BIOLOGICAL BULLETIN OF THE flDarine Biological Xaborator? 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. lE&itor FRANK R. LILLIE — The University of Chicago. VOLUME LI. WOODS HOLE. MASS. JULY TO DECEMBER, 1926 Contents of Volume LI No. i. JULY, 1926. Twenty-Eighth Annual Report of the Marine Biological Lab- oratory i No. 2. AUGUST, 1926. CASTLE, EDWARD S. Observations on MotUity in Certain Cyanophycece 69 HARMAN, MARY T., AND ROOT, FRANK P. Number and Be- liazior of the Chromosomes in Cavia cobaya (the Com- mon Guinea Pig) 73 HARVEY, E. NEWTON. On the Inhibition of Animal Lumi- nescence by Light 85 « HARVEY, E. NEWTON. Oxygen and Luminescence, with a Description of Methods for Removing Oxygen from Cells and Fluids 89 ESSENBERG, J. M. Complete Sex-Reversal in the J7ii'iparous Teleost Xiphophorus helleri 98 .UooRE, CARL R. Scrotal Replacement of Experimental Cryp- torchid Testes and the Recovery of Spermatogenetic Function (Guinea Pig} 112 LAWRENCE, WALTER. The Fate of the Germinal Epithelium of Experimental Crypt-orchid Testes of Guinea Pigs .... 129 No. 3. SEPTEMBER, 1926. FROBISHER, MARTIN, JR. Observations on the Relationship between a Red Tornla and a Mold Patlwgenic for Droso- phila niclanogaster 153 YOUNG, DONNELL B. Nuclear Regeneration in Stylonyehia ntytilus 163 MINNICH. D\\ K;IIT KL.MF.R. TJie Chemical Sensitivity of the Tarsi of Certain Mnseid 1'lies 166 ii CONTENTS OF VOUMK I.I. iii GREGORY, LOUISE H. Effects of Changes in Medium a U M 03 g X FIG. 4. Interior of one of the private biological laboratories overlooking Great Harbor and Nonamessett Island. Cement salt water table on left and usual furnishings, but special apparatus (14 x 20 ft.). FIG. 5. Interior of one of the private biochemical laboratories taken from the window end. Note chemical desk and hood in addition to the fittings of a biological laboratory (12 x 23 ft.). MARINE BIOLOGICAL LABORATORY. effects of the intervening brickwork. The main facade (Fig. 2) with its three high arched door-ways in the center, its rounded brick columns, and pediment ornamented with an intricate original marine composition are especially noteworthy. 2. The Dedication Exercises were held July 3, 1925. They were attended by official delegates of the following former and present subscribing and cooperating Institutions and Organi- zations, as well as by members of the Laboratory and neighbors. OFFICIAL DELEGATES. The Academy of Natural Sciences of Philadelphia Agnes Scott College The Department of Agriculture, Washington, D. C. University of Alabama The American Association of Anatomists American Association for the Ad- vancement of Science The American Chemical Society The American Society of Biologi- cal Chemists The American Society ralists The American Society of Zoolo- gists Amherst College Antioch College Barnard College Bermuda Biological Station for Research The Biological Laboratory, Cold Spring Harbor, L. I. Boston Society of Natural History Boston University Botanical Society of America Bowdoin College Boyce Thompson Institute for Plant Research Professor E. G. Conklin Professor C. E. McClung Miss Mary Stuart MacDougall Dr. E. D. Ball President George H. Denny Professor Henry McE. Knower Dr. J. McKeen Cattell Dr. H. E. Howe Dr. Albert P. Matthews of Natu- Professor S. O. Mast Professor G. H. Parker Professor Harold H. Plough Mr. Ondess L. Inman Professor Henry E. Crampum Professor \Y. J. Y. Osterhout 1 >r. Reginald G. Harris Dr. Joseph A. Cushman Rev. Zerna Vane Arthur Professor B. M. Duggar Professor J. R. Schramm Professor Manton Copeland I >i -. William Crocker THK REPORT OF THK DIKKCTOR. Bryn Ma\vr College Bureau of Fisheries, Washington, D. C. Butler College Carleton College Carnegie Corporation Carnegie Institution of Washing- ton Carnegie Institution, Station for Experimental Evolution The University of Chicago University of Cincinnati Clark University The College of the City of New York Columbia University Constantinople Woman's College Cornell University University of Delaware, Women's College DePauw University Doane College Elmira College University of Georgia Goucher College Hamilton College Harvard University Harvard University Medical School Haverford College Hood College Hope College Howard University The Johns Hopkins University and the Johns Hopkins Medical School Dr. Franz Schrader Dr. Willis H. Rich Dr. David Rioch Professor D. B. Young Professor T. H. Morgan Dr. A. F. Blakeslee Dr. E. C. MacDowell Professor Ralph S. Lillie Dr. Edward F. Malone Dr. William H. Cole Assistant Professor Earl A. Martin Professor Gary N. Calkins Professor Henry E. Crampton Professor Thomas H. Morgan Professor Edmund B. Wilson Dr. Mary Mills Patrick Professor Charles R. Stockhard Miss Margaret Walton Professor Walter Norton Hess Mrs. M. C. Bennett Miss Clara Dettmer Miss Elizabeth Humeston Dr. E. R. Clark Professor Clara L. Bacon Professor A. D. Morrill Professor W. J. V. Osterhout Professor George H. Parker Dr. A. C. Redfield Mr. William A. Wolff Dr. Mabel Bishop Professor Samuel O. Mast President J. Stanley Durkee Dr. E. E. Just Dr. Warren H. Lewis MARINE BIOLOGICAL LABORATORY. Eli Lilly and Company McGill University Marietta College University of Maryland Massachusetts Institute of Tech- nology Mercer University Miami University, Oxford, Ohio University of Michigan James Millikan University Milwaukee-Downer College University of Minnesota The Missouri Botanical Garden University of Missouri The Mount Desert Island Bio- logical Laboratory Mount Holyoke College National Research Council The National Academy of Sciences The New York Academy of Sciences The University of North Carolina Oberlin College Ohio Wesley an University Peabody Museum, Salem, Mass. University of Pennsylvania University of Pennsylvania School of Medicine Pomona College Randolph-Macon College Rhode Island State College Rutgers College Sheffield Scientific School, Vale University Shorter College Smithsonian Institution Stanford University Dr. G. II. A. Clowes Dr. I. Marlaren Thompson Professor II. R. Egglrston Professor P. \V. Zimmerman Professor Robert T. Bigelow Professor Gail L. Carver Dr. J. K. Breitenbecker Professor L. Y. Heilbrunn Dr. H. P. Agersborg Dr. Mary Edith Pinney Dean Klius P. Lyon Dr. Benjamin M. Duggar Dr. Charles H. Philpott Dr. Ulric Dahlgren Dr. Cornelia Clapp Dr. Maynard M. Metcalf Dr. Frank R. Lillie Professor Henry E. Crampton Dr. C. Dale Beers Professor Robert A. Budington Professor Charles G. Rogers Professor E. G. Conklin Professor Edward S. Morse Professor Clarence Erwin McClung Professor Henry Cuthbert Bazett Dr. Charles W. Metz President R. E. Blackwdl Dr. H. W. Browning Dr. F. Bauer Dr. Walter T. Marvin Professor Ross G. Harrison Dr. Ada K. Hall Dr. Austin II. Clark Mr. Charles Maurice Cram THE REPORT OF THE DIRECTOR. St. Louis University Syracuse University The Tulane University of Louisi- ana Union College Vassar College University of Vermont University of Virginia University of Washington Washington University Washington and Lee University Wesleyan University The Western College for Women Western Reserve University and Adelbert College Whitman College Williams College The University of Wisconsin The Wistar Institute of Anatomy and Biology Yale Universitv Professor Alphonse M. Schwitalla Professor C. W. Ilargitt Dr. Walter E. Garrry Professor James Watt Mavor Professor A. L. Treadwell Dr. E. G. Spaulding Dr. Ivey Foreman Lewis Mr. Louis G. Seagrave Mr. Alfred M. Lucas Professor W. D. Hoyt Professor Hubert B. Goodrich Dr. Ruth Laura Phillips Professor Francis H. Herrick Rt. Rev. Robert L. Paddock Professor James L. Kellogg Professor Charles E. Allen Professor Leon J. Cole Dr. Milton J. Greenman Professor Lorande L. Woodruff Members of the Laboratory and many invited guests were also present and the large new auditorium was completely filled. The afternoon addresses according to the subjoined program have been published in Science (Vol. LXIL, No. 1604, pages 271-280, Sept. 25, 1925). The Committee in Charge of the Exercises, under the able direction of Dr. H. E. Crampton, provided lunch and housing arrangements for delegates, and guides for inspection of the building, and arranged an afternoon tea and demonstrations of materials and experiments set up by workers at the Laboratory. The exercises were concluded by an evening lecture delivered by Professor W. J. V. Osterhout of Harvard University. 26 MARINE BIOLOGICAL LABORATORY. MARINE BIOLOGICAL LABORATORY. WOODS HOLE, MASSACHUSETTS. DEDICATION EXERCISES. FRIDAY, JULY 3. 2P.M. Daylight saving time. In the auditorium. Presiding Officer, THE HONORABLE CHARLES R. CRANE, President Board of Trustees. Address of Welcome: PROFESSOR FRANK R. LILLIE, Director. Address: PROFESSOR EDMUND B. WILSON, Columbia University. Address: 'The Changing Face of Nature and of Man at Woods Hole," by PROFESSOR EDWIN G. CONKLIN, Princeton Uni- versity. 4:00-6:00 P.M. Inspection of buildings and work. Afternoon tea will be served. 8:00 P.M. The first of the series of evening lectures will be delivered by PROFESSOR W. J. Y. OSTERHOUT, of Harvard University- Subject: "Absorption and Accumulation." 3. Report of the Building Committee (The Director, Assistant Director and Treasurer). — In the last Annual Report the esti- mates for construction of the new building and the sources of funds for the purposes were given. It is a source of gratification to the Committee that the building wras completed and equipped for a sum of $734,101.01 which is $14,485.70 within the estimates of $748,886.71. Ground was broken March, 20 1924 and the building practically fully equipped was ready for occupancy in June, 1925. In its final report presented to the Board of Trustees August ii, 1925 The Building Committee presented a summary of payments needed after July i, 1925 as follows: SUMMARY OF ADDITIONAL NEW BUILDING COSTS AFTER JULY i, 1925. July i, 1925 — Balance due Contractors and Architects. . . . $19,023.39 Balance due John Manville Co. for panel work in auditorium 1,500.00 Balance due on Supplementary Orders 22,321.70 $42.845.09 THE REPORT OF THE DIRECTOR. 2J Additional Amounts Needed. a. Apparatus and Chemicals $ 5,000.00 b. To complete Machine Shop 600.00 c. Additional items for completion of service and equipment 5,000.00 10,600.00 553,445-09 July I, Cash in Building Fund 30,094.48 $23,350.61 Anticipated savings on contracts: Cleghorn Company $817.00 Cronin Company 925.00 1,742.00 August 3, Amount required from Friendship Fund $21,608.61 In this accounting a fund of $10,600.00 was provided for minor items of completion. With this understanding the Com- mittee proposed that their accounts be closed after the payment of the outstanding items noted above and that the Committee be discharged as soon as this is accomplished. The report was accepted by the Board of Trustees, and the Secretary was instructed to notify the Friendship Fund that the Marine Biological Laboratory would consider their pledge fulfilled by a total payment of $221,608.61 instead of $248,886.71 as originally estimated. The subjoined analysis of the Building Fund Account was submitted by the Business Manager, Dec. 15, 1925. ANALYSIS, MARINE BIOLOGICAL LABORATORY, BUILDING FUND ACCOUNT, DECEMBER 15, 1925. Cash Received to date: Rockefeller Foundation $500,000.00 Friendship Fund 221,608.61 Interest on bank deposits: December, 1924 $ 8,721.67 June, 1925 459-28 9,180.95 Donation, Frank R. Lillie 3,311.45 -$734,101.01 Payments as follows: Geo. A. Fuller Co., Gen. Contractor. . . $484,210.56 C. H. Cronin, Inc., Plumbing 48,229.06 Cleghorn Company, Heating 30,658.95 Hixon Electric Co., Elec. work 66,555.36 F. S. Payne Co., Elevator 3-977-OO $633,630.93 28 MARINE BIOLOGICAL I. A I n >K \TOKV. Coolidge & Shattuck, Architects' ci>iumi-f Si 6, 750.00 annually will be available for the next five years for accessions to the library and probably a slight addition to cost of ad- ministration. These accessions added to present possessions should complete the foundations of a comprehensive research library in Biology. 8. Real Estate and Housing Conditions. — These problems have received considerable attention during the last year. The future of the Laboratory could not be regarded as secure until sufficient land for future extensions was secured. The supply of lots in the Gansett property is approaching exhaustion ; and the demand for real estate on the Cape is sending the price of land up very rapidly. Under these circumstances the Executive Committee decided to take advantage of an offer of the Fay Estate to sell a tract of land of about 105 acres, known as Devil's Lane tract, situated between the main Falmouth Road and the railroad, on very advantageous terms. This beautifully wooded tract is about one and a half miles from the Laboratory and it has the privileges to a right of way and a bathing reservation on the shore of Vineyard Sound. Part of the tract amounting to about 15 acres is good agricultural land, but most of it is hilly and well adapted to residence purposes. It is proposed to subdivide part for building purposes when the Gansett tract is sold out, re- stricting the sites to those connected with the Laboratory as in the case of the Gansett tract, and reserving the remainder for whatever purposes the Laboratory may serve in the future. During the year the Laboratory also purchased Dr. Drew's house and grounds, and repurchased Dr. Danchakoff's two lots and houses in the Gansett tract. These purchases are in line with the policy of controlling sufficient real estate to be independent of artificial prices and rise in values. They do not, however, solve the housing problem. The Laboratory now maintains seven rooming houses and dormi- tories with accomodations for 172 persons at prices ranging from $2.00 to $4.00 per week. This is by no means sufficient in the height of the season; moreover some of the houses are old, and all lack good sanitary conveniences and rooms for social pur- poses. The largest of the houses will probably soon have to be THE REPORT OF THE DIRECTOR. 33 abandoned. Village rentals either for separate rooms or for furnished houses tend to be unreasonably high. A very serious problem for the future of the Laboratory is that of the young married investigators especially if they have children. The Laboratory has no real provisions for family housing, and village rents are beyond the means of many of the most valued members of our organization. As a consequence in many cases they feel obliged to remain away from the Laboratory, which needs them, and which they need. This situation is being studied and plans are being prepared to provide for an apartment house and a modern dormitory building in the immediate neighborhood of the Laboratory and a few small bungalows to be placed on the Gansett tract. At present funds are not available for such a considerable under- taking, but it is doubtful if they could be secured without a well considered plan. We shall accordingly proceed hopefully until the problem is solved . 9. Changes in Personnel. — Coincident with the establishment of the Endowment Fund and the completion of the New Building the past year has witnessed very important changes in personnel. i. Resignation of Mr. C. R. Crane as President of the Corpo- ration. A. COPY OF MR. CRANE'S LETTER. TRUSTEES MINUTES, 1925, pp. 5-6. August 8, 1925. To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASS. Gentlemen: Twenty- two years have now elapsed since I became President of the Marine Biological Laboratory. I have enjoyed with you watching the growth of the Laboratory during that period. With the strong interest and support that is now assured, I feel that my own work has been completed and I hereby tender you my resignation which I ask you to accept. The Laboratory has one unique feature in that its Board of Trustees consists almost entirely of biologists and it seems to me desirable that your President should also belong to the Fraternity. I have talked informally with some of the members of the Committee on Future Administrative Organization over the plan which they will propose at your meeting and I heartily approve the plan. 3 34 MARINE BIOLOGICAL LABORATORY. The future progress and prosperity of the Laboratory will always be a matter of great interest to me quite as much as if I continued to be your President. Sincerely yours, (signed) CHARLES R. CRANE. B. LETTER OF ACCEPTANCE. August 21, 1925. HONORABLE CHARLES R. CRANE, Woods Hole, Massachusetts. Pi tir Mr. Crane: In regretfully accepting your resignation as Presi- dent of the Corporation of the Marine Biological Laboratory the Board of Trustees appointed the undersigned members of the Board a Committee to express to you their grateful appreciation of the invaluable services which you have rendered to the Laboratory. You have been a member of our Board since 1901 and President of the Corporation since 1905 and throughout this period you have been our constant supporter and friend. During your Presidency you have seen the Laboratory grow, from a relatively small beginning, to the largest and most complete biological laboratory in the world, with assets totaling more than two million dollars. Every year since your connection with our Board you have con- tributed most generously to the support, of the Laboratory and for nearly twenty years you have lifted from our shoulders the burden of a large deficit. Only those who were members of the Board before your advent can fully appreciate the relief from financial worries and fears of suspension which your support has brought to us. Almost every year of your Presidency you have made notable additions to our estate, among which are the Kideer lot, cottage and annex, the Whitman and Ritter cottages, and New Homestead and Mess Hall, the Bar Neck Property, the Gansett Property, our first permanent and fireproof laboratory which should be known as the Crane Building, and finally the completion of the fund for the building and equipment of the New Laboratory and the permanent endowment of your annual gift of twenty thousand dollars from the Friendship Fund. We are well aware that your interest in and support of the Labora- tory has been a powerful factor in enlisting the cooperation of other benefactors and of great Foundations. It we have to-day one of the finest biological institutions in the world we owe it in large part to your faith and foresight. Best of all has been the spirit of the support of yourself and Mrs. THE REPORT OF THE DIRECTOR. 35 Crane. Both of you have given generously and without any sugges- tion of conditions or interference. 'The gift without the giver is bare," but you have given yourselves. Your spirit of helpful, friendly cooperation is the very spirit of this Laboratory which you have helped to create and confirm. \Ye regret that you are to be our President no longer, but we rejoice to know that you will still be a member of our Board of Trustees and an ever cherished colleague and friend. Sincerely and cordially yours, (signed) E. G. CONKLIN, T. H. MORGAN, EDMUND B. WILSON, Committee. To this record there is nothing to add. The Director was elected President of the Corporation by the Board of Trustees in Mr. Crane's stead. 2. Resignation of Dr. Oilman A. Drew as Assistant Director. A. COPY OF DR. DREW'S LETTER OF DECEMBER 10, 1924. "To THE BOARD OF TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY. Gentlemen: Unfortunately I have found it necessary to quit Woods Hole before the new building is completed. This is a fitting time for me to sever my connection with the Labora- tory and I herewith tender my resignation as Assistant Director, as Vice-President, and as a member of all Committees on which I am serving. My membership on the Board is ex-officio and is automatically taken care of. Please do not elect me to fill a possible vacancy. I will not be able to attend meetings, and without direct connection my advice would rapidly fall in value. I wish to thank you all for your never-failing support and to assure you that the continued welfare of the Laboratory will ever give me pleasure. Sincerely yours, (signed) OILMAN A. DREW." In reply to this letter the Executive Committee granted Dr. Drew leave of absence until the next meeting of the Board, August n, 1925, and at this meeting Dr. Drew's resignation was 36 MARINE BIOLOGICAL LABORATORY. accepted as of December 31, 1925. In accepting the resignation of Dr. Drew, the Board of Trustees elected him Trustee emeritus and passed the following resolution which was ordered suitably engrossed on velum to be transmitted to Dr. Drew: The Board of Trustees of the Marine Biological Laboratory hereby expresses to Gilman Arthur Drew their great regret that he has found it necessary to resign as Assistant Director and their high appreciation of the invaluable services which he has rendered to the Laboratory as Instructor in Zoology and in Embryology from 1901 to 1916 and as Resident Assistant Director from 1910 to 1925. The evidences of his Scientific, Technical and Business ability are manifest everywhere in our Buildings, Equipment and Organization and his Zeal, Resourcefulness and Cooperation have helped to form the Spirit of this Institution. We extend to Dr. and Mrs. Drew our grateful thanks for all that they have done for the Marine Biological Laboratory and our best wishes for their future Health and Happiness. Dr. Drew's resignation had been contemplated by him for some years on account of continued ill-health, but his devotion to the Laboratory was such that he risked, and finally incurred, serious illness by remaining at his post until the more essential features of the construction of the new laboratory were com- pleted. The resolution expresses all too briefly the extent to which the Laboratory as an organization and a fully equipped plant is indebted to the long and devoted services of Dr. Drew. May his life on his citrus ranch in Florida to which he has retired bring him and Mrs. Drew good health and happiness! 3. Professor Merkel H. Jacobs of the University of Pennsyl- vania was elected by the Trustees on August 1 1, 1925 to succeed Dr. Drew and was given the title of Associate Director. For the present Dr. Jacobs will reside in Woods Hole five months in the year beginning May i. His training as Zoologist and General Physiologist fit him pre-eminently well to serve the Laboratory in his new capacity. 4. Dr. \Y. J. Y. Osterhout who has acted as head of the botanical research staff since 1919 also resigned for the reason that his research program will keep him away from Woods Hole for several years. For the great stimulus of his work and TIIK REPORT OF THE DIRECTOR. 37 example the Laboratory is both grateful and proud. Dr. Benjamin Duggar the noted plant physiologist of the Missouri Botanical garden has been elected as worthy successor to Dr. Osterhout. 5. Dr. Robert H. Bowen of Columbia University resigned charge of instruction of the course in Invertebrate Zoology after four years of conspicuously successful service in charge, and seven years in all, in order to devote his time more fully to research. Dr. J. A. Dawson of Harvard University an ex- perienced member of the staff of the course for several years was chosen to take charge. 10. Changes in the By-laws. — At the meeting of the Board of Trustees, August 12, 1924, a committee composed of the Treasurer, the Secretary and Dr. C. R. Stockard was appointed to consider and revise the By-laws and to report within six months to the Executive Committee. This report was ac- cordingly prepared and presented to the Executive Committee which considered and approved it, and recommended its adoption by the Board of Trustees at their meeting on August n, 1925. At this time it was considered and finally adopted. The following Report was presented by the Committee: Your committee has examined the Certificate of Incorporation, Articles of Association, the original By-laws of the Corporation, and the present By-laws. It finds that the By-laws are in a very simple form and that the absence of elaborate rules has been a benefit to the Corporation, and has made its business easy to administer. It does not recommend, therefore, any general change in the form of the By- laws. There are, however, certain minor changes which should perhaps be made, and to this end the members of the committee are agreed in recommending the following alterations: 1. By-law I. Change last sentence to read as follows: The President of the Corporation, the Director and Associate Director of the Laboratory shall also be Trustees ex-officio. 2. By-law V. Line 3. Change to read: They shall elect one of their number President of the Corporation who shall also be Chairman of the Board of Trustees; they shall appoint a Director of the Laboratory; and they may choose, etc. 38 MARINE BIOLOGICAL LABORATORY. 3. By-law VI I. provides for the appointment annually of two trustees to audit the account of the Treasurer and act as a Finance Committee. The auditing of the Treasurer's accounts has for a number of years been done by certified accountants, and the establishment of the trust funds in the hands of a Trust Company has made it probable that there will be no great amount of investing done by the Treasurer. It would therefore seem that By-law VI I should be amended to read as follows: The accounts of the Treasurer shall be audited annually by a certified public accountant. 4. By-law YIII. Amend the second sentence to read: In case of dissolution the property shall be disposed of in such manner and upon such terms as shall be determined by the affirmative vote of two-thirds of the Board of Trustees. These changes are incorporated in the By-laws printed with this report (p. 4). They do not affect in any essential way the original intent of the By-laws but they define more exactly existing practises and provide more broadly for the improbable contingency of dissolution. 1 1. The Board of Trustees. Enlargement of the Executive Com- mittee.— At their meeting held August u, 1925, the Executive Committee of the Laboratory was enlarged to include three members ex off. (the Director, Associate Director and the Treasurer) and four members at large to be elected by the Board from among their own number. At the meeting of the Board of Trustees held August 1 1, 1925, Professor Henry E. Crampton of Barnard College, Columbia University was elected to fill a vacancy in the Class of 1927 of the Board. Thus there returns to the service of the Laboratory a noted teacher and investigator whose earlier work on its behalf dates from over thirty years ago. The following resolution in memory of Lucius L. Xunn former member of the Board of Trustees and benefactor of the Labora- tory was adopted by the Board and by the Corporation at their meetings held August u, 1925: THE REPORT OF THE DIRECTOR. 39 RESOLUTION ON THE DEATH OF MR. L. L. NUNN. Dr. Lillie offered the following resolution (read at the Corpo- ration meeting) which was ordered spretid on the minutes. Mr. L. L. Nunn, Trustee of the Marine Biological Laboratory from 1897 to 1923 and Life Member of the Corporation died at his home in California, April 2, 1925. A mining engineer by profession, Mr. Nunn attained great success in the development of mines, power lines and banks in the State of Colorado. He was a man of deep human sympathies which found their outlet in his unmarried state in a school for boys, administered on very liberal lines, which he estab- lished at Deep Springs in California. In connection with the school scholarships were provided, and assigned on the election of the boys to the Telluride Association, which furnished a complete college and professional education with generous stipend for maintenance. Mr. Nunn also maintained a chapter house at Cornell University named Telluride House after his principal scene of operations in Colorado, for the use of members of the Telluride Association. Many young men owe their prospects in life to these benevolences of Mr. Nunn. He was a brother-in-law of Professor Whitman, first Director of this Laboratory, and showed constant sympathy with Professor Whitman's ideals and aims. He evinced this interest in a very striking way at the time of reorganization of the administration of the Laboratory in 1897, at which time he was elected to the Board of Trustees. He was present on several critical occasions in the history of the Labora- tory and always showed readiness to live up to the responsibilities of his Trusteeship. His largest gift to the Laboratory was a sum of about $5,600.00 in 1902 for the purchase of land important in con- nection with negotiations with the Carnegie Institution going on at that time. Although prevented by distance and illness from attending the meetings of the Trustees, Mr. Nunn maintained his interest in its progress. On December 31, 1918 he wrote the Director: My dear Dr. Lillie: I am just in receipt of your communication under date of December i6th, stating that you will not call a mid- winter meeting of the trustees of the laboratory. I think the decision a most wise one and will add one suggestion, viz: that the members of the Board send the laboratory the amount it would cost them to attend the meeting. Acung in accordance with the suggestion, I enclose my check for $200. 4O MARINE BIOLOGICAL LABORATORY. Earnestly hoping that I will be able to spend a few weeks with you at Woods Hole the coming summer, I remain Sincerely, L. L. XUNX. Again in August 1923 he telegraphed his regret at his inability to attend the meeting of the Board, and said that his interest in scientific research and devotion to the Laboratory were not lessened, and offered to donate Professor Whitman's collection of pamphlets and documents which were afterwards received. The Trustees of the Marine Biological Laboratory record their sorrow at the death of their old associate, and direct that this brief record be spread upon the minutes. 12. There are included as parts of this report the following ad- denda: 1. The Staff, 1925. 2. Investigators and Students, 1925. 3. Tabular view of attendance, 1921-25. 4. Subscribing and Cooperating Institutions, 1925. 5. Evening Lectures, 1925. 6. Members of the Corporation. i. THE STAFF, 1925. FRANK R. LILLIE, Director, Professor of Embryology, and Chairman of the Department of Zoology, The University of Chicago. (.ILMA.N A. DREW, Assistant Director, Marine Biological Laboratory. ZOOLOGY. I. INVESTIGATION. GARY X. 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. I. II. MORGAN, Professor of Experimental Zoology, Columbia Uni- versity. G. H. PARKER, Professor of Zoology, Harvard University. F. H. WILSON, Professor of Zoology, Columbia University. THE REPORT OF THE DIRECTOR. 4! II. INSTRUCTION. ROBERT H. BOWEN, Assistant Professor of Zoology, Columbia Uni- versity. HORACE B. BAKER, Instructor in Zoology, University of Pennsylvania. C. DALE BEERS, Graduate Student, Johns Hopkins University. RUDOLF BENNITT, Instructor in Biology, Tufts College. J. A. DAWSON, Instructor in Zoology, Harvard University. MADELEINE P. GRANT, Assistant Professor, Mount Holyoke College. CHRISTIANNA SMITH, Assistant Professor of Zoology, Mount Holyoke College. (Absent in 1925.) B. H. WILLIER, Assistant Professor of 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. (Absent in 1925.) FLORENCE DE L. LOWTHER, Instructor in Zoology, Barnard College. MARY S. MACDOUGALL, Head of the Biological Department, Agnes Scott 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, Associate in the Institute of Cancer Research, Columbia University. HAROLD H. PLOUGH, Associate Professor of Biology, Amherst College. CHARLES G. ROGERS, Professor of Comparative Physiology, Oberlin College. PHYSIOLOGY. I. INVESTIGATION. HAROLD C. BRADLEY, Professor of Physiological Chemistry, Uni- versity of Wisconsin. 42 MARINE BIOLOGICAL LABORATORY. WALTER E. GARREY, 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. II. INSTRUCTION. MERKEL II. JACOBS, Professor of General Physiology, University of Pennsylvania. WILLIAM R. AMBERSON, Assistant Professor of Physiology, Uni- versity of Pennsylvania. FRANK P. KNOWLTON, Professor of Physiology, Syracuse 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. IVEY F. LEWIS, Professor of Biology, University of Virginia. WINTHROP J. Y. OSTERHOUT, Professor of Botany, Harvard Uni- versity. (Absent in 1925.) JACOB R. SCHRAMM, Professor of Botany, College of Agriculture, Cornell University. II. INSTRUCTION. IVEY F. LEWIS, Professor of Biology, University of Virginia. ANSEI.M M. KEEFE, Fellow in Botany, University of Wisconsin. WILLIAM RANDOLPH TAYLOR, Assistant Professor of Botany, Uni- versity of Pennsylvania. LIBRARY. PRISCILLA B. MONTGOMERY (Mrs. Thomas 11. -Montgomery, Jr.), Librarian. — , Assistant Librarian. CIII.MK'AL SUPPLIES. OLIVER S. STRONG, Associate Professor of Neurology, Columbia Uni- versity, New York City, Chemist. THE REPORT OF THE DIRECTOR. 43 APPARATUS ROOM. SAMUEL E. POND, Assistant Professor of Physiology, Medical School, University of Pennsylvania, Philadelphia, Pa. Custodian of Appa- ratus. SUPPLY DEPARTMENT GEORGE M. GRAY, Curator. A. \Y. LEATHERS, Head of Ship- Assistant Curator: - — . ping Department . JOHN J. VEEDER, Captain. A. M. HILTON, Collector. E. M. LEWIS, Engineer. J. MC!NNIS, Collector. A. R. TRAVIS, Collector. F. M. MAcNAUGHT, Business Manager. HERBERT A. HILTON, Superintendent of Buildings and Grounds. THOMAS LARKIN, Superintendent of Machinery and Plumbing. LESTER F. Boss, Mechanician. WILLIAM HEMENWAY, Carpenter. ARNOLD H. Bisco, Storekeeper and head janitor. 2. INVESTIGATORS AND STUDENTS, 1925. ZOOLOGY — Independent Investigators. ADDISON, WILLIAM H. F., Professor of Histology and Embryology, University of Pennsylvania. BAKER, HORACE B., Instructor, University of Pennsylvania. BANT, ANTONI J., Lemberg University, Poland. BENNITT, RUDOLF, Instructor in Biology, Tufts College. BIGELOW, ROBERT P., Professor of Zoology, Massachusetts Institute of Technology. BISHOP, MABEL, Professor of Zoology, Hood College. BOSCHMA, HILBRAND, Chief Assistant, University of Leyden, Holland. BOWEN, ROBERT H., Assistant Professor of Zoology, Columbia University. BREITENBECHER, JOSEPH K., Trenton, Ohio. BRIDGES, CALVIN B., Research Assistant, Carnegie Institution of Washington. BUDINGTON, ROBERT A., Professor of Zoology, Oberlin College. CALKINS, GARY N., Professor of Protozoology, Columbia University. CAROTHERS, E. ELEANOR, University of Pennsylvania. CARVER, GAIL L,., Professor of Biology, Mercer University. CHAMBERS, ROBERT, Professor of Microscopic Anatomy, Cornell University Medical College. CLARK, ELIOT R., Professor of Anatomy, University of Georgia. CLARK, ELEANOR L., Research worker, University of Georgia. CLEVELAND, L. R., Fellow, National Research Council. CONKLIN, EDWIN G., Professor of Biology, Princeton University. COPELAND, MANTON, Professor of Biology, Bowdoin College. COWDRY, EDMUND V., Associate Member, Rockefeller Institute. DANCHAKOFF, VERA, Assistant Professor, College of Physicians and Surgeons. DAWSON, JAMES A., Instructor in Zoology, Harvard University. 44 MARINE BIOLOGICAL LABORATORY. DELLINGER, SAMUEL C., Professor of Zoology. University of Arkansas. DETLEFSEN, J. A., Associate Editor, Biological Abstracts. DOLLEY, WILLIAM L., JR., Assistant Professor of Biology, University of Buffalo. FORBES, WILLIAM T. M., Instructor in Entomology, Cornell University. FRY, HENRY J., Assistant Professor, Department of Biology, Washington Square College. GABRITSCHEVSKY, EUGEX, Research Fellow, International Education Board. GLASER, OTTO C., Professor of Biology, Amherst College. GOODRICH, HUBERT B., Professor of Biology, Wesleyan University. GRAVE, BENJAMIN H., Professor of Zoology, Wabash College. GRAVE, CASWELL, Professor of Zoology, Washington University. HANCE, ROBERT T., Associate Member, Rockefeller Institute. HANSTROM, BERTIL, Assistant Professor, University of Lund, Sweden. HESS, WALTER N., Professor of Zoology, DePauw University. HOADLEY, LEIGH, Fellow, National Research Council. JUST, ERNEST E., Professor of Zoology, Howard University. KAAN, HELEN W., 37 Prescott St., Reading, Mass. KNOWER, HENRY McE., Visiting Professor of Anatomy, University of Georgia. LILLIE, FRANK R., Chairman, Department of Zoology, University of Chicago. LOWTHER, FLORENCE DE L., Instructor in Zoology, Barnard College. 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 O., Professor of Zoology, Johns Hopkins University. MAYOR, JAMES W., Professor of Biology, Union College. MILLER, HARRY M., JR., Assistant Professor of Zoology, Washington University. DE MOL, W. E., University of Utrecht. Holland. MORGAN, THOMAS H., Professor of Experimental Zoology, Columbia University. MORGAN, LILIAN V., 409 W. nyth St., New York City. MORRILL, CHARLES Y., Associate Professor of Anatomy, Cornell University Medical College. NONIDEZ, JOSE F., Instructor in Anatomy, Cornell University Medical College. PACKARD, CHARLES, Associate in Zoology, Institute of Cancer Research, Columbia University. PAPANICOLAOU, GEORGE N., Assistant Professor in Anatomy, Cornell University Medical College. PARKER, GEORGE H., Professor of Zoology, Harvard University. PARMENTER, C. L., Assistant Professor, University of Pennsylvania. PHILLIPS, EVERETT F., Professor of Agriculture, Cornell University. PINNEY, MARY E., Assistant Professor of Zoology, Milwaukee-Downer College. PLOUGH, HAROLD H., Professor of Biology, Amherst College. RAND, HERBERT W., Associate Professor of Zoology, Harvard University. REDFIELD, HELEN, National Research Fellow in Zoology. SANDERS, ELIZABETH P., 2906 St. Paul St., Baltimore, Md. SAYLES, LEONARD P., Arnold Fellow, Brown University. SCHRADER, FRANZ, Associate Professor, Bryn Mawr College. SCHRADER, SALLY H., Instructor, Bryn Mawr College. SPEIDEL, CARL C., Associate Professor of Anatomy, University of Virginia. STARK, MARY B., Professor of Histology and Embryology, New York Homoeopathic Medical College. THE REPORT OF THE DIRECTOR. 45 STERN, CURT, Research Fellow, International Education Board. STOCKARD, CHARLES R., Professor of Anatomy, Cornell University Medical College. STRINGER, CAROLINE E., Head of the Department of Natural Science, Central High School, Omaha, Nebraska. STRONG, OLIVER S., Associate Professor of Neurology, Columbia University. STURTEVANT, ALFRED H., Member of Staff, Carnegie Institution of Washington. THOMPSON, I. MACLAREN, Assistant Professor of Anatomy, McGill University. UHLENHUTH, EDWARD, University of Maryland, School of Medicine. ULREY, ALBERT B., Professor of Biology, University of Southern California. WHEDON, ARTHUR D., Professor of Zoology, North Dakota Agricultural College. WIEMAN, HARRY L., Professor of Zoology, University of Cincinnati. WILDMAN, EDWARD E., Head of Science Department, West Philadelphia High School. WILLIER, BENJAMIN H., Assistant Professor of Zoology, University of Chicago. WILSON, EDMUND B., Da Costa Professor of Zoology, Columbia University. WILSON, J. WALTER, Assistant Professor of Biology, Brown University. WOODRUFF, LORANDE L., Professor of Protozoology, Yale University. YOUNG, DONNELL B., Assistant Professor of Biology, Carleton College. ZELENY, CHARLES, Professor of Zoology, University of Illinois. ZOOLOGY — Beginning Investigators. ANDERSON, CHAS. L., Student, University of Georgia Medical School. AUSTIN, MARY L., Columbia University. BAKER, WOODFORD B., Associate Professor of Biology, Emory University. BAKER, HERBERT N., Tonganoxie, Kansas. BEERS, CHARLES D., Johns Hopkins University. BERGNER, A. DOROTHY, Investigator, Columbia University. BLAKE, CHARLES H., Assistant, Massachusetts Institute of Technology. BROWN, ALAN W., Cornell University Medical College. BROWN, MURIEL W., Student, Johns Hopkins University. COOK, DANIEL, University of Cincinnati. DE FOREST, DAVID McC., Assistant, Union College. FOGG, LLOYD C., Graduate Student, Columbia University. FOSTER, KENDALL W., Instructor in Biology, Tufts Pre-Medical School. FRASER, DORIS A., Assistant, University of Pennsylvania. GRAND, CONSTANTINE G., Anatomy Assistant, Cornell University Medical College. GRANT, MADELEINE P., Assistant Professor, Mount Holyoke College. GRAVE, THOMAS B., Johns Hopkins University. GUERASIMOFF, HELEN, Columbia University. HARNLY, MORRIS H., Columbia University. HELWIG, EDWIN R., Instructor in Zoology, University of Pennsylvania. HICKMAN, CLEVELAND P., Assistant Professor of Zoology, DePauw University. HULPIEU, HAROLD R., Assistant, Johns Hopkins University. KARNS, HILDA E., Cornell University. KROPP, BENJAMIN, Graduate Student, Harvard University. Li, Ju CHI, Columbia University. LUCAS, ALFRED M., Assistant Instructor, Washington University. McCLUNG, DELLA E., University of Kansas. McCLUNG, ANNA A., 417 Harvard Avenue, Swarthmore, Pa. 46 MARINE BIOLOGICAL LABORATORY. MACDOUGALL, MARY S., Head of the Biological Department, Agnes Scott College. MATTHEWS, SAMUEL A., Student, Harvard College. NORTHRUP, FLORA E., Washington University. PARPART, ARTHUR K., Assistant in Biology, Amherst College. PIIILPOTT, CHARLES H., Professor of Zoology, Harris Teachers College, St. Louis, Missouri. PLUNKETT, CHARLES R., Columbia University. SANDISON, JAMES C., Assistant in Anatomy, Medical Department, University of Georgia. SCHULTZ, JACK, Assistant in Zoology, Columbia University. STURTEVANT, PHOEBE R., Assistant, Carnegie Institution. THOMPSON, RUTH C. McC., Brick Church, Orange, New Jersey. TILDEN, EVELYN B., Secretary and Laboratory Assistant, Rockefeller Institute. VARIAN, BASIL B., Laboratory Assistant, University of Pennsylvania. VICARI, EMILIA M., Sweet Briar College. WALLACE, EDITH M., Carnegie Institution. WIERDA, J. L., Instructor, Duke University. WOLFF, WILLIAM A., Greensboro, North Carolina. WRIGHT, SYDNEY L., JR., Chemist, Pennsylvania Hospital. PHYSIOLOGY— Independent Investigators. AMBERSON, WILLIAM R., Assistant Professor, University of Pennsylvania. BAZETT, HENRY C., Professor of Physiology, University of Pennsylvania Medical School. BERGLUND, HILDING, Assistant Professor of Medicine, Department of Biochemistry, Harvard University Medical School. BODANSKY, OSCAR, 124 East ii7th St., New York City. BRADLEY, HAROLD C., Professor of Physiological Chemistry, LTniversity of Wis- consin. BRONFENBRENNER, J. J., Associate Member, Rockefeller Institute. BRUNQUIST, ERNEST H., Instructor in Physiology, University of Colorado Medical School. CANNAN, ROBERT K., Assistant in Biochemistry, University College, London. CLOWES, G. H. A., Director of Research, Eli Lilly & Company. COHEN, BARNETT, Chemist, Hygienic Laboratory, Washington, D. C. COHN, EDWIN J., Assistant Professor of Physical Chemistry, Harvard University Medical School. CRACIUM, EMIL C., Assistant, Second Medical Clinic, University of Bucharest, Rumania. DEMBOWSKI, JAN, University of Warsaw. DEMBOWSKI, MRS. JAN, Warsaw, Poland. GARREY, WALTER E., Professor of Physiology, Vanderbilt University Medical School. HARVEY, EDMUND N., Professor of Physiology, Princeton University. HARVEY, ETHEL B.. 2 College Road, Princeton, New Jersey. HEILBRUNN, L. V., Assistant Professor, University of Michigan. HINRICHS, MARIE A., National Research Council Fellow, LTniversity of Chicago. INMAN, ONDESS L., Professor of Biology, Antioch College. D'IRSAY, STEPHEN, Seessel Fellow in Physiology, Yale University. THE REPORT OF THE DIRECTOR. 47 IRWIN, MARIAN, Research Associate, Rockefeller Institute. JACOBS, MERKEL H., Professor of General Physiology, University of Pennsylvania KNOWLTON, FRANK P., Professor of Physiology, College of Medicine, Syracuse University. LILLIE, RALPH S., Professor of General Physiology, University of Chicago. LOEB, LEO, Professor of Pathology, Washington University Medical School. LUCKE, BALDWIN, Assistant Professor of Pathology, University of Pennsylvania. LYON, ELIAS P., Professor of Physiology, University of Minnesota. McCuTCHEON, MORTON, Instructor in Pathology, University of Pennsylvania Medical School. MATHEWS, A. P., Professor of Biochemistry, University of Cincinnati Medical School. MORGULIS, SERGIUS, Professor of Biochemistry, University of Nebraska College of Medicine. POND, SAMUEL E., Assistant Professor of Physiology, University of Pennsylvania School of Medicine. RAEDER, OSCAR J., Instructor in Psychiatry, Harvard University. REDFIELD, ALFRED C., Assistant Professor of Physiology, Harvard University Medical School. REZNIKOFF, PAVL, Assistant in Anatomy, Cornell University Medical College. ROGERS, CHARLES G., Professor of Comparative Physiology, Oberlin College. SAMPSON, MYRA M., Associate Professor, Smith College. SIMPSON, GEORGE E., Assistant Professor, University of Pennsylvania. SNYDER, CHARLES D., Professor of Experimental Physiology, Johns Hopkins Uni- versity. WARREN, HOWARD C., Stuart Professor of Psychology, Princeton University. PHYSIOLOGY — Beginning Investigators. ALEXANDER, EDWARD G., Fellow in Biology, Princeton University. BASKERVILL, MARGARET, Instructor, Bellevue Medical College New York City. BLISS, CHESTER, I., Graduate Student, Columbia University. CATTELL, WARE, Garrison-on-Hudson, New York. COOLIDGE, THOMAS, Student, Harvard University Medical School. CROFTS, ELIZABETH, Assistant Professor, Mount Holyoke College. DARBY, HUGH H., Instructor, Columbia University. GENTHER, IDA T., Student, Mount Holyoke College. GLUSKER, DAVID, Investigator, University of Pennsylvania. HARTLINE, H. K., Johns Hopkins LTniversity Medical School. HAYWOOD, CHARLOTTE, Graduate Student, University of Pennsylvania. HENDRY, JESSIE L., Investigator, Harvard University Medical School. KURD, ARCHER L., Harvard University Medical School. KREDEL, FREDERICK E., Graduate Assistant in Zoology, LTniversity of Pittsburgh. KUPP, JOHN H., Johns Hopkins University Medical School. LANDIS, EUGENE M., Student, University of Pennsylvania. MULDER, ARTHUR G., Teaching Fellow, University of Minnesota. PAGE, IRVING H., Research worker, Eli Lilly & Company. RICE, KENNETH S., Graduate Student, Brown LTniversity. SALTER, WILLIAM T., Investigator, Harvard LTniversity Medical School. STEDMAN, HAZELTENE L., Assistant Professor, Mount Holyoke College. 48 MARINE BIOLOGICAL LABORATORY. WALDEX, GEORGE B., Research Chemist, Eli Lilly & Company. WALDEN, MRS. GEORGE B., Chemist, Eli Lilly & Company. BOTANY— Independent Investigators. BRIEGER, FRIEDIRCH G., Rockefeller Research Fellow, Bussey Institution. BROOKS, SUMNER C., Biologist, Hygienic Laboratory, Washington, D. C. BROOKS, MATILDA M., Associate Biologist, Hygienic Laboratory, Washington, D. C. CLELAND, RALPH E., Associate Professor of Biology, Goucher College. DUGGAR, BENJAMIN M., Missouri Botanical Garden, St. Louis, Missouri. LEWIS, IVEY F., Professor of Biology, L'niversity of Virginia. PRAT, SILVESTER, Assistant, Plant Physiological Laboratory, Charles University. SCARTH, GEORGE \Y., Assistant Professor, McGill University. SCHRAMM, J. R., Editor-in-Chief, Biological Abstracts. STOKEY, ALMA G., Professor of Botany, Mount Holyoke College. TAYLOR, WILLIAM RANDOLPH, Assistant Professor, University of Pennsylvania. BOTANY— Beginning Investigators. CARGILL, MIRIAM A., Valley Falls, Rhode Island. KEEFE, ANSELM M., Fellow in Botany, University of Wisconsin. MILLER, MRS. HARRY, Washington L'niversity. SVENSON, HENRY K., Assistant Professor of Biology, Union College. STUDENTS. 1925. Botany. FLIEDNER, FRIEDA, Teacher, Central High School, Cleveland. GORDESKY, TOBA, Teacher, Elementary School, Philadelphia, Pa. GRANT, JEAN F., Teacher, St. Mary's School, Raleigh, N. C. GRISWOLD, SYLVIA M., Teacher, Maryland State Normal School. HALL, ADA R., Professor of Biology, Shorter College. HASTINGS, HELEN, George Washington University. HORNE, EDITH L., Vassar College. LIEBERMANN, FANNIE, Science teacher, South Philadelphia High School for Girls. M< ( \i i ki:v, CHARLES F., JR., Student, Harvard College. MOORE, IMOGENS, Graduate Student, Vale University. ROWLEE, SILENCE, Instructor, Elmira College. TETZLOFF, LINA E., Instructor, Hunter College. TURNER, RICHARD G., Knoxville, Tennessee. Embryology. ARMSTRONG, PHILIP B., Assistant in Anatomy, Cornell University Medical College. BUNTEN, ISABEL, Student, George Washington University. CAMPBELL, MALCOLM D., Junior Master, Dorchester High School. CLARKE, HENRY G., Student, Amherst College. COBB, WILLIAM M., Amherst College. DETTMER, CLARA R., Elmira College. DOOLEY, EUNICE J., Student, Illinois Wesleyan University. THE REPORT OF THE DIRECTOR. 49 FABING, HOWARD D., Student, University of Cincinnati. FRIKDMAN, HILDA, Gouchcr College. GELBACK, ELIZABETH L., Student, Goucher College. GREENBERG, FRANCES, New York University. HALSEY, HARVEY R., Graduate student and laboratory assistant, Columbia Uni- versity. HINTZE, ANNA LAURA, Assistant, University of Wisconsin. HOLTON, RUTH G., Laboratory assistant, Wilson College. JOHNSON, HELEN R., Assistant in Biology, Brown University. KOSTIK, JOHN, Student, University of Wisconsin. LABARRE, ELIZABETH R., Student, Goucher College. LEICH, CHARLES F., Wabash College. LONDON, ISABEL M., Cornell University Medical College. MARKS, EDITH E., Student, Vassar College. MORRISON, HAROLD, Associate Entomologist, U. S. Bureau of Entomology. PALLETTE, EDWARD C., Leland Stanford University. PATTERSON, EDNA, Assistant Professor of Biology, Wesleyan College. PEARSON, NATHAN E., Assistant in Zoology, Indiana University. PYLE, MARY M., Graduate Assistant, University of Pittsburgh. THOMAS, MORRIS C., Wabash College. WARTERS, MARY, Ohio State University. WHEELER, PHILIP H., Wesleyan University. WILLIAMS, ELIZABETH T., Smith College. Physiology. BRANHAM, SARA E., Instructor in Bacteriology, The University of Chicago. CAMPOS, F. A. DEM., Assistant in Physiology, St. Pauls Medical School, Brazil. CHEN, TSE-YIN, Instructor in Biology, Peking University. CURRY, LALIAH F., Zoology Assistant, Washington University. DEGRAFF, ARTHUR C., Crete Research Fellow in Physiology, Western Reserve University. DILLER, WILLIAM F., JR., Instructor in Zoology, University of Pennsylvania. EMERSON, ETHEL, Student, College of Physicians and Surgeons. GRAHAM, ELEANOR P., Baltimore, Maryland. HERRICK, RUTH, University of Chicago. MILLER, RALPH E., Medical Student, Dartmouth College. NEAL, MARY E., Teacher of Science, Boston Schools. RAFFEL, DANIEL, Graduate Student, Johns Hopkins University. SANDS, JANE, Professor of Physiology, Woman's Medical College of Pennsylvania. SCOTT, WALTER J., University of Pennsylvania. STEWART, DOROTHY R., Hanover, New Hampshire. SUMWALT, MARGARET, Washington University. WEIMER, BERNAL R., Professor of Biology, Bethany College. WILLIAMS, HARLEY A., Oberlin College. YOUNG, WILLIAM C., Assistant in Department of Biology, Amhcrst College. ATTENDING MORNING LECTURES ONLY. BART, MAX S., 1685 University Ave., New York City. 4 50 MARINE BIOLOGICAL LABORATORY. Protozoology. BANKS. REBA, Kirbyville. Texas. BURT, FREDERICK A., Associate Professor, A. and M. College of Texas. COLEMAN, PETER F., 3314 Cortclyou Road, Brooklyn, New York. COOPER, ZOLA K., Graduate Student, Washington University. GORHAM, GRACE V., Assistant in Zoology, Columbia University. KAMENOFF, RALPH J., Columbia University. KEMPTON, RUDOLF T., Assistant, Washington Square College, New York Uni- versity. LICHTMAN, FRIEDA, Columbia University. MAN\VELL, REGINALD D., Senior Assistant in Biology, Amherst College. PAN, QUENTIN, Shanghai, China. PIERCE, MADELENE E., Radcliffe College. RICE, LUCINDA H., Assistant Instructor, Wellesley College. ROBERT, NAN L., Professor of Biology, Flora Macdonald College. SILBERBLATT, BERYL, Student, Columbia University. SPALUING, JANET E., Laboratory Assistant, College of the City of New York. VAN DUYN, EVELYN, Science Teacher, New York City. ZEMAN, MIRA, Assistant in Biology, New Y'ork University. ATTENDING MORNING LECTURES ONLY. KEITH, WILMA, 1229 Carr Avenue, Memphis, Tennessee. Zoology. ANDREWS, CECIL L., Butler College. BAUER, ETHEL C., H. Sophie Newcomb Memorial College. BOOTH, Lois, DePauw University. BOSWORTH, EDWARD B., Wesleyan University. BUEHLER, KATHERINE, Teacher of Biology, Albany High School. CARPENTER, ESTHER, Ohio Wesleyan University. CASTLE, WILLIAM A., University of Chicago. CHAMBERLAIN, HARRY, University of Rochester. CONKLIN, CECILE L., Teacher of Biology, Sweet Briar College. CORRINGTON, JULIAN D., Professor of Zoology, UTniversity of South Carolina. CROSBY, MARION R., Student, University of Wisconsin. CURTIS, MARY E., Student, Wilson College. DAHLBERG, RUTH M., Knox College. DEMPSTER, WILFRID T., Student, University of Michigan. FALES, DORIS E., Assistant in Biology, Western Reserve University. FISHER, CAROL A., Graduate Student, Yale University. FLEMING, JOAN, Student, Wellesley College. FRANZ, GERHARD E., Instructor of Zoology and Chemistry, Watertown College. FRIEND, ROGER B., Graduate Student, Y'ale University. GETCHELL, DONNIE C., Laboratory Assistant, Colby College. GORDON, KENNETH L., Graduate Assistant, University of Missouri. GREGERSEN, MAGNUS L, Teaching Fellow, Harvard Medical School. HARDY, JERRY D., Professor of Biology, High Point College. HENDEE, ESTHER C., Oberlin College. THE REPORT OF THE DIRECTOR. 51 HITCHCOCK, HAROLD B., Student, Williams College. HITCHCOCK, PAULINE D., Assistant Instructor in Biology, Simmons College. HUMESTON, ELIZABETH, Elmira College. HUNT, ARTHUR, Carleton College. HUTCHINSON, GEORGE A., Student, University of Rochester. ISELY, MARION F., Graduate Assistant, University of Missouri. JOHNSON, WILLIS H., Wabash College. KINNEY, ELIZABETH T., Assistant in Zoology and Graduate Student, Washington University. KIRK, MARGARET B., Radcliffe College. KNAPPEN, PHOEBE M., George Washington University. LADD, CAROLYN T., University of Vermont. LEACH, HARRIET P., Smith College. LEVIN, PAUL M., Johns Hopkins University. LONG, MARGARET E., Student, University of Pennsylvania. MILLER, HELEN M., Student, Goucher College. MITANI, FUMIKO, Student, Mt. Holyoke College. MUSTIN, MARGARET E., Assistant in Biology, Shorter College. QUINN, IRENE C., Carleton College. RYERSON, ROCKWELL T., Northwestern University. SARLES, MERRITT P., Graduate Assistant in Biology, Wesleyan University. SCHUETT, JESSE F., St. Louis, Mo. SHEVKET, FAZILE, Laboratory Assistant, Constantinople College. TAYLOR, PHILLIS, Hunter College. TEWINKEL, HELEN McG., Student, Oberlin College. TYLER, A. RANGER, Student, Rutgers College. VAN METER, IRENE, Student, Illinois Wesleyan University. WEATHERFORD, HAROLD L., Austin Teaching Fellow in Histology, Harvard Uni- versity. WEATHERBY, JESSE H., Graduate Student, Johns Hopkins University. WEICHERT, CHARLES K., Assistant in Zoology, University of Wisconsin. WELSH, JOHN H., JR., Harvard University. 3. TABULAR VIEW OF ATTENDANCE. 1921 1922 1923 1924 1925 iNVESTIGATORS-^Total IJ2 l82 176 194 2QJ Independent: Zoology 79 87 90 77 84 Physiology 26 28 23 33 40 Botany 13 15 13 *4 " Under Instruction: Zoology 34 34 41 47 45 Physiology 1 1 1 1 5 16 . 23 Botany 9 7 4 7 -1 STUDENTS — Total 120 126 146 134 132 MARINE BIOLOGICAL LABORATORY. Zoology 53 Protozoology 1 1 Embryology 28 Physiology 18 Botany 10 TOTAL ATTENDANCE 292 INSTITUTIONS REPRESENTED — Total. 95 By investigators 67 By students 53 SCHOOLS AND ACADEMIES REPRESENTED By investigators By students 59 12 28 19 8 308 104 68 59 50 54 16 17 17 3i 29 29 22 18 19 18 20 13 322 328 340 107 no 112 62 69 74 73 68 65 4- SUBSCRIBING AND COOPERATING INSTITUTIONS, 1925. Amherst College Antioch College Biological Abstracts Bowdoin College Brown University Bryn Mawr College Buffalo Society of Natural Sciences Butler College Carnegie Institution of Washing- ton Chinese Educational Mission College of Physicians & Surgeons Columbia University Cornell University Cornell University Medical Col- lege Dartmouth College DePauw University Elmira College General Education Board Goucher College I larvarcl Universitv Harvard University Medical School High Point College Hunter College Indiana University International Education Board Johns Hopkins University Johns Hopkins University Medical School Knox College Eli Lilly & Co. Massachusetts Institute of Tech- nology McGill University Mount Holyoke College New York University Oberlin College Ohio Wesleyan University Princeton University Radcliffe College Rockefeller Foundation Rockefeller Institute for Medical Research THE REPORT OF THE DIRECTOR. 53 Rutgers College University of Rochester Simmons College University of Vermont Smith College University of Virginia Sophie Newcomb College University of Wisconsin Trinity College Vassar College Tufts College Wabash College Union College Washington University University of Chicago Washington University Medical University of Cincinnati School University of Illinois Watertown College University of Michigan Wellesley College University of Minnesota Wesleyan University University of Missouri Western Reserve University University of Nebraska Wilson College University of Pennsylvania Wistar Institute of Anatomy and University of Pennsylvania Biology Medical School Yale University 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, 1925. Friday, July 3, PROFESSOR W. J. V. OSTERHOUT. . . ."Absorption and Accumula- tion." Tuesday, July 7, PROFESSOR G. H. PARKER "The Carbon Dioxide Ex- creted by Nerves." Friday, July 10, PROFESSOR E. B. WILSON " Protoplasmic Systems and Genetic Continuity" —Il- lustrated. Tuesday, July 14, PROFESSOR G. N. CALKINS "Organization and Variation in Protozoa.' 54 MARINE BIOLOGICAL LABORATORY. Friday, July 17, DR. F. M. CHAPMAN "The Origin and Distribu- tion of the Birds of Ecua- dor" -Illustrated. Tuesday, July 21, PROFESSOR C. R. STOCKARD "Analysis and General Bear- ings of the Ovulation Problem." Friday, July 24, DR. WILLIAM CROCKER. "Boyce Thompson Institute for Plant Research and Its Work" -Illustrated. Tuesday, July 28, PROFESSOR H. E. CRAMPTON "Among the Islands of the South Seas"- —Illustrated. Friday, July 31, DR. SEWALL WRIGHT "Genetic Studies with Three- and Four-toed Races of Guinea Pigs." Tuesday, August 4, PROFESSOR F.. NEWTON HARVEY. .. ."Animal Luminescence." Friday, August 7, PROFESSOR Ross G. HARRISON "Heteroplastic Grafting." 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. CLARKE, PROF. S. F., Williamstown, Mass. CONKLIN, PROF. EDWIN G., Princeton University, Princeton, N.J. CRANE, MR. C. R., New York City. 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. THE REPORT OF THE DIRECTOR. . 55 GARDINER, MRS. E. G., Woods Hole, Mass. GARDINER, Miss EUGENIA, 15 \V. 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. MORGAN, MR. J. PIERPONT, JR., \Yall 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. OSBORN, PROF. HENRY F., American Museum of Natural History, New York City, N. Y. 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. 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. 56 MARINE BIOLOGICAL LABORATORY. 2. REGULAR MEMBERS, AUGUST, 1925. ADAMS, Miss A. E., Mount Holyoke College, South Hadley, Mass. ADDISON, DR. \V. H. F., University of Pennsylvania Medical School, Philadelphia, Pa. . ADOLPH, DR. EDWARD F., University of Rochester, School of Medicine and Dentistry, Rochester, N. Y. AGERSBORG, DR. H. P. K., James Millikin University, Decatur, Illinois. ALLEE, DR. W. C., University of Chicago, Chicago, Illinois. ALLEX, PROF. CHAS. E., University of Wisconsin, Madison, Wisconsin. ALLEX, PROF. EZRA, Ursinus College, Collegeville, Pa. AI.LYX, DR. HARRIET M., Vassar College, Poughkeepsie, N. Y. AMBERSOX, DR. WILLIAM B., University of Pennsylvania, Phila- delphia, Pa. AXDERSOX, DR. E. G., University of Michigan, Ann Arbor, Michigan. ATTERBURY, MRS. RUTH R., Great Neck, Long Island, New York. BAITSELL, DR. GEORGE A., Yale University, New Haven, Conn. BAKER, DR. E. H., 5312 Hyde Park Boulevard, Hyde Park Station, Chicago, 111. I! \I.D\VIX, DR. F. M., Iowa State College, Ames, Iowa. BASCOM, DR. K. F., Medical School of Virginia, Richmond, Virginia. BECKWITH, DR. CORA J., Vassar College, Poughkeepsie, N. Y. BEHRE, DR. ELIXOR H., Louisiana State University, Baton Rouge, La. BEXXITT, DR. RUDOLF, Tufts College, Tufts College, Mass. BIGELOW, PROF. M. A., Teachers College, Columbia University, New York ( "ity. liH.i LOW, PROF. R. P., Massachusetts Institute of Technology, < .unbridge, Mass. HIXFORD, PROF. RAYMOND, Guilford College, Guilford College, N. C. BORIXG, DR. ALICE M., Yenching College, Peking, China. |'<>\VEN, DR. ROBERT H., Columbia University, New York City. THE REPORT OF THE DIRECTOR. 57 Box, Miss CORA M., University of Cincinnati, Cincinnati, Ohio. BRADLEY, PROF. HAROLD C, University of Wisconsin, Madison, Wis. BRAILEY, Miss MIRIAM E., 232 Maple St., Lynn, Mass. BRIDGES, DR. CALVIN B., Columbia University, New York City. BROOKS, DR. S. C., U. S. Public Health Service, Washington, D. C. BUCKINGHAM, Miss EDITH N., 342 Marlboro St., Boston, Mass. BUDINGTON, PROF. R. A., Oberlin College, Oberlin, Ohio. 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. CALVERT, PROF. PHILIP P., University of Pennsylvania, Phila- delphia, Pa. CARLSON, PROF. A. J., University of Chicago, Chicago, Illinois. CAROTHERS, DR. ELEANOR E., University of Pennsylvania, Philadelphia, Pa. CARROLL, PROF. MITCHEL, Franklin and Marshall College, Lancaster, Pa. CARVER, PROF. GAIL L., 613 Orange St., Macon, Georgia. CASTEEL, DR. D. B., University of Texas, Austin, Texas. CATTELL, PROF. J. McKEEN, Garrison-on-Hudson, New York. CATTELL, DR. McKEEN, Cornell University Medical College, New York City. CATTELL, MR. WARE, Garrison-on-Hudson, New York. CHAMBERS, DR. ROBERT, Cornell University Medical College, New York City. CHARLTON, DR. HARRY H., University of Missouri, Columbia, Missouri. CHIDESTER, PROF. F. E., West Virginia University, Morgantown, W. Va. CHILD, PROF. C. M., University of Chicago, Chicago, Illinois. CLAPP, PROF. CORNELIA M., Montague, Mass. CLARK, PROF. E. R., University of Georgia, Augusta, Georgia. CLELAND, PROF. RALPH E., Goucher College, Baltimore, Mary- land. CLOWES, DR. G. H. A., Eli Lilly & Co., Indianapolis, Indiana. COE, PROF. W. R., Yale University, New Haven, Conn. 58 MARINE BIOLOGICAL LABORATORY. COHN, DR. EDWIN J., 43 Kirkland St., Cambridge, Mass. COKER, DR. R. E., University of North Carolina, Chapel Hill, North Carolina. COLE, DR. LEON J., College of Agriculture, Madison, Wisconsin. COLLETT, DR. MARY E., Western Reserve University, Cleveland, Ohio. COLLEY, MRS. MARY W., 1712 Madison St., Madison, Wisconsin. COLTOX, PROF. H. S., Ardmore, Pennsylvania. COOLIDGE, MR. C. A., Ames Building, Boston, Mass. COPELAND, PROF. MANTON, Bowdoin College, Brunswick, Maine. COWDRY, DR. E. V., Rockefeller Institute, New York City. CRAMPTOX, 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 Uni- versity, 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, DR. ALICE R., 19 Ash St., Cambridge, Mass. DAWSON, DR. J. A., Harvard University, Cambridge, Mass. DEDERER, DR. PAULINE H., Connecticut College, New London, Connecticut. DETLEFSEN, DR. J. A., Swarthmore, Pennsylvania. 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., University of Buffalo, Buffalo, N. Y. DONALDSON, PROF. H. H., Wistar Institute of Anatomy and Biology, Philadelphia, Pa. DON AI .I>M>.\, DR. JOHN C., University of Pittsburgh, School of Medicine, Pittsburgh, Pa. DREW, PROF. OILMAN A., Eagle Lake, Florida. DUGGAR, DR. BENJAMIN. M., Missouri Botanical Garden, St. Louis, Missouri. THE REPORT OF THE DIRECTOR. 59 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, California. FINLEY, MR. CHARLES W., Lincoln School, 425 West i23d St., New York City. FORBES, DR. ALEXANDER, Harvard University Medical School, Boston, Mass. FRY, MR. HEXRY J., Washington Square College, New York City. GAGE, PROF. S. H., Cornell University, Ithaca, N. Y. GARREY, PROF. W. E., Vanderbilt University Medical School, Nashville, Tennessee. .GATES, PROF. R. RUGGLES, King's College, 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 Re- search, Princeton, N. J. GOLDFORB, PROF. A. J., College of the City of New York, New York City. GOODRICH, PROF. H. B., Wesleyan University, Middletown, Connecticut. GOWANLOCH, DR. J. N., Dalhousie University, Halifax, Nova Scotia. GRAVE, PROF. CASWELL, Washington University, St. Louis, Mo. GRAVE, PROF. B. H., Wabash College, Crawfordsville, Ind. GREENMAN, DR. M. J., Wistar Institute of Anatomy and Biol- ogy, Philadelphia, Pennsylvania. GREGORY, DR. LOUISE H., Barnard College, Columbia Uni- versity, New York City. 6O MARINE BIOLOGICAL LABORATORY. GUTHRIE, DR. MARY J., University of Missouri, Columbia, Mo. GUYER, PROF. M. F., University of Wisconsin, Madison, Wis. HAXCE, DR. ROBERT T., Rockefeller Institute, New York City. HARGITT, PROF. C. W., Syracuse University, Syracuse, New York. HARGITT, PROF. GEORGE T., Syracuse University, Syracuse, New York. HARMAN, DR. MARY T., Kansas State Agricultural College, Manhattan, Kansas. HARPER, PROF. R. A., Columbia University, New York City. HARRISON, PROF. Ross G., Yale University, New Haven, Connecticut. 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, California. HECHT, DR. SELIG, Harvard University Medical School, Boston, Mass. HEGNER, PROF. R. \V., Johns Hopkins University, Baltimore, Md. HEILBRUNN, DR. L. Y., University of Michigan, Ann Arbor, Michigan. HESS, PROF. WALTER N., DePauw University, Greencastle, Indiana. HINRICHS, DR. MARIE A., University of Chicago, Chicago, Illinois. HOADLEY, DR. LEIGH, Brown University, Providence, R. I. HOGUE, DR. MARY J., 503 N. High St., West Chester, Pa. HOLMES, PROF. S. J., University of California, Berkeley, Cali- fornia. HOOKER, PROF. DAVENPORT, University of Pittsburgh, Pitts- burgh, Pa. Hoi-Ki\>, DR. HOYT S., New York University College of Den- tistry, New York City. HOSKINS, MRS. ELMER R., University of Arkansas Medical School, Little Rock, Arkansas. THE REPORT OF THE DIRECTOR. 6l HOWE, DR. H. E., 2702 36th St., N. \V., Washington, D. C. HOYT, DR. WILLIAM D., Washington and Lee University, Lexington, Virginia. HUMPHREY, MR. R. R., University of Buffalo School of Medicine, Buffalo, N. Y. HYMAN, DR. LIBBIE H., University of Chicago, Chicago, Illinois. INMAN, PROF. ONDESS L., Antioch College, Yellow Springs, Ohio. 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. JOHNSON, PROF. GEORGE E., State Agricultural College, Man- hattan, Kansas. JONES, PROF. LYNDS, Oberlin College, Oberlin, Ohio. JORDAN, PROF. H. E., University of Virginia, Charlottesville, Va. JUST, PROF. E. E., Howard University, Washington, D. C. KEEFE, REV. ANSELM M., 424 North Murray St., Madison, Wisconsin. KENNEDY, DR. HARRIS, Readville, Mass. KINDRED, DR. J. E., University of Virginia, Charlottesville, Va. KING, DR. HELEN D., W^istar Institute of Anatomy and Biology, Philadelphia, Pennsylvania. KING, DR. ROBERT L., University of Pennsylvania, Philadelphia, Pennsylvania. KINGSBURY, PROF. B. F., Cornell University, Ithaca, New York. KINGSLEY, PROF. J. S., 2500 Cedar St., Berkeley, California. KIRKHAM, DR. W. B., Springfield College, Springfield, Mass. KNAPKE, REV. BEDE, St. Bernard's College, St. Bernard, Alabama. KNOWER, PROF. H. McE., University of Georgia, Medical De- partment, Augusta, Georgia. KNOWLTON, PROF. F. P., Syracuse University, Syracuse, New York. KOSTIR, DR. \V. J., Ohio State University, Columbus, Ohio. 62 MARINE BIOLOGICAL LABORATORY. KRIBS, DR. HERBERT, Ewing Christian College, Allahabed, North India. Kt'YK, DR. MARGARET P., Westbrook Ave., Richmond, Va. I.AXCEFIELD, DR. D. E., Columbia University, New York City. L AXGE, DR. MATHILDE M., \Vheaton College, Norton, Mass. LEE, PROF. F. S., College of Physicians and Surgeons, 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, Illinois. LILLIE, PROF. R. S., University of Chicago, Chicago, Illinois. LINTON, PROF. EDWIX, 1104 Milledge Road, Augusta, Georgia. 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. LUCRE, PROF. BALDWIN, University of Pennsylvania, Phila- delphia, Pa. LUND, DR. E. J., University of Minnesota, Minneapolis, Minn. LUSCOMBE, MR. W. O., Woods Hole, Mass. LVMAN, PROF. GEORGE R., College of Agriculture, University of West Virginia, Morgantown, \V. Va. LYNCH, Miss CLARA J., Rockefeller Institute, New York City. LYON, PROF. E. P., University of Minnesota, Minneapolis, Minn. MAC-CALLUM, DR. G. A., 925 St. Paul St., Baltimore, Md. MACDOUGALL, Miss MARY S., Agnes Scott College, Decatur, Georgia. 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. M< I \ DOO, DR. N. E., Bureau of Entomology, Washington, D. C. McMikkK ii, PROF. J. P., University of Toronto, Toronto, Canada. THE REPORT OF THE DIRECTOR. 63 McNAiR, DR. G. T., 1721 Grand Ave., Chickasha, Oklahoma. MACKLIN, DR. CHARLES C., School of Medicine, University of Western Ontario, London, Canada. MALONE, PROF. E. F., University of Cincinnati, Cincinnati, Ohio. 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, Ohio. MATSUI, PROF. K., Imperial College of Agriculture and Den- drology, Morioka, Japan. MAYOR, PROF. JAMES W., Union College, Schenectady, New York. MEDES, DR. GRACE, University of Minnesota, Minneapolis, Minn. 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, Ohio. 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. 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, Illinois. MOORE, PROF. J. PERCY, University of Pennsylvania, Phila- delphia, Pa. 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. 64 MARINE BIOLOGICAL LABORATORY. NABOURS, DR. R. K., Kansas State Agricultural College, Man- hattan, Kansas. NACHTRIEB, PROF. HEXRY F., 2523 Ridge Road, Berkeley, California. NEAL, PROF. H. V., Tufts College, Tufts College, Mass. NEWMAN, PROF. H. H., University of Chicago, Chicago, Illinois. 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, Michigan. OSBURN, PROF. R. C., Ohio State University, Columbus, Ohio. OSTERHOUT, PROF. W. J. V., Rockefeller Institute, New York City. PACKARD, DR. CHARLES, Columbia University, Institute of Cancer Research, 1145 Amsterdam Avenue, New York City. PAPAXICOLAOU, DR. GEORGE N., Cornell University Medical College, New York City. 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, Texas. PAYNE, PROF. F., University of Indiana, Bloomington, Ind. PEARL, PROF. RAYMOND, Johns Hopkins University, Baltimore, Md. PEARSE, PROF. A. S., University of Wisconsin, Madison, Wis- consin. PEEBLES, DR. FLORENCE, Pineville, Pennsylvania. PHILLIPS, Miss RUTH L., \Vestern College, Oxford, Ohio. PHILLIPS, DR. E. F., Cornell University, Ithaca, New York. PIKE, PROF. FRANK H., 437 West 59th St., New York City. PI\\I Y, Miss MARY E., Milwaukee-Downer College, Milwaukee, Wisconsin. PLOUGH, PROF. HAROLD H., Amherst College, Amherst, Mass. POND, DR. SAM TEL E., University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania. THE REPORT OF THE DIRECTOR. 65 PRATT, DR. FREDERICK H., Boston University School of Medi- cine, Boston, Mass. RANKIN, PROF. W. M., Princeton University, Princeton, N. J. RAPPORT, DR. ANNA YATES, Bryn Mawr College, Bryn Mawr, Pennsylvania. REDFIELD, DR. ALFRED C., Harvard University Medical School, Boston, Mass. REESE, PROF. ALBERT M., West Virginia University, Morgan- town, W. Ya. REINKE, DR. E. E., Vanderbilt University, Nashville, Tennessee. RHODES, PROF. ROBERT C., Emory University, Atlanta, Georgia. RICE, PROF. EDWARD L., Ohio Wesleyan University, Delaware, Ohio. RICHARDS, PROF. A., University of Oklahoma, Norman, Okla- homa. RIGGS, MR. LAWRASON, JR., 25 Broad St., New York City. ROBERTSON, PROF. W. R. B., 1505 Rosemary Lane, Columbia, Missouri. ROGERS, PROF. CHARLES G., Oberlin College, Oberlin, Ohio. ROMER, DR. ALFRED S., University of Chicago, Chicago, Illinois. 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, New York. SCHRADER, DR. FRANZ, Bryn Mawr College, Bryn Mawr, Pa. SCHRAMM, PROF. J. R., University of Pennsylvania, Philadel- phia, Pa. 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, Wyoming. SCOTT, PROF. WILLIAM B., 7 Cleveland Lane, Princeton, N. J. SHULL, PROF. A. FRANKLIN, University of Michigan, Ann Arbor, Michigan. SHUMWAY, DR. WTALDO, University of Illinois, Urbana, Illinois. SIVICKIS, DR. P. B., LIniversity of the Philippines, Manila, P. I. 66 MARINE BIOLOGICAL LABORATORY. SMITH, DR. BERTRAM G., 2015 University Ave., New York City. SMITH, Miss CHRISTIANNA, Mount Holyoke College, South Hadley, Mass. Sxow, DR. LAETITIA M., Wellesley College, \Vellesley, Mass. SNYDKR, PROF. CHARLES D., Johns Hopkins University Medical School, Baltimore, Maryland. SOLLMAN, DR. TORALD, Western Reserve University, Cleveland, Ohio. 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., Carnegie Institution, Baltimore, Maryland. 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. TASIIIRO, DR. SHIRO, Medical College, University of Cincinnati, Cincinnati, Ohio. TAYLOR, Miss KATIIERINE A., Cascade, Washington Co., Mary- land. TAYLOR, DR. WILLIAM R., University of Pennsylvania, Phila- delphia, Pa. Ti. \\KNT, PROF. D. H., Bryn Mawr College, Bryn Mawr, Pa. THARALDSEN, PROF. C. E., Northwestern University, Evanston, Illinois. TIIA i< IIKR, MR. LLOYD E., University of Mississippi, University, Mississippi. TIXKHAM, Miss FLORENCE L., 71 Ingersoll Grove, Springfield, TOMPKINS, Mi>- I.II/.ABETH M., 134 Linden Ave., Brooklyn, \. V. THE REPORT OF INK DIRECTOR. 67 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, Wisconsin. UHLEMEYER, Miss BERTHA, Washington University, St. Louis Mo. UHLENHUTH, DR. EDWARD, University of Maryland, School of Medicine, Baltimore, Maryland. VAN DER HEYDE, DR. H. C., Heemstede, Holland. VISSCHER, DR. J. PAUL, Western Reserve University, Cleveland Ohio. WAITE, PROF. F. C., Western Reserve University Medical School, Cleveland, Ohio. WALLACE, DR. LOUISE B., Constantinople Woman's College, Constantinople, Turkey. WANN, PROF. FRANK B., Cornell University, Ithaca, New York. WARD, PROF. HENRY B., University of Illinois, Urbana, Illinois. WARDWELL, DR. E. H., Chappaqua, New York. WARREN, PROF. HOWARD C., Princeton University, Princeton, New Jersey. WARREN, DR. HERBERT S., White Plains, New York. WENRICH, DR. D. H., University of Pennsylvania, Philadelphia, Pa. WHEDON, DR. A. D., North Dakota Agricultural College, Fargo, No. Dakota. WHEELER, PROF. W. M., Bussey Institution, Forest Hills, Mass. WHERRY, DR. W. B., Cincinnati Hospital, Cincinnati, Ohio. WHITE, DR. E. GRACE, Wilson College, Chambersburg, Pa. WHITESIDE, DR. BEATRICE, Detroit College of Medicine and Surgery, Detroit, Michigan. WHITING, DR. PHINEAS W., University of Maine, Orono, Maine. WHITNEY, DR. DAVID D., University of Nebraska, Lincoln, Neb. WIEMAN, PROF. H. L., University of Cincinnati, Cincinnati, Ohio. WILLIER, DR. B. H., University of Chicago, Chicago, Illinois WILSON, PROF. H. V., University of North Carolina, Cluprl Hill, N. C. \VOGLOM, PROF. WILLIAM H., Columbia University, New York City. MARINE BIOLOGICAL LABORATORY. WOODRUFF, PROF. L. L., Yale University, New Haven, Conn. WOODWARD, DR. ALVALYN E., University of Maine, Orono, Maine. YOUNG, DR. B. P., Cornell University, Ithaca, New York. YOUNG, DR. D. B., 617 North Park Avenue, Tucson, Arizona. ZELENY, DR. CHARLES, University of Illinois, Urbana, Illinois. Vol. LI August, 1926 No. 2 BIOLOGICAL BULLETIN OBSERVATIONS ON MOTILITY IN CERTAIN CYANOPHYCE^:.1 EDWARD S. CASTLE. The power of spontaneous movement in the blue-green algae has been stated by many authors to be confined to the family Oscillatoriaceae, occurring characteristically in the vegetative filaments of Oscillatoria, Phormidium, Spiridina and Arlhrospira, and occasionally in the hormogonia of related genera and even of such Heterocysteae as Nostoc, Scytonema, and Rivularia. In addition, DeBary (1863) and Phillips (1904) have described motion in vegetative filaments of the genus Cylindrospermum, a member of the Nostocaceae. Recently, however, two species of Anabcena occurring on the estate of Mr. E. F. Atkins at Soledad, Cuba, and studied by the writer in the Harvard Laboratory there, were found, although belonging to the Nostocaceae, to show consistent motility like that characteristic of the Oscillatoriaceae. These two species were both commonly collected, forming thin scums on damp earth in roadside ditches. In culture bottles they spread rapidly up the sides of the glass in a manner resembling an active Oscillaloria, finally covering the whole inside surface with filaments packed side by side in a single layer. Although exhibiting an active motility like that regarded as characteristic of members of the Oscillatoriaceae, the filaments of both these species were torulose and consistently contained heterocysts that conclusively excluded them from that family. The larger of the two forms (Fig. ib) regularly produced spores and in all essential characteristics justified being identified as a species of Anabana; the other, smaller form (Fig. id), although it never developed spores during 1 Studies from the Biological Laboratory in Cuba (Atkins Foundation) of the Harvard Institute for Tropical Biology and Medicine, No. i. 6 69 EDWARD S. CASTLF.. the two months it was under observation, nevertheless was judged an Anabcena on its general morphology. Exact determi- nation of the species was not attempted by the writer but will be referred later to specialists in the classification of the group. 10 FIG. I. Portions of the trichomes of the motile species of Anabcena studied at Soledad. (a) The smaller species showing its characteristic structure, (b) The larger species showing the cells, heterocyst, and spore in their characteristic arrangement. Both these species of Anabcena were consistently and actively motile, the motility resembling in general that customary in the Oscillatoriacea*. Flexion of the wrhole filament as well as translatory movement was seen in both species, but rotation was not observed. Filaments with heterocysts moved as actively as those without, contrary to what Borzi (1886) observed in the case of species of Nostoc. Speed of translation was, within limits, independent of the length of the filament, confirming the observations of Crozier and Federighi (1924) on Oscillatoria. At 28.9° C. (84° F.), the rates of translation of these species of Anabcena, compared with the rates of some rapidly-motile species of Oscillatoria, were: Anabxna sp. . . . av. 2.5 M per sec. Oscillatoria formosa ... 