- \\- ON BALANTIDIUM COLI (MALMSTEN) AND BALANTIDIUM SUIS (SP. NOV.), WITH AN ACCOUNT OF THEIR NEURO- MOTOR APPARATUS A THESIS ACCEPTED IN PARTIAL SATISFACTION OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE UNIVERSITY OF CALIFORNIA BY JAMES DALEY McDONALD 1922 ' ' *- UNIVERSITY OF CALIFORNIA PUBLICATIONS IN ZOOLOGY Vol. 20, No. 10, pp. 243-300, pis. 27-28, 15 figures in text May 8, 1922 ON BALANTIDIUM COLI (MALMSTEN) AND BALANTIDIUM SUIS (SP. NOV.), WITH AN ACCOUNT OF THEIR NEURO- MOTOR APPARATUS BY j. DALEY MCDONALD UNIVERSITY OF CALIFORNIA PRESS BERKELEY, CALIFORNIA 1922 UNIVERSITY OF CALIFORNIA 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 ali the publications of the University will be sent upon request. 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Mitosis in Giardia microti, by William C. Boeck. Pp. 1-26, plate 1. October, 1917 35 2. An Unusual Extension of the Distribution of the Shipworm in San Francisco Bay, California, by Albert L. Barrows. Pp. 27-43. December, 1917 .20 3. Description of Some New Species of Polynoidae from the Coast of Cali- fornia, by Christine Essenberg. Pp. 45-60, plates 2-3. October, 1917 20 4. New Species of Amphinomidae from the Pacific Coast, by Christine Esson- berg. Pp. 61-74, plates 4-5. October, 1917 15 5. Crithidia euryophthalmi, sp. nov., from the Hemipteran Bug, Eury aphtha! mus convivus Stal, by Irene McCulloch. Pp. 75-88, 35 figures in text. Decem- ber, 1917 15 6. On the Orientation of Erytliropsis, by Charles Atwood Kofoid and Olive Swezy. Pp. 89-102, 12 figures in text. December, 1917 .15 7. The Transmission of Nervous Impulses in Relation to Locomotion in the Earthworm, by John F. Bovard. Pp. 103-134, 14 figures in text. January, 1918 35 8. The Function of the Giant Fibers in Earthworms, by John F. Bovard. Pp. 135-144, 1 figure in text. January, 1918 10 9. A Rapid Method for the Detection of Protozoan Cysts in Mammalian Faeces, by William C. Boeck. Pp. 145-149. December, 1917 05 10. The Musculature of Heptanchus maculatus, by Pirie Davidson. Pp. 151-170, 12 figures in text. March, 1918 25 11. The Factors Controlling the Distribution of the Polynoidae of the Pacific Coast of North America, by Christine Essenberg. Pp. 171-238, plates 6-8, 2 figures in text. March, 1918 75 12. Differentials in Behavior of the Two Generations of Salpa democratica Relative to the Temperature of the Sea, by Ellis L. Michael. Pp. 239-298, plates 9-11, 1 figure in text. March, 1918 65 13. A Quantitative Analysis of the Molluscan Fauna of San Francisco Bay, by E. L. Packard. Pp. 299-336, plates 12-13, 6 figures in text. April, 1918 40 14. The Neuromotor Apparatus of Euplotcs patella, by Harry B. Yocom. Pp. 337-396, plates 14-16. September, 1918 70 15. The Significance of Skeletal Variations in the Genus Peridinium, by A. L. Barrows. Pp. 397-478, plates 17-20, 19 figures in text. June, 1918 90 ' * ' J J ON BALANTIDIUM COL I (Malmsten) AND BALANTIDIUM St WITH AN ACCOUNT OF THE i NEUROMOTOR APPARj by J.Daley McDonald - - Submitted in partial fulfillment of the requir* for the degree of Doctor of Philosophy Is Approved : ci-fr^ Pasadena, California UNIVERSITY OF CALIFORNIA PUBLICATIONS IN ZOOLOGY Vol. 20, No. 10, pp. 243-300, pis. 27-28, 15 figures in text May 8, 1922 ON BALANTIDIUM COLI (MALMSTEN) AND BALANTIDIUM SUIS (SP. NOV.), WITH AN ACCOUNT OF THEIR NEURO- MOTOR APPARATUS BY j. DALEY MCDONALD CONTENTS PAGE Introduction 244 Acknowledgments : 245 Material and technique 245 Occurrence and geographic distribution 246 Studies of living organisms 246 Systematic position of genus and species 248 Balantidium coli 249 Balantidium suis sp. nov 250 Method and use of measurements 254 Other specific characters 258 Balantidium from man 261 Morphology 262 Ectoplasmic structures 263 Pellicle , 263 Ectoplasm 266 Cilia 270 Basal apparatus of cilia 271 Ciliary movements 273 Cytostome 276 Oral plug 280 Contractile vacuoles 280 Endoplasmic structures 282 Endoplasm 282 Food vacuoles , '. 282 Macronucleus 282 Micronucleus 283 Neuromotor apparatus 284 Motorium .' 285 Circumoesophageal fiber 285 Adoral ciliary fiber 286 244 University of California Publications in Zoology [VOL. 20 PAGE Adoral ciliary rootlets 286 Radial fibers . 286 Discussion 288 Summary 293 Literature cited , 294 Explanation of plates 298 INTRODUCTION The earliest observation of Protozoa of the genus Balantidium has in several instances been accredited to Antony von Leeuwenhoek (1708). During an attack of dysentery he detected motile organisms in the discharges. At that time no discrimination had been made between ciliated and flagellated protozoa and his account of his obser- vations is not sufficiently complete to make possible the classification of the organisms which he found. However, he stated that they were about the size of red blood corpuscles, which would indicate that they were intestinal flagellates and not Balantidium, which is very much larger. Malmsten (1857) was the first to describe Balantidium coli. This species has become better known than the other species of the genus, due to its being the cause of a specific dysentery known as balantidiasis. Two persons suffering from this disease came to Malmsten for medical attention during 1856-57. Pie was assisted in the study of protozoans which he found in the excreta from these two patients by the zoologist Loven who believed that the parasites were new to science and so pre- pared a careful description of them accompanied by figures. For the organism they suggested the name Paramoecium ( ?) coli. Since that time infections with Balantiddum coli have been reported in increasing numbers and some cytological studies 'have been made, though much more attention has been given to the problems of prophylaxis and treatment of the disease which this species causes than to the parasite itself. The first record of Balantidium coli as a parasite of pigs was made by Leuckart (1861). Stein (1862) also studied these forms from pigs and he was the first to assign them to the genus Balantidium. The genus had been established by Claparede and Lachmann (1858) with Balantidium entozoon from the frog as the type species. More recently Strong (1904), Brumpt (1909), Walker (1913), and others have car- ried on investigations on this parasite of pigs in order to become acquainted with the problems involved in the infection of man. 1922] McDonald: On Balantidium eoli and Balantidium suis 245 ACKNOWLEDGMENTS It has been my privilege to study the morphology of Batantidium coli and Balantidium suis (sp. nov.) under the direction of Professor Charles A. Kof oid, to whom I am indebted for helpful suggestions and for oversight of the entire work. Acknowledgment is due Professor William W. Cort for many valuable criticisms. I also take this oppor- tunity to express my appreciation of the courtesy of Mr. E. B. Brown, superintendent of the Oakland Meat and Packing Company, who kindly granted me permission to work in the company's abattoir and also facilitated the work in every possible way. MATERIAL AND TECHNIQUE The material for these studies was obtained almost exclusively from pigs killed by the Oakland Meat and Packing Company, Stockyards, California. At their abattoir I was permitted to work in the room where the pigs were dressed, which made it possible to obtain the material from the intestine before it had cooled below the normal body temperature. To determine the presence of the balantidia a small slit was made in the caecum and a drop of the contents withdrawn with a pipette. This drop was quickly placed on a warm slide and exam- ined with a microscope. If the animals were present they would be detected very readily for they are exceedingly active ; in most cases they occurred in numbers sufficiently large that from one to ten could be seen in every field when a 16 mm. objective was used. This method was rapid enough to allow all pigs to be examined as fast as they were killed and dressed. A sample from the caecum was not relied upon as critical in the determination of infection until examination of the entire length of the intestine had been made in several instances. In order to discover the normal distribution throughout the intestine it was removed entire and taken to the laboratory of the abattoir. Incisions were made every one or two feet, beginning with the duodenum and continuing to the rectum, and samples examined from each of these incisions. In no cases were balantidia found more than three feet above the ileocaecal valve, and only in two or three instances were any at all present in 246 University of California Publications in Zoology [ VoL - 20 the small intestine. In the caecum and first three or four feet of the colon the balantidia were always more active and more numerous than elsewhere. Posteriorly from this region they were found in progressive stages of encystment until in the rectum the majority were completely encysted. OCCURRENCE AND GEOGRAPHIC DISTRIBUTION Approximately 200 pigs were examined. They had been raised in the Sacramento Valley, except for one lot from Los Banos, California, and a lot from the state of Nevada. Of the 200 pigs examined 68 per cent were infected. The examinations were made at nine separate times between September, 1913, and May, 1918, ten to sixty individuals being examined each