BIOLOGICAL BULLETIN OF THE fiDarine biological laboratory WOODS HOLE, MASS. EMtorial Staff E. G. CONKLIN Princeton University. GEORGE T. MOORE The Missouri Botanic Garden. T. H. MORGAN Columbia University. W. M. WHEELER Harvard University. E. B. WILSON Columbia University. JE&itor FRANK R. LTLLIE The University of Chicago. VOLUME XLIX. WOODS HOLE. MASS. JULY TO DECEMBER, 1925 LANCASTER PRESS, INC. LANCASTER, PA. Contents of Volume XLIX No. i. JULY, 1925. Twenty -seventh Annual Report of the Marine Biological Laboratory I SNYDER, CHARLES D. Egg-Volume and Fertilization Mem- brane 54 PEARCY, J. FRANK, AND KOPPANYI, T. The Effects of Dis- location of the Eye upon the Orientation and Equilibrium of the Goldfish (Carassius auratus] 61 No. 2. AUGUST, 1925. ANDREWS, JUSTIN M. Morphology and Mitosis in Tricho- monas termopsidis, an Intestinal Flagellate of the Termite, Termopsis 69 NELSON, THURLOW C. Recent Contributions to the Knowledge of the Crystalline Style of Lamellibranchs 86 LILLIE, RALPH S., AND CATTELL, WARE. The Conditions of Activation of Unfertilized Starfish Eggs by the Electric Current 100 NEWMAN, H. H. An Experimental Analysis of Asymmetry in the Starfish, Patiria miniata 1 1 1 No. 3. SEPTEMBER, 1925. WHEELER, WILLIAM MORTON. The Finding of the Queen of the Army Ant, Eciton hamatum Fabricius 139 WHEELER, WILLIAM MORTON. A New Guest-Ant and Other New Formicidx from. Barro Colorado Island, Panama. . 150 WILDER, H. H. Palm and Sole Studies 182 KATER, J. McA. Morphology and Life History of Poly- tomella citri sp. nov 213 No. 4. OCTOBER, 1925. KEPNER, WM. A., AND PICKENS, A. L. Trichodina steinii (C. and L.) from Planaria polychora (0. Schm.} 237 in IV CONTENTS OF VOLUME XLIX. HEILBRUNN, L. V. Studies in Artificial Parthenogenesis, V.. 241 KOEHRING, VERA. The Spermatheca of Eurycea bislineata . . 250 BREITENBECHER, J. K. The Inheritance of a Macula Muta- tion Concerned with Elytral Spotting and Latent Traits in the Male of Bruchus 265 TORREY, HARRY BEAL, AND HORNING, BENJAMIN. The Effect of Thyroid Feeding on the Moulting Process and Feather Structure of the Domestic Fowl 275 HYMAN, L. H. On the Action of Certain Substances on Oxygen Consumption 288 No. 5. NOVEMBER, 1925. HOLMES, FRANCIS O. The Relation of Herpetomonas elmas- siani (Migone) to its Plant and Insect Hosts 323 ROGERS, CHARLES G., AND COLE, KENNETH S. Heat Pro- duction by the Eggs ofArbacia punctulata during Fertiliza- tion and Early Cleavage 338 McEwEN, ROBERT S. Concerning the Relative Phototropism of Vestigial and Wild Type Drosophila 354 TORREY, HARRY BEAL, AND HORNING, BENJAMIN. Thyroid Feeding and Secondary Sex Characters in Rhode Island Red Chicks 365 TURNER, C. L. Studies on the Secondary Sexual Characters of Crayfishes, IV. Males with Two Sets of Super- numerary Male Characters 375 TURNER, C. L. Studies on the Secondary Sexual Characters of Crayfishes, V. Males with Female Characters 379 FINK, DAVID E. Physiological Studies on Hibernation in the Potato Beetle, Leptinotarsa decemlineata Say 381 No. 6. DECEMBER, 1925. BOSCHMA, H. On the Feeding Reactions and Digestion in the Coral Polyp Astrangia danas, with Notes on its Sym- biosis with Zooxanthellae 407 CLARE, M. R. A Study of Oxygen Metabolism in Drosophila melanogaster 440 HEILBRUNN, L. V. The Action of Ether on Protoplasm. . . . 461 Vol. XLIX July, 1925 No. i BIOLOGICAL BULLETIN THE MARINE BIOLOGICAL LABORATORY. TWENTY-SEVENTH REPORT, FOR THE YEAR 1924, THIRTY- SEVENTH YEAR. I. TRUSTEES AND EXECUTIVE COMMITTEE (AS OF AUGUST 12, 1924) i LIBRARY COMMITTEE 3 II. ACT OF INCORPORATION 3 III. BY-LAWS OF THE CORPORATION 4 IV. REPORT OF THE TREASURER 5 V. REPORT OF THE ASSISTANT LIBRARIAN 13 VI. REPORT OF THE DIRECTOR 20 Statement 20 Addenda: 1. The Staff. 1924 26 2. Investigators and Students, 1924 29 3. Tabular View of Attendance 37 4. Subscribing and Cooperating Institutions, 1924 38 5. Evening Lectures, 1924 39 6. Members of the Corporation 40 I. TRUSTEES (AS OF AUGUST 12, 1924). EX OFFICIO FRANK R. LILLIE, Director, The University of Chicago. OILMAN A. DREW, Assistant Director, Marine Biological Laboratory. LAWRASON RIGGS, JR., Treasurer, 25 Broad Street, New York City. GARY N. CALKINS, Clerk of the Corporation, and Secretary of the Board of Trustees, Columbia University. EMERITUS CORNELIA M. CLAPP, Mount Holyoke College. , TO SERVE UNTIL 1928 H. H. DONALDSON, Wistar Institute of Anatomy and Biology. W. E. CARREY, Tulane University. i l MARINE BIOLOGICAL LABORATORY. CASWELL GRAVE, Washington University. M. J. GREENMAN, Wistar Institute of Anatomy and Biology. R. A. HARPER, Columbia University. A. P. MATHEWS, The University of Cincinnati. G. H. PARKER, Harvard University. C. R. STOCKARD, Cornell University Medical College. TO SERVE UNTIL H. C. BUMPUS, Brown University. W. C. CURTIS, University of Missouri. GEORGE T. MOORE, Missouri Botanical Garden, St. Louis. W. J. V. OSTERHOUT, Rockefeller Institute for Medical Research. J. R. SCHRAMM, Cornell University. WILLIAM M. WHEELER, Bussey Institution, Harvard University. LORANDE L. WOODRUFF, Yale University. TO SERVE UNTIL IQ26 E. G. CONKLIN, Princeton University. OTTO C. GLASER, Amherst College. Ross G. HARRISON, Yale University. H. S. JENNINGS, Johns Hopkins University. F. P. KNOWLTON, Syracuse University. M. M. METCALF, Oberlin, Ohio. WILLIAM PATTEN, Dartmouth College. W. B. SCOTT, Princeton University. TO SERVE UNTIL 1 925 C. R. CRANE, New York City, President of the Corporation. I. F. LEWIS, University of Virginia. R. S. LILLIE, The University of Chicago. E. P. LYON, University of Minnesota. C. E. McCLUNG, University of Pennsylvania. T. H. MORGAN, Columbia University. D. H. TENNENT, Bryn Mawr College. E. B. WILSON, Columbia University. EXECUTIVE COMMITTEE OF THE BOARD OF TRUSTEES. FRANK R. LILLIE, Ex. off. Chairman. GILMAN A. DREW, Ex. off. IVEY F. LEWIS, to serve until 1927. E. G. CONKLIN, to serve until 1926. W. E. CARREY, to serve until 1925. ACT OF INCORPORATION. THE LIBRARY COMMITTEE. C. E. McCLUNG, Chairman, University of Pennsylvania. M. M. METCALF, National Research Council. J. R. SCHRAMM, Cornell University. E. E. JUST, Howard University. R. A. BUDINGTON, Oberlin College. 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 maintain- ing a laboratory or station for scientific study and investigation, and a school for instruction in biology and natural history, and have complied with the provisions of the statutes of this Commonwealth in such case made and provided, as appears from the 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 Massachusetts, do hereby certify that said A. Hyatt, W. S. Stevens, W. T. Sedgwick, E. G. Gardiner, S. Minns, C. S. Minot, S. Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, their associates and successors, are legally organized and established as, and are hereby made, an existing Corporation, under the name of the MARINE BIOLOGICAL LABORATORY, with the powers, rights, and privi- leges, 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. 4 MARINE BIOLOGICAL LABORATORY. III. BY-LAWS OF THE CORPORATION OF THE MARINE BIOLOGICAL LABORATORY. I.' The annual meeting of the members shall be held on the second Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 12 o'clock noon, in each year, and at such meeting the members shall choose by ballot a Treasurer and a Clerk, who shall be, ex officio, members of the Board of Trustees, and Trustees as hereinafter pro- vided. At the annual meeting to be held in 1897, not more than twenty-four Trustees shall be chosen, who shall be divided into four classes, to serve one, two, three, and four years, respectively, and thereafter not more than eight Trustees shall be chosen annually for the term of four years. These officers shall hold their respective offices until others are chosen and qualified in their stead. The Direc- tor and Assistant Director, who shall be chosen by the Trustees, shall also be Trustees, ex officio. II. Special meetings of the members may be called by the Trustees to be held in Boston or in Woods Hole at such time and place as may be designated. III. The Clerk shall give notice of meetings of the members by publication in some daily newspaper published in Boston at least fifteen days before such meeting, and in case of a special meeting the notice shall state the purpose for which it is called. IV. Twenty-five members shall constitute a quorum at any meeting- V. The Trustees shall have the control and management of the affairs of the Corporation; they shall present a report of its condition at every annual meeting; they shall elect one of their number Presi- dent and may choose such other officers and agents as they may think best; they may fix the compensation and define the duties of all the officers and agents; and may remove them, or any of them, except those chosen by the members, at any time; they may fill vacancies occurring in any manner in their own number or in any of the offices. They shall from time to time elect members to the Corporation upon such terms and conditions as they may think best. VI. Meetings of the Trustees shall be called by the President, or by any two Trustees, and the Secretary shall give notice thereof by written or printed notice sent to each Trustee by mail, postpaid. Seven Trustees shall constitute a quorum for the transaction of business. The Board of Trustees shall have power to choose an Executive Com- mittee from their own number, and to delegate to such Committee such of their own powers as they may deem expedient. THE REPORT OF THE TREASURER. 5 VII. The President shall annually appoint two Trustees, who shall constitute a committee of finance, to examine from time to time the books and accounts of the Treasurer, and to audit his accounts at the close of the year. No investments of the funds of the Corpora- tion shall be made by the Treasurer except approved by the finance committee in writing. VIII. The consent of every Trustee shall be necessary to dissolu- tion of the Marine Biological Laboratory. In case of dissolution, the property shall be given to the Boston Society of Natural History, or some similar public institution, on such terms as may then be agreed upon. IX. These By-laws may be altered at any meeting of the Trustees, provided that the notice of such meeting shall state that an alteration of the By-laws will be acted upon. X. Any member in good standing may vote at any meeting, either in person or by proxy duly executed. IV. THE REPORT OF THE TREASURER. 1 HARVEY S. CHASE & COMPANY Certified Public Accountants 84 State Street, Boston. February 9, 1925. MR. LAWRASON RIGGS, JR., 25 Broad Street, New York. Dear Sir: We have audited the accounts of the Marine Bio- logical Laboratory for the year ended December 31, 1924 and report thereon in the following exhibits and schedules, together with our comments, viz: Exhibit A Balance-Sheet, December 31, 1924. Exhibit B Income-and-Expense, Year ended December 31, 1924. Schedule I Securities held by Central Union Trust Company of New York as Trustees under Agreement dated January 22, 1924. Schedule II Lucretia Crocker Fund, December 31, 1924. 1 Schedules IV and V are not included in this report. The complete audit is on file at the Laboratory, and may be examined by any member. 6 MARINE BIOLOGICAL LABORATORY. Schedule III Cash Receipts and Payments, Reserve and Library Funds, January 8, 1924 to De- cember 31, 1924. Schedule IV Land, Buildings, and Equipment, December 31, 1924- Schedule V Supply Department Income-and-Expense Ac- count, Year ended December 31, 1924. During the year 1924 the Laboratory received endowment fund gifts amounting to $905,000.00 and a building fund gift of $500,000.00. The receipt of these large gifts made it advisable to change the arrangement of the balance-sheet and we have, therefore, set it up in the form that has been generally adopted by larger endowed institutions. We have also regrouped the items in the income- and-expense statement Exhibit B. We certify that the balance-sheet and income-and-expense statement shown in Exhibits A and B in our opinion set forth correctly the financial condition of the Marine Biological Labora- tory at December 31, 1924, and the results of its operations for the year ended on that date. Very respectfully, HARVEY S. CHASE & COMPANY, Certified Public Accountants. EXHIBIT A MARINE BIOLOGICAL LABORATORY BALANCE-SHEET, DECEMBER 31, 1924. Assets. Endowment Fund Assets: Securities in Hands of Trustee Schedule I $905,097.50 Cash in Hands of Trustee 12.00 $905,109.50 Lucretia Crocker Fund Assets, Securities Schedule II $ 4,005.17 Cash Schedule II 427.19 4.432-36 $ 909,541.86 Plant Assets: New Laboratory Building, Expenditures to December 31, 1924 $452,106.14 Cash in Building Fund 56,615.53 Total (See Contra) $508.721.67 THE REPORT OF THE TREASURER. Other Plant Assets Schedule IV, Land $102,094.89 Buildings 236,126.82 Equipment 108,837.49 $447,059.20 Less Reserve for Depreciation .. 69,768.47 377,290.73 886,012.40 Current Assets: Cash. In New York Bank $ 13,821.67 In Hands of Trustee 2,200.00 In Falmouth Bank 4,228.95 Petty Cash 500.00 $ 20,750.62 Accounts Receivable 19,586.38 Inventories, Supply Department $ 24,558.41 Biological Bulletin 3,483.21 28,041.62 Investments, Gansett Property Account $ 7,993.23 Stock in General Biological Sup- ply House, Inc 12,700.00 20,693.23 Prepaid Insurance 1,834.71 Items in Suspense 1,313.32 92,219.88 $1,887,774.14 Liabilities. Endowment Funds: Friendship Fund, Inc $405,000.00 John D. Rockefeller, Jr 400,000.00 Carnegie Corporation 100,000.00 Gain on Sale of Securities during Year 109.50 $905,109.50 Lucretia Crocker Fund 4,432.36 $ 909,541.86 Plant Funds: Rockefeller Foundation Contribution for New Laboratory Building $500,000.00 Interest Received on same during Year 8,721.67 $508,721.67 Other Investment in Plant from Gifts and from Current Funds 377.290.73 886,012.40 Current Liabilities and Surplus: Mortgage on Gansett Property $ 8,182.01 Accounts-Payable 398.85 Surplus, 8 MARINE BIOLOGICAL LABORATORY. Balance, January i, 1924 $427,309.99 Add, Proceeds of Library and Re- serve Fund Securities. . . . 9,715.24 Balance of Income for Year Exhibit B 23,904.52 $460,929.75 Deduct, Book Value of Plant Assets transferred to "Plant Funds" above 377.290.73 83,639.02 92,219.88 $1,887,774.14 EXHIBIT B MARINE BIOLOGICAL LABORATORY, INCOME AND EXPENSE, FOR YEAR ENDED DECEMBER 31, 1924. Total Net Expense Income Expense Income Income, Endowment Fund $ 33.656.58 $33.656.58 Donation for Expenses: Friendship Fund, Inc 4,880.00 4,880.00 Others 25.00 25.00 Instruction $ 7.516.39 10,095.00 2,578.61 Research 3.125.90 6,875.00 3,749.10 Biological Bulletin and Mem- bership Dues 5,127.14 4.943-79 $ I83-35 Supply Department, Schedule V 48,379.66 56.645.08 8,265.42 Mess Department 24,948.95 26,776.71 1,827.76 Dormitories Department 3,282.85 3,492.25 209.40 Interest and Depreciation charged to above Three Depts 10,221.99 10,221.99 Dividends on Stock in General Biological Supply House, Inc. 976.00 976.00 Rent of Microscopes 3*9-50 3I9-5Q Interest on Bank Deposits 103.54 103.54 Newman Cottage 122.85 150.00 27.15 Sundry Income 84.98 84.98 Maintenance of Plant: Maintenance of Buildings and Grounds $ 6,121.19 New Laboratory Expense.. 4,567.66 Chemical Department .... 2,776.50 Library Expenses 2,563.56 Sundry Expenses 1,322.32 THE REPORT OF THE TREASURER. Carpenter Department. . . . 1,080.28 Dexter House, Special .... 971-35 Truck Expenses 567.06 Bar Neck Property 375-QO Pumping Station 258.95 Evening Lectures 111.41 Janitor's House n-77 20,727.05 General Expenses: Administrative Expense. . .$ 10,981.15 Endowment Fund, Trustee 787.50 Interest on Loans 386.84 Bad Debts 346.51 12,502.00 Reserve for Depreciation $ 9,608.11 9,608.11 $149,023.43 $66,925.03 $125,118.91 125,118.91 $43,020.51 43,020.51 Balance of Income carried to Current Surplus Exhibit A $23,904.52 $23,904.52 SCHEDULE I MARINE BIOLOGICAL LABORATORY SCHEDULE OF SECURITIES HELD BY CENTRAL UNION TRUST COMPANY OF NEW YORK AS TRUSTEE UNDER AGREEMENT DATED JANUARY 22, 1924. DECEMBER 31, 1924. Bonds. Par Value Cost $ 25,000. Chesapeake & Potomac Telephone Company of Virginia, ist 30 Year S/F "A" 53, due May i, 1943; interest May and November I $ 23,812.50 *25,ooo. Great Western Power Company, ist Mortgage S/F 40 Year 5s, due July i, 1946; interest January and July i 25,000.00 *50,ooo. Government of the Dominion of Canada, 30 Year 53 due May i, 1952, interest May and November 1 50,000.00 25,000. Home Long Distance Telephone Company, ist S/F 20 Year 5s, due January 2, 1932; interest January and July 2.. . 24,031.25 *25,ooo. Joint Stock Farm Loan Bonds, The Lincoln Joint Stock Land Bank of Lincoln, Nebraska, 53 due November i, 1938; interest May and November 1 25,000.00 *50,ooo. Illinois Bell Telephone Company, ist and Refunding Mortgage "A" 53 due June i, 1956; interest June and December i 50,000.00 *io,ooo. Indiana Steel Company, ist Mortgage 53 due May i, 1952, interest May and November 1 10,000.00 *25,ooo. Indianapolis Gas Company of Indianapolis, Indiana, ist Consolidated Mortgage, 53 due April i, 1952; interest April and October i 25,000.00 10 MARINE BIOLOGICAL LABORATORY. 25,000. Lehigh Valley Harbor Terminal Railway Company, 1st 53 due February i, 1954; interest February and August i. 24,187.50 50,000. Mortgage Bond Company of New York, Series No. 4, 63 due November i, 1933; interest May and November i.. 49,500.00 *i 0,000. Nashville Florence & Sheffield Railway Company, 1st Mortgage 53 due August i, 1937; interest February and August i 10,000.00 *8,ooo. New Castle Company, Delaware Highway Improvement 458 due January i, 1932; interest January and July i.. 8,000.00 *5o,ooo. National Tube Company, ist Mortgage 53 due May i, 1952; interest May and November i 50,000.00 *io,ooo. New Castle Company, Delaware Highway Improvement 5th Series 458 due January i, 1931 ; interest January and July i 10,000.00 *io,ooo. New Castle Company, Delaware Highway Improvement 5th Series 458 due January i, 1930; interest January and July i 10,000.00 10,000. Philadelphia, Baltimore and Washington Railroad Com- pany, General "B" 53 due February i, 1974; interest February and August i 9,875.00 15,000. Reading Company, General and Refunding "A" 453 due January i, 1997; interest January and July i I3.4O3-75 *io,ooo. Southern Railway Company, ist Consolidated Mortgage 53 due July i, 1994; interest January and July i 10,000.00 *50,ooo. State of Louisiana Port Commission Serial Canal 5s due July i, 1958; interest January and July i 50,000.00 *io,ooo. Southern Public Utilities Company, ist and ref. mortgage 30 Year 53 due July i, 1943; interest January and July i. 10,000.00 *i2,ooo. U. S. A. 2nd Liberty Loan 4\s due November 15, 1942; interest May and November 15 12,000.00 *25,ooo. Virginian Railway Company, ist Mortgage, 50 Year "A" 53 due May i, 1962; interest May and November i... . 25,000.00 *25,ooo. Waco, Texas, Sewerage Disposal Plant, 5s due January I, 1948; interest January and July i 25,000.00 25,000. West Shore Railroad Company, Gtd. Registered ist 43 due January i, 2361; interest January and July i 19.687.50 Real Estate Mortgages 30,000. B/M Alva Realty Company, 1321/7 Amsterdam Avenue, 55 % due February i, 1929, interest February and August ! 30,000.00 30,000. B/M Alva Realty Company, 129 W. 56th Street. 55% due January 28, 1927; interest January and July 28 30,000.00 50,000. B/M Cordelia Realty Corporation, 47/5 1 E. 52nd Street, 55 % due December 5, 1928; interest June and December ! 50,000.00 50,000. B/M S & L Bldg. Company, N. E. Corner 82nd Street and Broadway, 55% due April 9, 1933; interest June and December i 50,000.00 * Securities received from Friendship Fund, Inc., taken at Par Value. Total, $405,000.00. THE REPORT OF THE TREASURER. II 50,000. B/M M & H Friedel, 328/38 E. 46th Street, 55% due January 23, 1929; interest January and July 23 50,000.00 50,000. B/M Canebrake Realty Company, 708-12 Greenwich Street, 55% due December 29, 1928; interest June and December 29 50,000.00 30,000. B/M Spruce & William Street Realty Company, N. E. Corner Spruce and William Streets, 5^ % due December 30, 1928; interest June and December 30 30,000.00 23,000. B/M W. T. Slevin, 47 Murray Street, 5^% due August i, 1926; interest February and August i 23,000.00 22,600. B/M Bertha Kahn, 594/6 Broadway. 5^% due March n, 1929; interest March and September n 22,600.00 $915,600. Total, December 31, 1924, Exhibit A $905,097.50 SCHEDULE II MARINE BIOLOGICAL LABORATORY, LUCRETIA CROCKER FUND, DECEMBER 31, 1924. Investments 1 8 shares Vermont & Massachusetts Railroad Company $2,416.50 3 shares American Telephone & Telegraph Company 504.04 4 shares Boston Consolidated Gas Company, Preferred 420.58 3 shares General Electric Company 349-55 165 shares General Electric Company Special @ $11.00 181.50 4^ shares General Electric Company Special (Received as Stock Dividend) i share Boston Elevated Railway Company, Second Preferred 133.00 Total, December 31, 1924 Exhibit A $4,005.17 Cash Transactions for Year Cash on Hand, January 8, 1924 $ 373.78 Received from Sale of Liberty Loan and American Telephone & Telegraph Company Bonds: Cost, as shown in Schedule I of Audit Report for 1923. . . .$494.75 Gain on Sale 4-98 499-73 Received for Interest and Dividends 189.87 Received from Sale of Rights 3.56 $1,066.94 Paid for Securities transferred from Reserve and Library Funds. $564. 75 Paid for 1924 Scholarship 75.00 639.75 Balance on Hand, December 31, 1924 Exhibit A. . . .$ 427.19 12 MARINE BIOLOGICAL LABORATORY. SCHEDULE III MARINE BIOLOGICAL LABORATORY CASH RECEIPTS AND PAY- MENTS RESERVE AND LIBRARY FUNDS JANUARY 8, 1924 TO DECEMBER 31, 1924. Reserve Fund: Cash on Hand, January 8, 1924 $ 782.93 Received from Sale of Securities, Cost as shown in Schedule I of Audit Report for 1923 . $5,895.76 Gain on Sale 95I-I5 6,846.91 Received for 1 2 shares General Electric Company, Special, transferred to Crocker Fund 132.00 Received for Interest and Dividends 226.93 $7.988.77 Transferred to Laboratory General Cash Account $7,988.77 Library Fund : Cash on Hand, January 8, 1924 $ 20.13 Received from Sale of Securities, Cost as shown in Schedule I of Audit Report for 1923 . $1,957.81 Gain on Sale 348.01 2,305.82 Received for Securities transferred to Crocker Fund: Cost.$ 362.38 Gain on Transfer.. 70-37 432-75 Received for Interest and Dividends 97-46 Received from Sale of Rights 10.65 $2,866.81 Transferred to Laboratory General Cash Account $2,866.81 The Reserve Fund and the Library Fund were, as shown above, closed out during the year by order of the Executive Committee, the proceeds of the Reserve Fund being used for the acquisition of new property, and the proceeds of the Library Fund being used for the purchase of books. Respectfully submitted, LAWRASON RIGGS, JR., Treasurer. REPORT OF THE ASSISTANT LIBRARIAN. 13 V. REPORT OF THE ASSISTANT LIBRARIAN DECEMBER 31, 1924. Along with the start of a new library building made a year ago, there has been a steady effort on the part of the Library Staff to so adapt their activities that, with the least expenditure of money and effort, our acquisitions and our new arrangements should fall into ready adjustment with the new and enlarged quarters. Even before the beginning of the year of 1924, a special sum had, at the request of the Assistant Librarian and of the Library Committee, been granted for the purchase of back sets a sum of $400.00, this entirely outside the appropriation for the years 1923 and 1924. At the same time $350.00 had been granted for the employment of a secretary for at least a part of the year 1924. A start in purchasing back sets of serials was therefore well begun by January I, 1924 and on the first of May the services of a secretary- stenographer were secured. With this kind of help, the Assistant Librarian has felt that work of real value to the Library has gotten under way. The Assistant Librarian was also authorized to anticipate the use of a sum of $4,000.00 that would be available before the expiration of the year 1924 from the accumulation of the endow- ment fund ; this was used in the purchase of back sets in the early spring that would, a few months later, have been unavailable. These were German sets for sale in this country, not as yet affected by the great jump made in German prices. During the late spring, extensive orders were placed in England with Heffer, and still later information showed France and French sets to be the best sales available. Certain sets of American journals were ordered at this same time, at high prices, but only where sets were out of print and no lessening of price could ever be anticipated. A detailed listing of the sets ordered and come into our actual possession during the year 1924 will follow later with the state- ment of other acquisitions. On September 20, the Secretary of the Executive Committee handed a statement to the Assistant Librarian authorizing a provisional budget of $6,500.00 for the year 1925 as well as the use of a further sum of something over $2,500.00 for the year 14 MARINE BIOLOGICAL LABORATORY. 1924-25 from the sale of the Library Fund securities. These actually realized $2,866.86. The Assistant Librarian was further assured by this same statement of the Executive Committee that the secretary would be retained and that early in the year 1925 the salary for an assistant would be available, besides a small sum for "man labor," over and above the sums stated on the budget and the $4,000.00 above referred to as a special sum. Along with this definite backing, the Assistant Librarian was also assured that any reasonable distribution for the two years of 1924-25 expenditures could be mapped out that seemed wise. For this reason and because of the practicability of running the two years together the provisional distribution of the total sum for 1924-25 of about $15,000.00 ($1,750.00 having been appropriated for expenditures aside from salaries for 1924) has been made as follows: current subscriptions, $3,000.00; books, $1,300.00; binding $2,000.00; back sets, $8,000.00; express, $200.00; sup- plies, $500.00. A feature as interesting as that of securing back sets of our current serials, is that of securing new current subscriptions. The sum for this purpose we propose to double in the year 1925 over 1924 which latter was $1,000.00, thus spending $3,000.00 for the two years. A list of those now already undertaken for the years 1924 and 1925 will be given later. The negotiation for new exchanges in current serials is being carried on with success most encouraging for the effort made. The new exchanges that have been secured for the BIOLOGICAL BULLETIN, already begun for the years 1924 and 1925, are also given below. With the larger funds available in these two years a really generous increase in the amount spent for books seemed right, especially since our attitude of favoring serials over books will for obvious reasons continue. And there are many gaps in our reference books in both systematic and morphological works as well as strictly physico-chemical lines. Important works in each of these lines have been purchased as is seen in the summary. There have been many gifts of books during the year. Very especial appreciation we hope may be conveyed to the authors of books who have remembered the users of the Library ; and to the publishers who have so wisely and generously seen the advantages that the Library offers for the spreading of news as to a good book. REPORT OF THE ASSISTANT LIBRARIAN. 15 These gifts have all been acknowledged personally and a full list follows at the end of the report. With the anticipated increase in space and especially the antici- pated assistance to be given by another member of the Library Staff, trained in science and language, a special plea for reprints was sent at the end of December to each member of the following societies: American Physiological Society; American Society of Biological Chemists; American Society of Pharmacology and Experimental Therapeutics; American Society for Experimental Pathology; Ecological Society of America ; Botanical Society of America; American Society of Zoologists; American Society of Anatomists; American Society of Naturalists. The return postal cards have shown how generous and co- operative the spirit of the scientist is. We feel great encourage- ment in regard to building up complete sets of reprints. We should have asked each author to send a bibliographic list of his reprints which could be filed in with such of his reprints as we have represented. This will have to be undertaken in the future. A count of the reprints acquired by the Library for 1924 will be deferred until the year 1925 and will be incorporated with the report for that year. It gives me pleasure to express here my appreciation of Miss Deborah Lawrence's assistance in the Library since September 1st as Secretary. She gives every promise of accurate and business-like executive. Miss Frances Childs performed this same duty well during the summer, and would have been con- tinued except that she undertook the summer work solely with the provision that she be released to pursue her studies in business training on September 1st. At this same time it is also a pleasure to announce that Miss Margaret Olmsted, Vassar, '24, with special training in physics and chemistry, as well as in French and German, will begin her duties as assistant in the Library on February 2nd, 1925. At this same time also I would greatly like to express my appreciation for the many suggestions that the Library Committee have given during the year. The sympa- thetic co-operation that each member has shown toward the work in the Library adds the authority as well as the balance of the scientist's point of view. The Library now contains some 13,000 volumes, about eight- 1 6 MARINE BIOLOGICAL LABORATORY. ninths of these being serial publications, mostly bound, and the remainder chiefly books. The reprints are about 9,800 with several hundreds besides these in our possession but so far uncatalogued. The number of currently received serials is about 332. Of these 132 are paid subscriptions and 200 are received by exchange for the Biological Bulletin, by gift or by loan. Forty- two subscriptions have been newly placed by purchase for the years 1924-25, and thirty-two newly secured by exchange. These are as follows: Paid subscriptions : new in 1924: Annales de Vlnstitut Pasteur; Archiv fur experimentelle Pathologic und Pharmacologie; Chemical Reviews; Genetica; Ecology; Journal of the Chemical Society; Journal of Immunology; Journal of Medical Research; Journal of Scientific Instruments; Revue algologique; Revue generate d'histo- logie; Science Abstracts; Zeitschrift fur Zellen- und Gewebelehre (Abteilung B. Zeitschrift fur wissenschaftliche Biologie}; Zeitschrift fur vergleichende Physiologic (Abteilung C. Zeitschrift fur wis- senschaftliche Biologie). Paid subscriptions: new in 1925: American Journal of Hygiene; Annals of Applied Biology; Annales de parasitologie humaine et compare; Archives de morphologic generate et experimentale; Archives de parasitologie; Brain; British Journal of Experimental Pathology; Centralblatt fur Bakteriologie; Ergebnisse und Fort- schritte der Zoologie; Flora; Allgemeine botanische Zeitschrift; Jahresberichte uber die Fortschritte der Anatomic und Entwicklungs- geschichte; Journal de V anatomic et de la physiologic; Journal de chimie physique; Journal de pharmacie et de chemie; Journal de physic; Journal of Botany; Journal of Experimental Pathology; Journal of Pathology and Bacteriology; Journal of the Washington Academy of Science; Kolloid-Zeitschrift: Beihefte; Quarterly Cumulative Index of Current Medical Literature; Ray Society Publications; Scientia; Tables annuelles de constantes; Virchow's Archiv fur pathologische Anatomic und Physiologic und fur klinische Medecin; Zeitschrift fur Immunitats-Forschung; Zoologischer Bericht. Exchanges: new in 1924: Acta hori bergiani; Annales scien- tifiques de VUniversite de Jassey; Archives internationales de pharmacodynamie; Archives of internal medicine; Australian journal of experimental biology and medical sciences; Biologisches REPORT OF THE ASSISTANT LIBRARIAN. 17 Centralblatt; Bulletin d'histologie appliquee a la physiologic, et ct la pathologic, et de technique microsco pique; Bulletin de I' Academic royale de Belgique; Bulletin de la societe beige de biologic; Bulletin de la societe royale des sciences medicates et naturelles de Bruxelles; Bulletin internationale de V Academic des Sciences; Internationale Revue der gesamten Hydrobiologie und Hydrographie; Journal of Metabolic Research; Journal of the Linnean Society: Zoology; Monitor e zoologico italiano; Nyt Magazinfiir Naturwidenskaberne; Osterreichische botanische Zeitschrift; Philippine journal of science; Recueil trauvaux botanique neerlandais; Publications of the Zoo- logical Museum of Russia; Studies from the Plant Physiological Laboratory of Charles University, Prague; Transactions and Reports of the Royal Society of Australia; Transactions of the Connecticut Academy of Arts and Science. Exchanges: new in 1925: Acta phytochimica; Botanisches Archiv; Dansk Botanisk Tidskrift; Fermentforschung; Japanese Journal of Biochemistry; Medical Science: Abstracts and Reviews; Naturae Novitates; Ofversigt af Finska Vetenskaps-Societates, Forhanlingar, and Commentationes, and Acta; Zeitschrift fur ivissenschaftliche Zoologie. Besides currently received serials, there are on the shelves, sets, more or less complete, of serials to the number of about two hundred and fifty-three that have now ceased publication. The following serials have been completed as sets by purchase as indicated, the expenditure for these having been about $4,251.80: Acta horti Bergiani, volumes 1-6, 1891-1919; Annales de Vlnstitut Pasteur, volumes 15-17; 24-38; Annales des sciences naturelles: Zoologie, series 4 to 9 filled in; (a few early volumes still missing) ; Annals of Applied Biology, volumes i-io, 1914-24; Annals of Botany, 3-21 ; I'Annee biologique, 1-21 ; Annual Report of the Progress of Chemistry, by volume 8; Archives d 'anatomic microscopique, 1-16; Archives de zoologie experimentel et generale, 32 scattered volumes; Archives inter nationales de physiologic, i- 14; Archivio di farmacologia sperimentale et scienze affini, 1-26; Bibliographia Zoologica, by volumes 30, 31, 32, and 34; (2 volumes still lacking) ; Biochemische Zeitschrift, 1-55, 1906-1913; 72-100, 1916-1919; British Journal of Experimental Pathology, 1-5, 1920-24; Bulletin de VInstitut Pasteur, 1-17, 1903-1909; Ecology, 1-4, 1920-23; Fauna und Flora des Golfes von Neapel, 1 8 MARINE BIOLOGICAL LABORATORY. monographs 26~35a, 1901-1923; Folia Neurobiologica, 2 and 3- 12, 1909-17 (volume I, 1908, still lacking); Harvey Lectures, 1-16, 1905-1922; Journal de physiologie et de pathologic generale, 1-15; 17 (volumes 16 and 18 still lacking); Journal of immunology, volumes 1-8; Journal of Physical Chemistry, volumes 1-24; Journal of the American Chemical Society, 32-42, 1910-1920; Journal of the Chemical Society (London), volumes 45-124, 1884- 1923; Nuova Notarisia, volumes 1-28, 1890-1918; Physical Review, I series 2, 16, 1893-1920; Proceedings of the Academy of Natural Sciences of Philadelphia, 41 volumes scattered thoughout the set; Proceedings of the Royal Society of London, 45-75, 1890- 1905; Psychobiology, 1-2; Quarterly Cumulative Index of Current Medical Literature, I and 5-9 (2-4 still lacking) ; Quarterly Journal of Microscopical Science, volumes 18 and 19; Revue generale d'histologie, 1-3, 1904-1911; Science Abstracts, 1-26, 1898-1923; Zeitschrift fur physikalische Chemie, 1-104, 1887-1923; Zentral- blatt fur Zoologie, Allgemeine und experimented Biologie, 1-6, 1912-1918. Many odd volumes besides these have been secured and paid under this sum above mentioned. Through exchange of back sets for the Biological Bulletin, these sets have been secured in 1924: Annales scientifiques de I'Uni- versitede Jassey, 112, 1900-1923; Anmiairedu museum zoologique de I 1 Academic Imperial des Sciences de la Russie, 9-13, 1914-20; Archives internationales de pharmacodynamie, volumes 1-29; Bulletin de la classe des sciences, l } Academic royale de Belgique, series 5, volumes 5-9, 1919-23; Fauna of Russia, volumes 1914- 19; Monitor e zoologico italiano, 1907-1924; Nouvelles archives du Museum d'Histoire Naturelle, Paris, series 2, I series 5, 6, 1878- 1914; Nyt magazin fur Naturvidenskaberne grumdlagt au den physio graphiske Forening, Christiania, volumes 46-60, 1909-1923; Ofversigt of Finska Venteskaps-Societetens; Forhandlingar, volumes 45-64; Osterreichische botanische Zeitschrift, volumes 71-74, 1922- 24; through exchange of duplicates of the Museum of Compara- tive Zoology publications and of the Boston Society of Natural History publications, the Library has, this year, also, filled in nearly all the bad gaps that existed, so that these sets are now practically complete. The more important year's purchases of books and sets of reference works as regarded from the view-point of cost, are as REPORT OF THE ASSISTANT LIBRARIAN. K) follows: Vergleichende Anatomic des Nervensy stems, by C. U. Ariens Kappers und Droogleever Fortuyn; Barnes and Heald: Analytic key to mosses of the United States; Berlese, A. : Gli Insetti; Daniel, John Franklin: The Elasmobranch fishes; De Toni: Sylloge Algorum, volume 6; Helmholtz: Handbuch der physio- logischen Optik; Jellinek, K. : Physikalische Chemie der homogenen und heterogenen Gasreaktionen; Leitgeb, H.: Untersuchungen uber die Lebermuse; Morgan, T. H.: Regeneration; Morse, H. N.: Investigations in Osmotic pressure; Ouvres de Pasteur : Dissymetrie moleculaire, Fermentations et generations dites spontanees, Etudes sur le vinaigre et sur le vin; Parker and Haswell: Text-book of Zoology jd edition; Plankton Expedition: Ergebnisse der in der Atlantisch Ozean, etc.; Sars: Crustacea of Norway, 1-3; United States Catalogue of 1912-1917; Wiesner, Julius von and Moeller, J.: Die Rohstoffe des Pflanzenreiches ; Williams, L. W. : Anatomy of the Common Squid; Wolle, Francis: Green Algae of the United States; Wood: Physical Optics. Through gifts we have received as follows : new current serials and to continue: American Journal of Photography (now issued as Camera], British Journal of Photography, Journal of the Photo- graphic Institute, Journal of the Optical Society of America, and Review of Scientific Instruments, Photographic journal of the Royal Society of London, from Dr. Pond; Public Health Report, from the United States Health Service; Monographs, from Cold Spring Harbor; Bulletin of the University of Wisconsin, and Wisconsin Studies in Science, at the request of Professor Guyer. From the publisher, Blakiston: Atwood, W. H.: Problems, Projects, and Experiments in Biology; and Comparative Vertebrate Dissection; Clark, A. J.: Applied Pharmacology: Scientific Evi- dence for Therapeutic Active Drugs; Hawk, Philip B.: Practical Physiological Chemistry; Palladin, Plant Physiology; Pratt, H. S.: Land and Freshwater Vertebrate Animals of the United States; from the publisher, Saunders: Howell: Physiology; Lusk, Graham : Elements of the Science of Nutrition (at the request of the author) ; Mallory and Wright : Pathological Technique; Wells, H. Gideon: Chemical Pathology; from the New Haven Yale University Press: Woodruff, L. L.: Development of the Sciences (at the request of the author) ; from the publisher, Wiley: Under- hill, Frank P.: Manual of Selective Biochemical Methods; San 20 MARINE BIOLOGICAL LABORATORY. Francisco Bay Marine Piling Survey Reports, from the Committee; from the publisher, Macmillan: Smith, Gilbert M.: A Textbook of General Botany; from Newman, Horatio Hackett: Outlines of General Zoology, and Evolution; from the Cape Cod Chamber of Commerce: Special Report on Population and Resources of Cape Cod; from H. S. Jennings: Life and Death, Heredity and Evolution in Unicellular Organisms; from Dr. Pond: Fischer, Max: Uber den Abbau von Chlorophyll und Blutf orb staff ; from Lusk, Graham: Science of Nutrition; from Fischer, M.: Oedema; from Lillie, R. S.: Protoplasmic Action and Nervous Action; from Mrs. Jacques Loeb : Regeneration from a Physico-chemical Viewpoint; from Price, Weston A.: Dental Pathology; through Professor Tower from the American Museum of Natural History, Dean, Bashford: A Bibliography of Fishes; from Loeb, Leo: Edema; from Winge, Herluf: Pattedyr-Slaegter; from Schroder, Paul: Introduction to the Histology and Histo pathology of the Nervous System; from the National Research Council: Atwood and Johnson: Marine Piling Investigations; from Hyman, L. H.: Laboratory Manual of Elementary Zoology; from Wheeler, Wm. M.: Social Life among the Insects; Professor Wheeler sent to us several boxes of reprints and odd volumes and numbers of journals also, which we were glad to get. Mrs. Montgomery presented some twelve books to the Library. VI. THE DIRECTOR'S REPORT. To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY. Gentlemen: I beg to submit herewith a report of the thirty- seventh session of the Marine Biological Laboratory for the year 1924. I. Attendance. As stated in the annual report for 1923 it was decided to set definite numerical limits to each of the courses of instruction and this was accordingly done and acted on in 1924. The result was the admission of only 134 students selected from almost twice the number of applicants. These students were distributed among the classes as shown in the tabular view of attendance (p. 37), each class being registered up to its full authorized number. The selection from among the applicants is THE DIRECTOR'S REPORT. 21 made by the head of each course of instruction on a given date, May 15 of each year, on the basis of a detailed application and letters of recommendation. The number of investigators was 194, the largest registration in the history of the Laboratory. It is interesting to note that 70 of these were "under instruction," that is for the most part graduate students from University laboratories. The oppor- tunity that is afforded these students of comparing the teachings and methods of the various University laboratories is a great benefit educationally for them. The large attendance of workers of this age augurs well for the future of the Laboratory which must depend for its growth on the development and maintenance of the devotion to its interests of successive generations of scien- tific workers. 2. Library. The report of the Acting Librarian is given in another place. It details the plans and the means available for enlargement of the library, which we should now set down as one of the principal ends to be emphasized in the development of the Laboratory during the next few years. Next to providing the materials for investigation, and facilities of modern and adequate type for the work of research itself, there is no material service that the Laboratory can render of greater importance than a thoroughly good working library. To that end a wing of the new building is devoted with stack space for 100,000 volumes. If this capacity be compared with the present possessions of the library, 13,000 bound volumes and about 10,000 reprints and pamphlets, it will be realized that our efforts on behalf of the library need not relax for lack of space for some years to come. The Acting Librarian is to be congratulated on getting an active policy of expansion into operation before the new quarters are entirely ready. 3. The Report of the Treasurer (pp. 5-12). Particular interest attaches to the report of the Treasurer this year because it includes the new endowment funds and the results of building operations as well as the customary current accounts. The assets of the Laboratory are thus of quite a different order of magnitude at the end of 1924 as compared with previous years: $1,887, 774. 14 on December 31, 1924 as compared with $445,504.69 on December 31, 1923. Of this amount $909,541.86 represent 22 MARINE BIOLOGICAL LABORATORY. income-yielding securities, the remainder being in plant assets and current assets. An additional sum of at least $200,000.00 is available from the Friendship Fund for completion of the building and its equipment, which will increase the assets of the Labora- tory to considerably over $2,000,000.00. The securities belong- ing to the "Reserve Fund" and the "Library Fund" of previous reports were sold during the year at considerable profit as com- pared with book value; the proceeds from the Reserve Fund were applied on the purchase of the Hubbard property, and the money from the Library Fund was used for purchase of books. These funds were accumulated from small savings of many years past for the purposes for which they were finally used. In the case of the "Reserve Fund" it was felt that the ownership of such a strategically placed piece of property as the Hubbard lot was worth much more to the Laboratory than the securities in the fund. The timeliness of conversion of the Library Fund into books requires no comment. It will be observed that the current accounts of the year 1924 show a considerable surplus ($23,904.52) of income over expense (Exhibit B of Treasurer's report). The operations of the year were planned on the usual scale, without reference to the new endowment fund, which was, however, paid in and invested early in the year. The income, which was considerably increased by an unusually good showing of the Supply Department (Exhibit B) was thus largely in excess of requirements in spite of an additional grant to the Library during the year, and repayment of loans totaling $6,100.00. This surplus enables us to start the operation of the enlarged plant with a considerable degree of confidence. It should be said also that the estimates for the year 1925 show a slight excess of income over expense including some unusual items. The net impression of a study of the Treasurer's report is that the Laboratory has an adequate financial foundation for its present needs, but that the same rigid economy will be needed in the future as in the past. 4. Building Progress. As stated in the last report the Rocke- feller Foundation made an appropriation of $500,000.00 for the new building; Mr. C. R. Crane also gave a pledge on behalf of the Friendship Fund to contribute whatever additional money was needed, according to the architects' plans and specifications, for THE DIRECTOR S REPORT. 23 completion and equipment of the building. When the bids for the building were received and the estimates for equipment added the total requirements were found to be $748,886.71 divided as follows : General Construction, Geo. A. Fuller Co $478,120.00 Plumbing, C. H. Cronin, Inc 54,860.92 Heating and ventilating, Cleghorn Co 3L534-OO Electric work, Hixon Electric Co 65,875.00 Elevator and book lift, Otis Elevator Co 6,970.00 Architects and engineers' commissions 36,526.79 Furniture, equipment, etc 65,000.00 Alterations in course of construction 10,000.00 $748,886.71 The Friendship Fund accordingly appropriated a sum not in excess of $248,886.71, and not in excess of the cost of the items enumerated, above the contribution of $500,000.00 for building by the Rockefeller Foundation and interest on this fund. The Building Committee has made every effort to keep within the available funds with the result now clearly in sight of entirely avoiding the necessity of any additional payment for alterations in the course of construction and of some probable slight savings in other directions. The great care with which the plans and specifications were prepared by the architects, and especially Dr. Drew's constant oversight of the building operations and purchase of equipment have been notable factors in the achievement of this result. It has certainly very rarely happened that an institution has received such signally generous treatment and confidence as the Marine Biological Laboratory has received from the donors of the building fund. The action of the Rockefeller Foundation in paying over their entire contribution in cash before building operations were begun amounted to an increase of their contri- bution by the interest received during construction. The action of the Friendship Fund in guaranteeing the completion and equip- ment of the Laboratory insured the hearty cooperation of the other contributors, created a notable spirit of confidence, and aroused our own organization to the highest state of morale. Ground was broken for the new building on March 20, 1924. No serious difficulties were experienced during the course of con- 24 MARINE BIOLOGICAL LABORATORY. struction which now seems likely to be completed within a year from the date of beginning. 5. Research Rooms. The charge for use of a research room during the summer session has remained at $100.00 since 1888. The Executive Committee reviewed the situation in 1924 and decided to make no change in the case of the wooden buildings, but in the case of the research rooms of the brick buildings, which are more than twice the size and much more amply supplied with fixtures, it seemed reasonable, and wise from the point of view of all members of our cooperative organization, to ask increased appropriations from the cooperating institutions using them. Accordingly a basic charge of $200.00 has been established for the summer season with the understanding that two persons may occupy such a room. This should be regarded as a first step towards an equitable arrangement that will permit the most efficient operation of the building for research purposes. 6. Biological Bulletin. The forty-seventh volume of the BIO- LOGICAL BULLETIN was completed in 1924. It had been main- tained at about a constant size since its establishment, two volumes with about 650-700 pages per year. Lately in com- mon with many other biological research journals it had been falling behind in promptness of publication so that about a year elapsed between receipt of manuscripts and their appearance in print. The cost of enlarging it so as to keep up with manuscripts offered was greater than the Laboratory could well afford. It has also become a very valuable medium of exchange for the library as stated in Mrs. Montgomery's report. It was therefore decided to enlarge it to 900 pages a year, about 75 pages per number and at the same time to raise the subscription price from $6.00 to $9.00 per year. Correspondingly the fees of members of the Corporation, who receive the BULLETIN without other sub- scription, were increased from $4.00 to $6.00 per year. The increase in size began with the first issue of 1925, and it is already apparent that the passage of a manuscript to publication will be reduced to at most six months, and it is hoped to a still shorter period. There has been no significant loss of subscriptions or of membership in the Corporation in consequence of the increased cost. 7. Membership of the Board of Trustees. At the meeting of the THE DIRECTOR S REPORT. 25 Corporation held August 12, 1924 Professor R. A. Harper of Columbia University was elected a member of the class of 1928, and the seven members of the class of 1924 were reelected to serve until 1928. The following resolution in memory of Jacques Loeb, member of the Laboratory since 1893 and of the Board of Trustees since 1897, was adopted by the Board of Trustees and by the Corpo- ration of the Marine Biological Laboratory at their meetings on August 12, 1924: By the death of Dr. Jacques Loeb the world has lost one of the great men of his generation; Biology has lost one of the finest intellects that has ever been devoted to this branch of science; this Laboratory has lost one of its most eminent members. He stood out among his fellows as an investigator, as a teacher, and as a cultivated gentleman broadly interested in all aspects of nature and all the activities of men. As an investigator he was tireless in energy, ingenious in experimentation and exceptionally gifted in insight. He lost little time on false leads, but rather blazed his trail straight into new territory and attained his objective by simple and crucial experiments. He brought to his work a broad knowledge of related sciences. In the latest advances of chemistry and physics he was always informed, and his researches showed the breadth of his reading and the solid character of his scholarship. He had a poet's imagination held in check by practical and mathematical faculties of high order. Consequently his hypotheses were at once brilliant and founded on the best physico-chemical data available. He was ready to change his theories as new facts were discovered. He believed that explanations of phenomena must be expressible ultimately in mathematical terms. He profoundly influenced General Physiology not only by his theories and experimental results but also, quite as much, by the emphasis he laid upon the quantitative method. Professor Loeb came to Woods Hole first in 1892. The epoch-making discovery of artificial parthenogenesis was made at this laboratory. The antagonistic action of ions was demonstrated here. Many other researches which have influenced biological thought were carried on here and may be noted in the long list of papers and books which constitute his best monument. He founded the course in General Physiology at Woods Hole in 1893 and drected it for several years. He was a trustee from 1897 to his death. From 1910 he directed the branch laboratory of the Rockefeller Institute in cooperation with Marine Biological Laboratory. As a teacher he was enthusiastic and inspiring. His lectures were in advance of the times and full of suggestions for research. With his graduate students he was helpful and friendly and at the same time critical and stimulating. Those who were his students know how enormously they profited by his inspiring personality. As a man his interests were well-nigh universal. He found time to make himself familiar with a large literature. He enjoyed music and all the arts. He was interested in economics, sociology and government. His "Mechanistic Conception of Life" is an important contribution to Philosophy and Psychology. He was a kindly man. He was a lovable man. He hated war and all sham. He had an incisive sense of humor and loved a harmless joke. He was the center of any company and he had many friends. 26 MARINE BIOLOGICAL LABORATORY. This is the man we have lost. Woods Hole is not the same without him, but the inspiration of his work remains in our midst. To his family we extend our sympathy and on the pages of our records we inscribe this memorial. There are included as parts of this report the following addenda: 1. The Staff, 1924. 2. Investigators and Students, 1924. 3. Tabular View of Attendance. 4. Subscribing and Cooperating Institutions, 1924. 5. Evening Lectures, 1924. 6. Members of the Corporation. i. THE STAFF, 1924. FRANK R. LILLIE, Director, Professor of Embryology, and Chairman of the Department of Zoology, The University of Chicago. GILMAN A. DREW, Assistant Director, Marine Biological Laboratory. ZOOLOGY I. INVESTIGATION GARY N. CALKINS, Professor of Protozoology, Columbia University. E. G. CONKLIN, Professor of Zoology, Princeton University. CASWELL GRAVE, Professor of Zoology, Washington University. H. S. JENNINGS, Professor of Zoology, Johns Hopkins University. FRANK R. LILLIE, Professor of Embryology, The University of Chi- cago. C. E. McCLUNG, Professor of Zoology, University of Pennsylvania. S. O. MAST, Professor of Zoology, Johns Hopkins University. T. H. MORGAN, Professor of Experimental Zoology, Columbia Uni- versity. G. H. PARKER, Professor of Zoology, Harvard University. E. B. WILSON, Professor of Zoology, Columbia University. II. INSTRUCTION ROBERT H. BOWEN, Assistant Professor of Zoology, Columbia Uni- versity. EDWARD F. ADOLPH, Instructor in General Physiology, University of Pittsburgh. HORACE B. BAKER, Instructor in Zoology, University of Pennsylvania. J. A. DAWSON, Instructor in Zoology, Harvard University. THE DIRECTOR S REPORT. 2J S. W. GEISER, Assistant Professor of Zoology, Washington Uni- versity. CHRISTIANNA SMITH, Assistant Professor of Zoology, Mount Holy- oke College. B. H. WILLIER, Instructor in Zoology, The University of Chicago. DONNELL B. YOUNG, Assistant Professor of Biology, Carleton College. PROTOZOOLOGY I. INVESTIGATION (See Zoology) II. INSTRUCTION GARY N. CALKINS, Professor of Protozoology, Columbia University. LOUISE H. GREGORY, Associate Professor of Zoology, Columbia Uni- versity. FLORENCE DE L. LOWTHER, Instructor in Zoology, Barnard College. EMBRYOLOGY I. INVESTIGATION (See Zoology) II. INSTRUCTION HUBERT B. GOODRICH, Professor of Biology, Wesleyan University. BENJAMIN H. GRAVE, Professor of Biology, Wabash College. CHARLES PACKARD, Assistant Professor, Peking Union Medical Col- lege. HAROLD H. PLOUGH, Associate Professor of Biology, Amherst Col- lege. CHARLES G. ROGERS, Professor of Comparative Physiology, Oberlin College. PHYSIOLOGY I. INVESTIGATION HAROLD C. BRADLEY, Professor of Physiological Chemistry, Uni- versity of Wisconsin. WALTER E. CARREY, Professor of Physiology, Tulane' University. RALPH S. LILLIE, Professor of General Physiology, The University of Chicago. ALBERT P. MATHEWS, Professor of Biochemistry, The University of Cincinnati. 28 MARINE BIOLOGICAL LABORATORY. II. INSTRUCTION MERKEL H. JACOBS, Professor of General Physiology, University ol Pennsylvania. WILLIAM R. AMBERSON, Assistant Professor of Physiology, Uni- versity of Pennsylvania. FRANK P. KNOWLTON, Professor of Physiology, Syracuse University. REYNOLD A. SPAETH, Associate in Physiology, School of Hygiene and Public Health, Johns Hopkins University. BOTANY I. INVESTIGATION S. C. BROOKS, Department of Public Health, Washington, D. C. EDWARD M. EAST, Professor of Experimental Plant Morphology > Harvard University. ROBERT A. HARPER, Professor of Botany, Columbia University. E. NEWTON HARVEY, Professor of Physiology, Princeton University. WINTHROP J. V. OSTERHOUT, Professor of Botany, Harvard Uni- versity. JACOB R. SCHRAMM, Professor of Botany, College of Agriculture, Cornell University. II. INSTRUCTION IVEY F. LEWIS, Professor of Biology, University of Virginia. TRACY E. HAZEN, Associate Professor of Botany, Barnard College, Columbia University. WILLIAM RANDOLPH TAYLOR, Assistant Professor of Botany, Uni- versity of Pennsylvania. WILLIAM H. WESTON, JR., Assistant Professor of Cryptogamic Bot- any, Harvard University. (Absent 1924.) LIBRARY , Librarian PRISCILLA B. MONTGOMERY (Mrs. Thomas H. Montgomery, Jr.), As- sistant Librarian, in charge. CHEMICAL SUPPLIES OLIVER S. STRONG, Associate Professor of Neurology, Columbia Uni- versity, New York City, Chemist. THE DIRECTOR S REPORT. 2Q SUPPLY DEPARTMENT GEORGE M. GRAY, Curator. A. VV. LEATHERS, Head of Ship- THOMAS M. DOUTHART, Assist- ping Department. ant Curator. MILTON B. GRAY, Collector. JOHN J. VEEDER, Captain. A. M. HILTON, Collector. E. M. LEWIS, Engineer. J. MclNNis, Collector. F. M. MACNAUGHT, Business Manager. HERBERT A. HILTON, Superintendent of Buildings and Grounds. 2. INVESTIGATORS AND STUDENTS, 1924. ZOOLOGY Independent Investigators. ADDISON, WILLIAM H. F., Professor of Histology and Embryology, University of Pennsylvania. APPLETON, JOSEPH L., Jr., Professor of Microbiology and Bacteriopathology, Evans Institute, University of Pennsylvania. AZUMA, RYOTARO, Physiological Laboratory, Tokio Imperial University. BAGG, HALSEY J., Associate in Biology, Cornell University Medical College. BAKER, HORACE BURRINGTON, Instructor, University of Pennsylvania. BIGELOW, ROBERT PAYNE, Professor of Zoology, Massachusetts Institute of Technology. BONNIER, GERT, University of Stockholm, Stockholm, Sweden. BOSCHMA, HILBRAND, Chief Assistant in Zoology, University of Leyden. BOWEN, ROBERT H. t Assistant Professor of Zoology, Columbia University. BREITENBECHER, J. K., Associate Professor of Zoology, University of Oklahoma. BRIDGES, CALVIN B., Research Assistant, Carnegie Institution of Washington. BUDINGTON, ROBERT A. Professor of Zoology, Oberlin College. BURNS, ROBERT KYLE, JR., Assistant in Zoology, Yale University. CALKINS, GARY N., Professor of Protozoology, Columbia University. CAROTHERS, E. ELEANOR, University of Pennsylvania. CHAMBERS, ROBERT, Professor of Microscopic Anatomy, Cornell University Medical College. CLARK, E. R., Professor of Anatomy, University of Georgia. CLARK, ELEANOR L., Research worker, University of Georgia. CONKLIN, EDWIN GRANT, Professor of Biology, Princeton University. COPELAND, MANTON, Professor of Biology, Bowdoin College. DAWSON, JAMES A., Instructor in Invertebrate Zoology, Harvard University. DETWILER, S. R., Assistant Professor of Zoology, Harvard University. DOLLEY, WM. LEE, JR., Professor of Biology, Randolph-Macon College. DONALDSON, HENRY HERBERT, Professor of Neurology, The Wistar Institute. DREW, GILMAN A., Assistant Director, Marine Biological Laboratory, Woods Hole, Mass. GLASER, OTTO CHARLES, Professor of Biology, Amherst College. GOLDFARB, A. J., College of the City of New York. GOODRICH, HUBERT BAKER, Professor of Biology, Wesleyan University. 3O MARINE BIOLOGICAL LABORATORY. GRAVE, BENJAMIN H., Professor of Zoology, Wabash College. GRAVE, CASWELL, Professor of Zoology, Washington University. GREGORY, LOUISE HOYT, Associate Professor of Zoology, Barnard College, Columbia University. HANCE, ROBERT T., University of Pennsylvania. HEILBRUNN, LEWIS V., Assistant Professor of Zoology, University of Michigan. HENTSCHEL, CHRISTOPHER CARL, King's College, London. HESS, WALTER N., Johnston Scholar, Johns Hopkins University. HIBBARD, HOPE, Associate Professor of Zoology, Elmira College. HOGUE, MARY J., Professor of Bacteriology, North Carolina College for Women. HUXLEY, JULIAN SORELL, Senior Demonstrator in Zoology, Oxford University. HYMAN, LIBBIE HENRIETTA, Research Assistant, University of Chicago. JENNINGS, HERBERT S., Professor of Zoology, Johns Hopkins University. JUST, ERNEST E., Professor of Zoology, Howard University. KINDRED, JAMES E., Assistant Professor of Histology and Embryology, University of Virginia. KNOWER, HENRY MCELDERRY, Professor of Anatomy, University of Cincinnati. KOMAI, TAKU, Assistant Professor of Zoology, Kyoto Imperial University. LANCEFIELD, DONALD E., Assistant Professor in Zoology, Columbia University. LANGE, MATHILDE M., Head of the Department of Biology, Wheaton College. LILLIE, FRANK R., Chairman, Department of Zoology, University of Chicago. LYNCH, RUTH STOCKING, Assistant in Graduate Zoology, Johns Hopkins University. McCLUNG, CLARENCE E., Director of Zoological Laboratory, University of Pennsyl- vania. MARTIN, EARL A., Assistant Professor, College of the City of New York. MAST, SAMUEL OTTMAR, Professor of Zoology, Johns Hopkins University. MAVOR, JAMES W., Professor of Biology, Union College. METCALF, MAYNARD M., 128 Forest St. Oberlin, Ohio. METZ, CHARLES W., Investigator, Carnegie Institution, Cold Spring Harbor. MORGAN, T. H., Professor of Experimental Zoology, Columbia University. MORGAN, LILIAN V., 409 W. ii?th St., New York City. MORRILL, CHARLES V., Associate Professor of Anatomy, Cornell University Medical College. PACKARD, CHARLES, Associate in Zoology, Crocker Laboratory, Columbia Uni- versity. PAPANICOLAO, GEORGE N., Assistant Professor in Anatomy, Cornell University Medical College. PLOUGH, HAROLD H., Professor of Biology, Amherst College. RAND, HERBERT W., Associate Professor of Zoology, Harvard University. RENYI, GEORGE S., Assistant Professor, University Medical College, Budapest. ROGERS, CHARLES G., Professor of Comparative Physiology, Oberlin College. SMITH, CHRISTIANNA, Assistant Professor, Mt. Holyoke College. SPEIDEL, CARL CASKEY, Associate Professor of Anatomy, University of Virginia. STARK, MARY B., Professor of Histology and Embryology, N. Y. Homoeopathic Medical College. STOCKARD, CHARLES R., Professor of Anatomy, Cornell University Medical College. STRONG, OLIVER S., Associate Professor of Neurology, Columbia University. STURTEVANT, ALFRED H., Member of Staff, Carnegie Institution of Washington. THOMPSON, IAN MACLAREN, Lecturer in Anatomy, McGill University. WATSON, ISABELLE, Research Assistant, Carnegie Institution. THE DIRECTOR'S REPORT. 