SS3) i UNIVERSITY OF CALIFORNIA PUBLICATIONS -^IN - ZOOLOGY Vol. 13, No. 4, pp. 43-122, pis. 3-7, 4 text figures May 4, 1914 DIPLODINIUM ECAUDATUM WITH AN ACCOUNT OF ITS NEUROMOTOR APPARATUS BY ROBERT G. SHARP A thesis presented to the Faculty of the College of Natural Sciences, in the University of California, in partial satisfaction of the requirements for the degree of the Doctor of Philosophy. May, 1914. UNIVERSITY OF CALIFORNIA PRESS BERKELEY UNIYEESITT OF OALIFOENIA PUBLICATIONS Note. The University of California Publications are offered in exchange for the publi- cations of learned societies and institutions, universities and libraries. Complete lists of all the publications of the University will be sent upon request. Tor sample copies, lists of publications or other information, address the Manager of the University Press, Berkeley, California, U. 8. A. All matter sent in exchange should be addressed to The Exchange Department, University Library, Berkeley, California, TJ. S. A. OTTO HAEEASSOWITZ, E. FEIEDLAENDEE & SOHN, LEIPZIG. BEELIN. Agent for the series in American Arch- Agent for the series in American Arch- aeology and Ethnology, Classical Philology, aeology and Ethnology, Botany, Geology, Education, Modern Philology, Philosophy, Geography, Mathematics, Pathology, Physi- Psychology, History. ology, Zoology, and Memoirs. ZOOLOGY. W. E. Eitter and C. A. Kofoid, Editors. Price per volume, $3.50; beginning with vol. 11, $5.00. This series contains the contributions from the Department of Zoology, from the Marine Laboratory of the Scripps Institution for Biological Eesearch, at La Jolla, California, and from the California Museum of Vertebrate Zoology in Berkeley. Cited as Univ. Calif. Publ. Zool. Volume 1, 1902-1905, 317 pages, with 28 plates ....., $3.50 Volume 2 (Contributions from the Laboratory of the Marine Biological Associa- tion of San Diego), 1904-1906, xvii + 382 pages, with 19 plates $3.50 Volume S, 1908-1907, 383 pages, with 23 plates $3.50 Volume 4, 1907-1908, 400 pages, with 24 plates $3.50 Volume 5, 1908-1910, 440 pages, with 34 plates $3.50 Volume 6, 1908-1911, 478 pages, with 48 plates $3.50 Vol. 7. (Contributions from the Museum of Vertebrate Zoology.) 1. Two New Owls from Arizona, with Description of the Juvenal Plum- age of Strix occidentals occidentals (Xantus), by Harry S. Swarth. Pp. 1-8. May, 1910 10 2. Birds and Mammals of the 1909 Alexander Alaska Expedition, by Harry S. Swarth. Pp. 9-172; plates 1-6; 3 text-figures. January, 1911. 1.50 S. An Apparent Hybrid in the Genus Dendroica, by Walter P. Taylor. Pp. 173-177. February, 1911 .05 4. The Linnet of the Hawaiian Islands: a Problem in Speciation, by Joseph Grinnell. Pp. 179-195. February, 1911 15 5. The Modesto Song Sparrow, by Joseph GrinneU. Pp. 197-199. Feb- ruary, 1911 03 6. Two New Species of Marmots from Northwestern America, by H. 8. Swarth. Pp. 201-204. February, 1911 Ofl 7. Mammals of the Alexander Nevada Expedition of 1909, by Walter P. Taylor. Pp. 205-307. June, 1911 1.0(1 8. Description of a New Spotted Towhee from the Great Basin, by J. Grinnell. Pp. 309-5*11. August, 1911 _. .05 9. Description of a New Hairy Woodpecker from Southeastern Alaska, by H. S. Swarth. Pp. 313-318. October, 1911 .05 10. Field Notes on Amphibians, Reptiles and Birds of Northern Humboldt County, Nevada, with a Discussion of Some of the Fauna! Features of the Begion, by Walter P. Taylor. Pp. 319-436, plates 7-12. February, 1912 1.00 Index, pp. 437-446. VoL 8. 1. The Vertical Distribution of Eumlanus elongatus in the San Diego Eegion during 1909, by Calvin O. Esterly. Pp. 1-7. May, 1911 .10 2. New and Eare Fishes from Southern California, by Edwin Chapin Starks and William M. Mann. Pp. 9-19, 2 text-figures. July, 1911. .10 3. Classification and Vertical Distribution of the Chaetognatha of the Sam Diego Eegion, Including Eedescriptions of Some Doubtful Species of the Group, by Ellis L. Michael. Pp. 21-186, pis. 1-8. December, 1911. 1.7fl 4. Dinoflagellata of the San Diego Eegion, IV. The Genus Gonyaulax, with Notes on Its Skeletal Morphology and a Discussion of Its Generic and Specific Characters, by Charles Atwood Kofoid. Pp. 187-286, plates 9-17. UNIVERSITY OF CALIFORNIA PUBLICATIONS IN ZOOLOGY Vol. 13, No. 4, pp. 43-122, pis. 3-7, 4 text figures May 4, 1914 DIPLODINIUM ECAUDATUM WITH AN ACCOUNT OF ITS NEUROMOTOR APPARATUS BY EGBERT G. SHARP CONTENTS PAGE A. Introduction 44 1. Review of literature 45 2. Present status 49 3. Validity of species 51 B. Technique 52 1. Living material 52 2. Methods of fixation and staining 55 C. General discussion of the genus 58 1. Diplodinium ecaudatum forma ecaudatum 62 (a) Ectoplasmic structures 63 (1) Cuticle 63 (2) Skeletal areas 64 (3) Ectoplasm 65 (4) Skeletal structures 65 (5) Boundary layer 67 Entoplasm 68 Organs of the body 69 (1) Macronucleus 69 (2) Micronucleus 71 (3) Organs of locomotion 72 (4) Organs of food-taking 77 (5) Organs of defecation 80 (6) Organs of excretion 82 (7) Neuromotor apparatus 82 (d) Retracted form 88 2. Diplodinium ecaudatum forma caudatum 90 3. Djiplodiuium ecaudatum forma bicaudatum forma nova 92 ' * 44 University of California Publications in Zoology [VOL. 13 PAGE 4. Diplodinium ecaudatum forma tricaudatum forma nova ........................ 92 5. Diplodinium ecaudatum forma quadricaudatum forma nova ................ 93 6. Diplodinium eeaudatum forma cattanei .................................................... 94 7. Table of dimensions ........................................................................................ 95 8. Observations on the living material .......................................................... 95 D. Conclusions .............................................................................................................. 101 E. Addendum. Discussion of Braune's paper ...................................................... 103 F. Bibliography ............................................................................................................ 110 INTRODUCTION During the winter of 1909 Professor C. A. Kofoid, of the University of California, called my attention to the papers of Fioren- tini (1889) and Eberlein (1895) dealing with the protozoan fauna of the stomachs of ruminants, and pointed out the fact that the life- history of these protozoans had not yet been traced. This appealed to me as an interesting bit of research, and acting upon Professor Kofoid 's further suggestion I first made certain that material was procurable and then consulted the literature upon the subject. The papers of Schuberg (1888), Fiorentini (1889), Eberlein (1895), and Giinther (1899, 1900), were at hand and from a hasty perusal of these it became evident that the genus Diplodinium offered the greater pos- sibilities as a research problem. Most of the described species of this genus were present in the stomach fluid which I was able to obtain, and especially abundant were the species which Fiorentini (1889) had described as Diplodinium ecaudatum, D. caudatum, and D. cattanei. As my observations proceeded it became evident that the Protozoa infecting the stomachs of western cattle presented many differences in structure from those figured and described both by Fiorentini (1889) and by Eberlein (1895). This was found to be especially true in the case of those species described as belonging to the genus Diplodinium. The present paper deals with the morphology of Diplodinium ecaudatum Fiorentini including D. caudatum Fiorentini, and D. cat- tanei Fiorentini, together with a description of three new forms of this species, viz., Diplodinium ecaudatum forma bicaudatum, Diplodinium ecaudatum forma tricaudatum, and Diplodinium ecaudatum forma quadricaudatum, all of which are found in the first and second divisions of the stomach of western cattle. 1914] Sharp: Diplodinium ecaudatum 45 ACKNOWLEDGMENTS Whatever there be of merit in the methods used and the results so far obtained is due to the kindly and helpful suggestions and interest of Professor Kofoid, under whose direction the work has been done. My acknowledgments are also due to Mr. R. B. Brown, Superin- tendent of the Oakland Meat and Packing Company, for his personal interest and assistance in procuring material for this study in the most advantageous manner. LITERATURE The literature upon this subject is not extensive, although covering a period of more than seventy years. A brief review follows. The first information regarding the presence of protozoans in the stomach of the ox was given by two French scientists, Grube and Delafond (1843). In this communication they presented a short general account of the stomach parasites of the horse, the dog, and the pig, as well as those of ruminants. This work is important because of the early date at which it was done and because it opened up a new field of investigation. The results, according to our modern conception, were by no means accurate, and the work, although carried out with great skill, considering the limited means of that period, is unfortunately unaccompanied by illustrations ; a fact which makes it difficult to determine which protozoans the investigators observed. In the case of the ox they gave descriptions of four species, from which it is almost certain that they had observed those protozoans now classified as belonging to the genera Ophryoscolex, Entodinium, Diplodinium and Isotricha. Even at this early date (1843) they called attention to the fact that in the ox these protozoans occur in the living condition only in the first two subdivisions of the stomach, the rumen and the reticulum; while in the third and fourth sub- divisions, the omasum and the abomasum, only dead and disintegrated animals are found. The next information regarding these protozoans was given by Colin (1854). In a discussion of the digestive processes which take place in the stomachs of the ruminants Colin reproduces eighteen draw- ings, but gives only brief descriptions. The figures demonstrate clearly, however, that Colin saw and recognized species subsequently described in the genera Diplodinium, Entodinium and Isotricha. 46 University of California Publications in Zoology [VOL. 13 Stein (1858, 1859, 1861, and 1867) followed with short but excel- lent descriptions. Although lacking illustrations, Stein's work was good; he gave model descriptions and a scientific classification. He described the genera Ophryoscolex, Entodinium and Isotricha. In 1861 he added to his earlier work, and again in 1867. In this later work he classifies for the first time the genera Ophryoscolex and Entodinium under the family name Ophryoscolecidae. In 1869 Weiss confirmed the presence of these infusorians in the stomachs of ruminants and contented himself principally with a report upon the writings of Delafond and Stein. Leuckart (1879-1886) only reviewed the researches of Stein. In 1872 Ziirn did a large amount of work, but owing to poor technique his material was bad, and consequently his descriptions were faulty and his figures inaccurate. In a second edition of his work (Ziirn and Plaut, 1887-1889), he abandoned his earlier figures and enlarged upon his descriptions by quoting from Schuberg's (1888) discoveries. Kent (1881) published, in his Manual of the Infusoria, a com- pilation of the work of Stein (1858, 1859, and 1861), but this com- pilation contained many errors. Kent, as a matter of fact, added nothing along this line to the work of his predecessors. List (1885) gave little that was new. The animals over which he worked were either dead or had been affected by the water. His work, in so far as it relates to the ciliates, is without present value. In 1888 Schuberg published the results of his work on Buetschlia, Isotricha, Dasytricha, and Entodinium. His work w r as the most scien- tific and most complete done up to his time and in some respects is still the best. He described two new genera, which he named Buetschlia (with two species) and Dasytricha (with one species). He added several species to Stein's genus Entodinium and divided the genus into two genera, i.e., Entodinium and Diplodinium, although he gives neither a description nor an illustration of the genus Diplo- dinium in fact nothing beyond the mere statement that the genus Diplodinium is provided with two sets of membranelles, one around the mouth opening and the other on the dorsal side. His methods he describes in detail and to these we shall have occasion to refer later. He intended to write a second lengthy paper, but never, so far as I am able to determine, was this published. In 1891 he pub- lished a short paper, which contains no illustrations and is occupied largely with a description of some of the structural relationships and Sharp: Diplodinium ecaudatum 47 a partial description of the process of division in the Ophryoscole- cidae and in Dasytricha. Fiorentini (1889) published a short paper dealing mainly with the genera Diplodinium and Entodinium. This paper is profusely illustrated and although the drawings are crude as compared to those of either Schuberg or of Eberlein, they are still, in some respects, more true to life than are those of either of the others. Fiorentini is apparently the first to describe individual species of the genus Diplodinium. He figures and briefly describes nine species (see table below). To the genus Entodinium he adds two new species and to the genus Buetscklia one new species (see table below) . It seems that Fiorentini contented himself with the discovery and naming of new species rather than with the careful and accurate description and illustration of the species upon which he worked. In general his methods were crude and his descriptions too brief. To these we shall refer again. It is interesting to note in this connection that Fiorentini (1890) in his paper dealing with the Protozoa parasitic in the intestinal tract of the horse, describes two new species which he refers to the genus Entodinium Stein, i.e., "Entodinium valvatum" and "Ento- dinium bipalmatum," and also two new species which he adds to the genus Diplodinium Schuberg, i.e., "Diplodinium uncinatum" and "Diplodinium unifasciculatum." Since, however, the descriptions of these new species are not in accord with the characteristics of the genera Entodinium and Diplodinium respectively as laid down by Stein (1858, p. 69), and Schuberg (1888, p. 404), and Biitschli (1888, p. 1783), it is evident that Fiorentini made a serious mistake in assigning these species to the genera Entodinium and Diplodinium, a fact which was very clearly pointed out by Bundle (1895, pp. 296- 298 and 309-312). In this paper Bundle founds the new genus Cycloposthium to which he assigns Entodinium bipalmatum Fioren- tini, which therefore properly becomes Cycloposthium bipalmatum (Fiorentini) and stands as the type species. In the same paper Bundle (1895) founded another new genus, Blepharocorys, to which he referred both the Entodinium valvatum Fiorentini which then becomes Blepharocorys valvatum (Fiorentini) type species, and also Diplodinium uncinatum Fiorentini which, therefore, becomes Blephar- ocorys uncinatum (Fiorentini). Bundle (1895) does not discuss Diplodinium unifasciculatum, but it can be seen at a glance that an animal such as Fiorentini has pictured as Diplodinium unifasci- 48 University of California Publications in Zoology [VOL. 13 culatum is not correctly referable either to the genus Diplodinium or to Entodinium. In my opinion it may be referred to the genus Blepharocorys Bundle. The next work of importance is that of Eberlein (1895), who published a rather voluminous account of his investigations as well as a resume of all that had been done in this field by previous investi- gators. His methods were much superior to those of his predecessors and his work as a whole bears the stamp of thoroughness, complete- ness, and scientific accuracy. He adds one new species, Ophryoscolex caudatus, and claims the discovery of another, which he names Diplodinium caudatum. We note that the name Diplodinium caudatum had been used by Fiorentini some five years previously and that Eberlein was aware of this fact, for he says, "Wenn ich trotzdem die Bezeichnung Diplo- donmm caudatum fur meine Form gewahlt habe, so geschah das nur deshalb, weil das Diplodinium caudatum Fiorentini 'identisch' ist mit dem von dem gleichen Forscher beschriebenen Diplodinium rostratum und desshalb in Fortf all kommt. ' ' Under the code of nomenclature this procedure is inadmissable. In my paper the term Diplodinium caudatum refers to the form described by Fiorentini under this name. To the work of Bundle (1895) reference has already been made. It might be well to add, however, that in this paper Bundle considers only those Protozoa which are found in the caecum of the horse and therefore his work is of interest in this connection only because it deals with animals more or less closely related to those found in the stomachs of ruminants, and because he describes from the caecum of the horse a single ciliate, Buetschlia postciliata, which may be correctly referable to the genus Buetscklia, described from the stomach of ruminants. Giinther (1899) published the results of his investigations ^on the manner of infection of ruminants with these protozoans, together with a very complete account of the process of division in Ophryoscolex caudatus, and describes for the first time a new structure in the body of this animal. This structure he terms the "Stiitzapparat" and suggests that it functions as a support for the retractile gullet. To this part of the paper reference will be made later. During the following year Giinther (1900) published a second paper, dealing this time with the finer structure of some of the ciliates both of the ruminants and of the horse, i.e., Ophryoscolex caudatus, Entodinium rostratum, and Diplodinium [sp. ?] from the sheep; and Cycloposthium bipalmatum from the horse. This paper is of especial 1914 J Sharp: Diplodinium ecaudatum 49 interest in that it contains a somewhat more complete description of the ' ' Stiitzapparat " in both Ophryoscolex caudatus and Entodinium rostratum, and gives the first account of an observed case of conjuga- tion in these ciliates, i.e., in Cysloposthium bipalmatum, a ciliate from the horse. He says (p. 659) : " Auch mir ist es leider nicht gelungen, mehr als sechs Exemplare, die in Konjugation waren, aufzufmden. Desshalb ist es mir auch unmoglich gewesen, die Vorgange wahrend der Konjugation zu verfolgen und ich kann daher auch nur wenig iiber das Faktum, dass ich Konjugation gefunden habe, hinausgehen. " Giinther also finds in Ophryoscolex caudatus and Entodinium ros- tratum certain structures which he describes at some length as "myo- nemes. ' ' These myonemes he finds especially at the bases of the mem- branelles, and at the bases of the spines. I can only say here that I have not been able to find such structures in the species Diplodinium ecaudatum, but that contractile fibers are undoubtedly present in the esophagus of this species. This point will be fully discussed under the description of the esophagus. . Some other European investigators and writers, Brandt (1909), Liebetanz (1910), and Doflein (1911), have, during the past few years, published on this group, but their communications have dealt largely with the physiological relations of these organisms and so have contributed little to the solution of the systematic or morphological problems of these interesting protozoans. PRESENT STATUS Up to the present time there have been described as existing in the stomach of ruminants some twenty-four species of protozoan ciliates, classified as follows : LIST OF SPECIES OF CILIATES DESCRIBED (a) from the stomach of ruminants Family A. Ophryoscolecidae Stein, 1858. Genus I. Ophryoscolex Stein, 1858. Species 1. Ophryoscolex inermis Stein, 1858. 2. Ophryoscolex purkynjei Stein, 1858. 3. Ophryoscolex caudatus Eberlein, 1895. Genus II. Diplodinium Schuberg, 1888. Species 4. Diplodinium vortex Fiorentini, 1889. 5. Diplodinium maggii Fiorentini, 1889. / 6. Diplodinium bursa Fiorentini, 1889. 50 University of California Publications in Zoology [VOL. 13 7. Diplodinium dentatum Schuberg, 1888, Fiorentini emend., 1889. 8. Diplodinium denticulatum Fiorentini, 1889. 9. Diplodinium ecaudatum Fiorentini, 1889. 10. Diplodinium caudatum Fiorentini, 1889. 11. Diplodinium rostratum Fiorentini, 1889. 12. Diplodinium cattanei Fiorentini, 1889. 13. Diplodinium caudatum Eberlein, 1895 (non caudatum Fioren- tini). Genus III. Entodinium Stein, 1858. Species 14. Entodinium bursa Stein, 1858. 15. Entodinium caudatum Stein, 1858. 16. Entodinium dentatum Stein, 1858. 17. Entodinium minimum Schuberg, 1888. 18. Entodinium rostratum Fiorentini, 1889. Family B. Isotrichidae Biitschli, 1888. Genus IV. Isotricha Stein, 1859. Species 19. Isotricha intestinalis Stein, 1858. 20. Isotricha prostoma Stein, 1859. Genus V. Dasytricha Schuberg, 1888. Species 21. Dasytricha ruminantium Schuberg, 1888. Genus VI. Buetschlia Schuberg, 1888. Species 22. Buetschilia parva Schuberg, 1888. 23. Buetschlia neglecta Schuberg, 1888. 24. Buetschlia lanceolata Fiorentini, 1889. (&) from the Caecum of the Horse Genus I. Cycloposthium Bundle, 1895. Species 1. Cycloposthium bipalmatum (Fiorentini), 1890. Genus II. Blepharyocorys Bundle, 1895. Species 2. Blepharyocorys uncinata (Fiorentini), 1890; Bundle, 1895. 3. Blepharyocorys valvata (Fiorentini), 1890; Bundle, 1895. 4. Blepharyocorys unifasciculatum (Fiorentini), 1890. 5. Blepharyocorys jubata Bundle, 1895. Genus III. Spirodinium Fiorentini, 1890. Species 6. Spirodinium equi Fiorentini, 1890. Genus IV. Triadinium Fiorentini, 1890. Species 7. Triadinium caudatum Fiorentini, 1890. 1914] Sharp: Diplodinium ecaudatum 51 Genus V. Paraisotricha Fiorentini, 1890. Species 8. Paraisotricha colpoidea Fiorentini, 1890. 9. Paraisotricha oblonga Fiorentini, 1890. 10. Paraisotricha ovalis Fiorentini, 1890. 11. Paraisotricha triangularis Fiorentini, 1890. 12. Paraisotricha ampulla Fiorentini, 1890. 13. Paraisotricha incisa Fiorentini, 1890. 14. Paraisotricha truncata Bundle, 1895. Genus VI. Didesmis Fiorentini, 1890. Species 15. Didesmis ovalis Fiorentini, 1890. 16. Didesmis quadrata Fiorentini, 1890. Genus VII. Buetschlia Schuberg, 1888. Species 17. Buetschlia postciliata Bundle, 1895. Genus VIII. Blepharoprosthium Bundle, 1895. Species 18. Blepharoprosthium pireum Bundle, 1895. Genus IX. Blepharosphaera Bundle, 1895. Species 19. Blepharosphaera intestinalis Bundle, 1895. Genus X. Blepharocodon Bundle, 1895. Species 20. Blepharocodon appendiculatus Bundle, 1895. Diplodinium mammosum Railliet (1890) is not included in the list, since we have not had access to its description. VALIDITY OP SPECIES Diplodinium vortex Fiorentini is undoubtedly identical with Ophryoscolex purkynjei Stein, and Diplodinium rostratum Fiorentini is unquestionably a recent division product of Diplodinium caudatum described by the same author. We have already referred to the validity of the species described by Eberlein (1888) as D. caudatum and shall refer to it again along with D. vortex Fiorentini, and D. rostratum Fiorentini. With the exception of Buetschlia neglecta Schuberg, Buetschlia lanceolata Fiorentini and Diplodinium eberleini nom. nov ( = D. caudatum Eberlein), all of the above named species (described from the stomachs of ruminants) are present in the stomachs of the sheep and cattle from the Pacific Coast, i.e., principally from California, Nevada, Arizona, and Mexico. 