van Deventer (1937) males slightly exceed the females in number and, during the egg-bearing period, greatly out-number the females in the active population. TABLE 1 Summary of molting and changes in the sexual cycles of the crayfish, C. propinquus, as observed in random field collections Form I males Form II males Females Date Total collected Condition Condition Condition Number Number of Number of Number of bearing exoskeleton exoskeleton exoskeleton eggs March 27 270 109 Hard 0 — 161 Hard 0 April 15 76 68 Hard 0 — 8 Hard 7 April 24 156 124 Hard 0 — 32 Hard 26 May 1 48 46 Hard 0 — - 2 Hard 2 May 13* 46 6 Hard 28 Soft 8 Hard 8 4 Soft 0 Soft June 3** 136 3 Hard 88 Medium • 26 Hard 0 Hard 1 19 Soft 0 July 6*** 69 6 Soft 26 Hard / 30 1 34 Soft Hard 0 0 Late July*** and Many Many Mostly Very Hard Many Hard None August hard few * Spring molt of males and non-reproducing females. !* Spring molt of egg-bearing females. '** Summer molt, both sexes. On April 15th only eight females were secured in a collection of 76 crayfish which was made with a dip net without searching under stones. Seven of the females were bearing eggs upon their swimmerets. All the males were still Form I. Col- lections of April 24th and May 1st consisted chiefly of hard-shelled Form I males. The majority of the females were carrying eggs and were found hidden under stones. Apparently they had not moved from their hiding places or eaten for several days because an examination of their alimentary canals revealed no food. The few fe- males not bearing eggs were moving around actively like the males. By May 13th there were many cast-off exoskeletons lying in the margins of the shallow waters and practically all of these had come from Form I males but a few- had come from non-reproducing females. Most of the males collected were soft- shelled and Form II, showing evidence of a recent molt. The four active females without eggs had molted recently. However, the eight females bearing eggs or young were concealed under rocks and had not molted. Most of the males collected on June 3rd were Form II, indicating that they had completed the spring molt. The young crayfish, which up to this time had been clinging to the pleopods of the females, were in an advanced stage and many had 232 HAROLD H. SCUDAMORE left the females altogether. Many of the females were soft-shelled as a result of a recent molt following escape of their young, and nearly all of the exuvia lying in the shallow water were those of females that had carried eggs. By July 6th most of the egg-bearing females had molted and some of the males had completed their second or summer molt. In late July and early August most of the males collected were Form I, indicating that the summer molt was completed. Some of the females apparently had undergone a second molt at this time and be- come sexually functional. In contrast to this observation, Van Deventer (1937) reported that adult females, which have borne eggs during the spring, undergo only a single molt. Although it is difficult to delineate accurately the various events because of the length of time between collections, certain generalizations may be made regarding the life cycle of C. propinqitus in southern Wisconsin. (1) Reproducing females deposit their eggs in early April and carry their eggs until the middle of May. The eggs hatch about mid-May and the young remain attached to the female for several days. (2) The spring-molting period of males and non-reproducing fe- males begins after May 1st with many animals molting by May 13th and the re- mainder before June 3rd. (3) The spring molt of egg-carrying females, which is delayed until after the young have left the female, occurs during the month of June or about three weeks after the male spring molt. (4) The second or summer molt of most mature males and at least some females takes place during July and early August. (5) Most mature males change from Form I to Form II during the spring molt and from Form II to Form I during the summer molt. MOLTING IN EGG-CARRYING FEMALES In order to determine the role of attachment of the eggs to the swimmerets in delaying molt, a number of egg-bearing female crayfish, C. propinquus, were placed in a large aquarium, closely simulating the natural environment, during the spring- molting period of males and non-reproducing females. The eggs were then removed from one group of females. Both those with eggs attached and those with eggs removed were given access to an abundant food supply. The egg-carrying females remained in their hiding places beneath stones in the aquarium, waving the mass of eggs attached to their swimmerets but not feeding. On the other hand, the cray- fish, from which the eggs had been removed, soon began to move about freely, fed activelv and molted within one or two weeks. •/ In experiments performed during the winter of 1941-42, twenty normal egg- carrying female crayfish, C. inimitnis, were placed in individual finger bowls and all of the eggs removed from the pleopods of ten of them; both eyestalks were extir- pated from another group of twenty egg-bearing females, and the eggs removed from ten of these animals. The crayfish from each of the four groups were sacrificed (or died) and examined for the presence of gastroliths at various intervals of time. One eyestalkless crayfish in each group died of operative injury before sufficient time had elapsed for gastroliths to form (Scudamore, 1947) and so were not in- cluded in tabulating the results. Only three of the normal egg-carrying crayfish were found to contain very small gastroliths and none molted (Table 2), even though these animals were observed for a period nearly three times longer than the eyestalkless crayfish (Table 3) . The MOLTING AND SEXUAL CYCLES IN THE CRAYFISH 233 experimental period was long enough to permit gastrolith formation and molting when compared to normal pre-molt periods (Scudamore, 1947). Minute gastro- liths have been observed in other normal crayfish during the winter, but their exact significance is not known. There was no essential difference in the results whether the eggs were removed or not. This experiment demonstrated that the mere removal of the eggs from the normal crayfish does not induce molting in a non-molting season. TABLE 2 Influence of removing the eggs from the pleopods of normal egg-carrying female crayfish, C. immunis, upon gastrolith formation and molting from December to February Number of animals Average duration of experiment (days) Number with gastroliths Average weight of both gastroliths (mgm.) Number molted Average carapace length (mm.) TABLE 3 Carrying eggs 10 34.5 2 0.25 0 30.6 Eggs removed 10 39.8 1 0.06 0 28.5 Effect of bilateral eyestalk extirpation upon gastrolith formation and molting of egg-carrying female crayfish, C. immunis, from December to February Number of animals Average duration of experiment (days) Number with gastroliths Average weight of both gastroliths (mgm.) Number molted Average carapace length (mm.) Carrying eggs 9 12.7 9 44.80 1 28.6 Eggs removed 9 15.2 9 54.19 2 27.8 All of the eyestalkless crayfish had formed large gastroliths in 5-19 days after eyestalk removal and three of them had molted between 15 and 17 days after opera- tion, even though winter is normally not a molting season (Table 3). The removal of the eyestalks resulted in gastrolith formation or molting whether the eggs were present or not. Although there was considerable individual variation, there was no significant difference in the rate of gastrolith formation in the two groups of eyestalkless crayfish (Table 4). The inhibition of molt seemed to be dependent upon both the presence of the eyestalks (sinus glands) and the attachment of the eggs to the pleopods of the female and not simply the attachment of the embryos to the female as concluded by Hess (1941). Although the evidence suggests that the sinus gland molt-inhibiting hormone is responsible for the delay in the spring molt of egg-bearing female crayfish, there are a number of possible factors that may operate in maintaining the sinus gland activity until after the eggs hatch and the young leave the female, namely : hormonal, meta- bolic or nervous factors. A female sex hormone elaborated in the ovaries or other tissues may cooperate with the sinus gland activity in the delay of molt. However, the lack of histological or experimental evidence for such glandular tissue (Brown, 1944) weakens this hypothesis. In this connection Turner (1935) reached the conclusion, on the basis 234 HAROLD H. SCUDAMORE of morphological studies, that the complete development of the annulus ventralis, a female secondary sex character, depends upon some ovarian tissue and the total absence of any testicular tissue. Yonge (1937) reported a cycle of histological changes in the oviducal epithelium and of secretion from the "cement" glands of the pleopods associated with egg-laying and attachment of the eggs to the pleopods of the lobster and suggested that, in the absence of nervous connections to the epi- thelium or the glands, the cycle of changes seemed to be controlled by hormones. TABLE 4 Rate of gastrolith formation in eyestalkless egg-carrying female crayfish, C. immunis, during the winter Day after eyestalk removal Carrying eggs Eggs removed Length of carapace (mm.) Weight of gastroliths (mgm.) Length of carapace (mm.) Weight of gastroliths (mgm.) 13 28.7 19.6 28.7 10.3 13 13 15 28.0 67.9 28.3 27.5 26.8 81.0 21.1 49.8 29.8 22.7 15 29.8 73.8 29.5 97.2* 16 16 17 19 23.2 40.2* 28.8 29.6 26.4 25.0 84.2 33.8 50.1* 60.2 31.5 69.7 * Indicates that animal molted. The annual cycle of metabolic changes, which normally may initiate molting di- rectly or through inhibition of the sinus glands, may be delayed in the egg-carrying females. The importance of metabolic factors is emphasized by the observation that egg-bearing female crayfish are largely inactive, hiding under stones and not feeding freely until after the young leave the pleopods. Furthermore, the egg- bearing females become active, feed and molt within a short period of time after the eggs are removed artificially during the male spring-molting period. Finally, prolongation of the molt-inhibiting action of the sinus glands by im- pulses over nerve-reflex pathways produced by the presence of the -eggs on the pleopods may explain the delay in molt. Welsh (1941) has demonstrated morpho- logically an innervation of the sinus glands of the crayfish from the "brain." More- over, the tracts followed within the central nervous system of the crayfish by sensory impulses from stimulation of proprioceptors and sensory hairs of the abdominal pleopods were traced functionally by Prosser (1935), confirming the neurone paths first described histologically by Retzius (1890). These observations suggest the nervous pathways which may be involved in the reflex stimulation of the sinus glands. However, the fact that removal of only the eggs and not the eyestalks did not initiate gastrolith formation in the winter even in the warmth of the laboratory, suggests that some factor or factors other than possible pleopod-sinus-gland reflexes are involved. Some combination of hormonal, metabolic and nervous factors seems like the most plausible explanation of this phenomenon. It is apparent that further experi- MOLTING AND SEXUAL CYCLES IN THE CRAYFISH 235 mentation is needed to establish the exact mechanism involved in the delay of the spring molt of egg-carrying females. MOLTING AND THE SEXUAL CYCLE OF MALES The cycle of changes in sexual form of the first pair of abdominal appendages is illustrated by observations on molting in a single male crayfish, C. immunis, be- tween April and September. This crayfish changed from Form I to Form II at the first or spring molt on May 15th and changed from Form II to Form I at the second or summer molt on July 15th. In order to study the changes in sexual form of crayfish during the fall and winter, observations were made of changes in sexual form following molting of mature crayfish, C. immunis, induced by bilateral eyestalk extirpation and these changes were compared with those occurring at normal spring and summer molts. As shown in Table 5, all the crayfish became Form II after the artificially induced winter molts whether they were Form I or II before molt. The normal crayfishes changed from Form I to Form II at the spring molt and from Form II to Form I at the summer molt.. Enough Form II males for this winter experiment were ob- tained by selection from a large number of animals, because most of the males were Form I. TABLE 5 Changes in sexual form of mature male crayfish, C. immunis, at the time of molt Period No. of animals Original sexual form Form after molt Spring molt 12 I II Summer molt 12 II I Winter molt, induced by eyestalk removal (12 I II (November to March) HO II II While studying spermatogenesis of the crayfish, Fasten (1914) found a seasonal variation in the size of the testes and in germ cell proliferation. The testes com- menced active proliferation and increased in size in June, reached greatest activity and size in July, remained large in August with their tubules filled with spermatozoa, decreased in size in September, and remained small until the following summer. This seasonal cycle of changes in the testes of mature males is illustrated in Figure 1 together with the duration of the spring- and summer-molting periods, the seasons during which Form I and Form II mature male crayfish predominate, and the time at which copulation occurs. The period of greatest testis activity (July-August) coincides exactly with the summer-molting period when males change from Form II to Form I. Copulation ensues a few weeks later at a time when the males have Form I gonopods and the testis tubules are filled with spermatozoa. During spring molts and during winter molts induced by eyestalk removal, when the testis size and spermatogenic activity are at a minimum, the male crayfish changes to Form II. These observations offer circumstantial evidence in support of an hypothesis 236 HAROLD H. SCUDAMORE that variations in the amount of a male sex hormone, produced in the testes or other body tissues, are responsible for the changes in sexual form of the male gonopods at the time of molt. The cyclical release of such a hormone could be influenced by other internal or by environmental factors. The greatest weakness of this hy- pothesis is the lack of conclusive histological or experimental evidence for the pres- ence of secretory cells within the testes. On the basis of morphological studies of the crayfish, Turner (1935) has considered that the development of aberrant sec- ondary sex characters is largely dependent upon genetic rather than hormonal fac- tors. However, the seasonal changes from Form I to Form II and the reverse obviously are not controlled genetically, since they occur in a single individual. MOLTING PERIODS FORM II FORM I COPULATION TESTIS ACTIVITY LARGE MEDIUM SMALL ANIMAL ACTIVITY J F M A MJJASOND ~~i 1 1 1 — i 1 1 — i 1 \ — i — SPRING SUMMER 1 _!_ _1_ F M A M J J A MONTH SOND FIGURE 1. Diagram of certain phases in the life cycle of the mature male crayfish, C. hnmimis, demonstrating the relationship of molting periods, duration of and time of change to each male sexual form, period of copulation, periods of animal activity, and cycle of changes in testis size and spermatogenic activity. The curve of testis activity is based on the results of Fasten (1914). The solid lines (- -) represent periods of predominant occurrence; broken lines (- -), periods of occasional occurrence. Proof for the existence of male, as well as of female, sex hormones must await histological evidence of secretory cells in the gonads or other tissues and the estab- lishment of definite endocrine functions of these gland cells by surgical extirpation, implantation and injection of specific gland substances. However, observation and experimentation investigating the seasonal changes in the male gonopods represents a promising method for studying the problem of the existence of male hormones in crustaceans — a problem which is far from satisfactorily settled at this time. SUMMARY 1. The phenomena of the delay in spring molt of egg-carrying females and of the changes in sexual form of males at the time of molt are described and illustrated by field observations on the crayfish, C. propinqnns. 2. Removal of the eggs from the pleopods of egg-bearing females, C. propinquus, during the male spring-molting period results in an earlier onset of molting. 3. The delay in the spring molt of egg-carrying female crayfish, C. immums and C. propinquus, is regulated by the action of the molt-inhibiting hormone of the sinus glands. MOLTING AND SEXUAL CYCLES IN THE CRAYFISH 237 4. Various factors, that may operate to maintain the sinus gland activity until after the eggs hatch and the young leave the female, are discussed. 5. The changes in sexual form of male crayfish, C. immunis, at the time of spring and summer molts, and during winter molts induced by eyestalk extirpation are described. 6. Evidence is presented supporting an hypothesis that a male sex hormone, elaborated in the testes or other tissues, may regulate the cycle of changes in sexual form at the time of molt. LITERATURE CITED BROWN, F. A., JR., 1944. Hormones in the Crustacea : their sources and activities. Quart. Rev. Biol, 19: 32-46, 118-143. BROWN, F. A., JR., AND O. CUNNINGHAM, 1939. Influence of the sinus gland of crustaceans on normal viability and ecdysis. Biol. Bull., 77: 104-114. FASTEN, N., 1914. Spermatogenesis of American crayfish, Cambarus virilis and Cambarus immunis (?), with special reference to synapsis and the chromatoid bodies. Jour. Morph., 25 : 587-649. HESS, W. N., 1941. Factors influencing molting in the crustacean, Crangon armillatus. Biol. Bull, 81 : 215-220. KYER, D. L., 1942. The influence of the sinus glands on gastrolith formation in the crayfish. Biol. Bull, 82 : 68-78. PROSSER, C. L., 1935. Action potentials in the nervous system of the crayfish. III. Central responses to proprioceptive and tactile stimulation. /. Comp. Ncnrol., 62 : 495-505. RETZIUS, G., 1890. Zur Kenntnis des Nervensystems der Crustaceen. Biol. Untcrs., 1 : 1-50. SCUDAMORE, H. H., 1942a. Hormonal regulation of molting and some related phenomena in the crayfish, Cambarus immunis. Anat. Rec., 84: 514-515. SCUDAMORE, H. H., 1942b. Hormonal influence on molting and the sexual cycle of the crayfish, Cambarus immunis. Anat. Rcc., 84: 515-516. SCUDAMORE, H. H., 1947. The influence of the sinus glands upon molting and associated changes in the crayfish. Physiol. Zool, 20 : 187-208. TACK, P. L, 1941. The life history and ecology of the crayfish, Cambarus immunis Hagen. Amer. Mid. Nat., 25 : 420-446. TURNER, C. L., 1926. The crayfishes of Ohio. Ohio Biol. Surv. Bull, 3: 145-195. TURNER, C. L., 1935. The aberrant secondary sex characters of the crayfishes of the genus Cambarus. Amer. Mid. Nat., 16: 863-882. VAN DEVENTER, W. C., 1937. Studies on the biology of the crayfish, Cambarus propinquus Girard. ///. Biol. Monogr., 15 : 7-57. WELSH, J. H., 1941. The sinus glands and 24-hour cycles of retinal pigment migration in the crayfish. Jour. Exp. Zool., 86 : 35-49. YONGE, C. M., 1937. The nature and significance of the membranes surrounding the developing eggs of Homarus vulgaris and other Decapoda. Proc. Zool. Soc. London, 107A: 499- 517. PROGRAM AND ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1948 JULY 6 Action pattern of crystalline muscle phosphorylase}- SHLOMO HESTRIN.2 Degradation of glycogen, amylopectin, and amylose by repeatedly recrystallized muscle phos- phorylase was studied. The reaction was found to be : polysaccharide + phosphate <=2 limit-dextrin + glucose-1-phosphate The limit-dextrin accounts for about 60 per cent of the weight of the parent polysaccharide in the case of glycogen and amylopectin, but must be a relatively minor reaction product in the case of amylose. The phosphorylase limit-dextrin of glycogen was isolated and further characterized. The properties of this substance, in particular its ability to undergo a limited hydrolysis (24 per cent) by beta-amylase, are in accord with the view that it is derived from glycogen by a shortening of outer chains only, and that the final length of the shortened chains is probably three glucose units. The phosphorylase limit-dextrin primes muscle phosphorylase and in suitable conditions can be shown to effect a shift in the equilibrium mediated by the enzyme. The findings thus further support Cori's theory that the primer is a stochiometric participant of the reaction. Recrystallized muscle .phosphorylase fails to degrade the beta amylase limit-dextrins of glycogen and amylopectin, and bacterial dextran. It is thus unable to cleave or by-pass an alpha 1—6 linkage and does not differ in this respect from potato phosphorylase as described by previous investigators. The enzyme may be regarded as a specific alpha 1-4 gluco-phosphorylase which acts on the 1-4 linkage only if the latter is terminal to a chain of sufficient length. Crude muscle extract, in contrast to purified phosphorylase, converts glycogen and amylo- pectin almost quantitively to hexose phosphate. The factor in crude extract which mediates this effect is being studied further by Professor G. T. Cori and the author. Vital Staining in ultraviolet and in ^vhite light combined. RUDOLPH KELLER. 3 Living animals, such as salamander larvae, tadpoles, insect larvae, fish, daphnia, are first stained in the usual way by methylene blue, neutral red, alizarin, congo red or china blue and, afterwards, illuminated with luminescent dyes, such as primulin and aesculin, which make the daylight dyes visible in ultraviolet light. With this method B. V. Pisha of this institution, using primulin, found that glands located in the distal end of the gut of daphnia showed a yellow greenish fluorescence. This, according to our former experiences, indicates the production of acid by the glands, which seems to neu- tralize the alkaline content of the gut. In other experiments we stained with blue dyes such as aesculin (1 : 1000), positively charged in a biological medium, and administered after ten minutes, and a yellow dye uranin (1 : 10,000), also positively charged. We observed that the yellow dye rapidly displaced the blue one (which left the brood pouch of the daphnia at the neck) and the yellow uranin entered at the distal end of the pouch. In further experiments we proceeded in the following way: To fish (Fundulus) in sea water in a vessel of 10 cc., three drops 1 per cent potassium ferrocyanide and two drops HC1 1/10 N 1 This work was carried out in the Department of Biological Chemistry, Washington Uni- versity School of Medicine, St. Louis, and was supported by a grant from the Corn Industries Research Foundation. The author is deeply indebted to Professor C. F. Cori for guidance in the conduct of the experiments. 2 Hebrew University Travelling Fellow. 3 Madison Foundation for Biochemical Research, New York. 238 PRESENTED AT MARINE BIOLOGICAL LABORATORY 239 were added. Later the animals were put into a solution of 1 : 800 thiazol yellow. The gills took up, first, in Prussian blue capillaries, only a little thiazol yellow. After killing the fish and adding new thiazol yellow, 1 : 800, to some gills on the slide, the space between some capil- laries became strongly yellow greenish fluorescent, particularly in the neighborhood of injuries, while spaces between others appeared violet or scarcely stained. The sulfhydryl metabolism of the beta cell and its relationship to the development of diabetes. ARNOLD LAZAROW. Since glutathione protects rats from a diabetogenic dose of alloxan (Lazarow, Proc. Soc. E.\-p. Biol. and Mcd., 61: 441 (1946)), and since alloxan reacts with the sulfhydryl group of glutathione, and of protein, to give a new compound with an absorption spectra maximum at 305 mM (Lazarow, Patterson, and Levey, Science, in press), it has been suggested that the glu- tathione, which is present in the pancreatic beta cell, normally serves to protect essential sulf- hydryl enzymes from alloxan. It was further suggested that a low beta cell glutathione content could explain why alloxan is selective, but not specific, for beta cells. It is estimated that the beta cells of man contain only 0.25 mgs. of glutathione [on the as- sumption that % per cent of the pancreatic weight (85 gms.) is islet tissue, and that the islet glutathione concentration is equal to that of the whole pancreas (60 mgs./lOO gms.)]. Since insulin contains 12 per cent cystine, it is estimated that if all the cysteine contained in the glutathione of the beta cells of man could be incorporated into insulin, less than one milligram (19 units) of insulin would be formed. This is but a fraction of the daily insulin requirement of man. Glutathione does not rapidly penetrate the cell membrane, for, on perfusion of liver, the glutathione is only slowly removed (Fabre and Simonnet, C. R. de I'Acad. des Sci., 185: 1628 (1927)). Following alloxan injection, the blood glutathione falls to near zero values, while the liver glutathione is only slightly affected. However, in spite of the large amount of glu- tathione in other tissues, the blood glutathione level has not returned to normal even after 6 hours (Leech and Bailey, /. Biol. Chcm., 157: 525 (1945)). Thus cellular glutathione is not rapidly restored, and local depletions may take place. It is therefore postulated that the synthesis of insulin, in physiological amounts, may produce a local depletion in beta cell glutathione, and thereby render these cells more susceptible to alloxan or to other sulfhydryl inactivators, which may appear in the body. Beta cell degeneration is also observed in (1) the pancreatic remnant following partial pancreatectomy, (2) following massive anterior pituitary hormone injections, and (3) after massive glucose injections. In all of these conditions, the beta cells are stimulated to an in- creased insulin production. It is further postulated that this increased insulin synthesis also sensitizes the beta cells to degeneration, because of a consequent local depletion in beta cell glutathione. If this theory of beta cell degeneration proves correct, then the glutathione metabo- lism of the beta cell will not only affect the etiology of alloxan, and other experimental diabetes, but it may also have an important bearing on the development of human diabetes. JULY 13 A partial separation of the cytochromes of mammalian heart muscle. B. EICHEL, S. J. COOPERSTEIN AND W. W. WAINIO. If successive amounts of sodium desoxycholate are added to an insoluble cytochrome com- plex preparation of mammalian heart muscle, the cytochromes can be partially separated. The desoxycholate is added at a concentration of 1 per cent and the undissolved residue in each in- stance is brought down by centrifugation at 20,000 X g for 1 hour. If 4 such successive frac- tions are prepared, the first fraction contains flavoprotein and cytochrome c, the second fraction contains cytochrome c and b, and the third and fourth fractions both contain cytochrome b and o.ridase. However, fraction 3 has more b than o.ridasc and fraction 4 has more oxidasc than b. The absorption maxima in these preparations containing sodium desoxycholate were found at 416, 520, and 550 m/" for ferrocytochrome c, at 408 mM for ferricytochrome c, at 429, 528 and 558 mM for reduced cytochrome b and at 441 and 601 nv* for reduced cytochrome oxidase. 240 PRESENTED AT MARINE BIOLOGICAL LABORATORY The identity of cytochrome a as a separate enzyme from cytochrome oxidase or o3 (Keilin and Hartree terminology — Proc. Roy. Soc. London, B127: 167-91, 1939) is being investigated. The role of cytochrome b as a carrier for the dehydrogenases must be clarified with respect to its relation to the other cytochromes and to flavoprotein. Alkaline phosphatase in demincralised mouse bones of different ages. ANITA ZORZOLI. The general objective of this study was to determine the histochemical localization of the enzyme alkaline phosphatase in the tibia of normal mice during developmental life, during the period of growth and after growth of the bones had ceased. The bones were demineralized, without attendant enzyme inactivation, in a sodium acetate-acetic acid buffer at pH 4.55, sec- tioned and incubated with sodium glycerophosphate according to the method of Gomori. Prior to the 15th day of gestation, the tibia was entirely cartilaginous and was completely devoid of enzyme. Phosphatase first appeared in the connective tissue surrounding a localized region of cartilage which was destined to become a center of ossification. Once the typical histological changes had begun, the enzyme appeared in the cartilage and was located in the nuclei, cytoplasm and to a slight extent in the matrix. Shortly later the bone salts became evi- dent. It was interesting that their appearance was always preceded by that of the enzyme. With the spread of the processes of ossification the enzyme increased in amount and distribution. In the early post-natal bones where rapid growth occurred, enzyme was prominent in the epiphyseal growth zone. The outermost cartilage cells of this region were small in size and were always phosphatase free. The adjoining cells, arranged in the form of columns parallel to the long axis of the bone, contained enzyme which increased in concentration with proximity to the hypertrophic zone. In the hypertrophic zone which was one of great activity, phosphatase occurred in both cells and cartilage matrix. It also appeared faintly in the calcified spicules and the newly formed bone. The osteoblasts were strongly phosphatase positive while osteo- clasts were never observed to contain enzyme. With increasing age the growth processes declined and the number of enzyme-containing cells of the hypertrophic zone decreased while phosphatase-free matrix increased. This change was already evident at 5% months of age and by 13 months only a few scattered cells remained. The order of ammo acids in silk: an application of isotopic derivative tcchnic. MIL- TON LEVY AND EVELYN .SLOBODiANSKY.1 The principles described by Keston, Udenfriend and Cannan (/. Am. Chem. Soc. 68 : 390, 1946) are applicable to complex mixtures of amino acids, dipeptides and higher peptides as present in partial hydrolysates of silk. We have estimated the peptides of alanine (A) and glycine (G) in hydrolysates produced by the action of concentrated HC1 at 39°. Thus, in a 48 hour hydroly- sate the per cent of the total nitrogen in each form was : G, 12.9 per cent ; A, 10.5 per cent ; AG, 27 per cent; GA, 8.3 per cent; and GG, 1.8 per cent. Random arrangement of the 42.3 per cent G and 28.2 per cent A in our sample would have led to a maximum of 12.3 per cent AG. The arrangement cannot therefore be random. It is suggested that a unit of silk structure may be -G--X-A-G-A-G-X-. In this structure two AG's are possible for each GA. No GG is possible. X stands for any other amino acid. Further analysis of complete hydrolysates indi- cates in per cent of the total nitrogen the following amino acids : Serine, 9.24 per cent ; glutamic acid, 1.07 per cent; aspartic acid, 1.54 per cent; hydroxyproline, 0.05 per cent. These analyses were done using the isotope derivative technic with separation by paper chromatography (Keston, Udenfriend and Levy, /. Am. Chem. Soc., 69: 315, 1947). It is noted that G, A and Serine are in the ratio of 9,6,2 and that glutamic and aspartic are in the ratio of 2 : 3. The radioactive isotopes used in this work were supplied by the Clinton Laboratories on Allocation from the U.S.A.E.C. The work was supported by the American Cancer Society on recommendation of the Committee on Growth of the National Research Council. 1 Stanley Tausend Foundation Fellow. PRESENTED AT MARINE BIOLOGICAL LABORATORY 241 JULY 20 On the specificity of cholinesterase. KLAS-BERTIL AucusxiNSSON.1 It has been demonstrated by different groups of investigators that the cholinesterase activities of various sources are not identical. A hypothesis has been proposed that two types of acetyl- choline splitting enzymes exist. There is no doubt, however, that many of the differences among the cholinesterases of various tissues cannot be accounted for by the fact that two types exist. This has been demon- strated in experiments with enzyme preparations from tissues and body fluids of various animals, vertebrates as well as invertebrates. A full account of these experiments has recently been pub- lished (Augustinsson, Ada physiol. Scand., 15: Suppl. 52, 1948). Cholinesterases are defined as esterases which split choline esters at a higher rate than other esters ; the specificity is not an absolute one. These esterases are regarded as a family of related enzymes with widely divergent properties. The following classification is based on the activity- substrate concentration relationships for the enzymic hydrolysis of acetylcholine. Group I is characterized by the inhibition of cholinesterase activity at high acetylcholine concentrations ; optimum activity at about 3 X 10~3 M acetylcholine. This group includes the cholinesterases of the nervous system, muscles, electric organs (Nachmansohn), erythrocytes, Helix blood, snake venom (Zeller). The enzymes of Group II follow the Michaelis-Menten formulation, which means that their activities are maximal only at infinite substrate concentra- tion. This group includes the choline-ester splitting esterases of certain sera (e.g., man, horse), dart sac (Helix pomatia), and pancreas (Mendel). The properties of the members within each group may then differ in certain -other respects. The optimum conditions in the hydrolysis of acetylcholine area priori not identical with those prevailing in the hydrolysis of other esters. When, for instance, the substrate concentration is arbitrarily chosen in the enzymic hydrolysis of a choline ester or a non-choline ester, the optimum conditions are not the same as those of the hydrolysis of acetylcholine, the rate of reaction may be lower, the same, or even higher than that of the acetylcholine hydrolysis. Effect of anticholinesterases on conduction. DAVID NACHMANSOHN. In 1942 the theory was proposed that the release and removal of acetylcholine are intra- cellular processes necessary for the conduction of the nerve impulse. This idea was based on a great variety of facts obtained by the study of the enzymes connected with acetylcholine metabo- lism, and their correlation with the electrical manifestations in conduction. If the rapid removal of acetylcholine is necessary for the propagation of the impulse, anti- cholinesterases should block conduction. This could be shown with eserine and other inhibitors of cholinesterase (/. N 'euro physiol., 9 : 9, 1946) . In this case, inhibition of the enzyme and block of conduction are reversible. Two years ago, this theory was assailed by several investigators based on observations with a new anticholinesterase, diisopropylfluorophosphate (DFP). This compound inhibits cholin- esterase irreversibly. All the objections raised, however, and the apparent difficulties have been overcome, and the necessity of cholinesterase for conduction has been demonstrated conclusively. Contrary to the original assumption, the irreversible inactivation of cholinesterase by DFP is not an immediate process but depends on a number of controllable factors. A striking parallel- ism has been established between the rate of irreversible abolition of conduction and that of ir- reversible inactivation of cholinesterase. This has been shown as a function of time as well as of temperature. The necessity of the enzyme for conduction has been established on a great variety of different types of nerves, and on striated muscle suggesting the same role of acetyl- choline in all conductive mechanisms throughout the animal kingdom (/. Neurophysiol., 10: 11, 1947). In no way is it possible to dissociate cholinesterase activity and conduction. Claims to the contrary were based on the use of inadequate techniques (/. Neurophysiol., 11 : 125, 1948). Having met the challenge, the theory emerged from these discussions stronger than before (Johns Hopkins Bull, in press). 1 Biochemical Institute, University of Stockholm. 242 PRESENTED AT MARINE BIOLOGICAL LABORATORY The ion permeability of the giant axon of squid. M. A. ROTHENBERG. The difference in concentration of ions between the inside and the outside of nerve fibers has long been assumed to be of importance in conduction (Ostwald, Bernstein, and many others). Most of the evidence suggesting ion movements during the passage of the impulse across the active membrane has been, however, of an indirect nature (see Hodgkin, /. Physiol., 106: 341, 1947). The exchange of ions across the nerve membrane of the giant axon of squid has now been measured by the direct determination of radioactive Na24 and K42 in the axoplasm of these fibers. In all cases, the outer environment was artificial sea water prepared according to Pantin (/. Exp. Biol., 11: 11, 1934), and no alteration in the concentration of ion species was made. The results appear to indicate that during rest the ions inside the fiber may be in dynamic equilibrium with the same ion species outside. Thus, within 20-30 minutes all of the Na23 inside the fiber (based on figures of Steinbach and Spiegelman, /. Cell, and Comp. Physiol., 22 : 187, 1943) exchanges for Na24 in the sea water. In the case of K42, only about 10 per cent of the total inside will exchange within the same period of time. A twofold increase in the K42 penetration rate could be obtained with a two-fold increase in the K42 concentration in the sea water. Attempts were made at determining the temperature coefficients for the Na24 and K42 ex- changes across the membrane. A ten degree difference in temperature of the bathing fluid (12° and 22° C. resp.) showed no marked alteration in the rates of exchange. This supports the assumption that at rest there are no important chemical reactions involved in this exchange other than a Donnan equilibrium. Electrical activity of the nerve increased markedly the rate of Na24 penetration. This same overall effect could be obtained by the addition of anticholinesterases to the sea water. For example, the addition of diisopropylfluorophosphate increased the Na24 penetration by about 50 per cent, and decreased the K42 penetration by about 35 per cent. This suggests an increase in membrane permeability and may, when studied more closely, throw some light on the immediate function of acetylcholine and cholinesterase in nervous conduction. Cocaine, unlike the anticholinesterases, had a very small effect on the membrane permeability. JULY 27 The extraction of purified squid ''visual purple." A. F. BLISS. The photochemistry of the squid retina has been studied by methods which have been suc- cessfully applied to vertebrate retinas. The common vertebrate visual pigment, rhodopsin, is replaced in the squid by a photostable homolog, cephalopsin. This pigment has been extracted in a purified state by a combination of Saito's and Lythgoe's methods for rhodopsin. Squid retinas are homogenized with 40 per cent sucrose and centrifuged at a high speed. This treat- ment separates the heavier melanin from the lighter red segments which contain the visual pig- ment. The red rod layer floats to the top and after separation is hardened with buffer at pH 4.5. It is then extracted with a mild detergent, aqueous digitonin. A clear red extract is ob- tained, whose absorption spectrum closely resembles that of rhodopsin. The maximum due to cephalopsin is at about 495 mM in the blue green, while that due to rhodopsin is at 502 mM. Cephalopsin becomes photosensitive in the presence of five per cent formalin and bleaches almost completely when exposed to light of a 100-watt lamp at a distance of one foot for 20 minutes. It is also bleached by strong acid and base in the dark. The bleaching products are indicator yellow and retinene, precisely as obtained from bleached rhodopsin. Cephalopsin apparently does not bleach in vivo. The increased retinene released from illuminated retinas shaken in petrol ether reported by Wald is probably a secondary physical effect due to pigment migration which increases the effective diffusion surface of the illuminated retina. Pigment migration fails in aerated retinas kept one hour after excision in complete darkness whereas Therman has shown that such retinas actually are more sensitive to light than freshly excised retinas. Retinas pre- pared according to Therman's procedure exhibited a light: dark retinene ratio of 1.02 ± 0.8, which does not differ significantly from unity. A detailed account of the purification of cephalopsin will appear in the Journal of Biological Chemistry. PRESENTED AT MARINE BIOLOGICAL LABORATORY 243 Biochemical and histochemical observations on the sc.viial dimorphism of mouse sub- maxillary glands. L. C. JUNQUEIRA, A. FAJER, M. RABINOVITCH AND L. FRANKENTAHL. Protease and amylase activity is demonstrated in extracts of mouse submaxillary glands. The protease content varies sexually but the aniylase content does not. In castrated adult mice steroid sexual hormones influence the protease content of the glands. Correlation of biochemical and histological data locates the site of protease production in the tubular portions of these glands, and amylase synthesis in the acinar regions. Evidence is presented for location of ribo- nucleoproteins in the basal regions of acinar and tubular cells, and for variations in its content with sexual variations in the tubular cells. The secretory granules of the tubular cells appear to be of protein nature, as they give a positive reaction for phenolic amino acids and for sulf- hydrilated proteins. "Acid" phosphatase is evidenced in. the apical portions of the tubular cells, and shows histochemical and biochemical sexual variations. "Alkaline" phosphatase is dis- tributed more diffusely throughout the gland. Our data suggest, but do not establish, sexual variation of the activity of this enzyme. Mucoproteins are present in the cytoplasm of the amylase producing acinar cells. pH estimation in reconstituting pieces of Tubularia stems. JAMES A. MILLER, JR.1 Phenol red, chlor phenol red, brom thymol blue, brom cresol purple and brom cresol green were injected into the coelenteron of Tubularia with the aid of a Chambers micromanipulator. Of these, phenol red proved the most satisfactory both because of low toxicity and appropriate range. The pH of uninjured stems ranged between 7.8 and 8.0. Cutting, crushing, inserting a pipette or even bending the stem sharply caused the release of an acid of injury the concentration of which depended upon the degree of injury. Recovery of normal pH required one to fifteen minutes, depending also on extent of injury. Reconstituting stems released acid metabolites which maintained the coelenteric fluid at 0.2 to 0.4 pH below that of the uninjured stem. The first morphological indications of hydranth reconstitution were accompanied by regions of increased acidity corresponding to the two rows of tentacles. Once formed, the tentacles remained acid (pH 6.8 to 7.0) as also did the ring of perisarc-secreting tissue just proximal to the hydranth. The pH of other parts could be increased by increasing the availability of oxygen. Stolon formation and growth was not accompanied by observable changes in pH. When reconstitution was blocked by placing stems in glass tubes, acidity was increased in all parts equally. Ligatured stems, on the other hand, showed the normal pH and in some cases developed regions of increased acidity at the ends indicating partial activation. One should therefore guard against considering that all ligatured stems are under truly basal conditions. These experiments demonstrate that acid metabolites are produced in the ectoderm and endoderm of Tubularia and are liberated into the coelenteron. If prevented from escaping they increase the acidity to a point which has been found to inhibit reconstitution when externally applied. The genetic block to jrcc oviposition in the chalcidoid ivasp Melittobia sp. — C. P. W. WHITING AND BERTINA M. BLAUCH. Melittobia females normally fail to oviposit freely unless mated. The very rare exceptions reported ("layers") produce large progenies (100 to 300) of haploid sons. From a stock de- rived from a layer, 100 virgin females were isolated. The test showed 15 layers and 85 non- layers, the latter producing not more than four sons each. Sons of the layers were crossed with their aunts. Of 150 daughters tested, 51 were layers. The trait is clear-cut with no intergrades. The genetic basis, however, is complex. Selection, inbreeding and crossing the descendents for seven generations showed layer females per fraternity varying from 0 to 100%. From sibling matings in one line, three fraternities totalled 6 layers, 355 non-layers (1.66% layers), three fraternities totalled 84 layers, 326 non-layers (20.49%), one fraternity totalled 73 layers, 77 non- 1 Emory University. 244 PRESENTED AT MARINE BIOLOGICAL LABORATORY layers (48.66%). It was noted that layer females have a shorter life than non-layers and that there is close association of the trait with sterility and short life of their sisters. One layer when self-crossed (mated to a haploid son) produced 27 layers, 31 non-layers and 29 dying sterile after ten days (33.33%' sterile). Another produced 22 layers, 4 non-layers and 20 dying sterile (43.48% sterile). Four produced 46 layers, no non-layers and 93 dying sterile (66.91% sterile). One self-crossed layer produced 6 layers, 240 non-layers (sterile not recorded). The trait is evidently not a recessive. Of these 240" non-layers, 15 were self-crossed. Grouped ac- cording to increasing percentage of layers and arranged as layers/non-layers/dying sterile, there were two fraternities, 3/73/12, with 13.64% dying sterile, five, 25/189/34, with 13.71%, one, 7/29/14, with 28.00%, five, 58/125/72, with 28.23%, and two, 34/37/28, with 28.28%. It is indicated that more than two factors may be involved with complementary effect and that the group dying sterile is genetically related to the layers. AUGUST 3 Inhibition of sea urchin egg cleavage by a scries of substituted carbamates. IVOR CORNMAN.1 Urethane (ethyl carbamate) is both carcinogenic, inducing lung tumors in rats and mice, and carcinostatic, diminishing tumor growth in rats and mice and lowering wbc count in murine and human leukemia. To correlate these properties with efficiency as a mitotic poison, eggs of Lytechinus and Tripncitstcs were exposed to a series of carbamates with methyl, ethyl, propyl, butyl, amyl and phenyl replacing the ethyl on the carboxyl or hydrogens on the amino end of urethane. These were supplied by Dr. C. D. Larsen of the National Cancer Inst. Exposure was begun 10 minutes after fertilization. Delay and blocking of cleavage were compared quanti- tatively. In general, effectiveness in blocking cleavage increased with increase in M.W. up to 8-10 carbon atoms. Isopropyl N-phenyl carbamate, Ethyl N,N-di-n-butyl carbamate and ethyl N-phenyl carbamate were the most efficient, blocking cleavage at 0.5-0.6 m Molar, while urethane required 56 mM/L. A cyclic configuration was the most effective, then straight-chain, * branched-chain (uo), and divided groups (N,N-dimethyl, etc.), in decreasing order of activity. This parallels the narcotic activity of carbamates, but is entirely divergent from efficiency of pulmonary adenoma induction or of tumor suppression. For both of these, urethane is reported as the most active of the carbamates tested. These remarkable influences on the neoplastic process appear not to devolve in any direct way from destruction of mitosis. A nuclear precursor to rlbo- and deso.ryribonucleic acids. A. MARSHAK.- Rats were given P32 Na^HPO4 intravenously and nuclei isolated from the liver three hours later. Incubation of the nuclei at 37° C. with ribonuclease, desoxyribonuclease and with no added enzyme showed that 90 per cent of the nuclear P3" was in nucleic and the remainder in lipid and acid soluble fractions. Very little or none of the P32 was in DNA in nuclei from normal liver (no mitosis) and also in nuclei from regenerating liver (rapid mitosis). Incuba- tion at 0°-2° C. releases no nucleic acid from the nuclei, although at 37°, 80 per cent is removed indicating the presence of an enzyme capable of splitting nucleoprotein or nucleic acid. The digestion products are not dialyzable and therefore are nucleic acid and not nucleotide. The native enzyme thus differs from ribonuclease which splits off nucleotide. The specific activity of the digestion products is 13 times as great as that of the RNA of the cytoplasm. The material extracted from the nuclei by the method for extracting RNA has a specific activity almost as high as that of the nuclei. This fraction may therefore be identical with the P32-containing nuclear substrate but both are very different in behavior from the RNA of the cytoplasm. Ex- traction of the nuclei for 24 hours with 1 M NaCl removes only 20 per cent of the P32 and the P32-containing material so extracted is not precipitated on dilution to 0.14 M NaCl. The P32- containing nuclear material thus differs in solubility from desoxyribonucleoprotein. On the basis of the orcinol and diphenylamine reactions the nuclear auto-digestion products contain "RNA" and an amount of "DNA" less than 1/10 of the "RNA." Analyses of uracil and thymine con- 1 Sloan-Kettering Inst. for Cancer Research. 2 New York University College of Medicine. Tuberculosis Control Division, United States Public Health Service. PRESENTED AT MARINE BIOLOGICAL LABORATORY 245 tent by preparing the p-iodophenylsulfanile derivatives with radioactive iodine show a thymine content less than 1/10 that of the uracil. In saline, desoxyribonuclease increases the rate of splitting of the PM-containing substrate but in carbonate buffer at pH 7.0 it reduces the rate, suggesting binding of substrate without splitting it. The nucleic acid in question thus appears to have some properties in common with both DNA and RNA. In both mitotic and non-mitotic nuclei the P32 appears first in this nucleic acid ; later, in mitotic cells, the P32 is accumulated in DNA, while in non-mitotic cells it passes into RNA of the cytoplasm, the P32 always being associated with nitrogenous base and sugar. Since it contributes to the formation of DNA and RNA and also since it has properties in common with each this nucleic acid is considered to be their precursor. These findings indicate that plasmagenes and other cytoplasmic constituents containing nu- cleic acid cannot be independent of nuclear activity. They also predict that cells may be found which contain no DNA. Strains of bacteria with no desoxyribose and with no thymine have been reported. The effect of "stabilising" and "unstabilizing" agents in relation to the metabolic mechanism supporting the resting potential of nerve.1 ABRAHAM M. SHANES.-" Certain agents which block conduction (e.g. calcium, cocaine, procaine) are known to en- hance the polarization of nerve fibers; others, which increase excitability (e.g. calcium precipi- tants, veratrine), have been observed to cause depolarization. The former have been designated as "stabilizing," the latter as "unstabilizing." It is now possible to characterize these two groups of substances by another effect, viz., their ability to modify the rate with which anoxia leads to depolarization. Thus, cocaine and procaine, in concentrations far below those which affect respiration (e.g. 0.001%), delay the decline of the resting potential in frog nerve during oxygen lack. Associated with this is a delay in the development of inexcitability. Spider crab (Libinia cinarginata) leg nerves also are rendered less sensitive to anoxia by procaine, but the concentration required (ca. 0.05%) causes de- polarization in oxygen. Veratrine, like calcium precipitants, in a concentration which causes negligible depolariza- tion in oxygen (1:200,000 for frog sciatic nerve, 1:2 million for crab nerve), speeds the de- polarization process during anoxia and, when depolarization has not been excessive, increases the potential rise upon return to oxygen. The exactly antagonistic effects of the stabilizing and unstabilizing compounds with respect to excitability, resting potential, and anoxia sensitivity suggest the involvement of a single basic locus. An explanation in terms of potassium permeability is in keeping with the available literature on the effects of these substances on potassium leakage and impedance ; this is sup- ported. further by (a) the reduced depolarizing action of KC1 on frog muscles treated with cocaine and (b) the ability of cocaine and procaine (0.2%) to slow the swelling of muscles in a Ringer in which some of the sodium has been replaced by potassium. f AUGUST 10 The relative rate of penetration of the lo^ver fatty acids into beef red cells. JAMES W. GREEN. Since the work of Overton the lipid solubility theory has been widely held to account for the penetration of lipid soluble compounds into cells. This theory may be tested by a study of the penetration rates of the lower fatty acids into mammalian erythrocytes. When a weak acid penetrates a mammalian red cell the oxyhemoglobin within the cell dissociates to a new equi- librium, the level of which appears to depend upon the amount and dissociation constant of the acid which entered the cell and the oxygen tension of the environment. Taking advantage of this dissociation of oxyhemoglobin, a modification of the Hartridge-Roughton rapid mixing 1 This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service, and from the American Philosophical Society. - Georgetown University School of Medicine. 246 PRESENTED AT MARINE BIOLOGICAL LABORATORY technique was used to measure spectrophotometrically the rate at which the lower, saturated, monocarboxylic acids penetrated beef red cells. The method is essentially chemical and does not depend upon volume changes of the cell. Dilute unbuffered red cell suspensions were mixed with dilute acid solutions made up in 1 per cent NaCl such that the pH of the final mixture was maintained in the range 4.2. The relative order of increasing rate of penetration of the acids studied for beef cells was found to be : formic, acetic, propionic, caprylic, heptylic, caproic, butyric, valeric. However, the rates of penetration of caproic, valeric and butyric acids could not be separated statistically. The actual time to 50 per cent penetration was calculated and found to vary from 0.138 seconds for valeric acid to 5.04 seconds for formic acid. From formic through caproic acids the relative rates of penetration are in agreement with results obtained by other methods and are in accord with the Overton theory. Heptylic and caprylic acids, although more lipid soluble than lower homologues, penetrate beef red cells more slowly than acids of smaller molecular volume. For this reason it is suggested that these two acids are limited in their penetration by reason of their larger molecular volumes. Hemolysis studies were made with these acids, using the Parpart Densimeter. The hemolysis curves obtained, taken as a measure of penetration of these acids, did not support the findings using the spectrophotometric technique. It is thought that the discrepancies were owing to the inability of the hemolytic technique to distinguish between osmotic and lytic hemolysis. Osmotic hemolysis in hypertonic solutions.* F. R. HuNTER.2 It has previously been noted in this laboratory that chicken erythrocytes standing for 12-24 hours in heparinized plasma at 37° C. become altered. Using a photoelectric apparatus to measure volume changes of these cells when they are placed in a hypertonic solution consisting of 0.3 M glycerol in Ringer Locke, a greater deflection of the galvanometer is noted when older cells (those which have stood at 37° C. for several hours) are used, as compared with new cells (those in freshly drawn blood). The apparent volumes, as measured photoelectrically, of both new and old cells in Ringer Locke, 2X Ringer Locke and 2X Ringer Locke to which an equal volume of water has subsequently been added are what would be predicted on the basis of the swelling curves. Spectrophotometric measurements of the amount of hemolysis in these various solutions show that as the cells stand in heparinized plasma at 37° C. there is an increase in their "fragility" as indicated by a large amount (up to 46 per cent) of hemolysis when old cells shrink and swell, the equilibrium medium being Ringer Locke. This hemolysis is noted whether the volume changes involve the penetration of the glycerol molecule or whether the cells shrink in 2X Ringer Locke and then swell again as a consequence of dilution of the medium with water. Hematocrit measurements show an increase in volume (5-20 per cent) as the cells stand in heparinized plasma. These cells also lose potassium and gain sodium. These experi- ments emphasize further the extreme sensitiveness of erythrocytes to their environment. Hippuric acid excretion in anxiety states. HAROLD PERSKY. The liver function of human subjects was determined by a variety of standard tests em- ployed by the clinician. Only the sodium benzoate tolerance test (intravenous) was significantly altered in patients suffering with anxiety states. Controls were normal persons and other psy- choneurotics hospitalized under identical conditions to the anxiety group but showing little visible anxiety. The experimental and control subjects were physically healthy adults between the ages of 16 and 58. They had no clinical indications of liver disease nor any previous history of liver disease. They were in good nutritional status as judged by clinical examination and by a three day nitrogen balance study performed just before the liver function tests. The subjects had good kidney function as indicated by a normal serum urea level and in some instances also, a normal urea clearance. 