4.2 n Oscillatoria princeps 5.0 ^ The rate of movement in Anabxna is thus seen to be half that of Oscillatoria princeps and somewhat more than half that of Oscillatoria formosa under the same conditions. Although motility has not been reported in Anal>;nia hitherto, the motility which has been recorded frequently in the hormogonia or such related Heterocystere as Nostoc, Scytonema, or Rivitlarin has in- OBSERVATIONS ON MOTILITY IN CYANOI'I! VCI I 7 I variably been described as of a very slow nature. The rate- given by West (1916, p. 23), as found by Brand for the hormogonia of species of Phormidium, vary from 0.004 to 0.05^ ^ per second, and are thus many times slower than those seen by the writer in the vegetative filaments of Anabxna; but it should be noinl that the temperature at which these slower rates occurred \\.i- not given. In view of the fact that during the movement of these species of Anabxna no rotation was ever observed by the writer, it seemed advisable for comparison to examine species of Oscillatoria in this respect, especially since Crozier and Federighi (loc. dt.) have expressed some doubt whether rotation of the filaments of Oscillatoria does indeed always take place during translation. Careful examination of a considerable number of favorable species of Oscillatoria at Soledad has convinced the writer most conclusively that .many species do exhibit rotation of the filament around the long axis. In species in which the ends of the fila- ments were characteristically hooked, this hook, which main- tained its peculiar shape, could be seen to rotate through the water in a manner precluding a mere circular bending of the tip. Moreover, in larger species, rotation could be observed very convincingly by following the change in relative position of granules lying at different depths within the cell content. Also, in view of the fact that West (1916 and 1904), when dis- cussing the motility of Arthrospira, states that rotation has not been observed in this genus, it may be of interest to record that in a species studied at Soledad, rotation was found to accompany progression, as in Spinilina, while flexion also was seen to occur at times. SUMMARY. Two species of Anabxna, of the family Nostocacea, studied in Cuba, were found to exhibit motion similar to that of the Oscil- latoriaceee. The rate of movement was independent of the presence of heterocysts or of the length of the filament, and was about one half that of actively motile Oscillatoria? under the same conditions, but much more rapid than that previmi>l> recorded for the hormogonia of Phormidium. No rotation was observed in these species of Anabxna but several species of 72 EDWARD S. CASTLE. Oscillatoria and one species of Arthrospira were seen to rotate customarily during free-swimming translation when observed under similar conditions. REFERENCES. Borzi. '86 Le Communicazioni intracellulari delle Nostochinee. Malpighia, i. (Re- viewed in Bot. Cent., 37, 1887.) Crozier, W. J., and Federighi, H. '24 Critical Thermal Increment for the Movement of Oscillatoria. Journ. Gen. Physiol., 7, 137-150. De Bary, A. '63 Beitrag zur Kentniss der Nostocaceae insbesonderbare der Rivularien. Flora. Phillips, O. P. '04 A Comparative Study of the Cytology and Movements of the Cyanophycese. Contrib. Bot. Lab. Univ. of Penna., Vol. II., No. 3. West, G. S. '04 British Freshwater Algae. '16 Cambridge Botanical Handbooks, Vol. i, Algae. NUMBER AND BEHAVIOR OF THE CHROMOSOMES IN CAVIA COBAYA1 (THE COMMON GUINEA PIG). MARY T. HARMAN AND FRANK P. ROOT. KANSAS STATE AGRICULTURAL COLLEGE.2 INTRODUCTION MATERIAL AND METHODS. ... -4 DESCRIPTION OF MATERIAL 1. Spermatogonial divisions 75 2. The growth period 3. Spermatocyte divisions 76 DISCUSSION SUMMARY -p LITERATURE CITED 7O, DESCRIPTION OF PLATES 82 INTRODUCTION. Since the guinea pig has been used extensively in genetic experiments and since investigation has revealed in many in- stances a close correlation between the genetic behavior and the number and behavior of the chromosomes of many organisms, the number and behavior of the chromosomes of the guinea pig is of interest alike to the cytologist and the geneticist. According to Harvey (1920) there is a wide difference of opinion concerning the number of chromosomes observed in Cavia. Von Bardeleben (1892) reports sixteen spermatogonial chromo- somes and eight chromosomes in the spermatids. Flemming (1898) says that there are probably twenty-four somatic chromo- somes. Moore and Walker (1906) give the number as thirty- two in the spermatogonial cells and sixteen in the primary and secondary spermatocyte cells. Stevens (1911) says that there are about fifty-six spermatogonial chromosomes and tweni\ eight chromosomes in both the primary and secondary spermatu- 1 Dr. H. L. Ibsen furnished the guinea pigs for this study. P. VV. Gregory assisted in the fixation and dehydration. The drawings were made by I Prince. 2 Contribution from the Zoological Laboratory, Kansas State Agricuhma! College No. 851. 73 74 MARY T. HARMAN AND FRAXK P. ROOT. cytes. She further states that the sex chromosomes are of the XY-type which are unequal in size and separate in the first spermatocyte division. Athias (1912) reports twenty-four to twenty-eight primary spermatocyte chromosomes in Cavia por- cellus and perhaps twenty-four secondary spermatocyte chromo- somes. Lams (1913) says that there are sixteen somatic chromo- somes, and eight primary and secondary spermatocyte chromo- somes and eight chromosomes in the spermatids. In most cases the material has been reported as very unfavorable or the count doubtful. Our counts do not agree with any of those reported previously. We may add, however, that in many instances our material is almost diagrammatically clear. We have found thirty-eight spermatogonial chromosomes and nineteen chromo- somes in the primary and secondary spermatocyte cells. Like Stevens, we have found an XY-chromosome which separates in the first maturation division. MATERIAL AND METHODS. The present paper is limited to the number and behavior of the spermatogonial and spermatocyte chromosomes. We have found some interesting things in connection with the trans- formation of the spermatid which we hope to give in another paper. Two male guinea pigs were used for this study. One of these animals was superactive sexually and the other was a normally active male. The testes were removed and cut into small sections. These were placed in cold Flemming's solution to which urea had been added. This was done according to the technique of Hance (1917) with the exception that the tempera- ture was 2° C. instead of 4° C. The fixing fluid had been out of doors several hours previous to using it. The material was fixed for about twenty-four hours. The temperature was almost constant during this time. The pieces of fixed testes were transferred to water at room temperature for twenty-four hours. The water was changed frequently. Dehydration was gradual, although the drop method was not employed. Xylol was used for clearing and paraffin was the embedding medium. The sections were cut from three to ten mirra. Those sections from CHROMOSOMES IN CAVIA COBAYA. 75 three to seven micra proved best for study. It was difficult to isolate individual cells in thicker sections. Sections three micra did not seem to be particularly advantageous over those five or six micra. The most favorable stain was Heidenhain's iron- alum hematoxylin. DESCRIPTION OF MATERIAL. It was found that the actively dividing cells are not distributed throughout the whole lining of the seminiferous tubules but are in very restricted areas. These areas are larger and more numerous in the superactive male. They are elliptical in shape. The greatest diameter is always lengthwise of the tubule. This dimension never exceeds two thirds of the circumference of the tubule and may be as small as only a few cells. I. Spermatogonial Divisions. Many and repeated counts have given us thirty-eight spermato- gonial chromosomes. Eight of these are distinctly U-shaped which may be arranged in four pairs. Twenty-eight chromo- somes are slightly bent rods of various sizes which, according to size and shape, may be arranged in pairs. The remaining two chromosomes are unequal in size and irregular in shape. We have designated these as the XY-pair. Fig. 2 is a polar view of a metaphase plate of a spermatogonial division. The chromo- somes which form the members of a given pair have been desig- nated by the same number. Reference to this figure shows that the chromosomes are not arranged merely around the periphery of the spindle as is so often the case in insects (Harman, 1915 and 1920), but fill in the entire diameter of the spindle. No one kind of chromosome occupies one area of the spindle exclusively. The large, slightly bent rods are deep in the spindle as well as around the periphery. The same may be said of the U-shaped chromo- somes. However, the relative arrangement of these various chromosomes is approximately the same. 2. The Growth Period. Evidently a longer period of time is spent in the stages between the last spermatogonial division and the first spermatocyte division than in any other stage, for there are always many times 76 MARY T. HARMAN AND FRANK P. ROOT. more cells in these stages than the other stages. This is true, not only for the animals used in this study, but also for other guinea pigs which we have examined. In the early prophase of the primary spermatocyte cells the chromatin is coarsely granular and is in an indistinct reticulum. There is no uniformity in either the size or the shape of these granules in a particular cell, but there are always a few which are distinctly larger than the majority and decidedly more compact. As the nucleus increases in size, the chromatin granules become smaller and the reticulum less distinct until the nucleus is almost clear, except for here and there masses of chromatin of loose construction. After this the nucleus does not get much larger but the chromatin material becomes more concentrated near one side of the nucleus until it becomes a tangled mass in which it is difficult to distinguish any particular parts or any definite arrangement. Gradually this mass loosens up and more nearly fills the entire nucleus. In addition to the reticulum there is a number of more compact particles of chromatin material. These seem to be the beginnings of the bivalent chromosomes. Long before all the chromatin material has been formed into definite chromosomes each of these masses appears double. Fig. 3 shows nineteen of these double chromosomes with the chromatin in so loose a condition that each body has a woolly appearance. Fig. 5 is a later stage in which the chromatin is more compact and the double condition is no longer apparent. Figs. 4 and 6 are fully formed primary spermatocyte chromo- somes. By reference to these figures it will be seen that no two chromosomes are of exactly the same shape or size. As was found in the spermatogonial divisions the spermatocyte chromo- somes are distributed through the entire diameter of the spindle. 3. Spermatocyte Divisions. Figure 7 is a lateral view of a metaphase plate of a primary spermatocyte division. It will be noted that one chromosome is almost divided and that the division is an unequal one. We have thought that this is the XY-chromosome. We have designated the larger component as the X and the smaller one as the Y. Fig. 8 shows the autosomes undivided while the XY has com- CHROMOSOMES IN CAVIA COBAYA. 77 pleted its division and each component is half way to the pol.- Fig. 10 is an anaphase of a primary spermatocyte division. Thi> figure shows a lack of synchronism in the division of the auto- somes. Figure 9 is an anaphase of a secondary spermatocyte division. It will be seen that some of the chromosomes divide much in advance of the others. Figs. 11 and 12 are polar views of meta- phase plates of the secondary spermatocyte divisions. In each of these figures are seen nineteen chromosomes. Fig. 12 is not a direct polar view but is a view of the metaphase plate at a tangent. DISCUSSION. The large number of chromosomes, the comparative small size and the tendency of the chromatin material to mass together and form into clumps in which the individual parts are not distinguish- able have made the study of mammalian chromosomes a difficult task. This may, in part, account for the differences in the interpretation of the chromosome complex in observations on the same species as was cited in the beginning of this paper. Other difficulties are the scarcity of cells in the various stages of active division, the lack of synchronism in the division of the chromo- somes and the distribution of the chromosomes throughout the entire diameter of the spindle. We are of the opinion that a selection of animals which show sexual activity at the time of securing the material greatly in- creases the size and number of areas of cells in active stages of division. We found this true not only for the animals used in this study but also for a number of other guinea pigs which we have observed. This also agrees with Painter's (Painter, 1924) observations on the monkey and other mammals. Since the chromosomes are closely packed together the selection of a fixing agent which does not swell the chromatin material is very desirous. We have found that the modification of Flem- ming's solution with urea used at a temperature slightly ab<>\» freezing gives the most satisfactory results. We have used Allen's modification of Bouin (Allen, 1919) at about bo v o • e> ** i-^ ^i * * •*" : - -i .•. * •— *^ FIG. i.1 Seminiferous tubules of normal guinea pig testis seventeen days after birth (127^-218). Many mitotic figures but germinal epithelium shows little advance over embryonic condition. 1 All drawings made by Kenji Toda. Il8 CARL R. MOORE. to show the structural condition of the testis shortly after birth. It can be seen that this 1 7-day normal guinea pig testis consists of seminiferous tubules that show little or no tendency to produce an active germinal epithelium. The testis is essentially of the embryonic type and differentiation into the different elements of the germinal line does not occur until considerably later. When such a testis as this is confined to the abdomen it tends to remain in about the same stage of differentiation for several months and in fact after two years abdominal retention quite a similar picture is presented, provided the opposite testis is a normal one. If, however, both testes are confined to the abdomen, this characteristic embryonic type is not retained. FIG. 2. Seminiferous tubules from a guinea pig testis (144/1-211) elevated to the abdomen thirty days after birth (unilateral cryptorchid with opposite testis in scrotum) and removed 45 months later. (Same magnification as Fig. i.) Fig. 2 (144^-211) is a reproduction of a few seminiferous tubules from the unilateral cryptorchid testis removed from animal No. 5 (see Table I.). It was elevated to the abdomen on the 3Oth day after birth and remained for 4^ months (opposite testis normal). Since this is approximately the average period of confinement in the abdomen for all cases reported here, it will serve to acquaint us with the grade of organization of all such testes at the time of their replacement in the scrotum. One can appreciate from Fig. 2 that although this testis was removed from the animal 5^ months after birth, its structural development has remained essentially that of the normal testis SCROTAL REPLACEMENT OF CRYPTORCHID TESTES. 119 of an animal seventeen days after birth; in other words the testis has shown practically no change from its condition at the time of elevation to the abdomen; it is prespermatic and has never produced a differentiated germinal epithelium (compare with Fig. i) nor would it ever build such an epithelium were it allowed to remain in the abdomen. Returned to the scrotum, however, such a testis becomes active, builds a normal germinal epithelium, and produces motile spermatozoa. In the nine animals (Table I.) in which the abdominally confined testis was replaced in the scrotum all but one (No. 6) subsequently produced spermatozoa. In two of the animals the testis was recovered after a period of but ninety days subsequent to replacement in the scrotum; two cases remained for 4^ months, one for 5 months, one for six, and two for ten months. Development under the scrotal influences has, therefore, produced a testis differentiating spermatozoa within ninety days after its return from the abdomen. The success in development varies with the success in scrotal replacement. In every replaced testis adhesions of variable extent had formed. This not only serves to modify even normal testis activity, but it has resulted in a variety of scrotal relation- ships. Some of the testes were firmly bound down to the bottom, or against the side of the scrotal pouch, and surrounded by the fat body normally attached to the anterior end of the testis; some were held firmly in the upper portion of the scrotum or in the inguinal canal; other testes were found to have been held firmly against the upper entrance to the scrotal pouch in a position more abdominal than scrotal, but in all at least one side of the testis projected sufficiently low to be palpable in the upper scrotum. Roughly paralleling the success of replacement in the scrotum is the success of the testis in developing normal spermatogenesis. Let us take for detail consideration, animal No. 7 (Table I.). The right testis was removed from the scrotum into the abdomen on the 3Oth day after birth and allowed to remain high up in the abdomen for 4} months; the left testis was undisturbed. Since the operation periods were the same for this animal as that from which Fig. 2 was made, we can appreciate its structural 120 CARL R. MOORE. condition at the time of scrotal replacement. Having remained in the abdomen for 4^ months the testis from animal No. 7 was returned to the scrotum and allowed to remain therein for a period of five months. When the animal was killed this testis vvas firmly adherent to the bottom of the scrotum and more or less surrounded by its fat body. It cannot, therefore, be con- sidered to have a normal environment on account of the con- <-•'.> ' • 5 4 •««'"<••-*'" •^i<^?"!SV\ fa'Tet-^.V '"«,V ,'• ?*W^^4^a -'••%'- '«'L*V"-i"?-i o"- r-^i ^ «VV .c o <&o • ^' :- -c V Ja » ^fo*--•'. :-"--, .V-v*-» P "- ^-•'^_- Fie. 3. Seminiferous tubules from guinea pig testis (145-213) elevated to abdomen thirty days after birth, remaining in abdomen 4^ months, replaced in scrotum for five months. Complete recovery in this area, si, seminiferous tubule with spermatozoa; ta, tunica albuginea. stricting adhesions and the encapsulating fat. It was many times larger than when replaced in the scrotum and compared rather favorably in size with the normal testis opposite. From entire cross sections at three different levels one can see that the majority of the seminiferous tubules are in active spermato- genesis and a large percentage of these contain spermatozoa. Fig. 3 is a reproduction of a few normal seminiferous tubules, SCROTAL REPLACEMENT OF CRVI'TORt IIII) TKSTKS. 121 some of which contain spermatozoa, and this figure should be carefully compared with Fig. 2 representing the structure of this testis at the time of scrotal replacement. H s v%^^*i; ET*!S> Kt •=•, m^ ' VJf' F-'.V''* '^t> ,;^"^ ^^te^l^r FIG. 4. Seminiferous tubules from same testis as Fig. 3 but from the side where adhesions were present. (Figs. 3, 4, 5 same magnification.) st, degenerate seminiferous tubules; ta. thickened tunica albuginea (adhesions). Despite the establishment of normal seminiferous tubules throughout this testis since its replacement in the scrotum, there are many tubules that have not been restored to normal con- 122 CARL R. MOORE. ditions. Such tubules are more or less restricted to certain regions of the testis and may be entirely devoid of cells in mitosis or cells that appear to be members of the germinal line. Fig. 4 shows a small area from the same microscopic section as that from which Fig. 3 was taken, but from the opposite side of the organ which had formed adhesions with the surrounding tissues; this portion of the testis, therefore, was not located in a suffi- ciently favorable position for resumption of germ cell activity. Here the tubules have not, and probably never would have, developed an active germinal epithelium. One cannot always account for the localized regions of degenerate tubules but these are almost invariably found on the side of the testis where adhesions of the tunica albuginea to surrounding structures have been formed, or on the side where the fat body has closely surrounded the organ. These degenerate areas within such a testicle do not interfere with the function of the normal areas. Spermatogenetic activity is continuous in the latter and spermatozoa are differentiated and transported to the epididymis; sections of the latter portion show the tubules filled with spermatozoa. Suspensions of these cells in 0.9 per cent. NaCl at the time of killing the animals revealed the spermatozoa to be as vigorously motile as those obtained from an entirely normal epididymis. The difference between an abdominal testis and one replaced in the scrotum for a few months is indeed striking (compare Figs. 2 and 3). The entire development of the active germinal epithelium in the case cited, has followed replacement of the testis in the scrotum within five months. In animals 3 and 4 the replaced testis were found to contain quantities of sperma- tozoa three months after scrotal replacement despite the fact that the testis had been confined to the abdomen for a slightly longer period than the one considered in detail above. Develop- ment of the entire germinal line follows, therefore, within three months after scrotal replacement and appears as well established quantitatively as after a period of ten months in the scrotum (animals 9 and 10). In the case of animal No. 7 the testis partner to the one replaced in the scrotum remained normal throughout the experiment but SCROTAL REPLACEMENT OF CRYPTORCHIU TESTES. 123 it is not necessary that it should be present for development of the replaced testis. This is well shown in the experiment inasmuch as in animals I, 2, 4, 8, 9, and 10 the normal testis was removed at the time of the second operation, and each of these six animals produced spermatozoa in the testis after replacement in the scrotum. Out of the nine cases of scrotal replacement only the testis in animal No. 6 failed to produce spermatozoa. This testis was successfully replaced in the scrotal position, but numerous constricting adhesions had been formed and the testis recovered at the termination of its three months in the scrotum was con- siderably smaller than others residing in the scrotum for a similar period of time after replacement. Sections of this testis showed an entire lack of dividing cells in the seminiferous tubules; all cells present appear of the Sertoli type. It appears that all cells of the germinal line had been removed from the tubules and it is doubtful if this testis would have shown recovery effects if it had been allowed to remain in the animal for a considerably longer period. The structure of the tubular portion of the testis in this case is typicakof those cryptorchid testes that have not been opposed by a normal testis. Fig. 5 shows, in comparison with Fig. 2, that the tubules have undergone a change from the embryonic type to a type where apparently only Sertoli cells are present. The tubules are larger in diameter and lack undifferentiated cells, and the intertubular spaces are decidedly different from those of the undeveloped testis; interstitial cells of Leydig are prominent. Just why this testis should have failed to develop normal seminiferous tubules as did the other eight of the experi- ment similarly operated is not known unless it can be attributed to the constricting adhesions that developed about it after scrotal replacement. The importance of the scrotum in controlling conditions that are essential for testicular activity can be shown by reference to conditions in which the testis was incompletely restored to the open scrotal pouch. In animal No. I scrotal replacement was incomplete inasmuch as the testis had developed adhesions and was held in a position essentially across the entrance into the 124 CARL R. MOORE. upper portion of the scrotum; in reality it was more nearly- abdominal than scrotal. On recovery, however, the testis was very evidently much larger than at the time of the replacement • • / ,*>•>- "* , ;*i .-.r m St JV -i*. f /o^r^cz. '26 FIG. 5. Seminiferous tubules from guinea pig testis (144-208) elevated to abdomen thirty days after birth, remaining in abdomen four months, replaced in scrotum for three months (no recovery of function). //, interstitial tissue; st, degenerate tubules. operation. Histological sections show that the majority of the seminiferous tubules were in active spermatogenesis, but very few of the tubules had developed to the stage of spermatozoon formation. Many of the tubules, though possessed of an active germinal epithelium, contained degenerating cells, multinuclear protoplasmic masses, and many vacuoles in the central portion of the epithelium. In other words, the testis was quite similar to testis grafts, heat applied testes, and subcutaneous testes in which spermatogenesis has continued but was usually never complete; the testis is not in an optimum environment for normal tubular activity. The few spermatozoon bearing tubules, however, show that the testis was in a position just on the effective borderline with a few tubules capable of completing spermatozoon formation. SCROTAL REPLACEMENT OF CRYPTORCHID TESTES. 125 DISCUSSION. A consideration of the observations here presented brings forcibly to our attention again the importance of the scrotum to the normal activity of the testes in those mammals for which a definite scrotum has been developed. It was pointed out earlier that testes caused to degenerate, after having once become active, by placing them in the abdomen for short periods of time would in some cases recover their normal function when replaced in the scrotum (Moore, '240). Such a set of conditions, however, are obviously different from abdominal confinement before differentiation of the embryonic cells had taken place or from congenitally retained testes found in nature. And in order to gain an insight into the relative powers of undescended testes to acquire normal function after replacement in the scrotum, this series of experiments has been made; it may give us a relative answer to the possibilities of replacement of unilateral cryptorchid testes and recovery of function in man, but it is obvious that the answer is not absolute. The conclusions are evident that a guinea pig testicle retained in the abdomen from shortly after birth until long after the establishment of sexual maturity, a condition essentially the same as a congenital retention of a testicle in man and a condition in which histologically similar testes are produced, can develop into a functional testis within ninety days after its replacement in the scrotum. Delay in the development of a testicle to its normal condition by abdominal confinement does not preclude its later development when it is brought into its proper environ- ment. In regard to the application of such a conclusion from the guinea pig to essentially similar conditions found in nature, one must be very hesitant indeed in making more of the com- parison than is justified. But if we may make so bold as to attempt a correlation between the findings here and their possible application to man, it would appear to be well indicated that we could expect scrotal replacement of a congenitally retained testis of a man long after the onset of sexual maturity, to produce a testis with normal spermatogenetic function within a relatively short period of time. In terms of years, it is more unsafe to predict the outcome. The guinea pig usually has produced 126 CARL R. MOORE. spermatozoa in its seminiferous tubules within sixty days after birth. By these operations we have prevented this and held the testis in an undifferentiated condition for a period longer than four months, yet have had recovery of function on scrotal replacement; the implications from testes confined longer in the abdomen are that similar results could have been obtained from testes confined in the abdomen for over a year. Since three years may fairly well represent the average duration of sexual function in the guinea pig, one who is interested may carry the implications to relative periods of retention in the human individual and perhaps gain some insight to possibilities existing in such conditions. From the fact that recovery in the guinea pig may follow many months after the onset of sexual maturity, we might expect that in the human individual of twenty-five to thirty years, and from implications one considerably older, a scrotal replacement might result in the production of a normal testicle. It is obvious that these implications, though basically sound, require exact study before they can be more than impli- cations. From the nature of the case it is obvious that since relatively few operations of this character are now attempted on the human individual, and since it is not frequent that such a replaced testicle will come to histological study, there is but little exact data in the literature of any significance in making such comparisons. Bevan ('18), among other surgeons, has given considerable study to the problems on the human indi- vidual, but it is well recognized that with one normal testis besides a scrotally replaced one, about the only criterion of effectiveness in its replacement would have to come from size and firmness determined by palpation or perhaps from hypo- dermic syringe samples. The nature of the influence on a prepubertal abdominally retained testis by the opposite normal testis is not clear; the spermatogenetic activity, however, does exert some influence on the character of the undescended testis. Lipschutz ('25) has recently shown that the expression of the ovarian influence in a male animal by an ovarian transplant is likewise conditioned by spermatogenetic activity; a normal testis prevents the expression of the hormone influences of a transplanted ovary whereas a SCROTAL REPLACEMENT OF CRYPTORCHID TESTES. 12 7 cryptorchid testis does not prevent it. The effect of the influence on a unilaterial abdominal testis is expressed by an almost entire suppression of any developmental capacities. In contrast to this the cryptorchid testis uninfluenced by spermatogenesis in another testis undergoes decided modification. In earlier reports (Moore, '240) it was noted that replacement of abdominal testes that had undergone this change was not followed by normal gametogenetic function. It is believed that the degeneration of such testes had proceeded to the extent that all germinal cells were removed from the tubules. The radical difference between the four or five month crypt- orchid testis here described and the condition following within 90 days after replacement in the scrotum, serves as a striking demonstration of the difference between the capabilities of a given testis retained in the abdomen and one replaced in the scrotum. Having remained embryonic in character and rela- tively quiescent for months, it rapidly becomes engaged on a constructive program and fulfills the primary function of the organ before the end of three months. Thus the scrotal influence is necessary for the expression of the potentialities of the organ and the reader is referred to previous articles for the many lines of evidence that have led to the conception that the effective principle involved is a heat regulatory function on the part of the scrotum. SUMMARY AND CONCLUSION. 1 . Guinea pig testes elevated from the scrotum to the abdomen shortly after birth (prespermatogenic) will, if opposed by a normal testis, retain an undifferentiated character practically throughout the life of the animal. If unaccompanied by a normal testis, they undergo changes leading to the enlargement of seminiferous tubules lined with cells of Sertoli, relative increase of interstitial cells, and a general condition unlike that of the embryonic type. 2. Replacement in the scrotum of the testis retained in the abdomen for five months was followed by normal activity and differentiation of spermatozoa in eight out of nine cases. Pre- sumably normal development of abdominal testes confined in the abdomen for much longer periods would follow scrotal replacement. 128 CARL R. MOORE. 3. Spermatozoa are produced in the testis within ninety days after scrotal replacement. 4. Development of the replaced testis occurs in the presence or absence of an opposite normal testis. 5. By implication we are led to believe that congenitally retained abdominal testes in man have a good chance of devel- oping into normal testes if they are replaced in the scrotum at a time considerably after the attainment of sexual maturity. 6. The function of the scrotum is beautifully shown by com- parison of the testis retained in the abdomen for several months with a similar testis replaced in the scrotum for ninety days. Only in the scrotum are spermatozoa produced in the testes. LITERATURE CITED. Bevan, A. D. '18 Undescended Testes. Surgical Clinic of Chicago, 2: 1101-1117. Griffiths, Joseph. '93 The Structural Changes in the Testicle of the Dog when it is Replaced within the Abdominal Cavity. Jour. Anat. and Physiol. (Lond.), 27: 482-499. '94 Retained Testes in Man and in the Dog. Jour. Anat. and Physiol. (Lond.), 28:209-220. Lipschutz, A., and others. '25 Experimenteller Hermaphroditismus und der antagonismus der geschlechts- drusen. I-III. Pflugers Archiv, 207: 548-595; IV, V, ibid., 208: 273-317. Moore, Carl R. '240 VI. Testicular Reactions in Experimental Cryptorchidism. '246 VIII. Heat Application and Testicular Degeneration; the Function of the Scrotum. Amer. Jour. Anat., 34: 337-358. '260 The Biology of the Mammalian Testis and Scrotum. Quart. Rev. of Biol., i: 4-50. '266 IV. Testis Graft Reactions in Different Environments (Rat). Amer. Jour. Anat., Vol. 37: 351-416. '26c The Activity of Displaced Testes and its Bearing on the Problem of the Function of the Scrotum. Amer. Jour. Physiol., Vol. 77: 59-68. '26d The Relation of the Scrotum to Germ Cell Differentiation in Gonad Grafts in the Guinea Pig. Amer. Nat., Vol. (in press). Moore, Carl R., and Quick, Wm. J. '24 The Scrotum as a Temperature Regulator for the Testes. Amer. Jour. Physiol., 68: 70-79. THE FATE OF THE GERMINAL EPITHELIUM OF EXPERIMENTAL CRYPTORCHID TESTES OF GUINEA PIGS.1 WALTER LAWRENCE, HULL ZOOLOGICAL LABORATORY, THE UNIVERSITY OF CHICAGO. The experimental work of Moore and his students on the biology of the testis has yielded many interesting results and has pointed the way to some further analyses of the activities of this organ. Moore ('246) has discussed at some length the reactions in a normal breeding guinea pig testis that has been removed from the scrotum and confined in the abdomen. The germinal epithelium rapidly becomes disorganized, undergoes degeneration, and the contents of the seminiferous tubules are almost completely removed within a very short time. Such a rapid degeneration is not followed by recovery of the generative portion as long as the testis remains in the abdomen; return to the scrotum, however, was followed by recovery to the extent that the testis again produced spermatozoa. This and many other determined facts emphasized the relation between a scrotal position and the germinal content of the organ and has led to the development of the idea that the scrotum is a local tempera- ture regulator for the testes and that such a regulation is necessary for the production of spermatozoa (Moore, '24^; Moore and Quick, '240). Despite our general knowledge of the progressive degeneration of a testis placed within the abdomen we yet have much of importance to determine in the ultimate fate of the cells of the germinal line. Are the contents of the epididymis affected in the same manner and as rapidly as those of the seminiferous tubules? How long can spermatozoa be found in the genital passages of cryptorchid testes, and what is the relationship 1 This investigation has been aided by a grant from the committee on sex research of the National Research Council; grant administered by F. R. Lillie. 10 129 130 WALTER LAWRENCE. between morphology and motility of spermatozoa? These and other questions are discussed in the following pages of this paper. It was at the suggestion of Professor Carl R. Moore that I attempted this phase of the sex problem, and I wish here to express my appreciation for this and for his kindly direction during the progress of the work. OBSERVATIONS. For the purpose of studying the progressive changes in the degenerating testis thirty-three guinea pigs have been used in which by operation one or both testes have been confined in the abdomen for relatively closely graded periods of from six to one hundred and thirteen days. The operation is simple and consists of opening the abdomen, freeing the testis attachment to the scrotum, and elevating the testis through the open inguinal canal into the abdomen ; to secure permanency in this position the inguinal canals have been closed by sutures. The blood supply, nerve connections and ductus deferens are thus subjected to no interference. A. SEMINIFEROUS TUBULE DEGENERATION. Six-day Cryptorchid Testes. — Observed at the end of six days retention in the abdomen the testis of an adult breeding guinea pig shows but minor macroscopic changes; the organ may appear somewhat hypenemic. Microscopical changes, however, are pronounced. The majority of the seminiferous tubules are abnormal, although the amount of alteration is variable. The diameters of the tubules are unchanged, and the intertubular spaces appear normal. In most cases the contents of the tubules are so shattered and disarranged as to bear little resemblance to the normal condition, while in a few the zones of spermato- gonia, spermatocytes, spermatids and spermatozoa can be made out. On more careful examination individual cells within the tubules are seen to be swollen and granular, and in many cases more or ess completely fragmented; numerous vacuoles appear among the cells constituting the germinal epithelium. From this it can be readily determined that some rapidly destructive process has involved all the tubules. The greater part of the FATE OF GERMINAL EPITHELIUM. lumen in most cases is filled with a mass of cells and debris, obviously in a state of parenchymatous degeneration. The intact cells are swollen and granular, and edges varying from perfectly smooth to a very irregular outline, and the nuclei likewise lose their identity; some of the latter appear eccentric, others fragmented, while in some of the degenerate masses no evidence of nuclei are to be seen. Of the degenerating cells in this central mass spermatids are most abundant, although many spermatogonia and spermatozoa may be seen. The latter, though least altered morphologically are most atypical in their position. Tufts of spermatozoa which normally border on or near the lumen are sometimes seen with their head almost touching the basement membrane of the tubule. Some of these masses are straight while others are adherent to the periphery of spheroidal masses of granular material. The other cellular elements have a tendency to coalesce and groups resembling typical giant cells of the myeloid type may be seen. The most common characteristic throughout all the tubules is the presence of large clear vacuoles in the germinal epithelium. These are most abundant near the periphery of the tubules, although they may be present in any portion of the cross section. Within some of these vacuoles fragments of more or less completely autolyzed cells are seen. There is little doubt that these vacuolar cavities represent areas of degeneration and liquefaction of epithelial cells. We see therefore that a normal adult guinea pig testis removed from the scrotum to the abdominal cavity is decidedly degenerate at the end of six days. Six days after operation is the earliest stage studied, but it is apparent that the effects could have been noted at least one, or possibly two days earlier. In a given cross section of the entire testis, approximately 95 per cent, of the tubules are atypical, the remainder approximating the normal. In the most pronounced degeneration the germinal epithelium is so radically affected that it is in complete disorganization, and the lumen of the tubule may be all but obliterated by whole and fragmented cells from the epithelium. Ten-day Cryptorchid Testes. — A testis retained in the abdomen for ten days is slightly smaller than the normal; this is clearly WALTER LAWRENCE. demonstrated where one testis is allowed to remain in the scrotum while its fellow is elevated to the abdominal cavity. In this case the cryptorchid testis is about four fifths the size of the normal. The beginning degeneration noted in testes of six-day abdom- inal retention is now greatly advanced. The vacuoles which characterized the preceding stage have become more or less confluent, and in many tubules the desquamated epithelium is not so abundant, so that the lumen of the tubules is now larger and more open than in the previous stage. The lumen is not completely reestablished in all cases; in many of them the central area is still occupied by wide vacuoles which have not as yet coalesced, while a few tubules are still cord-like in appearance. Cells showing advanced necrosis are present in the lumina of the seminiferous tubules and most of these are discrete, but some are agglutinated into masses which appear as a single large protoplasmic mass with multiple nuclei, the so-called giant cells. Of these degenerating elements within the lumen, many can be identified as spermatocytes and spermatids, but not as sperma- tozoa. Around the periphery, however, many spermatogonia and spermatocytes still remain, while an occasional spermatozoon may be seen not far from the basement membrane. The blood vessels are somewhat more dilated in this testis than in the normal and an extravasation of serous exudate into the intertubular spaces, and a diapedesis of leucocytes from the capillaries may be seen. A testis retained in the abdomen for ten days is, therefore, considerably more degenerate than one retained for six days, the chief difference being that in the ten-day retained testis there has been further degeneration of the cellular elements with a widening and often a confluence of the vacuoles. Sixteen-day Cryptorchid Testes. — The organ appears about two thirds the size of an unoperated testis, and presents no superficial evidences of congestion. Microscopically the tubules show a decrease in diameter, as well as a diminution in the germinal epithelium content. The lumen in most cases is narrowed or even filled in completely by a thin reticulum of Sertoli cell cytoplasm. The number of Sertoli cells is apparently increased, I ATE OF GERMINAL EPITHELIUM. 133 due in all probability to the smaller caliber of the tubules, and a concentration of these cells, and they appear here as a simple epithelial lining for the tubules. With the exception of a few spermatogonia situated near the periphery of the tubules, intact germinal epithelium is practically absent. A few cells of de- generating germinal epithelium are seen free in the lumen; the rete tubules also contain some degenerating material that is being transported to the epididymis as will be pointed out below. The intertubular spaces show slight evidences of vascular stasis; the interstitial cells show no obvious change. The tunica vaginalis is somewhat thickened. Thus by the end of the sixteenth day of abdominal retention, practically all of the former conspicuous germinal epithelium has been removed from seminiferous tubules, leaving chiefly a lining of Sertoli cells. Twenty-day Cry ptor chid Testes. — The testis confined to the abdomen for twenty days is smaller than the preceding organ, and measures slightly more than half the diameter of the un- operated testis in the same animal. The germinal epithelium is still more reduced here, so that only an occasional spermato- gonium may be identified. In about half of the tubules the lumen is distinctly widened at the expense of the Sertoli cell cytoplasm, so that the lumen occupies from one half to two thirds the diameter of the tubule. The interstitial cells are slightly more conspicuous here and the intertubular spaces contain considerable serous exudate. We see therefore that by the end of the twentieth day the process of evacuation of the germinal elements is complete, with the exception of a few spermatogonia, which retain their normal position between the bases of the Sertoli cells. Abdominal Retention for Longer Periods. — Despite the fact that my interest has been primarily in the direction of the disposition of the degenerating epithelium, and that by the end of the twentieth day of abdominal retention practically all of this has been transported from the body of the testis, it has been found necessary to study the epididymis after longer retention. A word or two in reference to the seminiferous tubules of these testes, retained for from twenty-five to one hundred thirteen days, will not be out of order. 134 WALTER LAWRENCE. Moore ('246) has already shown that a normal breeding guinea pig testis artificially retained in the abdomen loses practically all its germinal epithelium within twenty days and that it does not recover from this striking degeneration as long as it remains in the abdomen. My own observations confirm Moore's assertions that degeneration of the seminiferous tubules is marked after six days, that the degenerative process is pro- gressive, and that by the twentieth day the tubules contain only the Sertoli syncytium within which are a few spermatogonia. In my series of experiments testes retained from 25-113 days in the abdomen show no signs of seminiferous tubule recovery; indeed degeneration is more pronounced in that the testis becomes progressively smaller so that by the end of the second month the whole testis is but half the diameter of the opposite or normal organ. In these testes retained for two months or more in the abdominal cavity the diameter of the seminiferous tubules is greatly reduced and the interstitial cells occupy the greater part of the intertubular spaces; there is also more or less serous exudate between the tubules. The tunica albuginea is five or six times as thick as that of the normal testis. It is apparent therefore that active degeneration of the germinal epithelium begins some time before the sixth day, is most rapid from the sixth to the tenth, during which time the greater part of the germinal epithelium is shed; succeeding changes have to do chiefly with the Sertoli cells which seem to be increased, the diameter of the tubules, and the relative abundance of interstitial cells. The rapidity of the transformation seems to be quite constant for all animals studied. Vas Deferens Ligation of Abdominal Testes. — Seminiferous tubule degeneration, as pointed out above, is of a varied char- acter; some cells are thrown out into the lumen, some apparently undergo solution in situ, the autolyzed products being absorbed directly into the blood stream. The question arose in reference to this degeneration, whether a part of the fragmenting cells and general debris in the lumen of the tubules were carried into the epididymis and eliminated from the body through the genital passages, or whether it was wholly or partially absorbed into the blood stream from the testis. To demonstrate just what FATE OF GERMINAL EPITHELIUM. 135 elements are eliminated through the vas a number of animals were used in which both testes were elevated to the abdomen, and the vas deferens of one ligated in order to make certain the confinement of all testicular material to the organ in question. The degeneration of this vas-ligated, abdominally-retained testis was compared in its progressive stages with a similarly confined though unligated testis of the opposite side, and with testes with similar periods of retention in other animals. Macroscopically the two testes, one with ligated vas and the other with non-ligated vas when compared after any length of retention were entirely similar; one is unable to note difference in size or in vascularity. Microscopically also the two are similar. Degeneration begins as early in the testis with non-ligated vas as in the one with Tgated vas, and the tubules of each are cleared of germinal epithelium in approximately the same time. So far as the seminiferous tubule degeneration is concerned, therefore, ligation of the vas deferens of an abdominally retained testis is without effect. The significance of this added ligation of the vas is demonstrated in the following section where the contents of the epididymis are considered. I had found that some of the degenerating cells, and some cells apparently not so degenerate were transported to the epididymis. Are any of these cells eliminated from the testis through the vas deferens, or do they go into solution in the epididymis and pass out entirely in the blood stream? Comparison of epididymal contents in testes of equal retention where one vas is open and one closed should give us an answer to this problem. B. THE DEGENERATIVE PROCESS IN RELATION TO THE CONTENTS OF THE EPIDIDYMIS. The epididymis forms a cap-like extension on the posterior pole of the -testis, and is somewhat conical in contour, with the base adjacent to the body of the testis. Examined macro- scopically in a normal pig it is seen to consist of tubules of varying size, largest at the apex and smallest at the base, and covered with a transparent membrane, the tunica vaginalis, through which the tubules appear as small while cords. The larger tubules at the apex constitute the ampullae of the ductuli 136 WALTER LAWRENCE. efferentes, which become smaller toward the base of the epi- didymis. These tubules in a sexually mature male are normally distended with spermatozoa which are suspended in a viscous matrix. On opening one of these normal tubules the contents exudes as a thin white paste-like substance which on microscopic examination reveals innumerable spermatozoa; these cells are usually immotile until a drop of physiological saline solution is added, when they are activated to rapid motility. Anderson ('22) in a series of experiments on the spermatozoa of various domesticated animals finds that sperm are normally immotile until they are activated by the accessory secretions of the reproductive system, but that the same results may be obtained by adding normal saline. Armstead ('25) working with guinea pigs finds sperm are made motile by addition of exudate from the seminal vesicles which action he attributes to the alkalinity of the exudate. I have observed slight motility in freshly expressed spermatozoa, even before the saline was added but this was a weak vibratile motion. When the saline is added the spermatozoa became very motile, and are seen to swim about in the medium in groups of from two to eight or ten, the heads being cupped together, not unlike the condition seen in blood where the erythrocytes have a tendency to form rouleaux. In this stacked condition the flagella point in the same direction, but there appears to be no rhythm in the motion of the flagella, some vibrating vigorously and some little or none at all; as a consequence the locomotion of these sperm groups is slow and cumbersome, particularly so in the larger groups. In a cross section of the normal epididymis of the guinea pig the tubules are all seen to contain a greater or less number of spermatozoa with a marked tendency for agglutination as already described. The ductus epididymis contains a mass of sperm usually centrally located; the ductuli efferentes are more com- pletely filled and the ampulla; of these tubules are thin walled sacs well distended with sperm. The ductus deferens, likewise contains a large number of sperm throughout its course. Epididymis of Six Days Abdominal Retention. — Macroscopically the epididymis of a six-day abdominally retained testis shows slight differences in size and color from the normal. It is slightly FATE OF GERMINAL EPITHELIUM. 137 smaller and its base has a somewhat glassy appearance, whereas the normal epididymis is milky white throughout. The contents as seen in cross section of the distal portion are very similar to those of the normal epididymis, and are distinguished by a few more round cells than are to be found in the normal. Toward the base of the epididymis the number of round epithelial cells is seen to increase, and along with these a considerable amount of degenerating debris all intimately mixed with varying amounts of spermatozoa. These germinal epithelial cells, in various stages of degeneration, have been carried over from the testis as a result of the liberation of these elements from the germinal epithelium through abdominal retention. Spermatozoa in the distal portion of the ductus epididymis and the vas deferens were similar to those of normal testes. As it is important to know the effect of abdominal retention on the spermatozoa contained in the epididymis at the time of its removal from the scrotum, stained smears from the epididymis and vas deferens have been prepared in all cases after abdominal retention. Physiological saline has also been added to freshly expressed material from these localities in order to determine the duration of motility of spermatozoa. Mallory's triple stain has proven especially valuable here, as the head caps stain blue while the rest of the sperm body is stained red. Smears from the epididymis of a six-day abdominal testis show the sperm to be morphologically normal, and occurring in clusters as described above. On addition of saline the spermatozoa were seen to be as motile as the normal, moving about in the characteristic clusters, showing that as far as one can detect, the spermatozoa contained within the epididymis are in no way effected by the abdominal retention of six days. Epididymis of Eight Days Retention. — A testis retained in the abdomen for eight days shows the base of the epididymis slightly glassy in appearance. In section the distal portion of the ductus epididymis was seen to contain an abundance of sperm, while in the proximal portion of this tubule degenerating elements predominate. For the most part the ductuli efferentes contain no cellular elements, being filled with a homogeneous liquid which stains faintly. In certain areas of the ep.didymis, and 138 WALTER LAWRENCE. especially between the coils of the ductus epididymis are many polymorphonuclear leucocytes and indeed these are within the tubule in some areas. Ligation of the ductus deferens at the time of testis elevation produces no outstanding differences in the epididymal contents. Sperm expressed from the ductus epididymis and ductus deferens as well as sperm observed in the prepared material are seen to be morphologically intact, but they have lost some of their tendency toward agglutination. When physiological saline was added to these spermatozoa, only a slight amount of motility was observed and this was a weak vibratile motion. No differ- ence in the morphological or physiological characteristics in the sperm from the epididymis and vas could be discerned, nor was there any difference between the sperm from the ligated and non-1 igated sides. It should be remembered, as pointed out above, that the seminiferous tubules of the testes are at this time at the height of degeneration with the lumina packed with degenerating remains, and that but few sperm could be found among these; transfer has continued from the seminiferous tubules since the sixth day, many of the degenerating cells being present in smears and sections of the epididymis. The sperm seen here are undoubtedly at least eight days old, and their viability is a point not to be overlooked. Epididymis of Ten Days Retention. — The gross appearance of these epididymi is similar to that seen in the preceding stage. Where the vas is ligated the epididymis is somewhat larger and the basal portion shows a clear zone. At the end of ten days the ductus epididymis is fairly well distended with sperm, very little diluted with other constituents; this is true of both the ligated and un ligated sides. In the proximal portion of these tubules fewer sperm are present with an abundance of spherical epithelial cells from the seminiferous tubules, and are character- ized by their tendency to form spherical masses. These cells are identical with those described as giant cells in the degenerating seminiferous tubules. Some of the proximal tubules contain deep staining particules of irregular shape and dimensions immersed in a homogeneous coagulum. Sections show that in the proximal tubules in these epididymi the sperm have lost all FATE OF GERMINAL EPITHELIUM. 139 tendency toward clumping, but in the distal portion this tendency is still present; however, smears made from this stage show no tendency toward clumping, and no sperm motility could be observed when physiological saline was added. In neither the epididymis nor the vas did the sperm show any obvious morpho- logical change. It seems apparent therefore that as a criterion by which to judge of viability the tendency toward clumping is a better index than the morphology of the individual sperm. The Epididymis of Sixteen Days Retention. — The epididymis retained for sixteen days in the abdominal cavity shows a glassy appearing zone three or four millimeters wide at the base. The entire organ is smaller than the normal, whether the vas is ligated or not, and where not ligated it is smaller and presents a broader clear zone at the base than in the ligated one. In sections the distal tubules are seen to contain spermatozoa which appear normal except for the clumping tendency. Further toward the base of the epididymis the number of spermatozoa becomes less, and at the extreme base there are few or none; instead there is to be seen a coagulum of fragmenting elements, degenerate beyond identification. Between these extremes some of the tubules contain a few spermatozoa mixed with a large number of swollen epithelial cells of varying size, among which are characteristic globular masses of these epithelial constituents. Polymorphonuclear leucocytes are common within and between the tubules in the basal portion of the epididymis. In comparing the epididymis of the ligated and non-ligated vas, we find the latter somewhat smaller, the number of sperm less, and the amount of degenerating debris relatively more. This is easily explained since the ligature prevents the outward flow of sperm in the epididymis and vas, so that the degenerating constituents entering from the testis are able to occupy only the proximal tubules. The normal fellow of a cryptorchid epididymis is normal both as to appearance and contents and is apparently affected not at all by abdominal retention of the opposite testis. The smears of the vas and epididymal contents from the testes of sixteen-day retention show sperm which are to all appearances normal morphologically, but which exhibit no pronounced tendency to clump, and are not activated to motility by the addition of physiological saline. 140 WALTER LAWRENCE. The Epididymis of Twenty-day Retention. — There is little difference between this and the preceding; the tubules proximal to the testis contain either degenerating debris, or sperm highly diluted with epithelial cells all of which are products recently evacuated from the testis. The distal tubules contain chiefly spermatozoa, and although most of these are normal, some show separate head caps and stain slightly fainter than freshly killed spermatozoa. It is a noteworthy fact that although the germinal epithelium of the seminiferous tubules is markedly disarranged and shows considerable degeneration at the end of six days abdominal retention, the mature spermatozoa present in the epididymis at the time the organ was elevated to the abdominal cavity show noticeable changes only after twenty days, a time when the seminiferous tubules are almost completely denuded of their germinal epithelia. Twenty- five to One Hundred and Thirteen Days Retention.— With longer abdominal retention the vitreous area at the epi- didymis is increased, so that by the end of the second month the whole epididymis has lost its milky appearance. This difference in appearance is brought out by the fact that the 'iquefied and degenerating materials from the testis proper have mixed with the spermatozoa at the base and the latter have likewise slowly yielded to the degenerative process so that ultimately the entire organ contains only liquefied and frag- menting material which presents a vitreous appearance. That this material remains in the epididymis to be absorbed is shown by the fact that at the end of 113 days abdominal retention the tubules still contain a small amount of degenerating debris whether the vas is ligated or not. It should be mentioned here that the process of degeneration and absorption are much slower in the ductus deferens since sperm retained here proximal to the ligature are still identifiable after 113 days retention, whereas those retained in the efferent ducts are completely degenerated. It is reasonable to suppose that with the addition of the degenerating products from the seminiferous tubules to the already distended epididymis that the latter would be distended beyond its normal size where the vas was ligated, or that an overflow would occur with the vas unligated. Observation, FATE OF GERMINAL EPITHELIUM. 14! however, shows this to be true only in the early stages of degenera- tion, for in all cases of abdominal retention for longer than sixteen days the epididymis is actually smaller than the normal. This may be accounted for by the supposition that absorption rapidly removes the autolyzed portion of the degenerating material, and at the same time concentrates the contents already present in the tubules. On cutting one of these tubules and expressing some of the contents it is seen to be of much thicker consistency than that of the normal tubule. Sections through epididymi at various levels and with varying periods of abdominal retention bear proof that the spermatozoa degenerate very slowly; this process, however, varies somewhat in the same organ, being slower in the thicker walled portion of the ductus epididymis and more rapid in the efferent ducts. In the vas sperm retained their integrity for 113 days, while in the ampullae of the ductuli efferentes degeneration was apparent after 20 days. In the process of degeneration the spermatozoa first lose their head caps at which time they stain poorly, later the tail separates and the body undergoes granular degeneration. Spermatozoa in Other Passages. — As a part of the routine examination of the urogenital passages of the animal at autopsy, the seminal vesicles and urinary bladder were examined by making fresh smears for the presence of spermatozoa. This was done by aspirating some of the contents of the urinary bladder with a hypodermic syringe and by opening the seminal vesicles at various levels and making fresh mounts of the contents. In no case were sperm found in the urinary bladder. In five cases sperm were recorded from a seminal vesicle; in two cases a single sperm was seen in each; in two cases the number was described as "few;" and in one case they were abundant. This observation was made before it was realized how very easy it is to carry over sperm from a severed vas on scissors or instru- ments. My observations, therefore, confirm the findings of Fisher ('23) and Armstead ('25) who demonstrated that the seminal vesicles in guinea pigs are not sperm reservoirs. In two cases, when the animals were killed by a blow on the head there appeared on the phallus a mass of material the size of a pea, which to the feel was very rubbery, and which upon exam- 142 WALTER LAWREN'CE. ination was found to contain many motile spermatozoa, mixed with exudate from the seminal vesicles. As a result of this series of observations it becomes apparent that the degenerating products of the seminiferous tubules due to abdominal retention are removed chiefly by way of the blood stream. My study leaves little doubt that a great deal of cytolysis goes on in the early stages of degeneration in the seminiferous tubules, and it is highly probable that these materials are removed from the testis by the circulation; but it is also apparent that a great deal of the degenerating material is trans- ported to the epididymis. Since the tubules of the testis are cleared by the 2Oth day, and many times earlier, and since the epididymis, even with its vas normally open, contains much of its normal sperm content it is clear that the degenerating cells are not removed from the epididymis through the vas in any considerable amount. Indeed it is not apparent that the majority of the spermatozoa contained within the epididymis are so removed. My material leads me to believe that practically all the degenerating products from the seminiferous tubules are liquefied and absorbed in the blood stream, either from the testis proper, or the epididymis, and that a large part of the spermatozoa in the epididymis are likewise liquefied and absorbed. C. THE VITALITY OF SPERMATOZOA. The question of the. vitality of spermatozoa has been long debated, and extensive investigations have been made to deter- mine just how long spermatozoa will live in the uterus; less attention, however, has been paid to the life of the sperm in the male genital passages. De Lee ('21, p. 21) states that human spermatozoa will live for 21 days in the human uterus. Nurn- burger ('20) claims to have kept human spermatozoa alive in the laboratory for seven or eight days. Hoehne and Behne ('14) claim to have demonstrated that all sperm die in the human vagina in one and a quarter hours, and that in the uterus no live sperm may be found after three days. Lewis ('n) bred 20 sows to boars which were known to be fertile, after which the sows were slaughtered and the uteri examined. He states: "In only two cases were sperm found alive 24 hours after FATE OF GERMINAL EPITHELIUM. 143 breeding." Anderson ('22) has pointed out that sperm are very sensitive to chemical and temperature changes. He was also able to find a few live sperm in the uterus of a mare seven and one fourth hours after breeding; and in hens sperm were found six to eight hours after breeding, but after fifteen hours none could be found. Wolf ('21) was able to keep rabbit sperm alive for nine days by the use of properly balanced physiological saline and iso tonic glucose solution, and the required Ph concentration. Many more such references might be cited, but this will serve to show something of the diversity of the results obtained by different workers. Although there is doubtless a great variation in the viability of sperm from different species, doubtless some difference in results are attributable to the environment to which the sperm was subjected. In this series of experiments particular attention was paid to the vitality of spermatozoa. When an animal was killed the vasa and epididymi were examined for sperm and where found were tried for notility by adding physiological saline to the contents. As stated above spermatozoa were found alive in the epididymis as long as eight days after both testes were elevated to the abdominal cavity, and closely associated with this there was a strong tendency toward dissociation of the clumps so characteristic of viable guinea pig sperm. To recheck on this very important point five pigs were used. Both testes were elevated to the abdomen, both vasa ligated, the left with a single ligature near the posterior pole, and the right in two places, one near the epididymis and the other one inch below, so that on the right side, the sperm which were in the lumen at the time of operation were not allowed to escape, and no more sperm could enter. When the animals were killed the contents of the epididymi, and of the vasa above, between and below the ligatures were examined and in each case the results were the same. The animals were killed on the 7, 9, 11, 13 and I4th days after operation. In only the first two cases, that is after seven and nine days were motile sperm found, and in these cases no differ- ence in motility could be detected whether the sperm were taken from the region above, between or below the ligatures. In both these cases the motility was accompanied by the clumping 144 WALTER LAWRENCE. phenomenon described in a preceding paragraph. Although there was motility in some of the sperm after nine days abdominal retention the per cent, which were motile was considerably less than in the case just cited. With the loss in motility the tendency to clump is lost, so that in sperm from a ten-day abdominally retained epididymis there is little or no clumping, although the sperm bodies may appear approximately normal for a month longer in the ductus epididymis and vas. It is a surprising fact in view of the extremely rapid degenera- tion within the seminiferous tubule that spermatozoa morpho- logically normal are found in the epididymis for such long periods of time after all other elements have disappeared. Thus within twenty days the seminiferous tubules are approximately empty whereas the epididymis contains sperm apparently normal so far as morphology is concerned for thirty-seven days. It is also significant that the rapidity of degeneration of spermatozoa varies in different parts of the passages. Thus in the efferent ducts and in the proximal portion of the ductus epididymis degeneration is marked at twenty to thirty days, in the thicker walled distal portion of the ductus epididymis and vas sperm are considerably less altered after retention of 113 days. To account for this difference in rapidity of degeneration is beyond the scope of this paper, but it might be suggested that probably the cause is referable to the difference in vascularity of these parts. Since in the vas, degeneration is least marked, and since this is the real reservoir for mature sperm, it would be reasonable to suppose that vitality would be retained as long or even longer than any place inside or outside the animal body, as here they are surrounded by what would be supposed to be the most suitable environment. As sperm are capable of showing motility for nine days in this location after ligature, and as these sperm were present an indeterminate length of time before operation, we can state with assurance that the maximum life of the guinea pig sperm in the male genital tract is more than nine clays. D. VASCULAR CHANGES IN RELATION TO THE DEGENERATIVE PROCESS. Having noticed that testes made cryptorchid for from six to sixteen clays were somewhat hyperemic as evidenced by the FATE OF GERMINAL KI'ITIIKLIUM. 145 dilated condition of the superficial veins and the presence of serous exudate in the intertubular spaces, it occurred to me that perhaps degeneration might be due to such a vascular change. Was degeneration being brought about through a reflex dilation of the vessels supplying the testis and if so, might not the same changes be produced through ligation of the internal spermatic vein by inducing passive congestion within the whole organ? Moore ('246) also suggests that possibly some such relationship may occur. In an endeavor to demonstrate this relationship three pigs were operated; in two the internal spermatic veins were ligated and in the third the veins of the ductuli deferentes were ligated, care being taken not to occlude the ducts them- selves. After seven days one'of the pigs with internal spermatic vein ligature was killed. On the left side the ligature had not been made sufficiently tight to occlude the vessel, and the blood supply was found to be normal, but adhesions had followed handling the organ which was found within the abdominal cavity. In section this testis showed a very early stage in the degenerative process, earlier than that described for the six-day stage; and in this the contents were seriously deranged. Des- quamation of the germinal epithelium had begun and spermatids were forming into giant cells in abundance. In some cases as high as forty nuclei could be seen together. On the right side, however, the testis was in the scrotum and the attached fat body was firmly adherent to the inguinal ring, thus preventing the return of the testis to the abdominal cavity. This testis was deeply cyanosed, the spermatic vein being completely occluded while the veins of the ductus deferens were greatly dilated. Microscopically this testis showed a vastly greater amount of hyperemia than any of the cryptorchid testes exam- ined, yet there was nowhere the typical degeneration that was found in the abdominally-retained testes. There was no vacuol- ization nor emptying of the seminiferous tubules; instead, the tubular contents were considerably disarranged, swollen, and more or less coagulated in situ. The condition might well be compared to one of coagulation necrosis following infarction. The material stained very poorly with the stains which were being used. On the fourteenth day the other two pigs were 11 146 WALTER LAWRENCE. killed, and in the case of the second, operated as above, the left internal spermatic vein was also successfully occluded and here also a condition similar to the one just described was found. Many of the seminiferous tubules contained spermatozoa atypically arranged as were the other contents, with the whole staining poorly. In the case where the vein of the ductuli deferentes were ligated a new venous return was well established along the vas, and the testes were in full spermatogenic activity. These observations indicate that although the hyperemic state may be closely coupled with the process of degeneration it is certainly not the causative factor. It would be admissable to suppose that the hyperemia is the result of the degenerative changes, wherein the autolyzed products are more rapidly taken up by the blood stream. It was noticed repeatedly that leuco- cytes were abundantly distributed throughout the intertubular spaces, and in many instances they were present in large numbers in the tubules of the epididymis. They are here doubtless concerned with the phagocytic function, and the degenerating epithelium which has been liberated from the seminiferous tubules are in effect foreign proteins which have perhaps stimu- lated this phagocytosis. E. DISCUSSION. This work was undertaken to determine if possible, just how the germinal epithelium which is desquamated from the seminif- erous tubules in artifically cryptorchid testes is disposed of, and incidentally to ascertain something concerning the relative resistance of the various cells of the germinal epithelium to the degenerative process. As to the effect of abdominal retention upon the sexually mature male testis I have but to confirm the findings of Moore ably described in recent articles. The organ is effected in such a way as to cause the cessation of all spermatogenic activity by the end of six days; at twenty days abdominal retention the seminiferous tubules have been almost completely denuded of their germinal epithelium, and succeeding changes relate chiefly to the size of the testis, the diameter of the tubules, and the FATE OF GERMINAL EPITHELIUM. 147 Sertoli cell content. It has been abundantly demonstrated (Moore, '22, 24^; Moore and Oslund, '24; Fukui, '23) that heat of certain temperatures will produce in the testis a degenera- tion identical with that seen in abdominally retained testes, and that the degeneration is identical in nature and proportional within certain limits to the degree of heat, whether it be applied by hot water, hot air, parafine, electric lights, etc. And further it has been shown (Moore and Quick, *24a) that the temperature within the abdomen is definitely higher than intrascrotal tem- perature, all of which logically lead to the conception of the scrotum as a necessary thermo-regulator for the testis as devel- oped by Moore ('24^). Bouin and Ancel ('03) believe they have demonstrated tubular degeneration attributable to vas ligation. Others (Moore and Quick, '241:; Oslund, '24, '25) have been unable to produce degeneration by this method when the testes were carefully kept within the scrotum. Although in my work, testes, the vas of which were ligated, were elevated to the abdominal cavity where degeneration took place as a result of subjection to an elevated temperature, the testes so ligated were in all respect like those retained in the abdominal cavity the vas of which were not ligated. It is not proposed to offer this evidence in confirmation or derogation of the results obtained by any of these workers, but simply as a statement that in conjunction with abdominal retention ligation is without apparent effect. It was pointed out in the text of this paper that although many of the epithelial cells in the seminiferous tubules are partially or almost completely autolyzed in situ, many others are thrown into the lumen of the tubule almost unchanged. These cells were followed through the straight tubules and rete to the proximal tubules of the epididymis where they are seen intermixed with spermatozoa already present previous to the elevation of the organ of the abdominal cavity. Obviously these invading cells, together with the spermatozoa already present must become absorbed in the epididymis, remain there, or be evacuated through the genital passages. Were the latter to take place the spermatozoa would be pushed on ahead probably mixed with some of the degenerating cells, followed by the 148 WALTER LAWRENCE. newly acquired degenerating epithelium which should be found all along the passage. Were the whole to remain unchanged the epididymis would be ballooned up with both the old and newly acquired contents. The fact is the most of the sperma- tozoa present in the epididymis at the time of operation remain there, and are seen in various stages of degeneration within this organ. Likewise other germinal cells may be identified in the epididymis undergoing a more rapid degeneration than that seen in the case of the spermatozoa. We are left with little doubt that practically all the germinal epithelium of the testis and spermatozoa in the epididymis are absorbed directly from these organs into the blood stream after degeneration and liquefaction. As a further proof of this, if any is needed, sperma- tozoa which escape into the vas deferens which is markedly less vascular than the epididymis, require a much longer time for absorption than those retained in the ductuli efferentes. And what of the effect of all this absorption on the organism? McCartney ('23) states that he was able to produce sterility in rats through injection of spermatozoa, and in a second article he states: "In the male rat vasoligation may be followed by the development of antibodies for semen in the blood." It is admitted that if the first is true the second would seem plausible. In my work ten pigs were operated in which one testis was elevated to the abdominal cavity, the opposite retaining its normal relation in the scrotum. The operated testes were allowed to remain for from six to sixty-four days in the abdominal cavity. Varying amounts of degeneration had taken place with the absorption of these products together with the spermatozoa present at the time of operation, and in not a single case was the spermatogenic activity in the opposite testis affected, nor was there any noticeable change in the testicular or epididymal contents from the normal. Concerning the viability of spermatozoa so much has been said that any attempt to review the literature here would be futile. Be it said that reports vary from a few hours for the boar sperm in the sows uterus (Anderson, '22) to 21 days for the human sperm in the female uterus (DeLee, '21). In my observations I have found a fairly definite relation between FATE OF GERMINAL EPITHELIUM. 149 viability and a tendency toward clumping. Spermatozoa were found alive in the ductus epididymis, and the vas for nine days after elevation of the entire testis to the abdominal cavity, and since it is reasonably certain that spermatogenic activity ceases on elevation of the organ, and since the spermatozoa pent up in the vas and epididymis were in these locations some time previous to operation, it follows that the length of life of a fully mature spermatozoon is something more than nine days. Just how long a spermatozoon remains in the seminiferous tubule after maturity, and what time is required for its passage into the vas is a question which must be answered before we can state anything more definite concerning the length of life of fully developed sperm. As to the role played by the vascular changes in the degener- ative process it must be admitted that insufficient work has been done in this direction to permit of any definite statements. However, from the meager data here presented it would appear that haemostasis induced by ligation of the blood supply to the testis produces a coagulation necrosis of the germinal epithelium, not followed by a removal of the germinal epithelium at least within sixteen days. Hence we are lead to believe that the vascular changes seen in artifically cryptorchid testes is a case of active hyperemia, and that through this agency the degener- ating elements are rapidly taken into the blood stream. F. SUMMARY AND CONCLUSIONS. 