52 University, of California Publications in Zoology [VOL. 13 TECHNIQUE New facts are usually brought to light by the discovery of new or better methods. These afford a solid foundation for progress. Much can be judged of the scientific value of results by a knowledge of the methods used to obtain them. For this reason the methods employed in procuring, preparing, and studying these animals are given in some detail. Living Material. The material was obtained at the Oakland Meat and Packing Company's stockyards, which are a forty-five minute car-ride distant from the laboratory. The first problem was neces- sarily to devise a means of carrying the stomach fluid which contained these parasites from the slaughter-house to the laboratory without allowing a fall in temperature, for as is well known, a loss of three or four degrees Centigrade will cause the death of these animals. In this connection it is interesting to note that of all the twenty-odd species of ciliates described from the stomachs of ruminants the Diplo- dinium ecaudatum series is the most sensitive to changes of tempera- ture, a fact not heretofore recorded. Two points are kept in mind : the rapidity of obtaining the material after the animal has been killed, and prevention of loss of heat from the container while on the way to the laboratory. We go directly to the killing floor and as soon as the viscera are removed from an animal, which is usually within three to five minutes after its death, an assistant removes the reticulum or ' ' honeycomb, ' ' turns it inside out to remove the partly digested food material and then wrings it as one would wring a wet cloth, into the operator's hand, which is held cupped to receive the fluid. At the inner edge of the hand, pressed close to the flesh, is the opening of the container, held in such a manner that the hand forms a warm funnel which Collects and transfers the fluid to the container with the least possible loss of heat. By using a "Thermos" vacuum bottle contained in a well insulated warm box it is possible to convey the stomach fluid from the animal to the warm oven in the laboratory with a fall in tempera- ture of not more than one-half of a degree Centigrade. Schuberg (1888) after obtaining his material wrapped the glass container in a cloth and carried it thus in his pocket for half an hour before reaching the laboratory. Eberlein (1895) collected the stomach fluid in test-tubes, which he placed, without wrapping, in his trouser's pocket, where he carried them for an hour before reaching the labora- tory. Eberlein notes that the temperature may fall as low as 20 C 1914] Sharp: Diplodinium ecaudatum 53 without causing death, but, although this may be true for some of the species, it certainly does not hold for the species of Diplodinium. Fiorentini (1889) says nothing of the manner in which he conveys the stomach fluid to the laboratory except that it is in test-tubes, but he does explain how he keeps the liquid warm in the laboratory. He says : ' ' For keeping the test-tubes at the temperature indicated above (30 to 35 C) we have recourse to a system of immersion in a vase of hot water, which is renewed from time to time, or still better, we keep it in an oven at a constant temperature regulated at 35 C." Schuberg (1888) gives no detailed description of his method of keeping the animals alive while under observation. He speaks of a heated microscope stage ("geheizten Objecttisch"), but does not tell how he keeps this stage hot nor does he tell how long he is able to keep alive the animals under observation. Fiorentini says that he had recourse to the Schultze warm stage, and notes that he was by this method enabled to keep the animals under observation alive for a long time, "even for a whole hour at a time." Fiorentini also used another method which he explains as follows: "First I heat the glass slide at a small lamp until it is lukewarm; then I put a drop of the material to be observed upon it and cover it with a cover glass. Then with a pipette I take boiling water from a capsule which I keep near me and drop this hot water on the glass slide in such a manner that it will not mix with the fluid underneath the coverglass. The hot water thus placed upon the slide allows me to maintain for a long time the glass and the material being observed at a sufficiently high temperature so that I am permitted to make my observations on the living protozoans" (translated from the Italian original). And further he says: "Once the slide becomes cold one begins again with a new preparation." And again: "This method is indispensable dur- ing winter. In summer, however, it is sufficient to heat up the slide to get a preparation which keeps long enough to permit one to make a long observation." This method, Eberlein states, he was unable to use; instead he employed a glass plate which he laid on top of the stage and on top of this he placed his slides. The glass plate he heated by placing two small lamps under the projecting corners. Eberlein says that he soon became expert enough to tell the proper temperature of the glass plate through the sense of touch. Bundle (1895) in his report on the ciliates from the caecum of the horse says very little about the methods which he employed to the animals alive under observation except that he sometimes 54 University of California Publications in Zoology [VOL. 13 used the "heizbaren Objekttisch" of M. Schultze and sometimes did not, and that he was never able to keep the animals alive for more than two or three hours at the most. Giinther (1899 and 1900) says even less about his methods than does Bundle (1895). Both investigators found that the alcoholic corrosive-sublimate solution gave the best results as a fixing agent and were inclined to the use of haematoxylin as the most satisfactory stain. In my study of the living animals use has been made of an auto- matic constant-temperature warm oven slightly modified from the pattern used by Dr. J. A. Long (1912) in his study of the living eggs of rats and mice. The adjustment of the automatic temperature regu- lator is such that the temperature of the material on the slide may be kept constant to within 0.5 C. for an indefinite period of time. The great advantages that such an apparatus offer are: first, that not only the material under actual observation, but also the remaining material, the microscope stage, the slides, the cover glasses, the pipettes, etc. may all be kept at the desired constant temperature, and second, that the temperature under w r hich the animals are being studied, can be easily raised or lowered as desired, at the will of the observer. When the fluid to be studied is brought from the slaughter house to the laboratory, it is immediately put into this warm oven so that from the time of leaving the stomach to the time of being placed on the slide, the loss in temperature is not greater than 0.5 C. and once on the slide, the drop of fluid may be studied for from eight to ten hours without any apparent injury to the animals. A second impor- tant use, as noted above, to which this apparatus may be put and a most necessary one in studying the movements of the living animals is that of temperature control. Under normal conditions the exceeding great liveliness of these organisms makes their accurate study ex- tremely difficult. Various investigators have recommended the ad- dition of some substance such as a watery solution of cherry tree gum, a three or four percent solution of gelatin, Irish moss, quince seeds, etc., which would eliminate or at least diminish the constant motion of these animals. All of these methods have been tried by me with more or less success, but with the inevitable result that the longevity of the organisms has been decreased. By means of the above described con- stant-temperature oven, however, the operator can regulate the temper- ature to such a degree that the activity of the animals may be almost absolutely controlled without any apparent injurious effects. To 1914] Sharp: Diplodinium ecaudatum 55 avoid the evaporation of the fluid under the cover glass, small dishes of water are placed within the oven, a plan which serves to prolong the period of observation to some degree. Another plan which I use when long continued observations on the same animals are desired is to arrange on the slide a glass cell filled with a drop or two of the stomach fluid and from which a fine cotton thread leads to the fluid under observation. The thread acts as a siphon and serves to keep an ever fresh supply of fluid under the cover glass. In brief, it has been possible by means of this apparatus to control the rapidity of the movements of these animals, through temperature regulation, and to keep living Diplodinium under constant observation for from eight to ten hours at a time. In fact the animals have been kept alive for over forty-eight hours after removal from the stomach of the ox and, within this limit, i.e., forty-eight hours, the length of the period during which an individual animal may be kept under observa- tion, barring accidents, depends entirely upon the endurance of the observer. Fixation and Staining. The following fixing fluids have been used with good results; Schaudinn's alcoholic sublimate solution, Zenker's, Flemming's, Worcester's, and Bouin's fluids, formalin (4%), and os- mic acid (1%). Of these Schaudinn's, used hot, gave uniformly the best results. When it was desired to follow with Mallory's connective tissue stain Zenker's fluid was the best fixing agent. For the study of surface markings it was found necessary to fix in warm four per cent formalin and mount unstained in styrax. This method gives excellent preparations. The preparation of fixed material is done on the "killing floor" at the slaughter house. A table is arranged as near to the spot where the cattle are killed as possible. On the table are two pans of hot water, one maintained at 36 C. in which a shallow glass dish is placed and the other which is maintained at near the boiling point serves as a con- tainer for the tubes of fixing fluid. The minute the ox is opened and the stomach removed the assistant obtains the reticulum, turns it inside out in order to get rid of the superfluous food particles, then quickly wrings the fluid from the walls into the glass dish in the water at 36 C. temperature. Instantly the operator dashes the boiling Schaudinn's fluid into this dish. In this manner the protozoans are obtained with cilia extended and without contractions or contortions of the body. After fixing for five to ten minutes the mixture is shaken up with twice its volume of 50 per cent iodine alcohol. This is repeated until the 56 University of California Publications in Zoology [VOL. 13 iodine color persists. The mixture is then gradually run down to water when the process of staining is begun. In the case of formalin as a fix- ative it is best to use it at about 36 C. as the boiling formalin has a tendency to contract the endoplasm and ectoplasm and so leave the cuticle somewhat wrinkled. "When other fixatives are used the methods of handling are in accordance with those usually given for protozoan fixation. For in toto staining the most satisfactory results are obtained by the use of Heidenhain's iron-alum haematoxylin solution, as follows: From water the organisms are subjected to a 1 per cent iron-alum solution for twenty-four to thirty-six hours. Then thoroughly washed in distilled water, stained in a 0.3 per cent solution of haematoxlyn (Heidenhain's) for twenty-four hours and then washed in tap water. It is necessary then to differentiate with a 1 per cent iron-alum solution under the microscope to be sure that differentiation is carried on to the right degree. After differentiation the animals are again washed in tap water and distilled water, then passed up through the alcohols, xylol, to cedar oil, and mounted either in Canada balsam or styrax. Freshly made up iron-alum and freshly made up haematoxylin solutions have given uniformly better results than the ' ' ripened ' ' solutions. Eberlein (1895) suggests the freeing of the stomach fluid from excess food particles by straining through a warmed linen cloth. Such a method might result in the loss of many of the organisms. We have found that by careful manipulation of the centrifuge an almost pure culture of the animals may be obtained. By rotating the centrifuge at the proper speed the food particles heavier than the organisms will be thrown down first while those lighter than the organisms will remain nearer the top. The top and bottom portions may then be thrown away. This process is of course carried on simultaneously w'ith^hat of washing and staining and if the top and bottom portions are removed each time the fluid is changed the remainder will soon consist mainly of the desired organisms. Segregating. Both in making whole mounts and in sectioning the animals it has been desirable to segregate the species. This is accomp- lished by means of the following apparatus: A small hypodermic syringe, fitted with a glass tube drawn out to an almost microscopic cross section is clamped to the microscope stage in such a manner that the end of the glass tube is in the field of vision. The glass tube is ad- justed and held in position by a screw clamp. Then with the organisms in a cedar-oil medium and the glass tube and syringe filled with cedar Sharp: Diplodinium ecaudatum 57 oil the operator can by means of the mechanical stage bring a desired animal up to the end of the glass tube into which it may be sucked by a slight twist of the piston of the syringe. In this way the desired animals may be sorted out free from dirt and other animals to the number of from two hundred and fifty to five hundred in a single tube. By substituting a fresh slide on which is placed a drop of Canada bal- sam or styrax the animals may be easily ejected from the glass tube into the mounting substance. By mounting these animals between two thin cover glasses and then fixing the cover glasses in a brass frame which is constructed to hold them as a window frame holds the pane of glass, the same individual animal may be viewed from either side. This simple bit of apparatus has proved itself exceedingly useful in determining many otherwise difficult problems and is therefore recommended for consideration to those making similar investigations. Sectioning. When it is desired to section the animals they are segregated by the above method and ejected from the glass tube into small gelatin capsules (such as may be obtained at any pharmacy) which have been partially filled with imbedding paraffin. The capsule is placed in a warm oven at the proper temperature, the paraffin melts, the cedar oil diffuses through the paraffin, and the animals sink to the bottom. By soaking in water for a few minutes the gelatin capsule may be easily slipped off and by cutting off the bottom end each time and running this through another paraffin filled capsule for at least four times, a paraffin infiltration of the organisms, sufficient to allow of sectioning, is obtained. When the infiltration is complete the lower four or five drops of paraffin containing the animals are withdrawn by means of a warm capillary pipette and run into a mould which has been prepared in the following manner. A small brass rod is filed to a square cross section and fitted in an imbedding plate so that when the paraffin is poured around it, it will act as a core. When the paraffin hardens and the core is removed a perfect mould is obtained. Paraffin of a much higher melting point should be used for the moulds than that which is used for imbedding, and if the paraffin of the mould be slightly tinted by Sudan III., the liability of cutting into the imbed- ding paraffin is eliminated. When the imbedding paraffin containing the animals is run into the mould care should be taken that the animals are well distributed through the paraffin and that the paraffin is just above the melting point. It then hardens before the animals have time to sink to the bottom, and so results in their remaining well distributed 58 University of California Publications in Zoology [VOL. 13 throughout the block. By this method very little practice is necessary in order to obtain a perfect imbedding. The advantages of such a method are: (1) the certainty that only the desired animals are present; (2) the ease of handling; (3) perfect infiltration; (4) even distribution; (5) the production of a block with a smaller, truer, square cross section than it is possible to obtain by trimming by hand, a point which is very important when good ribbons are necessary. In some respects it is advantageous to stain in toto before sectioning, but the best results are obtained by staining the sections. Section Staining. For section staining Mallory's connective tissue stain not only gives excellent results giving four distinct colors but also reveals a very surprising structural differentiation which to my knowledge has not before been described. The slides should be run from xylol very gradually down to distilled water. Sec. Fuchsin S., aqueous solution (%%) 45 Distilled water 5 Phospho-molybdic acid (1%) - 60 Fresh distilled water 5 Anilin blue, orange G. and oxalic acid 60 Distilled water 10 95% alcohol 1 100% alcohol 1 Carbo-xylol 1 Xylol, Mount. If the above method is carefully followed preparations may be ob- tained in which the cilia show clear i.e., transparent, the ectoplasm a blue-red, the entoplasm pink, the macronucleus orange-brown and the micronucleus and some fibers which will be described later^how bright red by transmitted light. Heidenhain's iron-alum haematoxylin made up as described above for in toto staining also gives very perfect preparations. Genus Diplodinium Schuberg 1888 Schuberg (1888), p. 404. Fiorentini (1889), pp. 11-17, pi. 1, figs. 1-4; pi. 2, figs. 1-5; pi. 3, figs. 1-5. Eberlein (1895), pp. 251-264, pi. 17, figs. 8-17; pi. 18, figs. 18-20. The genus Diplodinium was separated from the genus Entodinium of Stein (1858) by Schuberg (1888). Although Schuberg set up this new genus he gives a very unsatisfactory characterization of it. He 1914] Sharp: Diplodinium ecaudatum 59 says (p. 404) : "Stein hat unter dem Gattungsnamen Entodinium drei Infusorienformen vereinigt, die ich mit ziemlicher Sicherheit auch angetroffen habe. Ein genaueres Studium ergab jedoch, dass eine dieser Arten, Entodinium dentatum, wie Ophryoscolex, eine zweite Wimperzone besitzt, und dass auch Thieren, die mit Entod. bursa Stein eine gewisse Aehnlichkeit haben, und die Stein weniger iiber- sehen, als mit dieser letztern zusammengeworfen zu haben scheint, eine solche zukommt. Ich trenne die Formen, die Entodinium im all- gemeinen gleichen, jedoch durch eine zweite Wimperzone ausgezeichnet sind, unter dem Namen Diplodinium ab ; dieselben sind iibrigens auch noch durch andere Merkmale als naher zusammen gehorig character- isirt, worauf aber hier noch nicht naher eingegangen werden soil. ' ' That this definition is insufficient will be seen when we consider the genus Opliryoscolex, the members of which also resemble Ento- dinium and possess two membranelle zones, but which are not members of the genus Diplodinium. Some of the more apparent differences between the three genera are given in the following table : *Mean dimensions of the body in mm. Examples Length Width O. inermis Stein 0.180 0.085 O. caudatus Eberlein 0.180 0.085 O. purkynjei Stein 0.175 0.085 D. bursa Fiorentini 0.120 0.065 D. caudatum (= eberleini nom. nov.) 0.105 0.065 D. eeaudatum Fiorentini 0.055 0.025 E. bursa Stein 0.090 0.050 E. caudatum Stein 0.080 0.040 E. dentatum Stein 0.075 0.040 * Eberlein (1895), p. 283. Dorsal membranelle zone Vacuoles Incomplete spiral en- circling % of the 5-6 entire body. Transverse, encircling less than % of the 2-4 entire body. Absent. Schuberg (1888), so far as I am able to determine, has never given any descriptions of individual species of this genus, but, as quoted above, merely cites Entodinium dentatum Stein as belonging to this new genus, along with another form which resembles Entodinium bursa and which, in my opinion, may probably be Diplodinium bursa Fiorentini (1889). Stein (1858) describes Entodinium bursa (type specie,? of the genus by location), E. dentatum, and E. caudatum, 60 University of California Publications in Zoology [VOL. 13 species which still hold good in that genus, but gives no figures, and Schuberg (1888) gives neither a description nor a figure of the species of Diplodinium to which he attached the name " dentatum." Stein (1858) explicitly states that "der Wimpergiirtel der Riickseite fehlt jedoch" in Entodinium. Schuberg (1888) founds Diplodinium upon a ciliate with a dorsal membranelle zone. There is, therefore, no ques- tion but that he had before him as the species upon which he founded his new genus a specimen different from Entodinium dentatum Stein and correctly referable to his new genus Diplodinium because it had a dorsal membranelle zone. Therefore E. dentatum Stein and Diplo- dinium dentatum Schuberg are two different species in different genera and both names are valid, although Schuberg 's opinion as to the identity of the two species implied in his statement above quoted and in his use of Stein's specific name is in error. This decision is in accord with the usage of Eberlein (1895). The question as to the type species of Diplodinium is a very complicated one. It seems wisest, however, in view of the subsequent history of the case, to accept Schu- berg 's D. dentatum as later described and figured by Fiorentini (1889) as the type species. Fiorentini (1889) utilized Schuberg 's name for this inadequately defined and still less adequately established genus and assigns to it nine species, which he figures and describes as Diplo- dinium vortex, D. maggii, D. bursa, D. dentatum Schuberg, D. denti- culatum, D. ecaudatum, D. caudatum, D. rostratum and D. cattanei. To these Eberlein (1895) added a new species which he named Diplo- dinium caudatum, thus making ten species described for this genus. Diplodinium vortex, however, as is pointed out by Eberlein (1895), is not a member of the genus Diplodinium at all, but is identical with Ophryoscolex purkynjei Stein (1858). There is some question also regarding the specific standing of D. maggii Fiorentini.^ Eberlein accepts it with some hesitation. He says of it: "Es ist ziemlicb gross, und diese Eigenschaft, besonders aber die unverhaltnismassige Breite unterscheidet es von Diplodinium bursa. Es bleibt zweifelhaft, ob es auf Grund dieser einen Eigenschaft berichtigt ist, Diplodinium maggii als selbstandige Art zu betrachten, oder ob es Diplodinium bursa zuzurechen ist. ' ' I have found it to be, however, in my material, a perfectly well-defined species. We also call attention to the fact that D. rostratum Fiorentini is merely an individual D. caudatum, described shortly after transverse division, and so drops into the synonymy of the latter. This leaves to the credit of Fiorentini seven species of the genus Diplodinium, all 1914 1 Sharp: Diplodinium ecaudatum 61 of which are present in my material and have been identified by me. We have already noted the inadmissible procedure of Eberlein (1895) in assigning the species D. caudatum Fiorentini to D. rostratum Fioren- tini and then appropriating the name D. caudatum for a species of his own. This species, D. caudatum Eberlein, I have not yet identified, but for purposes of conformity to the code of nomenclature I propose for it the name Diplodinium eberleini. Granted that this species, Diplodinium eberleini, is valid, then the genus Diplodinium contains up to the time of this paper eight valid species or forms, viz. : D. maggii Fiorentini, D. bursa Fiorentini, D. dentatum Schuberg (Fio- rentini emend.), D. denticulatum Fiorentini, D. ecaudatum Fiorentini, D. caudatum Fiorentini, D. cattanei Fiorentini, and D. eberleini nom. nov. My observations lead to the conclusion that D. denticulatum is merely a variant form of D. dentatum and that D. caudatum and D. cattanei are only forms of D. ecaudatum. In fact it is one of the purposes of the present paper to show that D. ecaudatum Fiorentini, D. caudatum Fiorentini and D. cattanei Fiorentini are forms of the species whose lawful name is Diplodinium ecaudatum Fiorentini. To this species (D. ecaudatum Fiorentini) I add three new forms to which I have given the descriptive names D. ecaudatum forma bicaudatum, D. e. forma tricaudatum, and D. e. forma quadricaudatum. Diplo- dinium caudatum therefore becomes D. e. forma caudatum, and D. cattanei, in accordance with facts w r hich will be given later, becomes D. e. forma cattanei. These three forms ( D. e. forma ecaudatum, D. e. forma caudatum, and D. e. forma cattanei), together with my three new forms, constitute a complete series ranging from D. e. forma ecau- datum without posterior spines up to D. e. forma cattanei with five spines. Allowing for normal variations in details of structure, the morphology of these six forms is identical, with the exception only of the presence and number of these spines and the necessary differences in the form of the posterior end w r hich their presence or absence occasions. From all the other species of Diplodinium, however, they differ considerably. These facts seem to warrant the placing of these six forms in a single species, which in accordance with the rules of nomenclature must be designated as Diplodinium ecaudatum, although many of the individuals have from one to five ' ' caudal ' ' appendages. 