1 This work was supported by grants from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service and the Faculty Research Fund, The University of Oklahoma. - Department of Zoological Sciences, The University of Oklahoma, Norman. PRESENTED AT MARINE BIOLOGICAL LABORATORY 247 The degree of anxiety was assessed by a variety of clinical and psychological tests with general agreement among them qualitatively for each subject. For quantitative correlation, a new method of scoring the Rorschach test for anxiety was employed. The anxiety group excreted an abnormally high amount of hippuric acid after i.v. injection of sodium benzoate. This effect was not observed in either of the two control groups. The excessive amount of hippuric acid in some instances was even greater than the theoretical yield of hippuric acid obtainable from the injected benzoate. The excretion of hippuric acid was directly related to the degree of anxiety for the anxiety state group. The relation is significant statistically. There is no correlation for the control groups. Following psychotherapy, the hippuric acid is significantly decreased when the anxiety is decreased. The psychotherapeutic techniques used were : electric shock, insulin shock and/or psychotherapy. The elevated hippuric acid excretion in anxiety states is due to an elevated endogenous hip- puric acid production in anxiety states over the control groups. This endogenous hippuric acid exceeds that of the control groups from two to ten times. The absolute amount is adequate to explain the supernormal values obtained when sodium benzoate is administered. The endogenous hippuric acid probably is derived from phenylalanine metabolism. It is postulated that the excessive hippuric acid obtained in anxiety states is due to diversion of phenylalanine employed for adrenaline synthesis resulting in an autonomic inbalance. Biological specificity and protein structure. DOROTHY WRiNCH.1 Studies on the relation between biological function and form are already so far advanced that it is universally recognized that biological specificities belong to the angstrom world. There is no longer any doubt that the fundamental questions involved can be formulated — and subse- quently studied and finally elucidated — only by recourse to the nature of the atomic patterns and electron density distributions. The studies of the crystal forms of about 170 different hemo- globins (Reichert and Brown, The crystalline hemoglobins, Washington, D. C., 1909) which have lain uninterpreted for nearly 40 years prove to yield material of the first importance for an understanding of biological specificities (Wrinch, Am. Mineral, in the press). The prevalence of twins and intergrowths among these protein, crystals and their pseudosymmetries in many cases are found to relate them closely to a number of minerals for which the atomic patterns are already established. Attention is called to the strong case which can be made out for an isostructural relation between the oxyhemoglobin of Ncctiirus maculatits and Staurolite (H2FeAl4SiJO,:;), between the oxyhemoglobin of Cai'itc cittleri and Tetrahedrite (Cu-,SbS3). To interpret these striking facts, which stem from the forms of the crystals and the cube and double cube nature of the twins, trillings and other intergrowths, it is necessary to find a gen- eralization of the structural essence of these minerals in terms applicable to proteins. Crystal- lographic studies have demonstrated how (e.g.) the carbon atoms in diamond may be replaced by molecules, in Sernarmontite or Arsenolite or the 29-hydrate of phosphotungstic acid (Wrinch, Phil. Mag., 38: 373, 1947: Wallerstein Communications in the press). By means of a basic principle of successive generalization, these molecules may, in turn, be replaced in appropriate circumstances by regular crystal-like arrays of molecules to give particles, crystals and inter- growths in general. It appears, therefore, that the major cubic theme in Staurolite and Tetra- hedrite should be interpreted for the hemoglobins as cubic skeletons of interlocked a-levo amino acid backbones. The accompanying minor theme, which may or may not be cubic, is then seen to mean the R-substituents on the skeleton — the "fluff" or "spines" on the surfaces of protein molecules — together with the accompanying foreign molecules or ions in the crystal. The postulate of a cubic arrangement of interlocked backbones in protein skeletons points the way to orderly arrangements of molecules into particles. Particles in the cubic and double cube systems, sometimes with disturbing non-cubic "fluff," containing 2, 3, 4, 5, 6, 7, 8, 12, 16, ... molecules are suggested for consideration in the cases of the hemoglobins, insulin, horse serum albumin, ribonuclease, etc. The picture suggested by the hemoglobin data thus includes (a) a general aspect embodying the protein essence in a cubic skeleton for the individual molecules, a number of molecules (possibly 12 with 48 residues apiece in many different hemoglobins) representing each individual particle and (b) a particular aspect embodying the protein specificity which resides in (bl) the 1 Smith College. 248 PRESENTED AT MARINE BIOLOGICAL LABORATORY pattern and associational complexes of the fluff or spines emerging from the skeletal surfaces and in (b2) the resulting characteristic differences in molecular patterns. In the forms of indi- vidual crystals and in the forms of their intergrowths in general lies a direct approach to the biological specificities of different hemoglobins at the angstrom level. (This work is supported by the Office of Naval Research under contract N8onr-579.) AUGUST 17 The incorporation of carbon dioxide into organic linkage by retina. R. K. CRANE, E. G. BALL, AND A. K. SOLOMON. The incorporation of carbon dioxide into organic linkage first demonstrated in bacteria by Wood and Werkman (Biochcm. J., 32: 1262, 1938) and in pigeon liver by Evans and Slotin (/. Biol. Chcm., 136: 301, 1940) has since been shown to proceed by the beta carboxylation of pyruvic acid (Wood- Werkman Reaction). The incorporation first observed in the whole rat by Hastings et al. (/. Biol. Chcm., 140: 171, 1941) has been assumed to occur by the same pathway. The present investigation confirms and extends the previous observations on avian tissues and provides a more direct demonstration of this reaction in the mammal. Tissues were incubated with pyruvate at 37° C. in a closed vessel containing carbon dioxide and bicarbonate labelled with carbon fourteen. Excess pyruvate was isolated as the 2,4-dinitro phenylhydrazone. The hydrazone was recrystallized and its radioactivity determined. In a survey of various tissues under standardized conditions the following rates of CO, incorporation were found: pigeon liver, 37; duck retina, 17; ox retina, 11; rat liver, 4; rat kidney, 3; pigeon heart, 0.6 ; rat heart, 0.5 ; and pigeon breast muscle, 0.3. Retinas were used whole, all others were sliced. The extensive damage on slicing may account for the low rate in muscular tissues. Since ox retina was the most active mammalian tissue, it was studied further. The rate of incorporation by this tissue was found essentially constant for at least four hours. Varying the COo-bicarbonate buffer system showed that optimum conditions exist when the bicarbonate ion does not exceed 20 millimols per liter. Increases above this decrease the rate whether the pH is or is not held constant by increasing the CO2 tension. Within the range studied, pH (7.1-7.7) and CO2 tension (5-20 per cent) appear to have little influence. Anaerobiosis reduced the in- corporation rate by 70 per cent with no additional effect on the addition of 0.01 molar iodoacetate. The same concentration of iodoacetate added aerobically caused a 60 per cent reduction. Am- monium ion (0.01 molar) produced a 70 per cent reduction under aerobic conditions. Ultrastructure of the nerve ax on. E. DEROBERTIS (no abstract submitted). Mechanisms of interaction of inhibitions with plasma cholinesterase. A. GOLDSTEIN (cf. Lalor Reports). The synthesis of nucleo proteins in developing Arbacia studied ^vith the aid of P32. CLAUDE A. VILLEE, M. LOWENS, M. GORDON, E. LEONARD, A. RICH (cf. Lalor Reports). GENERAL SCIENTIFIC MEETINGS AUGUST 24 Enzyme localisation in the giant nerve fiber of the squid. BENJAMIN LIBET (cf. Lalor Reports). Choline esterase choline acetylase ratio in invertebrate tissues. HAROLD PERSKY (cf. Lalor Reports). PRESENTED AT MARINE BIOLOGICAL LABORATORY 249 Non-integrativc synapses. THEODORE H. BULLOCK. l Synapses have been defined as valves which must change their conditions of openness from time to time in ordinary functions, or remain but partially open. That is they must integrate (make each presynaptic impulse count for more or less than one in eliciting an outgoing dis- charge Prosser). Until recently this concept could not be denied on grounds of known neuro- neural junctions. There are now several synapses found among invertebrates which behave somewhat like the classical vertebrate neuromuscular junction: they apparently act normally as simple relays, passing every impulse that arrives in a 1:1 manner. The giant synapse in the stellate ganglion of the squid may be such a case. Except in advanced stages of fatigue no summation, spatial or temporal, occurs, facilitation and after discharge are absent, though it is still possible that different paths as yet undetected physiologi- cally may alter this. The junctions between central giants and motor giants in the ventral cord of the crayfish act in this simple relay fashion (Wiersma). There are evidently commissural synapses between the two lateral giants in the same animal which normally conduct in a 1:1 and, furthermore, an unpolarized manner. Finally, the septal junctions which recur segmentally in these same crayfish laterals and in the earthworm giant fibers act likewise until severely fatigued. These sapta seem likely, on the basis of accumulating evidence (unpublished), to be real functional as well as anatomic barriers and therefore synapses on a less arbitrary definition. What biologic meaning can such relay junctions have? Unless new afferents are formed must they be regarded as present only for the occasions when fatigue is advanced, or as trophic boundaries, embryonic rests or the meeting of incompatible protoplasms (these same species have demonstrated that neuronal fusion can occur in suitable situations) ? Are they primitive or specialized junctions? Whatever their interpretation it seems necessary in the face of new evidence to broaden our conception of the synapse. Phosphagen in annelids (Polyehaeta}. ERNEST BALDWIN AND WARREN H. YUDKIN (cf. Lalor Reports). Crustacyanin, the bine carotenoid-protcin of the lobster shell. GEORGE WALD, NEAL NATHANSON, WILLIAM P. JENCKS AND ELIZABETH TARR.2 It has been suggested that the striking change in color which the lobster shell undergoes on boiling is caused by the splitting of the red carotenoid astaxanthin from a blue complex with protein (Kuhn and Sorensen, Bcr. dcntsch. chcm. Gcsel., 71: 1879, 1938). Kuhn and co- workers, however, were unable to extract such a complex from the shell, in which they believed it to be held by calcium deposits. We have extracted a deep blue astaxanthin-protein from the lobster shell with dilute citric acid. This substance, called crustacyanin (i.e., shell blue), possesses a broad absorption band maximal at about 625 mi*. It is precipitated from solution by 40 per cent saturation with am- monium sulfate, and dissolves in distilled water. Its isoelectric point lies below pH 4.5. On denaturation by heating at 100° C. or treatment with mineral acids, alcohols or acetone in the warm, the blue solutions turn orange-red, the absorption maximum moving to about 460 m^. The denatured solutions, on dilution with acetone or alcohol, yield all their color to neutral fat solvents in the form of astaxanthin. If crustacyanin is warmed to 60° C. in m/10 veronal buffer, pH 7, it exhibits a type of reversible denaturation. The color goes from blue to purple, the absorption maximum from 625 m/J- to about 530 m/*. On cooling, the color returns to blue, the absorption band to its previous position. Crustacyanin preparations frequently display a narrow absorption peak at about 412 1 University of California, Los Angeles. 2 This investigation was supported in part by the Medical Sciences Division of the Office of Naval Research. 250 PRESENTED AT MARINE BIOLOGICAL LABORATORY This is very variable in height and in our best preparations is completely absent. Some very close relation exists between the 625 and the 412 mn absorptions. When crustacyanin is brought to pH about 4.3 the 625 mM band falls while the 412 m/* peak rises proportionately. The final product possesses the 412 mM band alone. If this is heated it goes over to the orange-red color and 460 m/* absorption of heat-denatured crustacyanin. These relations are summarized in the following diagram : crustacyanin 100 412 mjjt acids ''530 mp, alcohols 460 xL alcohol astaxanthin (495 mu in aqueous digitonin) The activity and distribution of deso.vyriboniiclease and plwsphatases in tlic early development of Arbacia punctiilata.1 DANIEL MAZIA, GERTRUDE BLUMENTHAL, AND ELEANORE BENSON. The original aim of this investigation was to find enzymes that were associated with the nucleus and to follow their behavior through nuclear division. Desoxyribonuclease (DNase) seemed a logical choice because its substrate is restricted to the nucleus. There has also been evidence of nuclear localization of phosphatase (Pase). DNase was determined by the method of Barton and Mazia, involving separation of the high polymer DNA by precipitation with protamine. In the Pase measurements, glycerophosphate was used as substrate and inorganic P was determined. Nucleated and enucleated fragments of unfertilized eggs were obtained by the Harvey method. There was no clear indication of sharp nuclear localization of acid Pase (pH 5) or alkaline Pase (pH 9). Desoxyribonuclease was not restricted to the nucleated fragments. These generally had a higher activity per fragment, but not per unit volume. The sum of the activities of the fragments was 30-40 per cent higher than that of the whole eggs. It is indicated, therefore, that most of the DNase activity of the unfertilized egg is in the cytoplasm, though DNA has not been found there in measurable amounts. The DNase activity was followed through developmental stages to the 40 hour pluteus. Total activity and the activity of the "soluble" fraction (not sedimentable at 20,000 g.) were measured. Total activity did not increase significantly during development. The non- sedimentable fraction declined steadily, reaching at 40 hours a value 10-20 per cent of the total activity. The enzyme seems to be fixed on structural components of the cell, possibly nuclei, as development proceeds. In the unfertilized egg the acid Pase predominates. The activity is 3-5 times that of alka- line Pase. During development, acid Pase remains constant. Alkaline Pase remains constant until just before gastrulation, then rises steadily to a value 10 or more times that of the unferti- lized egg. These findings make no contribution to the original question of the behavior of nuclear enzymes in cell division but do show the existence of two different patterns of development of enzyme systems in morphogenesis. One is the formation of new enzyme (alkaline Pase) in the course of development. The other is the storage of adequate amounts of enzymes (DNase) 1 Work supported by grants from NRC Committee on Growth, acting for the American Cancer Society, and from University of Missouri Research Council. PRESENTED AT MARINE BIOLOGICAL LABORATORY 251 during oogenesis and the subsequent structural differentiation of the enzyme as morphogenesis proceeds. The effects of nitrogen, mustards on cleavage and development of Arbacia eggs. JOHN O. HUTCHENS AND BETTY PODOLSKY. Effects of tris(/3-chloroethyl) amine ; bis(/3-chloroethyl), methyl amine; bis (/3-chloroethyl), isopropyl amine; n-butyl, bis (/3-chloroethyl ) amine; benzyl, bis (/3-chloroethyl ) amine; bis- ( /3-chloroethyl), /3-niethoxyethyl amine; and bis (/3-chloroethyl) furfuryl amine on early cleavage and developmental stages attained at 12, 24, and 48 hours were studied using fertilized eggs of Arbacia punctulata. Amine hydrochlorides dissolved in 0.52 M NaCl were added to suspensions of eggs in sea water 20 minutes after fertilization. One hour after exposure the eggs were washed twice in sea water. Temperature was 22° C. Nitrogen mustards in non-cytolyzing concentrations slow, rather than completely block, divi- sion. At 3 hours only about 50 per cent reduction in number of cleavages can be effected with- out cytolysis. Concentrations of various compounds reducing cleavage to 75 per cent of the control value at 3 hours were therefore compared. Tris (/3-chloroethyl) amine is most effective, a dose of 2.8 X 10"5 M being required. The various bis (/3-chloroethyl) amines are all about one-tenth as effective, doses of 2-3 X 10~4 M being required. Cleavage in the presence of nitrogen mustards was frequently irregular, some blastomeres dividing more slowly than others. If cytolysis did not occur, apparently normal ciliated blastulae eventually developed. Gastrulation was blocked, however, even at concentrations which only slightly inhibited early cleavage. 10~5 M tris (/3-chloroethyl) amine or 10~4 M solutions of the other six compounds prevented normal pluteus development at 24 hours. That micromere for- mation and setting aside of proper proportions of animal and vegetal cells is not involved is indicated by the fact that ciliated blastulae (ca. 8 hrs. development) exposed to these same con- centrations were inhibited to about the same extent at 24 hours. Respiration of the fertilized egg is not inhibited significantly by these concentrations of nitrogen mustards. Respiration of treated embryos, in fact, increased with time, corresponding to the developmental stages attained. The effects of pressure on the insemination reactions of Arbacia eggs. DOUGLAS MARSLAND.1 A syringe placed inside the microscope-pressure chamber permits sperm to be ejected upon the eggs exactly 10 seconds before pressures up to 15,000 lbs./in.2 are applied. At higher pres- sures (6000 Ibs. and above) the egg gives no visible reaction to the sperm. Numerous active sperm may come into contact directly with the egg surface, but the fertilization membrane does not lift and the hyaloplasma layer does not appear so long as the pressure is maintained. But as soon as the pressure is released, the fertilization membranes and the hyaloplasma layers begin to appear ; and subsequently the eggs develop normally, at least to the free-swimming stage, provided the period of high pressure does not exceed about 15 minutes. However, the lifting of the fertilization membranes in the pressure treated eggs is slower and less complete than in untreated eggs ; and for pressures exceeding 8000 Ibs., no fertilization membranes can be distin- guished on most of the eggs, although the hyaloplasma layer becomes distinctly visible after the pressure is released, and the cleavages go ahead on schedule. Also pressures above 6000 lbs./in.2 must reversibly block the penetration of the sperm into the egg, since control eggs fertilized at the instant when the pressure is released undergo the first cleavages in exact synchrony with eggs which had been fertilized earlier and then immediately exposed to a 15 minute period of compression. Below 6000 lbs./in.2 the pressure inhibition of the insemination reactions is less complete. At 4000 Ibs. the sperm can penetrate the egg surface during the compression period and the hyaloplasma layer can form, although the lifting of the fertilization membrane is suppressed in almost all the eggs until the pressure is released. And while a pressure of 2000 Ibs. is main- 1 Professor of Biology, Washington Square College of Arts and Science, New York University. 252 PRESENTED AT MARINE BIOLOGICAL LABORATORY tained, the only observable effect is a delay in the penetration of the sperm, the formation of the hyaline layer, and the lifting of the fertilization membrane. Lactones as mitotic poisons, tested on sea urchin eggs. IVOR CORNMAN. The lactone ring is prominent in many physiologically active compounds produced by plants, unsaturated lactones being especially active. Experiments with the eggs of Lytcchinus varie- gatus, beginning exposure 10 minutes after fertilization, show that effectiveness in blocking cleavage increases 1000-fold with shifts in the position and number of the double bonds. Alpha angelica lactone (3-pentene-l,4-olide) slows the second cleavage 4 per cent at 0.11 mMolar, and blocks eggs in the 2-cell stage at 1.13 mMolar. Moving the double bond to carbons 2 and 3 (/3 angelica) decreases the activity by a factor of 10. However, adding a double bond (2,4- pentadiene-l,4-olide : protoanemonin) gives a compound which blocks all cleavage at 0.01 mMolar. Coupling two of these molecules at carbons 4 and 5, thereby destroying the second unsaturated linkage (anemonin) reduces the activity: blocking at 0.52 mMolar. A comparable decline in potency comes with moving the 4,5 double bond farther out a side chain (7-propenyl-7- crotonolactone) or hydrolysing it (2-pentene-4-hydroxy-l,4-olide). At these concentrations which block cleavage, there is first a development of the achromatic figure. It is then inactivated, as evidenced by the incomplete separation of anaphase chromo- somes in a-angelica lactone. Flakes of hyaline material in eggs treated with anemonin and protoanemonin suggest dispersal of the achromatic figure without its complete resorption. Stud- ies have already been made of the effects of lactones on plant mitoses (Erickson and Rosen, Science, in press). Penetration and effects of low temperature and cyanide on penetration of radioactive potassium into the eggs of Strong ylocentrotus purpuratus and Arbacia punc- tnlata. E. L. CHAMBERS, W. WHITE, NYLAN JEUNG AND S. C. BROOKS. x Radioactive potassium was added to 0.2 per cent suspension of (1) unfertilized, and (2) fertilized eggs of -S\ pnrpuratus at 15° C., giving a [K4'] of .015 Me/ml. Samples were removed at intervals for determinations of radioactivity, and for chemical analyses (cf. method, abstract presented by title, Penetration of Radioactive Phosphate, etc.). In the unfertilized egg the exchange rate is gradual. The total quantity of K42 entering reaches a maximum in 15 hrs. At this time the specific activity of the eggs: , 39 " j^y equals , . _ . \K*- outside] 20 per cent that of the suspension fluid: '39 • i - • [K outside] In the fertilized egg the exchange rate increases immediately, the maximum being reached in 30 minutes. At the time of first cleavage (110 minutes) the specific activity of the eggs had reached 50 per cent that of the suspension fluid. During this period no increase in content of K occurred. A maximum is reached in 15 hrs. when the specific activity approaches about 85-90 per cent that of the suspension fluid. During the first two hours of development (one cell stage) the total exchange rate of K in the fertilized egg was found to be 7-13 times that of the unfertilized. The tentative assumption is that the total quantity of readily exchangeable K approximates 20 per cent in the unfertilized egg and 85 per cent in the fertilized. By making the calculation on this assumption the rate of approach to equilibrium of the freely diffusible K is only 2-3 times more rapid in the fertilized egg. A similar relation was found in the eggs of Arbacia. Exposure of the fertilized purpuratus eggs for a short period to low temperature, and to cyanide, pronouncedly decreased the rate of exchange without altering the K content. The Q,0 (8.5° C.-18.50 C.) for cleavage time was 2.4, and for the exchange rate of potassium 2.0. NaCN was adjusted, immediately before use, to pH 8.4 and then added to hermetically closed flasks containing egg suspension. Concentrations ranging between 1 X 10^ to 1 X 10"3 M caused a 2-3 fold decrease in rate of exchange with complete inhibition of cleavage. The eggs showed 100 per cent recovery when returned to sea water. 1 Department of Zoology, University of California, and Eli Lilly Research Laboratories, M. B. L., Woods Hole, Mass. Under grant from N. C. L, U. S. P. H. S. PRESENTED AT MARINE BIOLOGICAL LABORATORY 253 In conclusion, before evaluation can be made of permeability differences between fertilized and unfertilized eggs, it is necessary to take into account tbe findings that the two types of eggs differ in their possession of relatively non-exchangeable potassium. Cartesian diver technique: a simplified mi.ring method in a new type of cartesian direr vessel. C. LLOYD CLAFF AND T. N. TAHMISIAN. Problems in manipulation of Cartesian divers where solutions must be mixed after an initial trend has been established are simplified. A new type of Cartesian diver vessel is introduced which has an "hour glass" shape. It can be fashioned without difficulty in the Diver Jig de- scribed by one of us (C. L. C.) in Science (Vol. 107, February, 1948). The lower chamber is used as an air expansion chamber, and the alkali drop is placed therein when necessary. The upper chamber is used for one of the reaction components and the lower end of the neck is used for the second reaction component, and when necessary a third component. The oil seal is placed in the usual position. The oil is colored with Sudan III. The KOH is colored with phenolphthalein. The dyes simplify observation: Sudan III in the oil makes it possible to see admixture of flotation medium should such an accident occur. The mixing of the reaction solutions is accomplished by over-pressure applied from a sphyg- momanometer bulb and controlled by the sphygmomanometer bulb valve. The overpressure is applied to the manifold system only. The manometer proper is closed off by a three way stopcock. Type experiments involving evolution of CO2 from quantitative mixture of NaHCO3 and HoSO4 ; normal respiration, toxicity of Chaos chaos to uranyl nitrate ; subsequent recovery by addition of phosphate-citrate to uranyl nitrate treated Chaos chaos; enzymatic catalysis of CO2 from pyruvate ; fertilization of Arbacia eggs; as well as respiration of Paramccium calkinsi, before and after mixing of Types I and II, were studied and graphs of results presented. Fixation and staining of plant nuclei in lacto-sitdan black b. ISADORE COHEN. Sudan Black B (SBB) is soluble in 85 per cent lactic acid, giving a deep reddish-brown solution (LSBB). Chromatin is stained brown by this mixture. SBB is a hydrophobic dye and precipitates out in the presence of excess water. Fixation and staining of freeha of onion bulb in LSBB showed that the resting nuclei in various tissues of the plant their fixation image. Nuclei with heavy coarse reticula represented one extreme and wfere related by a series of gradations to epidermal cell nuclei with remarkably sharp chromonematic structure. In the resting nuclei of root tips of the lima bean, the chromocenters stain red while the fine chromatin threads connected to them stain light brown. In onion root tip smash preparations made with LSBB, late prophase, meta- and anaphase stages were as a rule so poorly preserved that they were hardly recognizable as such. The spindle was not fixed and the chromosomes appeared to be despiralized and elongated. De- spiralization seems to occur also in resting and early prophase nuclei. It is suggested that this despiralization might explain the accentuated chromonematic fixation image given by 85 per cent lactic acid. Smash preparations of onion root tips, prefixed for one-half hour in Carney's fluid, were made using the mixture of 2 parts of LSBB and one part of N-butyl alcohol slightly under- saturated with about 8 per cent water. Large numbers of nuclei in various stages of the mitotic cycle are thus released into the medium. Frequently, in moderately despiralized chromo- somes there are seen exceptionally clear cut chromonematic structures. Nucleoli and cytoplasm do not stain. Onion epidermal cell nuclei fix and stain well in this LSBB and N-butyl alcohol mixture. When viewed with a blue daylight filter in combination with a Wratten X-l number 11 green filter the chromosomes and nuclei appear black against a green background. Urea reabsorption in the smooth dogfish kidney. RUDOLF T. KEMPTON. Urea reabsorption by the kidney tubules of the smooth dogfish, Mustclus cauls, has been studied by the use of inulin as a measure of glomerular filtration. In most of the experiments the reabsorption of water, urea and glucose was determined simultaneously. A total of 58 collec- 254 PRESENTED AT MARINE BIOLOGICAL LABORATORY tion periods in 17 different animals has dealt with urea at the normal blood levels; in 8 other collection periods with 4 animals the urea blood level was raised by the administration of very heavy doses of this substance. There has been a surprisingly wide range of urea levels in the plasma of freshly caught animals, varying from 745 mgm. per cent to 2100 mgm. per cent urea nitrogen (including any ammonia nitrogen) or in other words urea levels from approximately 1.6 to 4.5 per cent. When the average rate of reabsorption of urea per 100 cc. of nitrate is plotted against the plasma urea level, there results a straight line which is parallel to the filtration rate. On the average, there- fore, there is essentially a constant amount which is not reabsorbed from the filtrate. This does not result in a constancy of urea concentration in the urine because of the great variations in the concomitant reabsorption of water. It seems to be clear that the rate of urea reabsorption is therefore determined in some manner either by the amount of non-reabsorbed urea left in the tubule or by the plasma level. There are at present no data which give a clear indication of the proper choice of alternates, although some of the facts tend to point toward the latter as the controlling influence. In animals in which the urea level has been raised by heavy injections of urea, there is a decrease in the reabsorption of urea in relation to the plasma level, in spite of the fact that there is no similar decrease in water and glucose reabsorption. However, in seven of the eight collec- tion periods, if reabsorption per 100 cc. of filtrate is plotted against the plasma level prevailing before the urea injections were made, rather than afterward, the points fall in line with the other experiments. This would indicate, if further experiments now in progress substantiate this relation, that the rate of urea reabsorption has been "set" at the previous normal urea level ; and that the rate did not become markedly modified during the period in which the urea level was elevated. The influence of theophylline on the absorption of Mg-salts from the g astro -intestinal canal. A. FROELICH. Magnesium sulphate (MgSO4 + 7H2O) is an excellent laxative. When orally adminis- tered it is not absorbed ; no cases of Mg-poisoning have ever been reported. After injection (s.c., i.m., or i.v.) a condition of "Mg-Narcosis" develops (Meltzer and Auer) easily counteracted by intravenous injection of soluble Ca-salts. When MgSO4 + 7H2O was brought into the stomach of normal frogs (R. pipicns) in 25 per cent solution (ca. 0.5 cc. per 20 g. frog) nothing happened. But in frogs who had one hour before received a single injec- tion of Theophylline-sodium-acetate (0.15 mg. pro gm.) into a lymph sac, the same amount of Mg-sulphate produced within 10-20 min. the condition of "Mg-Narcosis." Frequently the frog died within the following 24 hours. The absorption took place chiefly from the stomach and to a minor part from the rectal part of the intestine, as could be shown in frogs whose pylorus had been previously ligatured. The Mg-chloride (MgCl2 + 6HoO) when given by mouth in a 20.5 per cent solution in a quantity similar to that of MgSO4 is already absorbed from the gastro-intestinal canal, without previous injection of theophylline. This is in accordance with the findings in large mammals (sheep, goat) : when big doses of MgCL + 6H.O were introduced into the stomach of normal animals, this led to "Mg-narcosis." However, experiments performed on rats during the winter of 1947-48 at the May Institute for Medical Research in Cincinnatti, O. (Director, Dr. A. Mirsky), gave results which differed in some respects from those obtained on frogs at the Marine Biological Laboratory, Woods Hole, Mass., during July and August 1948. Both MgSO4 4- 7H2O and MgCl2 + 6H.O were without any visible effect after introduction into the stomach of normal rats weighing from 100 to 250 gms [MgSO4 in doses of 2 or 3 mg. per 50 g. rat, MgCl in isoionic quantities (10 per cent)]. After previous s.c. injection of theophylline-sodium-acetate (0.15 mg pro gm.), the sulphate when given orally produced pro- nounced "Mg-Narcosis" leading in some cases to the animal's death. But the introduction of MgCL in isoionic quantities was found to be without this effect as well with or without previous treatment with theophylline. An explanation for this cannot be offered so far. For the occur- rence of "Mg-Narcosis" after introduction into the stomach of both frogs and rats under the action of theophylline, increased permeability leading to absorption undoubtedly is responsible (A. Froelich and E. Zak). PRESENTED AT MARINE BIOLOGICAL LABORATORY 255 The relative rate of penetration of the lower fatty acids into crythrocytes of the smooth dogfish. JAMES W. GREEN.1 The penetration of a weak acid into erythrocytes is accompanied by a dissociation of the oxyhemoglobin to a new equilibrium. This dissociation of the oxyhemoglobin may be detected spectrophotometrically. Using a modification of the Hartridge-Roughton rapid mixing tech- nique applicable to a spectrophotometer, a relative measure was made of the rate at which the saturated, monocarboxylic acids from formic through caprylic entered the red cells of the smooth dogfish. The method measures a chemical and not a volume change. Dilute, unbuffered red cell suspensions in sea water were mixed with dilute acid solutions also in sea water. The pH of the final mixtures was approximately 4.5 to 5.0. The relative order of increasing rate of penetration of these acids was found to be : formic, acetic, propionic, caprylic, heptylic, caproic, butyric and valeric. This was the same order as was found for beef red cells in earlier work. The time in seconds to 50 per cent penetration of the dogfish cells was found to be : formic, 5.72 ; acetic, 0.89 ; propionic, 0.21 ; butyric, 0.019 ; valeric, 0.019; caproic, 0.054; heptylic, 0.12; caprylic, 1.44. The times to 50 per cent penetration for acetic, propionic, butyric, valeric and caproic acids were markedly faster than the corresponding times in beef red cells. Since these acids, according to the Overton theory of lipid solubility, are thought to penetrate cells through the surface lipids it is suggested that the increase in penetration rate (over that found in beef cells for these acids) may be correlated with the greater lipid content of the fish cell ghosts as shown by the analyses of Dziemian. The penetration rate of the heptylic and caprylic acids is slower than expected on the basis of their lipid solubility. It is suggested that the molecular volume of these two acids offers a barrier to their entrance into these cells. The effect of bacterial toxins on the permeability of dogfish crythrocytes.2 F. R. HUNTER, JANE A. BULLOCK AND JUNE RA\VLEY.S As one aspect of the general problem of the action of bacterial toxins on the functioning of cells, the effect of a number of toxins on the permeability of dogfish (Mustclus canis) erythro- cytes was studied. Using a photoelectric technique to measure hemolysis times, little or no change in permeability to ethylene glycol was noted when dogfish erythrocytes were exposed to the following toxins for periods of time up to 24 hours: Clostridium scpticitin, Clostridiuin tctani, Staphylococcus aurcits and Coryncbactermui diphthcriac. The toxin of Clostridium perfringcns caused a marked decrease in hemolysis time in ethylene glycol almost immediately (less than 10 min.). The toxins of Bacillus ccrcus and Streptococcus pyogcncs caused a de- crease in hemolysis time after several hours (6-10 hours) exposure. It is hoped that a further analysis of these data and a comparison with comparable data obtained from studies on chicken erythrocytes, Astcrias and Arbacia eggs may give some information as to the action of toxins at the cellular level. New experiments and observations on sexual instability in Crcpidnla pi an a. HAR- LEY N. GOULD AND SIDNEY C. HSIAO. This report involves further attempts to clear up the mechanism of self-regulation of sex in communities of Crcpidula plana. In isolation-experiments, some of the small, sexless indi- viduals confined in separate glass tubes in the harbor water at Woods Hole have developed a temporarily functional male phase, as found by Coe, while control cultures in running sea water in the laboratory have disclosed at most only a partial male development. The difference does not appear to be of nutritional origin, as suggested by Coe, because (a) cultures in running sea water show equal or greater rapidity of growth compared with identical cultures in the 1 Physiological Laboratory. - This work was supported by grants from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service and the Faculty Research Fund, The University of Oklahoma. 3 Marine Biological Laboratory, Woods Hole, Mass., and the Department of Zoological Sciences, The University of Oklahoma, Norman. 256 PRESENTED AT MARINE BIOLOGICAL LABORATORY harbor, and (b) young sexless individuals confined in tubes with females in running sea water in the laboratory develop rapidly into adult males. Congenital differences in growth rate and sexual development appear in every culture of equal-sized young. This forms the basis for the presence of both sexes in the community, which is later assured by the effect of the more rapidly growing individuals on the smaller ones. Individuals of comparatively rapid growth under isolation, apparently tending toward direct female development, can be induced to develop into adult males by associating them with im- mature or mature females. Males which after long isolation have lost external male characters have regained full male development after eleven days association with females, and sections of the visceral sac demon- strate evidence of internal sexual atrophy followed by regeneration. In one-fourth of the cases, males from which the phallus has been removed by operation, when kept in association with females, regenerate the organ completely, and in another one- fourth, partially, after nine days. Males isolated after the operation show much less regenera- tive ability. These experiments are continuing, and investigations of starvation and of hormone effects are under way. The doubtful character of "break" excitation in skeletal muscle. PAUL G. LEFEVRE. It was previously reported that nerve-free regions of the frog sartor ius and pharyngeal retractor of Thyone failed to respond at "break" of a constant current. This was in accordance with absence of accommodation in these tissues, in light of excitation theory. Since this report, several papers from Dittler's laboratory at Marburg have dealt with response of curarized muscles to interruption of a steady current. For this reason, the matter was briefly reinvestigated on gastrocnemii from frogs, in some of which a section of the sciatic nerve in the lower thigh had been removed 2-4 weeks previously. These muscles were stimulated through non-polarizable calomel half-cells by means of a spe- cialized generator of pulses of about 2 seconds' duration; the automatic keying, tested oscil- lographically, was free from disturbing transients. The isometric response was observed oscil- lographically by amplification of current from a Statham strain gauge attached to the tendon of the slightly stretched muscle. Muscles containing intramuscular nervous elements responded in the classical manner, with a sharp development of tension at "make" and "break" of the current and some .degree of main- tained tension during its passage, depending on current intensity. "Break" response typically occurred with current intensities over 3-5 times threshold for "make" response. In denervated muscles, no "break" response was seen even at intensities up to 15 times "make" threshold. The "break" response was not, however, simply attributable to involvement of intramuscular nervous elements, as it persisted in muscles exposed to ^-tubocurarine in doses blocking neuro- muscular transmission. Since the capacity for "break" response appears to be neither intrinsic in denervated muscle nor dependent on neuromuscular transmission, it is suggested that it is a property of a spe- cialized region of the muscle at the neuromyal junction or of a special structure there interposed. Comparison of frog nerve and squid a.ron 7cr/Y// respect to the measurement of accom- modation. PAUL G. LEFEVRE. The previous report of the occurrence of "break" response in the absence of accommoda- tion, in decalcified frog nerves, was based on observation of the muscular response as index of nerve activity. Some difficulties of interpretation were avoided by use of a single-unit system, the squid giant axon, in an effort to check the phenomenon ; but it proved impossible to alter the natural rate of accommodation in this cell by decalcification. The unaltered accommodation rate in the face of pronounced autorhythmic activity in the decalcified state invalidates the simple interpretations of this rhythmicity suggested by Solandt and Katz. The pattern of response of the giant axon to alternating currents failed to conform with excitation theory. Curves relating liminal intensity with the logarithm of the frequency were symmetrical about the optimal frequency, in accordance with Coppee's observations on other PRESENTED AT MARINE BIOLOGICAL LABORATORY 257 tissues ; but there was consistent deviation from the predicted linearity in the relation between the squares of the liminal intensity and of the periods above the optimum. The accommodation rate was extremely rapid in the giant axon ; measured by stimulation with alternating currents, Hill's X was not definitely measurable due to the deviation from theory, but was certainly less than 7 msec. ; measured by stimulation with exponentially rising currents, X = 0.9 — 1.5 msec., or only 2-4 times the factor for the excitatory process ; whereas in frog nerve the two factors are of entirely different orders of magnitude. The disagreement between the rates of accommodation measured on the giant axon in these two ways (which yield identical results in frog nerve), and the non-conformity of the squid axon with theories fitting the behaviour of the frog nerve are probably associated with observa- tions on giant axons by Arvanitaki, Cole, and others, indicating that the response of the local excitatory state to subthreshold electrical disturbances is considerably more complex than as postulated in the excitation theories of Rashevsky, Hill, and Monnier. AUGUST 25 Do genes exist? P. W. WHITING. Johannsen named the gene and supposed it to be a "unit factor" or "element" "demon- strated by modern mendelian researches" and he suggested that it might be a side-chain of a large protein molecule. Recent speculation follows the same course, the gene being thus a corpuscular unit tracing back to the micella, the pangene, etc. It is here suggested that the word genes be used for terms in genetic formulae expressing mendelizing differences. It follows from this, somewhat in agreement with Goldschmidt's dissenting views, that the physical basis for genes is a considerable variety of chromosomal conditions. The germ plasma is genie mate- rial but it does not consist of genes. It produces genes by such structural reorganizations as may subsequently mendelize with the original condition. Usually this reorganization is very localized, giving rise to the linear order of the genes, but it may involve many regions provided these act as a unit of segregation in meiosis of the heterozygote. There is, however, no basis for assuming unified segregation in the homozygote. For example, the sex-differentiating gene pair of Drosophila mendelizes in the male, the heterozygote Mm (YX), but there is crossing- over between homologous parts of paternal m and maternal m in the female, mm (XX). The physical basis for the dominant gene M is the absence of one of the X chromosomes. Similar reasoning may be applied to gene pairs whose physical basis is known to be located in inver- sions, deletions, repeats, etc. Furthermore it seems preferable to extrapolate the nature of mendelizing pairs or series which are not microscopically demonstrable from those that are rather than to postulate the existence of a genie corpuscle by analogy with the subcellular units of past philosophies. Dominant IctJials induced by .v-rays in sperm- of the chalcidoid wasp Nasonia brc- vicornis Ashmead [= Mormoniella vitripennis (Walker) fide Mucscbcck in lit.}. D. T. RAY. In preliminary tests, 1287?$ and 249 dd (84.4% $2) were bred from 8 mated mothers, 2097 cW from 11 unmated. Later 2117 <3<$ were bred in 41 egg-laying days (51.6 per day) from 13 young unmated mothers and 1042 c?c? in 32 days (32.6 per day) from 42$ exposed to males. In the x-ray experiment males were treated as freshly cclosed adults and were then paired with untreated females. The females were set with several blow-fly pupae and transferred every two or three days for three or four transfers. 119 egg-laying days of the controls yielded 224213 (35.4 per day), <&?911 (7.7 per day) (82.2% 22). Data for the treated arranged as r units/ egg-laying days/daughters/sons were 500 r/40/1013/270; 1000 r/49/1 190/3 19; 1500 r/45/673/ 314; 2000 r/ 128/1326/903 ; 2500 r/16/95/133; 3000 r/49/341/404; 3500 r/24/84/245 ; 4000 r/90/ 200/700; 5000 r/60/50/540 ; 6000 r/128/24/1142; 7000 r/53/4/365 ; 8000 r/52/1/386; 9000 r/42/ 0/434; 10,000 r/34/0/342. Curve for 22/total progeny is much steeper than that for Habro- bracon (Heidenthal, Genetics, 30: 197-205) but not as steep as that for Melittobia (Kerschner, Anat. Rcc., 96 : 556) because sex ratio of the Nasonia controls is intermediate. A close fit to the Habrobracon curve is obtained by multiplying by 70/82, the percentage of Habrobracon 258 PRESENTED AT MARINE BIOLOGICAL LABORATORY females in controls divided by that of Nasonia. Sons, averaging 7.97 per day, are not directly affected by increasing dose, but with higher treatments of their "stepfathers" they show a slight increase in numbers and a considerable increase in size due to fewer sisters competing for food. Excluded from the above data are males produced after presumed exhaustion of the mothers' sperm supply. These totalled 2681 bred in 72 days, the average 37.2 contrasting with the low numbers appearing in earlier vials. Also excluded are 5 vials of diapause larvae pro- duced by 2 mothers preceding and by 3 mothers following production of progeny with normal life span. The use of dicthylstilbcstcrol in the production of eye mutations in drosophila melanogaster. BURTON L. STEKLER. The use of nitrogen mustard in the production of genetic mutations in Drosophila mela- nogaster is well known. However, there is no specificity in its action and like X-ray, produces the random type of mutation. In work along similar lines of investigation, it was found that diethylstilbesterol produced striking changes in the architecture of the Drosophila eye, and that these changes could be reproduced in succeeding generations. Due to the insolubility of diethylstilbesterol in water, the ordinary corn meal-molasses me- dium was not used. Instead, a synthetic medium containing sucrose, inorganic salts and agar was substituted. The drug, in varying concentrations (1 mg.%-20 mg.%), was then added, and the food allowed to harden. Three or four drops of a water-yeast mixture were added to this. Three different wild type stocks of Drosophila were used for the experiment. Two males and two females of each stock were allowed to mate on the drug-containing medium. Eggs were laid thereon and the larvae were forced to eat the drug-containing medium. The temperature was kept at 24° C. after the parents were put in with the experimental medium. The F-l generation was studied for mutations and it was found that the drug-treated Wood- bury and Florida wild type flies exhibited a fairly regular eye mutation. The eye characters resembled lobed eye, but as yet this fact has not been proven. The percentage of mutants in the two wild type stocks varied between 0.4-0.7%. The Oregon wild type stock did not pro- duce any mutations at 24° C. but did so at 29° C. Control groups of flies were run under the same conditions and the mutants were not observed to appear in 3500 individuals. These results substantiate previous data which were collected in 1946, in which the same experiment was tried. Mutants produced at that time have been carried on normal food since the F-l genera- tion and have so existed for 56 generations. There are indications pointing to the Stilbene nucleus as being the important factor in this chemically produced mutation. This is shown as a result of adding stilbamadine, which is closely allied in chemical structure to diethylstilbesterol, to the synthetic medium, the same eye changes are produced, only at a greatly reduced frequency. Much remains to be done in proving whether or not this drug is specifically directed and investigations will be continued toward this end. Predictable mutations in bacteria. E. RUTH WiTKUS.1 A colorless mutation has been produced in Sarclna lutea. The colorless mutation was ob- tained by mixing several different organisms in a liquid medium and growing the organisms together for twenty-four hours at 37° C. The following four species of bacteria were grown together in nutrient broth for twenty-four hours : Bacillus subtilis, Bacillus megatherium, Proteus viilgaris and Sarcina lutea. After twenty-four hours the organisms were reisolated by the dilution method and five dif- ferent strains instead of four were recovered. In addition to the four original species, a new non-pigmented form was always obtained. The new type is similar to Sarcina lutea in all morphological features except in its color. The yellow and white colonies appeared in a ratio of three to one. If Proteus vulyaris was omitted from the mixture, a white mutant was also produced. If either Bacillus megatherium or Bacillus subtilis was omitted, no white mutant was produced. No white mutant was obtained when mixtures of only two organisms were used. A mixture of four entirely different organisms was used — Bacillus ccrcus, Corynebacterium xerosc, Serratia marccsccns and Sarcina lutea. No white mutant was obtained in this instance. 1 Biological Laboratory, Fordham University. PRESENTED AT MARINE BIOLOGICAL LABORATORY 259 White mutants were also obtained by growing Sarcina lutca in a liquid medium containing a sodium salt of ribose nucleic acid. Method of origin of androgenetic males in Habrobracon. ANNA R. WHITING. 1.57 per cent of eggs x-rayed in first meiotic metaphase and fertilized by untreated sperm develop into normal fertile haploid androgenetic males. Although lethal dose for the egg nu- cleus in this stage is about 2,400 r, androgenetic males occur after treatments as high as 54,000 r. Of 291 eggs in which origin of cleavage nuclei could be determined, 3 or 1.03 per cent showed cleavage of sperm nuclei only. In 3 others, incipient androgenesis was strongly suggested. The maximum number of 6 or 2.04 per cent compared with 1.57 per cent of adults emerging shows that androgenetic larvae do not differ in viability from larvae developing in untreated eggs. Chromatin bridges in meiotic division II which occur following treatment in metaphase I allow androgenetic development when they retard the egg nucleus to such a degree that it does not reach the sperm nucleus before cleavage begins. The rarity of such bridges in eggs following treatment in prophase I will explain why no androgenetic males developing from these eggs have been found. X-Radiation effects on the restitution of dissociated Microciona. C. K. Liu.1 Dosages of 10,000 r, 25,000 r, 36,000 r and to some extent 50,000 r, given to Microciona imme- diately before squeezing it through bolting silk with meshes of 40 M, produced little effect on the process of restitution. Dosages of 100,000 r to 300,000 r were excessive. With 300,000 r, aggregation still occurred but stopped at the spheroid stage and the aggregates died after 4 days. With 100,000 r and 200,000 r aggregation proceeded to an ill-defined reticulum stage. The cell mass then retracted from the surrounding hyaline layer and collected into groups of rounded-up cells. Some of the masses survived for 3 weeks, but no differentiation took place. The dosage of 72,000 r produced significant results and was studied in more detail. Irradi- ation was done on: (1) sponges just before being dissociated; (2) suspension immediately after dissociation; (3) restituted sponges 10 days after dissociation when large numbers of flagellated chambers were formed. In (1) and (2) aggregation occurred normally but, after the reticulum stage, retracted in- stead of going into the stage of spreading. The retracted condition persisted until the llth day when the spreading out began to occur. Flagellated chambers appeared on the 14th day instead of on the 4th day as in the control. As an immediate result of (3) many flagellated chambers actually disappeared, presumably due to their breaking down into separate choanocytes. The remaining chambers collapsed. There was a decided loss of definition of all cells. Retraction of the colony soon followed and showed 50 per cent reduction in size at the end of 24 hours, leaving spicules behind. The colony retrogressed into a mass of cells with no structural differentiation. The nucleolated archeocytes became filled with coarse orange granules, presumably ingested choanocytes. The colony re- mained retracted for 10 days when it again spread out. Flagellated chambers began to form on the 14th day. Their subsequent development showed no superiority in number or size over (1) and (2). By comparing (1), (2) and (3), one can conclude that the regeneration of the flagellated chambers in the irradiated material is independent of their state of development at the time of irradiation. The fact that the flagellated chambers formed at the same interval of time after irradiation and increased at the same tempo suggests that the choanocytes are formed de novo from the archeocytes. Combined effect of ultraviolet light and heat upon first cleavage of Arbacia eggs. Lois M. HUTCHINGS. Ten minutes after insemination, Arbacia eggs were exposed for 1, 2, 3, 4, or 5 minutes to a temperature of 36° + 0.05° C. or were given 40, 80, 120, 160, or 200 seconds of 2537 A ultraviolet 1 Laboratory of Experimental Cell Research, M. B. L., Woods Hole. 260 PRESENTED AT MARINE BIOLOGICAL LABORATORY light. Other eggs were given serial dosages of heat preceded or followed by 10 seconds expo- sure to ultraviolet light, and still others received serial dosages of ultraviolet light preceded or followed by 120 seconds heat at 36° C. Thus, in addition to controls there were six phases to a complete experiment. All dishes were kept on a water table whose temperature variation during the experiment was + 0.15° C. The time at which 50 per cent underwent first cleavage was determined through several counts of 100 eggs per count. Thermal treatment applied to Arbacia eggs in serial dosages shows a continuous progression from mild to severe inhibition, which increases markedly after 3 ¥2 minutes of heating. Serial dosages of 2537 A ultraviolet light produce an intense initial inhibition of cleavage followed by a mild secondary inhibition which increases slowly in proportion to dosage. Treatments in- volving both irradiation and heat inhibit cleavage more than either agent alone. Curves of treat- ments involving irradiation and serial dosages of heat show no significant difference from a hypo- thetical curve representing the sum of the effects of each agent alone. Curves of treatments involving heat and serial dosages of ultraviolet light almost coincide with a hypothetical curve representing % of the sum of the effects of each alone. There is no evidence that irradiation preceding heat causes an exaggerated injury which application of the same agents in reverse order fails to do, i.e. there is no sensitization to heat through ultraviolet irradiation. *•<•! Streptomycin-induced chlorophyll-less races of Euglcna. LUIGI PROVAWS^I, S. H. AND ALBERT ScuAxz.2 Several aseptic clones of Emjlcna yracilis, E. t/racilis var. bacillaris and var. itrofihora be- came permanently apochlorotic in light through the action of streptomycin. Detailed investiga- tions were made of bleaching as a function of concentration and duration of exposure to strepto- mycin, sensitivity of proliferating and non-proliferating cells and comparative sensitivity in light and darkness. The following results were obtained. There was a smoothly progressive bleaching of the cultures with increasing duration of exposure and increasing concentration of streptomycin. There was no difference either in the intensity of bleaching between proliferating and non- proliferating cultures or in the time of exposure to streptomycin required, nor between light- and dark-treated cultures under proliferating conditions, in these respects. There was no evidence of development of streptomycin dependency. With streptomycin constant at 100 /*g/ml., an exposure of 1-8 hours was required for loss of roughly 50 per cent of the color, and 4 or more days for apparently complete bleaching. With constant time of exposure (15 days), 1 Mg/ml. gave roughly 50 per cent loss of color, and bleaching was nearly complete with 40 Mg/ml. Streptomycin did not interfere with the utilization of carbon sources in darkness. Preliminary microscopic observation of partially bleached mass cultures showed them to consist of both pale and almost fully green individuals. However a few green cells had less thick chloroplasts or only 1-2 ill-defined chloroplasts instead of the usual number. Aged white cells, derived both from primary treated cultures and from subcultures, accumulated an unusually large number of red granules. The stigma and paramylon grains remained permanently unaltered in all colorless cultures. The bleached clones have been carried through ten transfers in light without showing any resumption of color. The development of the basal mat in Hydr actinia. SEARS CROWELL. Small fragments of the basal mat of Hydractinia to which a few hydranths are attached can be isolated and fed with the nauplii of the brine shrimp, Artemia. These fragments some- times grow by the extension of stolons. However, in many cases, instead of producing stolons, the edge of the original mat grows out as a whole. Previous descriptions of the mat of Hydrac- tinia state or imply that it arises and grows by the production of stolons which later anastomose 1 Haskins Laboratories. - Sloan-Kettering Institute for Cancer Research. PRESENTED AT MARINE BIOLOGICAL LABORATORY 261 and broaden to give a solid mat. It is clear in my specimens that the mat may grow directly at its edge without an intermediate stage of stolon proliferation and subsequent fusion. Specificity in the fusion of stolons in hvdroids. SEARS CROWELL. The growth and fusion of stolons arising from small fragments of colonies of Hydractinia, Podocoryne, and Stylactis have been observed. When stolons growing out from the same frag- ment or from separate fragments taken from the same colony meet, they fuse in nearly every instance. However, fusion following contact does not occur between stolons of different genera, of different sex within a species, nor of different colonies of the same species and sex. It is clear that there is individual specificity in the fusion of stolons in these three hydroids. The distribution of the cerebrospinal fluid in the loiter vertebrates. H. P. K. ACERSBORG.1 Although a great deal of research has been done in anatomical laboratories in various countries on the distribution and function of the cerebrospinal fluid in some of the higher verte- brates (dog, cat, rabbit, man), the results of this type of research are not generally known among biologists. Since it is to the biologist the physician usually turns for information on basic biological problems, it was thought quite pertinent to tackle a study of this nature in a biological laboratory. It was also thought that if the cerebrospinal fluid is of such great impor- tance in man as the members of the healing arts think, it must be of like importance in not only mammals generally, to which the medical practitioner has turned for experimental enlighten- ment, but also to the vertebrates as a group of which the mammals including man are a part. For these reasons we have started a study of this problem in the lower vertebrates, using the same methods as have been used by the experimental worker in medical schools on higher vertebrates (mammals), and thinking that if the cerebrospinal fluid is of such great importance to mammals as the members of the healing arts claim, it must be of importance to verte- brates other than mammals, even to the lower vertebrates. If so, we would expect that such a system would be common to all just as the other visceral organs are not only analogous but homologous in all the vertebrates. Homologous organs are such that they are the same in origin and structure. Such is by and large the central nervous system in vertebrates. There- fore, this should hold, regarding the relationship of the nerves and their investments to the central nervous system on the one hand, and to the peripheral terminals on the other. This is exactly what we have established, using elasmobranch and teleost fishes, the frog, and turtle. There is a universal phylogenetic principle prevailing, a principle of common morphology, and perhaps also a common function. To ouT knowledge, no one has studied this problem in the lower Vertebrates before. Implications of cerebrospinal fluid distribution in the therapv of the healing arts. PAUL E. KIMBERLEY, D.O.1 The relationship between the cerebrospinal fluid and the rhythmical pulsations of the brain has been developed to formulate a functional unit. This unit, among other possibilities, aids the movement of cerebrospinal fluid from the intercellular spaces into the perivascular and sub- arachnoid spaces, thus simulating the mechanisms for moving intercellular fluids in other parts of the body. The distribution of cerebrospinal fluid, as it has been demonstrated, to the olfactory mucosa, the eye, the inner ear, the visceral nerves including their ganglia and the craniospinal nerves is enhanced and probably motivated by the pulsations of the central nervous system tissue. Thus a factor for promoting the biological requisites of fluid motion has been added which previously was not discussed for the nervous tissues. The possibility of stasis of cerebrospinal fluid naturally arises when considering the causes for the physiological perversions known as symptoms of disease. The effect of such a mechanism is seen as a* possible factor in the work of investigators using the vertebrates, especially mammals. 