31 WIEMAN, HARRY LEWIS, Professor of Zoology. University of Cincinnati. WILLIER, BENJAMIN H., Assistant Professor of Zoology, University of Chicago. WILSON, EDMUND B., Professor of Zoology, Columbia University. WOOD, FRANK ELMER, Head of the Department of Biology, Illinois Wesleyan University. WOODRUFF, LORANDE L., Professor of Protozoology, Yale University. YOUNG, DONNELL B., Assistant Professor of Biology, Carleton College. ZOOLOGY Beginning Investigators. ADOLPH, PAUL E., University of Pennsylvania. ANSON, MORTIMER L., Cambridge University. BECKER, ELERY R., Instructor, Princeton University. BEERMAN, HERMAN, Washington University. BEERS, CHARLES DALE, Student, Johns Hopkins University. BEERS, CATHERINE V., Assistant Professor of Biology. University of Southern California. BLAKE, CHARLES HENRY, Student, Massachusetts Institute of Technology. BOWEN, EDITH S., Instructor in Zoology, Wellesley College. BROWN, ALICE L., Student and Assistant, Cornell University Medical College. COLE, KENNETH, Instructor, Oberlin College. COOK, DANIEL, University of Cincinnati. COWPERTHWAITE, MARION, Instructor, Mt. Holyoke College. DEFOREST, DAVID M., Union College. FABER, ERWIN F., Instructor in Anatomical Drawing, University of Pennsylvania. FOSTER, KENDALL W., Austin Teaching Fellow in Zoology, Harvard University. FRY, HENRY J., Instructor, Department of Biology, New York University Washing- ton Square College. GRAND, CONSTANTINE G., Research, Cornell Medical College. GRAVE, THOMAS B., Johns Hopkins University. HARNLY, MORRIS H., Student, Columbia University. HARTWELL, RHODA A., Assistant, Illinois University. HELWIG, EDWIN R., Student, University of Pennsylvania. HERDMAN, EMMA C., Milet Road, Liverpool, England. HOPKINS, AUBREY E., Graduate Student, Harvard University. HOY, WILLIAM E., JR., Professor of Biology, Presbyterian College of South Carolina. KIERNAN, GRACE R., Instructor of Biology, Miami University. KNAPKE, BEDE, Teacher, St. Bernard College. LEWIS, RUTH W., Investigator, Harvard University. LUCAS, ALFRED M., Wilton, Connecticut. MACDOUGALL, MARY S., Head of the Biological Department, Agnes Scott College. MOSES, MILDRED S., Research Assistant, Carnegie Institution of Washington. PENNYPACKER, MIRIAM I., University of Pennsylvania. PLUNKETT, CHARLES R., Columbia University. RATCLIFFE, FRANCIS. N., Student, Oxford University. RICE, KENNETH S., Assistant Professor of Biology, Clark University. SANDISON, JAMES C., Assistant in Anatomy, University of Georgia. SCOTT, MIRIAM J., Student, University of Pennsylvania. STEWART, DOROTHY R., Bryn Mawr College. STRAUSS, MAURICE B., Amherst College. STURTEVANT, PHOEBE R.. Carnegie Institution. 32 MARINE BIOLOGICAL LABORATORY. TILDEN, EVELYN B., Technician, Rockefeller Institute. THILLAYAMPALAM, E. M., Graduate Student, Columbia University. VALENTINE, J. MANSON, Assistant, Yale University. VARIAN, BASIL B., Assistant, University of Pennsylvania. VICARI, EMILIA M., Research Scholar, Columbia University. VISSCHER, MAURICE B., Assistant, University of Minnesota. WALLACE, EDITH M., Carnegie Institution. WILLIS, JOSEPHINE, University of Pennsylvania. PHYSIOLOGY Independent Investigators. ADOLPH, EDWARD F., Instructor in General Physiology, University of Pittsburgh. AMBERSON, WILLIAM R., Assistant Professor, University of Pennsylvania. BAILEY, PERCIVAL, Director, Laboratory of Surgical Research, Harvard University Medical School. BAZETT, HENRY C., Professor of Physiology, University of Pennsylvania. BRADLEY, HAROLD C., Professor of Physiological Chemistry, University of Wis- consin. BRONFENBRENNER, J. J., Associate Member in Pathology and Bacteriology, Rockefeller Institute. CHOWN, BRUCE, Assistant Dispensary Physician, Johns Hopkins Hospital. CLARK, JEFFERSON H., Instructor in Pathology, Medical School, Temple University. CLOWES, G. H. A., Director of Research, Eli Lilly & Co. COHN, EDWIN J., Assistant Professor of Physical Chemistry, Harvard University Medical School. EDWARDS, DAYTON J., Associate Professor, Cornell University Medical College. GARREY, WALTER E., Professor of Physiology, Tulane University. HARVEY, EDMUND NEWTON, Professor of Physiology, Princeton University. HECHT, SELIG, Research Fellow, Harvard University. HINRICHS, MARIE A., Laboratory Assistant, Nela Research Laboratory. HOLT, L. EMMETT, JR., Instructor, Johns Hopkins University. JACOBS, M. H., Professor of General Physiology, University of Pennsylvania. KNOWLTON, FRANK P., Professor of Physiology, Syracuse University. LILLIE, RALPH S., Professor of General Physiology, The University of Chicago. LOEB, LEO, Professor of Pathology, Washington University Medical School. LYON, E. P., Professor of Physiology, University of Minnesota. MATHEWS, A. P., Professor of Biochemistry, University of Cincinnati. POND, SAMUEL E., Instructor in Physiology, Washington University. QUERIDO, ARIE, Harvard University Medical School. REDFIELD, A. C., Assistant Professor of Physiology, Harvard University Medical School. ROBERTSON, WILLIAM E., Professor of Medicine, Medical School, Temple University SCHWITALLA, ALPHONSE, Associate Professor of Biology, St. Louis University. SHAPLEY, HARLOW, Director, Harvard Observatory, Cambridge, Mass. SMITH, HOMER W., Harvard University Medical School. SPAETH, REYNOLD A., Visiting Professor of Physiology, Rockefeller Foundation. WARREN, H. C., Professor of Psychology, Princeton University. WOODWARD, ALVALYN E., Associate Professor, North Carolina College for Women. WULZEN, ROSALIND, Instructor, University of California. THE DIRECTOR'S REPORT. 33 PHYSIOLOGY Beginning Investigators. BAKER, HERBERT N., Johns Hopkins University. BROWN, D. E. S., University of Michigan. CATTELL, WARE, Garrison, New York. EGLOFF, WILLIAM C., University of Chicago. GENTHER, IDA T., Student, Mount Holyoke College. GLUSKER, S. DAVID, University of Pennsylvania. HARTLINE, H. K., Lafayette College. HURD, A. L., Harvard University Medical School. KREDEL, FREDERICK E., Graduate Assistant in Zoology, University of Pittsburgh. LANDIS, EUGENE M.. Student, University of Pennsylvania. NADLER, JACOB E., Assistant in Biology, Johns Hopkins University. PAGE, IRVINE H., Research, Eli Lilly & Co. STUDEBAKER, MABEL T., Chemist, Eli Lilly & Co., Indianapolis. THOMAS, GILES W., Research Assistant, Harvard University Medical School. WALDEN, EDA B., Research Chemist, Eli Lilly & Co. WALDEN, GEO. B., Research Chemist, Eli Lilly Si Co. BOTANY Independent Investigators. ALLEN, CHARLES E., Professor of Botany, University of Wisconsin. BROOKS, MATILDA M., Hygienic Laboratory, United States Public Health Service, Washington, D. C. BROOKS, SUMNER C., Hygienic Laboratory, United States Public Health Service, Washington, D. C. CLELAND, RALPH E., Associate Professor of Biology, Goucher College. COWDRY, NATHANIEL H., 412 Hawthorn Road, Roland Park, Baltimore, Md. GATES, REGINALD R., Professor of Botany, University of London, Kings College. HAZEN, TRACY E., Assistant Professor of Botany, Barnard College, Columbia University. INMAN, ONDESS L., Professor of Biology, Antioch College. LEWIS, IVEY F., Professor of Biology, University of Virginia. SCHRAMM, J. R., Professor of Botany, Cornell University. STEVENS, NEIL E., Pathologist, Department of Agriculture, Washington, D. C. STOKEY, ALMA G., Professor of Botany, Mount Holyoke College. TAYLOR, WILLIAM RANDOLPH, Assistant Professor, University of Pennsylvania. WANN, FRANK BURKETT, National Research Fellow, Cornell University. BOTANY Beginning Investigators. EMERSON, ROBERT, Harvard University. FOGG, JOHN MILTON, JR., University of Pennsylvania. HOP, ANNE, Watertown Arsenal, Watertown, Massachusetts. HOLDEN, ALAN NORDBY, Harvard University. KEEFE, ANSELM M., Fellow in Botany, University of Wisconsin. KEMP, MARGARET, Investigator, Radcliffe College. WILCOX, MARGUERITE S., Laboratory Assistant, Department of Agriculture, Washington, D. C. 34 MARINE BIOLOGICAL LABORATORY. STUDENTS. 1924. Botany. BEALL, RUTH, Assistant in Biology, Goucher College. BELL, HUGH PHILIP, Associate Professor of Biology, Dalhousie University. BETTS, EDWIN M., Student, University of Virginia. BOUGHTON, ESTHER MARIE, Graduate student, Elmira College. BUNTEN, EVA ISABEL, George Washington University. CANBY, MARGARET LESLIE, Student, Pomona College. DEGENER, OTTO, AMHERST, Mass. FLEXNER, WILLIAM WELCH, Student, Harvard College. FULLER, ANNIE MAE, Louisiana State University. HART, HELEN T., Assistant in Botany, Vassar College. KOCH, NINNA F., Graduate student, Tulane University. LEVY, CELIA D., Hunter College. NEAL, MARY EULALIA, Teacher of Science, Boston Schools. NICHOLSON, JAMES LAWRENCE, Student, Oberlin College. POOLE, JAMES P., Assistant Professor of Evolution, Dartmouth College. SABINE, JANET, Student, Radcliffe College. SCHIMIAN, GERVASE, High School Instructor, St. Vincent College. STUART, DOROTHY RHETT, Graduate student, Johns Hopkins University. SHOWERS, EDITH, Smith College. WALSH, LYDIA BOURNE, Wellesley College. Embryology. ALLEN, LOUISE RECTOR, Instructor, Vanderbilt University. BROWN, ALICE E., 924 Newington Ave., Baltimore, Md. CHASE, ELTON FLETCHER, Instructor, Tufts College. CLAPP, EUNICE E., Student, Smith College CURWEN, ALICE OSBORNE, Student, Smith College. DILLER, WILLIAM F., JR., Instructor, University of Pennsylvania. DOWLING, ALEXANDER SCOTT, Student, DePauw University. FALES, DORIS E., Student, Mount Holyoke College. GIANG, FREDERICA HSIEN TREN, Student, Vassar College. LEONARD, DONALD WILLIAM, Wesleyan University. MEAD, HAROLD TUPPER, Associate Professor Zoology, Tulane University. MEADER, RALPH G., Student Assistant in Zoology, Ohio Wesleyan University. MILLER, RALPH ENGLISH, Assistant Instructor, Dartmouth College. MULLIN, CATHARINE, Instructor in Zoology, State University of Iowa. ORR, PAUL RUDBERT, University of Pennsylvania. PARPART, ARTHUR K., Student, Amherst College. RANKIN, IVA LUCILLE, Assistant Instructor, University of Wisconsin. ROWLAND, VIRGINIA, Mount Holyoke College. ROXAS, HILARIO A., Student, University of Chicago. SANBORN, IRENE HANNAH, Student, Simmons College. SANDERS, ELIZABETH PERCY, Assistant in Biology, Goucher College. SCOTT, J. ALLEN, Instructor, University of Vermont. THE DIRECTOR'S REPORT. 35 SLEETER, VICTOR R., Asst. in Embryology Laboratory, Illinois Wesleyan Uni- versity. SPENCER, WARREN POPPING, Ohio State University. STEIN, KATHRYN F., Mount Holyoke College. URBANTKE, ELSIE, Instructor in Histology and Embryology, University of Texas, Medical branch. WENSTRUP, EDWARD JOSEPH, Teacher of Zoology, St. Vincent College. WILLIAMS, ELISABETH, Mount Holyoke College. WINKLEY, RUTH, University of Michigan. Physiology. ATLEE, J. L., JR., Lancaster, Pennsylvania. CASTLE, EDWARD SEARS, Harvard University. CLARKE, MIRIAM F., Assistant in Physiological Laboratory, Mount Holyoke College. GRAY, PHILIP LEWIS, Instructor in Biology, Hamilton College. HANGER, IRWIN C., Staunton, Virginia. HAYWOOD, CHARLOTTE, n Haewood St., Lynn, Mass. KENYON, WALTER A., Instructor in Biology. Hamline University. MERRITT, OLIVE ELEANOR, Instructor in Biology, Hunter College. MILES, ALICE L., Laboratory assistant, Yale University. MILLEMANN, RAYMOND JOSEPH, Student, Cornell Medical College. PEREIRA, JAYME REGALLO, Assistant in Clinical Medicine, Rio de Janeiro, Brasil. REED, LUCILLE LYMAN, Sophie Newcomb College. ROOT, WALTER STANTON, Assistant in Biology, Wesleyan University. TYCHOWSKI, DR. WIKTOR, Zbigniew, Poland, Europe. WAYMAN, MARGUERITE, Assistant Professor, Hunter College. WILLIAMS, S. CULVER, Assistant in Biology, Yale University. WRIGHT, SEWALL, Senior Animal Husbandman, Washington, D. C. WRIGHT, SYDNEY L., JR., Chemist, Pennsylvania Hospital. Protozoology. CASS, MILDRED FRANCES, Minor Assistant, Columbia, University. COLLIER, JANE, Technician, Harvard Medical School. EICKMANN, REINTRAUT W. M., Graduate Student, Columbia University. FOGG, LLOYD CLARKE, Graduate Student, Dartmouth College. GREINER, BRIGHT E., Instructor Zoology, Syracuse University. HOLE, FRANCES L., Instructor, Doane College. HALSEY, HARVEY RANDOLPH, Laboratory Instructor, Columbia University. KEITH, WILMA, High School teacher of Biology, 1229 Carr Ave. Memphis, Tenn. Li, Ju CHI, Student, Columbia University. LITTLE, MALCOLM EDGEWORTH, Laboratory Assistant, Columbia University. MESSER, HAROLD MADISON, Instructor in Biology, Washington Square College, N- Y. University. OAKES, MERVIN E., Instructor, Washington Sq. College, N. Y. University. PHILPOTT, CHARLES H., Professor of Biology, University of Missouri. PICKWELL, GAYLE B., Instructor in Zoology, Northwestern University. PIRKLE, RUTH JANETTE, Instructor in Biology, Agnes Scott College. SCHULTZ, JACK, Columbia University. TAFT, CHARLES H., JR., Assistant Professor, Tufts College. 36 MARINE BIOLOGICAL LABORATORY. Attending Morning Lectures Only. BERTSCH, MARGUERITE, Columbia University. Zoology. ABRAMS, ELEANOR L., Mount Holyoke College. ALBRO, HELEN TUCKER, Instructor in Zoology, Mount Holyoke College. APPEL, FRED W., Graduate Student, University of Chicago. BIRRELL, ETHEL JANE, Science Instructor in State Normal School, Danbury, Conn. BRADNER, JOHN, Student, Yale University. BRAY, C. RUSSELL, DePauw University. BRENNER, NANCY JANE, Hunter College. ERODE, MALCOLM D., Assistant, University of Chicago. BRUMMER, FREDA LOUISE, Student, University of Pennsylvania. CANBY, CAROLINE PRESCOTT, Student, Radcliffe College. CARR, RALPH WILLIAM, Wesleyan University. COOLMAN, RAYMOND, Student, Wabash College. CUMMINGS, ELIZABETH FRANCES, Student, Radcliffe College. CURRY, LALIAH FLORENCE, Wellesley College. DOBROSCKY, IRENE DOROTHY, Student, Cornell University. FORER, ROBERT, Student, Rutgers College. FOWLER, KATHARINE STEVENS, Bryn Mawr College. GOLDMAN, DOUGLAS, Student, University of Cincinnati. HAAS, EDNA L., Connecticut College for Women. HALL, HARRIS TREMAINE, Student, Princeton University. HARRIS, DOROTHY Lou, Wilson College. HAYDEN, CATHERINE SPENCER, Goucher College. KEYS, FLORENCE M., Assistant in Zoology, Washington University. HUNTER, GEORGE WILLIAM, JR., Illinois University. JALESKI, CLARENCE THOMAS, Butler College. KOTTMEIER, MARJORIE, Knox College. KROPP, BENJAMIN, Graduate Student, Harvard University. LEICH, CHARLES F., Evansville, Indiana. LYNCH, JAMES E., Assistant in Anatomy, Washington University. MACDONELL, THOMAS KENNERLY, Graduate Fellow, Emory University. McQuiLKiN, WILLIAM EVERETT, Student, Doane College. MILLER, DAVID K., Illinois Wesleyan University. NORTHRUP, FLORA E., Graduate Assistant, Washington University. PARMELEE, ELEANORE W., Graduate Fellow, Mt. Holyoke College. PIKE, RADCLIFFE BARNES, Bowdoin College. QUICK, MARY ELAINE, Geo. Washington University. RAFFEL, DANIEL, Student, Johns Hopkins University. ROBERTS, JOSEPH MARVIN., Laboratory Assistant in Zoology, Illinois Wesleyan University. RUSSELL, MARY ANNA, Goucher College. SAYLE, MARY HONORA, Assistant Instructor, University of Wisconsin. SCHULZE, LULA MAY, Assistant in Zoology, University of Missouri. STEPHENS. MARY ALLEN, Elmira College. SWAN, DOROTHY M., Colorado College. SWEETING, MARJORIE A., Barnard College, Columbia University. THE DIRECTOR'S REPORT. 37 TEWINKEL, Lois E., Oberlin College. TYSON, JOHN JOYNER, Ayden, North Carolina. WARREN, A. EMERSON, Student, and Assistant, Yale University. WENNER, WILLIAM FRANKLIN, Assistant Instructor, Yale University. WILLIAMS, ELIZABETH T., Smith College. ZEMAN, MIRA, New York University. 3. TABULAR VIEW OF ATTENDANCE. 1920 1921 1922 1923 1924 INVESTIGATORS Total 136 172 182 176 194 Independent: Zoology 69 79 87 90 77 Physiology 22 26 28 23 33 Botany 7 13 15 13 14 Under Instruction: Zoology 29 34 34 41 47 Physiology 7 n n 5 16 Botany 2 9 7 4 7 STUDENTS Total 120 120 126 146 134 Zoology 56 53 59 59 50 Protozoology 15 n 12 16 17 Embryology 26 28 28 31 29 Physiology 15 18 19 22 18 Botany 8 10 8 18 20 TOTAL ATTENDANCE 256 292 308 322 328 INSTITUTIONS REPRESENTED Total. 86 95 104 107 no By investigators 55 67 71 62 69 By students 57 53 68 73 68 SCHOOLS AND ACADEMIES REPRESENTED By investigators I By students 7 4 MARINE BIOLOGICAL LABORATORY. SUBSCRIBING AND COOPERATING INSTITUTIONS, 1924. Amherst College Antioch College Barnard College Bowdoin College Bryn Mawr College Butler College Carnegie Institution, Cold Spring Harbor Carnegie Institution of Washing- ton Clark University Columbia University Cornell University Cornell University Medical Col- lege Dartmouth College DePauw University Doane College Elmira College George Washington University Goucher College Harvard University Harvard University Medical School Hunter College Illinois Wesleyan University Johns Hopkins University Knox College Eli Lilly & Co. Massachusetts Institute of Tech- nology McGill University Miami University Mount Holyoke College Nela Research Laboratories New York University North Carolina College for Women Northwestern University Oberlin College Ohio Wesleyan University Presbyterian College of South Carolina Princeton University Radcliffe College Rockefeller Foundation Rockefeller Institute for Medical Research Rutgers College Simmons College Smith College Sophie Newcomb College St. Bernard College St. Louis University St. Norbert's College St. Vincent College Tufts College Union College University of Chicago University of Cincinnati University of Illinois University of Michigan University of Minnesota University of Missouri University of Pennsylvania University of Pennsylvania Medi- cal School University of Philippines University of Virginia University of Vermont University of Wisconsin U. S. Dept. of Agriculture Vanderbilt University Vassar College Washington University Washington University Medical School Wellesley College Wesleyan University W 7 ilson College Wistar Institute of Anatomy and Biology. Yale University THE DIRECTOR'S REPORT. 39 SCHOLARSHIP TABLES The Lucretia Crocker Scholarships for Teachers in Boston, Since 1888. Scholarship of $100.00, Supported by a Friend of the Laboratory, Since 1898. The Edwin S. Linton Memorial Scholarship of Washington and Jeffer- son College. 5. EVENING LECTURES, 1924. Thursday, July 3, DR. W. J. CROZIER "The Nature ot a Central Nervous Process." Friday, July n, DR. E. J. COHN "The Physico-chemical charac- terization of Proteins." Tuesday, July 15, DR. MERKEL H. JACOBS "Some introcellular changes de- pendent on differential cell permeability." Friday, July 18, DR. L. L. WOODRUFF "The Physiological Significance of Conjugation and Endo- mixis." Tuesday, July 22, DR. W. B. CANNON "Some Revelations of the De- nervated Heart." Friday, July 25, DR. C. R. MOORE "The Temperature Regulation of the Mammalian Testis and its Relation to Viability in Grafts and Displacements." Tuesday, July 29, DR. RALPH S. LILLIE "Structure and Function in Protoplasmic Systems with Special Reference to the Con- ditions of Transmission and Recovery." Friday, August I, DR. C. E. ALLEN "Studies of Inheritance in Sphaerocarpus." Tuesday, August 5, DR. J. K. BREITENBECKER "Genetics of Bruchus." 4O MARINE BIOLOGICAL LABORATORY. Tuesday, August 26, DR. JULIAN HUXLEY "Bird Courtship and the Prob- lem of Sexual Selection." 6. MEMBERS OF THE CORPORATION. i. LIFE MEMBERS. ALLIS, MR. E. P., JR., Palais Carnoles, Menton, France. ANDREWS, MRS. GWENDOLEN FOULKE, Baltimore, Md. BILLINGS, MR. R. C., 66 Franklin St., Boston, Mass. CAREY, MR. ARTHUR ASTOR, Fayerweather St., Boston, Mass. CLARKE, PROF. S. F., Williamstown, Mass. CONKLIN, PROF. EDWIN G., Princeton University, Princeton, N.J. CRANE, MR. C. R., Woods Hole, Mass. EVANS, MRS. GLENDOWER, 12 Otis Place, Boston, Mass. FAY, Miss S. B., 88 Mt. Vernon St., Boston, Mass. FOLSOM, Miss AMY, 88 Marlboro St., Boston, Mass. FOOT, Miss KATHERINE, Care of Morgan Harjes Cie, Paris, France. GARDINER, MRS. E. G, Woods Hole, Mass. GARDINER, Miss EUGENIA, 15 W. Cedar St., Boston, Mass. HARRISON, EX-PROVOST C. C., University of Pennsylvania, Philadelphia, Pa. JACKSON, Miss M. C., 88 Marlboro St., Boston, Mass. JACKSON, MR. CHAS. C., 24 Congress St., Boston, Mass. KIDDER, MR. NATHANIEL T., Milton, Mass. KING, MR. CHAS. A. LEE, MRS. FREDERIC S., 279 Madison Ave., New York City, N. Y. LOWELL, MR. A. LAWRENCE, 17 Quincy St., Cambridge, Mass. MARRS, MRS. LAURA NORCROSS, 9 Commonwealth Ave., Boston, Mass. MASON, Miss E. F., i Walnut St., Boston, Mass. MASON, Miss IDA M., i Walnut St., Boston, Mass. MEANS, DR. JAMES HOWARD, 15 Chestnut St., Boston, Mass. MERRIMAN, MRS. DANIEL, 73 Bay State Road, Boston, Mass. MINNS, Miss SUSAN, 14 Louisburg Square, Boston, Mass. THE DIRECTOR S REPORT. 4! MINNS, MR. THOMAS, 14 Louisburg Square, Boston, Mass. MORGAN, MR. J. PIERPONT, JR., Wall and Broad Sts., New York City, N. Y. MORGAN, PROF. T. H., Columbia University, New York City, N. Y. MORGAN, MRS. T. H., New York City, N. Y. NOYES, Miss EVA J. NUNN, MR. LUCIAN L., Telluride, Colo. OSBORN, PROF. HENRY F., American Museum of Natural History, New York City, N. Y. PHILLIPS, DR. JOHN C., Windy Knob, Wenham, Mass. PHILLIPS, MRS. JOHN C., Windy Knob, Wenham, Mass. PORTER, DR. H. C., University of Pennsylvania, Philadelphia, Pa. PULSIFER, MR. W. H., Newton Center, Mass. SEARS, DR. HENRY F., 86 Beacon St., Boston, Mass. SHEDD, MR. E. A. THORNDIKE, DR. EDWARD L., Teachers College, Columbia University, New York City, N. Y. TRELEASE, PROF. WILLIAM, University of Illinois, Urbana, 111. WARE, Miss MARY L., 41 Brimmer St., Boston, Mass. WILCOX, Miss MARY A., Wellesley College, Wellesley, Mass. WILLIAMS, MRS. ANNA P., 505 Beacon St., Boston, Mass. WILSON, DR. E. B., Columbia University, New York City, N. Y. WILSON, PROF. W. P., Commercial Museum, Philadelphia, Pa. 2. REGULAR MEMBERS, AUGUST, 1924. ADAMS, Miss A. E., Mount Holyoke College, South Hadley, Mass. ADDISON, DR. W. H. F., University of Pennsylvania Medical School, Philadelphia, Pa. ADOLPH, DR. EDWARD F., Johns Hopkins University, Baltimore, Md. AGERSBORG, DR. H. P. K., James Millikin University, Decatur, 111. ALLEE, DR. W. C., University of Chicago, Chicago, 111. ALLEN, PROF. CHAS. E., University of Wisconsin, Madison, Wis. ALLEN, PROF. EZRA, Ursinus College, Collegeville, Pa. ALLYN, Miss HARRIET M., Monticello Seminary, Godfrey, 111. 42 MARINE BIOLOGICAL LABORATORY. AMBERSON, DR. WILLIAM B., University of Pennsylvania, Phila- delphia, Pa. ANDERSON, DR. E. G., University of Michigan, Ann Arbor, Mich. ATTERBURY, MRS. RUTH R., Great Neck, Long Island, N. Y. BAITSELL, DR. GEORGE A., Yale University, New Haven, Conn. BAKER, DR. E. H., Cooper Carlton Hotel, Hyde Park Station, Chicago, 111. BALDWIN, DR. F. M., Iowa State College, Ames, la. BASCOM, DR. K. F., Medical School, Univ. of Pennsylvania, Philadelphia, Pa. BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, N. Y. BEHRE, DR. ELINOR H., Louisiana State University, Baton Rouge, La. BIGELOW, PROF. M. A., Teachers College, Columbia University, New York City. BIGELOW, PROF. R. P., Massachusetts Institute of Technology, Cambridge, Mass. BINFORD, PROF. RAYMOND, Guilford College, Guilford College, N. C. BLODGETT, DR. F. H., State Normal School, Danbury, Conn. BODANSKY, DR. MEYER, University of Texas, Galveston, Tex. BORING, DR. ALICE M., Yenching College, Peking, China. Box, Miss CORA M., University of Cincinnati, Cincinnati, O. BOWEN, DR. ROBERT H., Columbia University, New York City. BRADLEY, PROF. HAROLD C., University of Wisconsin, Madison, Wis. BRAILEY, Miss MIRIAM E., Mount Holyoke College, South Hadley, Mass. BRIDGES, DR. CALVIN B., Columbia University, New York City. BROOKS, DR. S. C., U. S. Public Health Service, Washington, D. C. BRUMFIEL, DR. DANIEL M., Henry Ford Hospital, Detroit, Mich. BUCKINGHAM, Miss EDITH N., 342 Marlboro St., Boston, Mass. BUDINGTON, PROF. R. A., Oberlin College, Oberlin, O. BUMPUS, PROF. H. C., Brown University, Providence, R. I. BYRNES, DR. ESTHER F., 193 Jefferson Ave., Brooklyn, N. Y. CALKINS, PROF. GARY N., Columbia University, New York City. THE DIRECTOR S REPORT. 43 CALVERT, PROF. PHILIP P., University of Pennsylvania, Phila- delphia, Pa. CARLSON, PROF. A. J., University of Chicago, Chicago, 111. CAROTHERS, DR. ELEANOR E., University of Pennsylvania, Philadelphia, Pa. CARROLL, PROF. MITCHEL, Franklin and Marshall College, Lancaster, Pa. CARVER, PROF. GAIL L., 258 Washington Ave., Macon, Ga. CASEY, COLONEL THOMAS L., Stoneleigh Court, Washington, D. C. CASTEEL, DR. D. B., University of Texas, Austin, Tex. CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, N. Y. CATTELL, DR. McKEEN, Cornell University Medical College, New York City. CHAMBERS, DR. ROBERT, Cornell University Medical College, New York City. CHARLTON, DR. HARRY H., University of Missouri, Columbia, Mo. CHIDESTER, PROF. F. E., West Virginia University, Morgantown, W. Va. CHILD, PROF. C. M., University of Chicago, Chicago, 111. CLAPP, PROF. CORNELIA M., Montague, Mass. CLARK, PROF. E. R., University of Georgia, Augusta, Ga. CLELAND, PROF. RALPH E., Goucher College, Baltimore, Md. CLOWES, PROF. G. H. A., Eli Lilly & Co., Indianapolis, Ind. COE, PROF. W. R., Yale University, New Haven, Conn. COHN, DR. EDWIN J., 43 Kirkland St., Cambridge, Mass. COKER, DR. R. E., University of North Carolina, Chapel Hill, N. C. COLE, DR. LEON J., College of Agriculture, Madison, Wis. COLLETT, DR. MARY E., Western Reserve University, Cleveland, O. COLLEY, MRS. MARY W., 1712 Madison St., Madison, Wis. COLTON, PROF. H. S., Ardmore, Pa. COOLIDGE, MR. C. A., Ames Building, Boston, Mass. COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Me. COWDRY, DR. E. V., Rockefeller Institute, New York City. COWDRY, PROF. N. H., 412 Hawthorne Road, Roland Park, Baltimore, Md. 44 MARINE BIOLOGICAL LABORATORY. CRAMPTON, PROF. H. E., Barnard College, Columbia University, New York City. CRANE, MRS. C. R., Woods Hole, Mass. CURTIS, PROF. W. C., University of Missouri, Columbia, Mo. CURTIS, DR. MAYNIE R., Crocker Laboratory, Columbia Univer- sity, New York City. DANCHAKOFF, DR. VERA, College of Physicians and Surgeons, New York City. DAVIS, DR. DONALD W., College of William and Mary, Williams- burg, Va. DAVIS, PROF. BRADLEY M., University of Michigan, Ann Arbor, Mich. DAVIS, DR. ALICE R., 19 Ash St., Cambridge, Mass. DAWSON, DR. J. A., Harvard University, Cambridge, Mass. DEDERER, DR. PAULINE H., Connecticut College, New London, Conn. DEXTER, DR. J. S., University of Porto Rico, Rio Piedras, Porto Rico. DODDS, PROF. G. S., Medical School, University of West Virginia, Morgantown, W. Va. DOLLEY, PROF. WILLIAM L., Randolph-Macon College, Ashland, Virginia. DONALDSON, PROF. H. H., Wistar Institute of Anatomy and Biology, Philadelphia, Pa. DONALDSON, DR. JOHN C., University of Pittsburgh, School of Medicine, Pittsburgh, Pa. DREW, PROF. OILMAN A., Marine Biological Laboratory, Woods Hole, Mass. DUNBAR, MR. WILLIAM H., 161 Devonshire St., Boston, Mass. DUNGAY, DR. NEIL S., Carleton College, Northfield, Minn. DUNN, DR. ELIZABETH H., Woods Hole, Mass. EDWARDS, DR. D. J., Cornell University Medical College, New York City. EIGENMANN, PROF. C. H., University of Indiana, Bloomington, Ind. ELLIS, DR. F. W., Monson, Mass. FARNUM, DR. LOUISE W., Hunan-Yale Hospital, Changsha, Hunan, China. FIELD, Miss HAZEL E., University of California, Berkeley, Calif. THE DIRECTOR S REPORT. 45 FINLEY, MR. CHARLES W., Lincoln School, 646 Park Ave., New York City. FISHER, Miss MARY J., Cornell University, Ithaca, New York. FRANKLIN, DR. CHRISTINE LADD, 617 West 113 St., New York City. FRY, MR. HENRY J., Columbia University, New York City. GAGE, PROF. S. H., Cornell University, Ithaca, N. Y. GARREY, PROF. W. E., Tulane University, Richardson Memorial, New Orleans, La. GATES, PROF. R. RUGGLES, University of London, London, England. GEISER, DR. S. W., Southern Methodist University, Dallas, Texas. GLASER, PROF. O. C., Amherst College, Amherst, Mass. GLASER, PROF. R. W., Rockefeller Institute for Medical Research, Princeton, N. J. GOLDFARB, PROF. A. J., College of the City of New York, New York City. GOODRICH, PROF. H. B., Wesleyan University, Middletown, Conn. GOWANLOCH, MR. J. N., Dalhousie University, Halifax, Nova Scotia. GRAVE, PROF. CASWELL, Washington University, St. Louis, Mo. GRAVE, PROF. B. H., Wabash College, Crawfordsville, Ind. GREENMAN, PROF. M. J., Wistar Institute of Anatomy and Biology, Philadelphia, Pa. GREGORY, DR. LOUISE H., Barnard College, Columbia University, New York City. GUNTHER, Miss MAUDE C., Business High School, Washington, D. C. GUTHRIE, DR. MARY J., University of Missouri, Columbia, Mo. GUYER, PROF. M. F., University of Wisconsin, Madison, W T is. HANCE, DR. ROBERT T., Rockefeller Institute, 66th St. and Ave. A. New York City. HARGITT, PROF. C. W., Syracuse University, Syracuse, N. Y. HARGITT, PROF. GEORGE T., Syracuse University, Syracuse, N. Y. HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kans. 46 MARINE BIOLOGICAL LABORATORY. HARPER, PROF. R. A., Columbia University, New York City. HARRISON, PROF. Ross G., Yale University, New Haven, Conn. HARVEY, PROF. E. N., Princeton University, Princeton, N. J. HARVEY, MRS. E. N., Princeton, N. J. HAYDEN, Miss MARGARET A., Wellesley College, Wellesley, Mass. HAZEN, DR. T. E., Barnard College, Columbia University, New York City. HEATH, PROF. HAROLD, Palo Alto, Calif. HECHT, DR. SELIG, Harvard Medical School, Boston, Mass. HEGNER, PROF. R. W., Johns Hopkins University, Baltimore, Md. HEILBRUNN, DR. L. V., University of Michigan, Ann Arbor, Mich. HESS, PROF. WALTER N., DePauw University, Greencastle, Ind. HINRICHS, DR. MARIE A., University of Chicago, Chicago, 111. HOADLEY, DR. LEIGH, University of Chicago, Chicago, 111. HOGUE, DR. MARY J., 503 N. High St. West Chester, Pa. HOLMES, PROF. S. J., University of California, Berkeley, Calif. HOOKER, PROF. DAVENPORT, University of Pittsburgh, Pitts- burgh, Pa. HOPKINS, DR. HOYT S., Iowa State College, Ames, la. HOSKINS, MRS. ELMER R., University of Arkansas Medical School, Little Rock, Ark. HOWE, DR. H. E., 2702 36th St., N. W., Washington, D. C. HOWLAND, PROF. RUTH B., Sweet Briar College, Sweet Briar, Va. HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Va. HUMPHREY, MR. R. R., University of Buffalo School of Medicine, Buffalo, N. Y. HYMAN, DR. LIBBIE H., University of Chicago, Chicago, 111. INMAN, PROF. ONDESS L., Antioch College, Yellow Springs, O. JACKSON, PROF. C. M., University of Minnesota, Minneapolis, Minn. JACOBS, PROF. MERKEL H., University of Pennsylvania, Phila- delphia, Pa. JENNINGS, PROF. H. S., Johns Hopkins University, Baltimore, Md. JEWETT, PROF. J. R., Harvard University, Cambridge, Mass. THE DIRECTOR'S REPORT. 47 JOHNSON, PROF. GEORGE E., State Agricultural College, Manhat- tan, Kans. JONES, PROF. LYNDS, Oberlin College, Oberlin, O. JONES, PROF. L. R., University of Wisconsin, Madison, Wis. JORDAN, PROF. H. E., University of Virginia, Charlottesville, Va. JUST, PROF. E. E., Howard University, Washington, D. C. KEEFE, REV. ANSELM M., 723 State St., Madison, Wis. KENNEDY, DR. HARRIS, Readville, Mass. KINDRED, DR. J. E., University of Virginia, Charlottesville, Va. KING, DR. HELEN D., Wistar Institute of Anatomy and Biology, Philadelphia, Pa. KING, DR. ROBERT L., University of Pennsylvania, Philadelphia, Pa. KINGSBURY, PROF. B. F., Cornell University, Ithaca, N. Y. KINGSLEY, PROF. J. S., 2500 Cedar St., Berkeley, Calif. KIRKHAM, DR. W. B., Springfield College, Springfield, Mass. KNAPKE, REV. BEDE, St. Bernard's College, St. Bernard, Ala. KNOWER, PROF. H. McE., University of Cincinnati, Cincinnati, O. KNOWLTON, PROF. F. P., Syracuse University, Syracuse, N. Y. KOSTIR, DR. W. J., Ohio State University, Columbus, O. KRIBS, DR. HERBERT, 5857 Cobbs Creek Parkway, Philadelphia, Pa. KUYK, DR. MARGARET P., Westbrook Ave., Richmond, Va. LANCEFIELD, DR. D. E., Columbia University, New York City. LANGE, DR. MATHILDE M., Wheaton College, Norton, Mass. LEE, PROF. F. S., 437 West 59th St., New York City. : LEWIS, PROF. I. F., University of Virginia, Charlottesville, Va. LEWIS, PROF. W. H., Johns Hopkins University, Baltimore, Md. LILLIE, PROF. FRANK R., University of Chicago, Chicago, 111. LILLIE, PROF. R. S., University of Chicago, Chicago, 111. LINTON, PROF. EDWIN, 1104 Milledge Road, Augusta, Ga. LOEB, PROF. LEO, Washington University Medical School, St. Louis, Mo. LOEB, MRS. LEO, 6803 Kingsburg Boulevard, St. Louis, Missouri. LOWTHER, MRS. FLORENCE DEL., Barnard College, Columbia University, New York City. LUCKE, PROF. BALDWIN, University of Pennsylvania, Phila- delphia, Pa. 48 MARINE BIOLOGICAL LABORATORY. LUND, DR. E. J., University of Minnesota, Minneapolis, Minn. LUSCOMBE, MR. W. O., Woods Hole, Mass. LYMAN, PROF. GEORGE R., College of Agriculture, University of West Virginia, Morgantown, W. Va. LYNCH, Miss CLARA J., Rockefeller Institute, New York City. LYON, PROF. E. P., University of Minnesota, Minneapolis, Minn. MACCALLUM, DR. G. A., 925 St. Paul St., Baltimore, Md. MACDOUGALL, Miss MARY S., Agnes Scott College, Decatur, Ga. McCLUNG, PROF. C. E., University of Pennsylvania, Phila- delphia, Pa. McGEE, DR. ANITA NEWCOMB., Stoneleigh Court, Washington, D. C. McGiLL, DR. CAROLINE, Murray Hospital, Butte, Montana. MCGREGOR, DR. J. H., Columbia University, New York City. MclNDOO, DR. N. E., Bureau of Entomology, Washington, D. C. McMuRRiCH, PROF. J. P., University of Toronto, Toronto, Canada. McNAiR, DR. G. T., 1721 Grand Ave., Chickasha, Okla. MACKLIN, DR. CHARLES C., School of Medicine, Western Univer- sity, London, Canada. MALONE, PROF. E. F., University of Cincinnati, Cincinnati, O. MARTIN, Miss BERTHA E., Lindenwood College, St. Charles, Mo. MARTIN, MR. E. A., College of the City of New York, New York City. MAST, PROF. S. O., Johns Hopkins University, Baltimore, Md. MATHEWS, PROF. A. P., University of Cincinnati, Cincinnati, O. MATSUI, PROF. K., Imperial College of Agriculture and Dendrol- ogy, Morioka, Japan. MAYOR, PROF. JAMES W., Union College, Schenectady, N. Y. MEDES, DR. GRACE, Wellesley College, Wellesley, Mass. MEIGS, DR. E. B., Dairy Division Experiment Station, Beltsville, Md. MEIGS, MRS. E. B., 1445 Rhode Island Ave., Washington, D. C. METCALF, PROF. M. M., 128 Forest St., Oberlin, O. METZ, PROF. CHARLES W., Carnegie Institution of Washington, Cold Spring Harbor, Long Island. MINER, DR. ROY W., American Museum of Natural History, New York City. THE DIRECTOR'S REPORT. 49 MITCHELL, DR. PHILIP H., Brown University, Providence, R. I. MOORE, PROF. GEORGE T., Missouri Botanical Garden, St. Louis, Mo. MOORE, DR. CARL R., University of Chicago, Chicago, 111. MOORE, PROF. J. PERCY, University of Pennsylvania, Phila- delphia, Pa. MOORE, DR. A. R., Rutgers College, New Brunswick, N. J. MORGAN, DR. ANNA H., Mount Holyoke College, South Hadley, Mass. MORRILL, PROF. A. D., Hamilton College, Clinton, N. Y. MORRILL, PROF. C. V., Cornell University Medical College, New York City. MULLER, DR. H. J., University of Texas, Austin, Texas. NABOURS, DR. R. K., Kansas State Agricultural College, Manhat- tan, Kans. NACHTRIEB, PROF. HENRY F., University of Minnesota, Min- neapolis, Minn. NEAL, PROF. H. V., Tufts College, Tufts College, Mass. NEWMAN, PROF. H. H., University of Chicago, Chicago, 111. NICHOLS, DR. M. LOUISE, Powelton Apartments, Philadelphia, Pa. NONIDEZ, DR. JOSE F., Cornell University Medical College, New York City. OKKELBERG, DR. PETER, University of Michigan, Ann Arbor, Mich. OSBURN, PROF. R. C., Ohio State University, Columbus, O. OSTERHOUT, PROF. W. J. V., Harvard University, Cambridge, Mass. PACKARD, DR. CHARLES, Columbia University, Institute of Cancer Research, 1 145 Amsterdam Avenue, New York City. PACKARD, DR. W. H., Bradley Polytechnic Institute, Peoria, 111. PAPPENHEIMER, DR. A. M., Columbia University, New York City. PARKER, PROF. G. H., Harvard University, Cambridge, Mass. PATON, PROF. STEWART, Princeton University, Princeton, N. J. PATTEN, PROF. WILLIAM, Dartmouth College, Hanover, N. H. PATTERSON, PROF. J. T., University of Texas, Austin, Tex. PAYNE, PROF. F., University of Indiana, Bloomington, Ind. 5O MARINE BIOLOGICAL LABORATORY. PEARL, PROF. RAYMOND, Johns Hopkins University, Baltimore, Md. PEARSE, PROF. A. S., University of Wisconsin, Madison, Wis. PEEBLES, PROF. FLORENCE, Pineville, Pa. PHILLIPS, Miss RUTH L., Western College, Oxford, O. PHILLIPS, DR. E. F., Cornell University, Ithaca, New York. PIKE, PROF. FRANK H., 437 West 59th St., New York City. PINNEY, Miss MARY E., Milwaukee-Downer College, Milwaukee, Wis. PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Mass. POND, DR. SAMUEL E., Washington University School of Medi- cine, St. Louis, Mo. PRATT, DR. FREDERICK H., Boston University School of Medicine Boston, Mass. PRICE, DR. WESTON A., 8926 Euclid Ave., Cleveland, Ohio. RANKIN, PROF. W. M., Princeton University, Princeton, N. J. RAPPORT, DR. ANNA YATES, Bryn Mawr College, Bryn Mawr, Pa. REDFIELD, DR. ALFRED C., Harvard Medical School, Boston, Mass. REESE, PROF. ALBERT M., West Virginia University, Morgan- town, W. Va. REINKE, DR. E. E., Vanderbilt University, Nashville, Tenn. RHODES, PROF. ROBERT C., Emory University, Atlanta, Ga. RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, O. RICHARDS, PROF. A., University of Oklahoma, Norman, Okla. RICHARDS, MRS. A., Norman, Okla. RIGGS, MR. LAWRASON JR., 25 Broad St., New York City. ROBERTSON, PROF. W. R. B., 1505 Rosemary Lane, Columbia. Mo. ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, O. ROMER, DR. ALFRED S., University of Chicago, Chicago, 111. RUDISCH, DR. J., Fifth Avenue Bank, 44th St. and Fifth Ave., New York City. SAMPSON, Miss MYRA M., Smith College, Northampton, Mass. SANDS, Miss ADELAIDE G., 348 N. Main St., Port Chester, N. Y. SCHRADER, DR. FRANZ, Bryn Mawr College, Bryn Mawr, Pa. SCHRAMM, PROF. J. R., Cornell University, Ithaca, N. Y. THE DIRECTORS REPORT. 5! SCOTT, DR. ERNEST L., Columbia University, New York City. SCOTT, PROF. G. G., College of the City of New York, New York City. SCOTT, PROF. JOHN W., University of Wyoming, Laramie, Wyo. SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, N. J. SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Mich. SHUMWAY, DR. WALDO, University of Illinois, Urbana, 111. SIVICKIS, DR. P. B., University of the Philippines, Manila, P. I. SMITH, DR. BERTRAM G., 2015 University Ave., New York City. SMITH, Miss CHRISTIANNA, Mount Holyoke College, South Hadley, Mass. SNOW, DR. LAETITIA M., Wellesley College, Wellesley, Mass. SNYDER, PROF. CHARLES D., Johns Hopkins University Medical School, Baltimore, Md. SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio. SPAETH, DR. REYNOLD A., Chulalongkorn Medical School, Bangkok, Siam. SPEIDEL, DR. CARL C., University of Virginia, Charlottesville, Va. SPENCER, PROF. H. J., 24 West loth St., New York City. STARK, DR. MARY B., N. Y. Homoeopathic Medical College and Flower Hospital, New York City. STOCKARD, PROF. C. R., Cornell University Medical College, New York City. STOKEY, DR. ALMA G., Mount Holyoke College, South Hadley, Mass. STREETER, PROF. GEORGE L., Johns Hopkins University Medical School, Baltimore, Md. STRONG, PROF. O. S., Columbia University, New York City. STRONG, PROF. R. M., Loyola University School of Medicine, Chicago, 111. STURTEVANT, DR. ALFRED H., Columbia University, New York City. SWETT, DR. F. H., Johns Hopkins University Medical School, Baltimore, Md. TASHIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati O. 52 MARINE BIOLOGICAL LABORATORY. TAYLOR, Miss KATHERINE A., Cascade, Washington Co., Md. TAYLOR, DR. WILLIAM R., University of Pennsylvania, Phila- delphia, Pa. TENNENT, PROF. D. H., Bryn Mawr College, Bryn Mawr, Pa. THARALDSEN, PROF. C. E., Northwestern University, Evanston, 111. THATCHER, MR. LLOYD E., University of Michigan; Ann Arbor, Mich. TINKHAM, Miss FLORENCE L., 71 Ingersoll Grove, Springfield, Mass. TOMPKINS, Miss ELIZABETH M., 134 Linden Ave., Brooklyn, N. Y. TRACY, PROF. HENRY C., University of Kansas, Lawrence, Kansas. TREADWELL, PROF. A. L., Vassar College, Poughkeepsie, N. Y. TURNER, PROF. C. L., Beloit College, Beloit, Wis. UHLEMEYER, Miss BERTHA, University of California, Berkeley, Calif. UHLENHUTH, DR. EDWARD, Rockefeller Institute for Medical Research, New York City. VAN DER HEYDE, DR. H. C., Heemstede, Holland. VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland, O. WAITE, PROF. F. C., Western Reserve University Medical School, Cleveland, O. WALLACE, DR. LOUISE B., Constantinople Woman's College, Constantinople, Turkey. WANN, DR. FRANK B., Cornell University, Ithaca, N. Y. WARD, PROF. HENRY B., University of Illinois, Urbana, 111. WARDWELL, DR. E. H., Chappaqua, N. Y. WARREN, PROF. HOWARD C., Princeton University, Princeton, N.J. WARREN, DR. HERBERT S., Columbia University, New York City. WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pa. WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, N. D. WHEELER, PROF. W. M., Bussey Institution, Forest Hills, Mass. THE DIRECTOR S REPORT. 53 WHERRY, DR. W. B., Cincinnati Hospital, Cincinnati, Ohio. WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pa. WHITESIDE, Miss BEATRICE, 4926 Forest Park Boulevard, St. Louis, Mo. WHITING, DR. PHINEAS W., University of Maine, Orono, Me. WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Neb. WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, O. WILDMAN, PROF. E. E., 47th and Walnut Sts., Philadelphia, Pa. WILLIER, DR. B. H., University of Chicago, Chicago, 111. WILSON, PROF. H. V., University of North Carolina, Chapel Hill, N. C. WOGLOM, PROF. WILLIAM H., Columbia University, New York City. WOODRUFF, PROF. L. L., Yale University, New Haven, Conn. WOODWARD, DR. ALVALYN E., North Carolina College for Women, Greensboro, N. C. YOUNG, DR. B. P., Cornell University, Ithaca, N. Y. YOUNG, DR. D. B., Carleton College, Northfield, Minn. YOUNG, PROF. ROBERT T., University of North Dakota, Univer- sity, N. D. ZELENY, DR. CHARLES, University of Illinois, Urbana, 111. EGG-VOLUME AND FERTILIZATION MEMBRANE. CHARLES D. SNYDER. The question of fertilization and egg-volume seems to come up periodically for renewed research and discussion in spite of the papers already written on the topic. 1 In view of the disagree- ment as to the facts of volume changes at the moment of fertili- zation the writer wishes to submit the following evidence that was put in a paper in December, 1904, from observations made in that year on two species of echinoderms, and that hitherto has not been published. The work was done at the Timothy Hopkins Sea-side Laboratory of Stanford University, California. The microscope images of the eggs were projected by a camera lucida, and outline drawings were made of their greatest diame- ters. Upon each sheet of drawings a stage micrometer scale was also projected and drawn. The magnification was uniform throughout. The diameters given in sections 1-4 inclusive, of this paper, are of these drawings, and should be divided by 1 10 in each case to get the approximate natural diameter. i. Echinarachnius eccentrical Eggs made to develop parthe- nogenetically by treatment with hypertonic salt solution are known to shrink and then to swell again upon return to normal sea-water. Mature eggs of Echinarachnius eccentrica put in optimum hypertonic solution for i| hours were observed to shrink from a mean normal diameter of 13.27 to a mean diameter of n.6 mm. After return to normal sea-water for I hour the eggs showed diameters ranging from 12.86 to 13.18 mm. By the time cleavage began, and the eggs were still in I and 2-cell stages, the diameters ranged between 14 and 16 mm. ; when in stages varying from the 4-cell to the morula stage, the diameters ranged between 14.5 and 18 mm. These measurements refer to the egg-cytoplasm, no membranes appearing on eggs so treated. 1 For review of the literature and discussion of the problem see Lillie, F. R., "Problems of Fertilization," Chicago, 1919, pp. 147-154; also Glaser, Otto: "Fertilization, Cortex and Volume," BIOL. BULL., Vol. XLVIL, pp. 274-283, 1924. 2 Thanks are due to Professor Harold Heath, who kindly identified this species of sand-dollar for the writer. 54 EGG-VOLUME AND FERTILIZATION MEMBRANE. 55 Of the eggs fertilized with sperm those in I to 4-cell stages had diameters of egg-cytoplasm ranging from 13 to 15, and of fertili- zation membrane from 14.7 to 18 mm.; those in stages from 8-cells to blastulse had diameters from 14.5 to 16 mm. for egg- cytoplasm, and 16.5 to 17 mm. for fertilization membrane. The number of eggs measured in each case was five only. But the extremes of a large number as well as mean sizes were taken in each case. The mean size of the hypertonic parthenogenetic eggs thus appear to reach nearly the same limit that the membrane does in eggs fertilized with sperm; the egg-cytoplasm of the former apparently takes up water as easily as the membrane of the latter. The writer's observations did not include a large enough number of these eggs at the moment of membrane for- mation to determine whether or not they showed a preliminary shrinking, as Otto Glaser 1 and others have observed in eggs of other species. One can only say that if the shrinking took place it must have occurred between the moment of the entrance of the spermatozoon and the moment just before the first cleavage. 2. Asterina miniata (Brandt) Perrier. 3 When mature, and just before fertilization, a sample of seven eggs of this species measured 19, 19, 18.5, 19, 20, 19 and 20 mm., giving a mean of 19.25 mm. and an average of 19.21 mm. in diameter. When treated with suitable amounts of citric acid for a while and then returned to normal sea-water the eggs began to go into cleavage and develop into blastulae. 4 A fertilization membrane could be seen reaching across the furrows between the cells of these eggs. They differed in this respect from the sand-dollar eggs that were made to go into cleavage by hypertonic solutions. But nevertheless the mem- branes did not free themselves from the outermost periphery of the egg-cytoplasm. The diameters, therefore, of egg-cytoplasm and fertilization membrane of Asterina miniata so treated under- went equal changes; they ranged between 19 and 21, giving a mean diameter of 20 mm. No measurements were made at the moment of treatment with the acid. 3 Thanks are due to Dr. W. K. Fisher, who kindly identified this species of Asterina for the writer. 4 The eggs of this species are known to be somewhat naturally parthenogenetic. In the series with the optimum concentration of the citrir acid 79 per cent, swimming larvae appeared at the end of 42 hours whereas in the control only 6 per cent, of the eggs were segmented and none were swimming. The average of all the series worked with showed a natural parthenogenesis to the extent of about 3.5 per cent. CHARLES D. SNYDER. When mature eggs of Asterina miniata are treated with sperm, the egg-cytoplasm during early stages of cleavage has an average diameter of 17.7 and the fertilization membrane one of 21 mm. This is shown in the table. EGGS OF Asterina Miniata FERTILIZED WITH NORMAL SPERM. Stage of Cleavage. Diam. of Egg Cytoplasm. Diam. of Fertilization Membrane. Stage of Cleavage. Diam. of Egg Cytoplasm. Diam. of Fertilization Membrane. i-cell 18 20 2-cell 18 20 i-cell 17 2O. "C 2-cell i8.<; 22 i-cell 17 20 2-cell 18.7 21 i-cell 17 21. S 2-cell 17. c 22 i-cell 18 22. 5 2-cell 18.5 22 i-cell 18 2S 2-cell 18.7 20.1 4-cell 18 21 2-cell IQ.2 22 2-cell I c 2O 2-cell IO.O 2O. 5 2-cell . . is 2O Average .... 17.7 21 The mean diameters in these observations are 17.1 for the egg-cytoplasm and 22.5 for the membrane. The averages thus show less deviation than do the mean numbers. The amount of shrinking of the egg-cytoplasm comparing the mean diameters before and after fertilization is n.i per cent.; comparing the average diameters it is 7.8 per cent. During the early stage of normal fertilization, then, the eggs of Asterina miniata may be said to show a marked shrinking of the egg-cytoplasm. These figures are of the same order as Glaser (1914) observed *or the reduction in diameters of just fertilized Arbacia eggs (from x.4 to 14.5 per cent.) and Asterias eggs (from 10 to 17 per cent.) ; and Okkelberg 5 in volume reduction of eggs of the brook lamprey, 13.4 per cent. Just lately (1924) Glaser 1 repeated the measure- ments of Arbacia, using an improved method in order to prevent possible flattening of eggs before the membrane has been elevated (first suggested by Reighard, see Okkelberg, loc. cit., p. 97, footnote 2), and finds that the percentage reduction of diameter is less than in his earlier work, but still a demonstrable mean of 3 per cent. 4. In spite of the meagerness of observations (the lack of measurements on the sand-dollar egg during the earliest stages following insemination, the lack of a more perfected treatment to 6 Okkelberg, "Volumetric Changes in the Egg of the Brook Lamprey . . . after Fertilization," BIOL. BULL., Vol. XXVI., pp. 92-99, 1914. EGG-VOLUME AND FERTILIZATION MEMBRANE. 57 induce parthenogenesis) the data still furnish one or two points of further interest. The shrinking of the eggs of Echinarachnius eccentrica when subjected to optimum hypertonic solution for parthenogenetic development was from the mean diameter 13.27 before, to one of 1 1.6 mm. at the end of the treatment, or a reduction of 12.6 per cent. After return to normal sea-water, and by the time cleavage completed the 2-cell stage, the mean diameter was 15 mm. an increase of 13 per cent.; by the time the eggs were in stages ranging from 4-cell to morulae the mean diameter was 16.2 mm. showing a total increase of 22 per cent. In the case of the inseminated eggs of this species the fertili- zation membrane (assuming it to be present on the unfertilized egg) showed an increase, while developing to the 4-cell stage, from a mean of 13.27 to one of 16.3 mm. an increase of 22.8 per cent. ; eggs in stages of 8-cells to blastulae showed an increase to a mean diameter of 16.75 mm. or a mean total increase of 26.2 per cent. In the case of Asterina miniata the increase in the average total diameter of fertilization membrane of inseminated eggs is from 19.21 to 21.0 scale divisions or one of 9.3 per cent, comparing averages the increase is 16.8 per cent. ; of the acid treated parthe- nogenetic eggs the increase of both egg-cytoplasm and fertilization membrane (as in the case of hypertonic parthenogenetic eggs of the sand-dollar) is equal, and is the difference between 19.25 and 20. o mm., or an increase of 3.9 per cent. 5. It may be of further interest to calculate the approximate mean actual diameters and actual volumes 6 of these eggs. If we assume them to have been spheres in all cases we have the following: The mature unfertilized egg of Echinarachnius eccentrica has a mean diameter of 120 ji, from which its volume must be about .0009 mm. 3 ; the optimum hypertonic shrinking gave a diameter of 105 /*, or a volume of .00061 mm. 3 , representing a volume reduction of 33 per cent. The mean actual diameter of mature Asterina miniata eggs is 174.6/1, representing a volume of .00278 mm. 3 ; the mean di- ameter of egg-cytoplasm just after fertilization is about 161 M, 6 These volumes are calculated by multiplying the cube of the radius by 4.18 in each case. The radius is found by dividing the mean projected greatest diameter of the egg (that given in the text) by 2 x no, no being the magnification. 58 CHARLES D. SNYDER. from which the volume must be about .00217 mm. 3 , representing a volume reduction of 21.9 per cent. The fertilization membrane on the other hand presented a diameter of 191/1 and therefore enclosed a volume of .00364 mm. 3 ; this represents an increase of volume capacity of 30.8 per cent. The above observations are summed up in the following sylla- bus for the purpose of ready comparison. It will be observed that the ratio of diameter and volume changes is roughly as 1 : 2.8, a purely geometrical ratio. SUMMARY. Percentage Dimensional Changes in Two Species of Echinoderm Eggs. Reduction in egg-cytoplasm: Of Echinarachnius eccentrica By hypertonic salt action in diameter, 12.6; in volume, 33-3- By insemination (not observed). Of Asterina miniata By citric acid treatment (not observed). By insemination- in diameter, 7.8; in volume, 22. Swelling of fertilization membrane: Of Echinarachnius eccentrica Parthenogenetic (hypertonic salt action) 7 in diameter, 22.8; in volume, about 62. Normally inseminated- in diameter 26; in volume, about 70. Of Asterina miniata Parthenogenetic (citric acid treatment) 4 in diameter 3.9; in volume, about n. Normally inseminated- in diameter 9.3; in volume, 31. No special technical procedure was resorted to, to make certain that the eggs in these experiments were always spherical. The writer cannot, therefore, be quite certain that the diameters given above are those of perfect spheres and accordingly that the volumes given are the exact volumes of the eggs observed. If, 1 The membrane is assumed to be present here and coextensive with the egg- cytoplasm. EGG-VOLUME AND FERTILIZATION MEMBRANE. 59 as has been pointed out by others, the eggs at one time may be disks flattened vertically (Reighard, Chambers 8 ) and at another time ellipsoids, or pear-shaped objects, suspended at one end of their long axes, and yet at another time perfect spheres, then there is an unrelated error among the observations that renders them worthless. In the cases here studied there exists only the possibility of the eggs being flattened vertically during the period before fertili- zation and then changing into spheres after the fertilization membranes are raised from the egg-cortex. This, however, affects only the reduction observed in the inseminated Asterina eggs. No one will doubt that the observed reduction in size of the sand-dollar eggs during the bath in hypertonic salt represents a real reduction in volume. If the effect of the osmosis is a gelation there may have been a hardening of the egg-cytoplasm, but this hardening of itself could not change the egg-mass from a spheroid to a sphere. If the effect of the osmosis is an increase in surface tension then such a change in form may well take place. The reduction in diameter of the inseminated Asterina eggs during the first stage of cleavage is of the same order of magnitude as was observed in the sand-dollar eggs in their hypertonic bath. While some of this reduction may have been due to reshaping, there also can be no doubt that some of it was due to loss of material on the part of the egg-cytoplasm. This material, as others have maintained, may be colloidal in part, but this obser- vation supports the view that considerable water is given off from the egg together with the colloid. It is remarkable that the percentage of swelling of the egg treated to the hypertonic salt bath, after return to normal sea- water, and the percentage swelling of the fertilization membrane of the inseminated egg should both be of the same order of magnitude. This swelling in neither case can be due to any considerable extent to a reshaping of egg-substance. However evident it is that the egg-cytoplasm of this parthenogenetic egg has undergone a change in permeability different from the egg- cytoplasm of the normally inseminated egg, it nevertheless appears that the limit to extension in the one case is the same as 60 CHARLES D. SNYDER. it is in the other. For since the beautiful demonstration of Chambers 8 there can be no doubt that the fertilization membrane is a preexistant structure. One may take the limit in the swelling, therefore, to be the degree of permeability and elasticity of the membranes in both cases. What has just been said of the eggs subjected to hypertonic solution appears also to be true of the eggs subjected to citric acid. For in this latter case the egg-cytoplasm also swells coextensively with the egg- membrane. It will be remembered that in these eggs the membrane could be seen bridging the furrows between the cells after cleavage, in this respect differing from the hyaline membrane that is observed to dip down and follow the furrow closely. 8 The limit to swelling in the artificially parthenogenetic Asterina egg thus also lies in the egg (fertilization) membrane. Only, the citric acid treatment seems to render the membrane somewhat less permeable to water or less elastic, or both less permeable and less elastic, than does the treatment with normal sperm. In conclusion it may be added that while the above observations contribute little or nothing of a decisive character to the problem, they nevertheless do add to the attractiveness of the space-time method. With the newer technique a thoroughly systematic application ought to yield results not only decisive but also important. THE JOHNS HOPKINS UNIVERSITY, December 18, 1924. 'Chambers. BIOL. BULL., Vol. XLL. pp. 318-350, 1921. THE EFFECTS OF DISLOCATION OF THE EYE UPON THE ORIENTATION AND EQUILIBRIUM OF THE GOLDFISH (CARASSIUS AURATUS). J. FRANK PEARCY AND THEODORE KOPPANYI. (From the Hull Physiological Laboratory of the University of Chicago). It has long been known that the eyeballs of various fishes show different movements during locomotion. It is also established that the body movements have an influence on the movements of the eyes. Lyon 1 has shown that the dogfish compensates rotation about a dorso ventral axis by moving its eyes in the opposite direction. "The eyes," states Lyon, "sometimes show the same motions when the animal moves voluntarily and normally." "A dogfish, for example, when swimming on its side may keep the upper eye to the ventral, the lower one to the dorsal side of the orbit. Compensatory motions are not, therefore, confined to passive rotation by external means." Lyon found that these compensatory motions of the eyeballs in fish are practically independent of the sense of light, for they cannot be abolished by blinding (transection of the optic nerves). He also found a causal relationship between the semicircular canals and these eye motions. The opposite question, whether the eye or visual impressions can influence the body orientation and movements in fish has also been investigated but only in reference to the positive and nega- tive heliotropism. Parker 2 has shown that in an unilluminated field dogfish will swim toward a single light, i.e., they are posi- tively phototropic. Thus the light has a stimulating effect on the progressive movements of the fish and Parker concludes that the retinal image is an important factor in guiding the locomotion of these fishes. Admitting the fact of positive phototaxis in fishes, there still remains the possibility that in diffused light visual impulses or the fields of vision as a whole may influence the orientation, despite 61 62 J. FRANK PEARCY AND THEODORE KOPPANYI. the fact as stated by Lee 3 that "the blinded fish swims normally in all respects." Numerous experiments by Loeb, Bethe, Bigelow, Lee, Parker, Maxwell, etc., have shown that the labyrinths are concerned with the equilibrium of the fish. The orientation of fish deprived of their labyrinths is very faulty or erratic. Comparing the striking effects on orientation of labyrinth extirpation with the absence of effects from blinding appears to indicate that the retinal image is not an important factor in equilibrium and orientation in diffused light. The usual method of studying the influence of the eye on orientation has been that of removal. The difference in the animal's behavior before and after blinding has been interpreted as being due to the removal of the visual function and therefore it is to be considered as representing the normal effect of vision upon orientation and locomotion. We attempted to approach the problem from a different angle. We tried to investigate the role of vision in orientation not by removal but by dislocation of the eye, hoping that while the dislocated eye would still perform its visual function, the animal's equilibrium and orientation could be observed and the problem, whether or not the eyes influence orientation, put to a crucial test. EXPERIMENTAL. Large goldfish (Carassius auratus), eight and twelve inches in length, were chosen for the work because of their availability and especial suitability. 1 The skull of the goldfish is high, forming a large intracranial space over the brain which permitted the operative work to be done readily. The method chosen was that of dislocation of one eye and removing the other. Dislocation was performed by inserting the eye with its nerve and blood supply intact into an artificial orbit placed higher in the skull. This was accomplished in the following way: A hole about the size of the eye was drilled in the top of the head at a point in the vertical plane of the eyes just to the left of 1 We acknowledge with pleasure our thanks to Mr. Parker and Mr. Young of the Lincoln Park Zoological Gardens, who kindly supplied us with the fish used in this experiment. EFFECTS OF DISLOCATION OF EYE UPON GOLDFISH. 63 the midsaggital line. In the skull and underlying tissue between the left orbit and the drilled hole a narrow channel was cut (including the orbital wall) and thus a communication between the natural and artificial orbit produced. The eyeball was slid along the prepared channel and into the drilled hole, which then served as an artificial orbit. This dislocation can be done very easily and without any force. Neither the eye muscles, nor the nerves of the dislocated eyeball were cut or obviously injured and even the major part of the conjunctiva bulbi and sclerae can be left intact. The artificial orbit is able to hold the dislocated eye in place indefinitely. At the replacement the dislocated eye showed some healing in the new orbit. The experiment was successful on two animals. In the third fish a fungus infection on the dislocated eye caused death five days after the operation. To prevent such infections a daily po- tassium-permanganate (weak solution) bath of the entire fish is necessary. Immediately following the operation (the first one performed on May 15, 1924) no abnormalities in the animal's behavior were observed. After keeping them under observation for a few days the other eye w r as removed (May 20, 1924). We were able thus experimentally to produce real cyclopy. These cyclops in their first week behaved exactly like normal blind fish, they swam and oriented themselves quite as before operation. However, after about the tenth day (in our first protocol: June I, 1924), the animal was observed to tilt the body somewhat to the left. When at rest it assumed a position with its dorsoventral axis several degrees to the left of the vertical. This position was maintained during swimming. The tilting of the body toward the side in which the eye was dislocated increased day by day, reaching its maximum in about four weeks (in the case of our first animal: June 27, 1924). This maximum tilting was about 45 and was permanent so long as the eye remained in its new orbit. The gross anatomy of the dislocated eye was quite normal. The media were clear. We observed also some oscillatory move- ments of the dislocated eyes, when we took the fish out of water. 