62 University of California Publications in Zoology [ VOL - 13 1. Diplodinium ecaudatum forma ecaudatum Fiorentini PI. 3, figs. 1, 2; pi. 4, figs. 3-5; pi. 6, figs. 11-19; pi. 7, figs. 20-33. Diplodinium ecaudatum Fiorentini (1889), pp. 15-16, pi. 3, fig. 1. Diplodinium ecaudatum, Eberlein (1895), pp. 263-267, pi. 18, fig. 19. Of all the various forms of Diplodinium ecaudatum found in the stomachs of western cattle the most numerous and the simplest morph- ologically is the forma ecaudatum. This form is almost universally present and when once identified may be easily distinguished from all other forms. It is the basis of the following full description, which is applicable to the other forms except only in the region of the pos- terior spines. The form of the body is constant (pi. 3, figs. 1, 2), somewhat over twice as long as wide, consistently circular in cross-section, obliquely rounded off at the anterior and pointedly at the posterior end of the body. In general the body somewhat resembles a rather short, plump banana, the dorsal side being convex and the ventral slightly concave. This resemblance would be still greater if the stem end of the banana, which corresponds in position to the oral opening, be held stationary while the posterior extremity is twisted slightly to the right of the median ventral line. The organs of food-taking and locomotion are situated in the anterior one-fourth of the body, which part is more or less flexible and decidedly contractile. The remaining three-fourths of the body is rigid, friable and comparatively smooth, i.e., free from appendages of any description. The anus is situated at the posterior extremity of the body close to the ventral side. The structure of the body is very complicated. It shows plainly (fig. B; pi. 4, figs. 3-5, and pis. 6, 7, figs. 11-33) a cuticle (cut.}, an ectoplasm (ect.}, and an entoplasm (ent.}, with a boundary layer (bd. I.) which separates the ectoplasm from the entoplasm. ^ The separate structures which are ectoplasmic in their origin and location are: three skeletal areas with underlying skeletal structures (I. sk. a., v. sk. a., and r. sk. a.), a macronucleus (mac.}, a micronucleus (mic.}, a motor mass (TO. m.}, motor fibers (d. m. str.}, a circum- oesophageal ring (circ. oes. ring}, oesophageal retractor strands (oes. retr. str.}, a dorsal row of membranelles (d. m.}, an adoral row of membranelles (ador. m.}, operculum (op.}, oral cilia (or. oil.}, mouth (or.}, oesophagus (oes.}, caecum (caec.}, rectum (rect.}, anus (an.}, and contractile vacuoles (ant. c. v. and post. c. v.}. The ento- plasm (ent.} is structureless with the exception of the contained food vacuoles (fd. vac.} and food particles. Sharp: Diplodinium ecaudatum 63 THE ECTOPLASMIC STRUCTURES Cuticle. The thin but resistant cuticle covers the entire body and is so highly specialized over certain regions (sk. a., pi. 3, figs. 1-2) as to demand special description. There are three such regions over which the cuticle shows a well-defined differentiation. (1) Over the dorsal (D.) and left surfaces of the body the cuticle is comparatively smooth, giving only faint evidence of longitudinal striations and in places traces of mottling. (2) The cuticle over the ventral surface (V.), extending from the middle to the posterior extremity of the body and over the spines when present, is faintly but distinctly mot- tled. This mottling is caused by minute diamond-shaped depressions in the cuticle which at certain levels do not transmit the light as readily as the non-depressed portions, and hence appear as little shadows. (3) The third region, embracing the anterior one-half of the left ventral and ventral surfaces and the anterior two-thirds of the right surface, is divided into three well-defined areas by under- lying ectoplasmic structures which appear to be skeletal in function. Eberlein (1895, p. 240) says in his description of Ophryoscolex iner- mis: "Die Ran der der Bauchflache sind beiderseits durch einen Streifen starker granulierten Plasmas begrenzt (fig. 1)," but makes no mention of a definite underlying structure. Erlanger (1890, p. 654) observed, in Chlamydodon mnemosyne Stein, a strange "Band" situated between the dorsal and ventral surfaces, surrounding the entire body with the exception of a small interruption at the posterior end, and which was clearly marked off by cross striations. And Levander (1894, pp. 66-67) in his description of Plagiopyla nasuta Stein calls attention to a previously unnoticed cross-striped band of differentiated ectoplasm situated on the right side of the body near to and parallel with the ventral border. The above-mentioned cases of ectoplasmic differentiation might be conceived as being similar to the peculiar ectoplasmic differentia- tions which are described in this paper as skeletal areas. In none of the above cases, however, is anything said about a specially differ- entiated underlying ectoplasmic structure. To GHinther (1899) belongs the credit for being the first to note and describe this underlying ectoplasmic structure. He says (p. 553) : "An jedem Ophryoscolex caudatus, schon bei der Untersuch- ung des lebenden Thiers, besser natiirlich an Konservirten gefarbten Thieren und Schnitten derselben, habe ich ein Organ (cf. figs. 1, 2, 5, 6, 7 st\) gefunden, im Ectoplasma liegend, iiber das bis jetzt noch kein 64 University of California Publications in Zoology [VOL. 13 Autor in der iiber die Infusorien des Wiederkauermagens handelden, mir zuganglichen Litteratur etwas bemerkt hat Nach meinen Beobachtungen stellt dasselbe einen Stiitzapparat fiir den Schlund dar. ' ' And again in his paper of 1900 he adds that he has also found a corresponding structure in Entodinium rostratum Fiorentini. But in neither of these papers does Giinther (1899 and 1900) make men- tion of a surface differentiation of the cuticle corresponding to the underlying ectoplasmic structures ("Stiitzapparat"). As a matter of fact these peculiar structures (sk. lam., pi. 4, figs. 3, 4, 5) with their overlying areas (1. sk. a., v. sk. a., and r. sk. a., pi. 3, figs. 1, 2) which I have found in Diplodinium, and which appear to be skeletal in function, and are so designated in this paper, are so different from anything heretofore mentioned that a rather complete description is deemed in order. Skeletal Areas. The skeletal, structures proper will be described in connection with the ectoplasm. The corresponding areas of cuticle, which will be designated as a left skeletal area, a ventral skeletal area, and a right skeletal area, show well-defined boundaries. The left skeletal area (1. sk. a., pi. 3, fig. 2), the smallest of the three, is triangular in shape with its base anteriorly marked off by a line drawn horizontally from the left extremity of the dorsal mem- branelle zone (d. m. z., pi. 3, fig. 2) to meet the adoral membranelle zone (ador. m. z., pi. 3, fig. 2) near its left extremity, and its apex extending obliquely posteriorly and toward the right to end on the ventral side about midway between mouth and anus. The ventral skeletal area (v. sk. a., pi. 3, figs. 1, 2), the largest of the three areas, is rectangular in shape and extends from the base of the outer adoral lip (pi. 3, figs. 1. 2) somewhat obliquely towards the posterior end and slightly towards the right to blend with the right skeletal a^ea just anterior to the middle of the body. The right skeletal area (r. sk. a., pi. 3, fig. 1), intermediate in size, also somewhat triangular in shape, with base marked off by a line extending from the right extremity of the dorsal membranelle zone to the dorsal side of the base of the outer adoral lip, and apex extending posteriorly, blends with the ventral skeletal area just anterior to the middle of the body. The two areas then extend posteriorly to terminate rather indistinctly at about the last fourth of the body. The relation of skeletal areas to underlying skeletal structures may be seen best by a consideration of plate 4, figure 4, and plate 7, figures 23-29. The cuticle over these areas is more transparent than elsewhere and through it may be seen 1914] Sharp: Diplodinium ecaudatum 65 the underlying skeletal structures. It is thrown into fine longitudinal ridges which mark off the little diamond-shaped depressions, which, as already pointed out, give the mottled appearance so characteristic of these areas. At the anus (an., pi. 4, fig. 3) the cuticle is continuous with the rectal sheath and at the mouth with the lining of the oral cavity and oesophagus (or., pi. 4, fig. 3). At the dorsal and adoral zones of cilia it dips down into the furrows (pi. 4, fig. 3) and covers the lips, disks, and operculum. Over the lips and operculum it is much thickened, but in the furrows and over the disks it is thin. Ectoplasm. The ectoplasm (ect., pi. 4, figs. 3-5), which is entirely covered by the cuticle and separated from the entoplasm by the very distinct boundary layer (bd. I.}, is not a homogeneous substance, but shows a rather definite alveolar stroma highly modified in certain regions, as noted above, to form skeletal structures. This layer of ectoplasm varies much in thickness in different parts of the body, being very much the thickest in the anterior region (ect., pi. 4, figs. 3, 4; pi. 6, figs. 14-16; pi. 7, figs. 23-27), and thinnest over the left side (ect., pi. 4, fig. 5.) In the anterior and posterior ends of the body the thick ectoplasm fills in the inequalities of the outer form of the body, so that the enclosed entoplasm is smoothly rounded off, and in these regions does not conform to the general contour of the body. Here also the reticular structure is coarser, the meshes appear larger, and may therefore be more easily studied. That part which lies close to the cuticle loses its irregular arrangement and forms a rather well- defined alveolar layer in which, in cross-sections, the alveoli appear irregularly quadrilateral. A similar layer in which the individual alveoli may be even more easily distinguished lies next to the boundary layer (bd. I., pi. 4, fig. 4). The ectoplasm also encloses the first part of the oesophagus (oes., pi. 4, figs. 3-4), the rectum (rect., pi. 4, fig. 3), macronucleus and micronucleus (mac. and mic., pi. 4, figs. 3, 5), and the two contractile vacuoles (ant. c. v. and post. c. v., pi. 4, fig. 3). It is noteworthy that in the immediate vicinity of the con- tractile vacuoles the alveolar structure is again modified, in that the meshes are larger and the interalveolar walls are finer in structure and stain less heavily than the remaining ectoplasm (c. v. r., pi. 4, fig. 3; see also microphotograph, pi. 7, fig. 27). No streaming whatever of the ectoplasm has been observed. Skeletal Structures. The skeletal structures noted in the descrip- tion >f the cuticle are undoubtedly of ectoplasmic origin, but if, as 66 University of California Publications in Zoology [VOL. 13 Eberlein (1895, p. 243) points out, the brittleness of the cuticle is due to the presence of silicic acid, there is probably silicic acid present in this skeletal structure, for of all the structures of the body this is at once the most rigid and the most brittle. The appearance in surface view of these areas has already been described. In cross- sections each of these regions is somewhat elliptical, transversely crossed by thin, paired laminae (sk. lam., pi. 4, figs. 3-5; see also microphotographs, pi. 7, figs. 23-29), extending from the cuticle per- pendicularly inward to the inner wall of the elliptical space. At their inner and outer attachments the members of each pair of laminae are in close juxtaposition, but in the middle they spread apart, leaving a minute elliptical interval between the two laminae. The appearance in cross-sections is as though the inner wall of the elliptical space were held away from the outer wall by a row of spindles, the longest of which is situated in the middle. These pairs of laminae extend ob- liquely posteriorly, corresponding with, and, as a matter of fact, causing the longitudinal ridges in the cuticle over the above-described skeletal structures. The central skeletal laminae of the left skeletal structure, passing from anterior to posterior, at first grow longer and longer, pushing the central portion of the inner wall of the ellipse farther away from the outer wall until what was an elliptical area becomes triangular in outline with apex pointed toward the longi- tudinal axis of the body (pi. 7, figs. 23-28). Just above the middle of the body, however, the reverse takes place, the central laminae grow gradually shorter and allow the inner wall of the ellipse to approach the outer wall until at about the middle of the body the left skeletal structure joins the ventral skeletal structure, which at about the same level joins the right skeletal structure, i.e., the three skeletal structures merge into one. The laminae of the left skeletal structure (pi. 7, fig. 29) are at this level much longer than those of the ventral and right skeletal structures, but they now become rapidly shorter, soon disappear altogether, and this elliptical space with its contained skeletal structure is entirely obliterated (pi. 7, figs. 30-32). The skeletal elliptical spaces of the ventral and right sides fuse immedi- ately anterior to the middle of the body (pi. 7, fig. 28). The laminae soon become shorter, are placed more closely together, and appear in cross-sections as a row of little pillars which support the oesophagus and keep it away from the right wall of the body (v. sk. lam. and r. sk. lam., pi. 4, fig. 5, and pi. 7, fig. 29). These laminae also grow narrower and narrower up to the point immediately anterior 1914] Sharp: Diplodinium ecaudatum 67 to the last one-fourth of the body, where they, too, disappear. It is to be noted that all traces of oesophageal structure are lost at about the same level as that at which the united ventral and right skeletal structures disappear. That the above described structure functions as a true skeletal (supporting) structure, not only for the retractile oseophagus but also for the entire body, seems altogether certain. In the consideration of the oesophagus and the motile anterior end of the body additional evidence will be given for this conclusion. Giinther (1899, p. 553) describes a homologous structure for Ophryoscolex caudatus, which is composed of two parts, at first sep- arated one from the other, partly embracing the gullet and then approaching each other quite closely, finally to separate again and end singly, deep down in the lower part of the animal. In the case of Ophryoscolex caudatus this structure is described as being com- posed of two layers, a fine, thin, structureless membrane and an en- closed substance which shows a definite structure, "mit grofien, meist zur Langsachse des Stiitzsapparates senkrecht gerichteten Waben." And in his description of a similar structure in Entodinium rostratum Giinther (1900, p. 644) says: "Dicht unter der Pellicula im oberen Theil des Thiers beginnend, rechts vom Kern liegend, zieht sich das- selbe (Fig. 13 st) in ziemlich breiter Ausdehnung bis zur Mitte, wo es sich in 3 Theile theilt (Fig. 14 st), von denen jeder einzeln tief unten im Thier endet, stets der Pellicula anliegend. " Of the finer structure of the apparatus in this case nothing is said. It is hardly necessary to point out that this description of the "Stiitzapparat" in Ophryoscolex caudatus has little in common with the above description of the skeletal structure in Diplodinium ecau- datum except in general indications of homology. Boundary layer. Separating the ectoplasm from the entoplasm is a constant and well-defined boundary layer (bd. I., pi. 4, figs. 3, 5, pi. 6, fig. 15, and pi. 7, fig. 25). This layer is probably ectoplasmic in nature; it stains very heavily either with iron haemotoxylin or Mallory's connective tissue stain. Even in well-stained whole mounts it may be clearly made out. In fact it may easily be mistaken for an external structure, it shows so clearly. From the alveolar ectoplasm this boundary layer is separated by a thin layer of regularly placed, small, cubical alveoli, and from the more homogeneous entoplasm by a similar alveolar layer consisting of large, more definitely cubical alveqji. This boundary layer with its two investing layers of alveoli 68 University of California Publications in Zoology [VOL. 13 forms a sort of sack enclosing the entoplasm, into which opens the oesophagus (oes., pi. 4, fig. 3) and out of which leads the rectum (rect., pi. 4, fig 3). This "boundary layer," as above described, refers only to the definite structureless membrane separating the ectoplasm from the entoplasm and bounded on either side by a layer of alveoli. Eberlein (1895, p. 244, 245) describes for this boundary layer ("Grenzschicht") a definite fibrillar structure and is inclined to assign to it also two or more alveolar layers. Giinther (1900, p. 643) states emphatically that, notwithstanding Eberlein 's description, he is unable to find any alveolar structure in the above-named layer. It must be noted, however, that the oesophagus contains within its walls, as will be described later, many fibrillae which are in this paper designated as oesophageal retractor strands (oes. retr. sir., figs. B, D ; pi. 4, fig. 3) and that in certain portions of the body the oesophagus comes to lie in such close contact with the boundary layer as to defy microscopic separation of the two. Hence, in these places, it is easy to see how a fibrillar structure might be assigned to the boundary layer. Careful investigation of this point, however, indicates an essen- tially non-fibrillar structure for the true boundary layer. The micronucleus and macronucleus, and the two contractile vacuoles, lie in depressions on the outside of this sack. At the anterior end of the body this boundary layer dips down, approaches the oeso- phagus as a funnel-shaped depression, accompanies it, and finally blends with the incoming cuticle (pi. 4, fig. 3). In the same way it approaches the rectum, and ascends with it to the caecum, on the sides of which it blends with the cuticle and becomes lost upon it. That this layer belongs to the ectoplasm rather than to the entoplasm is determined from the following facts. (1) AVhen these Protozoa are surrounded by an irritating chemical medium, or by disturbing physi- cal conditions, the entoplasm frequently flows out through the gullet, but in these cases the boundary layer always remains with the ecto- plasm. (2) While D. ecandatum is feeding the entoplasm is in con- stant, definite motion. The boundary layer takes no part in this movement but remains stationary with the ectoplasm. (3) After feed- ing, the entoplasm shows many changes, which, however, are not dis- tinguishable in either the boundary layer or in the ectoplasm. ENTOPLASM The entoplasm (ent., pi. 4, figs. 3-5; pi. 7, figs. 25-33), shows under the most powerful lenses (2600-3400 magnifications) no definite 1914 J Sharp: Diplodinium ecaudatum 69 structures with the exception of a single, or possibly a double, layer of alveoli adjacent to the boundary layer, but appears to be a more or less homogeneous mass containing food particles surrounded by food vacuoles. The entoplasm is entirely surrounded by the sack-like boundary layer, is kept from flowing out through the mouth, under normal conditions by the constriction of the oesophageal walls, and from flowing out through the anus by the boundary layer covering the rectum and caecum. Scattered throughout the entoplasm are food particles surrounded always by a food vacuole (fd. vac., pi. 4, figs. 3-5 ; pi. 6, figs. 14-19 ; pi. 7, figs. 25-33) . Eberlein (1895, p. 244) describes, in the case of Ophryoscolex inermis, "Waben" of the entoplasm, which group themselves around and enclose in a regular manner the bits of food which the animal has taken in. No such structure is present in D. ecaudatum, but it must be noted that, while in the case of 0. inermis the food consists almost wholly of cellulose fragments, in the case of D. ecaudatum and all of its forms the food consists almost entirely of bacteria. Evidence of a vegetable food ingestion in D. ecaudatum appears only in those cases in which the host (ox) has been fed just before slaughtering. After the ox has fed on alfalfa hay the entoplasm of D. ecaudatum contains, for two or three hours only, green chloroplastids, as well as the bac- teria, but no cellulose fragments. In living animals, during feeding periods, a definite streaming of the entoplasm is discernible. This streaming of the entoplasm will be described under observations on the living animals. ORGANS OF THE BODY Macronucleus. The macronucleus (mac., pi. 3, figs. 1, 2; pi. 4, figs. 3, 5; pi. 7, figs. 29, 33), is situated in the ectoplasm between the boundary layer and the cuticle on the right dorsal side of the body. In general the macronucleus has a rather constant size, shape, and position within the animal. Its anterior end is the larger, is some- what curved, and the longitudinal axis is slightly twisted from left to right. The size of the macronucleus is deserving of special mention. Its longitudinal dimension is equal to about five-eighths of the entire length of the body, and its transverse diameter throughout the greater part of its length is about one-fourth that of the cross-section of the body. Its anterior end is large, bluntly rounded off and situated just internal to and just below the base of the right extremity of the dorsal membranelle zone. It curves slightly dorsad and towards the posterior 70 University of California Publications in Zoology [VOL. 13 end. The diameter remains fairly constant until just below the mid- portion, whence it gradually diminishes, to end in a blunt point just below and to the right of the posterior contractile vacuole (post. c. v., pi. 4, fig. 3). At the mid-part on the dorsal side of the macronucleus is a shallow depression in which the micronucleus is held. The macro- nucleus is entirely surrounded by a definite nuclear membrane. This membrane conforms to the shape of the macronucleus and forms for it a well-defined capsule. Between the membrane and the macro- nucleus proper is a clear space in which no structures are visible even under the most powerful magnifications (2800 to 3400 diameters). The nuclear membrane is enclosed on the outside by ectoplasm, the reticular structure of which is somewhat more regularly arranged over the membrane (see pi. 4, figs. 3, 5). The position of the macro- nucleus within the body is absolutely fixed. No evidence of mobility such as has been suggested for the macronucleus of Dasytricha by Schuberg (1888), or of changes in its position as suggested for some species of Opkryoscolex by Eberlein (1895), and Giinther (1899), has been found in D. ecandatum. This absolute fixation of the macro- nucleus in D. ecaudatum is undoubtedly brought about through its close relation to the skeletal structure, to the right edge of which it appears to be firmly connected (pi. 3, fig. 1; pi. 4, fig. 5; pi. 7, figs. 26-30). The skeletal structure, therefore, in this place functions as a supporting structure for the macronucleus. The microphotographs (figs. 27, 28) show this especially well. It is desired to call attention in this place to the fact that a careful study of the best preparations gives no evidence which tends to show that there is any direct com- munication between the oesophageal wall and the nuclear membrane or that the macronucleus has any special supporting structures other than the right edge