time. In five of the nine lots every pig was found to be infected. The lowest percentage of infection was 13 per cent, in the lot shipped from Nevada. This indicates a very general infection of pigs with Balantidium in this region of the United States. Stiles (cit. Strong, 1904), Bel and Couret (1910), and others have previously found the organisms in pigs in the United States. Leuckart (1861), working in Germany, was the first to find Balantidium in pigs. Since then Stein (1862), Eckecrantz (1869), and Prowazek (1913) have reported them from the same country. In 1871 Wising noted their occurrence in pigs in Sweden. Grassi (1882) and Calandruccio (1888) have found the parasites in swine in Italy. Rapchevski (1882) reported the occurrence of balantidia in Russia. In France they have been found in pigs by Railliet (1886), Neumann (1888), and Brumpt (1909). Strong (1904), Walker (1913), and several others have noted the occurrence in pigs in the Philippine Islands. Similar reports from China have been made by Maxwell (1912), and Mason (1919) ; from Cuba by Taboadela (1911) ; and from South America by Bayana (1918). These citations indicate that BalantiMum coli is probably as widely distributed geographically as is its host, the pig. STUDIES OF LIVING ORGANISMS The balantidia are very sensitive to changes of temperature. When the medium in which they are swimming is cooled a few degrees they slow up their movements very decidedly. After a time they become almost perfectly spherical, in which form their activitiy is restricted to a rotary motion with little or no progression. In this condition they will live for six or eight hours at ordinary room temperature. 1922] McDonald: On Balantidium coli and Balantidium suis 247 In order to avoid the deleterious effects caused by cooling and by increased bacterial action, most of the studies on living organisms were made at the abattoir. When continuous observation over a long period was desired, however, the material was conveyed to the laboratory in thermos bottles and kept in the incubator at 37.5 C. In tfris~manner material could be kept for three days. Ultimate degeneration of the organisms seemed to be due more to the increase in the bacterial con- tent of the medium than to any other cause. During observation either an electric warm stage or the microscope warm oven designed by Long (1912) was employed. Of the several vital stains used, neutral red proved most satisfactory in the differentiation of the neuromotor apparatus. Fixation and staining. The following fixatives were used : Schau- dinn's fluid, Zenker's fluid, formalin, osmic acid, and picromercuric fluid (according to the formula by A. D. Drew, used by Yocom, 1912). Quick action was one of the most important factors in the fixation, and was usually obtained by having the killing fluid hot (60-80 C.) and using an amount at least equal to the amount of the material to be fixed. Frequently the action was so nearly instantaneous that the cilia on the killed animals retained their exact relative position (see fig. N). After fixation the material was thoroughly washed, iodine alcohol being used if mercurial salts were present. Material was pre- served in 70 per cent alcohol. Before staining, the preserved material was usually concen- trated by elimination of lighter debris by centrifuging and the heavier by sedimentation. Water was found to be a more satisfactory medium for these operations than either alcohol or salt solutions. In case sec- tions were to be made, additional care was taken in the concentration process and then the material was handled according to the methods employed by Metcalf (1909) and by Sharp (1914). Iron haematoxylin gave uniformly the best results in staining. For cysts, however, on account of their imperviousness, it was necessary to use Delafield's haematoxylin to which had been added a small amount of acetic acid. In addition to the first mentioned stain, Mallory's con- nective tissue stain was used on sections. 248 University of California Publications in Zoology [ V L - 20 SYSTEMATIC POSITION OF GENUS AND SPECIES Claparede and Lachmann (1858) removed Bursaria entozoon from the genus in which it had been placed by Ehrenberg (1838) and created for it the new genus Balantidium. This genus was of the family Bursaridae and the order Heterotricha. The twenty-two species of the genus that have been described to date are listed below. KNOWN SPEOIES OF THE GENUS BALANTIDIUM Species Balantidium entozoon Balantidium coli Balantidium duodeni Balantidium elongatum Balantidium medusarum Balantidium amphictenides Balantidium gyrans Balantidium viride Balantidium minutum Balantidium giganteum Balantidium helenae Balantidium graeile Balantidium Balantidium Balantidium Balantidium Balantidium Balantidium Balantidium Balantidium Balantidium Balantidium rotundum faleiformis ovale hyalinum littorinae testudinis hydrae piscicola caviae orchestia Original description by Ehrenberg, 1838 Malmsten, 1857 Stein, 1862 Stein, 1862 Mereschkowsky, 1879 Entz, Sr., 1888 Kellicott, 1889 Willach, 1893 Schaudinn, 1899 Bezzenberger, 1903 Bezzenberger, 1903 Bezzenberger, 1903 Bezzenberger, 1903 Walker, 1909 Dobell, 1910 Dobell, 1910 Chagas, 1911 Chagas, 1911 Entz, Jr., 1913 Entz, Jr., 1913 Neiva et al, 1914 Watson, 1916 Hosts Rana esculenta Rana temporaria Sus scrofa Homo sapiens Ban a esculenta Triton cristatus Triton alpestris Triton marmoratus Rana esculenta Rana temporaria Bougainvillea, Obelia, Eucope, Broda sp.? Amphictenis, Turbellaria marina Aquatic worm Columba sp.? Homo sapiens Rana esculenta Rana cyanophlyctis Rana tigrina Rana limnocharis Rana hexadactyla Rana cyanophlyctis Rana hexadactyla Rana esculenta Rana palustris Rana tigrina Rana tigrina Littorina Testudo graeca Hydra olygactis Piarectus brachypomus Cavia aperea Orchestia agilis Talorchestia longicornis 1922] McDonald: On Balantidium coli and Balantidium suis 249 The wide diversity of hosts, ranging from hydroids and crustaceans to the warm-blooded vertebrates, including man, must demand a wide versatility on the part of the parasite. Considerable structural varia- tion is apparent even on cursory examination, and some of these structural differences might be sufficiently marked to servTT for generic differentiation. A new generic division would seem desirable, but the suggestions of Biitschli (1884) and Schweier (1900) in this direction have not been generally accepted. BALANTIDIUM COLI MALMSTEN (1857) SYNONOMY: Paramoecium (?} coli Malmsten, 1857. Plagiotoma coli, Claparede and Lachmann, 1858. Leucophyra coli, Stein, 1860. Holophyra coli, Leuckart, 1861. Balantidium coli, Stein, 1862. Up to the present time only one species, Balantidium coli, has been described as parasitic in pigs. It was first described by Malmsten (1857) who, noting its likeness to Paramoecium colpoda (Ehrenberg), suggested the name Paramoecium (?) coli. During the following year, Claparede and Lachmann (1858) reproduced one of Malmsten 's orig- inal figures and after considering his description transferred the species to the genus Plagiotoma. In 1860, Stein, using the description by Malmsten (1857), pointed out that the organism was not a Para- moecium and Relieved that it properly belonged in the genus Leuco- phyra. Leuckart in 1861 discovered a ciliate in the intestine of pigs which he concluded was identical with the one already described, but he was not satisfied with the genus to which it had been assigned by Stein (1861) and believed that its closest relation was with Holophyra in which genus it should be placed. In. 1863 he still retained this view but suggested the appropriateness of the establishment of a new genus. But Stein (1862) had already recognized those characters of the species which showed its close relation to Balantidium entozoon and had placed it in the genus Balantidium. During the present investigation the following specific character- istics have been found very constant. The individuals of the species Balantidium coli are ovoid in form, the more pointed end being an- terior; length varies from 30/* to 150/t; breadth varies from 25,/A to 120ju, ; in the majority of individuals the length is 1.3 times the breadth ; the greatest transverse diameter intersects the longitudinal axis poster- 250 University of California Publications in Zoology [ VoL - 20 ior to its midpoint; the adoral zone is approximately terminal, and the anterior tip of the body lies within it; the plane of demarcation between the apical cone of the ectoplasm and the endoplasm is approx- imately at right angles to the long axis of the body ; the macronucleus is elongate but the length usually does not exceed three times the breadth; two contractile vacuoles are present, a smaller one located anteriorly and a larger one located posteriorly; a posterior cytopyge is usually distinctly visible. BALANTIDIUM suis SP. NOV. Early in the work of examining pigs for Balantidium coli it became evident that this protozoan showed extreme variation in shape. In many instances the diversity occurred among individuals from the same host. Further observations led me to believe that there were two fairly distinct types, the one, longer and more slender as compared with the other which was distinctly ovoid. Measurements have been recorded by various writers