1 Des Moines Still College of Osteopathy and Surgery, Des Moines 9, Iowa. 262 PRESENTED AT MARINE BIOLOGICAL LABORATORY PAPERS READ BY TITLE Studies of the chemical form of P32 after entry into the arbacia egg. DR. P. H. ABELSON.1 Further studies - have been made of the P32 uptake by eggs of the Arbacia punctulata, 1-5 hours after fertilization. When eggs are exposed to sea water containing 10~8 to 10"9 grams of inorganic phosphate and .02 MC. of P3" per ml., an uptake of P32 is observed. The new work shows that at least 97 per cent of the P32 appears in the egg as tri-chlor-acetic acid soluble compounds. An attempt was made to determine the chemical nature of these com- pounds. Procedures outlined by Umbreit 3 et al. were employed yielding barium soluble and barium insoluble fractions. The latter fraction was further divided into an inorganic phosphate component and a precipitate containing ATP and ADP. From hydrolysis studies of the barium soluble material, it is clear that this fraction contains at least two components. Thus one finds at least four chemical forms of P32 very soon after entry of the substance into the egg. The total P32 increases linearly with time from 1-5 hours after fertilization and the various acid soluble fractions likewise seem to increase linearly. The difficulties of performing P3" chemical analysis in the presence of echinochrome proved enormous. By using small amounts of eggs (.1-2 cc) and large amounts of carrier, 4 mg. P and 30 mg. ATP, it was possible to obtain more satisfactory separations. The average of twelve determinations performed without ATP carrier gave the following percentages of acid soluble components : PO4 ATP & ADP Ba soluble Missing ~42 23 21 14 With carriers for inorganic phosphate and ATP the results of six determinations gave : PQ4 ATP & ADP Ba soluble Missing ~16 41 41 2 It is possible that these results might be altered if carrier for the barium soluble components were available. Penetration of radioactive phosphate into the eggs of Strongylocentrotus purpuratus, S. franciscanus, and UrecJiis caupo. S. C. BROOKS AND E. L. CHAMBERS.* Samples of a 0.2 per cent suspension of (1) unfertilized and (2) fertilized eggs at 15° C., containing P32 as orthophosphate at concentrations of 0.001-.30 Me/ml., were centrifuged in Hop- kins vaccine tubes. The supernatant was withdrawn and the volume measured to an accuracy of ff = .00015 ml. The radioactivity of each sample was determined. Correction was made for the activity of the remaining supernatant. In the unfertilized egg the rate of penetration is slow and remains approximately constant for hours. In the fertilized egg a definite increase in penetration occurred within the first 6 min. and the rate rapidly rose after 15 min., reaching a maximum by 60 min. (cleavage at 110 min.). After 60 min. the rate remained constant throughout the first three cleavages. At total initial orthophosphate concentrations of 1.4 X 10~5, 4.3 X 10"6, and 2.3 X 10"6 GM /liter the "penetration" constants for the unfertilized eggs were respectively 2.2 X 10"12, 1.85 X 10~12 and 2.0 X 10"12 GM per ml. of eggs per second while for the fertilized eggs the values were 2.9 X 10~10, 2.0 X 10"10 and 1.65 X 10~10 GM/ml./sec. In 5". franciscamis the values at an initial suspension concentration of 1.7 X 10"6 GM/liter were: for the unfertilized eggs, 7.4 X 10~13 GM/ml./sec.; 1 Carnegie Institute. 2Abelson, P. H., Biological Bulletin, 93 j 203 (1947). 3 Manometric techniques, Burgess Publishing Co. 4 Department of Zoology, University of California. Under grant from the N. C. L, U. S. P. N. S. PRESENTED AT MARINE BIOLOGICAL LABORATORY 263 and for the fertilized, 8.2 X 10"" GM/ml./sec. Expressing results in terms of GM/ml. of eggs/ sec./GM per liter of suspension, the rate of entry into the fertilized eggs of both species was found to be 130-160 times that of the unfertilized. Continuous washing of fertilized eggs failed to remove more than 2-5 per cent of the con- tained activity. Therefore, the penetration constants for the fertilized egg essentially represent the rate of increase of total P in the egg, namely, only about 0.1 per cent increase in total P per hour. NaCN at 1 X 10~4 M decreased the rate of penetration threefold. Eggs of Urcchis caitpo show a similar phenomenon but the increase in rate. does not occur until after the second cleavage. The evidence indicates that (1) the rapid penetration of orthophosphate in the fertilized egg represents an accumulation rather than an exchange ; (2) the fraction of orthophosphate entering the fertilized eggs is greater the lower the suspension concentration; (3) during the first three cleavages the rate remains constant irrespective of the mitotic and cleavage cycle, and increase in surface area. A preliminary account of this work was given, at the 28th Annual Meeting of the Pacific Division of the A. A. A. S., San Diego, June 18, 1947. Distribution of radioactive phosphate in the eggs of the sea urchin Lytcchinus pictus. E. L. CHAMBERS, A. WHITELEY, R. CHAMBERS AND S. C. BROOKS. x A single layer of eggs was continuously perfused with sea water containing P32 at 0.0005 Me/ml. The eggs were microscopically observed from above, and counting rates taken with a Geiger-Muller tube placed under the chamber. Eggs fertilized in the chamber showed the same uptake curve as described above for 5\ purpuratus. Samples of membrane-free fertilized eggs in P32 sea water were washed in a NaCl/KCl mixture to remove the hyaline plasma layer, and the activity measured. The rate of penetration into these denuded eggs was the same as in the controls. Eggs containing P32 were centrifuged into light and heavy fragments. The equal sized por- tions of unfertilized eggs showed equal activity per ml. Membrane-less fertilized eggs separate into large, coarse granules and small heavy hyaline portions containing fine granules which stain with methyl green. The activity per ml. of the latter was twice that of the former. Eggs containing P3"' were washed and homogenized at different stages of development at 0° C. in 5 per cent trichloracetic acid and the activity of the acid soluble (AS) and acid insoluble (AI) fractions determined. In all cases (unfertilized and fertilized up to 16 celled stage) the activity in the AI fraction varied from 3.5^4.1 per cent of the total. Fertilized eggs exposed to P32 from 0' to 60', then washed free and homogenized at the blastula stage, showed 6.9 per cent activity in the AI fraction. Activity in the phospholipid fraction of the AI was negligible. Killed eggs homogenized in the presence of P32 showed 0.85 per cent of the total activity of the suspension in the AI. This is far below the percentage of activity accumulated in the AI fraction of living eggs. P32 accumulates progressively after fertilization and tends to be more intensely segregated in certain portions. After homogenization in trichloracetic acid the activity is found primarily in the AS fraction. A significant percentage of the activity is found in the AI fraction, which increases during development. On the combining weight of Cypridina luciferin. AURIN M. CHASE. Gifse and Chase (/. Cell. Comp. Physiol., 16 : 237, 1940) calculated a combining weight for partially purified Cypridina luciferin on the basis of its reaction with cyanide. Their value was between 800 and 2400, the lower value seeming more probable. Because of certain sources of error inherent in their method, a redetermination by some other method seemed desirable. The method chosen depends upon the oxidation of luciferin by ferricyanide (Harvey, /. Biol. Chcm., 78: 369, 1928; Anderson, /. Cell. Comp. Physiol., 8: 261, 1936). Having established that potassium ferricyanide in low concentrations was without signifi- cant effect upon the enzyme, luciferase, the luminescent reaction was measured over a range of ferricyanide concentrations from 10"5 to 10"9 Molar. The luciferin was from two cycles of puri- 1 University of California, California Institute of Technology, and M. B. L., Woods Hole, Mass, 264 PRESENTED AT MARINE BIOLOGICAL LABORATORY fication by Anderson's method (/. Gen. PhysioL, 19: 301, 1935). In one series of experiments the oxidant acted upon the luciferin for one-half minute ; in another series, for five minutes. In all cases 0.0016 mg. of the product from the purification procedure was present in 10 ml. of pH 6.70 phosphate buffer containing the desired concentration of ferricyanide. The percentage of reduced luciferin decreased progressively from 100 per cent, at a ferri- cyanide concentration of 10^s M, to zero per cent at a concentration of about 3 X 10"7 M. Using this latter figure and assuming that the luciferin was free of impurity (the degree of purity is not actually known but is probably fairly high) a value of about 500 was calculated for the combining weight. If the oxidation as measured involves a one electron change, which may be the case in the particular procedure used, the molecular weight would be equal to the calculated combining weight, about 500. For a two electron change it would be double this value. Fat cell sice in the mutant small-wings of Habrobracon. GROSCH. A. M. CLARK AND D. S. The presence of the gene small-wings (sw) reduces the length of wings and the size of wing tissue cells. In order to determine whether this gene also affects other types of cells in the body, measurements of "fat cells" were undertaken. Conditions of measurement as outlined by Grosch (1948 — Proc. N.C. Acad. of Science) were that the animals be equivalent in size, that they be used immediately upon emergence, and that isosmotic Ringer's solution be used as the suspending c 4- fluid. Five animals from each of the six genotypes obtained from -- 9 X + sw cf were c sw selected for the present study. The abdomen of each was removed and dissected on a slide containing a drop of Ringer's in order to permit dispersion of the fat cells. Diameters of one hundred fat cells were measured from each animal. Genotype c + rfi + iw c c sw + sw c + d1 c sw c? ±± 9 + sw _c sw $ + sw Mean Fat Cell Diameter ± S.E. 107.36 103.51 102.52 97.57 98.01 87.34 in microns 0.66 0.66 0.55 0.66 0.66 0.55 (C ~\~ \ I C SW \ - cT ) with diploid males \ c? ), haploids (c + cf) + sw / \ + sw / with haploids (c sw d"), and females ( -: 9 I with females 1 - - 9 ) indicates that the \ + sw / V + sw / mutant small-wings effects smaller fat cells. These size differences are similar in trend to those obtained for the wing cells (Clark and associates — Unpublished). Further, comparison of diploid males with haploid males indicates that the diploid males have larger fat cells. This also can be correlated with wing cell size. Comparison of males and females shows a sex difference in that females tend to have smaller fat cells. This sex difference is consistent with findings by Grosch on stock No. 25 and on stock No. 33 which are wild-type except for eye color markers . The relation of the plasma membrane, vitclline membrane and jelly in the egg of Nereis limb at a. DONALD P. COSTELLO. The relation of the plasma membrane to the extraneous coats and cortical layers of the Nereis egg may be ascertained by observation of the normal living egg and of the egg after certain experimental treatments. Evidence obtained from experiments with the centrifuge and by treating the egg with alkaline sodium chloride indicates that the plasma membrane of the unfertilized egg is external to the jelly precursor granules of the cortex, and just inside the vitelline membrane. Experiments with alkaline sodium chloride (pH 10.5) indicate that the perivitelline space of the fertilized egg is extra-ovular after jelly extrusion is complete. The behavior of the egg in alkaline sodium chloride and the normal cortical response of the egg to PRESENTED AT MARINE BIOLOGICAL LABORATORY 265 fertilization are attributed largely to the properties of the jelly. Preliminary data (determina- tions by Ferry, 1939) indicate that it contains less than 1 per cent nitrogen, and is at least 75 i per cent carbohydrate (estimated by the Tillmans-Phillipi method, and corrected for ash). The jelly is precipitated in fibrous form by barium ions at alkaline reactions but not at neu- trality. The evidence suggests that the jelly is a uronic acid polymer, occurring as the calcium (and perhaps magnesium) salt. Spiral cleavage is generally assumed to be limited to the polyclad Turbellaria, the Nemertea, the Annelida and all Mollusca except the Cephalopoda. Actually, spiral cleavage is considerably more widespread, if one considers certain modifications of the typical oblique cleavage taking place in four quadrants. In all forms, spiral cleavage becomes modified into bilateral cleavage at some stage in development. As described by Bresslau (1909, 1928) and Costello (1937), the Acoela, during early cleavage, are characterized by oblique spindles which alternate in their laeotropic and dexiotropic positions to give off three duets of micromeres. The developing egg thus consists of only two hemispheres, in contrast to the four quadrants typical of the Polycladia, Annelida, and Mollusca. A further deviation is exemplified by Lepas and other Cirripedia. Cleavage of Lepas may be looked upon as a cleavage by "monets," or as unit cleavage. If this unit is designated to correspond to the D-quadrant, three micromeres (Id, 2d, 3d) are given off in succession to give rise to ectoblast and secondary mesoblast, and the fourth micromere given off (4d) is the primary mesoblast. The earliest cleavages have spindles 'which are definitely oblique. Bigelow (1902) has used the notation of a quadrant system to designate the cleavage products, but he realized that he was not dealing with a four-quadrant system. This was in disagreement with the comments of Mark and Castle, who considered Lepas to have a slightly modified four- quadrant cleavage. Induction of autogainv in single animals of Paraineeinin calkinsi jolloiving mixture of tn'o mating types. WILLIAM F. DILLER. Mixtures of two mating types of P. calkinsi, obtained through the courtesy of C. B. Metz, give rise to autogamous reorganization in single animals, in addition to normal conjugating pairs and "pseudo-selfing" clumps of a number of reorganizing individuals, loosely joined to- gether. The latter are presumably similar to those resulting from mixture of living animals and dead animals of the opposite mating type of P. aurclia (Metz, '47). This race of P. calkinsi is characterized by a single micronucleus. A fungoid symbiont of the macronucleus is fre- quently found. Nuclear activity in autogamy in single animals of P. calkinsi closely resembles the nuclear phenomena of conjugation and both are much like the corresponding processes in P. aurelia. Typical crescent stages are rarely, if ever, found in single autogamous individuals. This is very different from the conditions in P. aurelia (Diller, '36) and P. polycaryum (Diller, unpublished data). Large swollen first meiotic prophase nuclei are often seen. Each of the two young macronuclear anlagen in both ex-conjugants and autogamous animals contains about thirty deeply staining granules which are probably enlarged, modified chromosomes. This point is being investigated further. An extra post-sygotic division in Paranieciuin caitdatiiin. WILLIAM F. DILLER. Three post-zygotic divisions following fertilization have been considered the normal proce- dure in the conjugation of P. candatuui. In a race from a Philadelphia pond in which the con- jugants separate normally in a late stage of the division of the synkaryon, evidence has been secured indicating that four post-zygotic divisions occur, though only one of the nuclear products of the first division takes part in all of them. One of these sister nuclei degenerates, while the other divides three additional times to produce eight nuclei, four of which become macronuclear anlagen. This extra division, with degeneration after the first, agrees with Chen's recent find- ings in P. bursaria. Further observations on the metabolism of elains' tissues in sea water at different salinities. HOYT S. HOPKINS. Earlier results (Anat. Record, 96: 522, 1946) indicated that the stimulating effect of diluted sea water upon oxygen consumption of excised gill tissue of Venus mercenaria was not primarily 266 PRESENTED AT MARINE BIOLOGICAL LABORATORY due to reduced concentration of any specific cation of sea water. The higher rate of respiration in % sea water was maintained when the concentration of K or Ca was restored to that in natural sea water, without raising the osmotic pressure. Replacement of Mg salts caused a reduction of one-third in the respiratory increment above that in normal sea water. Recent experiments with Van't Hoffs solution led to a similar conclusion : that the dilution effect is not the result of a reduction in concentration of particular salts. In this artificial me- dium the oxygen consumption of gill was slightly lower (9 per cent) than in natural sea water. The rate increased over 40 per cent in Van't Hoff's solution diluted to %, which was about as much as in diluted natural sea water. When Mg salts were omitted from the solution, maintain- ing the same osmotic pressure, there was an increase in oxygen consumption of about 20 per cent. If half of the Mg was added — giving a concentration approximating that in the diluted sea water experiments — the rise in oxygen consumption was negligible. Since the respiration of gill was augmented in diluted sea water, but not when isotonic NaCl, sucrose, or glucose solu- tion replaced water as the diluent, osmotic work may have been involved in the first case. Also favoring an osmotic interpretation is the finding that superficial tissues (gill, mantle) showed increased respiration in dilute sea water, whereas muscle showed a decrease. The stimulated respiration in hypotonic sea water, like that in isotonic NaCl, may involve hydration, since slightly macerated gill showed equally high rates in normal and diluted sea water. Studies on the red blood cells of fish. DR. BRUNO KISCH. In this paper investigations on erythrocytes of fish are reported. The hemoglobin content, red blood count, and size of the red blood cells have been measured in different species of Teleostae and Selachians. The investigations were performed in August 1948. The hemoglobin content was determined with a Leitz Haemoglobinometer with colored glass rods as standards for comparison (Sahli's method). The blood count was taken by the usual routine method. The size of the erythrocytes was measured by an ocular micrometer. TELEOSTS Name No. of animals investigated Hemoglobin per 1 cc. Red blood count in 1 mm.3 Diameter in n of red blood cells 1. Puffer 2 7.35 4,410,000 8.7 :6.9 2. Scup 1 10.6 3,990,000 8.4 : 6.9 3. Tautog 3 6.7 3,350,000 10.9:7.2 4. Porgy 4 13.4 3,170,000 9.8 : 7.2 5. Sea Robin 2 7.1 2,380,000 10.0 : 6.3 SELACHIANS Name No. of animals investigated Hemoglobin per 1 cc. Red blood count in 1 mm.3 Diameter in n of red blood cells 1. Mustelus canis 2. Carch?.rinas obsc 5 1 3.92 3.5 452,000 540.000 16.3 : 10.9 19.0 : 13.5 3. Raja eglant. 4. Raja stabilifor. 2 3 4.8 3.6 320,000 260,000 24.3 : 14.2 21.3 : 14.5 These figures show : Teleosts have a hemoglobin content of the blood 2 to 3 times higher than Selachians and a red blood count which is 5-8 times as high as that of Selachians, but the size of their erythrocytes is about V-2 that of the elasmobrarich fish. Among the latter a definite difference has been found between the quick sharks and the slow skates, not so much concerning the hemoglobin content as concerning the red blood cells. The red blood cells of two Mustelus embryos were much bigger than those of the grown PRESENTED AT MARINE BIOLOGICAL LABORATORY 267 animals, rather rounder than elliptic, and the nuclei showed a chromosome filament. The nuclei were less compact and bigger than those of grown animals. Among the teleosts, the swordfish showed small blood cells about the size of those of a puffer. The biggest erythrocytes among the bone fish, very similar to those of the elasmo- branch fish, were found in a specimen of the plectognate Mola Mola ; they were 12.5 : 9.4 in size. It is the impression of the author that the relation between size and number of red blood cells in fish is only a single instance of a general rule in biology. Whenever the same function is accomplished in different species, either by a few or by many functioning units, the size of each of the few units is big whereas the size of each of the many units is small. The set-up with the many small units seems to be the more efficient one, the set-up with the few big units the more primitive, less efficient one. It seems to be similar with the size and number of the glomeruli of the kidney for instance in the murida family and among the Chinoptera as can be judged by the data given by Denzer. Dr. R. Chambers was kind enough to call my attention to the chromosomes of different species of the family Cyclops where one species (Cyclops brcrispinosus) has four very big and another (C. viridis) twelve very small chromosomes. Further studies will show how far this rule applies to nerve ganglions and other functioning units of the living beings. The action of NH±Cl on the surface membranes of Arbacia eggs. M. J. KOPAC. A new granular layer can be revealed on centrifugation of unfertilized or fertilized Arbacia eggs immersed for several hours in 0.53 M NH4C1 (3 parts) + sea water (7 parts). This layer, less dense than the cytoplasmic matrix, is believed to contain released lipids (Heilbrunn, Biol. Bull., 71: 299, 1936). White halves, obtained by centrifugal splitting of unfertilized eggs, when treated with NH4C1 showed almost as much "lipid" substance as whole eggs. Much of this material, therefore, originates in the cytoplasmic matrix. Unfertilized Arbacia eggs, after treatment with NH4C1, were tested by the oil coalescency method (Kopac, Cold Spring Harbor Symposia, 8: 154, 1940). Following 90 to 120 minutes' immersion, the eggs readily coalesced with oil drops indicating the disappearance of jelly. The coalescency of the treated eggs progressively increased the longer the eggs were exposed to NH4C1, thereby indicating a weakening of the vitelline membrane. This was further suggested by the flimsy fertilization membranes that developed after insemination. Unfertilized eggs, after 4 to 6 hours' exposure to NH4C1, were stratified by centrifugation. Oil drops were applied to the surface adjacent to the lipid, matrix, yolk, and pigment zones where coalescence occurred readily. Immediately after coalescence near the matrix zone, a wave of peripheral disintegration encompassed the entire cell surface resulting in complete cytolysis of the cell. On occasion, this effect was obtained at the lipid zone. No cytolysis occurred on coalescence at the pigment zone nor at junction of pigment and yolk zones. Red halves from centrifuged, NH4Cl-treated cells sometimes retained a small portion of the matrix. Coalescence with oil drops at this region also induced the peripheral wave of disintegra- tion. No cytolysis occurred when yolk or pigment zones were similarly treated. A similar peripheral disintegration following coalescence was previously observed in com- pletely denuded eggs. This reaction always occurs when extraneous coats are not present to lend their mechanical support to the underlying protoplasmic surface layer. The absence of peripheral disintegration when oil drops penetrated near the pigment of yolk zones suggests a new role for packed cell inclusions. The packed granules and vacuoles can serve as an inner mechanical support for the protoplasmic surface layer, thus replacing the extraneous coats. These data suggest that, in addition to releasing bound cytoplasmic lipids, NH4C1 may alter the surfaces of Arbacia eggs. The principal action, however, appears to be on extraneous coats, e.g., jelly, vitelline membrane, and hyaline layer. The inhibition of development of Arbacia eggs by NH^Cl. M. J. KOPAC. Immersion of unfertilized Arbacia eggs in 0.53 M NH4C1 (3 parts) + sea water (7 parts) for several hours altered, inter alia, the density ratio between cytoplasmic matrix and nucleus. Following centrifugation of treated eggs, the nucleus assumed an equilibrium position near the yolk boundary rather than near the oil cap as in normal eggs. 268 PRESENTED AT MARINE BIOLOGICAL LABORATORY Nearly all cells immersed in NH4Cl-sea water up to 4 hours prior to insemination cleaved although there was considerable delay. About 50 per cent of the fertilized eggs divided following 7 hours' exposure. None divided following 13 hours' exposure. Before insemination, the cells were transferred to sea water. Subsequent development in normal sea water was slow and irregular. Following 2 to 3 hours' exposure to NH4C1, 50 per cent of the eggs developed into swimming blastulae. Follow- ing 5 to 6 hours' exposure, 10 per cent formed swimming blastulae. Gastrulation was com- pletely inhibited by only 15 minutes' exposure. The blastulae were sluggish and swam in tight circles near the bottom of the vessel. White and red halves obtained by centrifugal splitting of unfertilized eggs, previously ex- posed to NH4C1 for 4 hours, also cleaved following insemination. The red halves cleaved more rapidly than the white halves which contained all the displaceable low density substances. De- velopment was irregular and only a few of the red halves developed beyond the 12- or 16-cell stages. Fertilization membranes were weak and many ruptured as early as the 2- or 4-cell stage. Double and triple blastulae were common. Frequently, one part of the double blastula was well developed while the other part consisted of disorganized blastomeres which rarely held together. Some of these blastomeres had no hyaline layer as revealed by the oil coalescency method. Several examples were noted where one blastula, the size of the first blastomere, developed normally while attached to it were 2, 4, or more cells, apparently arising from the second blasto- mere formed during the first cleavage. The weakened extraneous coats are probably responsible for this irregular development. The fertilization membranes were weak and these did not elevate from the cell surface more than 2-3 microns. The hyaline layer was variable in thickness even during the first or second cleavages. Later, it frequently became so weak that bizarre aggregates of cells were produced instead of organized blastulae. The nature of the hcmolytic effect of silver. MARIAN E. LEFEVRE AND M. H. JACOBS. The hemolysis of certain fish erythrocytes by an impurity in some brands of C. P. NaCl, shown by Ball to be silver, has been further studied, using chiefly the erythrocytes of two sensi- tive species, the mackerel and the cunner. It has been determined that the mechanism of this hemolytic effect is an induced permeability of the erythrocytes to cations resulting in a swelling of the cells by the operation of the Donnan equilibrium. The evidence is: (1) that immediate and extensive shrinkage of the cells occurs in sucrose made isosmotic with the blood and con- taining a trace of calcium, (2) that swelling and hemolysis are rapid in NaCl of any strength, (3) that on the addition of concentrated NaCl- to a suspension of cells in an appropriate mixture of NaCl and sucrose there is an immediate shrinkage, quickly followed by a return to more than the original volume, (4) that hemolysis in NaCl is prevented by the addition of relatively low concentrations of sucrose, provided that Ca is present, and, (5) that under properly chosen conditions the rate of hemolysis after treatment with silver shows a minimum at a pH near the isoelectric point of hemoglobin. In the course of this work it was noted that the erythrocytes of the two species of fish in question, and some others, undergo hemolysis within an hour or less in pure isosmotic sucrose solutions. As in the case of the much less striking injury of mammalian erythrocytes in the non-electrolyte solutions studied by Wildbrandt, hemolysis of the fish erythrocytes was only feebly opposed by NaCl but very effectively by low concentrations of CaCL, BaCl., or MgCL. It was also noted that the well-washed erythrocytes of several species of fish, unlike mam- malian erythrocytes, may fail to hemolyze within several hours in a pure isosmotic solution of NH4C1. On the addition of a trace of bicarbonate, however, hemolysis in the same solution readily occurs. Reversible sphering of erythrocytes. WARNER E. LOVE AND M. H. JACOBS. The sphering action on human erythrocytes of sodium taurocholate and the fact that it is favored by alkalinity are well known. It has apparently not previously been reported, however, that sphering of taurocholate treated cells can be produced and reversed at will many times in succession merely by appropriate changes in the pH of the surrounding medium. PRESENTED AT MARINE BIOLOGICAL LABORATORY 269 A convenient way of showing this effect is to suspend well washed human erythrocytes in an isotonic NaCl solution buffered about neutrality with phosphate and lightly colored with phenol red, which in low concentrations has little effect on the cells. With a little NaOH the indicator is now made distinctly red and taurocholate is added until sphering just occurs. On the addition of HC1 until the indicator becomes yellow the cells immediately resume their bicon- cave shape. In the absence of phenol red the behavior of the cells themselves serves as a sensi- tive indicator of pH changes. Alkali and acid may now be added alternately as often as desired. We have in this way produced sphering and its reversal 15 times in succession. If only enough taurocholate be used to cause sphering, the cell may remain in good condition and be capable of showing reversal -of sphering for at least 24 hours. Several washings of the taurocholate-treated cells with isotonic NaCl do not destroy their ability to show the behavior just described. Under favorable condi- tions a pH change of 0.5 unit or less is sufficient to reverse the taurocholate effect, but even much larger changes have little influence on the sphering produced by sodium cetyl sulfate or sodium cetyl phosphate. Observations on the henwlytic effect of sodium dodcc\l sulfate. Lois H. LOVE AND M. H. JACOBS. The hemolysis of human erythrocytes by sodium dodecyl sulfate has several unusual aspects. The first is the effect of temperature on the rate of the process. At low temperatures with some concentrations complete hemolysis occurs within a few seconds, but as the temperature is in- creased, a point is reached where the process suddenly becomes very slow. Under some condi- tions a rise of temperature of 1° C. may change the time for complete hemolysis from less than a minute to more than an hour. With a further rise of temperature the rate of hemolysis may again become more rapid. In the temperature range of retardation two phases of hemolysis can be demonstrated, some of the cells hemolyzing immediately, the remainder very slowly. The slowly hemolyzing cells, however, show a normal susceptibility to the hemolytic action of hypotonic solutions. The second peculiarity of hemolysis by sodium dodecyl sulfate is that the addition of lecithin causes a remarkable acceleration of the process instead of the retardation reported in the case of certain other hemolytic agents. For example, in one experiment 80 per cent hemolysis by sodium dodecyl sulfate alone required 160 minutes ; in the presence of approxi- mately 50 7 of lecithin alone no hemolysis occurred in 180 minutes. But in a mixture of the two the time for 80 per cent hemolysis was 16 minutes. By a proper choice of conditions even an apparently non-hemolytic concentration of sodium dodecyl sulfate can be made hemolytic by addition of a non-hemolytic concentration of lecithin. Effects of hypertonic solutions on Nereis eggs. W. J. V. OSTERHOUT. Unfertilized eggs of Nereis limbata placed in 1.4 M MgSO4 or in 1.5 M dextrose in sea water lose water rapidly and appear collapsed with abnormal shapes. If after a few minutes they are replaced in sea water, they soon become normal in size and appearance. If sperm is promptly added many eggs extrude jelly, segment and develop trochophores. If the unfertilized eggs are placed in 2.7 M MgSO4 in sea water, they collapse at once, but in the course of time MgSO4 enters the eggs sufficiently to enable them to resume more nearly normal shape. The same thing happens in 2.8 M dextrose in sea water but here the penetration is much more rapid. When the unfertilized eggs are exposed for a short period to 2.7 M MgSO4 in sea water and are returned to sea water they swell rapidly and develop a clear zone just beneath the vitel- line membrane. Such eggs cannot extrude jelly nor segment nor produce trochophores when sperm is added. Solubility of the ritelline membrane of Nereis eggs. W. J. V. OSTERHOUT. The vitelline membrane of Nereis resists acid and alkali and all other reagents hitherto tried. But it dissolves quickly in a mixture of sodium dodecyl and tetradecyl sulfate. When unferti- lized eggs are placed in 1 per cent of this reagent in sea water the egg swells and soon shows a 270 PRESENTED AT MARINE BIOLOGICAL LABORATORY protrusion at one point as though the tough vitelline membrane were yielding to internal pressure. The membrane covering the end of the protrusion dissolves and this action spreads to sur- rounding regions and in a few minutes the membrane completely disappears. A similar process takes place in segmenting eggs. The dissolving of the vitelline membrane is more rapid and more easily observed if the eggs are first killed by placing them for a few minutes in 2.7 M MgSO4 in sea water and then leaving them for a short time in sea water before they go into the dissolving reagent. In sea water they swell and acquire a clear zone inside the vitelline membrane. In the reagent splits appear at several places in the membrane and at these spots the membrane dissolves and this action spreads to neighboring regions so that the membrane completely disappears. This process is accompanied by alterations in the appearance of the protoplasm. Experiments on chloroplasts and on photosynthesis. W. J. V. OSTERHOUT. • The chloroplasts of certain marine and fresh water algae and of some other plants show very interesting reactions when the cells are bathed in a saturated solution of hexylresorcinol. The chloroplasts shrink and clump together and in many cases form a more or less regular network. This continues to contract and may eventually form a single compact mass occupying only a small portion of the cell. There is great loss of water from the chloroplast and the minute drops of chlorophyll (grana) originally present in the chloroplast are evidently brought closer together and it seems possible that they may fuse to some extent. Chloroplasts exposed to "Sodium Lorol Sulfate" (which may be called S. L. S. for con- venience) also show great contraction. This reagent is a mixture of sodium dodecyl and tetra- decyl sulfate. When cells of Spirogyra are placed in 1 per cent S. L. S. in distilled water, the chloroplast contracts and eventually breaks up into small masses. The green color soon spreads uniformly throughout the cell as if in aqueous solution but is unable to pass out through the cellulose wall. Cells in this condition show no photosynthesis when well washed and placed in a solution of sodium bicarbonate with a trace of phenolphthalein as indicator. But cells subjected for 2 minutes to 0.01 per cent S. L. S. in which the chloroplast is still intact show photosynthesis though slightly delayed as compared with the control. When the cells are exposed for 8 minutes to the same reagent, the cells at first appear not to be seriously affected, but on washing thor- oughly and transferring them to the bicarbonate solution containing phenolphthalein, the injury progresses until the chloroplasts are affected. Such cells show no photosynthesis within 1 hour or longer. The control cells show rapid photosynthesis. A ncn' peritrich from Woods Hole. M. A. RUDZINSKA. Pachystomus Olistus (cf. Abstract Section Biological Bulletin, 93: 2, 1947). Intcr-myotome connections in early embryos of Mustclis canis. Lois E. TE\VINKEL. Embryos of the smooth dogfish, Mustclis canis, begin to flex rhythmically from side to side- at an approximate length of 3.5 mm. (20-23 somites). Such motion, observed in a number of elasmobranch embryos and shown by Wintrebert to be aneural, is exhibited for some time prior to the initiation of the heart beat, which, in the Mustelis embryos studied, occurred at the 5.5-6 mm. stage. Sections in the three usual planes show that the antero-posterior orientation of myoblasts has occurred in at least the anterior two-thirds of the somites in 3.5-4 mm. embryos. Cell boundaries are virtually impossible to distinguish but preliminary studies indicate that myoblasts of a given somite come into contact with those of immediately adjacent somites so that there is an interdigitation of their tapering ends. Fibrillar structures seen in the cells are identified tentatively as early myofibrillae. In general they are homogeneous but occasional faint signs of beading suggest the development of cross striations. In 5.8 and 8 mm. embryos, myoblasts of one myotome seem to be directly connected to those of neighboring myotomes at a more constricted area. Distinctly striated myofibrillae, which are now present, appear to be joined to those in cells of adjacent myotomes by means of fine light-staining homogeneous fibrils which extend across the inter-myotome bridge. At the 35 mm. stage, additional bundles of striated muscle cells have differentiated appar- PRESENTED AT MARINE BIOLOGICAL LABORATORY 271 ently from mesenchyme cells between the myotome bands and the notochord, but these bundles are separated inter-segmentally by numerous mesenchyme cells. The contrast, therefore, be- tween the longitudinally connected primary myotome bands and the segmented muscle bundles which develop later suggests that there may well be some relationship between myotome bridges and the rhythmic flexion of the early embryo. Properties of the surface coat in cinlvyos of Fundulns hcteroclitus. J. P. TRINKAUS. The eggs of Fundulus possess a surface layer similar to the surface coat of amphibian eggs. This coat constitutes the outer layer of the egg in all stages examined (immature, mature eggs, and developmental stages through blastopore closure). Microdissection demonstrates it to be an elastic gel, non-adhesive on the outer surface, sticky and less viscous at deeper levels. It solates in Ca-free SW and upon mechanical agitation. A wound in the coat covering the yolk first widens and then closes in 2-5 minutes (even though yolk is exuded throughout the process). During wound closure, radiating folds appear in the coat. When carbon particles are placed on opposite edges of a closing wound they move toward each other, indicating that wound closure is not due to the formation of a new surface, but to the expansion of an already present, elastic coat. Closure of a wound in the yolk coat near the marginal periblast apparently exerts tension, which not only stretches adjacent cells greatly, but also pulls a large sector of blastoderm toward the wounded area. A wound in the blastoderm itself initiates similar processes of wound healing, resulting in radial stretching of epiblast cells toward the wound closure. Carbon marking of normal cleavage and gastrula stages suggests a spreading of the surface layer in both cleavage and epiboly. At closure of blastopore of normal embryos, the cells at the blastopore lips elongate greatly, as if they are being pulled toward the point of blastopore closure. At an earlier stage (Oppenheimer, '14), this phenomenon can be simulated and blas- topore closure hastened, if the yolk coat is wounded at the point of future closure of blastopore. These preliminary studies suggest that the surface coat of the Fundulus egg, because of its remarkable elastic properties, plays a unifying and perhaps a causal role in gastrulation movements. Fertilizin of Nereis linibata. ALBERT TYLER. The fertilizins of eggs of sea-urchins and other marine animals are known to comprise the material of the gelatinous coat of the unfertilized egg. Since, in Nereis, a gelatinous coat forms after fertilization it is of interest to learn whether or not the material of this coat is fertilizin and to determine some of its chemical properties. Using the alkaline saline method of Costello (1945) for removal of the membrane, and extrusion and dissolution of the Nereis egg jelly, large quantities of this material were prepared with very little injury to the eggs. When added to sperm, under proper conditions of pH (ca. 9) the preparations gave strong agglutination, the titers being considerably greater than those given by ordinary egg water prepared from unferti- lized egg in the same time interval. While definitive proof is yet to be obtained, various tests favor the view that the jelly material is fertilizin and that that present in ordinary egg water is similar material that has diffused through the membrane in non-gelatinous form. The active material is found to be non-dialyzable, fairly heat stable and precipitable by 60 per cent alcohol. It has a nitrogen content of about 5 per cent and contains at least 18 per cent reducing sugar. From a total of approximately 20 cc. of eggs that have been obtained so far in the present season, the yield of alcohol precipitable material amounts to 300 mg., most of which is available for further analysis. Mating types and conjugation of four different races of Paramecium calkinsi and the effect of .v-rays on the mating reaction. RALPH WICHTERMAN. Four different races of Paramecium calkinsi have been cultivated up to two years in a medium consisting of 2 parts of lettuce infusion and one part of filtered sea-water. Opposite mating types I and II (Yale races) are unimicronucleate ; the remaining strains here designated as Ila and lib are unimicronucleate and bimicronucleate respectively as shown in Hematoxylin and Feulgen preparations. Micronuclei in all races are small and average only 2 M. Races Ila and bimicronucleate lib readily mate and conjugate with unimicronucleate type I. All races 272 PRESENTED AT MARINE BIOLOGICAL LABORATORY appear to be as stable as P. bnrsaria since no change in mating type has occurred during the period in culture. In all cases, the mating reaction occurs with the formation of clumps of a 100 or more specimens any time of day, followed by conjugation. Cultures containing P. calkinsi have, at approximately 24° C., a pH range of 6.5 at time of inoculation to pH 7.8 in older cultures. Greatest numbers of paramecia reactive for mating are found in cultures with a pH of 7.3 usually on the 7th day after inoculation. P. calkinsi is much more sensitive to x-rays than P. bursaria. When opposite mating types are irradiated with 100,000 r, 200,000 r and 300,000 r and mixed, the mating reaction occurs but the clumps thus formed are progressively smaller than the controls. Clumps then break down leaving only single specimens. However, mating types irradiated with 100,000 r and mixed will, 24-48 hours later, demonstrate pairs in conjugation. Those irradiated with 200,000 r and 300,000 r will demonstrate the mating reaction but do not enter into conjugation with the ex- ception of some survivors of 200,000 r dosage. When irradiated with 400,000 r and mixed, the slowly moving specimens do not show the mating reaction but die within a few hours. All unirradiated members of one sex type show the mating reaction with specimens of opposite type irradiated up to 400,000 r. Only those irradiated with 100,000 r and some survivors of 200,000 r will conjugate with unirradiated specimens of opposite type. Unirradiated specimens of one sex type will not mate or conjugate with irradiation-killed specimens of opposite sex type. The hydrogen-ion concentration in the cultivation and groivtJi of eight species of Paramecium. RALPH WICHTERMAN. A study was made of the pH changes occurring daily in 36 cultures of races of eight fresh and brackish-water species of Paramecium. Original cultures have been maintained continuously for 1-13 years in covered 250 ml. flasks with little if any variation in culture technique. P. aurclia, P. caudatiiin, P. tnitltiinicronuclcatuin and P. tricliiitin have been cultivated in hay me- dium consisting of 1% gm. of hay and 210 ml. of boiled distilled water which prior to inoculation had a pH of 6.2; P. bursaria and P. polycaryum in lettuce medium consisting of \Vi gm. desic- cated lettuce to 1 liter of boiled distilled water which was filtered then autoclaved and had a pH of 5.0 prior to inoculation ; P. calkinsi and P. woodruffi on lettuce-sea-water medium with a ratio of 2 : 1 respectively and a pH of 6.5 before inoculation. All media were prepared on one day and inoculated the following day with approximately 20 ml. from a rich culture containing paramecia and bacteria. Daily pH determinations, which in the course of the investigation totalled over 500, were made of the medium prior to inocula- tion, through the period of maximal growth and gradual decline of the population at about 24° C. with a Cambridge electronic pH meter having a sensitivity of 0.02 pH unit. Buffers were not added to cultures. Results are briefly summarized in the table. Optimum range in which Species Medium and pH before inoculation pH range of medium in cultures growth occurred and yielded greatest concen- tration of paramecia P. aurelia Hay: 6.2 6.2-7.3 7.0-7.2 P. caudatum Hay: 6.2 6.2-7.2 6.9-7.1 P. multimicronucleatum Hav: 6.2 6.2-7.5 6.5-7.0 P. trichium Hay: 6.2 6.2-7.1 6.7-7.1 P. bursaria Lettuce: 5.0 5.0-7.4 7.1-7.3 P. polycaryum Lettuce: 5.0 5.0-7.5 6.9-7.3 P. calkinsi Lettuce-sea H2O: 6.5 6.5-7.8 7.1-7.4 P. woodruffi Lettuce-sea H2O: 6.5 6.5-7.5 7.0-7.5 The cage hypothesis and a common feature of X-ray diffraction studies of crystalline proteins. DOROTHY WRiNCH.1 It is well recognized that X-ray diffraction data obtained from protein crystals constitute — potentially at least — crucial tests for any proposed theory of the atomic patterns of proteins. 1 Smith College. PRESENTED AT MARINE BIOLOGICAL LABORATORY 273 Fundamental difficulties however arise in using these data for this purpose in any detailed manner in that (1) a protein unit is in general an array of molecules, (2) a protein molecule comprises a skeleton plus a complement of substituents in most cases incompletely characterized and in all cases unknown as regards their spatial pattern, (3) protein crystals contain large numbers of "foreign" molecules and ions, (4) the diffraction patterns to be expected from vast arrays of atoms present scientific and technical problems of considerable complexity (Wrinch, Fourier transforms and structure factors, Am. Soc. for X-Ray and Electron Diffraction, 1946). In a hypothesis formulated in 1936 (Wrinch, Proc. Roy. Soc. London A161 : 505, 1937), protein molecules were formulated as cage structures, space-enclosing networks of multiply- connected a-levo amino acid backbones, with R-substituents emerging from Ca atoms. Natu- rally it has, as yet, not proved possible to allocate particular substituents to particular Ca sites, nor even to formulate the numbers or the spatial patterns of the molecules in various protein particles. The fact that this hypothesis gives a skeletal structure which is cubic leads to a pic- ture of protein particles with molecular patterns in which the orientations of the skeletons are structurally related, and thus to the possibility that the constructional principle common to pro- teins may prove, in some measure, recognizable in diffraction patterns, notwithstanding the fact that the skeletal atoms may constitute a third or less of the atoms in the crystal. This communication reports the fact that the series of cage structures, Ci, Ci, . . . Cn, . . . have one common feature in their diffraction patterns. The interaction of one pair of antipodal tetrahedral faces of the Cn structure yields maxima in diffraction space, at distances 8 an (1, 1/2, 1/3, . . . I/;;, . . .) A from the origin, where a — say ^ 1.5A — is the mean of the N-Ca, Ca-C, C-N bond lengths. Thus, for the whole series, these interactions produce a maximum at — 12A. The remaining three pairs of antipodal faces modify the maximum but slightly, moving them from — 12A to — 10A in d, from — ' 12A to >— 11.2A in C2, from — 12A to ^ 11.6A in C3, and so on. When the closely packed substituents on the cage faces are taken into account, these maxima may move to slightly shorter spacings (loc. cit., p. 55). It is well known that crystalline proteins as a class yield strong spacings in the neighborhood of 10-1 1.5A. It may therefore be claimed that the cage hypothesis meets the first challenge of the X-ray data in that it predicts one or more of a series of structures, all of which have pairs of antipodal faces which, with the remaining faces, produce high intensities at distances of this order of magnitude. It is pertinent to notice that the maxima derived from the predicted skeletons lie in octettes at the corners of a cube and that in the case of tobacco mosaic virus strong spacings at — 11. 3 A, close to cube corners, have been recorded (Bernal and Fankuchen, /. Gen. Physiol, 25 : 111, 1941). (This work is supported by the Office of Naval Research under contract NSonr-579.) REPORT ON THE LALOR FELLOWSHIP RESEARCH Phosphagen in annelids (Polyclmcta). ERNEST BALDWIN* AND WARREN H. YUDKIN. Kutscher and his school came to the conclusion, upheld by Andrew Hunter, that the creatine which is characteristic of vertebrate muscle is replaced by arginine in the muscles of 'inverte- brates. The discovery of creatine phosphate in the muscle of vertebrates by Eggleton and Eggleton (Biochcm. /., 21 : 185, 1927) and Fiske and Subbarow (/. Biol. Chan., 81 : 629, 1929) was shortly followed by that of arginine phosphate in invertebrates by Meyerhof and Lohmann (Biochem. Ztschr., 196: 22, 49, 1928). Comparative investigations by the Eggletons (/. Physiol., 65: 15, 1928) and by Meyerhof (Arch, di Sci. Biol., 12: 536, 1928) supported the broad principle of alternative occurrence. The wider comparative studies of Needham, Need- ham, Baldwin, and J. Yudkin (Proc. Roy. Soc. London B, 110: 260, 1932) upheld this general principle, but with certain notable exceptions : both phosphagens were found side by side in the body muscles of Balanoglossus and in the jaw muscles of an echinoid. These results provided chemical support for the echinoderm-enteropneust theory of vertebrate ancestry proposed by Bateson on morphological grounds. Our present investigation has demonstrated that the en- teropneust, Saccoglossus, contains only the single phophagen, creatine phosphate. * Lalor Fellow. 274 PRESENTED AT MARINE BIOLOGICAL LABORATORY One of the objects of our present study was to investigate the phosphagen of annelids more carefully than has hitherto been done for, while Meyerhof and Needham, Needham, Baldwin and J. Yudkin alike had demonstrated the presence in polychaetes and gephyreans of a labile sub- stance having the general properties of arginine phosphate, Arnold and Luck (/. Biol. Chem., 99: 677, 1933) were unable to find evidence for the presence of arginine itself in any of the marine worms they studied, though arginine was found in the terrestrial oligochaete Lumbricus and has in fact been isolated from this form by Kutscher and Ackermann (ZtscJir. physiol. Chem. 199: 266, 1931). Moreover, in the original experiments of Needham, Needham, Baldwin, and J. Yudkin the phosphagen of Nereis diversicolor showed somewhat anomalous behavior which was not further investigated at that time. Recently, Greenwald (/. Biol. Chem., 162 : 239, 1941), employing Jaffe's reaction, demonstrated the presence of chromogenic material indicative of creatine in the testes of several annelids. In the present investigation experiments were done on the atypical behavior of Neanthes phosphagen upon hydrolysis in the presence and the absence of the molybdate ion. These experi- ments showed the presence in this annelid of a labile phosphate compound having the properties of creatine phosphate, together with a phosphagen similar in behavior to arginine phosphate, which for the present we propose to call the "annelid phosphagen." Studies of other poly- chaetous annelids have now shown that the apparent creatine phosphate and the annelid phos- phagen may occur singly or together. From Table I we may generalize concerning the species examined : All the free swimming forms contain creatine phosphate, sometimes together with the annelid phosphagen ; all the sedentary forms, with the exception of Chaetoptcrus, contain the annelid phosphagen, sometimes together with creatine phosphate. TABLE I Occurrence of phosphagen in polychaetes The annelid phosphagen similar to arginine phosphate is signified by AP. CP indicates the phosphagen behaving like creatine phosphate. AP CP Free Swimming Forms (Errantia) Orbinia (Aricia) -f- Diopatra + Lumbrineris (Lumbrinereis) + A rabella + Glycera + Neanthes (Nereis) + + Lepidometria + + Sthenelais -f- -J- Sedentary Forms (Sedentaria) Chaetopterns + Cislenides + + A mphitrite -f- Pista + — Enoplobranchus + — Cirratulus + Maldane + Clymenella + These newer observations show that Needham, Needham, Baldwin, and J. Yudkin's former conclusions must be seriously modified. Whether they must actually be abandoned will depend on the results of further, thoroughgoing investigations of other annelid groups and of more representatives of other invertebrate phyla. We are pleased to acknowledge our indebtedness to Dr. Frank A. Brown, Jr., for procuring and identifying most of the annelids used in this study. PRESENTED AT MARINE BIOLOGICAL LABORATORY 275 The mechanism of interaction of inhibitors with human plasma choline st erase. AVRAM GOLDSTEIN.* The carbamic esters prostigmine, physostigmine, and carbaminoylcholine inhibit purified human plasma cholinesterase activity as determined by addition of acetylcholine to an enzyme- inhibitor mixture. During a period of two hours after addition of substrate there is a progres- sive relief of inhibition which is not the result of destruction of the inhibitor. Thus acetylcholine slowly displaces these inhibitors from combination with the enzyme. The dissociation that occurs on dilution of an enzyme-inhibitor mixture is also slow, requiring two to three hours for com- pleton. These compounds are reversible inhibitors (by dialysis). Determination of the nature of inhibition by varying substrate concentration by the method of Lineweaver and Burk (/. A. C. S., 56 : 658, 1934) reveals an apparent non-competitive inhibition, i.e., in a twenty minute period of determination no amount of substrate can reverse the combination. If, however, sub- strate is given full access to the enzyme (by its addition simultaneously with inhibitor) com- pletely competitive curves are found. ... In contrast to the above, choline, acetyl-b-methyl choline, procaine, methylene blue and other reversible inhibitors are competitive in the varying substrate experiments, but their displacement by substrate proceeds very rapidly. ... A curious paradox is presented by mercuric ion (chloride) which inhibits irreversibly (by dialysis) yet gives competitive, or partially competitive curves in the Lineweaver-Burk method — i.e., appears to be displaced immediately by acetylcholine. ... It may be inferred that while the carbamic esters described combine at the substrate-active center, mercuric ion does not ; but that combina- tion of substrate changes the affinity of the latter for the protein moiety of the enzyme. It is suggested that "competitive inhibition" need not imply competition with substrate for the same site of attachment to the enzyme. Complexes of hemocyanin and of hcmorythrin with small ions. I. M. KLOTZ * AND F. TlETZE. Previous investigations (Biol. Bull., 94: 40, 1948) have demonstrated that the serum of Limnlus polyphemus is capable of combining with small molecules other than those involved in its respiratory function. To establish that the binding is by the hemocyanin component of the serum, this protein has been isolated, and its ability to form complexes with several organic ions has been examined. Quantitative estimates of the binding of methyl orange by isoelectric hemocyanin have been obtained from equilibrium dialysis experiments. At dye concentrations as high as 7 X ICT5 molar, an average of about 0.1 mole of anion is bound by each unit (of 37,000 molecular weight) of the hemocyanin. This is of the same order of magnitude as was found for an equivalent quantity of this protein in the serum. Qualitative evidence of the binding of several other ions by hemocyanin has been obtained from spectrophotometric observations. Thus the spectrum of a mixture of Orange II and the protein differs from the sum of those for the pure components by a shift of 150 A in the region of the dye's peak near 4850A, as well as by an increased absorption in the 5800 A region of the chromoprotein. With salicylate ion, in turn, which has no spectrum in the visible region, only an increased absorption in the 5800 A band is observed. A small change in the absorption of solutions of the cationic dye, methylene blue, in the presence of hemocyanin has been found also, but its interpretation is doubtful, since the dye is aggregated, even at these low concentra- tions, and small shifts on addition of protein might be due merely to changes in the dielectric properties of the medium. Hemerythrin, isolated from a sample of serum of Phascolosoma gouldi kindly given to us by Dr. Ernest Baldwin, shows even more pronounced evidence of complex formation with small ions. Thus in dialysis experiments with the anionic dye, Orange II, an average of 2.5 moles of ion is bound, by each unit of 100,000 molecular weight, at a free dye concentration of slightly less than 1 X 10"4 molar. (The unit of 100,000 molecular weight was chosen arbitrarily, for purposes of comparison, since adequate data on this protein are not available.) Similarly, in spectrophoto- metric studies, the absorption peak at 5000 A is shifted over 500 A to shorter wave-lengths, and the optical density is doubled, on the addition of thiocyanate to the protein. Similar effects * Lalor Fellow. 