6 4 J. FRANK PEARCY AND THEODORE KOPPANYI. The vision of the fish with dislocated eyes was tested during the period of the last three weeks of the experiment and found to be very good. If a small rod was slowly moved toward the eye the animal quickly turned aside and avoided it constantly. 1 The animal showed in all respects the behavior of a fish in possession of its visual function it avoided all kinds of obstacles. The fishes did not show the phenomenon which Parker described concerning blind fish, i.e., they remain usually near the bottom and swim about in such a way as to be almost continually in contact with some solid surface, as though relying on its sense of touch for its location. FIG. i. Goldfish, with the dislocated eye. The other eye removed. i.When the'animal was near the surface it avoided the rod before it reached the water. EFFECTS OF DISLOCATION OF EYE UPON GOLDFISH. 65 On the 2yth of June the dislocated eye was removed from the artificial orbit and slid again along the channel into the original orbit. This operation is also a very easy procedure and can be done without obvious injury of the eye or of the animal. The animal, as most Anamnia, recovered very slowly from anesthesia and therefore we began our observations on the following day (June 28). The fish was in the normal position at rest and during locomotion. In the several weeks following, the orientation and locomotion were constantly normal, no tilted positions were observed. The other goldfish showed also two weeks after the operation the above described tilting and the tilting reached its maximum six weeks after the dislocation. The results obtained in our second experiment corroborate completely the observations on our first fish with dislocated eye, since in the second animal tilting and the return of vision occurred also synchronously. At no time during the experiment did the animal show any abnormalities other than the tilting. There were never any evidences of circus movements, etc., which may follow injuries of the midbrain, medulla and labyrinth (Steiner, 4 Loeb, 5 Bethe, 6 Bigelow, 7 ). DISCUSSION. "A normal fish has a delicate sense of the distance involved in swimming in a straight line. This is shown by the remarkable skill with which he avoids obstacles; in swimming around his aquarium constantly, he strikes his nose directly against the side of the tank comparatively rarely. This is not so with a fish deprived of all his otoliths or with all his macular nerves severed. Such fish seems to have little idea of the extent of a forward swim. He is often restless and frequently alters the direction of his progression" (Lee, 3 ). In his experiments Lee did not include the positive side of the role of vision in the orientation and equi- librium of the fish. Lee says, "When left to himself, the blinded fish swims normally in all respects, moving gracefully, easily, and without timidity, and shooting and diving like an uninjured fish." The first question which confronts us is, of course, the cause and mechanism of the tilting following the eye dislocation. There can be little doubt that the tilting is directly due to the dislocation 66 J. FRANK PEARCY AND THEODORE KOPPANYI. of the organ for light perception. We avoided injury to the brain and otic capsule and the immediate post-operative behavior of the fish proves that there were no lesions of those organs. Any continuous tilting of the fish is called a "forced position" by Loeb. Loeb discriminates in one of his earliest papers between two different types of forced responses: "forced movements and forced position." "Wir sprechen von Zwangsbewegungen, wenn die Tiere sich kontinuirlich oder sehr haufig in Bahnen bewegen, die von denen eines normalen Thieres unter den gleichen Um- standen in einem bestimmten einfachen Sinne abweichen." And on the other hand: "Wir bezeichnen als Zwangslagen die Abweichungen von der normalen Orientierung gegen den Schwer- punkt der Erde." According to Loeb the forced positions are due to geotropism. Moreover, he refers to the fishes as one of the clearest instances of such phenomena. The normal position of fishes in swimming or at rest is also, according to Loeb, a geotropic phenomenon. " Versuchen wir es einen solchen Fisch gewaltsam auf den Riicken zu legen, so widerstrebt er und bringt, so bald wir ihn wieder frei lassen, sich wieder in seine gewohnte Orientierung zuriick." Even the position of the eyes are influenced by the gravitation in the fish. "Bringen wir den Kopf eines Fisches gewaltsam in eine andere als die ihm zukommende Orientierung gegen die Schwerkraft der Erde, so gehen die Augen vollig oder theilweise in die alte Orientierung zuriick. . . . Das Licht hat mit diesen Erscheinungen nichts zu schaffen, sie treten auch, wie bekannt, im Dunkeln und bei vollig Erblindeten ein." (Loeb 5 ). As Loeb points out the orientation of the fish in relation to gravity takes place by means of the otolith apparatus, as first demonstrated by Mach and Breuer. Loeb cut (in Scyllium caniculd] the right VHIth nerve. He saw forced circus movements to the side of the lesion, forced tilting toward the side of the lesion, pleuro- thotonus, and compensatory positions of the eyeball and fins. Bethe does not deny the static function of the labyrinth, but he states that the geotropism of the Elasmobranches is not essentially changed by the removal of one labyrinth. He failed to observe in many animals the forced positions as after-effects of unilateral labyrinth -extirpation, but admits that forced move- ments and forced positions of the eyeballs and fins can often be EFFECTS OF DISLOCATION OF EYE UPON GOLDFISH. 67 seen in such animals. He says that the unbalanced tonus and innervation of the trunk, eye and fin muscles (Ewald) can explain satisfactorily these forced motions. But he admits that this tonus factor is also influenced by the geotropic function of the labyrinth. Although different authors describe somewhat differently the after-effects of labyrinth extirpation (this may be due to difference in technique, injury of other cranial organs, etc.), all seem to agree on the point that the geotropic function of the labyrinths is responsible for normal orientation and equilibrium. No other factor has been considered to play an important part in this behavior. Dogfish in which both labyrinths were removed as observed by Maxwell (8), could hardly be seen to differ from that of the normal fish except when greatly excited. The} 7 swim quietly around or settle on the bottom in normal position. Maxwell raised the question whether the orientation of labyrinth- less fish is due to retinal stimuli, but his experimental results showed to him that visual impulses did not play a role in the orientation of his operated fish. When he covered each eye with a large patch of heavy, black, rubber cloth, the animal swam about w r ith good orientation and never came to rest on the bottom in an abnormal position. This goes to show that in nature chronic or permanent tilting of the body of the fish occurs only from unilateral injury of the labyrinths and never from eye injuries. When both labyrinths are removed there remains no mechanism on which gravity can act in the way to induce orienta- tion, according to Loeb. In our fish the labyrinths were normal and the otoliths should have taken care of the normal position in swimming or at rest. Nevertheless the tilting of the body took place, as described. Evidently the influence of the labyrinth was modified by a new factor of vision. This factor is probably the influence of the visual field on the orientation mechanism. As long as the vision was impaired by the trauma of the oper- ation, no tilting of the body was noticed. The question is, whether this tilting is a phototropic reaction, a "forced" reaction or due to attempts of the animal to keep its usual visual field. Maxwell admits the possibility of such a mechanism, of course in a different type of experiment: "When the dogfish is rotated 68 J. FRANK PEARCY AND THEODORE KOPPANYI. around any one of these axes the eyes move as if to retain their original position in space, or to preserve the original visual field." We think that the tilting is due to so-called "voluntary" motor attempts of the fish to regain or retain the usual visual field. Heliotropic reactions involve different mechanism in fish than in the invertebrates, for Parker has shown that the dogfish, when only one optic nerve is cut, never moves in circles. Since in the tank, where the fish lived, there were diffused light conditions, there was no single source of light that could act as a phototropic stimulus. Interpretation of the tilting as a simple phototropic response seems therefore excluded. The tilting at 45 was the optimal condition for the animals to see the walls of the aquarium and most of the environment with which he can come in contact and our results indicate that the tilting is a quick reaction of the organism as a whole. 1 SUMMARY. 1. The dislocation of one eye into the top of the head and the removal of the other eye produce a tilting of the whole body with its dorsoventral axis 45 to the side of the vertical. The reposi- tion of the dislocated eye into its original orbit changes the orientation and equilibrium of the fish immediately, it regains its normal position in swimming and at rest. 2. The tilting is probably due to the attempts of the animal to keep its usual visual field. The writers wish to express their thanks to Professor A. J. Carlson for his constant help and encouragement. BIBLIOGRAPHY. 1. Lyon, E. P. Amer. Jour. Physiol., III., 86, 1900. 2. Parker, G. H. Bulletin of the Bureau of Fisheries, XXIX., 45, 1909. 3. Lee, F. S. Amer. Jour. Physiol., I., 128, 1898. 4. Steiner, T. Sitzungsberichte der K. preussischen Akademie d. Wiss., XXXII., 539. 1886. 5. Loeb, J. Pfliiger's Archiv, XLIX., 175, 1891. 5a. Loeb, J. Pfliiger's Archiv, L., 66, 1891. 6. Bethe, A. Pfliiger's Archiv, LXXVI., 470, 1899. 7. Bigelow, H. B. American Naturalist, XXXVIII., 275, 1904. 8. Maxwell, S. S. Labyrinth and Equilibrium. T. B. Lippincott Co., 1923. 1 If so, then the fish placed in the dark or by cutting its only optic nerve should regain its normal position. We contemplate to investigate this and related problems in further experiments. Vol. XLIX A llglist, IQ2 5 No. 2 BIOLOGICAL BULLETIN MORPHOLOGY AND MITOSIS IN TRICHOMONAS TERMOPSIDIS, AN INTESTINAL FLAGELLATE OF THE TERMITE, TERMOPSIS. 1 JUSTIN M. ANDREWS, INTRODUCTION. In recently published papers by Cleveland ('24 and '25) establishing the symbiotic relationship of termites and their intestinal protozoa, and the effects of starvation and oxygenation upon this association, he mentions that he has observed in the intestine of termites of the genus Termopsis, a flagellate possessing "four anterior flagella, axostyle, and undulating membrane" which he calls " Trichomonas termopsidis." The object of this paper is to describe the morphology and mitosis of this species as it occurs in the large Pacific Coast termites Termopsis nevadensis Hagen and T. an gus tic oil-is Hagen. MATERIAL. Sixteen colonies of Termopsis furnished the material for this study. Six of the colonies came from Oregon, and ten were from California. Winged forms appeared in two of the Oregon colonies, and were identified as T. nevadensis Hagen. Winged forms appeared in five of the California colonies which were identified as T. angusticollis Hagen. The insect material was identified by Dr. T. E. Snyder, Specialist in Forest Insects, Bureau of Entomology, U. S. Department of Agriculture, Washington, D. C. 1 From the department of Medical Zoology, School of Hygiene and Public Health, Johns Hopkins University. A portion of this work was done at the Bio- logical Laboratory, Cold Spring Harbor, Long Island, New York. 5 69 7O JUSTIN M. ANDREWS. ACKNOWLEDGMENTS. Many thanks are due Dr. R. W. Hegner for his valuable sug- gestions and criticisms. To Dr. L. R. Cleveland I am especially indebted for furnishing the material used, for suggestions as to methods, and for the oversight which he so generously gaVe. METHODS. In order to obtain preparations which contained more than the usual number of the flagellates per unit area, the termites were starved for eight days, at which time the two larger protozoa, Trichonympha campanula and Leidyopsis spliaerica, which had been previously present in large numbers, had entirely disap- peared (Cleveland '25). Then the termites were fed a pure cellulose diet (Whatman Filter Paper No. 43) which resulted in a much greater clarity of the intestinal fluid surrounding the flagellates and relieved the animals of many food inclusions (wood particles) that had previously obscured their internal morphology- Smears were made from the intestinal contents of termites treated as above. They were fixed in various fluids, Schaudinn's strong Flemming's, Gilson's, Bouin's, Zenker's, Carnoy's, osmic acid vapor, and chromic acid. These were stained, for the most part, with Heidenhain's iron alum hfemotoxylin ; a few were stained with Mallory's Tri-Stain. Others were dry fixed and stained with Wright's . stain. Sections of the intestines of termites both of individuals that had been partially defaunated as above, and of untreated hosts, were fixed in Schaudinn's fluid, Flemming's fluid, chromic acid, and osmic acid vapor. They were stained with iron haemotoxylin, and with Mallory's Tri- stain. The live animals were also studied under vaselined coverslips. MORPHOLOGY. Shape and Size of Body. Body shape in Trichomonas termopsidis is exceedingly variable' due to the absence of a restraining pellicle, and to the extreme lability of the cytoplasm. From a pyriform shape, it varies through a regular and elongated oval, to a spheroidal contour. The greatest width is usually near the middle of the animal, and varies, in proportion to the length, from I to 4.5, to I to i. MORPHOLOGY IX TRICHOMONAS TERMOPSIDIS. 7! The size is also very variable, owing perhaps, to division processes, the figures of which are exceedingly numerous. The majority, though by no means all, of the dividing forms are larger FIG. i. All figures of Trichomonas termo[>!>idis Cleveland, from material stained with iron alum haemotoxylin, drawn with the aid of camera lucida. Flagella are shown as they appeared in the preparation. A. Typical trophozoite in resting stage fixed in i per cent, chromic acid. X 2500. B. Multiple fission, of the type with the nuclei in a central position fixed in Carney's fluid. X 500. C. Multiple fission, of the type with nuclei at polar extremities fixed inCarnoy's fluid. X 500. than the resting individuals. Of one hundred measured as resting trophozoites, the length averaged 29.17 micra, and the width 14.49 micra. The extremes of length were 55.0 micra, and n.o micra, the majority of the individuals falling betw r een 25 and 35 micra. The extremes of width were 28.8 micra, and 6.78 micra, 72 JUSTIN M. ANDREWS. the majority falling between 12 and 20 micra. As these measure- ments were made from fixed, stained, individuals from partially defaunated hosts, the length measurement was checked by measuring one hundred live individuals from untreated termites. The average length in this series was 30.97 micra, indicating that the treatment has little, if any, effect on size. The above measurements, and those mentioned hereafter in the description of organelles, were all made from termites of three colonies which furnished most of the material used in the cyto- logical study. When other colonies (thirteen more) were investi- gated in order to observe the distribution of Trichomonas termopsidis, forms were encountered in twelve of the colonies that differed from those previously studied in size, which varied from 50 micra to 150 micra, most of the giant individuals lying between 70 micra and 90 micra in length. In all other morphological details, the two sizes of this Trichomonas are identical as far as we are able to observe. So, for the present, we are calling this a size race of Trichomonas termopsidis. It may be that further study will reveal specific differences rather than racial differences. CYTOPLASM. The cytoplasm, as stated above, is extremely labile; indeed it probably approaches fluidity. The shape is constantly changing, and the body assuming new proportions. Occasionly, in live specimens, one can see the animal slide some of the cytoplasm off the end of the axostyle in drops. The cytoplasm is kept in a state of agitation by the activity of the axostyle, which appears to function as an internal paddle, continually churning up the contents of the body. CYTOSTOME. The cytostome (Cytos., Fig. 2.) does not appear to be the comma-shaped vent usually pictured for the trichomonads, but extends as a spiral groove starting above the middle on the right- hand side of the animal, becoming more sharply indented as it proceeds ventrally, ending under the nucleus. Although an actual opening cannot be seen, the food particles, and fluid filled vacuoles are more numerous dorsal to the nucleus indicating the position of the opening. MORPHOLOGY IX TRICHOMONAS TERMOPSIDIS. 73 NUCLEUS. The nucleus is an ellipsoidal mass enveloped by a membrane which is sometimes closely applied to the darkly staining nucleo- plasm, but is more frequently found surrounding it a short Und. mem Ant f/g .Chr. has. Cerrtfobl Jt/iizo. /Vac. mem. _/7*o. chrom r Chrom. fil. chrom. Pxo. _toc. riarcj. fil. Post. fla N^ LjJ ^' ^ ^^^F FIG. 2. Diagrammatic representation of Trichomonas termopsidis. Abbreviations: Und. mem., undulating membrane; Ant. flag., anterior flagella; Chr. has. r., chromatic basal rod; Centrob., centroblepharoplast; Rhizo., rhizoplast; Nuc. mem., nuclear membrane; Cytos., cytostome; Axo. chrom., axostylar chro- midia; Par. b., parabasal body; Chrom. fil., chromatic filament; Cytop. chrom., cytoplasmic chromidia; Axo., axostyle; Vac., vacuole; Marg. fil., marginal filament; Post, flag., posterior flagellum. X 3000. distance away, leaving a clear, unstaining zone between the membrane and the nucleus (Fig. 4, B and C). Sometimes this zone is not regularly parallel to the contour of the nucleus, but in all such cases observed, the irregularity seems to be due to de- pressions or elevations in the outline of the nuclear mass, rather than to a collapse of the membrane (Fig. 4, A and D). The length of the resting nucleus is ordinarily about twice its width. It is always situated anterior to the middle of the body, except in dividing forms, where proportions are upset both in the body of 74 JUSTIN M. ANDREWS. the individual and in the nucleus. Its length varies from 5 to 10 micra. It stains a flat black unless the preparation is strongly decolorized, in which case it shows a granular composition, within which appears a karyosome-like unit contained in a clear unstaining vesicle (Fig. 4, A). In the resting stage, the nuclear membrane is connected to the centroblepharoplast by a single rhizoplast. NEUROMOTOR APPARATUS. In their paper dealing with Trichomitus termitidis, Kofoid and Swezy ('19) list the organelles composing this "complex, structur- ally integrated apparatus which links together the nucleus and motor organs" as: a centroblepharoplast, the anterior flagella, the undulating membrane, a posterior flagellum, a "parabasal body," and a nuclear rhizoplast. To that category, we would add, in describing Trichomonas termopsidis, an axostyle, and would call the organelle referred to as the parabasal body by Kofoid and Swezy, the chromatic basal rod, in view of the fact that a body first called the parabasal body by Janicki ('n), and later corroborated by Alexieff ('13), Kuczynski ('14), Janicki ('15), Cutler ('19), and Wenrich ('21), is plainly demonstrable in our preparations fixed in strong Flemming's fluid, osmic acid vapor, or chromic acid. The centroblepharoplast (Centra., Fig. 2.), apparently a single morphological unit while the trophozoite is in its resting stage, appears as a small, round, blackly-staining dot about a micron in diameter. It is closely applied to or embedded within the anterior end of the chromatic basal rod, so that except during division processes, its position is marked only by a bulbous enlargement of the extremity of the chromatic basal rod. The centroblepharoplast is always connected to the nucleus by one or more nuclear rhizoplasts. From the centroblepharoplast spring the four anterior flagella, the undulating membrane, the chro- matic basal rod, the axostyle, and the parabasal body. The four anterior flagella (Ant. flag., Fig. 2) are about equal in length, arise from the centroblepharoplast, and pass discretely out of the cytoplasm. At the point of emergence, the cytoplasm is usually raised to a small hillock. As the flagella leave the body, they are frequently so intertwisted and woven together, that it MORPHOLOGY IN TRICHOMONAS TERMOPSIDIS. 75 looks as if a single large flagellum proceeded from the body, and, at a short distance, became divided into four. This illusion seems particularly real as one observes the live specimens, but upon careful examination of the stained preparation, it is obvious that the flagella are not only separate outside the cytoplasm, but within it also. The anterior flagella are about three-quarters the length of the body. Specimens stained by Wright's method show the flagella very clearly, and it was not until this technique was employed that the number of flagella was unquestionably established. The undulating membrane, (Und. mem., Fig. 2) is a thin, rippling film, apparently composed of a fold of the pellicular covering, attached, at its outer edge, to a posteriorly directed flagellum, which, like the anterior flagella, originates in the centroblepharo- plast. The flagellum separates from the membrane at the pos- terior end of the body, as an inconspicuous trailing flagellum, which is apparently not as well developed as the anterior flagella. The membrane is hyaline, and persists after treatment which dissolves away the cytoplasm of the animal. It is best developed at the anterior end, and becomes less developed and effective tow r ards its posterior end. In preparations stained by Wright's method, the undulating membrane is differentiated into three distinct zones a distal, narrow, pink-staining line, which is probably the flagellum, then a pellucid, unstained zone approxi- mately half the remaining width of the membrane, and lastly a heavily staining (pink) zone which extends to the attachment (Fig. 3, Fig. 6). A similar differentiation was observed in several specimens stained with iron alum haemotoxylin that were more critically decolorized (Fig. 3, D}. The chromatic basal rod (Chr. has. r., Fig. 2) extends the length of the body, directly beneath the undulating membrane, in a curve that resembles either a capital "C" or "S." It takes its origin from the centroblepharoplast, and like that organelle, stains intensely black. It retains its stain, ordinarily, 'after the nucleus has become quite decolorized. Whether it functions as a source of energy for the motor activities of the undulating membrane (Kofoid and Swezy, '15) or serves as a skeletal support for that organelle, the fact is that the membrane seems to be JUSTIN M. ANDREWS. B MORPHOLOGY IX TRICHOMONAS TERMOPSIDIS. 77 attached directly to the chromatic basal rod. This observation is strikingly demonstrated in those cases where the cytoplasm of the animal has been destroyed, and the entire neuromotor system with, or without the nucleus, remains intact. In the majority of such cases, though not in every one, the undulating membrane remains adjacent to the chromatic basal rod suggesting that there may be some connection between them which is strong enough to withstand the treatment that destroys the cytoplasm, but which cannot, in a few cases, endure the chance violences of the smearing operation. The axostyle (Axo., Fig. 2) in Trichomonas termopsidis is one of its most conspicuous features when the animal is studied in the living state. As the flagellate moves about, turning and twisting, one of the first things to be noted is the projecting trunk of the axostyle. Occasionly it sticks out of the body for a distance as long as the length of the body, but more frequently it barely protrudes through the cytoplasm, the length of the extruded portion being varied both by changing the actual linear expanse of the organelle which is accomplished by bending it and curving it within the body but more often by changing the shape of the body, so that more of the axostyle is included at one time than at another. When the stained preparation is observed, the axostyle is not such a prominent feature. It does not stain well at all. Fre- quently it does not project beyond the body line, and in such cases it is easily overlooked. Certain fixing fluids show the FIG. 3. All figures of Trichomonas termopsidis sp. nov., from material stained with iron alum haemotoxylin, drawn with the aid of camera lucida. Flagella are shown as they appeared in the preparation. A. Trophozoite in early prophase blepharoplast just divided, and flagella sepa- lating into two bundles. Fixed in osmic acid vapor. X 2750. B. Prophase, showing new chromatic basal rod and undulating membrane growing out from daughter blepharoplast. Fixed in Schaudinn's fluid. X 1200. C. Later prophase, showing parabasal body split almost entire length. Fixed in i per cent, chromic acid. X 500. D. Late telophase, all structures duplicated, and animals ready to separate. Note flattened, twisted appearance of parabasal bodies also the pellucid margin of the undulating membrane. Fixed in I per cent, chromic acid. X 1000. E. Slightly earlier stage of telophase nuclei still connected by paradesmose. Fixed in Flemming's strong solution. X 1300. F . Isolated neuromotor system, dry fixed, and stained with Wright's stain. X 1150. 78 JUSTIN M. ANDREWS. axostyle, after the preparation is stained, much better than others. It is much more apparent in individuals fixed in osmic- acid vapor, chromic acid, and strong Flemming's fluid, than in Schaudinn's fluid, Carnoy's fluid, and Gilson's fluid. It shows very well in specimens stained with Wright's stain. At its anterior end, where it passes under the nucleus, it expands into a capitulum at least twice its lower width, and then tapers rapidly to a point where it is connected to the centroblepharoplast. Passing from its enlarged portion, the axostyle almost invariably takes a bend of at least 90 degrees. The particles of cytoplasm surrounding the organelle at this point stain more deeply than the rest of the proximal cytoplasm (Fig. I, A). The axostyle does not seem to be a cylindrical rod. It is flat, and, at its posterior end, it is sharply pointed. It has been observed in some prepa- rations to be folded back on itself after it has pierced the body. Frequently the trunk is not parallel for its entire length but gradually enlarges from its most narrow portion directly posterior to the capitulum to its maximum width near the posterior end (Fig. i, A). Then it tapers at once to an acute point. In some other cases, the width of the trunk is the same throughout its length. The granules of the axostyle axostylar chromidia (Axo. chrom., Fig. 2) stain intensely, and it is by their aid that the axostyle is recognized more frequently than by its own outline. They are sometimes arranged in a straight line (Fig. 3, A), occasionly they are arranged in spiral rows, (Fig. I, A), and not infrequently one finds them scattered throughout the organelle in an apparently hit-or-miss fashion. i The literature concerning the parabasal body (Par. b., Fig. 2) is so well reviewed in previous papers (Kofoid and Swezy, '15, Cutler, '19) that it is sufficient to state in this paper that Janicki ('n, '15), Alexieff ('13), Kuczynski ('14), Cutler ('19), and Wenrich ('21) have all reported the presence, in various tricho- monads, of darkly-staining, club-shaped organelles in addition to the elongated, slender, chromatoidal rod that lies under the undulating membrane. Kofoid and Swezy ('15) are inclined to regard this as the first step in the origin of the new chromatic basal rod in the prophase of fission. The figures of the developing .MORPHOLOGY IN TRICHOMONAS TERMOPSIDIS. 79 FIG. 4. These figures (Fig. 4 and Fig. 5.) are drawn to show the succession of nuclear changes during mitosis. The chromatic basal rod, undulating membrane, and anterior flagella are also indicated in order to orient the specimen. All material fixed in Schaudinn's fluid, drawn with the aid of camera lucida, and reproduced at a magnification of 2000. A. Resting nucleus, showing karysome. B. Chromatin beginning to condense and break up into irregular fragments that tend to arrange themselves linearly. Chromidia appearing in the cytoplasm. C. Linear arrangement of chromatin more pronounced, centroblepharoplast has released centrosome, which has just divided, as has the blepharoplast and rhizoplast. New chromatic basal rod appearing, accompanied by thin undulating membrane. Cytoplasmic chromidia disappearing. D. Flagella, chromatic basal rod, and undulating membrane completely dupli- cated; paradesmose has descended to nuclear membrane. E. Chromosomes strung over the subparadesmosal fiber judging from the complex appearance and probable number, they have already divided. 8O JUSTIN M. ANDREWS. chromatic basal rod published by Kofoid and Swezy ('15), pre- sumably in support of this view, differ from figures published by the other authors purporting to show parabasal bodies, both in size, shape, and spacial relationships. The parabasal body is an elusive organelle, and is apparently dissolved out by some fixatives, leaving nothing to suggest its shape or position. It is very sensitive to decolorization, and is seldom seen in those preparations which are decolorized suf- ficiently to permit analysis of mitotic phenomena. But in prepa- rations fixed with chromic acid, osmic acid vapor, or Flemming's fluid and perhaps others that are not too strongly decolorized, the parabasal body is a very conspicuous feature of Trichomonas termopsidis. If the parabasal is not carefully decolorized, it appears as a stout, club-shaped, serpentine organelle which takes its origin from the centroblepharoplast. After more critical destaining, a darker line appears, that is not always in the center, but which weaves from side to side, suggesting the probability that it does not lie within the parabasal body as is indicated by the previous writers, but is on the outside of it. This line the chromatic filament (Chrom. fil. Fig. 2) is undoubtedly the "parabasal thread" of Janicki. The body appears to have little rigidity of its own, as it sometimes loops itself back and forth on the axostyle (Fig. I, A), and occasionly surrounds it. The parabasal is about the same length as the axostyle, and about the same breadth, but is otherwise readily distinguishable from it. MITOSIS. Mitotic phenomena in Trichomonas termopsidis are strikingly different from any of the types described by Kofoid and Swezy ('15) for Trichomonas, but are identical with the type described for Trichomitus termitidis Kofoid and Swezy ('19). Briefly these are as follows: the nucleus darkens, and shortly thereafter the surrounding cytoplasm becomes charged with chromidia (Fig. 4, B] which soon disappear. Then the centroblepharoplast releases the centrosome (Fig. 3, A], and the remaining blepharoplast splits into two daughter blepharoplasts. Simultaneously the nuclear rhizoplast and the parabasal body split (Fig. 3, C). The centrosome maintains its connection with the two new blepharo- MORPHOLOGY IN TRICHOMONAS TERMOPSIDIS. 81 plasts by means of rhizoplasts (Fig. 4, C). As the blepharoplasts become separated, one remains with the old chromatic basal rod, retaining two of the original flagella, the old undulating mem- brane, and one of the nuclear rhizoplasts. It completes its locomotor apparatus by growing out two new anterior flagella. The other blepharoplast takes two anterior flagella and the other nuclear rhizoplast, but immediately starts to equip itself with two new anterior flagella, and a new chromatic basal rod, comple- mented by a new undulating membrane (Fig. 4, C and Fig. 3, B). C FIG. 5. A. Chromosomes have straightened themselves out and are seeking the polar extremities of the paradesmose they are still on the subparadesmosal fiber. B. Chromosomes beginning to congregate at the polar ends of the paradesmose. C. Chromosomes grouped in rosettes at ends of paradesmose, and beginning to pull apart. D. Chromosomes still in rosette formation, but widely separated, and ready to reorganize. JUSTIN M. ANDREWS. In the meanwhile the centrosome has split, and the two portions migrated apart, spinning out between them an intensely staining bar, the paradesmose. By the elongation of the rhizoplasts con- necting the centrosomes and blepharoplasts, the paradesmose descends to the nuclear membrane. As it approaches the mem- brane, the chromatin within begins to arrange itself in linear fashion perpendicular to the paradesmose, and shortly after, the paradesmose comes in contact with the membrane, and is greeted by a projection of chromatin that is thrust up from the central mass. At this time, the subparadesmosal fiber appears (Fig. 4, D) upon which the chromosomes seem to arrange themselves shortly after (Fig. 4, ). Probably the chromosomes divide at this time while they are in this position, for the next figures we are able to find show them being gradually drawn to the ends of the paradesmose, without any indication of spindle formation. The number of chromosomes is difficult to determine, but is somewhere around ten. . The chromosomes form irregular clusters or rosettes at the ends of the paradesmose, which always seems to maintain itself as a fairly straight line (Fig. 5, C). The two masses of chromosomes continue to migrate apart during anaphase (Fig. 5, D] and the paradesmose persists. In telophase, the nuclei assume their characteristic ellipsoidal shape, surrounded by hyaline zone and nuclear membrane, and the paradesmose gradually fades away. The two animals now separate by a plasmotomy, which is materially aided by the activity of the locomotor apparati of the two individuals. The division of the axostyle we have not been able to observe- but from its capricious staining reactions during prophase, it seems probable that it may be absorbed, and new ones grown out from the blepharoplasts. By telophase, the two axostyles are complete. Multiple Fission. Multiple fission seems to occur rarely in Trichomonas termop- sidis. In the material worked on, fifteen or twenty cases were observed, but they were all on one slide that is, from the in- testinal contents of one termite. There were two types of multiple fission, the one where the nuclei were in a polar position (Fig. i, C), and the other where the nuclei were in a central MORPHOLOGY IX TRICHOMONAS TERMOPSIDIS. 83 position (Fig. I, B). These two types occurred with approxi- mately equal frequency. In no case was the somatella composed of more than four prospective individuals, and was always found in the prophase of the second division. These somatellae were much larger than might be suspected. The greatest and least dimensions of ten of them (nuclei in central position) averaged 80.47 rnicra and 48.79 micra respectively. The extremes of the greatest dimensions were 1 1 1.80 micra and 62.68 micra, and of the least dimensions, 66.91 and 30.49 micra. It is quite possible that these multiple fission figures are related to the giant race of Trichomonas noted above that was found in some of the termites examined. Whether multiple fission represents a definite stage in the life cycle of Trichomonas termopsidis, or whether it is simply a chance happening stimulated by some particular complex of circum- stances, we do not know. It is significant, perhaps in favor of the latter hypothesis, that multiple fission was found only in one termite. CYSTS. Nothing was found on any preparations which could be identi- fied with certainty as being the cysts of Trichomonas termopsidis. The protozoon is probably transferred from host to host in its vegetative phases. RELATIONSHIPS. This flagellate has been assigned to the genus Trichomonas on purely morphological grounds that is, because of its possession of a cytostome, four anterior flagella, an undulating membrane, a chromatic basal rod, an axostyle, a parabasal body, and a centroblepharoplast. Its nearest relatives appear to be Trichomonas trypanoides Duboscq and Grasse, and Trichomitus termitidis Kofoid and Swezy. It is difficult to state all the points of difference of T. termopsidis from T. trypanoides because of the scantiness of the description of the latter (Duboscq et Grassi '24.). But it is certain that there is a difference in size "Les T. Trypanoides de courbure normale ont une taille assez constante de 16 micra." It is also found in a host, Reticulitermes lucifugus, which belongs to a different family (Rhinotermi tides) from that of Termopsis (Kalo- 84 JUSTIN M. ANDREWS. termitida). And finally, in T. trypanoides, there is a notable lack of constancy in the number of anterior flagella, which vary from one to four. Trichomonas termopsidis differs from Trichomitus termitidis primarily by the possession of an axostyle. Trichomitus termitidis is described from Termopsis angusticollis? (Kofoid and Swezy, '19). In our material, we are positive of five colonies of the sixteen studied as being Termopsis angusticollis, but it is very probable that of the nine remaining unidentified colonies (two were identified as Termopsis nevadensis] some are Termopsis angusticollis, as the distribution and frequency of occurence of these two species is the same in Oregon and California (Banks and Snyder, '20). In mitosis, Trichomonas termopsidis is identical with Trichomitus termitidis, which differs, as far as we are aware, from every other form of trichomonad division described. Inas- much as both forms are found in the same hosts, and as a size race of Trichomonas termopsidis agrees in measurements with those given for Trichomitus termitidis, and more particularly because the same peculiar type of phenomenon takes place at mitosis in both forms, which has not been described for any other forms, it would appear that Trichomitus termitidis should be suitably con- firmed before its validity is established. As Trichomitus termitidis differed from its type species, T. parvus Swezy ('15) in having the type of division where the centrosome is separated from the blepharoplast during the process, whereas division in the type species was of the kind described in Trichomonas and Eutrichomastix by Kofoid and Swezy ('15), the species of Trichomitus found in the termite was placed in a new subgenus, Trichomitopsis. Then, since Tri- chomonas termopsidis differs from the other trichomonads previ- ously described, in the same manner, it is proposed to assign this flagellate to Trichomonopsis subgen. nov. Trichomonas with centrosome separated from blepharoplast at mitosis. Type Species, Trichomonas termopsidis Cleveland, from Termopsis nevadensis Hagen, and Termopsis augusticollis Hagen. BIBLIOGRAPHY. Alexieff, A. '13 Systematisation de la mitose dite " primitive." Arch. f. Protist., 29, 344- 363. MORPHOLOGY IN TRICHOMONAS TERMOPSIDIS. 85 Banks, N., and Snyder, T. E. '20 A Revision of the Nearctic Termites. U. S. National Museum, Bui. 108. Cleveland, L. R. '24 The Physiological and Symbiotic Relationships Between the Intestinal Protozoa of Termites and their Host, with Special Reference to Re- ticulitermes flavipes Kollar. BIOL. BULL., 46, 177-226. '25 The Effects of Oxygenation and Starvation on the Symbiosis Between the Termite, Termopsis, and its Intestinal Flagellates. BIOL. BULL., 48, 309- 326. Cutler, D. W. '19 Observations on the Protozoa Parasitic in the Hind-Gut of Archotermopsis wroughloni Desn. Part I. Ditrichomonas (Trichomonas) termitis Imms. Quart. Jour. Mic. Soc., Vol. 63, 555-589. Duboscq, O, et Grasse, P. '24 Notes sur les Protistes Parasites des Termites de France. I. Trichomonas trypanoides, n. sp. Comptes rendu des seances de la Societe de Biologic, 90, 547-551- Janicki, C. 'n Die Parabasalapparat bei parasitischen Flagellaten. Biol. Cent., 31, 321- 330. '15 Untersuchungen an parasitischen Flagellaten. II. Teil. Die Gattungen Descovina, Parajoenia, Stephanonympha, Calonympha. Zeit. f. wiss. Zool. 112. 573-689. Kofoid, C. A., and Swezy, O. '15 Mitosis and Multiple Fission in Trichomonad Flagellates. Pro. Am. Acad. Arts Sci., 51, 289-379. "19 Studies on the Parasites of the Termites. II. On Trichomitus termitidis, a Polymastigote Flagellate with a Highly Developed Neuromotor System. U. of Cal. Pub. in Zool., 20, 21-41. Swezy, O. '15 On a New Trichomonad Flagellate, Trichomilus parvus, from the Intestine of Amphibians. U. of Cal. Pub. Zool., 16, 89-95. Wenrich, D. H. '21 The Structure and Division of Trichomonas muris (Hartmann). Jour. Morph., 36, 119-156. RECENT CONTRIBUTIONS TO THE KNOWLEDGE OF THE CRYSTALLINE STYLE OF LAMELLIBRANCHS. THURLOW C. NELSON. (From the Zoological Laboratory of Rtitgers University.) The crystalline style of pelecypod and of gastropod molluscs has long been a fascinating subject for investigation by students of these groups. In a paper (Nelson, '18) published six years ago the attempt was made to summarize and to analyse the more important contributions which had appeared up to that time, and by original observation to solve some of the problems regarding the origin, nature, and function of this structure. The con- clusions of Coupin, 'oo, Mitra, '01, and others, that the crystalline style contains strong amylolytic ferments, were confirmed. In addition the style was shown to have at least two other functions. The first and more important of these is the role played by the style in the separation of food materials from sand and other waste. The powerful cilia of the style sac spin the style around on its long axis while pushing it anteriorly into the stomach. The head of the rotating style gathers the food strings coming from the oesophagus and as the whole mass is whirled around in the stomach much of the sand and other non-digestible matter is sorted out mechanically by the ciliary tracts of the stomach wall, and passed on into the intestine. At the point where the head of the revolving style comes into contact with the anterior wall of the stomach there is developed a tough resistant covering which I named the "gastric shield" (fleche tricuspide of Poli, 1791). The importance of such a sorting and stirring mechanism in animals in which ciliary activity has completely replaced muscular peristalsis, was pointed out. In forms such as most of our common bivalves, where the style sac is incompletely separated from the intestine by two ridges or typhlosoles, an additional function is served. Food particles escaping from the stomach may be passed across the faces of the typhlosoles from the intestine and then be incorporated into the style. Most of such returned material enters the style sac near 86 CRYSTALLINE STYLE OF LAMELLIBRANCHS. 87 its base and is built into the soft core of the style. I have found oysters in which the entire core of the style was almost a solid brown mass of Glenodiniiim. In this position food materials at first rejected from the stomach in the separation of inert ma- terials may be recovered as the style is moved gradually forward into the stomach. In the interval since this paper, Nelson, /. c., was published there have appeared several noteworthy contributions to the knowledge of the physiology of digestion in bivalve molluscs, and of the function of the crystalline style. It is well therefore to review these at this time and to give such additional information as has been accumulated during the past seven years in order that the present status of the problem of the crystalline style may readily be ascertained. Edmondson ('20), in a detailed and very well illustrated paper, describes the reformation of the style of Mya arenaria following its extraction. Among the species of bivalves which occur on the northwest coast this investigator found in Cardium corbis, Saxidomus giganteus, S. mittallii, and Paphya staminea, that starvation or removal from the water resulted in dissolution of the style "within a few days at most." In other species, notably Siliqna patula, Schizothcerus nuttallii, Macoma nasuta, the style was found to be far more resistant to dissolution, being present even at death through starvation. In Mya only slight disso- lution of the style occurred even after 14 days out of water. (Contrast this with the oyster, in which dissolution of the style occurs while the molluscs are exposed during the low tide; Nelson, '18.) As Edmondson points out,' the forms with resistant styles possess a style sac nearly or completely separated from the intestine. Experiments were performed with Mya in which the mantle was cut in the midline along the ventral surface for a distance of 15 to 25 mm. posterior to the pedal opening. A transverse cut was made near the middle of the style sac and the style extracted. The clams were then planted out and the rate of regeneration of the style studied. No food was taken apparently until the style was sufficiently regenerated to project into the stomach, from which Edmondson concludes that ingestion and digestion of food THURLOW C. NELSON. depend upon the degree of development of the crystalline style. A period of approximately 74 days were required for complete reformation of the style following the extraction. This part of Edmondson's work and the conclusions he draws from it are open to some objection. As the experiments were carried out, there is no way of distinguishing the time actually required for style regeneration from the period of inactivity resulting from the effects of the operation. Out of 147 clams operated upon of which the observation period was in excess of three weeks, only 61, or approximately 42 per cent, survived the operation. Any operative procedure which results in the death of over one half of the animals should be carefully checked before conclusions are drawn from the results, and it seems that such checks were not made. The cut through the mantle alone must seriously have interfered with feeding. Bivalve molluscs are very sensitive to injury and will "sulk" without feeding for long periods after even slight disturbances. A much better method of determining the period required for regeneration of the crystalline style would seem to be to determine the rate at which this structure is pushed forward into the stomach and dissolved during the normal feeding of the mollusc. This may be determined readily in forms which like the oyster and fresh-water mussels have a style sac incompletely separated from *, > FIG. i. The crystalline style of Lampsilis luleola taken from an individual which had been allowed to siphon in water containing a suspension of fine carmine grains. The spiral bands are composed of carmine which was caught upon the gills, carried into the stomach, thence to the intestine from which it was fed across the typhlosoles at two points near the anterior end of the style sac and incorporated into the style. CRYSTALLINE STYLE OF LAMELLIBRANCHS. 89 the intestine. A weak suspension of finely divided carmine if added to a water rich in food materials will usually be ingested along with the food and may be incorporated into the style. In some instances the carmine is passed across the typhlosoles into the base of the style sac and results in a red style. In others, the carmine may be fed into the style sac at some point between the stomach and the base of the sac, in which case the carmine forms a spiral band about the style as it is rotated and pushed anteriorly, giving the effect of a barber's pole (Fig. i). Allen ('21) records a similar observation in a fresh-water mussel where minute green organisms took the place of the carmine. 1 Complete regeneration of the crystalline style in Ostrea, Modiolns, Anodonta, and Lampsilis will take place at summer temperatures in from approximately 15 minutes in the oyster to a few hours in the other genera. Allen ('21) finds style regener- ation in fresh-water mussels in about 24 hours. It is difficult to believe that a process which occurs in at the most a few hours in many of our common bivalves should require two and a half months in an active rapidly growing mollusc such as Mya. Although I have had no experience with this form, it would seem possible to inject by means of a hypodermic syringe a small amount of carmine in sea water near the base of the style sac and to determine the speed at which this pigment, incorporated into the style, is carried toward the stomach. If carefully performed such an operation should be far less drastic in its effects than was the technique employed by Edmondson. In their admirable work on the natural history and propagation of fresh-water mussels Coker and his associates ('21) accept the conclusions of my 1918 paper, but contribute no original obser- vations regarding the function of the crystalline style. Included in the former paper, pp. 88-91, are the observations of Dr. Franz 1 While this paper was in press I received a copy of the report of Dr. J. H. Orton '24 on the causes of unusual mortality among oysters on English oyster beds; Ministry of Agriculture and Fisheries, London. On page 54 of this report is figured the crystalline style of Ostrea edulis, bearing a spiral band of food organisms. In a footnote on the same page this author suggests that one function of the style is "the mechanical one of drawing the mucous strings enveloping the food material into the stomach by twisting the strings around the shredded revolving head of the style like a capstan." In my 1918 paper, pg. 101, it is observed that "so strong is the tractive force of the rotating style that strings of mucus from any part of the body if led to the stomach cavity, are at once drawn in and wound up in the food mass." 9O THURLOW C. NELSON. Schrader on the food of mussels. This investigator found that only about one half of the diatoms and green algae taken in were digested, and he concluded that these organisms play a com- paratively unimportant role in the food supply of mussels. He apparently was unaware that in the process of sorting over of the acquired food materials by the ciliary tracts of the stomach, aided by the rotation of the crystalline style, many food organisms are shunted off into the intestine along with the sand and dirt. Especially may this be true of relatively heavy forms such as the diatoms. Some of these escaped food organisms may be passed across the typhlosole into the style sac and thus eventually be returned to the stomach, but a considerable number escape undigested from the anus. Blegvad '15 likewise draws false deductions as to the "insignificant" role played by plankton organisms in nutrition, from the finding of living planktonts in the faeces of the European oyster. While at Madison, Wisconsin, I kept Anodonta and Lampsilis for months at a time in clear running water in which the chief food supply consisted of desmids, diatoms, and nannoplankton forms most of which were growing upon the sides of the tank and upon the shells of the mussels themselves (Allen, '14). Examination of the intestinal contents revealed many living organisms but also the empty tests of numerous diatoms which had been digested. Imperfect as may be the mode of separation of food from dirt by the ciliary mechanisms within the alimentary canal of bivalves, the wonder is that it functions as efficiently as it does. Such living organisms as are cast out in the faeces are not altogether lost since they accumulate on the shells or upon nearby objects, where they multiply rapidly within the faecal remains and form a rich growth which is continually contributing its quota of food to the siphons of the mollusc (Allen, '14; Martin, '23). Allen ('21) gives a detailed account of experiments on the effects of various food organisms in the formation of the crystal- line style of fresh-water mussels. The details of this work cannot be discussed here, but in general they confirm and extend the findings of his preliminary paper (Allen, '14) and my own con- clusions (Nelson, '18) regarding the role of food in style regener- ation. He further showed that nannoplankton is relatively more effective than is net plankton in effecting style formation. CRYSTALLINE STYLE OF LAMELLIBRANCHS. QI The only essential difference between Allen's results and mine is that in fresh-water mussels he found that relatively little escaped food was fed back into the stomach by way of the style, and he concluded that this reclaiming function of the style was of relatively little importance in these animals. The largest amounts of such material were found in the core of the style soon after regeneration of a new style had begun, an observation which I can confirm in Anodonta grandis. The relative efficiency of this retrieving mechanism in various bivalves will have to be ex- tensively studied before we shall be in a position to determine its importance to molluscs generally. The fact that Mya, Teredo, Martesia and many other genera with a style sac nearly or quite separated from the intestine are able to exist without such a mechanism indicates that on the whole it plays perhaps a minor role in nutrition. Phylogenetically it probably represents the development of a strong recurrent tract of cilia situated in the posterior part of the stomach of the ancestors of present-day types; such a group of cilia as may, for example, be demonstrated in the stomach of the larval oyster. Allen (/. c.) lays still further emphasis upon his conclusion of 1914, voiced likewise in my 1918 paper, that the crystalline style arises in response to the presence of food in the stomach. Evi- dence that such may not be the case is discussed further on in connection with the findings of Berkeley ('23). Nogouchi '21 examined the crystalline styles of various marine bivalves and gastropods for Cristispira, a large active spirochaete which was first discovered in the style of the oyster. The spirochaetes were found most frequently in Ostrea, next in Mya, then in Modiolus; but not at all in Venus, Ensis, Mactra, Mytilus, Pecten, Fulgur, and Nassa. Gross, however, has reported Cristispira pectenis from the crystalline style of Pecten. Nogouchi did not know of my 1918 paper in which is discussed briefly the occurrence of Cristispira in the styles of certain bivalves and its absence from others. Nogouchi observed that the style of the oyster quickly liquefied after extraction from the body, and that only oysters freshly removed from natural conditions contained this structure. He believes that the great abundance of C. balbiani in the oyster is due to the relatively soft consistency of 92 THURLOW C. NELSON. the style in this mollusc, and that the absence of spirochsetes in many molluscs is due to the very firm and resistant styles which they possess. Although the solidity and resistance to dissolution of the style may effect the distribution of Cristispira, they are certainly not the most important factors. As Nogouchi himself admits, and as Edmondson showed, the style of My a is very firm and relatively resistant to dissolution, yet it harbors Cristispira in an abundance second only to that of Ostrea. Martin ('23), studying the relative importance of the net plankton and of nannoplankton in the food of the oyster, found that water from which even the smallest nannoplankton organ- isms including bacteria had been removed would, if well aerated, permit reformation of the style in Ostrea. He concluded that although the appearance of the style in this mollusc is usually correlated with the taking in of food, this structure may arise in the complete absence of food, presumably as a response to the act of siphoning. 1 Three months after the publication of Martin's paper appeared an interesting communication from Berkeley ('23) regarding the function of the crystalline style as a possible factor in the anaerobic respiration of certain marine molluscs. This investi- gator attempts to account for the continued production of carbon dioxide by marine molluscs kept under anaerobic conditions, as demonstrated by Collip ('21). In seeking a possible explanation of this production of carbon dioxide Berkeley tested the reactions of various tissues and of the style of Saxidomus with an alcoholic solution of gum guaiacum. Only in the case of the style did he obtain a deep blue color, which suggested to him that this structure might be associated with anaerobic respiration. Mol- luscs kept under anaerobic conditions showed in all cases an absence of the style. In an earlier paper (Berkeley, '21) it was shown that a disappearance of glycogen accompanies anaerobiosis in Saxidomus gigantea, though not in Paphia nor in Mya. It is pointed out in a footnote of Berkeley's ('23) paper that the work was done in ignorance of the publications of Mitra, Allen, 1 Although an oyster has no siphons, this term has come into such general use for the process of passing water through the gills of a mollusc that it seems best to employ it here especially as no good substitute appears to be available. CRYSTALLINE STYLE OF LAMELLIBRANCHS. 93 Nelson, and Edmondson, and that in the light of the findings of these and of other workers a more critical series of experiments is needed to determine the relative importance of food and of oxygen in determining style formation. Berkeley concludes that the presence or absence of the style depends upon the presence or absence of oxygen, and he makes the astonishing assumption that food plays no part in the building up of this structure, on the ground that oatmeal added to the water caused no regeneration of the style. No examinations were made to determine whether the animals were eating the oatmeal, nor were any tests made using the natural plankton food of the molluscs. Taking the results of Martin and Berkeley together, however, I believe that Allen and I were mistaken in laying undue emphasis upon the role of food in stimulating style secretion. Although the presence of food in the stomach may play a part in the mechanism of style formation, Edmondson's finding that no food was taken by Mya until after the head of the regenerating style protruded into the stomach; Martin's results in obtaining style regeneration in aerated water devoid of all net and nanno- plankton; and Berkeley's conclusion that no style forms under anaerobic conditions, all point to the probability that secretion of the crystalline style may be a direct response to siphoning, regardless of whether the incoming water contains food organisms or not. 1 The chief criticism centers about the following conclusions of Berkeley; first, his assumption that since the style disappears when the bivalve is kept under anaerobic conditions it therefore represents a reserve of oxygen. As well might one conclude that all secretions contain reserve oxygen since secretion is diminished during decreased activity of the organism. Second, no corre- lation was shown between the size and persistence of the crystal- line style on the one hand and the degree of aeration of the environment on the other. As bearing upon the first of these assumptions Gray, '23, showed that for ciliary movement the degree of mechanical activity exactly parallels the relative amount of oxygen absorbed. Since the formation and movement of the 1 Orton, '24, I.e., pg. 55, observes that sound O. edulis will reform a stj'le in the absence of food, but from the text it is not clear that all nannoplankton was removed from the water. 94 THURLOW C. XELSOX. style are so largely dependent upon the activity of the powerful cilia of the style sac, it is to be expected that no style would form in an absence of oxygen. From what we know also regarding the oxygen consumption of the glands of mammals it may be assumed that relatively little secretion of style substance would occur in an absence of oxygen. Berkeley's work is open also to the objection that nowhere does he mention having watched his molluscs to determine whether they opened up and siphoned in the anaerobic water. Such observations are imperative owing to "sulking" on the part of some bivalves after handling, even when the sur- rounding water is plentifully supplied with oxygen and food. I find that the oyster will not open in oxygen-free water until it becomes too weak to remain closed. Dissolution of the style occurs when many species of bivalves remain tightly closed for a few hours, irrespective of whether the surrounding water is aerated. In connection with the relation between the degree of aeration of natural waters and style formation it is of interest to compare the crystalline style of Pisidium idahoense with that of Mactra. Pisidium occurs in abundance in the mud at the bottom of Lake Mendota, Wisconsin, where for two thirds of the year the water may be completely devoid of oxygen (Birge and Juday, 'n, Cole, '21). Mactra on the other hand lives in or close to the breaker line along sandy coasts, where the water at all seasons of the year is saturated with oxygen. The style of Pisidium is no larger than that of other Cyrenidae w r hich live in relatively well aerated creeks and ponds, whereas the crystalline styles of all Mactra which I have examined are relatively large, firm, and among the best developed of the styles I have found in any bivalve. In Ostrea the direct relationship of crystalline style secretion to feeding can readily be demonstrated since this process assumes a somewhat rhythmic character. During the flood tide when the bivalves are feeding actively the style is large and firm, but by the late ebb tide, at which time most of the sand has been sorted out and removed from the stomach and digestion is well under way, the style may be reduced to a soft amorphous mass of jelly. The crystalline style is usually thin or entirely lacking at sunrise before active feeding of the molluscs has commenced. I agree CRYSTALLINE STYLE OF LAMELLIBRANCHS. 