1. When the testes of sexually mature male guinea pigs are elevated to and allowed to remain in the abdominal cavity, a progressive desquamation and degeneration of the germinal epithelium takes place; at six days abdominal retention degener- ation is well marked, at ten days well advanced, and at twenty days practically all epithelium has been removed. This degener- ation can be directly attributed to the increased temperature of the abdominal cavity over that of the scrotum. 2. Neither the speed nor the nature of the degenerative process is effected by ligation of the ductus deferens of the cryptorchid testis. 3. All of the degenerating elements from the seminiferous WALTER LAWRENCE. tubules are absorbed in the testis proper, or are carried to the epididymis where they are absorbed along with the greater part of the spermatozoa which were present at the time the testis was elevated to the abdominal cavity. 4. Absorption of the germinal epithelium from a cryptorchid testis into the blood stream does not affect the integrity of the scrotally-retained normal testis. 5. Spermatozoa will remain alive in the germinal passages of the guinea pig for longer than nine days. Following their loss of motility they rapidly lose their tendency to clump, but degeneration of mature spermatozoa takes place slowly, and especially so in the ductus deferens. It has been determined that a given spermatozoon in the male genital passages though retaining its capacity for motility for nine days, may retain its morphological identity for over 100 days. 6. Venous stasis through ligature of the internal spermatic vein of testes allowed to remain in the scrotum does not produce the degeneration complex seen in testes retained in the abdominal cavity. FATE OF GERMINAL EPITHELIUM. LITERATURE CITED. Anderson, W. S. '22 Vitality of Spermatozoa. Ky. Agr. Exp. Sta. Bui. 239. Armstead, Reo B. '25 Structure Function and Regeneration of Seminal Vesicles of Guinea Pigs. Jour. Exp. Zool., Vol. 41, p. 215. De Lee, Jos. B. '21 Prin. and Practice of Obstetrics, p. 21. Fisher, N. F. '23 The Influence of the Gonad Hormones on the Seminal Vesicles. Am. Jour. Phys., Vol. 64, p. 244. Fukui, N. '23 On a Hitherto Unknown Action of Heat Ray on the Testicle. Japan Med. World, Vol. 3, No. 2. '23 Action of Body Temperature on the Testicle. Ibid., Vol. 3, No. 7. '23 On the Action of Heat Rays upon the Testicle: An Histological Hygienic and Endochrinological Study. Acta Scholse Medicinalis Universitatis Imperialis in Kioto, Vol. 6, Fasc. n, p. 225-228. Hoehne and Behne. '14 Uber die Lebensdauer homologer und heterologer Spermatozoen im weib- lichen Genitalapparat. Zentralblatt fur Gyn. (Quoted from Williams Obstetrics. 1923.) Lewis, L. L. 'u The Vitality of Reproductive Cells. Eul. 96, ign. Oklahoma Agri. and Mech. College. McCartney, J. L. '23 Studies on the Mechanism of Sterilization of the Female by Spermatoxins. Amer. Jour. Phys., Vol. 63, p. 207. '23 Further Observations on the Antigenic Effect of Semen. Ibid., Vol. 66, p. 404. Moore, Carl R. '22 Cryptorchidism Experimentally Produced. Proc. Soc. Zool. Anat. Rec., Vol. 24, p. 383. '240 The Behavior of the Testis of Transplantation, Experimental Cryptor- chidism, Vasectomy, Scrotal Insulation, and Heat Application Endocrin- ology, Vol. 8, No. 4. '246 Properties of the Gonads as Controllers of Somatic and Psychical Char- acters. VI. Testicular Reactions in Experimental Cryptorchidism. Am. Jour. Anat., Vol. 34. No. 2. '24C Properties of the Gonads as Controllers of Somatic and Psychical Char- acteristics, VIII. Heat Application and Testicular Degeneration, the Function of the Scrotum. Am. Jour. Anat., Vol. 34, No. 2. Moore, Carl R., and Oslund Robt. '24 Experiments on the Sheep Testis. Cryptorchidism, Vasectomy and Scrotal Insulation. Am. Jour. Phys., Vol. 67. pp. 595-607. Moore, Carl R., and Quick, Wm. J. '240 The Scrotum as a Temperature Regulator for the Testis. Am. Jour. Phys., Vol. 68, pp. 70-79. '246 The Properties of Gonads as Controllers of Somatic and Psychical Char- acteristics. VII. Vasectomy in the Rabbit. Am. Jour. Anat., Vol. 34, No. 2. 152 WALTER LAWRENCE. Niirnburger. '20 Klin und exp. Untersuchungen uber die Lebensdaur der menschlichen Spermatozoen. Monatschr. f. Geb. und Gyn. Quoted from Williams Obstetrics, 1923. Oslund, Robt. M. '24 A Study of Vasectomy of Rats and Guinea Pigs. Am. Jour. Phys., Vol. 67, pp. 422-443. '25 Vasectomy on Dogs. Am. Jour. Phys., Vol. 70, p. 117. Williams. '23 Obstetrics. P. 96. D. Appleton & Company. Wolf. '21 The Survival of Motility in Mammalian Spermatozoa. Am. Jour. Agri. Science, Vol. n, 1921. Quoted from Marshalls Physiology of Repro- duction, 1922. . Vol. LI. September, 1926. No. 3. h L I BIOLOGICAL BULLETIN OBSERVATIONS ON THE RELATIONSHIP BETWEEN A RED TORULA AND A MOLD PATHOGENIC FOR DROSOPHILA MELA NOG ASTER. MARTIN FROBISHER, JR., Sc.D. FROM THE DEPARTMENT OF PATHOLOGY AND BACTERIOLOGY, JOHNS HOPKINS UNIVERSITY, BALTIMORE, MD. In cultivating D. melanogaster at the Institute for Biological Research at Johns Hopkins University, a difficulty arose during the summer of 1925, which at times interfered with the growth of the flies. This difficulty was the appearance, in the flasks used for cultivation, of a growth that resembled the ordinary blue-green Penicillium but which appeared to form on the sur- face of agar, a thick, tough, basement membrane of a bright lemon-yellow color. This growth was apparently very obnoxious to the flies for they died in large numbers soon after being put into jars containing it. The phenomenon was brought to the attention of the author and the following studies made. Microorganisms pathogenic for flies of various species have been described (i, 2). It was thought, therefore, that the present phenomenon might also be due to the presence of some pathogenic organism in the jars. With this idea in mind an attempt was made to isolate such an organism from one of the jars in which the flies had died very rapidly, and in which the yellow growth was particularly thick. It has been observed by Pearl that flies might live in ves- sels containing growth of Penicillium of the ordinary varieties without any ill effects on the flies. Inasmuch as the mold in question was believed to be one of these ordinary types, it was thought that the obnoxious growth, if any were present, must be connected with the yellow pigmentation, since this had not been observed before and its appearance was connected with the death of the flies. 11 i53 154 MARTIN FROBISHER, JR. Material from the jar was therefore plated out on a medium similar to that used for the fly cultivation.1 To obtain the inoculum, a piece of the tough, yellow membrane covering the surface of the agar in the fly bottle, under the growth of the mold, was cut out with a loop of stiff sterile wire. The under side of this piece of membrane was carefully scraped and the scrapings spread upon the medium in petri plates. Smears of the scrapings were made at this time, and stained with Gram's stain and with Loeffler's methylene blue. These preparations showed the presence of mold hyphse, spores and yeast cells. No bacteria were seen. This is readily understood in view of the acidity of the medium. Plates streaked as above were incubated in the refrigerator at about 6° C., also in the laboratory at a temperature of about 25° C., and other plates were held at 37° C. Growth occurred at all three temperatures, but most abundantly and rapidly on the plates held at about 25° C. On all plates there appeared nothing but the white commercial yeast referred to and the mold. There was no yellow growth on any of the plates, even after long standing at different temperatures and in the dark as well as in the light. The attempt was regarded as a failure, since no organism appeared except those believed to be harmless to the flies. A second and third attempt yielded like results. No yellow organ- 1 This medium is prepared as follows: Solution A. Water Distilled 500 cc. Sucrose 83.5 grams CaCh 25 " MgS04 50 " (NH4hSC>4 2.00 " KH2P04 i.oo " Tartaric Acid 5.00 Rochelle Salt 8.40 " Solution B. Water Distilled 500 cc. Agar 30 grams Autoclave A and B separately. Mix aseptically in equal parts just before use. The solutions keep well separately. The reaction is about pH 3.2. RELATIONSHIP BETWEEN A RED TORULA AND A MOLD. 155 ism appeared and nothing even resembling the thick yellow growth in the original fly bottle could be seen. Other fly bottles were obtained, and finally, from one of these, a growth was obtained which had the typical yellow color and the leathery texture. Upon replating this growth repeatedly, however, nothing could be obtained except the ordinary yeast and the mold. After repeatedly smearing such material over plates and getting only the mold and ordinary yeast referred to, a final survey of all plates heretofore made was undertaken with a view to dis- carding most of them. During this survey, however, there was noted on one of the older plates several tiny colonies having a distinct, coral-red color. One of these was fished to Saboraud's agar and held at room temperature for three days. During this time there ap- peared a soft, creamy, coral colored growth, which, upon micro- scopical examination, was found to be a yeast or torula. It was later shown that this red torula occurred as a contaminant in cakes of the commercial yeast referred to above. After three or four more days, the pink growths on the plates from which the pink colony had been fished, were observed to be gradually turning yellow in places, and wherever the yellow color appeared there was also a growth of the mold. The yellow growth had also become tough and leathery. Smears made from these colonies showed the presence of a mass of yeast cells matted and closely bound together by mold hyphse. It appeared that the combined growth of the red torula and the white or unpigmented mold produced the bright lemon-yellow color, and the tough character of the colonies. It only remained to get pure cultures of the two organisms growing under controlled conditions to decide this point. This was easily done by inoculating one side of a plate with a pure culture of the mold and the other half with a pure culture of the red torulse. The plate was held at about 22° C. for four days and good growth of both organisms occurred. The two growths remained separate for some days, but soon the spreading mold began to overgrow the area streaked with the torulze. Wherever the mold encountered a colony of the torulae, the colony became yellow, hard and tough. I=;6 MARTIN FROBISHER, JR. One of these yellow colonies, smeared under a cover slip, showed that the mold had invaded the colony of torulae and matted and bound the whole colony into a solid mass. The yeast cells seemed to suffer somewhat from the contact, since when one of these yellow colonies was stained by methylene blue most of the entangled cells appeared to be wholly or partly disintegrated. The destruction of the red torula cells by the mold probably explains why so much difficulty was originally encountered in isolating the red organism. The mold does not appear to have such an effect upon the commercial yeast, for good growth of this yeast was obtained even after prolonged contact with the mold. Regarding the occurrence of a red torula in compressed yeast cakes it may be said that this is not a specially remarkable circumstance, and may have been observed before. The pink or red torulse appear to be fairly widely distributed in nature. They have been known for a long time and seem to have been first described in 1850 when Fresinius (3) first described Crypto- coccus glutinis. Pink torulae or yeasts have been isolated from various beer and wine musts, milk, cheese, sea water, plant diseases and other sources. The finding of such an organism by Fisher and Brebeck (4) in the stomach of a case of gastric fermen- tation is of interest in view of the ability of the organism herein described to grow well at an acidity similar to that of the normal gastric contents. A pink yeast or torula causing spoilage in oysters has been described by Hunter (5). Similar organisms have been isolated by the author from oysters taken from Chesa- peake Bay. The organism mentioned above resembles these varieties in many respects and probably belongs to the same family or genus, but also shows differences that indicate that it is not of the same species, the chief difference being that it is quite a strict aerobe. A cultural study of the red torula yielded the following data: Growth occurs at temperatures ranging from about 6° C. to about 37° C. The optimum is in the neighborhood of 25° C. Growth takes place on agar having a reaction ranging around Ph 7.4, and also as low as Ph 3.0. No very definite optimum seems to exist. RELATIONSHIP HKTWEEN A RED TORULA AND A MOLD. 157 In a fluid synthetic medium the reaction at which good growth ceases was found to be about Ph 2.8, while that at which even sparse growth ceases was found to be about Ph 2.0. Pigment formation seems to be slightly favored by the more acid medium. The organism will grow on all of the ordinary media, with good pigment production. Dextrose is fermented but slightly, even after 20 days growth. Acid is formed, but no alcohol could be detected by the method of distillation. No gas is formed. Lactose, saccarose, mannite and xylose are not fermented when used in extract bouillon. Growth in a suitable synthetic medium of about Ph 3.2 l occurs after five days as a dirty looking sediment. After standing for twenty-one days or more, however, heavy growth of coral pink sediment gradually appears, and, if left undisturbed, a thin pink scum forms, and a ring appears at the junction of the fluid surface and the vessel wall. The torula is an ae'robe. When an inoculated infusion agar slant was placed in a Brown (6) anaerobic jar, no growth occurred after ten days at room temperature. Upon removal of this slant from the jar, good growth occurred after forty-eight hours. A flask of the synthetic medium described above, which had been heated for some time in order to invert some of the sucrose, was also placed, after inoculation, in an anaerobic jar for one week. No growth occurred. Growth appeared quite promptly after removal of the flask from the jar. Morphologically, the organism is ellipsoidal, the cells being from 5 to 7 ju long by about 3 to 5 ^ wide. No spores have been observed. Typical budding occurs. A study of the pigment showed it to belong to the class of acid-alkali indicators. The growth on an agar slant was treated with nil acid. No change in color took place. On treatment of the same growth with n/i alkali however, the color turned to a brown-orange and the red color could be restored by further treat- ment with the acid. The change in color was not striking, but definite. 1 This medium is simply solution (A) of the agar described above, but containing 1,000 cc. of water instead of 500. '58 MARTIN FROBISHER, JR. The growth on a Buchner flask was suspended in 50 cc. of water and left in the incubator for four days. None of the color was imparted to the water. It was found that no pigment could be extracted from the intact cells with ether, alcohol, chloroform, glycerine, or water. Nor was any to be obtained with these solvents after grinding the dried cells with sand. Simultaneously with these cultural studies, attempts were being made to determine what had killed the flies in the original jars. Pure cultures were made of the red torulse and of the Penicil- lium-like mold in suitable flasks, and anaesthetized flies were introduced. The first three experiments are summarized in Table I. The red torula alone has since been used successfully to rear several vigorous batches of flies. TABLE I. FATE OF FLIES FED ON PURE CULTURES OF RED TORULSE AND Penicillium RESPECTIVELY. (Three experiments.) Organism No. Total Number of Flies Dead on Following Days: on which Exp. of Flies Were No. Flies Fed. Used. ISt 2d 3d 4th 5th 6th 7th 8th 9th ioth Red Torulae i 10 o o o o o i i i i i 2 IO o o o o o o 0 0 0 0 3 10 o o o 0 0 o 0 0 0 o Penicillium i IO o o I T, _1 8 IO IO 10 IO 2 IO o o o 5 6 6 9 9 9 9 3 10 o _1 6 7 _i 10 10 10 10 10 Ordinary Yeast (Control) . ... I IO o o o o o o o o o o 2 10 o o 0 o 0 o 0 0 o 0 3 10 0 0 0 o 0 0 o 0 o I 1 No observation. It has already been pointed out that the combined growth of torulac and mold results in the formation of a hard tough mass. This led to the idea that the mold might kill the flies by growing among the yeast cells in the alimentary passages and thus block elimination and engorgement. It was found that a combination of the mold, and of the commercial yeast upon which the flies RELATIONSHIP BETWEEN A RED TORULA AND A MOLD. 159 ordinarily engorge themselves with happy results, also produced a rather tough mass, but not nearly as hard and tough as the combination of the mold and the red toruhc. In cross sections of the gut of flies which had been allowed to feed first on torula and then on mold, and had then died, the gut appears somewhat distended with a matted mass of mold hyphac and torula cells. It seems improbable that the fly could have taken in through the proboscis such large filaments, and it is believed that they must have germinated and grown in the gut after the ingestion of spores, or smaller portions of filament. Sections show that the mold hyphse penetrate into the walls of the gut, either between the cells or into them. This suggests that the matted mass of torula cells may be firmly bound to the intestinal wall by these adhesions of mold hyphse. This would, it seems, add to the blocking effect. Further, the invasion of the intestinal wall might paralyze this organ so that elimination could not proceed normally on this account. A similar phenomenon has been described by Vincens (7) in the case of bees experimentally infected with a type of Aspergillus. That this may not be the only means by which the mold kills the flies, however, was shown by placing bacteriologically sterile flies on mold cultures. In preparing the flies for this purpose a number of young pupae were disloged from their moorings in one of the stock fly culture bottles and soaked for one minute in an aqueous solution of alcohol and mercuric chloride. They were then washed three times in sterile distilled water and placed, with a sterile loop, upon agar and kept three days at 37° C. and subsequently at about 22° C. After the adults emerged the vessels containing them and the agar were returned to the incubator for 48 hours and then removed. This was done to detect the presence of any bacteria which might have been inside the pupa cases and escaped the disinfectant. Most of the flies were found to be still contaminated, but some were not, and these were selected for use. The sterile flies were placed in a bottle containing a pure culture of the mold and in five days all were dead. This rapid death pointed to either an effect of the mold, or starvation, or death due to drought, or all three. i6o MARTIN FROBISHER, JR. In order to control the effects of starvation and drought on these flies, twenty-nine normal specimens were placed in a dry empty flask, ten in a flask containing nothing but a pledget of cotton wetted with distilled water, and thirty-four in a vessel containing sterile agar. The fate of these flies is shown in Table II. TABLE II. FATE OF FLIES UNDER CONDITIONS OF DROUGHT, STARVATION, ETC. Conditions Number1 Total Number of Flies Dead on Following Days: of Drought of Flies Starvation, at Etc. Start. ISt 2d 3d 4th 5th 6th 7th 8th 9th loth Dry, empty flask. . . . 29 1 29 29 29 29 29 29 29 29 29 Wet pledget of cotton 10 0 ' 6 10 10 10 10 10 10 10 IO Sterile agar 74 i I 3 2 9 0 17 17 17 17 Ordinary yeast cul- ture (control) 33 o o 0 O 0 0 0 0 0 O 1 Number varied because of activity of flies. 2 No observation. It appears that the flies die very rapidly as a result of drought and the absence of anything but water. The more prolonged survival of the flies on the sterile agar may be attributed either to an ability to metabolize this or its impurities or, more likely, to an automatic inoculation of the agar with the yeasts on their feet and in their feces. It appears to be shown by this experiment that the sterile flies survive in cultures of mold beyond the period within which it would be expected that they would die of drought or starvation and it therefore seems logical to assume that they eat the mold, but that the mold finally grows writhin them and kills them. Some of the dead flies were imbedded in paraffin within an hour after death and serial section made. Such sections show that the mold may actually invade the tissues of the fly. It should be noted that error due to variation in longevity of the flies is probably nil, since: i. The flies were all young (10-15 days old) progeny of standardized stock, originally furnished by Dr. Raymond Pearl, and deaths in these experiments always occurred well within the minimum of longevity. RELATIONSHIP BKTWKKN A RED TORULA AND A MOLD. l6l 2. The number of etherizations incident to these experiments never exceeded two in number nor more than 7 minutes in duration (8). 3. The conditions, under which the flies were reared and experimented upon, were practically ideal as to number in jars, ventilation, etc. (9, 10, u). SUMMARY AND CONCLUSIONS. 1. A red torula has been found as a contaminant of certain commercial yeast and a brief biological description given. 2. The combined growth of this torula and one of the group of Penicillium produces a bright lemon-yellow color. 3. Overgrowth of the torula by the mold transforms the creamy torula colony into a hard, tough mass, apparently by invading the colony and matting the cells together. The torula cells suffer by the contact. 4. The pigment of the torula has the properties of an acid- alkali indicator. Its sensitivity to these reagents is low. 5. The red torula may be used for raising batches of D. melanogaster. 6. A mold, probably closely related to Penicillium glaucum has been shown to have the power of killing D. melanogaster. The mechanism by which it injures the flies is shown to be possibly of two sorts. First, it appears to grow among the torula cells in the gut of the fly and by matting them together forms a more or less solid mass which may block both elimination and engorgement. By invasion of the intestinal wall, the mold hyphje are believed to bind this mass firmly in the intestine. Second, it is believed that the mold actually invades the tissues of the fly. BIBLIOGRAPHY. 1. Robaud, E., and Descazeau, J. '23 "Sur une agent bacterien pathogene pour les mouches communes. — Bad. delendcE-muscce, n. sp." Compt. Rend. Acad. Science, Paris. 177: 716. 2. Glaser, R. W. '24 "A Bacterial Disease of Adult House Flies." Am. Jour. Hyg., 4: 411, 3. Fresenius. '50 " Beitrage zur Morphologic," Frankfort, 63. 1 62 MARTIN FROBISHER, JR. 4. Fisher and Brebeck. '94 "Zur Morphologic, Biologic u. Systematik d. Kahmpilze, d. Manilla Candida, Hansen, u.d. Soorpilze." Jena, 1894. 5. Hunter, A. C. '20 "A Pink Yeast Causing Spoilage in Oysters." U. S. Dept. Agric- Bulletin No. 819, March 10, 1920. 6. Brown, J. H. '21 "An Improved Anaerobe Jar." Jour. Exp. Med., 36: 677. 7. Vincens, F. '23 Compt. Rend. Acad. Science, Paris, 177: 540. 8-1 1. Pearl, R. W., and Parker, S. '22 American Naturalist, 56: 174, 273, 312, 385. NUCLEAR REGENERATION IN STYLONYCHIA MYTILUS. DONNELL B. YOUNG, UNIVERSITY OF ARIZONA. In 1919 while working on the regeneration of lost parts in Uronychia which had been cut in various ways (cf. Young, Jour. Exp. Zool., Vol. 36, No. 3, Oct. 1922) it was discovered that one species, U. binudeata had two micronuclei. When this form was cut in such a way that the two micronuclei were in separate halves each part regenerated completely and sub- sequently divided. At that time, however, no stained prepara- tions were made to show the method of nuclear reorganization. In those species which had but a single micronucleus, U. setigera and U. transfuga, division never took place in the amicronucleate fragment although regeneration might do so. The question naturally arose, does the nuclear complex in those forms having two micronuclei regenerate at the same time that the cytoplasmic structures are being replaced and if so, how? Uronychia binudeata has not appeared in any of my cultures recently but as Stylonychia mytilus has two micronuclei so placed that a cut in the median transverse plane passes between them, they have been selected for this study. A wild mass culture was used and individuals selected from it at different times as needed. Several of the normal individuals were stained in order to record their nuclear complex. In every case two macro- and two micronuclei were found. Animals which have just completed division can usually be distinguished by their smaller size. In the following experiments individuals were selected which were of average size, indicating that they had not divided for four or five hours. As the formation of precocious cirri can be readily seen and as animals do not show any nuclear changes until such cirri do form, it is clear that individuals were not used which had already begun the process of division. The cutting was done free hand with a small sharp scalpel. The fragments were care- ts 164 DOXXELL B. YOUNG. fully studied under the microscope and any in which the plane of cutting was such that either part of the nuclear complex might have been injured were discarded. Seventeen animals were selected and cut in a median transverse plane, thus separating the two halves of the nuclear complex. The parts were kept under observation and the process of re- generation studied. In eight or ten hours each fragment had developed its missing structures. At the end of thirty-six hours most of the regenerated individuals had divided. Some of those which had already divided as well as some which had not yet started division were stained. In every case the nuclear com- plex was complete, that is, there were two macro- and two micronuclei. Ten individuals were next cut in the same plane and in sixteen hours the pieces were stained. Regeneration was complete in both nuclear and cytoplasmic structures. No division had taken place. There was no indication of the method of nuclear reorganization. Eight other individuals were stained about six hours after cutting. At this time the cytoplasmic regeneration was prac- tically complete. The body form was normal and the number and arrangement of cirri correct, although some of the cirri were not full size. In each animal studied, the micronucleus was in the process of mitotic division and the macronucleus had begun to separate into two parts. Eight individuals were stained three to five hours after cutting in the same plane as before. The cytoplasmic structures had begun to regenerate but the process was far from complete. The micronucleus showed various stages of division up to about a metaphase condition. The macronucleus in some cases had not begun to divide while in other cases it showed a slight con- striction. This study seems to indicate that in the race of Stylonychia mytilus used in this series of experiments the nuclear complex is constant and the whole complex is necessary for the normal life of the animal. Any cut which separates the two halves of the nuclear complex results in the regeneration of the normal complex by the amitotic division of the macronucleus and the NUCLEAR REGENERATION IN STYLONYCHIA MYTILUS. 165 mitotic division of the micronucleus. In every case studied, this process took place before the animal divided. LITERATURE CITED. Lewin, K. R. 'n The Behavior of the Infusorian Micronucleus in Regeneration. Roy. Soc. Proc., Ser. B, Vol. 84, p. 332. Young, D. B. '22 A Contribution to the Morphology and Physiology of the Genus Uronychia. Jour. Exp. Zool., Vol. 36, p. 353. THE CHEMICAL SENSITIVITY OF THE TARSI OF CERTAIN MUSCID FLIES. (Phormia regina MEIGEN, Phormia terrx-novx R.D. AND Lucilia sericata MEIGEN.) DWIGHT ELMER MINNICH. DEPARTMENT OF ANIMAL BIOLOGY, UNIVERSITY OF MINNESOTA. INTRODUCTION. Experiments previously published by the author (Minnich, '21, '220, J22b) have shown that the tarsi of certain butterflies possess sense organs which are exceedingly sensitive to contact chemical stimulation. These chemoreceptors are of such impor- tance that a more general knowledge of their occurrence and function in insects is highly desirable. The data presented in this paper are the results of experiments on three common muscid flies, Phormia regina Meigen, Phormia terrx-novx, R.D., and Lucilia sericata Meigen. For the identification of these flies I am indebted to Dr. J. M. Aldrich of the United States National Museum. The methods employed in these experiments are, in general, similar to those employed with butterflies, the results in both cases having been obtained by the use of proboscis exten- sion as an indication of stimulation. MATERIAL AND METHODS. The experiments were carried out in the spring and summer months. In the first experiments, therefore, the flies were indi- viduals which had hibernated, whereas in the later experiments they were individuals which had emerged the same season. All of the flies were captured, usually with a net, either from walls where they came to sun themselves in the early morning hours, or from waste cans to which they were attracted by the odor of decomposing organic matter. The flies were transferred in bottles to the laboratory where each animal was etherized, its species and sex determined, and 1 66 CHEMICAL SENSITIVITY OF TARSI OF CERTAIN FLIES. 1 67 its appendages carefully examined to determine their state of perfection. If all six legs were not perfect, the animal was usually discarded. If the legs were perfect, however, the wings were cut off close to the body with fine scissors, and the animal was mounted on a holder. The holder consisted of a straight piece of telephone wire, 19 to 20 cm. in length, and flattened out to form a small plate at one end. To this plate was attached a rectangular block of beeswax deeply notched in the middle, thus leaving two projecting tongues. By careful manipulation, it was possible to heat the tongues one at a time with a hot needle, and while the wax was still melted to attach the thorax of the fly to one and the abdomen to the other, the entire operation being com- pleted in about a minute. "Mounts" thus made are admirably adapted for experimental work and make possible the rapid handling of flies with the greatest ease (Fig. i). FIG. i. Side view of a mounted fly. b, block of beeswax notched at center; •w, wire flattened out at the end. Immediately after mounting, the legs of each animal were carefully rinsed with distilled water to remove any foreign matter adhering to them. Recovery from the anaesthesia usually re- sulted in a few minutes, but a minimum period of two hours was allowed from the time of anesthetization to the first trial. As far as could be determined without special experimentation, mounting and experimentation failed, in the great majority of cases at least, either to injure the animal or to shorten its life to any appreciable extent. The animals were usually kept under observation for at least 24 hours after the conclusion of the experiment before being discarded. In one experiment, however, involving eleven individuals belonging to the two species of Phormia, the animals were kept on after the experiment until 1 68 D\VIGHT ELMER MIXXICH. death, each animal being fed once daily with iM saccharose solution. When the solution failed to effect a proboscis extension through tarsal stimulation, it was found that a response could always be obtained by touching the oral lobes of the proboscis tip to the solution. It was, therefore, possible to force every animal to bring the extended proboscis in contact with the solution even though it drank little or none of it. Two of the eleven flies died within twenty-four hours. The remaining nine, however, lived many days. Exactly, their longevities were 9, 10, ii, 28, 30, 33, 35, 44 and 54 days. Six of the animals, three males and three females, thus lived a month or more, the greatest longevity extending to almost two months. It is doubtful whether these six animals would have lived any longer, if as long, in a state of nature. (Compare Glaser '23.). The use of proboscis extension as an index of tarsal stimulation entails a certain amount of care to avoid misinterpretation. Extensions of the proboscis are sometimes called forth by causes other than tarsal stimulation, e.g., cleaning operations, distance chemical stimulation not under the control of the experimenter, or hypersensitivity to mechanical stimulation induced by in- anition. In the last respect flies are like butterflies in that they sometimes become so sensitive during inanition that slight mechanical stimuli effect proboscis extensions, the proboscis even remaining permanently extended in some cases. Care was taken to avoid, in so far as possible, such sources of error, the animals being discarded if there was any regularly recurring question of interpretation. Occasionally, particularly with Lucilia, very slight movements of the oral lobes of the proboscis tip were noted which were apparently not coupled with any extensor movement. Such movements were counted as "no responses." Similarly, slight movements of the palp, noted in a few instances, were disregarded. The methods used in this paper are similar to those used in previous papers dealing with similar problems. Each trial lasted 30 seconds, at the end of which period "no response" was recorded if no extension of the proboscis had been observed. Minimum intervals of ten minutes were allowed between trials. Extensions of the proboscis judged as less than one half of com- CHEMICAL SENSITIVITY OF TARSI OF CERTAIN FLIES. 169 plete extension were weighted at .5, while extensions judged to be one half complete or more were weighted at I. As a matter of fact the great majority of responses were complete extensions. The figures which will be chiefly discussed are the percents of weighted responses, called the per cent, of response, computed by dividing the total weighted response obtained in a series of trials by the maximum weighted response possible in the trials. EXPERIMENTS. The questions to be answered by these experiments were two: 1. Do flies possess contact chemoreceptors in the tarsi similar to those present in butterflies? 2. If present, what are some of the substances discriminated by these sense organs? It is easy to demonstrate that mounted flies when placed with their tarsi in contact with absorbent cotton saturated with dis- tilled water or a iM saccharose solution will usually extend the proboscis. Such responses may conceivably be due chiefly or solely to: (i) tactile stimulation of the tarsi; (2) distance chem- ical stimulation of the antennae or other receptors by water vapor; (3) contact chemical stimulation of the tarsi. Experiments were accordingly devised to test out these possibilities. Two general types of experiments were devised, the first to test the ability of the tarsi to discriminate between water and paraffin oil; the second, to test the ability of the tarsi to dis- criminate between water and iM saccharose solution. Three sets of experiments of the first type were carried out, alike as to object and general procedure but differing slightly in apparatus and methods. Under the general conditions of these experiments it was found that the great majority of flies had become 100 per cent, responsive to distilled water by the time trials were begun, viz., two to four hours after anesthetization. A few individuals failed to become responsive in time to complete the experiment the same day, and still a few others failed to respond at all. Occasionally such animals were carried over to the second day and experimented upon, but, in general, they were discarded. The flies which did become responsive were given three trials with water, and alternating with these, three 12 I JO DWIGHT ELMER MIXXICH. trials with oil. Great care had to be exercised in trials with oil, not to allow the body to come in contact with the oil saturated cotton and further, to remove the oil from the legs after each trial with shreds of filter paper. Otherwise the oil spread over the body and quickly killed the animal. With the proper pre- cautions, however, flies could usually be given repeated trials with oil and yet be kept in good condition. FIG. 2. Apparatus used in testing the effect of tarsal contact with water. The apparatus consisted of a petri dish filled with water. Near the center are two rectangular pans: a, containing a cotton pad saturated with distilled water, and b, containing a cotton pad saturated with paraffin oil. The details of the first two sets of experiments were briefly as follows. To the bottom of a glass petri dish 14 cm. in diam- eter, two rectangular metal pans 2 cm. by 3.5 cm. by 1.5 cm. deep were fastened with wax. The petri dish was filled with distilled water nearly to the edge of the pans. In one pan (a, Fig. 2) was placed a smooth pack of absorbent cotton saturated with distilled water; in the other pan (b, Fig. 2) a similar pack, saturated with paraffin oil. The animals were tried in three positions: (i) with their feet in contact with the cotton of pan a, where they were subjected to the contact stimulus of water, and, the distance stimulus of water vapor; . (2) a similar position in CHEMICAL SENSITIVITY OF TARSI OF CERTAIN FLIES. pan b, where they were subjected to the contact stimulus of paraffin oil and the distance stimulus of water vapor; .(3) a position similar to 2, and far enough above pan b to prevent contact of the tarsi with the oil, in which position they were subjected to the distance stimulus of water vapor only. Posi- tions i and 2 differed only in the nature of the contact chemical stimulus afforded the tarsi, the tactile stimuli afforded by water and paraffin oil and the distance stimulus from water vapor being nearly identical in the two cases. Position 3 differed from i and 2 in lacking any contact tarsal stimulation, either tactile or chemical. TABLE I. SHOWING THE RESULTS OBTAINED WITH Two SPECIES OF Phormia UNDER THE FOLLOWING CONDITIONS OF STIMULATION: A, WATER VAPOR PLUS TARSAL CON- TACT WITH WATER; B, WATER VAPOR PLUS TARSAL CONTACT WITH PARAFFIN OIL. A. B. Number Water Vapor Water Vapor of Plus Tarsal Plus Tarsal Animals. Contact with Contact with Water. Paraffin Oil. 29 Phormia; l 6d", 23 9 Total number of trials. . . . 8? 87 Total number of responses 87 21 Total weight of responses . I 17-5 Per cent of response IOO 20 1 4cf and 19 9 , P. regina; id\ P. terras novas; lo71 and 49 belonging to these two species but not specifically identified. The first set of experiments was carried out on twenty-nine individuals belonging to the two species of Phormia. The results (Table I.) showed 100 per cent, response in position i, and but 20 per cent, response in position 2. The animals were not tried in position 3. In some of the trials in these experiments, however, a source of error was discovered. The cotton pad in the oil pan became accidentally contaminated with watei. In the second set of experiments, carried out on Lucilia sericata, care was taken to avoid the sources of error in the first group of experiments. In position I (Table II) Lucilia gave 95 per cent, response, in position 2, n per cent., and in position 3, 13 per cent. These animals, therefore, actually responded more to water vapor than they did to water vapor plus contact of the tarsi with oil. 172 DWIGHT ELMER MINNICH. TABLE II. SHOWING THE RESULTS OBTAINED WITH Lucilia sericata UNDER THE FOLLOWING CONDITIONS OF STIMULATION: A, WATER VAPOR PLUS TARSAL CONTACT WITH WATER; B, WATER VAPOR PLUS TARSAL CONTACT WITH PARAFFIN OIL; C, WATER VAPOR ONLY. Number of Animals. • A. Water Vapor Plus Tarsal Contact with Water. B. Water Vapor Plus Tarsal Contact with Paraffin Oil. C. Water Vapor Only. 14 Lucilia sericata; 6d", 89 Total number of trials 42 42 42 Total number of responses Total weight of re^pon^es 40 AO 5 A Z 8 C C Per cent, of re- sponse . . . oc 1 1 17 Because of the error involved in the first set of experiments with Phormia, another and third set was undertaken on these species. In these experiments the apparatus used in the first two sets was discarded for Syracuse watch glasses containing cotton pads soaked in water or oil. The animals were tested: (i) on water soaked cotton, where they were subjected to tarsal contact with water plus water vapor; (2) on oil soaked cotton, where they were subjected to tarsal contact with oil (no water vapor) ; (3) over the water soaked cotton but not in contact with it, where they were subjected to the stimulus of water vapor only. The results (Table III.) show 100 per cent, response in position 1 (column A, Table III.) and but 5 per cent, response in position 2 (column B}. After the trials in positions i and 2 were made, the animals were tested in position 3 where they gave 1.4 per cent, response (column C}. In order to show that the experi- mental procedure had not rendered the animals less responsive, each animal was finally tested in position I again immediately after each trial in position 3. The result (column D) was 100 per cent, response. The low per cent, obtained in position 3 could not, therefore, be attributed to any changed condition in the animals. The results of these experiments show two facts clearly. CHEMICAL SENSITIVITY OF TARSI OF CERTAIN FLIES. 173 TABLE III. SHOWING THE RESULTS OBTAINED FROM TWENTY-FOUR FEMALES OF Phormia regina UNDER THE FOLLOWING CONDITIONS OF STIMULATION: A, WATER VAPOR PLUS TARSAL CONTACT WITH WATER; B, TARSAL CONTACT WITH PARAFFIN OIL; C, WATER VAPOR ONLY; D, SAME AS B. THE LAST SET OF TRIALS WAS MADE AS A CHECK. A. B. C. D. Water Water Number of Vapor Plus Tarsal Contact Water Vapor Plus Animals. Tarsal Contact with Paraffin Vapor Only. Tarsal Contact with Oil. with Water. Water. 24 Phormia re- gina; 9 Total number of trials 12 72 662 83 Total number of i * / — responses 72 cl 10 58 Total weight of / * J 3 responses 72 o c Q t;8 Per cent, of re- y J1-* sponse IOO 1-4 IOO 1 Confined to 3 of the 24 animals. 2 Two of the animals were unfit for further experimentation after the first two sets of trials and had to be discarded, hence the number of animals here represented is only twenty-two. 3 Trials in which water vapor called forth a complete extension of the proboscis could not be followed immediately by another trial. This explains why there were only 58 instead of 66 trials. First, animals which gave about 100 per cent, response when their tarsi were brought in contact with water gave much less than 20 per cent, response when their tarsi were brought in contact with paraffin oil. Second, the response to water vapor alone was sufficient to account for at least part of the responses obtained with paraffin oil. In other words, in animals which were about 100 per cent, responsive to tarsal contact with dis- tilled water, the response to contact with paraffin oil was very small. The response to the oil was not zero, however, as the results of the third set of experiments show. Since the tactile stimuli of water and oil are nearly alike, the great difference in response can only be due to the difference in the contact chemical stimulation afforded the tarsi. The tarsi of these flies must, therefore, possess contact chemoreceptors. 174 DWIGHT ELMER MIXNICH. In the second type of experiment, distilled water and a iM saccharose solution were used as stimuli. In these experiments Syracuse watch glasses were used, each containing a layer of absorbent cotton, which was saturated with the solution to be tested. During the first phase of these experiments the flies were given neither water nor food. They were tested with distilled water until a complete extension of the proboscis was obtained in each of three successive trials. A few animals failed to meet this requirement and were discarded. The remainder were con- sidered as standardized animals, 100 per cent, responsive to distilled water, and were subjected to further experiment. Care was exercised in the first two trials with water to prevent the animals from drinking, but at the close of the third trial they were allowed to drink all the water they would. Beginning at this point, therefore, a period of water diet was initiated. Trials with distilled water were continued, each animal being allowed to drink each time it responded. On the average, how- ever, the animals ceased to respond after two administrations. \Yhen no response had been obtained in three successive trials the animals were considered to have become zero per cent, responsive to water, a standardization which proved to be very accurate. The animals on which this experiment was completed were, therefore, selected animals which had successfully met two preliminary tests: (i) They had, after several hours without access to water, become 100 per cent, responsive to water; (2) they had upon administration of water, become o per cent, responsive to this stimulus. Out of 27 flies which had met the first test only 5, 3 Phormias and 2 Lucilias, failed to meet the second test in the time covered by the experiment. For purpose of comparison, each time a "no response" to water was obtained, during the period of water diet, the animal was immediately tested with iM saccharose. Following such trials the legs were carefully rinsed in distilled water to prevent contamination in future trials. In the trial with \M saccharose following the third "no response" to water, each animal was allowed to drink all of this solution it would. Some individuals "drank" for only a few seconds while others remained with the proboscis extended for more than a minute. Thus, at this point CHEMICAL SENSITIVITY OF TARSI OF CERTAIN FLIES. 175 W Q 7. "2 a H t, o w CM O z 5 D a z o H D I O a; - 10 CM cs ro O 10 O O O ro 3 ^ CJ Q is H -1- •£ ty -t-i cj £ f'? ° W O\ O\ ro ^f ro ro ^^ cs w cs O\ "^ .2 w J ^ CD -O CM cj "o^ +j to 5 2 ro '> cd B ** 5 C tn K* 3 CS N O O O ^t O O O tM JO Period of Inanition. 2 ro '> cd W w M O H ^f *f ^1" O W CM CS O tc ec .% o cd — ^ ro "O II CJ fj3 -*-» < x T 3 a _£ O -^^ •*• •^ ^- _c ^ c ^ *J 'Z S co 3 Cj O ~ '-J O ej •*-* r- 5 § C SJ rv 4^ s o o C o *-* O cj ^ CJ — -_ — ^ >•• C C S O r" JD 3 . J3 « S cu *-i •£ cj o Si ^3 CJ C 3 " « E 8 o "• cu .ti a "S SJ o 5 § - _ >> — ' M •r J3 o s c o 'M*' t c >. cd ,0 « O. i r, to •^ c "5 ct° ^ t! iili^i cd ^: 3 .S to a o M .* £ 5 2 -s . a ,= o g ^ OJ y ^ fj ^3 O co 3 ~ g~ fe n C rt E-S O C *•- Q. .— O u ti •4J O CJ X _ t- ^ cj O -Q •b|2 .. S o 30^?.'^; — i- — P r ' -o • cw • ^ T5 O*" "cd O 00 E ~ 1'5-g § rt 2 o O en •«°^ Q, . U 1- Q £ "S O C> "O CJ o, -i — •* VJ ^ O 03 ^ O rf -^ ^ 5 -^ ES OF MEDIUM. 181 2. Experiments with Di-sodiurn Phosphate. Two parallel experiments were carried on with di-sodium phosphate as the initial treatment. In one set a N/iooo solution of Na2HPO4 was used for the 3O-minute initial dose, in the other set a N/7000 solution of the same salt was used. The latter strength was selected because it gives approximately the same percentage of sodium when added daily to the normal medium as that which caused a distinct increase in the rate of division of Paramecium (Packard) and of Uroleptus (Austin). Table II. gives the results of these experiments. TABLE II. THE EFFECTS OF AN INITIAL DOSE OF DI-SODIUM PHOSPHATE ON PROTOPLASM OF VARYING AGES. Av. No. Divisions Amt. of Variation per Line in Av. No. Divisions in Division Rate Series Age in lo-day Periods, per Line in per Line in No. Gen. Experimental Series. to-day Periods, lo-day Periods. Control Series. .ZV/iooo. N/IOOO. N/IQOO. N/TOOO. 1295 18 n.6 8.8 13.0 -i-4 -4.2 133 63 13-6 II. 0 + 2.6 133 74 II. O 13-8 -2.6 131 165 10.2 9-7 + -5 131 174 16.4 13-6 + 2.8 133 192 9-8 7.0 + 2.8 133 200 11.4 9.6 + 1.8 133 212 12. 0 13-2 12. 0 + O.O + 1.2 131 280 6.2 7.2 7-8 — 1.6 - .6 131 295 ii. 8 9-4 10. 0 + 1.8 - .6 At this time series 129 B was the only young series available for experimentation. This series was in the i8th generation and after the treatment with both the stronger and weaker solution, the division rate was lowered 1.4 divisions per line in the one case and 4.4 divisions per line in the other case as compared with the control series. The other nine experiments with individuals that varied in age from the 63d to the 295th generation showed the varying results that are characteristic of mature protoplasm, the majority being stimulated by the treatment. A few were slightly depressed and there was but little difference in the effects of the two solutions. 1 82 LOUISE H. GREGORY. These results when considered alone are too few to allow any definite conclusions to be drawn, but when they are con- sidered in the light of the preceding experiments with potassium phosphate and previous ones with beef-flour they indicate the same variations in the response of the protoplasm at different ages. B. THE EFFECTS OF DAILY STIMULATION WITH DI-POTASSIUM AND DI-SODIUM PHOSPHATE. i . Experiments with Potassium Phosphate. Initial experiments were conducted with series 133 at a period of early maturity in the iO-}.th generation in order to determine the most favorable amount of KoHPCX to be added to the normal medium. Five series of five lines each were kept for ten days in a medium consisting of normal hay-flour mixture to which potassium phosphate in different densities was added. The results showed that the individuals kept in a medium to which had been added daily N/$ooo KoHPCh gave the highest division rate averaging 5 divisions per line more than that of the control series. This strength of solution therefore was used in all of the experiments with potassium phosphate. Table III. shows the results of these experiments. TABLE III. EFFECTS OF POTASSIUM PHOSPHATE ADDED Daily TO NORMAL MEDIUM. Ave. No. Ave. No. Amt. of Divisions Divisions Variation Series Age in per Line in per Line in in Division No. Gen. io-day Periods, io-day Periods, Rate per Line Experimental Control in io-day Series. Series. Periods. 131 B 20 14.6 16.0 -1.4 129 B 58 14-8 16.0 — 1.2 129 B 80 14.6 10.2 +4-4 133 104 20.6 15-6 + 5-o 133 122 18.8 16.6 + 2.2 133 192 14.8 7.0 + 7.8 133 212 14.6 12. 0 + 2.6 131 22O 15-8 15-6 + .2 I3i 280 II. 8 7-8 + 4.0 131 300 9.6 9.2 + -4 128 3OO 8.6 8.8 — .2 EFFECTS OF CHANGES OF MEDIUM. 183 Two young series 131 B and 129 B in the 2Oth and 58th generation respectively were transferred daily for ten days to the potassium-hay-flour medium. Both series as a result of the treatment showed a lowered vitality and a slower division rate than the control series. In the 8oth generation however, 129 B was definitely stimulated when again treated daily with the potassium-hay-flour medium, dividing 4.4 divisions per line more than the control series. Series 133 was treated in the same way in the iO4th, I22d, I92d, and 2i2th generation for ten days each and at each period the division rate was increased over that of the control. Series 131 was treated three times with the potassium-hay-flour medium. In the 22oth generation it was but slightly stimulated during the ten days in the medium. In the 28oth generation the division rate rose to 4 divisions more per line than the control series and finally in the 3OOth generation, the rate dropped to but .4 divisions per line more than the control series. This was a vigorous series throughout all of the experiments and probably the usual old age reaction was only beginning in the 3OOth generation. As has been clearly shown by Calkins (5) in his study of this species, the various series differ in the length of life and also in general vitality. Some series live a long life with a moderate division rate which diminishes gradually during old age, others have a high division rate, respond definitely to changes in the environment and then die out quickly. Series 133 seems to have been more like the latter type while series 131 always showed a more moderate division rate which was less easily changed by variations in the environment. The oldest series (128) in the 295th generation showed a slight depression in its vitality when treated with the potassium-hay- flour medium. These results seem again to indicate that potas- sium used as a constant addition to the normal medium, causes the same changes in vitality as when it is used as an initial dose; the youngest lines are depressed, the mature ones are stimulated while the old lines are only slightly effected if at all. The hydrogen ion concentration of the potassium-hay-flour medium used in all the above experiments was 7.2 which is that of the normal medium. When testing for the most favorable 1 84 LOUISE H. GREGORY. W c H . Id U H z w tt a b, U, ta O S o 3! H, z o s H w ft o ta S I > u. o z c P Bi z z o U z 0 ^-> U o o a: a <: u. c in H tb U. W V rt u o "^ 03 c -o tfl n ^ .2 ^J- d M C* CS !>£ Q x ' t M^2 M H ei 1 1 ++ 1 c o .2 M ki -w r- .2 •- td • _rt g ^ o CC ^t N O O "o u . o •a '-5 O "^ to w *"^ ^^ 11 + 11 | S isions in •§ s o •- SJ If o o o o oo \O ^O 1 rt* 51 O II S II rt Tj- Tt CO "I XI M HI M M • HH O C $~ Ui a TJ OJ •S c U X 8-1 "o • CO U CA5 oo' .2 (-L| fc o N O 00 O 00 '" rt rt II H £? "2 2" ""' 5| -o ex 6 w a Z — CO 1 fi c 5. C O O OO M O ""> M IO N M O\ 1-1 (N CN a 6 cqcq M M l-l H-( M EFFECTS OF CHANGES OF MEDIUM. 185 amount of KaHPC^ to be added to the normal medium, as men- tioned above, the pH was taken of the different solutions and found to vary from 7.8 in the medium plus N/iooo K2HPO4 to 7.1 in the medium plus N/6ooo K2HPO4, and the greatest division rate was found among the individuals living in the medium to which had been added a solution of N[$ooo K2HPO4 and which showed a pH of 7.2. The results of experiments in this connection are shown in Table IV. Two experiments were conducted with five series of different ages. The experiments were identical except for the fact that in one the potassium solution was made up with distilled water and added to the normal medium, which then had a pH of 6.8, while in the other experiment the potassium solution was made as usual with spring water and the resulting medium showed the normal pH of 7.2. In every experiment the distilled water series showed a lower division rate than that of the spring water series and also than that of the control series except in one instance. The youngest series 131 B in the 2Oth generation divided 3.8 divisions less per line than the control series and the oldest series 128 in the 295th generation was also depressed and divided 3. divisions less per line than the control. Series 133 in the I22d generation was the only series that was able to live in the unfavorable medium without showing depres- sion. Here the protoplasm was in the early stage of maturity and apparently could adjust itself more easily to the changed conditions. Undoubtedly an acid condition creates an unfavor- able environment in which the youngest and oldest individuals are unable to maintain their normal activity. The method of distillation of the water may be an additional factor in causing depression. However, when the solutions are made with spring water and have the same hydrogen ion concentration as has been the case in all the experiments with this one exception, some other explanation must be sought to explain the variation in the response of protoplasm of different ages. 2. Experiments with Di-sodium Phosphate. In all of the experiments in which Uroleptus mobilis was kept in a medium to which sodium phosphate was added, the amount 13 1 86 LOUISE H. GREGORY. of sodium used was that found by Packard to cause a definite increase in the division rate of Paramecium. The same amount was also used by Austin in her experiments with the life history of Uroleptus. This amount was approximately a N/jooo solu- tion of NasHPO4 which was added to the spring water and used in diluting the hay-flour medium. Eleven experiments were carried out for ten day periods with protoplasm of different ages and the results are shown in Table V. TABLE V. EFFECTS OF SODIUM PHOSPHATE ADDED Daily TO THE NORMAL HAY-FLOUR MEDIUM. Av. No. Divisions Av. No. Divisions Amt. of Variation Series Age in per Line in per Line in in Division Rate No. Gen. lo-day Periods, lo-day Periods, per Line in Experimental Series. Control Series. 10 Days. 129 B 18 16.6 13.0 +3-6 131 B 40 15-2 IO.O + 5-2 129 B 80 14.4 IO.2 +4-2 133 90 21.4 17.0 +4.4 133 144 16.4 7.6 +8.8 131 179 19-7 16.0 +3-7 133 192 13-6 7-0 +6.6 133 212 14.8 12. 0 + 2.8 131 245 22.8 10.8 + 2.0 131 280 IO-4 7-8 + 2.6 131 295 ii. 8 9.2 + 2.6 In every experiment regardless of the age of the series there was an increase in the division rate. There was however, a marked difference in the amount of increase according to the age of the protoplasm. The youngest series, 129 B in the i6th generation, was stimulated to divide 3.8 divisions more per line than the control series. In the 8oth generation the same series increased its division rate to 4.2 divisions more than the control series. Series 131 B in the 4Oth generation was 5.2 divisions per line ahead of its control series. Series 133 in the QOth generation exceeded the control series by 4.4 divisions per line, in the I44th generation by 7.4 divisions per line, in the ig2d generation by 6.6 divisions and in the 2i2th generation by 1.8 divisions per line. Series 131 was stimulated in the lygth generation and showed a division rate of 3.1 divisions ahead of its control series. EFFECTS OF CHANGES OF MEDIUM. 187 In the 245th generation the increase over the control fell to 2. divisions per line above the control and maintained this same variation when stimulated in the 3OOth generation. Thus in every case the sodium phosphate when a part of the normal medium caused an increase in the vitality of the protoplasm but the amount of the increase varied, being greatest in the height of maturity and less in the period of youth and least in the commencement of old age. CONCLUSIONS AND SUMMARY. The foregoing experiments seem to indicate a variation in the response of Uroleptus mobilis to a single treatment with salts or to a constant addition of salts to the normal medium. Di- potassium and di-sodium phosphate when used as an initial dose and di-potassium phosphate when added daily to the normal medium cause a distinct depression of the vitality of the young series, in general a stimulation of the more mature individuals, and a slight depression as the protoplasm increases in age. These results agree in the main with those from the beef-flour treatment. If the hydrogen ion concentration of the medium falls much below that of the control, the depressing effects are more marked in both the young and old series and the stimulating effect on the immediately mature individuals is almost obliterated. The increase in acidity may not have been the only cause of the depression as Hartmann (6) found the method of distillation of the water used in his experiments to be an important factor in maintaining a normal culture of Eudorina elegans. When sodium phosphate is used as a constant stimulation there is a general increase in the division rate of all ages, but that exhibited by the young and ageing protoplasm is markedly less than that seen in the intermediate lines. The sodium must increase the permeability of the cells but if this were the whole explanation we should expect to have approximately the same increase in division rate for all the individuals. There seems to be a weaken- ing of the power of adjustment in both the young and old series, in the former possibly the derived organization has not yet become established and in the latter there is possibly an increasing stability or a loss in lability of the protoplasm with advancing age. I - 8 LOUISE H. GREGORY. The complete histories of several series showing their response to treatment at different periods in the life cycle would indicate clearly this variation in the activities of the protoplasm. Further experiments are planned to emphasize this point showing the instability of youthful protoplasm, the gradual increasing ability of maturing protoplasm to adjust itself to changes in its medium and finally the loss of this power as old age advances. These experiments with potassium and sodium phosphate using series of different ages as well as the same series at different periods in its life history, add further evidence to the theory of Calkins (5), that changes are taking place in the derived organiza- tion of the protoplasm of Uroleptus mobilis throughout the life cycle. These results can not be explained as ordinary variations in the division rate as the numbers of experiments are too many and the results too uniform; neither can they be due to varia- tions in the hydrogen ion concentration as that has been main- tained practically the same as that of the control save in the one experiment. Under certain conditions a low hydrogen ion con- centration may be a secondary cause of the variation in response, but the primary, underlying cause must be sought in the condition of the protoplasm itself, its organization changing as the series passes from the period of youth or immaturity, through one of maturity and finally into that of old age. BARNARD COLLEGE, COLUMBIA UNIVERSITY, NEW YORK CITY, April, 1926. REFERENCES. 1. Gregory, L. H. '25 BIOL. BULL., Vol. XLVIII., No. 3. 2. Woodruff, L. L. '05 Jour. Exp. Zool., Vol. 2, No. 4. 3. Packard, Charles. '26 Jour. Cancer Research, May. 4. Austin, Mary. Unpublished. 5. Calkins, Gary N. '26 Biology of the Protozoa. Lea and Febiger. 6. Hartmann, M. '21 Arch. f. Protistenkunde, Bd. 43. ON THE STERILIZATION OF DROSOPIIILA BY HIGH TEMPERATURE. WILLIAM C. YOUNG AND HAROLD H. PLOUGH, FROM THE BIOLOGICAL LABORATORY OF AMHERST COLLEGE, AMHERST. Breeding experiments with Drosophila melanogaster at high temperatures have demonstrated that at a point 5° C. to 9° C. above the normal optimum of 24° C. no offspring are produced after one generation. This fact was first noted by Northrop (i) for a single stock. Plough and Strauss (2) showed that the exact temperature at which complete sterilization occurred varied with different stocks, but was approximately constant for any particular one. In all cases a temperature was found which produced complete sterilization after an exposure of four days or more, even though adult flies continued to live for some time at this temperature, and newly laid eggs developed, pupated and went through metamorphosis normally enough. The additional fact was noted by all these workers that flies thus sterilized would usually recover normal fertility if they were replaced for a day or two at a temperature of 24 degrees. Northrop stated however: "If after they have emerged from the pupae they are left longer than ten days at the higher temperature the injury becomes permanent and they are no longer able to produce eggs capable of development at any temperature." Attempts to explain this sterility in terms of effects on the germ cells have not been entirely successful. Northrop stated, though without crucial evidence, that "Imagos raised and kept perma- nently at a temperature of 30 degrees, are unable to produce eggs capable of development at this temperature." Plough and Strauss demonstrated that this could not be the correct explana- tion by crossing both males and females hatched at 31 degrees to females and males respectively bred at 25 degrees, carrying the crosses at 31 degrees. Most of the 31 degree females mated to normal males gave offspring at 31 degrees, and about half of the 31 degree males likewise. The actual figures summarized from Plough and Strauss, Table II., are: 189 I9O WILLIAM C. YOUNG AND HAROLD H. PLOUGH. Fertile Cultures. Sterile Cultures. 31° 9 X 25° d" 13 2 25° 9 X 31° d" 6 5 In spite of the excess in the number of fertile cultures from females this result was taken to indicate that "neither the eggs nor sperm are injured by exposure to 31° C." Cytological exam- ination showed that apparently normal eggs and sperm were present in flies sterilized by heat, and copulation was observed between these flies at the high temperature. Plough and Strauss thus summed up the matter: 'The reason for the failure to produce offspring after the first generation seems to lie either in a failure of the sperm to reach the eggs, — that is unsuc- cessful copulation — or in the failure of the fertilization process itself." Further investigation of the question was delayed until the winter of 1924-5 when one of us — Young — was making a series of tests designed to determine whether strains of flies showing differing degrees of toleration of high temperature could be isolated from a single mass culture of a wild stock. The results of this series of tests will be reported elsewhere, but the experi- ments offered an excellent opportunity to settle the question raised above. An examination of the Plough and Strauss data quoted above shows only that eggs and sperm are not injured by exposure to 31 degrees in every case. Sterile cultures did occur, and a com- parison of the relative numbers suggests, in addition, that males are more likely to be sterilized than females. Since the 31 degree flies used were not actually tested and shown to be sterile at 31 degrees, but were taken from susceptible stocks (i.e., stocks which ordinarily failed to reproduce at 31 degrees), it is possible that some of the flies which gave offspring in crosses might have been fertile even if they had been mated inter se at 31 degrees. For these reasons we returned to the question of the possible injury to the germ cells of the flies by high temperature, and made more extended breeding tests, and more careful cytological examination of the gonads from flies sterile at 31 degrees. Most of the breeding tests were made by Young, while the remainder together with the cytological study were made by Plough. STERILIZATION OF DROSOPHILA. EXPERIMENTS. The Drosophila stocks used for the tests were for the most part taken from a wild stock culture collected at Canton, Ohio, in September 1924, but for certain of the experiments other stocks were used as indicated. They showed a varying degree of toleration of high temperature as recorded by Plough and Strauss, but even though the critical temperature was different for different stocks each could be rendered sterile at some particular temperature. Even when a culture showed sterility it was observed that copulation between. the flies takes place, and that eggs were laid in numbers roughly equal to those from similar flies at normal temperature. Since these eggs appear to be nor- mal and since virgin females are known to lay eggs under normal temperature conditions, the conclusion is again suggested that the sterility at high temperature is caused by some failure to function on the part of the males. Further data similar to those of Plough and Strauss were first collected. A series of lines of Canton stock were being run at 31 degrees. Since this temperature is close to the limit at which this stock can be bred, it was found that several strains usually showed sterility in each generation. There was a high degree of constancy in the reaction to temperature of these strains, some failing regularly after one generation, others running along for several generations normally. Matings were made at 31 degrees between both males and females from a number of these lines and flies of the opposite sex from the same strain carried continuously at 24 degrees. Normal flies of this stock are always fertile for at least one generation at 31 degrees. If a culture of the 31 degree stock showed sterility, then it might be supposed that the mating of sibs of this strain to the 24 degree stocks would indicate whether one sex rather than the other was responsible for the failure. The cultures in all cases contained five pairs of flies each, and a summary of the tests is given in Table I. A comparison of the totals in this table emphasizes the sug- gestion of the summaries of the similar tests of Plough and Strauss, namely that most of the males from a culture at 31 degrees are sterile, while most of the females are fertile. This 192 WILLIAM C. YOUNG AND HAROLD H. PLOUGH. TABLE I. TESTS RUN AT 31 DEGREES. Controls (Sibs of Tested Flies at 31°). cf1 Hatched at 31° X 9 at 24°. 9 from 31° X d" from 25°. Sterile. Fertile. Sterile. Fertile. Fertile — 16 8 ii 8 i o 6 16 5 Sterile — 12 Total Cultures 19 9 6 21 becomes still more significant when the figures are examined in comparison with the corresponding controls. Where these were fertile, all of the females were fertile, yet one half of the males were sterile. Apparently the males of a culture begin to be sterilized by heat, before there is any effect on the females whatever. When the controls were sterile, in only one out of twelve cultures did the males show fertility, while about half of the females are affected and half are still fertile. It would appear that high temperature causes at least partial sterilization of the males at a point where the females are largely unaffected. Eventually at least some of the females are sterilized. It was noticeable that in the strains showing a high degree of tolerance for 31 degrees, the males only showed sterility. In order to establish these facts beyond question it seemed wise to run a more extended series of matings to normal stock using cultures each of which was actually known to be sterile at high temperature. A series of cultures of flies of different strains was placed in the incubator at 32 degrees. This is sufficiently high to cause sterility in nearly all strains after the first generation, and in every one of the cases tabulated this was the result. Each fly was then mated to another of the opposite sex which had been hatched at the optimum temperature of 24 degrees, and all were replaced at 32 degrees. The results of these tests are given in Table II. The totals show that from cultures which gave no offspring at high temperature, 96 per cent, of the males were actually sterile, while only 50 per cent, of the females were affected. More detailed analysis makes plain the fact already demonstrated by STERILIZATION OF DROSOPIIILA. 193 TABLE II. FLIES STERILE AT 32°. REMATED AND CONTINUED AT 32°. The numbers given refer to individual flies. Stocks. d" from 32° X 9 from 25°. 9 from 32° X cf from 25°. Sterile. Fertile. Sterile. Fertile. Ci) Porto Rico 12 3 8 12 6 6 o o 2 o o o o o 8 6 6 6 16 3 4 3 0 o (2) Canton (3) Amherst (A) Prague (5) Brown (6) Spineless . . Totals 47 96% 2 04% 26 50% 26 50% %. Plough and Strauss, by Strauss (3), and by the unpublished work of Young mentioned above, that stocks show differences in their reactions to high temperature, some being much more tolerant than others. In the most tolerant stocks — Porto Rico and Canton — there is a clear differential effect such that the males are rendered completely sterile while the females are unaffected. In less tolerant stocks like the Prague and Amherst cultures the females show some effect, though it is not so great as in the case of the males. In mutant stocks like spineless and brown there is much less resistance and both sexes are rendered completely sterile. From these and other data there is evidence that at a temperature somewhat lower than 32 degrees even these stocks show the same differential sterilization of the males without any effect on the females. Turning now to experiments designed to test the length of time required for flies sterile at 31 degrees to recover fertility when returned to 24 degrees, we find this selective effect of high temperature on the male flies again clearly demonstrated. This recovery of fertility — first noted by Northrop as indicated above —was shown by removing cultures from the 31 degree incubator at intervals and placing them in new bottles at 24 degrees. Controls were furnished by cultures of normal flies bred con- tinuously at 24 degrees, and also by females from the sterile 31 degree cultures mated to normal males. In all cases the bottles 194 WILLIAM C. YOUNG AXD HAROLD H. PLOUGH. contained five pairs of flies each, so that individual differences are largely eliminated from the result. The time of the appear- ance of the first eggs and first larvae are noted in each case, and the data are tabulated in Table III. TABLE III. CANTON STOCK CULTURES STERILE AT 31°, REMOVED AND PLACED AT 24°. No. Average Average No. Days No. No. Days No. Days Cultures Description. at 31° Cul- before before Failed after tures. Eggs Larvse to Hatching. Appeared. Appeared. Recover. Flies bred at 24° continu- ously o I 7 I r 2 ^ Females from sterile cul- tures X males bred at 24°. . 10 7 T C -J o (9 only) Sterile 31° cultures 2 A T C ^ _ o 6 8 I c I (12^ %) t * * . * » 10 6 I C I (16.6%) (4 4 * H IT. IT I c o 5 (38.4%) For the cultures recorded in Table III. the date of the first adult flies was recorded also, but is not noted in the table since the average number of days between the appearance of larvae and the emergence of images was the same in all cases — about six days. The second line in the table affirms the result given in Tables I. and II., namely that females are not affected by a tem- perature of 31 degrees and their eggs develop as soon as fertilized. The remainder of the table therefore indicates the effect of the high temperature on the male flies. The females begin to lay eggs at once, but they do not lay eggs which develop until a period of seven to nine days has elapsed. The time when fertile eggs begin to be laid bears only a slight relation to the length of exposure to high temperature after hatching, but the last column showing the number of cultures which failed to recover, indicates that more males never regain fertility after a longer exposure. STERILIZATION OF DROSOPHILA. The experimental data apparently demonstrate that high temperature has a differential action on the germ cells of Droso- phila such that males are rendered completely sterile at a point at which females are uninjured. This effect is permanent as long as the temperature is maintained, but male flies will recover normal fertility if returned to the optimum temperature for a variable period. Longer exposures, however, tend to greatly increase the number of male flies which are permanently sterilized. Apparently raising the temperature still higher eventually sterilizes the females as well, and finally kills the flies. • CYTOLOGICAL OBSERVATIONS. Sections of testes and of ovaries from flies which failed to produce offspring at 31 degrees were made, and compared with similar sections from normal flies of the same age. In addition the spermathecae and ventral receptacles of females of the two sorts were examined both whole in Ringer's solution and in stained sections. Study of these preparations adds very little to the facts al- ready shown by experiment, though it confirms these. In none of the sections is it possible to discover any difference in the cytological picture between normal ovaries and ovaries of females from sterile cultures, except that the latter appear to be smaller, with fewer eggs at similar stages. Sections of the widened end of the testis just about at the point where it joins the vas deferens were studied in a sterile male and in a normal male respectively. The testes were taken from flies one group of which had been hatched and kept at 31 degrees for twelve days and the other at 24 degrees for the same time. The spermatozoa in the latter completely fill and distend the surrounding membrane. The same region in a sterile male is partially collapsed, and contains only a few scattered sperm. In other sections even these are absent. In portions of the testis slightly further up one can see a few cells which are probably spermatocytes together with some spermatozoa. At about the same place in testes from ster- ile flies only masses of spermatozoa appear. These seem to be shrunken away from the wall in masses and appear to be under- going degeneration for neither their size nor arrangement is nor- 196 WILLIAM C. YOUNG AND HAROLD H. PLOUGH. mal. In other sections from sterile males degeneration is also suggested for the sperms are decidedly reduced in number, have lost their orderly arrangement in masses in the testis, and seem shrunken in compact irregular aggregations. Apparently a few pass down into the vas deferens but these are probably not func- tional. Ventral receptacles of females from sterile and fertile cultures respectively were also examined. Actual dissection of such flies in Ringer's solution shows clearly that spermatozoa are present in the receptacles of normal flies, but they can not be found in the females from sterile cultures. The sections simply confirm these findings. Masses of sperm can be seen clearly to fill the lumen of the receptacles of flies from normal cultures while ducts of flies from sterile cultures are empty. These observa- tions prove that even though the male and female flies of sterile cultures copulate no transference of sperm to the receptacles of the females takes place. Beyond these facts cytological study reveals little. The testes of sterile flies are smaller on the average, and there is some suggestion of disorganization in the cells in the lower portion, but this is not constant. However it has been shown above that exposure of the adult males to 31 degrees for ten days or more produces sterility from which a certain number do not recover when they are placed at 24 degrees. If only the sperma- tozoa are affected, it is hard to see why this should happen. The data showing the recovery of the male flies suggests a pro- gressive sterilizing effect beginning with the sperms and even- tually reaching cells at earlier stages. Only when the latter occurred would the male fail to recover fertility when placed at 24 degrees. It is interesting that one pair of testes greatly reduced in size was found in a sterile male and sectioned separately. Both testes appear to have been but empty shells for neither spermato- zoa nor any clear germinal tissue whatever were present. This may have been a degenerative change produced by high tempera- ture, but such testes occasionally appear in normal stock. None did appear among the controls examined, however. The cytological observations therefore confirm the breeding STERILIZATION OF DROSOPHILA. 197 tests by showing that a temperature of 31 degrees sterilizes male flies, with little or no effect on the females. The sterility appears to be caused by some degenerative effect on the spermatozoa, such that all become non-motile, and eventually may be killed. Recovery apparently consists in the formation of spermatozoa anew from cells in the growth period or earlier. DISCUSSION. These facts are of especial interest when considered with others recently recorded. Cells of the male germ line are here shown to be much more sensitive than eggs or other tissues to a rise of 6 or 7 degrees in a cold-blooded animal, the fruit fly. C. R. Moore (4) has given similar evidence of the sensitiveness of the male germinal epithelium in warm blooded animals in his exten- sive studies on experimental cryptorchidism in mammals. He finds that when testes are exposed to the higher temperature of the peritoneal cavity rather than the scrotum there follows a rapid and progressive degeneration of the germinal cells. Such testes show progressive recovery when returned to their normal position. Bluhm (5) has given interesting evidence of the greater sensitivity of spermatozoa of mice to drugs (alcohol, caffein, etc.) since a portion of one class of spermatozoa, the female producing, become non-functional. Further the recent tests of Mavor and DeForest (6) on the sensitivity of eggs and sperm of Arbacia to X-rays show a much greater sterilizing effect on the sperms at all doses. Evidently the male germ cells are much more sus- ceptible than eggs or other cells of the animal body to external influences. SUMMARY. 1. High temperature has a differential effect on the germ cells of Drosophila such that males may be rendered completely ster- ile at a point at which females are still completely fertile. 2. This effect on the males is permanent as long as this tem- perature is maintained, but most males will recover normal fertility after being returned to the optimum temperature. 