of Balantidium coli from man. Malmsten (1857) in the original description gave the length as 60-10CV; breadth, 50-70/i. Solojew (1901) recorded the length as 65/*, the breadth as 40/A. Wising (1871) states that the length varies from 50-100/z, while the breadth varies from 40-50/*. Prowazek (1913) gave the length as 52-71/x, the breadth, 40-58/*. Leuckart (1861) measured balantidia from swine and found the length to be 75-110/x and the breadth 70/x. Still others give dimensions, but all are inadequate for the determina- tion of the occurrence of types with distinct proportions. First, with one or two exceptions all dimensions have been taken of balantidia found in man, and from these it might not be safe to draw conclusions regarding diversity among those found in pigs. In the second place, the measurements given are either averages or else represent extreme limits. In either case they are practically useless in determining indi- vidual variations, for even in the case of extreme types the range between limits is so great that two, or even more, distinct types, based on proportions of breadth to length, might be included. Nowhere has there been found a series of individual measurements which would make it possible to determine whether variations were continuous or discontinuous. To obtain such a series of measurements was the pur- pose of the phase of the work about to be described. Material which was to be used in taking the measurements was killed and preserved with all possible care. Hot Schaudinn's fluid was used in all cases, the material being quickly and thoroughly mixed 1922] McDonald: On Balantid/ium coli and Balantidium suis 251 into a large quantity of it so that action would be as nearly instan- taneous as possible, thus avoiding distortion. Osmic acid vapor was tried but Schaudinn's fluid gave equally good results and was more convenient for manipulation. Material was never allowed to cool before fixing, for on cooling the individuals tend to become spherical. Several attempts were made to measure living organisms but their ceaseless activity at normal temperature (.37.5 C) made this almost impossible and slowing them up by the use of Irish moss or by cooling, as mentioned above, caused them to become distorted. If there were changes due to fixation, the logical expectation would be that the error would be on the side of conservatism for such changes would tend to obliterate rather than accentuate the division into two groups ; for the shape of the elongate forms would be more changed by the fixative, the tendency being for them to shorten and broaden and thus approach the ovoid type. However, in the method of fixation used, I am sure that distortion was so slight as to be negligible. Following fixation the material was carefully washed and carried slowly through the lower grades of alcohol to 70 per cent in which the material was kept for measuring. A drop of the material from which measurements were to be taken was placed on a slide, covered with a coverglass, the excess of fluid removed, and the edges sealed with vaseline to prevent evaporation. Just enough fluid was removed from under the coverglass to reduce the depth of the medium so that the majority of the animals would lie flat, and yet not enough to allow the coverglass to exert any pressure. The exertion of pressure on the animals, however, would ordinarily be prevented by the presence of large particles of foreign material. The object of having animals lie flat on the slide was to avoid the error which would otherwise be caused by foreshortening. A slight elevation of one end would make con- siderable error in the determination of the length of the animal. The slide was then placed on the microscope and systematically examined by the use of the mechanical stage. Beginning at the upper left-hand corner and progressing as one would in reading a book, every individual encountered in the survey was measured. The only excep- tions made were in case the animal was not lying flat or showed marked signs of distortion. This procedure avoided selection which might unconsciously be made by the observer. For making the measurements a 4 mm. objective was used in combination with an ocular-micrometer inserted in 9x compensating ocular. With the magnification given by this combination the limit of error did not exceed one micron. 252 University of California Publications in Zoology [VOL. 20 The longitudinal axis and the longest transverse axis of each indi- vidual were measured, and the ratio of length to breadth computed (see Table I). TABLE I COMPARATIVE MEASUREMENTS OF BALANTIDIUM COLI INDIVIDUALS FROM PIG No. 1 (Bal. suis, with five exceptions) ONE HUNDRED INDIVIDUALS EACH OF AND BALANTIDIUM suis INDIVIDUALS FROM PIG No. 4 (Bal. coli, with one exception) Length in microns Breadth in microns Ratio of length to breadth Length in microns Breadth in microns Ratio of length to breadth 114 42 2.71 99 75 1.32 108 51 2.11 96 81 1.19 63 36 1.75 126 75 1.68* 90 45 2.00 118 87 1.36 93 48 1.94 111 87 1.28 72 39 1.85 87 75 1.16 126 69 1.83 90 66 1.36 87 48 1.82 105 75 1.40 108 57 1.90 78 66 1.18 102 54 1.89 90 63 1.43 117 51 2.30 87 72 1.21 120 57 2.10 87 72 1.21 96 42 2.29 99 75 1.32 84 39 2.12 105 75 1.40 120 48 2.50 89 75 1.19 117 48 2.42 81 63 1.28 93 51 1.83 81 66 1.23 96 51 1.89 75 63 1.19 72 57 1.26* 84 69 1.22 75 54 1.39* 69 60 1.15 111 51 2.14 90 66 1.36 111 54 2.03 93 75 1.23 84 36 2.31 87 69 1.26 69 39 1.77 90 81 1.11 78 51 1.52* 105 90 1.16 81 45 1.80 87 69 1.26 111 45 2.42 84 72 1.17 81 45 1.80 66 58 1.14 84 45 1.86 75 60 1.25 90 45 2.00 81 63 1.29 84 42 2.00 81 60 1.35 90 42 2.12 99 78 1.27 117 57 2.03 87 69 1.26 60 33 1.82 78 63 1.24 108 57 1.90 81 60 1.35 108 57 1.90 105 75 1.40 96 48 2.00 90 60 1.50 75 42 1.79 93 78 1.18 * Other characters showed that these individuals were of the other species rep- resented in the table. 1922] McDonald: On Bala/ntid/ium coU and Ealantidium suis 253 TABLE I (Continued} INDIVIDUALS FROM PIG No. 1 (Bal. suis, with five exceptions) INDIVIDUALS FROM PIG No. 4 (Bal. coli, with one exception) Length in microns Breadth in microns Ratio of length to breadth Length in microns Breadth in microns Ratio of length to breadth 105 57 1.85 69 60 _ JL.15 99 57 1.74 81 54 1.50 105 54 1.95 87 66 1.32 54 27 2.00 84 69 1.22 57 30 1.90 78 60 1.30 36 24 1.50* 66 51 1.29 81 31 2.23 66 54 1.22 75 42 1.78 96 66 1.45 66 33 2.00 81 54 1.50 78 36 2.08 84 66 1.28 93 39 2.39 114 87 1.31 63 36 1.74 87 69 1.26 102 48 2.06 93 72 1.29 78 39 2.00 84 72 1.17 81 42 1.93 102 65 1.58 87 48 1.82 87 63 1.38 93 45 2.03 99 66 1.50 84 42 2.00 81 72 1.13 81 39 2.04 78 63 1.24 75 39 1.93 102 75 1.36 78 45 1.74 66 48 1.38 75 37 2.01 75 57 1.32 100 45 2.11 84 66 1.27 81 39 2.04 93 66 1.40 72 39 1.85 72 58 1.24 84 40 2.06 96 72 1.33 90 42 2.07 90 68 1.32 78 36 2.08 60 39 1.54 102 48 2.06 87 69 1.26 81 37 2.10 69 54 1.28 66 37 1.78 90 69 1.31 87 36 2.41 69 54 1.28 78 42 1.86 72 60 1.20 84 42 2.00 75 57 1.47 90 38 2.37 75 62 1.2X 66 35 1.89 72 63 1.14 78 42 1.86 90 75 1.20 97 44 2.20 74 66 1.12 79 37 2.07 72 54 1.33 98 42 2.32 60 50 1.20 90 45 2.00 84 58 1.45 87 49 1.78 84 64 1.31 90 36 2.50 88 60 1.47 81. 35 2.31 99 75 1.32 69 48 1.44* 75 54 1.39 * Other characters showed that these individuals were of the other species rep- resented in the table. 254 University of California Publications in Zoology [ V L. 20 TABLE I (Continued} INDIVIDUALS FROM Pia No. 1 (Bal. suis, with five exceptions) INDIVIDUALS FROM PIG No. 4 (Bal. coli, with one exception) Length in Breadth in microns microns Ratio of length to breadth 90 54 1.68 66 33 2.00 76 38 2.00 78 42 1.86 90 57 1.58 66 40 1.65 81 39 2.04 96 39 2.43 75 38 1.98 84 39 2.18 51 30 1.70 75 39 1.92 84 40 2.05 116 47 2.48 84 36 2.32 81 39 2.04 75 Aver- 45 1.68 age 86 43 1.99 Length in microns Breadth in microns Ratio of length to breadth 81 69 1.18 66 52 1.27 93 63 1.48 81 75 ' 1.08 96 75 1.28 114 90 1.27 75 60 1.25 87 72 1.21 84 60 1.40 87 63 1.38 84 63 1.33 72 60 1.20 90 66 1.36 84 72 1.16 93 72 1.29 87 58 1.50 93 72 1.29 86 66 1.30 While taking the measurements of each individual, observations were made regarding the position of the mouth, the type and size of macro- nucleus, number and location of contractile vacuoles, and any other characters which might aid in differentiation. In the handling of the data on dimensions I have followed in a general way the method used by Jennings (1908) in differentiating races of Paratniaecium. For the purpose of this work, however, the results seemed more lucid if, instead of plotting length and breadth along separate axes, the ratio of length to breadth was computed for each individual and if these ratios were then plotted on the abscissa while the numbers of individuals having each of these ratios were plotted on the ordinate. In computing the ratios the quotient was carried to the second decimal place. But in the construction of the curves only intervals of tenths (or first decimal place) were used; thus, for example, all ratios occurring between 1.25 and 1.34 inclusive were grouped as if they were 1.3. This had two advantages : first, it produced a smoother, steeper curve than would result if smaller inter- vals were taken, using the same number of individuals measured, and emphasized group rather than individual