95 therefore with Berkeley that his conception of the respiratory function of the crystalline style rests upon insecure evidence; and I believe that this theory, interesting though it be, must be added to the long list of the purely suppositious functions of the style which have been proposed during the past two hundred and thirty years. Bokmann ('23) figures the crystalline style and the style sac of Mytilus chorus and his account agrees with the conclusions of List and of Mitra. From a histological study of the ciliated epithelium of the style sac he concluded that this must serve to put the style in rotation, although he did not observe the move- ment. This author apparently was not familiar with the work of recent American investigators. A paper of much interest and valuable for its collection of many observations under one head is that of Vonge ('23) in which for the first time is given in one place an adequate account of the mechanisms of feeding and of digestion in a bivalve mollusc. This investigator traces the fate of food particles from the time they enter the incurrent siphon until the waste is expelled from the anus. No new information regarding the function of the style is given in this paper, but the recent literature is well summarized and a clear description is given of the role of the crystalline style in digestion in My a. A study is made of the effects of varying concentrations of enzyme and of substrate, using the style and starch solutions. The optimum temperature for the reaction was found to lie at approximately 32 C., with complete destruction of the enzyme at 51 C. From the data thus collected it is concluded that the powerful amylolytic ferment of the style of Mya shows all of the characteristic properties of such an enzyme. It is pointed out that the lamellibranchs may be arranged into taxonomic groups on the basis of the morphology of the style sac and intestine, but that such a grouping does not follow the modern classification based on the gill structure. Yonge con- siders that either our present system of classification is faulty or that independent evolution has occurred within the digestive system. I believe the latter to be the case since the morphology of the digestive tract is to a high degree correlated with the food 96 THURLOW C. NELSON. habits and the habitat of the molluscs. This aspect of the problem is discussed likewise by Robson ('22) for the style sac and intestine of gastropods as well as of lamellibranchs. Criticisms of Vonge's work are, first, his failure to consider the important role of the leucocytes in digestion, especially of fat; 1 and second, an inadequate appreciation of the part played by the hepatopancreas in the digestion and absorption of food as demonstrated by List ('02). From microscopic examinations of this organ during the later stages of digestion in Ostrea and especially in Modiolus I am led to believe that in these forms even more food is digested in the hepatopancreas than in the stomach. I believe the functions of the stomach, in some lamellibranchs at least, to be chiefly those of sorting over of food materials and the final separation of dirt, and of mixing food and enzymes, rather than those of an organ where complete digestion is effected. Lazier ('24) in a valuable paper on the morphology of the digestive tract of Teredo navalis confirms for Teredo the con- clusions of my 1918 paper regarding the origin and function of the crystalline style. His investigation of the morphology of the stomach, style-sac, and intestine shows that these structures are essentially similar to those of other molluscs in which the style- sac is completely separated from the intestine. The style of Teredo is believed to be rotated in the manner I described for Modiolus, and in Anodonta, although the actual rotation was no observed by Lazier. In some of the marine borers, however, (Pholas and Martesiaj. for example) the style is of such proportions that if rotation occurs it must be very slow. In my 1918 paper is figured a transverse section of Martesia showing a style of a mass approximately equivalent to the entire remainder of the body exclusive of the gills. This aspect of the problem needs further investigation, especially as to the part played by the style in the acquisition and possible sorting over of the borings ingested by Teredo and its wood-boring allies. Churchill and Lewis ('24) make a valuable contribution to our knowledge of the mechanism of feeding in young bivalves. No 1 A recent paper by Vonk '24 gives a good summary of the work in this field and adds some original observations. CRYSTALLINE STYLE OF LAMELLIBRANCHS. 97 new data are given regarding the nature of use of the crystalline style, the authors accepting the conclusions of my 1918 paper. In conclusion it is apparent that the main facts regarding the origin, nature, and function of the crystalline style may be con- sidered as quite firmly established and that we are now in a position to attack with the aid of modern methods and the newer knowledge of general physiology some of the problems of nu- trition in molluscs. SUMMARY. The period of 74 days required for style reformation in Mya as determined by Edmondson ('20) represents not only the time during which a new style is regenerating after extraction, but also the period of recovery following the rather drastic operation performed by this investigator. From data procured from other species of lamellibranchs it would appear that the time necessary for actual reformation of the style is much less than the figure given. It was shown (Nelson, '18) that owing to the imperfect mode of separation of food particles from dirt and sand in the stomach of bivalve molluscs, some undigested food materials may escape down the intestine. In those forms in which the style sac is incompletely separated from the intestine a part of this rejected food material may be incorporated into the crystalline style, thus eventually being returned to the stomach. The remainder pass out undigested in the faeces. Failure to recognize this fact has led at least two recent investigators to conclude that such living organisms as are recovered from the faeces cannot be utilized by the molluscs as food. The degree of solidity and of resistance to dissolution of the crystalline style are suggested by Nogouchi ('21) as factors controlling the presence or absence of Cristispira. That the solidity and the resistance of the style are not the primary factors involved in the distribution of Cristispira is shown by the presence of these spirochaetes in the styles of Ostrea and of Mya which represent respectively minimum and maximum hardness. Martin ('23) showed that a style may arise in Ostrea as a direct response to siphoning in aerated water even when the incoming water is devoid of all net and nannoplankton or other sources of 98 THURLOW C. NELSON". food. Berkeley ('23) found no style reformation under anaerobic conditions. His conclusion, however, that the style represents a reserve of oxygen is open to serious objection as herein explained. The conclusions of Edmondson, '20, Alien, '21, Coker and others, '21, Bokman, '23, Churchill and Lewis, '24, and of Lazier, '24, agree in confirming the work of Coupin, 'oo, and the con- clusions of my 1918 paper. Except in so far as the latter are modified by the findings of Martin and of Berkeley, as set forth in this paper, it is believed that the conclusions of Coupin and of the writer represent the consensus of opinion today regarding the origin, nature, and function of the crystalline style of lamelli- branch molluscs. CITATIONS. Allen, W. R. '14 The Food and Feeding Nabits of Fresh-water Mussels. BIOL. BULL., 27, 127-139. '21 Studies of the Biology of Fresh-water Mussels. Experimental Studies qf. the Food Relations of Certain Unionidae. BIOL. BULL., 40, 210-241. Berkeley, C. '23 On the Crystalline Style as a Possible Factor in the Anaerobic Respiration of Certain Marine Molluscs. J. Exp. Zool., 37, 477-488. Vonk, H. J. '24 Verdauungsphagocytose bei den austern. Zeitschr. f. vergl. Physiol. Bd. i, H. 3, 4. Birge E. A., and Juday, C. 'n The Inland Lakes of Wisconsin. The Dissolved Gases of the Water and their Biological Significance. Bull Wis. Geol. and Nat. Hist. Sur., 22, p. x + 259. Blegvad, H. '15 Food and Conditions of Nourishment among the Communities of Inverte- brate Animals Found on or in the Sea Bottom in Danish Waters. Rept. Dan. Biol. Sta. for 1914, 22, 41-78. Bokman, F. '23 Ueber Stoffwechselorgane einiger Mytiliden. Jen. Zeit. f. Naturwiss. 59, 209-260. Churchill, E. P., and Lewis, S. I. '24 Food and Feeding of Fresh-water Mussels. Bull. U. S. B. F. 39, 439-471. Coker, R. E., Shira, A. F., Clark, H. W., and Howard, A. D. '21 Natural History and Propagation of Fresh-water Mussels. Bull. U. S. B. F. Cole, A. E. '21 Oxygen Supply of Certain Animals Living in Water Containing No Dis- solved Oxygen. J. Exp. Zool., 33, 239-320. Collip, J. B. '20 Studies on Molluskan Ccelomic Fluid. Effect of Change of Environment on the Carbon Dioxide Content of the Ccelomic Fluid. Anaerobic Respiration in Mya arenaria. J. Biol. Chem., 45, 23-49. CRYSTALLINE STYLE OF LAMELLIBRAXCHS. 99 '21 A Further Study of the Respiratory Processes in My a arenaria and other Marine Mollusca. J. Biol. Chem., 49, 297-310. Edmondson, C. H. '20 The Reformation of the Crystalline Style in Mya arenaria after extraction. J. Exp. Zool., 30, 259-291. Gray, J. '23 The Mechanism of Ciliary Movement. III. The Effect of Temperature. Proc. Roy. Soc., 95, Ser. B, No. 6664, 6-15. Lazier, E. L. '24 Morphology of the Digestive Tract of Teredo navalis. U. of Cal. Publ. Zool., 22, 455-474. Martin, G. W. '23 Food of the Oyster. Bot. Gaz., 75, 143-169. Nelson, T. C. '18 On the Origin, Nature, and Function of the Crystalline Style of Lamelli- branchs. J. Morph., 31, 53-111. Nogouchi, H. '21 Cristispira in North American Shellfish. J. Exp. Med., 34, 299-315. Robson, G. C. '22 On the Style Sac and Intestine in Gastropoda and Lamellibranchia. Proc. Malacal. Soc., 15. Yonge, C. M. '23 Studies on the Comparative Physiology of Digestion, i. The Mechanism of Feeding, Digestion, and Assimilation in the Lamellibranch Mya. Brit. J. Exp. Biol., i, 15-63. THE CONDITIONS OF ACTIVATION OF UNFERTI- LIZED STARFISH EGGS BY THE ELECTRIC CURRENT. RALPH S. LILLIE AND WARE CATTELL. (University of Chicago and Marine Biological Laboratory.) While sensitivity to the electric current seems universal in living matter, its degree varies greatly apparently in corre- spondence with the wide variation in general irritability. It is most highly developed in rapidly responding tissues such as muscle and nerve; but it can be shown to exist in supposedly insensitive cells like epidermal cells, which respond to electrical stimulation by increase of conductivity. 1 Specialized sensory receptors (retina, auditory or chemical senses) all respond to the electric current as well as to their own appropriate forms of stimulation. Electrical sensitivity thus appears to be the primary form of sensitivity. 2 In general, as the work of Nernst and his successors has shown, it is intimately connected with polarizability, which is dependent on the presence of diffusion- resisting or semipermeable partitions enclosing or pervading the protoplasmic system. Evidently the electric current acts within the living system by influencing the chemical reactions at its polarizable surfaces, as in electrode action in general; in living protoplasm the surfaces concerned are those of the protoplasmic structures, and especially of the films delimiting or separating the protoplasmic phases. The directive or stimulating action of the electric current on growth has been demonstrated in a number of cases, although much remains to be done in this field. The unfertilized egg-cell, however, seems usually to be relatively insensitive to the current. The earlier evidence of electrical parthenogenesis in the eggs of marine animals is inconclusive. Schiicking claims to have acti- vated starfish eggs by passing the current from two chromic acid 1 Ebbecke, U., Arch. ges. PhysioL, 1922, CXCV., pp. 300, 324. 2 For a fuller discussion cf. the recent volume of R. S. Lillie, "Protoplasmic Act!" and Nervous Action," University of Chicago Press, I9 2 3. PP- 273 el seq. IOO ACTIVATION OF UNFERTILIZED STARFISH EGGS. 101 elements for one to two minutes; l but the conditions (nature of electrodes, distance apart, quantity of sea-water, etc.) are not described. He does not consider the possibility that acid or alkali electrolytically produced, or heat, rather than the electric current as such, may have been responsible for the effect; and in the light of our own experiments it seems highly probable that this was the case. Schiicking found induction shocks to be ineffective, and we have repeated and confirmed this observation. In the condenser-like arrangement used by Delage, 2 the actual physical conditions were ill-defined. Delage's aim was to affect the eggs electrostatically (by induction); they were placed in a layer of sea-water separated by a thin sheet of mica from a sheet of tinfoil, the sea-water and the metal being connected with the poles of a battery. His results were irregular, and he himself expresses doubt as to the real nature of the conditions. The presence of currents and of products of electrolysis seems not to have been excluded. In the eggs of amphibia McClendon was able to start cleavage and development by passing the alternating current from the lighting circuit (no volts, 60 cycles) through the water containing the eggs. 3 In this case a current of considerable intensity acted for a brief period (i to 2 seconds), and the effect was probably not caused by electrolytic products or heat. Further and more precise investigation of the conditions of electrical partheno- genesis in these eggs seems desirable. Our aim in the experiments described in this paper has been (i) to ascertain whether in fact activation of starfish eggs by the direct electric current is possible, and to what degree, and (2) to determine more precisely the conditions, more particularly of current intensity, time of exposure and temperature, under which the effect is produced. The experiments were performed on the unfertilized eggs of As^erias forbesii at Woods Hole during the summers of 1922 and 1923. The direct current was used in all cases. Usually the eggs were exposed to the current during the interval between the breakdown of the germinal vesicle and the separation of the first polar body (prematuration period) ; 4 for 1 Schiicking, A., Arch. ges. Physiol., 1903, XCVIL, 86. - Delage, Y., Arch. zool. exper. et gen., Ser. 4, 1908, IX., p. xxx. 3 McClendon, J. F., Amer. Jour. Physiol., 1912, XXIX., p. 299. 4 The period most favorable for fertilization and artificial activation. IO2 RALPH S. LILLIE AND WARE CATTELL. comparison a number of observations were made with fully mature eggs. The procedure was simple ; the current was passed through a shallow layer of sea-water containing the eggs, and at regular intervals portions of eggs were transferred to dishes or watch-glasses containing sea-water. Afterwards they were ex- amined. Fertilized and unfertilized controls were kept in all cases. Our preliminary experiments with battery currents of moderate intensity (one to twelve storage cells) gave uniformly negative results, and in all of our later experiments we used the current from the direct current generator of the laboratory. We were not able at first to find suitable electrodes. The ordinary forms of non-polarizable electrodes ("boot" electrodes) proved unsatis- factory because of high resistance and the diffusion of ZnSO 4 into the sea-water. When platinum electrodes were used the eggs showed partial activation (membrane-formation) in some experi- ments; but it could be shown (by first passing the current through a layer of sea-water and then placing the eggs in the sea- water without the current) that this effect was due to or at least could be produced by the products of electrolysis. These results indicated the need for an arrangement by which strong currents could be passed through the layer of sea-water without contaminating the latter by the electrode solution or products of electrolysis; and after a number of preliminary experiments the following method was devised. The wires from the direct current line (no volts) were connected with two broad zinc plates each immersed in a dish of saturated ZnSO 4 ; these dishes with the plates constituted non-polarizable electrodes of low resistance. A rectangular glass vessel containing the sea-water in which the eggs were to be exposed was placed between the dishes. In the experiments of each summer a single vessel was used throughout the whole series; the dimensions in 1922 were 14 x 5.5 x 3.5 cm., in 1923, 13.1 x 6.6 x 3.5 cm. The depth of sea-water was kept constant as nearly as possible throughout each series, 0.4 cm. in 1922 and 0.5 cm. in 1923; the sectional area (from which the estimates of current density were made) was thus 2.2 sq. cm. in 1922 and 3.3 sq. cm. in 1923. The current was conveyed through the sea-water by massive bridges of agar jelly of the same width ACTIVATION OF UNFERTILIZED STARFISH EGGS. 103 as the rectangular vessel and connecting the latter with the electrode dishes. These bridges were made as follows : A concen- trated solution of agar-agar in sea-water was allowed to solidify in a large beaker; the mass of jelly was then removed and cut into two blocks of the general shape indicated in the figure; one end of each block stood in the ZnSO 4 solution and the other in the sea- water, as shown diagrammatically in the longitudinal section (Fig. i). By means of this arrangement strong currents (up to 2 FIG. i. or more amperes) could be passed through the sea-water. Ex- periment showed that the composition of the sea-water was not appreciably affected during the flow of the current for the period of an experiment. The temperature, however, rose rapidly unless controlled. In part of our experiments the control of temperature was effected by setting the whole system in a pan of ice water; this method proved satisfactory with currents of moderate in- tensity (up to 150 ma. /cm.). 1 In a number of experiments with stronger currents another method was used, to be described below. The strength of the current was regulated by two rheostats and measured directly by a Weston voltmeter provided with shunts so as to read as a milliammeter over the several ranges required. Intensities as high as 3 or 4 amperes were used for a brief period in some of the experiments with running sea-water described below (densities up to ca. 800 ma. /cm.). The usual procedure was as follows : A somewhat small quantity of eggs was placed with a pipette in the rectangular dish midway between the agar bridges (2 to 4 cm. from each). When the eggs had settled the circuit was closed, and at stated intervals, usually 2, 4, 8 and 12 minutes, successive portions of eggs were transferred to small dishes (usually Syracuse watch glasses or stender dishes) 1 Milliamperes per square centimeter of sectional area. The commonly used unit of current-density, 5 (microampere, i.e., .001 milliampere, per sqium- milli- meter), is one tenth of this unit. IO4 RALPH S. LILLIE AND WARE CATTELL. containing sea-water; these were kept covered except at times of examination, and the sea-water was changed several times. With small numbers of eggs this method is satisfactory and convenient. In each experiment the temperature of the sea- water in the rectangular dish was recorded at the end of. the longest exposure. The temperature was measured by a ther- mometer with the bulb placed near the eggs. A difficulty with this method is that the bulb was incompletely immersed when the layer of sea- water was shallow, as in most of our experiments; this was especially true of the earlier experiments, where the readings were too low and a correction of I to 3 degrees was found necessary; with later experiments a small thermometer with a short bulb was used which gave reliable readings. A large number of experiments (more than 50) were performed in which the vessel containing the eggs was immersed in ice water as described above; the current densities used ranged from less than 100 to 318 ma. /cm. The general results of these experi- ments may be summarized as follows. With currents of densities ranging from 136 to 242 ma. /cm., flowing from 2 to 12 minutes, activation was either absent or negligible, provided the temperature remained below 29. In all cases where the temperature rose to 30 or higher a variable degree of activation, usually incomplete, was obtained; and in some cases a considerable proportion of eggs developed to the swimming blastula stage. In the experiment showing the most striking effect of this kind (Aug. 22, 1922) a very typical picture of heat activation was presented; after 4 minutes exposure to a current of 227 ma. /cm., only a few eggs (ca. 3 per cent.) formed membranes; with exposures of 8 and 12 minutes almost all formed membranes and a large proportion formed blastulae (ca. 75 per cent, with 8 minutes and 25 per cent, with 12 minutes). The temperature at the end of the maximum period of exposure (12 m.), allowing for the error of measurement, was 30 or over. In such a case the activation caused by the current is mainly if not entirely an effect of the high temperature, and not of the current as such. This is shown by the fact that activation was never produced by the same current at lower temperatures (28 or ower) ; also by control experiments in \vhich eggs were activated ACTIVATION" OF UNFERTILIZED STARFISH EGGS. IO5 by sea-water which had been warmed to 30 or higher by the current, the latter being shut off before the eggs were introduced. It seems probable, nevertheless, that a part of the activating effect observed in this and similar experiments is to be attributed to the current; i.e., that there is a summation of the effects of heat and current, since the degree of activation was greater than would usually be produced by exposure to a temperature of 30 for the periods used. According to earlier observations, activation by warm sea-water (acting alone) requires a temperature of at least 29, and at 30 few eggs develop to a blastula stage after less than 15 minutes' exposure. 1 In other words, the effect of high temper- ature appears to be greater when a current is flowing through the sea-water containing the eggs than when no current is flowing. We have not, however, performed definite controlled experiments to determine with exactitude the degree of this additive effect. An analogous phenomenon is seen in the activation of starfish eggs by fatty acid; at temperatures of 26 and higher the effective times of exposure to the acid are much shorter than can be accounted for by the temperature coefficient of acid activation (Qio == ca. 3.0) shown at lower temperatures. 2 Apparently in warm sea-water the action of the fatty acid is accelerated by some condition dependent on temperature; i.e., there is a super- position of acid activation upon an incipient heat activation. Similarly, in the experiments with strong currents an effect resulting from the action of the current as such appears to be superposed upon that of the high temperature. With current-densities higher than 240 ma. /cm. the difficulty of evading the temperature effect was such that it was necessary to devise another means of compensating the heating action of the current. We therefore tried exposing the eggs to strong currents in running instead of stationary sea-water, and after some pre- liminary experimentation adopted the following procedure: In place of the rectangular glass dish a paper box of the same dimensions was used. This was reinforced and made an electric non-conductor by several coatings of paraffin. A rectangular slit (ca. 6 x 1.5 cm.) was cut at the base of this box along one of the longer sides. A small rectangular cloth basket (ca. 3x3x4 cm.), 1 Lillie, R. S., BIOL. BULL., 1915, XXVIII. , p. 260; cf. Table II., p. 269. 2 Lillie, R. S., BIOL. BULL., 1917, XXXII., p. 131; cf. Table II., p. 142. 106 RALPH S. LILLIE AXD WARE CATTELL. containing the eggs, was inserted between the two agar bridges at the center of the paper- paraffin dish. This basket consisted of a frame of slender wooden sticks reinforced with thread and paraffin, to which silk bolting cloth was sewn. The bolting cloth had 200 threads to the inch thus confining the eggs, yet allowing a free circulation of water. The apparatus was set on a wooden block in a large pan. A stream of sea water of considerable force was directed through a glass nozzle against the bottom of the paper- paraffin vessel on the opposite side of the basket from the slit. In this way a swift stream of water through the basket was ob- tained. After passing through the basket the water ran out of the slit down the side of the block into the pan where it was re- moved with a siphon. A small thermometer was p'aced with its bulb resting on the bottom of the basket. In carrying out these experiments the usual procedure was as follows. The sea w r ater and the current were started and the temperature was allowed to reach its position of equilibrium. Then with a pipette a quantity of eggs was placed in the cloth basket, and at the stated intervals the portions were removed to stender dishes of sea water for observation. The density of current was estimated from the ammeter reading and the average depth of the layer of running water in the paraffined vessel. Under these conditions, even with densities so high as 600 to 800 ma./cm., the current could be passed for several minutes without raising the temperature above 29, and with lower densities the temperature showed little increase over that of the sea water without the current. In one of the experiments with a strong current (ammeter reading 680-790 ma./cm.) passed for four minutes, the temperature reading was 29 for most of the period of flow but reached 3O-3i for a few seconds toward the end. In a second similar experiment, with a range of 648-810 ma./cm., in which eggs were removed to normal sea water at intervals of |, i, i| and 2 minutes, the temperature reading was unfortunately lost; probably 3O-3i was reached in this case also. With such strong currents the eggs showed marked deformation during the period of exposure, adopting shapes of the kind shown in Fig. 2. This effect is temporary; within a few minutes after ACTIVATION OF UNFERTILIZED STARFISH EGGS. removal from the current the eggs return to their original shape. Such eggs show a separation of fertilization-membranes in a considerable proportion of cases, but not in all. In the experi- ment just cited ca. 40 per cent, of the eggs exposed to the strong FIG. 2. current for if and 2 minutes showed well separated membranes and a small proportion developed to the blastula stage. The majority underwent disintegration inside the membrane without development. Of twelve experiments performed with this method (with good controls), using currents of densities ranging between 130 and 810 ma./cm., seven showed a varying degree of activation. Little or no activation was obtained with currents of less than 300 ma./cm. With higher intensities membrane-formation and activation occurred in a minority of eggs; the strongest currents in addition to deforming the eggs temporarily in the manner just described had a marked destructive effect. 1 In general the results of the foregoing experiments indicate that the electric current has little activating effect upon the starfish egg unless the intensities employed are sufficient to produce well marked structural changes in the egg-system. The evidence of these changes is deformation and subsequent breakdown of most eggs. A certain proportion of eggs, however, recover and show the usual phenomena of partial activation. These effects cannot be referred to the observed rise of temperature which produces no such definite deformation. Moreover, the highest temperatures reached (30 31) require a much longer period of exposure for activation of the degree observed. It should be remembered in considering the results of such experiments that the physical conditions are far from constant, and that the records of both temperature and current are subject 1 This destructive action of strong currents on egg cells has been noted by other observers; cf. the case of Crepidula as described by Conklin, Jour. Acad. Xat. Sciences, Philadelphia, 1912, XV., p. 521. IO8 RALPH S. LILLIE AND WARE CATTELL. to an error which may be considerable. In general the recorded current density is greater than that to which the eggs were actually exposed, since the current lines extend outside of the rectangular vessel into the overflow. There is also an error of measurement resulting from variability in the depth and contour of the layer of water between the agar bridges; this condition makes possible only an approximate estimate of the sectional areas. The usual procedure was to measure the depth of the water in the basket and on both sides of it and to calculate the cross section from these measurements. But even during a single 5-minute experiment the water level often varied considerably, sometimes because the eggs themselves clogged the silk bolting cloth thus raising the level of water inside the basket. Again, with regard to temperature, although the bulb of the thermometer was completely immersed the irregularity of the water stream is a source of uncertainty. Local temperatures may rise higher than the average temperature recorded by the thermometer; or the instrument may be actually registering the temperature of a stream of water of greater or less velocity than that to which the eggs are actually exposed. It may reasonably be assumed, however, that on the whole the errors in opposite directions compensate each other. It was thought possible that the physiological effect of the current might be changed (increased or decreased) by changing the balance of salts in the medium. This occurs, for example, in the electrical stimulation of muscle. 1 Eggs were suspended in pure isotonic NaCl solution (0.54 m), washed in this solution by gentle centrifuging and decantation, and exposed to the current (densities from 139 to 262 ma. /cm.) in the rectangular glass dishes under the conditions already described. A certain degree of activation results from the action of the pure NaCl solution in the absence of the current. 2 The result of passing the current through the NaCl solution containing the eggs was, however, essentially negative; four out of eleven experiments showed a slight increase in activation over that produced by the solution alone, five showed no difference, while two showed a decrease. 1 Cf. the observations of K. Lucas and G. W. Mines on the electrical stimulation of muscle in Journ. Physiol., 1908, XXXVII., p. 459. 2 Lillie, R. S., Amer. Journ. Physiol., 1911, XXVII., p. 289. ACTIVATION' OF UNFERTILIZED STARFISH EGGS. 109 The combined action of the NaCl solution and the current thus shows no significant difference from that of the pure solution alone. The increase noted in the first four experiments was probably the result of a slight rise of temperature; this increases the activating effect of the XaC'l solution, as control experiments showed. Decreasing the conductivity of the medium by adding isotonic sugar solution to the sea water was also found not to alter the effect of the current on the eggs. In conclusion brief mention should be made of similar experi- ments with Arbacia eggs. The results of these experiments were mainly negative. Little or no effect was produced by exposing the eggs in standing sea water to current-densities varying from 7.5 to 210 ma. /cm. No membranes were formed and no cleavage resulted. Eggs exposed to the current and immediately after- wards treated with hypertonic sea water showed no constant increase in the percentage of activation, above eggs treated with hypertonic sea water alone. The effect of exposing to currents of high density in running sea water was also essentially negative, although some cytolysis was caused by the longer exposures. In general the Arbacia egg is more resistant tg the current than the Asterias egg; this difference is probably to be correlated with the greater impermeability of the surface layer to water l (and pre- sumably to water soluble substances) and its greater resistance to alteration and the action of parthenogenetic agents in general. SUMMARY. 1. A new type of non-polarizable (Zn-ZnSO 4 ) electrode of low resistance is described by which strong electric currents (up to 2 amperes or more) can be passed for prolonged periods through a small quantity of sea-water without appreciably affecting its composition. 2. It was found that unfertilized starfish eggs can be readily and completely activated by moderate currents, of the density 200-300 milliamperes per square centimeter; but that the effect in.^uch cases is due almost entirely to the heating action of the current on the sea- water. When the temperature is kept below 29 such currents produce little or no effect upon the eggs. 1 Lillie, R. S., Amer. Jonrn. Pliysiol., 1918, XLV., p. 406; cf. p. 420. IIO RALPH S. LILLIE AND WARE CATTELL. Unfertilized eggs are thus insensitive to the current as compared with most irritable or active cells of other kinds. 3. When the eggs are exposed in a stream of running sea-water to stronger currents (600-800 ma. /cm.) for brief periods (| to 2 minutes), definite effects are produced. The eggs undergo marked deformation during the passage of the current, and a variable proportion afterwards show fertilization-membranes and partial activation. Complete activation of a large proportion of eggs was not possible in our experiments, although a few de- veloped to the blastula stage. 4. We conclude that the unfertilized starfish egg is insensitive to currents of moderate intensity, and exhibits activation only when currents are used of such intensity as to produce definite and well marked structural changes in the egg-system. 5. Similar exposure of unfertilized sea-urchin eggs (Arbacia) to strong currents, with and without after-treatment with hyper- tonic sea-water, gave inconstant or negative results. AN EXPERIMENTAL ANALYSIS OF ASYMMETRY IN THE STARFISH, PATIRIA MINI ATA H. H. XEWMAX. (From the Hull Zoological Laboratory, the University of Chicago.) I. INTRODUCTION. The present paper gives the results of a portion of the experi- mental work carried on at the Hopkins Marine Station of Leland Stanford University in the spring of 1923. The program of work was a continuation and an extension of the experiments begun in the same laboratory in 1920 and published during the next two years (Newman, '21, a; '21,6; '2i,c; '22). It became apparent in 1920 that not only twinning but other developmental aberrations could be induced by retarding the rate of development of eggs and embryos in a considerable variety of ways. Of all the agents used by various investigators for re- tarding development, that of low temperatures seemed to be the least open to objection and is perhaps the most readily managed. It was, therefore, with the program in mind, of subjecting Patiria and other echinoderms to various degrees of low temperatures at various stages of development and to observe and analyse the resulting developmental aberrations, that work was begun early in April, 1923. During some of the very first of the preliminary experiments it was found that subjection of Patiria blastulee to temperatures of about 2 C. for two or three hours brought about a marked in- crease in the percentage of larvae showing reversed or right-handed asymmetry. For over two months after this discovery it became a major consideration to gather an adequate mass of data on this subject and to work out the implications involved. Before it was possible to arrive at any justifiable conclusions as to the affects of low temperatures upon asymmetry it was neces- sary to study in some detail the normal development of Patiria. It seems likely that entirely normal development is unattainable under laboratory conditions, yet the culture methods used were experimentally determined to be a vast improvement upon those in 112 H. H. NEWMAN. commonly adopted'by experimentors upon like material. As an introduction to the experimental part of this paper we shall ask the reader to make with us a brief survey of the essential features of the normal embryonic and larval periods of Patiria, paying chief attention to the origin and development of asymmetry. II. THE NORMAL DEVELOPMENT OF Patiria. Methods of Securing and Handling Eggs and Larva. Patiria, miniata is a common Pacific Coast starfish with which I have done considerable work. During the spring of 1920 I had not dis- covered any better method of securing the eggs than that of shaking the excised ovaries in bowls of sea-water. This method had proven rather unsatisfactory because the great majority of eggs thus obtained were immature and incapable of fertilization even after standing for an hour or so. Furthermore, the presence of so many dying and disintegrating eggs tends to foul the water and to encourage the development of deleterious bacterial and fungoid growths, which are far from favorable for the living and active larvae. This difficulty was formerly obviated to some extent by taking advantage of the fact that there is a certain period in the early larval life when all healthy larvae swim to the surface and thus may be decanted off and placed in clean sea water. But many of the slightly subnormal larvae fail to reach the surface and either have to be picked out individually or left to develop amidst the debris of decaying eggs. The improved method takes advantage of the chance obser- vation that if ripe starfishes are allowed to stand for a few hours out of water they shed both eggs and milt in great volume. The procedure adopted was to place a considerable number of freshly collected starfishes upon a bed of seaweeds in a moist atmosphere. It is found that about ten per cent, of the individuals shed their genital products within from two to four hours and that both eggs and sperms are uniformly ripe and in prime condition, so that essentially all eggs are fertilized and develop normally. Many thousands of eggs may be shed within a few minutes by a single female, making it possible to conduct several experiments with a single batch of eggs. Variety may be secured by fertilizing one lot of eggs with the sperm of several males. It is necessary to use ASYMMETRY IN THE STARFISH. 113 the minimum sperm concentration in order to avoid polyspermy. The standard procedure adopted and used in all experiments was as follows: Five drops of solid sperm, just as it exudes from the genital pores, was mixed with 100 cc. of sea-water; and then ten drops of this sperm suspension was added to a finger-bowl con- taining a single layer of eggs and 100 cc. of sea-water. Eggs must stand for about an hour in sea-water before insemination is attempted. CLEAVAGE, BLASTULA, AND GASTRULA. Cleavage begins after about two hours and is entirely similar to that of other asteroids previously described. To get the best results it is necessary to keep the cultures in an unheated room in which the temperature ranges only a degree or so above or below 15 C. Especially is it important that the embryos should not be warmed; a few degrees of lower temperature may be beneficial. The cultures should also be shaded in such a way that no direct sunlight reaches them. When eggs are reared under these pre- cautions they reach a blastula stage in a little under or over 18 hours, and hatch out as swimmers in about 24 hours. The great majority of the blastulae are practically spherical in form. Even in the best of cultures there are always a few abnormal larva? that are wrinkled or nearly solid, but in spite of their deformity apparently more active than normal larvae. It has not been determined whether these abnormal larvae are the product of over- ripe eggs, of polyspermy, or of parthenogenesis. It is a simple matter to rid the cultures of all such abnormal larvae at an early stage, and thus to make conditions better for the normals. Gastrulation begins a few hours after the larvae have hatched, and is in no way different from that described for other asteroids. The various steps in the process are shown in Figures 1-6. It is to be especially noted that gastrulae reared in this fashion are quite free from any apical thickening such as Heath (1918) described for this species and which he considered homologous with the apical plate of the enteropneustan larva. Only sub- normal larvae show this structure, as was pointed out in a previous paper (Newman, 1922), but this becomes especially obvious when improved methods of obtaining eggs are used. 114 H. H. NEWMAN. The completed gastrula is somewhat elongated (Fig. 6) and the anterior end of the archenteron reaches about half way to the apical end. This stage is attained at about thirty hours after fertilization. ,c.p. ..c.p FIGS. 1-6. Early stages, blastulas and gastrulae, of Patiria. c.p., the thinned- out anterior portion of the archenteron in process of forming the coelomic pouches. Post-gastrula Stages. The changes leading up to the typical Bipennaria condition involve several distinct types of differenti- ation: (a) the development of well-defined dorsal and ventral surfaces, together with a relative shortening of the ventral surface, which thus becomes concave, and a relative lengthening of the dorsal surface, which becomes convex; (b) the differenti- ation of the external ciliated bands; and (c) the development of the cceloms. The larva at about fifty hours (Fig. 8) shows the blastopore moving slightly toward the ventral surface, the somewhat convex contour of the latter, the slight thickening and bulging of the region destined to form the preoral ciliated band, and the enlarge- ment and thinning out of the anterior end of the archenteron to form the pharynx and the anterior cceloms. The changes in larval form and the development of the ciliated bands are so nearly identical with those described for other asteroids that they ASYMMETRY IX THE STARFISH. 115 may be passed over, at least until more advanced stages are reached. The events associated with the formation of the system of coelomic pouches, however, need more detailed treatment for they seem to be unique in certain respects. Development of the Ccelomic Structures. As has already been said, the first change leading to the formation of coelomic struc- tures is the enlargement and thinning out of the free end of the archenteron. This is well shown in Fig. 5. The next stage involves a flattening out mushroom-fashion of this thin-walled vesicle, as shown in Fig. 6. Here the paired anterior coeloms are shown in process of being pinched off. This process completes itself, as shown in Fig. 7, by the end of the third day. I have 1. a.c... l.p.c.- f.a.c. 7 FIGS. 7-9. Early Bipennaria larvae, showing especially the formation of ccelomic structures, a.c., anterior coelom; c.b., ciliated band in optical section; h.p., hydropore; 1. a.c., left anterior coelom; l.p.c., left posterior ccelom; r.a.c. t right anterior coelom. never been able to assure myself, as other writers have done, that the left anterior coelom is from the first larger than the right. It appears to me that until the formation of the posterior coelom the larva is perfectly bilaterally symmetrical. The Posterior Ccelom. A very significant event, from the stand- point of the development of asymmetry, is the appearance of the posterior ccelomic vesicle. In Patiria, this vesicle is a conspicu- ous object for a considerable period of time and in the end becomes the hypogastric ccelom. This condition is like that described for Cribrella and Solaster and unlike that in most asteroids thus far described in which the hypogastric ccelom is derived from a Il6 H. H. NEWMAN. posterior outgrowth of the left anterior ccelom. In Patiria the posterior coelom typically arises on the left side of the archenteron at about the level of the middle of the prospective stomach. It is first seen in the form of a thick-walled evagination of the wall of the archenteron (Fig. 7). This pouch does not expand into a thin-walled vesicle, but merely constricts off a small solid ball of cells that later expands and forms a lumen. The vesicle is always a conspicuous object even under low powers of the microscope, and it affords a ready means of determining the asymmetry of the larva before there are any other positive indications of asymmetry Variations in the Position of the Posterior Ccelom. The typical point of origin of this organ is noticeably to the left of the median line of the archenteron. In many cases, however, it occurs exactly in the median line, under which circumstances it is not infrequently completely double or at least more or less constricted down the mid-dorsal line. Sometimes the double vesicle fuses into one and moves sometimes to the right, but usually to the left, and gives rise to right-handed or left-handed asymmetry. A statistical study of large numbers of larvae revealed the pro- portions in which right-handed and left-handed individuals occur. Out of 916 specimens examined, 811 had the posterior ccelom on the left, 98 had it on the right, and 9 had it in a median position or so nearly so that no asymmetry could be made out. This means that over 88 per cent, exhibit left-handed asymmetry, over 10 per cent, right-handed asymmetry, and a little over i per cent, show no asymmetry. This is in contrast with conditions described by Gemmill (1915) for Porania pulvillus, where "a rudimentary enteroccelic outgrowth arises dorsally by prolifer- ation of the stomach endoderm, usually on both sides of the median line, but sometimes in the median line, or on one side only. . . . When only one body is present it occurs rather of tener on the right than on the left side, a contrast to the condition in Asterias rubens, where the corresponding body (a still smaller one) appears most commonly on the left side." It seems evident from these observations that the presence among the asteroids of the posterior coelomic vesicle and its asymmetry exhibits a highly variable incidence. The body is one that seems to be relatively vestigial in the group as a whole, but appears in a Cribrella, ASYMMETRY IN THE STARFISH. I 17 Solaster, and Patiria (doubtless also in other species) to be rather large and of some functional importance. It should also be noted that (in Patiria) the asymmetry exhibited by this vesicle always coincides with that exhibited by other coelomic structures. Development of Ilydropores and Pore-canals. On about the fifth day the larva gives off, typically from the posterior end of the left anterior ccelom, a dorsally directed hollow process, which comes into contact with the larval body wall and breaks through to form an open hydropore (Fig. 9). In Patiria the pore canal and hydropore constitute a large and conspicuous landmark for the determination of asymmetry. Without a single observed exception the hydropore is formed on the same side as the already formed posterior ccelom. There were a few cases in which the posterior ccelom appeared to be median, while the hydropore appeared only on one side. Attention should be called here to the fact that, while in Poranus (Gemmill, '15) the hydropore appears a day or so earlier than the posterior ccelom, exactly the opposite time relation prevails in Patiria, the posterior ccelom appearing a day or so earlier than the hydropore. Probably the earlier appearance of the posterior ccelom in Patiria accounts for its larger size and its permanency. As was the case with the posterior ccelom, the hydropore also shows reversed asymmetry. The reversed or right-handed larvae are as large, as vigorous, and as healthy as are the more typical individuals. A census of 652 individuals, belonging to seven different lots of larvae, showed 589 with left-hand hydropores, 63 with right-hand hydropores, and none with both right-hand and left-hand hydropores; which means that over 90 per cent, are left-handed, and less than 10 per cent, are right-handed. We see then that the percentage of individuals with completed right hydropore is somewhat less than that of individuals with right posterior ccelom, but the difference is not significant, as both are very close to 10 per cent. Furthermore it may be said that the above censuses were made upon different lots of larvae from those given for controls in the experimental part of the paper, and strongly tend to confirm the latter. It should strongly be emphasized in this connection that the right-hand hydropores in Patiria are not merely temporary 8 Il8 H. H. NEWMAN. structures that subsequently regress and give place to left-hand hydropores, as Gemmill claims to be the case for Asterias rubens, but are permanent asymmetrical structures that remain up to the point of metamorphosis. In every culture observed the right- handed individuals maintained their reversed asymmetry and developed on the average as normally as did the more typical left-handed larvae; in fact one gets the impression that in some cases at least, the reversed larvse are a little larger and more active than the typical ones belonging to the same lot. This is, I believe, the first time that large numbers of indi- viduals with right-handed (reversed) asymmetry have been de- scribed for echinoderms. Only very rare and sporadic instances of reversal of asymmetry have been reported. In Poramis, a species of starfish studied intensively by Gemmill, double hydro- pores occurred in about 50 per cent, of the larvse, but he specific- ally states that but "a single instance was observed in which the hydropore occurred on the right side alone." It may be perti- nent, however, to recall that Oshima found both in control laboratory cultures and in experimental cultures of Echinus miliaris about 10 per cent, of specimens with right-hand asym- metry. In these experiments, however, it must not be forgotten that both controls and experiments were conducted under dis- tinctly abnormal conditions, since artificial sea-water was used and the food was unsatisfactory. The possibility is not to be denied, however, that this species of echinoid may exhibit normally a fairly high degree of reversed asymmetry. From what has been said, it may be inferred that Patiria miniata is in a somewhat delicate state of organic equilibrium with regard to its asymmetry, and should, therefore, furnish ideal material for an experimental analysis of the nature of this asymmetry. The Development of the Hydroccele and its Rays. Although one may observe by the end of the first week or ten days a posterior prolongation of the left (in reversed larvae, the right) anterior coelom, this structure does not thicken up and become obviously a hydroccele until about the end of the third week. In fact, a hydrocoele never develops unless the larvae are well fed and otherwise well cared for. The hydroccele appears at the same ASYMMETRY IX THE STARFISH. 119 time and in the same manner in right-handed as in left-handed larvae. The Form of Bipennaria and of Brachiolaria Larvce. The form of the Bipennaria on the fifth day is shown in Fig. 9. It may be noted that the blastopore or larval anus has moved around to a distinctly ventral position ; that the hydropore and pore-canal are well defined ; that the posterior coelom is now distinctly vesicular ; and that both preoral and postoral ciliated bands have begun to be differentiated. pr.l. /n. a c m r.a.c. l.p.C mt. -m oes. r.hyc. r.pc. IO II FIG. 10. Dorsal view of an advanced Bipennaria showing the ordinary left- handed asymmetry. FIG. n. A somewhat more advanced Bipennaria seen from the dorsal side and showing reversed or right-handed asymmetry- I'. p., hydropore; hyc., hydroccele; int., intestine; /. a. c., left anterior coelom; l.h.p., left hydropore; l.p.c., left posterior coelom; m, mouth; m.a.c., median anterior coelom; oes., oesophagus; pr.L, preoral lobe; r.a.c., right anterior coelom; r.hyc., right hydrocoele; r.p.c., right posterior coelom; st., stomach. At nine days the larva has undergone much change. The right and left anterior coeloms have grown forward beyond the mouth. The left (in reversed larvae, the right) anterior coelom has extended backward so as to be in contact with the posterior 120 H. H. NEWMAN. coelom; while the right anterior coelom has a posterior prolon- gation extending as far as the posterior end of the stomach. The preoral ciliated band is triangular and the posterior end of the postoral band is turned well forward toward the mouth. At two weeks (Fig. 10) the anterior coeloms of the two sides have fused above the pharynx to form a continuous vesicle. The left anterior coelom is still separate from the posterior ccelom, which is now quite large. The posterior prolongation of the right anterior coelom has become relatively small in caliber, in fact has begun the process of rudimentation. At twenty-four days (Fig. u) the left anterior ccelom has .pnl. m. a. c. oes -Ihyc. int.-, PC. f.h.p. rp.c. FIG. 12. Advanced Bipennaria larva seen from the ventral surface, showing left hydrocoele well developed. FIG. 13. Young Brachiolaria larva, seen from the dorsal surface, with reversed asymmetry. Primitive tentacles (p.t.) are shown arising from the hydrocoele of the right side. Right hydropore (r.h.p.) and right posterior ccelom (r.p.c.) The preoral lobe has become modified into the attachment organ of the simplified Brachiolaria, with median sucker (ra.s.) and lateral suckers (/.s.). The remaining lettered structures are explained in previous legend. ASYMMETRY IN THE STARFISH. 121 united with the posterior coelom; the hydrocoele has begun to undergo marked thickening of its walls; the median anterior part of the fused anterior coeloms has sent forward a long median process into the preoral lobe of the larva which has now a trilobed shape. At twenty-six days (Fig. 12) the median diverticulum of the anterior coeloms has branched terminally into three parts, a large median one and two small lateral ones. The preoral lobe is now decidedly a trilobed affair. At the end of a month the larva takes on the characteristic features of a simplified Brachiolaria (Fig. 13). The changes that signal the arrival of the larva at the Brachiolaria stage have to do with the differentiation of the median and the two lateral brachia of the preoral lobe, organs that were already foreshadowed in the trilobed form of the preoral lobe and in the three-branched condition of the median anterior coelomic process. The middle brachium develops a well-defined attachment disk (Fig. 16) on which are present three pairs of papillae. The lateral brachia remain simple knobs, slightly thickened and clubbed at the ends. One branch of the anterior extension of the coslom runs into each brachium, but the lateral branches remain relatively small. A ventral sucker appears between the paired lateral brachia. The hydroccele of the Brachiolaria has become lobed in some indi- viduals, and in others it has given off some or all of the radial branches, destined to form the radial water canals (Figs. 13, 14, 15). Further than this it was not possible to go with certainty. For one month the larvae grew normally on a diet of the diatom, Nitschia, but almost over night the best cultures underwent retrogressive changes and died. One individual was found which had rounded up into a thin-walled vesicle. At first it was hoped that this individual had undergone or was undergoing meta- morphosis, but in less than a day it also died. The same fate attended three other carefully reared lots of larvae, all dying after about a month, and none, so far as I was able to' determine with certainty, completing the process of metamorphosis. In spite of this ill fortune it should be emphasized that larvae with right- handed (reversed) asymmetry were found among the most 122 H. H. NEWMAN. advanced of the Brachiolarice, and would without doubt have developed the mouth and other oral structures of the adult from the right-hand side of the larva. If this type of symmetry /77. S. int. FIG. 14. Brachiolaria larva seen from the left side. The primitive tentacles of the prospective adult are numbered i, 2, 3, 4, 5. Other lettered structures the same as in previous legends. FIG. 15. Posterior portion of another Brachiolaria larva, showing another stage in the formation of the prospective adult. FIG. 16. Ventral external view of the preoral lobe of Brachiolaria shown in Fig. 14. The median brachium (m.br.) has a median sucker (m.s.) with six papillae (/>). There are two short lateral brachia (l.b.) each with a poorly developed lateral sucker (l.s.). Between the lateral suckers is a median thickening (m.th.) which may be another sucker. reversal takes place sometimes in nature, as it doubtless does, there appears to be nothing about the anatomy of the radially symmetrical adult to indicate whether it has been derived from a larva with the normal or one with the reversed asymmetry. In attempting to account for repeated failures to rear these asteroid larvae through the period of metamorphosis, it has occurred to me that in nature there doubtless takes place a radical change in the dietary of the young individuals, which up to metamorphosis had been herbivorous, feeding largely upon diatoms, and that after metamorphosis they change to a car- nivorous diet; for the adult is strictly carnivorous. Hence ASYMMETRY IN THE STARFISH. 123 failure to metamorphose may be due to the absence of the forms of animal food essential for this process. III. EXPERIMENTAL INDUCTION OF REVERSED ASYMMETRY AND OF BILATERALITY IN Patina. Materials and Methods. In all experiments dealt with in this paper the eggs of one female fertilized by the sperm of one male were used both for experi- mental and control lots. Large numbers of fully ripe eggs and sperm are readily obtained in the manner described in the intro- duction to this paper. After several preliminary trials it was found that the blastula stage is the one best adapted for the experimental induction of reversals of asymmetry. Consequently all experiments herewith presented were performed upon early, middle, and later blastulae, and early gastrulae. The experimental procedure was as follows: An ice box containing a large, flat layer of ice was the only unusual apparatus employed. A single lot of eggs was divided into two, three, or four equal lots, care being taken to avoid crowding. As a rule, a single layer of eggs on the bottom of the dish was found to be not too many to permit of normal development. One lot was always kept as a control, being placed from the time of fertilization on in an unheated room, resting upon a concrete table and shaded from too strong light. The experimental lots contained in covered glass vessels were placed directly upon the block of ice. After about half an hour temperature readings were taken and it was found that the water at the bottom of the dish where the embryos lay ranged from 2 to 3 C., this being about the temperature of the surface of the ice itself. At this temperature all progressive development comes to a complete standstill, but is resumed as soon as the normal growth temperatures are reinstated. In spite of the fact, which will be more fully dealt with in a subsequent paper, that development is inhibited at the experimental temper- atures, the ciliated larvae lived and remained active in some cases for as long as ten days, though kept continuously on ice for the entire period. This circumstance is mentioned here merely to 124 H - H - NEWMAN. emphasize the point that, though development ceases, certain phases of vital activity continue almost unabated. In presenting the experimental results it seems advisable to give in some detail the results of the first complete experiment, which afterwards proved to be typical, and to follow this with a relatively condensed account of several nearly identical experi- ments that serve to corroborate the findings of the more in- tensively studied first experiment. EXPERIMENT i. A fine batch of eggs, fertilized at 2.15 P.M., April 6, 1923, was divided into four equal lots. After 17 hours, when the embryos were still unhatched blastulae, but were rolling about within the vitelline membrane, three lots were put on ice and one lot kept under normal conditions as a control. One lot (i,A) was kept on ice for one hour, a second lot (i, B] was iced for two hours, and a third lot (i, C) was iced for three hours. After exposure to low temperatures the experimental lots were placed beside the control lot in a room with temperature of about 15 C. and allowed to develop without disturbance for five days, at which time the first definite signs of asymmetry may be observed. On the fifth day both the control and the experimental lots were carefully looked over with reference to the percentage of larvae showing reversal of asymmetry. It was obvious under casual observation that there were many more reversed larvae in the experimental lots than in the control, but the exact difference could be determined only by means of a census of a representative number of individuals from each lot. The plan adopted was that of random sampling. A few larvae at a time were picked up in a pipette and transferred to a Syracuse watch glass and all examined to determine the position of the posterior ccelom, a rather conspicuous object at this time. When the count reached 100 the census was considered complete. This might be considered too small a number to give significant data, but when this is repeated a great many times it gains statistical value. ASYMMETRY IN THE STARFISH. 125 First Census (5 days old). Control 87 % of larvae have left posterior ccelom 13% ' right i, A (iced i hr.) 56% left 44% " " " right i, B (iced 2 hrs.) 79%" " " left 21% " " " right i, C (iced 3 hrs.) 36% ' left 14% " " " right 50% median " Note: In the last lot it was impossible to decide in just half of the larvae whether the posterior ccelom was destined to go to the left or to the right. The culture was somewhat belated and at this time and the asymmetry matter was unsettled in many of the larvae. Yet those that have settled their asymmetry show 72 per cent, with left-hand asymmetry as compared with 28 per cent, with right-handed asymmetry, a result very closely consistent with that shown in the second census of the same lot. Second Census (six days old). Control 97% of larvae have left water pore and pore canal 3% right " i, A (iced i hr.) 71 %" " " left 29% ' right " i, B (iced 2 hrs.) 74%" " " left 26% " " " right " i, C (iced 3 hrs.) 72%" " " left 13% " " " right " 15% ' both right and left water pores and pore canals Third Census (ten days old). Control 98 % of larvae with left asymmetry 2% " " " right i, A (iced i hr.) 89%" " " left 11% right i, B (iced 2 hrs.) 88%" " " left 12 % " " " right i, C (iced 3 hrs.) 6i%" " " left 21% right 18% " " " bilateral symmetry Note i. At this time it was evident that the experimental larvae, especially those in Lot i, C, were smaller and less advanced than the control. It was necessary to discard from those chosen for the census all distinctly abnormal larvae and those in which no water pores or pore canals had developed, and there were many of these. The suppression of these structures in experimental lots is in itself an important result, and is considered simply a more marked expression of the same morpho- logical response that is shown in disturbances of symmetry. Note 2. In connection with the 18 per cent, of bilateral larvae listed for Lot i, C in the last census, it should be noted that the paired water pores and pore canals were rarely equal in size, sometimes that of the left side and sometimes that of the right side being larger. 