hiPiby in rte vias tireless tf dab dee tevin eps: olen Me Phe Phries bole} ai at ath; WML D pe e tities thel hehe woolen ys o— See: eee eee Lt ba eo vee ee Cee = title Serer Teste de ites 2 poet ats titiel et ecrar Si epeeeiyrece Piebe ‘ - ~9~9- p= rk eis ei tH teitiejertl x arate aie aero = ce ll ae — ih e E525 TOO TOEO oO HM/18WW oo a nf f sayin AR ee! i yn i Wh Mere eiys tis ti TU uf i (h Wi PROMOZOA IN BIOLOGICAL IES EF ASh CH PROTOZOA DN BTORwOGIC AL RESEARCH Edited by GARY IN. CALKINS and FRANCIS M. SUMMERS I Ltrs BSA New York: Morningside Heights COLUMBIA UNIVERSITY PRESS L941 COPYRIGHT 1941 COLUMBIA UNIVERSITY PRESS, NEW YORK Foreign agents; OxForD UNIVERSITY Press, Humphrey Milford, Amen House, London, E.C. 4, England, AND B. I. Building, Nicol Road, Bombay, India; MARUZEN COMPANY, LTD., 6 Nihonbashi, Tori-Nichome, Tokyo, Japan MANUFACTURED IN THE UNITED STATES OF AMERICA PREFACE NuMEROUS textbooks of varying degrees of excellence for the study of Protozoa are now on the market and should be consulted for a general treatment of these unicellular forms. This volume will not fill such a need, but has been prepared as a result of a discussion by a group of specialists assembled during the summer of 1937 at the Marine Biological Laboratory at Woods Hole, Massachusetts, for the purpose of ascertaining the best means to stimulate further research on these unicellular animals. As a result of these discussions it was decided that one good way to attain our end would be to have a group of specialists in the field of protozodlogy prepare a work on research in this field, each specialist to provide a chapter on the subject in which he is best known, and about which he can speak with authority. Our first real difficulty was to select a limited number of topics from a vast number of possibilities, and then to choose the biologists who, in our opinion, were the best men to write these chapters. As can be readily imagined, this opened up a long list of difficulties and led to many vexatious troubles, but the present work, finally, is the outcome. To our very great regret, one of the men chosen—Professor Noland, of the University of Wisconsin—who was most enthusiastic over the project, has been forced by reason of continued illness, to drop out. The loss of others, on the plea of pressure of work, and so forth, has also depleted our ranks. But the remainder have completed their labors, have come through, and the results here speak for their continued in- terest and assiduity. While the arrangement of our material does not make a great deal of difference, some order nevertheless is advantageous, and we have therefore arranged the chapters, according to the general character of their materials, into groups having a more or less common subject matter. Gary N. CALKINS FRANCIS M. SUMMERS New York City January 2, 1941 ACKNOWLEDGMENTS WE ARE INDEBTED to the American publishers of the following books or periodicals for permission to use figures or other illustrations which are reproduced in this book. In a few cases these have been slightly modified by the contributors. Also we are under obligation to numerous foreign publishers who, for obvious reasons, we have been unable to ask for permission. American Journal of Hygiene Figures 188, 191, 192 Biological Bulletin Figures 5, 10, 13, 74, 81, 82, 84, 96, 104, 181, 183, 185, 186, 187, 199A, 199B, 223 Biology of the Protozoa by G. N. Calkins, 1933. Lea and Febiger Co. Bigures 12; 3A 3B, 9 Genetics Figures 166, 168 Journal of Animal Behavior, Henry Holt and Co. Figure 103 Journal of Cellular and Comparative Physiology, Wistar Institute of Anatomy and Biology Eigures 119.120, 121, 122) amd: Table 2 Journal of the Elisha Mitchell Scientific Society Figures 202A, 202B Journal of Experimental Medicine, Rockefeller Institute for Medical Research Figures 189, 190 Journal of Experimental Zoology, Wistar Institute of Anatomy and Biology Figures 14, 94, 97, 98, 102, 105, 106, 149, 156, 162, 164, 179, 182 Journal of Morphology, Wistar Institute of Anatomy and Biology Figures 83, 91, 93, 154, 157, 160, 16%, 1163 Vill ACKNOWLEDGMENTS Journal of Preventative Medicine, John McCormick Institute for In- fectious Diseases Figure 193 Light and Behavior of Organisms by S. O. Mast, 1911. John Wiley and Sons, Inc. Figures 99, 107 Physiological Zoology, University of Chicago Press Figures 95, 113, 126, 180 Publications of the Carnegie Institute of Washington Figure 214 Science Figure 123 Transactions of the American Microscopical Society Bigutes 36,537 University of California Publications in Zoology, University of Cali- fornia Press Figures 15, 16, 39, 49, 50, 53, 54, 55, 70) 85. 86, 87, esmao 146, 148, 150, 151, 196, 219, 225 United States Geological Survey of the Territories Figure 221 Finally, it is a pleasure to recognize the care and patience on the part of the staff of the Columbia University Press in the preparation and distribution of this work. While all members have worked diligently and harmoniously with us, we are particularly indebted to Miss Georgia W. Read, Miss Ida M. Lynn, and Miss Eugenia Porter for the meticu- lous care with which they have sought to unify and, in some cases, to clarify the manuscripts on subjects unfamiliar to them. THE Epirors New York City January 2, 1941 CONTENTS Ieist: OL Ab eremiahionss ¢ Go on ke te es VO I. General Considerations By Gary N. Calkins, Pro- fessor Emeritus of Protozodlogy in Residence, Co- YW lumbia University Life and vitality—Fundamental and derived organiza- tion—Some ecological consideraticns—Some historical facts—The use of cultures—Factors influencing longev- ity—Changes with metabolism—Reorganization of the macronucleus and other derived structures in Ciliata— Reorganization by endomixis and by conjugation—Lit- erature cited II. Some Physical Properties of the Protoplasm of the Protozoa By H. W. Beams and R. L. King, State Di A TAN TOOT oe eae ol NES A Loe Se ae 43 Introduction—Properties of protoplasm as exhibited in Amoeba—Colloidal nature of protoplasm—Consist- ency—Surface properties—Specific gravity or density— Optical properties—Structural properties—Literature cited III. Cytoplasmic Inclusions By Ronald F., MacLennan, OoeriniEal Can Pay ot TNIIS Bhs maa) ae, os LE Mitochondria—The vacuome hypothesis—Digestive granules—Segregation granules—Golgi bodies—Excre- tory granules—Lipoid reserves—Carbohydrate reserves— Protein reserves—External secretion—The granular com- plex—The continuity of cytoplasmic granules—The classification of cytoplasmic granules—Comparison with cells of the Metazoa—Literature cited IV. Fibrillar Systems in Ciliates By C. V. Taylor, Stan- OCMUIZU CENTLY Aaya yeshi Ayia ee eel, wees: LDL Introduction—Examples of fibrillar systems—Structural 53684 WAL, VI. VIII. CONTENTS analysis—Interpretation—Fibrillar systems of other ciliates — Holotricha — Heterotricha — Oligotricha — Hypotricha—Conclusions—Literature cited Motor Response in Unicellular Animals By S. O. Mast, Director, Zoological Laboratory, Johns Hop- kins University . Introduction—Responses to light—Rhizopods—Flagel- lates—Ciliates—Colonial organisms—Responses to elec- tricity —Rhizopods—Flagellates—Ciliates—Colonial or- ganisms—Responses to Chemicals—Rhizopods—Ciliates —Literature cited Respiratory Metabolism By Theodore Louis Jahn, State University of Iowa Purposes of studying respiration-—Methods of measuring aérobic respiration—Aé€érobic respiration—Investigations which concern the source of energy—lInvestigations which concern the mechanism of respiration—The measurement of anaérobic metabolism and glycolysis— Occurrence of anaérobiosis and glycolysis—Why are anaérobes anaérobes, and aérobes aérobes ?—Oxida- tion-Reduction potential versus respiration and growth— Literature cited The Contractile Vacuole By J. H. Weatherby, Medr- cal College of Virginta Introduction—The origin of contractile vacuoles—The structure of contractile vacuoles—The function of con- tractile vacuoles—Contractile vacuoles and the Golgi apparatus—Conclusion—Literature cited The Technique and Significance of Control in Proto- zoan Culture By George W. Kidder, Brown University Introduction—The problem of protozoan sterilization— General material—General methods of sterilization— Special methods and manipulations—The importance of adequate sterility tests—Establishment of sterilized Pro- tozoa in culture—Literature cited Dik 352 404 448 CONTENTS xi IX. Food Requirements and Other Factors Influencing Growth of Protozoa in Pure Cultures By R. P. Hall, University College, New York University . . . 475 Food requirements of Protozoa—Photoautotrophic nu- trition—Photomesotrophic nutrition—Photometatrophic nutrition — Heteroautotrophic nutrition — Heteromeso- trophic nutrition—Heterometatrophic nutrition—Trophic specialization—Specific growth factors, or vitamins— Other growth factors—Growth stimulants—Growth in cultures as a population problem—The initial popula- tion—Growth in relation to waste products—Growth in relation to food concentration—Growth in relation to pH of the medium—Oxygen relationships—The redox potential—Growth in relation to temperature—Growth in relation to light and darkness—Acclimatization— Literature cited X. The Growth of the Protozoa By Oscar W. Richards, Research Biologist, Spencer Lens Company . . . 517 Methods for the measurement of growth—The growth of individual Protozoa—The growth of colonial Protozoa— Pedigree isolation culture and life cycles—Protozoan suc- cessions: nonlaboratory—Protozoan successions: labora- tory—Autocatalysis and allelocatalysis—Nutrition and growth—The growth of populations—The struggle for existence—Literature cited XI. The Life Cycle of the Protozoa By Charles Atwood Kofoid, University of California, Berkeley . . . 565 Introduction—Asexual reproduction in alternating binary and multiple fission (Type I)—Alternation of asexual and sexual reproduction (Type I1)—The life cycle of Eimeria schubergi—The life cycle of Plasmodium vivax—The life cycle of Paramecium caudatum—Litera- ture cited XII. Fertilization in Protozoa By John P. Turner, Univer- SO] MGHNESOldaeM EE Macronuclear reorganization—Endomictic phenomena— Autogamy—-Periodicity of endomixis—Genetical studies on endomixis—Conclusions—Literature cited XIV. Sexuality in Unicellular Organisms By T. M. Sonne- born, Uniwersiy of Indiana = 2 VS) 3 G66 Sexuality in Chlamydomonas—The kinds of gametic differences observed in Chlamydomonas—The nature of the physiological differences between gametes in Chlamydomonas—Interpretation of the Seal phe- nomena in Chlamydomonas—Sexuality in Paramecium and other ciliate Protozoa—Sexual differences between conjugant individuals—Mating types in relation to the Maupasian life cycle—The rdle of environmental con- ditions in determining conjugation—Sex differences be- tween gamete nuclei—Significance of the diversities be- tween conjugants and between gamete nuclei—Literature cited XV. Inheritance in Protozoa By H. S. Jennings, Univer- sity of (California LostAnccles ia. a vce eee Types of reproduction and inheritance—Inheritance in uniparental reproduction—Material processes—Inheri- tance of characteristics—Changes in inherited characters in uniparental reproduction—Age changes, sexual imma- turity, and maturity—Inherited degenerative changes re- sulting from unfavorable conditions—Inherited acclima- tization and immunity—Inherited environmental modifi- cations in form and structure—Variation and its inhert- tance occurring without obvious action of diverse en- vironments—Summary and interpretation—Inheritance in biparental reproduction—Biparental inheritance in haploids: Flagellata—Sex inheritance and sex-linked in- heritance—Biparental inheritance in diploids: Ciliata— Inheritance of mating type in Paramecium aurelia—In- heritance of mating type in Paramecium bursaria—Effect of the cytoplasm and its relation to nuclear constitution— Literature cited CONTENTS Xi XVI. The Protozoa in Connection with Morphogenetic Problems By Francis M. Summers, College of the CMON Of hs fod wid gk Eizieyoh, (os es «2 772 Physiological regeneration—Some of the factors in tfe- generation—External environment—Cyclical variations —Racial variations—Degree of injury and reorganiza- tion—The size factor—The nuclei in regeneration— Behavior of fragments: grafting and reincorporation— Regeneration and division—Polarity changes and proto- plasmic streaming—Physiological gradients—Regenera- tion in colonial forms—Literature cited XVII. Certain Aspects of Pathogenicity of Protozoa By Elery K. Becker, lowa State Gollege 5 29% *. 818 Problems of virulence and _pathogenicity—Amoebic dysentery and bacterial complications—Malaria: Plas- modium vivax—Variability in strains and in host re- sponse—Coccidiosis in poultry—Nutrition and resistance —Conclusions—Literature cited XVIII. The Immunology of the Parasitic Protozoa By Wil- liam H. Taliaferro, University of Chicago . . . 830 The physical bases of immunity—The cells involved in immunity—Antibodies and antigens involved in immu- nity—The cellular and humoral aspects of immunity— Role of immune processes in the development of proto- zoan infections—General methods—Malaria—Leishma- niasis—Nonlethal infection with the Trypanosoma lewitsi group of trypanosomes—Continuous fatal trypanosomi- asis in the mouse and sometimes in the rat—Intermittent fatal trypanosomiasis in various laboratory animals— Practical applications of immune reactions—Artificial immunization—Immunological reactions used in diagno- sis—Immunological reactions in relation to classifica- tion—Literature cited XIX. Relationships between Certain Protozoa and Other Animals By Harold Kirby, Jr., University of Cali- fornia, Berkeley A At hE, Scien, Mae OO) Accidental and facultative parasitism—Systematically related free-living and symbiotic Protozoa—Mastigo- X1V CONTENTS phora—Holotricha—Distributional host relationships and host-specificity in representative symbiotic faunules— General considerations—Ciliates of sea urchins—Proto- zoa of termites and the roach Cryptocercus—aAdaptive host relationships in morphology and life history— General considerations—Thigmotricha—Ptychostomidae —Astomata—Conidiophrys—aA postomea—Physiological host relationships illustrative of mutualism and com- mensalism—Flagellates of termites and Cryptocercus— Ciliates of ruminants—Literature cited XX. Organisms Living on and in Protozoa By Harold Kirby, Jr., University of California, Berkeley . Epibiotic schizomycetes—Schizomycetes on Mastigo- phora—Endobiotic schizomycetes—Associations of a constant character—Associations of an occasional char- acter—S phaerita and Nucleophaga—Historical account and distribution—Life history and structure—Effect on host—Parasites of the nucleus of Trichonympha— Phycomycetes other than Sphaerita and Nuacleophaga— Protozoa—Phytomastigophora—Zobmastigophora—Sar- codina—Sporozoa—Ciliophora—The genus 100 1000 locomotion in this form (Ludwig, 1928a, 1930). Certain Protozoa of floating habit frequently have hydrostatic devices which aid in flotation, such as the gas bubbles secreted in the protoplasm of Arcella and Difflugia; special layers of vacuolated protoplasm, such as the calymma of Radiolaria; or very highly vacuolated protoplasm, as in Noctiluca and in Heliozoa. According to Bles (1929) the gas bubbles of Arce/la are formed in the marginal protoplasm and are filled with oxygen. They are secreted when the oxygen tension is reduced experimentally and are adaptive, in that they reduce the specific gravity of the organism so that when oxy- gen tension is low the organism may float to the surface, where the oxy- gen tension is always somewhat higher. When Arcella is turned upside down so that the external pseudopods cannot adhere to the substratum, gas bubbles appear in from three to six minutes, before the animal be- gins to right itself and aid in this process by lowering the specific gravity. The bubbles disappear rapidly after the righting process is completed. When there is more than one bubble present all grow at the same time 78 PROTOPLASM OF PROTOZOA and decrease at the same time. In the Radiolaria periodic migrations take place to and from the surface layers of the sea; these are brought about by changes in the vacuolar contents of the hydrostatic layer, which, ac- cording to Brandt (1885), is lighter than water. Schewiakoff (1927) has described the presence of a clearly defined gelatinous hydrostatic layer in the Acantharia (Radiolaria). Although the specific gravity of Noctiluca (1.014) is less than that of sea water (1.026) in which it floats, according to Massart (1893), E. B. Harvey (1917) has shown that this form can lessen and increase its specific gravity in a regulatory fashion. Lund and Logan (1925) have shown that the increase in specific gravity, following strong mechanical shock or electrical stimulus, is caused by the coalescence of large vacuoles and the liberation of their contents, which diffuse through the pellicle. The density of the solution in the vacuoles is less than that of sea water; according to Ludwig (1928b) this is because of its lower salt content, its osmotic pressure being about half that of sea water. Most marine animals possess body fluids almost isotonic with the external fluid, but Noctéluca, together with the marine teleosts, are hypotonic and, to a great extent, osmotically independent. Such organisms must be impermeable to water, absorb water in some way without salts, or take in sea water and excrete salts. Marine teleosts apparently take in sea water and excrete the excessive salts, and, since the membrane of Noctiluca is permeable to water, os- motic work (negative osmotic force) must be done by the membrane (E. B. Harvey, 1917) in maintaining this steady state instead of os- motic equilibrium. The protoplasm of marine Protozoa frequently becomes much vacu- olated upon transfer to fresh water. The marine variety of Actinophrys sol, according to Gruber (1889), has thick, granular protoplasm poor in vacuoles and entirely lacking a contractile vacuole; during gradual transfer to fresh water the protoplasm becomes foamy with bubbles and a contractile vacuole appears, so that the organism is indistinguishable from the fresh-water variety. The formation of vacuoles and the entrance of water into them undoubtedly lowers the specific gravity in this form, when transferred from salt to fresh water. The spine-like pseudopodia of Heliozoa, Radiolaria, and other floating forms also serve as a protec- tion against sinking. The first estimation of the specific gravity of a protozoan apparently PROTOPLASM OF PROTOZOA 7D was that of Jensen (1893), who attempted to determine the energy rela- tions of the movement of P. aurelia. He obtained the value of 1.25 by suspending the organisms in solutions of potassium carbonate, a proce- dure which gave too high values because of excessive shrinkage, due to osmotic pressure. Later Platt (1899) suspended killed or anaesthetized Paramecium and Spirostomum in solutions of gum arabic and found their specific gravity to be 1.017. Lyon (1905) centrifuged living Para- mecium in solutions of gum arabic and obtained 1.048 or 1.049. This was repeated by Kanda (1914, 1918), who finally arrived at a value of 1.0382 to 1.0393 for Paramecium and 1.028 for Spirostomum. Fetter (1926) utilized approximately the same value, 1.038, which she ob- tained by centrifuging Paramecium in sugar solutions, in calculating the protoplasmic viscosity of that form. Leontjew (1927) has determined the density of various Protozoa (Fuligo, Stemonitis, slime molds; Naegleria, an amoeba; and Dunaliella, a flagellate) to be 1.020 to 1.065. Some of his readings on Fuligo varians, obtained with a micropyknometer, are interesting enough to be men- tioned in detail: in moist weather the density was 1.016; in dry, 1.040, and 11 hours before spore formation, 1.065. Heilbrunn (1929a, 1929b) used 1.03 as the specific gravity of the protoplasm in his studies on viscosity of A. dubia. Motile amoebae (Naegleria) have a density of 1.043, according to Leontjew (1926a), and cysts 1.060 to 1.070 (Joschida, 1920, cited by Leontjew, 1927); cysts of Hartmanella hyalina, a soil amoeba, have a specific gravity of 1.084 (Allison, 1924). It is, of course, generally recognized that the protoplasm of encysted Protozoa contains less water than that of active forms. Allison (1924) determined the specific gravity of cysts of Colpoda by the time required to fall through water. He finds that four-day cysts averaging 40.1 microns in diameter, have a density of 1.042; while twenty-day cysts, averaging 25.1 microns, have a density of 1.061. Similar results were found for cysts of Gonostomum. The decrease in size and increase in specific grav- ity are apparently caused by water loss. The specific gravity of protoplasm other than that of Protozoa has been found to vary from about 1.02 to 1.08, with average values about 1.045. The publication of Pfeiffer (1934) gives a résumé of the meth- ods and results of such studies. 80 PROTOPLASM OF PROTOZOA Relative specific gravity of cell inclusions and components.—It has long been known that the various inclusions of protozoan cells are of different specific gravities. In centrifuging cultures to obtain large num- bers of organisms for fixation previous to morphological studies, it is often noticed that certain crystals have been displaced centrifugally. Mc- Clendon (1909) was one of the earliest workers to fix and stain Parame- cium after long-continued centrifuging; he found that the crystals and nucleus were displaced centrifugally. Heilbrunn (1928) mentions that a centrifuged Ezglena loses its spindle-shaped contour and becomes spherical, with the granular inclusions packed at the centrifugal end. The same author (Heilbrunn, 1929b) has used the speed of movements of crystals centrifugally through the cytoplasm of A. dubia to estimate the absolute viscosity of the protoplasm; the specific gravity of the crystals was estimated to be approximately 1.10. E. N. Harvey (1931) records that the crystals of A. dubia fall down so rapidly that their velocity can hardly be determined in the microscope-centrifuge; and that the crystals of Paramecium were rapidly thrown down, as was the nucleus. He was also able to cleave living Stentor into two parts in the microscope-centrifuge; the lighter, oral half contained none of the Zodchlorellae which had been moved into the basal part. E. N. Harvey and Marsland (1932) observed the movement of cytoplasmic particles through the protoplasm of A. dzbia and found them to be layered out in the following order: coarse granules and crystals, most centrifugal; nu- cleus, a visibly empty zone, a zone of fine granules, and, most centripetal, the contractile vacuole. Mast and Doyle (1935b) have recorded as follows the relative specific gravities of the various cytoplasmic com- ponents in A. proteus, from centrifugal to centripetal: refractive bodies, beta granules (mitochondria) and food vacuoles containing little or no fat, nucleus and food vacuoles containing much fat, hyaline protoplasm, contractile vacuole, crystal vacuoles without crystals, and fat globules. The position of the crystal vacuoles varies with the size of the included crystals: those with large crystals are heavy and move centrifugally in the centrifuge; those with small crystals are lighter. The small alpha granules, which are about 0.25 micron in diameter, are not layered out. All the refractive bodies, a large proportion of the crystals, and all the fat may be removed, with no injurious effects, from a centrifuged amoeba by cutting off the light and heavy ends. However, removal of PROTOPLASM OF PROTOZOA 81 the beta granules (mitochondria) resulted in the death of the amoeba. Singh (1939) has also centrifuged A. proteus ‘Y,’ and found the order of layering to be: nutritive spheres, nucleus, crystals, neutral red bodies, mitochondria, cytoplasm, contractile vacuole, and fat. Patten and Beams (1936) centrifuged Evglena and found that the chloroplasts form a middle belt, having on the centrifugal side paramy- lum and neutral red bodies, while the clear cytoplasm containing the small spherical mitochondria is at the centripetal pole. In Menodium the heaviest inclusions are the paramylum and neutral red bodies; in Ch7- lomonas the starch and neutral red bodies are heaviest. Johnson (1939) has confirmed the results of Patten and Beams; in Evglena rubra, how- ever, hematochrome is present and is displaced to the centripetal pole with mitochondria. King and Beams (1937) ultracentrifuged Paramecium; in this form the various components and inclusions were layered in the following order from centrifugal to centripetal: crystals in vacuoles, compact chromatin of the macronucleus, food vacuoles and neutral red inclusions, achromatic matrix of the macronucleus, endoplasm, large clear vacuoles, and fat. Here the chromatin may be removed from the achromatic matrix of the macronucleus; the chromatin regenerates a macronucleus; the achromatic matrix persists for some time and apparently interferes with subsequent divisions. Browne (1938) has ultracentrifuged Sprrostomum and has found the contents of the cell to be layered as follows: centrifu- gally located are the mitochondria, food vacuoles, and macronucleus; cyto- plasm; Golgi bodies; and centripetally, vacuoles. Daniels (1938) has used the ultracentrifuge in a study of gregarines; here the paraglycogen and chromidial granules are heaviest; next, the mitochondria and nucleus; cytoplasm; then the larger Golgi bodies; and lightest the smaller Golgi bodies and fat globules. In the gregarines studied the karyosome moved centrifugally in the nucleus, and the con- tents of the deutomerite layered independently of those in the primite because of the presence of the transverse septum. It is obvious that the centrifuge may serve as an important research tool for the identification and study of the form, relative volume, and other characteristics of the components and inclusions found in proto- plasm. For example, Holter and Kopac (1937), by cutting amoebae in half after centrifuging, were able to demonstrate that the enzyme dipepti- 82 PROTOPLASM OF PROTOZOA dase is apparently associated with the cytoplasmic matrix, independent of all cytoplasmic constituents which could be stratified by centrifugal ac- celeration. OPTICAL PROPERTIES The ordinary optical characteristics of protoplasm, such as its trans- parency, color, and refractive index, do not seem to be of very great im- portance except that the observation of living protoplasm is conditioned by these properties. Too many have assumed that because a structure cannot be seen in living protoplasm, it is therefore nonexistent. TRANSPARENCY The: protoplasm of the protozoan cell is generally transparent or translucent, but in the presence of granular or other inclusions it may appear to be opaque or nearly so. Many of the differentiations are so nearly of the same index of refraction that special fixing and staining methods are necessary in order to study them. The state of aggregation of the colloids of the general protoplasm seems to be dependent, at least to some extent, on the salt content of the surrounding medium. Thus Actino phrys sol in sea water is densely granular, while in fresh water it is alveolar and translucent (Gruber, 1889). Spek (1921) has shown that Actinos phaerium becomes relatively opaque in certain salt solutions, and that P. bursaria, which is glass-clear, becomes dark brown in artificial salt solutions, owing to the collection of albuminoid substances into large aggregates. Certain observations of Schaeffer (1926) on marine Amoebae are of interest here: Flabellula pellucida, a most transparent marine amoeba, becomes densely granular in 25-percent sea water, while F. citata, another marine amoeba, is unusually transparent in 364-percent sea water. COLOR Protoplasm is usually observed to be colorless or grayish; many of the shades of blue, green, or yellow described for Amoebae are merely dif- fraction phenomena or subjective in nature. The color of the endoplasm of various amoebae has been described by Schaeffer (1926) as pale bluish-green, yellowish-green, bluish-gray; Hyalodiscus elegans has endo- plasm which is arange-yellow centrally and ashen-gray peripherally. The PROTOPLASM OF PROTOZOA 83 contents of the contractile vacuole are often slightly pink. This may be regarded as an optical illusion, since the color observed is complementary to the usual bluish-green of the endoplasm. Very frequently the color is obscured by the presence of colored inclusions of various kinds, or caused by colored inclusions of the same index of refraction as the proto- plasm and therefore difficult to differentiate. Ciliates may be colorless, gray, pink, blue, or violet. The blue color of S. coeruleus is caused by a coloring matter, called stentorin, diffused through the cytoplasm, but in Blepharisma the color, which may vary from none through pink and violet to purple, and varies with the cul- tural conditions and from individual to individual in the same culture, is apparently concentrated in the pellicle (Nadler, 1929). According to Jennings (1906), most colorless Infusoria do not react at all to a light of ordinary intensity; this has not been tested with forms such as Blepharisma, in which the color varies. In the plant-like flagellates color is usually caused by chromatophores, which may be green, blue (Lackey, 1936), brown, or yellow. The most interesting colored inclusion is the hematochrome, found in such forms as E. rubra as red granules from 0.3 to 0.5 microns in diameter. These euglenae form a green scum in shaded places; the green chloroplasts mask the hematochrome, which is centrally located; in direct sunlight the scum is red, the hematochrome being peripherally located and masking the chloroplasts. Control of the distribution of the hematochrome is so delicately balanced that if the euglenae are shaded for fifteen minutes, they change from red to green (Johnson, 1939). The mechanics of this control needs to be investigated. REFRACTIVE INDEX Even the finest strands of protoplasm can be seen in water, in spite of the fact that they may be transparent and colorless. This is because of their relatively high index of refraction. It is surprising that so little is known about the optical characters of protoplasm which may be seen to change during cell division. Schaeffer (1926) has shown that the nuclei of certain marine amoebae become much more prominent by dilu- tion of the sea water with fresh water, and Chalkley (1935) has shown that there is a change in the refractive index of the nuclei of A. proteus during division: the interkinetic nuclei can easily be seen at a magnifica- 84 PROTOPLASM OF PROTOZOA tion of 200 diameters; those in division cannot. Very few measurements of the refractive index of protoplasm have been made; Frederikse (1933b) has reported a value of 1.40 to 1.45 for A. verrucosa; Mackin- non and Vlés (1908) 1.51 for the cilia of Stentor, and 1.56 for the flagellum of Trypanosoma (Spirochaeta) balbiani. Mackinnon and Vlés made their determinations by immersing the organisms in media of different refractive indices; double refraction, due to depolarization, disappears in media of the same refractive index as the cilia and flagella. Fauré-Fremiet (1929) found the index of refraction for entire amoebo- cytes of Lumbricus to be 1.400, for the hyaloplasm 1.364; of Asterzas to be 1.446 and 1.385 respectively (for methods see Pfeiffer, 1931). STRUCTURAL PROPERTIES It has long been known that the polarity of a cell may persist after the relative positions of its various visible constituents have been changed; this has led to the idea that polarity has its basis somehow or other in the structure of homogenous cytoplasm, which remains unchanged in spite of exposure to high centrifugal forces (Conklin, 1924). Polarity and symmetry are generally present in the Mastigophora and Infusoria, in which an anterior-posterior axis is usually persistent throughout active life, and has been described as present in cysts (Lund, 1917, in Bursaria). However, in Sarcodina such as Amoeba, polarity may be thought of as continually changing, being bound up with the gel-sol process at the temporary posterior end, the flow of protoplasm forward, and the sol- gel process at the temporary anterior end. Hyman (1917) has demon- strated that the temporarily differentiated anterior end of Amoeba is the region of highest susceptibility to cyanide. Mast (1931) and others have reported that electrical currents have a solating action on the plasmagel, on the side directed toward the cathode. Recently Chalkley (1935) has studied the process of cytokinesis in A. proteus. He observed, with the onset of prophase, a loss in sensitivity, a swelling up of the organism, a decrease in activity of the contractile vacuole, and an increase in movement of the granules in the region of the nucleus. With the separation of the daughter chromosome plates, he observed a flow of the cytoplasm from the equator, in the same direc- tion as the separating daughter plates. As the daughter plates approach the surface of the cell and the new nuclei begin to form, a solation of PROTOPLASM OF PROTOZOA 85 the plasmagel takes place, resulting in the formation of numerous pseudopodia which become attached and undergo active ameboid move- ment. At the same time, because of the flow of the cytoplasm from the equator toward the two poles, the region of the Amoeba at the equator has become narrowed to a thin neck. Presumably a solation of the plasmagel at the equator, together with a pull exerted by the two ac- tively dividing ameboid daughter cells, produces the final separation of the organisms. The temporary polarity becomes immediately lost after the completion of division. The physical-chemical factors involving the change in polarity of the protoplasm at the equator and the forces responsible for the flow of the protoplasm from this point are unknown. However, Chalkley thinks the fundamental principles involved in these processes are the same as those described by Mast for ameboid movement, namely sol-gel trans- formation. Further, it has been shown by Chambers (1938) that if the nucleus is moved toward the cell surface, pseudopodia are induced in that region. Becker (1928) has demonstrated that the factor which de- termines the direction of streaming and hence polarity is located in Mastigina, a flagellated amoeba, in the region of the nucleus. If the nucleus is moved posteriorly, streaming ceases and is then resumed to- ward the nucleus. It should be pointed out that in the Protozoa nuclear division and cytoplasmic fission may be closely correlated or widely separated in tempo, and that they often exhibit a considerable degree of independence. How- ever, the plane of separation of the nucleus usually determines the plane of cytokinesis, in that they usually take place at or nearly at right angles to each other. In organisms like the Mastigophora, the plane of nu- clear division is parallel to the anterior-posterior axis and coincides with the plane of cytokinesis of the organism. In Ciliata the plane of nuclear division is perpendicular to the anterior-posterior axis and coin- cides with the plane of cytokinesis. In some ciliates there appears to be a definite and permanent divi- sion zone laid down early in the life of the organism, which 1s not disturbed by diverse multilations of the body (Calkins, 1926). Further- more, in Frontonia this zone differs so markedly from the surrounding cytoplasm that it can be easily seen in the living condition (Popoff, 1908). 86 PROTOPLASM OF PROTOZOA Multilation studies by Calkins (1911) and by Peebles (1912) on Paramecium have resulted in the production of numerous monsters. Peebles describes this condition as due to an upset in nuclear and cyto- plasmic division tempo; thus when the nucleus is ready to divide, the cytoplasm is not, and vice versa. If this be true, the mitotic and cyto- kinetic phenomena in this form must be closely integrated, and the division mechanism of the organism as a whole be dependent upon the proper coordination of both the nuclear and the cytoplasmic division processes. Child and Deviney (1926) and Child (1934), have shown that in ciliates generally there is an anteroposterior gradient, due to the existence of a physiological gradient in the longitudinal axis. The anterior end is more susceptible to many agents, and there is also an axial differential in the rate of reduction of methylene blue. Child is of the opinion that this metabolic gradient is the only basis of physiological polarity. Lund (1917, 1921) has found that reversal of polarity often occurs in cut halves of Bursarza undergoing regeneration; it may also occur in normal animals. An indication of this change in polarity was a reversed beat of the cilia. He further found that Paramecium showed a reversed beat of cilia, in direct electrical currents of proper strength. Verworn (1899) has shown that paramecia and other ciliates orient themselves with the anterior end of their bodies toward the cathode to which they swim. On the other hand, many flagellates show an opposite behavior. Schaeffer (1931) has presented evidence that the protoplasm of the amoebae, and presumably of other organisms, consists primarily of spe- cific molecules which are organized into definite patterns, and that most or all of the characteristics of the organisms are due to or correlated with positional relationships of the molecules. It is generally thought at the present time that adjacent protein mole- cules, because of their multipolar character, have an orienting effect upon one another and that the resulting configuration may be equivalent to a net-like structure, extended in three dimensions. That this is true may be inferred from the anomalous viscosity of solutions of proteins and protoplasm: they show non-Newtonian flow, i.e., their viscosity varies with the stress applied, although they may outwardly conform to true fluids in being free from rigidity. Bensley (1938) has recently isolated from the cytoplasm of liver PROTOPLASM OF PROTOZOA 87 cells a material called plasmosin, which he thinks is constituted of linear micelles. The formation of a fiber results from the end-to-end orientation of these linear micelles. In protoplasm in the liquid state these micelles are probably independent and irregularly arranged but in flowing protoplasm they would be oriented parallel to the axis of flow. . . . From this state by simple end-to-end combination all changes in viscosity are possible up to the forma- tion of a fibrous gel . . . or even discrete fibers. Bresslau (1928) has also shown that ‘“‘tektin,’” a material extruded by ciliates, has anisotropic properties somewhat similar to those of plasmo- sin. The fact that the chromosomes probably represent gene-strings has been of enormous importance in determining our ideas of significant pro- toplasmic structure. The chromosomes apparently reproduce themselves at each cell division, so that their individuality is retained in all cell generations. The demonstration of the presence of these linear aggregates of visible size, which are self-perpetuating, cleared the way for the micel- lar theory of protoplasm structure. Much of the evidence for the presence of linear aggregates in proto- plasm has been obtained by microdissection, by the use of dark-field and polarization microscopes, by studies on cohesion and swelling, and by X-ray diffraction methods. Some of these have already been considered and others will be discussed below. ELASTICITY According to Seifriz (1936), “elasticity is the best indication we have of the structure of living matter” and is evidence for the presence of linear aggregates. A body is said to be elastic if after having been strained it tends to return to its original form when the stress is removed. Volume elasticity is characteristic of fluids and solids; shape elasticity (rigidity ) of solids and colloids in the gel condition generally. The form assumed by the bodies of various flagellates and ciliates is characteristic and offers means of identification in many instances. Relative rigidity is of common occurrence among those Protozoa, such as Euplotes, which have a differentiated pellicle and a firm ectoplasm. C. V. Taylor (1920) demonstrated this elasticity by applying pressure with a microneedle; the body bent conspicuously over the needle but returned to normal shape 88 PROTOPLASM OF PROTOZOA upon release of the pressure. Animals cut two-thirds across may keep their shape; this argues for a stiff consistency of the ectoplasm, as well as a tough pellicle. Any apparent modification in the shape of E. patella occurs only from outside pressure, since the animal is unable to vary its shape. Other hypotrichs may be similarly armored, as is shown by the fact that they are broken in the ultracentrifuge (King and Beams, un- published work) so that fragments are found swimming around as though the whole animal were brittle rather than plastic. In other forms, such as Paramecium, the body may be constricted when the animal forces its way through obstacles. Upon ultracentrifuging in gum solutions paramecia become much elongated and thin, because of the presence of materials of different specific gravities in the cell. Such elongated animals may survive and return to their normal shape unless the pellicle and ectoplasm have been strained beyond the limit of their elasticity, in which case the structure responsible for return to normal shape has been destroyed and they die, permanently deformed (King and Beams, 1937). That the form of Paramecium is determined by the relatively firm outer layers has been shown by some observations of Chambers (1924), who tore the ectoplasm with a microneedle. The fluid interior pours out into the surrounding water and the ectoplasm soon disintegrates; but oc- casionally the fluid endoplasm forms a delicate surface film which main- tains the integrity of the extruded mass. Merton (1928) has studied these so-called autoplasmic paramecia which, deprived of pellicle, cilia, trichocysts, and ectoplasm, take on the form of a fan-shaped amoeba, which may live for some days, divide, and exhibit locomotor activities. Under unfavorable conditions a rayed stage, with long pseudopodia-like extensions reminiscent of a A. radiosa, may be assumed. That the pellicle is not the only element involved in rigidity has been shown for Blephayr- isma by Nadler (1929). The pellicle of this form may be shed after im- mersion in weak solutions of strychnine sulphate. The “naked” animal emerges from the old pellicle with the shape and elasticity characteristic of the species. Eventually the pellicle is reformed, and the process may then be repeated. That amoebae have elasticity of form to a considerable degree has been shown by Jennings (1904), who bent a pseudopod with a glass rod; when released the pseudopod sprang back into its original position. Whole amoebae were also bent, with subsequent return to original form PROTOPLASM OF PROTOZOA 89 after release; Dellinger (1906) and Hyman (1917) have repeated these experiments, as indeed may be done by anyone. Howland (1924c) has stretched the outer layer of A. verrucosa with microneedles; upon re- lease the animal recovers its normal shape, apparently unharmed. The protoplasm of plasmodia of slime molds (Seifriz, 1928) is at times poorly elastic, and at other times it may be stretched into very fine, long threads which snap back a goodly distance when released. Seifriz has also determined elastic values by inserting minute nickel particles into the protoplasm of slime molds and attracting these particles electromagnetically. On release of the current the metal particles return to their original position; the distance traveled is measured and used as an indicator of elasticity. A maximum stretching value of 4.4 microns was obtained for liquid, previously streaming protoplasm of myxomy- cetes, a maximum value of 292 microns for quiescent, highly viscous exuded masses of protoplasm from plasmodia. This latter value is slightly greater than that for gelatin solutions and slightly greater than that of fresh egg albumen. The long thread-like pseudopodia of Foraminifera, which usually pull the organism along by adhering to the surface terminally and then con- tracting, have been shown to be elastic by Schultz (1915), who cut these and observed them to snap back like a rubber band. The reticulose pseudopodia (myxopodia) of the Foraminifera are very different from the lobose and filose forms in other Rhizopoda. The former have a soft miscible outer protoplasm which leads to fusion on contact with one another and a relatively rigid inner axial structure which shortens without wrinkling when the pseudopod is withdrawn. As this denser core is formed as an elongation “in the direction of growth, strains will be set up during the process which will give rise to ordered and preferential arrangement tending toward the crystalline state” (Ewles and Speakman, 1930). Thus it will be seen that the axial solid protoplasm of these myxopodia, although it is not in the form of a fiber, serves the same function as the axial filament of the Heliozoa and Radiolaria. Up to this point elasticity has been considered principally in connec- tion with the protoplasm itself, or in its temporary completely reversible structures. There remain for consideration those differentiations which last the whole life of the organism and are usually irreversible, such as 90 PROTOPLASM OF PROTOZOA flagella, axial filaments, cilia, myonemes, and supporting fibers (mor- phonemes) of various kinds. The fact that these are elastic is too well. known to need more than mention here. Spindle fibers, on the other hand, are often thought to be artifacts, but Cleveland (1935), work- ing on the hypermastigote flagellates, has pulled the centriole out of position; the chromosomes were also displaced, but both centriole and chromosomes immediately sprang back into position when released. This argues not only for the reality of the chromosomal and spindle fibers, but also that they are structures of considerable elasticity. If one considers the aphorism of Needham (1936) “that biology is largely the study of fibers,” these fibrillar structures of the Protozoa are of great interest because they consist of parallel aggregations of the submicroscopic elon- gated particles (micelles) of protoplasm (see Taylor, 7vfra, Chapter IV). CONTRACTILITY We may distinguish between active contraction, as in muscle fibers, and elastic shortening after having been stretched, as in elastic fibers in the higher animals. Although there are many examples of contractility and elasticity, in the Protozoa, associated with differentiated myonemes or morphonemes respectively, there are also many instances of active con- traction in the absence of any optically differentiated structure. Accord- ing to Lewis (1926), theories of contractility must be based on the presence of a contractile molecule, because the fibrillae seen in heart muscle in tissue culture are not “true” cytological structures but are due to reversible gelation. Fauré-Fremiet (1930) also holds that the gelified condition is often bound up with the existence of internal fibrillar structures, which disappear when solution occurs. That such fibrillae appear and disappear may, of course, be caused by aggregation and disaggregation of smaller invisible fibrillae, or may even be due to changes’ of refractive index. It is well known that objects that are of the same refractive index, transparency, and color cannot be seen, even in the dark field (Schmidt, 1929). It is generally assumed that contraction of the gelled ectoplasmic cylinder in Amoeba forces the more fluid endoplasm forward (Schaeffer, 1920; Pantin, 1923; Mast, 1926b). The contraction is thought to be caused by the fact that the gel-sol process at the posterior end of the PROTOPLASM OF PROTOZOA 91 amoeba results in an increase of volume and so stretches the gelled cylin- der of protoplasm; the resulting elasticity forces the sol forward where a decrease in volume has occurred upon gelation of the plasmasol. How- ever, in shelled forms, such as Difflugia (Dellinger, 1906; Mast, 1931a) and Centropyxis, there is an active longitudinal contraction of the plas- magel cylinder which results in locomotion by pulling the shell along. That the circular elastic contraction in Amoeba and the longitudinal active contraction in Difflvgia are different mechanisms may be doubted. However, Mast (1931a) has shown that D/fflvgia deprived of their shells move much as does Amoeba. It is to be noted that the source of energy is in the ectoplasm, so that the streaming of the endoplasm in these forms must be of a different nature from cyclosis in other forms such as Paramecium ot Frontonia, where protoplasmic streaming is ex- tremely difficult to explain in terms of contraction. There are many examples of local contractility in the literature of ameboid movement. Swinging and revolving movements of lobose pseudopods when not in contact with the substrate have been described by Penard (1902), Jennings (1906), Hyman (1917), Kepner and Edwards (1917), and many others. These differential local contractions of the ectoplasm often approximate muscular activity, according to Kep- ner and Edwards. Schaeffer (1926) has described corkscrew-shaped pseu- dopodia in Astramoeba flagellipoda, with from two to eight spirals which wave about quite like flagella, often making a complete revolution in three seconds. The most spectacular instances of local contraction are those in which an Amoeba pinches a large ciliate in half. Mast and Root (1916) de- sctibe this process as taking ten seconds for Paramecium and show that it cannot be explained in terms of the surface tension of the Amoeba. Beers (1924) describes the constriction of Frontonia by Amoeba until the former was dumbbell-shaped, and ascribes the pinching to centripetal pressure exercised by an extending collar of protoplasm which pinched the prey in half in eight minutes. Kepner and Whitlock (1921) saw a partly ingested Paramecium constricted much as described by Beers for Frontonia, except for the loss of the cilia from the ingested part. The figures and descriptions of the two latter instances are very similar to those of Grosse-Allermann (1909) for ingestion by invagination in 92 PROTOPLASM OF PROTOZOA A. terricola, and of Mast and Doyle (1934) for the ingestion of water by various species of Amoeba in albumin solutions. Penard (1902) has described and figured the pinching off of an extensive injured portion by A. terricola as has Jennings (1906) for A. /7max. The process of egestion, as figured by Howland (1924c) for Amoeba verrucosa, seems also to involve extensive local contraction. The lobopodia and filopodia of the Amoebida and Testacea are solid peripherally with a central fluid region, while the pseudopods of the Radiolaria and Heliozoa (axopodia) and those of the Foraminifera (myxopodia) are more fluid peripherally and more solid axially. In axopods and myxopods there can be no flow of endoplasm caused by the elastic contraction of a gelled ectoplasmic cylinder. Roskin (1925) describes the origin of the axopods of Actinosphaerium by the flowing together and alignment of fibrillae, which eventually fuse into a hollow tube, filled with fluid which is associated with rigidity and contractility. That these axopods do contract rapidly has been shown for Acanthocystis, which, according to Penard (1904), may traverse twenty times its own diameter by rolling along the tips of the axopods, which must adhere, contract, and then release very rapidly. In the foraminiferan Astrorhiza, according to Schultz (1915), pseudopods stretch out five to six times the length of the body, make ‘‘feeling’” movements and may finally either adhere to the substratum or contract and be withdrawn. The organism is usually fastened by three bundles of pseudopods; if one of these be torn loose, the others contract rapidly and the animal is pulled forward. The axial, more solid stereoplasm of these pseudopodia is distinctly fibrillar. Schmidt (1929) has also described the formation of contractile pseudopo- dia in the Foraminiferan Rhuwmblerinella by the alignment and coales- cence of fibrillae. The minute size of cilia and flagella makes it extremely difficult to determine whether their movements are due to active contraction or to changes occurring in the cell which bears them. In addition to their usual method of locomotion by means of flagella, many Mastigophora such as the euglenoids show euglenoid movement, or metaboly. In these forms there are present in the pellicle more or less spiral striations, which apparently are elastic in nature and tend to pre- serve the form of the organism during and after the contraction of the superficial layers of the body. In some cryptomonads (e.g., Chrodmonas PROTOPLASM OF PROTOZOA 93 pulex) springing movements are brought about by the strong contrac- tion of the outer layer of the body, the resulting locomotion being inde- pendent of flagellar movement. Certain flagellates have an extraordinary superficial resemblance to medusae both in appearance and in method of locomotion: (Cystoflagellata: Leptodiscus, Craspedotella; Phytomona- dida: Medusochloris; Dinoflagellata: Clipeodinium). In these types ac- cording to Pascher (1917), the movements represent a special form of metaboly and two mechanical systems should be present, one radial and dilating, the other peripheral and contracting. The former could of course be elastic only and bring about passive return after the con- traction of the latter. In the Infusoria there are many examples of specialized retractile organelles: the tentacles of Suctoria may be retracted and extended much as may the axopods of the Heliozoa. The structure of these tentacles is quite similar to that of the axopods( Roskin, 1925). The remarkable tentacles of the ciliate Actinobolina may be extended to a length twice the diameter of the body or may be completely retracted. They are as- sociated internally with two groups of fibrils which seem to wind up to retract and unwind to extend them (Wenrich, 1929). In many ciliates, such as Stentor and Spirostomum, there are actively contractile myonemes in the ectoplasmic layer. In other related forms, such as Climacostomum, the corresponding structures are elastic only and have been referred to as morphonemes. The myonemes may even appear to be striated (Dierks, 1926, and others) but this has been denied (Roskin, 1923). A most unusual case of extension and retraction occurs in the ciliate Lacrymaria olor. Here the ‘“‘neck’’ may be extended to fifteen times the length of the body, the form of which remains unchanged. This “‘elas- ticity” is associated with the presence of what appears to be a series of spiral striations. However, upon complete extension of the neck, Penard (1922) has observed that there is only a single continuous spiral. No experimental work has been done upon this form, so that the nature of the extension and retraction is not understood. However, Verworn (1899) has cut the neck of L. olor free from the head and body. The neck retains its extensile and contractile properties exactly as when in connection with the body. The finer structure of the contractile stalks of the Vorticellidae has been studied by Koltzoff (1912), Fortner (1926), and many others. 94 PROTOPLASM OF PROTOZOA The stalk consists of an external wall, an inner liquid, and a spirally wound contractile cord. Within the contractile cord is an excentrically placed myoneme often called a spasmoneme, the contraction of which causes the stalk to become coiled like a spring. The distal end of the stalk is usually attached to the substratum, but in Vorticella natans and V. mayeri the organisms are never attached but swim, stalk first, through the water. In the former the stalk rolls up into narrow spirals, but in the latter the stalk swings in a wide loop on contraction somewhat like a flagellum. Bélehradek and Paspa (1928) have reconstructed a myogram from moving pictures of the stalk of a vorticellid and find that the spasmoneme does not function like a true muscle but like a modified flagellum. Various attempts have been made to explain the contraction and extension of the stalk of vorticellids as caused by the complex action of two opposing bundles of fibers, or in terms of internal pressure against coiling brought about by elastic fibers. Myonemes are also well developed in the gregarines, where longi- tudinal and circular myonemes are apparently responsible for bending and peristalsis-like movements. Myonemes called myophrisks are also present in certain Radiolaria. Here they are associated with the spreading out of the gelatinous cortical layer, previous to their decrease in specific gravity and subsequent rise to the surface (Schewiakoff, 1927). ROPINESS, OR THREAD FORMATION Living material is said to be ropy if it can be drawn out into threads; ropiness is thought to be due to the micellar structure of the material. The ability of a drop of a pure liquid to resist distortion is due to its surface tension only, and in heterogenous mixtures the surface film may approximate a solid consistency. A column of fluid breaks up into a number of smaller spherical bodies. The formation of pseudopodia, especially of filopodia, axopodia, and myxopodia, demonstrates the pres- ence of solid structures in protoplasm. Then, too, protoplasm may be drawn out into fine strands of considerable elasticity and tensile strength: the ectoplasm of Amoeba has been drawn out into fine strands by Hyman (1917) and many others; Schultz (1915) was able to draw out the protoplasm of the foraminiferan Astrorhiza into long threads; this was more marked in the outer layers than in the inner mass of protoplasm. PROTOPLASM OF PROTOZOA 95 Many other observers, using microdissection methods, have confirmed this for a whole series of animal and plant cells. The spinning of threads from protoplasm is generally assumed to be dependent upon the sub- microscopic fibrillar structure of the protoplasm, the latter being respon- sible for its elasticity and high degree of extensibility. The tensile strength of a strand of myxomycete protoplasm has been found by Pfeffer (quoted by Seifriz, 1936) to be 50 mgm. per square millimeter. The ropiness of protoplasm is of course conditioned by temperature, viscosity, hydrogen- ion concentration, and other factors which affect protoplasm (review by Jochims, 1930). Further evidence of the micellar nature of protoplasm may be deduced from the experiments of Seifriz (1936), who has examined the proto- plasm of slime molds with a Spierer lens. When the protoplasm is quiet it presents a mosaic appearance, but when in active flow or when formed into a thread is presents a striated appearance. Harvey and Marsland (1932) noted that the crystals of Amoeba fall in jerks when moving under centrifugal force; this may be due to the presence of structural elements in the protoplasm. Moore (1935) has found that slime molds in the plasmodial stage will flow through pores 1 y in diameter. He also forced this living material through bolting cloth of various mesh sizes. The plasmodia survived after being forced through pores 0.20 mm. in diameter or larger, but died when forced through smaller pores. Moore thinks that there are fibrillar elements in slime mold protoplasm which are destroyed if forced unoriented through a pore through which they could flow if the micelles were properly oriented. DOUBLE REFRACTION The great advantage of the use of the polarizing microscope is that characteristic structure which is otherwise imperceptible may be revealed, even in the living organism, without any alteration of the specimen. Amorphous and pseudoamorphous materials (i.e., materials in which the orientation of the particles is a random one) are dark when viewed between crossed nicol prisms. True double refraction is always the re- sult of an orientation of optically anisotropic elements; many substances, such as glass, become doubly refractive if deformed by external forces. Many sols when in movement or when placed in an electric or a magnetic 96 PROTOPLASM OF PROTOZOA field show double refraction, as do many gels when under pressure or when drawn out into threads. Mechanical stress may produce double refraction in gelatin even of 0.01 percent. Phenomena of double refraction in protoplasm often indicate the presence of mechanically and optically anisotropic elements oriented in some definite way, and are among the best evidences for the presence of micelles in protoplasm. See Schmidt, 1937, for a complete discussion. Valentin saw double refraction in the cilia of O palina ranarum in 1861 and Rouget in the stalk muscle of Carchesium in 1862. Engelmann (1875) carried out very extensive pioneering work on double refraction on the Protozoa and other forms. He saw anisotropy in the pellicle of large ciliates, such as Opalina, but was unable to distinguish double refraction in the myonemes of Stentor because the entire outer layer is doubly refractive. Mackinnon (1909) mentioned that the protoplasm of Actinosphaerium is quite generally anisotropic, and Schmidt (1937) saw faint but unmistakable traces of double refraction at the edge of the surface layers of Amoeba, which became distinctly greater as the amoeba became rounded up before encysting, the cyst wall showing it very distinctly. Engelmann (1875) also observed that the axopods of Actinosphaerium were doubly refractive in the living condition, the anisotropy being coextensive with the axial filaments which extend deep into the proto- plasm. With the withdrawal of the pseudopods the condition vanishes. Mackinnon (1909) confirmed the findings of Engelmann. The structure of the axial filaments of these pseudopods is known to be fibrillar (Ros- kin, 1925; Rumjantzew and Suntzowa, 1925). Schultz (1915) found the fibrillae in the rhizopods of the foraminiferan, Astrorhiza, to be doubly refractive, and Schmidt (1929) found weak anisotropy in the axopods of the radiolarian, Thalassicolla. Schultz (1915) and Schmidt (1929) report that the stereoplasmic axis of the pseudopods of the Foraminifera are formed by the parallel alignment of fibers. Cilia have been found to be doubly refractive by Valentin, Engel- mann (1875), and Mackinnon and Vlés (1908). The latter authors consider this to be caused by the depolarization of the light by reflection from surfaces of the cilia, because of the difference of refractive index. If air penetrates into the axis of a cilium, upon drying the anisotropy is greatly increased (Schmidt, 1937). The so-called rootlets of the cilia PROTOPLASM OF PROTOZOA . 97 are doubly refractive, but the basal granules are not (Engelmann, 1880.) Myonemes have been quite generally shown to be anisotropic, especially those in the stalk muscle of the vorticellids (Engelmann, 1875; Wrzesniowski, 1877; Mackinnon and Vlés, 1908). The myoneme of the stalk spreads out into fibrillae in the base of the animal. These, too, are doubly refractive. Engelmann (1875) found that the extensile neck of Trachelocerca (Lacrymaria) olor, when stretched out, was positively anisotropic in relation to its longitudinal axis. It is to be recalled that the neck may be extended as much as fifteen times the length of the body. Associated with this is a single spiral thread which becomes straight upon extension of the neck (Penard, 1922). Brandt (1885) has shown that the isospore nuclei of the Radiolaria are anisotropic, but not the vegetative nor the anisospore nuclei. This has been confirmed by Schmidt (1932) on living and preserved material of the same form. Schmidt (1929) had also observed double refraction of the nuclear membrane in living nuclei of a foraminiferan and of Amoeba; in the latter he also observed weak anisotropy of small visible granules (chromatin?) in the living nucleus. Finally, Kalmus (1931) has recorded that certain elements of the division figure of the nuclei of Paramecium show slight traces of double refraction during fission and conjugation. X-RAY DIFFRACTION AND ULTRACENTRIFUGATION Early studies on cohesion and swelling relations of organic fibrillar structures indicated that the finer structures of which they are composed are micellar in nature. Recently X-ray diffraction methods have sub- stantiated this view and have made it possible actually to measure the dimensions of these structural units. Much of this work has been done on keratin, elastin, chitin, myosin, cellulose, and other nonliving sub- stances. Some observations have been made on living nerve fibers (Schmitt, Bear, and Clark, 1935). The evidence from X-ray diffraction shows that animal fibers owe their anisotropic properties to the fact that they are composed of longitudinally oriented protein chains. However, in most protoplasm the configuration of such chains must be such that it may be altered rapidly and reversibly. For a review of X-ray diffraction, see Frey-Wyssling, 1933: 98 PROTOPLASM OF PROTOZOA Beams and King (1937) and King and Beams (1938) have shown that the complicated process of karyokinesis may occur in eggs of Ascaris, although cytokinesis does not usually take place at very high centrifugal forces. They believe that if stratification occurred, it would involve a breakdown of the normal, submicroscopic spatial relations, which are of importance for the maintenance of life. LITERATURE CITED Adolph, E. F. 1931. The regulation of size as illustrated in unicellular or- ganisms. Springfield. Alexander, G. 1931. The significance of hydrogen ion concentration in the biology of Exglena gracilis Klebs. Biol. Bull., 61: 165-84. Allison, R. V. 1924. The density of unicellular organisms. Ann. appl. Biol., 1: 153-64. Angerer, C. A. 1936. The effects of mechanical agitation on the relative vis- cosity of Amoeba protoplasm. J. cell comp. Physiol., 8: 329-45. Ball, G. H. 1927. Studies on Paramecium. Ill. The effects of vital dyes on Paramecium caudatum. Biol. Bull., 52: 68-78. Barnes, T. C. 1937. Textbook of general physiology. Philadelphia. Bayliss, W. M. 1920. The properties of colloidal systems. IV. Reversible gelation in living protoplasm. Proc. roy. Soc. B., 91: 196-201. Beams, H. W., and R. L. King. 1932. The architecture of the parietal cells of the salivary glands of the grasshopper. J. Morph., 53: 223-41. — 1937. The suppression of cleavage in Ascaris eggs by ultracentrifuging. Biol. Bull, 73: 99-111, Becker, E. R. 1928. Streaming and polarity in Mastigina hylae (Frenzel). Biol. Bull., 54: 109-16. Beers, C. D. 1924. Observations on Amoeba feeding on the ciliate Frontonia. Brit. J. exp. Bioly 335-41: Bélehradek, J. 1935. Temperature and living matter. Protop. Monog., vol. 8. Berlin. Bélehradek, J., and K. M. Paspa. 1928. Courbe myographique de la Vorticelle. Arch. Int. Physiol., 30: 70-72. Bensley, R. R. 1938. Plasmosin. The gel- and fiber-forming constituent of the protoplasm of the hepatic cell. Anat. Rec., 72: 351-69. Bles, E. J. 1929. Arcella. A study in cell physiology. Quart. J. micr. Sci., 72: 527-648. Bodine, J. H. 1923. Excystation of Colpoda cucullus, Some factors affecting excystation of Colpoda cucullus from its resting cysts. J. exp. Zool., 37: 115-25: PROTOPLASM OF PROTOZOA oD Brandt, K. 1885. Die koloniebildenden Radiolarien (Spaerozoen) des Golfes van Neapel. Fauna u. Flora Neapel, Monogr. 13. Bresslau, E. 1926. Die Stabchenstruktur der Tektinhiillen. Arb. Staatsinst. exp. Ther. Frankfurt, H. 21 (Festschr. f. W. Kolle): 26-31. Brinley, F. J. 1928. The effect of chemicals on viscosity of protoplasm as indicated by Brownian movement. Protoplasma, 4: 177-82. Brown, D. E. S., and D. A. Marshland. 1936. The viscosity of Amoeba at high hydrostatic pressure. J. cell. comp. Physiol., 8: 159-65. Browne, K. M. R. 1938. The Golgi apparatus and other cytoplasmic bodies in Sprrostomum ambiguum. J. R. micr. Soc., 58: 188-99. Brues, C. T. 1927. Animal life in hot springs. Quart. Rev. Biol., 2: 181-203. Butts, H. E. 1935. The effect of certain salts of sea water upon reproduction in the marine Amoeba, Flabellula mira Schaeffer. Physiol. Zodl., 8: 273-89. Calkins, G. N. 1911. Effects produced by cutting Paramecium cells. Biol. Bull., 21: 36-72. —— 1919 Uroleptus mobilis, Engelm. I. History of the nuclei during divi- sion and conjugation. J. Exp. Zool., 27: 293-355. 1933. The biology of the Protozoa. 2d ed., Philadelphia. Chalkley, H. W. 1929. Changes in water content in Amoeba in relation to changes in its protoplasmic structure. Physiol. Zodl., 2: 535-74. — 1930. On the relation between the resistance to heat and the mechanism of death in Paramecium. Physiol. Zo6l., 3: 425-40. —— 1935. The mechanism of cytoplasmic fission in Amoeba proteus. Proto- plasma, 24: 607-21. —— 1936. The behavior of the karosome and the “peripheral chromatin” during mitosis and interkinesis in Amoeba proteus with particular refer- ence to the morphologic distribution of nucleic acid as indicated by the Feulgen reaction. J. Morph., 60: 13-29. Chalkley, H. W., and G. E. Daniel. 1934. The effect of certain chemicals upon the division of the cytoplasm in Amoeba proteus. Protoplasma, 21: 258-67. Chambers, R. 1921. The effect of experimentally induced changes in con- sistency on protoplasmic movement. Proc. Soc. exp. Biol. N. Y., 19: 87- 88. —— 1922. A micro injection study on the permeability of the starfish egg. J. gen. Physiol., 5: 189-93. —— 1924, The physical structure of protoplasm as determined by micro- dissection and injection. Sec. V, General cytology, 234-309 (edited by E. V. Cowdry). Chicago. —— 1938. Structural aspects of cell division. Arch. exp. Zellforsch. 22: 252-56. 100 PROTOPLASM OF PROTOZOA Chambers, R., and J. A. Dawson. 1925. The structure of the undulating membrane in the ciliate Blepharisma, Biol. Bull., 48: 240-42. Chambers, R., and H. P. Hale. 1932. The formation of ice in protoplasm. Proc, Loy SOGHB11102336-52. Chambers, R., and P. Reznikoff. 1926. Micrurgical studies in cell physiology. 1. The action of the chlorides of Na, K, Ca, and Mg on the protoplasm of Amoeba proteus. J. gen. Physiol., 8: 369-401. Chase, A. M., and O. Glaser. 1930. Forward movement of Paramecium as a function of the hydrogen ion concentration. J. gen. Physiol., 13: 627-36. Chatton, E., and A. Lwoff. 1923a. Sur l’évolution des Infusoires des Lamelle- branches, Les formes primitives du phylum des Thigmotriches: le genre Thigmo., Phrya C. R. Acad. Sci. Paris, 177: 81-83. —— 1923b. Un Cas remarquable d’adaptation: Ellobiophrya donacis. n. g. n. sp., etc. C. R. Soc. Biol., Paris, 88: 749-52. Child, C. M. 1934. The differential reduction of methylene blue by Para- mecium and some other ciliates. Protoplasma, 22: 377-94. Child, C. M., and E. Deviney. 1926. Contributions to the physiology of Paramecium caudatum. J. exp. Zool., 43: 257-312. Cleveland, L. R. 1935. The centriole and its role in mitosis as seen in living cells. Science, 81: 598-600. Cleveland, L. R., in collaboration with S. R. Hall, E. P. Sanders, and J. Col- lier. 1934. The wood-feeding roach Cryptocercus, its Protozoa ,and the symbiosis between Protozoa and Roach. Mem. Amer. Acad. Arts Sci., 17, No. 2; 187-342. Conklin, E. G. 1924. Cellular differentiation. Sec. IX, General cytology, 539- 607 (ed. by E. V. Cowdry). Chicago. Daniel, G. E., and H. W. Chalkley. 1932. The influence of temperature upon the process of division in Amoeba proteus (Leidy). J. cell, comp. Physiol., 2: 311-27. Daniels, M. L. 1938. A cytological study of the gregarine parasites of Tenebrio molitor using the ultra-centrifuge. Quart. J. micr. Sci., 80: 293-320. Daugherty, K. 1937. The action of anesthetics on Amoeba protoplasm. Physiol. Zobl., 10: 473-83. Davenport, C. B. 1897. Experimental morphology. New York. Dawson, J. A., and M. Belkin. 1929. The digestion of oils by Amoeba proteus. Biol. Bull., 56: 80-86. Dellinger, A. P. 1906. Locomotion of amoebae and allied forms. J.. exp. Zool., 3: 337-58. — 1909. The cilium as a key to the structure of contractile protoplasm. J. Morph., 20: 171-209. Dierks, K. 1926. Untersuchungen tiber die Morphologie und Physiologie des Stentor coeruleus, Arch. Protistenk., 54: 1-91. Dobell, C. C. 1911. The principles of protistology. Arch. Protistenk., 23: 269-310. PROTOPLASM OF PROTOZOA 101 Dogiel, V., and M. Issakowa-Keo. 1927. Physiologische Studien an Infusorien. II. Der Einflusz der Salzlésungen auf die Ernahrung von Paramaecium. Biol. Zbl., 47: 577-86. Edwards, J. G. 1923. The effect of chemicals on locomotion in Amoeba. I. J. exp. Zool:, 38: 1-43. Engelmann, T. W. 1875. Contractilitét und Doppelbrechung. Pfliig. Arch. ges. Physiol., 11: 432-64. —— 1880. Zur Anatomie und Physiologie der Flimmerzellen. Pfliig. Arch. ges. Physiol., 23:: 505-35. Ephrussi, B., and R. Rapkin. 1928. Action des différents sels sur le Spirosto- mum. Protoplasma, 5: 35-40. Ewles, N. A., and J. B. Speakman. 1930. Examination of the fine structure of wool by X-ray analysis. Proc. roy. Soc. B., 105: 600-7. Fauré-Fremiet, E. 1905. L’appareil fixateur chez les Vorticellidae. Arch. Protistenk., 6: 207-26. —— 1908. L’Ancystropodium maupasi. Arch Protistenk., 13: 121-37. — 1910. La fixation chez les Infusoires cilies. Bull. Sci. Fr. Belg., 44: 27-50. — 1929. Caracteres physico-chimiques des choanoleucocytes de quelques invertébrés. Protoplasm, 6: 521-609. — 1930. The kinetics of living matter. Trans. Faraday Soc., 26: 779-93. —— 1932. Strombidium calkinsi, a new thigmotactic species. Biol. Bull. 62: 201-4. — 1935. L’Oeuvre de Félix Dupardin et la notion de protoplasma. Proto- plasma, 23: 250-69. Fauré-Fremiet, E., B. Ephrussi, and L. Rapkine. 1926. Lames minces formées par la diffluence du cytoplasma cellulaire. C. R. Soc. Biol. Paris, 94: 442-43, Fetter, D. 1926. Determination of the protoplasmic viscosity of Paramecium by the centrifuge method. J. exp. Zool., 44: 279-83. Fortner, H. 1926. Zur Morphologie und Physiologie des Vorticellenstieles. Zirwiss. Zool, 128° 114-32; Frederikse, A. M. 1933a. Viskositatsinderungen des protoplasmas wahrend der Narkose. Protoplasma. 18: 194-207. 1933b. Der Brechungsindex des Protoplasmas. Protoplasma, 19: 473-48. Frey-Wyssling, A. 1938. Submikroskopische Morprologie des Protoplasmas und seiner Derivate. Protop.-Monog. 15, Berlin. Gaw, H. Z. 1936a. Physiology of the contractile vacuole in ciliates. 2. The effect of hydrogen ion concentration. Arch. Protistenk., 87: 194-200. . —— 1936b. Physiology of the contractile vacuole in ciliates 4. The effect of heavy water. Arch. Protistenk. 87: 213-24. Gelei, J. V. 1928. Nochmals tiber den Nephridialapparat bei den Protozoen. Arch Protistenk., 64: 479-94. Ginsberg, H. 1922. Untersuchungen zum Plasmabau der Amében, im Hin- 102 PROTOPLASM OF PROTOZOA blick auf die Wabentheorie. Roux Arch EntwMech. Organ., 51: 150-250. Giese, A. C. 1935. The role of starvation in conjugation of Paramecium. Physiol. Zool., 8: 116-25. Gray, J. 1931. A textbook of experimental cytology. New York. Greeley, A. W. 1901. On the analogy between the effects of loss of water and lowering of temperature. Amer. J. Physiol., 6: 122-28. 1902. The artificial production of spores in Monas by a reduction of the temperature. Biol. Bull., 3: 165-71. —— 1903. Further studies on the effect of variations in temperature on ani- mal tissues. Biol. Bull., 5: 42-54. —— 1904, Experiments on the physical structure of the protoplasm of Paramoecium, etc. Biol. Bull., 7: 3-32. Grosse-Allermann, W. 1909. Studien tber Amoeba terricola Kreeff. Arch. Protistenk., 17: 203-57. Gruber, A. 1889. Biologische Studien an Protozoen. Biol. Zbl., 9: 14-23. Gruber, K. 1912. Biologische und experimentelle Untersuchungen an Amoeba proteus, Arch. Protestenk., 25: 316-76. Hahnert, W. F. 1912. A quantitative study of reactions to electricity in Amoeba proteus. Physiol. Zodl., 5: 491-526. Harvey, E. B. 1917. A physiological study of specific gravity and of lumines- cence in Noctiluca, with special reference to anesthesia. Publ. Carneg. Tnstita; INo: 251: 235-53. Harvey, E. N. 1931. Observations on living cells, made with the microscope- centrifuge. J. exp. Biol., 8: 267-74. — 1934. Biological effects of heavy water. Biol. Bull., 66: 91-96. —— 1936. The properties of elastic membranes with special reference to the cell surface. J. cell. comp. Physiol., 8: 251-60. 1938. Some physical properties of protoplasm. J. appl. Physics, 9: 68-80. Harvey, E. N., and J. F. Danielli. 1936. The elasticity of thin films in rela- tion to the cell surface. J. cell. comp. Physiol., 8: 31-36. Harvey, E. N., E. B. Harvey, and A. L. Loomis. 1928. Further observations on the effect of high frequency sound waves on living matter. Biol. Bull., 55: 459-69. Harvey, E. N., and D. A. Marsland. 1932. The tension at the surface of Amoeba dubia with direct observations on the movement of cytoplasmic particles at high centrifugal speeds. J. cell. comp. Physiol., 2: UaeMe Heilbrunn, L. V. 1928. The colloid chemistry of protoplasm. Berlin. 1929a. Protoplasmic viscosity of Amoeba at different temperatures. Protoplasma, 8: 58-64. 1929b. The absolute viscosity of Amoeba protoplasm. Protoplasma, 8: 65-69. — 1931. Anasthesia in Amoeba. Anat. Rec., 51: 27. —— 1937. An outline of general physiology. Philadelphia. PROTOPLASM OF PROTOZOA 103 Heilbrunn, L. V., and K. Daugherty. 1931. The action of the chlorides of sodium, potassium, calcium, and magnesium on the protoplasm of Amoeba dubia. Physiol. Zo6l., 4: 635-51. — 1932. The action of sodium, potassium, calcium, and magnesium ions on the plasmagel of Amoeba proteus. Physiol. Zodl., 5: 254-74. — 1933. The action of ultraviolet rays on Amoeba protoplasm. Protoplasma, 18: 596-619. — 1934. A further study of the action of potassium on Amoeba proto- plasm. J. cell. comp. Physiol., 5: 207-18. —— 1938. Fat release in Amoeba after irradiation. Physiol. Zodl., 11: 383-87. —— 1939. The electric charge of protoplasmic colloids. Physiol. Zodl., 12: ibe 4 Heilbrunn, L. V., and D. Mazia. 1936. The action of radiations on living protoplasm. Biological effects of radiation, (edited by B. M. Dugger) : 625-76. New York. Henderson, V. E. 1930. The present status of the theories of narcosis. Physiol. Revs. LOe 17 1-220° Holter, H., and M. J. Kopac. 1937. Studies on enzymatic histo-chemistry XXIV. Localization of peptidase in Ameba. J. cell. comp. Physiol., 10: Aa Hopkins, D. L. 1938. The vacuoles and vacuolar activity in the Marine amoeba, Flabellula mira Schaeffer and the nature of the natural red system in Protozoa. Biodyn., No. 34: 1-22. Howland, R. B. 1924a. On excretion of nitrogenous waste as a function of the contractile vacuole. J. exp. Zool., 40: 231-50. —— 1924b. Experiments on the contractile vacuole of Amoeba verrucosa and Paramecium caudatum. J. exp. Zool., 40: 251-62. 1924c. Dissection of the pellicle of Amoeba verrucosa. J. exp. Zool., 40: 263-70. Howland, R. B., and H. Pollack. 1927. Micrurgical studies on the contractile vacuole. I. Relation of the physical state of the internal protoplasm to the behavior of the vacuole. II. Micro-injection of distilled water. J. exp. Zool., 48: 441-58. Hyman, L. 1917. Metabolic gradients in Amoeba and their relation to the mechanism of amoeboid movement. J. exp. Zool., 24: 55-99. Jacobs, M. H. 1919. Acclimatization as a factor affecting the upper thermal death points of organisms. J. exp. Zool., 27: 427-42. — 1922. The effects of carbon dioxide on the consistency of protoplasm. Biol. Bull., 42: 14-30. Jennings, H. S. 1904. Movements and reactions of Amoeba. Publ. Carneg. Instn., No. 16: 129-234. 1906. Behavior of the lower organisms. New York. Jensen, P. 1893. Die absolute Kraft einer Flimmerzelle. Pflig. Arch. ges. Physiol., 54: 537-51. 104 PROTOPLASM OF PROTOZOA Jochims, J. 1930. Das Fadenziehen biologischer Substanzen. Protoplasma, 9: 298-317. Johnson, L. P. 1939. A study of Exglena rubra Hardy 1911. Trans. Amer. micr. Soc., 58: 42-48. Jordan-Lloyd, D. 1932. Colloidal structure and its biological significance. Biol. Rev., 7: 254-73. Joyet-Lavergne, Ph. 1931. La Physico-Chimie de la sexualité. Protop.- Monog., Vol. V. Berlin. Kahl, A. 1935. Wimpertiere order Ciliata (Infusoria), in Dahl: Die Tierwelt Deutschlands. 18 Teil, Jena. Kalmus, . 1931. Paramecium. Jena. Kanda, Sakyo. 1914. On the geotropism of Paramecium and Spirostomum Biol. Bull., 24: 1-24. —— 1918. Further studies on the geotropism of Paramecium caudatum. Biol. Bull., 34: 108-19. Kepner, W. A., and J. G. Edwards. 1917. Food reactions of Pelomyxa caro- linesis Wilson. J. exp. Zool., 24: 381-407. Kepner, W. A., and B. D. Reynolds. 1923. Reactions of cell-bodies and pseudopodia fragments of D/fflugia. Biol. Bull., 22-47. Kepner, W. A., and W. H. Taliaferro. 1913. Reactions of Amoeba proteus to food. Biol. Bull., 24: 411-22. Kepner, W. A., and C. Whitlock. 1921. Food reactions of Amoeba proteus. J. exp. Zool., 32: 397-425. King, R. L. 1928. The contractile vacuole in Paramecium trichium. Biol. Bull., 55: 59-64. — 1935. The contractile vacuole of Paramecium multimicronucleata. J. Morph... 58:9 555-74.. King, R. L., and H. W. Beams, 1937. The effect of ultracentrifuging on Paramecium, with special reference to recovery and macronuclear reor- ganization. J. Morph., 61: 27-49. — 1938. An experimental study of chromatin diminution in Ascarzs, J. exp. Zool., 77: 425-43. Kitching, J. A. 1936. The physiology of contractile vacuoles. II. The control of body volume in marine Peritricha. J. exp. Biol., 13: 11-27. 1938. Contractile vacuoles. Biol. Rev., 13: 403-44. Kite, G. L. 1913. Studies on the physical properties of protoplasm. I. The physical properties of the protoplasm of certain animal and plant cells. Amer. J. Physiol., 32: 146-64. Koltzoff, N. K. 1912. Untersuchungen tiber die Kontraktilitat des Vorticellens- tiels. Arch. Zellforsch, 7: 344-423. Lackey, J. B. 1936. Some freshwater Protozoa with blue chromatophores Biol. Bull., 71: 492-97. PROTOPLASM OF PROTOZOA 105 Leontjew, H. 1926a. Zur Biophysik der niederen Organismem. II. Mitteilung. Die Bestimmung des spezifischen Gewicht und der Masse von Naegleria. Pflug. Arch. ges. Physiol., 213: 1-4. —— 1926b. Uber das spezifische Gewicht des Protoplasmas Biochem. Z. 170: 326-29. —— 1927. Uber das spezifische Gewicht des Protoplasmas. III. Protoplasma, 2: 59-64. Lewis, W. H. 1926. Cultivation of embryonic heart muscle. Publ. Carneg. Instn., 363: 1-21. Lloyd, F. E. 1928. The contractile vacuole. Biol. Rev., 3: 329-58. Loeb, L. 1927. Amoeboid movement and agglutination in amoebocytes of Limulus and the relation of these processes to tissue formation and thrombosis. Protoplasma, 2: 512-53. —— 1928. Amoebocyte tissue and amoeboid movement. Protoplasma, 4: 596-625. Looper, J. B. 1928. Cytoplasmic fusion in Actinophrys sol, with special ref- erence to the karyoplasmic ratio. J. exp. Zool., 50: 31-49. Luce, R. H. 1926. Orientation to the electric current and to light in Amoeba. (Abss)\;Anats Rees 32155: Ludwig, W. 1928a. Der Betriebsstoffwechsel von Paramecium caudatum. Arch. Protistenk., 62: 12-40. —— 1928b. Permeabilitat und Wasserwechsel bei Noctiluca miliaris Suriray. Zool, Anz..76: 273-85. —— 1930. Zur Theorie der Flimmerbewegung. Z. vergl. Physiol., 13: 397- 504. Lund, E. J. 1917. Reversibility of morphogenetic processes in Bursaria. J. exp. Zool., 24: 1-33. —— 1921. Experimental control of organic polarity by the electric current. is JrexpsZool.,.34:,471-93,. — Lund, E. J., and G. A. Logan. 1925. The relation of the stability of protoplasmic films in Noctiluca to the duration and intensity of an ap- plied electric potential. J. gen. Physiol., 7: 461-72. Luyet, B. J., and P. M. Gehenio. 1935. Comparative ultra-violet absorption by the constituent parts of protozoan cells (Paramaecium). Biodyn., No. 7: 1-14. —— 1938. The lower limit of vital temperatures, a review. Biodyn. No. 33: 1-92; Lwoff, A. 1934. Die Bedeutung des Blutfarbstoffes fiir die parasitischen Flagellaten. Zbl. Bakt., 130: 498-518. Lyon, E. P. 1905. On the theory of geotropism in Paramaecium. Amet. J. Physiol., 14: 421-32. McClendon, J. F. 1909. Protozoan studies. J. exp. Zool., 6: 265-83. Mackinnon, D. L. 1909. Optical properties of contractile organs in Heliozoa. J. Physiol., 38: 254-58. 106 PROTOPLASM OF PROTOZOA Mackinnon, D. L., and F. Vlés. 1908. On the optical properties of contractile organs. J. R. micr. Soc., 553-58. Makarov, P. 1935. Experimentelle Untersuchungen an Protozoen mit Bezug auf das Narkose-Problem. Protoplasma, 24: 593-606. Marsland, D. 1933. The site of narcosis in a cell; the action of a series of paraffin oils on Amoeba dubia. J. cell. comp. Physiol., 4: 9-30. Marsland, D. A. 1939. The mechanism of protoplasmic streaming, etc. J. cell, comp. Physiol., 13: 23-30. Massart, J. 1893. Sur l’irritabilité des Noctiluques. Bull. sci. Fr. Belg., 25: 59-76. Mast, S. O. 1909. The reactions of Didinium nasutum. Biol. Bull., 16: 91-118. 1926a. The structure of protoplasm in Amoeba, Amer. Nat., 60: 133-42. — 1926b. Structure, movement, locomotion and stimulation in Amoeba. J. Morph., 41: 347-425. 1929. Mechanics of locomotion in Amoeba proteus with special refer- ence to the factors involved in attachment to the substratum. Protoplasma, 8: 344-77. —— 1931la. Movement and response in D7/flugia with special reference to the nature of cytoplasmic contraction. Biol. Bull., 61: 223-41. —— 1931b. The nature of the action of electricity in producing response and injury in Amoeba proteus (Leidy) and effect of electricity on the vis- cosity of protoplasm. Z. vergl. Physiol., 15: 309-28. —— 1938a. The contractile vacuole in Amoeba proteus (Leidy). Biol. Bull., 74: 306-13. 1938b. Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389-94. Mast, S. O., and W. L. Doyle. 1934. Ingestion of fluid by Amoeba. Proto- plasma, 20: 555-60. —— 1935a. Structure, origin and function of cytoplasmic constituents in Amoeba proteus. 1. Structure. Arch. Protistenk., 86: 155-80. —— 1935b. II. Origin and function based on experimental evidence; effect of centrifuging on Amoeba proteus. Arch. Protistenk., 86: 278-306. Mast, S. O., and C. Fowler. 1935. Permeability of Amoeba proteus to water. J. cell. comp. Physiol., 6: 151-67. Mast, S. O., and W. F. Hahnert. 1935. Feeding, digestion, and starvation in Amoeba proteus (Leidy). Physiol. Zodl., 8: 255-72. Mast, S. O., and F. M. Root. 1916. Observations on Ameba feeding on rotifers, nematodes and ciliates, and their bearing on the surface-tension theory. J. exp. Zool., 21: 33-49. Merton, H. 1928. Untersuchungen tiber die Enstehung amébenahnlicher Zellen an absterbenden Infusorien. Math.-nat. Kl. Heidelb. Akad. Wiss., 5 B. 1928, Nr. 3: 51-29. Metcalf, M. M. 1910. Studies upon Amoeba. J. exp. Zool., 9: 301-31. Moore, A. R. 1935. On the significance of cytoplasmic structure in Plasmo- dium. J. cell. comp. Physiol., 7: 113-29. PROTOPLASM OF PROTOZOA 107 Morita, Y., and R. Chambers. 1929. Permeability differences between nuclear and cytoplasmic surfaces in Amoeba dubia. Biol. Bull., 56: 64-67. Nadler, E. J. 1929. Notes on the loss and regeneration of the pellicle in Blepharisma undulans. Biol. Bull., 56: 327-30. Nassonov, D. 1924. Der Exkretionsapparat (kontraktile vakuole) der Protozoa als Homologon des Golgischen Apparats der Metazoazellen. Arch. mikr. Anat., 103: 437-82. Needham, J. 1936. Order and life. New Haven. Needham, J., and D. M. Needham. 1925. The hydrogen-ion concentration and oxidation-reduction potential of the cell interior: a micro-injection study. Proc. roy. Soc. B., 98: 259-86. —— 1926. Further micro-injection studies on the oxidation-reduction poten- tial of the cell-interior. Proc. roy. Soc. B., 99: 383-97. Noland, L. E. 1931. Studies on the taxonomy of the genus Vorticella. Trans. Amer, micr. Soc., 50: 81-123. Oliphant, J. F. 1938. The effect of chemicals and temperature on reversal in ciliary action in Paramecium. Physiol. Zo6l., 11: 19-30. Packard, C. 1923. The susceptibility of cells to radium radiations. Proc. Soc. exp, Biol. NUY., 20: 226-27. 1924. The susceptibility of cells to radium radiations. Biol. Bull., 46: 165-77. —— 1925. The effect of light on the permeability of Paramecium. J. gen. Physiol., 7: 363-72. Pantin, C. F, A. 1923. On the physiology of amoeboid movement. I. J. Mar. Biol. Assoc., 13: 24-69. ——— 1924a. Temperature and the viscosity of protoplasm. Jour. Mar. biol. Ass. U.K., 13: 331-39. —— 1924b. On the physiology of amoeboid movement. II. The effect of tem- perature. Brit. J. exp. Biol., 1: 519-38. -—— 1926a. On the physiology of amoeboid movement. III. The action of calcium. Brit. J. exp. Biol., 3: 275-95. 1926b. On the physiology of amoeboid movement. IV. The action of magnesium. Brit, J. exp. Biol., 3: 297-312. —— 1930. On the physiology of amoeboid movmeent: a.V. Anaerobic move- ment. Proc. roy. Soc. B. 105: 538-55. b. VI. The action of oxygen. Proc. roy. Soc. B., 105: 555-64. c.VII. The action of anaesthetics. Proc. roy. Soc. B.; 105: 565-79: Park, O. 1929. The differential reduction of osmic acid in the cortex of Paramecium, and its bearing upon the metabolic gradient conception. Physiol. Zodl., 2: 449-58. Parsons, C. W. 1926. Some observations on the behavior of Amoeba proteus. Quart. J. micr. Sci. 70: 629-48. Pascher, A. 1917. Von der merkwurdigen Bewegungsweise einiger Flagel- laten. Biol. Zbl., 37: 241-429. 108 PROTOPLASM OF PROTOZOA Patten, R., and H. W. Beams. 1936. Observations on the effect of the ultra- centrifuge on some free-living flagellates. Quart. J. micr. Sci., 78: 615-35. Peebles, F, 1912. Regeneration and regulation in Paramecium caudatum. Biol. Bull., 23: 154-70. Pekarek, J. 1930. Absolute Viskositatsmessung mit Hilfe der Brownschen Molekularbewegung. II. Viskositaétsbestimmung des Zellsaftes der Epi- dermiszellen von Allium cepa und des Amdben-Protoplasmas. Proto- plasma, 11: 19-48. Penard, E. 1902. Faune Rhizopodique due Basin du Leman. Geneve. 1904. Les Héliozoaires d’eau douce. Genéve. 1922. Etudes sur les Infusoires d’leau douce. Genéve. Pfeiffer, H. 1931. Uber mikro-refraktometrische methoden im Dienste der Protoplasma-Forschung. Z. wiss. Mikr., 48: 47-62. —— 1933a. Beitrage zur quantitativen Bestimmung von molekularkraften des Protoplasmas. I. Eine Methode zur messung der Adhisionsarbeit plasmatischer Oberflachen. Protoplasma, 19: 177-92. — 1933b. Beitrige zur quantitativen Bestimmung von molekularkraften des Protoplasmas. II. Eine modifikation der methode zur bestimmung des Reinbungswiederstandes nackter. Protoplasten. Protoplasma, 20: 73-78. —— 1933c. III. Die Bestimmung des Randwinkels aus der Form nackter Protoplasten. Protoplasma, 20: 79-84. — 1934. Versuche zur Bestimmung des spezifischen Gewichts nackter Protoplasten. Protoplasma, 21: 427-32. —— 1935. Literature on adhesiveness (stickiness) of protoplasm and related topics. Protoplasma, 23: 270-81. — 1936. Further tests of the elasticity of protoplasm. Physics, 7: 302-5. — 1937. Experimental researches on the non-Newtonian nature of Proto- plasma. Cytologia, Tokyo. (Fujii Jubilaei vol.) 701-10. Pitts, R. F., and S. O. Mast. 1934. The relation between inorganic salt con- centration, hydrogen ion concentration and physiological processes in Amoeba proteus. Il. J. cell. comp. Physiol., 4: 435-55. Platt, J. B. 1899. On the specific gravity of Spirostomum, Paramaecium, and the tadpole in relation to the problem of geotaxis. Amer. Nat., 33: 31-38. Pollack, H. 1928a. Intracellular hydrion concentration studies. III. etc. Biol. Bull., 55: 383-85. —— 1928b. Micrurgical studies in cell physiology. VI. Calcium ions in liv- ing protoplasm. J. gen. Physiol., 11: 539-45. Popoff, M. 1908. Experimentelle Zellstudien 1. Arch. Zellforsch., 1: 245-379. Piitter, A. 1903. Die Flimmerbewegung. Ergebn. Physiol., 2: 1-102. Reznikoff, P. 1926. Micrurgical studies in cell physiology. II. The action of the chlorides of lead, mercury, copper, iron, and aluminum on the proto- plasm of Amoeba proteus. J. gen. Physiol., 10: 9-21. PROTOPLASM OF PROTOZOA 109 Reznikoff, P., and R. Chambers. 1927. Micrurgical studies in cell physiology. III. The action of CO, and some salts of Na, Ca, and K on the proto- plasm of Amoeba dubia. J. gen. Physiol., 10: 731-55. Reznikoff, P., and H. Pollock. 1928. Intracellular hydrion concentration studies. II. The effect of injection of acids and salts on the cytoplasmic pH of Amoeba dubia. Biol. Bull., 55: 377-82. Rhumbler, L. 1898. Physikalische analyse von Lebenserscheinungen der Zelle. Roux Arch. EntwMech. Organ., 7: 103-350. Root, F. M. 1914. Reproduction and reactions to food in the suctorian, Podophrya collini, n. sp. Arch, Protistenk., 35: 164-96. Roskin, G. 1923. La Structure des myonemes des Infusoires. Bull. biol., 57: 143-51. —— 1925. Uber die Axopodien der Heliozoa und die Greiftentakeln der Ephelotiden. Arch Protistenk., 52: 207-16. Rumjantzew, A., and E. Suntzowa. 1925. Untersuchungen tiber den Proto- plasmabau von Actinosphaerium Eichhornit. Arch. Protistenk., 52: 217- 64. Saunders, J. T. 1925. The trichocysts in Paramecium. Biol. Rev., 1: 249-69. Schaeffer, A. A. 1916. On feeding habits of Ameba. J. exp. Zool., 20: 529-84. 1920. Amoeboid movement. Princeton. —— 1926. Taxonomy of the amebas. Publ. Carneg. Instn., No. 345. — 1931. On molecular organization in ameban protoplasm. Science, 74: 47-51. Schewiakoff, W. 1927. Die Acantharia des Golfes von Neapel. Fauna u. Flora neapel. Monogr., 37. Schmidt, W. J. 1929. Rheoplasma und Steroplasma. Protoplasma, 7: 353-94. 1932. Der submikroskopische Bau des Chromatins. II. Uber die Dop- pelbrechung der Isosporenkerne von Sphaerozoen. Arch. Protistenk., 77: 463-90. —— 1937. Die Doppelbrechung von Karyoplasma, Zytoplasma und Meta- plasm. Protop. Monog., 11. Berlin. Schmitt, F. O. 1938. Optical studies of the molecular organization of living systems. J. Appl. Physics, 9: 109-17. Schmitt, F. O., R. S. Bear, and G. L. Clark. 1935. X-ray diffraction studies on nerve. Radiology, 25: 131-51. Schultz, E. 1915. Die Hyle des Lebens. I. Beobactungen und Experimente an Astrorhiza limicola. Roux Arch. EntwMech. Organ., 41: 215-36. Seifriz, W. 1928. The physical properties of protoplasm. Colloid Chemistry (edited by J. Alexander), 11: 403-50. 1936. Protoplasm. New York. Singh, B. N. 1939. The cytology of Amoeba proteus “Y’.and the effects of large and small centrifugal forces. Quart. J. micr. Sci., 80: 601-35. Spek, J. 1921. Der Einfluss der Salze auf die Plasmakolloide von Actinosphae- rium eichhornit. Acta Zool. Stock., 2: 153-200. 110 PROTOPLASM OF PROTOZOA —— 1923. Uber den physikalischen Zustand von Plasma und Zelle der Opalina ranarum, (Purk. et Val.) Arch. Protistenk., 46: 166-00. —— 1924. Neue Beitrage zum Problem der Plasmastrukturen. Z. Zell. u. Gewebelehre, 1: 278-326. Spek, J., and R. Chambers. 1934. Neue experimentelle Studien tiber das Problem der Reaktion des Protoplasmas. Protoplasma, 20: 376-406. Stump, A. B. 1935. Observations on the feeding of Difflugia, Pontigulasia and Lesquereusia. Biol. Bull., 69: 136-42. 1936. The influence of test materials on reproduction in Pontigulasia vas (Leidy) Schouteden. Biol. Bull., 70: 142-47. Taliaferro, W. H., and L. G. Taliaferro. 1934. Complement fixation, pre- cipitin, adhesion, mercuric chloride and Wassermann tests in equine trypanosomiasis of Panama (murrina). J. Immunol., 26: 193-213. Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap- paratus in Ewplotes by the method of microdissection. Univ. Cal. Publ. Zool., 19: 403-70. —— 1923. The contractile vacuole in Evplotes: an example of the sol-gel reversibility of cytoplasm. J. exp. Zool., 37: 259-89. 1935. Protoplasmic reorganisation and animal life cycles. Biol. Rev., LOZ T11-22° Taylor, H. S., W. W. Swingle, H. Eyring, and A. A. Frost. 1933. The effect of water containing the isotope of hydrogen upon fresh water organisms. J. cell. comp. Physiol., 4: 1-8. Tchakhotine, S. 1937. Radiations, cell permeability and colloidal changes. Trans. Faraday Soc., 33: 1068-72. Thornton, F. E. 1932. The viscosity of the plasmagel of Amoeba proteus at different temperatures. Physiol. Zodl., 5: 246-53. —— 1935. The action of Sodium, Potassium, Calcium, and Magnesium ions on the plasmagel of Amoeba proteus at different temperatures. Physiol. Zool., 8: 246-54. Tiegs, O. W. 1928. Surface tension and the theory of protoplasmic move- ment. Protoplasma, 4: 88-139. Verworn, M. 1888. Biologische Protisten-Studien. Z. wiss. Zool., 46: 455-70. 1899. General physiology. London. Voegtlin, C., and H. W. Chalkley. 1935. The chemistry of cell division. IV. The influence of H,S, HCN, CO, and some other chemicals on mitosis in Amoeba proteus. Protoplasma, 24: 365-83. Wenrich, D. H. 1929. The structure and behavior of Actinobolus vorax. Biol. Bull., 56: 390-401. Winterstein, H. 1926. Die Narkose. Berlin. Wrzesniowski, A. 1877. Beitrige zur naturgeschichte der Infusorien. Z. wiss. Zool., 29: 267-323. CHAPTER III CYTOPLASMIC INCLUSIONS RONALD F. MACLENNAN ALL ACTIVE CELLS possess a large number of cytoplasmic granules which change in number, size, shape, and composition in accordance with the changes in the activities of the cell of which they are a part.* The fact that small granules are so constantly present in the living substance is an indication that such a fine suspension of material represents a colloidal condition favorable for the life process. It seems certain that as the physiology of the cell becomes more clearly understood there will be shown to be a definite dependence of vital phenomena on the granular nature of protoplasm, on the properties which it possesses by virtue of the fact that it is a suspen- sion (Heilbrunn, 1928, p. 20). The cytoplasmic granules are a visible part of the fundamental organi- zation of the cell, and the elucidation of their functions contributes not merely to a specialized branch of cytology but contributes directly to a solution of the fundamental problem of protoplasmic organization. The richness of the granular complex early attracted the interest of cytologists, and many studies were made on their chemical composition. The report of Biitschli’s discovery that the carbohydrate granules of gregarines differ from those in vertebrates is one of the classic papers in the group. During the first thirty years of this century the emphasis shifted from the earlier cytochemical methods to an interest in certain of the granules as permanent, self-perpetyating cytoplasmic organelles, which could be classified by certain empirical reactions such as osmic reduction or the segregation of janus green and neutral red. Dissatisfac- tion with the specificity of these methods has resulted recently in a re- newed emphasis on methods which yield specific information on the chemical and physical nature of the cytoplasmic granules and their cyclic changes. Too often, however, there has been a tendency to carry * This paper is a contribution from the departments of Zoélogy of the State College of Washington and Oberlin College. 12 CYTOPLASMIC INCLUSIONS on these two types of investigation separately, with the result that in many cases the morphological and functional studies of the cytoplasmic granules have become separated. The one group emphasizes the classifica- tion of granules into hard and fast categories of Golgi bodies, mitochon- dria, vacuome and so forth, with little specific consideration of function, while those engaged in functional studies tend to group all the granules together or to devise entirely new systems, which hinder the comparison of granules in several species. This review is an attempt to codrdinate these two angles of approach, so that both may contribute to our under- standing of the rdle of the cytoplasmic granules in the cell. Since summaries are available of the characteristics of single groups of granules, this review is not intended to provide an exhaustive catalogue of the facts of any one group. Particular emphasis is placed on those granules which have been described with sufficient completeness to fur- nish evidence as to reactions, classification, and function, as well as their relationship to other granules in the same cell. Specific directions on standard techniques for demonstrating the various granules are avail- able in the various books on microtechnique and histochemistry and so are not described in detail here. The more recent publications will be emphasized, since the specificity of methods has improved greatly and summaries of the earlier papers are available in the works of Calkins, Doflein-Reichenow, and others. The Protophyta have been omitted in most cases, since their inclusion would complicate the picture unneces- sarily. MITOCHONDRIA Undoubtedly many granules described in early cytological studies of Protoza were actually mitochondria, but their status as a separate group of cytoplasmic constituents in the unicellular organisms was not recog- nized until the publication of the monograph of Fauré-Fremiet (1910), which emphasized the concept that mitochondria are universal, self-per- petuating cytoplasmic constituents. The identification of mitochondria is not yet entirely satisfactory, since it depends upon stains and fixatives of the lipoid component, a material not restricted to mitochondria alone, or upon vital dyes which are not as effective in the Protozoa as in the Metazoa and which in certain cases stain other organelles as well. Typical mitochondria are refractile CYTOPLASMIC INCLUSIONS 113 in life, become grey brown, or black in osmic techniques, but usually bleach faster than the Golgi bodies, reduce pyrogallol, take basic stains after fixation in lipoid preservatives, stain weakly or not at all after fixa- tives containing acetic acid, and are stained vitally with janus green B. This last method is often considered to be the final criterion, but un- fortunately in many cases the mitochondria of Protozoa stain only a pale green (Subramaniam and Ganapati, 1938, and others), not the dark green described in metazoan cells. In addition, the stain is not always specific, since Lynch (1930) found that any concentration from 1:2000 to 1:500,000 tints the entire organism (Lechriopyla), although the mitochondria can be distinguished by their darker color. Hayes (1938) found that none of the granules in Dé/eptus stained electively with Janus green B. An additional source of difficulty is the fact that in flagellates, the parabasal bodies are often stained as darkly as the mitochondria. Although the parabasal bodies and mitochondria show additional simi- larities in staining reactions and composition, Volkonsky (1933) points out that the former are derivatives of the neuromotor system and cannot be considered as homologous with the mitochondria. Mitochondria present a wide variety of shapes, but most commonly they are spherules (Fig. 17), chains of spherules (Fig. 26), short rods (Figs. 22, 23), or dumb-bells. The filamentous structures found so often in metazoan cells are found rarely in the Protozoa, but a good example has been described in the phytoflagellate Polykrikos by Chatton and Grassé (1929). Lynch (1930), in his studies on the ciliate Lechriopyla, found a compound structure (Figs. 15. 16), composed of several discs. Each disc is composed of chromophobic material, with a rim of chromo- philic material which stains with Janus green and the other mitochondrial dyes. In cases of secretion (Fig. 17), the secretion granules often appear as a chromophobic center in the mitochondria (MacLennan, 1936), but the mitochondrial material itself usually appears to be homogeneous either in the living unstained ciliate or after any of the mitochondrial stains. It is probable that the chromophobic center of the discs of Lechriopyla represents material secreted by the rim, which is the mito- chondrial part of the complex discs. In some cases, however, mitochondria may have a true duplex structure, since Mast and Doyle (1935b) showed that the outer surface of the mitochondria of Amoeba stain more deeply than the center. This differentiation may be explained either as a definite a ee Wl 2 af "G4 ° Coat —"- Flite a : i : oR OY - earner - 2 t arash wali youn pdt tha ap Ta gaan yer 0 E "¢- a9 3 ie xf 27 Figure 15-16. From Lechriopyla mystax: Figure 15, end view after Hirschler’s mitochondrial technique; Figure 16, lateral view, after Champy-iron haematoxylin. (After Lynch, 1930.) Figure 17. From Ichthyophthirius multifiliis, series showing mitochondria and the secretion of paraglycogen, vital stain with janus green. (After MacLennan, 1936.) Figures 18-21. From Monocystis showing de novo origin of mitochondria: Fig- ure 18, sporozoite; Figure 19, trophozoite; Figure 20, conjugating gametes from a cyst; Figure 21, spores, Champy or Flemmings—iron haematoxylin. (After Horning, 1929.) Figures 22-25. From Amoeba proteus: Figures 22-23, free in cytoplasm; Figure 22, nor- mal: Figure 23, fixed in modified Regaud’s fluid; Figures 24-25, on surface of contractile vacuole. (After Mast and Doyle, 1935.) Figures 26-27, From Aggregata eberthi: Figure 26, mitochondria proper; Figure 27, mitochondria associated with protein reserves. (After Joyet-Lavergne, 1926.) Figures 28-29. From Bursaria truncatella: Figure 28, section of early conjugant; Figure 29, section of later conjugant. (After Poljansky, 1934.) In all cases, material which responds to mitochondrial stains is drawn in solid black; associated granules are stippled. CYTOPLASMIC INCLUSIONS 115 localization of stainable materials or as a dense surface with less dense centers, with the type of material the same in both places. The mitochondria in most Protozoa are fairly evenly distributed through the cytoplasm, sometimes alone and sometimes associated with various types of storage granules (see the discussion of function). Fauré-Fremiet (1910) found that if any localization occurs, the mito- chondria tend to concentrate beneath the pellicle and occasionally around the contractile vacuole. Horning (1927) extended these observations and contends that mitochondria tend to concentrate near all membranes— particularly around the food vacuoles during active digestion, beneath the pellicle, and around the nucleus. Hall and Nigrelli (1930) criticized Horning’s identification of mitochondria, which was based largely on dark-field observations, and showed that in Vorticella sp. the mito- chondria are not associated with the food vacuole. Volkonsky (1934) likewise rejected Horning’s identification and showed that the granules associated with the digestive vacuoles, in a large number of species, are stainable only with neutral red. MacLennan (1936) found a similar situation in Ichthyophthirius. In other Protozoa, however, the accumula- tion of mitochondria near membranes has been confirmed. Mast and Doyle (1935b) find that the mitochondria in Amoeba are occasionally associated with the gastriole (food vacuole) and they were able to cor- relate the association with the type and stage of digestion. They also demonstrated an association with the contractile vacuole, confirming the earlier work of Metcalf (1910). Volkonsky (1934) found a small ag- gregation of mitochondria around the food vacuole of Campanella and Paramecium during the alkaline phase of digestion. Chatton and Grassé (1929) showed that the filamentous mitochondria of Polykrikos tend to accumulate near the pellicle, but instead of being parallel to the surface, as in the ciliates described by Horning, are perpendicular to the surface. Poljansky (1934) studied the changes occurring in the life cycle of Bur- saria and found that the mitochondria of neutral individuals are uniformly dispersed throughout the cytoplasm, but during conjugation (Figs. 28, 29) the mitochondria migrate to the periphery and form a definite zone under the ectoplasm, and also around the micronuclear derivatives. The chondriosomes again scatter during the growth of the macronuclear pri- mordium. There seems to be neither universal nor permanent localization of mitochondria near membranes, as is to be expected according to Horn- 116 CYTOPLASMIC INCLUSIONS ing’s theory that mitochondria accumulate at the intracellular surfaces in accordance with the Gibbs-Thompson Law. Doyle (1935) suggests that mitochondria tend to collect at regions of active interchange, since he found that mitochondria are the only granules which flow out into the pseudopodia of the foraminiferan Irzdza. While this theory is attractive in certain cases, it is difficult to see how this would apply to the concentra- tions which occur in conjugating Byrsaria. Mitochondria are more widely accepted as universal, permanent, and self-perpetuating granules than any other cytoplasmic component, and for this reason it is worth while to consider in detail the proof upon which such statements rest. Rigid proof of this theory requires a demonstration of mitochondria only, with, of course, a lack of evidence of any de novo origin. Furthermore, it is obvious that proof of the continuity of any cytoplasmic component cannot be based on a study of only one stage in the life cycle, but must rest upon adequate studies of the whole life cycle. Mitochondria have been identified in a multitude of Protozoa of all groups by Fauré-Fremiet (1910) and later authors, and as a result these components are usually considered to be present in all Protozoa. How- ever, recent evidence shows that this assumption is unjustified. An ex- treme example is Trypanosoma diemyctyli, in which Nigrelli (1929) was not able to demonstrate any pre-formed mitochondria, although granules which satisfy the general criteria of mitochondria are induced by exposure of the organisms to Janus green B. These induced granules are not permanent, but disappear after about two hours. The marine amoeba Flabellula is another species in which mitochondria are normally lacking. Hopkins (1938b) found that the normal amoeba possessed no granules which can be classified as mitochondria, but that when the amoeba is disturbed in a variety of ways, granules are precipitated in small pre-formed cytoplasmic vacuoles, the contents of which are nor- mally a homogeneous fluid. After the recovery of the amoeba from the disturbing conditions, the granules are resorbed. These temporary granules possess the staining reactions of mitochondria, including the ability to segregate Janus green B, and are the only granules in this organism which can be classed in that group. As a contrast to these cases of induced mitochondria, Kirby (1936) reports the experimental destruc- tion of the mitochondria which are a normal component in the cytoplasm CYTOPLASMIC INCLUSIONS 17 of flagellates from termites. In normal Psewdodevescovina, large num- bers of mitochondria can be demonstrated by the Flemming-Regaud method but, after feeding for three days on filter paper soaked in one- percent Janus green B, no mitochondria can be demonstrated. Addi- tional examples of the lack of mitochondria are furnished by studies of the whole life cycle of certain Protozoa. Horning (1929) was able to demon- strate mitochondria in most stages of Monocystzs (Figs. 19, 20), but found that these granules disappear in the sporozoite stage (Fig. 21). Beers (1935) and MacLennan (1936) also found that mitochondria disappear in the later encysted stages of ciliates, although the same methods give positive results in other stages of the cycle. These observa- tions under both normal and experimental conditions demonstrate that mitochondria are neither universal nor permanent cytoplasmic constitu- ents. The crucial point in the classification of mitochondria as autonomous organelles is whether they always arise from preéxisting mitochondria. The occurrence of dumb-bell-shaped mitochondria and other possible division stages have been found so often in fixed and stained preparations that it is unnecessary to quote this evidence here. The observations on living material are few and perhaps the clearest is that of Horning (1926), who reported division stages of mitochondria in a living heterotrich and was able to confirm the descriptions based on fixed material. Thus division is a factor in the increase in numbers of mito- chondria, but returning again to the studies of the whole life cycle, we find that division is not the only method, since mitochondria must be formed de novo (Figs. 18-21) in those species in which the mito- chondria have disappeared during the quiescent phases of the life cycle. Mitochondria are not self-perpetuating organelles, but are differ- entiations which may endure for a longer or a shorter period during the cycle of the cell. In the cases considered above, mitochondria are cytoplasmic in their origin, but this is by no means the only possibility. Joyet-Lavergne (1926) states that the group of mitochondria attached to protein granules (Fig. 27) are derived from the nucleus along with the protein reserves (for discussion of this point, see p. 163), but Daniels (1938) in the same or related species could find no mitochondria attached to the protein granules. Calkins (1930) found in Uroleptus one set of cyto- 118 CYTOPLASMIC INCLUSIONS plasmic granules, which he traced back to macronuclear fragments dur- ing conjugation, interpreting these as mitochondria. Since the staining methods were not very specific and these granules do not stain with Janus green, the statement that these are mitochondria must be accepted with caution. Miller (1937) has endeavored to prove that mitochondria, Golgi bodies, and other cytoplasmic inclusions in A. proteus are “‘bac- teria spores, fungi, or yeasts, together with indigestible material of certain food organisms.” This idea is, of course, similar to that of Wallin and has been so thoroughly criticized by Cowdry and others that it need not be considered here in detail. Miller was not able to culture these cytoplasmic “bacteria’’ and his main argument seems to be based on the observation that mitochondrial stains and Golgi type impregnations will demonstrate granules in the culture medium. This merely shows that the stains used are not always specific under all conditions, a fact which has been pointed out many times. Miller does not present any evidence which can stand up against the observations and experiments of Mast and Doyle (1935a, 1935b), Holter and Kopac (1937), and Holter and Doyle (1938) on the same species. The mitochondria are specializations of the cell itself, probably in all cases from the cytoplasm, and are neither artifacts nor invaders. The composition of mitochondria is still incompletely known in any exact sense, in spite of the large amount of work done on these com- ponents. They have long been thought to be composed of both lipoids? and proteins, because of their staining reactions and solubilities (Fauré- Fremiet, 1910; Hirschler, 1924, 1927). Unfortunately no one has yet repeated in Protozoa the work of Bensley (1937), who isolated mitochondria from liver and was able to make both qualitative and quantitative analyses. These analyses confirmed the cytochemical analysis of lipoid and protein, but instead of the large amounts of phosphatids and so forth, which were predicted from the cytological reactions, he showed that the lipoid is largely neutral fat. Bensley also found that many reactions (for example the osmic-acid reaction) are much weaker * Lipoid is used here, as in most cytological works, in a very general sense. It includes all materials which are soluble in ether, absolute alcohol, and so forth, and which stain with Nile blue sulfate, the sudans, and other fat soluble dyes. Neutral fat, fatty acid, phosphatids, and the like respond to these tests. Lison (1936) suggests the rather awkward term sudanophil material, in order to emphasize the cytological side and to avoid false implications as to chemical nature. CYTOPLASMIC INCLUSIONS 119 in mitochondria in the cytoplasm than in mitochondria isolated from the cytoplasm. These observations confirm the validity of the interpretation of mitochondrial reactions in the Protozoa as indicative of a lipoid- protein mixture, but emphasize the need of caution in more specific interpretations before methods as specific as Bensley’s are applied to the Protozoa. It is clear that the evidence now available as to the nature of the lipoids and of the proteins in the mitochondria of Protozoa is sig- nificant largely as a lead for further work. Horning (1927) adheres to the view that the lipoid component of mitochondria is a phosphatid, but presents no conclusive evidence for this statement. Wermel (1925) found that the mitochondria (or lipo- somes) of Actinosphaerium react with Ciaccio’s method, but according to Lison the only valid interpretation is that unsaturated lipoids are present. MacLennan (1936) stated that the lipoid material in the mito- chondria of Ichthyophthirius ts a fatty acid, on the basis of a blue stain with Nile blue sulphate, used according to Lorrain Smith’s method. However, since Lison (1936) presents evidence against the specificity of this method, the above interpretation is perhaps too strict, but it is interesting to note that the original interpretation is in accord with Bensley’s analysis of the mitochondria of liver. There is likewise a lack of information on the nature of the proteins present. Hayes (1938) demonstrated a positive reaction to fuchsin-sulfurous acid reagent and claimed that nucleic acid is present. However, since lipoids may react in this manner in the “‘plasmal reaction,” it is possible that this test was concerned with the lipoid component rather than the protein portion. The metallic impregnation of mitochondria, or the depth of stain taken after the use of lipoid solvents, varies between the species of Protozoa and has usually been considered to be a rough indication of the pro- portion of lipoids present. Thus Scott and Horning (1932) find in O palina a large amount of lipoid; Lynch (1930) in Lechriopyla, Patten (1932) in Nyctotherus, MacLennan and Murer (1934) in Paramecium, find some lipoid; while Beers (1935) in Dédinium finds little if any evidence of lipoids in the mitochondria. Although this data is very crude, foundation is provided for the working hypothesis that there is a wide variation in the probable composition of mitochondria, rang- ing from practically pure lipoid to almost pure protein. Much of this difference represents constant differences between species and could be 120 CYTOPLASMIC INCLUSIONS detected by Bensley’s mass technique. However, Fauré-Fremiet (1910) found staining differences within the mitochondria of a single indi- vidual, and Peshkowskaya (1928) reports that the ectoplasmic chondrio- somes of Climacostomum are resistent to fixatives which usually dis- solve mitochondria, although the endoplasmic mitochondria are much more typical in their reactions. Pellissier (1936) found similar dif- ferences, not within the cell but between various individuals, and was able to show that all the mitochondria impregnate more deeply in the vegetative stages than in the stages just before reproduction. By the selection of species in which all granules are in the same stage at the same time, Bensley’s mass technique could be used very profitably in exploring the changes in mitochondrial composition. MacLennan and Murer (1934) found heavy deposits of ash in the typical mitochondrial rods, as well as in the other cytoplasmic granules of Paramecium. The presence of enzymes in mitochondria have been indicated in- directly in many cases by the morphological association of these bodies with structures in which digestive or synthetic activity is going on. The only direct demonstration of the localization of cytoplasmic enzymes is due to Holter and Kopac (1937) and Holter and Doyle (1938), who showed that dipeptidase is not present in mitochondria, but that amylase is. The method used was a combination of centrifugal localization of granules and micro-methods for the measurement of enzymatic activity. The mitochondria were concentrated in one end of an Amoeba, which was then cut and the enzymatic activity of the mitochondria-rich and the mitochondria-poor portions of the cytoplasm compared. Since both the centripetal and the centrifugal portions had the same amount of dipeptidase (measured by the ability to split alanylglycine) per unit volume of cytoplasm, Holter and Kopac con- cluded that the enzyme is in the matrix. They point out that this proves nothing as to the origin of the enzyme, which might diffuse out from a granule as fast as it is formed. Holter and Doyle found that the middle region of the centrifuged Amoeba had the most amylase (measured by the digestion of starch). The nucleus, crystals, digestive vacuoles, mitochondria, and matrix are found in this zone of the centri- fuged Amoeba. The enzyme could not be localized in the nucleus, since non-nucleated fragments show no significant diminution in amylase, CYTOPLASMIC INCLUSIONS 121 nor in the crystals since most of these are in the centrifugal end which would thus have the highest enzymatic activity. They also ruled out a localization in the digestive vacuoles by a demonstration that the enzyme content of hungry and feeding Amoeba, with a resultant difference in the number of vacuoles, is the same. The only structures the distribu- tion of which after centrifuging corresponds with the distribution of amylase are the mitochondria. The study of the centrifuged Amoeba presents many difficulties, since the stratification is never complete and there is always some mixing between the finishing of centrifuging and cutting the amoeba in parts, but the further use and development of these methods and their use in species in which stratification is complete will undoubtedly aid in the complete analysis of mitochondria and other cytoplasmic granules. The theory that mitochondria are concerned with cellular respiration has led to attempts to identify in the mitochondria the materials known to be active in this respect. One of these is glutathione, in which the physiologically active group is sulfhydril, demonstrable cytochemically by the sodium nitroprusside reaction. Joyet-Lavergne (1927-29) found that the mitochondria of Sporozoa give a positive reaction with sodium nitroprusside, and this was confirmed by Cowdry and Scott (1928) in Plasmodium. Chalkley (1937), however, found that the strongest re- action in vegetative Amoeba is in the nucleus and that at the metaphase this material is poured into the cytoplasm. Some granules in the nucleus give a particularly strong reaction, but in the cytoplasm the coloration is diffuse. These results of Chalkley’s extensive work on glutathione in Amoeba suggest the desirability of a reinvestigation of the Sporozoa, and certainly indicate that the materials containing the sulfhydryl group are not always localized in the mitochondria. Joyet-Lavergne (1934) has also shown that the mitochondria give a strong reaction with the anti- mony trichloride test for vitamin A, and concludes that this is a part of the respiratory mechanism along with glutathione. Although respiration cannot be discussed in detail here, it should be pointed out that the glutathione-vitamin A theory presents many difficulties and the system more usually accepted is glutathione-ascorbic acid (Holmes, 1937). Bourne and Allen (1935) and Bourne (1936) have demonstrated the concentration of ascorbic acid in cytoplasmic granules by the acetic- silver-nitrate method, but unfortunately have not correlated these with 122 CYTOPLASMIC INCLUSIONS any particular type of granules. Daniels (1938) used this method in gregarines, but although she was able to demonstrate granular accumula- tions in the cells of the gut, the gregarines remained clear. The functions ascribed to mitochondria in the Protozoa may be grouped under two main headings: synthesis (or segregation), and respiration. The first group includes a number of activities, all of which involve the accumulation of materials and in some cases the synthesis of new products from these raw materials. Examples of this type of function for which there is definite evidence are the secretion of reserve bodies, a digestive function in connection with the gastrioles, excretory function in connection with the contractile vacuoles, and the transport of materials from one organelle to another. Respiration also might be considered to fall in this category, since it would depend upon the ac- cumulation in the mitochondria of the substances responsible for the oxidation-reduction processes of the cell. The secretion of reserve bodies is cytologically the easiest phase of accumulation to demonstrate. Fauré-Fremiet described the formation of deutoplasmic granules by direct transformation of mitochondria, as well as by the more common method of segregation adjacent to or within the mitochondria which retain their identity. Joyet-Lavergne (1926a) occasionally found a relationship of this type between the mitochondria and paraglycogen and always found that protein granules possessed a mitochondrial cap (Fig. 27), but denied that this indicated anything more than a casual relationship. Horning (1925) described mitochondrial rims around the protein granules in O palina (this identi- fication of mitochondria is denied by Kedrowsky, 1931b) and accepts it as evidence of secretory activity. MacLennan (1936) described the origin of paraglycogen in the center of spherical mitochondria (Fig. 17). The identification of these granules was made not only with the usual permanent stains for mitochondria, but with specific microchemical stains (Sudan III, Nile blue sulfate, iodine, chlor-zinc-iodide), and their growth observed in live specimens stained with Janus green. The fact that the paraglycogen first appears as a center in a solid mito- chondrial sphere refutes the usual suggestions that the secretion merely happens to be in contact with the mitochondria and that there is no real connection between the two. There is no evidence as yet to show whether these visible secretions are actually synthesized by the mito- CYTOPLASMIC INCLUSIONS 3 chondria or are the result of the segregation by the mitochondria of materials synthesized at other points. Wermel (1925) found that certain mitochondria of Actinosphaerium have a high lipoid content, as shown by Ciaccio’s lipoid methods, 1.¢., a so-called liposome rather than an ordinary type of mitochondria, and concludes that they secrete the lipoid reserves. Except for the fact that these granules stain weakly Figure 30. The association of mitochondria with the gastriole in Amoeba proteus. (From Mast and Doyle, 1935b.) A, 2-6 hours; B, 6-8 hours; C, 8-16 hours; D and E, 16-30 hours; b and bi, mitochondria; f, fat; s, starch; v, vacuole refractive bodies; c, crystals; p, pellicle of Chilomonas. with Janus green, they are like the intermediate lipoid bodies and could perhaps be classed more conveniently with them. Zinger (1928) counted the “‘spherical inclusions” and the mitochondria in O phryoglena and found that they are roughly proportional in number. He concluded that the spherical inclusions are derived from mitochondria. Since many granules of entirely different origin will increase in number under favorable conditions, this conclusion cannot be considered as proved. The digestive function of mitochondria rests upon two types of evidence: the actual demonstration of enzymes in these granules (see p. 125), and the correlation between the periodic aggregation of mito- chondria around the gastriole and the type of digestion taking place within. Both types of evidence are available for A. proteus, so that the results of observations may be checked against a direct knowledge of the 124 CYTOPLASMIC INCLUSIONS enzymes actually present. In A. proteus, Mast and Doyle (1935b) find that the mitochondria accumulate around the food vacuole six to eight hours after its origin and then again at sixteen to thirty hours (Fig. 30). During the first contact, digestion begins, starch is changed to erythro- dextrine, and fat leaves the vacuole. In the second contact, vacuole re- fractive bodies and crystals disappear. These authors never found the mitochondria actually entering the vacuole. The relationship between mitochondria and starch digestion was directly demonstrated by Holter and Doyle, who showed that these granules contain amylase. Their function is restricted with respect to digestion, since they lack dipeptidase, according to similar studies by Holter and Kopac (1937). This situa- tion is not universal, since Hopkins (1938b) also working with Mast, showed that no formed granules are associated with digestion in the marine amoeba, Flabellula, although a material is dissolved in the vacuoles which when precipitated by disturbance, and so forth, forms granules which stain with Janus green B and other mitochondrial stains. Again, in many other Protozoa the mitochondria have no direct con- nection with digestion. Thus mitochondria may contain enzymes in some cases, but this is not a necessary association. Excretory granules associated with contractile vacuoles have been described many times, but only recently have mitochondria been proved to be associated with the excretory process. Mast and Doyle (1935a) have shown that the excretory granules of A. proteus (Figs. 24, 25, 47) correspond in their staining reactions to mitochondria. By centrifuging the majority of these bodies into one end of the amoeba and removing this part, these authors showed that the formation of the vacuole 1s dependent upon these granules and that if most of the mitochondria are removed, death follows. In one experiment, most of the mito- chondria were left, with the result that the average interval between pulsations was 3.46 minutes. If few mitochondria were left, the time was correspondingly longer, and when very few granules were left, the average time between pulsations increased to twenty-five minutes. Mast and Doyle interpreted these experiments as indicating that some excretory material, toxic to Amoeba, is eliminated by the vacuole, and that the mitochondria function as the means of transport to the vacuole. Doyle (1935) harmonized the apparently discrepant functions of digestion and excretion by pointing out that they may be united under CYTOPLASMIC INCLUSIONS 125 the general heading of transport, and pointed out further that mito- chondria tend to accumulate wherever exchanges are taking place, both within the individual and at the outer surface. Mast and Doyle (1935b) observed that the mitochondria accumulate around the crystal vacuoles while the crystals decrease in size, and with the surface of the refrac- tive bodies while the latter are increasing in size. The mitochondria of Amoeba seem to be a mechanism for intracellular transport and for carrying amylase to the food vacuoles, digested material from the food vacuoles and crystal vacuoles to the refractive bodies, and metabolic wastes to the contractile vacuoles. However, since Holter and Kopac (1937) have shown that dipeptidase is not associated with mito- chondria, and Volkonsky (1933) and MacLennan (1936) have shown cases in which vacuome alone touches the food vacuole, and Hopkins (1938b) cases in which no preformed granules are associated with the vacuole, it is clear that the mitochondria are not necessary in all cases either for transport function or a support for enzymes. The supposed universality and permanence of mitochondria have led to many suggestions that they are concerned with some vital part of cellular activity, some function more universal than secretion and storage. Some evidence to this effect has been presented by Joyet- Lavergne (1927-35), both from the standpoint of the presence of materials active in respiration (see p. 121) and from the standpoint of a direct demonstration of respiratory activity. He finds that vital dyes are reduced most strongly near mitochondria and that individuals which have large amounts of mitochondria reduce the dyes faster than individuals with less mitochondria. He was able to demonstrate gluta- thione and vitamin A in the mitochondria and attempted to show that these two form an oxidation-reduction system. Rey (1931a, 1931b) repeated Joyet-Lavergne’s staining experiments and obtained the same results, but criticized the latter’s interpretation of his findings. Rey also repeated the experiments using an electrometric method for deter- mining rH and found no significant differences. Wurmser (1932) like- wise criticized Joyet-Lavergne’s interpretations of the stain reactions as indications of oxidation-reduction differences. Bles (1929) found that the oxidation-reduction reactions which he studied in Arce/la were asso- ciated with the hyaloplasm, rather than with any granules. Since Joyet- Lavergne found morphological continuity between the mitochondria and 126 CYTOPLASMIC INCLUSIONS two types of reserve granules, the existence of a secretory function is possible, and it is not necessary to invoke respiration in order to find a function for these mitochondria. The respiratory function must be regarded as unproved, either from the standpoint of the proof of the presence of materials which could act as an oxidation-reduction system, or from the standpoint of the direct measurement of localized oxidation- reduction potentials. At best mitochondria as morphological entities cannot be necessary for respiration, since many species lack them at one time or another in the life cycle, and since in other cases they can be eliminated experimentally without fatal results (Kirby, 1936). All the various types of evidence—staining reactions, composition, function, and tracing through the life history—show that mitochondria in the Protozoa do not form a homogeneous group, but are actually a heterogeneous assortment which are associated merely by their ability to segregate Janus green B or by even less specific staining reactions. No one type is found in all Protozoa, and in all cases which have been carefully studied mitochondria are not self-perpetuating but arise de novo at some time during the life cycle. THE VACUOME HYPOTHESIS According to the vacuome hypothesis as applied in the Protozoa, there are only two fundamental cytoplasmic components in the Protozoa—the chondriome and the vacuome, since the Golgi bodies and vacuome are merely different aspects of the same thing. The term vacuome was substituted for the earlier term segregation granule as an indication of the supposed homology between the neutral red bodies in animal cells and the vacuoles of plant cells. Volkonsky (1929 on), Kedrowsky (1931- 33), Hall and his associates (1929 on), Lynch (1930), and others have upheld the general conclusion that the granules stainable with neutral red are identical with the Golgi bodies; but Kirby (1931), MacLennan (1933, 1940), Bush (1934), Kofoid and Bush (1936), Daniels (1938), and others have demonstrated many cases of neutral red granules which are not osmiophilic (for a more detailed discussion of this point, see p. 140). It should be pointed out that the acceptance of the vacuome hypothesis is by no means universal in the Metazoa or Metaphyta, according to Weier (1933) and Kirkman and Severinghaus (1938). CYTOPLASMIC INCLUSIONS 127 The ability to segregate neutral red has been ascribed to a specific reaction of the dye with a single material in the granule, the process being called the “neutral red reaction” by Koehring (1930) and in- cluded as a part of the “ferment theory of the vacuome”’ by Kedrowsky (1932b). The substance involved is supposed to be a proteolytic enzyme, a conclusion based on the work of Marston (1923), who showed that these enzymes are precipitated by combination with neutral red, Janus green, and other azine dyes. Le Breton (1931) has reviewed the rather voluminous literature resulting from this suggestion and concludes that the reaction is not specific, since ordinary proteins (an important con- stituent of most segregation bodies) also are precipitated. From the standpoint of cytology, the theory fails to explain how neutral red and Janus green have such different staining reactions in the granules of living cells. Hopkins’s (1938a) experiments with F/abellula show that precipitates are formed in vacuoles after either Janus green or neutral red. If Janus green was first used, and then neutral red added, a red precipitate formed around the original green one. “The small neutral red vacuoles are, then, the same as the vacuoles in which the condensation granules are formed, but it appears that Janus green B stains a different component of these vacuoles than does the neutral red.” Not one factor, but many are responsible for the segregation of neutral red by cytoplasmic granules. The rdle of pH in neutral red staining has been demonstrated by Chambers and Pollack (1927) and Chambers and Kempton (1937), who showed that neutral red tends to go from an alkaline region to an acid region so that ‘‘segregation granules’ would be those which are acid relative to the hyaloplasm. Kedrowsky (1931) demonstrated in Opalina that normally the segregation granules have this relationship with the hyaloplasm and that the staining reactions can be changed by altering the pH of the cytoplasm. He also showed that the granules will take up acid dyes in the presence of albumoses, which was confirmed by Volkonsky (1933) and included under the term “‘chromopexie.’’ Since neutral red has long been known as a lipoid stain (Fauré-Fremiet, Mayer, and Schaeffer, 1910) it is possible that this may play a rdle in the staining of bodies which contain lipoids, such as the dictyosomes of gregarinida and the digestive granules of Ichthyophthirius. From these brief examples it is clear that the segrega- tion of neutral red and other vital dyes is influenced by many internal 128 CYTOPLASMIC INCLUSIONS and external factors and that it cannot be considered specific in the sense that it combines with a single definite substance. Since many factors are involved in the segregation of neutral red, it is to be expected that more than one type of granule will be revealed by the use of this and similar dyes. Conclusive evidence of this lack of specificity is furnished by several Protozoa in which two or more types of granules are able to segregate neutral red at the same time, i.e., under identical conditions. Dangéard (1928) stained two types of granules in Evglena with neutral red—the vacuome and the mucous ap- paratus. The latter group may be extruded to form a mucous envelope or mucous hairs—an interesting example of true external secretion, and comparable to the staining of secretion granules in Metazoa by neutral red. However, Dangéard rejected the mucous apparatus as vacuome because it retains the neutral red after the death of the cell, while the other granules—the true vacuome—do not. Finley (1934) found four different groups of granules which segregate neutral red in Vorticella: pellicular secretions, pellicular tubercles, thecoplasmic granules, and re- fractile granules. He was careful to control the staining to avoid over- staining, so that his results cannot be questioned on that ground. Bush (1934) found in Haptophyra two sets of granules which are discon- tinuous in size and distribution. Mast and Doyle (1935b) complete the picture by showing that three groups of granules stain in Amoeba: vacuole refractive bodies, refractive bodies, and blebs on crystals, and they proved experimentally that these bodies are different in origin and in fate. The experiments of Kedrowsky (1931) on Opalina show the reverse picture—under certain feeding conditions, the growing segre- gation vacuoles lose their ability to take up neutral red, just as they do in vertebrate eggs. Hall and Loefer (1930) showed that the granules in Exglypha may vary in the same specimen from pink to bluish red or red violet. Since neutral red stains several different groups of granules and also does not stain all stages of the same granule, it does not of itself reveal fundamental homologies, and it seems to me to be unjustifiable to group all of these bodies as vacuome or under any other catchall term. As a preliminary step toward an accurate classification of this group, the digestive granules—i.e., those neutral red bodies associated with the gastrioles—are separated from the segregation granules which cor- CYTOPLASMIC INCLUSIONS 129 respond to the ‘‘vacuome de reserve” of Volkonsky and the segregation apparatus of Kedrowsky. The segregation granules of necessity still include a heterogeneous assortment and probably include granules other than those associated with synthesis and storage, but at present they cannot be classified properly because too many of them are known only by their ability to segregate neutral red. Kedrowsky and Volkonsky regard both types as carriers of enzymes, in one case acting to digest proteins and in the other case to synthesize them. However, each group appears de novo when the necessity arises, and the two groups show no direct continuity with each other. Further evidence of the independ- ence of these two groups of granules is furnished by Opalina, in which only the segregation bodies are present, and by Ichthyophthirius, in which only the digestive granules are present, these latter having no connection with the storage of the numerous protein granules. DIGESTIVE GRANULES The digestive granules may be briefly defined as cytoplasmic granules stainable with neutral red, which become associated with the newly formed vacuoles containing food. This does not include all neutral red granules in the food vacuole, since such granules as the vacuole refractive bodies of Amoeba are derived from the food (Mast and Doyle, 1935b) and thus are not cytoplasmic components. Volkonsky (1934) points out that the term vacuole has been applied to so many structures that it is a source of confusion, and he has substituted the term gastriole. The fluid vacuole which contains ingested food is a pro- gastriole, and with the addition of the digestive granules (vacuome in Volkonsky’s terminology) becomes a gastriole. In addition to these terms it is convenient to use postgastriole for the structures containing undigested remnants. There is no single pattern of the gastriole in the Protozoa. In A. proteus, mitochondria alone aggregate periodically around the gas- triole (Mast and Doyle, 1935b); in Paramecium and Campanella, the digestive granules enter the gastriole, and mitochondria cluster around the membrane in the alkaline phase; in Flabellula, the materials stain- able with Janus green or neutral red are normally dissolved in the fluid vacuole, which later forms the gastriole (Hopkins, 1938), and a some- what similar situation is found in hypermastigote flagellates (Duboscq 130 CYTOPLASMIC INCLUSIONS and Grassé, 1933); in Ichthyophthirius (MacLennan, 1936), the diges- tive granules (Fig. 31) enter the gastriole, but the mitochondria are never associated in any visible manner with the digestive mass. A more detailed description of digestion will not be given in this section, since attention is here centered on the granules. All careful descriptions agree that the digestive granules vary in size and shape during the gastriolar cycle, ranging from minute spherules to relatively large rods. Both vital stains and metallic impregnation show a homogeneous structure, and the deformation of these granules either GS) Gas 8 Figure 31. The association of the gastrioles and the digestive granules in Ichthyoph- thirius multifiliis. (From MacLennan, 1936.) from other cytoplasmic granules or from outside pressure show that they have a soft, semifluid consistency. Volkonsky (1934) has found that the morphology of the gastriole varies in the same species with the food used. In Acanthamoeba he was able to induce the formation of large granules, small granules, or a homogeneous rim stainable with neutral red, by varying the food used. No digestive granules were formed around vacuoles which contained only starch. The penetration of the digestive granules into the vacuole was not observed by Koehring (1930), Hall and Dunihue (1931), Dunihue (1931), or Hall and Nigrelli (1930), and the suggestion was made that the granules in the food vacuole are derived from food particles CYTOPLASMIC INCLUSIONS 131 rather than from the cytoplasm, a suggestion which later was proved to be true, in the case of vacuole refractive bodies of A. proteus, by Mast and Doyle (1935b). Volkonsky, however, checked his observations on many ciliates and ruled out exogenous granules in the gastrioles of Glaucoma by the use of bacterial free medium; he still observed diges- tive granules in the vacuoles and was able to trace them from the cyto- plasm. The migration of the digestive granules into the gastriole of Ichthyophthirius was found by MacLennan (1936), but it was shown that the granules do not penetrate any membrane. Instead, a new mem- brane is formed (Fig. 31) around the whole gastriole and the inner membrane, which is the original one, then disappears. The end result is exactly the same as in the cases described by Volkonsky, but the mechanism is somewhat different. Volkonsky’s interpretation of the digestive granules as vacuome 1s based upon their impregnation by the various Golgi-type methods. The question as to the identification of the granules as shown by entirely different methods is not present in this case, as it was in the case of the scattered cytoplasmic granules (segregation granules), since the digestive granules can be recognized independently of their staining reactions by their relationship to the gastriole. Hall and Nigrelli (1937) claim that the digestive granules are less consistent in impregnation than the scattered cytoplasmic granules and dispute Volkonsky’s claim that they can be considered as vacuome. MacLennan (1940) showed that the various types of osmiophilic granules could not be distinguished on the basis of impregnation alone; in particular the digestive granules of Ichthyophthirius show 100-percent impregnation. Few of the materials which occur in the digestive granules are known from direct evidence. The digestive granules of Paramecium are high in ash (MacLennan and Murer, 1934) and those in Ichthyophthirius contain lipoids (MacLennan, 1936). These latter bits of information are not as yet particularly useful and emphasize the need for more specific knowledge. The presence of enzymes is suggested by the morpho- logical evidence and this would be a fertile field for the use of the microenzymatic methods. The digestive granules are not permanent self-perpetuating structures, but appear to rise in the cytoplasm in response to the stimulus of feeding. Volkonsky (1934) found that when the preéxisting granules are utilized 132 CYTOPLASMIC INCLUSIONS in the formation of gastrioles, new granules are formed in the cyto- plasm. The digestive granules in Ichthyophthirius were observed by MacLennan (1936) to arise de novo in direct contact with the ingested particles of food. Final evidence of their de novo origin was furnished by the fact that no digestive granules are present in the encysted stage, after the gastrioles which were formed during the feeding stage have disappeared. Volkonsky suggests that the materials of the digestive granule exist in the cytoplasm in a diffuse state. In the hypermastigote flagellates this material is concentrated in dissolved form in the gastrioles; in Flabellula the materials are first concentrated in vacuoles, which con- tribute the fluid part of the gastrioles; in the more common cases, the materials are condensed to form digestive granules. We must not for- get, however, that not all the digestive reactions result in granules, since dipeptidase in Amoeba is independent of any granules. Whatever the specific morphology of the reaction, whenever the cytoplasmic equilib- rium is disturbed by the ingestion of food there is an effective mobiliza- tion of this material to cope with the ingested food. Volkonsky calls these varied changes the “vacuolar reaction,” an extremely useful term, but since we reject the term vacuome, “‘gastriolar reaction’ would be more appropriate. SEGREGATION GRANULES The segregation granules are bodies which are able to concentrate, accumulate, and store within themselves vital dyes, proteins, and other materials. Unlike the definitions of most cytoplasmic granules, this definition is based upon function rather than upon morphology or staining reactions. The evidence for this definition is due largely to the work of Kedrowsky (1931-33) on Opalina, Spirostomum, and other ciliates. Since the functions of most of the granules stainable with neutral red have not been demonstrated, the work on Opalina will be discussed first and granules in other Protozoa considered in the light of this work. The accumulation of vital dyes in higher concentration than they occur in the medium is, of course, one piece of evidence of the segregating ability, even if this has’ not been demonstrated with the materials which enter into the metabolism of the cell. The segregation bodies of Opalina (Figs. 32-35) are the external layer of granules or ectosomes and have been identified as Golgi bodies CYTOPLASMIC INCLUSIONS Ei) or as mitochondria by various authors, since they respond to some of the Golgi and mitochondrial techniques, although they are not stained specifically with Janus green B. Kedrowsky described four main morpho- logical types—fine dispersed granules, large granules, alveoli, and heteromorphic granules (Figs. 32-35). In some cases the dispersion of granules is so accentuated that they lose their identity as granules. These changes in morphology are associated under natural conditions with the seasons of the year; for instance, the heteromorphic types are common in the spring and early summer, and the large granular type is found in the early spring. There is some variation between populations of different frogs in the same season, but all members of the same popula- Figures 32-35. Basic morphological variations in the segregation granules of Opalina ranarum. Semischematic. (After Kedrowsky, 1931le): Figure 32. Dispersed type; Figure 33, homomorphic granular type; Figure 34, alveolar type; Figure 35, heteromorphic granular type. tion have essentially the same type of granules. These different forms can be produced experimentally by changing the culture medium. The colloidally dispersed type is typical of amino-acid cultures. In distilled water, each ectosome swells and becomes a watery vacuole. The hetero- morphic type is found in cultures which contain defibrinated and hemo- lysed blood. These changes, which occur both naturally and in artificial media, may well account for the disagreement among cytologists both as to the morphology and the identification of these granules. The segregation bodies of Opalina are not permanent organelles. As indicated above, they may disperse homogeneously through the ecto- plasm, which then takes a general pale stain with neutral red, and no method applied at this stage shows any indication of a remnant of the originally discrete granules. If the bodies are loaded with protein com- pounds of metals, such as silver, they may finally be extruded from the surface and be replaced by granules which arise de novo. 134 CYTOPLASMIC INCLUSIONS The composition of the segregation bodies is as varied as their morphology and results from the same causes—changes in the environ- ment. Under natural conditions they may contain proteins (Millon’s reaction), glycoproteins (Fischer's reaction), cholesterin (Schultz te- action, digitonin reaction), or be stained with bile pigments. The alveolar type of course contains mostly water. The segregation granules will store basic dyes in salt solution, acid dyes in the presence of pro- teins, silver in the form of kollargol or other similar compounds, iron albuminates, cholesterin, and so forth. According to the ‘ferment theory of the vacuome” of Kedrowsky (1932b), enzymes are present in the 36 a Figures 36-37. Segregation granules in Trypanosoma diemyctyli, neutral red stain. (After Nigrelli, 1929.) Figure 36, Preformed granules around the blepharoplast; Figure 37, preformed granules and granules induced by prolonged exposure to neutral red. segregation bodies, but, as is the case with the digestive granules, no direct proof of this is available. The segregation bodies obviously function in the concentration and storage of various materials, particularly proteins. This may be accounted for on a purely physical basis, as, for instance, the tendency of basic dyes to migrate to a more acid region, or the tendency of molecules to migrate toward bodies of opposite charge. The exact mechanism is, of course, a complex problem and cannot be taken up here, but the es- sential point at present is that the granules may play a purely passive role—the accumulation and storage of materials which originate else- where. Accumulation is merely one part of the function, according to the enzyme hypothesis of the segregation granules, advanced at nearly the same time in slightly varying forms by Koehring (1930), Kedrowsky (1931 on), and Volkonsky (1929 on). Koehring based her conclu- sions on the supposed specificity of the neutral red reaction for proteo- lytic enzymes (see p. 178). Kedrowsky considers that both the synthesis CYTOPLASMIC INCLUSIONS 155 of proteins and deaminization may occur in the segregation bodies. The evidence of synthesis may be summarized simply as the increase in size or number of segregation bodies in Opalima when immersed in various nutrient solutions, and the subsequent identification in the granules of materials from this culture medium. This certainly proves the segregat- ing ability, but the actual synthesis might take place almost anywhere and the increase in size be due simply to the segregation of these pre- formed materials. The evidence of deaminization is based on the appear- ance of glycoprotein in the late stages of the segregation bodies and the fact that the segregation granules are able to oxidize Rongalit white vitally. This evidence could hardly be called more than suggestive, but the processes which are indicated by these tests seem to be localized in the segregation granules. The neutral red granules of Protozoa other than Opalina, to be con- sidered in the rest of this section, excludes only the group which were discussed above as digestive granules. It is thus essentially the group called segregation apparatus by Kedrowsky (1931), or the vacuome of Hall (1929 on). Since neither the history nor the function of most of these granules is known, this is doubtless a heterogeneous group, but I believe that further splitting at this time would merely add names without increasing understanding. The segregation bodies are normal cytoplasmic constituents and are not induced by vital dyes, since they have been observed by many in- vestigators in normal, unstained specimens (Hall, 1929 on; Finley, 1934; MacLennan, 1933, 1936; Volkonsky, 1929 on; Kedrowsky, 1931 on, and others). Prolonged staining (Fig. 23), it is true, may induce the formation of new granules (Kedrowsky, 1931; Cowdry and Scott, 1928; Nigrelli, 1929), but this is not a universal phenomenon, since many species, in my own observations, show the general diffuse stain- ing of the cytoplasm and nucleus characteristic of severe overstaining, without the appearance of new granules. The normal origin of all types of segregation granules seems to be de novo. Mast and Doyle (1935b) removed most of the refractive bodies from A. proteus and observed the formation of new bodies several hours after feeding. The “blebs’’ on the crystals likewise clearly originate de novo. These cases are too few to justify any certain statements for all the many segregation granules, but it is indicative that in all the many descriptions of these granules, 136 CYTOPLASMIC INCLUSIONS there has been no clear case of division described—all the evidence indi- cates a de novo origin. Small homogeneous spherules are the commonest type of segregation body and they are found in all classes of Protozoa. There are often more than one group in a single individual, as shown by differences in size (Bush, 1934) or differences in localization (Finley, 1934). Complex bodies similar to those found by Kedrowsky in O palina have been found in flagellates from termites (Kirby, 1932). In some cases such complex ; y o =® f- SH ey a | A B Cc 02 E F Figure 38. Segregation granule (refractive body) of Amoeba proteus in successive stages of resorption. (From Mast and Doyle, 1935.) A, normal body; B-F, stages in resorption, optical sections; D, surface view. Outer layer cross-hatched, shell black, vacuole in outline. bodies may be the result of precipitation of dyes or other materials in an aqueous vacuole, but in the cases of the denser granules entirely, different methods show the duplex structure. In the refractive bodies of Amoeba (Fig. 38), microchemical tests show that the duplex struc- ture represents an actual difference in composition (Mast and Doyle, 1935a). The segregation granules are most often scattered at random through- out the endoplasm (Hall and his associates, 1929 on), but occasionally are more definitely localized. In Trypanosoma (Figs. 36, 37) they are concentrated around the blepharoplast (Nigrelli, 1929), in the Ophry- oscolecidae they are most common in the operculum and spines (Mac- Lennan, 1933), and in Lechriopyla a marked concentration is found just under the pellicle (Lynch, 1930). In Vorticella a group of discrete globular inclusions is found scattered in the cytoplasm, and another group is found in the stalk (Finley, 1934). The tubercles of the pellicle also stain, as do the secretions of the pellicle, but the latter have been consid- ered in the section on secretion granules and the former do not seem to me to fulfill the definition of cytoplasmic granules, or at least are clearly not in the same class as the ectosomes of O palina. As a contrast to these Protozoa with several types of segregation granules, the ciliate Ichthy- CYTOPLASMIC INCLUSIONS 137 ophthirius has none at all at any time in its life cycle (MacLennan, 1936), and the function of synthesis and storage of the protein re- serves is taken over by the macronucleus. The composition of the segregation granules of Protozoa other than Opalina is known only in the case of the refractive bodies and blebs of Amoeba. The osmiophilic shell of the refractive bodies of Amoeba has a protein stroma impregnated with a lipid substance (Mast and Doyle, 1935a). This portion stains with Sudan II only after Ciaccio’s method for “unmasking” the lipoids, and is intensely blue in Nile blue. These granules are not dissolved in alcohol in twelve hours, but they do lose their positive reaction to fat soluble dyes. They respond to the methylene-blue-sulphuric test for metachromatin and give a faint re- action with Millon’s reagent. Within this layer a brittle carbohydrate shell and an unknown fluid are found. The stainable blebs on crystals in the same protozoan, when first formed, contain only lipoids, but as they grow larger, protein is added so that their final composition is similar to the shell of the refractive bodies of the cytoplasm. These granules are therefore quite different from the segregation granules of O palina, since Kedrowsky showed that whatever else might be stored in these bodies, they do not segregate lipoids. However, since the re- fractive bodies do segregate proteins and stain with neutral red, they are included as segregation bodies. This is, of course, arbitrary, since they overlap on the Golgi granules and on the reserve granules. Sufh- cient mineral ash is present to mark the vacuome in incinerated speci- mens of Opalina (Horning and Scott, 1933) and Paramecium (Mac- Lennan and Murer, 1934). The only striking fact about the segregation bodies in most Protozoa is that with the exception of those in O palina and Amoeba, not even a sketchy outline of their composition is available. The function, in so far as it is known, agrees closely with the storage function described for the granules in Opalina. Variations of the num- bers of the segregation granules have been found in Paramecium (Duni- hue, 1931) and in Vorticella (Finley, 1934). Dunihue was able to correlate the decrease in numbers with starvation, thus indicating a storage function. Mast and Doyle (1935a) showed that both protein and lipoid materials are found in the blebs on the crystals and on the re- fractive bodies. Since the blebs appear shortly after feeding and since they in turn disappear as the refractive bodies are increasing, the blebs 138 CYTOPLASMIC INCLUSIONS seem to be temporary reserves, while the refractive bodies are the final reserves. In the blebs, the protein portion appears later than the lipoid portion. There is no evidence in these cases of anything more than the accumulation of materials. GOLGI BopDIEs The term Golgi body has been applied in Protozoa to organelles which differ fundamentally in both composition and function. These structures include contractile vacuoles (Ramon y Cajal, 1904-5), gran- ules (Hirschler, 1914), specialized regions of cytoplasm around the contractile vacuoles (Nassonov, 1924), segregation granules (Cowdry and Scott, 1928), osmiophilic nets (Brown, 1930), and the parabasal apparatus (Duboscq and Grassé, 1925). The controversies which have arisen between advocates of one or another of these structures have been due not so much to disagreement upon the actual facts involved as to disputes concerning criteria for the identification of Golgi material. The selection of criteria is thus a crucial point in codrdinating the in- vestigations of Golgi bodies; yet even after years of work on representa- tives of all the major groups of animals and plants, and notwithstand- ing periodic reviews of the field, few criteria seem to have unanimous approval—few, indeed, have majority approval. The most recent re- view (Kirkman and Severinghaus, 1938) after failing to demonstrate any universal and objective basis of identification, quotes as follows from Gatenby (1930): “modern workers in general have experienced no difficulty in identifying Golgi bodies.” This is very satisfactory as long as only one school of cytologists is considered, but such state- ments lose their attractive ring of authority when one tries to correlate the results presented by such experienced cytologists as Bowen, Parat, Gatenby, Canti, or Ludford, to mention only a few. This is particularly true when seeking criteria on which to base a reasonable identification of Golgi bodies in the highly specialized cells of the Protozoa. In the following paragraphs the major objective cri- teria which have been used are discussed with particular reference to their applicability to the Protozoa. In general these criteria involve two points—consistent impregnation, and occurrence in all types of cells. The first Golgi structures were discovered by the use of metallic im- pregnation methods, and ever since these methods have remained as CYTOPLASMIC INCLUSIONS Sy the primary criteria (Gatenby, 1930), particularly since the discovery that the supposedly typical reticular network is a relatively rare type and in many cases is due to a temporary aggregation of granules (Hirsch- ler, 1927). Both the osmic and the silver methods involve the same prin- ciple—the adsorption of the reduced metal on particular structures, although the actual reduction of the metallic compound may take place either where the deposits are located or in other parts of the cell (Owens and Bensley, 1929). Since the osmic acid methods have been most thoroughly investigated, this discussion will be restricted to these tech- niques, but the fact should be emphasized that the same general principles are involved in the silver techniques as well. The preliminary treatment of the cell strongly affects the subsequent impregnation (Lison, 1936). Thus direct exposure of the protozoan to osmic fumes (Hall, 1929) is not equivalent to the full technique which involves preliminary fixation in such mixtures as Champy’s fluid. Likewise, the blackening of granules by exposure to osmic fumes after they have been stained with neutral red is not the equivalent of the standard Golgi methods, since it has been shown in the case of Flabel- lula (Hopkins, 1938b) that the blackening is produced only in the presence of neutral red. The Golgi methods are not specific for any single type of material, since a series of different lipoids extracted from echinoderm eggs give a typical Golgi reaction (Tennent, Gardiner, and Smith, 1931). Further- more both lipoid and non-lipoid bodies of various types in the Protozoa react typically and identically to the Golgi techniques (MacLennan, 1940). Although the method is not specific in the sense that it reveals a single known substance, or some unknown Golgi material, the method is consistent in the sense that under proper conditions the results can be reproduced. In general, the reputation of osmic acid methods as carpricious and erratic is due to the slow penetration of tissues by osmium tetroxide, with the result that the cells in a block vary in ex- posure to the unreduced reagent because of their varying distances from the free surfaces. Another source of difficulty is the fact that the com- position of cytoplasmic granules changes during the growth of that granule (Kedrowsky, 193le; Mast and Doyle, 1935a, 1935b; Mac- Lennan, 1936), and it is to be expected that the reducing power will also vary. The Protozoa are admirably adapted to solve both of these 140 CYTOPLASMIC INCLUSIONS difficulties. The individual cells are either separate or in such small aggregates that all cells and all parts within the cell are exposed to essentially the same concentration of reagents. The variations in the stage of the Golgi granules can be observed directly in living Protozoa, so that the impregnation of the granules can be compared at equivalent stages, which can be identified with or without impregnation. In meta- zoan cells the Golgi nets are extremely difficult to demonstrate in the normal living cell, so that it is at present impossible in these cells to check the structures independently of impregnation. When standard conditions are achieved both around the cell and within the cell, abso- lutely consistent results are attained, even with osmic acid (MacLennan, 1940). Under these conditions, 100 percent of the digestive granules, fatty acid granules, excretory granules, and so forth, impregnate, with the result that these types cannot be separated on the basis of the Golgi- type reactions. The results of Hall and Nigrelli (1937), which seem to show that excretory granules and digestive granules are erratic in impregnation, is due to failure to allow for changes in composition and aggregation or for the occasional lack of these granules at certain times in the life cycle. All the stages have been lumped together, rather than equivalent stages compared. In view of the fact that osmic acid is reduced by many different sub- stances, it is highly important to prove that the impregnated bodies are not some other component (Hirschler, 1927; Bowen, 1928). Re- sistance to bleaching by turpentine or hydrogen peroxide is more pro- nounced in Golgi bodies than in most mitochondria, but unfortunately, even with this method, there are too many border-line cases in which the individual judgment of the observer is the determining factor. However, this individual judgment can be eliminated in comparing Golgi bodies and mitochondria by the use of such methods as the Alt- mann aniline acid fuchsin stain after osmic impregnation. Such a com- parison of Golgi bodies and segregation granules is not possible, since the major criteria of the one is a fixation method and of the other is a vital staining method. If the two components have a different distribution, a comparison of different cells is sufficient to establish the difference (MacLennan, 1933), but too often there is in both cases merely a random distribution. The most famous case of this sort is that of the gregarines, in which Joyet-Lavergne (1926b) described the Golgi CYTOPLASMIC INCLUSIONS 141 bodies as being identical with the neutral red bodies because of simt- larity in form and distribution, and this has been widely accepted in spite of objections presented by Tuzet (1931) and Subramaniam and Ganapati (1938). The question was not settled conclusively until Daniels (1938) applied the centrifuge to these species. She found that Golgi bodies always moved to the centripetal pole of the cell, but 42 43 Figures 39-45. Dictyosomes: Figure 39, Dictyosomes from Haptophrya michiganensis, Champy-osmic (after Bush, 1934); Figure 40, diagram of a dividing dictyosome of Lecudina brasili (after Subramanian and Ganapati, 1938); Figure 41, stages in the secretion of neutral fat in Ichthyophthirius multifiliis, Lorrain Smith Nile blue-sulphate method, black represents blue stain, stippling represents pink (after MacLennan, 1934) ; Figures 42-45, dictyosomes during the life cycle of Lecudina brasili; Figures 42 and 43, intracellular stages; Figure 44, growing trophozoite; Figure 45, ‘‘association’”’ stage, either daFano or Nassonow impregnations (after Subramanian and Ganapati, 1938). that the bodies stainable with neutral red were never displaced. Thus in this case the Golgi bodies and the neutral red granules are not identical. This does not mean that no osmiophilic granules segregate neutral red, since, for example, the digestive granules react to both impregnation and vital staining, but it does mean that the ability to segregate neutral red is not a characteristic of all the Golgi bodies of the Protozoa. 142 CYTOPLASMIC INCLUSIONS The theory that the Golgi apparatus is a universal organoid of the cell, as constant in its characteristics as is the nucleus, has given rise to a series of criteria requiring permanence during the whole cycle of the cell, as well as similarity in form and intracellular distribution. The presence of Golgi bodies in all stages of the life cycle has been demon- strated in Sporozoa (Joyet-Lavergne, 1926a) as well as their origin by the division of preéxisting Golgi bodies (Subramaniam and Gana- pati, 1938, Figs. 42-45). However, this is not universal, since neither cs Figures 46-47. The effect of centrifuging upon the distribution of cytoplasmic granules. Figure 46, diagram of a centrifuged gregarine, F, fat, GAa granular Golgi material, GAb larger Goli elements, N nucleus, K karyosome, M mitochondria, P paraglycogen, C ‘“‘chromidia,” neutral red bodies not shown; the paraglycogen mass marks the centrifugal pole. (From Daniels, 1938.) Figure 47, contractile vacuole of a centrifuged amoeba, v vacuole, b mitochondria; the mitochondria are thrown to the centrifugal surface of the vacuole. (From Mast and Doyle, 1935b.) of the types of Golgi bodies in Ichthyophthirius (MacLennan, 1936) nor Amoeba (Mast and Doyle, 1935a) are self-perpetuating or even present in all stages of the cycle, but arise de novo. Thus the Golgi bodies are not universally self-perpetuating and permanent. The criterion of similarity in form has received considerable support, but the evidence as to what this form is has been discordant. Nassonov (1924) described a net-like structure around contractile vacuoles and homologized this with the Golgi net. Hirschler (1927) finds that the typical Golgi bodies have an osmiophil cortex and an osmiophobe center, this duplex structure being called a dictyosome. The complex nets around the contractile vacuoles are, according to Hirschler, aggre- CYTOPLASMIC INCLUSIONS 143 gations of the dictyosomes. Subramaniam and Ganapati (1938) insist on this dictyosome structure, although they describe a homogeneous spherule in one stage of the Golgi cycle: a Golgi granule when it enlarges becomes differentiated into a vesicle having chromophile and chromophobic regions. Rupture of the vesicle gives rise to batonettes in which the chromophobic part is in relation with the cytoplasm. Since all of these structures—spherules, dictyosomes, aggregations—may be included in the cycle of a single granule (Kedrowsky, 1931; MacLen- nan, 1936; Mast and Doyle, 1935a) or as the result of the periodic ag- gregration of granules (Hirschler, 1927; MacLennan, OZ 33) nites impossible to insist on one type to the exclusion of the others, and we must conclude that morphology is not a criterion for the identification of various types of granules. Similarity in distribution within the cell has also been urged as a criterion. Hall (1931) holds that a random distribution throughout the cytoplasm is the typical configuration, while Subramaniam and Gopala- Aiyar (1937) consider that an excentric juxtanuclear position, similar to that found in spermatids or gland cells, is typical. In metazoan cells both types have been found, and both may be typical of the same cell if all stages of the life cycle are considered. Similar periodic aggregations and dispersal of Golgi granules have been described in connection with the pulsatory cycle of the contractile vacuoles of ciliates (MacLennan, 1933, 1936). These movements are comparable to the migration of mitochondria and of digestive granules, which have likewise been as- sociated with functional changes in the cell. The chief characteristic either of form or of distribution of Golgi bodies is a variability which is associated with functional changes, and even this variability is not a criterion since it is characteristic of all gran- ules which are actively concerned with the metabolism of the cell. The criteria based upon impregnation and upon separation from other granules which can be identified by more specific methods are the only truly objective criteria, while the criteria of universality in form, dis- tribution, and permanence are indefinite and are based upon various theories of the function, form, or derivation of Golgi bodies. The term Golgi body, being based upon nonspecific criteria (nor would retention of specific form or distribution make the definition more exact), in- 144 CYTOPLASMIC INCLUSIONS cludes a heterogeneous group of structures, including scattered endo- plasmic granules and granules associated with contractile vacuoles or fused to form heavy and permanent vacuolar membranes. A summaty of the known functions of these granules includes the excretion of ma- terials through the contractile vacuoles, the formation of granules of neutral fat, the storage of lipoids other than neutral fat, a secretory cycle which does not involve lipoids, as well as many functions as yet unknown. These functions are different, but in a broad sense they are all varieties of secretion and in this respect conform to the Nassonov-Bowen theory of the relationship between Golgi bodies and secretion. The restriction of criteria for Golgi bodies to that of impregnation alone thus does not do violence to the concept of Golgi bodies as originally developed in verte- brate tissues. The Golgi bodies are simply those secretory bodies (exclusive of mitochondria and segregation granules) which synthesize or store ma- terials which can be preserved by Golgi-type fixatives, and after this treatment are able to reduce (or adsorb the reduced metal) OsO, or silver nitrate. This is not a natural grouping, since on the one hand it includes several specific types of secretion, and on the other hand it does not include all types. In several cases the Golgi bodies can be classified according to function or composition, and in this discussion they are referred to as excretory granules, intermediate lipoid bodies (see p. 151), and, when the bodies aré secretory in nature but the type of secretion body unknown, secretory Golgi bodies. This leaves a miscel- laneous group of Golgi bodies which are known only by their ability to reduce osmium or silver and for which there is no evidence as to either composition or function. Since the impregnation reactions them- selves do not reveal homologies which must be based on composition and function, the retention of the term Golgi body is merely a con- venience to bridge the change from reliance on the nonspecific osmic techniques alone to reliance upon specific cytochemical and physiological criteria. EXCRETORY GRANULES Ramon y Cajal (1903-4) was the first to suggest that the contractile vacuole is equivalent to the Golgi reticulum of the cells of the Metazoa. The first confirmation of this view was the demonstration in several ciliates by Nassonov (1924) that this vacuolar region is osmiophilic. CYTOPLASMIC INCLUSIONS 145 Further work has extended the number of such cases in ciliates and flagellates, but at the same time it has been definitely proved that the vacuoles in several species of both classes are never osmiophilic. An examination of these cases shows that they form a closely graded series ranging from no impregnation at any time to complete impregnation at all times. Fabrea has no ectoplasmic Golgi bodies and no osmiophilic contractile vacuoles (Ellis, 1937). Lechriopyla has many ectoplasmic Golgi bodies, but they never form an aggregation around the contractile vacuole (Lynch, 1930). Epidinium, Eudiplodinium (Fig. 54), and oth- ets show an accumulation of granules only during diastole (Kra- scheninnikow, 1929; MacLennan, 1933). This same type is found in Ichthyophthirius in the parasitic stages (Fig. 48), but neither free osmio- philic ectoplasmic granules nor accumulations around the contractile vacuoles during encystment (MacLennan, 1936) are present. Me/a- dinium (Fig. 55) has a permanent granular nephridioplasm which waxes and wanes during the pulsatory cycle (MacLennan, 1933). Para- mecium caudatum and P. nephridiatum have a permanent osmiophilic shell around the radiating canals, but not around the contractile vacuole itself (Nassonov, 1924; von Gelei, 1928). Haptophrya possesses a vacuo- lar apparatus which consists of a permanent, homogeneous osmiophilic tube (Bush, 1934). This nicely graded series shows that the impregna- tion of parts of the vacuolar apparatus is due to aggregations of osmio- philic granules around the fluid vacuoles and their membranes. The only cases in which the membranes themselves impregnate are those ex- tremely specialized cases in which the osmiophilic material forms a permanent shell around the fluid vacuole. The highly complex osmio- philic apparatus in either Paramecium or Haptophrya is fundamentally no different from those vacuoles with a granular layer, and the homo- geneous osmiophilic shells are merely the result of the aggregation and specialization of the ordinary undifferentiated ectoplasmic Golgi bodies. This view is similar to that of Nassonoy (1924) and Hirschler (1927), except that these authors view the osmiophilic portion as the outer por- tion of the dictyosomes, and the fluid vacuole as the inner portion of the dictyosome structure. Both views are, of course, the same, if the osmio- phobic portion of the dictyosome be accepted as a secretion droplet and not as an essential part of the dictyosome itself, as held by Gatenby and Subramaniam and Ganapati. 146 CYTOPLASMIC INCLUSIONS The demonstration that the osmiophilic granules are an addition to the simple contractile vacuoles which may exist independently of the granules, even in the same species, is a close parallel to the union of the progastriole and digestive granules. The only difference is that no gastriole yet discovered presents a permanent, highly developed mem- 50 Sy, eye Figures 48-52. Excretory granules and contractile vacuoles. Figure 48, aggregation and disappearance of excretory granules during the pulsatory cycle, from Ichthyophthirius multifiliis, Champyosmic impregnation (after MacLennan, 1934); Figures 49-50, from Polyplastron multivesiculatum; Figure 49, cold impregnation; Figure 50, warm im- pregnation (after MacLennan, 1933) ; Figure 51, from Dogielella sphaerii, Champyosmic (after Nassonov, 1925); Figure 52, “nephridialplasm of Campanella umbellaria, Flemming-glychémalum (after Fauré-Fremiét, 1925). brane or a permanent granular region, as in the contractile vacuoles of Metadinium, Paramecium, and Haptophrya. The origin of a new vacuolar apparatus from the original structure of the parent has been described and compared with dictyokinesis in germ cells (Nassonov, 1924; von Gelei, 1928). The clearest case is that of Haptophrya, in which the vacuolar apparatus is a tube extending the full length of the ciliate. Studies of both live and fixed animals show that this tube is permanent and that the transverse fission of the cells CYTOPLASMIC INCLUSIONS 147 divides the tube in two parts, each of which continues to function in the daughter cells (Bush, 1934). The only possible exception would be the stages in which the parasite is transferred from one host to another. The new vacuolar apparatus of Paramecium is said to arise by the multipli- cation of canals and by the division of the whole vacuolar apparatus just prior to fission (Nassonov, 1924). The formation of extra vacuoles has been noted many times in living ciliates, but there is no recorded observa- tion of the actual division in a living Paramecium, and the interpretation of Nassanov’s figures of fixed material is susceptible to the difficulties inherent in building any cycle from fixed material alone. The majority of vacuolar systems, however, do not possess the thick, permanent wall similar to that in Haptophrya, but a temporary aqueous vacuole which certainly arises de novo (Taylor, 1923; Day, 1927; Mac- Lennan, 1933, 1936). These vacuoles and their membranes are not osmiophilic, the impregnation of the vacuolar system being due to the aggregation of the osmiophilic excretory granules. The fundamental question with respect to the origin of Golgi bodies is in these cases not the origin of the vacuoles, but the origin of the individual excretory granules. These granular aggregations in the Ophryoscolescidae have been observed in living specimens (Figs. 53-55) to be ectoplasmic Golgi bodies which migrate into the region of the vacuole during systole and the earliest stages of diastole (MacLennan, 1933). In specimens fixed during division of the ciliate, the newly arising vacuolar regions some- times overlap the old ones and give the appearance of a division of the old one as described in Didinium (von Gelei, 1938), but a study of similar stages in living ciliates shows that they originate independently. These granules are continually migrating toward the vacuoles and dis- solving there, and no granules migrate outward, so the question of origin is shifted to the granules at the time they are scattered in the ectoplasm. No cases of division were observed, either in fixed or in living material, at any place nor at any stage of the life cycle. This negative proof is not entirely satisfactory, since these granules are small (0.25—0.50 ) and a very rapid division might escape notice. This problem does not occur in Ichthyophthirius, since all ectoplasmic granules (whether scattered or around the vacuoles) are absent in the encysted stage (MacLennan, 1936), so that in this ciliate they must originate de novo in the young parasites, whether or not they continue from them by division as the 148 CYTOPLASMIC INCLUSIONS ciliate grows during the feeding stages. Persistence and genetic continu- ity is restricted to a few very highly specialized types of vacuoles, and in most cases there is no continuity either in whole or in part. The osmiophilic reaction of the differentiated cytoplasm around the contractile vacuoles has given rise to statements that this is a lipoid structure (Nassonov, 1924; Volkonsky, 1933; Haye, 1930), and to the interpretation of the vacuolar system as due to the accumulation of lipoids at the vacuole-cytoplasm interface, which is similar to Parat’s theory of the Golgi region around the vacuome. It is true that these structures are partially destroyed by lipoid solvents, but such evidence is not to be completely trusted and before acceptance must be corrobo- rated by more specific methods. In Ichthyophthirius these granules are negative to Sudan III and Nile blue sulphate. In Paramecium (unpub- lished work) I have used Ciaccio’s long unmasking process and find only a very faint reaction—only slightly more than in the hyaloplasm—and negative results with Nile blue sulphate. These experiments show that there is no concentration of lipoids, either in the excretory granules or in the cytoplasm around the vacuoles. The variations in impregnation during the pulsatory cycle and during the life cycle suggest that the osmiophilic reaction is due to some reduc- ing agent (not a lipoid) which is poured into the vacuole during di- astole. The excretory theory of the contractile vacuole is indicated by the name nephridialplasm (Fauré-Fremiet, 1925). The similarity between the cytological changes during the pulsatory cycle and in the glandular epithelium or renal tubules has been used in support of this theory (Nas- sanov, 1924, 1925). Further support was given by the demonstration that the osmiophilic granules dissolve in the vacuolar fluid which 1s then discharged (MacLennan, 1933). In Amoeba the activity of the contrac- tile vacuole is roughly proportional to the number of befa granules around it (Mast and Doyle, 1935b). It is certain in these cases that water plus some other material is being excreted. The question, then, is not whether excretion in a broad sense takes place, since water plus dissolved materials is certainly being excreted, but what are the dissolved substances which are carried to the vacuole in granular form? Nitrogenous excretion has often been assumed, but Weatherby (1927, 1929) found that the vacuolar fluid extracted by an application of the microdissection technique showed upon analysis too Figures 53-55. Excretory granules in living ciliates. Figure 53, from Polyplastron multivesiculatum ; Figure 54, from Eudiplodinium maggii; Figure 55, from Metadininm medium (from MacLennan, 1933). 150 CYTOPLASMIC INCLUSIONS little nitrogenous materials—probably not more than one percent—to account for any significant part of the total excretion of Paramecium. The application of these results to all Protozoa is by no means certain, since I have shown previously (1933) that the pellicle of the Ophryoscolecidae is highly impermeable and the only pathway for the excretion of kata- bolic wastes is through the contractile vacuoles. Frisch (1938) has demonstrated a similar impermeability of the pellicle in Paramecium itself, and suggests that Weatherby’s experiments be repeated, with the use of more delicate tests which have been devised recently. The materials concentrated in the osmiophilic structures of the con- tractile vacuole are not salts, since no significant accumulation is demon- strated in the nephridialplasm by microincineration (MacLennan and Murer, 1934). These materials may possibly be incidental in some species, but Amoeba dies if this elimination is prevented (Mast and Doyle, 1935b), demonstrating that these wastes are toxic and that in this respect they have the known properties of the nitrogenous wastes of metabolism. Evidence that these materials are the result of metabolism is found in Ichthyophthirius (MacLennan, 1936). The vacuoles are osmiophilic only during the active feeding phase, when large amounts of food are being used and converted into storage bodies; but during en- cystment, when these activities cease, the vacuoles are not osmiophilic. The food of this protozoan consists entirely of epithelial cells, which are largely proteins, but not more than a third of the reserves of the ciliate are proteins, the rest being carbohydrate and fat. During the feeding stage a large amount of the ingested protein would be deaminized to form other reserves, with the result that much larger amounts of nitroge- nous wastes would be formed during the feeding stage than in the en- cysted stage. Frisch (1938) suggests that the contractile vacuoles also function in respiration. This function cannot be correlated with the variations in the nephridialplasm in various Protozoa and thus, if this is a function of the contractile vacuoles, it is probably independent of the granules which are being considered here. LIPOID RESERVES The lipoid materials considered in this section include all lipoids which are visible as granules and which are laid down during active CYTOPLASMIC INCLUSIONS 151 feeding stages and are used during hunger or encystment. Zinger (1933) included all sudanophil particles as lipoid reserves, but digestive gran- ules, mitochondria, as well as other bodies respond to Sudan III because of their lipoid content. Zinger pointed this out, for in his conclusion he states that the sudanophil bodies are more than reserve materials. How- ever, until more is known of the functions of the intracellular lipoids, it is impossible to indicate accurately the boundary between reserve lipoids and those active directly in the metabolism of the cell. Lipoid reserves have been found in a large number of Protozoa (for a detailed list see von Brand, 1935). Usually, if not always, these gran- ules are in the endoplasm, either distributed at random, as in Ich- thyophthirius (MacLennan, 1936), or concentrated at one end, as in Anoplophrya (Eksemplarskaja, 1931). Although these visible lipoid granules occur in many Protozoa, they are not universal. Trypanosoma evansi lacks all lipoid reserves, a fact which is correlated with a lack of lipase (Krijgsman, 1936). The Ophryoscolecidae and Cycloposthiidae, noted for their tremendous glycogen reserves, have no important lipoid reserves. Mesnilella multispiculata has no lipoid reserves, although five other species of the same genus have many fat globules (Cheissin, 1930). The formation of droplets of neutral fat inside a granule of fatty acid has been demonstrated in O palina '(Kedrowsky, 1931) and Ich- thyophthivius (MacLennan, 1936) by the Nile blue sulphate method (Fig. 41). Since, after staining with Nile blue sulphate, very small quantities of fatty acid dissolved in neutral fat result in an intense blue color rather than the pink which is characteristic of pure neutral fats, the pink stain observed in the cases above indicates that there are no free fatty acids in the neutral fat granules, as would be expected if the fats were synthesized on the surface of these granules. The fatty acids and glycerine dissolved in the endoplasm are first segregated into granules, and in these granules the neutral fat is synthesized. Then this fat is segre- gated into the visible droplets of pure neutral fat inside the active gran- ules. These latter granules are typical Golgi bodies (MacLennan, 1936, 1940), as indicated by the name endoplasmic Golgi bodies. However, since these are functionally an intermediate stage in the development of the fat reserves, the descriptive term “intermediate lipoid body’’ is more appropriate in a functional classification. In A. proteus the fat droplets grow in the cytoplasm without any in- 152 CYTOPLASMIC INCLUSIONS termediate granules being visible. These cytoplasmic fat droplets are not derived directly from fat in the food vacuoles, but the ingested fat is absorbed as free fatty acid and glycerine, synthesized into neutral fat in the cytoplasm and then stored as granules (Mast, 1938). Free fatty acids were demonstrated in the food vacuoles when fat was being digested, but none were demonstrated in the cytoplasmic fat droplets or on their surface when they were being formed. In the cases in which this process is not visible, either the synthesis is carried on elsewhere and the fat trans- ported to the granules as such, or the fatty acids are never allowed to accumulate sufficiently to show under the microscope. The visible lipoids, i.e., those which are found in definite globules and demonstrable by the ordinary fat-staining technique, include only a part of the total lipoids of the cell. “The pathologists have known for many years that the fats and fat-like substances of protoplasm are so bound or united to proteins as to be for the most part non-recognizable in the living or stained cell” (Heilbrunn, 1936). Besides the factors of food and the formation of fat from other substances such as carbohy- drate or protein, changes from bound lipoids to free globules must be considered in any estimation of the reserve lipoids. Heilbrunn demon- strated an increase in lipoid globules in specimens of A. proteus kept in a dilute solution of ammoniumsalts. Three types of amoebae were found —those which show lipoids in culture, those in which lipoid globules appear after treatment with NH,Cl, and those in which no free lipoid appears even after treatment. In similar experiments with Arbacia eggs, Heilbrunn showed that the total lipoids of the protoplasm remained con- stant; therefore the newly visible bodies are derived from bound lipoids, not from new fat formation. Since ammonium salts in the culture medium raise the pH of the immersed cells, the results were attributed to alka- linization of the protoplasm. The fact that CO, bubbled in the medium (which would tend to lower the protoplasmic pH), inhibits the forma- tion of visible lipoids, confirms this hypothesis. Old cultures of Para- mecium show larger amounts of fat than new cultures, although the paramecia divide and show no ill effects (Zinger, 1933); and since such cultures contain ammonia (Weatherby, 1927), the presence of abnormal amounts of visible fats may be due to the resulting alkalinization of the protoplasm. Ultra-violet radiation causes a release of lipoid in Amoeba (Heil- CYTOPLASMIC INCLUSIONS iS brunn and Daugherty, 1938). The release of lipoids was greatly in- creased by a preliminary immersion in ammonium chloride solutions. “Further study is necessary in order to determine whether this fat re- lease is due to a direct action of the radiation on the protein-lipoid bind- ing or whether it may not be due indirectly to an alkalinization of the protoplasm” (Heilbrunn and Daugherty, 1938). In the same publica- tion it is stated that any stimulus in which localized increases in tempera- ture occur is efficient in the release of fat. This is also shown by the experiments of Sassuchin (1924), who compared the protoplasm of O palina kept at room temperature with the protoplasm of those kept at 35-38° C. In the first group he found elongate mitochondria in the endo- plasm (Kedrowsky’s endosomes), but in the heated group only fat spherules and protein spherules, and these results were interpreted as due to the separation of mitochondria into their two components. These latter experiments should be repeated in individuals with little or no fat, and in species as to which there is more agreement on the identification of mitochondria. In Paramecium (Zweibaum, 1921) and Stentor (Zhinkin, 1930) fat is stored under conditions of low oxygen tension and lost when the oxy- gen tension is restored. The rate of loss in this case is dependent upon the temperature. Pathological conditions are often marked by fatty degeneration in the Protozoa. Degenerating coccidial oocysts show an increase in fat globules (Thélohan, 1894), and in Awlacantha fatty vesicles are formed and the nucleus is finally replaced by fatty bodies (Borgert, 1909). Individuals of Actinophrys which show depression by a lowered division rate and otherwise, have an abnormal number of lipoid bodies, and’ in extreme cases show typical fatty degeneration. In the macronucleus of Paramecium parasitism by bacteria also results in tremendous quantities of visible lipoids in the cytoplasm and also of crystals (Fiveiskaja, 1929). CARBOHYDRATE RESERVES Granules containing carbohydrates are found in most Protozoa, al- though in a few species this reserve is in a diffuse form which is pre- cipitated as granules or irregular masses by fixation. The lack of any carbohydrate reserve at all has been proved in only a few species, such as Trypanosoma evansi. 