variations. Second, this grouping reduced any error which might result from the observer showing a preference for one graduation of the micrometer when 1922] McDonald: On Balantidium coli and Balantidium suis 255 an individual measured more than one but less than another whole division of the scale ; e.g., such a preference might result in the tabu- lation of several individuals having a length of 72/t, and of none with a length of 7 1/*,, though in reality all lay within these two limits and as many were as near to one as to the other. As previously mentioned, the graduations along the ordinate represent the number of individuals, each small interval representing one individual. In Jennings' (1908) work these intervals represent percentages of the total number of individuals. But it happens that I.I 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Fig. A. Graphic representation of the variation in the ratio of length to breadth among 200 Balantidium chosen at random from samples of material taken from several different pigs. The number of individuals is measured on the ordi- nate, the ratios on the abscissa. The dotted line is the curve resulting from the combination of the two curves shown in figure B, superimposed here to facilitate comparison. in the graphs shown in figures B and C, the number of individuals showing a certain ratio is identical with the percentage of the total, for in these cases the total is 100 individuals. Figure A represents graphically the result of the first attempt to determine the existence of different types. Measurements were made of 200 individuals. At least ten slides were used in getting these measurements and they were prepared from samples taken from nearly as many different pigs. It will be noted that the curve pro- duced by plotting the ratios of these individuals is decidedly bimodal. One mode represents those individuals which are approximately 1.2 times as long as wide, while the other represents those which are 1.6 to 1.8 times as long as wide. These findings seemed to fully justify my early suspicions that there were two very different types of balantidia parasitic in pigs. 256 University of California Publications in Zoology [VOL. 20 But it was decided to conduct one more experiment, for corroboration, under slightly different conditions and with especial care in fixation of the material. It had been noted that though often both types occurred in the same pig (which was the case in most of the samples used in the first measurements), still one type might be greatly in excess, or there might be only one type present. In getting material 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 Fig. B. Graphic representation of the difference in ratio of length to breadth between two carefully selected lots, of 100 individuals each, of Balantidium from separate hosts. The continuous line represents those from one pig (nearly all are Balantidium coli) ; the broken line, those from the other pig (nearly pure infection with Balantidium suis). Fig. C. This graph shows the variation in the ratio of length to breadth among 100 Balantidium secured from a case of balantidiasis in man. for this second set of measurements, it seemed best to take it from pigs which had, as nearly as could be determined, pure infections of the respective types. Fortunately these requirements were fulfilled in the next lot of pigs examined. Both samples of material, the one con- taining the ovoid and the one containing the elongate type, were treated in exactly the same way. They were killed at the same time 1922] McDonald: On Balantidium eoli and Balantidium suis 257 with the same fluid (in separate containers) and in the same water bath. Thence to 70 per cent alcohol the treatment continued identical. One hundred individuals from each sample of material were measured. From these measurements the graphs shown in figure B were con- structed in the same manner as the preyious one, except" that the curves of the separate samples were plotted separately on the same axis. The continuous line represents the individuals from one pig and the broken line those from the other. The mode of the first curve occurs at 1.3. If the ratios 1.2 and 1.4 be included with 1.3, it is found that 80 per cent came within these limits. Of the entire number of individuals in the lot only one showed a ratio of 1.6 and one as high as 1.7, while there were none with a higher ratio. The second curve reaches its highest point at 2.0, while 71 per cent of the entire number measured is included between 1.8 and 2.2. A number of individuals have ratios above 2.2, while one had a ratio as great as 2.7. Five indi- viduals have ratios below 1.6, at which point the curves begin to over- lap ; but in practically every one of these individuals there were observed characters (which are discussed below) that made it quite evident that they were really of the type represented by the other curve. Upon comparing figures A and B their likeness is very striking, the second being corroborative of the results shown by the first. That both are bimodal is evident. However, the low points, the point of demarcation of the two groups, do not occur at the same place ; in the first it is at 1.4, while in the second it occurs at 1.6. Also the median of the first mode in figure A occurs at 1.2 while in figure B it is at 1.3, and the median of the second mode in figure A is at 1.7 while in figure B it occurs at 2.0 ; that is, in figure A the entire curve is shifted to the left, meaning that all ratios are decreased or that all individuals approach nearer to the spherical shape. This shifting is greatest in the case of the second mode. This in conjunction with the greatest breadth of the second curve in figure B is what would be expected if the premise in regard to the effect of fixation discussed above (page 251) is correct. Extra care was used in the fixation of the latter lot of material whereas in the former only the ordinary pre- cautions were taken. At any rate these differences between the two groups do not detract from the evidence which they offer that there are two distinct types of balantidia parasitic in pigs. The value of these curves in showing race or species differentiation is directly proportional to the extent to which any other factors which 258 University of California Publications in Zoology [VOL. 20 might produce a bimodal curve are non-operative. Factors involved in faulty technique were eliminated as far as possible. Over the effect of growth or age variation, however, the observer has no control. The possibility of these variations producing such curves as the one above is precluded by reference to the data from which the curves are con- structed. Among the individual measurements recorded in Table I it will be noted that there are small individuals measuring 27 X 50/x, and others measuring 42 X 50/x; and that among the larger individ- uals, some measure 50 X 120/*, and some, 90 X 150/x. Further study of Table I shows that the two types are found among all sizes of individuals; consequently the variation of body proportions repre- sented by the graphs is not correlated with variations of size, and probably not with the age or growth of the individuals. That the variation could be accounted for by the occurrence of fission seems unlikely. Individuals might continue to elongate until binary fission occurred, and then by this process they might be shortened and the body proportion changed. Two considerations oppose this explanation. In figure A nearly equal numbers are of the respective types; in figure B each example contained almost exclusively one type of individual. In the former material very few dividing individuals were found, while in the latter not a single individual was seen in fission in either sample of material. But to accord with the above explanation one would expect to find many dividing forms among the elongate individuals represented by the broken line. In the second place, if this explanation were valid one would expect curves showing variation of body proportions to be continuous. Such is not the case, as is shown in figures A and B where the curve is bimodal due to a decided decrease of individuals having proportions intermediate between the two types. The possibility of any effect from gametic variation, was eliminated by the study of conjugating forms, through which it was determined that isogamy was the rule. Likewise the possibility of influence of the quality of intestinal content of the host was eliminated by the frequent occurrence of both types in the same host. Other factors it would seem must be of minor importance and should give way for more positively corroborative evidence which may be found in cor- related morphological difference. Other specific characters. In addition to the differences in relative lengths of the axes, one notes a distinct difference in the points of intersection, due to the variation in the shape of the types as pictured 1922] McDonald: On Balantidium coli and Balantidium suis 259 in figures D, E, F, G, and H. The one form resembles very closely a hen's egg, the small end being anterior. In this case the longest diameter crosses posterior to the midpoint of the longitudinal axis. In the elongate type, usually the posterior end is as much drawn to Figs. D-H. Camera lucicla drawings of Balantidium suis sp. nov. (figs. D and E), and Balantidium coli (figs. F, G, and H), showing specific differences. a point as is the anterior, and often more so. In these cases the longest diameter intersects the longitudinal axis at or anterior to its midpoint. Coincident with the taking of measurements a careful search was made to detect other morphological differences which might occur between the two forms and be of aid in distinguishing one from the other. The earliest difference to be noted related to the macronucleus. The macronucleus in the ovoid type is relatively short, being approxi- mately !