276 PRESENTED AT MARINE BIOLOGICAL LABORATORY are obtained with other known iron-complexing agents such as cyanide or hydroxylamine hydrochloride. Thus it seems probable that many of the respiratory pigments can act as ion-transport agents, in addition to their primary function as oxygen carriers. Further studies on the mechanism of allo.van action; the reaction of all o. van with sulfhydryl groups ; the glntathione content of islet tissue. ARNOLD LAZAROW.* It has been suggested that alloxan produces diabetes because of inactivation of essential sulfhydryl enzymes and that the selectivity of alloxan for the beta cells may be due to a low glutathione content (Lazarow, A., Proc. Soc. Exp. Biol. and Mcd., 61: 441, 1946). Alloxan has been shown to react with glutathione and protein at pH 7.4 to give a new com- pound with an absorption spectra maximum at 305 mM [Lazarow et al., Science (in press)]. When p-Cl-Hg benzoate is added to a glutathione solution prior to the addition of alloxan, no "305" is formed. Since p-Cl-Hg benzoate is a specific sulfhydryl reagent, the failure of forma- tion of "305" indicates that alloxan is reacting with the sulfhydryl group of glutathione. These in vitro studies suggest the possibility of protecting animals against alloxan diabetes by injecting p-Cl-Hg benzoate prior to a diabetogenic dose of alloxan. Following the disappearance of the injected alloxan (spontaneous decomposition occurs at pH 7.4 with a half-life of 1 minute at 37° C.), it may be possible to reactivate the sulfhydryl groups by the addition of BAL, which re- moves the mercury compound. Determinations have been carried out on the glutathione content of the islet tissue of fish. The ferricyanide method of Mason was adapted for micro-analysis in final volumes of 0.5 cc. The principal islet of the goosefish (Lophius piscatorius) contains 27-58 mgs. glutathione/ 100 gms. tissue (average glutathione of 9 fish was 50 mgs./lOO gms.). This value is lower than the glutathione content of liver or kidney, and greater than that of 'muscle. Although the islet glutathione values represent the average glutathione content of the alpha, beta, and gamma cells, it may be possible to determine the glutathione content of the beta cells by indirect means [using the glutathione content of alloxan diabetic fish (alpha and gamma cells) and the percentage com- position of cell types]. The insulin content of the islet tissue of alloxan diabetic fish. ARNOLD LAZAROW * AND JACK BERMAN. Diabetes was produced in the toadfish by injecting alloxan subcutaneously in doses of 600 mgs./kg. body weight. Serial blood sugar determinations were carried out by the Folin- Malmros microblood sugar method. After the removal of the islet tissue, it was homogenized in ice-cold saline and serials dilutions were prepared. Doses, ranging from .05 to 1.0 mgs. of islet tissue, were injected into mice which had been starved for 24 hours (three animals were used for each dilution). Blood samples were drawn from the tail vein at 0 and 30 minutes after injection. Using the islet tissue of normal fish, the percentage drop in' blood sugar was found to be proporportional to the log of the injected islet dose. When liver homogenates or saline were injected into mice, under similar conditions, they resulted in an elevation of blood sugar. The islet tissue of alloxan diabetic fish (sugars greater than 400 mgs./lOO cc.), assayed 48 hours after the injection of alloxan, showed considerable quantities of a blood sugar lowering factor (insulin). (The injection of 0.1 mg. or less of islet tissue per mouse produced a 25-40 per cent drop in blood sugar.) Although the limited number of analyses carried out so far do not permit accurate com- parison between the insulin content of the islets of normal and diabetic fish, they do indicate that both may be of the same order of magnitude. The presence of sizable quantities of insulin in the islet tissue of alloxan diabetic fish raises the question as to whether there may be a dissociation between insulin storage and insulin secre- tion, or whether factors other than insulin deficiency may play a role in the etiology of the hyperglycemia which persists after alloxan injection. * Lalor Fellow. PRESENTED AT MARINE BIOLOGICAL LABORATORY 277 A New Concept of the Action of Dicumarol. JOSEPH LniN.1 * All previous work with Dicumarol has tended to confirm the view that the drug acts by decreasing the prothrombin content of the blood by preventing its synthesis in the liver. The evidence for this view has been obtained by determining clotting times of Dicumarol treated plasma using a protein thromboplastic agent. Such plasma shows a markedly increased clotting time. In addition to protein thromboplastic agents there is also a lipid thromboplastic agent which has a considerably lower clotting activity than the protein with normal plasma. Experiments were carried out in which the clotting times of rabbits treated with Dicumarol were determined with the lipid and protein thromboplastic agents. Three day treatment of rabbits with 10 mg. of Dicumarol per kg. per day caused a marked increase in the clotting time of rabbit plasma using the protein thromboplastic agent, while the clotting time obtained with the lipid agent increased but slightly. Thus, in one case the protein agent did not clot the plasma in over three hours while the lipid clotted it in five minutes. Com- parison of the protein thromboplastic agent clotting times with control clotting times indicates further that the protein thromboplastic agent acts as an inhibitor of the clotting of Dicumarol plasma. - Addition of highly purified prothrombin brings the relationships to those found in normal plasma. Since reduction of prothrombin concentration of plasma by physical methods of dilution or absorption on aluminum hydroxide does not reverse the relative thromboplastic activities of the lipid and protein agents, it is believed that Dicumarol treatment does not prevent the synthesis of prothrombin by the liver but causes the synthesis of an altered prothrombin. This altered prothrombin is not converted to thrombin by the action of a protein thromboplastic agent but is converted by the action of a lipid agent. Enzyme localization in the giant nerve fiber of the squid. B. LiBET.2 * Last summer it was found that the enzyme ATP-ase is localized almost exclusively in the sheath of the giant axon, with practically none in the axoplasm. This sheath ATP-ase showed a rate of activity even greater than that of squid muscle, and also certain other similarities to muscle ATP-ase. A further analysis of the properties of this nerve ATP-ase shows the fol- lowing: (a) The rate of activity is increased by increasing concentration of either Ca or Mg ions, up to a maximum, (b) The maximum activity is greater with Mg and occurs at a lower concentration (about 0.003 M for Mg is almost maximal ; about 0.03 M for Ca). (c) Mg stimu- lates at all K ion concentrations (up to 0.58 M), unlike the situation in muscle, where it inhibits at high K. (d) When Ca and Mg are added together there is antagonism, but in the reverse direction from that reported for muscle ATP-ase; i.e., Ca inhibits the Mg activation in nerve. Neither sheath nor axoplasm splits any other phosphate esters tested at an appreciable rate. /3-glycerophosphate is split at a very low rate even at pH 9.1 in the presence of Mg and glycine, so that "alkaline phosphatase" is very low. ATP-ase seems to be the dominant phosphatase present. An attempt was made to localize further the ATP-ase within the sheath itself by separating the axolemma from the rest of the sheath, but this has yielded inconclusive results thus far. Since the sheath of the giant nerve fiber contains a high percentage of connective tissue (C. T.), ATP-ase was determined in muscle-free C. T. samples taken from the mantle where the latter faces the dorsal side of the pen of the squid.3 This muscle-free C. T. showed an ATP-ase ac- tivity of about 7 Mg. P/mg. wet wt./30 min. at 27° C. (compared to a usual figure of about 15-20 for the axon sheath) ; it is activated by Ca++ and does not split ^-glycerophosphate or hexose-di-phosphate appreciably. If one could assume that the C. T. of the axon sheath has an activity identical with that of this muscle-free C. T., considerable activity would still be left for the non-C. T. elements. An attempt at further localization will be made histochemically by * Lalor Fellow. 1 Department of Zoology, Syracuse University, Syracuse, New York. 2 This work was also supported in part by the Navy Nervous System Research contract with the University of Chicago. 3 I am indebted to Dr. Magnus Olson for identifying the muscle-free C. T. Only a trace of muscle, that in small blood vessel walls, was found. 278 PRESENTED AT MARINE BIOLOGICAL LABORATORY Dr. G. Goniori. Obviously results obtained by others on the distribution of other enzymes be- tween sheath and axoplasm in the giant nerve fiber must also be re-examined in the light of these findings. The choline acetylase and cholinc csterase content of some invertebrate tissues. HAROLD PERSKY * AND MARCIA GOLD. The hypothesis that acetylcholine plays the primary role in the generation of the nervous impulse requires that the enzymes synthesizing and decomposing acetylcholine (ACh) be present in significant amounts in all animals possessing a differentiated nervous system. In order to test this premise, the choline acetylase (ChAc) and choline esterase (ChE) content of representa- tives of the three phyla with the most primitive nervous systems were determined. Tubularia crocca (Coelenterate), Euplanaria maculata (Platyhelminthes) and Ncanthcs vircns (Annelida) were the organisms studied. The entire organism was employed in the first two animals be- cause of the difficulties associated with the dissection of the nervous system in these species. The first segment of Ncanthcs was used because it contains the "brain." The methods of assay for both enzymes were essentially those of Nachmansohn. All the results are expressed as milli- grams acetylcholine formed or split per hour per hundred milligrams protein nitrogen (Q). The data is summarized in the following table. Species QChAc1 QchE1 Q-ChE/O-ChAc Protein nitrogen , Wet weight Tubularia crocea Euplanaria maculata Neanthes mrens 0.03(0.002) 23. (0.25) 20. (0.3) 180(1.3) 2000(20.) 280(4.2) 00 85 14 0.75 1.1 1.5 1 Figures in parentheses are the Q values expressed per 100 mg. wet weight. All data at 20° C. 4 It is apparent from the data that choline esterase is present in all three species in amounts equal or greater to that in rat brain (QChE of 210). Since the ChE of these two species is the nerve-muscle type (Nachmansohn), the concentration of ChE in the nervous tissue of these two species may be from 102 to 104 times that of vertebrate brain. The problem of why such large amounts of ChE are present in such relatively poorly developed nervous systems requires greater study. The choline acetylase determinations posed greater technical problems than the ChE assays. Acetylcholine synthesis has not yet been shown to be proportional to enzyme content in verte- brate brain or nerve or even highly, purified choline acetylase. However, by employing low tissue concentrations, short incubation periods and low temperatures, first order responses be- tween enzyme activity and enzyme content were obtained in the case of Euplanaria and Neanthes. No such proportionality was obtained in the case of Tubularia. In all cases, the absolute amount of acetylcholine synthesized was small. In spite of these shortcomings, the ChAc content of Euplanaria and Ncanthcs exceeded vertebrate brain. Tubularia showed little ChAc content despite its high ChE content. The ratio of ChE to ChAc showed no constancy for the three species ; rather the ratio progressively decreased as the nervous system became more highly organized. The problem as to whether this change is related to the functional state of the nervous system is worthy of further investigation. Report of Investigations, Summer 1948. AVRAM GOLDSTEIN * AND DORA B. GOLD- STEIN. I. Non-nerve cholinesterases. No reasonable function can as yet be attributed to the cholin- esterases found in the plasma and liver of higher animals. This project sought to isolate and * Lalor Fellow. PRESENTED AT MARINE BIOLOGICAL LABORATORY 279 study the properties of a cholinesterase in a system where it would be known to have a definite metabolic function — e.g. in microorganisms. Fermenting cabbage, cucumber and algae (such materials had been shown to contain bacteria which produce acetylcholine — M. Stephenson, /. Gen. Microbiol., 2, No. 1, 1948) were used as sources for the isolation by enrichment culture, of organisms which might utilize acetylcholine. Forty pure cultures were obtained (representing at least twelve different species) which grew in several passages through acetylcholine in min- eral medium. These include Gram-negative bacilli, yeasts and molds. To date, growth and respiration studies reveal that some (but not all) metabolize added acetylcholine, grow on choline (but not on ammonia plus acetate) and hydrolyze acetylcholine anaerobically. One organism has yielded an acetylcholine-splitting enzyme in acetone powder. This esterase has not yet been characterized but it is insensitive to prostigmine. The work will be continued during the coming year. II. Modified Warburg technique. Theoretical work was completed and experimentally con- firmed for a variable-volume use of the standard Warburg apparatus. The method eliminates the necessity of repeatedly levelling the manometer fluid, increases the capacity for measuring gas uptake or liberation, and makes possible automatic recording of the progress of reactions. III. Further kinetic studies on the mechanism of inhibition of human plasma cholinesterase. See Seminar, presented August 17, 1948. The incorporation of P3- into the nuclco proteins and phosphoproteins of developing Arbacia cmbr\os. C. A. VILLEE,* M. LOWENS, M. GORDON, E. LEONARD, AND A. RICH. The quantitative conversion of ribonucleic acid (RNA) to desoxyribonucleic acid (DNA) in the developing sea urchin embryo was postulated by Brachet (1933) on the basis of an in- crease in DNA and decrease in RNA during the first 40 hours of development. Schmidt (1948) reinvestigated this with more precise analytical methods and found an increase in DNA but no decrease in RNA. Sea urchin embryos obtained at stages from 3 to 72 hours of development and analyzed by the method of Schmidt and Thannhauser (1945) showed an increase in DNA with time whereas the RNA remained constant or increased slightly. The major source of the DNA formed cannot be the RNA of the unfertilized egg because when fertilized eggs are allowed to develop in sea water containing P32, a great amount of radio-phosphorus is incorporated into DNA, which would not be the case if it were derived from the P32-free RNA of the unfertilized egg. A comparison of the specific activities of the two fractions showed that for DNA to be formed from an RNA intermediate, less than 5 per cent of the RNA must be active in forming DNA and this small fraction must be synthesized and broken down completely more than four times in 24 hours, A number of chemicals which have been found to have effects on phosphate metabolism were tested for their effects on phosphate uptake and on nucleoprotein synthesis by comparing the specific activity of the fraction of the treated with that of the control animals grown at the same time and under the same conditions. 5 X 10"5 M dinitrophenol and 10~2 M malononitrile inhibited cleavage so that after 12 hours only 1 or 2 cell stages were present, whereas the con- trol embryos were free swimmers. Both 10~2 and 10"4 M malononitrile inhibited respiration to about 25 per cent of the control value. Uranyl nitrate and tris (/3 chloroethyl) amine hydro- chloride inhibited cleavage but not, at the concentrations used, as much as dinitrophenol. The table gives the effects of these substances on the incorporation of P32 into the several fractions after 12 hours of incubation. Dinitrophenol appears to inhibit the uptake of phosphorus by the cell (the acid-soluble phosphorus fraction is low) more than the synthesis of DNA and RNA. Low temperature, which inhibited cleavage completely, inhibited the uptake of phos- phorus into the acid-soluble fraction about as much as dinitrophenol, but inhibited the synthesis of nucleoproteins much more. Malononitrile inhibited the uptake of phosphorus into the acid- soluble fraction to a lesser extent but inhibited DNA and RNA synthesis markedly. RNA synthesis appears to be more sensitive to malononitrile than DNA synthesis, which suggests that DNA is not formed from an RNA intermediate. Uranyl nitrate reduces the uptake of phos- phorus and inhibits both DNA and RNA synthesis. The nitrogen mustard, tris (/3 chloroethyl) * Lalor Fellow. 280 PRESENTED AT MARINE BIOLOGICAL LABORATORY amine inhibited both DNA and RNA synthesis, DNA more than RNA, without reducing the uptake of phosphorus by the cell to any marked extent. TABLE 1 Inhibition of the incorporation of P3- into the phosphate fractions of 12 hour Arbacia embryos The figures in the table represent the values of the ratio: Specific activity (counts per minute per mg. P) of treated embryos Specific activity (counts per minute per mg. P) of control embryos Dinitro- Uranyl Nitrogen Cold Malononitrile phenol nitrate mustard 10-< M 10-= M 5X10^ M IQ-i M 10-' M 0° C. Acid soluble P .69 .49 .18 .49 .83 .16 Total acid insoluble P .68 .10 .32 .40 .56 .06 DNA phosphorus .55 .04 .10 .27 .23 .03 RNA phosphorus .21 .03 .24 .40 .44 .03 Phosphoprotein P .24 29 .16 .55 2.57 .23 Studies on nucleoproteins from marine invertebrates.'1 C. A. VILLEE,* E. LEONARD, AND A. RICH. The studies on marine invertebrate nucleoproteins begun last summer were continued. Nucleoproteins were extracted from squid testis, spermatophore sac, and sperm receptacle using 2M NaCl. Purified solutions of these have a high relative viscosity and show strong flow bire- fringence. Measurements of the flow birefringence were made in, the Mehl and Edsall appa- ratus. Changing the pH of these solutions to pH 3.6 or 8.5 did not decrease the viscosity or flow birefringence. Heating to 100° C. for 40 minutes did not decrease viscosity or bire- fringence but heating for 90 minutes at that temperature did destroy birefringence and decreased the viscosity markedly. There was no change in these properties on the addition of guanidine hydrochloride up to 20 mM/ml. solution, which suggests that these properties do not depend on the presence of free nucleic acid. Observations from the viscosity and flow birefringence data indicate that the nucleoproteins are present as markedly asymmetric particles. Using as a model an ellipsoid of revolution, calculations show a rotary diffusion constant of 1.5 and a particle length of about 10,000 Angstroms. It is possible that the nucleoprotein molecules are associated into larger particles under these conditions. Analyses by the Schmidt and Thannhauser method for ribonucleic acid, desoxyribonucleic acid, and phosphoprotein were made on squid testis, spermatophore sac and on squid optic ganglion. The effect of continued stimulation on the nucleoprotein content of squid optic ganglia was investigated. Nitrogen : phosphorus ratios were determined on nucleoproteins and nucleic acids prepared from squid, starfish, and sea urchin tissues. * Lalor Fellow. 1 Aided by a grant from the Ella Sachs Plotz Foundation. PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GENERAL PHYSIOLOGISTS The wave of negativity produced by acetylcholine conducted over an oil-saline inter- face. T. C. BARNES AND R. BEUTNER.1 The wave of negativity generated by acetylcholine at a phase-boundary has been found to travel from the point of application of the drug to the recording non-polarizable electrodes placed a meter or more distant. An oil-saline interface is essential for the transmission of this wave since it will not cross a saline bridge. The aqueous medium was 0.9 per cent sodium chloride. In some cases sodium lauryl sulfonate or sodium benzoate was used to increase the sensitivity of the oil surface to the alkaloid. The elongated interface consisted of a glass tube containing the two layers with openings at one end for the introduction of acetylcholine and at the other for the electrodes leading to the electroencephalograph. The addition of 0.1 cc. of saline containing 0.1 mg. acetylcholine at the "entrance" produced a negative wave of 3 millivolts lasting 0.15 sec. recorded at the "exit" 80 or more cm. away. The glass tube can be replaced by two contiguous wicks, one containing oil and the other saline. A single wick of oil or a single wick of saline will not transmit the acetylcholine wave. The mechanism of transmission is being investigated. Controls of distilled water or saline applied to this artificial nerve do not produce the wave. An electrogenic organic base (acetylcholine mecholyl or thiamine) is essen- tial to establish the initial phase boundary potential. It will be noted that no "membrane" poten- tial is necessary for initiation or transmission of the wave of negativity. The acetylcholine potential was originally set up as a permanent "base-line shift" potential (Beutner and Barnes, Science, 104: 569, 1946), later developed into a wave (Barnes, Federation Proceedings 6: 73, 1947), and the experiments reported here describe the transmission of the wave. It is possible that nerve impulses and brain-waves are produced by a similar mechanism. Apyrase activity of invertebrate marine muscle. ARTHUR COHEN 2 (introduced b\< H. B. Steinbach) (by invitation). READ BY TITLE. A comparative study was made of the rates of hydrolysis of adenosine-triphosphate by un- purified homogenates of various marine invertebrate muscles. The results of these tests are shown in the table below in which the values are expressd as micrograms of phosphorus liberated from ATP by 1 mg. of fresh muscle in 5 and 15 minute incubation periods. No significant rela- tionship between smooth and striated muscles, the nitrogen content of the muscles, or their activity, could be derived from the wide range of enzyme activity. In each assay of enzyme activity approximately 0.5 g. of freshly excised muscle was homogenized in 5 cc. ice cold dis- tilled water and 0.1 cc. aliquot taken for analysis. The calcium salt of ATP was used with final concentration of 0.0015 M. The labile 7' P of the ATP was at least double the concen- tration of the P split by the enzyme at all times during the experiment. The pH of the medium was equal to 7.4 employing a veronal acetate buffer. The phosphate determinations were made by the Fiske-Subbarow method. For total nitrogen determinations the modified micro-Kjeldahl method of Ma and Zuazaga was followed. Incubation temperature of the enzyme mixture was 22° C. Muscle M P/mg./5 min. H P/mg./15 min. fj. Total N/mg. Thyone (retractor) 0.1 0.34 18.0 Phascolosoma (retractor) 0.5 . 0.90 21.2 Limulns (tail) 0.62 1.50 28.2 Pagurus (abdominal) 2.8 4.20 29.6 Pecten (adductor) 2.8 4.73 22.6 Mya arenaria (mantle) 3.6 5.88 29.5 Loligo Pealii (fin) 4.7 9.00 25.3 1 Department of Pharmacology, Hahnemann Medical College. - University of Minnesota. 281 282 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS On the nature of iron binding by siderophilin, conalbumin, hydroxylamine, asper- gillic add, and related hydro.raniic acids* SILVIO FIALA AND DEAN BuRK.1 Aspergillic acid, hydroxyaspergillic acid, N-hydroxy, 4-methylpyridene-2, N-hydroxy, 5- bromopyridene-2, N-hydroxy, 4,6-dimethylpyrimidene-2, and hydroxylamine have been found to bind ferric iron under appropriate conditions to yield complexes with the same absorption spec- trum maximum (460-465 IBM) and order of extinction at this maximum (0.025-0.05 per cm. at microgram complex-Fe per ml.) as do the specific salmon-pink complexes of iron with conal- bumin of egg white (Schade and Caroline, Science, 100: 14-15, 1944) and of iron with sideroph- ilin, the iron-binding j8i-pseudoglobulin of human plasma (Schade and Caroline, Science, 104: 340-341, 1946; Helmberg and Laurell, Acta Chcm. Scand., 1: 940-950, 1947; Surgenor, Strong, and Koechlin, /. Clin. Invest., in press; Schade, Reinhart, and Levy, Arch. Biochem., in press). Carbon dioxide (bicarbonate) is required to obtain the salmon-pink complex with iron and hydroxylamine, as Schade, Reinhart, and Levy first found for the iron complexes of conalbumin and siderophilin, the stoichiometric ratio of CO, to Fe in the latter being 1:1. With aspergillic acid and the related cyclic compounds listed, however, no carbon dioxide is required, consistent with the existence already of a carbonyl group adjacent to a hydroxylated nitrogen atom in the cyclic hydroxamic acid grouping that Dutcher (/. Biol. Chem., 171 : 341-353, 1947) has indi- cated to be responsible for iron binding. The ratio of C^O to Fe in the salmon-pink aspergillic acid iron compound is 1:1. Hydroxyl ion is also required to form the salmon-pink complexes of siderophilin and conal- bumin, but it is not yet required by hydroxylamine and the cyclic hydroxamic acids listed, which already contain OH groups attached to nitrogen. The shape of the titration curve of the salmon-pink siderophilin complex, in the presence of buffer such as phosphate or citrate, indi- cates a ratio of OH" to Fe of 1 : 1. The grouping in the siderophilin, conalbumin and other complexes yielding an absorption -c=cx \p spectrum maximum in the region 460 mM might thus be represented as _ _ / The spe- H cificity of the two proteins as compared with other proteins tends further to rule out primary involvement of the more common sulfhydryl and amino groups, which might otherwise be sus- pected of iron combination. Physiological implications will be discussed. The cytochrome system in relation to diapause and development in the Cecropia silk- worm.^ CARROLL M. WILLIAMS AND RICHARD C. SANBORN.2 As soon as the pupa is formed, the metamorphosis of the Cecropia silkworm is interrupted by a prolonged period of diapause, which, at room temperature, continues for not less than five months. During this period the animal persists in a state of developmental standstill. The termination of dormancy is under overall control, being evoked by a combination of two internal factors. One of these arises from the pupal brain, the other from the prothoracic glands. As a working hypothesis we may assume the existence of a biochemical defect in the tissues of the diapausing pupa that prevents their metabolism from contributing to morphogenesis. From this point of view, the brain and prothoracic glands should preside over some synthetic reaction whereby the tissues repair this defect. Certain properties of the metabolism during dormancy and during development indicate that the biochemical defect of diapause may involve iron-catalyzed oxidations. Thus, during diapause, the oxygen consumption is essentially insensitive to cyanide. But when the brain and prothoracic glands function to terminate diapause, the oxygen consumption increases and a progressively * The siderophilin (plasma fraction IV-7) employed in this study was kindly supplied by Professor E. J. Cohn and Dr. Douglas Surgenor, Department of Physical Chemistry, Harvard Medical School, the conalbumin by Dr. H. L. Fevold, Western Regional Research Laboratory, and the aspergillic acid, hydroxyaspergillic' acid, and related hydroxamic acids by Drs. J. D. Dutcher, O. Wintersteiner, and W. A. Lott of the Squibb Institute for Medical Research. 1 National Cancer Institute, National Institutes of Health, Bethesda, Maryland. f This study was aided by the Lalor Foundation. 2 The Biological Laboratories, Harvard University. PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 283 larger fraction becomes cyanide sensitive. Consequently, development seems to involve a pro- gressively larger utilization of the cytochrome system. This possibility has been tested by spectrophotometric assay of cytochrome C and manometric assay of cytochrome oxidase. A striking correlation was found between the titer of these en- zymes and the progress of adult development. Thus, cytochrome C is virtually absent from the diapausing pupa. During the period when development is dependent on the prothoracic glands, the concentration of cytochrome C increases from less than 1 to more than 50 gamma per gram live weight. Similarly, during the period of the brain's secretory activity, the titer of cytochrome oxidase increases from approximately 40 to nearly 700 units. Two correlations seem to emerge ; namely, that between the synthesis of cytochrome oxidase and the function of the brain, and that between the synthesis of cytochrome C and the function of the prothoracic glands. As a result of the combined functions of both the brain and protho- racic glands, the tissues of the dormant pupa, for the first time, come into possession of a com- plete cytochrome system. The relation of hcparin to protoplasmic clotting. L. V. HEILBRUNN AND W. L. WILSON. An essential part of the present-day theory concerning the colloidal behavior of protoplasm is the fact that the gelation of protoplasm is in many ways similar to the clotting of blood. The changes which protoplasm undergoes during its normal activities and as a result of drug action are frequently to be explained in terms of a clotting reaction similar to that which occurs in blood. Additional evidence in support of this point of view is the fact that at least in some cells heparin can prevent protoplasmic gelation. In the egg of the worm Chaetopterus, there is a gelation which precedes the appearance of the mitotic spindle. If eggs are exposed to dilute solutions of heparin and then fertilized, the mitotic gelation is inhibited and for the most part the eggs do not divide. In order to show this, it is necessary to inseminate the eggs with high concentrations of sperm, for the presence of heparin tends to inhibit fertilization by weaker sperm concentrations. The effect of heparin on cell division is reversible. The fact that heparin can actually prevent mitosis is perhaps of some significance in the interpretation of the effect of radiation in the treatment of cancer, for following irradiation of animals, there is a marked increase in the amount of heparin in the blood stream. Moreover, heparin may be an important factor in the control of protoplasmic clotting in various other types of cell activity. Enzyme activity and radiation sensitivity of enzyme-substrate films* DANIEL MAZIA AND GERTRUDE BLUMENTHAL.1 In a previous investigation (Mazia, Hayashi, and Yudowitch, Cold Spring Harbor Symp., 1947) the enzyme activity of mixed surface films of pepsin and albumin has been described. In all such experiments it has been difficult to exclude the possibility that the measured activity was not that of surface-spread enzyme molecules but of unspread molecules trapped in or adsorbed on the film. It has been found (Mazia and Blumenthal, P. N. A. S., 34: 328, 1948) that these enzyme-substrate films are extraordinarily sensitive to radiation, and a study of the relation between activity, surface pressure, and radiation sensitivity throws light not only on the mecha- nism of radiation sensitivity but also on the question of the activity of surface-spread enzyme. Methods are described in the publications cited. Surface pressure was measured by the vertical-pull balance. The activity of the films depends on the pressure against which they are spread. Films spread to zero pressure become inactive. Activity increases with initial pressure up to about 30 dynes/cm. There is some evidence of reversibility. The radiation-sensitivity is a function of the surface pressure. At a given dose, there is a discontinuous relationship be- tween inactivation and pressure. Dose-inactivation curves at various pressures yield a family of sigmoid curves which show that the effect of pressure is primarily on the "threshold" of the dose- effect relationship. A plot of pressure against dose for 50 per cent inactivation yields a straight line. The 50 per cent dose at 7 dynes/cm, is about 100 r; at 35 dynes/cm, it is 500 r. The * Work supported by National Research Committee on Growth acting for the American Cancer Society. 1 Department of Zoology,University of Missouri. 284 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS pressure effect on sensitivity is reversible. Sensitivity depends on the pressure at the time of radiation and is indpendent of previous pressure changes provided the pressure has not been permitted to fall to zero. Since not only the activity but also radiation-inactivation is a func- tion of surface pressure it is concluded ( 1 ) that the spreading of proteins at an air-water interface ("surface denaturation") is not necessarily an all-or-none phenomenon, but may be limited by the pressure against which the protein spread and (2) that if the unfolding of the molecule is not permitted to go to completion (i.e. to zero pressure) the enzyme activity of the protein may be preserved. A photo synthetic intermediate. A. H. BROWN, E. W. FAGER AND H. An intermediate of photosynthesis is by definition a substance which is transformed in one or more photochemical steps into the final product. This final product of photosynthesis must have the reduction level of a carbohydrate and be insensitive to further irradiation. The dis- covery of the dark fixation reactions in which free carbon dioxide becomes a carboxyl group in an organic molecule without the aid of light made it very probable that the first photosensitive intermediate in the course of photosynthesis is formed by a typical dark fixation and thus may be related to compounds which normally serve as intermediates of respiration. Consequently many biochemists expected photosynthesis to be revealed as a process in which intermediates known from the breakdown of carbohydrates are built up in steps which literally reverse the course of respiration. Benson and Calvin have reported that in studying photosynthesis with radioactive carbon as a tracer they have obtained labeled dicarboxylic acids, amino acids and very considerable quantities of glyceric acid and of glyceraldehyde. According to these authors, these substances have been formed by the direct photosynthetic reduction of carbon dioxide, that is, neither by the breakdown of a previously synthesized carbohydrate nor by way of an ordinary dark fixation. The intimate relationship of respiration and photosynthesis as well as the pathway of the latter process thus seems to have been conveniently established. In the course of our investigations with carbon 14 we could confirm most of the early obser- vations of Ruben, Kamen and Hassid, but could not find any indication that the substances men- tioned by Calvin et al. are present as intermediates of photosynthesis. Even in the case where these authors adopted the technique of short exposures and found glyceric acid and glyceralde- hyde as main products, the discrepancy persists. Under the same conditions we obtain 90 per cent of the assimilated carbon in one chemical fraction of the plant. This fraction contains the carbon in a photosensitive substance (or group of nearly related substances) which is not identi- cal with any one of the substances usually shown in the schemes representing the metabolism of carbohydrates. Labeled glyceraldehyde is absent, and glyceric acid is present, if at all, only in small amounts, which probably originate from secondary processes. The intermediate is ob- tained as a thermally and chemically very stable though impure syrup or its hygroscopic sodium or barium salts. It is easily adsorbed on all kinds of precipitates when these are produced in the solution. Its most interesting characteristic is its content of aromatic nitrogen. The ultra- violet spectrum is very similar to that of uracil, though this particular substance is not present. It seems that the radioactive carbon assimilated during the first moments of photosynthesis and a pyrimidine compound are closely associated. The chemical properties which set our photo- synthetic substance apart from the better known simple metabolites are paralleled by its behavior in the living cell. Instead of being rapidly transformed in the dark like any respiratory inter- mediate it is stable against attack by respiration. It also does not readily exchange its labeled carbon with free carbon dioxide, as it should if it were a primary dark fixation product. But the labeled carbon begins immediately to appear in other chemical fractions when the plant is exposed to light. These results do not support the idea that the photochemical reduction of carbon dioxide proceeds by a simple reversion of each step in the breakdown of carbohydrates. Synthesis reactions with acetic acid in isolated bone 'marro^v. RICHARD ABRAMS, J. M. GOLDINGER, AND E. S. G. BARRON. The metabolic activity of bone marrow has been the subject of much interest because of its role in blood cell production. It has been shown by Thorell (1947) that ribonucleic acid is 1 University of Chicago, Chicago, Illinois. PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 285 associated with protein formation in the proliferating marrow; by Goldinger, Lipton, and Barren (1947) that marrow can oxidatively utilize acetate, and by Buchanan, Sonne, and Delluva (1947) that acetate carboxyl can be a purine precoursor (uric acid in pigeons). Accordingly, we have incubated rabbit bone marrow slices and homogenates with acetate containing C14 in the car- boxyl group in an attempt to find an in vitro system for studying nucleic acid synthesis, as well as to survey the types of synthetic reactions in which acetate is involved. A small, but definite, incorporation of C14 was found in both ribonucleic and desoxyribonucleic acids. In addition there was observed a relatively large turnover in the phospholipid and protein fractions. In a typical experiment, rabbit marrow slices were incubated for 5 hours at 37° in Ringer- bicarbonate containing CH3C"OONa. Taking 100 as the specific activity of the substrate, the relative specific activities of the subsequently isolated fractions were: ribonucleic acid (RNA), 0.035; desoxyribonucleic acid (DNA), 0.014; CO. liberated from hydrolyzed protein by nin- hydrin, 0.61 ; lecithin, 0.90; and fatty acids from saponified fat, 0.10. A control experiment with C"O2 indicated that the startlingly high rate of protein formation from a fatty acid (as well as phospholipid synthesis) did not involve CO2 as an intermediate. The rate of incorporation of C14 was measured aerobically with slices and both aerobically and anaerobically with homogenates. With slices, there was a linear increase of specific activity with time in the DNA and amino acid carboxyl fractions, while the RNA activity, though reach- ing a level 3 to 4 times that of DNA, did so at a continuously diminishing rate. Homogenization caused a marked reduction in the rates of uptake of C14 by RNA and by protein. That respira- tion is necessary to furnish the energy for these synthetic processes is indicated by the fact that anaerobically C14 appeared in neither the nucleic acids nor the proteins. Thcnnodynainic theory of the contraction of actomyosin. A. SzENT-GvoRGYi.1 It has been shown in the speaker's laboratory that the contractile matter of muscle is built of a complex protein, actomyosin, composed of myosin and actin (F. B. Straub). Actomyosin contracts in a proper ionic milieu under influence of ATP. Evidence was obtained showing that the contractile matter is built of small functional units each of which consists of a certain amount of myosin, actin and one molecule of ATP. These units will be called "autones." Experiments indicated that these autones have but two stable states, the fully relaxed and fully contracted state ; that contraction is an all-or-none process ; and that it is an equilibrium-process, dependent on temperature. The equilibrium-constant was measured at different temperatures and free-energy changes calculated. It has been found that the AF, i.e. the free-energy spent by the single autone in contraction, rises with increasing temperature. In the rabbit it is 0 at about 0° C. and reaches 11,000 cal. at 53° C. In the frog-muscle, extracted with water, these values are reached a few degrees lower. The first question is whether this AF curve is correct. The theory involves that the system uses its own F in contraction and extraneous energy is needed for relaxation. The extraneous source of energy is the high-energy phosphate of ATP which has 11,000 cals. of F. It follows that the muscle has to go over permanently into the contracted state at the temperature where the F-expenditure reaches or exceeds 11,000 cal., which is 53° in the rabbit and 47° in the frog. This was actually found to be the case. In order to obtain further information about the correctness of the theory the total amount of work was measured in isometric and isotonic contraction. It was found that the curve of total work, if calculated for 35,000 gm. of myosin, agrees very closely with the AF curve. This is the case in isotonic contraction and in isometric contraction up to the point where the work corresponds to 5500 cal., % of 11,000. From this point on the tension developed remains con- stant and does not rise with increasing temperature. The thermodynamic reversibility of contraction could clearly be demonstrated. Having thus obtained evidence for the basic correctness of the theory an attempt was made to extend it. If we suppose that actomyosin, in absence of ATP, does not contract because the F of the contracted state is equal with that of the relaxed state, Fc — Fr, then contraction takes place with ATP because this latter makes FT < Fc which pushes the system towards a new equilibrium with a greater number of contracted units. Such assumptions can be tested by deriving their consequences and checking them in the experiment. 1 Marine Biological Laboratory, Woods Hole, Mass., and the National Institute of Health, Bethesda, Md. 286 PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS Fc — FT involves an equilibrium constant of 1 which means that at the temperature in ques- tion there is one contracted unit for every relaxed one, even in rest. The contracted units, being folded, can be expected to be highly elastic, the relaxed ones to be inelastic. The relative length of units in the folded and relaxed state is 1:7. The limit of elastic extensibility is reached thus when the folded units are fully extended which is reached at 175 per cent of the equilibrium length. The experiment showed that this is actually the case. The muscle is highly elastic only while it contains its normal amount of ATP. If this is de- composed the muscle becomes inelastic. That the change is actually due to ATP can be shown by adding ATP to the inelastic washed muscle fibers which, under influence of ATP, become highly elastic again. The elasticity of fresh muscle proves that ATP is present in an active form, linked to actomyosin. If the fully contracted autone is stretched it offers in the beginning little resistance. Then the resistance becomes proportional to stretching (Hooke region) and in the end tension exceeds stretching. If the uncontracted units contract in an isometric contraction the contracted units are stretched. If the muscle would be at equilibrium-length it could in the beginning develop no tension. Rest-length means a stretch of these units which brings the muscle into the Hooke region. The maximum of tension is reached when all units are half-extended and tension is one-half the maximum theoretical value as has actually been found in the experiment. No higher tension could be developed by the muscle without its permanent damage. If this one-half tension is exceeded, slipping begins, and if the full tension is reached, the muscle tears. The size of the autones can be approached in different ways. All observations indicate that it contains 18,000 gm. myosin, one molecule of ATP and 6000 gm. of actin. The actin and myosin in this unit are linked by one SH bridge. It is not impossible that these single autones also form higher units, containing one molecule actin of MW 70,000 gm. and thrice that amount of myosin. Usnic acid, an antibiotic, and sperm metabolism. LEONARD NELSON, by invitation. READ BY TITLE. The effect of Usnic acid on the respiration of Arbacia sperm was determined manometrically. This antibiotic had previously been shown to inhibit cleavage and P32 uptake in fertilized eggs of the same species, but had little effect upon their oxygen consumption (Marshak and Harting, J. C. C. P., 1948). However, the same concentration of Usnic acid pronouncedly affected the sperm respiration. This effect was found to be dependent upon the density of the sperm sus- pension. The sperm concentration was measured spectrophotometrically — the optical density being directly proportional to the logarithm of the number of sperm. A concentration of 1 mg. of Usnic acid per 100 cc. of 0.01 per cent gelatin-sea water was employed throughout, while the sperm concentration was varied from 100,000 to 1,000,000 sperm per cubic millimeter. At 25° C., the respiration was completely inhibited in the low concentra- tions and in the intermediate and high concentrations there was a marked increase in oxygen uptake. Per cent control Sperm count Sperm No./c.mm. 30 min. 90 min. 180 min. 100,000 0 0 0 180,000 100 0 0 250,000 470 375 280 410,000 565 590 660 1,120,000 470 500 500 Preliminary observations seem to show that these effects are not attributable to change in motility inasmuch as after a given interval, treated and control sperm had moved an equal dis- tance in capillary tubes. PRESENTED AT THE SOCIETY OF GENERAL PHYSIOLOGISTS 287 These experiments seem to indicate that some energetic process apparently independent of sperm motility and distinct from oxidative cycles in the egg is being affected. Similar results were obtained with Asterias sperm. Temperature coefficients of Apyrase systems from muscles of different animals. H. BURR STEINBACH. READ BY TITLE. The Apyrase activities of unfractionated homogenates of muscle were determined at various temperatures within the range zero to thirty degrees C. Reaction mixtures of 2 ml. volume contained 0.05 M barbital buffer, pH 7.4, ATP (as the Ca salt), 2X 10"4 M as well as the enzyme preparation. Checks with some of the tissues with glycerophosphate as substrate showed negligible activity toward this simple ester. Ten minutes reaction time was used, the reaction being stopped by adding trichloracetic acid to a final concentration of 5 per cent. Logarithms of rates at different temperatures plotted against either degrees C. (for Q]0 cal- culations) or the reciprocal of the absolute temperature approximated straight lines with a few instances which might be interpreted as breaks at critical temperatures near 15° C. Average results for QK> were as follows: for fi.sh (Cliuostonius elongatns and Lcpomis macrochiris} 1.4 to 1.6, frog (Rana pif>icns) 1.7, mouse 1.8 to 2.0, bird (Passer domesticus) 2.1 to 2.2, and turtle (Chryscmys marginata) 2.1 to 2.2. Further study would be necessary before concluding that these are real genetic differences between the animal groups. Preliminary experiments indi- cated that the Qio values bore no particular relationship to the environmental temperatures of the animals since fish kept at 22° C. for one month gave preparations having the same Qio as similar fish kept at 0 to 5° C. for a similar period. Likewise, tropical fish (Xiphophorus hellcrli Heckel) had temperature coefficients nearly like those of cold water minnows. A few observations of apyrase activity of brain homogenates of fish, mouse and frog showed uniformly low Qio values of about 1.4. Studies of the kinetics of potassium exchange bctzvecn cells and plasma of canine blood in -vitro using K*-. C. W. SHEPPARD AND W. R. MARTINA Freshly drawn heparinized canine whole blood is equilibrated in vitro (paraffin lined vessels) at 38.1° C. with a normal pulmonary atmosphere saturated with water vapor. It is then tagged by mixing with a small amount of plasma containing dextrose and radioactive KC1. The potas- sium content of the resulting plasma is thus raised by not more than 3 per cent of the normal and the blood sugar to about 400 mg. per cent. By this procedure cells are maintained in a healthy state for periods up to 10 hours as shown by minimal hemolysis, nearly constant hemato- crit and minimal potassium leakage. In whole blood the activity of the plasma decreases initially at a rapid rate which is corre- lated with the appearance of activity in the cells (red cells, white cells, and platelets). The initial decrease follows an exponential curve to an elevated base line, half of this change being complete in about 50 minutes. However, if the white cells and platelets be previously removed by repeated differential centrifugation the behavior is different. The plasma activity declines very slowly, at the rate of about 1 per cent per hour. The initial rapid decrease in plasma activity for whole blood is attributed to a rapid ex- change of potassium in the fraction containing the white cells and platelets. A typical value for the amount of this easily exchangeable potassium is about 30 per cent of the total potassium in the plasma. It is evident that studies of potassium exchange of erythrocytes must give equivocal results unless the white cells and platelets are removed in advance or otherwise taken into account. 1 Oak Ridge National Laboratory, Oak Ridge, Tennessee. Vol. 95, No. 3 December, 1948 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE LIFE CYCLE OF ASELOMARIS MICHAELI, A NEW GYMNOBLASTIC HYDROID N. J. BERRILL McGill University, Montreal The following account is of a hydroid not previously recorded, from the Atlantic coast of North America. It appears to be a form related to Rhizorhagium as redefined by Rees (1938), though not close enough to be included in that genus without a broadening of the definition and a reconsideration of the genera Garveia and Bimeria. Its closest relative appears to be Atractylis arenosa Alder (1862). According to Totton (1930), however, Atractylis is sunk in the synonymy of Bougainvillia, and A. arenosa is in any case in need of a new generic name. It is therefore proposed that the Atractylis arenosa of Alder become Aseloinaris arenosa, and that the hydroid described here, which lacks the striking pseudohydrotheca and gelatinous perisarc of A. arenosa, be known as Aselomaris michaeli. This is in ac- cordance with the views expressed by Rees and Totton (in personal correspondence). The genus Aselomaris is consequently defined as follows : bougainvillid hydroids with hydranths arisng singly from creeping stolons, with gonophores reduced to sporosacs and arising from the hydranth stalk, not from the stolons. The present species was found throughout the general region of Boothbay Harbor, Maine, attached to the sides of floats, occasionally on Fucus fronds, in widely separated localities, namely, Lobster Cove, Townsend Gut, and the Town wharves. In every place it was associated with Bougainvillia superciliaris which it resembles in some respects, such as color, size and form of the hydranths. Concerning its distribution, either it is an extremely local species or it has been extensively overlooked elsewhere. While a shallow water form, it is un- doubtedly most inconspicuous. At the same time it should have been found if it occurred in the intensively collected Woods Hole region. Collections to the north have been more sporadic and its presence is therefore in doubt. It is quite pos- sible that this is a northern species extending down to but not south of Cape Cod. ASELOMARIS MICHAELI This hydroid forms an encrusting mat often several centimetres square but with very little height. In fact most of the specimens obtained were collected only by shaving off the wood to which they were attached. The general nature of the hy- dranths is shown in Figure 1A. The hydranth grows vertically from the creeping stolon, the perisarc stopping short just above the base of the hydranth proper. It 289 290 N. J. BERRILL FIGURE 1. Aselomaris michaeli. A, large and small hydranths arising from the creeping hydrorhiza. B, somewhat older hydranth, at reduced scale, showing developing gonophores, gonophore with developing eggs and gonophore with planulae. C, maximum length hydranth, at same scale as B, showing succession of gonophores from distal juveniles to proximal stalk remnants, e, eggs; g, gonophore; gs, old gonophore stalk; p, planula ; r, hydrorhiza. LIFE CYCLE OF HYDROID ASELOMARIS 291 lias the form typical of the Bougainvillidae, with a conical manubrium and a single whorl of filiform tentacles. The tentacles are characteristically directed distally, laterally and proximally with virtually the same number in each category (Fig. 1A). New hydranths grow only from the creeping stolon. Gonophore buds grow laterally from the base of the hydranth immediately proximal to the junction of naked and perisarc-covered coenosarc. This is a region of growth in a double sense. Not only are gonophores initiated, but the hydranth stalk grows in length, in effect pushing the growing gonophores down the stalk ; that is, the new stalk that is pro- gressively added proximally secretes chitinous perisarc and carries with it the grow- ing gonophore, while new material and new gonophores are successively added be- tween the first formed gonophore and the hydranth base. Thus we get a continu- ally lengthening stalk bearing a series of gonophores, youngest nearest the base of the hydranth and oldest near the junction of the stalk and hydrorhiza (Fig. 1C). No branches are given off and the hydrorhiza forms a closely applied creeping stolon. The only outgrowths are the buds arising from the hydrorhiza that de- velop directly into hydranths ; that is, gonophores do not develop directly from the hydrorhiza, but only single hydranths; whereas hydranths only rarely form as lateral branches of the stalk, in place of a gonophore. In an old hydranth as many as fifteen gonophores may be present, the youngest near the hydranth base being in early stages of devolpment, those in the middle zone being close to or actually functional, and those near the base of the stalk being represented mainly by stumps within a persisting perisarc. FEMALE GONOPHORES Gonophores first appear at the base of the hydranth at the region where the hy- pertrophied gastrodermis of the hydranth thins down to that characteristic of the coenosarc. They arise one at a time as a two-layered protrusion of the body wall. The entocodon, which is apparently of epidermal origin, arises relatively early. By the time the next in series is starting to form, a given gonophore has its entocodon differentiated almost entirely into about eight or ten oocytes. The more opaque endodermal component becomes somewhat pointed at the center, foreshadowing the spadix (Fig. 2B). With further growth the spadix forms a high cone, reaching to the distal epidermis of the gonophore (Fig. 2C). At its maximum size (Fig. 2D), the oocytes are full grown with the germinal vesicle of each lying in the part farthest away from the spadix. The gonophore stalk is greatly lengthened and is about twice as long as the diameter of the gonophore proper. Rhythmical move- ments or a writhing of the gonophore commences at this stage, at least under the conditions of microscopic examination. The contained eggs change shape with the movements and may appear themselves to be responsible for them, but a close ex- amination reveals the activity to reside in the layer of the gonophore wall immedi- ately beneath the epidermis and derived from the entocodon. In mature gonophores the writhing movements and contractions of the wall culminate in its rupture distally (Fig. 2E). This rupture has two consequences. Distally, where the cells become greatly stretched and thinned out, rupture results mainly in their disaggregation, so that instead of withdrawing as a sheet they remain as isolated spherical cells scattered over the sticky surface of the ripe eggs. The proximal part of the wall 292 N. J. BERRILL however does withdraw towards the base of the spadix, leaving the latter naked and still holding on to the eggs by adhesion. At the same time the epidermis, with its tension now released, contracts down the stalk, and where the stalk narrows the epidermis becomes somewhat thickened and wrinkled (Fig. 2F, G). The eggs are fertilized and undergo their cleavage and development up to the planula stage ad- FIGURE 2. Female gonophores. A, young gonophore before appearance of entocodon. B, after segregation of ova from entocodon. C, D, fully formed gonophores with large ova and well developed spadix and long stalk. E, distal disintegration and rupture of epidermis, and basal epidermal contraction. F, G, matured gonophores with fully retracted epidermis, bearing fertilized eggs attached to the denuded spadix. e, contracted epidermis ; nsp, naked spadix ; o, ova ; sp, spadix. hering to the gonophore spadix, remaining there until able to move away as a result of their own efforts. Planulae on the point of departure are shown in Figure IB, C and Figure 4A. Throughout their development on the spadix, an internal active hydroplasmic streaming is maintained within the lumen of the spadix. Whether of course this streaming is of any value to the developing eggs is difficult to determine. LIFE CYCLE OF HYDROID ASELOMARIS MALE GONOPHORES 293 The male gonophores develop as do the female, and have essentially the same appearance. Early stages showing the entocodon and the formation of the spadix are shown in Figure 3 A and B. The lumen of the spadix alternately expands and B sp FIGURE 3. Male gonophores. A, B, young stage showing formation of entocodon and germ mass. C, later stage with arrows indicating expansion-contraction amplitude of spadix. D, late stage with germ cells present as spermatids, and broken lines indicating stalk diameter when dilated. E, ripe gonophore with active spermatozoa and expanding and contracting distal end. F, emission of spermatozoa, e, entocodon; sp, spadix. contracts in a regular manner, with an amplitude indicated by the arrows in Figure 3C. In the later stages (Fig. 3D) the dilatation and contraction is more obvious in the short stalk of the gonophore, the growing mass of male germ cells possibly inhibiting or at least reducing the freedom of movement of the spadix wall itself. 