of the skeletal structure and the surrounding boundary layer and ectoplasm. The microphotographs (pi. 6, fig. 13; pi. 7, figs. 26-30, 33) might indicate otherwise, but this is because the sections were cut somewhat obliquely and hence there is a slight super- imposition of some of the structures. Hence it can be emphatically stated that structures homologous with the "Kernstiele" of Schuberg (1888), Eberlein (1895), and Giinther (1899) are not present in D. ecaudatum. The macronucleus itself is distinctly granular. After iron-alum haematoxylin stain these granules stand out clearly and distinctly and may, in thin cross-sections, be counted (mac., pi. 4, figs. 3 and 5). The estimated total number (based on examination of three specimens) was approximately 25,000 granules. Interesting changes 1914] Sharp: Diplodinium ecaudatum 71 in the macronucleus in its preparation for and during division will be described in a subsequent paper. Micronucleus. The micronucleus may be clearly distinguished even in the living non-stained animals. Here it appears, by trans- mitted light, as a shining little body situated in a depression on the dorsal side of the macronucleus about midway between the anterior and posterior extremities. In the living condition it appears finely granular in structure and refracts light more strongly than does the macronucleus. In the stained preparations the micronucleus (mic., pi. 3, figs. 1, 2; pi. 4, figs. 3, 5; pi. 7, figs. 29, 33), is seen to be oblately spheroidal in shape, somewhat flattened dorso-ventrally, with its long axis placed longitudinally. The micronucleus, like the macronucleus, is encapsuled by a clear, well-defined membrane, between which and the nuclear substance is a structureless, clear space. The capsule of the micronucleus is firmly held in position by suspensory fibers (susp. f., pi. 4, fig. 3), which appear to arise from the nuclear membrane of the macronucleus above and below the depression for the micronucleus and its capsule. After iron-alum haematoxylin stain the micronucleus substance shows blue-black. After Mallory's connective tissue stain the micronucleus shows bright red. This is especially interesting when considered in connection with the fact that the micronucleus, together with some peculiar masses and fibers which are to be described later, takes this stain in the same intensity and they are the only structures which do show this peculiar, bright red. Since these peculiar masses and fibers are intimately connected with the organs of locomotion, and since they and the micronucleus invariably take the same stains in the same intensity it would seem that some relationship (chemical at least) must exist between the motor apparatus and the micronucleus, in which case the micronucleus might properly be termed a kineto- nucleus. This belief is strengthened by the fact that many cases have been observed by me in Diplodinium in which the macronucleus was engaged in dividing up into definite chromosomes, a function which in ciliates, except in patina, is generally supposed to be limited ex- clusively to the micronucleus. In other words, it appears that the macronucleus here may be analogous to the trophonucleus of try- panosomes and so the question is raised as to whether or not it might be held to contain the so-called "generative chromatin." This phe- nomenon will receive further consideration in my paper on conjuga- tion and reproduction in Diplodinium ecaudatum. The granules of the macronucleus are too small and too numerous to permit even an 72 University of California Publications in Zoology [VOL. 13 attempt at an approximation of their number. Changes in the micro- nucleus in preparation for and during division will also be described in a subsequent paper. ORGANS OF LOCOMOTION It has already been pointed out that one of the main characteristics of the genus Diplodinium is the presence of what Schuberg (1888 and 1891), Eberlein (1895), and Giinther (1899 and 1900) have termed a second, or dorsal, membranelle zone, and what Fiorentini (1889) calls a transverse crown of cilia. In Diplodinium ecaudatum the cilia of both the dorsal and adoral zones are grouped to form clumps or tufts of cilia. Normally all of the cilia composing each tuft adhere closely, just as do the hairs of an ordinary camel's-hair paint-brush when moistened so as to form a flexible pencil. The composition of these brush-like tufts of cilia will be more thoroughly considered below, but here attention is called to the structural difference between these ciliary brushes and true membranelles, i.e., "flapping or swinging membranes formed by fusion of two or more transverse rows of cilia implanted side by side and adhering to form a flat membrane" (Minchin, 1912, p. 55). Each ciliary brush is a perfectly definite unit, both struc- turally and functionally, and although structurally these ciliary brushes resemble cirri more closely than they do membranelles, still from the point of view of homology it seems best to retain the designa- tion membranelle, and hence in this paper each such tuft of cilia is referred to as a membranelle. Throughout the Ophryoscolecidae, as a matter of fact, these membranelles have the form of brushes and may be designated as brush or penicillate membranelles in contradistinction to those found elsewhere, as for example in the Vorticellidae, in which the cilia of the membranelle are arranged in the form of a plat$ of one or two lines of cilia fused in one locomotor unit of flattened type. Those of the dorsal region are termed dorsal membranelles and those of the adoral region, adoral membranelles. The complete row of dorsal membranelles, together with the inner and outer dorsal lips and intervening furrows, is termed the dorsal membranelle zone and likewise the row of adoral membranelles with its corresponding inner and outer adoral lips and furrows is designated as the adoral membranelle zone. The dorsal and adoral membranelle zones form the locomotor apparatus of the animal and since these two zones are not continuous the locomotor apparatus may be said to consist of two component parts, a dorsal locomotor apparatus or dorsal membranelle 1914] Sharp: Diplodinium ecaudatum 73 zone, which is locomotor only in function, and an adoral locomotor apparatus or adoral membranelle zone, which is both locomotor and nutritive in function. Considered from the point of view of its probable evolution, this adoral row of membranelles was undoubtedly primarily nutritive in function, but owing to developmental changes which have probably taken place, combined with the increasing neces- sity of greater speed or possibly the decreasing necessity of food- getting, this nutritive function has gradually been given over to that of locomotion, so that at the present time the function of this adoral row of membranelles may be regarded as primarily locomotor and secondarily nutritive. This conclusion has been reached after due consideration of the morphological position of the adoral membranelles combined with careful observations made of the living, active animals. Such observations invariably lead one to the conclusion that the adoral row of membranelles functions mainly as an organ of locomotion. And in this connection it is interesting to note that, of all the different species of the genus Diplodinium, the species ecaudatum is provided with the most powerful and the most complicated organs of locomotion. Dorsal locomotor apparatus. The dorsal locomotor apparatus, or dorsal membranelle zone, is placed transversely at the very anterior extremity of the dorsal surface of the body and consists of an outer and inner dorsal lip (o. d. lip and i. d. lip, figs. B, C ; pi. 4, figs. 3-4), an outer and inner dorsal furrow (o. d. fur. and i. d. fur., figs. B, C; pi. 4, figs 3, 4), and a row of twenty-six membranelles (d. m., figs. B, C ; pi. 4, figs. 3, 4; pi. 7, figs. 23-25). Beginning at a point just dorsal to and slightly posterior to the dorsal extremity of the base of the left skeletal area (I. sk. a., pi. 3, fig. 2) the dorsal row of membranelles extends transversely around the anterior extremity of the dorsal surface to the right side, where it makes a short curve anteriorly to end just dorsal to the dorsal ex- tremity of the base of the right skeletal area (r. sk. a., pi. 3, fig. 1 ; pi. 4, fig. 4) . The bases of these membranelles extend down into an inner dorsal furrow (i. d. fur., fig. B) and are there enclosed by a fold of the ectoplasm and cuticle which in cross-section resembles the human lower lip and has therefore been designated as the inner dorsal lip (i. d. lip, fig. B; pi. 4, figs. 3, 4). Outside of the inner dorsal lip is a second fold of ectoplasm and cuticle, not so high as the inner lip but much thicker and more substantial. This outer fold also resembles a lip and is therefore termed the outer dorsal lip (o. d. lip, fig. B ; pi. 4, figs. 3# 4) . The outer surface of the outer dorsal lip is continuous 74 University of California Publications in Zoology [VOL. 13 with the surface of the body and the bases and extremities of both lips are continuous with the ectoplasm of the body. Internal to the inner lip and between the inner and outer lips are deep furrows termed respectively the inner and outer dorsal furrows (i. d. fur. and o. d. fur., fig. B; pi. 4, figs. 3, 4). In the inner furrow, which is bounded ex- ternally by the inner dorsal lip and internally by a portion of the an- terior end of the body to be described later as the dorsal disk (d. disk, fig. B; pi. 4, fig. 3) are to be found the bases of the dorsal mem- branelles. These dorsal penicillate membranelles, strong and vigorous in life, measure from one-twelfth to one-eighth of the entire length of the body and number regularly twenty-six or twenty-seven. Each membranelle consists of from fifty to seventy-five cilia and has the appearance of a very long, fine camel 's-hair brush. It is almost certain that a part of the ciliary processes composing each membranelle springs from the ectoplasm of the dorsal disk. These origins are termed anterior ciliary roots (ant. cil. r., pi. 4, fig. 3). The larger number of these ciliary processes have their origin in the ectoplasm posterior to the outer dorsal groove, however, and these are designated as the posterior ciliary roots (post. cil. r., pi. 4, fig. 3). In heavily stained sections there appear at the junction of the anterior and posterior roots slight enlargements, which by iron-alum haematoxylin are stained more intensely than the root strands and by Mallory's connective tissue stain show the peculiar bright red which is characteristic of nerves stained by this method in amphibian tissue. In the living condition the cilia of each membranelle cling together as do the hairs of a wet camel's-hair brush, but in the fixed specimens which have been washed in alcohol these cilia may fluff out as do the hairs in a dry brush. In many cases the cilia composing each brush appear to be twisted spirally like the stripes on a barber's pole. The motion of these ciliary brushes or penicillate membranelles will be discussed later under observations on living material. Adoral locomotor apparatus. The adoral locomotor apparatus, or adoral membranelle zone (ador.m., figs. A, B, C ; ador.m.z., pi. 3, fig. 2; pi. 7, figs. 20-22, 33) is much the more complicated of the two zones. This apparatus, in so far as I am able to determine, has never been correctly figured nor described for the species ecaudatum. Briefly, the adoral locomotor apparatus consists of two rows or circles of cilia, an outer circle composed of larger, heavier membranelles along which the wave of contraction passes from left to right, and an inner circlet of smaller, finer cilia along which the waves pass from 1914] Sharp: Diplodinium ecaudatum 75 right to left. Thus these two circles, although continuous, run in opposite directions. In general the arrangement of the inner and outer lips and the inner and outer furrows is the same as in the case of the dorsal zone. The following differences, however, are to be noted. The outer lip (o. ador. Up, figs. A, B; pi. 3, figs. 1, 2) forms an almost complete circle, the plane of which is not horizontal but inclined pos- teriorly on the left side, and is therefore much higher on the right side. This lip reminds one of a stiff collar which is a little higher D. ador. i. ador. lip J ' o. ador. lip Fig. A. Anterior view of the oral region of Diplodinium ecaudatum to show peculiar recurved arrangement of adoral membranelles and oral cilia. X 1150. ador. m., adoral membranelle ; D., dorsal side; i. ador. lip, inner adoral lip; o. ador. lip, outer adoral lip; or., oral opening or cytostome; or. cil., oral eilia; or. disk, oral disk; V., ventral side; X, beginning of the adoral row of membranelles; Y, the point at which the adoral row of membranelles becomes recurved upon itself to form the row of oral cilia. on one side than on the other and the ends of which do not quite meet in front the "front" in this case being on the left dorsal side, i.e., just- above the left extremity of the dorsal membranelle zone. The inner adoral lip (i. ador. lip, fig. A; pi. 3, figs. 1, 2) is at first invisible, but gradually rises above the edge of the outer lip on the ventral side, and on the right and dorsal sides shows prominently above the outer lip. This inner lip might also be likened to a stiff collar, but in this case the two ends overlap, the terminal end passing internal to the origin,, where it may end gradually and indistinctly as in Figure A, 76 University of California Publications in Zoology [VOL. 13 or it may be continuous with the oral disk as in figure 2, plate 3, or it may end abruptly as in figures 6 and 7, plate 5, all depending upon the state of contraction and retraction or expansion and extrusion of the oral region. The adoral membranelles and the oral tufts of cilia are very different, both in their composition and in their arrangement. Starting from a point (x, fig. A) slightly anterior. and ventral to the left extremity of the dorsal row of membranelles, the row of adoral membranelles (ador. m., figs. A, B) circles at first ventrally and slightly posteriorly, then to the right and slightly anteriorly. Still ascending, it next curves dorsally and then to the left, reaching its highest level as it crosses the sagittal plane. Still curving to the left and ventrally, it gradually descends to a more posterior level, where it occupies a position internal and ventral to the starting-point and on a plane about midway between its highest and lowest levels. At this point (y, fig. A) the adoral zone proper may be said to terminate, but the row of membranelles turns suddenly inward, again ascends to a higher level, then recurving upon itself begins the secondary inner circlet, which runs parallel with, but in a direction exactly opposite to, that of the outer row of adoral membranelles. At the sudden turn (y, fig. A) the membranelles become much shorter and finer, lose their brushlike construction, become somewhat flattened in appearance, and may now be described as a row of less distinct groups of large cilia surrounding the mouth or oral opening, viz., oral cilia (or. cil., figs. A, B, C; pi. 3, figs. 1, 2; pi. 4, fig. 3). The membranelles of the adoral zone resemble those of the dorsal zone with the exception that they are neither so long nor do they individually contain so many ciliary processes. There are from thirty to thirty-six of these membranelles, each consisting of from forty to fifty separate cilia. Their bases are situated in the ectoplasm immediately posterior to the inner adoral furrow. Here also, as in the case of the dorsal membranelles, each membrarielle seem to be composed of two sets of roots, which, however, in this case must be designated as internal and external roots. The internal roots take their origin from the ectoplasm in the region of the boundary layer, and the external roots take their origin from the ectoplasm which lies close to the outer wall of the body. The oral cilia are too short and too fine and are located too centrally to be of much service as organs of locomotion, but since they are a direct con- tinuation of the adoral row of membranelles they will be described in this place. 1914] Sharp: Diplodinium ecaudatum 77 Oral cilia. The oral cilia (or. cil., figs. A, B, C; pi. 3, fig. 2; pi. 4, fig. 3) are not only exceedingly fine but are also exceedingly thick, thus making it difficult to arrive at any very definite conclusion regard- ing their size, number or arrangement. It is certain, however, that they are in some way connected with the same motor apparatus as is the adoral row of membranelles, of which they seem to be the direct continuation. In life they appear as minute tufts which are in almost constant motion. They completely surround the oral opening and extend down into the oesophagus for a short distance. They appear to have only one set of roots, which lie close to the oesophageal wall and end in or near the circumoesophageal ring (dr. oes. ring, figs. B, C; pi. 4, fig. 3; pi. 6, fig. 15; pi. 7, fig. 33). ORGANS OF FOOD-TAKING The organs of food-taking, ectoplasmic in nature, embrace a cyt- ostome, mouth, or oral opening (or., figs. A, B, C; pi. 7, figs. 20, 21), oral cilia (or. cil.}, oral disk (or. disk), to some extent the adoral mem- branelles (ador. m.), and an oesophagus (oes.). Cytostome. The cytostome, mouth or oral opening (or., figs. A, B; pi. 4, fig. 3). is an elliptical aperture almost entirely surrounded by the oral cilia and located at the very anterior extremity of the body, close to the ventral side and inclined somewhat ventrally and to the left. The structure of the oral cilia and adoral membranelles has been described above; their function we shall refer to later. The mouth opens directly into an oesophagus. Oesophagus. The oesophagus (oes., fig. B; pi. 4, figs. 3-5; pi. 7. figs. 20-33) extends, as a closed tube, from the mouth opening to a point slightly below the level of the anterior extremity of the macro- nucleus (pi. 7, fig. 27). At this level the inner wall disappears and the oesophagus descends as an open or one-sided tube to the extreme posterior limit of the entoplasm (pi. 7, figs. 31, 32). At the oral end it is exceedingly small and rather irregularly elliptical in cross-section, with the long axis of the ellipse extending transversely from right to left. It gradually grows larger as it descends posteriorly through the ectoplasm into the entoplasm, and swings obliquely to the right in such a manner that its ventral or outer wall, which is much the thicker of the two walls, approaches and finally comes to lie next to the right side of the body (oes., pi. 4, figs. 4, 5; pi. 7, figs. 23-30). Soon after entering the entoplasm the dorsal or inner wall, which, owing to the obliojue descent of the oesophagus, is now turned toward the left, dis- 78 University of California Publications in Zoology [VOL. 13 appears, and so leaves the left or internal side of the oesophagus in free communication with the entoplasm (oes., pi. 4, figs. 3-5; pi. 7, figs. 28-30). The walls of the oesophagus show, according to Eberlein (1895, pp. 245 and 255) three layers ("Gewebsschichten") : (1) an inner layer which is turned toward the lumen and \vhich is a thin continuation of the cuticle; (2) the middle layer, which is formed by the ectoplasm and is characterized by a closer and more regular formation of the reticulum; and (3) an outer layer which is formed by the boundary layer previously described. The definite arrangement of the " Gewebesschichten " which Eberlein (1895) describes for the "Schlund" of Ophryoscolex inermis and refers to as being the same for Diplodinium maggii (p. 255), I am sorry to say, I cannot confirm for D. ecaudatum. The oesophagus, as is shown in longitudinal sec- tions, does not come into contact with this third or boundary layer until it has descended some distance through the ectoplasm (oes., pi. 4, fig. 3; pi. 6, fig. 15). According to my observations, the walls of the oesophagus are composed (1) of a thin cuticular continuation from the cuticle of the body (pi. 4, fig. 3); (2) of definite longi- tudinal strands (oes. retr. sir., fig. B; pi. 4, figs. 3-5; pi. 7, figs. 29, 33), which, since they are attached posteriorly to the fused ventral and right skeletal structures, w r ould seem, both from their structural connections and their contractile nature, to function essen- tially as retractor strands; (3) of certain oesophageal fibers (oes. f., fig. B; pi. 4, fig. 4, and pi. 7, figs. 23-25), which are deemed neural in function and which will be described later; and (4) of the ground substance or matrix of the oesophagus, ectoplasmic in nature and to which or in which the above structures are attached. The number of these oesophageal retractor strands is so large, their extent so great, their arrangement so complicated, and their func- tion so important that it seems best to give them a further and more detailed consideration. Several estimations indicate that there are from 100 to 150 of these retractor strands in the oesophageal walls. They appear in cross sections (oes. retr. sir., pi. 4, fig. 5) as delicate radial lines joining the inner and outer lamellae of the oesophageal wall and in longitudinal sections as little ribbon-like bands which extend from the oral opening to the extreme posterior limit of the ento- plasm. In fact it seems probable that a number of these strands end in the vicinity of the anus. A satisfactory analysis of their arrange- ment has been made possible through a comparative study of the oesophageal retractor strands in Diplodinium bursa, which is a some- Sharp: Diplodinium ecaudatum 79 what larger form and one, moreover, in which these oesophageal struc- tures are especially clear. As the ventral or external wall of the oesophagus comes to lie against the skeletal structure (pi. 7, figs. 28, 29) it is separated from it only by the boundary layer, and, as the inner wall disappears, or, more accurately speaking, separates along its mid-line and its sides also become flattened against the boundary layer, it becomes more and more difficult to distinguish the latter from the oesophageal wall. As this oesophageal wall, if such it may still be called, approaches the posterior end it comes to extend over more and more of the circum- ference of the boundary layer until (compare figures 29 and 32, plate 7) at the posterior extremity it seems to completely encircle the ento- plasm and is distinct from the boundary layer only in the region of the rectum which seems to pass down between the two (pi. 7, figs. 31, 32). The manner in which the rectal sheath is formed will be considered below. It will suffice here to note that it contains fibrillae (red. f., pi. 4, fig. 3) which appear to be of the same origin and take the iron-alum haematoxylin stain in the same intensity as do the oesophageal rectractor strands. At the extreme posterior end of the entoplasm which is just dorsal to the anal opening all these fibrillae or retractor strands meet in a point which is probably the region of final constriction at the time of division. As noted above, the oeso- phageal wall comes to lie against the boundary layer and the skeletal structure at about the middle of the body, i.e., below the fusion of the right, ventral, and left skeletal structures. At just what point or points the oesophagus is attached to the skeletal structures has so far defied an exact determination, but that such attachments are made seems altogether certain. A study of the retracted forms (fig. D) justifies this belief and also furnishes evidence which tends to prove that the function of these oesophageal strands is one of retraction. This conception would at least explain how the whole oral and adoral region is pulled into the body when the organism is irritated. Either such a complicated oesophageal structure does not exist in the heretofore described ciliates from the horse and the ruminant, or it has been overlooked by previous investigators. We have seen that in the normal, active condition a large portion of the anterior end of the body is taken up with the organs of locomotion and nutrition. Three other structures, however, which are also situated at the>anterior end of the animal, deserve description. To these struc- 80 University of California Publications in Zoology [VOL. 13 tures we have given the names dorsal disk, operculum, and oral disk. Dorsal disk. The dorsal disk (d. disk, fig. B; pi. 3, figs. 1, 2; pi. 6, fig. 15; pi. 7, fig. 33), located between the dorsal membranelle zone and the operculum (op., fig. B), is an exceedingly elastic structure. Normally it has the shape of a spherical wedge, resembling one of the carpels of an orange. The equator corresponds in position to a line drawn from the middle of the operculum (op.