/3 the length of the entire organism. It is customarily bean- shaped in appearance, but may be almost straight or so sharply bent 260 University of California Publications in Zoology [ V L. 20 at its middle as to form a short V (fig. I), and its width averages about 0.4 to 0.5 of its length. In. the slender types the macronucleus is rela- tively long and slender, being approximately % of the entire length of the organism. It is ordinarily sausage-shaped, but it may also be in the form of a straight rod slightly enlarged toward the ends, or it may be so curved as to form an almost complete ring. In contrast to the form described above, the width in this case is about 0.2 to 0.3 of its length. Differences so great as these, viz., a length 2 to 3 times the breadth in one case and 4 to 5 times the breadth in the other are easily recognizable without actual measurement. These figures rep- resent the average and do not mean that the limits of the two never overlap. This difference in nuclei serves as one of the easiest and surest ways of distinguishing the two types, for though the organism during locomotion may modify its proportions tremendously this does not noticeably affect the nucleus. It has been impossible to determine any difference between the micronuclei of the two types. A very noticeable difference concerns the relative position of the cytostome. In the ovoid type the cytostome is almost, though never quite, terminal (see figs. F, G, and H). As mentioned previously, the anterior end is ordinarily drawn out to form a fairly decided point which lies within the area enclosed by the adoral cilia. In the slender type the cytostome is more laterally placed. The posterior limit of the right lip of the cytostome may extend ventrally to a point % of the length of the animal, in which case the dorsal portion of the adoral circlet of cilia may pass approximately through the terminal point of the body, but in normal form this point will never be within the adoral area. The parts which make up the adoral region of the animals show no fundamental differences except variation in the rela- tive position of parts due to the lateral displacement of the cytostome. For example, the plane of demarcation between ectoplasm and endo- plasm, which is approximately vertical to the long axis of the animal in the ovoid form, is at a decided angle, the ventral edge lying farther posterior in the elongate forms (see figs. D and E). In the opinion of some authors the ventral displacement of the mouth is very significant (Delage and Herouard, 1896; Minchin, 1912). These authors believe that the ventral displacement of the cytostome is progressive with evolution of the organism; i.e., that in the more primitive types the cytostome is terminal while in advanced groups it is successively displaced farther ventrally. Viewed in this light, the position of the mouth is here of considerable significance as 1922] McDonald: On Balantidium coli and Balantidium suis 261 a specific character. Attempts to discriminate between the two forms on the basis of cytopyge or vacuoles were without result. The specific differences just discussed have seemed to indicate a sufficient degree of separation of the two types to warrant iho~ division of the ciliates of the genus Balantidium which occur in the pig into two distinct species. The description by Malmsten (1857), in con- junction with the figures (Malmsten, pi. 1, figs. 1-6) which he pub- lished, make it practically certain that the ovoid type is the one originally described by him and to which he gave the name Para- moecium (?) coli. So far as I have been able to determine, the elongate species above described has never before been distinguished from Balantidium coli. For this new species I suggest the name Balantidium suis. As a sum- mary of the specific characters discussed above I give the following description : Balantidium suis sp. nov. Body elongate; length approximately twice the breadth and varies from 35 to 120/* ; breadth from 20 to 60/x ; usually tapers more posteriorly, is blunter anteriorly, longest diameter transects longitudinal axis anterior to its midpoint ; adoral region ven- trally placed, cytostome % of way posteriorly along ventral surface; nucleus rod or sausage-shaped, at least one-half the length of the entire organism, its width about one-fourth of its length ; the species is para- sitic in the pig. The specific name, Balantidium suis, seemed fitting since it indi- cated the common host, Su$ scrofa. Whether or not this species occurs in man it has not been possible to determine conclusively. A review of published case records of balantidiasis seems to show that it does not, but only a few of these records are accompanied by figures or descriptions of the organisms which are adequate for making positive discrimination. Fortunately, I have been able in two cases to make some direct observations. Through the kindness of Mr. W. H. Barnes, of the Department of Pathology of the University of California, I was permitted to study sections of the human intestine which he had obtained at an autopsy following a fatal attack of balantidiasis. Imbedded in the serous and subserous layers were numerous balantidia. Measurements to show proportions were of little value under the conditions, for the form of each organism was largely determined by pressure, exerted by sur- rounding tissues. But from other characters, the type of nucleus especially, it was conclusively determined that the species there present was Balantidium coli. No individuals of Balantidium suis were found. 262 University of California Publications in Zoology [ V OL. 20 Measurements of balantidia as they occur free in the human intes- tine were made possible through the kindness of Dr. E. L. Walker, of the Hooper Institute of Medical Research, who loaned me several slides which he had prepared while in the Philippine Islands. This material had been stained with haematoxylin. Using the same pre- cautions as in previous work, a total of 100 individuals was measured. The data were handled as before and the resulting graph is shown in figure C. In comparison with previous graphs it will be noted that this graph closely approaches coincidence with the curves representing Balantidium coli, for its mode is at 1.3 while the extreme portion of length and breadth is 1.5. In addition to slides of human material, there was also loaned material from one pig and from one monkey. It is interesting that each showed a pure infection with Balantidium coli. The monkey (Monkey No. 10, Table I; Walker, 1913) had been experimentally infected by feeding it cysts from a pig, but not the pig from which the above-mentioned material was taken. Therefore this material yields no evidence regarding the validity of the specific differentiation nor the possibility of Balantidium suis becoming established in mon- keys or in man. There is no likelihood of confusing the new species, Balantidium suis with Balantidium minutum (Schaudinn, 1899). The differences are very marked. The body of the latter is oval, pointed anteriorly, more like Balantidium coli. The peristome reaches to the equatorial plane. There is but a single vacuole while there are two in each of the species considered here. The macronucleus is spherical, whereas it is elongate in both Balantidium coli and Balantidium suis. MORPHOLOGY Balantidium coli (Malmsten) and Balantidium suis sp nov. are ciliated protozoans, barely visible to the unaided eye, and are in a general way sac-shape (balantidium, little bag). Viewed through the microscope they appear grayish green in color. The homogeneity of the cell contents is broken by the presence of the nuclei, the contractile vacuoles, the food vacuoles, and sometimes by the presence of highly refractile bodies, the paramylum bodies. The entire surface of the body, except that of the oral plug, is covered with fine cilia. The cell contents are retained by a thin transparent pellicle which is protective 1922] McDonald: On Balantidium coU and Balantidium suis 263 in function. The cytoplasm is distinctly differentiated into ectoplasm and endoplasm. The former constitutes a thin layer just underneath the pellicle and in it is situated the basal apparatus of the cilia. The layer of ectoplasm thickens greatly at the anterior end of the animal, to form, as it were, a matrix for the cytostome and its accessory appa- ratus. Within the ectoplasm, but not set off from it by a sharp line of demarcation, is the endoplasm. In the endoplasm are numerous food inclusions, often present in the form, of starch or paramylum bodies. The macronucleus and the micronucleus