3. Exposures to 31° C. of over ten days tend to increase greatly the number of flies which are permanently sterilized. 4. Examination of the testes of sterile males shows that high 198 WILLIAM C. YOUNG AND HAROLD H. PLOUGH. temperature causes loss of motility of the sperm, with progressive aggregation and degeneration. Few or no sperms are found in the lower end of the testis and the vas deferens. 5. Although such males copulate with females at high tem- perature, no sperms pass into the ventral receptacles of the females. LITERATURE CITED. 1. Northrop, J. H. 'i9-'2o J. Gen. Physiol., II, 313. 2. Plough, H. H., and Strauss, M. B. '23-'24 J. Gen. Physiol., VI, 167. 3. Strauss, M. B. '25 Am. Nat., LIX., July-August. 4. Moore, C. R. '24-'25 Am. J. of Anat., 34, 269, 337. 5. Bluhm, A. '24 Arch, fur Rass. u. Gesellschafts-Biol., 16, i. 6. Mavor, J. W., and D. M. DeForest. '25 Proc. Exp. Biol. Med., XXII, 19. ON THE LUMINESCENCE OF MICROSCOLEX PHOSPHOREUS DUG.1 STANISLAW SKOWRON. FROM THE ZOOLOGICAL STATION, NAPLES, ITALY, AND THE BIOLOGICAL LABORATORY OF THE UNIVERSITY, CRACOW, POLAND. In spite of several papers describing observations on the luminescence of different species of earthworms, there are still some uncertain points which deserve further investigation. In Mangold's ('14) article, which discusses thoroughly the power of luminescence in all groups of plants and animals, the conclusions lead to the assumption that the production of light in these animals is due to infection by luminous microorganisms or fungi, which may be present in places where earthworms obtain their food. Dubois ('14) and Linsbauer ('17) were unable, however, to show in the luminous slime any luminous microorganisms, which is a strong argument against the view of the bacterial origin of luminescence in this group of the animal kingdom. Gilchrist ('19) working on the South African earthworm species Chilota, describes many observations and experiments which seem to show that the luminescence in this species is not bacterial. Quite recently, however, Pierantoni ('24), according to his "hereditary symbiosis theory," believes that Microscolex phos- phoreus owes its luminescence to symbiotic bacteria living inside the cells of its body. It is the purpose of this paper to show how the luminescence in Microscolex originates, and which opinion rests upon a sounder basis. OBSERVATIONS AND EXPERIMENTS. Microscolex phosphoreus- a small earthworm with clitellum situated near the anterior part of the body is easily to be found in Naples and its environs. If the animal be kept undisturbed, one cannot observe the production of luminous slime, which is 1 This work was done while holding the International Education Board Fellow- ship. " I wish to thank Mr. G. E. Hutchinson for his help in identifying the earthworms. 199 200 STAN I SLAW SKOWRON. discharged only on mechanical, chemical or electrical stimulation. It may be mentioned here that all the specimens of Microscolex phosphoreus examined by me were found to be luminescent. In free living animals the small hard particles of the soil may pro- duce, by irritating the skin, the necessary stimulus for the secretion of the slime, which glows brightly with a yellow- greenish tinge. The slime is usually ejaculated from the anal aperture, and only in some specimens kept previously for a longer time undisturbed, did the slime flow also from the mouth opening. The posterior and the anterior end of the alimentary canal were the only two points at which the production of luminous slime was observed. As all these observations were carried out under the binocular microscope it is quite certain that the glands of the skin and the excretory system do not take part in the secretion of luminous slime. If the animal be wounded, however, on any point of the body, the luminescent slime flows from this part, the clitellum not being an exception. Nevertheless the luminous material does not arise from the alimentary canal, because in such cases the luminescence appears when the wound has not reached the intestinum. An injury of the body cavity is sufficient in itself for a production of luminous slime, which does originate, therefore, from the coelomic fluid. This fact was corroborated by microscopical observations. A freshly discharged slime is composed chiefly of round cells with many, strongly light-refracting granules, scattered inside the protoplasm. Cells of this kind are found abundantly in the body cavity. Besides the uninjured cells, a certain amount of free granules, liberated by breaking of the cells, is always sus- pended in the fluid. I wish to call attention to the fact that both the size of those cells, and the structure of the intestinum, excludes the possibility of any other way of reaching the alimen- tary canal by the cells, except by preformed openings at its posterior and anterior end. That the communication with the body cavity is possible in Oligochxta was also shown by Gil- christ in Chilota, though in Microscolex the posterior rather than the anterior part of the intestine is the chief point at which ejaculation of the luminous slime occurs. Benham ('99) found that in the New Zealand earthworm, Octochcstus mitltiponis, the LUMINESCENCE OF MICROSCOLEX PHOSPHOREUS DOUG. 2OI cells of the luminous slime take their origin from the body cavity. The fluid is discharged, however, "from the dorsal pores and from the mouth, which it reaches through the pepto- nephriclia opening into the buccal cavity." These observations show clearly that it is most improbable that the granules alone (Pierantoni's bacteria) may pass through the connective tissue and two layers of muscular system until they reach the glands of the skin as Pierantoni seems to believe. The next task was to find out whether the cells or the granules alone have the power of luminosity. To test this the luminous extract, obtained by grinding up the whole animals, which easily passes through ordinary filter paper without loosing its lumi- nescence, was centrifuged for 15 min. The centrifuged fluid in which only granules were seen could be made luminous on addition of a few drops of ether. After filtration through bac- teriological filters, however, no light was observed upon treat- ment with ether. The granules therefore must be regarded as a luminous material in Microscolex. This agrees with Gilchrist's observations on Chilola. It was noticed that the power of the ejaculation of the slime is directly proportional to the degree of the irritability of the nervous system. Luminous microorganisms, however, glow con- tinually, and are not affected by nerve stimulation when living in luminous organs of some other forms (in certain fishes and cephalopods). A lowered temperature also affects the lumi- nescence, probably by the lessened irritability which is especially noticeable if the animals be kept for a long time at 8°-io° C. In connection with this I should like to mention the day-night rhythm of luminescence in Microscolex. It was observed that if stimulated, the animal discharged more easily the luminescent slime during the night than in the daytime. It can be shown that this rhythm does not result from an inhibition by light, because the strong arc lamp and sunlight have no visible effect on luminescence. It is possible that this rhythm may be ex- plained by the general lower irritability of the animals during the daytime. Before the characteristics of the luminescence will be discussed, it must be stated that if the properties of the luminous slime be 14 202 STANISLAW SKOWRON. compared with those of an extract from the ground worms, it is easy to find a very striking difference. The slime continues to glow for about half an hour if kept moist, while on the contrary the light of the extract dies quickly, and on addition of water, immediately. Filter paper saturated with slime gives off light after an hour and a half if moistened ; the dried extract, however, prepared from the ground worms, does not show any lumines- cence. It is difficult to explain these facts by oxydation of lumi- nous material alone, especially as the experiments of Gilchrist show that pure luminous slime of Chilota mixed with water glows brightly for over two hours. Acidity does not seem to play an important role in the above mentioned observations, as solutions with different pH did not prolong the luminescence. It seems more probable that this difference may be due to some enzymes liberated from the cells by grinding, which have an inhibiting effect upon the luminous material. If to luminous bacteria some chloroform or ether be added, the light becomes fainter and dies out quicklv. On the contrary the extract from certain luminous animals such as Pelagia gives a bright flash of light on addition of fresh or distilled water, ether, saponin, etc. (E. N. Harvey, A. R. Moore). The extract of Microscolex reacts like that of Pelagia. Of the cytolytic sub- stances tried — ether, chloroform, saponin, sodium glycocholate— only ether gives positive results. The light was however never as bright as in the living animal, but still visible. Owing to the rather small size of the animals it was difficult to experiment with pure luminous slime, but a few observations showed that in this case the action of the cytolytic substances were more clearly seen. Ether and chloroform produce the brightest luminescence of luminous slime of Microscolex and the same effect was observed by rubbing it with a finger. E. N. Harvey supposes that the action of cytolytic substances may cause the cyto- or granulolysis of luminous bodies. In Microscolex it was difficult to observe directly the changes in the granules themselves, but after a longer time the slime treated previously with ether shows swelling of the luminous granules. It may be remarked that NaE in 1.5 per cent, concentration has no inhibiting effect on luminescence of the slime or the extract LUMINESCENCE OF MICROSCOLEX PHOSl'HOREUS DOUG. 2O3 of Microscolex. The light of free living luminous bacteria, how- ever, ceases quickly in this solution and the same was observed by E. N. Harvey on the symbiontes multiplying in the luminous organs of the two fishes, Photoblepharon and Anomalops, and by me in Sepiola intermedia. The presence of oxygen is necessary for the luminescence of Microscolex. It is obvious that the behavior of the earthworms does not differ on this point from many luminous organisms such as Cypridina, firefly, bacteria and others. In an atmosphere of hydrogen the luminescence disappears quickly and does not return on mechanical or electrical stimulation. After removing the animals from hydrogen the luminescence appears again. The luminous slime kept on a slide or the filter paper impregnated with slime glows brightly upon moistening with water even after an hour and a half. After this time no light can be observed. Under the cover-glass however, if the edges be covered with vaseline, the light appears after many days in the presence of oxygen and water. This observation confirms fully the data collected by Gilchrist in Chilota and shows that the disappearance of luminescence points to the complete oxidation of the luminous material. If the ground animals be mixed with water to which some sodium hyposulphite has been added to remove the oxygen, and a few drops of methylene blue which serves as an indicator for the presence of oxygen, the light which appears on addition of ether is visible after a longer time, and is brighter than in the control tube without the sodium hyposulphite. The flash of light is observed at the moment when on shaking, the oxygen dissolves in sufficient quantity so as to change the colorless methylene blue to the colored compound. This is apparently- due to the nonoxidation of some of the granules, which were not completely destroyed by the action of enzymes or perhaps to the lessened activity of these enzymes due to the modified composition of the solution. The different tissues of Microscolex contain a sufficient quantity of oxygen to allow luminescence as proved by vital staining with methylene blue. Nevertheless the light is not produced con- tinuallv as in luminous bacteria, which fact must be taken as 2O4 STANISLAW SKOWROX. contradictory to the bacterial origin of luminescence in this animal. It is well known that luminous bacteria cease to glow at a lower temperature than the secretions of animals which have luminescence of their own. Furthermore one of the constant characteristics of the bacterial light is the reappearance of the luminescence after cooling and of the same intensity as before heating. The living Microscolex kept in fresh water does not give off any light upon addition of ether or alcohol if previously heated to 60° C. The same was noticed if the dried material or the filter paper impregnated with luminous slime were tested for luminescence by pouring on them water heated to different temperatures from 55°-6o° C. It may be mentioned here that after cooling the luminescence did not return in any case. These observations agree with the results obtained in other luminous animals whose self-luminescence is beyond any doubt, and differ widely from the behavior of luminous bacteria. Different results were obtained, however, with solutions prepared from the whole worms. In this case the maximum of temperature at which the light was observed reaches but 40° C. This difference may be explained by the quicker destruction of the luminous granules by the substances (enzymes) liberated from the rest of the body. Should this explanation prove true one cannot expect to find in the solution at a higher temperature unaltered luminous material which may react with the added ether. In the older literature one finds statements that the dried luminous slime of the earthwrorms can be made luminous again by application of water. The same was found in my observa- tions and this experiment can be repeated many times until complete oxidation of the luminous material. If the whole animals be dried in an evacuated desiccator the ground powder behaves like the fresh discharged slime, the intensity of lumi- nescence being about the same. Even after three months the dry powder glows brilliantly if in water, the luminescence lasting for some time relatively to the amount of the powder. The dried bacteria however can be made luminous by moisture but only for a shorter time after desiccation, and the light is much fainter than that of the living microorganisms. LUMINESCENCE OF MICROSCOLEX PHOSl'HORKUS DOUG. 2C>5 Pierantoni states that the addition of sodium chloride to the water in which the living specimens of Microscolex phosphor eus are kept inhibits their luminescence. In spite of many trials I have not been able to demonstrate the effect of different salt solutions when added to the powder from desiccated earthworms. The observations on Microscolex agree therefore with those of E. N. Harvey on Cypridina where the effect of different ions was not to be found. Pierantoni in his paper distinguishes two types of luminescence in Microscolex. Besides the external secretion of luminous slime he describes a general luminescence of the whole body, first visible at the posterior and anterior part, which takes place within the different tissues. This internal lumi- nescence may be produced by the strong stimulating action of ammonia or alcohol. It seems to me that in many cases the ejaculated slime, through the movements of the animal, spreads over the whole body and sticks to the skin, thus imitating an internal luminescence. By rubbing the worm over the filter paper or cloth all the slime can be removed but in water, however, the viscous slime does not dissolve easily. In ammonia of the proper strength the motionless animal begins to glow, the pos- terior part glowing first. After some time the luminosity spreads over the whole body even if the animal be apparently dying. The same effect can be obtained with alcohol solution. With ether or chloroform I noticed only the external luminescence, the movements of the worm being for a longer time very energetic. Ammonia arrests almost immediately the muscular action. It is therefore to be understood that only in a few cases the ejacu- lation of the slime on application of ammonia was observed. The luminosity of the dying animal may be compared with the steady death glow as noticed in Noctiluca (E. B. Harvey, '17) and other forms. This internal luminescence may be hastened if the worm be rubbed gently with the finger when in ammonia or alcohol. If the animals are in a very poor condition they can also be made luminous by rubbing them, even without ammonia or alcohol. Sometimes the worms already luminescent can, if slightly pressed, throw the luminous slime through the anal or oral aperture. After the ejaculation of slime the animal loses its luminosity, which indicates that the internal luminescence cannot 2O6 STANISLAW SKOWRON. be taken as a different type from the external one. Pierantoni's view, which explains the internal luminosity as the continual glowing of the symbiontes inside the cells of the different tissues of the body, seems to me, therefore, a very improbable hypothesis. DISCUSSION. Summing up all the characteristics of the luminescence of Microscolex phosphoreus (the role of the nervous system, the effect of ether, temperature, the luminosity of desiccated worms) we can see that they do not agree with the observations on the properties of bacterial light but point to a luminescence of its own in this species of Oligochxta. It is very improbable that the luminous symbiontes may change their type of luminescence so completely as to behave like such animals as Cypridina, Pholas, Noctiluca and others, especially as the luminous bacteria living in luminous organs in some fishes and Cephalopods do not show such differences, though their shape may be modified. Microscolex and Chilota are therefore at least two earthworm species which are certainly not infected with luminous bacteria. Allolobophora Jcetida (Vejdowsky) however may be occasionally infected by microorganisms or fungi, as only a few luminous specimens were found. As I have mentioned before, the luminous material in Micro- scolex consists of small granules which usually begin to glow after the slime has been discharged. 11 the animal be dying however, they become luminous within the body cavity. It may be thought that the luminescence of the granules depends on an amount of oxygen which is not present in sufficient quantity in ccelomic fluid. If the worm be dying the amount of oxygen may be increased, but the experiments carried out by E. N. Harvey did not show any difference in the rate of permeability for oxygen between living and dead cells. One may suppose however that the luminescence begins when the cells disintegrate, which is corroborated by the fact that on rubbing the brightness of the slime increases. The process of breaking up the cells with granules within was observed by me several times after the slime was discharged. On one occasion Ciilchrist noticed that the li.uht was given off from the glowing particles situated in cells, LUMINESCENCE OF MICROSCOLEX PHOSPHOREUS DOUG. 2(>7 which were in process of breaking up. I do not think that the granules can be luminous only during the time when they begin to be thrown off from the cells, for the centrifuged extract con- taining the free granules alone gives off light if ether be added, but it may be that they become luminous after they are liberated. It is possible that if the worm be dying the cells break up inside the body cavity. This process never occurs if the animal is in good condition. The accelerating effect of the rubbing may be explained by the increase of the process of breaking up the cells. In favor of such a view it may be noticed that the ejaculated slime which glows brightly always contains free granules in abundance, and their amount increases with time when the cells dissolve. It was impossible to prove this directly as the light of the granules is not strong enough to be seen under the micro- scope. I wish to express my sincerest thanks to Professor E. N. Harvey for many suggestions and criticism. My thanks are also due to Professor R. Dohrn, Director of the Zoological Station in Naples and his staff for many courtesies shown me during my stay there. SUMMARY. Microscolex phosphoreus is characterized by an external lumi- nescence (except the steady death glow) which begins upon stimulation. All the properties of its light seem to show that this species has a luminescence of its own. The luminous material is represented by small granules situated in the protoplasm of the cells, which take their origin from the body cavity. The luminescence begins probably after the granules are liberated from the cells. BIBLIOGRAPHY. Beddard, F. E. '99 A Note upon Phosphorescent Earthworms. Nature, Vol. LX. London. Benham, B. W. '99 Phosphorescent Earthworms. Nature, Vol. LX. London. Dubois, R. '14 La lumiere et la vie. Paris. Friend, H. '93 Luminous Earthworms. Nature, Vol. XLVII. London. Gilchrist, J. D. F. '19 Luminosity and its Origin in a South African Earthworm (Chilota sp.?). Trans. Roy. Soc. South Afr., Vol. VII., part 3. 2O8 STAMSLAW SKOWRON. Harvey, E. N. '20 The Nature of Animal Light. Lippincott, Philadelphia. '22 The Production of Light by the Fishes Photoblepharon and Anomalops. Carneg. Inst. Wash. Pub. No. 312. '22 The Permeability of Cells for Oxygen and its Significance for the Theory of Stimulation. J. Gen. Physiol., Vol. V., No. 2. '22 Studies on Bioluminescence, XIV. J. Gen. Physiol., Vol. IV., No. 3. '24 Recent Advances in Bioluminescence. Physiol. Reviews, Vol. IV. '25 Studies on Bioluminescence, XVIII. J. Gen. Physiol., Vol. VII. '23 The Effect of Light on Luminous Bacteria. J. Gen. Physiol., Vol. VII. Harvey, E. B. '17 A Physiological Study of Specific Gravity and of Luminescence in Nocliluca, with Special Reference to Anesthesia. Carneg. Inst. Wash. Pub. No. 251. Heymans, C., and Moore, A. R. '24 Luminescence in Pelagia nocliluca. J. Gen. Physiol., Vol. VI. '25 Note on the Excitation and Inhibition of the Luminescence in Beroe. J. Gen. Physiol., Vol. VII. Linsbauer. '17 Selbstleuchtende Regenwurmer. Umschau. Mangold, E. '14 Die Produktion von Licht. Handb. Vergl. Physiol., Vol. III., T. 2. Moore, A. R. '24 Luminescence in Mnemiopsis. J. Gen. Physiol., Vol. VI. Mortara, S. '24 Sulla biofotogenesi e su alcuni batteri fotogeni. Riv. Biol., Vol. VI. Roma. Pierantoni, U. '24 La phosphorescenza e la simbiosi in Microscolex phosphoreus. Boll. Soc. Nat.. Vol. XXXVI. Napoli. Pratje, A. '23 Das Leuchten der Organismen. Ergb. Physiol., Vol. XXI. Skowron, S. '26 On the Luminescence of some Cephalopods. Riv. Biol. Roma (in press). Vejdowsky, F. '84 System und Morphologic der Oligochaeten. Praha. CHROMOSOMAL VESICLES AND THE STRUCTURE OF THE RESTING NUCLEUS IN PHASEOLUS. J. McA. KATER, ST. Louis UNIVERSITY SCHOOL OF MEDICINE. In 1885 Rabl formulated a theory which is essentially our modern theory of continuity of chromosomes. Since that time cytologists and geneticists have very thoroughly discussed this problem, with its bearing on our conception of the structure of the nucleus and the related problems of genetics. Unfortunately, even at the present time, a large part of the evidence for the individuality of chromosomes is purely inferential; consequently the subject still rests on a rather hypothetical basis. Only in one case have the chromosomal vesicles with clear outlines been traced through the resting nucleus and in this instance the condi- tions were such that the observations cannot be applied to other forms with any degree of safety (Richards, 1917). Other workers have identified chromophilic bands in the resting nucleus as chromosomes, but the outlines of the vesicles were not visible (Sharp, 1914; Wenrich, 1916). In addition to the above isolated cases a great deal of strong inferential evidence for the indi- viduality of chromosomes has been accumulated (Boveri, Conklin, etc.). While engaged in studying the cytological changes accompany- ing the ripening and germination of the seeds of the common bean, Phaseolus vulgaris, a number of observations were made which indicated that Phaseolus affords favorable material for studying the reconstruction of daughter nuclei and interpreting their resting structure. This material was even more promising because of the fact that the resting nucleus of Phaseolus is quite similar to that of some other plants. I wish to express my gratitude to Mr. G. T. Kline, artist, St. Louis University School of Medicine, for his skillful preparation of the plates, and to Professor Albert Kuntz for kindly reading the manuscript. 209 210 J. MCA. KATER. MATERIAL AND METHODS. The root tips of growing Phaseolus seedlings were used. Since a matter of considerable controversy was the subject of this work it was thought desirable to use a number of fixatives. Flemming's strong gave excellent results, though only the peripheral portion of the root could be used. Root tips treated with this fixative show three degrees of fixation. In the outer layers of cells the cytoplasm and nucleus appear quite normal, though the linin of the nucleus is not quite so accentuated as in Bouin's. The next few layers of cells appear to be poorly fixed, but are valuable for comparison with the outer cells. The innermost part of the root tip does not seem to have been penetrated at all. Bouin's proved to be very valuable since it slightly swells the chromatin and accentuates the linin. There wras no indication that the actual structure was hidden or changed, but rather that certain features of normal structure were made more evident. The cytoplasm was not as well fixed as in Flemming's strong. The tissue was fixed all the way through. Alcoholic Bouin's and a mixture of three parts Bouin's to one part one per cent, osmic acid gave only fair results, inferior in all respects to Flemming's and Bouin's. Zenker's, Carney's and corrosive-acetic-alcohol shrunk the cells to such an extent that they were of no value whatever. Very slow dehydration was found to be essential. Starting with thirty per cent, alcohol two to three days were consumed in the process of dehydration. The tips were embedded in hard paraffin and cut at 4 micra. The sections were stained in iron-alum-haematoxylin and lightly counterstained with eosin. OBSERVATIONS. Since the principal object of this work is the interpretation of the structure of the resting nucleus the logical starting point is the reconstruction of daughter nuclei. This will be followed by a description of the resting nucleus and the account will end with the prophasr. Reconstruction oj Daughter Nuclei. — One of the features prob- ably responsible for the value of Phaseolus for a study of this CHROMOSOMAL YI.MC1.KS IN H I ASKOLUS. 211 subject is the uniformly small size of the short rod-like chromo- somes. As is usually the case with chromosomes of this kind they migrate to the poles with their long axis parallel to the Ion- axis of the spindle and the advancing chn nii-^oines are so evenK placed on the spindle that an almost perfect ring is formed. This perfect order is maintained after the daughter chromosomes come in contact with each other at the poles and at the time when the apparent fusion occurs the chromosomal mass appears as a rectangle with its greatest dimension at right angles to the long axis of the spindle. At this time the outlines of the chrome- somes are not clear and we can recognize the individual chromo- somes only by the corrugation on the sides of the rectangle. The rounded ends of the chromosomes prevent their tips from coming in contact with each other and are responsible for the corrugations on the margins of the mass of chromosomes (Fig. i). Shortly after the appearance of globules of achromatic material in the chromosomes the notched edges of the chromatin mass disappear and the outline of the reconstructing nucleus becomes smooth. In rare instances the chromosomes are not perfectly parallel when they become contiguous writh each other at the poles. In such cases the chromatin mass is not as regular as described above. Figures 2 and 3 make a very important point for this descrip- tion. Fig. 2 illustrates the beginning of vacuolization in one of the daughter nuclei, and in the other the vacuolization has pro- ceeded to such an extent that distinctly clear areas are appearing in the proximal l ends of the chromosomes. This figure also shows that the achromatic globules occur between the indentation on the proximal surface of the nucleus and that a linin strand runs inward from the indentation and joins that part of the chromatin mass which has not yet taken up achromatic material. A little later in the process of recon- struction the proximal side of the nucleus becomes less chromatic, the vacuolized portion of the chromosomes being more extensive. The indentations, especially on the proximal surface, are still quite pronounced and linin strands extending inward from them are also prominent. 1 Throughout this paper the ends of the chromosomes towards the equator oi the spindle will be termed "proximal," while the opposite end will be called "distal." 212 J. MCA. KATER. It is well to emphasize at this point an interpretation of the linin strands which, doubtless has already occurred to the reader. The fact that they invariably run inward from the indentations on the surface of the chromatin mass which represent the points of contiguity of adjacent chromosomes strongly indicates that they as well as the superficial indentations, represent the line of contiguity of adjacent chromosomes. This also supports the conception of chromosome structure held by many zoologists, notably Conklin, that the chromosome consists of a linin bag tilled with chromatin. Achromatic globules appear in the chro- matin groundwork of the interior of the chromosomes while the linin bag remains unaltered, except for the swelling due to increase of material within it. At this early stage of reconstruction there is no evidence whatever for the existence of linin strands within the external chromosomal sheath (Fig. 3). The process of vacuolization normally continues until about three fourths of the length of the chromosome contains a large part of achromatic material, the so-called granules of chromatin. occurring principally along the linin strands. The distal fourth of the chromosome is still a solid mass of chromatin and so it remains until the reconstruction of the nucleus is complete (Fig. 4). The imbibition of achromatin by the proximal ends of the chromosomes has not yet been completed. This con- tinues and causes the increase in size of the daughter nucleus up to the time when it ceases swelling and enters the interphase or the resting period. Since the distal fourth of the chromatin mass does not take up achromatin, it does not increase in size in harmony with the proximal portion of the chromosomes, and the latter tends to> swell out over it and, as it were, to engulf it. This causes the [inin strands, representing fines of contact of chromosomes, to- change their position, andr ceasing to be parallel to the long, axis of the cell, they become radiating strands with the non- vacuolized chromatin mass as their center. At the same time the solid distal ends of the chromosomes tend to clump. Fig. 4 illustrates a cell in which the daughter nuclei still have the non- vacuolized portion of every chromosome distinctly separated. From this point on they start joining so that nuclei may be CHROMOSOMAL VESICLES IN PHASEOLUS. 213 found with only four, three or two masses of chromatin within them (Figs. 5 and 6). Fig. 6 illustrates two of these bodies in the act of joining. As pointed out above the vacuolization of the proximal portion of the chromatin mass tends to turn the outer ends of the linin strands outward from the long axis of the cell, while the clumping of the solid ends of the chromosomes drags their other end towards a somewhat central point in the nucleus, resulting in the strands radiating from the chromatin mass, which might now be termed the nucleolus or karyosome, like the spokes of a wheel from the hub. The above gives only a two dimensional view of the process which, naturally, is not complete. The chromosomes migrate to the poles of the spindle covering it all the way around, conse- quently, when the proximal ends of the chromosomes swell and the distal ends join to form the nucleolus the linin strands radiate from the nucleolus, not like spokes from a hub, but like spines from the body of a bur. When all of the small masses of chromatin first join to form the nucleolus the latter body is irregularly shaped, but it rapidly assumes the spherical form typical of nucleoli of resting nuclei. Fig. 8 represents a dividing cell in which the nucleolus in one of the daughter nuclei is almost spherical while that in the other daughter nucleus is irregular. Inter phase and Resting Nucleus. — The term "interphase" is used to signify the interkinetic period in the rapidly dividing cells of the root tip while the nuclei farther up the root that are divid- ing slowly or not at all will be called "resting" nuclei. The interphase nucleus exhibits a relatively large nucleolus which may contain one or more achromatic globules, and relatively little scattered chromatin. The linin strands which, as shown above, represent the line of contiguity of adjacent chromosomes, are quite evident in such nuclei. The few disperse chromatin granules are found principally around the periphery of the nucleus and even there are not sufficiently abundant to obscure the linin strands (Fig. 10). During the dormant period of the bean seed the scattered chromatin either enters the nucleolus or becomes closely appressed to the nuclear membrane, as has been previously shown (Kater, MSS.). In such nuclei there is nothing 15 214 J- MCA. KATER. to obscure the linin strands, and, consequently, they are very generally visible (Fig. 12). One might ask why the number of linin strands seen in the telophase and in resting nuclei is so small in comparison with the number of chromosomes in Phaseolus. It is only necessary to consider again that the nucleus has three dimensions and that, when observing a complete nucleus many of the linin strands are seen in end view, and it is only the ones in a plane parallel to the slide that are seen as definite lines. In the resting nucleus there is a much greater abundance of scattered chromatin than in the interphase. Likewise there is a compensatory decrease in the size of the nucleolus and the scat- tered granules come up almost to the nucleolus, leaving a very small perinucleolar zone. When tissue is fixed in Benin's small sections of the linin strands can be made out, but after fixation in Flemming's strong the linin strands are rarely seen in such nuclei. The abundance of scattered chromatin obscures them (Fig. 1 1). There can be no doubt as to their presence because in the same area of a ripe embryo, when the scattered chromatin has disappeared, they are quite evident and are invariably visible in interphase nuclei. In properly fixed cells the nuclei have no resemblance whatever to the perfect reticulated network figured for plant nuclei by Gregoire (1904) and Sharp (1914). A somewhat similar structure is found in the second zone of Flemming's fixation, mentioned above. Slight indications of the chromatin bands which Sharp identifies as chromosomes may also be seen (Fig. 13). It seems probable that such slight distortion does not actually mislead the observer, but merely clumps the chromatin of each individual chromosome and enables the observer to trace structures which otherwise he could not recognize. Before concluding this description of the resting nucleus we should emphasize the existing relationship between the chromo- somes, as outlined in the description of the reconstruction of daughter nuclei. Every chromosome in the resting nucleus must be essentially a pentagon with four of the sides equal triangles while the fifth side has a convex surface and serves as a part of the nuclear membrane. The inner point of this wedge-like CHROMOSOMAL VKSICLES IN PIIASKOLUS. 215 structure, where the four triangles come together, consists of non- vacuolated chromatin and makes up a part of the nucleolus. The outer part is principally achromatic and, consequently, much larger. Thus, every chromosome contributes to the forma- tion of the nucleolus and 'likewise to the so-called nuclear reticu- lum. As stated above in distinguishing between interphase and rest, when the meristematic region moves forward leaving behind it cells that cease dividing we find an increase of scattered chro- matin and a decrease in the size of the nucleolus. Since every chromosome enters into the formation of the nucleolus, this giving up of material by the latter body affects all of them and inasmuch as the nucleolus remains practically spherical the chromosomes apparently are equally active. Prophase. — Before attempting to interpret the structure of the resting nucleus it is necessary that one study the development of that body in the telophase. It is equally necessary that one check these results by studying the behavior of the chromosomes during the prophase. The onset of mitosis is first indicated by an increase of size and of the basophilic character of the scattered chromatin granules (Fig. 14). The next evident change is the appearance of con- siderable quantities of chromatin on the linin strands (Figs. 15). A little later the nucleus becomes smaller and consists almost entirely of chromatin. The linin strands are now covered with heavy bands of chromatin and the intervening spaces are well filled with large chromatin granules (Fig. 16). The next change is important and introduces a very revolu- tionary fact. The nuclear membrane does not dissolve and dis- appear as has been universally described previously. Breaks appear in its wall, but each break leads into a short channel which terminates before reaching the general outline of the nucleolus, and this channel is bordered on either side by the end of a chromo- some, now fully formed except that one end is still imbedded in the nucleolus. Fig. 17 shows a cell in which one side of the nuclear membrane is still intact and one can see that it appears to be continuous with the chromosomes projecting from the nucleolus on the other side. The spherical outline of the nucleus entirely disappears in the 2l6 J. MCA. KATER. manner described above and there remains in the place of the old nucleus a mass of chromosomes with one end of each still im- bedded in the apparently homogeneous nucleolus and the other end projecting freely (Fig. 18). In order to return to the point at which this description started the chromosomes have only to separate. This is a gradual process. Cases may be observed in which only one or two chromosomes have become entirely separated from the rest of the mass (Fig. 19). From this condi- tion we go, chromosome by chromosome, to the point where all or nearly all of them are separate (Fig. 20). The chromosomes of Phaseolus are too small to be favorable for a study of the process of splitting and since that is not the subject of this work no careful attempt has been made to follow that phenomenon. HISTORICAL AND DISCUSSION. Reconstruction of Daughter Nuclei. — Phaseolus has been used by several investigators in studies of mitosis and nuclear struc- ture. Wager (1904) has given a very excellent account of mitosis in this plant and, in view of the fact that he did not properly interpret his observations, the accuracy with which he figured them is remarkable. Although he did not follow the nuclear changes in the telophase closely enough to show conclusively that the linin strands, which apparently connect the nucleolus with the nuclear membrane in the resting stage, are the linin sheaths of chromosomes and that the chromosomes retain their individuality through the resting period, he, nevertheless, gives several very suggestive figures. Figs. 29 and 30 in his account are practically the same as Figs. 4 and 6 in the present work. The principal object of his study was to discover the origin of the nucleolus and his conclusion that it arises by the joining of non-vacuolated ends of chromosomes has been clearly verified by the present observations. Wager observed and figured telophase conditions which, if they had been carried a little further, would have shown conclusively that the chromosomes of this plant are continuous from one mitosis to the next. He merits credit for being the first to make such clear observations of this phenomenon even though his CHROMOSOMAL VESICLES IN I'll ASEOLUS. 2IJ interpretations were entirely beside the mark and his paper did not attract widespread attention. This author figures the linin strands in resting nuclei as well as their development in the telophase. His observations on both of these phases have been violently attacked by Martins Mano (1905). Immediately after the appearance of Wager's article Martins Mano undertook to repeat his work on Phaseolus. The latter concluded that the nuclear reticulum arises by the "branching and anastamosing " of telophase chromosomes and that the nucleolus appears independently and rather suddenly after the reconstruction of the daughter nuclei is almost complete. His figures of the telophase, which led to the above incorrect interpretations, can be explained on the basis of Fig. 7. This figure illustrates a cell through which the plane of the section passed in such a way that a very small portion of one of the daughter nuclei appears in the section. The non-vacuolized portions of the chromosomes are entirely absent and, since only a small part of the side of the reconstituting nucleus is present the proximal ends of the chromosomal vesicles appear in cross section and consequently fail to show the even division of the nucleus by parallel linin strands. The other nucleus in this dividing cell exhibits the normal picture. Martins Mano admitted that structures similar to Wager's description were found, but claimed that the ones which he figures were of more frequent occurrence. In the light of the present work it can be safely said that Martins Mano must have exercised a great deal of patience in searching for the abnormal and infrequently occurring cells which he figured and with which he tried to support the theory that the recon- struction of nuclei is by the branching and anastamosing of telo- phase chromosomes, a theory which is incorrect in the case of Phaseolus. Martins Mano says that the linin strands which Wager figures as running from the nucleolus to the nuclear membrane do not exist, but are a part of the reticulum above the nucleolus. Their presence in the dormant seed, when the reticulum disappears shows that \Vager was correct. The great majority of described cases of chromosomal vesicles appearing during the reconstruction of nuclei are from animal eggs. Such structures have been described and discussed so 2l8 J. MCA. KATER. frequently that it is not worth while to devote space to them here (Boveri, 1909; Moenkhaus, 1904; Conklin, 1912; Richards, 1917). The vacuolization of plant chromosomes has not been so fre- quently described and is not so easily studied because the chromo- somes do not take up as much achromatic material as do the chromosomes of animal eggs. The early vacuolization of the chromosomes of Vicia was studied by Sharp (1914). This plant has large chromosomes and affords very favorable material for the botanical approach to the subject. The chromosomes of Phaseolus are small and not suited to a study of this kind. Their great value for the present work lies in the fact that when the chromosomes come in contact with each other at the poles of the spindle they are perfectly parallel with each other. This fact, together with their rounded ends makes it possible to identify the linin strands as the linin sheaths of chromosomes. Similar linin strands have been figured in the resting nuclei of other plants (Martins Mano, 1904; Bergs, 1906; Lutman, 1925). These investigators as well as Derschau (1904) have also described the telophase in a manner which indicates that the condition in those forms on which they were working is very similar to that of Phaseolus. Prophase. — In the only case in which chromosomal vesicles have been traced through the resting stage of the nucleus (Richards, 1917) the figures are so clear and convincing that one has no cause whatever to doubt the endogenous formation of chromosomes. The new chromosomes are formed in the pro- phase vesicles and are set free by the dissolution of the old walls. After reading this account of the formation of chromosomes in Fundulus it was very surprising to find that they are not formed in the same way in Phaseolus, but that the chromosomal vesicles merely lose their achromatic content, contracting and giving rise to the new chromosomes, the old linin sheath apparently being continuous from one division through the next. Wager concluded that the nuclear membrane of Phaseolus dissolves. However, his figures do not entirely justify that con- clusion and indicate that his material was identical with that used in the present study. The author knows of no account at CHROMOSOMAL VESICLES IN PHASEOLUS. 2K) the present time which would justify the conclusion that in any other plants or animals the prophase chromosomes are formed in the same way as in the bean. Martins Mano states that the nucleolus of Phaseolus does not contribute to the formation of chromosomes. The figures of the present paper as well as those of Wager are quite conclusive on this point and show that he was mistaken. The Nucleolus or Karyosome. — In the many descriptions and discussions of chromatin nucleoli the various authors have not taken account of the fact that the nucleolus may be made up of a non-alveolized portion of every chromosome, as in the case in Phaseolus. If the chromatin nucleolus is a store-house for chro- matin it is quite natural that it should be found at the meeting point of all the chromosomes, since the latter remain distinct throughout the resting phase of the nucleus. Continuity of Chromosomes. — The significance of chromosomal continuity for various problems of cytology and genetics has been completely discussed previously, even before Richards had, for the first time, traced chromosomal vesicles through the inter- kinetic nucleus (Moenkhaus, 1904; Conklin, 1902; Wenrich, 1916; Richards, 1917; Wilson, 1925). A repetition of this discussion would be superfluous. SUMMARY. 1. When the chromosomes of Phaseolus reach the poles of the spindle they come in contact with each other, all lying parallel to the long axis of the spindle. The individual chromosomes can be traced by the indentations on the surface of the chromatin mass. 2. Vacuolization of chromosomes begins in the end nearest the equator of the cell (the proximal end). 3. The distal ends of the chromosomes do not become alveo- lized, but join to form the chromatin nucleolus. There is no plasmasome present in the root-tip cells of Phaseolus. 4. The linin sheath of the chromosomes becomes visible in conjunction with the telophasic vacuolization and can be traced through the interphase and resting condition, thus showing that the chromosomes remain distinct from one mitosis to the next. 220 J. MCA. KATER. 5. The chromosome is considered as a linin bag filled with chromatin. Therefore, in the interkinetic nucleus the chromosomes are contiguous and the chromatin of adjacent chromosomes is discrete, 6. The chromosomes arise in the prophase by the chromosomal vesicles losing the achromatic globules within them and con- tracting to form the apparently homogeneous metaphase chromo- somes. BIBLIOGRAPHY. Berghs, J. '06 Le noyau et la cinese chez le Spirogyra. La Cellule 23. Boveri, Th. '09 Die Blastomerenkerne von Ascaris megalocephala und die Theorie der Chromosomenindividualitat. Arch. f. Zcllforsch. 3. Conklin, E. G. '01 The Individuality of the Germ Nuclei during the Cleavage of the Egg of Crepidula. BIOL. BULL., 2. '02 Karyokinesis and Cytokinesis. Jour. Phila. Acad. Nat. Sci., 12. '12 Nuclear and Cell Division in the Eggs of Crepidula. Jour. Phila. Acad. Nat. Sci., 15. Derschau, M. '04 Wanderung Nucleolarer Substanz wahrend der Karyokinesis und in lokal sich verdichenden Zellen. Ber. d. D. hot. Ges., 22. Gregoire and Wygaerts. '04 La reconstitution du noyau et la formation des chromosomes dans les cineses somatique. La Cellule, 21. Kater, J. McA. A Cytological Study of Dormancy in the Seed of Phaseolus vulgaris. MSS. Lutman, B. F. '25 Senescence and Rejuvenescence in the Cells of the Potato. Bulletin 252 Vt. Agric. Exp. Station. Martins, Mano Th. '05 Nucleole et chromosomes dans le meristeme raidculaire de solannm tubero- sum et Phaseolus vulgaris. La Cellule, 22. Moenkhaus, Wm. J. '04 The Development of the Hybrids between Fundulus heteroclitus and Menidia notala with Especial Reference to the Behavior of the Maternal and Paternal Chromatin. Amer. Jour. Anat.. 3. Rabl, C. '85 Uber Zellteilung. Morph. Jahrb., 10. Richards, A. '17 The History of the Chromosomal Vesicles in Fundulus and the Theory of Genetic Continuity of Chromosomes. BIOL. BULL., 32. Rosenberg, O. '04 Uber die Individuality der Chromosomen in Pflanzenreich. Flora, 93. Sharp, L. W. '13 Somatic Chromosomes in Vicia. La Cellule, 29. "20 Somatic Chromosomes in Tradescantia. Amer. Jour. Bot., 7. CHROMOSOMAL VESICLES IN PHASEOLUS. 221 Wager, H. '04 The Nucleolus and NucleartDivision in the Root-Apex of Phaseolns. Ann. Bot., 18. Wenrich, D. H. '16 The Spermatogenesis of Phrynotettix magnus with 'Special Reference to Synapsis and the Individuality of the Chromosomes. Bull. Mus. Comp. Zool. Harvard, 60. Wilson, E. B. '25 The Cell. MacMillan Co., 1925. 222 J. MCA. KATER. EXPLANATION OF PLATES. All figures drawn from sections 4 micra thick with aid of Abbe camera lucida and Leitz 2 mm. oil immersion lens. Figs, i, 4, 7, 10, n, 13, and 15 to 22 from material fixed in Flemming's strong; the remaining figures from sections fixed in Bouin's. All sections stained with iron-alum-hjematoxylin and lightly counter- stained with eosin. With the exception of figures 21 and 22 all drawings represent structures in two dimensions. Magnification — 1900 X. PLATE i. FIG. i. The chromosomes have become contiguous at the poles of the spindle. Individual chromosomes can be traced by their rounded ends. FIG. 2. Vacuolization is beginning. Rounded ends still indicate limits of each chromosome. FIG. 3. Vacuolization continued. Disperse chromatin granules in chromo- somal vesicles. FIG. 4. Note non-vacuolized portion of each chromosome. Linin strands represent line of contiguity of different chromosomes. Chromatin granules in vesicles. FIG. 5. The linin borders of the chromosomes are still prominent and the non- vacuolized ends of the chromosomes are joining. FIG. 6. A continuation of the changes illustrated by Fig. 5. Note lower nucleus where the two last non-vacuolized portions of chromatin are joining to form chromatin nucleolus or karyosome. Disperse chromatin granules are located principally around the periphery of the nucleus. FIG. 7. Approximately the same stage of development as Fig. 6. The section was so cut that the nucleolus does not appear in one of the daughter nuclei. Note that the linin strands are not parallel in this one. FIG. 8. Completion of the joining of the non-vacuolized chromosome ends to form the nucleoli. Nucleolus approximately spherical in one daughter nucleus. Linin sheaths of chromosomes still prominent. FIG. 9. Polar view of reconstructing nucleus. This nucleus is at about the same stage of development as Fig. 5. The linin strands are not parallel as in side view. BIOLOGICAL BULLETIN, VOL. LI PLATE I P3H£ J. MCA. KATER. 224 J- MCA. KATER. PLATE n. FIG. 10. Interphase nucleus from the meristematic tissue at the tip of the root. Disperse chromatin granules around the periphery of the nucleus. Linin sheaths of chromosomes prominent. FIG. ii. Resting nucleus from slowly growing region of the root. Nucleolus smaller than in interphase and disperse chromatin so abundant that the linin strands are obscured. FIG. 12. Cell from the radicle of a dry mature bean embryo. Disperse chro- matin has disappeared. Linin sheaths of chromosomes are very prominent. FIG. 13. Resting cell illustrating poor fixation by Flemming's solution, except- ing a few peripheral layers of cells. FIG. 14. Early prophase. The disperse chromatin granules are becoming larger, fewer, and stain more darkly. FIG. 15. Prophase continued. The disperse granules are larger and a heavy band of chromatin is appearing along the linin strands. FIG. 16. A continuation of processes illustrated in Fig. 15. FIG. 17. The chromosomal vesicles on one side of the nucleus have lost all achromatic material and are contracting to form chromosomes. Note that the nuclear membrane of the other side is continuous with the nearest formed chromo- somes. FIGS. 18, 19 AND 20. Further steps in the contractions of chromosomal vesicles and separation of the chromosomes. FIGS. 21 AND 22. Drawings showing the chromosome elements in three dimen- sions. Compare 21 to 17 and 22 to 19. BIOLOGICAL BULLETIN, VOL. LI. PLATE II. J. MCA. KATER. Vol. LI October, 1926 No. 4 BIOLOGICAL BULLETIN GENETIC STUDIES IN POULTRY.1 I. THE SEX RATIO IN THE DOMESTIC FOWL. W. V. LAMBERT AND C. W. KNOX. INTRODUCTION. In comparison with mammals the sex 'ratio in birds has been studied by relatively few investigators. The first report on the sex ratio in birds appears to have been made by Darwin in 1871. He noted that the per cent, of males to females in 1,001 chickens raised by Stretch was 48.65. Field (1901) in 2,105 chickens reported a sex ratio of 44.63, Thomsen (1911) in 805 observations a sex ratio of 47.82, Pearl (1917) in 22,791 cases a percentage of 48.57 males, Crew and Huxley (1923) a sex ratio of 49.26 in a total of 753 chicks and embryos examined, Jull (1924) a sex ratio of 48.88 on 2,396 individuals sexed, and Mussehl (1924) a per- centage of 52.24 males from a total of 1,514 chicks upon which observations were made. In pigeons Cole and Kirkpatrick (1915) found that the per cent, of males to females was 51.06 on a total of 1,800 squabs and embryos examined. The above data represent the total ratios observed by the various investigators on living chicks and dead embryos. With respect to the prenatal sex ratio, however, the data are less extensive, although the sex of the chicken may be quite readily told macroscopically by the ninth day of incubation. Of those reporting on the prenatal sex ratio, Pearl (1917) has the most extensive data, and he gives a sex ratio of 48.3 from a total of 1,921 embryos dying between the tenth day of incubation and 1 Paper No. 12 from the Department of Genetics cooperating with Poultry Husbandry, Iowa State College, Ames, Iowa. The writers are indebted to Professor H. A. Bittenbender for permission to use the material for this study and to Doctor E. W. Lindstrom for valuable suggestions. IS 225 226 W. V. LAMBERT AND C. \V. KNOX. hatching. Jull (1924) found a sex ratio of 47.18 in embryos dying after the eleventh day of incubation taken as an average of the results of continuous hatches throughout a three-year period. Thomsen (1911) in a total of 805 embryos examined before hatching found a ratio of 47.82, while Crew and Huxley (1923) observed a prenatal sex ratio of 45.24 out of a total of 420 embryos examined. In the latter case, some of the parents of the embryos were subjected to treatment with a thyroid extract. MATERIAL AND METHODS. The material for the present study was obtained primarily from the FI and Fo generations and backcross chicks of crosses made between the Rhode Island Red and White Leghorn breeds, and also of FI data from crosses made reciprocally between the Black Langshan and White Plymouth Rock, the Black Langshan and Buff Orpington, and the White Plymouth Rock and Buff Orpington. In addition a number of observations were made on data from the White Leghorn breed. All eggs were candled on the sixth, twelfth, and eighteenth days of incubation, and embryos found dead on these respective days were classified as Di, D2 and D3. All embryos found dead between the eighteenth day of incubation and the day of hatching were listed as dead in the shell (DS). All chicks of the Dz and DS classes, and chicks dying before it was possible to deter- mine their sex from the external appearance, were dissected and the sex determined in that manner. The sex ratio on all living chicks was determined as soon as that was possible from their external appearance. In 1925 the total sex ratio from 121 hens between the hatching dates of March 4 and May 6 was obtained with the exception of losses due to straying, loss of bands, predatory enemies, and losses due to fire which destroyed a portion of the hatch of one week. Data are given showing the number of individuals upon which the sex was not determined, but there is no reason to believe that the sex ratio of these chicks would have deviated greatly from the ratios observed. In addition to the above, observations were made upon the live chicks and dead embryos of a number of hens during the years GENETIC STUDIES IN POULTRY. 227 1924 and 1925. These data are not continuous throughout the hatching season but represent only data secured at various inter- vals during the hatching season. The record on any one hen, however, is complete for the period in which the observations were made. The sex ratio as used herein expresses the percentage of males to females in the population. RESULTS. During the two years a grand total of 2,910 embryos and living chickens was examined. Of these 1,488 were males and 1,422 females or a sex ratio of 51.13. The results of the two years records are shown in Table I. TABLE I. THE TOTAL NUMBER OF MALES AND FEMALES OBSERVED AND THE SEX RATIO FOUND IN THE CHICKS HATCHING AND IN THE DEAD EMBRYOS. Year. Z>8. DS. Chicks Hatching. Totals. &3, DS and living chicks, is over fifty, it being 55.82 in the D3, 50.06 in the DS and 50.97 in the living chicks. The results in each of the two years are not in very close agreement although the same general trend exists in each case. From the 2,910 individuals shown in Table I. a complete sex ratio was secured on 2,266 chicks and embryos developing from the eggs produced by 121 hens during the season of 1925. Of all eggs used from this group of 121 hens and that developed beyond the twelfth day of incubation, barring those chicks lost due to the causes previously mentioned, a complete sex ratio was obtained between the hatching dates of March 4 and May 6. These data are shown in Table II. 228 W. V. LAMBERT AND C. W. KXOX. u C3 o b. ^ 'A 7. H H O £ H u a u O H H < a s = p c u ?p n ?. W o u Q a. o a s a O U u S * 55 £££ ff. « O f- 10 O »H H IO Totals. o 0 cs \O w 10 oj M N O M T^- CS N ro W HH Ov ro °.=d M Tj- "b 'b \O t"** TJ- O fO M o r^- (N 10 CS W CO N M O ro « M "b t^- OO O 00 W f^ 00 O W O\ 00 br £ ^o r^* ^f o co o IO is Hatchin o o HH l-< CS >H M ^ ro U •b ^" CO <*O O OO PO M ^ r^ ro M M W W M Tt°° 'b O Tf C* Tf O N Tf r- 10 O O\ * co Tf O\ r^ O 10 rj- 10 ^ T}* s, g 0 o M OO OO N 00 to • o i o o \o ^^ ^o o\ *o CN ro oo • "b 'b M OO IO ^O CO M N Tf M N ° 0 "? * . + c c c i 'i 4 C *- »- ) t t 1 f 1 t i 1 o M ^ W K^i T^ r™ 00 W t* ^j ^^ E rt *O C C rt oo c ^ rt o — • r- M M CN "u a — ~ ~ CO o t GENETIC STUDIES IN POULTRY. 229 • Of the 2,266 individuals shown in the above table 8.12 per cent, were embryos dead between the twelfth and eighteenth days of incubation, 22.11 per cent, between the eighteenth and twenty- first days, and 69.77 per cent, were chicks sexed after hatching. It is interesting to note in this connection that the most critical period of development is apparently between the eighteenth and twenty-first days of incubation. Of all eggs set and dying in the embryonic stage, 361 or 34.5 per cent, died before the twelfth day of incubation, 184 or 17.6 per cent, between the twelfth and eighteenth days, and 501 or 47.9 per cent, during the last three days of incubation. The sex ratio is listed each week for the three classes Ds, DS and chicks hatched respectively, together with the totals for each week and the total ratio of each class for the entire period with the corresponding ratio of males. These data in Table II. represent a total of 79.20 per cent, of all fertile eggs set during this time. Of the remaining 20.8 per cent, upon which the sex was not determined 12.62 per cent, or 361 died before the twelfth day of incubation and 8.18 per cent, or 234 were lost due to the causes listed. In the DZ class with the exception of the last two weeks in- terval the sex ratio is comparatively high, ranging from 52.83 to 64.70. However, no general trend is apparent during the con- secutive intervals as the highest sex ratio appears in the interval from March 4 to 1 1 and the next highest in the period of April 15 to 22. Of those embryos dying between the eighteenth and twenty- first days of incubation no general trend of the sex ratio is like- wise apparent. In general, however, the sex ratio is lower than among the D& class. Of the chicks sexed after hatching no particular trend is noted in the sex ratio from the beginning to the end of the hatching season. The per cent, of males of the chicks hatching varies from 46.37 to 54.00 with an average of 50.85. From the total of 2,266 chicks and embryos examined 1,170 or 51.63 per cent, are males and 1,096 or 48.37 per cent, females. Some of the data included in Table I. were not complete for the whole season. These were obtained in both 1924 and 1925 230 W. V. I.AMHKRT AND C. W. KXOX. and were tabulated without regard to the week in which they were obtained. On a few of the hens only data from the dead embryos were included, whereas, in others both data from dead embryos and chicks were included. In all cases, however, where the sex ratio of chicks was determined it represented a total hatch of those particular hens for that period. In other words it repre- sents the complete record of the hens for any one interval. These unclassified data appear in Table III. TABLE III. THE OBSERVED SEX RATIO IN SOME UNCLASSIFIED DATA OBTAINED DURING THE NORMAL HATCHING SEASONS OF 1924 AND 1925. Year. D3. DS. Chicks Hatching. Total. c?d" 9 9 Ro*c? rfc? 9 9 Rtfo* c?cf 9 9 RcTcf 0*0* 9 9 Rc?d* IO24 24 13 20 8 54-55 61.90 no 26 128 34 46.22 43-33 77 68 55 8 1 58-23 45-64 211 107 203 123 50-97 46.52 102=; . . Totals 37 28 56.92 136 162 45.64 145 136 51.60 318 326 49.38 The large number of chicks dead between the eighteenth and twenty-first days of incubation appearing in the 1924 data is due to the fact, as mentioned before, that in some cases only the dead embryos of certain hens were sexed. In this as in the preceding tables no very definite trend to the sex ratio is apparent. While there seems to be an excess of males in the DS column and an excess of females in the DS column the numbers are probably not large enough to indicate a selective mortality at the different stages of incubation. This would appear especially true since no similar condition appears for the more extensive data shown in Table II. In the chicks hatching listed in Table III. a high sex ratio occurred in 1924 and a low sex ratio in 1925. Hence it would appear that the variations noted in this table are probably due to the small numbers. From the methodological standpoint Pearl (1917) has indicated the advantage of basing the sex ratio on families of ten or more individuals. He has compared the mean sex ratio of families of various sizes and finds the following ratios in each : GENETIC STUDIES IN POULTRY. 231 Families of 10 and over 48.57 ± 0.28 Families of 4-9 inclusive 49-39 ± 0.84 Families of 1-3 inclusive 55-O7 ±2.11 Families of 4 and over 48.80 ± 0.33 Families all sizes 49-45 It becomes apparent from these figures that the smaller families are more likely to show extreme values of the sex ratio. In fact, according to Pearl, ratios above 80 and under 20 occur very rarely in families of ten or more. The ratio of males for different colonies in the present experi- ment together with the number of hens in each colony is shown in Table IV. TABLE IV. THE SEX RATIO FOR THE DATA SHOWN IN TABLE II. LISTED ACCORDING TO BREEDING AND COLONIES. Breeding. No. of Birds in Col- ony. Da. DS. Chicks Hatching. Total. Rc?o* -4-* qj -H q> -H q -H Ov -H s> 5 C/^ Q CN vO d O* Q Q M 2 a - >- t— 0 CN CN M M q\ H H QJ ^ d H CN M 0 « 'D ><_ 's 5fi o rt -H hH •u M 41 -H S o K — o '§ u >- VO M ro oo cs q CN 0 ^ N IO VO •o W X ro -O t— I o pj d d M M « t/5 rt -H -H -H 4i D W OJ 0 o o H 55 ^ o IO q M IS 1 OJ ro d q\ oo w » .. •^- i/-j 00 (_j ^ c 1 1 I 1 ffi [rf O > rt M M tH HH q ro rl ^ S! (/) O a w S J D ° Q H > u 5 Q z 2 a o CD I— I B< rt cu W *"* CO P H ^ V co M * THE AVERA ANTECEDEN Variables. 6 | CO iring hatchin; -!_J C CL) O CD O. C w S r^ i£ ^J •o « w ° BO o "> y M & 3 H H W < (0 & (IJ *rt 3 •M O •o O a C 5 W 2 a H H IS •3 •o 2 Q, bo O 4J CJ CLI O. H) .5 M CL* (d a; V •o .2 S bo rt "rt CLI U rt O IH 3 u u > O d S Id *5j *£ "^ OQ B ^ PQ U • 234 w- v- I-AMHKRT AND C. W. KNOX. different dates and the rate was determined by dividing the total number of eggs laid 1>\ the number of days between the date of first egg and the beginning of the hatching season. The correla- tion coefficient in this case was - - 0.048 ± o.i 1 1 . This correla- tion likewise is not significant and indicates, in this particular group of birds, that preceding production has not influenced the sex ratio. In a study of the sex ratio based on hatches throughout the entire year Jull (1924) found a correlation of - 0.704 ± 0.031 between the sex ratio and antecedent egg production. As the season advances the ratio of males decreases, and Jull concludes that the cause for this decrease is directly related to the ante- cedent egg production. The rate of egg production during the hatching season, like- wise, was found not to have influenced the sex ratio in this group of birds. The correlation coefficient between total pro- duction during the hatching season and the sex ratio was - 0.009 ± 0.108. This correlation, like the two preceding, is not significant being much smaller than its probable error. Since the correlation coefficients in all three cases were negative and smaller than their respective probable errors the conclusion is justified that neither mean egg weight, as measured for each bird nor the rate of production both preceding and during the hatching season has influenced the sex ratio. In other words there is no tendency for those birds laying the fastest or laying the heaviest eggs to produce more or less males than the birds producing the fewest or the lightest eggs. The correlations between these three variables together with their respective ranges, means, coefficients of variability, and standard deviations are shown in Table V. DISCUSSION. The data presented herein for the most part show a higher sex ratio than has been reported by other investigators. Mussehl (1924) reporting a sex ratio of 52.24 per cent, is the only one reporting a higher percentage of males. Cole and Kirkpatrick (1915) report approximately the same sex ratio in pigeons as is reported by the present writers, namely 51.06 per cent. All GENETIC STUDIES IN POULTRY. 235 other studies have indicated a ratio of less than fifty per cent, males, both for living chicks and dead embryos. No reason for this higher sex ratio is apparent. It is true that a number of the birds were lost due to various causes but the writers have no reason to believe that there was a selective mortality of the chicks that were lost. The proportion of males among the embryos dying between the twelfth day and eighteenth day of incubation is rather high, being 55.82. However, if reference is made to Table IV. it may be seen that it does not hold consistently true that the males exceed the females in the D3 class. In the DS class there is practically an equality of males and females, the ratio of males being 50.06. All previous investigators reporting on the prenatal sex ratio alone in birds have reported a sex ratio less than fifty per cent. They likewise have seemed agreed that a selective mortality of one sex or the other previous to hatching does not occur. The number of birds upon which the correlations have been calculated is small but the results found here are in general agreement with those of other investigators. More of the birds were not inc'uded in these studies because the mean egg weight varies for different breeds and in most cases the antecedent egg product on on the birds used in the various crosses was not known. SUMMARY. 1. The sex ratio for 2,910 chicks and embryos examined was 5I-I3- 2. The sex ratio for total living chicks was 50.97, for embryos dying between the eighteenth and twenty-first days of incubation 50.06, and for embryos dying between the twelfth and eighteenth days 55.82 per cent. 3. No tendency for an increase or a decrease in the sex ratio as the hatching season progressed was noted. 4. The sex ratio of hybrid birds is approximately the same as for that of the pure breeds observed. 5. No relationship between the sex ratio and the factors of mean individual egg weight, antecedent egg production, and actual egg production during the hatching season is apparent. 236 W. V. LAMBERT AND C. W. KXOX. The respective correlations found between these variables were - 0.060 ± 0.108; - 0.009 ± 0.108; and -- 0.048 ± o.i 1 1. LITERATURE CITED. Crew, F. A. E., and Huxley, J. S. '23 The Relation of Internal Secretion to Reproduction and Growth in the Domestic Fowl. I. Effect of Thyroid Feeding on Growth Rate, Feathering, and Egg Production. Vet. Jour., 79: 343-348. Cole, L. J., and Kirkpatrick, W. F. '15 Sex Ratios in Pigeons together with Observations on the Laying, Incuba- tion and Hatching of the Eggs. Rhode Island Agric. Exp. Sta. Bull., 162: 463-512. Darwin, Charles. '71 The Descent of Man. Vol. i, p. 296. D. Appleton and Co., New York. Field, G. W. '01 Experiments on Modifying the Normal Proportion of the Sexes in the Domestic Fowl. BIOL. BULL., 2: 360-361. Jull, M. A. '24 The Relation of Antecedent Egg Production to the Sex Ratio of the Domes- tic Fowl. Jour. Agr. Res., 28: 199-224. Jull, M. A., and Quinn, J. P. '25 The Shape and Weight of Eggs in Relation to the Sex of Chicks in the Domestic Fowl. Jour. Agr. Res., 29: 195-201. Mussehl, F. E. '24 Sex Ratios in Poultry. Poult. Sci., 3: 72-73. Pearl, R. '17 The Sex Ratio in the Domestic Fowl. Proc. Amer. Phil. Soc., 56: 416-436 Thomsen, E. 'n Die Differenzierung des Geschlechts und das Verhaltnis der Geschlechtei beim Hunchen. Arch. Entwick. Organismen 31: 512-530. LIFE CYCLE OF NASSULA ORNATA AND NASSULA ELEGANS: ARE THESE SPECIES VALID? EDNA McNALLY. I. DESCRIPTION. Nassula is a ciliated protozoan living in fresh water where there is an abundance of Oscillatoria and not too much sunlight. Laboratory cultures of Nassula for these experiments were maintained by placing them in ordinary square watch glasses with spring water and fresh Oscillatoria, and these in turn, placed in large covered glass vessels containing a small quantity of water. The cultures were kept in subdued light as direct sunlight was found to be fatal. All work on Nassula was done with pure lines started from one individual and maintained for nearly a year. The genus Nassula was first described by Eherenberg about 1830. His description includes the species Nassula ornata and Nassula elegans, the main difference between the two being color, shape, and size. N. ornata he describes as being ovate or cylindrical, dark green or violet in color and about 200-240 micra in length. N. elegans, he gives as varying in color from red to transparent, elongated, and about 160 micra in length (Eyferth, 1900). However there is great confusion in the classification of Nas- sula. Pritchard (61), describes N. ornata and N. viridis as the same species while Eyferth (oo), describes N. ornata and N. aurea as the same. The species N. elegans as described by Pritchard is exactly opposite to the description given by Cohn (53), for the same species. Not only the species are confused, but also the genus, as is shown by the following: "Stein hints it as probable that this species (N. aurea) and N. viridis, Chilodon aureas and Ch. ornatus are merely different stages of the same animal" (Pritchard, 61). The entire body of Nassula is covered with cilia appearing in rows, approximately longitudinally disposed, over its surface. 237 238 EDNA MCXALLY. The nuclear complex is peculiar in that it is not differentiated into micro- and macro-nuclei but is very similar to the nucleus of Amoeba proteus. This statement does not agree with Cohn (53), who says of N. elegans, "Nucleus elliptic, with nucleolus lodged at one end." And Pritchard (61), with N. elegans in mind, thinks the "nucleus is stoutly clavate, and terminated by a small oblong nucleolus at its narrower extremity." The nucleus does not occupy a fixed position but is moved around by the streaming of the protoplasm. The mouth opens into a pharyngeal apparatus composed of about twenty-five pharyngeal rods arranged in the form of a truncated cone, with the base anteriorly directed. This pharyn- geal apparatus is described by all of the above mentioned authors for all species with the exception of Cohn (53), who states that there is no such apparatus in N. elegans. About one third of the way posterior to the base of this basket-like cone appears to be a "band of refractive protoplasm," as described by Eherenberg (oo), for N. elegans. Nothing was said of this ring in N. ornata. However, I have been able to demonstrate this ring in prepared material of both species. Eherenberg probably overlooked it in living specimens of N. ornata because of its being obscured by the food vacuoles. This mechanism seems to serve as a support to the pharyngeal rods and also aids in drawing Oscillatoria filaments through the pharynx into the body of the animal. Food vacuoles are numerous and vary in number and color with the amount of food injested and the stage of digestion of this food. A freshly fed Nassula contains so many food vacuoles of such dark color that the other morphological details cannot be made out with certainty. Just after injestion of food the vacuoles are brown or dark green due to the color of the Oscilla- toria, but as digestion proceeds they are changed to a shade of purple, then pink and finally faint straw color. All these stages may be encountered at one time in the same individual. The contractile vacuole, of wrhich there is but one, is peculiar. It is stationary and is the point toward which the streaming of the protoplasm is directed. As metabolism ensues minute vacuoles of clear fluid are formed throughout the protoplasm. \Yith the streaming of the protoplasm these vacuoles are brought LIFE CYCLE OF NASSULA ORNATA AND N. ELEGANS. 239 into close contact with the point at which the contractile vacuole is formed. As the minute vacuoles come together they fuse to form a larger vacuole, which, when its maximum size is reached, is discharged. Thus there can always be seen a larger contractile vacuole with smaller vacuoles of various sizes surrounding it, while others are drifting through the cytoplasm toward it. At the point of excretion is situated a wedge-shaped pore connecting the contractile vacuole with the outside of the animal, and through which the waste material passes. Waste material is discharged at the rate of about eighty seconds. Nassula thrives only on Oscillatoria. Although I have ob- served Nassula containing a few desmids, I have never been able to maintain a culture using desmids as food. Nassula divides by transverse fission. The nuclear conditions during division have not yet been completely worked out but work is being done at the present time on this subject. Binary fission is found to occur more frequently in older cultures. 2. No WARRANT FOR Two SPECIES. The amount of food present plays a great part in the life history of Nassula. As long as there is an abundance of Oscil- latoria the animals, which were classified as N. ornata by Eheren- berg, live and multiply in the normal way and continue to present the color, size and contour of TV. ornata. But as the food supply becomes exhausted they undergo noticeable changes in these features. As stated above N. ornata is normally ovate or cylindrical, brown or dark green in color and about 200 micra long, but as the food supply diminishes the animal becomes very much smaller and is decidedly longer than it is wide. The color of the food vacuoles changes to a faint red. In this condi- tion it meets all descriptions of N. elegans. If, when this stage is reached, Oscillatoria is fed to the animals, they immediately attach themselves to the filaments to feed. Several hours later they will be seen to contain numerous food vacuoles of very dark color and their normal size is again reached, now meeting Eherenberg's description of N. ornata. Thus, by withholding food, N. ornata may be made to assume the characteristics of N. elegans. Age of the culture also, as over against lack of food, 240 EDNA MCXALLY. plays a small part in the converting of TV. ornata into the so-called N. elegans. Older cultures of N. ornata change a short while before those in which the water is fresher. This will be seen by referring to Table I. Hence we find that N. ornata and N. elegans are but metabolic phases of a single species based upon nutritional and toxic conditions. 3. ENCYSTMENT. As stated above, after food has been withheld from .V. ornata the animals develop the characteristics of N. elegans. If now, food is again added to these specimens a reversal will occur and the animals will again appear as N. ornata. This may be carried on indefinitely making alternate sequences of the forms so long as food is withheld and added at the proper time and in the proper sequences. However, if no food is added, the animals eventually become much lighter in color than even N. elegans, becoming almost transparent. Then encystment takes place. Thus it happens that they enter the cysts as TV. elegans, N. ornata has not been observed to encyst. Age of the culture aids in encystment but is not the basic cause as will be seen from the following specific experiment: Four cultures were started as follows: All the Nassulas being taken from the same culture which was a sub-culture of the original pure line. Culture No. I. — Water from a culture of Nassulas several weeks old was filtered and placed in a watch glass with approximately twenty-five TV. ornata. No Oscillatoria was present. Culture No. 2. — Fresh spring water, about twenty-five TV. ornata and no Oscillatoria. Culture No. j. — Water from the same vessel as No. I, about twenty-five TV. ornata and an abundance of Oscillatoria. Culture No. 4. — Fresh spring water, about twenty-five TV. ornata, and an abundance of Oscillatoria. All of the four cultures were kept in square watch glasses and these placed in a larger glass dish so that the external condi- tions were the same. The cysts are formed along the sides of the vessel or in the detritus on the bottom of the vessel. They are spheroidal in LIFE CYCLE OF NASSULA ORNATA AND N. ELEGANS. 241 W 16 ^""* ^^ y~v /— v ^-v CO CO CO CO CO -4-» 4-> -«-> *-* -4-> CO CO CO CO CO (J (J (J U (J O O O O O > I— < a a a a a « « a ca a a a a o o o o o o o o fe;!^^^^^fe; to 'In co 'w"co' -4-> -4-» •*-» -4-> •*-» CO CO CO CO CO U r* ,— % to to y CO >< >>H|B >. > > ^ f 1 H^ >** «J u 1> cu o O **" " J2 v_x^xv_xx_x ^ Co Co CO Co Co •*-» 5 a a a a a j? O ^ ^i ^i ^ ^ ^^^^^^^ I-H y N CO In P u H|N ^ *j "^ J2 ^"^^^ cu cu cu Co Co (o *-* -*-J -t-> 5 a a a ?