294 N. J. BERRILL With the attainment of full size, not only is the stalk relatively long, but the distal part of the gonophore also elongated. This is due on the one hand to the contractile property of the wall of the mature gonophore, and on the other to the pseudo-fluid quality of the mass of ripe germ cells. There is a rhythmical contrac- tion of the gonophore wall similar to that of the female gonophore, but here result- ing in an alternation between the stages indicated in Figure 3E. Finally the con- tractions culminate in rupture at the extreme distal pointed end, and the consequent escape of mature spermatozoa, shown escaping in Figure 3F. LIBERATION OF EGGS AND SPERM Both mature eggs and spermatozoa are liberated in essentially the same way, even though the eggs are not actually set free in the process. Rhythmical contrac- tions of the gonophore wall result in its rupture distally. The contractions in each case are due to the activity of the tissue immediately subjacent to the epidermis, which must be regarded as homologous with the muscle layer of the free-swimming medusae of species of Bougainvillia. Spawning in hydroids is usually associated with dawn (Lowe, 1926) or dusk, and not with darkness. Yet most of the ripe gonophores were examined at night and most of them ruptured within five minutes of first bt;tig exposed to the micro- B D FIGURE 4. Development of planula. A, planulae about to swim away from gonophore. B, free-swimming planula. C, planula after about 24 hours, changing shape and losing ciliation. D, 12 hours after attachment, with stolon and 4-tentacled hydranth. E, two individuals 24 hours after attachment, with bipolar stolon on hydrorhiza and 8-tentackd hydranth. g, gono- phore ; p, planula. LIFE CYCLE OF HYDROID ASELOMARIS 295 scope light. It appears probable therefore that the stimulus of light evokes the contractions of the muscle layer, and spawning inevitably follows. SETTLING OF THE PLANULA Planulae escape from their adhesion to the spadix only as they become ciliated and active. A set about to launch forth is shown in Figure 4A. A planula swims for about 24 hours as a ciliate organism, and then becomes progressively pear- shaped (Fig. 4C), at the same time 'resorbing the external coat of cilia. About 12 hours after settling, a hydranth and stolon are already differentiated (Fig. 4D), four tentacles emerging in the first place. Twenty-four hours after settling, four intermediate tentacles are usually well formed, or a total of eight, while stolonic growth is bipolar, growing in opposite directions from the base of the hydranth along the substratum (Fig. 4E). SUMMARY A new species of a hydroid genus not previously recorded from the Atlantic coast, Asclomaris inichaell, is described. The development and activity of both male and female gonophores are described in detail, together with settling of the planula and formation of the first hydranth. LITERATURE CITED ALDER, JOSHUA, 1862. Supplement to a catalogue of the zoophytes of Northumberland and Durham. Trans. Tyncside Nat. Field Club, 5: 225-247. LOWE, E., 1926. Embryology of Tubularia. Quart. Jour. Micr. Sci., 70 : 599-627. REES, W. J., 1938. Observations on British and Norwegian hydroids and their medusae. Jour. Mar. Biol. Assoc., 23 : 1-42. TOTTON, A. K., 1930. Hydroida. Brit. Antarct. ("Terra Nova"} E.vp., 1910, Nat. Hist. Rep., Zool., 5 : Coelenterata, 131-252. NOTE ON THE SPAWNING OF THE HOLOTHURIAN, THYONE BRIAREUS (LESUEUR) LAURA HUNTER COLWIN The Marine Biological Laboratory, Woods Hole, Massachusetts and the Department of Biology, Queens College, Flushing, New York Thyone briarcus is fairly abundant at Woods Hole, Massachusetts, yet very little has been recorded about its spawning. The present note is based upon observa- tions made in the laboratory in 1948. THE BREEDING SEASON June is the principal month in which shedding has been observed in the labora- tory at Woods Hole. Pearse (1909) noted spawning from June 22nd to July 5th, Ohshima (1925) reported it from June 21st to 24th in 1921, and Just (1929) ob- served it repeatedly during the month of June for several years. Mead (1898) found every animal full of nearly ripe eggs or sperm on April 24th, which would suggest a season beginning earlier than June, but Just (1929) claimed that eggs obtained in April and May were unripe ovocyteSj capable of responding to insemina- tion but unable to develop. There are no other data for the early part of the breed- ing season. As to the latter part, Pearse's observation of shedding on July 5th is the latest specific data published for Thyone at Woods Hole, although Bumpus (1898) remarked that the breeding season was probably June and July and Clark (1902) stated that Thyone apparently bred in the summer. A study of 314 animals was made during the second half of June, 1948. Forty-nine of these animals shed in the laboratory, and 215 which did not shed were dissected. The results are summarized in Table I. The table shows the number of animals collected on each date and their condition as determined by shedding or dissection, together with estimates of shedding capacity based on the findings in these two categories. Line 8 in the horizontal direction includes some cases of undetermined sex among spent animals. When spawning is over, the gonadal tubules are so small that a careful microscopical examination is necessary to determine the sex. Such examinations were not made in the cases indicated. The single case of undetermined sex among the partly spent animals of the June 19th group was simply the result of an oversight. It is quite easy to distinguish the sexes when partly spent animals are dissected. The gonadal tubules of the male are an opaque yellowish or orange color and are pointed at the distal end, while those of the female are more translucent, golden or mustard colored, and have blunt distal ends. The dates of collection shown in the table for the different groups of ani- mals do not necessarily indicate the dates on which spawning occurred. In fact, owing partly to experimental conditions, sheddings usually took place on subse- quent dates. Nevertheless, the sheddings are listed as of the date of collection inas- much as they indicate, no matter when they occurred, the shedding capacity of ani- mals collected at that time. For example, some animals collected on June 15th were 296 SPAWNING OF THYONE 297 t 8 a ' 8 » 8 C3 =0 ^ •3 O-X3 ^-* 6, a § -w s r^ s o 8 5 1x0 *» •§•§ a I "b o O CO LO O <-o' CO ON CN LO rt CN o ^ Tf r^. NO o ~* « CN ^ O o O O 0 O O O O % ^_ O -* -f CN C5 2 CO CN '"' "-1 LO 00 4) C O CN r*-, to CN 3 i—) ^— ( CO OO O o — i C NO CN ^ -H 2 'b ro O co ~* NO NO ">. ^ LO O CN ^ CO — 'O 0 C NO fv. co £ o CN O^JH CO ON 00 ON ON' o CN CO •b 0 O "^ ON NO ON « NO ON co rvi • ON *^f LO LO OJ 0 <->- 00 i—> CN ^ CO 'O 1 — i o o — 1 O LO CN i 2 5 U. 'b LO C l— O C5 CN LO ON' CN '-' Ol ^^ LO T* 0> "*- 00 ru. CN 2 NO 1—1 o - O LO CN C t» 00 00 OO o ON' CN •"•-1 1_ •b 2 0 01 -H 00 Os° 00 ON ON' O) •- tH CN CN fO s « £ fV. ^. 3 "" 11 — ' ^fe o ro O «-i CN 00 to' OO 5 1 " 1 rrj ON •b CO o •* LO NO CN ON CO to ON CD Tf ^ to C NO •sf • ,~* ^ CN 'f 0 CO LO t^* *— • ^ CN 0^ LO CN ^^ LO ^^ -*1 LO 'b 00 o LO o "^ co' o LO ,_, *-< •^ CN 11 •* ^^ CO C CN J^ ON CO ON o "H O Tt< — " 0 NO 4> ^ ^ \u X 4* a 3 O 6 4) ^"^ Number in group X Number shed Number dissected Full Partly spent Spent Remainder estimat sheddable Estimated spent Number of each se estimated in group Number of each se estimated sheddabl Per cent of each se estimated sheddabl - _ CN 1 CO* ^ LO O «>•' 00 ON S _ -' OJ OJ ^ C uj y^ M f- « 0) p 0 ° 'o U 1_ CU 4) 4> 4) s s 3 3 G C O O C C *•> 4) 4) 4) C J= CJ (J 0) *-- U U <-> w_ a) a; u o CX O. 4) ^ IO ON °" C ^0°°. O O r<5 t^ ^ 298 LAURA HUNTER COLWIN found shedding on the 18th, others not until the 22nd, 23rd and even the 26th of June, yet all of these sheddings indicate the shedding capacity of animals collected on the 15th. Compared with specimens actually collected on the 23rd or 26th, the June 15th group shows a much higher shedding capacity. The numbers collected were meager for some of the dates studied but an analysis of the table does show, in a general way at least, the probability of obtaining embryological material at this season. It may be mentioned that no single season is necessarily typical, since there is considerable variation in weather conditions during the spring months from year to year and this is probably reflected in the environment of the Thyone beds. A comparison of water temperatures at Woods Hole for the years 1902 through 1906 is given by Sumner, Osburn, and Cole (1911) and the air and water tempera- tures of recent years are on file at the Marine Biological Laboratory and the Woods Hole Oceanographic Institute (unpublished). It has not been found possible to determine the sex of Thyone briar eus by ex- ternal inspection. Certainly there are no obvious correlations between sex and size, color, general appearance, or behavior. Therefore it is assumed that the specimens selected for dissection represented a random sampling of the entire group and showed about the same proportions of the two sexes as would any other sampling, as, for instance, the specimens left undissected. From lines 5, 6, 7 and 8 it can be seen that a total of 115 females were found, through shedding and dissection, while there were 138 males. Obviously 253 animals are not enough for a very accurate study of the sex ratio but, for want of more, this number must be used at present. Hence, it is concluded that 45.4 per cent of the animals would be females and 54.5 per cent would be males, or a ratio of about five to six, in any group of Thyone col- lected as were the subjects of this study. Estimates of shedding capacity within a group were obtained in the following manner. (1) Animals which actually did shed were considered sheddable as of the date collected. (2) Some animals were probably prevented from shedding by the conditions to which they were subjected. If dissection showed the gonads full of seemingly mature gametes, the animals were considered sheddable. This seemed permissible since shedding did occur in the first group collected. (3) It was found subsequently that a given animal can shed more than once, at intervals of several days. Therefore, dissected animals found to be only partly spent were also con- sidered sheddable. (4) Some animals that did not shed were not dissected, but an estimate was made showing those that should have been able to shed. (The number not examined was multiplied by the proportion found sheddable among those that were examined.) The sum of these four categories gave the estimated number of animals able to shed, from which the percentage of shedding capacity for the group could be calculated. However, since there were more males than females and since, moreover, most groups showed more females than males to be already spent, the shedding capacity was estimated in terms of percent of each sex capable of shedding. This seemed worth while in spite of the inaccuracies bound to arise from a study of such small numbers (e.g. Group 3 shows 129.9 per cent sheddable males), because the low female shedding capacity would otherwise have been masked by the higher male activity. Table I shows that nearly half of the females examined in the middle of June had already shed their eggs. By June 21st more than half were spent, and as early SPAWNING OF THYONE 299 as June 23rd one group showed the exceptionally low shedding- capacity of only 7.6 per cent. In the last week of June, little more than a third of the females could shed and much lower percentages might he expected, such as the 13.8 per cent noted on June 29th. By July 15th there were no females ahle to shed, out of a group of 24 animals examined, although three of the males could still have shed some sperm. Aside from the numerical evidence of a waning season, the quantities of mature germ cells, either shed or found by dissection, dwindled as the month progressed. It seems, then, that the guess of Bumpus about shedding in June and July in 1898 would only have proved half accurate in 1948. And while one might have ex- pected an occasional shedding of eggs as late as July 5th, as Pearse reported in 1909, that date could not have been recommended for the purpose this year. In fact, it would not have been worth while to attempt a detailed examination of the eggs of Thyone from animals collected after the third week of June. Although this study was not begun until the middle of June, there are some in- dications of what may have occurred earlier. One might expect a higher per- centage of mature females to be capable of shedding at the height of the season than actually did shed at any time from mid-June on. Nearly half of the batch was spent as early as June 19th. As early as June 15th there were as many partially spent as there were shedding, and on the 19th and again on the 23rd, there were more par- tially spent than the sum of the shedding and fully ripe ones combined. Certainly the season was waning during the entire second half of June. All this suggests a peak reached before June 15th. If this is correct, then the spawning season must either have been very short or have begun before June 1st. We have Mead's ( 1898) observation to support the latter possibility and Just's (1929) to support the former. Until the question is settled, the most promising time for successful embryological collecting must be considered to be the first half of June or possibly the middle two weeks of this month. SHEDDING Before it begins to shed, an animal expands greatly, to perhaps twice its former length, and starts waving its tentacles gently in the water in "feeding movements," as noted by Pearse in 1909. The position of the body can vary from vertical to horizontal, attached to the bottom or the sides of its container. It needs enough free space for the extended tentacles because sudden contact with any object will usually cause contraction, which might possibly delay shedding. On the other hand, shedding is not impossible in cramped quarters and once begun, under whatever circumstances, the process can be resumed in spite of various interruptions, such as removing the animal from water, leaving it practically dry, placing it under a very bright light, rinsing it in cold tap water, and so on. However, the writhing movements of an animal out of water or in too small a container will disperse the germ cells and could injure them. As has been mentioned above, the sexes are indistinguishable externally. In both, the genital pore lies at the tip of an inconspicuous little stalk in the mid-dorsal line, between the bases of two large tentacles. It is easily located since it occupies the point on the tentacular ring diametrically opposite the only pair of small ten- tacles. The genital duct connects the pore with the gonads, which lie along the dor- sal body wall, about halfway between the mouth and anus. Kille (1939) has de- 300 LAURA HUNTER COLWIN scribed the gonads in post-spawning animals and discusses the tubules and their contents as found in July and August. It seems as if the entire gonadal knot need not be involved with spawning each year and some tubules may remain small and undistended. On the other hand this may be simply the appearance of late-season tubules. Ohshima (1925) described irregular masses of disintegrating yolk in the ovarian tubes, concluding that they were probably eggs which had failed to be laid in the previous spawning season and were undergoing degeneration. Brownish masses were especially notable in the ovarian tubules of spent females toward the end of the present study and these too were given Ohshima's interpretation, but it was thought that they probably represented degenerating eggs of the current season. Kille suggested that some large tubules may have been lost by some of his animals seen in July and August, an impression also gained from the present study, but not actually proved. Once when an animal was vastly expanded and undergoing the writhing move- ments that precede shedding, the body wall became semi-transparent and the genital duct could be seen, a straight, grayish line leading to the pore. It showed clearly even when the animal was removed from the water and held up to the light. Shedding finally did occur and the animal proved to be a female as had been pre- dicted. After the shedding the line was gone, but later a shorter line was noticed in the same region. Since the eggs are grayish and the sperm white, it is possible that some method of sex diagnosis might be worked out, but it probably would be practical only with full, mature animals. It was not effective this season. The germ cells are emitted in a slow, fountain-like stream, so that they rise up- ward for a very short distance and then fall toward the bottom of the container. Since the tentacles are waving, there is fairly rapid dispersal, especially of the sperm. Moreover, animals nearby in the same container will ingest the gametes being shed by a neighbor. A female which has been in a container with a shedding male can be partially freed of sperm by rinsing in running tap water ; but since the tentacles are withdrawn during this process, all the sperm cannot be reached and some of her eggs, if shed soon, are certain to be fertilized. Many changes of sea water with intervals for feeding movements in each would cut down the possibility of sperm contamination, but not eliminate it. Probably the best way to obtain unfertilized eggs under these circumstances is to hold a slender medicine dropper directly above the genital papilla. If introduced slowly and gently this is quite feasible, although tedious. It is much easier to keep prospective shedders in separate con- tainers. Segregation is desirable even if fertilized eggs are to be collected, since the relatively large amount of sperm, even in 6 by 9 inch battery jars, will literally smother the eggs and is to be avoided. The duration of the shedding process varied considerably, perhaps depending upon the age, and hence size, of an animal as well as on its degree of fullness. When watched, the process was sometimes as short as 10 or 15 minutes but extended up to four and a half hours in the largest shedding witnessed. Usually it was com- pleted within about a half hour of the time of starting. Sometimes shedding is not quite continuous even when apparently undisturbed, and will proceed intermittently with intervals of from a few minutes on up to nearly three days. These latter re- peated sheddings were noted several times, in one unusual case 7 and 9 days, re- SPAWNING OF THYONE 301 spectively, after the animal was collected. Repeated shedding has not yet been found in a female. The present study confirms the statements of Ohshima (1925) and Just (1929) that eggs obtained by means other than natural shedding connot be fertilized in the laboratory. It is easy to secure large eggs simply by mincing the ovarian tubules. The eggs which fall out are about as large as shed eggs. They contain a large germinal vesicle which has never been seen to break down except under pressure, applied externally. They have not been fertilized. FACTORS IN SHEDDING No reliable method has been found to induce shedding at the will of the in- vestigator, but various factors which might influence the phenomenon have been examined in connection with the shedding of the 35 males and 14 females which took place during the present study. The preponderance of males shedding was much greater than might be expected on the basis of the sex ratio. Perhaps they simply shed more easily or- more frequently. Perhaps their activity lasts longer during the declining season. Whatever the reason, it should be borne in mind that the factors considered were effective on males particularly. They cannot be clearly demonstrated to be shedding agents until they have been examined again, at a time when many ripe females are available. Time of Day Ohshima (1925) found that shedding always occurred late in the afternoon of the day the animals were brought into the laboratory. Once in 1948 a number of animals did shed around 6:00 P.M. of the day they were delivered, but it happened that this batch had been collected previously and kept on the water table in the sup- ply department for at least a day. By far the majority of sheddings watched this year took place in the evening, mostly around 8 :00 and 9 :00 o'clock, and some as late as 1 :00 or 2 :00 A.M. Of course, many of these sheddings occurred under ex- perimental conditions and could not be attributed entirely to a natural tendency. It did seem, however, as if there might have been a rather strong natural bent to- ward evening shedding which persisted despite external conditions. Nevertheless, at least two animals were seen shedding in the morning, one between 9 :00 and 10 :00 o'clock and the other at 12:00. Therefore, this part of the day need not be ruled out as a possible time for obtaining the germ cells. (2) Light Ohshima was able to induce animals to shed during the day by placing them in a dim light. Returned to bright light, they ceased shedding but would continue again if replaced in subdued light. Utter darkness did not cause shedding. Pre- sumably these observations w^ere made during the four days when Ohshima ob- tained eggs. During the present study sheddings were observed on at least ten different days and under many circumstances. They were seen to take place in natural, indirect daylight, in subdued daylight and in electric light of various in- tensities from rather subdued to bright lamplight directed right on the animals. They were also found to have occurred in complete darkness. Several times a 302 LAURA HUNTER COLWIN male which had started to shed was picked up unceremoniously and transferred to a new dish under a bright electric lamp. Soon it started to shed again. Some fe- males shed in bright light and their performance was watched in light sufficiently bright to allow one to see it easily, but it is true that at least 8 of the 14 females studied did shed in either complete or semi-darkness. The number is too small to be significant. This diversity of findings seems to rule out subdued light as a pre- requisite. Possibly Ohshima's observations can be explained in the following way. Thyone is very sensitive to changes in light which occur suddenly, as when, as Pearse showed (1908), a shadow passes abruptly between the animal and the light source. It also reacts immediately to jarring. The reaction to both stimuli is the same : contraction and in-drawing of tentacles, enough to interrupt shedding. It may take a minute or longer before the animal will expand again after such a con- traction. If the shedding time were a short one, interruptions such as these would be enough to appear to stop the process. With only four days of observations, and eggs to collect at the same time, Ohshima may have returned his specimens to the dim light before they had had time to start shedding in the other types of light investigated. On the dates of his collections, June 21st to 24th, he might also have been dealing with some animals that were partly spent and had little to shed, as in the present study. (3) Presence of a Male Finally, Ohshima found no females spawning spontaneously, but only after the emission of sperm by nearby males in the same container. This year it was incon- venient to isolate all the animals of a group because of the container requirements for animals of this size, but at least two females did start to shed while wholly isolated in separate bowls. Others, isolated as soon as shedding was noticed, would continue after isolation, and this was, incidentally, usually after a rinsing in cold tap water too. The male effect was tested conversely when a female which had shed two days earlier was placed in a jar of freshly shed sperm, in an attempt to induce fur- ther shedding. It did not occur, even though subsequent dissection showed at least some mature eggs in at least some ovarian tubes. It would be more conclusive to use a full, mature female, but as yet no way has been found to distinguish such ani- mals. At all events, isolated females are perfectly capable of initiating shedding. (4) Size The size of these animals is very hard to determine since so much depends upon their water content at the time of examination. However, any animals known to the supply department as medium or large will probably prove satisfactory as a source of germ cells, in season. It is not always the largest in a batch that will shed first, nor is the size any criterion of the sex of an animal. Very small speci- mens are to be eschewed for several reasons, (a) If mature, they will have only a small quantity of germ cells, (b) They may be mature animals already spent and hence reduced in bulk by the absence of distended gonads. (c) They may be small because they have eviscerated and hence are lacking in most of their viscera, usually including the larger gonad tubules. All three types have been encountered in the present study, (a) and (b) most frequently. SPAWNING OF THYONE 303 (5) Quantity of Sea Water Just (1929) observed spawning repeatedly among animals if kept in large quan- tities of sea water. This year, shedding would begin in any amount of sea water sufficient to hold the animal and allow it to expand, and once begun, several males were found able to continue shedding even when placed in practically dry finger bowls in the hope of stopping them temporarily. They provided a little water for themselves, from the cloaca. Sometimes an animal barely submerged in a four-inch, shallow bowl would undergo the expansion that usually precedes shedding and would virtually double up on itself in the cramped space. When noted, this was remedied out of sympathy and the animals went on to shed in larger quarters, but sometimes they were not moved, and shed in the small containers. Usually, how- ever, shedding was observed among animals in the 6 by 9 inch or 6 by 7y% inch cylindrical battery jars which proved to be the most suitable containers for them. Here there was room for maximum expansion but at the same time some hope of retrieving the shed cells. The water was usually changed several times a day to ensure freshness but it is questionable whether this is really necessary if the animals are stored in a cool place (15°-17° C). One sturdy specimen was found shedding after at least two days in unchanged sea water at about 20° C. Pearse (1908) and others have shown that Thyone can endure a good deal of variation in the environment, such as higher salt concentration, dilution with fresh water, excessive heat (some survived more than two hours' exposure to as high as 37° C.), or exposure to air. It is unlikely, then, that large quantities of sea water are essential to shedding. However, it is probably desirable for storage, since animals stored in running sea water did shed, even many days after collection. It would be very hard to gather eggs or sperm shed at random in a large aquarium and, as a matter of fact, no sheddings were ob- served among stored animals. Possibly they escaped notice because of rapid dissipa- tion of the gametes. (6) Freshness of Animals It is generally held that fresh, well-fed animals provide the best embryological material. In the case of Thyone there is one drawback to using animals just col- lected. Their habit of pumping mud through the gut soon clouds the water so that gametes would be very hard to see, if present. The waving of the tentacles pro- vides an excellent stirring system which keeps the debris from settling. An animal which has been stored in a large aquarium, or in running sea water, overnight or longer, has got rid of much of this material and is an easier one in which to watch shedding. Spawning often does occur several days after collection and in a few cases animals which had been in the laboratory as long as nine and ten days were found shedding. Normal larvae developed from eggs shed two or three days after the parents were collected. The problem, then, is to keep the animals from shed- ding until after they have emptied their digestive tracts. (7) Temperature Wanning. There is some evidence that temperature plays an important role in the spawning of Thyone. Several times when half of a group of animals was 304 LAURA HUNTER COLWIN kept at room temperature (20°-22° C.) while the other half remained at the tem- perature of the sea water (16.5°-18° C.), a number of sheddings occurred among the warmed animals, none among those kept cool. Precise tests of this were not feasible this year because of the increasing number of spent animals among those collected. The most suggestive results were obtained with 34 specimens collected on or before June 21st. At 3 :35 P.M. on that date, 17 animals, group A, were placed in water at room temperature while 17 others, group B, were left in the running sea water. Five and a half hours later the water was changed on group A, some of the animals being put into fresh cold sea water, the rest into fresh warm sea water. Within a half hour shedding began in the group returned to cool water, and an hour and a half later it began in the group continued warm. In all, there were 12 out of a possible 17 sheddings. Meanwhile the animals of group B, kept cool while the others were being warmed, were placed in containers of fresh sea water and under the same lighting conditions as group A (typical overhead electric light of this lab- oratory). Part were put into cool water and the others into water at 20° C. There was no shedding at all during the next three hours or indeed overnight after the animals had been returned to the cold water table. Yet subsequent examinations showed that at least some of these animals could have shed, though some were already spent. The warming, if this experiment is indicative, should be of longer duration than three hours and more in the vicinity of five and a half, or more, hours. Further support for the belief that warming induces shedding comes from the totals of the whole season's studies : No sheddings were ever observed in animals which had not been warmed for some time beforehand. Unfortunately, however, many animals which were warmed failed to shed, and others did not shed the first time they were warmed, but only after warmings on several days or after staying warm continuously for several days. Perhaps results such as these may be at- tributed to the waning season, or to the possibility that animals kept in confinement for many days, especially after being warmed and cooled spasmodically, may react with less predictability than fresher ones. Additional exploration of this aspect of shedding is certainly needed. Cooling after warming. A change to cool water following a period of warming often seemed to stimulate shedding, but since most of these cases occurred at the commonest shedding time, from 8:00 to 11 :00 P.M., time of day could not be ruled out as part of the stimulus. The best favoring evidence was this : A group of animals which had been in the supply house for at least a day was brought to the laboratory at 1 :20 P.M. on June 22nd. They were in water of 17.8° C. which rose to 18.8° C. by 2 :50 P.M. At that time all the jars of animals were placed in a cooler in which the temperature was about 19° C. but was gradually descending. At 5 :45 P.M. the temperature had reached 16.5° C. and a number of animals of both sexes were shed- ding. Warming followed by cooling had certainly occurred but the exact time of the two periods is not known. There were also other factors. The cooler was entirely dark while housing the animals and it was vibrating vigorously. The matter of light has been discussed above and may be dismissed here. In view of the animals' normal reaction to jarring (Pearse, 1908) it seems as if the shedding may have occurred in spite of this obstacle. The time at which the shedding took place is especially interesting. It was much earlier than usual. Could it be that the treat- ment had overcome another possible obstacle, namely, time of day? SPAWNING OF THYONE 305 Continued cooling. Just as warming, or warming followed by cooling, may possibly stimulate shedding, so continued cooling appears to prevent it from taking place. If freshly collected animals are not wanted for immediate use they should be kept as cool as the water they came from, that is, no warmer than 15°-17° C, to discourage shedding. On the other hand, once an animal has started to shed, sud- den cooling does not seem to stop it. (S) Other Factors A few attempts were made to stimulate shedding by other, more artificial, means. External mechanical stimuli such as jarring, squeezing or pricking merely resulted in quick contraction with tentacle withdrawal, wholly unfavorable to shedding. More drastic treatment, like cutting, either caused contraction as above or eviscera- tion which makes shedding quite impossible. Injections into the body cavity can be effected without harming the animal, the amount being gauged by the size of the body. Pearse (1909), studying the physiological effects of various substances, sug- gested that some of his results were influenced by the fact that the animals were spawning. Conversely, it might be that some of the shedding was induced by his treatment. For instance, he found that sodium chloride, in W% solution, resulted in "feeding movements" and otherwise caused no harm to the animals. The same reaction was obtained this year, using 0.5 to 1.5 cc. of lO^c NaCl. It seemed as if the animals might be just about to shed, but no shedding ensued. This treatment should be attempted again at the height of the spawning season. Palmer (1937) found potassium chloride a shedding stimulant in sea urchins. This was not ex- plored carefully in Thyone and should also be repeated earlier in the season. How- ever, injections of KC1 did not seem to be followed by the "feeding movements" that followed the NaCl injections and, moreover, some animals responded to it by eviscerating. SUMMARY 1. Spawning occurred in the sea-cucumber, Thyone briareits, during the month of June, 1948. The phenomenon appeared to wane as the month progressed. Probably early June and possibly even earlier months are the best time to find high percentages of ripe germ cells. 2. The process of shedding is described, together with various details concern- ing the handling of shedding animals and their gametes. 3. The following possible factors in shedding are discussed : Time of day, amount of light, presence of a shedding male to induce shedding by the female, size, quantity of sea water, freshness of animals, temperature, etc. It is shown that the first three are not essential prerequisites for shedding. LITERATURE CITED BUMPUS, H. C., 1898. The breeding of animals at Woods Holl during the months of June, July and August. Science, N.S., 8 : 850-858. CLARK, H. L., 1902. The echinoderms of the Woods Hole region. Bull. Bur. Fisheries, 22: 545-576. 306 LAURA HUNTER COLWIN JUST, E. E., 1929. The production of filaments by echinoderm ova as a response to insemina- tion, with special reference to the phenomenon as exhibited by ova of the genus Asterias. Biol. Bull., 57: 311-325. KILLE, F. R., 1939. Regeneration of gonad tubules following extirpation in the sea-cucumber, Thyone briareus (Lesueur). Biol. Bull., 76: 70-79. MEAD, A. D., 1898. The breeding of animals at Woods Holl during the month of April, 1898. Science, N.S., 7: 702-704. OHSHIMA, H., 1925. Notes on the development of the sea-cucumber, Thyone briareus. Sci- ence, 61 : 420-422. PALMER, L., 1937. The shedding reaction in Arbacia punctulata. Physiol. Zoo/., 10 : 352-367. PEARSE, A. S., 1908. Observations on the behavior of the holothurian, Thyone briareus (Lesueur). Biol. Bull., 15: 259-288. PEARSE, A. S., 1909. Autotomy in holothurians. Biol. Bull., 18: 42-49. SUMNER, F. B., R. C. OSBURN, AND L. J. COLE, 1911. A biological survey of the waters of Woods Hole and vicinity. Section 1. Chapter 2. Bull. Bur. Fisheries, 31 : 28-54. THE PHYSIOLOGY OF EXCRETION IN MOLGULA (TUNICATA, ASCIDIACEA) S. M. DAS The University, Lncknoiv, India INTRODUCTION Twelve years ago (Das, 1936) the author wrote: "The excretory function in ascidians is attributed to a number of organs, the renal nature of some of which is still imperfectly understood. The refringent organ of the Botryllidae, the pyloric gland of the Cynthiidae, the renal vesicles of the Alolgulidae and Ascidiidae, the parietal vesicles of the Cynthiidae (Pyuridae), and the neural gland of all ascidians, have been described by different workers as excretory organs." Although our knowledge of the subject has advanced considerably due to the contributions of Azema (1926, 1928, and 1929) and George (1936), so far as the fundamentals of the subject are concerned the above mentioned remarks still hold true. The present investigation was taken up by the author during his stay in Woods Hole, in the summer of 1947, with a view to elucidating the structure, nature and relationships of the excretory organs and their products in J\lolgnla manhattensis DeKay. Lacaze-Duthiers (1874, 1892) was among the very first to locate and describe the renal organs in Tumcata including the renal sacs in Pyuridae (Cynthiidae). Roule (1885) described renal organs in Phallusidae, while Herdman (1888, 1899) described excretory organs in Ascidiidae, Botryllidae, and other ascidians; Dahl- griin (1901) worked on the excretory organs of Botryllidae and Ascidiidae, and compiled an account of excretory organs in Tunicaia. Das (1936) gave an ac- count of the excretory organs in Hcrdmama, while George (1936) described blood cells bearing excretory matter in Twricata. Kiipffer (1872, 1874) was the first to demonstrate uric acid in Tunicata. Sulima (1914) attacked the problem of physiology of excretion in ascidians and showed that uric acid and other purine bases were the chief constituents of the excretory products. Schmidt (1924) confirmed the presence of birefringent purine derivative granules in the renal concretions of ascidians. Millot (1923) extended the investigation to other ascidians, and not only found purine bases as the chief ex- cretory product, but discovered that in Pyuridae (Cynthiidae) the excretory gran- ules are always made up of xanthine. Azema (1926-29) in a series of papers dealt with the mechanism of excretion in some ascidians, and demonstrated the presence of purine bases as granules inside certain blood and connective tissue cells. Finally, George (1936) has shown that certain blood cells have refractory granules of an excretory nature, and that these may circulate freely in the blood stream or be lo- calized in the connective tissue. From our present knowledge of the subject, therefore, Timciata can be divided into two groups, based on the presence or absence of definite renal vesicles or sacs 307 308 S. M. DAS which not only filter the nitrogenous excretory products from the blood, but also store them in a more or less insoluble form as solid renal concretions. These two groups of Tunic at a may be called : (a) the renal type — with a definitive renal sac, and (b) the arenal type — without any such sac. By far the greater number of tunicate species belong to the arenal type, the renal species being mainly confined to the Ascidiidae, the Molgulidae and the Pyuridae. In the arenal tunicates, the renal function is performed by the renal cells of the connective tissue and blood, as well as the central part of the neural gland (George, 1936; Das, 1936). The localization of the excretory granules may take place in the walls of the vas deferens (dona; Roule, 1885) ; the mesenchyme meshwork between oesophagus and stomach on the one hand and rectum on the other (Botryl- lidae; Dahlgrun, 1901) ; the immediate neighborhood of the gut (Ciona intestinalis ; Dahlgriin, 1901) ; the white pigmented patches on siphons (Ascidia pcllucida; Azema, 1929b). The renal tunicates, on the other hand, have a large well-marked vesicle (Mol- gulidae; Roule, Lacaze-Duthiers) or a number of small vesicles or sacs arranged in- side the body wall or in other regions of the body (Ascidiidae and Pyuridae; Lacaze-Duthiers, Dahlgrun, and Azema). These multicellular, blind, ductless, re- nal sacs not only store a fluid comparable with the urine of other chordates, but strangely enough continuously deposit inside them solid concretions that accumulate throughout life (Azema, 1926). It is remarkable that in the same family we have some species with definite re- nal vesicles and others which have only scattered renal cells ; e.g. in the family Pyuridae, Dahlgrun (1901) records renal vesicles in Cynthia dura and Micro- cosmus scrotits, while Azema (1929a) found no renal vesicles in a number of Pyurid species, but renal cells were present. Similarly George (1936) could find no renal sacs in Pyura vittata although renal cells were again present. Das (1936) could find no renal sacs in Herd-mania pallida (Pyuridae) but found renal cells with re- fractory granules not only in the visceral region, but also being discharged into the lumen of the neural gland, whence they pass out through the neural duct into the branchial cavity and thence to the outside. According to the present state of •our knowledge it is highly improbable, however, that individuals of the same species may possess both the renal vesicles as well as the localized renal cells, although scattered renal cells may often be found. MATERIAL AND METHODS The renal vesicle in the Molgulidae is a large bean-shaped sac in which waste matter is gradually deposited and stored in the form of concretions throughout the life of the animal. Molgula manhattensis was selected because of its abundance in the Woods Hole region, and because it is comparatively easy to expose or isolate the renal sac in the living condition. The structure of the renal sac was investigated both in the living and the fixed material, while the renal concretions were examined under reflecting as well as polarizing microscopes. The renal fluid was extracted from living renal sacs by means of a specially made micro-pipette. Care was taken to store the renal fluid under a layer of heavy PHYSIOLOGY OF EXCRETION IN MOLGULA 309 neutral mineral oil, and the method of Walker (1930 and 1937) used to determine the total molecular concentrations. The correlations between body weights and weights of renal vesicles were obtained by carefully weighing the entire live animal as well as the freshly isolated renal vesicles under standard conditions. The renal concretions were carefully separated from each vesicle, dried for a half hour at constant temperature, and weighed quickly when each batch was ready. STRUCTURE OF THE EXCRETORY ORGANS The main excretory organ in M. manhattensis is the large, blind, and ductless renal sac situated on the right side of the body, attached to the body wall or mantle just below the pericardium. It is a thin-walled, elongated, bean-shaped sac and contains a light, yellowish-brown fluid together with the solid concretions deposited in it. It is firmly attached to the wall of the mantle and is in such close proximity to the pericardial wall anteriorly, that it is difficult to separate the renal vesicle without destroying the heart. With each wave of contraction of the heart, the wall of the pericardium is pressed against the antero-dorsal wall of the renal sac, so that the waves of contraction appear to move over this part of the sac as a series of half-ring contractions. The renal sac lies at about two-thirds distance from the antero-dorsal border of the animal and about one-third from the postero-ventral. It lies across the mantle occupying about one third of its width at its middle (Fig. 1). In a full grown ani- mal measuring 4 X 3 X 1.5 cm., the sac is about 16 mm. long and about 6 mm. at its greatest width. One end of the sac is bluntly rounded while the other is taper- ing and bent, appearing like an elongated bean in outline (Fig. 2). It is circular in cross-section in the fresh condition but tends to become flattened from side to side in fixed material (Fig. 3). In very young specimens the length of the sac is comparatively short and it appears perfectly bean-shaped; but as the animals in- crease in size, the sac increases more in length than in width. The wall of the sac consists of a single layer of cells (one cell thick) which is bounded by a thin basement membrane. Each cell is almost like a cubical epithelial cell although the width is greater than the height. The nucleus lies nearer the basal end of the cell, while the cytoplasm contains one, two, or more vacuoles of different sizes (Fig. 4). The structure of each cell was best seen in the living condition, when a light methylene blue intra-vitam stain was used. The largest vacuole lying on the distal periphery of the cell always takes up the maximum stain, and con- tains a small mass (or two or three masses) of solid birefringent concretions that appear very much like the ones seen by George (1936) in the blood cells of Poly- androcarpa tincta. When kept under continuous observation, these concretions are seen to mimic "Brownian movement," increase in size, and be evacuated into the cavity of the renal sac, while a fresh vacuole soon takes the place of the one dis- charged. These cells are therefore primarily instrumental not only in the absorp- tion of excretory material from the body fluid bathing the outside of the sac, but also in the isolation and final deposition of solid excretory products into the renal sac. The cavity of the sac is filled with the transparent, yellowish-brown, renal fluid. The renal concretions lie free in the cavity of the sac. They are located in con- centrated clumps or masses more at the rounded end of the sac, and spread out in a more or less continuous chain, growing towards the tapering end the the sac. The 310 S. M. DAS FIG. 1. FIG. 2. FIGURE 1. Diagrammatic sketch of right side of animal after removal of test to show position of renal vesicle (bottom) in relation to pericardium (middle) and right gonad (top). FIGURE 2. a-f : Stages in growth of renal sac and renal concretions, (a) being the young- est and (f) full grown. FIG. 3. FIGURE 3. a and b : shape of fresh renal sac in T.S. ; c : flattened outline after fixation. FIGURE 4. a : the renal epithelium, X 120 ; b : one renal epithelial cell showing a main vacuole containing concretions, and two secondary vacuoles being formed, X 250 ; c : the main vacuole enlarged to show formative renal concretions, X 750. chain can be broken up into clumps even in the living renal sac, by merely shaking it. Each clump consists of a mass of discrete rounded or oval bodies, the bire- fringent granules, some of which are large and others small. The smallest ones measure about 3 to 4 /x across, while some of the largest ones were 30 to 40 p. in diameter. The larger granules show definite concentric lamellae or layers of growth, che central part of the granule showing radial striations. Two, three, or more con- FIGURE 5. a and b : mass of renal concretions, showing attachment of later depositions on the previously deposited concretions in the renal sac ; c : two concretion-bodies showing charac- teristic concentric layers; d: T.S. of a concretion-body showing radiating lines. PHYSIOLOGY OF EXCRETION IN MOLGULA 311 2 3 4 S WEIGHT OF BODY FIGURE 6. Correlation between body weight and weight of renal sac. centric layers can be observed under the higher powers of the microscope (Fig. 5). This deposition clearly occurs from the renal fluid, without the wall of the sac taking any direct part in its formation. Smaller animals have definitely smaller excretory granules, while the largest ones show three, four, or more concentric lamellae. This appears to indicate that the rings or layers are formed by different rates of deposition of excretory matter, dependent on' the differences in rates of metabolic activity at different periods in the life of the tunicate. The neural gland opens by a neural duct into the branchial cavity, as in other tunicates. The cilia of the duct are long and lash away from the gland ~L towards 1 Huus (1937) stated that "the ciliary pit whose function is not as yet known, may prob- ably be an organ for the reception of sexual stimuli, and that the neural gland which is closely connected with the ciliary pit, responds to these stimuli with a hormone production which in turn calls forth spawning." Similarly, Butscher (1930), and Bacq and Florkin (1935) have shown that the neural gland in ascidians produces a hormone with similar physiological effects as pituitrin. Now a hormone is always produced by a ductless gland. The neural gland has not only a duct with cilia that beat outwards, but the entire central part of the gland consists of branching ductules joining to form the main duct. The outward beating cilia would not allow passage of fluids into the gland, although the dorsal tubercle itself may receive sexual stimuli. Pituitrin is probably produced by the peripheral part of N. gland. 312 S. M. DAS the opening of the duct as seen in ducts dissected in Ringer's fluid. Cells bearing birefringent granules were seen to be extruded into the lumen of the gland and car- ried out by the "stream" produced by the cilia of the neural duct. This process is similar to the one observed in Herdmania (Das, 1936). CORRELATION BETWEEN BODY WEIGHT AND WEIGHT OF RENAL SAC Dahlgriin (1901), Azema (1926-29), and George (1936) state that waste matter is gradually deposited in the renal sac and stored in the form of concretions through- out life. They have not given any quantitative estimates of the concretions, nor 9C I sc o 40 .5 30 10 3 4 WEIGHT or BODY 8 j>-ff) FIGURE 7. Correlation between body weight and weight of renal concretions. have they established any correlations between the weight of concretions and the body weight, which alone would elucidate the rate and mode of disposal of ex- cretory products. The present author isolated over one hundred renal sacs from animals of all possible sizes available at Woods Hole, ranging from a body weight of about half a gram to as large as seven grams. The weights of a series of indi- viduals of graded sizes were taken and the weight of the renal sac of each was deter- mined. The results are shown in graphical form in Figure 6. It is apparent from PHYSIOLOGY OF EXCRETION IN MOLGULA 313 the nature of the curve that the growth in weight of the renal sac is not directly pro- portionate to the growth in body weight. Up to a body weight of about three grams, the rate of growth of the sac is proportionate to that of the body; above this size, however, the rate of growth of the sac outstrips that of the body until a body weight of about five grams is reached. Thereafter the rate of growth of the sac becomes slower than that of the body, little growth taking place above a body weight of seven grams. This correlation may be interpreted as that between the body of the tunicate and the renal fluid, since the latter constitutes over 80 per cent by weight of the renal sac. It can thus be concluded that comparatively more renal fluid is present in the sac in half to full grown tunicates than in the younger or older stages. The correlation between body weight and weight of renal fluid is a dynamic one, as explained later. CORRELATION BETWEEN BODY WEIGHT AND WEIGHT OF RENAL CONCRETIONS If, as found by past workers on the subject, the solid concretions in the renal sac are stored throughout life, it was surmised that there should be a straight line relationship between the body weight and the weight of renal concretions in ani- mals of different sizes. Actually, however, the data from the present study yield a curve which is far from a straight line. From a body weight of about 0.4 gm. up to about 3 gm., the increase in weight of the concretions is rather small ; but above this the weight increases rapidly and the correlation curve shows a steep ascent (Fig. 7). This means that instead of the accumulation of excretion being proportional to the body weight (i:e. instead of the increase in excretory material being proportional to the growth of the animal), the excretory material is deposited at a faster rate as the animal grows older. CORRELATION BETWEEN WEIGHT OF RENAL VESICLE AND WEIGHT OF RENAL CONCRETIONS This correlation is shown in Figure 8. It appears to be almost a "straight line relationship in its first half, meaning that the rate of accumulation of concretions is directly proportional to the increase in weight of the renal sac. In other words in the first part of the life of the tunicate, the increase of renal concretions is directly proportional to the increase in renal fluid inside the sac. As the tunicate grows older, however, the rate of increase of the renal sac is overtaken by the rate of depo- sition of renal concretions ; and we find the upper part of the correlation line- curving steeply upwards (Fig. 8). This increase falls rapidly as the maximum size of the tunicate is reached ; and finally the weights of both the renal sac and the concretions remain almost stationary even with an increase in body weight. The final stage, when no further accumulation of concretions is possible, may be called the saturation stage. The tunicate could not live much longer once this stage has been reached. The renal sac from large living Molgula manhattensis was removed and the animals kept under observation for a week or more, but no regeneration of the sac or formation of secondary renal sacs was observed. This is exactly the reverse of what obtains in multivesicled animals like Ascidia and Ascidiella, where a number of renal sacs are present instead of only one as in Molgula. In A. mentitla (Azema, 1926) renal sacs are formed quite early in development ; but instead of all the sacs 314 S. M. DAS being formed simultaneously, they are formed successively in the regions around the oesophagus, stomach, and intestine. New vesicles arise as the old ones are used up. It is apparent, therefore, that these multivesicular tunicates have a more effective excretory system than the univesicular ones. 14 z o it/ 0 10 10 SO 40 50 (,0 70 SO WEIGHT OF RENAL VESICLE FIGURE 8. Correlation between weight of renal vesicle and weight of renal concretions. THE RENAL FLUID The renal fluid, when examined in fresh condition, is a clear, pale, yellowish- brown fluid which turns darker on exposure to air. The pH of this fluid ranged from 7 A to 7.6 as determined by the Cambridge glass-electrode potentiometer. Tunicate urine is, therefore, slightly alkaline in nature. That this is caused by free ammonia was determined by the permutit method. No urea was present in any of the renal fluid samples. A full quantitative estimation of all the constituents of PHYSIOLOGY OF EXCRETION IN MOLGULA 315 the fluid could not be undertaken in the short time available at Woods Hole, but will no doubt be attempted in the near future. Total molecular concentration and tonicity Pure samples of the renal fluid were obtained from freshly dissected-out renal sacs by inserting a chemically clean, dry, glass micropipette into several sacs in turn. The pooled renal fluid was collected under mineral oil in order to avoid evaporation, absorption of water, or any changes due to exposure in air. The total molecular concentration of these fluids was compared with that of known NaCl solu- tions in "Barger tubes" (glass capillary tubes) using the method of Walker (1930 and 1937). This method involves sealing a series of micro-drops of the two fluids to be compared, each separated from its neighbours by a tiny air-gap, into chemically clean, dry, glass capillaries of very uniform internal diameter. Relative volumes of the drops are determined by measuring the lengths of the columns in the capillaries under a compound microscope, using a filar micrometer. These measurements are repeated at intervals for a period of two or three days, the capillaries being stored in a water bath between times. Consistent lengthening of a drop and shortening of its neighbours, indicates its higher total molecular concentration and vice versa. Thus by using saline solutions of known strength, the molecular concentration of the unknown fluid can be determined. Microdrops were prepared and read for : (a) renal fluid against known saline solution (b) body fluid against known saline solution (c) sea water against known saline solution (d) renal fluid against sea water (e) body fluid against sea water The results may be summarized in tabular form : SPECIMEN APPROX. CORK. NA€L CON.C. Pooled body fluid 2.75 gm./lOO cc. Sea water 3.10 gm./lOO cc. Pooled renal fluid 3.45 gm./lOO cc. One sample of renal fluid Hypertonic to sea water This comparison of the total molecular concentration of the renal fluid, the sea water, and the body fluid, has, as will be presently seen, led to very interesting re- sults. The renal fluid was thus found to be hypertonic to sea water in which the animal lives, while the body fluid was hypotonic to sea water, i.e., renal fluid > sea water > body fluid. Osmoregnlation The occurrence of hypertonic urine in Tunicata throws new light on osmoregula- tion in this group, little work on this subject having been done in the past. The body of the tunicate is closed to all exchanges between the surface and the sea water due to the covering of the test. But the current of sea water passing through the branchial siphon into the pharynx and out through the atrial siphon, brings the extensive branchial wall and vessels into close contact with it. The CX is absorbed 316 S. M. DAS and CO2 is thus excreted through the branchial vessels. The body fluid, as estab- lished above, is hypotonic to sea water ; and thus it is apparent that an osmotic gradient is maintained in the tunicate between the internal and the external environment. Whether water required by the animal is taken from the sea water into its alimentary canal like the marine teleostean fishes, is not definitely established but is quite probable. The role of the hypertonic renal fluid is, however, quite defi- nite. There is a constant flow of fluids from the blood into the renal sac, the ex- cretory products being extracted mainly by the cells constituting the wall of the sac. The renal cells not only maintain an active osmotic gradient, but allow more or less water to pass out of the sac according to the requirements of the animal. Experi- mental animals were kept in grades of sea water ranging from 40 per cent to 80 per 90 to z ul X 30 40 60 70 HO PiRCENTAGE 110 SALINITY 130 J_ _L _L 150 ito no NOX FIGURE 9. Correlation between weight of renal sac and different concentrations of sea water. Three experimental series are represented, the animals in each series weighing about the same. cent (hypotonic), and from 110 per cent to 160 per cent (hypertonic), for a fixed pe- riod of five hours each. Each animal was weighed and a "twin" found weighing about the same. The "twins" were kept as controls in 100 per cent sea water for the same period. The renal sacs of each living experimental animal and its "twin" were rapidly dissected out and both weighed against each other. It was observed that renal vesicles from animals in diluted sea water weighed more than their con- trols, and that the extra weight was inversely proportional to the concentration of sea water in which the animals were kept ; that is, the lower the concentration of sea water, the higher the weight of the renal sac. On the other hand, renal vesicles from animals kept in concentrated sea water weighed less than their controls ; that is, the higher the concentration of sea water, the lower was the weight of the sac PHYSIOLOGY OF EXCRETION IN MOLGULA 317 (Fig. 9). It can therefore be concluded that the renal sac is an active osmoregula- tory organ as well as an efficient extractor and storer of excretory material. THE RENAL CONCRETIONS As stated above, the concretions are dark brown in color, are birefringent in polarized light, and acquire a yellowish tinge by reflected light when spread out thinly on a glass slide. The concretions are insoluble in water and in dilute acids, but easily dissolved in diluted alkalies (NaOH, KOH). A qualitative analysis of the concretions was made to test for uric acid, xanthine, guanine, adenine, and creatinine. Folin's test yielded faint traces of creatinine ; the murexide test and microscopic ex- amination showed that a part of the concretions was made up of uric acid ; Weidel's reaction indicated the presence of moderate amounts of xanthine ; while the picrate test showed that even guanine was present. The zinc test for adenine was en- tirely negative, and the presence of hypoxanthine was also extremely doubtful. No allantoin could be traced in the concretions. As shown in Figure 8, the rate of accumulation of concretions is directly pro- portional to increase in weight of the renal sac in the young tunicate. As it grows older, however, the rate of increase of the renal sac is overtaken by the rate of depo- sition of the concretions ; and finally, when the saturation stage is reached, no more can be deposited. THE MECHANISM OF EXCRETION According to the findings of Ktipffer (1872, 1874), Sulima (1914), Millot (1923). Schmidt (1924), and Azema (1926-1929), the main excretory products are : uric acid in some Ascidiidae, xanthine in Pyuridae, and un-named purine deriv- atives in other ascidians. This picture becomes more confusing if we now add guanine (found in Molgulidae) to this list. In all animals the ultimate source of the excretory nitrogen derivatives are the a-amino-N and the nucleoproteins. A list of the main nitrogen end products of various chordates may be given here : sharks, dogfish— urea bony fishes (Teolosts) — ammonia frogs, newts- — urea turtles — urea snakes, lizards — uric acid birds — uric acid mammals — urea In tunicates, however, it appears that different families have, as the main nitrogen end product of excretion, either uric acid or one of the purine derivatives. In chordates, where we find uric acid, urea, or ammonia as the main nitrogen end product, the conversions stop at these respective steps. In the tunicate, although some of the nitrogenous matter gets converted into uric acid, a large part of it appears to stay as xanthine and guanine. We thus find in Tunicata a unique case of incomplete conversion, which accounts for the presence of xanthine and guanine, as well as uric acid. The author hopes to make exact quantitative estimations of these three constituents, in different tunicates, in the near future. 