are also within it, but seem to have no constant position in the cell. The .macronucleus is either bean-shaped (as in Balantidium coli) or elongate and sausage- shaped (as in Balantidium suis). There are two contractile vacuoles, the larger being situated anteriorly and the smaller posteriorly. They lie closely beneath or may be entirely surrounded by ectoplasm, thus belonging really within that layer. So far as can be determined the animals show no modification with respect to a substratum, yet the lateral and posterior displacement of the cytostome has lead to the designation of that side, toward which displacement occurs, as ventral, and the opposite surface as dorsal. This terminology is very nearly universal in the literature on Balan- tidium, and the correlated terms of right and left are used in the original description of the family Bursaridae (Stein, 1867) and the genus Balantidium (Claparede and Lachmann, 1858). The dorsal side may be somewhat more convex than the ventral ; this occurs not infrequently in Balantidium suis, though, due to the plasticity of the organism, this is by no means constant. No part of the body is differ- entiated for skeletal purposes. The anal aperture or cytopyge is at the posterior tip and may be present as an actual aperture or only as an extreme thinning of the ectoplasm and pellicle at this point. In connection with it there is usually a rectal vacuole which serves as a storage reservoir for solid waste awaiting extrusion. ECTOPLASMIC STRUCTURES Pellicle. The entire body is covered by an extremely thin but resistant pellicle (pel., figs. I and L). The pellicle seems to be some- what thickened, as shown by a higher degree of refractibility, along the margin of the lips of the cytostome where it turns in to form the lining of the oesophagus and the groove in which the oral cilia are set ; otherwise, it is nowhere noticeably specialized. It shows alternating 264 University of California Publications in Zoology E V L. 20 post. c. v Pig. I. Balantidium ooli. Ventral view showing principal structures. The adoral cilia, with exception of one at either side, are indicated by basal granules only. The adoral membranelles are also represented solely by the basal apparatus. Ectoplasm is shaded, and ciliary rootlets are shown on one side only. X 1250. ad. oil., adoral cilia; ad. cil. /., adoral ciliary fiber; ad. mcmb., adoral membranelles; ant. c. v., anterior contractile vacuole; bg., basal granule; bg. ad. cil., basal granules of adoral cilia ; cil., cilia ; oil. r., ciliary rootlet ; dr. oes. /., circumoesophageal fiber ; cytpg., eytopyge ; ect., ectoplasm ; enl. ad. cil. r., enlargement of adoral ciliary root- let; end., endoplasm; gr. b., granular band of ectoplasm; long. /., longitudinal fibers; moo., macronucleus ; mic., micronucleus ; mot., motorium; nuc.memb., nuclear membrane; oes., oesophagus; or.pl., oral plug; or.pl.f., oral plug fibers; pel., pellicle ; per., margin of peristome ; post. c. v., posterior contractile vacuole ; rad. /., radial fiber ; ret. v^ rectal vacuole. 1922] McDonald: On Balantidium coli and Balantidium suis 265 ridges and grooves, the cilia passing through the bottom of the latter, but this condition is not due to longitudinal thickenings in the pellicle itself, but to the fact that it is closely applied to the ectoplasm which is thus furrowed. It can often be separated in " blisters 'J_ from the ectoplasm by tannic acid or weak alcohol, and when thus removed it shows regular longitudinal rows of perforations through which the cilia pass out from the ectoplasm. In this condition its transparency is very evident. The pellicle is not extremely impervious. For instance, when the active animals are introduced into normal salt ad. memb. _ cyst. dr. oes. f. II / or. pi. f. per. Fig. J. The neuromotor apparatus of the adoral region, anterior view. X 2000. ., adoral ciliary fiber; ad. memb., adoral membranelles ; ~bg.ad.cil., basal granule of adoral eilia; dr. oes. f., circumoesophageal fiber; oytst., cytostome; mot., motorium; or.pl., oral plug; or. pi. f., oral plug fibers; per., margin of peri- stome. solution plasmolysis takes place very quickly, and they present a grotesque appearance as they swim about with several huge depres- sions in their surfaces due to the shrinkage. Intra vitam stains such as neutral red and Janus green also penetrate very quickly. Resist- ance to pressure and mechanical change, however, is very marked, and is due to its tenacity and flexibility, both of which qualities are shown when the animal forces itself through an opening much smaller than the normal diameter of its body (fig. K) and also by the extreme flat- tening which it withstands under the increasing pressure of the cover- glass when evaporation of the preparation is allowed. As previously mentioned, these qualities indicate that the pellicle is protective and retentive in function rather than supportive or skeletal. 266 University of California Publications in Zoology [ V L - 20 These organisms show remarkable mobility when observed under conditions as nearly normal as possible. I have attempted to depict something of this plasticity in figure K. As they travel amid the debris in the intestinal contents, which has been removed with them, a tendency to penetrate is much more noticeable than any avoiding reaction. Instead of reversing the ciliary action, backing away and taking a new direction as would paramaecia, the balantidia, when they come in contact with a solid object, rather appty themselves to the surface, round up, and seem to roll along it. After a moment of such slow contortion, they may swim away in a new direction, Fig. K. Diagrammatic illustration of the plasticity of the organism, resulting in ability to pass through remarkably small openings. determined by the direction of the anterior end. They avail them- selves of the slightest opportunity to force their way through or between any obstacles. The anterior end, especially the thickened ectoplasmic portion, becomes at such times decidedly elongate and conical (fig. K, 6). The cilia of this region beat spirally producing a boring action as this anterior tip is protruded into any slight opening. This action has in many instances been observed to cause two obstacles, either of which was larger than the organism itself, to separate sufficiently to allow it to pass between. The aperture need not be one-half of the diameter of the animal for the latter will constrict (fig. K, c) and the fluid contents flow through anteriorly as it progresses, resembling the process of putting a bag of beans through a small hole in a board. Throughout observations of the activity of these organisms, one is impressed with their fitness for penetrating the mucous lining of the intestine and the underlying tissues. Its thigmotropic response, its boring action, and its extreme plasticity, all seem to be adaptations for the function of penetration. Ectoplasm. Immediately underneath the pellicle, the cytoplasm is differentiated to form the ectoplasm (ect., fig. I; pi. 27, figs. 1-7). The ectoplasmic layer, except at the anterior end, does not exceed two 1922] McDonald: On Balantidium coli and Balantidium suis 267 microns in thickness. The anterior end of the animal, that is, all anterior to a transverse plane which would transect the body at a point % to y 5 of the way to the posterior end, is composed entirely of ectoplasm. In this cone-shaped area is located the cytostome and a large part of the neuromotor apparatus. The protoplasniTof this region seems to be homogeneously granular in fundamental structure.. It stains very deeply with haematoxylin ; so deeply in fact, that in differentiation it is necessary to destain other parts of the body almost completely before this part reaches a degree of transparency suitable for study. With Mallory's connective tissue stain this region also stains very densely, taking on both the brilliant red and the deep blue elements of the stain, in different structures, as will be explained below. This extensive thickening of the ectoplasm at the anterior end is clearly shown in the figures by Leuckart (1861) and has been noted by nearly all who have studied the animal more recently, but I have failed to find any discussion of its significance. This same phenomenon occurs in the Ophyroscolecidae, as pointed out by Sharp (1914) in his work on Diplodinium and by Braune (1913) in Ophyroscolex. In these cases the change seems to be correlated with the high degree of activity and specialization of the anterior end of the animal ; in Diplodinium in connection with its selective feeding, and in Balantidium in con- nection with both feeding and activity of this entire region in pene- trating the mucosa of the intestine. The centering of the neuromotor apparatus in this region gives additional evidence in regard to this question which will .be discussed further in connection with the de- scription of that apparatus. Throughout the entire investigation of the minute structure of this animal, I have been unable to demonstrate the presence of any definite plane of demarcation between endoplasm and ectoplasm, such as the " ectoplasmic boundary layer" described by Sharp (1914) in Diplo- dinium, and shown in plate IV, figure 3 of his paper. Many of the