126 H. H. NEWMAN. Summary of Experiment i. 1. There was a fairly high percentage of control larvae in the first census showing reversed asymmetry of the posterior coelom, but the numbers of control larvae with reversed asymmetry in the later censuses was very small, only 3 per cent, and 2 per cent, respectively. 2. In every census (9 in all) the experimental cultures showed a much higher percentage of reversed larvae than the controls. The average per cent, of reversed larvae in experimental lots was 30.44, as compared with 4.66 for the controls. Thus there was over six times as high a percentage of symmetry reversal in experimental lots as in controls. 3. In Lot i, C (iced 3 hours) there were many bilateral larvae, only one of these appearing in any of the other lots. 4. The conclusion seems obvious that in this experiment the percentage of instances of symmetry reversal has been very markedly increased by retarding the development of the blastulae. In other words, reversal of asymmetry and bilateral symmetry have both been induced experimentally. 5. To determine whether this experiment is exceptional or typical, eight other experiments were carried out using the same materials and methods, but with some variations in the stages at which the larvae were iced and in the length of exposure to low temperatures. EXPERIMENT 2. Twenty-one hours after fertilization, when all larvae were hatched and swimming about as late blastulae and early gastrulae, they were divided into three lots. One was left as a control, a second iced for one hour, and a third iced for two and one half hours. First Census (5 days old). Control 94 % of larvae with left posterior coelom 6% " " " right 2, A (iced i hr.) 81% left 6% " " " right 13% " " " median 2, B (iced 2.5 hrs.) 63%" " " left 31%) right 6% " " " median ASYMMETRY IX THE STARFISH. 127 / Second Census (One week old). Control. . . .92% of larvae with left water pore and pore canal 8% ' right 2, A (iced i hr.) 86% left 13% right i% " " " bilateral " 2, B (iced 2.5 hrs.) 76%" " " left 24% right EXPERIMENT 3. Twenty hours after fertilization, when all larvae were free- swimming blastulae or early gastrulae, they were divided into three lots: one the control, one iced one and a quarter hours, and the third iced tw r o and a quarter hours. First Census (5 days old). Control 84% of larvae with left posterior coelom 16% " " " right 3, A (iced 1.25 hrs.) 69% left 24% ' right 7% " " " median 3, B (iced 2.25 hrs.) 77%" " " left 19% " " " right 4% " " " median Note: There was one specimen in Lot 3, B (iced 2.25 hrs.), which had two full- sized posterior cceloms on the left side, one behind the other. This might be interpreted as incipient metamerism. Second Census (one week old). Control 87 % of larvae with left asymmetry 13% ' right 3, A (iced 1.25 hrs.) 8i%" " " left 16% ' right 3 % ' bilateral symmetry 3, B (iced 2.25 hrs.) 83% left asymmetry 14% right 3% ' bilateral symmetry Note: This experiment shows only a slight increase in the percentage of reversed asymmetry through icing. It should be noted, however, that a large percentage of the larvae, though not quite so old as those in Experiment 2, were beginning gastru- lation and thus past the period of maximum susceptibility to symmetry reversal. This will be shown more clearly in Experiments 8 and 9. EXPERIMENTS 4, 5, 6, 7, 8, 9. Six different lots of eggs from six different females, fertilized by the sperm of one male, were dealt with exactly alike except that some w T ere allowed to develop a few hours longer before icing, and 128 H. H. NEWMAN. varying times of exposure to low temperatures were tried. Each culture was divided evenly into two lots, one for control and one for experiment. As all lots were allowed to develop for a week or more before the census, the results may be readily tabulated as though belonging to one census though it took about three days to complete the count. TABLE I. No. of Exper. Stage at which Iced. Time Iced. Symmetry of Larvae when Examined. A Early blastulae 2 hrs. Control 89% left 11% right Iced 76% left 24% right asymmetry 1 1 t < t 1 C Early blastulae 3 hrs. Control 83 % left 17% right Iced 73 % left 27% right 6 Advanced blastulae 7 hrs. Control 87% left 13% right Iced 72% left 24% right 4% bilateral symmetry 7 . . Advanced blastulae 1 8 hrs. Control 93 % left asymmetry 7% right Iced 69% left 21% right 10% bilateral symmetry 8 Early gastrulae i hr. Control 88% left 12% right Iced 87% left 13% right asymmetry o ' Later gastrulae 6 hrs. Control 91 % left 9% right Iced 93 % left 7% right SUMMARY OF TABLE I. (a) In Experiments 4, 5, 6, and 7, in which blastulae were used, the iced lots showed a marked increase of symmetry reversal over the controls. Including those showing bilaterality among the reversed types, there are over 27 per cent, on the average, of reversed larvae, as compared with 12 per cent, for the controls. (6) In Experiments 8 and 9, where gastrulae were used, there was no significant increase or decrease in the percentage of larvae with reversed asymmetry as the result of low temperatures. This is in accord with Experiment 3, in which a large proportion of the larvae were gastrulae and in which the increase in reversed larvae was relatively slight. (c) Only in Experiments 6 and 7, where the larvae were exposed for long periods to low temperatures (7 and 18 hours, respectively) were any larvae found with water pores and pore canals on both sides. It might then be said that bilaterality is harder to induce than reversal of asymmetry. ASYMMETRY IN THE STARFISH. I2Q IV. DISCUSSION OF EXPERIMENTAL RESULTS. Patiria miniata is a species of starfish that either exhibits in nature a fairly high degree of reversed asymmetry or else is so susceptible to influences tending to affect the symmetry system that even control cultures cannot be reared without the induction of some degree of symmetry reversal. Three experiments were tried with the idea of determining to what extent symmetry reversal could be reduced by taking every conceivable precaution against conditions that might be subnormal. Using only the freshest eggs and a minimum sperm concentration, allowing only a few eggs to a dish, keeping the dishes in running sea-water, feeding the larvae on a pure culture of the diatom, Nitschia, and introducing some fresh sea-water daily, there was found to be on the average about 8 per cent, of larvae showing reversed asym- metry, which is not apprecially smaller than that seen in the controls in the various lots previously dealt with. One may conclude then that Patiria is a species in which left-handed asymmetry is not fully fixed, but that the symmetry system is in a somewhat plastic state, a state well adapted for experimental purposes. Left-handed asymmetry is at the present time normal for echinoderms. Considerable work has been done which tends to prove that this asymmetry is present even in the unsegmented egg, and also that the first cleavage divides the egg into the prospective right- and left-hand sides. When twins are produced by the isolation of the blastomeres at the two-cell stage it has been found that one twin develops faster and becomes a more advanced larva at any given time than does the other. It seems then that one half of the egg, one of the two blastomeres, and one half of the larva is physiologically superior to the other in the sense that it has a higher general metabolic rate as expressed in more rapid growth. As a rule the superior side is the left side. Studies of the development of double monsters, which are incompletely divided twins, have shown that, in these bilateral systems, whenever one component for any reason gains a physio- logical ascendancy over the other, there is a strong tendency for the superior component to remain normal and for the inferior individual to become subnormal and to be reduced to a condition 130 H. H. NEWMAN. of a mere parasite upon the normal component. Thus arises the condition known as that of the "autosite and parasite" type of double monsters. In extreme cases of this sort the "parasite" component is reduced to a rudimentary cyst embedded in the tissues of the parasite. This condition I have already interpreted as one of dominance and subordination in growth. According to this principle, which has been shown to hold good in large numbers of cases in both animals and plants, a rapidly growing or differentiating region, or apical region, in an organism tends to inhibit the growth or differentiation of like regions within the realm of its influence. This is true of equivalent bilateral regions, which may be conceived of as intense rivals. If one rival region gains an ascendancy over the other, the inferior region tends to be suppressed. Applying this principle to echinoderm development, we can readily recognize the analogy to the "autosite and parasite" situation in double monsters. Commonly the left side acts as the superior or "autosite" component, and the right side as the inferior or "parasite" component. The result is that certain important and actively differentiating structures (the posterior coelom, the hydropore, and the hydroccele) appear first on the more rapidly developing left side, and that these structures sup- press the development of equivalent structures on the right side and tend to reduce to vestiges certain other coelomic structures of the right side that have already appeared. In other words, the typical adult echinoderm is morphologically the surviving left- hand component of a twin or double monster, and its radial symmetry is merely a sort of mechanical adjustment necessary in order to avoid too pronounced a one-sidedness. If, however, the rate of growth of a larva be sharply checked in such a way that the physiological state of the right and left components are reduced to a parity, it should be possible to get an organism both halves of which are equivalent. As a matter of fact, this result is not infrequently realized in the bilateral larvae that have arisen as the result of rather prolonged icing. The commoner result, however, is that the right side usurps the ascendancy typically belonging to the left side and gives rise to larve with reversed asymmetry. If the physiological condition ASYMMETRY IN THE STARFISH. of the two sides were really reduced to parity, we should always have, as the result of icing, bilateral larvae; but we should not, a priori, expect a retarding agent to effect the two sides equally. On the contrary, the expectation would be that the superior side would be more seriously affected than the inferior, so that when recovery took place the originally inferior side would emerge from the growth retarding influence first and would then behave like a physiologically superior side throughout development. Two difficulties immediately present themselves. Why, it may be asked, are not all larvae reversed? And why do not the right- handed larvae, about ten per cent, of which seem to be present in each lot, reverse and become left-handed? The second of these queries may best be answered first by admitting that there is no ground for denying that at least some of the right-handed individuals, under the conditions of the experi- ment, do reverse their already reversed asymmetry back to the specific left-handed asymmetry. If this be taken into account, we w r ould have to add materially to the observed percentages of reversed larvae, for a certain percentage of the left-handed larvae that are counted as unreversed are doubtless reversed right- handers. But there is still a large percentage of larvae that have failed to undergo symmetry reversal, and our problem is to account for their failure to reverse. The only solution of the problem is one that takes into consideration the well-known fact that no two eggs or embryos are alike in the degree of their susceptibility to inhibiting agents or in their responses to given agents. It is probable that only a limited number of larvae at any one time are in just that state of susceptibility the normal response to which is symmetry reversal, and that these unsus- ceptible types are responsible for the surplus of left-handed larvae in all experimental cultures. Other larvae show other expressions of growth inhibition, while still other particularly hardy forms show no effects at all. The problem that confronts us in this connection is in no essential respect different from the one that has faced experi- mental embryologists ever since the first eggs or embryos were subjected to conditions other than normal. It makes no difference whether one uses toxic chemicals, anaesthetics, low 132 H. H. NEWMAN. temperatures, or hybridization, the result is always the same: some individuals are affected in one way, some in another, and some not all. A lot of Fundulus eggs, for example, are exposed for a given time to a given intensity of X-rays. Some of the radiated individuals, as Miss Hinrichs has shown, develop into two-headed monsters, others become cy- clopic, others show lesions of the heart or circulation, not to mention many other typical teratological conditions. Our pre- sent problem does not concern itself with any attempt to account for the individual differences in susceptibility among eggs of the same batch : this is a problem of heredity, not one of development. To revert specifically to the present experiments, it should now be said that, although our attention has been focused upon symmetry reversal to the exclusion of any other effects of exposure to low temperatures, it would be unfortunate to leave the reader with the impression that everything else about the experimental larvae was strictly normal. The truth is that some larvae develop no ccelomic structures at all ; that others show a tendency toward a metameric duplication of anterior or posterior coeloms; that occasionally a larva has a hydropore connected with the posterior ccelom as well as the anterior. It should also be added that in all experiments in which the icing of larvae lasted more than about two hours, many other expressions of growth inhibition appeared, sometimes associated with symmetry reversal and sometimes not. My strong impression was that on the whole the larvae with reversed asymmetry were rather more normal in other respects than those with typical asymmetry. In conclusion then, it may be said that symmetry reversal, and that particular phase of it which we may call induced bilaterality, are merely some of the specific effects resulting from arresting the growth of the larvae at the blastula stage. If the arrest takes place much earlier or much later than the blastula stage other expressions of differential inhibition prevail and there is no evi- dence of symmetry reversal. If eggs are iced during early cleavage or even before cleavage the particular response is twinning, while icing at other times give equally striking but different results. These matters, however, must be left for another study and another report. ASYMMETRY IX THE STARFISH. 133 One of the principal values accruing from experimental embry- ology is that it often throws light upon the mechanism of normal development. It would be strange, then, were we to fail to find in this study no suggestion as to a more complete understanding of the significance of asymmetry in the echinoderms and of that remarkable series of changes involved in metamorphosis. I venture, therefore, to conclude this paper with an attempt to reinterpret in physiological terms the phenomenon of echinoderm metamorphosis in general. V. A PHYSIOLOGICAL INTERPRETATION OF ECHIXODERM METAMORPHOSIS. There are many remarkable ontogenetic transformations in nature, but none so radical as the metamorphosis of the echino- derm larva into the adult. In the metamorphosis of the cater- pillar into the butterfly, the axes of polarity and of symmetry are carried over unchanged from larva to adult ; in the metamorphosis of the tunicate tadpole larva into the adult sea-squirt there is a relative inhibition of the apical regions of both axes, yet the polarity and symmetry of the larva are maintained in the adult; but in the metamorphosis of the echinoderm both polarity and symmetry of the larva are, as it were, entirely ignored, and the axes of the adult are established practically de novo and without much reference to previously existing axiate relations. In terms of the physiological gradients involved, it may be said that what apparently happens is, that the original gradients of the egg and of the larva become practically obliterated and a new major axis arises approximately at right angles to the larval major axis with its apical region at a point somewhere near the middle of the left side of the larval stomach at the point where the hydroccele arises. The crucial event in this shifting of axiate relations is, morphologically speaking, the differentiation of the hydroccele. This structure, as all know, arises as an outpouching of the posterior end of the left anterior ccelom (enterocoele). There comes a time in late larval life when all other growth changes cease and the enlargement and differentiation of the hydroccele seem to be the main changes taking place. This rapidly growing region then assumes dominance over the rest of 9 134 H. H. NEWMAN. the organism, becomes the apical point of a new axis, and assumes control of the entire subsequent development. For some time after the new or adult axis has arisen the original lacval axes are maintained in so far as the general external form and behavior of the individual are concerned, but sooner or later these yield to the increasing dominance of the new apical region, as may be seen by the casting off or resorption of the most characteristic features of the larva, such as the ciliated arms or bands, the preoral lobe with its mouth and accompanying structures, and in the rudimentation of the ccelomic structures of the right side. The developmental history of the hydroccele may briefly be reviewed. Physiologically, it represents a region of rapid cell proliferation in a larva which is, at the time in question, practi- cally at a developmental standstill. As is well known, any rapidly growing region in an organism has the capacity of domi- nating the development of other regions about it and of sup- pressing the differentiation of similar or homologous regions else- where in the organism. It may also, in the course of time, inhibit the development of rival regions and cause their atrophy or resorption. Morphologically speaking, the events are as follows: The hydroccele, at first a small lobe of the left anterior enteroccele, buds off five primary tentacles destined to become the five radial water canals. These canals assume a radiate position about a central ring through which the new mouth passes. It is about this ring and its five more in some species primary tentacles that the new adult radial organization centers. The primary axis of the adult passes through the center of the water-vascular ring. This new axis can be called neither an antero-posterior axis nor an axis of symmetry; it is something quite sui generis, as is recog- nized by those who have given to the two poles the names "oral " and "aboral." The secondary axis is also quite unique, for there is no true symmetry, but merely a superficial semblance of radial symmetry. Symmetry Relations of Larva and Adult. From the standpoint of the original larval axes of symmetry, it is clear that the events that lead up to metamorphosis occur typically only on the left side. On this account the situation has long been thought of as an outstanding example of asymmetrical development, and there ASYMMETRY IX THE STARFISH. 135 has been much speculation as to its morphological and its phylo- genetic significance. Prominent among the speculations that have arisen in connection with this phenomenon are those that concern themselves with the phylogeny of the echinoderms. It is generally held that the ancestral echinoderm was, like the larva, bilaterally symmetrical and that hydrocceles and their derivatives occurred on both sides. The objection to this view is that such an organism would be biaxiate, with two mouths and oral surfaces, two water-vascular systems and nerve rings. In view of this serious objection, it would seem best to look upon the develop- ment of the new axis as an evolutionary process resulting from the establishment of a pronounced asymmetry, and that it was only in connection with this newly established asymmetry that the development of such structures as the new mouth, the nerve ring, the Aristotle's lantern in echinoids, and similar structures, takes place. This view does not preclude the possibility of an originally bilateral ancestor, but merely denies to such an ancestor the bilateral possession of such specialized structures as the hydroccele and its derivatives that arise only in connection with the apical region of an entirely new axis. When, however, larvae are found with hydrocceles and kindred structures on both sides, evidence seems to be afforded for the theory that the ancestral echinoderm had two sets of hydroccele structures bilaterally arranged. If such anomalous larvae are to be considered as atavistic, how can we explain the occurrence of larvae in which the hydroccele and its appurtenances appear exclusively on the right side? Is the right-handed or reversed larva also an atavistic reminiscence of an ancestor that had right- handed asymmetry? Even the most obdurate phylogenist would hesitate to go so far ; yet the occurrence of reversed asymmetry is commoner than is bilaterality, and both can be induced by the same experimental means. Moreover, the two conditions grade into each other; for there are many larvae in which hydropores and such structures occur on both sides but in which one or the other side shows these structures in all states of partial rudi- mentation. How then can we explain the occurrence both in nature and in experiments of reversed and bilateral larvae? The previous parts 136 H. H. NEWMAN. of this paper have indicated that all anomalies of this kind are merely induced developmental disturbances and that neither bilateral nor reversed larvae have any more phylogenetic signific- ance than has the occurrence of occasional two-headed fishes or than the sporadic occurrence of sinistral asymmetry in a species of snail typically dextral. The Nature and Origin of Asymmetrical Development. The question naturally arises as to why one side of an echinoderm larva should develop structures not duplicated on the other. Two possible explanations suggest themselves: first, that the cells constituting one half of the larval body have a higher rate of metabolism and hence a somewhat more rapid rate of pro- liferation and of differentiation than those of the slower side; second, that the asymmetry is due to some environmental factor that influences one side only or the two sides differently. In sup- port of the first explanation it should be said that the cells destined to form the two bilateral halves of the larva are set apart from a very early period. Driesch (1906) found that bilaterality was definitely established at the eight-cell stage, and presented data that tended to show that the first cleavage plane coincides with the sagittal plane of the larva. In a number of cases he found that when twin gastrulae were formed from the two blasto- meres these twins showed mirror-image symmetry. This obser- vation I have been able to confirm in connection with studies of twinning in Patiria (Newman, '22). Bilateral symmetry then appears to be established at the beginning of cleavage and is probably predetermined in the unsegmented egg. More significant still for our problem is the fact that, in a large proportion of twins produced from the physiologically isolated blastomeres of the two-cell stage, the rate of development of the two larvae is markedly different. Almost invariably one of a pair of such twin blastulae or gastrulae within a common vitelline membrane is well in advance of the other. In an earlier stage it can be seen that one twin blastula has fewer cells and a smaller blastoccele than the other. In later stages one gastrula is relatively normal and quite active, while the other is not in- frequently subnormal and shows signs of disintegration. These data indicate that the asymmetry which is in later stages ASYMMETRY IN THE STARFISH. 137 expressed in the unilateral appearance of the hydrocoele and its derivatives, is traceable to a very early stage of development and is probably present in the unsegmented egg. Moreover, it appears merely to consist of an inequality of metabolic rate between the antimeric halves of egg and embryo. The difference between the two sides is in no sense qualitative, as some writers prefer to believe, but is purely quantitative; for, as we have seen, all the structures that characterize the superior side may be inhibited on that side and may be thus allowed to appear on the other side, or they may appear on both sides at once in case the physiological state of the two sides is experimentally equalized. The basis of asymmetry in the typical echinoderm is then the result of a dynamic imbalance between the earliest primordia of the prospective antimeric halves of the embryo. On this account one side develops more rapidly than the other, especially in the region where the hydroccele arises, and the differentiation of the hydrocoele and its derivatives tends to suppress the growth of equivalent structures on the opposite side. This view is con- sistent with the experimental work reported in this paper and with a large mass of morphogenetic work of other sorts. No claim of finality is made for the conclusions reached in this study, but it is hoped that some of the ideas expressed may form a starting point for future investigation. BIBLIOGRAPHY. Gemmill. J. F. '14 The Development and Certain Points in the Adult Structure of the Starfish. Asterias rubens. Phil. Trans. Royal Soc., Series B. '15 Twin Gastrulae and Bipennariae of Luidia sarsi. J. Marshall Biol. Ass., Vol. 10, No. 4. '16 Double Hydroccele in the Development and Metamorphosis of the larva of Asterias rubens. Q. J. M. S., Vol. 61, No. 241. MacBride, E. W. '11 Two Abnormal Plutei of Echinus, etc. Q. J. M. S., Vol. 57, Xo. 226. '18 The Artificial Production of Echinoderm Larvae with Two Water-vascular Systems. Proc. Roy. Soc., B, Vol. 90. Newman, H. H. '21, a On the Development of the spontaneously Parthenogenetic Eggs of Asterina (Patiria) miniata. Biol. Bull., Vol. XL., No. 2. '21, b On the Occurrence of Paired Madreporic Pores and Pore Canals in the Advanced Bipennaria Larva? of Asterina (Patiria) miniata, etc. BIOL. BULL., Vol. XL., No. 2. 138 H. H. NEWMAN. '21, c The Experimental Production of Twins and Double Monsters in the Larvse of the Starfish Paritia miniala, etc. Jour. Exper., Vol. 33, No. 2. '22 Normal Versus Subnormal Development in Patiria miniata, etc. Biol. Bull., Vol. XLIII., No. i. '23 The Physiology of Twinning. Univ. of Chicago Press. Oshima, H. '21 Reversal of Asymmetry in the Plutei of Echinus miliaris. Vol. XLIX September, No. 3 BIOLOGICAL BULLETIN THE FINDING OF THE QUEEN OF THE ARMY ANT ECITON HAMATUM FABRICIUS l WILLIAM MORTON .WHEELER. After the peculiar, large-bodied, wingless females, or queens of the army ants of the American tropics belonging to the typical subgenus Eciton had been sought in vain for many years by many collectors I finally succeeded in July, 1920, in securing two queens of Eciton burchelli Westwood in British Guiana, and the same year published an account of their capture. 2 Since that time the queens of three other typical Ecitons have been discovered in rapid succession. In August, 1920, Mr. F. M. Gaige took the queen of E. vagans Roger in Colombia, but has not yet described the specimen. In 1923 Dr. Carlos Bruch published an account with fine figures, of a queen of E. dulcius Forel, subsp. jujuensis Forel, which he took in the Argentine. 3 He has also described a queen of Eciton hetschkoi Mayr, captured by Weiseran July 1923 in the same country. 4 The latter is not, however, a true Eciton, but belongs to the subgenus Acamatus, which occurs also in our Southern United States, from which I have described the queens of three species (schmitti Emery, opacithorax Emery and caro- linense Emery). More recently Reichensperger has published a description of the queen of E. quadriglume Haliday, from four specimens taken by Franciscan brothers on the Rio Negro, Brazil in the fall of 1923. 5 This Eciton is very closely related to 1 Contributions from The Entomological Laboratory of the Bussey Institution, Harvard University, No. 244. 2 "Observations on Army Ants in British Guiana," Proc. Amer. Acad. Arts and Set., 56, 1921, p. 291-328, 10 figs. 3 "Estudios Mirmecologicos," Rev. Mus. de la Plata, 27, 1923, p. 172-179, i pi. 8 text-figs. 4 "Descripcion de la reina 'Eciton (Acamatus) Hetschkoi' Mayr," Rev. Soc. Argent. Cienc. Nat., 7, 1924, p. 232-235, 2 pis., 4 figs. 5 "Das Weibchen von Eciton quadriglume Hal., einige neue ecitophile Histeriden und allgemeine Bemerkumgen," Zoo/. Anzeig., 60, 1924, p. 201-213, 5 figs- 10 139 I4O WILLIAM MORTON WHEELER. dulcius. Among the few remaining species of true Eciton of which the queen has persistently eluded capture is E. hamatum. During the past summer (1924) I was fortunate enough to secure a beautiful specimen of this insect from a bivouacking colony just behind the new tropical laboratory on Barro Colorado Island in the Panama Canal Zone. This find is, perhaps, the more important because hamatum is the type of the genus and because it has such a wide distribution (from Brazil to Mexico) and is so common that its raids have been noticed by nearly every zoolog- ical explorer in tropical America. It is, in fact, among the more than one hundred described species of Eciton, the one which has been longest known and most often cited in the literature since Fabricius first described the soldier in 1781. At 8.00 A.M. on August 1st, after a heavy rain on the preceding day, while collecting behind the laboratory I encountered an army of Eciton hamatum foraging along the central trail (Fig. i) and in the adjacent jungle. The workers were plundering numerous nests of ants (Pheidole, Acromyrmex and Camponotus species) and carrying away their helpless larvae and pupae. On one of the tall trees they had found a large nest of a yellowish wasp (Polybia sp.) and for some hours were bringing in the brood in great quantities. Dr. Curt Richter devoted the morning to watching the files, computing their rate of movement and the nature of their prey. By following the various converging columns we eventually located the colony which was bivouacking less than a hundred yards from the laboratory near the edge of the jungle. The great mass of ants presented an astonishing spectacle (Fig. 2). They had selected the base of a small tree, which, about 15 inches above the ground, was joined to the trunk of a young stilt palm by a looped liana and some twigs, together forming a horizontal frame. The larger tree trunk was inclined to the north so that the surface of the bark and the ground beneath were quite dry. In this spot, which had evidently been sheltered from the heavy rains for several days, the ants had congregated in a compact, cuboidal mass, 13 to 15 inches high and broad, and suspended from the frame above mentioned. Dozens of large workers hung by their claws from the twigs and supported solid clusters and curtain-like sheets of workers and soldiers, the THE FINDING OF THE QUEEN OF THE ARMY ANT. 14! FIG. i. Entrance to the central trail through the jungle on Barro Colorado. The Ecitons were foraging along this trail. Their suspended swarm (shown in Fig. 2) was situated in the jungle a few hundred feet to the left. Photograph by Dr. David Fairchild. 142 WILLIAM MORTON WHEELER. latter dotting the somber mass of intertwined brown-red bodies, legs and antennae with their large shining, ivory-white heads. The mass hung down to the ground but left a small opening at the bottom on the west side, a kind of portal, through which the FIG. 2. Bivouacking colony of Eciton hamalum. The minute white dots are the heads of the soldiers scattered through the suspended mass of workers. Photo- graph by Dr. Frances G. Smith. converging bootyladen files of workers and soldiers were con- tinually pouring to deposit their burdens in the center of the mass and on the ground immediately beneath it. At my request, Miss F. G. Smith, who happened to be visiting the laboratory, kindly undertook to photograph the colony, and later Dr. Fairchild took a more enlarged flash-light picture. Considering the fact that the ants were clustered in a dark spot, the two photographs, which are shown in Figs. 2 and 3, give a good likeness of the shape of the cluster and of its composition. The blurred areas in Fig. 3 are, of course, due to the movements of several workers on its surface. THE FINDING OF THE QUEEN OF Till-: ARMY ANT. 143 FIG. 3. Part of the suspended bivouacking colony of Eciton hamalum Fabr. on Barro Colorado Island. The interlacing legs and antennae of the thousands of ants are distinctly shown. The white spots are the heads of the soldiers scattered among the swarm. About j natural size. Photograph by Dr. David Fairchild. 144 WILLIAM MORTON WHEELER. Throughout the day the colony showed no essential change. It seemed very probable that the queen was concealed somewhere in the cluster, and although my enthusiastic young friend, Dr. Richter, wished to keep the colony under observation for several days, if possible, my arguments, inspired by fear that it might decamp during the night and disappear in the jungle and a desire FIG. 4. Soldier and small worker of Eciton hamatum Fabr. Dr. David Fairchild. Photograph by to secure the unknown female and any guests, or ecitophiles that the mass might contain, eventually convinced him and my other companions, Mr. Nathan Banks, Dr. David Fairchild, his son Graham, and Mr. Frederick Burgess that it would be advisable to kill the whole mass and sort it over carefully at our leisure. The opportunity was, indeed, exceptional, because bivouacking colonies of army ants are rarely seen and when one is encountered it is almost always in some inaccessible place, in or under a large log, in a hollow tree-trunk or a hole in the ground. The de- struction of the colony, however, seemed to be a serious matter, both because it involved eliminating one of possibly only a few THE FINDING OF THE QUEEN OF THE ARMY ANT. 145 colonies of the species on the island, which is an animal and plant reservation, and because it is not an agreeable task to tackle a populous colony of one of the larger Ecitons. Fortunately Tiamatum is far from being as vicious or from stinging as badly as E. burchelli, though the soldiers (Fig. 4) are able to run their recurved mandibles into one's skin in such a manner as to make them as difficult to remove as an equal number of fish-hooks. Just before dusk we matured and carried out the following plan : A towel saturated with ether was tightly wedged into the bottom of a five gallon gasoline tin from which the top had been removed. After cautiously cutting away the main supports of the frame- work of twigs, the tin was suddenly inverted over the mass of ants, and at the same time one of the party sprayed those left outside the receptacle with "Komo," a preparation used for killing house-flies and mosquitoes. The whole mass of asphyxi- ated insects, which measured two and one-half gallons, and comprised many thousands of individuals, was then examined. The queen was found, together with a great number of nearly full grown worker larvae, but no pupae, a considerable amount of prey, mainly ant and wasp larvae and pupae, and a series of interesting ecitophiles. On the following morning the hamatum workers and soldiers, which had been out foraging when the main body of their colony was captured, were found to have formed four small compact clusters, each about the size of an orange at widely separated points in the jungle. As usual under such circum- stances these meager remnants of a vigorous population had a demoralized and dejected appearance. In the course of a few hours they decamped and disappeared in the undergrowth. The E. hamatum queen (Figs. 5, 6 and 7) measures 15 mm. (head, thorax and petiole 6.5 mm., gaster 8.5 mm.) and differs from the queen of E. burchelli Westwood in the following par- ticulars: The color is uniformly ferruginous red, of a distinctly paler tint than in burchelli, and there are no markings on the gaster, except some brownish clouds on the middle of the second to fourth segments, near their posterior borders, and a few brownish spots on the fifth segment. The mandibles are shorter and slightly broader, the antennal funiculi longer and stouter, the cheeks more inflated, the antennal foveae larger and more sharply 146 WILLIAM MORTON WHEELER. FIG. 5. Eciton hamalum Fabr. Female, dorsal view. Photograph by Prof. C. T. Brues. FIG. 6. Eciton hamalum Fabr. Female, dorsolateral view. Photograph by Prof. C. T. Brues. THE FINDING OF THE QUEEN OF THE ARMY AXT. 147 defined and the eyes slightly larger than in biirchelli. The pronotum is longer and more narrowed anteriorly, the mesonotum less convex and not distinctly grooved in the middle, the tubercles FIG. 7. Eciton hamatum Fabr. female; a, head, dorsal aspect; b, thorax and petiole in profile; c, same, dorsal aspect; d, hypopygium. on the sides of the metanotum more pronounced. The horns on the epinotum are much longer and more pointed, those on the 148 WILLIAM MORTON WHEELER. petiole much shorter, blunter and more rounded than in West- wood's species. The surface of the body, even of the sides of the epinotum and petiole are subopaque, the coarse punctures on the thoracic dorsum are much as in burchelli, but those on the convex dorsal portion of the first gastric segment are larger and more scattered. The hairs are shorter, finer, less golden and decidedly less abundant, especially on the legs, which in burchelli are very pilose. Other structural details may be gleaned from the ac- companying figures (Figs. 5, 6 and 7). This queen is obviously in the same physiological stage as the two queens of E. burchelli described in my former paper. Owing to the small size of her gaster she must be either a young indi- vidual or, if the mother of the numerous larvae, workers and FIG. 8. Histerid ecitophile Euxenister wheeleri Mann, from bivouacking colony of Eciton hamatum Fabr. Photograph by Prof. C. T. Brues. soldiers among which she was living, must have passed through a fecund period. The freshness of her color, integument and pilosity seems to support the former alternative. That the colony from which she was taken was not as large as some hamatum colonies I have seen, might also indicate that she was a young individual or one in which the gaster had temporarily returned to a contracted state after a first period (perhaps seasonal) of fecundity. THE FINDING OF THE QUEEN OF THE ARMY ANT. 149 Of the eleven species of insects found in the bivouacking mass of hamatum workers and soldiers nine are Coleoptera, one a large Thysanuran (Atelura sp.) and one a mite resembling Discopoma. The beetles have been studied by Dr. W. M. Mann and Mr. H. S. Barber of the National Museum. The former writes me that there are five species of ecitophiles, namely two Staphylinids and three new species of Histeridae. One of these is a Trog- lostermis, one a Synodites and the third, the remarkable form represented in Fig. 8, has been described by Dr. Mann as Euxenister wheeleri. The four remaining forms are bark- inhabiting or fungus beetles which were evidently brought in as prey by the foraging columns of ants. Mr. Barber identifies the specimens as a species of Staphylinus (sensu lato], two species of Erchomus and a Rhymbus (Endomychidae). He sends the following note concerning the last, of which several specimens were taken : ' ' The Rhymbus seems to be Rh. hemisphcericus Gerst. 1858, but not the species treated under that name by Gorham 1873 (Biol. C-A) although his piceus is perhaps a synonym of Gerstackers species. Unfortunately an earlier generic and specific name (Bystus coccinelloides Guerin 1857, from Colombia) is listed under Rhymbus Gerst. 1858, and from its original de- scription I cannot see why this latter name should not apply to the Barro Colorado specimens. The species is new to the National Collection.' A NEW GUEST-ANT AND OTHER NEW FORMICID^ FROM BARRO COLORADO ISLAND, PANAMA. 1 WILLIAM MORTON WHEELER. The researches of the past twenty-five years have shown that the number of ants which regularly live in more or less intimate symbiotic or parasitic relations with other ants is considerable, and that the behavior exhibited under these conditions is re- markably diverse. Until recently, however, such social parasites were known only from north temperate and subtropical regions. Several very interesting forms have now been discovered in the tropics and even in the south temperate zone. The following is a list of the workerless parasites (permanent social parasites) resembling the European Aner gates atratiilus Schenck and the North American Epcecns pergandei Emery that have been de- scribed from paleotropical and neotropical localities: (1) Wheeleriella wroughtoni Forel (1910, 1911), described from female and male specimens found living in the nests of Mono- morium solomonis indicum Forel at Poona, India. (2) Parapheidole oculata Emery (1900, 1914-15), described from a female specimen from Madagascar and supposed to be a workerless parasite of some species of Pheidole. (3) Anergatides kohli Wasmann (1915). Males and females taken in nests of Pheidole megacephala melancholica Santschi in the Belgian Congo. (4) Plagiolepis (Anoplolepis] nuptialis Santschi (1917). Males taken in Cape Province by Dr. H. Brauns in nests of P. (A.} custodiens Sm. (5) Pseudoatta argentina Gallardo (1916). Female and male described from specimens taken in Argentina and supposed to be parasitic in the nests of some fungus-growing ant of the genus Mcellerius, probably M. bahani Emery. (6) Xenometra monilicornis Emery (1917, 1921), described 1 Contributions from the Entomological Laboratory of the Bussey Institution, Harvard University, No. 241. 150 NEW FORMICID/E FROM BARRO COLORADO ISLAND. 15! from female specimens taken on the island of St. Thomas, W. I., in a nest of Cardiocondyla emeryi Forel. (7) Bruchomyrma acutidens Santschi (1923), described from female specimens taken by Carlos Bruch in Argentina in the nest of Pheidole str obeli richteri Forel. Besides the accounts of these extreme, workerless, permanent parasites numerous scattered and more or less incomplete notes have been published on other types of social parasites within the tropics. No slave-making ants have been recorded, but certain African and Malagasy species of Crematogaster of the subgenera Oxygyne and Atopogyne are probably temporary parasites in the nests of species of the typical subgenus Crematogaster, and the phenomenon known as "parabiosis," as Forel (1898), Mann (1912), and I (1913, 1 92 1 a) have shown, is well-developed among certain neotropical ants belonging to several genera (Dolichoderus, Crematogaster, Odontomachus, Camponotiis] . There is, moreover, in the tropics of both hemispheres a long series of tiny "thief," or lestobiotic ants, which belong to the Myrmicine genera Solenopsis, Oligomyrmex, Aeromyrma, Pcedalgus, Carebara, Erebomyrma, Tranopelta, Liomyrmex, Pheidole, Xenomyrmex, Monomorium, etc. and live in or very near the nests of other ants or of termites. During late July and early August, 1924, while studying the exuberant ant-fauna about the new tropical laboratory on Barro Colorado Island, in the Panama Canal Zone, I repeatedly came upon a small and peculiar Megalomyrmex living in the fungus gardens of a Sericomyrmex. Since the behavior of these insects represents a new type of symbiosis or xenobiosis, I here describe them, prefacing my account of each with a few historical notes. The taxonomic descriptions of the two ants and of some small lestobiotic species associated with the Sericomyrmex colonies are placed at the end of the paper. Our knowledge of the habits of the Attine ants of the genus Sericomyrmex is rather meager. The earliest and best account is that of Urich published as early as 1895. It refers to a Trinidad species, later described as 5. urichi Forel, but at the time of Urich's writing supposed to be opacus Mayr. 'The nests of these ants," he says, "are found commonly about Port of Spain, in gardens, in the grass as a rule, but sometimes in the flower beds, 152 WILLIAM MORTON WHEELER. and from their peculiar raised entrance can readily be recognized. They are always excavated in clayey soil, and the raised entrances, which are more or less cylindrical, are constructed with the particles of earth resulting from their mining operations and are about an inch in height. In young colonies this entrance leads into a small chamber, about six inches below the surface of the ground, situated, not at the end of the gallery but either to the left or right of it. As the colony increases the ants do not enlarge this original chamber, but, piercing its side, form another chamber near it with a small entrance hole. In large colonies, which never consist of more than about 200 individuals, a nest consists of two or three chambers which open on the original excavation. This is no longer used for growing the fungus in, but forms a sort of antechamber which generally contains material brought in by the ants to grow their mushrooms on, which is deposited here and gradually made use of. The chambers adjoining are more or less round, with a diameter of about 2-3 inches, and any small roots of plants growing through them are not cut away but used by the ants to hang their mushroom gardens on. These fill the interior of the chamber and consist of a gray spongy mass consisting of a great number of little irregular cells and resembling a coarse sponge, amongst which are scattered larvae, pupae, and ants. The walls of the cells consist of small round pellets resembling dust shot and are penetrated by and enveloped in white fungus hyphae, which hold the mass together. Strewn thickly upon the surface of the garden are to be seen round white bodies about a quarter of a millimeter in diameter. These are what Moller terms "Kohl- rabi" clumps, and consist of an aggregation of hyphae with spherical swellings on their ends. It is on this that the ants feed. The fungus found by Moller in the nests of the Brazilian fungus growers (Acromyrmex) is the Rozites gongylophora Moller, and if it is not the same species cultivated by S. opacus it is, at any rate, very nearly related to it. As material to grow their mushrooms on the ants make use of particles of fruit, flowers, and leaves, but prefer the fruit. They do well in artificial nests and are easy to watch. I have tried them with all kinds of vegetable products; they have taken orange, banana, rose petals and leaves, and once they even made use of the dried glue from the back of an old book NEW FORMICID^E FROM BARRO COLORADO ISLAND. 153 lying near their nest, but that day they had nothing else; if the choice be left to them they invariably take fruit and seem to prefer the orange among these. Very small particles of the white skin of the oranges are torn off, and, after undergoing a slight kneading process in the ants' mandibles, are placed in the nest. The neutres are all of the same size, varying but slightly and never exceed 4 mm. in length. They are more diurnal in their habits than other species of fungus growers, but also work a little at night. -I have found winged forms in the nests in the month of July." The following year Forel, while recording his observations on the Attini of Colombia, published the following remark (1896, p. 406): 'The fungus gardens of the large A tta species, of the subgenera Trachymyrmex For. and Mycocepurus For., as well as of the genus Sericomyrmex were previously unknown and were discovered by me. The gardens of the three latter groups seem to resemble those of Apterostigma, and these small ants are never seen on the trees in the act of cutting leaves. They bring into their nests small, desiccated vegetable particles; their fungus garden lies very deep in the earth and is very imperfect." The Colombian species of Sericomyrmex (S. diego Forel) observed by Forel was not described till 1912 (p. 193). He then added the following note: "Don Diego, at the foot of the Sierra Madre de Santa Marta, Colombia, the third of March, 1896, in the forest; nest in the humus, with a crater of coarse granules. A beautiful fungus garden at a depth of 2 decimeters in the earth. The worker feigns death like the species of Cyphomyrmex. They collect little green vegetable particles resembling an alga and make their fungus garden of them and other debris." Essentially the same account was published by Forel in the " Biologia Centrali-Americana" (1899-1900, p. 37). It will be noticed that Urich and not Forel was the first to observe the fungus gardens of Sericomyrmex and that the latter's various accounts contain some glaring discrepancies. In one account the garden is described as "very imperfect," in another as "beautiful." Furthermore, he could not have seen the fungus garden of Mycocepurus, which cultivates a peculiar fungus very similar to if not the same as the Tyridiomyces formicarum culti- vated by Cyphomyrmex rimosus (Wheeler, 1907, p. 771). 154 WILLIAM MORTON WHEELER. For many years Sericomyrmex has been the one genus of Attine ants which I have not had an adequate opportunity to study. These insects are local or sporadic in their occurrence and very unobtrusive and timid in their behavior. Even on the few occasions when I have encountered them I could make but a superficial examination of their nests. My note-books contain only the following jottings: FIG. i. Laboratory of the Institute for Tropical Research on Barro Colorado Island, in Gatun Lake, Panama Canal Zone. Photograph by Mr. James Zetek. Dec. 15, 1911, I happened on a number of nests of a small Sericomyrmex, which I have since described as 5. zacapanus (1924) on the clay banks of a small irrigating ditch in an orchard at Zacapa, a very arid locality in Guatemala. The nests had small craters 2-3 inches in diameter, which were either single or in rows, like those of Solenopsis geminata nests, and were covered with the ejected fragments of exhausted fungus substratum. The soil was so hard that I could not reach the chambers and gardens which must have been some distance beneath the surface. The few workers that were abroad were bringing in small vegetable NEW FORMICID^E FROM BARRO COLORADO ISLAND. 155 particles. During the summer of 1920 I examined some of the colonies of S. urichi, which were nesting in the lawn near Mr. Urich's laboratory in Port of Spain, but time to make a careful investigation was lacking. During the same summer I took in the sandy area adjoining the Tropical Laboratory of the New York Zoological Society at Kartabo, British Guiana, a few workers of a Sericomyrmex which I have recently described as impexus (1924), but I failed to reach the chambers in the very few nests that were excavated. My sojourn on Barro Colorado Island finally yielded the desired opportunity to study not only Sericomyrmex but also several other Attini. During the height of the rainy season this locality is a veritable myrmecological and mycological paradise. Within a few hundred yards of the laboratory (Fig. i) numerous colonies of at least 14 species of fungus-growing ants could be found, all with their gardens close to the surface of the ground and easily accessible. I recognized two species of A tta, one of Acromyrmex, three of Trachymyrmex, three of A ptero stigma, three of Cyphomyrmex, one of Myrmecocrypta and one of Sericomyrmex. Thus nearly all the known genera of Attini were represented. Further search will probably reveal the presence of Mycocepurus on the island. Nor were the ants the only fungus-cultivating insects. The trunks of the trees that had been felled during the dry season (spring of 1924), when the small clearing was made around the laboratory, had reached a stage when they attracted thousands of ambrosia beetles of the family Platypodidae. During June and July these insects were everywhere making their long tubular fungus-lined galleries in the dead wood and covering the logs with their frass. It would, indeed, be difficult to find a more favorable locality for mycological investigations, not only on account of the interesting fungi cultivated by so many ants and beetles but also of the extraordinary number and variety of other fungi, which during the rainy season flourish in all parts of the jungle. Leaving an account of the other Attini for consideration at some future time, I will here confine my remarks to the Seri- comyrmex which harbors the Megalomyrmex in its gardens. This Sericomyrmex seems to represent an undescribed species, which I 11 156 WILLIAM MORTON WHEELER. shall call amabilis. It is very closely related to 5. impexus Wheeler of British Guiana, but the worker is somewhat larger, the external borders of the mandibles are less convex, the meso- thoracic tubercles are more acute and the silky hairs and pu- bescence, especially on the upper surface of the body, are longer and more conspicuous. The worker averages a little over 3 mm. in length, the female somewhat more than 5 mm., the male about 3 mm. All the phases are ferruginous brown, the female being decidedly darker and less reddish than the worker. Like most of the smaller Attini, the workers are very timid and inoffensive. When rudely touched they at once curl up and feign death. Owing to their gentle disposition and graceful and deliberate movements they are among the most fascinating ants to observe in artificial nests. .S. amabilis is probably common in many parts of the jungle on Barro Colorado Island but I was able to detect its nests only in the recently made clearing and trails, where the red clay, which gives its name to the island, is exposed to the sunlight, and there only some hours after one of the almost daily, heavy showers. As soon as the rain ceased the silky, mouse-like workers began to bring up small pellets of earth and carefully deposit them in the form of a loose crater around the entrance, which was about 3 mm. in diameter. These craters, which are completely obliter- ated by each rain, are probably large and noticeable during the dry season. As soon as one approaches the nest, the excavating workers, like those of Trachymyrmex, either feign death and become indistinguishable from the soil or hastily withdraw within the nest entrance, so that the observer must remain motionless for several minutes before they resume their labors. Most of the nests observed had a single crater and entrance, with a slender gallery descending more or less obliquely to a chamber about 4 to 6 inches beneath the surface, but a few large nests resembled A tta nests, on a very diminutive scale in having several entrances and craters and as many as 3 to 5 chambers, scattered over an area of about a quarter of a square yard. The chambers varied in size from that of a pigeon's to that of a hen's egg, and were each filled with a fungus garden which was either entirely built up on the floor and walls or partly suspended from NEW FORMICID^E FROM BARRO COLORADO ISLAND. 157 rootlets left intact by the ants during their excavations. The substratum of the gardens consisted of small, dull-yellow pellets of uniform size, held together and covered by a dense white mycelium, bearing minute clusters (bromatia) of spherical FIG. 2. Fungus garden of Sericomyrmex amabilis sp. nov. built in a Petri dish. X i%. Photograph by Dr. David Fairchild. swellings ("kohlrabi") like those of Atta, Acromyrmex and Trachymyrmex. Urich's description of the gardens of S. urichi applies accurately to those of amabilis. The pellets of the sub- stratum are undoubtedly particles of soft, chlorophylless vege- table matter collected by the workers in the immediate vicinity of the nest but I was unable to identify them more closely or to observe the ants in the act of gathering them. 158 WILLIAM MORTON WHEELER. In order to study the ants I placed them with their brood and fungus gardens in large Petri dishes. These made excellent artificial nests in which the insects could be kept in perfect health for two to three weeks and conveniently observed under a strong pocket-lens. Within 24 to 36 hours the frail fungus gardens, which inevitably fell to pieces when extracted from the earthen chambers, were completely reconstructed by the workers as an elaborate and rather regular sponge-work with polygonal crypts a quarter to half an inch in diameter. Of course, the flat space, less than half an inch in thickness, to which the ants were confined, compelled them to rebuild their garden in the form of a disc instead of a sphere or ovoid, but this was very advantageous, since it permitted the observer to scrutinize all parts of the structure through the glass cover. (See figures 2 and 3 from photographs by my friend Dr. David Fairchild.) The ants placed their eggs, larvae and pupae on the fungus-covered surfaces and in the crypts. The queen is a very sluggish insect and remained for long hours in a somnolent attitude near the center of the garden, or moved about very slowly and scattered her eggs in the immediate vicinity. These were rather large and broadly elliptical and were permitted to lie where they were laid till sometime after the larvae had hatched. The workers then carried them to other parts of the garden and placed them in contact with fresh hyphae. Since I never saw the workers ad- ministering hyphae or "kohlrabi" to the larvae as described for Atta cephalotes by Tanner (1892), I infer that the latter, when hungry, merely reach out and crop the fungus. The larvae are short, thickset and beset with sparse, long, flagellum-like hairs. The head is large and subrectangular, bearing small, acute mandibles covered with acute points. This type of mandible, which I find to be peculiar to the Attini, seems to be adapted to puncturing the delicate fungus hyphae and expressing their juices. Worker pupae were common in the nests, but pupal males and females were much less numerous. A few of the winged adult sexual forms emerged during the last week of July and the first ten days of August. The workers were frequently observed in the act of building and rearranging the particles of the substratum of the garden and NEW FORMICID.E FROM BARRO COLORADO ISLAND. 159 feeding on the "kohlrabi." They cropped the delicate hyphae with their maxillae, and not with their mandibles, without dis- turbing or shaking the substratum. On several occasions I saw FIG. 3. Fungus garden of Sericomyrmex amabilis sp. nov. built in a Petri dish X 2. Photograph by Dr. David Fairchild. them devouring injured larvae or pupae, and they greedily fed on the pulp of various fruits, such as mangos and bananas. They also tore off small bits of the inner rind of oranges and bananas and incorporated them in the garden. When such materials l6O WILLIAM MORTON WHEELER. were not available, they manured the garden with numerous golden yellow droplets of their own feces. In two of my nests the gardens were suddenly blasted in a peculiar and unexpected manner. Bits of mango had been left in the Petri dishes and had decomposed during the night. This decomposition seemed to be due to some bacterium which was accidentally transferred to the gardens, probably on the feet and mouthparts of the ants, and at once overwhelmed the fungus, so that within a few hours it shrivelled up and turned black. The ants, apparently quite unable to prevent the inroads of the lethal microorganism or to restore their fungus to its normal condition, became demoralized and eventually wandered away from it to other parts of the dishes. The population of the Sericomyrmex colonies which I examined, resembled that of Trachymyrmex colonies, the smaller nests con- taining between 100 and 200, the largest (a single nest) about 300 ants. No incipient colonies were seen, but there can be no doubt that the recently fecundated queens establish their colonies and gardens in the manner described by von Ihering, Huber, Goeldi and Bruch for various South American species of Atta and Acromyrmex. The guest-ant, which I found in ten out of the twelve Seri- comyrmex nests excavated in the immediate vicinity of the laboratory on Barro Colorado Island, is obviously a species of Megalomyrmex, a peculiar neotropical genus established by Forel in 1884 for a Colombian ant, M. leoninns and now known to comprise some 15 species which range from Bolivia to Mexico. While the generic name was appropriate to the type and several other species, which measure nearly a centimeter in length, it is a misnomer for several much smaller species gceldii Forel, pusillus Forel, ivallacei Mann, silvestrii Wheeler and sjostedti Wheeler which have been recently described. The known species of the genus are divisible into two groups, one of which, including the type, has convex, coarsely 5- or 6-toothed mandibles, with a sharp angle between their apical and basal borders, whereas in the other group, comprising only two species, silvestrii and sjostedti, the mandibles are narrower and more flattened, with a rounded angle between the basal and apical borders and the latter with two large terminal teeth and a series of very minute basal denticles. This NEW FORMICID/E FROM BARRO COLORADO ISLAND. l6l group is regarded by Mann as a distinct subgenus, for which he has proposed the name Wheelerimyrmex. I find that there is also a difference in the number of palpal joints in these two groups. In Megalomyrmex sens. str. (M. bittiberculatus Fabr.) the maxillary palpi are 4-jointed, the labial palpi 3-jointed. 