318 S. M. DAS SUMMARY 1. The present contribution includes a review of past work on excretion in Tunicata. 2. In M. manhattensis the single-celled wall of the renal sac absorbs the ex- cretory products, develops the concretions^ and discharges them into the cavity of the sac, where they are stored throughout life. 3. The concretions consist of granules, some of which show three or four con- centric lamellae caused by further deposition of excretory matter on top of the originally secreted granule. 4. The correlation between body weight, weight of renal sac, and weight of con- cretions is given. It is shown that accumulation of concretions is not uniformly maintained throughout the life of the tunicate. 5. If the adult renal sac be entirely removed, no regeneration or formation of secondary sacs takes place. 6. The renal fluid is hypcrtonic to sea water, whereas the body fluid is hypotonic. 7. The molecular concentration of the renal fluid is 3.45 gm./lOOcc. ; of the sea water at Woods Hole 3.10 gm./lOO cc. ; and of the body fluid 2.75 gm./lOO cc., corresponding to NaCl concentrations. 8. The renal concretions contain xanthine, guanine, and uric acid. ACKNOWLEDGMENT I am indebted to Dr. Parpart, director of the physiology course at Woods Hole during 1947, for allowing me a table in the physiology laboratory and giving me facilities for work. My thanks are due to Dr. Kempton, professor in charge of the section on excretion and osmoregulation, for his kind advice during the course of the investigation. I should like to express my gratitude to Dr. Kellogg, my col- league in the physiology course, for confirmation of my finding, arrived at by experiments on osmoregulation, that the renal fluid is hypertonic. to sea water; and also for estimation of the total molecular concentrations of renal fluid, sea water, and body fluid of Molgula manhattensis by the "Barger tube" method. Finally, I must thank the staff of the Woods Hole Laboratory for supplying animals when required and for the all-round kindness shown me during my stay in the summer of 1947. LITERATURE CITED AZEMA, M., 1926. Sur la formation des vesicules renales et le developpement du rein chez Ascidia mentula. C. R. Acad. dcs Sciences, 183. AZEMA, M., 1928. Quelques aspects de 1'excretion chez les Ascidies. C. R. Assoc. dcs Anat., Prague, 23 (reunion). AZEMA, M., 1929a. Note sur les cellules excretrices des Cynthiadae. Bull. Soc. Zoo!., France, 54 (13). AZEMA, M., 1929H. Sur les cellules excretrices d'Ascidia pellucida. Bull. Soc. Zoo!., France, 54 (13). BACQ, Z. M., AND M. FLORKIN, 1935. Mise en evidence, dans le complexe-ganglion nerveux, gland neurale d'un Ascidie — analogues a ceux du lobe posterieur de hypophyse des vertebres. Arch. Internal, de Physiol., 40. BUTSCHER, E. O., 1930. The pituitary in the Ascidians. /. E.r/>. Zoo/., Philadelphia, 57. PHYSIOLOGY OF EXCRETION IN MOLGULA 319 DAHLGRUN, W., 1901. Untersuchungen iiber den Bau der Excretionsorgan der Tunicaten. Arch. f. Mikr. Anat., 58. DAS, S. M., 1936. Herdmania, the monascidian of the Indian Seas. Indian Zool. Memoirs, 5 : 1-103. GEORGE, W. C, 1936. The role of blood cells in excretion in Ascidians. Biol. Bull., 71 (1). HERDMAN, W. A., 1888. Report on the Tunicata collected by H.M.S. Challenger during the years 1873-1876. Report on the scientific results of the voyage of H.MS. Challenger, Zoology, Edinburgh, Vols. 6, 14, 27. HERDMAN, W. A., 1899. Ascidia. L.M.B.C. Memoirs, 1. Huus, J., 1937. Tunicata. Kukcnthal's Handbuch der Zoologie, Berlin and Leipzig, 5 (2). KUPFFER, C. W., 1872. Zur Entwickelung der einfachen Ascidien. Arch. Mikr. Anat., 8: 358-396. KUPFFER, C. W., 1874. Tunicata. Die Zweite Deutsche Nordpolfahrt, Leipzig, 2. LACAZE-DUTHIERS, H. DE, AND Y. DELAGE, 1874. Les Ascidies simples des cotes de France. Arch. Zool. E.rp. Gen., 3: 119-174. LACAZE-DUTHIERS, H. DE, AND Y. DELAGE, 1892. Faune des Cynthiadees de Roscoff et cotes de Bretagne. Mem. Pres. Acad. des Sc., France, 45: 1-319. MILLOT, J., 1923. Le pigment purique chez les Vertebres inferieurs. Biol. Bull., France et Belgique, 57. ROULE, L., 1885. Recherches sur les Ascidies simples des cotes de Provence. Ann. des Sc. Nat. Zool. et Palaeont., 20 (6). SCHMIDT, W. J., 1924. Die Baustcinc des Tierkoerpcrs in polarisirten Licht. Cohen, Bonn. SULIMA, A., 1914. Beitrage zur Kenntnis der Harnsaurstoffwechsels niederen Tiere. Zeitschr. f. Biologic, 63. WALKER, A. K., 1930. Comparisons of total molecular concentrations of glomerular urine and blood plasma from the frog and from Necturus. /. Biol. Chem., 87 : 499. WALKER, A. K., 1937. The total molecular concentration and the chloride concentration of fluid from different segments of the renal tubule of Amphibia. Am. J. Physiol., 118: 121. THE LIFE HISTORY AND BIOLOGY OF A MARINE HARPAC- TICOID COPEPOD, TISBE FURCATA (BAIRD) 1 MARTIN W. JOHNSON AND J. BENNET OLSON Scripps Institution of Oceanography of the University of California, La JoUa, California INTRODUCTION Only a few free-living marine copepods have been reared with a view to deter- mining the details of their complete life histories. This can no doubt be attributed to the difficulty experienced in culturing most species through the entire life cycle. Considerable literature has accumulated on the morphology of developmental stages and on certain aspects of the biology, particularly of the more important planktonic calanoid species, especially Calanus finmarchicus. This species has also been reared through its entire life cycle in cultures (Lebour, 1916), and its biology studied un- der laboratory conditions more recently by Raymont and Gross (1942). The cyclopoid, Oithonina nana, has also been reared through the developmental stages (Murphy, 1923). But pertinent questions relative to the age at maturity, fecundity, and life span of individuals have received scant attention for other marine species. The fresh-water copepods on the other hand have been the subject of a large amount of investigation and much detail is known about their life histories and reproduction (see especially Gurney, 1931; Ewers, 1930, 1936). Of the marine harpacticoids, Longipedia coronata Claus, L. scotti G. O. Sars, and L. minor T. A. Scott have been reared sufficiently to reveal specially significant as- pects of their biology (A. G. Nicholls, 1935). Tigriopits fulvus (Fischer) has also been studied in considerable detail by Fraser (1936) and Shaw (1938). The mor- phology of the larval stages of a number of other marine harpacticoids has been de- scribed by various investigators, especially Chappuis (1916), Brian (1919, 1922), Gurney (1930, 1932), and Nicholls (1941). Many of the littoral species, though less conspicuous than planktonic copepods, fill an important niche as microscav- engers on the bottom, and therefore warrant a close study for ecological reasons. Tisbe furcata, also known under the generic names of Idya and Idyae (see Wil- son, 1932), is a littoral cosmopolitan species. It thrives in cultures and therefore often occurs as a contaminant in laboratory cultures of other organisms. At Scripps Institution it is constantly present in the salt-water system, aquariums, etc., to which it gains entrance from the sea through the pumping system. In 1934 it occurred in such numbers that it contributed materially to formation of flocculent detritus in the pipes. This detritus when matted together with loosened calcareous tubes of Spirorbis (also established in the system) caused serious clog- ging of water delivery jets in the aquariums. There is some possibility that "culture forms," characterized by small differences in proportions of appendage segments and strength of setae, may develop in Tisbe in these cases of isolation or semi-isolation from the "wild" population. However, 1 Contributions from Scripps Institution of Oceanography, New Series, No. 399. 320 LIFE HISTORY AND BIOLOGY OF TISBE 321 Monk (1941) considers the present form to be a new variety, johnsoni. Controlled tests were not made of the factors involved in development of culture forms, but the temperature range (about 17-21° C.) over which the animals were reared is small compared to that used by Coker (1934) in experiments affecting the form of fresh- water Cyclops. In nature Tlsbe jurcata is usually found in shallow water among algae or on the bottom where it is frequently the dominant species, but being capable of swimming, it is sometimes found also in the coastal plankton. Sars ( 1903-191 1 ) , who has given a full description of the adult, found it to be the most widespread of the harpacti- coids in Norway wherefrom he also reports a large deep-water form of the species. THE DEVELOPMENTAL STAGES During its life cycle Tisbe jurcata passes through six naupliar and six cope- podid stages, the last of which is the adult. The female is about 1.0 mm. and male 0.7 mm. long. The instars are separated by only one molt as in other copepods that have been studied. The female carries the eggs in a single egg-sac attached to the genital segment. The early cleavage and embryological stages have been studied by Witschi (1934). All drawings have been made from specimens reared in the culture experiments to be described later. The assistance of Dr. Cecil R. Monk is gratefully acknowl- edged in connection with the 1938 cultures and with the developmental stages. Naupliar Stages (Plate I, Figs. 1 to 6) In all these stages the larvae are normally benthonic, but occasionally they are taken in the plankton near shore where they have been swept off the bottom by water currents. They are colorless, subcircular in outline and possess a small red eye spot. Nauplius Stage I ( Fig. 1 ) . — Average length 0.062 mm. ; labrum circular flap ; first antennae three-segmented, the distal segment with three terminal setae; the second antennae strongly built, the endopod terminating in a prehensile hook, exo- pod normal. The first basis of the second antennae is provided with a large but rudimentary masticatory blade or hook with a small seta at its base, and the second basis bears two strong spines. The mandibular palp possesses a splender exopod of three indistinct segments, the distal segment bearing one short and one very long seta ; the endopod bears two strong short hooks and two setae. The caudal armature consists of two rather long flaccid setae. Nauplius Stage II (Fig. 2). — Average length 0.082 mm. The obvious structural advancements over Stage I consist of increase in strength of the masticatory blade of the second antennae and the appearance of two strong setae, one each on either side of the posterior ventral side. These setae are the earliest visible fundaments of the first maxillae. This early appearance of the maxillae is characteristic of the harpacticoids. In the strange nauplius larvae of Longipedia the first maxillae are present already in the first stage (Nicholls, 1935). In this respect the development of harpacticoids is similar to that of many cyclopoids. In calanoids the rudiments of these appendages first appear in the third or fourth nauplius stage. 322 MARTIN W. JOHNSON AND J. BENNET OLSON PLATE I Development of Tisbe furcata FIGURES 1-6. Nauplius stages I to VI — ventral : all drawn to same scale with aid of camera lucida. LIFE HISTORY AND BIOLOGY OF TISBE 323 PLATE II Development of Tisbe furcata (Camera htcida drawings) FIGURE 7. Copepodid stage I — dorsal. FIGURES 8 and 9. First and second feet — Copepodid stage I. FIGURES 10, 11, AND 12. First, second and third feet — Copepodid stage II. FIGURES 13, 14, 15, 16, AND 17. First, second, third, fourth, and fifth feet — Copepodid stage V — female. FIGURE 18. Posterior ventral portion of urosome of male Copepodid stage IV to show sixth feet as represented by spines. 324 MARTIN W. JOHNSON AND J. BENNET OLSON Nauplius Stage III (Fig. 3). — Average length 0.110 mm. As in II, but with three marginal setae on the distal segment of the first antennae, one additional seta on the endopod of the mandibular palp, and one short seta issuing from the outer angle of each caudal ramus. The original caudal setae are now much longer and more rigid than before. Nauplius Stage IV (Fig. 4). — Average length 0.130 mm. As in III, but posterior segment of body more clearly defined. The distal segment of first anten- nae with increased number of marginal setae that can be discerned only with diffi- culty and are therefore not useful in identification as in calanoid larvae. The endo- pod of mandibular palp with four setae ; the first maxillae, each showing one segment with three spines and a long seta; each caudal ramus with two short outer setae at the base of the long seta. Nauplius Stage V (Fig. 5). — Average length 0.156 mm. As in IV, but the rudimentary first maxillae have two segments each, the distal segment with two strong curved spines and the proximal with one spine and one seta. Caudal ramus each with an additional short seta set at the inner base of the long seta making a total of three short setae. Nauplius Stage VI (Fig. 6). — Average length 0.178 mm. As in V, but distal segment of first antenna with five marginal setae; one-segmented rudiment of sec- ond maxilla is evident with one short seta; rudiments of first and second pairs of legs present but the maxillipeds were not evident. Caudal armature as in V, but the formerly short setae are much longer, especially the inner pair. Copepodid Stages (Plate II, Figs. 7 to 18) The sixth nauplius stage metamorphoses to the first of six copepodid stages, the last of which is the adult. The period required to pass from hatching of the first nauplius to the last copepodid is about 16 days at temperatures of 17° to 21° C. The first copepodid stage is 0.20 mm. long and resembles the adult in general ap- pearance, but there are present only the first two pairs of feet with a rudiment of the third pair and the metasome and urosome have respectively only three and two divisions, using the terminology of Sars. In subsequent stages one additional pair of feet is added for each molt except the fifth. The order of segmentation of the feet follows the same pattern as that found in calanoid copepods. Each pair of feet at the time of its appearance possesses only one segment in each ramus, and in the next following molt each ramus becomes two- segmented and remains thus until the fifth molt (Copepodid V) when the rami of all the feet, 1 to 4, acquire three segments each (Plate II, Figs. 13-16). The new segment is derived from the proximal portion of the distal segment. This is il- lustrated in the first pair of feet which already in the fifth copepodid stage furnish diagnostic features of the genus. The fifth pair of feet which furnish the specific characters are not completely diagnostic until the sixth copepodid stage. The sexes are readily separable morphologically in the fourth copepodid stage. The male in this stage is not only smaller in body size but the sixth pair of feet is already clearly developed, each foot represented by a small spine (Plate II, Fig. 18). LIFE HISTORY AND BIOLOGY OF TISBE 325 In the female these feet are wanting or indistinguishable from the row of fine spines on the urosome. THE BIOLOGY OF TISBE FURCATA Culture experiments Syracuse watch glasses and low stender dishes of 50 ml. capacity were used for culture dishes in order to facilitate direct examination under the low-power microscope. The water used in the dishes was filtered to eliminate the possibility of contamination with foreign eggs or nauplii from the general sea-water supply. A fairly constant temperature was maintained by keeping the culture dishes on a table of running sea water, though the animals thrive in dishes kept at ordinary room temperatures. A variety of marine foods was tested including ground dried mussels and scal- lops, dehydrated kelp, fresh seaweed and fecal pellets of Nereis. Each of these given individually in very small amounts was quite satisfactory as food. Shavings of fresh brown seaweed or dehydrated kelp appeared to be the best single food. Fresh seaweed has the disadvantage that the slime secreted tends to entrap the or- ganisms. In general, however, the cultures were fed various mixtures of the above items so that some measure of selection was possible for the animals. About twice a week the dishes were cleaned by removing the debris and about one half of the water with a pipette, and a fresh supply of water and food added. Life history series were obtained in four separate years, i.e., March-May 1935, June-July 1938, April-June 1942, and Sept.-Dec. 1947. During this time 30 or more broods and some of their progeny for two generations were studied more or less completely. During these periods no marked seasonal changes were observed in the breeding habits, or rate of reproduction, though a somewhat higher average number of eggs per egg-sac was noted in December. Since observations on the 1942 series were the most continuous, they will form the main basis for discussing the salient features in the biology and life cycle of this species. Where data can be drawn from the earlier or later cultures, they will be made a part of the summary of time involved in the various phases or periods of the life cycle. The lapse of time given for the various periods is the minimum time, or the average of several observed minima. For example, the first appearance of a nauplius in a culture was set as the beginning of the naupliar period and the first appearance of a copepodid marked the end of the naupliar period for any one brood. Number of eggs produced In order to obtain an estimate of the average number of eggs produced by each female per brood, counts were made of the eggs in the brood sacs of ten ovigerous females selected at random from the general population in March 1942. The num- ber ranged from 29 to 82, with an average of 42.9 eggs per sac. There is obviously a very wide range in the number of eggs produced per egg-sac depending upon how long the individual has been laying eggs, and perhaps also upon the season. A check count was made in December 1947 in which the numbers ranged from 45 to 93 with an average of 72 eggs in each of 12 egg-sacs. Kunz (1935) reports 30 to 41 eggs for the species in Kiel Bay. 326 MARTIN W. JOHNSON AND J. BENNET OLSON A group of ten ovigerous females was selected at random from the same popu- lation from which the 1942 egg counts were made and isolated for experimental culture, two females per dish. Each female was removed from the dish as soon as her eggs had all hatched. Thus each of the five culture dishes contained the nauplii from two egg-sacs. Careful account was kept of each population. As they matured and the adults mated, each mating pair was removed, recorded, and placed in an- other dish. In this way an accurate census was obtained. Success of hatching and survival From the ten broods 343 individual animals reached maturity. This indicates about 80 per cent survival, if we assume that the average egg-sac contains 43 eggs. Incubation period The duration of the incubation period was approximated by noting the first ap- pearance of the egg-sac and the subsequent hatching of the nauplius larvae from the sac. The minima obtained ranged from two to four days. On the basis of the average of 21 cases the period was 2.5 days. Usually the cultures were examined every one to three days. Naupliar period The time required for completion of the separate stages was not studied. Dur- ing the naupliar stages the animals creep about actively over the bottom or cling to detritus, etc., by means of prehensile hooks on the second antennae (Plate I, Figs. 1-6). The duration of this period is from three to eight days, with an average minimum of five days based on 22 brood histories. Sex ratios Of the 343 individuals reaching maturity 114 or only 33.3 per cent were fe- males. One culture dish produced 34 females and 33 males, but in all of the rest the males were in great majority. In order to compare this unequal sex ratio in the culture dishes with that of the general population an examination was made of 44 adults from the water system. Of these only 41 per cent were females. Additional analysis involving 98 specimens reared in December 1947 also showed a ratio of one female to two males. The numerical dominance of males is of considerable interest since usually in other species of copepods the females are the more numerous, sometimes over- whelmingly so. This inequality of the sexes is often seasonal, but no seasonality has come to light in the study of Tisbe. In a study of the tide pool harpacticoid Tigriopus julvus, Tsen-Hwang Shaw (1938) found 58.8 per cent females in a collection of 318 specimens. Sexual maturity A study of the rate at which sexual maturity was attained by 87 pairs of the 343 individuals hatched from the ten egg-sacs mentioned above, showed that two LIFE HISTORY AND BIOLOGY OF TISBE 327 pairs began mating (as indicated by clasping) just 14 days after they had hatched. The last four pairs mated in 25 days. The average time required to reach mating maturity after hatching was 16 days. The minimum period observed in earlier ex- periments at Scripps Institution was ten days. The period of clasping varied from a few hours up to one or more days, but there was no evidence that the actual trans- fer of the spermatophore to the female is delayed until she molts to the last or sixth copepodid stage. Clasping was rarely observed before the female had reached the sixth copepodid stage, and on only one occasion was there evidence of molting during the clasping period. This point deserves further study. Williams (1907) found that for the harpacticoids, Harpacticus uniremis, H. gracilis, and Tachidus Httoralis, "every successful copulation must be prolonged until the female molts." Length of generation The lapse of time between the hatching of one generation and the first appearance (hatching) of the next generation resulting from the above matings varied from 17 to 24 days at temperatures of 17° to 18° C. during the spring months. The maxi- mum period observed in one case was 31 days. In 1935, two individuals required 20 days and one individual only 16 days. MacGinitie (1937) in an incidental statement records that this species completes its life cycle in ten days at Corona del Mar, California. The time required for Tisbe to develop from egg to production of eggs is com- parable to that reported for various species of the freshwater cyclopoid Cyclops. Ewers (1936), for example, found that for 12 species under observation the time varied from 8 to 50 days. It appears that Tisbe furcata may complete its life cycle in about one-half the time required for Tigriopits fulvus which under favorable conditions in the labora- tory at University College of Hull required about two months (Fraser, 1936). However Tsen-Hwang Shaw (1938) states that for this species at Pacific Grove, California, the adult stage is reached in about one month. It is probable that the Pacific Grove species is distinct from T. fulvus, since Monk (1941) mentions only T. californicus from the coast. Number of broods produced In order to determine the approximate number of broods produced by a single female, four of the above 87 pairs in copula were isolated in individual dishes. Several additional males were added to each dish to afford ample opportunity for additional matings, and the males were replaced from time to time with younger ma- ture males. (Lowndes, 1933, and Fraser, 1936, infer that the presence of males stimulates egg production.) Nattplii were removed from the dishes as soon as they appeared. Records were kept of the number of egg-sacs produced by each female under these conditions. One female produced 12 broods between May 9 and June 10. Two other females each produced nine broods in the same period and one produced five broods in 20 days before dying, apparently prematurely. The first egg-sacs produced were the largest, and the last two or three produced were about half the size of the first. The egg-sacs appeared with regularity, two to five days elapsing between hatchings with three days as the average period. <-u 328 MARTIN W. JOHNSON AND J. BENNET OLSON Although males were kept in the presence of the females during the whole pe- riod, only one mating was observed to take place and this preceded the entire period of egg laying. In order to test further this observation in a more crowded con- dition as often occurs in nature, four separate cultures were set up each containing from 8 to 20 pairs of newly matured adults in copula. A total of 54 pairs were in- volved. All pairs in any one dish were from the same brood and therefore of the same age. At two day intervals all adults were transferred to fresh culture dishes so that their generation could be kept separate from the younger generation con- stantly being produced. These adults were maintained from May 12 to June 12, and during the time following the separation of the pairs originally mated, only one in- stance of a second clasping was observed. This instance occurred 21 days after the original mating, and it is not known if a spermatophore was transferred. The pair concerned was isolated from the rest of the culture in order to determine if the fe- male would continue to produce eggs for a longer time than the other females as a result of this second mating. However, no additional eggs were carried by her. This is in agreement with the earlier observations where in one case a succession of seven broods was produced by a female that had been reared in culture and isolated following one mating. She lived 70 or 71 days but produced no eggs after the 53rd day. In another test 11 ovigerous females isolated from the general population produced two to five broods, but it is not known if previous broods had been pro- duced before isolation. Nicholls (1935) has reported a case in which a female speci- men of Longipedia scotti had produced at least nine broods during a period of iso- lation from contacts with males. The question naturally arises as to whether or not the male of Tisbe produces a succession of spermatophores and remains fertile after the first mating. To test this, a single male was isolated from a group of newly matured virgin males. Sev- eral virgin females were added to the dish at intervals, and records were kept of the fertility of eggs subsequently produced by each female. Out of a total of 13 virgin females brought into contact with this one male, six produced fertile eggs which hatched. The other seven females produced eggs which failed to hatch. In another test one male fertilized eight females which produced fertile eggs. Virgin females kept in isolation have produced full egg-sacs, but in no instance was there any evidence of parthenogenesis as reported by Roy (1931) for the harpacticoid Canthocaniptus bidens. Pine (1934) has reported parthenogenetic reproduction in Cyclops viridis. This, however, appears to be a misinterpretation resulting from failure to note that several egg-sacs with fertile eggs may be produced following only one mating. The females with which she worked were doubtless already fertile since they were isolated in the gravid condition from the wild. DISCUSSION » Rather little is known regarding the kinds of predators that feed directly upon the littoral copepods. That this type of animal food constitutes an appreciable source of nourishment for littoral animals is suggested by the fact that only a moderate population of Tisbe has been observed in the sea despite a relatively rapid and steady rate of replenishment as suggested by the present study. Being detritus feeders with a wide range of acceptable food, it is probable that in favorable LIFE HISTORY AND BIOLOGY OF TISBE 329 habitats Tisbe population density is controlled largely by predators. Among these predators are no doubt small fish capable of capturing small prey of this kind by pursuing and picking up the copepodid stages individually, and coelenterates hav- ing tentacles supplied with nematocysts and viscid surfaces that enable capture by chance contact. Various detritus feeders scooping up particles of detritus would consume together with this many Tisbe, especially the nauplii, that are prone to cling to particles upon which they are feeding. From the present study it appears that an average of about 513 eggs is produced by each female in her lifetime. This is calculated from an average of 57 eggs per brood (using the averages of March and December) and a total of nine broods. For unknown reasons only about 80 per cent of the larvae hatched in the cultures survived to adult state. Thus, using the above figure for one female, 410 survive, of which only about 135 (approximately one third) are females. Considering a suc- cession of generations each 25 days, a prodigious number of progeny is possible, for in about 100 days (fourth generation) over 1 billion 55 million individuals of both sexes would be produced. Ten males plus 5 females make up a mass of about 1 mm.3. Hence in 100 days 70 liters of copepods would have been produced. TABLE I Potential rate of reproduction in Tisbe furcata and Calanus finmarchicus in cultures No. of broods or spawnings No. of eggs per brood or spawning Total no. of eggs in life- time of one 9 Interval between generations Survival to adult Tisbe 7-12 average 9 29-93 average 57 513 15-31 days usually 19-24 days 80% Calanus 1-3 1-120 Maximum Minimum 41 ? (4?) usually 15-70 observed 120 days These figures can of course have no significance other than to emphasize that the animal substance which Tisbe could supply is considerable and that a very large number of copepods must be consumed regularly by enemy predators in order to keep a balance between consumption and reproduction. It is instructive to compare certain vital aspects of the life history of this im- portant littoral species with similar aspects of Calanus finmarchicus, the most widely studied of planktonic-copepod species. In view of the vast swarms of Calanus which sometimes occur in the sea, one might expect that studies of its rate of reproduction would reveal aspects suggesting possibilities of greater individual fecundity than occurs in Tisbe, a seemingly less abundant species. In no instance, however, is a greater rate of reproduction shown for Calanus on the basis of studies that have thus far been made in cultures. Table I compiled mainly from Nicholls (1933), Clarke and Zinn (1937) and Raymont and Gross (1942) shows the great discrepancies that are brought out when comparing these two genera of widely different ecological habits. 330 MARTIN W. JOHNSON AND J. BENNET OLSON The life span of Calanus is greater than that of Tisbe, but this appears to have no bearing on the fecundity since the number of spawnings is not shown to be affected thereby. Raymont and Gross found that under laboratory conditions Calanus was capable of spawning throughout the year, similar to our observations for Tisbe, but field observations by numerous investigators show that the rate of re- production for Calanus in the sea varies greatly with season, there being a marked minimum in autumn and winter when the animals survive in copepodid Stage V. No similar field data are available for Tisbe. The usually great preponderance of females over males in Calanus should en- hance the reproductive rate provided this preponderance obtains also for the hatch- ing eggs and provided further that there are always sufficient males in the population. That the latter is probably true is indicated by the study of Gibbons (1933). " There is some evidence that in Calanus, as in Tisbe, one mating may suffice for continued spawning, since females have been observed to produce fertile eggs three or four weeks after isolation. The greater ratio of males to females in Tisbe seems to have no logical explana- tion, since it is shown that one mating suffices for all the broods the female can pro- duce, and each male produces a sufficient number of spermatophores to fertilize seven or eight females. In considering this anomalous situation with respect to the relative reproductive rate of the two genera in cultures contrasted with their abundance in nature, the answer might be found in the greater ease with which Tisbe can be reared in culture. Hence, a truer picture might be obtained for that genus, but one that is difficult to check in the field. It is probable that in nature Calanus actually reaches reproductive maturity at a younger age and produces more eggs than have thus far been shown in culture experiments or deduced from field observations. An obvious alternative is that the survival rate is greater for Calanus (possibly because of greater oppor- tunity for dispersal both vertically and laterally). The conclusion must then be that Tisbe is heavily preyed upon in its natural habitat. SUMMARY 1. Tisbe jurcata is a littoral copepod that commonly invades salt-water systems connected with the sea. It is readily reared through all of its developmental stages. Being a scavenger, it thrives on various types of food, but thin slices of fresh sea- weed and dehydrated kelp were especially acceptable. 2. Following the egg, there are six naupliar and six copepodid stages, the last of which is the adult. Each stage is separated by one molt. 3. The incubation period is from two to four days, usually about 2.5 days. 4. The total duration of the naupliar stage is three to eight days, usually about five days. 5. The first indication of sexual maturity as shown by clasping by the male oc- curred between the 10th and 25th days, usually about the 16th day. 6. The minimum time between generations (i.e., from egg to egg production) was 15 days, but usually between 19 to 24 days. 7. The span of life of individuals varied greatly. It was studied mainly in the females, some individuals of which lived for 40 to 50 days. The oldest specimer had a life span of 70 or 71 days, but no eggs were produced after the 53rd day. LIFE HISTORY AND BIOLOGY OF TISBE 331 8. The number of broods indicated by egg-sacs produced by isolated females varied from 7 to 12, with an average of about 9. Following the first egg sac, the subsequent ones appeared at intervals of two to five days, usually about three days. 9. The number of eggs in a brood varied from 29 to 93 with an average of 43 in one sampling and 72 in another. 10. Each female mated but once, and this mating sufficed for fertilization of all of the eggs to be produced. Males were capable of several matings. 11. About 80 per cent of the larvae hatched survived to adult state. 12. There is no evidence of parthenogenesis. LITERATURE CITED BRIAN, ALESSANDRO, 1919. Sviluppo larvale della Psamathe longicauda Ph. e dell' Harpacticus uniremis Kroy. Atti delta Societa Italiana di Sciense Naturali, LVIII : 29-58. BRIAN, ALESSANDRO, 1922. The Alteutha depressa Baird (harpacticoid copepod) and its larval stages. Monitore Zoologico Italiano, Anno XXXIII, N. 1-3 : 8-14. CHAPPUIS, P. A., 1916. Die Metamorphose einiger Harpacticidengenera. Zoologischer An- zeigcr, 48: 20-31. CLARKE, G. L., AND DONALD J. ZINN, 1937. Seasonal production of zooplankton off Woods Hole with special reference to Calanus finmarchicus. Biol. Bull., 73 : 464-487. COKER, R. E., 1934. Influence of temperature on form of the freshwater copepod, Cyclops vernalis Fischer. Intern. Rev. dcr gcsam. Hydrobiol. und Hydrog., 30: 411-427. EWERS, LELA A., 1930. The larval development of freshwater Copepoda. Ohio State Univ., F. T. Stone Lab. Contrib. No. 3. EWERS, LELA A., 1936. Propagation and rate of reproduction of some freshwater Copepoda. Trans. Amer. Micros. Soc., 55: 230-238. FRASER, J. H., 1936. The occurrence, ecology and life history of Tigriopus fulvus (Fischer). Jour. Mar. Biol. Assoc., 20 : 523-536. GIBBONS, SYDNEY G., 1933. A study of the biology of Calanus finmarchicus in the north- western North Sea. Fisheries, Scotland, Sci. Invest. No. 1 : 3-23. GURNEY, R., 1930. The larval stages of the copepod Longipedia. Jour. Mar. Biol. Assoc., N. S., 16 : 461-474. GURNEY, R., 1931. British fresh-water Copepoda. Ray Society, London, 1 : 1-230. GURNEY, R., 1932. British fresh-water Copepoda. Ray Society, London, 2 : 1-326. KUNZ, HELMUT, 1935. Zur Oekologie der Copepoden Schleswig-Holstein und der Kieler Bucht. Schr. Naturiviss. Ver. f. Schlesu'ig-Holstein, 21 : 84-127. LEBOUR, M., 1916. Stages in the life history of Calanus finmarchicus. Jour. Mar. Biol. Assoc., 11: 1-17. LOWNDES, A. G., 1933. Sexual reproduction in copepods. Nature, 131 : 240-241. MACGINITIE, G. E., 1937. Notes on the natural history of several marine Crustacea. Am. Midland Naturalist, 18: 1031-1037. MONK, CECIL R., 1941. Marine harpacticoid copepods from California. Trans. Amcr. Micros. Soc., 60 : 75-99. MURPHY, H. E., 1923. The life cycle of Oithona nana, reared experimentally. Univ. Calif. Publ. Zool., Berkeley, 22 : 449-454. NICHOLLS, A. G., 1933. On the biology of Calanus finmarchicus. I. Reproduction and seasonal distribution in the Clyde Sea-Area during 1932. Jour. Alar. Biol. Assoc. U. K., N. S., 19: 83-110. NICHOLLS, A. G., 1935. The larval stages of Longipedia coronata Claus, L. scotti G. O. Sars, and L. minor T. and A. Scott, with a description of the male of L. scotti. Jour. Mar. Biol. Assoc. U. K., 20 : 29-45. NICHOLLS, A. G., 1941. The developmental stages of Metis jousseaumei (Richard) (Copepoda, Harpacticoida). Annals and Magazine Nat. Hist., Ser. 11: 317. PINE, ROSE L., 1934. Metamorphosis of Cyclops viridis. Trans. Amer. Micro. Soc., 53 : 286- 292. 332 MARTIN W. JOHNSON AND J. BENNET OLSON RAYMONT, J. E. G., AND F. GROSS, 1942. On the feeding and breeding of Calanus finmarchicus under laboratory conditions. Proc. Roy. Soc. Edinburgh, Sec. B, 61 : 267-287. ROY, JEAN, 1931. Sur 1'existence de la parthenogenese chez une espece de Copepodes (Elaphoidella bidens). C. R. Acad. Sci. Paris, 192: 507-508. SARS, G. O., 1903-1911. Crustacea of Norway. Copepoda, Parts I and II Harpacticoida, Bergen Museum, 5 : 1-449. SHAW, TSEN-HWANG, 1938. Some observations on the life history of a tide-pool copepod, Tigriopus fulvus (Fischer). Bull. Fan Memorial Inst. Biol., Zool. Ser., 8: 9-16. WILLIAMS, L. W., 1907. The significance of the grasping antennae of harpacticoid copepods. Science, N. S., 25 (632) : 225-226. WILSON, C. B., 1932. The copepods of the Woods Hole region, Massachusetts. U. S. Natl. Mus. Bull, 158 : 1-635. WITSCHI, E., 1934. On determinative cleavage and yolk formation in the harpacticoid copepod, Tisbe furcata (Baird). Biol. Bull, 67: 335-340. FURTHER CHEMICAL ASPECTS OF THE SENS1TIZATION AND ACTIVATION REACTIONS OF NEREIS EGGS PAUL G. LEFEVRE Marine Biological Laboratory and College of Medicine, University of Vermont The peculiar effects of 2,4,6-trinitrophenol (picric acid) in relation to the artificial activation of the eggs of Nereis limbata have been described in an earlier re- port (LeFevre, 1945). Discovery of these effects grew from a reinvestigation of a few experiments by Heilbrunn (1925) concerning the enhancing effect of acidifica- tion on the heat-activation of these eggs. Among a series of rather unrelated or- ganic acids tested, only picric acid exerted a reliable activity of this sort, and further experimentation with this compound revealed a rather paradoxical set of properties with regard to the egg activation. Thus, at a concentration of about M/1000 in sea water, trinitrophenol prevented breakdown of the germinal vesicle, and ac- companying cytoplasmic reorganization, ordinarily produced in these cells by ex- posure to heat (Just, 1915), excess potassium ion, or decalcifying agents (Wilbur, 1941). At the same time, exposure of the eggs to the same concentration of trinitrophenol rendered them subsequently hypersensitive to these same activating agents, so that upon removal from the acid they could be activated by exposure to these agents in doses too small to activate untreated eggs. This development of hypersensitivity was progressive with continued exposure to trinitrophenol, until after several hours the eggs were activated simply by removal to sea water, without application of any additional chemical or physical stimulant. The eggs remained in this state of hypersensitivity in the trinitrophenol for as long as three days, by which time in sea water they would long since have cytolyzed. Various hypotheses were considered in an effort to integrate these diverse in- fluences of trinitrophenol on the reactions of activation "into a simple coherent pat- tern, in relation to some of the established factors relating to this process in the Xci'ds egg. The importance of the calcium ion in this connection (Heilbrunn and Wilbur, 1937; Wilbur, 1939, .1941) directed attention to the question of a calcium picrate complex formation, but there seemed to be no physicochemical basis for this ; complications confronting interpretation of the effects of trinitrophenol in terms of a calcium-release theory are considered in the previous report (LeFevre, 1945). The least involved interpretation capable of explaining the behavior of the trinitrophenol seemed to be that this substance might form a reversible combination with some sub- stance produced in the course of the cell's metabolic activities; that this substance could precipitate the breakdown of the germinal vesicle, but that it is normally re- moved by chemical reaction or by diffusion from the cell before activating concentra- tions are attained. This hypothesis would attribute the stimulating effects of the various agents to an increased rate of production of the activating metabolite, which can then take part in the critical reactions. Inhibition of this stimulation by trini- trophenol was attributed to formation of an inactive complex between the acid and the hypothetical activating substance. This interpretation accounted for the pro- 333 334 PAUL G. LEFEVRE gressive development of hypersensitivity to activating agents during exposure to trinitrophenol, since removal from the acid bath to ordinary sea water, with ac- companying rapid loss of picrate, would then release the accumulated activator, and would be equivalent to the application of an activating agent. The original report may be consulted for a more complete consideration of this hypothesis in the light of the earlier experiments. The present report is an extension of the above in- vestigation in an attempt to elucidate the nature of the hypothetical reactions sug- gested by the original work. MATERIALS AND METHODS Two main methods of approach have been employed : chemical disturbance of the egg-suspension medium, either during the sensitizing procedure with trinitrophenol or during application of chemical activating agents ; and treatment of the eggs with substituted phenols other than picric acid, to ascertain the molecular specificity of the activities described for the latter. The materials and general handling procedures have been previously described (LeFevre, 1945). In those experiments reported below involving treatment of egg-suspensions with gas mixtures, the appropriate quantities of carbon dioxide, un- purified commercial nitrogen, and air were drawn into a glass vessel of several liters capacity, by removal of water through a siphon ; from this vessel the mixture was similarly forced in fine bubbles through 25-35 ml. of the egg-suspension in a 10--15 cm. column in an ordinary test tube. In addition to gas mixtures, specific metabolic inhibitors tested included potassium cyanide, sodium azide, sodium iodoacetate, hy- droxylamine, /'-chloromercuribenzoic acid, cupric chloride, urethane, and diethyl ether. Substituted phenols tested included 2,4,6-trinitrophenol, 2,4-dinitrophenol, o-nitrophenol, ^-nitrophenol, 2,6-dichlor,4-nitrophenol, o-chlorophenol, and />-chlo- rophenol. The artificial sea water medium used in some of the experiments was made up by mixing of several pure salt solutions isotonic with sea water : NaCl, 0.52 M, 500 volumes; KC1, 0.53 M, 10 volumes; MgCU, 0.37 M, 40 volumes; MgSO4, 0.96 M, 15 volumes ; NaHCO3, 0.52 M, 2 volumes j" CaCL, 0.34 M, 15 volumes. For those experiments involving upset of the normal proportions of the ingredients of sea water, corresponding changes were made in the relative volumes of the isotonic sol- utions mixed, so that the resultant surplus or deficit in total electrolyte content was taken up by all the other ingredients in their usual proportions. RESULTS I. Experiments concerning reactions accompanying activation a. Effects of anoxia The dependence of the germinal vesicle breakdown response on aerobic processes was tested only in the case of activation by removal to sea water from prolonged exposures to trinitrophenol (10~3 M in sea water). There was no evidence of activation when the eggs were transferred to sea water through which a mixture of 95 per cent N2, 5 per cent CO2 had been passed for about thirty minutes, while 100 per cent activation was ordinarily obtained in undisturbed sea water or in sea water EGG SENSITIZATION AND ACTIVATION 335 gassed with air or 95 per cent air, 5 per cent CO,. This inhibition of activation in a nitrogen atmosphere was reversible for several minutes ; Table I shows a typical instance of this, in which the eggs, upon removal from the anoxic bath to ordinary sea water, were activated in progressively diminishing numbers as the interval in the anoxic medium was extended. b. Effects of metabolic inhibitors The usual chain of events following removal of eggs from trinitrophenol baths to sea water was similarly inhibited by ether, potassium cyanide, or sodium azide ; these inhibitors, however, did not prevent a slight elevation of the membrane, and the very first indications of the onset of nuclear reorganization, as previously de- scribed in eggs exposed to mixtures of sea water and isotonic KC1 or sodium citrate in the presence of trinitrophenol (LeFevre, 1945). This initial disturbance was "frozen" at a very early stage, so that the germinal vesicles retained their identity, and none of the cytoplasmic rearrangements consequent to vesicular breakdown oc- curred. If, subsequently, the inhibitor was removed, even after several hours of TABLE I Reversible inhibition of activation by anoxia Sea water under atmosphere of: Per cent activation Air 98 95% N,, 5% C02 0 Removed, exposed to air after 1 min. 76 3 min. 60 5 min. 54 10 min. 32 Activating procedure: eggs removed to sea water, under atmosphere indicated, after exposure for three hours to trinitrophenol, M/1000, in sea water. such suspended activation, the usual processes continued normally; i.e., the in- hibition was entirely reversible. In the case of ether, in fact, gradual evaporation of the narcotic led ultimately to a resurgence of the activation reactions, without actual removal of the eggs to fresh sea water. A typical set of experiments is pre- sented in Table II. It may be noted that this reversibility outlasted that observed in anoxia by a wide margin, but this may have been attributable to the more rapid onset of irreversible injury to the eggs in the absence of oxygen. The critical concentra- tions for inhibition were those used in the experiments cited in Table II. The same concentrations of ether, azide, or cyanide were equally effective in preventing activation in a few tests made with mixtures of sea water and isotonic KC1 or sodium citrate (Table II) ; reversibility of this inhibition was not tested, but the high percentages of "incipient activation" observed would indicate the same state of affairs as seen with these inhibitors following the sensitization procedure. Hydroxylamine, at 10~2 M, also prevented activation by these agents, but the in- hibition was irreversible, and although there was no immediate obvious morpho- logical change, inhibition in this instance probably indicated nothing more than com- plete inactivation of the cells. 336 PAUL G. LEFEVRE The other inhibitors tested in this same connection, rather than preventing ac- tivation, proved to be themselves activating agents. The same nuclear and cyto- plasmic changes accompanying treatment with excess potassium, heat, etc., were seen when the eggs were exposed to sodium iodoacetate, at ca. 2-10~2 M, or to urethane, at ca. 5 • 10~3 M. At these concentrations, and throughout a fairly ex- tensive range above these figures, 90-100 per cent germinal vesicle breakdown was regularly observed and was frequently followed by extensive irregular cleavage, though no swimming forms were found. Activation by these means resembled that induced by other agents in that it failed in the presence of trinitrophenol at 10~3 M. />-Chloromercuribenzoate or CuCU (like iodoacetate, inhibitors of sulfhydryl ac- tivity) also activated the eggs, at concentrations in the neighborhod of 10~5 M ; but this reaction was less easily reproducible than with the other stimulating "inhibitors," as the threshold concentration for activation was only slightly lower than the lytic TABLE II Reversible inhibition of activation by metabolic inhibitors Activating agent Inhibitor Per cent activation Without inhibitor With inhibitor Inhibitor removed after 7 hours Sea water, after 5 hours in trinitrophenol, M/1000, in sea water Ether, 1% KCN, 10-3 M NaN3, lO-2 M 100 100 100 0, 80* 0, 100 0, 50 100 100 100 KC1, 20 vol. isotonic, to 80 vol. sea water Ether, 1% KCN, lO-3 M NaN3, lO-2 M 100 100 100 0, 70 0, 75 0, 100 Sodium citrate, 25 vol. iso- tonic, to 75 vol. sea NaN3, 10~2 M 94 0, 90 water * The second figure in this column is the approximate percentage of eggs in the inhibitor showing the condition of "incipient activation" (elsewhere described). concentrations, and there was some variability in the effective concentrations for dif- ferent batches of eggs. A further difference in the activation in these instances is that that induced by Cu++ or />-chloromercuribenzoate was not interfered with by trinitrophenol. Thus activation by these agents appears not to be comparable to that otherwise induced, but more analogous to the prelytic activation seen in other instances (Loeb, 1913; Lillie, 1926). c. Effects of ionic components of sea ^vater The regular response of the eggs upon removal to sea water after several hours' exposure to trinitrophenol, 10~3 M in sea water, raised the question of what components of sea water were essential to this response. If the eggs were removed from the conditioning bath not to sea water, but to isotonic solutions of either NaCl, KC1, CaCl2, or MgCU, no stimulatory reaction was ever observed. Mixtures of EGG SENSIT1ZATION AND ACTIVATION 337 various proportions of these basic ingredients were then tested. In the absence of magnesium, there was always such a distortion of the egg contours, with extreme membrane elevation and discoloring, that it was difficult to analyze other differences that might appear. It was however evident that the addition of calcium, in the ab- sence of magnesium, greatly augmented these disruptive changes, leading to de- cided cellular deformation and nuclear disarrangement resembling in some respects the changes that occur in activation, and frequently to extensive cleavage, but with- out any change in the appearance of the cytoplasm. The deleterious effects were further exaggerated in the presence of bicarbonate buffer, with a characteristic red- brown discoloration and vesiculation of the protoplasm. In an isotonic solution containing MgCl,, NaCl, and NaHCCX, with the Na+, Mg++, and HCCr in pro- portions similar to those found in sea water, the eggs remained perfectly normal in appearance. But this mixture was not capable of inducing the reactions of activa- tion in eggs transferred from a conditioning bath with trinitrophenol. Activation re- quired the presence of at least a reasonable trace of calcium ion ; and at a given cal- TABLE III Action of calcium and potassium ions in initiation of activation Per cent activation with calcium ion concentration of: Concentration of potassium ion 0* 0.01 M 0.02 M 0.04 M 0* 0 0 50 66 0.005 M 0 93 92 98 0.01 M 0 100 100 100 0.02 M 3 100 100 100 * On basis of absolute freedom of reagents from Ca++, K+-contamination, and ignoring small amount carried over in sea water with eggs. Medium was made up of NaCl, 0.47 M; MgCb, 0.037 M; NaHCO3, 0.0026 M; plus CaCl2, KC1 as indicated for each case. Eggs removed to medium indicated after exposure for 7 hours to trinitrophenol, M/1000, in sea water. cium ion level the reaction was augmented by increased potassium ion concentration, as in the typical experiment shown in Table III. If both calcium and potassium were present at M/100, the reaction was as easily elicited as in sea water. To some extent, increased concentration of magnesium ion antagonized this stimulatory ac- tivity, so that more calcium and potassium were required to elicit the same percent- age of response. The specific necessity for calcium was verified by varying its con- centration in the otherwise fairly complete artificial sea water medium described in an earlier section ; reduction of the calcium level to half the normal figure produced a noticeable diminution in the percentage of response, although there was still some degree of response even with only one-tenth of the normal calcium concentration. The necessity of calcium, in initiating the stimulatory changes upon removal from exposure to trinitrophenol, was immediate, as shown in Table IV. Addition of calcium to the system only 60 seconds after the removal of the trinitrophenol did not result in a significant amount of activation. Thus in this sense the inhibition of activation by calcium-lack may be said to be irreversible. This effect is not, how- ever, attributable to general injury to the cells because of the lack of calcium, as the 338 PAUL G. LEFEVRE same cells may subsequently be activated by any of the usual procedures. Also, the eggs remained fertilizable for as long as eight hours in artificial sea water as nearly Ca-free as the purity of the reagents permitted. Such eggs, however, al- ways lost their fertilizability and cytolyzed some time before those in the control medium.1 TABLE IV Immediate necessity of calcium in activation following sensitization Condition Per cent activation In artificial sea water 50 In Ca-free artificial sea water 0 Removed, to complete artificial sea water, after 15 sec. 51 30 sec. 36 60 sec. 10 90 sec. 0 120 sec. 0 180 sec. 0 Eggs removed to medium indicated after exposure for 24 hours to trinitrophenol, M/1000, in sea water. In a similar manner, depletion of the calcium content of the medium prevented activation of the eggs by Wilbur's methods, addition of isotonic KC1 or sodium ci- trate to the sea water medium. Citrate activation was especially sensitive to low calcium concentration, failing entirely if as little as % of the calcium was removed ; this was particularly curious, since the citrate in itself would be expected to render unavailable most of the calcium in the medium. //. Experiments concerning reactions accompanying sensitization a. Effects of anoxia Comparisons were made of the rates of development of sensitivity to sea water, during exposure to trinitrophenol, in the presence of various gas mixtures. Gassing the trinitrophenol solution (10~3 M in sea water) with nitrogen, or with 95 per cent nitrogen, 5 per cent carbon dioxide, led to a decidedly earlier development of sen- sitivity (Table V) ; but with more prolonged exposures, the eggs in the anoxic baths became irreversibly inactivated, and began to cytolyze, long before any evi- dence of damage appeared in the control aerated dishes. Increased percentage of CO2 in mixtures with air, at least up to 50 per cent CO2, similarly enhanced the development of sensitization, and did not appear to injure the eggs subsequently. It was frequently observed in other experiments that the packing of eggs in consider- able thicknesses at the bottom of the container, during exposures to trinitrophenol, led to more rapid development of sensitivity than appeared when more dispersed, thinner layers of eggs were used. It seems likely that this effect is to be attributed 1 On the other hand, less pronounced depletion of the calcium, down to about 1/100 the normal level, progressively delayed the onset of cytolysis ; this effect may be due simply to the calcium requirements of micro-organisms responsible for the disintegration of the eggs, but Schechter (1937a, b) has described similar effects of Ca++-reduction in a number of species of commonly used marine eggs. EGG SENSITIZAT1ON AND ACTIVATION 339 TABLE V Enhancement by anoxia of sensitization in trinitrophenol Duration of sensitizing bath min. 4 16 41 59 Per cent activation following sensitization under atmosphere of: 95% N2 5% 0 7 100 100 95% air 5% CO2 0 1 6 63 Eggs removed to fresh sea water after indicated exposure to trinitrophenol (10~3 M in sea water) under atmosphere described. to the higher CO2 or the lower O2 tension (or both) in the immediate environment of the cells packed in greater density. b. Effects of metabolic inhibitors Short of blocking subsequent activity by killing the cells (as indicated by loss of fertilizability, rapidly followed in most instances by disintegration), no reliable in- fluence on the rate of sensitization in trinitrophenol was observed in the presence of ether, urethane, or sodium iodoacetate. (See section d below.) Hydroxylamine, at 10~2 M, or potassium cyanide, at ca. 10~3 M,2 markedly enhanced the rate of sensi- tization, the more so as the concentration was increased ; a less reliable similar ac- tivity was seen with sodium azide, in the neighborhood of 5-10"3 M. Table VI shows a typical set of results with various concentrations of KCN. In all of these cases, the stimulatory action was superseded by lethal effects, more readily at the higher-concentrations, the maximum stimulating action then passing progressively to the lower concentrations, as in the instance presented in Table VI. This cytolysis occurred following the transfer to sea water, not in the trinitrophenol baths con- taining the inhibitor ; this is as would be expected if a great excess of the activator led to cytolysis rather than activation, as was also suggested in the older experiments. TABLE VI Enhancement by cyanide of sensitization in trinitrophenol Duration of Per cent activation when sensitized with KCN at concentration of: bath mm. 5 -10-3 M 10-3 M 2 •!