fixed preparations used in the search for such a layer were sections of the animal, treated as nearly as possible according to the technique used by him and stained, as were his preparations, with Mallory 's con- nective tissue stain. So far as it was possible for me to determine, any sharp boundary line between ecto- and endoplasm is lacking. On the contrary, they merge into one another and only in a general way can it be said where one terminates and the other begins. Prowazek (1913, fig. 2) describes, in Balantidium coll, "ein Art von Zwischenmembran " which appears as wavy lines in optical sec- 268 University of California Publications in Zoology [VOL. 20 tion. According to his interpretation this "Querlinie" separates the protoplasm of the cell body into two regions, the * * apical zone, ' ' which I have described above as a thickening of the ectoplasm, and the rest of the cell protoplasm. The extent of this * * Zwischenmembran " he does not note, but his text figures (1 and 2) do not show it as extending quite to the pellicle, but instead as stopping short of the pellicle at a distance about equivalent to the thickness of the ectoplasm at that point. This is significant in my interpretation of this region, namely, that what appears as a continuous line or plane when viewed from the side is in reality, as shown in cross-sections (pi. 27, figs. 6 and 7), a set of diverging fibers. These fibers take origin from dark-staining oil cil. r. end. Fig. L. Portion of the peripheral region of a cross-section of Balantidium coli, showing the structure of the ectoplasm and arrangement of cilia, somewhat dia- grammatic. X 1500. b. ff., basal granule; oil., body-cilia; oil.r., ciliary rootlet; end., endoplasm; gr. &., granular band of ectoplasm; hy. &., hyaline band of ecto- plasm; pel., pellicle. enlargements on the longitudinal fibers in the wall of the gullet and, diverging, pass peripherally until they turn posteriorly at the very inner edge of the thin layer of ectoplasm which covers the remainder of the body (pi. 28, figs. 9-12). Even in lateral view careful focusing will often show that the apparent ' ' membrane ' ' is really discontinuous, showing breaks and irregularities as one focuses on different levels and hence can not be considered as a true membrane. The arrange- ment of these fibers will be described more exactly under the discussion of the neuromotor apparatus. The ectoplasm which constitutes a layer less than 3 microns in thickness around the remainder of the periphery of the cell shows a definite and somewhat complex structure. In tangential sections of the surface, which are so thin that they do not include much of the underlying endoplasm, one detects alternating light and dark longi- tudinal spiral bands (gr.b., hy.b., fig. N). These bands are parallel to the rows of cilia and very nearly equal in width. In cross-sections (fig. L) they are seen to extend nearly, if not quite the full depth of 1922] McDonald: On Balantidium coli and Balantidium suis 269 the ectoplasm. As one follows these bands (or "stripes," as they are named by Johnson (1893, in his work on 8 tent or) anteriorly they seem to lose their distinctness when they become continuous with the apical cone. In some individuals, however, one can follow them some distance into this cone, but never is there the same degree of differentiation of the two areas in this region. In the living animals, which are often quite opaque due to inclu- sions, it is nearly impossible to distinguish these longitudinal light and dark bands. With neutral red the dark or granular band stains faintly. With the haematoxylin stains used in thin sections of fixed material the dark band seemed to be finely granular in fundamental structure. The granularity in this case must be determined largely by the general appearance and stainability, for the individual granules are so small as to defy identification. There is no indication, however, of alveolar structure, so that the term granular is probably the more applicable and will be used to distinguish this from the light band. The latter takes only faintly the stains used and seems hyaline in structure. The granular bands lie directly beneath the ridges in the cuticle which occur between the rows of cilia. Or more correctly, the ridges on the surface of the animal are produced by the projection of these granular bands outwardly beyond the hyaline bands. These latter are directly beneath the grooves of the surface where the cilia pass through the cuticle and attach with the basal granules which lie in longitudinal rows; a single row in each hyaline band. The ciliary rootlets (cil. r., figs. I and L) extending in from the basal granules proceed diagonally inward and pass into the interior margin of the granular band. Stein as early as 1876 pointed out these alternating dark and light stripes in Stentor. To the granular and bright stripes, Biitschli (1889) gave the names ' ' Riffenstreif en " and " Zwischenstreif en, " respectively. Johnson (1893) gives a careful description of these bands as they occur in Stentor coeruleus. He notes that they vary greatly in width, and this is true in Balantidium; but both bands are much narrower than in Stentor, the combined width of the two not exceeding two microns. In Stentor coeruleus Johnson (1893) gives the width of the granular band as 22/x and that of the bright band as 7/x, these measurements being taken just under the adoral zone. It is interesting to note that in Balantidium coli the granular band is also slightly wider than the bright band. These bands become narrower from the region of the greatest circumference of the animals toward 270 University of California Publications in Zoology [ VoL - 20 each end, which is comparable with the arrangement in Stentor, though in the latter the narrowing must necessarily take place in the posterior direction only. The above author mentions the branching of stripes, but this does not occur in Balanticfrium so far as I have been able to determine. As the bands pass posteriorly, however, they become less distinctly differentiated and are hard to follow, and it might be that further study with more intensive stains would reveal a union in the region of convergence at the posterior end. More recent work has added to the number of Heterotricha that show this sort of differentiation of ectoplasm. Maier (1903) shows the striped nature of this layer in Prorodon and Spirostomum, while Neresheimer (1903) confirms the structure found by Johnson ( 1893 ) in Stentor. Schuberg (1887) also indicates a comparable plan of structure in Bursar la. The granular ridges of ectoplasm between the furrows in which the anal cirri are situated in Euplotes patella, discovered by Yocom (1918), may be comparable with the bands which occur in Hetero- tricha. Cilia. The entire surface of Balanticbium coli, with the exception of the oral plug, is thickly beset with cilia (cil., ador. c., fig. I). These are of two kinds, viz., those which make up the adoral row of cilia and which measure from 8 to 12/A in length, and those covering the body, which vary from 4 to 6ju. Those covering the apical cone form an intergradation between the two. On this surface the cilia which occur immediately posterior to the adoral row are only slightly shorter- and slightly more slender than the adoral cilia themselves. Passing posteriorly they gradually become shorter and less cirrus-like until they reach the base of the apical cone. Thence posteriorly they retain the uniform size. The body cilia are comparatively short and very slender. So small are they in fact that to observe a single one is nearly impossible. In slides prepared by the usual methods no stain remains in the cilia if destaining is carried sufficiently far to differentiate other structures. Iodine (Weigert's solution) gives a fairly satisfactory stain for tem- porary mounts. The arrangement of cilia may be determined most readily by using a heavier stain and then observing the distribution of basal granules. Neutral red proves very satisfactory for this pur- pose. The cilia occur in longitudinal, slightly spiral rows, following the grooves between the ridges in the pellicle. These rows originate immediately posterior to the groove in which the adoral cilia are set and for a very short distance pass almost meridionally ; very soon, 1922] McDonald: On Balantidium coli and Balantidium suis 271 however, they turn toward the left, that is, in a counter-clockwise direction when the animal is viewed from a point exactly in front. They continue their spiral direction until almost to the posterior end when they again follow a meridional path to their termination. In passing the entire length of the body any single row of cilia twists to the left approximately 120, or one-third the entire circumference. Whether some rows terminate or become continuous with contiguous rows before reaching the posterior tip of the animal, I have been unable to determine, for both basal granules and granular bands become very indistinct in this region even in the best preparations. The number of rows was counted with difficulty in several cross- sections from the equatorial regions of different animals, and it was found to vary from about 60 in small individuals to 120 in larger ones. No correlation