} to the middle of the dorsal membranelle zone, and the axis, or diameter, corresponds to a line connecting the right and left extremities of the inner dorsal lip (i. d. lip). The size and shape of the dorsal disk depend entirely upon the degree of contraction or relaxation of the operculum and dorsal membranelle zone. Operculum. The operculum (op., fig. B; pi. 3, figs. 1-2; pi. 7, fig. 33), also elastic and contractile in its nature, functions as a protective structure to the organs of locomotion and nutrition, when these are retracted. It is located between the dorsal disk and the dorsal curve of the outer adoral lip and is attached to the bases of the right and left skeletal areas respectively by its right and left extremities, which thus serve as skeletal attachments. The size and shape of the operculum depend largely upon its state of contraction. This structure will be further considered under observations on the living animals. Oral disk. The oral disk (or. disk, figs. A, B; pi. 4, fig. 3; pi. 7, figs. 20, 21), resembles the dorsal disk only slightly. It is circular in form when viewed from above and dome-shaped when viewed from the side. The oral disk almost completely surrounds the mouth when that organ is open and entirely surrounds -it when it is closed. It is sur- rounded externally by the adoral membranelles and bounded internally by the oral cilia which separate it from the mouth opening. The oral disk is thicker on the right side than on the left, thus throwing the mouth opening rather to the left of the center of the disk. Tfie func- tion of this disk is to support the oral cilia, give shape to the mouth opening, and act as a valve to close the oral aperture. ORGANS OF DEFECATION The organs of defecation are the caecum (caec.), rectum (rect.), and anus (an.). Caecum. The caecum (caec., pi. 4, fig. 3; pi. 7, fig. 33), is situated in the posterior one-fourth of the body close to the ventral wall and slightly to the left of the median plane. The size and shape of the caecum depend entirely upon the amount of excreta which it contains. 1914] Sharp: Diplodinium ecaudatum 81 When empty the caecum cannot be distinguished in the living animals, but when well filled it may be discerned as a round or balloon-shaped structure whose walls become more and more definite as they approach the rectum. We noted (p. 68) under the description of the boundary layer that in the stained specimens this layer, together with its asso- ciated alveolar layers, is lost upon the sides of the caecum. The caecum empties directly into the rectum. Rectum. The rectum (rect., pi. 4, fig. 3; pi. 6, figs. 16-19; pi. 7, figs. 31-33), short, but well defined, elliptical in cross-section, leads from the caecum to the cytopyge or anal opening (an., pi. 4, fig. 3). In the case of the rectum the three layers, i.e., (1) cuticular, (2) alveolar, and (3) boundary layer, may be seen. It is intensely interesting to note, just at this point, that during the process of organ formation, in the predivision stage, the new oral cilia form in a little cavity which is situated in the ectoplasm between the ventral edge of the oesophageal wall and the ventral surface of body at about the level of the posterior contractile vacuole. As development progresses this ventral edge of the oesophageal wall is forced more and more toward the central axis of the body. When division is completed the little pocket in which the oral region of the posterior animal was developed now becomes the caecum of the an- terior animal, and the right and dorsal wall of this caecum is formed by that part of the ventral edge of the oesophageal wall which was pushed in by the developing oral cilia as described above. The ventral wall is formed by the boundary layer. This becomes all the more interesting when, as will be described under observations on the living animals, it will be noted that the internal posterior current (current No. 3) of the entoplasm is directed obliquely from the left ventral side above towards the right ventral side below, i.e., towards the most open side of the caecum. Further it is to be noted that lower down the whole dorsal wall of the rectum is formed by what was once the ventral edge of the oesophagus, hence this wall is richly supplied with fibrillae which, moreover, before division were oesophageal retractor strands. That these strands or fibrillae (rect. f., pi. 4, fig. 3) have undergone a certain amount of atrophy or degeneration owing to disuse, or possibly a change in function is indicated by the fact that with Mallory's modified connective tissue stain they no longer stain as distinctly and as intensely as the oesophageal fibers from which they were derived. After iron haematoxylin, however, they are quite distinct. Whether these strands still retain some of their retractile 82 University of California Publications in Zoology [VOL. 13 nature and function and assist in the process of defecation, or whether they serve merely as supporting structures to the rectum it is not yet possible for me to say. Anal opening. The anal opening (an., pi. 4, fig. 3; pi. 6, figs. 18, 19; pi. 7, figs. 32, 33), which is a mere slit, is located at the pos- terior extremity of the body close to the ventral side. The caecum, rectum, and anus have to do with the discharge of the more solid particles, i.e., the undigested remnants of the bacteria upon which the animal feeds. The fluid excreta are gotten rid of by means of the contractile vacuoles. ORGANS OF EXCRETION These are the contractile vacuoles (ant. c. v. and post. c. v., pi. 4, fig. 3; pi. 6, figs. 15, 17; pi. 7, figs. 28, 30), which are also ectoplasmic structures. They are two in number and anterior and posterior in location. The anterior contractile vacuole (ant. c. v., pi. 4, fig. 3), is located close to the dorsal wall in the median sagittal plane about mid- way between the dorsal membranelle zone and the micronucleus and just to the left of the macronucleus. The posterior contractile vacuole (post. c. v., pi. 4, fig. 3), is similarly located in the posterior half of the body close to the mid-dorsal wall, half way between the micronucleus and the posterior end of the body and just to the left of the macronu- cleus. Each vacuole when distended is ellipsoidal in shape, slightly larger than the micronucleus, and is surrounded by a slightly differ- entiated ectoplasm previously described. Each vacuole opens on the dorsal surface through a small canaliculus and a minute pore (ant. c. v., pi. 4, fig. 3). For the species D. ecaudatum these contractile vacuoles have proved almost absolutely constant both in number and in position. In fact, out of the large number of individuals of this specias which have been observed by me during the past three years, not more than a dozen have shown the single vacuole as figured by Fiorentini (1889, pp. 15, 16, pi. 3, figs. 1-2) and by Eberlein (1895, pp. 262, 263, pi. 18, figs. 18-19). NEUROMOTOR APPARATUS We now come to the description of what is believed to be the most interesting structure in the anatomy of this organism, a structure so intimately and so peculiarly connected with the motile parts of the body that its function as a neuromotor apparatus is strongly indicated. 1914] Sharp: Diplodinium ecaudatum 83 Here again attention is called to the fact that the literature on these animals is devoid of any suggestion of the structure which is here described. The possibility of this structure functioning as a motor apparatus or even possibly as a neuromotor apparatus is suggested and for purposes of description the designation neuromotor apparatus will be used, and its constituent parts will be described as a motorium or motor mass (m. m., figs. B, C; pi. 4, fig. 3; pi. 6, figs. 14-16; pi. 7, figs. 21-24, 33), a dorsal motor strand (d. m. sir.), a ventral motor strand (v. m. sir.), opercular fibers (op. /.), an adoral lip strand (ador. lip str.), oesophageal fibers (oes. /.), and a circumoesophageal ring (dr. oes. ring). The term motorium, as applied here, is used in its anatomical or neurological sense, i.e., the common center of motor influences. It may be, however, that we have here a condition in which nervous, contractile, and supporting elements are in so primitive a stage of evolution as to be incapable of separation into purely nervous, purely contractile, or purely supporting structures. The structural conditions and the observations on the living animal in activity, how- ever, suggest emphasis upon the neural rather than upon the con- tractile, or supporting nature of these structures, though not excluding the latter two. They are probably comparable to the simple fibres of Stentor figured by Neresheimer (1903) and regarded by him as ' ' neurophanes. ' ' Motorium. The motorium or motor mass (m. m., figs. B, C ; pi. 4, fig. 3; pi. 6, figs. 14-16; pi. 7, fig. 33), is a very small mass of chem- ically differentiated tissue located rather deeply in the ectoplasm, just above the base of the left skeletal area (1. sk. a., figs. B, C ; pi. 4, fig. 3) and between the left extremities of the dorsal and adoral membranelle zones. This motor mass was first noted in sections stained with my modification of Mallory's connective tissue stain. It was discovered that here, in the region just described, was a mass of tissue which had stained rather intensely and showed by transmitted light the same bright red color which was noted in the case of the micronucleus. Further investigation along this line revealed the fact that not only was this mass constant but (1) that it was connected dorsally, by means of a delicate strand, i.e., dorsal motor strand (d. m. str., figs. B, C; pi. 4, fig. 4), with the bases of the dorsal membranelles, also a branch strand ran along the base of the inner dorsal lip, i.e., the dorsal lip strand (d. Up str.) ; (2) that a fine strand, the ventral motor strand (v. m. str.), ran from it to the bases of the adoral membranelles, alsoxthat a branch strand left this ventral motor strand and passed 84 University of California Publications in Zoology [VOL. 13 along the base of the inner adoral lip, the adoral lip strand (ador. lip str.), and that many well-defined fibers passed from it, following the contour of the opereulum towards the right to become lost in the im- mediate vicinity of the base of the right skeletal structure. These are the opercular fibers (op. /.). Most interesting of all, however, was the apparently perfectly definite connection with a ring of substance surrounding the oesophagus at just about the level of the outer adoral furrow. This ring, which is designated as the circumoesophageal ring (circ. oes. ring, figs. B. C; pi. 4, fig. 3; pi. 6, figs. 14-16; pi. 7, fig. ador. m. - r i. ador. fur. ~ '. ador. lip } r o. ador. fur. ! o. ador. lip tn. m. Fig. B. Diplodinium ecaudatum. Semi-diagrammatic representation of an- terior half to show arrangement and relations of neuromotor apparatus. X 1150. ador. lip str., adoral lip strand; ador. m., adoral membranelles ; ant. c. v., anterior contractile vacuole; dr. oes. r., circumoesophageal ring; D., dorsal surface; d. dislc, dorsal disk; d. m., dorsal membranelles; d. m. str., dorsal motor strand; i. ador. Up, inner adoral lip; i. d. fur., inner dorsal furrow; i. d. lip, inner dorsal lip; 1. sk. a., left skeletal area; mac., macronucleus; mic., micronucleus; m. -m., motor mass or motorium; o. ador. -fur., outer adoral furrow; o. ador. lip, outer adoral lip; o. d. fur., outer dorsal furrow; o. d. Up, outer dorsal lip; oes., oesophagus; oes. f., oesophageal fibers (neural fibers) ; oes. retr. str., oesophasreal retractor strands (contractile strands); op., opereulum; op. f., opercular fibers; or., oral opening (cytostome; or. cil., oral cilia; or. disk, oral disk; sk. lam., skeletal laminae; V., ventral surface; v. m. str., ventral motor strand; v. sk. a., ventral skeletal area. 1914] Sharp: Diplodinium ecaudatum 85 33) as well as all of the fibers described as leaving the motorium, showed in all regions the same bright red color. Other fibers also staining bright red are found in the oesophageal walls. These are called oesophageal fibers (oes. f., fig. B; pi. 4, figs. 3, 4), but thus far it has not been definitely decided whether they take their origin from the motorium or directly from the circumoesophageal ring, probably the latter, however. It was noted in the description of the oral cilia that the root-fibers of these cilia end either in, or very close to, this D. d. m. o. d. lip - t d. m. sir. r. sk. a.- ador. m.*-~- dr. oes. r.< > j dr. oes. r. sir -> V. . a. 1 - - - ador. lip. sir. Fig. C. Diplodinium ecaudatum. Diagrammatic representation of the moto- rium or neuromotor mass and its neuromotor strands seen from the anterior end. X 1150. ador. lip sir., adoral lip strand; ador. m., adoral membranelles ; dr. oes., circumoesophageal ring; dr. oes. r. sir., cireumoesophageal ring strand; D., dorsal surface; d. lip str., dorsal lip strand; d. m., dorsal membranelles; d. m. sir., dorsal motor strand; i. d. fur., inner dorsal furrow; i. d. lip, inner dorsal lip; 1. sk. a., left skeletal area; m. m., motorium or neuromotor mass; o. d. fiir., outer dorsal furrow; o. d. lip, outer dorsal lip; op. f., opercular fibers; or., oral opening or cytostome; r. sic. a., right skeletal area; V., ventral side; v. m. str., ventral motor strand. circumoesophageal ring. These root-fibers also show the bright red color characteristic of w r hat is here called the neuromotor apparatus. In particularly well-stained whole mounts (stained with iron-alum haematoxylin) the motorium, the dorsal motor strand, the ventral motor strand, and the oesophageal ring show r very clearly Special attention is here called to the microphotographs of this apparatus (pi. 6, figs. 14-16; pi. 7, figs. 21-26, 33). 86 University of California Publications in Zoology [VOL. 13 The facts which indicate a co-ordinating (i.e., nervous) function rather than a contractile or a supporting function for the above de- scribed structure may be summed up as follows : 1. The size, shape, position, and absence of direct connection with surrounding structures make the possibility of the motorium function- ing as an organ either of contraction or of support seem highly im- probable. For in order to function as an organ of contraction it would necessarily need to have as its attachments on the one hand structures which are fixed, and on the other structures which are movable, or it would need to be located between two structures both of which were to be moved. This however, is not the case, for the motorium seems to have no direct connections with the fixed structures of the body, nor does it lie in the direction of contraction of the oesophagus and oral region, which upon retraction, descend posteriorly into the body (compare figures B and D). Neither would the assign- ment of a supporting function to the motorium be feasible, first because of its relatively diminutive size, second because of its shape which does not conform particularly to that portion of the animal in which it is located, and third because of its location, i.e., it is situated in the anterior flexible and retractile end of the body surrounded by the nonresistant, semifluid ectoplasm. 2. The strands which leave the operculum are likewise not at- tached to fixed structures but lie in the semifluid ectoplasm of the opercular region and in the inner dorsal and inner adoral lips which are both highly mobile. Also it is to be remembered that both the inner dorsal and inner adoral lips which are mobile are well pro- tected and well supported by the outer dorsal and outer adoral lips respectively both of which are fixed and rigid. 3. There is never a translation of the parts in the direction^ of the strands leaving the motorium, but rather in a direction at right angles to the course of the fibers, thus militating against a contractile function for the fibers. An apparent exception to this general statement is to be found in the case of the oesophagus with its oesophageal fibers, but here it is to be noted; first, that these oesophageal fibers end in the vicinity of the micronucleus without any discoverable connection with a fixed structure, and second that the oesophagus is richly sup- plied with another set of fibers (the oesophageal retractor strands), which do not take the red stain, and which are apparently attached to the skeletal structure as described above. 1914 J Sharp: Diplodinium ecaudatum 87 4. Every mobile territory is supplied by strands from the central mass (motorium) and especially are the bases of the membranelles, both dorsal and adoral, well supplied by these fibers. 5. All parts connected by this neuromotor system act in perfect co-ordination. For example, on being surrounded by an irritating medium, mouth, oesophagus, and oral disk are retracted, not in the direction of the motor strands, but posteriorly into the body, the inner adoral and inner dorsal lips are shot forward completely en- closing the adoral and dorsal membranelles (fig. D), the motion of these membranelles and of the oral cilia is suspended, and for the time being, the animal remains in an apparently inactive condition. On being again surrounded by a favorable medium, however, the cilia once more take up their activity, even while retracted within the body. Finally the oral region is protruded, the inner adoral and inner dorsal lips return to their original position, and the animal exhibits all its former liveliness of motion. Not only do the two zones of membranelles act in unison when the animal is swimming freely, but during semi-quiescent periods as when the animal is feeding on a mass of bacteria, the adoral membranelles and oral cilia may be particularly active while the dorsal membranelles remain motionless or only slightly active. Or again only a few of these membranelles may be active while the others are entirely motionless, thus suggesting the innervation of each single membranelle by a separate fiber. Not least in significance for this conception, is the fact that this neuromotor apparatus is located in the most advantageous position possible to function as a center of motor co-ordination in an animal which is exceedingly active, exceedingly sensitive, and exceedingly responsive to external stimuli, and one, moreover, which exhibits a high degree of selective feeding. Also in this connection it is to be noted that that portion of the animal which first comes in contact with new media, viz., the operculum, is the most richly supplied with fibers radiating from the motorium (op. /., figs. B, C, D ; pi. 6, figs. 13-15). A consideration of the location and the distribution of the opercular fibers suggests the interesting question of the possibility of these fibers having a sensory function. Such a view is not advanced to the exclusion of other possible functions. If, however, these opercular fibers do serve as conductors of sensory impulses, then their location in the most anterior, most exposed portion of the body, is one of advantage, especially when the membranelle zones are enclosed. Such S 88 University of California Publications in Zoology [VOL. 13 a view helps to explain not only the number and distribution of the opercular fibers, but also the fact that the oral cilia and the mem- branelles of either or of both zones may be set in motion within the body, i.e., before the protrusion of the oral region and the return of the inner adoral and inner dorsal lips to their normal positions, when- ever the animal is again surrounded by a favorable medium. Such a hypothesis would also help to explain the sometimes sudden retraction of the membranelle zones when the animal bumps into an obstruction or swims into an irritating medium, and upon no other hypothesis can all these phenomena of retraction, protrusion and won- derful co-ordination of membranelles and membranelle zones be so easily and so satisfactorily explained. I 1. ador. lip retr.or.cil. 1 I . ador. m. i. ador. Up , i c. a dor. Up post. oil. r. _^ ant . c. v. oes. retr. str. - mac. mic. Fig. D. Diplodinium ecaudatum. Eetracted form constructed from camera hicida drawings. X 1150. ador. m., adoral membranelles; ant. c. v., anterior contractile vaeuole; bd. I., boundary layer; circ. oes. r., circumoesophageal ring; D., dorsal surface; d. m., dorsal membranelles; i. ador. lip, inner adoral lip; i. d. lip, inner dorsal lip; I. sk. a., left skeletal area; mac., macronucleus ; mic., micro- nucleus; o. ador. lip, outer adoral lip; o. d. lip, outer dorsal lip; oes., oesophagus; oes. f., oesophageal fibers (neural in nature) ; oes. retr. str., oesophageal retractor strands (contractile in nature); op., operculum; op. f., opercular fibers (nervous in nature); or. disk, oral disk (retracted); post. cil. r., posterior ciliary roots; retr. or. cil., retracted oral cilia; sk. lam., skeletal laminae; V., ventral surface. 1914] Sharp: Diplodinium ecaudatum 89 RETRACTED FORM A detailed description of the mechanism of the retraction of the oral zone and the resulting encasement of the adoral and dorsal mem- branelles will be given later in the account of observations on the liv- ing material. At this point will be given only a brief description of figure D, which is a reconstruction of three camera lucida drawings made from three paramedian sagittal sections each five microns thick. The noteworthy points may be summed up as follows: (1) The whole oral region is retracted within the body. (2) The oral cilia come to lie within the oesophagus (retr. or. cil.). (3) The attachment of the boundary layer to the oesophagus is pulled posteriorly to a considerable extent, thus showing that both the point of .attachment of the oseopha- geal retractor strands (oes. retr. sir.) and the region of actual con- traction of these strands is below the point of junction of boundary layer with oesophageal wall (compare with fig. B). (4) The inner adoral lips (i. ador. lips) and the inner dorsal lip (i. d. lip) are ex- tended in such a manner as to become direct continuations of the outer and more rigid adoral and dorsal lips (o. ador. lip and o. d. lip) re- spectively and at the same time to meet in the epioral line in the case of the adoral lips and to meet the dorsal edge of the operculum (op.) in the case of the dorsal lip, thus forming a complete protective encasement for the delicate membranelles. (5) The circumoesopha- geal ring (dr. oes. r.) appears to be somewhat enlarged and stands out even more clearly than in the extended animals. (6) The indi- vidual fibers (oes. /'.) and individual retractor strands (oes. retr. str.) are also very distinct indicating shortening and thickening. This figure does not show the motorium and its connections as that struc- ture lies to the left of the plane here depicted. The cut ends of the opercular fibers (op. /.), however, show very clearly. In the descrip- tion of the neuromotor apparatus it will be remembered, strands were described which passed to and ran along in the inner adoral and inner dorsal lips respectively. These strands showed fairly well in those sections stained with the modified Mallory's connective tissue stain. No sections of animals in the retracted condition, however, have been prepared with the Mallory stain. But in the sections of the retracted animals stained with Heidenhain's iron-alum haematoxylin no trace of such strands can be distinguished within the extended inner lips. Whether this fact is due to the extended condition of the lip with a corresponding separation of the fibers so as to make them too minute for identification, or whether it is due to a lack of "affinitv" for the 90 University of California Publications in Zoology [VOL. 13 haematoxylin stain, I am unable to say. One is here working at the limit of microscopical vision and the possibility of error in interpre- tation is not excluded as a third contingency. 