<)-< M. 0 72 100 27 39 4 127 52 68 61 2 432 0 97 75 62 1554 Cytolyzed Cytolyzed 100 99 Eggs removed to fresh sea water after indicated exposure to trinitrophenol (10~3 M in sea water) with added KCN as shown. 2 Cyanide and trinitrophenol reacted slowly in these preparations, progressively developing an amber color of unknown significance, over a period of several hours. With higher concen- trations of cyanide, the rate of development of this discoloration was correspondingly higher. 340 PAUL G. LEFEVRE c. Effects of ionic components of sea water Following the experiments described above, in which it appeared that the cal- cium ion was the specifically essential component of sea water in permitting the in- itiation of activation, possible involvement of this ion in the reactions of sensitization was tested. Removal of calcum ion from the artificial sea water medium, contain- ing trinitrophenol at M/1000, produced no evident change in the rate of sensitiza- tion ; this absence of effect was noted with calcium contents as low as 1/60 the nor- mal level. More complete removal was impracticable, since in such low-Ca++ media the trinitrophenol became rapidly toxic to the cells and led to cytolysis before the sensitization had gotten well under way. This protective effect of calcium against destruction of the eggs by the acid was evident in comparison of the normal medium with that containing as much as 1/10 the normal amount of calcium ion. Since it was evident from experiments considered above that increased CO2 ten- sion hastened sensitization in trinitrophenol, it seemed expedient to test the effects of alterations in the related buffer system. But simple neutralization of the trinitro- phenol (readjusting the pH from 6.7-7.0 back to 8.0 as in sea water) had no demon- TABLE VII Effect of bicarbonate on sensitization in trinitrophenol Duration of sensitizing bath min. Per cent activation following sensitization in trinitrophenol, 10 3 M, in artificial sea water with bicarbonate content of: 3 X usual Usual (0.0018 M) 0.3 X usual None 25 0 0 0 0 92 0 0 0 0 156 2 0 0 0 212 38 4 3 1 405 100 77 3 0 1307 100 100 100 100 strable effect on the rate of sensitization. In nearly all of the experiments with trinitrophenol, this neutralization was routinely performed. In a single experiment, the amount of bicarbonate in equilibrium with atmospheric CO2 was varied in arti- ficial sea water media, from zero to three times the normal amount, and eggs were exposed to neutralized trinitrophenol, 10~3 M, in each of these mixtures. Through- out this range, the sensitization rate was more rapid, the more bicarbonate present (Table VII). The effect of CO2 is thus probably not to be attributed to its acidify- ing effects in the medium, but to its action after passing intracellularly, as in the effects described by Jacobs (1920). d. Effects of stimulating agents One deduction from the hypothesis of the activator-substance (LeFevre, 1945), subject to experimental test, is that the activator should accumulate more rapidly if the eggs in the trinitrophenol bath are simultaneously exposed to an agent which would bring about activation in the absence of the inhibitor. As previously re- EGG SENSITIZATION AND ACTIVATION 341 ported, this proved to be the case in only about ^ of the tests when heat was used as the activating agent, the remaining % of the results showing no significant differ- ences in either direction. The same procedure applied to mixtures of sea water and isotonic KC1 or sodium citrate gave different results with the two agents. With addition of extra potas- sium to the sensitizing picrate baths, there was invariably a more rapid development of the capacity to react upon removal to sea water, similar to that shown with other agents in Tables V, VI, and VII. This was in accord with the predictions of the hypothesis ; in no case, however, did such a difference appear in the presence of the citrate stimulant. Also, as noted above, the stimulants urethane and iodoacetate did not regularly enhance the development of sensitivity in trinitrophenol ; each of these substances exerted this effect in a few instances, and in no case produced a slowing of the sensitization, so that their position in this regard is the same as that of the heat stimulus originally investigated. ///. Experiments concerning the effectiveness of substituted phenols other than picric acid a. Blocking of activation Several substituted phenols differing from trinitrophenol in the nature or posi- tion of the substituted groups on the phenol were compared, all at 10~3 M, the con- centration at which standard experimentation with trinitrophenol was carried out.3 2,4-Dinitrophenol and /'-nitrophenol were more effective than trinitrophenol in blocking activation by either KCl-sea water mixtures or sodium citrate-sea water mixtures ; in one case only, /'-nitrophenol allowed a small number of eggs to reach the stage previously designated as "incipient activation." o-Nitrophenol was almost as effective against citrate activation, and was superior in this respect to trinitro- phenol ; in spite of this fact, o-nitrophenol had absolutely no influence on activation by excess potassium. 2,6-Dichlor,4-nitrophenol was approximately equivalent to 2.4,6-trinitrophenol in its inhibitory action, usually allowing a moderate percentage of "incipients." o-Chlorophenol and />-chlorophenol usually prevented activation, but these effects appeared to be attributable to irreversible damage to the eggs. Pro- longed exposure to the chlorophenols led to cytolysis, the initial stages of which, as noted by many investigators in artificial parthenogenesis, resemble in some re- spects the early stages of activation, so that it at times appeared upon cursory exami- nation that these chlorophenols augmented the stimulating action of the chemical agents used. Phenol itself had no inhibitory effect whatsoever at this concentra- tion. In summary, the inhibitory effect of picric acid was duplicated by each tested phenol which had a nitro-group in the para-position ; the only comparable action 3 If neutralized, trinitrophenol at concentrations as high as 10~2 M were tolerated for many hours, and were very effective in blocking and sensitizing, but the concentration of 10~3 M gave the most prolonged effects without cell damage. Increasing the concentration past 1(T3 M did not increase the rate of development of sensitivity to sea water in eggs exposed to the trinitro- phenol ; this independence of the rate of sensitization from the trinitrophenol concentration is in harmony with the hypothesis advanced in the discussion concerning the mode of action of the trinitrophenol. 342 PAUL G. LEFEVRE seen in the absence of this group was that of o-nitrophenol with respect to citrate activation. b. Sensitization to sea water At 10~3 M, o-nitrophenol, 2,4-dinitrophenol, o-chlorophenol, or />-chlorophenol had no sensitizing effect on the eggs in exposures up to 24 hours or until the onset of cytolysis. Cytolysis occurred within a few hours in />-chlorophenol, after about 20 hours in o-chlorophenol or dinitrophenol, but no sooner in o-nitrophenol than in sea water. />-Nitrophenol showed some sensitizing activity, as indicated in 100 80 60 o < 40 o 20 cr: 0 CYTOLYSIS DCNP CYTOLYSIS 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 LOG OF DURATION OF BATH, IN MIN. FIGURE 1. Comparison of sensitization rates in the three effective substituted phenols found. Eggs removed to sea water following bath in either 2,4,6-trinitrophenol (TNP), />-nitro- phenol (/>-NP), or 2,6-dichlor,4-nitrophenol (DCNP); all at 1(T3 M, in sea water. Each point is the average of all experiments (usually three or four) performed in the logarithmic time interval marked at the base-line. Each individual experiment compares the action of the three phenols on the same batch of eggs. Figure 1, but only after about 10 hours, and this effect disappeared rather rapidly, so that cytolysis began to be evident at about 20 hours. The dichloronitrophenol sensitized the eggs to sea water as rapidly as, or perhaps a little more rapidly than, trinitrophenol, but, like /j-nitrophenol, led to cytolysis within about 20 hours (Fig. 1). EGG SENSITIZATION AND ACTIVATION 343 Thus, it seemed apparent that again the — NO2 group in the para-position on the phenol was the primary essential, the groups in the ortho-positions exerting only modifying influences on the sensitizing reactions. However, unlike the situ- ation observed in the study of inhibition of activation, there was no sensitizing ac- tivity evident with 2,4-dinitrophenol, which contains the apparently critical group. This is especially odd in view of the fact that either the addition of another — NO2 in the 6-position, or removal of the — NO2 from the 2-position, produces a highly active substance (many swimming "larvae" were obtained from the use of trinitro- phenol or />-nitrophenol). Also, substitution of — Cl for both of the — NO2 groups in the ortho-positions did not interfere wth the sensitization activity. However, of all these, only 2,4,6-trinitrophenol (picric acid) was effective in the lengthy preservation of the eggs against death and cytolysis ; the others were all, in fact, somewhat toxic, leading to cytolysis within the first day of exposure. This may mean that the two nitro-groups in the ortho-positions serve to detoxify the molecule, perhaps preventing other reactions not basically related to those involved in the reversible inhibition and sensitization under study. DISCUSSION None of the newly acquired data is antagonistic to the hypothesis developed from the results previously reported, involving the concept of the metabolite acti- vator-principle. However, no crucial experimental test of this hypothesis has as yet been conceived ; the present data concern the general nature of the reactions ac- companying chemical activation and sensitization of the eggs, insofar as this is re- vealed by aberrations in these reactions with changes in the chemical environment. Thus it is apparent that some phase of the reactions accompanying the process of germinal vesicle breakdown involves an oxidation employing molecular oxygen, probably through a cytochrome system, since this process was reversibly prevented by anoxia, cyanide, or azide. However, as noted by Barren (1932), in the activa- tion of Nereis eggs by actual fertilization by sperm in very complete anoxia, this inhibition affects not the initiation of activation, but the later nuclear changes and consequent development. Diethyl ether also prevented these reactions, but there is less specificity in its action, so that interpretation of this inhibition is more indefinite. With the exception of ether, the same inhibitors which prevented activation also hastened the onset of sensitization of the eggs exposed to trinitrophenol, so that the cells were activated, upon removal from the acid to plain sea water, following a shorter stay than required in the absence of the inhibitors. This fact is readily incor- porated into the general hypothesis, since inhibition of the activating reactions would be expected to lead to more rapid accumulation of the activator-trinitrophenol com- plex, by eliminating one of the routes by which the free activator might be other- wise removed. Interpretation might also be sought on the basis of the acidifying effects of these inhibitors intracellularly, with consequent release of Ca++ from com- bination with cellular proteins. The fact that the inhibitors never appeared to in- duce sensitization in themselves, together with the temporal characteristics of the sensitization, would necessitate a rather unwieldy complex of assumptions about the behavior of the Ca++ and trinitrophenol, in application of these interpretations (LeFevre, 1945). 344 PAUL G. LEFEVRE The observed results would however be expected if there were any alternative pathway by which the activator could be removed, either by chemical reaction or by diffusion from the cell ; or simply if the reaction in which the activator is released were reversible and governed by mass action. In the latter case, accumulation of the activator in the presence of these metabolic poisons would be self-limited, whereas large amounts could be accumulated in inactive form with trinitrophenol, and this process would be accelerated by addition of the inhibitors ; this would ac- count for all the relevant experimental results with a minimum of independent hypotheses. As noted above, the metabolic inhibitors which prevented activation exert their influence not on the immediate activating disturbance (visible at the egg surface) but on the immediately subsequent cellular reorganization. On the other hand, in- hibition of the same over-all process ("activation") by removal of calcium ions from the medium acted at the very earliest stages in the chain of events. The absence of calcium ion at the critical instant of potential initiation of activation was thus an irreversible disturbance, and the cell did not react upon replacement of the missing element unless a second stimulus was applied. Heilbrunn (1925), Heilbrunn and Wilbur (1937), and Wilbur (1939, 1941) concluded from experiments along vari- ous lines that the reaction in question is dependent on rearrangement of the intra- cellular calcium with respect to the protoplasmic colloids, with accompanying changes in viscosity. The modifying influences of potassium and magnesium ions, as re- ported above, are in keeping with the general pattern of these cations in affecting colloidal reactions with calcium ion, as described by Heilbrunn in numerous cellular reactions. Other aspects of the effects of metabolic poisons on the reactions of activation are not so readily interpreted ; it is particularly odd, though not entirely without parallel, that germinal vesicle breakdown is initiated by exposure to iodoacetate or urethane (or, in a different manner, by application of Cu++ or /?-chloromercuri- benzoate), which substances are generally recognized as inhibitors or narcotics. The most evident interpretation of these results in the light of the related observa- tions is that these inhibitory agents may prevent some alternative reactions of the activator substance or its precursors, so that the activator concentration is increased by the presence of the inhibitors. Obviously no specific characterization of the hypothetical reactions involved can be made, except that it seems likely that some enzyme concerned contains active sulfhydryl groups. Some special mention should be made of the fact that there is not complete agreement in the results obtained with the various procedures employed. Some of these discrepancies are easily dismissed as quantitative differences in effects of the activators and inhibitors on the critical reaction rates. However, the distinct in- hibition of citrate activation by o-nitrophenol is entirely out of line with all other relevant data ; citrate in stimulatory concentrations also invariably failed to hasten sensitization of the eggs in trinitrophenol. Beyond the considerations outlined, the present data do not permit identifica- tion of the hypothetical substances or their reactions ; perhaps some clue is af- forded in the apparent specificity of the />-nitrophenol grouping in the reversible formation of an inactive complex with the activator. The author is not prepared to interpret this finding ; innovation in experimental approach is probably necessary EGG SENSITIZATION AND ACTIVATION 345 before a more coherent pattern will emerge from the diverse observations reported in this paper and in the earlier report. The interpretations offered seem the least in- volved and most comprehensive of the facts available at this time. SUMMARY 1. Inhibition of activation of Nereis eggs by trinitrophenol, with concurrent sensitization of the eggs to subsequent stimulation, appears to depend on the nitro- group in the para-position on the phenol. 2. The rate of the sensitization process is enhanced by anoxia, CCX, inhibitors of the cytochrome system, or increased potassium ion concentration, but is in- sensitive to several other inhibitors, narcotics, stimulating agents, and to calcium ion deprivation. 3. The immediate initiation of activation by various chemical procedures re- quires the presence of the calcium ion, is assisted by the potassium ion, and slightly depressed by increasing magnesium ion concentration, but is not affected by anoxia or by various metabolic poisons. 4. Subsequent nuclear and cytoplasmic reorganization, ensuing some minutes after the initial disturbance, is reversibly inhibited by anoxia, inhibitors of the cytochrome system, or diethyl ether. 5. Urethane and iodoacetate activate the eggs; this activation is inhibited by trinitrophenol. Cupric ion and />-chloromercuribenzoate also activate the eggs, but only at nearly lytic concentrations, and the activation is not affected by trinitrophenol. 6. These data are partly interpreted in relation to the hypothesis of an activator metabolite produced within the egg. LITERATURE CITED BARRON, E. S. G., 1932. The effect of anaerobiosis on the eggs and sperm of sea urchin star- fish and Nereis and fertilization under anaerobic conditions. Biol. Bull., 62 : 46. HEILBRUNN, L. V., 1925. Studies on artificial parthenogenesis. IV. Heat parthenogenesis. Jour. Exp. Zool, 41 : 243. HEILBRUNN, L. V., AND K. M. WILBUR, 1937. Stimulation and nuclear breakdown in the Nereis egg. Biol. Bull., 73: 557. JACOBS, M. H., 1920. The production of intracellular acidity by neutral and alkaline solutions containing carbon dioxide. Amer. Jour. Physiol, 53 : 457. JUST, E. E., 1915. Initiation of development in Nereis. Biol. Bull., 28: 1. LEFEVRE, P. G., 1945. Certain chemical factors influencing artificial activation of Nereis eggs. Biol Bull, 89 : 144. LILLIE, R. S., 1926. The activation of starfish eggs by acids. Jour. Gen. Physiol., 8 : 339. LOEB, J., 1913. Artificial parthcnogcnsis and fertilization. The University of Chicago Press. SCHECHTER, V., 1937a. Calcium reduction and the prolongation of life in the egg cells of Arbacia punctulata. Biol Bull, 72 : 366. SCHECHTER, V., 1937b. Calcium and magnesium in relation to longevity of Mactra, Nereis and Hydroides egg cells. Biol. Bull, 73: 392. WILBUR, K. M., 1939. The relation of the magnesium ion to ultra-violet stimulation in the Nereis egg. Physiol Zool, 12 : 102. WILBUR, K. M., 1941. The stimulating action of citrates and oxalates on the Nereis egg. Physiol Zool, 14: 84. THE ACTION OF CHOLINE AND RELATED COMPOUNDS ON THE HEART OF VENUS MERCENARIA * JOHN H. WELSH AND RAE TAUB Biological Laboratories, Harvard University Since the demonstration by Prosser (1940) of the unusual sensitivity to acetyl- choline (Ach) of the isolated heart of the bivalve molusc, Venus mercenaria, we have extensively employed this preparation for the bio-assay of Ach in tissue extracts (e.g. Welsh, 1943; Welsh and Hyde, 1944a and b; Prajmovsky and Welsh, 1948). In certain respects it is superior to the classical Ach assay preparations such as the dorsal muscle of the leech, rectus abdominis of the frog, isolated frog heart, and blood pressure of cat. For example, it is more sensitive to Ach, with complete in- hibition occurring at about 50 times the threshold inhibitory concentration ; it is rela- tively unaffected by changes in pH, inorganic ions, and tissue constituents other than Ach ; it recovers quickly, thereby allowing more rapid estimation than the above- mentioned preparations. While employing the Venus heart for bio-assay, its responses to a variety of drugs, organic compounds, and inorganic ions have been studied and, in particular, to a series of choline esters and analogs — this in the hope of obtaining evidence to- ward a better understanding of the fundamental mode of action of Ach. An organ with a high specificity for choline esters, exhibiting a response which is easily quan- tified, and which has so little self-contained cholinesterase that blocking of this enzyme is not necessary when working at great dilutions of the unstable esters, provides a suitable object for studying certain aspects of the mechanism by which Ach acts on cells. The present paper has two purposes : ( 1 ) to indicate the methods of preparing and employing the Venus heart for the bio-assay of Ach, and (2) to compare the effects of other choline esters and related compounds which differ in greater or less degree from the Ach molecule. METHODS OF PREPARING AND USING THE HEARTS FOR ACH ESTIMATION An earlier paper by Wait (1943) covers some of the necessary procedures for preparing the isolated Venus heart for Ach determinations. For convenience, how- ever, the steps which we employ from the securing of appropriate test animals to the quantitative estimation of Ach in tissue extracts will be outlined. Venus mercenaria (the hard shell clam or quahog), being an important com- mercial shellfish along the Atlantic Coast, are usually available where shellfish are sold. They remain edible for some weeks after digging or dredging, if maintained under refrigeration, but. after one to two weeks the hearts of such animals tend to beat with an irregular rhythm ; it is important, therefore, to obtain experimental 1 This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, U. S. Public Health Service. 346 CHOL1NE AND THE HEART OF VENUS 347 material from a source where the previous history is known - and to use this ma- terial within one to two weeks after removal from the sea. Venus with a shell length of 8 to 12 cm. have been found of most convenient size. In the laboratory they may be stored dry at 5°-10° C, or preferably kept in shallow tanks of aerated sea' water, at 15°-18° C. The heart is exposed by breaking and removing the dorsal portion of the shells (umbos and hinge) and then cutting away the mantle and pericardium dorsal to the heart. The heart consists of a single, median ventricle with laterally disposed, thin- walled atria (auricles). Anterior and posterior blood vessels leave the heart in close association with the intestine which passes through the heart. Threads for attaching to a support in the bath and to the writing lever may be passed under the atria and tied close to the ventricle in order to include some of the thicker-walled, ventricular muscle. Cutting the atria distal to the threads, and cutting the blood vessels and intestine, isolates the ventricle which may then be placed in an appro- priate heart bath. Only the outer surface of the heart is directly exposed to mate- rials introduced into the bath, but cannulation of the heart and introduction of Ach into the ventricle does not increase its sensitivity. The bath figured by Wait (1943) is satisfactory unless temperature control is desired (e.g. when working in a room above 20° C., or when maximum sensitivity is required) ; then a bath with a water jacket through which water of an appropriate temperature (15°-18° C.) is circulated may be employed, or the heart bath may be placed in a larger temperature-controlled vessel. It is necessary that provision be made for changing the fluids of the heart bath without draining the bath and sub- jecting the heart to undue mechanical disturbance. A bath holding 10 ml. when filled has been found appropriate. An analysis of the inorganic salts of the blood of Venus mercenaria by Cole (1940) showed only small differences in comparison with sea water. It is not sur- prising, therefore, that sea water is an adequate perfusion fluid for the isolated heart, allowing a regular beat to be maintained for 2-3 days. Where natural sea water is not available, an artificial perfusion fluid may be used and several have been tried, with differing ratios of the common ions, without noting any appreciable effects on the heart until radical departures from the normal concentrations of the common ions are made. A fluid found satisfactory has the following composition : 30 gm. NaCl; 0.9 gm. KC1 ; 1.1 gm. CaCU ; 3.5 gm. MgSO4-3H2O in one liter of water with a phosphate or bicarbonate buffer (pH 7-7.5). Changes of pH between 6 and 8.5 have little or no effect on the amplitude or frequency of beat, or on the response of the heart to acetylcholine for periods of time up to several hours. Oxygen may be supplied by air or a mixture of 95 per cent O2-5 per cent CO2 passed through the bath. The bubbles should be small to avoid mechanical disturb- ance to the heart. The gas mixture or air may be admitted to the bath through the hooked support for the lower attachment of the heart if this is made from glass tub- ing drawn out to a fine tip. The heart lever should be counterweighted to give a pull of 200-300 mg. A kymograph speed of about 2 cm. per minute is desirable. Substances to be tested may conveniently be added at the bottom of the bath by 2 E.g. Supply Department, Marine Biological Laboratory, Woods Hole, Mass., or a whole- sale dealer in shellfish. 348 JOHN H. WELSH AND RAE TAUB C -O i •- •" *-> ij 2 y u •£ tp .S 13 rt "° JS C t> -— •— C ' •"' o o X P o ft g >.^-° bo 1> E .S -a o :s H 0- > rt £0^0 t? (L> -S -5 O § c (/) 3 *-• . « 2 I.S •" s S ^ r J - • l- cj rt fcfi « a 2*3 S a > rt rt rt ^ T3 U .tJ *S •a 4; tn .JJ •5 O -M ^ ^ «J JH E^ | "rt ^ 'X O bfl,^ .S ? u« n B "3 C 3 PQ > "O 3 (L> U » l«8| sill ii Co <" _ S ™ N -S -S be be ^ 3. 0 p f iri u i« o o *^ t— i C u , o c M-l en C y (/) ^P K — rt d> O r cj M-H •— _, o| i^-i g «- o . *o n O O rf " <-" «u w II t: C'a; •a ccq R •£ C "™~" fi rt i.^ ,_ flj c QJ Q P "T* j_» -t cu -a u o u JJ rt 11 ft r: n !_ S C *^ rt o< S w rt o r^ rt Illli "E in a i-S is s t C 3 « fo n o ,, (1J -j-i O) o II n 3 •£ •S « 8-3-g - a c c o o E 1 | 8 QJ O ^ ^* rt »— c ^^ rt CHOLINE AND THE HEART OF VENUS 349 means of a long hypodermic needle bent at a right angle ; the volumes added should be small (1 ml. or less). In estimating the Ach content of a tissue extract, a dilution should be found that gives between 20 and 80 per cent decrease in amplitude of beat at the end of one or two minutes. This should be matched, preferably twice, with known concentra- tions of Ach, after which appropriate calculations will give the Ach equivalent per gram of tissue. A sample record is shown in Figure 1. If it is suspected that sub- stances in the tissue extract other than Ach are affecting the heart, it may be de- sirable to treat a portion of the extract by the addition of NaOH and warming in order to destroy the Ach present, and after neutralizing with HC1, to use this to make up the last dilution of Ach prior to adding to the bath. Treatment of a heart with an anti-cholinesterase (physostigmine, neostigmine or di-isopropyl fluorophosphate) may potentiate the action of Ach two to five times. This small degree of potentiation is undoubtedly due to the low level of cholinesterase activity in these hearts (Smith and Click, 1939; Jullien et al., 1938). Because the untreated heart is so sensitive to Ach (threshold for inhibition is usually between 10~10 and 10"11 gm. per ml.), and because recovery after Ach is slowed by treat- ment with an anti-cholinesterase, it is normally undesirable to employ this means for increasing sensitivity. Occasionally hearts are encountered which fail to beat or which beat with a low amplitude. Although adrenalin and tyramine have been found to be excitants in relatively high concentrations (5 X 10~5 to 10~4 M) their effects are quickly abol-' ished by washing. On the other hand ergotoxine, ergotamine, and ergonovine have been found to have a remarkably persistent excitatory action. For example, one part per million of ergotoxine ethanesulfonate will frequently cause renewal of heart beat, or an increase in amplitude of two to three times, in a heart with an abnormally low amplitude. Treatment for a few minutes with one of these ergot alkaloids pro- duces a change in the physiology of the heart which persists for many hours in spite of repeated washings, while the response to Ach is affected but slightly. In this connection it should be noted that the Venus heart is composed of smooth muscle and its pharmacology is not unlike that of certain types of vertebrate smooth muscle. There is some seasonal variation in the sensitivity of Venus hearts to Ach, with maximum sensitivity in the late winter and spring months (cf. Prosser, 1940; Wait, 1943), but the change is probably not as great as indicated by Prosser. More important to note is that in late summer there is a tendency toward irregularity in beat. This is often so pronounced that accwaTe estimates of small differences in Ach levels cannot readily be made in August and early September in the region of Massachusetts. THE ACTION OF CERTAIN CHOLINE DERIVATIVES AND RELATED COMPOUNDS ON THE VENUS HEART The greatest gap in our knowledge concerning Ach is the precise manner in which this physiologically active substance affects the excitability of cells. More de- tailed studies of the mechanism of action of Ach are needed, and it would appear to matter little what type of Ach-sensitive tissue or organ is used in these studies. For many reasons the isolated heart of the quahog appears to be peculiarly suitable 350 JOHN H. WELSH AND RAE TAUB for such a study, and in this section of the present paper it will be shown that no compound related to Ach has yet been found having as great an inhibitory action on this organ as does Ach. It will also be shown that the methyl grouping around the onium element is, in many respects, the most significant portion of the Ach molecule. The typical effects of Ach on 'the Venus heart will first be described, and then the relative activities of a number of common choline derivatives and certain re- lated compounds will be discussed. When Ach is added to a bath containing a beating Venus heart, which has re- ceived no previous drug treatment, to give a concentration in the vicinity of 10"11 to 10~10 M, a small increase in amplitude is sometimes observed. A similar stimu- lating action of low concentrations of Ach on vertebrate hearts has been observed by AlcDowall (1946) and others. At concentrations of Ach in the vicinity of 10~9 to 10 8 M a negative inotropic effect is seen ; and with increasing amounts of Ach the amplitude of beat decreases until the heart stops in diastole at a concentration of Ach about 50 times that which gives a just measurable decrease in amplitude. Thus the range of concentrations from the threshold of inhibition to complete inhibition is relatively narrow. The log-concentration-response curve is sigmoid, with the por- tion between 20 and 80 per cent inhibition approximating a straight line. The in- hibitory action of Ach on the Venus heart is more prominent and consistent than the excitatory action of lower concentrations, but it seems probable, as pointed out else- where (Welsh, 1948), that Ach first excites and then in higher concentrations in- hibits or paralyzes this organ as it may do to all tissues or organs which respond to Ach. In Table I a summary is given of the relative inhibitory activities of a number of compounds related to choline or Ach. The data shown in this table were obtained by finding a molar concentration of Ach that would produce between 20 and 80 per cent decrease in amplitude of beat of a given heart, and then the molar quantity of a related compound that would produce a degree of inhibition exactly matching that produced by the Ach. Each value shown for a given compound was obtained on a different heart. The average values may be taken as a fairly precise indication of the relative inhibitory effectiveness of these several compounds on the Venus heart. Since cholinesterase activity in this organ is extremely low, anti-cholinesterases were not employed, but the complications which may arise when stable choline esters are compared with unstable in the presence of active cholinesterases are believed to be minimal. In commenting on certain of the more interesting facts given in Table I attention may first be called to choline. Choline affects the isolated Venus heart in a manner very much like that of Ach, except that it is far less active. When first applied in low concentrations, the amplitude of beat may increase (Fig. 2, Curve 1). Greater variation in the response of different hearts to choline was observed than in the case of any other compound. This is illustrated by Figure 2, where con- centration response curves for three different hearts are shown. The wide range of values obtained when choline was compared with Ach may be accounted for by individual variation in the response to choline, for it is obvious that if a match of molar concentrations of Ach and choline producing 25 per cent inhibition were made on the heart represented by curve 3, the relative value for choline might be 1000; while a match of 25 per cent inhibition made on the heart represented by curve 2 would yield a value showing Ach to be perhaps 50,000 times as active as choline. CHOLINK AND THE HKART OF VENUS 351 TABLE 1 Relative molar quantities required to produce a decrease in amplitude of beat equivalent to that produced by a molar quantity of acetylcholine chloride equal to 1 Choline derivative Structural formula Molec- ular weight Values obtained Averages Carbamylcholine chloride (CH.,)3NCH2CH2OCNH.. 1 II CI 0 182.5 20 20 25 40 40 50 80 100 100 150 250 80 «-Propionylcholine chloride (CH3)3NCH,CH2OCCH.>CH3 1 II Cl O 195 100 100 100 120 105 Ethoxycholine bromide (CH3)3N— CH2CH2OCH2CH3 Br 212 100 100 100 130 200 200 200 250 160 Butyrylcholine chloride (CH,)jN— CH.CHsOC(CHs)sCH, i, A 209 200 500 800 1,000 625 Chloracetylcholine chloride (CH,),NCH.CHsOCCH.Cl i, H 215.9 500 750 1,000 1,600 960 Acetyl /3 methyl choline chloride (CH3)3N— CH2CHOCCH;( 1 1 II Cl CH3 O 195 400 1,000 1,000 2,000 1,100 Benzoylcholine chloride (CH3)3NCH2CH2OC— 243 10,000 10,000 12,000 13,000 20,000 15,000 Cl O Choline chloride (CH3)3NCH2CH2OH Cl 139.5 400 1,000 1,000 1,000 1,000 2,000 4,000 5,000 7,000 15,000 40,000 100,000 14,000 Betaine ethyl ester chloride (CH3)3NCH2COCH2CH3 1 l! Cl O 181.5 1,000 1,000 1,000 Betaine hydro- chloride r(CH3)3NCH2CCT| [ 4 J-HCL 117 3,300 5,000 10,000 20,000 25,000 25,000 15,000 Triethylcholine chloride (C2H5)3NCH2CH2OH Cl 272 10~2 M — no inhibition Triethylacetyl choline iodide (C2H6)3NCH2CH2OCCH3 I O 315 10~2 M — no inhibition However, the average value showing that 14,000 times as many molecules of choline are required than of Ach to produce a given effect, is probably a close approximation to the relative activities of these two compounds on the Venus heart. Thus the acetic acid ester of choline is far more active than is the parent compound. This is an observation that has been made by many workers on many tissues and organs. 352 JOHN H. WELSH AND RAE TAUB The several esters of choline which were tested and the one ether (ethoxycholine) were all far more active than choline, with the exception of benzoylcholine which has approximately the same level of activity. The presence of the ring structure at the non-polar end of the molecule obviously affects the activity greatly. It is of in- terest to note that the substitution of chlorine for a hydrogen atom of the terminal methyl group in Ach to yield chloracetylcholine reduces the activity approximately one thousand fold. 100 - 8 -7 -6 - 5 ~ LOG MOLAR CONC. Of CHOLINE Cl. FIGURE 2. Showing the extreme variation in response of three different isolated Venus hearts to choline. Heart number 1 responded by an increase in amplitude to low concentrations of choline while hearts 2 and 3 showed only a decrease in amplitude, but their sensitivities differed markedly. Betaine, a naturally occurring compound closely related to choline, is approxi- mately equivalent to choline in its ability to depress the beat of the Venus heart, while the ethyl ester of betaine was found to be considerably more active than betaine. A clear indication of the importance of the methyl groups attached to the nitrogen was seen when triethylcholine and triethylacetylcholine were tested. In the highest concentrations employed (10~2 M), neither of these produced the slightest degree of inhibition of heart beat. We have observed a similar striking difference in the actions of the tetramethyl ammonium ion, which decreases the amplitude of beat of the Venus heart, and tetraethyl-, tetra-n-propyl-, and triethyl-n-octyl ammonium CHOLINE AND THE HEART OF VENUS 353 ions, all three having an excitatory action only. These results obtained with the quaternary ammonium ions will be reported more extensively in a separate paper. Importance of the methyl groups attached to the onium element directs atten- tion to this portion of the Ach molecule. It is apparent from the present study and from similar earlier studies that different choline esters differ in their degree of pharmacological activity. This is also true of the Venus heart, but they all produce a characteristic decrease in amplitude. The substitution of ethyl for methyl groups in choline and Ach yields molecules which are completely lacking in inhibitory ac- tivity when applied to the isolated Venus heart. It has been indicated elsewhere (Welsh, 1948) that this specificity of the (CH3):tN group, the rapidity of action of and recovery from Ach, and its activity in small amounts, suggest that Ach acts at the surface of cells as a "trigger" to set off a reaction or chain of reactions in the manner of an unstable coenzyme. Thus, the condition of the cell membrane is al- tered and the cell excited and then depressed depending on the concentration and time of action of the Ach. In further testing of this hypothesis, it is believed that the isolated Venus heart will continue to provide an ideal experimental object. SUMMARY 1. A method of preparing and employing the isolated heart of the quahog, Venus mercenaries, for the bio-assay of acetylcholine (Ach) is described. 2. The activities of choline and certain choline esters ; of betaine and its ethyl ester; and of triethylcholine and triethyl-acetylcholine on the isolated Venus heart are compared. In further understanding the fundamental mode of action of Ach, the most significant observation was that the substitution of ethyl groups for methyl on the nitrogen of choline and Ach resulted in a complete loss of activity determined by observation on the amplitude of heart beat. LITERATURE CITED COLE, W. H., 1940. The composition of fluids and sera of some marine animals and of the sea water in which they live. /. Gen. Physiol., 23 : 575-584. JULLIEN, H., D. VINCENT, M. BOUCHET, AND M. VIULLET, 1938. Observations sur 1'acetyl- choline et la choline-esterase du coenr des Mollusques. Ann. Phys. et Ph\s. Biol., 14 : 567-574. McDowALL, R. J. S., 1946. The stimulating action of acetylcholine on the heart. /. Physiol., 104: 392-403. PRAJMOVSKY, M., AND J. H. WELSH, 1948. Total and free acetylcholine in rat peripheral nerves. /. Neurophysiol, 11 : 1-8. PROSSER, C. L., 1940. Acetylcholine and nervous inhibition in the heart of Venus mercenaria. Biol Bull, 78 : 92-102. SMITH, C. C., AND D. CLICK, 1939. Some observations on cholinesterase in invertebrates (abstract). Biol. Bull, 77: 321-322. WAIT, R. B., 1943. The action of acetylcholine on the isolated heart of Venus mercenaria. Biol. Bull, 85 : 79-85. WELSH, J. H., 1943. Acetylcholine level of rat cerebral cortex under conditions of anoxia and hypoglycemia. /. Neurophysiol, 6 : 329-336. WELSH, J. H., 1948. Concerning the mode of action of acetylcholine. Bull Johns Hopkins Hospital (in press). WELSH, J. H., AND J. E. HYDE, 1944a. The distribution of acetylcholine in brains of rats of different ages. /. Neurophysiol., 7 : 41-50. WELSH, J. H., AND J. E. HYDE, 1944b. The effects of potassium on the synthesis of acetyl- choline in brain. Am. J. Physiol, 142 : 512-518. INCIDENCE AND ORIGIN OF ANDROGENETIC MALES IN X-RAYED HABROBRACON EGGS * ANNA R. WHITING Ihiii'ersity of Pennsylvania INTRODUCTION The terms androgenesis and merogony are sometimes used interchangeably. The former is denned by Wilson (1925) as "the activation of the egg by the sperm followed by development without the participation of the egg nucleus ;" the latter as the "development of an egg fragment devoid of a nucleus fertilized by a normal sperm." This distinction is kept in this paper. The study was undertaken with two objects in view: first, to determine whether low incidence of androgenetic males or high embryonic mortality is responsible for the low ratio of their occurrence as adults; and second, to work out the cytological mechanism underlying androgenesis in Habrobracon. The literature dealing with androgenesis is considerable. Much of it is sum- marized in Wilson (1925), Sharpe (1934) and Darlington (1937). Three papers have been selected for discussion here. Packard (1918) exposed unfertilized Chactopterus eggs in first meiotic metaphase to radium, and fertilized them with untreated sperm. When exposure was relatively long (35 to 50 minutes) the egg nucleus remained attached to the second polar body, the sperm nucleus divided, and development was androgenetic. Hasimoto (1934) identified some silkworm (Bom- byx) males as androgenetic. These had developed from eggs which had been ex- posed to high temperature at time of oviposition when the eggs were undergoing the maturation divisions. These males were diploid, and by means of appropriate genetic combinations, he was able to demonstrate that they arose from the "union of two sperm nuclei in the egg cytoplasm without fertilization with the egg nucleus." Polyspermy is the rule in Bomby.v. The third paper (Astaurow, 1937) describes the production of androgenetic males, likewise in Bomby.v. They were produced along with the expected classes either by thermo-activation (40° C. for one hour) after fertilization, or by irradiation of the egg followed by fertilization with un- treated sperm, or by both. At X-ray doses lethal to the egg nucleus, androgenetic males developed only after thermo-activation of the fertilized eggs. Heat treat- ment is used in this form to break diapause. MATERIAL AND METHODS For X-ray treatments a dual-tube self-rectifying outfit with a simultaneous cross- firing technique was used. The secondary voltage was 182 kv. and the tube cur- 1 This investigation was completed with the aid of a research grant from the National Cancer Institute of the National Institute of Health, U. S. Public Health Service. The author is also grateful to the University of Pennsylvania and to the Marine Biological Laboratory, Woods Hole, Massachusetts, for use of laboratory facilities, and to Mr. L. R. Hyde for admin- istering the X-ray treatments. The drawings were made by Mrs. Jean Wilson. 354 X-RAYS AND ANDROGENESIS 355 rent on each tube was 25 ma. The heavy glass of the tube walls and 5 mm. of bakelite of the tube shields gave the filtering value of 0.2 mm. copper shield. The output intensity was 7210 r per minute, distance 9.5 cm. All breeding was carried on at 30° C. Well-fed wild type females of the parasitic wasp Habrobracon juglandis were X-rayed and mated to untreated males which differed from wild type by one or more recessive traits. Eggs laid by these females during the first six hours after treatment had been X-rayed in late metaphase of the first meiotic division (meta- phase I) ; the majority of those laid after this time had been treated in first meiotic prophase (prophase I) (Whiting 1938). Lethal dose2 for the former is about 2400 r; for the latter, about 54,000 r (Whiting, 1941). From all control crosses of the type used in this study, only diploid biparental females and haploid gynogenetic males are produced. Therefore, when wild type fe- males are mated to males with traits recessive to wild type, daughters are wild type, heterozygous for the recessive traits, while sons have maternal wild type genes only. If this kind of cross is made after the females have been X-rayed, there appear occasionally males which show all the recessive paternal traits (Whiting, 1946a). These males are normal in appearance, fully fertile and transmit paternal traits only. They are, therefore, androgenetic. Their fertility is proof that they are haploid since diploid males which may arise from certain crosses in Habrobracon are always sterile or nearly so. Repeated tests have shown that androgenetic males arise from eggs treated in metaphase I, and of 381 such eggs observed, six only, 1.57 per cent, developed into these exceptional males (dose 14,420 r-28,840 r). Although lethal dose for the nuclei of these eggs is 2400 r, androgenetic males have developed in eggs X-rayed with dose as high as 54,000 r (Whiting 1946b). Eggs laid during the first six hours after treatment ( 14,420 r-36,050 r) were col- lected at one hour intervals, punctured, and fixed in Kahle's fixative. They were stained with the Feulgen technique and mounted whole in Canada balsam. OBSERVATIONS Speicher (1936) found that the most advanced eggs in Habrobracon egg sacs are in "early anaphase of the first maturation" (the author prefers to call this late metaphase I), and described normal oogenesis following oviposition. After the egg is laid, the maturation spindle passes into telophase I. The second division follows immediately. The four haploid groups of chromosomes (la, Ib, 2a, 2b) lie in a row roughly perpendicular to the egg surface. During anaphase II, polar nuclei la and 2a remain stationary, Ib moves close to 2a, and 2b (functional nucleus) sinks deeper into the egg, a membrane forming as it moves. Nucleus la soon disintegrates, Ib and 2a unite and form a metaphase plate which divides and then disintegrates. Cleavage is of the usual insect type, with nuclei moving about until blastoderm formation, when cell membranes first appear. - Apparent inconsistencies in lethal doses in successive papers dealing with Habrobracon eggs are due to changes in method of calibration at the Marine Biological Laboratory. Condi- tions of treatment have not varied. In this paper all doses have been corrected for the latest measurements. 356 ANNA R. WHITING PLATE I -r; v-^- ;::"*'•-••".••.;:.;:; ;••. .-.'• '-i' . ''• ,-••' '-.,' ' '*••'•'.'&'•'. " .. • ::. • '• • : • - •'?! . • -•.'. • -> -•' ;-t- "':,.::.•-.*- '•'•'•' K:-.:- X-RAYS AND ANDROGENESIS 357 After treatment with X-rays in prophase I, chromatin fragments, bridges, or both may occur in division I, or in division II, or in both divisions (Whiting, 1945a). Bridges occur but rarely in division II and when present appear to be single. After treatment in metaphase I, chromatin fragments (never bridges) may be seen between chromosome groups in telophase I and chromatin bridges appear in division II. These bridges may occur between nuclei la and 2a, or Ib and 2b, or between both pairs, never between 2a and Ib. They are made up of several chro- matin threads and are present in both regions when close is high. The attached egg pronucleus can move a long distance without breaking the bridges, and under these conditions it is pulled out into a "tear-drop" (Whiting, 1945b). Controls, of course, do not show fragments or bridges. These bridges may retard the female pronucleus, but that they do not often stop it completely at relatively low doses is demonstrated by the fact that after lethal dose (2400 r) all but 2.4 per cent of unfertilized eggs develop well beyond first cleavage before death, and that these 2.4 per cent advance to first cleavage before dying (Whiting, 1945a). These facts suggested to the author that the chromatin bridges formed during the second meiotic division of eggs X-rayed in metaphase I might sometimes retard the attached egg pronucleus to such a degree that the untreated sperm pronucleus would cleave before the egg pronucleus could reach it. There were 702 eggs prepared and studied. Of these, 58.55 per cent were use- less, either because they had been fixed at stages previous to syngamy or cleavage, or were not clearly stained ; 41.45 per cent were of significance. They included all eggs undergoing syngamy as well as those in cleavage, where either chromosome number or presence or absence of chromosome aberrations demonstrated which pronuclei had taken part in cleavage. Of the 291 eggs fulfilling these requirements, three (1.03 per cent) only were found in which androgenetic development had begun (Plate 1, Figs. 6, 7 and 8). Three eggs not included in the 291 suggested incipient androgenesis. In each the male pronucleus was preparing for first cleavage while the female pronucleus was greatly retarded (Plate 1, Fig. 2). If these are accepted as androgenetic, a maximum of six among 294, or 2.04 per cent, is obtained. This does not differ significantly from 1.57 per cent of adult survivors, and demonstrates PLATE I All illustrations were drawn with aid of a camera lucida from whole mounts of eggs which had been X-rayed in first meiotic metaphase. Fertilization was accomplished by untreated sperm. Anterior end and lateral view of each egg is shown. Cytoplasm is somewhat conven- tionalized. The lenses employed were a Spencer 2 mm. n. a. 1.3 apochromatic oil immersion objective and a X 5 or X 10 compensating ocular. 1. Attached pronucleus of unfertilized egg. Dose 28,840 r. X 650. 2. Attached and greatly retarded female pronucleus with normal male pronucleus. Egg X-rayed with 28,840 r. K 650. 3. First cleavage spindle of unfertilized egg. Chromatin bridges still attached to some cleavage chromosomes. Dose 14,420 r. X 370. 4. Cleavage spindle of Figure 3 in detail. X 650. 5. Syngamy. Egg X-rayed with 36,050 r. X 650. 6. Second cleavage of androgenetic development. Egg pronucleus greatly retarded. Egg X-rayed with 36,050 r. < 370. 7. Second cleavage of androgenetic development. Egg X-rayed with 28,840 r. X 370. 8. First cleavage of androgenetic development. Egg X-rayed with 14,420 r. X 370. 358 ANNA R. WHITING that there is little or no death of androgenetic embryos in spite of their development in cytoplasm irradiated with doses from six to fifteen times that lethal for the egg nucleus. A study of Plate I will illustrate some points of cytology. It should be recalled here that Habrobracon chromosomes are extremely small. This will explain why they are not always represented exactly as to form and number (n— 10). In Plate I, Figure 1, is shown a typical "tear-drop" pronucleus. Polar nuclei are de- generating and suggest by their condition that the egg pronucleus is considerably re- tarded. Chromatin at the outer end of, as well as within, the egg pronucleus, is at- tached to bridges. This egg was not fertilized. Figures 3 and 4 show what can happen to such a tear-drop as that "just described. The spindle is that of the first cleavage, and bridges with chromatin thickenings can be seen, still attached to chromosomes on the spindle. This is the only egg studied in which the chromatin connections could not be followed continuously from polar nuclei to egg pronucleus. What happens to a tear-drop pronucleus in the majority of cases when an X-rayed egg is fertilized after treatment, is shown in Figure 5. It unites with the normal male pronucleus and, in doing so, ultimately kills the embryo because of upset in chromosome balance due to chromatin loss. Figure 2 strongly suggests an incipient androgenetic male. The egg pronucleus is so retarded and the male pronucleus so advanced that subsequent syngamy seems highly improbable. Figures 6, 7, and 8 represent the only eggs found in which androgenetic develop- ment had begun. The isolation of the egg pronucleus from cleavage figures, with no evidence of any chromatin connection or remains between them and the normal appearance of the cleavage chromosomes, are to be noted. Figure 8 is especially convincing. These three eggs have one thing in common which is rare in control eggs and not the rule in X-rayed ones : cleavage is taking place more posteriorly than one would expect. This suggests that some cytoplasmic factor, perhaps greater fluidity of the cytoplasm, may alter action of the sperm pronucleus so that it has moved "beyond the reach" of the impeded egg pronucleus. DISCUSSION It is definitely established that androgenetic development (haploid) occurs in Chaetopterus and in Habrobracon after irradiation in metaphase I. Concerning Bombyx, Kawaguchi (1928) states, "Die Kerne in den Ovarialeiern der Schmet- terlinge nach ihrem Ausschliipfen aus der Puppe stehen fast immer im Stadium der Metaphase der ersten Reifteilung." This indicates that in Bonibyx also treatment was given in metaphase I, since adult females were irradiated and then mated to untreated males. That there is some special cytological response of tetrads to irradiation which causes chromatin bridges to be formed in division II, is apparent in both Chaetopterus and Habrobracon. The author (1945b) has discussed this in some detail but has been prevented from checking the theory completely by the small size of Habrobracon chromosomes. Chaetopterus chromosomes are relatively large and distinctive in character and should be analyzed in detail from this viewpoint. Packard describes chromatin bridges (dicentrics) in cleavage in eggs irradiated with doses low enough X-RAYS AND ANDROGENESIS 359 to permit the egg pronucleus to function in syngamy, a fact consistent with the con- ditions found in Habrobracon. No cytological study of Bomby.v eggs after treatment has been made. Hasi- moto's conclusion, derived from genetic data, that androgenetic males (diploid) arise from the union of two sperm pronuclei, is not inconsistent with the suggestion that, in this form also, chromatin bridges retard the egg pronucleus. That some viscosity change may be involved in Habrobracon androgenesis as well as chromatin attachment of the egg pronucleus to the polar nucleus, has been mentioned above. Hasimoto and Astaurow obtained androgenetic males in Bombyx by thermo-activation alone during meiotic divisions and after fertilization, and this may be evidence for viscosity change and movement of sperm pronuclei from their usual position. However, in Chactoptcrus the sperm chromosomes remain in their normal position, close to those of the egg, yet syngamy does not occur. A comparison of other factors in the two insect genera shows further similarities. In both, the percentage of eggs developing into androgenetic adults is low — 1.57 per cent in Habrobracon; never higher than 0.273 per cent in Bomby.v (Astaurow, 1937). In the former, 54,000 r is the highest dose at which androgenetic males were obtained ; in the latter, 27,000 r was the highest dose tested, and they were produced after this treatment. In the Chactopterus study, treatments were measured in minutes of exposure to radium bromide. After 50 minutes' exposure, about 70 per cent of treated eggs underwent haploid cleavage. At longer treatments the per- centage dropped, until prolonged exposure stopped cleavage altogether. In Chaetopterns, incidence of androgenesis but not adult survival ; in Bomby.v, adult survival but not incidence; and in Habrobracon. both incidence and adult sur- vival, have been determined. SUMMARY 1. 1.57 per cent of Habrobracon eggs X-rayed in first meiotic metaphase (14,420 r-28,840 r) and laid by treated females mated to untreated males develop into androgenetic males. These will develop after any dose up to 54,000 r although lethal dose for the egg chromosomes in this stage is 2400 r. Cytological study of 294 such eggs (14,420 r-36,050 r) shows that three were undergoing androgenetic cleavage while three others were possibly preparing for it. A maximum of six, or 2.04 per cent, does not differ significantly from androgenetic survivors, and it must be concluded that androgenetic embryos at doses used are as viable as embryos de- veloping in untreated eggs. 2. Chromatin bridges which appear in meiotic division II after treatment in first meiotic metaphase retard and distort the egg pronucleus, occasionally to such a degree that the sperm pronucleus -cleaves and develops into a normal fertile haploid male with paternal traits only. The almost complete absence of these bridges after treatment in first meiotic prophase will explain the failure of androgenetic males to develop in these eggs. LITERATURE CITED ASTAUROW, B. L., 1937. Versuche iiber Experimentelle Androgenese und Gynogenese beim Seidenspinner (Bombyx mori L.). Biologicheskij Zhurnal, 6: 3-50. DARLINGTON, C. D., 1937. Recent advances in cytology. Philadelphia, Blakiston. 