154 CYTOPLASMIC INCLUSIONS The carbohydrate reserves in Paramecium (Rammelmeyer, 1925) and in the cysts of Bursaria (Poljansky, 1934) are probably dissolved in the protoplasm, since they are visible in fixed specimens only as cloudy masses, not in regular granules. Homogeneous vacuoles, granules, or platelets visible in the living normal Protozoa are very common. They are well known in lodamoeba and other intestinal amoebae. Large num- bers of these granules are found in the flagellates from termites and wood-eating roaches (Cutler, 1921; Kirby, 1932; Cleveland, 1934; Yamasaki, 1937a). The carbohydrate granules of Stentor tend to be localized in a peripheral sheath of the endoplasm (Zhinkin, 1930) and just beneath the pellicle. In Arce//a these granules are embedded in the chromidial net. In Ichthyophthireus these smaller granules are always associated with mitochondria (MacLennan, 1936). Glycogen granules are often associated with the parabasal bodies in flagellates (Duboscq and Grassé, 1933). Carbohydrate granules with definite internal structure are by no means uncommon. The granules of Sporozoa (Fig. 60) have a cross or star- shaped center (Joyet-Lavergne, 1926a; Daniels, 1938), the general ap- pearance of which and ability to accumulate iodine suggest vacuoles. Identification of a lipoid center (Erdmann, 1917) is based on insufficient evidence and, in view of the later work quoted above, seems unlikely. Vacuolated bodies are also found in Balantidium (Fig. 57) with the added feature of crystals floating in some of the vacuoles (Jirovec, 1926). Two types of granules are found in D7fflzgza, small homogeneous spherules and larger elliptical bodies with a center granule which stains a pale blue after hematoxylin and a rim which is rose-colored after Best’s stain (Rumjantzew, 1922). In the Ophryoscolecidae, the granules possess a spherical center (Fig. 56) denser than the rest of the granule (MacLennan, 1934). The most spectacular of the carbohydrate reserves are the skeletal plates of the Cycloposthiidae, Ophryoscolecidae, and related families. The plates themselves are probably supporting structures, but in their meshes are platelets of the same type as the scattered cytoplasmic granules. The plate- lets in the Cycloposthiidae (Fig. 61) are roughly spool-shaped with slen- der strands connecting the flanges of adjacent granules (Strelkow, 1931), but in the Ophryoscolecidae (Fig. 62) the polygonal plates are unconnected (MacLennan, 1934). CYTOPLASMIC INCLUSIONS 153 The formation of paraglycogen bodies has been followed in only a few cases. The bodies of Pelomyxa behave like permanent bodies with a protein stroma and with the paraglycogen being built up or released as the case demands (Leiner, 1924). The paraglycogen granules of Poly- plastron are likewise independent of other formed components and are apparently self-perpetuating (Fig. 56), since they show regular division (MacLennan, 1934). The dense centers may be naked or, more often, surrounded by an envelope of varying thickness. In the largest of these compound granules, the centers are dumb-bell-shaped or double, and in the latter the envelope also is constricted. These stages probably repre- sent growth or utilization stages and division stages, although this was not confirmed by following a single granule in live ciliates. The com- plex granules of Amoeba hydroxena (Fig. 58, 59), in which a varying number of glycogen granules are imbedded in glycoproteid (Wermel, 1925), suggests a conversion of glycogen into glycoprotein for storage and the reversal of this process in utilization. Some paraglycogen granules are formed in association with mito- chondria instead of being independent bodies. The paraglycogen in Ichthyophthirius first appears as a minute vacuole (Fig. 17) in the center of a sphere of mitochondrial material (MacLennan, 1936). As the gran- ules grow, this mitochondrial shell breaks into short rods fused to the surface of the paraglycogen. The mitochondria disappear after the gran- ule has attained full size. Joyet-Lavergne (1926b) also noticed a morpho- logical relationship between mitochondria and paraglycogen of greg- arines, but says “il y a la un simple rapport de contact et nous n’avons aucune raison de suppose une intervention dans da génése du para- glycogéne.” However, in the case of a granule in the center of an un- broken sphere, as in Ichthyophthirius, it is difficult to list the relationship as merely an incidental contact. Duboscq and Grassé (1933) show that the glycogen granules of Cryptobia helicis are not found scattered in the cytoplasm, but are formed in close contact with or in the strands of the parabasal bodies (Fig. 65). The glycogen is not laid down in the summer, but only in the winter, a fact which, they point out, would explain the negative results of other authors. This formation of glycogen by the parabasal body parallels the secretion of protein granules by the macronucleus of ciliates—a part of the segregation function which, in most species, is performed by isolated Figures 56-64. Carbohydrate reserves, Figure 56, stages in the paraglycogen granules of Polyplastron multivesiculatum, Champy-osmic impregnation followed by Sudan III in hot paraffin (after MacLennan, 1934); Figure 57, growth of crystals in paraglycogen granules of Balantidium elongatum, Zenkers-dahlia (after Jirovec, 1926); Figures 58- 59, glycogen droplets in a glycoproteid granule, from Amoeba hydroxena, Carnoy-Best (after Wermel, 1925) ; Figure 60, vacuolated paraglycogen body from Sporozoa (after Joyet-Lavergne, 1926) ; Figure 61, skeletal platelets of Cycloposthium edentatum, Lugol (after Strelkow, 1929); Figure 62, skeletal platelets of Polyplastron multivesiculatum, Champy-osmic-Sudan JII in hot paraffin (after MacLennan, 1934); Figures 63-64, glycogen reserves in Trichonympha agilis, Best’s stain, 63 normal, 64 showing loss of glycogen just before death under conditions of lowered temperature and raised oxygen pressure (after Yamasaki, 1937). CYTOPLASMIC INCLUSIONS 157 cytoplasmic granules, is performed in the one case by a nuclear structure and in the other case by a neuromotor structure. The differentiation between the various carbohydrates found in the Protozoa is based on their staining reactions and solubility, since the exact nature of the sugars involved in the formation of protozoan poly- saccharides is unknown. Zhinkin (1930) and von Brand (1935) pointed out that this is unsatisfactory and contend that no separation should be made from glycogen until this is known. However, the differences are so pronounced that it is convenient to retain the name paraglycogen. Soluble glycogen as found in vertebrate liver cells is relatively rare. The diffuse materials found in Paramecium and Bursaria are probably of this type. The commonest carbohydrate is paraglycogen, distinguished by Buitschli (1885) from glycogen on the basis of its relative insolubility in water as compared with true glycogen. It is digested by ptyalin and diastase and the sugar produced reduces Fehling’s solution. It stains a light brown in todine and brown or brown purple in todine-sulphuric acid or chlor-zinc-iodide. Probably all of the granular reserves of carbo- hydrate in Protozoa are paraglycogen or some similar relatively insoluble compound. The reserve granules of the flagellates of termites have been identified as glycogen (Yamasaki, 1937a; Kirby, 1932); but in the re- lated flagellates of the wood roach, since the Protozoa contain no enzyme capable of breaking down glycogen, it has been suggested that the gran- ules which stain with iodine consist of some other product which results from the breakdown of cellulose (Cleveland, 1934). The material in the platelets of the Ophryoscolecidae has been named ophryoscolecin on the ground that it is unique in this family and is more like cellulose than paraglycogen (Dogiel and Fedorowa, 1925). It was later identified as a hemicellulose (Strelkow, 1929). This interpretation is based on slight variations in solubility and color reactions, but other authors, using some of the same methods and some different methods, were not able to find any difference between the reactions of paraglycogen and the platelets (Schulze, 1922, 1924, 1927; Weineck, 1931, 1934; MacLennan, 1934). However, such arguments cannot be settled, as von Brand suggests, until the exact structure of these polysaccharides is known, and the term para- glycogen in this discussion is used in a rather general sense for carbo- hydrates more insoluble in water than glycogen and differing in color reactions from starch and cellulose. 158 CYTOPLASMIC INCLUSIONS The presence of more than one type of material in the same granule has been demonstrated in several cases, in spite of the relative crudity of the cytochemical methods for the demonstration of carbohydrates. The oval carbohydrate bodies of D7fflugza are not completely dissolved in ptyalin, and their staining reactions suggest the presence of a glyco- proteid (Rumjantzew, 1922). Two types of carbohydrate reserves have been reported from Sporozoa by Dobell (1925). Chakravarty (1936) Figure 65. The associa- tion between glycogen and the parabasal body in Cryptobia helicis, winter forms stained with iodine. (After Duboscq and Grassé, 1933.) also differentiated two sets of granules by differences in the speed of destaining after treatment in iodine. These authors refer to one set as glycogen, the other as paraglycogen. Two types of carbohydrate have been found in Actinosphaerium (Rumjantzew and Wermel, 1925), and were identified as glycogen and a glycoprotein on the basis of their re- action to Best’s and Fischer’s stains. Pelomyxa loses its paraglycogen dur- ing prolonged starvation, but since the remnants of these granules may be stained with haematoxylin (Leiner, 1924), it is probable that the carbohydrate is here associated with a protein. The carbohydrate bodies CYTOPLASMIC INCLUSIONS 159 of Amoeba hydroxena contain two different materials (Figs. 58, 59) and on the basis of Best’s and Fischer's methods have been interpreted as granules of glycogen embedded in glycoprotein. The decrease in glycogen or paraglycogen during hunger or encyst- ment, and its storage during the feeding stages, has been noted so often that detailed descriptions of observations under controlled conditions are rare. The glycogen in Stentor is deposited during low temperatures and utilized at higher temperatures, and this process is accelerated by starvation (Zhinkin, 1930). Fat, rather than glycogen, is deposited, if the oxygen tension is lowered. Under such conditions some of the car- bohydrate is probably converted into fat. In Trichonympha also, avail- able food, oxygen tension, and temperature affects the amount of glyco- gen present (Yamasaki, 1937b). The cytoplasm of this species is divided into two parts by a fibrillar basket, which suspends the nucleus from the anterior cone of the body and separates this portion from the rounded posterior part in which the food vacuoles are formed. Both regions normally contain glycogen (Fig. 63), but during starvation the glycogen in the posterior part disappears first, the glycogen in the anterior part then diminishes and disappears, and the death of the organism follows shortly. Similar results are observed at high temperatures, or with oxy- genation at room temperature. However, when the termites are oxygen- ated at low temperatures (Fig. 64), the glycogen in the posterior por- tion often shows little change, but the portion anterior to the nucleus disappears rapidly. As soon as the glycogen in the corbula disappears the protozoan dies, even though glycogen remains in the body region. Yamasaki states that the posterior region is simply one of synthesis and storage, while the anterior region is the region of consumption. He concludes that defaunation by oxygen is due not only to toxicity but also to a depletion of the glycogen available for the nucleus and motor or- ganelles. Trypanosoma evansi possesses no glycogen and, since it possesses no amylase, is not able to synthesize it (Krijgsman, 1936). Other trypano- somes do deposit glycogen, but at best it forms an insignificant reserve, since trypanosomes may use three times their body weight in sugar in twenty-four hours (von Brand, 1938). In this case the glycogen re- serves of the trypanosomes are the liver glycogen of the host. 160 CYTOPLASMIC INCLUSIONS PROTEIN RESERVES This term is one of convenience and, as in the case of the term lipoid, cannot be taken in a strict sense, but is used here to include, besides true proteins, bodies which contain lipoids or carbohydrates, as well as proteins, amino acids, nucleic acid, and so forth. Since most of the fixing agents precipitate at least the protein portions of such granules, many have been described, although relatively few have been identified by acceptable microchemical methods. For this reason they have been de- scribed under a variety of names, many of which mention incidental staining properties. Some of the names which are most securely em- bedded in the literature are chromidia, volutin, metachromatic granules, basophilic granules, chromatoidal bodies, and albuminoid reserves. The confusion in these terms is best illustrated by chromidia. This was orig- inally used to designate chromatin bodies which are extruded into the cytoplasm from the nucleus (Hertwig, 1902) and which have the abil- ity to reaggregate to form new nuclei. Although this interpretation has been disproved, the name may be retained to designate these granules (Meyers, 1935). In other cases it is used to designate nonchromatin ma- terial which is supposed to be extruded from the nucleus (Daniels, 1938). Other authors use it even more loosely to designate basophilic and metachromatic cytoplasmic bodies which are secretory in nature (Campbell, 1926). The elimination of the original meaning was due to the improvement of both cytoplasmic and nuclear methods, accompanied by detailed studies of life cycles. The last stronghold of this theory— the Foraminifera—was eliminated by the tracing of the nuclear history in live Patellina throughout the vegetative and sexual stages, with a complete demonstration of the cycle with moving pictures (Meyers, 1935). The exclusion of chromidia in the original sense, with respect to the cells of the Metazoa, has already been accepted (Wilson, 1928). Many chromidia are actually mitochondria (Fauré-Fremiet, 1910) which contain a high percentage of protein and are therefore resistant to routine fixatives. This probably led to one revival of the chromidial theory, according to which all cytoplasmic structures are formed from mitochondria, which in turn originate from the nucleus as chromidia. Alexeieff in a series of works on the Flagellata strives to prove that all cell structures are formed at the expense of mitochondria. The latter, according to Alexeieff, in their turn are not autonomic, as the majority of investigators CYTOPLASMIC INCLUSIONS 161 suppose, but originate from the nucleus as chromidia. . . . In cases where the autonomy of the mitochondria and of the blepharoplast is indisputable, this author always attributes to them a nuclear origin though phylogenetic. After this summary of Alexeieff’s theory, Milovidov (1932) rejects it. Certain cytoplasmic granules of Uroleptus are derived from the nuclei during reorganization (Calkins, 1930), and these granules were de- scribed as mitochondria, but since they do not stain with janus green they do not seem to be typical mitochondria. The term chromidia, as now accepted, includes cytoplasmic granules supposed to be derived from the nucleus (but not necessarily chromatin), particularly in the Sporozoa. It also includes granules in the rhizopods, at one time supposed to be examples of the chromidial theory, but now retained without any such implication. The chromidial net, characteristic of many of the rhizopods with shells, is a definite morphological entity which may be recognized independently of particular staining methods. The net itself is negative to Feulgen’s stain in Arcella and Chlamydophrys, either with or without hydrolysis, and is digested more rapidly than the nucleus by pepsin or trypsin (Reichenow, 1928). Since the net in D/fflugia gives a positive reaction with Ciaccio’s lipoid method (Rumjantzew, 1922), it probably has a lipoid component in addition to the protein component in this species. Although basophilic, it is not directly related to the nuclear material. On the other hand, the net of Patellina is positive to Feulgen’s method, but complete studies show that it is independent of the nuclei (Meyers, 1935). In both cases the net is a specialized mass of reserve protein, and within it may be found two other types of reserve, volutin and glycogen granules. This is not true in all species, since no glycogen is found in the net of Difflugia (Rumjantzew, 1922). The chromidia of gregarines are similar in ordinary staining reactions to the karyosome and to the pro- tein reserves (Daniels, 1938). The chromidia of several Sporozoa (Joyet-Lavergne, 1926a) are posi- tive to Millon’s reagent and are therefore certainly protein and they appear to be associated with mitochondria. In gregarines from meal- worms, on the other hand, these granules are negative to both Millon’s reagent and Feulgen’s reagent, and show no morphological relationship with mitochondria (Daniels, 1938). Daniels found chromidia and volu- tin similar in shape, distribution, and so forth, but found fewer black 162 CYTOPLASMIC INCLUSIONS granules in haematoxylin preparations than blue granules after the methylene blue method. She concludes that they are separate types of granules, although there is a close relationship. Joyet-Lavergne was not able to decide whether chromidia and volutin are really separate. Daniels observed buds on the surface of the karyosome, then bodies in the nuclear sap, and finally in the cytoplasm near the nucleus, but she found no direct evidence as to how they penetrate the nuclear mem- brane. On the basis of these suggestive observations, she concludes that these bodies are derived from the karyosome. With regard to the validity of this conclusion, the comment of Wilson (1928, p. 96) with regard to a similar case in oogenesis is highly pertinent: ‘“To the writer none of these cases yet seems to be satisfactorily demonstrated, and the question is a most difficult one to be settled by studies on fixed material alone.” Joyet-Lavergne (1926a) calls these protein granules albuminoid reserves, a name far more appropriate than chromidia, which at least implies a nuclear origin. Volutin granules are basophilic granules which are also metachromat- ic. Because of their pronounced basophilia, volutin granules have often been linked with chromatin. However, they are negative to Feulgen’s stain after hydrolysis, but give a positive reaction when the preliminary hydrolysis is omitted (Reichenow, 1928), a characteristic of free nu- cleic acid. The full Feulgen reaction apparently dissolves this type of volutin granule, so that in Arcella there results a diffuse Feulgen reac- tion in the chromidial net. This is a possible explanation of the positive Feulgen test by the chromidial net of Patellina. The volutin granules of Trypanosoma melophagium contain no nucleic acid (van Thiel, 1925), while those of T. equinum do (Reichenow, 1928). The volutin bodies of T. evansi were not tested in this respect (Krijgsman, 1936), although they are listed as containing nucleic acid. Since reserve bodies are not the same in all species of the genus (some trypanosomes are able to store glycogen while others are not, according to von Brand, 1938), both analyses of the basophilic granules may be correct. Volutin granules increase and nuclear granules decrease in trypano- somes which have been treated with atoxyl (Swellengrebel, 1908). This fact in conjunction with the staining reactions of volutin, were inter- preted as indicating a direct nuclear origin—in other words, a type of chromidia. In these experiments the results are probably a degeneration CYTOPLASMIC INCLUSIONS 163 phenomenon, since in Pelomyxa the expulsion of chromatin into the cytoplasm is found just prior to death (Schirch, 1914). Hindle (1910) thought of this as a degeneration phenomenon in T. gambiense. In various phytoflagellates division stops when volutin is lost, and the volu- tin was interpreted as a nuclear reserve (Reichenow, 1928), although no direct connection between the two was demonstrated. A dehydrase has been demonstrated in Trypanosoma by the leucomethylene blue method and localized in the volutin granules (Krijgsman, 1936). Krijgsman, however, holds to Reichenow’s views of volutin as a nuclear reserve. Protein bodies in Oxymonas dimorpha, which are negative to Feul- gen’s stain and stain with either basic or acid dyes (i.e., not metachromat- ic), have been called volutin granules (Connell, 1930), although they are not volutin in the sense used by Reichenow. However, in Oxymzonas, as in the phytoflagellates of Reichenow’s experiments, division ceases when these granules are exhausted. Since the division stages of Oxymonas are also the flagellated stage, the protein granules could be explained as reserve bodies for the expenditure of energy by these organelles. Neither explanation has adequate proof, since each merely correlates obvious phenomena. Volutin is thus a term which has no standard usage, but wherever microchemical tests have been made volutin has been found to contain proteins, nucleic acid, or other similar materials. Since the available evi- dence shows that it behaves as a reserve material, it seems to me to be convenient to include it as one of the various types of protein reserves and to eliminate the terms volutin and metachromatin, neither of which seems to have been used consistently by protozodlogists. The macronuclei of many ciliates contain one or more large, intensely basophilic bodies lodged in vacuoles among the closely packed granules of chromatin (Chakravarty, 1936; MacLennan, 1936). Since their num- ber and size vary, it has been suggested that these are reserve materials (Kazancev, 1928). In Ichthyophthirius these granules have been traced in living ciliates from the macronucleus through temporary breaks in the macronuclear membrane into the cytoplasm (Fig. 66) where they are stored until resorption and utilization occurs in the encysted stages (Mac- Lennan, 1936). These bodies take both acid and basic dyes even more strongly than chromatin and, unlike the chromatin, are negative to Feulgen’s reaction and Macallum’s tests for iron. These granules first 164 CYTOPLASMIC INCLUSIONS appear as minute bodies at the lower limits of visibility, embedded in the chromatin net during the feeding stages of the ciliate but not during encystment. The macronucleus is positive to Feulgen’s reagent without preliminary hydrolysis during the formation of these granules, but at no other time. It seems probable that food materials are built up in the macronucleus into chromatin, which is then split into a group contain- ing iron and nucleic acid and another protein group which lacks these substances. The first group is used to rebuild more chromatin and the latter group is segregated into the granules which are ejected into the Figure 66. The forma- tion and release of protein granules from the macro- nucleus of Ichthyophthi- rius multifiliis, Feulgen- light green. (After Mac- Lennan, 1936.) cytoplasm. Since granules of this type are found in both the macto- nucleus and the cytoplasm of so many ciliates, this is probably quite a general phenomenon. The Protociliata lack macronuclei, but perform this same function by the segregation apparatus. The balls of chromatin and other macronuclear fragments which are extruded during the various types of macronuclear reorganization are only incidentally reserve material, if at all, and will be considered in detail in the chapters on nuclear phenomena. The crystals which are common in various Protozoa are often con- sidered to be excretory products, and in some cases have been identified as uric acid (fora discussion of this work, see Reichenow, 1929). Recent work (Mast and Doyle, 1935b) shows, however, that some of the crystals must be regarded as reserve material. A. proteus contains two types of crystals, a bipyramidal type and a plate-like type, which are suspended in vacuoles containing an alkaline fluid. A careful study of spectroscopic CYTOPLASMIC INCLUSIONS 165 analysis, solubility, and form shows that the bipyramidal type probably consists of a magnesium salt of a substituted glycine. The plate-like crystals are insoluble in a saturated solution of leucine, and in structure resemble leucine crystals. If the crystals are removed by centrifuging and the Amoeba is then put in a solution which contains amino acids and egg albumin, the platelets are formed in the vacuoles which con- tain leucine, while the bipyramidal crystals are formed in all solutions. “Crystals are normally formed from amino acids derived from food dur- ing digestion” (Mast and Doyle, 1935b). These crystals decrease in number just before the refractive bodies increase in number, indicating that the crystals are an intermediate stage in the transfer of food from the food vacuoles to the lipoprotein refractive bodies. The chromatoidal bodies of various parasitic amoebae, are intensely basophilic structures, the fixing and staining reactions of which suggest a protein composition. They possess neither chromatin nor free nucleic acid (Reichenow, 1928), so are not volutin; but they are similar in their reactions to the protein bodies of ciliates. Since they disappear dur- ing encystment they are reserve bodies. The reserve proteins are often found in combination with other ma- terials. In Amoeba, lipoids and proteins are bound together in the re- fractive bodies (Mast and Doyle, 1935a). In Actinosphaerium, granules of glycoproteid are present (Rumjantzew and Wermel, 1925). Similar inclusions are found in Ophryoglena (Zinger, 1928). In the Foet- tingeriidae the protein reserves have the characteristics of the vitellin of the hen’s egg and in Polyspira there is a single central mass consisting of protein associated with a carotenoid (Chatton, Parat, and Lwoff, 1927). The protein portion alone is used, the carotenoid remaining in the old cyst, and finally disappearing during encystment. The protein bodies which are found throughout the Protozoa vary greatly in their specific structure and composition. This variation, with the resulting variation in staining reactions, has resulted in a complicated nomenclature with the usage of terms proposed by each author. The term chromidia is so definitely bound up with disproved theories that it should be dropped. Volutin should either be dropped or definitely restricted to metachromatic granules which respond to Feulgen’s stain when used without hydrolysis. Whenever the function is that of a re- serve, as in the majority of known cases, I believe these granules should 166 CYTOPLASMIC INCLUSIONS be called simply protein reserve bodies, a usage found convenient by Joyet-Lavergne (1926a). At the same time, it should be recognized that a reserve function is the one most easily identified by morphological methods, and that other functions must be investigated. The presence of dehydrogenase (Krijgsman, 1936) in protein aie of Try pano- soma evansi is one definite lead. The protein reserve bodies are as catholic in origin as in structure and may result from the activities of the segregation granules, macronucleus, mitochondria, or food vacuoles, or may be independent of other formed bodies. The crudity of our knowledge of cytoplasmic granules is illus- trated by the fact that no suggestion of the significance of these differ- ences can be made. EXTERNAL SECRETION This important cytological subject has been greatly neglected in the Protozoa and is generally ignored in a discussion of the protozoan cyto- plasm, except as the vacuolar apparatus is considered to be a secretory or- ganelle. It is an important subject in itself and is most nearly comparable to secretion studied in the Metazoa. The attaching organs, or at least the cementing portion, of sessile Protozoa are secreted structures. Just preceding the formation of the peduncle of Campanella, granules are found in the basal region (Fauré- Fremiet, 1905). T7ntinnopsis nucula is cemented to the lorica by a mucus secretion which is derived from basophilic granules in the stalk (Camp- bell, 1926). The lorica of Favella is likewise derived from cytoplasmic granules (Campbell, 1927). Granules which are to form the new lorica accumu- late near the mouth, in dividing animals. After division these are forced out through the cytostome, expand, fuse, and harden. At the same time fecal pellets are molded into this secreted material and the whole lorica is shaped by the activities of the motor organelles. There is in this form a local zone of secretion, as in gland cells, not a general secretion over the whole surface. The shell of Exg/ypha is formed from separate shell plates, which are secreted within the cytoplasm (Hall and Loefer, 1930). They appear first as small refractive spheres in vacuoles, then enlarge and elongate to become typical shell plates. (Fig. 67). The finished plates lie free in the CYTOPLASMIC INCLUSIONS 167 cytoplasm. It was demonstrated that the reserve plates have no connection with either the mitochondria or the neutral red globules. The cyst of Ichthyophthirius is secreted in two parts: first a homo- geneous clear membrane is formed (Fig. 68) and then individual fibrils are extruded, apparently between the bases of the cilia (Mac- Lennan, 1937). These sticky fibrils are stroked into rope-like fibers, which adhere to the under side of the outer membrane (Fig. 69) by the activities of the cilia. Although seven types of granules were demon- 50 eo gh i Figures 67-69. External secretion. Figure 67, inclusions in Evglypha alveolata, prob- ably representing the formation of reserve shell plates (after Hall and Loefer, 1930) ; Figures 68-69, secretion of the cyst wall in Ichthyophthirius multifiliis; Figure 68, section of early stage showing only the homogeneous layer; Figure 69, section of later stage showing the addition of the fibrillar layer (from MacLennan, 1936). strated, no granules could be associated with the secretion of this cyst. In Nyctotherus, variations in the thickness of the secreted cyst may be correlated with the distribution of ectoplasmic structures (Rosenberg, 1937), but no granules responsible for the secretion were noted in this form either. The lorica of Folliculina ampulla is likewise secreted in the form of a clear fluid, which hardens to form a membrane just beyond the tips of the somatic cilia (Fauré-Fremiet, 1932). The secretion of vacuoles of oxygen in Arcel/a is not associated with granules, but with nongranular regions of hyaloplasm, and is probably a result of oxidative and reductive processes of the cell (Bles, 1929). The most striking aspect of these examples of protozoan secretion is 168 CYTOPLASMIC INCLUSIONS that none of them has been traced to Golgi bodies, mitochondria, or segregation bodies—1.e., they do not react with osmic acid, Janus green B, nor neutral red. It is evident that not all important segregation nor synthesis is revealed by these stains. The mucus granules of Evglena (Dangéard, 1928) and the pellicular secretions of Vorticella (Finley, 1934) stain with neutral red and thus might be classed the segregation granules of Opalina and others, the only difference being that in the former is segregated mucus, which is not used within the cell but is extruded in the normal functioning of the protozodn, while in the latter are segregated proteins, which are normally used within the cell. It is interesting, however, that Kedrowsky found that when the segregation granules were filled with foreign ma- terials, such as the organic silver compounds, the granules are extruded. The expulsion of droplets containing neutral red may be induced in Paramecium (Frisch, 1938) and other ciliates. These examples indicate that the formation of the segregation granules and the secretion granules is comparable, the only difference being that in one the material is used internally and in the other externally. THE GRANULAR COMPLEX The detailed consideration of each of the types of cytoplasmic gran- ules has resulted in the conclusion that there are no universal cyto- plasmic components and that each of the terms mitochondria, Golgi bodies, neutral red granules, and so forth has been applied to a hetero- geneous assortment of granules of widely different functions. This con- clusion, derived from a consideration of the types of granules separately, becomes inescapable if we consider the whole granular complex. The problem is on the surface one of classification, but fundamentally it 1s one of function—what functions are performed by cytoplasmic gran- ules, and is the same function always performed by the same type of granule in different Protozoa? Since these granules are not independent units but are part of a granular complex which in turn is a part of the whole cell, this whole complex must be considered in seeking an answer to these problems of function. The investigations which seem to be suitable for this comparison are those of Mast and Doyle (1935a, 1935b), Holter and Kopac (1937), Holter and Doyle (1938), all on Amoeba proteus; Hopkins (1938a, 1938b) on Flabellula mira; Ked- CYTOPLASMIC INCLUSIONS 169 rowsky (1931-33) on Opalina ranarum; MacLennan (1936, 1937) on Ichthyophthirius multifiliis; and Joyet-Lavergne (1926 on) on several Sporozoa, supplemented by the observations and experiments of Daniels (1938) on similar species. Since a summary of the individual granules has been given in the previous sections, only a general account of each granule will be given in this comparison. The number of types of granules ranges from two in the marine amoeba Flabellula to at least six in Ichthyophthirius and some of the Sporozoa. In Flabellula there are only digestive granules and small gran- ules of unknown composition and function. This small number contrasts sharply with A. protews, which has four types of granules of cytoplasmic origin: refractive bodies (dictyosomes), alpha granules unknown in composition and function, mitochondria (beta granules), and neutral fat granules. In addition to these granules there are two types of crystals, blebs on these crystals, and vacuole refractive bodies, all of which arise in connection with the food vacuole. The two ciliates Opalina and Ich- thyo phthirius also show marked differences in number of granules—the former with only four types and the latter with seven. O palina has segre- gation bodies, endosomes (mitochondria?), intermediate lipoid bodies (endoplasmic Golgi), and neutral fat, while Ichthyophthirius has inter- mediate lipoid bodies (endoplasmic Golgi), neutral fat, excretory gran- ules (ectoplasmic Golgi), mitochondria, paraglycogen, and protein bodies. Gregarines and Coccidia have at least one type of Golgi body, neutral fat, one or two types of mitochondria, paraglycogen, one or two types of protein reserves, and neutral red bodies, a total of six to eight types of granules, allowing for differences in the accounts of Daniels and Joyet-Lavergne. Not all the Sporozoa present such a complicated picture, since there are probably not more than three types of granules in Plasmo- dium: mitochondria, segregation granules, and pigment. These marked differences, which appear even with a crude comparison based only on number of types of granules, show clearly that at best only very few gran- ules could be universal. Furthermore, the number of granules varies in- dependently of the relationships of the Protozoa involved, since both large and small numbers of granules are found in Protozoa of the same class. The immediate facts which stand out with respect to the staining re- actions of these five species of Protozoa are that in each species are granules or vacuoles which are stained specifically by Janus green B 170 CYTOPLASMIC INCLUSIONS and other mitochondrial stains, in each are granules which segregate neutral red, and in each, with the exception of Flabellula, are granules which may be specifically impregnated by the Golgi methods. If we do not press the comparison any further, we can say that chondriome and vacuome are universal cell constituents but, even with the very crude definition based on impregnation alone, Golgi bodies are lacking in one of the five Protozoa discussed. However, this apparent uniformity is reduced if these granules are compared with respect to other character- istics than the so-called specific staining methods, which are in reality quite crude in spite of brilliant contrasts. With respect to mitochondria, the bodies range from one extreme of temporary induced granules in Flabellula to two separate types in some of the Sporozoa. The neutral red bodies are even less comparable: in Ichthyophthirius they are lipoid- containing bodies, which are found only in association with gastrioles. In Opalina they range from watery vacuoles to dense bodies of proteins and are obviously not connected with any gastrioles. In Flabellula they are vacuoles in which is also dissolved the material stainable with Janus green. In Amoeba there are two types of neutral red bodies which are of cytoplasmic origin, and both contain large amounts of lipoids and at some stages proteins as well. One of these types, the refractive bodies, are apparently built up from material derived from the blebs and the crystals. In a structural sense also the refractive bodies are unique and are much more complex than any of the other neutral red bodies. In the Sporozoa, lipoid dictyosomes are weakly stainable with neutral red, but in addition there are non-lipoid bodies which stain much more specifically with neutral red. With respect to Golgi bodies, even if we ignore Flabellula, harmony is not achieved. In Amoeba the two types of neutral red bodies, as well as the mitochondria, respond to impregna- tion and bleach with difficulty. A comparison between these lipoid bodies and the endoplasmic Golgi bodies (intermediate lipoid bodies) of Ichthyophthirius, O palina, and the dictyosomes of Sporozoa seems quite logical, until we consider that in these latter three species, these fatty acid bodies are an intermediate step in the formation of neutral fat, while the refractive bodies of Amoeba are finished bodies, the granules of neutral fat being morphologically independent of them. None of these lipoid bodies is comparable with the non-lipoidal ectoplasmic Golgi CYTOPLASMIC INCLUSIONS nA bodies (excretory Golgi) around the contractile vacuoles of Ichthy- ophthirius. These comparisons demonstrate that with respect to actual composition not even one single type of granule is found throughout these five species of Protozoa, and the apparent universal presence of certain types of granules is due to the lack of specificity in Janus green, neutral red, osmic acid, and other stains. This is demonstrated in spite of the fact that the known composition of these bodies can be stated only in qualitative terms which are actually very broad—t.e., lipoid, non-lipoid, protein, and so forth. A comparison on the basis of composition alone is open to criticism if it is not checked from other angles. The segregation apparatus of O palina may be aqueous vacuoles, dense protein bodies, or it may contain bile pigments, depending on the medium and the temperature. Using composition alone as a criterion (or the neutral red reaction, for that matter), these granules would be separately classified, actually Ke- drowsky showed they are the same granules with the same function— segregation and synthesis. In this case the stain reactions and the compo- sition are incidental, and they are important in the classification of the segregation apparatus only if they can be used to reveal the function. Can groups of granules be demonstrated if the granules are compared, not on the basis of structure nor of composition, but on function? If so, are any of these groups represented in all of these five species which we are considering? This comparison cannot be in any way as complete as the comparison based on staining reactions and composition, since for the most part this knowledge is restricted to those functions which have a definite morphological expression—digestion, storage, external secre- tion, and so forth. The apparent emphasis on these functions should not be considered as an implication that these are the only functions in which the cytoplasmic granules may play a rdle, but as an inverse expression of the difficulties of localizing functions which do not produce visible structures. The segregation and storage of protein reserves is obvious morpho- logically and the materials which are stored can be identified by adequate cytochemical methods. All the five Protozoa, again with the exception of Flabellula, have visible stores of proteins or derivative substances. i/2 CYTOPLASMIC INCLUSIONS A. proteus forms leucine and glycine crystals in the gastrioles (they are therefore not strictly speaking cytoplasmic bodies), and these are then separated from the gastriole and the materials are transported from the crystal vacuoles to the growing refractive bodies and are there stored in the form of the protein stroma of these lipoid-protein bodies. In Opalina proteins are stored in the ectoplasmic segregation bodies. In both these cases, the final structures are of cytoplasmic origin and al- though they are so different they could perhaps be harmonized on a functional basis. In Ichthyophthirius, on the other hand, the protein spherules are stored and utilized in the cytoplasm, but originate in the macronucleus by splitting from the chromatin a portion which contains nucleic acid and iron, leaving a reserve protein in the form of large gran- ules which are then discharged as completed bodies into the cytoplasm. There is some evidence in the Sporozoa also of a nuclear origin of some of the protein reserves, although it is entirely possible that they are connected with mitochondria, since Joyet-Lavergne noted a morpho- logical relationship between the two in the Sporozoa he studied. How- ever, even if we disregard the somewhat questionable case of the Sporo- zoa, we find that an identical function—the storage in the cytoplasm of proteins—is accomplished in two cases by cytoplasmic structures, but in a third case by the macronucleus. Digestion, except in the astomatous species, is accomplished by the gastriole, a structure formed by the union of a vacuole which contains the food particles with granules or vacuoles of cytoplasmic origin. In Ichthyophthirius the granules involved are cytoplasmic in origin, but become enclosed within the vacuole; the cytoplasmic vacuoles of Fla- bellula apparently furnish the fluid in the gastriole; while in A. proteus the granules merely aggregate around the gastriole. From morphological evidence, the granules in Ichthyophthirius and Flabellula are concerned with all types of digestion, but in A. proteus both morphological and mi- croenzymatic studies show that the mitochondria are concerned with the digestion of carbohydrates and with the transport of digested materials from the gastriole to such bodies as the refractive granules. There 1s thus some variation in the digestive function, but clearer evidence of differences is the fact that in both Ichthyophthirius and Flabellula the diffusion of materials outward from the vacuole is accomplished without the intervention of any visible granules. The granules of Amoeba, there- CYTOPLASMIC INCLUSIONS 173 fore, have a transport function, but none of the granules of either Ichthyophthirius or Flabellula give evidence of a relationship with the gastriole which would permit such a function. The storage of lipoids is accomplished in almost identical fashion in O palina, Ichthyophthirius, and Gregarina by the formation of inter- mediate lipoid bodies which are converted into neutral fats. A. proteus also stores neutral fats, but in this case no visible intermediate bodies are formed. This is not necessarily in conflict with the facts observed in the other species, since it is very possible that the intermediate bodies of fatty acid might be present but never get as large as the lower limits of microscopic visibility. However, not all the visible lipoid reserves of Amoeba are in the form of neutral fat; some are in the form of masked lipoids in the refractive bodies, and none of the other Protozoa in this group have granules which are strictly comparable with these complex structures. Carbohydrate reserves are found in the form of paraglycogen gran- ules in Ichthyophthirius and the Sporozoa, and in both cases are secreted by mitochondria. In O palina, glycoprotein is found in certain cases in the segregation apparatus, but according to Kedrowsky, there are no im- portant stores of carbohydrates in this species. For Amoeba likewise this statement holds, the only carbohydrate being the shell between the fluid and the lipoid-protein rim of the refractive bodies. No carbohydrate reserves are found in Flabellula. From these cases it would appear that the function of carbohydrate storage is largely accomplished by mito- chondria, but it must be remembered that this is not a general rule, since these reserves may be formed by independent bodies, as in the Ophryo- scolecidae, or by the parabasal body, as in certain flagellates. The vacuolar apparatus performs at least two functions—the excretion of water to maintain the proper water balance, and the excretion of other materials which are probably metabolic wastes. The first function may be performed without the intervention of granules, as in the encysted stages of Ichthyophthirius, but the excretion of other materials is accom- panied by the periodic aggregation of granules around the vacuole in Amoeba and Ichthyophthirius. In the former function the granules con- form to the definitions of mitochondria, and in the latter to the Golgi bodies. These bodies are certainly a group having to do, in Ichthyoph- thirius, only with the contractile vacuoles; but in Amoeba they are appar- 174 CYTOPLASMIC INCLUSIONS ently also associated with the food vacuoles, the refractive bodies, and so forth, in a general transport function. The two groups are comparable, but the granules of Ichthyophthirius perform only a part of the functions assigned to them in Amoeba. It may be possible that the mitochondria or beta granules of Amoeba include more than one type, but in view of the detailed experiments which have been made on this form, this explan- ation is hardly more than a possibility. This survey of functions which have a granular basis fails to reveal any general uniformity, even in this restricted group of five Protozoa. The most clear-cut case of two different mechanisms having the same function is that of the segregation of reserve protein in Ichthyophthirius and Opalina. No matter how broad the definitions are made, the fact remains that the identical result is attained through the mediation of two different cellular mechanisms—in the one case the mechanism is the macronucleus, in the other it is strictly cytoplasmic, the segregation bodies. The general concept of transport introduced by Doyle helps in several cases to group apparently diverse functions within a single func- tional concept; but here again none of Kedrowsky’s published obser- vations in the case of Opalina, nor my own observations in Ichthyoph- thirius, would support this. The stored fat, paraglycogen, and proteins merely decreased in size during encystment, and no intermediate bodies aid in the redistribution of this material. The granules which are produced in a particular species are typical of that species, but in other species the same function may be accomplished by granules different in composition and relationships from those of the first species, or the same function may be accomplished without the form- ation of visible granules. The cell is not restricted in the accomplishment of its functions by any system of universal and invariable cytoplasmic components. THE CONTINUITY OF CYTOPLASMIC GRANULES The failure to find evidence of universal cytoplasmic components by the use of either composition or function as criteria, brings the discus- sion to a much more general concept—the distinction between granules as permanent organelles and as temporary reserve granules. This dis- tinction can be traced back to Altmann’s bioblast theory, but it has been applied more recently in a modified and refined form to the mitochondria, CYTOPLASMIC INCLUSIONS 175 Golgi bodies, and the vacuome. The rejection of the universality of any one of these components still leaves the possibility that in each cell there are two sets of granules—the group which is permanent in organization, and a group of temporary granules, usually “passive’’ reserve bodies, which may be derived from the activities of the first. This is not a restatement of Altmann’s theory, in the sense that it implies that the first group are living units as such, nor even that these granules are regarded in any strict sense as independent, since their maintenance obviously depends upon their interaction with the other parts of the cell. The question is whether any set of granules are present during the whole life cycle, and further whether new granules of the same group arise directly from the old granules and never arise de novo. The morphological studies of Fauré-Fremiet (1910), Joyet-Lavergne (1926a), MacLennan (1934), Subramaniam and Ganapati (1938), and others have shown that mitochondria, Golgi bodies, glycogen granules, and so forth, in various Protozoa, undergo division in such a manner as to retain the original organization of the granules, and that these bodies are found in all stages of the life cycle. On the other hand, Horning (1929), Volkonsky (1929 on), MacLennan (1936), and Kedrowsky (1931 on) find that one or more of the supposedly fundamental com- ponents arise de novo either continuously or at some stage of the life cycle. A de novo origin has been proved by Mast and Doyle (1935b) not only for granules which are simple morphologically, but even for the complex tripartite refractive granules of A. proteus. In Ichthyophthirius apparently none of the granules are retained through the life cycle, thus clearly eliminating in this protozoan any distinction based on continuity. These morphological studies show that there may be a genetic continuity with respect to some granules in some of the Protozoa, but that it is not a general thing. The observation that in some Protozoa all of the granules arise de novo at some time or other, raises the question whether the observed divi- sions are significant or are merely incidental. Kedrowsky was able to in- duce typical division figures in the endosomes of O palina by altering the culture medium. Horning (1929) showed that dividing mitochondria are found in the trophozoite of Monocystis but that these granules dis- appear completely during the spore stages and form de novo in the newly liberated sporozoite. The digestive granules of Ichthyophthirius 176 CYTOPLASMIC INCLUSIONS divide inside the gastriole to form a larger number of small granules, and both the mitochondria and the intermediate lipoid bodies (endoplas- mic Golgi) fracture and split into rods as the granule within the sphere grows; yet in none of these cases is the division more than an incident in the cycle of the original granule, which arises in all cases de novo. The division of cytoplasmic granules is merely an indication that the granule is unstable under the particular conditions of size, surface tension, and so forth. The term “‘vacuolar reaction’? was introduced by Volkonsky to de- scribe the relationship between the formation of new digestive granules (his vacuome) and the presence of food. The pattern of the reaction depended both upon the species of cell and upon the type of food present. This formation of granules as a response to a specific stimulus is by no means restricted to the single case of the digestive granules. The forma- tion of secretion granules in the Tintinnidae is a specific response to the factors which require a new lorica, and these granules are present at no other time. The formation of the complex refractive bodies in Amoeba is a specific response of that particular protozodn to excess food; when this condition no longer holds, the granules are resorbed. The excre- tory granules of Ichthyophthirius are the response of this protozodn to the presence of metabolic wastes, which result from active feeding and growth and which disappear in encystment, when the original condition no longer holds good. The segregation bodies of O palina likewise exhibit changes which are specific responses to the particular food which is available. Horning (1929) points out that the disappearance and re- appearance of mitochondria in Monocysizs 1s correlated with the decrease and increase of metabolism resulting from encystment and excystment. Volkonsky’s vacuolar reaction is one case of general response, or “granu- lar reaction,” of the cell to a host of stimuli. If the stimulus is always present, the particular granules which are characteristic for the stimulus and for the particular cell under consideration are always present, but if the stimulus is intermittent, the particular granules involved are present only for the corresponding period. Continuity is of no significance in the evaluation of the granules, but is rather a criterion of the continuity of the stimulus which induces the formation of the granule. This, together with the demonstration that the division of the granules is purely incidental, shows that it ts not possi- CYTOPLASMIC INCLUSIONS U7 ble to distinguish between permanent organelles and temporary com- ponents, nor between active and passive granules. THE CLASSIFICATION OF CYTOPLASMIC GRANULES The cytoplasmic granules are a visible reaction of the cell to various stimuli, with the result that they show as great a variety as do the func- tions of which they are the visible expression and as the cells which form these granules. Any final classification must be based, then, on function, composition, and origin, rather than on a few nonspecific stains which give the impression of universal components, or on a distinction between permanent organelles and temporary reserves. Since function, composi- tion, and relationship vary widely from one cell to another, the cyto- plasmic granules, even of the Protozoa alone, cannot be divided into three or four sharply defined types, but must be separated into more types, with a classification sufficiently flexible to allow for the combina- tion of several functions in the same granule. Such an ideal classification may be defined briefly as functional. A functional classification is impossible at the present time, since the usual cytological or cytochemical methods reveal only those functions which result in the accumulation of visible masses of material—the segregating functions. The general type of material which is segregated has been identified in many cases, but usually there is insufficient evidence to determine whether this is a simple segregation process or whether there is actual synthesis involved. A functional classification on such a narrow base would lack permanent value, but it is necessary to readjust the present classification, in order to separate granules which are obvi- ously unlike, even on the relatively scanty evidence now available. This separation has been outlined in the previous sections with the detailed evidence, but it is worthwhile to assemble these suggested changes here in one place. The mitochondria are those granules which respond to mitochondrial methods, such as those employed by Regaud, Benda, and so forth, and which usually segregate Janus green. This is admittedly a heterogeneous group, but there is insufficient information at present to separate any groups on a logical basis. The term Golgi body is used to designate granules or structures which impregnate specifically with the classic reduction methods, but excepts 178 CYTOPLASMIC INCLUSIONS those bodies which are mitochondria, or which segregate neutral red vitally. The term as used here is thus merely a convenient way to indicate briefly certain techniques. Included in this group of granules are the fatty acid bodies, which are simply a stage in the formation of neutral fat granules and which have been called sntermediate lipoid bodies. The granules or membranes which are associated with the contractile vacuoles are a separate group in composition and function, and are called excre- tory granules. There is a third group which display a characteristic secre- tory cycle but which are neither lipoidal in composition nor excretory in nature (Ellis, 1937). For these bodies and other unknown granules, the term Golgi body is appropriate, since it merely designates them ac- cording to the techniques used and implies nothing as to their composi- tion or functions. The term neutral red granule refers to any body which segregates neutral red or similar basic dyes in the living normal protozo6n. Again, it is a term which indicates only the technique used and is a convenience when there is no evidence as to function. In this group are the segrega- tion granules, which accumulate and perhaps synthesize proteins and similar materials, as in Opalina. The refractive bodies of Amoeba may be included here because of the neutral red reaction and because of the protein stroma, or they could be listed in the lipoid reserves because of their high lipoid content. The d/gestive granules are a separate group and are associated with the gastriole. One of the problems in this con- nection is how these digestive granules differ in function and composi- tion from the mitochondria, which may also be associated with digestion. Many different types of granules, some of them with the power to segregate neutral red, are expelled from the cell in the formation of shells, cysts, cement, and so forth, and are named secretion granules. These granules should be given more attention, since they indicate a situation similar to the secretion granules of gland cells. The reserve bodies have been separated on the basis of the material stored—protein, lipoid, and carbohydrate—which also allows for the various combinations which do occur, and which will permit further subdivision when justified by an increase in the precision of cytochemical methods. This is convenient in summarizing the reserves, but for final classification it is unsatisfactory, since it ignores the differences in origin— whether they are independent bodies, as the segregation granules of O palina and the paraglycogen granules of Polyplastron, or whether they CYTOPLASMIC INCLUSIONS 179 are products of the macronucleus, mitochondria, and so forth. This is a difficulty which cannot be overcome until the fundamental processes which are involved have been worked out. In addition to these granules are a heterogeneous group of wnknown granules such as the alpha granules of Amoeba and the accessory bodies formed by the neuromotor system of Haptophrya (Fig. 70); various pigment granules, which may in some cases be part of the lipoid reserves, or in some cases residues of food, as in Plasmodium and Ichthyophthirius, this latter type of course not being true cytoplasmic granules. Crystals also are often present, the ones in Amoeba being classed as a part of the protein reserves (although here there is a question, since they orig- Figure 70. Accessory bodies being formed from the neuromotor ring in Haptophrya michiganensis, Zenker’s haematoxylin. (After Bush, 1934.) inate in the food vacuoles) on the evidence of Mast and Doyle. Other crystals, according to Reichenow, may be excretory granules. COMPARISON WITH CELLS OF THE METAZOA Cytological investigations in the Protozoa have always been influ- enced by the transfer of concepts originally developed from a study of the cells of the Metazoa, particularly the vertebrates, with the result that the division of granules into mitochondria, Golgi bodies, vacuome, and passive reserve bodies are as common in the literature of the Protozoa as in that of the Metazoa. In spite of Dobell’s denial of the cellular nature of the Protozoa, any consideration of the granules of the Protozoa necessitates a comparison with the cytoplasmic granules in other animal cells. The lack of a “‘typical’ reticular net of Golgi in the Protozoa, the infrequency of filamentous mitochondria, and other striking morpho- logical differences between the protozoan and metazoan cells have been stressed so often that it is well to present several cases of equally striking similarities in both structure and function. Volkonsky (1934) found 180 CYTOPLASMIC INCLUSIONS that the digestive granules and gastrioles of Protozoa, choanocytes, and leucocytes are entirely comparable, and included all of these cells in his vacuolar reaction. Fauré-Fremiet (1909) and later Kedrowsky (1932, 1933) compared the segregation granules of Protozoa and vertebrate tissues and found they were similar in appearance, staining reactions, and function. Chatton, Parat, and Lwow (1927), on the basis of specific microchemical reactions, compared the protein reserves in certain of the Foettingeriidae with the vitellin of hen’s eggs. Kedrowsky (1931) and MacLennan (1936) have given figures of the formation of neutral fat bodies in the Protozoa which are almost identical with the descrip- tion and figures of Bowen (1929) in the relationship between Golgi bodies and lipoid secretion in cells of the mouse. Examples could be multiplied, but these are sufficient to emphasize the fact that there are similarities as well as differences between protozoan and metazoan cells, with respect to their cytoplasmic granules. The same difficulties with respect to the so-called specific staining reactions arise in both Protozoa and Metazoa. In a study of echinoderm eggs, Tennent, Gardiner, and Smith (1931) showed that not one ma- terial, but many reduce osmium in the Golgi techniques. The presence of more than one type of osmiophilic granules has been proved by Mast and Doyle (1935) and MacLennan (1936) in Protozoa. Although Kirkman and Severinghaus (1938) hold to the idea of a particular Golgi substance, they bear witness to the occurrence of additional osmio- philic materials: “One often finds small osmiophilic granules of uncet- tain significance in Kolatchev sections, but they are present in addition to the Golgi apparatus and appear to bear no relation to the latter struc- ture.” In neither group is there any evidence that there is a particular Golgi substance, any more than a particular Golgi structure. There is also at times an embarrassing overlapping in the results ob- tained from the use of Janus green and neutral red. These dyes were found by Hopkins (1938) to stain the same vacuoles in the marine amoeba, Flabellula, and Uhlenhuth (1938) reports similar results in the thyroid cells of amphibia. The mitochondria turn out to be not a simple group but a complex group, as indicated by the distinction between mitochondria and active mitochondria, or mitochondria proper, by Parat (1927) and by Joyet-Lavergne (1926). Evidence is accumulating in both Protozoa and Metazoa that no type of cytoplasmic granule (with the the exception of the centriole) can be CYTOPLASMIC INCLUSIONS 181 considered as permanent, self-perpetuating structures. Wilson and Pol- lister (1937), in connection with an investigation on sperm formation in scorpions, review the division and distribution of mitochondra, Golgi bodies, and vacuome, and show that the supposed accurate division 1s actually an incidental fragmentation of large masses and that the distri- bution is random during the division of the cell. They state: ‘There 1s, however, little ground for the contention that either Golgi bodies or chondriosomes can be regarded as permanent individuals having the power of self perpetuation by growth and regular division.” Exactly the same situation has been shown in this discussion with respect to the cytoplasmic granules of the Protozoa, from studies of both fixed material and of living normal cells. The permanence of mitochondria in metazoan cells has been summed up recently by Bensley (1937): “The disappear- ance and reappearance of mitochondria in living cells under observation, as described by Chambers, however repugnant the idea may be to those who would elevate mitochondria to the dignity of living, self-reproduc- ing units, must be definitely entertained as probable.” With respect to the de novo origin of Golgi apparatus, Kirkman and Severinghaus (1938) assert that “there is little to favor such a view,” although they quote at least a dozen authors who have advanced evidence of a de novo origin in one form or another, admitting in several cases that the evidence presented has not been refuted. For a detailed discussion, the reader is referred to the review of the subject by these authors and to the original publications, but it is clear that not all cytologists agree with these authors on the permanence of the Golgi bodies. The concept that the cytoplasmic granules arise or are resorbed as a result of specific conditions of metabolism in the cell—in other words that there is a granular reaction—is a logical result of the evidence that the granules are neither permanent nor self-perpetuating, and it 1s therefore no surprise to find that this interpretation has been made with respect to the cytoplasmic constituents of the Metazoa as well as of the Protozoa. One of the clearest statements of this concept has been made by Tennent, Gardiner, and Smith (1931): ‘‘The results of this research have been the conviction that neither Golgi bodies nor Chondriosomes are structural elements in the cellular architecture, but that both are the chemical products of physiological processes.” Nahm (1933) like- wise states that “they are the visible products of chemical reactions that occur in the cell.” 182 CYTOPLASMIC INCLUSIONS LITERATURE CITED Beers, C. D. 1935. Structural changes during encystment and excystment in the ciliate Didininm nasutum. Arch. Protistenk., 84: 133-55. Bensley, R. R. 1937. On fat distribution in mitochondria of the guinea pig liver. Anat. Rec., 69: 341-54. Bles, E. J. 1929. Arcella, a study in cell physiology. Quart. J. micr. Sci. n. s., 72: 527-648. Borgert, A. 1909. Ueber erscheinungen fettiger Degeneration bei tripyleen Radiolarien. Arch, Protistenk., 16: 1-25. Bourne, G. 1936. The role of vitamin C in the organism as suggested by its cytology. Physiol. Rev., 16: 442-49. Bourne, G., and R. Allen. 1935. The distribution of vitamin C in lower or- ganisms. Aust. J. exp. Biol. med. Sci., 13: 165-74. Bowen, R. H. 1928. The methods for the demonstration of the Golgi ap- paratus. VI. Protozoa. The vacuome. Plant tissues. Aust. J. exp. Biol. med. Sci., 40: 225-76. 1929. The cytology of glandular secretion. Quart. Rev. Biol., 4: 484-519. Brand, T. von. 1935. Der Stoffwechsel der Protozoen. Ergebn. Biol., 12: 161- 220. — 1938. The metabolism of pathogenic trypanosomes and the carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13: 41-50. Brown, V. E. 1930. The Golgi apparatus of Pyrsonympha and Dinenympha. Arch, Protistenk., 71: 453-62. Bush, M. 1934. The morphology of Haptophrya michiganensis Woodhead. Univ. Cal. Publ. Zool., 39: 251-76. Bitschli, O. 1885. Bemerkungen ueber einen dem glikogen verwandten Koerper in Gregarinen. Z. Biol., 21: 603-12. Cajal, Ramon y, Santiago. 1904. Variaciones morfologicas del reticulo nervioso de invertebrados y vertebrados sometidos 4 la accién de condiciones naturales. Trab. Lab. Invest. biol. Univ. Madr., 1: 3. Calkins, G. N. 1930. Uroleptus halseyi n. sp. I. The origin and fate of the macronuclear chromatin. Arch. Protistenk., 69: 151-74. Campbell, A. S. 1926. The cytology of Tintinnopsis nucula (Fol) Laakman. Univ. Cal. Publ. Zool., 29: 179-236. 1927. Studies on the marine ciliate Favella, with. special regard to the neuromotor apparatus and its rdle in the formation of the lorica. Univ. Cal. Publ. Zool., 29: 429-52. Chakravarty, M. 1936. On the morphology of Balantidium depressum Ghosh from a mollusc Pila globosa, with a note on its nucleal’ reaction and cytoplasmic inclusions. Arch. Protistenk., 87: 1-9. Chalkley, H. W. 1937. The chemistry of cell division VII. Protoplasma, 28: 489-97. Chambers, R., and R. T. Kempton. 1937. The elimination of neutral red by the frog’s kidney. J. cell. comp. Physiol., 10: 199-221. CYTOPLASMIC INCLUSIONS 183 Chambers, R., and H. Pollack. 1927. Micrurgical studies in cell physiology. IV. Colorimetric determination of the nuclear and cytoplasmic pH in the starfish egg. J. gen. Physiol., 10: 739-55. Chatton, E., M. Parat, and A. Lwow. 1927. La formation, la nature et |’evolu- tion des réserves chez les Spirophrya, les Polyspira, et les Gymnodinioides (Infusoires Foettingeriidae). C. R. Soc. Biol. Paris, 96: 6-8. Chatton, E., and P. P. Grassé. 1929. Le chondriome, le vacuome, les vésicules osmiophiles, le parabasal, les trichocystes, et les chidocystes du Dinoflagellé Polykrikos Schwartzi Biitschli. C. R. Soc. Biol. Paris, 100: 281-85. ; Cheissin, E. 1930. Morphologische und systematische Studien tiber Astomata aus dem Baikalsee. Arch. Protistenk., 70: 531-618. Cleveland, L. R. 1934. The wood-feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: 185-331. Connell, F. H. 1930. The morphology and life cycle of Oxymonas dimor pha sp. nov. from Neotermes sim plicicornis (Banks) . Univ. Cal. Publ. Zool., 36: 51-66. Cowdry, E. V. 1924. General cytology. Chicago. Cowdry, E. V., and G. H. Scott. 1928. Etudes cytologiques sur le paludisme. III. Mitochondries, granules colorable au rouge neutre et appareil de Golgi. Arch. Inst. Pasteur Afr. N., 17: 233-52. Cutler, M. 1921. Observation on the Protozoa parasitic in Archotermopsis wroughtoni Desn. Part III. Pseudotrichonympha pristina. Quart. J. micr. Sci., 65: 248-64. Dangéard, P. 1928. L’Appareil mucifére et le vacuome chez les Eugléniens. Ann. Protist., 1: 69-74. Daniels, M. L. 1938. A cytological study of the gregarine parasites of Tenebrio molitor, using the ultra-centrifuge. Quart. J. micr. Sci., 80: 293-320. Day, H. C. 1927. The formation of contractile vacuoles in Amoeba proteus. J. Morph. 44: 363-72, 6 figs. Dobell, C. C. 1925. The life history and chromosome cycle of Aggregata eberthi, Parasitology, 17: 1-136. Doflein, F., and E. Reichenow. 1929. Doflein, Lehrbuch der Protozoenkunde. Sth ed., Jena. Dogiel, V., and T. Fedorowa. 1925. Uber den Bau und die Funktion des inneren Skeletts der Ophryoscoleciden. Zool. Anz., 62: 97-107. Doyle, W. L. 1935. Distribution of mitochondria in the foraminiferan, Iridia diaphana, Science, n.s., 81: 387. Duboscq, O., and P. Grassé. 1925. Notes sur les protistes parasites des Termites de France. Appareil de Golgi, mitochondries, et vésicules sous- flagellaires de Pyrsonympha vertens Leidy. C. R. Soc. Biol. Paris, 93: 345-48. —— 1933. L’Appareil parabasal des flagellés, avec remarques sur le tropho- 184 CYTOPLASMIC INCLUSIONS sponge, l'appareil Golgi, les mitochondries, et le vacuome. Arch. zool. exp. gén., 732 381-6217. Dunihue, F. W. 1931. The vacuome and the neutral red reaction in Para- moecium caudatum, Arch. Protistenk., 75: 476-97. Eksemplarskaja, E. V. 1931. Morphologie und Cytologie von Anoplophrya sp. aus dem Regenwurmdarm. Arch. Protistenk., 73: 147-63. Ellis, J. M. 1937. The morphology, division and conjugation of the salt marsh ciliate Fabrea salina Henneguy. Univ. Cal. Publ. Zool., 41: 343-88. Erdmann, R. 1917. Chloromyxum leydigi und seine Bezeichnung zu anderen myxosporidien. Arch. Protistenk., 37: 276-326. Fauré-Fremiet, E. 1905. Sur l’organisation de la Campanella umbellaria. C. R. Sec. Biol. Patiss 582 205-1. —— 1909. Vacuoles colorables par le rouge neutre chez un infusoire. C. R. Ass. Anat., 11: 286-88. —— 1910. Etude sur les mitochondries des protozoaires et des cellules sexuelles. Arch. Anat. micr., 11: 457-648. — 1925. La Structure permanente de l'appareil excréteur chez quelques vorticellides. C. R. Soc. Biol. Paris, 93: 500-3. —— 1932. Division et morphogenése chez Folliculina ampulla OFM Bull. biol., 66: 79-109. Fauré-Fremiet, E., A. Mayer, and G. Schaeffer. 1910. Sur la microchimie des corps gras. Arch. Anat. micr., 12: 19-102. Finley, H. E. 1934. On the vacuome in three species of Vorticella. Trans. Amer. micr. Soc., 53: 57-64. Fiveiskaja, A. 1929. Einfluss der Kernparasiten der Infusorien auf den Stoff- wechsel. Arch. Protistenk., 65: 275-98. Frisch, A. 1938. The rate of pulsation of the contractile vacuole in Paramecium multimicronucleatum. Arch. Protistenk., 90: 123-61. Gatenby, J. B. 1930. Cell nomenclature. J. R. micr. Soc., 50: 20-29. Gelei, J. von. 1928. Nochmals ber den Nephridialapparat bei den Protozoen. Arch. Protistenk., 64: 479-94. — 1938. Das Exkretionsplasma von Déidinium nasutum in Ruhe und Teilung. Arch. Protistenk., 90: 368-82. Hall, R. P. 1929. Modifications of technique for demonstration of Golgi ap- paratus in free-living Protozoa. Trans. Amer. micr. Soc., 48: 443-44. —— 1931. Vacuome and Golgi apparatus in the ciliate, Stylonychia. Z. Zellfotsch., 13: 770-82. Hall, R. P., and F. W. Dunthue. 1931. On the vacuome and food vacuoles in V orticella. Trans. Amer. micr. Soc., 50: 196-205. Hall, R. P., and J. B. Loefer. 1930. Studies on Exglypha. I. Cytoplasmic inclusions of Exglypha alveolata. Arch. Protistenk., 72: 365-76. Hall, R. P., and R. F. Nigrelli. 1930. Relation between mitochondria and food vacuoles in the ciliate, Vorticella, Trans. Amer. micr. Soc., 49: 54- aya CYTOPLASMIC INCLUSIONS 185 — 1937. A note on the vacuome of Paramecium bursaria and the contractile vacuole of certain ciliates. Trans. Amer. micr. Soc., 56: 185-90. Haye, Ans. 1930. Uber den Exkretionsapparat bei den Protozoen, nebst Bemerkungen uber einige andere feinere Strukturverhaltnisse der unter- suchten Arten. Arch. Protistenk., 70: 1-87. Hayes, M. L. 1938. Cytological studies on Dileptus anser. Trans. Amer. micr. S0G315 75 11225: Heilbrunn, L. V. 1928. The colloid chemistry of protoplasm. Berlin. —— 1936. Protein lipid binding in protoplasm. Biol. Bull., 71: 299-305. Heilbrunn, L. V., and K. Daugherty. 1938. Fat release in Amoeba after itra- diation. Physiol. Zodl., 11: 383-87. Hertwig, R. 1902. Die Protozoen und die Zelltheorie. Arch. Protistenk., 1: 1-40. Hindle, E. 1910. Degeneration phenomena of Trypanosoma gambiense. Parasitology, 3: 423. Hirschler, J. 1914. Uber Plasmastrukturen (Golgi’scher Apparat, Mito- chondrien, u.a.) in den Tunicaten-, Spongien, und Protozoen-zellen. Anat. Anz., 47: 289-311. —— 1924. Sur les composants lipoidiféres du plasma des Protozoaires. C. R. Soc. Biol. Paris, 10: 891-93. — 1927. Studien uber die sich mit Osmium schwarzenden Plasmakompo- nenten (Golgi-apparat, Mitochondrien) einiger protozoenarten, nebst Bemerkungen uber die Morphologie der ersten von ihnen im Tierreiche. Z. Zellforsch., 5: 704-86. Holmes, E. 1937. The metabolism of living tissues. Cambridge. Holter, H., and W. L. Doyle. 1938. Uber die lokalisation der Amylase in der Amében. C. R. Lab. Carlsberg (Sér. Chim.) , 22: 219-25. Holter, H., and M. J. Kopac. 1937. Studies in enzymatic histochemistry. XXIV. Localization of peptidase in the Amoeba. J. cell. comp. Physiol., 10: 423-37. Hopkins, D. L. 1938a. The mechanism for the control of the intake and out- put of water by the vacuoles in the marine amoeba, Flabellula mira Schaeffer. Biol. Bull., 75: 353. —— 1938b. The vacuoles and vacuolar activity in the marine amoeba Flabellula mira Schaeffer and the nature of the neutral red system in Protozoa. Biodyn., No. 34, 22 pp. Horning, E. S. 1925. The mitochondria of a protozo6n (Opalina) and their behavior during the life cycle. Aust. J. exp. Biol. med. Sci., 2: —— 1926. Studies on the mitochondria of Paramoecium. Aust. J. exp. Biol. med. Sci., 3: 91-94. —— 1927. On the orientation of mitochondria in the surface cytoplasm of infusorians. Aust. J. exp. Biol. med. Sci., 4: 187-90. —— 1929. Mitochondrial behavior during the life cycle of a sporozoén (Monocystis) Quart. J. micr. Sci., 73: 135-43. 186 CYTOPLASMIC INCLUSIONS Horning, E. S., and G. H. Scott. 1933. Comparative cytochemical studies by micro-incineration of a saprozoic and an holozoic infusorian. J. Morph., 54: 389-94. Jirovec, O. 1926. Protozoenstudien I. Arch. Protistenk., 56: 280-90. Joyet-Lavergne, P. 1926a. Recherches sur les cytoplasmes des Sporozoaires. Arch. Anat. micr., 22: 1-128. — 1926b. Sur la coloration vitale des elements de Golgi des gregarines. C. R. Soc. Biol. Paris, 94: 830-32. —— 1927. Sur les rapports entre le glutathion et le chondriome. C. R. Acad. SciParis; 1s4ato srs —— 1928. Le Pouvoir oxydo-reducteur du chondriome des Gregarines et les procédés de recherches du chondriome. C. R. Soc. Biol. Paris, 98: 501. —— 1929. Glutathion et chondriome. Protoplasma, 6: 84-112. —— 1931. Le Potentiel d’oxydo-reduction et la sexualisation cytoplasmique des Gregarines. C. R. Soc. Biol. Paris, 107: 951-52. —— 1932a. Sur le pouvoir oxydant du chondriome dans la cellule vivante. GOR Soe Biol’ Paris; 11107552255: — 1932b. Sur la mise en evidence des zones d’oxydation dans la cellule animale. C. R. Soc. Biol. Paris, 110: 663-64. — 1934. Une Théorie nouvelle sur le mécanisme des oxydo-réductions intracellulaires. C. R. Acad. Sci. Paris, 199: 1159. 1935. Réchérches sur la catalyse des oxydo-reductions dans la cellule vivante. Protoplasma, 23: 50-69. Kazancev, V. 1928. Beitrag zur Kenntnis der Grosskerne der Ciltaten. Trav. Lab. zool. Sebastopol, Ser. II, 13: 1-30. Kedrowsky, B. 1931a. Die Stoffaufnahme bei Opalina ranarum. I. Methodik der Kultur in kiinstlichen medien, pH regulieren und Ionen Gleich- gewichte im Kulturmedien. Protoplasma, 12: 356-79. —— 1931b. II. Struktur. Wasseraufnahme und Wasserzustand in Proto- plasma von Opalina. Protoplasma, 14: 192-255. — 1931c, HI. Aufnahme und Speicherung von Farbstoffen. Z. Zellforsch., 12: 600-65. —— 193ld. IV. Die synthetische Fettspeicherung. Z. Zellforsch., 12: 666- 714. —— 193l1e. V. Der Segregationsapparat. Z. Zellforsch., 13: 1-81. —— 1932a. Vitalfarbungsstudien an Infusorien, Z. Zellforsch., 15: 93-113. — 1932b. Uber die Natur des Vakuoms. Z. Zellforsch., 15: 731-60. —— 1933. Neue Probleme im Studium des Etweissstoffwechsels der Zelle. Arch. exp. Zellforsch., 14: 533-54. Kirby, H. 1931. Trichomonad flagellates from termites. II. Eutrichomastix and the subfamily Trichomonadinae. Univ. Cal. Publ. Zool., 36: 171-262. — 1932. Flagellates of the genus Trichonympha in termites. Univ. Cal. Publ. Zool., 37: 349-76. CYTOPLASMIC INCLUSIONS 187 —— 1936. Two polymastigote flagellates of the genera Pseudodevescovina and Caduceia. Quart. J. micr. Sci., n.s., 79: 309-35. Kirkman, H., and A. E. Severinghaus. 1938. A review of the Golgi apparatus. Anat. Rec., 70: 413-32, 557-74; 71: 79-104. Koehring, V. 1930. The neutral red reaction. J. Morph., 49: 45-130. Kofoid, C. A., and M. Bush. 1936. The life cycle of Parachaenia myae, gen. nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Fran- cisco Bay, Calif. Bull. Mus. Hist. nat. Belg., 12: 1-15. Kraschenninikow, S. 1929. Uber den Exkretionsapparat einiger Infusorien- atten der Familie Ophryoscolecidae. Z. Zellforsch., 8: 470-83. Krijgsman, B. J. 1936. Verleichend physiologische Untersuchungen tiber den stoffwechsel von Trypanosoma evansi in Zusammenhang mit der Anpas- sung an das Wirtstier. Z. vergl. Physiol., 23: 663-711. LeBreton, E. 1931. Mitochondries et ferments protéolytiques. Examen de ’hypothése de Robertson-Marston. Arch. Biol. Paris, 42: 349-63. Leiner, M. 1924. Das Glycogen in Pelomyxa palustris Greef mit Beitragen zur kenntnis des Tieres. Arch. Protistenk., 47: 253-307. Lison, L. 1936. Histochimie Animale. Paris. Lynch, J. E. 1930. Studies on the ciliates from the intestine of Strongylocen- trotus. Il. Lechriopyla mystax, gen. nov., sp. nov. Univ. Cal. Publ. Zool., J. 907-50: MacLennan, R. F. 1933. The pulsatory cycle of the contractile vacuoles in the Ophryoscolecidae, ciliates from the stomach of cattle. Univ. Cal. Publ. Zool., 39: 205-50. — 1934. The morphology of the glycogen reserves in Polyplastron. Arch. Protistenk., 81: 412-19. — 1936. Dedifferentiation and redifferentiation in Ichthyophthirius. Il. The origin and function of cytoplasmic granules. Arch. Protistenk., 86: 404-26, —— 1937. Growth in the ciliate Ichthyophthirius. 1. Maturity and encystment. J. exp. Zool., 76: 423-40. —— 1940. A quantitative study of the osmic acid reaction in Protozoa. Trans. Amer. micr. Soc., 59: 149-59. MacLennan, R. F., and H. K. Murer. 1934. Localization of mineral ash in the organelles and cytoplasmic components of Paramecium. J. Morph., 55: 421-33. Marston, H. R. 1923. The azine and azonium compounds of the proteolytic enzymes. Bio-chem. J., 17: 850-59. Mast, S. O. 1938. Digestion of fat in Amoeba proteus. Biol. Bull., 75: 389-94. Mast, S. O., and W. L. Doyle. 1935a. Structure, origin, and function of cytoplasmic constituents in Amoeba proteus. I. Structure. Arch. Pro- .tistenk, 86: 155-80. —— 1945b. Structure, origin, and function of cytoplasmic constituents in 188 CYTOPLASMIC INCLUSIONS Amoeba proteus with special reference to mitochondria and Golgi sub- stance. II. Origin and function based on experimental evidence; effect of centrifuging on Amoeba proteus. Arch. Protistenk., 86: 278-306. Metcalf, M. M. 1910. Studies upon Amoeba, J. Exp. Zool., 9: 301-32. Meyers, E. H. 1935. The life history of Patellina corrugata Williamson, a foraminifer. Bull. Scripps Instn. Oceanogr. tech., Ser. 3: 355-92. Miller, E. D. 1937. A comparative study of the contents of the gelatinous accumulations of the culture media and the contents of the cytoplasm of Amoeba proteus and Arcella vulgaris. J. Morph., 60: 325-54. Milovidov, P. F. 1932. Independence of chondriosomes from nuclear matter. Cytologia, 4: 158-73. Nahm, L. J. 1933. A study of the Golgi elements. J. Morph., 54: 259-301. Nassonov, D. 1924. Der Exkretionsapparat (kontraktile Vakuole) der Pro- tozoa als Homologen des Golgischen Apparats der Metazonzellen. Arch. Mikr. Anat., 103: 437-82. —— 1925. Zur Frage tber den Bau und die Bedeutung des lipoiden Excre- tionsapparates bei Protozoa. (Chilodon, Dogielella). Z. Zellforsch., 2: 87-97. Nigrelli, R. F. 1929. On the morphology and life history of Trypanosoma diemyctyli and the relation of trypanosomiasis to the polynuclear count. Trans. Amer. micr. Soc., 48: 366-87. Owens, H. B., and R. R. Bensley. 1929. On osmic acid as a microchemical reagent, with special reference to the reticular cipipeleans of Golgi. Amer. J. Anat., 44: 79-110. Parat, M. 1927. A review of recent developments in histochemistry. Biol. Rev., Za 3 5 -OY Patten, R. 1932. Observations on the cytology of O palina ranarum and Nycto- therus cordiformis. Proc. R. Irish Acad., 41: 73-94. Pellissier, M. 1936. Sur certains constituants cytoplasmiques de I’infusoire cili€, Trichodinopsis paradoxa Clap. et Lach. Arch. zool. exp. gén, 78: 32-36. Peshkowskaya, L. 1928. On the biology and the morphology of Climacosto- mum virens. Arch, russ. protist., 7: 205-35. Poljansky, G. 1934. Geschlechtsprozesse bei Bursaria truncatella OFM. Arch. Protistenk., 81: 420-546. Rammelmeyer, H. 1925. Zur Frage ueber die Glykogendifferenzierung bei Paramecium caudatum. Arch. Protistenk., 51: 184-88. Reichenow, E. 1928. Ergebnisse mit der nuclealfaerbung bei Protozoen. Arch. Protistenk., 61: 144-66. 1929. Doflein, Lehrbuch der Protozoenkunde. Sth ed., Jena. Rey, P. 1931a. Potentiel d’oxydo-reduction et sexualité chez les Gregarines. GARY Soe BioleParis: M07 Glee —— 1931b. Coloration vitale et potentiel d’oxydo-reduction chez les gre- garines. C. R. Soc. Biol. Paris, 107: 1508-11. CYTOPLASMIC INCLUSIONS 189 Rosenberg, L. E. 1937. The neuromotor system of Nyctotherus hylae. Univ. Cal. Publ. Zool., 41: 249-76. Rumjantzew, A. 1922. Uber den Bau der Chromidial substanz bei Dzfflugia pyriformis. Arch. russ. protist., 1: 87-105. Rumjantzew, A., and E. Wermel. 1925. Untersuchungen ueber den Proto- plasmabau von Actinosphaerium Eichhorni. Arch. Protistenk., 52: 217- 64. Sassuchin, D. 1924. Zur Kenntnis der Plasmaeinschliisse bei den Opalinen. Arch. russ. protist. 3: 147-54. Schrich, P. 1914. Beitrige zur Kenntnis des Lebenscyclus.von Arcella vulgaris und Pelomyxa palustris. Arch. Protistenk., 33: 247-71. Schulze, P. 1922. Uber Beziehungen zwischen tierischen und pflanzlichen Skelettsubstanzen und tiber Chitinreaktionen. Biol. Zbl., 42: 389-94. —— 1924. Der Nachweis und die Verbreitung des Chitins mit einem Anhang iiber das komplizierte Verdauungssystem der Ophryoscoleciden. Z. Morph. Okol. Tiere, 2: 643-66. — 1927. Noch einmal die ‘‘Skelettplatten’”’ der Ophryoscoleciden. Z. Morph. Okol. Tiere, 7: 670-89. Scott, G. H., and E. S. Horning. 1932. The structure of Opalinids as revealed by the technique of microincineration. J. Morph., 53: 381-88. Strelkow, A. 1929. Morphologische Studien uber Oligotriche Infusorien aus dem Darme des Pferdes. Arch. Protistenk., 68: 503-54. — 1931. II. Cytologische Untersuchungen der Gattung Cycloposthium Bundle. Arch. Protistenk., 75: 191-220. Subramaniam, M. K., and P. N. Ganapati. 1938. Studies on the structure of the Golgi apparatus. I. Cytoplasmic inclusions in the gregarine Lecudina brasili n.sp. parasitic in the gut of Lwmbriconereis. Cytologia, 9: 1-16. Subramaniam, M. K., and R. Gopala Aiyar. 1937. The Golgi apparatus and the vacuome in protozoa—some misconceptions and the question of terminology. Proc. Indian Acad. Sci., Sec. B., 6: 1-18. Swellengrebel, N. H. 1908. La Volutine chez les trypanosomes. C. R. Soc. Biol. Paris, 64: 38-40. Taylor, C. V. 1923. The contractile vacuole in Evplotes an example of the sol-gel reversibility of cytoplasm. J. exp. Zool., 37: 259-82. Tennent, D. H., M. S. Gardiner, and D. E. Smith, 1931. A cytological and biochemical study of the ovaries of the sea-urchin Echinometra lucunter. Pap. Tortugas Lab., 27: 1-46. Thélohan, P. 1894. Nouvelles réchérches sur les coccidies. Arch. zool. exp. gén., 3: 541-73. Tuzet, O. 1931. Une Grégarine parasite de Bythinia tentaculata. Gonospora duboscqui nov. sp. Arch. zool. exp. gén., 71: 16-21. Uhlenhuth, E. 1938. A quantitative approach to the secretion process of the thyroid. Coll. Net, 13: 76-87. 190 CYTOPLASMIC INCLUSIONS Van Thiel, P. H. 1925. Was ist Rickettsia melophagi? Arch. Protistenk., 52: 394-403, Volkonsky, M. 1929. Les phénoménes cytologiques au cours de la digestion intracellulaire de quelques ciliés. C. R. Soc. Biol. Paris, 101: 133-35. —— 1933. Digestion intracelluaire et accumulation des colorants acides. Etude cytologique des cellules sanguines des Sipunculides. Bull. Biol., 67: 135-286. —— 1934, L’Aspect cytologique de la digestion intracellulaire. Arch. exp. Zellforsch., 15: 355-72. Weatherby, J. H. 1927. The function of the contractile vacuole in Paramecium caudatum; with special reference to the excretion of nitrogenous com- pounds. Biol. Bull., 52: 208-22. —— 1929. Excretion of nitrogenous substances in the Protozoa. Physiol. Zobl., 2: 375-94. Weier, T. E. 1933. A critique of the vacuome hypothesis. Protoplasma, 19: 589-601. Weineck, I. 1931. Die chemische Natur der Skelettsubstanzen bei den Oph- ryoscolec iden. Jena. Z. Natur., 65: 739-51. —— 1934. Die Celluloseverdauung bei den Ciliaten des Wiederkduermagens. Arch. Protistenk., 82: 169-202. Wermel, E. 1925. Beitrage zur Cytologie der Amoeba hydroxena Entz. Arch. russ. protist., 4: 95-120. Wilson, E. B. 1928. The Cell. 3d ed., New York. Wilson, E. B., and A. W. Pollister. 1937. Observations on sperm formation in the centrurid scorpions with especial reference to the Golgi material. J. Morph., 60: 407-44. Wurmser, M. 1932. Sur l'emploi de certain colorants pour l’évaluation des proprietés oxydantes du cytoplasme. C. R. Soc. Biol. Paris, 111: 690. Yamasaki, M. 1937a. Studies on the intestinal Protozoa of termites. III. The distribution of glycogen in the bodies of intestinal flagellates of Termites Leucotermes and Coptotermes. Mem. Coll. Sci. Kyoto, B 12: 212-24. —— 1937b. IV. Glycogen in the body of Trichonympha agilis v. japonica under experimental conditions. Mem. Coll. Sct. Kyoto, 12: 225-35. Zhinkin, L. 1930. Zur frage der Reservestoffe bei Infusorien (Fett und Gly- kogen bei Stentor polymorphus). Z. Morph. Okol. Tiere, 18: 217-48. Zinger, J. A. 1928. Morphologische Beobachtungen an Ophryoglena flava Ehr. Arch. russ. protist., 7: 179-204. —— 1929. Beitriige zur Morphologie und Cytologie der Siisswasserinfusorien. Arch, russ. protist., 8: 51-90. ——— 1933. Beobachtungen an Fetteinschliissen bei einigen Protozoen. Arch. Protistenk., 81: 57-87. Zweibaum, I. 1921. Ricerche sperimentali sulla conjugazione degli Infusori. II. Influenza della coningazione sulla produzione dei materiali di riserva nel Paramecium caudatum. Arch. Protistenk., 44: 99-114. CHAPTER IV FIBRILLAR SYSTEMS IN CILIATES Gir TAMIOR INTRODUCTION THE ESSENTIAL NATURE of Leeuwenhoek’s “‘little animals’ remained obscure for more than 150 years, evidently because the methods of ob- servation which characterized that ingenious microscopist of Delft were replaced largely by fruitless speculation. Otherwise, man’s epochal dis- covery of the cellular nature of living things might have been realized sooner. Meantime, it is true, a prodigious diversity of macroscopic forms had been examined and classified. But the disclosure of cellularity, which eventually unified all of this diversity in organic form, had to depend upon the detailed analysis of organic structure. During the hundred years that have now intervened, that common denominator of organic form and function has come to be regarded, for multicellular plants and animals, as a sort of master key to the solution of their fundamental problems. And for the major advances in biology during that memorable century, we are surely indebted primarily to this cellular concept of the organization of living things. For the microdrganisms, however, the concept of cellularity, although generally conceded, has encountered not infrequently some confusing difficulties. With von Siebold’s pronouncement in 1845 of the unicellu- larity of the Protozoa, the way at first seemed clear toward simplifying and unifying all forms of life, in terms of the cell as the universal unit. Eventually, however, it was evident that, for the Protozoa, this concept did not simplify matters so satisfactorily. The chief difficulty here arose in trying to equate the protozoon cell with a tissue cell of the Metazoa. And even in recent times this comparison has again been challenged by Dobell (1911) and others, who would maintain that Protozoa are not cells at all and so should be regarded as non-celluar organisms. 192 CILIATE FIBRILLAR SYSTEMS But similar difficulties in comparing microscopic with macroscopic forms of life had confronted investigators several years before von Sie- bold’s pronouncement and, in fact, before the concept of cellularity had been definitely formulated. As is well known, this all culminated in the Ehrenberg-Dujardin controversy, beginning in 1835. Obviously for these investigations, the issue was not one of cellularity, but it had to do with complexity versus simplicity in the organization of the Infusoria. It seems probable that Ehrenberg defended his thesis of “complete or- ganisms’’ partly in refutation of the theory of spontaneous generation, then vigorously championed for microorganisms. At any rate, he sought to identify in the Infusoria all the organs common to other animals. Much of his adduced evidence, it will be recalled, was successfully re- futed by Dujardin, who described among other things, his newly dis- covered “‘sarcode”’ in support of his contentions for uniqueness and sim- plicity in the organization of the Infusoria. The essentials of these contrasting views of Ehrenberg and Dujardin on the nature of infusorian organization have recurred, in varied guise, many times in the literature since their day. These opposing viewpoints have, of course, become translated into terms of the concept of cellu- larity, so that now the nature of unicellular organization, or “‘proto- plasmic differentiation,” is commonly contrasted with ‘“‘cellular differ- entiation” of multicellular organisms. Accordingly, in the following review of literature on fibrillar systems in ciliates, it will become evident that some discrepancies in both the analysis of structure and the interpretation of functions may owe their origin largely to contrasting points of view on the essential nature of “protoplasmic differentiation” in the Protozoa and ‘‘cellular differentia- tion” in the Metazoa. Before beginning that review, however, the fact should be emphasized that, as Maupas (1883) has pointed out, the Ehrenberg-Dujardin con-. troversy marks a turning point in protistological investigations. Not only did it enlist a wider interest in these microdrganisms, but it made clear the necessity of a critical structural analysis of their greatly diversified types of organization and of a comparative study of such types before any satisfactory interpretations were possible. The literature resuiting from those analyses is so voluminous that when one undertakes to review the accounts of a given system of organ- CILIATE FIBRILLAR SYSTEMS 193 elles, such as the fibrillar system, and to condense that review within reasonable bounds, the difficulties soon become evident. For this reason it has seemed advisable in the review that follows, in the interests of students and laity as well as of specialists, to present a fairly detailed account of the structural analysis, together with interpretations of the fibrillar differentiations of a well-known representative of each of four major groups of ciliates. This is followed by a brief review of other published work, mostly since 1920, on fibrillar systems in other ciliates, with some suitable illustrations; and finally, a few paragraphs of general discussion are added under the caption “Conclusions.” The discussion of the structural analysis of the fibrillar systems of the four representative ciliates, Paramecium, Stentor, Euplotes and Vorti- cella, is offered first and separate from the interpretations for these four ciliates, whose order is then, for convenience, reversed. This separate treatment was decided upon primarily for the sake of accuracy and clarity. Often in the literature the author’s interpretations are so intermixed with his factual descriptions that it is sometimes very difficult to make certain just what he observed and undertook to describe. EXAMPLES OF FIBRILLAR SYSTEMS A. STRUCTURAL ANALYSIS 1. Paramecium.—vThis familiar representative of the holotrichs has doubtless been more generally used in both teaching and research labora- tories than has any other of the numerous kinds of ciliates. Probably its apparent simplicity, more than its smaller size, tended to discourage a search for a fibrillar system, such as had been found in Stentor and other forms. In 1905, however, Schuberg described for both Paramecium and Frontonia fibrillar differentiations which, running close under the pel- licle, united the basal granules in the longitudinal rows of cilia. By means of a bichromate-osmic fixative and Loefflet’s stain, not only was this relationship of fibril and basal granules clearly defined, but also, because of their staining properties, they could be well differentiated from the hexagonal, or rhomboidal, pattern of the pellicle, as was well illustrated in Schuberg’s several figures. In 1925, J. von Gelei described in Paramecium nephridiatum a periph- eral network of fibrils which was not connected with the familar po- 194 CILIATE FIBRILLAR SYSTEMS lygonal pattern observable in the living organism. For fixation, he used Apathy’s sublimate-osmium and stained with toluidin blue. In the following year Klein (1926a) reported the results of his studies on a peripheral fibrillar complex in certain ciliates by means of a new silver-nitrate technique which involved no previous fixation. The method, thus employed, is now well known as the “dry method,” in contrast to von Gelei’s (1932a) “‘wet method,” and the resulting silver-impregnated fibrillar complex is quite commonly referred to as the “silverline system.” The several subsequent publications of these two authors on the fibril- lar system of Paramecium, using especially the silver-nitrate technique but also other methods, admit of useful comparisons for this brief re- view, so that their results will now be considered together. In most of these various articles, the author’s account of the structures that were clearly observed is at times so involved with his avowed inter- pretations of their functions that it has been found difficult to sift out the essential data for which this review is intended. Their structural analyses of the fibrillar system of Paramecium have, nevertheless, several important points in common which may now be fairly, and as simply as possible, presented. On the basis of these com- mon points, certain discrepancies will then be indicated. To this end, it will be convenient to recall the findings of Schuberg (1905). He observed (1) a differentially stainable pellicular pattern, which was hexagonal over the body and rhomboidal on each side of the mouth, and below this (2) a longitudinal fibril connecting (3) the basal granules in each row of cilia—each such granule appearing below the center of each pellicular polygon (Fig. 71). In outline, the descriptions of both von Gelei and Klein present this same general picture, which may now serve to simplify a brief com- parison of their essential findings. In their later papers, both of these authors agree that the fibrillar system of Paramecium is entirely sub pel- licular. Bearing this in mind, we may note that: 1. Schuberg’s pellicular pattern corresponds im general outline to von Gelei’s “‘Stiitzgitter System’ and to Klein’s ‘“Indirekt verbindung System” (““Meridiaan II. Ordnung’’). 2. Both the Stiitzgitter System and the Indirekt verbindung System, lying under the pellicle, comprise each: (1) a longitudinal fibril, between CILIATE FIBRILLAR SYSTEMS 195 the rows of cilia; (2) cross fibrils, connecting adjacent longitudinal fibrils; (3) opening for a trichocyst midway on the cross fibril; and (4) a suture line at the anterior and the posterior poles. The resulting lattice, therefore, has its counterpart in Schuberg’s pattern. 3. Beneath von Gelei’s Stiitzgitter System lies his “Neuronem Sys- tem.” 4, Beneath Klein’s Indirekt verbindung System lies his ‘“Direkt ver- bindung System.” 5. Both Neuronem System and Direkt verbindung System comprise Lfio: ms i bgr. -—--4 Nye ges AL q ie 4 za Figure 71. Pellicular Pattern and Longitudinal Fibrils Connecting Basal Granules in Paramecium. (Modified from Schuberg, 1905.) b. gr.—basal granule 1. fib—tlongitudinal fibril pell. p.—pellicular pattern each: (1) an /nterciliary fibril, connecting the ‘“Basalapparaten” in each longitudinal row of cilia; (2) cross fibrils, connecting the interciliary fibrils; and (3) Relationskérner, which include the Basalap parat and the “Trichocystenkorn.” The interciliary fibril and the Basalapparaten, therefore, find their counterpart essentially in Schuberg’s longitudinal fibril and its connected basal granules. For both von Gelei and Klein, accordingly, it is evident that the fibrillar system of Paramecium includes two subsystems, or fibrillar com- plexes. For present convenience and especially for later discussion, I shall refer to these as von Gelei’s outer fibrillar complex and inner fibril- lar complex and Klein’s outer fibrillar complex and inner fibrillar com- plex respectively. DO Geet h (‘dioy ‘n 344g UsyIsIMZ) (aAoqe) Z ‘T uI— AIOYIITT, JO pus Jayno ye a[nuvss v—usoyuaysA0yIIIT, “q ‘JrepnIIIZ = {JauloyuaqaNN OMY, (Z) Ug usoxeseg (1) (uoddnisiaiaiq ) yereddeyeseg ‘ev (ua}yyUndSsso}¢ ) TJOUIOYSUOTILIAY “¢ a] qyorejuswe]q s]iiqy Jo uorjounf }e duTpIoATIs WoIfZ astie (sunyyds Aq "[ wosz paatiap) aypiqyrepnoirz *z YUM “TSeJIVIIIIOJUT “7 ‘PIO TIL FO YA AWNUTUOD UF s[Isqry (wo}shg “pulgioA JYxPIP) SunupsiO "[ ueiprioy_ ‘TT (e870 spiuqy yySIIpUT “Nn yyAIIP) s19}UT_Y “gq atury JvjodumIny—y]og aapioA ‘ve SHOTRMENE UJoyUe}sADOYIII}OId JIvJUIWIpPNY “q ysAD0YIII} JOF Furuado YW “ve puesys | UPY} STOUT S spIIqY *jSUOT UIdMJagG—uOYTeqIaNEd °*Z puvyjs T Wey} STOUT :¥ITID JO SMOI UaaMJoq—JosLJSULT *T SUNUPIOP'T ut “PHO Wo1Z paarsop ‘apoijod ay} Japun yIOMjOU INL}suOU YW (Wia}shg “pUIqIoA }YPIIpUr) SunupsiO “[] uempusyy ‘T ul21N (7Z61 ‘seay) woysds IeT[IIqy d1}ewWse[desjuy uaTIqIy apeuosviq “¢ VaTTqgIINO °Z Ud] [IIqyssueyT *] (é) UorUNF UF d9]1}9vI}UOD ‘ sSuIssoID ye AjIMUT}UOD UT ‘ssaUydIY} jO Sopris JUaIayIp Jo somqy ‘aynuvis [eseq YUM oad] dures ay} 3 pue Y}eaUaq pa}ed0] s[lIqy jo xatdurod dtwsedowo uy (TaJ2H) UOA *D Jo) wayshsr9}IH orerpoeIjuy Jasejierfiosjoyuy puv jeseddeyeseg surpunosins vusejdoman °*9 IWIVOINIUILI[III9JUI UO UIOYUa}sADOYIIIT, “q uloyuaqeaN, (¢) osejUY UIOX[eseg WN SulI jeseq (Z) uloyyeseg (T) yereddeyeseg ev awauOINeU Uo MoU asIIe JO UOIssy SuIINp aplAIp—souIOYsuOT}LAY *¢ ‘Iqy € JO Z Jo—PIBeH PN Jodsayuryy ‘p UIWIUOINIUSSULPT UddM} -2q S[IIqy-sso1d jo woajsks J[vInsstwuwo0I—jYeNT [OdIapI0OA *¢ UasUNpUIqIaAIANG) *Z noysnosy} eee “q snonunuod nq SorelIovig “e ‘uvIs ysAIOYINI} Y}IM—JOSvJIEIIDIOWU] *T I YIM payauUO0) jou Ss]IIqy JO YIOM}JIN wajsk§ aWaUOININ Jola}sog “q JOMa}Uy ‘eB arurpyeN *¢ pus Yove je UIOYIIVI YU “q ysAI0YIIIT, JOJ Surusdo yA “ev pueijs [—uor}I9s “ur Area [| Suneredas ‘spriqy ‘ysuoy U39M}9G—Uoy[eqIand) *Z pues}sS [—PI]ID JO SMOI UIIMJOG—UIasv]JssuLT “T UOTLIVUIIOYIP Jtuwsvpdoyea {aypotyJad ayy Jopun ApaIIp s[qy Jo YIOMJoU |. YsTURYOW-YDsTWV}s,, W wayshg 19}}13Z)NIS 12/25) uo A WIMDAWNVAVd -T ATAV YE ‘AI ‘Ill ll CILIATE FIBRILLAR SYSTEMS 197 The several components of these fibrillar complexes, which were listed above, constitute the main structural features shared by Klein and von Gelei in their various accounts of the fibrillar system of Paramecium. But certain discrepancies appear in their descriptions of these and espe- cially of some other components. Space does not permit a discussion of all of these discrepancies, but most of them will be found listed in Table 1, which comparatively sum- marizes all the structural components ascribed to Paramecium’s fibrillar system by von Gelei and by Klein in their various publications. Some of Figure 72. Diagram of G/tter (lattice) with attached trichocysts and of the neuro- nemes connecting bases of the cilia. (Von Gelei, 1925.) Git—Gitter neu.—neuroneme tri.trichocyst the discrepancies may very possibly be attributable to species differences, since von Gelei studied P. nephridiatum and P. caudatum, while Klein’s descriptions are of P. awrelia. A few discrepancies should, however, here be pointed out. The first and most important of these has to do with any structural integration between the two fibrillar complexes. Klein especially emphasizes the fact that his outer fibrillar complex and inner fibrillar complex are a con- tinuum through interfibrillar connectives. Von Gelei, on the contrary, denies that any connection exists and so states that his outer and inner fibrillar complexes are only contiguous (Fig. 72). A further discrepancy concerns the basal apparatus of the cilium. According to von Gelei (1932a), this apparatus consists of (1) a basal 198 CILIATE FIBRILLAR SYSTEMS ring surrounding (2) the basal granule of the cilium, and (3) the “Nebenkorn” occurring at the right of the junction of the basal ring and the interciliary fibril (p. 158). According to Klein (1931), the elements of the basal apparatus include (1) a ring (Zirkularfibrille) within which are usually three granules (Drierkérner). The central granule is the basal granule, and the other two are Nebenkérner. The discrepancy here is not only in the relative number of granules in the basal apparatus but in their relationship, since von Gelei regards his ‘“Nebenkorn”’ as identical with Klein’s basal granule, and accounts for Klein’s third granule as being only a thickening of the basal ring. Finally, mention may be made in this connection of an additional system of fibrils described by Gabor von Gelei (1937) in three species of Paramecium,—P. caudatum, P. multimicronucleata, and P. trichinum. This third fibrillar complex, in addition to the two noted above, was found at the level of and below the basal granules. Its fibrils spread throughout the entire body surface, including the vestibule, where it sends a thickened fibril into the cytopharynx between the membranelles. The general pattern of this complex, made up of longitudinal and cross fibrils, resembled that of the outer fibrillar complex, the meshes of the former being smaller and more numerous, however, than those of the latter. Also, the course of the fibrils of this third complex were more irregular. Its longitudinal fibrils become fewer in the middle region of the body and at times are directed diagonally, even spirally. A splitting of fibrils was occasionally observed, as well as variations in their thick- ness. They may alsc anastomose and form a ‘‘Schaumgiter.” The author could discover no connection between this fibrillar complex and either of the other two. Apparently the most recent detailed account of the fibrillar system of P. caudatum, including that of its cytostome, was made by Lund (1933), working in Kofoid’s laboratory. After comparing the descrip- tions of earlier workers (Engelmann, 1880; Maupas, 1883; Schuberg, 1905; Rees, 1922; von Gelei, 1925-32; Klein, 1926-31; Jacobsen, 1931) with his own findings, Lund concluded that previous investigators had confused “parts of at least two and possibly three quite different aggre- gations of structures, namely, the pellicle, the trichocysts and the periph- eral portion of the neuromotor system. In addition” they had failed CILIATE FIBRILLAR SYSTEMS 199 “completely to demonstrate the great pharyngeal complex,” which is an integral part of this system. Lund was able to differentiate between these “‘different aggregations of structures” by means of the silverline technique (Klein’s and von Gelei and Horvath’s) on the one hand and, on the other, by the use of iron-haematoxylin and Mallory’s stain. The former method demonstrated von Gelei’s ‘‘Stiitzgitter System” and Klein’s ‘‘Indirekt verbindung System,” i.e., their “outer fibrillar complex’’ noted above. It also revealed essentially their “inner fibrillar complex,’’ also as noted above. But, according to Lund, these are separate and distinct “aggregates.” The “outer fibrillar complex’ is not subpellicular, as both von Gelei and Klein maintain, but represents rather the sculptural polygonal pat- tern of the pellicle itself. A similar interpretation was made by Brown (1930). The “inner fibrillar complex’ of von Gelei and of Klein comprises the basal granules, their connecting longitudinal body fibrils, transverse fibrils connecting the longitudinal fibrils, and others which include the “radial fibrils.” These last mentioned “originate as longitudinal fibrils in the cytopharynx and oesophagus, spread radially out from the oral opening over the body surface and terminate a short way from the cyto- stome.”’ This inner fibrillar complex may be clearly demonstrated by the silver- line techniques, especially by the wet method. There is, however, a portion of the fibrillar complex within the cytopharynx and cytoesopha- gus which is not wholly demonstrable by these techniques. This was well differentiated by the iron-haematoxylin and Mallory’s methods, and described as “seven major parts, namely: (1) the pharyngo-esophageal network, (2) the neuromotorium, (3) the penniculus, (4) the oesopha- geal process, (5) the paraesophageal fibrils, (6) the posterior neuromotor chain, and (7) postesophageal fibrils.’ For the descriptive details of this very elaborate complex of fibrils and associated parts, obviously the original account must be read. It is evident, however, from this brief review of the results of these several workers on the fibrillar system of Paramecium that a number of discrep- ancies need to be cleared up and perhaps further structural analysis of 200 CILIATE FIBRILLAR SYSTEMS this system made before we can hope to have a complete understanding of the parts that are, or are not, structurally integrated. In recent publications Chatton and Lwoff (1935, 1936) have de- scribed a fibrillar complex in several ciliates, which has long been known (Chatton and Lwoff, 1936) but has not been clearly distinguished from Klein’s silverline system. The fibrils are visible 72 vivo and may be clearly differentiated in preparations fixed in Bouin’s or Champy’s solution and stained in iron-haematoxylin. Each fibril (c/nétodesme) has connected, always along its left side, the basal granules (c/nétosomes) of a longitudinal row of cilia. The fibrils, together with their adjoined basal granules (the so-called infra- ciliature), are each essentially an independent entity. They are never united by anastomosis or otherwise at either body pole, and so include no transverse or other fibrillar connectives throughout their course. The fibrils of this 7fraciliature are entirely superficial and adhere to the pellicle as rectilinear (never sinuous) threads. Other granules, as well as the ciliary basal granules, appear likewise attached, and these represent successive stages of the multiplication of the basal granules. Fibrils and granules stain alike, but in some species the fibrils cannot be impregnated with silver by the usual techniques. After fixation (osmic acid, Da Fano, Champy) and covering with gelatin or gelose, the fibrils may show, upon silver-nitrate treatment, the basal granules connected to a sinuous thread which, with its various connectives, represents Klein’s silverline ‘‘plexus.” This plexus, according to Chatton and Lwoff (1935), is acid labile and cannot be stained. The selective staining properties and relations of the infraciliature show that it is quite distinct from the silverline fibrils, and is comparable with the flagellar ridges of the Hypermastigidae, marking the place of formation and of the insertion of the cilia. It would appear that Chatton and Lwoff’s infraciliature may be identt- fied with the longitudinal fibrils and basal granules of the inner fibrillar complex reviewed above. The left lateral attachments of the czmétosomes to the cinétodesmes is evidently a new finding. 2. Stentor—In his search for organs that would account for the well-known contractile behavior of Stentor, Ehrenberg (1838) saw in its conspicuous longitudinal bands the seat of that contractility. This interpretation of its contractile mechanism was accepted by several later CILIATE FIBRILLAR SYSTEMS 201 investigators including K6lliker (1864) and, according to Neresheimer (1903), Haeckel’s (1873) “‘Myophanen”’ should be so construed. It was between these longitudinal stripes, within the clearer non- pigmented meridians (Biitschli’s ““Zwischenstreifen”), that Lieberkthn, in 1857, found a distinct contractile fibril coursing from the basal disc forward to the adoral zone. Greeff (1870) confirmed these findings and Engelmann (1875) made detailed studies on the refractive and contractile properties of the fibers which have come to be commonly referred to as myonemes. Four authors may be cited, among many others, for the descriptive details of the fibrillar system of Stentor: Schuberg (1890), Johnson (1893), Neresheimer (1903), and Dierks (1926). These have been the main sources for the following brief review of this system. Schuberg (1890) made several important observations on the arrange- ment of the myonemes of Stentor coeruleus and an analysis of the basal apparatus of the membranelles. He found that the course of the body myonemes, from the basal disc to the peristome border, was not con- stant. Instead, some showed bifurcations, with occasional re-branching. This branching of myonemes followed consistently a corresponding branching of the longitudinal rows of cilia and their adjacent, non- pigmented bands. Similar relations of bands, ciliary rows, and myonemes obtained also for the peristome field. Schuberg further observed that the double row of cilia, comprising a membranelle, was seated in an ectoplasmic basal platelet (‘“Basal- saum’’), itself bipartite, below which appeared a triangular Jamella (“‘Basallamelle”). The inwardly directed apex of this triangle was continued as a fibril (““Endfadchen’’) which was, in turn, united to all other such end fibrils by a basal fibril. The latter then ran rather deep below and parallel to the entire series of membranelles. Schuberg’s account of Stentor’s myonemes and his analysis of its membranelles have been generally confirmed, with the exception of the basal fibril. The latter was identified by Johnson (1893), Maier (1903), and Schréder (1906). But Neresheimer (1903) and Dierks (1926) are certain that, as such, it does not exist. It is worth noting that Schu- berg’s ‘‘Basalfibrille’”’ has been widely cited in the literature. Johnson’s (1893) work is not concerned primarily with a structural analysis of Stentor’s fibrillar system, but his observations were thorough 202 CILIATE FIBRILLAR SYSTEMS and critical and, for the most part, they have remained valid. Reference now may be made to his search for the so-called myoneme canal, de- scribed by Biitschli and Schewiakoff (1889, p. 1297). Beneath the “Zwischenstreifen” they found a fairly spacious fluid- filled canal which surrounded the myoneme throughout its course. Johnson looked in vain for this canal, finally deciding that he was “unable to find the least evidence of such a structure, either in optical or actual sections.’’ The majority of authors—including Delage and Herouard (1896), Maier (1903), Neresheimer (1903), and especially Dierks (1926)—agree with Johnson that the canal does not exist except as an artefact. Schroder (1907), on the contrary, affirms its form to be oval or circular in cross-section, its shape and position varying with the degree of body constriction. Roskin (1918) and von Gelei (1929) claim also to have definitely identified it. The latter regards it as an “organic part’’ of the myoneme, “‘solid and elastic.” Neresheimer’s chief contribution to the microanatomy of Stentor was his discovery of another complex of fibrils to which he gave the name ‘‘Neurophanen.” These were associated contiguously with the myonemes, but coursed usually peripheral to them. In suitable preparations which had been differentiated with Mallory’s triple stain, the myonemes were distinctly red, whereas the neurophanes were colored a dark violet. The Zwischenstreifen remained unstained. Schréder (1906) maintained that these fibrils were rather only a structural feature of the “Zwischen- streifen,” which, according to his results, also with Mallory’s stain, did show an intense purple color. More recently, however, von Gelei (1925) and Dierks (1926) have identified similar fibrils, as will be noted further on. Neresheimer traced these neurophanes as coursing, each fibril directly over a myoneme, from the aboral plate to about halfway up the body. Here some ended in a knob and all others disappeared before reaching the peristome border. While the myonemes became shorter and thicker in fully contracted Stentors, the neurophanes appeared sinuous but otherwise remained unchanged. It is not clear, however, how Neres- heimer could make sure of the changed or unchanged appearance of these fibrils, since he stated that he was not able to fix Stentor in an uncontracted state. Evidently the myonemes may be visible in the living organism (Biitschli, 1889; Johnson, 1893), which may have been CILIATE FIBRILLAR SYSTEMS 203 Neresheimer’s means of observing a ‘‘three-fold increase” in the thick- ness of the contracted myonemes. Perhaps the most complete structural analysis of the fibrillar differ- entiations of S. coeruleus is that by Dierks (1926). His work, which was carried out in Korschelt’s laboratory, considerably revised and extended earlier accounts of the fibrillar system of this heterotrich. He noted a gradual thickening of the myonemes from the peristome border down to the aboral pole, where the fibers do not end abruptly, Figure 73. Connecting branch from neuroid to myoneme in Stentor. (Dierks, 1926.) myo. str.—striation of myo- neme neu.—neuroid neu. br.—neuroid branch to myoneme as Johnson (1893) thought, but bend sharply inward and revert an- teriorly to form a pencil-like bundle (see also Schréder, 1906a). This bundle soon becomes fimbriated, its component fibrils branch, and their tapering ends disappear in the cytoplasm ‘“‘near the center of the con- tracted animal.” Dierks confirmed Neresheimer’s (1903) findings of a second fibril coursing parallel and usually peripheral to the myoneme, both of which also stained differentially by Mallory’s method. But the relationship of these two sorts of fibrils was found by Dierks to be evidently more intimate than Neresheimer had observed. In various sectioned and stained preparations, the smaller fibril, or “‘neuroid,” gave off one or 204 CILIATE FIBRILLAR SYSTEMS more branches (Fig. 73) to its adjacent myoneme, with which it apparently united (cf. von Gelei, 1929b). He observed also the knob-like endings of these neuroids, as described by Neresheimer for his neurophanes, but Dierks apparently could ac- count for such knobs as being merely the cross sections of fibrils that happened to be bent near the plane of section. The cross striations of the myonemes, described by Butschli and Schewiakoff (1889), were observed by Dierks in the living organism as well as in his preparations. Johnson (1893) had regarded these as artifacts due possibly to wrinkling of the myonemes, but the regularity of their recurrence and spatial relations seemed to preclude this. The myonemes were usually elliptical in cross section, with the longer axis of the ellipse directed toward the center of the body. This cross section revealed definitely an outer cortex (Plasmahiille) and a medulla (Plas- mamark) (cf. Roskin, 1918). Dierks’ analysis of Stentor’s membranelle apparatus differs in several points from most earlier descriptions. The membranelle platelet, sup- porting each membranelle, represented essentially the aggregate of basal granules of the component cilia. Continuintg from these granules into the cytoplasm was a basal lamella, the outline of which was clearly rectangular and not triangular as Schuberg (1890) had claimed for his “‘Basallamelle.’’ Dierks’ rectangular lamella could appear as a tri- angle, whose apex might be directed either toward or away from the basal granules, depending upon their position when viewed. For these lamellae were as ribbons, each about three times as long as broad, and each alike was slightly twisted on its long axis. This, according to Dierks, accounted for the erroneous interpretation of Schuberg (1890), Schroder (1906), and others. Not only might the lamellae appear as triangles, but also their inwardly directed “apexes’’ might then seem to be con- tinued as a fibril (‘‘Endfadchen’’). To account for the basal fibril, which Schuberg thought united all of the end fibrils (“Endfadchen’”’), Dierks observed that his basal lamellae overlapped in such a way that their ends could give the impression of a continuous fiber, comparable in appearance, direction, and extent to Schuberg’s described “Basalfibrille.” 3. Euplotes——As a major group of ciliates, the hypotrichs probably mark the acme of highly differentiated motor organelles (undulating membranes, membranelles, and cirri) the related fibrillar system of CILIATE FIBRILLAR SYSTEMS 205 which may appear correspondingly specialized. Another structural dif- ferentiation, the pellicle, assumes in this connection a significant im- portance in maintaining the flattened bodily form so characteristic of the hypotrichs. The often remarkable rigidity of this pellicle has long been recognized. Euplotes, the representative whose fibrillar system will now be reviewed, suitably illustrates these well-known characteristics of the hypotrichous ciliates. Maupas (1883) was apparently the first to identify fibrillar differ- entiations in this genus. He described in Ewplotes patella var. a fibril extending anteriorly from the basal plate of each of the five anal cirri. These five fibrils united into a single fiber, which continued anteriorly and disappeared near the bases of the adoral membranelles. Maupas’s findings were essentially confirmed by Prowazek (1903) in his brief account of protoplasmic reorganization in E. harpa. He further observed the ‘‘solide und fest’’ nature of fixed anal cirri fibrils, as indicated upon sectioning, when they might be pulled and bent thread-like by the microtome knife. Similar fibrils were seen to extend radially from the bases of the other cirri. Prowazek also described and figured still finer fibrillar lines (‘‘Fibrillenziige’’) going in parallel to the adoral membranelles. These finer fibrils have apparently not been identified as such by later workers. Some years later Griffin (1910) gave a fairly detailed description of the fibrillar system which he discovered in E. worcesteri. From the base of each of its five anal cirri, he observed a fiber extending ante- riorly. All five fibers converged toward the adoral membranelles, near which they disappeared ‘“‘close to each other.” Unlike similar fibers described by Maupas (1883) and by Prowazek (1903) for other species of Exzplotes, these of E. worcesteri apparently did not unite to form a common strand and were not traceable to the membranelles. Several finer fibers were found associated with the bases of some of the other cirri. Their number and direction varied, however, and they had no connection with those of the anal cirri. Griffin suggested that all of these fibers might be comparable to myonemes, the number of which had become reduced with a reduction in rows of cilia, as postulated for the hypotrichs generally; but he noted also that some of the fibers may be directed even transverse to the hypothetical original ciliary rows. 206 CILIATE FIBRILLAR SYSTEMS Yocom (1918), working in Kofoid’s laboratory, found and described in E. patella a fibrillar system much more extensive than that delineated in other Explotes by Maupas, Prowazek, and Griffin, as noted above. In addition to the anal cirri fibers, such as they had found, Yocom discovered in E. patella ‘“‘a fiber connecting the inner ends of the cytostomal membranelles’’ (“anterior cytostomal fiber’), and a “‘mo- torium’’ (after Sharp, 1914) which united the membranelle fiber with those from the anal cirri. A structural integration was traced, therefore, between the cytostomal membranelles and the anal cirri. Similar fibers, radiating from the bases of the other cirri, were also described, but no connection was found between them and the others above mentioned. In the “anterior lip” of this species, Yocom depicted a fibrillar lattice- work which was united by “‘short rodlike projections” to the mem- branelle fiber. The intimate contiguity between this fibrillar system of E. patella and its motor organelles was clearly detailed by Yocom. Certain minor modifications and additions to his account were made by Taylor (1920), from studies especially of dissected and slowly disintegrating organisms. Following Sharp’s (1914) terminology for a comparable fibrillar system which he had found and elaborately described in Diplodinium ecauda- tum, Yocom designated this system in E. patella a “neuromotor ap- paratus.”’ The neuromotor apparatus discovered by Yocom is to be distinguished from an additional fibrillar system in this same species, which was carefully worked out by Turner (1933) by means of his modification of Klein’s (1926) and von Gelei and Horvath’s (1931) methods. His technical procedure is here worthy of note. After fixing the organisms in osmic acid vapor for about three seconds, and before the material was quite dry, Turner added two or three drops of 2-percent silver nitrate. Within four to eight minutes the nitrate was poured off and the slide placed in distilled water, barely covering the preparation. Over a white background, the slide was then exposed to the sun until the reduction of the nitrate had progressed as desired, according to occa- sional microscopic examinations. The preparation was then thoroughly washed in distilled water, dehydrated, and mounted. “The method gives strikingly clear-cut results.’’ For this study, various other techniques were also employed, on both whole mounts and sections. CILIATE FIBRILLAR SYSTEMS 207 By these several methods Turner was able to disclose an ‘‘external fibrillar network,” which included: 1. The “dorsal network,’ comprising (1) seven to nine longitudinal rows of granular rosettes, from each of which protruded a central bristle; (2) seven to nine longitudinal fibrils, uniting the bristles of the rosettes and designated primary fibrils; (3) longitudinal secondary fibrils, running midway between the primary fibrils; (4) commissural fibrils connecting, midway between the rosettes, the primary fibril and on either side its secondary fibril, “pulling the latter slightly out of line.” The square meshes of this dorsal network, which appeared remarkably constant, averaged about four microns across. 2. The “collar,” anterior to this dorsal network, comprising, (1) the inclined row of parallel basal plates of the adoral membranelles, the lower ends of which “‘rest’’ on the oral lip; (2) a posterior membranelle fibril, connecting the “upper” ends of the membranelle basal plates; (3) an anterior POSE SiON TID) pie Sau lene, ie Se AE AY a VEO TINVIO. ea sey VANARAAD i. SPY __. sen. Bf. von een plate vay los. rer eyy TAs fa COUP TID BOK ARG ee ae ao Figure 74. Euplotes patella: dorso-lateral view of external fibrillar system. (Turner, 1933.) ant. m. fib—anterior membranelle fibril com. fib.—commissural fibril med. m. f.—median membranelle fibril mem. pl.—membranelle plate post. m. fib.—posterior membranelle fibril pri. fib.—primary fib. ros.—rosette sec. fib—secondary fibril sen. br.—sensory bristle membranelle fibril, connecting short commissures at the “lower” ends of these basal plates; (4) a median membranelle fibril, attached on the basal plates between the anterior and posterior membranelle fibrils. These anterior, median, and posterior membranelle fibrils continue this same relationship with the membranelle basal plates throughout the course of the membranelles along the ventral ‘‘lapel’’ down to their ending in the cytopharynx (Fig. 74). Of primary importance is Turner’s observation that his anterior mem- branelle fibril is identical with Yocom’s anterior cytostomal fiber, because this obviously integrates structurally the nmeuromotor apparatus described by Yocom and the external fibrillar system described by Turner. 3. The “ventral network,’ which is entirely comparable, in its general features, with the dorsal network, and includes also primary and secondary longitudinal fibrils and rosettes, with their central bristles. 208 CILIATE FIBRILLAR SYSTEMS Its pattern, although “constant and characteristic,” is less regular and “reminds one of badly treated chicken wire.” Sectioned material showed the fibrils of both dorsal and ventral networks “to be immediately wzder the pellicle and in contact with it.” The fibrils stained zntra vitam were distinctly more delicate than those impregnated with the silver. Turner confirmed Yocom’s observations on the neuromotor apparatus, excepting the motorium. In the E. patella which he studied, he was unable to detect this cited organelle. Instead, the single fiber, formed by fusion of the five anal cirri fibrils, was traceable to the “‘collar,” without a break, where it continued as the anterior membranelle fibril (Turner’s designation) noted above. 4, Vorticella—The vorticellids, by their size and quick reactions, caught the eye of the earliest microscopists, including, of course, Leeuwenhoek. The sudden contraction of the spiraling stalk along with the inversion and closure of the adoral membranelles naturally invited speculations on the kinds of mechanisms that might account for such reactions. Geza Entz (1893) cites Wrisberg (1765) as among the first to describe “mit recht treffenden Worden” this surprising behavior and to point out its elastic nature. To Ehrenberg (1838), however, apparently should go the credit for the earliest detailed studies of the fibrous nature of the contractile stalk and the detection of longitudinal and circular fibers in the body of several vorticellids. He attributed to all of these fibers a contractile function and described in the stalk “muscle’’ cross striations comparable to those of the striated muscles of other animals. This fibrillar complex in these peritrichs came to be a favorite object of investigation by many able workers, especially during the latter half of the past century: Dujardin (1841), Czermak (1853), Lachmann (1856), Lieberkithn (1857), Kithne (1859), Rouget (1861), Cohn (1862), Haeckel (1863), Metschnikoff (1863), Kollicker (1864), Greeff (1871), Everts (1873), Engelmann (1875), Wrzesniowskt (1877), Forrest (1879), Maupas (1883), Brauer (1885), Biitschli (1889), Schewiakoff (1889), and Entz (1893). The literature for this period has been reviewed by Greeff (1871), Wrzesniowski (1877), and Biitschli (1889). Similar investigations on the vorticellids have been relatively meager during the present century, and the most detailed and CILIATE FIBRILLAR SYSTEMS 209 careful analysis of the finer structure of their fibrillar system, so far as I have found, is that of G. Entz (1893) on “Die elastischen und con- tractilen Elemente der Vorticellen.” In the brief review here presented of this system, I have followed chiefly this excellent account. In this review of the fibrillar system of Vorticella and its relatives, Figure 75. Stalk of Zoothamnium arbuscula. (Entz, 1893.) axo.—Axonem cyt.—Cytophane spr.—Spironem sps.—Spasmonem it will be more convenient to consider first the fibrils of its contractile stalk, then those of its body and its peristome. Previous to the critical investigations of Entz (1893), the “Svzel- strang”’ in the contractile stalk of the vorticellids, which had been identified by Ehrenberg (1838) as the ‘“‘Stielmuskel,’ was found by later analysts to be composed of two parts: (1) the “Stielmuskel,” a cylindrical, or band-like, strongly refractive fiber, and (2) an adjacent, granular “Proto plasmastrang,” which accompanied the former through- 210 CILIATE FIBRILLAR SYSTEMS out its course. Both were surrounded by a delicate membrane, the “Strangscheide,”” in the same manner that the whole Stielstrang is en- closed within the outside membrane, ‘‘Stielscheide,”’ of the stalk. The Stelmuskel, so most authors (e.g., Engelmann, 1875; Wrzesniow- ski, 1877) agreed, was made up of distinct fibrils running variously transverse or parallel to its long axis. For Biitschli (1889), however, this composition of the Stielmuskel represented rather (and probably more in line with his alveolar hypothesis) an attenuated meshwork. And Ehrenberg (1838), Leydig (1883), ef al. could apparently see cross striations in the Size/muskel of some vorticellids, comparable with those of metazoan muscle. Entz (1893) described for the giant stalk of Zoothamnium arbuscula, Figure 76. Spasmonem (cross section) in Zoo- thamnium. (Entz, 1893.) and as typical for all the Contractilia, a Stielstrang comprising three well-defined fibers: a “Spasmonem,” a “Spironem,” and an “Axonem”’ (Fig. 75), all enclosed within the Strangscheide. He identified the Spasmonem with the Stielmuskel (noted above), and the Spironem and Axonem with the Proto plasmastrang. In the smaller branches of Z. arbuscula, the Spasmonem (Stielmuskel) is a round fiber which, in the main stalk, becomes compressed by the adjacent Spironem so that a cross section of the former appears crescent- shaped or, since one edge of the crescent is swollen, rather like the form of a comma (Fig. 76). As shown by Engelmann (1875), it is bire- fringent and may frequently be tinted a steel-gray or appear faintly greenish or yellowish. It is stainable in carmine and disintegrates in alkali and in mineral salts—teactions which are more rapid in the younger stems, and which, according to Entz, would indicate that the Spasmonem is not cellulose; neither is it chitin nor keratin, although least unlike chitin. This age difference shows itself also in the finer structure of the CILIATE FIBRILLAR SYSTEMS pAligt Spasmonem. In the younger branches it is fairly homogeneous, but in the older stems and in the main trunk one may distinctly observe, in its hyaline ground substance, parallel longitudinal fibrils that gradually disappear toward the distal end. Nodal interruptions in these parallel fibrils apparently account for the cross striations cited by earlier workers. Outside these longitudinal fibrils is a single, or composite, spiraling fibril, and centrally placed along the Spasmonem are ovoid discs, each containing a central granule or “nucleus.” Entz’s Spironem and Axonem constitute, as noted above, the Proto- Figure 77. Three components of Spironem of Zoothamnium, (Entz, 1893.) cir. filb.—circular fibril cyto.—cytophanes 1. filb—longitudinal fibrils plasmastrang. This structural duality had been previously overlooked, perhaps because the Spironem is wound closely around the Axonem. Also, the spirals of the former, which are contiguous when the stalk is fully contracted, separate increasingly as the stalk is extended, so that the two fibers may easily appear as one in the completely extended stalk. In its finer details, the Spironem (Fig. 77) shows beneath its investing membrane a spirally wound fibril, under which are several longitudinal fibrils. All these fibrils are birefringent and, as noted for those in the Spasmonem, nodes of less refringence give here also the effect of cross striations. Along the axis of the Spironem are oval bodies (Cytophanes) containing each a central granule (Caryophane). These Cytophanes are connected by a longitudinal fibril (Fig. 75) similar to a string of pearls. In the Axonem (Fig. 75) longitudinal fibrils comparable to those in the Spironem also occur, but their course is apparently completely inter- 212 CILIATE FIBRILLAR SYSTEMS rupted at regular intervals by Cytophanes which are relatively much larger than those found in the Spzronem. It seems to be generally agreed that the stalk of all the vorticellids, both the Contractilia and the Acontractilia, is a direct continuation of the body. Bearing this in mind, we may now briefly review the fibrillar Figure 78. Pellicular structure, and branching of longitudinal myonemes in Zootham- nium. (Entz, 1893.) lon. myo.—longitudinal myoneme pell.—pellicle system of the body proper and later note how the parts of this system are related to those in the stalk. For convenience in description and with special reference to the genus Vorticella, we may regard the body as divisible into three fairly well- defined regions: (1) the funnel, lying between the stalk and the ciliary ring; (2) the bell, that part of the body above the ciliary ring; and (3) the disk, which includes the peristomal border, adoral zone, and cytostome. The entire body, like the stalk, is covered by a pellicle which, accord- CILIATE FIBRILLAR SYSTEMS 213 ing to Entz (1893), is not homogeneous but distinctly sculptured (Fig. 78) as if composed of “Stabchen” that overlap, somewhat as tile on a roof. The fibrillar system of the body lies immediately beneath the pellicle and comprises an outer and an inner complex of fibrils, or myonemes. Each such complex is in turn composed of (1) an outer circular layer, and (2) an inner longitudinal layer, making in all, then, four fibrillar layers. Lachmann (1856) was first to describe the outermost circular layer. Figure 79. Myonemes of stalk sheath of Vorticella. (Entz, 1893.) lon. my.—longitudinal myoneme 0. sp. my.—outer spiral myoneme It was later recognized also by Stein (1867), but apparently overlooked by other investigators previous to Entz (1893). As a single fibril, it spitals directly beneath the pellicle and may be followed from the attachment of the stalk uninterruptedly to the center of the disc. Its spiral course accounts for the annular appearance of the pellicle. Entz (1893) thinks also that the birefringence of the pellicle may be due to this underlying fibril, since, as shown by Engelmann (1875), all the fibrils are refractive in polarized light. The fibrils of the next layer, the longitudinal fibrils, lie immediately below layer (1), noted above, and likewise pass from the style’s attach- ment to the center of the peristomal disc. On this disc they of course are radially arranged. This layer was found by Greeff (1871), but Butschli (1889) questioned its existence. It should further be noted also that this outer fibrillar complex is continued uninterruptedly into the protoplasmic lining of the style sheath (Fig. 79). This will be referred to again in later discussion. 214 CILIATE FIBRILLAR SYSTEMS i mer. go. Figure 80. Arrangement of second complex of body fibrils in Epistylis. (Entz, 1893.) fnl. msc.—funnel muscle mer. myo.—meridional myoneme per. bdr.—peristome border Of the second complex of fibrils, its outer component, unlike the outer single fibril of the first complex, behaves rather as a large fiber that may become split into several fibrils. This set of fibrils courses spirally around only the lower half of the fannel portion of the body (Fig. 80). Toward the ciliary ring it disappears and, according to Entz (1893), reappears actually to form this ciliary ring “out of a number of fine fibrils (Myone- CILIATE FIBRILLAR SYSTEMS 215 men).” This would account for the interlaced appearance of the ciliary ring. Above the ring, in the be// part of the body, this set of fibrils could not be detected except in the peristome border, where it forms a fairly strong spiral, narrowly wound. Its occurrence there was described by earlier authors as a ‘‘sphincter ring’ of the peristome border (Fig. 80). On the disk, this fiber appears as a fairly thick strand, one end of which originates in the center of the disk and, after following at their base the several turns of the adoral membranelles along the outer margin of the disk and through the mouth into the gullet, it continues down the inner surface of the gullet the whole length. Along the gullet wall Entz thought that, in some preparations, he could observe this fiber branching into many increasingly fine fibrils. The inner, longitudinal layer of the second fibrillar complex is a direct continuation of the Spasmonem (Stielmuskel) of the contractile stalk. Upon entering the body this Spasmonem breaks up into its com- ponent fibrils, which diverge and so form the ‘funnel muscle” (Fig. 80) of earlier authors. Higher in the funnel, these fibrils may rebranch, then anastomose into a network. At the ciliary ring, the fibrils curve round it as crescent-like spans, which are somewhat larger and apparently more dense, and continue directly up the wall of the be// to the peristome border. These meridional myonemes (Fig. 80) may end singly on this border or, as Engelmann (1875) noted, they may bifurcate and unite, each of a pair with its adjacent neighbor, to form “‘arcades”’ just below the peristome border. The fibrils then span this projected border, much as they curved over the ciliary ring, and thereupon proceed as radii to the center of the disc. Toward this center, these fibrils may sometimes branch and anastomose to form varied and striking patterns. Finally, it may be mentioned that, in addition to the four layers of fibrils noted above, Entz (1893) describes a fairly thick strand of other fibrils which originate from the middle of the disc and project down- ward through the endoplasm and finally disappear in the region of the cytostome. B. INTERPRETATION The essential structural components of the fibrillar systems of several representative ciliates have been briefly reviewed in the foregoing para- graphs. It remains now to consider the several functions that have been 216 CILIATE FIBRILLAR SYSTEMS ascribed in the literature to each of these fibrillar mechanisms, as illustra- tive for similar systems that have been studied thus far in a large number and variety of other ciliates. It will be recalled that Ehrenberg (1838) saw in the “‘stalk muscle”’ of Vorticella cross striations which he regarded as comparable to those of the striated muscles of other animals. Also, he detected both longi- tudinal and circular fibers in the body of several other vorticellids. Quite in line with his search for such comparisons, he attributed to all of these fibrils a contractile function. For him they were literally miniature muscles which had their structural and functional counterparts in the muscles of macroscopic organisms (Maupas, 1883). Evidently, Ehren- berg’s comparison between microérganisms and macroérganisms led him to look for a one-to-one correspondence between microérgans and macroorgans. It will be recalled that what Ehrenberg regarded as the stalk muscle (Stielmuskel) was analyzed into several components by later investi- gators. These, according to Entz (1893), included: a cross-striated, rod or band-like Spasmonem, and another rod-like strand, the Axonem, around which was coiled the Sprronem. Each of these was, in turn, analyzable into longitudinal fibrils, whose properties apparently ac- counted for their birefringence and cross striations. The Spasmonem and Spironem showed also, spiraling within their own spiral strand just outside its longitudinal fibrils, a fine fibrillar coil coursing throughout their length. Also, both Spironem and Axonem had, centrally located, the ‘“‘cytophanes” which, with their longitudinal interconnections, ap- peared like a string of pearls. Thus Ehrenberg’s “Stielmuskel” turns out to be a highly complex mechanism which is structurally quite different from the “‘muscles of other animals’ to which he had compared it. Finally, mention was made also of the circular and longitudinal myonemes of the protoplasmic lining of the style sheath, which are continuations of the owter fibrillar complex of the body myonemes. Taking into account this assemblage of highly differentiated com- ponents within the stalk of the Contractilia, together with a few rather inconclusive experimental results, various interpretations were advanced to account for both the contraction and the extension of this stalk. These may be summarized as follows: 1. The majority of investigators agree that the quick spiraling con- CILIATE FIBRILLAR SYSTEMS JAF traction of the stalk is referable to contractile properties of some one or more of its components. Dujardin (1841) was apparently the only one to regard the stalk sheath as the seat of that contractility. Ehren- berg’s Stzelmuskel was for him the contractile component, but also the only component that he had recognized within the stalk sheath. Of the three fibers into which this Stzelmuskel was later analyzed, viz., the Spasmonem and Spironem and Axonem, Kiuhne (1859) considered the latter two, or Protoplasmastrang, as the contractile organelle. This he likened to the sarcoglia of metazoan muscle. Other proponents of this contractile theory (Czermak, 1853; Engelmann, 1875; Wrzesniow- ski, 1877; and others) regarded the Spasmonem as the contractile fiber which, for later authors, represented a bundle of fibrils or myonemes that continued without interruption into the body of the vorticellid. 2. Apparently there is general agreement among all the investigators that the mechanism of sfalk extension inheres in the elastic properties of the pellicle (Bitschli, 1889; Entz, 1893). Only Kithne (1859), who alone regarded the Spironem and Axonem as the contractile organelles, attributed elasticity to both the pellicle and the Spasmonem, thus to account for the stalk’s extension. 3. While the majority, as noted, support the contractile theory of stalk contraction, there is a minority group who maintained that the vorticellid stalk, in its quick spiraling retraction, actually does not con- tract as such but instead recoils somewhat as a coiled spring. A chief proponent of this elastic theory is Entz (1893), who sees the Spasmonem not as a contractile fiber but, like Kiihne, as an elastic fiber. This organelle is, according to Entz, primarily responsible for the sudden recoil of an extended stalk. He likens this elastic fiber to a curly hair which when stretched and then released will resume its spiral form. The opposing force, tending to “stretch” this normally spiral Spas- monenz, 1s inherent in the elastic pellicle. Thus pellicle and Spasmonem constitute a pair of “antagonistic elements.” Associated with this pair is another pair of antagonistic elements, viz., (1) the longitudinal myonemes in (a) the stalk sheath and (b) the Spironem; and (2) the circular myoneme in (a) the stalk sheath and (b) the Sp/ronem. These opposing pairs of fibrils are relatively weak, but of a strength sufficient to determine by their antagonistic contractions whether the stalk ‘‘contracts’”’ with the recoil of the elastic 218 CILIATE FIBRILLAR SYSTEMS Spasmonem or extends by means of the elastic pellicle. Accordingly the longitudinal fibrils of the stalk sheath and the Spironem tend to reinforce the elasticity of the Spasmonem, and the spiral myoneme of both sheath and Spironem, upon relaxation of the longitudinal pair of myonemes, tend to reinforce the elasticity of the pellicle. Entz’s rather elaborate thesis at least makes evident the elaborate differentiation of the vorticellid’s stalk, and so provides one working hypothesis which might be experimentally tested. With modern tech- niques, this should not prove very difficult. And, in the writer’s opinion, the vorticellids offer extraordinary possibilities for some clear-cut and fruitful experiments which should help toward a better understanding of their fibrillar mechanisms. It seems to be more generally agreed that the fibrillar complexes of the funnel, bell, and disc are primarily contractile. If so, and if the Spasmonem of the stalk is, as Entz and others claim, continued without interruption into the funnel and bell as the longitudinal fibrils of the “inner complex,” then it should follow that if these fibrils of the inner complex are contractile, one should expect the Spasmonem also to be contractile. Entz (1893) apparently does not discuss this point. Finally, it may be noted that Entz’s thesis attributes no specific function to the Axonem. Both he (1893) and Stein (1854) suggest that it might function as a neuroneme. Observations on the fibrillar system of Stentor have provided a basis for some interpretations which appear more plausible than those cited above for the vorticellids. These interpretations have to do exclusively with the myonemes. The other fibrils are poorly understood. Before discussing these, however, the essential components of this system should be recalled to mind. As was pointed out, and as will doubtless be familiar to the reader, the myonemes of Stentor are band- like fibers which course beneath and slightly lateral to the longitudinal rows of body cilia and the curved ciliary rows on the oral disc. Associated with these band-like myonemes, but lying peripheral to and mostly contiguous to them, are other fibrils which Neresheimer (1903) described as “neurophanes” and which Dierks (1926) recently further described and named “‘neuroids,” since he could not fully identify these with Neresheimer’s neuronemes. Dierks added one very significant observation, which, if verifiable, CILIATE, FIBRILLAR SYSTEMS 219 would definitely suggest a conductive function for his neuroids. He saw, in several preparations, that the smaller fibril gave off one or more branches to its adjacent myoneme. As will be noted further on, this claim has recently been challenged by von Gelei (1929b). Another fibril, found and described by Schuberg (1890), connected the basal lamellae of the entire series of membranelles. This basal fibril was noted also by Johnson (1893), Maier (1903), and Schréder (1906), but Neresheimer and Dierks maintain that such does not exist. Dierks (1926) does describe a fibril coursing along the platelets of the membranelles, which he thinks both Schuberg (1890) and Meyer (1920) may have seen but misinterpreted. The evidence supporting the interpretation that the myonemes are contractile organelles has been supplied from many sources, beginning with their discoverer, Lieberkithn (1857). Ehrenberg (1838) claimed to have seen in a living Stentor that in the extended state the myonemes were serpentine, while with the contraction of the body they became shortened and straight. But since he misidentified the pigmented meridi- ans as myonemes, the significance of his observation is uncertain. Probably the most significant evidence for the contractile nature of Stentor’s myonemes was provided by Johnson (1893) and verified by Dierks (1926). The former investigator compressed the living organism beneath the cover slip and could then observe the myonemes “alter- nately to extend and contract,’ concluding that ‘“‘no one who has once observed them under these conditions can doubt that they are responsible for the contractions of the animals.” Biitschli (1889) apparently made similar observations on living Stentor upon applying an electric stimulus. Merton (1932) has confirmed these former evidences of the contractile properties of Stentor’s myonemes. Dierks (1926) states further that “these fibrils are definitely shorter and thicker in the contracted animal and longer and shorter’? when the organism is extended. He also noted, however, that this behavior was lacking in the myonemes toward the posterior end of the body, and supposed that here the body protoplasm contracted more or less inde- pendently, suggesting a progressive differentiation in the myonemes anteriorly along their course. The assigned contractile function of the myonemes of Stentor appears to be agreed upon without exception. Schroder (1906) added to the 220 CILIATE FIBRILLAR SYSTEMS knowledge of their contractile function probably also that of conducting. But for the other fibrils of this ciliate, evidence regarding their actual function or functions is mostly wanting. Interpretations based on mor- phological evidence concern chiefly the relations of these fibrils (neuro- phanes or neuroids) to other organelles. Neresheimer’s observation on the parallel course of the neurophanes with the myonemes and the differential stainability of these two kinds of fibrils, suggested for the neurophanes a conductive function. By means of a drop-weight apparatus (Fallmachine) he tested the responses of Stentor to a variety of narcotics: morphine, strychnine, atropine, caffein, and so forth, and to other chemicals which are known to affect the nerves of higher animals. On the basis of an assumed selective action of these reagents, he derived evidence which seemed to favor assigning a nervous function to the neurophanes. The technique for these tests was ingeni- ously devised and might prove useful also for others. But owing to lack of adequate controls and because of other possible interpretations, his results do not seem very convincing. Schroder (1906) considered Neresheimer’s interpretation of the neurophanes invalid. He believed that Neresheimer had mistaken a struc- tural feature of the “mid-stripes’”’ for his neurophanes, as indicated upon comparing the latter’s Figures 7 and 8 with Schréder’s Figures 1-5. From Dierks’ more extensive and detailed studies, however, there can be no doubt about the identity of another fibril coursing with each of the myonemes. The occurrence of these was consistent and their relation- ship with the myonemes apparently significant. As formerly noted, Dierks found that in good preparations branches were given off from the neuroids to the myonemes which they evidently joined. He considered several possible functions which this connection between neuroids and myonemes might indicate, and finally, as a plausible hypothesis, sug- gested for them a conductive function. Von Gelei (1926c) rejects this interpretation. From his own previous studies (1925) on Stentor, he concludes that Neresheimer’s neurophanes and Dierks’s neuroids are real and are identical structures. But, according to von Gelei, these neuroids are not fibers but bands composed of fibrils, which are quite wide, especially in the aboral region. They are not a structural feature of the pellicle (Schroder, 1906), but are swbpellicular and fused to the pellicle. This was indicated in contracted Stentors by CILIATE FIBRILLAR SYSTEMS 221 the body groove overlying the neuroid, as produced by the contraction of the myoneme under the neuroid. The attachment of the pellicle to the neuroid and the latter, in turn, to the myoneme would, upon contraction of the myoneme, cause an ingrooving of the pellicle. Studies on the hypotrichous ciliates yielded apparently the first re- corded example of fibrils directly associated with ciliary locomotor organelles. Engelmann (1880) traced such fibrils from the bases of the marginal cirri of Stylonychia ‘‘nach der Mittellinie des Leibes.” He postulated for these fibrils a conductive function in transmitting im- pulses, as in nerves of higher animals, from the ventral region of this hypotrich to the cirri, the movements of which might thereby be regulated. Engelmann’s interpretation was formulated entirely by analogy and he offered no substantial evidence to support it. It seems to have been generally rejected by his contemporaries. Maupas, who three years later (1883) described similar fibrils in E. patella var., said, in a brief foot- note (p. 622) concerning his own findings: ‘Quant a la signification physiologique de ces racines ciliaires, j’avoue ne pas la connaitre.’”’ And with reference to Engelmann’s discovery of ‘“‘fibrilles nerveuses’’ in Stylonychia mytilus, Maupas adds: “‘Je ne sais comment concilier des interpretations aussi divergents.’’ Biitschli regarded Engelmann’s inter- pretation as untenable and proposed, on equally meager evidence, a contractile function for these fibrils. Maier (1903), who attributed a contractile function to the basal fibril associated with the basal lamellae of membranelles in Stentor, concluded that the lateral cirri fibers in Stylonychia were “required” for the support of these cirri. Prowazek (1903), on the other hand, suggested that the anal cirri fibers which he found in E. arpa might perform the dual functions of contractility and conductivity. He noted, however, that E. arpa could move each anal cirrus independently and could flex the tip of these cirri quite at will. He observed, moreover, that a detached anal cirrus might continue its contractions for a time. These important observations have apparently been overlooked by later investigators. It will be recalled that Griffin (1910) compared the anal cirri fibrils of E. worcesteri, which he found and described, with myonemes whose number might have become reduced phylogenetically during reduction in the hypotrichs’s rows of cilia. He pointed out a difficulty in this 222 CILIATE FIBRILLAR SYSTEMS interpretation, viz., that some of the few fibrils of other cirri in this species were not aligned longitudinally, but might be even transverse to the longitudinal axis. Griffin based his concept of contractility for these fibrils in E. worces- ter? only partly, however, on this comparison with myonemes in other ciliates. He accepted Biutschli’s interpretation of a contractile function for the marginal and anal cirri fibrils in Stylonychia as a much more reasonable view, and also noted that ‘‘Every detail of arrangement and structure indicates that the fibrils are, principally at least, contractile in function.”” He observed, also, that the fibrils were developed around the bases of the cirri in such a way as to assist in producing the ordinary motions. ‘As the anal cirri have only a single strong motion, a vigorous kick directed backward, each needs but a single strong fibril.” Evidently Griffin had not seen any reversal in the effective stroke of these anal cirri. In several other species of Ezp/otes this reversal is not uncommon in both swimming and creeping movements. Should that be the case in E. worcesteri, then a contractile function for the anal cirri fibers is scarcely conceivable. The effective function of a contractile fibril is obviously a pulling but not a pushing function. Moreover, Prowazek’s account of the contractile behavior of the anal cirri in E. harpa, cited above, largely vitiates any claims for contractility in the cirri fibrils of this species, and similar behavior has been observed by the writer in the anal cirri of E. patella. It was also noted (Taylor, 1920) that cutting these fibrils apparently did not impair the effective stroke of the anal cirri, whether that stroke was directed backward or forward. The latter evidences were thus cited against the assumption that these fibrils in E. patella were contractile. It was further shown by those experiments that cutting the anal cirri fibrils interrupted the codrdinated movements between anal cirri and adoral membranelles. Also, severing the membranelle fiber likewise interrupted the codrdinated movements of the membranelles on opposite sides of the incision. Incisions in other parts of the body did not impair the codrdination of these organelles. It should be pointed out, however, that those incisions which did inter- rupt codrdinated movement of organelles cut not only the anal cirri fibrils or the membranelle fiber, but also the peripheral fibrils which have since been described especially by Turner (1933). Whatever rdle, if any, these peripheral fibrils may have in E. patella’s co6rdinated CILIATE FIBRILLAR SYSTEMS 223 behavior was not demonstrated by the writer's (1920) experiments. Reinvestigation of this problem, especially on a more favorable form such as Lichnophora (Stevens, 1891), ought, therefore, to be undertaken in order to determine what relative rdles the so-called introplasmic fibrils and the peripheral fibrils each perform in the codrdinated movements of the organelles with which such fibrils are demonstrably associated. Several investigators (Bélaz, 1921; Jacobson, 1931; Peschowsky, 1927) have maintained that all such cirri fibrils in the hypotrichous ciliates and the fibrillar systems in various other ciliates are primarily or exclusively supporting in function. Jacobson (1931), for example, studied by means of various techniques, including the silver-nitrate methods, the fibrillar systems of some twenty-seven ciliates. These com- prised representatives of all the major groups and included among the hypotrichs E. patella and E. charon. She concluded from the results that no evidence was found in support of a conductive function for any of the fibrillar systems studied. It was pointed out that in the hypotrichs whose motor organelles are localized, fibrils are nevertheless present, as on the dorsal side where cilia are wanting. Reference on this point should be made to Turner’s (1933) studies on E. patella, which showed, as previously mentioned, that longitudinal fibers connect the bases of the dorsal and ventral rows of bristles, whose function is not known. But they may have a function and if that function is, as has been suggested, sensory in nature, it is surely not inconceivable that the associated fibrils may facilitate its performance. Another and more significant observation was made by Jacobson, viz., that in Sciadostoma difficile, where three ciliary rows surround the anterior body pole, no silverline connection exists between the basal granules. An impulse, therefore, originating at the anterior ciliary ring would need to pass nearly to the posterior body pole and back again in order to affect adjacent cilia. Since our assumptions should be, first of all, plausible, one would justifiably regard this morphological evidence contradictory to the thesis that these silverlines in S. difficile are con- ductive in function. It may be pointed out, however, that Chatton and Lwoff (1935) apparently demonstrated that the fibrils (cinétodesme ) described for several holotrichous ciliates, which alone were connected with the basal granules, could not be stained by silver impregnation, whereas other adjacent fibrils (interpreted by them as silverline fibrils ) 224 CILIATE FIBRILLAR SYSTEMS could be silver impregnated but apparently were not in contact with the basal granules. The thesis that all fibrils of all ciliates are only supportive in function is, of course, not tenable. One would at once except myonemes. But why limit the exceptions to myonemes? If, in the eons of time, protoplasmic fibrils have become differentiated so as to facilitate contractility in protistan organisms, who can deny them the capacity to have become differentiated also to facilitate conductivity or some other function in these unicellular forms of life? All our assumptions should be both plausible and reasonable assumptions, the validity of which may, in the last analysis, be demonstrated only by experiment. In conformity with the less specialized differentiation of its motor organelles, the fibrillar system of Paramecium is also relatively less specialized, as compared with those of the other three representative ciliates reviewed in foregoing paragraphs. From the accounts especially of Schuberg (1905), von Gelei (1925- 31), and Lund (1933), it appears that two separate and distinct com- plexes have been described in the literature, which may be represented by the outer fibrillar complex of von Gelei and of Klein (1926-32), and by their inner fibrillar complex. For these latter authors, both com- plexes are swbpellicular. According to Schuberg (1905) and Lund (1933), howeyer, the above-mentioned outer fibrillar complex is not subpellicular, but actually represents the polygonal pattern of the pellicle itself. Lund emphasizes the fact, therefore, that the essential fibrillar system of Paramecium 1s exclusively the complex which is associated with the basal granules of the entire motor mechanism, including the cilia of the mouth, cyto- pharynx, and cytoesophagus, as well as the body cilia. Lund’s fibrillar system would essentially include, therefore, the inner fibrillar complex of von Gelei and of Klein. The discrepancies just noted in the structural interpretations of Paramecium’s fibrillar system are obviously crucial, since they go hand in hand with discrepancies in the functional interpretations of that system. This holds, of course, not only for the investigators cited above, but for various others also. Referring now to these diverse functional interpretations, Schuberg (1905) suggested that the fibrils connecting the basal granules, such as CILIATE FIBRILLAR SYSTEMS 225 he had described for Paramecium and Frontonia, might function in the well known metachronism of ciliary movement. He was not inclined, however, to compare these fibrils with Neresheimer’s neurophanes nor with the neurofibrils of the Metazoa. Obviously, since it had not yet been proved that the neurofibrils were the conductive elements of nerves, then an analogy between these neurofibrils and the fibrils found in Paramecium would not add to our understanding of either the one or the other. Mention may here be made of the system of fibrils described for Paramecium by Rees (1922). These have an arrangement and relation- ship quite different from any of the fibrils referred to above. According to Rees, all of the fibrils he observed connected the basal granules of the cilia of the body and cytopharynx with the motorium, located just anterior to the cytostome, by coursing through the cytoplasm in several graceful whorls. From the results of his few experiments, Rees concluded that these fibrils were conductive in function. By severing with a microneedle the fibrils connecting the cytopharyngeal membranelles with the motorium, the codrdinated movements of these membranelles was interrupted. Like- wise, the codrdinated movement of body cilia was interrupted when the neuromotor center was destroyed. In view of the later descriptions of the more peripheral system of fibrils in Paramecium, Rees’s experiments should be repeated. It may be noted also that Jacobson (1931) reinvestigated this fibrillar complex described by Rees, and concluded that he had observed not fibrils but internally discharged trichocysts, as effected by the killing agents used. Her figures of fixed Paramecium illustrating such trichocysts are com- parable to some of Rees’s figures, except for certain regularities in the “fibrillar whorls” depicted by Rees. One could suppose that the in- ternally discharging trichocysts might tend to follow the course of these whorls of fibrils and so reproduce that course in fixed material, which, through differential staining, revealed the trichocysts but not the fibrils. Only further careful investigation can clarify this discrepancy. The interpretations of von Gelei (1925-31) and of Klein (1926-32) agree in ascribing a conductive function to the inner fibrillar complex of Paramecium, but their views on the relations and functions of its outer fibrillar complex are not in accord. The basis for their interpreta- 226 CILIATE FIBRILLAR SYSTEMS tions is confined primarily to morphological evidence. This evidence is derived, however, not only from their studies on the fibrillar system of Paramecium, but also from comparative studies of a considerable number and variety of other ciliates. According to von Gelei’s interpretation, Klein’s strongly birefringent “indirekt System” (Meridian II. Ordnung) is nothing more than the well known “‘pelliculare Gittersystem” of the ciliates, which functions as a supporting network to maintain body form. As such, it has no differentiated structural connections with the less refractive inner fibrillar complex (von Gelei’s neuroneme system), which alone, therefore, 1s the peripheral conductive mechanism. Von Gelei sees in the longitudinal and transverse fibrils of his neuroneme network a mechanism which structurally integrates the ciliary basal granules, the trichocysts, and even the contractile vacuole pore into a codrdinated whole (“einer koordi- nierten Einheit’’). This integrated mechanism codrdinates automatically the metachronous effective strokes of successive cilia. The direction of the stroke of one cilium activates conditionally that of the next by way of the basal apparatus (basal granule, basal ring, and ‘‘Nebenkorn’”’), which repre- sents a sensory organelle. For the codrdinated activity of the organism as a whole, however, von Gelei recognizes in his peripheral neuroneme system the absence of a neuromotor center. Accordingly, he regards the “intraplasmatic’’ fibrillar complex described by Rees as a centralized mechanism which may complement the peripheral neuroneme complex, thus to provide reasonably a unified neuromotor system. Von Gelet (1929b) observed by means of his silver-osmium-formol method a “platte’”” beneath the basal apparatus, which, he thought, might serve as a sort of end plate connecting Rees’s intraplasmic fibrils with the peripheral neuroneme complex. As has been previously indicated, Klein’s (1926-32) interpretation of his inner fibrillar complex (Meridian I. Ordnung) agrees essentially with von Gelei’s concept of a conductive function for his peripheral neuroneme network. Among their minor differences, Klein would ac- count for the commonly observed reversal in the effective stroke of cilia, not by means of Rees’s intraplasmic complex as von Gelei supposed, but by way of a “‘primitive reflex arc.” This reflex arc includes the axial filament of the cilium as the receptor, the basal apparatus as the “‘relator,”’ CILIATE FIBRILLAR SYSTEMS 227 and the protoplasmic sheath as the effector. The basal apparatus com- prises the basal granule and two “Nebenkorner.” The latter serve to spread the proximal part of the reflex arc and, with the basal granule, may function as a kind of commutator that regulates the direction and change in direction of the effective stroke of the cilium. We may note that while such a mechanism might conceivably account for a reversal in the effective stroke of a given cilium, obviously it does not, as such, provide for the synchronous reversal of the many other cilia, which is an essential part of the problem of codrdinated movement. In this connection, reference may be made to a fairly recent paper by J. C. Hammond (1935), who would refer the phenomenon of synchronous and metachronous ciliary behavior to an anterior-posterior physiological dominance in the organization of the cell, as opposed to the concept of a neuromotor mechanism. This thesis might help to account for this more general codrdinated behavior (Rees, 1921), but it would presuppose a reversal in anterior-posterior dominance of the physiological axis in order to explain the reversal of ciliary stroke so common in the swimming or creeping behavior of ciliates. Moreover, the well known localized reversal of a few or many of the cytostomal membranelles in the intake or ejection of solids would obviously require a similar presupposition. The chief discrepancy between Klein’s and von Gelei’s interpretations of the fibrillar system of Paramecium concerns the structural and func- tional relations between their outer fibrillar complex and their inner fibrillar complex. Klein (1928, p. 203) regards these two fibrillar com- plexes as a continuum. His ‘‘Meridian II. Ordnung’” is a derivative of his ““Meridian I. Ordnung,” as are also all cilia, basal granules, ‘‘Neben- kérner,”” trichocyst granules, and protrichocyst granules (secretory, or “Tektin” granules). In its re-genesis, observed during reorganization, many more fibrils (Profibrille) are formed than are retained, varying with the species and genus, and of those that persist some may unite to form composite fibrils (Biindelfibrillen), as occurs in Paramecium (Klein, 1932). Klein’s silverline system incorporates as a unit, therefore, both struc- turally and functionally, the outer and inner fibrillar complexes. In its re-genesis, it functions as a ‘‘form-building system,’’ and some of its fibrils become more resistant and rigid by the addition of a secondary 228 CILIATE FIBRILLAR SYSTEMS substance, the “‘fibrillare Komponente”’ (1928, p. 255). By virtue of its integral relation with the basal apparatus of the cilia and with the tricho- cysts, he attributed to his silverline system also a specifically conductive, coordinative function. Chatton and Lwoff’s (1935-36) criticisms of Klein’s interpretations bear mainly upon the structural relations of the fibril to which are ad- joined, always on its left s7de, the basal granules. The former authors think Klein’s silverline system is quite separate and distinct from their infraciliature. It appears to the writer, however, that this discrepancy might be completely resolved by identifying Chatton and Lwoff’s infra- ciliature with the longitudinal fibrils and basal granules of Klein and von Gelei’s inner fibrillar complex. Further critical investigation would be needed, of course, to establish such identity. FIBRILLAR SYSTEMS OF OTHER CILIATES A. HOLOTRICHA Ancistruma (Kidder, 1933).—The fibrils in Ancistruma mytili and A. isseli are of three types—the Jongitudinal fibrils of the ciliary rows, the irregularly distributed transverse fibrils, and the net complex of fine fibrils in the peristomal region. The latter fibrils seem to connect di- rectly with the basal bodies of the peristomal cilia, and they are coarser and less numerous in A. /ssel7 than in A. mytilz. The longitudinal fibrils in A, mytil? are continuous around the posterior end, but in A. ssselz they center about a posterior suture. A number of fine fibrils connect the inner dorsal row of peristomal cilia to the outer dorsal row. These seem to be distinct from the net complex and probably are in the nature of concentrated transverse fibrils. In A. zsseli such fibrils are absent, and instead the fibril of the outer ciliary row joins that of the inner dorsal row. Fibrils resembling the interstrial fibrils of Boverza (Pickard, 1927) are sometimes seen in A. mzytili, but these may represent a deep-lying network of the same type regularly seen in the peristomal region. METHODS Fixatives: Schaudinn, sublimate-acetic, Bouin’s, Zenker’s, Champy’s. Stains: Heidenhain’s and Delafield’s haematoxylin, crystal violet-sulph- alizarinate (Benda’s). CILIATE FIBRILLAR SYSTEMS 229 Klein’s silver method, modified to include fixation with osmic acid fumes and impregnation period of one to three hours in 2-percent solution of silver nitrate. Boveria teredinidi Nelson (Pickard, 1927).—Surrounding the cyto- stome is an oral rng which begins and ends in the motorium. From the motorvium arise the anterior and the posterior adoral fibrils, which bound the adoral zone. The posterior fibril joins the anterior fibril distally, and the latter continues as the pharyngeal fibril. This enters the endo- plasm in the region of the pharynx and spirals around the potential gul- let. A fibril from a point on the ring opposite the motorium enters the pharyngeal fibril near the margin of the peristome. Within the peristomal field delicate fibrils connect the anterior adoral fibril with the ring. The longitudinal lines of the body surface consist of contractile mzyo- nemes and basal granules of the ciliary rows. Myonemes arise at the anterior end directly from the posterior adoral fiber, or indirectly from fibrils of the posterior granular line. The myonemes pass posteriorly and in somewhat oblique parallel lines. They gradually converge in the pos- terior field. Basal granules of cilia rest on myonemes, oral ring, and the anterior and posterior adoral fibrils (except the free end of anterior adoral fibril). A deep nerve net, consisting of longitudinal interstrial and transverse fibrils, interconnect the area “between the myonemes and their basal granules.” METHODS Fixatives: Schaudinn’s (60° C.), Bouin, Zenker, formalin, osmic acid, Da Fano. Stains: Delafield’s iron haematoxylin, Mallory’s triple, alum carmine, and Yabroff’s silver-gold method. Clearing: Xylol, oil of cedar; equal parts of bergamot, oil cedar, and phenol, and sometimes before imbedding, in synthetic oil of wintergreen. Chlamydodon sp. nov. (MacDougall, 1928) .—The structural analysis of this new species of Chlamydodon disclosed “a complex neuromotor apparatus, including a codrdinating center, and systems of fibers con- nected with cilia, the mouth opening, the pharyngeal basket, and the ‘railroad track.’ ”’ The motorium was identified as a bilobed mass located just below the anterior end of the large pharyngeal basket. This mass and all the 230 CILIATE FIBRILLAR SYSTEMS fibrils of the neuromotor system stained bright red by Mallory’s method. When dislodged along with the basket by means of microdissection, the motorium was ‘a refringent body.” In stained sections, however, it ap- peared granular. ‘Fans of fibrils” join the motorivm at both its anterior and posterior ends. Those at the anterior end mark the convergence of longitudinal frbrils (Fig. 81A) of the body cilia, of ventral fibrils from the posterior region of the body and of fibrils from the mouth. The circular myonemes of the mouth are traversed by many fine fibrils that continue on toward the “‘railroad track.” A fibrillar fan from the posterior end of the moto- rium continues as the dorsal fibers to the “railroad track,” which, in turn, show a very complex system of fibrils associated with its trichites. The basal bodies of the cilia are connected not only by the longitudinal fibrils but also by cross fibrils (Fig. 81B). By methods of microdissection it was shown that (1) after destruc- tion of the motorium “there is a marked disturbance in the action of the cilia, in no way comparable to the disturbance of the cilia if other parts of the body are injured”; (2) the cilia may still exhibit a wave-like motion, but they do not reverse after the moforium is destroyed. This seemed to suggest a codrdinating function for the fibrillar system of this Chlamydodon, whose inconspicuous motor organelles are not favorable for an experimental study of modifications of their codrdinated activity. METHODS Fixatives: Schaudinn’s, Bouin’s, and strong Flemming’s. Stas: Iron-haematoxylin, Mallory’s (after Zenker’s or picromercuric fixation) . Microdissection. Concho phthirus mytili De Morgan (Kidder, 1933).—The peripheral longitudinal fibrils linking the ciliary basal bodies originate from a transverse fibril in the anterior ventral region. These almost parallel rows of fibrils pass around the posterior end of the organism uninterrupted, continue over the anterior end, and again return to the transverse fibril of the ventral surface. Each basal body is furnished with a ciliary rootlet extending toward the endoplasm. An elongate mass of homogeneous material, the motorium, follows the posterior line of the cytopharynx and continues into the endoplasm as a CILIATE FIBRILLAR SYSTEMS 231 fib. f. ees ab 1B: Figure 81. Chlamydodon sp. (MacDougall, 1928.) A. c. myon.—circular myoneme and traversing fibrils b. gr.—basal granule B. fib. f—fibrillar fan c. fib—cross fibril strand which appears to be made up of fibrils. This strand follows the posterior margin of the gullet and frays out in the endoplasm near the left side of the organism. From the outer end of the otorium originate two fibrils: one joins the basal elements of the posterior brush, while the other is the fiber of the dorsal peristomal row of cilia. The latter pro- ceeds anteriorly to join the anterior fibril and thus to the transverse fibril mentioned above. 232 CILIATE: FIBRILLAR] SYSTEMS The fused basal elements of the large oral brush of cilia form a deeply staining band or plate at right angles to the long axis of the motorium. From its anterior end two fibrils are given off which join the anterior and middle oral brush plates. Fine fibrils arise from the basal elements of the large oral brush line of the cytopharynx and connect with the motorium along its anterior side. Finally, a ventral peristomal fiber runs under the basal plate of the long ventral peristomal cilia, curves slightly to the outside, and ends just anterior to the oral brush. The brushes of cilia about the mouth seem to connect directly with the motorium but not with the peristomal fibers. This lack of connection is noted in the movements of the cilia, the peripheral and peristomal cilia beating regularly, continuously and metachronously while the beating of the oral brushes is non-continuous and synchronous. METHODS Fixatives: Flemming’s, Zenker’s (for whole mounts), Bouin’s, Zenker’s, strong Flemming’s (for sections). Stains: Heidenhain’s haematoxylin, Mallory’s triple. Concho phthirus (Kidder, 1934).—The “well integrated and closely interconnected neuromotor systems” of three species of Concho phthirus —C. anodontae Stein, C. curtus Engl., and C. magna sp. nov.—are quite comparable. The description is given of the external, internal, and peri- stomal fibrillar complexes, with reference especially to C. magna. Most of the numerous, closely set ciliary rows originate in an antero- ventral suture and terminate in a dorsal suture near the posterior end. The ventral suture comprises two fibrils which are united at their ends and are connected irregularly by cross fibrils. This ventral suture is con- tinued posteriorly as the pre-oral connecting fiber, ftom which arise two fibrils: (1) connecting the rows of basal granules of the dorsal lip, and (2) connecting the basal granules of the pharyngeal ciliary row. The pre-oral connecting fiber itself becomes the peristomal net fiber. The latter gives off secondary fibrils to the peristomal field. On the left, these secondary fibrils are bounded by a longitudinal znner net fiber (Fig. 82) from which arise numerous fine fibrils that line the ventral side of the peristomal basket. On the floor of this basket, these fine fibrils join the nner basket fiber, which, in turn, gives off many branches that line the dorsal surface of the basket. These branches then unite with the CILIATE FIBRILLAR SYSTEMS 233 fiber (noted above) that connects the basal granules of the dorsal lip. At the anterior, inner end of the pharynx is a large pharyngeal ring fiber that fuses with the snner basket fiber, previously noted, to form the fibrillar bundle. This point of fusion is regarded as comparable to the motorium of C. mytil7, From the fibrillar bundle a gullet fiber extends inward and courses throughout the floor of the gullet, finally fraying out at the posterior end. The inner basket fiber is united posteriorly with the posterior basket connecting fiber. The latter, bending dorsally and to the right, comes to join the post-oral connecting fiber. Thus a direct connection is made between the peristomal region and the dorsal suture. From this post-oral fiber, numerous cross fibrils connect with adjacent ciliary rows. “This neuromotor system is thought to be mainly conductive but some parts of it may possibly be contractile or even supportive.” METHODS Fixatives: Klein’s (1926), von Gelei-Horvath’s (1931), strong Flem- ming’s for four hours. Stains: Heidenhain’s haematoxylin, destained with hydrogen peroxide. Dallasia frontata Stokes (Calkins and Bowling, 1929).—The most conspicuous part of the neuromotor system of Dallasza frontata was found to be the complicated apparatus of the mouth. This is composed of a tongue running through the buccal cavity, supported by bars which are anchored in long strands of dense material lying on the floor of the buccal cavity. There are two of these /ongitudinal strands and two series of bars from the tongue, one on each side. On the right and left sides are undulating membranes. A Jadder-like organ originates anteriorly just below the membrane of the buccal cavity and at the right side of the mouth, and runs into the gullet. A discoidal mass on the left side of the gullet is interpreted as the motorium. It is connected by fibers directly to the proximal end of the tongue and strands. Similar fibrils connect the outer and the inner margins of the /adder-like structure with the motorium, and these fibers appear to form the outer and inner margins of this organ. Minute granules are present at the ends of each bar, at the points where the bars join with the longitudinal fibers. Posteriorly two fine fibers run from the motorium 234 CILIATE FIBRILLAR SYSTEMS x a <>) el ph. r. fib. er ee LY) Fi DOB: ay TESTS Se a m Ary CLS 4 SS LY ANNA Gf prom se Figure 82. Conchophthirus magna. (Kidder, 1934.) Diagram of the fibrillar system of peristomal region. d. f. p. b.—dorsal fibers of peristomal basket d. I. c. r—dorsal lip ciliary row fib. b—fibrillar bundle gul. fib—gullet fibril i. n. fib.—inner net fibril in. b. filb—inner basket fibril 1, 2, 3, 4—rows of body cilia p. b. c. f.—posterior basket connecting fibril p. o. c. f—pre-oral connecting fibril p. o. c. f'.—post-oral connecting fibril ph. c. r—pharyngeal ciliary row ph. r. fib.—pharyngeal ring fibril v. f. p. b—ventral fibril of peristomal basket deep into the endoplasm, where they are lost, one in the vicinity of the macronucleus, the other near the contractile vacuole. Basal granules are described, but no connecting fibrils mentioned. The function of this enigmatical organ is purely conjectural; possibly it has something to do with the opening and closing of the mouth. . . . What- ever the specific function may be there is little reason to doubt that it is intimately connected with the irritability of the mouth region. CILIATE FIBRILLAR SYSTEMS 235 METHOD (whole mounts and sections) Fixatives: Schaudinn’s, made up in 95-percent ethyl alcohol, followed by prolonged treatment with turpentine. Stains: Iron haematoxylin; solution of acid fuchsin and methyl green. Dileptus gigas (Visscher, 1927).—From a rod-like motorium found near the base of the gullet, several sets of fibrils extend to different parts of the body, as follows: (1) one set supplies the wall of the fun- nel-shaped gullet, (2) two more distinct fibrils course anteriorly along the proboscis, one on either side of a row of trichocysts; and (3) a set of branching finer fibrils spreads posteriorly over the body, where they Figure 83. Dileptus gigas. Neuromotor apparatus (camera drawing). (Visscher, 1927.) a. fib.—anterior fibril f. fib—fine fibrils i. fib—inner fibrils mot.—motorium o. fib.—outer fibrils t.—trichocysts 236 CILIATE FIBRILLAR SYSTEMS are probably associated with the longitudinal rows of cilia running paral- lel to the contractile fibrils (Fig. 83). METHODS Fixative: Schaudinn’s. Stains: Iron-haematoxylin, acid borax carmine. Entodiscus borealis (Powers, 1933) .—The following distinct groups of fibrils and associated structures characterize the fibrillar system of Entodiscus borealis: (1) the stomatostyle, with its dorsal and ventral anterior horns and their adoral fibrils; the labial fibrils; the pharyngeal fibrils; and the circum pharyngeal rods; (2) the anterior fibrillar center, or motorium, with its anterior, posterior, and marginal strands; the pos- terior auxiliary fibrillar center with its associated strands; and (3) the peripheral transverse commissural fibrils interconnecting the basal gran- ules. Closely associated with the commussural fibrils are the distal branch- ing ends of most of the internal fibrils. The ventral peripheral layer is further complicated by the long (6-12 y) célzary rootlets, which ex- tend into the endoplasm from the basal granules of the cilia. The pellicle of the ventral surface is thicker than elsewhere and is highly differentiated owing to pellicular fibrils accompanying each longitudinal ciliary row. These fibrils are conspicuous only at the anterior end. They are interpreted as either supporting or contractile in nature (Fig. 84). METHODS Fixatives: Schaudinn’s, Da Fano’s, 25-percent osmic acid, Flemming’s without acetic acid (the two latter fixatives for sections). Stains: Iron alum haematoxylin, Yabroff’s silver method. Entorhipidium echini (Lynch, 1929).—One fibrillar system of Ento- rhipidium echini consists of a motorium which is connected to a network of peripheral fibrils linking all the basal granules. The other set of fibrils is developed in the pellicle, chiefly on the ventral surface. The motorium is located to the left of the buccal cavity. It is com- posed of five heavy strands united into a rod-like body by /ongitudinal fibrils which appear to be continuous with the newrofibrils of the basal granules. These delicate Jongitudinal fibrils unite the basal granules of each peripheral row of cilia, and other fibrils encircle the body, uniting od. ad. #6. Ger DNC. eBwela@ Zon Tio: Figure 84. Entodiscus borealis. (Powers, 1933.) A. Reconstruction of neuromotor system, dorsal surface (modified from author). B and C. Relations of body cilia and their basal granules with fibrils of neuromotor system. a. fib. c-—anterior fibrillar center b. gr.—basal granule cil. r—ciliary rootlet d. ad. fib.—dorsal adoral fibril 1. m. fib.—left marginal fibril 1. pell—tlongitudinal pellicular thickening li. cyt—lips of cytostome p. fib.—posterior fibril p. fib. c.—posterior fibrillar center ph. fib—pharyngeal fibril r. m. fib.—tright marginal fibril stom.—stomatostyle t. f—transverse fibril v. ad. fib.—ventral adoral fibril 238 CILIATE FIBRILLAR SYSTEMS the basal granules with perfectly regular transverse commissures. In the region posterior to the frontal lobe, the commissural fibrils branch, form- ing numerous collaterals extending in various directions. These unite with transverse and longitudinal fibrils. The pellicular thickenings of the frontal lobe are present in the form of heavy, deeply staining fibers which alternate with the ciliary rows. Be- tween the anterior ends of these and the anterior ends of the dorsal rows of cilia is a delicate fretwork, or polygonal area. The boundaries of polygons correspond with the boundaries of a double row of large vacuoles. Posterior to the frontal lobe, the eavy fibrils become fine and lie so close to the xewrofibrils that they are almost indistinguishable from them. Similar fibrils are evident on the dorsal surface of the organism only at the anterior end (Fig. 85). METHODS Fixatives: Schaudinn’s, Da Fano’s, 2-percent osmic acid, Flemming’s with- out acetic acid (the two latter for sections). Stains: Iron-haematoxylin, Yabroff’s silver method. Eupoterion pernix (MacLennan and Connell, 1931).—Most of the longitudinal ciliary fibrils of the body take their origin from a heavy bar, the anterior connective fibril, that lies dorsoventrally across the anterior tip, whence they extend to the posterior end, where they fuse. Additional fibrils arise in pairs along the pre-oral suture line and these fuse mostly also at the posterior end. Each basal granule gives off one or two com- missural fibrils to adjacent ciliary rows, resulting in a fairly regular lat- ticework (Fig. 86). The neuromotorium lies beneath the wall of the cytostome. The fibrils of the four pairs of oral ciliary rows along the oral groove are fused in a V-shaped figure at the apex of the suture line. All of these end directly in the motorium or are closely connected to it by the transverse fibril or by the longitudinal ciliary fibrils. The transverse fibral lies actoss the end of the neuromotorium, its right end joins the pharyngeal fibrils, thus forming a pharyngeal strand; and its left end fuses with the two outer- most peristomal ciliary fibrils and ends farther left in a connective fibril of adjacent outer rows of cilia. The two rows of cilia arising from the anterior ends of the outer peristomial rows (of the ordinary peripheral pp Te 4... br fib ERre aN Figure 85. A. Entorhipidium echini. (Lynch, 1929.) Cross section, showing periplast of the anterior dorsal fibril. B. Extorphidinm echini, tangential section of anterior ventral surface. A. b. gr.—basal granule B. comm.—commissural neuro-fibril 1. p. fib—longitudinal pellicular fibril |. fib—longitudinal fibril t.—trichocyst p. fib.—pellicular fibril tr. fib—transverse fibril 240 CILIATE FIBRILLAR SYSTEMS type) are connected to the rest of the body rows by commissural fibrils, thus uniting the peristomial cilia with the rest of the body cilia. METHODS Fixative: Schaudinn’s (60° C.). Stain: Heidenhain’s iron haematoxylin. Haptophrya michiganensis “Woodhead (Bush 1934).—Haptophrya michiganensis has an integrated system of fibrils that center in a moto- Figure 86. Expoterion pernix, optical section. (MacLennan and Connell, 1931.) a. c. fib.—anterior connective fibril n. mot.—neuromotorium b. gr.—basal granule ph. fib—pharyngeal fibril cyto.—cytostome ph. str—pharyngeal strand ectop].—ectoplasm tr. fib.—transverse fibril rium. Within the sucker at the anterior end is a fibrillar ring, homologous with the esophageal ring of stomatous ciliates. The motorium is located in the center of this ring and is connected with it by radzal connectives. Accessory bodies of the motorium are suspended from various points on the inner edge of the ring. (These are not evident during fission.) Nu- merous myonemes radiate from the inner edge of the fibrillar rng and extend to the opposite walls, posteriorly and laterally, dividing into fine fibrils at their outer ends. Equally spaced perspheral myonemes arise from the external edge of the fibrillar ring, adhere to the inner layer of CILIATE FIBRILLAR SYSTEMS 241 the ectoplasm, and extend to the posterior end of the animal. These myonemes are “‘closely associated with the basal granules.” Commissures connecting the basal granules form a close network over the entire body. Supporting fibrils from the nuclear membrane, the endoplasmic cone, and the contractile canal extend to the peripheral ectoplasm (Fig. 87). The deeply staining mass was interpreted as a motorium because *' (1) it is connected, directly or indirectly, with all parts of the fibrillar system; (2) it is near the anterior end of the ciliate; (3) if, with this mass, the sucker is removed, the animal loses its power of worm-like forward movement even though the cilia continue to beat; and (4) a toxic sub- stance acts first upon the anterior part, particularly the sucker, where- upon the animal ceases its forward movement.” METHODS Fixatives: Schaudinn’s and Zenket’s. Stains: Delafield’s and Heidenhain’s haematoxylin; also Kolatschev’s osmic impregnation, as outlined by Bowen; Yabroff method. Ichthyophthirius (MacLennan, 1935).—The longitudinal fibrils con- necting the basal granules beneath the ciliary rows of the body surface ate linked together anteriorly and to some extent posteriorly by small centers, the anterior and the posterior fields. The centers are connected by a “‘suture fibril” which marks the ventral side. The suture fibril is interrupted by the oral region, thus dividing it into pre- and post-oral segments. Concentric accessory suture fibrils lie on the sides of the oral region and terminate anteriorly and posteriorly in the suture fibrils. The lip of the oral opening is bounded by (1) the outer peristomal fibrils, which are also linked to the suture fibril. (2) Circular fibrils line the walls of the oral cavity and radial fibrils intersect the two sets of fibrils at right angles. These transverse connections between the ciliary fibrils are present only in the oral region. Two heavy basophilic rods, each attached to a heavy esophageal fibril are located near the esophageal plug. An inner peristomal fibril runs from this bilobed newromotorium to the basal granules. Ciliary rootlets are developed in the region of the inner peristomal fibrils; they are less well developed in the region of the outer peristomal fibrils, and not found in the region of ordinary body cilia. About 50-100 individual ciliary rootlets combine to form numerous esophageal strands, Pell. 2-==n= NUC. Me : i --- -- endopl. 116. cont.can. f16. ia. a 3 = Se. Biel & CONG. Cal. eG fre eR aiho: io) ola a comm. - Figure 87. Haptophrya michiganensis. (Bush, 1934.) A. Diagrammatic section show- ing one-fourth of the animal body with part of the macronucleus. B. Diagram of anterior part of neuromotor system. acc. mot.—accessory motorium mot.—motorium b. gr.—basal granule macro.—macronucleus comm.—commissures nuc. fib—nuclear fibrils c. fib.—coarse fibrils nuc. m.—nuclear membrane cont. can.—contractile canal pell.—pellicle cont. can. fib.—contractile canal fibrils per. my.—peripheral myoneme endopl. c.—endoplasmic cone rad. my.—tradial myonemes endopl. fib—endoplasmic fibrils rad. conn.—radial connectives fibr. r.—fibrillar ring ret. fib.—reticulate fibrils CILIATE FIBRILLAR SYSTEMS 243 each of which turns sharply and continues to the endoplasm, parallel to the main axis of the oral pit. METHOD Fixatives: Schaudinn’s, Zenket’s. Stains: Heidenhain’s iron-haematoxylin, Delafield’s (for fibrils), Klein’s (1926) (for ciliospores), Lund’s wet silver method (for adults). Lechriopyla mastax (Lynch, 1930).—The cilia of Lechriopyla mastax can be divided into three areas: (1) the cilia of the general surface of the body, (2) the cilia of the peristome, and (3) a transverse band of cilia known as the supraoral band. The basal granules of all these cilia are connected by delicate longitudinal fibrils without transverse con- nectives. Below the ciliary lines of the peristome and vestibule are verti- cal pellicular lamellae, which are fused to the furcula described below. A crescent-shaped neuromotorium lies beneath the pellicle at the left end of the peristome. Its ends are continued as long fibers, the anterior and posterior adoral fibers, which form a complete (or nearly complete) ring about the peristome. From the ring arise the ciliary lines which extend over the surface of the body, or pass into the peristome and the pharyngeal involution. A variable number of fibrils arising from the outer border of the motorium extend through the cytoplasm for vary- ing distances, to fuse with the pellicle just beneath a ciliary line. They may branch or anastomose, and they become more delicate distally. The furcula, shaped much like a heavy tuning fork, partly surrounds the vestibule. The ends fuse with the walls of the pharynx, and delicate fibrils from the pharyngeal wall extend to the furcula. This organelle may be an additional element of the neuromotor system. A long pellicular fiber extends from the left end of the internal open- ing of the cytopharynx to the middle of the posterior end of the organism and occasionally curves along the right side for varying distances. This fibril is not included in the author’s description of the neuromotor sys- tem. METHODS Fixatives: Bouin’s, Schaudinn’s, Da Fano’s cobalt nitrate-formalin. Stains: Iron alum haematoxylin, carmines, cochineals, Yabroff’s silver- nitrate method (no success), Klein’s silver method. Sections prepared in a variety of ways. 244 CILIATE FIBRILLAR SYSTEMS Ptychostomum chattoni Rossolimo (Studitsky, 1932) .—The mouth of this parasitic ciliate is at its posterior end. At the anterior end is a horse- shoe-shaped sucker with a projecting rim for attachment. The sucker (Frxationsapparat) is provided with a system of fibrils. The largest fibril, the perspheral cord, borders the sucker and gives off at its ends fine fibrils that extend into the cytoplasm. The sucker’s disc has four sets of fibrils: (1) the deeper set comprises two groups of paral- lel fibrils that cross each other at a sharp angle as they traverse the disc, both groups coming to adhere to the per/pheral cord; (2) the uppermost set, visible in the living organism, courses from right to left and from anterior to posterior; (3) a third set of fine fibrils are attached to the peripheral cord by their anterior ends; and (4) the fourth set, com- posed of sixteen or seventeen strands that run from right to left and from anterior to posterior, frays into four or five fine fibrils at the anterior end of each strand, to become attached to the peripheral cord and to other adjacent fibrils; the posterior ends of these strands become fimbri- ated also into fine fibrils. All apparently serve for support. METHODS Fixatives: Schaudinn’s, Carnoy’s, Bouin’s, Champy’s and Benda’s (for whole mounts), Altmann-Kull’s (for sections). Stains: Heidenhain’s haematoxylin, safranin, von Gelei’s toluidin blue, Mallory’s triple. B. HETEROTRICHA Balantidium coli Malmsten and B. sus sp. nov. (McDonald, 1922) .— The motorium of Balantidium suis lies within the ectoplasm of the apical cone, close to the right ventral wall of the esophagus. A fibril encircling the esophagus arises and ends at the anterior end of the moto- rium, where the ddoral ciliary fiber also arises. The circumesophageal frbril has irregular enlargements, from which fibers pass both posteriorly and anteriorly into the ectoplasmic mass of the anterior end. These fibrils appear to fade out in the ectoplasm. The adoral fiber connects the basal granules of the adoral cilia. The remainder of the fibrils are not directly connected with the motorium. The basal granules of the peripheral longitudinal spiral rows of cilia are so Closely set that it has been impossible to see a fibrillar connection. No transverse fibrils connecting the rows were observed. A ciliary rootlet CILIATE FIBRILLAR SYSTEMS 245 extends from each basal granule, and a second small granule is found at the junction of the ectoplasmic and endoplasmic layers. The ectoplasmic layer is quite thin, except at the anterior end of the organism, where it is deep and the distance between the granules of each ciltum correspond- ingly long. The rootlets of the row of adoral cilia around the margin of the peristome, the “radial fibrils,” are exceedingly long, ending in about the posterior third of the body without connection or attachment. The cilia immediately posterior also have long rootlets, but they become shorter as they approach the base of the apical cone (Fig. 88). METHODS Vital stain: Neutral red (differentiates the neuromotor apparatus). Fixatives: Schaudinn’s, Zenker’s, Formalin, osmic acid, picrocuric, 60-80° C. Whole mounts and sections Stains: Iron haematoxylin, Mallory’s triple stain (particularly for sections). Figure 88. Balantidium coli, cross section of peripheral region. (McDonald, 1922. b. gr.