1 In Wheelerimyrmex I find the maxillary palpi to be 3-jointed, the labial palpi 2-jointed. The guest ant, which really represents a new subgenus and species and is described in the sequel as Cepobroticus symmetochus, has the same number of palpal joints as Wheelerimyrmex, but the mandibles are intermediate between the two other subgenera, having a sharp angle between the basal and apical border, and the latter with a large terminal and five or six small basal teeth. The antennae, moreover, are short, all the funicular joints, except the last being decidedly broader than long and the clava absent. The promesonotal suture is obsolete but this character occurs also in one species of Wheelerimyrmex (silvestrii). In sjostedti the suture is as distinct as it is in the species of Megalomyrmex sens. str. The eyes of the worker Cepobroticus are rather small. It is, perhaps, significant that Emery (1921) has placed the genus Megalomyrmex in his tribe Monomorii, in the midst of a series of Old World genera Hagioxenus, Wheeleriella, Phacota, Xenomyrmex and Liomyrmex which are known to have xeno- biotic or parasitic habits. Unfortunately very little is known concerning the habits of the described species of Megalomyrmex. The only data I have been able to secure are a few notes by Mann on M. tuberculatus and M. (W.) silvestrii. Of the former he says (1916, p. 445): "This form, which is confined to the upper Amazonian region, attends Membracidse and the workers were generally found in company with these on shrubs in the dense forest. The nest is subterranean, the entrance nearly always at the base of a tree. The living insect is slow in its movements." Concerning silvestrii, which he observed in Honduras, he says: "A good series of workers were taken at Ceiba and San Juan Pueblo, nesting in the ground and in rotten logs. It is a timid species and very active when disturbed." These notes indicate that the species of Megalomyrmex sens. str. and the subgenus 1 Forel and Emery give 3 joints for each palpus. 1 62 WILLIAM MORTON WHEELER. Wheelerimyrmex are nonparasitic and epigaeic. The occurrence of Cepobroticus only as a hypogaeic inquiline, or guest in the fungus gardens of Sericomyrmex may be regarded as an ethological character which still further emphasizes its subgeneric status. The Cepobroticus symmetochus worker measures only 3-3.5 mm., the female 3.8 mm., the male 3 mm. It is therefore the smallest known species of its genus. The body is very smooth and shining in all three phases and in the worker and female yellowish red, with the appendages scarcely paler and the dorsal median third of the gaster dark brown or blackish. The male is paler and more yellow throughout. All the castes, and especially the worker and female, are covered with rather coarse, long, golden yellow hairs. For other characters the taxonomic de- scription and figures may be consulted. The colonies of the Cepobroticus so frequently found living with Sericomyrmex amabilis were decidedly less populous than those of their host. The largest comprised less than 75 individuals, and often the number did not exceed 40 or 50. In every nest a dealated mother queen was present. She usually took up her station, surrounded by a group of her workers, in one of the crypts of the fungus garden a short distance half to three quarters of an inch from the Sericomyrmex queen. The guest ants kept their brood in small clusters scattered through the garden and each cluster was cared for by a few workers. Al- though the ants and their brood were thus intermingled, the workers of each species lavished their attention exclusively on their own eggs, larvae and pupae and were never seen even to transport the progeny of the other species from one part of the garden to another. The workers and queens of Cepobroticus are rather alert and move about more rapidly than their hosts. They devote so much time to licking and fondling one another that the observer is some- what astonished to find them paying little or no attention to the fungus-growers. As a rule the two species are indifferent to one another. One may watch them for hours without observing anything more than rather distant, mutual antennal salutations. On rare occasions a worker Cepobroticus may be seen licking the gaster of a Sericomyrmex worker or of the sluggish queen. More NEW FORMICID/E FROM BARRO COLORADO ISLAND. 163 frequently one of the host workers may be observed in the act of lavishing similar but more elaborate attentions on a Cepobroticus worker. The fungus-grower begins by licking the feet or tarsi, the tibiae and femora, then the thorax or abdomen and finally the head and even the mandibles of the guest. During this operation the latter remains motionless and inclines its body somewhat to one side. The Sericomyrmex never feed their guests by regurgitation. This is not surprising because they never feed one another thus, but resort individually to the growing fungus bromatia. When hungry the Cepobroticus workers and queen also crop the fungus mycelium, but they do this rather roughly, using their mandibles and even shaking or disturbing the substratum. The guests very rarely transport or rearrange the particles of the substratum or take the slightest interest in the garden, except as a source of nourishment. Only on one occasion did I see a Cepobroticus carry a particle of the substratum to another spot, insert it and pat it down with her fore feet. When fresh fruit was introduced into the nest, it was much less frequently visited and eaten, by the guests than by their hosts. The larvae and pupae of the Cepobroticus can be readily dis- tinguished from the Sericomyrmex brood. The larvae are more slender and more cylindrical and have smaller heads, with flat, 3-toothed mandibles. The hairs on the body are more numerous, shorter and stouter, though rapidly tapering at their tips. I was unable to determine whether the larvae are nourished by regurgi- tation or feed directly on the fungus hyphae. The fact that they usually lie in the crypts in small clusters and in less intimate contact with the fungus than the Sericomyrmex larvae would seem to indicate that they are fed by their nurses with regurgitated liquids. The inquilines evidently lead a purely hypogaeic life. Only the males and winged females leave the fungus chambers and come to the surface to mate. I took a few of the sexual forms which had thus escaped, and Prof. W. C. Alice, who collected on Barro Colorado Island during the spring of 1924, sent me among a number of miscellaneous ants a few winged females and several males of Cepobroticus which he had evidently taken on the ground 1 64 WILLIAM MORTON WHEELER. or vegetation. The small size of the eyes in the worker as com- pared with other species of Megalomyrmex indicates that this hypogaeic mode of life is beginning to affect the visual organs. Other obvious adaptive characters are the dentition of the mandibles, which is well suited to cropping the fungus hyphse, and the investment of long, golden yellow hairs, which suggest a trichomal function like the golden tufts of many symphilic myrmecophiles. Some experiments were conducted in mingling the personnel from different Sericomyrmex and Cepobroticus colonies. The former were so gentle and tolerant that when workers and queens belonging to different colonies were placed in the same Petri dish little animosity and that of very short duration was exhibited. Similarly, when inquilines from an alien colony were introduced, they were adopted at once without hostility, but the members of different colonies of the inquilines were much more hostile to one another. Frequently workers or queens would be dragged about for days and eventually mutilated or even killed by workers of their own species. This behavior was, perhaps, to be expected from what is known of the mutual animosity of parasites of the same species when confined with a single host. The foregoing observations make it seem probable that the Sericomyrmex- Cepobroticus colonies are not established by a con- sociation of fecundated queens of the two species immediately after their nuptial flight, but that the Cepobroticus queen enters a well-established Sericomyrmex nest in which the fungus garden is already large and flourishing and being cultivated by a lot of workers. The development of the garden by the recently fecundated Sericomyrmex queen, as already suggested, evidently takes place in the same manner as in A tta, Acromyrmex, Mcellerius, Apterostigma and other Attini, and is such a slow and delicate operation that the presence of a fungus-devouring inquiline at the inception of colony formation would, to say the least, seriously interfere with the welfare of both queens. On the other hand, the intrusion of the Cepobroticus queen at a later stage, when the garden is well established, would not seriously affect the life and development of both colonies, especially as the inquiline is by no means a very fecund ant. This is shown by the small size of her NEW FORMICID/E FROM BARRO COLORADO ISLAND. 165 own colony, her diminutive stature, and the small size of her gaster, which scarcely exceeds that of the worker. The fact that the workers show only a beginning in the reduction of the eyes would seem to indicate that the hypogaeic and inquilinous habit is of rather recent phylogenetic origin. This supposition is also supported by the consideration that the Attini themselves consti- tute a young, or recent tribe of Myrmicine ants. It is evident that Cepobroticus is merely a single aberrant species of Megalomyrmex which has abandoned an independent life, has associated itself permanently with Sericomyrmex and has taken to feeding on the fungus which it cultivates. The associ- ation thus established is a type of "compound nest," as defined by Wasmann, but differs from all the known types in certain important particulars. The relationship between the two species is somewhat like that obtaining between the xenobiotic Lepto- thorax emersoni and Myrmica canadensis in the mountains of our northern states and British America, but is in certain respects much less intimate. Although the Cepobrotici look after their own brood, they do not, like the Leptothorax, construct special chambers communicating with those of the host. Mutual feeding by regurgitation has not been developed, because both species feed on a delicate plant which is carefully provided and cultivated by one of them. We may, therefore, regard the relations of the Cepobroticus to the Sericomyrmex as a case of what the Germans call "Futterparasitismus," a case to which we might, perhaps, apply the term "mycetometochy." With the possible exception of the Pseudoatta described by Gallardo, we know of no other example of this relation among ants, but further investigation may reveal its occurrence among the termites of Africa and Southern Asia. In the soil immediately surrounding the fungus chambers of some of the Sericomyrmex nests I found five minute species of ants which are described below as Pheidole (Hendecapheidole) mendi- cula sp. nov; Oligomyrmex panamensis sp. nov. ; Solenopsis conjurata sp. nov.; Tranopelta gilva Mayr var. columbica Forel and Rhizomyrma sp. With the exception of the last these seem all to be "thief," or lestobiotic ants, but further observations will be required to establish their precise relations to the fungus 1 66 WILLIAM MORTON WHEELER. growers. The most interesting species is the Oligomyrmex, because no representative of this genus, which is widely dis- tributed over the warmer portions of the Old World Southern Europe, Asia Minor, Africa, Madagascar, India, Indonesia, Papua, Australia has been taken hitherto in any part of the New World. The new Hendecapheidole is also of interest, because only two species of the subgenus have been described, tachigalice Wheeler and enter soni Wheeler (1922), both from British Guiana. TAXONOMIC DESCRIPTIONS. Sericomyrmex amabilis sp. nov. (Fig. 4.) Worker. Length 3-3.5 mm. Very close to S. impexus Wheeler but differing in its somewhat larger size and darker color and in the following structural details : The head is more deeply excised posteriorly, the eyes are distinctly larger and more convex, the posterior angles of the frontal lobes a FIG. 4. Sericomyrmex amabilis sp. nov. a, head of worker, dorsal aspect; b, thorax and pedicel of same in profile. more acute, the continuations of the frontal carinae which form the inner boundaries of the scrobe-like depressions for the an- tennae, more pronounced, the mandibles with less convex external borders and more pronounced striae. Their surfaces are at the same time more shining. The median joints of the antennal NEW FORMICID.E FROM BARRO COLORADO ISLAND. 167 funiculi are distinctly more transverse. The thorax, abdomen and legs are very similar to those of impexus, but the inferior angles of the pronotum, the pair of tubercles on this segment and the two pairs of tubercles on the mesonotum are somewhat larger and more acute and the lateral marginations of the gaster are more pronounced, as are also the three broad longitudinal depressions near its base on the dorsal side. The pilosity and pubescence are decidedly more abundant than in impexus. The former is longer and more completely covers the integument, the latter is also longer and more conspicuous, especially on the dorsal surface of the body. The hairs are blackish at the base, with long, slender, flexuous, pale grayish or yellowish tips. The color of the body and appendages in mature specimens is rich ferruginous brown, with somewhat darker mandibles. Female. Length 5-5.5 mm.; wings 6.3 mm. Similar to the worker. Backward extensions of the frontal carinae and of the carinae of the cheeks more acute, so that the antennal scrobes are more strongly developed. Mandibles coarsely striatopunctate. Pronotum with a blunt tubercle on each side, the inferior angles not very distinct. Mesonotum subrectangular, slightly longer than broad, flattened above, with a feeble Y-shaped impression. Scutellum less than twice as broad as long, bluntly bidentate behind. Epinotum declivous, with a pair of blunt longitudinal ridges terminating in blunt teeth. Petiole and postpetiole each with a pair of blunt longitudinal ridges above. The large first gastric segment is broader behind than in front, with straight sides, which are marginate; the three longitudinal impressions on the dorsal surface somewhat more distinct than in the worker. Pilosity and especially the pubescence even longer than in the worker. Color darker, more brown and less reddish. Wings rather strongly and uniformly infuscated ; veins and pterostigma pale, but very narrowly outlined with blackish. Male. Length nearly 3 mm. Head, including the eyes, as long as broad, somewhat narrowed behind, with straight posterior border and rounded posterior corners. Eyes rather large and convex, the ocelli small and 1 68 WILLIAM MORTON WHEELER. widely separated. Mandibles well-developed but narrow, their long apical borders finely denticulate. Antennae slender, the scapes extending well beyond the posterior border of the head. Thorax large, the mesonotum convex anteriorly, with distinct Mayrian furrows. Scutellum trapezoidal, nearly as long as broad, feebly impressed in the middle, with entire posterior border. Petiole and postpetiole similar to those of the worker but the former more pedunculate anteriorly. Gaster small, oval; legs long and slender, the femora feebly bent. Surface of the body smoother than in the worker and female, the mandibles and gaster somewhat shining, the remainder of the body subopaque. Pilosity and pubescence very short and meager, only the sides of the petiole and postpetiole with tufts of hairs like those of the worker and female. Antennae and legs destitute of hairs, with fine, indistinct pubescence. Brownish yellow, head, a spot on the posterior portion of the mesonotum and a line on each side of it, brown. Wings colored as in the female. Described from numerous workers, five females and a male taken on Barro Colorado Island, C. Z. during late July and early- August. This form is so close to 5. impexus of British Guiana that it might be regarded as a subspecies. I have given it specific rank, however, because it now appears that there are several forms impexus Wheeler, urichi Forel, diego Forel, morierai Santschi, lutzi Wheeler, zacapanus Wheeler, opacus Mayr, pusillus Forel and aztecus Forel which are so closely related that they may be merely geographical races, or subspecies of one or a few highly variable species. At present our knowledge of these various forms and of their phases is so meager that it seems best to regard them as specifically distinct. Megalomyrmex (Cepobroticus Subgen. nov) symmetochus sp. nov. (Fig. 5-) Worker. Length 3-3.5 mm. Head subrectangular, very slightly narrower behind than in front, with straight posterior and very feebly convex lateral NEW FORMICID^: FROM BARRO COLORADO ISLAND. 169 borders; the posterior corners rounded. Eyes small, feebly convex, at the middle of the sides. Minute ocelli sometimes present in large workers. Clypeus convex, its anterior border broadly and evenly rounded. Frontal carinse short, parallel; frontal area indistinct, convex in the middle. Mandibles rather narrow and not very convex, 7-8 toothed, the apical tooth longer FIG. 5. Megalomyrmex (Cepobroticus) symmetochus sp. nov. a, head of worker, dorsal aspect; b, thorax and pedicel of same, lateral aspect; c, head of male, dorsal aspect; d, fore wing of female. than the others which are subequal, the most basal forming the angle between the basal and apical borders. Maxillary palpi 3-jointed; labial palpi 2-jointed. Antennae robust, the scapes extending less than one third their length beyond the posterior corners of the head; funiculi thickened apically, but not forming WILLIAM MORTON WHEELER. a distinct club ; all the joints except the first and last broader than long; joints 2-4 transverse, nearly twice as broad as long. Thorax slender, narrower than the head ; the pro- and mesonotum forming an even convexity above, without promesonotal suture ; the mesoepinotal impression distinct but shallow; the epinotum in profile rising steeply for a short distance in front, then becoming straight and horizontal in the middle and gradually passing into the short sloping declivity; the metasternal angles rather large, lamellate and rounded. Seen from above the epinotum has a ridge on each side, bounding a large median longitudinal im- pression for the accomodation of the petiole. The latter is nearly as high as long, the peduncle shorter than the node, which rises rather abruptly in front and is rounded above, with a posterior slope like the anterior. Seen from above the node is somewhat broader than long. The ventral surface of the peduncle bears a small, blunt tooth anteriorly. Postpetiole lower than the petiole and about half again as broad, with bluntly subangular sides, the node very convex above and inclined somewhat forward. There is a small acute tooth at the anterior end on the ventral side. Gaster elliptical, its anterior border feebly excavated. Legs rather slender. Mandibles subopaque, densely striated; remainder of body very smooth and shining, with minute, sparse, piligerous punc- tures. Cheeks, mesopleurse and sides of epinotum longitudinally rugulose. Hairs long, erect or suberect, golden yellow, somewhat bristly and rather abundant on the body, legs and antennae; pubescence absent, except on the funiculi. Yellowish red; mandibles, funiculi, the posterior half of the first segment of the gaster and the sutures of the thorax and pedicel, brown; tip of gaster yellowish. Female. Length nearly 4 mm. Very similar to the worker, with larger eyes and distinct ocelli. Thorax as broad as the head through the eyes, the mesonotum convex and rounded above, subhexagonal, as broad as long. Epinotum sloping, without distinct base and declivity. Gaster as in the worker. Wings with a distinct discoidal cell, a single elongate cubital cell and the submarginal cell open at the tip. NEW FORMICID.E FROM BARRO COLORADO ISLAND. Sculpture, pilosity and color as in the worker. Each ocellus with a black margin internally. Wings yellowish hyaline, iri- descent, with pale yellow veins and pterostigma ; their membranes distinctly pubescent. Male. Length nearly 3 mm. Head without the eyes longer than broad, with rounded pos- terior corners and somewhat convex posterior border. Eyes and ocelli very large. Anterior border of clypeus produced and rounded. Mandibles well-developed, with triangular denticulate blades. Antennae slender; scapes nearly as long as in the worker; first funicular joint small, as broad as long, remaining joints, except the last, subequal, twice as long as broad, terminal joint somewhat longer. Thorax resembling that of the female. Peti- olar node much lower than in the worker and female. Gaster elliptical, not excavated at the base. Legs very slender. Sculpture very similar to that of the worker and female, but the sides of the thorax are smooth. Pilosity also similar, but the wings with longer pubescence than in the female. Brownish yellow, gaster a little darker, antennae and legs slightly paler; eyes and a spot along the inner border of each ocellus black. Described from numerous workers and females and two males taken from several colonies living in the fungus gardens of Sericomyrmex amabilis on Barro Colorado Island, C. Z. I have made this ant the type of a new subgenus largely on account of the dentition of the mandibles and structure of the antennal funiculus. One unfamiliar with the smaller species of Megalomyrmex, especially those of the subgenus Wheelerimyrmex would be inclined to regard the new species as a Monomorium, mainly because the stature is so small, the mesoepinotal con- striction so feeble and the lateral ridges of the epinotum are so poorly developed as compared w r ith other species of Mega- lomyrmex, but I believe that there can be no doubt concerning the natural affinities of the insect. Emery states (1921) that there is no discoidal cell in the fore wing of Megalomyrmex, but I have found it present in all the species I have examined. Ap- parently this cell may be either present or absent in the species of Monomorium. 12 172 WILLIAM MORTON WHEELER. Pheidole (Hendecapheidole) mendicula sp. nov. (Fig. 6.) Soldier. Length 1.3 mm. Head very large, subrectangular, rather convex above, about i longer than broad, as broad in front as behind, with nearly straight, subparallel sides and the posterior border rather deeply and semicircularly excised in the middle. The occipital and frontal grooves are rather shallow. Eyes small, convex, sub- FiG. 6. Pheidole (Hendecapheidole) mendicula sp. nov. a, head of soldier, dorsal aspect; b, thorax and pedicel of same, in profile; c, head of worker, dorsal aspect; d, head of male; e, antenna of same. triangular. Mandibles not very convex, with rather straight external borders and two large apical teeth. Clypeus somewhat flattened, its anterior border straight and entire in the middle, sinuate on each side. Frontal area small and indistinct; frontal carinae rapidly diverging, half as long as the head and forming sharp inner borders to rather deep scrobes for the accomodation of the antennae, which are small. Scapes reaching to the middle of the sides of the head, the club as long as the remainder of the funiculus; joints 2-7 of the latter small and transverse. Thorax NEW FORMICID^; FROM BARRO COLORADO ISLAND. 173 short and robust, the pro- and mesonotum forming a mass which is very convex and subangulate above in profile, the posterior surface of the mesonotum descending perpendicularly to the pronounced mesoepinotal constriction. From above the meso- notum is semicircular in front, with rather prominent humeri, behind which the sides are straight and converge to the meso- epinotal constriction. Epinotum as long as broad, much lower than the promesonotum, with subequal base and declivity and two backwardly directed spines which are slightly longer than the width of their bases and nearly as long as the base of the epinotum. Petiole small, about i| times as long as broad, the peduncle distinct and parallel-sided, the node strongly compressed antero- posteriorly, with abrupt anterior and more sloping posterior surface and entire, distinctly transverse superior border. Post- petiole nearly half again as broad as the petiole; transversely elliptical, convex and rounded above and on the sides. Gaster smaller than the head, elongate-elliptical, with slightly concave anterior border. Femora and tibiae distinctly thickened and clavate. Shining; mandibles and clypeus smooth and very sparsely and finely punctate. Remainder of head densely punctate and trans- versely rugulose, the rugules most distinct on the front. Thorax and petiole densely and finely punctate, somewhat more coarsely on the promesonotum. Postpetiole and gaster very smooth and shining, the latter with a small punctate area near the insertion of the postpetiole. Hairs yellowish, rather long and coarse, erect or suberect, moderately numerous, longest on the abdomen, sparser and some- what shorter on the legs. Brownish black, mandibles, sides of clypeus, antennae and legs piceous; tarsi brownish yellow. Worker. Length I mm. Head as broad as long, subrectangular, with distinct but rounded posterior corners, nearly straight posterior border and feebly convex sides. Mandibles with 7. small teeth, the second, fourth and sixth from the apex very minute. Clypeal border with four or five minute denticles which are the anterior termi- nations of longitudinal rugules. Eyes moderately convex, as long 174 WILLIAM MORTON WHEELER. as their distance from the anterior border of the head. Frontal carinae very short. There are no scrobes. Antennal scapes reaching to the posterior corners of the head. Thorax shaped much as in the soldier but the promesonotal mass is smaller and the humeral angles are less prominent, though the mesonotum is distinctly angular, with perpendicularly descending posterior surface. The epinotal spines are well developed but more erect than in the soldier. Postpetiole transversely rectangular, nearly i J times as broad as long and less than half again as broad as the petiole, which is similar to that of the soldier. Gaster truncated anteriorly. Legs as in the soldier. In sculpture, pilosity and color very similar to the soldier, except that the head is merely densely and evenly punctate. The tips of the mandibles and the f uniculi are paler and more brownish yellow in some specimens. Male. Length nearly 2 mm. Slender; head, including the eyes, as long as broad, narrowed behind, with straight sides and concave posterior border. Eyes and ocelli large. Mandibles and clypeus small, the former tridentate, the latter convex in the middle, with rounded anterior border. Antennae 12-jointed; scape very small and slender, scarcely longer than the swollen, ovoidal first funicular joint; joints 2-6 about twice as long as broad; 7-10 somewhat longer, the terminal joint slender and elongate. The funiculus tapers gradually to its tip. Thorax broader than the head, the meso- notum large, convex in front, as broad as long. Epinotum convex, with subequal base and declivity, rounding into each other. Petiole slender, parallel-sided, with very low and indistinct node; postpetiole somewhat broader, campanulate, as long as broad. Gaster and legs slender. Shining ; head subopaque and very finely and densely punctate ; pronotum also finely punctate but more shining. Pilosity yellowish, similar to that of the soldier and worker but shorter, especially on the legs, where the hairs are also more reclinate. Yellowish brown; dorsal surface of body darker; head black; mandibles, mouthparts, antennae, legs, insertions of wings and genitalia, pale yellow. Wings hyaline, with colorless veins and pterostigma. NEW FORMICID^E FROM BARRO COLORADO ISLAND. 175 Described from two soldiers, numerous workers and two males taken from a colony that was nesting in the soil immediately around the fungus chamber of a Sericomyrmex amabilis nest on Barro Colorado Island, C. Z. This is quite distinct from the two other known species of Hendecapheidole, tachigalice Wheeler and emersoni Wheeler. The soldier of mendicula can be at once distinguished from that of tachigalice by its dark color and the very different sculpture of the head, the worker by its color and much stouter epinotal spines. The soldier emersoni is unknown, but the worker is paler than that of mendicula, much less pilose, with less developed epinotal spines. The male emersoni has a broader and differently shaped head, stouter petiole, coarser sculpture, darker wings and 1 1- instead of 12-jointed antennae. The types of tachigalice were found in- habiting the petiolar swellings of a myrmecophyte (Tachigalia panicidata Aublet), those of emersoni a small cell within a termite nest (see Wheeler, 1921, p. 148, and 1922, p. 4). Oligomyrmex panamensis sp. nov. (Fig. 7.) Soldier. Length 1.3 mm. Head large, rather flat, fully i| times as long as broad, very slightly broader in front than behind, with straight, subparallel sides and deeply, semicircularly excised posterior border. A well- developed anterior ocellus is present. Eyes very small, situated about ^ the distance from the anterior to the posterior corners of the head. In the specimen the right eye is larger and pigmented, the left very minute and colorless. Mandibles short and convex, with about five blunt teeth. Clypeus very short and abrupt, its anterior border bluntly bidentate, sinuately emarginate in the middle and on the sides. Frontal carinae short but well-de- veloped, rapidly diverging. Antennae small and slender, 9- jointed; the scapes reaching the lateral border of the head at points two fifths the distance from its anterior to its posterior corners. The 2-jointed club is as long as the remainder of the funiculus, the terminal joint large and swollen, fully three times as long as the penultimate, which is distinctly longer than broad ; joints 2-4 subequal, broader than long; 5-6 nearly as long as broad, the basal joint as long as 2-5 together. Thorax narrower 1 7 6 WILLIAM MORTON WHEELER. than the head, elongate, broadest through the pronotum, which in profile is rounded in front and straight and horizontal behind, where its outline is continued into the straight, horizontal outline of the mesonotum. There is no mesoepinotal constriction but there are very small though distinct scutellar and metanotal a d FIG. 7. Oligomyrmex panamensis sp. nov. a, head of soldier (or ergatoid?), dorsal aspect; b, thorax and pedicel of same, dorsal aspect; c, same, in profile; d, head of worker, dorsal aspect. sclerites. The epinotum is subrectangular in profile, with an abrupt declivity shorter than the straight horizontal base. The angle on each side is formed by a ridge which is most distinct on the declivity. The surface between the two ridges is slightly concave. Petiole with a very short peduncle, which bears a strong, forwardly directed anteroventral tooth. The node is large and rounded, with rather steep, straight anterior and more abrupt, rounded posterior slope; from above it is transversely elliptical and nearly as long as broad. Postpetiole lower than the petiole, convex above, with a large, rounded tubercle on each side below and a minute anteroventral denticle. From above this segment is nearly if times as broad as the petiolar node and of a similar shape. Caster about the size of the head, elliptical, NEW FORMICID/E FROM BARRO COLORADO ISLAND. 177 somewhat flattened, its basal border rather straight. Legs short and slender. Mandibles somewhat shining, finely punctate. Head subo- paque, densely, finely and evenly longitudinally rugulose, the rugules straight and feebly diverging from between the frontal carinae to the posterior corners. Thorax, petiole and postpetiole also subopaque but the pro- and mesonotum, and especially the scutellum, more shining; the pronotum indistinctly and very finely longitudinally striate. Gaster shining, with rather numer- ous, minute, piligerous punctures. Hairs yellowish, suberect, sparse, more numerous on the gaster, fine and subappressed on the appendages. Ferruginous red; legs, funiculi, except the articulations, and gaster paler and more yellowish; borders of mandibles and clypeus brown or blackish. Worker. Length 0.9 mm. Head shaped somewhat as in the soldier, but much smaller, with evenly convex sides and more feebly excised posterior border. Eyes and ocelli absent. Mandibles less convex, with oblique blades, bearing three large apical teeth and a small basal tooth. Clypeus resembling that of the soldier. Frontal carinae very short. Antennae g-jointed, the scapes reaching to the middle of the sides of the head ; the terminal joint of the club proportionally longer than in the soldier, joints 2-6 of the funiculus decidedly shorter and more transverse, fully twice as broad as long. The thorax lacks the scutellar and metanotal sclerites and has a small but distinct mesoepinotal constriction. Epinotum small, with subequal base and declivity, the former slightly convex, the latter sloping, the angle between the two obtuse and rounded. Petiolar and postpetiolar nodes subequal, the latter nearly as long as broad, rounded on the sides, without tubercles. Gaster much smaller than the head, with somewhat concave anterior border. Smooth and shining, with scattered piligerous punctures, which are most distinct on the head, especially on its sides. Pilosity much as in the soldier but the hairs are decidedly shorter and of more uniform length. Clypeus with four stout bristles. Hairs on the front directed transversely, on the sides of the head forward. 178 WILLIAM MORTON WHEELER. Yellow; legs and antennal funiculi somewhat paler. Described from single soldier and worker specimens found in the soil surrounding a fungus chamber of Sericomyrmex amabilis on Barro Colorado Island, C. Z. This minute ant, the first Oligomyrmex to come to light in the New World, closely resembles its Old World cousins, except in the shape of the thorax in the soldier. The fact that the eyes on the two sides of the head are differently developed indicates that it is somewhat abnormal, and since the thorax is somewhat like that of a female in possessing scutellar and metanotal sclerites the specimen may prove to be an incomplete ergatoid or pseudogyne. Tranopelta gilva Mayr var. columbica Forel. A small colony of workers with larvae of what I take to be this form, originally described as a variety of T. heyeri Forel, was found in the earth immediately surrounding a fungus-chamber of Sericomyrmex amabilis on Barro Colorado Island. It is obviously very close to the var. albida Mann of Matto Grosso, Brazil, but the eyes are even smaller. The mesoepinotal impression is a trifle less pronounced, the color is whitish as in albida and the pilosity is the same. It is interesting to note that Forel found the types of columbica at the bottom of the nest of a fungus-grower Mycocepurus smithi Forel. Another colony, however, was taken by him "in a subterranean nest, beneath dried cow-dung." Solenopsis conjurata sp. nov. (Fig. 8.) Worker. Length 1.4-1.5 mm. Head subrectangular, distinctly longer than broad, with feebly convex sides and slightly concave posterior border. Eyes minute, consisting of 5 or 6 abortive but pigmented ommatidia, placed one-third the distance from the anterior to the posterior corners of the head. Mandibles narrow, with oblique 4-toothed apical borders. Clypeus with the two median teeth stout, acuminate and turned inward, the lateral teeth short, broad and blunt. Antennas rather slender; scapes reaching the posterior fifth of the head; basal funicular joint as long as the three succeeding joints together; joints 2-7 subequal, distinctly broader than long; the 2-jointed club somewhat longer than the remainder of the NEW FORMICID/E FROM BARRO COLORADO ISLAND. 179 funiculus; the terminal joint fully three times as long as the penultimate, which is distinctly longer than broad. Thorax rather slender, the promesonotum longer than broad, somewhat depressed above, its outline in profile rather straight in the middle ; mesoepinotal constriction distinct but not very deep; epinotum small, as long as broad, in profile convex, rounded and sloping, FIG. 8. Sole-no psis conjurata sp. nov. a, head of worker, dorsal aspect; b, thorax and pedicel of same in profile. without distinct base and declivity. Petiole small, the peduncle short, with a blunt anteroventral tooth, the node rather conical, rounded, as long as broad when seen from above. Postpetiole globular, a little broader than the petiolar node, somewhat broader than long. Caster as large as the head, elliptical, with straight anterior border. Legs rather slender. Smooth and shining throughout, with very fine, sparse, pi- ligerous punctures. Pilosity whitish, moderately long and abundant, erect on the body, more reclinate on the legs and scapes. Uniformly pale yellow throughout, only the teeth of the mandi- bles reddish and the minute eyes black. Described from numerous specimens belonging to a populous colony which was living in the earth surrounding a fungus chamber of Sericomyrmex amabilis on Barro Colorado Island, C. Z. These minute ants were kept for several days in an artificial ISO WILLIAM MORTON WHEELER. nest with the Sericomyrmex and their guest ants but though they mingled freely with the large ants remained quite unnoticed. It would seem therefore that 5. conjurata may be a true thief-ant like many other species of the genus (S. molesta Say,fugax Latr., etc.). I have described this Panamanian ant as new because it does not agree with any of the neotropical species of which I have seen specimens or descriptions. In Emery's key it runs down to S. Helena Emery, but this species, judging from his figures, has a much more rectangular head and very different teeth on the clypeus. Rhizomyrma sp. A single pale yellow, dealated female, clearly referable to this difficult genus and measuring only 2.3 mm. was found in the same situation as the preceding species. It is very probably unde- scribed but it seems best not to give it a name till the cospecific worker comes to light. Bibliography. Emery, C. '15 Definizione del Genere Apheenogaster e partizione di esso in Sottogeneri. Parapheidole e Novomessor nn. gg. Rend. Accad. Sc. 1st. Bologna, 1915, ii pp., 2 figs. '21 Myrmicinse in Wytsman's " Genera Insectorum," 1921, 397 pp., 7 pis. Forel, A. '96 Zur Fauna und Lebensweise der Ameisen im columbischen Urwald. Mitth. schweiz. Ent. Gesell., 9, 1896, p. 401-410. '98 La Parabiose chez les Fourmis. Bull. Soc. Vaud Sc. Nat., 34, 1898, p. 380-384. '99-'oo Formicidae in " Biologia Centrali-Americana." Hymen., 3, 1899-1900, 169 pp., 4 pis. '10 Glanures Myrmecologiques. Ann. Soc. Ent. Belg. 54, 1910, pp. 6-32. 'n Ameisen aus Ceylon. In Escherich's " Termitenleben auf Ceylon," 1911, pp. 215-228. '12 Formicides Neotropiques. Part 2. Mem. Soc. Ent. Belg., 19, 1912, pp. 179-209. Gallardo, A. '16 Notes Systematiques et Ethologiques sur les Fourmis Attines de la Re- publique Argentine. Anal. Mus. Nac. Hist. Nat. Buenos Aires, 28, 1916, PP- 317-344. 3 figs- Mann, W. M. '12 Parabiosis in Brazilian Ants. Psyche, 19, 1912, pp. 36-41. Mann, W. M. '16 The Ants of Brazil. The Stanford Expedition to Brazil, 1911, John C. Branner, Director. Bull. Mus. Comp. Zool., 60, 1916, pp. 399-490, 7 pis. '22 Ants from Honduras and Guatemala. Proc. U. S. Nat. Mus., 61, 1922, p. 1-54, 22 figs. NEW FORMICID^E FROM BARRO COLORADO ISLAND. l8l Santschi, F. '17 Fourmis Nouvelles de la Colonie du Cap, du Natal et de Rhodesia. Ann. Soc. Ent. France, 85, (1916) 1917, pp. 279-296. '22 Description de Nouvelles Fourmis de L'Argentine et Pays Limitrophes- Anal. Soc. Cient. Argentina, 94, 1922, pp. 241-262, i fig. Tanner, J. E. '92 Oecodoma cephalotes. The Parasol or Leaf-cutting Ant. Trinidad Field Naturalists' Club, i, 1892, pp. 68, 69, 123, 127. Urich, F. W. '95 Notes on the Fungus growing and eating habit of Sericomyrmex opacus Mayr. Trans. Ent. Soc. London, 1895, pp. 77-78. Wasmann, E. '15 Anergatides Kohli, eine neue arbeiterlose Schmarotzerameise vom oberen Congo. Ent. Mitteil. Deutsch. Ent. Mus. Berlin, 4, 1915, pp. 279-288, 2 pis. Wheeler, W. M. '07 The Fungus-Growing Ants of North America. Bull. Amer. Mus. Nat. Hist., 23, 1907, pp. 669-807, 5 pis., 31 text-figs. '13 Observations on the Central American Acacia Ants. Trans. 2d Ent. Congr. Oxford, (1912) 1913. PP- 109-139. '2ia A New Case of Parabiosis and the " Ant Gardens " of British Guiana. Ecology, 2, 1921, pp. 89-103, 3 figs. '2ib A Study of Some Social Beetles in British Guiana and of Their Relations to the Ant-plant Tachigalia. Zoologica, 3, 1921, pp. 35-183, 4 pis., 16 text-figs. Wheeler, W. M. '22a A New Genus and Subgenus of Myrmicinae from Tropical America. Amer. Mus. Novitates, No. 46, 1922, pp. 1-6, 2 figs. '22b Neotropical Ants of the Genera Carebara, Tranopelta and Tranopeltoides, new genus. Amer. Mus. Novitates, No. 48, 1922, pp. 1-14, 3 figs. '25(?) Neotropical Ants in the Collection of the Royal Museum of Stockholm. Part i. Ark. Zool. (in press). PALM AND SOLE STUDIES. VIII. OCCURRENCE OF PRIMITIVE PATTERNS (WHORLS). l H. H. WILDER, SMITH COLLEGE. INTRODUCTION. If we are correct in our morphological interpretation of the friction-skin patterns of the human palms and soles (Miss Whipple, 1904; H. H. Wilder, 1916) considering them the vestiges of the former walking-pads, we should expect to find upon each of these surfaces the typical set of eleven patterns, each in its proper topographical position. These are, in either hand or foot, Five Apical or terminal, upon the balls of the terminal phalanges of the five digits, fingers or toes. Four I nter digital , upon the more distal portion of palm or sole, proximal to the bases of the separate digits, and corre- sponding to the intervals between them. One Thenar, upon the thenar eminence, i.e., the more proximal portion of the palm or sole, on the side of digit I., radial or tibial. One Hypothenar, upon the hypothenar eminence, i.e., the more proximal portion of the palm or sole, on the side of digit V., ulnar or fibular. All eleven of these may be represented in a single palm or sole (H. H. Wilder, 1908, Figs. I and 2), but this is the greatest of rarities, only two cases, and these duplicate twins, have been thus far recorded. In the overwhelming majority of individuals the palm and sole patterns present but a small part of the complete set, and exhibit these in every stage of degeneracy down to a slight convergence of ridges in one spot, which indicates the final disappearance of the last triradius. Some of these are much more constant than others, as illustrated by the five apical patterns, the "finger-prints" of the professional dactyloscopists, 1 Contributions from the department of Zoology, Smith College, No. 130. 182 PALM AND SOLE STUDIES. 183 although even here there is a well-known type, the "simple arch," in which the original pattern is reduced to its lowest terms, and shows merely the position, or "core" of the former pattern without more than the last vestiges of a single triradius. On the other hand, others, like the second interdigital of the hand, or the thenar of the foot, are seldom found, and when they do occur, it is usually in the form of a mere vestige, where the closer ap- proximation, or the change of direction, of a few ridges, are the only indication of the former presence of a pattern that has become lost. Again, a given pattern, when compared in different individuals in w r hich it occurs, may show every stage of degeneracy, from a concentric whorl, the most complete and ancestral form, through those showing the loss of one or more of the triradii, to a wholly vestigial condition, where a scarcely perceptible disturbance in otherwise parallel ridges is indicative of the last traces of its presence. In this difference in the liability of occurrence of the different patterns, and in the sort of pattern when it does occur, whether more frequently a primitive Whorl or simply a vestige, we find a perfect correlation with the physiological use of the region involved. In general those patterns which are situated upon the more prominent surfaces, and which are therefore more often in contact with external objects, are far more constant in their occurrence, and appear more frequently in the form of whorls, than are those which, during the normal course of human activi- ties, lie in more retired spots, and are somewhat more shielded. Whorls are, for instance, by no means uncommon on the finger- balls, where a pattern of some sort is seldom entirely lacking, precisely upon those surfaces which are subject to the most wear and tear, and which press the most constantly upon external surfaces; while the four interdigitals are far less constant, and among these the second interdigital, especially protected both by the activity of the thumb, and by that of the cooperating index finger, seldom appears. On the foot the most common place for a whorl is upon the ball of the great toe (the first interdigital pattern) which bears the weight of the body at each forward step, and in the bare foot is constantly in contact with the ground. 184 H. H. WILDER. Here the pattern is not only huge in area, and composed of coarse ridges, but is very often still in the form of the primitive whorl, composed of concentric circles, and embraced with three triradii. The thenar, on the other hand, lying on the inner side of the foot, is of all foot patterns the most obscure and frequently entirely overlooked. It seldom or never shows more than one or two triradii, and has not yet been reported in the whorl form. In short, the correlation between the occurrence of the various patterns of the human palm and sole and their varied experiences during the customary activities of everyday life are so great that, could we suppose the entire species to become extinct, and to have left behind absolutely nothing but the records of numberless palms and soles, the customary uses of the lost hands and feet, even to the differentiation between the separate fingers, .could readily be surmised and described with much detail. While it is undeniable that this sounds strongly Lamarckian, there is nothing here intended other than to state the actual facts; whatever one's personal beliefs are with regard to the inheritance of acquired characters, it is certain that there is a strong corre- lation between the occurrence of patterns and the amount of habitual use of the regions where they occur, and between the habitual use and the percentage of occurrence of the more primitive types of patterns. We feel that we have established the following correlations between the surface relief, the size of the ridges, the occurrence and type of patterns, and the use of the various regions of the surface. I. Over the raised areas of the palm or sole, which come conse- quently into more constant contact with external objects, i.e., have the hardest use: (a) The ridges are the largest and coarsest. (b) Patterns occur more frequently. (c) The patterns show a greater percentage of whorls. II. Over the hollowed areas, which are thus shielded from contact with external objects: (a) The ridges are the finest and least prominent. (b) Patterns are less likely to occur. (c) When they occur they are more likely to be vestigial, i.e.', either arches or loops, seldom whorls. PALM AND SOLE STUDIES. 1 85 III. In the more primitive patterns the underlying surface often presents something of the shape of a conical mound, and the center of the cone coincides with the exact center or core of the pattern. The hypothenar pattern of the hand, of frequent oc- currence in the European-American race, often shows this particu- larly well, and in the occasional cases in which this pattern is in the form of concentric circles, it forms a definite mound, rising with each circle as one approaches the center, and at the apex is pointed, like a papilla, so that it may be noticeable in profile (Figs. I and 2). FIG. i. Photograph of the author's right hand, seen in profile and showing the hypothenar pad covered with a whorl, plainly showing its mound-like relief and the papilla at the core of the pattern. In spite of the great difference in the percentage of occurrence of the different morphological patterns it is probable that in the human race at the present time no one of the twenty-three patterns of the hand and foot has been allowed to degenerate so completely as never to occur in the primitive form, that of a whorl, although in the more than 1,700 individuals which I have thus far examined, there are at least three places out of the twenty-three in which such a pattern has not yet been found. These, as may be expected, are all in places where there is little contact. Two are in the foot, the thenar, and the hypothenar, and one is in the 186 H. H. WILDER. hand, the second interdigital. Of those in the foot the thenar occurs in the hollow on the inner side of the foot, where only occasionally a piece of clay, a stone, or a wad of grass will ever FIG. 2. A very primitive hypothenar of the right hand. Note the three embracing triradii; of these (a) is distal, (b) is the outer one, and (c) the proximal one, on or near the wrist. touch this region, and the hypothenar lies on the outside, and does not meet much contact, although it would be expected, and might well be looked for in the larger Anthropoids. PALM AND SOLE STUDIES. 187 Hands. Thus, to begin with, the apical patterns, the ones which are found on the balls of the fingers, that is, the "finger-prints" of the identification experts, are frequently found in the form of whorls. The formulation according to the Henry system, of a given hand as 32/32, means that there is a whorl on every finger. This type is not rare in a large collection, like the one in New York, although to the professional finger-print expert a number of patterns might easily be determined as whorls, which are not typical enough for the morphologist, and would not consist of concentric circles, although they probably would have two triradii. There is some feeling that the presence of whorls upon all the fingers is a racial characteristic of the Jewish race, although definite sta- tistics are not as yet available. Certain fingers are more apt to show whorls than others; for example the thumb and index are very apt to have whorls, while they are seldom found in the ring and little fingers. If one stop to consider the relative amount of independent use, and the variation to which they are subjected, he will see that here, also, as elsewhere, there is a direct correlation between the occurrence of whorls and the amount of use. Still, if one consult a large collection he will have little difficulty in finding even a perfect whorl on a little finger. Morphologically we have the right to expect four interdigital patterns, placed along the distal border of the palm, beneath (proximal to) the four intervals between the five fingers. Oc- casionally we find the last two, III. and IV., beneath the sepa- rations between the middle and ring fingers, and between the ring and little fingers respectively. Thus in Fig. 3, which shows the print of the left hand of a man in New York, one of the finger- print experts at the Headquarters office, 300 Mulberry St., interdigital IV. is particularly well shown, a whorl with a center of concentric circles, and with three triradii, upper, outer, and inner. In formulating this palm either the upper or the outer of these could be used equally well as the starting point of line D, with an exponent letter (/) to signify an extra triradius. Inter- digital III. is also indicated, but not so completely, and with only one triradius, the pattern being simply a loop. Fig. 4, enlarged photographically from the same, gives these same patterns in 13 1 88 H. H. WILDER. FIG. 3. Hand-print of one of the finger-print experts in the New York office, showing the fourth interdigital in the form of a primitive whorl with three triradii. The third interdigital also is well indicated, but is not an actual whorl, and lacks two of the three necessary triradii. PALM AND SOLE STUDIES. 189 more detail. In this is seen better the almost perfect condition of the fourth interdigital, with its three triradii. Fig. 5 shows a J^ FIG. 4. Detail taken from Fig. 3, enlarged. Here the third interdigital pattern is very typical, and compares well with the primitive hypothenar shown in Fig. 2. detail of the right palm of the author's wife (No. 70) with inter- digitals III. and IV., the first with two triradii, the latter a rudi- ment. In the actual hand these two areas are especially promi- nent, appearing almost like minute papillae, with the patterns on their apices. In the left hand of the same individual (Fig. 6) the same two interdigitals are shown, but only one of them is a whorl, the 1 1 Id, while the IVth is simply a loop, a very common pattern in the European-American race, and almost usual enough to serve as a racial characteristic in Japanese and Chinese. Occasionally one meets with either the third or the fourth interdigital as perfect as in Figs. 3 and 4, but it is always a surprise. My first experience of this kind was in one of two twin girls, relatives of the Director of the Eugenics Record Office at Cold Spring Harbor, L. I., but not found in her sister. This was in the right hand, and showed a whorl with three triradii, in the position of the Hid interdigital (Fig. 7). In enlargement (Fig. 8) it shows the three triradii. Fig. 9, a sketch taken with an Abbe camera, shows the details of the separate ridges. The corre- 190 H. H. WILDER. u - 1 1 2 rt =3 . cu I-H CS 1-1 3 aj O C rt 6 aj cu ft 45 ' (-< O J5 s 13 > a _ o *J O <-, J2 O co 4-J pC c . o . 3 r- 1 1 PALM AND SOLE STUDIES. 191 . *- = s x j > .5 . -*-> r^ .2 ^ -5 nj . O< S O a g i O a a *o 1-. 4) II &o c p. "3 -g o 2 "> S "3 o a c . o rt . C 03 PI C ed 192 H. H. WILDER. spending 1 1 Id interdigital of the left hand of the same person (Fig. 10) shows an indication of the same pattern, but not nearly FIG. 7. Tracing of the right hand of No. 1056, with a conspicuous third inter- digital pattern. The left hand of this same individual shows a good whorl on the same place (third interdigital) but smaller, and not so clear. Compare, for details Figs. 8 and 9. as complete. Other cases of the occurrence of these interdigital patterns, the 1 1 Id and the IVth, are shown in Figs. 1 1 and 12, and include nearly every case I have met with. Occasionally, as in Fig. ii the pattern is minute, but may still retain two out of the three triradii; in other cases, the pattern is still more shrunken, and can be identified only by its position on the palm, as in Fig. 12. Even here, however, in its reduced state of one PALM AND SOLE STUDIES. 193 ' &NS & FIG. 8. Enlarged photograph of the third right interdigital pattern of No. 1056. The entire hand, with position of this pattern, is seen in Fig. 7. FIG. 9. Detail of the third Interdigital pattern of the right hand of No. 1056- Compare with the photograph of the same, shown in Fig. 8 and the tracing of the entire hand of Fig. 7. Drawn from an original print by a drawing camera. 194 H. H. WILDER. ridge forming a circle, with a dot in the center, the reduction to its lowest terms, there is at least one of the three triradii, and a possibility of treating a prominent fork as another one. -'-' v- ' fjiVw^ &! ;iS^ ^ vf *, A^nr- * , '- f v ' C**4-" " "-'''-"" '"- - ; " '- site FIG. 10. Photographic enlargement of the left interdigital pattern of No. 1056. Compare with Figs. 6 and 7. Interdigital II., between the index finger and the middle one, is always rare and never more than a loop at best. In hands looked over for this very pattern, my collection gives the following: European- Americans (females) 200 hands 5 cases Japanese (males) 166 hands 7 cases Japanese (females) 224 hands. . 2 cases Chinese (equal number of males and females) 200 hands i case Fig. 13, taken from my Japanese collection, gives as good an example of this rare pattern as I have. This hand is interesting as showing also the 1 1 Id and a rudiment of a IVth, indicated by the convergence of the ridges in the proper place. This rudimentary condition seen here is also quite likely to occur on the area desig- nated for this pattern. Fig. 14 also from a Japanese male, shows PALM AND SOLE STUDIES. 195 FIG. ii. Photographic enlargement of the fourth interdigital pattern of No. 1212. This pattern is minute, but accurately placed directly below the interval between the ring and little fingers. Of its three triradii, the upper one forms the starting point of line D. Line C has no triradius in this hand. The photograph was given by Mr. Bert Wentworth, and was taken by the owner of the hand. FIG. 12. Photographic enlargement of the right third interdigital pattern of a female, not in my collection. This photograph was given me by Mr. Bert Wentworth. 196 H. H. WILDER. 6 M c J3 1 o. 4-i O fJ G .= . 05 " Si, HI 60 D. T3 _0 'C 1) > ^ D O O c a; bfl .SP ' M C i < I -t_> - - OJ IH -3 41 Co 4_> s .s OJ C C cS t^ M "> gfl A A O i S -i o c o o PALM AND SOLE STUDIES. 197 the convergence of ridges quite definitely and may be considered as a case having this lid interdigital pattern in the condition of a rudiment. 1 > . ,"- *. ''-'.-' . ''^ ' "" FIG. 14. Detail of print of No. 1585, (Japanese male) showing vestige of inter- digital II., between index and medius; also interdigital III. in the form of the more usual loop. Hasebe finds the percentage of occurrence for this pattern as 2 per cent, among the Japanese, and 4 per cent, among the Aino, corresponding fairly well with my figures. Interdigital I. has long been, in my experience, in the same 198 H. H. WILDER. class as interdigital II., that is, that no whorl pattern has yet been known, but within a few years a quite perfect whorl, although small, appeared in my collection of prints of college students. The normal close association of this with the definite thenar will FIG. 15. Tracing from the print of No. 788, a Smith College student of Euro- pean-American race. This is the only case thus far known of a complete whorl in the position of the first interdigital. This has apparently the full quota of three triradii, but the triradius below, between this pattern, and the thenar, which is here, as usual, represented by a loop, evidently belongs to the thenar pattern. be remembered, how in the majority of cases with any disturbance at all in the thenar region, there are apt to be found two loops back to back, and facing in opposite directions; the true thenar and the first interdigital. In the case in which the first inter- PALM AND SOLE STUDIES. 199 digital appears as a whorl, as shown in Figs. 15 and 16, the core or center of the pattern is marked by a complete circle, beyond which there are surrounding ridges which assume the usual loop shape, and bear the usual relationship to a thenar. This is certainly not a large or conspicuous pattern, but still answers to FIG. 16. Detail of the first interdigital pattern, from the left hand of No. 788, a Smith College student. This pattern here forms a complete whorl, though small. It is the only case yet reported in this position. the requirements of a definite whorl. It is to be remembered in this case that it occurs in one of the so-called "better classes" of our own race, and that there is otherwise nothing especially primitive in this individual. The case is a bit different in the only case of a thenar whorl which I have seen, as it occurs in a native Liberian soldier, one "Jimmy" Kamo, of the Bande tribe, collected for me by Prof. Frederick Starr (Fig. 17). This print is in my possession and although it is very dense, as too much ink was used, it is unmistakable. By treating the print with turpentine, as may always be done in such cases, the sepa- rate ridges can be well brought out. A second case like this has been published and figured by Hasebe (his Tafel IV., Fig. 10). This author states that he has met a thenar in form of a whorl twice, both times in Japanese. Hasebe finds the occurrence of any form of thenar pattern as 5 per cent, of all palms in both Japanese and Ainos, which may be compared with what I have found in European-Americans, 7 per 200 H. H. WILDER. cent, in one investigation, and II per cent, in another. The percentage is much higher in negroes, and very much higher (50 per cent.) in Maya Indians. FIG. 17. Tracing of the right hand of No. 571, showing a complete whorl on the thenar eminence. The subject is a Liberian soldier, Jimmie Kamo, of the Bande tribe. The print was taken by Prof. Frederick Starr. For a time this case of the Liberian soldier, which I published in the American Anthropologist, 1913, p. 202, Fig. 35 was the only one known, but Hasebe has recently (1918) found a second one. Thus, with the unique case of my No. 788, the two patterns on the thenar side of the thumb, which represent the closely associated PALM AND SOLE STUDIES. 2OI first interdigital and the true thenar, are both occasionally repre- sented as whorls. Feet. The study of toe patterns, doubtless owing to the universal deformation of the toe-balls through shoes and stockings, has scarcely been studied in this country or in Europe, but Hasebe, whose countrymen universally wear clogs (geta) which protect the plantar surface from the roughnesses of the soil, yet allow perfectly natural freedom in walking, and encourage the habit of dispensing entirely with all such artificial covering when in the house, has taken advantage of the material thus provided, and gives a careful report on the figures on the balls of the toes, the apical patterns of the foot. He studied the toes of 100 individuals (1,000 toes) and finds the distribution of whorls on the toe-balls as follows: Right: Left: ist (great) .' 4 ist (great) 10 2d 1 8 2d 13 3d 53 3d. . 50 4th ii 4th 12 5th o 5th i Hasebe further expresses these in the form of percentages, putting both feet together, as follows: Percentage of Whorls (both feet) : ist (great) 7.0 % 2d 15-5% 3d.. ..5-5% 4th , n.5% 5th .5% From these figures there will be seen a general similarity to the condition of the fingers in respect to the occurrence of whorls, except for the small percentage in the great toe when compared with the thumb; yet we would expect this very difference when we consider the amount of difference in the habitual action of the two digits. The thumb, with its extreme flexibility, and the short and rigid great toe, show, in this difference in the occurrence of whorls, at least a correlation between use and morphology, of the same sort as elsewhere in the friction-skin configuration. The 202 H. H. WILDER. large occurrence of whorls on the third toe is unexpected, but may be accounted for by the prominence of that digit in ordinary walking, and its extreme projection in the average foot. FlG. 1 8. Apical pattern of the right great toe of a European-American. Typical whorls do occasionally occur in the apical pattern of the great toe (Fig. 18), but a loop, usually fibular, is much commoner. Unlike the hand, the foot, owing to the greater equality in its digits, and especially to the evenness of the intervals between them, disposes of its four interdigital patterns in a straight row across the sole, occupying the mounded region commonly spoken of as the "ball." Remembering that the first interdigital pattern is here in line with the others, and is neither dropped out of place, FIG. 19. First interdigital (hallucal) of right foot of a Japanese girl, a student at the Doshisha school in Kyoto, showing the concentric whorl of a primitive pattern. The outer triradius was not printed but its position is indicated by the convergence of the ridges. It would doubtless have been printed if the foot had been rolled a little. nor approximated to the thenar, as in the hand, there is occasion- ally a sole in which all four interdigital patterns appear as either loops or whorls, but in a number of cases, perhaps the majority, the interdigital areas except the first are not marked by patterns, PALM AND SOLE STUDIES. 