360 ANNA R. WHITING HASIMOTO, HARUO, 1934. Formation of an individual by the union of two sperm nuclei in the silkworm. Bull. Impcr. Scricult. Exp. Sta., 8: 463-464. KAWAGUCHI, E., 1928. Zytologische Untersuchungen am Seidenspinner und seinen Verwandten. Z. f. Zcllforsch. u. mikr. Anatomic, 4 : 519-552. PACKARD, CHARLES, 1918. The effect of radium radiations on the development of Chaetopterus. Biol. Bull., 35: 50-71. SHARPE, LESTER W., 1934. Introduction to cytology. New York, McGraw-Hill. SPEICHER, B. R., 1936. Oogenesis, fertilization and early cleavage in Habrobracon. Jour. Morph., 59: 401-421. WHITING, ANNA R., 1938. Sensitivity to X-rays of stages in oogenesis of Habrobracon. Rcc. Genetics Soc. Am., 7 : 89. WHITING, ANNA R., 1941. X-ray sensitivity of first meiotic prophase and metaphase in Habrobracon eggs. Rcc. Genetics Soc. Am., 10 : 174. WHITING, ANNA R., 1945a. Effects of X-rays on hatchability and on chromosomes of Habro- bracon eggs treated in first meiotic prophase and metaphase. Amcr. Naturalist, 79: 193-227. WHITING, ANNA R., 1945b. Dominant lethality and correlated chromosome effects in Habro- bracon eggs X-rayed in diplotene and in late metaphase I. Biol. Bull., 89: 61-71. WHITING, ANNA R., 1946a. Motherless males from irradiated eggs. Sci., 103: 219-220. WHITING, ANNA R., 1946b. Androgenetic males from eggs X-rayed with dose many times lethal. Rcc. Am. Soc. Zool, 96: 11. WILSON, E. B., 1925. The cell in development and heredity. New York, Macmillan. ERRATUM PAPERS PRESENTED AT GENERAL SCIENTIFIC MEETINGS, MARINE BIOLOGICAL LABORATORY, SUMMER OF 1948 OCTOBER, 1948, ISSUE. PAGES 264-265 The relation of the plasma membrane, vitelline membrane and jelly in the egg of Nereis limbata. DONALD P. COSTELLO. This article contains abstracts of two papers presented by Dr. Costello. The title "Spiral Cleavage" should be inserted following line 6, page 265. 361 INDEX A BELSON, P. H. Studies of the chemical form of P32 after entry into the Arbacia egg, 262. ABRAMS, RICHARD, J. M. GOLDINGER, AND E. S. G. BARRON. Synthesis reactions with acetic acid in isolated bone marrow, 284. Abstracts of scinntific papers presented at the Marine Biological Laboratory, summer of 1948, 238. Accommodation, measurement of; comparison of frog nerve and squid axon, 256. Acetylcholine, wave of negativity produced by, conducted over an oil-saline interface, 281. Action of choline and related compounds on the heart of Venus mercenaria, 346. Action of NH4C1 on the surface membranes of Arbacia eggs, 267. Action pattern of crystalline muscle phos- phorylase, 238. Activity and distribution of desoxyribonu- clease and phosphatases in the early de- velopment of Arbacia punctulata, 250. Actomyosin, thermodynamic theory of con- traction of, 284. Addresses at the Lillie Memorial Meeting, Woods Hole, August 11, 1948, 151. AGERSBORG, H. P. K. The distribution of the cerebrospinal fluid in the lower verte- brates, 261. Alkaline phosphatase in demineralized mouse bones of different ages, 240. Alloxan action, mechanism of: reaction of alloxan with sulfhydryl groups; gluta- thione content of islet tissue, 276. Amino acids, order of, in silk, 240. Androgenesis, incidence and origin of, in X-rayed Habrobracon eggs, 354. Annelids, phosphagen in, 273. Annual report of the Marine Biological Labo- ratory, 1. Anticholinesterases, effect of, on conduction, 241. Anxiety states, hippuric acid excretion in, 246. Apyrase activity of invertebrate marine muscle, ' 281. Apyrase systems, temperature coefficients of, from muscles of different animals, 287. Arbacia, inhibition of fertilization in, by blood extracts, 69. Arbacia eggs, action of NH4C1 on surface membranes of, 267. Arbacia eggs, combined effect of ultraviolet light and heat upon first cleavage of, 259. Arbacia eggs, effects of pressure on insemina- tion reactions of, 251. Arbacia eggs, inhibition of development of, by NH4C1, 267. Aselomaris michaeli, a new gymnoblastic hybroid, life cycle of, 289. Asterias, respiration of oocytes, unfertilized eggs and fertilized eggs from, 124. AUGUSTINSSON, KLAS-BERTiL. On the speci- ficity of cholinesterase, 241. DACTERIA, predictable mutations in, 258. Bacterial toxins, effect of, on permeability of dogfish erythrocytes, 255. BALDWIN, ERNEST AND WARREN H. YUDKIN. Phosphagen in annelids (Polychaeta), 273. BALL, E. G. See R. K. CRANE AND A. K. SOLOMON, 248. BARNES, T.. C. AND R. BEUTNER. The wave of negativity produced by acetylcholine conducted over an oil-saline interface, 281. BARRON, E. S. G. See ARNOLD LAZAROW, 276. Basal mat, development of, in Hydractinia, 260. BENSON, ELEANORE. See DANIEL MAZIA AND GERTRUDE BLUMENTHAL, 250. BERMAN, JACK. See ARNOLD LAZAROW, 276. BERRILL, N. J. The life-cycle of Aselomaris michaeli, a new gymnoblastic hydroid, 289. BERRILL, N. J. A new method of reproduction in Obelia, 94. BEUTNER, R. See T. C. BARNES, 281. Biochemical and histochemical observations on the sexual dimorphism of mouse sub- maxillary glands, 243. Biological specifity and protein structure, 247. BLACK, VIRGINIA S. Changes in density, weight, chloride, and swimbladder gas in the killifish, Fundulus heteroclitus, in fresh water and sea water, 83. BLAUCH, BERTINA M, See P. W. WHITING, 243. BLISS, A. F. The extraccion of purified squid "visual purple," 242. 362 INDEX 363 Blood (canine), kinetics of potassium exchange between cells and plasma of, in vitro, using K42, 287. Blood extracts, inhibition of fertilization in Arbacia by, 69. BLUMENTHAL, GERTRUDE. See DANIEL MAZIA AND ELEANORE BENSON, 250. BLUMENTHAL, GERTRUDE. See DANIEL MAZIA, 283. Bone marrow, synthesis reactions with acetic acid in, 284. .Bones (mouse), alkaline phosphatase in de- mineralized, of different ages, 240. BOREI, HANS. Respiration of oocytes, un- fertilized eggs, and fertilized eggs from Psammechinus and Asterias, 124. BROOKS, S. C. See E. L. CHAMBERS, W. WHITE, AND NYLAN JEUNG, 252. BROOKS, S. C. See E. L. CHAMBERS, A. WHITELEY, R. CHAMBERS, AND S. C. BROOKS, 263. BROOKS, S. C. AND E. L. CHAMBERS. Pene- tration of radioactive phosphate into the eggs of Strongylocentrotus purpuratus, S. franciscanus, and Urechis caupo, 262. BROWN, A. H., E. W. EAGER, AND H. GAFFRON. A photosynthetic intermediate, 284. BULLOCK, JANE A. See F. R. HUNTER AND JUNE RAWLEY, 255. BULLOCK, THEODORE H. Non-integrative syn- . apses, 249. BURK, DEAN. See SILVIO FIALA. 282. GE hypothesis and a common feature of X-ray diffraction studies of crystalline proteins, 272. Carbon dioxide, incorporation of, into organic linkage by retina, 248. Cartesian diver technique: a simplified mixing method in a new type of Cartesian diver vessel, 253. Cells (larval epidermal), Golgi material in, of Drosophila, 163. Cerebrospinal fluid, distribution of, in lower vertebrates, 261. Cerebrospinal fluid, implications of distribu- tion, in healing therapy, 261. Chaetopterus, protoplasmic viscosity changes * during mitosis in egg of, 57. CHAMBERS, E. L. See S. C. BROOKS, 262. CHAMBERS, E. L., W. WHITE, NYLAN JEUNG, AND S. C. BROOKS. Penetration and effects of low temperature and cyanide on penetration of radioactive potassium into eggs of Strongylocentrotus purpuratus and Arbacia punctulata, 252. CHAMBERS, E. L., A. WHITELEY, R. CHAMBERS, AND S. C. BROOKS. Distribution of radioactive phosphate in the eggs of the sea urchin Lytechinus pictus, 263. CHAMBERS, R. See E. L. CHAMBERS, A. WHITELEY, AND S. C. BROOKS, 263. Changes in density, weight, chloride, and swimbladder gas in the killifish, Fundulus heteroclitus, in fresh water and sea water, 83. CHASE, AURIN M. On the combining weight of Cypridina luciferin, 263. Choline, action of, on heart of Venus mer- cenaria, 346. Choline acetylase and choline esterase content of some invertebrate tissues, 278. Cholinesterase, specificity of, 241. Cholinesterase (human plasma), interaction of inhibitors with, 275. Cholinesterases: Report of investigations, sum- mer 1948, 278. CLAFF, C. LLOYD AND T. N. TAHMISIAN. Cartesian diver technique: a simplified mixing method in a new type of Cartesian diver vessel, 253. Clam tissues, further observations on metab- olism of, in sea water at different salinities, 265. CLARK, A. M. AND D. S. GROSCH. Fat cell size in the mutant small-wings of Habro- bracon, 264. Cleavage (sea urchin egg), inhibition of, by a series of substituted carbamates, 244. Cleavage, spiral, 265. [See Erratum, 361.] COHEN, ARTHUR. Apyrase activity of in- vertebrate marine muscle, 281. COHEN, ISADORE. Fixation and staining of plant nuclei in lacto-sudan black b, 253. COLWIN, LAURA HUNTER. Note on spawning of the holothurian, Thyone briareus (Lesueur), 296. Comparison of frog nerve and squid axon with respect to the measurement of accommo- dation, 256. Conduction, effect of anticholinesterases on, 241. COOPERSTEIN, S. J. See B. EICHEL AND W. W. WAINIO, 239. CORNMAN, IVOR. Inhibition of sea urchin egg cleavage by a series of substituted carba- mates, 244. CORNMAN, IVOR. Lactones as mitotic poisons, tested on sea urchin eggs, 252. COSTELLO, DONALD P. The relation of the plasma membrane, vitelline membrane and jelly in the agg of Nereis limbata, 264 COSTELLO, DONALD P. Spiral cleavage, 265' [See Erratum, 361.] Crab (blue), fungus Lagenidium callinectes Couch on eggs of, in Chesapeake Bay, 214 Crabs (grapsoid), role of sinus glands in retina . pigment migration in, 169. 364 INDEX CRANE, R. K., E. G. BALL, AND A. K. SOLOMON, The incorporation of carbon dioxide into organic linkage by retina, 248. Crayfish, molting and sexual cycles in, 229. Crepidula plana, new experiments on sexual instability in, 255. CROWELL, SEARS. The development of the basal mat in Hydractinia, 260. CROWELL, SEARS. Specificity in the fusion of stolons in hydroids, 261. Crustacyanin, the blue carotenoid protein of the lobster shell, 249. Cytochrome system in relation to diapause and development in the Cecropia silkworm, 282. T^AS, S. M. The physiology of excretion in Molgula (Tunica ta, Ascidiacea), 307. Desoxyribonuclease, activity and distribution of, in early development of Arbacia, 250. Diabetes, development of; sulfhydryl metab- olism of beta cell and relationship to, 239. Diabetic fish (alloxan), insulin content of islet tissue of, 276. Diapause and development in Cecropia silk- worm, cytochrome system in relation to, 282. Dicumarol, new concept of action of, 277. Diethylstilbesterol in the production of eye mutations in Drosophila melanogaster, 258. DILLER, WILLIAM F. An extra post-zygotic division in Paramecium caudatum, 265. DILLER, WILLIAM F. Induction of autogamy in single animals of Paramecium calkinsi following mixture of two mating types, 265. Distribution of radioactive phosphate in the eggs of the sea urchin Lytechinus pictus, 263. Dogfish (smooth), tooth succession in, 100. Dogfish (smooth), urea reabsorption in kidney of, 253. Dominant lethals induced by X-rays in sperm of the chalcidoid wasp Nasonia brevicornis Ashmead, 257. Doubtful character of "break" excitation in skeletal muscle, 256. Drosophila melanogaster, Golgi material in larval epidermal cells of, 163. Drosophila melanogaster, use of diethylstil- besterol in production of eye-mutations in, 258. Drosophila, utilization of sugars and other substances by, 114. pFFECTof "stabilizing" and "unstabilizing" agents in relation to the metabolic mech- anism supporting the resting potential of nerve, 245. Effects of pressure on the insemination reactions of Arbacia eggs, 251. Egg (Chaetopterus), protoplasmic viscosity changes during mitosis in, 57. Eggs (Blue Crab), fungus Lagenidium calli- nectes Couch on, in Chesapeake Bay, 214. Eggs (Habrobracon), X-rayed, incidence and origin of androgenetic males in, 354. Eggs (Nereis), .chemical aspects of sensitization and activation reactions of, 333. Eggs (Psammechinus and Asterias), respiration of unfertilized and fertilized, 124. ElCHEL, B., S. J. COOPERSTEIN, AND W. W. WAINIO. A partial separation of the cytochromes of mammalian heart muscle, 239. Embryos (Arbacia), incorporation of P32 into nucleoproteins and phosphoproteins of, 279. Embryos (Mustelus canis), inter-myotome connections in early, 270. Enzyme activity and radiation sensitivity of enzyme-substrate films, 283. Enzyme localization in the giant nerve fiber of the squid, 277. Erythrocytes (dogfish), effect of bacterial toxins on permeability of, 255. Erythrocytes, reversible sphering of, 268. Euglena, streptomycin-induced chlorophyll- less races of, 260. Excretion, physiology of, in Molgula, 307. Experiments on chloroplasts and on photo- synthesis, 270. Extra post-zygotic division in Paramecium caudatum, 265. Extraction of purified squid "visual purple," 242. UACTORS influencing molting and sexual cycles in the crayfish, 229. FAGER, E. W. See A. H. BROWN AND H. GAFFRON, 284. FAJER, A. See L. C. JUNQUEIRA, M. RABINO- VITCH, AND L. FRANKENTAHL, 243. Fat cell size in the mutant small-wings of Habrobracon, 264. Fatty acids (lower), relative rate of penetration of, into beef red cells, 245. Fatty acids (lower), relative rate of penetration of, into erythrocytes of smooth dogfish, 255. Fertilization, inhibition of, in Arbacia by blood extracts, 69. Fertilizin of Nereis limbata, 271. FIALA, SILVIO AND DEAN BURK. On the nature of iron binding by siderophilin, conalbumin, hydroxylamine, aspergillic acid, and related hydroxamic acids, 282. INDEX 365 Fish, red blood cells of, 266. Fixation and staining of plant nuclei in lacto- sudan black b, 253. FRANKENTAHL, L. See L. C. JUNQUEIRA, A. FAJER, AND M. RABINOVITCH, 243. FROELICH, A. The influence of theophylline on the absorption of Mg-salts from the gastro- intestinal canal, 254. Fundulus heteroclitus, changes in density, weight, chloride, and swimbladder gas in, in fresh water and sea water, 83. Fundulus heteroclitus, properties of surface coat in embryos of, 271. Fungus Lagenidium callinectes Couch (1942) on eggs of the blue crab in Chesapeake Bay, 214. Further chemical aspects of the sensitization and activation reactions of Nereis eggs, 333. pAFFRON, H. See A. H. BROWN AND E. W. FAGER, 284. Gastro-intestinal canal, influence of theo- phylline on absorption of Mg-salts from, 254. Genes: Do genes exist, 257. Genetic block to free oviposition in the chal- cidoid wasp Melittobia sp.-C, 243. Glands (mouse submaxillary), biochemical and histochemical observations on sexual dimorphism of, 243. Glands (prothoracic), of Leucophaea maderae (Orthoptera), 186. Glands (sinus), role of, in retinal pigment migration in grapsoid crabs, 169. GOLD, MARCIA. See HAROLD PERSKY, 278. GOLDIXGER, J. M. See RICHARD ABRAMS AND E. S. G. BARRON, 284. GOLDSTEIN, AVRAM. The mechanism of inter- action of inhibitors with human plasma cholinesterase, 275. GOLDSTEIN, AVRAM AND DORA B. GOLDSTEIN. Report of investigations, summer 1948, 278. Golgi material in larval epidermal cells of Drosophila, 163. GORDON, M. See C. A. VILLEE, M. LOWENS, E. LEONARD, AND A. RICH, 279. GOULD, HARLEY N. AND SIDNEY C. HSIAO. New experiments and observations on sexual instability in Crepidula plana, 255. GREEN, JAMES W. The relative rate of pene- tration of the lower fatty acids into beef red cells, 245. GREEN, JAMES W. The relative rate of pene- tration of the lower fatty acids into ery- throcytes of the smooth dogfish, 255. GROSCH, D. S. See A. M. CLARK, 264. Growth changes (postembryonic) in Pentidotea resecata (Stimpson), 107. LJ ABROBRACON, fat cell size in the mutant small-wings of, 264. Habrobracon, method of origin of androgenetic males in, 259. Habrobracon eggs (X-rayed), incidence and origin of androgenetic males in, 354. HASSETT, CHARLES C. The utilization of sugars and other substances by Droso- phila, 114. Heart, action of choline and related compounds on, of Venus mercenaria, 346. Heart muscle (mammalian), partial separation of cytochromes of, 239. HEILBRUNN, L. V. AND W. L. WILSON. Proto- plasmic viscosity changes during mitosis in the egg of the Chaetopterus, 57. HEILBRUNN, L. V. AND W. L. WILSON. The relation of heparin to protoplasmic clotting, 283. Hemocyanin and hemorythrin complexes with small ions, 275. Hemolytic effect of silver, nature of, 268. Hemolytic effect of sodium dodecyl sulfate, observations on, 269. HESTRIN, SHLOMO. Action pattern of crystal- line muscle phosphorylase, 238. Hippuric acid excretion in anxiety states, 246. HOPKINS, HOYT S. Further observations on the metabolism of clams' tissues in sea water at different salinities, 265. HSIAO, SIDNEY C. See HARLEY N. GOULD, 255. Hsu, W. SIANG. Some observations on the Golgi material in the larval epidermal cells of Drosophila melanogaster, 163. HUNTER, F. R. Osmotic hemolysis in hyper- tonic solutions, 246. HUNTER, F. R., JANE A. BULLOCK, AND JUNE RAWLEY. The effect of bacterial toxins on the permeability of dogfish erythro- cytes, 255. HUNTER, S. H. See LUIGI PROVALOSI AND ALBERT SCHATZ, 260. HUTCHENS, JOHN O. AND BETTY PODOLSKY. The effects of nitrogen mustards on cleav- age and development of Arbacia eggs, 251. HUTCHINGS, Lois M. Combined effect of ultraviolet light and heat upon first cleav- age of Arbacia eggs, 259. Hydractinia, development of basal mat in, 260. Hydrogen-ion concentration in the cultivation and growth of eight species of Paramecium, 272. Hydroid (gymnoblastic), life cycle of a new, Aselomaris michaeli, 289. Hydroxamic acids, iron binding by, 282. 366 INDEX Hypertonic solutions, effects of, on Nereis ^ eggs, 269. Hypertonic solutions, osmotic hemolysis in, 246. TFFT, JOHN D. AND DONALD J. ZINN. Tooth succession in the smooth dogfish, Mustelus canis, 100. Incidence and origin of androgenetic males in X-rayed Habrobracon eggs, 354. Incorporation of carbon dioxide into organic linkage by retina, 248. Induction of autogamy in single animals of Paramecium calkinsi following mixture of two mating types, 265. Inhibition of development of Arbacia eggs by NH4C1, 267. Inhibition of fertilization in Arbacia by blood extracts, 69. Inhibition of sea urchin egg cleavage by a series of substituted carbamates, 244. Insulin content of the islet tissues of alloxan diabetic fish, 276. Intermediate, photosynthetic, 284. Inter-myotome connections in early embryos of Mustelus canis, 270. Invertebrates (marine), studies on nucleopro- teins from, 280. Ion permeability of the giant axon of squid, 242. Ions (small), complexes of hemocyanin and hemorythrin with, 275. Iron binding by siderophilin, conalbumin, hydroxylamine, aspergillic acid, and re- lated hydroxamic acids, 282. Islet tissue, glutathione content of, 276. Isotopic derivative technic, application of; order of amino acids in silk, 240. JACOBS, M. H. See MARIAN E. LEFEVRE, J 268. JACOBS, M. H. See WARNER E. LOVE, 268. JACOBS, M. H. See Lois H. LOVE, 269. JENCKS, WILLIAM P. See GEORGE WALD, NEAL NATHANSON, AND ELIZABETH TARR, 249. JEUNG, NYLAN. See E. L. CHAMBERS, W. - WHITE, AND S. C. BROOKS, 252. JOHNSON, MARTIN W. AND J. BENNETT OLSON. The life history and biology of a marine harpacticoid copepod, Tisbe furcata (Baird), 320. JUNQUEIRA, L. C., A. FAJER, M. RABINOVITCH, AND L. FRANKENTAHL. Biochemical and histochemical observations on the sexual dimorphism of mouse submaxillary glands, 243. I7ELLER, RUDOLPH. Vital staining in ultra- violet and in white light combined, 238. KEMPTON, RUDOLPH T. Urea reabsorption in the smooth dogfish kidney, 253. KIMBERLEY, PAUL E. Implications of cere- brospinal fluid distribution in the therapy of the healing arts, 261. KISCH, BRUNO. Studies on the red blood cells of fish, 266. KLOTZ, I. M. AND F. TIETZE. Complexes of hemocyanin and of hemorythrin with small ions, 275. KOPAC, M. J. The action of NH4C1 on the surface membranes of Arbacia eggs, 267. KOPAC, M. J. The inhibition of development of Arbacia eggs by NH4C1, 267. T ACTO-SuDAN BLACK B, fixation and stain- ing of plant nuclei in, 253. Lactones as mitotic poisons, tested on sea urchin eggs, 252. Lagenidium callinectes Couch (fungus) on eggs of blue crab in Chesapeake Bay, 214. Lalor Fellowship Research, report on, 273. Larval epidermal cells (Drosophila), Golgi material in, 163. LA.ZAROW, ARNOLD. Further studies on the mechanism of alloxan action: the reaction of alloxan with sulfhydryl groups; the glutathione content of islet tissue, 276. LAZAROW, ARNOLD. Sulfhydryl metabolism of the beta cell and its relationship to the development of diabetes, 239. LAZAROW, ARNOLD AND JACK BERMAN. The insulin content of the islet tissue of alloxan diabetic fish, 276. LEFEVRE, MARIAN E. AND M. H. JACOBS. The nature of the hemolytic effect of silver, 268. LEFEVRE, PAUL G. Comparison of frog nerve and squid axon with respect to the meas- urement of accommodation, 256. LEFEVRE, PAUL G. The doubtful character of "break" excitation in skeletal muscle, 256. LEFEVRE, PAUL G. Futher chemical aspects of the sensitization and activation reac- tions of Nereis eggs, 333. LEIN, JOSEPH. A new concept of the action of dicumarol, 277. LEONARD, E. See C. A. VILLEE, M. LOWENS, M. GORDON, AND A. RICH, 279. LEONARD, E. See C. A. VILLEE AND A. RICH, 280. Leucophaea maderae (Orthoptera), prothoracic glands of, 186 LEVY, MILTON AND EVELYN SLOBODIANSKY. The order of amino acids in silk: an ap- INDEX 367 plication of isotopic derivative technic, 240. LIBET, B. Enzyme localization in the giant nerve fiber of the squid, 277. Life cycle of Aselomaris michaeli, a new gym- noblastic hydroid, 289. Life history and biology of a marine harpacti- coid copepod, Tisbe furcata (Baird), 320. Lillie Memorial Addresses, 151. Liu, C. K. X-radiation effects on the restitu- tion of dissociated Microciona, 259. Lobster shell, blue carotenoid-protein of (crustacyanin), 249. LOVE, Lois H. AND M. H. JACOBS. Observa- tions on the hemolytic effect of sodium dodecyl sulfate, 269. LOVE, WARNER E. AND M. H. JACOBS. Re- versible sphering of erythrocytes, 268. LOWENS, M. Ses C. A. VILLEE, M. GORDON, E. LEONARD, AND A. RICH, 279. Luciferin (cypridina), combining weight of, 263. V/f ANN, ELIZABETH ROGERS. See THEODOR VON BRAND AND M. O. NOLAN, 199. Marine Biological Laboratory, annual report of, 1. MARSHAK, A. A nuclear precursor to ribo- and desoxyribonucleic acids, 244. MARSLAND, DOUGLAS. The effects of pressure on the insemination reactions of Arbacia eggs, 251. MARTIN, W. R. See C. W. SHEPPARD, 287. Mating types and conjugation of four different races of Paramecium calkinsi and the effect of X-rays on the mating reaction, 271. MAZIA, DANIEL AND GERTRUDE BLUMENTHAL. Enzyme activity and radiation sensitivity of enzyme-substrate films, 283. MAZIA, DANIEL, GERTRUDE BLUMENTHAL, AND ELEANORE BENSON. The activity and distribution of desoxyribonuclease and phosphatases in the early development of Arbacia punctulata, 250. Mechanism of interaction of inhibitors with human plasma cholinesterase, 275. MENZIES, ROBERT J. AND RICHARD J. WAID- ZUNAS. Postembryonic growth changes in the isopod Pentidotea resecata (Stimp- son) with remarks on their taxonomic significance, 107. Metabolism of clams' tissues in sea water at different salinities, further observations on, 265. Method of origin of androgenetic males in Habrobracon, 259. Mg-salts, influence of theophylline on absorp- tion of, from gastro-intestinal canal, 254. Microciona (dissociated), X-radiation effects on restitution of, 259. MILLER, JAMES A., JR. pH estimation in re- constituting pieces of Tubularia stems, 243. Mitosis, protoplasmic viscosity changes during, in egg of Chaetopterus, 57. Mitotic poisons, lactones as, tested on sea urchin eggs, 252. Molgula, physiology of excretion in, 307. Molting, factors influencing, in crayfish, 229. Muscle (invertebrate marine), apyrase activity of, 281. Muscle (skeletal), doubtful character of "break" excitation in, 256. Muscle, temperature coefficients of apyrase systems from, of different animals, 287. Muscle phosphorylase (crystalline), action pattern of, 238. Mustelus canis, tooth succession in, 100. VTACHMANSON, DAVID. Effect of anti- cholinesterases on conduction, 241. Nasonia brevicornis Ashmead (chalcidoid wasp), dominant lethals induced by X-rays in sperm of, 257. NATHANSON, NEAL. See GEORGE WALD, WILLIAM P. JENCKS, AND ELIZABETH TARR, 249. NELSON, LEONARD. Usnic acid, an antibiotic, and sperm metabolism, 286. Nereis eggs, chemical aspects of sensitization and activation reactions of, 333. Nereis eggs, effects of hypertonic solutions on, 269. Nereis eggs, solubility of vitelline membrane of, 269. Nereis limbata, fertilizin of, 271. Nereis limbata egg, relation of plasma membrane, vitelline membrane, and jelly in, 264. Nerve, effect of "stabilizing" and "unstabiliz- ing" agents in relation to metabolic mech- anism supporting resting potential of, 245. Nerve fiber (giant), enzyme localization in, of squid, 277. New concept of the action of dicumarol, 277. New experiments and observations on sexual instability in Crepidula plana, 255. New method of reproduction in Obelia, 94. Nitrogen mustards, effects of, on cleavage and development of Arbacia eggs, 251. NOLAN, M. O. See THEODOR VON BRAND AND ELIZABETH ROGERS MANN, 199. Non-integrative synapses, 249. Note on spawning of the holothurian, Thyone briareus (Lesueur), 296. Nuclear precursor to ribo- and desoxyribo- nucleic acids, 244. Nucleoproteins from marine invertebrates, 280. 368 INDEX /~\BELIA, new method of reproduction in, 94. Observations on the respiration of Atistralorhis glabratus and some other aquatic snails, 199. OLSON, J. BENNET. See MARTIN W JOHNSON, 320. On the combining weight of Cypridina luciferin, 263. On the nature of iron binding by siderophilin, conalbumin, hydroxylamine, aspergillic acid, and related hydroxamic acids, 282. On the specificity of cholinesterase, 241 Oocytes, respiration of, from Psammechinus and Asterias, 124. Osmotic hemolysis in hypertonic solutions, 246. OSTERHOUT, W. J. V. Effects of hypertonic solutions on Nereis eggs, 269. OSTERHOUT, W. J. V. Experiments on chloro- plasts and on photosynthesis, 270. OSTERHOUT, W. J. V. Solubility of the vitel- line membrane of Nereis eggs, 269. Oviposition, genetic block to, in chalcidoid wasp Melittobia sp.-C, 243. p32, chemical form of, after entry into Arbacia egg, 262. P32 incorporation into the nucleoproteins and phosphoproteins of developing Arbacia embryos, 279. pH estimation in reconstituting pieces of Tubularia stems, 243. Papers presented at the meeting of the Society of General Physiologists, 281. Paramecium, eight species of, hydrogen-ion concentration in cultivation and growth of, 272. Paramecium calkinsi, induction of autogamy in, 265. Paramecium calkinsi, mating types and con- jugation of four races of, and effect of X-rays on mating reaction, 271. Paramecium caudatum, an extra post-zygotic division in, 265. Partial separation of the cytochromes of mammalian heart muscle, 239. Penetration and effects of low temperature and cyanide on penetration of radioactive potassium into the eggs of Strongylocen- trotus purpuratus and Arbacia punctulata, 252. Penetration of radio-active phosphate into the eggs of Strongylocentrotus purpuratus, S. franciscanus, and Urechis caupo, 262. Pentidotea resecata (Stimpson), postembryonic growth changes in, 107. PEQUEGNAT, WILLIS E. Inhibition of fertiliza- tion in Arbacia by blood extracts, 69. PERSKY, HAROLD. Hippuric acid excretion in anxiety states, 246. PERSKY, HAROLD AND MARCIA GOLD. The choline acetylase and choline esterase content of some invertebrate tissues, 278. Physiology of excretion in Molgula (Tunicata, Ascidiacea), 307. Phosphagen in annelids (Polychaeta), 273. Phosphatases, activity and distribution of, in early development of Arbacia, 250. Photosynthesis, experiments on, and on chloroplasts, 270. Photosynthetic intermediate, 284. PODOLSKY, BETTY. See JOHN O. HUTCHENS, 251. Postembryonic growth changes in the isopod Pentidotea resecata (Stimpson) with re- marks on their taxonomic significance, 107. Potassium exchange between cells and plasma of canine blood in vitro using K42, studies of kinetics of, 287. Predictable mutations in bacteria, 258. Properties of the surface coat in embryos of Fundulus heteroclitus, 271. Protein structure, biological specificity and, 247. Proteins (crystalline), X-ray diffraction studies of; cage hypothesis, 272. Prothoracic glands of Leucophaea maderae (Orthoptera), 186. Protoplasmic clotting, relation of heparin to, 283. Protoplasmic viscosity changes during mitosis in the agg of the Chaetopterus, 57. PROVALOSI, LUIGI, S. H. HUNTER, AND ALBERT SCHATZ. Streptomycin-induced chloro- phyll-less races of Euglena, 260. Psammechinus, respiration of oocytes, un- fertilized and fertilized eggs from, 124. T> ABINOVITCH, M. See L. C. JUNQUEIRA, A. FAJER, AND L. FRANKENTAHL, 243. Radiation sensitivity and enzyme activity of enzyme-substrate films, 283. Radioactive phosphate, distribution of, in eggs of sea urchin Lytechinus pictus, 263. Radioactive phosphate, penetration of, into eggs of Strongylocentrotus purpuratus, S. franciscanus, and Urechis caupo, 262. Radioactive potassium, low temperature and cyanide effects on penetration of, into eggs of Strongylocentrotus and Arbacia, 252. RAWLEY, JUNE. See F. R. HUNTER AND JANE A. BULLOCK, 255. RAY, D. T. Dominant lethals induced by X-rays in sperm of the chalcidoid wasp Nasonia brevicornis Ashmead, 257. Red blood cells of fish, studies on, 266. INDEX 369 Relation of heparin to protoplasmic clotting, 283. Relation of the plasma membrane, vitelline membrane and jelly in the egg of Nereis limbata, 264. Relative rate of penetration of the lower fatty acids into beef red cells, 245. Relative rate of penetration of the lower fatty acids into erythrocytes of the smooth dog- fish, 255. Report of investigations [on cholinesterase], summer 1948, 278. Reproduction, new method of, in Obelia, 94. Respiration, observations on, of Australorbis glabratus and other aquatic snails, 199. Respiration of oocytes, unfertilized eggs and fertilized eggs from Psammechinus and Asterias, 124. Retinal pigment migration, role of sinus glands in, in grapsoid crabs, 169. Reversible sphering of erythrocytes, 268. Ribonucleic and desoxyribonucleic acids, nu- clear precursor to, 244. RICH, A. See C. A. YILLEE, M. LOWENS, M. GORDON, AND E. LEONARD, 279. ROGERS-TALBERT, R. The fungus Lageni- dium callinectes Couch (1942) on eggs of the blue crab in Chesapeake Bay, 214. Role of the sinus glands in retinal pigment migration in grapsoid crabs, 169. ROTHENBERG, M. A. The ion permeability of the giant axon of squid, 242. CANBORN, RICHARD O. See CARROLL M. WILLIAMS, 282. SCHARRER, BERTA. The prothoracic glands of Leucophaea maderae (Orthoptera), 186. SCHATZ, ALBERT. See LUIGI PROVALOSI AND S. H. HUNTER, 260. SCUDAMORE, HAROLD H. Factors influencing molting and sexual cycles in the crayfish, 229. Sensitization and activation reactions, chemical aspects of, of Nereis eggs, 333. Sexual cycles in crayfish, factors influencing, 229. SHANES, ABRAHAM M. The effect of "stabiliz- ing" and "unstabilizing" agents in relation to the metabolic mechanism supporting the resting potential of nerve, 245. SHEPPARD, C. W. AND W. R. MARTIN. Studies of the kinetics of potassium exchange be- tween cells and plasma of canine blood in vitro using K42, 287. Silver, hemolytic effect of, 268. Sinus glands, role of, in retinal pigment migra- tion in grapsoid crabs, 169. SLOBODIANSKY, EVELYN. See MILTON LEVY, 240. SMITH, RALPH I. The role of the sinus glands in retinal pigment migration in grapsoid crabs, 169. Snails (Australorbis glabratus and others), respiration of, 199. Sodium dodecyl sulfate, hemolytic effect of, 269. SOLOMON, A. K. See R. K. CRANE AND E. G. BALL, 248. Solubility of the vitelline membrane of Nereis eggs, 269. Some observations on the Golgi material in the larval epidermal cells of Drosophila me- lanogaster, 163. Spawning of Thyone briareus (Lesueur), 296. Specificity in the fusion of stolons in hydroids, 261. Sperm metabolism, usnic acid and, 286. Spiral cleavage, 265. [See Erratum, 361.] Squid, extraction of purified "visual purple," 242. Squid, ion permeability of giant axon of, 242. Staining, vital, in ultraviolet and in white light combined, 238. STEINBACH, H. BURR. Temperature coeffici- ents of apyrase systems from muscles of different animals, 287. STEKLER, BURTON L. The use of diethylstil- bestero! in the production of eye mutations in Drosophila melanogaster, 258. Stolons, specificity in fusion of, in hydroids, 261. Streptomycin-induced chlorophyll-less races of Euglena, 260. Studies of the chemical form of P32 after entry into the Arbacia egg, 262. Sugars, utilization of, by Drosophila, 114. Sulfhydryl groups, reaction of alloxan with, 276. Sulfydryl metabolism of the beta cell and its relationship to the development of dia- betes, 239. Synapses, non-integrative, 249. Synthesis reactions with acetic acid in isolated bone marrow, 284. SZENT-GYORGYI, A. Thermodynamic theory of the contraction of actomyosin, 285. ^PAHMISIAN, T. N. See C. LLOYD CLAFF, j. 253. TARR, ELIZABETH. See GEORGE WALD, NEAL NATHANSON, AND WILLIAM P. JENCKS, 249. TAUB, RAE. See JOHN H. WELSH, 346. TEWINKEL, Lois E. Inter-myotome con- nections in early embryos of Mustelus canis, 270. Temperature coefficients of apyrase systems from muscles of different animals, 287. 370 INDEX Theophylline, influence of, on absorption of Mg-salts from the gastro-intestinal canal, 254. Thermodynamic theory of the contraction of actomyosin, 285. Thyone briareus (Lesueur), spawning of, 296. TIETZE, F. See I. M. KLOTZ, 275. Tisbe furcata, life history and biology of, 320. Tissues (invertebrate), choline acetylase and choline esterase content of, 278. Tooth succession in the smooth dogfish, Mustelus canis, 100. TRINKAUS, J. P. Properties of the surface coat in embryos of Fundulus heteroclitus, 271. Tubularia stems, pH estimation in reconstitut- ing pieces of, 243. TYLER, ALBERT. Fertilizin of Nereis limbata, 271. TJLTRAVIOLET light and heat, combined effect of, upon first cleavage of Arbacia eggs, 259. Urea reabsorption in the smooth dogfish kidney 253. Usnic acid, an antibiotic, and sperm metab- olism, 286. Utilization of sugars and other substances by Drosophila, 114. WENUS mercenaria, action of choline and related compounds on heart of, 346. VILLEE, C. A., E. LEONARD, AND A. RICH. Studies on nucleoproteins from marine invertebrates, 280. VILLEE, C. A., M. LOWENS, M. GORDON, E. LEONARD, AND A. RICH. The incorpora- tion of P32 into the nucleoproteins and phosphoproteins of developing Arbacia embryos, 279. Vital staining in ultraviolet and in white light combined, 238. VON BRAND, THEODOR, M. O. NOLAN, AND ELIZABETH ROGERS MANN. Observations on the respiration of Australorbis glabratus and some other aquatic snails, 199. vyAIDZUNAS, RICHARD J. See ROBERT J. MENZIES, 107. WAINIO, W. VV. See B. EICHEL AND S. J. COOPERSTEIN, 239. WALD, GEORGE, NEAL NATHANSON, WILLIAM P. JENCKS, AND ELIZABETH TARR. Crust- acyanin, the blue carotenoid-protein of the lobster shell, 249. Wave of negativity produced by acetylcholine conducted over an oil-saline interface, 281. WELSH, JOHN H. AND RAE TAUB. The action of choline and related compounds on the heart of Venus mercenaria, 346. WHITE, W. See E. L. CHAMBERS, NYLAN JEUNG, AND S. C. BROOKS, 252. WHITELEY, A. See E. L. CHAMBERS, R. CHAMBERS, AND S. C. BROOKS, 263. WHITING, ANNA R. Incidence and origin of androgenetic males in X-rayed Habro- bracon eggs, 354. WHITING, ANNA R. Method of origin of andro- genetic males in Habrobracon, 259. WHITING, P. W. Do genes exist, 257. WHITING, P. W. AND BERTINA M. BLAUCH. The genetic block to free oviposition in the chalcidoid wasp Melittobia sp.-C, 243. WICHTERMAN, RALPH. The hydrogen-ion con- centration in the cultivation and growth of eight species of Paramecium, 272. WICHTERMAN, RALPH. Mating types and con- jugation of four different races of Para- mecium calkinsi and the effect of X-rays on the mating reaction, 271. WILLIAMS, CARROLL M. AND RICHARD C. SANBORN. The cytochrome system in relation to diapause and development in the Cecropia silkworm, 282. WILSON, W. L. See L. V. HEILBRUNN, 57. WILSON, W. L. See L. V. HEILBRUNN, 283. WITKUS, E. RUTH. Predictable mutations in bacteria, 258. WRINCH, DOROTHY. Biological specificity and protein structure, 247. WRINCH, DOROTHY. The cage hypothesis and a common feature of X-ray diffraction studies of crystalline proteins, 272. V-RADIATION effects on the restitution of dissociated Microciona, 259. X-rayed Habrobracon eggs, incidence and origin of androgenetic males in, 354 yUDKIN, WARREN H. See ERNEST BALD- WIN, 273. V INN, DONALD J. See JOHN D. IFFT, 100. ZORZOLI, ANITA. Alkaline phosphatase in demineralized mouse bones of different ages, 240. Volume 95 Number 1 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board E. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden E. N. HARVEY, Princeton University G. H. PARKER, Harvard University LEIGH HOADLEY, Harvard University A. C. REDFIELD, Harvard University L. IRVING, Swarthmore College F. SCHRADER, Columbia University M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University H. B. STEINBACH, University of Minnesota Managing Editor AUGUST, 1948 I Marine Biolefical SEP 7 -1948 WOODS HOLE, MASS. Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. BACK ISSUES 1 HE Laboratory needs early numbers of the BIOLOGICAL BULLETIN to replenish its stock, nearly depleted after meeting the needs of biologists here and abroad during the last 25 years. Members willing to contribute any of the numbers listed below should send them, express collect, to the Marine Biological Laboratory, Woods Hole, Mass. Vol. Nos. Vol. Nos. Vol. Nos. 1 1-6 25 1-6 35 1-6 2 1-6 26 1-6 36 1-6 81 27 4, 5, 6 37 1-6 17 5 28 1, 3, 6 38 1-6 18 5, 6 29 1-6 39 1, 2, 4-6 20 1 30 1-6 40 1-6 21 6 31 1-4, 6 41 1^6 22 1-6 32 1-6 42 1-6 23 2, 5, 6 33 1-6 47 3 24 1-6 34 1-5 BOOKS AND WORLD RECOVERY IHE desperate and continued need for American publications to serve as tools of physical and intellectual reconstruction abroad has been made vividly apparent by appeals from scholars in many lands. The American Book Center for War Devastated Libraries has been urged to continue meeting this need at least through 1948. The Book Center is therefore making a renewed appeal for American books and periodicals — for technical and scholarly books and periodicals in all fields and particularly for publications of the past ten years. We shall especially welcome complete or incomplete recent files of the BIOLOGICAL BULLETIN. The generous support which has been given to the Book Center has made it possible to ship more than 700,000 volumes abroad in the past year. It is hoped to double this amount before the Book Center closes. The books and periodicals which your personal or institutional library can spare are urgently needed and will help in the reconstruction which must preface world understanding and peace. Ship your contributions to the American Book Center, c/o The Library of Congress, Washington 25, D. C, freight prepaid, or write to the Center for further information. BIOLOGICAL ABSTRACTS COVERS THE WORLD'S BIOLOGICAL LITERATURE How do you keep abreast of the literature in your field? No individual possibly could accumulate and read all of the biological contributions in the original — yet some relatively obscure journal might publish a revealing paper on the very subject in which you are most interested. Biological Abstracts now publishes concise, informative abridgments of all the significant contributions from more than 2,500 journals. As well as the complete edition, it also is published in nine low-priced sectional editions which are specially designed for individuals who are interested only in one or more closely related fields. Production costs have increased to such an extent that the active support of all biologists is needed to maintain this important service. Write for full details and a sample copy of the sectional edition covering your field. BIOLOGICAL ABSTRACTS UNIVERSITY OF PENNSYLVANIA PHILADELPHIA 4, PA. MICROFILM SERVICE • The Library of The Marine Biological Laboratory can supply microfilms of ma- terial from periodicals in- cluded in its list. Requests should include the title of the paper, the author, peri- odical, volume and date of publication. Rates are as follows: $.50 for papers up to 50 pages, and $.10 for each additional 10 pages or fraction thereof. LANCASTER PRESS, Inc. LANCASTER, PA. THE EXPERIENCE we have gained from printing some sixty educational publica- tions has fitted us to meet the standards of customers who demand the best. We shall be happy to have workers at the MARINE BIOLOGICAL LABORATORY write for estimates on journals or monographs. Our prices are moderate. INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts papers on a variety of subjects of biologi- cal interest. In general, a paper will appear within three months of the date of its acceptance. The Editorial Board requests that manuscripts conform to the requirements set below. Manuscripts. Manuscripts should be typed in double or triple spacing on one side of paper, BYz by 11 inches. Tables should be typewritten on separate sheets and placed in correct sequence in the text. Explanations of figures should be typed on a separate sheet and placed at the end of the text. Footnotes, numbered consecutively, may be placed on a separate sheet at the end of the paper. A condensed title or running page head of not more than thirty-five letters should be included. Figures. The dimensions of the printed page, 5 by 7% inches, should be kept in mind in preparing figures for publication. Illustrations should be large enough so that all details will be clear after appropriate reduction. Explana- tory matter should be included in legends as far as possible, not lettered on the illustrations. Figures should be prepared for reproduction as line cuts or half- tones; other methods will be used only at the author's expense. Figures to be reproduced as line cuts should be drawn in black ink on white paper or blue- lined co-ordinate paper; those to be reproduced as halftones should be mounted on Bristol board and any designating letters or numbers should be made di- rectly on the figures. The author's name should appear on the reverse side of all figures. The desired reduction should be specified on each figure. Literature cited. The list of literature cited should conform to the style set in this issue of The Biological Bulletin. Papers referred to in the manuscript should be listed on separate pages headed "Literature Cited." Mailing. Manuscripts should be packed flat. Large illustrations may be rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must be accompanied by photographic reproductions or tracings that may be folded to page size. Reprints. Authors will be furnished, free of charge, one hundred reprints without covers. Additional copies may be obtained at cost; approximate figures will be furnished upon request. THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. Subscriptions and similar matter should be addressed to The Biologi- cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, $1.75. Subscription per volume (three issues), $4.50. Communications relative to manuscripts should be sent to the Manag- ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 15 and September 1, and to the Department of Zoology, University of Minnesota, Minneapolis, Minnesota, during the remainder of the year. Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page Annual Report of the Marine Biological Laboratory 1 HEILBRUN, L. V., AND W. L. WILSON Protoplasmic viscosity changes during mitosis in the egg of the Chaetopterus . 57 PEQUEGNAT, WILLIS E. Inhibition of fertilization in Arbacia by blood extracts 69 BLACK, VIRGINIA S. Changes in density, weight, chloride, and swimbladder gas in the killifish, fundulus heteroclitus, in fresh water and sea water 83 BERRILL, N. J. A new method of reproduction in obelia 94 IFFT, JOHN D., AND DONALD J. ZINN Tooth succession in the smooth dogfish, mustelus canis 100 MENZIES, ROBERT J., AND RICHARD J. WAIDZUNAS Postembryonic growth changes in the isopod pentidotea re- secata (stimpson) with remarks on their taxonomic significance 107 HASSETT, CHARLES C. The utilization of sugars and other substances by drosophila 114 BOREI, HANS Respiration of oocytes, unfertilized eggs and fertilized eggs from psammechinus and asterias 124 Volume 95 Number 2 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board £. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden E. N. HARVEY, Princeton University G. H. PARKER, Harvard University LEIGH HOADLE7, Harvard University A. C. REDFIELD, Harvard University L. IRVING, Swarthmore College F. SCHRADER, Columbia University M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University H. B. STEINBACH, University of Minnesota Managing Editor :Efelc|;cs1 l:::.r«iory NOV12tf'~ WOODS HOLE, MASS. OCTOBER, 1948 Printed and Issued by LANCASTER PRESS, Inc. PRINCE 8C LEMON STS. LANCASTER, PA. BACK ISSUES iHE Laboratory needs early numbers of the BIOLOGICAL BULLETIN to replenish its stock, nearly depleted after meeting the needs of biologists here and abroad during the last 25 years. Members willing to contribute any of the numbers listed below should send them, express collect, to the Marine Biological Laboratory, Woods Hole, Mass. Vol. Nos. Vol. Nos. Vol. Nos. 11-6 25 . 1-6 35 1-6 2 1-6 26 1-6 36 1-6 81 27 4, 5, 6 37 1-6 17 5 28 1, 3, 6 38 1-6 18 5, 6 29 1-6 39 1, 2, 4-6 20 1 30 1-6 40 1-6 21 6 31 1^, 6 41 1-6 22 1-6 32 1-6 42 1-6 23 2, 5, 6 33 1-6 47 1-6 24 1-6 34 1-5 BOOKS AND WORLD RECOVERY IHE desperate and continued need for American publications to serve as tools of physical and intellectual reconstruction abroad has been made vividly apparent by appeals from scholars in many lands. The American Book Center for War Devastated Libraries has been urged to continue meeting this need at least through 1948. The Book Center is therefore making a renewed appeal for American books and periodicals — for technical and scholarly books and periodicals in all fields and particularly for publications of the past ten years. We shall especially welcome complete or incomplete recent files of the BIOLOGICAL BULLETIN. The generous support which has been given to the Book Center has made it possible to ship more than 700,000 volumes abroad in the past year. It is hoped to double this amount before the Book Center closes. The books and periodicals which your personal or institutional library can spare are urgently needed and will help in the reconstruction which must preface world understanding and peace. Ship your contributions to the American Book Center, c/o The Library of Congress, Washington 25, D. C, freight prepaid, or write to the Center for further information. BIOLOGICAL ABSTRACTS COVERS THE WORLD'S BIOLOGICAL LITERATURE How do you keep abreast of the literature in your field? No individual possibly could accumulate and read all of the biological contributions in the original — yet some relatively obscure journal might publish a revealing paper on the very subject in which you are most interested. Biological Abstracts now publishes concise, informative abridgments of all the significant contributions from more than 2,500 journals. As well as the complete edition, it also is published in nine low-priced sectional editions which are specially designed for individuals who are interested only in one or more closely related fields. Production costs have increased to such an extent that the active support of all biologists is needed to maintain this important service. Write for full details and a sample copy of the sectional edition covering your field. BIOLOGICAL ABSTRACTS UNIVERSITY OF PENNSYLVANIA PHILADELPHIA 4, PA. MICROFILM SERVICE • The Library of The Marine Biological Laboratory can supply microfilms of ma- terial from periodicals in- cluded in its list. Requests should include the title of the paper, the author, peri- odical, volume and date of publication. Rates are as follows: $.50 for papers up to 50 pages, and $.10 for each additional 10 pages or fraction thereof. LANCASTER PRESS, Inc. LANCASTER, PA. THE EXPERIENCE we have gained from printing some sixty educational publica- tions has fitted us to meet the standards of customers who demand the best. We shall be happy to have workers at the MARINE BIOLOGICAL LABORATORY write for estimates on journals or monographs. Our prices are moderate. INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts papers on a variety of subjects of biologi- cal interest. In general, a paper will appear within three months of the date of its acceptance. The Editorial Board requests that manuscripts conform to the requirements set below. Manuscripts. Manuscripts should be typed in double or triple spacing on one side of paper, 8l/z by 11 inches. Tables should be typewritten on separate sheets and placed in correct sequence in the text. Explanations of figures should be typed on a separate sheet and placed at the end of the text. Footnotes, numbered consecutively, may be placed on a separate sheet at the end of the paper. A condensed title or running page head of not more than thirty-five letters should be included. Figures. The dimensions of the printed page, 5 by 7% inches, should be kept in mind in preparing figures for publication. Illustrations should be large enough so that all details will be clear after appropriate reduction. Explana- tory matter should be included in legends as far as possible, not lettered on the illustrations. Figures should be prepared for reproduction as line cuts or half- tones; other methods will be used only at the author's expense. Figures to be reproduced as line cuts should be drawn in black ink on white paper or blue- lined co-ordinate paper; those to be reproduced as halftones should be mounted on Bristol board and any designating letters or numbers should be made di- rectly on the figures. The author's name should appear on the reverse side of all figures. The desired reduction should be specified on each figure. Literature cited. The list of literature cited should conform to the style set in this issue of The Biological Bulletin. Papers referred to in the manuscript should be listed on separate pages headed "Literature Cited." Mailing. Manuscripts should be packed flat. Large illustrations may be rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must be accompanied by photographic reproductions or tracings that may be folded to page size. Reprints. Authors will be furnished, free of charge, one hundred reprints without covers. Additional copies may be obtained at cost; approximate figures will be furnished upon request. THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. Subscriptions and similar matter should be addressed to The Biologi- cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, $1.75. Subscription per volume (three issues), $4.50. Communications relative to manuscripts should be sent to the Manag- ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 15 and September 1, and to the Department of Zoology, University of Minnesota, Minneapolis, Minnesota, during the remainder of the year. Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page ADDRESSES AT THE LILLIE MEMORIAL MEETING WOODS HOLE, AUGUST 11, 1948 151 SIANG Hsu, W. Some observations on the Golgi material in the larval epi- dermal cells of Drosophila melanogaster 163 SMITH, RALPH I. The role of the sinus glands in retinal pigment migration in grapsoid crabs 169 SCHARRER, BERTA The prothoracic glands of Leucophaea maderae (Orthoptera) 186 VON BRAND, THEODOR, M. O. NOLAN, AND ELIZABETH ROGERS MANN Observations on the respiration of Australorbis glaubratus and some other aquatic snails 199 ROGERS-TALBERT, R. The fungus Lagenidium callinectes Couch (1942) on eggs of the blue crab hi Chesapeake Bay 214 SCUDAMORE, HAROLD H. Factors influencing molting and the sexual cycles in the crayfish 229 ABSTRACTS OF SCIENTIFIC PAPERS PRESENTED AT THE MARINE BIOLOGICAL LABORATORY, SUMMER OF 1948 238 PAPERS PRESENTED AT THE MEETING OF THE SOCIETY OF GEN- ERAL PHYSIOLOGISTS. 281 Volume 95 Number 3 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board £. G. CONKLIN, Princeton University CARL R. MOORE, University of Chicago DONALD P. COSTELLO, University of North Carolina GEORGE T. MOORE, Missouri Botanical Garden £. N. HARVEY, Princeton University G. H. PARKER, Harvard University LEIGH HOADLEY, Harvard University A. C. REDFLELD, Harvard University L. IRVING, Swarthmore College F. SCHRADER, Columbia University M. H. JACOBS, University of Pennsylvania DOUGLAS WHITAKER, Stanford University H. B. STEINBACH, University of Minnesota Managing Editor DECEMBER, 1948 Marine Biological LI SH A JR-5T JAN 9-1949 WOODS HOLE, MASS. _ Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. BACK ISSUES iHE Laboratory needs early numbers of the BIOLOGICAL BULLETIN to replenish its stock, nearly depleted after meeting the needs of biologists here and abroad during the last 25 years. Members willing to contribute any of the numbers listed below should send them, express collect, to the Marine Biological Laboratory, Woods Hole, Mass. Vol. Nos. Vol. Nos. Vol. Nos. 1 1-6 25 1-6 35 1-6 2 1-6 26 1-6 36 1-6 81 27 4, 5, 6 37 1-6 17 5 28 1, 3, 6 38 1-6 18 5, 6 29 1-6 39 1, 2, 4-6 20 1 30 1-6 40 1-6 21 6 31 1-4, 6 41 1-6 22 1-6 32 1-6 42 1-6 23 2, 5, 6 33 1-6 47 1-6 24 1-6 34 1-5 BOOKS AND WORLD RECOVERY 1 HE desperate and continued need for American publications to serve as tools of physical and intellectual reconstruction abroad has been made vividly apparent by appeals from scholars in many lands. The American Book Center for War Devastated Libraries has been urged to continue meeting this need at least through 1948. The Book Center is therefore making a renewed appeal for American books and periodicals — for technical and scholarly books and periodicals in all fields and particularly for publications of the past ten years. We shall especially welcome complete or incomplete recent files of the BIOLOGICAL BULLETIN. The generous support which has been given to the Book Center has made it possible to ship more than 700,000 volumes abroad in the past year. It is hoped to double this amount before the Book Center closes. The books and periodicals which your personal or institutional library can spare are urgently needed and will help in the reconstruction which must preface world understanding and peace. Ship your contributions to the American Book Center, c/o The Library of Congress, Washington 25, D. C, freight prepaid, or write to the Center for further information. BIOLOGICAL ABSTRACTS COVERS THE WORLD'S BIOLOGICAL LITERATURE How do you keep abreast of the literature in your field? No individual possibly could accumulate and read all of the biological contributions in the original — yet some relatively obscure journal might publish a revealing paper on the very subject in which you are most interested. Biological Abstracts now publishes concise, informative abridgments of all the significant contributions from more than 2,500 journals. As well as the complete edition, it also is published in nine low-priced sectional editions which are specially designed for individuals who are interested only in one or more closely related fields. Production costs have increased to such an extent that the active support of all biologists is needed to maintain this important service. Write for full details and a sample copy of the sectional edition covering your field. • BIOLOGICAL ABSTRACTS UNIVERSITY OF PENNSYLVANIA PHILADELPHIA 4, PA. MICROFILM SERVICE • The Library of The Marine Biological Laboratory can supply microfilms of ma- terial from periodicals in- cluded in its list. Requests should include the title of the paper, the author, peri- odical, volume and date of publication. Rates are as follows: $1.00 for papers up to SO pages, and $.10 for each additional 10 pages or fraction thereof. LANCASTER PRESS, Inc. LANCASTER, PA. THE EXPERIENCE we have gained from printing some sixty educational publica- tions has fitted us to meet the standards of customers who demand the best. We shall be happy to have workers at the MARINE BIOLOGICAL LABORATORY write for estimates on journals or monographs. Our prices are moderate. INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts papers on a variety of subjects of biologi- cal interest. In general, a paper will appear within three months of the date of its acceptance. The Editorial Board requests that manuscripts conform to the requirements set below. Manuscripts. Manuscripts should be typed in double or triple spacing on one side of paper, 8% by 11 inches. Tables should be typewritten on separate sheets and placed in correct sequence in the text. Explanations of figures should be typed on a separate sheet and placed at the end of the text. Footnotes, numbered consecutively, may be placed on a separate sheet at the end of the paper. A condensed title or running page head of not more than thirty-five letters should be included. Figures. The dimensions of the printed page, 5 by 7% inches, should be kept in mind in preparing figures for publication. Illustrations should be large enough so that all details will be clear after appropriate reduction. Explana- tory matter should be included in legends as far as possible, not lettered on the illustrations. Figures should be prepared for reproduction as line cuts or half- tones; other methods will be used only at the author's expense. Figures to be reproduced as line cuts should be drawn in black ink on white paper or blue- lined co-ordinate paper; those to be reproduced as halftones should be mounted on Bristol board and any designating letters or numbers should be made di- rectly on the figures. The author's name should appear on the reverse side of all figures. The desired reduction should be specified on each figure. Literature cited. The list of literature cited should conform to the style set in this issue of The Biological Bulletin. Papers referred to in the manuscript should be listed on separate pages headed "Literature Cited." Mailing. Manuscripts should be packed flat. Large illustrations may be rolled in a mailing tube, but all illustrations larger than 9 by 12 inches must be accompanied by photographic reproductions or tracings that may be folded to page size. Reprints. Authors will be furnished, free of charge, one hundred reprints without covers. Additional copies may be obtained at cost; approximate figures will be furnished upon request. THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. Subscriptions and similar matter should be addressed to The Biologi- cal Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain: Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers, $1.75. Subscription per volume (three issues), $4.50. Communications relative to manuscripts should be sent to the Manag- ing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 15 and September 1, and to the Department of Zoology, University of Minnesota, Minneapolis, Minnesota, during the remainder of the year. Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page BERRILL, N. J. The life cycle of Aselomaris michaeli, a new gymnoblastic hydroid 289 COLWIN, LAURA HUNTER Note on the spawning of the holothurian, Thyone briareus (Lesueur) 296 DAS, S. M. The physiology of excretion in Molgula (Tunicata, Ascidiacea) 307 JOHNSON, MARTIN W. AND J. BENNET OLSON The life history and biology of a marine harpacticoid copepod, Tisbe furcata (Baird) 320 LEFEVRE, PAUL G. Further chemical aspects of the sensitization and activation reactions of Nereis eggs 333 WELSH, JOHN H. AND RAE TAUB The action of choline and related compounds on the heart of Venus mercenaria 346 WHITING, ANNA R. Incidence and origin of androgenetic males in X-rayed Habrobracon eggs 354 Papers Presented at General Scientific Meetings, Marine Biological Laboratory, Summer of 1948: Erratum 361 »" 'V $