—basal granule gr. b.—granular band cil. r.—ciliary rootlet hy. b.—hyaline band endo.—endoplasm pell.—pellicle Balantidium sushilii (Ray, 1932).—Fibrils associated with the con- tractile vacuoles have been described in Balantidinm sushili, Each of the two lateral vacuoles has a fibril running from the wall of its outer half to the neighboring pellicle. The neck of the terminal vacuole 1s sur- rounded by a diaphragm of fibrils running from the wall of the neck to the surrounding pellicle. These unusual fibrils are described and figured in the extended as well as the contracted condition. An axial and a peripheral system of fibrils can be seen also in the living organism. Stained preparations show that the former consists of 246 CILIATE FIBRILLAR SYSTEMS three or four fibrils, parallel or twisted after the manner of a rope, which originate below the pellicle at the anterior end and extend posteriorly a short distance beyond the mouth. The individual fibrils gradually be- come thinner toward the posterior end, where they come in contact with the limiting membrane formed by some of the fibrils of the peripheral system. At the anterior termination of these fibrils is found a knob-like structure which may be imbedded in the epithelium of the host. The peripheral system of fibrils is arranged in two conspicuous arches along the left anterior border of the organism. Anteriorly, fibrils extend both to the right and to the left peristomal lips. Posteriorly, the two arches converge. Some of the fibers continue mesially and come to form a kind of limiting membrane beyond which no fibrils are traceable. The group of fibers forming the axial system, together with the borer attached at its anterior end, is called the boring apparatus, as the first of its kind to be noted. It may be compared with the axostyle of some flagellates. The peripheral system is believed to represent morphonemes. METHODS Fixatives: Brasil’s modification of Bouin-Duboscq’s (for whole mounts), Bouin’s alcoholic, twenty-four hours (for sections 5 wp). Stam: Heidenhain’s haematoxylin (whole mounts and sections). Fabrea salina Henneguy (Ellis, 1937).—In Fabrea there are numer- ous longitudinal rows of closely set body cilia (in pairs). These are interrupted on the ventral side by a coiling adoral zone. The basal gran- ules are connected by fine longitudinal fibrils. No transverse connections or ciliary rootlets were observed. Each membranelle consists of two rows of basal granules whose ciliary rootlets fuse into a single plate, the basal lamella. Each longitudinal fibril of the dorsal and ventral surfaces, with the exception of those that merge with one another, is connected at the adoral zone with the basal lamella of a membranelle. The basal lamella is connected with the adoral fibril by fibril running across the peristomal groove. The adoral fibril starts at the anterior tip and follows the course of the inner border of the adoral zone. The peristomal fibril is continued beyond the end of the adoral zone on the wall of the funnel and ends in a ganglion-like body on the left wall of the ventral lobe. From this motorvium arise several fibrils—the adoral fibril, which follows the course CILIATE FIBRILLAR SYSTEMS 247 of the adoral zone, and other fibrils which appear to end blindly in the endoplasm of the ventral lobe. Anteriorly the fibrils of the frontal field tend to converge and end very obliquely on the adoral fibril. No pellicular pattern was demonstrated by any of the silver methods. but they did show the longitudinal fibrils connecting the cilia. METHODS Fixatives: Schaudinn’s, Bouin’s and Flemming’s. Stains; Iron-haematoxylin, Mallory’s triple. Silver method: Yabroft’s modification of Da Fano’s. Metopus circumlabens (Lucas, 1934).—The motorium of Metopus circumlabens lies posterior to the cytostome. From its left side it gives rise to a pair of ventral adoral fibrils which follow the peristomal curvature outward to the oral margin and there end in a sort of arborization. Each row of peristomal membranelles arises immediately in contact with a con- nective between these two fibrils. A dorsal adoral fibril extends from the right side of the motorium. At slightly irregular intervals is gives rise to from ten to twenty heavy connectives, which may partially fuse in pairs as they curve beneath the dorsal wall of the peristome to its left side. There they turn ventrad and unite with the ventral adoral fibers. A fibrillar pharyngeal strand arises from the posterior end of the moto- rium. Its course varies in different organisms, but it usually lies along the right lateral wall of the organism to the right of the cytopharynx. Near the posterior end of the latter structure it forms a large spiral coil. The entire body surface, except the right lateral margin, is covered with rows of cilia. Longitudinal ciliary fibrils are present, but no com- missural fibrils were observed. Each basal body in the most dorsal of the five rows of the crest cilia gives rise to rootlets which end freely in the cytoplasm. A relation between these peripheral fibrils and the fibrils of the motorium seems to be suggested by the numerous fine branches which arise from the ventral adoral fibrils. These extend indefinitely into the cytoplasm toward the longitudinal ciliary rows of the ventral surface. In view of the contrastingly striking and obvious specialization in the fibrillar structure of the neuromotor system about the peristomal, pharyngeal, and central endoplasmic regions of the cell, one is inclined to believe that the neuromotor system of this ciliate is vitally, though not exclusively, con- cerned in the conductile functions related to the metabolic activities of the organism. It is possible that, because they are located within the mobile 248 CILIATE FIBRILLAR SYSTEMS cytoplasm of the protozoan, the stouter of these various fibres may serve in addition some function in the nature of support. METHODS Fixatives: Schaudinn’s, Bouin’s, Jorgensen’s, Van Rath’s. Stains: Heidenhain’s iron haematoxylin, Regaud’s haematoxylin, Mallory’s triple (whole mounts and sections). Nyctotherus hylae (Rosenberg, 1937).—A detailed description of the neuromotor system of Nyctotherus hylae is given, including two centers and a group of special fibrils believed to control the reversal of ciliary action. An incomplete account of some of these structures was given by Kirby (1932) for N. sylvestrianus. The movements of N. Aylae were studied by cinematographic methods. From the main moforium located at the distal end of the cytopharynx arise two sets of unbranching fibrils. One set extends along the anterior border of the pharynx, eventually terminating at the end of the peristome. The other set follows the posterior border and arm of the ectoplasmic thickening. Some of the latter fibrils unite at the cytostomal border with the peripheral per7stomal fibril. The so-called “reversal fibrils’ originate from the posterior part of the moforium, radiate through the endoplasm, and at their distal ends unite with the ciliary lines at several points, not including the presutural ciliary lines. The membranelles have each a basal plate and two rows of basal gran- ules, from which fine fibrils connect with the circum pharyngeal fibrils. The latter become the transverse peristomal fibrils, of which there are at least two for each membranelle of the series. A number of fibrils from the motorium directly connect with the anterior neuromotor center. From this structure arise many ciliary lines that connect rows of basal granules. Commissural fibrils between these lines are present only in the apical post-sutural region. The lateral and sagittal sutures which divide the ciliation into definite regions were interpreted as probable conductors between ciliary lines. The pharyngeal terminus, a deeply staining structure, gives rise to a post-pharyngeal bundle of fibrils that have no apparent distal attach- ment. Kirby (1932) described a similar “‘band formed structure” in N. silvestrianus which may extend beyond the cytopharynx, its course in the endoplasm varying in different individuals. He considers it “ho- CILIATE FIBRILLAR SYSTEMS 249 mologous with the ‘continuation tube’ of the ‘subpharyngeal canal’ de- scribed by Higgins (1929) in N. cordiformis” (Kirby, 1932, p. 298). Whether or not this strand is a part of the neuromotor system is an open question. Fibrils interpreted as mor phonemes are as follows: (1) those extend- comm. fib. tr. perist. 116. per. perist fib. _1, q CieiOu) os ana moet. sh, B fib. Af 4 0s, a aoe oa Figure 89. Nyctotherus hylae, fibrillar system, diagrammed. (Rosenberg, 1937.) ant. neur. cen.—anterior neuromotor cen- ter c. b. b.—ciliary basal body c. 1—ciliary line ca. fib—caryophore fibril cir. fib—circumpharyngeal fibril comm. fib——commissural fibril 1. ph. fib—longitudinal pharyngeal fibril lat. sut.—lateral suture mot.—motorium n. env.—nuclear envelope p. |. ph. fib—posterior pharyngeal fibril p. ph. b.—post-pharyngeal bundle ph. term.—pharyngeal terminal per. perist. fib—peripheral peristomal fi- bril re. fib.—reversal fibrils sh. s. fib—shelf supporting fibrils tr. perist. fib.—transverse peristomal fibril longitudinal 250 CILIATE FIBRILE ARTS SiEMS ing from the right to the left of the body, (2) the caryophore fibrils, and (3) the shelf-supporting fibrils (Fig. 89). METHODS Fixatives: Schaudinn’s (5-percent acetic), Flemming’s. Stain: Heidenhain’s iron haematoxylin, aqueous and alcoholic. Silver techniques: Klein, Gelei-Horvath, Yabroff (negative). Spirostomum ambiguum Ehrbg. (Bishop, 1927).—The ridges and furrows in the ectoplasm of Sprrostomum ambiguum follow a sinistral spiral course from the anterior to the posterior end of the body. Be- neath the furrows lie thread-like myonemes, somewhat beaded in ap- pearance, but without light and dark alternating bands. The myonemes taper gradually as they approach either end of the body and finally disap- pear from view. They are not attached to any structure. Longitudinal myonemes were found on either side of and running parallel to the band of peristomal membranelles, except along those membranelles nearest the cytostome. No evidence was obtained confirming the presence of other fibrils such as neurophanes. On the anterior side of each myoneme lie the basal granules of the body cilia. The rows of granules are parallel to and slightly above the level of the mzyonemes. No ciliary rootlets nor connections between basal granules or myonemes were discovered. The system of fibrils underlying the membranelles (Fig. 90) of S. ambiguum includes: an anterior basal fibril extending from the anterior end of the body to the beginning of the peristomial depression; a middle fibrillar system which varies in its course in different individuals, but which collects the end-threads of the membranelles lying on the left side of the peristomial depression; and a posterior basal fibril into which the end-threads of the membranelles at the posterior end of the peristomial depression and in the cytopharynx join. A connection between the posterior basal fibril and the middle fibrillar system is seldom found, and there is always a break between the middle fibrillar system and the anterior basal fibril. A central body to which the fibrils join was found in no case. METHODS Fixatives: Schaudinn’s, picro-mercuric (hot). Stains: ron-haematoxylin (alcoholic and aqueous), Mallory’s triple (Sharp’s modification), Fuchsin S. GIEVATE FIBRILLAR SYSTEMS 25 1 Figure 90. Spirostomum ambiguum. Diagram of membranelles and __ their intracytoplasmic structures. (Bishop, 1927.) b. fib.—basal fibril b. 1.—hbasal lamella b. pl.—basal plate e. thr.—end thread memb.—membranelle 52 AA b. fb. C. OLIGOTRICHA Diplodinium ecaudatum (Sharp, 1914).—The motorium is located in the ectoplasm above the base of the left skeletal area and between the left extremities of the dorsal and the adoral membranelle zones. Dorsally it is connected with the bases of the dorsal membranelles by a dorsal motor strand, which also sends a branch along the base of the inner dorsal lip. A ventral motor strand connects the motorium with the bases of the adoral membranelles and its branch and passes along the base of the inner adoral lip. Numerous fibers from the motorium follow the contour of the operculum and disappear near the base of the right skeletal structure. A circumesophageal ring surrounds the esophagus at the level of the outer adoral furrow, from which a fibril connects with the 70- torium, Certain fibrils in the wall of the esophagus appear to unite with the circumeso phageal ring; others are attached to skeletal structures and are considered contractile fibrils. Rootlets from the oral cilia end in, or close to, the ring. A codrdinating (conductive) function was ascribed to this fibrillar system of D. ecaudatum, because of its initimate relation- ships with the motor organelles and its complete structural integration through the motorium. METHODS Fixatives: Schaudinn’s (alcoholic, hot), Zenker’s, Flemming’s, Worcestet’s, Bouin’s, formalin 4 percent, osmic acid one percent (formalin 36° C.) Stains: Heidenhain’s haematoxylin and Mallory’s triple. 22 CILIATE FIBRILLAR SYSTEMS Diplodinium medium (Rees, 1931).—The structure of D. medium is compared with D. ecaudatum as described by Sharp. The disagreement between Rees’s interpretation and the interpretation of Sharp has to do primarily with the ectoplasmic layer directly under the pellicle, except in the region of the adoral lip and the inner boundary layer of the ecto- plasm. In D. medium this layer, according to Rees, is more prominent than in D. ecawdatum and consists, instead of fine alveoli, of an znter- woven network, or complex system of fibrils. Serial cross and longitudi- nal sections, 3 p. in thickness, made it possible to trace the ectoplasmic layers. Cross sections of D. medium show a fold of this middle layer of the ectoplasm which corresponds in its position to Sharp’s motorium. Further- more, the esophageal ring described by Sharp is interpreted as section of the inner boundary layer of ectoplasm. The fibers connecting the mortorium with the membranelles and esophagus could not be differ- entiated. The ciliary rootlets are attached to membranes composed of sheets of the fused middle and inner layers of ectoplasm, which in turn are at- tached to the fibrillar system of the ectoplasmic layers The structures are membranes, according to Rees, because “‘one or the other of them occurs in all longitudinal and oblique sections, whether cut with reference to the parasagittal plane or to a plane at right angle or at any other angle to it.” The esophageal tractor strands are considered to be a part of the ectoplasmic network of fibrils. The membranes, instead of strands, are believed by Rees to function in the retraction of the adoral and the dorsal cilia. The occurrence of fine fibrillae in the non-ciliated ectoplasm of Diplodinium is of interest in connection with other papers on the neuromotor system. It is obvious that in the latter ciliate the fibrillae of the ectoplasmic layers have no relationship to a neuromotor system. METHODS Fixatives: Not listed. Stains Iton-haematoxylin, Zirkle’s N-butyl alcohol method of dehydration. Diplodinium Schbg. (Kofoid and MacLennan, 1932).—In addition to the neuromotor apparatus as described by Sharp (1914) for D. ecauda- tum and incidentally confirmed in this systematic investigation, a fibrillar CILIATE FIBRILLAR SYSTEMS 253 complex also was found to occur in the caudal spines of D. dentatum, in contrast to the apparently structureless spines in Entodinium. Along the bases of the caudal spines appeared a heavy marginal fibril from which finer anchoring fibrils extended into each spine, terminating under the cuticle of its outer margin. A very heavy main anchoring fibril bordered the inner edge of each spine. From the anchoring fibril of the ventral spine smaller fibrils branched toward the anus, where they ended, one on each side. Small branches from these coursed in the wall of the rectum, parallel to its main axis. No connection was evident between this fibrillar complex of the caudal spines of D. dentatum and its neuromotor system. The location and re- lationships of the former suggested a supporting function similar to that of the longitudinal surface fibrils. Also, since these spines undergo a change in their curvature such as might obviously be facilitated especially by the main anchoring fibril together with the marginal fibrils, these caudal fibrils were considered to be myonemes. A similar fibrillar system had been described by Bélaf (1925) in Epidinium caudatum, on the basis of which Reichenow (1929) denied a neuromotor function for all fibrils of the Ophryoscolecidae. The clear difference in the morphological relationships of the two fibrillar systems in D. dentatum, however, indicated that these systems have quite differ- ent functions: The caudal fibrils are admirably situated to serve as supporting and contractile structures. The motor fibrils are so situated as to be of little or no use either as supporting or as contractile fibrils. The caudal fibrils show no con- nection to the motor organelles. The motor fibrils link together (through the neuromotorium) all the motor organelles of the individual. METHODS Fixative: Schaudinn’s. Stain: Iron-haematoxylin. Favella jorgensen (Campbell, 1927).—The neuromotorium 1s a spindle-shaped body in the ventral ectoplasmic wall in the mid-region of the gullet. This organelle gives rise to five intracytoplasmic fibrils as follows: (1) the adoral fibril, extending to and interconnecting the mem- branelles; (2) the circumesophageal fibril, with branches surrounding the gullet; (3) a dorsal fibril which appears to connect with the striations 254 CILIATE. FIBRILLAR SYSTEMS of the oral plug; and (4, 5) two ventral fibrils extending downward and ending freely in the endoplasm. It was observed that the mem- branelles not only serve the organism in feeding and in locomotion, but, during periods of binary fission, function in the building of the lorica. In addition to their fibrillar connection with the motorium, each mem- branelle is supplied with three large basal bodies. METHOD Fixative: Schaudinn’s (aqueous and alcoholic), 90° C. Stain: Iron-haematoxylin (whole mounts and sections). Tintinno psis nucula (Campbell, 1926).—The somatic ciliation is con- fined to the column and forms in longitudinal rows along the myonemes. These showed basal granules, but without fibril connection. The myo- nemes are ectoplasmic structures, arranged longitudinally and un- branched. Anteriorly, they extend to the reflexed margin of the collar and possibly connect with basal granules of the adoral membranelles; posteriorly, they fade out. At the base of each adoral membranelle are three basal granules con- nected by fibrils. Through this triangular base passes the adoral motor fibril. Three oral membranelles (flat plates of fine cilia) follow the spiral of the gullet. Each oral membranelle ends in a distinct basal body. The ciliary membrane (undulating membrane of unusual construction), which functions in house-building and repair, is connected through its basal granules to the adoral fiber. A retractile tentaculoid is found be- tween each adoral membranelle. Tentaculoids, accessory combs, and trichocysts have no known fibrillar connection. The motorium is located in the ectoplasm of the ventral wall of the column. From it arise directly (1) the adoral fiber (granular), which connects with the adoral membranelles, the oral membranelles, and the ciliary membrane; (2) two dorsal fibers, extending into the ectoplasm adorally, where they end freely; and (3) the ventral fiber, which extends downward and also ends freely in ectoplasm. The circumesophageal ring is connected to the motorium indirectly by a single fiber. Short fibers from the r7vg surround the gullet. METHODS Fixatives: Schaudinn’s, 90° C. Stain: Heidenhain’s iron-alum haematoxylin, aqueous and alcoholic. GILTATE FIBRIELAR SYSTEMS 255 D. HYPOTRICHA Oxytricha (Lund, 1935).—The parts of the neuromotor system of Oxytricha apparently are confined to the more specialized organelles. No fibrils were found in connection with most of the ventral cirri. A long membranelle fibril connects the inner ends of the membranelle plate. The frontal membranelles, in addition, have célzary rootlets which arise only from the most proximal basal granules of each of the three rows. These combine into a stouter fibril for each membranelle, which ends free in the endoplasm. Along the ventral margin of the peristome are numerous fibrils (Fig. 91). One of these connects the basal granules of the undulating mem- 4 EE CEOS \ \ R S S> SSS > Figure 91. Oxytricha. Diagram of fibrillar complex of cytostome, ventral view. (Lund, 1935.) b. fib. u. memb.—basal fibril of undulating membrane b. pl. memb.—basal plate of membranelle d. cy. fib.—dorsal cytostomal fibril ect. f.—ectoplasmic fold memb. r.—membranelle rootlet mg. fib. memb.—marginal fibril of membranelles mg. u. memb.—position of marginal undulating membrane p. e. fib—post-esophageal fibril term, fil—terminal filament v. cy. fib—ventral cytostomal fibril 256 CILIATE FIBRILLAR SYSTEMS brane. Twenty-two originate near the anterior end of this fibril. Their posterior destinations are as follows: ten terminate in ten small granules attached to the marginal fibril in the region of the posterior seven or eight membranelles; six pass along the dorsal wall of the gullet and extend into the endoplasm to a point near the right side of the body; and the other six continue into the gullet to form the ventral post-esopha- geal fibrils. These fibrils along the right side of the peristome were ob- served to be lax, apparently nonelastic, and capable of individual move- ment. A delicate fibril extends anteriorly from each of the five anal cirri. In the region at the left of the posterior macronucleus they disappear from view. Their position suggests, however, that they may join the other parts of the neuromotor system, in the region of the posterior wn- dulating membrane fibril. METHODS Fixatives: Schaudinn’s (with 5-percent acetic), Zenker'’s. Figure 92. Uroleptus halseyi, section through anterior end. (Calkins, 1930.) b. gr.—basal granule c. fib—codrdinating fibrils b. pl.—basal plate mot.—motorium CILIATE FIBRILLAR SYSTEMS Zoy, Stains: Iron-haematoxylin, Mallory’s triple (Sharp’s modification) . Microdissection Uroleptus halseyi Calkins (Calkins, 1930).—The conspicuous parts of this kinetic system are the motorium near the right side of the gullet and, leading from it, the longitudinal anterior fibril which links a row of endoplasmic granules. Each basal plate of the membranelle series is con- nected by a short fibril to one of these basal granules, in regular order, and additional connectives unite the basal granules of each frontal cirrus with the chain. One short anterior fibril from the motorium extends to the margin of the peristome; the other leads to the undulating membrane, and two posterior fibrils are soon lost in the endoplasm. (Fig. 92). METHODS Fixatives: HgCl, saturated in 95-percent alcohol. Stain: Iron-haematoxylin. CONCLUSIONS It is evident from the review presented in foregoing paragraphs that the differentiation of fibrils in ciliates has been established for various representatives of their major groups beyond any doubt. Such differ- entiations, as revealed in fixed and stained material, are not artifacts, for many may be seen in living or in slowly disintegrating organisms (Worley, 1933). It is not certain, however, that all of the structures thus identified are actually fibrillar. Some may represent rather a sculptural, fibrillar-like pattern in the pellicle. It is also clear that the fibrils are not all alike, either structurally or as related to other protoplasmic differentiations of the cell. This was well illustrated in the several complexes of fibrils in the contractile stalk of the vorticellids. Here the structural elements composing the Spzronem, for example, differed partly in kind, but especially in arrangement, from those of the Axonem. Also, their relations to the fibrillar com- plexes of the be// were found to be different. Again, it is known that many fibrillar complexes of various ciliates are intimately associated with the basal apparatus of the motor organ- elles. This was shown for the inner fibrillar complex, or the ifracilza- 258 CILIATE FIBRILLAR SYSTEMS ture, of holotrichs such as Paramecium; for the membranelle fibril of Stentor and of Euplotes; and for most of the fibrillar systems of the other ciliates, the accounts of which were more briefly reviewed. Also, the literature contains numerous records of fibrils, in a variety of ciliates, which are structurally integrated into a so-called fibrillar system. It is with such fibrillar systems that this review has been chiefly concerned. Having established the identity of these fibrils and fibrillar systems and, for many, their structural continuity or contiguity with other organ- elles of the cells, especially the motor organelles, the investigators’ further interest has, of course, been concerned with the function or functions which may be performed by such definitely related and integrated fibrillar systems. It was previously pointed out that the interpretation of the function or functions of these fibrillar systems has been based largely on the evi- dences of their structural integration and their relationship to other organelles. Relatively few of the interpretations have been made from experimental evidence. From both kinds of evidence, it was noted that at last four elementary functions have been ascribed by the many in- vestigators to these various fibrils or fibrillar systems: (1) elasticity, (2) mechanical support, (3) contractility, and (4) conductivity. Some examples of these included (1) Elasticity, the Spasmonem and pellicle in the contractile stalk of the vorticellids (Entz, 1893), the axial filament of cilia (Koltzoff, 1912); (2) Mechanical support, Stitzgitter system of Paramecium (von Gelei, 1929); fibrils generally (Jacobsen, 1931); (3) Contractility, myonemes of Stentor (Johnson, 1893; and other authors), and of Boveria (Pickard, 1927); (4) Conductivity, neuromotor system of many ciliates (Sharp, 1914; Yocom, 1918; and other authors). A fifth function, ‘‘metabolic influence,” not previously noted, has re- cently been proposed by Parker (1929) for the fibrillar complex in Paramecium (Rees, 1922) and in other ciliates, comparable to the func- tion of fibrils in nerve cells. The neurofibrillar hypothesis for conduc- tivity in nerves was regarded as untenable by Parker (1929). After an extensive review of the evidences for and against this thesis, including Bethe’s (1897) experiment on the brain neurones in the crab Carcinus, which showed that the nerve impulses did not have to traverse the fibrils CILIATE FIBRILLAR SYSTEMS 259 of the cell body, Parker suggested that neurofibrils generally, and possi- bly the fibrils described for certain ciliates, may serve to transmit, from the metabolic center or nucleus, metabolic influences “‘essential for the continued life of the whole neurone.’’ How these transmissions might be made was not clear. He thought they might involve “chains of ionic readjustment such as have been proposed as an explanation of the nerve impulse.”’ Aside from whether or not such might apply to the function of the fibrils in ciliates, however, he rightly observed that these fibrils may not be intimately associated with the nucleus, as seems to be the case 1n neurones. Entz’s (1893) interpretation of an elastic function for the Spasmonem in the recoiling stalk of the vorticellids was discussed under the caption “Interpretations.” In addition to this, reference may be made briefly to Koltzoff’s (1903, 1906, 1912) similar interpretations for elastic fibrils in cilia and in cells generally. He would ascribe elasticity to all fibrils in maintaining all organic form other than spherical. Since proto- plasm is liquid, as shown by the sphericity of its enclosed vacuoles, then elastic elements must be postulated to counteract the physical forces of inner and outer osmotic pressure and surface tension, which tend always to effect spherical form. Such elements are fibrillar, as observed in the many kinds of cells investigated. The amount of evidence adduced by Koltzoff is impressive, but his interpretation obviously cannot apply exclusively to all fibrils. Similar claims for a supporting function for fibrils are rather wide- spread in the literature. Thus Jacobson (1931), as already stated, is disposed to attribute a supporting function to all noncontractile fibrils. These few citations, together with many others previously noted, may serve to indicate the diversity of functions that have been variously attributed to fibrillar differentiations in ciliates. In so far as they suggest that these fibrils and fibrillar systems may differ in their structure, func- tions, and relationships among the manifold kinds of ciliated Protista, certainly no one could present conclusive evidence to the contrary. But when, in the absence of proof, an investigator seriously contends that in these unicellular organisms any and all fibrillar differentiations per- form only one elementary function, whether it be that of elasticity, mechanical support, contractility, conductivity, or “metabolic transmis- sion,’ or when he assigns to these fibrils or fibrillar systems one or two 260 CILIATE FIBRILLAR SYSTEMS functions to the exclusion of another possible function or functions, then surely that investigator thereby adopts a point of view which is incon- sistent and indefensible as well. In such instances we begin to sense a recrudescence of the opposing claims advanced in the Ehrenberg-Dujardin controversy and of the non- cellular theory of protistan organization proposed by Dobell and others. Once having denied the validity of Ehrenberg’s extreme contention that the organs of the Infusoria are essentially miniature counterparts of those of macroscopic organisms, a comparably extreme viewpoint is sub- stituted, which would maintain that the Protista represent a complete de- parture in the organization of living things and so belong in the wholly exclusive category of non-cellular organisms. Thus the claims of these counter extremists would have us search for identities in organization, on the one hand, or only for differences in protistan and multicellular or- ganization on the other hand. In Ehrenberg’s day similar extreme points of view were quite irrec- oncilable, but in our day they can scarcely represent anything less than rash inconsistencies. Obviously the thesis of non-cellular organization tends to place exclusive emphasis on d/fferences between protozoan and metazoan organization and, if one is still inclined to accept that thesis, one might well refer to Bélat’s (1926) excellent monograph on the protistan nucleus. Variable as are the nuclei, in form and behavior, of the many kinds there described and illustrated—where they appear to differ from one another more than some differ from metazoan nuclei— surely one cannot fail to recognize that their numerous modifications do not represent discrete differences, but clearly betray the indelible marks of a common origin. They are like the musical variations of some great motif. They demonstrate irrefutably that living nature has been both labile and stable in its evolutionary history, so that we are amply justified in searching out and emphasizing not merely differences but also, and more fundamentally, similarities, in both the structural and functional processes of protoplasmic differentiation. And since all cellular differentiation is referable in its last analysis to protoplasmic differentiation, then certainly the fibrils and fibrillar sys- tems of multicellular tissues, such as those described by Grave and Schmitt (1925), may belong in the same category, both structurally and functionally, as some fibrillar differentiations that have been described and some that we may afford further to search for, in unicellular organ- CILIATE FIBRILLAR SYSTEMS 261 isms. Knowing today the general properties and behavior of the long- chain protein molecules, if such fibrils are proteinaceous, as evidently they are, then fibrillar differentiation is one of the most likely kinds of protoplasmic differentiation that might be expected. But by the same token, we would not expect all such proteinaceous fibrils to be alike, either structurally or functionally. Both by virtue of their intrinsic properties and their relations to other organelles, some fibrils of protistan cells, or of metazoan cells, may serve for support, others for contraction, and still others for conduction of impulses to and from motor or other organelles. Or any one fibrillar complex may per- form more than one of these, or of other yet unkown, functions. And this duality or plurality of fibrillar functions may obtain for protistan cells and for tissue cells of multicellular organisms. Certainly we know of no evidences contradicting this posszbil7ty. The actual function or functions of most fibrils or fibrillar systems are not as yet finally known. There can be no doubt about the contractile properties of the myonemes of Stentor, and some experimental evidences indicate a codrdinating (conductive) function for some fibrils in several ciliates and in epithelial tissue. The outer fibrillar systems of Paramecium and other ciliates may be fibrillar, or only apparently fibrillar, as an integral part and pattern of the pellicle. If of the pellicle, then at least one of its functions would evidently be that of support. Much mote study and more critical analysis of these fibrillar systems are greatly needed by both improved old and newly devised observational methods, perhaps such as that of the recently developed electron micro- scope. Then complementing these observational and comparative studies will be required indispensably, as the crucial test of all of our hypotheses, exceedingly refined and precise tools and methods of experimentation. Even today there are many devices suitable for this purpose, if properly adapted and fully utilized by the ingenious well-trained hands and eyes of thoroughly informed, exceptionally endowed minds. It is a mistake to suppose that such microtechniques are peculiarly difficult and that such problems are really unapproachable. It is rather that these techniques are different and that their use requires special training. With such training, it may be easier to transect a ciliate or a marine ovum, with much more accuracy, than to perform ‘‘free hand” some of the disections on macro- scopic organisms. By micromanipulative methods and with the aid of other modern 262 CILIATE: FIBRILLAR SYSTEMS devices, such as the ultracentrifuge, and by micromethods of irradiation, and the like, we may expect for the future, once the world has recovered its sanity, notable advances in protistological investigations such as may not have been dreamed of, even by the most sanguine of our predecessors. No other group of organisms may offer more than the unicellular forms toward the solution of some of our most fundamental problems. The re- sults, therefore, will provide a better understanding not only of these unicellular organisms, but of all forms of life. In their last analysis, how- ever, all such problems must surely depend for any final solution upon unique exacting methods of biological experimentation. ‘Belief uncon- firmed by experiment is vain’’ (Francesco Redi, of Florence, 1668). LITERATURE CITED Alverdes, F. 1922. Untersuchungen tber Flimmerbewegung. Pfliig. Arch. ges. Physiol., 195: 243-49, Bélat, K. 1921. Protozoenstudien III. Arch. Protistenk., 43: 431-62. — 1925. Hartmann, Allgemeine Biologie. Jena. —— 1926. Die Formwechsel der Protistenkerne. Ergbn. Zool., 4: 235-664. Belehradek, Jan., 1921. Rozbor pohybu Vorticell. Biol. Listy, 8: 49-53. Beltran, Enrique. 1933. Gruberia calkinsi sp. nov., a brackish-water ciliate (Protozoa, Heterotrichida) from Woods Hole, Mass. Biol. Bull., 64: De Bethe, A. 1897. Das Nervensystem von Carcinus maenas. 1. Arch. mikr. Anat., 50: 589-639. Bishop, Ann. 1927. The cytoplasmic structures of Spirostomum ambiguum (CEhrbe)sOuatts | mict. Sci 7-47-72" Brauer, A. 1885. Bursaria truncatella unter Beriicksichtigung anderer Hetero- trichen und der Vorticellinen. Jena. Z. Naturw., 19: 489-519. Bresslau, E. 1921. Die Gelatinierbarkeit des Protoplasmas als Grundlage eines Verfahrens zur Schellanfertigung gefarbter Dauerpraparate von Infusorien. Arch. Protistenk., 43: 469-80. Bretschneider, L. H. 1931. Beitrige zur Strukturlehre der Ophryoscoleciden. I. Ekto- und Entoplasma. Fibrillen. Zool. Ans. Suppl., 5: 324-30. —— 1934. Beitrage zur Strukturlehre der Ophryoscoleciden. II. Arch, Pro- tistenk., 82: 298-330. Brown, V. E. 1930. The neuromotor apparatus of Paramecium. Arch. zool. exp. gén., 70: 469-81. Bush, Mildred. 1934. The morphology of Haptophrya michiganensis Wood- head, an astomatous ciliate from the intestinal tract of Hemidactylium scutatum (Schlegel). Univ. Cal. Publ. Zool., 39: 251-76. Butschli, O. 1887-89. Protozoa. Bronn’s Klassen, Bd. 1-3. CILIATE FIBRILLAR SYSTEMS 263 Calkins, Gary N. 1930. Uroleptus halseyi Calkins, HI. The kinetic elements and the micronucleus. Arch. Protistenk., 72: 49-70. Calkins, Gary N., and R. Bowling. 1929. Studies on Dallasia frontata. II. Cytology, gametogamy, and conjugation. Arch. Protistenk., 66: 11-32. Campbell, A. S. 1926. The cytology of Tntinnopsis nucula (Fol) Laackmann, with an account of its neuromotor apparatus, division, and a new intra- nuclear parasite. Univ. Cal. Publ. Zool., 29: 179-236. —— 1927. Studies on the marine ciliate Favella (Jorgensen), with special regard to the neuromotor apparatus and its rdle in the formation of the lorica. Univ. Cal. Publ. Zool., 29: 429-52. Chakravarty, M. 1936. On the morphology of Balantidium depressum (Ghosh) from a mollusc, Pla globosa, with a note on its nuclear reac- tions and cytoplasmic inclusions. Arch. Protistenk., 87: 1-9. Chatton, E., and S. Brachon. 1935. Discrimination, chez deux Infusoires du genre Glaucoma, entre systeme argentophile et infraciliature. C. R., Soc. Biol. Paris, 118: 399-402. Chatton, E., and A. Lwoff. 1930. Impregnation, par diffusion argentique, de l'infraciliature des ciliés marins et d’eau douce, apres fixation cytologique et sans desiccation. C. R., Soc. Biol. Paris, 104: 834-36. 1935. I La Constitution primitive de la strie ciliare des Infusoires. La desmodexie. C. R. Soc. Biol. Paris, 118: 1068-71. —— 1936. Les Remaniements et la continuité du cinétome au cours de la scission chez les Thigmotriches Ancistrumidés. Arch. zool. exp. gén., 78: 84-91. Cohn, F., and C. V. Siebold. 1862. Ueber die contractilen Staubfaden der Dis- teln. Z. wiss. Zool., 12: 366-71. Czermak, J. 1853. Uber den Stiel der Vorticellen. Z. wiss. Zool., 4: 438-50. Delage, Y., and Ed. Herouard. 1896. Traité de zoologie concrete. 1. La Cellule et les protozoaires. Paris. Dierks, K. 1926. Untersuchungen tiber die Morphologie und Physiologie des Stentor coereuleus mit besonderer Beriicksichtigung seiner kontraktilen und konduktilen Elemente. Arch. Protistenk., 54: 1-91. D'Udekem, M. J. 1864. Description des Infusoires de la Belgique. I. Ser. Les Vorticelliens. Mém. Acad. roy. Belg., 35: 1-52. Dujardin, F. 1835. Sur les prétendus estomacs des animalcules infusoires et sur une substance appelée sarcode. Ann. Sci. nat., 2: 4. —— 1841. Histoire naturelle des (Zoophytes) Infusoires. Suites a Buffon. Paris. Ehrenberg, C. G. 1838. Die Infusionsthierchen als Vollkommene Organismen. Leipzig. Atlas, 64 pls. Ellis, John. 1937. The morphology, division, and conjugation of the salt-marsh ciliate Fabrea salina Henneguy. Univ. Cal. Publ. Zool., 41: 343-88. Dobell, C. C. 1911. Principles of protistology. Arch. Protistenk., 23: 269-310. 264 CILIATE FIBRILLAR SYSTEMS Doflein, F., and E. Reichenow, 1929. Doflein, Lehrbuch der Protozoenkunde. 5th ed., Jena. Engelmann, T. W. 1875. Contractilitét und Doppeltbrechung. Pflug. Arch. ges. Physiol., 11: 432-64. —— 1879. Physiologie der Protoplasma und Flimmerbewegung. Hermann’s Handbuch d. Physiol., 1: 343-408. —— 1880. Zur Anatomie und Physiologie der Flimmerzellen. Pflig. Arch. ges. Physiol., 23: 505-35. Entz, G. 1893. Die elastischen und contractilen Elemente der Vorticellen. Math. naturw. Ber. Ung., 10: 1-48. Everts, E. 1873. Untersuchungen tiber Vorticella nebulifera. Z. wiss. Zool., 23:5592-622. Fabre-Doumergue, P. 1888. Recherches anatomiques et physiologiques sur les infusoires ciliés. Ann. Sci. nat. (b) Zool., 1: 1-140. Forest, H. E. 1879. The natural history and development of the Vorticellidae. Mid]. Nat., 2: Gelei, G. von. 1937. Ein neues Fibrillensystem im Ectoplasma von Paramecium. Arch. Protistenk., 89: 133-62. Gelei, J. von. 1925. Ein neues Paramaecium aus der Umgebung von Szeged, Paramaecium nephridiatum n. sp. Allatt. Kézlem., 22: 121-59 (résumé in German, 245-48). | 1926a. Zur Kenntnis des Wimperapparates. Z. Anat. Entw. Gesch., 81: 530-53, —— 1926b. Cilienstruktur und Cilienbewegung. Zool. Anz. Suppl., 2: 202- i: —— 1926c. Sind die Neurophane von Neresheimer neuroide Elemente? Arch. Protistenk., 56: 232-42. — 1927. Eine neue Osmium-Toluidinsmethode fiir Protistenforschung. Mikrokosmos, 20: 97-103. —— 1929a. Sensorischer Basalapparat der Tastborsten und der Syncilien bet Hypotrichen. Zool. Anz., 83: 275-80. —— 1929b. Uber das Nervensystem der Protozoen. Allatt. Kézlem. (Zool. Mitt.) , 26: 186-90. —— 1932a. Die reizleitenden Elemente der Ciliaten in nass hergestellten Silber-bwz. Goldpraparaten. Arch. Protistenk., 77: 152-74. —— 1932b. Ein Neue Goldmethode fiir Ciliatenforschung und eine neue Ciliate: Colpidium pannonicum. Arch. Protistenk., 77: 219-30 —— 1933. Uber den Bau die Abstammung und die Bedeutung der sog. Tast- borsten bei den ciliaten. Arch. Protistenk., 80: 116-27. — 1934. Der Cytopharynx der Paramecien. Matemat. és termész. értesit6, Is 750-50! —— 1934. Die Differenzierung der Cilienmeridiane der Ciliaten und der Begriff des Richtungsmeridians. Matemat. és termész. €rtesit6, 51: 632-44. —— 1934, Das Verhalten der ectoplasmatischen Elemente des Parameciums wahrend der Teilung. Zool. Anz., 107: 161-77. CILIATE FIBRILLAR SYSTEMS 265 —— 1934. Die Vermehrung der Sinneshaare von Emplotes wahrend des Teilungsprozesses. Zool. Anz., 105: 258-66. — 1934. Eine mikrotechnische Studie ber Farbung der subpellicularen Ele- mente der Ciliaten. Z. wiss. Mikr., 51: 103-78. — 1934. Der feinere Bau des Cytopharynx von Paramecium und seine systematische Bedeutung. Arch. Protistenk., 82: 331-62. —— 1935. Eine neue Abanderung der Klein’schen trockenen Silbermethode und das Silberliniensystem von Glaucoma scintillans. Arch. Protistenk., 84: 446-55. —— 1935. Der Richtungsmeridian und die Neubildung des Mundes wahrend und ausserhalb der Teilung bei den ciliaten. Biol. Zbl., 55: 436-45. — 1935. Historisches und Neues wtber die interciliaren Fasern und ihr morphologische Bedeutung. Z. Zellforsch., 22: 244-54, — 1935. Colpidium glaucomaeformae n. sp. (Hymenostomata) und sein Neuronensystem. Arch. Protistenk., 85: 289-302. —— 1936-37. Der schraubige K6rperbau in der Ciliatenwelt im Vergleich zu Symmetrieverhaltnissen der vielzelligen Tiere. Arch. Protistenk., 88: 314-38. — 1937. Pori secretorii am Ciliatenkérper. Biol. Zbl., 57: 175-87. —— 1938. Schraubenbewegung und Ko6rperbau pei Paramecium. Arch. Protistenk., 90: 165-77. and P. Horvath. 1931. Eine nasse Silber-bzw. Goldmethode fur die Herstellung der reizleitender Elemente bei den Ziliaten. Z. wiss. Mikr., 48 9-29. Grave, C., and C. O. Schmitt. 1925, A mechanism for the codrdination and regulation of ciliary movement as revealed by micro-dissection and cyto- logical studies of ciliated cells of molluscs. J. Morph., 40: 479-515. Greeff, R. 1870-71. Untersuchungen tiber den Bau und die Naturgeschichte der Vorticellen. Arch. Naturgesch., I-II Abt., 36: 37. Griffin, L. E. 1910. Explotes worcesteri sp. nov. I. Structure. Philipp. J. Sct., a: Col S52: Haeckel, E. 1873. Zur Morphologie der Infusorien. Jena. Z. Naturgesch., 7: 516-60. Hall, R. F. 1923. Morphology and binary fission of Menozdium incurvum. Univ. Cal. Publ. Zool., 20: 447-76. Hammond, D. M., and C. A. Kofoid. 1936. The continuity of structure and function in th neuromotor system of Explotes patella during its life cycle. Proc. Amer. phil. Soc., 77: 207-18. Hammond, J. C. 1935. Physiological dominance as a factor in ciliary codrdina- tion in the Protozoa. Ohio J. Sci., 35: 304-6. Hartmann, M. 1925. Allgemeine Biologie. Eine Einleitung in die Lehre vom Leben. Jena. Heidenhain, M. V. 1899. Beitrige zur Aufklirung des wahren Wesens der faserformigen Differenzierungen. Anat. Anz., 16: 97-131. 266 CIEIATE FIBRIELARYSYSTEMS — 1911. Plasma und Zelle. Jena. Heidenreich, E. 1935. Ptychostomum lumbriculi n. sp. Arch. Protistenk., 85: 205-D): Higgins, H. T. 1929. Variations in Nyctotherus found in frog and toad tad- poles and adults. Trans. Amer. Micr. Soc., 48: 141-57. Hofker, J. 1928. Das neuromotorische Apparat der Protozoen. Tijdschr. Nederlandsch. 1:34-38. Horvath, J. 1938. Eine neue Silbermethode fiir die Darstellung des Stiitz- gitters und der erregungsleitenden Elemente der Ciliaten. Z. wiss. Mikr., Dole Jacobson, Irene. 1931. Fibrillaére Differenzierungen bei Ciliaten. Arch. Protis- tenk., 75: 31-100. Johnson, H. P. 1893. A contribution to the morphology and biology of the stentors. J. Morph., 8: 467-562. Kate, C. G. B. ten. 1926. Uber das Fibrillensystem der Ciliaten. Dissertation Univ. Utrecht. Zutphen. —— 1927. Uber das Fibrillensystem der Ciliaten. Arch. Protistenk., 57: 362- 426. —— 1928. Uber das Fibrillensystem der Ciliaten. 2. Das Fibrillensystem der Isotrichen. Arch. Protistenk., 62: 328-54. Kidder, G. W. 1933a. On the Genus Ancistruma Strand (Ancistruma Mau- pas). I. The structure and division of A. mytili Quenn. and A. isselz Kahl. Biol. Bull. 64: 1-20. —— 1933. Studies on Conchophthirus mytili De Morgan. I. Morphology and division. Arch. Protistenk., 79: 1-24. — 1934. Studies on the ciliates from fresh water mussels. I. The structure and neuromotor system of Conchophthirus anodontae Stein, C. curtus Engl., and C. magna sp. nov. Biol. Bull. 66: 69-90. Kirby, Harold. 1932. Protozoa in termites of the Genus Amztermes. Parasitol- Ogy, 24: 289-304. Klein, B. M. 1926a. Uber eine neue Eigentiimlichkeit der Pellicula von Chilodon uncinatus Ehrbg. Zool. Anz., 67: 160-62. —— 1926b. Ergebnisse mit einer Silbermethode bei Ciliaten. Arch. Protistenk., 56: 243-79. — 1927. Die Silberliniensysteme der Ciliaten. Ihr Verhalten wahrend Teilung und Conjugation, neue Silberbilder, Nachtraige. Arch. Protistenk., 58: 55-142. —— 1928. Die Silberliniensysteme der Ciliaten. Weitere Resultate. Arch. Protistenk., 62: 177-260. 1929. Weitere Beitrage zur Kenntnis des Silberliniensystems der Ciliaten. Arch. Protistenk., 65; 183-257. 1930. Das Silberliniensystem der Ciliaten. Weitere Ergebnisse. IV. Arch. Protistenk., 69: 235-326. CILIATE FIBRILLAR SYSTEMS 267 1931. Uber die ZugehGrigkeit gewisser Fibrillen bzw. Fibrillenkomplex zum Silberliniensystem. Arch. Protistenk., 74: 401-16. —— 1932. Das Ciliensystem in seiner Bedeutung fiir Locomotion Coordina- tion und Formbildung usw. Ergebn. Biol., 8: 75-179. —— 1933. Silberliniensystem und Infraciliatur. Eine kritische Gegenuberstel- lung. Arch. Protistenk., 79: 146-69. —— 1935. Die Darstellung des Silberlinien oder neuroformativen Systems nebst Griindsatzlichem zur Silbermethodik. Z. wiss. Mikr., 52: 120-57. —— 1936. Wirkung von Schlangengiften auf Leben und Silberliniensystem von Infusorien. Arch. Protistenk., 87: 299-313. —— 1936. Beziehungen zwischen Maschenweite und Bildungsvorgingen im Silberliniensystem der Ciliaten. Arch. Protistenk., 88: 1-22. —— 1937. Uber die Eigenk6rplichkeit des Silberliniensystems. Arch. Protts- tenk., 88: 188-91. —— 1937. Regionire Reaktionen im Silberlinien- oder neuroformativen Sys- tem der Ciliaten. Arch. Protistenk., 88: 192-210. —— 1938. Miss-bzw. Doppelbildung am Silberliniensystem von Ciltaten. Arch. Protistenk., 90: 292-98. Kofoid, C. A., and R. F. MacLennan 1932. Ciliates from Bos mdicus Linn. II. A revision of Diplodinium Schuberg. Univ. Cal. Publ. Zool., 37: 3-152: Kolliker, A. 1845. Die Lehre von der tierischen Zelle. Z. wiss. Bot., 1: 2. —— 1864. Icones histiologicae 1. Leipzig. Koltzoff, N. K. 1903. Uber formbestimmende elastische Gebilde in Zellen. Biol. Zbl., 23: 680-96. —— 1906. Die Spermien der Decopoda, als Einleitung in das Problem der Zellengestalt. Arch. mikr. Anat., 67: 364-571. —— 1909. Studien iiber die Gestalt der Zelle. I]. Untersuchungen tuber das Kopfskelett des tierschen Spermiums. Arch. Zellforsch., 2: 1-65. — 1912. Studien tiber die Gestalt der Zelle. II]. Untersuchungen tber die Kontraktilitat des Vorticellenstiels. Arch. Zellforsch. 7: 344-423. Kihne, W. 1859. Untersuchungen tiber Bewegung und Verinderungen der contractilen Substanz. Arch. Anat. Physiol., 1859: 748-835. —— 1859. Uber sogenannte idiomuskulare Kontraktion. Arch. Anat. Physiol., 1859: 418-20. Lachmann, J. 1856. Uber die Organization der Infusorien, besonders der Vorticellen. Arch. Anat. Physiol., 1856: 340-98. Lang, A. 1901. Lehrbuch der vergleichenden Anatomie der wirbellosen Tiere. (Protozoa). 2. Aufl., Abt. 1. Berlin. Lebedew, W. 1909. Uber Trachelocerca phoenicopterus (ein marines Infusor). Arch. Protistenk., 13: 70-114. Leiberman, P. R. 1929. Ciliary arrangement in different species of Paramecium ——~ irans, Aimer, Micr. Soc, 48: 1-11. AGS i % LY ee LSSYAvDS la LO" 4 ¥ ty Ga > RAR 2 f 2 oN ° \ 268 CILIATE FIBRILLAR SYSTEMS Leydig, F. 1883. Untersuchungen uber Anatomie und Histologie der Tiere. Bonn. Lieberkihn, N. 1857. Beitrage zur Anatomie der Infusorien. Arch. Anat. Physiol., 1857: 20-36. Lucas, M. S. 1934. Ciliates from Bermuda sea urchins. I. Metopus. J. R. mict. Soc., 54: 79-93. Lithe, M. 1913. A. Lang, Handbuch der Morphologie der wirbellosen Tiere. 1. Protozoa. Berlin. Lund, E. E. 1933. A correlation of the silverline and neuromotor systems of Paramecium. Univ. Cal. Publ. Zool., 39: 35-76. 1935. The neuromotor system of Oxytricha, J. Morph., 58: 257-77. Lynch, J. E. 1929. Studies on the ciliates from the intestine of strongylocentro- tus. 1. Entorhipidium Gen. nov. Univ. Cal. Publ. Zool., 33: 27-56. —— 1930. Studies on the ciliates from the intestine of Strongylocentrotus. Il. Lechriopyla mystax, Gen. nov., sp. nov. Univ. Cal. Publ. Zool., 33: 307-50. McDonald, J. Daley. 1922. On Balantidium coli (Malmsten) and Balantidium suis (sp. nov.), with an account of their neuromotor apparatus. Univ. Cal. Publ. Zool., 20: 243-300. MacDougall, Mary S. 1928. The neuromotor system of Chlamydodon sp. Biol. Bull., 54: 471-84. MacLennan, R. F. 1935. Dedifferentiation and redifferentiation in Ichthy- ophthirius. 1. Neuromotor system. Arch. Protistenk., 87: 191-210. MacLennan, R. F., and F. H. Connell. 1931. The morphology of Expoterion pernix Gen. nov., sp. nov. Univ. Cal. Publ. Zool., 36: 141-56. Maier, H. N. 1903. Uber den feineren Bau der Wimperapparate der In- fusorien. Arch. Protistenk., 2:73-179. Maupas, E. 1883. Contribution a l’etude morphologique et anatomique des infusoires ciliés, Arch. zool. exp. gén., (2) I: 427-644. Merton, H. 1932. Gestalterhaltende fixierungsversuche an besonders kon- traktilen Infusorien nebst Beobachtungen tiber das verhalten der lebenden Myoneme und Wimpern bei Stentor, Arch. Protistenk., 77: 491-521. Metschnikoff, E. 1863. Untersuchungen tiber den Stiel der Vorticellen. Arch. Anat. Physiol., 1863: 180-86. —— 1864. Nachtragliche Bemerkungen tiber den Stiel der Vorticellen. Arch. Anat. Physiol., 1864: 291-302. Meyer, A. 1920. Analyse der Zelle. Jena. Neresheimer, E. R. 1903. Die Hohe histologischer Differenzierung bei hetero- trichen Ciliaten. Arch. Protistenk., 2: 305-24. 1907. Nochmals tiber Stentor coeruleus. Arch. Protistenk., 9: 137-38. Parker, G. H. 1929. What are neurofibrils? Amer. Nat., 63: 97-117. Pensa, A. 1926. Particolarita strutturale di alcuni protozoi cigliati in rapporto con la contratilia. Monit. zool. ital., 37: 165-73. Peschkowsky, L. 1927. Skelettgebilde bei Infusorien. Arch. Protistenk., 57: aii CILIATE FIBRILLAR SYSTEMS 269 Pickard, E. A. 1927. The neuromotor apparatus of Boveria ternedinidi Nelson, a ciliate from the gills of Teredo navalis. Univ. Cal. Publ. Zool., 29: 405-28. Powers, P. B. A. 1933. Studies on the ciliates from sea urchins. II. Envtodiscus borealis (Hentschel). Behavior and morphology. Biol. Bull., 65: 122-36. Prowazek, S. 1903. Protozoenstudien. III. Ewplotes PEN, Arb. zool. Inst. Univ. Wien., 14: 81-88. Pitter, A. 1904. Die Reizbeantwortung der ciliaten Infusorien. Z. allg. Physiol., 3: 406-54. Raabe, Zdz. 1932. Untersuchungen an einigen Arten des Genus Concho ph- thirus Stein. Bull. Int. Acad. Cracovie, Cl. Sci. Math et Nat. Ser. B. Sci. Nat. (II) (Zool.) 1932 (8/10): 295-310. Ray, Harendranath. 1932. On the morphology of Balantidium sushilii n. sp., from Rana tigrina Daud. J. R. micr. Soc., 52: 374-82. Rees, C. W. 1921. The neuromotor apparatus of Paramecium. Amer. Nat., 55: 464-68. —— 1922. The neuromotor apparatus of Paramecium. Univ. Cal. Publ. Zool., 20: 333-64. —— 1930. Is there a neuromotor apparatus in Diplodinium ecaudatum? Sci- ence, 71: 369-70. 1931. The anatomy of Diplodinium medium. J. Morph., 52: 195-215. Reichenow, E. 1929. Doflein, Lehrbuch der Protozoenkunde. Sth ed., Jena. Rosenberg, L. E. 1937. The neuromotor system of Nyctotherus hylae. Univ. Cal. Publ. Zool., 41: 249-76. Roskin, G. 1915. La Structure des myonémes contractiles de Stevtor coeruleus. Tirage a part des memoires scientifique des Chaniavsky Université de Moscou. 1: — 1918. Sur la structure de certains elements contractiles de la cellule. Arch. russ. d’Anat., —— 1923. Die Cytologie der Kontraktion der glatten Muskelgellen. Arch. Zellsforsch., 17: 368-81. Rouget, C. 1861. Sur les phenomenes de polarisation qui s’observent dans quelques tissus vegetative et des animaux. J. Physiol. Path. gén., 5: Schewiakoff, W. 1889. Beitrige zur Kenntnis der holotrichen Ciliaten. Bibli- oer.) zo0lf> Hd, 1-77. Schmidt, W. J. 1924. Die Bausteine des Tierk6rpers im polarisierten Lichte. Bonn. Schréder, O. 1906a. Beitrage zur Kenntnis von Campanella umbellaria. Arch. Protistenk., 7: 75-105. —— 1906b. Beitrige zur Kenntnis von Epistylis plicatilis. Arch. Protistenk., TN) 3-S5. —— 1906c. Beitrage zur Kenntnis von Vorticella monilata. Arch. Protistenk.. 7: 395-410. — 1907. Beitrage zur Kenntnis von Stentor coeruleus und Stentor roeselii Ehrbg. Arch. Protistenk., 8: 1-16. 270 CILIATE FIBRILEAR SYSTEMS Schuberg, A. 1890. Zur Kenntnis des Stentor coeruleus. Zool. Jahrb., Abt. Anat. Ontog., 4: 197-238. —— 1905. Uber Cilien und Trichocysten einiger Infusorien. Arch. Protistenk.., 6: 61-110. Seifriz, W. 1929. The contractility of protoplasm. Amer. Nat., 63: 410-34. Sharp, R. G. 1914. Diplodiminum ecaudatum with an account of its neuro- motor apparatus. Univ. Cal. Publ. Zool., 13: 42-122. Siebold, C. Th. von. 1849. Uber einzellege Pflanzen und Tiere. Z. wiss. Zool., Le 270: Siebold, C. Th. von, and H. Stannius. 1845. Lehrbuch der vergleichenden Anatomie, H. 1. Berlin. Stein, F. 1854. Die infusionsthiere auf ihre Entwicklung untersucht. Leipzig. — 1859. Der Organismus der Infusionsthiere nach einigen Forschungen in systematischer Reihenfolge bearbeitet. I. Abtheilung Allgemeiner Theil und Naturgeschichte der hypotrichen Infusionsthiere. Leipzig. — 1867. Ibid., 11. Abtheilung Naturgeschichte der heterotrichen Infusorien, pp. 140-352. Leipzig. Stevens, N. M. 1891. Studies on ciliate Infusoria. Proc. Calif. Acad. Sci., 3: 1-42. Studitsky, A. N. 1932. Uber die Morphologie, Cytologie u. Systematik von Ptychostomum chattoni Rossolimo. Arch. Protistenk., 76: 188-216. Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap- paratus in Explotes by the method of microdissection. Univ. Cal. Publ. Zool., 19: 403-70. —— 1929. Experimental evidence of the function of the fibrillar system in certain Protozoa. Amer. Nat., 63: 328-45. Turner, J. P. 1933. The external fibrillar system of Evp/otes, with notes on the neuromotor apparatus. Biol. Bull., 64: 53-66. Verworn, Max. 1889. Protisten-Studien. Jena. Visscher, J. P. 1927. A neuromotor apparatus in the ciliate Dileptus gigas. J. Morph., 44: 373-81. Worley, L. G. 1933. The intracellular fibre systems of Paramecium. Proc. nat. Acad. Sci. Wash., 19: 323-26. Wrisberg, H. A. 1765. Observationum de animalculis infusoriis satura, Got- tingae. Wrzesniowski, A. 1877. Betrage zur Naturgeschichte der Infusorien. Z. wiss. Zool., 29: 267-323. Yabroff, S. W. 1928. A modification of the Da Fano technique. Trans. Amer. micr. Soc., 47: 94-95. Yocom, H. B. 1918. The neuromotor apparatus of Emplotes patella. Univ. Cal. Publ. Zool., 18: 337-96. Zon, Leo. 1936. The physical chemistry of silver staining. Stain Tech., 11: 53-65. CHAPTER: V. MOTOR RESPONSE IN UNICELLULAR ANIMALS SO MASA: RESPONSES consist of changes in structure, composition, form, or move- ment in organisms, which, in turn, are correlated with changes in the constituents of the environment or the organisms. Responses are found in all living systems and are among the most fundamental distinguish- ing characteristics of life. Motor responses consist of changes in tate or direction of movement of organisms or their constituents. They facilitate the control of the environment by the organisms involved and are consequently of great importance to them. Knowledge concerning these responses and their relation to the factors correlated with them makes it possible to control the activities of organisms, it throws light on the distribution of pain and pleasure (consciousness) which pro- foundly affects the attitude of man toward his fellow creatures, and it illuminates the processes involved in instincts and in learning. Such knowledge is therefore very valuable. The unicellular organisms are in many respects extraordinarily favor- able for the study of the more fundamental characteristics of these re- sponses. They are relatively simple in structure. Many of them can be readily procured and maintained in great numbers, and the factors in their environment can be accurately controlled or changed as desired; and, in addition, details concerning the responses can be readily seen. Moreover, the motor responses in these organisms are very favorable for the study of adaptation, as pointed out long ago by Jennings (1906). In the following pages are presented the more important facts in hand concerning the motor responses of the rhizopods, flagellates, ciliates, and colonial forms to light, electricity, and chemicals. These are presented with the view of encouraging the use of these organisms in further work on various biological problems. Important results have also been ob- tained on responses to contact, temperature, and gravity, but limitation in space prevents the consideration of these. 272 MOTOR RESPONSES RESPONSES TO LIGHT A. RHIZOPODS Many of the rhizopods respond to light and some orient if they are exposed in a beam of light. Some of the responses are correlated with the rate of change in intensity, others are not. They are fundamentally the same in all the species which have been investigated, but they have been more thoroughly studied in Amoeba proteus than in any of the other species. The following considerations therefore refer largely to this species. A, proteus consists of a thin elastic outer membrane, the plasmalemma, a central relatively fluid granular mass, the plasmasol, surrounded by a Figure 93. Camera sketch of horizontal optical section of Amoeba proteus. Ps, plas- masol; Pg, plasmagel; Pl, plasmalemma; HC, hyaline cap; Pgs, plasmagel sheet; L, Liquid layer; S, region of solation; G, region of gelation. (After Mast, 1926.) relatively solid granular layer, the plasmagel, and a thin fluid hyaline layer between the plasmagel and the plasmalemma (Fig. 93). During locomotion, the plasmalemma is attached to the substratum and to the adjoining plasmagel; the plasmagel at the posterior end is transformed into plasmasol, which flows forward to the anterior end and is there transformed into plasmagel. The forward flow of the plasmasol is due to contraction of the plasmagel at the posterior end and expansion at the anterior end, owing to difference in its elastic strength in these two re- gions. In some species surface tension is probably also involved in locomo- MOTOR RESPONSES 212 tion. In A. mira, for example, the anterior portion consists of a thin sheet of hyaline cytoplasm in close contact with the substratum (Hopkins, 1938). The surface tension at the water-substrate interface is probably greater than the combined surface tensions at the cytoplasm-substrate and cytoplasm-water interfaces. This would result in a spreading of the cyto- plasm over the substrate (like oil over water) wherever the two are in contact, i.e., at the anterior end of the organism. With reference to a marine amoeba, Pantin (1923-31) contends that the cytoplasm at the anterior end swells and extracts water “from the posterior protoplasm of the amoeba itself, and that this will cause a stream from the posterior to the anterior end.”’ Presumably he holds that the cytoplasm shrinks there and gives it off. However, that would neces- sitate absorption of water during gelation at the anterior end, and elimina- tion during solation at the posterior end, which is contrary to what is ordinarily observed in the process of gelation and solation of gels. Marsland and Brown (1936) suggest that the forward flow is due to increase in volume during solation at the posterior end, and decrease in volume during gelation at the anterior end. They give no direct evi- dence in support of this suggestion, but simply say that “The magnitude of these volume changes in relation to the observed rate of flow is prob- lematical.”’ Responses of Amoeba to light are therefore probably due to localized changes in (1) the elastic strength of the plasmagel, (2) the rate of transformation of plasmasol to plasmagel and vice versa, or (3) the firmness, extent, or region of attachment to the substratum (Mast, 1923, 1926a,) 193 ba); Shock-reactions —Engelmann (1879) long ago observed that if strong light is flashed on an amoeba, movement stops suddenly, but that if the intensity is gradually increased, movement continues. This response therefore depends upon the rate of change of light intensity. Such re- sponses are usually designated ‘‘Schreckbewegungen,” or shock-reactions. They are closely correlated with adaptation. The shock-reaction in Amoeba produced by light varies greatly. It may consist merely of mo- mentary retardation in streaming in a localized region in a pseudopod, of total cessation throughout the entire animal with reversal in direction of streaming after recovery, or of any one of an endless number of modifica- 274 MOTOR RESPONSES tions between these extremes. The character of the response is correlated with the amount of light received, as well as with the rate of reception. There is no fixed threshold and the “‘all-or-none” law does not apply (Mast, 1931a). If an amoeba is intensely illuminated for a very short time only, move- ment does not cease until some time after the light has been cut off. The period between the beginning of illumination and the response ts Figure 94. Curves showing for Amoeba proteus the : relation between luminous intensity, reaction time \ (RT), stimulation period (SP) and latent period (LP), and a hyperbola (H). Note that the curve for re- action time simulates a hyperbola, but that the curve for the stimulation period does not. This shows that the amount of light energy necessary for response varies with the intensity and that the Bunsen-Roscoe law does not apply. (Modified after Folger, 1925.) -— . -_--—- UMINOUS INTENSIT known as the “reaction time’’; the time illumination must continue, the “stimulation period’; and the time it need not continue, the “latent period” (Folger, 1925). There are therefore two processes involved in producing this response. The first occurs only in light, the other in light or in darkness. The action of light probably results in the formation of a substance which acts to produce, independent of light, another sub- stance which induces the response. MOTOR RESPONSES 275) After an amoeba has responded to rapid increase in illumination, some time must elapse before it will respond again to the same increase in illumination. There is therefore a refractory period, a period during which the amoeba recovers from the effect of the stimulation. During a part of this period the amoeba may remain either in light of the same intensity, such as that which induced the response, in light of lower intensity, or in darkness; but during the remainder of the period it must be in light of lower intensity or in darkness. There are therefore two processes which occur during the refractory period, one (1 to 2 minutes) which proceeds with or without any change in luminous intensity, and another (10 to 20 seconds) which proceeds only if the intensity is de- creased. These processes result in the production of the physiological state which existed before the exposure; that is, in recovery (Folger, 1925). The latent period and the amount of light energy required to induce cessation of movement vary with the intensity of the light used (Fig. 94). Figure 94 shows that as the intensity increases, the latent period increases rapidly from about one second at 500 + meter-candles to a maximum of about 6 seconds at 1,000 ++ meter-candles, and then decreases gradu- ally to about 0.75 seconds at 11,000 + meter-candles; and that the light energy required to induce cessation of movement decreases from about 7,000 ++ meter-candle seconds at 500 -+ meter-candles to a maximum of about 24,000 -— meter-candle seconds at 1,500 + meter-candles, and then increases to about 30,000 + meter-candle seconds at 11,000 + meter-candles. These results are, however, only rather crude approxima- tions. They were obtained by a method of calculation which yields results with a large probable error and they have not been confirmed. The data are, however, sufficiently accurate to substantiate Folger’s conclusion that the Bunsen-Roscoe law does not hold. This work should be repeated, and the latent period established by direct observation in all luminous intensities, instead of by calculation. This is especially desirable since recent experience makes it possible to select specimens of A. proteus in which the responses are much more consistent than they were in those used by Folger. No explanation has been offered for the mode of variation in the latent period, with variations in luminous intensity during the period of stimulation. However, it has been suggested that the variation in the 276 MOTOR RESPONSES amount of light energy required to induce cessation of movement is due, at least in part, to adaptation (Mast, 1931a). For if the light is rapidly increased and then held, streaming soon begins again, t.e., the organism recovers from the effect of the increase in light. In other words, it be- comes adapted (Mast, 1939; Folger, 1925). This shows that the effect of rapid increase in light is eliminated while the organism is continuously Figure 95. Camera drawings of Amoeba sp. illustrating the response to localized illumination. Rectangular areas, regions of high illumination; arrows, direction of proto- plasmic streaming; dotted lines in B, C and D, positions and forms shortly after the illumination of the parts indicated; , nucleus; v, contractile vacuole. E and F, same specimen; F, form and direction of streaming assumed by E after the anterior end had been illuminated for a few minutes. (After Mast, 1932.) exposed to the light. It also indicates that there are two opposing processes involved, i.e., that increase in light induces certain changes in the or- ganism and that internal factors tend continuously to oppose and to eliminate these changes. If this is true, the more rapidly a given amount of light is received, the less time there is for recovery, and consequently the greater will be the effect of a given quantity of light. This probably accounts for the increase in the amount of light energy required (with decrease in intensity) when observations are made in weak light; but it MOTOR RESPONSES ZF, does not account for the increase in the amount required (with increase in intensity) if the observations are made in strong light. The quantity of light energy required to induce cessation of movement depends upon the chemical composition of the surrounding medium. In- crease in HCL, for example, causes an increase in the quantity of light required. On the other hand, an increase in CO, causes decrease in the quantity required. In solutions of KCI, CaCl,, and MgCl,, respectively, the quantity of energy required increases as the salt concentration de- creases, but in solutions of NaCl there is no consistent correlation be- tween the quantity of energy and the concentration of the salt. In gen- eral, the quantity of energy required appears to vary directly with the viscosity of the cytoplasm (Mast and Hulpieu, 1930). The observations made by these authors extended, however, over only a very limited range of environmental variation. The conclusions reached are therefore not applicable to wide ranges of variations in the environment (Mast and Prosser, 1932). Increase in the illumination of any localized region of an amoeba re- sults in an increase of the thickness of the plasmagel in this region (Fig. 95). Increase in the illumination of the entire amoeba results in an increase in the thickness of the plasmagel at the tip of the advancing pseudopods. In turn, this causes a cessation of movement (shock-reac- tion). The shorter waves of light are more efficient in inducing this response than the longer waves (Harrington and Leaming, 1900; Mast, 1910). According to Inman, Bovie, and Barr (1926), ultra-violet light is prob- ably more efficient than visible light. Although the distribution in the spectrum of stimulating efficiency has not been precisely ascertained, Folger (1925) maintains that it is not closely correlated with tempera- ture. He did not thoroughly investigate the problem, however. Kinetic ves ponses.—lf an amoeba is kept for some time in very weak light it becomes inactive; if the light is then increased, the organism gradually becomes active again. This response is similar to the response to change in temperature. It is primarily correlated with the magnitude of the change, not with the rate of change in intensity. It is probably due to the effect of light on the rate of transformation of gel to sol and vice versa. This type of response occurs also in D7fflagia (Mast, 1931c), but Ona HT CO % @ 15000 m.c. Rate of locomotion, micra per minute 26600 m.c. 7.5 15 DIS les) 15 22.5 30 Time in light in minutes Figure 96. Relation between adaptation to light of different intensities and rate of locomotion in Amoeba proteus. Each point in the figure represents the average for one measurement on each of from fourteen to twenty-three specimens. The time in light is the time from the beginning of movement after exposure until the measurements were made. (After Mast and Stahler, 1937.) MOTOR RESPONSES 249 the observations on it should be repeated and extended under carefully controlled conditions. Mast and Stahler (1937) made a thorough study of the relation be- tween luminous intensity and rate of locomotion in A. proteus. They found that if dark-adapted amoebae are exposed to light, the rate of locomotion gradually increases to a maximum and then remains con- stant; that the time required to reach the maximum decreases from 15 minutes at 225 meter-candles to a minimum of 7 minutes at 15,000 meter-candles, and then increases to 30 minutes at 40,000 meter-candles; and that the rate of locomotion at the maximum increases from 128.8 ++ 10.8 micra per minute at 50 meter-candles to 219.3 -+ 11.4 micra per minute at 15,000 meter-candles, and then decreases to 150.2 + 8.5 micra per minute at 40,000 meter-candles (Fig. 96). They present evi- dence which indicates that the increase in rate of locomotion with in- crease in light intensity is due to the action of the longer waves, and that the decrease in rate in intensities beyond the optimum is due to the action of the shorter waves. This action of light on rate of locomotion 1s similar to the action of temperature. It is probably due to changes in the rate of sol-gel and gel-sol transformations. If this is true, both of these trans- formations must be augmented by the longer waves and retarded by the shorter. Orientation —Davenport (1897) found that A. proteus orients fairly precisely in a beam of direct sunlight and that it is photonegative, but he did not ascertain the processes involved in orientation. Mast (1910) demonstrated that if an amoeba is unilaterally illu- minated, pseudopods develop more freely on the shaded side than on the illuminated side, and that this results in gradual turning from the light (Fig. 97). He concludes that orientation is due to retardation in the formation of pseudopods on the more highly illuminated side, owing to increase in the thickness of the plasmagel on this side caused by the gelating effect of light. There is some evidence which indicates that A. proteus is photopositive in very weak light (Schaeffer, 1917; Mast, 1931a). More carefully con- trolled observations concerning this are highly desirable. It is possible that the kinetic responses in Amoeba are due to changes in the rate of sol-gel transformations at the anterior end, and gel-sol 280 MOTOR RESPONSES transformations at the posterior end. Moreover, the shock reactions ap- pear to be associated with rapid local increases in the sol-gel transforma- tion. If the views concerning the process of orientation as presented —oo 3:52 3:49 3:48 P.M. 3:54 <¢——— iF — 3257°5 Ga 3:55 3:57 a 3:58°5 kK 05 mn. a] Figure 97. Camera outlines representing different stages in the process of orientation in Amoeba proteus. 1, Amoeba oriented in light I’; 2-9, successive positions after ex- posure to light /, time indicated in each. Arrows represent the direction of streaming of protoplasm in the pseudopods. In those which do not contain arrows there was no perceptible streaming at the time the sketch was made. / and /’ direction of light; mm, projected scale. (After Mast, 1910.) above are correct, it is obvious that orientation is the result of shock reactions rather than kinetic reactions. B. HEAGELLATES Response to rapid changes in the intensity of light is very widespread among the flagellates, and many of them orient fairly precisely. The processes involved are essentially the same in all. Ezglena is representa- tive of those which orient, and Peranema trichophorum is representative of those which do not. Euglena rotates continuously on its longitudinal axis as it swims. The flagellum extends backward along the ventral or abeyespot surface. This causes continuous deflection of the anterior end toward the opposite MOTOR RESPONSES 281 surface, resulting in a spiral course. Its direction of movement is changed by the shifting of the distal end of the flagellum from the surface of the body so as to increase the angle between it and the surface. This in- creases the deflection of the anterior end (Fig. 98). Shock reaction and aggregation —Engelmann (1882) observed that if the intensity of the light in a field in which euglenae are swimming about at random is rapidly decreased, they stop suddenly, then turn and pro- A B Figure 98. A. Diagrams showing the position of the flagellum as seen in a viscid medium; a, when Exglena is swimming forward in a narrow spiral; 5, when swerving sharply towards the dorsal side; c, when moving backwards. B. Dotted area, shows the position of the moving India-ink particles. a, when Evglena is swimming forward in a narrow spiral; 5, when swerving toward the dorsal side during a shock-movement. (After Bancroft, 1913.) ceed in various directions. He designated the response as a ‘‘Schreck- bewegung” (fright movement, or shock reaction), because the re-ori- ented organisms gave the impression of having been frightened. It was found that if the intensity is slowly changed this response does not occur. It is therefore dependent upon the rate of change in intensity. He says that if there is a spot of relatively strong light in the field, it acts just like a trap; owing to random movements, the euglenae get into this spot, but as they reach the boundary on the way out, the rapid reduction in intensity induces the shock reaction and consequently pre- vents their exit. Under some conditions the euglenae respond to rapid increase in in- 282 MOTOR RESPONSES tensity and aggregate in a spot of relatively weak light in the field (Mast, 1911). Orientation —lf euglenae are exposed in a beam of light, they usually swim toward or away from the source of light, 1.e., they may be either photopositive or photonegative. Verworn (1895) postulated that if the euglenae are not directed to- ward or away from the light, so that one side is more strongly illuminated than the other, the flagellum beats more effectively in one direction than in the opposite, that this causes the euglenae to turn until both sides are equally illuminated, and that the flagellum then beats equally in op- posite directions and the organism moves directly toward or away from the source of light. The above hypothesis is, in principle, essentially the same as that formulated by Ray (1693), in reference to orientation in plants, and later accepted by de Candolle (1832). According to this idea, the effect of light on the activity of the motor mechanism, or upon the photo- receptors connected with it, is dependent upon the intensity (not upon change of intensity) of the illumination. The light acts continuously after orientation has been attained, as well as during the process of orientation. During the process of orientation, the illumination on op- posite sides 1s unequal, which results in quantitatively unequal action in the motor mechanism; but after orientation, it is equal on opposite sides, and consequently the action of the mechanism on opposite sides is equalized. Verworn applied this theory to ciliates as well as flagellates. In his earlier work, Loeb (1890) strenuously opposed the theory out- lined above, accepting Sachs’s “‘ray-direction theory” as the alternative. He adopted it later (1906), however, and applied it to higher animals, introducing the idea that the action of the locomotor appendages is quantitatively proportional to the intensity of the light on the photo- receptors connected with them. He maintained that this is due to the effect of light on muscle tonus. This theory has been designated the “difference of intensity theory,” the ‘‘continuous-action theory,” the “tropism theory,” and ‘““Loeb’s muscle-tonus theory” (Mast, 1923). Engelmann (1882) demonstrated that only the anterior end of Exglena is sensitive to changes in luminous intensity. Jennings (1904) contends that because of this, all turning from the light results in a reduction of illumination, whereas all turning toward the light results MOTOR RESPONSES 283 in an increase in illumination of photosensitive substance. The photoposi- tive specimens consequently turn until they face the light, whereas photo- negative specimens turn until they face in the opposite direction. When the stimulus which induces turning ceases, the organisms continue either directly toward or from the light. Mast (1911) made a very intensive study of the process of orientation in a species of Evglena which crawls on the substratum but continuously rotates on the longitudinal axis as it proceeds. This Evglena orients very precisely in light, it has a well-developed eyespot, and it moves so slowly that the different phases of its responses can readily be followed in detail. It is therefore very favorable for the study of the process of orientation. If the intensity of the light is rapidly decreased in a beam in which specimens are proceeding toward the source of light, they stop suddenly and bend in the middle toward the abeyespot surface until the two halves form nearly a right angle; then they begin again to rotate on the longi- tudinal axis; and, while rotating, they gradually straighten and proceed once more toward the light source. If the intensity of the light is in- creased, or if it is slowly decreased, there is no perceptible response. The cessation of movement and the bending are therefore dependent upon the rate of decrease in the intensity of the light in the field, i.e., it 1s a shock reaction. The decrease in the intensity of light in the field neces- sarily results in decrease in intensity of light on all the substance in the field; it therefore must cause decrease in the illumination of the photo- sensitive substance. The response, then, is dependent upon the rate of decrease in the light on the photosensitive substance. If the direction of the beam of light is changed through 90° without alteration in intensity, the specimens oriented in it are illuminated laterally. Those in which the eyespot surface faces the light after the direction of the rays has been changed, stop at once. They bend in the middle toward the abeyespot surface, then rotate, and gradually straighten to resume their crawling movements. Those in which the eye- spot surface does not face the light after the direction of the rays has been changed, do not respond to the changed direction of the rays, until, in the process of rotation on the longitudinal axis, this surface faces the light; then they also stop, bend, rotate, straighten, and proceed. Thus they continue until, in the process of rotation, the eyespot surface again 284 MOTOR RESPONSES faces the light, when they again respond. The gradual straightening during rotation results in greater deflection of the anterior end toward, h m en 0-03 seed Figure 99. Ezglena sp. in a crawling state, showing details in the process of orienta- tion; v, contractile vacuole; es, eyespot; 7, 0. direction of light; a-c, positions of Euglena with light from » is intercepted; c-m, positions after light from » is turned on and that from o cut off, so as to change the direction of the rays. If the ray direction is changed when the Ezglena is in position c, there is no reaction until it reaches d. Then it suddenly reacts by bending away from the source of light to e, after which it continues to rotate and reaches position f, where it gradually straightens to g, and rotates to 4, when the eyespot again faces the light and the organism is again stimulated and bends to 7, from which it proceeds to j, and so forth. If the ray direction is changed when the Ezglena is at d, it responds at once and orients as described above. If the intensity from » is lower than that from o the organism may respond at once when the ray direction is changed, no matter in which position it is. (After Mast, 1911.) rather than away from the light. Thus the anterior end becomes directed more and more nearly toward the light source, until an axial position is reached in which changes in illumination of the eyespot surface, owing MOTOR RESPONSES 285 to rotation, disappear. The organism 1s then oriented (Fig. 99). The response induced by changing the direction of the rays, or by rotation in lateral illumination of uniform intensity, is precisely the same as the response induced by a decrease in the intensity of the light in the field Figure 100. Side view of anterior end of Exglena viridis. e, pigmented portion of eyespot; f, flagellum; e.f, enlargement in flagellum; c.v, contractile vacuole; e.s. eye- spot surface of the organism; a4.s. abeyespot surface of the organism. (After Wager, 1900.) without a change in the surface illuminated. The change from illumina- tion of the anterior end or the abeyespot surface to illumination of the eyespot surface therefore must, in some way, result in a rapid decrease in the illumination of the photosensitive substance. How is this brought about? Wager (1900) demonstrated that the eyespot in Exglena consists of a spoon-shaped portion containing red pigment and a small globular enlargement of one of the roots of the flagellum in the concavity of the pigmented portion (Fig. 100). The eyespot is situated near the eyespot 286 MOTOR RESPONSES surface, a short distance from the anterior end, with the convex surface directed outward and backward. When the anterior end, or the abeyespot surface, is directed toward the light, the enlargement in the eyespot 1s fully exposed; but when the eyespot surface faces the light, the enlarge- ment is in the shadow cast by the pigmented portion. It 1s evident, then, that rapid change from illumination of the anterior end, or the abeyespot surface, to illumination of the eyespot surface causes rapid decrease in illumination of the enlargement in the eyespot, and that if the en- largement is photosensitive, change in the direction of the rays or rota- tion on the longitudinal axis has the same effect as decrease in the in- tensity of the light in the field. It is therefore highly probable that the enlargement in the eyespot is photosensitive and that the pigmented por- tion functions in producing changes in intensity of light on it, when the axial position of the organism changes and when it rotates on the longi- tudinal axis in lateral illumination. This contention is supported by the facts that the region of maximum stimulating efficiency in the spectrum is in the blue for Evglena (Mast, 1917) and that blue is absorbed by the yellowish-red pigmented portion of the eyespot (Fig. 102). In photonegative specimens the responses to changes in light intensity in the field and to changes in the surfaces illuminated are precisely like those of photopositive specimens, except that the responses are induced by (1) increase rather than decrease in light intensity and (2) by change from illumination of the eyespot surface to illumination of the abeyespot surface. The process of orientation in free-swimming specimens ts, in prin- ciple, precisely the same as it is in crawling specimens. Orientation in Evglena is, then, clearly due to a series of responses dependent upon the rate of change in the intensity of the light on the photosensitive substance, which 1s probably situated in the concave sur- face of the pigmented portion of the eyespot. The light does not act continuously, and there is no evidence whatever indicating anything in the nature of balanced or antagonistic action of locomotor appendages on opposite sides, in accord with the Ray-Verworn theory. The evidence in hand indicates, in short, that the photosensitive sub- stance is confined to the concavity in the pigmented portion of the eye- spot; that rotation on the longitudinal axis results in alternate shading and exposing of this substance, if the organisms are not directed toward MOTOR RESPONSES 287 or from the light; that this induces shock reactions which result in orientation; and that the organisms remain oriented and proceed directly toward or away from the light, because, after they have attained either of these two axial positions, rotation no longer produces changes in the illumination of the photosensitive substance in the eyespot, and they therefore continue in the direction assumed. In other words, the orienting stimulus ceases after the organism has become oriented. The organism then continues directly toward or away from the light because (1) owing to internal factors, it tends to take a straight course, and because (2) if for any reason it is turned from this course, the orienting stimulus immediately acts, and induces shock reactions which bring it back on its course. Bancroft (1913) presented evidence against the contention that photic orientation in Evglena is due to shock reactions and concluded that it is due to tonus effects brought about by “the continuous action of the light,” in accord with his conception of Loeb’s tropism theory. Mast (1914) demonstrated, however, that if the evidence presented by Bancroft is valid, it proves that his explanation of orientation in Exglena is not correct. Moreover, the fact that after Evglena is oriented, the rate of locomotion is practically independent of the luminous in- tensity (Mast and Gover, 1922) also militates against his explanation. Orientation in light from two sources —In a field of light consisting of two horizontal beams crossing at right angles, Evglena orients and goes toward or away from a point between the two beams. The location of this point is related to the relative intensity of the two beams in such a way that the tangent of the angle between the direction of locomotion and the rays in the stronger beam is approximately equal to the intensity of the weaker divided by that of the stronger (Fig. 101) (Buder, 1917; Mast and Johnson, 1932). Buder maintains that this demonstrates that there is a quantitative proportionality between the stimulus and the re- sponse. Mast and Johnson conclude that “‘it has no bearing on the prob- lem concerning the quantitative relation between stimulus and response,” but that it can be explained on the assumptions that the eyespot is a photoreceptor and that the stimulating efficiency of light varies with the angle of incidence. Wave length and stimulating efficiency —The shorter waves in the visible spectrum are more efficient than the longer in stimulating Euglena Theoretical 1 Observed Figure 101. Graphs showing the relation between the direction of locomotion ob- served in a field of light produced by two horizontal beams crossing at right angles and that demanded by the ‘‘Resultantengesetz.’’ Abscissae, angles between the direction of locomotion and the direction of the rays in the stronger beam observed with dif- ferent ratios of intensities in the two beams, ranging from 0 at 0° to 1 at 45°; ordinates, angles between the direction of locomotion and the direction of the rays in the stronger beam demanded by the ‘‘Resultantengesetz.”” @ Euglena rubra; © Gonium pectorale; @ Volvox minor; ©) Volvox globator. (After Mast, 1907.) Note that if the observed direction of locomotion were the same as the theoretical all the points would fall on the broken line, and that this practically obtains for Evglena but not for Volvox and Gonium. Note also that for the latter, as the ratio between intensity in the two beams decreases from 1, the difference between the theoretical and the observed results in- creases to a maximum, then decreases to zero, after which it increases in the opposite direction. (After Mast and Johnson, 1932.) MOTOR RESPONSES 289 and other flagellates. Strasburger (1878) concluded that stimulation is confined to violet, indigo, and blue in the solar spectrum, with the maxt- mum in the indigo. Engelmann (1882) maintains that for Euglena the maximum is in the blue between 470 my, and 490 mu, and Loeb and | | ALA ZA | ABISh MAN I 1) ©} | S| LH cad S =— a — is ——— PYLON RI FB BSe =a ie =] Eee NR S aoe aS -—+— =! me: ~~ anny ie) ~ Pes Vee ce Rahss A x LJ Z| a LL ia Ef ll om rsa amas ays F Pl Blo N L Be a a se a ll al BSE Seba Res rex es Je eSB ERReSGERaee SIAR Sees esas JASE 2S2eares JAR SVAe sashes ARS SRBs [A > f=) Sisuss! eS eS | |Z {@ ACies 19% iE @| | Ultra Violet Violet Blue Green Yellow Red = > o e e °o o Figure 102. Curves representing the distribution in the spectrum of stimulating efficiency, constructed from data given in Table 15 (See Mast 1917). A, Pandorina (nega- tive); B, Pandorina (positive) ; C, blowfly larvae; D, Euglena viridis (negative); E, Euglena viridis (positive) F, Euglena tripteris (negative); G, Avena sativa (oat seed- lings). (Constructed from data obtained by Blaauw.) The circles represent points experi- mentally established abscissae, wave lengths; ordinates, relative stimulating efficiency on the basis of equal energy. The curves for Eudorina and Spondylomorum, not represented in the figure, are in position and form essentially like those for Pandorina; the curve for Chlamydomonas is much like that for blowfly larvae; those for Euglena gracilis, E. minima, E. granulata, Phacus, Trachelomonas, Gonium, Arenicola, and Lumbricus are nearly like those for E. viridis and E. tripteris. (After Mast, 1917.) Maxwell (1910) assert that in the carbon-arc spectrum it is between 460 and 510 my. The unequal distribution of energy in the spectrum was not considered in these conclusions. Mast (1917) made corrections for un- equal distribution of energy and ascertained the relative stimulating effi- ciency of negative and positive orientation at intervals of 10 #2 through- 290 MOTOR RESPONSES out the visible spectrum. He found that as the wave length increases, the stimulating efficiency also increases very rapidly from zero at about 410 my to a maximum of 21 arbitrary units at 485 mu, and then de- creases equally rapidly to zero at about 540 my, (Fig. 102). He holds, however, that the limits of the stimulating region depend upon the luminous intensity. Kinetic responses—Ilf Euglena is subjected for long periods to low illumination or to darkness, it gradually becomes less active; and if the illumination is then increased, it gradually becomes more active. The rate of change in activity varies with the magnitude of the change in intensity. But this response is never so sudden and abrupt as the shock reaction. There are therefore two types of responses to light in Exglena, one depending primarily upon the rate of change in luminous intensity, the other primarily upon change in the amount of light received. The one results in orientation and aggregation, the other in change in activity. Mast and Gover (1922) measured the rate of locomotion in several different flagellates in different intensities of light and found very little correlation between rate and intensity. The environmental factors were, however, not accurately controlled, and adaptation was not considered. The measurements should therefore be repeated, with the methods used by Mast and Stahler (1937) in their observations on Amoeba. Reversal in response —Euglena 1s ordinarily photopositive in weak light and photonegative in strong light. The orienting response therefore tends to keep it in light of moderate intensity, indicating that these re- sponses are fundamentally adaptive. This has not been demonstrated, however, because the direction of orientation is not specifically correlated with luminous intensity. For example, euglenae which are strongly photo- positive in a given intensity of light at room temperature may become equally strongly photonegative if the temperature is rapidly decreased 10 to 15 degrees, the extent of the requisite decrease depending upon the state of adaptation (Mast, 1911). This problem is much in need of thorough investigation. It is a very important problem because it concerns the biological significance of response to light in these organisms. II. Peranema tricophorum.——Peranema is a colorless flagellate with- out an eyespot. It is usually in contact with the substratum and moves slowly with the flagellum extending forward (Fig. 103). If the luminous MOTOR RESPONSES Zo intensity is rapidly increased, it stops suddenly and then deflects the anterior end sharply to one side. If the intensity is slowly increased, or if it is decreased, there is no response. The entire organism is sensitive to Ha Os Tr 2, oy Figure 103. Camera drawings illustrating the response of Peranema to contact or to rapid increase in luminous intensity. Al, normal locomotion; 2, immediately after re- sponse; 3, 4, recovery from response; B, response to contact with grain of sand, 0, and recovery. Note that response results in a change in the direction of motion of approxi- mately 90 degrees. (Mast, 1912.) light, but the flagellum is most sensitive and the posterior end least sen- sitive (Shettles, 1937). Dark adaptation—Mast and Hawk (1936) demonstrated that if light-adapted peranemae are subjected to darkness, the time required in light of 2,000 meter-candles to induce the response decreases from 30.95 seconds after 15 minutes in darkness to a minimum of 4.54 seconds after one hour in darkness, then increases to a maximum of 63.46 seconds 292 MOTOR RESPONSES after 6 hours in darkness, and then remains nearly constant. As the time in darkness increases, the sensitivity to light rapidly increases to a maxi- mum, then decreases to a minimum, before it becomes constant (Fig. 104). ae. ee ee (2 4 6 8 /0 /2 /4 TIME IN DARKNESS IN HOURS 60 3 A 9 wy 9 REACTION TIME IN SECONDS NY is} JO aA Figure 104. Graph showing the effect of dark-adaptation on sensitivity to light in Peranema trichophorum. Each point on the curve, except the last two, represents the average reaction time for from fifteen to seventeen tests. (After Mast and Hawk, 1936.) Light adaptation—Shettles (1937, 1938) made a much more exten- sive and thorough investigation of this response. He confirmed the con- clusions reached by Mast and Hawk (1936). A brief summary of other results obtained, and the conclusions reached, follows: If dark-adapted peranemae are subjected to light, their sensitivity to light increases rapidly to a maximum, then decreases considerably, after which it remains nearly constant (Fig 105). The reaction time consists MOTOR RESPONSES 295 of an exposure period and a latent period. With increase in intensity of illumination, the exposure period decreases, at first rapidly, then more slowly, until it becomes nearly constant; the latent period increases to a maximum and then decreases, and the amount of light energy re- quired during the exposure period increases from 22,970 meter-candle seconds at 538 meter-candles to 54,315 meter candle seconds at 2,152 meter-candles, and then decreases to 13,498 meter-candle seconds at INTENSITY J 2l52m.c. REACTION TIME [N SECONDS INTENSITY INTENSITY 86// m.c. 6458 =m.c. / e 4 TIME IN LIGHT IN HOURS Figure 105. Graphs showing rate of light-adaptation. Dark-adapted peranemas were exposed to light (intensity given in the graph) for the time indicated, then subjected to darkness one half hour, then exposed to 2,152 m.c., and the reaction time measured. Each point on the curves represents the average reaction time for ten tests, different individuals being used in each test. (After Shettles, 1937.) 6,458 meter-candles (Fig. 106). He concludes that ‘the amount of light energy required to induce a shock-reaction in Peranema varies greatly with the intensity of the light and that the Bunsen-Roscoe law conse- quently does not hold.” The latent period decreases from 39.68 seconds at 10° C. to 24.3 seconds at 30° (15.38 seconds), but the exposure period decreases from 27.87 to 22.97 seconds (only 5.8 seconds). This indicates that there 294 MOTOR RESPONSES 70 60 WY = S 50 5 Q wh = 40 4 ly = re Ww S a SANe } FERIOD EXPOSURE PERIOD” s /O LIGHT ENERGY IN 10,000 METER CANDLE SECONDS % 538 107% 2/52 3227 4305 $382 6458 &6// 10764 LUMINOUS INTENSITY IN METER CANDLES Figure 106. Graphs showing relation between luminous intensity and reaction time, exposure period, latent period, and energy. Each point on the curves of the reaction time and the exposure period represents the average for ten tests, different individuals being used in each test. Each point on the curve of the latent period represents the dif- ference between the reaction time and the exposure period. Each point on the energy curve represents exposure period luminous intensity. (After Shettles, 1937.) are at least two processes involved in the response of Peranema to light. One of these 1s nearly independent of temperature and is therefore prob- ably photochemical. The other is closely correlated with temperature and is therefore not photochemical. After Peranema has responded in light of a given intensity, it must be subjected to light of a lower intensity or to darkness before it will again respond. The time required for recovery varies directly with the MOTOR RESPONSES 29) luminous intensity in which the response occurred, and inversely with the temperature. Wave length and stimulation Stimulating efficiency of light 1s closely correlated with wave length. There are two maxima in the spec- trum, one in the ultra-violet at 302 m1 and one in the visible at 505 my. The latter is nearly twice as great as the former. The absorption of light by Peranema in the violet remains nearly constant as the wave length decreases from 450 my to 325 my, then increases rapidly and extensively to a maximum at 253 my. The maxi- mum injuring efficiency is also at 253 my. Injury is therefore closely correlated with the amount of light absorbed, but stimulating efficiency is not, for the maximum is at 302 my in place of 250 nz. The processes involved in stimulation therefore differ from those involved in injury. Injury, in Peranema, is due to coagulation of the protoplasm, whereas stimulation is probably not due either to coagulation or to increased viscosity. Peranema tesponds very precisely and very consistently. Its move- ments are very slow and its reaction time long. Since it can be grown under fairly accurately known environmental conditions in total dark- ness, it is well suited for quantitative work of a high order. Cy GClEVAES Very few of the ciliates respond to light. Only one of these, Stentor coeruleus, has been investigated extensively. If the luminous intensity is rapidly increased, this organism stops, turns toward the aboral surface, and then proceeds. This is a shock reaction, because if the intensity is slowly increased there 1s no response. If the intensity is decreased, no matter whether rapidly or slowly, there is no response. If Stentor is exposed in a beam of light, it orients fairly precisely and swims away from the light, i.e., it is photonegative. It rotates on the longitudinal axis as it swims, so that if it is not oriented, the oral and the aboral surfaces are alternately shaded and illuminated. The oral surface is much more sensitive than the aboral; therefore every time that this surface is carried from the shaded to the illuminated side, the result is the same as an increase in the illumination of the entire organism, and it consequently responds, i.e., it turns toward the aboral surface. This continues until it is directed away from the light, and Figure 107. Stentor coeruleus in the process of orientation. Curved line, spiral course; arrows m and », direction of light from two sources; a-f, different positions of Stentor on its course; 0, oral surface; ab, aboral surface. At a the Stentor is oriented in light from m, n being shaded. If » is exposed and m shaded simultaneously when the Stentor is in position 4, there is usually no reaction until it reaches ¢ and the oral side faces the light; then the organism may respond by suddenly stopping, backing, and turning sharply toward the aboral side (dotted outline), and become oriented at once; or it may merely swerve toward the aboral side without stopping. At e the oral side is again exposed, and the organism is again stimulated and it again swerves from the source of light. This process continues until the oral side is approximately equally exposed to the light in all positions on the spiral course. If the Stentor is at c when » is exposed, it responds at once and orients as described above. If the light from » is more intense than that from m, or if the organism is very sensitive when » is exposed and m shaded, it re- sponds at once, no matter in which position it is. If it is at 4, it turns toward the source of light, but now repeats the reaction, successively turning in various directions until it becomes oriented. (After Mast, 1911.) MOTOR RESPONSES 297 rotation no longer produces changes of intensity on the opposite sur- faces (Fig. 107). Photic orientation in S/entor is therefore the result of a series of shock reactions, as is the case in Evglena. There is no evi- dence in support of the view that it is the result of a continuous quanti- tative difference in the activity of the cilia on opposite sides, in pro- portion to the difference in the illumination of these sides. The process of orientation in Stentor is therefore not in accord with the Ray-Verworn theory. If stentors are exposed in a field of diffuse, non-directive light which contains a dark spot, they aggregate in this spot. The process of ag- gregation is, in principle, precisely the same as the process of aggrega- tion of photonegative euglenae in a dark spot. They reach the dark spot by random movements. No reaction occurs when the organisms enter the unilluminated area. However, at the periphery on the way out, as the light intensity rapidly increases, they stop suddenly, turn sharply to- ward the aboral surface, and then proceed in a different direction. The dark spot therefore acts like a trap (Jennings, 1904; Mast, 1906, 1911). The relative stimulating efficiency of different regions in the spectrum has not been investigated; no observations have been made on the quantitative relation between the different phases of the shock reaction, the state of adaptation, and the extent of change in luminous intensity. Indeed, very little is known about the body processes involved in stimula- tion and response. D. COLONIAL ORGANISMS Response to light is essentially the same in all of the colonial forms in which it has been studied, but it has been more intensively investigated in Volvox globator than in any of the other species. Volvox is a slightly elongated, globular colonial organism somewhat less than one mm. in diameter. It consists of numerous cells (zodids), each of which contains two flagella and an eyespot. The zodids are ar- ranged in a single layer at the surface of the colonies. The eyespot in each zodid is directed toward the posterior end of the colony, but those at the anterior end are much larger than the rest (Fig. 108). Mast (1927a) presented evidence which demonstrates that the eye- spots consist of a cup-shaped pigmented portion, a lens-like structure near the opening of the cup, and photosensitive substance between this 298 MOTOR RESPONSES and the inner surface of the cup. The evidence also indicates that the lens-like structure brings the longer incident waves of light to focus in the wall of the cup; and that the shorter wave lengths, after being reflected from the inner surface of the cup, are focused in the photosensitive sub- stance (Fig. 109). ! ! 1 i ' ) 1 i} ' 1 1 ' 1 1 1 | ' 1 ' ' ' ' ! ' ' ' ' ' 1 i} ' ' ' ' ' ( 1 ' ' 1 1 1 ' ' ' ' Figure 108. Camera drawing showing the zodids in about one half of an optical sec- tion through the longitudinal axis of a colony of Volvox. l-a, longitudinal axis of colony; a, anterior end; z, zodids, f, flagella; e, eyes. Note that the eyes are located at the outer posterior border of the zodids and that they become larger as the anterior end of the colony is approached. (After Mast, 1927.) Movement, response, and orientation in V. globator have been thor- oughly studied by Mast (1907, 1926b, 1927b, 1932b). The more im- portant of the results obtained in this study lead to the following con- clusions. Shock reaction.—V olvox colonies rotate on the longitudinal axis as they swim. This is due to the diagonal stroke of the flagella. In a beam of light they usually orient and go almost directly either toward or away from the light, i.e., they may be photopositive photonegative, or neutral. If, while the colonies are swimming toward the light, the intensity ts rapidly decreased without any change in the direction of the rays, rota- tion on the longitudinal axis stops and forward movement increases MOTOR RESPONSES 299 greatly. On the other hand, if the intensity is rapidly increased, the for- ward movement stops and the rate of rotation increases. If the colonies are swimming away from the light, the reverse occurs, i.e., forward move- ment decreases if the intensity is increased, and increases if it is de- creased. If the colonies are neutral, there are no such responses to changes of intensity. These responses consist chiefly, if not entirely, of rapid changes in the direction of the stroke of the flagella. In other words, a rapid decrease in the illumination of photopositive colonies changes the Figure 109. Sketches showing the structure of the eyespot in Volvox and its action on light entering the pigment-cup at different angles. p, pigment-cup; /, lens; y, yel- low focal spot; 4, bluish green focal spot; 55, photosensitive substance; large arrows, incident rays of light. Note that the longer waves of the incident light are brought to focus in the wall of the pigment-cup and that the shorter waves are brought to focus in the cup, after being reflected from the inner surface, and then continue in the form of a concentrated beam of bluish-green light. Note also that the more obliquely the incident light enters the pigment-cup, the nearer the edge of the cup the yellow focal spot is located. (After Mast, 1927.) stroke of the flagella from diagonally backward to straight backward. An increase in the illumination causes it to change from diagonally back- ward to sidewise. In photonegative colonies precisely the reverse obtains. These responses continue for only a few seconds, although if the luminous intensity is slowly changed they do not occur at all. They are therefore dependent upon the rate of change in intensity, i.e., they are shock- reactions which are somewhat similar to those observed in Euglena. Kinetic res ponses.—If Volvox is kept in weak illumination or in dark- ness for several hours, it becomes inactive; but if the illumination 1s afterwards increased, it gradually becomes active again. These responses consist chiefly, if not entirely, in changes in the rate or the efficiency of 300 MOTOR RESPONSES the stroke. Changes in the direction of the stroke of the flagella are not involved. They are relatively slow responses which occur, even if the luminous intensity is gradually changed. The responses are primarily dependent upon change in luminous intensity, not upon the rate of change. Consequently, there are, in Volvox, two different types of re- sponse: (1) typical shock reactions, and (2) responses which consist merely in changes in activity. Holmes (1903) maintains there is no consistent correlation between luminous intensity and rate of locomotion in Vo/vox. But his methods did not exclude the effect of adaptation. Further work concerning this correlation is therefore highly desirable. Orientation —lf a colony of Volvox in a beam of light is laterally illuminated, it turns gradually until it is oriented, and then proceeds either toward or away from the light source. When it is laterally illumi- nated, the zodids, owing to rotation of the colony on the longitudinal axis, are continuously transferred from the light side to the dark side, and vice versa. As the zodids pass from the light side to the dark side, the photosensitive substance in the eyespots becomes shaded by the pigment cup. As they pass from the dark side to the light side, this substance be- comes fully exposed. A rapid decrease in the illumination of the sensitive substance on the dark side of photopositive colonies induces shock reac- tions on this side, and the flagellar stroke increases in its backward phase. A rapid increase in illumination of the sensitive substance on the light side of a colony induces shock reactions. The latter consist of increase in the lateral phase of the stroke of the flagella (Fig. 110). This difference in the direction of the stroke of the flagella causes the colonies to orient gradually, until they are directed toward the light, after which all sides are equally illuminated. Rotation on the longitudinal axis then no longer produces changes in the illumination of the photosensitive substance, and the shock reactions cease. The Volvox colonies continue directly toward the light because, in the absence of external stimulation, they tend to take a straight course. Furthermore, if they are forced out of their course, opposite sides immediately become unequally illuminated, the intensity of the illumination of the photosensitive substance in the eyespots changes, and consequently reorientation occurs. If photopositive colonies are exposed in a field of light consisting of two horizontal beams which cross at right angles, they orient and swim toward a point between the two beams. The location of this point de- Figure 110. Diagrammatic representation of the process of orientation in Volvox. A, B, C, D, four zodids at the anterior end of the colony; /-a, longitudinal axis; large arrows, direction of illumination; small arrows, direction of locomotion; curved arrows, direction of rotation; f, flagella; e, eyes, containing a pigment-cup represented by a heavy black line and photosensitive tissue in the concavity of the cup. Note that when the colony is laterally illuminated, the photosensitive tissue in the eyes on the side facing the light is fully exposed and the flagella on-this side beat laterally. Those on the opposite side, shaded by the pigment-cup and the flagella on this side, beat directly backward. The difference in the direction of the beat of the flagella on these two sides is due to alternate decrease and increase in the luminous intensity to which the photo- sensitive tissue in the eyes is exposed, owing to the rotation of the colony on its longi- tudinal axis—an increase causing, in photopositive colonies, a change in the direction of the stroke of the flagella from backward or diagonal to lateral; and a decrease, a change from lateral or diagonal to backward. In photonegative colonies, precisely the opposite obtains. In photopositive colonies, this results in turning toward and in photonegative colonies turning from the source of light. In both, the turning continues until opposite sides are equally illuminated, when changes of intensity on the photosensitive tissue are no longer produced by rotation and the orienting stimulus ceases. (After Mast, 1926a.) 302 MOTOR RESPONSES pends upon the relative intensity of the light in the beams. The higher the intensity in one of the beams in relation to that in the other, the nearer to the former the point is. If the intensity in the two beams 1s equal, the point is halfway between them. The colonies are oriented under these conditions, opposite sides are equally illuminated, both in reference to intensity and direction of the rays, i.e., the angle of inci- dence at the surface of the colony. If the intensity in the two beams 1s not equal, the illumination of the oriented colonies is higher and the angle of incidence greater on one side than on the other. However, when a colony is oriented in a field of light, no matter how unequal the in- tensity from different directions may be, transfer of the zodids from side to side in consequence of rotation on the longitudinal axis causes no responses. In other words, the effect of unequal illumination on opposite sides is equal. This obviously must be correlated with the difference in the angle of incidence. Mast (1927a) and Mast and Johnson (1932) demonstrated that the location of the point of focus in the eyespot varies with the angle of incidence. By ascertaining the location of these points in the eyespots on opposite sides of the colonies, in relation to the relative intensity of the two beams, they calculated the distribution of sensitivity and found that the photosensitive substance is much more sensitive in the central regions of the eyespot than at the periphery (Fig. 109). The stimulating eff- ciency of light, therefore, depends upon the location of the point of focus; this, in turn, depends upon the angle of incidence. The equal effect of light on the sides of colonies which are unequally illuminated on op- posite sides when they are oriented, is therefore due to the fact that the point of focus in the eyespots is more nearly centrally located on the side which receives the least light than on that which receives most. In photonegative colonies the process of orientation is precisely the same as it is in photopositive colonies, except that decrease in intensity causes increase in the lateral phase, and increase in the light intensity increases the backward phase of the stroke of the flagella. In consequence, the illuminated side moves more rapidly than the shaded side. The colonies therefore turn away from the light source. Orientation of Vo/vox in light is the result of qualitative differences in the action of the locomotor appendages on opposite sides. These dif- ferences are due to shock reactions induced by rapid change in the 1n- MOTOR RESPONSES 303 tensity or the location of the light in the photosensitive substance in the eyespot, by virtue of colony rotation on the longitudinal axis. It should be noted that the responses observed are not the result of quantitative differences due to continuous action of the light. The explanation offered is therefore not in accord with the Ray-Verworn theory. Wave length and res ponse.—The distribution of stimulating efficiency in the spectrum for Volvox (Laurens and Hooker, 1920) and Gonium (Mast, 1917) is essentially the same as it is for Evglena; but for the closely related forms Pandorina and Spondylororum (Mast, 1917) the maximum is at 535 my in place of 485 my, and the effective region ex- tends from this wave length much farther in either direction than it does for Euglena, Gonium, and Volvox (Fig. 102). The orange, pigmented portion of the eyespot in these forms is opaque in reference to the light of all those regions of the spectrum which have the highest stimulating efficiency. The distribution of stimulating efficiency for these forms con- sequently supports the conclusions reached concerning the structure of the eyespots, the distribution of photosensitive substance in them, and their function in the process of orientation. Threshold.—Mast (1907), on the basis of quantitative results, con- cludes that the minimum difference in light intensity on opposite sides of a colony which is necessary to induce a response varies greatly with the physiological state of the colony; but that with colonies in a given physiological state, the response varies directly with the intensity, and the ratio is nearly constant, 7.e., nearly in accord with the Weber-Fechner law. His observations, however, covered such a small range (2-27 meter- candles) and the probable error in the results is so large that further observations concerning this relation are highly desirable. Reversal 1n res ponse.—V olvox is usually positive in weak, and nega- tive in strong light. However, the reverse obtains under some conditions. It may be positive, negative, or neutral in every condition of illumination in which orientations occurs. If it is positive, a shadow on the photo- sensitive substance in the eyespots causes a change in the direction of the stroke of the flagella of the zodids from diagonal to backward. A flash of light on this substance causes a change of stroke from diagonal to side- wise. If the colony is photonegative, the reverse obtains; and if it is neutral, there is no response unless the changes in luminous intensity are great. 304 MOTOR RESPONSES Reversal in the direction of orientation from positive to negative is therefore due to internal changes of such a nature that shock reactions which were produced by decrease are produced by increase in the il- lumination of the photosensitive substance. The shift from negative to positive is due to the reverse. The nature of the response to light in Volvox depends upon the state of adaptation and upon the intensity of the illumination. If Volvox is fully adapted in a given intensity, it be- comes positive if the intensity is increased or negative if it is decreased. If the colony is not fully adapted, it becomes negative if the intensity ts increased or positive if it is decreased. The time required for colonies of Volvox to become negative or posi- tive after the luminous intensity has been changed (the reaction time) depends upon the degree of adaptation and the extent of the change. If colonies which have been subjected first to strong light (1-2 hours) and then to a variable period in darkness are exposed to strong light, the time required to become positive (reaction time) increases with in- crease in the length of the period in darkness (dark adaptation) from 0.04 minutes (with 2 minutes in darkness) to a maximum of 0.52 min- utes (with 16 minutes in darkness) and then decreases to 0.18 minutes (with 25 minutes in darkness). If the colonies are kept longer in strong light and are then subjected to darkness, the reaction time decreases to a minimum and then increases as the time in darkness increases. If they are left in darkness until they are fully dark-adapted, and are then ex- posed to light of different intensities, the reaction time (as the intensity increases) decreases from 29 minutes in 5.24 meter-candles to a mini- mum of 0.098 minutes in 7.5 meter-candles, and then increases to 0.358 minutes in 62,222 meter-candles. The energy required to make the colo- nies positive varies directly with the light intensity, over the whole range tested. Over most of the range this variation is nearly proportional to the variation in intensity. No satisfactory explanation of this relation is avail- able. If colonies are kept in a given intensity or in darkness, they become adapted, 7.e., they lose the ability to respond to light. Their responsive- ness is regained if, after dark adaptation, the intensity is changed. The processes associated with adaptation and those induced by change in il- lumination are therefore antagonistic. The rate of these antagonistic processes varies greatly, depending upon the magnitude of the change MOTOR RESPONSES 305 in intensity. For example, if dark-adapted colonies are exposed to light of 22,400 meter-candles for 0.05 minutes then returned to darkness, it takes 20 minutes or more in darkness to eliminate the effect of the light. This indicates that, under these conditions, the processes which occur in light proceed at least 400 times as fast as the reverse processes which occur in darkness. To account for the phenomena described, it is necessary to postulate at least three interrelated processes, some of which must be directly corre- lated with light in such a way that change in illumination of very short duration can cause complete reversal in the nature of the response. It is altogether probable (1) that some of these processes are photochemical reactions; (2) that others are dependent upon the results of these; and (3) that all are closely correlated with the physiological state of the organism as a whole (Mast, 1932b). The evidence now available clearly indicates that such simple processes as those postulated by Mast (1907) in his first publication dealing with this problem, and those postulated by Luntz (1932) are very inadequate (Mast, 1932b). A considerable number of other facts have been established concerning reversal in Volvox and related forms. For example, increase in tempera- ture of hydrogen-ion concentration, and some anesthetics (especially chloroform) cause photonegative colonies to become strongly photo- positive. However, they usually remain positive only a few moments, then become negative again (Mast, 1918, 1919). There is also a very interesting correlation between reversal in light and response to elec- tricity, in that photopositive colonies always swim toward the cathode and photonegative colonies toward the anode (Mast, 1927c). These facts show that reversal in light is not due to direct action of environ- mental factors. They also indicate that it is correlated with the rate of metabolism; but there is no clue to the nature of the processes involved. RESPONSES TO ELECTRICITY A. RHIZOPODS All the rhizopods which have been investigated (Amoeba, Pelomyxa, Difflugia, Arcella, Actinosphaerium, and others) respond to electricity. Kithne (1864) and Engelmann (1869) observed that if they are sub- jected to a series of induction shocks (alternating current), streaming in them stops and they then round up. Verworn (1895), from observa- 306 MOTOR RESPONSES tions on rhizopods in a direct current, maintains that immediately after the circuit is closed, there is marked contraction at the anodal side and then movement toward the cathode, and that if the current is strong enough, disintegration begins on the anodal surface of the organism. Greeley (1904), in referring to Amoeba, says that ‘‘on the anodal side of the cells the protoplasm is coagulated .. . and on the cathode side it is liquefied.” Bayliss (1920) maintains, however, that the current causes only gelation. According to the careful observations of Luce (1926), hyaline blisters appear on pseudopods oriented with their longi- tudinal axis perpendicular to the direction of the current. With the aid of superior optical apparatus, he observed the transformation of these blisters into pseudopodia. Since there was no indication of gelation, the phenomenon must have been due to a liquefaction of the plasmagel at the cathodal surface. More details concerning response of rhizopods to electricity were ob- tained by Mast (1931b) in observations on A. proteus in direct and alternating currents of various intensities. The results obtained are as fol- lows. Direct current.——In direct current of low density, movement continues no matter how the amoebae are oriented in the field, but the formation of pseudopods is inhibited on the anodal side, resulting in gradual turn- ing toward the cathode. In stronger currents movement ceases imme- diately after the circuit is closed, then in a few moments one or more pseudopods appear on the cathodal side and movement continues directly toward the cathode. In still stronger currents there is marked contraction on the anodal side immediately after the circuit is closed. This is soon followed by disintegration which begins at this side. If the anterior end of the amoeba faces the cathode when the current is made, there is, in the lowest density that produces an observable effect, merely a slight momentary increase in the rate of flow in the plasmasol immediately back of the hyaline cap. No change in the rate of flow ts seen elsewhere. If the current is stronger, this increase extends back farther, the hyaline cap disappears, the plasmasol extends to the tip, the anterior end becomes distinctly broader, and the plasmagel becomes very thin (Fig. 111). If the current is strong enough, this is followed by violent contraction at the posterior end, slight contraction at the an- MOTOR RESPONSES 307 Figure 111. Sketches illustrating the effect of a galvanic current on a monopodal Amoeba moving toward the cathode. g, plasmagel; s, plasmasol; /, plasmalemma; ¢, hyaline cap; 4, hyaline layer; —, cathode; +, anode; arrows, direction of streaming. A, very weak current, B, C, D, progressively stronger current. Note that in a current of moderate density the hyaline cap disappears and the plasmasol extends to the plasmalemma and that in stronger current the cathodal end expands and the anodal end contracts and finally breaks, after which the granules in the plasmasol flow toward the anode, indi- cating that they are negatively charged. If the surrounding medium is acid, the amoebae do not break. (After Mast, 1931a.) terior end, and, finally, by disintegration beginning at the posterior end, i.e., that which is directed toward the anode. If the anterior end of the amoeba is directed toward the anode and the current is weak, there is merely a momentary retardation of stream- ing at the posterior (cathodal) end. With successively stronger currents, 308 MOTOR RESPONSES the streaming at this end (1) stops a few moments, then begins again, and continues in the original direction; (2) it stops a bit longer, then begins, and continues in the reverse direction, i.e., the plasmasol then streams toward the cathode at one end and toward the anode at the other (Fig. 112); (3) the reversal extends to the anodal end, and a new hya- line cap forms at the original posterior end, which now becomes the an- terior end; (4) the reversal is followed by marked contraction at the Figure 112. Sketches illustrating the effect of a galvanic current on monopodal amoebae moving toward the anode in a weak current. Labels are the same as in Figure 111. Note that the direction of streaming reverses, that it begins at the cathodal end and proceeds toward the anodal end, and that this results in movement in opposite directions at the two ends during one phase in the process of reversal. (After Mast, 1931a.) anodal end, which is followed by partial or complete disintegration, al- ways beginning at the anodal end. The fact should be emphasized that no matter how extensive a reversal in the direction of streaming may be, it always begins at the cathodal end of the amoeba. When the contraction at the anodal end begins, the hyaline layer in this region becomes thicker here and there, resulting in the formation of several small blisters and in numerous minute papilla-like foldings in the plasmalemma (Fig. 111). As contraction continues, some of the blisters, containing fluid and a few granules in violent Brownian move- MOTOR RESPONSES 309 ment, round up and are pinched off; and others together with the plasmagel break, after which granules in the plasmasol stream out and proceed rapidly toward the anode. This continues until frequently there is nothing left intact except a crumpled membranous sac, the plasma- lemma. The plasmagel changes entirely into plasmasol and is carried away. The fact that the granules are carried toward the anode shows that they are negatively charged. McClendon (1910) came to the same conclusion concerning the gran- ules in the eggs of frogs and the cells in root tips of onions. But Heil- brunn (1923) says: ‘Particles in the interior of living cells bear a posi- tive, whereas the particles in the surface layer have a negative charge.” If a small amount of HCl is added to the culture solution, the amoeba does not disintegrate, regardless of the current strength. Its plasmagel turns distinctly yellow at the anodal end immediately after contraction begins, after which it increases in thickness until the entire amoeba has solidified and is dead. If the current is broken before more than about one-fourth of the amoeba has gelated, the gelated portion is usually pinched off. The rest of the amoeba then proceeds normally. If the amoeba is moving toward the anode when the current is made, streaming of the plasmasol reverses before it stops at the anodal end. This behavior demonstrates conclusively that the effect of the current begins at the surface directed toward the cathode. The fact that before the reversal occurs, the thick plasmagel at the cathodal end is replaced by a very thin plasmagel sheet and a hyaline cap strongly indicates that the first effect of the current is solation of the plasmagel at the cathodal surface. This conclusion is supported by the facts that if the anterior end is directed toward the cathode when the current is made, the plasmagel sheet disappears entirely, the anterior end enlarges, and the plasmasol extends to the plasmalemma. It is also true that if the current passes through the amoeba in a direction perpendicular to the longitudinal axis, the forward streaming stops, and pseudopods are formed on the cathodal side. The contraction of amoeba at the anodal end, and the increase in the thickness of the plasmagel—especially in specimens directed toward the anode—seem to show that the current causes gelation at the anodal sur- face. The facts that the end directed toward the cathode enlarges, that the 310 MOTOR RESPONSES opposite end decreases in size, breaks up, and becomes yellow just as do amoebae killed in an acid solution, and the granules in the amoeba then move toward the anode, all indicate (1) that the granules bear a negative charge, (2) that water in the amoeba is carried toward the cathode, and (3) that the plasmagel at the anodal surface becomes acid. In very accurately controlled observations on A. proteus, in direct current, Hahnert (1932) found that there is at first a momentary in- crease in rate of locomotion and then a gradual decrease. He also noted 25 20_* ahr : (@) AS} am 15 (@) () 4 LY) — () 0 ©) @ ~ Ore 10 8s Oe | B ORLA | 2050, WEE 4 OG) AB Oy 5 eee | Figure 113. Graphs showing comparative effect of different densities of current on the rate of locomotion. Abscissae, time in minutes, (A) before and (B) during the passage of current; ordinates, apparent rate of locomotion. To obtain actual rate in millimeters per minute, divide the apparent rate by 85. (After Hahnert, 1932.) that the rate of decrease varies directly with the current density (Fig. 113), and that the time required to induce cessation of movement in specimens directed toward the anode varies nearly inversely with the square of the current density, “the time-intensity relation being nearly in accord with the equation 7\/¢— (x/) = K in which 7 is the intensity of the current and ¢ its duration.” Alternating current —If an active Amoeba mounted in culture fluid is subjected to a weak alternating current, locomotion ceases at once. The pseudopods are then retracted partially or entirely, and the animal be- MOTOR RESPONSES ey comes somewhat rounded. It remains in this condition a few moments, then a pseudopod appears on one of the sides between the two surfaces facing the poles and projects at right angles to a line connecting the two poles. Almost immediately after the first pseudopod begins to form, an- other usually appears on the opposite surface and extends in the opposite direction. Thus the amoeba becomes oriented perpendicularly to the direction of the current, i.e., at right angles to the direction in which orientation occurs in a direct current. These two pseudopods usually con- tinue to stretch out in opposite directions until the amoeba becomes greatly elongated. Then one is withdrawn, and the amoeba continues in the opposite direction, soon moving out of the field (Fig. 114). These pseudopods usually contain no hyaline cap and no plasmagel at the tip, and the plasmagel elsewhere is very thin. Sometimes such a large portion of the distal end is without plasmagel that the plasmasol very definitely streams back at the surface. If the circuit is opened shortly after these pseudopods have begun to form, no change in movement 1s seen; if it is closed again, there is still no response. This indicates that pseudopods in which the plasmagel is very thin, or absent, do not re- spond to electricity; and that the response to electricity consequently is due to its action on the plasmagel. If the current is stronger, movement ceases and the pseudopods re- tract just as they do in weak currents. The contraction which follows the retraction of the pseudopods is much more marked, especially on the surfaces directed toward the poles. Here the plasmagel fairly shrinks up and becomes yellowish in color. This is apparently precisely what occurs at the anodal end of an amoeba subjected to the action of a direct current. If the circuit is opened immediately after the violent contraction has occurred, the amoeba soon recovers. But the two masses of plasma- gel that have become yellowish are usually pinched off. An irreversible transformation takes place in them, which results in the death of a por- tion of the cytoplasm. Occasionally, however, these masses, especially if they are relatively small, are taken into the plasmasol and are there di- gested. If observations are made under an oil immersion objective on a speci- men in an alternating current, the plasmagel can be seen to contract and become yellowish. Fluid is squeezed out on either side of the organism and the adjoining plasmalemma is thrown into folds and papillae of 312 MOTOR RESPONSES various sizes. The latter are filled with hyaline substance containing a few scattered granules which exhibit violent Brownian movement, show- ing that the substance in which they are suspended is a fluid with low viscosity. Some of the folds and papillae round up and pinch off, to Figure 114. A series of camera sketches of an Amoeba, showing the effect of an alternating current. A, before current was made; B-F, successive stages after it was made; g, plasmagel; s, plasmasol; /, plasmalemma; /, hyaline layer; c, hyaline cap; arrows, direction of streaming; double headed arrows, direction of the current. Note that the Amoeba orients perpendicularly to the direction of the current, that the plasmagel in the pseudopods is at this time very thin or absent, that the plasmagel contracts violently at the surface directed toward the poles, that blisters are formed on these surfaces, and that the Amoeba eventually breaks here and then disintegrates. If the surrounding medium is acid, the Amoeba does not break and disintegrate. (After Mast, 1931a.) form spherical bodies filled with granule-containing fluid observed in the folds (Fig. 114). If the current is strong enough, the plasmagel usually breaks after it has thus contracted. The plasmasol flows out and the entire amoeba soon disintegrates and dissolves. Sometimes breaks occur in the pseudo- MOTOR RESPONSES 313 pods before there is much contraction, and then the plasmasol flows out through these. As the plasmasol flows out, it collects about the amoeba; and the granules and fluid in it do not stream toward the poles as they do in direct current. Cataphoresis and electroendosmosis are thus neutral- ized, owing to the reversal in the direction of flow of the current. There is no change in the responses if acid is added to the culture fluid. However, instead of disintegrating as usual, the plasmasol coagulates after the amoeba breaks up. The essential phenomena observed in the effect of the alternating current on Amoeba appear to be: (1) mild contraction in extended pseudopods, beginning at the tip; (2) violent contraction on the two surfaces facing the poles, with the formation of blisters in these regions; (3) formation of highly fluid pseudopods between these two surfaces; and, finally, (4) rupture at the surfaces directed toward the poles, fol- lowed by disintegration of the organism. Contraction in the pseudopods, as was repeatedly observed, occurs first in those directed toward the poles and last in those in which the longitudinal axis is perpendicular to the direction of the current. In- deed, there is some indication that there is no contraction at all if the axis is actually perpendicular to the direction of the current. Contraction in the pseudopods under these conditions results in retraction which, in all respects, appears to be the same as retraction of pseudopods in normal locomotion. The retraction appears to be due to increase in the elastic strength of the plasmagel, owing to reversible gelation of ad- joining plasmasol. It may be concluded, then, that the first effect of the alternating current is reversible gelation of the plasmasol adjoining the plasmagel at the tip of the pseudopods directed toward the poles. This results in an increase in elastic strength of the plasmagel in this region, and in a retraction of the pseudopods. The contraction at the surfaces facing the poles is, in the beginning, doubtless due to the same phe- nomena; but the facts (1) that the plasmagel in this region later changes in color, (2) that it does not become thicker, (3) that fluid is squeezed out of it, and (4) that it is killed and then breaks, show that contraction here is associated with profound changes in the plasmagel itself. These changes result in such marked decrease in the strength and elasticity of this structure that it breaks readily. These changes are also associated with simultaneous increase in fluidity of the plasmasol, as indicated by 314 MOTOR RESPONSES the structure of the pseudopods which form at this time. The evidence presented above shows that the described changes are brought about by the action of the current. Alsup (1939) measured the time required in alternating current and in light respectively to cause cessation in streaming (reaction time) and that required for recovery after this response (recovery period). He found in both that the reaction increased as the recovery period decreased. This indicates that after the plasmasol has gelated, owing to the action of electricity or light, and then solated, owing to the recovery processes, it no longer gelates so readily as it did. Alsup also found that subminimum exposure to an alternating cur- rent followed by a subminimum exposure to light, or vice versa, may induce a response, indicating that the effects of these two agents are additive. In order to account for the essential phenomena observed in amoebae when subjected to the action of an alternating current, it is then neces- sary to explain reversible gelation of the plasmasol adjoining the plasma- gel on the sides of the organisms facing the poles. It should be noted that the gelation of the plasmasol is followed by changes in the plasmagel in the adjoining regions—changes which result in violent contraction, loss of fluid, decrease in elasticity, and rupture, and by increase in the fluidity of the plasmasol. Mechanics of response.—In rhizopods all of the responses to direct current appear to be due primarily to solation at the cathodal surface, followed by gelation at the anodal surface. The question then arises as to what causes this. If an electric current is passed through a culture solution containing amoebae, the negative ions in the solution and in the amoebae move to- ward the anode, whereas the positive ions migrate toward the cathode. If the surface layers of the amoebae are semipermeable, as they un- doubtedly are, there will be an accumulation of positive ions (e.g., Na) on the inside, and negative ions (e.g., Cl) on the outside of the surface of the amoebae directed toward the cathode. Positive ions will accumulate on the outside and negative ions on the inside of the surface directed toward the anode (Ostwald, 1890). The positive ions will, however, unite with the hydroxyl ions of the water, forming bases (e.g., NaOH); and the negative ions will unite with the hydrogen ions of the water, MOTOR RESPONSES ZiLD forming acids (e.g., HCl). The cathodal surface layer of the amoebae should therefore become alkaline on the inside and acid on the outside. The anodal surface layer should react in the opposite manner. Numerous observations were made on specimens stained with neutral red and subjected to direct current under various conditions. No differ- ence whatever was observed in the color of different regions in any of these specimens. The neutral red staining in A. proteus is, however, confined to granules and vacuoles. It was observed that if the specimens are crushed, the color of these granules and vacuoles does not immedi- ately change, in accord with the hydrogen-ion concentration of the solu- tion in which they are immersed. It is obvious, then, that the fact that no difference in color was observed in the vacuoles and granules does not prove that the hydrogen-ion concentration of the cytoplasm was the same. Moreover, there is indirect evidence which indicates that it was not the same. Kihne (1864) long ago observed in certain epidermal cells of the leaves of Tradescantia subjected to a galvanic current that the ends of the cells directed toward the cathode become alkaline, and that those directed toward the anode become acid. These cells contain a natural indicator which is bluish in neutral solutions, red in acid solutions, and green in alkaline solutions. Habenicht (1935) came to the same conclusion in experiments on the effect of the galvanic current on cylinders of egg white. Mast (1931b) repeated and extended Kihne’s experiments and ob- tained results which confirm his contentions. It may then be assumed with considerable confidence that when an amoeba is subjected to a direct current, the hydrogen-ion concentration in the cytoplasm decreases at the cathodal end and increases at the anodal end. Edwards (1923) demonstrated that if an alkaline solution is locally applied to the surface of an amoeba, the plasmagel in this region dis- integrates; and that if acid is applied, it becomes thicker, owing to gela- tion of the adjoining plasmasol. This has been confirmed indirectly by Pantin (1923), Chambers and Reznikoff (1926), and others. If, then, the direct current produces a decrease in hydrogen-ion concentration at the cathodal end and an increase at the anodal end, one would expect the plasmagel to become thinner at the cathodal end and thicker at the anodal end. This is precisely what was observed. And if the elastic 316 MOTOR RESPONSES strength of the plasmagel varies directly with its thickness, as is doubt- less true, this would result in formation of pseudopods at the cathodal surface. This has been confirmed by observation. Since streaming toward the cathode begins at the cathodal surface before it does at the anodal surface, movement toward the cathode must be due primarily to the solation of the plasmagel at the cathodal surface. The accumulation of positive ions at this surface therefore must produce the solation. But this obviously does not account for the disintegration of the entire organism. Neither does it account for the violent contraction preceding disintegra- tion at the anodal side. It will be remembered that violent contraction and disintegration beginning at the anodal surface were observed only in relatively strong currents. Furthermore, the reactions were observed to occur only after large pseudopods develop and begin to advance to- ward the cathode. An amoeba disintegrates only if the direct current applied is sufh- ciently strong. After the circuit is closed, there is, on the inner surface of the plasmagel or in the plasmagel, an accumulation of positive ions at the cathodal side, and of negative ions at the anodal side. The former produces a decrease in the elastic strength of this layer, which results in the formation of a pseudopod directed toward the cathode—a pseudopod in which the plasmagel extends to the plasmalemma. Local disintegra- tion occurs at first; but, as the current continues, more and more of the plasmagel in this pseudopod disintegrates. The accumulation of nega- tive ions and consequently of hydrogen ions at the anodal end causes a thickening of the plasmagel, as well as gelation of the adjoining plas- masol. This results in violent contraction and finally in the rupture ob- served at the anodal end. Cataphoresis and electroendosmosis are probably also involved. The granules in Amoeba are negatively charged in relation to the fluid. The fluid consequently tends to flow from the anodal toward the cathodal end. This would facilitate contraction at the former and expansion at the latter end, which is precisely what was observed. There is, however, some evidence to indicate that transfer of water is of little importance in the rupture and disintegration at the anodal end. This will be presented later. It is therefore fairly clear how, in a direct current, substances accumu- late locally; and how this can produce most of the processes associated with the responses of amoebae in it. But in an alternating current the MOTOR RESPONSES RT situation is quite different. There is an equal movement of all substances in opposite directions. Consequently there can be no accumulation of different substances in different parts of the organism, unless there is some process which makes the movement in one direction greater than that in the other. The essential phenomena observed in Amoeba as a consequence of exposure to alternating current may be summarized in the order of their appearance. A mild contraction begins first at the tips of extended pseudo- podia. This is followed by violent contraction on the two surfaces fac- ing the poles (blisters appear on these surfaces). Then highly fluid pseudopods form between these two surfaces. Finally, the surfaces di- rected toward the poles rupture and the organism disintegrates. Numerous observations with the best lens system obtainable were made on the movement of microscopic particles, both in the field of the alternating current and in the amoebae in this field. There was no indi- cation of a drift of these particles nor of their accumulation in any part of the organisms. It therefore is evident that cataphoresis and electroendos- mosis cannot be involved in the observed contraction. It seems necessary, then, to conclude that the phenomenon of contraction is associated with the movement of the ions produced by the electric current; and, further, that ion movements are accompanied by processes which result in the accumulation of ions in certain regions of the organism. Dixon and Bennet-Clark (1927) and others maintain that alternat- ing current causes increase in the permeability of the plasmamembrane in cells. If so, then may not the contraction observed in the plasmagel be due to the action of substances which enter from the surrounding medium, since localized accumulation of ions at the surface of the amoeba 1n- creases its permeability owing to the action of the current? Two facts suggest that the contraction of Amoeba during exposure to alternating current cannot be due to the entrance of substances from the outside. In the first place, the contractions are known to occur in both alkaline and acid solutions. Secondly, alkaline solutions tend to produce solation in the plasmagel, thus decreasing its elastic strength. Possibly the accumulation of ions in or near the plasmagel causes the contrac- tion. It is well known that the positive ions, Na, K, Ca, and others ordi- narily pass through membranes more readily and more rapidly than the 318 MOTOR RESPONSES negative ions, SO,, PO,, NO,, and others. In a structure like the plas- magel, the movement of ions is undoubtedly hindered. Nevertheless, the positive ions may move farther from their initial positions toward the pole than do the negative ions. It may be assumed that the nega- tive ions tend to remain in the plasmagel, whereas the positive ions tend to leave it and return again as the direction of the current reverses. If the return movement of the positive ions is inhibited, there may be a momentary preponderance of negative ions within the plasmagel, and of positive ions in the adjoining substances, i.e., in the plasmasol, the hyaline layer, and the plasmalemma. If the movement of the posi- tive ions away from the negative ions is extensive enough, the negative ions remaining in the plasmagel will unite with the hydrogen ions of the water surrounding them, to form acids. The hydroxyl ions thus lib- erated will, owing to the fact that they pass rapidly and freely through tissues, move out and unite with the positive ions, to form bases in the substance adjoining the plasmagel. It is possible that this union retards the return movement of the positive ions during the next reversal in the direction of the current. Consequently the effect would be cumulative, gradually increasing the acidity within the plasmagel and the alkalinity of the substance on either side. The increase in acidity in the plasmagel would produce gelation in this layer and probably also in closely applied plasmasol. This would in- crease the thickness and elastic strength of the plasmagel. Contraction would be the result. If the accumulation of negative tons were great enough, the increase in acidity in the plasmagel would cause irreversible coagulation (death), accompanied by violent contraction and dehydra- tion, thus making the coagulated plasmagel so brittle that it would break readily. The increased alkalinity in the plasmasol and plasmalemma would tend to make the former more fluid, and it would tend to break up the latter. The postulated action of the current is precisely in accord with the observations. It also accounts for the fact that no response was observed in pseudopods which contained no plasmagel. According to the explanation offered, an accumulation of ions occurs in or near the plas- magel; there could be no action in structures which have none. Moreover, on the basis of this hypothesis, it is possible to account for the well-known fact that in many organisms the effect of a current varies inversely with the frequency of reversal. It is necessary only to as- MOTOR RESPONSES 319 sume that the higher the frequency of reversal, the more restricted the movements of the ions toward the poles, and the shorter the period of separation of positive from negative ions; and that the shorter this period, the more restricted the union of the negative ions with hydrogen tons, and the positive ions with hydroxyl ions. The more restricted these unions, the less the increase in acidity in the plasmagel, and the less the increase in alkalinity in the plasmasol, and the less the stimulating and the in- jurious effect. The hypothesis, then, that the action of the electric cur- rent on organisms is due to localized increase in acidity and alkalinity in different regions of the cell is in full accord with the fact that the effect in alternating currents varies inversely with the frequency. What bearing has all this on the problem concerning the observed con- traction at the anodal side of amoebae subjected to direct current? Carlgren (1899), as previously stated, holds that this is due to elec- troendosmotic extraction of water by the current, owing to negative charge of the solid substance. However, the fact that the same phe- nomenon occurs in alternating current, in which electroendosmosis 1s neu- tralized, strongly indicates that Carlgren’s conclusion is not valid. It also seems to show that the anodal contraction in direct current must be due, as appears to be the case in alternating current, to the action of the cur- rent on the movement of ions. The evidence presented in reference to the effect of both direct and alternating current on A. proteus indicates that the assertion of Bayliss (1920), Weber and Weber (1922), Taylor (1925), and others that electricity gelates cytoplasm, is misleading, for it shows that if an electric current causes gelation in a cell, it probably always causes simultaneous solation, each being confined to a portion of the cell. It was demonstrated above that this does not obtain for light. The implication frequently found in the literature that the action of electricity on protoplasm is the same as the action of light, appears therefore to be erroneous. Heilbrunn and Daugherty (1931) found that if ammonium hydroxide or chloride is added to the culture fluid, A. protews becomes anopositive. They maintain that the ‘‘protoplasmic granules” are ordinarily positively charged and are consequently carried (cataphoretically) toward the cathode, and that ammonium hydrate or chloride causes a change in the charge to negative and a consequent reversal in the direction in which 320 MOTOR RESPONSES they are carried. They contend that the contact of these granules with the inner surface of the plasmagel causes it to liquefy, and that this results in the formation of a pseudopod which is directed toward the cathode if the granules are positively charged and toward the anode if they are negatively charged. These authors say: As the granules move either toward cathode or anode, they must tend to break down the thixotropic gel on the side toward which they move. . . If this gel is liquefied in any local region, such a region becomes pushed out to form an advancing pseudopod. These are interesting views, but they obviously do not account for the direction of movement of pseudopods in alternating current (Fig. 114), nor for the direction of movement of pseudopods under some conditions in direct current (Fig. 112). For this would require cata- phoretic movement of the granules perpendicularly to the direction of the current in the former, and in opposite directions at opposite ends of the amoeba in the latter. Moreover, the fact that the granules stream toward the anode after the amoeba disintegrates, indicates that they are normally negatively (not positively) charged, and that this charge is consequently not involved in the cathopositive response. How, then, can the observed reversal in the direction of orientation be explained? The cathopositive response is probably brought about as described above. The ammonium hydrate or chloride added to the culture fluid probably results in the liquefaction of all the plasmagel, and conse- quently in free movement toward the anode of the negatively charged granules suspended in it. According to this view, the reversal in the direction of galvanic orientation is due to liquefaction of the plasmagel caused by the ammonium compounds used, not to change in the electric charge on the “protoplasmic granules.” B, FLAGELLATES Some of the flagellates orient very precisely in a direct current. Some are cathopositive, others anopositive, and still others both or neutral, depending upon the environmental conditions (Verworn, 1889; Pearl, 1900; Bancroft, 1913). Verworn maintains that orientation is brought about by differences in the effective stroke of the flagellum in opposite directions. Pearl believes that it is the result of typical avoiding reac- MOTOR RESPONSES BAL! tions, whereas Bancroft thinks that it is identical with the process of orientation in light. Further details concerning the processes involved are much desired. Moreover, it is noteworthy that some of the flagel- lates have proved to be excellent material for quantitative study of the relation between stimulus and response. C. CILIATES The responses to electricity have been more intensively and extensively studied in the ciliates than in any of the other groups of Protozoa. Jennings (1906) presents an excellent review of all the earlier in- vestigations concerning these responses. He maintains that the results obtained show the following: The principal feature in the response of all of the different species studied consists of reversal in the direction of the effective stroke of the cilia on the cathodal surface. In those species in which other ciliary actions are only slightly or not at all involved (e.g., Paramecium), this results in direct turning until the anterior end is directed toward the cathode. The organism then moves toward this pole. The extent of the cathodal surface affected varies directly with the strength of the current. If the current is strong enough to produce reversal over more than half of the surface of the Paramecium, it swims backward toward the anode. A still stronger current causes marked swelling of the anterior end and contraction of the posterior end, changes which are followed by disin- tegration beginning posteriorly (Fig. 115). The effect of the reversal on the cathodal surface is variously modified by the normal action of the cilia in other regions, in such a way that ‘‘with different strengths of current, and with infusoria of different action systems, this results some- times in movement forward to the cathode; sometimes in movement forward to the anode; sometimes in cessation of movement, the anterior end continuing to point to the cathode; sometimes in a backward move- ment to the anode; sometimes in a position transverse to the current, the animal either remaining at rest or moving across the current.” Jennings holds that all the responses of the ciliates to electricity are due to stimulation at the cathodal surface, resulting in local reversal in the direction of the effective stroke of the cilia on this surface. Bancroft (1906), in his experiments with Paramecium, observed that if certain salts (especially potassium, sodium, or barium salts) are added 322 MOTOR RESPONSES to the solution, the organisms will swim forward toward the anode. He maintains that this is due to stimulation at the anodal surface, and that consequently it is an exception to Pfliiger’s law. It is well known that some salts cause paramecia to swim backward, Figure 115. Progressive cathodic reversal of the cilia and change of form in Para- mecium as the constant electric current is made stronger. The cathode is supposed to lie at the upper end. The current is weakest at 7, where only a few cilia are reversed; 2-6, successive changes as the current is gradually increased. (After Statkewitsch, 903.) owing to the forward stroke of all the cilia (Jennings, 1899; Mast and Nadler, 1926; Oliphant, 1938). Obviously, if under such conditions there is reversal in the direction of the stroke at the cathodal surface, the paramecia will turn and swim forward toward the anode (Mast, MOTOR RESPONSES 323 1927c). If this reaction obtains, the forward swimming toward the anode is in full accord with Pfliiger’s law. Kamada (1929) made a study of the correlation between the effect of many different salts on reversal from forward to backward swimming, and the direction of orientation in a direct current. He maintains that some salts which induce forward swimming toward the anode do not induce backward swimming. He consequently supports Bancroft’s views. Kamada (1931) also maintains that in paramecia which are ano- positive there is ciliary reversal at the anodal surface, in place of the cathodal, and that with increase in current density this is modified in various ways. The evidence he presents is, however, by no means con- clusive. Further observations are therefore needed. Paramecia are most sensitive if the anterior end is directed toward the cathode, less sensitive if it is directed toward the anode, and least sensi- tive if the longitudinal axis is perpendicular to a line connecting the two poles (Statkewitsch, 1903; Kinosita, 1936). The same holds for Spv- rostomum (WKinosita, 1938a). Statkewitsch (1907) subjected to direct and to alternating currents paramecia which had been stained with neutral red. He maintains that the stained structures in them became violet (acid) in weak currents and distinctly yellowish (alkaline) in strong currents. He apparently did not observe any difference in the color at the two ends. In Kinosita’s (1936) experiments with paramecia stained with either neutral red or Nile blue sulphate, the color of opposite ends differed. He says that the changes in color observed show that the paramecia become acid at the cathodal and alkaline at the anodal end, but that the alkaline portion rapidly ex- tends forward and soon includes the entire body. There is consequently a diversity of opinion concerning the effect of the electric current on the hydrogen-ion concentration of the cytoplasm in Paramecium. This also obtains for other cells, since, as previously stated, Kine maintains that cells of Tradescantia become acid at the anodal end and alkaline at the cathodal end. Mast confirmed this, but could observe no difference in this respect between the two ends in Amoeba. It is there- fore obviously desirable to have further observations concerning this problem, for it is theoretically very important. Kinosita measured the time required to make the anodal end alkaline 324 MOTOR RESPONSES in different current densities, extending over a wide range. He maintains that the results indicate that 7\/f— a) ==K. This equation is similar to the one obtained by Kamada in observations on the destruction of the surface membrane in paramecia by a direct current. It is also related to the one obtained by Hahnert (see above) in his observations on the cessation of streaming in Amoeba in a constant current. Internal processes involved in response.—Several theories have been formulated to account for the responses of ciliates to an electric current. Loeb and Budgett (1897) contend that these are in reality the result of responses to changes in the chemical constitution of the environment produced by the electric current. Pearl (1901) asserts that the direction of the stroke of the cilia is specifically correlated with the direction of protoplasmic streaming directly below the surface. Coehn and Barratt (1905) hold that the movements are purely cataphoretic; Bancroft (1906) maintains that the galvanic responses are due to local changes in the calcium content of the tissue in relation to that of other ions, especially monovalent cations. Although Carlgren (1899) lays especial stress on localized changes in water content within the organisms, due to endosmotic streaming. Some assert that the movements are due to direct action of the electric current on the cilia, others that they must be due to action on a codrdinating center. Ludloff (1895), Verworn (1895), and Koehler (1925) postulate functional division of the organ- isms into anterior and -posterior halves, such that one responds in one way that the other responds in another way. The views of Nernst (1899), Lucas (1910), Lillie (1923), and others regarding stimulation in higher organisms would lead to the idea that local changes in permeability of the surface membrane is the all-essential in controlling the movements of the lower organisms in an electric field. The relation between these hypotheses and the facts established has been very illuminatingly discussed by Jennings (1906) and Koehler (1925). Both conclude that the facts in hand are not adequately ac- counted for by any of the hypotheses presented. Jennings contends, as previously stated, that the most important perceptible characteristic of the response is reversal in the direction of the stroke of the cilia on the cathodal side. Koehler holds that the processes involved cannot be solely dependent upon surface phenomena, that somehow the current results in a division of the organism into cathodal and anodal portions which MOTOR RESPONSES 2045) function differently, owing to different internal factors. But neither Jen- nings nor Koehler offers any explanation of how the responses are reg- ulated. Among the most important of the known facts concerning the re- sponses in Protozoa to an electric current are those discovered by Lud- loff in observations on Paramecium. Ludloff (1895) found, as has been abundantly confirmed, that when the circuit is closed, the direction of the stroke of the cilia on the surface of the paramecia directed toward the cathode reverses; but that if the longitudinal axis of the organisms is directed obliquely to the direction of the current, reversal occurs on Figure 116. Paramecium showing reversal in the direction of the stroke of the cilia in a galvanic current. A, weak current; B, strong current; +, anode, —, cathode. (After Ludloff, 1895.) all sides of the end of the body nearest the cathode, extending to a line around the body produced by passing a plane through it at right angles to the direction of the current. The extent of the portion of the body on which such reversal occurs depends upon the strength of the current: the stronger the current, the larger the portion affected (Fig. 116). The fact that the cilia in different regions on the same side of the paramecia are not always affected equally by the current seems to show that the responses observed cannot, as Koehler points out, be due to direct action on the cilia or to surface phenomena alone; for if they were, all the cilia on either side should act alike, with the possible exception 326 MOTOR RESPONSES of those in the oral groove. If this is true, it is evident that the responses in this form must be associated with internal changes. The fact that in specimens with the longitudinal axis directed ob- liquely to the direction of the current, the entire end nearest the cathode is affected, and the fact that the size of the portion affected varies directly with the strength of the current, indicates not only that the current re- sults in a functional division of the organism (as maintained by Ludloff, Verworn, and Koehler), but also that whatever the factors involved may be, they act on a structure which is well distributed through the entire body and which is located some distance below the surface— probably the neuromotor apparatus. For only in a structure which is some distance from the surface, could a current produce the same changes in the distribution of substances on the anodal and the cathodal sides of the portion affected, resulting in reversal in the direction of the stroke 1n all the cilia on this portion. It is well known that momentary reversal in the stroke of the cilia can be induced in Paramecium by almost any sudden environmental change (Jennings, 1906), and that more prolonged reversal can be in- duced by transfer from culture fluid to distilled water or from distilled water to solutions of monovalent cation salts, but not usually by transfer to solutions of bivalent cation salts (Mast and Nadler, 1926; Oliphant, 1938). These changes, therefore, produce the same result as is produced by an electric current on the cathodal surface, indicating similarity in action. Greeley (1904) maintains that paramecia drift toward the anode, i.e., that they are negatively charged, indicating that there is a negative layer at the surface. Statkewitsch (1903) observed that if one end is directed toward the anode and the other toward the cathode, the former shrinks and the latter swells, indicating that the more solid substance is negative in relation to the more fluid substance. If all this obtains, a direct cur- rent will result in a decrease in the concentration of the positive, or an increase in the concentration of the negative ions on the cathodal side of the surface of the organism and on each semipermeable structure within. There will also be a decrease in the concentration of the negative, or an increase in the concentration of the positive ions on the opposite side. As a result, water will drift toward the cathodal side, and the solid particles will drift in the opposite direction. MOTOR RESPONSES 27, Since reversal in the stroke of the cilia begins on the cathodal side, it would seem that it must be associated either with an increase in the concentration of the negative ions or with a decrease in the concentration of the positive ions, on the cathodal side of the semipermeable structures on this side of the organisms. Either a decrease of polarization or an increase in the water content of the cytoplasm may be involved. If this is true, then transfer from culture fluid to distilled water and from dis- tilled water to solutions of monovalent cation salts should, since this re- sults in reversal of the stroke of all the cilia, produce similar changes in the concentration of ions or water. In other words, the transfer from culture fluid to distilled water should produce a decrease in the con- centration of positive ions on the outside of the semipermeable struc- tures, or an increase in the water content at the surface. A transfer from distilied water to solutions of monovalent cation salts should produce like changes. Whether or not this obtains is at present unknown, but one would expect it to obtain, if, as is frequently asserted, permeability is increased by monovalent cation salts. D. COLONIAL ORGANISMS No detailed observations have been made on the response to electricity of any of the colonial organisms except Volvox. Volvox orients very precisely in direct current. It swims toward the cathode under some condi- tions and toward the anode under others. Carlgren (1899) maintains that reversal in the direction of orientation is correlated with the dura- tion of exposure to the current. Terry (1906) and Bancroft (1907) contend that it is correlated with the intensity of the light received and the duration of exposure to it. Mast (1927c) found that Volvox swims toward the cathode when it is photopositive, and toward the anode when it is photonegative, i.e., that the response to electricity is specifically cor- related with the response to light. In photopositive colonies in which rotation on the longitudinal axis is inhibited by means of pressure, the flagella on the cathodal side stop beating immediately after the circuit is closed. They remain inactive 4.5 to 6 seconds and then begin to beat again (Fig. 117). If the circuit is now opened, those on the anodal side stop for a few moments and then beat again. If the colonies are swimming and rotating on the longi- tudinal axis in the normal way, but are not proceeding directly toward 328 MOTOR RESPONSES either pole when the current is made, flagellar inactivity on the cathodal side is continuous, owing to the continuous transfer of the zooids from the anodal to the cathodal side. The colonies therefore turn toward the cathode until they face it directly, and the transfer of zodids from side to side ceases. Orientation.—In photonegative colonies precisely the opposite occurs. The flagella on the anodal side stop beating after the circuit is closed, \} Figure 117. Sketch showing in a stationary photopositive colony of Volvox the effect of a galvanic current on the currents of water produced by the flagella. A, outline of colony oriented in light; B, same colony immediately after the circuit was closed; a, anterior end; straight arrows, direction of illumination; curved arrows, currents pro- duced by the flagella; +, positive pole; —, negative pole. (After Mast, 1927.) and the colonies turn toward the anode until they face it and then swim toward it (Mast, 1927c). : Galvanic orientation is consequently correlated with photic orientation, but the processes involved differ, for, as previously stated, photic orienta- tion is due to a change in the dzrection of the stroke of the flagella on opposite sides, while galvanic orientation is due to decrease or cessation in the activity of the flagella on one side. Electric charge on the colonies —Galvanic orientation in Volvox also differs from that in Paramecium, for while the one is due to decrease or cessation in activity on one side, the other, as previously stated, is due to reversal in the direction of the effective stroke of the cilia on one side. Since a given colony of Vo/vox may be either photopositive or photo- negative in the same environment, the difference in response to the light MOTOR RESPONSES 329 in this environment must be due to changes in the colony itself. Since photopositive response is specifically correlated with cathopositive re- sponse, and photonegative response with anopositive response, the differ- ence in the response to electricity must be due to like changes in the colony. The only difference observed in the colonies in connection with the response to light concerns the electric charge. Referring to this, Mast (1927c) says: Most of the photopositive colonies observed drifted toward the anode and most of the photonegative ones drifted toward the cathode, indicating that the former were negatively and the latter positively charged. However, owing to the negatively charged glass bottom of the aquarium in which the observations were made, there was produced in the solution near the bottom an endosmotic current of water toward the cathode and this current produced at the upper surface a current in the opposite direction, i.e., toward the anode. In making the observations it was impossible to ascertain pre- cisely the location of the colonies in relation to these currents, resulting fre- quently in uncertainty as to whether the drift was due to cataphoresis or to endosmosis. The results obtained are consequently somewhat equivocal. These observation should therefore be repeated under more favorable conditions, for the results are of fundamental importance in the analysis of the mechanics of the response to electricity, as will be shown pres- ently. Mechanics of response.—The outstanding characteristics of the re- sponses to the electric current of the colonial forms, exemplified in Volvox, consist in momentary decrease in the action of the flagella, cor- related with the direction and the density of the current and the nature of the response to light. In photopositive colonies this occurs in such a way that the flagellar activity decreases on the cathodal side after the current is made. In photonegative colonies the activity decreases on the anodal side, continues a few seconds, and then increases again. But if the current is broken, it decreases on the opposite side, continues a few seconds, then begins again. The extent of the region affected under all conditions varies directly with the density of the current. The action of the current must be due to movement of ions, parti- cles, or fluid in the colonies or the surrounding solution, and to differ- ences in the responses in photonegative and photopositive colonies to differences in the effects produced by the movements of these substances. 330 MOTOR RESPONSES There is considerable evidence which indicates that when colonies are photopositive and cathopositive, they are negatively charged; and when they are photonegative and anopositive they are positively charged (Mast, 1927c). If this obtains, the movement of ions in photopositive colonies in a direct current would result in decrease in the negative ions it | + + ++ ++ > + + >~ >~ + ++ > > +t ++ ' a > > 0 Figure 118. Diagrams illustrating the effect of direct current on the distribution of ions in colonies of Volvox and their response. —, negative charge on colony; +, positive; o, no charge; A, anode; K, cathode; large arrows, beam of light; small arrows, direction of movement of colonies in the beam of light; curved arrows, direction the colonies turn after the current is made and broken respectively. Note that there is in negatively charged colonies, after the circuit is closed, a decrease in potential at the cathode, and a decrease at the anode after it is opened, that the opposite obtains for positively charged colonies, and that there is no change in potential in neutral colonies. (Modified after Mast, 1927b.) on the cathodal side. In turn, this would cause decrease in potential and increase in permeability here. It would also result in increase in the negative ions at the anodal side and increase in potential and a decrease in permeability there (Fig. 118). The movement of ions in photonega- tive colonies would result in increase in positive ions, in the potential, and increase in permeability on the cathodal side. The reverse would MOTOR RESPONSES 3) occur on the anodal side. The ion movement in photoneutral colonies would result in increase in positive ions, and increase in potential and decrease in permeability on the cathodal side. On the anodal side there would be equivalent increase in negative tons and increase in potential and decrease in permeability. After the current 1s broken, the change in distribution of ions, and its effect, would be precisely opposite in all respects) :( Figs 99))r If the decrease in flagellar activity is due to local decrease in polariza- tion and increase in permeability, it accounts for the observed direction of movement of photopositive and photonegative colonies in a direct current. And if the decrease in flagellar activity is correlated with rate of change in these characteristics, it also accounts-for the fact that the decreased activity induced by making or breaking the current continues only a few seconds, for the change in polarization and permeability undoubtedly lasts but a few seconds. How does the fact that strong currents cause decrease in the activity of the flagella simultaneously on all sides of the colonies, harmonize with this view? It is well known that a galvanic current will produce cytolysis if it is strong enough, and that cytolysis is associated with increase in perme- ability and decrease in polarization. It is therefore not difficult to see that such a current could cause decrease in polarization simultaneously on all sides. It is probable, however, that the processes involved in galvanic stimula- tion, resulting in orientation, are not the same in all organisms. For example, Ludloff (1895) and Statkewitsch (1903) found that in a galvanic current the fluid in the body of Paramecium is carried endosmoti- cally toward the surface, on which the stroke of the cilia reverses, 1.e., in the specimens which swim forward toward the cathode, there is, if the current is strong enough, contraction at the anode and expansion at the cathode end. In Volvox precisely the opposite obtains. In the one, stimulation appears to be associated with increase, in the other, with de- crease in water content. In both, however, as pointed out above, it ap- pears to be associated with decrease in polarization. In general, it may be said that a galvanic current usually induces in the lower organisms chemical and physical changes which differ at op- 332 MOTOR RESPONSES posite sides, the one facing the anode, the other the cathode; and that either or both of these two sets of changes may result in what is usually called a response, although not necessarily an orienting response. Thus while the motor responses in these organisms usually occur at the catho- dal side when the circuit is closed, Kihne (1864), Verworn (1895), and McClendon (1911) observed in Amoeba and other rhizopods con- traction at the anodal side. In some of these organisms, the anodal con- traction appears to be involved in streaming toward the cathode; but there are other anodal responses which obviously have nothing to do with locomotion. For example, Loeb and Budgett (1897) assert that there is, in Amblystoma, copious secretion of mucus on the anodal side. Moore (1926) obtained bioluminescence and contraction on the anodal side of the Ctenophores, Mvemiopsis, and Berde. Lyon (1923) and Lund and Logan (1925) observed, in Noctdluca, a sort of contraction first at the anodal side and later also at the cathodal side, and sometimes the reverse. In all of the organisms referred to above, except Noctiluca, the anodal responses differ radically from the cathodal responses. This is very evident from the results of observations on Amoeba, in which it can be clearly seen that, after the circuit is closed, there is first local liquefaction of the plasmagel on the cathodal side, then contraction, and finally cytolysis on the anodal side. In Vo/vox, however, the response on the cathodal side in colonies which are positive to the cathode, is, in all perceptible characteristics, precisely the same as the response on the anodal side in those which are positive to the anode. Here, then, is an actual reversal in the action of the current, i.e., an actual reversal of Pfliger’s law. Closing the circuit apparently produces the same effect on the anodal side of colonies in certain physiological states as it does on the cathodal side of colonies in other physiological states. These physiological states are, as set forth above, specifically associated with those involved in reversal in the direction of photic orientation. As demonstrated above, these are dependent upon illumination, tempera- ture, and chemicals in the environment, which apparently control the electric charge carried by the colonies and the bodies within them. What is much needed now is a more comprehensive study of these charges, in relation to the chemical and the physical content of the environment as well as the character of the responses of the colonies. MOTOR RESPONSES 333 RESPONSES TO CHEMICALS A. RHIZOPODS None of the rhizopods except Amoeba has been studied with refer- ence to motor responses to chemicals. Many of the observations made on Amoeba are so indefinite that further work under more carefully con- trolled conditions is highly desirable. In this work the large form, Pelomyxa carolinensis, which can now be readily procured, would doubt- less be very favorable. Using a capillary pipette, Edwards (1923) applied various chemicals locally to the surface of active specimens of A. proteus. With a few of these chemicals he obtained fairly definite results, which lead to the following conclusions: If an alkali comes in contact with the side of an active amoeba, stream- ing stops and a local protuberance is formed at the point of contact. If the solution is weak, the protuberance develops into a normal pseudopod, which continues indefinitely toward the source of the solution. If it is strong, the protuberance breaks at the tip and the central portion of the amoeba flows out, leaving nothing but a crumpled membrane. If an acid is applied, streaming stops and a similar protuberance is formed but it does not become large and does not develop into a nor- mal pseudopod. If the acid is weak, the streaming soon begins again and the protuberance gradually disappears. If it is strong, the protuber- ance is very small and pseudopods form on the region opposite the point of application. If acid is applied after a rupture in the surface of an amoeba has been produced by local application of an alkali and after the central portion begins to flow out, the flow immediately stops and the amoeba soon proceeds normally. These and other facts show that alkalies cause the cytoplasm of Amoeba to solate and that strong acids cause it to gelate; but what is involved in the formation of a protuberance by weak acid at the region of application is not clear. A strong solution of sodium chloride results in formation of pseudo- pods opposite the region of application; a weak solution results only in the formation of a protuberance in the region to which it is applied. Thus Amoeba is negative to a solution of this salt if it is strong, and positive if it is weak. This indicates that the former induces gelation of 334 MOTOR RESPONSES the cytoplasm, and the latter, solation. Whether or not this obtains for other salts has not been ascertained. Strong alcohol produces a blister at the point of application, followed by formation of pseudopods on the opposite surface. The effect of all these substances is correlated with the kind of chem1- 220 Gel/Sol Ratio /40 Rate of Locomotion (Mu/min) ~ Figure 119. The relation between rate of locomotion, gel/sol ratio, and hydrogen-ion concentration in a balanced salt solution. A, Ai, rate of locomotion and gel/sol ratio in solutions containing salts in concentrations given in Table 2; B, B,, rate of locomotion and gel/sol ratio in salts in concentration five times as great as those given in Table 2. Curve A, mean rates of movement of images of amoebae given in Table 2; B, means of the rates of movement of seventeen to twenty-four individuals for from five to seven minutes each in each hydrogen-ion concentration; Ai and B,, mean gel/sol ratios cal- culated from results obtained in measurements made on seventeen to twenty-four indi- viduals in each hydrogen-ion concentration as described above. (After Pitts and Mast, 1933.) cals, with their concentration in the medium surrounding the amoebae, and with the length of exposure to them. If these conclusions are valid, one would expect amoebae in a culture medium to aggregate in regions which are alkaline or which contain relatively little salt. Hopkins (1928) made some observations concern- ing the former. He put specimens into a drop of solution which was MOTOR RESPONSES 335 pH 7.1 and then joined this drop with other drops which were respec- tively pH 6 and 8. He found that the amoebae at the border between the drops formed pseudopods which protruded toward the drop added, no matter whether it was alkaline or acid. This indicates that amoebae in Rate of Locomotion (Nu/min) 0/2°Y /0G//99 Figure 120. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con- centration, and sodium-ion concentration. Each of the curves represents a series of ex- periments conducted at constant sodium-ion concentration. The four solid curves are based on the measurement of the rate of locomotion of an average of 14.8 different specimens for an average of 88.1 minutes in each hydrogen-ion concentration tested. The two broken curves are based on the measurement of the gel/sol ratio in an average of 19 different individuals in each hydrogen-ion concentration tested. (After Pitts and Mast, 1934b.) a neutral solution tend to aggregate, either in acid or alkaline regions. The observations made were, however, not extensive enough to war- rant any definite conclusions. There is, then, nothing definite known concerning the relation between differences in the chemical composition of the medium and aggregation in any of the rhizopods, and but little concerning the action of chemicals in relation to changes in direction of 336 MOTOR RESPONSES movement. On the other hand, there are some very definite results con- cerning the relation between the rate of locomotion and the chemicals in the surrounding medium, especially hydrogen ions. Rate of locomotion and H-ion concentration —Hopkins (1928) ob- served that A. protews, in an ordinary hay culture fluid, is inactive if the fluid is neutral, but active if the fluid is either acid or alkaline, and that the rate of locomotion is maximum and nearly equal at about pH 6.5 Gel/Sol Ratio a ae Od : at Te 001 MN 005 62 68 74 60 Al Rate of Locomotion (Mu/min) 30 I6 Figure 121. The relation between rate of locomotion, gel/sol ratio, hydrogen-ion con- centration, and calcium-ion concentration. Curve ©, mean rate of locomotion in 0.001 M, based on measurements on an average of 22.1 different specimens for 11.6 minutes in each hydrogen-ion concentration tested; curve @, mean rate of locomotion in 0.005 M, based on an average of 18.7 different specimens for a total average of 96.6 minutes in each hydrogen-ion concentration tested; curve @, mean gel/sol ratio in 0.001 M, based on measurements on an average of 21.3 different specimens in each hydrogen-ion con- centration tested; curve ©), mean gel/sol ratio in 0.005 M, based on measurements on a total average of 14.6 different specimens in each hydrogen-ion concentration tested. (After Pitts and Mast, 1934b.) and pH 8 respectively. Mast and Prosser (1932) confirmed these results. Pitts and Mast (1933), in a much more thorough study on A. proteus, demonstrated that the inactivity at neutrality is correlated with the rela- tion between the amount of sodium or potassium and calcium present; and that the relation between activity and the concentration of hydrogen ions varies greatly with the kind, the concentration, and the proportion of salts in the surrounding medium. Some of the results obtained in this study are presented in figures 119, 120, 121, and 122. These figures show the following: MOTOR RESPONSES 357 In a balanced salt solution the activity is minimum at neutrality and maximum on either side; but the activity at any given hydrogen-ion concentration varies with the salt concentration (Fig. 119). In sodium or calcium salt solutions (Table 2) the maximum rate of locomotion is nearly as high as in a balanced salt solution. Furthermore, the rate at Rate of Locomotion(u/min) ONeY /OC//25 a7 53 49 pH 65 7 77 Figure 122. The effect of adding calcium in different concentrations to 0.005 N sodium solutions, on the relation between hydrogen-ion concentration, rate of locomotion, and gel/sol ratio. The solid curve for the solutions containing calcium 0.001 M is based on measurements of the rate of locomotion, on an average of 14.75 different specimens for a total average of 89.9 minutes; that for calcium 0.0005 M, on an average of 18.6 different specimens for a total average of 95 minutes; and that for calcium 0.001 M, on an average of 20.5 different specimens for a total average of 104.6 minutes in each hydrogen-ion concentration tested. The broken curve for the solution containing cal- cium 0.001 M is based on measurement of the gel/sol ratio, on an average of 19.8 different specimens; that for calcium 0.0005 M, on an average of 17.9 different speci- mens; and that for calcium 0.0001 M, on an average of twenty different specimens in each hydrogen-ion concentration tested. (After Pitts and Mast, 1934c.) any given hydrogen-ion concentration varies with the salt concentration, although the relation between the rate and the hydrogen-ion concen- tration in the one differs greatly from that in the other, and there is no indication of inactivity at neutrality in either (Figs. 120, 121). If cal- cium salt is added to a solution of sodium salt, the activity decreases greatly at neutrality (Fig. 122). The ratio between the amount of plas- magel and the amount of plasmasol in Amoeba varies with the hydro- gen-ion concentration; but the rate of locomotion is not specifically cor- related with this ratio. 