between the variation in the number of rows of cilia and the species of the animal could be determined, but this is possibly due to the limitation of observation. Basal apparatus. The cilia perforate the pellicle and attach to the basal granules which lie immediately underneath (fig. L). The latter are small and apparently spherical or oval. They stain very deeply black or blue with haematoxylin. In the living animal they are readily emphasized by the use of neutral red, and less so by Janus green. In preparations stained with Mallory's connective tissue stain these granules show brilliantly red with the acid fuchsin, as do the other parts of the neuromotor apparatus and also the micronucleus. Longitudinally the granules are so closely placed that it is impossible to observe whether they are actually connected by a fibre. Cross- sections show that the cilia of one row have no transverse connection by any sort of stainable fiber with those of the next row. The rows of basal granules lie close beneath the depression in the pellicle in the hyaline or bright band of the ectoplasm. A ciliary rootlet (cil. r., figs. I and L) extends from each basal granule centrally toward the endoplasm. It does not proceed in an exact radial line but rather diagonally toward the right until it enters the granular band near the inner surface of the latter. The ciliary rootlets in some cross- sections appear to have an exactly radial direction. In such cases, however, the granular band is somewhat diagonal in the opposite direction. This variation is probably produced either by torsion of the animal or by the direction of the effective beat of the cilia at the instant of fixation. The diagonal direction of the rootlets is readily detected in tangential sections. In focusing down through such a sec- 272 University of California Publications in Zoology [ V OL. 20 tion, the rootlet is invariably seen to run from the basal granule toward the observer's right into the contiguous granular band on that side. Thus, to avoid any confusion that might arise from the terms right and left, in a cross-section of the animal viewed from the anterior surface (such a view is shown in fig. L) the ciliary rootlets swerve in the counter-clockwise direction and enter the granular band lying immediately in that direction. As the rootlet enters into the granular band it apparently enlarges thus forming a secondary basal granule. In some cases this may stain even more deeply than the basal granule itself, and appear as a definite body somewhat elongated in the direction of the circumference of the animal. It was thought at first that this might be the cross-section of a longitudinal fiber or myoneme. But the study of numerous tan- gential sections has failed to show the presence of any longitudinal fiber within the granular band. The stainability varies greatly and in preparations which have stained lightly the ciliary rootlets appear to fray out and merge into the granular band, while still retaining deeper color than the rest. Which interpretation is correct it is difficult to say, but the latter seems the more probable, especially in view of certain relations with the neuromotor apparatus which will be discussed later. Putter (1903) reproduces a figure from Studnicka (1899) showing in a schematic way five types of attachment of the cilia with their basal apparatus. Of these, two, at least, represent cases in which two basal granules or a diplosome are present. Saguchi (1917) in his studies on ciliated cells of Metazoa says in part regarding the basal granules of certain ciliated cells from amphibian larvae, ' * With favor- able staining the basal corpuscles appear as diplosome or dumbbell shaped granules. One of these is situated at the upper the other at the lower border of the cuticle." In Balantidium coli the arrange- ment with respect to protoplasmic layers is quite different, though the cilia seem to follow somewhat the same plan of structure even to the presence of rootlets. The most fruitful comparison may be made with the .basal appa- ratus of cilia in Isotricha prostoma as described and pictured by Braune (1913). In this organism he describes diplosomic structure of the basal apparatus, in which the basal granule lies directly beneath the pellicle. The cilia, however, extend beyond the basal granule into the underlying layer the * * Zwischenschicht " of Eberlein, and ter- minate in the "Grenzschicht" with a second granule, which upon 1922 J McDonald: On Balantidium coli and Balantidium suis 273 maceration remains attached to the basal end of the cilium. So the basal apparatus in Balantidium coli even to the relative location of the basal granules is almost identical with that in Isotricha prostoma. In Balantidium, however, as mentioned above, the "Grenzschicht" seems to be lacking. The comparison becomes more significant in view of the fact that both ciliates are parasitic in the digestive tract of mammals, and both are in much the same state with reference to the degree of specialization and degeneration correlated with habits of the parasitic mode of life. So that in general morphology they seem to be more alike, though they are in separate orders, than do Diplodinium ecaudatum and Balantidium coli, which are in separate suborders only. Ciliary Movements. Locomotion is the chief function of the cilia except for those of the adoral zone. The balantidia swim in approximately a straight line and not in a spiral course as do paramoecia. They do, however, rotate on their axis as they progress. This rotation is generally from left to right, that is, in a counter-clockwise direction when viewed from a point in front of the animal. A few instances of reversal of the direction have been seen, but it is not at all common. The direction of rotation, i.e., from left to right, seemed at first inexplicable, since this was not compatible with the direction of the rows of cilia. The rows of cilia, as described above, are comparable to the threads of a left-hand screw. In order to penetrate, such a screw must be turned in a clockwise direction (when viewed from the point, not from the head). Such, also, is the direction of rotation, of balantidia which one would expect to find if the arrangement of the cilia were the con- trolling factor, but the rotation is in the reverse direction. In the further study of the problem, I fortunately obtained some very thin tangential sections of animals on which the fixative had acted so quickly that the cilia were stopped instantly and left in the relative positions assumed in normal ciliary action. Figure N is a camera lucida drawing of such a section. By analysis of the position of the cilia on this and other like sections, it was possible to determine the complete cycle of a single cilium. This cycle is diagrammatically represented in figures M and 0. Figure N includes about two and one-half cj^cles of action as represented by the waves. The dark portions are produced by the prostrate position of the cilia at the termination of the effective stroke. The lighter 274 University of California Publications in Zoology t v L - 20 areas between are due to the fact that the cilia are recovering their vertical position and hence are viewed very nearly endwise. The position of consecutive cilia in any single row, from one point in a wave to a similar point in a following wave will fairly represent the successive positions taken by a single cilium in making one complete cycle. Figure M was made in this way. The arrow represents the long axis of the animal. From this diagram it is seen that the cilium at the end of the effective stroke lies rather close to the surface of the body and not along the row but decidedly to the left from it. In -a Fig. M Fig. N Fig. O Fig. M. Diagrammatic representation of the successive steps in one complete beat of a cilium of Balantidium coli. It also illustrates the positions of the respec- tive cilia of a single row between the points a and & in fig. M, at which points the cilia are in a prostrate position at the end of their effective stroke. The arrow indicates the long axis of the animal. Fig. N. Tangential section of Balantidium coli. The cilia still retain respec- tive positions which they had in the normal swimming movements of the organism. X 1500. Fig. O. Diagram illustrating the effect of the ciliary action in the rotation of the organism. The arrow represents the long axis of the animal; ab., the direction of the rows of cilia ; cd., the direction of effective stroke of a cilium attached at the point of intersection of the three lines; the ellipse is described by the tip of the cilium. recovery it straightens up and passes anteriorly, thence to the right, crossing the row, of which it is a part, at right angles. At this point the cilium leans anteriorly only slightly from the vertical. The effective beat is produced by the quick stroke of the cilium posteriorly and to the left, and continues until the cilium has crossed the row again and lies close to the surface and extends to the left as represented by the position of the last cilium shown in figure M. According to the classification given by Putter (1903), the movement of the cilia of Balantidium would be called^ infundibular. As will be noted from 1922] McDonald: On Balantidium eoli