2. Diplodinium ecaudatum forma caudatum Fiorentini PI. 5, fig. 6 Diplodinium caudatum Fiorentini (1889), pp. 15, 16, pi. 3, fig. 2. Diplodinium rostratum, Fiorentini (1889), p. 16, pi. 3, fig. 3. Diplodinium rostratum, Eberlein (1895), pp. 262-263, pi. 18, fig. 18. The forma caudatum of the species Diplodinium ecaudatum was first described and figured by Fiorentini (1889). Although he gives only a very brief and entirely inadequate description of the shape and structure of the body and fails to interpret correctly the things which he saw, and although his drawing is not only crude and inade- quate, but also in some respects absolutely erroneous, still he was the first to describe this form and in many ways his drawing is a better representation of the living animal than is that of Eberlein (1895). It is not desired to criticize too harshly the work of either Fiorentini or Eberlein, but merely to point out the fact that the previous work on this form is entirely inadequate. After a careful study of the description and figure of D. rostratum by Fiorentini (1889) it seems certain, as has already been pointed out (p. 51), that the individual which Fiorentini describes as D. ros- tratum is nothing more than D. e. forma caudatum observed shortly after division. If one were to draw the anterior portion of a dividing individual of D. ecaudatum forma caudatum in which the division was just com- pleted, the result would resemble Fiorentini 's figure of D. rostratum. In such a case the relatively great width of body, the blunt, rpunded- off posterior end, and the short, stumpy tail would all be accounted for. Even the two small, unequal contractile vacuoles, placed close together, are characteristic of the recently divided individual. If this interpretation is correct, then D. rostratum falls into the synonymy of D. e. forma caudatum. Eberlein 's (1895, pp. 262-263) description of Diplodinium ros- tratum Fiorentini covers also the form which Fiorentini describes as Diplodinium caudatum. To quote: "Ferner ist das von dem gleichen Forscher in seiner Abhandlung (1889) auf Taf. Ill, fig. 2, abgebildete und das Diplodinium caudatum Fiorentini beschriebene Thier zwei- felsohne mit dieser Form 'identisch' und tauscht nur einzelne Ver- 1914] Sharp: Diplodinium ecaudatum 91 schiedenheiten dadurch vor, dass es mehr vom Riicken gesehen darge- stellt wurde. ' ' Hence the Diplodinium rostratum Fiorentini described by Eberlein also falls into the synonymy of Diplodinium ecaudatum forma caudatum. The action of Eberlein (1895) in using the name Diplodinium caudatum for a new species discovered by him has already been dis- cussed. After an exhaustive study of the living animals, whole mounts, and transverse, frontal, sagittal, and oblique sections of all the forms of Diplodinium ecaudatum, both from the originals, camera drawings, and microphotographs, it is certain that with the exceptions of normal variations and of the changes in form and structure of the posterior extremity occasioned by the presence of one or more spines, 'the mor- phology of all of these forms is identical. Hence it is only necessary in this place to consider these spines and the changes in form and structure which their presence occasions. Forma caudatum (pi. 5, fig. 6) appears in almost as great num- bers as does forma ecaudatum. This form may be immediately distin- guished from all the other forms of this species by the fact that a portion of the posterior end of the body is prolonged in the form of a tail-like continuation, or spine (sp. 1, pi. 5, fig. 6). This spine, which is designated as the primary spine, takes its origin from the whole of that portion of the posterior end of the body which lies ventral to the anal opening. Cross-sections of the spine at or near its base appear bean- or kidney-shaped, with the convex margin directed ventrally and the concave side directed dorsally ; as the distal extremity is ap- proached, the cross-sections become more nearly circular. Eberlein (1895, p. 262) describes this spine as being "von beiden Seiten etwas zusammengedrucktes, " a description which is not substantiated by cross-sections of the spine. In life this spine is equal to from one- third to one-half of the length of the body and is either straight or slightly curved distally toward the dorsal side. In fixed specimens it is universally more or less curved toward the dorsal side. The cuticle, somewhat thickened, completely covers the spine and the ectoplasm is prolonged down into it. In no other way does the presence of this spine affect the morphology of the animal. In swimming this spine seems to function as a rudder, for it is to be noticed that D. e. forma caudatum is able to advance in a more nearly straight line than is D. e. forma ecaudatum. Dimensions of this form^are given below, page 95. 92 University of California Publications in Zoology [VOL. 13 3. Diplodinium ecaudatum forma bicaudatum forma nova PI. 5, fig. 7 This is the least abundant form of the species. It is characterized by the presence of a secondary spine (sp. 2, pi. 5, fig. 7) or tail-like continuation of the posterior extremity of the body. The base of this spine is located to the left of the median plane and close to the dorsal side of the body and the spine itself curves ventrally and inward. This secondary spine varies in size from a mere nodule situated just to the right and dorsal to the anal opening up to a spine one-half to two- thirds the size of the primary spine. The morphology of the secondary spine is identical with that of the ventral or primary spine. The pos- session of a secondary spine in nowise affects the position or mor- phology of the ventral or primary spine, but does, when large, affect to some extent the shape of the dorsal portion of the posterior end of the body. When more than one spine is present the posterior end of the body is relatively enlarged for their accommodation and the body appears more nearly cylindrical. These changes in size relationships may be seen by a comparison of figures 6-10, plate 5. In no other way does forma bicaudatum differ from forma caudatum. Dimensions of this form are given on page 95. 4. Diplodinium ecaudatum forma tricaudatum forma nova PI. 5, fig. 8 This also a relatively rare form, is distinguished by the presence of a third spine. This third or tertiary spine (sp. 3, pi. 5, fig. 8) is located rather to the right of the median plane and, as in the case of the secondary spine, curves ventrally and inward. The tertiary spine may also be present as a mere nodule situated rather close to the right extremity of the anal slit or may be quite as large as the secondary spine. I have never found either the secondary or the tertiary spine to be as large as the primary one, although the secondary and tertiary sometimes equal each other in size. The secondary spine, however, is generally the larger of the two. The presence of this third spine (sp. 3} does not in any way affect the primary (sp. 1) and secondary spines (sp. 2}. The posterior end of the body, however, is necessarily a little larger. Dimensions of this form are given on page 95. 1914] Sharp: Diplodinium ecaudatum 93 5. Diplodinium ecaudatum forma quadricaudatum forma nova Pi. 5, fig. 9 This is again a very abundant form, occurring not only in the majority of cattle but also in great numbers in the individual hosts. In fact this form is almost as abundant as is D. e. forma caudatum. This form is characterized, as its name would indicate, by the pos- session of four spines on the posterior end of the body. This fourth or quaternary spine (sp. 4, pi. 5, fig. 9) occurs normally on the right side of the body about midway between the primary (sp. 1} and ter- tiary (sp. 2) spines. The tertiary spine is then crowded dorsally until it occupies a position somewhat more dorsal than does the secondary spine (sp. 2}. In about four per cent of the animals examined the quaternary spine (sp. 4) was located on the left side between the primary (sp. 1} and secondary (sp. 2} spines. When this is the case the secondary spine (sp. 5) is crowded so far dorsally as to appear to be almost exactly opposite to the ventral or primary spine. On which- ever side it may occur this quaternary spine (sp. 4) is almost invari- ably the smallest and ranges in size from a mere protuberance to a spine almost as large as the secondary spine. In some cases, however, this fourth spine was even larger than either the secondary or tertiary spines. It has been pointed out that in the fixed material the primary spine ordinarily curves dorsally while the secondary and tertiary spines almost invariably curve ventrally and inward. In the case of the quaternary spine this curvature may be either ventrally and in- ward, dorsally and inward, or merely toward the main axis. It is to be noted that the designation of these spines as primary, secondary, etc., has been according to their position rather than to their size. Up to the form under discussion a definite relationship has seemed to exist between size and position, but with D. e. forma quadricaudatum this definite relationship no longer holds, except that the primary spine is always the largest, and the first three spines retain their relative positions. In this connection it is to be noted that considerable varia- tion exists as to the relative lengths of the spines both in relation to each other and in relation to the body. That is to say, each animal may present any one of the three general conditions: (1) all of the spines may be short, (2) all of the spines may be long, or (3) some of the spines may be short and the others long. Cross-sections through the bases of these spines (forma quadricaudatum} show normally a quack-angular arrangement and in many cases the bases of the sec- 94 University of California Publications in Zoology t v L - 13 ondary, tertiary, and quaternary spines appear exactly equal in size, with the primary always, however, somewhat larger. Here again we note the enlargement of the posterior end of the body to accommodate the added number of spines. In all other respects this forma is identi- cal with D. e. forma ecaudatum. The body dimensions are given on page 95. 6. Diplodinium ecaudatum forma cattanei Fiorentini Diplodinium Cattanei Fiorentini (1889), pp. 16-17, pi. 3, figs. 4, 5. PI. 5, fig. 10 This form is not very abundant. Not only is its presence in cattle the exception, but even when present it occurs only in small numbers. The distinguishing feature of this form is the possession of five pos- terior spines. The quintary spine (sp. 5) occurs on the right side just dorsal to the primary spine and curves dorsally and inward. In every case of D. e. forma cattanei examined the arrangement of spines was as shown in the figure (pi. 5, fig. 10), i.e., a very large ventral spine (sp. 1), a large secondary spine (sp. 2} normally placed, a very much smaller tertiary spine (sp. 3) also normally located, a very broad, somewhat flattened quaternary spine (sp. 4} on the left side, and a second very small quintary spine (sp. 5) on the right side. In every case the two spines of the right side were so small and the two spines of the left side so large that it was necessary to view the spines from the right side in order to see and draw the small spines. In all other respects the morphology of the spines and of the body was what would be expected. The dimensions of this form are given below. Considerable hesitation was at first experienced in assigning Diplo- dinium cattanei, as described and pictured by Fiorentini (1889), to the Diplodinium ecaudatum series. As a matter of fact, it was not at all certain that the five-spined form described by this investigator was identical with the one occuring in my material. But with the dis- covery of all the intermediate forms of the series, viz. : the two, the three, and the four-spined forms, coupled with the fact, on the one hand, that the five-spined form which was present in my material was so surely a member of the ecaudatum series, and on the other hand, that with the exception of the number of vacuoles pictured for D. cattanei by Fiorentini (1889, plate 3, fig. 5), my five-spined form corresponded very closely to his, it became evident that the two were 1914 ] Sharp: Diplodinium ecaudatum 95 identical. D. cattanei was therefore the end member of the Diplo- dinium ecaudatum series and as such was to be designated as D. ecau- datum forma cattanei. This conclusion is materially strengthened by the fact that no other five-spined Diplodinium has been described, although several investigators have been working over this same field since Fiorentini's (1889) first communication. Apparent discrepan- cies between Fiorentini's figures and descriptions of Diplodinium cattanei and my figure and description may be explained upon the assumption that one of his figures (pi. 3, fig. 5) and his description of the same was based upon an abnormal or pathological individual. The possession of two contractile vacuoles is an exceedingly constant characteristic of this species and, as a matter of fact, in his figure 4, plate 3, Fiorentini pictures only two such vacuoles. The slight differ- ence in size is easily accounted for when it is remembered that the normal variations in the size of individuals of this group are consider- able and that the natural tendency of the observer is to select the larger and therefore more easily figured individuals. TABLE OF DIMENSIONS FOR ALL FORMS OF Diplodinium ecaudatum Five animals in each case Animals ecaudatum measured L. W. caudatum bicaudatum L. W. L. W. tricaudatum L. W. quadri- caudatum L. W. cattanei L. W. 1st 122 40 122 45 126 44 126 46 138 52 138 53 2nd 132 45 126 45 122 42 122 42 112 38 126 46 3rd 126 44 132 48 126 45 132 45 138 54 132 52 4th 132 45 132 50 130 48 132 48 138 54 138 54 5th 122 43 138 53 126 45 138 45 132 50 122 44 Average 127 43 130 48 126 45 129 47 131 50 131 50 L. Length of body from mouth to anus in microns. W. Width of body at level of micronucleus in microns. These measurements were taken from preparations which repre- sented the average size. Exceptionally, preparations from other stomachs were examined, in which all of the animals were either undersized or oversized, thus suggesting the occurrence in Diplodinium of races or pure lines similar to those described by Jennings (1909) for Paramecium. OBSERVATIONS ON THE LIVING MATERIAL One of the first things that the observer notices when studying these interesting little animals under conditions made as nearly normal as possible is the terrific rate of speed at which they travel. Several observations led to the conclusion that at the normal temperature, 96 University of California Publications in Zoology [VOL. 13 i.e., 35?5 C, an individual of the species D. ccaudatum could easily travel a distance equal to twenty times its own length in less than one second. It must be admitted that no accurate measurements were taken, but the most careful and conservative estimates led to the above conclusion. In fact we are convinced that this species holds the speed record for the genus Diplodinium and probably for all of the genera described thus far from the stomachs of ruminants. The normal course taken by a member of this species is not in a straight line, but, like so many of the asymmetrical protozoans, it advances in a right spiral as does the point of a corkscrew when penetrating a cork. This fact becomes doubly interesting when we consider the build of the anterior extremity of the body. We noted under the description of the organs of nutrition that, owing to the greater thickness and greater height of the adoral membranelle zone and oral disk on the right and dorsal sides of the mouth than on the left and ventral sides, the plane of the mouth was directed toward the left and ventrally. Thus we see that by the clockwise rotation and the spiral course of the body the mouth opening is brought into contact with a greater amount of the surrounding medium and more directly than could possibly be accomplished in any other manner of locomotion. Keeping in mind that all the evidence points toward a bacterial diet for this species, and therefore the probable necessity of great numbers of these small food particles, we are struck with the wonderful co-ordination of locomotor and nutritive organs, which makes for efficiency in food getting. Another interesting fact was one day forcibly brought to my attention when, after returning to the laboratory with samples from the contents of ten stomachs, I was absolutely unable to find a single member of the species D. ecaudatum. As all of these samples had been taken from the same herd of cattle, the question arose, Does geographical environment play any part in infecting cattle with this protozoan? Careful records kept from that time on have furnished the following information: (1) In the same herd some cattle may be heavily infected with ciliated protozoans, others very slightly. (2) In the same herd some cattle may be heavily infected with some or all of the species of one genus and not with another, while other cattle reverse the conditions and contain heavy infections of those species and genera which the first cattle lacked. (3) In the same herd some cattle contain only certain forms of a species, while other cattle contain only other forms of the same species. (4) In the case of Diplodinium 1914 1 Sharp: Diplodinium ccaudatum 97 ecaudatum in any one stomach certain groupings of forms seemed to be the rule ; that is to say, we have here a general condition in which (a) forma ecaudatum may be the only form present, (6) forma can- datum may be the only form present, (c) forma ecaudatum and forma caudatum may be present in about equal numbers, (d) forma quadri- caudatum may be the only form present, (e) forma quadricaudatum may be associated with any or all of the other forms. In other words, almost any combination may exist, with this exception the forms bicaudatum, tricaudatum, and cattanei have never been found except in the presence of the forma quadricaudatum. As noted above, under technique, satisfactory observations of the activities of the living animals could be obtained only by reducing the temperature a few degrees, which reduction and control was made possible by the automatic constant-temperature oven to which refer- ence has been made. In this oven at a temperature of about 30 C., the following observations were made, for at that temperature, al- though only 5?5 C. below normal, the restless activities of the mem- bers of this species are slowed down sufficiently to permit of satis- factory study. The cuticle shows clear and transparent, the skeletal areas are easily defined; in fact these areas are much more plainly seen in the living animals than in the fixBd material. The boundary layer be- tween ectoplasm and entoplasm is very clearly marked off, and at certain levels the macronucleus and the micronucleus can be easily distinguished, the macronucleus having the characteristic granular appearance and the micronucleus appearing as a bright, shining little body, refracting the light strongly. The contractile vacuoles show up much more clearly in the living animals than they do in the fixed material. These contractile vacuoles do not contract suddenly and disappear as in the case of Paramecium, but, on the contrary, contract slowly and only slightly, then gradually enlarge to their former size. Their action is more of a true pulsation. The caecum and rectum may be distinguished just before and during the process of defecation. In a few cases this process has been observed. The streaming of the entoplasm referred to previously may usually be observed during quiescent periods in the animal's locomotor activity. For purpose of description this streaming may be roughly divided into three main currents, according to the general direction assumed by each: (1) a peripheral posterior current, (2) an anterior current, anctr(3) an internal posterior current. (1) The direction of the 98 University of California Publications in Zoology [VOL. 13 posterior peripheral current, beginning immediately posterior to the level at which the oesophagus passes through the boundary layer, is obliquely posterior and to the right, i.e., following the general direction of the oesophagus. At the posterior limit of the sack the direction of the current changes so as to flow towards the left dorsal wall. (2) The anterior current beginning at this point follows the left wall of the sack, passing obliquely anteriorly and somewhat ventrally, i.e., in exactly the opposite direction to the posterior current. When the current reaches the anterior extremity of the entoplasmic sack, which extremity, as will be remembered, is anterior to the opening of the oesophagus into the sack, it is again directed posteriorly. (3) The internal posterior current flowing internally to the first described posterior current passes posteriorly to the region of the caecum, where it becomes lost in the anterior current. Thus we see that the two posterior currents, i.e., peripheral and internal, pass in the same direction as does the oesophagus, and so may assist in drawing food particles into the entoplasm ; also that by reason of its flowing directly toward the caecum the internal current may assist in carrying waste products to the organ of defecation. Most interesting of all the observations, however, were those upon the action of the dorsal and adoral membranelle zones together with that of the operculum. First of all, it was noted that in swimming the organism uses both zones of membranelles and that normally the contractions take place as waves passing from one extremity to the other. In the case of the dorsal row of membranelles these waves usually started at the left extremity in the following manner : The first membranelle is made to circumscribe a conical space, the base of which corresponds to the distal extremity of the membranelle and the apex of which corresponds to its attachment to the bo(|y, i.e., circumduction. The direction of this movement is clockwise and the movement has no sooner started in the first membranelle than it is begun in the second, and so on. Even when the animal is swimming slowly a second wave may be started before the first wave has reached the opposite extremity. Thus two or three waves of contraction may be passing along the row of membranelles at the same time. In the case of the adoral membranelles the movements are made out with much more difficulty and it is only when the adoral region faces the observer that satisfactory results can be obtained. It is sometimes possible to bring the animal into this position by a careful manipu- lation of the cover glass. In general the movements of the adoral Sharp: Diplodinium ecaudatum 99 membranelles resemble those of the dorsal zone. Normally the wave of contraction starts at the junction of the heavier adoral membra- nelles with the finer oral cilia and passes first to the right and then ventrally and to the left, to end at the left extremity of the adoral row of membranelles. The movement of the individual adoral mem- branelle is the same as in the case of the dorsal membranelles, i.e.. circumduction. A second interesting observation was to the effect that any single membranelle or any set of membranelles of either zone could be moved at the point of stimulation, by simple contact, or even independently of any apparent stimulus, and this without disturbing the other mem- branelles, either of the same zone or of the other zone, a phenomenon which reminds the observer of the result obtained by stimulating a single tentacle or set of tentacles of the sea anemone. This fact leads to the belief that each penicillate membranelle is supplied by an individual ' ' nerve fiber. ' ' As the oral cilia have never been observed in a quiescent state, it is