338 MOTOR RESPONSES TABLE 2: RATE OF LOCOMOTION OF A. PROTEUS IN SODIUM AND CALCIUM SALT SOLUTION* Composition of the solution used to ascertain the effect of hydrogen-ion concentration on rate of locomotion and gel/sol ratio in a balanced salt solution. To obtain different hydrogen-ion concentrations, the acid and the alkaline components were mixed in dif- ferent proportions. Note that the concentration of Na, K, Ca, and Mg remains constant, no matter what the proportion of the two components is. In the second series of experiments the concentration of all the salts was increased five times (after Pitts and Mast, 1933). Acid Component Alkaline Component Molar Ratio NaH:PO, 0.00150N NaOH 0.00150N Na 60 KH2PO, 0.00010N KOH 0.00010N K 4 CaH4(POs.)2 0.00010N Ca(OH), 0.00010N (Gat Dy MgCl. 0.00005N MgCl, 0.00005N Mg I * After Pitts and Mast, 1933, by permission of the Journal of Cellular and Comparative Physiology, Wistar Institute of Anatomy and Biology. Mechanics of response —Concerning the action of chemicals produc- ing these responses on Amoeba, Pitts and Mast make the following state- ments: It is obvious that a substance in the environment may influence processes which occur in a cell either by entering the cell and acting directly on substances in the cell, or by acting on the surface of the cell in such a way as to retard or facilitate the passage into or out of the cell of other sub- stances which, owing to their presence or absence, induce alterations in internal processes. . . . It may be assumed, then, either (1) that the gel/sol ratio depends upon the entrance of salts into the cell and reaction between these and internal substances, and that the rate of entrance of salts varies with the hydrogen ion concentration; or (2) that the gel/sol ratio varies with the rate of en- trance of hydrogen and hydroxyl ions into the cells and reaction between these and internal substances, and that the rate of entrance of these ions varies with the concentration of the salts and the hydrogen ions; or (3) that the gel/sol ratio depends upon the exit of substances from the cell, e.g., water, and that this depends upon the hydrogen ion concentration, and the salt concentration and the kinds of salts present. Let us now attempt to ascertain if the processes in question are in accord with any of these groups of assumptions. . If they are in accord with the first of these assumptions, the gel/sol ratio must vary directly or indirectly with the amount of salt that enters the cell and this must vary directly or indirectly with the hydrogen ion con- MOTOR RESPONSES be) centration. If it varies indirectly with the amount of salt that enters and this varies either directly or indirectly with the hydrogen ion concentra- tion practically none of the results obtained are in accord with the assump- tions. If it varies directly with the amount of salt that enters and this varies directly with the hydrogen ion concentration the results obtained with balanced salt solutions and some others are in full accord with the assump- tions; but the assumption that the entrance of salts varies directly with hydrogen ion concentration is not in harmony with the results obtained by practically all who have investigated this problem. Moreover, the assump- tions do not account for the independence or inverse variation between hydro- gen ion concentration and gel/sol ratio in calcium solutions. . . . If they are in accord with the second of the three assumptions made above, the gel/sol ratio must depend upon the hydrogen ion concentration with- in the cell and this must vary with the concentration of the salts and the hydrogen ions in the surrounding medium. If it varies indirectly with the hydrogen ion concentration of the surrounding medium and directly or indirectly with the salt concentration, few if any of the results obtained are in accord with the assumptions. If it varies directly with the hydrogen ion concentration and the salt concentration of the surrounding medium, the results obtained with balanced salt solutions and some others are in ac- cord with the assumptions, but those obtained with calcium solutions are not. There is, moreover, no evidence which indicates that the hydrogen ton concentration within Amoeba varies appreciably with variation in the hydro- gen ion and the salt concentrations of the surrounding medium (Chambers, 1928)... | In accord with the third group of assumptions, the gel/sol ratio must vary with the rate of exit of substances from the cell, and this must vary d1- rectly or indirectly with the salt and the hydrogen ion concentration, and it must also vary with the kind of salts present in the environment. Without entering upon a detailed analysis of the correlation between these assump- tions and the results under consideration, it is evident that, no matter what combination is selected, there are between them and the results inconsist- encies of the same nature as those presented above. . It is consequently obvious that the results in hand cannot be consistently explained by any one of the three groups of assumptions made, and that there must be a fairly complicated interaction between the various factors involved. If this is true, the statement, without qualification, that any given factor facilitates gelation or solation is obviously so incomplete that it is without value. This conclusion is in full harmony with that reached by Mast and Prosser (1932). ... In reference to the relations between rate of locomotion and kind of salts, salt concentration and hydrogen ion concentration, the results ob- tained are, on the basis of any one of the groups of assumptions considered 340 MOTOR RESPONSES above, even less explicable than are those in reference to the gel/sol ratio. The outstanding difficulty here concerns the remarkable decrease in rate of locomotion as neutrality is approached either from the acid or from the alkaline side. The results presented show that this decrease occurs in balanced solutions; that is that it is specifically correlated with the Na/Ca ratio; that the higher this ratio within the limits of the concentrations tested, the greater the decrease; and that it does not occur in solutions con- taining only one salt... . The results show that if calcium is added to a solution containing only sodium salts, the rate of locomotion in the alkaline range increases greatly, with but little change in the acid range and in the region of neutrality; and that if sodium is added to solutions containing calcium salts, the rate de- creases greatly in the region of neutrality, with but little change else- wheres: 2) The questions now arise as to why addition of calcium to solutions con- taining only sodium salts causes great increase in rate of locomotion in the alkaline range, and why addition of sodium to solutions containing only calcium salts causes great decrease in the rate in the region of neutrality. Similar questions have arisen in reference to the bimodal curves obtained by a number of other investigators in plotting the rate of various physiological processes against hydrogen ion concentration, e.g., by Robbins (1926) and Farr (1928) im various processes in plants; by Ephrussi and Neukomm (1927) in the resistance to heat in the eggs of a sea urchin; by Hopkins (1928) in the rate of locomotion in Amoeba; by Mast (1928) in the rate of assumption of stellate forms in Amoeba; by Eisenberg-Hamburg (1929) in the rate of increase in water content in infusoria; by Chalkley (1929) in water content and gel/sol ratio in Amoeba; and (1930a, 1930b) thermal death rate in Paramecium, by Chase and Glaser (1930) in rate of locomo- tion in Paramecium, and by Mast and Prosser (1932) in rate of locomotion in Amoeba. ... Only a few of these investigators attempted to elucidate the phenomenon. Mast and Prosser (1932), as previously stated, concluded that it is cor- related with salt concentration. We have already considered this view. Robbins (1926) contends that the hydrogen ion concentration, at which the median minimum in the plant processes studied occurs, coincides with the isoelectric point of the principal proteins in the plant. Farr (1928) main- tains, however, that this view is not tenable. He found in observations on the relation between hydrogen ion concentration and rate of growth in root hairs of collards that the hydrogen ion concentration at which the median minimum occurs varies greatly with salt concentration and he concludes that it therefore cannot be specifically correlated with the isoelectric point of any given protein in the organism... . In reference to Amoeba, the constancy of a median minimum rate of MOTOR RESPONSES 341 locomotion at pH 7.0 in various solutions indicates some fixity of mechanism determining this median minimum. This mechanism might be correlated with the behavior of the membrane in the neighborhood of an ampholyte isoelectric point near neutrality in accord with the view of Robbins (1926). But arguing against such an isoelectric point are the pertinent facts that, (1) the median minimum is lacking in solutions of single salts, though locomotion in the dilute solutions occurs at hydrogen ion concentrations in which it is usually found; (2) the difference between the maximum and the median minimum rate of locomotion depends on the sodium/calcium ratio and only slightly if at all on the total salt concentration; (3) the cations have marked effect on the rate of locomotion on the acid side of the neutral point as well as on the alkaline side; (4) the anions (so far as chloride and phosphate are concerned) have little effect down as far in the acid range as the observations were made... . We are at present unable to suggest a satisfactory explanation for this median minimum. Whatever the cause of it may be, the relation between the rate of locomotion in Amoeba and the factors in its environment is doubt- less fairly complex, for it is probable that these factors influence locomotion in it by their action on the surface, affecting adhesiveness and other prop- erties of the surface, as well as by their action on permeability of the sur- face membrane (Mast, 1926a). B. MARINE AMOEBAE ' Pantin (1923-31) made observations on the relation between the rate of locomotion in a marine amoeba and various chemicals. He found that as the hydrogen-ion concentration increases, the rate increases rapidly from zero at pH 10 to a maximum at about pH 8, and then decreases rapidly to zero at pH 5.5. He observed no indication of decrease in activity at neutrality. He maintains that the rate is closely correlated with the relative concentration of sodium, potassium, magnesium, and cal- cium salts, that more than one of these salts is necessary for locomotion, and that calcium is required in all combinations. He maintains that cal- cium functions primarily in the contractile mechanism, and the others in the regulation of permeability. The evidence presented in support of these conclusions is, however, not unequivocal. Oxygen is necessary for locomotion in Amoeba (Hulpieu, 1930; Pan- tin, 1930) but only in very low concentrations. Pantin maintains that none is immediately necessary, and that it functions in recovery some- what as it does in the contraction of muscles. This, however, has not been demonstrated. 342 MOTOR RESPONSES Ce GILTARES The most prominent response of the ciliates to chemicals consists in reversal in the direction of the effective stroke of the locomotor cilia, and consequent backward swimming. This reversal may be so brief that _ it results in scarcely perceptible backward movement, or it may continue for several minutes. It has been investigated in some detail in Paramecium, but not in any of the other ciliates. The rate of locomotion in all the ciliates is doubtless correlated with the chemical composition of the surrounding medium, but no measurements concerning this correlation have been made in any of them. The reversal in the direction of the stroke of the cilia of paramecia in response to chemicals extends to the entire surface of the body, ex- cept the oral groove. It consequently results in backward swimming. It is the same as the reversal induced by contact and by rapid changes in temperature or osmotic concentration, and it is usually followed by turn- ing toward the aboral surface and forward movement in a new direction, i.e., it usually constitutes the first stage in the avoiding reaction. Jennings (1906) describes in detail how this response results in aggre- gation of paramecia in regions which contain ineffective chemicals in re- lation to those in adjoining regions, e.g., regions which are slightly acid, surrounded by regions which are slightly alkaline. If this obtains, he says, paramecia do not respond as they enter the acid region, but do when they reach the edge of this region and are about to enter the alkaline region. They consequently remain in the acid region. As more paramecia enter, owing to random movements, an aggregation is formed. He maintains that these responses usually result in aggregations in re- gions which are favorable for the organisms, but that there are exceptions. That is, he holds that these responses are, in general, adaptive. However, he has made no suggestions concerning the processes in the organism in- volved in producing these responses. Merton (1923) found that sodium and potassium salts induce reversal in Stentor and that calcium and magnesium do not, but he offers no suggestions concerning the nature of the action of the former nor the cause of inaction of the latter. Mast and Nadler (1926) ascertained the effect of fifty-six different chemicals on the direction of the effective stroke of the cilia in Parame- cium. They maintain that all of the monovalent cation salts and hydrates MOTOR RESPONSES 343 tested (thirty-one), except (NH,),SO, and NH,C,H,0.,, induce rever- sal; but that none of the bivalent and trivalent cation salts tested (nine- teen), except CaHPO, and MgHPO,, induce it. Also that Ba(OH).,, H,PO, and H,C.O, induce reversal, while HCL and lactose do not. These authors contend that the duration of reversed action is closely correlated with the concentration of the salts; that it varies with the kind of salt at any given concentration; and that bivalent and trivalent cation salts neutralize the effect of monovalent salts. Concerning the physiological processes involved in reversal in ciliary action, they make the following statement: Copeland (1919, 1922) and Grave and Schmitt (1925) demonstrated that the cilia in higher forms are closely connected with nerve fibers and that their action is in all probability controlled by nerve impulses. The results obtained in the investigations of Yocum (1918), Taylor (1920), Rees (1922), Visscher (1927) and others on the neuromotor apparatus in the protozoa, indicate that the action of the cilia in these forms is similarly con- trolled. If this is true, the question arises as to how environmental changes which have been observed to induce reversal in ciliary action influence the neuromotor apparatus. This may be conceived to be either through chemical changes produced in the receptors or elsewhere in the organism or through changes produced in the electric potential at the surface, or in the permeability and the consistency of the surface layer. . The results obtained by Mast and Nadler indicate that reversal in ciliary action is largely dependent upon the cations, that it is induced by monovalent but usually not by bi- and tri-valent cation salts, and that it depends upon the concentration of the salts... . It is well known that adsorption of the cations is usually relatively greater in bi- and tri-valent than in monovalent cation salt solutions and that the adsorption varies with the concentration of the salts in the solution. This seems to indicate that the difference in the action of the monovalent and the bi- and tri-valent cation salts and the difference in the action of different concentrations of the monovalent cation salts is at least in part associated with differential adsorption of the ions in the various solutions, resulting in changes in the electrical potential at the surface of the paramecia, which directly or indirectly produce impulses in the neuromotor apparatus which pass to the cilia and influence their action. The facts, however, that Ba(OH), produces reversal while BaCl,, CaCl,, MgCl, do not, that H,C,O, and H,PO, produce reversal while HCI does not, that CaHPO, and MgHPO, produce reversal while Ca,(PO,), and Mg(PO,), do not, all indicate that there must be other factors involved in reversal aside from differential adsorption of the cations. 344 MOTOR RESPONSES Oliphant (1938) made a much more extensive and thoroughly con- trolled study of the effect of monovalent and bivalent cation salts on ciliary reversal in Paramecium. The results obtained support the contentions of Mast and Nadler (1926) that the monovalent salts induce reversal, whereas bivalent ca- tion salts do not, and that the duration of the reversed action varies with the kind and the concentration of the salts; but they do not support their contentions as to the nature of this variation. The results show that the effect of the salts is due primarily to the action of the cations, and that the anions have little, if any, effect. They show that the order of effectiveness of the cations is K>Li>Na>NH,, and that the duration of their effect varies inversely with the temperature. Oliphant (1938) cites work which indicates that in Paramecium, Amoeba, Actinosphaerium, Spirogyra, root hair of Trianea, and eggs of Arbacia, monovalent cations induce increase, and that bivalent cations decrease the viscosity of the cytoplasm (Spek, 1921; Heilbrunn, 1923, 1931, Cholodnyj, 1923; Weber, 1924). He concludes that this indicates that reversal in ciliary action is correlated with increase in viscosity of the cytoplasm, and contends that this conclusion is supported by the fact that “reversal in response to temperature occurs only at temperatures almost immediately lethal,” i.e., at temperatures which cause marked increase in viscosity. He holds that the action of the cilia is controlled by the neuromotor apparatus and that increase in viscosity produces im- pulses in this structure which cause reversal in ciliary action, but he thinks that changes in electric potential, in permeability, in the consistency of the surface layer, or in the chemical composition of the receptors or other structures in the organism may be involved. It is obvious from the above discussion that there is still much to be learned concerning the processes involved in the responses of the ciliates to chemicals. LITERATURE CITED Alsup, Fred W. 1939. Relation between the responses of Amoeba proteus to alternating electric current and sudden illumination. Physiol. Zool., 12; 35-95. Alverdes, F. 1923. Der Sondercharakter der von den Ciliaten gezeigten Gal- . vanotaxis. Pfliiger’s Archiv ges. Physiol., 198: 513-42. MOTOR RESPONSES 345 Bancroft, F. W. 1906. The control of galvanotropism in Paramecium by chemical substances. Univ. Cal. Publ. Physiol., 3: 21-31. —— 1907. The mechanism of the galvanotropic orientation in Volvox. J. exp. Zool., 4: 157-63. —— 1913. Heliotropism, differential sensibility, and galvanotropism in Euglena. J. exp. Zool., 15: 383-428. Bayliss, W. M. 1920. The properties of colloidal systems. IV. Reversible gelation in living protoplasm. Proc. roy. Soc., B, 91: 196-201. Buder, J. 1917. Zur Kenntniss der phototaktischen Richtungsbewegungen. Jb. wiss. Bot., 58: 105-220. Carlgren, O. 1899. Uber die Einwirkung des konstanten galvanischen Stromes auf niedere Organismen. Arch. Anat. Physiol. Lpz., Physiol. Abth., 49-76. Chalkley, H. W. 1929. Changes in water content in Amoeba in relation to changes in its protoplasmic structure. Physiol. Zodl., 2: 535-74. ——1930a. Resistance of Paramecium to heat as affected by changes in hydro- gen ion concentration and in inorganic salt balance in the surrounding medium. Pub. Hlth. Rep., Wash., 45: 481-89. —— 1930b. On the relation between resistance to heat and the mechanism of death in Paramecium. Physiol. Zodl., 3: 425-40. Chambers, R. 1928. Intracellular hydrion concentration studies. I. The relation of the environment to the pH of the cytoplasm and of its inclusion bodies. Biol. Bull., 55: 369-76. — Chambers, R., and P. Reznikoff. 1926. Micrurgical studies in cell physiol- ogy. I. The action of the chlorides of Na, K, Ca, and Mg on the proto- plasm of Amoeba proteus. J. gen. Physiol., 8: 369-401. Chase, A., and O. Glazer. 1930. Forward movement of Paramecium as a func- tion of the hydrogen-ion concentration. J. gen. Physiol., 13: 627-36. Cholodnyj, N. 1923. Zur Frage uber die Beeinflussung des Protoplasmus durch mono und bivalente Metallionen. Beih. Bot. Zbl., 39: 231. Coehn, A., and W. Barratt. 1905. Uber Galvanotaxis vom Standpunkte des physikalischen Chemie. Z. allg. Physiol., 5: 1-9. Copeland, M. 1919. Locomotion in two species of the gastropod genus Alectrion with observations on the behavior of pedal cilia. Biol. Bull., 37: 126-38. —— 1922. Ciliary and muscular locomotion in the gastropod genus Polznices. Biol. Bull., 42: 132-42. Davenport, C. B. 1897. Experimental Morphology. Vol. 1, New York, 280 pp. De Candolle, A. P. 1832. Physiologie vegetale. Paris. Dixon, H. H., and T. A. Bennet-Clark. 1927. Responses of plant tissues to electric currents. Sci. Proc. R. Dublin Soc., 18: 351-72. Edwards, J. G. 1923. The effect of chemicals on locomotion in Amoeba. J. exp. Zool., 38: 1-43. 346 MOTOR RESPONSES Eisenberg-Hamburg, E. 1929. Recherches comparatives sur le fonctionnement de la vacuole pulsatile chez les infusoires parasites de la grenouille et chez les infusions d’eau douce. Influence de la pression osmotique, des electrolytes et du pH. Arch. Protistenk., 78: 251-70. Engelmann, T. W. 1869. Beitrige zur Physiologie des Protoplasm. Pflug. Arch. ges. Physiol., 2: 307-22. —— 1879. Uber Reizung contraktilen Protoplasmus durch plotzliche Beleuch- tung. Pflug. Arch. ges. Physiol., 19: 1-7. —— 1882. Uber Licht- und Farbenperception niederster Organismen. Pflug. Arch. ges. Physiol., 29: 387-400. Ephrussi, Boris, and Alex Neukomm. 1927. Resistance a la chaleur des oeufs d’oursin (Paracentrotus lividus, Lk). Protoplasma, 2: 34-44. Farr, C. H. 1928. Studies on the growth of root hairs in solutions. IV. The pH molar rate relation for collards in calctum chloride. Amer. J. Bot., 15: 6-31. Folger, H. T. 1925. A quantitative study of reactions to light in Amoeba. J. exp. Zool., 41: 261-91. Grave, C., and F. O. Schmitt. 1925. A mechanism for the codrdination and regulation of ciliary movement as revealed by microdissection and cyto- logical studies of ciliated cells in mollusks. J. Morph., 40: 479-513. Greeley, A. W. 1904. Experiments on the physical structure of the protoplasm of Paramecium and its relation to the reactions of the organism to thermal, chemical and electrical stimuli. Biol. Bull., 7: 3-32. Habenicht, Wilhelm. 1935. Die Wirkung des galvanischen Stromes auf den Eiweisszylinder nach Du Bois-Reymond. Protoplasma, 22: 321-36. Hahnert, W. F. 1932. A quantitative study of reactions to electricity in Amoeba proteus. Physiol. Zool., 5: 491-526. Harrington, N. R., and E. Leaming. 1900. The reactions of Amoeba to light of different colors. Amer. J. Physiol., 3: 9-16. Heilbrunn, L. V. 1923. The colloid chemistry of protoplasm. I. General con- siderations: IH. The electrical charges of protoplasm. Amer. J. Physiol., 64: 481. Heilbrunn, L. V., and K. Daugherty. 1931. The action of sodium, potassium, calcium, and magnesium on the protoplasm of Amoeba dubia. Physiol. Zodl., 4: 635. Holmes, S. J. 1903. Phototaxis in Volvox. Biol. Bull., 4: 319-26. Hopkins, D. L. 1928. The effects of certain physical and chemical factors on locomotion and other life processes in Amoeba proteus. J. Morph., 45: 97-119. — 1929. The effects of the substratum, divalent and monovalent cations on locomotion in Amoeba proteus. J. Morph., 48: 371-83. —— 1938. The vacuoles and vacuolar activity in the marine Amoeba, Flabel- lula mira Schaeffer and the nature of the neutral red system in Protozoa. Biodyn., No. 34: 1-22. MOTOR RESPONSES 347 Hulpieu, H. R. 1930. The effect of oxygen on Amoeba proteus. J. exp. Zool., 56: 321-61. Inman, O. L., W. T. Bovie, and C. E. Barr. 1926. The reversal of physiologi- cal dominance in Amoeba by ultraviolet light. J. exp. Zool., 43: 475-84. Jennings, H. S. 1899. Studies on reactions to stimuli in unicellular organisms. Il. The mechanism of the motor reactions of Paramecium. Amer. J. Physiol., 2: 311-41. —— 1904. Contributions to the study of the behavior of lower organisms. Publ. Carneg. Instn., No. 16: 256 pp. 1906. Behavior of the Lower Organisms. New York, 366 pp. Kamada, T. 1928-31. Control of galvanotropism in Paramecium. J. Fac. Sa. Tokyo Univ., 2: 295 123239) 285-307. ——— 1934. Some observations on potential differences across the ectoplasm membrane of Paramecium. J. exp. Biol., 11: 94-102. Kinosita, H. 1936. Electric stimulation of Paramecium. Jour. Fac. Sci. Tokyo Univ., 4: 137-94. —— 1938a. Electric stimulation of Spirostomum. Jour. Fac. Sci. Tokyo Univ., D2 71-105, —— 1938b. Electric stimulation of Paramecium with two successive subliminal current pulses. J. cell. comp. Physiol., 12: 103-17. 1939. Electrical stimulation of Paramecium with linearly increasing cur- rent. J. cell. comp. Physiol., 13: 253-61. Koehler, O. 1925. Galvanotaxis. Handb. d. Norm. u. Pathol. Physiol., 11: 1027-49. Kiihne, W’. 1864. Untersuchungen iiber das Protoplasma und die Contractilitat. Leipzig, 158 S. Laurens, H., and H. D. Hooker, 1920. Studies on the relative physiological value of spectral lights. II. The sensibility of Volvox to wave-lengths of equal energy content. J. exp. Zool., 30: 345-68. Lillie, R. S. 1923. Protoplasmic Action and Nervous Action. Chicago, 417 pp. Loeb, J. 1890. Der Heliotropismus der Tiere und seine Uebereinstimmung mit dem Heliotropismus der Pflanzen. Wurzburg, 118 pp. — 1906. The Dynamics of Living Matter. New York, 233 pp. Loeb, J., and S. P. Budgett. 1897. Zur Theorie des Galvanotropismus. IV. Mitt. Uber die Ausscheidung elektropositiver Ionen an der ausseren Anodenflache protoplasmitischer Gebilde als Ursache der Abweichungen vom Pfliigerschen Erregungsgesetz. Pfliig. Arch. ges. Physiol., 66: 518-34. Loeb, J., and S. S. Maxwell. 1910. Further proof of the identity of helio- tropism in animals and plants. Univ. Cal. Publ. Physiol., 3: 195-97. Lucas, K. 1910. An analysis of changes and differences in the excitatory process of muscles and nerves based on the physical theory of excitation. J. Physiol., 40: 225-49. Luce, R. H. 1926. Orientation to the electric current and to light in Amoeba. Anat. Rec., 32: 55. 348 MOTOR RESPONSES Ludloff, K. 1895. Untersuchungen uber den Galvanotropismus. Pflig. Arch. ges. Physiol., 59: 525-54. Lund, E. J., and G. A. Logan. 1925. The relation of the stability of protoplas- mic films in Noctéluca to the duration and intensity of an applied elec- tric potential. J. gen. Physiol., 7: 461-71. Luntz, A. 1931-32. Untersuchungen uber die Phototaxis. Z. vergl. Physiol. I, 14: 68-92; II, 15: 652-78; III, 16: 204-17. Lyon, E. P. 1923. Effects of Electricity on Noctiluca. Proc. Soc. exp. Biol. N.Y., 20: 284-85. McClendon, J. F. 1910. On the dynamics of cell division. I. The electric charge on colloids in living cells in the root tips of plants. Archiv. f. Entw. mech. d. Organismen. 31: 80-90. McClendon, J. J. 1911. Ein Versuch amdboide Bewegung als Folgeerscheinung wechselnden elektrischen Polarisationszustandes der Plasmahaut zu erklaren. Pflig. Arch. ges. Physiol., 140: 271-80. Marsland, D. A., and D. E. S. Brown. 1936. Amoeboid movement at high hydrostatic pressure. J. cell. comp. Physiol., 8: 167-78. Mast, S. O. 1906. Light reactions in lower organisms. I; Stentor coeruleus. J. exp. Zool., 3: 359-99. 1907. Light reactions in lower organisms. II. Volvox. J. comp. Neurol., ti 99-180: —— 1910. Reactions in Amoeba to light, J. exp. Zool., 9: 265-77. —— 1911. Light and the Behavior of Organisms. New York, 410 pp. —— 1912. The reactions of the flagellate Peranema. J. Anim. Behav., 2: 91-97. —— 1914. Orientation in Evglena with some remarks on tropisms. Biol. Zbl., 34: 641-74. —— 1916. The process of orientation in the colonial organism, Gonimm pectorale, and a study of the structure and function of the eyespot. J. exp. Zoole:202 1-172 —— 1917. The relation between spectral color and stimulation in the lower organisms. J. exp. Zool., 22: 471-528. —— 1918. Effects of chemicals on reversion in orientation to light in the colonial form, Spondylomorum quaternarium. J. exp. Zool., 26: 503-20. 1919. Reversion in the sense of orientation to light in the colonial form, Volvox globator and Pandorina morum. J. exp. Zool., 27: 367-90. — 1923. Photic orientation in insects with special reference to the drone- fly, Eristalis tenax and the robber fly, Erax rufibarbis. J. exp. Zool., 38: 109-205. —— 1926a. Structure, movement, locomotion and stimulation in Amoeba. J. Morph., 41: 347-425. —— 1926b. Reactions to light in Volvox, with special reference to the proc- ess of orientation. Z. vergl. Physiol., 4: 637-58. MOTOR RESPONSES 349 —— 1927a. Structure and function of the eyespot in unicellular and colonial organisms. Arch. Protistenk., 60: 197-220. —— 1927b. Reversal in photic orientation in Volvox and the nature of photic stimulation. Z. vergl. Physiol., 5: 730-38. —— 1927c. Response to electricity in Volvox and the nature of galvanic stimulation, Z. vergl. Physiol., 5: 740-61. —— 1928. Factors involved in changes in form in Amoeba. J. exp. Zool., 51: 97-120. —— 1931a. The nature of response to light in Amoeba proteus (Leidy). Z. vergl. Physiol., 15: 139-47. —— 1931b. The nature of the action of electricity in producing response and injury in Amoeba proteus (Leidy) and the effect of electricity on the viscosity of protoplasm. Z. vergl. Physiol., 15: 309-28. —— 1931c. Movement and response in D/fflvgia with special reference to the nature of cytoplasmic contraction. Biol. Bull., 61: 223-41. —— 1932a. Localized stimulation, transmission of impulses, and the nature of response in Amoeba. Physiol. Zodl., 5: 1-15. —— 1932b. The rate of adaptation to light and to darkness in Volvox glo- bator. Z. vergl. Physiol., 17: 644-58. —— 1936. Motor responses to light in the invertebrate animals. Biological Effects of Radiation (ed. by B. M. Dugger). New York. Pp. 573-623. —— 1939. The relation between kind of food, growth, and structure in Amoeba. Biol. Bull. 77: 391-98. Mast, S. O., and Mary Gover. 1922. Relation between intensity of light and rate of locomotion in Phacus pleuronectes and Euglena gracilis and its bearing on orientation. Biol. Bull. 43: 203-9. Mast, S. O., and Brainard Hawk. 1936. Response to light in Peranema tricho phorum. 1. Relation between dark-adaptation and sensitivity to light. Biol. Bull. 70: 408-12. Mast, S. O., and H. R. Hulpieu. 1930. Variation in the response to light in Amoeba proteus with special reference to the effects of salts and hydrogen ion concentration. Protoplasma, 11: 412-31. Mast, S. O., and P. L. Johnson. 1932. Orientation in light from two sources and its bearing on the function of the eyespot. Z. vergl. Physiol., 16: 252-74. Mast, S. O., and J. E. Nadler. 1926. Reversal a ciliary action in Paramecium caudatum. J. Morph., 43: 105-17. Mast, S. O., and C. L. Prosser. 1932. Effect of temperature, salts, and hydrogen ion contentrition on rupture of the plasmagel sheet, rate of locomotion, and gel/sol ratio in Amoeba proteus. J. cell. comp. Physiol., 1: 333-54. Mast, S. O., and N. Stahler. 1937. The relation between luminous intensity, adaptation to light, and rate of locomotion in Amoeba proteus (Leidy). Biol. Bull., 73: 126-33. 350 MOTOR RESPENSES Merton, H. 1923. Studien tiber Flimmerbewegung. Pflug. Arch. ges. Physiol., 198: 1-28. Moore, A. R. 1926. Galvanic stimulation of luminescence in Pelagia noctiluca. J. gen. Physiol., 9: 375-79. Nernst, W. 1899. Zur Theorie der electrischen Reizung. Nachr. v. d. Kgl. Gesell. Wiss. Gottingen, Math. physik. KI., 104-8. Oliphant, J. F. 1938. The effect of chemicals and temperature on reversal in ciliary action in Paramecium. Physiol. Zodl., 11: 19-30. Ostwald, W. 1890. Elektrische Eigenschaften halbdurchlassiger Scheidewande. Z; phys. Chem=63\ 71-82. Pantin, C. F. A. 1923-31. On the physiology of amoeboid movement. J. Mar. piols AssmiUigiew3> 24-69-SBrit) exp: Biol., e519). "3: aye oe 8: 365-78; Proc. roy. Soc., 105:-538-79. Pearl, R. 1900. Studies on electrotaxis. I. On the reactions of certain infusoria to the electric current. Amer. J. Physiol., 4: 96-123. ——— 1901. Studies on the effects of electricity on organisms. II. The reactions of Hydra to the constant current. A. Jour. Physiol., 5: 301-20. Pitts, R. F., and S. O. Mast. 1933. The relation between inorganic salt concen- tration, hydrogen ion concentration and physiological processes in Amoeba proteus, 1. Rate of locomotion, gel/sol ratio and hydrogen-ion concentration in balanced salt solutions. J. cell. comp. Physiol., 3: 449-62. — 1934a. II. Rate of locomotion, gel/sol ratio and hydrogen ion concentra- tion in solutions of single salts. J. cell. comp. Physiol., 4: 237-56. —— 1934b. III. The interaction between salts (antagonism) in relation to hydrogen-ion concentration and salt concentration. J. cell. comp. Physiol., 4: 435-55. Ray, J. 1693. Historia Plantarum, 2: 985-1944. London. Rees, C. W. 1922. The neuromotor apparatus of Paramecium. Univ. Cal. Publ. Zool., 20: 333-64. Robbins, W. J. 1926. The isoelectric point for plant tissues and its importance in absorption and toxicity. Univ. Mo. Stud., 1: 3-60. Schaeffer, A. A. 1917. Reactions of Amoeba to light and the effect of light on feeding. Biol. Bull. 32: 45-74. Shettles, L. B. 1937. Response to light in Peranema trichophorum with special reference to dark-adaptation and light-adaptation. J. exp. Zool., 77: 215-49. — 1938. Effect of ultraviolet light and x-rays on Peranema tricho phorum. J. cell. comp. Physiol., 12: 263-72. Spek, J. 1921. Der Einfluss der Salze auf die Plasmokolloide von Actinosphae- rium Eichhorni. Acta Zool. Stock., 2: 153. Statkewitsch, P. 1903. Uber die Wirkung der Induktionsschlige auf einige Ciliata, Physiol. russe, 3: 1-55. — 1907. Galvanotropismus and Galvanotaxis der Ciliata. Z. allg. Physiol., 6: 13-43. MOTOR RESPONSES 351 Strasburger, E. 1878. Wirkung des Lichtes und der Warme auf Schwarm- sporen. Jena. Z. Naturw., n. f., 12: 551-625. Taylor, C. V. 1920. Demonstration of the function of the neuromotor ap- paratus in Evplotes, by the method of microdissection. Univ. Cal. Publ. Zool., 19: 403-470. —— 1925. Cataphoresis of ultramicropic particles in protoplasm. Proc. Soc. exp. Biol. N.Y722:153336, Terry, O. P. 1906. Galvanotropism of Volvox. Amer. J. Physiol., 15: 235-43. Verworn, M. 1889. Psycho-physiologische Protistenstudien. Jena, 218 pp. 1895. Allgemeine Physiologie. Jena, 615 pp. Visscher, J. P. 1927. A neuromotor apparatus in the ciliate Dileptus gigas. J. Morph. and Physiol., 44: 373-81. Wager, H. 1900. On the eyespot and flagellum of Ezglena viridis. J. linn. Soc. (Zool.), 27: 463-81. Weber, E. B., and I. Weber. 1922. Reversible Viskositatserhohung des Cyto- plasmas unter der Einwirkung des elektrischen Stromes. Ber. dtsch. bot. Ges., 40: 254-58. Weber, F. 1924. Plasmolyseform und Protoplasmaviskositat. Ost. Bot. Z., 73: 261. Yocum, H. B. 1918. The neuromotor apparatus of Ezplotes patella. Univ. Cal. Publ. Zool., 18: 337-96. CHAPTER VI RESPIRATORY METABOLISM THEODORE LOUIS JAHN STUDIES OF THE RESPIRATION of the Protozoa have, for the most part, been fragmentary, and our information on the subject resembles an ac- cumulation of a number of more or less isolated data, rather than a unified body of knowledge. This situation is due to a variety of causes, chief among which is probably the fact that most studies of respiration have been made by physiologists who chose, among the members of the animal kingdom, the organisms which seemed to be the most suitable for a particular type of experiment. From this viewpoint the study of protozoan respiration has suffered a severe handicap, in that a consider- able mass of protozoan protoplasm, free from bacteria and other organ- isms, has not always been easy to obtain, and in that our methods for measuring very small rates of respiratory exchange have not been nearly as accurate or as convenient as we might desire. However, with gradual technical advances, it seems probable that in the near future we shall see the development of an organized account of protozoan respiration, and it also appears probable that this development will take place among investigators who are primarily interested in the Protozoa. Therefore, it seems advisable to combine a review of data on protozoan respiration with a discussion of the general problems of respiratory metabolism in other biological materials, and to outline for the student not thoroughly trained in the lore of respirometry some of the purposes, methods, and possible interpretations of such a study. Among the Protozoa, the intake of oxygen does not require compli- cated respiratory mechanisms. Apparently diffusion, high rate of water exchange, and protoplasmic movements (cyclosis, amoeboid streaming, and ‘‘metabolic’” movements) are sufficient to maintain a suitable level of O, tension in the protoplasm and to prevent the accumulation of toxic amounts of CO,. The mechanisms which are responsible for protoplasmic movements and the high rate of water exchange are more properly treated RESPIRATORY METABOLISM 353 under the subjects of movement, permeability, and excretion. Therefore the problem of respiratory metabolism of a protozoan organism, at least for the present discussion, is easily resolved into a problem comparable to that of cellular respiration in the Metazoa. PURPOSES OF STUDYING RESPIRATION One of the first questions to be considered is: “What can be learned by studying respiration?’ The measurement of gaseous exchange is not an end in itself, but it is a tool which, when used singly or in combina- tion with other tools, may help us to obtain answers to the following types of questions. 1. What is the rate of energy expenditure of the organism? How does the metabolic rate (basal and otherwise) of one species compare with that of another? How does it change during starvation? Or conjugation? Is this rate dependent upon the O, or CO, tension of the environment? How is it affected by narcotics? How does it vary with temperature? Or with other chemical and physical factors of the normal environment? 2. What is the source of this energy? Is it obtained by oxidation of fats, carbohydrates, or proteins? Or by anaérobic oxido-reductions? Can this source be shifted by changing the chemical or physical environment of the organism? What are the intermediate products formed during oxidation of the substrate? Is oxidation of the substrate complete (1.e., to CO, and H,O), or may these intermediate products be excreted by the cell? 3. What is the mechanism by which energy is obtained from the sub- states? Is this brought about through the intermediary action of dehydro- genase, cytochrome, and Warburg’s respiratory enzyme? Or is it brought about through dehydrogenase and reversible oxidation-reduction systems, such as yellow pigment or pyocyanine? Or by glutathione? Can these substances be replaced by artificial oxidation-reduction systems? Can a shift in this mechanism of respiration be induced by changing the avail- able substrate? How is this mechanism related to the degree of anaérobio- sis which the organisms can endure? Is the organism capable of synthesiz- ing the respiratory enzymes from simple substances, or must they be obtained from complex outside sources, i.e., from vitamins? Measurements of the respiratory metabolism of an organism may be used as indices of the rate at which it uses energy, the substrate from 354 RESPIRATORY METABOLISM which this energy is derived, and the mechanism by which it ts obtained from the substrate. In addition to these factors, there is a possibility of a direct relationship between certain metabolic processes and the patho- genicity of parasitic forms. METHODS OF MEASURING AEROBIC RESPIRATION Methods that have been or might be used for measurement of aérobic protozoan respiration fall naturally into two groups—those applicable TABLE 3: SENSITIVITY OF RESPIROMETERS Nearest Unit to Approximate Sensitivity Type of Respirometer Which Meniscus in Terms of Scale Can Be Read* Divisions Standard Warburg (Warburg, 1926) o.2 mm. I mm.=1.0-2.0 mm.’ O; Microsemidifferential (Duryée, 1936) 9-2 mm. Imm.=o.5 mm.’ Oz Microdifferential (Fenn, 1928) 9:1 mm. I mm.=o.3 mm.3 Og Microdifferential (Described in text) 9:2 mm. I mm.=o.2 mm.3 Op, Straight capillary tubes | (Howland and Bernstein, 1931) oor mm. 0.01 mm.=o.oo1 mm.3 Oy Straight capillary tubes in closed air chamber (Gerard and Hartline, 1934) 9006 mm. 0.006 mm.=0.0013 mm. Op Microdifferential (Schmitt, 1933) : micron I micron=0.0005 mm.? Oy Cartesian diver (Needham and Boell, 1938) 5.2 mm. I mm.=o.oo1 mm.’ Og * It is assumed that the meniscus is read to the nearest 0.2 mm., unless otherwise stated by the authors. In those cases where the author claims greater reading accuracy, the smallest unit of change which can be detected is given in this column. If stability is adequate, the sensitivity of several models may be considerably increased by the use of special reading devices. to concentrated suspensions of organisms, and those applicable to a few or to single cells. For concentrated suspensions, titration, gas analysis, and standard manometer methods have been used; and for studies of RESPIRATORY METABOLISM 595 single protozoan cells, micromanometric methods have been devised (Kalmus, 1927; Howland and Bernstein, 1931). More recently, still better micromanometric methods have come into existence, but these have not yet been applied to the respiration of protozoa. Table 3 gives the sensitivity of various types of manometers, some of which have not yet been used to measure the respiration of Protozoa. It should be remem- bered that whenever a respirometer is made more sensitive to changes in gas volume produced by organisms, it simultaneously becomes more sensitive to slight changes produced by the environment (thermal and barometric effects) and to inaccuracies arising from imperfect design and construction (ground-glass connections and stopcocks, surface phe- nomena at the meniscus of manometer fluid, inaccuracies of capillary bore and so forth). Therefore, stability of the apparatus, on which final ac- curacy must depend, becomes more and more difficult to obtain. For that reason the most sensitive types should be reserved solely for those prob- lems in which concentrated suspensions are undesirable or unobtainable. By comparison of Tables 3 and 4, it should be possible to determine ap- proximately the type of respirometer necessary for any one of a variety of problems. 1.TITRATION METHODS a. Dissolved O, determinations. For any aquatic animal it is possible to measure O, consumption by placing the organisms in a closed chamber filled with water of known O, content and by measuring the amount of O, left after a definite period of time. For this purpose a modified Win- kler titration method is usually used (Standard Methods of Water Anal)- sis, 1936). Special precautions are necessary whenever the animals can- not be removed from the solution, or if iron is present. This method has been used by Lund (1918a, 1918b, 1918c) and Leichsenring (1925) on Paramecium and Col poda. b. Measurement of CO, production. Production of CO, may be measured by placing the organisms in a small amount of solution in a small open container. This is placed inside of a larger closed container in which an alkali, preferably Ba(OH),, is present. The CO, given off by the organisms is absorbed by the alkali, which can then be titrated with acid in the presence of an indicator. This method was recom- mended by Lund (1918d) for use with Paramecium. 356 RESPIRATORY METABOLISM The above titration methods are applicable only to quite large num- bers of organisms. It seems possible that these methods could be im- proved by the use of accurately controllable microburettes, smaller vol- umes of liquid, and so forth, but it is doubtful whether they could be made as accurate and reliable as some of the manometric procedures discussed below. Also, respiratory quotients are not easily obtained by titration methods. i 2. GAS ANALYSIS Soule (1925), Amberson (1928), and Root (1930) have applied the standard Haldane-Henderson methods of gas analysis to respiration of the Protozoa. These methods are adequate for use with rather con- centrated suspensions and pressures, and possess certain definite ad- vantages when O, or especially CO, tension is being varied experimen- tally and would have to be determined separately if manometric methods were used. Whenever gases other than CO, are evolved by an organism, gas analysis seems to be the only satisfactory method of measurement. The details of gas analysis methods are discussed by Peters and van Slyke (1932). Soule (1925), for studies of the metabolism of Lessh- mania tropica and Trypanosoma lewisi, used gas analysis, supplemented by readings of an insensitive manometer, the purpose of which was principally to indicate when gas exchange was taking place (method described in detail by Novy, Roehm, and Soule, 1925). 3. STANDARD MANOMETRIC METHODS The principle of the manometric method is somewhat as follows: the organisms, in a suitable immersion medium, are placed in a closed flask large enough so that a considerable air space is present. The flask is connected to a capillary manometer tube partially filled with a liquid. Alkali may be present in a separate small container inside of the flask. If so, then CO, is absorbed, and the amount of O, consumed may be measured by means of the movement of fluid in the manometer tube as changes in volume (Haldane, Thunberg, Winterstein, Duryée, and Dixon types), or in pressure at a given volume (Warburg), or as the resultant of simultaneous changes in both (Barcroft differential type). Manometric methods, although very simple in outline, are filled with pitfalls for the inexperienced investigator, and a careful reading of the RESPIRATORY METABOLISM Soy), excellent treatise of Dixon (1934) is recommended. In this publication the theory and the more common forms of the apparatus are described in detail. The Barcroft differential type can be made sufficiently small and sensitive for the study of respiration of Protozoa, when moderately con- centrated mass cultures are available. A manometer designed by Dr. T. C. Evans (modified from that of Bodine and Orr, 1925), which has been in use in this laboratory for some time, seems to be quite suitable. It resembles the standard Barcroft (Dixon, 1934, p. 37) ex- cept that the two stopcocks are replaced by one double stopcock, which insures the simultaneous opening and closing of both flasks. The cups may be relatively small (about 5 cc.), and the U-shaped portion of the capillary (0.3 mm. bore) is placed at an angle of about twenty degrees from the horizontal. This design combines a high degree of sensitivity with ruggedness and dependability. The Duryée (1936) modi- fication of the Thunberg-Winterstein principle and the Fenn (1928) form of microdifferential respirometer also seem to have a sensitivity adequate for moderately concentrated suspensions. Schmitt (1933) has devised an extremely sensitive form of microdifferential manometer, in which the gain in sensitivity and accuracy is due chiefly to an elaborate reading device and a system of temperature control which is stated to make meniscus movements of as little as one micron both detectable and significant. 4, CAPILLARY MANOMETER The use of capillary tubes for measuring respiration of single protozoan cells was introduced by Kalmus (1927, 1928a). This method was improved by Howland and Bernstein (1931), who by means of a mictro- injection device drew small amounts of oil, air, KOH solution, and water containing an animal into small capillary tubes, so that they were finally arranged in the following order: oil, KOH, air, animal in water, oil. As the animal consumed O,, the distance between the oil-water and the oil-KOH interfaces decreased. This change was measured mictro- scopically by means of an ocular micrometer and a calibrated mechanical stage. By using control tubes made in a similar manner but without an animal, it was possible to correct for slight movements due to thermo- barometric changes and to osmotic differences between the water and 358 RESPIRATORY METABOLISM KOH. Gerard and Hartline (1934) have improved the method by enclosing the tubes in an air tight chamber to eliminate barometric dis- turbances, and by using a screw micrometer to increase accuracy of reading. 5. CARTESIAN DIVER ULTRAMICROMANOMETER (NEEDHAM AND BOELL, 1938) This is an application of the principle of the Cartesian diver for use as a constant-volume manometer. The ‘‘diver’” chambers are constructed from capillary tubing and consist of a bulb partially filled with gas, an open capillary neck, and a solid glass tail to ensure that the diver floats upright. The diver is placed in a closed chamber partially filled with a strong salt solution, the specific gravity of which is such that the diver maintains a position below the surface of the salt solution, and that a small amount of this solution enters the neck. If the amount of gas in the diver is changed by a reaction, more salt solution will be drawn in or forced out of the neck, the specific gravity of the diver will change, and the level of flotation will also change. By changing the pressure on the salt solution, the diver may be brought back to any given level. Therefore, the diver may serve as a constant-volume ma- nometer, and changes in the amount of gas in the chamber may be calcu- lated from the changes in the external pressure which are necessary to maintain the diver at a definite level. This application of the Cartesian diver was suggested by Linderstrom-Lang (1937), and has been used for parts of amphibian embryos by Needham and Boell (1938), who describe the use of this instrument for measurement of O, consumption, anaérobic glycosis, and respiratory quotient. From Table 3 it may be noted that this instrument when read only to 0.2 mm. has a sensitivity as great as those which employ special reading devices, and that it therefore has the possibility of being made more sensitive. AEROBIC RESPIRATION 1. THE NORMAL RATE OF RESPIRATION One fundamental essential in measuring the respiration of any bio- logical material is that other material, also capable of respiratory ac- tivity, be absent or very well controlled. This means that bacteria must be absent, or at least must contribute only a negligible amount to the RESPIRATORY METABOLISM 359 measured respiration. Various workers have tried removing bacteria by washing and filtering, or have corrected the figures for O, consump- tion by running controls of bacteria without Protozoa. Data obtained by these methods are extremely difficult for a reviewer to evaluate critically, and usually one may either accept them at face value until they can be checked with bacteria-free cultures or ignore them entirely. In the present discussion the tendency has been to accept all data in which the magnitude of the error is not obviously large, and to point out possible difficulties involved. In view of the fact that some bacteria have a respiratory rate per gram many times that of other types of cells (the rate for Azotobacter is stated by Burk [1937] to be equivalent to that of a 200-pound man consuming one ton of glucose per hour), the present viewpoint may be considered far from conservative. In some cases (e.g., the papers on cyanide insensitivity of Paramecium) the data on Protozoa seem quite adequate to prove the principal conclusions of the author, but are not accurate enough to afford detailed comparisons of respiratory rate. In such cases only the main points (e.g., insensitivity to cyanide) are given serious consideration. In only a few cases have investigators used bacteria-free cultures for measurement of respiration (cf. Table 4). For comparative purposes in work with metazoan tissues, it is custom- ary to express oxygen consumption in cubic millimeters (at normal tem- perature and pressure denoted as N.T.P.) per hour per milligram of dry weight of the tissue (symbolized by Qo,). In the protozoan literature, where the rate of O, consumption is expressed in absolute units, this unit is sometimes the Qo, but is more usually mm® per hour per organism, principally because the counting of organisms is simpler than measuring dry weight. Some authors use the symbol Qo, for O, consumption per 1,000,000 or per 100,000,000 organisms (e.g., von Fenyvessy and Reiner, 1928; Hall, 1938), but it seems preferable to avoid confusion by retaining this symbol for its original meaning and using a new symbol for consumption per 1,000,000 organisms, perhaps Qo, as used in Table 4. However, since not dry weight, nor wet weight, nor number of or- ganisms affords the possibility of comparing the oxygen consumption per unit of respiring protoplasm, these discrepancies are not as important as one might at first suppose. In the case of flagellates such as Astasia and Chilomonas, in which a high percentage of the weight may be in the 360 RESPIRATORY METABOLISM form of carbohydrate reserves, either dry or wet weight would be a poor index of the amount of respiring protoplasm. If one wishes to compare the rate per unit of protoplasm, the best index is probably the nitrogen content, because protein and other nitrogen-containing compounds are not ordinarily stored as reserve food. However, in the case of Protozoa which secrete nitrogen-containing tests, this criterion might also be very poor. Therefore any comparison of the absolute rate of different species, even after all differences in technique, immersion fluid, and physiological condition of the animal have been overcome, must usually be made with reservations, or at least with an adequate understanding of the limitations involved. Some authors have chosen to calculate O, consumption in terms of cubic millimeters of organisms, but the errors in- herent in the methods of packing the animals for measurement (usually centrifuging) or in calculating volume from linear dimensions are too great to allow close comparison of data for different types of organisms. However, for an extended series of experiments on the same or very similar organisms, the use of dry weight (A. Lwoff, 1933) or of volume measurements (Elliott, 1939) seems to be entirely satisfactory. If concentrated suspensions of organisms are used, the rate of shaking should be carefully controlled. The importance of this factor is demon- strated by the data of Hall (1938). If ammonia is produced by an organism, it is necessary to maintain acid within the respiring chamber in order to obtain true values of O, consumption. This very important procedure is discussed by Specht (1935). Another question which arises in expressing results in absolute form is that of measuring basal metabolism, i.e., the metabolism of rest. In a mammal, for instance, there are certain well-defined limitations of condi- tions under which O, consumption may be termed a measurement of basal metabolism. In a protozoan it is more difficult, if at all possible, to apply these criteria, and in all known measurements we have a sum of the total metabolic processes, i.e., of those to which we refer as “basal,” those due to movement of the organism, and, if the medium is nutrient, those due to the manufacture of reserve food material and to growth. The energy expended for each of these purposes will probably vary with the species, the physiological state, and the environmental condi- tions. If the metabolic substrates of an organism undergo complete oxida- RESPIRATORY METABOLISM 361 tion, we are able, by means of measurements of O, consumption, to determine directly the amount of energy available to the organism. If oxidation is incomplete, a further knowledge of the oxidation products is necessary. It is usually assumed, unless we have knowledge to the contrary, that oxidation is complete in the a€robic Metazoa (cf. intes- tinal nematodes, von Brand and Jahn, 1940). Among the bacteria and also among the Protozoa this is not always true, even in the presence of normal O, tension. However, since carbohydrate cleavage and intra- molecular oxidation, even in the presence of O,, may be considered an anaérobic process, that question will be discussed under ana€robiosis. From studies on the heat of combustion, we know that complete oxidation of glucose yields 677,000 calories per gram molecule, or about 3,700 calories per gram. Complete combustion of protein yields 5,700 calories per gram, and fats yield 8,000-9,000 calories per gram. There- fore, if we know the substrate being oxidized and the rate at which O, is consumed, we can calculate the energy made available by oxidation. According to the equation CHO: = GOs GiCO, | 6 H,O —- 677,000 cal. one gram molecule of glucose requires six gram molecules of O,. The volume of O, consumed (at N.T.P.) is 6 & 22.4 liters, or 134.4 liters. The ratio of O, consumed to calories released is 134 liters/677,000 calories, or about one calorie for each 200 mm? of O, consumed. Similar calculations may be made for fats and proteins. Table 4 contains most of the known data for respiratory rates for the Protozoa which can be expressed in absolute terms—either as mm* O, per organism per hour, or mm’ O, per gram dry or wet weight per hour. Similar tables are given by von Brand (1935) and Hall (1938). 2. THE EFFECT OF O, TENSION ON O, CONSUMPTION For many types of biological material it has been quite well established that, under usual experimental conditions, O, consumption is inde- pendent of O, tension, within very wide limits (exceptions cited by Tang, 1933, McCoy, 1935). Recently, however, Kempner (1936, 1937) demonstrated that this is not true for several species of bacteria, for human leucemic leucocytes, for red blood cells of man, fowl, and allt- gator, and for pine needles if CO, is present or if the temperature is 362 RESPIRATORY METABOLISM above 25° C. With these same materials in CO,-free alkaline media below 25° C., O, consumption was independent of O, tension. The effect of O, tension apparently varied with pH, CO, tension, salt con- TABLE 4: MEASUREMENTS OF PROTOZOAN RESPIRATION 3 M*mi O) per aoe Hour per Tem- Bac- : as per mg. Dry ; : Species Million Weizh perature, | teria- Investigator eight C free Qu, Qo, Paramecium caudatum 120 (CO) De No | Barratt (1905) 140 Tis No_ | Lund (1918c) 2,250 Re No | Zweibaum (1921) 3,900 19° No_ | Necheles (1924) 5,600 Dp No_ | Kalmus (1928b) 500 Mtg ae No | Howland and Bern- stein (1931) Paramecium multimicro- nucleatum THODT Dre No | Mast, Pace, and Mast (1936) Colpidium campylum 200 24.0° No | Pitts (1932) 112.5 19.8° Yes | Hall (1938) Colpidium colpoda 200 17.0° No | Wachendorff (1912) 200 No | Peters (1929) Colpoda sp. 600-1, 200] 19.7° No_ | Adolph (1929) Glaucoma piriformis 35 22m Yes | M. Lwoff (1934) Blepharisma undulans (0.5)* 20.8° No_ | Emerson (1929) Spirostomum ambiguum 2,590 25.0° No_ | Specht (1935) Strigomonas oncopelti 0.4 62 28.0° Yes | A. Lwoff (1933) Strigomonas fasciculata 0.4 55 28.0° Yes | A. Lwoff (1933) Leptomonas ctenocephalus 0.3 40 28.0° Yes | A. Lwoff (1933) Trypanosoma equiperdum 0.05 37.0° Yes | Von Fenyvessy and Reiner (1928) Chilomonas paramecium 17-26 25.0° Yes | Mast, Pace, and Mast (1936, 1937) Astasia sp. 2, 400 Yes | Jay (1938) Khawkinea halli 2,050 Yes | Jay (1938) Actinosphaerium eichhornii, 1, 100 20.0° No | Howland and Bern- stein (1931) Amoeba proteus (0.2)* 20.0° No_ | Emerson (1929) * Not Qoo but mm* O. per hour per mm%. tent, and temperature. Since O, consumption of the yellow pigment of respiration (see below) varies with O, tension, it would not be sur- prising to find a similar relationship in organisms with this mechanism. However, this is not always the case (Schlayer, 1936). Clarification of RESPIRATORY METABOLISM 363 the relationship between O, tension and O, consumption in Protozoa will apparently require much more data than is now available. A dis- cussion of the theoretical relationship between oxygen tension and oxygen consumption is given by Marsh (1935). For Protozoa, the available evidence indicates that within wide limits O, tension has little or no effect on the rate of O, consumption for Paramecium and Col poda, and that it does have an effect on Spirostomum. Lund (1918a) found that the rate of O, consumption for Paramecium was independent of O, tension between 0.04 cc. and 2.2 cc. O, per 137 cc.—a 55-fold range. This was determined by placing thick sus- pensions of Paramecium in stoppered bottles and measuring the dis- solved O, content of the water by the Winkler method, until the animals died. Lund’s conclusion was confirmed by Amberson (1928), who placed the organisms in a closed vessel, in contact with an atmosphere of known O, content. By gas analyses he demonstrated a uniform rate of O, consumption, with O, partial pressures which varied from 50 to 220 mm. Hg, and only a slight decrease (about 20 percent) at pressures as low as 11 mm. Hg. Adolph (1929) found that the O, consumption of Colpoda did not vaty significantly with O, tension between 155 and 750 mm. Hg. In a single experiment at 4-8 mm. Hg, O, consumption decreased to 31 percent of its previous value. However, Adolph did find that low O, tension (40 mm.) was correlated with smaller size of the progeny of cultures. Specht (1935) measured the respiration of Spirostomum in pure oxygen, in air, and in 0.5 percent O, in N,. He found that O, consumption in these gases was in the ratio of 151 to 100 to 71, and that CO, production was in the ratio of 175 to 100 to 70. When considering the effect of low O, tensions on O, consumption for any of the larger Protozoa, one should consider the O, tension at various points within the organism as well as at the surface. This can be calculated by the diffusion equations of Harvey (1928) and others, on the assumption that the rates of cyclosis and water exchange are low. The O, tension at the center of an ellipsoid which is consuming O, uniformly throughout its substance, will be zero when the shortest radius Be A a 364 RESPIRATORY METABOLISM where D is the diffusion coefficient of O,, c is the O, tension at the surface, and A is its rate of O, consumption. In the case of a cylinder (e.g., Spzrostomum) the factor 5 should be 4. For Colpoda, Adolph (1929) calculated the value of “a” to be 148 y at atmospheric O, tension, and 72 y at 40 mm. Hg partial pressure of O,. In large ciliates this factor might be important at low O, tensions, even with a rather low rate of O, consumption. 3. EFFECT OF CO, TENSION ON O, CONSUMPTION Root (1930) showed that when CO, tension was raised from 1 mm. Hg to 15-20 mm. Hg, the respiration of Paramecium increased slightly (less than 15 percent), and it was believed that this increase might be caused by increased activity of the organisms. As the CO, tension was increased above 60 mm. Hg, O, consumption decreased continuously to about 40-60 percent of the control when CO, tension reached 220- 360 mm. Hg. Similar experiments on fertilized Arbacia eggs did not show increase at low concentrations, and all CO, tensions greater than 30 mm. Hg produced a decrease to less than 40 percent of normal. Paramecium apparently was much more resistant to increase of CO, tension than Arbacia. For both Paramecium and Arbacia only slight effects were obtained with HCl, at pH values comparable to those present during the CO, experiment (4.5 to 7.5 for Paramecium). From the information available, it is not possible to determine the mechanism of action of CO, on protozoan respiration, and results with other organisms are few and variable. Apparently CO, is not involved as an inhibitor or accelerator of any of the known mechanisms of respiration (to be discussed below), and at present we can only say that the effect on respiration seems to be indirect, and that the results are not due to pH changes in the external fluid. However, internal pH changes, as suggested by Root (1930), might account for the effect. This is an explanation comparable to that given by Jahn (1936) for the effect of the lack of CO, on growth of Chilomonas and bacteria. It is well established that certain bacteria will not grow in the absence of CO,. Jahn (1936) studied the effect of CO,-free media on growth of Chilomonas and Colpidium and found a distinct inhibition with Chilomonas and none with Colpidium. The explanation was offered that the inhibition of growth might be caused by inadequate intracellular buffering in those species which were affected by lack of CO,, and that RESPIRATORY METABOLISM 365 organisms whose normal environment is high in CO, might depend more on CO, buffering than those the normal environment of which is low in CO,. (For possible application of this idea to culture of in- testinal forms, see Jahn, 1934). 4, THE EFFECT OF THE PHYSIOLOGICAL STATE ON ©; CONSUMPTION It seems as if the effect of various factors which influence the physio- logical state of an organism may be reflected in measurements of O, consumption. Factors which have been investigated for the Protozoa are starvation, age of the culture, and conjugation. Lund (1918c) starved Paramecium in tap water and noted an ap- preciable decrease in respiration during the first twenty hours. This was simultaneous with the disappearance of deutoplasmic food reserves from the protoplasm. Upon feeding starved animals with boiled yeast sus- pensions, the rate of oxygen consumption could be increased two to three times. This increase was independent of cell division. Leichsenring (1925) demonstrated a decrease of 23 percent after twenty-four hours of starvation and 29 percent after seventy-two hours. The effect of the age of the culture on O, consumption was first studied by Wachendorff (1912), who found that for Colpidium colpoda the O, consumed per organism diminished from 191 mm* per hour the first day to 151 mm* the tenth day, and to 59 mm‘* on the thirtieth day. Reidmuller (1936) reported a higher rate of O, consumption for young cultures of Trichomonas foetus than for older cultures. Twenty- four to forty-eight-hour-old cultures consumed 4.3 mm? O, per mm‘* of organisms, sixty-hour cultures, 2.8 mm,? and three-day-old cultures only 0.81 mm* O,. Andrews and von Brand (1938) reported a decrease in sugar consumption per organism for this species, with increasing age of the culture, and their data indicate that the differences observed by Reidmuller were real, in spite of objections to the method used be- cause of the possibility of hydrogen or methane evolution. Zweibaum (1921) studied the rate of O, consumption of Paramecium caudatum in relation to conjugation. He found that the rate just before conjugation was about 0.73 mm? O, per thousand organisms per hour. During conjugation this rate rose to 3.4 mm*/1,000/hour, and immedi- ately after conjugation decreased to about 0.73. During the first eight or nine days following conjugation, the rate rose slowly to 2.0 mm?/1,000/hour, and remained at this value from four to five months. 366 RESPIRATORY METABOLISM 5. THE EFFECT OF TEMPERATURE ON © CONSUMPTION Data concerning the effect of temperature on O, consumption are not numerous, and in most cases are incomplete. Barratt (1905) determined the CO, production of Paramecium at various temperatures and found that the rate at 27°-30° C. was more than twice that at 15° C. Wachen- dorff (1912) found that C. col/poda respired about four times as fast at 17° as at 7° C. Leichsenring (1925) demonstrated that Paramecium, when transferred from a temperature of 20° C. to one of 35° C., showed a respiratory increase of 35 percent, and that when transferred to a temperature of 15°, 10°, 5°, 0° C. respiration was decreased 30 per- cent, 34 percent, 50 percent, and 58 percent respectively. These effects were not completely reversible. The data of Kalmus (1928b) showed a Q,, value (temperature coefficient) of 1.5 for Paramecium respira- tion between 23° and 32° C. A. Lwoff (1933) found a Q,, value of 2.1 between 13° and 23° C., and about 1.5 between 23° and 32° C. The temperature characteristic (1 value) was 9,830 calories for the range 13° to 34° C. —_ Lwoff also calculated a j value of 21,350 for the synthesis of respiratory enzyme (oxidase, see below) by the organism between 18° and 31.5° C., and a value -of —52,000 between 31.5° and SOME 6. THE EFFECT OF ANESTHETICS AND POISONS ON O, CONSUMPTION The effect of various toxic agents (e.g., KCN, CO, N;H, arsenite, urethanes, and so forth) which are supposed to exert a specific effect on the normal functioning of certain respiratory enzymes, will be dis- cussed in connection with the mechanism of respiration. The effect of other anesthetics on respiration has not been extensively studied. Leich- senring (1925) found that ethylene and nitrous oxide had no effect on the respiration of Paramecium, and that ether and chloroform produced decreases of as much as 25 percent (after two and one hours respec- tively). The effect was reversible with ether, but not with chloroform if the exposure was more than one-half hour. Co/poda was more sensitive to these substances than Paramecium. Von Fenyvessy and Reiner (1928) found no decrease in respiration of Trypanosoma equiperdum when one- percent Germanin was added. This was surprising, in view of the fact that Germanin is very toxic for trypanosomes 7” vivo. RESPIRATORY METABOLISM 367 7. THE EFFECT OF NUTRITIVE SUBSTANCES AND OTHER MATERIALS The effect of various nutritive substances on Protozoa has been demon- strated by a number of investigators, but the criteria used are usually growth or the accumulation of food reserves, and not respiration. In some cases it is possible to determine that the substance is oxidized directly (e.g., glucose), or that it contributes toward the synthesis of the respiratory enzyme (A. Lwoff, 1933). These data will be dis- cussed below. The effect of various substances on the respiration of Paramecium was studied by Leichsenring (1925), who found that cap- rine, glutamic acid, peptone, and aminoids increased respiration 12-18 percent; that glycocoll and succinic acid increased respiration 8-9 per- cent; and that tyrosine and cystine produced little effect. Lactose gave an increase of 16 percent, and other sugars and polysaccharides gave in- creases of 3-10 percent. Thyroxin gave an increase of 13 percent. No explanation was made of the mechanism of these effects. Mast, Pace, and Mast (1936) found that Chilomonas grew well, formed considerable starch but little fat, and consumed 0.17 mm* of O, per 10,000 organisms per hour, in a solution of MgSO,, NH,Cl, K,HPO,, Na-acetate, and silicon. When sulphur was omitted, the starch remained constant, fat accumulated, O, consumption decreased, and the animals finally died. When acetate was omitted, the organisms decreased in size, starch and fat decreased, and O, consumption decreased to 0.07 mm?/10,000/hour. When both sulphur and acetate were omitted, starch decreased to zero, fat accumulated, O, consumption decreased, and the organisms died. These authors conclude that starch is normally changed to fat, that sulphur induces oxidation of fat, thereby increasing respira- tion and preventing accumulation of fat, and that fat oxidation is probably associated with a cystine-cysteine mechanism (see glutathione, below). Mast and Pace (1937) reported a 25 percent increase in res- piration of Chilomonas when Na,SiO, was added to inorganic media. This was supposedly caused by the catalytic action of Si on organic syntheses. 8. EVOLUTION OF GASES OTHER THAN CO, The possibility that Protozoa evolve gases other than CO, was first shown by Cook (1932) for the flagellates of termites (Termopszs neva- densis). A gas which was not absorbable by hydroxide was evolved by 368 RESPIRATORY METABOLISM normal termites, under anaérobic conditions. However, this gas was not evolved if the intestinal flagellates had been removed by oxygenation, and it was suggested that it might have been evolved by the flagellates. The evidence is incomplete, however, in that oxygenation probably also changed the bacterial flora. Witte (1933) observed the production of gas bubbles by Trichomonas foetus. This was confirmed by Andrews and von Brand (1938), who found that this gas was not absorbable by alkali and that when mixed with O, it burned with indications of ex- plosiveness. Final identification was not made. The formation of gas vacuoles has been reported for several organisms, but in most cases there is little evidence regarding the identity of the gas. Bles (1929) believed that the gas vacuoles of Arcella contained O,,. The chlorophyll-bearing flagellates, of course, might give off O, in the presence of strong light, because of photosynthesis, and this might also be true of the ciliates which harbor zodchlorellae. There is con- siderable indirect evidence that this is true, but no direct measurements are available. INVESTIGATIONS WHICH CONCERN THE SOURCE OF ENERGY Whenever an oxidizable material is subjected to complete combustion, the ratio of CO, given off to O, consumed will vary with the type of material. This ratio (CO,/O,) is called the respiratory quotient (R.Q.). Carbohydrates (relatively rich in oxygen) have an R.Q. of 1.0; fats (relatively poor in oxygen) have an R.Q. of about 0.71; and proteins have an R.Q. of 0.83 if the nitrogen is eliminated as urea, and 0.93 if it is eliminated as ammonia. By measurements of the respiratory quotient we may obtain an index of the type of material which is being oxidized by the organism, at least as to whether it is predominantly carbohydrate or predominantly fat. If the excreted nitrogenous material can be identi- fied and measured, the amount of protein and consequently the amounts of carbohydrates and fats consumed can be calculated. Under these con- ditions the measurement of R.Q. becomes more significant. These interpretations are based on the assumptions that complete oxidation of metabolic substrates is the sole cause of gaseous exchange, and that gases other than O, and CO, are not involved. If these assump- tions are unsound, then any interpretation of the R.Q. is necessarily more difficult. If carbohydrate is being converted into fat within the RESPIRATORY METABOLISM 369 organism for purposes of storage, the R.Q. may reach a value of 1.4. If fat or protein is being converted to carbohydrate, there will be a cor- responding tendency for the R.Q. to be lowered. A high value for the R.Q. can also arise whenever CO, is removed from a compound with- out the consumption of O,, or whenever an oxygen debt accumulates (during heavy exercise). Whenever an oxygen debt is being removed, the R.Q. may fall to extremely low values (during rest after exercise). Unusual values of R.Q. may be obtained if substrates other than carbo- hydrates, fats, or proteins are being consumed. In the case of very rapid protozoan growth on substrates of organic acids, the R.Q. should vary with the oxygen content of the molecule being oxidized (acetic acid, 1.0; proprionic acid, 0.85; butyric acid, 0.80). The respiratory quotient of an organism may be calculated with almost any of the manometric methods described above, if separate measure- ments are made with and without a CO,-absorbing alkali in the respira- tory chamber. In this case, one set of readings (with KOH) will give a measure of the O, consumed, and the other set (without KOH) will be an index of the difference between CO, given off and O, consumed. From this the R.Q. may be calculated, provided no NH, is evolved and no CO, is retained in the immersion fluid. If these complications arise, suitable modifications may be introduced. The use of somewhat more complicated manometer chambers allows measurements of simultaneous O, consumption and CO, production to be made on the same material (Dixon, 1934). For the Protozoa this method seems preferable because, in addition to its usual advantages, it prevents the results from being affected by the possible secretion of ammonia (Specht, 1935) and other bases (e.g., sodium carbonate from oxidation of Na-acetate, Jahn, 1935a). However, even this method does not correct for the possible evolution of hydrogen or methane (cf. Trichomonas foetus, Andrews and von Brand, 1938). For the ciliates, several measurements of R.Q. have been made. Wa- chendortf (1912) reported R.Q. values of about 0.3 for Colpidium, but in view of later developments it seems as if this material should be re- examined with more modern methods. Emerson (1929) studied the respiration of Blepharisma undulans and found an R.Q. slightly less than 1.0. Daniel (1931) obtained an R.Q. of 0.84 for Balantidium colt, but the possible effects of bacteria were not well controlled. Amberson 370 RESPIRATORY METABOLISM “ (1928) reported an R.Q. of 0.69 for Paramecium, and Root (1930) in a number of experiments obtained an R.Q. value of 0.62. Root also found that the R.Q. varied somewhat irregularly with changes in CO, tension. However, there was a definite trend toward high R.Q. values in media of high CO, tension, and the average R.Q. at 238-423 mm. Hg. was 1.43. This apparently was caused by a decrease in O, consump- tion (see above) without a corresponding decrease in CO, production, thereby giving a high R.Q. According to Root, “It is possible that the suppression of oxidations under these conditions results in the produc- tion of acid metabolites which drive out carbon dioxide from the bi- carbonate contained in the cells and in the surrounding medium.” Similar experiments on Arbacia eggs did not show an increase in R.Q., and it was assumed either that acid substances were not produced or that they were rapidly converted into a non-acid form and did not accumulate in appreciable amounts. Apparently CO, tension is a factor which should be considered when making measurements of R.Q. However, if ex- periments are conducted with standard manometric techniques, this factor is probably not important. Specht (1935) measured the R.Q. of Sprrostomum in manometers, both with and without the presence of acid in a side arm of the manom- eter flask. He found an R.Q. of 0.24 without the acid and 0.84 when acid was present. This discrepancy was explained as being caused by the elimination of NH, by the organisms, and the value of 0.84 is therefore accepted as more nearly correct. However, it was also demon- strated that the R.Q. was 0.98 in an atmosphere of O,, and that this value was not affected by the presence or absence of acid. Apparently NH, is not produced at high O, tension. These experiments indicate very clearly that ammonia secretion is a possible source of error in measurements of protozoan respiratory quotients, and therefore one should be suspicious of the validity of low R.Q. values unless adequate precautions have been taken against the ammonia error. For the free-living flagellates, very low values of R.Q. have been reported. Jay (1938) found R.Q. values of 0.34 and 0.56 for Astasia and Khawkinea, respectively. Mast, Pace, and Mast (1936) reported R.Q. values of 0.28 to 0.37 for Chilomonas, and 0.72 for Paramecium multimicronucleatum under similar conditions. The possible explana- tions mentioned by Jay for the low R.Q. value are conversion of pro- RESPIRATORY METABOLISM 371 tein to carbohydrate, or incomplete oxidation of carbohydrate. The ex- planation of Mast ef a/. is that carbohydrate was being synthesized from carbon dioxide. This explanation is based on previous nutritional studies, but these are open to question (review, Hall, 1939). One obvious possi- bility is that CO, may be retained in the immersion fluid, but Mast and his coworkers obtained only a slightly higher R.Q. value when the bound CO, was liberated by acid (single experiment only). In these cases the explanations offered must be considered as only tentative, until the possibilities of NH, production and CO, retention are positively eliminated. Mast and his coworkers also reported for Chilomonas that under certain conditions starch was converted to fat, and that fat oxida- tion could be decreased by depriving the organism of sulphur. However, values obtained for the R.Q. were variable and showed no definite cor- relation with these conditions. Values of the R.Q. reported for members of the family Trypano- somidae are within the normal range. Soule (1925) obtained an R.Q. of 0.84-0.91 for Lesshmania tropica and 0.74-0.89 for Trypanosama Jewisi in blood agar medium. When glucose was present, the R.Q. rose to 0.95 for L. tropica and 0.94 for T. lew7s7. Novy (1932) reported res- piratory quotients of 0.93 to 1.0 for T. Jew7s7, L. tropica, L. donovani, L. infantum, Strigomonas oncopelti, S. culicidarum, S. culicidarum var. ano phelis, S. lygaeorium, S. media, S. muscidarum, and S. parva, when grown on glucose-blood agar. When grown on glycerol-blood agar or plain blood agar, the R.Q. was about 0.8 to 0.87 for the four species of Leishmania. Von Fenyvessy and Reiner (1924) found an R.Q. of 0.60 for Trypanosoma equiperdum in diluted blood. A. Lwoff (1933) ob- tained R.Q. values of 1.0 for Strigomonas oncopelti and S. fasciculata, and a value of 0.88 for Leptomonas ctenoce phali. Apparently the only R.Q. measurement on a rhizopod is that of Emerson (1929) on Amoeba proteus, which gave a value slightly less than 1.0. Another method, in addition to that of the respiratory quotient, which might be used as an index of the source of energy in an organism is the calorific quotient. Since the ratio of heat produced to oxygen con- sumed differs with carbohydrate, fat, and protein (3.5, 3.3, and 3.2, respectively), it is possible to measure heat production and O, con- sumption and to use this ratio as an index of the substrate being utilized. S72 RESPIRATORY METABOLISM However, because of the small differences in the ratios and because of the complications of the technique of heat measurement, the calorific quotient has not been found to be very useful in determining the energy source for Metazoa (Needham, 1931), and apparently has not been tried for Protozoa. INVESTIGATIONS WHICH CONCERN THE MECHANISM OF RESPIRATION 1. GENERAL THEORY For a general consideration of the mechanism of respiration, the reader is referred to the monographs of Meldrum (1934) and Holmes (1937), to standard textbooks of general physiology, to several excel- lent discussions in recent volumes of the Annual Review of Biochemistry, and to the forthcoming volume of the Cold Spring Harbor Symposia in Quantitative Biology (Vol. VII). The present discussion of the mecha- nism of respiration will include only those portions of a bare outline which are necessary for an understanding of the data and interpretations which are to follow. The first step in oxidation of a substrate is the removal of hydrogen from the substrate molecule, and the addition of this hydrogen to any other molecule which will serve as a hydrogen acceptor. After de- hydrogenation is accomplished, the resulting molecule is supposed to be very unstable and easily undergoes oxidation by molecular oxygen, to form CO, and water. The enzymes necessary for these final stages in respiration are not well known, but the enzymes and respiratory pig- ments responsible for dehydrogenation and the subsequent transfer of the hydrogen to O, with formation of water are listed below. (Any distinction between respiratory enzymes and pigments is purely arbi- trary. ) (1) Dehydrogenases are enzymes which bring about activation of the substrate, so that it may be oxidized by oxygen or intermediate hydrogen acceptors such as cytochrome. These enzymes are highly specific, in that they react with only one or a few substrates. Dehydrogenases are divided into two groups: anaérobic dehydrogenases, which cannot reduce molecu- lar O, in the presence of their substrates, and aérobic dehydrogenases which can do so. Cytochrome and cytochrome oxidase are important factors in the completion of oxidation by anaérobic dehydrogenases. RESPIRATORY METABOLISM BS, (2) Cytochrome is a group of pigments or enzymes, which in the living cell are oxidized under aérobic, and reduced under anaérobic conditions, but which cannot be oxidized directly by molecular O,. These serve as hydrogen acceptors for anaérobic dehydrogenase systems. (3) Oxidase—The term oxidase includes all enzymes which are capable of performing oxidations in the presence of molecular oxygen. To this group belongs the respiratory enzyme of Warburg, which ts perhaps identical with the oxidase of cytochrome, which in turn is also referred to as indophenol oxidase because of one method of detect- ing its activity. This enzyme brings about the oxidation of reduced cytochrome by molecular oxygen, and water is supposed to be oxidized to hydrogen peroxide during the process. Aérobic dehydrogenases are sometimes classified as oxidases. (4) Catalase is an enzyme present in aérobic organisms and usually absent in anaérobes. This enzyme converts hydrogen peroxide to water and molecular oxygen, and its place in the respiratory chain is given below. (5) Peroxidases are enzymes which in the presence of an oxidizable substrate convert hydrogen peroxide to water and activated oxygen, thereby causing oxidation of the substrate. The exact rdle of peroxidases in cellular respiration is not understood. The peroxidases are iron com- pounds, and other iron compounds, such as cytochrome and methemo- globin, exhibit some peroxidase-like activity. (6) Yellow respiratory pigment, or enzyme, is a flavo-protein capable of reversible oxidation and reduction which may be reduced in a re- action involving oxidation of substrate (through the intermediary action of a co-enzyme) and which can then be reoxidized in the presence of molecular O, (with formation of H,O,) or other hydrogen acceptors. (7) Glutathione is an amino-acid complex capable of reversible oxt- dation and reduction, and which may act as a hydrogen acceptor through the reduction of an -S-S- group to two -SH groups (cysteine to 2 cysteine) which are auto-oxidizable in the presence of molecular O,. The details of how these substances function in the living cell are subject to considerable controversy, but for our present purpose we may regard the general outline for the first four items listed above as follows: (1) substrate + 2 oxidized cytochrome ———== dehydrogenase oxidized substrate +- 2 reduced cytochrome 374 RESPIRATORY METABOLISM (2) 2 reduced cytochrome + O, == 2 oxidized cytochrome + H,O, oxidase (3) H,O, == H,O+ 4 O, catalase The substrate may be activated by “anaérobic’’ dehydrogenase, and it is then oxidized by cytochrome, the cytochrome itself being reduced in the process (equation 1). Cytochrome is, in turn, oxidized by an oxidase system which may be identical with Warburg’s respiratory en- zyme (equation 2). During this process H,O, is formed and is then broken down to water and molecular O, by catalase (equation 3). The oxidase and catalase systems are inhibited by the presence of HCN and H,S, and the oxidase system is also inhibited by CO. In the presence of any of these reagents, reaction (1) can proceed but not reactions (2) or (3). Therefore all of the cytochrome becomes reduced, and respiration by means of this mechanism is stopped. The dehydrogen- ase systems are inhibited by narcotics (e.g., the urethanes), by warm- ing and cooling, and these agents leave all of the cytochrome in the oxidized state. These two general methods of treatment, therefore, may be used as tools in studying the above respiratory mechanisms. There are also a€robic dehydrogenases which, in addition to activating the sub- strate, can react directly with molecular oxygen without the mediation of cytochrome and oxidase. Respiration which is brought about by this type of system is not supposed to be affected by HCN. It is possible to demonstrate that in some systems certain reversible oxidation-reduction indicators (e.g., methylene blue) can replace the cytochrome-cytochrome oxidase system, and that in this capacity the action of these indicators may or may not be affected by HCN and CO (e.g., grasshopper embryos, Bodine and Boell, 1937; Escherichia coli, Broh-Kahn and Mirsky, 1938). If HCN and methylene blue are added to such a respiratory system and inhibition does not occur, the ensuing reactions might be visualized as follows: oxidized substrate (4) substrate + methylene blue dehydrogenase + leuco-methylene blue (5) leuco-methylene blue + oxygen = methylene blue ++ H,O, Since catalase is inactivated by HCN, the hydrogen peroxide presumably RESPIRATORY METABOLISM 3715 accumulates and in E. co/7 cultures can be measured experimentally. In anaérobes which normally do not possess catalase, this mechanism might explain the bacteriostatic effect of oxidation-reduction indicators. It is also known that there are certain pigments of bacteria and yeasts (e.g., yellow enzyme, or pyocyanine) which are capable of bringing about a similar result, and other naturally occurring oxidation-reduction indicators have been described (echinochrome, hermidin, and pigments from B. violaceus and Chromodoris zebra) which apparently might func- tion in a similar fashion. The reactions involving yellow pigment and its coenzyme may be indicated as follows: (6) substrate + coenzyme oxidized substrate + reduced coenzyme (7) reduced coenzyme + yellow pigment = coenzyme +- leuco-yellow pigment (8) leuco-yellow pigment + oxygen = yellow pigment + H,O, (9) H,O, = H.0+ 40, | catalase dehydrogenase In this case only the action of catalase is prevented by HCN, and there- fore H,O, accumulates. In the absence of O, the leuco-yellow enzyme may be oxidized by other substances (e.g., by methylene blue). The yellow enzyme has been found to be a combination of protein and vitamin G, and it is believed that while this sort of system is present in a€robic organisms, it assumes its greatest importance in anaérobic species. In anaérobic organisms (yellow enzyme can be prepared from bottom beer yeast or lactic acid bacilli) we have, then, a respiratory system which is quite independent of cytochrome and Warburg’s oxidase, and which therefore is insensitive to HCN and CO. Perhaps when we say that the respiration of a given species is cyanide insensitive, we may be inferring that that species has a respiratory system more suited to anaérobic conditions (temporary or otherwise). Under anaérobic condi- tions the leuco-yellow pigment is probably oxidized by substances other than molecular oxygen, and H,O, is not formed. The known respiratory enzymes of bacteria are summarized by Frei (1935) and Stephenson (eae The relationships between the various respiratory enzyme systems 376 RESPIRATORY METABOLISM are not so well known nor so clear-cut as, for the sake of clarity and brevity, they have been made to appear in the above outline. Whenever we conclude, on the basis of the action of certain reagents on respiration, that one respiratory mechanism is very important and that another is not, we should do so only with certain mental reservations, and the conclusions should not be considered final, but merely indicative. 2. EXPERIMENTS WHICH CONCERN THE CYTOCHROME-CYTOCHROME OXIDASE SYSTEM OF HYDROGEN ACCEPTORS We have, through the action of HCN and CO on respiration, a tool for determining how much of the respiratory activity of a given organism is carried on by means of the cytochrome-respiratory enzyme system and how much is not. Respiration which is not cyanide and CO sensitive may be due to aérobic dehydrogenases, or to anaérobic dehydrogenases plus an enzyme of the yellow-pigment type or perhaps to the action of peroxidases. Such analyses have been made for several types of bio- logical material. It has been determined that respiration of some cells is extremely sensitive to HCN (e.g., yeast, B. col, most bacteria, and mammalian tissues), while that of others is quite resistant (Chlorella, Paramecium, Sarcina, Pneumococcus, B. acidophilus, Streptococcus, Staphylococcus); also, the same organism may differ in sensitivity at different periods during its life history (grasshopper eggs, Robbie, Boell, and Bodine, 1938). One technical precaution which should be ob- served in cyanide experiments is the use of a KOH-KCN absorbing fluid for CO, (van Heyningen, 1935). By the selection of the proper KOH-KCN mixture, the osmotic transfer of KCN through the air from the experimental material to the KOH solution can be prevented. This is apparently one possible source of error in all work on the effect of cyanide on protozoan respiration—that none of the authors has used balanced KOH-KCN solutions. Among the Protozoa the effect of cyanide has been studied on several ciliates and flagellates. It is quite well established that the respiratory mechanism of Paramecium is insensitive to cyanide (Lund, 1918b; Shoup and Boykin, 1931; Gerard and Hyman, 1931), and the work of Shoup and Boykin (1931) shows that the addition of iron salts does not increase respiration and that very little or no iron is present in Para- mecium. These results may be interpreted to mean that the cytochrome- RESPIRATORY METABOLISM Sigh respiratory enzyme system plays no part in the respiration of Paramecium. Petets(1929) obtained no inhibition with M/500 KCN on Colpidinm colpoda. The data of Pitts (1932) for C. campylum shows that less than 20 percent of the respiration is cyanide sensitive and that this depression is only temporary, and that while still in cyanide the respiration may rise to a rate which is as much as 25 percent above normal and drop again to 80 percent of the normal rate. M. Lwoff (1934) found that the respiration of Glaucoma piriformis in peptone solution was depressed as much as 80 percent during the first half hour, but that by the third hour the rate had returned to normal (M/1,000 KCN), or almost normal (15 percent below in M/450 KCN), and then decreased. In a weaker KCN solution (M/4,500) this latter decrease did not occur. The organisms were able to live twenty-four hours in M/450 KCN and eight days in M/1,000, but they did not divide. In M/2,000 and M/5,000 KCN multiplication of the organisms occurred slowly. In glucose-Ringer solution, M/450 KCN did not inhibit, but produced an acceleration of as much as 36 percent during the first half hour. The conclusion to be drawn from these data is that the ciliates, as far as we know, are relatively insensitive to the action of cyanide, and we might consider the temporary inhibitions produced in some cases as secondary effects rather than direct effects upon the respiratory mechanism (cf. another explanation mentioned below). It would be interesting to rein- vestigate the effect of KCN on some of these ciliates by the use of balanced KCN-KOH solutions. It seems possible that the data, especially in the case of the temporary effects, may be complicated by the loss of KCN from the experimental solution to the KOH solution. Among some of the flagellates, however, there seems to be quite a different respiratory mechanism. A. Lwoff (1933) found that M/3,000 KCN inhibited respiration of Str7gomonas oncopeltz 90 percent, of S. fasciculata 83 percent, and of Leptomonas ctenocephali 95 percent. With M/1,000 KCN, both species of Str7gomonas were inhibited 90 percent, and L. ctenocephali 95 percent. The latter species was extremely sensitive and was inhibited 92 percent by M/20,000. Growth was also decreased in those concentrations which inhibited respiration, and the organisms were killed only by much greater concentrations. M. Lwoft (1934) reported an inhibition of 90 percent for Polytoma uvella, and Jay (1938) an inhibition of 60-65 percent for Khawkinea and Astasia 378 RESPIRATORY METABOLISM at a concentration of M/100 KCN. Von Fenyvessy and Reiner (1928), however, reported no effect with 0.1 percent KCN (M/65) on either oxygen consumption or acid production of Trypanosoma equiperdum in glucose-bicarbonate-Ringer solution. These results demonstrate that the respiratory mechanisms of various Protozoa are probably not the same. The respiratory mechanisms of some Protozoa seem to resemble those of Ch/orella and Sarcina, while those of other species resemble the mechanisms of yeast and mammalian and other tissues. This question is one which should be studied carefully in a wide variety of organisms, and with a wide concentration range of cyanide solutions. The taxonomic position of the Protozoa should make such an investigation doubly interesting. It would also be of interest to know if the cyanide insensitivity of Paramecium 1s still main- tained in the presence of glucose and other substances, or if an apparent change in the respiratory mechanism is brought about by the presence of glucose. Emerson (1929) found that the respiration of Chlorella was cyanide sensitive only in the presence of glucose; Gerard (1931) found that glucose had no effect on the cyanide sensitivity of Sarcima, but M. Lwoff (1934) found that the respiration of Glaucoma was accelerated by HCN when glucose was absent. These divergent results should have a final explanation in terms of the respiratory or other metabolic mecha- nisms involved. An alternative theory to the supposed coexistence of cyanide sensitive and insensitive fractions in the normal cell is that all normal respiration is CN sensitive, and that in the presence of CN an entirely new respiratory mechanism is called into existence. This interpretation would indicate that among many bacteria, algae, and ciliates (but not among certain flagellates) there is a greater adaptability of the respiratory mechanism than among the more specialized cells of the Metazoa. Such generalizations are probably premature, but it does seem possible that aérobic protozoa which can live anaérobically for considerable periods of time might have a dual respiratory mechanism. It has been demonstrated for several biological materials that respira- tion sensitive to cyanide is also sensitive to CO (because of CO-inhibi- tion of cytochrome oxidase), and that respiration insensitive to cyanide is not depressed and may even be stimulated by CO (literature cited by Bodine and Boell, 1934). A. Lwoff (1933) showed that the KCN RESPIRATORY METABOLISM ous) sensitive fraction (90 percent) of S. fasciculata respiration was also sensitive to CO. In an 80/20 mixture of CO/O, inhibition was 61 percent, in 95/5 mixtures 85 percent, and in 98/2 mixtures 90 percent. Values of K for the Warburg-Negelein equation A CO == anes where A is O, consumption in the CO/O, mixture and A, is O, con- sumption in the control, varied from 2.58 in 80 percent CO to 5.3 in 98 percent CO. M. Lwoff found that carbon monoxide (2-5 percent O, in CO) produced the same effect on Glaucoma as KCN in both peptone and in glucose-Ringer solutions. In peptone there was a marked inhibi- tion for the first half hour and then a return to normal or almost normal, and in glucose-Ringer there was an increase of 20 percent. Reidmuller (1936) reported no appreciable effect of CO on O, consumption for Trichomonas foetus in 95/5 mixtures of CO and O,. The effect of KCN was not investigated. The effect of CO on other Protozoa should be in- vestigated together with the effect of cyanide. Recently azide (HN;) has been found to have an effect on respiration which is similar to but not identical with that of HCN and CO (Keilin and Hartree, 1936; and others), and it would be interesting to make comparisons of these re- agents on protozoan material. For the purpose of inhibiting the cytochrome-cytochrome oxidase sys- tem, CO is apparently much more specific then HCN or azide. This is especially true if inhibition accurs in the dark but not in the presence of bright light, because the inactive compound formed by CO and cytochrome oxidase is dissociated upon illumination into CO and active oxidase. The reversibility upon illumination of CO inhibition has not been investigated for Protozoa. K The distribution of cytochrome among the Protozoa is a relatively untouched subject. A. Lwoff (1933) found two absorption bands in Strigomonas fasciculata, one at 530, and another rather broad band at 555 my. These bands disappeared upon passage of O, through the solu- tion. Upon addition of KCN no other bands became visible, and the question arises as to whether the 555 band was the b and c bands of cyto- chrome or the b band of the hemochromogen, as has been found for various bacteria. Lwoff found the 555 band also in S$. oncopelti, Glau- 380 RESPIRATORY METABOLISM coma piriformis, and Exglena gracilis. By treating G. piriformis and Polytoma uvella with sodium hydrosulphite and pyridine, he obtained the bands of pyridine-hemochromogen. The observations on Glaucoma are especially interesting because respiration is KCN and CO insensitive, and the explanation of the KCN and CO experiments therefore needs further clarification. Perhaps these organisms contain both cytochrome and a KCN insensitive system (e.g., yellow pigment) which may func- tion interchangeably. This would explain their adaptability to both aérobic and anaérobic conditions, the presence of cytochrome and KCN and CO insensitivity, and perhaps also the somewhat oscillatory character of Colpidium and Glaucoma respiration in the presence of KCN (Pitts, 1932; M. Lwoff, 1934). This, of course, is pure speculation. However, the possibility of any discrepancies in the supposed parallelism between CN, CO, and HN, insensitivity and the absence of cytochrome should warrant an intensive investigation. Reidmuller (1936) was unable by spectroscopic methods to find either cytochrome or hemochromogen in Trichomonas foetus, and this result should be expected because of the CO-insensitivity of Tr7chomonas respiration. 3. EXPERIMENTS WHICH CONCERN OTHER SYSTEMS OF HYDROGEN AC- CEPTORS If we assume that the Warburg-Keilin system is not present in the ciliates, then we must seek another respiratory mechanism. Is this to be found in the action of glutathione? According to M. Lwoff (1934), the effect of arsenious acid on respiration offers a tool for detecting the action of glutathione because it is not supposed to affect the Warburg-Keilin respiratory system and because it does combine with -SH groups, thereby inhibiting the normal functioning of glutathione. In Glaucoma piriformts M. Lwoff found that M/1,900 arsenious acid (neutralized sodium ar- senite) inhibited 75-80 percent of the respiration and that M/1,150 inhibited 90 percent. The organisms moved slowly in M/400 to M/2,000 and remained alive more than thirteen days in M/6,000, but did not multiply. The inhibition of respiration was entirely reversible (recovery in 1 1/2-2 hours from M/2,000). Monoiodoacetic acid, at least in some cases, is supposed to be similar in its action to arsenious acid, that is, it combines with -SH. (In other cases its action may be different, e.g., in the prevention of lactic-acid production from glycogen in muscle extracts RESPIRATORY METABOLISM 381 in which glutathione is not present.) Therefore we may use it as an ad- ditional indicator in detecting the action of glutathione in respiration. M. Lwoff found that monoiodoacetic acid produced 61-82 percent in- hibition of respiration of Glaucoma in concentrations of one part in 121,000 to 77,000, while the ciliates appeared normal and moved slowly. These results are interpreted to indicate that glutathione seems to be quite important in the respiration of Glaucoma. Another interpretation which has been used for work on other material (Korr, 1935; Cohen and Gerard, 1937) is that arsenites inhibit dehydrogenases (Szent-Gyorgyi and Banga, 1933). It is interesting that the accepted dehydrogenase in- hibitors (urethanes, see below) do not result in as great an inhibition with Glaucoma as atsenites and monoiodoacetic acid. Another respiratory mechanism which might exist among the cyanide- insensitive Protozoa is the yellow pigment found in yeast and other an- aérobic organisms. It seems as if an investigation of the distribution of enzymes of this type should be made among the Protozoa, especially with those species in which respiration proves to be cyanide insensitive. Since a large number of Protozoa are presumably facultative anaérobes, it might be possible to poison the normal aérobic mechanism and study the anaérobic mechanisms under various conditions, as has been done for Escherichia coli by Broh-Kahn and Mirsky (1938). 4. INHIBITION OF THE DEHYDROGENASE SYSTEM The dehydrogenases are apparently a part of the respiratory chain in- volved in several of the enzyme mechanisms. Therefore one would ex- pect any substance which inhibits the dehydrogenases to inhibit respiration. Such is the case with the urethanes. M. Lwoff (1934) found that in the respiration of Glaucoma one percent methyl urethane pro- duced 9 percent inhibition of respiration, 2 percent inhibited 38 percent, 2.5 percent inhibited 52 percent, and 3.5 percent inhibited 55-63 percent. Ethylurethane in concentration of 1.66 percent inhibited 44 percent, and 2 percent inhibited 57-61 percent. Propylurethane in 0.5 percent solu- tion produced an inhibition of 47 percent. The ciliates appeared normal, movement was slow, but the effect on respiration was reversible. There- fore we may conclude that dehydrogenase systems are probably involved in the respiration of Glaucoma. It might be interesting to try the com- bined effects of urethane and arsenious acid, in order to obtain evidence 382 RESPIRATORY METABOLISM for or against the idea that both dehydrogenase and glutathione are part of the same respiratory chain. 5. SYNTHESIS OF RESPIRATORY ENZYMES—VITAMINS After we have determined that a certain organism has a certain type of respiratory system, the question arises of how the enzymes are formed. Is the organism capable of synthesizing them from relatively simple compounds, or must certain prosthetic groups be present in the nutritive substrate? For certain flagellates this question has been answered very definitely by A. Lwoff (1933). Strigomonas oncopelti, S. fasciculata, and Le ptomonas ctenocephali have respiratory systems which are 90 percent dependent upon cytochrome (as demonstrated above with KCN and CO). It was found that S. oncope/ti could live indefinitely in peptone solutions without the addition of hematin compounds. For the other two flagellates, hematin compounds were found to be necessary. L. ctenocephali would not grow unless rabbit blood (or an equivalent amount of hematin) were present in concentrations of one part to 1,200. S. fasciculata showed growth in blood dilutions as great as 1/1,000,000, and within limits the amount of growth was directly proportional to the amount of blood. Hemoglobin disappeared rapidly from the culture medium, and it apparently was being used to form more respiratory enzyme. When small amounts of blood were added, the Qo, increased linearly for several hours, until apparently all of the hematin was con- vetted into respiratory enzyme; then the Qo, remained constant. This constant level varied with the amount added. It was found that for each gamma of blood (between one and 5 gamma) added to 1 mgm. of flagellate (dry weight) the Qo, was increased about 3 units. After 8.5 hours 5 gamma of blood raised the Qo, from 19.5 to 37.0. Blood could be replaced with hematin, prohemin, and protoporphyrin, but not by cyto- chrome C nor by a wide variety of synthetic hematin and porphyrin com- pounds, chlorophyll, peroxidase, or active iron (Lwoff, 1938). Ap- parently only the porphyrin compound which contained the vinyl (-CH = CH.) radical (protoporphyrin) was effective. Deuteroporphyrin, which differs from protoporphyrin in having hydrogen in place of the vinyl groups, was not effective. More recent investigations of the chem- ical structure of cytochrome indicate that the vinyl groups may be neces- sary for linking the prosthetic group (iron porphyrin) to the protein RESPIRATORY METABOLISM 383 portion of the cytochrome molecule through a pait of sulphur atoma. Lwoff calculated that each flagellate required 520,000 molecules of pro- toporhyrin in order to bring the Qo, to 55, and that each organism must contain about 700,000 molecules of protoporphyrin before division would take place. Although cytochrome C was ineffective alone, the action of proto- porphyrin was increased when cytochrome was present (A. Lwoff, 1936). A. Lwoff (1933) found that the organisms had absorption bands at 555 and 530 mn, and this indicated the presence of cytochrome. There- fore we may conclude that protoporphyrin is necessary for the building of (1) cytochrome or (2) cytochrome oxidase. Since cytochrome C can- not be substituted for protoporphyrin, it seems as if the reaction proto- porphyrin > porphyrin C is irreversible and that protoporphyrin is neces- sary for the synthesis of something other than cytochtrome—probably cytochrome oxidase. On the assumption that all of the iron is used to build the respiratory enzyme, we may calculate the rate of catalysis: one gramatom of iron at 28° carries 4.83 grammolecules of O, per second. For yeast, Warburg obtained a value of 100. Therefore, on the basis of these assumptions, it seems as if the respiratory enzyme of yeast is 20.8 times as active as that of S/rigomonas. It has been demonstrated by M. Lwoft (review, A. Lwoff, 1938) that hematin is necessary for the growth of S. muscidarum, S. culicidarum vat. ano phelis, L. tropica, La donovani, L. agamae, L. ceramodactyli, and Schizotrypanum cruzi, as well as for the organisms discussed above. The mechanism of its action has not been intensively studied, but presumably it may serve a purpose similar to that it serves in S. fascculata. We may conclude from the above experiments that protoporphyrin 1s necessary for the normal metabolism and growth of Strigomonas fascicu- Jata and that the organism is not capable of its synthesis. Therefore proto- porphyrin may be considered a vitamin. Comparable examples are known for other organisms: e.g., cholesterol for Trichomonas columbae, T. foetus, and Eutrichomastix coluborum; aneurine (vitamin B,) for Glaucoma pivriformis, S. oncopelti, S. fasciculata, S. culicidarum, cet- tain bacteria, and fungi; pyrimidine and thiazol (parts of the aneurine molecule) for Polytomella caeca and Chilomonas paramecium; ascorbic acid for Schizotrypanum cruzi; and lactoflavine, nicotinic acid, and phospho-pyridine-nucleotides for various bacteria. In all cases where the 384 RESPIRATORY METABOLISM function of these essential compounds is known or indicated, they seem to be necessary for the formation or at least for the normal functioning of the respiratory enzymes (review, A. Lwoff, 1938). Recent work on the supposed function of most of these substances has been reviewed by Burk (1937) and Stern (1938). 6. THE DETECTION OF OXIDASE, PEROXIDASE, AND CATALASE No extensive investigation of isolated enzyme systems of the Protozoa has been made. Certain enzymes, especially oxidase and peroxidase, are supposed to react with certain stains so that the position of the enzyme may be located in the cell. The methods used involve the Nadi, Dopa, benzidine-H,O,, and pyronine-gnapthol-H,O, reactions (methods given by Roskin and Levinsohn, 1926; Guyer, 1936; McClung, 1937). These reactions are important in studying vertebrate blood and nerve cells, but apparently no correlation has been made of the presence of “oxidase” or “peroxidase” granules, detectable by these methods, and the respiratory mechanisms discussed above, and it has not been demon- strated that these reactions are specific for oxidase or peroxidase. Several observations have been made on the Protozoa (Roskin and Levinsohn, 1926; Bles, 1929), and when more is known about the respiratory mechanisms of the Protozoa it might be possible to correlate the results of staining and of manometric methods. However, such attempts are omitted in the present discussion. Perhaps a certain degree of localization of the enzymes could be obtained by centrifuging the organisms and mak- ing activity tests on cell fragments, as has been done for peptidase in marine ova (Holter, 1936). The presence of catalase can be detected by adding hydrogen peroxide to a cell suspension and measuring (chemically or manometrically) the oxygen evolved. Burge (1924) studied the catalase action of Paramecium and Colpoda and found that it was decreased by ether and chloroform, but not by ethylene or nitrous oxide. A much more accurate method was used by Holter and Doyle (1938), who found that the average catalase activity per individual of Frontonia, Paramecium, and Amoeba was in the ratio 190:30:5. Considerable variation was found between different cultures and even between different individuals from the same culture. Reidmuller (1936) reported only a trace of catalase, no peroxidase, and no indophenol oxidase for Trichomonas foetus. RESPIRATORY METABOLISM 385 THE MEASUREMENT OF ANAEROBIC METABOLISM AND GLYCOLYSIS The measurement of anaérobic metabolism is somewhat more complex than the measurement of aérobic. The standard criterion of O, con- sumption does not exist, and the auxiliary criterion of CO, production indicates only the carbon which is completely oxidized. Sometimes this may comprise only a small percentage of the total metabolic changes, and in some cases measurements may be complicated by the presence of hydrogen, methane, and other gases. Therefore we must usually attempt to trace anaérobic metabolism by measuring changes in the concentration of several substances in the liquid phase, instead of one or two substances in the gaseous phase; and this is more difficult. In some cases it is possible to give an organism a known substrate, for example, carbohydrate, and to measure the decrease in the quantity of the substrate and the increase in the amount of decomposition products at various intervals. From carbohydrate decomposition these may be alcohols, aldehydes, and or- ganic acids. From protein decomposition we might expect a wide variety of nitrogen-containing amino-acid fragments, and by deaminization of amino acids a wide variety of organic acids may be produced. From de- composition of lipoids we may expect products somewhat similar to those from carbohydrates. Methods for the final identification of these compounds usually take one into the field of microanalytical biochemistry (see Peters and van Slyke, 1932; Friedemann, 1938; and publications by von Brand, Reiner, and others, cited below). The identification of the acid formed is important in any study of the energetics of anaérobic carbohydrate metabolism, because the processes which yield the various acids release quite different amounts of energy. For many purposes, however, it is customary, if not adequate, to measure acid production by manometric measurement of the amount of CO, which is released from a bicarbonate buffering system, as CO, or stronger acids are produced by the organism. This gives an index of acid production, but leaves us ignorant of the nature of the acid. Changes in total titratable acidity or alkalinity are also used, and it seems as if, under certain conditions, accurate titration curves might be obtained which would give a fair index of the kind and amount of acids present. In metazoan metabolism it is usually assumed that oxidation of glu- cose is preceded by a molecular rearrangement which results in the formation of lactic acid. 386 RESPIRATORY METABOLISM Glucose = 2 lactic acid + 43,000 cal. (A H) 2 lactic acid 4. 60, > 6CO, + 6H,O + 634,000 cal. (A H) The first reaction is referred to as glycolysis, or cleavage. Glycolysis is reversible, and it occurs under both aérobic and anaérobic conditions, but the rate of the reverse reaction (lactic acid + glucose) is very much less under anaérobic than under a€robic conditions. Consequently, in some tissues (or in the tissue medium) lactic acid may accumulate aérobically, but usually it accumulates only during anaérobiosis. If lactic acid does tend to accumulate, it can be measured by allowing it to displace CO, from a bicarbonate immersion medium (usually glucose-bicarbonate- Ringer). If it is assumed that the CO, given off by oxidation is equal to the O, consumed, then the amount of lactic acid can be calculated as the “excess CO,,” 1.e., the CO, evolved in addition to that released by oxidation. This is expressed as QO for the unit one cmm. of CO, per mg. dry weight of tissue per hour. (Older authors use Q2, which is easily confused with Q¢o,, the respiratory CO,, and recent German au- thors use QO: for the same quantity.) If O, is replaced by N,, all of the CO, evolved must come from glycolysis, and the unit is expressed as Qe (or QR, or QX’). For comparative studies on various organisms, it has been found to be useful to calculate the Meyerhof quotient (M.Q.), which is defined as Qe Qo. This is an index of the amount of lactic acid reconverted to glucose per unit of oxygen consumption, 1.e., a measure of the resynthesis of glucose. Recent work on the interpretation of Meyerhof quotients is reviewed by Burk (1937). Recent investigations of glycolysis in vertebrate tissue indicate that glucose is converted into pyruvic acid, part of which 1s oxidized and part of which is resynthesized into glucose, and that lactic acid is a step in the resynthesis, rather than the end product of glycolysis. It seems as if this may also be true for Protozoa. OCCURRENCE OF ANAEROBIOSIS AND GLYCOLYSIS Many Protozoa live in media which are almost if not entirely devoid of oxygen. Examples are those which inhabit the bottom of stagnant ponds RESPIRATORY METABOLISM 387 (especially if a considerable amount of decaying organic matter, and consequently hydrogen sulphide, is present), those which are found in sewage-disposal plants, those which appear near the bottom of putrid laboratory cultures, and those which inhabit the lumen of the lower in- testine of Metazoa. These organisms, because of the characteristics of their environment, are deprived of one of the chief sources of energy available to other animals—the reduction of molecular oxygen, and they must be able to obtain energy by other methods, such as molecular re- arrangements (e.g., glucose to lactic acid) or oxido-reductions (€.g., glucose to CO, and alcohol). A summary of the early theories of an- aérobic fermentations is given by Slater (1928), and a review of the data pertaining to anaérobic life of Protozoa and other invertebrates 1s given by von Brand (1934). Some of the anaérobic Protozoa seem to be obligatory anaérobes and are quickly killed by aération (e.g., Tre pomonas agilis, Lackey, 1932). Therefore one might expect them to have a type of metabolism comparable to those of the anaérobic bacteria. Other organisms, such as certain intestinal forms, are certainly not strict anaérobes, but are facultative, or amphibiotic. Measurements of the intestinal gases (reviewed by von Brand and Jahn, 1940) and of the oxt- dation-reduction potential of the digestive tract (Jahn, 1933a) indicate that the lumen of the intestinal tract is largely devoid of oxygen. How- ever, organisms which live at the surface of the epithelium (e.g., Gzar- dia) and within the villi, and especially those such as Endamoeba histo- lytica and Balantidium coli which invade the tissue, do have access to molecular oxygen. The O, tension of the environment of the rumen in- fusoria of ruminants must be extremely variable, but, for considerable pe- riods of time, almost devoid of oxygen. The question then arises as to what kind or kinds of respiratory mechanisms are present in these faculta- tive organisms. The same question arises with such organisms as Para- mecium, Which are normally aérobic but can withstand lack of oxygen for a relatively long period of time. Do the facultative anaérobes of the phylum Protozoa have respiratory mechanisms comparable to those of bacterial facultative anaérobes? This question, although interesting and suggestive, is unanswerable at present because we know nothing about the respiratory mechanisms of anaérobic Protozoa, and not very much about those of bacteria. However, recent investigations indicate that among bacteria the respiratory mechanism of the strict anaérobes is 388 RESPIRATORY METABOLISM probably different from the anaérobic mechanism of the facultative an- aérobes (Broh-Kahn and Mirsky, 1938). There is considerable evidence that carbohydrate decomposition takes place in Protozoa under anaérobic conditions. It was found by Piitter (1905) that the glycogen content of Paramecium decreased under an- aérobic conditions. He also found that Paramecium poor in glycogen could live anaérobically for a considerable length of time, probably at the expense of albumen. A. Lwoff (1932) found that Glaucoma piri- formis could live three days without oxygen only if sugar were present. M. Lwoff (1934) obtained a value of 10 for the QN: of G. pirtformis in peptone broth (Qo, == 35). Emerson (1929) found that under an- aérobic conditions 80 mm* of Blepharisma released 12.5 mm.* CO, per hour from a bicarbonate buffer mixture; negative results were obtained with Amoeba proteus. Zhinkin (1930) demonstrated that the glycogen content of Stentor decreased under anaérobic conditions and that visible fat increased. Upon exposure to O, that fat disappeared. This apparent conversion of glycogen to fat was observed in experimental cultures and under natural conditions in winter when the O, content of ponds was negligible, but it did not occur in experimental cultures in the presence of light because of the photosynthetic action of zoéchlorellae. Some data of this type was also obtained for Prorodon teres and Loxodes (Zhinkin, 1930) and for Paramecium (Pacinotti, 1914). The possible changes which take place in the glycogen content of intestinal amoebae and ciliates should also be investigated in this connection (see discussion by von Brand, 1934). The trypanosomes are a group of organisms which live under con- ditions of high O, tension, but they apparently have a high degree of anaérobic metabolism (glycolysis). At least, they use much more sugar than they could possibly oxidize with the O, which they consume, and apparently the amount of acid produced by glucose destruction does not differ much under aérobic or anaérobic conditions. According to the data of von Fenyvessy and Reiner (1924), the O, consumption for a billion trypanosomes (T. equiperdum) suspended in diluted blood was about 0.07 mg. per hour. The sugar consumed under similar conditions (Yorke, Adams, and Murgatroyd, 1929) was about 5 mg. Since complete oxida- tion of 5 mg. of sugar requires about 5 mg. of O,, it appears as if the major portion. of the sugar destruction was anaérobic. This is discussed RESPIRATORY METABOLISM 389 by von Brand (1934, 1935). On the basis of an assumed R.Q. of one, von Fenyvessy and Reiner (1928) subtracted the Qo, from the rate of CO, evolution from a bicarbonate solution and found that the resulting value was equal to the QN:. Therefore the rate of glycolysis (acid pro- duction) is the same in O, or N, and is independent of O, consumption. The data of von Fenyvessy and Reiner showed that the amount of CO, evolved when the organisms were in bicarbonate-glucose-Ringer was so high under aérobic conditions that apparent R.Q. values of 1.7 to 3.6 were obtained. Therefore we must conclude that glucose —> acid conver- sion is very high in T. eguiperdum. According to the experiments of von Brand, Regendanz, and Weise (1932), this acid production is appar- ently not a true glycolysis, because lactic acid could not be detected in the medium by chemical methods. However, if we consider lactic acid to be a step in the resynthesis of pyruvic acid to glucose (see above), the ab- sence of lactic acid may merely mean that resynthesis does not occur. In this case the Meyerhof quotient should be zero. It was shown by Reiner and Smythe (1934) and Reiner, Smythe, and Pedlow (1936) that aérobic sugar destruction by T’. equiperdum was as follows: 1 glucose — 1 glycerol 4 1 pyruvic acid 1 glycerol + O, — 1 pyruvic acid +. 2H,O Apparently lactic acid and CO, were not produced. For T. Jew/s7, under aérobic conditions, the end products were identified as succinic, acetic, and formic acids, ethyl alcohol, and carbon dioxide. It has been suggested that the large amount of acid produced by trypanosomes might be the mechanism by which toxic effects are pro- duced. This possibility was investigated for T. evans by Kligler, Geiger, and Comaroff (1929), who analyzed the blood of infected rats and concluded that death was caused by lactic acid acidosis. In subsequent publications (Kligler, Geiger, and Comaroff, 1930; Geiger, Kligler, and Comaroff, 1930) they reported the measurement of glycolysis of T. evansi, having obtained even higher values (9.2 mgm./billion/hour) than Yorke, Adams, and Murgatroyd had obtained for T. equiperdum. Von Brand, Regendanz, and Weise (1932) measured the glucose, lactic acid, and alkali reserve of animals infected with T. gambiense, T. brucei, and T’. equiperdum, and found no evidence of low glucose, high lactic acid, or low alkali reserves, and therefore no support for the acidosis 390 RESPIRATORY METABOLISM theory of death. Von Brand (1933) measured the rate of sugar destruc- tion for various trypanosomes, and obtained high values (8.0 mgm. /bil- lion/hour) for the pathogenic trypanosomes T. bruce7, T. gambiense, T. rhodesiense, and T. congolense, and very low values for the nonpatho- genic T. /ew7s7 (about 1.4), and still lower values for the pathogenic Schizotrypanum cruzi. The results of these investigators indicate that although sugar destruction and formation of acid may be a contributing factor, it will not explain all of the pathological effects of the trypano- somes. This is reviewed by von Brand (1938). It is interesting to compare the high aérobic glycolysis rate of trypano- somes with that of malignant tumors. The Warburg quotient (aérobic glycolysis/Qo,) for normal tissues is usually less than 0.3 (except retina and placenta), while that for benign tumors is about one, and that for malignant tumors is 3.1-3.9 (review, Needham, 1931). The Warburg quotients calculated from the data of von Fenyvessy and Reiner (1928) for T. eguiperdum are 0.78 to 2.67. It has been found that KCN may change the Warburg quotient of chick embryos from 0.1 to 3.4, but the quotient for T. equiperdum, in the presence of KCN, showed no significant change (0.78 and 0.80 in two experiments). These compari- sons may be taken to indicate that the relative glycolytic rate of the trypanosomes 1s different from that of normal tissues and resembles in certain respects that of benign or malignant tumors, but the fact that pyruvic and other acids are formed by trypanosomes instead of lactic acid invalidates this comparison. Meyerhof quotients were calculated by A. Lwoff (1933) for S¢r7- gomonas fasciculata, S. oncopelti, and Leptomonas ctenocephali and were found to be 1.20, 1.38, and 0.125 respectively. The first two are within the normal range of metazoan tissues (Needham, 1931), but that of Leptomonas is very low. Values of the M.Q. calculated from the data of von Fenyvessy and Reiner (1928) on trypanosomes are approxi- mately zero, indicating no reversal of glycolysis, and this conclusion agrees with the chemical equations given above. WuHy ARE ANAEROBES ANAEROBES, AND AEROBES AEROBES? One question which arises in any treatment of anaérobiosis is, “Why does oxygen prevent growth of obligatory anaérobes?”’ There are several explanatory theories: RESPIRATORY METABOLISM 59 1. Oxygen is directly lethal to the cell. 2. Anaérobes do not contain catalase and therefore are incapable of destroying the toxic H,O, which is formed by reduction of oxygen (see equations given above). 3. Growth of anaérobes is dependent upon the presence of a low oxidation-reduction potential in the medium, the attainment of which is prevented by oxygen. 4. O, forms a loose chemical complex with the respiratory system of obligatory anaérobes, and thereby inhibits its activity. The relative merits of these theories, as applied to bacteria, are dis- cussed by Hewitt (1936) and Broh-Kahn and Mirsky (1938). The first theory is certainly not true for those anaérobic organisms which will grow under anaérobic conditions after exposure to oxygen. The second theory is supported by considerable evidence, in that most anaérobes do not contain catalase, and in that some bacteria (e.g., pneumococci) will grow aérobically until they are killed by the accumulation of H,O, re- sulting from their metabolism (so-called “suicide” of cultures). How- ever, some anaérobes do contain catalase, and apparently it has not been definitely demonstrated that strict anaérobes consume O, in order to produce H,O,, or even that obligatory anaérobes do produce H,O, (Broh- Kahn and Mirsky). The theory, however, might still be applicable to organisms such as penumococci and hemolytic streptococci, and to Escherichia coli in the presence of HCN and methylene blue. In these cases appreciable amounts of H,O, can be detected. Among the Protozoa we have very little evidence of the relative merits of the first two of these theories. It is shown by the work of Cleveland on termites and on xylophagous cockroaches (Cleveland, Hall, Sanders, and Collier, 1934, include citations of earlier papers) that the symbiotic Protozoa which inhabit the digestive tracts of these organisms are prob- ably strict anaérobes. At least the O, tension of their normal environment is extremely low, and they are rapidly killed by appreciable quantities of molecular O,. Cleveland found that at 23° C. the time necessary for death of the symbionts of termites was an inverse function of oxygen tension (e.g., in Termopsis, all Protozoa were dead in 72 hours at one atmosphere, in 30 minutes at 3.5 atmospheres). This is apparently due to an increase in O, concentration in the digestive tract, with increased O, pressure in the atmosphere, and this could easily be explained on the 392 RESPIRATORY METABOLISM basis of either of the above two theories. Additional evidence, however, might be gained for the second theory from the fact that one atmosphere of O, is more toxic at low temperatures (4-5° C.) than at high (23- 25° C.), and that four atmospheres of O, are more toxic at high tempera- tures than at low. The greater solubility of O, at 4-5° C. can account for the greater toxic effect with one atmosphere pressure. However, the re- verse effect at four atmospheres O, pressure must, as suggested by Cleve- land, be connected in some manner with metabolic processes. Super- ficially, at least, these results seem to be explicable on the basis of the second of the above theories, i.e., the organisms grew more rapidly and produced more H,O, at the higher temperatures. An examination of the protozoa for catalase, or of the digestive contents of oxygenated insects for H,O,, might yield pertinent information. The data of Cleveland (1925) on the toxicity of oxygen for the in- testinal Protozoa of earthworms, salamanders, frogs, and goldfish, are possibly open to this explanation, but here also we lack experimental evidence. Where such defaunation procedures failed, as in the rat, we might assume that the O, tension of the digestive tract was not raised in a manner comparable with that which occurred in the smaller or- ganisms, or that the Protozoa present were more resistant. The former theory seems much more probable. (For review of the chemistry of the intestinal contents, see von Brand and Jahn, 1940.) The theory that the growth of anaérobes is dependent upon a low oxidation-reduction potential in the medium was proposed by Quastel and Stephenson (1926), and has gained considerable support among bacteriologists (review, Hewitt, 1936) and some dissent (literature cited by Broh-Kahn and Mirsky, 1938). Positive evidence consists mainly of the facts that (1) during the growth phases of anaérobic cultures, especially the sporulating anaérobes, much lower oxidation-reduction potentials are produced than during the growth phases of cultures of aérobes; and (2) anaérobic forms do not start growing until the potential is quite low (E, < + 100 mv.). The first type of evidence does not help to distinguish between cause and effect, and there are some exceptions to the general trend. Most of these, however, are due to the fact that the organism is only one factor which tends to determine the E, of the medium; the chemical composition of the medium certainly determines, to a great extent, what potentials may be attained. The second RESPIRATORY METABOLISM 393 type of evidence is well founded in fact—anaérobes do not grow in media of high E,, value. However, if the E, value of a suitable medium is low- ered through displacement of air with H, or N,, or by various chemical reagents, or by the growth of an aérobic organism, then the ana€robic forms are capable of growth. According to this theory, anaérobes and aérobes differ in their ability to grow at various points along the E, scale, in a manner comparable to that which is exhibited by various acido- philic and basiphilic forms in growing at various points along the pH scale. Of course, there are intermediate-range and wide-range forms in respect to both pH and E,. The fact that the toxic effects of lack of O,, or of supernormal O, tensions, are not equal in all species supports this idea, but these data, of course, are subject to other interpretations. Investigations of the rdle of oxidation-reduction potentials among the Protozoa have never passed the preliminary stages. The possible im- portance of such a study was pointed out by Jahn (1933b, 1934), in connection with experiments on -SH compounds, on the toxic action of methylene blue and on possible relationships with auto- and allelocataly- sis. Measurements of the E, of Chilomonas cultures (Jahn, 1935b), of hay infusions (Efimoff, Nekrassow, and Efimoff, 1928), and of digestive contents (Jahn, 1933a) have been made. However, until more data be- come available, most of these results are difficult to interpret. It was de- termined that Chilomonas would grow in mixtures of NaSH and H,O, only if the concentrations of these were balanced so that the medium just failed to reduce methylene blue. This might indicate a microaéro- philic tendency for Chilomonas. Other experiments with Chilomonas indicated that it could live, but could grow only slowly, however, in media in which methylene blue was reduced. Neither of these ideas is contradictory to its known habits in laboratory cultures. It was also demonstrated (Jahn, 1935b) that casein-acetate broth cultures of C/v- lomonas, when exposed to the air, developed potentials of -20 mv. at pH 7.55, a point at which methylene blue is about half reduced. The chief difficulty in interpreting experiments pertaining to the effect of Ey on growth is that it is necessary to change O, tension in order to change E,,. This makes an experiment containing only one variable seemingly im- possible to execute, and the theory, therefore, has not been amenable to experimental approach. The fourth theory of the effect of O, on anaérobes—that of an inac- 394 RESPIRATORY METABOLISM tivation of the respiratory mechanism of obligatory anaérobes by O,— is mentioned by Broh-Kahn and Mirsky (1938), but at present is unsupported by experimental evidence. The bacteriostatic effect of dyes might also be interpreted to mean that these have an inactivating effect on the respiratory mechanism. In connection with the effect of oxygen on anaérobes, it should be mentioned that the converse problem also exists. Why do aérobes die in the absence of oxygen? On this question there has been considerably less discussion in the literature than on the former. It can be seen from the respiratory equations given above that activation of the substrate and partial oxidation can proceed without O,. Even in the presence of O, a considerable destruction of substrate, in some forms (e.g., trypano- somes, cited above), seems to be incomplete. Poisoning of the aérobic cell under anaérobic conditions is supposedly caused by the accumulation of toxic products of carbohydrate cleavage or of incomplete oxidation, or by the conversion of all of the respiratory pigment to the reduced state. In facultative anaérobes the former, and in obligatory aérobes the latter theory seems more probable. The observation of Fauré-Fremiet, Léon, Mayer, and Plantefol (1929) that Paramecium withstands lack of O, longer at 4° C. than at higher temperatures, is open to either inter- pretation. The data of Piitter (1905), which indicate that Paramecium can live longer under anaérobic conditions when the ratio of volume of medium to cells is higher, can be explained on the basis of accumulation of toxic products. What these toxic products are probably depends to a large extent upon the organism, the substrate, and the conditions of the experiment, and the most likely possibilities include lactic and lower fatty acids. OXIDATION-REDUCTION POTENTIAL VERSUS RESPIRATION AND GROWTH Another question which arises is why we might or might not expect the oxidation-reduction potential of the medium to affect the respiration and growth of the organism. It is obvious from the outline of the respir- atory processes given above, and from many other types of data, that oxidation-reduction phenomena are involved in respiration. The respira- tory pigments and perhaps also the respiratory enzymes are reversible oxidation-reduction systems. Therefore their action should be affected RESPIRATORY METABOLISM 395 by the potential of the medium in which they are found, that is, by the oxidation-reduction potential of protoplasm, and they, in turn, must to a large extent determine this potential. If we were to mix numerous half-reduced, completely reversible oxidation-reduction indicators in a homogeneous solution, an equilibrium would be reached, and the po- tential attained would depend upon the E, values of the substances and upon their relative amounts. Substances with E, values far from the result- ing E,, value of the solution would be either completely reduced or com- pletely oxidized, and would be unable to contribute much toward oxidizing or reducing small amounts of added materials unless the E, of the mixture were appreciably changed. Obviously such a simple system is not present in protoplasm. Because of the presence of irreversible systems and of the continual introduction of new substrate and the removal of certain end products, a true equi- librium is never attained. Also, the colloidal nature of protoplasm makes possible the existence of different E, values in different phases of the substance, and the differential adsorption of oxidized and reduced ma- terial at interfaces may produce a potential different from that in any of the phases. Therefore the term ‘‘oxidation-reduction potential of proto- plasm” may be without any interpretable significance (for discussion, see Jahn, 1934; Korr, 1938). But there must certainly be a significance to the Eo values of the respiratory pigments, and the possibility of an indi- vidual expression of these values may be maintained by the polyphasic nature of protoplasm. The oxidation-reduction potential, which can be measured with indicators, is probably an index of the potential developed by one or more of these pigments (for summary of such measurements, see Chambers, 1933; Cohen, 1933). It is known that the apparent E, value of protoplasm, as measured by indicators, varies with the E, of the external medium when the external O, tension is changed. Therefore, why cannot the E, of the external medium determine the degree of re- duction of the respiratory pigments and therefore the rate of respiration? This mechanism might be used to explain the inhibition which is pro- duced by oxidation-reduction indicators in cultures of bacteria (Dubos, 1929) and in cultures of Chilomonas (Jahn, 1933b). One difficulty in predicting what reactions would occur in protoplasm, even if we had a thorough knowledge of the oxidation-reduction systems involved, 1s the fact that such knowledge can tell us only what reactions might or might 396 RESPIRATORY METABOLISM not occur if all of the reactions were reversible. Since many of the re- acting substances are changed irreversibly and since the rates of reactions are dependent not only upon Eo values but upon enzymes, knowledge of E, and E, values cannot indicate what reactions will occur. Much of this discussion of oxidation-reduction potentials is pure speculation, but it is the type of speculation (often unexpressed) which has spurred investigators to a study of the naturally occurring oxidation- reduction systems, of the apparent oxidation-reduction potential of proto- plasm, and of the E, values developed in bacterial and protozoan cultures (reviews, Needham and Needham, 1927; Wurmser, 1932; Chambers, 1933; Clark, 1934; Hewitt, 1936). Interpretations of the data have not always been as fruitful as one might expect, and one is led at times to suspect that the modes, if not the points of attack on the problem, are in need of revision. However, since the necessity of some such relationship as Outlined above seems sound, it is more probable that merely the time for the harvest has not yet arrived. Clark (1934) estimated that another half century will be necessary for the solution of these problems. Another means by which the oxidation-reduction potential of the medium is supposed to affect metabolism is described by the surface catal- ysis theory suggested by Quastel (1930), Kluyver (1931), and others (discussed by Hewitt, 1936). It is suggested that many oxidative pro- cesses of bacteria take place at the surface of the cell (Quastel, 1930), and it seems as if for these reactions the E, of the medium would be more im- portant than that of the protoplasm. It is also very probable that oxida- tion-reduction enzymes are merely surface catalysts, which produce their effect by nature of intense interfacial electrical fields (Kluyver, 1931). These fields might be affected by the potential of the medium, whether they occur at the cell surface or within the protoplasm. An intriguing speculation would be that the respiratory pigments are distributed among the various phases of protoplasm, and that the enzymes are actually the interfaces of the emulsion. Unfortunately, such ideas are difficult to check experimentally. However, since oxidation-reduction enzymes are proteins which in all probability exert their catalytic properties through surface action, it is possible that the catalytic interfaces of the cells, dis- cussed by Kluyver (1931), are merely the surfaces of the protein mole- cules. RESPIRATORY METABOLISM 397 LITERATURE CITED Adolph, E. F. 1929. The regulation of adult body size in the protozoan Col poda. J. exp. Zool., 53: 269-312. Amberson, W. F. 1928. The influence of oxygen tension upon the respiration of unicellular organisms. Biol. Bull., 55: 79-91. Andrews, Justin, and Th. von Brand. 1938. Quantitative studies on glucose consumption by Trichomonas foetus. Amer. J. Hyg., 28: 138-47. Barratt, J. O. W. 1905. Die Kohlensdureproduktion von Paramecium aurelia. Z. allg. Physiol., 5: 66-72. Bles, E. 1929. Arcella. A study in cell physiology. Quart. J. micr. Sca., 72: 527-648. Bodine, J. H., and E. J. Boell. 1934. Respiratory mechanisms of normally de- veloping and blocked embryonic cells (Orthoptera) . J. cell. comp. Physiol., Bis eis, —— 1937. The action of certain stimulating and inhibiting substances on the respiration of active and blocked eggs and isolated embryos. Physiol. Z00\.,. 10:5 245-572 Bodine, J. H., and P. R. Orr. 1925. Respiratory metabolism. Biol. Bull., 48: 1-14. Brand, Th. von. 1933. Studien tiber den Kohlehydratstoffwechsel parasitischer Protozoen. II. Der. zuckerstoffwechsel der Trypanosomen. Z. vergl. Physiol., 19: 587-614. — 1934. Das Leben ohne Saurstoff bei witbellosen Tieren. Ergebn. Biol., 10:37-100. —— 1935. Der stoffwechsel der protozoen. Ergebn. Biol., 12:161-220. —— 1938. The metabolism of pathogenic trypanosomes and the carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13: 41-50. Brand, Th. von, and T. L. Jahn, 1940. Chemical composition and metabolism of Nematode parasites of vertebrates, and the chemistry of their en- vironment. In An introduction to Nematology (ed. by J. R. Christie). (In press.) Baltimore. Brand, Th. von, P. Regendanz, and W. Weise. 1932. Der Milchsduregehalt und die Alkalireserve des Blutes bei experimentellen Trypanosomeninfek- tionen. Zbl. Bakt., I. (Orig.) 125: 461-68. Broh-Kahn, R. H., and I. A. Mirsky. 1938. Studies on anaerobiosis. I. The nature of the inhibition of growth of cyanide-treated E. coli by reversible oxidation-reduction systems. J. Bact., 35: 455-75. Burge, W. E. 1924. The effect of different anaesthetics on the catalase content and oxygen consumption of unicellular organisms. Amer. J. Physiol., 69: 304-6, Burk, Dean. 1937. On the biochemical significance of the Pasteur reaction and Meyerhof cycle in intermediate carbohydrate metabolism. Some Funda- mental Aspects of the Cancer Problem. Occ. Publ. Amer. Ass. Adv. Sci. 298 RESPIRATORY METABOLISM Chambers, R. 1933. An analysis of determinations of intracellular reduction potentials. Cold Spring Harbor Symp. Quant. Biol., 1: 205-13. Clark, W. M. 1934. The potential energies of oxidation-reduction systems and their biochemical significance. Medicine, 13: 207-50. Cleveland, L. R. 1925. Toxicity of oxygen for Protozoa in vivo and in vitro. Biol. Bull. 48: 455-68. Cleveland, L. R., S. R. Hall, E. P. Sanders, and Jane Collier. 1934. The wood- feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: 185-342. Cohen, Barnett. 1933. Reactions of oxidation-reduction indicators in biological material, and their interpretation. Cold Spring Harbor Symp. Quant. Biol., Ls 24223) Cohen, R. A., and R. W. Gerard. 1937. Hyperthyroidism and brain oxida- tions. J. cell. comp. Physiol., 10: 223-40. Cook, S. F. 1932. The respiratory gas exchange in Termopsis nevadensis. Biol. Bull. 63: 246-57. Daniel, G. E. 1931. The respiratory quotient of Balantidinm coli. Amer. J. Hyg., 14: 411-20. Dixon, M. 1934. Manometric methods as applied to the measurement of cell respiration and other processes. London. Dubos, R. 1929. The relation of the bacteriostatic action of certain dyes to oxidation-reduction processes. J. exp. Med., 49: 575-92. Duryée, W. R. 1936. A modified microrespirometer. Z. vergl. Physiol., 23: 208-13. Efimoff, W. W., N. J. Nekrassaw, and Alexandra W. Efimoff. 1928. Die Einwirkung des Oxydationspotentials und der H-Ionenkonzentration auf die Vermehrung der Protozoen und Abwechselung ihrer Arten. Biochem. Zi, 1972 VO5"0s Elliott, A. M. 1939. A volumetric method for estimating population densities of Protozoa. Trans. Amer. micr. Soc., 58: 97-99. Emerson, R. 1929. Measurements of the metabolism of two protozoans. J. gen. Physiol., 13: 153-58. Fauré-Fremiet, E., C. Léon, A. Mayer, and L. Plantefol. 1929. Recherches sur le besoin d’oxygéne libre. L’oxygéne et les mouvements des _para- mécies. Ann. Physiol. Physicochim. biol., 5: 633-41. Fenn, W. O. 1928. A new method for the simultaneous determination of minute amounts of carbon dioxide and oxygen. Amer. J. Physiol., 84: 110-18. Fenyvessy, B. von, and L. Reiner. 1924. Untersuchungen uber den respira- torischen Stoffwechsel der Trypanosomen. Z. Hyg. Infektkr., 102: 109- 169) —— 1928. Atmung und Glykolyse der Trypanosomen. H. Biochem. Z., 202: 75-80. RESPIRATORY METABOLISM 399 Frei, W. 1935. Atmungssysteme der Bakterien. Zbl. f. Bakt., 134: 26-35. Friedemann, T. E. 1938. Metabolism of pathogenic bacteria. J. Bact., 35: 527-46. Geiger, A., I. J. Kligler, and R. Comaroff. 1930. The glycolytic power of trypanosomes (Trypanosoma evanst) in vitro. Ann. Trop. Med. Parasit., 24: 319-27. Gerard, R. W. 1931. Observations on the metabolism of Sarcina lutea. II. Biol. Bull., 60: 227-41. Gerard, R. W., and H. K. Hartline. 1934. Respiration due to natural nerve impulses. A method for measuring respiration. J. cell. comp. Physiol., 4: 141-60. Gerard, R. W., and L. H. Hyman. 1931. The cyanide sensitivity of Para- mecium. Amer. J. Physiol., 97: 524-25. Guyer, M. F. 1936. Animal micrology. Chicago. Hall, Robert H. 1938. The oxygen-consumption of Colpidium campylum. Biol. Bull., 75: 395-408. Hall, R. P. 1939. The trophic nature of the plant-like flagellates. Quart. Rev. Biol., 14: 1-12. Harvey, E. N. 1928. The oxygen consumption of luminous bacteria. J. gen. Physiol., 11: 469-75. Hewitt, L. F. 1936. Oxidation-reduction potentials in bacteriology and bio- chemistry. 2d ed. (London County Council) London, Heyningen, W. E. van. 1935. The inhibition of respiration by cyanide. Bio- chem; J).,, 29° 2036-39: Holmes, Eric. 1937. The metabolism of living tissues. London. Holter, H. 1936. Studies on enzymatic histochemistry. XVIII. Localization of peptidase in marine ova. J. cell. comp. Physiol., 8: 179-200. Holter, H., and W. L. Doyle. 1938. Studies on enzymatic histochemistry. XXVIII. Enzymatic studies on protozoa. J. cell. comp. Physiol., 12: 295- 308. Howland, R. B., and A. Bernstein. 1931. A method for determining the oxy- gen consumption of a single cell. J. gen. Physiol., 14: 339-48. Jahn, Theo. L. 1933a, Oxidation-reduction potential as a possible factor in the growth of intestinal parasites in vitro. J. Parasit., 20: 129. —— 1933b. Studies on the oxidation-reduction potential of protozoan cul- tures. I. The effect of —SH on Chilomonas paramecium. Protoplasma, 20: 90-104. —— 1934. Problems of population growth in the Protozoa. Cold Spring Har- bor Symp. Quant. Biol., 2: 167-80. —— 1935a. Studies on the physiology of the euglenoid flagellates. VI. The effects of temperature and of acetate on Evglena gracilis in the dark. Arch. Protistenk., 86: 251-57. — 1935b. Studies on the oxidation-reduction potential of protozoan cul- 400 RESPIRATORY METABOLISM tures. II. The reduction potential of cultures of Chilomonas paramecium. Arch. Protistenk., 86: 225-37. — 1936. Effect of aeration and lack of CO, on growth of bacteria-free cultures of protozoa. Proc. Soc. exp. Biol. N.Y., 33: 494-98. Jay, George, Jr. 1938. Respiration of Astasia sp. and Khawkinea halli. Anat. Rec., 72 (Suppl.): 104. Kalmus, H. 1927. Das Kapillar-Respirometer: Eine neue Versuchsanordnung zur Messung des Gaswechsels von Mikrodrganismen. Vorlaufige Mit- teilung demonstriert an einem Beispel: Die Atmung von Paramecium caudatum. Biol. Zbl., 47: 595-600. —— 1928a. Die Messung der Atmung, Garung und CO,-Assimilation kleiner Organismen in der Kapillare. Z. vergl. Physiol., 7: 304-13. — 1928b. Untersuchungen tiber die Atmung von Paramecium caudatum. Z. vergl. Physiol., 7: 314-22. Keilin, D., and E. F. Hartree. 1936. On some properties of catalase hematin. Proc..toy. Soc. By i213 173-91. Kempner, W. 1936. Effect of low oxygen tension upon respiration and fer- mentation of isolated cells. Proc. Soc. exp. Biol. N.Y., 35: 148-51. — 1937. Effect of oxygen tension on cellular metabolism. J. cell. comp. Physiol., 10: 339-64. Kligler, I. J., A. Geiger, and R. Comaroff. 1929. Susceptibility and resistance to trypanosome infections. VII. Cause of injury and death in trypano- some infected rats. Ann. Trop. Med. Parasit., 23: 325-35. — 1930. Effect of the nature and composition of the substrate on the develop- ment and viability of trypanosomes. Ann. Trop. Med. Parasit., 24: 329- 45. Kluyver, A. J. 1931. Chemical activities of microorganisms. London. Korr, I. M. 1935. An electrometric study of the reducing intensity of lumi- nous bacteria in the presence of agents affecting oxidations. J. cell. comp. Physiol., 6: 181-216. — 1938. Oxidation-reduction potentials in heterogeneous systems. J. cell. comp. Physiol., 11: 233-45. Lackey, J. B. 1932. Oxygen deficiency and sewage Protozoa: with descriptions of some new species. Biol. Bull., 63: 287-95. Leichsenring, J. M. 1925. Factors influencing the rate of oxygen consumption in unicellular organisms. Amer. J. Physiol., 75: 84-92. Linderstrom-Lang, K. 1937. Principle of the Cartesian diver applied to gasometric technique. Nature, 140: 108. Lund, E. J. 1918a. Relation of oxygen concentration and the rate of intra- cellular oxidation in P. caudatum, Amer. J. Physiol., 45: 351-64. 1918b. Rate of oxidation in P. cawdatum and its independence of the toxic action of KCN. Amer. J. Physiol., 45: 365-73. —— 1918c. III. Intracellular respiration, relation of the state of nutrition of RESPIRATORY METABOLISM 401 Paramecium to the rate of intracellular oxidation. Amer. J. Physiol., ASE SET —— 1918d. A simple method for measuring carbon dioxide produced by small organisms. Biol. Bull., 36: 105-14. Lwoff, A. 1932. Recherches biochimique sur Ja nutrition des Protozoaires. Monogr. Inst. Pasteur, 1932. —— 1933. Die Bedeutung des Blutfarbstoffes fiir die parasitischen Flagellaten. Zbl. Bakt. Abt. 1 (Orig.), 130: 498-518. —— 1938. Les Facteurs de croissance pour les microdrganismes. Premier Congrés des Microbiologistes de Langue francaise, 4 Paris. Lwoff, M. 1934. Sur la respiration du Cilié Glaucoma piriformis. C. R. Soc. Biol. Paris, 115: 237-41. Marsh, G. 1935. Kinetics of an intracellular system for respiration and bio- electric potential at flux equilibrium. Plant Physiol., 10: 681-97. Mast, S. O., and D. M. Pace. 1937. The effect of silicon on growth and respi- ration in Chilomonas paramecium. J. cell. comp. Physiol., 10: 1-14. Mast, S. O., D. M. Pace, and L. R. Mast. 1936. The effect of sulfur on the rate of respiration and on the respiratory quotient of Chilomonas para- mecium. J. cell. comp. Physiol., 8: 125-39. McClung, C. E., Ed. 1937. Handbook of microscopical technique. New York. Meldrum, N. U. 1934. Cellular Respiration. London. Necheles, H. 1924. Unpublished experiments. Cited by Kestner and Plant, in WWinterstein’s Handbuch der vergleichenden Physiologie. Bd. 2/2. Needham, Joseph. 1931. Chemical embryology, Vol. II. Cambridge Univ. Press, London. Needham, Joseph, and E. J. Boell. 1938. Metabolic properties of the regions of the amphibian gastrula. Proc. Soc. Exp. Biol. N.Y., 39: 287-90. Needham, J., and D. M. Needham. 1927. The oxidation-reduction potential of protoplasm: a review. Protoplasma, 1: 255-94. Novy, F. G. 1932. Respiration of microorganisms. J. Lab. clin. Med., 17: 731- 47. Novy, F. G., H. R. Roehm, and M. H. Soule. 1925. Microbic respiration. I. The compensation manometer and other means for study of microbic respiration. J. Infect. Dis., 36: 109-67. Pacinotti, G. 1914. Infusorien, welche Glykogen in Fett umwandeln. Boll. Soc. eustach. No. 3. Peters, J. P., and D. D. Van Slyke. 1932. Quantitative clinical chemistry Vol. II. Methods. Baltimore. Peters, R. A. 1929. Observations on the oxygen consumption of Colpidium Pitts, R. F. 1932. Effect of cyanide on respiration of the protozoan, Colpidimm campylum, Proc. Soc, exp. Biol. N.Y., 29: 542. Putter, A. 1905. Die Atmung der Protozoen. Z. allg. Physiol., 5: 566-612. 402 RESPIRATORY METABOLISM Quastel, J. H. 1930. The mechanism of bacterial action. Trans. Faraday Soc., 26: 853-64. Quastel, J. H., and M. Stephenson. 1926. Experiments on “‘strict’’ anaerobes. I. The relation of B. sporogenes to oxygen. Bio-chem. J., 20: 1125-37. Reiner, L., and C. V. Smythe. 1934. Glucose metabolism of the Trypanosoma equiperdum in vitro. Proc. Soc. exp. Biol. N.Y., 31: 1086-88. Reiner, L., C. V. Smythe, and J. T. Pedlow. 1936. On the glucose metabolism of trypanosomes. J. biol. Chem., 113: 75-88. Riedmuller, L. 1936. Beitrag zum kulturellen Verhalten von Trichomonas foetus. Zbl. Bakt. (Orig.), 137: 428-33. Robbie, W. A., E. J. Boell, and J. H. Bodine. 1938. A study of the mechanism of cyanide inhibition. I. Effect of concentration on the egg of Me/anoplus differentialis. Physiol. Zo6l., 11: 54-62. Root, W. S. 1930. The influence of carbon dioxide upon the oxygen con- sumption of Paramecium and the egg of Arbacia. Biol. Bull., 59: 48-62. Roskin, Gr., and L. Levinsohn. 1926. Die Oxydasen und Peroxydasen bei Protozoa. Arch. Protistenk., 56: 145-66. Schlayer, C. 1936. The influence of oxygen tension on the respiration of pneumococci (Type I). J. Bact., 31: 181-90. Schmitt, F. O. 1933. The oxygen consumption of stimulated nerve. Amer. J. Physiol., 104: 303-19. Shoup, C. S., and J. T. Boykin. 1931. The insensitivity of Paramecium to cyanide and effects of iron on respiration. J. gen. Physiol., 15: 107-18. Slater, W. K. 1928. Anaerobic life in animals. Biol. Rev., 3: 303-28. Soule, M. H. 1925. Microbic Respiration. II]. Respiration of Trypanosoma lewisi and Leishmania tropica. J. Infect. Dis., 36: 245-308. Specht, H. 1935. Aerobic respiration in Spirostomum ambiguum and the production of ammonia. J. cell. comp. Physiol., 5: 319-33. Standard Methods of Water Analysis. 1936. New York. Stephenson, M. 1939. Bacterial Metabolism, London. Stern, Kurt G. 1938. The relationship between prosthetic group and protein carrier in certain enzymes and biological pigments. Cold Spring Harbor Symp. Quant. Biol. 6: 286-300. Szent-Gyorgyi, A., and I. Banga. 1933. Uber das Co-ferment der Milchsauer- oxydation. Z. phys. Chem., 217: 39-49. Tang, Pei-Sung. 1933. On the rate of oxygen consumption by tissues and lower organisms as a function of oxygen tension. Quart. Rev. Biol., 8: 260-74. Wachendorff, T. 1912. Der Gaswechsel von Colpidinm colpoda. Z. allg. Physiol., 13: 105-10. Warburg, O. 1926. Uber den Stoffwechsel der Tumoren. Berlin. Witte, J. 1933. Bakterienfreie Ziichtung von Trichomonaden aus dem Uterus des Rindes in einfachen Nahrbéden Zbl. Bakt. Abbt. I. Orig., 128: 188-95. RESPIRATORY METABOLISM 403 Wurmser, R. 1932. La Signification biologique des potentiels d’oxydoréduc- tion: Biol. Reva 7350-81. Yorke, W., A. R. D. Adams, and F. Murgatroyd. 1929. Studies in Chemo- therapy I. A method for maintaining pathogenic trypanosomes alive in vitro at 37°C. for 24 hours. Ann. trop. Med. Parasit., 23: 501-18. Zhinkin, L. M. 1930. Zur Frage der Reservestoffe bei Infusorien. Z. Morph. Okol. Tiere, 18: 217-48. Zweibaum, J. 1921. Richerche sperimentali sulla coniugazione degli Infusori. I. Influenze della coniugazione sull’ assorbimento dell’ O, nel Para- mecium caudatum. Arch. Protistenk., 44: 99-114. CHAPTER Vil THE CONTRACTILE VACUOLE J. H. WEATHERBY INTRODUCTION SINCE THE FIRST description of the protozoan contractile vacuole, prob- ably made by Spallanzani in 1776, few structures in these organisms have received such intensive investigation. Unfortunately, solutions of many of the perplexing questions which have arisen as a result of these studies are not yet at hand. Indeed, much of the more recent work has given rise to entirely new questions which are no less insistent in their demands for answers than were the earlier ones. In the literature claims and counterclaims are abundant; important discoveries have been made only to be discarded because of lack of confirmation, or, in some instances, because of direct contradiction. In view of the somewhat confused state of the evidence concerning contractile vacuoles a re-survey of some of the more important questions seems to be in order. Probably the first question asked by the first investigator to see a con- tractile vacuole was ‘“What is its function?’ Needless to say, this first investigator did not learn the answer, and, in the opinions of many, the most recent investigation probably does not supply the complete answer. Following this question there have been others hardly less interesting. Is it essential to life? Is it a permanent structure, or does it arise anew at the beginning of each new cycle? Does it always occupy the same position in the organism with respect to other structures? Is the vacuole surrounded by a permanent membrane? Is its discharge to the exterior through a permanent excretory pore? If there is no pre-formed excretory pore, how may one explain the formation even of a temporary pore, and once formed how is it closed again? What natural forces operate to expel the contents of the vacuole? There are many other equally interesting questions, but only a few can be considered at this time. The most promising order for discussing these problems seems to be THE CONTRACTILE VACUOLE 405 to deal first with those pertaining to origin of the vacuole—to see, if possible, just where this organelle comes from, if it does not exist in the cell as a permanent structure. Then, having traced its origin, questions dealing with structure and function will follow in more logical order. An attempt will be made to follow this general plan, but the very nature of the subject will necessitate digressions from time to time. While a conscientious review of the literature has been attempted, it is quite possible that important publications have been overlooked. It is hoped that this will prove not to be true, not only for the sake of com- pleteness, but also for the sake of giving credit where credit is due. The author herewith offers his apologies to anyone whose labors have not been acknowledged. THE ORIGIN OF CONTRACTILE VACUOLES Metcalf (1910) noticed in amoeba of the proteus type that the vacu- ole is surrounded by a layer of granules of the same approximate size and appearance as the “microsomes” of the general cytoplasm. When the vacuole is of moderate size, these granules form a layer on its surface one granule thick; when the vacuole is fully distended, as just before systole, there are spaces between the granules; but when the vacuole is small the layer may be several granules thick. At systole the vacuole usually collapses completely, and the granules may be seen clumped to- gether in the region of the cytoplasm previously occupied by the vacuole. The new vacuole arises in the midst of these granules, and is formed by the fusion of several small vacuoles. According to Metcalf, who reported observations which sometimes lasted for as long as several hours on a single organism, the vacuole never arises in any other part of the body under normal conditions, except among the granules which surrounded it before its last contraction. From these observations he concludes that the granules are associated in some way with the origin and the function of the vacuole, and for this reason calls them “‘excretory granules.” How- ever, he decides that the granules are not essential for life, since most of them, together with the vacuole, may be removed from an Amoeba by operation without a fatal result. Under these conditions a new vacuole develops, although there are few if any granules to be seen surrounding it when it first appears. Metcalf reaffirmed his statement concerning these observations in 1926, 406 THE. CONTRACTILE: VACUOLE Mast (1926) agrees with Metcalf concerning the frequent presence of granules around the vacuole, but does not interpret this as indicating a physiological association between them. This opinion is based on his having observed vacuoles functioning perfectly normally without the presence of a single granule in the immediate vicinity of the vacuole. To these granules Mast applies the name “‘beta granules,” to distinguish them from others of a different nature which are also present in the cytoplasm. Mast and Doyle (1935) reinvestigated the relationship be- tween granules and vacuole. By centrifuging amoebae it is possible to cause stratification of various cytoplasmic constituents. Organisms treated in such a manner can be operated on so as to remove all or any desired portion of almost any one of the constituents, including these granules. It was found by Mast and Doyle that removal of all or most of the gran- ules resulted in the death of the organism. Removal of fewer granules caused a decrease in pulsation frequency of the vacuole, which was di- rectly proportional to the relative number of granules removed; that is, pulsation frequency was found to be directly proportional to the number of granules remaining. Removal of the contractile vacuole alone resulted in the prompt formation of another. Concerning this same question, Mast (1938, p. 312) more recently states: The beta granules around the contractile vacuole vary greatly in number and the layer of substance in which they are embedded varies greatly in thickness, without any apparent variation in the function of the vacuole. These facts indicate that neither the granules nor the layer of substance 1s involved in the function of the contractile vacuole, at least not directly. Howland (1924a) found that there is no concentration of granules on the surface of the vacuole in Amoeba verrucosa, but she considers it likely that the vacuole arises from the coalescing of small hyaline globules, which in turn are derived from the dissolving of granules. In any case, Howland traces the ultimate origin of the vacuole back to granules in somewhat the same manner that Metcalf does, although in A. verrucosa these granules are probably dispersed throughout the cyto- plasm. On the other hand, Haye (1930) found in fixed and stained preparations of A. verspertilio essentially the same relationship between granules and vacuole as described by Metcalf and later by Mast; that is, the filled vacuole is more or less covered by granules, and after systole the new vacuole arises in the midst of these granules. THEJGONTRACTILE) VACUOLE 407 Hall (1930a) studied the cytoplasmic inclusions in Tr7chamoeba after osmic and silver impregnation. In a few instances he observed the adher- ence of blackened globules to the outer surfaces of vacuoles. At first glance these appeared to be vacuoles with heavily impregnated walls, but close observation revealed the granular or globular nature of the blackened material. It must be remembered that these granules are not confined to the im- mediate vicinity of the contractile vacuole, but usually are scattered throughout the entire cytoplasm as well. If the origin of the vacuole is associated with and dependent on the presence of these granules, then one would expect other parts of the organism to be at least potentially capable of giving rise to vacuoles, since some granules are present in other parts. That such a phenomenon actually occurs in Amoeba has been ob- served by various authors, among whom are Day (1927), as well as Howland and Mast and Doyle. In this connection it 1s interesting to note that Dimitrowa (1928) was able to induce formation of extra vacuoles in Paramecium caudatum by interfering mechanically with the normal function of those already present. These extra vacuoles usually appeared to be entirely normal, although in a few instances there were no radial canals. The customary number of vacuoles was restored at fission by failure of the organism to form new ones if two extra ones had been in- duced, or by the formation of one new vacuole if only one had been in- duced artificially. In the event that there were three extra vacuoles, one daughter cell received three and formed a single new one when it in turn divided. Haye (1930) investigated eight species from two orders of flagellates. In Phacus pleuronectes he found that the walls of contractile vacuoles contain lipoid granules which are arranged in a net-like fashion. In Euglena pisciformis and Trachelomonas hispida the surfaces of both the reservoir and the vacuoles show a granular structure. In Persdinium steinii no granules were observed, nor were accessory vacuoles seen, ex- cept in organisms obtained from a laboratory aquarium in which con- ditions were thought to have been abnormal. A differentiated plasma zone, reminding one of the ‘excretory plasma” of fresh-water Protozoa, was noted around the pusule. In the wall of the pusule of P. divergens were observed lipoid granules similar to those in the wall of the contrac- tile vacuole of fresh-water Protozoa. Besides the two sac pusules, a col- 408 THE CONTRACTILE VACUOLE lecting pusule with daughter vacuoles and numerous accessory vacuoles were observed in Phalacroma sp. In Goniodoma sp. there were, besides a sac pusule, a collecting pusule with daughter pusules and an accessory vacuole. Only one large pusule was noted in Ceratzum hirundinella. \n both orders of flagellates Haye believes that emptying of accessory vacu- oles is accomplished by diffusion through the walls into the contractile vacuole, rather than by coalescence with it. Hall (1930b) found that in Menoidium, stained according to the Da Fano silver method, the contractile vacuole is formed by the fusion of several smaller vacuoles arising near the gullet. The mode of origin of contractile vacuoles has been studied in a greater variety of ciliates than in either rhizopods or flagellates, and in- formation on this subject is proportionally more abundant. Taylor (1923) observed in Explotes that the vacuole (V,), in its final form immediately before contraction, is the result of the fusion of several smaller vacuoles, and that these smaller vacuoles (designated as group V.) in turn are formed by the fusion of still smaller vacuoles (group V,). The smallest vacuoles in the series are thought to arise as the result of the dissolving of granules, or to arise de novo. Thus Taylor suggests granules as a possible source of vacuolar fluid, and he observed formation of the vacuole by the fusion of several small accessory vacuoles. King (1933), who studied Ezp/otes after impregnation with osmic acid, found that the smallest visible accessory vacuoles (V,) have their origin at the distal ends of a very large number of collecting canals, located just under the ectoplasm on the dorsal surface of the ciliate. These canals radiate like a sun-burst from the vicinity of the vacuoles, and seem to end blindly in the protoplasm of the organism. These canals have a diameter of ap- proximately 0.5 micron at their distal ends, and become relatively much narrower as they pass away from the region of the vacuoles. The canals are not visible in living organisms, but may be clearly demonstrated by proper impregnation with osmic acid. On the basis of information now available, it is difficult to tell whether the canals described by King and the granules mentioned by Taylor represent different interpretations of the same structures, observed under different conditions, or whether the canals merely provide a means for the transport of fluids which have originated in more distant parts of the body as a result of the activity of granules. THE CONTRACTILE VACUOLE 409 Of particular interest are the observations of MacLennan (1933) on the Ophryoscolecidae, ciliates from the stomachs of cattle. The cycle of the contractile vacuole was studied in both living and fixed material, in- cluding the following genera: Ophryoscolex, Epidinium, Ostracodinium, Polyplastron, Eudiplodinium, and Metadinium. In all these genera the contractile vacuole is formed by the coalescence of small accessory vacu- oles, just as in Ezplotes, as described by Taylor and also by King, and in Amoeba, as described by Metcalf. These accessory vacuoles arise from the dissolving of granules which are found in sharply defined regions around the contractile vacuole in Exdzplodinium and Metadinium, in a narrow dorsal strip of the ectoplasm in Ostracodinium, and in the whole ectoplasm in Ophryoscolex and Epidinium. If one may be permitted to assume that the canals and granules in Evp/oftes are identical, then the mode of origin of the contractile vacuole in this form is quite similar to that described by MacLennan for the Ophryoscolecidae; and in its funda- mental features it also resembles that reported for amoebae as well as for some of the flagellates. In addition to the flagellates previously mentioned, Haye (1930) also studied representatives of thirteen genera from four orders of ciliates. In Opalina dimidiata, Isotricha prostoma, Spirostomum ambiguum, and Nyctotherus cordiformis little was observed which suggests the mode of origin of contractile vacuoles. Except for the fact that the walls of the canals were found to contain lighter and darker zones—probably because of the presence of lipoid granules—little was observed in Para- mecium caudatum which may be associated with origin of vacuoles or their contents. Rod-shaped entosomes were found closely packed about the wall of the vacuole in Lionotus fasciola. In Stentor polymorpha the wall of the vacuole is very delicate and shows only here and there a granular structure; several secondary vacuoles are usually present. In Blepharisma undulans, Balantidium entozoén, Polyplastron multivesi- culatum, Ostracodinium gracile, and O phrydium versatile are to be found granules (entosomes) within the wall, or closely associated with the wall, of the contractile vacuole. The vacuole in Epistylis plicatilis is formed from numerous secondary vacuoles; no granules or entosomes are to be observed. Von Gelei (1933) states that the vacuole system in Spathidium con- sists of a primary vacuole, located usually in the posterior end of the 410 THE CONTRACTILE VACUOLE organism. Around this are one or two rows of smaller secondary vacuoles, which fuse and give rise to a new primary vacuole following systole. Whether or not these secondary vacuoles originate from granules was not ascertained by von Gelei. Essentially the same relationship between primary and secondary vacuoles in Ble pharisma was described by Moore (1934), who made the further statement that excretory granules could not be observed. Both Wenrich (1926) and King (1928) found the vacuoles of P. trichium to be vesicle-fed, although neither author men- tioned the origin of these vesicles. Day (1930) found that the vacuolar fluid reaches the elongated canal of Spirostomum by the fusion of small vacuoles with the canal throughout its entire length. A similar source of fluid in canals of P. caudatum was also reported, but in neither in- stance was the origin of the accessory vacuoles mentioned. Fauré-Fremiet (1925) observed the filling of contractile vacuoles in several species of Vorticella by the discharge of small vesicles into the vacuole. These vesicles originate in the wall of the vacuole, and cor- respond to the “mural vacuoles’’ described by Haye (1930) for Cam- panella, Chilodon, Dogielella, and some of the Ophryoscolecidae. Nas- sonov (1925) also investigated Chilodon and Dogielella, and found structure and mechanism of filling to be somewhat different from that described by Haye. According to Nassonov, vacuoles in these forms do not appear to have the thick walls described by Haye, nor even to have any sort of membrane, but lie directly in the cytoplasm. However, there is a strongly osmiophilic structure closely associated with them, which, for Chilodon at least, and possibly also for Dogzelella, may be mistaken for a thick wall or membrane under certain conditions. In both forms the osmiophilic structure remains essentially unaltered in appearance after collapse of the vacuole. Nassonov observed the origin of accessory vacuoles (the mural vacuoles of Haye) in these osmiophilic structures, and believes them to contribute to the filling of the contractile vacuole. Many authors hold that in certain Protozoa, typified by Paramecium caudatum, the question of origin of the contractile vacuole does not arise, since in these forms the vacuole system is a permanent structure. This view 1s not universally accepted, as will be pointed out later. But, whether permanent or temporary, there still exists a no less fundamental question as to the origin of the fluid which finds its way into these organelles. If the origin of this fluid is associated with granules in many diverse organ- THE CONTRACTILE VACUOLE 411 isms, as much of the evidence implies, then it would be somewhat un- expected if such granules are not to be found distributed generally throughout the Protozoa. MacLennan called attention to the fact that many investigators have demonstrated a more or less solid membrane surrounding vacuoles in a variety of Protozoa, these demonstrations having been made by osmic-acid impregnation. Most of these workers, according to MacLennan, used the warm method of impregnation advo- cated by Nassonov. Hirschler showed that this method tends to produce overimpregnation, resulting in the production of a heavy black band or membrane in what is actually a granular zone. MacLennan found this to be true in the Ophryoscolecidae, while impregnation by the cold method shows this same region to be granular, a condition which can be seen in living material. He further calls attention to the fact that figures showing solid impregnation of the vacuolar walls of Chilodon and Dogielella, published by Nassonov, of Balantidium by Bojewa-Petrus- chewskaja, and of the Cyclopostheiidae by Strelkow, indicate a marked granular roughening of the outer margin of the osmiophilic layer. He takes this to indicate that what has been interpreted by these authors as a solid membrane may, in fact, be only the result of overimpregnation of a granular zone. Among the Ophryoscolecidae alone MacLennan found various degrees of aggregation of these granules—from virtually none to a very pronounced aggregation—around the contractile vacuole. He fur- ther showed that localization of the origin of contractile vacuoles in these forms is correlated with the degree of aggregation of the granules. Of interest in this connection are the observations of Lloyd and Scarth (1926) on the origin of vacuoles in Spirogyra. These authors found that in sufficiently high concentrations even the most innocuous plasmolytes may by themselves cause subsidiary vacuoles to arise in the cytoplasm. It is not only by plasmolytes that this effect is produced however, but also by other more readily penetrating substances such as the narcotics, chloro- form, and ether, and by very low concentrations of salts. But without any artificial influence, similar vacuoles may form in normal cells. Their ‘constant occurrence was demonstrated in the gametes during conjugation in Spirogyra, and their excretory function in the taking up of water from the central vacuole and its discharge to the exterior in typical ‘‘contrac- tile” fashion was proved. The authors state that these vacuoles originate from peculiar “‘lecithin-like” bodies already present in the cytoplasm. 412 THE CONTRACTILE VACUOLE Scarth and Lloyd (1927) claim that the vacuolar wall arises from the “‘kinoplasm” of Strasburger. They observed a reciprocal quantitative re- lation between kinoplasm and mitochondria. The activity of kinoplasm resembles that of lecithin, which is abundant in mitochondria. On the basis of this resemblance, they conclude that water at least may accumu- late in the vacuoles without the visible interaction of any other struc- ture. The observations and opinions reported in the foregoing pages, while somewhat contradictory at times, point to two general conclusions con- cerning the origin of contractile vacuoles. First, in the great majority of forms, perhaps in all forms, fluid reaches the contractile vacuole through the fusion of small vesicles, or accessory vacuoles, with the con- tractile vacuole or its filling canals. The vesicles arise within what often appears to be the wall of the vacuole; the accessory vacuoles usually originate at a greater or less distance from the contractile vacuole, and coalesce to form the latter. Second, vesicles originate within walls of vacuoles which have been shown in many instances to be granular in nature or to be intimately associated with granules; accessory vacuoles have been reported by various authors as originating among granules which may be closely associated with the vacuole, or occasionally re- moved some distance from it. Thus it appears that in spite of their great variety of shapes and general appearances under the microscope, con- tractile vacuoles originate in a remarkably similar manner in all forms so far investigated with this problem in mind. In certain instances authors have reported the absence of granules in the vicinity of the contractile vacuole, and from this have concluded that granules are not concerned in the origin of vacuoles. In this connection it must be remembered that in certain forms, e.g., Euplotes, some of the Ophryoscolecidae, and apparently in others as well, the accessory vacuoles which ultimately give rise to the contractile vacuole originate at some distance from the ultimate site of the final vacuole. Also, gran- ules frequently are visible only after osmium or silver impregnation. Keeping in mind the greater vulnerability of negative evidence, one is justified in the thought that perhaps a reéxamination of organisms for which the absence of granules in the vicinity of the vacuole has been re- ported, may reveal the presence of granules in other parts of the body, either scattered or in aggregates. Such scattered granules, which are known THE CONTRACTILE VACUOLE 413 to exist in some amoebae, may be the site of origin of new vacuoles when the function of the original vacuole is disturbed by artificial means or removed by operation. While such granules have not been demonstrated to be scattered about in the cytoplasm of P. caudatum, their presence would explain the origin of extra vacuoles in this form, when func- tion of the original vacuoles is interfered with mechanically, as reported by Dimitrowa. The origin of new vacuoles at fission would have a similar explanation, since, as proposed by Dimitrowa, during fission the greater abundance of metabolites would impose a necessity on the organism essentially similar to interference with normal function. After fission, when the daughter cells are smaller than the parent cell was immediately prior to fission, and the metabolic rate is lowered, there no longer exists a stimulus for the formation of extra vacuoles, and the daughter cells appear quite normal, with the usual number. THE STRUCTURE OF CONTRACTILE VACUOLES The question of the structure of the contractile vacuole and its as- sociated parts has occupied the attention of protozodlogists for many years. As a result the main question has been broken up into several parts, each concerned with a limited phase of this main question. Is the ~ vacuole surrounded by a permanent membrane? Is its discharge to the exterior through a permanent excretory pore? If there is no permanent pore, how may one explain the formation even of a temporary pore, and once formed how is it closed again? Is the vacuole a permanent structure, or does it arise anew at the beginning of each new cycle? The dispute as to the presence or absence of a permanent membrane surrounding the vacuole began over a hundred years ago, and continues, with little to indicate that is will end within the near future. According to Taylor (1923), to whom we may refer for a more detailed account of the history of this question, the following investigators have written in support of the idea of a permanent membrane: Ehrenberg, Siebold, Claparéde, Lachmann, Degen, and Stempell. Those who believe that the vacuole possesses no permanent wall are: Dujardin, Meyen, Stein, Wrzesniowski, Perty, Schmidt, Zenker, Maupas, Rhumbler, Biitschli, Lan- kester, and Khainsky. Taylor himself holds this view, at least for Ez- plotes. Without reflecting unfavorably in any way on the researches of those who worked on this subject prior to 1900, or possibly as late as 414 THE CONTRACTILE VACUOLE 1920, one must admit that only limited importance can be attached to their opinions. Unquestionably most of these investigators were careful observers, and expressed opinions only after due consideration of all the factors which they were able to recognize. But the microscope of today is a far different instrument from that of a hundred years ago, or even fifty years ago; and chemical procedures, particularly those dealing with colloids, have undergone extensive development. However highly one may regard this or that early investigator, the fact remains that none could have been better than the tools with which he worked, and ad- mittedly the tools were poor. Consequently, the author maintains that it is neither unkind nor unappreciative to propose that these various early opinions be considered mainly as of historical interest, and of little worth in settling the question as to the presence or absence of permanent membranes, or of any kind of membrane for that matter, around con- tractile vacuoles. The employment of the best of modern instruments and techniques leaves the question in an unsatisfactory state. Before presenting the more recent evidence concerning this question of membranes, perhaps it will not be unwise to present briefly the more fundamental question of what constitutes a membrane. Most textbooks either avoid the issue more or less completely or describe the structure and properties of the artificial membranes so often used in the laboratory for experimental purposes. Although reliable information concerning living membranes is scant, there is sufficient evidence to justify the divi- sion of membranes into two types: morphological membranes and physi- ological membranes. Morphological membranes are permanent struc- tures which are frequently visible in living material viewed through the microscope, and usually may be demonstrated more or less clearly by suitable staining techniques. Apparently they consist mainly of a reticu- lum, or framework, which is described by some authors as being com- posed largely of protein. Such membranes are usually thought to possess an appreciable amount of rigidity, and to serve primarily as supporting structures. Free permeability in both directions is usually assigned to them. Physiological membranes are entirely different from morphological membranes in many important respects. Usually they are considered to be so thin as to be invisible even with the highest magnification. They may possess a certain degree of rigidity, but probably much less than THE (CONTRACTILE VACUOLE 415 morphological membranes with which they are often associated in liv- ing material. Semipermeability, or more properly selective permeability, is a property of all living physiological membranes. Colloid-chemists, as well as many physiologists, are agreed that a physiological membrane is simply a phase boundary, an interface between two different fluids. In order that such phase boundary may be more or less permanent, it is necessary that the two phases be only slightly miscible at most—the more complete the immiscibility the more nearly perfect and permanent the membrane. A very wide variety of molecules show polar phenomena; that is, the two ends of the molecules are electrically and chemically different. This results in orientation of molecules with respect to one another and to various other molecules, in much the same manner that a compass needle becomes oriented with respect to the magnetic poles of the earth. This phenomenon of orientation is associated with organic acids, alcohols, aldehydes, lipoids, fats, proteins, and many other so- called “physiological” compounds. Thus at the interface of the two- phase system, oil-water, the oil molecules (glycerol-esters of fatty acids) become oriented in such a manner that the hydrocarbon ends of the molecules project into the oil phase, whereas the glycerol ends project into the water. Fat molecules undergo much more nearly perfect orienta- tion than water molecules, although with the latter there appears to be a certain degree of orientation. Such an aggregation and packing to- gether of oriented polar molecules at an interface represents a physio- logical membrane. Since protoplasm contains a variety of polar molecules, the membrane formed between protoplasm and water is composed of various types of molecules apparently arranged in the form of a mosaic. The thickness of such a membrane has not been definitely established. Some authors maintain that it is only a single molecule thick, or at most only one or two milli-micra thick, but at least one author (Peters, see Clark, 1933, p. 40) has advanced a theory according to which the cell is composed of a three-dimensional protein mosaic, with the molecules in the interior of the cell oriented on the surface film. Since the interior of many cells is known to be fluid, the structure must be regarded as an orientation rather than as an anatomical skeleton. This theory of Peters’s agrees fairly well with certain evidence concerning the action of drugs on cells. Without entering into the question as to how far orientation extends beneath the surface layer, suffice it to say that it is well established 416 THE CONTRACTILE VACUOLE that orientation of surface molecules occurs at the interface between two different phases (provided, of course, that at least one phase contains polar molecules), whether the system is composed of oil and water or protoplasm and water. The converse of this is equally true; since the _ivery existence of this oriented layer depends on the presence of two different phases, the removal of one phase necessarily results in disintegra- tion of the membrane. Whether or not this surface layer of oriented molecules actually comprises the true physiological membrane may be subject to debate, but the importance of such a membrane is obvious, since it not only separates the organism from its surroundings but at the same time provides the only means of communication between the interior and the exterior of the cell. If one accepts the idea of a physiological membrane as a layer of oriented molecules at a phase boundary, as outlined briefly above, then it necessarily follows that any cell vacuole which contains a fluid differ- ent from cytoplasm must be surrounded by such a membrane. In the light of the information available at the present time, membranes around protozoan contractile vacuoles probably should be considered as tem- porary; although it is not inconceivable that in some forms the new vac- uole may be formed with such rapidity, in the midst of oriented mole- cules remaining after systole, that dispersion of the membrane does not have time to occur before the second phase is present again. If such condi- tion obtains, then the membrane may be considered as having a greater or less degree of permanence. There are numerous references to such physiological membranes in the literature on contractile vacuoles, so it appears that the idea has gained rather wide acceptance. The controversy is not so much concerned with such membranes as with the presence of absence of morphological, and hence permanent, membranes. Before leaving the subject of physiological membranes, there are sev- eral phenomena which may be discussed profitably with this concept in mind. Repeatedly authors speak of the coalescence of accessory vacuoles to form contractile vacuoles. Taylor (1923) refers to this, and considers coalescence to be due to a reversion of the gel state of the surrounding film to the sol state. He further states that vacuoles are surrounded by highly viscous boundaries of endoplasm, and that the consistency of the papilla pulsatoria strikingly resembles that of the endoplasmic boundaries. Without raising the question as to whether or not coalescence of accessory THE CONTRACTILE VACUOLE 417 vacuoles and the simultaneous rupture of vacuole wall and papilla pul- satoria represent sol-gel reversibility, it is obvious that exactly these phenomena must be anticipated, on the basis of the concept of physiologi- cal membranes such as described above. When two accessory vacuoles lie touching each other, the cytoplasmic phase of the two-phase system is pushed aside, at the same time removing the basic forces on which the presence of these membranes depends. In the absence of these forces, the membranes disintegrate at the site of contact, and the two vacuoles fuse into one. Likewise, when the filled vacuole comes in contact with the papilla pulsatoria, one phase (the cytoplasmic phase again) is pushed aside, the membranes disintegrate at the site of contact, and the contents of the vacuole are discharged to the exterior. After discharge the two- phase system is again established, since there is cytoplasm on one side of the pore (within the organism) and water on the other (outside the organism), whereas prior to discharge there was water on the outside as well as a fluid composed chiefly of water within the vacuole. The papilla is formed in this manner from the vacuole wall, which readily accounts for the similarity noted by Taylor. Other phenomena which can be explained in like manner by the pres- ence of physiological membranes are easy to find in the Protozoa. The ingestion of food by Amoeba is essentially the result of fusion of the walls of the organism after they have been extruded around the food particle in such a manner as completely to enclose it. The two-phase system exists as long as there is water on one side of the cell membrane and cytoplasm on the other; but when the engulfing process is complete and cell membrane is in contact with cell membrane with no water separating the two portions, the membrane disintegrates at the site of contact, and continuity of cell structures as well as of the vacuole mem- brane is established. The food-vacuole membrane persists as long as there is water within to maintain the two-phase system. In a similar man- ner one may explain the readiness with which an amputated fragment of an amoeba unites with the parent body when the two portions come together, although it has no bearing on the fact that a fragment from a diverse strain is refused. It has also been observed that occasionally an amoeba attempts to engulf a relatively large organism, such as P. cauda- tum, but is unable to accomplish this completely. The Paramecium is squeezed in two, apparently, with half inside the amoeba and half out- 418 THE CONTRACTILE VACUOLE side. Calculations have been made of the physical force necessarily ex- erted by the amoeba to accomplish this, but they have not taken into account some of the properties of physiological membranes, such as spon- taneous disintegration when membrane comes into direct contact with membrane. The adherence of conjugating organisms may be dependent, likewise, on these same properties of membranes. While it is interesting to speculate on such matters, it must be admitted that these remarks on feeding amoebae are purely speculative, with little other than superficial observation to support them. Concerning the presence of a permanent (morphological) membrane surrounding the contractile vacuole, there are diverse opinions. These diverse opinions apply not only to different species but even to the same species. Howland (1924a) found that the contributory globules as well as the vacuole may be removed from the organism to the surrounding water, where they retain their identity for an indefinite period of time. This may be taken to indicate a considerable degree of permanence of the vacuole wall, such as would be possessed by a morphological membrane, although Howland 1s of the opinion that these vacuole membranes are temporary. A temporary physiological membrane, formed of oriented molecules in a compact layer, may be expected to retain its identity for an appreciable period of time before dispersion of the molecules occurs, but it hardly seems probable that this “appreciable period of time’’ can be more than a few minutes. Day (1927) expresses the opinion that the vacuole wall in A. proteus is a ‘condensation membrane,” or gel, disap- pearing with each contraction. By © likely implies such a structure as has been described above as a physiologi- cal membrane, so that the two terms may be taken as synonymous. Con- cerning A. proteus, Mast (1938, p. 307) states: “At the surface of the contractile vacuole under the layer of substance containing the beta gran- ules, there is a layer or membrane about 0.5 micron thick which is opti- cally well differentiated from the adjoining substance on either surface, for under favorable conditions a line indicating an interface can be clearly seen at both these surfaces.’’ Whether this membrane is a permanent structure or is formed anew with each successive vacuole was not sug- gested by Mast. After an examination of fixed, stained, and sectioned material, Haye (1930) concluded that vacuole membranes are lacking in Amoeba, although it is probable that this author referred to morpho- condensation membrane” Day very THE CONTRACTILE ‘'VACUOLE 419 logical and not to physiological membranes, since these latter cannot be demonstrated in this manner. Among the flagellates, Nassonov (1924) found an osmiophilic mem- brane surrounding the vacuole in Chilomonas paramecium. Haye (1930) could distinguish no vacuole wall in the Euglenoidina, but in many other flagellates examined by him distinct walls were visible in stained ma- terial. Among the ciliates, morphological membranes are reported in a great variety of organisms by many investigators. Nassonov (1924) reports osmiophilic walls for the vacuoles in Paramecium caudatum, Lionotus folium, Nassula laterita, Campanella umbellaria, Epistylis gallea,Z ootham- nium arbuscula, and Vorticella sp. Fauré-Fremiet (1925) confirmed the findings of Nassonov, using several species of Vorticella, in which osmiophilic walls were observed, even after collapse of the vacuole. Young (1924) concludes from studies on P. caudatum stained with iron hematoxylin that the vacuole system is a permanent and continuous struc- ture. King (1928) arrived at essentially the same conclusion concerning the vesicle-fed system of P. trichium. Wenrich (1926) observed definite vacuole walls in P. ¢richium stained with Mayer's hemalum or Heiden- hain’s iron-alum-hematoxylin. Concerning this Wenrich states (p. 89): It was somewhat surprising to find how distinctly the vacuolar walls showed in fixed and stained specimens. The relative thickness of the wall is note- worthy and it usually appears to be laminated. In sectioned material the walls contained strands of more or less intensely staining material, suggesting the presence of contractile fibers. Von Gelei observed osmiophilic walls in P. caudatum (1925, 1928) and in Spathidium giganteum (1935). Haye (1930) found thin vacuole walls in the following forms: Blepharisma undulans, Lionotus fasciola, Ophrydium versatile, Stentor polymorphus, Spirostomum ambiguum, Balantidium entozo6n, and Isotricha prostoma. Thick walls were ob- served in Campanella, Chilodon, Dogielella, Paramecium, and the Oph- ryoscolecidae. As previously mentioned, Nassonov (1925) examined Chilodon and Dogielella after osmium impregnation and concluded that the vacuoles possess no membranes, but lie directly in the cytoplasm. In these latter organisms, structures considered by Nassonov to be the | Golgi apparatus surround the vacuole in such a manner that they may be mistaken for vacuole walls in certain preparations. 