> r- y S ao cu: DO y y y . OJ rt J2 J3 J! J3 J3 -"-> * Q 10 vO t- OO O W M 242 EDNA MCNALLY. shape, brown and the wall is rather thick, presenting a scalloped appearance. They are a great deal smaller than the normal animals and in a newly formed cyst a light spot, the nucleus can be seen. Excystment can be brought about by the addition of fresh water and Oscillatoria. After excystment the animal has the characteristics of N. elegans until it encounters food. A few hours after feeding it again presents the characteristics of N. ornata. 4. CONJUGATION. In old cultures low in food supply, in which because of their age and food scarcity, encystment might be expected, conjuga- tion has been encountered. Conjugation has only been observed under such conditions and always occurring after the animals have undergone the change from N. ornata to N. elegans. Thus, these adverse conditions of the culture seem to play the greatest role in conjugation. The animals approach each other and fuse end to end. The nuclear stages during conjugation have not yet been worked out, but observations of living specimens as well as prepared materials show that the nucleus is not broken down in the process. Calkins (26) states that in many of the protozoa which have been thought to possess a single nucleus, there are in reality both micro- and macro-nuclei. These two nuclei are so closely associated that the micro-nucleus is not visible until conjugation takes place. I am convinced that this is not the case in Nassula, for during conjugation of protozoa where there are both types of nuclei, the macro-nucleus breaks down and the micro-nucleus takes the active part. In Nassula, as stated above, there is no such process, the conspicuous nucleus, which according to Calkins statement would be the macro-nucleus, functioning as does a micro-nucleus during conjugation. Many thanks are due Dr. W. A. Kepner of this Laboratory, for his valuable suggestions and aid in carrying on this work, and Miss Margaret Haase for the drawing. SUMMARY. i. Nassula ornata and Nassula elegans represent not two species, but two metabolic stages of a single species. LIFE CYCLE OF NASSULA ORNATA AND N. ELEGANS. 243 2. The well nourished phase, known as Nassula ornata, does not encyst. Lack of food, first, converts Nassula ornata into Nassula elegans, and, in time, drives the forms as Nassula elegans into encystment. ^Sj-^inary fission occurs more frequently in the poorly fed forms (Nassula elegans) than in the well-nourished forms (Nassula ornata} . 4. There is but one nucleus in this ciliate, there being no differentiation of the nuclear complex into micro- and macro- nuclei. 5. Conjugation involving this single nucleus takes place in stocks that have aged. BIBLIOGRAPHY. 1. Calkins, G. N. '26 Biology of the Protozoa. 2. Cohn, R. S. '53 from Pritchard, A. "Infusoria," 1861. 3. Eyferth, B. 'oo Einfachste Lebensformen. 4. Pritchard, A. '61 "Infusoria." 244 EDNA MCNALLY. DESCRIPTION OF PLATE. Right aspect of Nassula in the intermediate phase, i.e., a Nassula elegans not yet sufficiently nourished to assume the larger, spheroidal contour of Nassula ornata. PH. pharynx: NU, nucleus; CV, contractile vacuole. X 250. BIOLOGICAL BULLETIN, VOL. II. PLATE I. NU- •-PH -cv EDNA MACNALLY. THE MORPHOLOGY OF THE COPULATORY STRUC- TURES IN SOME CASES OF GYNANDRO- MORPHISM IN LEPIDOPTERA. N. J. KUSNEZOV. In spite of many excellent investigations on the gynandro- morphism in Lepidoptera (Goldschmidt, 1914-1923; Cockayne, 1915, 1916; Meisenheimer, 1921; Morgan and Bridges, 1919; Poppelbaum, 1909, 1913; Gerould, 1925) and some ingenious speculations about its causes and origin, the perplexing phe- nomena of intersexuality and gynandromorphism are still far from being sufficiently elucidated. One may say that we now possess many facts, a good many hypotheses and visual schemes, but few experiments (Kosminsky, 1924) and a great lack of physiological interpretation, confined exclusively to the theory of sexual hormones. Moreover, the facts, so numerous in this order of insects, are described insufficiently and based mostly on the superficial in- spection of the external features, like the structure and pigment- ation of wings and the structure of antennse. The sexual organs have been examined in a few cases only. But the question of the correlation of these superficial, secondary sexual characters with the primary reproductive system is, of course, of very great interest. In my previous paper l on this subject I have had occasion to describe in full the genital or copulatory apparatus of seven gynandrous individuals (Pieris rapx L., Gonopteryx rhamni L., Dendrolimus pini L., Pygxra timon Hb. and three Porthetria dispar L.), halved or mosaic, and I came to the conclusion that "there is evident a considerable independence, in existence and evolution, of different structures of the sexual sphere in the Lepidoptera; this independence is most sharply defined between the gonads (primary) and the secondary (tertiary and so on) systems; but, 1 Kusnezov, N. J., "Contributions to the Morphology of the Genital Apparatus in Lepidoptera. Some Cases of Gynandromorphism," Revue Russe d'Entomologie, XVI., 1916, pp. 151-191. 245 246 N. J- KUSNEZOV. on the other hand, the existence of a connecting influence between the secondary (copulatory) and tertiary (pigmentation, markings, etc.) sexual characters is also indisputable (cases of gynandro- morphism)." The task of my present article r is to contribute further data regarding the dependencies and correlations between the copu- latory organs and external sexual characters in lepidopterous gynandromorphs. I have had for examination five gynandrous specimens, all typically halved, viz., of Argynnis paphia L., Gonopteryx rhamni L., Bupalus piniarius L., Malacosoma neustria L., and Lycsena argus L., preserved dry and pinned. I am indebted for this interesting material to the generosity of Messrs. A. P. Tshernyshev (Kaluga), A. I. Iljinskij (Kaluga), A. M. Djakonov (St. Petersburg) and V. J. Fridolin (St. Petersburg) who most kindly put it at my disposal. Argynnis paphia L. — Wings: typical male on the right, valesina-female on the left. Left labial palpus female in shape and color. (Legs broken off.) Hairs on the thorax and abdomen fulvous or male on the right, and greenish-gray or female on the left side of the body. No observable differences in the antennae. Captured at Kaluga. Copulatory Apparatus (Figs. 2 and 3).2 Nearly perfectly male. Valvae, with their harpae in the form of two hooked processes, as well as the processus superior and inferior and so-called crista obliqua, have nearly the same shape and size as in the normal male (Fig. i). The differences seen in preparations, like the incisions and emarginations on the valvae and the length of the processus inferior, or the size of the penis, are quite unimportant and lie within the limits of individual variability. The main abnormality in the copulatory apparatus of this specimen is confined to the tegumen, i.e., to the complex of the Qth and loth tergites fused together, and to the uncus, the dorsal appendage of the latter segment. The right side of the tegumen (Fig. 3, tg. 10 c?) is weaker and less developed in comparison with that of 1 Read at the December meeting of the Russian Entomological Society in 1925. 2 In the description of the genital armature I use the terminology elaborated by myself in my introduction to the study of Lepidoptera (Kusnezov, N. J., "Faune de la Russie. Lt-pidopteres. Introduction," Vol. I., 1915, published by the Russian Academy of Sciences). GYNANDROMORPHISM IN LKI'IDOl'TKRA. 247 the normal (Fig. i, teg.), and, in particular, its uncus is small, reduced and deprived of the processes which adorn its dorsal sac FIG. i. Copulatory apparatus of the normal male of A rgynnis paphia L. : teg., tegumen; unc., uncus; pr. sup., processus superior valvae of Petersen (cercina of Fruhstorfer) ; pr. inf., processus inferior valvae of Petersen; h., harpae (clinopus of Fruhstorfer) ; ft. inf., fultura inferior; pn., penis; sac., saccus; cr. obi., crista obliqua of Petersen; v. d., valva dextra. — Zeiss, obj. as, oc. I. surface and have the form of a cock's comb. Moreover, this uncoid is situated asymmetrically, being clearly displaced to the right side. In one word, this uncoid appears to be a right half cr Sctc FIG. 2. Copulatory apparatus of the gynandrous Argynnis paphia L. : p. an., papilla analis; unc., uncoid; pn., penis; v. d., valva dextra; v. s., valva sinistra; h., harpae; //. inf., fultura inferior; sac., saccus; cr. obi., crista obliqua; pr. inf., processus inferior; pr. sup., processus superior. — Zeiss, obj. as, oc. I. 248 X. J. KUSNEZOV. only of the normal uncus. On the left side of the tegumen an elongate hairy prominence is situated, which, without hesitation, should be identified as the left papilla analis of the female (Fig. 2, p. a.}. Lastly, on the pleural regions of the 8th and 9th abdomi- nal segments there are some deformities rather exactly repro- ducing the pleural portions of the female abdominal segments (Fig. 3, pi. 7 9 and pi. + st. 7 9). ..un c. FIG. 3. Copulatory apparatus of the gynandrous Argynnis paphia L., side view: tg. 8, 8th tergite; pi. 7 9 . ?th pleurite of the female; tg. 9, Qth tergite; tg. xocf, loth tergite of the male; p. a., papilla analis; tine., uncoid; v. s., valva sinistra; pi. + si. 7 ? , 7th pleurite and sternite of the female fused together. — Zeiss, obj. ac, oc. I. Thus, in this case, the structural and pigmental female charac- ters of the wings, labial palpi and hair clothing show a corre- spondence with the copulatory apparatus on the same side of the body. In other terms, the tertiary sexual characters (structure and pigmentation of the wings and palpi) are strongly divided and separated by the symmetry plane, while the secondary ones (copulatory structures) are to some extent independent of this plane. Further, the male genital structures are markedly dominant and the female ones repressed. Gonopteryx rhamni L. — Wings: female on the left, male on the right. (Antennae broken off.) Both labial palpi yellow. No differences in the legs. Captured in the region under the Kaluga government. Copulatory Apparatus (Fig. 4). — Valvae, penis, with its vallum (Fig. 4, v. p.}, as well as the saccus (Fig. 4, sac.) nearly normal. GYNANDROMORPHISM IN LEFIDOPTERA. 249 Tegumen and uncus absent, and replaced by a pair of rather normal double papillae anales of the female. Moreover, the bursa copulatrix, with its appendix (Fig. 4, b. c. and a. b.} and the lamina -v.s. Sin. and. FIG. 4. Copulatory apparatus of the gynandrous Gonopteryx rhamni L. : tg. 7. and tg. 8., 7th and 8th tergites; p. a., papillae anales; v. d. and v. s., valva dextra and sinistra; pn., penis; pi. 9., pleurite of the Qth segment; o. b., ostium bursae; sin. and., sinus androconialis; sac., sacQis; I. d., lamina dentata; b. c., bursa copulatrix; a. b., appendix bursae; d. b., ductus bursae; v. p., vallum penis. — Zeiss, obj. AA, oc. I. dentata (I. d.}, are well developed and provided with a ductus bursae which opens in the ostium bursae (o. b.) lying normally, between the 7th and 8th sternites, but immediately on the left of the invagination of the saccus. Below this latter an androconial pouch (Fig. 4, sin. and.) is situated, representing in miniature the much larger structure of this kind in the normal female. Thus, in this case we find in the structure of the copulatory apparatus no correspondence with the disposition of the wing pigments. The latter, male and female, are strongly divided and separated by the plane of bilateral symmetry, while the copu- latory appendages are divided rather by a frontal plane, the fe- male papillae lying above, and male penis, saccus and valvae below it. But at the same time the internal reproductive organs, the bursa copulatrix of the female with its appendages and, probably, the ductus ejaculatorius of the male exist side by side, with their openings in normal position. 250 X. J. KUSNEZOV. Malacosoma neustria L. — Wings: female on the left, male on the right. Antennae: right male, left female. Specimen captured in the region under the Kaluga government. l-.8. pn. -v.s. pr. S FIG. 5. Copulatory apparatus of the gynandrous Malacosoma neustria L., lateral view: tg. 7. and tg. 8., 7th and 8th tergites; pn., penis; pr. pi. 8., processus of the 8th pleurite; teg., tegumen; unc., uncus; v. s., valva sinistra; pr. sac. , processus sacci; st. 79?, ?th sternite of the female (probably); sac., saccus. — Zeiss, obj. AA, oc. I. Copulatory Apparatus (Figs. 5 and 6).— Almost normal male, except the hind margin of the yth abdominal sternite which bears slightly stronger indentations than those in the normal male. The female structures absent. FIG. 6. Copulatory apparatus of the gynandrous Malacosoma neustria L., ventral view; abbreviations the same as in Fig. 5. — Zeiss, obj. AA, oc. I. Thus, in this case, the pigmental and structural sexual differ- entiations of the wings and structural sexual features of the antennae, strongly divided by the plane of bilateral symmetry, do not correspond with the differentiation of the genital armature. GYNANDROMORPHISM IN LEPIDOPTERA. 251 Bupalus piniarius L. — Wings: left fore wing ill-developed, curled; left hind wing not developed and rudimentary; on the v.s FIG. 7. Copulatory apparatus of the gynandrous Bupalus piniarius L., right side view: c. pn., ccecum penis; a. p., apophyses posteriores; v. d . and v . s. , right and left valvoids; a. a., apophyses anteriores; sac.?, saccus (probably); d. b., ductus bursae; 1. p., lamella postvaginalis; o. b., ostium bursae; p. a., papillae anales; Ig. Q., 9th tergite; pn., penis. — Zeiss, obj. AA, oc. 2. right side the fore and hind wings normally developed. Color of all wings pale yellowish, more feminine than that of the yellowish forms of males; but, as far as appearances go, wings on both sides V.ei. P' o.'S. •V. 3. FIG. 8. Copulatory apparatus of the gynandrous Bupalus piniarius L., left side view: v. d. and v. s., right and left valvoids; c. pn., coecum penis; pn., penis; a. p., apophyses posteriores; tgm., tegumen; p. a., papillae anales; ssph., subscaphium; o. b., ostium bursae; d. b., ductus bursae; b. c., bursa copulatrix; a. a., apophyses anteriores. — Zeiss, obj. AA, oc. I. 252 X. J. KUSNEZOV. are uniform in color and markings. Antennae: left bipectinated, male; right filiform, female. Bred specimen, emerged at St. Petersburg January 27, 1901. Copulatory Apparatus (Figs. 7 and 8). — 8th and 9th segments female, with nearly normal apophyses anteriores and posteriores (Figs. 7 and 8, a. a. and a. p.}. Papillae anales well developed, symmetrical. Ostium bursae quite normal, strongly chitinized. Bursa copulatrix normal, but lamella antevaginalis absent, the postvaginalis in the form of a chitinous band (Fig. 7, /. p.}. On the other hand, there is a penis, with its ccecum (Fig. 7, c. pn.}, rather well developed and lying inside of the body cavity, but capable of being protruded through a very narrow orifice in the diaphragm. Uncus absent. Rudiments of tegumen in the form of a more strongly chitinized left portion of the 9th tergite (Fig. 8, tgm.}. A well-developed left portion of the subscaphium (Fig. 8, ssph.} lying below the left papilla analis. As to the valvae, they are both present, but deformed, lying within the body cavity and protruding only partially outside (Figs. 8 and 9, v. s. and v. d.}. Lastly, an oviform structure lying freely in the body cavity on the external surface of the right valvoid (Fig. 7, sac.?) may be regarded as a rudiment of the saccus. a a Si- 7. ' FIG. 9. Copulatory apparatus of the normal female of Lycana argus L.: Ig. 8. and tg. 9., 8th and pth tergites; a. p., apophyses posteriores; p. a., papillae anales; o. b., ostium bursae; d. b., ductus bursae; pr. d. b., processus ductus bursae; I. a., lamella antevaginalis; st. 7., ?th sternite; a. a., apophyses anteriores. — Zeiss, obj. AA. oc. I. GYNANDROMORPHISM IN LEPIDOPTERA. 253 Thus, in this case almost all male and female genital structures, except the male uncus, coexist, though the male ones are deformed ; they are disposed, with the exception of the rudimentary sub- scaphium, on both sides of the plane of bilateral symmetry. This case is similar to that of the G. rhamni L. in the respect that here also the paired female papillae and the paired male valvae are divided from one another by the frontal plane. Lycxna argus L. (xgon Schiff.).1 — Wings: male on the left and female on the right. Antennae uniform. Both fore legs with female tarsus. Copulatory apparatus (Fig. 10) predominantly female. Tergite and sternite of the 8th segment nearly normal except for the distal angles of the tergite being obtusely shortened. loth tergite FIG. 10. Copulatory apparatus of the gynandrous Lyccena argus L. : v., valvoid »' pr. v., processus valvae of the normal male; other abbreviations as in Fig. 9. — Zeiss, obj. AA, oc. I. split on its dorsal surface. Apophyses anteriores and posteriores, as well as papillae anales, normal. Membranous tube (Fig. 10, pr. d. b.) bearing on its tip the ostium bursae, developed, but about three times stouter than normal. A pair of lamellae antevaginales a little wider and shorter than normal ones. Ostium bursae present, its ductus indistinct. Bursa copulatrix bag-like, long, plicated, nearly normal. A deformed, strongly chitinized, plate lying in the body cavity between the 8th and Qth tergites may be considered as male valvoid, because on its distal extremity it 1 Species with claws on the fore tibiae. 254 N- J- KfSXEZOV. bears a process having three teeth quite similar to those on the tips of the normal valva (Fig. 10, pr. v.}. Under this valvoid there are some minute sclerites disseminated over the inter- segmental membrane of the tube of the ductus bursae. From their consistence and structure they also may be considered as crushed rudiments of the male valva. Thus, in this case we observe a predominance of female charac- ters in the form of both fore tarsi and almost all parts of the copulatory apparatus which are but slightly deformed. Male features, represented only by rudiments of valvae, are nevertheless situated on the same side of the plane of bilateral symmetry as the male wings. In all described gynandrous individuals, however similar in their external appearance which is that of common halved gynandro- morphs, quite different interrelations in the degree of development and disposition between the secondary and tertiary * sexual structures are observed. My present observations, as well as previous (1916), mentioned above, permit me, I think, to draw the following conclusions. The division of the tertiary sexual structures into two halves by the plane of bilateral symmetry may be associated : (1) With a division of the paired copulatory structures by this plane into two pretty normal halves, the unpaired organs, like penis, bursa and their ostia being developed nearly normally; the case of Pieris rapx L.; (2) With a more or less complete unisexual! ty of the genital structures: male, as in my cases of Malacosoma neustria L. (complete unisexuality) and Dendrolimus pini L., or female, as in Lycxna argus L. ; (3) With an intermediate position, where the structures of one sex are intermingled with those of the opposite sex on both sides of the body; these are the cases of Pygxra timon Hbn., Bupalus piniarius L., Gonopteryx rliamni L., one sex being rather more strongly developed than the other; (4) With a division of the paired copulatory organs in respect to a frontal plane of the body, with the presence of the unpaired 1 Under the term of secondary sexual structures is meant the copulatory organs or genital ia, under that of tertiary the locomotor apparatus, pigments, sense organs and their accessories. GYNANDROMORPHISM IN LEPIDOPTERA. 255 ones in a rather normal state; these are the cases of Bupalus piniarius L. and Gonopteryx rhamni L. No case of purely crossed gynandromorph, i.e., having the tertiary characters of a given sex situated precisely on the sexually opposite side of the body, has been, as far as I know, yet observed. It is doubtful whether attempts to elucidate these various combinations by referring to their connection with the geometrical planes of the organism will have any success. The plane of bilateral symmetry, as is true of each other plane of the body, is by no means a real physiological factor; and physiology has nothing to do with these abstract morphological concepts. Very possibly, in general, no architectonic rules are followed by the developing organism. That is why the chimsera hypotheses fail also totally in their attempts to explain the confusion observed in the distribution of the secondary genital rudiments in certain gynandromorphs. Far more probable is the explanation (Morgan 1922) of these irregularities by a simple anatomical shifting of the diversely determined body cells during the embryonic develop- ment. So is also the hypothesis of the precocious development of one sex tissue in comparison with the other, growing across the plane of bilateral symmetry and encroaching upon another half differently determined (Gerould, 1925; partly Goldschmidt). The cellular and blastomeric hypotheses (Boveri, 1888, 1902, 1915; Lang, 1912; Cockayne, 1915) fail also to explain the multitude of facts when the body planes are nearly never main- tained but are in different ways transgressed. Lastly, it is a rather discordant fact for the hypothesis of polyspermy (Morgan, I9°5> J9O9) that only a quite insignificant number of cases of the plurality of the same organ have ever been observed.1 The generally accepted hormonal theory of the chemical stimulation also meets great difficulties in the explanation of asymmetrical, halved, gynandromorphism connected with the body planes. My opinion is that the phenomena of the "irregular" spacial distribution of sexually determined parts of the body should be interpreted as cases of organic regulation (in the sense of Driesch) 1 Those described by Goldschmidt (1921) need further examination. A "super- numerary" right valva described by Gerould (1925), more than possibly, will prove to be but a deformed portion of the right valva. 17 256 N. J. KUSNEZOV. in which the "normal" conditions are altered in the gynandrous embryo by the interruption of the normal chemical stimulation of somatic cells which have already obtained their sexual determi- nation. It is a custom to unite all sexual structures, other than gonads, under a general term of "somatic" structures and to subordinate them to the hormones of the gonads. But (i) for insects this subordination is yet to be proved (Oudemans, 1898; Meisen- heimer, 1909; Kellogg, 1904; Kopec, 1908-1913); and (2) it is evident that the somatic sexual structures are to be classed among several rather independent groups, each group being influenced by its own regulating factor, or, perhaps, possessing its own determination (Abderhalden, 1911). The idea of a sexual determination preexisting in all somatic cells grows more and more acceptable. But, on the other hand, the division of the reproductive system into primary, secondary organs, and so on, can be admitted only theoretically. Physiologically speaking this system is a unity. Perhaps, we should not speak, therefore, of dependencies of one group upon another, but only of the degrees observed in the display of the whole system in its parts collectively or individually. And, it may be, the sexual chemical factors, though different in their manifestations, are in reality the same, differing only quantitatively. ZOOLOGICAL MUSEUM OF THE RUSSIAN ACADEMY OF SCIENCES, May 18, 1926. A NOTE ON THE OCCURRENCE AND HABITS OF A LUMINOUS SQUID (ABRALIA VERANYI) AT MADEIRA. S. STILLMAN BERRY. REDLANDS, CALIFORNIA. Originally made known in very incomplete fashion (vide Riippell, 1844) from individuals taken in the neighborhood of Messina, our knowledge of this extremely interesting species of squid is based on the information gleaned from the very limited number of specimens which have now and then fallen into the hands of students of the group, principally systematists, since that time. These specimens have been entirely Mediterranean in origin, the majority of them, like the type, from Messina, but a few from Nice, Toulon, and so on. Until quite lately it has happened that almost every captured specimen has found its way into the collections of one or another of the German museums. It has therefore somewhat curiously come about that whereas neither Gray three quarters of a century ago, nor Tryon, nor even Hoyle seem to have had any actual material of the species to work upon, Pfeffer's recourse to the Hamburg and other German collections, especially that at Leipzig, yielded him no less than fifteen specimens (Pfeffer, '12, p. 136). More recently some further specimens, likewise taken at Messina, have been the subject of a short but beautiful memoir by Dr. Silvia Mortara ('22) on the histology of the photophores. Practically the whole of our real knowledge regarding Abralia veranyi, from whatever aspect, is to be found in this paper, and in the monograph of Pfeffer, although there is some information to be had from an older paper by Steenstrup ('80) and another by Joubin ('95), where the luminous organs of some specimens from Nice which are probably referable to this species are described under the name Abralia Oweni. It is of interest to remember that of the seven described species of Abralia, A. veranyi as at present understood is the only 257 258 S. STILLMAX BERRY. occidental one. One species comes from the Red Sea. The remainder are all Indo-Pacific, two of them being Hawaiian. From the waters of the western hemisphere, even in what would seem entirely appropriate latitudes, the genus is as yet unknown. Thus from the entire Atlantic beyond the gates of the Mediter- ranean no member of the genus has been reported hitherto. Furthermore in regard to the behavior of the animal in life or concerning even so much as its appearance at the time of capture I have been able to discover no published information in as nearly an exhaustive search as has been possible to me. What we know or think we know of the natural history of this interesting animal is almost entirely inferential or presumptive. In view of the situation outlined it is therefore a matter of no small satisfaction to be able to report not alone a considerable and important extension of the known geographic range of this genus and species, but to record a few observations made by the captors of the specimens, which throw a certain light on the habits of the animal, are a welcome contribution after the nearly blank record of the past eighty years, and are themselves withal full of lively interest. For this my acknowledgment is due to the gentlemen to be indicated, as well as to others later mentioned in the course of this paper. When Professor T. D. A. Cockerell of the University of Colo- rado returned to the United States from his Madeiran trip in 1921, he brought with him various specimens of invertebrates, among them as by no means the least of the treasures, three small squids. These he generously turned over to me, with the word that they were given him by Senhor A. C. de Noronha of Funchal. The bottle containing them bore the following label in Sr. de Noronha's hand: "Luminous. Caught during the night near the shore rocks. Funchal. June 19, 1917." The three specimens proved to include a beautifully preserved male and two females of the rare Mediterranean enoploteuthid referred to in our preface, Abralia veranyi. Through the kindness of Dr. Silvia Mortara I have also been the fortunate recipient of one of her precious Messina specimens, captured in November, 1921. A direct comparison of the Funchal form with authentic Mediterranean material has therefore been made possible. A LUMINOUS SQUID. 259 The principal references covering this species in the literature are given below. Those who desire a more complete synonymy are referred to Pfeffer, '12. Abralia veranyi (Riippell, 1844). (Dates of more important references italicized.) 1844. Enoploteuthis Verany Riippell. — Giorn. Gab. Messina, 26, p. 3, f. 2. 1851. Enoploteuthis Veranyi Verany. — Ceph. medit., p. 83, pi. 30, f. b. 1879. Enoploteuthis Veranyi Tryon. — Man. Conch, (i), v. I, p. 173. pl- 76, f. 3*8-319 (after Verany). 1880. Abralia veranii and Veranyi Steenstrup. — Overs. K. D. Vid. Selsk. Forh. 1880, p. 92, no [22, 40], pl. 3, f. 2-6. 1886. Abralia veranyi Hoyle. — Ceph. Challenger Exp., p. 38, 217 (merely catalogued). 1895. Abralia Oweni Joubin. — Mem. Soc. Zool. France, v. 8, p. 220 [9], f. 6-1 1 (photogenic organs). 1899. Abralia Veranyi and Enoploteuthis Verany "Riippel" Ficalbi. — Monit. Zool. Ital., v. 10, p. 80-82, text f. 2 (after Riippell). 1900. Abralia armata (pars) Pfeffer. — Synops. cegops. Ceph., p. 167. 1900. Asteroteuthis veranyi Pfeffer. — Teuthol. Bemerk., p. 289. 1912. Asteroteuthis Veranyi Pfeffer. — Monogr. (Egops., p. 129, 785, 794, Pi- 16. IQI2. Abralia Veranyi Pfeffer. — id., p. 785, 794. 1922. Abralia veranyi Mortara. — R. Com. Talass. Ital., Mem. 95, p. 1-20, text f. 1-2, pl. (photogenic organs). A careful comparison of the Madeiran specimens with that from Messina and with the lengthy account given by Pfeffer has brought to light no points of difference thought to be in any respect essential from the taxonomic standpoint. As will be seen from the appended table of measurements, the dimensions of the Madeiran examples are as a rule well in excess of those of the individual from Messina, but this is very decidedly more true of the two females than of the single male and may possibly prove to be, in part at least, a secondary attribute of sex. The wider, 26O S. STILLMAN BERRY. more flaring mantle possessed by all three Madeiran examples is doubtless but an incident of the mechanics of their preservation. The same is doubtless true of the apparent great number of chromatophores resulting in the consequent darker color of these specimens. The greater conspicuousness of the chromatophores seems to carry a concomitant accentuation of the photophores so that at first sight they appear much more numerous than in the Messina specimen. The attempt to count the photophores in corresponding areas, however, has not led to proof of any actual critical difference in their number. The male indeed does show a few slight differences in the structure of its beautifully preserved hectocotylus, especially in the fact that the tip of the modified arm is so produced and attenuated beyond the curious fleshy folds which are a feature of the distal part of this arm in the present species, and regarding the exact function of which we are still quite in the dark except that there seems good reason to believe that they may serve in some fashion or other in the manipulation and fixation of the spermatophores. This male and one of the females show but three hooks on each tentacle club. The other female has three hooks and a possible remnant of a more minute proximal one on the left club, while the right club bears four hooks in agreement with both clubs of the Messina specimen. These seem but normal variations. I have noticed no record of any specimens with clubs bearing less than two nor more than four hooks. A certain degree of sexual dimorphism is, as already noted, indicated by the present specimens, but it is not conspicuous, being chiefly manifested by the somewhat smaller body and fins of the male. The differences noted appear both absolute and relative. The fins are almost equally wide in proportion to mantle length in both males and females, but in the former they are relatively somewhat shorter, giving an index of about 60 as against 66 in the females. The arms of the male on the other hand are somewhat longer in relation to the body than those of the female. As was discovered by Steenstrup ('80, p. no), the spermato- phores become attached in a rosette-like cluster to the inner A LUMINOUS SQUID. 261 surface of the mantle of the female, this being in the median line just back of the nuchal cartilage. These clusters are very conspicuous in both of the females before me, but there is present in each of them in addition to the principal rosette, a much smaller cluster of 4 to 8 spermatophores or remains of the same adhering to the visceral mass at its mesial junction with the collar, a position not quite opposite to that occupied by the main rosette, but more anterior. Possibly therefore this smaller cluster is to be regarded as a fragment of the larger one, adhering here accidentally at the time of the emplacement of the latter. In both of these specimens the ovaries are swollen and packed with developing ova. Just how soon the ova would be ready for extrusion is at present problematic. TABLE OF MEASUREMENTS. Mes- sina. Funchal. Sex ^ ?, rt S O IH O •s aJ •O "o C fe PQ H cj O 264 S. STILLMAN BERRY. vidual which swam rapidly near the surface, giving out its phosphorescence, and then my friend from the top of the wall dexterously manipulated his long-handled 'peneiro' (dip-net). I obtained in this manner a dozen examples, and I could have had many more if I had had need of them. It was an evening warm and calm, and it seems to me that it is for this fine weather that the Abralia shows preference. At least it is i-n summer and autumn that the charming cephalopod arrives near the walls of the harbor of Pontinha. This locality is very sheltered, almost closed to currents and winds, and it is perhaps this circumstance which draws it to this nook of the shore, attracted furthermore by the lamp which illumines the quay. Indeed, as I believe, this cephalopod has never been seen in the five less protected places on the south coast of Madeira, where, during the night, they drag the fish seines, and where it would be easy to capture them if they were there. I myself have investigated these places, but I have seen taken in the nets only the common cephalopods of Madeira: a Loligo, a Sepia, and a Polypus. " I would also broach the opinion that the Abralia veranyi is an abyssal species which at night in summer migrates vertically and horizontally to attain the shore line, and by day betakes itself anew to the depths of the ocean." Under date of February I, 1922, Sr. Nunes wrote me further: "The cephalopod in question has been captured by myself in the sheltered quay of this city of Funchal, called the Quay of Pontinha, during the months of July, August, and September.— Almost every year one may capture them in this harbor during the night where they approach the steps of debarcation, following the lighting of the electric lamps of the above- mentioned steps. With a certain alacrity one may catch them with the aid of a little wire basket, because these animals come almost to the surface of the water, being distinguishable by the brilliancy ('6clat') of a bluish phosphorescence which they cause to gleam from their eyes." From these notes it would appear that the phenomena described are distinctly seasonal in character. This fact, the exceeding vigor and activity evinced by the animals when captured, and A LUMINOUS SQUID. 265 finally my own observations on the physiological condition of both males and females, affords convincing evidence that the inshore nocturnal migration of the Madeiran Abralia is essentially of reproductive significance and has to do with either the mating or the spawning, or very possibly indeed with both. Such a con- clusion finds the strongest possible confirmation in the detailed observations of Ishikawa ('13) and Sasaki ('14) on a somewhat nearly allied species of squid, Watasenia scintillans, the famous Firefly Squid ("Hotaru-ika") of Japan. The parallel extends to still further particulars, but so little information of consequence concerning the habits and life histories of the smaller squids has accumulated that the Japanese observations are almost the only ones of any relevance to be found in the literature. Like the Abralia, Watasenia "is a deep-sea animal, living during the day in a depth of 100 or more fathoms, and when the night is at hand, they approach to the coast, and after sunset they lay the eggs, and as soon as they finish their spawning, go back to the deep sea" (Sasaki, '14, p. 95). After the fixation of the spermatophores in the nape of the female (in quite a different position, as it would appear, from what is to be observed in Abralia veranyi}, the male Watasenia is thought to perish. The season of this extraordinary migration varies somewhat in different parts of the Japanese Empire, but in Toyama Bay on the west coast it is late April and May. Here the firefly squid comes inshore in such enormous num- bers that their fishery is a considerable industry, the total catch be- ing given by Sasaki as 1,000 tons. Due to the disappearance of mated males, nearly the entire catch seems to be comprised of fe- males (one count given is I cf to 79 9 9). No juvenals are found with them, and only a small per cent, of those taken were found to have food in the stomach. A net drawn up at 9 or 10 P.M. is said to be better filled than one hauled in at 3 or 4 A.M., a circum- stance which Sasaki suggests may be largely due to the fact that the schools swim in from the deep when sunset approaches, lay their eggs towards evening, and become entrapped in the nets on their way back. The fact that the Abralia is to be seen soon after the lights on the quay are illuminated may indicate that something of a nearly similar nature goes on here. Sr. de Noronha's allusion to the enraged state of the animal 266 S. STILLMAN BERRY. when captured is curiously in accord with the behaviour of Watasenia at the height of its own (spring) migration, at which time Sasaki states that "they attacked us violently, biting our hands with their jaws." Such as the Japanese catch in late summer or autumn, on the other hand, are quiet and show little vitality. From the relative emphasis laid upon the sources of the animal's illumination by both Madeiran observers, but especially by Nunes, it would seem that the ocular photophores of this Abralia irradiate a conspicuously brighter light than the more abundant organs of the body surface. This is very much what one would superficially expect from the general appearance of these organs in the dead animal. Turning again to the Japanese species we find that Ishikawa ('13, p. 168) likewise states that of the three types of photophores found in Watasenia scintillans, at least two of which are entirely homologous with those of Abralia veranyi, the organs of the outer integument come last in the intensity of their light. Yet it must be remembered further that Sasaki, working more extendedly on the same species, was unable to make out any difference between the light of the ocular and that of the integumentary photophores. However this may be, the function of light production in both species would seem to be essentially the same. The brilliance of the display stressed by both Madeiran and Japanese observers, coinciding as it does in each case with the schooling habit, the nocturnal migration, and the period of sexual activity, is most readily interpreted as a mating phenomenon, at least in very large part. I do not mean by this that the sexes actually recognize one another's different nature by means of corresponding differences in the luminosity of male and female, although at the same time the possibility of such recognition should not be too quickly excluded from con- sideration merely because Sasaki, who inquired into this aspect of the question quite particularly, found himself entirely unable to distinguish the sexes merely by the light of the animals at night. For there is another important way in which the photogenic function could serve an animal behaving as Abralia veranyi does during the reproductive season, and that is by simply furnishing a visual method by which the schools can assemble or keep together A LUMINOUS SQUID. 267 during the vicissitudes of migration. If this be supplemented either by slight visible sex differences, or by some chemical or other means of inter-attraction, such an arrangement might be sufficiently adequate to insure the maintenance of the species. Further field observations with this point in mind would doubtless yield some valuable and entertaining information. At first thought it seems passing strange that so conspicuous a phenomenon has not been observed more commonly. However, for the occurrence of species possessing such habits at stations convenient for observation near the shore there would seem to be required not alone shelter from heavy waves but an abrupt slope from the shore-line to the loo-fathom mark so that the contingent requirement for lateral migration be not too great, or similarly a near approach on the part of the deeper regions of the sea either by the agency of a submerged valley or some other consider- able depression. Such conditions are perhaps not always found in appropriate combination in the regions inhabited by these species. Whatever the said conditions may be, Funchal evidently satisfies them, and so affords nearly ideal opportunity for the study of Abralia veranyi. It is hoped that possibly this will prove by no means the only enoploteuthid squid to be found there. Whether the animal occurs in the near neighborhood of Madeira at all seasons of the year, or whether its nightly ap- pearance in summer is but the visible culmination of a more extended series of migrations from much farther afield, is a final interesting problem which must be left for some future deep- sea expedition to solve. Acknowledgment is due to Senhores M. O. Perestrello & Fos. of Madeira for the use of the accompanying photograph, and to Sr. de Noronha for the original of the small sketch map appended. A grant from the Rumford Committee has also been a con- tributing factor in the preparation of this report. SUMMARY. I. Abralia veranyi (Riippell) is a somewhat rarely captured Mediterranean cephalopod, possessing interesting luminous qualities due to the presence of numerous and exceedingly complicated photogenic organs. 268 S. STILLMAN BERRY. 2. It is here reported from the Bay of Funchal, Madeira, this constituting, it is believed, the first record of the species from the open Atlantic. 3. The specimens appear to differ in only very minor respects if at all from those of Mediterranean origin. 4. The species comes into the shallow water of the harbor at night during the summer months, supposedly for the purpose of mating, spawning, or both. • 5. Owing to their comparative abundance at this time of year and the ease with which they may be observed and captured, Funchal is evidently an unusually favorable locality for the study of the light production or general bionomics of this interesting little squid. BIBLIOGRAPHY. Ficalbi, E. '99 Una pubblicazione poco conosciuta di Riippel intitolata: " Intorno ad alcuni Cefalopodi del mare di Messina (Messina, 1844)." Monitore Zoologico Italiano, Anno X, pp. 79—84, Text Fig. 1-2, 1899. Ishikawa, C. '13 Einige Bemerkungen liber den leuchtenden Tintenfisch, Watasea nov. gen. (Abraliopsis der Autoren) scintillans Berry, aus Japan. Zoologischer Anzeiger, Bd. 43, p. 162-172, Text Fig. 1-6, Dec. 1913. Joubin, L. '95 Note sur les appareils photogenes cutanes de deux cephalopodes: Histiopsis atlantica Hoyle et Abralia oweni (Verany) Hoyle. Memoires Societe Zoologique France, v. 8, p. 212-228 (1-17), Text Fig. i-n, 1895. Mortara, S. '22 Gli organi fotogeni di Abralia veranyi. R. Comitato Talassografico Italiano, Mem. 95, p. 1-20, Text fig. 1-2, PI., 1922. Pfeffer, G. '12 Die Cephalopoden der Planktonexpedition. Zugleich eine monographische Ubersicht der oegopsiden Cephalopoden. Ergebn. Planktonexpedition der Humboldt-Stiftung, Bd. II., p. i-xxi, 1-815, Atlas of 48 pis., 1912. Ruppell, E. '44 Intorno ad alcuni Cefalopodi del Mare di Messina. Giornale del Gabi- netto lettcrario di Messina, Fasc. 26, p. 129-135 [1-7], 2 figs., 1844 (or, according to some authors, 1845). Sasaki, M. '14 Observations on Hotaru-ika Watasenia scintillans. Journal College Agri- culture, Tohoku Imperial University, Sapporo, Vol. 6, p. 75-105, 2 Text figs., PI. 1-3, Nov. 1914. Steenstrup, J. '80 De Oinmatostrephagtige Blaeksprutters indbyrdes Forhold. Oversigt over d. K. D. Vidensk. Selsk. Forhandl. 1880, p. 73-110 (1-40), PI. 3, 12 figs, in text, 1880. CONVERGENCE OF COLORATION BETWEEN AMERICAN PILOSE FLIES AND BUMBLEBEES (BO MB US). E. GABRITSCHEVSKY, RESEARCH FELLOW OF THE INTERNATIONAL EDUCATION BOARD, COLUMBIA UNIVERSITY, NEW YORK. THE EUROPEAN AND ASIATIC GROUP OF Vohicella Bombylans. In a previous paper, in which the heredity of color variations in the European fly Vohicella Bombylans has been described, an attempt has been made to show the striking resemblance in coloration between various flies of the families Syrphidae, Asilidse, Tabanidae, (Estridx and Bombylidx with various bumblebee species which inhabit the same geographical regions. The study of those analogous color patterns which appear both amongst flies and bumblebees is interesting from a genetic point of view and bears on the problem of mimicry in animals. The black V. Bombylans (PI. L, Figs, i, d\ 9 ; 2, 9 ; 3, 9 ) with its fulvous bands on the last abdominal segments has exactly the same coloration as the common European Bombus lapidarius, B. confusus, B. rajellus, B. mastrucatus, etc. The other variety, V. hxmoroidalis (PI. L, Figs. 4, 5), is almost indistinguishable from many other Bombus species, as for example B. agrorum, B. vorticosus. The third variety of the V. Bombylans, the V. B. var. plumata (PL L, Figs. 6, 6 , 9 ; 7, 9 ) with its characteristic white hairs on the last abdominal rings, is extremely like B. hortorum, B. lucorum. Vohicella bombylans (and its varieties), if really pro- tected by her mimetic coloration, may be supposed to creep unobserved into a Bombus nest to lay her eggs there on the wax- combs. More often however the fly puts her eggs on the grass and moss, which covers the Bombus nest. This of course can only be performed with a Bombus species which builds its nest on the ground, not under the ground. In localities where many Bombus colonies of this kind are present the Vohicella flies are also found in large quantities. The larvae of Volucella feed on wax, 269 2/O E. GABRITSCHEVSKY. pollen and decayed or injured Bombus larvae (fresh larvae are never attacked). The "Mimicry' of Volucella offers a fascinating field for research and one of the steps which must be taken in the pre- liminary study of this case is to find out how close a resemblance there is between a variety of Volucella and the various species of Bombus in the same geographical area. Such a resemblance has been described in my previous paper for the European group of Volucella. I was interested therefore to see if this was true for the American varieties of Volucella. Before dealing with this question I wish to refer to some new details on the coloration of the European Volucella Bombylans which must be also taken into consideration in the study of the North American group. Many flies of these species have been collected recently in the central parts of Russia, to see if there are any other variations than those which have been generally described. Specimens of V. Bombylans have been found with an entirely rufous abdomen (PI. I., 2, 9 ), which corresponds exactly to the color of the abdomen of the northern B. laponicus and other allied species. This variety has been collected in Russia only twice in a region where the B. laponicus does not occur. Another variety of V. Bombylans, which may be named V. Bombylans var. flava, is not uncommon. This fly has a spot of yellow hairs on the thorax (PI. I., Fig. 3, 9 ), but the rest of the fly is typical Bomby- lans. (Only females of this type have been collected.) The yellow spot of hairs of this black variety is located in the same place as the black spot of yellow varieties of male V. hasmoroidalis and male V. plumata. It is significant from a genetic point of view that in the European Volucella group of flies only this little area of thoracic hairs is limited to one sex, whereas the entire body coloration is otherwise alike in both sexes. Thus the yellow spot of the black variety seems to appear only in the females, the black spot of the yellow varieties on the contrary is limited to the males (V. plumata and V. hxmoroidalis] . It is, however, possible to find in some localities also females of V. plumata and V. hxmo- roidalis which have a very reduced black thoracic spot of hairs. The black spot itself has often a brown color in the females, but never in the males. The V. altaica from Asia is extremely AMERICAN PILOSE FLIES AND BUMBLEBEES. 271 dimorphic in regard to this black spot. The male (PI. I., Fig. 9) has a perfectly black thorax, the female has the usual black spot in relation to its size and coloration like the males of V. hxmo- roidalis (PI. I., Fig. 4) and V. plumata (PI. I., Figs. 6, 7). The yellow spot of V. Bombylans var.flava is inherited. I had in one culture (unpublished data) 6 females in Ti of V. Bombylans var. flava from a female V. Bombylans var.flava (PI. I., Fig. 8). The V. Bombylans var. caucasica, which lives in bumblebee nests of the Caucasian mountains, has a white thorax (the black spot is present in the males), and the upper part of the abdomen is also white, but the third to sixth abdominal rings are exactly the same as in the European V. Bombylans and V. hxmoroidalis (compare Figs. I, 2, 3, 4, 8, PI. I.). On looking at this fly one gets the impression that only the yellow hairs of the European variety V. plumata and V. haemo- roidalis have changed into white, but that the black and fulvous hair covering the end of the abdomen remained unaffected. A i parallel color change occurs in almost all black bumblebee species which inhabit the Caucasus. The Volucella Caucasica is thus similar to the Caucasian B. eriophorus, B. niveatus and other species of this region, which have white patches of hair on the thorax. The black and rufous parts of the bumblebees' abdominal rings, which correspond to the black and rufous bands of the endemic Volucella flies remained also unaffected and similar in coloration to those of the European Bombus lapidarius, B. confusus and Volucella Bombylans (type) . Many other Caucasian flies (Cheilosia cestracea, Tabanus gudaurensis, T. tricolor, etc.) have similar color changes parallel to those described above. The appearance of white spots or bands is characteristic for both bumblebees and flies of this mountain region. Summarizing these data we can say that there is a striking phenomenon of color convergence between European Syrphidx (especially Volucella, and its varieties), Tabanidx, Asilidx and (Estridx and bumblebees, which are distributed in the same geographical area. A similar parallelism in coloration can be also noticed for the Caucasian region. It must be also pointed out that there is a predominance of certain color patterns for the European group of bumblebee-like flies and bumblebees. The 18 2J2 E. GABRITSCHEVSKY. combination of black and rufous, white and yellow, and black and white stripes is more often found than black and yellow. This is a characteristic feature of both insect genera in the European part of the old world. An entirely different situation is found in the United States of America. Even a superficial survey of the collections of American (Syrphidae and Asilidae) flies and bumblebees shows distinctly, that both insect genera have, strictly speaking, only one pattern, namely, black and yellow striped individuals. (Only two species, B. occidentalis and B. borealis, have parts of their body covered with white hairs.) The combination of black and rufous, yellow and white, black and white, is absolutely absent or very rare on this continent. The overwhelming numbers of black and yellow striped specimens among these flies and bumblebees is a peculiar feature throughout the United States. The phenomenon of color convergence has reached its climax in North America. THE NORTH AMERICAN GROUP OF^Volucella Bombylans. The systematic relationship of the American varieties of V. Bombylans has been recently studied by Johnson ('16, '25) who divides these flies into two large groups. To the first (the black- faced group) belongs the eastern V. evecta, var. V. evecta americana var. V. evecta sanguined, and the boreal V. evecta arctica. To the second (the yellow-faced group) belongs the western V. facialis, the Canadian V. facialis lateralis and the Vol. facialis var. nifomaculata, which is found in Colorado and along the Rocky Mountains. Excepting the coloration of the pleura, which may be yellow or black in both groups, and excepting differences in the color patterns of the thorax and abdomen, no other distinguishing characters have been detected. It is very probable that all European and American varieties might interbreed. CONVERGENCE OF COLORATION BETWEEN FLIES OF THE EASTERN STATES AND BUMBLEBEES OF THE SAME REGION. Volucella bombylans var. evecta-americana (PL. I., 15). The eastern Volucella bombylans var. evecta (PI. I., 15) americana is entirely pale yellow except the last two to four posterior abdom- AMERICAN PILOSE FLIES AND BUMBLEBEES. 273 inal segments, which are covered with black hairs. The same distribution of yellow and black hairs on the thorax and abdomen is characteristic for all the eastern species of pilose flies. Mallota posticata (PI. I., 18, 19), Mallota cimbiciformis (PI. I., 20-22), Eristalis flavipes (PI. I., 23-24), Eristalis bastardii (PI. I., 26-27), Dasyllis lata (PI. II., 9), Dasyllis marquarti (PL II., 9), Das. sacraton (PI. II., 9), Das. champlaini (PL II., 8). All these Syrphidx and Asilidx are very common in the eastern states and can be found on the same flowers with Bombus. Six bumblebee species of the eastern states, namely Bombus vagans (PL III., i), B.affinis (PL III., 7), B.impatiens (PL III., 3), B. separatus (PL III., 2), B. bimaculatus (PL III., 5), B. perplexus (PL IV., 4) have a coloration exactly corresponding to Volucella evecta americana (PL I., 15) and to the other Syrphidx and Asilidx which are mentioned above. (Compare PL I., 15, with PL III., i, 7, 3, 2, 5, 4. Compare also PL II., 7, 8, 9, with PL III., !» 7» 3) 2> 5. 4 and then PL II., 10, n, with PL III., 13, 14, 15.) Four other eastern bumblebee species, namely B .pennsylvanicus (PL IV., 13, i/[),B.auricomus (PL IV., 13, 14), B. terricola (PL IV., 14) have a slightly different distribution of black and yellow hairs on the thorax and abdomen. This group of bees has its corre- sponding group of flies. It is hardly possible to detect any difference between the eastern Eristalis flavipes (PL I., 24) var. b., Dasyllis dithoracica (PL II., 10, n), Das. grossa (PL II., n) and the bumblebees enumerated above. The flies are generally like the females, workers and males of the corresponding bumble- bee species, but this is only true when the Hymenoptera has no polymorphic females or males. B. bimaculatus (PL III., 5) of this first eastern group, which is like V. evecta americana, has a male variety colored like Eristalis flavipes var. b (PL I., 24), Eristalis flavipes, on the other hand, has just been compared with the bumblebees of the second eastern group. (The groups are divided here only in relation to their color patterns.) We find the same phenomenon in B. affinis (PL III., 7), and B. pennsyl- vanicus, both of which have three types of males; (PL IV., 10, 1 1. 15-18) one of these is like the female, but the two others are entirely different. The latter two are extremely like Criorhina verbosa (PL II., 14) and Criorhina Kinkaidi var. b (PL II., 16). 2J4 E- GABRITSCHEVSKY. As a rule, it is possible to maintain that most of the bumblebees' males are like the Criorhina flies. This is due to the fact that the males, like these flies, have reduced black bands which make them almost indistinguishable. From the above we might infer that there is a very close resemblance between bumblebees and flies of the eastern parts of the United States. Yet it would be a mistake to conclude from this that there is mimicry between both genera; it must not be forgotten that there is also the same convergence in coloration, even as regards details, within the group of bumblebees and also within the group of flies or, more exactly, there is not only a correspondence between the flies and bumblebees of a given district, but the several species of Hymenoptera found living in a region resemble one another closely, and species of flies are also much alike among themselves. For example, when the bumble- bees are compared with one another, we see that the coloration of the variety of B. auricomus (PL III., 13, 14), with the yellow scutellum, reappears in the same pattern in B. pennsylvanicus (PI. III., 13, 14). One of the males of B. bimaculatus (PI. III., 9), is similar in this respect to one of the males of B. affinis (PI. III., n). Many other coincidences are also evident and not uncommon in these groups. (Compare figures.) The coloration of the European B. lapidarius reappears in B. confusus, in many other species and in Volucdla Bombylans (type) (PI. I., i). The distribution of V. b. evecta americana is probably by far more restricted than that of the above mentioned bumblebees, which may be found further to the south. This can be explained through the fact that all large Volucella are circumboreal species and never appear farther to the south than the 42d parallel, unless there are mountains which extend to the south. Favorable conditions are thus produced and the bumblebee nests may be found again infested with Volucella larvae in more elevated zones. Volucella evecta americana is not to be found much farther to the north. According to Johnson this variety can be collected along the Atlantic coast as far north as Mount Desert, but there this fly is already replaced by Volucella bombylans facialis lateralis (PI. I., ii), which belongs to the Canadian zone. AMERICAN PILOSE FLIES AND BUMBLEBEES. 275 Volucella bombylans evecta (Walker) (PI. I., 14). The thorax, scutellum and the three first abdominal rings of this fly correspond to V. B. evecta americana, (PI. I., 15), but the posterior 4-6 segments may be either yellow or reddish. Evecta is the upper austral form, extending through the transition zone. This fly resembles closely the males of the eastern bumblebees. Its coloration has altogether the aspect of these Hymenoptera, but a strict parallelism in color patterns with any of the eastern bumblebees is not traceable. Volucella evecta is a rare form when compared with V. evecta americana. It is possible that a detailed study of its distribution in the east might explain its color patterns. Volucella bombylans evecta sanguined, (PI. I., 16). This variety is an extremely rare variation of V. B. evecta americana. It has been recorded in the eastern states. This fly has yellow hairs on thorax, scutellum and abdomen, except the third abdominal ring, which is covered with fulvous pile. The sides of this segment have some black hairs. This Volucella represents, in a way, a parallel variation to that of V. rufomaculata in the subspecies facialis, which is distributed along the more elevated portions of the Rocky Mountains. V. sanguined is not comparable to any bumblebee species of the eastern United States. A third and analogous parallel variation is found in Eristalis flavipes var. melanostoma (PI. I., 25) of the east, and a fourth in Dasyllis fernaldi (PI. II., 18) an Asilid fly of the Rocky Mountains. The sporadic reappearance of parallel variations in V. sanguinea and Eristalis flavipes, which are analogous to the variety of V. rufomaculata and Dasyllis fernaldi (PI. II., 18) of the west, suggests that the genie mutations producing these color changes in some individuals are probably of a related chemical structure, in the sense of their end effect. It is also interesting that they affect the same parts of the insect's body. The same argument can be applied to the bumblebees. It would be more difficult to explain, however, the way in which a new mutation could replace the more abundant form for a given geographical zone. 276 E. GABRITSCHEVSKY. CONVERGENCE OF COLORATION BETWEEN FLIES OF THE ARCTIC ZONE. Volucella bombylans arctica (PI. I., 17). This syrphid of the evecta group is entirely dark yellow, with a short and unfinished black band on the thorax. This Volucella is extremely like in coloration to the arctic bumblebee — Bombus gelidus (PI. IV., i), and B. borealis (not figured). CONVERGENCE OF COLORATION BETWEEN FLIES OF THE ROCKY MOUNTAINS AND BUMBLEBEES OF THIS REGION. The Volucella facialis Group, Volucella Bombylans facialis var. rufomaculata (PI. I., 13). Volucella rufomaculata, which is a parallel variation to the V. evecta sanguinea of the evecta group is limited to the mountain region of Colorado, Utah and New Mexico. The dorsum of the thorax is black pilose-like in all varieties of the facialis group. This is a characteristic which is entirely absent in the evecta group (compare PI. II., 14-17). In this respect the male and female flies of the evecta group are like the females of the European varieties of Volucella Bombylans, and the male and female flies of the facialis group can be compared to the males of the European varieties of Volucella plumata and hxmoroidalis . The pleura of the facialis flies are black as in the European varieties. It is not improbable that the western American Volucella group is more closely related to the European group. It is again striking that V. rufomaculata has a coloration resembling that of most of the bumblebees of this mountain region. An exact coincidence in color patterns can be observed in Bombus sylvicola (PI. IV., 5), B. huntii (PI. IV., 1-2), B. melanopygus (PI. IV., 4), B. eduardii (PI. IV., 13-17), B. ternarius (PI. IV., 6). The same is true for the Asilid fly Dasyllis fernaldi (PI. II., 18). Five other bumblebee species of the Colorado area, namely, Bombus centralis (PI. IV., 18), B. borealis (PI. IV., 12), B. gelidus (PI. IV., i), B.flavijrons (PI. IV., 7, 8), B. rufocinctus (PI. IV., 9), B. kirbuellus (not figured), B. appositus (PI. IV., 12) have also the same type of coloration with the characteristic rufous abdominal segment like that of V. rufomaculata. The coincidence in AMERICAN PILOSE FLIES AND BUMBLEBEES. 2/7 coloration of the segments is not so exact as in the first four bumblebee species enumerated above, but the reappearance of some segments with red pile must be regarded as a characteristic feature in both insect genera of this region. It is strange that the Caucasian mountain species of flies and bees are partly changing their pile into a shining white color, whereas the American mountain species tend to turn into a bright vermilion coloration. Another group, consisting of bumblebees with a shining wax yellow coloration is also present in the Colorado region. This includes B. sonorus (PI. IV., 3), B. morissoni (PL IV., 4), B. nevadensis (PI. IV., 5), B. terricola (PL III., 14). As regards the distribution of black and yellow bands, the first three species are like the first group of the eastern bumblebees, and the fourth one is like the bumblebees of the second eastern group. The main difference is observed in the tint of the yellow pile, which is extremely bright in B. morissoni, nevadensis, sonorus and terricola. It is apparently again a case of color convergence, but which is present only in the hymenoptera group, as no flies of this kind have been collected in this mountain zone. Occasionally it is possible to find Volucella flies with the more brilliant wax yellow pile, but it must be pointed out that the yellow hairs of the flies vary in intensity of pattern from light ochre to swarthy brown, or more exactly from maize yellow through wax yellow to deep colonial buff. Many slight variations can also be noticed in the red pile. The black color on the contrary is always constant. Many Asilidae are like these bumblebees; an exact distribution of the same colored bands can be observed in Dasyllis lata var. a. D. champlaini, D. macquarti, D. grossa 9 (PL II., 7» 8, 9, 12). Mallota posticata (PL I., 19). CONVERGENCE OF COLORATION BETWEEN FLIES OF THE PACIFIC COAST AND THE Bombus SPECIES WHICH INHABIT THE SAME REGION. The Volucella bombylans facialis (PL I., 12). This fly is one of the darkest when compared with the other pilose species of this family. The pleura are black pilose; the dorsum of the thorax has a larger area covered with black hairs than in all other Volucella flies. The black hairs of the third 2/8 E. GABRITSCHEVSKY. abdominal segment extend also partly onto the second ring, and sometimes on to the upper quarter of the fourth abdominal ring. The last segments are yellow pilose or reddish. An increase of black hairs on thorax, scutellum and abdomen is also charac- teristic for the endemic bumblebees. Bombus vosnesensky (PL IV., 6), B. Crotskii (PI. IV., 7), B. californicus (PI. IV., 8-11) are good examples of this. Volucella facialis is distributed along the Pacific coast and in Alaska. The coincidence in the distribution of black and yellow bands in Volucella flies and bumblebees of this zone is not absolutely exact, but yet the increase of the black hairs and the distinct and parallel process of melanization in both insect genera is strikingly evident. Volucella B. facialis lateralis (PI. I., n). This fly is extremely like the former one, but the pleura are yellow pilose, and the black spot on the dorsum of the thorax has a more reduced area than in V. facialis. V. lateralis belongs to the Canadian zone (Alaska to Newfoundland, Mt. Desert) and is, so to speak, the eastern representative of facialis. I have no data as to other flies and bumblebees of this large area, and a com- parison has thus been impossible. SUMMARY. 1. The European Syrphid fly Volucella Bombylans and its varieties, V. haemoroidalis and V. plumata have a coloration which corresponds to the coloration of the majority of European bum- blebees. The same is true of many other pilose flies of Europe. 2. The Caucasian Volucella bombylans caucasica has a color- ation which corresponds to the coloration of various bumblebees of this mountain region. The same is true for other pilose flies of this zone. 3. The American Volucella bombylans evecta-americana of the eastern states and 8 other species of Syrphid flies have exactly the same color patterns as six different Bombus species of this region; four different Bombus of the same area have' the same type of coloration as three other beelike flies of the eastern states. 4. Volucella bombylans evecta sanguinea, and Eristalis flavipes var. melanostoma of the eastern states are rare and parallel AMERICAN PILOSE FLIES AND BUMBLEBEES. 279 variations which have analogous coloration to that of Volucella bomb. var. rufomaculata of the facialis group and of the Rocky Mountain region. 5. Volucella bombylans arctica of the evecta group is similar in coloration to the arctic bumblebee Bombus gelidus and to other species. 6. Volucella bombylans var. rufomaculata of the Rocky moun- tains has a similar coloration to twelve different bumblebee species which occur in the same region. The Asilid fly Dasyllis fernaldi of the same zone has a corresponding coloration to V. rufomaculata and to the twelve bumblebee species. 7. Volucella bomb, facialis of the Pacific coast has analogous color patterns to three bumblebee species of this zone. Volucella bomb, facialis lateralis of the Canadian zone and V. bomb, evecta of the eastern states have not been studied on account of absence of data as to their distribution. I am greatly indebted to Dr. A. Sturtevant, Dr. Johnson and Dr. Lutz, for the use of their collections of flies and of Bombus, also for their help and advice in this work. LITERATURE. C. W. Johnson. '16 The Volucella Bombylans group in America. Psyche, Vol. XXIII., No. 6. '25 The North American Varieties of Volucella bombylans. Psyche, Vol. XXII., April 1925. E. Gabritschevsky. '24 Farbenpolymorphismus und Vererbung mimeticher varietaten der Fliege V. Bombylans und anderes Zweifliigler. Zeitschr. fur Induktive Abst. und Vererbungslehre, Bd. XXXII., Vol. i. 2. C. E. Keeler. '26 Recent work by Gabritschevsky on the Inheritance of Color Varieties in Volucella Bombylans. Psyche (A Journal of Entomology), Vol. XXXIII., No. i, February 1926. EXPLANATION OF PLATES I-IV. All figures represent flies in a schematized way. The upper square is the thorax; the second one is the scutellum; the third is the abdomen. The dotted line between the abdomens of different flies shows the segments of the insects. The head is not drawn. In Plate I. the black color corresponds to the black pile, the white to white hairs, the spotted parts are of a yellow coloration, and the vertical parallel lines correspond to a cadmium orange tint in the flies. In all the other plates, (I. (11-27) and III., IV.) the uncolored parts of the schemes correspond to different tints of yellow (the exact color stands in the explanation of the plates; these colors have been compared with Ridgway's Color Standards — R. Ridgway, "Color 280 E. GABRITSCHEVSKY. Standards and Color Nomenclature," 1912, Washington). The black parts are again black in the flies and bumblebees; the striped individuals have on those segments a cadmium orange coloration. The exact shades of these different red tints are given under every figure in the explanation of the plate. EXPLANATION OF PLATE I (Nos. i-io). The European and Asiatic Groups of Volucella Bombylans. 1. Volucella Bombylans, 6, 9. 2. Volucella Bombylans, var. rufa 9 . 3. Volucella Bombylans, var. flava. 9. 4. Volucella B. hcemoroidalis 6 . and Volucella B. altaica 9 . 5. Volucella B. hcemoroidalis 9 . 6. Volucella B. plumata 6 • 7. Volucella B. plumata 9 . 8. Volucella caucasica 6 . 9. Volucella altaica 6 . 10. Volucella plumata var. a 9 . The yellow pile is variable. Specimens of these flies are found with a maize yellow (19-40, y. /., PI. IV.), wax yellow (21, o-yy PI. XVI.), and deep colonial buff yellow (21, o, PI. XXX.). The red pile is less variable, yet specimens with a cadmium orange (13, o-o, PI. III.), or Mars yellow (15-00, PI. III.), or ochraceus tawny (15-00, PI. XV.) are found. The white pile is snow white, or with a slight yellow tint. EXPLANATION OF PLATE I. (Nos. 11-27). The North American Group of Volucella bombylans. 11. Volucella bombylans facialis lateralis, 6. 9 . 1 ,, The facialis group — maize 12. V. B. facialis, 6.9- yellow. 13. V. B. facialis var. rufomaculata, 6.9. 14. V. B. evecta, 6.9. 15. V. B. evecta-americana, 6 . 9 . 16. V. B. evecta var. sanguinea, 9 • 17. V. B. evecta artica, 6.9. The evecta yellow. group — maize The North American Bumblebee-like Syphida 11-27. The yellow pile is maize yellow (19-40, y. /., PI. IV.), the red pile is cadmium orange (130-0 PI. III.). 18. Mallota poslicata, 6.9. 19. Mallota posticata var. a, 6 , 9 . 20. M. cimbiciformis var. a, 6 . 9 . 21. M. cimbiciformis var. b, 6 . 9 . 22. M. cimbiciformis var. c, 6 . 9 . 23. Erislalis flavipes var. a, 6 . 9 • 24. Er. flavipes var. b, 6 . 9 • 25. Er. flavipes var. melanostoma, 9 26. Er. baslardii, 9 . 27. Er. bastardii, 6 . 9 • !> Wax yellow or maize yellow. \ Gray pile. Wax yellow pile. Wax yellow or maize yellow. > Maize yellow. BIOLOGICAL BULLETIN, VOL. L. PLATE E. GABRITSCHEVSKY. 282 E. GABRITSCHEVSKY- 9 ; D. grossa, 6 , 9 . EXPLANATION OF PLATE II. The North American Bumblebee-like Asilidce, and Flies of the Criorhina Group. 1. Dasylus unicolor, 6.9. 2. Dasylus insignis, 6.9. 3. D. posticata, 6.9. 4. D. divisor, D. cinerea, 6.9. 5. D. sp. ?. 6. 9. 6. D. flavicola, 6 . 9 ; D. virginica, 6 7. £>. lala var. a, 6 . 9 . 8. D. champlaini, 6.9. 9. D. lata var. 6, 6 . 9 ; D. marquarli, 6 . 9 ; D. sacraton, 6 . 9 10. D. dithoracica, var. a, 6 . 9 . 11. D. dithoracica var. 6, 6 . 9 ; D. grossa, 9 . 12. £>. grossa var. a, 9. 13. Criorhina nigripes, 6.9. 14. Criorhina verbosa, 6.9. 15. Criorhina kincaidi var. a, 6. 9. 16. Criorhina kincaidi var. fe, 6 . 9 . 17. Cephenomya abdominalis, 6. 9. 18. Dasylus fernaldi, 6.9. Wax yellow of deep co- lonial buff. Deep colonial buff. Cadmium orange, maize yellow. BIOLOGICAL BULLETIN, VOL. LI. PLATE II. /3 L J V" - 6 E. GABRITSCHEVSKY. 19 284 E. GABRITSCHEVSKY. EXPLANATION OF PLATE III. Bumblebees of the Eastern Stales of North America. First Group i-u. 1. Bombus vagans, 9 , 8 , 6 • (Deep colonial buff 21", o — PI. XXX.) 2. B. seperatus, 9 , 8 , 6 • (Deep colonial buff — PI. XXX.) 3. B. impatiens, 9 , $ , 6 . (Deep colonial buff— PI. XXX.) 4. B. perplexus, 9 , S , 6 • • (Deep buff yellow — PI. IV.) 5. B. bimaculatus, 9 , 8 , 6 • (Deep colonial buff, 21", o — PI. XXX.) 6. B. bimaculatus, 9 , 3 , 6 . (Deep colonial buff— PI. XXX.) 7. B. affmis, 9 , 8 , 6 • (Deep colonial buff— PI. XXX.) 8. B. vagans, —.—, 6 • (Deep colonial buff— PI. XXX.) 9. B. bimaculalus, — , — , 6 • (Deep colonial buff — PI. XXX.) 10. B. affinis, — , — , 6 . (Deep colonial buff — PI. XXX.) 11. B. affinis, —,—, £ . (Olive buff. 21", o— PI. XL.) Second Group 12-18. 12. B.fralernus. 9, 8, 6- (Deep colonial buff 21", o— PI. XXX.) 13. 14. B. pensylvanicus, 9,9. (Mustard yellow 19', 6 — PI. XVI.) 13. 14. B. auricomus, 9,9. (Deep colonial buff, 21"— PI. XXX.) 14. B. lerricola, 9 , Q , 6- (Apricot yellow — PI. IV.) 15. B. pensylvanicus, 6- (Mustard yellow, 19' — PI. XVI.) 16. B. pensylvanicus, 6. (Mustard yellow, 19' — PI. XVI.) 17. B. pensylvanicus, 6- (Pyrite yellow, 23' — PI. IV.) B. fervidus, 9 . 8 , 6 . 18. B. pensylvanicus, 6. (Pyrite yellow, 23' — PI. IV.) BIOLOGICAL BULLETIN, VOL. LI PLATE III. 6.7. fcH BP // H / i 3 ejC E. GABRITSCHEVSKY. 286 E. GABRITSCHEVSKY. EXPLANATION OF PLATE IV. Bumblebees of the Rocky Mountains. 1. B. hunlii, 9, 8, 6. (Olive ochre, 21"— PI. XXX.) 2. B. huntii, —,—, 6 . (Amber yellow, 21"— PI. XVI.) (3d and 4th abd. segments cadmium orange, 130 — PI. III.) 3. B. melanopygus, 9. (Cartridge buff 19" — PI. XXX.) (3d and 4th abd. segments cadmium orange 130 — PI. III.) 4. B. melanopygus, $ . (Segments cadmium orange — PI. III.) 5. B. sylvicola, 9, 8, 6- (Straw yellow, 21' — PI. XVI.) (3d and 4th abd. segments orange rufous, n', i — PI. II.) 6. B. lernarius, 9, 8, 6- (Straw yellow, 21'— PI. XVI.) (3d, 4th and 5th abd. segments cadmium orange — PI. III.) (130— light buff, 17'— PI. XV.) 7. B. flavifrons, 9,8. (Thorax light buff, 17', o — PI. XV.) (Abdomen ochraceus tawny, 15 — PI. XV.) 8. B. flavifrons, 6- (Antimony yellow, 17', b — PL XV.) 9. B. rufocinctus, 9, 8. (Thorax, colonial buff, abdomen olive ochre, 21" — • PI. XXX.) 10. B. rufocinctus, 6 • (Colonial buff.) 11. B. rufocinctus, £. (Colonial buff.) 12. B. apositus, 9 . 8 , 6 • (Thorax white with gray.) B. borealis, 9 . 13. B. edwardsii, 9 , 8 . (Maize yellow, 19 — PI. IV.) 14-15. B. edwardsii, 6 . (Maize yellow — PI. IV.) 16. B. edwardsii, 6 • (Cadmium orange, 13 — PI. III.) 17. B. edwardsii, £. (Cadmium orange, 13 — PI. III.) 1 8. B. centralis, 9,8. (Maize yellow, 19 — PI.—.) (Cadmium orange, 13 — PI. II.) EXPLANATION OF PLATE IV. The Arctic Species of Bumblebees (Labrador). 1. B. gelidus, 9,8. 2. B. frigidus, 9,8. The Wax Yellow Group of Rocky Mountain Bumblebees. 3. B. sonorus, 9, 8. (Wax yellow, 21' — PI. XVI.) 4. B. morrissoni, 9, 8. (Wax yellow— PI. XVI.) 5. B. nevadensis, 9,8. (Antimony yellow, 17' — PI. XV.) EXPLANATION OF PLATE IV. Bumblebees of the Pacific Coast (California- Alaska). 6. B. -voznosenskii, 9, 8, 6- (Pinard yellow, 21' — PI. IV.) 7. B. crotshii, 9. 8, 6- (Deep colonial buff, 21'— PL XXX.) (4th, 7th segments flame scarlet — PL II.) 8. B. californicus, 9. (Chartreuse yellow, 25" — PL XXX.) 9. B. californicus, 9 . (Chartreuse yellow — PL XXX.) 10. B. californicus, 6- (Yellow ochre, 17' — PL XV.) 11. B. californicus, $. (Yellow ochre — PL XXX.) BIOLOGICAL BULLETIN, VOL. LI. -- PLATE IV. E. GABRITSCHEVSKY. Vol. LI. November, 1926. No. 5. BIOLOGICAL BULLETIN THE FLAGELLATE FAUNA OF THE CCECUM OF THE STRIPED GROUND SQUIRREL, CITELLUS TRIDECEMLINEATUS, WITH SPECIAL REFERENCE TO CHILOMASTIX MAGNA SP. NOV. ELERY R. BECKER, IOWA STATE COLLEGE. The intestinal protozoan fauna of the rat, mouse, guinea pig and rabbit has been investigated quite extensively. Not so much has been done on field rodents as on those which lend themselves to laboratory use. In the autumn of 1925 and spring of 1926 the writer made a study of the protozoa harbored in the alimentary tract of the striped ground squirrel, Citellus tridecemlineatus . This rodent was found to be quite as rich a field for investigation of protozoan parasites as the aforementioned animals. The protozoa so far found to be residents of the ccecum of the striped ground squirrel comprise one species of amoeba, Endamceba citelli; one species of Chilomastix; two species of Trichomonas , one of them probably new; one species of Tetratrichomastix, subgenus of Eutrichomastix; one species of Hexamitus; and a flagellate which is either a species of Hexamitus or Urophagus. In the small intestine a Giardia was found. Endamceba citelli, and its pathogenic parasite, Sphxrita endamosbss, were described by the writer (1926) in a previous paper. The Giardia was sent for identification to Dr. R. W. Hegner, who found it to be a new species.* The Chilomastix, Trichomonas, Tetratrichomastix, and Hexamitus will be reported upon in this paper in the order named. The preparations of the other flagellate were not entirely suitable * Since this paper went to press a paper by Dr. R. W. Hegner has appeared in which this Giardia is described. See Hegner, R. W. 1926. Giardia beckeri n. sp from the ground squirrel and Endamceba dipodomysi n sp. from the kangaroo rat Jour. Paras. 12: 203-206. 19 287 288 ELERY R. BECKER. for critical study, and the specimens were very scarce. Conse- quently a description of this species (Hexamitus or Urophagus) must be delayed until more suitable material is obtained. Twenty ground squirrels in all were examined for protozoa. All save one harbored infinite numbers of protozoa in the coecum. This one had only a light infection. The technique consisted of a careful microscopic examination of thin emulsions of the coecal contents in normal salt solution. Permanent prepared slides were made by Heidenhain's iron- haematoxylin staining after fixation in Schaudinn's solution. Chilomastix magna sp. nov. This protozoan was found in all twenty ground squirrels. The shape is essentially like that of Chilomastix mesnili from man, pyriform with three flagella at the broadly rounded anterior end. There is often a tuft of short bristly cuticular projections in the region of the flagella (Fig. i). It may be present in either living or stained material, and is not a fixation artefact. Fixed and stained individuals measured from 10 to 21.6 micra in length and from 6 to u micra in width, the average width being about 9 micra. The length of Chilomastix mesnili, as given by Kofoid and Swezy (1920) is from 9.6 to 15 micra in stained preparations. The writer measured a number of Chilomastix mesnili on slides which he had prepared, and found a range of from 7 to 1 1 micra. When one considers that twenty out of thirty specimens of Chilomastix magna measured from 15 to 21.6 micra, it will be seen that this flagellate is almost twice as long as the one found in the intestine of man. A study of the cytology of this protozoan promised to be particularly interesting because of the discrepancies between the accounts of Chilomastix mesnili by Kofoid and Swezy (1920) and Dobell and O'Connor (1921). The latter authors are particularly outspoken in flatly denying the existence of certain cell structures which the former claim to have seen. Without desiring to become in any way involved in the controversies of these writers, the writer must state that his observations agree more nearly with those of Kofoid and Swezy, although his interpretations may differ in certain respects. The morphology of the free form, cyst, and dividing individuals will be described. The nucleus of the free form lies in the extreme anterior end of FLAGELLATE FAUNA OF CCECUM OF SQUIRREL. 