2O3 but in these cases the areas where they belong are sufficiently well indicated by the triradii, especially the upper or distal ones. The first interdigital area is often marked by a whorl, frequently a typical one (Fig. 19) but the three others are either in the form of a loop, which may open either up or down, or, perhaps most FIG. 20. Tracing from a sole print (No. 82) showing the areas of the four inter- digital patterns. The first and the third are seen as whorls. frequently of all, are crossed by approximately parallel lines with no suggestion of a pattern. The second interdigital pattern often gives the suggestion of having been squeezed laterally, and is often in the form of a narrow loop, opening downward. Aside from the first, the third interdigital is the most likely to be in the form of a whorl (Fig. 20), and in Fig. 21, there is a large whorl, concerning whose identification there is some doubt, not cleared up by the tracing of the entire sole (Fig. 22), although it is probably the fourth. 14 2O4 H. H. WILDER. Thus, if this last be taken as the fourth interdigital, and the whorl of Fig. 20 be considered the third, which is very evident, then, with the frequent occurrence of a whorl pattern on the first interdigital, or hallucal, this leaves the second alone which has not yet been seen in the primitive form, and the squeezed up condition of this renders it very unlikely that it has in modern men any FIG. 21. Sole print of No. 712, in which the third interdigital pattern, a whorl, is present, and large. Compare with Fig. 22, a tracing of the same sole. longer the chance to express itself. It is quite likely that some- time the impress of a naked foot of some paleolithic man may be found on the clay floor of a European cave, and it will be then with the most breathless interest that we will look to see if the second interdigital area was then as pressed laterally as now, or whether it ever expressed itself in the form of a whorl. There is still some little doubt concerning the identity of the proximal part of the foot, that proximal to the line of interdigital PALM AND SOLE STUDIES. 205 patterns which runs across the ball, but, after the analogy of the hand the hypothenar pattern is to be looked for somewhere along the outer, or fibular, edge of the sole, a little back of (proximal to) the interdigital pattern of the little toe and the one next to it. There is frequently found a loop in precisely this place, which runs FIG. 22. Tracing of the sole print of No. 712, shown in Fig. 21. The whorl is probably that of the fourth interdigital pattern. across the entire sole, the core of which is so far out that it is usually beyond the tread area, and requires a slight outward roll of the foot to get wholly into a print. Generally, too, the loop is a narrow one, with a straight axis, but occasionally one may be met with that has a definite bend in the axis, making the loop into a hook, as in the Negro boy in Fig. 23. In one case I have found this bending of the loop so extreme that the core assumes almost the appearance of a whorl (Fig. 24) . This occurred on the foot of a small son of a university professor. 206 H. H. WILDER. The thenar pattern is to be expected on the inner side of the foot, proximal to the first interdigital pattern. This would place it on the hollow of the foot, precisely where the surface is seldom touched by foreign objects. We might expect, a priori, not to find the pattern developed, but to find, at most, a triradius or FIG. 23. Tracing of No. 178, a Negro boy, with an unusually primitive hypo- thenar pattern. The loop, which is unusual, has become bent backwards, or recurved, leading towards the still more primitive one shown in Fig. 24, which has almost become a whorl. two, and a patch of ridges in a different direction. That is precisely what we do find, if we take the precaution to print a separate piece of paper, by first inking the inner curve of the foot directly, and covering it with the piece of paper. In the majority of cases the result is negative, but occasionally there is some remnant of a pattern, and this is exactly what one would expect, quite frequently a loop, either with or without a triradius, and PALM AND SOLE STUDIES. 207 always drawn with very fine and soft ridges. The difference in this feature between members of the European race, on the one hand and Japanese and Chinese on the other is sufficient to serve as a racial criterion, for the almost entire absence of any trace of such a pattern in the latter, and the frequency of some trace in the FIG. 24. Left hypothenar pattern of the foot of No. 1107. In this boy who is of the European-American race, the pattern has gone in the same way as in the Negro boy (No. 23), and has produced the nearest pattern to a whorl that has yet been found in any race. former is very striking, as is shown in tabular form in my recent paper on the Chinese and Japanese (Journ. Phys. Anthrop., Vol. V., No. 2, p. 203, 204). The calcar pattern completes the study of the feet. This is of rare occurrence in any race, but has been observed in several 208 H. H. WILDER. cases. It occurs about once in every hundred individuals among our people, and is usually in the form of a large loop, covering almost the entire heel, opening to the tibial side (Fig. 26). A triradius is usual, though not necessary, lying near the curve of the loop. I have met with this pattern a few times, notably in each of a pair of duplicate twins, as was to be expected. Natu- FiG. 25. Foot of No. 87, showing the thenar pattern in the form of a closed loop. One triradius is plainly visible, the upper one belongs on the curve of the loop, and its position is sketched in (dotted line). The third, which is necessary to transform the figure into a whorl, is indicated by the convergence of the lines of the loop. That this figure is not a misplaced calcar pattern is shown by the fact that in this same foot there is a normal calcar pattern, of which the end is shown by the convergence from the other side of the ridges that come around the heel. The friction-ridge configuration is taken directly from a print, but the foot outlines are conventional, and serve to show the position of the pattern. rally it would be most unexpected to find this pattern in the form of a complete loop, but one such case has been found, in the foot of a university professor (Fig. 27). The morphology of the calcar pattern is still uncertain. It occurs on the calcar projection of the heel, which is distinctly human, and is unrepresented in the foot of any of our other Primates, even the anthropoid apes. One theory identifies it PALM AND SOLE STUDIES. 2O9 with a pattern in the form of a loop, which is quite constant in the lower anthropoids, but the feet of these forms, although carefully studied, has not been treated with the basis of the walking-pads as a background and is not wholly satisfactory. Another theory links it with the hypothenar, and treats it as a component part of an enormously extended pattern, like the one reported in a few FIG. 26. A typical calcar pattern in its usual form, that of a loop, with the head of the curve fibular, and opening (i.e., converging) towards the tibial side. From life, No. 1128, pattern drawn in with help of prints. cases, mainly of negroes, which covers almost the entire sole, back of the ball. It is also conceivable to connect it with the vestiges of the thenar. The finding of a complete calcar pattern, as in Fig. 27 goes a long way towards the determination of the calcar as an independent pattern, distinct from all others, and developed secondarily upon the extensive human addition. The study of comparative morphology is replete with instances of survivals ; the persistence of organs of former usefulness later allowed to lapse from a growing lack of importance until, no longer of value, they become vestigial. The original eleven walking pads of the primitive mammalian paw, still useful and constant in pentadactylous forms like squirrels and mice, and retained in the form of whorls in the pictures sketched by the Primates in friction-ridge patterns, and faithfully representing a 2IO H. H. WILDER. former relief of pads with their surrounding skin folds and embracing triradii, and these, in spite of the high intellectual development of man, with its accompanying change in life and habits, will still occasionally crop out, not especially in those races FIG. 27. Outlines taken directly from sole prints of No. 887. This sketch is taken from several prints, lapped over so that they will meet, and spread out flat. This device is rendered necessary in order to express on a flat surface the details of a curved one. commonly considered low and bestial, but are found quite as frequently in peoples of the highest culture, and appear in primitive form now in the foot of a university professor, or in the hand of the daughter of a New Hampshire bank president, of a PALM AND SOLE STUDIES. 211 New York finger-print expert, or of the author of this paper. It is true that one of the most primitive illustrations was found in a native Liberian soldier, but equally primitive cases, both in hands and feet, occur among the students of Smith College. In my rather limited collection of peoples generally considered primitive, and including Ainus, Bontoc Igorots, Negritoes, and African Pygmies, I have found no cases that compare for primi- tiveness with those I have figured, which, with a few exceptions, were all of the European-American race. In some cases, if not in all, however, the lack of primitive characters has been un- doubtedly due to the small number of individuals in these exotic cases, although, in the cases of the Japanese and Chinese, I was enabled to consult a sufficient number of cases to bring out unusual cases if there were any. Still, we know as yet too few individuals of any race, even of our own, to draw any sweeping conclusions from them, and it is best to treat the cases here presented as individual rather than racial. BIBLIOGRAPHY. In my paper of 1916, published in this magazine, I endeavored, as was fitting a relative new subject, to make the bibliography as near complete as possible. Since then, with the added interest in this field, the task of keeping even the titles complete is quite beyond me, and nowhere near as possible as it was nine years ago. Here I may content myself with adding a few of the most recent titles, from the bibliographies in which one may obtain the later literature. Bonnevie, Kristine. '24 Studies on Papillary Patterns of Human Fingers. Journal of Genetics, Vol. XV., No. L., Nov. 1924- Cummins, Harold, and Joseph Sicomo. '22 A Case of Hyperdactylism: Bilateral Duplication of the Hallux and First Metatarsal in an Adult Negro. Anat. Rec., Vol. XXIII., No. 3, March 1922. '23 Plantar Epidermal Configurations in Lower Grade Syndactylism (Zygo- dactyly) of the Second and Third Toes. Anat. Rec., Vol. XXV., No. 6, July 1923. Cummins, Harold. '23 The Configuration of Epidermal Ridges in a Human Acephalic Monster. Anat. Rec., Vol. XXVI., No. i, Aug. 1923. 212 H. H. WILDER. Hasebe, K. '18 Ueber die Hautleistensystem der Vola und Planta der Japaner und Aino. Arbeit. Anat. Inst. Kaiserl. Japan. Univ. Sendai., H. i, 1918. Kubo, T. '18 Beitrage zur Dactyloskopie der Koreaner. Mitteil. d. med. Fachschule zu Keijo, 1918. Stockis, E. '22 Les Characteres ethniques du dessin papillaire. Rev. Anthrop. Ann., 32, 1922. Widler, H. H. '22 Racial Differences in Palm and Sole configuration; Palm and Sole Prints of Japanese and Chinese. Journ. Phys. Anthropol., Vol. V., No. 2, Apr.- June 1922. MORPHOLOGY AND LIFE HISTORY OF POLYTO- MELLA CITRI SP. NOV. J. McA. KATER, UNIVERSITY OF CALIFORNIA AND PRINCETON UNIVERSITY. The organism dealt with in this paper was first found in Berkeley, California, during January, 1924, in a culture medium for Euglena gracilis made after Zumstein \vhich contained the following parts: peptone .5, citric acid .2, grape sugar .5, MgSO 4 -7H 2 O .02, KH 2 PO 4 .05, water 100. In September, 1924, a culture was shipped to me in New Jersey, from Berkeley and the cultures made from this material are still in good condition. A little observation showed that this flagellate reproduces very rapidly during the active stage and also that an abundance of resting forms are periodically present. These facts seemed to indicate possibilities for studying the life history. Since no previous account of this organism has appeared, the name Polytomella citri is proposed for it. I wish to thank Professor C. A. Kofoid for the many courtesies extended to me while working in his laboratory, Professor E. Newton Harvey for many valuable suggestions, and Professor E. G. Conklin for his constant guidance and advice. METHODS. Polytomella citri thrives on the medium mentioned above, but it does not make a lasting culture, two weeks usually being the limit. The best results were obtained from uncooked timothy hay in distilled water, to which a little sugar was occasionally added. An old culture can easily be renewed by dilution with distilled water. Successful cultures can be made on media with Ph value ranging from 3.5 to 9. The P h of old hay cultures is always around 5.5 which seems to be the ideal value for this protozoan. On several occasions fresh cultures were placed in a refrigerator. After remaining there for two months the jars were fairly teeming with Polytomella. 213 214 J- McA - KATER. During the early part of the work the cultures contained in addition to Polytomella, Glaucoma scintillans, Chilomonas para- mecitim, Euglena gracilis, Bodo sp. and some mold, but after several months everything disappeared except Polytomella and the mold. Zumstein's medium showed only E. gracilis mold, and Polytomella citri, the acidity of this solution being too great for the others. The active forms were fixed in hot Schaudinn's fluid and stained in hot iron-alum-haematoxylin. Methyl green and Dela- field's haematoxylin were also used, but with rather poor results. For counterstaining Bordeaux red, eosin, methylene blue, and orange G were employed, the first two yielding by far the best results. Eosin was used in the 95 per cent, alcohol, while when Bordeaux red was the counterstain the slides were placed in a o.i per cent, solution of the dye for twenty-four hours, immedi- ately after applying the haematoxylin. Bonney's triple stain was tried, but was found to have little value for this work. Most of the preparations were made by pipetting a drop of the culture fluid onto a slide, which had been previously smeared with a little albumen fixative, permitting this to evaporate until it did not run and then dropping slide in Schaudinn's for two minutes. Although this technique was quite successful even better results were gotten by centrifuging the material and fixing before pipetting onto the slides. It was found that the encysted forms did not fix well in Schaudinn's nor was it possible to get them to take any kind of stain, even aceto-carmine failed to leave any trace of color. The successful technique finally developed was fixation in Bouin's or McClung's modification of Bouin's for from twelve to twenty-four hours, followed by dehydration and imbedding in paraffin. Sections were cut from two to four micra thick, and stained as above. Schaudinn's fluid was tried in place of Bouin's, but usually resulted in shrinkage of the protoplasm away from the cellulose wall. MORPHOLOGY. Polytomella citri is a colorless flagellate of pyriform shape, the anterior end being rounded, the posterior rather bluntly pointed. The size varies from 14 by 10 micra to 10 by 7, while the average POLYTOMELLA CITRI, SP. NOV. 215 dimensions are 12 by 8 micra. Although the body of living individuals is usually pear-shaped, the form can readily be changed. Thus we find some whose width is greater than their length, others with their posterior ends split into two or more pointed processes (Figs. 2, 3 and 4). In the latter case a groove extends forward for some distance from the notch between these processes. An optical cross-section of the typical form would be circular. The cell is enclosed in a very thin pellicle. This covering is not visible in life and it divides with the body at fission. It is also to be noticed that it is not sufficiently rigid to prevent considerable changes of shape (Figs. 2 to 4). However, its presence can be demonstrated by dissolving the contents of the cell with dilute NaOH. When this is done the pellicle can be seen, providing the light is cut very low. In some cases plasmolysis causes the protoplasm to shrink away from the wall, thereby serving as a demonstration of its existence, but it requires very careful manipulation. The composition of this covering could not be determined, but since the heavy wall which develops around the cyst was found to be cellulose it is presumed that this has the same chemical constitution. At the center of the anterior end are two very noticeable basal granules from which arise four equal flagella. These are slightly shorter than the body and do not taper at all, being as large at the free end as at the point of insertion. The two pairs of flagella are well separated in the living organism by a small cytoplasmic protuberance which extends forward between the basal granules (Fig. i). This prominence is never preserved after fixation. The two contractile vacuoles are located at the anterior end. They pulsate alternately. The spherical nucleus is about 3 micra in diameter and is situated at any point along the long axis of the cell from the posterior fourth to the anterior fourth. All of the chromatin is concentrated in a central sphere, the karyosome. This body is anchored to the nuclear membrane by an indefinite number of achromatic strands. Six is the greatest number of these supports that have been seen, four the least. Aside from the strands .the space between the karyosome and membrane is optically empty. 216 J. McA. KATER. Surrounding the nucleus is an area of very granular, slightly chromatic cytoplasm. This is of very variable extensiveness, sometimes filling half of the cell, while in others it is hardly noticeable. Examination of living specimens reveals longitudinal striations which number about eighteen. It was not possible to make sure of the exact number and consequently their constancy could not be determined. Superficial examination would lead one to think that these were on the outside of the pellicle, but careful focusing shows that they are slightly below the surface and that they do not quite reach either the anterior or posterior ends. They, consequently, must be either on the inside of the pellicle or in the outermost layer of the ectoplasm. After devoting considerable study to this question I rather lean towards the latter, and think that the striated effect is produced by some stringy arrangement of the protoplasm. Living mounts or iodine-eosin preparations show this structure very well, but fixed and stained material gives no indication of it. In the living cell all of the internal structure is effectually screened by relatively large starch bodies which are located peripherally. They may be so large and numerous that they are packed closely together from one end to the other or they may be entirely absent. By the use of a medium which does not contain any carbohydrate (peptone 0.5 part, citric acid 0.2 part, mag- nesium sulphate hydrate .02, KH 2 PO 4 .05 part, and water 100 parts) the organisms may be freed from starch for several days, when it reappears again. No explanation of this fact is known. The addition of cane-sugar to a timothy hay culture will increase the amount of reserve starch in the cells. If they are only partially filled with these bodies it is generally the posterior end which is free from them. The following reactions are the justification for identifying this material as starch ; it gives a deep blue color when treated with iodine; it is digested by ptyalin; when treated with iodine and heated it loses its color, the blue returning when cooled; when treated with iodine followed by NaOH no color is found. The presence of a centriole could not be demonstrated in the interkinetic cell of the active form. However it was seen in POLYTOMELLA CITRI, SP. NOV. 217 several of the division stages figured in this paper (Figs. 13-15). Sections of cysts showed a centriole very clearly. In the active forms an indication of rhizoplasts connecting the basal granules with the nucleus was seen in a few cases (Figs. 20 and 24). This, how r ever must remain purely tentative until more evidence can be produced. There is no doubt as to the presence of this structure in the cyst just previous to excystment (Figs. 38-40). Whether it degenerates after excystment or merely was not demonstrated because of faulty technique, or for other reasons, cannot be decided. Scattered through the cytoplasm of the active form there are sometimes seen a few very basiphilic granules. These stain even darker with iron-alum-haematoxylin than does the nuclear chro- matin, and also give the typical reaction with Bonney's triple stain and methyl green. They increase to such an extent in the cyst that during part of that stage they effectually obscure the entire contents of the cell. For reasons which will be discussed later these are termed metachromatic granules. Seven individuals with two nuclei have been seen. They probably represent cases where fission was prevented, by some unknown reason, from following mitosis. When the resting condition is entered the flagellates cease movement, lose their flagella, round up into a perfect sphere and secrete an extremely thick wall (Fig. 26). From this early spherical stage they pass into an indefinitely wrinkled condition, which must result from drying of the cell contents (Figs. 28 and 29). A gradual disappearance of the starch bodies can be traced and correlated with the wrinkling of the cyst. Chloro-zinc- iodide and sulphuric acid followed by iodine both indicate the wall to be composed of cellulose. LIFE HISTORY. So far as could be determined the life history consists of two phases, (i) active life and (2) rest and reorganization through encystment. During the time of activity Polytomella reproduces by means of longitudinal fission. No multiplication whatever takes place during the period of rest. This is well established since hundreds of stained specimens have been examined and 2l8 J. MCA. KATER. many living ones watched continuously during the process of excystment without the slightest evidence of propagation. There is no indication of a sexual phase in any part of the life cycle. Mitosis. By far the greater number of flagellates in which mitosis has been studied possess within the nucleus an endosome and, surrounding this body, scattered chromatin granules. It is from this scattered material that the chromosomes are usually formed. In Polytomella citri we have a different story, all of the chromatin being located in a single central body, the karyosome * (Parapolytoma satura has a very similar nucleus, Jameson, 1916). The orderly behavior of the karyosome during the early part of the prophase is very interesting. The first indication of mitosis is the bisection of this body in a plane transverse to the long axis of the cell-body. The two parts appear to be equal and they pull slightly apart, leaving between them a light cloud. A second division follows the first and forms at right angles with it. The karyosome now consists of four parts which still have the chro- matic cloud between them. In the next stage that could be found eight karyosomal bodies, imbedded in the chromatic cloud, were visible. Since the karyosome is spherical we would expect a division, corresponding to the third cleavage of a fertilized egg, between the last two. It may be that such is the case, but the minute size of the objects made it impossible to determine. The division of the karyosome continues until more than twenty particles can be made out, still imbedded in the chromatic cloud. It is interesting to note that the anchoring strands are still per- sisting. Careful observations were made in an attempt to de- termine whether they connected, internally, with individual granules or were a part of some achromatic supporting structure within the karyosome, but without results. In the next step the chromidial cloud lightens, the nucleus becomes transversely elongate to a slight extent, there is a conden- sation of the numerous small granules and a spireme appears. This consists of seventeen or eighteen chromatin bodies connected by slender achromatic strands. When the nucleus lengthens 1 There is no question concerning the nature of the chromatic body within the nucleus of Polytomella and since karyosome has a more restricted meaning than endosome it can well be used here, though I consider that endosome, as suggested by Minchin, is a valuable term for the nucleoli of many protozoan nuclei. POLYTOMELLA CITRI, SP. NOV. 219 more and an indication of a spindle appears the chromatin knots on the spireme have been reduced in number to nine and have increased considerably in size. It cannot be said whether this results from the side to side pairing of the eighteen earlier granules or the contraction of alternate connecting strands as evidence was found for both. The achromatic connections still exist between these, the definitive chromosomes. The spireme is now near the equator of the spindle, and in all later stages the strands between the chromosomes have disappeared. The last figure which can be classed as prophase shows the chromosomes lining up on the equator to form a horse shoe around the spindle. The chromo- somes which are first freed from each other by the disappearance of the strands are considerably longer than they are broad. The length is quickly reduced, so that when they split at the meta- phase the two daughter chromosomes resemble two balls that are in contact. Several very clear polar views of the metaphase were found which indicated the number of chromosomes to be nine. These are arranged in the horseshoe-shape which was noted in side view. As noted above a centriole could not be demonstrated in the resting cell. With the elongation of the nucleus and formation of the spindle in the late prophase a granule appears at the poles of the spindle (Figs. 13-15). On the anterior side of the nucleus a line connecting the poles is seen on the nuclear membrane. This, which we shall tentatively term a paradesmose, becomes much more striking in the later stages. Hesitancy in definitely naming this structure is due not to doubt concerning its occurrence, but to the inconclusive demonstrations of a constant centriole, which, presumably, would give rise to the paradesmose. With the progress of the chromosomes towards the poles the spindle widens to equal the diameter of the horse shoe of chro- matin until the band has traversed two thirds of the distance to the poles. Some of the chromosomes now advance ahead of the rest, which show a tendency to clump together. At the same time the nuclear membrane is beginning to constrict at the equator. A little later the dividing nucleus is drawn out into a cylinder with pointed ends, and the chromosomes are clumped into several masses at the ends of the slender cylinder. With the IS 22O J. MCA. KATER. cylindrical shape of the nucleus comes an enlargement of the basal granules, preparatory to their division which will take place just before constriction of the nucleus is complete. At this time the chromatin is clumped into a single mass. The two basal granules divide to form four, each of which gets one of the old flagella. Although no unquestionable figures were found which showed the outgrowth of new flagella it is presumed that this is the method of formation, because if the old flagella split we would expect that to happen at the same time that the basal granules divide and that is not the case. The duplication is by outgrowth in P. agiles (Doflein, 1916), Dunaliella (Teodoresco, 1905), Stephana pter a (Dangeard, 1910), all belonging to this same family. The history of the chromatin from the beginning of the telo- phase, when there are single masses at the poles, until the con- struction of the daughter nuclei is complete is a rapid return through the same stages in reverse order that were passed through in the prophase. The solid masses break up to form a spireme which goes back through the quadri- and bi-partite karyosome to the single condition with which we started. By the time the quadri-partite condition of the karyosome is reached the cell shows marked furrows on both the anterior and posterior ends. In most flagellates fission has been described as beginning at one or the other end. The present case is an example of equal furrowing all around the cell-body. The resting condition is reached by the nuclei, or is very closely approximated, by the time fission is complete. It is interesting to note that the two daughter nuclei are frequently not advanced to the same degree. We must now return to the first elongation of the nucleus and find what happens to the area of dense cytoplasm which surrounds the nucleus. A glance at the figures from metaphase on will show that there is a fairly even distribution of this material to the daughters. Sometimes it surrounds the nucleus in an even layer, in others it is almost all on one side, and in rare cases it is slightly drawn away from the nucleus. A band of this substance can be seen stretching between the daughter nuclei just before the cells separate. The chromatic nature of this material probably indi- cates that, although the nuclear membrane remains intact during POLYTOMELLA CITRI, SP. NOV. 221 mitosis, there is some substance of the nature of oxychromatin that is dissolved and passes through the nuclear membrane. This would make the cytoplasmic area under discussion corre- spond to the archoplasm of Boveri and the archiplasm of Conklin and Wilson. There was no indication of granules of chromatin going bodily through the nuclear membrane. Encystment. When the cell enters encystment the cellulose wall is evidently formed very rapidly. Only a few individuals were found that did not have a perceptible membrane and in these cases it was only a matter of several hours until they looked quite old and sturdy. While the cyst is still spherical the starch bodies are well under way to disappearance (Figs. 26 and 27). W 7 hen the wrinkling of the cyst has reached the degree which typically makes the general outline triangular the starch is all gone. Observation of many stained sections of early spherical cysts did not reveal any remnant of the basal granules. As soon as the organism has become encysted the region im- mediately surrounding the nucleus becomes filled with spherical bodies which are more basiphilic than the nucleus itself. These bodies increase in extent with the aging of the cyst and soon come to fill the entire cell. In many cases the nucleus, which has now decreased to half its original size and stains very faintly, is obscured. Unless a study of this stage is made very critically one might be led to believe that the nucleus has broken up to form chromidia. Such is not the case. Whenever the knife cut through the nucleus or near it the karyosome, anchoring strands, and nuclear membrane make a perfect miniature, in both size and staining character, of the nucleus of the active form. In the hundreds of such sections examined there was no suggestion of chromatin particles leaving the nucleus. Since this material does not come from the nucleus and stains more deeply with haema- toxylin, Bonney's triple stain, and methyl green than does chromatin the name metachromatic granules is justified. It is of interest that the increase of metachromatic granules is highly correlated with the disappearance of starch. After remaining in this condition for some time the granules gradually drain out of the central part of the cell and collect around the periphery, mostly in the angles. The previous 222 J. MCA. KATER. spherical form is lost and they assume various shapes. In most cases they entirely disappear before excystment. When the metachromatic granules are distributed through the entire cell with only small dots of cytoplasm visible, acid dyes, such as Bordeaux Red and eosin, have no effect on what cyto- plasm can be seen, even when extra high percentages are used. With the migration of the granules to the periphery the cytoplasm regains its affinity for acid dyes. After the metachromatic granules have left the immediate vicinity of the nucleus and the cytoplasm has become quite clear we get the first indication of a new centriole and basal granule, in the form of a thickening at the base of one of the anchoring strands. This thickening next appears as a chromatic ball on the strand, a part of which can be seen on each side of the ball. Later it continues to the outside of the nuclear membrane. The basiphilic material that has been drawn out of the karyosome leaves the latter body without disfiguring it. Thus we see that the centriole and basal granules are not formed from a slice of the karyosome, but are taken from that structure by some definitely organized scheme. The body on the outside of the nuclear membrane divides, leaving one portion, supposedly the centriole, while the other portion migrates out through the cytoplasm, spinning a rhizoplast between it and the centriole. Before TEXT FIGURE A. Diagrammatic representation of stages in excystment and the activity of a newly excysted individual. Arrows indicate direction of movement. POLYTOMELLA CITRI, SP. NOV. 223 reaching the periphery the single basal granule divides, the rhizoplast dividing with it, to form the two basal granules. The flagella do not develop until the organism is partly excysted. Excystment is ushered in by the dissolution of the cyst wall on one side (Text Fig. Aa). Many stages of this thinning process can be seen before the living cell comes in contact with the outside. The first step in escaping from the cyst seems to be due to the absorption of water, which swells the cell and causes it to protrude through the dissolved opening. Complete freedom from the cyst wall is attained by amoeboid movement of the newly formed flagellate. Outgrowth of flagella begins just prior to flowing away from the old wall. For periods varying from one to fifteen minutes the newly excysted individual shows only amoeboid movement, without regard for the polarization caused by the presence of flagella (Text Fig. A e, f, g, h) . With the cessation of amreboid movement they assume the typical form and swim off. DISCUSSION. Affinities. The presence of starch within the cell-body of Polytomella citri and the typical Phytomonad symmetry are sufficient to establish its position with the order Phytomonadina. Further, the fact that the pellicle divides with the cell at fission places this organism within the family Polyblepharididae. This family was suggested by Dangeard in 1887 to include, at that time, Pyramimonas tetracynchus Schmarda, Chloraster gyrans Ehrenberg, C. agilis Kent and Polyblepharides singularis Dangeard. In 1905 Teodoresco placed Dnnaliella salina in this group. This is a marine protozoan, as the name would indicate. Griffith (1909) described Pyramimonas delicatulus, taken from a pond in England. In 1910 Dangeard made another contribution to the Polyblepharididae with his description of Stephanoptera fabrece, a salt water form. This species is one of the largest of the family, reaching 35 micra in length. Pyramimonas, 40 micra, being the only one exceeding that size. All of the members of this group possess the ability to change their shape to some extent and Spermatozopsis exsultans (Korchikoff, 1913) has the dis- 224 J- McA - KATER. tinction of leading the family in this respect. Astermonas was the last holophytic genus described (Artari, 1913). Dangeard instituted this family to include holophytic, uni- cellular algae and all of the species mentioned above would fit in with that scheme. They possess a chloroplast, a pyrenoid for the formation of starch, and a red pigment spot may or may not be present. In Dunaliella the green is sometimes obscured by another pigment which gives the organism a vermillion color. A phylogenetic series might be made, beginning with the six or eight flagellated Polyblepharides, going through the five flagel- lated Chloraster, Pyramimonas, which possesses four flagella, Stephanoptera, with two flagella, and culminating with the bi- flagellate Dunaliella, the nearest approach to Chlamydomonas. With the exception of Dunaliella all species have four longitudinal ridges. One might suggest that the scheme could be arranged in the opposite direction, but since the specialization of the Chla- mydomonad type is much greater than that of the Polyble- pharididae this order seems the more reasonable. In 1910 Aragao described Polytomella agilis and suggested that ts relationship was with the Protomonads. Doflein (1916) demonstrated the presence of starch in the same species and consequently, reclassified it under the order Phytomonadina, and since its pellicle divides at fission its family is Polyblepharididae. This colorless form, possessing four flagella, stands in the same relation to Pyramimonas that Polytoma and Paraplytoma do to Chlamydomonas. Dangeard 's family no longer includes only holophytic algae, and the green color must be omitted from a characterization of it. The protozoan dealt with in this paper differs from Aragao's species in the following points: an eye-spot is present in agilis while citri possesses no such structure; the nucleus of agilis contains scattered chromatin while in citri it is concentrated in a single karyosome, the four flagella of agilis arise from four blepharoplasts and citri has only two basal granules. These differences are not sufficient for generic distinction, but justify the formation of a new species. It is to be regretted that so little study has been made of the finer points of structure of the members of this group, the POLYTOMELLA CITRI, SP. NOV. 225 importance of which must be realized when we consider its position on the threshold of the Phytomonadina. LIFE HISTORY. With the exception of Polytomella agilis and Dunaliella salina the descriptions of the members of this family would lead us to believe that the life history consists of two phases, binary fission during the active stage and rest and reorganization through encystment. Dangeard (1889) describes the cysts of Polyble- pharides as spherical bodies with a gelatinous wall. This is the only one which has such a covering. To the same author is due the observation that Pyramimonas forms cysts with a very tough membrane. Germination of the latter was not observed, but in the former it gave rise to a single organism, which, after exhibiting typical amoeboid movement for several minutes would assume the usual shape and swim off by the activity of their flagella. This same observation was made on Polytomella citri. Hamburger (1905) noted the formation of cysts by Dunaliella and also saw numerous empty shells but did not observe any in the process of excystment. The cysts of Stephanoptera are spherical and have a very heavy wall. Dangeard mentions the fact that a few of these had two nuclei, and suggests that there may be some autogamous process during encysted life. The binucleate condition, however, probably does not have any more significance here than it does in the active stage of Polytomella citri, where we have interpreted it as representing cases of cytodierisis being prevented from following nuclear division. Conjugation has been figured by Aragao for Polytomella agilis and by Teodoresco for Dunaliella. In both cases the figures and description are very incomplete and unconvincing. There are none of the few figures given that could not more easily be interpreted as stages in fission than as representing conjugation. This is especially true for the work of Aragao. In spite of the fact that sexual reproduction probably plays no part in the life of most of the members of this family, we would not be surprised to find sexual processes in Dunaliella because of its near relationship to Chlamydomonas . However, until a more adequate study has been made with this problem in view it is well to be hesitant in 226 J. MCA. KATER. accepting the Polyblepharididae as the group which gave rise to the sexual phenomena which are so well developed in the two higher families of the Phytomonadina. Mitosis. The nucleus of most flagellates contains, in addition to the endosome, scattered chromatin granules which seem to be suspended on a linin net-work. The role of the endosome in nuclear division is generally one of two types. The first, typified by the Euglenoids, is the case where this body is drawn out into a dumbbell shape, in the interior of the spindle, with the two knobs forming polar caps. With the progress of the chromosomes to the poles this structure is severed at the equator and forms the endosomes of the daughter nuclei. Some writers (Belar, 1916^4, Berliner, 1909, Schussler, 1917) have termed this central body of the Euglenoid a "centrocaryosome," assigning to it the function of a centrosome. Hall, however, has questioned this view. Another type of behavior is illustrated by the endosome of Polytoma (Entz, 1918) and Chlorogoninm (Hartmann, 1916) which disappears during the prophase. Aragao described the formation of two sets of chromosomes in Polytomella agilis, one from the scattered chromatin, the other from the endosome. Doflein (1916), repeating his work, found that the endosome disappeared during the prophase without contributing to the formation of the chromosomes. It is very evident that a comparison cannot be made of the mitotic phenomena of P. citri and those of any form just men- tioned, not even the other species of its own genus. However, a similar form is found in Parapolytoma satura (Jameson, 1914) which has all of the chromatin gathered into a single body, the karyosome, which is suspended from the nuclear membrane in much the same way as in P. citri. Unfortunately Jameson's account begins rather late in the prophase, his first figure showing the karyosome broken into a number of bodies which are con- nected by linin strands. He states that the greatest number of these ever found is sixteen or eighteen, which fuse in two's or three's to form the eight definitive chromosomes. The same behavior has been found in P. citri, with the addition of the early stages in the fragmentation of the karyosome. Although the chromosomes of P. agilis are formed from scattered chromatin the POLYTOMELLA CITRI, SP. NOV. 227 five definitive ones arise in much the same way, by the pairing of ten chromatin granules which form a ring around the endosome. Doflein analogizes this fusion with the conjugation of homologous pairs of chromosomes in metazoan and metaphytan cells. Thus we see that the greatest similarity in the activity of two cell-organs is not to be found in two organisms whose general structure is most nearly alike, but in two that have the nearest approach to identity in that particular organ. Encystment. There is a remarkable correspondence of the encysted forms of the various species of this family. Pyrami- monas delicatnlus and Spermatozopsis exsultans are the only ones in which cysts have not been described. There is only one case where division takes place within the cyst (Doflein, 1916). Aragao did not find division in this same species. This author met with the same difficulty that was encountered in the present work, namely, that the cyst could not be stained. This fact is probably responsible for the fragmentary study of this important phase of the life history of the Polyblepharididae, as no adequate account has heretofore appeared. In Polyblepharides and Polytomella citri the living organisms have been watched during excystment and in both cases amoeboid movement was observed for some minutes before any use was made of the developing flagella. Dunaliella and Polytomella citri, and probably all of their near relatives, leave the cyst wall behind without dissolving it. The latter dissolves on one side in order to gain freedom, but the remainder of the shell stays in the culture for some time. Since the pioneer work of Guillermond and Meyer metachro- matic granules have been described from bacteria to metazoa. The fact that these granules not only increase greatly during the early days of encystment, but also are dissolved, not thrown off, before excystment indicates that they have some important role to play in the life of Polytomella citri. CENTRIOLE AND BASAL GRANULES. The observations made on this subject in the present work are as follows: There are two basal granules from which arise four flagella. These granules divide during the telophase of mitosis. 228 J. MCA. KATER. The poles of the spindle are lodged in centrioles which are located on the outside of the nuclear membrane. An indication of rhizoplasts connecting the basal granules with the nucleus have been seen in both resting and dividing cells. In the early cysts no basal granules can be seen. Before excystment a centriole and new basal granules arise from the karyosome. This material is drawn out of the latter body without disfiguring it. There is, at first, a single basal granule which divides before reaching the periphery. A rhizoplast is very evident during this phase. The new flagella grow out after the organism is partly excysted. The questions not answered are: is a centriole present in the resting cell of the active form ; what becomes of the rhizoplasts that are so evident before excystment ; are the basal granules drawn into the karyosome at the beginning of encystment or do they degenerate? In view of the fact that a centriole can be seen in division stages, is very pronounced in the cysts and that Doflein has demonstrated an extra-nuclear centriole in Polytomella agilis I think we can conclude that such a center is present in the inter- kinetic cell of P. citri. In addition the paradesmose, which is quite evident, would, presumably, come from a centriole. Jame- son (1914) says that Parapolytoma has no centriole. He mentions Chilomonas and Polytoma as other examples of flagellates without this division center. It is to be noted that Belar (1916) demon- strated a centriole in Chilomonas and Entz (1918) showed that Polytoma has a very definite one on the inside of the nuclear membrane, with a heavy centrodesmose appearing at division. Aragao figures rhizoplasts connecting the basal granules of P. agilis with the nucleus. Hamburger did likewise for Dunaliella. In view of these observations, coupled with what has been seen in P. citri I believe we are justified in assuming that the rhizoplasts are constant structures in P. citri. Berliner states that the basal granules of Copromonas are drawn into the endosome at division. His figures, however, are not at all complete. Jameson finds that the basal granules of Para- polytoma never divide, but three of the four individuals resulting from division receive new basal granules from the karyosome. Entz finds that the centriole and blepharoplasts of Polytoma arise POLYTOMELLA CITRI, SP. NOV. 22Q in the same way as in P. citri with the exception that the centriole remains inside the nuclear membrane. Although it is impossible to definitely answer the third question suggested above, it seems more probable that the granules degenerate because if they entered the karyosome we would expect them to maintain their integrity to sufficient extent to prevent the karyosome from appearing perfectly homogeneous. Such is not the case. SUMMARY. The flagellate herein described belongs to the genus Polyto- mella (Aragao, 1910). The specific name "citri" is proposed. The size ranges from 10 by 7 micra to 14 by 10 micra. Outside of the absence of chlorophyll the structure is typical for the family Polyblepharididse. Although it is a colorless, saprophytic organism the cell-body contains numerous starch bodies. The life history consists of active stage, in which reproduction is by binary fission, and encystment. No reproduction takes place during encystment. There is no indication of any sexual process at any point of the life cycle. Conjugation is not well established in any species of the family. The chromosomes arise as a result of the fusion of chromatin particles which have come from the karyosome. The cysts are filled with metachromatic granules. They usually disappear before excystment. A new centriole and basal granules are given off from the karyosome previous to excystment. Flagella do not grow out until the cell comes in contact with the outside. Amoeboid movement is the means of locomotion for several minutes following excystment. 230 J. MCA. KATER. LITERATURE. Aragao, H. deB. '10 Untersuchungen iiber Polytomella agilis n.g., n. sp. Memorias do Instituto Oswaldo Cruz, Tomo 2. Belar, K. 'i6A Protozoenstudien I. 'i6B Protozoenstudien II. Arch. f. Protistenkunde, 36. Berliner, E. '09 Flagellatenstudien. Arch. f. Protistenkunde, 15. Dangeard, P. A. '89 Memoire sur les Algues. Le Botaniste, premiere serie. '10 C. R. Acad. de Science, Paris, 151. Dill, O. '95 Die Gattung Chlamydomonas und ihre nachten Verwandten. Jabrbiicher fur Botanik, 28. Doflein, F. '16 Polytomella agilis. Zoologischer Anzeiger, 47. '18 Beitrage zur Kenntnis von Bau und Teilung der Protozoenkerne. Zoologi- scher Anzeiger, 49. Entz, G. '18 Ueber die mitotische Teilung von Polytoma uvella. Arch. f. Protistenkunde, 38. Griffith, B. M. '09 On Two New Members of the Volvocaceae. New Phytologist, 8. Guilliermond, A. '10 A propos des corpuscles metachromatiques ou grains de volutin. Arch. f. Protistenkunde, 19. Hamburger, Clara. '05 Zur Kenntnis der Dunaliella salina. Arch. f. Protistenkunde, 6. Hartmann, Max. '16 Die Kernteilung von Chlorogonium elongatum. Sitzungsber. Ges. nat. Freunde, Berlin, 1916. Hartmann und Prowazek. '07 Blepharoplast, Caryosom und Centrosom. Arch. f. Protistenkunde, 10. Jameson, A. P. '14 A New Phytoflagellate and its Method of Nuclear Division. Arch. f. Protistenkunde, 33. Korchikoff, A. '13 Spermatozopsis exsultans nov. Gen. et sp. aus der Gruppe der Volvocales. Berichte der Deutschen Botanischer Gessellschaft, 31. Merton, H. '08 liber den Bau und die Fortpflanzung von Pleodorina illinoisensis . Zeit- schrift fur Wissenschaftliche Zoologie, 90. Minchin, E. A. '14 Remarks on the nature of Blepharoplasts or Basal Granules of Flagella. Arch. f. Protistenkunde, 34. POLYTOMELLA CITRI, SP. XOV. 23! Schussler, H. '17 Cytologische und entwicklungsgeschichtliche Protozoen studien. I. Ueber die Teilung von Scytomonas pusilla Stein. Arch. f. Prot., 38. Teodoresco, E. C. '05 Organisation et developpement du Dunaliella, nouveau genre de Volvocacee- Polyblepharidee. Beihefte zum Botanischen Centralblatt, 19. West, G. S. '16 Algae I. Cambridge University Press. 232 J. MCA. KATER. EXPLANATION OF PLATES. Figures I and 25 to 29 made from living material. Figures 2 to 24 made from material fixed in hot Schaudinn's fluid and stained in hot iron-alum-haematoxylin, counterstained with Bordeaux red. Figures 30 to 41 were made from specimens fixed in Bouin's, stained in hot iron-alum-haematoxylin and counterstained with either Bordeaux red or eosin. All drawings made with Abbe camera lucida. Magnification 2500 X. PLATE I. FIG. i. Diagrammatic camera lucida drawing of living individual pressed between two mold hyphae. cv, contractile vacuole; s, starch bodies. FIGS. 2, 3 AND 4. Showing extreme changes of shape. FIG. 5. Binucleate individual. FIG. 6. Resting nucleus. Extensive area of slightly chromatic cytoplasm surrounding nucleus and filling posterior end. FIGS. 7 TO 24. Stages in the division of the nucleus and cell-body. Fig. 7. The transverse splitting of the karyosome, the first indication of mitosis. FIGS. 8 TO ii. Continued fragmentation of the karyosome resulting in over twenty chromatin particles imbedded in a chromatic cloud. Anchoring strands persisting. FIG. 12. Nucleus becomes slightly elongate, anchoring strands disappear, and seventeen or eighteen chromatin particles connected by slender achromatic strands become visible. FIG. 13. The chromatin particles of the preceding one have paired to produce the nine definitive chromosomes. An indication of a spindle appears and the poles are lodged in centrioles which are connected by a paradesmose on the anterior side of the nucleus. BIOLOGICAL BULLETIN, VOL. XLIX PLATE I mmml ' -- ' '*'- .; faXri* \i n J. MCA. KATER. 16 234 J- McA - KATER. PLATE II. FIG. 14. Chromosomes lining up on equator to form horse shoe around the spindle. Paradesmose visible above spindle. FIG. 15. Metaphase. FIG. 16. Polar view of metaphase. FIGS. 17 AND 18. Migration of chromosomes to poles of spindle. FIG. 19. The nucleus has become elongated into a cylinder with pointed ends and the chromosomes are fusing together. The paradesmose is seen on anterior side of nuclear membrane and the basal granules are enlarging. FIG. 20. The chromosomes have fused into a single mass at the poles, and the basal granules have divided. FIG. 21. The chromatin masses of the preceding one have broken up to form a spireme comparable to that of Fig. 13. FIGS. 22 AND 23. Reconstitution of the daughter nuclei and division of the cell. FIG. 24. A case where fission was either delayed or prevented by some unknown cause. FIG. 25. Beginning of encystment. The flagella have been lost and the cell is packed with starch bodies. BIOLOGICAL BULLETIN, VOL. XLIX. PLATE II. J. MCA. KATER. 236 J. MCA. KATER. PLATE III. FIGS. 26 TO 29. Stages in the formation of wrinkled cyst wall and the disap- pearance of the starch bodies. FIGS. 30 TO 40. Made from sections four micra thick. FIG. 30. Early cyst. Metachromatic granules are forming around the nucleus. FIG. 31. The wall has become thicker than in the preceding case and the metachromatic granules are scattered through the entire cell. Nucleus obscured. FIG. 32. The metachromatic granules are evenly distributed through the cytoplasm. Wall becoming wrinkled. FIG. 33- The metachromatic granules are migrating to the periphery where they will be dissolved. FIG. 34. The first indication of budding of the karyosome. A thickening appears at the base of one of the anchoring strands. FIG. 35. The material which is going to form the new centriole and basal granules appears as a chromatic ball on one of the anchoring strands. FIG. 36. The chromatic ball has reached the outside of the nuclear membrane. FIGS. 37, 38 AND 39. Stages in the migration of the basal granule towards the periphery. FIG. 40. The basal granule has divided, the rhizoplast dividing with it. FIG. 41. Drawing made from whole mount. The organism is partly excysted and the flagella are growing out. BIOLOGICAL BULLETIN, VOL. XLIX. PLATE III. 37 J. MCA. KATER. Vol. XLIX October, 1925 No. 4 BIOLOGICAL BULLETIN TRICHODINA STEINII (C. AND L.) FROM PLAN ARIA POLY CHORA (O. SCHM.). WM. A. KEPNER AND A. L. PICKENS, UNIVERSITY OF VIRGINIA. In the spring of 1924 the junior author secured specimens of Planaria polychora O. Schm. from a pool several hundred yards west of the Biological Laboratory of the University of Virginia. The Planaria, in some cases, carried upon their surfaces speci- mens of Trichodina Steinii. These specimens afforded us an excellent opportunity to study the habits and anatomy of this species of large ectozoic peritrichous ciliate. Our observations led us to recognize two interesting facts concerning this species first described by Claparede and Lachmann (58). The genus Trichodina is so closely related to Vorticella that it might be placed in the subfamily Vorticellidae. Indeed Fulton (23) said of the subfamily Urceolariidae, to which Minchin assigned Trichodina, "It is a matter of personal preference whether one considers the Urceolariidae a subfamily of the Vorticellidae ... or as a separate family." With this close systematic relationship in mind, certain homologies between the anatomical details of Trichodina and those of Vorticella are of interest. The peristome and nucleus complex are clearly the homologues of the peristome and nucleus complex of Vorticella. In the free swimming Trichodina Steinii, the peristome remains closed (Fig. 3) in a manner that causes it to greatly resemble the closed peristome of a contracted Vorticella. A free swimming Vorticella has a posterior zone of cilia (Fig. 4). The homologue of these adoral cilia become fused at their bases to form the "velum" of Trichodina. Trichodina Steinii, in correlation with its habit as an ectozoon, has developed a horny flexible ring 17 237 .c I*'' V ^~* ,..- ^** x FIG. i. Dorsal aspect of specimen as seen when fixed to or creeping over host. v, velum; c.v., contractile vacuole; f.v., food vacuoles; p.c., cilia of peristome; m.n., meganucleus (U-shaped). Observe the small micronucleus lying at outer margin of the U-shaped contour of meganucleus. X 1000. FIG. 2. Transverse optical section through diameter of striated horny ring or ring band of adhesive organ, r.b., horny ring or striated ring band; d, denticles or hooks. X 1000. FIG. 3. Free swimming Trichodina Steinii. p, peristome closed. Note posi- tion of micronucleus and meganucleus with reference to the animal's swimming posteriorly, as indicated by the arrow. FIG. 4. Free swimming form of Vorticella. Note position of micronucleus and meganucleus with reference to the animal's swimming anteriorly, as indicated by arrow. TRICHODINA STEINII (C. AND L.). 239 (ring band) as a conspicuous feature of its "adhesive organ." In this complex adhesive organ, with its velum, Louis Agassiz (50) saw homologies between it and the details of an hydro- medusa. Because of this he looked upon the Trichodina as being the medusa of Hydra. Attempting to establish analogies may, therefore, lead one into strange ventures. But a further homol- ogy may be seen in the horny ring of the adhesive organ of Trichodina Steinii. This ring is of a chitinous texture and lies at the posterior extremity of the body of Trichodina. Faure- Fremiet (05) studied the adhesive substance that Vorticella elaborates when it fixes itself to a surface. He found that this adhesive material was a "secretion chitineuse." If now the reader will look at our figures 3 and 4, he will see that in the free swimming Vorticella and Trichodina at the posterior end of each of these ciliates is a region concerned with the secretion of a horny material. In Vorticella the horny substance is the adhesive material, whereas the homologue of this horny substance becomes the horny ring or "striated ring band" of the adhesive mechanism in Trichodinii Steinii. Perhaps a more interesting comparison may be drawn between Vorticella and Trichodina than that based upon homologies. This contrast presents itself in the polarity of the two protozoa. Vorticella has its anterior-posterior polarity normal. When a Vorticella moves about it swims with its peristome forward as indicated by the arrow in Fig. 4. The careful studies of Jennings (04) indicate that it is a matter of great importance to an animal like Paramoscium that its peristome lies towards the line of travel. To reverse this attitude would appear, therefore, to be a radical change. This, however, is what has taken place in Trichodina Steinii. These ciliates become quite active when their host is placed under the compound microscope's light and creep about over the surface of the host freely. In all cases they advance posteriorly as they cling lightly to the surface of the host with their adhesive organs. If the disturbance be continued long enough, they will leave the host and swim off into the water. Even the free swimming specimens travel posteriorly. When thus traveling, they have the peristome closed and inactive while the velum and horny ring are expanded (Fig. 3). There is thus a complete reversal of polarity of this animal both when it creeps over a 240 \VM. A. KEENER AND A. L. PICKENS. surface and when it swims. This reversal of polarity has been overlooked by the earlier observers of Trichodina Steinii. Kent (81) implies that Trichodina pediculus (Ehr.) has the usual polarity. Fulton (1923) in making a thorough review of the literature of this genus and in his own observations, seems not to have encountered this reversal of physiological and morpho- logical polarity. This author, as well as earlier investigators, has also overlooked the homologies that we have draw r n between this ectozoic peritrich and Vorticella. This animal should, therefore, serve as the basis of some interesting work done upon its physiological axial gradient. From what we have seen of its morphology we expect to find that its axial gradients have been reversed in their sequence; for it is seen that there has been a morphological reversal, despite the homologies that we have indicated. In Vorticella there is an emphasis placed upon the anterior region in that the loop of the meganucleus and the micronucleus lie in that region. In Trichodina Steinii the morphological emphasis is reversed; for in this form the loop of the meganucleus and the micronucleus lie in the posterior region of the cell. Thus it appears that in Trichodina rather complex adaptive structures are but the homologues of simpler structures found in Vorticella; but that correlated with the ectozoic mode of living Trichodina has undergone a reversal of polarity that involves both its physiological axis and its nuclear complex. LITERATURE. Agassiz, L. '50 Remarks on the Little Bodies seen on Hydra, which have been Described as Parasites. Proc. Bost. Soc. Nat. Hist., Vol. 3, p. 354. Claparede, Ed., and Johannes Lachmann. '58 Etudes sur les Infusoires et les Rhizopodes. (Extrait des tomes V, VI, et VII des " Memoires de 1'Institut Genevois.") Faure-Fremiet, Emmanuel. '05 La Structure de 1'appareil fixateur chez les Vorticellidae. Arch. Protisten- kunde. Bd. 6, p. 207. Fulton, John F. '23 Trichodina pediculus and a New Closely Related Species. Proc. Boston Soc. of Nat. History, Vol. 37, pp. 29. Jennings, H. S. '04 Contributions to the Study of the " Behavior of the Lower Organisms." Carnegie Institution, Washington, D. C. Kent, W. Saville. '81 Manual of the Infusoria. London. STUDIES IN ARTIFICIAL PARTHENOGENESIS. V. THE ANOMALOUS ACTION OF MERCURIC CHLORIDE. L. V. HEILBRUNN, UNIVERSITY OF MICHIGAN. Traube 1 in 1909 made the claim that substances of low surface tension were more effective in producing parthenogenesis than those of higher surface tension. In 1913 and again in 1915, Heilbrunn 2 proposed a theory of membrane elevation in the sea-urchin egg, according to which the lifting off of the membrane (in this egg) is the direct result of a lowering of its surface tension. If this surface tension theory of membrane elevation is correct, only those substances which cause a decrease in surface tension can produce typical membrane elevation. So far as previous experiments have gone, this is true. There is no case in which membrane elevation has been produced by an agent which does not sharply lower surface tension (Heilbrunn, '24). During the past summer an apparent exception was dis- covered. In the course of some work which we were doing with dilute solutions of mercuric chloride in sea-water, my assistant Mr. D. E. S. Brown called my attention to what was apparently true membrane elevation in the egg of the sea-urchin Arbacia. Beautiful wide membranes arose from the egg surface. That these were truly elevated membranes (not swollen membranes) was shown by the fact that they could be made to collapse on the addition of egg albumin to the sea-water. The parthenogenetic action of mercuric chloride was known to F. R. Lillie and he mentions it briefly in a paper published in 1921 3 (see p. 140). It seems certain that mercuric chloride as such could have no great effect on surface tension. Apparently, therefore, the fact that the mercury salt causes membrane elevation can not be made to fit in with the surface tension theory. Because of this apparent conflict, it was thought worth while to further investi- 241 242 L. V. HEILBRUNN. gate the action of the mercuric chloride, with a view to the possible abandonment of the surface tension theory. Although many experiments were performed, only a few of them will be considered and these rather hastily. They suffice to show that the action of mercuric chloride is not a simple one. If an m/io solution of mercuric chloride is diluted a thousand times with sea-water, then an " w/io,ooo solution of mercuric chloride in sea-water " is obtained. A solution of this sort is very favorable to membrane elevation. Solutions decidedly more concentrated than m/io,ooo do not act as well. In an ra/iooo solution, the membranes, if they lift off at all, become only partially elevated, apparently they stick to several points of the egg surface as they attempt to lift away from it. On the other hand extreme dilutions are also ineffective, although it is possible to obtain good membrane elevation in solutions as weak as w/ioo,ooo. Thus there is a wide range of concentration in which the mercuric chloride is effective. Most of the experi- ments were made with an w/io,ooo solution (in sea-water). On exposure to the mercuric chloride, membrane elevation does not occur immediately. After 3 or 4 or 5 minutes, the membranes can be seen rising from the surface of the eggs. The effect of the sublimate is not solely a cortical effect. After eggs have been exposed for about 12 minutes other changes may begin to appear. In many cases the cell undergoes what is apparently a cell division. At any rate it becomes constricted. Usually this constriction divides the egg unequally, a large cell and a small cell are formed. In such cases of unequal division the smaller cell usually contains a mass of pigment and the larger cell is comparatively colorless. Now and then instead of an unequal division there is a furrow around the very center of the egg with an accumulation of pigment granules underneath the furrow. No attempt was made to discover whether nuclear changes accompanied the changes in the cytoplasm. If the constrictions of the cytoplasm do represent anything at all comparable to a normal cell division, it is surprising that they occur so soon after the eggs are exposed to the mercuric chloride. Typically the Arbacia egg does not divide until 50 or 60 minutes have elapsed after fertilization. The cytoplasmic constrictions STUDIES IN ARTIFICIAL PARTHENOGENESIS. 