420 THE’ CONTRACTILE VACUOLE The presence of definite vacuole walls or morphological membranes around the vacuoles of ciliates is not accepted by all authors. Thus, Taylor (1923) believes the vacuole in Ezp/lotes to disappear completely at sys- tole, and to be replaced by an entirely new structure. If a morphological membrane were present, this could hardly obtain, although Taylor was able to distinguish a ‘‘highly viscous boundary’’ of endoplasm surround- ing the vacuole. Moore (1931) was unable to cause osmication of the vacuole wall in Blepharisma undulans, using the technique of Nassonov, and from this she concludes that the vacuole lacks a permanent wall. This opinion was again expressed (1934) after further observation. The findings of Moore afte in opposition to those of Haye, who re- ported thin vacuole walls in the same species. Day (1930) concludes from his observations on Paramecium caudatum, Spirostomum ambi- guum, and S. teres that vacuoles in these forms are temporary structures which disappear at systole. King (1935, p. 564) found that: The permanent components of the contractile vacuole system in Paramecium multimicronucleata include the pore with its discharging tubule, and the feeding canals, each made up of a distal excretory portion, an ampulla and an injection tubule. . . . The membrane of the contracting vacuole is a temporary structure, disappearing at systole. The pore is closed by the remnant of the old vacuole which ruptures at the next systole. Essentially the same was found in P. aurelia. These observations were made on material osmicated at 38° C. It is somewhat surprising that the vacuole proper of the contractile vacuole systems in P. multimicro- nucleata and P. aurelia is a temporary structure, replaced anew after each contraction, whereas that of the closely related species P. caudatum is commonly believed to be a permanent structure. Perhaps the stainable vacuole wall described by Young for P. cavdatum represents the same kind of material which King believes closes the excretory pore follow- ing systole in P. multimicronucleata and P. aurelia. After examining both living and stained material, Fortmer (1926) concludes that the membrane of the vacuole in Protista is a temporary structure, which, after fulfilling its purpose, closes the excretory pore during the period of diastole. He further believes that all surface layers sharing in the excretion process have the property of fusing together again merely on contact. The present state of the knowledge concerning the presence or absence THE CONTRACTILE VACUOLE 421 of membranes around contractile vacuoles is exceedingly unsatisfactory. Aside from the fact that many investigators admit the probability of, or in some instances the necessity for, a physiological membrane of the type described above as composed of molecules oriented and more closely packed together at the phase boundary, very little of a positive nature is known. It is difficult to understand how a vacuole without any sort of membrane can retain its identity in cytoplasm with which its contents ap- pear to be freely miscible. That most of the cellular contents are freely miscible with water is indicated by the fact that discharge of the cell contents into the surrounding water, following rupture of the cell wall, is soon followed by dispersion of most of the cytoplasm into the sur- rounding medium; usually only granules, of one sort or another, re- main to indicate the original position of the extruded material. If the vacuole content is mostly water, a belief quite generally if not universally held, how can a vacuole ever be formed in the absence of any kind of membrane to prevent this water from flowing back into the cytoplasm as rapidly as it is mobilized? As indicated above, a great many investigators have demonstrated structures which were interpreted as vacuole “walls.” In some material these walls were visible in living organisms as layers of substance, opti- cally different from substances on either side of it. In other material the walls were visible only after fixation and staining. One is more or less obliged to accept an author’s description of structures in material ex- amined by him; but an observation sometimes is subject to two entirely different interpretations. This is clearly shown by MacLennan (1933) in his work on the Ophryoscolecidae, as pointed out earlier. In several in- stances the presence of morphological membranes is claimed by various. authors, on the basis of observations on what may have been overim- pregnated material. Because of the extreme thinness of physiological membranes, it is doubtful if they ever can be demonstrated visually, but evidence obtained from the study of other colloidal systems indicates that they almost certainly exist. THE FUNCTION OF CONTRACTILE VACUOLES During the years that have intervened since the discovery of the con- tractile vacuole, an extensive literature concerning its function (or func- tions) has accumulated. Excellent reviews of this literature have been 422 THE CONTRACTILE VACUOLE published from time to time (Howland, 1924b; Day, 1927; Lloyd, 1928). Therefore no attempt will be made to present another review at this time, except in so far as the works to be mentioned have a direct bearing on one or the other of the two functions generally conceded to be most probable. Of the various functions assigned to the contractile vacuole those of excretion of metabolic waste products and regulation of hydrostatic pres- sure within the cell have received most frequent support. Some authors prefer to limit “metabolic waste products’ to nitrogenous substances, although others include carbon dioxide as well. In view of the scarcity of evidence bearing directly on the subject, it hardly seems advisable at this time to distinguish between different kinds of metabolic wastes. On the other hand, if one is to understand excretion to mean the expulsion of any sort of waste material from the organism, then the function was defi- nitely established as excretory when Stokes (1893), and later Jennings (1904) proved the discharge of the vacuole to the exterior. But such a generalization offers little satisfaction. Probably the earliest suggestion that the vacuole is an excretory organ- elle was made by Stein and Schmidt (see Kent, 1880, p. 69), who stated that “the functions discharged by the contractile vacuole are ex- cretory and correspond most nearly with that of the renal organs of the higher animals.” Griffiths (1888) made the statement, based on his own experiments, that the vacuole performs the function of a kidney, and that its secretions are “capable of yielding microscopic crystals of uric acid.”” As material for these experiments he used Amoeba, Parame- cium, and Vorticella. In describing these experiments, Griffiths says (p. 132) After the addition of alcohol minute flakes could be distinctly seen floating in the fluid of certain vacuoles. Bearing in mind the murexide reaction, there is every reason to believe that these flakes are nothing more or less than minute crystals of uric acid. These experiments were repeated many times, generally with positive results, indicating the presence of uric acid. At times, however, the vacuole was found not to contain the slightest trace of uric acid. Howland (1924b) repeated these experiments using Paramecium, Centro pyxts, and Amoeba, but always with negative results. However, uric acid was found in cultures of Paramecium and Amoeba by Howland, and the TEECCON TRAC TILE) VACUOLE 423 concentration was observed to be roughly proportional to the age of the culture. From this she concludes that uric acid is excreted by these forms, though probably not by the vacuole. Experiments of Nowikoff (1908), Shumway (1917), and Riddle and Torrey (1923), in which the effects of thyroid feeding and the re- sponse of Paramecium to thyroxin were observed, offer further though indirect evidence in favor of the excretory function. Flather (1919) found that epinephrine, posterior pituitary extract, and pineal gland ex- tract produce similar results—an acceleration in pulsation frequency, and a dilatation of the vacuole. Since these drugs cause diuresis in vertebrates, the action on vacuoles may be interpreted as resembling stimulation of excretion. Weatherby (1927) found that urea is excreted by Paramecium cauda- tum, but was unable to detect urea in the fluid of the contractile vacuole | by means of the micro-injection of his own modification of the xanthydrol reagent of Fosse (1913). This reagent yields positive results with dilu- tions of urea as great as one part in 12,000. Calculations based on the volume of fluid eliminated by vacuoles and the quantity of urea excreted by known numbers of organisms in mass cultures indicate that the con- centration in fluid of the vacuole would be of the order of one part in 2,000 or 3,000, if all the urea were excreted via this route. It therefore appears that at most only a small part of the total urea is excreted in this manner. After removal of the fluid from the contractile vacuole of Spirostomum by means of micro-manipulation apparatus, and subsequent hydrolysis with urease, Weatherby (1929) found urea to be present in the vacuolar fluid in a concentration of about one part in 100,000. Cal- culations of the rate of excretion of urea by known numbers of Sperosto- | mum in mass cultures indicate that this amount of urea accounts for only about one percent of the total urea excreted. Parnas (1926) concludes from observed differences in pulsation fre- quency that the vacuole is mainly excretory in marine Protozoa, and both excretory and osmotic-pressure-regulatory in fresh-water forms. The ex- cretory function is accepted apparently without reservation by von Gelet (1925, 1928), who homologizes the various parts of the vacuole system in Paramecium with the vertebrate kidney, ureter, bladder, and urethra, although he admits the possibility that this system may aid in removing excess water from within the organism. In Paramecium, von Gelet states 424 THE CONTRACTILE VACUOLE that the vacuole removes approximately ten times as much water as is taken in with food, a fact which he fails to correlate with his claim of a predominantly excretory function. Day (1927) suggests that vacuoles in Amoeba originate in “the fusion and coalescence of ultramiscroscopic droplets of soluble katabolic waste which may include water of osmosis.”’ He observed that conductivity water increases size, number, and pulsation frequency of vacuoles. Essentially the same observations and conclusions were extended by him to Paramecium and Spirostomum (1930). Mac- Lennan (1933) observed in the Ophryoscolecidae that granules accumu- late around the vacuole during the early part of diastole and then are gradually reduced in number. The formation of accessory vacuoles in these granular regions involves a solution of granules in the vacuolar fluid. He suggests this as a possible method for the elimination of kata- bolic wastes. Since he found the pellicle of these organisms to be rela- tively impermeable, MacLennan believes an excretory function to be all the more probable in these forms, since the vacuole is the only visible means for the removal of wastes. Adolph (1926) found that no change of external conditions alters significantly the rate of elimination of fluid by vacuoles of Amoeba, and from this concludes that water is not elimi- nated merely because it has unavoidably diffused into the body. Dimitrowa (1928) observed (as mentioned earlier) that mechanical interference, as by pressure on the cover glass, induces the development of extra vacuole systems in Paramecium. In most instances these vacuoles assumed normal structure, size, and pulsation frequency, although in some cases there were actively pulsating vacuoles with no radial canals. Dimit- rowa explained the formation of extra vacuoles, as well as of those nor- mally formed at fission, by the assumption that if for one reason or an- other the excretory organs become inadequate to remove wastes, extra organs are formed. If one vacuole is rendered ineffective by mechanical interference, then another is formed to take over its function. Likewise, since metabolism is thought to be increased during fission, new vacuoles are formed to care for the increased production of wastes. Extra vacuoles, induced artificially, obviate the necessity for the formation of a like number at fission, since an ample excretory function is already present. Somewhat contradictory evidence has been presented by various authors concerning the nature of nitrogenous end products of metabolism in the Protozoa. As previously mentioned, Griffiths (1888) reported uric acid LAE IGONTRACTILE: VACUOLE 425 in the vacuolar fluid of several forms. Howland (1924b) was unable to confirm this, but found uric acid in mass cultures of Amoeba and Parame- cium. Weatherby (1929) found urea to be excreted by Paramecium and Spirostomum, but detected no ammonia nor uric acid; ammonia, as well as a questionable trace of uric acid, were found to be excreted by Didz- nium. Specht (1934) found that Spzrostomum exctetes ammonia, the amount being augmented by lack of oxygen and minimized by abundance of it. Weatherby noticed that cold aqueous extracts of many substances commonly used in culture media (hay, wheat, barley, rye, oats, malted milk, beef extract, blood fibrin, and blood albumen) yield positive tests for uric acid, and suggested this as a possible source of the uric acid found by Howland in cultures of Paramecium and Amoeba. Lwoff and Roukhelman (1926) found amino-nitrogen as well as additional nitro- gen, which they report as ammonia plus amide-nitrogen, in pure cultures of Glaucoma. No urea nor uric acid was present. Doyle and Harding (1937) analyzed the food (in the form of Psewdomonas) supplied Glau- coma, and found that most of the nitrogen present was excreted as am- | monia approximately six hours after ingestion of food. No urea was detected. If the contractile vacuole is active in excretion of nitrogenous wastes, as is frequently maintained, then one would expect it to be able to ex- crete certain dyes which had been injected into the cytoplasm. Many at- tempts doubtless have been made to demonstrate such a phenomenon, but few accounts of such experiments are to be found in the literature. Apparently negative results have discouraged publication. A personal com- munication from one investigator reports complete failure to demon- strate elimination of dyes by way of the contractile vacuole, although the dyes used in these experiments are known to be excreted readily by the kidney of higher forms. Howland and Pollack (1927) found that Picric acid, injected into the cytoplasm of Amoeba dubia, is picked up and excreted by the contractile vacuole. Ludwig (1928) studied gaseous metabolism in Paramecium, and found that the amount of oxygen dissolved in water taken with food is insignificant, compared with the respiratory requirement of the organism. For the satisfaction of the oxygen requirement, there must be a quantity of water, saturated with oxygen, equivalent to 260 to 30,000 times the amount taken in through the gullet. Oxygen intake must also occur 426 THE CONTRACTILE VACUOLE through the cell surface. The amount of water expelled by the vacuole corresponds within reasonable limits to that necessary for the excretion of carbon dioxide, if it is assumed that this water is saturated with the gas. From this Ludwig concludes that the vacuole is of special significance not only in the regulation of osmotic pressure within the cell, but also in excretion of carbon dioxide. Evidence bearing directly on the excretory nature of the vacuole func- tion is exceedingly scant, and for the most part negative. The reason for the relatively few observations is immediately apparent to all who have attempted experiments of this nature. Perhaps a more thorough investigation of the nature of nitrogenous waste products in other Protozoa will suggest more effective methods for answering the question, by reveal- ing other chemicals which may be detected more readily. Hartog (1888), Degen (1905), Zuelzer (1910), Doflein (1911), and others maintain that the contractile vacuole is concerned primarily in the regulation of hydrostatic pressure within the cell, or the preven- tion of overdilution of the cell contents by water taken into the cell in feeding as well as through the cell membrane by osmosis. Harvey (1917) found that Noctiluca, which normally lives near the surface of sea water, sinks when transferred to diluted sea water, but ultimately rises to the surface again. Meanwhile, expansion takes place, owing to the taking up of water by the organism. This passage of water from exterior to interior is from a region of higher concentration to one of lower concentration, and therefore contrary to the laws of osmosis in simple systems. When organisms are transferred from diluted sea water to pure sea water, they shrink, vacuoles are formed, and these appear to discharge to the exterior in somewhat the same manner as contractile vacuoles. This appears to aid in reéstablishing the normal salt concentra- tion within the organism. Hance (1917) made extensive observations on a race of Paramecium possessing extra contractile vacuoles. He found that these animals cannot withstand immediate immersion in water containing 0.5 percent sea salt, but can be acclimated gradually to this concentration. The number of vacuoles is not reduced by this treatment, but the pulsation frequency is reduced. This response may be taken to indicate a decreased rate of entry of water into the cell, presumably because of the higher external osmotic pressure; but Hance observed also an increased viscosity and toughness of the pellicle, which may indi- THE CONTRACTILE VACUOLE 427 cate that a decreased permeability of the cell wall is partly responsible. Herfs (1922) investigated the effects of changes in tonicity of the external medium on several kinds of organisms, both free-living fresh- water forms and parasitic forms. He found the pulsation frequency in Paramecium to be decreased to about one-fourth the normal when the organism is transferred from fresh water to 0.75 percent NaCl solution. Lower concentrations of salt produce less marked changes. Gastrostyla Steini’ showed essentially the same reaction, except that organisms kept for about fourteen days in one-percent NaCl solution were found to contain no contracting vacuoles. With Gastrostyla the vacuole seems to disappear at a NaCl concentration of 1.1 percent to 1.3 percent, and to reappear at a concentration of about 0.5 percent. The pulsation fre- quency of Nyctotherus cordiformis, an intestinal parasite of the frog, was found to vary between wide limits, presumably because of cor- responding variations in the water content of the medium. Graded pulsa- tion frequencies were observed zz vitro when the exterior medium varied from tap water to one-percent NaCl solution. Opalina ranarum, which possesses no contractile vacuole, can adapt itself to relatively wide varia- tions in tonicity of the exterior medium without developing a vacuole, if the changes are made gradually. From this Herfs was led to doubt whether or not a vacuole is necessary for the prevention of overdilution of the cytoplasm. He noted further that the lack of a vacuole in O palina goes hand-in-hand with the lack of a cell mouth; whereas Nyctotherus possesses both a cell mouth and a vacuole. From this he assumes that it is the water taken in through the mouth that is pumped out by the vacuole. Herfs does not seem to be altogether consistent in this idea, since he further states, as his opinion, that in ordinary cases at least the water taken in through the entire cell surface is of decisive signifi- cance for the appearance of the vacuole. With respect to the adaptation of organisms possessing no vacuoles to variations in tonicity of the ex- terior medium, Herfs seems to have overlooked as an explanation, the possibility of an interchange of salts between cell and medium, a pos- sibility which will be mentioned again later. Eisenberg (1926), assuming the volume of Paramecium to be ap- proximately equal to that of an ellipsoid of rotation having the same dimensions, found that the two vacuoles discharge a volume of liquid equal to that of the organism in 20 minutes, 51 seconds. The average 428 THE CONTRACTILE VACUOLE amount of fluid expelled in three experiments, each of 10 minutes’ dura- tion, was 45,000 cubic micra; and the average amount of water taken into the organism with food was 11,700 cubic micra, or about one- fourth the total amount expelled. In animals not feeding, the entire amount of fluid expelled entered the body otherwise than with food. Basing his theory on the work of Nirenstein as well as on his own observations, Eisenberg concludes that water penetrates the body by way of the peristome, even when a food vacuole is not in process of forma- tion. It was further observed that an increase in the osmotic pressure of the exterior medium results in a decrease in pulsation frequency, and that equi-osmotic solutions of different chemicals may cause different degrees of slowing. Fortner (1926) concludes, largely on the basis of theoretical con- siderations, that the vacuole operates for the preservation of vital cell turgescence, since there must be an accumulation of water in the proto- plasm because it is surrounded by a membrane impermeable to water and aqueous solutions. Eisenberg (1929) investigated the relationship between the osmotic pressure and the pulsation frequency of the vacuole in Balantidium entozoon. He found that the frequency of the formation of vacuoles depends on the osmotic pressure, and is all the greater the more the pressure is reduced below that of the usual environment. A pulsation frequency accelerated by the removal of the organism to a medium of lower osmotic pressure does not remain accelerated, but returns to normal after a certain period of time. The rapidity and extent of this return to normal are proportional to the osmotic pressure of the medium. Frisch (1935) was unable to adapt Paramecium caudatum and P. multimicronucleata to sea water, the organisms dying when the concen- tration reached 40 percent. However, among other marked changes in the organisms was a pronounced decrease in pulsation frequency of the vacuoles. Day (1930) concludes from his observations on Spirostomum and Paramecium that the vacuole is a hydrostatic organelle, which functions also in elimination of metabolic wastes. He found conductivity water to increase the size, number, and rate of pulsation of vacuoles. The lowering of the temperature of the culture medium slows the organisms and retards the contraction rate of vacuoles, while the raising of the THE CONTRACTILE VACUOLE 429 temperature increases movement and pulsation frequency. Kitching (1934) found that the rate of output of fluid from the contractile vacuole of a fresh-water peritrich ciliate is decreased to a new steady value immediately, when the organism is placed in a mixture of tap water and sea water. The rate of output returns to its original value immediately, when the organism is replaced in tap water. Pulsa- tion is stopped when the medium contains more than 12 percent of sea water. Transference of marine peritrich ciliates from sea water to mixtures of sea water and tap water leads to an immediate increase in body volume, to a new and generally steady value. Return of the organ- isms to pure sea water results in an immediate return of body volume to normal, or less. When the concentration of sea water is less than 75 percent, the pulsation rate increases, and then generally falls off slightly to a new steady value which is still considerably above the normal in sea water. The maximum sustained increase in rate observed by Kitch- ing was 80-fold. From these observations it is concluded that the vacuole is probably a regulator of hydrostatic pressure in the fresh-water Protozoa, | but in those marine Protozoa which possess vacuoles the functions remain obscure. Hyman (1936) believes that the vacuole in Amoeba vers pertilio serves to discharge water which has necessarily entered the cell from a hypotonic medium. One of the most remarkable instances of adjustment of a protozoan to abnormal media is shown in the experiments of Hopkins (1938) on the marine amoeba, Flabellula mira. He found that this amoeba can be cultured in any concentration, from sea water diluted twenty times with fresh water to sea water concentrated ten times by evaporation. It never forms contractile vacuoles such as are typical for fresh-water Protozoa. The food vacuoles, when extruded from the cell, contain large quantities of water as well as fecal material. The rate of elimination of fluid by means of these vacuoles is inversely proportional to the concentration of the medium, and directly proportional to the volume of the amoeba. When the concentration of the medium is decreased, the organism swells at first, and then shrinks to its original volume. During shrinkage, elimination of fluid by food vacuoles does not nearly account for the volume loss. If the concentration of the medium is increased, the amoeba shrinks at first, and then swells to its original volume. Only a small 430 THE CONTRACTILE VACUOLE incfease in concentration is necessary to cause shrinkage, indicating an osmotic value for the cytoplasm, after adjustment, only slightly above that of the medium. Hopkins concludes from these observations that when the medium is either diluted or concentrated, the organism auto- matically loses or gains osmotically active substances to or from the medium respectively, in such proportion that when adjustment is com- pleted the osmotic value of the cytoplasm is but slightly higher than that of the medium, and that this is accomplished independently of the action of vacuoles. Herfs, whose observations on Opalina have been described previously, may find such an explanation applicable to the unexpected behavior of this organism. It is interesting to speculate as to whether or not such adjustment to external osmotic-pressure differences as postulated for Flabellula, and possibly Opalina, represents the most primitive type of mechanism for this type of adjustment with Noctiluca (see Harvey 1917), which develops contractile vacuoles when the tonic- ity of the external medium is greatly reduced, occupying a position inter- mediate between Flabellula and those forms which possess vacuole sys- tems. In spite of the quite extensive literature dealing with the question, one is obliged to admit that virtually nothing has been proved beyond question concerning the function or functions of contractile vacuoles. Car- bon dioxide and nitrogenous wastes of one sort or another are undoubtedly excreted by Protozoa. It is reasonable to suppose that at least a part of these highly soluble wastes finds its way into the fluid of the vacuole and is excreted in this manner. Many authors hold that it is not only reasonable to suppose this, but that it is unreasonable to suppose that it does not occur. But, be that as it may, the contractile vacuole certainly has not been proved an organelle whose main function is excretion of metabolic wastes. Likewise, there is indisputable evidence that many fresh-water Protozoa show a decreased pulsation frequency when the tonicity of the exterior medium is increased; and there is equally valid evidence indicat- ing that the reverse occurs when many marine and parasitic Protozoa are transferred to a medium having a decreased tonicity. One may regret the fact, but it is none the less true, that these observations prove nothing more than the bare statement which describes the observations. They strongly suggest that the vacuole operates to prevent excessive dilution of the cytoplasm, or to regulate osmotic pressure within the THE CONTRACTILE VACUOLE 431 cell, but beyond this the interpretation is subject to criticism. As stated by Calkins (1926), these supposed functions are not necessarily exclu- sive, and the possibility still exists that other functions, as well as these, are performed by the contractile vacuoles. CONTRACTILE VACUOLES AND THE GOLGI APPARATUS Few publications within recent years on the general subject of con- tractile vacuoles have aroused as much interest or stimulated as much constructive research as that of Nassonov (1924), in which he sug- gests that the vacuole in Protozoa is homologous with the Golgi ap- paratus in metazoan cells. Neglecting for the moment the ultimate status of this proposed homology, one must admit that this article is responsi- ble, either directly or indirectly, for valuable work which otherwise might have been delayed indefinitely. Before attempting a discussion of the literature bearing on this pro- posed homology, a few words concerning the general nature of the Golgi apparatus may be of benefit. Its discovery in 1899 is attributed to the man whose name it bears. For approximately twenty-five years after the first description of such a structure its actual existence was doubted by many competent cytologists. Demonstration of the Golgi apparatus in most cells requires a somewhat rigorous treatment of the tissue with various chemical agents, some of which may reasonably be suspected of leaving in the cytoplasm chemical or physical changes of such a nature as to be visible after the Golgi technique, when in reality no such structures exist, pre-formed, in the cell. The problem is probably complicated even further by the multiplicity of forms and shapes which the Golgi apparatus is observed to assume in different cells. At present there seems to be little doubt but that such structures exist, pre-formed, in most cells. Many investigators go so far as to state that the Golgi apparatus is one of two or three cytoplasmic constituents which are in- variably present in all cells, both plant and animal. If this is true, then it is probable that the rdle of the Golgi apparatus in the life history © of the cell is of very great importance. Demonstration of the Golgi apparatus, or Golgi bodies as the struc- tures are frequently called, depends on the reduction of certain metallic compounds to the free metals, the compounds most frequently used con- taining either osmium or silver. The reduced metal results in blackening 432 THE CONTRACTILE VACUOLE of the structure. It has been observed that treatment of stained material with turpentine or hydrogen peroxide results in the bleaching of most structures other than the Golgi apparatus, which may have been black- ened by the procedure; Golgi bodies resist even prolonged bleaching effects of these agents. Structures which normally are blackened by the Golgi technique, or any of its modifications, are not blackened if the cell or tissue is first subjected to alcohol or dilute acetic acid. For these reasons, and others which need not be mentioned here, the Golgi ap- paratus is thought to be composed largely of lipoid substances. Together with the Golgi apparatus, mitochondria are generally con- ceded to be invariably present in all cells. These structures, variously called chondriome, chondriosomes, cytomicrosomes, and so forth, are frequently present in the form of short rods or ovoid granules, although the shape is not constant for different types of cells. Some authors main- tain that besides the Golgi apparatus and mitochondria a third invariable cytoplasmic constituent, the vacuome, is also present. Whether invari- ably present or not, the vacuome is at least frequently found in cells. The literature dealing with these structures is exceedingly confusing, owing largely to the lack of a uniform nomenclature. Repeatedly several authors have written of the same structure under different names, or different structures under the same name. The lack of standard techniques also contributes to the confusion. One of the most commonly used tech- niques for differentiating between the Golgi apparatus and mitochondria is staining of the tissue with a mixture of neutral red and Janus green; mitochondria readily stain with the latter, and some authors maintain that the Golgi apparatus is stainable with neutral red. The vacuome also stains readily with neutral red, and on this basis it has been proposed that the two structures, Golgi apparatus and vacuome, are identical. Others have found within the same cell neutral-red stainable inclusions which are not osmiophilic, and osmiophilic inclusions which are not neutral-red stainable; so it appears that the two structures are not identi- cal in all cells, but in some exist as separate entities. Furthermore, it is sometimes claimed that osmiophilic bodies (the Golgi apparatus) are derived from mitochondria (Janus-green stainable, but not osmiophilic). If this latter is true, then one might expect to find in occasional cells structures which are both osmiophilic and Janus-green stainable, although such a situation has not come to the attention of the author. Unfortu- THE CONTRACTILE VACUOLE 433 nately, no method or group of methods has been devised for the identifi- cation of these structures, which is acceptable to all concerned. However, for the protozoan Golgi apparatus it is generally conceded that in most instances it exists as granules, globules, spherules, short rods, or ovoid structures; but there appear to be many exceptions. These bodies reduce certain osmium and silver compounds to the free metals, thereby causing a blackening of the structures which resists bleaching with turpentine and hydrogen peroxide. Most stains commonly used in cytological studies are ineffective, although in some instances neutral red is found to stain some structures which answer other requirements for the true Golgi apparatus. Except for the occasional positive reaction to neutral red, the protozoan Golgi apparatus reacts in a manner practically identical with that of the metazoan Golgi apparatus. Nassonoy (1924) demonstrated the presence of osmiophilic mem- branes around the vacuoles in Paramecium caudatum, Lionotus folium, Nassula laterita, Campanella umbellaria, Epistylis gallea, Zoothamnium arbuscula, V orticella sp., and Chilomonas paramecium. These membranes he found to be permanent structures, merely collapsing at systole of the vacuole—not disappearing, to be reformed anew during the next period of diastole. In Paramecium the vacuole system was found to consist of a thin-walled reservoir and filling canals, the latter composed of the short injection canal, the ampulla, and the distal section. The distal section Nassonov found to be surrounded by a specially differentiated plasma, from which hypertonic fluid is secreted into the lumen of the canal. This hypertonicity results in the passage of water into the canal, and ultimately into the vacuole. The vacuole wall in Paramecium is considered not to take part in secretion, but to serve only as a temporary reservoir or bladder. In other forms which possess no filling canals, the osmiophilic vacuole wall is considered capable of performing the secre- tory function as well. The formation of small droplets of fluid within this wall was sometimes seen to occur, following partial systole of the vacuole. Subsequent observations by Nassonov (1925) on Chilodon and Dogielella necessitated a modification of the original view so as to include conditions which were not observed in the organisms men- tioned in the earlier article. In Chilodon the osmiophilic material appears as a heavy black ring, although this ring is not always complete. If 434 THE CONTRACTILE VACUOLE fixation occurs at diastole, the vacuole appears to lie within the ring. The ring does not collapse at systole, but remains more or less un- changed. The vacuole is believed to be formed by the flowing together, or coalescing, of small droplets (Sammelvacuolen) which form within the substance of this ring. In Dogielella the osmiophilic material is in the form of a ring around the vacuole, resembling, as Nassonov de- scribes it, the rings around the planet Saturn. On contraction of the vacuole, the ring remains essentially unaltered, showing a certain amount of elasticity. The vacuole seems to arise as a result of the coalescing of numerous droplets of fluid, just as in Chilodon. These two forms, as well as many others described by other authors, represent a separation of the Golgi apparatus from the vacuole, although the close functional associa- tion remains. Nassonov’s conception of this close functional association is expressed in a third publication (1926), in which he states that the Golgi apparatus serves as a mechanism for collecting certain materials from the cell substance and preparing them in such a way that they can be discharged from the cell by the vacuole. To do this the Golgi apparatus need not be a part of the vacuole system, nor even in direct contact with it. This conception represents an important departure from the first, in so far as morphology is concerned, but does not alter the essential physiological relationship. Further evidence that such is the function of the metazoan Golgi apparatus was obtained from experiments in which the dye, Trypan blue, was injected into mice. On examination of sections taken from the livers and kidneys of these mice, it was found that the dye was concentrated in that region of the cells of the liver and of the convoluted tubules of the kidney in which the Golgi apparatus is situated. Distribution of mitochondria in these cells was found to be quite different, indicating that these structures are not intimately assoct- ated with the collection of the dye. Some authors summarily reject the idea of a relationship between vacuole and Golgi apparatus, solely on the ground that the wall of the vacuole proves not to be osmiophilic. However, some of these same authors present evidence which supports the idea of a physiological relationship, even though the actual identity of the two structures is dis- proved. Nassonov himself was among the first to demonstrate that by no means all contractile vacuoles have osmiophilic walls, but this does DHE ICONTRACTILEE VACUOLE 435 not alter the possibility of such a functional relationship as he sug- gested. Brown (1930) found that the Golgi apparatus of Amoeba proteus is the characteristic protozoan type of globules and spherules, with clear centers and dark rims. From a central focus these spherules appear under the microscope to be crescent-shaped structures. He suggests that the minute vacuoles which occur in the endoplasm of Amoeba are as- sociated in some way with these crescent-shaped structures, and that they unite to form the contractile vacuole. Brown further suggests this as the reason that the vacuole in this form is not blackened by osmic acid, as it is in Paramecium. Hall (1930a) found small globular inclusions in Trichamoeba which are osmiophilic, and which resist bleaching by either hydrogen peroxide or turpentine. These inclusions are similar in size and distribution to those which are stained vitally by neutral red. In material impregnated by the Kolatchev method, the contractile vacuoles are not blackened. In material prepared according to the Mann-Kopsch method, small globules, similar to those seen in the Kolatchey material, are blackened. These globules likewise resist bleaching by turpentine and hydrogen peroxide. In the Mann-Kopsch material, small vacuoles—two, three, or more in number—are blackened in many amoebae. In a few instances a number of blackened globules were seen adherent to the wall of the contractile vacuole, which, on casual examination, gave the appearance of a vacuole with blackened walls. Hall suggests that in material less effectively bleached, such a condition might easily be mistaken for heavily impreg- nated vacuoles. Nigrelli and Hall (1930) report the presence of small osmiophilic and neutral-red stainable granules in Arcella vulgaris. Mast and Doyle (1935) apply the name “‘beta granules’ to small structures, usually spherical but sometimes ellipsoidal or rod-like in shape, which have a diameter of about one micron. These granules are distributed more or less uniformly throughout the cytoplasm, except at the surface of the contractile vacuole, where they tend to become con- centrated in a layer. Aggregation of granules on the surface was described by Metcalf (1910), as previously mentioned. These granules, accord- ing to Mast and Doyle, are stained vitally by Janus green, but only on the surface, indicating that they have a differentiated surface layer similar to that in mitochondria. In addition to beta granules, these authors in- 436 THE CONTRACTILE VACUOLE vestigated other more or less spherical cytoplasmic inclusions, which they call “cytoplasmic refractive bodies.” The outer layers of these bodies are readily stained by neutral red and osmium, whereas the central portions react negatively to osmium and stain but faintly with neutral red. Apparently these are the same structures studied by Brown, who believes them to give rise to minute vacuoles which are precursors to contractile vacuoles. The beta granules are not blackened by osmium. Many of these granules are usually situated close to the contractile vacuole, while others are scattered throughout the entire cytoplasm. The pulsation frequency of the vacuole is proportional to the number of beta granules remaining after some have been removed by operation, indicating a close relationship between granules and vacuole function. Removal of most of these granules results in the death of the organism. The relationship between the contractile vacuole and cytoplasmic in- clusions in Amoeba is puzzling. One would be inclined to accept, at least tentatively, the idea of the origin of vacuoles in the beta granules which surround it, were it not for the fact that in a variety of other Protozoa the vacuole has been seen to originate as minute droplets in the region of the cell occupied by osmiophilic granules. Yet in A. proteus the granules among which the vacuole apparently arises are not osmio- philic nor stainable by neutral red, but are stainable by Janus green. It might be suggested that the situation in Azzoeba is the reverse to what it appears to be in other Protozoa, but such a suggestion offers no satis- faction. A more likely explanation lies in the uncertainty of identification of these cytoplasmic inclusions. Some authors (e.g., Hall, 1930a) consider the vacuome, which is neutral-red stainable, identical with the Golgi apparatus, which is osmiophilic; this Hall observed to be true in Trich- amoeba. Others (e.g., MacLennan, 1933) have identified both neutral- red stainable and osmiophilic granules as separate structures within the same organism. Apparently Dunihue (1931) finds the same in Para- mecium. Further, MacLennan observed that the only granules in Evdz- plodinium which can be impregnated with osmium are those found in the vacuolar region; yet in a study of living material, it was shown that this region is composed of granules which originate in the surrounding ectoplasm. Therefore, these granules, as they assemble in the vacuolar region, undergo some change, either chemical or physical or both, which makes them osmiophilic. It has been suggested at one time or another THE CONTRACTILE VACUOLE 437 that the Golgi apparatus is derived from mitochondria. Until this puz- zling situation is clarified, it seems necessary to assume that in some organisms neutral-red stainable granules (vacuome), osmiophilic gran- ules (the Golgi apparatus), and Janus-green stainable granules (mito- chondria) exist as separate and distinct entities, whereas in others the Golgi apparatus may be combined with one or the other of the two remaining types of granules. Hirschler (1924) found only one kind of lipoid body in Gregarina and Spirostomum, and suggested that these represent a primitive type of organism in which Golgi apparatus and mitochondria are combined in a single type of granule. Until the identity and function of the various types of granules in A. proteus have been investigated further, it is difficult to arrive at any reasonable conclusion concerning the relationship of the contractile vacuole to them. Hirschler (1927) examined a variety of organisms after fixation and staining with several dyes, as well as impregnation with osmium and silver. From these studies he concludes that both Golgi apparatus and mitochondria are present in Bodo lacertae, Lophomonas blattarum, L. striata, Try pano plasma dendrocoeli, Entamoeba blattae, Monocystis agilis, Trypanoplasma helicis, Diplocystis phryganeae, Gregarina polymorpha, and Clepsidrina blattarum. In these organisms the Golgi apparatus and the mitochondria were shown to have the same staining reactions as corresponding structures in metazoan cells. Hall (1929) found osmiophilic granules which resist bleaching with hydrogen peroxide in Peranema trichophorum, in Menoidium and Eu- glena (1930b); in Chromulina sp., Astasia sp., and Chilomonas para- mecium (1930c); and in Stylonychia (1931). Hall and Dunihue (1931) found similar granules, or globules, in Vorticella. In many of these ex- periments two or more methods of osmium impregnation, as well as silver impregnation, were used. In some of them the osmiophilic bodies were found to be stainable with neutral red also. In some species the wall of the contractile vacuole was found to be osmiophilic after pro- longed osmication, but generally this was readily bleached by hydrogen peroxide or turpentine. Janus green and neutral red were used as vital stains for several organisms; in these the osmiophilic granules were identified as the neutral-red stainable material, whereas smaller granules were stained with Janus green. Fauré-Fremiet (1925) observed in several species of Vortzcella es- 438 THE ‘CONTRACTILE. VACUOLE sentially the same type of structure as that described by Nassonov. The vacuole wall was found to be in the form of a ring deeply blackened by osmium. Following systole, the vacuole collapses, but the wall re- mains quite evident. Small vesicles or droplets appear within the thickness of the wall, fuse together, and thus give rise to the new vacuole. On the other hand, Finley (1934) demonstrated, by means of recognized osmium and silver-impregnation techniques, discrete globular inclusions in the cytoplasm of Vorticella convallaria, V. microstoma, and V. cam- panula. These globules were readily distinguishable from the rod-shaped mitochondria by staining with a mixture of Janus green and neutral red, the globules reacting positively to neutral red and negatively to Janus green, whereas with mitochondria the reverse was true. Moore (1931) found distributed through the entire endoplasm of Ble pharisma globules with osmiophilic cortices and osmiophobic centers. These structures resist bleaching with turpentine. Only in instances of overimpregnation is the contractile vacuole blackened in this form, al- though paramecia, mixed with the Blepharisma uniformly show black- ened vacuole systems. Where impregnation of the vacuole is produced in Ble pharisma, it is readily bleached with turpentine. No evidence was noted by Moore that in these osmiophilic globules lay the origin of the contractile vacuole. In a later investigation, Moore (1934) found that the secondary vacuoles do not empty their contents into the primary vacuole, and thus contribute to its filling; but as contraction of the primary vacuole occurs, the secondary vacuoles move into the place it had occupied, where they coalesce to form a new primary vacuole. No “excretory granules” were observed, but in the earlier work Moore de- scribed osmiophilic globules scattered throughout the cytoplasm. On the basis of these observations, Moore rejects the Nassonov homology for Ble pharisma. King (1933) found in Ezplotes that the vacuoles termed group V, by Taylor (1923) have their origin at the distal ends of a very large number of collecting tubules, located just under the ectoplasm on the dorsal surface of the organism. The presence of these tubules was demon- strated by impregnation with osmium. King believes that these tubules, or canals, like those in Paramecium, are responsible for collection of fluid which ultimately reaches the contractile vacuole. In a comprehensive series of observations on the Ophryoscolecidae, THE CONTRACTILE VACUOLE 439 MacLennan (1933) found that the osmiophilic granules contribute di- rectly to the formation of accessory vacuoles, which in turn form the contractile vacuole. With respect to the possible function of the vacuole, he states (p. 236): The vacuolar region found in these ciliates shows definite evidence of the elimination of materials by means of the vacuolar fluid and corresponds to the secretary region or “region of Golgi’ in gland cells. The nature of the materials eliminated by the vacuolar region was not determined in this in- vestigation. Since, however, the pellicle in the Ophryoscolecidae has been shown to be relatively impermeable and since the vacuolar region is the only demonstrable path by which materials are constantly being passed to the exterior, it is likely that the katabolic wastes of these ciliates are eliminated by this organelle rather than by direct diffusion through the pellicle. Dunihue (1931) found that the vacuole system in Paramecium cauda- tum is osmicated only after the neutral-red stainable globules. These globules and Janus-green stainable elements, he believes, represent the vacuome and chondriome (mitochondria) respectively. King (1935) noted a “specialized excretory protoplasm’? surrounding the feeding canals in P. multimicronucleata, but denied that this material is ho- mologous with the Golgi apparatus of metazoan cells. The opinions of von Gelei (1925, 1928) concerning the structure and function of the contractile vacuole in Paramecium are of special interest. He described essentially the same structures in stained Para- mecium as those mentioned by Nassonoy. The zone of specialized plasma around the distal portion of the canals, particularly, was described in detail, and an excretory function assigned to it. This specialized plasma von Gelei calls “‘nephridial plasma,” its excretory function being implied by its name. This “excretory” function von Gelei believes is entirely dif- | ferent from the ‘‘secretory’”’ function assigned by Nassonov, when the latter considers the specialized plasma to be the Golgi apparatus. This disagreement appears to be imaginary rather than real, since an analysis of their respective views indicates that the two authors observed struc- tures which are identical in practically every respect; but to describe the function, they selected different words. Moreover, these different words, when translated into terms of physiological processes, are practically iden- tical. Von Gelei pictures two different arrangements of the deeply stain- ing material in this zone of specialized plasma; in one the stained ele- 440 THE CONTRACTILE VACUOLE ments are in the form of short rods, which lie with their long axes at right-angles to the long axis of the filling canal, in much the same manner as the bristles of a test-tube brush are arranged with respect to the wire handle to which they are attached; and in the other, these elements are in the form of a net surrounding the filling canal. It is interesting to note that this net-like arrangement is commonly seen in the Golgi apparatus of many metazoan cells, as well as in Dogielella. Von Gelei (1933) ob- served in Spathidium giganteum that not only the contractile vacuole and the smaller vacuoles in its immediate vicinity possess osmiophilic walls, but also others further removed. One can but wonder if this represents the origin of contractile vacuoles by the coalescence of secondary vacuoles, which have arisen in more or less remote parts of the organism. In Monocystis agilis and M. ascidiae, Hirschler (1924) identified two kinds of lipoid bodies. The smaller of these he considers mitochondria, the larger the Golgi apparatus. In Gregarina polymorpha, G. blattarum, and Spirostomum ambiguum only one kind of lipoid body was ob- served. From this Hirschler concludes that the latter are representatives of a more primative state, in which lipoid bodies are not yet differenti- ated into mitochondria and Golgi apparatus. In most instances in which description of structures are given in sufh- cient detail, and in which organisms have been subjected to a variety of stains as well as osmium and silver impregnation, very strong evi- dence has been presented to indicate that contractile vacuoles derive the fluid which they expel to the exterior from granules which are osmio- philic, argentophilic, and sometimes neutral-red stainable. In some cells the osmiophilic granules are aggregated around the vacuole or in that part of the cell in which the vacuole ordinarily arises; this is usually associated with the origin of the vacuole in a restricted portion of the cell. In other cells the osmiophilic granules are dispersed to a greater or less extent, sometimes apparently uniformly throughout the cytoplasm, this is usually associated with at least the potential origin of vacuoles in almost any part of the cytoplasm. Evidence bearing on the subject indi- cates that these osmiophilic granules may represent at least one type of the “excretory granule” so frequently mentioned in the literature. Several authors have reported osmiophilic substances in the form of relatively broad bands, or rings, which may or may not be in direct contact with the vacuole wall. Most authors seem to agree that the usual THE ‘CONTRACTILE VACUOLE 441 form for the protozoan Golgi apparatus is that of granules, globules, or short rods. Overimpregnation of a granular region, which occurs when the process is carried out at too high temperatures, has been shown to produce heavy black bands, or rings, in certain organisms. This fact suggests the possibility that the Golgi apparatus may be of the usual form, even in those organisms in which the band, or ring, type has been observed. Identification of osmiophilic substances answering the known criteria for the Golgi apparatus has been extended to include representatives of the four classes of Protozoa: Mastigophora, Sarcodina, Sporozoa, and Ciliata. This substantiates the idea that the Golgi apparatus is a cytoplasmic inclusion of all living cells. CONCLUSION In spite of the multiplicity of claims, counter claims, theories, and suggestions, a few generalizations seem to be established well enough to indicate at least some of the fundamental processes associated with ac- tivity of the contractile vacuole. It is not intended that these shall be accepted as proved beyond question, but rather that the evidence points in their direction more consistently than in any other. Further investiga- tion may necessitate a complete revision of opinion concerning these processes, but in the light of the information available at the present time the following conclusions seem to be justified. 1. Contractile vacuoles originate as a result of the activity of certain cytoplasmic inclusions, which may be aggregated in the immediate vicin- ity of the vacuole in some species, or distributed more or less generally throughout the cytoplasm in others. Temporary contractile vacuoles are formed by the fusion or coalescence of small accessory vacuoles, which in turn originate by the fusion of still smaller accessory vacuoles, the last and smallest vacuoles being formed in or associated with the cyto- plasmic inclusions mentioned above. More or less permanent contractile vacuoles (e.g., those of Paramecium) receive fluid as small droplets, or accessory vacuoles which fuse with some portion of the filling canals; these droplets originate in or on cytoplasmic inclusions in the same man- ner as those mentioned above. 2. On the basis of known physicochemical laws and processes, it is necessary to postulate the existence of a physiological membrane sur- rounding the contractile vacuole. In some organisms, particularly those 442 THE CONTRACTILE VACUOLE possessing more or less permanent vacuole systems, these organelles ap- pear to be surrounded by morphological membranes. 3. Direct evidence concerning the function of contractile vacuoles is almost entirely lacking. Indirect evidence indicates that in fresh-water forms the vacuole protects the organism against excessive dilution of its cytoplasm. In marine and parasitic forms such a function would seem to be largely superfluous, although even in these the elimination of at least a small quantity of water by some mechanism appears to be neces- sary. Direct evidence indicating the presence of waste products of me- tabolism in the vacuolar fluid is very scant, although, in those forms possessing relatively impermeable surface structures, the vacuole is the only visible means by which such wastes may be passed to the exterior. 4. In some Protozoa three types of cytoplasmic inclusions have been identified, in others only two types. In all Protozoa so far examined with this in view, at least some of the inclusions are osmiophilic. In some oth- ers these osmiophilic inclusions are also stainable by neutral red, but not by Janus green. Osmication of certain Protozoa by one technique or an- other frequently shows more than one type of inclusion to be osmiophilic, but generally one of these resists bleaching by hydrogen peroxide or turpentine more completely than the others. Such inclusions are generally recognized as the Golgi apparatus. By comparing living organisms with those stained vitally with various dyes, as well as with others impregnated with osmium or silver, identity of the Golgi apparatus and the cyto- plasmic inclusions concerned with the origin of the contractile vacuole has been established for many forms. The usual form of protozoan Golgi apparatus is granular, globular, or rod-like. In a few species (Para- mecium, Dogielella, Chilodon, and others), it frequently appears as a network, while in others (Lonotus, Nassula, Campanella, and others) it is in the form of a thick ring, or membrane, surrounding part or all of the vacuole. Evidence has been presented which indicates that in some of these, if not all, a granular structure has been overimpregnated, this causing it to assume the appearances mentioned. It therefore appears that fluid which is expelled from the organism by the contractile vacuole originates as droplets in association with the Golgi apparatus, although the Golgi apparatus is not necessarily in intimate contact with the vacu- ole. Concerning the origin of secretions in metazoan gland cells, Bowen (19297 pai )estates:: THE CONTRACTILE VACUOLE 443 Secretion is in essence a phenomenon of “granule” or droplet formation. Starting with a single such secretory droplet about to be expelled from the cell, we find it possible to trace its origin step by step to a minute vacuole, which has thus from the beginning served as a segregation center for a specific secretion-material. The primordial vacuole is found to arise in that zone of the cell characterized by the presence of the Golgi apparatus, and the evidence indicates, if it does not demonstrate, that the primordial vacuole arises through the activity of the Golgi substance and undergoes a part at least of its development in contact with, or imbedded in, the Golgi ap- paratus. The idea of Nassonov, as developed in 1925 and 1926, as well as that of MacLennan (1933), concerning the origin of protozoan vacuoles could hardly be expressed more exactly. The outstanding features of contractile vacuoles, taken collectively, then, do not lie in differences among them, but rather in similarities, both morphological and physiological. Another fundamental link in the kinship between all cells seems to be established by the apparent homol- ogy, both structural and functional, between the protozoan and the meta- zoan Golgi apparatus. LITERATURE CITED Adolph, E. F. 1926. The metabolism of water in Amoeba as measured in the contractile vacuole. J. exp. Zool., 44: 355-81. Bowen, R. H. 1929. The cytology of glandular secretion. Quart. Rev. Biol., 4: 299-324 and 484-519. Brown, V. E. 1930. The Golgi apparatus of Amoeba proteus Pallas. Biol. Bull. 59: 240-46. Butschli, O. 1887. Die Contraktilen Vacuolen. Bronn’s Thierreich, Protozoa, 1: 1411-59. Calkins, G. N. 1926. The biology of the protozoa. Philadelphia. Clark, A. J. 1933. Mode of action of drugs on cells. London. Day, H. C. 1927. The formation of contractile vacuoles in Amoeba proteus. J. Morph., 44: 363-72. —— 1930. Studies on the contractile vacuole of Spzrostomum and Para- mecium. Physiol. Zool., 3: 56-71. Degen, A. 1905. Untersuchungen uber die Contractile Vacuole und die Wabenstruktur des Protoplasmas. Botan. Ztg., 63: 160-202. Dimitrowa, Ariadne. 1928. Untersuchungen iiber die tiberzahligen pulsier- enden Vakuolen bei Paramecium caudatum. Arch. Protistenk., 64: 462- They, Doflein, F. 1911. Lehrbuch der Protozoenkunde. “tt THE CONTRAGIILE, VACUOLE Doyle, W. L., and J. P. Harding. 1937. Quantitative studies on the ciliate Glaucoma. Excretion of ammonia. J. exp. Biol., 14: 462-69. Dunihue, F. W. 1931. The vacuome and the neutral red reaction in Para- mecium caudatum. Arch. Protistenk., 75: 476-97. Eisenberg, E. 1926. Recherches sur le fonctionnement de la vesicule pulsatile des Infusoires dans les condition normales et sous I’action de certains agents experimentaux. Arch. Biol. Paris, 35: 441. —— 1929. Recherches comparatives sur le fonctionnement de la vacuole pulsa- tile chez les Infusoires parasites de la grenouille et chez les Infusoires d’eau douce. Influence de la pression osmotique, des electrolytes et du pH. Arch. Protistenk., 68: 451-70. Fauré-Fremiet, E. 1925. La Structure permanent de l’appareil excréteur chez quelques vorticellides. C. R. Soc. Biol. Paris, 93: 500-3. Finley, H. E. 1934. On the vacuome in three species of Vorticella. Trans. Amer. micr. Soc., 53: 57-65. Flather, M. D. 1919. The influence of glandular extracts upon the contractile vacuoles of Paramecium caudatum. Biol. Bull., 37: 22-39. Fortner, H. 1926, Zur Frage der diskontinuierlichen Excretion bei Protisten. Arch. Protistenk., 56: 295-320. Fosse, R. 1913. Sur I’Identification de l’urée et sa précipitation de solution extrément diluées. C. R. Acad. Sci. Paris, 137: 948-57. Frisch, J. A. 1935. Experimental adaptation of fresh water ciliates to sea water. Science, 81; 537. Gelei, J. von. 1925. Nephridialapparat bei Protozoen. Biol. Zbl., 45: 676-83. 1928. Nochmals tber den Nephridialapparat bei den Protozoen. Arch. Protistenk., 64: 479-94. —— 1933. Wandernde Exkretionsvakuolen bei den Protozoa. Arch. Protis- tenk., 81: 231-42. Griffiths, A. B. 1888. A method of demonstrating the presence of uric acid in the contractile vacuoles of some of the lower organisms. Proc. roy. Soc. Edinb., 16: 131-35. Hall, R. P. 1929. Reaction of certain cytoplasmic inclusions to vital dyes and their relation to mitochondria and Golgi apparatus in the flagellate, Pera- nema trichophorum. J. Morph., 48: 105-18. —— 1930a. Cytoplasmic inclusions of Trichamoeba and their reaction to vital dyes and to osmic and silver impregnation. J. Morph., 49: 139-51. —— 1930b. Cytoplasmic inclusions of Menoidium and Euglena, with special reference to the vacuome and ‘“'Golgi apparatus” of Euglenoid flagellates. Ann. Protist., 3: 57-68. —— 1930c. Osmiophilic inclusions similar to Golgi apparatus in the flagel- lates, Chromulina, Chilomonas, and Astasia. Arch. Protistenk., 69: 7-22. —— 1931. Vacuome and Golgi apparatus in the ciliate, Stylonychia. Z. Zell- forsch., 13: 770-82. THE GONTRAGHEE VACUOLE 445 Hall, R. P., and F. W. Dunihue. 1931. On the vacuome and food vacuoles in Vorticella. Trans. Amer. micr. Soc., 50: 196-205. Hance, R. T. 1917. Studies on a race of Paramecium possessing extra con- tractile vacuoles. J. exp. Zool., 23: 287-333. Hartog, M. M. 1888. Preliminary notes on the functions and homologies of the contractile vacuole in plants and animals. Rep. Brit. Assoc. Ady. Sci., Bath, pp. 714-16. Harvey, E. B. 1917. A physiological study of specific gravity and of lumines- cence in Noctiluca, with special reference to anesthesia. Pap. Tortugas Lab., 11: 237-53. Haye, Ans. 1930. Uber den Exkretionsapparat bei den Protisten, nebst Be- merkungen tiber einige andere feinere Strukturverhaltnisse untersuchten Arten. Arch. Protistenk., 70: 1-86. Herfs, A. 1922. Die pulsierende Vakuole der Protozoen, ein Schutzorgan gegen Aussiissung. Arch. Protistenk., 44: 227-60. Hirschler, J. 1924. Sur une Méthode de Noircissment de l’Appareil de Golgi. C. R. Soc. Biol. Paris, 90: 893. — 1927. Studien uber die sich mit Osmium schwarzenden Plasmakom- ponenten einiger Protozoenarten, Z. Zellforsch., 5: 704-86. Hopkins, D. L. 1938. Adjustment of the marine amoeba, Flabellula mira Schaeffer, to changes in the total salt concentration of the outside medium. Biol. Bull 7525337 a(@Abstr:): Howland, R. B. 1924a. Experiments on the contractile vacuole of Amoeba verrucosa and Paramecium caudatum. J. exp. Zool., 40: 251-62. — 1924b. On excretion of nitrogenous waste as a function of the contractile vacuole, J. exp. Zool., 40: 231-50. Howland, R. B., and H. Pollack. 1927. Expulsion of injected solute by the contractile vacuole of Amoeba. Proc. Soc. exp. Biol. N.Y., 25: 221-22. Hyman, Libbie H. 1936. Observations on Protozoa: The impermanence of the contractile vacuole in Amoeba verspertilio. Quart. J. micr. Sci., 79: 43-56, Jennings, H. S. 1904. A method of demonstrating the external discharge of the contractile vacuole. Zool. Anz., 27: 656-58. Kent, W. S. 1880. A Manual of the Infusoria. London. Khainsky, A. 1910. Zur Morphologie und Physiologie einiger Infusorien (Paramecium caudatum) auf Grund einer neuen histologischen Methode. Arch. Protistenk., 21: 1-60. King, R. L. 1928. The contractile vacuole in Paramecium trichinm. Biol. Bull., DDI 9-69: —— 1933. Contractile vacuole of Explotes. Trans. Amer. micr. Soc., 52: 103-6. —— 1935. The contractile vacuole of Paramecium multimicronucleata. J. Morph., 58: 555-71. 446 THE CONTRACTILE, VACUOLE Kitching, J. A. 1934. The physiology of contractile vacuoles: osmotic relations. J. exp. Biol., 11: 364-81. Lachmann, K. F. 1891. Uber einiger neu entdekte Infusorien und ttber Con- tractilen Blasen bei den Infusorien. Verh. Naturh. Ver. preuss. Rheinl., 16: 66-68, 91-93. Lloyd, F. E. 1928. The contractile vacuole. Biol. Rev., 3: 329-58. Lloyd, F. E., and G. W. Scarth. 1926, Origin of vacuoles. Science, 63: 459-60. Ludwig, W. 1928. Der Betriebsstoffwechsel von Paramecium caudatum. Arch. Protistenk., 62: 12-40. Lwoff, André, and Nadia Roukhelman. 1926. Variations de quelques formes d’azote dans une culture pure d’infusoires. C. R. Acad. Sci. Paris, 183: 156-58. MacLennan, R. F. 1933. The pulsatory cycle of the contractile vacuoles in the Ophryoscolecidae, ciliates from the stomach of cattle. Univ. Cal. Publ. Zool., 39: 205-50. Mast, S. O. 1926. Structure, movement, locomotion, and stimulation in Amoeba. J. Morph., 41: 347-425. —— 1938. The contractile vacuole in Amoeba proteus (Leidy). Biol. Bull., 74: 306-13. Mast, S. O., and W. L. Doyle. 1935. Structure, origin, and function of cyto- plasmic constituents in Amoeba proteus with special reference to mito- chondria and Golgi substance. Origin and function based on experimental evidence; effect of centrifuging on Amoeba proteus. Arch. Protistenk., 86: 278-306. Maupas, E. 1883. Contribution a I’Etude Morphologique et Anatomique des Infusoires Ciliés. Arch. zool. exp. gén., 2 serie, 1: 427-664. Metcalf, M. M. 1910. Studies upon Amoeba. J. exp. Zool., 9: 301-31. —— 1926. The contractile vacuole granules in Amoeba proteus. Science, 63: 523-24. Moore, Imogene. 1931. Reaction of Blepharisma to Golgi impregnation methods. Proc. Soc. exp. Biol. N.Y., 28: 805-6. —— 1934. Morphology of the contractile vacuole and cloacal region of Blephavisma undulans. J. exp. Zool., 69: 59-104. Nassonoy, D. 1924. Der Exkretionsapparat der Protozoen als Homologen des Golgischen Apparats der Metazoazellen. Arch, mikr. Anat. 103: 437-82. —— 1925. Zur Frage tiber den Bau und die Bedeutung des lipoiden Exkre- tionsapparates bei Protozoa. Z. Zellforsch., 2: 87-97. ——— 1926. Die physiologische Bedeutung des Golgi Apparats im Lichte der Vitalfarbungsmethode. Z. Zellforsch., 3: 472-502. Nigrelli, R. F., and R. P. Hall. 1930, Osmiophilic and neutral-red-stainable inclusions of Arcella. Trans. Amer. micr. Soc., 49: 18-25. Nowikoff, M. 1908. Uber die Wirkung des Schildrusen Extrakts und einiger anderer Organstoffe auf Ciliaten. Arch. Protistenk., 11: 309-26. THE GONTRACTILE VACUOLE 447 Parnas, J. K. 1926. Allgameines und Vergleichendes des Wasserhaushalts. Handbuch. d. norm. u. pathol. Physiol., 17, correlationem 3: 137-60. Riddle, M. C., and H. B. Torrey. 1923. The physiological response of Para- mecium to thyroxin. Anat. Rec., 24: 396. Scarth, G. W., and F. E. Lloyd. 1927. The réle of kinoplasm in the genesis of vacuoles. Science, 65: 599-600. Shumway, W. 1917. Effects of thyroid diet upon paramecia. J. exp. Zool., 22: 529-62. Specht, Heinz. 1934. Aerobic respiration in Spirostomum ambiguum and the production of ammonia. J. cell. comp. Physiol., 5: 319-33. Stempell, W. 1914. Uber die Funktion der Pulsierende Vacuole und einen Apparat zur Demonstrationen derselben. Zool. Jahrb., Abt. Allg. Zool. Physiol. Tiere, 34: 437-78. Stokes, A. C. 1893. The contractile vacuole. Amer. mon. micr. J., 14: 182-88. Taylor, C. V. 1923. The contractile vacuole in Evplotes: an example of the sol-gel reversibility of cytoplasm. J. exp. Zool., 37: 259-90. Weatherby, J. H. 1927. The function of the contractile vacuole in Paramecium caudatum ; with special reference to the excretion of nitrogenous com- pounds. Biol. Bull., 52: 208-18. — 1929. Excretion of nitrogenous substances in Protozoa. Physiol. Zodl., 2: 375-94, Wenrich, D. H. 1926. The structure and division of Paramecium trichinm Stokes. J. Morph., 43: 81-103. Young, R. A. 1924. On the excretory apparatus in Paramecium. Science, 60: 244, Ziilzer, Margaret. 1910. Uber den Einfluss des Meerwassers auf die pul- sierende Vacuole. Roux Arch. Entw. Mech. Organ., 29: 632-40. CHAPTER WAIT THE TECHNIQUE AND SIGNIFICANCE OF CONTROL IN| PROTOZOAN CULTURE GEORGE W. KIDDER INTRODUCTION DURING THE LAST FEW YEARS there has come to be an appreciation of methods of culturing Protozoa which will permit the investigator to_ determine the conditions under which his study is being made. Studies of populations and the various interesting and important factors in- volved, mass physiology, nutrition, and numerous other phases of cellu- lar activity may be profitably dealt with by the student of the Protozoa only when he can be sure that the effects noted are due to the conditions under investigation. The science of protozodlogy has passed through the phase of “pure-mixed” methods of culture. This term simply means that a single strain of Protozoa is grown in association with a chance combination of other microdrganisms, usually bacteria. Many valuable and thought-provoking contributions, based upon this method, have been made, and these contributions have paved the way to the more pre- cise evaluations of the present. In the culture of practically any species of Protozoa, it may be safely said that the bacteria as a group offer the most serious obstacle to con- trolled conditions. Experimental modifications of factors such as nutritive materials, temperature, oxygen or carbon dioxide tensions, oxydation- reduction potentials, and so forth, may produce effects, but whether these effects are the result of changes in protozoan activity per se, or are sec- ondary through the change of activity of the bacteria, is usually nearly or totally obscure. These facts are recognized, and there has been built up a body of literature reporting progress in methods which will allow for the control or, better still, the elimination of bacteria. Numerous 1n- vestigators have succeeded in sterilizing various species of Protozoa and have made great strides in advancing our knowledge of cellular activities through the use of pure cultures. GONTROL OF CULTURES 449 It is the purpose of this section to devote some time to methods or techniques of protozoan sterilization, in order to bring before the reader some of the many problems which must be dealt with in work of this kind. As may be supposed, the nature of such a discussion makes it necessary to assume at least a rudimentary knowledge of bacteriological technique. And above all there must be a thorough appreciation of the potentialities of many different types of bacteria to resist even the most careful methods of irradication, potentialities which express themselves in some cases only after prolonged periods of apparent sterility. In addition to an outline of techniques for sterilization, the question of acceptable tests for the sterility of cultures will be considered, and finally some of the problems and conditions arising from the establish- ment of sterile Protozoa in culture. THE PROBLEM OF PROTOZOAN STERILIZATION 1. GENERAL MATERIAL Protozoa from natural waters, soil, and so forth, are, and have been throughout their existence, in association with bacteria. This does not mean that the bacterial flora of their surroundings has remained constant either as to numbers or types. The flora is probably continually changing. This very change is one of the most important factors in the succession of microscopic animals in ponds and streams. The variety of bacterial types one would expect to encounter in any extended survey of natural ponds is practically limitless. Therefore it is impossible to do more than discuss the general factors to be taken into account in dealing with bac- teria. To attain successful sterile cultures of Protozoa, it is, of course, de- sirable to have rather large numbers of healthy organisms with which to work. It is usually possible to isolate single organisms into fresh infusion and obtain from fair to good growth. If they are bacteria-feeders (the great majority of free-living ciliates are), enough food organisms are brought over in the isolation to insure, at least in a high percentage of cases, against starvation. As the bacteria multiply they, in turn, are uti- lized by the Protozoa. In dealing with ciliates from the wild, a partial substitution method may be attempted. For a number of species, this method facilitates later sterilization. If the ciliate to be used will feed on living yeast (and this 450 CONTROE OF CULTURES can be determined only by experimentation), then cultures may be estab- lished by suspending yeast cells in spring water, distilled water, or bal- anced salt solutions, and introducing the desired ciliate. Often abundant growth will result and after a few subcultures have been made, the ratio of bacteria to Protozoa will be reduced. When serious attempts at sterili- zation are then carried out, the yeast cells will be found relatively easy to eliminate. It should not be supposed that it will be possible to elimi- nate the contaminating bacteria in this way, as they will be multiplying slowly all the while. In fact, long-continued cultures of this type are apt to show a decided increase in non-nutritive bacteria over those which were present at the start (see Kidder and Stuart, 1939). Therefore it is advisable to make from one to three subcultures only, and then to start the sterilization procedure. In general it can be said that the larger the protozoan the more diffi- cult it will be to sterilize. This fact becomes apparent from an examina- tion of the literature, and was noted by Hetherington (1934). Physical properties likewise play a rdle in ease of sterilization. Holotrichous and heterotrichous ciliates, possessing large numbers of closely set cilia, are apt to retain a few of their associated bacteria even after repeated wash- ing, while hypotrichous ciliates may be washed free of bacteria more readily. Flagellates, being for the most part smooth in surface, are rela- tively easy to sterilize. Activity is also important, both as to movement and metabolism. Highly motile forms may usually be freed of bacteria more readily than sluggish types. Those possessing a high rate of me- tabolism tend to utilize or defecate the contents of their food vacuoles more rapidly than the slow-growing types, and do not tend to carry over viable spores to contaminate later cultures. Many other characters which will influence the facility with which sterilization may be accomplished might be mentioned, but these will become apparent when we examine some of the procedures. 2. GENERAL METHODS OF STERILIZATION In order to rid the Protozoa of their associated bacteria, workers have made use of three principles. The first and most generally useful is simple washing in sterile fluid. This is the dilution method whereby the bacteria are diluted out of the solution. The Protozoa must be retained of course, and a number of different manipulations have been devised to insure this. CONTROL OF CULTURES 451 The principle of dilution takes for granted that the bacteria either are suspended in the fluid or that they may be caused to become suspended. The second principle is one of migration. The Protozoa to be sterilized are allowed or caused to swim through sterile fluid or semisolid medium or over the surface of solid medium, !eaving the bacteria behind. This method has been used with success on a number of different types of ciliates and flagellates and a few amoebae. The Protozoa may be induced to migrate laterally by introducing them into one side of a flat dish of sterile fluid. Or, if they happen to be negatively geotropic, they may be introduced into the bottom of a vessel of sterile fluid and taken off at the top. Those that are positively geotropic may be introduced at the top and taken off at the bottom. Extremely active types may be able to migrate through a semisolid medium and literally scrape off their adher- ing bacteria. Combinations of the above two principles have been used with marked success, and a number of ingenious pieces of apparatus have been de- signed to facilitate the manipulations and to reduce the chance of ex- traneous contamination. These will be described in detail later. The third principle that has been applied to this problem is that of bactericidal agents. This method has met with questionable success and then usually only in cases where resistant phases (cysts) could be ob- tained. As might be expected, any agent which would kill the bacteria in a culture would most surely kill trophic Protozoa. It has been shown many times that the various species of Protozoa are much more suscep- tible to the usual toxic agents than many of the common bacteria. (An exception to the foregoing statement is indicated in the work of Brown, et al., 1933, using X-rays as a sterilizing agent.) 3. SPECIAL METHODS AND MANIPULATIONS This section will be devoted to a description of the procedures which have been used by various investigators to rid the different types of Proto- zoa of their associated bacteria. Considerable pains will be taken to de- scribe the apparatus used, the manipulations performed, and the results obtained. It is hoped that by so doing the reader will be able to gain constructive ideas which will allow him either to utilize one of the de- scribed methods or to formulate a modification which will meet his needs. A. Dilution —One of the first reports of the sterilization of Protozoa 452 CONTROL OF CULEURES by this method is that of Hargitt and Fray (1917), using Paramecium aurelia and P. caudatum. They experimented with a number of modifi- cations of the washing technique and followed their results by plating on nutrient agar. Their first procedure was dilution by centrifugation, wherein they centrifuged down the paramecia and then quickly with- drew the supernatant fluid with a sterile pipette. The paramecia were then covered again with sterile fluid, and the process repeated five times. At the end of the fifth wash they found that the number of bac- teria “‘per drop” had decreased from 500 colonies (per plate) in the first wash to 3 colonies in the fifth. These results were not satisfactory, however, and the method was abandoned. The authors offer the follow- ing objections to the method: a great deal of time was consumed, the wash waters had to be drawn off immediately after the centrifuge stopped or the paramecia rose in a body and prevented the removal of the wash water. . . . Another serious drawback to the centrifuge method is the difficulty of keeping the wash waters free from contamination by bacteria from the air. The air may contain such enormous numbers of bacteria that instruments and media which are sterile to start with will be contaminated unless precautions are taken to prevent the contact of air bacteria. A sterile pipette laid down on the table is no longer sterile, a wash water left unprotected is soon contaminated by air bacteria [p. 435}. It will become obvious from later discussions of these points that success of the centrifuge method in the hands of Hargitt and Fray was pre- vented by two principal faults in their manipulations—too few washes, and failure to keep their centrifuge and wash tubes plugged at all times. These authors next attempted to reduce the chances of outside con- tamination by the transfer method, using watch crystals enclosed within Petri dishes, and transferring single ciliates through five separate dishes. They again failed to effect sterility, but succeeded in reducing the num- ber of colonies ‘“‘per drop” from 2,500 in the first wash to one colony in the fifth wash. They blame their lack of success with this procedure upon the fact that the amount of wash fluid was so large that consider- able time was required to locate the ciliate between transfers. Successful sterilization was accomplished by transferring individual paramecia through five successive washes of sterile tap water in sterile depression slides. The transfer pipettes were sterilized by dry heat be- fore using, as were the Petri-dish-contained depression slides. The tap GONTROL OF CULTURES 453 water was autoclaved. They claim to have effected sterilization in a high percentage of their trials, with a total time consumption per ciliate of not more than five minutes. If their bacteriological tests are accepted (de- velopment of bacterial colonies on agar plates, time of incubation not given) then we can only conclude that they were extremely fortunate in avoiding ciliates with ingested viable spores. A chief criticism of the work of Hargitt and Fray was given by Par- part (1928). He pointed out that most of their sterility tests were con- fined to the washing fluids and not to the supposedly sterilized paramecia. In reinvestigating their results, he found that five washes gave sterile fluid at the end, but that even after ten washes in six out of eight trials the animals themselves were contaminated. He ascribed this fact to the probability of the carrying over of viable spores within the vacuoles of the ciliates. He suggested a simple modification of the method of Hargitt and Fray, which yielded fifty sterile paramecia out of fifty trials. Instead of five washes he employed ten, thereby increasing the dilution factor. Time was allowed (five hours) for the ciliates to void their vacuoles of possible spores in the fifth wash. His method ts essentially as follows: A single Paramecium was trans- ferred with a sterile pipette from a wild culture to a sterile Petri-dish- enclosed depression slide containing about six drops of washing fluid. After about one minute, the animal was transferred to the next similar bath. At the fifth bath the Paramecium was allowed to swim about for five hours and was then carried through five further washes. All of the manipu- lations were carried out under a rather elaborate hood to minimize the possibility of contamination from the air. This modification of the simple washing method has probably been more generally used than any other, owing principally to its simplicity of manipulation. It is admirably adapted to large ciliates which can be followed with ease under the low powers of the dissecting microscope. The smaller the organism the more difficult this method becomes. Of course failure will most surely follow any deviation from absolutely aseptic technique. In an attempt further to simplify the technique as outlined by Par- part, especially regarding the hood under which the transfers were carried on, the following procedure has yielded extremely satisfactory results in our laboratory (Kidder, Lilly, and Claff, 1940). Syracuse 454 CONTROL) OF CULGURES watch glasses are enclosed in cellophane bags, the ends of which are folded over, and the whole sterilized in the autoclave. After cooling, the bags are carefully opened and 5 ml. of sterile wash fluid is placed in each watch glass by means of sterile serological pipettes. The protozoan to be sterilized is placed in the first bath by means of a micro-pipette inserted through the open end of the bag. There are three obvious ad- vantages in this modification, aside from simplicity of apparatus. The opening of the bag is at the side of the dish and at some distance from it. The top of the dish and therefore the fluid is never exposed to the air from above. The same situation is here repeated as obtains when making tube inoculations in ordinary bacteriological technique, where the tube is always held at a slant. This system is less dangerous than one in which the top of a Petri dish must be removed, and obviates the necessity for a hood or drape. The second advantage is that the ob- server may follow the movements of the protozoan at all times and may then readily draw it up in the transfer pipette. This is usually impossible or difficult when using a Petri dish with the cover in place, as the water of condensation reduces the visibility markedly. Water does not condense on the cellophane, and observations are therefore not hampered. The third advantage is simply one of choice of containers. The Syracuse watch glasses holding 5 ml. of fluid raise enormously the all-important dilution factor. When it is possible to obtain large numbers of Protozoa in heavy con- centrations, sterilization may be accomplished by centrifugation. This method, although unsuccessful in the hands of Hargitt and Fray (1917), has been used to advantage recently (Kidder and Stuart, 1939). It 1s recommended for use with those species of Protozoa which are so small as to make them difficult to follow under the powers of a dissecting microscope. By choosing a washing fluid favorable for the species to be used and carrying the number of washes far enough, it is usually possible to recover large numbers of sterile Protozoa after the final wash. The method which was finally adopted for the sterilization of Colpoda steinii (see Burt, 1940, for species designation) is quoted below: After excystment had occurred the ciliates were concentrated by slow centrifugation and the concentrate removed to a single, sterile, cotton-plugged centrifuge tube. This ciliate concentrate was diluted with 10 ml. of sterile distilled water and recentrifuged at a speed which would just throw down CONTROL OF CULTURES 455 the majority of ciliates in 3 minutes. It was found that the most satisfactory speed for this purpose was 2000 revolutions per minute. As soon as the centrifuge stopped the supernatant fluid (9 ml.) was immediately withdrawn with a sterile 10 ml. pipette and the tube was allowed to stand for about two minutes in order that the ciliates might swim to the top of the remaining milliliter of water. With a sterile 1 ml. pipette, 0.5 ml. of ciliate suspension was withdrawn and placed in an empty sterile centrifuge tube. This suspen- sion was again diluted, and the process repeated until the ciliates had gone through an average of fifteen such transfers with the accompanying dilutions (a dilution factor of approximately 10'). This method entails a great loss of ciliates but was found necessary inasmuch as, without removal to fresh tubes with the consequent discarding of the residue, contaminations were invariable. It was demonstrated that the contaminations resulted from the fact that ciliates died or became immobilized during centrifugation and were carried passively to the bottom of the tube with their adhering bacteria. How- ever, by discarding the dead forms we were able to completely sterilize several hundred ciliates at each attempt and these gave us the necessary organisms with which to work [Kidder and Stuart, 1939, p. 332]. The washing fluid which was used in this case was sterile Pyrex- distilled water. All pipettes were paper-wrapped and autoclaved. The centrifuge tubes were closed with large cotton plugs and autoclaved. During centrifugation the cotton was folded over and fastened with a rubber band, to prevent the plug from being drawn into the tube. B. Migration —Probably the first report of a technique for obtaining Protozoa free from bacteria by the utilization of migration was that of Ogata (1893). He reports that he was able to recover as many as fifty- two sterile phytomonads (Polytoma uvella) within five to thirty minutes after the start of the migration. The apparatus he employed was a capil- lary tube 10 to 20 cm. in length, with a 0.3 to 0.5 mm. bore. This tube was filled to within one to 2 cm. from the end with a sterile fluid, and then inserted into a culture of the flagellates and allowed to fill com- pletely. Care was taken to avoid aid bubbles between the layers. Both ends of the capillary were sealed by heat and the whole allowed to stand for from five to thirty minutes. Eventually some of the flagellates were found to have migrated away from their associated bacteria, and when a number had collected in the upper end of the tube, this end was broken off and the flagellates inoculated into nutrient media. A refinement of the same technique is reported by Stone and Rey- nolds (1939) for the sterilization of the parasitic flagellate Tv7chomonas 456 CONTROE OF CULTURES hominis. Their capillary tube was made from a piece of 6 mm. Pyrex tubing about eight inches in length, which, before sterilization, had been plugged at both ends with cotton. The capillary was then drawn from one end of the tube, its tip broken off with sterile forceps, and a series of () \c(! ~ Figure 123. Capillary tube used for the steriliza- tion of Trichomonas ho- minis, The whole tube is filled with sterile fluid, the lower end sealed in a flame, and the Protozoa to be ster- ilized are layered on to the fluid at the large end. The Protozoa eventually migrate through the capillary por- tion, but the associated bac- teria are trapped at the first or second bend. (Redrawn from Stone and Reynolds, 1939.) loops constructed (Fig. 123). All of these manipulations were carried on with care not to contaminate the outside of the tube, for after the loops were made the whole tube was filled to within one inch of the top with sterile fluid (in this case, one part Ringer’s, eight parts horse serum) by suction, applied to the large end. The capillary end was then sealed off and the tube, in a vertical position, was incubated forty-eight hours €ONTROE? OF? CULTURES 457 as a check on sterility. If no turbidity developed, contaminated Ti- chomonas were layered onto the fluid in the large end of the tube. Within forty-eight hours many flagellates had migrated down the tube and could be seen in the last inch or two of the capillary portion. The authors state that the bacteria failed, for the most part, to migrate past the first loop, and never passed the second. The last portion of the capil- lary was cut off and sealed by means of a flame and the cut-off portion was submerged in tincture of iodine (7 percent) for one hour. Then one end was grasped in the fingers and the tube held upright to drain. When dry, pieces of the tube were broken off with sterile forceps and dropped into selected culture media. The authors state that they have repeatedly isolated T. hominis bacteria-free by this method, but have not tested it with other Protozoa. The above method appears to be applicable to many types of Protozoa, and should receive serious consideration. The manipulations offer some difficulty, however, and extreme care will have to be exercised to insure against outside contamination, especially during the filling of the tube with the sterile fluid and again during the breaking of the sections of capillary into nutrient culture media. Probably the simplest method which takes advantage of the migration of Protozoa in fluid media is the Petri-dish method. A sterile Petri dish is partially filled with sterile fluid and placed on the stand of the dissect- ing microscope so that one edge is under the objective. After all motion of the fluid has ceased, the Petri dish cover is raised and a drop of con- centrated protozoan culture is placed very near the edge opposite the one under the lens. This manipulation must be done with great care, so that the fluid is disturbed as little as possible. The cover is then gently lowered and sufficient time (five to ten minutes) is allowed for the Protozoa to swim to the opposite edge of the dish. The cover is again raised, and single organisms are picked out with sterile pipettes and transferred to selected media. Minimum time for the migration is im- portant, so that none of the highly motile bacteria will reach the area from which the Protozoa are being taken. Enough Protozoa should be separated singly in this way to allow for the law of averages. The greater the motility of the Protozoa, the smaller their size, and the smoother their bodies, the greater the chance for successful sterilization by this method. 458 CONTROL OF CULTURES We have used the above method to sterilize a number of flagellates (Euglena, Astasia, Chilomonas). With a single migration across a Petri dish and the selection of twenty-five organisms at each trial, the per- centage of sterile to contaminated cultures was very high (80 to 90 percent). We used a Plastocoel (transparent) shield over the microscope and always worked in a draft-free room. Tetrahymena geleii (Furgason, 1940) was also sterilized by a single migration, but with about only 10- percent efficiency. Although the Tetrahymena were more motile than the flagellates, they proved harder to rid of their bacteria, probably because of the tendency of the bacteria to become lodged among the cilia. Oehler (1919) states that he was able to free various Protozoa of bac- teria by allowing them to migrate over the surfaces of agar in Petri dishes. This technique may be applicable to a few types which are able to swim in a very thin film of moisture. The utilization of large tubes of sterile fluids for migration purposes was first mentioned by Purdy and Butterfield (1918). In their studies on the growth of bacteria in sewage, they state that they obtained on one occasion sterile Paramecium after allowing the ciliate to swim through thirty feet of sterile water. They call this the “marathon bath,” but give no details of its construction, use, or (and this appears to be important from the practical standpoint) means of sterilization of so long a tube. Glaser and Coria (1930) carried out a rather exhaustive study on methods of sterilization. One of the methods they used with success was the large-tube migration, used with negatively geotropic Protozoa. Their apparatus consisted of a tube fourteen or more inches long, with one- fourth inch bore and a fine tapering point. The large end was plugged with cotton and the whole sterilized in a container. Sterile fluid was drawn up to within two inches of the top by applying suction to the large end through a rubber tube. About 2 ml. of contaminated protozoan culture was then drawn up, and this formed a layer beneath the sterile fluid. The fine end was sealed in the flame and the tube mounted upright in a rack (Fig. 124). After periods of time varying from five minutes to eighteen hours, depending on the species of Protozoa, samples from the top of the tube contained many organisms which had “washed themselves free of most other microorganisms” (p. 790). It was usually necessary to repeat the migration, and this was accomplished by filling a second tube as before, and then drawing up two inches of fluid from the top CONTROL OF CULTURES 459 of the first tube. Occasionally a third wash was necessary to render the Protozoa bacteriologically sterile. Glaser and Coria were successful in sterilizing three species of ciliates and three species of flagellates by this method, and later (1935) three other species of ciliates were added to this list. The identifications of the organisms sterilized are uncertain, except for the well-known types, Paramecium and Chilomonas. Another method of migration described by Glaser and Corta in their Fig. 124. Migration tube. The tube is filled with a sterile fluid to within about two inches from the top. Protozoa to be sterilized are drawn up under the sterile fluid and the small tip sealed in a flame. The Protozoa migrate upward and are taken off at the top. (Redrawn from Glaser and Coria, 1930.) 1930 paper was one employing a V shaped tube (Fig. 125), filled with Noguchi’s semisolid medium. The larger arm of the tube measured 12 cm. in length and had an inner diameter of 28 mm. The smaller arm was 9 cm. in length with an inside diameter of 8 mm. After sterilization, the tube was filled with 15 ml. of sterile melted medium and this was al- lowed partially to solidify. Then the contaminated culture was placed at the bottom of the tube by injection through the small arm with a long, fine pipette. Air bubbles were excluded. The tube was then allowed to stand at room temperature for a sufficient time for some of the Protozoa to reach the top of the large arm, from the surface of which they were re- covered. 460 CGONTROE OF CULTURES This method, employing the use of semisolid media, appears to be applicable for many types of Protozoa. The consistency of the medium through which the Protozoa migrate seems to favor the removal of bac- teria, in that vigorous motion is necessary. The bacteria, on the other hand, would be largely prevented from dispersing far from the point of inoculation, at least for some time. This method, or some modification of it, should receive serious consideration from future investigators, in- Figure 125. V migration tube for semisolid media. The Protozoa to be steril- ized are injected through the small arm and deposited at the bottom of the V. They migrate up through the semisolid medium and are removed at the top of the large arm. (Redrawn from Glaser and Coria, 1930.) terested in the problems arising from the use of bacteriologically sterile protozoa. C. Combinations of dilution and migration —Cleveland (1928) de- scribed in detail the various manipulations which he employed to effect the sterilization of Tritrichomonas fecalis, a flagellate parasitic in the hu- man intestine. The method which yielded consistently satisfactory results was a combination of washing and migration. The flagellates to be steril- ized were concentrated by centrifugation, and the supernatant fluid drawn off with sterile pipettes. The packed flagellates were than layered onto the surface of sterile fluid (serum-saline) in centrifuge tubes, and the centrifuging process repeated. This procedure was continued through twenty sets of tubes, at which time Cleveland states that the ratio of Tritrichomonas to bacteria was about fifty to one. This constituted the CONTROL OF CULTURES 461 dilution part of the technique, and by this part of the procedure alone he was able to obtain many sterile flagellates. Higher percentages of sterile flagellates resulted, however, when he added a final migration to the above washing. After washing, a drop of the packed flagellates was placed in the center of a large Petri dish filled with sterile fluid. The Protozoa migrated in all directions, and after various intervals of time loops of medium, taken two to three inches from the center, were found to contain ten to fifteen trichomonads. These loops were inoculated di- rectly into tubes of media and the majority proved to be sterile. Hetherington (1934) described a much simpler technique, wherein he alternated the dilution method with migration. He employed Colum- bia culture dishes in Petri dishes, micropipettes for the transfer of the organisms, and 10 ml. serological pipettes for filling the dishes. The procedure was as follows: A drop of concentrated protozoan suspension was placed in the left margin of the first dish, care being taken not to disturb the one ml. of sterile fluid. By observing the activity of the Proto- zoa, it was seen that numbers of them migrated to the right edge of the fluid. Fifteen to twenty-five Protozoa were picked up in sterile micro- pipettes and transferred to the left side of a second dish. These were allowed to migrate. Those Protozoa which migrated were then placed in a third dish and allowed to remain there for three hours. They were transferred to the left side of the fourth dish and allowed to migrate, then transferred to the fifth dish for a second three-hour period. The sixth and seventh dishes were again used for migration, the Protozoa from the seventh being placed in culture medium. By exercising care in the handling of the fluids, the pipettes, and the covers of the Petri dishes, this method gave excellent results. Spores were defecated during the two three-hour periods. Especially adherent bacteria were lost during the migrations and washing, according to Hetherington, owing to the fact that the medium was nutritive (Bacto yeast extract in Peter's medium), resulting in the heightened activity of the bacteria. Recently Claff (1940) has described an apparatus designed to sterilize negatively geotropic Protozoa, which employs both the principles of dilution and migration and at the same time reduces the chances of air contamination. This apparatus consists of six flasks in series (Fig. 126). The Protozoa are injected into the bottom of flask 1 through a rubber vaccine cap with a hypodermic needle. From this point on, until they are 462 CONTROL’ OF CUETURES finally recovered from the sixth flask, they are in an entirely closed sys- tem. After injection, the Protozoa are allowed to migrate to the narrow top of the flask and, when large numbers have collected, they are forced over into the bottom of flask 2 by a volume (1-2 ml.) of sterile medium from the liter reservoir. After each migration the process is repeated, and the fluid drained from the system at the top of flask 6 is kept in Vaccine Porr Vaccine Porr Figure 126. Migration-dilution apparatus drawn to show construction. All flasks are filled with fluid and sterilized. After cooling, the Protozoa to be sterilized are injected through the vaccine port of flask 1. Successive migrations result, and the Protozoa are finally collected in the test tube from flask 6. (From Claff, 1940.) sterile test tubes and used as bacteriological controls on the fluid going before the Protozoa. The first Protozoa collected in the sixth tube are then separated into the various culture media. The chief advantages of this apparatus are the simplicity of operation and the reduction of chance contamination. The whole apparatus ts as- sembled in a compact unit and can be sterilized partially full of medium, Completion of the filling of the flasks is carried out after cooling, ex- treme care being taken to expel all air bubbles. No air bubbles may be allowed to enter during the injection of the contaminated Protozoa, as these will practically always rise ahead of the migration and contaminate every flask in order. Claff gives experimental evidence for the sterilization of Paramecium, Tillina, Tetrahymena and Glaucoma in this apparatus. It has been used on numerous occasions in this laboratory and found to be very satisfac- GONTROL OF CULTURES 463 tory. Perhaps its chief drawbacks are that it is limited to negatively geo- tropic organisms and to those types which are relatively powerful swimmers, and that the flasks must be specially built, as well as the car- riage, and that the whole is rather expensive. However, in those labo- ratories in which an extensive program of investigation requiring sterile Protozoa is being carried on, the purchase or the building of this appara- tus will be found advantageous. D. Bactericidal agents—Many methods have been tried in which agents were employed to kill bacteria without killing the Protozoa under investigation. Inhibition of bacterial overgrowth has been reported on numerous occasions by the use of various chemicals (Zumstein, 1900; Kofoid and Johnstone, 1929; and others) but sterility has rarely been obtained. Cleveland (1928) states that he used numerous chemicals in the hope that some would prove less injurious to Tritrichomonas than to the associated bacteria. His results were entirely negative, and he states that this type of investigation “appears to be almost a hopeless under- taking” (p. 256). We do have, however, a few reports which indicate the possibility of obtaining sterile Protozoa after treatments with various chemicals which are more toxic to the bacteria than to the Protozoa. In all cases the bac- tericidal agents were used on protozoan cysts, not on the trophic forms. Frosch (1897) was the first to report the sterilization of cysts by chemi- cal means. By the immersion of old cysts of Amoeba nitro phila in satu- rated sodium carbonate for a period of three days, Frosch claims to have killed all the associated non-spore-forming bacteria and to have recov- ered some of the amoebae. This method was repeated by Walker (1908) and the results confirmed. Oehler (1924) examined the possibility of treating ciliate and amoebae cysts with a variety of disinfectants, acids, alkalies, and salts but these results were far from encouraging. Severt- zoff (1924) investigated the action of toluene, chlorine, and calcium sulphide on the cysts of “‘soil amoebae” and found that the cysts were able to withstand the deleterious effects of these chemicals better than the associated bacteria (non-spore-forming types). He claims to have ef- fected complete sterility by the use of calcium sulphide, but was unable to establish pure cultures from the resulting cysts. Glaser and Coria (1930) were unable to obtain sterile Evglena proxima by their washing methods, but succeeded by treating ‘round or encysted stages’ (p. 803) 464 CONTROL OF CULTURES with a solution of potassium dichromate (1.25 to 2.5 percent) for from fifteen to thirty minutes. Perhaps the most exhaustive study on the rela- tive effects of chemicals on bacteria and protozoan cysts was carried out by Luck and Sheets (1931). They investigated the lethal concentrations of some eighteen substances on two-day-old cysts of Explotes taylori and their associated bacteria. They appear to have obtained sterile ciliates in some cases, when silver nitrate was used in high concentrations of glu- cose and sucrose. All other substances either were more toxic to the protozoan cysts than to the bacteria or were uniformly lethal to both. This line of approach to protozoan sterility does not seem to be too encouraging. As might well be supposed, the investigator is limited in any case to cyst-forming types. Even these are not uniformly resistant to chemical action, so that it appears that little can be learned at this time from the experiences of others. It seems that the methods referred to above, while they may have produced the desired results in some cases, are not to be recommended for routine work. There is always the prob- ability that any type of protozoan collected from the wild will have as- sociated with it spore-forming bacteria, which are always highly resistant to disinfectants. If a cyst-forming type of protozoan is first sterilized by washing or some other method and established in culture with a single known bacterium, then some of these disinfectants might be used to ad- vantage later for obtaining large numbers of sterile organisms for ex- perimentation. Of the physical bactericidal agents which have been employed, heat, used in different ways but always upon cysts of various species, has been most generally used. Walker (1908) reports that he was able to obtain sterile “Amoeba intestinalis’ by inoculating an agar plate, first with concentric rings of B. col7 and then, in the center, with amoebae cysts. The plate was heated to 70°-75° C. for one hour, which was sufficient to kill the Bac#llus coli but did not kill the cysts. He then added fresh bac- teria to the center of the plate and the amoebae excysted, fed, and mi- grated through the rings of dead B. co/7, Walker states that in their migra- tion through the dead bacteria they freed themselves of all living bacteria (by scraping them off?). He was able to recover sterile amoebae at the periphery of the plate. This report is surprising, in that moist heat was used, and cysts of Protozoa in general, under these conditions, are usually killed. CONTROL OF CUETURES 465 Oehler (1924) reports that he was able to obtain sterile Colpoda from cysts which had been heated either at 37° C. for six weeks or at 60°-64° C. for several hours. While it is true that certain types of bac- teria are killed by desiccation for considerable lengths of time, it would be nothing short of a miracle if only these delicate forms happened to make up the associated flora from the wild. In this laboratory we have employed a modification of Oehler’s method for obtaining large numbers of C. steinii, but a short description of the results will indicate that the method is not to be relied upon for initial sterilization. Figure 127. Details of construction of the sponge- and-glass plunger for the collection of cysts. The cen- ter rod is solid, and a piece of tubing holds the sponge down. We maintain cultures of C. ste/nii, sterilized by centrifugation, as de- scribed in a preceding section, in suspensions of the common nonspore- forming coliform bacterium, Aérobacter cloacae. From time to time we have needed large numbers of sterile ciliates for experimental work, and these were obtained from cysts collected as follows. A ring of sponge is placed at the bottom of a glass plunger which will reach well into a culture tube (Fig. 127). These sponge and glass plungers, with the top of the rods wrapped in cotton, are placed in test tubes of water and autoclaved. When cool, the plunger is transferred aseptically to a culture of Colpoda in Aérobacter. After the food organism is largely depleted, the ciliates encyst on and within the sponge. The plunger is then with- drawn and placed in a dry sterile test tube and set aside to dry. At first 466 CONTROL OF CULTURES it was hoped that prolonged desiccation, which we knew would not harm the cysts, would cause the death of the bacteria. Tests were made after one, two, three, and six months (duplicate preparations being placed by desiccators) by placing the plungers into tubes of sterile ex- cysting fluid (yeast extract). While the length of time required to show turbidity increased with the time of drying, viable Aérobacter were pres- ent in every case. Heating the dry sponge to 80° C. for three hours did not kill the cysts, nor did it kill the Aérobacter. The heat resistance is very different in the dry and the wet states, as evidenced by the fact that one hour of heating at 50° C. is enough to kill a suspension of this bacterium. It was found, however, that a simple manipulation could be employed with eight-months’-old sponge-glass plunger preparation to obtain large numbers of sterile ciliates. U-shaped tubes were partly filled with yeast extract and sterilized, and the sponge-glass plunger was inserted aseptt- cally into one arm. The tube was placed in an upright position and the minimum time was allowed for the ciliates to excyst. Then, without disturbing the fluid, the plug from the other arm was lifted and some of the fluid containing the freshly excysted ciliates was withdrawn. These ciliates were sterile, provided not too long a time had elapsed since the sponge was introduced and provided the fluid was not unduly disturbed. What appears to happen is that the dry bacteria take a considerable time to become active and to disperse through the medium, while a few sec- onds after the ciliates leave their cyst walls they migrate to the top of the fluid. It should be remembered that this method has been used only when the associated bacteria have been reduced to a single species. By careful manipulation and by having the conditions just right, it might be possible to use this method on wild, cyst-forming material, but this 1s only a conjecture as it has not been done to date. The possibility of using radiation for sterilization was investigated by Brown, ef al. (1933). They found that E. taylor7, trophic or cysts, with- stood a much longer exposure to X-rays (2,110 Roentgen units per second) than did the associated bacteria (in this case Pseudomonas fluorescens and B. coli). They were able to obtain many sterile ciliates by this method. Here again, the success of the method appears to be due to the specific type of bacteria present, and it is extremely doubtful if, CONTROL OF CULTURES 467 under the conditions of wild cultures, sterile Protozoa could be obtained with any regularity by this method. THE IMPORTANCE OF ADEQUATE STERILITY TESTS It is, of course, obvious that any method for ridding Protozoa’ of bac- teria must be carried out under the rigid rules of bacteriological tech- nique. Bacteria are so varied in form and activity that special pains must be taken to check the results of any method before the treated Protozoa may be pronounced sterile. Microscopic examination is of little or no use, at least until time has been given for any accompanying bacteria to multiply. We must therefore give any possible contaminant every conceivable chance to multiply, thereby revealing its presence. Sterility tests are usually made in two ways, by inoculation into fluid media and by spreading on nutrient solid media. In the fluid media (broth) contamination shows itself when the broth becomes turbid. The turbidity test is sufficient, when the contaminant is such a one as will distribute itself through the media. In other words, turbidity denotes contamination. But lack of turbidity does not always denote sterility. Some bacteria grow very slowly in broth and form small clumps which sink to the bottom of the test tube, leaving the broth clear. This experi- ence was reported by Hetherington (1933, 1934) and by Stuart, Kidder, and Griffin (1939). A macroscopic examination of a tube was not enough to reveal the presence of these organisms. Plating, either by pipetting fluid from the culture to be tested or by streaking the surface of solid media (nutrient agar) with a needle dipped into the culture, is usually more satisfactory than the turbidity test. The plates are allowed to incubate, and the surface is examined for bacterial colonies. The usual contaminants from wild infusions will appear in from twenty-four to forty-eight hours. But this method has its limitations. Some bacteria grow very slowly at room temperatures, but well at higher temperatures. Others are the reverse. Duplicate sets of plates should always be made, one set to be incubated at room temperature and the other at temperatures from 30° to 37° C. The time factor should be carefully considered. The slow-growing types (such as the Mycobac- tertum reported by Stuart, Kidder, and Griffin, 1939) may not appear until many days after the inoculation. It is necessary in all tests with agar 468 CONTROL OF GUETURES plates to keep the plates for at least ten days and it is safer to keep them two weeks. The plating method is usually a better criterion of conditions within a culture than the turbidity test, for another reason. When dealing with ciliates, normally bacteria-feeders, it is often the case that the bac- teria are eaten out of the media almost as fast as they multiply. This is more likely to happen when the ciliate is a voracious feeder and multi- plies rapidly and when the contaminant is one of the slow-growing va- riety. It should not be supposed, however, that all of the bacteria will be eaten, although two such cases are on record (Elliott, 1933; Johnson, 1935). Some of the bacteria will almost invariably escape and be carried along from transplant to transplant. On the solid media, however, the ciliates do not move about, and colonies of bacteria develop unhampered. Although they are not prevalent in wild infusions, tests should always be conducted for anaérobic bacteria. The simplest test and one which will usually determine their presence or absence is the following: Tubes containing not over 3 ml. of nutrient broth plus a two to two-and-a-half- inch layer of paraffin oil, are plugged with cotton and autoclaved for twenty minutes at fifteen pounds’ pressure. Rubber stoppers are sterilized at the same time. Immediately after sterilization the rubber stoppers are fitted into the tubes, which are then allowed to cool. When the broth is cool, inoculations are accomplished by injecting the material to be tested through the paraffin oil into the broth, the rubber stopper being im- mediately replaced. This type of culture will allow even obligatory an- aérobes to multiply, although not necessarily to the height of their capacity. Enough growth is obtained, however, to determine the pres- ence of anaérobic contaminants. The obligatory anaérobic bacteria, it must be admitted, do not form an important group for our consideration, as they occur so infrequently. The facultative anaérobes may be detected by more common procedures. ESTABLISHMENT OF STERILIZED PROTOZOA IN CULTURE The sterilization of Protozoa is, after all, only a means to an end. It is of very little value to the investigator if, after going to the trouble to rid a species of Protozoa of their associated bacteria, the Protozoa fail to live. For the most perfect control of a protozoan culture for experimental work, pure cultures are necessary. This means that the protozoan under investigation must be established in a medium containing no other living CONTROL OF CULTURES 469 organism. This is easily accomplished in a number of cases, even to the establishment in media containing only dissolved proteins. A large num- ber of flagellates probably exist in nature by the utilization of dissolved substances and, when sterilized, continue to employ this type of nutri- tion. Among the free-living ciliates, all species are known to possess oral openings into which solid foods are drawn. Some of these, however, are able to live on dissolved proteins in pure culture, e.g., Tetrahymena. Other types may be able to obtain only a small amount of nutriment from the dissolved proteins, but are able to feed on nonliving particu- late matter. This was found to be the case in this laboratory with Glaz- coma scintillans (unpublished work). When sterilized ciliates were placed in a wide variety of media containing dissolved proteins, very little multiplication took place. However, good growth resulted in un- filtered Yeast Harris, containing quantities of broken-down yeast cells. Still other types of free-living ciliates appear to be unable to exist with- out living organisms as food. This may be the case with the true carni- vores, and here there is an excellent opportunity to make some interesting studies on the ‘“Zweigliedrige Kulture” without employing bacteria. It is only necessary to be able to grow the food Protozoa in pure culture and to supply it to the sterile carnivores. Some work along these lines has already been started in our laboratory, using pure cultures of Tetra- hymena as the food organism and studying the effects of such a diet on G. vorax (Kidder, Lilly, and Claff, 1940). Similar cultures of Stylony- chia pustulata are being studied by D. M. Lilly (Lilly, 1940), and the nutritional requirements of a Evglena-feeding Perispira is being investi- gated by the author and V. C. Dewey in this laboratory. Oehler (1919) stated that Colpoda steinii was unable to live on dissolved nutrients but would live on particulate matter, including dead bacteria. Kidder and Stuart (1939) were unable to confirm Oehler’s results, but found that C. stesniz was dependent upon living organisms. They remark, however, that the possibility does exist that some combination of food substances and conditions, as yet not known, may possibly allow this important ciliate to reproduce in the absence of living organisms. It may be inferred from what has already been said that the estab- lishment of a sterile protozoan in culture is not a routine matter. Of course the goal is a pure culture, for more precise control is then possible. With some types it has been found that growth follows when they are 470 CONTROL OF CULTURES placed immediately in a medium containing dissolved proteins (tryptone, proteose-peptone, yeast extract, and so forth). These types are the true saprozoic forms. Others must have particulate matter, so it is best, espe- cially when dealing with a new type, to inoculate into a wide variety of media. In this laboratory it is the practice to start our newly sterilized Protozoa in five different types of media, usually ten isolations into each. Our standard five types for first tests are 0.1-percent proteose-peptone; 5-percent yeast autolysate; 0.5-percent yeast extract; 0.5-percent malted milk; and 0.5-percent unfiltered Yeast Harris. It is sometimes necessary to have quite a range of pH values within the different media, in order to obtain growth in even one or two of the tubes. A number of species of Protozoa appear to be dependent upon living organisms as a source of food. This is true not only of the carnivores, but seems to hold for a number of bacteria-feeders as well. With the carnivores it is usually sufficient to observe their diet in nature to decide upon a suitable food animal. If the food animal can be grown in pure culture, then the chances are good that it will be possible to establish the carnivore in “Zwiegliedrige Kulture.” Some carnivores have been found to be very selective, while others are able to feed on any one of a number of organisms. Occasionally a natural bacteria-feeder will turn carnivorous and then can be established without bacteria. With the obligatory bacteria-feeders the best that can be done, as far as we now know, is to establish them on a single species of favorable food bacteria. Here again it is absolutely necessary to start with sterile Protozoa, as even in the so-called non-nutritive fluids (salt solutions, distilled water, and so forth) many extraneous bacteria, which are not favorable as food, will multiply and be continually present from trans- plant to transplant. Results may be entirely misleading under these con- ditions, as a number of common bacteria prove to be deleterious to many Protozoa (see Kidder and Stuart, 1939). One method of setting up cul- tures containing a single protozoan species in a suspension of a single species of bacteria is simply to try out a number of known bacteria until one is found which will support growth. However, some ciliates prove to be extremely selective, even as to specific bacteria. If poor growth or no growth results after all the known bacteria have been used, then the investigator must try to isolate from the wild culture the type of bacteria upon which the ciliate was originally feeding. This procedure is tedious, CONTROL OF CULTURES 471 but sometimes necessary. A wild culture is selected in which the Protozoa under investigation are multiplying rapidly. From the fluid of this cul- ture, surface-streak plates are prepared and pure cultures of all the different types of bacteria are obtained. From these pure cultures, sus- pensions are systematically prepared and inoculated with the sterile Protozoa. This was the method used by Johnson (1933) in his work on Oxytricha. Johnson states that his selection was made on the basis of prevalence, in a thriving wild culture. In other words, the type of bac- teria found in the greatest abundance he supposed to be the type upon which the Protozoa were most likely to be feeding. We have found that this is not always the case. In our work on the ciliate T7/lina (T. canalt- fera, obtained from Dr. J. P. Turner and described by him in 1937) we were able to obtain growth on one species only, out of twenty-six types isolated from a thriving culture. This one species (a Zopfius) was the least prevalent of all on our plates. The reason for this appears to be that T7//7na being so very selective, the Zopfws were eaten out of the culture by the time we took our samples, while the other twenty-five species were left to multiply. We have found this situation to hold in a number of cases, so that we are of the opinion that the results obtained by Johnson were due to the fact that he was dealing with a ciliate which was not rigidly selective. The work tending to show that supplementary factors (viz., thiamin and the like) are necessary for the growth of several Protozoa in pure culture has been reviewed in a subsequent chapter, but several observa- tions regarding the same theme may be given here, as they apply to “Zwiegliedrige Kulture.” These supplements may make the difference between success and failure to establish a protozoan in bacteria-free cul- ture. Investigations are now going on in this laboratory on the supple- ment question, but they are as yet far from complete. Therefore little can be said as to the exact nature of the substances or factors to be de- scribed. In order to present this problem clearly to the reader, a descrip- tion of a typical example will be given. D. M. Lilly, working in this laboratory, has studied the nutritional requirements of two hypotrichous ciliates, Stylonychia pustulata and Pleurotricha lanceolata. Both of these forms are bacteria-feeders in na- ture, but will also become carnivorous in the presence of other small ciliates. Sterilization was carried out, with the use of the dilution method, 472 CONTROL OF CULTURES in Syracuse watch glasses enclosed in cellophane bags, as previously described. In the case of Stylonychia, it was found possible to establish them on living yeast cells, suspended in distilled water, in the absence of any other food material. Sterile ciliates would not live on autoclaved yeast, however. Sterile ciliates would eat quantities of living Tetrahymena (taken from agar slants and suspended in distilled water), but would not divide. Sterile ciliates, placed in suspensions of autoclaved yeast, and living Tetrahymena grew well and established flourishing cultures. Sterile ciliates in dead yeast and dead Tetrahymena, failed to multiply. Additions of none of the known water-soluble vitamins changed the situation. The inference is, as Lilly points out (1940), that Stylonychia requires, among other things, two unknown factors—one found in yeast (even after autoclaving), but not present, at least in sufficient quantities, in Tetra- hymena; the other what might be called a living factor, present in living yeast and Tetrahymena. Both of these factors are present in certain favor- able species of bacteria when the bacteria are alive, but the “living factor” is destroyed with the death of the bacteria. The so-called living factor is not a surprising requirement among Protozoa, as experience has shown that many different types will not live without being supplied with some type of living organism. The yeast factor seems to belong to the water- soluble, heat-stabile group, but is not identifiable with any one of the known B complex. While this factor is present in dried and pasteurized yeast (Brewer's Yeast Harris), it is not present in sufficient quantities in Difco dehydrated yeast extract. Concentration and partial purification of the yeast factor have been carried out, but until this work is further along we must content ourselves with these few facts. This example is one of many similar cases and serves to point out that several conditions must be recognized and fulfilled, if the investigator is to be successful in establishing sterile Protozoa in culture. The possi- bility of supplementary factors must be considered before it can be said of any type that it cannot be grown bacteria-free. Somewhat the same situation was encountered by Glaser and Coria (1933) in their work on Paramecium. They finally announced a complicated medium which proved to be successful, and this medium contained pieces of fresh rabbit kidney (possibly supplying the living factor during the early growth phases of the ciliate). It is not the purpose of this chapter to consider in detail all of the CONTROL OF CULTURES 473 interesting work which has been done regarding accessory growth fac- tors and nutritional supplements. A large number of these are consid- ered in the chapter on pure cultures (Chapter IX). It might be sug- gested, however, that one of the most fertile fields of protozoan investi- gation has been opened up with the development of bacteria-free tech- niques, and our knowledge of unsuspected requirements in the nutrition of carnivores should be extended greatly in the near future. The possi- bilities are many along these lines, and therefore considerable time has been devoted to the methods which will have to be employed in the be- ginning of any such studies. LITERATURE CITED Brown, M. G., J. M. Luck, G. Sheets, and C. V. Taylor. 1933. The action of X-rays on Explotes taylori and associated bacteria. J. gen. Physiol., 16: 397-406. Burt, R. L. 1940. Specific analysis of the genus Colpoda with special reference to the standardization of experimental material. Trans. Amer. Micros. Soc. (in press.) Claff, C. L. 1940. A migration-dilution apparatus for the sterilization of Protozoa. Physiol. Zo6l. (in press. ) Cleveland, L. R. 1928. The suitability of various bacteria, molds, yeasts and spirochaetes as food for the flagellate Tritrichomonas fecalis of man as brought out by the measurement of its fission rate, population density, and longevity in pure cultures of these microorganisms. Amer. J. Hyg., 8: 990-1013. Elliott, A. M. 1933. Isolation of Colpidium striatum Stokes in bacteria-free cul- tures and the relation of growth to pH of the medium. Biol. Bull., 65: 45-56. Frosch, P. 1897. Zur Frage der Reinziichtung der Amében. Zbl. Bakt., Orig., 21: 926-32. Furgason, W. H. 1940. The significant cytostomal pattern of the “Glaucoma- Colpidium group,” and a proposed new genus and species, Tetrahymena geleii, Arch. Protistenk. (In press.) Glaser, R. W., and N. A. Coria. 1930. Methods for the pure culture of certain Protozoa. J. exper. Med., 51: 787-806. —— 1933. The culture of Paramecium caudatum free from living micro- organisms. Jour. Parasit., 20: 33-37. 1935. The culture and reactions of purified Protozoa. Amer. J. Hyg., PABA VIMIEP AU Hargitt, G. T., and W. W. Fray. 1917. The growth of Paramecium in pure cultures of bacteria. J. exper. Zool., 22: 421-54. Hetherington, A. 1933. The culture of some holotrichous ciliates. Arch. Protistenk., 80: 255-80. 474 CONTROL OF CULTURES —— 1934. The rdle of bacteria in the growth of Colpidium colpoda. Physiol. Zool. 7: 618-41. Johnson, D. F. 1935. Isolation of Glaucoma ficaria Kahl in bacteria-free cul- tures, and growth in relation to pH of the medium. Arch. Protistenk., 86: 263-77. Johnson, W. H. 1933. Effects of population density on the rate of reproduc- tion in Oxytricha. Physiol. Zo6l., 6: 22-54. Kidder, G. W., D. M. Lilly, and C. L. Claff. 1940. Growth studies on ciliates. IV. The influence of food on the structure and growth of Glaucoma vorax, sp. nov. Biol. Bull., 78: 9-23. Kidder, G. W., and C. A. Stuart. 1939. Growth studies on ciliates. I. The role of bacteria in the growth and reproduction of Colpoda. Physiol. Zool., 12: 329-40. Kofoid, C. A., and H. G. Johnstone. 1929. The cultivation of Endameba gingivalis (Gros) from the human mouth. Amer. J. publ. Hlth., 19: 549-52. Lilly, D. M. 1940. Nutritional and supplementary factors in the growth of carnivorous ciliates. (MS.) Luck, J. M., and Grace Sheets. 1931. The sterilization of Protozoa. Arch. Protistenk., 75: 255-69. Oehler, R. 1919. Flagellaten- und Ciliatenzucht auf reinem Boden. Arch. Protistenk., 40: 16-26. —— 1924. Weitere Mitteilungen tiber gereinigte AmGdben- und Ciliaten- zucht. Arch. Protistenk., 49: 112-34. Ogata, M. 1893. Uber die Reinkultur gewisser Protozoen (Infusorien). Zbl. Bakt., Orig., 14: 165-69. Parpart, A. K. 1928. The bacteriological sterilization of Protozoa. Biol. Bull., 113-20: Purdy, W. C., and C. T. Butterfield. 1918. Effect of plankton animals upon bacterial death rates. Amer. J. publ. Hlth., 8: 499-505. Severtzoff, L. B. 1924. Method of counting, culture medium and pure cultures of soil amoebae. Zbl. Bakt., Orig., 92: 151-58. Stone, W. S., and F. H. K. Reynolds. 1939. A practical method of obtaining bacteria-free cultures of Trichomonas hominis. Science, n.s., 90: O1-92: Stuart, C. A., G. W. Kidder, and A. M. Griffin. 1939. Growth studies on ciliates. III. Experimental alteration of the method of reproduction in Colpoda. Physiol. Zoél., 12: 348-62. Turner, J. P. 1937. Studies on the ciliate Ti/lina canalifera, n. sp. Trans. Amer. micr. Soc., 56: 447-56. Walker, E. L. 1908. The parasitic amebae in the intestinal tract of man and other animals. J. med. Res., 17: 379-459. Zumstein, H. 1900. Zur Morphologie und Physiologie der Evglena gracilis Klebs. Jb. wiss. Bot., 34: 149-98. CHAPTER IX FOOD REQUIREMENTS AND OTHER FACTORS INFLUENCING GROWTH OF PROTOZOA IN PURE CULTURES Rege. ALT Ir Is OBVIOUS that the growth of Protozoa is influenced by many differ- ent factors. The importance of some of these is well recognized and the relationships to growth are partially understood in a few instances, but there is little or no detailed information bearing on other factors. Here and there, investigations have suggested possible solutions to certain problems, but just as frequently have uncovered new problems which in turn must be solved in the approach to an understanding of protozoan growth. The present lack of information extends to such questions as the list of essential elements, the nature of the simplest organic foods adequate for various species, “‘growth factor’ or vitamin requirements, and the combined effects of various environmental factors on growth. Furthermore, Protozoa in cultures constitute populations and presumably are subject to general laws of population growth. Hence the final inter- pretation of many experimental results demands further knowledge of the behavior of populations. From the experimental standpoint, several types of protozoan popu- lations may be distinguished. (1) The pure culture contains a single protozoan species with no other microérganisms. In most cases such cultures have been started from pure lines and are thus genetically homo- geneous. The number of bacteria-free strains now in existence is un- certain, although an estimate of 100 may be fairly accurate. Many strains of Phytomastigophora are maintained by Pringsheim (1930), while ad- ditional species belonging to various groups of Protozoa are to be found in several other laboratories. (2) The species-pure culture contains a single protozoan species, usually in pure line, with bacteria, algac, or other microérganisms as sources of food. Populations of this type have been maintained on known species of microérganisms (e.g., Ocehler, 476 FOOD REQUIREMENTS 1916, 1919; Philpott, 1928; Geise and Taylor, 1935; D. F. Johnson, 1936; W. H. Johnson, 1933, 1936; Loefer, 1936d) or on mixtures of bacteria. (3) Mixed populations, as described in the work of Gause (1935), contain two species of Protozoa feeding on other microérgan- isms, or perhaps one upon the other. This technique presents interesting possibilities. (4) Wild populations are mixtures of species as obtained from natural sources. Such populations have been studied particularly in relation to succession of species in cultures (e.g., Woodruff, 1912). The present discussion deals primarily with investigations on pure cultures, which, with their obvious advantages, afford favorable ma- terial for the study of many problems. With the exclusion of other microorganisms, it is possible to control the food supply and to deter- mine, more accurately than by other methods, the relation of environ- mental factors to growth. Detailed investigation of metabolic activities is possible with pure cultures, whereas allowance must be made for other microorganisms when bacteria-free material is not used. The pure- culture technique and scrupulous cleanliness of glassware are essential in studies on food requirements. This is true particularly of investiga- tions on autotrophic nutrition, since protein contamination, to the ex- tent of one part in millions, may influence growth. Likewise, pure cultures are a prerequisite to investigations on specific growth factors, or vitamins. Some of the methods used by various investigators have been described elsewhere (Pringsheim, 1926; Hall, 1937a). The technique is not par- ticularly difficult and, while the preparation of glassware is somewhat laborious and constant precaution against contamination must be exercised, the results more than justify the additional time and effort. Food REQUIREMENTS OF PROTOZOA Food requirements of the various groups of Protozoa differ in certain general respects. The chlorophyll-bearing species may utilize carbon dioxide, while other types require a more complex carbon source. Nitro- gen requirements also vary. Some forms thrive on ammonium salts or on nitrates; growth of other species is supported by nothing simpler than an amino acid; while that of a third group is dependent upon peptones or comparable protein-cleavage products. On the basis of such criteria, a number of different methods of protozoan nutrition have been recog- nized (Lwoff, 1938a; Pringsheim, 1937d). A somewhat simplified classi- fication (Hall, 1939b) is presented below: FOOD REQUIREMENTS 477 I. Phototrophic nutrition is characteristic of chlorophyll-bearing species, which utilize the energy of light in photosynthesis. Some appear to be obligate phototrophs, while others may be grown in darkness under suitable conditions. On the basis of nitrogen requirements, several va- rieties of phototrophic nutrition may be recognized: (1) Photoautotrophic nutrition is characteristic of species which can grow in inorganic media; Chlorogonium euchlorum (Loefer, 1934; Hall and Schoenborn, 1938a) is typical. No obligate photoautotroph is known. (2) Photomesotrophic nutrition 1s that in which one or more amino acids serve as nitrogen sources. In Euglena deses (Dusi, 1933b) this seems to be the simplest possible method of nutrition. Photomesotrophic nutrition may also be carried on by facultative photoautotrophs. (3) Photometatro phic nutrition is characteristic of species which grow in peptone solutions or comparable protein media. Exglena pisciformis (Dusi, 1933b) has been described as an obligate photometatroph. This type of nutrition may also be carried on by facultative photoautotrophs and photomesotrophs. II. Heterotrophic nutrition is characteristic of species which have no chlorophyll and hence require an organic carbon source. Some chloro- phyll-bearing species have been grown in darkness and may, in this sense, be considered facultative heterotrophs. On the basis of nitrogen require- ments, three varieties of heterotrophic nutrition may be distinguished. (1) Heteroautotrophic nutrition involves utilization of inorganic nitrogen compounds in the presence of an organic carbon source. Poly- toma uvella (Pringsheim, 1921; Lwoff and Dusi, 1938a) and Astasia sp. (Schoenborn, 1938) are examples. (2) Heteromesotrophic nutrition: growth requirements may be satis- fied by one or more amino acids as sources of nitrogen and carbon. Growth is usually much more vigorous with an additional carbon source, such as acetate. Polytomella caeca (Pringsheim, 1937a, 1937c) is repre- sentative. ; (3) Heterometatrophic nutrition is characteristic of organisms which grow in peptone solutions or similar media. Obligate heterometatrophs, such as Hyalogonium klebsi (Pringsheim, 1937b) and Glaucoma piri- formzs (A. Lwoff, 1932), cannot be grown in amino-acid solutions or simpler media. This type of nutrition is exhibited by various holozoic Protozoa (ciliates, amoebae) which have been grown in pure culture. Among the parasitic flagellates, certain Trypanosomidae (M. Lwoff, 478 FOOD REQUIREMENTS 1930, 1933a, 1936) have been grown under comparable conditions; other parasitic flagellates (M. Lwoff, 1929a, 1929b, 1929c, 1929d, 1933a, 1933b, 1937, 1938a; Glaser and Coria, 1935b; Cailleau, 1936a, 1936b, 1937a, 1937b, 1938a, 1938b) apparently require, in addition, blood, serum, tissue extracts, or special growth factors. PHOTOAUTOTROPHIC NUTRITION Photoautotrophic nutrition is generally attributed to the chlorophyll- bearing plant-like flagellates and is, by definition, limited to this group of Protozoa. On the other hand, there is no evidence to support the as- sumption that all chlorophyll-bearing species are photoautotrophic, since several green flagellates have been grown only in amino acid or peptone media. Furthermore, in the absence of pure cultures, there is no conclu- sive evidence that any member of the Chrysomonadida, Heterochlorida, Cryptomonadida, Dinoflagellida, or Chloromonadida is capable of carry- ing on photoautotrophic nutrition. While it may be expected that such flagellates will be found in each of these orders, speculation must re- main subject to experimental verification. The known facultative photoautotrophs are: Chlamydomonas agloé- formis (M. Lwoff and A. Lwoff, 1929), Chlorogonium elongatum (Loefer, 1934), C. eachlorum (Loefer, 1934; Hall and Schoenborn, 1938a), Haematcoccus pluvialis (M. Lwoff and A. Lwoff, 1929), and Lobomonas piviformis (Osterud, 1938, 1939), representing the Phyto- monadida; and Euglena anabaena (Dusi, 1933b; Hall, 1938b), E. gracz- lis (Pringsheim, 1912; Dusi, 1933a; Hall and Schoenborn, 1939a), E. klebsii and E. stellata (Dusi, 1933b) and E. viridis (Hall, 1939a), representing the Euglenidae. The establishment of autotrophic strains has often encountered difh- culties, and conflicting results have sometimes been reported for the same species. Some of the apparent contradictions may be the result of differ- ences in culture media and in technique. In addition, the technical diffi- culties may sometimes be augmented by a selective action of inorganic media, as observed in Evglena (Hall and Schoenborn, 1938b). The present knowledge of food requirements in photoautotrophic nutrition is far from complete. In fact, it is not yet possible to list all the elements which are essential to growth, and little or nothing 1s known about quantitative food requirements. However, the following elements, FOOD REQUIREMENTS 479 which are found as general constituents of protoplasm, may be listed as probably essential to growth: C, H, O, N, P, S, Ca, Fe, K, Na, Mg, Ck Additional possibilities include Cu, Sr, Al, Mn, Zn, Ni, B, Rb, Ba, Si, Ti, V, As, Co, and Cr, since these have all been demonstrated in plant or animal tissues and some appear to be essential to the growth of higher organisms. By a process of successive eliminations, it should be possible to deter- mine which elements are and which are not essential to growth. Such investigations, however, are entirely dependent upon adequately purified chemicals. In certain investigations (Hall, 1938b, 1939a; Hall and Schoenborn, 1939a; Osterud, 1938) analyzed reagents have been used in the preparation of culture media and, within such limits, the composi- tion of each medium is known. One of these media (EF) contains the following elements: C, H, O, N, P, K, Mg, S, Ca, and Cl in appreciable amounts, and traces (1 & 10 to1 X 10 gm. percc.) of Cu, Ba, Fe, As, Mn, Na, Zn, and Pb. This medium has supported growth of Evglena gracilis, E. viridis, E. anabaena, and Lobomonas piriformis. Another medium (EC) has supported growth of E. gracilis, E. viridis, and Chlo- rogonium euchlorum. So far as the component elements are concerned, this medium differs from EF in the absence of Ba, in lower concentrations of Ca, Cl, Mg, and Mn, and in higher concentrations of P and K. Media EA and EAB, which have supported growth of E. gracilis, E. viridis, and C. euchlorum, contain a trace of Al, but no Ba; except for concentrations, the list of elements is otherwise the same as in EC and EF. Just how many of the “‘trace’’ elements are actually essential to growth has not been determined. The omission of Ba from three media and of Al from two media seems to be of little significance, and the status of these two elements as essential substances is questionable. By comparable methods of elimination, it may be possible to determine whether various other elements are actually essential in photoautotrophic nutrition. In a few cases there is evidence that particular elements exert signifi- cant effects on growth. Calcium requirements of Ezglena stellata (Dust, 1933b) are much greater than those of other Euglenidae investigated, and manganese (Hall, 1937c) has been found to accelerate growth of E. anabaena. In addition, a few similar observations on heteroautotrophic flagellates have been reported. For instance, A. Lwoff (1930) has re- ported that Fe is essential to growth of Polytoma uvella. Similarly, Mast 480 FOOD REQUIREMENTS and Pace (1935) found that Chilomonas paramecium survived for only a few transfers in media without S. For example, one S-free line died on the seventh day and others on the third, while several lines in media containing S were maintained for from twenty to twenty-four days. An- other example is that of Hyalogonium klebsu, which requires relatively large amounts of calcium (Pringsheim, 1937b). It is possible, of course, that the action of certain elements may not be specific; in other words, comparable effects on metabolism may be exerted by several different elements, one of which may be substituted for another. This possibility should be considered in investigations on food requirements of photoautotrophs and heteroautotrophs. PHOTOMESOTROPHIC NUTRITION Euglena deses (Dusi, 1933b) may be considered an obligate photo- mesotroph, a flagellate which has lost the primitive photoautotrophic ability characteristic of various other green flagellates. In addition to this species, several facultative photoautotrophs among the Euglenidae are known to carry on photomesotrophic nutrition: E. anabaena (Dust, 1933b; Hall, 1938b), E. gracilis (Dusi, 1933a), E. klebsii, and E. stellata (Dusi, 1933b). An interesting feature of these Euglenidae 1s that a particular amino acid may support growth of one species but not another (Dusi, 1931). For example, phenylalanine was satisfactory for E. anabaena, E. gracilis, and E. stellata, but not for E. deses and E. klebsii, while serine was adequate for growth of all except E. anabaena. Comparable differences were noted for several other amino acids. Among the Phytomonadia, photomesotrophic nutrition has been dem- onstrated in Chlamydomonas agloéformis and Haematococcus plu- vialis (A. Lwoff, 1932), and also in Lobomonas piriformis (Osterud, 1939). In addition, Loefer (1935b) observed, in Chlorogonium elonga- tum and E. euchlorum, acceleration of growth by glycocoll and several other amino acids, added separately and in mixtures, to an inorganic medium and to a salt solution containing sodium acetate. The growth of photomesotrophic species may be accelerated by the addition of various carbon sources (e.g., sodium acetate) to an amino- acid medium. Concerning mineral requirements in photomestrophic nutrition, nothing is known beyond the fact that amino acids have often FOOD REQUIREMENTS 481 been added to salt solutions comparable, except for the omission of inorganic nitrogen, to the media used for photoautotrophic nutrition. PHOTOMETATROPHIC NUTRITION Photometatrophic nutrition can be carried on by all of the chlorophyll- bearing flagellates which have been established in pure culture. Certain species, such as E. pisciformis (Dusi, 1933b), may prove to be obligate photometatrophs, although recent observations (Dusi, 1939) indicate that E. pisciformis should not be so classified. Peptones of one type or another have usually furnished the food supply, and in at least a few cases a solution of peptone in distilled water has supported growth. In addition to peptones, gelatin (Hall, 1938b) may also support growth, and certain species are known to produce proteolytic enzymes (Mainx, 1928; Jahn, 1931; Hall, 1937b). Many of the flagellates grow well on agar slants, provided the agar is enriched with a suitable peptone and sometimes with an additional source of carbon; such cultures are con- venient for the maintenance of laboratory stocks. Although various peptone media are satisfactory for all the species which have been studied, growth may be accelerated by the addition of salts of certain fatty acids, various carbohydrates, and several alcohols. Acceleration of growth by carbohydrates has been noted in E. gracilis (Jahn, 1935b) and in two species of Chlorogonium (Loefer, 1935a). Fermentation of dextrose by E. proxima was reported by Glaser and Coria (1930, 1935a), but other workers have failed to note such changes in cultures of Euglenidae. Furthermore, Loefer (1938b), using Bene- dict’s colorimetric method, failed to detect utilization of dextrose by C. elongatum and C. euchlorum, in spite of the accelerating effect on growth. Acceleration of growth by ethyl alcohol has been reported by Loefer and Hall (1936) in E. deses and E. gracilis, and similar effects of several alcohols on the latter species have been described by Provasoli (1938c). Acceleration of growth by fatty acids, in cultures exposed to light, has been reported for E. gracilis (Jahn, 1935d) and E. stellata (Hall, 1937d). Furthermore, macroscopic observations on cultures in various stock-culture media have indicated such effects in approximately thirty species maintained in our laboratory. However, most of the quanti- tative studies on carbon sources have been based upon cultures main- 482 FOOD REQUIREMENTS tained in darkness (heteromesotrophic and heterometatrophic nutrition) , as described below. Hutner (1936) failed to note acceleration of growth by fatty acids or carbohydrates in E. anabaena or by carbohydrates in E. gracilis. Since Hutner’s conclusions apparently were based upon the macroscopic ap- pearance of his cultures, he may have overlooked effects comparable to those reported by other workers. HETEROAUTOTROPHIC NUTRITION The utilization of inorganic nitrogen compounds in the presence of acetate or another organic carbon source, has been attributed to several colorless Phytomastigophora: Chilomonas paramecium (Mast and Pace, 1933), Polytoma uvella (Pringsheim, 1921; Lwoff and Dusi, 1938a), P. obtusum (Lwoff, 1929b, 1932), and Astasza sp. (Schoenborn, 1938, 1940). The results of Mast and Pace have not been duplicated by Loefer (1934) nor by Hall and Loefer (1936). Pringsheim (1935a) reported growth of C. paramecium in an ammonium-salt and acetate medium, but Lwoff and Lederer (1935) and Pringsheim (1935b) have pointed out that Pringsheim’s medium contained “‘extract of soil,” without which the flagellates failed to grow. Hence, Pringsheim did not confirm the observations of Mast and Pace. More recently, Lwoff and Dusi (1937a, 1938a, 1939b) have grown a strain of this species in an ammonium acetate medium, but only in the presence of either thiamine or thiazole and pyrimidine. Since Lwoff and Dusi added organic nitrogen com- pounds to their medium, application of the term heteroautotro phic to C. paramecium may be inappropriate. The contradictory results obtained by various workers with this species have not yet been explained. It is possible that different strains may vary in their nutritional requirements. Or it is conceivable that the strain of Mast and Pace was established through a selective process, similar to that reported in several species of Euglena (Hall and Schoenborn, 1938b). The first known instance of heteroautotrophic nutrition in Euglenida is that described by Schoenborn (1938, 1940) in Astasia sp. This strain has now passed the nineteenth transfer, so that the peptone carried over from the original stock culture has been reduced, through serial dilu- tion alone, to a calculated concentration of less than 1.8 & 10? gm. perce According to Pringsheim (1937b), Chlorogonium euchlorum may be FOOD REQUIREMENTS 483 grown in darkness as a facultative heteroautotroph, provided glucose caramel is added to the medium. Osterud (1939) has reported growth of Lobomonas piriformis in an ammonium-nitrate and acetate medium for three transfers (twelve weeks) in darkness. Likewise, in a medium similar to that of Osterud, growth of Ezglena gracilis has been observed (Schoenborn, 1939) through four successive transfers, covering a period of eighteen weeks. These suggestive observations indicate that certain chlorophyll-bearing flagellates may retain the ability to grow in inorganic nitrogen media, even after suppression of photosynthesis. HETEROMESOTROPHIC NUTRITION Heteromesotrophic nutrition has been demonstrated in several color- less Phytomastigophora. The Cryptomonadida are represented by CAzlo- monas paramecium, which has been grown in an amino-acid and acetate medium by Mast and Pace (1933) and by Hall and Loefer (1936). This type of nutrition has not yet been demonstrated in colorless Eugle- nida, and E. gracilis has been grown in darkness for only a few transfers in a medium containing asparagin and acetate (A. Lwoff and Dusi, 1929, 1931). Such results are modified by the addition of thiamine, as de- sctibed below. Several heteromesotrophs have been identified among colorless Phytomonadida. Pringsheim (1921) found glycocoll an ade- quate nitrogen source for Polytoma uvella, and comparable results were later obtained for P. obtusum (A. Lwoft, 1929b, 1932; A. Lwoff and Dusi, 1934). In addition, P. caudatum var. astigmata (A. Lwoff and Provasoli, 1935), Polytomella agilis (A. Lwoff, 1935b), and P. caeca (Pringsheim, 1935, 1937c; A. Lwoff and Dusi, 1937a) seem to be capable of heteromesotrophic nutrition. On the other hand, the related species, Hyadlogonium klebsii (Pringsheim, 1937a), appears to be an obligate heterometatroph. The chlorophyll-bearing phytomonad, Chloro- gonium euchlorum, has been grown in darkness in an asparagin medium (A. Lwoff and Dusi, 1935b); likewise, Lobomonas piriformis is capable of growth under similar conditions in a glycocoll and acetate medium (Osterud, 1939). HETEROMETATROPHIC NUTRITION All of the colorless Phytomastigophora which have been investigated appear to thrive in simple peptone media, although growth is always accelerated by the addition of a suitable organic carbon source. In addi- 484 FOOD REQUIREMENTS tion, a few of the chlorophyll-bearing species have been maintained in darkness in such media (Jahn, 1935c, 1935d; A. Lwoff and Dusi, 1929, 1931, 1935a, 1935b; M. Lwoff and A. Lwoff, 1929; Loefer, 1934; Provasoli, 1938b), especially with added acetate. Certain Trypanosomi- dae, such as Strigomonas oncopelti, have been grown in peptone media (M. Lwoff, 1930, 1933a, 1936), but growth of other Trypanosomidae (M. Lwoff, 1929a, 1929b, 1929c, 1929d, 1933a, 1933b) and of Polymas- tigida (Glaser and Coria, 1935b; Cailleau, 1935, 1936a, 1936b, 1937a, 1937b, 1938a, 1938b, 1939) seems to be supported only by more complex organic media containing blood, serum, tissue extracts, or particular growth factors. Several of the ciliates—Colpidium campylum and C. striatum (Elliott, 1933, 1935b), Glaucoma ficaria (D. F. Johnson, 1935a), G. piriformis (Lwoff, 1924, 1925, 1929a), Paramecium bursaria (Loefer, 1934b, 1936b, 1936c), and certain Sarcodina—Acanthamoeba castellanii (Cailleau, 1933, 1934), Mayorella palestinensis (Reich, 1935, 1936), have been grown in peptone media comparable to those which support growth of the heterometatrophic Phytomastigophora. On the other hand, Glaser and Coria (1930, 1933, 1935a) have used somewhat more complex media for several free-living ciliates. At present little is known of the nitrogen requirements in heterometa- trophic nutrition, and definite conclusions regarding the saprophilic or saprogenic nature of particular species are not always possible. Enzymes which hydrolyze gelatin and casein are produced by C. stratum (Elliott, 1933), by G. piriformis (A. Lwoff and Roukhelman, 1929; Lawrie, 1937), and by Saprophilus oviformis, Trichoda pura, and Chilodon cucullus (Glaser and Coria, 1935a); hence these ciliates cannot be con- sidered saprophilic organisms. A. Lwoff (1924, 1925) found that com- plete peptones were satisfactory for growth of G. piriformis, whereas silk peptone, gelatin, and fibrin, each supposedly lacking certain amino acids, were inadequate. Recently, however, several strains of C. campylum have been grown in the writer's laboratory for twenty-four transfers in silk peptone media and for eighteen transfers in gelatin media. In a compari- son of various peptones, Elliott (1935b) noted that C. striatum and C. campylum grew most rapidly in the peptones containing high percentages of free amino N. and Van Slyke amino N. Growth of the same ciliates (Hall and Elliott, 1935) was also accelerated by certain amino acids, added singly to a medium which supports slow multiplication. As in FOOD REQUIREMENTS 485 Col pidium, Loefer (1936c) found for P. bwrsaria that the least satisfac- tory of several peptones were those containing the smallest amounts of amino N. Hence preliminary partial hydrolysis of peptones appears to be advantageous, especially in the early growth of ciliate populations. Nitrogen metabolism of G. piriformis has been investigated by A. Lwoff and Roukhelman (1929), who have traced the quantitative changes in total N, peptone N, amino N, ammonia N, and amide N. In Witte peptone medium, peptone N decreased steadily. Amino N in- creased for the first two weeks, and then gradually decreased for two or three weeks; later, a secondary increase sometimes followed the death of many ciliates. Somewhat comparable results have been reported for Acanthamoeba castellanii (Cailleau, 1934), although hydrolysis was always less extensive than in cultures of G. piriformis and much less ammonia N was produced. Fermentation of carbohydrates and the acceleration of growth by carbo- hydrates and other carbon sources have been reported in many species. Colas-Belcour and A. Lwoff (1925) observed fermentation of dextrose and levulose by Leptomonas ctenocephali, Leishmania tro pica, and L. donovani (var. infantum). More recently, M. Lwoff (1936) has reported fermentation of fourteen carbohydrates by Strigomonas muscidarum and of a smaller number by S. media and S. parva. These three flagellates showed specific differences in their fermentation reactions. Likewise, Cailleau (1937b) has described fermentation of several monosaccharides and disaccharides by Eutrichomastix colubrorum, and the fermentation of dextrin, starch, and inulin, as well as some of the simpler carbohydrates, by Trichomonas foetus and T. columbae. Utilization of dextrose by try- panosomes (for review, see von Brand, 1938) has been known for some years. Recently, utilization of dextrose by T. foetws — the strain of Glaser and Coria (1935b)—has been measured by Andrews and von Brand (1938), who found that rate of utilization was correlated with growth rate. Although growth of C. paramecium is accelerated by dextrose (Loe- fer, 1935a), utilization of the sugar could not be detected (Loefer, 1938b) by means of Benedict’s colorimetric method. Acceleration of growth by starch has been reported for Polytoma caudatum (A. Lwoft and Provasoli, 1935), P. obtusum (A. Lwoff and Provasoli, 1937), and Polytomella agilis (A. Lwoff, 1935b). Few observations have been reported for the Sarcodina. Fermentation ‘ 486 FOOD REQUIREMENTS of carbohydrates by Acanthamoeba castellanii was not observed by Cail- leau (1933), who also obtained no evidence that sugars are actually con- sumed by this species. On the other hand, dextrose accelerates growth of Mayorella palestinensis (Reich, 1936), maximal effects being pro- duced by concentrations of 0.5 to 1.0 percent. In Colpidium campylum and Glaucoma piriformis, Loefer (1938b) has measured dextrose consumption over a short pH range. In general, the rate of utilization followed the growth-pH relationship. Fermenta- tion of carbohydrates has been demonstrated previously in several species. G. piriformis produces acid from dextrose, levulose, galactose, and mal- tose (Colas-Belcour and A. Lwoff, 1925), and also from dextrin and sol- uble starch (D. F. Johnson, 1935b); G. ficaria (D. F. Johnson, 1935b) ferments the same carbohydrates, with the apparent exception of levulose. The reactions of C. campylum and C. striatum (Elliott, 1935a) are sim- ilar to those of G. pirtformis, except for fermentation of mannose and failure to ferment galactose. Growth of C. campylum is accelerated by several carbohydrates, in addition to those which are fermented. Ac- cording to Glaser and Coria (1935a), dextrose and maltose are fer- mented, and starch and cellulose are hydrolyzed, by Saprophilus oviformts and Trichoda pura; starch and cellulose are attacked also by Chilodon cucullus, Paramecium caudatum, and P. multimicronucleatum. Growth of P. bursaria (Loefer, 1936c) is increased by dextrose, mannose, maltose, dextrin, and melizitose, while little or no effect is produced by other carbohydrates. No marked change in pH occurred in any case. Other carbon compounds known to accelerate growth of heterometa- trophs include various alcohols and salts of certain organic acids. The effects of several fatty acids on the growth of C. paramecium have been compared quantitatively by Loefer (1935a): the greatest acceleration was produced by acetate, butyrate, and valerate. Recently, Provasoli (1937a, 1937b, 1938a, 1938b, 1938c) has completed more extensive investigations on nine colorless flagellates (C. paramecium, Hyalogo- nium klebsii, Polytoma obtusum, P. caudaltum, P. uvella, P. ocellatum, Polytomella caeca, Astasia quartana, A. chattoni). Acetate and butyrate accelerated growth of all, while the effects of other fatty acids varied with the species. Provasoli has pointed out that the negative results previously obtained with certain salts probably resulted from their use in toxic concentrations. The effects of sodium acetate on the growth of several colorless species had been described previously by A. Lwoff (1929b, FOOD REQUIREMENTS 487 1931, 1932, 1935a, 1935b, 1938a), who proposed a class of “‘Oxy- trophes”’ to include organisms showing marked acceleration with acetate. Acceleration of growth by several alcohols has been reported by Provasoli (1938c) in Astasia chattont, A. quartana, Polytomella caeca, Polytoma ocellatum, and Chilomonas paramecium. For chlorophyll-bearing flagellates maintained in darkness, the effects of various carbon sources on growth seem to be much the same as for the colorless species. The growth of several species of Evglena (A. Lwoff and Dusi, 1929, 1931; Dusi, 1933a; 1933b; Jahn, 1935c, 1935d; Hall, 1937d) is accelerated by salts of certain fatty acids, particularly acetic, just as in the case of Astasia (A. Lwoff and Dusi, 1936). Jahn (1935d), in comparing the effects of certain salts on growth of E. gracilis in dark- ness and in light, found that butyrate and acetate were most effective in either case, while lactate produced a much greater acceleration in dark- ness than in light. Succinate was toxic in light, but produced a slight ac- celeration in darkness. Comparable effects of fatty acids have been re- ported in several Phytomonadida—Chlamydomonas agloéformis and Haematococcus pluviatis (M. Lwoff and A. Lwoff, 1929), and Chloro- gonium elongatum and C. euchlorum (Loefer, 1935a). Accelerating effects of carbohydrates on the growth of Evglena anabaena in darkness have also been noted (Hall, 1934). Elliott (1935b) has described acceleration of growth by acetate and butyrate in the ciliates Colpidium campylum and C. striatum, the effects being limited to the pH range 6.5 to 7.5, approximately. The increases ranged from about 15 percent to 300 percent at different pH values. Accelerating effects of pimelic acid on C. campylum also have been reported (Hall, 1939c), but the substance was used in low concentra- tions and may have been important as a catalyst, rather than as a carbon source. TROPHIC SPECIALIZATION In this brief survey of the food requirements of Protozoa, it has been pointed out that different methods of nutrition may be exhibited by different members of the same family or even of the same genus. Such varying degrees of specialization are particularly interesting, in that they afford a basis for speculation concerning the evolution of the more animal-like Protozoa from the plant-like flagellates. Theoretically, the evolution of animal-like flagellates from chlorophyll- 488 FOOD REQUIREMENTS bearing facultative photoautotrophs may have proceeded as follows: (1) Certain flagellates lost the ability to use inorganic compounds as the sole source of nitrogen, except in the presence of a suitable organic carbon compound. This type of specialization may or may not have in- volved the loss of chlorophyll in the beginning. (2) The ability to grow in inorganic-nitrogen media was lost completely, so that a single amino acid represented the simplest adequate nitrogen source. (3) The ability to grow in an amino-acid medium was lost, as the degree of specialization approached that of the animal-like flagellates. On the other hand, the existence of heteroautotrophic flagellates, which can utilize inorganic nitrogen sources without carrying on photo- synthesis, suggests the possibility that primitive colorless flagellates may have appeared before the origin of chlorophyll. Chlorophyll, with the attendant power of photosynthesis, would thus have been acquired dur- ing the evolution of plant-like flagellates. This hypothesis would gain additional support from the demonstration of chemoautotrophic nutri- tion in flagellates, and the suggestive report of such a phenomenon in Chilomonas paramecium (Mast and Pace, 1933) is particularly inter- esting. Further evidence may eventually necessitate revision of the cur- rent view that the chlorophyll-bearing flagellates are the most primitive of all the Protozoa. Even in adhering to the concept of a primitive chlorophyll-bearing stock, it must be admitted that in the known cases of heteroautotrophic nutrition, the presumed “‘loss’’ of chlorophyll has introduced only one new food requirement, a simple organic carbon source (e.g., acetate). A second stage of specialization is represented by such types as the chlorophyll-bearing Exglena deses and the colorless Polytomella caeca, each of which requires a simple organic nitrogen source. The third step in specialization also appears in the Phytomastigophora, and is illus- trated by the chlorophyll-bearing Exglena pisciformis, described by Dust (1933a) as an obligate photometatroph, and the colorless Hyalogonium klebsii, reported to be an obligate heterometatroph (Pringsheim, 1937a). If the types of nutrition described for these various species are taken for granted, it must be admitted that the presence of chlorophyll is no handicap to progressive physiological specialization. Furthermore, the mere absence of chlorophyll has not necessitated specialization beyond the first degree, although it may be accompanied by the assumption FOOD REQUIREMENTS 489 of heteromesotrophic or heterometatrophic nutrition. Hence the case for primitive chlorophyll-bearing forms as the ancestors of all the Protozoa may not be so strong as is generally assumed. The plant-like flagellates as a group, however, furnish a logical start- ing point for the evolution of other groups of Protozoa. Primitive meth- ods of nutrition are not the only methods to be observed in Phytomastt- gophora, and it is obvious that some of these flagellates approach in their growth requirements the Zodmastigophora, Sarcodina, and Ciliata, repre- sentatives of which have been grown in peptone media comparable to those required by E. pisczformis and H. klebsiz. Accordingly, it seems that, so far as physiological modifications are concerned, the evolution of animal-like flagellates and other groups of Protozoa from an ancestral stock of plant-like flagellates could have presented few problems. SPECIFIC GROWTH FACTORS, OR VITAMINS A concise definition of the term, growth factor, is not yet available. The term is now usually restricted to an essential substance which the organism in question cannot synthesize, or perhaps cannot synthesize rapidly enough to meet the normal requirements for growth. Such a growth factor may exert its characteristic effects, even when present in low concentration. By general agreement, the concept excludes the es- sential food substances and elements necessary for synthesis of proto- plasm. While a growth factor may in itself accelerate growth, it is to be distinguished from nonessential growth stimulants, which also pro- duce noticeable effects when present in low concentrations. A survey of the rapidly growing literature reveals that growth-factor requirements may differ among the species of a single protozoan genus, and that some species can be grown in media apparently containing no growth factors, while related types are much more exacting. At present, there is no sound basis for generalization. Lack of information concern- ing food requirements makes it impossible in some cases to decide whether or not a specific growth factor is necessary, and occasionally a postulated need for growth factors has disappeared after further in- vestigation. Thus Dusi (1936) suggested that growth of E. viridis in inorganic media might be impossible without a growth factor, but the species has since been grown as a photoautotroph (Hall, 1939a). Simi- larly, Hutner (1936) concluded that a vitamin-like substance is a neces- 490 FOOD REQUIREMENTS sary constituent of media for the growth of E. anabaena and E. gracilis in light. The former has been grown in inorganic media by Dusi and by Hall; the latter, by Dusi and by Hall and Schoenborn. It is obvious, therefore, that the inorganic food requirements of a given species should be satisfied, as a prerequisite to the evaluation of specific growth factors. Aneurin, or thiamine (vitamin B,).—It must be admitted that the facultative photoautotrophs and heteroautotrophs are capable of synthe- sizing aneurin, if this substance is actually essential to the growth of such organisms. Various other Protozoa, however, apparently show a definite need for aneurin, or for one or both of its constituents. Among the Cryptomonadida, the thiamine requirements of C. para- mecium have been investigated by A. Lwoff and Dusi (1937b, 1938a). In their first publication these workers stated that growth of the flagellate in asparagin medium is supported by thiamine, or by thiazole alone. In their later article, they have concluded that for growth in an ammonium acetate medium, thiamine can be replaced by thiazole and pyrimidine, but not by either one separately. Without the growth factor, growth in the control medium was always negative. Recently, A. Lwoff and Dusi (1938b, 1938c) have shown that these substances are not specific; so far as C. paramecium is concerned, several thiazoles and pyrimidines are satisfactory for growth. In the Euglenida, the existence of photoautotrophic species (E. gracilis, and others) and the occurrence of heteroautotrophic nutrition (Astasza sp., Schoenborn, 1938, 1940) seem to belie a need for thiamine in cer- tain species. Furthermore, Elliott (1937a) observed no accelerating ef- fect of this substance on the growth of E. gracilis in light. However, it has been assumed that such flagellates are capable of synthesizing thia- mine in light, and this hypothesis receives indirect support from re- ports that the growth of E. gracilis in darkness is possible in an asparagin and acetate medium only when thiamine (Lwoff and Dusi, 1937c) or pyrimidine (Lwoff and Dusi, 1938a) is present. On the other hand, Dusi (1939) has concluded that E. pisciformis requires such a growth factor even in light, since an asparagin medium containing thiamine (or both thiazole and pyrimidine) supported growth, while the same medium without a growth factor was unsatisfactory. In this connection, it has been noted (Hall, 1938b) that growth of E. anabaena in light is little, if any, better in an asparagin medium FOOD REQUIREMENTS 491 than in an ammonium-nitrate medium, the two media differing only with respect to the nitrogen source. Hence asparagin may actually be a poor nitrogen source for Euglenidae, even in light, and any growth stimulant might produce an effect comparable to that noted by Dusi in E. pisciformis. It should be noted, also, that the serial-transfer technique, in which Dusi apparently used two-drop inocula, might require an increase of as much as a hundred times in each transfer, if the original density of population is to be maintained. In the writer's experience with E. anabaena, the increase in asparagin medium was never greater than twenty-five times in any tranfer, and was often less. Hence the possibility exists that Dusi’s rate of dilution in serial transfers was much more rapid than the growth of his flagellates in media without growth factors, and that the use of larger inocula might reveal E. pzsczformzs to be capable of slow growth in asparagin media. If growth factors are actually essential, all the chlorophyll-bearing Phytomonadida which have been investigated appear to synthesize such substances from the constituents of suitable inorganic media. The color- less species, P. uvella and P. obtusum (Lwofft and Dusi, 1938a), show the same synthetic ability in salt solutions to which acetate has been added. Polytoma ocellatum and P. caudatum (Lwoff and Dusi, 1937b, 1937c) apparently require thiazole for growth in such media, while Polytomella caeca (A. Lwoff and Dusi, 1937a, 1938a, 1938b, 1938c) re- quires both thiazole and pyrimidine. A. Lwoff and Dusi (1937a) have shown that P. caeca grows fairly well in an asparagin medium, and much more rapidly after the addition of thiamine or of thiazole and pyrimidine. They have assumed, accordingly, that the growth in asparagin alone was dependent upon a trace of thiamine in the asparagin itself. The same interpretation is also applied to several other flagellates, on the basis of similar evidence. Just as in the case of C. paramecium, several dif- ferent pyrimidines and thiazoles accelerate the growth of P. caeca (Lwoft and Dusi, 1938b, 1938c), and several thiazoles are also effective with P. ocellatum. Among the Zo6dmastigophora, Strigomonas oncopelti (M. Lwoff, 1937), S. culicidarum, and S. fasciculata (M. Lwoff, 1938b) appear to require thiamine, which cannot be replaced by thiazole and pyrimidine. The last two species require hematin in addition to thiamine. Of the Sarcodina, Acanthamoeba castellanii (A. Lwoff, 1938b) ap- 492 FOOD REQUIREMENTS parently requires thiamine, or both pyrimidine and thiazole. Accord- ingly, Lwoff has concluded that this species is capable of synthesizing this growth factor from the two components, although such a synthesis has not been demonstrated. With respect to growth-factor requirements, A. castellanii thus seems to resemble Polytomella caeca and Chilomonas paramecium. So far, only a few investigations have been completed on the ciliates. Hall and Elliott (1935) noted that the addition of yeast extract in low concentration to a gelatin medium would support growth of Colpidium campylum and C. striatum, whereas gelatin medium alone was unsatis- factory. A. Lwoff and M. Lwoff (1937) have since found that Glau- coma piriformis will grow in a silk-peptone-dextrose medium containing thiamine, while the control cultures failed in the second or third transfer. Likewise, Elliott (1937a, 1939) noted a marked acceleration of growth in C. striatum when thiamine was added to a standard peptone solution and to a peptone medium autoclaved at pH 9.6. In the latter case, the controls showed very little growth in the first transfer. Observations of the Lwoffs (1937, 1938) indicate that G. pzr7formis requires the entire thiamine molecule, and is presumably unable to synthesize the substance from the thiazole and pyrimidine constituents. Various other related compounds cannot be substituted for thiamine. Other Growth Factors —Vitamin B, (riboflavin) apparently will not replace thiamine in meeting the growth requirements of Colpidium striatum, although a moderate acceleration of growth by this factor has been noted (Elliott, 1939). A vitamin B, concentrate has produced even less noticeable effects on the growth of the same species (Elliott, 1939). Vitamin C (ascorbic acid) requirements have been investigated in several species. M. Lwoff (1938a, 1939) has reported that ascorbic acid is one of the factors essential to growth of Trypanosoma cruzt, Leishmania tropica, and L. donovani in cultures; and Cailleau (1938a, 1938b, 1939) has reached the same conclusion for Trichomonas foetus, Eutrichomastix colubrorum, and T. columbae. Nicotinic acid and nicotinamide both seem to serve as growth factors for certain bacteria (for review, see Koser and Saunders, 1938). None of the investigations on Protozoa has yet been completed. Hematin has been found essential for the growth of certain Tryp- anosomidae—for example, Trypanosoma cruzi, Leishmania donovani, FOOD REQUIREMENTS 493 L. tropica (M. Lwoff, 1938a, 1939), Leptomonas ctenocephali, and Strigomonas fasciculata (M. Lwoff, 1933a). Protohemin and protopor- phyrin have been substituted for hematin in the case of T. cruzi. The significance of such growth factors has been discussed by A. Lwoff (1934, 1936), who suggested that these substances may enter into the composition of respiratory catalysts (cytochrome). Cholesterol_—The investigations of Cailleau (1936a, 1936b, 1937a, 1937b, 1938a, 1938b) indicate that cholesterol and certain other sterols serve as grown factors for the parasitic Polymastigida, Trichomonas columbae, T. foetus, and Eutrichomastix colubrorum. The physiological significance of these substances has not yet been determined for Protozoa. Extract of soil—aAn aqueous extract of soil has been used extensively by Pringsheim, who found it to accelerate the growth of a number of the plant-like flagellates and also to facilitate the growth of certain species in simple media. Accordingly, Pringsheim has considered this extract a source of unknown growth factors. The accelerative action has been verified by A. Lwoff and Lederer (1935), whose results suggest that soil extracts contain organic nitrogen in concentrations sufficient for growth of Polytomella agilis. Hence the status of soil extracts as a source of growth factors is yet to be evaluated. GROWTH STIMULANTS Growth stimulants differ from growth factors in that they are not essential to life. Like growth factors, however, they may be effective in low concentrations. So far as their relation to Protozoa is concerned, pantothenic acid and the plant “hormones” (auxins) may be placed in this category. Elliott (1935c) has shown that pantothenic acid accelerates the growth of Colpidium campylum, the maximal effect being noted at pH 6.0. Above 7.0 there was either no acceleration, or else a slight decrease in growth rate. Similar experiments with Haematococcus pluvi- alis, within the pH range 4.5 to 8.5, showed no acceleration. Addition of pantothenic acid to gelatin, gliadin, and zein media, which in themselves did not support the growth of Colpidiumm (Hall and Elliott, 1935), was without effect. These results indicate that pantothenic acid is not a substitute for thiamine. The effects of several plant hormones, or auxins, on the growth of Euglena gracilis, Khawkinea halli, and C. striatum have also been in- 494 FOOD REQUIREMENTS vestigated by Elliott (1938). The growth of E. gracilis was markedly accelerated at pH 5.6, while effects at lower and higher pH values were much less significant. In the colorless euglenoid, K. alli, and in C. striatum no acceleration of growth was observed at any pH. Thus the effects of the auxins may be correlated with the presence of chlorophyll, as well as the pH of the medium. Elliott (1937b) has shown further log of numbers numbers TIME TIME Figure 128. Growth phases in a hypothetical population. In the curve at the left, logarithms of numbers are plotted against time; on the right, numbers are plotted against time for a comparable population. Successive growth phases are numbered from 1 to 7. that such acceleration may also be dependent upon light, since there was no effect on growth of E. gracilzs in darkness. An accelerating effect of pimelic acid upon the growth of Colpidinm cam pylum has been noted by Hall (1939c); concentrations ranging from 10° to 10+ gm. per cc. were effective in gelatin and in peptone media. These results are comparable to the findings of Mueller (see Koser and Saunders, 1938) with the diphtheria bacillus. Certain preliminary ob- servations (Hall, 1938a) may indicate a possible growth-factor status for pimelic acid, but a definite conclusion is not yet warranted and pimelic acid may be considered, at least for the present, a growth stimulant for C. campylum. Glucose caramel, as used by Pringsheim (1937b, 1937c), may also be classified as a growth stimulant. Pringsheim insists that this substance FOOD REQUIREMENTS 495 does not serve as a carbon source in his cultures of plant-like flagellates, and that it should be considered a “growth factor.’ On the other hand, it has not yet been demonstrated that glucose-caramel is essential to the life of Protozoa, and until such evidence is available the substance should not be classified as a growth factor. GROWTH IN CULTURES AS A POPULATION PROBLEM The growth of microdrganisms in cultures has been described by Buchanan (Buchanan and Fulmer, 1928) in terms of seven phases: (Fig. 128): (1) Initial stationary phase, during which there is no in- crease in population; (2) lag phase (phase of positive growth accelera- tion), in which the division rate increases to a maximum; (3) logarithmic growth phase, during which the maximal rate is maintained; (4) phase of negative growth acceleration, in which the division rate decreases steadily; (5) maximum stationary phase, in which the population re- mains practically constant; (6) phase of accelerated death, in which the total population begins to decrease; and (7) a so-called logarithmic death phase, during which the population decreases at a more or less constant rate. Little is known about the history of protozoan populations, and com- plete growth curves seem to have been traced for only two species in pure cultures—Paramecium bursaria (Loefer, 1936b) and Polytoma (Provasoli, 1938c). Loefer’s growth curves, comparable to the numbers curve in Figure 128, show in general the phases recognized by Buchanan. Since counts were made at intervals of twenty-four hours or more, an initial stationary phase was not detected in several of the cultures. Total population histories covered from twenty to forty days in different media. Provasoli’s curve for Polytoma also shows the general growth phases. Phelps (1935, 1936) traced Glaucoma piriformis well into the maximal stationary phase and observed in most cases the first five of the con- ventional growth phases. On the basis of such evidence, it may be assumed that the growth of Protozoa in pure cultures follows the general trends observed in populations of bacteria and yeasts. More information concerning growth of protozoan populations is needed, since interpretation of experimental results may depend upon such knowledge. For example, the addition of a given substance to a 496 FOOD REQUIREMENTS logarithms of numbers TIME Figure 129. Hypothetical modifications (curves B-G) of the normal growth of a population (curve A), from the initial stationary to the maximal stationary phase. certain medium might produce any one of several effects: (1) The maximal population might be increased without any appreciable effect on the growth rate; that is, the length of the logarithmic phase might be increased without any change in the division rate (curve B, Fig. 129), as compared with that in the control medium (curve A). (2) The growth rate might be increased without a change in density in the maximal stationary phase (curve C). (3) Both the growth rate and the maximal density of population might be increased (curve D). (4) The FOOD REQUIREMENTS 497 maximal density of population might be decreased, with no appreciable effect on the early growth rate (curve E). (5) The growth rate might be decreased, without any effect on the maximal density of population (curve F). (6) Both the growth rate and the maximal density of popula- tion might be decreased (curve G). These possibilities will serve as illustrations. Curves A, B, and E would show no significant differences in the early histories of the cultures; yet each reaches a different maximal stationary phase, one higher and one lower than that of the control (curve A). On the other hand, early examination of cultures A, C, and F would show significant differences in population density and in growth rate, although each culture eventually reaches the same maximal density. Hence without detailed information concerning the behavior of popula- tions, there is obvious need for caution in interpreting experimental results. In an analysis of the conditions which might influence the population- growth curve, the concentration of available food, the general condition and density of the initial population, the pH of the medium, the tempera- ture of incubation, the accumulation of waste products, the oxygen ten- sion, and the redox potential of the medium—to mention some of the more apparent factors—all seem to be significant. Although the impor- tance of such factors may seem obvious, their detailed relationships to growth are mostly unknown. THE INITIAL POPULATION Both the physiological condition and the size of the initial population may influence the rate of growth. Phelps (1935) has reported that the length of the initial stationary phase and the lag period bears a rela- tion to the age of the inoculum. With inocula taken from the logarithmic- growth phase of a stock culture, the length of the combined lag and initial stationary phases was usually reduced to zero. With inocula from older cultures, these two phases were usually detectable and were often quite pronounced. Obviously, therefore, age and other qualities of the inoculum must be considered in comparative studies on population growth and on the effects of environmental factors. The relation between the initial density of population and the growth rate in bacterized cultures has been disputed for many years. Robertson and others have described an allelocatalytic effect, in which the rate of 498 FOOD REQUIREMENTS population growth is correlated more or less directly with the size of the initial population. A number of investigators, however, either have noted no significant correlation, or else have found that the growth rate varies inversely with the initial density of population (e.g., Woodruff, 1911). W. H. Johnson (1936, 1937) has pointed out that these results must be interpreted in relation to the concentration of bacteria in the cultures. Jahn (1929), who partially eliminated bacteria by growing Ezglena sp. in inorganic media and washing the flagellates before inoculation, found that the growth rate varied inversely with the initial density of the popu- lation. Most of the literature on bacterized cultures has been reviewed by Jahn (1929), W.H. Johnson (1933, 1936, 1937), and Petersen (1929). In contrast to the studies on bacterized cultures, very little work has yet been done with bacteria-free strains. Evidence bearing on the growth of such populations has been presented by Phelps (1935), Mast and Pace (1938), Reich (1938), and Hall and Schoenborn (1939b). Phelps concluded, for Glaucoma piriformis, that the density of popula- tion at the end of the logarithmic phase is, within wide limits, inde- pendent of the initial density of population. This may mean that the logarithmic phase is prolonged in the cultures with small inocula; or that the growth rate is higher in the cultures with low initial density; or perhaps that both the growth rate and the length of the logarithmic phase are increased. Phelps did not consider this question in detail, but some of his data (ser. I], Figs. 4, 5) indicate, at the end of the loga- rithmic phase, an average generation time of about four hours in the low- initial-density cultures and approximately five hours in the cultures started with larger inocula. Such data suggest an inverse relationship be- tween the initial density of population and the rate of population growth. In Chilomonas paramecium (Mast and Pace, 1938) the rate of repro- duction varies directly with the initial density of population under some conditions, but inversely under others. Some sort of optimal relationship is indicated by the findings of Mast and Pace, since the growth rate in- creased to a maximum with decreasing volumes of medium per flagellate and then decreased to zero. Reich (1938) observed that in Mayorella palestinensis the division rate varies directly with the initial density of population in cultures started with less than 3,000 amoebae per cc., although the population “eventually attained is largely independent of the quantity of inoculum.” FOOD REQUIREMENTS 499 These results are interpreted as supporting Robertson’s concept of allelo- catalysis, although Reich does not subscribe to the theory of an auto- catalyst of growth. In observations on Ezg/ena, Hall and Schoenborn (1939b) have noted that the population tends to reach a concentration which is more or less independent of the initial density of population under the conditions described, and thus that the increase in the population varies inversely with the initial density of population. These conclusions were based upon counts made after specified periods of incubation, and population curves were not traced. At present it is impossible to correlate the results which have been obtained with bacteria-free cultures, although the cited observations all indicate that the initial density of population influences the rate of population growth. Since the experiments on various species have been carried out under different conditions and in different media, it is pos- sible that some of the puzzling contradictions may eventually be traced to differences in technique, rather than differences in the nature of popu- lation growth. In fact, different relationships between the density of population and the rate of growth might reasonably be expected under different experimental conditions. GROWTH IN RELATION TO WASTE PRODUCTS Investigations on so-called waste products of Protozoa have led to con- flicting opinions. Woodruff (1911, 1913) concluded that waste prod- ucts inhibit growth of the homologous species, although growth of a different species may be relatively unaffected by the same substances. The other extreme is represented by Dimitrowa’s (1932) observation that the growth of Paramecium caudatum is accelerated by the addition of small amounts of old medium to the experimental cultures. More recently, W. H. Johnson and Hardin (1938) have observed no significant effects of old culture medium on the growth of P. multimicronucleata. Very little work on this problem has been carried out with pure cultures. The observations of A. Lwoff and Roukhelman (1929), that growth of G. périformis ceases long before the food supply is exhausted, has lent some support to the view that growth may be inhibited by accumulated waste products. The results of later investigations are not so readily interpreted. Mast and Pace (1938) have noted that old culture 500 FOOD REQUIREMENTS medium, in high concentrations, inhibits the growth of Chilomonas paramecium, while small amounts accelerate growth. Reich (1938) found that the addition of culture filtrate produced no effect on the growth of M. palestinensis. However, Reich’s filtrate was obtained from young cultures (twenty-four-hour cultures in one experiment, for example), and his technique is not entirely comparable to that of Mast and Pace. Hall and Loefer (1940), working with C. campylum in peptone medium, found that the addition of old culture filtrates (one part in ten, to five parts in ten) markedly increased the population yield, as compared with that in control cultures. Furthermore, the growth rate, after the first or second day of incubation, was noticeably higher in the cultures containing old-culture filtrate. Acceleration of comparable magnitude was also produced by the addition of aged sterile medium to fresh pep- tone medium. In view of the latter observation, it now seems impos- sible to attribute the effects of old-culture filtrates solely to a ‘‘biological conditioning’ of the medium, or entirely to a product or products elaborated by the organisms growing in the medium. A basic explana- tion for these various phenomena is not yet available, and it is possible that any single explanation may be inadequate. Thus the ‘‘factor” of Mast and Pace is said to be heat-labile, whereas the effects noted by Hall and Loefer were produced by culture filtrates and aged sterile medium which had been sterilized in the autoclave. At any rate, these results are not only interesting in themselves, but they may also furnish important clues in untangling the conflicting opinions concerning alle- locatalytic and autocatalytic phenomena. For instance, some preliminary observations of the writer have already shown that the “accelerating factor” in old cultures may have a definite bearing on the growth of C. campylum, in relation to initial density of population. GROWTH IN RELATION TO FOOD CONCENTRATION It seems obvious that, within reasonable limits, the density of a protozoan population should vary more or less directly with the con- centration of available food until an optimal concentration is reached, although the relationship might not be evident in the early history of the culture. Such a generalization is supported by studies on pure cul- tures. Cailleau (1933) noted that peptone concentrations of 3.0 percent sup- ported abundant growth of Acanthamoeba castellanii, while lower con- FOOD REQUIREMENTS 501 centrations were much less favorable. In Colpidium campylum, Bond (1933) observed comparable relationships between growth rate and concentration of yeast autolysate. Optimal concentrations of peptone for C. striatum (Elliott, 1935b) lie between 1.0 and 3.0 percent, while for Glaucoma ficaria (D. F. Johnson, 1935a) the optimum ts about 1.5 per- cent. In both species of Colpidium the effects were apparent after twenty- four hours of incubation and became more marked in older cultures. Loefer’s (1936b) observations on Paramecium bursaria indicate similar relationships, although the limits are somewhat narrower than for Col pidium and Glaucoma. For example, one of the peptones tested was optimal in 0.5-percent solution, whereas no growth of P. bursaria oc- curred in a concentration of 1.4 percent. Phelps (1936) observed that in the logarithmic phase the growth rate of G. piriformis was, within wide limits, practically independent of the food concentration. In later history of the cultures, however, the relationships were comparable to those ob- served by Elliott and D. F. Johnson. Rottier (1936b) described, in Polytoma uvella, a direct relation between the growth rate and the con- centration of peptone (0.2 to 1.0 percent), and of asparagin (0.2 to 2.0 percent), significant differences being noted after about five days of incubation. The effect of a substance added to an adequate medium also varies with concentration, as would be expected. Johnson (1935a) reported for Glaucoma ficaria in peptone medium, maximal acceleration by 0.5- percent yeast extract and inhibitory effects of concentrations above 2.0 percent. In Paramecium bursaria (Loefer, 1936b), the optimal concen- tration of the same yeast extract was 0.03 percent, and growth was defi- nitely inhibited in a 0.5-percent solution. Dextrose was most effective for P. bursaria in a concentration of 0.5 percent. Reich (1936) has obtained similar results with Mayorella palestinensis. The effects of added sodium acetate on P. wvella (Rottier, 1936b) vary in the same fashion, the maxi- mal effect being produced by 0.8-percent acetate. The optimum is much lower in Euglena stellata (Hall, 1937d), in which 0.05-percent sodium acetate was most effective in both light and darkness. GROWTH IN RELATION TO pH OF THE MEDIUM It has been known for many years that the acidity or the alkalinity of the medium bears some relation to the growth of Protozoa, and investi- gations on bacterized cultures have determined the optimal pH and pH 502 FOOD REQUIREMENTS range for a number of species (for reviews, see Loefer, 1935c; D. F. Johnson, 1935a). More recently, similar investigations have been carried out with bacteria-free material. Dusi (1930) has shown that each of six species of Euglena has a characteristic pH range in certain media, and that the optimum varies somewhat for the different species. Jahn (1931), using quantitative methods, has studied that pH relationships of E. graczlzs in detail, and similar relationships have been determined for E. anabaena and E. deses (Hall, 1933a) and for two species of Astasza (Schoenborn, 1936). Among the Cryptomonadida and Phytomonadida, the pH-growth rela- tionships of Chilomonas paramecium, Chlorogonium elongatum, and C. euchlorum have been investigated by Loefer (1935c). Growth of the two phytomonads, with an optimum slightly above pH 7.0, was more or less comparable to that of several Euglenidae; C. paramecium, on the other hand, showed a bimaximal pH-growth curve with peaks at pH 4.9 and 7.0 and an intermediate low point at pH 6.0. Relationships between growth and pH have also been determined for several ciliates. Elliott (1933, 1935b) has described the pH ranges and optima for Colpidium campylum and C. striatum, and has pointed out that the pH relationships vary with the type of medium. In one peptone medium (Difco tryptone) a bimodal curve, with peaks at pH 5.5 and 7.5, was noted; in certain other peptone media, a unimaximal pH-growth curve was observed. The addition of sodium acetate or a carbohydrate (e.g., maltose) to tryptone medium changed the shape of the curve from bimaximal to unimaximal. The extent of the pH range also varied with the type of medium. D. F. Johnson (1935a), in similar fashion, has com- pared the pH-growth curves of Glaucoma ficaria and G. piriformis in dif- ferent types of media. Appreciable differences between the two species were noted, and the pH range and general form of the growth curves were found to vary with the type of medium, much as in Colpidium. More recently, Loefer (1938a) has studied the growth rate and general morphology of Paramecium bursaria in relation to the pH of the medium. Conditions known to be optimal for the symbiotic Chorella (Loefer, 1936a) did not coincide with those most favorable to the growth of P. bursaria containing the algae. The pH optimum for the ciliate was approximately 6.8, and growth occurred within the range 4.9 to about 7.8. The size of the ciliates varied with the pH, but independently of the growth rate. FOOD REQUIREMENTS 503 In addition to the presumably direct influence upon growth rate, the pH of the medium has been found to modify the effects of other fac- tors. For example, Elliott (1935a) has shown that the maximal acceler- ating effect of certain carbohydrates on the growth of C. striatum 1s exerted below pH 7.0, with little or no acceleration above that point. Some of Jahn’s (1935b) results with E. gracilis in inorganic medium also seem to show a correlation between pH and the effect of several carbohydrates. Elliott noted also that the effects of sodium acetate and - butyrate varied with the pH. The former inhibited growth of C. striatum more or less completely at pH 6.0 and lower, but produced moderate acceleration near the neutral point. Butyrate was toxic at pH 6.5 or below, but showed an accelerating effect at pH 7.0-7.5. Jahn (1934) has suggested that such effects of acetate and butyrate may be explained on the basis that only the undissociated organic-acid molecule is toxic. A. Lwoff (1935a), in reviewing Elliott’s work, stated that acetate and butyrate inhibit the growth of Colpidium; this is true for only a certain pH range. Another indirect effect of the pH is the influence on tempera- ture relationships, as indicated in Jahn’s (1933a) observation that the susceptibility of E. gracilis to relatively high temperature is lowest at pH 5.0 and greatest above pH 7.0. The growth-accelerating effects of panto- thenic acid on C. striatum (Elliott, 1935c) and of plant auxins on E. gracilis (Elliott, 1938) are also dependent upon the pH of the medium. The evidence already accumulated shows that the pH relationships of Protozoa are exceedingly complex, and that they vary not only with the individual species but also with the composition of the medium and with other environmental conditions. Furthermore, such relationships may vary with time, since Jahn (1931) has observed that the optimal pH differs in young and in old cultures of E. gracilis. To some extent, the pH-growth relationships may be correlated with the activities of enzymes, which may show characteristic pH optima; for example, the protease of G. piriformis (Lawrie, 1937) shows maximal activity at pH 6.0. But this may represent only one of many ways in which growth is related to the pH of the medium. OXYGEN RELATIONSHIPS That oxygen tension of the medium influences growth of Protozoa is obvious, but relatively little detailed information has been accumulated 504 FOOD REQUIREMENTS in experimental studies. Observations on the natural occurrence of Protozoa indicate definite differences in oxygen requirements. Some species appear to be strict aérobes, others are perhaps comparable to the microaérophiles among the bacteria, and many intestinal parasites are probably to be regarded as facultative anaérobes. Investigations on proto- zoan respiration are discussed in another chapter of this volume (Chap- ter Vib): Growth in relation to oxygen requirements has been investigated for only a few bacteria-free strains, and no attempt has been made to corre- late definite oxygen tensions with growth rate. G. piriformis, according to Lwoff (1932), is incapable of growth under anaérobic conditions. Likewise, Hall (1933b) found that under reduced oxygen tension (Buchner pyrogallol method), growth of C. campylum was approxt- mately 50 percent less than in aérobic controls in peptone medium. With added dextrose, however, growth was greater than in the aérobic con- trols in peptone medium. The results suggest a certain degree of simi- larity between C. campylum and the facultative anaérobes among the bacteria. Phelps (1936) demonstrated that aération of flask cultures in yeast autolysate produces a much heavier population of G. pirzformzs than in unaérated flasks. These results are somewhat comparable to the findings of Jahn (1936), who compared G. piriformis and Chilomonas paramecium with respect to effects of aération. Growth of the ciliates was most rapid at first in unaérated flasks, but after three days of in- cubation the aérated flasks showed heavier populations. In C. para- mecium, however, growth was consistently more rapid in the unaérated flasks. Rottier (1936a) has reported that the growth of Polytoma uvella in flasks is more rapid than in tubes, after approximately forty hours of incubation. Likewise, aérated tube cultures showed heavier growth than unaérated ones. THE REDOX POTENTIAL As applied to culture media, the redox potential may be considered an indication of the oxidizing or reducing power of such an oxidation- reduction system. In other words, the more positive the redox potential, the more highly oxidized is the medium; the more negative the po- tential, the more highly reduced will be the medium. In effect, the potential is a measure of intensity rather than of oxidizing or reducing capacity, and hence is somewhat analogous to the pH, which gives no FOOD REQUIREMENTS 505 indication of the amount of acid or alkali necessary to change the re- action by a given amount. In addition, the redox potential varies with the pH of the medium. A number of investigators have correlated the redox potential of culture media with the growth and metabolism of bacteria, but very little work along this line has yet been done in proto- zoology. So far, the only detailed investigations are those of Jahn (1933b, 1935a), who has studied growth of Chilomonas paramecium in relation to the redox potential of the medium. In his first publication, Jahn found that growth is accelerated by NaSH, while the addition of H,O, to a peptone and acetate medium inhibited growth. On the other hand, relatively rapid growth occurred when both peroxide and a high con- centration of -SH were added to the medium. These results were ex- plained on the basis of the redox potential. In his second article, Jahn traced the continuous changes in the pH and Eh in cultures of C. para- mecium. The Eh of different media was found to drop as much as 300-460 my. during the first few days of incubation, and Jahn suggested that such changes may involve not only a lowering of the oxygen ten- sion but also the accumulation of reducing substances in the medium. After three to five days, depending upon the type of medium, the Eh began to rise; this change was attributed to a sharp decrease in the growth rate of the flagellates, with a corresponding decrease in oxygen consumption. So far as the Protozoa are concerned, Jahn’s results indicate that there is much to be learned concerning detailed relationships between growth and the redox potential of culture media. The exact effects of changes in the redox potential are still unknown, and the relative im- portance of the redox potential and the oxygen tension in different cases is yet to be determined. Possible relationships to growth have been dis- cussed by Jahn (1934). GROWTH IN RELATION TO TEMPERATURE The importance of temperature relationships is obvious, and rigid control of temperature is essential in many types of experimental in- vestigations. The actual relationships between growth and temperature are undoubtedly complex, since changes in temperature may not only affect metabolic activities of the organism directly, but may also modify 506 FOOD REQUIREMENTS the action of other environmental factors. Conversely, changes in vari- ous environmental conditions may modify the temperature relationships of a given species. In one of the few investigations carried out on pure cultures, Jahn (1935c) has demonstrated an interesting temperature relationship in Euglena gracilis. In darkness, the optimal temperature for this species in a peptone medium was about 10° C. When sodium acetate was added to the medium, not only was growth accelerated, but the optimal tem- perature was shifted to about 23°, a point approaching the optimum for growth in light. Another instance in which the temperature rela- tionships vary with other environmental conditions is represented by the thermal death time of E. gracilis (Jahn, 1933a), which appears to be a function of the pH, the greatest resistance to a temperature of 40° C. being noted at pH 5.0, and a greater susceptibility above pH 7.0 than below. GROWTH IN RELATION TO LIGHT AND DARKNESS Little or nothing is known concerning the relation between light and the growth of colorless Protozoa. On the other hand, the importance of light is obvious in the case of the chlorophyll-bearing species, and the relation to photosynthesis probably accounts for a number of the known effects of light. Dusi (1937) has noted certain interesting peculiarities of several Euglenidae. In constant light, E. gracil7s grows well in pep- tone medium, but poorly in inorganic media. On the other hand, E. klebsi grows perfectly in inorganic medium under constant illumina- tion, while E. viridis is incapable of growing under such conditions, even in peptone medium. No explanation for such specific differences in light relationships is yet available. An apparent relationship between light and the optimal temperature for growth has been noted by Jahn (1935c), who reported that the optimal temperature for growth of E. gracilis in darkness lies near 10° C., whereas the optimum in light for the same species is approximately 25°. The presence or absence of light is also a factor which must be considered in interpreting the effects of carbon compounds on growth. Thus Jahn (1935d) has observed that the accelerating effects of several organic acids on the growth of E. gracilis are relatively much greater in darkness than in light. Succinate, on the other hand, produced a slight acceleration in darkness, but was FOOD REQUIREMENTS 507 mildly toxic in light, while oxalate exerted no effect in darkness and a slight acceleration in light. Hall (1937d) observed also that in E. ste/- lata tolerance to concentrations of acetate above 0.2 percent was much less in light than in darkness. Furthermore, Jahn (1936b) has obtained some evidence that intensity of light may influence the effects of carbo- hydrates on the growth of E. gracilis in an inorganic medium. Another instance involving light relationships is the effect of plant ‘“hormones”’ on E. gracilis, in which Elliott (1937b) has shown that growth 1s ac- celerated in light, but not in darkness. ACCLIMATIZATION Acclimatization of Protozoa to various experimental conditions has been reported in many instances, ranging from acclimatization to toxic chemicals to the development of resistance to antibodies. A few cases have been described in bacteria-free cultures of free-living species. Such a process may occasionally be involved in the establishment of pure cul- tures, as reported by Elliott (1933) for Colpidium striatum and by Johnson (1935a) for Glaucoma ficaria. More recently, Loefer (1938c) has studied the acclimatization of several species (C. campylum, G. pirt- formis, Chlorogonium euchlorum, Euglena gracilis, and Astasia sp.) to progressively increased salt concentrations. E. gracilis developed no ap- preciable tolerance, but, after a series of transfers, the other species all showed the ability to grow in salt concentrations which were lethal in the initial exposures. The salinity finally tolerated by C. campylum was higher than that of ordinary sea water. Further investigations on acclimatization should prove interesting, and may throw some light on various experimental results which at present seem very puzzling. LITERATURE CITED No attempt has been made to include all the literature on bacteria-free cultures of Protozoa. Among the papers cited, those which contain good bibliographies are indicated by an asterisk. Andrews, J., and T. von Brand. 