289 the body. Measurements indicate a diameter of from 3 to 4.5 micra. Anterior to the nucleus lie a number of deeply staining granules, concerning the affinities of which there is so much controversy. Kofoid and Swezy's account of the arrangement of these granules based largely upon their observations of the cyst is somewhat as follows. There lies upon the nuclear mem- brane a centrosome, which is connected by the "nuclear rhizo- plast" with a "primary blepharoplast." This blepharoplast is joined to the "secondary blepharoplast" by means of the "transverse rhizoplast.". Finally, another thin thread-like line connects the secondary and tertiary blepharoplast. Directed somewhat posteriorly from the secondary and tertiary rhizoplast are two deeply staining fibrils, the one (parastyle) supporting the left lip of the cytostome, the other (parabasal body) supporting the right lip. Two of the three anterior flagella take their origin in the primary blepharoplast, one from the secondary. From the tertiary rhizoplast there arises, in addition to the "parabasal body," the cytostomal flagellum and a thread enclosing the mouth, the "peristomal fiber." The cytostomal flagellum vibrates along the edge of an undulating membrane. Dobell and O'Connor deny the existence of any integrated fibrillar system (neuromotor system), or connections between the granules at the anterior end, claiming in addition that there are six granules instead of four. Furthermore, in regard to the peri- stomal fiber they state, "We believe there is no such fibre, and that the mouth is not situated in this position." Despite long and painstaking study the writer was not able to make out all the details of the granule complex with the positive- ness of Kofoid and Swezy. That there is an integrated fibrillar system connecting the granules, however, the writer has no doubt. The rhizoplast connecting the blepharoplasts designated by Kofoid as secondary and tertiary is especially prominent in most specimens because of its thickness and deeply-staining qualities (Fig. 12). A fine rhizoplast running from a granule on the nuclear membrane was occasionally recognized. The best evi- dence for the fibrillar system, however, comes from the dividing stages, which will be described below. The writer was not able to make out the exact affinities of the flagella, because of the ELERY R. BECKER. impossibility of resolving the granules in the granule complex. Fig. 12 represents one of the best observations which the writer \vas able to make. A structure was noted which Dobell and O'Connor neither mention nor figure, but which resembled very much Kofoid and Swezy's figures of the peristomal fibril enclosing a more deeply- shaded granular area, the cytostome. In Chilomastix magna, this structure takes its origin in the blepharoplast complex, and passes posteriorly just dorsal to the cytostome and right supporting fibril (Kofoid's "parabasal body"), if we consider the cytostome to be on the ventral side. It usually extends far beyond the posterior curvature of this fibril into the posterior region of the body (Fig. i). It appears to be surrounded by a fibril. This structure, however, has three dimensions, and is from one third to one half as deep as broad, as proved by polar views of a number of flagellates standing on end. So what appears to be a fibril, is in reality a membrane marking the boundary between this structure and the remaining cytoplasm. Rarely this structure appears to adhere to the right supporting fibril (Fig. 2). In others, no connection between the two rs evident (Fig. i). There is no connection between this body and the cytostomal flagellum, which lies between the right and left supporting fibrils (Fig. i, 2, 12). \Yhat is the function of this organelle? Is it an oral structure associated with the mechanism for the intake of food? It is not visible in the unstained living specimens, so this point could not be determined. Is it a reserve food supply? Failure to stain it either with iodine or intra vitam with Janus green gives no clue. For the present the writer prefers to call it a "parabasal body," because of its non-committal significance of anything as to function, although it does not stain with Janus green, which Shipley (1916) found would color the parabasal body of Trypano- soma lewisi and Becker (1923) found would color the parabasal bodies of Crithidia and Herpetomonas, suggesting their mitochon- drial nature. Besides this structure, no other organelle re- sembling a peristomal fiber was seen. Might it not be that this body is much shorter in Chilomastix mesnili, and that the sharpness of its limiting membrane led Kofoid and Swezy to call it a peristomal fiber? FLAGELLATE FAUNA OF CCECUM OF SQUIRREL. 29 1 The cyst is typically lemon-shaped (Fig. 6). The size is markedly constant, every mature cyst measuring almost exactly 10 by 7.5 micra except one, which measured 12 by 8 micra. By way of comparison the writer measured twelve cysts of Chilo- mastix mesnili. These measured from 6 by 5 micra up to 8 by 7 micra. This indicates that the cyst of Chilomastix magna is somewhat larger than that of Chilomastix mesnili, but there is not the difference in size that there is in the free stages. The cyst contains a nucleus, the cytostomal structures, and the "parabasal body." There is nowhere in the literature anything like a complete account of the cell division of Chilomastix. For this reason it was thought to be quite worth while to spend a large amount of time searching for dividing specimens, although they were extremely rare. As a result a fairly complete account of the process can now be given, although not all points have been cleared up. What the writer conceives to be the resting nucleus is repre- sented in Figs, i, 7, and 12. There may be one blob of chromatin on the inner anterior surface of the nuclear membrane, or there may be one blob here and another in the posterior portion of the nucleus (Figs, i, 12), or there may be a large number of smaller granules lying on the nuclear membrane (Fig. 7). There is a cloud of chromatin granules scattered thruout the entire nucleus. Very often at this time a small deeply staining spot, an endobasal body, or intranuclear kinetic element is visible (Figs. 7, 12). A little later the granules become more concentrated and form a large endosome (Figs. 2, 9, 10). Careful study of this endosome shows that it is sometimes a concentration of granules (Fig. 2) and sometimes apparently a "chromatin knot" (Figs. 9, 10). Often a deeply-staining intradesmose is visible at the points where it passes from the endosome to the blobs of chromatin at one or both poles of the nucleus. The endosomal chromatin becomes resolved into chromosomes, which appear to be dumbbell-shaped. It was not possible to count them exactly in any case, but the number is near ten. Strangely enough, the intradesmose could not be observed at this stage. Perhaps it lay too close to the nuclear membrane, for which there is some evidence to be given later. 292 ELERY R. BECKER. The chromosomes arrange themselves in an equatorial plate, and a spindle forms. Fig. n is an exact drawing of the spindle at the time it first comes into evidence. The poles of the spindle are the blobs of chromatin at the poles of the nucleus in which the intradesmose had previously terminated. There is some evidence that these blobs of chromatin are not the centrioles, but that the centriole lies within them; e.g., Fig. 2 shows what appear to be two centrioles lying against the chromatin blobs. As mitosis progresses the spindle elongates considerably, the nucleus elongating simultaneously and the nuclear membrane commencing to dissolve. The polar blobs of chromatin disap- pear, leaving the tiny centrioles at the poles of the spindle (Fig. 3). The chromosomes of the dividing equatorial plate of the early anaphase become massed together so that it is not possible to count them, although in some cases they may appear as a mass of discrete granules. The oral apparatus of the parent cell (right and left supporting fibrils, blepharoplasts, etc.) has degenerated. The centrioles divide, leaving connecting rhizoplasts between each centriole and its daughter blepharoplast. The daughter blepharoplast divides at least once (perhaps more), and from the products of the division the left and right supporting fibrils of the mouth grow out. The intradesmose is in evidence along the nuclear mem- brane, connecting the centrosomes at the poles of the spindle. The intrazonal spindle fibers disappear in the late anaphase, and the two halves of the equatorial plate are disconnected from one another (Fig. 4). The remains of the spindle attaches the chromatin mass to the centrioles, while a fine rhizoplast connects the centriole with the blepharoplast complex. Fig. 4 appears inconsistent with Fig. 3 in regard to the points of attachment of the intradesmose which traverses the cell. It is extremely difficult to resolve the granules exactly, and the writer has put the intradesmose in each case exactly where it appeared to be. At this stage the "parabasal body" has appeared, and lies in its permanent position, just dorsal to the right supporting fiber. The cell constricts in the middle (Fig. 5). The two daughter cells then reconstruct themselves, each with a complete new set of cell organelles. During division no food particles can be seen in the cell protoplasm. FLAGELLATE FAUNA OF CGECUM OF SQUIRREL. 2Q3 Trichomonas muris (Hartmann) var. citelli. This flagellate was encountered in only three out of twenty ground squirrels. It is a common inhabitant of the coecum of albino, house, and wild mice. The form from the ground squirrel, however, so closely resembles in its important features Wenrich's (1924) description of T. muris that it is considered to be at least a variety of this species. Furthermore, the chromosome count agrees with the number which Wenrich found in T. muris. The size of T. muris on prepared slides, according to Wenrich, ranges from 8 to 22 micra, with an average of 12.9 micra. The average of the body length of 100 individuals from the ground squirrel, not including the protruding axostyle, was 17 micra, somewhat larger than Wenrich's measurements. The reader is referred to Wenrich's excellent account for the detailed description of this species. A rhizoplast connecting the nucleus and blepharoplast was seen by the writer in some cases. Andrews (1925) also saw it in T. termopsidis. One other rather unimportant respect in which the form from the ground squirrel differs from Wenrich's description is in the arrangement of the "inner row of chromatic granules." In the form studied by the writer the granules in this area constitute a field rather than a linear series (Fig. 13). In some specimens a small granular body was noted near the anterior end, although Schaudinn's fixative was used (Fig. 13). This was interpreted to be the parabasal body, as described by Wenrich. What the writer saw was evi- dently a residue left after the mitochondrial substance had been dissolved out. This parabasal body, however, does not stain intra vitam with Janus green. Large numbers of dividing individuals were found. The writer has nothing to add to Wenrich's account. Several good chromosomes counts were made at the anaphase (Fig. 14) and prophase (Fig. 15). The number was six, in agreement with Wenrich. There seemed to be in most cases a fairly definite size range for the prophase chromosomes from one large chromosome down to a very small one (Fig. 15). Multiple mitosis or somatella formation of the nature of that observed and described by Kofoid (1915) was not seen in this material. Trichomonas sp.? This small flagellate was present usually in 294 ELERY R. BECKER. large numbers in all ground squirrels examined. The body is elliptical, with a pointed posterior end, due to the protruding axostyle (Fig. 19). Length is from 6.5 to 10 micra without the axostyle which protrudes from 2 to 3 micra beyond the body. The nucleus lies at the anterior end. In the resting stage there is a round central chromatic endosome, an achromatic nuclear membrane, with a space between the endosome and membrane which is clear except for a sprinkling of fine granules. The endosome is prominently visible even in living unstained speci- mens. A large deeply staining basal body lies anterior to the nucleus. From it arise an axostyle which curves about the nucleus, and passes to the posterior end of the body; three an- terior flagella; and a fourth flagellum which is directed toward the posterior. It lies in close contact with the body for about half the length of the cell, and the remaining length trails behind. Typically, members of the genus Trichomonas possess an undulating membrane with a chromatic basal rod; e.g., Tricho- monas muris. This flagellate possesses no true undulating membrane, since the posterior flagellum which is the homologue of the marginal flagellum lies close to the body. It vibrates only slightly in the anterior region, while the free posterior end is quite active. Stained preparations show no chromatic basal rod. Whether these differences are sufficient to create a new genus is doubtful. Stained preparations often show a large open cyto- stome (Fig. 17). In living preparations it is usually closed. Very few dividing forms were found, and hence no chromosome counts were made. Sometimes this flagellate is parasitized by a Sphaerita (Fig. 19). Tetratrichomastix citelli sp. nov. This flagellate (Figs. 16, 18) was present in nineteen of the twenty ground squirrels. The body is irregularly pyriform, but approximately bilaterally symmetrical, due to a slight indentation just behind the anterior tip of the body from which the flagella leave the body. The axostyle protrudes from the posterior tip of the body. The body length, exclusive of axostyle, ranges from 7 to 13 micra. The axostyle often extends 2 to 4 micra beyond the body. The flagella are five in number, four anterior ones which beat back- wards in unison, with lashing strokes, and a schleppgeisel, which FLAGELLATE FAUNA OF CCECUM OF SQUIRREL. 295 does not come so far forward as the others in its lashings. The flagella arise from a blepharoplast situated on the anterior surface of the nucleus. It is almost impossible to count the flagella in the living cell, because of their peculiar movements. A large number of favorable observations in the stained specimens makes it fairly certain that the number is five. The nucleus is round, and possesses a tiny endobasal body embedded in a mass of granules (Fig. 16). Sometimes only a larger deeply staining endosome is seen (Fig. 18). About the nucleus is sometimes seen a cloud of granules (Fig. 16). The axostyle passes from the anterior end to the posterior tip. It stains lightly. A number of favorable observations show that it originates in the basal granule. A cytostome is often indistinctly seen at the anterior end. Hexamitus pulcher sp. nov. This Hexamitus, found in eighteen of the twenty ground squirrels, is characterized by the presence in the living cell of a large number of slightly refringent spherules which stain intra vitam green with Janus green at first, then later are reduced to the red. These granules dissolve out for the most part in Schaudinn's fixative, although the cytoplasm of the stained cells is markedly granular (Figs. 20, 21). It was because of the wealth of mitochondria and the beauty of the vital stain with Janus green that the specific name pulcher was given. The body is irregularly oval, bilaterally symmetrical, and somewhat plastic. The size does not vary much from eight to ten micra in length by six or seven micra in width. The flagella are eight in number, the anterior pair projecting almost at right angles to the body and vibrating actively in the living cell. Two other pairs are inclined to wrap themselves about the body giving it the deceptive appearance of possessing several undulating membranes. The posterior pair leaves the body at the posterior tip. Between them the posterior tips of the axostyles protrude. Stained slides show the cell structures more plainly (Figs. 20, 21). In the anterior region on each side of the median line is an elongated, deeply staining, nucleus. Between the nuclei lie a pair of blepharoplasts from which the flagella arise, altho some- times there seem to be two pairs. A pair of axostyles in the axis 296 ELERY R. BECKER. of the body terminate at about the location of the blepharoplasts. In the anterior tip of the cell is a dark spot connected to each axostyle by a fine fiber. SUMMARY. The intestines of twenty ground squirrels (Citellus tridecemli- neatus) were examined for protozoa. The following species of protozoa were found to be present: Giardia sp.?, Endamceba citelli, Chilomastix magna sp. nov., Trichomonas muris var. citelli, Trichomonas sp.?, Tetratrichomastix (Eutrichomastix) citelli sp. nov., and Hexamitus pulcher sp. nov. The first was found in the small intestine, all the others in the coecum. In this paper all the above species except the first two are described, particular attention having been given to the morphology of the free Chilomastix magna and to its division phases. So far as is known, all these protozoa are commensals. Whether or not they assist in the digestion of cellulose, which is said to take place very largely in the cceca of certain animals, is unknown. BIBLIOGRAPHY. Andrews, J. M. '25 Morphology and Mitosis in Trichomonas lermopsidis, an Intestinal Flagellate of the Termite, Termopsis. BIOL. BULL., 59: 69-85. Becker, Elery R. '23 Observations on the Life Cycle of Crithidia gerridis Patton in the Water- strider, Gerris remigis Say. Jour. Paras., 9: 141-152. "23 Observations on the Life History of Herpetomonas musca-domesticce in North American Muscoid Flies. Jour. Paras., 9: 199-213. Dobell, C., and O'Connor, F. W. '21 The Intestinal Protozoa of Man. London. Kofoid, C. A., and Swezy, O. "15 Mitosis and Multiple Fission in Trichomonad Flagellates. Proc. Am. Acad. Arts and Sc., 51: 289-378. '20 On the Morphology and Mitosis of Chilomastix mesnill (.Wenyon). Univ. Calif. Pub. Zool., 20: 117-144. Shipley, P. G. '16 Vital Staining in Trypanosoma lewisi. Anat. Rec., 10: 439-445. Wenrich, D. H. '21 The Structure and Division of Trichomonas muris (Hartmann). Jour. Morph., 36: 119-147. 2Q8 ELERY R. BECKER. EXPLANATION OF PLATE. X ca. 1950. FIG. i. Chilomaslix magnet, free form, showing a tuft of cortical projections in flagellar region, and dorsal view of the cytostomal structures. Large granular body dorsal to cytostomal organelles is the "parabasal body." FIG. 2. Chilomaslix magna, free form, dorsal view with "parabasal body" apparently following right supporting fiber of cytostome. FIGS. 3, 4, 5. Chilomastix magna, free form, in division. FIG. 6. Chilomastix magna, cyst. FIGS. 7 TO 11. (Not according to scale.) Various nuclear appearances found in Chilomaslix magna. FIG. 12. (Not according to scale.) Dorsal view of nucleus, integrated fibrillar system and cytostomal structures of Chilomaslix magna. FIG. 13. Trichomonas muris var. cilelli. FIG. 14. Trichomonas muris var. cilelli, dividing, nucleus in anaphase. FIG. 15. Trichomonas muris, prophase nucleus of Trichomonas muris showing six chromosomes. FlGS. 16, 18. Telratrichomaslix cilelli, free forms. FIGS. 17, 19. Trichomonas sp.? Individual in Fig. 19 infected by a Sphcerila. FIG. 20. Hexamitus pulcher, side view. FIG. 21. Hexamitus pulcher, front view. BIOLOGICAL BULLETIN. VOL. LI. PLATE I. ELERY R. BECKER. VARIATION IN THE NUMBER OF FIN-RAYS IN THREE SPECIES OF FUNDULUS OF THE WOODS HOLE REGION. ROBERT PAYNE BIGELOW. While engaged in the study of the feeding habits of young fishes in August, 1924, the author collected in Woods Hole Harbor a number of young Fundulus having a total length of from 9 to 51 mm. All except the youngest of these fish showed the darkly pigmented vertical bands, 9 to 1 1 in number, characteristic of the young of all three of the species of Fundulus known to occur in the vicinity of Woods Hole. (Jordan & Evermann, '96 ; Newman, '07; Sumner et al., '13.) The color markings give no clue to specific identity at this stage, and the anatomical proportions, changing as they do with growth, are equally useless for identification. On the other hand, certain supporting structures, such as vertebrae and fin-rays, are developed at an early stage and remain constant in number throughout life. Therefore it was hoped that the number of fin- rays would furnish a means of assigning these young fish to their proper species. The number of fin-rays in the dorsal and anal fins of the smallest fishes in the collection (total length 9 and 12 mm.) were found to be D 9, A 7 and 9. The two next in size (14 and 20 mm.) had in both D n, A 10. All the others had D 13 to 15, A 9 to n, of these seven out of ten had more than 13 dorsal rays. Turning to Jordan and Evermann '96 for identification, the fin-ray formulae for the species known to occur at Woods Hole were found to be given as follows : F. heteroclitus , Dn,Aioorn; F. majalis, D 12, A 10; F. diaphanus, D 13, A n. From the fin- formulse alone it might have been supposed that the specimens in question were F. diaphanus. But this was improbable because they were taken in the pure sea water of Woods Hole Harbor, whereas F. diaphanus is supposed to inhabit only brackish or fresh water. On the other hand, the number of rays in the dorsal fins was much greater than the numbers given for the two purely marine species. 299 300 ROBERT PAYNE BIGELOW. Therefore it was decided to make a fresh examination of the fin structure in the adults of the three local species, and a thorough search of the literature on this subject. The results of this investigation are given in the present paper. The divergence of the present results from those obtained by previous investigators may be due to the difficulties of obser- vation. The fin-rays of Fundulus are all of the soft kind. Each ray consists of two parallel columns of segments bilaterally arranged. In the anal fin the first and last of the rays are unbranched. The other rays are branched dichotomously, one half to one third of the distance from the base, and usually the longer branches are bifurcated again. In both the dorsal and the anal fins the ray bordering the posterior margin is frequently very slender and easily overlooked. In some specimens there are two very slender posterior rays closely united at the base. In preserved specimens that have been hardened with the fins flexed, the folding of the fin makes it very difficult to observe the posterior rays. In the present investigation observations were made with a binocular microscope and transmitted light. Some of the specimens were examined while fresh. Most of them were killed and preserved in 5 per cent, neutral formalin in sea water, after being anaesthetized by a few crystals of chloral hydrate inserted in the mouth. The specimens to be killed were placed in the formalin one at a time and the dorsal and anal fins were quickly grasped with forceps and held in an extended position until sufficiently hardened. In order that the specimens should lie flat in the dish during the remainder of the hardening process, the air-bladder was punctured by an incision in the body-wall just above the tip of the pectoral fin and parallel to its dorsal edge. By this method specimens were preserved with the dorsal and anal fins in the best posture for observation. In the transparent fins of F. diaphanus prepared in this way and viewed with the binocular microscope, it was easy to count the fin-rays when illuminated by transmitted light. In the two other species pigmentation of the fin often obscured the rays. This is especially true of the densely pigmented dorsal fin of F. hetero- clitus, on which it was at times necessary to use a dissecting needle. FIN RAYS IN THREE SPECIES OF FUNDULUS. 301 The anal fin of the females of F. heteroclitus presents another difficulty. On the anterior edge of this fin, and extending from its base nearly half way to the tip, is a thick fold of skin enclosing the oviduct (as stated by Jordan and Evermann '96). The thickened skin extends backward over the bases of the first and second rays, at least, and may make a count impossible without dissection. The adult material for this study consisted of 40 males and 37 females of F. heteroclitus and 10 males and 22 females of F. majalis, both species from a small salt water pond connected with the head of Woods Hole Great Harbor, and also of 10 males and 1 6 females of F. diaphanus from Oyster Pond (fresh water) in Falmouth, Massachusetts. Although the number of specimens is too small for statistical accuracy, the results obtained show that the populations repre- sented have considerably more variation in the number of fin-rays than is indicated by the previously published descriptions of these three species. Full descriptions have been published by Bean, '92 and '03, Garman '95, Jordan and Evermann '96, Smith '07. In the following table the results of the present investigation are given in comparison with those of the authors just mentioned. Present Results. Previous Authors. Species. Number. c £ Number of Fin-rays. M o\ . ro c j= 1* Garman. Jordan & Evermann. .c -4-» a en V w> cd ti +j •<-> 1 C! § 0 OJ s£ & C a 3°)- The new macro-nucleus at first is spherical, but later may assume different shapes as indicated above. The conjugants become separated during the formation of the new nuclei. According to Enriques ('08), in Chilodon uncinatus there is a single division of the conjugation nucleus and that the two daughter cleavage nuclei become differentiated into the new macro and the new micro-nucleus respectively. THE OLD MACRONUCLEUS. The macronucleus of the free swimming forms, as indicated above, is rather variable, ranging in shape from a circular form to an elongated condition, or at times it is somewhat curved. During early conjugation the nucleus when elongated shortens slightly and frequently divides into two or more parts (Figs. 16 and 17). But in most cases it does not divide at all (Figs. 20 and 21). The chromatin becomes more and more condensed and stains as a dark homogeneous body. The border of the dark staining nucleus fades out, leaving a dark center within a clear boundary (Figs. 23 and 25). Finally the dark center fragments 314 GEORGE W. TANNREUTIIER. into many small bodies and disappears within the cytoplasm leaving a clear unstained outline (Figs. 28-30), which is later absorbed. The old macronucleus sometimes persists in the exconjugants after the formation of the new micronucleus and macronucleus are complete, before the beginning of its dis- integration. Occasionally when the macronucleus of one of the conjugants is situated at the anterior end in close proximity to the cytoplasmic bridge as in figure fifteen, it would be carried over to the opposite conjugant, due to the movement of the cytoplasm within. The two macronuclei within the same conjugant however, pass through the usual stages in their disintegration. This passing over of the macronucleus through the cytoplasmic bridge from one conjugant to the other is quite common in one of the species of Chilodon, where its passage can be followed in the living forms. In Chilodon, however, not only the macronucleus may pass in toto from one conjugant to the other, but the macronucleus of either conjugant divides, one of the resultant daughter nuclei acting as the moving nucleus and the other as the stationary nucleus, comparable to the micronuclear activities in normal conjugation. GENERAL REMARKS. Conjugation and binary fission of the ciliate P. griseus, to my knowledge, has never been described. The general activities however, in reproduction for the most part agree with the results of investigators as found in other ciliates. One of the chief points of interest in studying the different stages in the life history of the ciliate P. griseus, is the direct association of binary fission and conjugation with encystment. The entire process of cell division may occur within a given cyst. While the first steps in conjugation always occur (as far as my observations go), in the free swimming forms and encysts later when the union of the two conjugants is complete. Temporary encystment however, can not be considered as essential for binary fission and conjugation. Since both may occur in the free forms. Encystment here acts more as a protection in unusual or adverse conditions. In no instance observed did the daughter cells pro- duced by binary fission within the cyst, undergo conjugation before their escape. LIFE HISTORY OF PRORODON GRISEUS. 315 P. griseus differs from most Holotrichs, in the first stages of conjugation, in that each conjugant contracts, becomes sperical, and rotates vigorously. The sides in contact are somewhat flattened, with their sub-terminal oral apertures facing each other. They remain in this spherical shape until a point of cytoplasmic union is complete (Fig. 70). Then they elongate (Fig. 3), and move freely through the culture until conjugation is complete. A greater number of the paired conjugants as a rule remain contracted, rotate on their long axes and secrete a temporary cyst in which they remain for the greater part or the entire period in the completion of conjugation (Fig. 26). In this connection it may be of interest to note that no conju- gating individuals were found out of doors or under wild con- ditions. Although many of the pools were found in unfavorable conditions due to evaporation. In fact very few free-swimming forms were found at any one time when collections were made. Most of the individuals were encysted. But when placed under laboratory conditions, within a few hours the greater number of individuals escaped from the cysts, and became quite active. This was followed immediately by a regular epidemic of conju- gation. Very few single individuals could be found in the cultures. Again within three to five hours very few conjugating individuals were present. The exconjugants during their free- swimming period fed quite actively and reproduced by binary fission. In about five hours encystment again occurred. Thus beginning a new cycle. A few active forms however, can be found in the cultures at all times. It is maintained by Calkins and others that encystment (especially where cellular reorganization occurs), and conjugation are both conducive to renewed vitality. While by Mast and others it is held that neither encystment nor conjugation have any appreciable effect in producing new vigor. In P. griseus however, we have conjugation immediately following encystment or even the conjugants may encyst during their nuclear exchange and reorganization. Here we might conclude that the exconjugants would receive unusual renewed vigor and increase the fission rate. This however, is not what we find in P. griseus. It is true that an epidemic of cell division GEORGE \V. TANNREUTHER. takes place in the exconjugants. But if we isolate an equal number of the exconjugants that encysted during conjugation, and an equal number of those that failed to conjugate immedi- ately after encystment. We find that the rate of fission per given number is about the same in the two isolated lots. Hence it may be safe to conclude that simultaneous encystment and conju- gation do not produce renewed vigor or at least increase the fission rate in P. griseus. FIG. 31. Prorodon griseus in the early completion of the permanent cyst with all the organs in tact. FIG. 32. Later stage of encystment with the disappearance of the cilia and the breaking up of the pharynx. The micronucleus and the contractile vacuole(?) persist. The macronucleus is in its early stage of metamorphosis, c. v., contractile vacuole. FIG. 33. The macronucleus in its later stage of metamorphosis. Several cytoplasmic vacuoles are present. FIG. 34. Final stages of metamorphosis of macronucleus or cytoplasmic reorganization of structures during encystment. The missing organs have re- formed. The new macronucleus is a direct metamorphosis of the old. Encystment in the ciliates according to Calkins may serve a threefold purpose. That is for protection, reproduction and reorganization. In P. griseus there are two types of cysts which we have designated as thin or temporary and thick or permanent. The two types differ in the thickness and the compactness of their walls. The temporary cysts are quite flexible and yield to the moving ciliate within. The temporary cyst serves two purposes: first for protection against sudden adverse conditions such as LIFE HISTORY OF PRORODON GRISEUS. 317 temperature, scarcity of food and evaporation. The ciliate if disturbed may remain in the temporary cyst less than an hour, and immediately after escape form a new cyst. The organism during temporary encystment does not undergo any internal changes. It maintains its spherical shape and continues to rotate. The second purpose is for reproduction by transverse division. Likewise conjugation, except the early fusion of the conjugants, may occur within the cyst. The third purpose is for the reorganization of the cell. The cyst in this case is of the permanent type, its walls being thick and dense. The individual becomes quiescent, and loses its cilia and pharynx during internal reorganization (Figs. 31-33). In this latter case the organism can not be recovered from the cyst within a period of at least four or more days or until its reorganization is complete. The micro-nucleus and contractile vacuole persist throughout the reorganization without undergoing any appreciable change. The macro-nucleus however passes through a distinct series of changes as indicated in Figs. 31 to 34. Fig. 31 represents the condition at the beginning of encystment when all of the organs are present. The macro-nucleus is horse-shoe shaped, but in many individuals it is spherical. In the first steps of macro- nuclear metamorphosis the chromatin becomes ragged (Fig. 32), with a clear border beneath the nuclear membrane. The central region of the chromatin remains dark. Fig. 33 shows a third stage in which the chromatin has taken a more spherical shape with the ragged edges fraying out. Finally in Fig. 34 the macro- nuclear change is complete, the more central portion of the chromatin persisting as the new nucleus. The organs which were lost have reformed. The macro-nuclear changes in P. griseus are similar to the conditions found by Stolte, in Blepharisma undulans and by Brand, in Vorticella microstoma and in Stylonychia mytilus. The macro-nuclear activities during encystment are considered by them as a cytoplasmic reorganization process since no new nuclei are formed. According to Stolte cytoplasmic depression, oc- casioned by adverse external conditions leads to encystment, and nuclear depression leads to conjugation. In Didinium nastutum however, as reported by Calkins and in Stylonychia pustulata as 21 318 GEORGE \V. TAXNREUTHER. reported by Fermor, the macro-nucleus disintegrates and a new micro-nucleus and a new macro-nucleus are formed from the old micro-nucleus, thus undergoing a complete nuclear reorganization comparable to conjugation. GENERAL SUMMARY. 1. Prorodon griseus is a Holotrichous ciliate. It is recognized by its sub-terminal oral aperature, its rod like structure enclosing the pharynx, and its terminal contractile vacuole. 2. P. griseus has a single inicronucleus and a single macro- nucleus. 3. Binary fission and conjugation occur either while encysted or in the free forms. 4. Encystment serves for protection, reproduction and re- organization. 5. Cysts are of two kinds: (a) temporary for protection during short intervals. (£) permanent for reorganization of cell and protection for long intervals, from one cycle to the next. 6. Conjugation is terminal, fusion occurring at their anterior ends. Conjugation occurs immediately after the animals escape from the cysts. 7. The aniir/als are found encysted during the greater part of their life history. The greater number of free forms are found immediately after conjugation, during the active feeding period. 8. Encystment and conjugation in P. griseus have little or no effect in the production of new vigor and in the change of fission rate. 9. The old macronucleus persists during early conjugation, then disintegrates and disappears by absorption within the cytoplasm. 10. The new micronucleus and macronucleus are formed from the daughter nuclei, produced by a single division of the conju- gation nucleus. 1 1 . The reduction of chromosomes occurs in the second maturation division by a pairing of the eight chromosome in the formation of the four bivalent structures. 12. In the third division the pronuclei at first are connected by a drawn out fiber. LIFE HISTORY OF PRORODON GRISEUS. 13. The pronuclei elongate slightly during their process of fusion. 14. In cellular reorganization within the permanent cyst, the cilia and pharynx disappear. The micronucleus and contractile vacuole (?) persist. The macronucleus passes through a distinct metamorphosis. 15. Reorganization during permanent encystment in P. griseus is of the cytoplasmic or passive type. Nuclear reorganization or formation of a new micronucleus and a new macronucleus from the old micronucleus was not found to occur. COLUMBIA, Mo., JULY 6, 1925. LITERATURE CITED. Brand, Th. v. '23 Die Encystierung bei Vorticella microstoma und Hypotrichen Infusorien. Arch. Protist., Vol. 47. Calkins, G. N. '07 The Conjugation of Paramcecium caudatum. Arch. Protist., Vol. 10. '12 The Pcedogamous Conjugation of Blepharisma undnlans, Journ. Morph., Vol. 23. '15 Didinium nasutum. I. The Life History. Jour. Exp. Zool., Vol. 19. '16 Encystment of Didinium nasutum. Science, N. S., Vol. 43. "19 Uroleptus mobilis. I. History of the Nuclei During Division and Conju- gation. Jour. Exp. Zool., Vol. 27. Dogiel, V. '25 Die Gesclechtsprozesse bei Infusorien (special bei den Ophryoscoleciden), neue Tatsachen und theoretische Erwagungen. Archiv. Protist., Vol. 50. Enriques, Paolo. '08 Die Conjugation und sexuelle Differenzierung der Infusorien. Arch. Protist., Vol. 12. Fermor, H. '13 Die Bedeutung der Encystierung bei Stylonychia pustulala. Zool. Anz., Vol. 42. Gregory, Louise H. '23 The Conjugation of Oxtricha fallax. Journ. Morph., Vol. 37. Hamburger, C. '04 Die Conjugation von Paramcecium bursaria Foche. Arch. Protist., Vol. 4. Kalterboch, R. '16 Die Conjugation von Ophrydium versalite. Arch. Protist., Vol. 36. Kent, W. S. '80-82 Manual of Infusoria. 3 Vols., London. Mast, S. O., and Yasushi, Ibara. '23 The Effect of Temperature, Food and Age of the Culture on the Encystment of Didinium nasulum. BIOL. BULL., Vol. 45, No. i. Mayer, Martin. '19 Zur Cystenbildung von Trichominas muris. Arch. Protist., Vol. 40. 320 GEORGE W. TANNREUTHER. Moore, E. Lucile. '24 Endomixis and encystment in Spathidium spathula. Jour. Exp. Zool., Vol. 39, No. 2. Prandtl, H. '06 Die Konjugation von Didinium nasulum. O. F. M. Arch. Protist., Vol. 7. Stolte, Hans-Adam. '24 Morphologische und Physiologische Untersuchungen an Blepharisma undulana Stein, (studien iiber den Formwechsel der Infusorien). Arch. Protist., Vol. 48. Swellengrebel, N. H. '17 tiber die Cystenbildung des Chilomastrix mesnili Wenyon. Arch. Protist., Vol. 38. STUDIES IN THE LIFE HISTORY OF EUGLENA* I. EUGLENA AGILIS, CARTER. WOOLFORD B. BAKER, COLUMBIA UNIVERSITY, ZOOLOGICAL LABORATORY, AND EMORY UNIVERSITY, DEPARTMENT OF BIOLOGY. CONTENTS. I. INTRODUCTION 321 II. ACKNOWLEDGMENTS 322 III. MATERIAL AND METHODS 322 IV. GENERAL MORPHOLOGY 324 1 . Diagnostic Characters 324 2. Gullet and Reservoir 325 3. Nucleus 326 4. Chromatophores 327 5. Motor Apparatus 327 V. REPRODUCTION 328 1. Nuclear Division 329 (a) Changes in the Endosome 329 (&) Formation of Chromosomes 330 (c) Migration of the Nucleus 331 (d) Metaphase 331 (e) Anaphase 332 (/) Telophase 332 2. Division of Gullet and Reservoir 333 3. Division of the Body 334 4. Behavior of Chromatophores During Mitosis 335 5. The Flagellum and History of Kinetic Elements 336 VI. DISCUSSION 337 1 . The Resting Nucleus 338 2. The Endosome 339 3. Mitosis 347 4. The Residual Mass from the Kinetic Complex 350 VII. SUMMARY AND CONCLUSIONS 352 VIII. LITERATURE CITED 354 IX. DESCRIPTION OF PLATES 360 INTRODUCTION. Attention has been called many times to the importance of research on the Flagellates because of their relatively simple structure, their apparent position at the bottom of the scale of * Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University. 321 322 WOOLFORD B. BAKER. living organisms, their partaking in many instances of character- istics of both plants and animals, their universal distribution and their contributions to our knowledge of general principles of biological phenomena. Many workers have engaged in such investigation and much progress has been made. The present investigation was undertaken on the Euglenoidina because of the primitive character of the organisms composing the group, their wide distribution, their general use as types in fundamental courses in biology and the fact that the literature does not show a complete and critical account of the cycle of any one of them with attention given to all of the organoids of the cell during the successive stages in the cycle. Solutions have been sought to four main problems through the study of: (1) the origin and behavior of the chromatic elements of the cell; (2) the nature and behavior of the endosome of the nucleus; (3) the origin and behavior of the locomotor apparatus during mitosis; (4) the location of the division center. ACKNOWLEDGMENTS . The desirability of critical work on Euglena was originally suggested to me by Professor Robert C. Rhodes of Emory Uni- versity in 1919. I desire to express my appreciation to him for much of the earlier criticism and suggestions as well as for the original plan of the work. The investigation was completed under the direction of Professor Gary N. Calkins of Columbia University. I am indebted to him for his suggestive interpre- tations and criticisms, both constructive and destructive, as well as for his continued encouragement and inspiration, beginning at Woods Hole in 1922, continuing at Columbia during 1924-25 and at Woods Hole in the summer of 1925. I am especially in- debted to my wife, Bernice Hall Baker for her painstaking care in the preparation of the figures. MATERIAL AND METHODS. Several species of Euglena from wild cultures have been col- lected and identified. Attempts have been made to cultivate all STUDIES IN THE LIFE-HISTORY OF EUGLENA.. 323 of them in order to secure material for detailed study. It has been found however, that a wide variation exists in the type of culture medium required for the different species, and that when one says a culture medium has been discovered for Euglena the statement means little unless the species is indicated. A small amount of green scum known to contain Euglena, found on the surface of a culture of pond water, leaves and grass, was placed in a large petri dish containing one liter of tap water and one gram of malted milk. An abundance of active Euglenx were produced in a few days and identified as Euglena agilis. From this original culture all the material for my sub-cultures used in the investigation were secured. The most satisfactory culture medium for this species has been found to be split pea infusion made by boiling forty split peas in one liter of tap water. Sufficient citric acid is added to keep down extensive growth of bacteria. For keeping cultures in an inactive condition, quince seed jelly as recommended by Turner (1917), has been used. This medium has also been satisfactory for the study of the animals in the living state under the micro- scope. Inoculation of split pea infusion with material from the quince seed jelly produces abundant active forms within a few days. Various killing fluids have been used, the best results being obtained with Schaudinn's fluid heated to 56 degrees Centigrade. Other fluids used were Flemming's strong, Bouin's picro-formol solution, chrom-acetic-osmic mixture, Zenker's fluid and osmic acid vapor. For staining, aqueous iron-alum hsematoxylin, long method, has given the most satisfactory preparations. The use of a counter stain has aided in the study of the motor apparatus. Eosin and bordeaux red (.1 per cent, solution for 24 hours before mordanting) have given the best results. All figures have been drawn from material counter-stained with eosin. Other stains used were Delafield's haematoxylin, safranin and light green, Flemming's triple, Borrell's stain, and iron alum haematoxylin counter stained with light green or orange G. The organisms were killed at different times during the day, the most satisfactory material being secured from eight to twelve 324 WOOLFORD B. BAKER. P.M. During that period the divisions stages are the most numerous. Twenty cc. of the material was taken at thirty minute intervals, centrifuged, killed, and either stained en masse or taken from the centrifuge tube, placed on cover glasses and stained according to that method. The first method gives good results when the material is abundant. Material killed at ten P.M. gave the greatest number of stages of mitosis. After twelve P.M. most of the divisions initiated around eight P.M. have been completed, and from twelve P.M. to six A.M. very few dividing forms can be found in the culture. The animals in a culture appear to be active during the day but usually about five P.M. they become less active, some assuming a spherical shape then extending the body again and again as if undergoing intense metabolic activity. This difference in the cyclical phases of Euglena agilis as observed at different times of the day, and especially the nocturnal incidence of dividing forms may be explained perhaps by the difference in the carbon dioxide content of the medium at different times. At evening and during the night the photosynthetic processes which have gone on during the day are retarded and cease. The normal activity of the animals would then increase the relative amount of carbon dioxide present and perhaps influence the changes which are observed. Sufficient data have not yet been obtained to prove the validity of such an assumption. GENERAL MORPHOLOGY. The description of Euglena agilis as given by Carter and quoted from Kent is as follows: "Body somewhat flask-shaped, inflated and widest posteriorly, attenuate anteriorly; a short, blunt caudal prolongation some- times present, but not essential; multiplying in the active condition by longitudinal and transverse fission, and in its passive or encysted one by crucial or by linear segmentation ; color green, movements very active. Length 1/600 inch." The species used in the present study conforms to this de- scription except for the fact that it does not divide by transverse fission. In addition there are certain characters observed which seem to be diagnostic: STUDIES IN THE LIFE-HISTORY OF EUGLENA. 325 (1) three to seven oblong, flattened chromatophores present, each with an uncovered pyrenoid in the center; (2) the periplast is more rigid than in many other forms studied so that the body does not show typical euglenoid move- ment when in the active condition ; (3) flagellum about two thirds the body length; (4) paramylon bodies small and not very abundant. Gullet and Reservoir. This structure has been described and figured for many flagel- lates, including Astasia ocellata and Euglena viridis (Khawkine, 1886); E. viridis (Wager, 1899); E. sanguined (Haase, 1910); E. Ehrenbergii (Hamburger, 1911); Astasia levis (Belar, 1916); Peranema (Hartmann and Chagas, 1910); Eutreptia (Steuer, 1904); Copromonas (Dobell, 1908); Menoidium (Hall, 1923). In E. agilis a somewhat funnel like depression is located at the anterior end and leads directly into the gullet which curves as it passes inward toward the side of the animal on which the stigma is located. The reservoir seems to be in direct connection with the gullet; it is asymmetrical in shape, the largest portion being toward the inner side of the body (Fig. i). The vacuole system in connection with the reservoir has not been made out in E. agilis but in some of the larger species examined, E. polymorpha, E. caudata, E. sanguinea and E. spirogyra, a series of small pulsating vacuoles have been seen discharging their contents into the reservoir along its periphery. The function of the gullet-reservoir system is not definitely known. Khawkine (1886) suggested that it was used for the admission of liquid food, giving as evidence the accumulation of small granules of paramylon near the gullet when the animals were fed potato starch in the dark. Kent describes observations made on Euglena viridis kept in carmine suspension, where he observed the passage of fine particles of the carmine into the body by way of the gullet. Wager (1899) considers it an excretory system. Rhodes and Kirby (Mss.) find that Hetronema, a related form, lives on a diet of Euglena proxima, which they have observed being taken in through a specially modified cytostome supported by a horseshoe-shaped lip and two deeply staining pharyngeal 326 WOOLFORD B. BAKER. rods. This structure is separate and distinct from the gullet- reservoir system as described in Euglena. They conclude that the reservoir functions primarily in the reception of the discharge of contractile vacuoles which are located on its periphery. Our own observations indicate that all these functions may be exer- cised by the system in Euglena. The Nucleus. The nucleus of Euglena agilis lies at the center of mass of the body, being located in the region of the center of the posterior flask shaped enlargement. It is spherical to oblong or ovoid in shape. Its chief characteristic is the presence of a large, deeply staining, central body or endosome, the "Binnenkorper' of Doflein and Tschenzoff. Immediately surrounding the endosome is a light hyaline area apparently devoid of chromatin material. The presence of this hyaline layer enables one to observe more readily the budding off of the motor complex, as described below, from the endosome during the early stages of mitosis. In the resting nucleus, chromatin granules arranged apparently at the nodes of a linin network, surround the hyaline area. During the early prophase of division distinct chromosomes are formed by the growth and aggregation of these chromatin masses. When preparations stained in iron-alum hajmatoxylin and counter stained with eosin, are destained considerably, the endosome retains the hsematoxylin so as to appear quite dark while the chromosomes appear red with the eosin. This seems to indicate that there is a great deal of linin in the network on which the chromatin masses are lodged. In most instances the masses seem to occur in pairs giving the appearance of dumb-bells. Wenrich describes somewhat the same appearance of the chromatin in Trichomonas. He says that in the anaphase each chromosome becomes constricted and passes into the resting nucleus as a paired structure. A nuclear membrane can be distinguished with difficulty with the stains used, yet the shape of the nucleus and its apparent separation from the cytoplasm certainly indicates its presence as well as its persistence during mitosis. The endosome appears homogeneous, although vacuoles or STUDIES IN THE LIFE-HISTORY OF EUGLENA. 327 light areas which appear as vacuoles, of varying number and distribution, can be observed at all times during the cycle. No centriole has been observed within the mass. The Chromatophores. The presence of chromatophores is characteristic of all but two species of Euglena heretofore described. They occur in varying numbers in the different species and are usually described as being of characteristic shape for each species. In E. agilis they appear differently at different stages in the cycle. In the resting stages they are rod shape, arranged parallel to the long axis of the body, and with a distinct pyrenoid in the center. During the stages preparatory to division as well as during the reconstruction of the daughter cells after division, they appear to be more rounded or oblong, much larger and with the pyrenoid quite indistinct or apparently absent. The number likewise is variable, since they are apparently distributed to the daughter animals in some cases before the cell divides and in other cases they divide before cell division and thus give a larger count in the body. These facts lead one to believe that this feature of the cell of Euglena is not a satisfactory basis for the classification of the group. A few preliminary observations on the effect of varying H-ion concen- tration in the culture medium indicates that the form, number and arrangement of the chromatophores is affected by changes in the culture medium. More work needs to be done on this point. The Motor Apparatus. This consists of a single flagellum and related structures as described below. It extends through the gullet, usually lying on the side nearest the stigma or eye-spot, and ends at one side of the lower border of the reservoir in two distinct branches, each terminated by a granule. The two branches are unequal in size and structure and perhaps also in function. The branch nearest the side of the gullet and reservoir is larger than the other and has a lens shaped enlargement at the point where the gullet widens into the reservoir, apparently underneath the stigma. This arrangement is similar to that described by Wager for E. viridis. The continuation of this branch appears to form the external 328 WOOLFORD B. BAKER. flagellum. The smaller branch passes from its anchoring granule through the reservoir to a point opposite or above the lens-shaped enlargement on the larger and there fuses with it. In the early stages of development after division the two branches appear to be entirely separate, though the larger grows more rapidly and extends from the cytostome before fusion with the smaller. In some few cases observed there is an indication that the two branches may remain separate not only at the outset but perma- nently. The granule at the base of the larger branch seems to originate first from the kinetic center and is therefore interpreted as the blepharoplast. This divides and gives rise to the granule at the base of the smaller branch which granule is here designated the basal granule. During the late prophase and continuing for some time after division is completed there may be seen a rhizoplast extending from the blepharoplast into the cytoplasm and ending on the nuclear membrane in a heavily staining body which originates in the early prophase from the endosome. In older animals such a connection cannot be detected, indicating that direct connection with the nucleus is not necessary for the proper functioning of the motor apparatus. The early connection of the various parts seems sufficient grounds however, to designate the motor appa- ratus of Euglena agilis as a neuro-motor system as defined by Kofoid and his students. Their definition involves an integrated motor system connecting with the nucleus. (Kofoid and Christiansen, 19150, b; Kofoid, 1916; Swezy, 1916; Boeck, 1917; Kofoid and Swezy, 19190, b, 1920, 1922; Hall, 1923.) It is true that only during the later division stages and for a short time after binary fission there is a complete integration of nucleus and motor apparatus, even to the presence of an intra-nuclear rhizoplast (Fig. 13). The presence of these rhizoplasts is merely reminiscent of the process of development of the parts of the motor apparatus, as will be described later, since they are not permanent structures of the cell. REPRODUCTION. The typical method of reproduction as described for Euglenidae is by longitudinal fission, accompanied by division of the nucleus STUDIES IN THE LIFE-HISTORY OF EUGLENA. 329 and varying behavior of other organoids of the cell. This division occurs in the motile state in some species, in a quiescent but not encysted condition in others while in some a true cyst is formed before the organism divides. In the encysted condition the division may be binary or multiple. Some of our observations indicate that some species may divide in the free swimming condition under proper conditions but when the conditions arise which induce encystment, these conditions being either internal or external or both, the divisions may take place in the cyst. The special problems of encystment are now under investigation and will constitue the subject matter for a later paper. In this paper we are concerned with the details of the division process in Euglena agilis. Nuclear Division. Three principal changes characterize the beginning of nuclear division : (a) changes in the endosome and the budding off of the kinetic complex; (6) formation of the chromosomes^ (c) migration of the nucleus anteriorly. (a) Changes in the Endosome and the Budding off of the Motor Complex. — The endosome seems to become more vacuolated than in a typical vegetative stage, increases in size, and at the same time a small knob or bud appears on one side (Fig. 2). The bud usually appears on the side of the endosome toward the anterior portion of the animal but in several instances it has been observed first in other positions becoming turned anteriorly during the migration of the nucleus. The bud increases in size, becomes a spherical or slightly elongated, deeply staining mass and passes through the hyaline area surrounding the endosome, thence through the chromatin of the outer nucleus to the nuclear membrane. In most instances it can be seen definitely connected to the endosome by a rhizoplast (Figs. 3, 4). On the nuclear membrane, the mass, now a distinctly spherical body, divides and the daughter bodies separate and pass gradually to opposite sides of the nucleus (Figs. 4, 5, 6). Later the portion of the nuclear membrane which lies between the bodies is drawn into a fairly definite line when viewed in optical section (Fig. 8). 33O WOOLFORD B. BAKER. As they separate the bodies again divide giving rise to the blepharoplasts of the two future cells. From each blepharoplast a new flagellum arises. When the nucleus has passed anteriorly until it comes into contact with the lower border of the reservoir, each blepharoplast divides, giving rises to a basal granule which serves as the basis for the smaller branch of the bifurcated flagellum (Fig. 6). In some cases the mass from the endosome does not follow this sequence, but both roots of the flagellum begin to grow out directly, the blepharoplast and basal granule forming later. This is much as described by Hartman and Chagas for Spongomonas. (b) Formation of Chromosomes. — The chromatin which in the resting nucleus is observed at the nodes of a linin network in the form of granules and masses of various sizes, now collects in a number of separate rods or filaments, the chromosomes (Figs. 3, 5). The entire nucleus stains more heavily at this time than in the vegetative stages, and gives the appearance of containing more chromatin. It is perhaps true that the chromatin masses increase in bulk and underg^phemical transformation so as to give the changed appearance. The dumb-bell shape observed in the resting nucleus may still be observed, as the chromosomes become outlined. Each is seen more or less constricted at first, later becoming almost uniform in width. All the chromosomes do not form simultaneously, hence the areas where the separate chromatin masses are beginning to fuse to form the chromosomes show what might be interpreted as a spireme structure. Free ends may be distinguished however, so that one is led to believe that a continuous spireme is not formed but rather each chromo- some retains a certain degree of continuity or individuality which has persisted throughout the resting stages, the chromosome as such reforming in the prophase from the chromatin network. As the filaments or rods assume definite outlines they are seen to be more or less looped. This appearance is perhaps due to the fact that the chromatin has increased in bulk while the nuclear membrane has persisted and has not enlarged in proportion. .The looped shape might easily be mistaken for a paired arrangement of separate units, especially as the chromosomes come to lie in the equatorial region and the ends of the loops become pressed close together (Fig. 7). STUDIES IN THE LIFE-HISTORY OF EUGLENA. 33 1 (c) Migration of the Nucleus. — While the changes described above are taking place, the body of the animal tends to become alternately rounded and extended, during which movement the nucleus passes anteriorly until it comes into contact with the lower border of the reservoir. Here the nuclear membrane and the border of the reservoir present a thickened appearance and because of the presence of the kinetic elements at opposite sides of the nucleus might easily be mistaken for a paradesmose (Fig. 8). Such an interpretation has been given to Euglena agilis by Hall (1923). A careful study of these stages has been made and in no case has a definite line been detected other than would be the natural consequence of the coming together of two surfaces such as the nuclear membrane and the border of the reservoir. By the time the nucleus has reached the reservoir, the endosome has elongated, passing from spindle to rod shape, then to dumb- bell shape. In the meantime the chromosomes collect in a more or less haphazard fashion along the middle portion of the endo- some. In the main they assume a radial arrangement as in Fig. 7. In this condition each chromosome^hows a definite longitudinal split. (d) The Metaphase. — The chromosomes, many of which show a longitudinal split during the prophase, and most of which are in the form of loops, now show a distinct separation along the plane of the longitudinal split. The separation of the split halves produces a complicated figure. Those which appear as loops open out from the apex first, gradually assuming a diamond or double V shape. Those not as loops begin to separate at one end first and gradually form elongated single V shape structures which extend the entire length of the equatorial cylinder. As the splitting proceeds the halves come to lie more nearly parallel to the elongating endosome and produce an apparent equatorial cylinder (Fig. 10). This parallel arrangement of the chromo- somes with their subsequent complete separation in the midline might easily be mistaken for a transverse division. The endosome assumes a single bend or curve in its middle region with its convex side toward the posterior part of the body of the animal. This is perhaps caused by the increase in its length beyond the width of the body of the animal, the bend 332 WOOLFORD B. BAKER. being the means of adjustment to the width of the body. On the other hand the bend may be the first indication of the plane of separation of the daughter animals and brought about by the metabolic changes in this plane (Fig. 12). (e) The Anaphase. — During the anaphase the endosome con- tinues to lengthen, accompanied by the nuclear membrane and motor complex on the outside. The ends of the endosome increase in size, perhaps at the expense of the mid-portion or centrodesmose. This region becomes much attenuated and is seen to lose its staining capacity for haematoxylin and to take the eosin in its place. The nuclear membrane can be followed distinctly between the groups of daughter chromosomes and seems to inclose a plastin like spindle. Material counter stained with light green does not show definite fibers in this spindle but indicates, rather, protoplasm that has been drawn out under the influence of the division forces. The opposite ends of the endosome are now seen to be quite unlike in shape. One end is club shaped and rounded, as in the preceding stages, while th^other is elongated anteriorly and flattened on the outer side. A projection can be detected pointing toward the granule on the periphery of the nucleus on that side (Fig. n). In many figures a rhizoplast may be traced from the granule to this projection. This marks the path of the budding off of the original motor complex as observed in the early prophase. The daughter chromosomes formed in the late metaphase or early anaphase now pass toward opposite ends of the nucleus and group themselves in a sort of barrel shaped cylinder around the enlarged ends of the endosome. The ends of the endosome project beyond the cylinder of chromosomes into a clear hyaline area (Fig. n). The number of chromosomes seems to be equal at both ends and can be counted with reasonable accuracy. A number of counts from figures in end view disclose approximately 14 distinct chromatin rods. (/) The Telophase. — The telophase begins after the endosome has elongated until the thin mid-portion of the centrodesmose becomes constricted. The nuclear membrane is likewise con- stricted and the daughter nuclei begin to round up. Here some STUDIES IN THE LIFE-HISTORY OF EUGLENA. 333 interesting information can be had regarding the development of the motor or kinetic complex. Each nucleus is seen to be some- what pear shaped, its apex turned toward the border of the new reservoir and usually in contact with it (Fig. 13). At the apex of each nucleus there appears the deeply staining granule which has given rise to the blepharoplast of the new flagellum, as described above. These granules have arisen by division of the bud which came from the endosome in the early prophase. The rhizoplast which marks the path of origin of the bud from the endosome may be seen as a persistent structure in one of the nuclei. From each granule can be seen a rhizoplast passing upward to the lower border of the reservoir and ending in the blepharoplast. From the blepharoplast a connecting rhizoplast is seen passing to the basal granule of the smaller branch of the flagellum. From both the blepharoplast and basal granule the new branches of the flagellum may be seen passing upward through the reservoir into the gullet. The endosomes of the daughter nuclei become rounded; the chromosomes take on a somewhat spiral arrangement in the peripheral region of each nucleus; condensed nodes appear in each chromosome, giving to them a beaded appearance. Each definite chromosome loses its distinct outlines and gradually becomes a part of the rapidly forming network of chromatin characteristic of the vegetative nucleus. During these reorganization stages of the telophase, the chromosomes seem to change their staining capacity. Each appears more diffuse and broader. This appearance may be variously interpreted. In the first place it is conceivable that the change is due to a longitudinal splitting of each chromosome, the halves remaining close together without separation before passing into the resting condition. This is the interpretation which Tschenzoff gives for Euglena viridis. Another explanation is that the metabolic changes in the chromosomes during the reorgani- zation period, especially the relative change from gel to sol, is sufficient to give the diffuse appearance. Division of the Gullet and Reservoir. As has been mentioned above, the nucleus migrates anteriorly during the early prophase and comes into contact with the lower 22 334 WOOLFORD B. BAKER. border of the reservoir. The mass of the kinetic complex has in the meantime divided and the daughter halves have passed away from each other on the upper margin of the nuclear membrane. The lower border of the reservoir is seen to broaden as the separation of the masses or granules continues, much as if they were the centers of the division process. A small lobe is seen to form on each side of the widening reservoir in contact with the nucleus (Fig. 6). As the nucleus elongates the lobes increase in size and eventually become separated in the anaphase (Fig. 10). This separation continues upward along the gullet until two structures are formed from the original one, remaining connected however, at the peripheral opening of the original gullet. In the early telophase the external opening becomes oval shaped and gradually divides. No indication has been observed of the disappearance of the old gullet-reservoir and the rebuilding of a new one as Dobell de- scribes for Copromonas. Neither has one daughter animal been seen to take over the old system while the other animal builds a new one, as described by some authors. Division of the Body. The first indication of the longitudinal split of the body is seen in the region of the reservoir (Fig. 13). The split continues posteriorly along the median plane of the body until the two daughter animals are completely separated. The flagella appear extended from the gullets at an early stage after separation begins, and their movement perhaps aids in the eventual pulling apart of the bodies. Observations of living animals at this stage shows continued twisting and writhing of the anterior ends as separation progresses, until the two bodies are connected by a small strand of protoplasm. Then usually both animals take a position in a straight line with their anterior ends pointing in opposite directions, in which position a veritable tug of war takes place until the bodies are completely separated. Immediately after separation the bodies round up slightly, forming elongated oval shapes and remain more or less quiescent for some minutes. In this quiescent condition the final processes of reorganization seem to occur. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 335 Observations of the pairs of daughter animals in this quiescent period immediately after separation shows interesting changes in the relationship between the nucleus and the kinetic complex. The deeply staining granule at the apex of the pear shaped nucleus becomes surrounded by a lightly staining halo, increases in size, and begins to show indications of breaking away from the nuclear membrane (Fig. 15). In the meantime the nucleus begins to rotate slightly and passes backward toward the posterior region of the body. When this posterior migration begins the rhizoplast connecting the granule with the blepharoplast, and in one of the pair of daughter animals an intranuclear rhizoplast as well, can usually be distinguished. As the animals begin to elongate after the quiescent period the granule becomes disconnected entirely from the nuclear membrane and passes with its surrounding halo into the cytoplasm. The rhizoplasts may be seen for some time after the separation but they eventually disappear and the granule becomes entirely separated from the motor organs and the nucleus (Figs. 16, 17). The separated granule seems to have no definite position in the cytoplasm, but is found in various places. It remains as a definite structure until the later stages of the next successive division when it is observed to gradually fade out of view. In some cases it has been observed as late as the quiescent period after binary fission, so that two such bodies are then found in one cell. These cases however, are exceptional. Behavior of Chromatophores During Mitosis. When the division process is first initiated in the animal, the chromatophores are quite definitely delineated. A pyrenoid lies in the center of each one and takes a fairly intense stain with the haematoxylin. The body of the chromatophore stains with the eosin rather than the haematoxylin. In material counter-stained with Orange G the pyrenoids become a light orange yellow. In Borrel's stain the body of the chromatophore becomes green while the pyrenoid is blue. As the division process goes on the pyrenoids show definite changes in contour and apparently in composition. The chromatic substance becomes first aggregated around the periphery in the form of distinct masses, which later 336 WOOLFORD B. BAKER. become elongated and thread-like. In the meantime the body of the chromatophore loses its definite outline, becomes more rounded and apparently increases in size. The individuality of the pyrenoid becomes less distinct, until the later stages show the chromatophore as more less homogeneous with a slightly denser central region. This central region increases in size, elongates, becomes constricted in the center and forms two regions from which the pyrenoids seem to form in the reorganization period of the cell. This division of the chromatophores is not synchronous with the division of the nucleus and the cell, since in many cases the number of such bodies is seen to be halved in the division process, and division of each occurs later. In almost all instances however, the internal changes in the pyrenoid accompany the changes in mitosis. It is planned to make a detailed study of the behavior of the chromatophores and pyrenoids, with special reference to their relation to the mitochondria of the cell, the subject of a later paper. Behavior of the Flagellum and History of the Kinetic Elements. In the early prophase the external flagellum seems to become more viscous and sticky so as to adhere readily to external objects or to the oral region of the animal. Many stained specimens show the flagellum much shortened and thickened at this stage, with abundance of foreign particles adhering to it. Frequently the end adheres to the body near the cytostome and with the foreign particles attached obscures the opening into the gullet. This evidence of dissolution progresses inward, eventually involving the two branches in the reservoir, as well as the blepharoplast and basal granule on the lower border. In a few instances remains of the old flagellum have been observed in the late metaphase (Fig. 7). As a rule however, the flagellum seems to have been absorbed or sloughed off at a much earlier stage. Berliner has described a somewhat similar degeneration in Copromonas major. Other observers describe the disappearance of the motor apparatus during division; Wallengren (1905); Dobell (1908); Hartman and Chagas (1910). In other instances however, the flagella are said to persist for one daughter cell and new ones are developed in the other: Cutler (1919); Jameson (1914); Whitmore (1911); Awerinzew (1907). STUDIES IN THE LIFE-HISTORY OF EUGLENA. 337 In Euglena agilis, the origin of the motor apparatus can be traced to the budding off of the kinetic complex from the endo- some during the early prophase. As a rule, the new flagella do not begin to grow until the nucleus has migrated anteriorly to the lower border of the reservoir, and the blepharoplasts have been produced from the kinetic mass. In a few cases what appear to be the new flagella have been observed previous to this time. The growth of both branches of each new flagellum progresses rapidly and by the time the body begins to separate into two daughter animals the main branch at least, has pene- trated to the exterior. It has been impossible to trace the origin of the lens shaped thickening on the main branch of the flagellum. It seems to arise gradually from the blepharoplast while the latter remains in connection with the kinetic mass on the nuclear membrane. When first observed during the quiescent period after division, it appears as a thick fiber extending toward the blepharoplast. Later it seems to condense at about the region of the stigma, increasing in size as the reorganization process is completed. The significance of this thickening has not been determined. It appears to have no connection with the mitotic phenomena, but disappears and a new one is reconstructed with each division. Its association with the stigma suggests that it functions in some way as a sensitive area to perhaps both heat and light. It perhaps initiates the motor reflex in Euglena as described by Mast, Jennings, Bancroft and others. Steuer describes a similar thickening on both the flagella of Eutreptia, in approximately the same position with relation to the stigma. This may represent a mere duplication of the structure as found in Euglena and serve the same function for the two flagella, the one directed anteriorly and the other posteriorly. DISCUSSION. The importance of the flagellates as objects of study has long been recognized by students of the Protozoa. Biitschli (1878), Klebs (1883), Fisch (1883), and many other earlier workers did much to stimulate such research. The Euglenoid nucleus has proved to be a very favorable object for study of nuclear phe- 338 WOOLFORD B. BAKER. nomena during mitosis and yet not until comparatively recent time were the nuclear phenomena here critically described and figured. Much controversy has arisen because of superficial observation as regards both the structure of the resting nucleus and the process of nuclear division. Tschenzoff (1916), presented one of the most complete and critical studies of Euglena viridis recorded and attempted to interpret the nuclear phenomena. Belar likewise, gave a good account of the process in Astasia, although his descriptions and figures seem to be somewhat incomplete. Other workers, Rhodes and Kirby (MSS.) on Heteronema, Hall (1923) on Menoidium have given more detailed accounts. Much further study must be made before many of the disputed points can be settled. The Resting Nucleus. The nuclei of flagellates present varied appearances. Prowazek (1903), attempted to classify the various types and Dobell (1908) grouped these types under the following four classes, adding a fifth class himself: (1) Simple nuclei, with an evenly distributed chromatic network, and no internal structures (karyosomes, division centers, etc.), e.g., Ilerpetomonas. (2) Vesicular nuclei, with direct division; with a central chro- matin mass surrounded by a clear zone, across which a more or less distinct network extends outward to the nuclear membrane. Such a nucleus may be seen in some species of Bodo, and is well represented by Copromonas. (3) Centronuclei containing a "nucleolo-centrosome" (Keuten, 1895) and separate chromatin masses. This type of nucleus is characteristic of Euglena and its allies. (The centronucleus, as defined by Boveri, is a nucleus which contains a cyto-center, either in consolidated or diffuse form. In the case of Euglena, etc., the cyto-center is the nucleolo-centrosome, i.e., is of the consolidated type.) (4) Vesicular nuclei, with karyokinetic division, e.g., Polytoma, Chlamydomonas , etc. ( 5) Nuclei in which the achromatic division center lies freely in the cell, whilst the chromatin is diffuse in the form of chromidia. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 339 According to this system of classification, the nucleus of Euglena agilis is of type three, yet the fact of the apparent karyokinetic division would indicate a modification of the type. Various descriptions have been given of the Euglenoid nucleus: Biitschli (1889), Steuer (1904), Haase (1910), Hamburger (1911), Tschenzoff (1916), Hall (1923), considered it similar to the nuclei of other plant and animal cells so far as the chromatin material was concerned. Klebs (1883), Dangeard (1902), Dehorne following Dangeard (1920), record a spireme structure in the resting nucleus. It is quite probable that the latter descriptions are of nuclei in the early prophase stages of division rather than in the resting condition. Keuten (1895), described well formed chromosomes in the resting nucleus of Euglena viridis, but here again it seems probable that prophase stages were observed. The description as given by Tschenzoff (1916), seems to be typical for many of the Euglenoid nuclei, that of Euglena agilis included. He described a central " Binnenkorper " around which is found " chromatische substanz in Form von feinsten Brockchen und Fadchen. Manchmal tritt mehr brockliche Beschaffenheit des Chromatin, manchmals feinfadige Zutage. Manchmal die Chromatin konnen auf einer Netwerk aufgereiht zu sein, . . . Ein geschlossenes Webenwerk konnte ich nicht festellen, wenn auch das Vorhandsein eines solches zu vermuten ist." This description agrees with that of Butschli (1883-1889) for nuclei of Euglenoidina in general and with the descriptions by many other observers. It might well be added that in several species of Euglena examined the endosome is fragmented, the fragments often being indistinguishable from the granules of chromatin when stained with haematoxylin . On the whole, there seems to be no single distinct type of nucleus characteristic of the Euglenidse. We must face the fact therefore, that much more detailed work needs to be done on the group before an accurate classification of the nuclei can be made. The Endosome. This structure has led to much discussion among various observers as to both structure and function. It was first defined 340 WOOLFORD B. BAKER. by Minchin as being the chromatin mass at or near the center of a "vesicular' nucleus, the precise nature of which not being determined in the definition. There may or may not occur in such a nucleus other granules of chromatin. If they do occur they are always smaller than the central mass or endosome. Some observers describe the structure as a body of homogeneous character comparable to the nucleoli of other plant and animal cells. Others say it consists of a matrix of plastin impregnated with chromatin, arranged more or less in granules, and plays a part in the formation of chromosomes (Berliner, 1909; Haase, 1910; Schiissler, 1917; Hartmann, 1919, 1921). Still others, Keuten (1895) compare it with the centrosome and central spindle of diatoms and speak of it as the "nucleolo-centrosome." Schaudinn (1896) thought he had demonstrated this to be the nature of the endosome in Oxyrrhis when by placing the animal in dilute sea-water the endosome migrated from the nucleus and functioned as an extranuclear centrosome. Hall (1924), after critical study of Oxyrrhis, thinks Schaudinn's supposed endosome was a small food mass. Steuer (1904) describes a "nucleolo- centrosoma" in the center of the nucleus of Eutreptia. In the work of TschenzofT and in the general work of Doflein the body is spoken of as the "Binnenkorper," which term is quite non- committal both as to structure and function. The question of the presence of a centriole in the endosome has also been the subject of much discussion. Schiissler (1917) believes that both nuclear components are located in the endo- some of Scytomonas , and that from this type as a starting point all degrees of differentiation of the kinetic and generative com- ponents may be found. Belar (1916) figures definite centrioles in the endosome of Astasia, tracing them through their entire cycle. In Rhynchomonas, he describes a centriole which passes from the karyosome to the nuclear membrane where it divides. In the same paper (1916) he concludes that "the basal body of Flagel- lates is doubtless a centriole." A good summary of the relation between the kinetic and generative components of the nucleus, bearing upon the problem of the endosome, is to be found in the work of Hartmann (1918) and of Jollos (1917): STUDIES IN THE LIFE-HISTORY OF EUGLENA. 34! " Denn wir haben ja eingehend gesehen, wie in dem Telophasen bei Chlorogonium elongatum zunachst aus dem gesamten Kernmaterial, den Chromosomen (der generative Komponente) wie Centriole (der Teilungskomponente) ein einheitlicher Binnenkorper, also ein primares, echtes Caryosome gebildet wird. Erst sekundar trat das Chromosomenmaterial (sowie offenbar das Centriol) in den aussenkern iiber und es bleib nur ein einfacher Binnenkorper oder Nucleolus zuriick, der nun weder die gener- ative noch die Teilungskomponente enthalt." Page 28, Hartmann. "Der Binnenkorper kann vielmehr, je nachdem bei den verschiedenen Protisten oder in verschiedenen Entwicklung- stadien ein und derselben Form, entweder beide Komponenten enthalten, oder die eine oder die andere, oder schliesslich iiber- haupt keine. Das Wesenlichte unserer Anschauung, mit den alle neuren Tatschen iibereinstimmen, ist 'die Lehre, das bei der Teilung alien Protisten, der einfachsten wie der hochstenwickelten zwei Komponenten hervortreten, die lokomotorische und die idiogenerative, und dass die mannigfaltige Konstitution der Protistenkerne durch die wechselnde Anordnung und Ausbildung dieser beiden Komponenten bedingt wird.' Jollos, 1917." Hartmann, page 28. In Euglena agilis certain phases of this intranuclear body seem to shed some light on the endosome problem. One of the first evidences of approaching mitosis is the budding off from the endosome of a deeply staining granule of unmistakable character (Figs. 2,3). This granule passes to the nuclear membrane where it divides, the division products taking their place at opposite sides of the nucleus, although not at the exact poles. Throughout the division process a rhizoplast may be seen connecting the original granule with the endosome. From each of the daughter granules are budded off the blepharoplast and basal body for the motor apparatus. The entire system remains integrated as a distinct unit until the division is completed and the animal goes into the quiescent period. The portion of the endosome remaining in the nucleus, lengthens out during division, becoming first rod-shape, then spindle-shape and finally dumb-bell shape, but through all the changes in shape the opposite ends are seen to be different. One 342 WOOLFORD B. BAKER. is rounded or knob-like while the other is blunt and possesses an elongation on the anterior side, from which the intra-nuclear rhizoplast may be traced to the kinetic complex. As division progresses and the centrodesmose elongates, the opposite ends of the endosome seem to be drawn upward toward the two kinetic centers, or rather the two masses from which the motor elements have been developed. The mass of the endosome appears as a passive frame work or support serving as a central spindle for the division process, and not as a kinetic center for the division. Whitmore (1911) describes in Proivazekia asiatica a process quite similar to that observed in Euglena agilis. According to him a body is pinched off from the karyosome which has loosened up during the early stages of division. From this body arise the centrioles while the remainder of the karyosome together with the outer chromatin of the nucleus forms the spindle. He concludes that the body pinched off is the locomotor-generative component, the karyosome residue constituting the idio-chromatin. In diphasic amebse which combine amoeboid and flagellated phases in one life cycle, e.g., Nxgleria gruberi (Wilson, 1915), a granule in the endosome gives rise to the blepharoplast by division. One daughter-granule destined to become the ble- pharoplast, migrates to the nuclear membrane, remaining con- nected with the granule in the endosome by an intra-nuclear rhizoplast. The flagellum grows out from the blepharoplast, hence the motor apparatus in these forms arises directly or indirectly as an outgrowth from the endosome. The process in Euglena agilis is not quite as simple as this since the mass budded from the endosome contains blepharoplasts, basal bodies, and a chromatoid residue, which remains in close connection with the motor apparatus until the daughter animals become fully re- organized after division. McCulloch (1917) describes the origin of a chromatoid mass from the endosome in Crithidia euryophthalmi. This mass later is seen connected with the blepharoplast and the endosome by means of rhizoplasts. She identifies the structure as the para- basal body. A somewhat similar body is figured in Cryptobia by Swezy, who likewise speaks of it as the parabasal body. The resemblance between these figures and certain stages in the cycle of Euglena agilis is readily seen (Text-figure A}. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 343 s.r. TEXT-FIGURE A. Diagrams showing the origin and development of the motor apparatus in Euglena agilis. A, vegetative stage; B-D, stages in the prophase; E, metaphase; F-H, anaphase; I-J, telophase; K, cells in quiescent period; L, daughter animal reorganized, (bl.) Blepharoplast; b. b. basal body; end., endo- some; ch. m., chromatoid mass left as residue after formation of kinetic elements; «, nucleus; gr., granule which arises from the endosome, divides and gives rise to the kinetic elements; r, rhizoplast. 344 WOOLFORD B. BAKER. The so-called "kineto-nucleus" of trypanosomes appears in approximately the same relation to the rest of the cell as does the deeply staining chromatoid mass of the kinetic complex in Euglena agilis. When one remembers the importance given to the "kineto-nucleus" and the emphasis placed upon homologous structures in other forms, in the development of the group Binucleata, the interest attached to the comparable body in Euglena agilis is increased. It is generally agreed by Proto- zoologists the "kineto-nucleus" is neither a nucleus nor an active kinetic center and the name should therefore be replaced by one of greater significance. Among the terms suggested is " parabasal body," first used by Janicki but redefined and used more ex- tensively by Kofoid. Examination of the descriptions given of parabasal bodies and their behavior by Janicki and others, shows striking similarity to that of the chromatoid mass of the kinetic complex in E. agilis. The parabasal body is closely connected with the kinetic elements of the cell, and according to Kofoid serves as a reservoir of substances which are used by the organism in its kinetic activities. During mitosis the body behaves variously in different species. In Devescovina striata, for example, it divides and the two daughter parabasals are shown in constant relationship to the poles of the extra-nuclear, rod-like, division spindle, where centrioles can often be determined. In other cases, e.g., Lophomonas blattarum, the parabasal body degenerates with approaching mitosis and a new one is formed for each of the daughter animals produced. In Euglena agilis, the granule which migrates from the endosome, divides, the daughter halves separate and occupy positions at the approximate poles of the division figure. From each granule is freed a blepharoplast-basal body complex which becomes the base of the bifurcated flagellum. A chromatoid residue remains on the nuclear membrane, connected by a rhizoplast to the ble- pharoplast and in one animal to the endosome. The residue retains this intimate connection with the motor apparatus until the animals are completely reorganized, at which time it breaks away from the nuclear membrane, the rhizoplasts disappear and it passes into the cytoplasm. As the next mitosis approaches, it begins to degenerate as a rule, and finally disappears entirely. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 345 Such a type of organization and behavior of related and kinetic elements is what one might expect in so primitive an organism as Euglena. We have many valid reasons for believing that within this group are to be found the progenitors of the other groups of Protozoa. As evolution progressed toward the rhizopod type, exflagellation might easily be conceived as a process of inhibition of the development of the kinetic complex from the endosome of the nucleus. When conditions in the cycle of an amoeba, e.g., Nagleria gruberi, are appropriate, flagellation is stimulated and we see the complex arising from the endosome as in Euglena. In those flagellates which have a more diverse series of life habits and hence greater complexity of structure, such as Trypanosomes, Devescovina, Crithidia, Lophomonas , Calonympha, etc., we find a type of organization easily derived from some stage shown in the development of Euglena. In these parasitic flagellates which show the presence of a permanent parabasal body in connection with the motor apparatus, we can easily conceive that the chromatoid residue which remains in connection with the motor apparatus in Euglena agilis, only during the late division stages and the reorganization period, becomes a permanent organelle of the cell. Such a change might be brought about through the greater demands made upon the motor apparatus when the organisms become adapted to a dense medium. In E. agilis the residue serves as a temporary reserve only, for the kinetic elements of the cell and becomes disconnected ffom them as soon as the new flagellum begins to function actively. • A permanent reservoir of substances to be used by the organisms in its kinetic activities is required by the parasitic flagellates. This need is met by the parabasal body. We may draw the following conclusions from the preceding discussion : (1) The endosome in Euglena agilis is the ultimate source of all the kinetic elements of the cell, but does not contribute to the formation of the chromosomes. (2) An endobasal body is located in the endosome and gives rise by division to a chromatoid mass, the kinetic complex. This migrates to the periphery of the nucleus where it divides, the daughter halves passing to the approximate poles of the division 346 WOOLFORD B. BAKER. D TEXT-FIGURE B. Diagrams showing the suggested homology between the kinetic elements of Euglena agilis and other flagellates. A, Cryplobia sp.; B. Herpetomonas musca-domesticae; C, Trypanosoma cruzi; D, Euglena agilis. (b) Blepharoplast; (r) rhizoplast; (p) parabasal body; (en) endosome; (n) nucleus; (par. ho.) parabasal homologue. (A and B from Calkins after Swezy;) C from Calkins after McCulloch.) STUDIES IN THE LIFE-HISTORY OF EUGLENA. 347 figure. In this position the bodies seem to be homologous to the centro-blepharoplasts of Trichomonas and other forms as de- scribed by Kofoid. (3) The chromatoid residue left on the nuclear membrane after the blepharoplast-basal body complex is freed, as described above, is homologous to the parabasal body of many parasitic flagellates as well as to the so-called "kinetonucleus" of Trypanosomes. This homology is indicated by the position of the residue with relation to the poles of the division figure and its connections with the endosome and blepharoplast. Examination of Text-Fig. B indicates the homologies suggested. Mitosis. Many cases of "amitosis" were described by the earlier observers of division stages in the Protozoa. More refined methods of technique have enabled later workers to determine, however, that amitosis is of rare occurrence in this group as it is in Metazoa. Nagler (1909) designated what had been previously called "amitosis" in amoeba and many other Protozoa, as "Promitosis," in order to differentiate this type from mitosis as found in Metazoa and Metaphyta. He says: "Fiir die soge- nannte Amitose der Protozoen fiihrt man daher am besten eine neue Bezeichnung ein und definiert sie als eine Kernteilung, die weder ausgesprochene Mitose, noch Amitose ist und sich char- akterisiert durch die Teilung eines Nucleolocentrosoms, des Cary- osoms. Ich schlage deshalb fiir diese Teilungsform den Namen Promitose vor." Chatton (1910) distinguished three types of mitosis, charac- terized as follows: (1) Promitosis — Division center within the karyosome, nuclear membrane persistent, chromosomes formed from the peripheral chromatin. (2) Mesomitosis — Division center outside the karyosome, nuclear membrane persistent, chromosomes from the karyosome. (3) Metamitosis — Division center outside the nucleus, nuclear membrane disappears, chromosomes from the nuclear chromatin. 348 WOOLFORD B. BAKER. This system is based on a study of Amoeba and hence could scarcely be expected to apply to other forms equally as well. Euglena agilis seems at first sight to fit into type one or promi- tosis, and yet the division center cannot be definitely located within the endosome. Alexeieff (1913) elaborates a system of classification which goes into great detail, but which even so, does not cover all cases. According to his system, Euglena agilis seems to show haplo- mitosis, since no centriole can be positively identified and yet distinct polar masses are formed. Prophase. — The formation of chromosomes in Euglena agilis does not occur as was described by Tschenzoff for E. viridis. In the resting stage the chromatin is arranged in masses, frequently dumb-bell shaped, along an achromatic network. In the early prophase these separate masses appear to enlarge and increase in staining capacity, at the same time fusing along the linin threads of the network to form chromosomes. Tschenzoff describes the formation of a number of granules around the periphery of the nucleus which eventually become arranged in rows or files. These become the chromosomes. It is an easy matter to make out granules around the periphery of the nucleus in E. agilis at this stage, but upon close examination they prove to be optical cross sections of chromosomes. Also, the granules of the motor complex may be found in a similar position, staining with the same intensity as does the endosome. I have not been able to make out the beaded structure of the chromosomes, such as have been figured by some investigators. It is true that each chromosome appears to consist of two masses in its earlier stages of formation and later these masses seem to run together to form the smooth chromosome. It is difficult to interpret this apparent double structure of the chromosomes. It is possible that it represents a doubling or splitting at some previous stage in the cycle. Wenrich describes such an ap- pearance of chromosomes in Trichomonas and traces it back to a constriction in the previous anaphase. He does not connect this constriction with the split chromosomes of the prophase however, nor does he attach any particular importance to the phenomenon. Kofoid describes a pseudo-reduction of chromosomes in STUDIES IN THE LIFE-HISTORY OF EUGLENA. 349 Trichonympha, brought about by a pairing of single chromosomes in the prophase. He suggests the possibility of a pregametic doubling of chromosomes in Protozoa such as occurs in the Metazoa, but does not consider this to be the most plausible explanation of the observed facts. Stevens describes the association of chromosomes in pairs during the vegetative stages of Boveria subcylindrica. The significance of this apparent reduction in vegetative mitosis is problematical and the subject requires much more work before a correct interpretation and correlation can be made. In Uroleptus mobilis Calkins finds eight chromosomes which unite in four pairs during the first maturation division, the pairs splitting at the second maturation division so that the daughter nuclei have four single chromosomes. He concludes that this is undoubted synapsis arid reduction. Tschenzoff describes a telophasic split in Euglena viridis, which doubles the number of chromosomes entering the resting vege- tative stage. Such a precocious split has not been observed in Euglena agilis, although the double appearance of the chromatin masses may be indicative of such a process. On the other hand, a definite longitudinal split has been observed in the prophase. It is not known what changes take place in the nucleus during the encystment in Euglena, but it is entirely possible that some phenomena will be found here to throw light on the apparently paired arrangement of the chromatin masses. Metaphase. — Tschenzoff describes in the metaphase, a sepa- ration of daughter chromosomes formed by a splitting in the telophase of the preceding generation. He states that the elements may be seen paired on the spindle at this stage and then separating without further split. Structures may be observed in Euglena agilis which might easily be interpreted in this manner but which show on critical examination an entirely different situation. The network of chromatin in the resting nucleus becomes enlarged at the nodes or at the points where the chro- matin masses are fusing to form chromosomes. This increase in bulk of chromatin does not seem to be compensated for by a corresponding increase in size of the nucleus. Since the nuclear membrane remains intact during the entire process of division the 23 35O WOOLFORD B. BAKER. chromosomes, as soon as formed, become quite tortuous in the limited space. The enlarging endosome with the budding off of the kinetic complex tends to force the chromosomes toward the periphery of the nucleus thus forming many twists and loops. When these chromosomes come to lie radially around the elongated endosome, preceding the longitudinal splitting, they appear as loops whose arms are rather closely pressed together. Thus at one focus one can distinguish a number of pairs of distinct chromosomes, but as the focus is changed the ends may be found to be connected into the loop. The longitudinal split begins at the end next to the endosome and proceeds toward the periphery. Thus, if an apex is inward, as is frequently the case, a double V-shaped figure is produced as the split halves separate. An X shape or a figure 8 shape is produced if the free ends are turned inward. In either case, the pulling apart of the split halves forms the equatorial plate figure, or as it might be better expressed, the "equatorial cylinder." Anaphase. — From the equatorial cylinder stage the ends of the split chromosomes pull apart and an appearance of a transverse splitting of chromosomes is the result. This perhaps accounts for some of the earlier descriptions and widely quoted figures which show a transvers splitting of chromosomes in Euglena. In the late anaphase, the chromosomes begin to show evidence of a return to the typical vegetative condition. Dehorne (1920) describes them as going back into the continuous spireme stage of the resting nucleus. It is true that the exact identity of the chromosomes tends to be lost as they begin to condense into the granular condition, but little evidence is found that a continuous spireme is constructed. Telophase. — Careful search was made for a precocious splitting at this stage in order to determine whether the description as given by Tschenzoff for Euglena viridis held also for Euglena agilis. Aside from the apparently diffuse nature of the chromo- somes and their change in intensity of staining reaction, no definite evidence of a split was observed. The Residual Mass from the Kinetic Complex. Blochmann (1894) found in Euglena velata a body in the cytoplasm, often lying near the nucleus but sometimes far from it. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 351 He first regarded it as a centrosome but because of its appearance in different parts of the cell, and because it appeared to persist after division was completed, he did not commit himself to this interpretation. Keuten (1895) likewise found bodies lying near the nucleus during the division of Euglena viridis, which persisted after division and could be found surrounded by a halo in various parts of the cell in the vegetative condition. He did not think these were centrosomes nor in any way related to the division phenomena. Haase (1910), in Euglena sanguined, traces the flagellum from the lower border of the reservoir into the cytoplasm where she finds and figures both branches ending in a basal body (diplosome?). Tschenzoff (1916) describes granules in the cytoplasm of Euglena viridis but explains them as artifacts resulting from his methods of killing. One is led to believe from the above descriptions that the granule observed in the different species mentioned is the same as that traced through its entire development in Euglena agilis. It has been seen to arise from the endosome, pass to the periphery of the nucleus, divide into two equal daughter granules which pass to opposite of the nucleus. In this position the granules seem to be homologous to the centro-blepharoplasts as described by Kofoid. In the early prophase stages each granule produces a blepharoplast which passes to the border of the reservoir and later gives rise to the main branch of the flagellum. The chromatoid residue left on the nuclear membrane increases in size during the telophase, and during the quiescent period when the nucleus migrates posteriorly to its original position in the cell, breaks away from the nuclear membrane, becomes surrounded by a clear hyaline vesicle and becomes a cytoplasmic structure. During the vegetative stages no change can be observed in this granule, but as the stages of the next successive mitosis proceed it is seen to lose its staining capacity and as a rule disappears before the late anaphase. In some cases it has been seen to persist throughout the division and to be distributed to one of the daughter animals. In this case a single cell would be seen to contain two such 352 WOOLFORD B. RAKER. granules during the early stages after division. This would account for the fact that Blochmann records three instances where he found two granules in the body of an animal. In one case he records three as being present. The question arises as to the significance of this bit of nuclear material sent into the cytoplasm at each division period. It seems clearly demonstrated that it acts as the vehicle for the transfer of the kinetic elements from the endosome to the cyto- plasm. After the kinetic elements are freed it remains in close connection with both the endosome and the blepharoplast for sometime after division, in fact until the daughter cells are fully reorganized. In this position we may conclude that it is homolo- gous, both in structure and function, to the parabasal body of parasitic flagellates. It seems to serve as a reserve of kinetic energy until the newly developed motor apparatus is fully formed and functioning normally. \Yith this function completed, does it play any further role in the physiology of the cell? Only conjecture can be made at present. It seems reasonable to suppose however, that there is sufficient chromatin material in the mass to bring about some sort of reorganization in the cytoplasm of the body after each division. Thus the protoplasm might be kept in a proper state of physiological differentiation so as to make process of conjugation, autogamy or endomixis unnecessary. The disappearance of the mass in the early stages of each division is suggestive that in Euglena we find a mechanism which functions as a sort of regulator of the division cycle. It is possible that changes in this mass of chromatin, this kinetic reserve, may be the stimulus which incites the division of the cell with such striking regularity. Although it does not function as a division center, yet it sets up a series of concatenated events which result in the final production of two fully vigorous individuals from the single parent cell. SUMMARY AND CONCLUSIONS. i. Mitosis in Euglena agilis is accomplished in a manner quite similar to that described for higher plants and animals. Definite chromosomes are formed, divide longitudinally and are dis- tributed equally to the daughter cells. The chromosomes are STUDIES IN THE LIFE-HISTORY OF F.UGLENA. 353 formed from the chromatin of the outer nucleus and not from the endosome. 2. Division of the nucleus is preceded by its migration anteri- orly in the body until it comes into close contact with the lower border of the reservoir. 3. A chromatoid mass, the kinetic complex, originates from the endosome during the prophase, migrates to the periphery of the nucleus where it divides. The division products pass to opposite sides of the anterior border of the nucleus, in which position they suggest homology with the centroblepharoplasts of Trichomonas. No paradesmose has been detected between them however. 4. From the kinetic complex a blepharoplast is freed and produces the main branch of a new flagellum. Later, a basal body arises from each blepharoplast, and from it is produced the secondary branch or anchoring root of a flagellum. 5. The residue from the kinetic complex, after the formation of the blepharoplast, remains on the nuclear membrane until division of the animal is completed and the daughter cells have reorganized. A rhizoplast passes from it to the blepharoplast and in one of the daughter animals an intra-nuclear rhizoplast is seen making connection with the endosome. After reorganization of the daughter animals is completed, the residual mass is cast out into the cytoplasm where it remains during the vegetative stages of the organism as a deeply staining granule surrounded by a lightly staining halo. It usually disappears during the early phases of the next successive mitosis. Its behavior and relation- ship to the motor apparatus suggests a homology with the parabasal body of parasitic flagellates. 6. The division process is a function of the cell as a whole, being initiated not by the division of a specific body that might be called a centrosome, but rather by simultaneous changes in all parts of the cell. The centroblepharoplast homologue, together with an endobasal body in the endosome seem to constitute the principal dynamic centers. The mass of the endosome seems to constitute a sort of passive framework or spindle for the division process. 7. The entire motor apparatus of the parent animal, consisting of flagellum, basal body and blepharoplast disappears during division and a new one is built up in each daughter animal. 354 WOOLFORD B. BAKER. 8. The mitotic and related phenomena in Euglena agilis give additional evidence of the primitive character of the Phyto- mastigoda. The development of the motor organoids indicate relationship with diphasic amoeba on the one hand and with the complex parasitic flagellates on the other. 9. Changes in the chromatophores during the division and vegetative periods, as well as their changes when the Euglense are subjected to different cultural conditions indicate that these structures are not satisfactory criteria upon which to base the classification of the group. LITERATURE CITED. Alexeieff, A. 'n Haplomitose chez les Eugleniens et dans d'autres groupes de protozoaires. C. R. Soc. Biol., Paris, 71, 614, 8 figs, in text, 'n Sur le stade flagelle dans 1'evolution des Amibes Umax. I. 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'14 Bau, Teilung und Infektion verhaltnisse von Trypanoplasme dendrocceli Fantham. Arch. f. Prot., 32, 171-204, i pi. Gicklhorn, J. '21 Methode zur Darstellung der Geissel mit Basalkorn. Zeit. f. Wiss. Mikr.. 38, 123-129. Glaser, H. '12 Untersuchungen iiber die Teilung einiger Amoben. Arch. f. Prot., 25, 127- 152., 5 pis., 5 figs, in text. Guillermond, A. 'i2a Recherches sur le mode de formation de 1'amidon et sur les plastes des vegetaux (leuco-chloro et chromoplasts). Contribution a 1'etude des mitochondries chez les vegetaux. Arch. Anat. Micr., 14, 309-428, pis. 13-18, ii figs, in text. Haase, Gertrude. '10 Studien iiber Euglena sanguined. Arch. f. Prot., 20, 47-59, pis. 4-6. Hall, R. P. '23 Morphology and Binary Fission of Menoidium incurvum (Fres.) Klebs. Univ. Calif. Publ. Zool., 20, 447-476, pis. 40-41, 2 figs, in text. 356 WOOLFORD B. BAKER. '23 Binary Fission in Oxyrrhis Marina Dujardin. Ibid., 26, 281-324, pis. 26-30, 7 figs, in text. Hamburger, C. 'ii Studicn iiber Euglena Ehrenbergii, inbesondere iiber die Korperhulle. Sitz.-Ber. d. Heidelburg. Akad. wiss. Math.-naturw. Kl., Jarhg. 1911, 1-22, pi. I. Hartmann, M. and Prowazek, S. von. '07 Blepharoplast, Caryosome und Centrosom. Arch. f. Prot., 10, 306-333. Hartman, M. '19 Untersuchungen iiber die Morphologic und Physiologic des Formwechsels (Ent\vicklung, Fortpflanzung, Befruchtung und Vererbung) der Phytomo- nadinen (Volvocales). I Mitt. Arch. f. Prot. 39, 1-33, 3 pis. '21 III Mitt. Die dauerend agame Zucht von Eudorina elegans, experimentale Beitrage zum Befruchtungs und Todproblcm. Ibid, 43. Hartmann, M., and Chagas, C. '10 Flagellatenstudien. Mem. Inst. Osw. Cruz, 2, 64-125, pis. 4-6. Jameson, A. Pringle. '14 A New Phytoflagellate. Arch. f. Prot. 32, 21-44., p'- 3- Janicki, C. 'n Zur Kenntniss des Parabasalapparatus bei parasitischer Flagellaten. Biol. Cent., 31, 321-330, 8 figs, in text. '15 Untersuchen an parasitischer Flagellaten. II. Die Gattungen Devescovina, Parajcenia, Stephanonympha, Calonympha. Zcit. wiss. Zool. 112, 573- 689, pis. 13-18., 17 figs, in text. Jollos, V. '17 Untersuchungen zur Morphologic der Amobenteilung. Arch. f. Prot., 37, 229—275, pis. 13—16, 4 figs, in text. Kent, W. Saville. "80-82 A Manual of the Infusoria. (London, Bogue), i, 1-472; 2,473-913; 3, pis. 1-50. Keuten, J. '95 Die Kernteilung von Euglena viridis Ehrenb. Zeit. wiss. Zool., 60, 215-235. pi. n. Khawkine, W. '86 Recherches biologiques sur 1'Astasia ocellata n.s., et I' Euglena viridis Ehr. Sci. Nat., Zool. ?e Ser., i, 319-376, pi. 16. Klebs, G. '83 Uber die Organization einiger Flagellatengruppen und ihre Bezeihungen zu Algen und Infusorien. Unters. bot. Inst. Tubingen, i, 233-325. '92 Flagellatenstudien. Theil II. Zeit. f. wiss. Zool., 55, 353~445. pis. 17-18. Kofoid, C. A. '23 The Life Cycle of the Protozoa. Sci., 57, 397-408. Kofoid, C. A., and Christiansen, E. B. '150 On the Life-history of Giardia. Proc. Nat. Acad. Sci., i, 547-552. i fig. in text. '156 On binary and multiple fission in Giardia muris (Grassi). Univ. Calif. Publ. Zool., 16, 30-54, pis. 5-8, i fig. in text. Kofoid, C. A., and Swezy, O. '150 Mitosis in Trichomonas. Proc. Nat. Acad. Sci., i, 315-321, 9 figs, in text. '156 Mitosis and Multiple Fission in Trichomonad Flagellates. Proc. Am. Acad. Arts, and Sci., Boston, 2, 289-378, pis. 1-8, 7 figs, in text. STUDIES IN THE LIFE-HISTORY OF EUGLENA. 357 'iga Studies on the Parasites of Termites. II. On Trichomitus termitidis, a polymastigote flagellate with a highly developed neuromotor system. Univ. Calif. Publ. Zool., 20, 21-40, pis. 3-4, 2 figs, in text. 'igb Studies on the Parasites of Termites. III. On Trichonympha campanula, sp. nov. Univ. Cal. Publ. Zool., 20, 41-98, pis. 2-12, 4 figs, in text. Maier, H. N. '03 Uber der feineren Bau der Wimperapparate der Infusorien. Arch. f. Prot., 2, 73-179- 4 Pis. McCulloch, I. '17 Crithidia euryopthalmi sp. nov. from the Hemipteran Bug Euryoplhalmus convivus. Univ. Calif. Pub. Zool., 18. Minchin, E. A. '12 An Introduction to the Study of the Protozoa. (London, Arnold), xi, 517, 194 figs, in text. '14 Remarks on the Nature of the Blepharoplasts or Basal Granules of Flagella. Arch. f. Prot., 34, 212-216. Moroff, Theodor. '03 Beitrage zur Kenntnis einiger Flagellaten. (Euglena quartana, n.s.). Arch. f. Prot. 3, 96-103, pi. 8. Prowazek, S. von. '03 Die Kernteilung von Entosiphon. Arch. f. Prot., 2, 325-328, 12 figs, in text. '03 Flagellatenstudien. Fibrillare Strukturen der Vorticellinien. Arch. f. Prot., 2, 195-212, 2 pis. Rhodes, R. C. '19 Binary Fission in Collodictyon triciliatum, Carter. Univ. Calif. Publ. Zool. 19, 201-274, pis. 7-14, 4 figs, in text. Rhodes, R. C., and Kirby, H. Mitosis in Heteronema acus. MSS. Robertson, Muriel. '09 Further Notes on a Trypanosome found in the Alimentary Tract of Pont- obdella mnricata. Q. J. M. S., 54, 119, pi. 9. 5 figs, in text. Schiissler, H. '17 Cytologische und entwicklungsgeschichtliche Protozoenstudien. I. Uber die Teilung von Scytomonas pusilla Stein. Arch. f. Prot., 38, 117—125, pl- 5- Steuer, A. '04 tiber eine Euglenoide (Eutreptia) aus dem Canale Grande von Triest. Arch. f. Prot., 3, 126-137, 13 %s. in text. Swezy, O. '150 Binary and multiple fission in Hexamitus. Univ. Cal. Publ. Zool., 71-88, pis. 9-11. Ternetz, Charlotte. '12 Beitrage zur Morphologic und Physiologic der Euglena gracilis Klebs. Jahrb. f. wiss. Botanik, 51, 435-513, i pl. Tschenzoff, B. '16 Die Kernteilung bei Euglena viridis Ehrb. Arch. f. Prot., 36, 137-173, pis. H-I2, 2 figs, in text. Wager, H. '99 On the Eyespot and Flagellum in Euglena viridis. Jour, of Linn. Soc. London, Botany, 27, 463-481, pl. 32. 358 WOOLFORD B. BAKER. Wallengren. '05 Zur Kenntniss der Flimmerzellen. Zeit. f. Allg. Physiol., 5, 351-414, pi. 12-14. Wenrich, D. H. '21 Structure and Division of Trichomonas. Jour. Morph., 36, 119-156, pis. 4. Wenyon, C. M. 'n Oriental Sore in Bagdad, Leishmania tropica. Parasitology, 4, p. 273. Whitmore, E. R. 'n Prowazekia asiatica. Arch. f. Prot., 22, Studien iiber Kulturamoben aus Manila. Trimastigameba philippinensis. Arch. f. Prot. 33, 80-95, 2 pis. Wilson, C. W. '16 On the Life-history of a Soil Ameba. Univ. Cal. Pub. Zool., 16, 241-292, pis. 18-23. Wilson, E. B. '25 The Cell in Development and Heredity. New York, xxxvii + 1232, 529 figs, in text. Woodcock, H. M. '06 The Hsemoflagellates: A Review of Present Knowledge Relating to the Trypanosomes and Allied Forms. Q. J. M. S., 50, 151-233. Zumstein, H. "99 Zur Morphologic und Physiologic der Euglena gracilis. Jahrb. f. wiss. Bot., 34, 149-196, pi. 6. 360 WOOLFORD B. BAKER. , EXPLANATION OF FIGURES. PLATE I. All sketches were made with camera lucida with Leitz compens ocular (20) and Leitz apo. obj. 1.5. They have been reduced one half. FIG. i. Euglena agilis in vegetative condition showing nucleus, endosome, chromatophores with pyrenoids, blepharoplast, basal body, flagellar enlargement, reservoir, gullet or cytopharynx, and kinetic residue. The stigma or eyespot does not show with the stains used. The chromatin is arranged in a net work. FIG. 2. Early prophase drawn in optical section showing first stages in for- mation of chromosomes and early budding of the kinetic complex from the endosome. FIG. 3. The chromosomes appear as elongated rods or loops and give the appearance of a spireme. The bud from the endosome is definitely connected by a rhizoplast. FIG. 4. The granule budded from the endosome has divided, the daughter halves passing along the periphery of the nucleus. One is connected by rhizoplast to the endosome. FIG. 5. Another view of the daughter granules separating on the nuclear membrane. The animal is beginning to round up and the nucleus is approaching lower border of the reservoir. FIG. 6. The endosome has elongated, each daughter half of the kinetic complex has given rise to a blepharoplast and this in turn to a basal body. From both the blepharoplast and basal body a flagellar element has grown out. The remainder of the old flagellum is seen in the right side of the figure. The pyrenoids of the chromatophous are undergoing change in organization. The chromosomes are arranged around the elongated endosome. FIG. 7. An optical section of a late prophase or metaphase with chromosomes arranged somewhat radially around the endosome. Each chromosome shows a longitudinal split and the looped nature of each gives the appearance of a pairing. FIGS. 8-10. Stages in the anaphase, showing separation of the daughter chromosomes and the formation of an equatorial cylinder. The rhizoplast con- necting the blepharoplast to the residue of the kinetic complex is visible in figure eight. BIOLOGICAL BULLETIN, VOL. LI. PLATE I. WOOLFORD B. BAKER. 362 \YOOLFORD B. BAKER. PLATE II. FIGS. 11-12. Late anaphases showing chromosomes grouped around ends of elongated endosome. In Fig. n the integrated kinetic elements show on both sides of the nucleus while they are visible on one side only in Fig. 12. The com- ponents of the other side are at a much lower level. FIGS. 13-14. Telophase stages showing enlargement of residue from kinetic complex and beginning of formation of halo around it. FIG. 15. Division completed and the daughter organisms in quiescent period. The nucleus is receding from the reservoir. FIG. 16. The chromosomes have begun to go back into vegetative condition. The kinetic residue is cast into cytoplasm. FIG. 17. Further stage in reorganization. FIG. 18. Organism reorganized. BIOLOGICAL BULLETIN, VOL. LI. 18 WOOLFORO B. BAKER. HYDROGEN ION CONCENTRATION IN THE BLOOD OF CERTAIN INSECTS (ORTHOPTERA). JOSEPH HALL BODINE, ZOOLOGY LABORATORY, UNIVERSITY OF PENNSYLVANIA. Inasmuch as most accurate quantitative results on the hy- drogen ion concentration of blood and body fluids have been largely confined to higher animals it seems highly desirable to extend these observations to lower forms, and especially insects. Since only small amounts of fluids are usually available from single insects, an accurate micro-electrometric method is neces- sary for the successful carrying out of such investigations. A simple micro hydrogen electrode and vessel, particularly designed for this purpose, have been described by the author (Bodine and Fink, i). The present paper contains data obtained by the use of this method for the blood of different species of grasshoppers. With the exception of two papers (Bishop, 2; Brecher, 3), all previous work on insect blood seems to have been carried out by means of colorimetric methods (Jameson and Atkins, 4; Crozier, 5; Bodine, 6; Glaser, 7). Since the blood of most insects is colored it seems highly desirable that other methods than micro- colorimetric ones be used and especially since the indicator most used in these methods is brom-thymol-blue. The blue-green to % yellow-green color of insect blood seems to introduce an unde- sirable factor in micro-colorimetric methods using this indicator. Results obtained by the use of colorimetric methods by different authors are somewhat variable and it therefore seemed highly desirable to check them by means of an electrometric method. In the present investigation different species of grasshoppers, reared under controlled food, temperature and other laboratory conditions, have been used as well as other animals taken from their normal out-of-door environments. Individual studies have been made on the blood of certain species from the day of hatching until death or in other words during their entire life cycle. Blood was taken from the grasshoppers in a variety of ways, — by 24 363 364 JOSEPH HALL BODINE. cutting off the tip of the feet, puncturing the animal by means of needles, insertion of electrode vessel directly into the body cavity, etc. No marked differences in pH of the blood obtained by these different methods was noted. The method found most satis- TABLE i. SPECIES: Chorlophaga viridifasciata. Age, Color, Sex. pH of Blood- nymph — brown — cT 6.83 nymph — green — 9 6.83 nymph — brown — $ 6.84 nymph — brown — 9 6.86 adult — cf 6.62 adult — cf 6.43 adult — cf 6.67 adult — o* 7.05 adult — d"1 6.85 adult — 9 6.65 adult — 9 6.94 adult — 9 6.51 adult — 9 6.95 adult — 9 6.73 adult — 9 6.93 Average 6.79 Range 6.43-7.05 Explanation of Table I. Data taken at random from many individuals showing pH of blood of Chorto- pkaga viridifasciata. Animals all taken from out-of-doors. Some bloods tested at once. Other animals kept in laboratory and fed lettuce over different periods. Samples of blood tested regularly. Average given for all individuals tested. factory and least harmful to the grasshopper, however, was a small needle puncture into the mid-lateral abdominal wall. Only a very small amount of blood is thus lost by the insect and the wound is quickly sealed. Sufficient blood (approximately o.oi cc.), however, is obtained in this way for electrometric determi- nations of pH values. The grasshopper was usually punctured and the blood immediately drawn up into the electrode vessel with practically no exposure to air. As a matter of fact, identical results were obtained with blood taken from the interior of the animal by the insertion of the electrode vessel directly into the animal as by the method of puncturing and then immediately HYDROGEN ION CONCENTRATION IN BLOOD OF INSECTS. 365 TABLE 2. SPECIES: Melano plus femur rubrum. Age, Sex. pH of Blood. Nymph 7.00 Nymph 6.23 Nymph 6.95 Nymph 6.36 Nymph 6.70 Nymph 6.59 Nymph 6.71 Nymph 7.07 Nymph 6.79 Nymph 6.96 Nymph 6.55 Nymph 6.70 Nymph 6.45 Nymph 6.66 Adult— cf 6.86 Adult— d" 6.86 Adult — d" 6.58 Adult — d"1 6.80 Adult — 9 7.00 Adult — ? 6.73 Average 6.70 Range 6.23-7.11 Explanation of Table 2. Data selected showing range of pH for blood of different individuals. All animals laboratory raised and fed lettuce. Average and range of pH for all indi- viduals tested. TABLE 3. SPECIES: Melanoplus differ enlialis. Age. pH of Blood. Nymph 6.85 Nymph 6.43 Nymph 6.52 Nymph 6.56 Nymph 6.50 Nymph 6.75 Nymph 6.98 Nymph 6.93 Average 6.68 Range 6.42-6.98 Explanation of Table 3. Data selected from several hundred individuals showing range of pH values. Adult values are similar to those for nymphs. Animals reared in laboratory and fed lettuce. Average and range of pH for all individuals tested. 366 JOSEPH HALL BODINE. drawing the blood up into the vessel. Individual adult grass- hoppers can be punctured at least two to three times daily without any apparent injury. TABLE 4. Romalca microptera.1 Average Date. Stage. pH of Blood. Feb. 19, 1926 Nymphs 6.83 26, 1926 Nymphs 6.88 Mar. 5, 1926 Nymphs 6.72 12, 1926 Nymphs 6.74 19. 1926 Nymphs 6.84 26, 1926 Nymphs 6.79 Apr. 9, 1926 Nymphs 6.66 16, 1926 Nymphs 6.87 23, 1926 Nymphs 6.56 28, 1926 Nymphs 7.10 (?) 30, 1926 Adults 6.82 May 7, 1926 Adults 6.88 14. 1926 Adults 6.84 21, 1926 Adults 6.68 28, 1926 Adults 6.46 June i, 1926 Adults 6.77 2, 1926 Adults 6.67 3, 1926 Adults 6.55 4, 1926 Adults 6.53 5, 1926 Adults 6.72 7, 1926 Adults 6.50 9, 1926 Adults 6.59 ii, 1926 Adults 6.82 15, 1926 Adults 6.87 Average 6.73 Range 6.4-6.8 Explanation of Table 4. Detailed average pH values for grasshoppers from day of hatching until death. Data for each day represents average readings for from 5 to 20 individuals. All animals laboratory raised and fed lettuce. Average and range of pH given for all individuals tested. All laboratory animals were hatched from eggs laid in the laboratory and were fed lettuce. Animals were also collected 1 The author is indebted to Dr. H. Fry for his generosity in supplying some of the eggs used in these experiments. These eggs were from a cross between Romalea microplcra and Romalea microptera, variety macri. No differences in pH of the blood from this cross and the R. microplera were noted. HYDROGEN ION CONCENTRATION IN BLOOD OF INSECTS. 367 from the outside and comparisons made with those reared entirely under laboratory conditions. Temperature during the course of the experiments ranged from 22 to 24° C. TABLE 5. Temp. 24.° C. — June 15, 1926. Samples — E.M.F. Average pH. Buffer (5.65) (a) -57573 5-6i (b) -57580 (c) .57560 Romalea adult (a) .65250 6.90 (b) -65555 Buffer (5.65) (a) .58210 5.69 (b) .58088 (c) .58006 Romalea adult (a) .64553 6.79 (b) .64553 Buffer (5.65) (a) .58173 5.67 (b) -57953 Romalea adult (a) .65203 6.90 (b) .65273 Buffer (5.65) (a) .58265 5.67 (b) .57963 (c) .57963 Romalea adult (a) .65226 6.89 (b) .65163 Buffer (5.65) (a) .58200 5.67 (b) .57742 Explanation of Table 5. Part of daily record for June 15, 1926, to show method of procedure in making determinations of blood pH values. Note that standard or buffer solution is always read before and after every reading on blood as check on electrode. The results of the determinations are summarized in Tables 1-5. From an examination of these it is evident that considerable variation exists in the hydrogen ion concentration of the blood of the same and different species. No marked species, sex or age differences have been detected. The variations shown can hardly be due primarily to differences in food conditions since the laboratory animals were all fed the same food, lettuce, and the out-of-door animals had varied diets depending on the locality from which they were taken. The data in the tables are summa- rized and do not show individual variations which are to a degree 368 JOSEPH HALL BODINE. closely similar to those indicated for groups. Readings for the same individual, however, are quite constant for any one day. An explanation for these consistent variations is not at hand. It is of some interest to compare the present results with those obtained with colorimetric methods by Glaser (7), for grass- hoppers (Melanoplus differ entialis'}. This author found the range of pH values for the blood of these grasshoppers to be 7.2 to 7.6. In previous experiments (6), using a colorimetric method the average pH for the blood of many species of grasshoppers was found to be 6.8. It seems to the author that the results of Glaser (7) are too high, due doubtless to difficulties in the colorimetric method already indicated above. Preliminary experiments on the CO2 tension of grasshopper blood indicate a rather low value, which seems consistent with an acid pH for the blood and the mechanism of tracheal respiration. Grasshopper blood seems to be slightly buffered, since upon dilution no marked changes in pH are detected until a dilution of two to four times is reached. Most of the buffer effects are apparently due to the fairly large amounts of phosphates which the blood contains. In contrast to mammalian blood there seems to be present rather small amounts of carbonates. Results for pH values of blood for other insects obtained by means of electrometric methods (Bishop, 2 ; Brecher 3) 6.8, agree rather closely with those obtained in the present work for grass- hopper blood. SUMMARY. The pH values for the blood of different species of grasshoppers have been determined by a micro-electrometric method. Con- siderable variation exists in the pH values for blood of the same species of animals. No correlations seem to exist between blood pH and age or sex. The range and average pH values for the blood of the different species of grasshoppers examined are as follows: Species. Range of pH. Average pH. Melanoplus femur rubrum 6.4-6.8 6.73 Melanoplus differentialis 6.42-6.98 6.68 Chortophaga viridifasciata 6.43-7.05 6.79 Romalea microptera 6.4-6.8 6.73 HYDROGEN ION CONCENTRATION IN BLOOD OF INSECTS. 369 LITERATURE CITED. 1. Bodine, J. H., and Fink, D. E. '25 J. Gen. Physiol., VII., 73$- 2. Bishop, G. H. '23-24 J. Biol. Chem., LVIIL, 543. 3. Brecher, L. '25 Zeits. f. verg. Physiol., 2 Band, 6. Heft, 691. 4. Jameson, A. P., and Atkins, W. R. G. '21 Biochem. J., XV., 209. 5. Crozier, W. J. '23-24 J. Gen. Physiol.. VI., 289. 6. Bodine, J. H. '25 BIOL. BULL., XLVIIL, 79. 7. Glaser, R. W. '25 J. Gen. Physiol., VII., 599. Vol. LI December, 1926 No. 6 BIOLOGICAL BULLETIN INFLUENCE OF AGE OF MOTHER ON APPEARANCE OF AN HEREDITARY VARIATION IN HABROBRACON. P. W. WHITING, UNIVERSITY OF MAINE. Contentions about the inheritance of acquired characters have frequently involved questions of the age of the parents. Thus in trotting horses older sires or dams which have undergone con- siderable training have been supposed to transmit some of the benefits of this training to their offspring. Similarly, acquired knowledge, acquired immunity to disease, etc., have been stated to be transmitted. When these matters are investigated scien- tifically it is found that unintentional selection of parents may account for the bulk of cases. Nor is character of offspring which may be found to be correlated with parental age to be taken as proof of change in germ plasm of parents. Some non-germinal influence may be transmitted. Thus it has been shown (Hart, 1923) that Mongolian idiots are at their birth twenty-three times as likely to have mothers forty years of age or over as mothers from twenty to twenty-four. There is no good evidence that Mongolian idiocy is hereditary. The material considered in the present paper presents clear evidence of change in average traits of offspring according to age of mothers. Discussion as to whether this difference is germinal will be reserved for a later part of the paper. In the parasitic wasp Habrobracon juglandis (Ashmead) which has been bred under constant environmental conditions, ab- normalities in antennae, external genitalia or posterior part of the digestive tract occur. Abnormal antennas may be branched, bent or clubbed. Gonapophyses of the female (sensory appendages forming sheath for the sting) may be swollen at the end or twisted from 25 371 372 P. W. WHITING. their normal position; extra gonapophyses may appear in addition to the two normally present. These abnormalities which consist in increase in size or number of parts or in malformation without reduction are to be dis- tinguished from deficiency. DEFICIENCY. Antennal deficiency is usually evidenced by tapering and fusion of terminal joints as in right antenna of specimen shown in Plate i, Fig. H (Male from nyi^c). There is, in general, a tendency toward symmetry but much irregularity. Fusion without tapering may occur or one antenna may be normal (Figs. A and H). Males may have genitalia missing and abdomen rounded off at tip, or the normal parts may be reduced in size, twisted out of shape or lacking on one side of the body while present in almost normal condition on the other. In Plate I., Fig. D is shown the ventral side of the abdomen of a normal male. Fig. E shows the abdomen from posterior view, somewhat oblique, with genitalia extruded. The tip is curved ventrally so that it is seen in dorsal aspect. Figs. A, B, and C are from a male (Freak 60) illustrating deficiency in antennae and genitalia on the right side of the body both anteriorly and posteriorly. Fig. B shows the tip of the abdomen ventrally and slightly oblique while Fig. C is an aspect comparable with Fig. E. Genitalia are completely lacking on right side, including even the right side of the penis, while posterior sternites and tergites taper off dextrally. Organs on the left are somewhat bent toward the right but otherwise normal. Sections of abdomen showed right testis and vas deferens completely lacking, left apparently normal. Fig. A , ventral view of head, shows left antenna normal. It is drawn displaced from the head for economy of space. Right antenna is shortened by reduction and fusion of joints toward the tip. Right labial palpus is missing. Figs. K, L, and M, show dorsal, ventral, and right aspect respectively of abdomen of normal female. Figs. / and / show ventral and right aspect respectively of INFLUENCE OF AGE OF MOTHER. 373 abdomen of a deficient female (Freak 6). Great reduction and fusion of parts posteriorly, especially of gonapophyses, is to be noted. Sections show large eggs and nurse cells developing, but no trace of poison apparatus; deficiency therefore extended internally to posterior organs. Reduction in the posterior part of the digestive tract also occurs. This may be indicated by a swollen appearance of the abdomen with an abnormally dark color, due to failure of elimination of fecal matter. Sections show different degrees of defect ranging from an apparent failure of the proctenteron to connect with the mid-gut, to a complete disappearance of the former and considerable reduction of the latter posteriorly. This reduction may shorten the Malpighian tubules until they are little knobs on the posterior end of the mid-gut sack, or the latter may be rounded off smoothly. Fig. F shows a specimen of this type from a female (Freak 21) with no external genitalia. There were no hind-gut, no Malpighian tubules, no poison apparatus. In the ovaries nurse cells were present but ova were small. The posterior compound nerve ganglion was normal. Fig. G is from a male (Freak 20) with no external genitalia, and no hind-gut posterior to the Malpighian tubules which are short and thick. The mid-gut is much swollen with fecal matter. Testes were present, rilled with mature sperm but there were no vasa deferentia. The posterior compound nerve ganglion was separated into its three elements. RARE OCCURRENCE OF ABNORMALITIES IN NORMAL STOCKS. Among 53,148 males and 16,036 females of normal stocks the following abnormalities in antennae, genitalia, and digestive tract have appeared. Only twelve females had abnormal gonapophyses. Abnormal antennae were recorded in only thirty males and twenty-six females. These may be classified as having one or more antennae short, sixteen; absent, twenty-eight; clubbed, one; branched, three; and unclassified, eight. Two of those having short antennae were males with deficient genitalia. Association between deficiency in genitalia and digestive tract is much closer. Males deficient in genitalia alone were twenty- 374 p- w- WHITING. four; in digestive tract alone, fifty-five; and in genitalia and digestive tract, fifteen. Females deficient in genitalia alone were nine; in digestive tract alone, twenty-two; and in genitalia and digestive tract, nine. These figures illustrate the rare occurrence of these abnor- malities in normal stocks. Deficiencies in genitalia necessarily entail sterility. Mating reactions are in all cases quite normal. A male having no trace of external genitalia and internal much reduced will mount a female and beat its antennae in the usual manner. Females with reduced genitalia or abdomen swollen and blackened by fecal matter show characteristic responses to caterpillars. Although wasps with deficient digestive tract live only a few days and females lay but few eggs or none it has sometimes been possible to obtain offspring from them. These offspring are usually normal. STRAIN SHOWING HEREDITARY DEFICIENCY IN GENITALIA AND DIGESTIVE TRACT. Hereditary deficiency in genitalia and in digestive tract arose from the combination of material from Lancaster, Pennsylvania, and Iowa City, Iowa. No deficient occurred in the direct line of Iowa City ancestry which included 598 males and 543 females running back to the original female. An I male was crossed to an L female, stock 6. An F2 male from this cross was mated to an L female, stock 10. This resulted in fifty-three normal males and 130 normal females, besides one female with deficient digestive tract. This is the first occurrence of the character in the ancestry. A sister of this female produced 224 normal males one of which was mated to an I female. Fifty normal males and ninety-six normal females resulted. One of these females pro- duced 139 normal males, five deficient males and one impaternate female. (Female from unfertilized egg — from 934-5-) Descendents of this impaternate female include in all 209 fraternities. Inbreeding was carried on by simply isolating individual females after they had had a chance to mate with their brothers. Ratios of deficients in these fraternities varied er- ratically and a large number of insects died immature. The latter were recorded without reference to sex. INFLUENCE OF AGE OF MOTHER. 375 Ratios of deficients among the males in the 209 fraternities were arranged in a frequency distribution. Grouped to the nearest 2 per cent, they ranged from o to 58 per cent, with frequencies 57, 6, 4, 9, n, 21, 19, 8, 6, 5, 7, 5, 3, 8, 6, 6, 5, 5, o, 6, 2, o, o, 2, o, 2, o, i, i, 2, besides two fraternities each consisting of a single deficient individual. 131 of these fraternities included females with ratios of deficient ranging from o to 34 per cent. Corresponding frequencies were 80, i, 10, 9, 7, 2, 4, 7, 2, 2, i, o, o, 3, i, o, o, 2. There were also two small fraternities, one with one deficient to one normal and the other with four deficient to two normal. Percentage of wasps dying immaturely were calculated for the 209 different fraternities. These ranged from o to 32 with frequencies 125, n, 17, 19, 12, 5, 6, 2, 3, i, 3, o, i, i, 2, o, i. There were 10,335 males and 2,165 females in the entire group. Frequency According to Age of Mother. In a special line of this deficient stock consisting of thirty-seven cultures there were 2,094 males with 540 or 25.788 per cent, deficient, 468 females with 43 or 9.188 per cent, deficient, and among the total of 2,732 there were 170 or 6.223 Per cent, dying immature. Females are transferred every four or five days and culture vials labelled a, b, c, etc. Thus if offspring be summarized according to letter of vial average changes in progeny according to age of mothers may be obtained. Table A and Chart I show TABLE A. OFFSPRING OF STRAIN DEFICIENT IN GENITALIA AND DIGESTIVE TRACT ARRANGED ACCORDING TO AGE OF MOTHER (VIALS). \ rial. a b C d e / g Total. MaleSt Total . . 604 4^8 370 3i8 148 7O ^6 2 OOA Deficient 109 i^s I2Q I2O 74 IO T. ^AO Females, Total Deficient . . 168 12 196 17 96 14 8 o 468 A1 Total 888 706 513 358 1 60 71 36 2 7"*2 Immature deaths.. . 26 52 47 32 12 I 0 170 GENr-l IDT. 99 ,GEN DTVJ CHART i. Percentage of deficient offspring according to age of mothers. Culture vials through which mothers were successively transferred are denoted by a, b, c, d, e, f, g. Mothers remained in each vial between four and five days. Gen. Ant. and Gen. Dt. denote offspring from mothers of strains in which deficiency in genitalia was associated with deficiency in antennae and in digestive tract respec- tively. For actual numbers see Tables A and D. INFLUENCE OF AGE OF MOTHER. 377 W I/I 3 H O % O o o i s w a. O H O g s OS o u D W O z OS O z 1-4 H O Q EC, c "t3 o H I— N O 'fr ro io Oi HI ON »/> O oo O N w-> ro r*3 M 10 O ro M ts ro N ^* 10 M ro t- <-> 6*3 O OOrOOOOOO O - O O O r— M N 13 NO M N - CN 00 N 00 OO t— N M » •sf M O\ O r^ o ro N M I- M OO M •<*• •O M O t— MM M o ro t> M ^r oo rO O M 10 M r~» « r- o M N *£ oo o w O O M « % . p 42 u *-> *-• o , 72 in 2"? 48 <; Deficient . ... .... AZ 16 S8 -58 CT 12 222 It may be concluded that age of mother is an effective factor in determining this type of deficiency. DISCUSSION AND SUMMARY. Deficiency or reduction in antennae, in external genitalia, and in posterior part of the digestive tract occurs occasionally in individuals of normal stocks. Two cases of marked hereditary deficiency present some interesting points of difference. In one, deficiency in genitalia was associated with deficiency in digestive tract and many immature deaths occurred. The trait was increased and main- tained by selection but very few fraternities approximated 50 per cent, even among the males. Great variability in ratios in the different fraternities and failure to recover the stock by breeding normal up to males of selected deficient indicate that the character is genetically complex. In the other case, deficiency in genitalia was associated with deficient antennae. There was no increase in immature deaths over those normally occurring but much female sterility. The line originated from a female from stock supposedly normal. Fraternities fell into at least two groups. In one the deficient averaged 2.6 per cent, of the total and were deficient either in genitalia or in antennae but not in both. In the other they averaged 45.8 per cent, of the total, the majority of them being deficient both in antennae and in genitalia. The facts indicate genetic complexity. Percentage of deficient offspring varies according to age of the mother in both cases. It increases up to approximately the tenth 382 P. W. WHITING. or fourteenth day of adult life and then decreases. No consistent change in type of deficiency according to mother's age could be detected. Possible explanations of this rise and subsequent fall of ratio of deficient during life of mother may be considered. Cytoplasm or yolk of eggs might differ according to some rhythm in the life of the female. The influence would have to be carried through the larval period and up to the time of the formation of the organs in the pupa. Something in the food of the larva might vary according to the age of the mother, some ingredient of the fluid injected at time of stinging the caterpillars. Neither of these theories involves genetic change in maternal contribution. A third explanation may be suggested. It has been shown that linkage in Drosophila varies with age of mother. If this is true in Habrobracon a difference in genetic composition of offspring according to the mother's age may be brought about. In order to test this possibility records on a lethal factor linked with orange were reinvestigated (Whiting, P. W., 1921) and the five fra- ternities involving linkage were summarized according to vials. Results are given in Table E. The difference between vials a and TABLE E. CROSS-OVERS OF A LETHAL LINKED WITH ORANGE ARRANGED ACCORDING TO AGE OF MOTHER. Vial. a b c d-f Total. Cross-overs 14/73 18/108 !9/fi3 8/57 59/301 Total . . . Per cent . 19.2 ± 3.1 16.7 ± 2.4 30.2 ±3.9 14.0 ±3-1 b is of no significance. There is an increase in cross-overs from b to c, 13.5 ± 4.6 per cent., which is probably significant and a decrease after c, 16.1 ± 5.0 per cent, which is also probably significant. If this rise and fall of crossing-over according to age is proved when further linked factors are found, it may be the most reasonable explanation of change in ratio of deficient. INFLUENCE OF AGE OF MOTHER. 383 LITERATURE CITED. Hart, Hornell. '23 Mongolian Amentia and the Ages of the Mothers at the Births of the Aments. Quart. Pub. Am. Statis. Assoc. Sep. Whiting, P. W. '21 A Lethal Factor Linked with Orange. BIOL. BULL., Vol. 41, pp. 153-155. 384 P. W. WHITING. EXPLANATION OF PLATE I. All figures have been drawn with camera lucida. Drawings have been reduced two thirds. Final magnifications are X 32 for Figs. A-H, X 15 for Figs. I-M. FIGS. A, B AND C. Ventral view of head, ventral and posterior views of tip of abdomen of deficient male (Freak 60). FIGS. D AND E. Ventral and posterior views of abdomen of normal male. FIGS. F AND G. Abdominal portion of digestive tracts of deficient female (Freak 21) and deficient male (Freak 20). FIG. //. Anterior view of head of deficient male (from 1171.3*:). FIGS. I AND J. Ventral and right views of abdomen of deficient female (Freak 6). FIGS. K, L AND M. Dorsal, ventral and right views of abdomen of normal female. BIOLOGICAL BULLETIN, VOL. LI. PLATE I P. W. WHITING. GAMETIC MEIOSIS IN MONOCYSTIS. GARY N. CALKINS AND RACHEL C. BOWLING. The monocystids of the earthworm, probably the commonest and most frequently found type of gregarine, have never been completely monographed, although an excellent start in this direction was made by Hesse (1909). Cytological studies have been made by Wolters (1891), Cuenot (1901), Shellack (1912), and Mulsow (1911), the latter finding typical gametic meiosis or chromosome reduction in a species which he regarded as new and to which he gave the name Monocystis rostraia. In this form which he named from the presence of an attenuated proboscis-like end of the cell, he found eight chromosomes of characteristic form and size. This number of chromosomes was found in the first nuclear spindle of the gamont after metamorphosis of the vege- tative nucleus, and the same number was present in the successive spindles of the succeeding progametic nuclei and until the final division when the gametes are formed. In this ultimate division Mulsow found that whereas eight chromosomes are present in the nuclear plate of the mitotic figure at the metaphase only four chromosomes are present in the daughter plates of the anaphase stage and only four chromosomes are present in each of the two resulting gametes. Here then the haploid number of chromo- somes is characteristic of only one set of cells, viz. the gametes, of the entire life cycle. Upon fusion of two gametes the diploid number is restored and this diploid number is characteristic of the entire vegetative cycle including all of the progametic nuclei. The actual reduction in number of chromosomes according to this account agrees in point of time with the corresponding meiotic phase in the great majority of the metazoa, a type which Wilson describes as gametic meiosis. A very different time of reduction in chromosome number was described by Dobell in connection with gamete formation, fertilization, and metagamic divisions of the coccidian Aggregate, eberthi, and by Jameson in connection with the gregarine Diplo- 26 385 386 GARY N. CALKINS AND RACHEL C. BOWLING. cystis sclmeideri (Dobell and Jameson, 1915). This joint paper was followed later by individual papers on each of the two types (Dobell, 1925; Jameson, 1920). In both of these types the authors found that the progametic divisions are all alike including the last or gamete-forming division, in having nuclei with the same number of chromosomes, and that this number is the same in all of the vegetative stages. The zygotes, alone of all cells of the vegetative cycle, have the double or diploid number of chromosomes. Meiosis or reduction in number of chromosomes takes place with the first mitotic division of the zygote, a type known as zygotic meiosis. The individuals which one ordinarily encounters of these two species are therefore haploid as regards chromosome number. From these findings Dobell and Jameson make the generali- zation that zygotic meiosis is probably universal in Gregarinida and Coccidia and they interpret Mulsow's account of reduction in Monocystis rostrata as evidence of confusion by him of two species of Monocystis, one with eight the other with four chromosomes. It seems a little premature to say the least to sweep away contra- dictory evidence in this fashion when a re-examination of Monocystis in the light of Dobell and Jameson's work would clear up the matter. The cytological results of such a re- examination are presented herewith. There are many species of Monocystis in earthworms some of which are apparently limited to certain species of worms. We have restricted our investigation to the parasites of the seminal vesicles of one species — Lumbricus terresiris. Here several species of monocystid gregarines are found, some almost invariably present, others less frequently. We have frequently encountered the large form with a proboscis-like end which undoubtedly corresponds with Mulsow's Monocystis roslrata (450 fj. long by 220 fj. broad). We have not seen the long thread-like form which Hesse names Nematocyslis magna (Monocystis magna Schmidt) and is said to be from 4.5 mm. to 5 mm. long and only 50 ju in diameter. We have not seen Monocystis striata Hesse with its longitudinal grooves and striations which are described as frequently prolonged posteriorly to form a tuft of filaments as in the genus Zygocystis. We often find Monocystis agilis which can GAMKTIC MEIOSIS IN MONOCYSTIS. 387 be easily identified and we often find another species which might be regarded as one of the varieties of Monocystis lumbrici which are not precisely described in the literature. Cuenot (1901) Brasil (1905) and Hesse (1909) all remark on the difficulty of tracing a pair of pseudo-con jugants of Monocystis back to the individual agamonts which form the subjects of the specific descriptions. Where only one type of gregarine is present in an organ the assumption is safe that the cysts found in the organ come from that one type. When, however, there may be as many as five different species present at the same time, as is the case in the seminal vesicles of Lumbricus terrestris, then the problem is real. There is indeed a real danger of confusing sporocysts from different species and of getting a false interpre- tation of the life history of any one. On the other hand, if the species of Monocystis were based upon the size and structure of the sporocysts and sporoblasts instead of upon the agamont stages the difficulties of identification attendant upon multiple infection would be materially lessened. While an ideal condition would be the indisputable association of a given agamont with its definitive sporocyst in these mono- cystids, for our purpose such an ideal is unnecessary. In all gregarines two individuals upon reaching maturity become associated in pairs (pseudo-conjugation) and a capsule, called the sporocyst capsule or simply sporocyst, is formed about them (Fig. i). These gamonts then proceed to form gametes by repeated divisions of the originally single nucleus in each, the gametes ultimately appearing as a zone of small nucleated cells budded out from the periphery of the gamonts. The gametes from one parent cell then meet and fuse with the gametes from the associated cell. The zygotes thus formed by fusion two by two are practically equal in number to the total number of gametes formed by either one of the associated parent cells. Each zygote forms its individual capsule termed the sporoblast capsule within which the zygote nucleus, an amphinucleus, divides a certain number of times, usually three. The eight resulting nuclei become the nuclei of the definitive germs termed sporozoites. It is no more difficult to recognize different specimens of the GARY N. CALKINS AND RACHEL C. BOWLING. .. ,- - and sporoblasts than it is to recognize different individuals of the same species of Monocystis. The structure of protoplasm, the nature of the sporocyst capsule, the same type of cysts individuals c the agamont A-m"; VC. ^AI>-':;fX,;yW^Wv-^l %p: %::. . \ x >/;;; *-.••£.-<'•. ' .A \ fe::^M - 2 . .XV>;.A V4-, >-i •---x ' r- ••-.-• Vj.'&s^s'.v.y • v.j ../. , •-V-.-v'^.^ ;X ^^**%j-;:: s^vf "''' •' \ XI*: ii-i. :--.-t FIG. i. Monocystis species. Two gamonts in pseudo-conjugation in sporocyst capsule. Nuclei beginning metamorphosis. Minute spindles in substance of centrosphere. Smear preparation. Camera lucida. X 900. appearance of the nuclei in size and character together with the number of chromosomes, are factors involved in the recognition of a cyst of a certain species. We have been no more fortunate than our predecessors in tracing the indisputable connection between a specific agamont and the sporozoites which it ultimately forms and for this reason we beg the question of the particular species of Monocystis with which we are dealing. We are confident however that we have followed the history of one single species. We have chosen for particular study the cysts containing the largest sporoblasts and the largest gametes because their nuclei GAMETIC MEIOSIS IN MONOCYSTIS. 389 give the clearest pictures of the nuclear contents. The total number of gametes formed by both gamonts in this species is approximately 130 and the number of sporoblasts is approxi- mately 65. The protoplasm is uniformly alveolar, clear, and with alveoli of medium size. The nucleus of the gamont is spherical and contains a single spherical endosome. FJG. 2. First pregametic spindle with 10 chromosomes in one gamont; portion of disintegrating nucleus in the other. Section. Camera lucida. X 2650. The material was fixed in hot Schaudinn's fluid consisting of a saturated solution of bichloride of mercury in absolute alcohol without and with 5 per cent, of glacial acetic acid. Seminal vesicles were removed entire from richly infected earthworms and immersed after puncturing, in the killing fluid where they were left for from two to four hours. They were then treated for twenty-four hours with iodized alcohol, embedded in paraffine and cut in sections from three to four microns thick. The sections were stained with iron haematoxylin without or with a counter stain. Other stains were tried but the best results were obtained with iron haematoxylin. The metamorphosis of the gamont nucleus begins with the condensation of the chromatin and formation of the chromosomes. At this stage the latter are feebly staining and even in the metaphase of the first division they do not stain with the same intensity as in the later progamous divisions or as they do in the 390 GARY X. CALKINS AND RACHEL C. BOWLING. developing zygote. An astrosphere is condensed from the cytoplasmic reticulum at one pole of the nucleus and the nuclear membrane disappears at this region. We did not observe a stage in which a single centriole could be found in the astrosphere, the earliest stage seen being a minute spindle which appeared to be formed in the substance of the astrosphere in the region which had been occupied by the now dissolved nuclear membrane (Fig. i). This spindle enlarges until it occupies fully one half of the diameter of the cell (Fig. 2). The short, rod-shape or slightly curved chromosomes are ten in number. The daughter nuclei when fully divided may be separated by the full diameter of the cell, and remains of the spindle substances trail out behind them marking the path of the spindle (Fig. 3). FIG. 3. Two gamonts after first pregametic nuclear division. Smear. Camera lucida. X 900. GAMETIC MEIOSIS IN MONOCYSTIS. 39! The subsequent spindle figures are characterized by the increasingly intense staining capacity of the chromosomes while the size of the nuclei decreases. There is a tendency for the FIG. 4. Third pregametic division of nuclei with 10 chromosomes. Section. Camera lucida. X 1750. daughter nuclei after each division to approach the periphery and the final progamous nuclear spindle lies in a plane tangential to the surface (Figs. 4 and 5). In some cases the spindle occupies a bud which stands out from the surface in characteristic fashion 392 GARY N. CALKINS AND RACHEL C. BOWLING. (Fig. 7). Such tangential nuclear figures are typical of the final progamous division, and their products become the gamete nuclei. FIG. 5. Detail of a spindle during the third pregametic division. Section. Camera lucida. X 2650. In the tangential nuclei as in the earlier pregametic nuclei we have seen no evidence of a prophase skein stage. Metaphase stages of division are frequently found (Fig. 6) and early and late :-c v^.'"/:vj ;:sB'!- %'>• FIG. 6. Group of spindles in the metaphase stage of the last pregametic division, one showing polar view of a nuclear plate. From different individuals; all with 10 chromosomes. Sections. Camera lucida. X i?50- anaphase stages. In these the number of chromosomes is always ten (Figs. 7 and 8). In the metaphase stage of the tangential GAMP;TIC MEIOSIS IN MONOCYSTIS. 393 spindles there are likewise ten chromosomes, but these ten do not divide longitudinally. They are separated into two groups of five chromosomes each, easily distinguishable in the anaphase f ^mi',) i-; . ' &./^:-/'\..;-'\.:; ^Ti-'-f '•'-:'•. >•..•''"•:..'.-• •::vr-v< 4 v--'--.^^.r.;i >• /f V^j:-/-^^^ rl FIG. 7. Group of spindles in the anaphase stage of the last pregametic division ; from different individuals. 5 chromosomes in the daughter plates. Sections- Camera lucida. X 1750. groups (Figs. 7 and 8). Reduction in number of chromosomes from 10 to 5 therefore takes place with the last progamous division. The chromosomes are not sufficiently different in shape and size to furnish any evidence of coupling. Two daughter groups are represented in Fig. 8; some are U-shape others are usually somewhat sigmoid in character, but the differences are not so 394 GARY N. CALKINS AND RACHEL C. BOWLING. Sfe, ii-vA - ffS eKpaBs^K^^ » i* ' "-i.^. L--.-iiiL-V- ""'•.'.•• ;. '•. .; .;. -••..t'C ••;?"*'••• V ';' ,^'7-->-**V!£& -,/pj . .•fVT"--^**| FIG. 8. Anaphase stage of last pregametic division and 3 groups of chromo- somes from other spindles in the same stage. Sections. Camera lucida. X 2650. marked as in the case of Aggregate, or as in Mulsow's Monocystis rostrata. The gametes are relatively large, spherical to ovoidal in shape and without constant dimorphism although minor variations in FIG. 9. Characteristic grouping of gametes around periphery of gamont. Section. Camera lucida. X 600. size may be observed even in gametes from the same gamont (Fig. 9). The nuclei are usually near the anterior end of the cell GAMETIC MEIOSIS IN MONOCYSTIS. 395 (Fig. 10) and when two gametes come together, fusion begins at this nucleated end. The nuclei fuse and the zygote secretes a delicate plastic membrane which is transformed later into the rigid sporoblast capsule (Figs, n, 12 and 13). At this early Skdlp^ '"*'•'.' .' >r--j:' -*!*"»«"«::. V--"",^ ^ llr „ i :-i ifc . •• ..*=*».. '•• \v-~"- -r^. FIG. 10. Group of gametes. Smear. Camera lucida. X 900. period of the zygote the form is quite variable owing to the plasticity of the membrane (Fig. 15). The amphinucleus swells and becomes more conspicuous by reason of the more intensely staining chromatin. The chromomeres become arranged in a loose spireme which appears to be continuous (Fig. 14). The f*^£\ .-••:.«,. -^....'•*.:.'i. "C X ...v-' •..'•-/VV.l flfcV -y. f"v. •.*..:.-..•«; "•'. '••,.,^. $&•'*'•*••:•. -.'•'•. ^"N/«. W i , |-;-4i-%,. ':•'•. . •?. ,:••••• / • . . /:, ~\'!$ 1 V,*^ . *,•-:.?•;. .•*!.••.••••,•••• .'.•-:'•':/'•<: A:' .&S-iiivf%z&p FIG. ii. Characteristic grouping of gametes and zygotes about gametocyte. Section. Camera lucida. X 600. threads are double and break up into 10 chromosomes. A spindle is formed with definite poles and fibrils but the latter are not as sharply defined as in the progamous divisions (Fig. 15). In the anaphase stage 10 chromosomes are present in each daughter plate (Fig. 14). After this first metagamic division, which is not accompanied by cell division, we were unable to make out any definite spindle 396 GARY X. CALKINS AND RACHEL C. BOWLING. , •-. ;W 'H "- -v<- - •"..- " x^¥ - '- AV if4 t jf^f. . §£•• .••"•>•:££" ''ii;'- '•-,. ^^ , ' '• ., ^•;v^kS ^ FIG. 12. Group of gametes undergoing fusion to form zygotes. Section. Camera lucida. X 900. ^^•'?'