impossible to be certain of the direction of their wave contractions. When the temperature drops too low or the animal is otherwise irritated, either mechanically or chemically, the oral region, viz., the oral cilia, the oral disk, and the oral opening, is retracted pos- teriorly into the body. Simultaneously with this the inner adoral and inner dorsal lips are extended in such a manner as to become directly continuous with the outer adoral and outer dorsal lips respectively, that is to say, the outer furrows are obliterated and the two lips are smoothly continuous one with the other. When the oral region is sud- denly retracted the popping out of these inner lips reminds one of the popping out of the inverted finger tips of the surgeon's rubber glove when everted by air pressure. And a similar explanation is applicable to both. In other words, the protrusion of the inner lips is a mechanical occurrence brought about through the contraction of the oesophageal fibers, which pulls the oral region into the anterior end of the body and thus increases the pressure in the semifluid ectoplasm. According to the laws of physics, pressure is transmitted equally in all directions. Hence when the increased pressure due to the inward pull of the oesophageal retractor strands is sufficient to overcome the resistance offered by the weakest portion of the contain- ing wall that weakest portion will yield sufficiently to bring again the pressure relations to an equilibrium. In this case the weakest portions of the retaining wall are the inner adoral and the inner dorsal 100 University of California Publications in Zoology [VOL. 13 lips and the equilibrium is again established, in the one case, when the different portions of the inner adoral lip meet in the epioral region, and in the other case, when the inner dorsal lip meets the dorsal edge of the operculum. If it can be assumed that these inner adoral and inner dorsal lips exhibit any degree of inherent elasticity, and such an assumption is entirely within the bounds of probability, then an explanation of the return of these extended inner lips to their normal positions to- gether with the simultaneous protrusion of the oral region is also to be made along mechanical lines as follows; the contraction of the oesophageal fibers is relaxed and the elasticity of the inner lips and possibly of the whole anterior end of the animal, i.e., the tendency to return to the normal position, now exerts a pressure upon the enclosed ectoplasm in a direction opposite to that which caused the protrusion of these inner lips, with the result that as the inner lips return to their original positions the oral region is again protruded and the animal once more presents the normal appearance and re- sumes its activity. It is desired in this place to call attention to the fact that previous observers have described the membranelle zones as retractile structures. Both the study of the preparations of fixed material and the observa- tions on the living animals lead to the conclusion that the dorsal mem- branelles, and probably also the adoral membranelles, are not retracted within the body, but that the picture presented by these so-called re- tracted forms (fig. D) is brought about, as described above, by the pro- trusion of the lips. In other words, the evidence goes to show that during the process of oesophageal retraction the membranelles remain stationary and become encased by the protrusion of the inner adoral and dorsal lips respectively. ^ In watching these phenomena of retraction and expansion in the living, active animals one cannot help but be impressed with the won- derful co-ordination of parts, the simple and yet efficient mechanism by means of which the encasement and protection of the delicate mem- branelles is effected and withal the probable presence of at least the rudiments of a nervous system. 1914] Sharp: Diplodinium ecaudatum 101 CONCLUSIONS 1. One result of the present study has been the discovery of three new forms of Diplodinium ecaudatum, namely forma bicaudatum (two posterior spines), forma tricaudatum (three posterior spines), and forma quadricaudatum (four posterior spines). These three forms, together with D. caudatum Fiorentini (one posterior spine), and D. cattanei Fiorentini (five posterior spines) because of their structural similarity have been assigned to the species ecaudatum (no posterior spines). 2. Diplodinium ecaudatum, therefore, consists of a series of six forms ranging from D. ecaudatum, without posterior spines, up to D. cattanei, a form with five posterior spines. No other structural characteristics distinguish these forms from each other. 3. The reasons for assigning Diplodinium caudatum and the forms bicaudatum, tricaudatum, quadricaudatum, and cattanei to the species D. ecaudatum are as follows: First, with the exception of the presence or absence of the spines, the dimensions and structures of all these forms are practically identical. Second, the series of spines from D. e. forma ecaudatum, without spines, up to D. e. forma cattanei, with five spines, is complete. Third, with the exception of the primary spine, spines of all sizes are to be found, ranging from mere nodules up to spines which are equal to one-third of the entire length of the body. 4. The reasons why each of these types of this organism has been designated as a "forma" according to the number of spines present are as follows : First In every case of division observed animals with a certain number of spines gave rise to two daughter animals, each of which was provided with the original number of spines. Attention is called to the fact, however, that none of these cases of division, so far as the evidence is at hand, followed at once after conjugation, and that it is possible that division immediately following conjugation might have resulted differently. Second That the presence of one of these forms in the stomach of the ox in no wise necessitates the presence of other forms. The forms bicaudatum, tricaudatum, and cattanei, however, have never been found except in the presence of forma quadricaudatum. 5. The genus Diplodinium has been revised, as a result, in part, oMhe discoverv of the three above-named forms, with the result that 102 University of California Publications in Zoology [VOL. 13 the number of valid species in the genus has been reduced from ten to five. 6. The body is covered by a very resistant cuticle, divided into definite areas, characterized by peculiar surface markings. Three of these areas, because of their relation to underlying skeletal structures, are designated as left, ventral, and right skeletal areas. These three areas with their underlying skeletal structures are separate at the anterior end of the animal, but merge together as they approach the posterior extremity. They afford attachment for the internal retractor structures. 7. The arrangement of the oral cilia and the adoral membranelles differs from that previously described for this genus. Starting from a point on the left side of the animal, near to the anterior extremity, the adoral row of membranelles circles from left to right around the adoral region until it reaches a point inside of and opposite to that at which it started, then turning upon itself it reverses its direction and now as oral cilia circles from right to left around the oral opening. 8. There is present in D. ecaudatum, a complicated structure, the neuromotor apparatus, which is probably nervous in function. This apparatus consists of a central motor mass or motorium, from which definite strands radiate: one to the roots of the dorsal membranelles (dorsal motor strand) ; one to the roots of the adoral membranelles (ventral motor strand) ; one to the circumoesophageal ring (circum- oesophageal ring strand) ; and several pass out into the ectoplasm of the operculum (opercular fibers). Each of these strands may send off one or more branches. In the walls of the oesophagus both nervous and contractile fibers may be distinguished. The structural and func- tional relations of these parts are such as to indicate that they con- stitute a neuromotor apparatus. The Protozoa have often been defined as simple, one-celled animals. Calkins (1909, p. 1) says of them: "Their beauty, their varied modes of life, the suddenness of their appearance and disappearance, the simplicity of their structure and modes of reproduction combine to make them, even to the superficial observer, a fascinating group." From the present study of these ciliated protozoans of the stomach of the ox we may conclude that in the various forms of the species Diplodinium ecaudatum are to be found some of the most interesting and also the most complex of all known Protozoa. Transmitted May 10, 1913. Sharp: Diplodinium ecaudatum 103 ADDENDUM This paper was accepted for publication by the University Press, May 10, 1913. The receipt of a very generous gift. May 22, 1913, made possible the publication of plate 4 in colors and also the addi- tion of the microphotographs, plates 6 and 7. The preparation of these plates has delayed publication. On December 12, 1913, after this paper had gone to press, the Archiv fiir Protistenkunde, of November 11, 1913, containing Braune's excellent paper, ' ' Untersuchungen tiber die im Wiederkauermagen vorkommenden Protozoen, " was received at this laboratory. Since Braune has worked on the same family, the Ophryoscolecidae, as myself, and has figured and described for Opkryoscolex purkynjei Stein structures which are apparently homologous with those described by me for Diplodinium ecaudatum, and since our interpretations, not only of the morphology but also of the functions of several of these structures, differ to some considerable extent, it seems necessary to add a word here. It must be kept in mind that although our observa- tions have been made upon somewhat similar organisms, yet notwith- standing their close relationship these organisms may present many dissimilarities. Having made no comparative study of the minute structure of the form which Braune describes I must content myself with a brief discussion of the more obvious points wherein we differ. Although the distinction between ectoplasm and entoplasm may be a more or less arbitrary one depending upon the definitions of the observer, still the separation by Braune (1913, p. 151, and pi. 6, figs. 38-41) of the " Ectoplasma, " " Grenzschicht, " and "Entoplasma" of Schuberg (1888), Eberlein (1895), and Giinther (1899, 1900) into his so called "Entoplasma a," " Fibrillenschicht, " and "Entoplasma b" is not in harmony with the evidence obtained by me from Diplo- dinium ecaudatum. Nor is it altogether in accord with Braune's own work, for he suggests (p. 152) the correspondence with similar layers in Isotricha prostoma and yet in this form both in his description (p. 140) and in his figures (pi. 5, figs. 32, 33) he holds that the ciliary roots penetrate only as far as the "Grenzschicht" which separates ectoplasm from entoplasm while for Opkryoscolex purkynjei, he states (p. 158, pi. 6, fig. 37) that the membranelle roots pass through not only the ectoplasm and ' ' Grenzschicht, ' ' but also penetrate the ' ' Ento- plasna a." 104 University of California Publications in Zoology [VOL. 13 That neither his description of the anatomy nor his interpretation of the function of the ' ' Stiitzapparat " of Ophryoscolex purkynjei will hold, even in the main, for the skeletal structure of Diplodinium ecaudatum may easily be seen from a glance at the microphotographs (pi. 7, figs. 20-33). Braune (1913, pp. 152-154, and pis. 6, figs. 38-40) states in the first place that the "Stiitzapparat" is a unit organization, a struc- ture ("einheitliches Gebilde") situated in the "Entoplasma a," and filling the entire ventral side. Microphotographs 23-29, plate 7, show very clearly that for D. ecaudatum the skeletal structure is much more complicated, that it consists of three component parts, well de- fined at the anterior end (figs. 25-26) and merging near the middle of the animal (figs. 28-29) ; that these component parts are situated in the ectoplasm, and that at the anterior end of the animal this structure extends over the entire ventral half of the circumference of the body, but as the posterior half of the body is approached the structure comes to lie more and more to the right side. A second point made by Braune is that the "Stiitzapparat" is a plate with its lateral edges bent in towards the inner part of the body and its anterior ends drawn out to points thus better to surround the oesophagus. An examination of the microphotographs 22-29, plate 7, shows conclusively that in the case of D. ecaudatum the skeletal structure cannot be described as a plate with its lateral edges bent in towards the inner part of the body nor are the anterior ends drawn out to points, for it will be noted that figure 23, plate 7. which repre- sents the fifth section in the series, and one which is the most anterior section showing this skeletal structure, gives absolute evidence, by measurement, that each of the three component parts is actually broader at its most anterior extremity than at any other level in its^ entire length. Also in these first five or six sections which represent the anterior one fourth of the animal it is plainly evident that the oesophagus has no definite connection with the skeletal structure other than with the surrounding ectoplasm. In the third place Braune maintains that this structure may be divided into three layers, (a) an outer layer composed of fine, long, interlacing fibrillae, (b) a middle, alveolar layer (described by Giinther, 1899, 1900), composed of very large alveoli which at times occupy the whole thickness of the supporting structure, and (c) an inner " Fibrillenlage " situated between "Entoplasma a" and "Ento- plasma b" which exhibits the large, parallel fibrillae. Again exam- 19 14 ] Sharp: Diplodinium ecaudatum 105 ining microphotographs, figures 23-29, plate 7, it will be seen that no fibrillar layer exists between the skeletal structure and the cuticle nor is there any structure which might correspond to the "Fibril- lenlage. ' ' Braune also states that the thin right edge of the ' ' Stiitzapparat ' ' separates the macronucleus from the "Entoplasma b." That this does not hold for D. ecaudatum is evidenced by figures 27-29, plate 7. He further holds that the importance of the "Stiitzapparat" lies in its relations to the internal structures and that it serves as a sup- port not only for the gullet, but also for the many longitudinal and transverse fibrillae. As to the relation between skeletal structure and fibrillae in D. ecaudatum reference will be made later, and as to the skeletal structures serving as supports for the oesophagus, the micro- photographs, figures 20-23, 33, show that the oesophagus extends 20-24 microns further anteriorly than do the skeletal structures, that there is no direct connection between oesophagus and skeletal struc- ture for another 30-40 microns. Microphotographs, figures 28-30, however, show pretty conclusively that soon after the oesophagus does become attached to the skeletal structure, this latter structure dis- appears. In other words the skeletal structure serves as a fixed organ for the posterior attachment of the contractile or retractile oesophageal strands. Observations made upon the living animals and examinations of the stained sections lead to the conclusion that the important func- tions of the skeletal structure in D. ecaudatum are first of all, to give the characteristic shape and rigidity to the body, secondly to provide a fixed posterior attachment for the retractile oesophagus and a sub- stantial support for the operculum and the macronucleus, and thirdly by a combination of the above to afford protection to all of the body structures. Braune 's account of a most remarkable network of fibrillae ("Fibrillenapparat") in 0. purkynjei, is worthy of a more compre- hensive discussion than it is possible to give it here. Only the more important differences between this "Fibrillenapparat" and the neuro- motor apparatus described for D. ecaudatum will be discussed. He says (p. 156), "Mit dem Nachweis dieses auBerordentlichen Fibrillen- reichtums ergibt sich aber die Schwierigkeit, ihrer in der Beschrei- bung gerecht zu werden und von ihrer Schonheit und Harmonic zu berechten, ' ' and his description is, in the main, as follows : 1. Separating the "Entoplasma a" from the "Entoplasma b" is a saeli-like "Fibrillenschicht (Fig. 38, Fb. sch.) " which is an "auBer- 106 University of California Publications in Zoology [VOL. 13 ordentlich verzweigten Fibrillensystem, " but which represents only a part of the complicated "Fibrillenapparates" and is to be regarded as a particular structure in the entoplasma. These "Fibrillen" are longitudinal and are internal to the transverse "Fibrillen" next described. 2. The whole body is surrounded by a large number of almost parallel transverse "Fibrillen" which take their origin from one edge of the ' ' Stiitzapparat " (his fig. 36, qu. fibr.) and pass around the dorsal side of the body to their attachment in the opposite edge of the "Stiitzapparat." They assume considerable size when they have a particular function to perform. To quote (p. 158), "So finden wir oberhalb, der Membranellenzone zwei quer Fibrillenbiindel (Fig. 36a, &), die in einem bestimmten Abstand, durch mehr oder weniger regel- maBig angeordnete Langsfibrillen verbunden, stehen. ' ' Although the same fixing and staining methods (viz., Schaudinn's alcoholic sublimate solution followed by Heidenhain's iron-alum haematoxylin) that Braune used for 0. purkynjei have been used on D. ecaudatum it was not possible to demonstrate, in this organism, either the "Filbrillenschicht" or the transverse "Fibrillen." Ah ex- amination of the microphotographs (pis. 6, 7, figs. 11-33) will show that although in many of these sections in which, even in the prints, the separate granules of the macronucleus (pi. 7, figs. 27-29), the separate cilia in the membranelles (pi. 6, figs. 14, 15, 19 ; pi. 7, fig. 25), and the individual bacteria within the food vacuoles (pi. 7, fig. 33) may be fairly well made out (and certain it is that all details may be seen to much better advantage in the original sections) there is no evidence of the presence of these above described "Fibrillen." The fine parallel lines of the lower ends of figures 12-14, plate 6, might at the first glance be confused with the " Fibrillenschicht " of Braune, but on a closer study it will be noted that these lines are surface mark- ings (cf. fig. 11). Also an examination of figures 26 to 32 will show that in the case of D. ecaudatum the layer separating the internal entoplasm from the more external layers (ectoplasm and cuticle) is a continuous, homogeneous membrane rather than a layer of "Fibril- len," as pictured and described by Braune for 0. purkynjei, with the possible exception, as noted in the main body of my paper, of the extreme posterior end of the body in which the oesophageal wall lies so close to the boundary layer as to defy a microscopic separation and identification of the two layers. 3. In his description of the lips of the dorsal membranelle zone (p. 157), he says, "In den Wanden aiiBern Saumes trifft man regel- 1914] Sharp: Diplodinium ecaudatum 107 maBig einzelne starkere quere Fibrillen (Fig. 37a)." Evidently there is some mistake here for in "Fig. 37" the "a" refers to the inner lip and apparently the walls in this region are structurally the same as the walls in practically all other parts of the body. And in speaking of the double fastenings of the dorsal membranelles (p. 158) he notes, that, after penetrating the outer layer, their inner ends are connected with the " Fibrillenschicht " by short inner supports, while to the outer boundary layer decidedly longer "Fibrillen" proceed (Figs. 36, 37, In. st.: aus. st.). And to quote from his description of the outer adoral lip (pp. 158-159), "Die sie umgebenden Wiilste zeigen noch starkere Fibrilleneinlagerung wie die des queren Membranellenzugs. ' ' He says further, the insertions of adoral membranelles are similar to those of the dorsal membranelles, viz., the double fastening, to the "Fibrillen- schicht" internally and to the "Grenzschicht" externally. Then fol- lows a detailed description of a very complicated fibrillar system which may be rather briefly summed up as follows : These Stiitzfibril- len," as Braune terms the ciliary roots, which here lie in the so-called "Entoplasma a," are extraordinarily lengthened out. The innermost set of these parallel "Fibrillen" (Fig. 39, St. fbr.) extend anteriorly beyond the " Stiitzapparat " and in consequence of its spine-like pro- longations are brought together in a circle. Near to the mouth opening these fibrillae are in turn encircled by a " Fibrillenschlundring (Fig. 39, Schlr.)." On their oral ends are imposed the oral cilia and there- fore they may be counted as "inneren Stiitzen" of the oral mem- branelles. The outer supports (AuBenstiitzen) of these membranelles likewise unite by threes or fours into " Stiitzfibrillen " which again combine to form larger groups. Each of these larger groups has only one point of attachment, which is found either in the "Stiitzapparat" itself or upon the two " Fibrillenstammen " situated just above the dorsal membranelle zone (Fig. 39, v auf &). This double fastening of the membranelles, viz., anterior and pos- terior roots in the case of the dorsal membranelles and internal and external roots in the case of the adoral membranelles, has been de- scribed for D. ecaudatum in the main body of my paper, but these fastenings were regarded by me as ciliary root-filaments rather than as supporting fibrillae. An examination of figures 33 and 23 to 29 will show that in D. ecaudatum the only fibrillae present in the adoral region are those which are imbedded in the oesophageal walls, and according to Braune 's description such a picture as is obtained in the contracted forms (text fig. D) would be absolutely impossible. 