1938. Quantitative studies on glucose con- sumption by Trichomonas foetus. Amer. J. Hyg., 28: 138-47. Bond, R. M. 1933. A contribution to the study of the natural food cycle in aquatic environments. Bull. Bingham oceanogr. Coll. 4 (Art. 4), 89 pp. Brand, T. von. 1938.* The metabolism of pathogenic trypanosomes and the carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13: 41-50. 508 FOOD REQUIREMENTS Buchanan, R. E., and E. I. Fulmer. 1928. Physiology and biochemistry of Bacteria. Vol. I. Baltimore. Cailleau, R. 1933. Culture d’ Acanthamoeba castellanii sur milieu peptone. Action sur les glucides. C. R. Soc. Biol. Paris, 114: 474-76. —— 1934. Utilization des milieux liquides par Acanthamoeba castellanit. C. R. ‘Soc Biol? Paris’ 1G 39729=23; —— 1935. La Nutrition de Trichomonas columbae en culture. C. R. Soc. Biol. Paris, 119: 853-56. —— 1936a. Le Cholestérol, facteur de croissance pour le flagellé Trachomonas columbae. C. R. Soc. Biol. Paris, 121: 424-25. 1936b. L’ Activité de quelques stérols envisagés comme facteurs de croissance pour le flagellé Trichomonas columbae. C. R. Soc. Biol. Paris, 122: 1027-28. —— 1937a. Nouvelles Recherches sur I’activité de quelques stérols con- siderées comme facteurs de croissance pour le flagellé Trichomonas columbae. C. R. Soc. Biol. Paris, 124: 1042-44. —— 1937b.* La Nutrition des flagellés Tetramitides. Les stérols, facteurs de croissance pour les Trichomonades. Ann. Inst. Pasteur, 59: 137-293. —— 1938a. Le Cholestérol et I’acide ascorbique, facteurs de croissance pour le flagellé tetramitide Trichomonas foetus Riedmiiller. C. R. Soc. Biol. Paris, 127: 861-63. —— 1938b. L’Acide ascorbique et le cholestérol, facteurs de croissance pour le flagellé Extrichomastix colubrorum. C. R. Soc. Biol. Paris, 127: 1421-23. —— 1939. L’Acide ascorbique, facteur de croissance pour le flagellé Tricho- monas columbae. C. R. Soc. Biol. Paris, 130: 319-21. Colas-Belcour, J., and A. Lwoff. 1925. L’Utilisation des glucides par quelques Protozoaires. C. R. Soc. Biol. Paris, 93: 1421-22. Dimitrowa, A. 1932. Die Férdernde Wirkung der Exkrete von Paramecium caudatum Ehrbg. auf dessen Teilungsgeschwindigkeit. Zool. Anz., 100: 127-32. Dusi, H. 1930. Limites de la concentration en ions H pour la culture de quelques euglénes. C. R. Soc. Biol. Paris, 104: 734-36. —— 1931. L’Assimilation des acides aminés par quelques eugléniens. C. R. Soc. Biol. Paris, 107: 1232-34: —— 1933a. Recherches sur la nutrition de quelques euglénes. I. Euglena gracilis. Ann. Inst. Pasteur, 50: 550-97. —— 1933b. Recherches sur la nutrition de quelques euglénes. II. Evglena stellata, klebsii, anabaena, deses et pisciformis. Ann, Inst. Pasteur, 50: 840-90. —— 1936. Recherches sur la culture et la nutrition d’Euglena viridis. Arch. zool. expr. gén., 78 (M. et R.): 133-36. —— 1937. Le Besoin de substances organiques de quelques eugléniens a chlorophylle. Arch. Protistenk., 89: 94-99. FOOD REQUIREMENTS 509 —— 1939. La Pyrimidine et le thiazol, facteurs de croissance pour le flagellé a chlorophylle, Ezglena pisciformis. C. R. Soc. Biol. Paris, 130: 419-22. Elliott, A. M. 1933.* Isolation of Colpidium striatum Stokes in bacteria-free culture and the relation of growth to pH of the medium. Biol. Bull., 65: 45-56. —— 1935a. Effects of carbohydrates on growth of Colpidinm. Arch. Protistenk., 84: 156-74. — 1935b.* Effects of certain organic acids and protein derivatives on the growth of Colpidium. Arch. Protistenk., 84: 225-31. — 1935c. The influence of pantothenic acid on growth of Protozoa. Biol. Bull., 68: 82-92. — 1937a. Vitamin B, and growth of Protozoa. Anat. Rec., 70 (Suppl.): 27: — 1937b. Plant hormones and growth of Evg/ena in relation to light. Anat. Rec., 70 (Suppl.): 128. —— 1938.* The influence of certain plant hormones on growth of Protozoa. Physiol. Zo6l., 11: 31-39. — 1939. The vitamin B complex and the growth of Colpidinm striatum. Physiol. Zo6l., 12: 363-373. Gause, G. F. 1935. Experimentelle Untersuchungen uber die Konkurrenz zwischen Paramecium caudatum und Paramecium aurelia. Arch. Protistenk., 84: 207-24. Geise, A. C., and C. V. Taylor. 1935. Paramecia for experimental purposes in controlled mass cultures on a single strain of bacteria. Arch. Protistenk., 84: 225-31. Glaser, R. W., and N. A. Coria. 1930. Methods for the pure culture of certain Protozoa. J. exp. Med., 51: 787-806. —— 1933. The culture of Paramecium caudatum free from living micro- organisms. J. Parasite., 20: 33-37. 1935a. The culture and reactions of purified Protozoa. Amer. J. Hyg., 2S VA1=20: —— 1935b. Purification and culture of Tritrichomonas foetus (Riedmiller) from cows. Amer. J. Hyg., 22: 221-26. Hall, R. P. 1933a. On the relation of hydrogen-ion concentration to the growth of Euglena anabaena vat. minor and E. deses. Arch. Protistenk., 79: 239-48. —— 1933b. Growth of Colpidium campylum with reference to oxygen re- lationships. Anat. Rec., 57 (Suppl.): 95. —— 1934. Effects of carbohydrates on growth of Euglena anabaena vat. minor in darkness. Arch. Protistenk., 82: 45-50. —— 1937a.* “Growth of free-living Protozoa in pure cultures.” In Culture Methods for Invertebrate Animals. Ithaca. Pp. 51-59. —— 1937b. Certain culture reactions of several species of Euglenidae. Trans. Amer, mice. Soe. 563-285-387. 510 FOOD REQUIREMENTS —— 1937c. Effects of manganese on the growth of Euglena anabaena, Astasia sp. and Colpidium campylum. Arch. Protistenk., 90: 178-84. 1937d. Effects of different concentrations of sodium acetate on growth of Exglena stellata, Anat. Rec., 70 (Suppl.): 127. 1938a, Pimelic acid as a growth factor for the ciliate, Colpidium campylum. Anat. Rec., 72 (Suppl.): 110. —— 1938b. Nitrogen requirements of Evglena anabaena var. minor. Arch. Protistenk., 91: 465-73. —— 1939a. The trophic nature of Euglena viridis. Arch. zool. expr. gén., 80 (N. et R.): 61-67. —— 1939b. The trophic nature of the plant-like flagellates. Quart. Rec. Biol., 14: 1-12. —— 1939c. Pimelic acid as a growth stimulant for Colpidinm campylum. Arch. Protistenk., 92: 315-19. Hall, R. P., and A. M. Elliott. 1935. Growth of Colpidium in relation to certain incomplete proteins and amino acids. Arch. Protistenk., 85: 443-50. Hall, R. P., and J. B. Loefer. 1936. On the supposed utilization of inorganic nitrogen by the colorless cryptomonad flagellate, Chilomonas paramecium. Protoplasma, 26: 321-30. 1940. Effects of culture filtrates and old medium on growth of the ciliate, Colpidium campylum. Proc. Soc. exp. Biol. N. Y., 43: 128-33. Hall, R. P., and H. W. Schoenborn. 1938a. Studies on the question of auto- trophic nutrition in Chlorogonium euchlorum, Euglena anabaena and E, deses. Arch. Protistenk., 90: 259-71. —— 1938b. The selective action of inorganic media in bacteria-free cultures of Euglena. Anat. Rec., 72 (Suppl.) : 129-30. 1939a. The question of autotrophic nutrition in Exglena gracilis. Physiol. Zo6l., 12: 76-84. —— 1939b. Fluctuations in growth rate of Euglena anabaena, E. gracilis and E. viridis and their apparent relation to initial density of population. Physiol. Zo6l., 12: 201-08. Hutner, S. H. 1936. The nutritional requirements of two species of Euglena. Arch. Protistenk., 88: 93-106. Jahn, T. L. 1929.* Studies on the physiology of the euglenoid flagellates. I. The relation of the density of population to the growth rate of Euglena. Biol. Bull., 57: 81-106. —— 1931. Studies on the physiology of the euglenoid flagellates. III. The effect of hydrogen-ion concentration on the growth of Ezglena gracilis. Biol. Bull., 61: 387-99. —— 1933a.* Studies on the physiology of the euglenoid flagellates. IV. The thermal death time of Exglena gracilis Klebs. Arch. Protistenk., 79: 249-62. —— 1933b.* Studies on the oxidation-reduction potential of protozoan cul- FOOD REQUIREMENTS ul tures. I. The effect of -SH on Chilomonas paramecium. Protoplasma, 20: 90-104. —— 1934. Problems of population growth in the Protozoa. Cold Spring Harbor Symp. Quant. Biol., 2: 167-80. —— 1935a. Studies on the oxidation-reduction potential of protozoan cul- tures. II. The reduction potential of cultures of Chilomonas paramecium. Arch. Protistenk., 86: 225-37. —— 1935b. Studies on the physiology of the euglenoid flagellates. V. The effect of certain carbohydrates on the growth of Exglena gracilis Klebs. Arch, Protistenk., 86: 238-50. —— 1935c. Studies on the physiology of the euglenoid flagellates. VI. The effects of temperature and of acetate on Ezglena gracilis cultures in the dark. Arch. Protistenk., 86: 251-57. —— 1935d. Studies on the physiology of the euglenoid flagellates. VII. The effect of salts of certain organic acids on growth of Evglena gracilis Klebs. Arch. Protistenk., 86: 258-62. —— 1936. Effect of aeration and lack of CO, on growth of bacteria-free cultures of Protozoa. Proc. Soc. Exper. Biol. N. Y., 33: 494-98. Johnson, D. F. 1935a.* The isolation of Glaucoma ficaria in bacteria-free cultures, and growth in relation to pH of the medium. Arch. Protistenk., 86: 263-77. —— 1935b. Fermentation of carbohydrates by Glaucoma and effects of carbohydrates on growth of two species. Anat. Rec., 64 (Suppl.) : 106-07. 1936.* Growth of Glaucoma ficaria Kahl in cultures with single species of other microorganisms. Arch, Protistenk., 86: 359-78. Johnson, W. H. 1933.* Effects of population density on the rate of reproduc- tion in Oxytricha. Physiol. Zoél., 6: 22-54. —— 1936.* Studies on the nutrition and reproduction of Paramecium. Physiol. Zodl., 9: 1-14. —— 1937.* Experimental populations of microscopic organisms. Amer. Nat7 135220! Johnson, W. H., and G. Mardin. 1938. Reproduction of Paramecium in old culture medium. Physiol. Zodl., 11: 333-46. Koser, S. A., and F. Saunders. 1938. Accessory growth factors for bacteria and related microorganisms. Bact. Rev., 2: 99-160. Lawrie, N. R. 1937. Studies in the metabolism of Protozoa. III. Some prop- erties of a proteolytic extract obtained from Glaucoma piriformis. Bio- chem. J., 31: 789-98. Loefer, J. B. 1934. The trophic nature of Chlorogonium and Chilomonas. Biol. Bull., 66: 1-6. —— 1935a.* Effect of certain carbohydrates and organic acids on growth of Chlorogonium and Chilomonas, Arch. Protistenk., 84: 456-71. —— 1935b. Effects of certain nitrogen compounds on growth of Chloro- gonium and Chilomonas. Arch. Protistenk., 85: 74-86. 12 FOOD REQUIREMENTS —— 1935c.* Relation of hydrogen-ion concentration to growth of Chilo- monas and Chlorogonium. Atch. Protistenk., 85: 209-23. —— 1936a. Isolation and growth characteristics of the ‘“‘zoochlorella’” of Paramecium bursaria. Amer. Nat., 70: 184-88. — 1936b. Bacteria-free cultures of Paramecium bursaria and concentration of the medium as a factor in growth, J. exp. Zool., 72: 387-407. —— 1936c. Effect of certain “peptone’’ media and carbohydrates on the growth of Paramecium bursaria. Arch. Protistenk., 87: 142-50. —— 1936d. A simple method for maintaining pure-line mass cultures of Paramecium caudatum on a single species of yeast. Trans. Amer. micr. SOC., 552)254-56; —— 1938a. Effect of hydrogen-ion concentration on the growth and morphology of Paramecium bursaria. Arch. Protistenk., 87: 142-50. —— 1938b.* Utilization of dextrose by Colpidium, Glaucoma, Chilomonas and Chlorogonium in bacteria-free cultures. J. exp. Zool., 79: 167-83. —— 1938c. Effect of osmotic pressure on the motility and viability of fresh- water Protozoa. Anat. Rec., 72 (Suppl.) : 50. Loefer, J. B., and R. P. Hall. 1936. Effect of ethyl alcohol on the growth of eight protozoan species in bacteria-free cultures. Arch. Protistenk., 87: #23-50. Lwoff, A. 1924. Le Pouvoir de synthése d’un protist hétérotrophe: Glaucoma piriformis. C. R. Soc. Biol. Paris, 91: 344-45. —— 1925. La Nutrition des infusoires au dépens des substances dissoutes. C. R. Soc. Biol. Paris, 93: 1272-73. —— 1929a. Milieux de culture et d’entretien pour Glaucoma piriformis (cilié) . C. R. Soc. Biol. Paris, 100: 635-36. —— 1929b. La Nutrition de Polytoma uvella Ehrenberg (flagellé Chlamydo- monadinae) et le Pouvoir de synthése des protistes hétérotrophes. Les protistes mésotrophes. C. R. Acad. Sci. Paris, 188: 114-16. —— 1930. Le Fer, élément indispensable au flagellé Polytoma uvella Ehr. C. R. Soc. Biol. Paris, 104: 664-66. —— 1931. La Nutrition carbonée de Polytoma uvella. C. R. Soc. Biol. Paris, 1072 1070372? ——— 1932.* Recherches biochimiques sur la nutrition des protozoaires. Le pouvoir synthése. Monogr. Inst. Pasteur. —— 1934. Die Bedeutung des Blutfarbstoffes fiir die parasitischen flagellaten. Zbl. Bakt., Orig. 130: 498-518. —— 1935a. L’Oxytrophie et les organisms oxytrophes. C. R. Soc. Biol. Paris, 119: 87-90. —— 1935b. La Nutrition azotée et carbonée de Polytomella agilis (Polyblépharidée incolore). C. R. Soc. Biol. Paris, 119: 974-76. —— 1936. La Fonction de la protohémin pour les protozoaires et les bactéries parahémotrophes. C. R. Soc. Biol. Paris, 122: 1041-42. FOOD REQUIREMENTS Biles —— 1938a. Remarques sur la physiologie comparée des protistes eucaryotes. Les Leucophytes et l’oxytrophie. Arch. Protistenk., 90: 194-209. —— 1938b. La Synthése de l’aneurine par le protozoaire, Acanthamoeba castellanii. C. R. Soc. Biol. Paris, 128: 455-58. Lwoff, A., and H. Dusi. 1929. Le Pouvoir de synthése d’Euglena gracilis cultivée a l’obscurité. C. R. Soc. Biol. Paris, 102: 567-69. — 1931. La Nutrition azotée et carbonée d’Evglena gracilis en culture pure a l’obscurité. C. R. Soc. Biol. Paris, 107: 1068-69. —— 1934. L’Oxytrophie et la nutrition des flagellés leucocophytes. Ann. Inst. Pasteur, 53: 641-53. —— 1935a. La Supression expérimentale des chloroplastes chez Euglena mesnilz, Ann. Inst. Pasteur, 119: 1092-95. —— 1935b. La Nutrition azotée et carbonée de Chlorogonium euchlorum a l’obscurité; l’acide acétique envisagé comme produit de |’assimilation chlorophylliene. C. R. Soc. Biol. Paris, 119: 1260-63. — 1936. La Nutrition de l’euglénien Astasia chattoni. C. R. Acad. Sci. Paris, 202: 248-50. -—— 1937a. La Pyrimidine et le thiazol, facteurs de croissance pour le flagellé Polytomella caeca. C. R. Acad. Sci. Paris, 630-32. ——— 1937b. Le Thiazol, facteur de croissance pour les flagellés Polytoma caudatum et Chilomonas paramecium. C. R. Acad. Sci. Paris, 205: 756-58. ——— 1937c. Le Thiazol, facteur de croissance pour Polytoma ocellatum (Chlamydomonadine). Importance des constituants de l’aneurine pour les flagellés leucophytes. C. R. Acad. Sci. Paris, 205: 882-84. —— 1938a. Culture de divers flagellés leucophytes en milieu synthétique. GoRs See Biol Pans 127: 53-56. —— 1938b. L’ Activité de diverses pyrimidines, considérées comme facteurs de croissance pour les flagellés Polytomella caeca et Chilomonas para- mecium. C. R. Soc. Biol. Paris, 127: 1408-11. —— 1938c. Influence de diverses substitutions sur l’activité de thiazol con- sidéré comme facteur de croissance pour quelques flagellés leucophytes. C. R. Soe: Biol. Paris, 128: 238-41. Lwoff, A., and E. Lederer. 1935. Remarques sur |’‘‘extrait de terre’ envisagé comme facteur de croissance pour les flagellés. C. R. Soc. Biol. Paris, WD) STATS Lwoff, A., and M. Lwoff. 1937. L’Aneurine, facteur de croissance pour le cilié Glaucoma piriformis. C. R. Soc. Biol. Paris, 126: 644-46. —— 1938. La Specificité de l’aneurine, facteur de croissance pour le cilié Glaucoma piriformis. C. R. Soc. Biol Paris, 127: 1170-72. Lwoff, A., and L. Provasoli. 1935. La Nutrition de Polytoma caudatum vat. astigmata (Chlamydomonadine incolore), et la synthése de |’amidon par les leucophytes. C. R. Soc. Biol. Paris, 119: 90-93. —— 1937. Caractéres physiologiques du flagellé Polytoma obtusum, C. R. Soc. Biol. Paris, 126: 279-80. 514 FOOD REQUIREMENTS Lwoff, A., and N. Roukhelman. 1929. Variations de quelques formes d’azote dans une culture pure d’infusoires. C. R. Acad. Sci. Paris, 183: 156-58. Lwoff, M. 1929a. Culture de Leptomonas ctenocephali var. chattoni Laveran et Franchini, en milieu privés de sang frais: milieux liquides au sange chauffé. Bull. Soc. Path. exot., 22: 247-51. —— 1929b. Milieu d’isolement et d’entretien pour Schizotrypanum cruzi Chagas. Bull. Soc. Path. exot., 22: 909-12. —— 1929c. Action favorisante du sang sur la culture due Leptomonas ctenocephali (flagellé trypanosomide). C. R. Soc. Biol. Paris, 99: 472-74. —— 1929d. Culture de Leptomonas ctenocephali Fanth. (flagellé trypanoso- mide) en milieu privé de sang frais: les organes stérilisés. C. R. Soc. Biol. Paris, 99: 1133-35. —— 1929e. Influence du degré d’hydrolyse des matiéres protéiques sur la nutrition de Leptomonas ctenocephali (Fantham) in vitro. C. R. Soc. Biol. Paris, 100: 240-43. —— 1930. Une Flagellé parasite hétérotrophe: Leptomonas oncopelti Noguchi et Tilden (Trypanosomidae). C. R. Soc. Biol. Paris, 105: 835- aie —— 1933a.* Recherches sur la nutrition des trypanosomides. Ann. Inst. Pasteur, 515i: —— 1933b. Remarques sur la nutrition des trypanosomides et des bactéries parahémotrophes. Le “fer actif’ de Baudisch. Ann. Inst. Pasteur, 51: 707-13. ——— 1936, Le Pouvoir de synthése des trypanosomides des muscides. C. R. Soc. Biol. Paris, 121: 419-21. ——— 1937. L’Aneurine, facteur de croissance pour le flagellé trypanosomide Strigomonas oncopelti (Noguchi et Tilden). C. R. Soc. Biol. Paris, 126: Were —— 1938a. L’Hématine et l’acide ascorbique, facteurs de croissance pour le flagellé Schizotrypanum cruzi. C. R. Acad. Sci. Paris, 206: 540-42. —— 1938b. L’Aneurine, facteur de croissance pour le Strigomonas (flagellés Trypanosomides). C. R. Soc. Biol. Paris, 128: 241-43. ——— 1939. Le Pouvoir de synthése des leishmanies. C. R. Soc. Biol. Paris, 130: 406-8. Lwoff, M., and A. Lwoff. 1929. Le Pouvoir de synthése de Chlamydomonas agloéformis et d’'Haematococcus pluvialis en culture pure, 4 l’obscurité. C. R. Soc. Biol. Paris, 102: 569-71. Mainx, F. 1928.* Beitrage zur Morphologie und Physiologie der Eugleninen. II. Teil. Untersuchungen tiber die Ernahrungs-und Reizphysiologie. Arch. Protistenk., 60: 355-414. Mast, S. O., and D. M. Pace. 1933. Synthesis from inorganic compounds of starch, fats proteins and protoplasm in the colorless animal, Chilomonas paramecium, Protoplasma, 20: 326-58. —— 1935. Relation between sulphur in various chemical forms and the FOOD REQUIREMENTS 515 rate of growth in the colorless flagellate, Chilomonas paramecium. Proto- plasma, 23: 297-325. —— 1938. The effect of substances produced by Chilomonas paramecium on rate of reproduction. Physiol. Zodl., 11: 359-82. Oehler, R. 1916. Amébenzucht auf reinem Boden. Arch. Protistenk., 37: 175-90. —— 1919. Flagellaten- und Ciliatenzucht auf reinem Boden. Arch. Protistenk., 40: 16-26. Osterud, K. L. 1938. The nitrogen requirements of Lobomonas piriformis. Anat. Rec., 72 (Suppl.) : 128-29. —— 1939. The nitrogen and carbon requirements of Lobomonas piriformis. Anat. Rec., 75 (Suppl.) : 150-51. Petersen, W. A. 1929. The relation of density of population to rate of re- production. Physiol. Zodl., 2: 221-54. Phelps, A. 1935. Growth of Protozoa in pure culture. I. Effect upon the growth curve of the age of the inoculum and of the amount of the inoculum. J. exp. Zool., 70: 109-30. 1936. Growth of Protozoa in pure culture. II. Effect upon the growth curve of different concentrations of nutrient materials. J. exp. Zool., 72: 479-96. Philpott, C. H. 1928. Growth of Paramecium in pure cultures of pathogenic bacteria and in the presence of soluble products of such bacteria. J. Morph., 46: 85-129. Pringsheim, E. G. 1912. Kulturversuche mit chlorophyllfiihrenden Mikro- organismen. II. Zur Physiologie der Euglena gracilis. Beitr. Biol. Pfl., 12: 1-48. —— 1921. Zur Physiologie saprophytischer Flagellaten (Polytoma, Astasia und Chilomonas) . Beitr. allg. Bot., 2: 88-137. 1926. Kulturversuche mit chlorophyllfihrenden Mikroorganismen. V. Mitt. Methoden und Erfahrungen. Beitr. Biol. Pfl., 14: 283-312. — 1930. Algenreinkulturen. Eine Liste der Stamme welche auf Wunsch abgegeben werden. Arch. Protistenk., 69: 659-65. —— 1935a. Uber Azetatflagellaten. Naturwissenschaften, 23: 110-14. — 1935b. Wuchstoffe im Erdboden? Naturwissenschaften, 23: 197. —— 1937a. Assimilation of different organic substances by saprophytic flagellates. Nature, 139: 196. —— 1937b.* Beitrage zur Physiologie saprophytischer Algen und Flagellaten. 1 Mitt.: Chlorogonium und Hyalogonium. Planta, 26: 631- 64. —— 1937c.* Beitréige zur Physiologie saprophytischer Algen und Flagellaten. 2 Mitt.: Polytoma und Polytomella. Planta, 26: 665-91. —— 1937d.* Beitrige zur Physiologie saprotropher Algen und Flagellaten. 3 Mitt.: Die Stellung der Azetatflagellaten in einem physiologischen Ernahrungssystem. Planta, 27: 61-92. 516 FOOD REQUIREMENTS Provasoli, L. 1937a. La Nutrition carbonée du flagellé Polytoma uvella. C. R. Soc. Biol. Paris, 126: 280-82. —— 1937b. La Nutrition carbonée du flagellé Polytoma ocellatum. C. R. Soc. Biol. Paris, 126: 847-49. —— 1938a. La Nutrition carbonée de l’euglénien Astas7a quartana (Moroft). GRP Soc Biel Paris 1272 sl-s3: —— 1938b. Remarques sur la nutrition carbonée des eugléniens. C. R. Soc. Biol. Paris, 127: 190-92. —— 1938c. Studi sulla nutrizione dei Protozoi. Boll. Lab. Zool. agr. Bachic. Milano, 9 (rpr.), 124 pp. Reich, K. 1935. The cultivation of a sterile amoeba on media without solid food. J. exp. Zool., 69: 497-500. —— 1936. Studies on the physiology of Amoeba. I. The relation between nutrient solution, zone of growth and density of population. Physiol. Zool., 9: 254-63. —— 1938. Studies on the physiology of Amoeba. II. The allelocatalytic effect in Amoeba cultures free of bacteria. Physiol. Zodl., 11: 347-58. Rottier, P. B. 1936a. Recherches sur les courbes de croissance de Polytoma uvella. L’influence de l’oxygénation. C. R. Soc. Biol. Paris, 122: 65-68. —— 1936b. Recherches sur la croissance de Polytoma uvella, L’influence de la concentration des substances nutritives. C. R. Soc. Biol. Paris, 122: 776-80. Schoenborn, H. W. 1936. Growth of two species of Astasia in relation to pH of the medium. Anat. Rec., 67 (Suppl.) : 121. 1938. Growth of Astasia sp. and Exglena gracilis in media containing inorganic nitrogen. Anat. Rec., 72 (Suppl.) : 51. —— 1939. Growth of Evglena gracilis on inorganic nitrogen sources in the absence of light. Anat. Rec., 75 (Suppl.) : 151. —— 1940. Studies on the nutrition of colorless euglenoid flagellates. I. Utilization of inorganic nitrogen by As/asia in pure cultures. Ann. N. Y. Acad. Sci.,-40: 1-36. Woodruff, L. L. 1911. The effect of excretion products of Paramecium on its rate of reproduction. J. exp. Zool., 10: 557-81. 1912. Observations on the origin and sequence of the protozoan fauna of hay infusions. J. exp. Zool., 12: 205-64. —— 1913. The effect of excretion products of Infusoria on the same and on different species, with special reference to the protozoan sequence in infusions. J. exp. Zool., 14: 575-82. CHAPTERYX THE GROW Ti OF eiHE -PROTOZOA: OscaR W. RICHARDS GROWTH is a fundamental attribute of living organisms, manifested by a change of size of the individual, or in the number of organisms in a unit of environment. Negative growth may occur during adverse con- ditions or in certain dimensions when growth involves change of form. The analysis of population growth requires knowledge of the environ- ment, the individuals, and the interactions of each on the other. Growth is determined by measurement, and the information gained from any single measure is delimited by the nature of the measuring unit chosen. Rarely is a single measure adequate for the study of growth, even though it may be useful for practical application. Analytical studies require the simultaneous use of as many different measures as are neces- sary to give a picture sufficiently complete for the analysis. When there is no change in form, certain dimensions may be related directly, as length with volume or weight, but in allometric growth the conversion constants may change during the course of the growing period. These problems will be illustrated and discussed in this chapter, in so far as numerical data are available. METHODS FOR THE MEASUREMENT OF GROWTH Individual Protozoa have been measured to show growth changes in length and breadth, but these two dimensions may not permit very exact calculation of volume if the shape of the animal departs much from that of a sphere, cube, ellipsoid or other simple geometrical form. The area of the animal may be calculated by the use of a planimeter, from an enlarged photomicrograph or a tracing of the outline of the animal. The softer animals may be gently compressed between a slide and a cover glass, and the area measured. Multiplication of this figure by that of the thickness of the preparation gives the volume. The three- halves power of the area obtained from planimetric measurement may 518 GROWTH give the volume of some species fairly accurately. This is true for only a few solids, such as the cube. The method has an error of about 33 percent when used with spherical organisms. Chalkley (1929) measured the volume of Amoeba by gently drawing it into a capillary tube of known diameter and calculating the volume from the length of tube filled plus the two hemispherical ends. Populations of Protozoa are usually measured by counting a sample of the population in a Sedgewick-Rafter cell with a Whipple disc in the eyepiece of the microscope, or with a hemocytometer (cf. Woodruff, 1912; Hall et a/., 1935). The chief source of error of this method de- pends on how closely the sample represents the population. Care must be used that none of the animals are lost by sticking to the transfer pipette and to make sure that all are counted once only. Berkson ef al. (1935) has given a quantitative treatment of the errors of counting red blood cells with a hemocytometer, and their evaluation might be applied to estimates of protozoan populations. Tippett (1932) has suggested that the mean number may be esti- mated by counting the squares containing 0, 1, 2, and so forth animals and using the tables prepared for the Poisson distribution. With Proto- zoa, greater precision may be obtained by killing the animals before making the count. Many killing fluids are hypertonic, and animals may be lost from the osmotic effects of the killing fluid. Jennings (1908) and others have found that Worcester’s fluid causes little change with paramecia when a sufficient amount is used to overwhelm the animals. Hardy’s (1938) method of estimating numbers by comparing with standards containing a known number of dots might be used when high precision is not required. Protozoa may be centrifuged into a tube with a calibrated capillary bottom, similar to that used by Carlson (1913) for yeast. Elliott (1939) obtains greater precision and convenience by fusing a hematocrit tube toa 10 ml. centrifuge tube. When the animals are killed before centrifug- ing, it is necessary that the volume of the animals not be changed by an anisotonic killing fluid. Commercially made tubes should be carefully calibrated, as errors as great as 12 percent have been reported for some makes of Hopkins vaccine tubes. Solid packing may not be possible with the usual laboratory centrifuges, but for given conditions constant pack- ing may be obtained in equal time intervals. If the values are to be used GROWTH 519 for other than intercomparison, the centrifugal force used should be stated. The nomogram of Shapiro (1935a) simplifies this computation. If the distribution of animals of different sizes changes, e.g., just after a large proportion of them have divided or during endomictic reorgani- zation, the total volume may not indicate the number present. Size changes of yeast cells and failure to obtain constant packing have been reported by Richards (1934). Shapiro (1935b) has discussed the valid- ity of the centrifuge method with respect to marine ova. Simultaneous counts and volume determinations of Colpidium campylum have been made by Bond (1933). The population density of pigmented forms may be estimated from the optical density of the suspension, by means of a nephelometer (Richards and Jahn, 1933). A beam of light is passed through the suspension and the amount of light absorbed by the organisms is meas- ured by a photoelectric cell and a microammeter. When I is the micro- ammeter reading with a given tube and medium and J; is the reading of the suspension at time ¢, then the optical density, D = Jog I, — log I. In this way the small variations in transmission of different test tubes may be canceled out, and it is not necessary to open the tube, a factor which may be important if the organisms are reared in a bacteria-free cul- ture. The optical density depends on the number of organisms present, the distribution of organisms of various sizes, and their metabolic condition. It is sometimes difficult to relate measurements with this criterion to the number of organisms present, because changes in internal cell struc- ture (e.g., storage products) may alter their transparency. With proper care and control, the nephelometer may give a useful measure of the amount of protoplasm present in the population. For technical informa- tion the following may be consulted: Kober and Graves (1915), Mestre (1935), Russell (1937), and Miiller (1939). Difficulties in the use of the method have been summarized by Loofbourow and Dyer (1938) and by Stier, Newton, and Sprince (1939). Miss Wright (1937) has measured the turbidity of bacterial suspensions by passing the light beam through the suspension at right angles to the axis of the photo- electric cell. The dry weight of Protozoa may be obtained by filtering them from the culture medium with filter paper of fine porosity, a sintered glass 520 GROWTH filter, an alundum crucible, or an asbestos mat; washing them rapidly to remove the culture fluid, but not to burst the cells, and drying them to a constant weight. A vacuum desiccator containing sulphuric acid or phosphorous pentoxide at room temperature may give better results than a drying oven. This is a difficult method to control, so as to get consistent results. Bacteriological methods of diluting and plating are not often ap- plicable to Protozoa, but may be useful for testing the culture medium to make certain that it is bacteria-free. Standard texts should be con- sulted for methods. The ordinary nutrient agar is not a certain medium for estimating the bacteria found in water, and special media must be used. The number of colonies on an incubated plate may be less than the number of bacteria unless care has been used to prevent clumping of the bacteria. The errors of plate counts have been evaluated by Mattick et al. (1935) and by Ziegler and Halvorson (1935). Gordon (1938) has questioned these probability tables. Other methods which might be useful to protozodlogists are the measurement of the suspension in terms of viscosity (Shapiro, 1937) and the determination of the velocity of sedimentation (Nielsen, 1933). These would be used with killed or nonmotile animals. THE GROWTH OF INDIVIDUAL PROTOZOA Simpson (1902) measured the length and breadth of Paramecium caudatum with an ocular micrometer at a few and at many hours after fission. Jennings (1908) supplied the first detailed measurements, and the data from his summary table are plotted in Figures 130 and 131. At division the animal decreases in breadth and increases in length. After the separation the increase in both dimensions is increasingly rapid, then proceeds at a nearly constant relative rate, and finally slows until the cycle is repeated. The graphs of the growth are made on arithlog, or semilogarithmic paper, to facilitate analysis. This equivalent to plotting the logarithm of the size against a linear time axis. The slope of the growth curve at any point is the relative rate of growth (dy/ydt). Two growth curves, parallel to each other, are changing at the same relative rates. When no change in form occurs, the curves for area and volume will be corre- spondingly above and have slopes two and three times as great as a linear dimension. GROWTH S21 A year later Popoff (1908) measured the growth of P. caudatum by killing a sister cell immediately and the other member of the pair at a given time after fission. His results are expressed in micrometer units which have been converted into microns. The average of the cells killed Figure 130. Growth in length and in area of Paramecium caudatum. Data: M from Mizuno (1927), S from Schmalhausen and Synagajewska (1925), E28 and E38 from Estabrook (1910), J from Jennings 1908), and P from Popoff (1909). at fission was taken as the standard size at zero time, and the average differences for the intervals were added successively to obtain the data plotted in Figures 130, 131, and 132. He measured length (L), breadth (B), and thickness (T) and computed the volume as — 4xLBT/24, assuming the form to be ellipsoid, and he also gives similar measure- ments of the growth of the nucleus and of the nucleocytoplasmic ratio. Estabrook (1910) investigated the effect of various chemicals on the 522 GROWTH growth of P. caudatum and some of his control series, with more fre- quent measurements, are plotted. Measurements of growth in length by Schmalhausen and Syngajewskaja (1925) and by Mizuno (1927) are also available for P. caudatum. Mizuno also determined the area of the animal with a planimeter, from a camera-lucida tracing. x x f= fa) < WW a ao Figure 131. Growth in breadth and thickness (TH) of Paramecium caudatum. Data: P from Popoff (1909), M from Mizuno (1927), J from Jennings (1908), E21, E28, and E38 from Estabrook (1910). All of the investigators took precautions to prevent any change in the dimensions of the organism from killing the organism. The curves for growth in length are quite similar. All were grown in hay infusion medium and the temperature, when stated, was 24-26° C. Mizuno’s ani- mals grew at first at a more rapid relative rate than the others. Esta- brook’s and Schmalhausen’s animals were appreciably larger than Jennings’s, Mizuno’s, and Popoft’s Paramecia. The decrease in the early GROWTH D29 logarithmic growth is more rapid in the data from Jennings’s measure- ments; however, his animals continued to grow for a longer period and reached a slightly greater length than did those measured by Mizuno and Popoff. The variations may be due to differences in nutrition or to race. The results are all in terms of averages from different animals, and do not show the continuous change of size of a given animal. This type of averaging of cross-sectional data is known to give variation. The measurements of breadth (Fig. 131) are not as consistent as those of length. With the exception of one of Estabrook’s series, the breadth decreases following fission, and growth in this dimension com- mences later, in the measurements given. Thickness and breadth meas- urements, plotted from Popoft’s observations, show no change in size for the first two hours. The lack of agreement of the different series of measurements suggests differences in the pattern of growth for the different races. Growth in thickness occurred later than growth in breadth with Popoff’s P. caudatum. Growth in area (Fig. 130) is negative during the time that the breadth is decreasing, after which the increase continues for most of the cycle at about the same relative rate. Growth in volume, from Popoft’s calculations (Fig. 132), does not show the early negative phase, because his animals apparently did not change form during division as the oth- ers did. With minor fluctuations the increase in volume continues in a suitable environment until the time of the next fission. The growth of Paramecium aurelia (Fig. 132), plotted from the meas- urements of Erdmann (1920), is quite similar to that of P. caudatum. Erdmann’s three races differed from one another in size. No decrease in breadth was reported for this species. The growth in volume of the first individual and its progeny is given in Figure 132 for the soil amoeba, Hartmanella hyalina (Cutler and Crump, 1927). The growth curve is very similar to that of P. caudatum. Ten drawings were made rapidly at each time, and the volume was ob- tained from the average areas, on the assumption that the animals were 1 thick. The temperature was 21° C. The growth in volume of Amoeba proteus was measured by Chalkley (1929). The animals were pipetted until they assumed a spherical form, and the diameters of the cell and nucleus were then measured. The growth of the Amoeba is slow, and Chalkley’s observations were not continued until the equilibrium size 524 GROWTH #43 - 00000 OMITTED it} 55 > —] oO > Figure 132. A. Growth in volume of Paramecium caudatum (P), Frontonia leucas (F), from Popoff (1907, 1908), and of Hartmanella hyalina (H) from Cutler and Crump (1927). B. Growth in length of P. aurelia. Data from Erdmann (1920). was reached. The rate of growth is influenced by the number of nuclet present in the animal. Popoff (1908) measured the growth of Frontonia leucas in length, breadth, and thickness, and estimated the volume as the product of these three factors. The growth of the nucleus was measured similarly. His data are given in arbitrary size units, which were converted into micra. The growth of Frontonia was much more variable than Popoft’s growth data indicated to be the case for Paramecium, except for the volume changes. The average volume of the cytoplasm and the nucleus of the control animals killed at fission, was used as the value for zero GROWTH 525 time. To these values were added successively the average increments, in order to obtain the values at each time. The sum of the nuclear and the cytoplasmic volumes is plotted in Figure 132. The increments were read from Popoft’s summary graph. The growth in volume of F. /ewcas is more rapid during the first hour; the rate then decreases, until the relative rate is nearly linear, that ts, until about one hour before the next division. The rate increases before division, mainly owing to the increase in nuclear volume. The volume of the cytoplasm increases continually. The volume of the nucleus de- creases for the first two hours after division to about 86 percent of its size at fission, then increases slowly, until it shows the customary rapid increase in the three hours preceding a new fission. Entz (1931) has followed the growth of populations and of indi- vidual dinoflagellates, Ceratium hirudinella, in their natural habitats. The largest animals were found in April. The highest rate of division occurred in late June, July, and August, which coincides with the period of maximum temperature. The growth was measured in three dimen- sions, and the time, in hours, for the growth stages is: nucleal and cyto- plasmic division, one; horn regeneration, 2; slim stage, 27; indifferent stage, 72; compacted stage, 18; total, 120 hours. Extensive studies have been made of the variation in the sizes of Protozoa, and this information has been collated by Adolph (1931). A change in form during growth is reported by MacLennan (1935) for Ichthyophthirius. This and other Protozoa would furnish excellent ma- terial for the study of allometric growth. Cf. Huxley (1932), Need- ham (1934), Teissier (1934), Richards (1935), and Huxley and Teis- sier (1936). The size of Paramecium bursaria was modified by changing the pH of the culture medium (Loefer, 1938a). Cell size and nuclear size in Oxytricha fallax was found by Woodruff (1913a) to be least during the periods of rapid reproduction, and to become larger as the division rate decreases. The nucleocytoplasmic ratio was highest during the period of greatest reproductive activity, and this was interpreted as an incidental result rather than as a cause of the rate of division. The first evidence of division is a slight groove encircling the animal, and separation occurs in Paramecium about one-half hour later (Jen- nings, 1908). The nucleocytoplasmic ratio increases for the first three hours, and then decreases for the next three hours (Popoff, 1909). 526 GROWTH During the first hour, Paramecia are little affected by chemicals, and food plays no rdle (Estabrook, 1910). Increase in length is predomi- nant at first, and then the animals fill out in breadth. Negative correla- tions of length and breadth were found by Mizuno at one, four, and six hours after fission. Erdmann (1920) found changes in the sizes of Paramecia which were related to the endomictic rhythm. Individual growth has been measured in relatively few species, mostly Infusioria. Data on other forms would be useful for comparison. Growth studies should be made successively on the same living animals, to avoid the difficulties of averaging and of using information from different individuals. A promising method for obtaining the measurements would be to take pictures of the animal in isolation culture, at frequent and regular intervals, through the microscope, with a motion-picture camera. The images could be measured from the film, which would provide a permanent record for the analysis of the growth. The growth of individual Protozoa is quite similar to that observed with other plants and animals. If we disregard the first decrease in breadth, the growth curves are sigmoid. Sufficient information is not available to indicate to what extent there is a general change of form during growth. Many of these studies were not made with a constant culture fluid. In order to compare the growth patterns of different species, the culture conditions should be known to be optimal and reproducible. Such studies would establish standards for the further study of environ- ment on the individual growth pattern. The application of geometrical and metabolic considerations, similar to those used by Schmalhausen and Syngajewskaja (1925) with bacteria, will contribute to the theory of growth. THE GROWTH OF COLONIAL PROTOZOA The colonial Protozoa are believed by Fauré-Fremiet (1930) to constitute an intermediary step between the unicellular and the multi- cellular organisms. The colony grows by regular division from a free- swimming cell, until a size characteristic for the species is reached. When the environment is unfavorable, the growth is restricted. In favorable cultures two types of growth are found. In certain species, e.g., Epzstylis arenicolae and E. Perrieri, the first divisions are dichotomous and equal, and the mass growth of the colony follows in geometrical progression. Later, the sister cells divide unequally, and the growth becomes arith- GROWTH 527 metical, until the final size of the colony is reached. The time interval between cell divisions also gradually increases. The growth of Zodthamnium alternans depends on the common stalk, according to Fauré-Fremiet (1925). The cell initiating the colony has a given component of granular material which is distributed un- equally during division and, as it is used up, the growth of the colony ceases through transformation of material into the stalk. This may be analogous to the accumulation of nonliving material in multicellular organisms, a phenomenon which some biologists believe results in the limited growth of such organisms. The growth curves are sigmoid (Fauré-Fremiet, 1925, 1930), and the growth is believed to be auto- catalyzed by the granular material. PEDIGREE ISOLATION CULTURE AND LIFE CYCLES The culture of a single cell in a drop of a suitable food and the iso- lation of one of the divided cells, shortly after division, into a new drop of medium, effectively maintains the environment constant, and the growth is then potentially unlimited. With a constant medium and suit- able bacterial food, the rate of growth is very nearly constant, as has been demonstrated by Woodruff and Baitsell (1911), Darby (1930, 1930a, 1930b), Beers (1929), and others. If the growth curve for the sum of the individuals be plotted on arithlog paper, a straight line will result, because the growth (7) is exponential, y == yoe*t, when o is the amount of the seeding, or the growth at time, ¢ — O and e, is the Naperian base. The proportionality constant k — (/n 2)/G.T., or 0.639/G.T. The generation time (G.T.) is the time between divisions. Tabel 5 summarizes the growth rates under fairly constant conditions for certain Protozoa. The study of the variation in cell division has been obscured by the unfortunate practice of plotting the division rates in the form of a histo- gram. The bars of the plot commonly give the average division rate for ten-day periods. The histogram is used properly to show unit events which have no intermediate values, e.g., the results of tossing dice which cannot assume a position intermediate between two of the num- bered sides. The average division rate is no such discrete, mutually ex- clusive attribute, but may take any fractional value within the limits of the experiment. An example showing how information on the division 528 GROWTH TABLE 5. DIVISION RATES OF PROTOZOA WITH CONSTANT CONDITIONS Reference Organism Divisions AG Remarks Chilomonas 3.5/day 24 paramecium o.14/hour 26-30.5 | NaAc-mineral salts Didinium nasutum | 3.6/day 2I Fed on Paramecia Euglena gracilis RO was 10 In dark no NaAc 0.47" 23 In dark with NaAc 3.5 day 25 In wheat infusion Glaucoma pyriformis | 6.86/day 24.2—| Yeast extract 7.65 to 25.2 | Whole yeast+ yeast 8.02/day extract or peptone 5.62/day Paramecium aurelia | 0.72/day 20+ Lettuce and bacteria 1.2+/day 1.4/day 26.8 2.02/day 28 Paramecium 2.1/day 25? Over 200 days caudatum 1.8/day 25-28 | 51-day av. Min. salt +B. subtilis 2.3/day 26 Oaten medium-+bac- teria Polytoma uvella 4.4/day 22 Aérated peptone me- dium 1.85/day 22 Unaérated peptone me- dium Stentor coeruleus 2.1-0.7/hour | 18-20 | Modified Peters’ me- dium-+ ciliates 0.65 /hour 22 Hetherington medium +Blepharisma Stylonychia pustulata| 4.5-5/day 25? 3.2/day 24 .7/da Di 2} 3.7/day 5 Re * Divisions per day, per organism. From Darby (1930a). Mast and Pace (1934) Smith (1938) Beers (1929) Jahn (1935) Sweet (1939) Hetherington (1936) Phelps (1934) Phelps (1934) Woodruff and Baitsell (1911a) Phelps (1934) Phelps (1934) Darby (1930b) W.H. Johnson (1936) Gause (1934) Rottier (1936) Rottier (1936) Hetherington (1932) Gerstein (1937) Darby (1930) Baitsell (1912) T Maupas rate may be obscured by incorrect histogram plots was given by Richards and Dawson (1927). The changes in division rate may be plotted to advantage as a running average. The three-day running average is readily Gal@ulated: slit xn ens oo ee for the first is ¥, — (2x, ++ x,) /3; the second, x, = (x, + x, - x3) /3; . X» are the daily rates for ” days, the value GROWTH 529 and the last is (x,-1-+ 2x,)/3. If further smoothing is desired, a five-day instead of a three-day running average may be used, and it is computed in a corresponding manner. Phelps (1934) gives an example of the running average plot. In many of the earlier studies, the culture medium was inadequate, and after a time the division rate approached zero. Unless the animals were transferred to a favorable medium, the strain then died out. Such a growth period has been termed a “cycle” by Calkins. During a cycle, or, with some Protozoa during periods of nearly constant growth, small fluctuations in the growth rate occur. These minor variations are termed “rhythms” (Woodruff, 1905; Woodruff and Baitsell, 1911). Rhythms are associated with cellular reorganization (endomixis). The constant culture of Didinium nasutum without rhythms led Beers (1928) to be- lieve that rhythms were due to food, temperature, and the condition of the culture medium. Rhythms may have a function in some species and be merely effects of the environment in others. That the Calkins cycle depends on the adequacy of the culture medium has been demonstrated by a number of experimenters, e.g., Woodruff and Baitsell (1911a), Mast (1917), Beers (1928b), Darby (1930a), and Gerstein (1937). A medium that may be adequate for a few weeks may not be suitable for long periods. Dawson’s Paramecium and Ble pha- v7sma showed gradual negative trends during the three years of the cul- ture. At this rate the cycle would not end for several years (Richards and Dawson, 1927) and, in the meantime, some slight change in the medium might reverse the trend by supplying the cultural inadequacy, thus prolonging the cycle. Competition may bring out more rapidly the effects of the environment with populations than with individuals. The study of Protozoa, maintained for some time in an effectively constant culture medium, should add materially to our knowledge of growth. Peters (1901) gives useful methods; yeast techniques are summarized by Richards (1934). Dawson kept pedigreed isolation cultures of Histrio com planatus, Blepharisma undulans, and a mutant P. avrelia for three years. A sta- tistical analysis of the division rates removed the long-time trends, and established a seasonal cycle, with a maximum division rate in the sum- mer and a minimum rate in winter (Richards and Dawson, 1927). The statistical methods used were those used in economics in the study of 530 GROWTH cyclic phenomena. Further study suggested that the seasonal cycle was associated with sunlight (Richards, 1929). The pigmented Blepharisma followed, more closely than the others, the seasonal variation of radiant energy. A recent graphic method of Spurr (1937) could be used to advantage in the analysis of seasonal cycles in the division rate of Pro- tozoa. Properly controlled studies should be made to determine just how much effect light has, over a considerable period of time, on the growth of Protozoa. Such seasonal effect appears reasonable, as it is known that the reproductive cycles of some birds and other animals are initiated by the increased amount of light during the early part of the year. Conjugation restricts variation, which aids in survival during ad- verse conditions, according to Pearl (1907). Endomixis occasioned large variations in size, which Erdmann (1920) believed aided in sur- vival. She advised that attempts at selection be made during or immedi- ately after endomixis. Changes that aid in survival of a species through an unfavorable period are important in population studies; they might even effect the growth of the individual, and they deserve further in- vestigation by protozodlogists. Selection of rapidly dividing Amoeba proteus by Halsey (1936) did not produce a permanent race of rapidly dividing individuals. Burnside (1929) failed to change the size of Stentor coeruleus by fragmenting animals with large and small amounts of nuclear material. When the animals regenerated, the regulatory pro- cesses produced the same sized biotype. Variations in the cell, at the time of division or during periods of intercellular reorganization, may aid in the adaptation of the cell to a new environment and may account for the success or failure of investi- gators in acclimating an organism to life in a synthetic liquid culture medium. Hegerty (1939) has shown that young Streptococcus lactis, at the end of the lag period and just before the period of logarithmic growth, can produce new enzymes which permit the use of a new sub- strate, to which the bacteria could not adapt themselves at any other period of their life cycle. Do comparable changes occur in Protozoa? If so, pure culture methods would be facilitated. Thus the nature of the life cycles, as demonstrated by the earlier in- vestigators (M. Robertson, 1929), may now be studied effectively by physiological methods, as well as by post-mortem cytology. The de- tailed discussion of reproduction must be left for consideration in the GROWTH 531 other chapters. The critical periods of binary fission, conjugation, and intercellular reorganization are important to the study of growth, and further information on these phenomena will facilitate our understand- ing of growth. PROTOZOAN SUCCESSIONS: NONLABORATORY The variety of Protozoa and the numbers of each vary in time, and the abundance of individuals is usually inversely correlated with the diversity of kinds. At Geneva, Roux (1901) found the largest variety of species in January and in October and found that in the same loca- tions there was considerable variation at the corresponding time in two successive years. The sequence of Protozoa on sewage filtration beds (New Jersey) was followed by Crozier (1923) and Crozier and Harris (1923). A maximum number of rhizopods was found in August and of ciliates in May-June and November-December. Paramecium had a sharp maxi- mum in December-January, Vorticella in late December and in May, and Co/poda in the first third of the year. The sequence was attributed to the amount of anaérobiosis and to the formation and sloughing of the film. In this environment the abundance was directly correlated with the diversity of types. Noland (1925) found the sequence of Protozoa related to the tem- perature, oxygen, and carbon-dioxide concentrations in natural ponds. The hydrogen-ion concentration was not believed to be a controlling factor. Most of the Protozoa found were not those usually studied in the laboratory, but when samples were transferred to the laboratory, Col- poda cucullus, Glaucoma pyriformis, and Paramecium caudatum ap- peared, showing that these animals may thrive better in the laboratory than in natural habitats. Changes in the concentration of Protozoa in a Philadelphia pond were followed for a year by Wang (1928), who measured also the temperature, oxygen concentration, pH, and relative amount of dis- solved gases. The surface forms showed the greatest variation, which was believed due to the dissolved oxygen, depending on temperature and on the activity of the plants. A marked increase of acidity could be a limiting condition. The maximum number of forms was found in September-October. Since the amount of sunlight was greatest at this 532 GROWTH time and the temperature was not at a maximum, Richards (1929) has suggested that sunlight may have had more effect than temperature on numbers. The kinds of Mastigophora and Infusoria were inversely correlated with the abundance of individuals during the seasonal varia- tions. Coe (1932) found Protozoa attached to cement blocks suspended in the Pacific Ocean at La Jolla, from June to October. Protozoan sequences and numbers have been used by Lackey (1938a) for the study of sewage pollution of streams. Sufficient information is not yet available to explain the sequence of protozoan population growths, or the declines and succession by other species in nature. Many of the factors are interrelated, since the solubility of dissolved gases is a function of temperature, and the oxygen produc- tion of aquatic plants depends on the amount of light. The solution of these ecological problems promises to be of considerable practical value to man, as well as an aid in the elucidation of the growth processes. PROTOZOAN SUCCESSIONS: LABORATORY Cultures maintained in the laboratory are more readily followed than those in natural habitats, and there are many records of the growth of populations of Protozoa, their decline and succession by a compar- able growth of another species. Woodruff (1912) reported that near the surface of mass cultures the sequence was monads, Colpoda, Hypotrichida, Paramecium, Vorticella, and then Amoeba. The sequence of increase and disappearance was identical with appearance, except that the Amoeba advanced from the sixth to the fifth, and then to the fourth place. A definite succession was not apparent at the center or the bottom of the cultures, and a second cycle was rarely observed. The maximum rise and fall was about equal, but the final disappearance might be long delayed. The differences in the relative potential of division were be- lieved to establish the sequence, which was determined by the food and waste products secreted by the animals. The waste products were shown to be toxic, and the toxicity was species-specific and did not effect other species (Woodruff, 1913b). No relation was found between the titrable acidity and the sequence of the Protozoa (Fine, 1912). The acidity was related, rather, to the activities of the bacteria present. Fifteen series of two-liter cultures, made to imitate natural conditions, GROWTH 533 were followed by Eddy (1928). Counts were made with a Sedgewick- Rafter cell, but the results were not published, beyond general statements of sequence and dominance. Light had no effect on the sequence. Tem- perature exerts its influence by way of the bacteria serving as food for the Protozoa. Oxygenation of the culture increased the growth, espe- cially at the bottom of the culture. Too great concentrations of carbon dioxide were deleterious and could be buffered by including soil in the culture. The sequence was effected by the quantity and type of the in- fusion material. Dominance of a species was believed to depend on favor- able growing conditions for that species, rather than on the rate of re- production (cf. Woodruff, above). Unger (1931) has listed the sequence of Protozoa for two years in five cultures started from five different plants. Laboratory cultures, not restricted to a single species, show a regular series of population growths and declines for different species. The na- ture of the culture, its bacterial flora, and the reproductive potential of each species regulate the period of intensive growth, and the accumulat- ing excretion products of the animals bring about the decline of the population. The growth cycle of a species may modify the medium so that it becomes favorable for the growth of the next following species. Limiting conditions are oxygen and carbon-dioxide concentrations, pH, and temperature, and these will be discussed later. Remarkable flower- ings of Algae and Protozoa in the ocean and in lakes have been re- ported and are apparently on a more intense scale than occurs in labora- tory cultures. Some bacteria are inadequate as food sources; others are poisonous for some Protozoa; and the rise of a population of these bacteria would eliminate the susceptible Protozoa in the culture. Poisonous bacteria have been reported by Hargitt and Fray (1917) and by Kidder and Stuart (1938). The Protozoa commonly studied in the laboratory are apparently less frequently found in natural habitats. The growth of protozoan populations in mixed mass cultures is different from that of most other organisms, as no equilibrium is reached and maintained; instead, extinction seems to be the rule. AUTOCATALYSIS AND ALLELOCATALYSIS The theories of T. B. Robertson have greatly influenced the study of growth, and the first of these has been concerned primarily with the 534 GROWTH growth of Protozoa. Robertson (1923) believed that the growth of an organism, or of a population of organisms, was awtocatalytic, because the growth curves were sigmoid and could be fitted by the equation for a monomolecular, autocatalytic, chemical reaction. The slowest chemical reaction in the growth process was believed by Robertson to be the con- trolling master reaction for the process which established the form of the growth curve, and this could be discovered from the shape of the growth curve. His particular choice of chemical reaction was not sat- isfactory, and later he and other investigators have found difficulties which have, for the most part, led to the abandonment of the autocata- lytic theory. Cf. Robertson (1923), Snell (1929), Jahn (1930), Kava- nagh and Richards (1934). The sigmoid nature of the growth curve is the inevitable result of the regular geometrical increase during the time that the environment 1s favorable, and the slowing of this increase when the environment be- comes unfavorable as a result of the growth in it (decrease of foodstuffs and accumulation of excretion products). As long as the environment 1s maintained effectively constant, the rate of growth is constant and the growth curve is exponential. However, it eventually becomes impossible to maintain this constancy, and the growth is thus ultimately arrested. In this sense Bernard’s “milieu interieur’’ is part of the environment. The granular material which Fauré-Fremiet believes to limit the growth of some colonial Protozoa is one of the few reported examples of limita- tion in growth which apparently follows the appearance of a single substance. Such a substance might be considered a catalyst in the Robertson sense. Teissier (1937) has questioned this conclusion, and Snell’s (1929) objections are also applicable. Such substances, however, are rare. Allelocatalysis, according to Robertson (1924a), is “‘the acceleration of multiplication by the contiguity of a second organism in a restricted volume of medium.’ Robertson reported (1921b) that two Enchelys farcimen, ot two Col pidium colpoda in a drop of culture medium divided more rapidly than twice the division rate of one individual in an en- vironment of equal volume. It was shown later that his Colpidium was Colpoda cucullus. Other publications followed, reporting that some un- known substance, the allelocatalyst, stimulated cell division, and Robert- son believed this was formed during nuclear division and effected the permeability of the cells. GROWTH 535 Cutler and Crump (1923), using Colpidium, were unable to con- firm Robertson; and Greenleaf (1924) failed to demonstrate allelocataly- sis with Paramecium aurelia and P. caudatum, and with Pleurotricha lanceolata. Peskett (1924) could not demonstrate allelocatalysis with yeast. Robertson (1924) attributed their failures to the fact that they had not washed their cultures free from the catalyst present in the me- dium from which the cells were removed for inoculation. Cutler and Crump (1925) and Peskett (1925) repeated their work, but were un- able to demonstrate allelocatalysis either with washed or unwashed cul- tures. Yocom (1928) found the division rate of Oxytricha higher in cul- tures of four drops of medium than in ten-drop cultures, and attributed the difference to an allelocatalyst. Petersen (1929) found that division of P. caudatum was accelerated in volumes of culture of 0.83 ml., but not in volumes of less than 0.21 ml. Dimitrowa (1932) obtained better growth in “conditioned” medium which had previously supported the growth of Paramecium than in medium which had not been “‘condi- tioned.” Colpidium campylum grew better when some sterile filtrate from an old culture was added to a synthetic medium, according to Hall and Loefer (1938). Garrod (1936) reported that small inocula of Staphylococcus aureus did not grow in broth, but that large inoculations would grow. Mast and Pace (1937, 1938b) give evidence in support of an unknown substance produced in cultures, which, in low concentra- tions, stimulates the growth of Chilomonas paramecium, but which in high concentrations retards the growth of the animals. A soil amoeba, Mayorella, grown bacteria-free in mass cultures by Reich (1938), di- vided less when the initial populations were small. His data, replotted in the form of Figure 134, shows that the populations were proportionate to the seeding in rate of growth, within the large errors of observation, and do not support the allelocatalytic theory. Yeast populations grew at the same rate when the inoculation was varied from 5 to 8 & 10° cells per ml. (Clark, 1922); and from 12 to 1,200 cells per cu. mm. (Richards, 1932). Peskett (1927) found no difference when one yeast cell was introduced into volumes from 0.008 to 40 cu. mm. Meyers (1927) failed to demonstrate allelocatalysis with P. caudatum and found that conditioning the medium lessened the growth of the animals. Increasing inoculations of Glaucoma up to 536 GROWTH 70,000 times gave no allelocatalysis (Phelps, 1935). Darby (1930) maintained that allelocatalytic effects were due to the pH of the medium, and Jahn (1933) believed them due to the oxidation-reduction poising of the medium. When the medium was optimum, there would be no increased rate of reproduction; but if the medium was suboptimum, two or more organisms might modify it enough to permit growth whereas one organism could not do so and would grow slowly or fail to survive. Johnson and Hardin (1938) reported that medium conditioned by the growth of Psewdomonas fluorescens inhibits the reproduction of Paramecium micronucleatum. With the saline medium, used old-cul- ture medium was as efficient as fresh medium. The difference between these and Woodruff’s conclusions may be due to the effects of mixed bacteria in the natural medium used by Woodruff. Kidder (1939) stud- ied the effect of conditioning with a bacteria-free Colpidium campylum culture in proteose-peptone, dextrose broth. He believes that there is an accelerator and an inhibitor in the conditioned medium for growth. These were separated by absorption and filtration. Caution should be exercised in the use of filtered media, as some kinds of filters make the filtrate toxic (Richards, 1933). Sweet (1939) reinvestigated the volume seeding relation, using Ew- glena gracilis, and found that seedings of one and two individuals grew better in four drops of about 0.05 ml. each and inoculations of four and eight individuals in slightly larger, five-drop environments. This au- thor’s methods and technique illustrate survival, rather than growth, and while a volume effect of the environment was found, her results did not support the Robertson theories. The observations of these investigators and others focused attention on the suitability of the culture medium and suggested that the allelo- catalytic effects found by some biologists and discredited by others might be explained on this basis. Woodruff’s (1911) demonstration that the waste products limited growth was recalled and clarified some of the volume effects on growth, wherein the yield of cells depended on the volume of the culture medium rather than on the size of the inocula- tion. Johnson (1933) explained allelocatalytic effects on the relation of the bacterial food concentration to the number of Protozoa in the culture. An allelocatalytic effect on P. caudatum and on Moina macro- copa was found with a high nutrient concentration, and the reverse of GROWTH 5), 7 this with media of low nutrient concentrations (McPherson, Smith, and Banta, 1932). Another possible interpretation depends on the presence or absence of essential elements, both organic and inorganic, or on vitamin or hormone-like effects. This field has hardly been touched, and investi- gations here may clear up many problems concerning the nutritional requirements and the responses of the organisms to various culture fluids. The present tendency is to look in this direction for an under- standing of variations in the reproductive rate, rather than to attribute them to special allelocatalysts. Cf. Elliott, (1936), Hammond (1938), Koser and Saunders (1938), Hall (1939), and other chapters of this book. Another explanation of the effect of the volume of the culture on the reproduction rate of the organisms might be that in larger volumes the organisms use more energy swimming about, which would leave less energy for reproduction. This view could be tested by the use of cine- photomicrographic films in measuring the amount of activity of animals in large and small isolation cultures, and correlating this figure with the rate of multiplication. The relation might be different in rich and in poor nutrient media and, if so, this would elucidate some of the contradictory observations in the literature. NUTRITION AND GROWTH Protozoa (Ciliophora) feed naturally on bacteria, and with mixed population of both it is difficult to analyze the growth. Maupas recog- nized this difficulty in the nineteenth century and recommended that Pasteur’s methods be applied to the pure culturing of Protozoa. How- ever, for some time little was done, other than to insure a uniform and adequate supply of bacteria in the medium by cross culturing. Hargitt and Fray (1917) isolated and identified a number of bacteria from protozoan cultures and endeavored to grow Paramecium on pute cultures of bacteria, but found that no single species of bacteria was as satisfactory food as mixed cultures. Bacillus subtilis was the nearest satis- factory single species. Some species of bacteria were found to be toxic to the paramecia, and other poisonous bacteria have been reported by Kidder and Stuart (1938). Phillips (1922) extended the work of Har- gitt and Fray and was unable to find a single species of bacteria suit- 538 GROWTH able for the maintenance of P. aurelia. She concluded also that the paramecia could not live on dissolved substances, but were dependent on particulate food. Glaucoma ficaria was grown on a number of single species of bacteria, yeast, and flagellates by D. E. Johnson (1936). B. prodigiosus was the most satisfactory food organism. The results depended largely on the food being small enough for ingestion. Recent studies have been directed toward determining the food ele- ments required by Protozoa and toward devising synthetic media in which the Protozoa could be grown in bacteria-free, pure cultures. While it it not possible to separate studies on growth and nutrition except for con- venience, this chapter will be limited to studies occupied primarily with the analysis of growth. The broad problem of nutrition will be covered elsewhere. Different species have different nutritional requirements, and the failure of some protozodlogists to realize this fully has led to confusion in the literature on growth. Very few data are available which give the growth of the bacteria, as well as that of the Protozoa, present in mixed cultures. Considerably more labor would be involved in secur- ing this information, but the methods have been worked out and the information gained would justify the work. It is now possible to grow pure cultures of a variety of Protozoa in bacteria-free synthetic media. Some of the nutrient conditions limiting growth will be examined briefly. Tolerance to changes of osmotic pressure was found by Loefer (1938), in attempts to adapt fresh-water Protozoa to artificial sea water, to be limited. Yocom (1934) was more successful. Loefer (1939) found that tolerance to diluted Van Hoff solution developed over several generations. Changes in the oxidation-reduction potential have been measured in Chilomonas paramecium caltures by Jahn (1933), and his results suggest that when the medium is poised at the optimum rH, growth will be most rapid. The increased growth of Protozoa at the surface of mass cultures shows their sensitivity to oxygen. Aération will often extend the growth to deeper levels. Inadequate amounts of oxygen limit the growth of Polytoma uvella, and sufficient oxygen must be provided before the effects of other nutrients may be evaluated (Rottier, 1936; Mond, 1937). Reich (1936) believes oxygen concentration more important in Amoeba cultures than acidity. Jahn (1936) aérated bacterta- free cultures of Glaucoma pyriformis and Chilomonas paramecium in a hydrolyzed casein medium with air, and air freed of carbon dioxide. GROWTH De) The Chilomonas grew best in unaérated cultures and not so well in the cultures aérated with CO, free air. The Glaucoma grew equally well with and without CO,, but better than in unaérated cultures. Jahn believes that CO, is necessary to some organisms to avert the weakening of the buffer systems within the cell. The anaérobes are believed less sensitive to CO, removal because the amino acids and other weak acids may re- place the carbonic acid. The lag period in bacterial growth varies with the CO, concentration (Walker, 1932), and increased production is associated with physiological changes in the bacterial cells (Huntington and Winslow, 1937; Gladstone et al., 1935). Similar effects should be watched for in protozoan populations. Temperature has long been known to affect growth. Woodruff and Baitsell (1911b) found that the Q,, for the cell division of P. aurelia was 2.7, over a range of 21.5° to 31.5° C., and that the optimum range for them was 24° to 28.5° C. Individual pedigree cultures and mass cul- tures were measured by Mitchell (1929) over a range of 12° to 27° C,, and the thermal increment (1) for cell division was found to be 23,000 calories. A lag was found in the isolation cultures, and a method is given for calculating the division rates from data covering several days. Pos- sibly with a different culture medium the lag might have been avoided or changed. Daniel and Chalkley (1933) found y to equal 16,500 for the whole division process of Amoeba proteus (4° to 30° C). For nu- clear division ,, equals 16,600 (4° to 35° C.); for cytoplasmic division, 20,500" (11° to 20°C )R75300 {21° to. 26° C:); prophase 11,700, and anaphase 20,200 (13° to 26° C.). The increments suggest that oxida- tive processes control cell division. Jahn (1935) found a maximum growth rate for Euglena gracilzs, grown in a hydrolyzed casein medium at 10° C., but the addition of sodium acetate changed the temperature of maximum growth to 23°. Motility and the occurrence of encystment and palmella stages were related to the temperature and food. Smith (1938) reported that CAz/o- monas paramecium grew in a sodium acetate-mineral salts medium from 9.5° to 35° C., with an optimum range of 26° to 30.5° C. Prolonged exposure to the lower temperatures decreased the resistance of the ani- mals to the cold. Adaptation to changed temperature required at least forty-eight hours. The synthesis of fat and starch is a result of tempera- ture and in turn may control the division rate. The chlorophyll-containing Protozoa vary in their light requirements. 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NUMBER.. Figure 134. A. Population growth curves plotted on arithlog coordinates of Exglena (E, and E,) from Jahn (1929) ; Paramecium aurelia (PA), P. caudatum (PC), Stylony- chia pustulata (SP) from Gause (1934); and M. Mayorella palestinensis from Reich (1938). B. Population growth curves plotted on Cartesian coérdinates (same data). 550 GROWTH initial densities up to 70,000 times failed to show any allelocatalytic effect. The number of animals present at the end of the logarithmic phase was independent of the number in the seeding. In comparing the phases of Glaucoma population growth with those of bacterial and yeast populations, he found the following differences: the initial station- ary and lag phases in G/awcoma populations are much shorter, in propor- tion to the optimum generation time; the stationary phase is independent of the size of the seeding; and the change from the logarithmic to the equilibrium phase of growth is more abrupt. No period of decreasing population size appeared within 120 hours. Changing from yeast extract to yeast autolysate increased the yield (Phelps, 1936). The rate of growth was found to be independent of the food concentration within wide limits, but the total number of animals was proportional to the amount of food. The concentration of excretion products did not inhibit the growth until very great population densities were reached. This again is quite different from yeast cells, which are adversely effected by low amounts of excretion and fermentation prod- ucts. A more favorable food medium and the use of aération flasks, as well as differences in the species of animals used, may account for the lesser effect of waste products observed by Phelps than by Wood- ruff (1911). The G. pyriformis used by Phelps is identified now as Tetrahymena glaucomiforma. The growth of populations of Colpidium campylum was measured by Bond (1933). With small amounts of yeast autolysate, the growth was slight and the lag period was greatly prolonged. With greater amounts of food, the equilibrium population was greater, the logarithmic phase was longer, the rate of growth greater, and the transition from the logarithmic phase to the equilibrium phase of the growth curve less abrupt. Bond’s evidence suggests that the yield of animals depends more on the amount of food available than on an inhibitory effect of excretion products. Gause (1934) presented the growth of a population of P. caudatum on an oatmeal infusion, with bacteria. He fitted the S-shaped growth curve with the logistic equation, and his analysis of the curve has been mentioned before. The growth curve of Stylonychia pustulata, Fig. 134, illustrates rapid growth, with a short equilibrium phase, followed by a period of negative growth leading to a lower equilibrium level. The GROWTH Ba! second equilibrium level decreased slightly from the eighth day to the sixteenth day, when a second and shorter growth cycle commenced. The second cycle passed through a brief equilibrium period and then de- clined to about the same level as that which followed the first growth cycle. Population growth curves are given for S. mytdllus, P. aurelia and, in a later monograph (1935), for Glaucoma scintilans, Didinium nasu- tum, Bursaria truncatella, and P. bursaria. Some of these will be dis- cussed in the next section. One set of data is interesting from the view- point of population growth, that for P. awrelia and P. caudatum, grown separately in a standardized medium which was changed every twenty- four hours (Fig. 134). The equilibrium phases showed that there were over twice as many P. awrelia produced as P. caudatum. Gause then measured the sizes of the animals and computed the mean volume of each and the total volume of population. The volume curves showed that very nearly the same volume of protoplasm was produced by each species, with the same medium and conditions of culture. P. caudatum was grown in a balanced salt medium, with one unit of concentration, and with five units’ concentration of bacteria, by Johnson (1935). The growth curves are sigmoid and show no stationary phase and only a short lag phase. The equilibrium number was maintained with no decline for seven days. The number of animals produced in the greater concentration was more than five times the number in the lesser concentration. In the lower concentration a single animal divided more times than did a group of animals, while in the greater concentration a group divided more rapidly, for about three days, when the population figure from the single animal seeding passed the group curve to reach a higher equilibrium level. Mond’s (1937) estimates of both the bacteria and the infusorian populations point the way to more adequate studies of protozoan growth. Populations of Colpoda duodenaria were maintained in aération flasks for four months by Taylor and Strickland (1938). By continuous feed- ing, densities of 6 > 10° per milliliter were produced. The size of the population fluctuated with the amount of food available and could be modified as the experimenters wished. Over the whole period the num- ber of Protozoa produced from a given amount of food was constant. Excretion products did not limit the growth, but the continuous aération may have ameliorated the effects of the waste products, so that the conditions are not comparable with unaérated cultures. Doe GROWTH The growth curves of protozoan populations are sigmoid and te- semble closely in form those of other populations. The growth curves of some Protozoa show all phases. The growth of different Protozoa depends on environmental conditions, and for details the reader should consult the original publications. The size of a protozoan population depends primarily on the amount of available food. Waste products do not limit the growth, as they do with yeast populations, and are in- hibitory only in very dense populations. However, yeast populations contain more organisms than the protozoan populations—Paramecium (Jones, 1928) 10°; Glaucoma (Phelps, 1936) 7.25 & 10°; Colpoda (Taylor and Strickland, 1938) 60 10°; yeast (Richards, 1932) 335 X 10° per ml.; bacteria (Steinhaus and Birkeland, 1939) to 2.5 x 10° —and laboratory populations of yeast are far less dense than those produced in aérated and cooled commercial fermenters. The total volume of protoplasm (number of individuals, x mean size) should be con- sidered, and metabolic rates known, when comparing populations of different organisms. Under identical conditions P. awrelia and P. cauda- tum produced nearly the same total volume, although there were over twice as many of the smaller P. aurelza. So far no selective mortality has been reported for protozoan popula- tion growth, although this is well known in yeast populations. The decline may occur because fewer of the Protozoa reproduce or it may be due to a slowing of the rate of cell division. Jahn (personal communica- tion) believed the latter true for his Evg/ena populations. Jones reported a disruptive mortality in his P. multimicronucleata populations. No evidence of differences in the sizes and their distribution among Protozoa —which would reveal how homogeneous the populations are from time to time during the population growth—has been given in recent studies, with carefully controlled conditions (e.g., bacteria-free cultures, on syn- thetic media). Can Protozoa become resistant to an unfavorable medium and remain abie to reproduce? Is encystment always governed by food concentration (Taylor and Strickland, 1938), or do other factors have a role? To what extent can an equilibrium population be maintained by en- and excystment? The lack of information on these and many other problems should attract more students of physiology and of growth to protozodlogy. GROWTH 99 THE STRUGGLE FOR EXISTENCE The mathematical analysis of the question of survival by Volterra, Lotka, Haldane, and others has established certain principles. Gause (1934, 1935) has contributed to both the experimental and the theoreti- cal advancement of the subject. The mathematical analyses are compli- cated, even though in the state of first approximations, and the interested reader should consult the original articles. Cf. Lotka (1925, 1934), Kostitzin (1934), Gause (1934, 1935). Chapman (1931) gives a translation of part of Volterra’s work. Protozoan populations have been used to test the hypothesis, and some of the experiments of Gause are here summarized to illustrate the beginning of a quantitative attack on the problems of struggle for existence and survival of the fittest. Separate and mixed populations of Paramecium caudatum and Sty- lonychia mytilus wete grown on an oatmeal infusion inoculated with B. subtilis. Neither species grew as well in mixed populations, but the influence of Stylonychia on Paramecium is about forty times as great as the effect of the latter on the former. With more food, provided by mixed, wild bacteria, Paramecium grew to about the same level in mixed populations as it did in pure population. Stylonychia grew only to about half the number when competing in the same environment with Para- mecium as it would have alone, and its population soon declined, while that of the Paramecium maintained itself despite the competition. Paramecium caudatum and P. aurelia may be grown together, and will compete for the same food. It is necessary to make comparisons in terms of volume of protoplasm, as discussed in the previous section. In mixed populations the growth curves for the two populations are quite similar for the first eight days, after which the P. aurelia popula- tion continues to grow, while that of the P. caudatum declines, reaching the point of extinction in about sixteen days. P. caudatum has an ad- vantage in a greater coefficient of geometrical increase, but requires 1.64 times as much food as P. awrelia. Consequently, the greater rate of growth is a liability in competition. P. aurelia is less affected by excretion prod- ucts, as it can live twice as long in the presence of a strong concentration of waste excretion products as P. caudatum. With the amount of food available and the medium used, only the P. awrelia could survive the competition of the mixed population. Glaucoma scintillans, growing in competition with P. awrelza, will survive when the latter perishes. 554 GROWTH A more complicated series of experiments was made on P. aurelia or P. caudatum and P. bursaria with food supplied by bacteria and yeast. The P. busaria could eat the yeast, but the two other species could not. Varying equilibria of populations could be established, depending on the initial concentrations of the four organisms. In this case the com- petition is in different niches. Populations of predators and prey are interesting and have been studied in epidemiology, notably by Ross and Lotka working with the malarial parasite. A simpler case, of less personal interest to man, is the compett- tion of mixed populations of bacteria, Paramecium, and Didinium nasu- tum. The latter consumes a Paramecium every three hours. In such a mixed population, Gause found that at first both the Paramecium and the Didinium populations grew, but later the didinia ate all of the para- mecia and then promptly starved. With medium with sediment in which some of the paramecia moved about and thus were not available as food for the didinia, the didinia ate the available paramecia and then starved while the remaining paramecia grew. Another experiment utilized Bur- sarta truncatella, which preyed on P. bursaria. The experiments may be grouped in three classes: (1) two species in the same ecological niche, competing for the same food; (2) two species in different niches, competing for the same food; or (3) two species, one eating the other. Gause (1935) has given mathematical analyses of the equilibria, depending on the variables involved. Much progress has been made in this phase of biological science, even though it 1s less than a quarter of a century old, and well-planned experiments or heuristic theoretical analysis may be expected to contribute to an under- standing of the growth of the Protozoa, to ecology, and to historical (evolutionary) biological science. LITERATURE CITED Adolph, E. F. 1931. The regulation of size. Springfield, Ill. 235 pp. Allee, W. C. 1934. Recent studies in mass physiology. Biol. Rev., 9: 1-48. Beers, C. D. 1928a. The regulation of dietary insufficiency to vitality in the ciliate Didinium nasutum. J. exp. Zool., 51: 121-33. 1928b. Rhythms in infusoria with special reference to Didinium nasutum. J. exp. Zool., 51: 485-93. —— 1928c. Some effects of dietary insufficiency in the ciliate Didininm nasutum. Proc. Nat. Acad. Sci., Wash., 14: 132-37. GROWTH IID —— 1929. On the possibility of indefinite reproduction in the ciliate Didinium nasutum without conjugation or endomixis., Amer. Nat., 63: 125-29: Berkson, J., T. B. Magath, and M. Hurn. 1935. Laboratory standards in relation to chance fluctuations of the erythrocyte count as estimated with the haemocytometer. J. Amer. statist. Ass., 30: 414-26. Bodine, J. H. 1917. Hydrogen ion concentration of protozoan cultures. Biol. Bull., 41: 73-77. Bond, R. M. 1933. A contribution to the study of the natural food-cycle in aquatic environments. Bull. Bingham oceanogr. Coll., 4: 1-89. Bowen, W. J. 1938. The effects of copper and vanadium on the frequency of division. Biol. Bull., 75: 361. (Cf. also Amer. J. Physiol., 1939, 126: Aes) Buchanan, R. E., and E. I. Fulmer. 1928. Physiology and biochemistry of bacteria. Vol. I. Baltimore. Burnside, L. H. 1929. Relation of nuclear size to body size in Stentor coeruleus. J. exp. Zool., 54: 473-83. Carlson, Tor. 1913. Uber Geschwindigkeit und Grésse der Hefevermehrung in Wirze. Biochem. Z., 57: 313-35. Chalkley, H. W. 1929. Changes in water content of Amoeba in relation to change in its protoplasmic structure. Physiol. Zo6l., 4: 535-74. — 1931. The chemistry of cell division. II. The relation between cell growth and division in Amoeba proteus. Publ. Hlth. Rep. Wash., 46: 1736-54. Chapman, R. N. 1931. Animal ecology. New York. Clark, N. A. 1922. Rate of formation and yield of yeast in wort. J. phys. Chem., 26: 42-60. Coe, W. R. 1932. Season of attachment and rate of growth of sedentary marine organisms at the pier of the Scripps Institution of Oceanography, La Jolla, Calif. Bull. Scripps Instn. Oceanogr. tech. 3: 37-86. Crozier, W. J. 1923. On abundance and diversity in the protozoan fauna of a sewage ‘‘Filter.”’ Sczence, 58: 424-25. Crozier, W. J., and E. S. Harris. 1923. Animal population of a sewage sprinkling filter. Stat. Rep. N. J., 503-16. Cutler, D. W., and L. M. Crump. 1923. The rate of reproduction in artificial culture of Colpidium colpoda. Bio-chem. J., 17: 878-86. 1924. The rate of reproduction in artificial culture of Colpidinm col poda. Bio-chem. J., 18: 905-12. —— 1925. The influence of washing upon the reproduction rate of Col- pidium col poda. Bio-chem. J., 19: 450-53. —— 1927. The qualitative and quantitative effects of food on the growth of soil Amoeba. Brit. J. exp. Biol., 5: 155-65. Daniel, G. E., and H. W. Chalkley. 1933. The influence of temperature on 556 GROWTH the process of division in Amoeba proteus (Leidy). J. cell. comp. Physiol., 2: 311-27. Darby, H. H. 1930a. The experimental production of life cycles in ciliates. J. exp. Biol., 7: 132-42. —— 1930b. Studies on growth acceleration in Protozoa and yeast. J. exp. Biol., 7: 308-16. Dimitrowa, A. 1932. Die fordernde Wirkung der Paramaecium caudatum. Ehrbg. auf dessen Teilungsgeschwindigkeit. Zool. Anz., 100: 127-32. Eddy, S. 1928. Succession of Protozoa in cultures under controlled conditions. Trans. Amer. micr. Soc., 47: 283-339. Elliott, A. M. 1935a. The influence of pantothenic acid on growth of Proto- zoa. Biol. Bull., 68: 82-92. 1935b. Effects of certain organic acids and protein derivatives on the growth of Col pidium. Arch. Protistenk., 84: 472-94. 1936. Nutritional studies on Protozoa. Proc. Minn. Acad. Sci., Rept. 6 pp. — 1938. The influence of certain plant hormones on growth of Protozoa. Physiol. Zo6l., 11: 31-39. —— 1939. A volumetric method for estimating population densities of Protozoa. Trans. Amer. micr. Soc., 58: 97-99. Entz, G., Jr. 1931. Analyse des Wachstums und der Teilung einer Popula- tion sowie eines Individuums des Protisten Ceratium hirudinella unter den natiirlichen Verhialtnissen. Arch. Protistenk., 74: 311-61. Erdmann, R. 1920. Endomixis and size variations in pure bred lines of Paramecium aurelia. Roux Arch. EntwMech. Organ., 46: 85-148. Estabrook, A. H. 1910. Effect of chemicals on growth in Paramecium. J. exp. Zool., 8: 489-543. Fauré-Fremiet, E. 1925. La Cinétique du développement. Paris. 1930. Growth and differentiation of the colonies of Zoothamnium alternans. Biol. Bull., 58: 28-51. Fine, M. S. 1912. Chemical properties of hay infusions with special reference to the titratable acidity and its relation to the protozoan sequence. J. exp. ZOO 2A265-8ile Garrod, L. P. 1936. Allelocatalysis. J. Path. Bact., 42: 535-36. Gause, G. F. 1932. Ecology of populations. Quart. Rev. Biol., 7: 27-46. —— 1934. The struggle for existence. Baltimore. —— 1935. Verifications expérimentales de la théorie mathématique de la lutte pour la vie. Act. Sci. Indust., No. 277. Paris. Gerstein, J. 1937. The culture and division rate of Stentor coeruleus. Proc. Soc, €xp: Biol. Ni¥-4137; 210=1de Giese, A. C. 1939. Ultraviolet radiation and cell division, I. Effects of 4 2654 and 2804 A upon Paramecium caudatum. J. cell. comp. Physiol., 13: 139-50. GROWTH Spy Gladstone, G. P., P. Fildes, and G. M. Richardson. 1935. Carbon dioxide as an essential factor in the growth of bacteria. Brit. J. exp. Path., 16: 335- 48. Gordon, R. D. 1938. Note on estimating bacterial populations by the dilution method. Proc. nat. Acad. Sci. Wash., 24: 212-15. Gray, J. 1929. The kinetics of growth. Brit. J. exp. Biol., 6: 248-74. Greenleaf, W. E. 1924. Influence of volume of culture medium and cell proximity on the rate of reproduction of Protozoa. Proc. Soc. exp. Biol. ING Ye, 2ill 405263 Gregory, L. H. 1925. Direct and after effects of changes in medium during different periods in the life history of Uroleptus mobilis, Biol. Bull., 48: 200-8. — 1926. Effects of changes in medium during different periods in the life history of Uroleptus mobilis, Biol. Bull., 51: 179-88. — 1928. The effects of changes in medium during different periods in the life history of Uroleptis mobilis and other Protozoa. Biol. Bull., 55: 386-94. Hall, R. P. 1933. On the relation of hydrogen-ion concentration to the growth of Exglena anabaena vat. minor and E. deses. Arch. Protistenk., 79: 239- 48. 1938a. Effects of manganese on the growth of Euglena anabaena, Astasta sp. and Colpidium campylum. Arch. Protistenk., 90: 178-84. 1938b. Pimelic acid as a growth factor for the ciliate, Colpidium campylum. Anat. Rec., 72 (Suppl.) : 110. 1939. The trophic nature of the plant-like flagellates. Quart. Rev. Biol., 14: 1-12. Hall, R. P., D. F. Johnson, and J. B. Loefer. 1935. A method for counting Protozoa in the measurement of growth under experimental conditions. Trans. Amer. micr. Soc., 54: 298. Hall, R. P., and J. B. Loefer. 1938. Effect of the addition of old culture medium on the growth of Colpidinm campylum. Amat. Rec., 72 (Suppl.) : 50. Hall, R. P., and H. W. Schoenborn. 1938. The selective action of inorganic media in bacteria-free cultures of Evglena. Anat. Rec., 72 (Suppl.) : 129. Halsey, H. R. 1936. The life cycle of Amoeba proteus Pallas, Leidy and of Amoeba dubia Schaeffer. J. exp. Zool., 74: 167-203. Hammett, F. S. 1929. Chemical stimulants for growth by increase in cell number. Proto plasma, 7: 297-322. Hammond, E. C. 1938. Biological effects of population density in lower organisms. Quart. Rev. Biol., 13: 421-38; 14: 35-59. Hardy, A. C. 1938. Estimating numbers without counting. Nature, 142: 255- 56. Hargitt, G. T., and W. W. Fray. 1917. The growth of Paramecium in pure cultures of bacteria. J. exp. Zool., 22: 421-55. 558 GROWTH Hegerty, C. P. 1939. Physiological youth as an important factor in adaptive enzyme formation. J. Bact., 37: 145-52. Hetherington, A. 1932. The constant culture of Stentor coeruleus, Arch. Protistenk., 76: 118-29. —— 1936. The precise control of growth in a pure culture of a ciliate Glaucoma pyriformis. Biol. Bull., 70: 426-40. Huntington, E., and C.-E. A. Winslow. 1937. Cell size and metabolic activity at various phases of the bacterial culture cycle. J. Bact., 33: 123-44. Huxley, J. S. 1932. Problems in relative growth. London. Huxley, J. S., and G. Teissier. 1936. Terminology of relative growth. Nature, 137: 780-81. Jahn, T. L. 1929. Studies on the physiology of the Euglenoid flagellates. I. The relation of the density of population to the growth rate of Evglena. Biol. Bull., 57: 81-106. —— 1930. Studies etc. II. The autocatalytic equation and the question of an autocatalyst in the growth of Evglena. Biol. Bull., 58: 281. —— 1933. Studies on the oxydation-reduction potential of protozoan cul- tures. I. The effect of -SH on Chilomonas paramecium. Protoplasma, 20: 90-104. —— 1934. Problems of population growth in the Protozoa. Cold Spring Harbor Symp. Quant. Biol., 2: 167-80. —— 1935. Studies etc. VI. The effects of temperature and of acetate on Euglena gracilis caltures in the dark. Arch. Protistenk., 86: 251-57. —— 1936. Effect of aeration and lack of CO, on growth of bacteria-free cultures of Protozoa. Proc. Soc. exp. Biol. N. Y., 33: 494-98. Jennings, H. S. 1908. Heredity, variation and evolution in Protozoa II. Proc. Amer. phil. Soc., 47: 393-546. Johnson, D. E. 1936. Growth of Glaucoma ficaria Kahl in cultures with single species of other organisms. Arch. Protistenk., 86: 359-78. Johnson, W. H. 1933. Effects of population density on the rate of reproduc- tion in Oxytricha. Physiol. Zo6l., 6: 22-54. —— 1935. Isolation of Glaucoma ficaria Kahl in bacteria-free cultures and growth in relation to the pH of the media. Arch. Protistenk., 86: 263-77. —— 1936. Studies in the nutrition and reproduction of Paramecium. Physiol. Zool., 9: 1-14. —— 1937. Experimental populations of microscopic organisms, Amer. Nat., 7-20: Johnson, W. H., and G. Hardin. 1938. Reproduction of paramecium in old culture medium. Physiol. Zodl., 11: 333-46. Jones, E. P. 1928. Population curves of Paramecium. Proc. Pa. Acad. Sci., 2% O27 =28, —— 1930. Paramecium infusion histories. I. Hydrogen ion change in hay and hay-flour infusions. Biol. Bull., 59: 275-84. GROWTH r po Kavanagh, A. J., and O. W. Richards. 1939. The autocatalytic growth curve. Amer. Nat., 68: 54-59. — 1937a. The unusual mortality which characterizes a Paramecium cul- ture. Anat. Rec., 70 (Suppl.) : 39. — 1937b. The potential longevity of a Paramecium culture. Anat. Rec., 70 (Suppl.) : 39. Kidder, G. W. 1939. The effects of biologically conditioned medium on the growth of Colpidium campylum. Biol. Bull., 77: 297-98. Kidder, G. W., and C. A. Stuart. 1938. The role of chromogenic bacteria in ciliate growth. Biol. Bull., 75: 336. Kober, P. A., and S. S. Graves. 1915. Nephelometry (Photometric Analysis) . I. History of method and development of instruments. J. industr. Engng. Chem., 7: 843-47. Koser, S. A., and F. Saunders. 1938. Accessory growth factors for bacteria and related microorganisms. Bact. Rev., 2: 99-190. Kostitzin, V. A. 1934. Symbiose, parasitisme et évolution. Act. Sci. Indust., No. 96. Paris. 47 pp. Lackey, J. B. 1938a. Protozoan plankton as indicators of pollution in a flowing stream. Publ. Hlth. Rep. Wash., 53: 2037-58. —— 1938b. A study of some ecologic factors affecting the distribution of Protozoa. Ecol. Monogr., 8: 501-27. Loefer, J. B. 1935. Effects of certain carbohydrates and organic acids on growth of Chlorogonium and Chilomonas. Arch. Protistenk., 84: 456-71. —— 1938a. Effects of hydrogen ion concentration on the growth and morphology of Paramecium bursaria. Arch. Protistenk., 90: 185-93. —— 1938b. Effect of osmotic pressure on the motility and viability of fresh-water Protozoa. Anat. Rec., 72 (Suppl.) : 50. —— 1938c. Growth of Chlorogonium tetragonium at different hydrogen- ion concentrations. Anat. Rec., 72 (Suppl.) : 129. —— 1938d. Bacteria-free culture of Paramecium bursaria and concentration of the medium as a factor in growth. J. exp. Zool., 72: 387-407. —— 1939. Acclimatization of fresh-water ciliates and flagellates to media of higher osmotic pressure. Physiol. Zodl., 12: 161-72. Loofbourow, J. R., and C. M. Dyer. 1938. Relative consistency of weights and counts in determining microorganisms by photoelectric nephelom- eters. Studies Inst. Divi Thomae, 1: 129-35. Lotka, A. J. 1925. Elements of physical biology. Baltimore. 1934. Théorie analitique des associations biologiques. Act. Sci. Indust., No. 187. Paris. Lwoff, A., and H. Dusi. 1937. La Pyrimidine et le thiazol, facteurs de croissance pour le Flagellé Polytoma caeca. C. R. Soc. Biol. Paris, 126: 630-32. —— 1938. Culture de divers flagelles leucophytes en milieu synthétique. C. R. Soc. Biol. Paris, 127: 53-56. 560 GROWTH McCay, C. M. 1933. Is longevity compatible with optimum life? Science, 77: 410-11. MacDougall, M. S. 1929. Modifications in Chilodon uncinatus produced by ultraviolet radiation. J. exp. Zool., 54: 95-109. MacLennan, R. F. 1935. Dedifferentiation and redifferentiation in Ichthyoph- thirius. 1. Neuromuscular system. Arch. Protistenk., 86: 191-210. McPherson, M., G. A. Smith, and A. M. Banta. 1932. New data with pos- sible bearing on Robertson’s theory of autocatalysis. Anat. Rec., 54 (Suppl.) : 23. Mast, S. O., and D. M. Pace. 1933. Synthesis from inorganic compounds of starch, fat, proteins and protoplasm in the colorless animal Chilomonas paramecium, Protoplasma, 20: 326-58. —— 1935. Relation between sulphur in various chemical forms and the rate of growth in the colorless flagellate Chilomonas paramecium. Proto- plasma, 23: 297-325. —— 1937. The relation between the number of individuals per volume of culture solution and rate of growth in Chilomonas paramecium. Anat. Rec., 70(Suppl.) : 40. —— 1938a. The relation between the hydrogen ion concentration of the culture medium and the rate of reproduction in Chilomonas paramecium. J. exp. Zool., 79: 429-31. —— 1938b. The effects of substances produced by Chilomonas paramecium on the rate of reproduction. Physiol. Zodl., 11: 360-82. 1938c. The relation between the age of the cultures from which Chilomonas is taken and the rate of reproduction in fresh culture fluid. Anat. Rec., 72 (Suppl.) : 62. Mattick, A. T. R., J. McClemont, and J. O. Irwin. 1935. The plate count of milk. J. Dairy Sci., 6: 130-47. Mestre, H. 1935. A precision photometer for the study of suspensions of bacteria and other microorganisms. J. Bact., 30: 335-58. Meyers, E. C. 1927. Relation of density of population and certain other factors to survival and reproduction in different biotypes of P. cawdatum. J. exp. Zool., 49: 1-43. Mitchell, W. H., Jr. 1929. The division rate of Paramecium in relation to temperature. J. exp. Zool., 54: 383-410. Mizuno, F. 1927. Sur la croissance du Paramecium caudatum. Sci. Rep. Tohuku Univ., 4th Ser., 2: 367-81. Mond, J. 1937. Réaction d’entretien et ration de croissance dans les popula- tions bactériennes. C. R. Acad. Sci. Paris, 205: 1456-57. Mottram, J. C., 1939. An increase in the rate of growth of paramecium sub- jected to the blastogenic hydrocarbon 3:4-benzpyrene. Nature, 144: 154. Miller, R. H. 1939. Photoelectric methods in analytical colorimetry. Industr. Engng. Chem., 11: 1-17. Needham, J. 1934. Chemical heterogony and the ground-plan of animal growth. Biol. Rev., 9: 79-109. GROWTH 561 Nielson, N. 1933. A method for determining the velocity of sedimentation of yeast. C. R. Lab. Carlsberg, Nr. 19. Noland, L. E. 1925. Factors influencing the distribution of fresh water ciliates. Ecology, 4: 437-52. Park, T. 1939. Analytical population studies in relation to general ecology. Amer. Midl. Nat., 21: 235-55. Pearl, R. 1907. A biometrical study of conjugation in Paramecium. Biometrika, 213-078 —— 1925. Biology of population growth. New York. — 1928. The rate of living. New York. Peskett, G. L. 1924. Allelocatalysis and the growth of yeast. J. Physiol., SO} SSxiit: —— 1925a. Studies on the growth of yeast. I. Influence of the volume of culture medium employed. Bio-chem. J., 19: 464-73. —— 1925b. Studies etc. IJ. A further note on allelocatalysis. Bio-chem. J., 19: 474-76. — 1927. Studies etc. III. a further study on the influence of volume of media employed. Bio-chem. J., 21: 104-10. Peters, A. W. 1901. Some methods for use in the study of Infusoria. Amer. Nat., 35: 553-59. — 1904. Metabolism and division in Protozoa. Proc. Amer. Acad. Arts. Sci., 39: 441-516. — 1907. Chemical studies on the cell and its medium. Amer. J. Physiol., 17: 443-77 ;-18: 321-46. Peters, R. A. 1920. Nutrition of the Protozoa. J. Physiol., 54: L. 1921. The substances needed for the growth of a pure culture of Colpidium colpoda. J. Physiol., 55: 1-32. Petersen, W. A. 1929. Relation of density of population to rate of reproduc- tion of Paramecium caudatum. Physiol. Zodl., 2: 221-54. Phelps, A. 1931. Effects of H-ion concentration on the division rate of Paramecium aurelia. Science, 74: 395-96. — 1934. Studies on the nutrition of Paramecium. Arch. Protistenk., 82: 134-63. 1935. Growth of Protozoa in pure cultures. I. Effect upon the growth curve of the age of the inoculum and of the amount of the inoculum. J. exp. Zool., 70: 109-30. —— 1936. Growth etc. II. Effect upon the growth curve of different con- centrations of nutrient materials. J. exp. Zool., 72: 479-96. Phillips, R. L. 1922. The growth of Paramecium in infusions of known bacterial content. J. exp. Zool., 36: 135-83. Popoff, M. 1908. Experimentelle Zellstudien. I. Arch. Zellforsch., 1: 246- asthe: 1909. Experimentelle Zellstudien. I]. Arch. Zellforsch., 3: 124-80. Pruthi, H. S. 1926. On the hydrogen ion concentration of hay infusions with 562 GROWTH special reference to its influence on the protozoan sequence. Brit. J. exp. Biol., 4: 292-300. Reich, K. 1936. Studies on the physiology of Amoeba. I. The relation be- tween nutrient solution zone of growth and density of population. Physiol. Zo6l., 9: 254-63. —— 1938. Studies etc. I. The allelocatalytic effect in Amoeba culture free of bacteria. Physiol. Zodl., 11: 347-58. Richards, O. W. 1929. The correlation of the amount of sunlight with the division rate of ciliates. Biol. Bull. 56: 298-305. —— 1932. The second cycle and subsequent growth of a population of yeast. Arch. Protistenk., 78: 263-301. —— 1933. The toxicity of some metals and Berkefeld filtered sea water to Mytilus edulzs. Biol. Bull., 65: 371-72. —— 1934. The analysis of growth as illustrated by yeast. Cold Spring Harbor Symp. Quant. Biol., 2: 157-66. —— 1935. Analysis of the constant differential growth ratio. Pap. Tortugas Lab., 29: 173-83. Richards, O. W., and J. A. Dawson. 1927. The analysis of the division rate of ciliates. J. gen. Physiol., 10: 853-58. Richards, O. W., and T. L. Jahn. 1933. A photoelectric nephelometer for estimating population density of microdrganisms. J. Bact., 26: 385-91. Richards, O. W., and A. K. Kavanagh. 1937. The course of population growth and the size of seeding. Growth, 1: 217-27. Robertson, M. 1929. Life cycles in the Protozoa. Biol. Rev., 4: 152-79. Robertson, T. B. 1921a. Experimental studies on cellular multiplication. II. The influence of mutual contiguity upon reproductive rate and the part played therein by the “x-substance’’ in bacterial infusions which stimu- lates the multiplication of Infusoria. Bio-chem. J., 15: 612-19. -—— 1921b. The multiplication of isolated Infusoria. Bio-chem. J., 15: 595- Glie —— 1922. Reproduction in cell communities. J. Physiol., 56: 404-12. ——— 1923. The chemical basis of growth and senescence. Philadelphia. —— 1924a. The influence of washing on the multiplication of isolated Infusoria and upon the allelocatalytic effect in cultures initially contain- ing two Infusoria. Aust. J. exp. Biol. med. Sci., 1: 151-73. —— 1924b. Allelocatalytic effect in cultures of Colpidium in hay infusion and in synthetic media. Bio-chem. J., 18: 1240-47. —— 1927. On some conditions affecting the viability of cultures of Infusoria and the occurrence of allelocatalysis therein. Aust. J. exp. Biol. med. Sci., 4: 1-23. Rottier, P.-B. 1936a. Recherches sur les courbes de croissance de Polytoma uvella, L'influence de l’oxygénation. C. R. Soc. Biol. Paris, 122: 65-67. GROWTH 563 —— 1936b. Recherches sur la croissance de Polytomella uvella. L’influence de la concentration des substances nutritives. C. R. Soc. Biol. Paris, 122: 776-79. Roux, J. 1901. Faune infusorienne des eaux stagnantes des environs de Genéve. Mém. Inst. nat. genév., Vol. 19. 142 pp. Russell, J. 1937. A photoelectric cell circuit with a logarithmic response. Rev. sci. Instrum., 8: 495-96. Schmalhausen, I., and E. Syngajewskaja. 1925. Studien tber Wachstum und Differenzierung. I. Die Individuelle Wachstumskurve von Paramecium caudatum. Roux Arch, Entwkmech. Organ., 105: 711-17. Shapiro, H. 1935a. Nomogram for centrifugal speed. Industr. Engng. Chem., U3 235 —— 1935b. The validity of the centrifuge method for estimating aggregate cell volume in suspensions of the eggs of the sea-urchin Arbacia punctulata. Biol. Bull., 68: 363-77. —— 1937. The viscosimeter method for determination of cell concentration in suspensions of living cells. Anat. Rec., 70 (Suppl.) : 110. Simpson, J. Y. 1902. The relation of binary fission to variation. Biometrika, 1: 400-7. Smith, J. A. 1938. Some effects of temperature on the reproduction of Chilomonas paramecium. Biol. Bull. 75: 336-37. —— 1939. Effects of temperature on starch & fat in Chilomonas. Coll. Net, 1473-30: Snell, S. D. 1929. An inherent defect in the theory that growth rate is con- trolled by an allelocatalytic process. Proc. nat. Acad. Sci. Wash., 15: 274-81. Spurr, W. A. 1937. A graphic method for measuring seasonal variation. J. Amer. statist: Ass., 32: 281-89. Steinhaus, E. A., and J. M. Birkeland. 1939. The senescent phase in ageing cultures and the probable mechanisms involved. J. Bact., 38: 249-61. Stier, T. J. B., M. I. Newton, and H. Sprince. 1939. Relation between the increase in opacity of yeast suspensions during glucose metabolism and assimilation. Science, 89: 85-86. Sweet, H. E. 1939. A micro-population study of Evglena gracilis Klebs in sterile, autotrophic media and in bacterial suspensions. Physiol. Zodl., 12 173-200. Taylor, C. V., and A. G. R. Strickland. 1938. Reactions of Colpoda duodenaria to environmental factors. I. Some factors influencing growth and encystment. Arch. Protistenk., 90: 396-409. Teissier, G. 1928. Croissance des populations et croissance des organismes. Ann. Physiol. Physicochim. biol., 4: 342-86. ——— 1934. Dysharmonies et discontinuités dans la croissance. Paris. —— 1937. Les Lois quantitatives de la croissance. Paris. Tippett, L. H. C. 1932. A modified method of counting particles. Proc. roy. Soc., A137: 434-46. 564 GROWTH Unger, W. B. 1931. The protozoan sequence in five plant infusions. Trans. Amer. micro. Soc., 50: 144-53. Walker, H. H. 1932. Carbon dioxide as a factor affecting lag in bacterial cultures. Science, 76: 602-4. Wang, C. C. 1928. Ecological studies of the seasonal distribution of Protozoa in a fresh water pond. J. Morph., 46: 431-78. Wetzel, N. C. 1937. On the motion of growth. XVII. Theoretical foundations. Growth, 1: 6-59. Woodruff, L. L. 1905. An experimental study on the life history of hypotrichous Infusoria. J. Exp. Zool., 2:585-632. 1911. The effect of excretion products of Paramecium on its trate of reproduction. J. exp. Zool., 10: 557-81. 1912. Observations on the origin and sequence of the protozoan fauna of hay infusions. J. exp. Zool., 12: 205-64. 1913a. Cell size, nuclear size and the nuclear cytoplasmic relation during the life of a pedigreed race of Oxytricha fallax. J. exp. Zool., 15: 1-22. 1913b. The effect of excretion products on the same and on different species with special reference to the protozoan sequence in infusions. J. exp. Zool., 14: 575-82. Woodruff L. L., and G. A. Baitsell. 1911a. The reproduction of Paramecium aurelia in a “constant” culture medium of beef extract. J. exp. Zool., Lis 135-42. —— 1911b, The temperature coefficient of the rate of reproduction of Paramecium aurelia. Amer. J. Physiol., 29: 147-55. —— 1911c. Rhythms in the reproductive activity of the Infusoria. J. exp. Zool., 11: 339-59. Wright, E. V., and H. Kersten. 1937. An apparatus for measuring turbidity of bacterial suspensions. J. Bact., 34:581-83. Yocom, H. B. 1928. The effect of the quantity of culture medium on the division rate of Oxytricha. Biol. Bull., 54: 410-16. —— 1934. Observations on the experimental adaptation of certain fresh water ciliates to sea water. Biol. Bull., 67: 273-76. Zhalkovskii, B. G. 1938. The differences in biological action of transmitted and reflected light. I. Experiments with Paramecium caudatum. Bull. biol. exp. med. U. R. S. S. 493-95. From Chem. Abstr., 1939, 33: 2544. Ziegler, N. R., and H. O. Halvorson. 1935. Application of statistics to prob- lems in bacteriology. IV. Experimental comparison of the dilution method, the plate count, and the direct count for the determination of bacterial populations. J. Bact., 29: 609-34. GHAPTER XI THE RES GYGLE OF THE PROTOZOA CHARLES ATWOOD KOFOID INTRODUCTION THE ORGANISM has the fourth dimension of time. In the course of its life cycle, its three spatial dimensions change. The fourth changes also, interacting with the three. It may be measured by metabolic rate, by struc- tural results of growth, or by organismal cyclic changes which follow one after the other in sequences. These may be regular, interrupted, repeated, or in some other way responsive to or dependent upon internal environmental conditions, or to external conditions, such as changing quantity or quality of food supply; rise or fall in temperature, of seasonal origin, or due to migration; inciting or deterrent chemical or physical factors, such as pH, intensity and duration of light, and changes of host. The Protozoa* differ from the Metazoa because of their smaller size and the resulting more highly significant and potent surface-volume re- lations, as these affect the rate and intensity of the impact of environ- mental factors upon the organism and the changes they initiate and induce. It is therefore to be expected that the Protozoa will be relatively more susceptible to the modification of the individual and to the dis- tortion and interruption of its normal life cycle than are the Metazoa, thus obscuring and complicating the evidence of the existence of life cycles among them. The factors of time, volume, and season enter more or less definitely into the life cycles of Metazoa such as hydroids, flukes, tapeworms, crustaceans, insects, and tunicates. Among the Protozoa, on the other hand, the time units required for the various cyclic changes may be very brief, and these changes very often have little or no de- pendence upon cosmic cycles, with the result that the evidence of their * Assistance in preparing this chapter, rendered by the personnel of Work Projects Administration Official Project No. 65-1-08-113, Unit C1, is acknowledged. 566 THE EIBESCYGLE occurrence and continuity is more difficult to organize than is that for cycles in the larger, longer-lived Metazoa. Furthermore, a certain hesitancy about life cycles in the Protozoa has arisen historically because of the fact that skilled workers in this field have been caught in error by reason of the difficulties above noted. Following upon Biitschli’s (1876) and Hertwig’s (1889) fundamental analyses of conjugation in the Ciliata and its resemblance to maturation and fertilization in other organisms, there arose a Munich school of protozodlogists whose labors brought forth an array of protozoan life cycles fitted to the metazoan pattern. Under the brilliant leadership of Fritz Schaudinn, most of the major groups of Protozoa were subjected to this pattern of analysis, with resulting marvelous conformity to type. Some of these, notably those of Trypanosoma, Endamoeba, and Mastv- gella, have not stood the test of subsequent critical reexamination. Others, such as those of Plasmodium, Coccidium, and Paramecium, have, on the other hand, survived and have proved the validity of the basic assump- tion that there are life cycles in the Protozoa, though not necessarily all of the same type. The life cycle in the Metazoa starts with the diploid or polyploid zygote, a unicellular stage whose genes, derived from the haploid gametes, determine the characters of all of the varied subsequent stages unfolded in the ensuing life cycle. This cycle in many instances is marked by indirect development with one or more larval stages, followed by metamorphosis into the adult, sexual maturity, gametogenesis, senes- cence, and death. In other instances the development is direct, with adolescence replacing metamorphosis. In both types asexual repro- duction may intervene at different periods in embryonic, larval, and even adult life, giving rise by budding, binary and multiple fission, and sporulation to two or many different functional individuals, all with the original genetic constitution. Parthenogenesis may also intervene and alternate with normal sexual reproduction. There is often considerable change in the external appearance of the successive stages, as in larva, pupa, and imago of the Lepidoptera, though a striking similarity, even continuity, may occur in various organ systems from stage to stage. The stages occurring in the metazoan life cycle are brought about by the processes of cleavage, gastrulation, organogenesis and histogenesis, growth, adolescence or metamorphosis, gametogenesis, senescence, and THEY BIBE(CYGLE 567 death. Asexual reproduction may be interjected into the midst of any one of these processes, resulting in from one to many repeated genera- tions of functional individuals. Not infrequently these individuals are heterogonous, with marked differences in structure from the parent, as for example in the larval stages of the Trematoda. This alternation of sexually and asexually produced generations is widely distributed in the living world, ranging from some of the lower algae to the Quints. The ease with which regeneration occurs after mutilation and with which experimental asexual multiplication of func- tional individuals may be imposed upon the genetic individual is indica- tive of the fundamental organic basis of asexual reproduction, perhaps as a corollary of the still more fundamental capacity of growth on the part of the organism. The Protozoa, from the evolutionary point of view, are of excep- tional interest among the phyla, since it is among these primitive organ- isms that most of the basic biological properties, structures, and functions of the organism have had their evolution. Within these microcosms all of the basic functions of living must be performed. As one surveys their diversities and complexities of pattern, one is impressed with the evidence that among these minute organisms, adapted to so many eco- logical niches and exhibiting so many types of behavior, a vast deal of evolutionary experimentation has been enacted. It is among the Protozoa and Protophyta that the following have been evolved: nuclear structure, sex, sexual dimorphism, sexual reproduction, mitosis, chromo- somes, gametogenesis, histogenesis, multicellularity, sex and somatic cells, asexual reproduction by the various methods of binary and multiple fission, budding and sporulation, and the beginnings of the organization of organ systems. Varying combinations and sequences of these evo- lutionary accomplishments are exhibited in the diverse patterns of life cycles to be detected among the Protozoa. Cycles of comparable type, in some instances apparently independently of one another, have emerged to a varying degree in the different classes and orders of Protozoa. These cycles fall into two major groups. The first is the simpler and the more primitive. It consists merely of recurrent rhythms of homo- geneous asexual reproduction, in which mitosis produces a multicellular (== multinuclear) body of from two to many cells, forming a plas- modium, coenobium, sporocyst, or cyst. Fission of binary, multiple, or 568 THEO IRE ICYGLE budding type breaks up this body into functional individuals of the ancestral type. Although nuclear division is essential in the accomplish- ment of this cycle, it does not initiate it. This is shown strikingly in the Polymastigophora, in which the entire neuromotor complex of centro- some, blepharoplast, flagella, undulating membrane, and axostyle of the individual is duplicated by new growth, accompanied by extensive dedifferentiation of the parental equipment before the nuclear phe- nomena of mitosis ensue. Asexual reproduction is thus profoundly an organismal phenomenon involving a rejuvenation of the organelle sys- tems of the body of the individual. This type of life cycle seemingly exists without any evidence of sex, sexual reproduction, or sexual dimorphism. Efforts to establish sexuality on the basis of the relative size of supposedly male and female individuals and upon interpretations of behavior are will-o’-the-wisps of wishful thinking. The only basis is gametogenesis, verified by reduction of the diploid to the haploid number of chromosomes, and fertilization, with the resulting return to the diploid state. The juxtaposition or even fusion of motile individuals among flagel- lates and rhizopods may occur when adverse conditions or internal states induce an adhesive periphery; sometimes cannibalistic feeding of rhi- zopods resembles fusion; and changes in position from divergence to lateral contact in sister schizonts among Mastigophora resemble conjuga- tion, all of which evidence is never to be accepted as sexual behavior unless confirmed by critical cytological evidence. The not uncommon opinion that sex is an inherent characteristic of organisms and that sexual reproduction is to be expected in all animals and plants and even in bacteria, is as yet without convincing cytological evidence among the more primitive forms. It has, however, been clearly demonstrated in the Sporozoa, Ciliata, Foraminifera, and the Volvocidae, all representatives of the more highly evolved Protozoa. The present evidence, negative though it be, lends support to the view that sex was evolved in the Protozoa, perhaps independently in the different classes. It may well be that its origin rests ultimately on differential metabolism within the species, leading in time to more favorable condi- tions for permanent fusion of gametes, though this alone makes no provision for gametogenesis. The fact that some flagellates and rhizo- pods have an odd number of chromosomes suggests that they are not THE EEE CYCLE 569 zygotes nor derived from zygotes, but primitive haploids. While it is to be expected that the cases of critically proved instances of sexual reproduction will increase in both number and systematic range with further investigation, even this will be far from establishing the unt- versality of sexual differentiation among the Protista. Haploid (odd) numbers of chromosomes in primitive species will still require a solu- tion. There are three chromosomes in Trichomonas buccalis (Hinshaw, 1926) and five in lodamoeba biitschli, according to unpublished obser- vations made in my laboratory by Dr. Dora P. Henry. In the absence of sex and sexual reproduction among primitive Proto- zoa, this first type of a merely asexual life cycle is the only one feasible. It is, however, incorporated into the second type of cycle, in which it alternates in varying irregularity with sexual reproduction and may even exhibit several forms with structurally different functional individuals within the same cycle, as in Plasmodium. ASEXUAL REPRODUCTION IN ALTERNATING BINARY AND MULTIPLE FIssION (TYPE I) An example of the first type of the protozoan life cycle among the Mastigophora is seen in Trichomonas augusta, in which asexual repro- duction by binary fission prevails, but 1s interrupted at unknown inter- vals by the formation of an eight or sixteen-celled somatella with a common cytoplasm, each cell of which has its own neuromotor ap- paratus. Within this plasmodium paired schizonts, temporarily joined to each other by the paradesmose, ceaselessly tug at this tether until they are disunited except by the common cytoplasm. Serial plasmotomy releases each schizont, to start again the cycle with binary fission. There is in this type of cycle no clue to sexual reproduction. Another example from the Rhizopoda is found in Cowncilmania la- fleuri, usually called Endamoeba coli, in which there is an alternation between a unicellular free motile phase and a multicellular encysted one. During the motile phase binary fission prevails, and reversion to the unicellular condition follows each mitosis. This is interrupted from time to time by the encysted phase, in which, following reduction in volume, the body rounds out and secretes about itself an impervious membrane or cyst wall of elastin, with a differentiated exit pore closed by a plug. Encystment follows feeding and the accumulation of food reserves, which Multiple frssion Figure 135. Diagram of the life cycle of Trichomonas augusta from the intestine of the frog, Rana boylei, including an alternation of binary fission of the two-cell soma- tella and of multiple fission of the eight-cell plasmodium. THE, LIFES CYCLE 71 in the cyst are stored in a large central glycogen vacuole. The glycogen is transformed into chromatoidal structures of unknown composition, staining deeply and formed on the surface of the glycogen vacuole. These progressively disappear as mitotic divisions ensue. Soon after encystment is completed, a series of mitotic nuclear di- visions occur, resulting in two, four, eight, and sixteen-cell stages, rarely thirty-two-cell, and in one observed instance approximately a sixty-four-cell stage, thus running the rhythm of normal cell division in a metazoan egg. Plasmotomy, however, does not attend the nuclear divisions. Cyst formation, in this instance, serves the function of assimila- tion and growth. Measurements of cysts in the one, two, four, eight, and sixteen-cell stages show a slight progressive increase in diameter. Excystment occurs normally in the bowel, as shown by the occurrence of cysts free from glycogen or chromatoidals, with reduced numbers of nuclei from fifteen down. It can also be followed in fresh stools, as the small mononucleate amoebulae escape singly out of the exit pore. Excystment is a form of asexual reproduction, of budding, or progres- sive multiple or serial fission. In this type of life cycle we find an alterna- tion of a unicellular free phasé with reproduction by binary fission, with the formation of a multicellular encysted somatella, with reproduction by multiple fission and a return to the unicellular motile phase. ALTERNATION OF ASEXUAL AND SEXUAL REPRODUCTION (TYPE II) The second major type of the protozoan life cycle is that in which asexual and sexual reproduction alternate. It may or may not be accom- panied by sexual dimorphism, as exhibited by differential reaction to aniline stains in Nina, by structural differentiation of gametes in Evmeria, of gametocytes in Plasmodium, or of conjugants in Vorticella. It seems probable that sex has become a genetic characteristic of the individual throughout the whole cycle, in all life cycles having sexual reproduc- tion, even though structural features indicative of sexual dimorphism cannot be detected. From the biological point of view, it is unfortunate that the life cycles of parasitic Protozoa have been arranged, in illustrations, in se- quences as parasitic cycles, rather than biological life cycles. They are usually designated as beginning with the infection of the host, or in the case of a parasite with two hosts with that of the primary host, or SF Le cap &. -Pseudopodium Motte phase Binary fission --4-Peripheral is chromatin \-Glycogen vacuole Encysted phase Multiple serial f/S5/0N Figure 136. Diagram of the life cycle of Exdamoeba coli (= Councilmania lafleuri, Kofoid and Swezy, 1921) from the intestine of man, including an alternation of binary fission in the motile phase and of budding, or serial multiple fission, of the eight-cell encysted somatella. THE sLIBEP Gy CLE Bile, with that of the more significant host, for example, with the infection of man in the case of Plasmodium. The psychological effect of this is to deflect interest from the significant biological aspects of such cycles. In order to follow these life cycles in their true biological sequences, we have rearranged them and will now proceed to discuss three of the most widely known ones, viz., those of Ezmeria, Plasmodium, and Para- mecium. THE LIFE CYCLE OF Esmeria schubergi The life cycle of Evmeria schubergi, a parasite in the intestinal epi- thelium of Lithobius forficatus, is a typical one with an alternation of asexual and sexual reproduction, and of a sexual phase with asexual ones. In this cycle no less than five different structural types of functional individuals appear, each with a distinctive pattern of shape, size, struc- ture, and activity. Four of the five appear but once, but one is subject to numerous repetitions under favorable conditions. As rearranged, the biological cycle begins with the zygote formed in the lumen of the intestine of the host by the fusion of a flagellated spermatozoan with a yolk-laden egg, recently emerging from an intestinal epithelial cell of its host. Even before the pronuclei fuse, the fertilized egg forms a fertilization membrane and secretes a cyst wall around its spherical body (Fig. 137, 1). Two nuclear divisions bring the organism to the cleavage stage of a four-celled somatella, the sporoblast. There- upon there ensues the first asexual reproduction, when this somatella divides into four unicellular spores. Unlike their spherical parent, these functional individuals are ellipsoidal, and they, too, secrete about their respective bodies a resistant ellipsoidal spore wall. There then ensues the second asexual reproduction, when each spore cell divides into two spindle-shaped, naked unicellular sporozoites, re- tained within the spore case and the enveloping cyst wall of the sporo- blast. At about this stage of the cycle, the sporocyst with its eight sporo- zoites in four spores, is discharged from the intestine of its host, and further development ceases until this infective stage is eaten by a Litho- bius. Here the digestive fluids unstopper the cyst, the sporozoites are released from the spores, each escapes singly through the pore and enters an epithelial cell of the intestine of the host where it develops as a trophozoite, changing in pattern from a spindle shape to a spherical one. The organism at this stage is devoid of any special protecting cover and Crow th EDD Sere 3 Asexual reproduction cycle xN és ARES \ Be A ut 2”? Asexual reproduction Lt Asex ual reproduction Figure 137. Diagram of the alternating sexual and asexual reproduction in the life cycle of Eimeria schubergi, from the intestine of Lithobius forficatus. For convenience, the first two asexual divisions within the sporoblast cyst wall are figured in the sexual cycle. They are transitional to the repeated multiplicative multiple fissions attending the infection of the intestinal cells of the host. Owing to post-zygotic reduction, both phases are haploid except for the diploid zygote. (Modified from Schaudinn, 1900.) HEE LiPEAG VCLE 575 grows into a somatella of about sixty-four cells, in a rhythm of repeated mitoses. Then follows the third asexual reproduction, in which the organism divides by plasmotomy into motile unicellular spindle-shaped merozoites, similar in size and pattern to the sporozoites. These in turn infect other intestinal epithelial cells, and this phase of the cycle is repeated an un- known number of times so long as susceptible host cells are available for infection. This phase ends the asexual part of the life cycle, as gametogenesis approaches, except in the male. The sexual phase is marked by developing sexual dimorphism among the merozoites. Presumably sex is determined at fertilization, and all functional individuals derived from one zygote will accordingly be of one sex only, and the myriapod host must have acquired an infection by spores of each sex, in order that both male and female gametes of Eimeria, fertilization, and spore formation may ensue in the intestine. The sexual dimorphism of the gametocytes is determined by two factors, the metabolic rate and probably also the chemical nature of the food reserves on the one hand, and the structure and number of gametes pro- duced on the other. Both male and female gamete mother cells grow to the size and spherical form of the trophozoite, but do not run its type of rhythm of cleavage, mitoses, and plasmotomy. The female 1s early differentiated from the male by the internal elaboration of spheri- cal granules of food reserves or yolk, whereas in the male none appear. This functional and structural dimorphism is accompanied by a differ- ence in nuclear behavior. In the male there appear to be as many as six successive mitoses, as in the trophozoite, producing up to sixty-four gametes. These are elongated slender, deeply staining bodies, largely of nuclear substance, with one trailing flagellum and a second one lat- erally attached to the anterior half of the body and free posteriorly. The female gamete mother cell, on the other hand, undergoes no divi- sions, and transforms directly into the egg, though indications of metab- olic activity appear in deeply staining spherules adjacent to the parasite in the host’s cytoplasm. This absence of divisions in the female gameto- cyte, and their superabundance in the male, not only emphasizes a metabolic contrast, but also on cytological grounds offers cytological difficulties to the existence of maturation in these phases. These ob- stacles, which Schaudinn (1900) left unresolved, were removed by the 576 THE AEIBPENGY GEE discovery by Jameson (1920)—later extended by Dobell (1925), Na- ville (1931), Yarwood (1937), and Noble (1938)—that the matura- tion division takes place in the first division of the zygote and that, aside from the diploid zygote itself, the rest of the cycle is a haploid one. It is obvious that in the case of the male there is a fourth asexual reproduction by multiple fission of a sixty-four-celled somatella, and that this does not occur in the female. This life cycle is typical in having an alternation of sexual and asexual reproduction upon which are superposed certain features, in part adap- tive and in part more fundamentally a part of the cycle. The first of these features is the building up of multicellular somatellas numbering respectively four, two, + sixty-four (x n), and + sixty-four in male cells only, prior to multiple fission. The body thus formed is temporary, lacking both nervous and hormonal mechanisms of integration to insure the maintenance of interacting relations. The adaptive aspect lies in the fact that these multiplicative reproductions make possible, with the least expenditure of individuals, the quick utilization of the food supply in the host’s intestinal cells. This cycle from the cytological point of view, as well as the general biological one, is atypical in the animal kingdom, though less so in the plant kingdom, in that only the zygote is diploid and all of the rest of the cycle is haploid. The fact that other Coccidiomorpha are known to have the same limitation and that this subclass has affinities with the flagellates, in some of which an odd number of chromosomes are known, suggests that the primitive Protozoa are haploid and that the diploid phase, like the polyploid, is a secondary evolutionary acquisition, de- pendent, in part at least, on the union of individuals or gametes in sexual reproduction. Thus both sex and sexual reproduction have had their origin in the Protozoa. The limitation of the diploid phase to the zygote only in Evmeria thus has a basic evolutionary significance. Another feature of this life cycle which also has a basic significance is the fact that every one of the four phases of asexual reproduction results in the formation of a somatella of from two to sixty-four cells, and that the sexual phase also leads to a four-celled somatella. This evidence clearly indicates that these Protozoa are as truly multicellular, as are the early stages of the Metazoa. They undergo asexual reproduction as do Metazoa, from Porifera to Quints, but with this difference: that the U shicn \/ 27 Asexual” Wy Reproduction Jy 14 Pa f & Wh [r IN tS 1502] Asexual (2 Reproduction Para Min Ne ) eS NWS GO Hy SX W We X= ee uu NV /N MAN SeSe52 /N ANOPHELES Figure 138. Diagram of the life cycle of Plasmodium vivax, parasitic in Anopheles and in the red cells of man. For convenience, the asexual reproductions of the sporoblast are figured in the sexual cycle, since they are preparatory to the repeated multiple fissions of the asexual cycle in man. By analogy with E/meria, maturation is post-zygotic and both sexual and asexual phases are haploid except for the zygote. There is possibly an asexual reproduction. (Modified from Schaudinn, 1902.) 578 THE LIFE ICYCEE units into which they split are single cells, instead of flagellated cham- bers or axial organizers. THE LIFE CYCLE OF Plasmodium vivax A second example of the same general pattern, with added speciali- zations due to parasitism in two hosts, is found in the malarial parasite, Plasmodium vivax, with the sexual and one (or two?) asexual phases in the mosquito, Anopheles, and oft-repeated merogony in the red cells of the blood of man, and a second asexual phase in the gamete mother cells of the male only on transfer of these cells to a lower temperature than that of the blood of man, as on a microscope slide or in the stomach of the mosquito. THE LIFE CYCLE OF Paramecium caudatum The life cycle of Paramecium caudatum makes a definite evolutionary advance in the Ciliata in two mutually interdependent features. The first is the differentiation of sex and somatic cells in the same individual, and the second is a permanent multicellular condition of two cells, de- rived from an undifferentiated eight-celled cleavage stage. The original description of the cell, the selection of its name, the focusing of attention on total cleavage, with plasmotomy in embryology, rather than upon mitosis, all have combined to emphasize the separation of one cell from another by a wall or dividing structural boundary. These are all minor considerations. On the other hand, the significance of derivation, continuity in time, physiological functions, and above all of genetics, focus attention on the nucleus and the cytoplasm associated with it or brought in in the normal sequence of growth, fertilization, appropriation, or experiment under its control. These are all major con- siderations. In this modern sense it is biologically medieval to refer, as do many textbooks and other works, to Paramecium as a unicellular organism. It is biologically quite as logical to call a whale unicellular. Both start their cycles as one cell and both achieve multicellularity. No great biological significance attaches to the particular number of cells in the multicellular body, except during maturation. The significant achievement is the differentiation of sex and somatic cells. One of the primary distinctions in function, as well as in embryological origin, in the multicellular metazoan is this differentiation. Cc uU V Asexual cycle xN Binary fission x] Fertilizatio | I Figure 139. Diagram of the life cycle of Paramecium caudatum exhibiting an alterna- tion of asexual reproduction, or binary fission of the two-cell somatella, and sexual re- production with mutual fertilization of conjugants. The first and second divisions of the eight-cell somatella, or exconjugant, are for convenience included in the diagram of the sexual cycle, since they are preparatory to the asexual phase. 580 THE LIRE, GYGLE The life cycle of Paramecium is further complicated by the fact of conjugation and mutual fertilization of the conjugants. Sexual dimor- phism is not evident between the conjugants as a whole, but appears in the behavior of the gamete nuclei. The migrant one is assumed to be male because of its motility, and the resident one female because of the lack of this quality. Dimorphism of the conjugants is structurally evi- dent in Vorticella, in which the males are small and the females large. The biological life cycle in Paramecium starts with the zygote, formed from the body of a conjugant by the fusion of the two haploid nuclei, one from the immigrant male gamete and the other the resident nucleus of the egg. The cleavage nucleus thus formed utilizes the cytoplasm of the egg, as in the Metazoa, with only a small amount from the male gamete. The old macronucleus in each continues (Fig. 139, I-IV) to disintegrate and is soon entirely metabolized into cytoplasm as food. This is the death of the soma of the conjugant, the future of which ts henceforth under a new genetic control. There then ensue three succes- sive mitotic divisions (Fig. 139, II-VI), representing the cleavage of the egg to an eight-celled somatella, when cleavage abruptly stops (Fig. 139, VI) and differentiation into four somatic and four sex cells occurs by the enlargement of the nuclei of the former and an increase in their chromatin. The four sex cells do not all survive. Three of them dis- integrate at once, leaving a somatella of five cells, four somatic and one sex cell. Then begins asexual reproduction which in two peculiar binary fissions distributes the four macronuclei among the four daughter schi- zonts, with an accompanying division of the sex cell or micronucleus at each of the two fissions. In the diagram these two asexual fissions have been included in the sexual cycle, since they are necessary to restore the organism to the pattern in which regular asexual reproduction prevails. They otherwise belong in the asexual period. The precise period in which maturation occurs in the sexual cycle ts perhaps undetermined. It has been generally assumed that it occurs in the first two divisions of the micronucleus in the conjugant, in which case its third division would be an asexual reproduction of the gamete. This view does not rest upon exact chromosome count. The occurrence of post-zygotic maturation in the Sporozoa suggests the possibility of its occurrence in the Ciliata also. This view is further supported by the death of three of the post-zygotic sex nuclei and in Paramecium by the un- THES PIRE CYCLE 581 necessary third mitosis of the pronuclei in the conjugants. The evidence for chromosome reduction in the conjugants (Calkins and Cull, 1907) is inconclusive, because of the small size and the large number of the chromosomes in the three divisions prior to the formation of the zygote. A cytological examination of chromosome number during conjugation, in some ciliate with a small number of large chromosomes, may throw light on this problem. Both pre and post-zygotic alternatives should be explored. The asexual cycle proper of Paramecium is one of oft-repeated simple binary fission, prior to which the two-celled somatella may grow, and by nuclear division become a four-celled one for a brief period. This cycle is one of indefinite duration. This survey and interpretation of life cycles among the Protozoa ex- hibit the basic similarity of this fundamental characteristic of organ- isms among them to those emergent among the Metazoa. Peculiar to the Protozoa is the absence of sexual reproduction in the life cycles of the more primitive forms, among which it appears that they live a haploid life and that sexual reproduction has not as yet been evolved. Rare cases among Metazoa and the Metaphyta of the seeming absence of sexual reproduction are obviously secondary phenomena, but this interpretation is less defensible for the primitive Protozoa. In the higher Protozoa, as in the Metazoa, the life cycle includes maturation, fertilization, cleavage to a multicellular stage, histogenesis of organelles, asexual reproduction with resulting functional individuals of differing structure in the different asexual phases, sexual dimorphism, adolescence, gametogenesis, senescence, and death. The emphasis so generally placed upon the unicellular phase of the Protozoa, as against all Metazoa, tends to obscure their basic similarity in life cycle to that of the Metazoa, and thus to minimize the biological significances of the varied evolutionary accomplishments which have oc- curred in this primitive phylum. Similarities in biological phenomena are the bases on which an integrated concept of the evolution of life can be erected. LITERATURE CITED Biitschli, Otto. 1876. Studien tiber die ersten Entwicklungsvorginge der Eizelle, die Zelltheilung und die Conjugation, Abhl. senckenb. naturf. Ges., 10: 213-452, 15 pls. 582 THE LIE FeYCLe Calkins, G. N., and Sarah W. Cull. 1907. The conjugation of Paramoecium aurelia (caudatum). Arch. Protistenk., 10: 375-415, pls. 12-18. Dobell, Clifford. 1925. The life-history and chromosome cycle of Aggregata eberthi, Parasitology, 17: 1-136, 6 pls., 3 figs. in text. Hertwig, Richard. 1889. Uber die Conjugation der Infusorien. Abh. bayer. Akad. Wiss., Math.-Natur. KI., 17: 151-234, 4 pls. Hinshaw, H. C. 1926. On the morphology and mitosis of Trichomonas buccalis (Goodey) Kofoid. Univ. Cal. Publ. Zool., 29: 159-74, 1 pl. 2 figs. in text. Jameson, A. P. 1920. The chromosome cycle of gregarines, with special refer- ence to Diplocystis schneideri Kunstler. Quart. J. micr. Soc., London, 64: 207-66, pls. 12-15. Kofoid, C. A., and Olive Swezy. 1915. Mitosis and multiple fission in trichomonad flagellates. Proc. Am. Acad. Arts and Sci. Wash., 51: 290-371, 8 pls., 7 figs. in text. — 1921. On the free, encysted, and budding stages of Councilmania lafleuri, a parasitic amoeba of the human intestine. Univ. Cal. Publ. Zool., 20: 169-98, pls. 18-22, 3 figs. in text. Naville, André. 1931. Les Sporozoaires (cycles chromosomiques et sexualité) . Mem. Soc. Phys. Genéve, 41: 1-223, 3 tables, 150 figs. in text. Noble, E. R. 1938. The life cycle of Zygosoma globosum, sp. nov., a gregarine parasite of Urechis caupo. Univ. Cal. Publ. Zool., 43: 41-66, pls. 7-10, 3 figs. in text. Schaudinn, Fritz. 1900. Untersuchungen uber Generationswechsal bei Coccidien. Zool. Jb. Abt. Anat., 13: 199-292, pls. 13-16. — 1902. Studien tber krankheitserregende Protozoen. II. Plasmodium vivax (Grassi and Feletti) der Erreger des Tertianfiebers beim Menschen. Arb. GesundAmt. Berl., 19: 169-250, pls. 4-6. Yarwood, Evangeline A. 1937. The life cycle of Adelina cryptocerci sp. nov. a coccidian parasite of Cryptocercus punctulatus. Parasitology, 29: 370-90, pls. 15-19, 1 fig. in text, CHAPTERS XIE FERTILIZATION IN PROTOZOA JOHN P. TURNER MUCH HAS BEEN WRITTEN on the phenomena which accompany fertili- zation in the Protozoa. For detailed analyses of this literature the reader is referred to texts by Minchin (1912), Doflein-Reichenow (5th Ed. 1929), and Calkins (1933). The aim of this chapter is to give a bird’s- eye view of the subject, to present some of the more significant facts al- ready discovered, and last and most important to point out the need for investigation to determine those facts and principles still awaiting dis- covery. If we consider sex to be essentially the formation of gametes and the fusion of those gametes in the fertilization process, we are using the term sex in a somewhat broader sense than if we limit it to the differ- ence or distinction between the two sexes. In the Metazoa these phe- nomena seem to be fairly uniform for all groups; consequently, when the basic principles of the process are understood for one animal, those same principles may be applied to all the higher animals. Until very recently, however, the Protozoa were thought to belong in a different category, and one did not apply to them the general laws which were considered applicable to all other animals. With recent discoveries, more and more of these preconceived dif- ferences have disappeared and we are now faced with the question of how close we can draw the analogies in sex phenomena between the Protozoa and the Metazoa. In other words, are the fundamentals of sex, 1.e., maturation of gametes and fertilization, common to all animals, both metazoan and protozoan? If so, how similar or how dissimilar are the processes, and if not, just how do they differ? No final answer can be given to these questions in our present state of knowledge, but con- siderable evidence may be pointed out that is extremely significant. In the Metazoa fertilization is accomplished by a small, active micro- gamete (spermatozo6n) penetrating and fusing with a large, nonmotile 584 FERTILIZATION macrogamete (ovum). The difference in appearance between the male and the female gametes, as well as between the two kinds of animals which produce them, is so clearly recognizable in most cases that we have come to think of sex in terms of the differences between maleness and femaleness. Among the Protozoa we find some species which also show a clear differentiation between male and female gametes, even though the sex differences between the organisms producing them are not so apparent. The protozoan organism is unicellular, and in many cases this single cell produces both male and female gamete nuclei in a kind of hermaphroditism (e.g., wandering and stationary pronuclei in ciliates). This complication makes the homologies between Metazoa and Proto- zoa less easily understandable. In a great many Protozoa there is no apparent differentiation between gametes, yet their formation and fusion are accompanied by the same fundamental processes as is the case with differentiated gametes. Isog- amous reproduction, therefore, is considered a sexual process. The difference between the individual and the gamete is not always clear in Protozoa. Perhaps the most primitive kind of sexual phenomena is exemplified by two Protozoa, apparently identical to the vegetative forms, coming together and fusing in a fertilization process. According to Dobell (1908), this occurs in the flagellate Copromonas subtilis. Nuclear “reduction” occurs after partial fusion of the cell bodies and before nuclear fusion (Fig. 140). COPULATION GAMETIC MEIOSIS AND FERTILIZATION Copulation, the complete and permanent fusion of gamete cells, is the type of sexual activity found generally in the Plasmodroma. In cases in which the parent organism gives rise to specialized cells which per- form in fertilization, the process is known as fertilization by union of gametes, or simply gamogamy. Both isogamy, the union of similar gametes, and anisogamy or heterogamy, the union of dissimilar gametes, are found in this group. In cases in which gametes are as extremely dis- similar as spermatozoa and ova, the union is sometimes referred to as odgamy. In cases in which the organism itself fuses with another or- ganism in permanent union, the whole organism functions as a gamete and the process is called hologamy. FERTILIZATION 585 In all of these cases, presumably, maturation of nucleus occurs to prepare it for union with its mate. In most cases maturation takes place in the last two divisions of the nucleus, before formation of the fusion nuclei or pronuclei. These two meiotic divisions are similar to those characteristic of spermatogenesis and odgenesis in Metazoa (see Sharp, 1934). The result of meiosis is the halving of the chromosome number to the haploid condition, so that fusion of the two haploid Figure 140. Copromonas subtilis in hologamous copulation. A, vegetative form; B, two individuals beginning to fuse anteriorly; C, cytoplasmic fusion well under way, nuclei in heteropolar, second ‘‘reduction” division; D, zygote with synkaryon and single flagellum. (After Dobell, 1908.) gametes will reéstablish the diploid number which is characteristic of the species. In the Mastigophora, syngamy has been described in very few forms except among the Phytomastigina, in which it seems to be the general rule. A typical case of hologamous copulation was described by Dobell (1908) in the colorless phytomonad Copromonas subtilis (Fig. 140). Two organisms which appear identical to each other and to ordinary vegetative forms come together and partially fuse. This partial union evidently acts as a stimulus to the nucleus of each gamont, for it pro- ceeds to undergo two “maturation divisions’ before fusing with its mate. Of the two products of the first progamic division, one degenerates and 586 FERTILIZATION the other divides again into two very unequal parts. The smaller de- generates and the larger is the functional pronucleus, which fuses with the pronucleus of the other member of the pair. Superficially, this type of reduction is strikingly similar to polar body formation in metazoan odgenesis, although the actual reduction in chromosome number was not established by Dobell. The assumption that the two nuclear divisions preceding syngamy are reduction divisions seems reasonable, consider- ing the almost universal occurrence of two divisions in the maturation of gamete nuclei. However, we are not justified in concluding that re- duction in chromosome number occurs, unless positive determinations can be made of the chromosome number before and after reduction. In Copromonas there is obvious reduction in the amount of chromatin when all but one product of the two divisions disintegrate; but reduc- tion in chromosome number has not been demonstrated, although it must occur somewhere in the life cycle, if chromosomes exist in this species. From the genetic angle, this is a critical point and should be determined if possible. After syngamy the zygote may encyst or it may develop directly into a vegetative form. Hologamous fertilization has been reported in a few other members of the Plasmodroma, but is not confined to this group if Brumpt’s (1909) description of fertilization in the parasitic ciliate Balantidium coli is correct. In this case two individuals come together and are in- vested by a common membrane, as in pseudoconjugation of gregarines. But the two balantidia then fuse completely and permanently. Other workers have not supported Brumpt’s description and, although the details differ with species and author, Jameson (1927), Scott (1927), and Nelson (1934) agree that conjugation and not copulation is the form of sexual union found in this ciliate. The occurrence of fertilization has been reported for a number of flagellates, but in very few cases has the evidence been convincing ex- cept for the Phytomonadida. Goldschmidt (1907) gave a detailed de- scription of a sexual cycle in the animal flagellate Mastzgella, similar in type to those described for Arcel/a and other Rhizopoda. If substanti- ated, another close link between the Pantostomatida and the Rhizopoda will be established. According to Goldschmidt, vegetative forms de- velop into macrogametocytes and microgametocytes, the nuclei of which give off chromatin into the cytoplasm. These chromidia in turn produce FERTILIZATION 587 secondary nuclei, each of which appropriates some cytoplasm, forms a gamete, and undergoes reduction. Active flagellated macrogametes seek out and fuse with nonmotile microgametes, in contrast to the usual method. The zygote retains the flagellum and reproduces its monad-like self for several generations by fission. Then the offspring develop into the adult vegetative Mastzgella. Heterogamy seems to be clearly established in this amoeboid flagellate by the differences in the activity of the gametocytes as well as in the dif- ferences in size and motility of the gametes. Another point of interest is the pedogenic reproduction of the zygote. The interpolation of this asexual cycle into the life cycle bears a strong similarity to sporogony in Plasmodium and other Sporozoa. In both cases it is the asexual multi- plication of the zygote before the adult stage, or trophozoite, is formed. A point of particular interest to cytologists and geneticists alike is the origin of the gamete nuclei from chromidia. Not only here but also in a number of the Sarcodina, the origin of gamete and vegetative nuclei from chromidia has been reported. If the chromidial origin of nuclei is a fact, what of the genetic and structural continuity of the chromo- somes and of the genes? Must we accept Hartmann’s (1911) ‘“‘poly- energid”’ interpretations that the nucleus is really an aggregate of many small nuclei, each with its sphere of influence, and that chromidia repre- sent the scattered little nuclei or energids? At present we can only specu- late. The problem is one of fundamental significance and is in great need of further investigation. Chatton (1927) described a case of gametic meiosis in the flagellate Paradinium poucheti which is ‘exactly comparable in its progress and complexity’’ to spermatogenesis in certain insects. Included in his out- line of this process are leptotine and pachytine stages, and diakinesis with tetrad rings and crosses. It is surprising that such highly developed processes should be found in such a primitive flagellate, although similar stages are not uncommon in ciliates and also occur in some Sporozoa and Sarcodina. The wide distribution of typical meiotic phenomena indi- cates that they are fundamental in nature, and it is probable that proc- esses of comparable nature also occur in all forms in which fertilization takes place. Meticulous examination with improved techniques will throw much light on this question. In the Dinoflagellata a few cases of syngamy have been reported, but 588 FERTILIZATION most of them are too fragmentary or are supported by too little evidence to be discussed here. An extremely interesting case has recently been reported by Diwald (1938) in Glenodinium lubiniensiforme. In this form, four flagellated isogametes are produced by the subdivision of each parent protoplast, and these gametes will copulate only with gametes of certain other clones. The obvious question is, of course, what is the nature of the difference between these ‘“-” and ‘‘—”’ strains, a difference which in- hibits their copulating among themselves or stimulates them to copulate with gametes of the other strain? This problem is yet to be solved, but it is similar to that found in Paramecium aurelia by Sonneborn (1937) and in P. bursaria by Jennings (1938). Diwald states that after fertili- zation the zygotes rest, then germinate and undergo two reduction divi- sions to form a ‘‘tetrad”’ of four potential individuals, only one of which, however, usually persists. This is the only described case of zygotic reduction outside the Telosporidia, with the possible exception of the amoeba Sappinia diploidea (see p. 595 below). A reasonable doubt re- mains, however, as to Diwald’s interpretations. He gives no chromo- some counts that would support his contention, and any assumption of chromosome reduction not based on determinations of chromosome numbers before and after reduction, especially in such an unusual case, is Open to serious question. Dinoflagellates are not popular subjects for cytological investigation at the present time, but perhaps the work of Diwald will stimulate further research in this group. In Ceratium hirundinella fertilization is accomplished in a way simi- lar to that of the filamentous algae, according to the description of Zeder- bauer (1904). Two flagellates come together, the protoplasm of each extrudes from the lorica and makes contact with that of the other. The two masses now copulate, forming a zygote outside the loricas. Zeder- bauer observed these protoplasmic fusions only in the living state, so his account leaves much to be desired in the way of cytological details on which to base sound conclusions. Chatton and Biecheler (1936) have more recently reported fertili- zation by slightly anisogamous gametes in the parsitic form Coccidinium mesnili. In Noctiluca scintillans (miliaris) gamete formation has been re- peatedly reported. In recent accounts Pratje (1921) could find no FERTILIZATION 589 conclusive evidence for copulation, while Gross (1934) described copu- lation of isogametes. It seems odd that the life history of this abundant and spectacular species should still be a matter of such uncertainty. Sexual reproduction is widespread among the Phytomonadida. These plant-like flagellates illustrate so nicely the gradations in sexual develop- ment and differentiation that they have been favorite material for class- room instruction. 2 Chlamydomonas is a non-colonial genus among the species of which both isogamy and heterogamy are found. In C. Stemi, according to Goroschankin (1891), the flagellate divides into many isogametes within a cyst. The gametes fuse, beginning at their flagellated ends, and zygotes are formed which develop into resistant cysts. In C. brauniz, the same author (1890) describes gametes of different sizes fusing in anisogamous copulation. Besides being definitely though not pronouncedly smaller, the microgamete is slenderer and more pyriform than the macrogamete. In still another species the differentiation is still more striking. Some individuals of C. coccifera (Goroschankin, 1905) are transformed di- rectly into large nonmotile, egg-like macrogametes, while others divide into relatively very small, flagellated, sperm-like microgametes. Chlamydomonas, therefore, illustrates possible stages in the evolution of sex from isogamy, in which the gametes are smaller than vegetative individuals but otherwise similar; through early differentiation of gam- etes, wherein the gametes are only slightly though clearly differentiated in size and therefore exhibit the very beginnings of anisogamy, to ex- tremely well-differentiated heterogamy or odgamy, in which the micro- gametes and macrogametes are almost typical sperms and eggs. Only one more fundamental advance has been made in the evolution of sex in the Metazoa, and that is the differentiation of the adult forms into male and female individuals. Structural developments for the production and care of offspring belong to a different category. It is unfortunate that so little is known of the maturation processes in Chlamydomonas. It is not known where reduction occurs, much less what the nature of the chromosomes and their behavior in reduction are. Some investigators assume that because of the way in which the zygotes of Chlamydomonas, Gonium, Pandorina, and so forth, behave at divi- sion, reduction is zygotic. Dangeard (1898) found no nuclear reduction taking place before fertilization in Chlamydomonas and suggested that 590 FERTILIZATION it occurs during the division of the zygote. Pascher (1916), in his work on Mendelian inheritance in Chlamydomonas, presented genetic evidence that reduction is zygotic—that is, reduction in chromosome number occurs in the first two divisions of the zygote, which produce four swarmers. This would mean that only the zygote is diploid and that all other stages in the life cycle are haploid (see p. 611 below). Here, indeed, is a peculiarly promising opportunity for the correla- tion of cytological and genetic evidence of chromosome behavior, if only the cytological data were available. Meiosis, not complicated by subsequent fusion of gametes, and the attendant bringing together of homologous chromosomes would offer some interesting possibilities. Another excellent example of a series of organisms exhibiting pro- gressively advancing stages in the evolution of sex is found in the colonial phytomonad flagellates. This series is so well known that it is usually discussed even in textbooks on general biology. At one end of the series is Goniwm pectorale. At certain times the sixteen cells making up this flat colony function as gametocytes by pro- ducing isogametes, which copulate in pairs to form zygotes. The gametes may vary somewhat in size, but the manner in which they copulate— apparently at random with any of the others—indicates that the slight variation in size of the gametes is without significance. In Stephano- Sphaera pluvialis, a colony of eight cells, the gametes are all identical. The chief advance which these isogamous Volvocidae exhibit over the Copromonas type of fertilization is that the gametes are different from the vegetative form. In other words, vegetative or asexual forms may be distinguished from sex cells. In Pandorina morum (Pringsheim, 1869), a subspherical colony of sixteen cells, two distinct sizes of gametes are produced, and two combt- nations are possible. Small gametes may fuse with other small gametes, and small gametes may fuse with large ones. Large gametes, however, never fuse with other large ones. Here, then, in a single species is ex- hibited both isogamy and heterogamy, for the failure of the large gam- etes to fuse with each other indicates that the size difference is significant. The critical factor may be the size itself, or it may be some less obvious factor associated with size. It might be argued from this that primitive heterogamy is associated with hermaphroditism. The same colony produces both large and small FERTILIZATION BION gametes; so, if the size difference means a step in the direction of male- ness and femaleness, then the colony is monoecious or hermaphroditic. This obviously leads to the conclusion that, in the evolution of sex in these forms, gametes became differentiated into male and female types before the parent organisms did. The fact that a larger percentage of primitive Metazoa exhibit hermaphroditism than do the higher forms lends weight to the assumption that this is a general truth. In Exdorina elegans differentiation of gametes has become very marked, and in Volvox the series is climaxed by such extreme differentia- tion between the microgametes and the macrogametes as is seen in meta- zoan sperms and eggs. Furthermore, the vegetative cells of the Volvox colony are comparable to the somatic cells of the Metazoa, while rela- tively few cells of the colony carry on the germ line. Another advance seen in Volvox is that some species have developed the dioecious condition, wherein some colonies produce only microgametes and others produce only macrogametes. Among the Infusoria are found a few cases in which gametes are formed that unite in complete and permanent fusion. This process is therefore copulation, rather than the usual ciliate conjugation (see p. 617). In most cases of copulation in ciliates, the gamonts undergo re- peated divisions, which result in the production of numerous small “microgametes’’ which copulate with each other. Thus copulation of gametes has been described for Trachelocerca phoenicopterus (Lebe- dew, 1909), in the Opalinidae (Neresheimer, 1907; Metcalf, 1923), and in Glaucoma (Dallasia) frontata (Calkins and Bowling, 1929). While these gametes may differ a little in size and be called ‘‘micro- gametes’’ and “macrogametes’”’ by some, the differences do not appear to be very significant. In general, they bear considerable resemblance to the trophic individuals except in size. In the Vorticellidae, however, gametes are formed which are truly anisogamous and which fuse per- manently, although cytologically they more nearly resemble anisogamous conjugants (see p. 621 below). In the ciliate Metopus sigmoides, Noland (1927) described a sexual process which is somewhat intermediate between copulation and conju- gation. Conjugants come together and join anteriorly, but instead of exchanging pronuclei as conjugants usually do, most of the cytoplasm and both pronuclei of one member of the pair pass over into the body 592 FERTILIZATION of the other member, leaving behind only a shrunken remnant of the donor, which then detaches itself from the recipient and dies. This process is functionally very similar to that which occurs in the Vorticelli- dae, while the differences in structural details serve as a connecting link to typical ciliate conjugation. Sexual phenomena seem to be fairly common in the Sarcodina, but they are not so characteristic of the group as was thought by many of the earlier workers. In spite of the many reports of sexual stages in Amoeba proteus, sev- eral recent investigators have failed to observe any type of reproduction other than fission. Johnson (1930), whose article includes a review of the literature, believes that parasites and aquatic fungi have led to many misinterpretations of the life cycle of Amoeba proteus. Liesche (1938) carried A. proteus through 800 generations and observed no sexual stages and no cysts. It is quite possible that there is a sexual stage that occurs only at long intervals, or only under conditions which ordinarily do not obtain in the laboratory. It is obvious that if sexual stages occurred very often in this form, it would be reported more frequently and more convincingly than it has been, in view of the fact that this species is cultivated and studied so constantly in scores of biological laboratories. For instance, at the University of Minnesota A. proteus has been cultivated continuously for nine years, during which period thousands of observations have been made and several hundred permanent slides have been prepared from time to time. In spite of this prolonged search, no sexual stages have ever been found. Binucleate forms, presumably early dividing stages, have been observed frequently, but nothing suggesting gamete formation has ever been noted. It is true that sexual stages could have occurred and escaped observation, for examinations have not been made daily, but it seems reasonable to sup- pose that they would have been discovered at some time if they occurred at any but the rarest intervals. However, Jones (1928) confirms the earlier work of Calkins (1907) and others, with descriptions and photomicrographs of gamete forma- tion by fragmentation of the primary nucleus. He further claims that fertilization is accomplished by means of flagellated gametes. A skeptic might point out that his photomicrographs of gametes and zygotes (his Figs. 13, 15, Plate 11) are strikingly similar to the figures FERTILIZATION 593 of the parasite Sphaerita in Amoeba limax, as pictured by Chatton and Brodsky (1909, Figs. 2, 3). The possibility of confusing sporulation of Sphaerita with gamete formation in Amoeba is not too remote to be considered; although, as Calkins has pointed out, a parasitologist is in- clined to see parasites in everything, and the parasite explanation has probably been over emphasized. The contradictory reports leave us in the peculiar position of not being very sure of the life cycle of our best known and most widely used protozodn, “the common laboratory Amoeba.” Fertilization processes have been described for a number of other amoebae, including Pelomyxa palustris (Bott, 1907) and Sappinia (Amoeba) diploidea (Hartmann and Nagler, 1908). Bott’s account of fertilization in Pelomyxa is unusual indeed. The nuclei of this multi- nucleated plasmodium extrude vegetative and generative chromidia into the cytoplasm. The chromidia form secondary nuclei, which in turn cast out the vegetative chromatin. The secondary nuclei, which now contain only generative chromatin, undergo the first maturation division, in which the chromosome number is reduced from eight to four. In the second maturation division four chromosomes appear and split, so that four go to each pole. Now each granddaughter nucleus divides into two compact masses of chromatin and a vacuole is formed near-by. The chromatin of the two masses then migrates into the adjacent vacuole, in the form of minute granules. After receiving the chromatin, the vacu- ole forms a membrane and becomes the definitive pronucleus of the gamete. The pronuclei which have arisen in this unique manner ap- propriate some cytoplasm and wander out as heliozodn-like gametes, which copulate in pairs to form zygotes. Each zygote grows into a new multinucleate Pelomyxa. Aside from the peculiar role played by the vacuole in this maturation process, which introduces a sort of modified autogamy into the cycle just before the regular fertilization process, the cycle is worthy of further examination. Formation of secondary nuclei from chromidia, which in turn have resulted from the extrusion of chromatin from primary nuclei, has been described in many Sarcodina, several Sporozoa, at least one flagellate (Mastigella, see above) and one ciliate (Trachelocerca phoent- copterus, Lebedew, 1909). Many protozodlogists remain skeptical of the entire proposition of 594 FERTILIZATION the chromidial origin of nuclei (Doflein, 1916). Kofoid (1921) says “The evidence thus far presented of the de novo chromidial origin of protozoan nuclei is wholly inadequate to establish this hypothesis.” Intra- cellular parasites are held responsible for some of the misinterpretations. More recent investigations have clearly refuted at least some of the earlier reports of the chromidial origin of nuclei. The reports of Myers (1935, 1938) and of Le Calvez (1938) on Foraminifera are good ex- amples of this. However, many other reports must be reinvestigated before we can establish any very firm basis for our views. Calkins (1933, p. 70) points out that the chromidial net of Arcella stains green with the Borrel mixture and usually gives a negative re- action to the Feulgen treatment. This supports Hartmann’s experiments, in which the chromidia were dissolved out by pepsin, while the chromatin of the secondary nuclei remained conspicuous. Bélat (1926) believes this is conclusive evidence that chromidia are not composed of chromatin. However, Calkins shows that by omitting the strong hydrolysis of the Feulgen reaction, the chromidia are positively stained and therefore are composed of chromatin, or at least that nucleic acid is present in them. Nucleic acid becomes more concentrated in the nuclei, and this may explain why the nuclei resist pepsin digestion while the residue is dis- solved. The author can confirm Calkins’s positive results in staining Ar- cella chromidia with Feulgen. This organism has been stained with Feulgen at many stages in its life history by omitting strong hydrolysis, and intense staining of both chromidia and nuclei has resulted. Chromidia are colored an intense purple in forms containing nuclet, as well as in forms in which no detectable nuclei are present. It is not impossible that in the latter forms, some of the larger chromidia are actually minute nuclei which are lineal descendants by mitosis of the original nuclei. According to Elpatiewsky (1907) and Swarczewsky (1908), the life cycle of Arcella vulgaris is extremely complicated. In addition to several methods of asexual reproduction, both chromidiogamy and anisogamous syngamy occur. In chromidiogamy two Arce/la, the nuclei of which are degenerating into chromidia, come together. The protoplasm of one passes over into the shell of the other and, after the intermingling of the chromidia, half of the protoplasm passes back into the first shell, and the two organisms pull apart. After separation, the chromidia of FERTILIZATION 595 each individual give rise to the nuclei of amoebulae, which bud off and grow into new adults. Zuelzer (1904) described chromidiogamy in D/f- flugia urceolata, but in this case all the chromidia are said to fuse into a single mass, and the united protoplasmic bodies condense and form a cyst. New nuclei form from the chromidia. The significance of chromidiogamy has never been satisfactorily ex- plained; in fact, the existence of the process itself remains in consider- able doubt. While it is true that specimens of Arcel/a may frequently be found in which no typical nuclei are visible and the cytoplasm of which may contain numerous chromidia, these may be degenerating forms, and only a thoroughgoing reinvestigation of the life history of this interesting organism will convince the skeptics or disillusion the credulous. In Elpatiewsky’s account of anisogamy, some individuals form macro- gametes by repeated nuclear division, while others form microgametes. The gametes are amoebulae, and the difference between male and fe- male is one of size. After copulation between large and small gametes, the zygotes grow up into adult arcellae. A remarkable type of sexual process was described by Hartmann and Nagler (1908) in Sappinia (Amoeba) diploidea, a binucleate form (Fig. 141). The active organism contains two nuclei, derived originally from two parents. It is therefore a kind of adult prezygote. Two such binucleate amoebae come together and develop a common cyst, but their bodies do not fuse. In each amoeba the two nuclei now fuse in a long- delayed fertilization, or karyogamy, after first giving off “vegetative chromidia.” The cytoplasms of the two amoebae now fuse completely. Each synkaryon undergoes two “‘reduction divisions,” after which three products of each degenerate, leaving one reduced nucleus from each synkaryon. These two are the nuclei of the vegetative form. If these two divisions are in reality meiotic divisions, the organism lives a haploid existence, and constitutes the only known case of zygotic reduction in the Sarcodina. If not, some other interpretation must be found for the two divisions which follow syngamy. Since chromosome number and behavior are not known in this form, no satisfactory conclusions may be drawn. It may be argued, of course, that the two haploid nuclei, lying close together in the cytoplasm, are the equivalent of one diploid nucleus, but such speculation must await the positive determination of the chromo- some behavior. 596 FERTILIZATION Another noteworthy point here is that if the two amoebae which en- cyst together are derived from the same parent, the process is a case of autogamy or pedogamy; if not, it is delayed hologamy. Figure 141. Sappinia (Amoeba) diploidea. A, the binucleate vegetative form; B, two such individuals (sister cells ?) encyst within a common capsule and in each amoeba the two nuclei fuse together; C, the bodies of the two amoebae now unite, and the two fusion nuclei undergo two “reduction divisions,’ C, D, after which the nuclei lie side by side, as in A, throughout the vegetative period. (After Hartmann and Nagler, 1908.) The life history of the Foraminifera has been a subject of controversy for many years. Since the pioneer researches of Lister (1895), which were confirmed by Schaudinn (1903) and others, it has been generally believed that the life history of Polystomellina crispa is fairly repre- sentative of the group. According to these investigators, alternation of FERTILIZATION 597 sexual (macrospheric generation — gamont) and asexual (microspheric generation — agamont) generations occurs, and the two generations may be distinguished morphologically, chiefly on the basis of the rela- tive size of the original chamber of the shell. The protoplasm of the two adult generations was said to fragment, to produce flagellated iso- gametes from the gamont and agamete amoebulae from the agamont. Fertilization occurs free in the water, and the zygotes develop into agamonts, while the amoebulae develop directly into new gamonts. The nuclei of both the agametes and the gametes were said to arise from chromidia which are derived from the fragmentation of the primary nuclei. In recent studies on the Foraminifera, Myers (1935, 1936, 1938) has confirmed the earlier work of Lister and Schaudinn, except for the origin of the gamete and agamete nuclei. In Patellina corrugata, Polystomellina crispa, Spirillina vivipara and Discorbis patelliformis, the nuclei of all stages, according to Myers, are derived by an orderly process of mitotic divisions from preéxisting nuclei. He believes that the chromidia are “concerned with feeding and metabolic activities’’ and in no case give rise to nuclei. This is another blow to those who hold to the chromidial origin of nuclei in Protozoa. Myers (1935) further states that gametic reduction occurs in Pa- tellina corrugata and that the haploid number of chromosomes 1s twelve. These observations differ from those of Schaudinn on the same species. In P. corrugata and in S. vivipara, the isogametes are amoeboid, but in D. patelliformis and Polystomellina crispa they are biflagellated, as indi- cated by the earlier workers. In some forms, two or more gamonts become more or less closely as- sociated in a kind of pseudoconjugation known as a syzygy, wherein the pseudopodia may temporarily fuse with those of close neighbors, while in other species they may encyst in a common capsule. This intimate association possibly has a synchronizing effect on gamete formation. Le Calvez (1938) supports Myers’s contention that gamete nuclei are not derived from chromidia, but arise by mitotic divisions from pre- existing nuclei. In Iridia lucida, he states, the secondary nuclei ‘‘disinte- grate’ by rapid divisions which at first are typically mitotic. Later, be- cause they are so small and the character so obscure, the mitoses are recognizable more by the centrosome than by the clarity of the chromo- 598 FERTILIZATION somes. Concerning the origin of the secondary nuclei, Le Calvez states that he “has not been able to discover the chain of processes which, from the disintegration of the vegetative nuclei, lead to the formation of a well defined micronucleus” (secondary nucleus). He believes that the hypothesis of generative chromatin ought to be completely abandoned. In the Actinopoda, sexual reproduction has been reported for both Radiolaria and Heliozoa, but in only two forms has the process been reliably described. The classical case is that of Actinosphaerium eich- hornii (Hertwig, 1898). The multinucleated vegetative individual forms a ‘mother cyst’ and absorbs all but a few (up to 20) of its nuclei. The cytoplasm divides into as many primary cysts (cytospore number one) as there are nuclei. Each primary cyst divides into two distinct secondary cysts (cytospore number two), the nuclei of which undergo two succes- sive “reduction” divisions, resulting in one pronucleus and two “polar bodies’? each. The matured secondary cysts reunite with their sisters as gametes, and the nuclei fuse to complete fertilization. This is obviously a type of autogamy. Hertwig’s claim that in both reduction divisions the chromosomes (numbering between 120 and 150) are divided in the metaphase seems open to question. If this were true, the divisions would not be reductional in character, so that the chromosome number would have to be reduced in some other manner than the usual gametic meioses. According to Schaudinn (1896), Actinophrys sol undergoes isoga- mous macrogamy, or hologamy. He stated that two full-grown similar individuals come together and form a common cyst. The nucleus of each divides twice, and at both divisions one nuclear product degenerates and is expelled. The two cells, with their matured pronuclei, then fuse. The resulting zygote soon divides into two individuals, which later es- cape from the common cyst as vegetative animals. The more recent and detailed investigations of Bélat (1923) have demonstrated in this species a type of sexual activity similar in many respects to that described by Schaudinn, except for the significant differ- ence that the two original gametocytes within the cyst are sister cells, since they are derived by a progamous division of the original gamont (Fig. 142). The process, therefore, is a type of autogamy (pedogamy ) similar to that occurring in Actinosphaerium, except that in the latter case the palmella produces several pairs of sister gametocytes. Incipient Figure 142. Actinophrys sol in autogamous fertilization. A, progamous division of original gamont; B, the two daughters of this division within a common envelope, their nuclei showing looping chromatin threads; C, pairing and twisting of thread-like chromo- somes (left), and shortening and thickening of chromosomes (right); D, first matura- tion (reduction) division, with bivalent chromosomes on the equatorial plate (left), and disjunction and separation of homologous chromosomes (right) ; E, second matura- tion (equational) division, with first “polar bodies” below; F, pseudopodium of 6 gamete making contact with @ gamete; G, fusion of cell bodies; H, fusion of nuclei to form zygote. (After Bélar, 1923. D is a composite.) 600 FERTILIZATION heterogamy is seen in Actinophrys sol. When the gametes unite, one of them sends out a pseudopodial process to the other, to initiate the fusion. This pseudopodium is formed by only one member of the pair, and the maturation processes in this one seem to occur a little ahead of those in the other. These slight differences between the two gametes are inter- preted as the beginnings of differentiation toward maleness and female- ness. In rare cases the pseudopodium of the male fails to make contact with the female, and then the female sends out a pseudopodium which brings about fusion. The indication here is that whatever the degree of differentiation of the gametes is, this differentiation is reversible. Perhaps the potentiality for pseudopodial formation is retained in all gametes, but only the one completing maturation first ordinarily exhibits it. When neither gamete succeeds in connecting with its pseudopodium, no sexual differentiation is demonstrable. In such cases both gametes form par- thenogenetic cysts. The most noteworthy phase of gamete formation in Actmophrys sol is the striking similarity of the meiotic stages to those of the Metazoa. Following the progamous division of the gamont into the two gameto- cytes, two maturation divisions occur which reduce the chromosome number from the diploid forty-four to the haploid twenty-two. In the prophase of the first maturation division, the chromatin forms into slender looping threads (leptonema) which pair off (parasynapsis), become thicker (pachynema), and are obviously twisted around each other (strepsinema). Then they shorten (diakinesis) into compact chromosomes on the metaphase spindle, and the two parts of the bi- valent chromosomes separate in the anaphase, twenty-two univalent chromosomes going to each pole. One product of this division degen- erates, and the other undergoes the second maturation division, which is equational. The twenty-two chromosomes split longitudinally, so that the pronucleus and the two polar bodies of each gamete have twenty- two chromosomes. It seems that this relatively simple heliozo6n has developed a matura- tion process that is as highly specialized and clear-cut as any found in the Metazoa. It is probably safe to say that further diligent search will undoubtedly reveal other species of Protozoa with equally well developed meiotic phenomena. FERTILIZATION 601 SPOROZOA Among the Sporozoa fertilization is almost universally present. As would be expected in such a heterogeneous group, all kinds of fertili- zation processes are known. Isogamy, heterogamy of all degrees of dif- ferentiation, pseudoconjugation, gametic meiosis, zygotic meiosis, and many other variations of the fertilization process have been described. Naturally only a few typical examples, illustrating the chief types of these phenomena, can be mentioned here. Monocystis, the gregarine parasite in the seminal vesicles of the earth- Figure 143. Monocystis rostrata. A and B, metaphase and anaphase of early progamous divisions of pseudoconjugant, eight chromosomes splitting, eight going to each pole; C and D metaphase and anaphase of last progamous (reduction) division, paired chromosomes disjoining, four going to each haploid pole. (After Mulsow, 1911.) worm, illustrates typical pseudoconjugation, gametic meiosis, and isog- amous fertilization. Two adult gregarines come together and are en- closed in a common cyst, but do not fuse. This intimate association with- out protoplasmic union is pseudoconjugation, and the members of this chaste betrothal are now gametocytes. The nucleus of each gametocyte divides again and again to form a large number of small nuclei, which migrate to the periphery and eventually become the gamete nuclei. Ac- cording to Mulsow (1911), reduction occurs in the last of these divi- sions before formation of the pronucleus, in Monocystis rostrata. The earlier mitoses (Fig. 143) show eight thread-like chromosomes which split longitudinally, eight halves going to each daughter nucleus. In the last division the eight chromosomes associate in four pairs. In the ana- phase that follows, members of the pairs separate and pass to different poles, thus reducing the number of chromosomes from eight to four. The surface of the gametocyte produces many small buds, each contain- 602 FERTILIZATION ing a pronucleus. These pinch off as gametes, and the walls between the associated gametocytes break down, allowing the gametes of one to fuse with those of the other. Thus cross-fertilization occurs and the process is isogamous, as there is no differentiation between gametes in this species. Calkins and Bowling (1926), working on a species of Monocystis, have confirmed Mulsow’s interpretations and have furnished additional critical evidence in support of Mulsow’s belief. They found the early progamous divisions with the diploid number of chromosomes (ten) in each daughter plate and also the final progamous divisions with the haploid number (five) in each daughter plate. Naville (1927a) has shown that in three types of Monocystis reduc- tion is gametic. In types “A” and “B” early divisions of the pseudocon- jugants show eight chromosomes and type “‘C” shows four as the diploid number. Anaphases of the last two divisions preceding gamete forma- tion show four chromosomes going to each pole in types A and B, and two in type C. The next to the last division, therefore, is the reduction division. The first amphinuclear division is not reductional, and the sporoblast is diploid during its subsequent development. Naville (1927b) also showed that in Urospora lagidis reduction of chromosome number from a diploid four to a haploid two occurs in the formation of its anisogamous gametes. A noteworthy occurrence here is that synaptic conjugation of chromosomes takes place in the synkaryon. The subsequent division of the synkaryon is equational, but the phe- nomenon serves to illustrate the possibility, in other forms, of an ex- tremely precocious synapsis being prolonged throughout the life cycle until the next sexual stage appears, when the two members of the syn- aptic pairs would separate in a progamic division. Such a condition, if it exists, would explain in terms of gametic reduction the few known examples of zygotic reduction. This hypothesis seems worth investigat- ing. Valkanov (1935) found pairing of chromosomes (synapsis) and condensation of chromosomes (diakinesis) into rings and crosses, in the zygotes of Monocystella arndti; and he believes that reduction oc- curs in the first zygotic division (see p. 613 below), although he was not able to follow the subsequent behavior of the chromosomes. His figures show eleven pairs in the zygote and eleven single chromosomes in the early divisions of the pseudoconjugants. These numbers indicate Figure 144. Ophryocystis mesnili, Isogamous gamete formation and fertilization. A and B, two trophic forms attached to ciliated cells of host; C, gamonts pairing in pseudoconjugation; D, E, F, two nuclear divisions, probably meiotic; G, mature gamont; H and I, formation of gametes by internal budding; J, K, L, fusion of gametes in fertilization; M, N, O, divisions of the zygote to form eight sporozoites in the single spore. (After Léger, 1907.) 