\ v • .. Ml ! £ ^;: ^ j FIG. 13. Group of zygotes with stages in fusion of the nuclei. Section. Camera lucida. X 1750. formation. The two daughter nuclei soon divide again, an inference formed by reason of the comparatively rare occurrence of the binucleated sporoblast. The two nuclei give rise to four GAMETIC MEIOSIS IN MONOCYSTIS. 397 FIG. 14. Young sporoblasts, one with nucleus in spireme stage, the other with nucleus in anaphase stage of division with 10 chromosomes in each daughter plate. Section. Camera lucida. X 2300. FIG. 15. Group of young sporoblasts in different stages. Sections. Camera lucida. X 1750. 398 GARY N. CALKINS AND RACHEL C. BOWLING. \vithout division of the cell and such sporoblasts with four nuclei are much more common than are the binucleated ones (Fig. 16). FIG. 16. Group of developing sporoblasts, 2 with 2 nuclei, 2 with 4 nuclei and one with 8 nuclei — no cytoplasmic division. Section. Camera lucida. X i7S°- A third division gives rise to the eight nuclei of the sporozoites and at this stage the cytoplasm divides into eight elongated cells each bearing a nucleus; each is a sporozoite (Figs. 16 and 17). FIG. 17. Group of late sporoblasts from one sporocyst, each with 8 sporozoites In one the sporoblast suture is clearly indicated. Section. Camera lucida. X 1750. During this later period the sporoblast capsule becomes definitely formed and rigid ; near each pole a distinct ring of more GAMETIC MEIOSIS IN MONOCYSTIS. 399 intensely staining substance is conspicuous. The origin and significance of these rings will be considered in a later paper. In old sporoblasts a definite suture or groove runs down the sporo- blast capsule on both sides from pole to pole, indicating the presence of two valves and a prospective opening by dehiscence (Fig. 17). The chromosome history of this gregarine thus does not bear out the generalization made by Dobell and Jameson that zygotic meiosis is probably characteristic of all gregarines. It is more probable that both zygotic and gametic meiosis occur in this group, the former probably in forms like Echinomera hispida (Shellack, 1907) which has 5 chromosomes, in Nina gracilis (Leger and Duboscq, 1909) which has 5 chromosomes, and in Monocystis ovata (Shellack, 1912) which has 3 chromosomes, and in other forms with similar odd numbers of chromosomes. LITERATURE CITED. Cuenot, L. '01 Recherches sur 1'evolution et la conjugaison des Gregarines. Archiv. d. Biologic, XVII., 4. Dobell, C. C., and Jameson, A. P. '15 The Chromosome Cycle in Coccidia and Gregarines. Proc. Roy. Soc. Biol.. Vol. 89. Hesse, E. '09 Contribution a 1' etude des Monocystidies des Oligochetes. Arch, de Zool. Exp. et Gen. (5) Vol. 43. Jameson, A. P. '20 The Chromosome Cycle of Gregarines with special Reference to Diplocystis schneideri Kunst. Q. J. M. S. 64 No. 2. Leger, L., and Duboscq, O. '09 Etudes sur la sexualite chez les gregarines. Arch. Prot. 17. Mulsow, K. 'n Uber Fortpflanzungserscheinungen bei Monocystis rostrata n. sp. Arch. Prot. 22. Shellack, C. '07 Uber die Entwicklung und Fortpflanzung von Echinomera hispida. Arch. Prot. Vol. 9. Shellack, C. '12 Die Gregarinen, in Prowazek's Handbk. d. path. Protoz., Vol. I. Wolters, M. '91 Die Conjugation etc. bei Gregarinen. Arch. mik. Anat., Vol. 37. PRELIMINARY NOTE ON A NEW PROTOZOAN PARASITE OF EARTHWORMS OF THE GENUS EUTYPHCEUS. G. E. GATES. JUDSON COLLEGE, RANGOON, BURMA. In the course of a recent study of the earthworms of Burma a large protozoan parasite of unusual form was frequently en- countered. The writer can discover no reference to any parasitic animal of similar form in the literature. The following descrip- -V— -n FIG. i. Part of a trophozoite. (Camera lucida.) s.r., secondary ramus; p.r., primary ramus; n., nuclear membrane; e., endosome. tion will therefore be of interest to zoologists, although it should be understood that the present paper attempts nothing more than the presentation in a preliminary way of data gathered during a study of the host animal. The parasites may be as long as 4 mm. To the unaided eye 400 PRELIMINARY NOTE ON NEW PROTOZOAN PARASITE. 40! the animal presents a smooth creamy-white appearance, or very rarely, a light brownish tinge. The latter color, however, is probably due to a staining action on the parasite of some sub- stance from the host. The large unattached end of the animal is bluntly rounded. At a greater or less distance from this end the body branches into two rami each of which is narrower than the main trunk (Fig. i). Each of the two primary rami branch again to form two smaller secondary rami. This branching continues until eight or sixteen small ramuli are produced. The region at which ramification begins is not constant in position. FIG. 2. Sucker-like organs of attachment. (Camera lucida.) X ca. 475. In one specimen which is 1.4 mm. long the first branching occurs at a distance of about .9 mm. from the blunt end, the second 1.2 mm., and the third 1.32 mm. In other specimens the trunk portion is often much shorter proportionately, rarely even shorter than the primary rami. The ramuli at the attached end are the shortest, the rami usually increasing in length towards the free end of the parasite. The dichotomy is regular in all specimens examined. The ramuli bear groups of irregularly-ovoid, sucker- like objects by means of which the parasite is attached to the host (Fig. 2). Longitudinal and transverse striations are visible in the ecto- plasm with the high powers of the microscope. The transparent fluid endoplasm of the trunk and primary rami is densely packed with ovoid paraglycogen granules. These granules are widely- separated in the secondary rami and only very sparsely dis- tributed in the ultimate, transparent branches. The nucleus (Fig. 3) is a large, ovoid body sometimes visible to the unaided eye in cleared specimens as a minute spot. In large specimens it is .64-.S6 mm. in length. The contents of the nucleus are perfectly transparent except for a single, ec- centrically located, spherical body of light bluish appearance. 27 402 C. E. GATES. This endosome has either one large, or several smaller, trans- parent, vacuole-like areas. The nuclei of dead parasites are always found in the unhranched portion. O o C A B FIG. 3. A. Nucleus from trophozoite 1.3 mm. in length. (Camera lucida.) X ca. 335. e., endosome; n.m., nuclear membrane. B. Nucleus from larger trophozoite. (Camera lucida.) X ca. 350. C. Paraglycogen granules. (Camera lucida.) X ca. 350. Live attached parasites have not been studied but in living detached forms two series of movements are noticeable. The first series may be described as waves of peristaltic contraction passing along the trunk and rami of the first order. These peristaltic contractions produce a back and forth churning of the endoplasmic contents in the trunk, from the trunk into the primary rami, and back and forth in the rami. By these con- tractions the nucleus is squeezed to and fro from one end of the trunk to the other. In two of the living specimens examined the nucleus remained in the unbranched portion of the parasite during the whole period of observation. In the other animal the nucleus frequently passed into one or the other of the primary rami. In the primary ramus the nucleus was either rolled back and forth several times or at once passed back into the trunk. The nucleus moved with equal ease and frequency into either one of the primary rami but never passed into the secondary rami which were narrower than the nucleus. It should be noted that in this third animal the ramification began unusually near the blunt end so that the trunk portion was less than one third of the total length of the parasite. The second series of movements may be called rotation move- ments and result in a spiral twisting of the primary rami and their branches back and forth on their own axes. PRELIMINARY NOTE ON NEW PROTOZOAN PARASITE. 403 The parasites occur in the coelomic cavities of one genus of Burmese earthworms, Eutyphceus and most commonly in E. foveatus. They are found in smaller numbers and more rarely in E. spinulosus, rams, and peguanus. One hundred per cent, of the E. joveatus examined during a period extending over four years have been infected. In the majority of worms of the last named species, large numbers of parasites are found, masses containing 20-40 individuals being not uncommon. The largest groups of parasites occur in segment three, the majority of which are attached to the large nephridial masses of this segment. Other locations are: under the nerve cord in segment four, and on the septa or parietes of segments four to twenty. Those found posterior to segment nine are almost always less than one millimeter in length. AIKINETOCYSTIS siNGULARis, n. gen., n. sp. Diagnosis. Uninucleated protozoa parasitic in the ccelomic cavity of certain earthworms of Burma; body cylindrical or columnar with a characteristic, regularly dichotomous branching at the at- tached end; fixation to the host by means of sucker-like bodies borne on the ultimate branches; nucleus with a single ec- centrically placed endosome. Additional Observations. Pairs of animals are frequently found attached to each other near the rounded free ends. Among the larger sized forms such pairs are nearly as common as single animals, but do not occur among the smaller parasites. Groups of three to eight animals similarly adherent to each other are also found. Attempts to pull apart the attached animals have always resulted in breaking one or both of the animals near the place of attachment. How- ever no evidence of organic connection between members of a pair or group has been found. Monocystid-like spores of typical pseudo-navicella form in masses visible to the naked eye are sometimes present in segments three to five. In other specimens in which such masses are not visible similar spores can almost always be found by scraping the body wall of the segments just mentioned. The spores are of 404 C. E. GATES. two sizes: the larger .O2-.O23 mm. long, the smaller .ooj-.ooS mm. long. The contents of the spores are coarsely granular. A single transparent vesicular body is present, centrally placed in the small spores, eccentrically placed in the large spores. As no other parasites with a cyst- or spore-forming habit are found in segments three to five and as the spores are practically always present with the trophozoites, it may be inferred, at least tenta- tively, that they are produced by the trophozoites with which they are always associated. Numerous specimens of the host have been dissected in every month in which the worm can be obtained but only three cysts from as many worms have been found in proximity to the trophozoites. The cyst in each case was ovoid in shape and about .62 mm. long. A tough membrane enclosed a fluid in which floated two hemispherical bodies in contact with each other on a flattened surface, nearly filling the cavity of the cyst. After rupturing the membrane the gamonts were easily separated from each other. The single hemisphere retained its shape and opacity in clearing fluid. Rupturing the hemisphere by pressure on the cover-glass released granules similar in appearance to those found in the endoplasm of the trophozoite, but no nucleus or spores could be seen. Spore-containing sporocysts have not been found in the anterior segments but cysts about .75 mm. long and .6 mm. wide, densely packed with spores similar in size and appearance to those occurring in the anterior segments are sometimes present in the whitish masses filling the ccelomic cavities of the anal segments. Worms of the genus Eutyphceus occurring in Rangoon suddenly disappear at the end of the rainy season and are not to be found again until after the beginning of the next rainy season. The failure to find the sporocysts in spite of the constant presence of the spores may be due to a coincidence of the sporocyst formation with this winter hibernation period of the host. SUMMARY. A new protozoan parasite of the ccelomic cavities of certain tropical earthworms, with a remarkable branched structure of the trophozoite is described. Mention is made of associated spores and sporocysts possibly belonging to the trophozoites. A CYTOLOGICAL STUDY OF SECRETORY PHE- NOMENA IN THE SILK GLAND OF IIYPHANTRIA CUNEA. ELIZABETH KINNEY, WASHINGTON UNIVERSITY, ST. Louis. INTRODUCTION. The general anatomy and histology of the silk glands of lepidopterous larvae have been carefully studied and described by numerous investigators beginning with the work of Helm ('76), but little purely cytological study of the cells with an aim toward giving an account of the mechanism of secretion was reported before the investigations of Maziarski ('n) and later of Nakahara ('17). Since that time, few papers have appeared based upon studies of the silk gland, with the exception of an account by Yamanouchi ("22) describing a morphological study of silk secretion. In previous investigations of silk secretion, studies have been made only upon glands of caterpillars which spin a cocoon at a certain period in their life history. Such study is of value in observing glandular function and development during the pro- gressive stages preparatory to and at the time of activity. In order to best study the actual process of secretion, the optimum material would naturally be found in a form which secretes actively over a comparatively long period of time. The fall webworm, Hyphantria cunea, is ideally adapted for the study of active secretion, for the caterpillars live together in colonies, secreting the web continually throughout larval life in the process of securing food. This form, therefore, was selected for the present investigation and by the application of cytological methods, an attempt has been made to determine the cellular organization which is concerned with secretory phenomena in the silk gland. Work upon this problem was for the most part carried on during 1925 and 1926, at Washington University, St. Louis, 405 406 KLIZABETH KIXNEY. under the direction and supervision of Prof. Caswell Grave. It is with great pleasure that I take this opportunity to express to Dr. Grave my sincere appreciation for his advice, criticisms, and valuable suggestions. MATERIAL. The spinning-glands of the larval Hyphantria cunea, like those of all lepidopteran larvae, consist of two long tubes, easily distin- guishable by their glassy appearance, situated on either side of the nerve cord and below the digestive tract. Each tube is a cylindrical, folded body, averaging between 6 and 8 mm. in length, or slightly less than the entire length of the caterpillar which averages about i cm. In total mounts of a spinning-gland, three arbitrary divisions may be distinguished, characterized by differences in appearance, and, in section, by structure (Text- Fig. D) ; the posterior, or secreting portion lying between the eighth and eleventh segments and terminating blindly; the middle portion or reservoir, bending posteriorly to the ninth or tenth segment, then folding anteriorly to the fifth segment; the anterior, or conducting portion extending straight forward to the head where the two tubes converge and unite to open at the apex of a median cylindrical organ, the spinneret. Each tube consists of a single layer of epithelial cells (Imms, '25, p. 407), varying in shape and size according to position in the tubule. The cells are arranged in two longitudinal rows around a central lumen. The nucleus of each cell is charac- teristically branched and very irregular. Externally, the gland is covered by a thin membrane, the tunica propria, and internally, the lumen is lined by the thicker tunica intima. The cells of the posterior region are relatively large and irregular in form and contain a greatly branched nucleus. The inner cell wall is irregular in surface contour, dm- to numerous drops of secretion in passage between the cell and lumen. The tunica intima is not readily distinguishable (Text-Fig. A, B). The cells composing the middle region are greatly flattened with .1 < onsequent elongation of the nucleus, and possess a clear hyalin, inner border. The cavity of the lumen has increased in diameter and is filled by secreted material (Text-Fig. C, D). SKCRKTORY PHENOMENA IN SILK GLAND. The cells of the anterior conducting portion are reduced in size, being compressed but less elongated than cells of the reservoir. The nuclei tend to be rounder and less branched, and the intima can be definitely distinguished (Text-Fig. E, F). The tracheae that supply the posterior and middle regions penetrate through the tunica propria into the substance of the cell. It is significant that no intracellular tracheae are present in the anterior conducting portion of the silk glands. COLOR REACTIONS OF SILK GLAND CELLS WITH VARIOUS STAINS AND FIXATIVES. Fixative and Stain. Chro- matin. Nuclear Bodies. Nucleo- loid Bodies. Mito- chondria. Benda: Delafield's Haem., Acid Fuchsin. . Ehrlich's Haem., Acid Fuchsin. . . . Ehrlich-Biondi Blue Blue Pink Black Blue Pink Black Blue Orange I Iron Haem., Erythrosin Black Black Black Black Bouin: Delafield's Haem., Acid Fuchsin. . Ehrlich's Haem., Acid Fuchsin. . . . Iron Haem., Acid Fuchsin Blue Blue Pink Blue Blue Black Blue Black Black Champy: Altmann, Acid Fuchsin, Methyl gr.. Green Red Red Red Delafield's Haem., Acid Fuchsin. . Ehrlich's Haem., Acid Fuchsin. . . . Ehrlich-Biondi Blue Blue Pink Black Blue Red Black Blue Orange Iron Haem., Acid Fuchsin Black Black Black Black Gatenby: Delafield's Haem., Acid Fuchsin. . Iron Haem., Acid Fuchsin Black Black Black Black Black Black Black Mann-Kopsch: Unstained Yellow Yellow Yellow Altmann, Acid Fuchsin, Methyl gr. . Reddish Reddish Brown Regaud: Altmann, Acid Fuchsin, Methyl gr. . Green Red Green Red Iron Haem., Erythrosin Pink Black Black Larval H. cunea furnished the entire supply of material with the exception of a few glands from A patella americana and Diacrisia virginica which were preserved in Bouin and DaFano preparations for comparison. The age of the webworms could not be determined as they had been actively secreting for some time previous to collection as evidenced by the size of the webs. An abundant supply of material was secured from bayberry 408 ELIZABETH KIXXEY. * c c c -^ (-" o 4-> s E £ E c c c 0 ^ ^ C/ ^ S c c c c = = 1 £ 0 ^ >> U £ £ 'J £ £ S £ O £ S O £ £ £ >• o C £ '5 £ _ | 3* 3 C S C E J^ c c c c C tf ""^ O OH OH OH OH . K J^" J^1 •T^ J^ ^,1 n . n ^ p . n . PH fX< ^* ed E E r^ C »>i ^ i^ V ' 11 ^! ^ O ^ »y i> £ AJ p. •_jj C •- S C 03 Jj C C £ E .S .£ £ 2 V J3 HI 0, 3 OH C- 23 O £ £ C £ OH OH CO C P5CQ U c5' | — rt ¥ *"* 1 r^ OJ Ol c 4, <0 ^ ^ -^ ^ ££«£•£ +> X V M^i • HM £» j^ CJ CJ •Sj - > 4! « — ^ ^> T"* *c ~™^ C ._ ..-, 3 c3 c c- e 5JO Cy •— ^j i> £% 4-1 3 3 ea oa o. C E _; 03 3 C 03 J CQ C ^ C<03 O * o e o tH .Q -c E o rt u u o 2 ^j '<* Q c ^ fS »-— '** .^ ^_ /^ V^ ' I- b> OJ o M C • — C r3 rt — — i, rt S £i J5 J OH 3 OH 03 IH OH Q 0 'J 03 C a o V 2 -5 _^ ^ r* h£ •*^ y1^ i> ^ ^j ±e >, o oca c c rt "c "= rt „ .2 Jid u w J5 c ^H O O S nJ cS .- •gj OH PH 03 OH OH 03 P^ no p, rQ pa 03 3 95 03 tt Cv c • : jj [3 X C c : E 8 u 41 — '7. c '7. c M'K C IH ^- r* '"T MJ= •:? .£• E« O i' u -r •^ CJ to 3 to S « •^'to s S to.E 5 £ •_- •o to g — to S£ " -n to !2 ^ — 1 ^ Jj cd O "O »i •y T3 0 S 'o ~ y 'u y ^ l^t 'f. < 'o c to -< i . s (^ Q 2 c/i 1/1 C = E O z J O / z o u u PH 3! O o U SECRETORY PHENOMENA IN SILK GLAND. 409 bushes on Nonamessett Island, Mass., between August 13 and 16, and brought into the laboratory at Woods Hole where the animals were kept supplied with food and moisture until killed- METHODS. The caterpillars were killed by piercing the head with a pin after which they were immediately fastened in a paraffined dish containing Locke's solution, opened by a dorsal incision, and the glands removed directly with glass needles and placed in fixing fluids. Several fixing solutions were used, namely ; Bouin's, Zenker's, Gilson's, for general structure, Benda's, Gatenby's modified " Flemmings" for cell inclusions, Champy's method for preserving and differentiating Golgi bodies and mitochondria, and Regaud for mitochondria. Transverse and longitudinal sections, 4 micra in thickness, were made of the glands and stained by various methods. The results of different combinations of fixatives and stains may be seen from the chart. OBSERVATIONS. i. The Nucleus. (a) Historical. — The nucleus of larval spinning-gland cells has been found to be characteristically large and greatly branched; the branches extending toward all sides of the cell. This peculi- arity of shape early attracted investigators and led them to study the nucleus and its contents in an effort to find some possible relation between its unusual form and the active secretion of silk; but it was not until Korschelt in 1896 published the results of his observations upon living and fixed material that a detailed account of the nuclear content of spinning-gland cells appeared in the literature. From the results obtained through use of a modification of the Ehrlich-Biondi stain, Korschelt concluded that the larger bodies or macrosomes found in the nucleus correspond to the chromatin, and the smaller bodies, or microsomes to nucleoli. Meves ('97) used other staining reagents and came to opposite conclusions. He identi- fied the macrosomes with the nucleoli and the microsomes with chromatin. More recent investigations have seemed to support 410 ELIZABETH KINNEY. the conclusions of Meves (Marshall and Vorhies, '06; Yorhies, '08; Maziarski, 'n; Nakahara, '17). (b) Observations. — Within the nuclear membrane, which can usually be distinguished with little difficulty in the spinning- gland cells of //. cunea, bodies of two sizes are always present (except in the anterior conducting region) and with great regu- larity and frequency a larger, third body is also found (Fig. 5). The smallest bodies are by far the most abundant and tend to nil the entire nucleus. With certain fixatives, Bouin, Zenker, a reticulum can be distinguished, upon which are arranged these smallest bodies. This is not the case, however, after fixation in Regaud, Champy, Benda, or Gatenby, for in the middle and posterior regions of the gland the bodies always appear to be free in the nucleus, forming a compact mass of small granules, and take a basic stain. (See chart.) These bodies have been considered as chromatin. In the anterior region of the gland, these bodies are consistently present although considerably more scattered. The second group of bodies present in the nucleus exhibits considerable variability both of size and distribution. These are the macrosomes or nucleoli of previous workers, but in this paper, they will be referred to as "nuclear bodies." It is only rarely that these nuclear bodies are found in the nuclei of the conducting cells, and when they chance to be present, are considerably smaller than similar bodies in the posterior or middle regions. No general rule can be given for the shape or dimensions of these nuclear bodies as they tend to vary greatly. Their usual shape is spherical or ellipsoid, and this is typical of their form in the middle region, but toward the posterior end of the gland where there are evidences of active secretion indi- cated by droplets in the lumen and on the edge of the lumen, these bodies are often elongated and irregular in shape. In size, there is variation in length between 0.5 micron and 2 micra and occasionally a few are larger. Even within the same nucleus, such variation in si/e is present. The bodies have no special arrangement in the nucleus but tend to be dispersed throughout ilx- area, very often approaching the nuclear membrane. They have even been seen penetrating the nuclear wall into the cyto- SECRETORY PHENOMENA IN SILK GLAND. 4! I plasm. Critical observation with high magnification definitely showed nuclear bodies partly within the nucleus and partly in the cytoplasm (Fig. 4). The passage of nuclear bodies into the cytoplasm is a fact that has been described with great care by Maziarski ('11), Nakahara ('17), and earlier workers, as of regular occurrence in the spinning-gland cell; by Saguchi ('20) as occurring in nuclear function of the pancreas cell; and by Ludford ('25) in tissue cultures of fibroblasts of the rats' kidney. The staining reaction of these nuclear bodies is of interest because of the fact that although it is more or less variable, yet it tends to show greater affinity for acid stains. (See Chart.) It has been upon reactions to stains that all the conclusions in regard to the relation between nucleus and secretion have been made, since it has been regularly found that the nuclear bodies (nucleoli) and apparently related bodies in the cytoplasm exhibit the same staining reactions. The third class of nuclear bodies found in //. cunea has not been described in the literature so far as it has been possible to determine. Because of the similarity between these bodies and the true nucleoli, they will be referred to throughout this paper as "nucleoloid bodies." Meves ('97) mentions the fact that nucleoli may contain several small vacuoles, but in the figures of nucleoli in which he shows this structure, they are no larger than numerous other bodies in the nucleus. Korschelt ('97) in replying to the criticism of Meves, attempts to explain the presence of vacuoles by the hypothesis that the nucleoli or micro- somes are derived from the chromatic substance by some trans- formation evidenced, no doubt, by these vacuoles. Marshall and Yorhies ('06) definitely state that no plasmosome or special structure is formed in the nucleus during secretion in spinning- gland cells of Platyphylax, but mention the fact that sometimes the nucleoli may contain vacuoles but no further significance is attached to the observation; nor did Nakahara ('17) consider as important the presence of vacuoles in nucleoli. In the nuclei of H. cunea, vacuolated bodies are found to be sufficiently definite to attract especial attention. After carefully measuring a large number of such bodies in all parts of the silk gland, it was found that the average diameter is about 4 micra regardless 412 ELIZABETH KIXNEY. of position in the gland. Several measured 3 micra in diameter and a very few in the more actively secreting region are as large as 5 micra or 6 micra, while in the anterior region, these bodies average about 2 micra in diameter. This size relation is constant regardless of the fixation used. Each nucleoloid body contains two or more vacuoles or clear spaces which either do not stain or, if so, only faintly. In a section of a nucleus, usually only one of these bodies is present, rarely two (Figs. 3, 5, 6), and the frequency with which they occur in serial transverse sections 4 micra in thickness leads to the conclusion that not more than two are usually present in a nucleus. This point has been very difficult to determine ac- curately on account of the uncertainty in deciding where one nucleus ends and the next begins. From longitudinal sections, the evidence was more definitely in support of this conclusion. These nucleoloid bodies are either centrally or just eccentrically placed, and are never found in close approximation to the nuclear wall. Their usual shape is spherical, thus distinguishing nucleoloid bodies from nuclear bodies which tend to vary in shape. From careful study, it has appeared that the presence of nucleoloid bodies is usually associated with an increase in the number of nuclear bodies (macrosomes) either in the same section or the next in series. In the immediate vicinity of the nucleoloid body, the nuclear bodies are smaller, increasing in size toward the periphery of the nucleus (Figs. 3, 5, 6). The periphery of the nucleoloid bodies is not always regular, for often protrusions apparently in the process of pinching off may be seen (Fig. 5). That the vacuolated nucleolus may give rise to nuclear bodies is not without precedent in observations. Saguchi ('20) in the pancreas cell, describes a very similar process which gives rise to nucleolar corpuscles: p. 355: "As regards the genesis of the nucleolar corpuscles, I am fully convinced that they are derived from the main or side nucleoli." Montgomery ('99) concludes that the nucleolus may act as a nuclear organ either of excretion or storage and that when it has enlarged to a certain extent, it constricts pieces from itself. 1 lie nucleoloid bodies usually react more specifically to basic SECRETORY PHENOMENA IN SILK GLAND. 413 stains (see chart), but occasionally with Altmann's anilin acid fuchsin, methyl green after fixation by the Regaud method, bodies can be found staining deeply green at the periphery and pink in the center. (c} Critical. — From observations upon silk gland cells of //. cunea, it would seem that the bodies which migrate from the nucleus into the cytoplasm are not nucleoli, but are rather products of nuclear activity. That this is placing too great a burden upon a single small organ of the nucleus may be argued, but it need not necessarily follow that all the products of secretion are derived from the nucleoloid bodies, but rather that the latter contribute bodies which enlarge in the nucleus, pass out into the cytoplasm where they undergo further modification and later become discharged into the lumen. In order to verify the concluding part of this statement, a general survey of the cyto- plasm and its contents is necessary. 2. The Cytoplasm. (a) Cytoplasm. — The cytoplasmic structure of the silk gland cell as of any cell, is difficult to describe in concrete terms for it is more or less affected by fixation and the exact or true nature is hard to determine. After fixation with the Champy, Gatenby, Benda, or Regaud methods, the cytoplasm is homogeneous and granular, but after Bouin and Mann-Kopsch fixation, a definitely striated appearance may be distinguished, the striae extending from the lumen toward the periphery. No evidence of fibrils have been found in any case. Yamanouchi ('22) definitely describes fibrillse in the protoplasm between which are situated granules, especially toward the lumen. These, he explains, are cross sections of fibrillae and that the entire structure is composed of minute fibrillae arranged in rows. The usual staining reaction is acidophilic. (See chart.) (b) Granules. — In the posterior region of the gland, throughout the cytoplasm, there appear spherical bodies either enclosed in vacuoles or apparently free in the protoplasm (Fig. i). Maziarski ('n), Nakahara ('17), and others, noted this condition and from the fact that the bodies are often found in close proximity to the nucleus and exhibit the same staining reactions 414 ELIZABETH KIXNEY. as the nuclear bodies drew the conclusion that the cytoplasmic granules are derived from the nuclear bodies (nucleoli) by migration of the latter. Tanaka ('u) believes the fibroin, or silk core, to be a product of the gland cell substance, visible only when the secretion has accumulated to give rise to fine fibroin droplets which unite to form larger droplets. Yamanouchi ('22) likewise believes that secretion is produced in the cell. Whether or not these cytoplasmic bodies are produced in the cell or in the nucleus, it seems to be generally concluded that they are directly connected with the products of secretion. From observation of cytoplasmic granules in the cells of //. cunea, it was found that they average between i micron and 2 micra in diameter, or approximate the size of the nuclear bodies. The cytoplasmic granules tend to increase in size as they approach the lumen and finally, the droplets of secretion when passed into the cavity coalesce and become of considerable size (Fig. 16). Evidence of active secretion is correlated with an abundance of cytoplasmic bodies present in the cell, but not, as might be expected, with a decrease in the number of nuclear bodies present. It seems, therefore, that if the nucleus does play an active part in the formation of secretion that there is always an excess of nuclear bodies stored in the branched nucleus, and while the drain upon one part may be great, the reserve from another part of the nucleus may supply the demand. The cytoplasmic bodies are found in all parts of the cell, but usually most abundantly between portions of a nucleus or between the nucleus and the lumen (Fig. i). These bodies usually react to acid stains, but are generally stained black with iron hsematoxylin. (See chart.) That no cytoplasmic bodies are present in the middle section is difficult to determine with absolute certainty. If the nucleus with its nucleoloid bodies and nuclear bodies is responsible for secretion, surely secretion should occur in this region, for both types of bodies are present in the nuclei. Material fixed by the Gatenby method and stained with iron ha>matoxylin and acid fuchsin furnished the only indication that cytoplasmic bodies may be present in the reservoir region. The bodies took a light pink stain very similar to that of the clear, structureless substance SECRETORY PHENOMENA IN SILK GLAND. 415 surrounding the silk core. These bodies were so indistinct that they may have been the result of poor technique. Yamanouchi ('22) finds evidence that sericin, or the outer layer of the silk core, is secreted in the posterior portion of the middle region. Blanc ('89) considers that mucous is secreted in the anterior portion of the reservoir in the form of numerous granules, but this point has received little support in subsequent literature. (c) Mitochondria. — Throughout the whole of the literature dealing with the silk glands of various lepidopteran larvae, no reference has been found which even suggests that the workers suspected the presence of mitochondria in the cells. This may be due to the fact that no investigator has employed specific mitochondrial fixatives. Maziarski ('i i) describes certain bodies that are found occasion- ally in the protoplasm as having the form of short "batonnets," and staining deeply. These have a parallel arrangement along the shorter axis of the cell and unite with one another by fine protoplasmic fibrils. The substance of these batonnets is re- fractile and stains with acid stains. Gilson noticed these bodies and thought they were related to functional processes of the protoplasm in that they have the appearance of little tubules filled with secretory substance. Maziarski did not find these bodies consistently present in all of his material. After fixation with Regaud, and staining with Altmann's anilin acid fuchsin, methyl green, clear-cut evidence of the existence of mitochondria in the cells of //. cunea has been obtained. i. Posterior Region. — The mitochondria in the posterior region are very numerous, especially between portions of a nucleus and toward the lumen. In whatever part of the cell the mitochondria may appear, they always show a characteristic orientation with their longitudinal axis toward the lumen. There is no evidence that these bodies have any direct relation to the nucleus for they do not radiate from it or tend to cluster around it (Figs. 1,2). Their usual shape is filamentous, rod-shaped or granular. Where active secretion is taking place, long filaments are present, ELIZABETH KINNEY. averaging between 3 and 5 micra in length, and about 0.5 micron in diameter. In the more anterior portions of the gland, the mitochondria are found to be shorter, but equally numerous. Mitochondria do not appear to have any direct relation to the cytoplasmic bodies, but the latter are found situated among the mitochondria! filaments. Material stained with iron haematoxylin after fixation in either the Champy or Gatenby method exhibits the presence of structures similar in size, shape and distribution to those ob- served with Regaud fixation (Figs. 5, 6). 2. Middle Region. — In the middle region, mitochondria are always present but their shape is somewhat different from that of the mitochondria in the posterior region. A gradual transition may be seen from the long filaments in the secreting region to the shorter rods in the anterior portion of this region (Fig. 7) and finally, in the true reservoir, the mitochondria become shortened to granules or spherules. The latter have the properties of mitochondria but are somewhat larger in diameter than the mitochondrial granules. It is not probable that the larger spherules represent secretion since in appearance, they more nearly resemble mitochondria than secretion bodies. The distribution of mitochondria is fairly disperse through- out all parts of the cell, but with a tendency to be more abundant toward the lumen. No definite orientation can be distinguished in the longer rods (Fig. 12). 3. Anterior Region. — Bodies taking a stain characteristic of mitochondria are present in the anterior region, but these are always granular and arranged with little definite order (Figs. «, 9). 4. Critical. — From tin- preceding observations, it is seen that mitochondria are present in the cytoplasm throughout the entire extent of the tubule. Their shape and to a less extent their number varies, however, in the three arbitrary divisions of the gland. Cowdry ('24, p. 318) suggests that mitochondria may serve to some extent as indicators of secretory polarity- In the case of silk secretion, it seems that this is truly the- conclusion to be drawn. Secretion can be traced from the cell into the lumen, and mitochondria appear to be streaming toward the SKCRKTOKY I'll KNO.M ICNA IN SILK (.LAND. 417 lumen in a very definite- way. There is no evidence that mito- chondria actively produce secretion but only the indication that they may participate in the process. That mitochondria are present in the reservoir and conducting regions but that here they do not show polarity seems to indicate that mitochondria may be present to perform other functions than that of partici- pating in secretory activity. Activity is occurring in both the middle and anterior regions, although not secretory activity. The middle portion appears to be a storage place for secretion where possible modifications of the secreted material may take place, for example, a condensation and compression. The secreted material then passes through the anterior conducting portion . Tanaka (' 1 1 ) is the only investigator who has suggested any possibility of the existence of secretory function in the anterior region, and this probably only in the embryonic stages. The usual interpretation is that this region serves as a duct for the passage of secretion. In this case, the presence of mito- chondria would seem to indicate that some activity exists within the cell, possibly of general metabolic nature. (d) Golgi Bodies. — In preparations of glands fixed by the Mann-Kopsch, and by the Champy method, no structures have been found which could be interpreted as true Golgi bodies. In silk gland cells of H. cunea impregnated with osmic acid, numerous fine dark granules appear (Fig. n), that are dis- tributed very abundantly along the periphery of the cell-body and scattered more generally toward the lumen. These fine granules are present in all regions of the gland and always characteristically more abundant toward the periphery of the cell. In the extreme posterior tip of the gland, the granules appear grouped as a dense border which gradually becomes more disperse toward the lumen. In cells of the secreting region, groups of large, black bodies, spherical in shape and usually situated toward or near the lumen or the periphery are common and may be followed through several sections of a series. Miss Murray ('26) describes a condition in the follicle cells of the cricket which seems quite similar and her figures compare closely with the condition found in the spinning-gland cell. With regard to her observations, she states (p. 223) that "In 28 41 8 ELIZABETH KINNEV. the proximal region of some of these cells (follicle cells) there has been seen a nebulous structure composed of very finely divided black particles, which might be considered to indicate an excessive deposition of fat in the tissue. This fine emulsion might conceivably be coarsened at the boundary of the cell and coalesce into fatty globules." In silk gland cells of A. americana and D. virginica which were fixed with DaFano's modification of the Cajal method, the same condition was found, but in these cases, there is a greater tendency toward an arrangement of the granules in rows, thus presenting the appearance of striations extending from the lumen toward the periphery, and likewise a greater grouping of granules around the edge of the cell toward the lumen. These bodies tend to concentrate along all sections of tracheae, thus outlining the course of tracheids very definitely. In these two species, it must be remembered, active secretion of silk only visibly appears at the time of forming the cocoon. It seems possible that these fine spherical granules may represent by products of cellular activity that are being elimi- nated. Their condensation along the tracheids and the proximal border of the cell may indicate that such may be the case. In the two species in which the process of secretion is only in the preparatory stage, an accumulation of waste products may be imagined to be occurring in the cell. 3. The Membranes. (a) Historical. — Before the work of Helm ('76), the existence of a tunica propria, or external membrane, and a tunica intima, or internal membrane was known and had been described, but little was known concerning their structure or function. The tunica propria was quickly dismissed by Helm and those following him as a structureless membrane fitting very closely to the l-a-al membrane throughout the entire extent of the gland that roists to a considerable degree the penetration of fixing fluids. But the tunica intima could not be so easily defined. Helm describes its structure in the anterior region as containing fine radial tubes which he refers to as "Porencanale." The entire intima, according to his observations, represents a cuticular SKCRIiTORY IMIKNOMKNA IX SILK GLAND. 419 layei which is a modified secretion of the outer body wall and is, in consequence, shed at each moult. The fine canaliculi are arranged perpendicular to the surface and are interpreted to serve as conduction paths through the cuticular intima for the liquid secreted by the cells to the lumen. Blanc ('89) working on the silk gland cells of Bombyx mori concluded that the intima was a chitinous structure composed of very fine parallel threads encircling the lumen at intervals of from 3-4 micra and showing occasional anastomosis. Gilson ('94) confirmed the observations of Blanc. Maziarski ('n) describing the intima, pictures the structure as a thin membrane composed of a great number of filaments passing in spiral lines around the lumen of the canal and joined by fine transverse fibrillae, thus producing the appear- ance of a network of threads. These filaments are described as staining deeply with iron hsematoxylin and having the appearance of rigid fibrils located in the protoplasm of the cell nearest the lumen, and were thought to resemble organs of support but not to serve as passageways for substances elaborated in the cells. Tanaka ('u) describes the intima as a striated, filamentous- appearing structure of chitinous nature, thickest in the anterior division and thin in the middle region. The filamentous stri- ations appear to have a parallel arrangement except toward the posterior region where anastomosis may occur. This chitinous substance which gives rise to the intima is thought by Tanaka to be secreted in the anterior region and never to undergo ecdysis, but rather is added to in thickness with age. This is (p. 16), he concludes, "a fact which goes a long way in proving the secretion of this chitinous substance throughout the whole larval life." Yamanouchi ('22) found the intima to be a glassy-like cuticular layer most conspicuously present in the anterior region where it has a thickness twice that in the other parts of the gland. This structure appears to be hyalin in nature, reacting to stains very similarly to sericin but more intensely in the anterior portion of the middle region, this structure is not homogeneous but gives the appearance of containing concentric bands whereby it is possible to distinguish two or three layers of intima. This arrangement in layers disappears anteriorly. ELIZABETH KINNEY. (b) Observation. — In the gland of //. cunea after fixation by the Regaud method, the presence of a clear, hyalin layer of material between lumen and cell-wall is found to be quite generally present in both the middle and anterior regions. (Figs. 7, 8, 9, 12.) This layer is always found to be very closely connected with the cell-wall, and yet to be perfectly distinct from the protoplasm. Oftentimes large vacuoles may be seen in the structure (Fig. 7), but these are always on the edge toward the cell and may be explained as the result of shrinkage at the time of fixation. This vacuolated condition occurs with greater frequency in the posterior portion of the middle region where the layer is thicker and less regular in form. Posteriorly, in the secreting region, no indication of an intima is found except for the presence of a thin membrane along the inner edge of the cells. The cells usually have an irregular surface on the side toward the lumen, the protoplasm seeming to be in very close connection with the cavity (Fig. 2), but after fixation with Champy, Gatenby, or Benda preparations, this membrane is quite definite (Fig. i). Toward the more anterior portion of the middle region, fibrillae may be seen extending through the clear hyalin substance and around the lumen. In longitudinal sections through the edge of the lumen, these structures appear to extend from the edge of the cell on one side of the lumen to the cell on the other side (Fig. 10), and to take their origin along the margin of the proto- plasm. These fibrilhe stain darkly with iron haematoxylin and dark red with Altmann's acid fuchsin after fixation by the Champy method. In general, the fibrilhc tend to be arranged in parallel series and some evidence has been found of slight anastomosis between fibril he in the more posterior region. After the tube has become narrowed to form the conducting portion, fibrillae disappear leaving the intima clear and structureless. (c) Critical. — The fact that the intima is present in the anterior and middle portions of the gland as a thickened layer -eems to indicate that this structure is not connected with active secretion but must serve some function related to conduction of secreted products to the outside. A copious supply of secretion i- being poured continually into the reservoir from the posterior SECRETORY PHENOMENA IN SILK GLAND. 42! region. To strengthen the cell against the resulting pressure and to prevent it from distortion, a thin layer of hyalin material might be conceived to serve as a support. At the point in the tube where it begins to narrow anteriorly, the pressure becomes greater and more support is necessary. That fibrillae should appear in the transition region between the middle and anterior regions, is, therefore, to be expected. It is quite possible, moreover, that these fibrillae may be more than merely supporting structures. Their shape and position around the lumen seems to indicate that they may have a contractile function, serving, perhaps, as a simple musculature in pushing the products of secretion into the long conducting tube. It is evident that some regulatory mechanism is necessary to prevent the small tube from becoming clogged by the ever accumulating secretion, and likewise, to regulate the amount of material passing through in order to produce a silk thread of uniform diameter. 4. The Secretion. (a) Historical. — The morphological appearance of the secreted material has received considerably more attention throughout the literature than the mechanism of the secretion process, and yet little is definitely known about the real nature of the secreted substance. Staining reactions have been used almost exclusively as criteria upon which to base conclusions. Little detailed work has been done upon the actual chemical composition of the silk fiber further than a general analysis of the chemical components, the result of which has been to show that the fiber is made up of two slightly different substances; an outer, acidophilic layer of sericin, and a central core of basophilic fibroin. The former is quite similar in composition to the latter except that, according to Blanc ('89), it contains more oxygen, dissolves more readily in alkaline solutions and is more granular in appearance. Tanaka ('n, p. 18-20) has reviewed very completely the results of previous workers and has arranged their views con- cerning the origin of fibroin and sericin into four principal groups. He then attempts to show that these theories have failed to satisfy all the conditions and proceeds to modify and combine parts of each theory into one more in accord with 422 ELIZABETH KINNEV. his own conclusions which he states as follows: 'The fibroin is • produced by virtue of the physiological function of the gland cells, without reference to any part of the gland tube; the sericin is, in its formation from fibroin, quite indifferent from the physiological function of the cells; on the contrary this transformation goes on merely physico-chemically." Maziarski ('u) in his enthusiasm for the nuclear origin of secretory products, states that the nucleoli which leave the nucleus become the fibroin and that chromatin, likewise, may be extruded into the protoplasm, there to undergo quite extended changes and finally pass out into the lumen as the sericin or gummy covering of the silk thread. This idea was not entirely new, for Vorhies ('08) had suggested that chromatin material may have a secretory function in addition to serving as the "bearer of heredity." Nakahara ('n) recognizes only the formation of silk from transformed nucleoli, but Yamanouchi ('22) describes the pro- duction of sericin and fibroin in great detail and concludes that fibroin secretion takes place only in the posterior region, but that sericin secretion occurs in the posterior and middle parts of the middle region. The two kinds of secretion appear alter- nately and, due to the fact that each kind tends to remain separate, two layers are formed, distinguishable by their different staining reactions. Blanc ('89) has described a third product of secretion, called by him " mucoidine," which envelops the silk fiber and stains more intensely with methyl green than either the fibroin or sericin. This substance, he thought, is formed in the anterior portion of the reservoir and may serve the function of a lubricant, thus facilitating the passage of the silk. (b) Observations.- — From a comparative study of silk gland cells prepared in different fixatives, it is evident that there exist in the lumen, substances which show different types of reactions to stains. After Regaud fixation, the silk thread appears to be perfectly homogeneous throughout its entire extent except in the .interior region of the gland where a slight ly darker band may be distinguished around the thread. (Fig. 9.) The secretion found in the posterior and middle portions takes a reddish-blue stain SKCRKTORY I'UKXOMKX A IN SII.K (iLAND. 42,} with Altmann's acid fuchsin, methyl green, and in the anterior portion, is decidedly green. The intima takes a bright red stain and appears to be a homogeneous, hyalin substance. The condition of the secretion product after fixation with Champy, Benda, or Gatenby solutions is considerably different and leads to doubt concerning the identity of the various structures present in the lumen. With iron haematoxylin and acid fuchsin, the core of secretion is stained black. In the middle region, the secretion does not fill the lumen as is the case after fixation with Regaud, but between the secretion and the intima, now distinguished as a thin black membrane at the cell border, there is present a compact mass of finely granular substance that takes the acid stain. This substance, however, is stained light green in material fixed by the Champy, and stained by the Altmann method. Usually, the material appears to be homogeneous, but after Benda, and sometimes after Champy fixation, the substance is arranged in thread-like strands that extend from the cell-wall toward the secretion. That this band of substance is separate from the core of secretion is quite evident, for the secretion may be found entirely removed from the substance and be free in the lumen. The substance is never free from the edge of the gland cell. It would seem that either the amount of secretion present is determined by the wridth of this band which fills the remainder of the lumen or that the size of the band of substance is dependent upon the amount of secretion present, since after fixation, little or no shrinkage apparently occurs and the core of silk is contained in the center of the lumen closely surrounded by a band of this substance. The former hypothesis seems more probable since some regulatory mechanism for producing a thread of uniformly decreasing dimensions must be present. Material fixed by the Champy, and stained with the Altmann method affords evidence of the presence of two substances in the thread. Secreted substance in the posterior portion of the gland stains light pink on the periphery while the center is either dark pink or green. This condition is not seen as sections are selected in the more anterior part of the gland. After fixation of the gland in Bouin, the silk thread exhibits two 424 ELIZABETH KIXNEY. distinct staining reactions. The core stains a clear pink and the periphery forms a black cortex around this core. This reaction of the secreted substance is consistent throughout the tube. In silk glands of D. virginica fixed in Bouin and stained in iron hsematoxylin and acid fuchsin, the silk thread has a central core of clear pink substance surrounded by a black peripheral cortex. A wide region filled with a reticulum or meshwork of fine pink granules surrounds the thread. Bordering this region and extending to the inner cell-wall, a wide compact band of fine granular substance arranged in concentric rings occurs. Throughout this band, numerous large drops of black substance appear which probably represent secretion material. By com- parison of the condition just described, with that found in H. cunea, and also with conditions described by Yamanouchi, it appears that some difference exists between the type of gland which secretes continuously and that which secretes at definite periods only. The presence of two diverse staining reactions in the thread seems to indicate that two substances are consistently coexistent in the two different types of silk gland cell, but the exact relationship between these two substances has not been determined with certainty. (c) Critical. — The question as to the identity of the substances within the lumen has presented a problem which cannot be definitely settled without further study both of preserved material from a variety of caterpillars and also of living material. Ob- servation of the material at hand seems to show that in the posterior portion of the gland, the secreted substance is usually homogeneous, and this is likewise true in the middle region. But some modification of the secreted substance takes place in the anterior conducting region which gives the silk thread the appearance of being composed of two substances. It is evident that even in forms which secrete silk during tin- entire larval life, the process is not a continuous one, for often within the lumen of the silk gland, the end of one thread « .in be seen and, close upon the posterior part of this thread, but -t [i. n.itcd from it by the substance filling the lumen, the end of another discrete cylinder of secretion begins (Fig. 7). SECRETORY PHENOMENA IN SILK (iLAND. 425 The substance which fills all available space in the lumen not occupied by silk secretion, may well be similar in its properties to mucus, lubricating the tube for the passage of the silk fiber. No origin for this substance has been found. It does not appear to be secreted by the cells of the gland, nor does it seem to be a dislodged part of the bodies of the cells. DISCUSSION. i. Relation of the Nucleus to Secretion. The nucleus and cytoplasm of the cells of the silk gland are closely associated in the production of the silk secretion. By many investigators, the nucleus has been considered the center of secretory activity in that the nucleoli migrate from the nucleus into the cytoplasm and there undergo modification. Study of the silk gland cells of II. cunea has given further indication of nuclear participation in silk production through the presence of nucleoloid bodies. These nucleoloid bodies do not themselves pass into the cytoplasm but appear to constrict pieces from their periphery that increase in size within the nucleus, finally passing through the nuclear wall into the cytoplasm where further modification of their substance occurs. 2. Relation of Mitochondria to Secretion. Mitochondria having a characteristic arrangement in the cyto- plasm of the secreting region are present in great numbers. It is quite possible that they take some part during the transitory stage when the secretion droplets are present in the cytoplasm, in modifying the material and transmitting it to the lumen. Mitochondria may act as catalysts, as suggested by Emberger ('25) or they may take a more direct part in secretion, but their presence and orientation toward the lumen seem to indicate that they are not passive cytoplasmic inclusions. Mitochondria are present throughout the extent of the gland but in the middle and anterior portions, they are shorter than in the secretory portion and show little definite orientation. In these portions, they may aid in the process of conduction and in general metabolic activity. 426 ELIZABETH KIXXEY. 3. The Nature of the Secretion. The column of silk secretion is apparently composed of two slightly different substances as evidenced by diverse staining reactions, but further study is necessary before the exact origin and nature of each is determined. Since in the posterior region, the silk thread appears to be a homogeneous cylinder when preserved in several different fixa- tives, it would seem that there is but one kind of substance secreted and that this substance undergoes partial modification, possibly oxidation, during its progress down the tube. A substance similar to mucous appears in the lumen as an envelop surrounding the silk column, but no account of its origin can be given. In life, it is evidently a fluid substance because it is found to be present wherever a break in the con- tinuity of the fiber occurs. The distinction between this sub- stance that is finely granular in appearance after certain fixations and the clear hyalin substance present in the same relative position, following fixation in Regaud's solution, cannot be determined. The latter substance has been considered as com- posing the intima but the former cannot be so accounted for. SUMMARY. 1. In the silk gland of Hyphantria cunea, three regions are distinguishable; a posterior, or secreting region; a middle region, or reservoir; and an anterior, or conducting region. 2. The nuclei of silk gland cells are greatly branched in the posterior region, but tend to be more flattened and less branched in the more anterior regions. 3. Three types of bodies are found to be present in each nucleus; the small basophilic granules, or chromatin; the inter- mediate acidophilic bodies generally termed nucleoli, but here called nuclear bodies; the relatively large, spherical body con- taining vacuoles and corresponding to a true nucleolus, hence called a "nudeoloid body." 4. The nudeoloid bodies appear to give rise to the nuclear bodies which pass out of the nucleus into the cytoplasm. 5. Cytoplasmic granules exhibit the same staining reactions as nuclear bodies and are approximately the same in size when they occur in the region of the nucleus. SECRETORY PHENOMENA IN SILK GLAND. 427 6. Mitochondria are present in all parts of the gland, but only in the posterior region do they show definite orientation. 7. Two membranes are present along the course of the gland, and external tunica propria, and an internal tunica intima. 8. Two substances regularly appear to constitute the silk thread in the anterior region. This condition is found to con- tinue more posteriorly after fixation with the Champy, Gatenby, Benda, and Bouin methods. 9. A substance similar to mucous surrounds the silk thread in the middle region but does not extend into the anterior portion. The origin of this substance has not been determined. LITERATURE. Blanc, M. Louis. '89 Etude sur la secretion de la soie et la structure du brin et de la bave dans le Bombyx mori. Lab. d'Etudes de la Soie, pp. 53~98- Cowdry, E. V. '24 General Cytology. Univ. Chic. Press, Chicago, 111., p. 318. Emberger, L. '25 Les mitochondria sont-elles des catalyseurs? Bull. d'Histologie (Physiol. and Path.), Vol. 2:12, pp. 3O9~374- Gilson, G. '94 La soie et les appareils sericigenes-I. Glandes sericigenes des Lepidopteres. La Cellule, Vol. X., pp. 39-42. Helm, F. E. '76 Uber die Spinndriisen der Lepidopteren. Zeit. fur wiss. Zool., Vol. 26, pp. 434-469. Imms, A. D. '25 A General Text-book of Entomology. Methuen & Co., Ltd., 36 Essex St., W. C. London, p. 407. Korshelt, E. '96 tiber die Struktur der Kerne in den Spinndrusen der Raupen. Arch. f. Mikroskop. Anat., Vol. 47, pp. 500-569. '97 Uber die Bau der Kerne in den Spinndrusen der Raupen. Arch. f. Mikro- skop. Anat., Vol. 49, pp. 798-803. Ludford, R. J. '25 Nuclear Activity in Tissue Cultures. Proc. Roy. Soc., Vol. 98: 692 (B), pp. Marshall, W. S., and Vorhies, C. T. '06 Cytological Studies on Spinning-glands of Plalyphylax designatiis Walker. Intern. Monatschr. f. Anat. u. Phys., Vol. 23, pp. 397-420. Maziarski, S. 'n Recherches cytologique sur les phenomenes secretaires dans les glandes filieres des larves des Lepidopteres. Arch. f. Zellforsch., Vol. 6, pp. 397-433- Meves, F. '97 Fiir Structur der Kerne in den Spinndrusen der Raupe. Arch. f. Mikroskop. Anat., Vol. 48, pp. 573-578. 428 ELIZABETH KINNEY. Montgomery, T. H., Jr. '99 Comparative Cytological Studies with Especial Regard to the Morphology of the Nucleolus. Jour. Morph., Vol. 15, pp. 265-582. Murray, Margaret. '26 Secretion in the Amitotic Cells of the Cricket Egg Follicle. BIOL. BULL., Vol. 50: 3, pp. 210-228. Nakahara, Waro. '17 Physiology of Nucleoli as Seen in Silk Gland Cells of Certain Insects. Jour. Morph., Vol. 29, pp. 55-68. Saguchi, S. '20 Studies on the Glandular Cells of the Frog's Pancreas. Am. Jour. Anat., Vol. 26, pp. 348-402. Tanaka, Yoshimara. 'ii Studies on Anatomy and Physiology of Silk-Producing Insects. Jour, of Coll. Agr., Tohoku Imp. Univ., Vol. IV.: 2, pp. 1-28. Vorhies, C. T. '08 Development of Nuclei of Spinning Gland Cells of Plaiyphylax designates Walker. BIOL. BULL., Vol. 15, pp. 54-61. Yamanouchi, M. '22 Morphologische Beobachtung iiber die Seidensekretion bei den Seidenraupe Jour. Coll. Agr., Hokkaido Imp. Univ., Vol. X.: 4, pp. 1-49. 43O ELIZABETH KIXXEY. EXPLANATION OF PLATES. PLATE I. Fit;. /. Diagrammatic outline of silk gland of H. cunea. Anterior, middle, and posterior regions of gland numbered I, 2, and 3 respectively. Letters corre- spond to sections taken at these levels. FIGS. A-F. Camera lucida outlines of sections from the three regions of a tubule fixed by the Regaud method. Cross sections cut 4 micra in thickness; magnification, oc. 10 X, obj. 4 mm. Note: Stippling represents cytoplasm; solid lines represent secretion; broken ines represent intima. BIOLOGICAL BULLETIN, VOL. LI. PLATE I. .- . ^^1 • ::':&% r^i&f&ipsyt - .. / \ " • \ • •-^ •" v ELIZABETH KINNEY. 29 432 ELIZABETH KIXXEY. PLATE II. All figures made from sections cut 4 micra in thickness, and drawn with camera lucida, magnification, oc. 12.5 X, obj. 1.8 mm., oil immersion. FIG. i. Section of cell from posterior region of tubule fixed by the Champy method and stained with iron haematoxylin, acid fuchsin. Cross section. Cyto- plasmic granules appear to be grouping toward lumen. FIG. 2. Section of cell from secreting region of tubule fixed by Regaud method and stained with acid fuchsin, methyl green. Cross section. Mitochondria show definite orientation toward lumen. FIG. 3. A section of a nucleus from material fixed by the Champy method and stained with iron haematoxylin, acid fuchsin. The nucleoloid body is surrounded by nuclear bodies that are smaller than those toward the nuclear wall. FIG. 4. Section of nucleus from material fixed by the Gatenby method and stained with iron haematoxylin, acid fuchsin. Two nuclear bodies are in the process of passing through the nuclear wall into the cytoplasm. FIG. 5. Longitudinal section of cell from posterior region of gland fixed by the Champy method and stained with iron haematoxylin, acid fuchsin. Nucleoloid body in process of constricting pieces from periphery. FIG. 6. Longitudinal section of cell showing two nucleoloid bodies present in nucleus. Regaud fixation. Symbols: eg. cytoplasmic granules; /. lumen; mf. mitochondrial filaments; ni. chromatin; n2. nuclear bodies; nb. nucleoloid body; s. secretion. BIOLOGICAL BULLETIN. VOL. LI. PLATE II. 8 ^^^T^T, 7^^~^ X^/ ^/x^^i i V/i"--*^ A \ . • •>. \- Afc Ji 4' if '^ <' . -v .v C;.u -ivfr^»>^ ••r.*> H 3^^^^S^'^ ELIZABETH KINNEY. 434 ELIZABETH KIXXEY. PLATE III. FIG. 7. Section of cell taken from posterior part of middle region of gland fixed by the Regaud method and stained with acid fuchsin, methyl green. Cross section. Hyalin layer appears irregular and contains vacuoles along the border toward cell. Mitochondria slightly shorter. Two columns of secretion chance to be present at this level. FIG. 8. Cross section of tubule from anterior conducting region of gland fixed by the Regaud method and stained with acid fuchsin, methyl green. Mito- chondria present as granules. Intima definite. FIG. 9. Cross section from anterior conducting region of tubule fixed by the Champy method and stained with iron haematoxylin, acid fuchsin. Mitochondria appear as granules. Intima well defined, two types of secretion present. FIG. 10. Longitudinal section of tubule fixed by the Champy method and stained with iron haematoxylin, acid fuchsin. Section taken from anterior part of the middle region at edge of lumen. Fibers appear extending from the edge of one cell to the edge of the cell opposite. FIG. ii. Longitudinal section of cell from secreting region of tubule fixed by the Mann-Kopsch method, unstained. Numerous fine black granules fill cytoplasm and tend to concentrate toward the outer cell boundary. FIG. 12. Cross section of cell from middle region of tubule fixed by the Regaud method, stained with acid fuchsin, methyl green. Mitochondria are shorter and less definitely orientated. Clear hyalin intima present along the edge toward lumen. Symbols: /., fibers; g., granules; mg., mitochondrial granules; p., tunica propria; si., inner layer of secretion; so., outer layer of secretion. BIOLOGICAL BULLETIN. VOL. LI. PLATE III. B ELIZABETH KINNEY. THE LINKAGE DISTURBANCE INVOLVED IN THE CHROMOSOME TRANSLOCATION I. OF DROSO- PHILA, AND ITS PROBABLE SIGNIFICANCE. G. W. DELUZ HAMLETT, DEPARTMENT OF ZOOLOGY, UNIVERSITY OF TEXAS. The chromosomal abnormality in Drosophila known as "trans- location I." was found by Bridges in 1917, and was first referred to by Morgan (1919); a preliminary account of it was given by Bridges in 1923. The case is a very remarkable one, unique in the literature to date, and it raises certain new problems con- cerning the arrangement which genes may have in a chromosome as well as concerning the changes in arrangement which they may undergo. The abnormality arose by a portion of the right hand end of chromosome II. breaking off and becoming attached near the middle of the right half of chromosome III. Bridges' data showed the size of the transposed piece and its approximate point of attachment on the third chromosome. It was known that the break in chromosome II. was between arc (ar) and plexus (pi), making the fragment about 8 units long, and that the transposed piece was attached somewhere between ebony (e) and rough (ro) in chromosome III. The deficient second chromo- some was known as "Pale," from its effect on eosin-eyed flies, and the deficiency was lethal unless accompanied by trans- location. In the hope of getting further light on the chromosome change involved, the writer undertook to test more extensively the third chromosome linkage values in flies which carried the trans- location. I wish to take this opportunity to express my in- debtedness to Dr. H. J. Muller, who suggested the work, and whose aid and suggestions have made it possible. In these experiments the homozygous as well as the heterozygous condition of translocation was studied, and all of the left hand end of Chromosome III. was under genetic observation. Bridges had already shown that crossing over was greatly reduced in the region close to the locus of translocation ; but his data included 435 436 G. W. DKLUZ HAMLETT. only flies heterozygous for the transposed piece. The comparison of the homozygous and the heterozygous conditions with each other is of especial interest because of its bearing on the problem of the mode of attachment of the second chromosome fragment, as will be shown later. The third chromosome mutant genes used in the experiments are, in their order from left to right along the chromosome: roughoid (ru), hairy (h), scarlet (st), pink (p), spineless (ss), delta (A), hairless (H), ebony (e), rough (ro), and claret (ca). Their locations are shown in the map of Fig. i. Translocation is represented by Tr, and the deficient second chromosome by PI. Curly (Cy) is a second chromosome gene used in balancing stocks. The linkage in flies homozygous for Tr was tested in two experi- ments. The summarized results for these crosses are given below, in Tables I. and II. TABLE I. A H e Tr PI ru h st p ss Tr 9 X ru h st p ss e cf1. Regional Crossover Frequencies. Total. ru-h. h-st. st-p. p-ss. ss-A. A-H. 285 202 45 86 61 10 1,097 26.0% 18.4% 4-1% 7-8% 5-6% •9% TABLE II. Cy A H e Tr PI Tr ro ca 9 X e ro ca cf • Regional Crossover Frequencies. A-H. H-e. e-ro. ro-ca. 8 13 27 1 6 463 T -I Of 1-7/0 2.8% 5-9% 3-5% These figures show a remarkable drop below normal in t he- crossover values as the locus of translocation is approached. This may be seen by comparing their map values (see Fig. i, TRANSLOCATION I OF DROSOPHILA. 437 b and c) with the normal third chromosome map (Fig. I, a). The map length of the left-hand third will be seen to be slightly increased, but the genes between pink and claret are crowded much closer together. There is an apparent lengthening of the B 0.0 Tru 26.5- 43.8 47-8 585- 65.0 69.5. 707 ' o.o ru 26.0 St Ss A H 44.4 485 563 61.9 628 St -P SS A 61.9 63-6 66.4 72.3 75.8 o.o mru 28.0 43.7 47.5 54.4 59.1, st SS A H 6L3 68.5 -ro CO ro 61.3 66.8 69.9 ro CO 91.1 TO 100.7 FIG. i. (a) The normal third chromosome map. (b) and (c) Maps of third chromosome homozygous for translocation. (d), (e) and (/) Maps of third chromo- some heterozygous for translocation. distance between hairless and ebony, but this distance is so short, and the probable error so large, that the increase is probably not significant. Between ebony and rough, the region where the second chromosome fragment is attached, the per cent, of crossing over is less than one third of the normal value. These 438 G. W. DELUZ HAMLETT. relations are shown in Fig. 2, a, which shows the ratios of the values obtained in these experiments to the standard values of each of the major regions studied. At the same time that these crosses were being made, the linkage in flies heterozygous for the abnormality was being tested. The same stocks were used in making up the crosses, and the characters used were introduced into the cross in as near the same way as possible. Both the homozygous and heterozygous lines were kept under the same conditions, so that any possible variations in viability would affect both lines alike and allow us to draw valid comparisons between them. As will be seen in Tables III. and IV., the results were almost identical with those of the first tests. TABLE III. A H e Tr PI ru h st p ss 9 X ru h st p ss e c?. Regional Crossover Frequencies. ru-h. h-st. st-p. p-ss. ss-A. A-H. 162 91 22 40 27 6 578 28.0% TC 1 (>7 I5-7 /o 3-8% 6.9% 4.7% 1.0% TABLE IV. PI ru h st p ss Tr p ss e ro 9 X p ss e ro Crossovers. Total Flies. (ru h st) p ss e (ru h st) p ss ro 1,155 42 4i 1 Crossovers 83 7-2% In Table V. are results for a test involving the ebony-rough and rough-claret distances. Only those flies which did not show i< (logical Bulletin, Woods Hole, Mass., June I5th to Sept. I5th. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. BIOLOGICAL BULLETIN • OF THE fiDavine Biological Uaboraton? WOODS HOLE, MASS. VOL. LI SEPTEMBER, 1926 No. 3 CONTENTS FROBISHER, MARTIN, JR. Observations on the Relationship Between a Red Ton/la and a Mold I'athogeuic for Drosophila melano'gaster 153 YOUNG. DONNKLL B. Xuclear Regeneration in Styloiiychia my- tilus I'M MINNICH, DWIGIIT ELMER. The Chemical Sensitivity of the Tarsi of Certain Muse id Flics ifi6 GREGORY, LOUISE H. Effects of Changes in Medium During Dif- ferent Periods in the Life History of Uroleptiis mobilis 179 YOUNG, WILLIAM C., AND PLOUGH, HAROLD H. On the Sterili-atinn of Drosophila by High Temperature 189 SKOWRON, STANISLAW. On the Luminescence of Microscolcx phos- phoreus Dug 100 KATER, J. McA. Chromosomal Vesicles and the Structure of the Resting Nucleus in Phaseolus 209 PUBLISHED MONTHLY BY THE MARINE BIOLOGICAL LABORATORY PRINTED AND ISSUED BY LANCASTER PRESS, INC. LANCASTER, PA. AGENT FOR GREAT BRITAIN WHELDON & WESLEY, LIMITED 2, j and 4 Arthur Street, New Oxford Street, London, W. C. 2 Single Numbers, $1.00. Per Volume (6 numbers), S4.50 •BBBI^JBiBlH^^^^^^^^^KH9H&BHHBHBSflMB^^BBSBBHMBKHBHiBH3BB^^^BBDHM^I^BBBH^BHi^HHBI^HllHBBHHB9EB^^^^^^D^^9^^BI^^B^EE9^^IB Entered October 10, 1902. at Lancaster, Pa., as second-class matter under Act of Congress of July 16, 1894 lEMtorial Staff GARY N. CALKINS — Columbia University. E. G. CONKLIN — Princeton University. M. H. JACOBS — University of Pennsylvania. FRANK R. LILLIE — University of Chicago. GEORGE T. MOORE — The Missouri Botanic Garden. T. H. MORGAN — Columbia University. W. M. WHEELER — Harvard University. E. B. WILSON — Columbia University. EMtor C. R. MOORE — The University of Chicago. All communications and manuscripts should be sent to the Man- aging Editor, the University of Chicago, Sept. I5th to June I5th, or to Biological Bulletin, Woods Hole, Mass., June I5th to Sept. I5th. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. BIOLOGICAL BULLETIN OF THE (lOarine Biological Xaboraton> WOODS HOLE, MASS. VOL. LI OCTOBER, 1926 No. 4 CONTENTS LAMBERT, W. V., AND KNOX, C. W. Genetic Studies in Poultry 225 McNALLY, EDNA. Life Cycle of Nassula ornata and Nassula ele- gans: Arc these Species Valid? 237 KUSNEZOV, N. J. The Morphology of the Copulatory Structures in Some Cases of Gynandromorphism in Lepi- doptera 245 BERRY, S. STILLMAN. Note on the Occurrence and Habits of a Luminous Squid (Abralia veranyi) at Madeira 257 GABRITSCHEVSKY, E. Convergence of Coloration Between American Pilose Flies and Bumblebees (Bombus) 269 PUBLISHED MONTHLY BY THE MARINE BIOLOGICAL LABORATORY PRINTED AND ISSUED BY LANCASTER PRESS, INC. LANCASTER, PA. AGENT FOR GREAT BRITAIN WHKI.DON & WESLEY, LIMITKD 2, j and 4 Arthur Street, New Oxford Street. London, \\'. C. 2 Single Numbers, $1.00. Per Volume (6 numbers), $4.50 Entered October 10. 1902. at Lancaster, Pa., as second-class matter under Act of Congress of July 16, 1894 lEfcitorial Statf GARY N. CALKINS — Columbia University. E. G. CONKLIN — Princeton University. M. H. JACOBS — University of Pennsylvania. FRANK R. LILLIE — University of Chicago. GEORGE T. MOORE — The Missouri Botanic Garden. T. H. MORGAN — Columbia University. W. M. WHEELER — Harvard University. E. B. WILSON — Columbia University. BMtor C. R. MOORE — The University of Chicago. All communications and manuscripts should be sent to the Man- aging Editor, the University of Chicago, Sept. I5th to June I5th, or to Biological Bulletin, Woods Hole, Mass., June I5th to Sept. isth. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. BIOLOGICAL BULLETIN OF THE flDarine Biological Xaborator^ WOODS HOLE, MASS. VOL. LI NOVEMBER, 1926 No. 5 CONTENTS BECKER, ELERY R. The Flagellate Fauna of the Ccecum of the Striped Ground Squirrel, Cilellus tri- decemlineatus, -with Special Reference to Chilomastix magna sf>. nov 287 BIGELOW, ROBERT PAYNE. Variation in the Number of Fin-Rays in Three Species of Fundulus of the Woods Hole Region 299 TANNREUTHER, GEORGE W. Life History of Prorodon griseus 303 BAKER, WOOLFORD B. Studies in the Life History ofEuglena. 321 BODINE, JOSEPH HALL. Hydrogen Ion Concentration in the Blood of Certain Insects (Orthoptera) 363 PUBLISHED MONTHLY BY THE MARINE BIOLOGICAL LABORATORY PRINTED AND ISSUED BY LANCASTER PRESS, INC. LANCASTER, PA. AGENT FOR GREAT BRITAIN WHELDON & WESLEY, LIMITED 2,3 and 4 Arthur Street, New Oxford Street, London, W. C. 2 Single Numbers, $1.00. Per Volume (6 numbers), S4.50 Entered October 10, 1902. at Lancaster, Pa., as second-class matter under Act of Congress of July 16, 1894 EMtorial Staff GARY N. CALKINS — Columbia University. E. G. CONKLIN — Princeton University. M. H. JACOBS — University of Pennsylvania. FRANK R. LILLIE — University of Chicago. GEORGE T. MOORE — The Missouri Botanic Garden. T. H. MORGAN — Columbia University. W. M. WHEELER — Harvard University. E. B. WILSON — Columbia University. Managing EMtor C. R. MOORE — The University of Chicago. All communications and manuscripts should be sent to the Man- aging Editor, the University of Chicago, Sept. I5th to June I5th, or to Biological Bulletin, Woods Hole, Mass., June i5th to Sept. I5th. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. BIOLOGICAL BULLETIN OF THE noartne Biological laboratory WOODS HOLE, MASS. VOL. LI DECEMBER, 1926 No. 6 CONTENTS WHITING, P. W. Influence of Age of Mother on Appear- ance of an Hereditary Variation in Habrobracon 37 l CALKINS, GARY N., AND BOWLING, RACHEL C. Game-tic Meiosis in Monocystis 385 GATES, G. E. Preliminary Note on a New Protozoan Parasite of Earthworms of the Genus Eutyphceus 400 KINNEY, ELIZABETH. A Cytological Study of Secretory Phenom- ena in the. Silk Gland of Hyphantria cunea 405 HAMLETT, G. W. DELUZ. The Linkage Disturbance Involved in the Chromosome Translocation I. of Dro- sophila, and its Probable Significance 435 HANCE, ROBERT T. The Chromosomes of the Chick Soma . . 443 PUBLISHED MONTHLY BY THE MARINE BIOLOGICAL LABORATORY PRINTED AND ISSUED BY LANCASTER PRESS, INC. LANCASTER, PA. AGENT FOR GREAT BRITAIN WHELDON & WESLEY, LIMITED 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2 Single Numbers, $1.00. Per Volume (6 numbers), S4.50 Entered October 10, 1902, at Lancaster, Pa., as second-class matter under Act of Congress of July 16 , 1804 EDitortal Staff GARY N. CALKINS — Columbia University. E. G. CONKLIN — Princeton University. M. H. JACOBS — University of Pennsylvania. FRANK R. LILLIE — University of Chicago. GEORGE T. MOORE — The Missouri Botanic Garden. T. H. MORGAN — Columbia University. W. M. WHEELER — Harvard University. E. B. WILSON — Columbia University. B&itor C. R. MOORE — The University of Chicago. All communications and manuscripts should be sent to the Man- aging Editor, the University of Chicago, Sept. I5th to June I5th, or to Biological Bulletin, Woods Hole, Mass., June I5th to Sept. I5th. Subscriptions and other matter should be addressed to the Biological Bulletin, Prince and Lemon Streets, Lancaster, Pa. Notice to contributors. 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