108 University of California Publications in Zoology [VOL. 13 4. A rich fibrillar supply is also described for the walls of the rec- tum and for the region of the bases of the spines. One or more of these fibrillae are described as extending down into each spine as a sort of axial rod. A fibrillar layer is present in the internal wall of the rectal sheath of Diplodinium ecaudatum, as has been described in the body of my paper, but as was there pointed out, these rectal fibrillae (rect. f., pi. 3, fig. 3), are, in the ontogeny of D. ecaudatum, derived from the ventral edge of the preexisting oesophageal wall and are not exactly comparable to the "quere Fibrillenziige " described for the correspond- ing region in Ophryoscolex purkynjei. These rectal fibers, however, as well as those fibers in that portion of the oesophageal wall which I have described as lying so close to the boundary layer, in the posterior end of the animal, as to defy microscopic separation from the boundary layer, are undoubtedly the homologues of the "Fibrillen" described by Braune for the corresponding region in 0. Purkynjei. Other than this no general fibrillation corresponding to that described for 0. Pur- kynjei has been discovered in the forms investigated by me. In regard to the function of this "Fibrillenreichtum," Braune concludes, "daB die Fibrillen keine Myoneme, sondern einfache elas- tische Stabchen sind, denen nur die Stiitzfunktion zukommt." And further he suggests that the retraction of the peristome and the mem- branelle zones is easily brought into harmony with his view. To quote again (p. 161), "Das Protoplasma bleibt nach wie vor ein scheinbar homogenes, zahlfliissiges Medium, daB bei unseren Formen durch die Fibrillenanhaufungen an eine starre Gestalt gebunden wird. Ver- moge der Elastizitat der einzelnen Fibrillen ist das Protoplasma im- stande, irgendwelche Reize durch schwache Lageveranderung der beiden Wimperzonen zu beantworten. Aus dem Gesagten geht hervor, dass die bi- zarre Korpergestalt der Ophryoscoleciden nur durch das Fibrillensystem erhalten wird.' The arguments against the neuromotor apparatus of D. ecaudatum serving merely as a supporting structure, the interpretation which Braune places upon the "Fibrillenapparat" of 0. purkynjei, have been given rather fully in my paper. It is unfortunate indeed that no microphotographs accompany Braune 's article and that his drawings are so fragmentary. As neither his description nor his figures give any evidence that he saw the motor mass which shows so clearly in microphotographs of D. ecaudatum (pi. 6, figs. 15, 16; pi. 7, figs, 22, 23, 33), it would seem either that a homologous structure is lacking in Sharp: Diplodinium ecaudatum 109 0. purkynjei or that it was overlooked by the observer. Also, as has just been noted, such fibrillar structures as the " Fibrillenschict, " " Querfibrillen, " " Stiitzfibrillen, " "queren Korperfibrillen, " and the "Vereinigung der AuBen stiitzen des inneren kleinen Bogens, " and "des auBeren groBen Bogens" described for 0. purkynjei are lacking in D. ecaudatum. And even if they did occur as figured by the above named author it would be hard to interpret them as being merely sup- porting structures, for according to Braune's figures (pi. 6, figs. 36- 43) these "Fibrillen" occur most abundantly, first in those regions of the body which are otherwise well supported by the definite and ample skeletal structures, and secondly in that region which is the most markedly retractile, viz., the oesophageal region. In the former case these fibrillae, in the role of supporting structures, would be super- fluous while in the latter case it would seem as though a contractile function might be more logically assumed for them. In regard to the role played by the spines Braune suggests that by means of these, the ciliates which he considers to be normally boring rather than swimming animals, are enabled to keep from slip- ping back while forcing their way through the more solid masses of food. Attention has already been called to the fact that in D. ecaudatum the spines, when present, curve in towards the central axis of the body. This seems to be true for 0. purkynjei as well. Such being the case it is difficult to imagine how these spines may be of any assistance whatever in serving to keep the animal from slipping back through the mass of food particles and especially would this be so, if, as Braune points out for 0. purkynjei, that portion of the body just anterior to the spines is greater in diameter than that portion upon which the spines are situated. Also in looking at the matter from the viewpoint of evolution, according to Braune's interpretation the presence of these spines being of advantage to the animal, it might be expected that those species or forms so provided with posterior spines would contain the greater number of individuals. Such, however, is not the case, at least this does not hold true for D. ecaudatum. Also it must be noted that many of the forms which are provided with posterior spines are flat, do not habitually rotate about the longitudinal axis and are essentially free swimming and not boring forms. Zoological Laboratory, University of California. Transmitted February, 1914. 110 University of California Publications in Zoology [VOL. 13 BIBLIOGRAPHY BRANDT, H. 1909. Beitrage zur Biologie der Infusorien im Digestionstractus der Herbi- voren. Inaug. Diss., Bern., 38 pp. BUNDLE, A. 1895. Ciliate Infusorien im Cocum des Pferdes. Zeit. f. wiss. Zoo]., 60, 284-350, pis. 15-16. BtlTSCHLI, O. 1888. "Ciliata" in "Protozoa" (1887-1889) in Bronn, Klass. und Ordn. des Thierreichs, 1, Abth. 3, pp. 1228-1841, pis. 56-86. CALKINS, G. N. 1909. Protozoology (New York and Philadelphia, Lea & Febiger), x + 349, 4 pis., 125 figs, in text. CERTES, A. 1880. Sur la glycogenese chez les infusoires. Compt. Rend. Acad. Sci., Paris, 90, 77-80. 1889. Note sur les microorganismes de la panse des ruminants. Bull. Soc. Zool. France, 14, 70-73. Also Jonrn. de Microgr., 13, 277-279. 1891. Sur le precede de M. Joseph Eismond pour 1 'etude des infusoires vivantes. Bull. Soc. Zool. France, 16, 93-94. COLIN, G. 1854. Traite de physiologic comparee des animaux domestiques (Paris, Bailliere), 2, 667 pp., 57 figs, in text. Pp. 607, 657, figs. 49, 56, 57. DOFLEIN, F. 1911. Lehrbuch der Protozoenkunde (Jena, Fischer), 3 Aufl., xii + 1043, 951 figs, in text. EBERLEIN, E. 1895. Ueber die im Wiederkauermagen ciliaten Infusioren. Zeit. f. wiss. Zool., 59, 233-304, pis. 16-18. ERLANGER, E. v. 1890. Zur Kenntnis einiger Infusorien. Zeit. f. wiss. Zool., 49, 649-662, pi. 29. FlORENTINI, A. 1889. Intorno ai protisti dello stomaco dei bovini (Pavia, frat. Fusi.), 27 pp., 6 pis. 1890a. Intoruo ai protisti dell' intestine degli equini (Pavia, Bizzoni), 24 pp., 5 pis. 1890b. Intorno ai protisti dell' intestine degli equini. Boll. Sci. (Maggi), 12, 7-17, 2 pis. 1890c. Intorno ai protisti dello stomaco dei bovini. Boll. Sci. (Maggi), 11, 87-91. 1890d. Sur les protistes de 1'estomac des bovides. Journ. de Microg., 14, 23-28, 79-83, 178-183, 3 pis. 1891. Intorno ai protisti dell' intestino degli equini. Boll. Sci. (Maggi), 12, 51-60, 3 pis. GRUBE ET DELAFOND. 1843. Sur des animalcules se developpant dans 1'estomac et dans les intes- tins pendant la digestion des animaux herbivores et carnivores. Compt. Bend. Acad. Sc. Paris, 17, 1304-1308. 1914] Sharp: Diplodinium ecaudatum 111 GtJNTHER, A. 1899. Untersuchungen iiber die im Magen unserer Hauswiederkauer vor- kommenden Wimperinfusorien. Zeit. f. wiss. Zool., 65, 529-572, pis. 28-29, 2 figs, in text. 1900. Weitere Beitrage zur Kenntnis des feinern Baues einiger Infusorien aus dem Wiederkauermagen und dem Coecum des Pferdes. Zeit. f. wiss. Zool., 67, 640-662, pis. 36-37. JENNINGS, H. S. 1909. Heredity and variation in the simplest organisms. Amer. Nat., 43, 321-337, 5 figs, in text. KENT, W. S. 1881. A Manual of the Infusoria (London, Bogue), 2 vols. and atlas, x + 913, front, and 51 pis. LIST, A. 1885. Untersuchungen iiber die in und auf dem Korper des gesunden Schafes vorkommenden niederen Pilze. Inaug. Diss., Leipzig, 66 pp., 4 pis. LONG, J. A. 1912. Studies on early stages of development in rats and mice, by E. L. Mark and J. A. Long, no. 3. The living eggs of rats and mice, with a description of apparatus for obtaining and observing them. Univ. Calif. Publ. Zool., 9, 105-136, pis. 13-17, 11 figs, in text. LEVANDER, K. M. 1894. Beitrage zur Kenntniss einiger Ciliaten. Inaug. Diss., Helsingfors, 87 pp., 3 pis. LEUCKART, E. 1879-1886. Die Parasiten des Menschen und die von ihnen herriihrenden Krankheiten (Leipzig und Heidelberg, Winter), 2 Aufl., 1, Abth. 2, pp. xxxi + 1000, 410 figs, in text. LlEBETANZ, E. 1910. Die parasitischen Protozoen des Wiederkauermagens. Arch. f. Prot., 19, 19-80, pis. 1-2, 1 fig. in text. MlNCHIN, E. A. 1912. An introduction to the study of the Protozoa (London, Arnold), xi + 520, 194 figs, in text. NERESHEIMER, E. E. 1903. Ueber die hohe histologische Differenzierung bei heterotrichen Cili- aten. Arch. f. Prot., 2, 305-324, 1 pi., 1 fig. in text. 1907. Nochmals iiber Stentor coeruleus. Arch. f. Prot., 9, 137-138. SCHUBERG, A. 1888. Die Protozoen des Wiederkauermagens 1. Biitschlia, Isotricha, Dasy- tricTia, Entodinium. Zool. Jahrb., 3, 365-418, pis. 12-13. 1891. Ueber einige Organisationsverhaltnisse der Infusorien des Wieder- kauermagens. Sitzber. d. phys.-med. Ges. Wiirzburg, 1891, 122- 137. STEIN, R 1858. Ueber mehrere neue im Pansen der Wiederkauer lebende Infusions- thiere. Abh. d. Kais. Bohm. Ges. Wiss., 10, 69-70. 1859. Characteristik neuer Infusorien-Gattungen. Lotos, 9, 2-5, 57-60. 1861. Ueber d. Conjugation d. Infusionsthiere u. iiber d. geschlechtliehe Fortpflanzung d. Stentoren. Sitzber. d. Kais. Bb'hm. Ges. d. Wiss., 1861, 62-77. 112 University of California Publications in Zoology [VOL. 13 1867. Der Organismus der Infusionsthiere nach eigenen Forschungen in systematischer Keihenfolge bearbeitet. (1) Darstellung der neues- ten Forschungsergebnisse iiber Bau, Fortpflanzung und Entwicke- lung der Infusionsthiere. (2) Naturgeschichte der heterotrichen Infusorien (Leipzig, Englemann), viii + 355, 16 pis. ZiiRN, F. A. 1872. Die Schmarotzer auf und in dem Kb'rper unserer Haussaugetiere, sowie die durch erstere veranlassten Krankheiten, deren Behand- lung und Verhiitung. II. Teil: Die pflanzlichen Parasiten (Weimar, Voigt), 1 Aufl., pp. xvi + 474, 4 pis. ZURN, F. A., und PLAUT, H. . 1887-1889. Die Schmarotzer auf und in dem Korper unserer Haussauge- tiere, sowie die durch erstere veranlassten Krankheiten, deren Behandlung und Verhiitung. II. Teil: pflanzliche Parasiten (Wei- mar, Voigt), 2 Aufl., xvi -(- xvi + 837, 4 pis., 2 figs, in text. WEISS, D. C. F. H. 1869. Specielle Physiologie der Haussaugethiere fur Thierarzte und Land- wirthe (Stuttgart, Metzler), xii + 548, 80 figs, in text. EXPLANATION OF PLATES PLATE 3 Diplodinium ecaudatum forma ecaudatum Fiorentini (Camera lucida drawings) Fig. 1. View of right side of body, showing surface markings, underlying structures shown in outline. X 1400. Fig. 2. View of left side of body, showing surface markings, underlying structures shown in outline. Same animal as figure 1, viewed from opposite side. X 1400. ABBREVIATIONS ador. m. s. adoral membranelle zone. ant. c. v. anterior contractile vacuole. D. dorsal surface of the body. d. in. z. dorsal membranelle zone. 1. sic. a. left skeletal area. mac. macronucleus. mic. micronucleus. post. c. v. posterior contractile vacuole. r. sk. a. right skeletal area. V. ventral surface of the body. v. sic. a. ventral skeletal area. [114] PLATE 4 Diplodinium ecaudatum forma ecaudatum Fiorentini (Fixed in Zenker's fluid and stained with modified Mallory's connective tissue stain. Camera lucida drawings, colored to match the stain.) Fig. 3. Median sagittal section constructed from superimposed camera lucida drawings from three sections, each 5 microns thick. X 1500. Compare with microphotographs, plate 6, figures 12-18, and plate 7, figure 33. Fig. 4. Cross-section through region of dorsal membranelle zone from three sections, each 6 microns thick. X 1500. Compare with microphotographs, plate 7, figures 23-25. Fig. 5. Cross-section (same series as fig. 4) through the micronueleus (cf. fig. 3). X 1500. Compare with microphotograph, plate 7, figure 29. ABBREVIATIONS ador. m. adoral membranelles. In figure 3 the leader is carried beyond the mem- branelle. an. anus. ant. cil. r. anterior ciliary roots. ant. c. v. anterior contractile vacuole. bd. 1. boundary layer (ectoplasmic). dr. oes. r. circumeosophageal ring. caec. caecum. cut. cuticle. c. v. r. region about contractile vacuole. D. dorsal side of body. d. disk dorsal disk. d. fur. dorsal furrow. d. m. str. dorsal motor strand. d. m. dorsal membranelles. ect. ectoplasm. ent. entoplasm. fd. vac. food vacuoles. i. ador. lip inner adoral lip. i. d. lip inner dorsal lip. L. left side of body. 1. sic. a. left skeletal area. mac. macronucleus. mic. micronueleus. m. m. motor mass (motorium). o. ador. fur. outer adoral furrow. o. ador. lip outer adoral lip. o. d. fur. outer dorsal furrow. o. d. lip outer dorsal lip. oes. oesophagus or cytopharynx. oes. f. oesophageal fibers. oes. retr. str. oesophageal rectrator strands. op. operculum. op. f. opercular fibers. or. oral opening, mouth, or cytostome. or. cil. oral cilia. or. disk oral disk. post. cil. r. posterior ciliary roots. post. c. v. posterior contractile vacuole. JR. right side of body. rect. rectum. red. f. rectal fibers. r. sk. a. right skeletal area. sk. lam. skeletal laminae. susp. f. suspensory fibers. V. ventral side of body. v. and r. sk. lam. ventral and right skeletal laminae. v. sk. a. ventral skeletal area. 11. m. nuclear membrane. [116] PLATE 5 (Camera lucida drawings from whole mounts) Fig. 6. Diplodinium ecaudatum forma caudatum Fiorentini. From left side. X 700. Fig. 7. Diplodinium ecaudatum forma bicaudatum forma nova. From left side. X 700. Fig. 8. Diplodinium ecaudatum forma tricaudatum forma nova. Posterior one-fourth of body from left side. X 700. Fig. 9. Diplodinium ecaudatum forma quadricaudatum forma nova. From left side of body. X 700. Fig. 10. Diplodinium ecaudatum forma cattanei. From right side of body. X 700. ABBREVIATIONS sp. 1 primary spine. sp. 2 secondary spine. sp. 3 tertiary spine. sp. 4 quaternary spine. sp. 5 quintary spine. [118] UNIV. CALIF. PUBL. ZOOL. VOL. 13 [SHARP] PLATE 5 Sp. 1. if w) Sp. 3. Sp.2. Sp.l. '8 Sp. 1. Sp.2. Sp.4. Sp.3. Sp.3. Sp.2. \ Sp.4. Sp.5. Sp. 1. 10 PLATE 6 Microphotographs. Diplodinium ecaudatum forma ecaudatum fixed in Schau- dinn 's alcoholic sublimate solution, stained in Heidenhain 's iron-haexmatoxylin. imbedded in paraffin, and sectioned at 5/j.. Nine sections in series. X 600. Fig. 11; Tangential section, right side of the anterior one-half of animal nearer to the ventral than to the dorsal side; shows surface markings of the right skeletal area. Fig. 12. Note the anterior end of the macronucleus, which lies close to the right dorsal wall of the body. Fig. 13. Shows right extremity of dorsal membranelle zone, right side of adoral membranelles, outlines of oesophagus and micronuclens. Fig. 14. Note especially the cut ends of the opercular fibers (see op. f., pi. 4, fig. 3) and the faint outline of the circumoesophageal ring. Fig. 15. Almost a median sagittal section at anterior end, but comes to sur- face of body on right side at posterior end. Shows dorsal neuromotor mass (motorium), oesophageal retractor strands, circumoesophageal ring, boundary layer between ectoplasm and entoplasm, anterior contractile vacuole, posterior limit of macronucleus, and ventral skeletal laminae. Fig. 16. Shows food vacuoles in entoplasm, ventral skeletal laminae, faint outline of posterior contractile vacuole between entoplasm and ectoplasm just dorsal to posterior end of macronucleus; and at the same level, near the ventral wall, may be seen the darkly stained granular faecal mass filling the caecum. Fig. 17. Note posterior contractile vacuole. Fig. 18. Shows rectum and anus. Fig. 19. Adoral membranelles and rectum. [120] UNIV. CALIF, PUBL. ZOOL, VOL 13 [SHARP] PLATE 6 ie 17 19 PLATE 7 Figs. 20-32. Cross-sections of Diplodinium ecaudatum, fixed in Zenker's fluid, stained in modified Mallory's connective tissue stain, embedded in paraf- fin, and sectioned at 6 microns. Microphotographs. X 750. This series (series A-2-c-l) contains twenty sections, of which only eleven are here reproduced, figure 20 being the second section of the series and figure 32 the nineteenth section; figures 29 and 30 are taken from a second series (series A-2-C-2). Section 1, although clear on the slide, could not be satisfactorily photographed. Fig. 20. Section 2 of series A-2-c-l is cut somewhat obliquely with refer- ence to the longitudinal axis of the body, but in an almost exact transverse plane with reference to the oral and adoral region of the animal. Note the oral opening (cf. fig. A) and adoral membranelles. Fig. 21. Section 3, series A-2-c-l. Although cut more deeply than figure 20, this section shows the oral and adoral arrangement to even better advan- tage (cf. figs. A, C). Pig. 22. Shows the left extremity of the adoral row of membranelles. Figs. 23-25. Sections 5-7, series A-2-c-l. Taken through the dorsal mem- branelle zone. Compare with plate 4, figure 4. Note especially the darkly stained oesophageal fibers (cf. oes. f., pi. 4, fig. 4) and the dorsal motor strand (cf. d. TO. sir., pi. 4, fig. 4). Fig. 26. Note outer and inner dorsal lips (cf. o. d. lip and i. d. lip, pi. 4, tig. 4) and the three skeletal regions. Fig. 27. Section 9, series A-2-c-l. Shows anterior end of macronucleus and areolar, contractile vacuole region (cf. c. v. r., pi. 4, fig. 3). Fig. 28. Shows especially well the anterior contractile vacuole. Fig. 29. Section 11, series A-2-C-2. This series is from a somewhat larger animal and was taken from an ox which had not been fed for some 18 hours. Note that there are no food vacuoles in the entoplasm. Shows macronucleus and micronucleus and proximity of oesophageal wall to the micronucleus (cf. pi. 4, fig. 4). Fig. 30. Section 15, series A-2-C-2. Shows posterior contractile vacuole. Fig. 31. Section 17, series A-2-c-l. Through posterior extremity of macro- nucleus and posterior contractile vacuole, and through junction of rectum and caecum. Fig. 32. Section 19, series A-2-c-l. Shows rectum enlarging into anal slit. Fig. 33. Paramedian section of D. ecaudatum forma ecaudatum, just to right of median sagittal plane. Microphotograph of 15 y. section. X 600. (Cf. pi. 4, fig. 3.) [122] UNIV. CALIF. PUBL. ZOOL VOL. 13 [SHARP] PLATE 7 UNIVERSITY OF CALIFORNIA PUBLICATIONS (Continued) 6. On the Skeletal Morphology of Gonyaulax r.aienata (Levander), by Charles Atwood Sofoid, Pp. 287-294, plate 18. 6. Dinoflagellata of the San Diego Eegion, V. On Spiraulax, a New Genus of the Peridinida, by Charles Atwood Kofoid. Pp. 295-300, plate 19. Nos. 4, 5, and 6 in one cover. September, 1911 IJHJ 7. Notes on Some Cephalopods in the Collection of the University of Cali- fornia, by S. S. Berry. Pp. 301-310, plates 20-21. September, 1911. .10 8. On a Self -Closing Plankton Net for Horizontal Towing, by Charles Atwood Kofoid. Pp. 311-348, plates 22-25. 9. On an Improved Form of Self-closing Water-bucket for Planktoa In- vestigations, by Charles Atwood Kofoid. Pp. 349-352. Nos. 8 and 9 in one cover. November, 1911 _. M Index, pp. 353-357. Vol. 9. 1. The Horned Lizards of California and Nevada of the Genera Phryno- soma ad Anota, by Harold C. Bryant. Pp. 1-84, plates 1-9. Decem- ber, 1911 70 2. On a Lyinphoid Structure Lying Over the Myelencephalon of Lepisos- teus, by Asa C. Chandler. Pp. 85-104, plates 10-12. December, 1911. .25 5. Studies on Early Stages of Development in Eats and Mice, No. 3, by E. L. Mark and J. A. Long. The Living Eggs of Eats and Mice with a Description of Apparatus for Obtaining and Observing Them (Pre- liminary paper), by J. A. Long. Pp. 105-136, plates 13-17. February, 1912 30 4. The Marine Biological Station of San Diego, Its History, Present Con- ditions, Achievements, and Aims, by Wm. E. Sitter, Pp. 137-248, plates 18-24, and 2 maps. March, 1912 _ l.Ofl 6. Oxygen and Polarity in Tubularia, by Harry Beal Torrey. Pp. 249- 251. May, 1912 OS 6. The Occurrence and Vertical Distribution of the Copepod* of the San Diego Eegion, with particular reference to Nineteen Species, by Cal- vin O. Esterly. Pp. 253-340, 7 text-figures. July, 1912 1.0Q 7. Observations on the Suckling Period in the Guinea-Pig, by J. Marlon Eead. Pp. 341-351. September, 1912 _ .10 8. Haeokel's Sethocephnlus eucecryphalus (Eadiolaria), a Marine Oiliate, by Charles Atwood Kofoid. Pp. 353-357. September, 1912 .05 Index, pp. 359-365. VoL 10. (Contributions from the Museum of Vertebrate Zoology.) 1. Eeport on a Collection of Birds and Mammals from Vancouver Island, by Harry S. Swarth. Pp. 1-124, plates 1-4. February, 1912 1.00 2. A New Cony from the Vicinity of Mount Whitney, by Joseph GrinneU. Pp. 125-129. January, 1912 05 5. The Mole of Southern California, by J. Grinnell and H. S, Swarth. Pp. 131-136, 2 text-figures. 4. Myotis orinomus Elliott, a Bat New to California, by J. GrinneU and H. S. Swarth. Pp. 137-142, 2 text-figures. Nos. 3 and 4 in one cover. April, 1912 12 5. The Bighorn of the Sierra Nevada, by Joseph Grinnell. Pp. 143-153, 4 text-figures. May, 1912 10 8. A New Perognafhus from the San Joaquin Valley, California, by Walter P. Taylor. Pp. 155-166, 1 text-figure. 7. The Beaver of West Central California, by Walter P. Taylor. Pp. 167-169. Nos. 6 and 7 in one cover. May, 1912 15 8. The Two Pocket Gophers of the Eegion Contiguous to the Lower Colo- rado Eiver, in California and Arizona, by Joseph Grinnell. Pp. 171- 178. June, 1912 15 9. The Species of the Mammalian Genus Sorcx of West-Central Cali- fornia, with a note on the Vertebrate Palustrine Faunas of the Eegion, by Joseph Grinnell. Pp. 179-195, figs. 1-6. March, 1913 15 10. An Account of the Birds and Mammals of the San Jacinto Area of Southern California, with Remarks Upon the Behavior of Geographic ^ Races on the Margins of Their Habitats, by J. Grinnell and H. S. Swarth. Pp. 197-406, pis. 6-10. October, 1913 2.00 Index, pp. 407-417. UNIVERSITY OF CALIFORNIA PUBLICATIONS (Continued) Vol. 11. 1. Birds in Relation to a Grasshopper Outbreak in California, by Harold C. Bryant. Pp. 1-20. November, 1912 20 2. On the Structure and Relationships of Dinosphaera palustris (Lemm.), by Charles Atwood Kofoid and Josephine Rigden Michener. Pp. 21- 28. December, 1912 10 3. A Study of Epithelioma Contagiosum of the Common Fowl, by Clifford D. Sweet. Pp. 29-51. January, 1913 25 4. The Control of Pigment Formation in Amphibian Larvae, by Myrtle E. Johnson. Pp. 53-88, plate 1. March, 1913 85 6. Sayitta calif ornica, n. sp., from the San Diego Region, including Remarks on Its Variation and Distribution, by Ellis L. Michael. Pp. 89-126, plate 2. June, 1913 .35 6. Pycnogonida from the Coast of California, with Description of Two New Species, by H. V. M. Hall. Pp. 127-142, plates 3-4. August, 1913. .20 7. Observations on Isolated Living Pigment Cells from the Larvae of Amphibians by S. J. Holmes. Pp. 143-154, plates 5-6. 8. Behavior of Ectodermic Epithelium of Tadpoles when Cultivated IB Plasma, by S. J. Holmes. Pp. 155-172, plates 7-8. Nos. 7 and 8 in one cover. September, 1913 30 9. On Some Californian Schizopoda, by H. J. Hansen. Pp. 173-180, pi. 9. November, 1913 10 10. Fourth laxonomic Report on the Copepoda of the San Diego Region, by Calvin O. Esterly. Pp. 181-196, pis. 10-12. November, 1913 .15 11. The Behavior of Leeches with Especial Reference to Its Modifiability, A. The General Reactions of the Leeches Dina microstoma Moore and Glossiphonia stagnates Linnaeus; B. Modifiability in the Behavior of the Leech Dina microstoma Moore, by Wilson Gee. Pp. 197-305, 13 text figures. December, 1913 _. 1.00 12. The Structure of the Ocelli of Polyorchis penicillata, by Etta Viola Little. Pp. 307-328, plates 13-15. February, 1914 20 13. Modifications and Adaptations to Functions in the Feathers of Circus , Jmdsonius, by Asa C. Chandler. Pp. 329-376, plates 16-20. March, 1914 50 14. A Determination of the Economic Status of the Western Meadowlark (Sturnella neglecta) in California, by Harold Child Bryant. Pp. 377- 510, plates 21-24, 5 text figures. February, 1914 1.25 15. Parasynaptic Stages in the Testis of Aneides lugubris (Hallowell), by Harry James Snook and J. A. Long. Pp. 511-528, plates 25-26, 1 text fig. April, 1914 25 Vol. 12. 1. A Study of a Collection of Geese of the Urania canadensis Group from the San Joaquin Valley, California, by Harry S. Swarth. Pp. 1-24, plates 1-2, 8 text figs. November, 1913 SO 2. Nocturnal Wanderings of the California Pocket Gopher, by Harold C. Bryant. Pp. 25-29, 1 text fig. November, 1913 05 5. The Reptiles of the San Jacinto Area of Southern California, by Sarah Rogers Atsatt. Pp. 31-50. November, 1913 .20 4. An Account of the Mammals and Birds of the Lower Colorado Valley, with Especial Reference to the Distributional Problems Presented, by Joseph Grinnell. Pp. 51-294, plates 3-13, 9 text figs. March, 1914. 2.40 5. Aplodontia chryseola, a New Mountain Beaver from the Trinity Region of Northern California, by Louise Kellogg. Pp. 295-296. 6. A Previously Undescribed Aplodontia from the Middle North Coast of California, by Walter P. Taylor. Pp. 297-300. Nos. 5 and 6 in one cover. April, 1914 05 7. A Second Species of the Mammalian Genus Microdipodops from Cali- fornia, by Joseph Grinnell. Pp. 301-304. April, 1914 05 Vol. 13. 1. The Schizopoda of the San Diego Region, by Calvin O. Esterly. Pp. 1-20, plates 1-2, April, 1914 15 2. A Study of the Occurrence and Manner of Distribution of the -Cteno- phora of the San Diego Region, by Calvin O. Esterly. Pp. 21-38. April, 1914 15 3. A New Self -Regulating Paraffin Bath, by C. W. Woodworth. Pp. 39- 42, 2 text-figures. April, 1914 05 4. Diplodinium ecaudatum, with an Account of Its Neuromotor Apparatus, by Robert G. Sharp. Pp. 43-122, plates 3-7, 4 text figures. May, 1914 .80 5. The Vertical Distribution and Movements of the Schizopoda of the San Diego Region, by Calvin O. Esterly. Pp. 123-145. May, 1914 20 U.C.BERKELEY LIBRARIES NON-CIRCULATING BOOK 27V448 UNIVERSITY OF CALIFORNIA LIBRARY