243 just described make their appearance 12 or 15 minutes after the eggs are placed in the mercury solution. The constriction of the cytoplasm is not followed by anything approaching normal development, even if the egg is removed from the solution of mercuric chloride and placed in normal sea-water. Ordinarily when Arbacia eggs are exposed to an m/ 10,000 solution of mercuric chloride in sea-water, only about 10 per cent, of the eggs undergo membrane elevation. Why 10 per cent, of the eggs should throw off perfect membranes and the other 90 per cent, should show no change at all, was for a time a mystery. After a number of experiments under diverse conditions, it was found that the percentage of eggs undergoing membrane elevation could be greatly increased by allowing the eggs to age. If eggs are taken from the ovary and placed directly into dilute solutions of mercuric chloride, no membrane elevation occurs. On the other hand if they are allowed to lie in sea-water in shallow dishes for 5 or 6 hours, practically a hundred per cent, will undergo membrane elevation when exposed to the mer- curic chloride. In working with this reagent it is therefore advisable to allow eggs to age before starting to experiment with them. The effect of aging is shown in the following table. The first column of this table gives the number of minutes the eggs were allowed to remain in sea-water before they were treated with m/io.ooo mercuric chloride. Only the eggs of a single female were used but these were very plentiful. On removal from the ovary they were placed in 50 cc. of sea-water in a fingerbowl. After a time, as a result of the constant removal of material, the eggs at the bottom of the fingerbowl were no longer evenly distributed, some being more closely clustered than others. In the denser masses of eggs, the aging process apparently proceeded at a slower rate and this accounts for the irregularities in the table. The second column shows the percentage of eggs under- going membrane elevation when subjected to mercuric chloride. To obtain this percentage two hundred eggs were counted in every instance except in the case of the eggs aged for 268 minutes. Only a hundred of these were counted. 244 L. V. HEILBRUNN. Time Eggs Remained Percentage of Eggs in Sea-water before Showing Membrane Treatment with Elevation. HgCh, Minutes. o o 5 6J 10 7 2 15 I0 20 10 30 US 40 13 50 142 60 17 81 103 . 128 158 16 193 l8 i 238 3oi 268 21 300 - 625 345 -942 Although the table is not as regular as might be wished, it is clear that as the eggs lie in sea-water they become increasingly susceptible to mercuric chloride. This fact is interesting for at least two reasons. In the first place it apparently does not harmonize very well with the fertilizin theory of fertilization as developed by F. R. Lillie. 3 Aging of eggs, according to this theory, involves a loss of fertilizin, a material which is regarded as essential to fertilization, and yet in the experiment described above, such aging favored a cortical change characteristic of fertilization. But the case of mercuric chloride is certainly a special one, and it is doubtful if any general conclusions for or against the fertilizin theory can be derived from it. This will be more obvious from the later discussion. Our main interest is elsewhere. Why do eggs after standing in sea-water respond more readily to treatment with mercuric chloride? Possibly in or around eggs fresh from the ovary there is some substance present which exerts an interference. This substance escapes on standing; it may be gaseous. On a priori grounds one would suspect carbon dioxide. Experimental evidence favors this conjecture. When eggs STUDIES IN ARTIFICIAL PARTHENOGENESIS. 245 were allowed to stand until they became highly susceptible to mercuric chloride and were then treated with a solution of the sublimate in sea-water which contained a trace of hydrochloric acid, they did not lift off membranes. If now the solution of mercuric chloride in acidified sea-water was shaken vigorously for a few minutes it again became potent as a stimulant to membrane elevation. In one experiment 4 cc. of n/io HCl was added to 200 cc. of sea-water. To this was added n cc. of m/iooo HgClo. The resultant solution had a p H below 6.8 and caused no membrane elevation. It was shaken until the p H rose to above 7, but it still remained ineffective. It was then shaken vigorously for several minutes, and the p H rose well above 7. After such shaking it produced membrane elevation generally. The effect of the shaking is to remove carbon dioxide from the solution. It seems certain from this experiment that the presence of carbon dioxide interferes with the production of membrane elevation by mercuric chloride. Our first point then is the fact that mercuric chloride causes membrane elevation only in the absence of any considerable quantity of carbon dioxide. A second point, which was not investigated as closely as it might have been, is the fact that sublimate treated eggs, if centrifuged one or two minutes after the exposure begins, do not lift off membranes. In one experiment no membranes were lifted off when eggs were centrifuged either one or two minutes after they had been placed in a w/io,ooo HgCl 2 solution. Those centrifuged 3? minutes after being placed in the solution showed membrane elevation in 91 out of 100 cases. The centrifugal force used was 4,968 times gravity and in this experiment the eggs were centrifuged for 40 seconds in each instance. Appar- ently it is not necessary to centrifuge the eggs for as long a time as 40 seconds. In other experiments it was found that centrifugal treatment for only 5 seconds was enough to prevent all but a small percentage of the eggs from undergoing membrane eleva- tion. The reason for the effect of the centrifuge is not certain. A plausible hypothesis, but by no means the only one, is that the mercuric chloride reacts with the jelly of the egg to form mer- curous chloride and chlorine, and that it is the chlorine which is 246 L. V. HEILBRUNN. important for membrane elevation. Centrifuging removes the jelly from proximity to the egg surface. In favor of this view is the fact that mercuric chloride regularly does break down into calomel (or mercuric oxychloride) and chlorine in the presence of organic substances (Mellor, 4 Gmelin-Kraut 5 ). On the other hand it should be pointed out that if the jelly is removed from the eggs either by shaking or centrifuging or both, and the eggs are then subjected to mercuric chloride solution, they undergo membrane elevation just as well as when the jelly is present. This fact need not interfere with our hypothesis. We can assume that although ordinarily the mercuric chloride reacts with the jelly to form chlorine, in the absence of chlorine it may react with the cortex of the egg itself. A test of this point might be made by removing the jelly, treating with mercuric chloride and then centrifuging. If the above interpretation is correct then centrifuging of jelly-free eggs should not interfere with membrane elevation produced by the mercury salt. Unfortunately such a test was not made. But there is other evidence in favor of the view that mercuric chloride reacts with some part of the egg. Sublimate solutions lose their effectiveness when allowed to stand over eggs. In one instance, after an m/io,ooo solution had stood over eggs for 10 hours it in large measure lost its effectiveness as a stimulant to membrane elevation. Although a control of the same solution produced typical membrane elevation, the solution which had stood over eggs caused at best only a partial and slight membrane elevation. Even in the absence of eggs an m/ 10,000 mercuric chloride solution slowly loses its power to cause membrane elevation. The loss of this power is much more rapid in the presence of eggs. If we assume that mercuric chloride gives off chlorine and it is this substance which causes membrane elevation, it should be possible to duplicate the action of the mercury salt with chlorine itself. Chlorine gas was manufactured by adding hydrochloric acid to potassium permanganate and the gas was then passed through sea-water. If two drops of the solution of chlorine in sea-water are added to 20 cc. of sea-water the resultant solution is then STUDIES IN ARTIFICIAL PARTHENOGENESIS. 247 effective in inducing membrane elevation. The results with chlorine itself are not as good as those with mercuric chloride. It is hard to regulate the amount of chlorine and only a very narrow range of concentrations may be used. One drop more or less of chlorine water added to 20 cc. of sea-water would often determine whether the resultant solution would be effective or not. Moreover the number of eggs introduced into the chlorine solution was an important factor. Thus in one experiment two stender dishes were each filled with 20 cc. of sea-water plus 2 drops of chlorine water. Many eggs were placed in one of the dishes, only a few in the other. In the latter case 78 per cent, of the eggs underwent partial or complete membrane elevation; in the dish containing many eggs no membrane elevation occurred at all. It is easy to suppose that the addition of mercuric chloride to eggs furnishes a mechanism for supplying approximately the proper dose of chlorine to the egg surface. This is probably the reason that solutions of chlorine alone are not as effective as the mercuric chloride. An argument that might be advanced against the idea that the action of mercuric chloride is due to chlorine is the fact that mercuric nitrate is also effective in causing membrane elevation. In order to exclude the presence of chlorine, the mercuric nitrate was made up in a 0.9 m sugar solution. A solution roughly w/iO,ooo was prepared. The action of such a solution is very peculiar. Extremely wide membranes are lifted off, membranes which are quite different in appearance from the normal-looking ones produced by mercuric chloride. It would be easy to show that a reaction occurs between the sugar and the mercuric nitrate, but no effort was made to determine the products of such a reaction. However it is believed that one of the reaction products causes the membrane elevation. From the evidence cited it is obvious that the action of mercuric chloride is complex. It appears certain that the reagent does not act in its original form but undergoes some sort of a chemical transformation. There is good reason to believe that chlorine is produced, and that this substance acts on the eggs and incites membrane elevation. 248 L. V. HEILBRUNN. If this view is correct then the fact that mercuric chloride solutions are effective in causing membrane elevation can not be used as an argument against the surface tension theory. If the sublimate forms chlorine we know that a substance of extremely low surface tension must be present. It is possible that many more facts with regard to the action of mercuric chloride on egg cells or cells in general might easily be obtained by a further study of the sea-urchin egg in the presence of this reagent. Our only interest in the subject lay in the possibility of overthrowing the surface tension theory of membrane elevation. As soon as it was evident that no such overthrow was possible on the basis of the mercury evidence, no additional experiments were planned. SUMMARY. 1. Dilute solutions of mercuric chloride in sea-water cause typical membrane elevation in the sea-urchin egg in spite of the fact that they presumably do not lower surface tension. 2. The action of the mercuric chloride is favored by aging the eggs. Eggs fresh from the ovary are not acted upon, and the percentage of membrane elevation on treatment with the subli- mate solution increases in proportion to the time the eggs have stood in sea-water before being subjected to the reagent. 3. The favorable effect of aging is apparently due to the removal of carbon dioxide. The addition of carbon dioxide prevents membrane elevation by mercuric chloride. 4. If eggs are centrifuged one or two minutes after the treat- ment with mercuric chloride is begun, membrane elevation is generally prevented. 5. Solutions of mercuric chloride in contact with eggs lose their power of provoking membrane elevation. 6. The facts cited in 4 and 5 are regarded as evidence in favor of the view that mercuric chloride reacts with the jelly or cortex of the egg to form chlorine. Such a reaction is in accord with the usual behavior of mercuric chloride in the presence of organic materials. 7. Chlorine gas is effective in producing membrane elevation. 8. The action of mercuric chloride in causing membrane eleva- STUDIES IN ARTIFICIAL PARTHENOGENESIS. 249 tion is probably due to the formation of chlorine. Since chlorine has a very low surface tension, the fact that mercuric chloride causes membrane elevation can not be used as an argument against the surface tension theory. REFERENCES. 1. Traube. '09 Biochem. Zeitsch., XVI., 182. 2. Heilbrunn. '13 BIOL. BULL., XXIV., 343; 1915, ib., NNIX., 149; 1924, ib., XLVI., 277. 3. Lillie. '21 BIOL. BULL., XLI., 125. 4. Lillie. '19 Problems of Fertilization. Chicago, 1919. 5. Mellor. A Comparative Treatise on Inorganic and Theoretical Chemistry, Vol. IV., London, 1923. 6. Gmelin-Kraut. '14 Handbuch der anorganischen Chemie. Bd. V., Abt. 2. 7te Aufl. Heidelberg, 1914. THE SPERM ATHECA OF EURYCEA BISLINEATA. 1 VERA KOEHRING, SMITH COLLEGE, NORTHAMPTON, MASS. Since 1785 it has been known, first through Spallanzani, that fertilization in salamanders is internal. Later there were found at intervals in several species "receptacula seminis" into which spermatozoa are received and stored. So far as is known in most species of salamanders the spermatozoa are transferred some time before egg-laying to the cloaca of the female by means of spermatophores deposited by the male and received by the female either actively or passively, the method of deposition and re- ception varying somewhat in the few species which have been noted at breeding. The "receptacula seminis" or spermathecse 2 of several forms has been noted by Rathke (1820), Leydig ('53), Siebold ('58), Jordan, Fisher and Stieda ('91), and Kingsbury ('95). The work of Dr. Kingsbury is especially important as a comparative study of the spermathecee of Necturns maculatus, Diemyctylus viridescens, Desmognathus fuscus, Amblystoma punctatum, Ple- thodon glutinosus and Spelerpes bislineatus (Eurycea bislineata). In all of these forms the spermatheca is dorsal to the cloaca and consists of a number of tubules. These tubules open severally into the dorsal wall in Necturus, Diemyctylus, and Ambystoma and cover comparatively large areas in this region. In Des- mognathus, Plethodon and Eurycea the organ is more compact and opens by a single central tubule into the cloaca. Kingsbury describes but one specimen of Eurycea bislineata taken in October and containing no spermatozoa. It is the purpose of this paper to continue the work on Eurycea, describing the mature spermatheca at various seasons and in the course of its development. 1 Contributions from the department of Zoology, Smith College, No. 131. 2 Dr. H. H. Wilder calls attention to the fact that the correct word is Sperma- totheca, using the root of the word (spermato-) instead of the nominative stem. 250 THE SPERMATHECA OF EURYCEA BISLINEATA. 251 I. THE MATURE SPERMATHECA. The spermatheca of Eurycea bislineata is a compact, definitely bounded organ. Lying in a median dorsal position with reference to the cloaca it extends from the opening of the organ, a point cephalad to the vent of the cloaca, to the posterior wall which lies above the vent of the cloaca. A mature spermatheca is usually from 1/2 mm. to 2/3 mm. in antero-posterior extent, approximately as wide as it is long and slightly higher, dorso- ventrally, than it is wide that is, somewhat conical in shape. The spermatheca is a sac of tubules. The storage tubules, where the spermatozoa lie in season, are flask-shaped, from seven or eight to sixteen in number, and they converge into one central tubule which bends abruptly and opens into the cloaca. The sac is of heavily pigmented connective tissue. In the posterior wall and the floor of the sac this is thickest, most coarse and black, and the flask ends of the tubules are imbedded in the mass as in sockets. The coarse, pigmented tissue thins out in the anterior region and there is none of it in proximity to the necks of the flask-shaped tubules or the central tubule. The extreme caudal end of the sac is but loosely connected with the cloacal wall and surrounding tissues, being held in place by scattered coarse strands of connective tissue. But approaching the mouth of the organ, where the pigmented floor thins out, it becomes more a part of the cloacal structure, the smooth musculature of the cloacal walls merging with the similar fibers of the interior of the spermatheca. The arrangement of the tubules is noteworthy. The flask- shaped ends lie in the floor of the sac imbedded in pigmented tissue. Some are quite anterior, being anterior to the plane of the opening of the central tubule into the cloaca. But these most anterior tubules, one on each side of the median line, have their sockets of pigmented tissue though pigment is otherwise sparse in this region. The necks of the flasks converge in the posterior end of the sac and form the central tubule which runs dorsally anterior. Thus some of the flasks have very long and narrow necks. The convergence of the necks occasions a large reservoir which narrows into the central tubule proper. 252 VERA KOEHRING. The lining of the whole system of tubules is of most delicate columnar epithelium which extends into the cloaca in the region around the mouth of the central tubule. It is a markedly different tissue from the mucous lining of the cloaca. There is no mucous secretion from the spermatheca; nor at times is there any secretion of any kind as far as can be ascertained by staining reactions. Save for the spermatozoa during the breeding season the lumina may be clean and clear. Some series, however, show a faint indefinite substance, rather stringy, in the flask ends. The necks and central tubule may show slight traces of the matter also, though it is rare in the central tubule which is generally unusually clear. There is a slight differentiation in the lining tissue in different parts of the system. In the blind ends the cells are long. The bases are densely granular with large nuclei. The apices stain faintly; they seem filled with minute, transparent globules. The apices are irregular they do not form an even border for the lumen. They gi\ T e a picket-fence appearance. In some series these inner halves of the cells are shrunken so that their ends appear as amceba-like processes, or as if they were in an exhausted state. Yet these conditions occur during the fall and winter months during which it is to be supposed that there is no reason for activity of the cells as no spermatozoa are present. The cells of the necks of the flask tubules are shorter than those of the blnid ends and produce a much more even border- that is, there is no appearance of shrinkage of the cells. The apices are rounding. It is in the central tubule, however, from the region of the convergence of the necks to the area surrounding the mouth of the cloaca that there is the most marked difference from the lining of the flask ends. The appearance of the central duct is constant at all seasons. These cells do not differ as to bases but the apices are very slightly longer and stain even more delicately than do those of the flask ends there is no globular appearance. The apices fit perfectly together and form a smooth, even surface for the lumen. No variation of this condition has been noted in any mature spermatheca. The size of the lumen of the central tubule varies it is a reservoir at the convergence of the necks. In what are presumed THE SPERMATHECA OF EURYCEA BISLINEATA. 253 to be very old animals there are two of these reservoirs formed by the necks of each side; these join, narrow and become the central tubule. The lumen of this tubule near the opening into the cloaca is often very small about i/ioo mm. but it may be as wide as 4/100 mm. The lumina of the necks vary around i/ioo mm. and those of the flask ends are usually about 6/100 mm. in diameter. The dense coat of pigmented tissue, covering and binding in place the tubules, has been spoken of. This coat is thin, some- times of but a few strands on the roof of the spermatheca. It is along this dorsal region that the central duct lies. This duct and the necks are surrounded by massed smooth muscle fibers which do not differ from the smooth muscle of the walls of the cloaca. Unless injected, it is difficult in amphibian material to study the blood supply of an organ. Red blood corpuscles stain vividly with eosin and when present in numbers indicate blood vessels and capillaries even when dense pigment is present. In one series of a spermatheca which is on the verge of maturity many blood corpuscles have remained in this region. It is evident that the blood supply is rich. Corpuscles running single file everywhere throughout the region of the spermatheca indicate a close network of capillaries and some larger blood vessels are present. Only injection would show certainly, however, just where these vessels branch from the dorsal aorta which lies immediately above the spermatheca. The mating of Eurycea takes place in the spring. There have been no spermatozoa found in the spermatheca during the fall and winter months. April may be taken as the average mating season though there must be as variable a season for mating as there is for egg-laying and many early and late dates have been recorded for Eurycea eggs. Spermatozoa are found in the flask tubules usually in dense, orderly whorls similar to the groups found in the vas deferens of the male. They may be, however, scattered and tangled. No spermatozoa have been found in the central tubule in any series I have sectioned and in but one are there any in the necks of the tubules. This is a sagittal series and some sections, 18 254 VERA KOEHRING. in one side of the spermatheca, pass longitudinally through a flask and its neck. The spermatozoa are streaming in a mass from the flask into the neck. But other sections show that they have not proceeded as far as the central duct. Flask tubules containing spermatozoa show a modification of the epithelium. The tall columnar cells are unrecognizable. There is instead a narrow row of flat cells utterly different in appearance from columnar cells. The delicate part of the cells has completely disappeared and no ragged nor shrivelled edges even indicate its former presence. The width of these cells is i/ioo mm. in comparison with measurements of 3/100 and 4/100 mm. during the non-breeding periods. Some of this difference might be accounted for by presuming that the tubules are stretched by the mass of spermatozoa contained within them. However, there does not seem to be any such stretching. Measurements of the diameter of the tubules during the breeding season are normal corresponding to the size of the spermatheca. Also when the flasks are cut squarely through, the whorl of spermatozoa is shown lying in the lumen with no adherence to the walls. There is no evidence of crowding or packing. II. DEVELOPMENT. The above description is of a typical mature spermatheca following at least one season of egg-laying. The organ in some specimens which are larger and presumably older differs in some details and will be discussed at the end of this part on the development. The development of the spermatheca is to be considered with reference to the growth of the cloacal region also. There is a great deal of change in the whole region from the time of meta- morphosis to sexual maturity. The youngest specimen prepared was a 45 mm. female identi- fied by Mrs. Wilder as a stage in early metamorphosis. There is no indication of any spermathecal tubules though the beginning of some cloacal glands may be apparent. In this paper the term "cloacal glands" is applied to the mucous glands of the THE SPERMATHECA OF EURYCEA BISLIXEATA. 255 cloaca. Kingsbury speaks of "ventral glands" but since many of the tubules are in the lateral walls of the cloaca and far dorsal, some beginning near the walls of the spermatheca, the more general term "cloacal glands" may be accurate enough. The cloaca at this stage is very simple; the walls are smooth and the only projection or fold is a small median papilla. Other young animals prepared are 58 mm., 59 mm., 64 mm., 66 mm. and 68 mm. total length. But after metamorphosis there is no definite guide to the age of the animals as length is not a criterion and the size of the gonads is variable in all ages at various seasons, and within this range of lengths mature organs are found. A 67 mm. female killed in April was functional with spermatozoa in the flask tubules. A 68 mm. animal killed in June had a mature spermatheca which undoubtedly had been functional at least during the recent breeding season. Another April specimen, 69 mm. long, contained spermatozoa. Roughly speaking, size up to 68 or 70 mm. indicates sexual immaturity. Animals longer than 70 mm. will generally have the mature organ. Exceptions must be numerous considering the variable length of individuals at metamorphosis (I. Wilder '24). In a 58 mm. animal identified by Mrs. Wilder as in advanced metamorphosis there are four distinct tubules in the dorsal region of the cloaca. They are in pairs two on each side of the median line and one pair posterior to the other. These tubules are made up of a very small group of cells closely grouped and darkly stained with barely perceptible lumina and short ducts. The ducts very nearly reach the thick walls of an invagination of the cloacal wall which is the anlage of the central tubule. Several smaller but otherwise similar groups of cells appear more ventral but are most probably not part of the spermatheca group. A 59 mm. animal in the incipient metamorphic stage also shows two pairs of tubules, one posterior to the other. Although this animal is not as far advanced in metamorphosis as the 58 mm. individual, the spermatheca anlage is in a more advanced condition. The lumina of the tubules are definitely open, the necks curve somewhat dorsally toward the median line and reach the walls of the central tubule. The opening of the central 256 VERA KOEHRING. tubule into the cloaca is minute and the very short duct above is broader in diameter. Columnar epithelium is already present lining this little chamber. There is no sign of the columnar epithelium in the necks or dense cells of the two primary tubules- A 64 mm. animal, an adult probably recently transformed, shows another pair of tubules and otherwise little change in the spermatheca development, though the cloacal glands are greater in number and length and the cloaca is beginning to form folds of the walls. In a 66 mm. animal there is another pair of tubules thus making four tubules on a side. The tubules seem to be all of the same size; the blind ends show an open lumen; the necks are solid cords. The central tubule is no larger in size and extent that it was in the 58 and 59 mm. animals but it is further removed from the cloacal wall more dorsal. There seems to be no opening at all into the cloaca. It is not probable that any sections are lost in this region which might account for not finding an opening. The organ at this time is functionless and is developing slowly ; on the other hand the cloaca is developing steadily, that is, the walls are becoming folded and glands are enlarging. The spermatheca group of tubules is left behind until the approach of sexual maturity. A 68 mm. animal was sectioned horizontally and badly torn by inability to fix the contents of the rectum, but there is dis- cernible the same primitive condition as of the 66 mm. female. The number of tubules has increased; five pairs are certain but six may be present. All are in the same stage. In all these series there is no way of determining the primary pair of tubules nor the latest formed. From the first few rudimentary tubules with their cord-like necks and small central tubule and the whole without any sign of pigment, to the pigment-walled mature spermatheca which occurs in so many 68-72 mm. females and in practically all of them of greater length, seems a great jump and implies rapid growth in a short period most probably just preceding the first egg-laying season. The author regards it as a piece of good-luck to have found, simply by chance sectioning, as there seems to be no other method of determining age at this period, a series THE SPERMATHECA OF EURYCEA BISLINEATA. 257 which is intermediate between the young and the mature sperma- theca. This was a 71 mm. female caught and killed in September. In this spermatheca the average number of tubules found in a mature organ is not complete (though at least one functional organ in this study was observed with no more) and only one pair, one tubule on each side of the median line, is mature in size and cell structure. The necks of these two largest tubules are well developed: the lumina appear at first sight to be open but upon higher magnification they are seen to be so filled with the faintly stained apices of the columnar cells as to be virtually closed tubes. The remainder of the tubules and their necks are in various stages of development. A most marked development of the central tubule is evident. This seems much out of proportion to the same duct in the fully mature organ. The extent is less, that is, the tube is short and does not extend far posterior, but the diameter is wide. Above the short, stout entrance the duct immediately widens into a very broad chamber into which the necks empty. Most of the necks being rather immature their confluence is not so graceful as is typical in older spermathecae. Also the cells of the columnar lining of the central tubule have not the length and delicacy of apices peculiar to this duct in all later stages, and consequently they are not so conspicuous. Lastly pigment is present in quantities but not in the arrange- ment of the mature organ. Sockets around the ends of the two largest tubules are being formed. Around some of the smaller tubules also it has gathered in tangled networks, but around the largest pair it is more concentrated coarse, thick strands. Aside from these foci the pigment is scattered and the pigment producing cells still visible. The definitely bounding walls of the organ are not formed. The spermatheca just described measures slightly less than 1/3 mm. in length and must suffice to complete the develop- mental series. But a word may be said about the largest spermatheca observed during this investigation. The animal was a 95 mm. female killed in September. Unfortunately for the series as a whole it was sectioned horizontally but the sperma- theca is perfect. Eighteen tubules were counted and, although 258 VERA KOEHRING. there is no apparent symmetry in the bilateral arrangement of the tubules, the necks from each side converge separately which causes two reservoirs and these, joining, form the large chamber of the central tubule. This narrows abruptly into the long, narrow dorsal passage which dips into the cloaca. In this largest spermatheca as in some others which are presumed to be several breeding seasons of age, the central tubule extends quite to the posterior wall of the organ and, coincident, are the exaggerated curves of the necks of the flask tubules. All of the tubules lie in the floor of the organ but the most anterior have their blind end walls faced anteriorly. The necks then curve posteriorly and dorsally. The epithelium in this oldest series is particularly conspicuous and the whole thing is an organ of striking beauty, in size approximately 2/3 mm. long. III. RELATION TO CLOACA. The development of the cloaca from the simple smooth-walled stage of larvae to the complications of sexual maturity cannot well come within the scope of this paper, but a brief summary and a short description of the mature conditions should not be out of place in explaining some of the relations of the spermatheca. The most distinctive feature of the cloaca after metamorphosis, aside from the development of the mucous glands, is the formation of a short, dorsal papilla midway between the vent and the opening of the oviducts. This lengthens antero-posteriorly becoming a fold and also deepens until it appears in sections like an icicle pendent from the roof of the cloaca. Until a period shortly preceding sexual maturity the walls of this fold are comparatively smooth. Then they become folded and especially modified at the tip. The tip broadens into a flat surface with two wings which extend close to the lateral walls of the cloaca so as to form two virtually closed dorsal passages through the cloaca up to the opening of the spermatheca where the entire fold ends. The fold undoubtedly functions as an egg guide causing a kind of continuation of the oviducts into the cloaca, but the function of the two small blind pouches in the posterior region of the fold is unknown. THE SPERMATHECA OF EURYCEA BISLINEATA. 259 Although deep folds and spurs in the walls of the cloaca admit of great expansion of the region it does not seem possible that more than one egg could pass at a time, and during the progress of this egg down one side of the cloaca corresponding to the oviduct from which it has proceeded the other passage must be entirely occluded by the pressure of the central fold against the opposite wall. Thus each egg after being squeezed through this crowded region of the cloaca arrives in the larger, freer space at the opening of the spermatheca. The distance from the opening of the oviduct to the mouth of the spermatheca is not over one half a millimeter. The posterior wall of the fold and an extended area of the dorsal wall of the cloaca surrounding the opening of the sperma- theca is lined with the delicate columnar epithelium otherwise singular to the central tubules of the spermatheca. The peculiar function of these cells can be guessed at; they secrete a substance which attracts spermatozoa. During egg-laying the smooth musculature of the spermatheca is probably affected by the convulsions of the entire region and spermatozoa are forced out from the necks of the flasks into the central tubule. Thus disturbed they may swim about and into the cloaca within the secretions of the columnar cells, however, thus preventing any loss. This secretion, as has been stated before, seems not to be mucin or any staining substance but is probably acid. Pfeffer has shown that malic acid is a common attraction for spermatozoa in ferns and Jordan ('91) believes it must be responsible for the attraction of spermatozoa to the spermatheca. But the substances surrounding the egg as it is released into this region of the cloaca must in some manner offer greater attraction and the egg is probably surrounded with a numerous gathering of spermatozoa. It is not known whether one of these will penetrate the egg membrane immediately before it leaves the cloaca, or whether the whole group will promptly be expelled from the vent, one sperm gaining entrance and the rest perishing during attachment. There is no record of newly laid eggs being examined for impotent spermatozoa adhering to them. The theory of the attraction of the columnar cells of the area around the mouth of the spermatheca also accounts for the 260 VERA KOEHRING. spermatozoa being drawn up into the spermatheca at the time of reception instead of attempting the larger passages of the cloaca, oviducts or even rectum. IV. SUMMARY. The spermatheca of Eurycea bislineata is a compactly walled-in set of tubules in the arrangement of a miniature "cat-o'-nine- tails"; the tails are the bulbed or flask-like storage tubules and the long curved handle is the central tubule which is both entrance and exit for spermatozoa. The organ develops slowly in the median dorsal wall of the cloaca during and after meta- morphosis until the autumn before the first egg-laying season, when, more rapidly, the majority of the tubules are formed, pigment appears forming the walls and sockets for the tubules and the columnar lining of the system is completed, proliferating back, probably, from the central tubule. The author expresses profound appreciation for the help and advice of Dr. Harris Hawthorne Wilder and Mrs. Inez Whipple Wilder in the preparation of this paper. MATERIAL AND TECHNIQUE. Aside from dissections of fresh and preserved material, fifteen female Eurycea were sectioned. In size these vary from 45 mm. to 95. mm. total length. Specimens were killed in September, October, November, February, April and June. All were de- calcified and sectioned entire from the ovary to a point caudal to the vent of the cloaca to preclude any distortion of the cloacal region. Bouin's fixative was generally used though some of the young animals were preserved in formalin. Fourteen were stained with hematoxylin-eosin and one with muci-carmine. Most of the series are transverse; two are frontal and two sagittal. The drawings are made with a projection apparatus. THE SPERMATHECA OF EURYCEA BISLINEATA. 26 1 ABBREVIATIONS USED. ant anterior nc nerve cord ap anterior pouch nt notochord bl bladder ov oviduct eg cloacal glands post posterior cl cloaca pu pubis ct central tubule pw posterior wall of spermatheca dor dorsal re rectum / dorsal fold of cloaca sp spermatheca ft flask tubule spz spermatozoa nts mesonephros ur ureter n neck vent ventral BIBLIOGRAPHY. Fischer, G. '91 Beitrage zur Kenntniss des Geotriton fuscus. Hay, O. P. '88 O bservations on Amphiuma and its Young. Am. Nat., Vol. XXII. Jordan, E. O. '91 The Spermatophores of Diemyctylus. Jour. Morph., Vol. V. Kingsbury, B. F. '95 The Spermatheca and Methods of Fertilization of some American Newts and Salamanders. Pro. Am. Micr. Soc., Vol. 17. Leydig, F. '53 Anatomisch-histologische Untersuchungen iiber Fische und Reptilien. Rathke. 1820 Beitrage zur Geschichte der Thierwelt. Siebold, C. T. von. '58 Ueber das Receptaculum seminis der weiblichen Urodelen. Zeitsch. f. wiss. Zool., Vol. IX. Smith, B. G. '06 The Life History and Habits of Cryptobranchus allegheniensis. BIOL. BULL., Vol. XII. Spallanzani, L. 1785 Experiences pour servir a 1'histoire de la generation des animaux et des plantes; avec une ebauche de 1'histoire des etres organises avant leur fecondation. Wilder, I. W. '13 The Life History of Desmognathus fusca. BIOL. BULL., Vol. XXIV. Wilder, I. W. '24 The Relation of Growth to Metamorphosis in Eurycea bislineata (Green). Jour. Exp. Zool., Vol. XL. 262 VERA KOEHRING. EXPLANATION OF FIGURES. PLATE I. FIG. i. (Mag. X c. 85.) Fig. i is a diagrammatic drawing of a young functional spermatheca with part of the pigment sac removed. The diagram is based upon the study of the spermatheca of the 69 mm. female of Fig. 3 and the immature spermatheca of a 71 mm. specimen described in the text. An average of eight tubules would be typical of this stage. The necks bend but slightly posterior and some are anterior in direction. At their convergence the central tubule is extremely large but short, antero-posteriorly, and narrows as it dips into the cloaca. The posterior wall and floor of the spermatheca are densely pigmented and sockets enclose the bulb of each flask tubule. FIG. 2. (Mag. X c. 85.) Fig. 2 is a diagrammatic drawing of an older sperma- theca. The sac wall is removed and half of the tubules are shown, bisected to show their contents the coiled spermatozoa in the flask tubules. These tubules lying in the floor of the sac in pigmented sockets bend posteriorly and dorsally to converge into the central tubule. The reservoir formed by this convergence narrows and the duct passes anteriorly through the dorsal part of the sac where it bends to pass into the cloacal wall. The diagram shows this portion of the central tubule anterior to all the flask tubules but in many cases it passes between the two most anterior flask ends. See Fig. 7. (Mag. X c. 85.) FIG. 3. (Mag. X 19.) Fig. 3 is a para-sagittal section through the region of the rectum and cloaca of a 69 mm. female to show the orientation of the sperma- theca. The plane of sectioning is not quite true so that the anterior region of this drawing is very nearly median-sagittal showing the nerve cord, notochord, rectum and bladder. The posterior half of the drawing shows the end of the mesonephros, one oviduct, the spermatheca and the cloaca almost obscured by the many folds of its walls. It is to be noted in this figure that the mesonephros does not extend as far posterior as the spermatheca. This may be compared with transverse sections shown in other figures. In some individuals the mesonephros lies dorsal to the spermatheca and in others it ends more anteriorly. It is not certain whether these variations are due to changes or varying lengths in the mesonephros itself or if they might be due to the influence of the spermatheca. An "/" placed below a fold separating the oviduct from the lower part of the cloaca is part of the fold represented in Fig. 10, transverse section. The spermatheca in this specimen is young, of the type represented in Fig. i. It is higher, dorso-ventrally, than it is long, antero-posteriorly. There are few tubules and the necks of these do not bend posteriorly. Through a median section the central tubule is large as in Fig. i, but this section is through one side of it. The flask ends are filled with spermatozoa and in this section is the tubule described in the text from which the spermatozoa are streaming into the neck. The lining epithelium of these tubules is flat, unlike the delicate columnar cells pictured in the one flask tubule of Fig. 9, or in Fig. 5. "ap" on this figure indicates an interesting fact about the mature cloaca that is very puzzling in transverse sections through the region an anterior pouch, although this section is only through the wall of it. The anterior pouch is a ventral region of the cloaca separated from the anterior dorsal region by a band of muscle between the bladder and the cloaca. The region is left blank in the figure and indicated by "m." Ventral dissections of the cloaca did not show this particular and in transverse sections it appeared so startling that its function was not guessed. BIOLOGICAL BULLETIN. VOL. XLIX. PLATE I. dor post vent spz VERA KOEHRING. 264 VERA KOEHRING. From a sagittal view, however, it is very simply explained. During egg-laying the rectum and bladder are pushed anteriorly, the folds of the cloaca smooth out and the anterior pouch is drawn down as the vent is stretched open. It is but another rather larger fold of the cloaca. FIG. 4. (Mag. X 175.) Fig. 4 is a transverse section through a 68 mm. female midway through the spermatheca. The mesonephros lies above the spermatheca. In the floor of the spermatheca lie the flask tubules. A few necks are evident. The central tubule is near the dorsal wall. This is an animal killed in June; the tubules are empty and the columnar cells long. Although this animal is short in body length the spermatheca and cloaca show the maturity of several seasons of breeding. PLATE II. FIG. 5 (mag. X 40), FIG. 6 (mag. X 41?). FIG. 7 (mag. X 42). Figs. 5, 6 and 7 are regions of the spermatheca of a 70 mm. female. This organ has not as many tubules as that of the 68 mm. spermatheca of Fig. 4 nor is the cloaca as complex. On the other hand, there are more tubules and more pigment than in the 69 mm. spermatheca of Fig. 3. The significance of these variations after maturity is not known. Fig. 5 is a transverse section through the posterior wall of the spermatheca. Fig. 6 is through the region where the necks are converging into the central tubule. Fig. 7 is through the anterior part of the organ; the central tubule bends to open into the cloacal wall and the most anterior flask tubules lie on either side. FIG. 8. (Mag. X 50.) Fig. 8 is a transverse section through the anlage of the spermatheca in a 59 mm. female. One pair of flask tubules is shown and the central tubule in which the columnar epithelium has developed. The short, cord-like necks do not show in this section. FIG. 9. (Mag. X 86.) Fig. 9 is a frontal section through a 95 mm. female. The necks converge into two groups which join to form the central tubule. One flask tubule is shown with the characteristic ragged-looking but nevertheless very delicate epithelium. The necks are similar to the epithelium of the central tubule. The oviducts and one ureter are in the anterior part of the drawing. FIG. 10. (Mag. X 265.) Fig. 10 is the region through the cloaca of the 68 mm. individual of Fig. 4 several sections anterior to the opening of the sperma- theca. The central fold pendent from the wall of the cloaca divides the dorsal region, and serves as an egg guide. BIOLOGICAL BULLETIN, VOL. XLIX. PLATE II. ,8 1 ant post 10 pw ant VERA KOEHRING. THE INHERITANCE OF A MACULA MUTATION CONCERNED WITH ELYTRAL SPOTTING AND LATENT TRAITS IN THE MALE OF BRUCIIUS. 1 J. K. BREITENBECHER, MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASS. A conspicuous, bilateral, three-character pattern is found on each elytron of the normal wild type female. It consists of two circular black spots placed one anterior and the other posterior, and a thin horizontal line of white pubescence along the inner edge. The elytron of the wild male is unmarked, the four black spots and the two white lines being absent. This condition enables the sexes to be easily distinguished and makes the wild Bruchus quadrimaculatus , Fabr., sex-limited. In this culture, on October 3, 1922, at the University of Oklahoma, there appeared a male insect bearing indistinct black spots and white pubescence, a pattern similar to that of the female described above although less perceptible. This mutation was designated "macula." Approximately a year of inbreeding and selection was required before homozygous macula cultures could be assuredly separated from the wild type in which they originated, because the macula female and the wild female are identical in both homozygous and heterozygous cultures. The difference between the unmarked wild type male and the spotted, macula male was therefore the only visible criterion available for these selection tests. The macula mutation is represented homozygously by the genes, MM, and its recessive allelomorph, spotted females and non-spotted males of the normal insect, homozygously by the factors, mm. In the first test a (MM) female, homozygous for spotted males and females was crossed with a normal (mm) male, homozygous for non-spotted males and spotted females. The offspring from 1 It is a pleasure to acknowledge my indebtedness to Dr. Frank R. Lillie, Director, for the privilege of utilizing the many excellent facilities of the Marine Biological Laboratory. 19 265 266 J. K. BREITENBECHER. 22 separate pairs, totaled 422 heterozygous, spotted (Mm) females and 412 heterozygous, spotted (Mm) males. This proves that the macula trait, spotted males and females, is dominant to the normal character, non-spotted males and spotted females. Complete data for this test is tabulated in Table I. TABLE I. Pi: i MM female, homozygous for spotted males and females X i mm homozygous for non-spotted males and spotted females. Fi: Offspring: male, Pair No. Females Spotted. Males Spotted. Pair No. Females Spotted. Males Spotted. 3 (Mm) I c; (Mm) 16 08 (Mm) I r (Mm) T C 2 20 2Q QO . . 72 42 17 28 14 104 IO 19 77 10 no. . I I 36 e 17 116 6 c 44 38 12 c i -i IO 51 ii 13 9 . . o -1 54 24 27 IO IO o 59 24 38 1 06 . . 10 18 60 2S 16 112 27 7? 94 18 14 120 74. 7O Totals 422 412 TABLE II. RECIPROCAL CROSS. Pi: i mm female, homozygous for non-spotted males and spotted females X I MM male, homozygous for spotted males and females. Fi: Offspring: Pair No. Females Spotted. Males Spotted. Pair No. Females Spotted. Males Spotted. i (Mm) ii (Mm) 8 70 . (Mm) "?! (Mm) T.A IO i 2 80 77 29 34 1 c 3O 81 77, 16 39 II 1 1 88 18 16 47 24 10 90 . 24 27 48 7 6 92 CI c6 49 I c 9 I O9 IO 27 63. 28 46 12"? 22 2? 64. 6 7 6 2 o 68 7 6 9 c 69. . I e 16 7 40 27 78 44 46 8 12 9 Totals. . . . 488 483 INHERITANCE OF A MACULA MUTATION. 26 7 TABLE III. Fi: Mm heterozygous spotted female X Mm heterozygous spotted male. Fz: Progeny: Pair No. Females Spotted. Males. Spotted. Non-spotted. 4 (MM, 2 Mm, mm) 23 37 15 45 30 14 26 7 48 9 34 36 22 32 4 63 16 17 8 17 21 9 33 54 13 49 37 7 75 21 20 26 43 55 27 69 34 3i 79 21 24 7 19 (MM. 2 Mm) 19 30 10 33 19 16 15 6 3i ii 20 27 . 16 25 5 44 13 18 4 12 17 7 29 37 ii 39 22 5 58 17 15 12 39 45 28 59 35 20 68 17 20 8 12 (mm) 9 7 3 15 4 4 6 I 13 3 5 8 3 7 2 15 3 5 i 3 8 2 9 14 4 16 9 2 20 2 5 3 19 16 8 22 10 5 16 4 6 2 4 87 126 127 128 II t.l 3.4 3.7 ip.l IQ.O . 44.7 . S4.i => . 54.31 71. T . 87.3 QQ.^ . 104.3 116.1 116.3 2.6 2.7 . 54.20 59.6 60.6 s 2O 62 63.1 60. 1 69. ^ . 81.5 . 91.2 108.4 109.3 109.4 . 123.2 123.3 123.7 7.2 . 7.3 7.4 6.2 Totals 1,079 994 323 The second experiment was the reciprocal of the previous one. A (mm) female, homozygous for spotted females and non-spotted 268 J. K. BREITENBECHER. TABLE IV. BACK CROSS TEST Pi: Mm heterozygous spotted female X mm homozygous non-spotted male. Fi: Progeny: Pair No. Females Spotted. Males. Spotted. Non-spotted. 4 (Mm, mm) 23 3i 32 10 o 22 21 26 32 IO 30 12 18 3 36 20 45 25 7 37 28 131 3i 27 43 30 34 74 126 24 13 3i 29 23 103 4 96 ii 37 52 ii (Mm) 13 19 16 4 i 7 17 13 9 7 16 7 ii 2 18 22 2O 14 4 18 IO 47 ii 14 22 13 20 40 68 7 4 5 10 17 34 3 44 7 18 24 3 (mm) 10 20 17 3 2 7 16 IO 10 6 10 4 15 3 13 23 12 13 3 18 9 69 19 14 23 19 16 52 50 ii 6 6 12 IO 34 3 42 9 ii 24 4 71 86 ... 06 ios I IQ 12=; 7.1 1-2 . i.c . 3.6 . 17.7 108 Si- 4 . Si.6 . S4.I =54.2 . S4-4 . S4-I4 . 54.16 54.30 60. 1 60.7, . 60.4 . 60.8 60.0 . 8s. 3 . 86.2 87.1 Qd.d 94-S . 98.2 Q8.7. . OQ.I 00.2 QQ.1 99.4 119.1 I2S.S . I27.I 128.4 . Totals 1,375 679 658 males was bred to a (MM) male, homozygous for spotted males and females. The FI offspring from 24 separate pairs gave 488 INHERITANCE OF A MACULA MUTATION. 269 heterozygous, spotted (Mm) females and 483 heterozygous, spotted (Mm) males. This test further shows the dominance of the spotted trait to the normal. Furthermore this experiment, when compared with the previous one, proves that the character is not sex-linked. Table II. gives the data. The third test concerned heterozygous, spotted (Mm) females and heterozygous, spotted (Mm) males. The F 2 progeny from 43 FI single pairs totaled 1,079 spotted (MM, Mm, mM, mm) females, 994 spotted (MM, Mm, mM) males, and 323 non- spotted (mm) males. The result approaches a 4:3:1 sex- limited ratio, which shows that the spotted females appear identical, and that macula is dominant to normal. This data is tabulated in Table III. The fourth experiment is a back cross with the FI hybrids, (Mm), heterozygous insects, and the normal type (mm) weevils. The parents were heterozygous, spotted (Mm) females and homozygous, non-spotted (mm) males. From 41 single pair matings 1,375 spotted (Mm, mm) females, 697 heterozygous, spotted (Mm) males, and 658 homozygous, non-spotted (mm) males, were obtained. The ratio, therefore, approximates a sex-limited 2:1 : I. This result indicates further that normal spotting is recessive to macula. The data is listed in Table IV. The fifth experiment was another back-cross test, the reciprocal of the previous one. Homozygous, normal, spotted (mm) fe- males were mated with heterozygous spotted (Mm) males. From the 43 different pairs a total of 1,415 spotted (Mm, mm) females, 786 heterozygous spotted (Mm) males and 798 homo- zygous non-spotted (mm) males were obtained. This gave a 2 : I : i sex-limited ratio, or actually a I : I ratio, since the females appeared alike. Spotting is dominant to the normal type. Complete information is found in Table Y. In the sixth test heterozygous spotted (Mm) females and homozygous spotted (MM) males were used. The offspring from 26 separate pairs totaled 701 spotted (MM, Mm) females and 654 spotted (MM, Mm) males. This demonstrates the dominance of the macula character to its recessive normal trait, spotted females and non-spotted males. This data is compiled in Table 6. 270 J. K. BREITENBECHER. TABLE V. BACK CROSS TEST: RECIPROCAL CROSS. Pi: mm homozygous wild type female X Mm heterozygous spotted male. Fi: Offspring: Pair No. Females Spotted. Males. Spotted. Non-spotted. 6 (Mm, mm) 33 2 27 33 20 24 4i 34 16 3i 16 ii 25 3i 17 29 48 13 33 62 15 3i 95 34 IS 73 157 5 67 40 6 8 33 64 42 3 30 25 29 27 8 15 (Mm) 30 4 20 18 8 10 24 14 8 19 9 3 16 33 ' 4 ii 19 6 15 33 7 16 37 60 7 22 83 7 28 19 7 10 13 37 19 2 13 15 12 23 4 10 (mm) 27 4 15 19 9 13 19 15 9 26 6 4 19 38 4 17 19 4 18 33 ii 14 34 5i 12 28 78 12 29 16 5 ii 16 3i 23 3 I? ii 10 i? 5 10 6.1 31 01 108 100 IO^? 1.2 20. 7 . 31.6 . . -14.7 . ^4.2 . ^O.I 78.1 78.^5 . 7O I 70.7 . 70.7 . 81.4 . 88.2 . 88 7 01 I 01.3 . QI.6 . 02. 1 02.=; 02 6 108 i 12"?. I 12"?. 4 . 64.1 64.2 . 112. 1 112. 2 Q.IO. . 14-H 54.28. . 82.2 116.=; . 128.1 IO.IO IOI.2 Totals . ... I.4I5 786 798 The seventh experiment was the reciprocal of the previous one. The parents were homozygous, spotted (MM) females and INHERITANCE OF A MACULA MUTATION. 271 TABLE VI. BACK CROSS TEST. Pi: Heterozygous, Mm, spotted female X homozygous, MM, spotted male. Fi: Offspring: Pair No. Female Spotted. Male Spotted. Pair No. Female Spotted. Male Spotted. 20 i (MM, Mm) I (MM, Mm) i 47-S - (MM, Mm) 19 (MM, Mm) 17 48 2 4 =; 49.1 . 21 24 62 4 17 1C 49.2 . 3 4 60 A o 4 47.7 . . 5 9 78 6 70 56 47.8. . 30 34 80 <; -1 7 48.1 . II 6 81 i -!-! 16 68. S. TI 32 88 s 7 i 68.6 28 40 88 6 22 z. 68.7 29 25 O2 7 1^1 149 79. "5 37 32 A1 O 77 2 "5 80.2 21 30 47 T. AT -14 80.4 36 49 A7 A 28 20 81.8 29 18 Totals. . . . 701 654 heterozygous spotted (Mm) males. The offspring from 15 pairs, gave 236 spotted (MM, Mm) females and 213 spotted (MM, Mm) males. Hence, both sexes were homozygous for the macula (MM) genes, and heterozygous for the macula (M) gene and normal (m) factor. The macula mutation is dominant to the wild type. Complete data is presented in Table VII. TABLE VII. BACK CROSS TEST. Reciprocal Cross. Pi: Homozygous, MM, spotted female X heterozygous, Mm, spotted male. Fi: Offspring: Pair No. Female Spotted. Male Spotted. Pair No. Female Spotted. Male Spotted. 17 I (MM, Mm) 28 (MM, Mm) 27 QA 6 (MM, Mm) IO (MM, Mm) 5 17 2 A I ^i 8 6 9 ";i 2 21 IO 54*12 . . 3 ci -i 2 2 "54.21 . 36 33 ci II 18 18 126.4 9 i? <;o 2 26 18 126.8 18 18 CO. 7 27 27 IOI. 29 3i 71.4 3 I Totals 2"?6 213 272 J. K. BREITENBECHER. The above series of experiments prove that the two elytra traits, normal and macula, differ in the male phenotypes only, since the factors for the macula (MM) mutation and the wild (mm) type appear identical for both female traits. These females are alike phenotypically but deviate genotypically, since the normal female has the same black four spotted pattern as its mutant, the macula female. Again, normal is recessive to macula. Many sex-differences are detected in insects, occurring most frequently in Diptera; Drosophila probably furnishing the greatest number, with Lepidotera next, then Coleoptera. This sequence appears, as the result of the amount of genetic study directed upon these species. Many of the sex-limited traits in Drosphila are less distinct in one sex than in the other, thus differing somewhat from Bruchus, in which there is no visible manifestation of such characters in the male, except for the macula mutation. Examples of these sex-limited traits for Bruchus, in which the male elytra is a non-spotted tan, have been previously demon- strated, hence they will be merely mentioned in this relation. The first mutations discovered (Breitenbecher, 1921) consisted of red, black, white, and tan elytra colors apparent only in the females. The respective male for each of these four female cultures was a non-spotted tan, the wild type elytra color. The four traits are multiple allelomorphs. A second non-visible trait in the male occurs with mosaic females (Breitenbecher, 1922). These females have elytra of different colors, often combined with varied spotting. In a third instance the males remained non-spotted tan, although the females displayed four red spots on the elytra (Breitenbecher, 1923). This character was dominant to the normal. Another mutation, in which the male showed a complete absence of the trait, was that in which all females were apterous. The males were fully winged. This was a recessive character (Breitenbecher, 1925). Lastly, another mutation, never visible in the male, was called "piebald." Here bilateral asymmetry was manifested since the females were of two types, about equal in numbers. One group had two red spots on the left elytron and two black spots on the right; the INHERITANCE OF A MACULA MUTATION. 273 other group had two black spots on the left elytron and two red ones on the right. When the two types of females were added, the trait was found to be inherited as a recessive to normal (Breitenbecher, 1925). Every male, for the entire list of char- acters enumerated, had non-spotted tan elytra similar to the wild type. It is improbable that such latent characters in the male are caused by developmental differences, because in sex-limited, as well as other Bruchid mutations, the male emerges a fraction of a day before the female. Inhibitors associated with the male complex may produce this peculiarity. Or, since the female has two X-chromosomes, and the male only one X-chromosome, the sex-limited traits may be caused by pattern, or normal, genes within the X-chromosome. These may be associated with factors in other chromosomes in a degree enabling the character to be completely manifested in the female. The one sex- chromosome of the male may not be sufficient for complete manifestation of the trait. This conception is similar to that of Bridges (1922) for Drosophila. The macula and normal patterns of Bruchus, illustrating sex differences, may be considered as the result of identical pheno- types for the female, since the females for each trait have dupli- cate spotting patterns on the elytra. The mm genes in the wild male are non-visible, while the MM factors for the normal four black spots are visible in the macula male. It is to be hoped that some mutation may occur, in which the gene concerned with the entire pattern or non-pattern trait may appear. This might assure a definite solution for these sex- limited differences in Bruchus. CONCLUSIONS. 1. The macula mutation is dominant to the wild type. 2. Genes, mm, represent the wild type, having spotted females and non-spotted males. 3. Factors, MM, represent the macula mutation, in which both males and females are spotted. 4. The