604 FERTILIZATION zygotic reduction, but actual separation of chromosomes in the reduction division must be observed before the case is considered to be proved. M. arndti may have zygotic reduction, as the evidence indicates, but Na- ville’s interpretation may apply to this case, so that gametic reduction remains a possibility. Gamete formation by endogenous budding was found by Léger (1907) to occur in Ophryocystis mesnili (Fig. 144). In this form two gamonts adhere in pseudoconjugation, and the nucleus to each divides twice, one product of each division being destined to degenerate. These are presumably reduction divisions, although cytological evidence for this is lacking. One product of the two divisions becomes the pro- nucleus of the single large gamete which is formed inside the gamont as a loose internal bud. The walls between the gamonts break down and the two isogametes fuse. The zygote thus formed develops a spore wall, and eight sporozoites are produced by metagamic divisions. Whiie isogamy is most frequently observed in the gregarines, as for instance in the species already named and in Diplocystis schneideri (Jameson, 1920), Gregarina cuneata (Milojevic, 1925), Actinocephalus parvus (Weschenfelder, 1938), and others, several species show various degrees of anisogamy. Species other than Urospora lagidis, already men- tioned, are Echinomera hispida (Schellack, 1907), Stylocephalus longi- collis (Léger, 1903), and Nini gracilis (Léger and Duboscq, 1909), the last of which shows a marked degree of differentiation between the microgametes and macrogametes. This differentiation approaches that usually seen in the Coccidiomorpha. An extreme differentiation of gametes (odgamy) is seen in Esmeria as well as in other Coccidia and in many Haemosporidia. The type of syngamy observed by Schaudinn (1900) in E/meria schubergi will serve to illustrate this group. Here, as in other cases in which accurate chromo- some determinations have not been made, it is assumed that reduction is gametic. The sexual phase starts with some of the merozoites develop- ing into gametocytes, instead of repeating their asexual cycle. It is diff- cult to explain on a purely environmental basis why some merozoites repeat the asexual cycle while others, obviously in the same environ- ment (intestinal epithelium) develop into gametocytes. If external factors play the chief rdle in determining whether a protozoan will con- tinue asexual multiplication or enter a sexual phase, then it will be neces- FERTILIZATION 605 sary to look further for the effecting stimulus in the Coccidiomorpha. The phenomenon is more easily explained in these forms by the inter- pretation of Maupas, which has since been developed especially by Cal- kins, that internal factors play the determining role. This would mean that when the protoplasm had reached a certain degree of maturity in its cycle of development, the sexual phase would be initiated, even though the external conditions remained unchanged. Whatever the cause, gametocytes appear and are differentiated into male and female gametocytes. The macrogametocytes are said to eliminate their karyosomes to accomplish reduction. By this process they are trans- formed into large, yolk-filled, egg-like macrogametes. The nuclei of the microgametocytes are said to give off chromidia and then degenerate. The chromidia condense into a number of clusters to form the nuclei of small, sperm-like, flagellated microgametes. A macrogamete is found and fertilized by a microgamete, and the resulting zygote forms an odcyst. The synkaryon divides twice to produce four sporoblasts, each of which now develops two sporozoites. A more thorough cytological study of the cycle may eventually reveal chromosome reduction taking place in the two divisions of the syn- karyon, in which case meiosis would be zygotic; or in nuclear divisions prior to gamete formation, in which case it would be gametic. Karyo- some extrusion and the formation of gamete nuclei from chromidia cannot be accepted today as conclusive evidence of meiotic reduction. If, indeed, no chromosomes are formed in E/meria schubergi, then we shall be forced to modify our concept of meiosis. Here again we find urgent need for the application of improved techniques in cytological studies of a fundamental nature. The sexual processes in the Adeleidea differ from those of the other Coccidia in several interesting respects. In Adelina dimidiata, according to Schellack (1913), two gametocytes of different sizes unite in a pseudo- conjugation process similar to that of the gregarines. The nucleus of the microgametocyte divides twice, and one of the nuclei enters and fertilizes the macrogamete. In this species only one macrogamete is formed and it is fertilized by one pronucleus of the microgamete in a way similar to anisogamous conjugation in the Vorticellidae. The peculiar behavior of the ciliate Metopus sigmoides (see p. 622 below) in conjugation also resembles the sexual union of A. dimidiata. 606 FERTILIZATION Fertilization in the Cnidosporidia is typically autogamous, and will be dealt with under the subject of autogamy. Little is known of the fertilization phenomena in the Acnidosporidia, but Crawley (1916) de- scribes gamete formation in Sarcocyst7s murzs, similar in general to odgamy in Ezmeria except that the microgametocyte gives rise to the microgametes by a peculiar kind of nuclear fragmentation. AUTOGAMY Autogamy, or self-fertilization, is accomplished in several ways in the Protozoa, but the result in all cases is the fusion of two gamete nuclei, both of which have been derived from the same parent cell. In some cases the two pronuclei have been separated by cytoplasmic divisions into separate cells which later fuse. In other cases the two pronuclei re- main in the undivided cytoplasm and fuse after casting out part of the chromatin, with or without visible meiotic reduction. Whatever benefit there may be to the individual or the race in ex- ogamy, or cross-fertilization, in the way of renewing the vigor of the protoplasm and in propagating the race, this benefit is also a property of autogamy. There is no apparent reason why autogamy in these re- spects should not be as efficacious as exogamy. In two respects, however, the processes differ greatly. In the uniparental inheritance of autogamous individuals, meiosis will shuffle and sort out whatever genes are pres- ent; and if the allelomorphs are different, the resulting gametes and offspring will vary in their characteristics. However, the tendency in this kind of inbreeding would be overwhelmingly toward the production of homozygous races. Therefore the pronuclei and the resulting offspring would be less variable than in races in which exogamy brings together two sets of genes from two different parents, in the production of a heterozygous individual (see Jennings, 1920). From the genetic and the evolutionary standpoint, it is evident that exogamy would tend to produce a more heterozygous race; and, if natural selection is the criti- cal factor in evolving organisms by the selection of favorable variants, then the advantage of exogamy over autogamy 1s apparent. Another difference is the advantage the autogamous species has over the exogamous species in accomplishing fertilization. It is obvious that in exogamous species either the sexual organism or the gamete must seek out and find a mate before syngamy may occur; and if the organ- Fig. 145. Diagrammatic life cycle of Sphaeromyxa sabrazesi, 1, mononuclear zygote (sporozoite) ; 2, multinuclear plasmodium (schizogony), large outline represents plas- modium during subsequent development; 3-6, 17-19, multiplication of diploid nucle; 7, 20, differentiation of nuclei into large and small; 11-12, 21-23, reduction division in macro- and microgametocytes; 14-15, 25-26, equational division; 16, macrogamete; 29, microgamete; 30, plastogamy; 31, pansporoblast; 32-37, mitoses of haploid nuclei to form fourteen; 38, two sporoblasts formed; 39, two pronuclei remaining in each spore; 40, fertilzation within spore. (After Naville, 1930b.) 608 FERTILIZATION isms are too widely dispersed or too effectively isolated by barriers, there will be no progeny. This difficulty simply does not exist for autogamous organisms, as the isolated individual can reproduce itself sexually with- out recourse to others of its kind. Several cases of self-fertilization have already been pointed out (Ac#7- nos phaerium, Actinophrys, Sap pinia?). In these cases fertilization is ac- complished by the fusion of sister cells, which have undergone nuclear reorganization (perhaps reduction). This type of autogamy has been called pedogamy. Another type of autogamy is common in the Cnidosporidia and is known best in the Myxosporidia. Here the nucleus divides to form several nuclei, without division of the cytoplasm. The nuclei then reunite in pairs, to form amphinuclei. In the myxosporidian Sphaeromyxa sabrazesi, according to Schroder (1907, 1910), the plasmodial body contains two kinds of nuclei. Small areas become differentiated from the surrounding protoplasm. Each area, or pansporoblast, contains two nuclei, one large and one small. Both of these nuclei divide to form seven, so that there are fourteen nuclei pro- duced in each pansporoblast. The pansporoblast divides into two halves, the sporoblasts, which are destined to become the two spores. The daughter sporoblasts receive six nuclei apiece, and the other two nuclei are expelled at the fission of the pansporoblast and degenerate as “‘re- duction nuclei.”” Of the remaining six in each sporoblast, two form the capsule and shell, two form the polar capsules, and two presumably one of each original kind, remain as the pronuclei and later fuse with each other in autogamous fertilization. More recently Kudo (1926) has described a somewhat similar case of autogamy in Myxosoma catostomt. Debaisieux (1924) found six chromosomes in the early mitoses of the plasmodium of Sphaeromyxa sabrazesi. In later stages he found the number reduced to the haploid three. Naville (1930b) is very specific in his account of this species (Fig. 145) and of S. balbianii. In both forms four chromosomes are reduced to the haploid two in the plasmodium, just before the formation of the pansporoblast and after the differentiation of the nuclei into large and small types. The two types he calls macrogametocytes and microgameto- cytes, because they are surrounded by a zone of condensed cytoplasm. The union of two of these zones of cytoplasm in plastogamy brings a large FERTILIZATION 609 and a small nucleus together in the formation of the pansporoblast. A considerable portion of the life cycle of these organisms is therefore passed in the haploid state. Naville also describes a similar diploid- haploid cycle of four and two chromosomes for Myxidium incurvatum. In this case there are more variations in the method of spore formation, but reduction from four to two chromosomes occurs in the plasmodium, as in Sphaeromyxa, and fertilization occurs between the two remaining nuclei of the spore. Many variations of this process occur in other forms, but Chloromyxum leydigi (Naville, 1931) is of particular interest, because of its two hap- loid-diploid cycles. In this multinucleated plasmodium, the nuclei divide by mitosis, showing a diploid chromosome number of four. Then a heteropolar reduction division occurs, producing large and small nuclei with two chromosomes each. Internal buds are formed, wherein large and small nuclei fuse in pairs (first union). The difference in size of these fusing nuclei makes this an anisogamous fertilization. Several divi- sions of the fusion nuclei follow, each showing the diploid four chromo- somes. The young plasmodium grows until the advent of spore forma- tion, which is marked by the appearance of groups of four nuclei, two large and two small, each with chromosomes again reduced to the hap- loid two. The two small nuclei degenerate, while the larger two divide twice, to form a group of eight which become enclosed in a wall. Of the eight nuclei thus formed, six function in the formation of the spore complex and the remaining two fuse in the second fertilization of the cycle. This second fusion is comparable to fertilization in other forms, but the first fusion of nuclei in the plasmodium is a secondary develop- ment interpolated in the life cycle. Its significance is a matter of specula- tion. The phenomenon is actually a double autogamy and is difficult to harmonize with meiotic processes of either Metazoa or other Protozoa. Little is known of chromosome behavior in other Cnidosporidia. Among the Actinomyxida and Microsporidia, autogamous fertiliza- tion is said to occur in a manner broadly similar to that of the Myxo- sporidia. One member of the Actinomyxida, Gayenotia sphaerulosa, has been shown by Naville (1930a) to undergo chromosome reduction in the second of three gametogenic divisions. In this case the development of the pansporoblast occurs as described in the case of other Actino- myxida. The two nuclei of the sporozoite divide mitotically, forming 610 FERTILIZATION two large central germinal cells which ultimately give rise to eight male and eight female gametes respectively, and two smaller peripheral cells which divide again to form the four enveloping cells of the cyst wall. Of the three gametogenic divisions, the second reduces the chromosome number from the diploid four to the haploid two. The gametes are differentiated on the basis of size, the male gametes being smaller than the female. The eight microgametes unite with the eight macrogametes to form eight zygotes and reéstablish the diploid condition. The origin of the two nuclei in the sporozoites is not known, but it is presumably by division of an original single nucleus. As the male and female gametes are produced by a single original sporozoite, it may be regarded as a hermaphroditic animal. This condition is rare in the Proto- zoa except in ciliate conjugation, in which the two pronuclei produced by one conjugant behave differently and in a few cases are morphologi- cally differentiated (see p. 622 below). While a number of other life cycles of Cnidosporidia have been worked out, knowledge of the fertilization process is fragmentary and data on chromosome behavior in meiosis are almost entirely lacking. Descriptions of “reduction” generally refer to the loss of chromatin by the degeneration of some of the nuclei which are sisters of the func- tional pronuclei. In other cases the extrusion of the karyosome 1s inter- preted as reduction. Such loss of nuclear elements is not to be confused with reduction in the chromosome number from the diploid condition to the haploid. It is possible that the two processes are similar in func- tion, but until such time as that is demonstrated, we are not justified in assuming that one is the equivalent of the other. Autogamous fertilization has also been described for a few ciliates. According to Buschkiel (1911), the parasitic form Ichthyophthirius multifiliis becomes encysted and the micronucleus divides twice produc- ing four, of which two degenerate while the remaining two fuse au- togamously. Fermor (1913) described a reorganization process within the cyst of Stylonychia pustulata, wherein the old macronuclei degenerate and the micronuclei fuse and produce a new nuclear complex. The evi- dence in support of these two cases is not conclusive. Diller (1936) has recently given a detailed account of autogamy in Paramecium aurelia. Vhe process is similar to conjugation except that there is no pairing nor cross-fertilization. The macronucleus disintegrates FERTILIZATION 611 during the process, as in conjugation and endomixis, and the micro- nucleus produces two pronuclei, as in conjugation. The pronuclei fuse autogamously and the synkaryon divides twice to form four nuclei; two are macronuclear An/agen which separate at the first fission, and two be- come micronuclei. Diller challenges the very existence of endomixis in this species, on the grounds that stages of autogamy and irregular reorganization processes called ‘‘hemixis’’ have been mistaken for endo- mixis. Certainly this challenge must be met by careful reéxamination of the studies already made on endomixis (see Woodruff, Chapter XIII; also Sonneborn, Chapter XIV). ZYGOTIC MEIOSIS In nearly all animals that have two parents, the two sets of chromo- somes that are contributed to the offspring remain in the nuclei of all cells derived by mitosis from the zygote. This diploid number 1s charac- teristic of all cells except the gametes, in which case the chromosomes are separated out again by one or two meiotic divisions, giving to each gamete one half the diploid number. This haploid number is found only in the gamete, because the union of gametes reéstablishes the diploid condi- tion. There is evidence to show that in a few Protozoa, notably among the Telosporidia, reduction in chromosome number occurs in the division of the zygote. This means that the organism lives a haploid existence and only the zygote is diploid. Bélat (1926) argued that since the com- plete reduction process is known only in Aggregata and Karyolysus among Coccidia and in Diplocystis among gregarines, and that in all these forms reduction is zygotic, therefore reduction in all members of the two groups is probably zygotic. He suggested that Mulsow (1911) confused two species in obtaining his results, but the later work of Calkins and Bowling (1926) and of Naville (1927a) on Monocystis has made that conclusion untenable. Bélat further points to the frequent occurrence of odd numbers of chromosomes in these groups as evidence of the haploid condition, which implies zygotic reduction. Odd chromo- some numbers could be explained by postulating a supernumerary or a sex chromosome, or by interpreting each chromosome as in reality a pair in close and prolonged synapsis; but these assumptions are unsat- isfying, in the absence of more adequate evidence. 612 FERTILIZATION In their preliminary report, Dobell and Jameson (1915) gave the main features of their later detailed descriptions of the life cycles of the gregarine Diplocystis schneideri (Jameson, 1920) and the coccidian Aggregata eberthi (Dobell, 1925). In both cases meiotic reduction occurs in the first division of the zygote, and the organism lives all the rest of its life as a haploid animal. In Diplocystis the nucleus of each pseudoconjugant divides many times, showing the haploid three chromosomes at each division. The dividing nuclei migrate to the periphery and eventually form club-shaped gametes, which pinch off from the gametocyte and fuse with those of the other pseudoconjugant. After a synaptic clumping of bead-like chromatin threads, six chromosomes appear in the prophase of the zygo- tic division and three go to each pole. The diploid number of chromo- somes in the zygote is therefore reduced at the first amphinuclear dtvi- sion to the haploid three, a number which is also observed in the later divisions of the sporoblast. Agegregata eberthi, like other Coccidia, has well differentiated male and female gametes. The female gametocyte is transformed bodily into a macrogamete after a complicated nuclear reorganization, which does — not, however, involve reduction, though a spindle is formed and the six haploid chromosomes are seen. The nucleus of the male gametocyte divides by a complicated method, showing six chromosomes at each division. When the small flagellated microgamete fertilizes the macro- gamete, a fertilization membrane appears. The diploid number of twelve chromosomes appears on the spindle of the first zygotic division. Pairing of homologous chromosomes follows, and the bivalent chromosomes be- come closely applied to each other. They later disjoin and six go to each pole, thereby reducing the number to the haploid condition. All other divisions of the nuclei are mitotic and six chromosomes appear and are divided at each mitosis. It is possible that in both Aggregata and Diplocystis disjunction does not occur in the metaphase of the first zygotic division, and that, instead of this the bivalent chromosomes divide equationally, with six bivalents going to each pole. If at each subsequent mitosis the bivalent chromo- somes divided until just before gamete formation and then disjoined, then reduction would be gametic instead of zygotic. However, the evi- dence seems conclusive enough to convince most biologists that in these two cases meiosis is truly zygotic. FERTILIZATION 613 In 1898 Dangeard found no reduction taking place during gamete formation in Chlamydomonas and suggested that it occurs during the germination of the egg. Pascher (1916) made no chromosome counts, but presented genetic evidence for zygotic meiosis in Chlamydomonas. Hartmann and Nagler (1908) indicated that reduction is zygotic in Sappinia (Amoeba) diploidea, because three nuclei disintegrate, out of the four that are formed by two zygotic divisions. Diwald (1938) very recently stated that, because he could see no meiosis in the formation of the four gametes of Glenodinium lubiniensiforme and because a tetrad of four potential individuals are produced by two divisions of the zygote, reduction occurs in the two zygotic divisions. It does not seem justifiable to base an assumption of zygotic meiosis on such indirect and questionable evidence. Genetic evidence must be considered, in the absence of cytological data; but only positive determina- tion of chromosome number and identification of the stage in the cycle in which the number is reduced from diploid to haploid can be accepted as conclusive evidence of this phenomenon. Valkanov (1935) has presented fragmentary cytological evidence of zygotic reduction in Monocystella arndti. He shows eleven long chromo- somes in the early divisions of the pseudoconjugants. In the first zygotic division, eleven synaptic pairs condense into short, fat Ys and Xs. He concludes that reduction is zygotic, but his evidence is admittedly incom- plete, as he was unable to follow the subsequent behavior of the chromo- somes. Whether this is truly zygotic meiosis or whether the zygotic pair- ing of chromosomes is a phenomenon similar to that found in Urospora lagidis (see p. 602 above) remains an open question. The odd number of chromosomes lends some support to Valkanov’s belief. Weschenfelder (1938) has just published what appears to be a clear- cut case of zygotic meiosis in the gregarine Actinocephalus parvus. In the early nuclear divisions of the pseudoconjugants, four long, rod- shaped, haploid chromosomes are repeatedly observed. Isogametes bud off the mother cell and fertilization occurs as in other gregarines. At the first division of the zygote, eight chromosomes develop from the syn- karyon as four synaptic pairs. These pairs disjoin in the anaphase and four go to each pole, reducing the number of chromosomes to the hap- loid condition again. Subsequent mitoses in the sporoblast reveal the haploid four chromosomes, now globular in shape, appearing in the prophase and passing to each pole in the anaphase. 614 FERTILIZATION Weschenfelder’s observations have confirmed the suspicions of many protozodlogists that there exist other gregarines besides Diplocystis schneideri which undergo zygotic meiosis. The problem of inheritance in these forms presents some interesting possibilities to the geneticist. All genes possessed by the two parent organisms are passed to the zygote; therefore, if odcyst (sporoblast) characters can be differentiated and mated, the immediate and direct effect of those genes may be observed in the resulting zygote. Furthermore, a haploid organism whose charac- teristics are controlled by a single set of chromosomes presents a rare opportunity for unusual genetic and cytological studies. SIGNIFICANCE OF FERTILIZATION The causes and effects of fertilization in Protozoa are subjects upon which a great deal has been written and some significant data obtained. The three conditions cited by Maupas (1889) as necessary for conjuga- tion in ciliates are sexual maturity, diverse ancestry, and hunger. All three of these contributing factors have been supported by evidence from some later investigations and all three have been discounted by other investigations. In many cases the investigators have been dealing with different species of Protozoa. This in itself is probably responsible for many of the conflicting conclusions that have been reached. As the evidence accumulates, it becomes increasingly clear that different Proto- zoa require different conditions for conjugation and copulation, and that we are not justified in applying to all Protozoa conclusions derived from one or two or even several species. Among the flagellates and rhizopods there are many organisms in which sexual phenomena have never been reported and in which prob- ably none exists. These forms, then, are able to reproduce indefinitely by asexual means. Inherently, therefore, protoplasm does not seem to require sexual union. At the other end of the sexual scale are found those Sporozoa and Foraminifera in which the life cycle is an obvious fact, and in which a sexual stage develops as one sector of that cycle, without which they could not continue their existence. If generalizations were made from these two kinds of organisms, there would be contradictions too obvious to relate. For similar but less obvious reasons, we may partially account for FERTILIZATION 615 the different schools of thought regarding the conditions necessary for conjugation in ciliates, upon which most of this work has been done. In regard to ancestry, Calkins (1904) found that in Paramecium caudatum there are fully as many conjugations between closely related individuals as between individuals of diverse ancestry. He further indi- cates (Calkins 1933) that similar results have been obtained through isolation cultures of Didinium nasutum, P. aurelia, P. bursaria, Styl- nychia sp., Blepharisma undulans, Spathidium spathula, Oxytricha fal- lax, Chilodonella cucullus, and Uroleptus mobilis. Sonneborn and Cohen (1936) found that under identical conditions, ‘“The Johns Hopkins stock R of Paramecium aurelia can invariably be induced to conjugate,” while “the Yale stock of the same species cannot.’ This difference ap- pears to be clearly racial. Sonneborn’s (1937) discovery of two ‘“‘sex reaction types’’ in a race of P. awrelia may throw considerable new light on this question. Members of one type readily conjugate with those of the other type, but do not conjugate among themselves. At first this looked as though something resembling the two sexes of other organisms had been found in the reaction of one ciliate to another. However, the discovery by Jennings (1938) of as many as nine sex reaction types in P. bursaria seems to remove these types from the cate- gory of sexes and indicates that they are simply strains which will not inbreed. The significance of these discoveries is not yet clear, but they do show that in some cases, at least, diverse ancestry is a potent factor in conjugation. In regard to the relative importance of external conditions and in- ternal conditions in ciliate conjugation, we again find contradictory evi- dence if we generalize from specific instances. The inductive method of reasoning is certainly stimulating and productive, but its misuse has led to some unjustifiably broad propositions. There is a rapidly accumulat- ing array of evidence that external conditions, such as food, tempera- ture, pH, population concentration, light, seasons, chemicals, condition of host in some parasitic forms, and so forth, do play an important rdle in inducing conjugation in some ciliates. However, there is valid evi- dence to indicate that in some forms, at least, conjugation can be in- duced only at certain times in the life cycle of the organism—in other words, only when the protoplasm is sexually “mature” for conjugation. Calkins (1933, p. 286) states that “One unmistakable conclusion can be 616 FERTILIZATION drawn from the many diverse observations and interpretations of the conditions under which fertilization occurs in ciliates, viz., the proto- plasmic state with which conjugation 1s possible is induced in large part, but not wholly, by environmental conditions.” It is a matter of common observation that when conjugation occurs in mass cultures, all the ciliates do not conjugate, but only a certain proportion of them. The proportion may vary with the culture and the species, but in any case if the conditions in the mass culture are favorable for inducing conjugation in some individuals, why do they not all con- jugate? The fact that some do and some do not conjugate under condi- tions that appear to be identical, would indicate the existence of internal differences. Calkins (1933, p. 290) summarizes the evidence and concludes “that environmental stimuli are without effect in producing conjugations un- less the protoplasm is in a condition where such conjugations are pos- sible.” Two examples illustrate different phases of this proposition: Uroleptus mobilis will conjugate only after a period of from five to ten days after fertilization, and stock R of Paramecium aurelia (Sonne- born, 1936) will conjugate only in descendants of animals which have recently undergone conjugation or endomixis. The time factor is ob- viously different in these two animals, but both clearly indicate a strong cyclical differentiation which affects conjugation. For more detailed analyses of this subject, reference should be made to Calkins’s Biology of the Protozoa (1933) and to Chapter XIV below, by Sonneborn. There is, perhaps, even less agreement concerning the effects of con- jugation than concerning the causes. In some ciliates, e.g., Urole ptus mobilis (Calkins, 1919), conjugation results in a definite renewal of vitality, as indicated by an increase in the fission rate. Calkins interprets this as a fundamental process, which is an integral and normal part of the life cyle. Woodruff and Spencer (1924) found a similar renewal of vitality following conjugation in Spathidium spathula, but Woodruff (1925) interprets this as a rescue process to “meet the emergency of physiological degeneration induced by environmental conditions which are not ideal.’’ Beers (1931) shows that conjugation increases vitality in Didinium nasutum which has been depressed by inadequate feeding, but that no depression occurs in well-fed animals. FERTILIZATION 617 In other ciliates, however, conjugation apparently reduces vitality. Thus in Blepharisma undulans Calkins (1912) found that all excon- jugants died, although Woodruff (1927) concluded from his investiga- tions that conjugation in this species accelerates the division rate. Jen- nings (1913) concluded that conjugation reduced vitality in Paramecium as indicated by a reduction in the average rate of fission in exconjugants. At the present time the problem as regards ciliates seems to be: does increased vitality following conjugation mean that conjugation is a nor- mal and essential part of the life cycle, or is it merely an emergency measure called into play when unfavorable environmental conditions have resulted in physiological degeneration? This problem is not easy to solve, because it is difficult to know what optimum or even “normal” environmental conditions are, and the two are probably not identical. Another complication is that in some species endomixis may be substt- tuted for conjugation as a revitalizing process. Another angle from which this problem may be approached is that of comparison with the plant kingdom. Many plants are able to repro- duce themselves indefinitely by asexual methods, but at the same time sexual stages occur which, though not indispensable to their continued existence, are nevertheless certainly an integral part of their normal life cycle and valuable to the organism in other ways. In other plants, sexual processes must occur at regular intervals under “normal” condi- tions, or the species will die out. Further investigation may disclose a similar situation in the ciliates, wherein some ciliates cannot continue to exist without periods of con- jugation, while in others endomixis may be substituted for conjuga- tion, and in still others asexual reproduction will carry on the line in- definitely. The final answer to this problem will come only through con- tinued investigation. CON JUGATION Conjugation has been defined as the temporary union of two proto- zoan cells for the exchange of nuclear elements. It is a sexual process, differing from ordinary sexual union in that it is not directly related to reproduction. Two organisms enter into the relationship and the same two functional units leave the relationship; no third party—no progeny —has come into being. The two conjugants have been genetically 618 FERTILIZATION changed by conjugation into new genetic entities, but this is actually genetic transformation rather than reproduction. Before the exconju- gants return to their normal vegetative condition, they undergo one or more divisions in most cases, but these divisions are reproduction by binary fission, an asexual phenomenon. Although these divisions are modified by the previous sexual union, they are none the less asexual reproduction. | Conjugation is peculiar to the Ciliata and the process is strikingly uniform, with but few exceptions, in all ciliates which have been studied. The general course of the maturation phenomena in conjugation was first described by Maupas (1889), who studied the process in a number of ciliates and divided it for convenience into eight stages. Calkins (1933) states that ‘“With one or two exceptions (Trachelacerca phoene- copterus, Spirostomum ambiguum, etc.) all of the free living ciliates thus far described agree in the general course of their maturation phe- nomena.” Several parasitic species, however, exhibit some important dif- ferences from the usual course, and recent investigations have revealed a few interesting deviations among free-living forms. With a few noteworthy exceptions, the union of two ciliates in con- jugation takes place longitudinally and symmetrically (Fig. 146). The first sign of approaching conjugation in a mass culture is frequently a tendency to agglomerate in dense masses. Individuals appear to stick together on contact, even though they may separate soon after. Even- tually, two individuals will adhere side by side, or with ventral surfaces together, and become more intimately connected in the anterior region. The extent of union varies from a thin protoplasmic bridge at the time of cross-fertilization to an intimate fusion of more than half the body length in other species. Two individuals of Ezplotes patella will come into contact, spiral about each other for a few moments, and then apply themselves to- gether at their left peristomal margins, so that the appearance is similar to two turtles stuck together by their left ventral halves (Fig. 146). They swim forward together in a well codrdinated manner, rotating on an axis which, owing to the symmetry of the pair, is straighter than the spiraling axis of a single individual. At this stage the pairs are joined only by their cirri. After remaining together a short time, they may separate and repeat the process with the same or with other individuals, until finally FERTILIZATION 619 a union is made which involves an insecure adhesion of the bodies in the anterior left peristomal region. The peristome is distorted by the fusion, but the mouth continues to feed until it degenerates in the reor- ganization process. While the majority of ciliates become attached along their ventral or ventro-lateral margins and fuse anteriorly, several exceptions are note- Figure 146. A pair of Euplotes patella in conjugation. The micronuclei have under- gone preliminary division and are now in the first meiotic division; the C-shaped macro- nuclei are beginning to degenerate. (Turner, 1930.) worthy. Didinium nasutum (Prandtl, 1906; Mast, 1917) and members of the Ophryoscolecidae (Dogiel, 1925) join end to end anteriorly, the latter forming an oral chamber by the juxtaposition of the two deep peristomal pockets. Dogiel states that the conjugants are smaller than the ordinary forms, owing to special progamic fissions. In Parachaenia myae, Kofoid and Bush (1936) found conjugants attached by their posterior 620 BERTILIZATION ends, the anterior ends pointing in opposite directions. Kidder (1933b) describes the anterior tip of one member of a pair of Ancistruma isseli uniting asymmetrically with the peristomal groove part way back on its mate, though the two are of equal size. Miyashita (1927) shows that radically asymmetrical union occurs in Lada tanishi, in which the micro- conjugant, smaller than its mate, attaches its anterior end to the posterior ventral surface of the macroconjugant. In Kzdderia (Concho phthirius) mytili, Kidder (1933a) shows an almost tandum association, with the anterior peristomal region of the slightly smaller member joined by a wide protoplasmic bridge to the aboral surface of the larger member of the pair. In Dileptus gigas (Visscher, 1927) fusion takes place along the ventral surfaces of the proboscides, and the mouth remains in evi- dence during the entire period of conjugation. The varied methods of joining of the conjugants suggest that the location of the fusion bridge is not significant. If ciliate conjugation evolved from a process similar to pseudoconjugation as seen in present- day gregarines, as many protodlogists believe, it seems reasonable that one location would serve as well as another, provided the cortex were not too firm for a protoplasmic bridge to be formed. It is interesting in this connection to observe that in Explotes patella (Turner, 1930), which has a rigid cuirass, no true cytoplasmic bridge is formed. The wandering pronucleus of one conjugant breaks out of the left anterio- ventral margin of the one conjugant and passes backwards through a tube formed by the local separation of the applied surfaces of the two conjugants, and finally enters the cytostome of the other conjugant. This method of entering the apposed conjugant probably developed simply because it was easier to penetrate the soft cytostomal membrane than the rigid cuirass. The mouth-to-mouth migration of the male pronucleus in Cycloposthium bipalmatum (Dogiel, 1925) is a simpler example of the same process. In Polyspira and other members of the Foettingereidae, there occurs a remarkable combination of conjugation, fission, and chain formation called “‘syndesmogamie” by Minkiewicz (1912), and recently renamed “zygopalintomie”’ by Chatton and Lwoff (1935) in their comprehensive work on the Apostomea. The two conjugants unite by their lateral sur- faces rather than by the usual ventral method, then proceed to undergo a series of synchronous, partial, transverse divisions, until a chain of con- FERTILIZATION 621 jugating zodids is formed, resembling superficially a double tapeworm. After a time the fissions cease, and conjugation proceeds between mem- bers of each pair of zodids according to the “‘classical scheme,” although the nuclear details have not been worked out. Eventually fission is com- pleted, and the exconjugants soon separate, reorganizing themselves in the usual way. These fissions of the paired conjugants appear to be related in kind to the special preconjugation fissions, observed in several other ciliates, which result in conjugants that may be distinguished from the vegetative forms chiefly by their smaller size. Specialized conjugants are observed in Nicollella cteriodactyli and Collinella gundi (Chatton and Pénard, 1921); the Ophryoscolecidae (Dogiel, 1925); Déleptus gigas (Visscher, 1927); Balantidium sp. (Nelson, 1934); Nyctotherus cordiformis (Wichterman, 1937), and in the microgametes of pert- trichs. The preliminary division of the micronucleus in Ezplotes charon and E. patella (Maupas, 1889; Turner, 1930), without fission of the body, is a modification of this same tendency of the conjugants to differ from the vegetative forms. Conjugants of many other ciliates are in some degree smaller than vegetative individuals, and this may be the result of reduced feeding or of other factors as yet unknown. It is among the copulating ciliates that the greatest difference occurs between the vegetative forms and the mature gametes (see p. 610 above). Sexual differences are difficult to elucidate in the ciliates because the picture is confused by two kinds of possible differences. The two con- jugants may show differences in size, shape, or other characteristics. These differences between the two conjugants entering the union may be interpreted as indicating maleness and femaleness. In a number of forms the differences are slight but fairly constant, as in Miyashita’s (1927) ‘“‘macroconjugants’ and “microconjugants’” of Lada tanishi. In the Vorticellidae, on the other hand, the difference in size and behavior of the microconjugant and the macroconjugant is very striking, far greater, in fact, than could be explained on the grounds of fluctuating variation. The small free-swimming form that seeks out and fertilizes the large sessile form could reasonably be called the male conjugant, and the large form may be considered a female conjugant. In the Vorticellidae and in Metopus sigmoides (Noland, 1927), mutual fertilization 1s not accom- 622 FERTILIZATION plished, because both pronuclei of one member—the microconjugant in the Vorticellidae—pass over with the cytoplasm into the other conjugant. One pronucleus of the donor and one pronucleus of the recipient fuse, to form the functional synkaryon. The other two pronuclei may or may not fuse, but in either case they eventually disintegrate. In Metopus the conjugants separate and the remnant of the donor dies; in the Vorti- cellidae the microconjugant fuses completely with the macroconjugant. These are obviously fertilization types intermediate between copulation and conjugation. The other category of differences is that exhibited between the wander- ing and the stationary pronuclei which are produced in the same con- jugant. They are usually considered to be male and female pronuclei respectively. Here the only apparent difference may be in their behavior, as is the case in the majority of ciliates studied. In Explotes patella there is a slight difference in size between the wandering and the stationary pronuclei, and there is a special zone of cytoplasm which accompanies the wandering pronucleus in its migration. In Cycloposthium bipalma- tum, however, Dogiel (1925) has described a spermatozo6n-like wander- ing pronucleus, which is in striking contrast to the rounded stationary pronucleus. These illustrations may be considered as representing stages in the evolution of distinct sexual differences between pronuclei of ciliates. If we assume that differences between members of a conjugating pair indicate sexual differentiation, then we would have male and female individuals both producing structurally isogamous but functionally an- isogamous pronuclet, as in Chilodonella (Chilodon ) uncinatus (Enriques, 1908; MacDougall, 1925). In other cases we would see male and female conjugants both producing pronuclei which are functionally and structurally differentiated as male and female, as in Cycloposthium (Do- giel, 1925). If we consider the differences in behavior and structure between the wandering and the stationary pronuclei as indicating sexual differences, then we must consider the parent conjugants as hermaphrodites, and any differences between conjugants would then be a leaning toward male- ness or femaleness on the part of an hermaphroditic organism. Viewed in this light, members of the Vorticellidae have lost their double nature, and the microconjugant has come to produce only male functional pro- FERTILIZATION 623 nuclei and the macroconjugant only female functional pronuclei. The other pronuclei are produced as usual, but fail to develop. Similarly, in Metopus sigmoides (Noland, 1927) one conjugant contributes all of its potentialities to the other, in what may be interpreted as a male ani- mal contributing its life and all its potential gametes to the female. THE MACRONUCLEUS DURING CONJUGATION In ciliates, the micronucleus is concerned with sexual activity and reproduction and is therefore frequently referred to as the generative or reproductive nucleus and represents the “germ plasm” of the Metazoa. The macronucleus, on the other hand is concerned with metabolism or vegetative activity and is considered the trophic or vegetative nucleus and represents in part the somatoplasm of the Metazoa. The chromatin of these two types of nuclei is combined in the single nucleus of other cells and in the one kind of nucleus found in the multinucleate O palina. The disintegration of the macronucleus at the time of conjugation, in all ciliates with dimorphic nuclei, represents the death of the soma and the end of the genetic unit. Differentiation of the new macronucleus in exconjugants similarly represents the development of the new somatic individual from the zygote. In ciliates generally, the old macronucleus shows signs of disintegra- tion during the maturation divisions, and, by the time of crossing of the pronuclei, fragmentation or other evidences of disintegration are well under way. It is during the differentiation of the new macronucleus from the synkaryon that the most rapid breakdown and the final absorption of the old macronucleus occur. This is probably due to the withdrawal from the cytoplasm of all chromatin-building elements by the developing macronuclear An/lage or “‘placenta.’’ The old macronuclear remnants are possibly used as “‘fertilizer,” or reserve of chemical elements in about the right proportion, for replenishing the cytoplasm and maintaining the equilibrium. Before disintegration, the old macronucleus exhibits strange activity in several species of Anoplophrya (Schneider, 1886; Collin, 1909; Brumpt, 1913; Summers and Kidder, 1936), and in two species of Chilodonella (MacDougall, 1936). At about the time of crossing of the pronuclei, the macronucleus in each conjugant elongates and con- stricts in the middle, as one half pushes across the protoplasmic bridge 624 FERTILIZATION into the apposed conjugant. This macronuclear exchange results in each exconjugant possessing half of both macronuclei. Their eventual de- composition makes the exchange difficult to explain on functional grounds. Summers and Kidder suggest that it may represent a ‘“‘remi- niscence of a more primitive protozoan condition before the separation of trophic nuclear materials from the germinal materials.” The odd elongations of degenerating macronuclear chromatin into ribbon or rod-like fragments in Paramecium may be an abortive attempt at a similar process. CON JUGANT MEIOSIS Because the general course of conjugation, as outlined by Maupas (1889) is followed by the vast majority of ciliates so far studied, it 1s convenient to use this outline in reviewing the process. His eight stages are as follows: Stage A, in which the micronucleus swells and prepares for division; Stage B, the first meiotic or maturation division; Stage C, the second meiotic division; Stage D, the third nuclear division, which produces the pronuclet; Stage E, that of mutual exchange and the union of pronuclet; Stage F, the first metagamic (amphinuclear) division; Stage G, the second metagamic division; Stage H, subsequent reorganization. Stages A, B, C, and D are concerned with preparation for syngamy. This preparation includes meiosis and the formation of pronuclei (see Fig. 147). Stage E is the climax of the entire process, wherein the act of fertilization is consummated. Stages F, G, and H are concerned with the reorganization of the body and the reéstablishment of the usual vege- tative form. Among ciliates that normally possess more than one micronucleus there is little uniformity in the number of nuclei that undergo the two meiotic divisions. In many forms all micronuclei enter the first meiotic division. Then all products of this division may divide again, or various numbers of them may be resorbed (see Calkins, 1933, p. 295). In Dileptus gigas, however, Visscher (1927) has shown that only one of the large number of micronuclei undergoes maturation. Two to eighteen micronuclei have been described as entering the first — <— 10 11 Figure 147. Diagram of ciliate conjugation. 1, two ciliates joined ventrally, micro- nuclei in prophase parachute stage; 2, first meiotic (equational) division; 3, second meiotic (reduction) division in which the chromosome number is reduced from diploid to haploid, and the macronucleus begins to degenerate; 4, third maturation division, involving only one of the four nuclei in each animal, the other three degenerate; 5, migration of the wandering (¢) pronuclei into the apposed animals, 6 and @ pro- nuclei of left conjugant stippled to indicate common origin; 6, fusion of wandering and stationary pronuclei to form synkaryon and restore diploid condition; 7, conjugants separate, first division of amphinucleus in exconjugants; 8, second amphinuclear division; 9, four nuclei produced by the two amphinuclear divisions, the old macronucleus dis- integrates; 10, two of the four new nuclei develop into new micronuclei, two into new macronuclei; 11 and 12, first fission of the exconjugant separates out one micronucleus and one macronucleus to each daughter cell, reéstablishing the vegetative condition. 626 FERTILIZATION maturation division of different ciliates. Variation in the number of nuclei involved in the maturation divisions occurs within the same species, as well as among different species. As only two pronuclei function in fertilization, and as these two are known to be sister nuclei in many ciliates, only one micronucleus really needs to undergo maturation. STAGES A AND B, THE FIRST MEIOTIC DIVISION In the earlier accounts of conjugation, the first maturation division in several ciliates was said to be not greatly different from ordinary vegetative mitosis. Recent accounts, based on careful cytological studies, show marked peculiarities in the prophase of the first maturation division. It seems possible, therefore, that more detailed studies, with improved techniques, may reveal these distinguishing prophase stages in all ciliates. The fact that such a careful worker as Maupas failed to observe the highly characteristic changes that occur in Ezplotes patella (Turner, 1930) lends weight to this possibility. j In the vast majority of ciliates, the prophase of the first maturation division is highly characteristic and presages the coming reduction. In some ciliates the micronucleus takes on the form of a crescent, or comma, during the prophase, and this appearance is sufficiently characteristic to be recognized as a general type. Among ciliates that exhibit the crescent formation are various species of Paramecium and the Vorticellidae. There 1s little agreement as to the number of chromosomes in Para- mecium or even as to the method by which the crescent is transformed into the metaphase spindle. The chromosome number in all species 1s surely larger than the 8 or 9 given by Hertwig (1889) for P. aurelza, and probably less than 150, which has been attributed to P. caudatum. Calkins and Cull (1907) suggested that the 165 or more small chroma- tin rods or fibers seen in P. caudatum are comparable to the physical counterpart of the individual genes of higher animals. Aggregates of these would represent a chromosome in cases where chromosomes are formed. Perhaps the 32 chromomeres of Exzplotes patella (Turner, 1930) also represent 32 genes, although one would expect this highly specialized hypotrich to have more genes than the more primitive holo- trich. Whatever the nature of the chromatin elements in Paramecium may be, these investigators, as well as Dehorne (1920), show that the first FERTILIZATION O27 maturation spindle is formed at right angles to the prophase crescent by the migration of the division center from the apex of the crescent to the middle of the crescent, and by the pushing out of the other pole across the crescent. Earlier workers believed the spindle was formed by a shortening of the crescent. Dehorne finds no chromosomes at all, but, instead, a simple convoluted thread. In a wide variety of other ciliates, the prophase develops a “‘cande- labra”” (Collin, 1909) or “parachute” (Calkins, 1919) stage. It is noteworthy that the parachute prophase occurs in most of the ciliates in which reasonably complete chromosome studies have been made and reduction definitely located. Kidderia (Concho phthirius) mytili may be an exception to this, but Kidder (1933a) admits he might have missed finding it. Tannreuther (1926) describes a simple type of chromosome formation in Prorodon griseus, in which chromosomes arise directly out of a central chromatin mass upon the equator of the spindle. In Explotes patella a typical parachute is formed, which is seen as a stage in the transformation of resting chromatin into the chromosomes of the metaphase spindle. The events transpire synchronously in both nuclei produced by the preliminary, or pre-maturation division of the micronucleus occurring in this species. Each nucleus swells to several times its original size, as the faintly granular chromatin becomes more basophilic, and is arranged in a reticulum filling the nuclear space (Fig. 148). The reticulum condenses in the center and becomes polarized, with most of the chromatin at one pole. Further condensation forms a dense club-like structure, which presently loosens up and is transformed into a parachute, with most of the chromatin forming the “‘cloth”’ at one pole, the spindle fibers forming the ‘‘rope,” and an endosome at the other pole forming the ‘‘weight.’’ The chromatin then forms thirty-two discrete chromatin granules, the chromomeres, which soon migrate to the equa- torial plate in groups of four. These eight groups of four chromomeres apparently correspond to the eight diploid chromosomes found in other stages of the life cycle. In the anaphase of this division, sixteen chromo- meres pass to each pole, and one may frequently observe them associated in pairs as loosely connected dumb-bells. The sixteen chromomeres, or eight dumb-bells, which pass to each pole represent the eight diploid chromosomes and identify this as an equational division. In Pleurotricha lanceolata, Manwell (1928) found chromomeres that 628 FERTILIZATION fuse to form about eighty dumb-bells. Since the diploid chromosome num- ber is forty in this species, four granules (two dumb-bells) evidently represent a chromosome, just as they do in E. patella, and forty dumb- bells pass to each pole in the anaphase. In Kidderia (Conchophthirius) mytili, Kidder (1933a) found thirty- Figure 148. Stages in the first maturation division of Euplotes patella. A, early nucleus with finely granular chromatin; B, chromatin reticulum condensing in center; C, para- chute stage; D, later parachute showing chromatin granules migrating from upper pole, endobasal bodies and intradesmose visible; E, metaphase stage with thirty-two chromo- meres arranged in eight chromosome groups; F, anaphase stage with sixteen chromo- meres (eight chromosomes) passing to each pole. (Turner, 1930.) two granules forming on the spindle and sixteen passing to each pole, as in E, patella. Sixteen is the diploid number in this species, so the thirty-two granules represent half a chromosome each, although there is no visible association between the halves. Gregory (1923) described forty-eight chromomeres appearing in the prophase and fusing to form twenty-four dumb-bell chromosomes in Oxytricha fallax. In this case twelve dumb-bells pass to each pole. If FERTILIZATION 629 twelve were the diploid number, this would correspond exactly to the condition existing in Ewplotes patella and in Pleurotricha lanceolata. However, Gregory believes that twelve is the haploid number and that the separation of the twenty-four dumb-bells into two groups of twelve each, in the first maturation division, means that this is the reduction division, a condition unusual in ciliates. This interpretation is weakened somewhat by the fact that twenty-four dumb-bells are formed in the prophase of the second maturation division, twelve passing to each pole in the anaphase. It is possible that the twenty-four dumb-bells which separate into two groups of twelve each in the first maturation division are actually tetrads, and that the twelve going to each pole are diads. This would mean that the joining of the original granules is synaptic in character and that their passing to the same pole indicates splitting, or equational division. This explanation is not completely satisfying, in view of the events of the second maturation division. Each of the twenty- four dumb-bells which are formed in the prophase of the second division would have to be derived from one granule of the first anaphase dumb- bells. If that occurred, then the second maturation division would be reductional; but if they are formed by the splitting of entire dumb-bells, as believed by Gregory, then the second division would be equational and the first would be reductional. Whichever interpretation is correct, one thing seems clear: in all these forms the chromosomes of the first maturation division are composed of a definite number of loosely associated chromomeres. It is in their method of distribution that interpretations differ, and further investiga- tion in this field will be welcomed by those interested in meiotic phe- nomena in the Protozoa. No parachute stage was found by Noland (1927) in Metopus sig- moides. Instead, the chromatin forms a spireme, which condenses into a single large sausage-shaped ‘“‘chromosome”’ on the spindle. This divides, and one part goes to each pole. The interpretation of this condition is difficult, because of the obscurity of later stages. From the appearance of the synkaria in Noland’s drawings, one would judge that there are two large chromosomes in some, and four in others. If two were the diploid number, the four would represent splitting for fission. Then the single chromosome of the first maturation division could be interpreted as a synaptic pair in close union. This speculation may not be justified 630 FERTILIZATION by further investigation, but it seems reasonable on the basis of the avail- able facts and is in line with current theory. Calkins (1919) describes a peculiar situation in Uroleptus mobilis, in which two types of metaphase stages are found (Fig. 149), one in which about twenty-four chromosomes appear and twelve go to each pole, and another in which eight chromosomes appear and eight go to each pole in an obviously equational division. Although the number 1s not strictly homologous, the first type is similar to that which occurs in Euplotes patella and other species, and the second type is what would be expected if all chromosomes were compact. Intermediate forms are conceivable, in which some chromosomes are compact and others are Figure 149. First maturation spindles of Uroleptus mobilis. A and B, two types of metaphase stages; C and D, two types of anaphase stages found in this form. Both types are equational divisions, since the diploid eight chromosomes appear in the sub- sequent division, in which they are reduced to four. (After Calkins, 1919.) dispersed as several loosely associated chromomeres. This would explain many irregular counts, which otherwise seem chaotic. In Chilodonella (Chilodon) uncinatus a parachute is formed after the division of an endobasal body, according to MacDougall (1925). Enriques (1908) failed to see the parachute in the same species, but described a peculiar rod formation. In MacDougall’s material, four strands of chromomeres are formed from the spireme of the late para- chute stage and condense into four dumb-bell chromosomes on the spindle. This author states that the exact number of granules in each strand was not determined, but her Figure 23 shows four on each strand. This is interesting, since four chromomeres to a chromosome has been found in E. patella (see Fig. 148 E) and other ciliates. The four chromo- somes then split longitudinally and four halves migrate to each pole, at FERTILIZATION 631 which point they fuse in pairs, forming diads before entering upon the resting stage. A tetraploid strain arose spontaneously in a pure culture of Mac- Dougall’s Chilodonella uncinatus. Maturation phenomena in the tetra- ploid form were similar to those of the usual diploid form, except that there were twice the number of chromosomes in every stage. Investiga- tions, presented and reviewed in a later article by MacDougall (1936), show meiotic processes which are similar in six species of Chilodonella. In all species the diploid number of chromosomes is four. A parachute stage is followed by the formation and the synaptic pairing of chroma- tin threads, as in “‘classic leptotine and zygotine’’ stages, which condense to form the pachytene chromosomes. MacDougall’s descriptions reveal the striking similarity of meiosis in Chilodonella to the general scheme of meiosis in the Metazoa. Messiatzev (1924) reported synapsis occur- ring in the first maturation division and again in a fifth amphinuclear division of Lionotus lamella, but Poljansky (1926) believes that Mes- siatzev confused his stages in the latter case. The small number and the large size of the chromosomes in Chilodo- nella make this a very favorable form for study of meiotic phenomena. It seems unfortunate that more of the recent studies that have genetic sig- nificance were not made on this animal, in which chromosome behavior is clear-cut and well known, instead of on Paramecium, in which it is practically impossible to determine any of the significant stages in meiosis. STAGE C, THE SECOND MEIOTIC DIVISION The second meiotic division is the reduction division in all ciliates thus far studied, except in Oxytricha fallax, according to Gregory (1923). Prandtl (1906), in his work on Didinium nasutum, was the first to present conclusive evidence on chromosome reduction in ciliates when he described reduction from sixteen to eight chromosomes in the second meiotic division. There seems to be no general rule for the num- ber of nuclei that enter this division. In the species of Chilodonella studied by MacDougall (1936), only one nucleus is involved in any of the three progamous divisions; the other products degenerate. In perhaps the majority of ciliates both products of the first division enter the second division. In O. fallax (Gregory, 1923) and in forms 632 FERTILIZATION with multiple micronuclei such as Uroleptus mobilis (Calkins, 1919), a variable number of nuclei may divide a second time. In Ezplotes patella all micronuclear products undergo a second meiotic division. Because of the preliminary division, there are four in each conjugant. No resting stage occurs between divisions here, in contrast to MacDougall’s (1936) account of Chilodonella. The daughter nuclei of the previous division are still connected by their respective drawn-out nuclear membranes when the chromatin begins to resolve itself into a reticulum in each nucleus and the granules on the reticulum condense into eight discrete, ovoid Figure 150. Second meiotic (reduction) division in Explotes patella. A, eight ovoid chromosomes appearing on the spindle; B, synaptic pairing and lengthening of chromo- somes; C, disjunction and separation of homologous chromosomes in the anaphase, four passing to each pole. chromosomes (Fig. 150 A). The chromosomes now conjugate in four pairs, in what is probably a delayed synapsis, elongate somewhat, and disjoin longitudinally, four haploid chromosomes passing to each pole. Calkins (1919) described a similar pairing and separation of the eight chromosomes in Uroleptus mobilis. Tannreuther (1926) presents evidence of chromosome pairing in the reduction division of Prorodon griseus, but in most cases where synapsis has been observed, it occurs in the first meiotic division. STAGE D, THE THIRD DIVISION, AND THE FORMATION OF PRONUCLEI In all ciliates thus far studied, a third division occurs. This division is equational in character and usually involves only one nucleus, while the rest degenerate. The two products of this division are the pronuclei which take part in fertilization. In a few ciliates, two, three, and four micronuclei have been reported to divide at this stage, but in no case has it been demonstrated that the FERTILIZATION 633 two functional pronuclei are derived from different spindles. In all cases in which only one nucleus is involved, and possibly also in those where two or more are involved, the two pronuclei must be genetically identical if the third division is equational. In Uroleptus (Calkins, 1919), two or three nuclei divide, but the two pronuclei are always sister nuclei. As this occurs in both members of the pair, the exconjugants should theo- retically be genetically identical. This appears to be the significant feature in the third maturation division. Figure 151. Explotes patella. A, B, C, third maturation division, in which the four haploid chromosomes in A split longitudinally and the halves slip past each other in B, and four go to each pole in C; D, fertilization nucleus in which the ¢ and @ pronuclei have just joined, but their chromosome groups have not yet mingled. In Ex plotes patella (Turner, 1930), two nuclei enter the third division. In each nucleus (Fig. 151) the chromatin reticulum condenses into four strands of chromatin granules, which condense into four compact sausage- shaped chromosomes lying lengthwise of the spindle. The chromosomes split longitudinally, and the halves slip past each other as they migrate to separate poles in this equational division. The chromosomes are all lying in the same axis, so that as the chromosomes slip past each other in the early anaphase, they appear end to end, and the figure might easily be misinterpreted as a transverse division of chromosomes. It 1s possible that the descriptions of the transverse division of chromosomes in the third division, given by Enriques (1908), Calkins (1919, 1930), MacDougall (1925), and others were based on some such artifact. Cal- kins points out that if each chromosome represents one gene, the method of division is of no consequence. This interpretation would doubtless serve for Paramecium, which has a large number of chromosomes, but it is hardly conceivable that Chilodonella would have only two pairs of genes. It seems more probable that the apparent transverse division of some chromosomes in mitosis is due to our inability to demonstrate by 634 FERTILIZATION present techniques some of the finer structural changes which occur within the chromatin mass. Since Prandtl (1906) first noted a difference in size between the wan- dering and the stationary pronuclei of Didininm nasutum, slight differ- ences have been reported in a number of other cases. Calkins and Cull (1907) showed that this is due to a heteropolar third division, in Paramecium caudatum, Maupas (1889) was the first to record a struc- tural difference between pronuclei, when he observed the area of dense cytoplasm in front of the migrating pronucleus of Ezplotes patella. The most striking dimorphism appears in Cycloposthium, according to Dogiel (1925). The wandering pronucleus is spermatozoén-like in hav- ing an elongated tail. All these differences between pronuclei must be cytoplasmic in origin, for the nuclei, as has been pointed out, are genetically identical, if our present concepts are correct. STAGE E, MIGRATION OF PRONUCLEI AND FERTILIZATION Migration of the wandering nucleus occurs synchronously in the two conjugants, so that they generally pass each other in the cytoplasmic bridge which joins the two conjugants. In Cycloposthium (Dogiel, 1925), the spermatozodn-like male pronucleus passes out of the mouth of the parent body and into the mouth of the recipient, by way of the juxtaposed peristomal cavities (Fig. 167). In Explotes patella (Turner, 1930), the wandering pronucleus breaks out of the left anterior tip of the parent body, which is pressed into the peristomal field of its mate (see p. 620, above), passes backward be- tween the appressed conjugants, and finally enters the cytostomal area of the recipient. Both pronuclei form spindles as the male approaches the female, and four chromosomes can be seen in each. As the pronuclet touch, their membranes dissolve and the two groups of four chromo- somes mingle, as fertilization is completed and the diploid condition restored (Fig. 151 D). In Chilodonella, MacDougall (1925) shows that the two haploid chromosomes are visible throughout the migration period (Fig. 152), but lose their identity soon after fertilization. The appearance of the pronuclei at the time of union varies with the species. In a number of ciliates, they are in the form of a spindle similar to those of Euplotes, although few show chromosomes. In other ciliates, the pronuclei are spherical and vesicular at the time of union. In still others, intermediate conditions have been reported. FERTILIZATION 635 Figure 152. Chilodonella uncinatus. Migration of the pronuclei across the protoplasmic bridge. Each pronucleus contains two haploid chromosomes still attached by strands to their sister halves. Also visible in each conjugant are old and new oral baskets and the granular remnants of the old macronucleus. (After MacDougall, 1925.) STAGES F, G, AND H, THE EXCON JUGANTS The subject of reorganization is dealt with elsewhere in this volume, but we may consider briefly some of the cytological aspects of the re- organizing exconjugant. After fusion of the pronuclei, the fertilization nucleus divides one or more times, and from the products of division the new micronuclear and macronuclear elements are formed, while extra products disintegrate. The number of divisions the synkaryon undergoes before differentia- tion of the macronuclei and micronuclei in various ciliates is reviewed by Kidder (1933b) in his work on Ancistruma. Kidder lists eight species in “group A,” in which the micronucleus and the macronucleus are differentiated after the first amphinuclear division. To these we may add Chilodonella cucullulus (Ivani¢, 1933); C. chattoni, C. labiata, C. caudata, C. faurii (MacDougall, 1936); and N yctotherus cordiformis (Wichterman, 1937). In other ciliates, differentiation occurs after the second amphinuclear division. Kidder lists twenty-one species in this “group B,” which includes a majority of the best-known ciliates. To this list may be added Balantidium (Nelson, 1934), from the Chim- panzee. In about half of these species, all four products remain functional, 636 FERTILIZATION and in the others two or three develop into micronuclei and macro- nuclei, while the remaining one or two degenerate. In Ezplotes patella, the macronucleus and the micronucleus are never sister nuclei, so that the first amphinuclear division evidently separates the macronuclear line from the micronuclear line. However, there is no apparent difference, except one of position, between the two products of the first division. In “group C’ Kidder lists twelve ciliates, in which differentiation occurs after the third amphinuclear division. To this list, which includes the familiar Paramecium caudatum, may be added Parachaenia myae (Kofoid and Bush, 1936). The number of products, if any, which de- generate appears to vary with the author, as well as with the species. In about half of these species, all nuclei remain functional. In Paramecium caudatum, four of the eight amphinuclear products become macronuclei and are distributed by fission to the four grand- daughters, while four become micronuclei. According to Calkins and Cull (1907), all of these micronuclei remain functional and are dis- tributed by fission; but Maupas (1889), Jennings (1920), and Doflein- Reichenow (1928) indicate that three degenerate, while the fourth remains as the functional micronucleus and divides at each subsequent fission, just as it does in P. putrinum (Doflein, 1916). In some material, both of these schemes are represented in P. cauda- tum; but, perhaps because the dividing micronucleus is more easily identified, the latter type is more clearly and unquestionably demon- strable. In “group D,” in which differentiation occurs after the fourth amphi- nuclear division, Kidder lists only P. mu/timicronucleata (Landis, 1925), Kidderia (Conchophthirius) mytili (Kidder, 1933a), and Bzrsaria truncatella, according to Prowazek (1899). But Bwrsaria belongs in “group C,”’ according to Poljansky (1928, 1934). After the final division of the synkaryon, which separates macro- nuclear elements from micronuclear elements, the micronuclei return to the normal condition. This involves shrinkage in size and the restora- tion of the chromatin to the homogeneous condition. If more than the normal number are formed, they are separated by the subsequent body fissions until the normal number is established, as in P. caudatum, according to Calkins and Cull (1907). If the normal number were FERTILIZATION 637 differentiated, they divide at every subsequent body division, as in Euplotes patella. The macronuclei grow to their normal size and are distributed by body fissions to the daughter and granddaughter cells, if more than the vegetative number are formed, as in Paramecium. Thus the ordinary vegetative form is reéstablished. During their development, the young macronuclei undergo some striking and significant changes. Several investigators have reported marked enlargement of the macronuclear Av/agen in ciliates, since the early description of the “ball-of-yarn” stage in Nyctotherus cordiformis by Stein (1867). Calkins (1930) showed that the macronuclear Anlage of Uroleptus halseyi at first contains no chromatin, if chromatin be defined as a nucleic acid containing substance. As the young macronucleus grows, minute chromatin granules are formed within the matrix and grow in size and number until the nucleus is filled with large, intensely staining chromatin granules. Subsequent divisions of this “‘placenta’’ and of the cell body restore the normal vegetative nuclear complex. A number of other ciliates show enlargement of the placenta to a size greater than that of the ordinary condition, after which shrinkage, or condensation, reduces it to its vegetative size. According to MacDougall (1925), the young Anlage of Chilodonella uncinata stains very faintly at first, but more intensely later. The enlargement continues until the young macronucleus nearly fills the cell. In Explotes patella (Turner, 1930), as in Uroleptus, the early macro- nuclear An/age contains little, if any, demonstrable chromatin. After several hours of growth, a fine chromatin reticulum develops and is transformed into a broken spireme. The chromatin spireme enlarges with the nucleus and resembles the “ball of yarn” in Nyctotherus cor- diformis, as pictures by Stein (1867) and Wichterman (1937), except that the spireme is more tortuous. Earlier stages resemble those of Meto pus sigmoides as drawn by Noland (1927), and of Paraclevelandia simplex (Kidder, 1938) in cystic reorganization. The spireme finally becomes shorter and very much thicker, as chromatin granules are formed along its periphery. At this stage a fine thread is discernible run- ning through the center of the spireme. The granules become scattered throughout the macronuclear Anlage as the structure of the spireme 638 FERTILIZATION disappears. The large granular ball thus formed condenses somewhat and then elongates, starts bending into its normal C-shape, and com- bines with one or two sizeable remnants of the old macronucleus which have persisted through the process. When the elongating macronuclear Anlage approaches the remnants of the old macronucleus, the latter lose their pycnotic appearance and the chromatin reorganizes itself into dis- crete, dispersed granules, again resembling the normal condition. The new and the old portions then unite, end to end, to produce the C-shaped nucleus of the trophic form, the reconstituted portion forming the pos- terior portion. Examination of hundreds of exconjugants at this stage convinces one that the proximity of the elongating Av/age is the influ- ence which brings about the reorganization of the old remnants. The old chromatin is so thoroughly reorganized before joining the new Anlage that it possibly has little more effect on the nature of the new nucleus than if it had been dissolved and re-formed within the new nuclear membrane. Ikeda and Ozaki (1918) first reported fragments of the old macro- nucleus being incorporated into the developing macronuclear An/age in Boveria labialis. Kidder (1933a, 1933b) has described an interesting phenomenon oc- curring in the macronuclear Anlagen of Kidderia (Concho phthirius) mytilt and in Ancistruma isseli, At each of the two or three exconjugant fissions which separate the seven or eight new macronuclei, all un- separated macronuclei cast out in an orderly manner a sphere of chroma- tin. According to Kidder, this “may represent the sloughing off of the germinal chromatin contained in the amphinucleus, a substance that 1s superfluous for the further activity of a purely trophic cell element (Reichenow, 1927).” Diller (1928) suggested this as an explanation of a similar occurrence during endomixis in Trichodina. Chromatin ex- trusions from developing macronuclear An/agen would appear to be even more closely analogous to the Ascaris type of chromatin diminution, as brought out by Boveri, than is the chromatin diminution described by MacDougall in the division of the macronucleus in Chilodonella. Until recently the behavior of the macronucleus in conjugation has been given scant attention. The disintegration of the old macronucleus and the differentiation, number, and distribution of new macronuclear Anlagen have been noted, but no great significance has been attached to FERTILIZATION 639 these processes. It now seems probable that more intensive investigation of the réle of the macronucleus in conjugation will be extremely profit- able. LITERATURE CITED Beers, C. D. 1931. Some effects of conjugation in the ciliate Didinium nasutum, J. exp. Zool., 58: 455-70. Bélat, K. 1923. Untersuchungen an Actinophrys sol Ehrenberg. I. Die Morphologie des Formwechsels. Arch. Protistenk., 46: 1-96. —— 1926. Der Formwechsel der Protistenkerne. Ergebn. Zool., 6: 235- 654. Bott, K. 1907. Uber die Fortpflanzung von Pelomyxa palustris. Arch. Protistenk., 8: 120-58. Brumpt, E. 1909. Demonstration du réle pathogéne du Balantidinm coli. Enkystment et conjugaison de cet infusore. C. R. Soc. Biol. Paris, 67: 103-5. —— 1913. Etudes sur les Infusoires parasites. I. La Conjugaison d'Anoplophrya circulans Balbiani, 1885. Arch. parasit., 16: 187-210. Buschkiel, A. L. 1911. Beitrige zur Kenntnis des Ichthyophthirius multi filis Fouquet. Arch. Protistenk., 21: 61-102. Calkins, G. N. 1904. Studies on the life history of Protozoa. IV. Death of the A series. J. exp. Zool., 1: 423-61. ——— 1907. The fertilization of Amoeba proteus. Biol. Bull., 13: 219-30. ——— 1912. The paedogamous conjugation of Blepharisma undulans. J. Morph., 23: 667-88. —— 1919. Uroleptus mobilis, Engelm. 1. History of the nuclei during division and conjugation. J. exp. Zool., 27: 293-357. —— 1930. Uroleptus Halseyi Calkins. II. The origin and fate of the macro- nuclear chromatin. Arch. Protistenk., 69: 151-74. —— 1933. The Biology of the Protozoa. Philadelphia. Calkins, G. N., and R. Bowling. 1926. Gametic meiosis in Monocystis. Biol. Bull., 51: 385-99. — 1929. Studies on Dallasia frontata Stokes. II. Cytology, gametogamy and conjugation. Arch. Protistenk., 66: 11-32. Calkins, G. N., and S. W. Cull. 1907. The conjugation of Paramecium aurelia (caudatum ). Arch. Protistenk., 10: 375-415. Chatton, E. 1927. La Gamétogénése méiotique du flagellé Paradininm pouchett. G. Re AcadsSets Patisnt 85: 553-55. Chatton, E., and B. Biechler. 1936. Documents nouveaux relatifs aux Coccidinides (Dino flagellés parasites). La Sexualité du Coccidinium mesnili n. sp. C. R. Acad. Sci., Paris, 203: 573-76. Chatton, E., and A. Brodsky. 1909. Le Parasitisme d’une Cytridinee du genre Sphaerita Dangeard chez Amoeba limax Dujard. Etude compara- tive. Arch. Protistenk., 17: 1-18. 640 FERTILIZATION Chatton, E., and A. Lwoff. 1935. Les Ciliés Apostomes. I. Apercu historique et général; étude monographique des genres et des especes. Arch. zool. exp. gén., 77, Protistologica L: 1-453. Chatton, E., and C. Pénard. 1921. Les Nicollellidae. Infusoires intestinaux des Gondis et des Damans, et le “cycle évolutif’”’ des ciliés. Bull. biol., 59: 87-153: Collin, B. 1909. La conjugaison d’Anoplophrya branchiarum (Stein) (A. circulans Balbiani.). Arch. zool. exp. gén., Series 5, 1: 345-88. Crawley, H. 1916. The sexual evolution of Sarcocystis muris, Proc. Acad. nat. Sci. Philad., 68: 2-43. Dangeard, P. A. 1898. Sur les Chlamydomonadinées. C. R. Acad. Scie., Paris, 127: 736-38. Debaisieux, P. 1924. Sphaeromyxa sabrazesi Laveran et Mesnil. Cellule, 35: 267-301. Dehorne, A. 1920. Contribution a l’étude comparée de l’appareil nucleaire des Infusoires ciliés (Paramecium caudatum et Colpidium truncatum), des Euglenes et des Cyanophycees. Arch. zool. exp. gén., 60: 47-176. Diller, W. F. 1928. Binary fission and endomixis in the Trichodina from tadpoles (Protozoa, Ciliata). J. Morph., 46: 521-61. — 1936. Nuclear reorganization processes in Paramecium aurelia, with descriptions of autogamy and “hemixis.” J. Morph., 59: 11-67. Diwald, K. 1938. Die ungeschlechtliche und geschlechtliche Fortpflanzung von Glenodinium lubiniensiforme spec. nov. Flora, Jena, 32: 174-92. Dobell, C. C. 1908. The structure and life-history of Copromonas subtilis. Quart. J. micr. Sci., 52: 75-120. —— 1925. The life-history and chromosome cycle of Aggregata eberthi. Parasitology, 17: 1-136. Dobell, C. C., and A. P. Jameson. 1915. The chromosome cycle in Coccidia and gregarines. Proc. roy. Soc., B., 89: 83-94. Doflein, F. 1916. Lehrbuch der Protozoenkunde. 4th ed., Jena. Doflein, F., and E. Reichenow, 1928. Doflein, Lehrbuch der Protozoenkunde. 5th ed., Jena. Dogiel, V. 1925. Die Geschlechtsprozesse bei Infusorien (speziell bei den Ophryoscoleciden) , neue Tatsachen und theoretische Erwagungen. Arch. Protistenk., 50: 283-442. Elpatiewsky, W. 1907. Zur Fortpflanzung von Arcella vulgaris Ehrbg. Arch. Protistenk., 10: 441-66. Enriques, P. 1908. Die Conjugation and sexuelle Differenzierung der In- fusorien. Arch. Protistenk., 12: 213-76. Fermor, X. 1913. Die Bedeutung der Encystierung bei Stylonychia pustulatg. Zool. Anz., 42: 380-84. Goldschmidt, R. 1907. Lebensgeschichte der Mastigamében Mastzgella vitrea n. sp. u. Mastigina setosa n. sp. Arch. Protistenk., Suppl. 1: 83-168. FERTILIZATION 641 Goroschankin, J. N. 1890. Beitrige zur Kenntnis der Morphologie und Systematik der Chlamydomonaden. I. Chlamydomonas Braunii (Mihi). Bull. Soc. imp. Natur., Moscou, pp. 498-520. — 1891. Beitrige zur Kenntnis der Morphologie und Systematik der Chlamydomonaden. II. Chlamydomonas Reinhardii (Dang.). Bull. Soc. imp. Natur., Moscou, pp. 101-42. — 1905. Beitrage zur Kenntnis der Morphologie und Systematik der Chlamydomonaden. III. Chlamydomonas cocifera (Mihi). Flora, Jena, 94: 420-23. Gregory, L. H. 1923. The conjugation of Oxytricha fallax. J. Morph., 37: 555-81. Gross, F. 1934. Zur Biologie und Entwicklungsgeschichte von Noctiluca miliaris, Arch. Protistenk., 83: 178-96. Hartmann, M. 1911. Die Konstitution der Protistenkerne und ihre Bedeutung fiir die Zellenlehre. Jena. Hartmann, M., and K. Nagler. 1908. Copulation bei Amoeba diploidea n. sp. mit. Selbstandigbleiben der Gametenkerne wahrend des ganzen Lebenszyklus. S. B. Ges. naturf. Fr. Berl., Nr. 5, 112-25. Hertwig, R. 1889. Uber die Konjugation der Infusorien. Abh. bayer. Akad. Wiss. Munchen, II. KI. 17: 1-83. 1898. Uber Kernteilung, Richtungskérperbildung und Befruchtung von Actinosphaerium eichhornii, Abh. math.-phys. bayer. Akad. Wiss. Munchen, 19: 631-734. Ikeda, I, and Y. Ozaki. 1918. Notes on a new Boveria species, Boveria labialis n. sp. J. Coll. Sci. Tokyo, 40: 1-25. Ivani¢, M. 1933. Die conjugation von Chilodon cucullulus Ehtbg. Arch. Protistenk., 79: 313-48. Jameson, A. P. 1920. The chromosome cycle of gregarines, with special refer- ence to Diplocystis schneideri Kunstler. Quart. J. micr. Sci., 64: 207-66. —— 1927. The behaviour of Balantidium coli Malm. in cultures. Parasitology, 19: 411-19. Jennings, H. S. 1913. The effect of conjugation in Paramecium. J. exp. Zool., 14; 279-391. —— 1920. Life and death; heredity and evolution in unicellular organisms. Boston. —— 1938. Sex reaction types and their interrelations in Paramecium bursaria. 1, Il. Clones collected from natural habitat. Proc. Nat. Acad. Sci. Wash., 24: 112-20. Jehnson, P. L. 1930. Reproduction in Amoeba proteus. Arch. Protistenk., 71: 463-98. Jones, P. M. 1928. Life cycle of Amoeba proteus (Chaos diffluens) with special reference to the sexual stage. Arch. Protistenk., 63: 322-32. Kidder, G. W. 1933a. Studies on Conchophthirius mytili De Morgan. II. Conjugation and nuclear reorganization. Arch. Protistenk., 79: 25-49. 642 FERTILIZATION —— 1933b. On the genus Avcistruma Strand (= Ancistrum Maupas). II. The conjugation and nuclear reorganization of A. isseli Kahl. Arch. Protistenk., 81: 1-18. — 1938. Nuclear reorganization without cell division in Paraclevelandia simplex (Family Clevelandellidae), an endocommensal ciliate of the wood-feeding roach, Panesthia, Arch. Protistenk., 91: 69-77. Kofoid, C. A. 1921. Symposium on fertilization. Anat. Rec., 20: 223-325. Kofoid, C. A., and M. Bush, 1936. The life cycle of Parachaenia myae gen. nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Fran- cisco Bay, California. Bull. Mus. Hist. nat. Belg., 12: 1-15. Kudo, R. 1926. On Myxosoma catostomi Kudo 1923, a myxosporidian para- site of the sucker, Catostomus cammersonii. Arch Protistenk., 56: 90- fit). Landis, E. M. 1925. Conjugation of Paramecium multimicronucleata, Powers and Mitchell. J. Morph., 40: 111-67. Lebedew, W. 1909. Uber Trachelocerca phoenecopterus Cohn, Ein marines Infusor. Arch. Protistenk., 13: 70-114. Le Calvez, J. 1938. Recherches sur les Foraminiféres. I. Développement et reproduction. Arch. zool. exp. gén., 80: 163-333. Léger, L. 1903. La Reproduction sexuée chez les Stylorynchus. Arch. Protistenk., 3: 303-57. —— 1907. Les Schizogregarines des Trachéates. I. Le genre Ophryocystis. Arch. Protistenk., 8: 159-202. Léger, L., and O. Duboscq. 1909. Etudes sur la sexualité chez les grégarines. Arch. Protistenk., 17: 19-134. Liesche, W. 1938. Kie Kern- und Fortpflanzungsverhaltnisse von Amoeba proteus (Pall.). Arch, Protistenk., 91: 135-86. Lister, J. J. 1895. The life-history of the Foraminifera. Philos. Trans., B. 186: 401-53. MacDougall, M. S. 1925. Cytological observations on gymnostomatous cilt- ates, with a description of the maturation phenomena in diploid and tetraploid forms of Chilodon uncinatus. Quart. J. micr. Sci., 69: 361-84. —— 1935. Cytological studies of the genus Chilodonella Strand, 1926 (Chilodon Ehrbg., 1838). I. The conjugation of Chilodonella sp. Arch. Protistenk., 84: 199-206. —— 1936. Etude cytologique de trois espéces du genre Chilodonella Strand. Morphologie, conjugaison, réorganisation. Bull. biol., 70: 308-31. Manwell, R. D. 1928. Conjugation, division, and encystment in Plewrotricha lanceolata. Biol. Bull., 54: 417-63. Mast, S. O. 1917. Conjugation and encystment in Didinium nasutum with especial reference to their significance. J. exp. Zool., 23: 335-59. Maupas, E. 1889. Le Rejeunissement karyogamique chez les ciliés. Arch. zool. exp. gén., Ser. 2, 7: 149-517. FERTILIZATION 643 Messiatzev, J. 1924. The conjugation of Lionotus lamella (in Russian). Arch. russ. protist., 3, no. 1-2. Metcalf, M. M. 1923. The opalinid ciliate infusorians. Bull. U. S. nat. Mus., 120. Milojevic, B. D. 1925. Zur Entwicklungsgeschichte der Gregarina cuneata (F. St.), mit besonderer Beriicksichtigung der Entstehung des Geschlechtskerns. Arch. Protistenk., 50: 1-26. Minchin, E. A. 1912. An Introduction to the Study of the Protozoa. London. Minkiewicz, R. 1912. Un cas de reproduction extraordinaire chez Polyspira Delagei. C. R. Acad. Sci., Paris, 155: 733. Miyashita, Y. 1927. On a new parasitic ciliate Lada tanishi n. sp., with preliminary notes on its heterogamic conjugation. Jap. J. Zool., 1: 205- 18. Mulsow, K. 1911. Ueber Fortpflanzungserscheinungen bet Monocystis rostrata n. sp. Arch. Protistenk., 22: 20-55. Myers, E. H. 1935. The life history of Patellina corrugata Williamson, a foraminifer. Univ. Cal. Publ. Zool. tech., series, 3: 355-92. —— 1936. The life cycle of Spzrillina vivipara, with notes on morphogenesis, systematics and distribution in the Foraminifera. J. R. micr. Soc., 56: 120-46. —— 1938. The present state of our knowledge concerning the life cycle of the Foraminifera. Proc. nat. Acad. Sci. Wash., 24: 10-17. Naville, A. 1927a. Le Cycle chromosomique et la meiose chez les Monocystis. Z. Zellforsch., 6: 257-84. —— 1927b. Le Cycle chromosomique d’Urospora lagidis (de Saint Joseph). Parasitology, 19: 100-38. —— 1927c. Le Cycle chromosomique, la fécondation et la réduction chro- matique de Chloromyxum leydigi Mingazz. Ann. Inst. océanogr. monaco, 4: 177-208. —— 1930a. Le Cycle chromosomique d'une nouvelle Actino myxidie: Guyenotia sphaerulosa n. gen.; n. sp. Quart. J. micr. Sci., 73: 547-75. —— 1930b. Recherches sur la sexualité les myxosporidies. Arch. Protistenk., 69: 327-400. 1931. Les Sporozoaires. Mém. Soc. Phys. Genéve, 41, No. 1. Nelson, E. C. 1934. Observations and experiments on conjugation of the Balantidium from the chimpanzee. Amer. J. Hyg., 20: 106-34. Neresheimer, E. R. 1907. Die Fortpflanzung der Opalinen. Arch. Protistenk., (Suppl.) 1: 1-42. —— 1908. Fortpflanzung eines parasitischen Infusors (Ichthyophthirius). S. B. Ges. Morph. Physiol., Miinchen, 23. Noland, L. E. 1927. Conjugation in the ciliate Metopus sigmoides C. and L. J. Morph., 44: 341-61. Pascher, A. 1916, Uber die Kreuzung einzelliger, haploider Organnismen: Chlamydomonas. Ber. dtsch. bot. Ges., 34: 228-42. 644 FERTILIZATION Poljansky, G. 1926. Die Conjugation von Dogielella sphaerii (Infusoria Holotricha, Astomata). Arch. Protistenk., 53: 407-34. — 1928. Uber die Konjugation von Bursaria truncatella. Zool. Anz., 79: D1-58. — 1934. Geschlechtsprozesse bei Bursaria truncatella O. F. Mill. Arch. Protistenk., 81: 420-546. Prandtl, H. 1906. Die Konjugation von Didinium nasutum. Atch. Protistenk., 7: 229-58. Pratje, A. 1921. Noctiluca miliaris Suriray. Beitrage zur Morphologie, Physiologie und Cytologie. I. Morphlogie und Physiologie. (Beobach- tungen an der lebenden Zelle.) Arch. Protistenk., 42: 1-98. Pringsheim, E. 1869. Uber Paarung von Schwarmsporen, die morphologische Grundform der zeugung im Pflanzenreiche. Monatsber. preuss. Akad. Wissensch., 721-38. Prowazek, S. 1899. Protozoenstudien. I. Bwrsaria truncatella und thre Con- jugation. Arb. zool. Inst. Univ. Wien. 11: 195-286. Schaudinn, F. 1896. Uber die Copulation von Actinophrys sol Ehrbg. S. B. preuss. Akad. Wiss., 83-89. —— 1903. Untersuchungen tiber die Fortpflanzung. Arb. GesundhAmt., 19: 547-76. Schellack, C. 1907. Uber die Entwicklung und Fortpflanzung von Echinomera hispida A. Schn., Arch. Protistenk., 9: 297-345. — 1913. Coccidien-Untersuchungen II. Die Entwicklung von Adelina dimidiata A. Schn., einem Coccidium aus Scolopendra cingulata Latr. Arb. GesundhAmt. Berl., 45: 269-316. Schneider, A. 1886. Anoplophrya circulans (Balb.) Tabl. Zool., 1:31-88. Schroder, O. 1907. Beitrage zur Entwicklungsgeschichte der Myxosporidien. Sphaeromyxa sabrazesi (Laveran et Mesnil). Arch. Protistenk., 9: 359-81. 1910. Uber die Anlage der Sporocyste (Pansporoblast) bei Sphaeromyxa Sabrazesi Laveran et Mesnil. Arch. Protistenk., 19: 1-5. Scott, M. J. 1927. Studies on the Balantidium from the guinea-pig. J. Morph., 44: 417-65. Sharp, R. W. 1934. Introduction to Cytology. New York. Sonneborn, T. M. 1936. Factors determining conjugation in Paramecium aurelia. 1. The cyclical factor: the recency of nuclear reorganization. Genetics, 21: 503-14. — 1937. Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. nat. Acad. Sci. Wash., 23: 378-85. Sonneborn, T. M., and B. M. Cohen. 1936. Factors determining conjuga- tion in Paramecium aurelia. 1. Genetic diversities between stocks or races. Genetics, 21: 515-18. Stein, F. 1867. Der Organismus der Infusionsthiere. I]. Abt. Leipzig. Summers, F. M., and G. W. Kidder. 1936. Taxonomic and cytological FERTILIZATION 645 studies on the ciliates associated with the amphipod family Orchestiidae from the Woods Hole district. Il. The coelozoic astomatous parasites. Arch. Protistenk., 86: 379-403. Swarczewsky, B. 1908. Uber die Fortpflanzungserscheinungen bei Arcella vulgaris Ehrbg. Arch. Protistenk., 12: 173-212. Tannreuther, G. W. 1926. The life history of Prorodon griseus. Biol. Bull., 51: 303-20. Turner, J. P. 1930. Division and conjugation in Ezplotes patella Ehrenberg with special reference to the nuclear phenomena. Univ. Cal. Publ. Zodl., 35, 193-258. Valkanov, A. 1935. Untersuchungen tber den Entwicklungskreis eines Turbellarienparasiten (Monocystella Arndti). Z. Parasitenk., 7: 517-38. Visscher, J. P. 1927. Conjugation in the ciliated protozoén, Dileptus gigas, with special reference to the nuclear phenomena. J. Morph., 44: 383- 414, Weschenfelder, R. 1938. Die Entwicklung von Actinocephalus parvus Well- mer. Arch. Protistenk., 91: 1-60. Wichterman, R. 1937. Division and conjugation in Nyctotherus cordiformis (Ehr.) Stein (Protozoa, Ciliata) with special reference to the nuclear phenomena. J. Morph., 60: 563-611. Woodruff, L. L. 1925. The physiological significance of conjugation and endomixis in the Infusoria. Amer. nat., 59: 225-49. 1927. Studies on the life history of Blepharisma undulans. Proc. Soc. exp. Biol. N.Y., 24: 769-770. Woodruff, L. L., and H. Spencer. 1924. Studies on Spathidium spathula. Il. The significance of conjugation. J. exp. Zool. 39: 133-96. Zederbauer, E. 1904. Geschlechtliche und ungeschlechtliche Fortpflanzung von Ceratium hirundinella. Ber. dtsch. bot. Ges., 22: 1-8. Zuelzer, M. 1904. Beitrage zur Kenntnis der Difflugia urceolata Carter. Arch. Protistenk., 4: 240-95. CHAPTER XIII ENDOMIXIS LORANDE Loss WOODRUFF DURING RECENT YEARS the attention of students of the Ciliophora has been focused increasingly on macronuclear changes during the life of the individual cell and the life history of the species. This has revealed that the macronucleus is by no means a relatively passive agent in the nuclear complex, but rather a product of micronuclear activity which undergoes various radical transformations in contributing to the “somatic” functions of the cell until another is provided from the same source. MACRONUCLEAR REORGANIZATION As early as 1859, Stein noted a clear band, or Kernspalt, in the mactro- nuclei of hypotrichous ciliates, but chiefly within the past decade this has Figure 153. Stages in the progress of the reorganization bands in Aspidisca lynceus from the center of the horseshoe-shaped macronucleus to its tips. (From Summers, 1935.) been intensively studied by several investigators who have shown that Kernspalten are but a part of the regions now called reorganization bands, reconstruction bands, and so forth, which have an important function in the transformation of macronuclear substance at the time of division (see p. 21). Thus in certain cases material visibly passes from this region into the surrounding cytoplasm, as, for example, in ENDOMIXIS 647 Uroleptus (Calkins, 1919, 1930), Explotes patella (Turner, 1930), and As pidisca (Summers, 1935), while in others, such as Ez plotes worcesteri (Griffin, 1910), “extrusion bodies” are either absent or not described. Again, in some ciliates reorganization bands may not appear so clear-cut as in hypotrichous forms. Nevertheless the macronuclear material 1s eliminated as a residual mass, left between the two parts of a dividing macronucleus, as in Ancistruma and Conchophthirius (Kidder, 1933a, 1933b), Colpidium, Glaucoma, and Urocentrum (Kidder and Diller, 1934), and Blepharisma (D. Young, 1939); or beside the macronucleus D Fig. 154. Macronuclear dissolution in Blepharisma undulans. A, vegetative individual with dumb-bell-shaped macronucleus; B, early stage in the formation of the central bulb; C, central bulb fully formed; D, dissipation of the central bulb almost accomplished. (From D. Young, 1939.) during or after cell division, as in Chilodonella (MacDougall, 1936) and Colpoda (Kidder and Claff, 1938) (Figs. 153, 154). Variations in the details of such intrinsic macronuclear reorganiza- tion processes are indeed legion—reaching their climax, perhaps, in one or more of the types of “hemixis’’ described by Diller (1936)—but the accumulating data seem to justify the belief that they may be universal in the ciliates and perhaps represent at once a “‘purification” (Calkins) of the macronucleus, a regulation of the nucleo-cytoplasmic ratio, and a contribution of significance to the metabolic activities of the cyto- plasm. For summaries, reference may be made to Summers (1935) and Kidder and Claff (1938). Apparently, intrinsic macronuclear reorganization phenomena dur- ing the division of the cell suffice, in most species at least, for the con- 648 ENDOMIXIS tinued well-being of the race, but provisions are also made for the periodic destruction of the macronucleus and its replacement from the micronuclear reserve: in some species by endomixis and autogamy, and probably in all by conjugation. ENDOMICTIC PHENOMENA A petiodic replacement of the macronuclear apparatus, without syn- karyon formation, was described in Paramecium aurelia, and named en- A Figure 155. General plan of the usual nuclear changes during endomixis in Para- mecium aurelia. A, typical nuclear condition; B, degeneration of macronucleus (chro- matin bodies not shown) and first division of micronuclei; C, “climax’’: second division of micronuclei; D, degeneration of six of the eight micronuclei; E, division of the cell; F, first reconstruction micronuclear division; G, second reconstruction micro- nuclear division; H, transformation of two micronuclei into macronuclei; I, micro- nuclear and cell division; J, typical nuclear condition restored. (Constructed from the description and figures of Woodruff and Erdmann, 1914.) domixis, by Woodruff and Erdmann (1914). Following their account, in summary, endomixis in this species involves the resolution of the old macronucleus into chromatin bodies, which disintegrate in the cytoplasm, ENDOMIXIS 649 and the transformation of one or two of the products of the micronuclear divisions into new macronuclei, to reconstitute the normal vegetative apparatus when distributed by cell division (Figs. 155, 156). Immediately after this announcement, Hertwig (1914) described similar phenomena in P. aurelia as parthenogenesis, induced, he believed, by degenerative changes, and emphasized the fact that in his study of conjugation in this species (1889), he had noted stages in certain non- conjugants that were open to a similar interpretation. Thereafter en- AA MA iN Mh ME hi Figure 156. Possible methods of micronuclear and cell division at the climax of endo- mixis in Paramecium aurelia. tb is typical. (From Woodruff and Erdmann, 1914, p. 448.) domixis, or, if one prefers, diploid parthenogenesis, was reported by many investigators, including Erdmann and Woodruff (1916) in P. caudatum, Calkins (1915, 1919) in Didinium nasutum and Uroleptus mobilis, Moore (1924) in Spathidium spathula, Erdmann (1925) in P. bursaria, Woodruff and Spencer (1923) in P. polycaryum, Klee (1925) in Explotes longipes, Ivanié (1928, 1929) in Chilodonella uncinatus, Vorticella nebulifera, Euplotes charon, and E. patella, Man- well (1928) in Pleurotricha lanceolata, Diller (1928) in Trichodina sp., Chejfec (1928, 1930) in P. caudatum, Fauré-Fremiet (1930) in Zoothamnium alternans, Stranghoner (1932) in P. multimicronucleatum, Tittler (1935) in Urostyla grandis, Kidder (1938) in Paraclevelandia Simplex, and Gelei (1938) in Paramecium nephridiatum. In most of 650 ENDOMIXIS these studies, it must be admitted, the authors failed to follow the sequence of nuclear events in series of pedigreed animals, but fitted their findings in isolated animals into the picture of endomixis as originally portrayed. Three of these investigations are of particular significance at the moment. Diller (1928), in a study of “binary fission and endomixis in Tricho- dina from tadpoles,” gives a categorical account of the reorganization process. He shows the resolution of the macronucleus into chromatin bodies: ‘In most cases the macronucleus breaks up completely by form- ing numerous spherical bodies of varying sizes.” And the origin of the primordium of the new macronucleus is from a residual micronucleus: “The final eight products of the micronuclear divisions are originally all apparently similar. Seven of them, however, rapidly differentiate into macronuclear Anlagen, while one remains the functional micronucleus.” The process is ‘characterized by the absence of maturation spindles and synkaryon formation” (Fig. 157). Stranghéner (1932), in a detailed description of endomixis in Parame- cium multimicronucleatum, emphasizes the fact that “Im Gegensatz zur Conjugation bildet der Macronucleus bei der Auflosung keine wurst- formigen Schlingen,’’ and describes and figures the incorporation of chromatin spheres from the old macronucleus into the new one (Fig 158)e Kidder (1938) observes a “nuclear reorganization without cell divi: sion in Paraclevelandia simplex,’ in which the details of the process are unique but the end result is the same. The old macronucleus elim1- nates a large part of its chromatin and the remainder then becomes 1n- corporated with one product of a single micronuclear division, to con- stitute the primordium of the new macronuclear apparatus (Fig. 159). In this codperation between macronucleus and micronucleus, de- scribed by Strangh6ner and Kidder as endomixis, we seem to have, as it were, stages in the evolution of macronuclear metamorphosis intermedi- ate between the intrinsic changes evidenced by reorganization bands, direct elimination of material, and so forth, and the complete compe- tence of the micronucleus alone to form a new macronucleus during endomixis as it has been described in other species. That intrinsic reorganization is adequate for the continued life of the race appears to be evident from the study of Dawson (1919) on an Ta Tb 8a 8b Figure 157. Diagram of the normal process of endomixis in Trichodina sp. 1, normal vegetative animal; 2, macronucleus fragmented and disintegrating, and not shown in sub- sequent diagrams (the micronucleus has migrated to the other end of the body) ; 3, micronucleus divided the first time; 4, micronucleus divided the second time; 5, micro- nucleus divided the third time; 6, seven of the nuclei differentiating into macronuclear Anlagen, while the eighth remains the functional micronucleus which divides before each cell division; 7a and 7b, daughters resulting from the first cell division and having four and three macronuclear Anlagen respectively; 8a and 8b, daughters resulting from the second cell division (the 8b monomacronucleate individual is completely reorganized ) ; 9a and 9b, daughters resulting from the third cell division (growth of the macronucleus will reconstruct them into normal vegetative individuals). (From Diller, 1928.) Fig. 158. Endomixis in Paramecium multimicronucleatum, (From Stranghéner, 1932.) ENDOMIXIS | 653 amicronucleate race of Oxytricha hymenostoma, in which, obviously, a micronuclear reserve could play no part. Woodruft’s work (1935) on a race of Blepharisma undulans, without endomixis or autogamy, and D. Young's findings (1939) on the same race, showing the elimination of material from the macronucleus during division, support this thesis. However, these results might be anticipated because several species have been cultured for long periods without showing any evidence of a Figure 159. Endomixis in Paraclevelandia simplex. 1, pte-cystic form (the macronucleus differentiating into degenerating posterior, and reorganizing anterior chromatin; the mi- cronucleus in prophase) ; 2, later stage (the micronucleus in anaphase; further differentia- tion of macronucleus) ; 3, telophase of the micronucleus (note the smoothly granular an- terior half of the macronucleus) ; 4, fusion of daughter micronucleus with reorganized por- tion of macronucleus (posterior portion of macronucleus shrinking away from the old nuclear membrane; enlarged condition of the daughter micronuclei quite characteristic). (From Kidder, 1938.) reorganization process, either intrinsic or endomictic, in the free-living animals, and probably none occurs. As examples, reference may be made to the work on Spathidium spathula by Woodruff and Moore (1924), on Paramecium calkinsi by Spencer (1924), and on Didinium nasutum by Beers (1929). In regard to reorganization by endomixis, the culture of P. caudatum studied by Metalnikov (1937), and the (to date) thirty-three-year-old culture of P. awrelia at Yale University may be cited, unless the future should prove that autogamy, to the exclusion of en- domixis, occurs in these species (Woodruff, 1932). Thus in the species in which reorganizational phenomena occur, it 654 ENDOMIXIS appears that intrinsic reorganization, as well as endomixis, meet the nor- mal exigencies of existence and keep the race on the even tenor of its way. For significant possibilities of genetic change in heterozygous indi- viduals, however, reorganization is accompanied by synkaryon forma- tion, either in autogamy or conjugation. AUTOGAMY The first definite statement of autogamy in the ciliates was given in a brief article by Fermor (1913), who described the degeneration of the macronucleus and the origin of a new one from a synkaryon of micro- nuclear origin, during the encystment of Stylonychia pustulata. But the problem was not emphasized until Diller (1936) described autogamy in P. aurelia and stated: I have not been able to confirm the micronuclear behavior which Woodruff and Erdmann have described for endomixis in P. awrelia. In the failure of such verification I am inclined to deny the existence of endomixis as a valid reorganization process. I feel that Woodruff and Erdmann have combined stages of hemixis and autogamy into one scheme, “‘endomixis,” overlooking the maturation and syncaryon stages in autogamy. And in regard to his own earlier description of endomixis in Trichodina sp., Diller remarks, “It may be that hemixis and exconjugant stages were lumped together as ‘endomixis’ in this account’ (Figs. 160, 161). In enthusiasm for the concept of autogamy, it may be well not to exclude endomixis in P. aurelia—or Trichodina sp.—without careful consideration, although there is no inclination to deny that autogamy occurs in P, aurelia, in view of the combined data presented in the cytological study by Diller and the genetical studies of Sonneborn (1939a, 1939b, 1939c). However, Sonneborn’s observations were not made on the Yale race of P. awrelia nor on the mating-type variety which it represents, because there are as yet no known genes in this variety, and such tests therefore cannot be made. In the opinion of the writer, the crucial cytological stages are not absolutely demonstrated, in part because most of the animals were taken from mass cultures and relatively few from isolated lines or from pedigreed lines, and therefore the sequence of events was not determined from pedigreed serzes. To demonstrate satisfactorily the exact sequence, it is necessary to follow critically the nuclear behavior in series of pedi- Figure 160. Nuclear changes during autogamy in Paramecium aurelia. (From Diller, 1936.) TYPICAL INDIVIDUAL t ihre! | Figure 161. Hemixis. Diagram of the macronuclear behavior (exclusive of conjugation and autogamy) in Paramecium aurelia. The macronuclei are represented by large solid ovals; macronuclear fragments by smaller circles; micronuclei by small round dots; “Anlagen-like’’ macronuclei by stippled circles. The interrelationships of the various forms are indicated by arrows. (From Diller, 1936.) ENDOMIXIS 657 greed animals from day to day, as emphasized by Woodruff and Erd- mann (1914, pp. 457-72) and Beers (1935). The study of pedigreed series of animals, for example, precluded, it is believed, the possibility of “combining stages of hemixis and autogamy in one scheme.” Indeed, Erdmann and Woodruff (1916), contrasting endomixis in P. caudatum and P. aurelia, stated that they had ‘some data which suggest that under certain conditions merely a partial re- organization, not involving the formation of macronuclear Av/agen, Figure 162. Climax of endomixis in Paramecium aurelia. The old macronu- cleus is merely in the form of a membrane from which the numerous chromatin bodies have been ejected and are free in the cyto- plasm. Eight so-called re- duction micronuclei. (From Woodruff and Erdmann, 1914, plate 2.) may lead, at least temporarily, to the continuance of the life of the line.” This would appear to be Diller’s ““hemixis.” On the other hand, ‘overlooking the maturation and synkaryon stages’ is a different matter, as will be appreciated by anyone who has worked on the cytology of Paramecium. This may have occurred, even though Woodruff and Erdmann naturally ‘“‘expected”’ to find autogamy when they observed the primordia of macronuclei in non-conjugants. Their inability to find maturation spindles and synkaryon of course led them to coin the name endomixis for the process. But an equally plausible explanation, at least in the mind of the writer, as to why these investi- gators did not find such stages, nor even the paroral lobe, in which the synkatyon is characteristically located, according to Diller, is that these 658 ENDOMIXIS did mot occur in their material. None have been observed by other in- vestigators studying what they interpret as endomixis in various species. As already stated, none were found by Diller in endomixis in Trichodina. Certainly none occur in Paraclevelandia simplex, according to the clear- Figure 163. 1, Autogamy, Woodruff race (gamete nuclei in contact in the paroral cone at the right; five or six degenerating nuclei are visible; macronucleus in skein) ; 2, autogamy, Woodruff race (synkaryon formation; gamete nuclei enclosed within a common membrane; paroral cone; no degenerating micronuclei visible; macronucleus in skein) ; 3, autogamy, isolation, Philadelphia race (synkaryon, in paroral cone, in meta- phase of first division; no degenerating micronuclei seen; macronuclear skein frag- menting; a number of macronuclear bodies of various stages of degeneration present in the cell). Animals 1 and 2 are from mass cultures. (From Diller, 1936.) cut description of Kidder (1938). So from the latter account alone it 1s evident that endomictic phenomena actually do occur. Synkaryon forma- tion is not a necessary antecedent to the formation of a macronuclear primordium (Figs. 162, 163). But confining attention to Paramecium aurelia, the fact must be emphasized that almost the entire picture, and not merely the crucial detail of the presence or absence of a synkaryon, differs in the endomixis of Woodruff and Erdmann and the autogamy of Diller. In endomixis, ENDOMIXIS 659 maturation ‘“‘crescents’’ were not observed, and the elimination of chro- matin bodies was found to be the typical method of macronuclear de- struction. Only one among the many hundreds of endomictic animals studied by Woodruff and Erdmann showed even a slight simulation of the macronuclear ribbon-formation so characteristic of conjugating ani- mals, and also of autogamy according to Diller. This single animal, of the four-thousand-and-eighty-seventh generation, was figured as atypi- cal. However, eight years later Woodruff and Spencer (1922) found, on one single day in a subculture from this same pedigreed race at about the eight-thousand-nine-hundredth generation, several animals with rib- bon-like degenerating macronuclei. The publication of this exception brought a protest from Erdmann, who was convinced that conjugation must have occurred in the subculture. Now much of Diller’s work has been done on this same Yale race, and therefore it is clear that ribbon-formation does occur, other than at conjugation, in this race, under certain conditions. It is not clear how Diller’s culture conditions differ from those in the Yale Laboratory, where ribbon-formation has not been observed since the instance in 1922, referred to above. A clue may be afforded by De Lamater (1939), who found that different kinds of bacteria in the culture medium of this same race of Paramecium had marked effects on the macronuclear changes. It is possible that other types of bacteria or other environmental changes may underly the differences between endomixis and autogamy. PERIODICITY OF ENDOMIXIS Another important point is the rhythmic periodicity of endomixis ob- served by Woodruff and Erdmann (1914, 1916) and Woodruff (1917a, 1917b), which, according to Diller, is absent in autogamy. He says: “Under the conditions of my experiments, no regular periodicity in the incidence of autogamy was evident.” Woodruff and Erdmann (1914) and Woodruff (1917) definitely stated that the interendomictic periods in both P. awrelia and P. caudatum showed some variation in length and furthermore were somewhat modi- fied by environmental factors, but nevertheless were strikingly periodic— endomictic periods and interendomictic periods affording the rhythms in the division rate of pedigreed cultures. And this rhythmicality has appeared throughout the years in the culture of P. awrelia in the Yale 660 ENDOMIXIS Laboratory, whenever tests have been made; but now, in the thirty- third year of its life, with unimpaired vitality, the interendomictic periods seem to be slightly more variable in length (Fig. 164). A number of other investigators have studied the question of peri- odicity in this and other races of P. aurelia and P. caudatum, among them R. T. Young (1918), Jollos (1916, 1920), Erdmann (1920), Chejfec (1930), Galadjieff (1932), and, in particular, Sonneborn (1937a). Figure 164. Graph of the division rate of Paramecium aurelia, line III, subculture IE, averaged for five-day periods. Endomixis occurred during the periods indicated by an X. Note that the interendomictic periods exhibit some variation in length, and the final endomixis shown is deferred. (From Woodruff and Erdmann, 1914.) The latter compared the endomictic period in the Yale race of P. aurelia with that of another race under identical environmental condi- tions. Sonneborn shows that great variations may exist in the interendo- mictic interval, not only in different races, but even in the same race under carefully standardized conditions of daily isolation culture. So in regard to the periodicity of endomixis, it now appears that the limits of approximately 25 to 30 days and 40 to 50 fissions for P. aurelia, and 50 to 60 days and 80 to 100 fissions for P. caudatum, as originally announced, are somewhat too narrow and stereotyped. En- vironmental and racial factors play a still greater part than these investt- gators believed. But withal, the endomictic process does recur with con- siderable regularity when the environmental and genetic factors are uniform, and so must still be regarded as periodic. GENETICAL STUDIES ON ENDOMIXIS Genetical sttidies on endomixis include those by Erdmann (1920), Jollos (1921), Parker (1927), Caldwell (1933), Kimball (1937, 1939), and Sonneborn (1937b, 1939a, 1939b, 1939c). The results ENDOMIXIS 661 obtained by Sonneborn (1939), in particular, afford evidence that the preciseness of the ratios and the segregation of sex or mating types, following the reorganization process, is quite as regular and exact as after conjugation. Thus it would appear that autogamy and not endo- mixis is involved. Indeed, Sonneborn states that in one race of P. aurelia —not the Yale race nor the mating-type variety which it represents— the alternative between endomixis and autogamy was tested genetically by determining the genotypes following reorganization in clones of type Aa (genes determining mating types). Genetic analysis of the reorgani- zations showed that all the resulting lines are homozygous, half of them dominant and half recessive. From any one reorganizing individual both catyonides are of the same genotype. Thus, under these conditions (mass cultures at 31° C. and isolation lines at 27° C.), autogamy, not endo- mixis, takes place. Accordingly the combination of genetical and cytological data at pres- ent available justifies the conclusion that autogamy occurs, under cer- tain circumstances at least, in some races of P. aurelia. Granting this raises the question whether all the investigations reported on the physiology and genetics of endomixis actually are on autogamy, because Paramecium is the form that has been almost universally employed in such studies. If the accumulated data are really all in regard to autogamy, then the question is essentially one of name. On the other hand, if both endomixis and autogamy occur in Paramecium, then, for a time, confu- sion is worse confounded. Obviously, at present it is useless to attempt to generalize in regard to reorganization in Paramecium—that must await far more extended in- vestigation. However, the personal judgment of the writer, at the mo- ment, is that both endomixis and autogamy do occur in Paramecium— an opinion reached, it is believed impartially, from a consideration of the picture of the micronuclear divisions and the macronuclear destruc- tion, as he saw them in the original work on the reorganization process and as demonstrated in the cytological preparations of Diller. Certainly the two pictures presented are quite different; indeed, in many ways as different as the stages in endomixis and conjugation appeared in the original study. And Sonneborn (1939a) remarks: ‘Probably it will be found that autogamy and endomixis take place in different races or under different conditions.” The occurrence of these two processes, either in 662 ENDOMIXIS different races or in the same race, synchronously or otherwise, thus adds to the known repertoire of the versatile Paramecium. CONCLUSIONS A synoptic view of the rapidly accumulating data on macronuclear reorganization phenomena in the Ciliophora justifies, it is believed, the statement that these processes include intrinsic reorganization (reor- ganization bands, and so forth) , coéperation of macronucleus and micro- nucleus (endomixis), a new macronucleus of micronuclear origin (en- domixis), and a new macronucleus of synkaryon origin (autogamy and conjugation). These constitute a series of macronuclear metamorphoses of increasing complexity, affording progressively greater possibilities for the organism. LITERATURE CITED Beers, C. D. 1929. On the possibility of indefinite reproduction in the ciliate Didinium without conjugation or endomixis. Amer. Nat., 63: 125-29. — 1935. Structural changes during encystment and excystment in the ciliate Didinium nasutum. Arch. Protistenk., 84: 133-55. Caldwell, L. 1933. The production of inherited diversities at endomixis in Paramecium aurelia. J. exp. Zool., 66: 371-407. — 1915. Didinium nasutum: \. The life history. J. exp. Zool., 19: 225- 39. — 1916. General biology of the protozoan life cycle. Amer. Nat., 50: 257-70. Calkins, G. N. 1919. Uroleptus mobilis Engelm. I. Renewal of vitality through conjugation. J. exp. Zool., 29: 121-56. —— 1930. Uroleptus halseyi Calkins. Il. The origin and fate of the macro- nuclear chromatin. Arch. Protistenk., 69: 151-74. Chejfec, M. 1928. On the nuclear reorganization of Paramecium caudatum. Acta Biol. exp., 2: 89-121. —— 1930. Zur Kenntniss der Kernreorganizationsprozesse bei Paramecium caudatum. Arch. Protistenk., 70: 87-118. Dawson, J. A. 1919. An experimental study of an amicronucleate Oxytricha. I. A study of the normal animal with an account of cannibalism. J. exp. Zool., 29: 473-511. De Lamater, A. J. 1939. Effect of certain bacteria on the occurrence of endomixis in Paramecium aurelia. Biol. Bull. 76: 217-225. Diller, W. F. 1928. Binary fission and endomixis in the Trichodina from tadpoles. J. Morph., 46: 521-52. —— 1936. Nuclear reorganization processes in Paramecium aurelia, with descriptions of autogamy and ‘“‘hemixis.” J. Morph., 59: 11-67. ENDOMIXIS 663 Enriques, P. 1916. Duemila Cinquecento generazioni in un infusorio, senza coiugazione né partenogenesi, né depressioni. R. Accad. Sci. Bologna, Ser. VII. Erdman, R. 1920. Endomixis and size variation in pure bred lines of Para- mecium aurelia, Roux Arch. EntwMech. Organ., 46: 85-148. — 1925. Endomixis bei Paramecium bursaria. S. B. Ges. natur. Fr. Berl., Jahrgang, 1925: 24-27. Erdmann, R., and L. L. Woodruff. 1916. The periodic reorganization process in Paramecium caudatum. J. exp. Zool., 20: 59-97. Fauré-Fremiet, E. 1930. Growth and differentiation of the colonies of Zoothamnium alternans. Biol. Bull., 58: 28-51. Fermor, X. 1913. Die Bedeutung der Enzystierung bei Stylonychia pustulata. Zool. Anz., 42: 380-84. Galadjieff, M. 1932. Sur le probleme de l’immortalité des Protozoaires (Vingt ans de culture de l’infusoire Paramecium caudatum sans conjugation). Bull. Acad. Sci. U.R.S.S., VII série, Classe Sci. Math. Nat. Gelei, J. von. 1938. Beitrage zur Ciliatenfauna der Umgebung von Szeged. VII. Paramecium nephridiatum. Arch. Protistenk., 91: 343-56. Griffin, L. E. 1910. Ewplotes worcester sp. nov. II. Division. Philipp. J. Sct., I SLD=3 6: Hertwig, R. 1889. Uber die conjugation der Infusorien. Abh. Bayer. Akad. Wiss. Cl 2ai7e —— 1914. Uber Parthenogenesis der Infusorien und die Depression- szustande der Protozoen. Biol. Zbl., 34: 557-81. Ivani¢, M. 1928. Uber die mit den Parthenogenetischen Reorganisations- prozessen des Kernapparates verb. Vermehrungscysten v. Chilodon uncinatus. Arch. Protistenk., 61: 293-348. —— 1929. Zur Auffassung der sog. bandférmigen Grosskerne bei Infusorien ; zugleich ein Beitrage zur Kenntnis der sog. parthenogenetischen und thnen ahnlichen Reorganisationsprozesse des Kernapparates bei Proto- zoen. Arch. Protistenk., 66: 133-59. Jennings, H. S. 1929. Genetics of the Protozoa. Bibliog. Genet., 5: 105-330. Jollos, V. 1916. Die Fortpflanzung der Infusorien und die potentielle Un- sterblichkeit der Einzellen. Biol. Zbl., 36: 497-514. —— 1920. Experimentelle Vererbungsstudien an Infusorien. Z. Indukt. Abstamm.- u. VererbLehre, 24: 77-97. Kidder, G. W. 1933a. Studies on Concophthirius mytili, 11. Conjugation and nuclear reorganization. Arch. Protistenk., 79: 25-49. 1933b. On the genus Ancistruma Strand. Il. The conjugation and nuclear reorganization of A. isseli. Arch. Protistenk., 81: 1-18. 1938. Nuclear reorganization without cell division in Paraclevelandia simplex, an endocommensal ciliate of the wood-feeding roach, Panesthia. Arch. Protistenk., 91: 69-77. 664 ENDOMIXIS Kidder, G. W., and C. L. Claff. 1938. Cytological investigations of Colpoda cucullus. Biol. Bull., 74: 178-97. Kidder, G. W., and W. F. Diller. 1934. Observations on the binary fission of four species of common free-living ciliates, with special reference to the macronuclear chromatin. Biol. Bull., 67: 201-19. Kimball, R. F. 1937. The inheritance of sex at endomixis in Paramecium aurelia. Proc. Nat. Acad. Sci., Wash., 23: 469-74. —— 1939. Change of mating type during vegetative reproduction in Para- mecium aurelia. J. exp. Zool., 81: 165-79. Klee, E. E. 1925. Der Formwechsel im Lebenskreis reiner Linien von Explotes longipes. Zool. Jarhb., 42: 307-66. MacDougal, M. S. 1936, Etude cytologique de trois espece du genre Chilodo- nella strand. Bull. biol., 70: 308-331. Manwell, R. D. 1928. Conjugation, division, and encystment in Pleurotricha lanceolata. Biol. Bull. 54: 417-63. Metalnikov, S. 1937. Le Role et la signification de la fécondation. Scientia, Milano, Mars, 1937: 167-76. Moore, E. L. 1924. Endomixis and encystment in Spathidium spathula. J. exp: Zool) 39: 317237 Parker, R. C. 1927. The effect of selection in pedigree lines of Infusoria. J. exp. Zool., 49: 401-39. Sonneborn, T. M. 1937a. The extent of the interendomictic interval in Paramecium aurelia and some factors determining its variability. J. exp. Zool., 75: 471-502. — 1937b. Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. Nat. Acad. Sci., 23: 378-385. 1939a. Sexuality and related problems in Paramecium. Coll. Net, 14: 77-84. —— 1939b. Genetic evidence of autogamy in Paramecium aurelia. Anat. Rec:,. 7585..(Supp!s) —— 1939c. Paramecium aurelia: mating types and groups; lethal interac- tions; determination and inheritance. Amer. Nat., 73: 390-413. Spencer, H. 1924. Studies on a pedigree culture of Paramecium calkinsi. J. Morph., 39: 543-551. Stein, F. R. 1859. Der Organismus der Infusionsthiere. I. Abth. Leipzig. Stranghéner, E. 1932. Teilungsrate und Kernreorganisationsprozess bei Para- mecium multimicronucleatum. Arch. Protistenk., 78: 302-60. Summers, F. M. 1935. The division and reorganization of Aspidisca lynceus, Dio phrys appendiculata, and Stylonychia pustulata, Arch. Protistenk., 85: 175-208, Tittler, J. A. 1935. Division, encystment and endomixis in Urostyla grandis, with an account of an amicronucleate race. Cellule, 44: 189-218. Turner, J. P. 1930. Division and conjugation in Evplotes patella, with special ENDOMIXIS 665 reference to the nuclear phenomena. Univ. Cal. Publ. Zool., 33: 193-258. Woodruff, L. L. 1917a. Rhythms and endomixis in various races of Para- mecium aurelia. Biol. Bull., 33: 51-56. —— 1917b. The influence of general environmental conditions on the periodicity of endomixis in Paramecium aurelia. Biol. Bull., 33: 437-62. —— 1932. Paramecium aurelia in pedigree culture for twenty-five years. Trans. Amer. micr. Soc., 51: 196-98. —— 1935. Physiological significance of conjugation in Blepharisma undulans. J. exp. Zool., 70: 287-300. Woodruff, L. L., and R. Erdmann. 1914. A normal periodic reorganization process without cell fusion in Paramecium. J. exp. Zool., 17: 425-517. Woodruff, L. L., and E. L. Moore. 1924. On the longevity of Spathidium spathula without endomixis or conjugation. Proc. Nat. Acad. Sci. Wash., 10: 183-86. Woodruff, L. L., and H. Spencer. 1922. On the method of macronuclear dis- integration during endomixis in Paramecium aurelia, Proc. Soc. exp. Biol. N.Y., 19: 290-91. —— 1923. Paramecium polycaryum, sp. nov. Proc. Soc. exp. Biol. N.Y., 20: 338-39. —— 1924. Studies on Spathidium spathula. Ul. The significance of conju- gation. Jour. exp. Zool., 39: 133-96. Young, D. 1939. Macronuclear reorganization in Blepharisma undulans. J. Morph., 64: 297-353. Young, R. T. 1918. The relation of rhythms and endomixis, their periodicity and synchronism in Paramecium aurelia. Biol. Bull., 35: 38-47. CHAPTER XIV. SEXUALITY IN UNICELLULAR ORGANISMS T. M. SONNEBORN AMONG UNICELLULAR ORGANISMS, many different sexual conditions have long been known; descriptions of these are readily accessible (e.g., Calkins, 1926). In recent years, surprising discoveries concerning sexuality have been reported in some of the commonest and most studied unicellular organisms, such as Polytoma and Chlamydomonas among the flagellates, and Paramecium and Euplotes among the ciliates. In this chapter an attempt will be made to set forth this recent work and to examine critically the current interpretations of it. The work on the flagellates began earlier and has been carried fur- ther than the work on the ciliates; it will therefore be presented first. Although in both classes of organisms investigations of similar nature have been pursued on a number of forms (see especially Moewus, 1935a, 1935b, 1935c, 1937a), they have been carried further on Chlamydo- monas among the flagellates and on Paramecium among the ciliates. As these show essentially the same general relations as do other species of the same classes, the following account will be confined in the main to these two genera. SEXUALITY IN Chlamydomonas The recent work on Chlamydomonas has appeared in a series of ex- tensive and detailed studies by Moewus since 1932 (Moewus, 1933, 1934, 1936, 1937b, 1938a, 1938b, 1939a, 1939b, 1939c; Hartmann, 1932, 1934). From the beginning, its great importance with relation to problems of sexuality has been apparent and, as the work progressed, these relations have been repeatedly emphasized by Hartmann, Moewus, and others. However, in order to approach the facts without theoretical bias, they will be restated here in a purely descriptive way. Six species of Chlamydomonas have been most fully investigated: C. braunit, C. dresdensis, C. eugametos, C. paupera, C. paradoxa, and C. SEXUALITY 667 pseudo paradoxa. In all species the vegetative cells and gametes have a haploid set of ten small dot-like chromosomes. Under appropriate con- ditions, differing in different species and races, the vegetative cells pro- duce or become gametes that copulate and form a diploid zygote cyst. (In some species, vegetative cells function as gametes; in others, gametes differ from vegetative cells.) Under certain conditions, maturation divi- sions, restoring the haploid condition, take place in the cyst. The reduced cells emerge from the cyst and each gives rise by vegetative multiplica- tion to a clone. THE KINDS OF GAMETIC DIFFERENCES OBSERVED IN Chlamydomonas The basic problem of sexuality in unicellular organisms is whether the copulating or conjugating cells regularly differ from each other. As will appear at once, certain differences are found only in some species or races, not in others; while other differences seem to be of general oc- currence. Morphological differences between copulating cells or gametes.—In C. coccifera (Moewus, 1937b), the copulating pairs invariably consist of a large, nonflagellated gamete and a small, flagellated one. In C. braunii, both copulants are flagellated, but one is always much smaller than the other. In the remaining species, there is no regular morpho- logical difference between copulating gametes. Nevertheless, in particu- lar pairs of at least certain species (e.g., C. ewgametos; Moewus, 1933), one gamete may be as much as twice as large as the other, while in other pairs of the same species no size difference appears. All possible kinds of gamete combinations are found: large with large, large with small, and small with small. Finally, in species like C. pseudo paradoxa (Hartmann, 1934), the gametes are regularly smaller than vegetative cells, though the two gametes do not ordinarily differ from each other. Functional differences between gametes—In C. coccifera (Moewus, 1937b), the large gametes lack flagella, and the small ones retain them. Consequently, the small gametes are more active and must move toward the larger ones to accomplish copulation. Further evidence of the greater activity of the smaller gamete appears during copulation, for its contents regularly pass into the larger gamete. Less functional differentiation appears in C. braunii; here both gametes are flagellated and active, but during copulation the smaller gamete regularly empties into the larger 668 SEXUALITY one. This same functional difference occurs also in those pairs that differ markedly in size in C. ewgametos; but not in the usual pairs, in which the gametes are alike in size (Moewus, 1933). Nor does it appear in other species in which the gametes are morphologically isogamous. Func- tional differentiation thus appears to be strictly correlated with morpho- logical differentiation, occurring only when one gamete is flagellated and the other not, or when one is much larger than the other. Further, both morphological and functional differentiation may exist between gametes in some copulating couples and not in others of the same species (C. eugametos, Moewus, 1933). Physiological differences between gametes.—In this section will be given only the general evidence of physiological differentiation between gametes, reserving for later consideration the question of the nature of such differentiation. If copulating gametes are not diverse physiologically, then any two gametes can copulate with each other; but if they are regu- larly diverse, there must be at least two kinds of gametes, with copula- tion taking place between gametes of different types. The basic question, therefore, is simply whether or not any two gametes of a species can copulate with each other. In some species and varieties (C. coccifera, C. braunu, C. paupera, C. eugametos f. typica and simplex, C. paradoxa, and C. pseudopara- doxa from Coimbra, Portugal; Moewus, 1933; Hartmann, 1934; Moewus, 1936, 1937b, 1938a), the answer to this question is simple and definite. Gametes produced within a clone do not copulate with each other, but they do copulate with gametes produced by certain other clones. In these races, therefore, the copulating gametes must always be physiologically diverse. Moreover, this diversity is not invariably associated with morphological or functional diversity, for five of the seven races showing this phenomenon have morphologically and func- tionally isogamous gametes. To the same category belongs a race of C. pseudoparadoxa, from Giessen; but the physiological difference is less apparent, requiring special methods to bring it to light. In this race, copulation does not normally occur either between gametes of the same clone or between gametes of different clones. Gametes of C. pseudoparadoxa are recog- nizable by their small size. Moewus (in Hartmann’s article, 1934) found that the noncopulating gametes of the Giessen race could be rendered SEXUALITY 669 capable of copulating by subjecting them to filtrates from gamete cul- tures of the Coimbra race of the same species. (See 675 ef seq. for a further account of these filtrates.) However, filtrates from any one Coimbra clone would activate some of the Giessen clones, but not all. The remaining Giessen clones required for activation treatment with a filtrate from a different Coimbra clone, one which would copulate with the first Coimbra clone. The Giessen gametes would now copulate only if a clone activated by the one filtrate was mixed with a clone activated by the other filtrate. Thus the Giessen clones are of two diverse physio- logical types, and copulation occurs only between the two types, not between gametes of the same clone. In all the races thus far considered, there are regularly physiological differences between copulating gametes, which are invariably members of different clones. In the remaining three races investigated by Moewus (1934, 1938a), C. eugametos f. subheteroica and f. synoica and C. dresdenis, copulation occurred regularly among gametes of any one clone. Among these three races, the situation in C. eugametos f. sub- heteroica is unique. In any culture relatively few cells copulate. If the cells left over after copulation has ceased in a culture are mixed with the left overs from cultures of other clones, some of the mixtures will exhibit typical copulation. Exhaustive analysis of many clones shows that the same results hold here for the left overs as for entire clones of the species previously discussed. The left overs of any one clone are always of the same physiological type, but other clones yield left overs of a different type. There are just two kinds of clones, differing in the type of left overs they produce. Left overs of one type copulate only with left overs of the other type. From these observations, Moewus (1934) concludes that each clone produces both types of gametes, but always one in much greater fre- quency than the other. Some clones regularly produce mostly one kind of gamete; other clones regularly produce mostly the other kind of gamete. Copulation then takes place within a clone until all the rarer type of gametes have found partners, so that all the left-over cells are of the prevailing type. Moewus (1934) reports that the behavior of this race can be made to simulate that of those previously discussed by subjecting the cultures to very dilute formalin or acetaldehyde. With this treatment, the cultures no longer yield copulation within a clone, 670 SEXUALITY but the clones are divisible into two physiological types, with copula- tion taking place only between the two types of clones. Under these conditions, the type of each clone is identical with the type of its left- over cells under normal conditions. These results of Moewus show that different clones of C. evgametos f. subheteroica produce different kinds of gametes and that in mixtures of left-over cells and in mixtures of chemically treated cultures copula- tion is between physiologically diverse types of gametes. His further conclusion that the copulation occurring under normal conditions within each clone is also between the same two types of gametes has not been directly demonstrated, though it appears a reasonable inference. The possibility that copulation is here taking place between gametes of the same type has not been excluded. Convincing evidence on this important question calls for direct tests with “split pairs,” as performed by Kimball (1939a) on Paramecium (see p. 697). The other two races showing copulation within a clone, C. ewgametos f. synoica and C. dresdensis, differ from C. ewgametos £. subheteroica in three respects: (1) copulation occurs presumably on a much larger scale within a clone, the proportion of left overs being relatively small; (2) in different cultures of the same clone the left overs may be of different types; (3) no environmental means of suppressing or decreasing copula- tion within the clone has been reported. Otherwise the observations re- ported for these two races agree with those reported for C. ewgametos f. subheteroica. Mixture of the left overs from different cultures in all possible combinations of two shows that in each race there are two kinds of left-over gametes, with copulation occurring only between the two kinds. The same uncertainty attaches here to Moewus’s conclusion that the copulations within a clone are likewise between gametes of different type. However, the interpretation is here rendered more probable, for it has been shown that both types are producible within a single clone, different cultures of the same clone yielding left overs of different type. The question at issue here is of theoretical importance. Does copula- tion ever take place between cells that are physiologically as well as morphologically and functionally identical? The preceding survey of the conditions in various species and races of Chlamydomonas shows that morphological and functional differences are frequently lacking, but that at least in some of these cases physiological differences do exist when no SEXUALITY 671 others are apparent. The only cases about which a reasonable doubt may still be entertained are those in which copulation occurs within a clone. This matter ‘has been intensively studied by Pascher (1931) in C. pau pera and by Pringsheim and Ondraéek (1939) mainly in Polytoma. Their observations are in fundamental disagreement with those of Moewus, leading them to conclude that these forms show copulation without any physiological sex differentiation. Further, Pringsheim and Ondracek could not confirm Moewus’s observation that the cells left over after copulation were unable to copulate with each other because they were all of one physiological type. They attribute the cessation of copulation to a change in the chemical conditions in the culture, rendering it un- suitable for copulation. Appropriate modification of the conditions leads to resumed copulation. They therefore deny the validity of the left-over technique for the analysis of the question at issue. The reader is referred to their article for a detailed criticism of numerous points in Moewus’s work. Moewus (1940) has replied to these criticisms in an article that appeared too late for inclusion in this review. THE NATURE OF THE PHYSIOLOGICAL DIFFERENCES BETWEEN GAMETES IN Chlamydomonas As the union of gametes in Chlamydomonas is obviously a sex act, the physiological differences that usually, if not always, characterize the gametes may be considered sex differences. This section will set forth the number and interrelations of these sexes, their chemical characteristics, and their possible relation to male and female. The number of sexes and their interrelations —The system of breed- ing relations in Chlamydomonas was discovered by mixing together, in combinations of two, cultures of the sexes isolated from the species and varieties of Chlamydomonas examined. The two species C. paradoxa and C. pseudo paradoxa constitute one interbreeding system, and the four species C. engametos, C. paupera, C. braunii, and C. dresdensis consti- tute another interbreeding system; but these two systems of species will not breed with each other. Among the first group of species, Moewus (Hartmann, 1934) found two sexes in each of two races (from Giessen and from Coimbra) of C. pseudoparadoxa and in C. paradoxa. In order to discover whether the two sexes were alike in the three races, they were matched up in all 672 SEXUALITY possible combinations. As appears in Table 7, no two of the sexes are exactly alike. For example, the sexes I have designated A and B differ in that A will copulate with C while B will not; A and C differ in that they copulate with each other, although they are alike in their re- actions to the other four sexes; and so on. The direct inference naturally drawn from these observations is that there are six diverse sexes in this group of races, and I have therefore designated them by six different letters. The question of whether such multiple sex systems can be reduced TABLE 7: BREEDING RELATIONS IN Chlamydomonas paradoxa AND C. pseudo paradoxa* Species oe Pola C. pseudoparadoxa a y i Source Coimbra | Giessen | Giessen | Coimbra Sex A B Cc D E Nis C. para- doxa A = _ a i. ae we Coimbra | B — = = = ae a C. pseudo. | Giessen | C + — - + + + paradoxa | Giessen | D + + + — = ss Coimbra | E + + + _ = on C. paradoxa le + + + + = = * +. = copulation; — = no copulation. Data by Moewus (Hartmann, 1934). The designations of the sexes differ from those used by Moewus. to two sexes, male and female, will be taken up later. Moewus holds that they can and designates them otherwise than I have done in Tables 7, 8, and 9. A similar system of multiple sexes is indicated by the breeding rela- tions in the second group of species, as shown in Table 8, constructed from the data of Moewus (Hartmann, 1934; Moewus, 1936, 1937b, 1938a). Two sexes have been isolated in C. sp. (coccifera?), C. braunit, and dresdensis, six in C. paupera, and eight in C. eugametos. Not all of these are diverse, however. The two in C. dresdensis are the same as two in C. paxpera and two in C. eugametos; four in C. engametos are reduci- ble to two diverse sexes identical with two others in C. paupera; and the remaining two in C. paupera are identical with two others in C. SEXUALITY 673 eugametos. Altogether, there are eight diverse sexes designated in the table by the letters G to O. Two of these occur in C. braunii, two in C. dresdensis, six in C. paupera, and six in C. ewgametos. Their interrela- tions are shown in condensed form in Table 8. G copulates with all others but H, H with all others except G and J, and so on. The breeding relations in this system of eight sexes is in general similar to the rela- tions shown by the six sexes of the first group of races (Table 7): any TABLE 8: BREEDING RELATIONS IN Chlamydomonas sp. (coccifera?), C. braunii, C. dresdensis, C. eugametos AND C. paupera* Sx G H J K L M N O Se a ee ee eee es | a ee ree eae | 2 Sees ee eae ae ae ae ee Se |, TS ae eae ag es i a areas er ee eee eT eon Ges: | Le a aa Se ee | i ee eo) een Tees Sea ea Sse * + = copulation; — = no copulation. In each species the sexes found are as follows: in both C. sp. and C. braunii, sexes G and O; in C. dresdensis, sexes H and N;; in both C. exgametos and C. paupera, sexes H, J, K, L, M, and N. In C. exgametos sexes H and N occur in form typica, J and M in form simplex, K and L in both forms subheteroica and synoica. Data from Moewus, 1936, 1937b, 1938a; and Hartmann, 1934. The designations of the sexes in this table differ from those used by Moewus and Hartmann. Not all combinations between the sexes in different varieties and species have been reported (e.g., the sexes in C. sp. were tested only with the sexes of C. braunii; and C. braunii was tested with all others except C. paupera), but every possible combination of sexes was made with at least one representative of the sexes. sex copulates with any other sex in the group except that sometimes copulation will not take place with the sex next above or below it in the table. Nature of the sex differences——tIn the preceding section the sexes were defined in terms of the sexes with which they copulate. Two cul- tures are of the same sex if they do not copulate with each other and if each does copulate with all the sexes with which the other copulates. Two cultures are of different sex if they copulate with each other, or if one 674 SEXUALITY copulates with one or more sexes that the other does not copulate with. Thus the primary differences among the sexes lie in these breeding rela- tions. There are at least two further kinds of sex differences that throw much light on the nature of the sexes in Chlamydomonas. The first of these involves the intensity of the mating reaction. It is known that algal gametes of certain species (including Chlamydo- monas) form groups as a preliminary to copulation (Fig. 165). When ripe cultures of gametes that can copulate with each other are mixed together, the gametes at once form clusters of as many as 100 or more a = Rb ot Y iam + fe % Figure 165. Group for- mation in Chlamydomonas, showing the groups formed in a mixture of cells dif- fering in sex.- (From Moewus, 1933.) gametes. Within the clusters the gametes pair off, with the result that the cluster disintegrates into copulating pairs. The size of the initial clusters is partly determined by the number of gametes per unit of volume. When this concentration is uniform (e.g., 2 < 10° gametes per cc.), the size of the clusters depends upon which two sexes are present in the mixture. Certain combinations of sexes yield groups of 100 or more gametes, others give groups of but 10 to 20 gametes, others give only pairs, and of course some do not even give pairs. These four grades of reaction have been designated as 3, 2, 1, and 0 respectively, and the intensities of the reaction of the sex mixtures shown in Table 8 are given in Table 9. As shown in Table 9, the same sex may give different grades of reaction in mixtures with different sexes: thus G reacts to L, M, N, and O with SEXUALITY 675 intensity 3, to K with intensity 2, to J with intensity 1, and not at all to H. Moreover, the weak reaction of G with J is not due to the weak- ness of J, for J reacts with intensity 3 in mixture with L, while the latter gives but a weak reaction with N. Consequently the strength of the reac- tion is not a general characteristic of a given sex, but depends in some way on the particular combination of sexes. When the sexes are arranged as in Tables 8 and 9, the differing intensities of reaction fall into a defi- nite system: the strength of reaction between sexes increases with their TABLE 9: GRADES OF SEX REACTION IN MIXTURES OF SEXES G TO O FROM THE Chlamydomonas SPECIES C. braunii, C. dresdensis, AND C. eugametos (FORMS TYPICA, SIMPLEX, SUBHETEROICA, AND SYNOICA) Sex G H I K 1G M N O D fo) fo) I 2 3 3 3 3 ical fo) fe) fe) I 3 3 3, 3 I I fo) fo) O 3 3 3 3 K 2 I fo) O 2 3 3 3 L 3 3 3 2 ) o I 2 M 3 3 3 3 fo) ) O I N 3 3 3 4 I fo) fo) fe) O 3 3 3 3 2 I o o * Mixtures made from cultures with cells in concentration of 2 x 108 per cc. O = no reaction; 1 = pairs only; 2 = clumps of 10 to 20 cells; 3 = clumps of 100 or more cells. Data from Moewus, 1938a. The designations of the sexes differ from those used by Moewus. distance apart in the table until the maximum reaction (grade 3) 1s reached. These quantitative differences in intensity of sex reaction sug- gest that the fundamental differences among the sexes are also quantita- tive, a suggestion strikingly confirmed by studies of Moewus on the chemical basis of the sex reaction, as will now be set forth. The culture fluid in which ripe gametes are living has been shown, in a number of algae, to contain material (‘‘sex stuffs’) capable of af- fecting the sexual behavior of other gametes. In Chlamydomonas, Moewus (1933, and later) obtained this material free from the or- 676 SEXUALITY, ganisms that produce it, by means of filtration and centrifugation, and found it to have two striking effects. Gametes grown in the dark are incapable of copulating, but treatment with the sex stuff from a ripe culture of gametes of the same sex rendered them capable of copulation. It will be recalled that activation of non-reactive gametes by filtrates from cultures of reactive gametes has previously been referred to (pp. 668- 669) as the method employed in activating the peculiar gametes of the Giessen race of C. pseudo paradoxa, which are always normally nonreac- tive. This situation differs from most of the others described by Moewus in that activation is here brought about by the sex stuff from gametes of a different sex. Reference to page 672 and Table 7 will show that the sex stuff from sexes B and E were used to activate gametes of sexes C and D respectively. Similarly Moewus (1934) states that filtrates from sex K can activate gametes of sex H. This raises the question of how wide a range of sexes can be activated by sex stuffs from any one sex. Moewus (1939a), in a discussion of those sexes which I have designated G to O, states that each sex can be activated by filtrates only when they are derived from active gametes of the same sex. The earlier results with C. pseudo paradoxa and C. eugametos (Hartmann, 1934; Moewus, 1934) do not agree with this generalization. The second effect is observed when reactive gametes of one sex are added to sex stuffs obtained from gametes of certain other sexes. The introduced gametes form groups or clusters as if they were about to copulate, but eventually the clusters disintegrate without copulation tak- ing place. This happens when reactive gametes of one sex are added to sex stuffs from filtrates of reactive gametes of the other sex in the same race, as, for example, when gametes of sex J are added to filtrates from gametes of sex M, for sexes J and M are the two found in C. exgametos f. simplex. Whether similar effects of one sex stuff are producible on more than one other sex is not stated. The important point here is that sex stuffs can induce a sex reaction between cells alike in sex, but cannot induce them to copulate with each other. This indicates that there are two distinct processes in the sex act: the agglutinative sex reaction, and the actual fusion of cells and nuclei. The sex stuffs function in the for- mer but not in the latter process. The existence of sex differences with- out sex stuffs (or with sex stuffs in ineffective concentrations) is also shown by the gametes of C. pseudoparadoxa from Giessen (see p. 668 SEXUALITY 677 above). These observations by Moewus are perhaps subject to a very different interpretation. The agglutinative reactions observed between gametes of the same sex are weak and transient. Failure to copulate might well be due to this, rather than to the absence of an additional factor such as a sex difference. Similar weak mating reactions between cells of the same mating type were observed by Sonneborn (1937) in Para- mecium aurelia after the cells had been in contact with animals of an- other mating type. As in Chlamydomonas, the mating reaction was tran- sient and did not lead to copulation. Similar behavior was also observed by Sonneborn (1938a) when cultures of mating types II and V, belong- ing to non-interbreeding varieties, were mixed together. Here the mating reaction occurs between animals of different mating types, and yet they fail to conjugate. Further, cultures known to belong to two mating types that will interbreed under favorable conditions will, under other conditions, give a weak and brief mating reaction without proceeding to conjugate. In view of these observations, it appears to be still an open question whether the failure of copulation to take place between cells of the same sex in Chlamydomonas that have given a weak sex reaction with each other is due to the weakness of the reaction or to some other aspect of sex, different from the production of diverse sex stuffs. In later publications, Moewus (1938b, 1939a) reported the discovery of the chemical nature of the sex stuffs in the group of sexes G to O. The active stuffs for these eight sexes are all diverse percentage combi- nations of the cis and trans forms of the dimethyl ester of crocetin. The proportions are as follows: See ere ee yin 7 JG ae pen heyy mg AR MG Percentage Cis 95 85 UD 65 35 25 15 a5 Percentage trans 5 1S: 25 35 65 Wp) 85 95 The chemical nature of the sex stuffs aids greatly in understanding the breeding relations summarized in Tables 8 and 9. The order of sexes from G to O in the table is in the order of decreasing percentages of cis and increasing percentages of trans dimethyl crocetin. The dif- ference in percentage of either cis or trans between any two successive sexes in the table is always 10 percent, except between sexes K and L which show a difference of 30 percent. Copulation occurs between any 678 SEXUALITY two sexes differing by 20 percent or more in the production of either cis or trans dimethyl crocetin, but not if they differ less than this. Fur- ther, the intensity of the sex reaction (Table 9), as measured by the size of clusters, also depends upon the difference in proportions of cis and trans dimethyl crocetin produced by the two sexes under examination: a difference of 20 percent results in the formation of pairs only (grade one reaction); a difference of 30 percent yields clusters of 10 to 20 cells (grade 2); a difference of 40 percent or more yields clusters of 100 or more cells (grade 3). By introducing capillary tubes filled with known mixtures of cis and trans dimethyl crocetin into one edge of a drop of culture fluid and adding gametes of a known sex to the opposite edge of the drop, Moewus (1939b, 1939c) observed that the gametes aggregated at the open end of the capillary tube whenever it contained cis and trans dimethyl cro- cetin in proportions differing from those produced by the gametes by 19 percent or more, but not when the difference was less than this. More- over, the time required to obtain at the mouth of the tube an aggrega- tion of from 18 to 22 cells was from 200 to 254 seconds when the dif- ference in proportions was 20 percent, 140 to 180 seconds when the difference was 30 percent, and 80 to 109 seconds when the difference was 40 percent. The speed of aggregation increased with increasing cis/trans difference to 22 to 37 seconds with a difference of 90 percent; hence the sex stuffs are chemotactic substances, and the grades of sex reaction are indices of the speed of chemotaxis. Moreover, in any com- bination of gametes that will copulate, each sex secretes chemicals that attract the other and each reacts to the chemicals secreted by the other: both gametes thus attract and both respond. INTERPRETATION OF THE SEXUAL PHENOMENA IN Chlamydomonas The sexual phenomena in Chlamydomonas have been interpreted by Moewus and by Hartmann in accordance with Hartmann’s (1929) theory of sexuality. This theory may be formulated in the following series of propositions: 1. Sex is a universal biological phenomenon. 2. There are always two and only two sexes. 3. These two sexes are always male and female. 4. Male and female are qualitatively diverse. SEXUALITY 679 5. Every cell has the full Anlagen, or potencies, of both male and female. 6. These potencies are not localized in any one cell component, but are general properties of all the living material. 7. The sex manifested by a cell is the result of a weakening or strengthening of the expression of either the male or female potency. 8. This weakening or strengthening may be determined by outer conditions, or by developmental conditions, or by genetic factors. 9. The degree of weakening or strengthening depends upon the ef- fectiveness of the determiners listed in proposition 8. "10. This quantitative variation results in the appearance of each sex in a series of strengths called valences. 11. Sexual union takes place only under one or the other of two conditions: (a) when the gametes differ in sex; i.e., when one manifests a stronger male than female potency, the other a stronger female than male potency; (b) when the gametes are alike in sex, but very different in sex valence; e.g., when one is strong female, the other weak female; or when one is strong male, the other weak male. 12. Sexual union equalizes or reduces the tension resulting from dif- ference in sex or sex valence. The work on Chlamydomonas shows that physiological sex differences may exist in cases in which morphological sex differences are lacking. This is most clearly evident in those species and races in which each clone consists exclusively of one sex type. Here sexual union takes place only between gametes from different clones, the physiological sex dif- ference of which has been demonstrated. Moewus and Hartmann fur- ther hold that similar physiological sex differences distinguish the unit- ing gametes in species and races manifesting copulation among the members of a single clone. The evidence for this, drawn from experiments employing the “‘left-over’’ technique, has been set forth on pages 670- 671, along with the contrary evidence of Pascher and of Prings- heim and Ondra¢ek. There thus remains some doubt, even within the genus Chlamydomonas, as to whether sex union is invariably accom- panied by sex differences. Hartmann’s contention that sex differences are always male and fe- male could not at first be applied to Chlamydomonas. Moewus, there- fore, simply classified the sexes as plus (+) and minus (—). In the 680 SEXUALITY group of species shown in Table 7, sexes A, B, and C were called +, sexes D, E, and F, —. The three sexes of each type were assigned arbi- trary strengths or valences: A and F were assigned a valence of 3; B and E, 2; and C and D, 1. Copulation was thus held to take place either TABLE 10: SYSTEM OF MATING RELATIONS IN Chlamydomonas braunii, C. dresdensis, AND C. eugametos, SUMMARIZING THE OBSERVATIONS AND INTERPRETATIONS OF MOEWUS, 1937B, 1938A, 1938B, 1939A* Sex (Later View) Female Male Sex (Earlier View) = = Valence 4 3 3 I I 2 3 4 Sex as Designated in This Review G H J K L M N O Percentage cis 95/5 |85/15/75/25 65/35)35/65 25/75 15/85) 5/95 Percentage trans 4 | G | 95/5 ch I 2 3 3 3 3 3 | H | 85/15 OF" Or Por | Pray |eea eninge lemma (ame Female | + |——|—— ————_ $<, ——__ | ———. 2) Je 75/25 Ty fl) 2Op sO wait One nas 9.) 8 alas re | IS YRS 2 I fe) fo) 2 4 3 3 tr | L | 35/65 3 3 3 2 fo) fo) I 2 2 |M | 25/75 Bo 1 lee Sill Ole lela Masai lame Male = 3 | ING 157/85 34 S51) (SPB Ba Ko i eome amo AP OL 5/05 ae elie lie ea me eh) * The numbers 0, 1, 2, 3 in the body of the table give the intensity of the sex reaction; 0 = no copulation; 1 = pairs which form directly; 2 = preliminary clusters of 10 to 20 cells; 3 = pre- liminary clusters of 100 or more cells. Percentage cis/Percentage trans = the proportions of the sex stuffs, cis and trans dimethyl crocetin, produced by the gamets. between gametes differing in sex (i.e., between any + and any —), or between two gametes of the same sex differing by as much as 2 in valence (for example, between gametes of A and C, both of which are held to be +, because A is + 3 and C is + 1). Similar interpretations were put forth for the mating relations summarized in Tables 8 and 9. The sexes G, H, J, and K were denominated +- in sex, with valences of 4, SEXUALITY 681 3, 2, and 1, respectively; and the sexes L, M, N, and O were said to be — in sex, with valences of 1, 2, 3, and 4 respectively. In this group, Moewus (1937b, 1938a) later held that the sex previously called +- was female, the one called — male. These designations, together with other pertinent information on the strength of sex reaction and sex stuffs, are shown in Table 10. (Identification of male and female in the first group of races has not yet been reported. ) The remainder of the interpretation is largely genetic and will be dis- cussed here only insofar as appears necessary for a satisfactory under- standing of the general phenomena of sexuality and their relation to the theory of Hartmann. For further details the reader should consult Chapter XV, “Inheritance in Protozoa,” by H. S. Jennings. Moewus gives evidence for two series of multiple alleles affecting sex in the braunii, dresdensis, paupera, eugametos group of species: at one locus is a series of genes M1, M2, M3, and M4, determining the four valences of male gametes; at another linked locus is a series of genes F1, F2, F3, and F4, determining the corresponding four valences of female gametes. When crossing over takes place between these two loci, nuclei with a chromosome lacking both an M and F gene die, while those with both M and F genes survive. In the latter, if the valences are equal, sex is determined by nongenetic factors, the valence is unchanged, and both male and female gametes arise within a single clone; but if the valences are unequal, sex is determined by the gene of stronger valence and the resulting valence is the arithmetic difference between the valences of the two genes. In races such as C. exgametos f. subheteroica, in which each clone is always prevailing of one sex, another pair of genes determines which sex shall prevail. The genetic relations have not been worked out so fully in the paradoxa-pseudoparadoxa group of races, but there also multiple alleles are held to operate. Although evidence as to whether the + and the — genes are alleles has not been reported, observations on regular non-disjunction showed that the sex and valence resulting from the presence of two or more alleles was their algebraic sum. The various genes affecting sex are considered to be the sex realisators, in agreement with Hartmann. They are held to operate by acting on the underlying sexual Anlagen, or potencies, A and G, the genes of dif- fering valence acting on A and G to different extents. Sexual union then results when gametes differ in sex or in sex valence by as much as 2. 682 SEXUALITY Further, the grades of reaction shown in Table 10 are presumably indices of the magnitude of sex tension between the gametes. Difference of sex always results in a grade 3 reaction, except between gametes of the lowest valence. When alike in sex, a difference of 2 in valence is re- quired for a grade 1 reaction and a difference of 3 for a grade 2 reaction. Certain features of Moewus’s interpretation are of special interest: (1) his reduction of what appeared superficially to be many interbreed- ing sexes to but two, assumed to be qualitatively diverse; (2) his iden- tification of these two sexes with male and female; (3) his distinction between unions resulting from (qualitative) difference in sex and those resulting from (quantitative) difference in sex valence. The evidence and reasoning involved in these views is set forth in the following. The original basis for holding that only two sexes are present in each interbreeding system appears to be partly that the sexes were discovered in pairs. For example, in the paradoxa-pseudoparadoxa group of species (Table 7), Moewus found the two sexes here called A and F in C. paradoxa, B and E in the race of C. pseudo paradoxa from Coimbra, and C and D in the race from Giessen. Similar pairs of sexes were found in the other group of species: in C. brauniz, G and O; in C. dresdensis, H and N; in C. exgametos f. typica, H and N; in C. ewgametos f. sim- plex, J and M; in C. e~gametos f. subheteroica and f. synoica, K and L in each. Only in C. paw pera did an exception appear; the six types H, J, K, L, M, and N were all found together in a single natural source. From this point on, it appears to be simply assumed that the two sexes in one race are qualitatively the same as the two in any other race with which it can interbreed. If this assumption be accepted, then the remain- ing interpretation follows naturally. For example, if in C. paradoxa (Table 7) the two sexes A and F are designated + and — respectively, then in the Coimbra race of C. pseudo paradoxa B must be + and E —, for B copulates with F (—), not with A (-+); and E copulates with A (-++) not with F (—). Similarly, C and D in the Giessen race are + and — respectively. This is clear from their mating relations with B and E. The exceptional copulations between like-sexed gametes (A- with C+ and D— with F—) are interpreted as follows: A and C must both be the same sex (++) because of the mating relations set forth above; yet they must also be unlike in some sexual way, for they copulate with each other, though neither will copulate with others like itself; hence SEXUALITY 683 they must differ in degree of sex, or valence. In a similar way, the eight types G to O (Table 10) are reduced to two sexes, +- and —,, each ap- pearing in four valences. Here K and L are recognized as +- and — of the lowest valence because they give a weaker sex reaction (grade 2) with each other than do J and M (in C. evgametos f. simplex), or H and N (in C. dresdensis), or G and O (in C. braunii). Of the four grades of + gametes, G is most diverse from K because it gives the strongest reaction with it; hence G has the highest valence among the ++ gametes. Similarly, O is the — gamete of highest valence, and H and N are the next strongest + and — types (for they react less strongly with K and L than do G and O, while the others do not react at all with them). This leaves J and M intermediate between H and K and between L and N; and this is confirmed by their grades of reaction with G. The identification of -+ and — with female and male (in the ewga- metos—paupera group of species) is based on differences in morphol- ogy, activity, and function between the gametes in certain species, and on the assumed identity of the sex differences in all the species. In C. coccifera and C. braunii, as set forth on pages 667, 668, the two kinds of gametes differ markedly in size and behavior during copulation: the smaller gamete empties into the larger one. Further, in C. cocczfera the large gametes lack flagella and are nonmotile, while the small gametes have flagella and are motile. Moewus therefore holds that the large, nonmotile gametes of C. coccifera are eggs and so female, while the small, motile gametes are comparable to sperm and so are male. If this be admitted, then the large and the small gametes of C. brawnii are also female and male, even though both are flagellated, because in combina- tions between the two species copulation occurs only between large and small gametes. On the same grounds, the gametes of isogamous species are female and male, because of the two physiological kinds of gametes in C. ewgametos f. typica (types H and N), H will copulate only with the small gametes of C. brawnii while N will copulate only with the large ones. Thus the + sex has been identified with female and the — with male in C. e~gametos f. typica. And, since + and — were assumed to be the same in all races and species, female and male must be the same in all races and species. The copulations between female gametes (or between male gametes) of different races must then be consequences of difference of sex valence. 684 SEXUALITY Critique of the works of Moewus on Chlamydomonas.—Attention should be called to certain difficulties in some of the important features of Moewus’s interpretations and observations. 1. Identification of +- and — with female and male. Moewus’s iden- tification of + and — with female and male is based, as set forth above, on two points: the two sexes in anisogamous species, especially in C. coccifera, are male and female; the two sexes are the same in all races and species. The point has already been emphasized that the latter is an assumption, not a fact of observation. The interpretation of the two sexes in C, coccifera as male and female is based on the proposition that female gametes are distinguishable from male gametes by their passive role in copulation, their larger size, and their nonmotility. Though these criteria are widely accepted as valid, one may question whether the evi- dence warrants this. The passive rdle of the “female” gamete in copula- tion is shown by the fact that the ‘‘male” gamete empties its contents into the “female” gamete. Nevertheless, the same behavior takes place in a certain race of C. exgametos, in which Moewus (1933) showed that it is of no sexual significance for both the ++ and the — gametes may play either rdle in copulation. The same holds for difference in size: either the + or the — gamete of this race of C. eugametos may be twice as large as its mate. The difference in behavior is correlated with the difference in size, but neither is correlated with sex. One may doubt, then, whether these two criteria are of sexual significance in C. coccifera, since they are clearly not significant in C. ewgametos. The difference in motility is perhaps stronger evidence, for only the + gametes of C. coccifera are non-flagellated and these are generally considered to be comparable to eggs. It is important to keep clearly in mind that the use of the terms male and female for the gametes of all the races and species of Chlamydomonas rests finally on the single fact that the -+- gametes of C. coccifera lack flagella. Whether this is sufficient ground for holding they are female in the same sense as the eggs of higher organisms and for extending the terms male and female to the gametes in all other species of Chlamydomonas that interbreed with C. coccifera must be left to the judgment of the critical reader. The present author, in agreement with Kniep (1928) Mainx (1933) and others, holds that such facts constitute too slender a basis to justify an interpretation of such general theoretical significance. SEXUALITY 685 2. Reduction of systems of multiple gamete types to two sexes. As earlier set forth, the reduction of the multiple gamete types in an inter- breeding system to two sexes is based on the assumption that the two sexes in any one race or species are fundamentally the same as the two in any other race. In the case of C. paw pera, in which six types of gametes were found in the same natural source, it is presumably assumed that three races, each with the same two sexes, were here living together. It is important to recognize clearly that this view is based on Hartmann’s theory; it is not an observation or an induction from observation. Chem- ical analysis of the sex stuffs shows that reduction of the eight gamete types in the e~gametos-paupera group of species to two qualitatively di- verse sexes cannot be made on this basis, for the differences among the eight sex stuffs are exclusively quantitative. The ‘‘tension” assumed to bring the gametes together is held to be of two kinds. One kind is purely chemotactic and due to the sex stuffs; this brings the gametes into con- tact. It is clearly a quantitative phenomenon, dependent upon differences in relative proportions of cis and trans dimethyl crocetin. The other kind of tension determines whether gametes that have been brought into contact will unite in copulation. The evidence for this, together with considerations that render the conclusion less certain, was set forth on page 682. However, if an unknown factor determining union in copulation exists, it appears to act in the same quantitative way as the sex stuffs, for copulation takes place between any two gamete types that produce sex stuffs sufficiently diverse to attract each other. Consequently, there are no observations justifying or even suggesting the introduction of the concept of two qualitatively diverse sexes; all the observations point directly to a system of multiple, quantitatively diverse sexes. In one respect the preceding account may not fairly represent Moe- wus’s views. The two sex stuffs may be taken as indices of two qualita- tively diverse sex tendencies or potencies, cis demethyl crocetin being the manifestation of the + sex potency, and trans dimethyl crocetin of the — sex potency. In four of the eight types of gametes, the + sex potency prevails, for these types produce more cis than trans; and this prevails to different degrees in each type. In this sense these four types of gametes may be considered as different strengths or valences of the + sex. Cor- respondingly, the remaining four types could be considered four diverse valences of the — sex. This view is in accord with that part of Hart- 686 SEXUALITY mann’s theory which holds that both sex potencies reside in all kinds of gametes and that the sex of the gamete 1s simply the potency that prevails. Thus the qualitative sex difference is not segregated into different gam- etes and has nothing to do with copulation; all gametes have both quali- tative sex characters and differ only in the quantitative manifestation of one or the other. These quantitative differences alone determine copula- tion and sex reactivity. Conceivably two qualitatively diverse sexes might exist, one producing only cis, the other only trans dimethyl crocetin. But these have not been found. The observed gamete types are all quantita- tively diverse grades of intersexes, some prevailingly +_, others prevail- ingly —. Viewed in this way, the observations are in accord with part of Hartmann’s theory. 3. Difficulties in Moewus’s observations. There are certain difficulties in Moewus’s observations that raise serious questions concerning the reliablity and accuracy of his reports. Two of these must be mentioned. The first involves the apparently irreconcilable conflict between observa- tions of the consequences of non-disjunction of the sex chromosomes in crosses between C. paupera and C. ewgametos and the later discoveries of the sex stuffs. Moewus (1939a) reports that copulation takes place between gametes of the same sex when there is at least a difference of 2 in valence. By definition, gametes of valence 5 would copulate with gametes of valence 3, but not with gametes of valence 4; and gametes of valence 6 would copulate with those of valence 4, but not with those of valence 5. In a series of crosses and back crosses involving C. ewga- metos £. subheteroica (valence 1) in C. paupera (valence 3), Moewus (as reported by Hartmann, 1934) obtained through nondisjunction of the sex chromosomes clones that yielded gametes of valences 5 and 6, presumably recognized as such through the breeding tests mentioned above. Moewus (1939a) shows that copulation will take place only when there is a difference of at least 20 percent in the cis or trans dimethyl croe- tin produced. Valence 5, by definition, copulates with valence 3; but va- lence 3 produces 85 percent cis or trans dimethyl crocetin. This leads to the impossible conclusion that the valence 5 gametes produced 105 per- cent cis or trans dimethyl crocetin. Similarly, the valence 6 gametes would be required to produce 115 percent cis or trans dimethyl crocetin. This apparently irreconcilable contradiction in the reports raises the serious question of whether the reporting is accurate and reliable. SEXUALITY 687 The same question has been raised by Philip and Haldane (1939) from an analysis of data in many experiments by Moewus on crossing over and segregation in both Chlamydomonas and Protosiphon. These authors calculated that the chance of getting such close numerical agree- ment among the 22 experiments analyzed was once in 3.5 >< 10°? trials. According to them “if every member of the human race conducted a set of experiments of this type daily, they might reasonably hope for such a success once in 50,000 million years.’ They suggest that this im- plies a conscious or unconscious adjustment of observations to fit a theory and they call for repetition of the experiments by an independent worker. The failure of Pringsheim and Ondracek (1939) in their at- tempts to confirm certain parts of Moewus’s work, their numerous criti- cisms, the criticisms of Philip and Haldane, the internal inconsistencies in Moewus’s data, and the great theoretical importance of the work, all make independent repetition of the work an urgent need. SEXUALITY IN Paramecium AND OTHER CILIATE PROTOZOA The ciliate Protozoa differ from Chlamydomonas and the flagellates in their nuclear condition and in some features of the sexual phenomena. There are two kinds of nuclei: macronuclei and micronuclei. Ordinarily the macronucleus disappears during the sexual processes and a new one is formed from a product of the micronucleus. The micronuclei alone contain recognizable chromosomes and play the leading rdle in the nuclear changes involved in sexual processes. The vegetative individuals contain diploid micronuclei that undergo maturation with reduction of the chromosomes to the haploid condition during mating. In each con- jugant two reduced nuclei are formed. In most ciliates, both of these are functional: one remains within the animal in which it is formed and is known as the stationary pronucleus, or gamete nucleus; the other goes into the mate of this animal and is known as the migratory gamete nucleus, or pronucleus. The two nuclei present in each conjugant after exchange of pronuclei unite to form a synkaryon. Conjugation thus in- volves a reciprocal fertilization, both conjugants being fertilized, each by the other. The conjugants then separate and each reconstitutes a new nuclear apparatus and gives rise to progeny by repeated fissions. (See Chapter XII. ) The mating process is somewhat different in the peritrichous ciliates. 688 SEXUALITY Unlike most other ciliates, in the peritrichs the two mates differ greatly: one is sessile and large, the other is motile and much smaller. Of the two reduced nuclei formed in each mate, only one is functional: one of those formed in the microconjugant wanders into the macroconjugant and unites with one of its nuclei. The other nuclei degenerate, as does the remainder of the microconjugant. Thus only one individual results from the mating act and this one then reproduces by repeated fissions. Obviously the phenomena of sexuality are different in the Peritrichida from what they are in other ciliates. In the following, attention will be directed chiefly toward these other ciliates, of which Paramecium is an example. For both kinds of ciliates, however, the problems of sexuality are essentially the same: (1) Are the conjugant individuals sexually diverse? That is, can any two individuals conjugate with each other, or do the individuals differ morphologically or physiologically so that con- jugation can occur only between individuals of these different types? (2) Are the two gamete nuclei, formed in each conjugant, sexually di- verse? (3) Do conjugants differ from non-conjugants? This question involves the problem of the ciliate life cycle, with possible periods of immaturity and maturity. SEXUAL DIFFERENCES BETWEEN CON JUGANT INDIVIDUALS As already indicated, in one order of ciliates, the Peritrichida, the conjugants show a clear-cut differentiation into two sex types. One type, the macroconjugant, is sessile and large; the other, the microconjugant, is small and free-swimming. Conjugation takes place only between these two types, never between two individuals of the same type. In these re- spects the Peritrichida and a few Holotrichida (e.g. Opalina, Trachelo- cerca, Ichthyophthirius) differ from all other ciliates. In Metopus, Noland (1927) observed that although the conjugants are at first morphologically indistinguishable, only one mate is fertilized and the other one degenerates. Whether this difference in behavior and fate of the two conjugants of a pair is determined by preéxisting physio- logical differences between them, or whether it arises first in the process of conjugation is not known. In another order of ciliates, the Oligotrichida, a few species have been reported by Dogiel (1925) and others to show an equally clear-cut dimorphism, which is not, however, so clearly or simply viewed as a SEXUALITY 689 sex difference. In Opisthotrichum, as in the peritrichs, there are large and small individuals that differ considerably in structure, though both are motile. About 85 percent of the conjugant pairs include one large and one small member, 15 percent include two large members, and none include two small members. The small conjugants are thus sexually specialized for conjugation with large animals only; but the large type is only to a slight degree sexually specialized: it conjugates more readily with the small than with the large type, though it can conjugate with either type. Indications of differences between the conjugants in some pairs are often observed. Doflein (1907) observed differences in size between the two conjugants in many pairs of Paramecium putrinum, and Mulsow (1913) observed the same thing in about 70 percent of the pairs of Stentor. Calkins and Cull (1907) reported frequent differences in via- bility between the two members of pairs of P. caudatum. Zweibaum (1922) found that in about 70 percent of the conjugant pairs the two members differed in the amount of glycogen they contained. These ob- servers suggested that the larger size, greater viability, and higher glycogen content were female characters, and the reverse characters male. On the other hand, Jennings (1911) showed by thorough statistical analysis that while the two members of a pair did sometimes differ in their characters, on the whole there was a high degree of assortative mating, or tendency for like to mate with like; and, further (Jennings and Lashley, 1913a, 1913b), that after conjugation there was remark- able agreement in character between the two members of a pair (bi- parental inheritance), even with respect to vigor and viability. It was generally held, therefore, that in most ciliates regular or frequent dif- ferences between the two members of conjugant pairs were lacking. Two observations made long ago raise the question of whether after all there might not be, beneath the usual superficial morphological simi- larity of the conjugants, a deeper-lying physiological difference. In Chilodonella, Enriques (1908) found that although the two conjugat- ing individuals are indistinguishable at the start of mating, they become diverse as mating progresses: the left conjugant changes form so as to appear shorter, and its mouth migrates to the opposite side of the body. However, it is not clear whether this is an indication of a prior physio- logical difference between the mates, or whether it is a direct conse- 690 SEXUALITY quence of their method of union. The second observation is one made by Maupas (1889). He observed that in certain species conjugation never occurred in cultures containing animals all from a single natural source; it was necessary, in order to get conjugation in a single culture, to have animals from different natural sources. He concluded that di- versity of ancestry was a necessary condition for conjugation. Many later observers found that conjugation occurred abundantly among the progeny of a single individual and so turned attention away from Maupas’s con- tention, with its implication of physiological difference between con- jugants. Until recently it was generally supposed that, in most species of ciliates, any two individuals of the same species could conjugate with each other if they were capable of conjugation at all. The interpretations given the various observations just set forth will be deferred until the newer knowledge of sexuality in Paramecium has been outlined. In describing this newer work, there will be mentioned first the usual typical relations, and later, certain instructive exceptions. The facts on which the following account is based are to be found in recent articles by Sonneborn (1937, 1938a, 1938b, 1939a, 1939b, and 1939c), Kimball (1937, 1939a, 1939b, 1939c), Jennings (1938a, 1938b, 1939a, 1939b), Gilman (1939), Giese (1938, 1939), and Giese and Arkoosh (1939). In P. aurelia, individuals containing macronuclei descended from one original macronucleus do not as a rule conjugate with each other. Such a group of individuals is called a caryonide. Caryonides terminate and new ones are formed when the macronuclei are destroyed and replaced by products of the micronucleus, during the reorganization following conjugation and during endomixis or autogamy. At such times usually two new macronuclei arise in each reorganizing individual, and these go into different cells at the first fission. The fact that individuals of the same caryonide do not conjugate with each other agrees with Maupas’s observation that closely related individuals do not interbreed. But if several caryonides are present in the same culture, even though all come from a single original individual, they may conjugate. This agrees with the observation of the opponents of Maupas, who found conjugation within a clone. When several caryonides are cultivated in different dishes and samples of each are mixed with samples of each of the others, in some of the SEXUALITY 691 combinations nothing happens—each individual moves about inde- pendently of the others; but in other mixtures the animals quickly unite in large clusters. The animals stick together as they collide in their ran- ” e : os a © ge. 2 > \ Se TB tye A Figure 166. The mating reaction in Paramecium bursaria. Upper left, single mating type with individuals scattered singly. Upper right, the clusters formed six minutes after mixture of cultures of two different mating types. Lower left, a later stage of the mating reaction (after five hours). Lower right, the final conjugating pairs as they appeared twenty-four hours later. (From Jennings, 1939.) dom movements. Animals not in contact do not attract each other; nor are they in a specially sticky condition, as has been so often maintained, for neither caryonide shows the least trace of stickiness until the animals are mixed, and then only when animals of different caryonides collide. The clusters begin with just two individuals and build up into larger 692 SEXUALITY aggregations by the repeated addition of other individuals, as these col- lide with those already united. In the course of an hour or so, the clusters break down into conjugating pairs. A detailed account of this mating reaction (Fig. 166) is given for P. bursaria by Jennings (1939a). The final pairs always consist of one individual from each of the two caryonides. When animals of the two caryonides differ in size, each pair consists of one large and one small animal. In P. bursaria (Jennings, 1938a) the two members of each pair differ in color when a normal green culture is mixed with one made pale as a result of recent rapid multipli- cation. When all possible combinations are made among a group of caryo- nides, they are classifiable, on the basis of their reactions, into two groups (Table 11); no two members of the same group will conjugate with each other, but any two caryonides from different groups will. These TABLE 11: RESULTS OF MIXING TOGETHER ANIMALS FROM DIFFERENT CARYONIDES OF STOCK F, Paramecium aurelia* Caryonides 2b1 | 2b2 | 3a2 | gbr | 4b2 | rb2 | 2ar | 2a2 | 3a1 | sbr | sb2 5 op am: = = = ae ata ate fo ae ieee oo | 2 ea | ee ee S| - S (— See et SS | — | ee eee (ele Se |+\ eee Joe Sse \- 3 var | + re an at as ee en es aaa | |] a Paes | = |) eS ee el ie ee | eee soll SA 5 ES a A | ee a * + = conjugation; — = no conjugation. Data from Sonneborn, 1938a. The caryonides 2b1, 2b2, 3a2, 4b1, and 4b2 are of mating type I; caryonides 1b2, 2a1, 2a2, 3a1, 5b1, and 5b2 are of mating type II. SEXUALITY 693 two groups are said to be of different mating types, and in one group of races are designated as I and II. Conjugation occurs between types I and II, never between individuals of the same type, whether they be mem- bers of the same or different caryonides. In order to ascertain the type of any unknown caryonide, some of its animals are mixed with type I and some with type II; conjugation occurs in one of the mixtures, not in the other. The type of the new caryonide must then be the same as the type TABLE 12: THE SYSTEM OF BREEDING RELATIONS IN Paramecium aurelia, DATA FROM SONNEBORN, 1938A* Variety I 2 3 Mating Type I tee a IV V VI I - + - ~ - - I eee atatee | e I a = - - — ~ ll - _ - = - — 2 | IV — — + - - - Vv ie eS b= = “ S , | VI ~ ~ — - + - * The three varieties (1, 2, and 3) do not interbreed; conjugation occurs only between the two mating types within each variety. + = conjugation; — = no conjugation. with which it did not conjugate, different from the one with which it did. For example, if a culture fails to conjugate with type I, but does with type II, it is type I. Sonneborn (1938a, 1939a, 1939b) has analyzed some fifty stocks of P. aurelia, collected from various regions between Canada and Florida and from the Atlantic to the Pacific Coast. Nearly all showed a similar system: in each stock all caryonides were classifiable into one or the other of two mating types. The few remaining stocks consisted exclusively of but one mating type: e.g., in stock B all caryonides conjugated with type II from another stock, none with type I; so stock B consists exclusively of type I. Studied alone, stock B would be considered non-conjugating, because it never conjugates among its own members. All so-called non- conjugating stocks behave like this; they consist of only one mating type 694 SEXUALITY and conjugate readily when mixed with the proper type from another stock. Mating types appear to be of universal occurrence in P. aurelia. Although not more than two mating types occur in any one stock, more than two must exist in the species, for both mating types in some stocks fail to conjugate with either of the types in certain other stocks. Altogether, six different mating types have been found (Table 12). One group of stocks contains types I and II; a second group contains types III and IV; a third group, types V and VI. Conjugation takes place only between the two types in the same group, never between types in different groups. This sexual isolation of the three groups of stocks makes them distinct genetical species or varieties; but they appear to be morphologically alike, all conforming to the description of the taxo- nomic species P. awvrelia. However, they are physiologically diverse in a number of ways. Each mating type is uniquely defined by the type with which it mates. The mating type of a culture can be ascertained by mixing some of its animals with standard cultures of each of the six mating types. With one, and only one, of these it will conjugate. Its mating type is the other one in the variety with which it mates. For example, if it mates with type V, it belongs to variety 3 and is of mating type VI. In P. bursaria, Jennings (1938a, 1938b; 1939a, 1939b) reports some- what different mating-type relations. Each stock of this species shows as a rule only one mating type. As nuclear reorganization is extremely rare, a stock is practically equivalent to a caryonide of P. aurelia. The mating types fall into three different groups, or genetical species (a fact first found in P. bursaria), with no conjugation between types in different groups (Table 13). In group I occur the four mating types A to D; in group II, the eight types E to M; in group III, the the four types N to Q. In each group each mating type conjugates with all the other types in that group. This system of multiple interbreeding types is in marked contrast to the system of paired types in P. awrelia. To discover the group to which a new stock belongs, it must be mixed with at least two types from each of the three groups. It will conjugate with one or both of the types from one group, not with any of the others. It belongs to the group with which it conjugates. To discover its mating type, it must now be mixed with all the types of this group until one is found with which it will not conjugate. It is then of the same type as this one. For example, SEXUALITY 695 TABLE 13: THE SYSTEM OF BREEDING RELATIONS IN Paramecium bursaria, DATA FROM JENNINGS, 1939A* Variety I II III Mating Type A B | | | | | | | | | | | Sa@) || + +{+| 1 | | a +/+]/4+][+]/4+]0]4+]+ ate tee) tes ee et | | | +/+][+)4+]4+]4+]4 42 | ae fae | ae tb | | | | | | | | | —/-|-|-) 4/4 + pet ae Se = Ill +/+] 4+ -|-]-|-|-/-|-|-]-|-|-|-|+|+]- Q |-|-|-]|-/-|-|-|-|-]-|-|-|+]4+]4]- * The three groups (or varieties) 1, 2, and 3 do not interbreed; conjugation occurs only among the four or eight mating types within each variety. -- = conjugation; — = no conjugation. if it mates with A, B, and C, but not with D, it is of mating type D. Five of the seven species of Paramecium found in the United States have been examined for mating types and all have shown them. P. durelia and P. bursaria have already been discussed. In P. caudatum, Gilman (1939) finds a system of the same kind as found in P. awrelia: six mating types occurring in three groups, with only two interbreeding mating types in each group, and no conjugation between types in dif- 696 SEXUALITY ferent groups. Sonneborn (1938a, 1939a) found two mating types in P. calkinsi and three interbreeding types in P. trichium, indicating a sys- tem of multiple types such as Jennings found in P. bursaria. Giese (1938, 1939) and Giese and Arkoosh (1939) have found mating types in P. multimicronucleatum and P. caudatum. Kimball (1939c) found in Ezplotes, one of the hypotrichous ciliates, a system of mating types like the one in P. bursaria. There are five groups of non-interbreeding types, with morphological differences between some of the groups, indicating that these may be taxonomically as well as genetically different species. In each group occur multiple interbreeding mating types, six in the group most fully studied, any one of these con- jugating with any of the other five. The striking agglutinative mating reaction so characteristic of Paramecium appears to be lacking: conjuga- tion first occurs several hours after mixture of the different types, and then pairs form directly without the prior formation of clusters. In view of our present knowledge, it seems allowable to include Mau- pas’s (1889) old evidence for the necessity of diverse ancestry as evi- dence for diversity of mating type. If so, at least four more species must be added to the list of those in which mating types are known: Stylony- chia pustulata, Leucophyrs patula, Onychodomus grandis, and Loxophyl- lum fasciola. This brings the number of species now known to have mating types to about a dozen. These belong to six different genera and two different orders of ciliate Protozoa. It appears, therefore, that mating types will be found to be widely distributed among the ciliates. The view that any two individuals of the same species can conjugate with each other, if capable of conjugating at all, is demonstrably false; on the contrary, in general, conjugation can take place only between individuals of diverse mating types. Are there ever exceptions to this general rule? Does conjugation ever take place between animals of the same mating type? In nearly all the species examined in detail, conjugation has been observed in cultures containing only one caryonide, and, as members of the same caryonide are presumably of the same mating type, this appears to be conjugation between animals of the same mating type. Can individuals of the same caryonide ever differ in mating type? And is this the explanation of these exceptional conjugations within a caryonide? There is only one method SEXUALITY 697 (Kimball, 1939a) of answering these questions directly. The two ani- mals that come together for conjugation must be separated before they become too tightly united, and the mating types of the two members of such a split pair must be directly ascertained by placing each of them separately in standard cultures of the different types, to discover with which ones they will react sexually. If one reacts only with type I and the other only with type I, they must be of different types; but if both react with the same type, then they are alike in mating type. This problem has been most fully studied by Kimball (1939a, 1939b). In P. aurelia, he found that conjugation within a caryonide occurred un- der two very different kinds of conditions. One kind is very common; it occurs in caryonides genotypically of type I, when the last preceding caryonide in the direct line of ancestry was of type II. Under these condi- tions conjugation may occur in the caryonide during the first few days of its existence. Kimball split some of these conjugant pairs, tested them directly for mating type, and showed that in each pair one animal was of type I, the other of type II. Thus both mating types can be present in a single caryonide, and the mating is between the two types only. Kimball now obtained clone cultures from the two members of such split pairs and found in every pair that both cultures were of type I and showed no further conjugation among their own members. Hence the type II ani- mals originally present in the caryonide changed to type I. The early oc- currence of type II was due to the type II character of the immediate ancestors. This phenotypic or cytoplasmic ‘“‘hang-over”’ fades out, as the new genotype comes into action. Not all individuals accomplish this at the same speed, so for a short time some are still type II while others have completed the change to type I; at this moment conjugation may occur. A little later all have changed to type I, and conjugation is no longer possible. Similar “cytoplasmic lag” in the inheritance of other characters in Paramecium had been reported by both De Garis (1935) and by Sonneborn and Lynch (1934). The other type of conjugation within a caryonide is of much rarer occurrence. In the race of P. aurelia examined by Kimball (1939b), less than 3 percent of the caryonides showed it. In these, conjugation oc- curred not only when the caryonide was young, but probably through- out its whole history. Moreover, any individual in the caryonide gave rise to progeny that conjugated with each other. Even the members of a split 698 SEXUALITY pair both gave rise to cultures in which conjugation took place. Never- theless, Kimball found that the two members of a split pair were always of diverse mating types at the time they conjugated: one was type I, the other type II. Hence such caryonides are unstable in mating type. The type changes back and forth repeatedly; but when conjugation occurs, it is always between animals of different mating type. In the Vorticellidae, the invariable morphological and functional dif- ference between conjugants has already been mentioned. Finley (1939a, 1939b) shows clearly that both types of conjugants not only arise within a caryonide, but at a single unequal cell division. The macroconjugant and the microconjugant preduced at this fission can then copulate with each other or with other similarly differentiated conjugants of the same caryonide. Here it is obvious that conjugation within a caryonide is never- theless invariably between different mating types or sexes. There are, however, a number of known instances of conjugation within a caryonide which require further investigation. Foremost among these are species, without morphological difference between the conju- gants, in which conjugation regularly occurs within a clone or a caryo- nide. This has been reported as common in P. multimicronucleatum by Giese (1938, 1939), less common in P. caudatum by Gilman (1939), and very rare in P. bursaria by Jennings (1938a, 1938b, 1939a, 1939b). An especially interesting situation is reported for Explotes by Kimball (1939c). Fluid from a culture of one mating type, added to a culture of a different mating type, induces the latter to conjugate among them- selves. Likewise, in mixtures between normal animals of one mating type and double animals of another type, some of the resulting con- jugant pairs are unions of singles with singles, a few are doubles with doubles, though most are, as would be expected, singles with doubles. The relations here raise the question of whether subjection to fluid from another mating type makes animals acquire a type corresponding to the fluid, as Jollos (1926) showed happens in the alga Dasycladus. If so, it may be difficult or impossible to analyze it satisfactorily, because in ascertaining the types of members of split pairs they have to be sub- jected to the very fluid that would change their type. This may be one of those exasperating problems, like attempting to determine the posi- tion and the velocity of an electron at the same time, in which the meth- ods of investigation essentially alter the things being investigated. SEXUALITY 699 Are any of these observations of conjugation within a caryonide evi- dence of conjugation between animals identical in mating type? The direct test has not been made in most cases; but, in the few examples in which it was made, it was demonstrated that conjugants were always of different types, in spite of the fact that they were members of the same caryonide. The evidence is therefore strongly against the occurrence of conjugation between animals of the same mating type, though final judgment must await further analysis. MATING TYPES IN RELATION TO THE MAUPASIAN LIFE CYCLE According to the well-known theory of Maupas (1889), the ciliate exconjugant is conceived as being a young individual producing by re- peated fissions immature cells unable to mate; the cells produced after many youthful fissions become sexually mature and are capable of con- jugating; after many more fissions, the cells grow old, losing their power of conjugating and showing other signs of senescence; and they finally die. If conjugation occurs during the period of maturity, the conjugants are rejuvenated and the cycle is renewed. In some ciliates, such as Uro- leptus mobilis, investigated by Calkins (1920), this Maupasian life cycle is clearly shown. In Paramecium, however, there are striking spe- cific and racial differences in presumably so fundamental a matter as the life cycle. Many races of P. aurelia (Sonneborn 1937, 1938a) show a definite period of immaturity: during the first week or two after conjugation, cultures do not give the mating reaction and cannot conjugate. In a few more days, the power of conjugating rapidly develops to full strength, inaugurating a period of maturity. But the organisms remain mature indefinitely; no period of senescence appears. Only for a day or so during the periodically recurring processes of nuclear reorganization, are they unable to conjugate. As soon as reorganization is completed, the mating reaction reappears in full strength. Why the reorganized cells fail to begin again with a period of immaturity, as they do after conjugation, is at present a puzzling and probably a significant fact. Other races of P. aurelia (Sonneborn, 1938a) not only lack a period of senescence, but also a period of immaturity: they are able to conjugate immediately after conjugation. Eight successive conjugations have been obtained in a period of seventeen days (Sonneborn, 1936). As the 700 SEXUALITY process of conjugation and nuclear reconstitution require one day, there could have been only about a day between successive conjugations, a period in which at most only three or four fissions could take place. In P. bursaria, Jennings (1939a, 1939b) reports a regularly occurring period of immaturity. In group I it lasts for from two weeks to several months; in group II all clones under investigation were still immature at last reports, eight months after their origin at conjugation. Periods of immaturity have also been found regularly in P. caudatum by Gilman (1939) and in Ezplotes by Kimball (1939c). In none of these species has there as yet been any report that maturity is followed by a period of senescence, with loss of ability to conjugate. Many of Jennings’s clones of P. bursaria have been mature for over two years, without loss of sexual vigor; and in this species endomixis is so rare as scarcely to account for the results. Thus age sometimes is and sometimes is not a factor in determining conjugation; the Maupasian life cycle is not an invariable feature of ciliate life. Immaturity may be absent, short, or long; maturity may be coextensive with life, or it may be simply preceded by a period of im- maturity; or it may be delimited on either side by periods of immaturity and senescence. THE ROLE OF ENVIRONMENTAL CONDITIONS IN DETERMINING CON- JUGATION Maupas (1889) recognized the importance of environmental condi- tions in determining conjugation, and most subsequent workers have been in more or less agreement on this point; but some have carried this view to the extreme of ascribing to environmental conditions alone the determination of conjugation. The preceding account has shown that this cannot always be true, for hereditary and developmental internal factors have been demonstrated as playing a decisive role in many of the races and species. Nevertheless, environmental conditions, such as nutti- tion, temperature, and light, do have marked limiting effects on the occurrence of conjugation. In P. aurelia (Sonneborn, 1938a), the mating reaction does not take place in cultures that are either overfed or completely starved. Inter- mediate nutritive conditions are most favorable for its occurrence. More- SEXUALITY 701 over, the cultural conditions must be good in other respects: when dele- terious bacteria or other unfavorable conditions injure the paramecia, the mating reaction is weak or lacking. In variety 1, mating types I and II will react sexually at any tempera- ture within the range examined, 9° C. to 32° C.; but mating types IH and IV of variety 2 will not react above 24° C. and types V and VI of variety 3 not above 27° C. Similar differences appear in the time of day in which reactions will occur: variety 1 will react at any time; but variety 2 reacts only between 6 P.M. and 7 A.M., while variety 3 reacts only between 1 A.M. and 1 P.M. As might be supposed, this periodicity is an effect of the daily alternation of light and dark. In variety 3, sexual reactivity has been completely suppressed by exposing the organisms to continuous illumination, and they have been made to react at all hours by keeping them in continuous darkness. These effects have been shown (Sonneborn, 1938a) to be due to the suppression of reactivity by light, not to its stimulation by dark- ness. Similar diurnal periodicities in mating occur in P. bursaria (Jen- nings, 1938a, 1939a, 1939b). The environmental conditions thus determine whether conjugation will occur when the proper mating types are brought together. Ordinarily the mating types themselves are hereditary characters (see Chapter XV, “Inheritance in Protozoa,” Jennings); but in the exceptional unstable caryonides studied by Kimball (1939b), genetic determination seems excluded, for the mating types change repeatedly during vegetative re- production. Here environmental conditions probably determine even the mating types themselves, and similar relations may be the rule, instead of the exception, in species in which conjugation within a caryonide oc- curs regularly. Thus investigations of possible genetic, developmental, and environmental factors determining conjugation show all to be in- volved, as might have been expected. SEX DIFFERENCES BETWEEN GAMETE NUCLEI Careful observations on the form and behavior of the gamete nuclei during conjugation were made by Maupas (1889) and by R. Hertwig (1889). These and nearly all subsequent investigators have agreed that in most ciliates the two gamete nuclei formed in each conjugant differ in 702 SEXUALITY behavior: one, the stationary gamete nucleus, remains in the conjugant that produces it; the other, the migratory gamete nucleus, passes into the mate and unites with the stationary gamete nucleus located in that ani- mal. As a rule, the gamete nuclei are morphologically indistinguishable; but in some species differences in size and form have been reported. The most extreme example of morphologically different gamete nuclei is in Cycloposthium (Dogiel, 1925). The spindle resulting in the formation of the gamete nuclei is heteropolar: one pole, destined to produce the Figure 167. Conjugation in Cycloposthinm bipalmatum, showing the sperm-like mi- gratory pronuclei differing from the spherical stationary pronuclei. (After Dogiel. ) stationary gamete nucleus, is larger and rounder than the other smaller and more pointed pole, destined to yield the migratory gamete nucleus. The latter arises from the anterior pole of the spindle and develops a long tail-like appendage at the proximal end, and a small, pointed distal end, functional in piercing the cuticle in its passage from one mate into the other (Fig. 167). In other ciliates, lesser differences between the gamete nuclei have been observed: slight differences in size in Didinium (Prandtl, 1906), in Paramecium caudatum (Calkins and Cull, 1907), and in P. multimicronucleatum (Landis, 1925). Calkins and Cull (1907) concluded that the two gamete nuclei in P. caudatum differ in their chromatin content, as a consequence of transverse chromosomal division at the nuclear division which gives rise to them. In most ciliates, however, no morphological differences between the two gamete nuclei have been observed. SEXUALITY 703 SIGNIFICANCE OF THE DIVERSITIES BETWEEN CONJUGANTS AND BE- TWEEN GAMETE NUCLEI There is great diversity of opinion regarding the significance of the observed differences between conjugants and between gamete nuclet. This is due partly to the variety and complexity of the observed phenom- ena, and partly to confusion as to the meaning of the concepts employed, particularly concepts developed primarily with relation to phenomena in higher organisms. An attempt will be made to summarize the more prominent views concerning the main types of observed relations and to set forth some general considerations concerning them. In some ciliates, the Vorticellidae and a few others, in which the conjugants are always morphologically diverse and the gamete nuclei morphologically alike, with fertilization of only the larger conjugant, it is usually agreed that the conjugants differ sexually. Further, the gamete nuclei in each conjugant are sometimes said to be of the same sex as the conjugant. Some authors hold that the sexes here are female and male. In Chilodonella (see p. 689), in which the conjugants become morphologically diverse during conjugation and fertilization 1s recip- rocal, Enriques (1908) concluded that although both conjugants were functionally hermaphroditic (producing two sexually diverse gamete nuclei), the conjugants also showed a partial, incompletely developed sex diversity, for which he devised the term ‘‘hemisexes.’”’ In Opisthotrichum (see p. 689) fertilization is also reciprocal, involving gamete nuclei with strongly marked morphological sex dif- ferences. Nevertheless, in a majority of the conjugant pairs, the two members differ in size. When the two are alike in size, both are large. Dogiel (1925) interprets these facts as follows: the conjugants are all functionally hermaphroditic, each producing male and female gamete nuclei; but the conjugants also show the beginnings of sexual differentia- tion, the small ones being more differentiated, for they can mate only with large individuals, while the large ones can mate either with small or large, though more commonly with the former. The small conjugants are viewed as considerably differentiated toward the male condition, in spite of their functional hermaphroditism. In Paramecium and Euplotes, fertilization is reciprocal, the gamete nuclei show little or no morphological difference, and the conjugants 704 SEXUALIDY show no significant morphological differences. Yet the conjugants are regularly differentiated physiologically into diverse mating types. The gamete nuclei in these and similar forms are often considered to be sexually diverse; frequently the migratory gamete nucleus is viewed as male, the stationary one as female. This introduces the same difficulty as in Chilodonella and O pisthotrichum. How reconcile sex differences between the gamete nuclei with the differences between the ‘“‘hermaph: roditic’” conjugants? Jennings (1939a) inclines toward interpreting the mating types as manifesting phenomena of self-sterility, or incom- patibility, of the kind found in certain higher plants (Stout, 1938) and animals (Morgan, 1938), in the sense that the single clone or caryonide, like the single self-sterile plant, ordinarily does not fertilize itself. Jen- nings points out the features in which the two sets of phenomena are different, as well as those in which they are alike. More recently, Sonne- born ( 1939¢) has shown that the periodic nuclear reorganization in variety 1 of P. aurelia is regularly a self-fertilization, as maintained by Diller (1936). Consequently, P. avrela is not self-sterile, but regularly self- fertile. The failure of individuals of the same mating type to conjugate with each other is thus not related to any incompatibility between their gametes, for such appears not to exist. It seems, therefore, more compar- able to the failure of two individuals of the same sex to unite in copula- tion. In higher organisms, self-sterility serves to prevent self-fertilization; in P. aurelia the mating types serve to bring together for cross-fertilization diverse sex types, each of which regularly undergoes self-fertilization. The present author, therefore, concludes that the mating-type phenomena are not properly to be viewed as self-sterility or incompatibility. If by sexual differentiation is meant the differentiation of the individuals of a species into diverse kinds, so that mating occurs regularly between dif- ferent kinds, not between two of the same kind, then the mating types of Paramecium are diverse sexes. As in O pisthotrichum, the sex differ- ences between the conjugants are of a different kind from those existing between the gamete nuclei: one serves to bring together the mates, the other to bring together their gamete nuclei. Multiple sex systems, such as those in P. bursaria and Explotes, offer serious difficulties to those who, like Hartmann (1929), hold there can be but two sexes. Whether or not one agrees with this contention, the SEXUALITY 705 work of Moewus (1939a) on Chlamydomonas shows how what appears, through biological analysis, to be a multiple sex system may be reduced, through chemical analysis, to a fundamentally dual system. Further in- vestigation is, of course, required to ascertain whether the multiple sex systems in ciliates are, in fact, similar in this respect to the system in Chlamydomonas. A number of the interpretations of sex relations in ciliates employ the concepts male and female, as set forth above. Many authors follow Hartmann (1929), who holds, as has been pointed out on page 678, that sex differences, wherever found, are always male and female. The characters by which the female is ordinarily recognized are larger size, lesser activity, greater storage of nutritive reserves, and egg-like form; and the male by the corresponding opposed characters. In attempt- ing to apply these views, however, numerous difficulties are encountered. In the Vorticellidae, both gamete nuclei in the microconjugant are held to be male; yet only one of them shows the “male” character of activity by migrating into the macroconjugant. In O pisthotrichum, the migratory gamete nucleus has the form of a sperm, but it has the “female” char- acter of much greater size than the stationary gamete nucleus. The diffi- culty of using size as an index of femaleness is clearly shown in the work of Satina and Blakeslee (1930) on certain bread molds. In a number of strains, two sexes were observed and found to be the same in all strains. One sex was distinctly larger than the other in each strain, yet the larger sex in one strain was shown to be identical with the small sex in others. Geitler (1932) found similar difficulties in identifying the sexes in diatoms by their activity. These and other difficulties have led Kniep (1928), Mainx (1933), and others to abandon the concepts of male and female in unicellular organisms and to view sexual union as brought about by copulation-conditioning factors, some of which operate to bring together the cells, others the nuclei. In the present state of knowledge, this point of view appears to be preferable to one that appeals to such abstract, ill-defined, and confusing concepts as fundamental maleness and femaleness. From this point of view, the conflicts between sexual differentiation in the gamete nuclei and sexual differentiation in the conjugant indi- viduals present far less difficulty than from the point of view which 706 SEXUALITY, requires identification of all sex differences with male and female. There may be two kinds of copulation-conditioning factors: one functioning in bringing together the cells, the other in bringing together their nuclei. In Chlamydomonas and the Vorticellidae, the two kinds of nuclear fac- tors operate in different kinds of cells; in Paramecium and most other ciliates, both kinds of nuclear factors operate in each of the kinds of cells. Thus by abandoning the pure assumption that sex differences, wherever found, must always be fundamentally the same (male and female), the conflict between sex differences in nuclei and sex differences in cells disappears. LITERATURE CITED Calkins, G. N. 1920. Uroleptus mobilis Engelm. U1. A study in vitality. J. exp. Zool., 34: 449-70. 1926. The Biology of the Protozoa. Philadelphia and New York. Calkins, G. N., and S. W. Cull. 1907. The conjugation of Paramecium aurelia (caudatum). Arch. Protistenk., 10: 375-415. De Garis, C. F. 1935. Heritable effects of conjugation between free individuals and double monsters in diverse races of Paramecium caudatum. J. exp. Zool., 71: 209-56. Diller, W. F. 1936. Nuclear reorganization processes in Paramecium aurelia, with descriptions of autogamy and “hemixis.” J. Morph., 59: 11-68. Doflein, F. 1907. Beobachtungen und Ideen tiber die Konjugation der In- fusorien. S. B. Ges. Morph. Miinchen, 23: 107-14. Dogiel, V. 1925. Die Geschlechtsprozesse bei Infusorien (speziell bei den Ophryoscoleciden) , neue Tatsachen und theoretische Erwagungen. Arch. Protistenk., 50: 283-442. Enriques, P. 1908. Die Conjugation und sexuelle Differenzierung der In- fusorien. Zweite Abhandlung. Wiederconjugante und Hemisexe bei Chilodon. Arch. Protistenk., 12: 213-76. Finley, H. E. 1939. Sexual differentiation in Vorticella microstoma. J. exp. Zool., 81: 209-29. —— 1939b, Further observations upon sexual differentiation in Vorticella microstoma. Anat. Rec., 75, (suppl.), p. 85. Geitler, L. 1932. Der Formwechsel der pennaten Diatomeen (Kieselalgen). Arch. Protistenk., 78: 1-226. Giese, A. C. 1938. Race and conjugation of Paramecium. Physiol. Zoél., 11: 326-32. — 1939. Studies on conjugation in Paramecium multimicronucleatum. Amer. Nat., 73: 432-44. Giese, A. C., and M. A. Arkoosh. 1939. Tests for sexual differentiation in SEXUALITY 707 Paramecium multimicronucleatum and Paramecium caudatum. Physiol. Zodl., 12: 70-75. Gilman, L. C. 1939. Mating types in Paramecium caudatum. Amer. Nat., 73: 445-50. Hartmann, M. 1929. Verteilung, Bestimmung und Vererbung des Geschlechtes bei den Protisten und Thallophyten. Handb. d. Vererbungswiss. II. —— 1932. Neue Ergebnisse zum Befruchtungs-und Sexualitatsproblem. (Nach Untersuchungen von M. Hartmann, J. Hammerling und F. Moewus.) Naturwissenschaften, 20: 567-73. —— 1934. Beitrige zur Sexualititstheorie. Mit besonderer Beriicksichtigung neuer Ergebnisse von Fr. Moewus. S. B. preuss. Akad. Wiss., Phys. Math. KI., 379-400. Hertwig, R. 1889. Uber die Conjugationen der Infusorien. Abh. bayer. Akad. Wiss. Ll Kile ya toieoa2e Jennings, H. S. 1911. Assortative mating, variability and inheritance of size in the conjugation of Paramecium. J. exp. Zool., 11: 1-134. —— 1938a. Sex reaction types and their interrelations in Paramecium bursaria. 1. Proc. Nat: Acad: Sci. Wash., 24: 112-17. —— 1938b. Sex reaction types and their interrelations in Paramecium bursaria, I. Clones collected from natural habitats. Proc. Nat. Acad. Sci., 24511 17-20; —— 1939a. Genetics of Paramecium bursaria. I. Mating types and groups, their interrelations and distribution; mating behavior and self sterility. Genetics, 24: 202-33. —— 1939b. Paramecium bursaria: mating types and groups, mating behavior, self sterility ; their development and inheritance. Amer. Nat., 73: 414-31. Jennings, H. S., and K. S. Lashley. 1913a. Biparental inheritance and the question of sexuality in Paramecium. J. exp. Zool., 14: 393-466. —— 1913b. Biparental inheritance of size in Paramecium. J. exp. Zool., 15: 193-99. Jollos, V. 1926. Untersuchungen uber die Sexualitatsverhaltnisse von Dasycladus clavaeformis. Biol. Zbl., 46: 279-95. Kimball, R. F. 1937. The inheritance of sex at endomixis in Paramecium auvelia. Proc. Nat. Acad. Sci. Wash., 23: 469-74. —— 1939a. A delayed change of phenotype following a change of genotype in Paramecium aurelia. Genetics, 24: 49-58. -—— 1939b. Change of mating type during vegetative reproduction in Paramecium aurelia. J. exp. Zool., 81: 165-79. 1939c. Mating types in Euplotes, Amer. Nat., 73: 451-56. Kniep, H. 1928. Die Sexualitat der niederen Pflanzen. Jena. Landis, E. M. 1925. Conjugation of Paramecium multimicronucleata, Powers and Mitchell. J. Morph., 40: 111-67. Mainx, F. 1933. Die Sexualitat als Problem der Genetik. Jena. 708 SEXUALITY; Maupas, E. 1889. La Rajeunissement karyogamique chez les ciliés. Arch. zool. exp.) gén. (2), 72 149-517. Moewus, F. 1933. Untersuchungen uber die Sexualitét und Entwicklung von Chlorophyceen. Arch. Protistenk., 80: 469-526. —— 1934. Uber Subheterdzie bei Chlamydomonas eugametos. Arch. Protistenk., 83: 98-109. —— 1935a. Uber den Einfluss ausserer Faktoren auf die Bestimmung des Geschlechts bei Protosiphon. Biol. Zbl., 55: 293-309. —— 1935b. Die Vererbung des Geschlechts bei verschiedenen Rassen von Protosiphon botryoides. Arch. Protistenk., 86: 1-57. —— 1935c. Uber die Vererbung des Geschlechtes bei Polytoma Pascheri und bet Polytoma uvella. Z. indukt. Abstamm. u. VererbLehre, 69: 376-417. —— 1936. Faktorenaustausch, insbesondere der Realisatoren bei Chlamy- domonas-Kreuzungen. Ber. dtsh. bot. Ges., 54: 45-57. —— 1937a. Methodik und Nachtrige zu den Kreuzungen zwischen Polytoma-Arten und zwischen Protosiphon-Rassen. Z. f. indukt. Abstamm.- u. VererbLehre, 73: 63-107. ——— 1937b. Die allgemeinen Grundlagen der Sexualitat. Biologe, 6: 145-51. —— 1938a. Vererbung des Geschlechts bei Chlamydomonas eugametos und verwandten Arten. Biol. Zbl., 58: 516-36. ——— 1938b. Carotinoide als Sexualstoffe von Algen. Jb. wiss. Bot., 86: 753-03. —— 1939a. Untersuchungen uber die relative Sexualitét von Algen. Biol. ZbI., 59: 40-58. —— 1939b. Carotinoide als Sexualstoffe von Algen. Naturwissenschaften, 2297 OAs —— 1939c. Uber die Chemotaxis von Algengameten. Arch. Protistenk., 92: 485-526. —— 1940. Carotinoid. Derivate als beschlecktsbestimmende Stoffe von Algen Biol. Zbl., 60: 143-66. Morgan, T. H. 1938. The genetic and physiological problems of self-sterility in Crona. I and I. J. exp. Zool., 78: 271-334. Mulsow, W. 1913. Die Conjugation von Stentor coeruleus und Stentor polymorphus. Arch. Protistenk., 28: 363-88. Noland, L. E. 1927. Conjugation in the ciliate Metopus sigmoides. J. Morph., 44: 341-61, Pascher, A. 1931. Uber Gruppenbildung und ‘‘Geschlechtswechsel’” bei den Gameten einer Chlamydomonadine (Chlamydomonas paupera). Studien und Beobachtungen uber die geschlechtliche Fortpflanzung und den Generationswechsel der Griinalgen. I. Jb. wiss. Bot., 75: 551-80. Philip, V., and J. B. S. Haldane. 1939. Relative sexuality in unicellular Algae. Nature, 143: 334. Prandtl, H. 1906. Die Konjugation von Didinium nasutum, O. F. M. Atch. Protistenk., 7: 229-58. SEXUALITY 709 Pringsheim, E. G., and K. Ondracek. 1939. Untersuchungen uber die Geschlechtsvorginge bei Polytoma. Beih. bot. Zbl., 59A: 117-72. Satina, S., and A. F. Blakeslee. 1930. Imperfect sexual reactions in homothallic and heterothallic Mucors. Bot. Gaz., 90 (3): 299-311. Sonneborn, T. M. 1936. Factors determining conjugation in Paramecium aurelia. 1. The cyclical factor: the recency of nuclear reorganization. Genetics, 21: 503-14. — 1937. Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. Nat. Acad. Sci. Wash., 23: 378-85. — 1938a. Mating types in Paramecium aurelia: diverse conditions for mating in different stocks; occurrence, number and interrelations of the types: Proc; Amer: Phil Soe, 79:41 1-34. —— 1938b. Mating types, toxic interactions and heredity in Paramecium aurelia, Science, 88: 503. —— 1939a. Sexuality and related problems in Paramecium. Coll. Net, 14: 77-84. —— 1939b. Paramecium aurelia: mating types and groups; lethal inter- actions; determination and inheritance. Amer. Nat. 73: 390-413. — 1939c. Genetic evidence of autogamy in Paramecium aurelia. Anat. Rec.) 7/5; (suppl) capes): Sonneborn, T. M., and R. S. Lynch. 1934. Hybridization and segregation in Paramecium aurelia. J. exp. Zool., 67: 1-72. Stout, A. B. 1938. The genetics of incompatibilities in homomorphic flowering plants. Bot. Rev., 4: 275-369. Woodruff, L. L., and R. Erdmann. 1914. A normal periodic nuclear reorgani- zation process without cell fusion in Paramecium. J. exp. Zool., 17: 425-518. Zweibaum, J. 1921. Richerche sperimentali sulla conjugazione degli Infusori. II. Influenza della conjugazione sulla produzione dei materiali di riserva nel Paramecium caudatum. Arch, Protistenk., 44: 375-96. CHAPTER XV INHERITANCE IN PROTOZOA H. S. JENNINGS IN His Genetics of the Protozoa (1929), the author has reviewed some- what fully the investigations and literature on inheritance in Protozoa, up to 1929. No attempt is made to repeat here these detailed reviews; the plan is rather to summarize the present state of knowledge on the subject. Very great advances have been made since 1929, particularly in the knowledge of biparental inheritance, largely through the work of Moewus (1932-38). The question dealt with in the study of inheritance is: To what extent and how are the constitutions and characteristics of later genera- tions affected by the constitutions of their ancestors, particularly by the constitutions of the*immediate parents? Certain subordinate questions arise in connection with this: To what extent and how are characteristics affected by environmental conditions? What are the relations between environmental modifications and genetic constitution? By genetic constitution is meant the constitution insofar as it affects descendants. The genetic constitution is known from studies of multi- cellular organisms to be embodied in certain genetic materials. These are, mainly or entirely, found in the chromosomes. General genetics has shown (1) that in the chromosomes there are great numbers of diverse genetic materials (known commonly as genes or factors), having dif- ferent effects on development and characteristics; and (2) that genetic materials are transferred bodily from parents to offspring. In the present account the term ‘‘factors’”’ will usually be employed in place of the term “genes,” since the latter has acquired, of late, certain doubtful theoretical implications. Genetic materials have two essential properties: (1) the genetic ma- terials received from parents affect the development and characteristics of the descendants; (2) the many different kinds of genetic materials INHERITANCE 711 (genes or factors) reproduce themselves true to type, in development and reproduction. Each kind of genetic material assimilates, producing more material of its own type, and each unit of material, or gene, pro- duces at division new units like itself. The Protozoa have chromosomes that are similar to those in other organisms (see Chapter XII). One question that arises in protozoan genetics is this: Are there in the Protozoa other genetic materials in ad- dition to those in the chromosomes, having the two essential properties just mentioned? TYPES OF REPRODUCTION AND INHERITANCE In the Protozoa, as in some other organisms, there are two main types of relation of offspring to parents: 1. Uniparental reproduction; offspring arise from a single parent, as in the various types of vegetative reproduction. 2. Biparental reproduction; offspring are formed from the combined parts of two parents, as in sexual reproduction in the Protozoa by copu- lation, conjugation, and the like—followed by division. The two kinds of reproduction differ fundamentally in their relation to the genetic constitution, or genetic materials (chromosomes and their genes). In uniparental reproduction, typically each of the genetic mate- rials of the parent is divided and duplicated, so that the genetic consti- tutions of offspring are like those of their parents. In biparental repro- duction, the complex of genetic materials present in each of the two parents is taken apart, and a new combination is made from parts of these. The genetic materials of the offspring are a new combination of those of the two parents. In consequence of these differences, uniparental and biparental repro- duction give very different consequences in inheritance. The two will therefore be dealt with separately. INHERITANCE IN UNIPARENTAL REPRODUCTION MATERIAL PROCESSES Details as to the material processes in uniparental reproduction are dealt with in other chapters (see Chapters XIII and XIV). The essential features, for genetics, are that the nuclei divide, each chromosome di- 712 INHERITANCE vides, each gene divides—one product from each going to each of the two offspring. In the ciliate Infusoria, the macronucleus not only divides but is in many cases reorganized (see Chapter XIII). The cytoplasmic body divides and is to a great extent (or entirely) reorganized. The general upshot 1s that the constitution of nucleus and cytoplasm is typi- cally the same in the offspring as in the parent (exceptional conditions are dealt with in later pages). INHERITANCE OF CHARACTERISTICS Clones—All the individuals produced by uniparental reproduction from a single individual are known collectively as a clone. The general rule for inheritance in uniparental reproduction {s that all members of the clone are alike in genetic constitution and in inherited characteristics. That is, the new individuals (clone) produced from a single parent are like the parent and like one another in their characteristics, structural and physiological. Taken together, they form the equivalent of a set of iden- tical twins. There are numerous exceptions to this rule of the genetic identity of parent and offspring in uniparental reproduction, and these are among the most important and interesting phenomena of genetics. They are dealt with fully on later pages. But the relation of identity of genetic con- stitution in parent and offspring holds for perhaps 99.9 percent of all cases; it is the most striking feature of uniparental reproduction. Certain manifestations of this principle of identity in genetic consti- tution between parent and offspring require special consideration: 1. Biotypes. In all Protozoa fully studied, any species consists of a great number of diverse biotypes—races differing in inherited character- istics. The different biotypes may differ in size, form, structure, and physiology (rate of multiplication and the like). Such diverse biotypes in Paramecium, Difflugia, Arcella, and other Protozoa are described and illustrated in the present author’s Genetics of the Protozoa (1929). When individuals of diverse biotypes reproduce uniparentally, as by fission, the general rule is that each biotype retains its characteristics. The offspring are like the parents in all conspicuous respects. Thus all members of a single clone belong to the same biotype and have the same inherited characteristics. In biotypes of large individuals, each individual INHERITANCE FAB produces a clone of large individuals; biotypes of small individuals give clones with small individuals; rapidly multiplying biotypes produce rapidly multiplying descendants; and so on. Such inheritance is shown with respect to vigor or weakness, to resistance and lack of resistance, and to structural and physiological characteristics of all sorts. Members of a given biotype, having the same genetic constitution, may differ in ways induced by different environments, or resulting from dif- ferent periods in the life of the individual. Such differences are, as a rule, not inherited in uniparental reproduction (exceptions are dealt with later). The main classes of non-heritable differences among the indi- viduals of a single biotype are: age differences; nutritional differences, and environmental diversities resulting from differences in temperature, chemical conditions, and the like. In addition to these, there are in some species non-heritable diversities of unknown origin between members of the same biotype, the same clone. Thus in Di fflagia corona, which has a silicious shell bearing spines, there are within the same clone differences as to the number and size of the spines borne by the shell. In this case the differences arise at repro- duction, presumably under the influence of environmental diversities. They follow the same rule as known environmental differences; they are not as a rule inherited. If parents with many spines produce descendants, the mean number of spines in these descendants is the same as in the descendants of individuals of the same clone that have few spines (excep- tions noted in later pages). Thus, as a rule, racial or inherited characters are not altered in unt- parental reproduction. This is the most striking and obvious feature of such reproduction. Yet it does not hold absolutely; there are important limitations and exceptions to this rule. A large proportion of our dis- cussion of uniparental inheritance will deal with these exceptions. They are taken up next. CHANGES IN INHERITED CHARACTERS IN UNIPARENTAL REPRODUCTION In a number of different categories of cases, inherited differences arise during uniparental reproduction, so that the members of a single clone are not all alike in characteristics that are inherited in vegetative repro- duction. Some of these phenomena are of great interest for general genetics. They may be classified in various ways. 714 INHERITANCE AGE CHANGES: SEXUAL IMMATURITY AND MATURITY Beginning with an individual that has recently conjugated, if the lines of descent by vegetative reproduction are followed for great numbers of generations, certain characteristics of the individuals are found gradu- ally to alter. The offspring produced at different periods differ. In Uro- leptus mobilis (Calkins, 1919) or in Paramecium bursaria (Jennings, 1939), the individuals are at first sexually immature; they do not con- jugate under any conditions. This continues for many generations of vegetative multiplication. The offspring during this period are like the parents in this respect. But after many generations have passed, the descendants gradually become sexually mature. They now conjugate when mixed with indi- viduals of different mating type. These descendants are thus different in this respect from their earlier ancestors. In this period their own off- spring inherit from them the mature condition. In Paramecium bursaria, and presumably in other species, the mature condition comes on slowly and gradually. There is for many generations partial maturity, in which the tendency to conjugate is but slight. The tendency becomes stronger as generations pass, until full maturity is reached. The period of full maturity lasts for a great number of genera- tions, during which the mature condition is inherited in vegetative repro- duction. Such periods of immaturity and maturity were described fifty years ago by Maupas (1889) for a number of species of ciliates. In some species, however, they hardly exist, or the period of immaturity if it occurs at all is very short. Such is the situation in P. awrelia (Sonne- born, 1936). At a late period in the life history, in some species the individuals are found to become less vigorous as generations pass. They multiply less rapidly, become “‘depressed,’”’ degenerate. Whether this is an additional period in the life history, beyond the periods of immaturity and ma- turity; whether, in other words, it is an age change, constituting a period of senescence and final senility, or whether it is only a degenerate condi- tion arising in consequence of living long under unfavorable conditions, appears as yet unsettled. This period of decline will therefore be con- sidered in the next section. In some of the Protozoa, particularly among parasitic forms, in dif- INHERITANCE VALS, ferent periods of the life history there are very great differences in form, structure, and physiology, constituting an “alternation of generations.” Each condition is transmitted from parent to offspring for many genera- tions, yet each in time transforms into a later condition. All these phenomena are commonly thought of as matters of “life history,” rather than of inheritance. Yet they represent fundamental fea- tures in those relations of successive generations that are called inherit- ance. The single cell, reproducing vegetatively, produces a great number of other free cells that are like itself in their special peculiarities. Later the character of the cells changes; and again the resulting condition 1s for a long period inherited in vegetative reproduction. In these respects the phenomena are like modifications resulting from environmental action, as shown in later paragraphs. Are the diverse conditions that are vegetatively inherited in different periods—such as sexual immaturity and maturity—the result of changes in chromosomal materials, or changes in the cytoplasm? Dobell (1924) shows that the chromosomes do not visibly change in the series of diverse forms passed through in the life history of certain haploid Sporozoa; throughout all the changes the same set of chromosomes in the same number are present. Tartar and Chen (1940) have found that in the period of sexual maturity, in P. bursaria, parts of the individual consisting only of cytoplasm react sexually. Neither of these observations proves conclusively that the chromosomal materials are not altered in the different periods, but they perhaps make it probable that the different periods in the life history result rather from such interactions between chromosomes and cytoplasm as must occur in producing the bodily differ- entiations of a developing multicellular organism. Whatever the seat of the different inherited conditions in different periods of the life history, it is clear that the material on which the different conditions depend must multiply itself, for long periods remain- ing true to type. An immature individual contains a certain small amount of the material on which immaturity depends. In ten generations this material has multiplied to more than a thousand times its original quan- tity, still remaining immature. Later, having attained the mature condi- tion, it again multiplies in that condition to thousands of times its original quantity. 716 INHERITANCE INHERITED DEGENERATIVE CHANGES RESULTING FROM UNFAVORABLE CONDITIONS In many cases, when ciliate Infusoria are cultivated for long periods in isolation cultures, in which great numbers of successive generations are produced, the organisms are found in the later generations to decline in vigor and vitality. This change is progressive; it becomes greater in later generations. The vital processes become “‘depressed,” slow, inefh- cient; in particular the rate of multiplication decreases. In time the ani- mals become degenerate—abnormal in form and structure, reduced in size. As an index of this decline in vigor and vitality, the changes in the rate of multiplication are commonly employed. Graphs of the daily num- ber of fissions show a curve gradually descending from a high point at the beginning of the isolation culture, to nearly zero at a later period. A large number of such graphs, based on the work of many different investigators on many species, are published in the author’s Genetics of the Protozoa (1929). It has been held by many investigators that this decline is a matter of age; that these graphs are curves of senescence. The earlier periods of immaturity and maturity were believed to be followed inevitably by a period of senescence. Whether this is, indeed, true for some species is still uncertain. But for a number of species it has been shown that the decline need not and does not occur if the conditions are kept entirely favorable (a summary of investigations on this matter is found in the author's Genetics of the Protozoa, 1929). In these latter species, there- fore, the decline and degeneracy are consequences of life under unfavor- able conditions. Thus unfavorable environmental conditions, acting for many succes- sive generations, cause changes in the characteristics of the individuals, and cause them to produce in the later periods offspring that differ from those produced in the earlier periods. In the earlier periods, parents and offspring are vigorous, multiplying rapidly. Later, under the same environmental conditions as before, parents and offspring are weak, nonresistant, multiplying slowly. The effects of the unfavorable environ- ment become cumulative as generations pass, and in vegetative repro- duction they are transmitted to the offspring. The inheritance of the depressed condition is demonstrated in the INHERITANCE TAG following way. Individuals from the depressed later generations are cultivated side by side, under the same conditions, with individuals from the same clone that have not lived under unfavorable conditions. One of the two sets—the latter—multiplies rapidly and at a high level of vitality. But the former set, that has lived under unfavorable conditions, multiplies slowly, at a low level of vitality, in a degenerate condition. Thus in these Protozoa we find realized what some have held must occur in mankind: the production of inherited degeneration, by long- continued bad living conditions. Discussion of the nature of these changes will be reserved until other inherited environmental modifications have been considered. INHERITED ACCLIMATIZATION AND IMMUNITY The changes in inherited characters induced by unfavorable environ- mental conditions are not always degenerative in character. In the uni- cellular organisms, as in multicellular organisms, long exposure to un- favorable conditions may result in the production of acclimatization or immunity. In the Protozoa, after removal from the unfavorable condi- tions, the acquired acclimatization or immunity is inherited in vegetative reproduction for many generations. Cases are on record in which such inheritance continued for many months, including hundreds of vegetative generations. But in the course of many generations under the favorable conditions, with the injurious agent no longer present, the acquired immunity becomes gradually less marked; it slowly decreases, and, finally, in a sufficiently long period it is lost. But this may not occur until months after removal from the immunizing agent, during which time the acquired immunity is inherited. Such acquisition of inherited immunity is most extensively known in parasitic Protozoa and in pathogenic bacteria, since in these organisms it is of medical importance. Detailed accounts of the knowledge in these fields will be found in the treatise of Taliaferro (1929). But acquisition and inheritance of acclimatization or immunity occurs also in free-living Protozoa. A somewhat detailed review and discussion of investigations in this field will be found in the author’s Genetics of the Protozoa (1929). Here only brief summaries of some of the more important investigations in this field can be presented. 718 INHERITANCE In a famous investigation by Dallinger (1887), published fifty-two years ago, three species of flagellates were acclimatized, in seven years, to a high degree of heat. At first the organisims could not tolerate a temperature higher than 26° C. If subjected to this temperature for some time, death occurred. By raising the temperature a half degree at a time, at the average rate of two degrees a month, the tolerance was in the seven years raised to 70° C. When the temperature was raised at any period, usually many of the individuals died. Others lived and multiplied, replacing those that died. There was thus a selective action of the heat; the individuals that did not become acclimatized died. At certain periods, the cytoplasm of the organisms became filled with small vacuoles, which lasted for a month or more, then disappeared. The temperature could not be raised further until the vacuoles had disap- peared. It has been suggested that this vacuolization is a process of getting rid of water, since it is known that protoplasm containing little water can tolerate higher temperatures than protoplasms containing much water. In cases of acclimatization to high temperatures, it is obvious that all parts of the organism must be altered. If any part—nucleus, chromo- somes, cytoplasm—failed to acquire the increased resistance to heat, the organisms would die. Extensive experimental studies in this field were carried on for many years by Jollos (1913, 1920, 1921). He investigated the acclimatization of Paramecium caudatum to arsenic (solutions of arsenious acid), to certain other chemicals, and to high temperatures. The most extensive work was on acclimatization to solutions of arsenic. It was found that P. awrel7a could not be acclimatized to arsenic, and that some biotypes of P. cawdatum were likewise refractory. But in other biotypes of P. caudatum acclimatization was successfully produced. In the successful experiments, a method combining selective action with subjection to gradually increasing concentrations of arsenic was employed. Using as a standard concentration a one-tenth normal solution of arsenious acid, the animals were first placed in a very weak concen- tration, the maximum tolerance at the beginning being about one per- cent of the standard solution. The organisms were left for a long time (days or months) in a solution too weak to destroy them. Then the con- INHERITANCE 719 centration was slightly increased, until many were killed. The few that survived were restored to a weak solution and allowed to multiply. Again there was an increase in concentration until most died; then a restoration to a weak solution. As this continued, it was found that the animals became able to tolerate higher concentrations. In three or four months, the toleration was thus increased from about one percent to 2.5 percent of the standard solution. In other cases the tolerance was raised in several months to 5 or 6 percent. When the organisms that had thus acquired a higher resistance to arsenic were restored to water containing no arsenic, the increased resist- ance was for long periods not lost. Tests at intervals showed that they still retained the higher tolerance to arsenic. The acquired higher resist- ance lasted in some cases for eight months or more. As the animals were reproducing at about the rate of one fission daily, the acquired resistance was inherited for about 250 generations. But during this time in water the acquired resistance to arsenic gradu- ally decreased. The rate of decrease was very slow, so that in such a case as that mentioned above, the tolerance had not returned to its original low level until after a period of eight months. The course of events may be illustrated from the history of Jollos’s clone A of P. caudatum. In this clone the original maximum tolerance to arsenic was to 1.1 percent of the standard solution. By cultivation in gradually increasing concentrations for four months, the maximum toler- ance was raised to 5 percent. Upon restoration to water containing no arsenic, the tolerance was, in tests for successive periods after the restora- tion, as. follows: 6 days, 5 percent; 22 days, 5 percent; 46 days, 4.5 percent; 53 days, 4.7 percent; 60 days, 4.7 percent; 75 days, 4.7 percent; 130 days, 4.5 percent; 144 days, 4.2 percent; 151 days, 4.0 percent; 166 days, 3.0 percent; 183 days, 2.5 percent; 198 .days, 1.25 percent; 255 days, 1.0 percent. Thus the acquired resistance persisted in a very marked degree for more than five months, but had been entirely lost at the end of eight and a half months. Jollos acclimatized Paramecium also to high temperatures, continuing the experimental cultures in some cases as long as two and a half years. The tolerance to high temperatures was increased, and the increased toler- ance lasted in some cases to six months after removal from the high 720 INHERITANCE temperatures. As in the case of resistance to arsenic, the acquired toler- ance slowly decreased and finally disappeared when the organisms were cultivated at moderate temperatures. To such long-lasting modifications, inherited for many generations but finally disappearing, Jollos gave the name Dauermodifikationen. This designation is much employed, even in languages other than German. Jollos interprets these modifications as affecting only the cytoplasm, not the chromosomes; a matter to which we shall turn later. Phenomena of a similar character have been described by Jollos and others in multicellu- lar organisms. Hammerling (1929) has published an extensive sum- mary of what are believed to be Dawermodifikationen in many organisms. Certain additional features of acclimatization in free-living Protozoa are brought out in the work of Neuschloss (1919, 1920). He investi- gated the acclimatization of P. cawdatum to certain chemicals, particularly to quinine, methylene blue, trypan blue, fuchsin; also to arsenic and anti- mony. He found that he could induce increased resistance in about a month by gradually subjecting the animals to increasing concentrations of the substances. Neuschloss investigated the nature of the changes in the organisms in the following way. After acclimatizing the organisms to an injurious substance, two equal samples of that substance were taken, in concen- tration somewhat greater than that to which the animals were resistant. To one of these was added a large number of the acclimatized paramecia, to the other an equal number of the unacclimatized animals. The animals were in each case left in the samples until death occurred. After death, the amount of injurious substance that had been removed by the organ- isms from the solution was quantitatively determined. In all cases it was found that the acclimatized animals removed from solution a much greater proportion of the injurious substance than did the unacclimatized. In the case of the four organic compounds, the per- centage removed from the solutions by the acclimatized and the unacclima- tized animals were as follows: Injurious Acclimatized Unacclimatized Substance Animals Animals Quinine 80.0 4.5 Methylene blue SS) 0.5 Trypan blue 54.0 0.75 Fuchsin 60.0 1.00 INHERITANCE 721 The substances removed from solution were found not to exist in the bodies of the animals. It appears, therefore, that the acclimatized animals in some way destroy the injurious organic compounds. Presumably they produce some secretion that has this effect. In the case of the inorganic poisons, arsenic and antimony, the acclima- tized animals were found to have acquired the power to transform the highly injurious trivalent compounds of the two elements into the rela- tively harmless pentavalent compounds. The acclimatization was found to be, as a rule, specific for the substance to which the animals had been exposed. Acclimatization to one of the organic substances did not increase resistance to the other three. But in the case of the related inorganic materials arsenic and antimony, acquire- ment of increased resistance to one induced acclimatization to the other. INHERITED ENVIRONMENTAL MODIFICATIONS IN FORM AND STRUCTURE Reynolds (1923) and Jollos (1924) studied extensively the inherit- ance of certain abnormal forms of the shell in Arce//a; an extended review of this work is given in the author’s Genetics of the Protozoa (1929). The work of Jollos shows that these abnormalities are favored by certain environmental conditions, and perhaps makes it probable that they originate as environmental modifications. Inheritance of the abnor- mal conditions continued for many generations. By selection, the grades or degrees of abnormality could be changed. By long selection of the most nearly normal individuals, a stock that was nearly or quite normal could be produced. According to Jollos, the length of time required to bring the animals by selection back to a normal con- dition is proportional to the length of time that the ancestors have lived under the environmental conditions that favor the abnormality. When the abnormal stock is allowed to multiply without selection, the proportion of individuals with but slight degrees of abnormality increases. Jollos holds that this indicates that the abnormal condition is essentially transient, a Dauermodifikation, in which the cytoplasmic tendency to abnormality is in time overcome by the nuclear tendency toward normality. On the other hand, the higher degrees of abnormality are harmful to the organism, so that natural selection tends to weed these out, causing the stock to become less abnormal in later generations. See the extended presentation of the evidence and the discussion on this 722 INHERITANCE point in the author’s Genetics of the Protozoa (1929, pp. 261-70). Moewus (1934b) has made extensive studies of inherited environ- mental modifications in structural characters in Chlamydomonas de- baryana. This species is known to occur in many varieties differing slightly in form, size, and structure; Moewus describes and figures twelve of these. The different varieties are found in nature; also they occur under different cultural conditions in the laboratory. Moewus found that he could transform the different varieties one into another by the use of different culture media. But the transformation did TABLE 14: ENVIRONMENTAL MODIFICATIONS, Chlamydomonas debaryana; RELATION OF THE NUMBER OF Days CULTIVATED IN PEPTONE MEDIUM TO THE NUMBER OF Days IN SALT-SUGAR MEDIUM REQUIRED TO TRANS- FORM THE ORGANISMS FROM TYPE 1 TO TYPE 5 (FROM Moewus, 1934b) Daysonibepione Days in Salt-Sugar Medium T Required for Transformation as Type I Teper 28 28 140 49 273 133 441 175 567 231 609 379 644 459 672 531 690 534 not occur at once, on transfer to the different culture medium; on the contrary, many generations in the new culture medium were necessary to induce the transformation. An extremely important relation was observed to hold as to the time required for the transformation. The longer a stock has remained under conditions producing a given type, the greater the time and the number of generations required to transform it to a new type, when placed under new conditions. An example will illustrate the operation of this principle. Individuals in peptone culture are of type 1. If transferred to a “‘salt- sugar” infusion, they transform to type 5. If they have been only a few generations in the peptone culture, the transformation in the salt-sugar medium occurs very quickly. But if they have lived for a long time in the peptone medium, a long time is required for transformation to type 5. INHERITANCE 723 Table 14 from Moewus shows the number of days required for the transformation to type 5, in relation to the number of days the organisms had lived in peptone medium. In interpreting this table, it is important to keep in mind the fact that reproduction occurs at about the rate of one generation a day, or more rapidly. In the last examples of Table 14, it required a year and five months in their new conditions to induce in the organisms the transformation to type 5. During this period hundreds of generations were produced. The longer the organisms remain in peptone culture as type 1, the longer it requires for the new conditions to transform them to type 5. Moewus presents many other cases illustrating the same principle. The different types or varieties found in nature usually require a very long period of culture in a given medium to induce them to transform to the type characteristic for that medium. Thus a certain stock 4 was found in nature as type 10, having no papilla. Placed in peptone culture, it remains as type 10 for 450 generations, then the organisms begin to transform to type 1, having a papilla; in time all transform to type 1. The type 10 was seemingly a Davermodifikation in nature; it transforms to type 1 only after many generations under artificial conditions. Moewus describes many other cases in which types found in nature resist, for many generations, changes due to new conditions, but finally transform into types characteristic for the medium to which they are transferred. Thus the indications are, as Moewus points out, that many of the diverse types (‘varieties’) found in nature are in fact long-lasting modifications resulting from the long-continued action of certain environmental condi- tions. VARIATION AND ITS INHERITANCE OCCURRING WITHOUT OBVIOUS ACTION OF DIVERSE ENVIRONMENTS In addition to inherited changes produced by diverse environments, already described, there occur in some cases during vegetative reproduc- tion inherited changes, without apparent action of diverse environments. Such cases possibly differ in principle from those described above, illus- trating the occurrence of inherited variations that are not brought about by environmental action. If so, they are of much theoretical interest. On the other hand, they may involve concealed action of diverse environ- mental conditions, as urged by Jollos (1934). The phenomena are in 724 INHERITANCE themselves of interest, illustrating certain methods of action not thus far described. The author’s studies on Difflugia corona may serve as an example (Jennings, 1916, 1929). In D. corona (Fig. 168) the individuals differ greatly in a number of strongly marked structural features. They have a silicious cell, produced Figure 168. Difflugia corona. Members of four different clones, showing diversities in characteristics. (After Jennings, 1916.) at the time of reproduction. The shell bears conspicuous spines, which differ greatly in number and in length in the different individuals. The number of teeth surrounding the mouth differs in different cases. The size of the shell shows wide variation. Many diverse biotypes occur in nature, each characterized by a definite combination of characteristics. Some have few spines, some many. Some have long spines, others short spines. Some have few teeth, others many teeth. Some are large, others small. INHERITANCE #25 Even within a single clone there is variation in characters, though much less than when different clones are taken into consideration. Different individuals of the same clone may differ greatly in number and length of spines, slightly in size of the body, very slightly in the number of teeth. In vegetative reproduction, the offspring resemble the parent, but not completely. The general rule is that parent and offspring are alike in respect to characters that distinguish different biotypes or races, but they need not be alike with respect to characters in which different indi- viduals of a clone are diverse. The type of inheritance will be seen from examples of the numbers of spines in successive generations in certain clones: Clone — > —_1—-6 Clone 22 =v 4 Gloners 6 o— o_o —_4— 5 _7 © Clone 4: O0—0—0O—O—1—0—0O—1—0O Thus clones differ from each other in the fact that some usually have few spines or none, others a slightly larger number, others a still larger number—though within each clone there is variation. A given biotype, or clone, is characterized by a certain average number of spines; another by a different average number. In respect to the average numbers of spines, many different biotypes may be distinguished. When from a single clone individuals differing in number of spines are allowed to multiply until each has produced many descendants, the usual result is that the average number of spines in the two sets of descendants is the same. Thus in clone 1 above, the individual with 2 spines and that with 6 spines would, as a rule, produce descendants having the same average number of spines. The individual differences within the clone are usually not inherited; the mean differences between biotypes are inherited. To what are due the differences in numbers of spines or in other char- acteristics, between the different members of the same clone, illustrated above? The shell is produced complete at the time that reproduction occurs. The individual about to reproduce buries itself in a mass of soft debris. The protoplasm swells, projects from the mouth of the 726 INHERITANCE parental shell, and takes on the form of the protoplasmic body. A mass of silicious particles, which have earlier been collected within the parent, passes to the surface of the new protoplasmic body and is there molded into the final form of the shell of the offspring. Meantime the nucleus has divided and one of the products passes into the young individual. Parent and offspring now separate. During this process are formed the spines in their final form, length, and number. The parent shell may have four spines, the shell of the new individual five spines or some other number; there may be differences in the length of the spines. The differences between the characteristics of parent and offspring might conceivably be due to slight and obscure differences in the environmental conditions at the time of reproduction. Or they might be due to diversities in the internal condition or consti- tution of the individuals at the time of reproduction, however such diversities had been produced. Results of long-continued selection From the results thus far set forth, it appears that the individual diversities within a clone of D/f- flugia are not usually or strongly inherited. Is there any tendency what- ever for such inheritance? In other words, does the difference of condi- tion or constitution that results in diversity of characters ever or in any degree persist through vegetative reproduction? This may be tested by long-continued selective breeding within a clone. Suppose that the basis of selection is the number of spines. From a single clone two groups are segregated, one containing only individ- uals with high numbers of spines, the other only individuals with low numbers of spines. In the former group are retained only descendants with high numbers of spines; from the second group only descendants with low numbers of spines. In this way there are obtained in later gen- erations from the first group individuals descended for many generations from ancestors all of which had high numbers of spines, and from the second group individuals descended for many generations from ancestors with low numbers of spines. Selective breeding of this kind may be car- ried on with any of the varying characteristics. When such selection is practiced for many generations, usually for considerable periods no difference in the offspring of the two groups is to be discovered. But as the number of generations becomes greater, an INHERITANCE T2Y. effect in the direction of selection appears. The offspring of the group whose ancestors have had high numbers of spines have a greater average number of spines than the offspring of the group whose ancestors have had low numbers of spines. Such results may be illustrated from the clone 326, in which selective breeding for high and low numbers of spines was carried on for a long period (see Table 15). TABLE 15: EARLY RESULTS OF SELECTION FOR LOW AND HIGH NUMBERS OF SPINES, Difflugia corona (FROM JENNINGS, 1916) Low Group HicH Group Beisod Dies Parental Mean Spines Parental Mean Spines Spines of Descendants Spines of Descendants I 36 1-4 5.56 5-9 5.46 2 60 1-4 5.13 6-11 5.19 3 60 I-4 : 6-11 5.46 4 120 ih 5.29 4-11 5.79 In the fourth period there is a distinct difference in the two groups. To test whether this is significant, the fourth period (four months) was divided into six successive periods, and the numbers of spines in the offspring of the two groups determined for each. They were as shown in Table 16. TABLE 16: LATER RESULTS OF SELECTION FOR Low AND HIGH NUMBERS OF SPINES, Difflugia corona; NUMBERS OF SPINES IN THE PARENTS (1-5 IN THE Low Group, 7-11 IN THE HIGH GROUP) WITH THE MEAN Num- BERS OF SPINES ON THEIR OFFSPRING IN SUCCESSIVE PERIODS (FROM JEN- NINGS, 1916) PARENTS I-§ 7-11 Period Offspring I 5.59 6.11 3) op) 5.71 3 5.49 6.53 4 5.23 ao 5 Helo} 5.38 6 4.63 5-59 728 INHERITANCE Thus in each of the six periods of the last four months, the parents with high numbers of spines produced offspring with higher mean num- bers of spines than did parents with low numbers of spines, though the differences were not very great. Selective breeding of this type was practiced also with relation to the length of the longest spines. The results here show clearly a phenomenon of much interest, namely inherited variation with “regression toward the mean’’ of the biotype as a whole. This appeared also in the results of TABLE 17: INHERITANCE OF SPINE LENGTH, WITH REGRESSION TOWARD THE MEAN, Djifflugia corona, CLONE 326; MEASUREMENTS OF SPINE LENGTHS, IN UNITs OF 4 2/3 MICRONS EACH (FROM JENNINGS, 1916) Parents Mean Lengths of Length of Longest Number of Offspring Longest Spine in the Spines, in Units Offspring, in Units 4-6 21 10.38 7-9 | 162 I1.O1 10-12 | 451 11.85 13-15 367 12.90 16-18 129 14.39 19-21 26 14.34 22-24 15 16.34 25 and above 18 17.06 Mean length for all, 12.54 selective breeding with respect to other characters. The results of selection for length of spines are given in Table 17. (The spines were measured in units, each of which was 424 microns. ) Table 17 shows that parents with longer spines produce, on the average, offspring with longer spines, so that there is a distinct tendency to inheritance in spine length, even within the single clone. But another relation is equally evident. Parents that are above the mean of the biotype (12.54) produce offspring that are above the mean, but not so much above the mean as ate the selected parents. Parents that are below the mean produce offspring that are below the mean, but not so much below the mean as are the selected parents. That is, inheritance of the parental peculiarities occurs, but always with regression toward the mean of the biotype. On the average, the offspring diverge from the INHERITANCE 729 racial mean in the same direction as do the selected parents, but not so far. Only a small part of the selected parents’ divergence from the mean is inherited by the offspring. To what is due such inheritance, with regression toward the racial mean? The interpretation is not clear, but the most natural conception appears to be the following. The divergence from the racial mean in the case of the selected parents is partly a matter of environment, partly a matter of genetic constitution. The latter is inherited by offspring, the former not. The fact that a part of the parent’s peculiarity is inherited shows that variation in the genetic constitution itself occurs in vegetative reproduction and is inherited. It is, however, in large degree masked by variations due to environmental conditions, and not inherited. As a consequence of the inheritance of some portion of the parents’ peculiarities, in time the single clone or biotype may by selective breeding become differentiated into a number of biotypes, differing slightly in inherited characters. Five such hereditarily different biotypes of D/fflagia corona, produced through selective breeding from a single one, are described and figured in the paper of Jennings (1916). For such heritable change in the genetic constitution two types of interpretation may be suggested. On the one hand, the heritable changes may be due to obscure alterations in environmental conditions, so that these changes are comparable to those in acclimatization or other Dawer- modi fikationen. On the other hand, the changes may be attributed to irregularities occurring in the genetic materials—either ‘“‘mutations”’ or slight irregularities of distribution—when reproduction occurs. At pres- ent it appears not possible to decide between these two interpretations. SUMMARY AND INTERPRETATION To recapitulate, in Protozoa, a number of different types of inherited changes may occur during vegetative reproduction, giving rise to indi- viduals or lines of descent that have different characteristics which are transmitted to descendants in further vegetative reproduction. First, there are changes that occur in the course of the normal life history—changes as to sexual maturity, and, in some species, profound alterations in structural and physiological characters (“‘alternation of generations’). Each stage in such a life history includes many successive 730 INHERITANCE generations, during which the distinctive features of that stage (imma- turity, maturity, or the like) are transmitted to descendants. Second, there are inherited degenerative changes, resulting from life under unfavorable conditions. Third, there are adaptive changes, fitting the organism to a changed environment—inherited acclimatization, or immaturity. Fourth, there are inherited changes in form and structure, apparently neither adaptive nor degenerative, occurring under the influence of spe- cific environmental conditions. Fifth, forming perhaps an additional type, there are inherited varia- tions in form, size, and other characters, that are not obviously due to environmental conditions. Some of these five types fall under Jollo’s concept of Dauermodifi- kationen: changes produced under the influence of environmental condi- tions, inherited for many generations after removal from those conditions, but gradually fading out to final disappearance. This includes the third and fourth types above, and, according to Jollos’s view, also the fifth. The first type obviously appears not to fall under this concept, and the second type is not known to disappear upon restoration to a more favor- able environment. Most of thest types of modification may disappear or be altered upon the occurrence of sexual reproduction. In type 1, the condition of matur- ity changes at sexual reproduction into one of immaturity. In type 2, the inherited degenerative changes in many cases disappear at sexual repro- duction (“‘rejuvenescence through conjugation’’), but it is a notable fact that in some cases they do not. In type 3, the inherited acclimatization and immunity likewise often disappear at sexual reproduction, though again in some cases they do not. In types 4 and 5, the effect of sexual repro- duction is not known. These inherited modifications, so far as they can be brought under the concept of Dauermodifikationen, are held by Jollos to have their seat in the cytoplasm only, the genetic materials of the chromosomes being unchanged. This is based in the main on the fact that the inherited modifications grow less and finally disappear, when the organisms are restored to environments that do not produce the modifications. A change in the genetic materials of the chromosomes (“‘mutation’’), it is held, would be permanent; it would not disappear after many generations in INHERITANCE on an altered environment. But a change in the cytoplasm, it is urged, would in the course of time be overcome and dominated by the unchanged con- stitution of the nucleus, bringing about a return to the original charac- teristics—as actually occurs. These inherited modifications are, on this view, essentially transitory conditions, forming no part of the system of genuinely inherited characters. The fact that these modifications usually (though not always) dis- appear at sexual reproduction is likewise held to be due to the fact that they have their seat in the cytoplasm, though the logic of this is not entirely clear. As will be shown later, it is the nucleus, not the cytoplasm, that is directly changed in constitution at conjugation. The disappearance at conjugation is seemingly attributed to the profound making over of the cytoplasm that has been believed to occur at conjugation, producing rejuvenescence. The long-continued inheritance of the modifications dur- ing vegetative reproduction is held to be an example of cytoplasmic in- heritance. If we are here indeed dealing with cytoplasmic inheritance, this appears to demonstrate that in the Protozoa the cytoplasm partakes of the essential features of genetic material. These essential features, as before set forth are: (1) the fact that the material in question affects the inherited characteristics, and (2) the fact that this material multiplies true to type. The environmental modifications are, as we have seen, at times inherited for more than 200 generations after removal from the conditions that induced them. In 10 generations, the originally modified cytoplasm has been diluted to one-thousandth of its volume, in any indi- vidual; in 20 generations, to less than one-millionth of its volume. The rest of the cytoplasm of the individual is a product of cytoplasmic growth. Yet the environmental modification still persists. If the cytoplasm 1s the seat of the modification, the modified cytoplasm must reproduce itself in its modified condition at fission; otherwise its effect would have dis- appeared under the great dilution that it has undergone. On the question as to whether cytoplasmic inheritance indeed occurs in these organisms, evidence will be presented in the section on biparental reproduction. There also a method will be indicated by which it may be determined whether the seat of these modifications is in the cytoplasm or in the nucleus. ere INHERITANCE INHERITANCE IN BIPARENTAL REPRODUCTION Biparental or sexual reproduction includes the processes that lead to the formation of new individuals from the united parts of two earlier individuals. Biparental reproduction in the Protozoa occurs in two main types, known respectively as copulation and conjugation. Copulation is characteristic of haploid species; in it two haploid individuals unite com- pletely to form a diploid zygote, which later divides with reduction, to form haploid individuals again. This method occurs in the Flagellata. In conjugation two diploid individuals exchange halves of their nuclei, including a haploid set of chromosomes; then the two separate and each continues thereafter to multiply by fission. This is the characteristic method in the Ciliata. BIPARENTAL INHERITANCE IN HAPLOIDS: FLAGELLATA Knowledge of the genetics of flagellates is largely due to the recent work of Moewus (1932-38). In flagellates the single motile individual is haploid. In copulation two such haploid cells unite completely, to form a diploid cell. The two hapoid cells thus correspond functionally to two gametes, while the dip- loid cell is the zygote. The zygote is inactive; it secretes a wall about itself and becomes a cyst. Later, under favorable conditions, the diploid cyst divides twice by the two “maturation divisions.’ At one of these divisions chromosome reduction occurs, so that the four cells formed are haploid. At times additional cell divisions occur before the cells emerge from the cyst. The cyst wall dissolves and the haploid cells are freed. Each develops flagella and swims about as a free individual. On emerging from the cyst, the individuals are often called swarmers or swarm cells; each is potentially a gamete. These free cells commonly multiply vegetatively for many generations, the descendants of each original swarm cell forming a clone. In any species or variety the haploid individuals or gametes are dif- ferentiated into two sexes; details as to this will be found in the chapter on “Sexuality in Unicellular Organisms’’ (Chapter XIV). In some spe- cies or varieties all members of the same clone are of the same sex, the different sexes being in different clones (dioecious species or races). In others both sexes are found among the individuals of a single clone INHERITANCE 125 4 C D Figure 169. Polytoma uvella (A, B) and P. pascheri (C, D); the four races used in the breeding experiments of Moewus. (After Moewus, 1935b.) (monoecious species or races). Details as to sex determination will be found in Chapter XIV; here sex will be dealt with only insofar as it plays a rdle in the general system of inheritance. The flagellates exemplify in admirably simple form the system of in- heritance in haploid organisms. The system is well exhibited in the studies of Moewus (1935b) on inheritance in Polytoma. In this work 734 INHERITANCE two species were employed, and of each species there were two varieties. Polytoma uvella is ellipsoidal in form, with no papilla at the anterior end (between the places of attachment of the two flagella). In one variety there is a large eyespot, in the other there is no eyespot (see Fig. 169A, B). In P. pascher7 the form is a long oval, there is a papilla between the flagellar attachments, and there is no eyespot. One variety is large, the other very small (Fig. 169 C, D). In the four varieties there are thus differences in four pairs of char- acters. Moewus designates these characters as follows: Form of body: ellipsoidal, F (as in P. avella) long oval, f (as in P. pascherz) Papilla: present, P absent, p Eyespot: present, S absent, s Size: large, D small, d Using these designations, the four varieties may be represented by formulae as follows: P. uvella, with eyespot, FpSD without eyespot, FpsD P. pascheri, large, fPsD small, fPsd Any of these four varieties may be crossed with any other, so that six different crosses are possible. Each of the varieties has eight chromo- somes, so that any cross forms a zygote with sixteen chromosomes. From each zygote arise, by the two maturation divisions, four swarm cells. These four, after becoming free, multiply vegetatively to form clones. All the individuals of each clone show the same characteristics as does the swarm cell from which the clone is derived, so that we may speak indifferently of the characteristics of each of the four swarm cells, or of each of the four clones derived from them. When two varieties that differ in a single pair of characters are crossed, two of the four swarm cells produced show one of these characters, the other two the other character. Thus P. wvella with eyespot, S, is crossed with P. wvella without eyespot, s. Of the four swarmers arising from the INHERITANCE 735 zygote, two have the eyespot (S), two are without it (s). The cross may be represented as follows: SX s=S-+s It is obvious that the segregation of the two characters must have oc- curred at the reduction division. One of the sets of eight chromosomes includes the conditions for producing S, the other the conditions for producing s. The results of the crosses in which the parents differ in two pairs of characters may be exemplified in the mating of P. wve/la without an eyespot (FpsD) with the large variety of P. pascheri (fPsD) (Fig. 170 A, B). The two differ in form (F and f), and in presence or absence of the papilla (P and p). The zygote thus carries both these sets of factors; it is FpsD fPsD The zygote divides into four swarm cells, with reduction. The results are as follows: 1. From any single zygote two and only two types or combinations are produced. Two of the four cells are of one combination, two of the other. 2. Different pairs of types are produced in different cases (Fig. 170). About half the zygotes yield again the two parental types, FpsD and fPsD (Fig. 170 C, D). The other half yield FPsD (ellipsoidal with papilla, Fig. 170 E) and fpsD (oval without papilla, Fig. 170 F). Thus the characters, form, and papilla are inherited independently. The factors F and f are in the two members of one pair of chromosomes, the factors P and p in another pair of chromosomes. When crosses are made in which the individuals differ in three pairs of characters (as FpsD x fPsd) or in all the four pairs of characters (FpSD x fPsd), all the four pairs are found to be independent in their distribution. From any single zygote only two types of offspring are produced. But from different zygotes of the three-pair cross, eight dif- ferent combinations are produced; from those of the four-pair cross, sixteen different combinations. The different combinations occur in ap- proximately equal numbers. Thus the factors for the four pairs of char- acters are distributed in four chromosome pairs, which at reduction are assorted independently. F Figure 170. Results of a cross between two species of Polytoma. (Combined from figures of Moewus, 1936) A and B, the two parents ; C and D, types produced by half the zygotes; E and F, types produced by the remainder of the zygotes. INHERITANCE Hew! In consequence of the independent inheritance of these four pairs of characteristics, there result from crosses certain curious combinations in which there is lack of harmony in the parts of the individuals. Thus the eyespot, S, is inherited independently of the size of the cell (D or d). The eyespot is originally in the large race, D, where its length is about one-sixth the length of the cell. By crosses it may be transferred to the small race, d. Here it retains its large size, so that there are produced small individuals with eyespots about half the length of the individual. The fact that from any single zygote but two types or combinations appear among the offspring shows that the reduction of the chromo- somes must occur at the first division of the zygote. If it occurred at the second division, there would be in some cases four different types or combinations from a single zygote. For suppose that we have a two-factor cross, such as Fp fP. Then the zygote has the combination FpfP. If reduction occurs at the first division, in some zygotes the two cells produced are Fp and fP. Each now divides equationally, giving two cells that are Fp, two that are fP. In other zygotes the reduction division yields FP and fp; again the second division yields two cells of each type. In either case but two types are produced from any one zygote. But if reduction occurs at the second division, then after the first divi- sion there are two cells present, both with the combination FpfP. Now by reduction at the second division, one of these may yield Fp and fP, the other FP and fp, so that four different combinations would be pro- duced from a single zygote. Linkage and crossing over.—Besides the four independent pairs of characters just described, there are in Polytoma others that are linked with some of the four. Such a character is length of the flagella. In the original types, the length of the flagella is proportional to the length of the body; large cells (A, B, C) have long flagella, small cells (D) have short flagella (Fig. 169). This proportionality usually holds among the crosses; those that have large bodies have long flagella, those with small bodies have short flagella. This indicates either that the factors for size (D and d) and those for length of flagella are close together in the same chromosome, and so linked, or that the relation is merely a physiological one, cells of a given size always producing flagella of length propor- tional to the size. 738 INHERITANCE To determine which of these alternatives is correct, Moewus made 1,400 crosses between the large and the small types (D and d). Of these, 1,357 yielded as usual zygotes which gave 50 percent large cells with long flagella, 50 percent small cells with short flagella. The remaining 73, or 5.2 percent of all, yielded zygotes that gave 50 percent large cells with short flagella, and 50 percent small cells with long flagella (Fig. 171). In other words, these 73 zygotes gave only cells carrying the new combinations. It appeared, therefore, that the factors for size of body and length of flagella are merely linked, through the fact that the two a b < Figure 171. New combinations resulting from crossing over. a and c, large cells with short flagella, b, small cell with long flagella. (After Moewus.) are close together in the same chromosome. Their distance apart is such as to yield crossing over in about 5.2 percent of the zygotes. This was confirmed by making 600 crosses between the new combinations, one parent having large body cells with short flagella, the other small body cells with long flagella (Fig. 171). Of the zygotes, 570 gave equal num- bers that were like the parents, while 30 (or 5 percent) gave equal numbers of the original combinations—large body with long flagella, and small body with short flagella. These 30 therefore gave 100 percent crossovers. In these cases an extraordinary situation appears, seemingly unique in crossing over. In all these cases, any zygote that yields any crossover combination yields exclusively crossovers. The 73 zygotes of the first 1,400 mentioned in the preceding paragraph yielded 50 percent of one INHERITANCE ey) of the crossover combinations (large cells with short flagella), 50 per- cent of the other crossover combination (small cells with long flagella). The same relations hold for the 30 crossover zygotes out of 600 in the recrossing of the new combinations; they yield 100 percent crossovers. Such results can occur only if crossing over takes place between the two entire chromosomes that are in synapsis, that is, only if crossing over occurs in the so-called two-strand stage of the synapsed chromo- some pair. In other organisms, so far as the matter has been analyzed, crossing over occurs only after the two synapsed chromosomes have split, so that there are four strands instead of two. Crossing over, then, occurs between but two of the four strands, with the result that two strands remain without crossovers. If this were the case in Polytoma, the zygotes that yield crossovers would yield but two crossover cells and two that were non-crossovers, in place of yielding only crossovers. The same situation is found in all the accounts of crossing over in Flagellata given by Moewus, up to his article of 1938. In this publication he states that he has observed in Ch/amydomonas the occurrence of crossing over in accordance with the four-strand schema (details to be given later). None of the four pairs of characters thus far considered was found to be linked with sex. That is, any of the alternative characters occurs equally frequently with either sex. If, therefore, sex depends on a chromosome pair, it is a fifth pair, not one of the four that carry the characters above discussed. In an article of 1936, Moewus presents the results of extensive studies of inheritance in crosses of Chlamydomonas eugametos with C. pau pera. Here eleven pairs of characters were distinguishable, some morphologi- cal, others physiological. In addition, there were sex differences, making twelve pairs of characters in all. In the species of Chlamydomonas there are ten chromosomes, so that some of the twelve pairs of characters must have their factors in the same chromosome, and in fact Moewus discovered that some of the char- acteristics are linked. He reports that in the many crosses made, he analyzed the 8,000 haploid individuals derived from 2,000 zygotes re- sulting from crosses, and that he obtained 1,024 diverse types in such proportions as to show that each of the ten chromosomes bears the fac- tors for one or more pairs of characters. Of most of these combinations no detailed accounts are given. Linkage was found to exist between two physiological characters, (1) 740 INHERITANCE adaptation to acid or alkaline medium, with (2) differences in the num- ber of cells into which the zygote divides before the cells are set free. Here, as in the former case, the results are such as to indicate crossing over in the two-strand stage. Any zygote that yields crossovers yields nothing but crossovers. Other cases of linkage involved factors for sex; these will be mentioned in the account of sex inheritance. Certain general relations may be pointed out in the method of inheri- tance of non-sexual characters thus far presented, particularly as illus- trated by Polytoma: 1. The inheritance is the typical Mendelian inheritance for haploid organisms. The characters manifested in the haploid descendants are combinations of characters that were manifested in the two parents. No characters appear in the offspring that were not manifested in the two parents, that is, no recessive characters occur in haploid organisms. In them all characters for which factors exist are manifested. This is, of course, a consequence of the fact that in haploids the chromosomes are not in pairs, but single. Hence heterozygotes cannot oc- cur (except in the diploid zygotes). In haploid inheritance, the combina- tions of characteristics that occur in the offspring depend wholely on the characteristics manifested in the immediate parents. 2. The inheritance, like that in multicellular organisms, follows the course that is to be expected if the different pairs of characters depend on factors present in the different pairs of chromosomes. Independent segregation, linkage, and crossing over are demonstrated in Protozoa. Crossing over is, however, as before mentioned, of an exceptional type. SEX INHERITANCE AND SEX-LINKED INHERITANCE In any species or race of the flagellates examined by Moewus, there are two sexes, or mating types. An account of these will be found in Chapter XIV of the present volume, ‘‘Sexuality in Unicellular Organ- isms.” Moewus designates the two sexes on his earlier reports as plus and minus, in later publications as male and female, those earlier called minus being male, while the plus types are female (Moewus, 1937a). Here the different types of sex inheritance will be summarized, the ac- count being based upon the work of Moewus. In the flagellates investigated, some stocks are dioecious, others monoecious. Both types may occur in different races of the same species. INHERITANCE 741 Dioecious races.—tIn these the sexes are in separate clones, all mem- bers of any one clone being of the same sex. The diploid zygote is formed by the union of two haploids of opposite sex and from different clones. By the two maturation divisions, the zygote divides into four cells. Two of these are always of one sex, two of the other. Thus the sexes are segregated at the reduction division, as if sex were determined by a single chromosome pair, one member of which produces one sex, the other the other sex. Sex is determined by the genetic constitution of the clone; sex determination is genotypic. Such dioecious stocks occur in certain races of Chlamydomonas eugametos, of Polytoma pascheri, and of Protosiphon botryoides. Subdioecious races—In certain races of Chlamydomonas eugametos there is found a modification of the dioecious condition. In any single clone, most of the individuals belong to one sex, a few to the opposite sex. Some clones are prevailingly plus, others prevailingly minus. A num- ber of different matings are possible: 1. Plus gamete from a prevailingly plus race, minus gamete from a prevailingly minus race. Two of the cells from the zygote yield prevail- ingly plus clones, two yield prevailingly minus clones. The segregation of “‘prevailingly plus” from “prevailingly minus” therefore occurs at the reduction division; the difference is genotypic. 2. Plus and minus gametes from a prevailingly plus race. All clones from the zygote are prevailingly plus. 3. Plus and minus gametes from a prevailingly minus race. All clones from the zygote are prevailingly minus. In other crosses, clones from a dioecious race, in which the clones are exclusively of one sex, are crossed with prevailingly plus or prevailingly minus clones from subdioecious races. The former will-be spoken of as “pure” for sex, the latter as ‘‘mixed’’ for sex. 4. Plus gamete from a race pure for sex, minus gamete from a pre- vailingly minus clone of mixed race. Offspring: two pure plus, two prevailingly minus mixed. In other crosses of this type, the results were similar; the “pure”’ condition segregates from the “mixed” condition at the reduction divi- sion. In general, the two types that unite to produce the zygote separate again at the reduction division; the differences are genotypic. The fact that in single clones of the subdioecious races some indi- 742 INHERITANCE viduals are plus and others minus is held by Moewus to be due to some- thing in the surrounding conditions; the sex determination within the clone is phenotypic instead of genotypic, as in the dioecious races. But in the “prevailingly plus” clones the constitution is such that minus gametes are not so readily or numerously produced by the conditions as is the case in “prevailingly minus” clones. Moewus found that subjec- tion to dilute formaldehyde or acetone causes the zygotes of the sub- dioecious races to produce gametes of only one sex, that sex which would have been in the majority if these substances had not been used. The precise constitution of subdioecious races, together with that of other types, is considered in a later section. Monoecious races ——In monoecious races both sexes occur in a single clone, so that such clones may be spoken of as ‘‘mixed”’ as to sex. This is the situation in certain races of various species of Chlamydomonas; in Polytoma uvella, and in somes races of P. pascheri and of Protosiphon botryoides. In monoecious stocks obviously the fission of a single individual gives rise to both sexes. The determination of sex in such stocks is largely or entirely phenotypic; that is, through external conditions. Such pheno- typic sex determination is not dealt with in the present chapter. When from monoecious stocks plus and minus gametes are mated, the descendant clones are all monoecious, that is, mixed as to sex. Crosses between dioecious and monoecious stocks —A number of dif- ferent types of crosses may be made between dioecious and monoecious stocks, as follows: 1. Plus gamete from a “mixed” (monoecious) clone; minus gamete from a “‘pure’’ (dioecious) clone (cross of two diverse races of Polytoma pascher7). Result, two of the four descendant clones are mixed, two pure minus. 2. Plus gamete from pure clone, minus gamete from mixed clone (cross of two races of P. pascheri). Result, two clones mixed, two pure plus. In these two crosses, two results are notable. (1) The pure condition segregates from the mixed condition at the reduction division, the two depending apparently on the two different chromosomes of a pair. The difference is genotypic. (2) The “pure” condition emerges with the same sex (plus or minus) as that with which it enters the cross. If the INHERITANCE 743 plus parent is pure for sex, it is the plus offspring that are pure; if the minus parent is pure, the minus offspring are pure. Linkage and crossing over.—Certain crosses yield a small proportion of exceptional results, which are held to be due to crossing over. Such are the following: Polytoma pascheri: pure plus clone by pure minus clone. Result, out of 2,000 zygotes, 1,843 gave four cells each (as usual), two of which were pure plus, two pure minus. The other 157 (7.9 percent) gave but two cells each, and these all produced clones mixed as to sex. The results given by the 1,843 zygotes are those to be expected from P ce ae ooo se TC ORAM RT SOE SM TREE EB TE ar SRE SME SE Te ae | Figure 172. Diagram of the sex chromosome of Polytoma pascheri plus (upper line, with factor P), and of P. wvella minus (lower line, with factors P and M). The two together show the sex pair in the zygote of a cross between the two species. the principles thus far set forth. How are the 157 exceptional zygotes to be accounted for? The exceptions might be produced either by non-disjunction of two sex chromosomes, or by crossing over between them. If they were the re- sult of non-disjunction, the 157 individuals would have received both the plus-producing and the minus-producing chromosomes of the sex pair; this would account for their mixed sex condition. There would be a pair of sex chromosomes, in place of the usual single chromosome. The exceptional individuals would therefore contain nine chromosomes instead of the eight usual for Polyfoma. But cytological observations showed that only eight were present. The exceptional cases are there- fore not the result of non-disjunction. The alternative explanation is that they are due to crossing over. But how could crossing over between plus-producing and minus-producing chromosomes yield clones mixed as to sex? Moewus concludes that the plus-producing chromosomes must con- 744 INHERITANCE tain a factor (‘‘realisator’) for the plus sex condition, the minus-pro- ducing chromosomes a factor for the minus sex condition, and that by crossing over, both must come into the same chromosome, which there- fore produces the mixed sex condition. The cells containing such a chromosome may become either plus or minus, depending on external conditions. But if the plus and minus factors may by crossing over come into the same chromosome, these factors are not alleles; they are not at the same locus. The condition of the chromosomes may then be represented as in Figures 172 and 173, in which P represents the plus-producing factor, M the minus-producing factor. If crossing over occurs between the P and the M chromosomes, half of p M rr jee. Ge P Figure 173. Diagram of the sex chromosomes produced by crossing over between the chromosomes of Polytoma pascheri plus (light line) and P. zvella minus (heavy line). The upper chromosome has the factor P from P. pascheri, the factor M from P. uvella. the resulting chromosomes in this case contain both P and M, while the other half contains neither. Moewus holds that the cells carrying neither P nor M die. Thus is accounted for the fact that each of the 157 ex- ceptional zygotes produces but two cells in place of the usual four. It is to be noted that here, as in former cases, the results are such as would be given by two-strand crossing over; a zygote produces either no cross- over cells or exclusively crossover cells. From these considerations and others of similar character, Moewus concludes that in clones mixed as to sex (monoecious races), the sex chromosome contains both the sex factors—the factor P for producing the plus sex, and the factor M for producing the minus sex. Moewus (1936) reached similar results in a cross of Chlamydomonas paradoxa and C. pseudoparadoxa. Both species are dioecious, any clone being pure for one sex or the other. When plus clones of one species are INHERITANCE 745 mated with minus clones of the other, the majority of the zygotes yield four clones, of which two are pure plus, two pure minus. But in a par- ticular case, out of 1,000 such zygotes, 117 gave exceptional results, all the four clones from each zygote being mixed as to sex. These excep- tions are held to be due to crossing over. One of the haploid parents had the factor P, the other the factor M in the sex chromosome. By crossing over, the two factors are brought into one chromosome; the cells that receive this chromosome yield clones that are mixed as to sex. Half of the sex chromosomes in which crossing over occurred would lack both the factors P and M. Moewus holds that the cells that receive such chromosomes die, while the cells that receive both P and M divide twice before escaping from the zygote. Thus the zygote produces four cells as usual, all having the crossover combination P and M. Here, as in former cases, the results are those characteristic for two-strand crossing over. Many other cases of crossing over of the sex factors (with results that require two-strand crossing over) are described by Moewus (1936) in crosses of C. evgametos and C. paupera—these all indicating that clones which are mixed as to sex carry a chromosome which has both sex fac- tors, P and M. Other cases will be mentioned in later paragraphs. Sex-linked inheritance.—In crosses of the two species of Chlamydo- monas just mentioned, Moewus observed sex-linked inheritance. The species C. eugametos has an eyespot, while C. paupera has none. When C. eugametos of one sex is crossed with C. paupera of the other sex, in the descendants the gametes that are of the same sex as the C. e~gametos parent have the eyespot, while those that are of the same sex as the C. pau pera parent have none. The eyespot is thus linked with sex. There are, however, a few exceptional cases, due to crossing over, in which the eyespot no longer goes with the parental sex. The results are complex and will not here be presented in detail. In all cases the re- sults, as given by Moewus, are those that would be characteristic for two- strand crossing over. By analysis of the results, Moewus believes that he is able to establish the order in the chromosome of the two sex factors (P, M) and that for eyespot (S), as P-M-S. Relative sexuality, in crosses between different species——In crosses between different species, in some cases gametes of like sex may copulate and yield descendants (see Chapter XIV). Plus gametes from one spe- cies may unite with either plus or minus gametes from the other species, 746 INHERITANCE and minus gametes from one with plus or minus from the other. This phenomenon, known as relative sexuality, is accounted for by Moewus (following Hartmann) by assuming that the gametes of the different species differ in the strength, or “valence,” of their sex tendency and that if two gametes differ sufficiently in the strength of the sex tendency they copulate, whether of the same or of different sex. Exemplifying this situation is the fact that any gamete of Polytoma uvella may copulate with any gamete of P. pascheri, irrespective of the sex of the gametes. Hence it is held that in one of these species the sex tendency of the gametes is stronger than in the other species. The relations as to crossing over described in the preceding section offer an opportunity for determining whether the sex factors present in the chromosomes of the two species differ in the strength of the sex tendency; also for determining in which species the sex factors are stronger. By crossing over, the plus factor of one species may be brought into the same chromosome with the minus factor of the other species. It is then possible to determine which of these prevails, and thus to discover in which species the sex factor is stronger. Moewus crossed plus P. pascher7 gametes from a clone pure for sex with minus P. wvella gametes from a race mixed for sex. According to the hypothesis, the P. wvel/a gametes, being from a race mixed for sex, contain a sex chromosome which carries both the plus and the minus factors, P and M. The P. pascher7 gametes have a sex chromosome con- taining only the P factor. The situation as to sex chromosomes in the zygote may therefore be represented as in Figure 172. From this cross 625 zygotes were obtained. Of these, 582 gave the results that are usual without crossing over. Each zygote gave two clones that are pure plus, as in the P. pascheri parent, two that are mixed, as in the P. wvella parent. The remaining 43, or 6.9 percent, gave exceptional results, presumably due to crossing over. In these, two of the four cells from each zygote yield pure plus clones, like the P. pascheri parent, while the other two yield pure minus, unlike either parent. No mixed clones are produced. How are these results accounted for? The original condition of the chromosomes is that shown in Figure 172. By crossing over (the break occurring between P and M), the con- dition shown in Figure 173 is produced. One chromosome contains only the plus P. wvella factor (P); it is obviously the gametes containing this INHERITANCE 747 that yield the pure plus clones. The other chromosome contains both the P from P. pascheri and the M from P. wvella. The gametes that contain such chromosomes act, as above mentioned, like pure minus gametes. Therefore the effect of the M P. wvella factor completely overcomes the effect of the P factor from P. pascher7. The sex factors of P. mvella are thus stronger 1n sex tendency than those of P. pascheri. From this the further conclusion is drawn that the gametes of P. wvella are stronger in sex tendency than the gametes of P. pascheri. Since P. uvella has only monoecious races (mixed as to sex), these gametes, ac- cording to the assumption, contain both a strong P and a strong M factor. It might be anticipated that the two would partly or entirely counteract each other, leaving the gametes weak or neutral in sex tend- ency. This, however, does not occur; if Moewus’s assumption is correct, one of the two fully prevails, the other having no effect, so that the gametes are strong. Moewus carried out numerous other crosses of these two species, which gave results that are in accord with those just set forth. They all indicate that the P. wvella factors have a stronger sex tendency than the P. pascheri factors. They agree further with the idea that the P and M factors are in different loci, and that by crossing over they may be brought into the same chromosome. One further result of these experiments is of special interest. In cer- tain cases there were crosses of two dioecious races (produced by hy- bridization), in which the gametes were of the same sex (both plus or both minus). In such crosses between plus gametes, according to the theory, each gamete contains only the P factor; no M factor is present. All the offspring in such cases are then of the plus sex, as the theory would lead one to expect. Similarly, if the two gametes each contain only the minus factor, all the offspring are of the minus sex. An elaborate investigation of these matters was carried out by Moewus (1935a, 1935c) on diverse races of the unicellular alga Protosiphon botryoides. The relations here, while differing much in details, are con- cordant with those above set forth for Po/ytoma in the matters of cross- ing over of sex factors, different strengths of sex tendencies in different races, segregation at the reduction division, and the like. The results will therefore not be taken up in detail here. It is to be remarked that in this species, as in the others, crossing over (as the data are reported by 748 INHERITANCE Moewus) took place in accordance with the two-strand scheme; any zygote that gave crossover combinations at all gave only crossover com- binations. In Protosiphon an investigation was made on the determination of sex by external conditions. Protos7phon includes dioecious races, in which the determination of sex is genotypic, as described in earlier pages; monoecious races, in which a single clone contains individuals of both sexes, the determination of sex being mainly phenotypic; and certain other races that agree in some respects with one type, in some respects with the other. The phenotypic determination of sex is not here dealt with. Of interest for the nature of inheritance are the races which partake of the features of both types. Such is the race d, described by Moewus (1935c). At a certain stage in its life history, Protos7phon is a small club- shaped “‘haplont,’’ containing many haploid nuclei imbedded in a mass of cytoplasm without cell walls. Such a haplont may be produced by a zygote resulting from the union of two gametes. In the diploid zygote, the reduction division occurs and the haploid nuclei multiply, giving rise to the haplont. Or haplonts may be produced each from a single haploid swarm cell, which comes to rest on the surface of a solid (as agar) and produces many nuclei by division of its single nucleus. Haplonts pro- duced in either of these ways give rise later to swarm cells (gametes). The nuclei separate, each is surrounded by a small mass of cytoplasm and each such cell transforms into a flagellate swarm cell. In the race d, as in dioecious races, all the swarm cells produced by a single haplont are of the same sex. But, in the race d, the sex of the swarm cells produced by the haplont depends on the conditions under which the haplont was produced. Haplonts grown on acid agar give rise only to plus swarm cells. Those grown on alkaline agar yield only minus swarm cells. Of those grown on neutral agar, some yield only plus swarm cells, others only minus swarm cells. The sex of the swarm cells and of all their descendants by vegetative fission is determined once for all by the conditions prevail- ing in the development of the haplont from which they arise. This is true not only for haplonts produced from single swarm cells, but also for haplonts produced from zygotes. Since in the zygote the reduction divi- sion occurs, yet all the four cells resulting from the maturation divisions INHERITANCE 749 are of the same sex, it is clear that sex segregation does not occur at the reduction division. Moewus determined by careful experimentation at exactly what stage in the development of the haplont sex determination occurs. It is at the first division of the single nucleus of the swarmer that is dividing to produce a haplont. If at this time the conditions are acid, the swarm cells later produced from the haplont are plus; if the conditions at that time are alkaline, the later swarmers are minus. In this case, therefore, the sex is determined by an external condition, and this sex is inherited by all the descendants of the cell in which it 1s thus determined. The consequences induced by an environmental condi- tion are inherited. In his article of 1938, Moewus sums up the conclusions to which his investigations lead as to inheritance and determination of sex, and par- ticularly as to the constitution of the sex chromosomes in the different sex conditions. In pure dioecious races, each sex chromosome carries but one sex factor, P or M, and sex is segregated at the reduction division. In different dioecious races, the strength or valence of the sex factors may be diverse. In monoecious races, the sex chromosome of each individual carries both sex factors, P and M, in equal strength; which of these shall prevail in determining the sex of the gamete is decided by external conditions. In the subdioecious cases, in which any clone is prevailingly of one sex but produces also a few cells of the other sex, the sex chromo- somes are held each to contain both sex factors, P and M, but every cell contains an additional factor which inclines it toward one sex or the other. The prevailingly plus clones contain a factor which inclines them toward the plus sex, the prevailingly minus cells a factor which inclines them toward the minus sex. But the tendencies of these additional fac- tors are in some cases overcome by special outer conditions, so that a few cells of the opposite sex are produced. In addition to these three types, there are other types in which the sex chromosome carries both the sex factors, P and M, but one of these is stronger than the other, so that it prevails and determines alone the sex of the clone. If P is the stronger, the sex of the clone is plus; if M is stronger, the sex of the clones is minus. Each clone is therefore pure for sex, and the race is dioecious. Such are called by Moewus complex dioe- cious races. They are not found in nature, but occur as a result of crossing 750 INHERITANCE over between dioecious races having sex factors of different strengths. In this article of 1938, Moewus discusses the difficulties arising in his data as to crossing over. In all the data presented by him up to this time, crossing over was reported as occurring in accordance with the two- strand schema. Any zygote that gave any crossover combination gave such combinations exclusively. Moewus now concludes that this is not the normal state of affairs; he characterizes it, indeed, as pathological. Under certain conditions, he reports, crossing over occurs in the normal manner, according to the four-strand schema. He promises a future ac- count of detailed investigations showing this. Certainly there is great need for clearing up the confused situation as to crossing over in these organisms. In hundreds of detailed earlier re- ports, Moewus has given data that are consistent only with two-strand crossing over. A further serious criticism, based on other grounds, has been made as to the accuracy of Moewus’s data on crossing over, by Philip and Haldane (1939). Aside from this difficulty, the work of Moewus has placed the genetics of the Protozoa on a new footing. It has brought the phenomena of inheritance in these organisms into the same system that is manifested in the Mendelian inheritance of higher organisms. It has brought to light in the flagellate Protozoa instances of most of the phenomena in such inheritance, as before known in multicellular organisms. BIPARENTAL INHERITANCE IN DIPLOIDS: CILIATA The genetics of diploids is necessarily more complex than that of haploids. The individuals have during active life two sets of chromo- somes instead of one. In consequence, in the sexual process two indi- viduals do not normally unite completely, to form a diploid zygote, as in the copulation of the flagellates. In the diploid ciliates, two individuals merely come into intimate contact and exchange pronuclei that contain each a haploid set of chromosomes, a process known as conjugation. The two then separate, each carrying two haploid sets of chromosomes, one from each of the conjugant individuals. Each then multiplies vegetatively, forming clones, all members of any clone having the same diploid com- bination of chromosomes. Conjugation thus is like fertilization in higher organisms, in that it produces new diploid combinations of chromosomes. Important for the understanding of inheritance in conjugation are the following: INHERITANCE foe 1. The macronucleus or macronuclei of each individual disappear, being absorbed into the cytoplasm. 2. If more than one micronucleus are present, usually all but one are absorbed and disappear. 3. The single micronucleus divides three times in succession (the “maturation divisions”). By the first two divisions (Fig. 174, 1 and 2) four nuclei are produced; of these, three are absorbed and disappear. The remaining one divides once more into two (Fig. 174, at 3). ae D Figure 174. The three maturation divisions (1, 2, 3) and the exchange of pronuclei in the micronuclei during conjugation. A and D are the micronuclei of the two con- jugants respectively. Reduction at the second division, represented by the separation of XX and XY. The diagram illustrates the fact that the two diploid nuclei produced (above) are alike in constitution. 4, The two nuclei produced by the third division are haploid, so that one of the three maturation divisions is a reduction division. 5. One of the two haploid nuclei (the “migrating pronucleus’’) in each individual passes over into the other individual, where it unites with the remaining one (‘‘stationary pronucleus’) of that individual. Thus there is produced again a diploid nucleus in each individual. 6. This diploid nucleus divides, some of its products becoming large as macronuclei, others remaining small as micronuclei. These macro- nuclei and micronuclei are distributed by fission to different individuals, the exact processes differing in different species. 7. In conjugation, only nuclei are exchanged; each individual retains its cytoplasm complete. Hence after conjugation each individual has a 752 INHERITANCE new combination of chromosomes, half derived from each conjugant, but has the same cytoplasm as before. This gives an opportunity to com- pare the relative rdles of chromosomes and cytoplasm in inheritance, since in the ex-conjugants the chromosomal combination is changed, but the cytoplasm is not. The only qualification required by the statement that cytoplasm is not changed is the fact that a minute bit of cytoplasm carrying an aster pre- cedes the migratory pronucleus into the opposite conjugant. The results show, as will be seen, that this minute bit of cytoplasm is not effective in determining inheritance. The two individuals that conjugate are, in some species at least, physio- logically differentiated into diverse “‘mating types,’ which play the same physiological role in bringing about conjugation as do diverse sexes. For an account of these, Chapter XIV is to be consulted. Knowledge of biparental inheritance is much less exact and extensive in the ciliates than in the flagellates. The recent discovery of diverse mating types furnishes an opportunity for exact analysis; but the novelty of this discovery has not yet given opportunity for its full investigation. The earlier work on inheritance in conjugation before the discovery of mating types, is summarized in the author's Genetics of the Protozoa (1929). In the early work numerical ratios were not obtained, but qualli- tative relations of importance were demonstrated. Most generally ex- pressed, the work showed that conjugation results in the production of many hereditarily diverse biotypes from the two involved in the conju- gation. Production of hereditary diversities at conjugation was demon- strated by the work of Jennings in respect to the following types of characteristics: rate of fission, rate of mortality, presence of abnormali- ties. This demonstration of the production of inherited differences at conjugation was extended in 1930 by the work of Raffel, and in 1932 by Jennings, Raffel, Lynch, and Sonneborn to various other characteristics, including size and form, vigor, resistance, and degeneration. It was fur- ther shown that conjugation causes the descendants of the two members of a pair to become similar in fission rate, in mortality, in the occurrence of abnormalities, and probably in size (see Jennings, 1929, pp. 181-85). Later, more exact work on inheritance in ciliates deals with the inherit- ance of mating type (Sonneborn, 1937-39; and Jennings, 1938-39) INHERITANCE HS) and with the inheritance of size (De Garis, 1935). That on mating type will be presented first. INHERITANCE OF MATING TYPE IN Paramecium aurelia In Paramecium aurelia, according to the work of Sonneborn (1937, 1938, 1939), there are in any variety but two mating types, members of which unite in conjugation as the two sexes unite in multicellular ani- Figure 175. The four clones (al, a2, b1, b2) produced from the two ex-conjugants (a and b) of a pair, in the experiments of Sonneborn and of Jennings. mals. Three varieties or “groups” are known; individuals of one variety do not conjugate with individuals of the other varieties. In Variety or Group 1, the two mating types are known as types I and II. These unite in conjugation. In studying the types produced by them, each ex-conjugant after separation was allowed to divide once, and from each of the products a clone was produced by vegetative reproduc- tion. Thus four clones were derived from each pair, two from each ex- conjugant, as shown in Figure 175. 754 INHERITANCE All members of any one of the four clones belong to the same mating type, and that type is inherited within the clone until endomixis or a new conjugation occurs. In a certain experiment described by Sonneborn (1937), there were fifty-six pairs formed by union of mating types I and II. The four clones from each pair were constituted as shown in the accompanying table. In this table the following relations are seen: 1. In some cases (4 out of 56) all the descendants of the two parents (which were of diverse type) are of the same type. In these cases the type of one of the conjugants and its descendants was changed by con- PARENTS, I X II Number of pence ne Mating Types of the Four Pairs iypell anpeul Descendant Clones I 4 eel 18 3 I Teale 21 2 2 Ie le JOG 3! 13 I 3 I JU JOT, WL 3 4 OE TOG We Tt jugation. That ts, an individual of type I, receiving a pronucleus from type II, becomes changed to type II, and vice versa. 2. In some cases the two ex-conjugants of a pair give clones of dif- ferent mating types. 3. In most cases a single ex-conjugant gives rise to two clones of the same type. 4. But in some cases a single ex-conjugant gives clones of different mating type. Do ex-conjugants that were of a given type before conjugation tend after conjugation to produce descendant clones of that type, or do they produce both types with the same frequency? Sonneborn tested this by mating two clones that were different in appearance, one of each type, then determining the mating type of the descendants. The results were as follows: Of 22 ex-conjugants originally of type I, 5 gave descendants of type I only, 7 of type II only, and 10 half of type I, half of type II. Of 25 ex- INHERITANCE eS, conjugants originally of type I, 4 gave descendants of type II only, 3 of type I only, and 18 half of type I, half of type II. In summary, there were 47 ex-conjugants, which give 94 descendant clones. Of these 94, 46 were of the same type as their parent’s before conjugation, 48 of different type. Thus it is clear that ex-conjugants originally of a given type produce descendants of both mating types with equal frequency. The reception in conjugation of a pronucleus from an individual of different type changes the type as frequently as it leaves it unchanged. There is no tendency for the descendants to be of the same type as was the cytoplasmic body from which they are derived (certain exceptions will be described later). Of the 47 ex-conjugants just considered, 28 gave two clones of dif- ferent types, while 19 gave two clones of like type. There is no indica- tion of a tendency for the two clones descended from a single ex-con- jugant to be alike in type. Thus the first fission after conjugation separates the ex-conjugant into two individuals which may be of different mating type, giving rise to two clones of different types; or they may be of the same type. Segre- gation of the diverse mating types (in cases in which it occurs) takes place at the first fission after conjugation. What is it that decides the mating type of each clone? Sonneborn 1s disposed to believe that the segregation is the result of the separation of the two macronuclei, one macronucleus tending to produce type I, the other type II. How the two became diverse (if this is the case) is not known. There appears to be no evidence of a reduction division at this point, such as might give rise to nuclei differing in chromosomes. It appears equally difficult to suppose that the two nuclei of the same cell are subjected to differing conditions, such as to cause one to be of type I, the other of type II. Sonneborn (1938) has discovered, however, that the temperature during conjugation affects the proportion of the types produced in a group of ex-conjugants. In variety or group 1, higher temperatures cause the appearance of a greater proportion of type I; in group 3, higher tempera- tures favor the production of type VI (rather than type V). But it is not evident how the difference of type in the two products of fission of a single individual could be induced in any such manner. The segregation of types at the first fission after conjugation remains a riddle. 756 INHERITANCE Segregation of mating types at endomixis. —It is of great interest that at the first fission after endomixis segregation of the mating types may occur, just as it does after conjugation. The single individual, of a definite mating type, divides after endomixis into two that may be diverse in mating type. The nuclear processes in endomixis are not yet fully cleared up, but are known to be in many respects similar to those which occur at conjugation. According to the recent work of Diller (1936), the simi- larity goes so far that there is in endomixis a union of two micronuclei (presumably haploid), just as occurs in conjugation. In this autogamy the two micronuclei that unite are of course both from the same indi- vidual. After their union, the single (diploid) nucleus divides into two, then into four. Two of these become macronucei and two remain micro- nuclei, just as in conjugation. The two macronuclei are separated at fis- sion, just as occurs after conjugation, and the same is true of the micro- nuclei. As will be seen later, there is genetic evidence that autogamy does indeed occur at endomixis. In variety or group 1 of Paramecium aurelia, Kimball (1937) deter- mined the mating type of the two clones produced by each of 181 indi- viduals that had undergone endomixis. Of the individuals, 96 gave 2 clones of the same type (both I or both II), while 85 gave two clones of different types (one type I, one type II). In other respects also the re- sults of endomixis in relation to the mating types are like those of con- jugation (see Kimball, 1937). As a rule, in both endomixis and conjugation the segregation of the mating types occurs at the first fission after completion of the process. But in a certain stock there was a small proportion of cases in which segregation into two mating types occurred at the second fission after conjugation. In this same stock, cytological examination showed that in about the same small proportion of cases the ex-conjugants had three or four macronuclear Anlagen in place of the usual two. It of course re- quires two fissions to separate these into different individuals. Sonneborn is disposed to consider this the cause of the occasional segregation of the mating types at the second fission, in place of the first. Single-type clones.—There exist clones in which there is no segrega- tion into different mating types at endomixis. In such clones endomixis makes no change in the mating type; the clone remains throughout of the same type (Sonneborn, 1938c). Of twenty-six clones examined by INHERITANCE 757 Sonneborn, six showed no change of type at endomixis. All these six were of mating type I. Such clones may be designated as single-type clones, as compared with the more common double-type clones, in which from a single clone both sexes are produced at endomixis. Crosses between single-type and double-ty pe clones.—Crosses between the two types (Sonneborn, 1939) show that they are inherited in typical Mendelian fashion, double type being dominant over single type. The factor for double type may therefore be designated A, that for single type a. The two original diploid clones are AA and aa. Crosses of the two (AA X aa) gave in the 149 pairs examined, all double-type (Aa) offspring. Mating together these heterozygotes yielded, in 120 pairs, 88 of the dominant double type, 32 of the recessive single type, so that the results approximate the typical three-to-one ratio (Aa & Aa == AA + 2A + aa). The hybrids Aa were back crossed to the single-type parents (aa) in 165 pairs; these yielded 88 double-type and 77 single-type de- scendant clones, an approximation to the expected one-to-one ratio (Aa aa —=- Aa + aa). Here we have diploid Mendelian inheritance, one of the characters being recessive. Genetic evidence of autogamy.—In the further study of the heterozy- gotes Aa, a discovery of great interest was made. At endomixis, some of these heterozygotic individuals produce (at the first fission after endo- mixis) double-type clones, others single-type clones. Is the double-type clone the heterozygote Aa or the homozygote AA? This was tested by mating them with the normal single-type individuals aa. All the de- scendant clones are the heterozygotic double-type Aa. This shows that the double-type clones produced at endomixis are the homozygotes AA; correspondingly, the single-type clones produced at endomixis are neces- sarily aa. From the heterozygotes Aa there are produced at endomixis two types, both homozygotic: AA and aa. How is this result brought about? It is the natural consequence of the occurrence at endomixis of a reduction division with subsequent union of two of the reduced nuclei (autogamy), as described cytologically by Diller (1936). The heterozygote nucleus before reduction is Aa. By re- duction are produced haploid nuclei A and a: by a second division these give rise to four nuclei A, A, a, a. Three of these haploid nuclei de- generate, leaving but one, A or a. This remaining nucleus now under- 758 INHERITANCE goes the third division, yielding in one case two nuclei A and A, in the second case a and a. These two nuclei now reunite (autogamy), yielding in the one case the homozygote dominant AA, in the other case the homozygote recessive aa. Thus after endomixis has occurred in any stock, the clones are all homozygotes, as Sonneborn points out. These results are obviously strong genetic evidence for the occurrence of autogamy at endomixis in Paramecium aurelia. Certain rare and exceptional conditions in the genetics of the mating types in P. avrelia are discussed in Chapter XIV, ‘Sexuality in Unicellular Organisms.” INHERITANCE OF MATING TYPE IN Paramecium bursaria In Paramecium bursaria, according to the work of Jennings (1939a, 1939b), there are in one of the varieties four mating types, A, B, C, and D. Another variety has eight mating types (E, F, G, H, J, K, L, and M), a third variety four types (N, O, P, and Q) distinct from the four of the first variety. Inheritance of mating type has been examined only in the first variety (some of the relations are here published for the first time). In variety 1 (as in the other varieties), clones of any one of the types may conjugate with clones of any of the other types, but not with clones of their own type Thus in variety 1 six different matings occur, yielding pairs AB, AC, AD, BC, BD, CD. Very rarely in this species a single clone, in which all the individuals belong originally to the same type, may differentiate into two types, which thereupon conjugate. This phenomenon is parallel to the segrega- tion of different types from one type at endomixis in P. awrelia. It is presumably the result of endomixis in P. bursaria; in this species endo- mixis is known to occur very rarely (Erdmann, 1927). Thus the occa- sional ‘“‘self-fertilization’’ of a clone is in fact the conjugation of two diverse mating types. A clone of the mating type D has been observed to differentiate part of its individuals into the mating type A; these then conjugate with the D individuals, giving the cross AD. The results of “‘self-fertilization” may therefore be considered with those of other mat- ings between two different types. In studies of mating-type inheritance, four descendant clones are ob- tained from each pair, two from the first fission of each ex-conjugant, INHERITANCE 79 as indicated in the diagram of Figure 175. After the attainment of sexual maturity, the mating type of each of the four clones is discovered by testing them with standard clones representing the four known mating types. The fullest data as yet available are for the cross A & D. Of this cross the mating types have been determined for the clones descended from 61 pairs, including 175 clones. The 4 original clones did not survive in all the pairs. They did all survive in 26 pairs (104 clones). The remain- ing pairs had each but 1, 2, or 3 surviving clones. But in all cases all TABLE 18: INHERITANCE OF MATING TYPES (THE FouR TYPES ARE A, B, C, D), Paramecium bursaria Parents, A X D Typical Constitution of the Four Descendant Clones of Each Pair 30 Pairs give type A only A+A+A+A 4 Pairs give type B only B+B-+B+B 4 Pairs give type C only C+C+C+C 23 Pairs give type D only D+D+D+D clones descended from any pair were of the same mating type. The re- sults have been summarized as in Table 18. In Table 18 the following general relations appear: 1. All the descendants of any one pair are of the same mating type, though the parents were of two different types. 2. Among the descendants of the cross of the two types A and D occur all four types A, B, C, and D. 3. The majority of the descendant clones are like one or the other of the two parents in type; only a few differ. In this case 53 of the 61 clones are either A or D (the parental types); only 8 are of types differ- ent from those of the parents. 4. By conjugation the mating type has become changed in one or both of the ex-conjugants and their descendants. In the above table, the type is changed in both ex-conjugants in 8 pairs out of 61; it is changed in but one of the ex-conjugants in the remaining 53 pairs. The change in type is due to the exchange of migratory pronuclet, since the cytoplasm remains unmixed. Individuals of type A, receiving a micronucleus from type D, are changed to type D in nearly half of the 760 INHERITANCE cases; to B or C in few cases. In about half the cases they remain un- changed. Parallel statements may be made for the individuals originally of type D. It is clear that although all four mating types are present in the de- scendants of a cross of but two types, the statistical make-up of the de- scendant population is influenced by the constitution of the two parents. TABLE 19: MATING TyPES OF DESCENDANT CLONES FROM 131 Pairs (INCLUDING 279 DESCENDANT CLONES), IN THE SIX POSSIBLE CROSSES OF THE FouR MATING TYPES OF VARIETY 1, Paramecium bursaria Matinc Types OF THE DescENDANT CLONES: NUMBERS C Number or Pairs YietpiInc Eacu Type TOSS : elena eae ea Ho 2 of Pairs IN NOE VID) Like Parents Unlike Parents ASGB 25 1405} fo) fo) 25 fo) ASC 6 3 I I 4 2 AeD 61 30 4 HB 53 8 I< 10 ©). 36) fo) fo) 10 fo) BX D 15 5 8 fo) 2 10 5 Cx D 14 fo) fe) 6 8 14 ° Totals 131 ye ah ay 116 15 The majority of the descendant clones belong to one or the other of the two parental types. This is evident in the results of all the crosses that have been made, as shown in Table 19. As Table 19 shows, of 131 pairs in which the original type of the conjugants was known, 116, or 88.5 percent, gave descendant clones that were of the same type as one or the other of the two parent individuals, while but 15, or 11.5 percent, gave descendants that were of different mating type from either of the two parents. What determines the type to which a particular clone belongs? In all the 131 pairs of Table 19, all surviving clones from any pair (1 to 4 clones per pair, in different cases) were of the same mating type. The four clones descended from the two members of any one pair were culti- vated separately and tested separately. The fact that all four turn out to be of the same mating type shows that the type is determined at the time that the two ex-conjugants separate and before they divide. It might be determined at that time either by internal or external conditions. The fact that in the great majority of cases the descendant clones are of the INHERITANCE 761 same type as one or the other of the two parents shows that the constt- tution of the two parents is one important factor in determining the type of the descendants. But why in the same cross the descendants from some pairs should be of the same type as one parent, those from other pairs of the same type as the other parent, while a few are of different type from either parent—this is as yet quite unknown. In extremely rare cases the four clones produced by a particular pair are not all of the same type. Thus in a certain case a pair composed of the types A X C yielded four clones of the types A, B, B, B. In another case A X C gave B, D, D, D. The great rarity of such cases indicates that such results are due to irregularities in the cytological processes, comparable to cases of non-disjunction of chromosomes. Immaturity and partial maturity—The relations thus far described are those that exist after the descendant clones have reached sexual or reproductive maturity. But for a long period after conjugation, varying in different clones from a few weeks to more than a year, the descendant clones are in Paramecium bursaria sexually immature. During this period the descendant clones do not mate at all, with any of the mating types. In group 1, if they are mixed with mature individuals of any of the four mating types A, B, C, D, there is no mating reaction, no formation of pairs. In this period descendant clones show the characters of no mating type. Later begins a period of partial maturity, in which a few members of the clone pair with mature members of certain of the mating types. During the early part of this period of partial maturity there are in variety 1 only two mating types instead of four. Some of the descendant clones form pairs with the types A or B, but not with C or D; others with C or D, but not with A or B. The former may now be said to con- stitute the type CD, the latter the type AB. Later these two young types become further differentiated. Some of the clones that thus far do not react with A or B acquire the power to react with A, but still do not react with B; these now belong to the definitive mating type B. Others acquire the power to react with B but not with A; these belong to the definitive type A. A similar differentiation occurs among the clones of the young type CD; some of these become type C, others type D. Thus the types A and B are closely akin, being for a time one type AB; similarly, types C and D at first constitute one type, CD. The interest of these gradual changes in maturity and type during vegetative reproduction has been emphasized in an earlier section (p. 714 above). 762 INHERITANCE EFFECT OF THE CYTOPLASM AND ITS RELATION TO NUCLEAR CONSTITUTION Certain phenomena in the biparental reproduction of ciliates throw light on the relative role of the nucleus and the cytoplasm in inheritance. Such phenomena are shown in the inheritance of a number of different types of characteristics, but are best seen in crosses of individuals of different inherited sizes, as set forth in the work of De Garis (1935). By ingenious methods De Garis obtained in Paramecium caudatum > Figure 176. Change of size resulting from conjugation of individuals of large and small races, in the experiments of De Garis. Upper left, A and B, the large and small individuals of the pair, showing their relative sizes. The columns headed A and B respectively show (read- ing from above downward) the average sizes of the descendants of the two at successive intervals of two days each. (Diagram based on the measurements of De Garis, 1935.) val 3 represents the growing \l ANY oe point of the primary axis. > Dice, © The numerous common nu- Fw ) aD tritive cells of each branch Ae 1S ey are division products of the & Av VA <= terminal cell of that branch. TF Re FY On th th branch, 10g” =A. n the seventh branch, 10g (= SK, 5g? 59° 7g? Ar designates the terminal a4 2) branch cell of the tenth generation. The lateral cell of the first branch genera- ion (Gis, IAD) fr its La A) : : CA ml Sm v7) two immediate descendants S law (e.g., 1G" and 1G’) repre- ( KY a sent the potential cilio- Q eN ey, spores; several stages in the RSs: i 2a? differentiation of these cells Ol y j on several of the branches Tees a Je are shown. (From Summers, oo 1938a.) in position along the primary axis, lying alternately on the right and left sides of the axis at successive nodes. Cells of the branch strain de- scend from the initial branch cell by a series of equal divisions. Here again each division results in a median and a lateral individual, with the MORPHOGENESIS 807 former remaining in the terminal position on the laterally growing branch axis. Thus the primary developmental functions of a growing colony are almost exclusively limited to the terminal cells of the primary and branch axes (Fig. 185). A painstaking cytological analysis of normal development in this species led Fauré-Fremiet (1930) to postulate that the two daughters resulting from the division of one initial cell are never equivalent as to their developmental potentialities. He assumed that quantitatively differential divisions of the apical cell series restrict the subsequent power of division in the branch strains; and, similarly, the qualitatively differ- ential nature of the first division of the initial branch cells effects a segregation of potencies for ciliospore formation. Such a hypothesis of embryonic segregation by division apparently covers the facts of normal development. A terminal branch cell, for example, produces fewer gen- erations than the apical cell (see Fig. 185). And, for the most part, a ciliospore differentiates only from the lateral cell of the first branch generation or from its two immediate descendants on certain of the branches. The facts derived from regeneration studies are not in accord with Fauré-Fremiet’s assumptions. Common branch cells above or lateral to the supposedly differential divisions retain, for a time at least, po- tentialities for regenerating large portions of the colony. An apical cell, terminal branch cells, common nutritive cells, ciliospores, and sometimes gamonts differentiate at appropriate positions on the regenerate. Further- more, the ciliospore-forming cells can be induced to differentiate as new apical cells, which continue axial development according to the normal pattern (Fig. 186). If the well-defined apical cell is removed, the terminal cell of the topmost branch usually differentiates as a new apical cell, the subse- quent development of which is identical with that of the original. But if the apical cell and the first terminal branch cell are destroyed, the functions of the former are assumed by either the subterminal cell of the topmost branch or the terminal cell of the penultimate branch, more frequently the latter. A variety of operations performed by the writer (1938b) upon large and small colonies show that subordinate cells—terminal branch cells or merely the common nutritive cells, the complete developmental potentialities of which are never otherwise 808 MORPHOGENESIS expressed—can be induced to assume the dominant generative functions. The regenerative behavior following simple, compound, or successive operations is another illustration of what Child (1929) referred to as physiological correlation: the relations of dominance, or control and subordination between parts. The single apical cell of Zoothamnium colonies exercises the controlling influence over growth and differentia- tion in subadjacent cells. Bsarean Figure 186. A, a seventy-two-hour regenerate produced from a lateral cell of the first branch generation (a cell which ordinarily represents the presumptive ciliospore) ; B, schematic diagram of the apical portion of the colony at the time of cutting (see arrow) ; C, a similar diagram of the regenerating colony seventy-two hours later, or as shown in A. (From Summers, 1938b.) Regional coérdination, according to Child, depends primarily upon quantitative rather than specific differences in the protoplasmic condi- tion of the dominant region. Evidence that this is not necessarily the MORPHOGENESIS 809 case comes from another phase of development in Zoothamnium. In the sexual process, the apical cell becomes the sessile macrogamont. The fusion of a free-swimming microgamont with the sexually differentiated apical cell arrests axial development for several days, pending the origin of a new apical cell from one of the ex-conjugants. In the meantime, all of the cells on three or four of the youngest (uppermost) branches begin to divide precociously. The terminal branch cells are aroused to unusual mitotic activity, producing twice as many generations as when they comprise a part of the vegetative colony. The common branch cells are likewise activated to produce secondary and even tertiary branches. This precocious development never occurs when the apical cell presides over a vegetative colony. Neither does it occur as a result of decapita- tion—when the apical cell is destroyed. The phenomenon appears to be initiated by qualitative changes in the co6drdinating mechanism, which arise in consequence of reorganizational activities in the single apical cell. The growth relations are likewise altered by conjugation in Z. arbus- cula (Furssenko, 1929). Each of the several primary axes in this species bears an apical cell which becomes the macrogamont during the sexual period. Conjugation on one axis stops further apical extension of that branch until two new vegetative axes spring from the two “stem cells” of the fourth ex-conjugant generation. One daughter cell from each of the first four generations differentiates into a very large macrozodid (immature ciliospore). A single conjugant therefore produces two grow- ing points and a cluster of from four to six bulbous macrozodids. As is the case in Z. alternans, several of the small secondary branches below the conjugant-bearing node develop to the proportions of primary axes. Under the influence of a vegetative apical cell, these branches do not hypertrophy. Furssenko accounts for the changed relations between apical and subordinate regions in terms of local variations in the food-energy re- quirements. In the light of the above observations, he supposed that the cluster of huge non-feeding macrozodids at the tip of the stalk, to- gether with the two developing apical cells, have energy needs in excess of the apical requirements in non-conjugating colonies. Multiplication of the actively feeding cells on neighboring branches presumably occurs, in order to compensate for the unusual metabolic needs at the apex. 810 MORPHOGENESIS In Z. alternans, however, hypertrophy of the inferior branch cells begins before the conjugant undergoes its first division. It is therefore doubt- ful whether any increase in the energy requirements, coincidental with the conversion of an apical cell into an exconjugant, is adequate to account for the far-reaching alterations of the normal growth pattern. It is more than likely that the combined energy demands of the ac- tively dividing cells on subordinate branches exceed those of the single conjugant or its first few non-feeding descendants. It is probable, then, that the flux would be directed away from the cell or cells in the apical position. Figure 187. A, branch C of a colony fifty-six hours after injury to the neuromuscular cord (drawn from above) (the original colony of six branches was pinched in the mid- region, isolating ABC from DEF and the apical cell; the terminal cell on branch C dif- ferentiated into a new apical cell, which produced two new branches as illustrated) ; B, schematic representation of branch C, as drawn in A. We are thus confronted with two divergent interpretations relative to the specificity of form-regulating factors in Zoothamnium. The evi- dence presented by Summers suggests that qualitative physiological changes in one cell play a greater part in the development of neighbor- ing cells than heretofore suspected. Furssenko’s hypothesis, on the other hand, emphasizes the directive influence of metabolic fluctuations. It is therefore consistent with the metabolic-gradient theory, inasmuch as the nutritive factors may be quantitative and continuous. The stalk structure of Z. alternans, when viewed in section, somewhat resembles a sheathed nerve fiber. There is an elastic surface membrane, a thick cortical region of hyaline, gelatinous material, and a core of protoplasm, the neuromuscular cord. The latter is continuous from branch to branch and from cell to cell. Unlike the axis cylinder of a . MORPHOGENESIS 811 nerve fiber, a break in the neuromuscular cord does not cause degeneration in either of its separated parts. The motor reactions of the cells joined by the distal segment of the cord are well integrated, but independent of those connected by the proximal portion. The cord alone, rather than the entire stalk structure, appears to be the medium through which regenerative functions are codrdinated. Preliminary experiments have shown that a local injury to the protoplasmic portion of the stalk physio- logically divides a colony. The distal part, with its original apical cell, continues to develop as before, whereas one of the cells on the proximal (basal) portion differentiates into an apical cell the activity of which produces another dominant growth axis (Fig. 187). How the single apical cell regulates developmental functions in the distantly separated cells, through the agency of the slender protoplasmic thread, remains as one of the more important problems that invite attention to these colonial Protozoa. We are far from having more than an elementary notion of what goes on within differentiating cells, but it is probable that the recent developments in other more specific phases of cell physiology presage a period of keener interest in the age-old problems of growth and form determination. Most of the publications on regeneration now extant are important in substance, but lack the specificity of detail which characterizes the newer contributions on nutrition, respiration, and so forth. They also indicate (1) the advisability of more carefully planned pre- and post-operative culture techniques, wherein a better evaluation of extrinsic factors is possible; and (2) the value of critical distinction between the failure of cells to survive an experimental procedure and their failure to regenerate. LITERATURE CITED Alverdes, Friederick von. 1922. Zur Lokalisation des chemischen und therm- ischen Sinnes bei Paramecium und Stentor, Zool. Anz., 55: 19-21. Balamuth, W. 1939. Studies on regeneration in Protozoa. I. Cytology and regeneration of Lichnophora macfarlandi. Univ. Cal. Library, MS, 1939. Balbiani, E. G. 1888. Recherches expérimentales sur la mérotomie des In- fusoires ciliés. Rec. zool. suisse, 5: 1-72. —— 1891. Sur la régénérations successives du peristome comme characteré d’age chez les Stentors et sur la rdle du noyau dans ce phénoméne. Zool. Anz., 14: 312-16. 812 MORPHOGENESIS —— 1892. Nouvelle Recherches expérimentales sur la mérotomie des In- fusoires ciliés. Ann. Microg., 4: 369-449. 1893. Mérotomie des infusoires ciliés. Ann. Microg., 5: 49-84. Bauer, E. S., and A. M. Granowskaja. 1934a. Die Rekonstruktion des Kerns und die Atmungsprozesse bei Hypotricha im Ergebnis operativer Ein- wirkungen auf das Protoplasma und ihre Abhangigkeit vom Alter. Biol. Zbl., 3, 457 (Berichte ti. wiss. Biol., 33: 282). — 1934b. Abhangigkeit der experimentellen “‘individuellen Unsterblich- keit’’ vom Alter. Biol. Zbl., 3: 609 (Berichte ti. wiss. Biol., 33, 282). Becker, E. 1928. Streaming and polarity in Mastigina hyale. Biol. Bull., 54: 109-16. Bills, C. E. 1922. Inhibition of locomotion in Paramecium and observations on certain structures and internal activities. Biol. Bull., 42: 7-13. Bozler, E. 1924. Uber die Morphologie der Ernahrungsorganelle und die Physiologie der Nahrungsaufnahme bei Paramecium caudatum Ehtbg. Arch. Protistenk., 49: 163-215. Brandt, K. 1877. Uber Actinosphaerium eichornii. Dissertation, Halle. Brehme, K. S. 1933. An investigation of the question of cytoplasmic fusion in Amoeba proteus. Arch, Protistenk., 79: 303-10. Bullington, W. E. 1925. A study of spiral movement in the ciliate infusoria. Arch. Protistenk., 50: 219-74. Burch, P. R. 1930. The effect on the division rates of Arcella vulgaris and A. rotunda 1. of the injury caused by the excision of cytoplasm; 2. of the loss of cytoplasm; 3. of the gain of cytoplasm. Arch. Protistenk., 71: 307-22. Burnside, L. H. 1929. Relation of body size to nuclear size in Stentor coeru- leus. J. exp. Zool., 54: 473-83. Calkins, G. N. 1911a. Regeneration and cell division in Uronychia. J. exp. Zool., 10: 95-116. — 1911b. Effects produced by cutting Paramecium cells. Biol. Bull., 21: 36-72. — 1919. Uroleptus mobilis, Engelm. 1. History of the nuclei during divi- sion and conjugation. J. exp. Zool., 27: 293-357. —— 1921. Uroleptus mobilis, Engelm. IV. Effect of cutting during conjuga- tion. J. exp. Zool., 34: 449-70. —— 1934, Factors controlling longevity in protozoan protoplasm. Biol. Bull., 67: 410-31. Causin, M. 1931. Régénération du Stentor coeruleus. Arch. Anat. mict.27: 107-25. Chejfec, M. 1932. Regulacja i regeneracja u Paramecium caudatum. Acta Biol. exp; 75, 115-34. Child, C. M. 1914. The axial gradient in ciliate infusoria. Biol. Bull., 26: 36-54, MORPHOGENESIS 813 1919. Demonstration of the axial gradients by means of potassium permanganate. Biol. Bull., 36: 133-47. — 1920. Some considerations concerning the nature and origin of physio- logical gradients. Biol. Bull., 39: 147-87. —— 1929. Physiological dominance and physiological isolation in develop- ment and reconstitution. Roux Arch. EntwMech. Organ., 117: 21-66. Child, C. M., and E. Deviney. 1926, Contributions to the physiology of Paramecium caudatum. J. exp. Zool., 43: 257-312. Claparéde, E., and J. Lachmann. 1858-60. Etudes sur les Infusoires et les Rhizopods. Mém, Inst. nat. genev., 5-7. Conklin, E. G. 1912. Cell size nad nuclear size. J. exp. Zool., 12: 1-98. Dawson, J. A. 1919. An experimental study of an amicronucleate Oxytricha. J. exp. Zool., 29: 473-508. Dembowska, W. S. 1925. Studien iiber die Regeneration von Stylonychia mytilus, Arch. mikr. Anat., 104: 185-209. — 1926, Studies on the regeneration of Protozoa. J. exp. Zool., 43: 485- 504. — 1938. K6rperreorganisation von Stylonychia mytilus beim Hungern. Arch. Protistenk., 91: 89-105. Fauré-Fremiet, E. 1930. Growth and differentiation of the colonies of Zoothamnium alternans (Clap. and Lachm.). Biol. Bull., 58: 28-51. Fortner, H. 1933. Uber Kernresektion bei einem Hypotrichen (nov. spec.?). Arch. Protistenk., 81: 284-307. Furssenko, A. 1929. Lebenscyclus und Morphologie von Zoothamnium ar- buscula Ehrenberg. Arch, Protistenk., 67: 376-500. Garnjobst, L. 1937. A comparative study of protoplasmic reorganization in two hypotrichous ciliates, Stylonethes sterkii and Euplotes taylori, with special reference to cystment. Arch. Protistenk., 89: 317-80. Gregory, L. H. 1909. Observations on the life history of Tillina magna. J. exp. Zool., 6: 383-431. Gruber, A. 1885. Uber kunstliche Teilung bei Infusorien. Biol. Zbl., 4: 717- D2: —— 1886. Beitrage zur Kenntniss der Physiologie und Biologie der Pro- tozden. Ber. naturf. Ges. Freiburg i. B., 1: 1-33. Hartmann, M. 1924. Der Ersatz der Fortpflanzung von Amében durch fort- gesetzte Regenerationen. Weitere Versuche zum Todproblem. Arch. Protistenk., 49: 447-64. —— 1928. Uber experimentelle Unsterblichkeit von Protozoen-Individuen. Ersatz der Fortpflanzung von Amoeba proteus durch fortgesetzte Re- generationen. Zool. Jahrb., Abt. allg. Zool. Physiol. Tiere, 45: 973-87. Hegner, R. W. 1920. The relations between nuclear number, chromatin mass, and shell characteristics in four species of the genus Arcella. J. exp. Zool., 503 1-95; 814 MORPHOGENESIS Hertwig, R. 1903. Uber Korrelation von Zell-und Kerngrésse und ihre Be- deutung fiir die geschlechtliche Differenzierung und die Teilung der Zelle. Biol. Zbl., 23: 49-62; 108-19. 1908. Uber neue Probleme der Zellenlehre. Arch. Zellforsch., 1: 1-33. Hetherington, A. 1932. On the absence of physiological regeneration in Stentor coeruleus. Arch. Protistenk., 77: 58-63. Hewitt, J. H. 1914. Regeneration of Plewrotricha after merotomy with refer- ence especially to the number of micronuclei and the occurrence of un- inucleated cells. Biol. Bull., 27: 169-76. Hofer, B. 1890. Experimentelle Untersuchungen tber den Einfluss des Kerns auf das Protoplasma. Jena. Z. Naturw., 24: 105-76. Holmes, S. J. 1907. The behavior of Loxophyllum and its relation to regenera- tion. J. exp. Zool., 4: 399-430. Horton, F. M. 1935. On the reactions of isolated parts of Paramecium cauda- jum, |. exp. Biola 2213-16. Hosoi, T. 1937. Protoplasmic streaming in isolated pieces of Paramecium. J. Fac. Sci. Tokyo Univ., Zool., 4: 299-305. Howland, R. B. 1928. Grafting and reincorporation in Actinosphaerium eichornit Ehr. Biol. Bull., 54: 279-88. Hyman, L. H. 1917. Metabolic gradients in Amoeba and their relation to the mechanism of amoeboid movement. J. exp. Zool., 24: 55-99. Ishikawa, H. 1912. Wundheilungs- und Regenerationsvorgange bei In- fusorien, Roux Arch. EntwMech. Organ. 35: 1-29. Jennings, H. S. 1901. On the significance of the spiral swimming of organ- isms. Amer. Nat., 35: 369-78. Jennings, H. S., and C. Jamieson. 1902. Studies on reactions to stimuli in unicellular organisms. Biol. Bull., 3: 225-34. Jensen, P. 1896. Uber individuelle physiologische Unterschiede zwischen Zellen der gleichen Art. Pflig. Arch. ges. Physiol., 62: 172-200. Kepner, W. A., and B. D. Reynolds. 1923. Reactions of cell bodies and pseudopodial fragments in D/fflugia. Biol. Bull., 44: 22-46. Landis, E. M. 1920. An amicronucleate race of Paramecium caudatum. Amet. Nat., 54: 453-57. LeDantec, F. 1897. La Régénération du micronucleus chez quelques infusoires Giliéss |G Re Acad=SciPariswe2snoi5 2. Lewin, K. R. 1910. Nuclear relations of Paramecium caudatum during the asexual period. Camb. Phil. Soc. Proc., 16: 39-41. — 1912. The behavior of the infusorian micronucleus in regeneration. Proc. roy. Soc., B, 84: 332-44. Lillie, F. R. 1896. On the smallest parts of Stentor capable of regeneration. J. Morph., 12: 239-49. Looper, J. B. 1928. Cytoplasmic fusion in Actinophrys sol, with special ref- erence to the karyoplasmic ratio. J. exp. Zool., 50: 31-49. MORPHOGENESIS 815 Ludwig, W. 1929. Untersuchungen tiber die Schraubenbahnen neiderer Or- ganismen. Z. f. vergl. Physiol., 9: 734-801. Lund, E. J. 1917. Reversibility of morphogenetic processes in Bursaria. J. exp. Zool., 24: 1-19. Luntz, A. 1936. Unsterblichkeit von Protozoenindividuen, erhalten durch periodische Reizungen. Arch. Protistenk., 88: 23-26. Lynch, V. 1919. The function of the nucleus of the living cell. Amer. J. Physiol., 48: 258-83. Mast, S. O., and F. M. Root. 1916. Observations on Amoeba feeding on rotifers, nematodes and ciliates, and their bearing on the surface-tension theory. J. exp. Zool., 21: 33-49. Maupas, M. E. 1888. Recherches expérimentelles sur la multiplication des infusoires ciliés. Arch. zool. exp. gén., II, 6: 165-277. Miller, E. De Witt. 1932. Reappropriation of cystoplasmic fragments. Arch. Protistenk., 78: 635-45. Minchin, E. A. 1912. An introduction to the study of the Protozoa. London. Monod, J. 1933. Mise en évidence du gradient axial chez les Infusoires ciliés par photolyse a l’aide des rayons ultraviolets. C. R. Acad. Sci. Paris, 196: 212-14. Moody, J. E. 1912. Observations on the life history of two rare ciliates, Spathidium spathula and Actinobolus radians. J. Morph., 23: 349-99. Moore, E. L. 1924. Regeneration at various phases in the life history of Spathidium spathula and Blepharisma undulans. J. exp. Zool., 39: 249- 316. Morea, L. 1935. Régénération chez Spirostomum ambiguum. C. R. Soc. Biol. Paris, 119: 235-37. Morgan, T. H. 1901. Regeneration of proportionate structures in Stentor. Biol. Bull. 2: 311-28. Nadler, J. E. 1929. Notes on the loss and regeneration of the pellicle in Blepharisma undulans. Biol. Bull. 56: 327-30. Nussbaum, M. 1884. Uber spontane und kiinstliche Zelltheilung. Verh. Nat- uth. Ver, Bonne 259: Okada, Y6 K. 1930. Transplantationsversuche an Protozoen. Arch. Protistenk., 69: 39-94. Patten, M. 1921. The life history of an amicronucleate race of Didinium nasu- tum. Proc. Soc. Exp. Biol. N.Y., 18: 188-89. Peebles, F. 1912. Regeneration and regulation in Paramecium caudatum. Biol. Bull., 23: 154-70. Phelps, L. A. 1926. Experimental analysis of factors concerned in division in Ameba. Trans. Amer. micr. Soc., 45: 133-45. Poljansky, G. 1938. Die Rekonstruktion des Kernapparates der Bursaria trun- catella bei experimentellen Trennung der konjugierenden Paare. Biol. Zbl., 7: 123 (Berichte ii. d. wiss. Biol., 48: 608). 816 MORPHOGENESIS Popoff, M. 1907. Depression der Protozoenzelle und der Geschlechtszelle der Metazoen. Arch. Protistenk., (Suppl.) 1: 43-82. Prowazek, S. 1904. Beitrage zur Kenntnis der Regeneration und Biologie der Protozoen. Arch. Protistenk., 3: 44-59. Reynolds, B. D. 1924. Interaction of protoplasmic masses in relation to the study of heredity and environment in Arcella polypora. Biol. Bull., 46: 106-40. Reynolds, M. E. 1932. Regeneration in an amicronucleate infusorian. J. exp. Zool., 62: 327-61. Rosel von Rosenhof, A. S. 1755. Insekten Belustigung. 3 vols., Nurnberg. Runyan, E. M., and H. B. Torrey. 1914. Regulation in Vorticella. Biol. Bull., 27: 343-45. Schwartz, V. 1934. Versuche tiber Regeneration und Kerndimorphismus der Ciliaten. Nachr. Ges. wiss. Gottingen, math.-physik., N.F. 1: 143-55. — 1935. Versuche tber Regeneration und Kerndimorphismus bei Stevtor coeruleus Ehrbg. Arch. Protistenk., 85: 100-39. Seyd, E. L. 1936. Studies on the regulation of Spirostomum ambiguum. Arch. Protistenk., 86: 454-70. Sokoloff, B. 1922. Le Noyau est-il indispensable a la régénération des Proto- zoaires? C. R. Soc. Biol. Paris, 87: 1144-47. — 1923. Hunger and regeneration. J. R. micr. Soc., London, 1923, 183- 39: —— 1924. Das Regenerationsproblem bei Protozoen. Arch. Protistenk., 47: 143-252. Stevens, N. M. 1903. Further studies on the ciliate infusoria, Lichnophora and Boveria. Arch. Protistenk., 3: 1-43. Stol¢, A. 1910. Uber kernlosen Individuen und kernlose Teile von Amoeba proteus. Roux Arch. EntwMech. Organ., 29: 152-68. Summers, F. M. 1938a. Some aspects of normal development in the colonial ciliate Zoothamnium alternans. Biol. Bull., 74: 117-29. — 1938b. Form regulation in Zoothamnium alternans, Biol. Bull., 74: 130-54. Tartar, V. 1939. The so-called racial variation in the power of regeneration in Paramecium. J. exp. Zool., 81: 181-208. Taylor, C. V. 1928. Protoplasmic reorganization in Uronychia uncinata, sp. nov., during binary fission and regeneration. Physiol. Zo6l., 1: 1-25. Taylor, C. V., and W. P. Farber. 1924. Fatal effects of the removal of the micronucleus in Evplotes. Univ. Cal. Publ. Zool., 26: 131-43. Thon, K. 1905. Uber den feineren Bau von Didinium nasutum O. F. M. Arch. Protistenk., 5: 281-321. Tittler, I. A. 1935. Division, encystment, and conjugation in Urostyla grandis. Cellule, 44: 189-218. 1938. Regeneration and reorganization in Uroleptus mobilis follow- ing injury by induced electric currents. Biol. Bull., 75: 533-41. MORPHOGENESIS 817 Verworn, Max. 1889. General Physiology. 2d ed., London. 1892. Die physiologische Bedeutung des Zellkerns. Pfliig. Arch. ges. Physiol., 51: 1-118. Willis, H. S. 1916. The influence of the nucleus on the behavior of Amoeba. Biol. Bull., 30: 253-70. Woodruff, L. L. 1905. An experimental study of the life history of hypo- trichous infusoria. J. exp. Zool., 2: 585-632. —— 1913. Cell size, nuclear size and the nucleocytoplasmic relation dur- ing the life of a pedigreed race of Oxytricha fallax. J. exp. Zool., 15: 1-22. —— 1921. Micronucleate and amicronucleate races of infusoria. J. exp. Zool., 34: 329-37. Young, D. B. 1922. A contribution to the morphology and physiology of the genus Uronychia. J. exp. Zool., 36: 353-90. —— 1926. Nuclear regeneration in Stylonychia mytilus. Biol. Bull., 51: 163-65. Young, Dixie. 1939. Macronuclear reorganization in Blepharisma undulans. J. Morph., 64: 297-353. CHAPTER XVII CERTAIN ASPECTS OF PATHOGENICITY OF PROTOZOA ELERY R. BECKER IT Is CUSTOMARY to recognize three functional categories of parasitic Protozoa: (1) commensals, which neither harm nor abet the host; (2) symbionts (= symbiotes), which aid the host; and (3) true parasites or pathogenes, which disarrange the host organism to a greater or less degree. This practice may be defended on academic grounds, since it serves to clarify concepts and to attract students’ interest to animal microérganisms and the rdles they play in the lives of other animals and plants, but it is in reality highly artificial. The ensuing discussion will be developed principally about this point, with the deliberate intention of provoking wide consideration of the subject, particularly as regards the “pathogenic” aspects of parasite activity, as was done with the subject of host-specificity of parasites a number of years ago (Becker, 1933). Such terms as commensalism, symbiosis (symbiotism), and pathogenicity can represent no more than an expression of the state of adjustment between two separately functioning entities, the host and the parasite, co€éxisting in one of the most intimate relationships, and as such are subject to analysis. PROBLEMS OF VIRULENCE AND PATHOGENICITY The functional categories have no counterparts in the zodlogical scheme: that is to say, there are no classes, orders, or families which have as their distinguishing character that they are pathogenic or other- wise. The statement applies also to genera, for, as a matter of fact, we recognize ‘‘pathogenic”’ and ‘“‘non-pathogenic’” members of Trypanosoma, Trichomonas, Entamoeba, and other genera. The situation is seen, at the outstart, to limit itself almost entirely to a consideration of ‘“‘patho- genic species,”’ but it is actually still more complicated than that. There is indisputable evidence that many species of pathogenic Protozoa are PATHOGENICITY 819 made up of a number of strains. Entamoeba histolytica, for example, is believed to be a composite of many races differing both in cyst size (see Dobell and Jepps, 1918) and virulence (see Meleney and Frye, 1935, pp. 431-32). The evidence for the latter is indisputable, especially since the appearance of the work of Meleney and Frye (1933, 1935), although Craig (1936) is still skeptical regarding the existence of avirulent strains. The latter point can be conceded for the present (though it is still a live issue), without impugning the significance of observations on human cases and experimental infection in kittens and puppies point- ing to the existence of strains of low virulence, medium virulence, and high virulence. Meleney and Frye adopted a standardized procedure. Recognizing the doubtful validity of experiments performed on too few animals and conducted without due allowance for variability of individual re- sponse, they made it a practice to test each strain in a large series of kittens of standard size. Each strain was isolated in culture, young trans- plants were used for inoculations, and inoculations were made directly into the caecum after laparotomy incision. Furthermore, the history of the human patient was known, and there were records regarding the character of the community and the prevalence of amoebiasis in the com- munity in which each patient resided. The criteria of pathogenicity were success or failure in infecting, extent and intensity of lesions produced, and duration of the infection. The results of the experiment showed conclusively that certain strains of Entamoeba histolytica of human origin exhibited more “‘pathogenic activity” in kittens than other strains. Furthermore, by correlating the experimental data with field observa- tions, they were led to the following conclusion regarding the relative pathogenicity of the strains for man: “The more pathogenic strains (i.e., in kittens), whether they were obtained from acute cases of amoebic dysentery or from so-called ‘healthy carriers’ were associated geographically and epidemiologically with acute dysentery, whereas the less pathogenic strains were associated both individually and epidemi- ologically with very little evidence of acute dysentery.” AMOEBIC DYSENTERY AND BACTERIAL COMPLICATIONS The problem of virulence has had its reflection in matters of specificity. Brumpt (1925) described Entamoeba dispar as an amoeba of the 820 PATHOGENICITY histolytica-type dwelling in man, incapable of producing symptoms of dysentery in its human host, and producing no definite macroscopic ulcerations in the cat, but capable of penetrating the intestinal wall of the latter animal so far as the muscularis mucosae. Simi¢, in several papers, has corroborated Brumpts’s claim for the validity of E. dispar, but his last (1935) paper presents the strong argument that E. dispar infection in dogs lasts only from 6 to 8 days, while E. /Azstolytica infection lasts from 60 to 120 days. E. dispar infection in dogs is quite benign, while E. histolytica produces characteristic amoebic ulcerations, and amoebae with ingested red cells may be found in the stools of the infected dog. Wenyon (1936), however, in commenting on the strong case built up by Simié (1935) states: It still seems futile to attempt to separate E. dispar from E. histolytica on the grounds of pathogenicity. It seems that all the differences described can be more reasonably accounted for by the supposition that races of E. histolytica of varying virulence occur and that hosts vary in their susceptibility to the one species. Meleney and Frye (1935) likewise prefer to consider E. dispar as a strain of E. histolytica, possessing a low degree of pathogenicity. Even the least virulent strains encountered by the latter authors, though not producing clinical symptoms in the persons in whom they had their origin, were capable of producing lesions in some kittens. Hence the skepticism of these authors regarding totally avirulent strains of E. hzs- tolytica. Is virulence-level retained by a strain of E. histolytica, or is it sub- ject to modifying factors, such as attenuating effects of artificial culture media or exaltation by animal passage? Meleney and Frye (1933) first noted the contrast in pathogenic activity in kittens between “A”’ strains of low virulence from the hill country, where symptomless carriers and persons with mild symptoms were the rule, and “‘B” strains of high virulence from severe cases of amoebic dysentery in the bottom lands, where acute cases were much more common than in the hills. Later (1935), they were able to report, after adequate testing in kittens, that two “A” strains and two “B” strains had retained their respective patho- genic indices after a period of three years of artificial cultivation. The highly virulent strains did not decline in pathogenicity. But what about the effect of animal passage? Meleney and Frye (1936) state that their PATHOGENICITY 821 efforts to step up the virulence of less pathogenic strains by serial passage through kittens and dogs have always failed at the first transfer, but Cleveland and Sanders (1930) have made some experiments bearing on this point, except that they ascribe the effects observed to bacteria rather than to amoebae. Using for the first passage in kittens a strain that had been carried on in culture for from 460 to 540 days, they found that only 2 out of 26 animals became infected in the first passage, 5 out of 5 in the second, 3 out of 7 in the third, and 2 out of 2 in the fourth. They conclude: ‘‘An increase in the percentage of animals that became infected with passage is demonstrated in these experiments, but this may be due, as in the liver passages, to an increase in virulence by the bacteria rather than the amoebae.” The need for further work on the possibility of exalt- ing the virulence of less pathogenic strains by animal passage is appar- ent, but efforts along this line, in order to obtain results of significance, will first have to eliminate the effects of bacteria accompanying the Protozoa. The comments of Cleveland and Sanders regarding bacteria suggest the next point, the effect of bacteria on pathogenicity of E. histolytica. Their criterion for virulence was principally infectivity for the liver in kittens, when inoculated with a hypodermic needle directly into this organ. Pure cultures in liver-infusion, agar-horse serum saline medium lost most of their ability to establish infection in the liver after a year or more. Such a strain was reduced to an infectivity of 20 percent in the first passage. The infectivity increased, however, with succeeding pas- sages, until by the sixth passage it amounted to 73 percent. Was the ap- parent increase in virulence to be attributed to the amoebae or to the bacteria accompanying them? Which had lost virulence during the year of life in the artificial media? An attempt to settle the issue was made in a crisscross experiment. Bacteria from the fifth passage were inoculated with the culture amoeba that had not been passed; and, conversely, the passed amoebae were inoculated with bacteria that had not been passed, the latter being the nonpathogenic Bacillus brevis. The experiments showed that fifth-pas- sage bacteria increased the virulence of amoebae in culture for a year, and that amoebae were not able to maintain themselves in the liver of the cat unless accompanied by bacteria capable of damaging the liver. Thus Cleveland and Sanders concluded that it was the bacteria accompanying 822 PATHOGENICITY E. histolytica in culture, and not the amoeba, which lost virulence during the year in the artificial medium and regained virulence after repeated liver passage. They add the precautionary remark that failure to infect livers with cultures containing only nonpathogenic bacteria might not be repeated with more virulent strains of E. histolytica. Frye and Meleney (1933) attacked the same problem, using severity of intestinal infection as the criterion. They, too, tried a crisscross tech- nique, interchanging the bacteria in a culture of proved high virulence with those in a culture of proved low virulence. The interchange did not materially alter the incidence or severity of infection of the two strains of amoebae; hence their conclusion that the difference in pathogenicity of the two cultures was really due to the amoebae themselves. Thus differences in pathogenicity of strains of E. Aistolytica claimed by Mel- eney and Frye was shown to be due to inherent qualities of the proto- zoon, and not to accompanying microdrganisms. Since they had previously not been able to detect any alteration of pathogenicity in artificial me- dium, the question of alteration of virulence of bacteria in such a medium does not enter in. MALARIA: Plasmodium vivax It is inescapable that there are strains of intestinal Protozoa differing in virulence, but what is the situation regarding the pathogenic blood Protozoa? Since the behavior of the trypanosomes in animal passage 1s complicated by differences in behavior of “‘passage’’ and ‘‘relapse”’ strains, the author prefers to evade discussing this subject. Human malaria, however, lends itself more readily to discussion, as becomes evident after reading the chapter entitled “The Complexity of the Ma- laria Parasite” in Hackett (1937). Malaria therapy in general paralysis (paresis) has made it possible to determine definitely whether there are strains of the human malarias differing in morphology, pathogenicity, or other behavior, and the facts learned have been rather surprising. Plasmodium vivax is the species commonly employed in malaria ther- apy. Using infected Anopheles for inoculation, Boyd and Stratman- Thomas (1933a) showed that during an attack of malaria induced by a particular strain of this species, a patient acquires a ‘“‘tolerance’’ which makes him refractory to reinoculation with that strain, but not with a different strain of the same species. They concluded that a person in- PATHOGENICITY 823 fected with benign tertian malaria acquires a homologous but not a heterologous tolerance to P. vivax. Later, Boyd, Stratman-Thomas, and Muench (1934) discovered that superinfections with heterologous strains appear to result in clinical attacks of milder intensity than the original attacks. Manwell and Goldstein (1939) have discovered a similar situation in P. circum flexum infection in birds. Using six strains, they concluded that immunity was strain specific rather than species specific, although all strains conferred at least partial protection against the others. It should be added that certain strains of P. vivax do have the ability to immunize (or premunize) the patient toward certain other strains. VARIABILITY IN STRAINS AND IN Host RESPONSE Morphological differences between strains of P. vivax have been ob- served. Two strains of this species widely used in Europe for malaria therapy are the so-called Dutch and Madagascar strains. Buck (1935) has found that the Dutch strain consistently exhibits between twelve and thirteen merozoites in both mosquito-inoculated and blood-inocu- lated malaria, while the Madagascar strain exhibits between seventeen and eighteen. The incubation period of the former is twenty-one days, while that of the latter is but twelve days. Whether there is a relationship between merozoite number and incubation period in these cases is some- what of a problem, especially since the discovery of extracellular schi- zogony of malaria organisms in the internal organs. Strains of P. vivax likewise differ exceedingly in pathogenicity. Some strains are too low in virulence to be useful in malaria therapy of gen- eral paralysis. The Dutch strain referred to above is said by Hackett (1937) to give higher fever, to be less susceptible to treatment with salvarsan, and to be less virulent than the Madagascar strain. Further- more, it often produced no immediate attack, but in 40 percent of the cases went into a long latency of several months, a phenomenon that occurred with the Madagascar strain in only 6 percent of the cases. There appear to be likewise multiple strains of the other human malarias, viz., P. falciparum and P. malariae (see Hackett, 1937; Boyd and Kitchen, 1937). Every case of parasitism exhibits three aspects—the parasite, the host, and the effect of the impinging of the one on the other. It has been 824 PATHOGENICITY shown that parasitic Protozoa differ not only in the response they evoke from the host according to their standing as species, but also according to strain properties. There is likewise abundant evidence that individual hosts differ in their response to the same strain of parasitic protozo6n. The latter is in reality a statistical concept. It has been a general experi- ence that when an attribute of an unselected group of individuals was measured, the plotted measurements fell into the well-known frequency distribution curve, either normal or skewed. The writer knows of no data which have been plotted to demonstrate that quantitative data on either individual resistance or susceptibility to adverse effects of parasit- ism could be presented in a similar sort of graph, but there are many facts, to support such a supposition. COCCIDIOSIS IN POULTRY For several years the writer (see Becker and Waters, 1938, 1939b) has been testing the effect of the ration on the course of caecal coccidiosis in chicks. While, in general, fatality was used as the criterion for com- paring the effects of two rations, it has been possible to make a number of additional hitherto-unpublished observations bearing on the variability of host response to the disease. In one lot of thirty-three White Leg- horn chicks experimentally infected with the same dosage, there were three deaths by eight o’clock in the morning of the fifth day, and five more during the remainder of that day. The next day nine succumbed, making a total of seventeen. Nine others were noted to be in an ex- tremely precarious condition, missed succumbing only by a narrow mar- gin, but recovered to a considerable degree. Five others were observed to be severely affected, but continued to move about and eat some feed during the entire ordeal. One was quite active throughout, though its comb paled significantly. One, a cockerel, continued to eat and move about with undiminished vigor, and its comb did not pale perceptibly, though the droppings were streaked slightly with blood. Similar observa- tions have been common, and justify the assertion that fowls differ sig- nificantly in the morbidity they exhibit in response to uniform dosage with the same strain of Coccidinm. The literature is replete with evidence that similar variability of host response exists in the case of other protozoan infections. Walker and Sellards (1913) early distinguished between ‘“‘contact”’ carriers of En- PATHOGENICITY 825 tamoeba histolytica (who did not develop dysentery) and ‘‘convalescent”’ carriers (who have suffered with dysentery, but have become convales- cent). In fact, they passed a strain of the amoeba from a convalescent carrier serially through three other men, two of whom became contact carriers, i.e., did not develop dysentery, and one of whom became a victim of an acute attack of amoebic dysentery. Meleney and Frye, in the experiments previously mentioned, found that kittens inoculated with the same strain differed as to whether or not they became infected, as to the extent and severity of the lesions in the colon, and as to the pe- riod of survival of the diseased kittens. Individuals differ also in the degree of resistance offered to the multi- plication of the malaria parasite in their blood and tissues, and in their reaction to parasite density. The existence of racial tolerance or resist- ance of Negroes to inoculation with Plasmodium vivax was pointed out by Boyd and Stratman-Thomas (1933b), though it was by no means absolute. The same authors later (1934) reported their finding that Caucasians appear to be universally susceptible. Wilson (1936, quoted by Hackett) made the observation that Bantu babies in Tanganyika Ter- ritory were all infected with the three species of human malaria by the fifth month of life, and commented as follows: One of the striking features of this period of acute infestation, lasting about eighteen months, is the difference in degree of infestation in different in- dividuals. These babies were constantly being reinfected by fresh invasions of sporozoites. The difference cannot therefore be due to variations in the parasites, but rather to a variation in individual resistance. Hackett (1937) discusses the variability in the incubation period ex- hibited by different individuals, and states that in some cases there were as few as one parasite per cubic millimeter at the onset of symptoms, while in others there were 900. Thus it is evident that the clinical aspects of protozoan infections may differ, owing to inherent basic characters of both the parasite and the host. The reaction of the host may be governed further by another factor that we shall designate the physiological state. Admittedly very little is known concerning the relationship between the physiological state and pathogenicity, but one would conclude a priori that a far-reach- ing relationship should prevail here. As a striking concrete example, nursling rats usually succumb to the long-supposed ‘non-pathogenic’ 826 PATHOGENICITY Trypanosoma lewisi, while older rats undergo a response to this micro- organism, in behavior of leucocytes and monocytes, that confers on them sufficient resistance for survival. NUTRITION AND RESISTANCE Nutrition may have a far-reaching effect on physiological state, and indirectly on resistance. The following hitherto-unpublished experiment is useful in illustrating the point. Forty young rats of about fifty grams’ average weight were divided into two equal groups. One group was fed the following mixture (parts by weight): Beet sugar, 67; casein, unextracted, 10; normal salt mixture, 3; lard, 3; cod liver oil, 2; bright green alfalfa meal, 15. The other group was fed the same mixture, ex- cept that alfalfa meal was replaced with whole oats ground to a fine flour. After two weeks on these rations, the lot receiving the ground oats had made slightly greater weight gains than the other. On the fif- teenth to the eighteenth days each rat was fed 10,000 recently sporulated oocysts of Ezmerza nieschulzi, a coccidium that develops in enormous numbers in the mucosa of the small intestine. On the sixth day the al- falfa-fed rats were obviously affected with diarrhoea, while the oat-fed animals were not showing distress. Strangely enough, on the seventh day the alfalfa-fed lot appeared to be recovering, with formed stools and return of appetite, but the oat-feds were off their feed and passing liquid stools. On the eighth and ninth days, 16 out of 20 of the latter died, a marked contrast to what happened in the alfalfa-fed lot all of which re- covered. The result was rather surprising, in view of the biological assays of Becker and Derbyshire (1937, 1938) and Becker and Waters (1939a), which showed that alfalfa meal in the ration in some manner or other stimulated the development of several times as many odcysts of Eimeria nieschulzi in its rat host as either oat hulls or hulled oats. What is the explanation of the observed effects? The early develop- ment of diarrhoea in the alfalfa-feds appears to have been due to the preponderance of the parasite population, but there is a possibility that it lies in the superior accessory food factors of alfalfa meal. The follow- ing experiment suggests that vitamin B may have had something to do with it. Twenty young rats were fed the following ration: beet sugar, 71; soy-bean oil meal, expeller process, 10; casein, commercial medium fineness, 10; normal salt mixture, 4; lard, 3; cod-liver oil, 2. Another PATHOGENICITY 827 lot of 20 was fed the same mixture with 10 micrograms of thiamin chloride (vitamin B) per rat daily. The second lot made much greater weight gain during the next ten days than the first. On the tenth to the fifteenth days each rat was inoculated with daily doses of 6,000 sporu- lated odcysts of E. nieschulzi. Twelve rats out of 20 in the first lot succumbed to the infection, and the remaining 8 all lost weight upon recovery. The recipients of vitamin B all lived and, by the time the in- fection had cleared up, all had gained in weight. Thus it is evident that the physiological state may be of prime im- portance in determining whether or not an animal survives an infection. In one state it may show few or no outwardly visible symptoms, while in another it may be seriously affected, or even succumb. CONCLUSIONS Such terms as commensalism and true parasitism lose their significance when a comprehensive analysis is made of the circumstances surround- ing an infection with any particular protozoan species. Pathogenicity in the generally accepted sense is a matter of degree, subject in the first place not only to the species, but also to the strain, of the microorganism concerned in the infection. The degree of pathogenicity exhibited by a particular strain in its host may vary from nil to fatal termination, depending upon the inherent defense mechanisms and the other condi- tions affecting the resistance of the host. The effectiveness of this resist- ance, in turn, may vary according to changes in the physiological state of the host. These considerations are of fundamental importance to the investigator who conducts researches on the reaction of any host to the invasion of a protozoan parasite. LITERATURE CITED Becker, E. R. 1933. Host-specificity and specificity of animal parasites. Amer. J. lrop.- Med 13)505-23) Becker, E. R., and R. C. Derbyshire. 1937. Biological assay of feeding stuffs in a basal ration for coccidium-growth-promoting substance. I. Procedure, yellow corn meal, oats, oat hulls, wheat, linseed meal, meat scraps. Iowa St. Coll. J. Sci., 11 (1938): 311-22. II. Barley, rye, wheat bran, wheat flour middlings, soy bean meal. Ibid., 12: 211-15. Becker, E. R., and P. C. Waters. 1938. The influence of the ration on mortality from caecal coccidiosis in chicks. Iowa St. Coll. J. Sci., 12: 405-14. —— 1939a. Biological assay of feeding stuffs in a basal ration for coccidium- 828 PATHOGENICITY growth-promoting substance. III. Dried fish meal, alfalfa meal, white wheat flour. Iowa St. Coll. J. Sci., 13: 243. —— 1939b. Dried skim milk and other supplements in the ration during caecal coccidiosis of chicks. Soc. Exp. Biol. N.Y., 40:439. Boyd, M. F., and S. F. Kitchen. 1937. The duration of the intrinsic incuba- tion period in Falciparum malariae in relation to certain factors affecting the parasites. Amer. J. Trop. Med., 17: 845-48. Boyd, M. F., and W. K. Stratman-Thomas. 1933a. Studies on benign ter- tian malaria. I. On the occurrence of acquired tolerance to Plasmodium vivax, Amer. J. Hyg., 17: 55-59. 1933b. Studies on benign tertian malaria. IV. On the refractoriness of Negroes to inoculation with Plasmodium vivax. Amer. J. Hyg., 18: 485-89. — 1934. Studies on benign tertian malaria. V. On the susceptibility of Caucasians. Amer. J. Hyg., 19: 541-44. Boyd, M. F., W. K. Stratman-Thomas, and H. Muench. 1934. Studies on be- nign tertian malaria. VI. On heterologous tolerance. Amer. J. Hyg., 20: 482-87. Brumpt, E. 1925. Etude sommaire de |’“Entamoeba dispar’ n.sp. Amibe a kystes quadrinucleés, parasite de "homme. Bull. Acad. Med., Paris, 94: 943. Buck, A. de. 1935. Ein morphologischer Unterschied zwischen zwei Plasmo- dium vivax-Stammen. Arch. Schiffs- u. Tropenhyg., 39: 342-45. Cleveland, L. R. and E. P. Sanders. 1930. The virulence of a pure line and several strains of Entamoeba histolytica for the liver of cats and the relation of bacteria, cultivation, and liver passage to virulence. Amer. i: Tyg 12569-6005» Craig, C. F. 1936. Some unsolved problems in the parasitology of amebiasis. Parasitology, 22: 1. Dobell, C., and M. W. Jepps. 1918. A study of the diverse races of Entamoeba histolytica. Parasitology, 10: 320-51. Frye, W. W., and H. E. Meleney. 1933. Studies of Endamoeba histolytica and other intestinal Protozoa in Tennessee. VI. The influence of the bacterial flora in cultures of E. Aiéstolytica on the pathogenicity of the Amoebae. Amer. J. Hyg., 18: 543-54. Hackett, L. W. 1937. Malaria in Europe. London. Manwell, R. D., and F. Goldstein. 1939. Strain immunity in avian malaria. Amer, J. Hyg. sec. Cy 30315222: Meleney, H. E., and W. W. Frye. 1933. Studies of Endamoeba histolytica and other intestinal Protozoa in Tennessee. V. A comparison of five strains of E. histolytica with reference to their pathogenicity for kittens. Amet:.JaHyg.,'17: 637-55. — 1935. Studies of Endamoeba histolytica and other intestinal Protozoa PATHOGENICITY 829 in Tennessee. IX. Further observations on the pathogenicity of certain strains of E. Aistolytica for kittens. Amer. J. Hyg., 21: 422-37. 1936. The pathogenicity of Endamoeba histolytica. Trans. R. Soc. trop. Med. Hyg. 29: 369. Simi¢, T. 1933. L’Infection du chien par l’Entamoeba dispar Brumpt. Ann. Parasit. hum. corp., 11: 117-28. — 1935. Infection expérimentale du chat et du chien par Entamoeba dispar et Entamoeba dysenteriae. Ann. Parasit. hum. corp., 13: 345-50. Walker, E. L., and A. W. Sellards. 1913. Experimental entamoebic dysentery. Philipp. J. Sa. (B., Trop. Med:), 6: 259. Wenyon, C. M. (“C. M. W.”) 1936. Trop. Dis. Bull., 33:534. CHAPTER XV iil THE IMMUNOLOGY OF THE PARASITIC PROTOZOA WILLIAM H. TALIAFERRO THE CENTRAL theme of the science of immunology is the study of the defense mechanisms of the host against the invasion of parasitic or- ganisms or against the introduction of their products or of other 1n- animate materials. In the present chapter emphasis is placed almost entirely on the defense mechanisms against living parasites. A complete analysis of these mechanisms involves such widely diverse subjects as the origin, nature, and developmental potencies of the cells and tissues of the host, the physiological action and chemical nature of the humoral forces marshaled by the host in defense, the activity of the invading parasite, the chemical nature of the products of the parasite which stimulate the immune processes in the host, and the effects of the various immune proc- esses on the parasite. As protozoan immunity is just one aspect of the general field of immunology, most of the general principles of immunity can be applied directly to the protozoan parasites. Work on protozoan immunity itself, however, has been restricted more or less to the biologi- cal aspects, such as the study of the cellular and the serological mecha- nisms of the host and the effects of resistance on the parasite, with very little emphasis on chemical phases. THE PHYSICAL BASES OF IMMUNITY Immunity or resistance, in the broad sense, denotes various mecha- nisms of the host which counteract the invasion and the activities of a parasite. It may be manifested as hindrances to the action of invasion, as conditions arising in the body of the host adverse to the parasite, as efforts on the part of the host to make good the deleterious effects of the parasite (as evidenced by the hyperactivity of hematopoietic organs after the destruction of red cells in malaria), or the production of antitoxins in those infections in which toxins are formed. It may be natural (in- nate) or acquired. Natural immunity is generally correlated with non- IMMUNOLOGY 831 specific factors which are incompatible with or unfitted to the life of the parasite in the unimmunized host. The specificity of parasites for various hosts (Chapter XVII) is largely an expression of natural immunity. Acquired immunity, on the other hand, denotes the various conditions arising in a host as a result of infection or other immunizing procedure and is generally thought of as resulting in large measure from the pro- duction of antibodies in the host. Immunity is the reciprocal of virulence, which in this sense is an ex- pression of the ability of the parasite to invade and parasitize the host. Both immunity and virulence are relative and represent the resultant of the invasive activities of the parasite and the defense activities of the host; they may, therefore, vary in degree from zero to 100 percent. THE CELLS INVOLVED IN IMMUNITY The defense of the vertebrate body against invading parasites, or even against inanimate foreign material introduced parenterally, appears to be taken care of predominantly by some of the cells of the connective tissue and is a specialized or accentuated aspect of their normal functions. The connective tissue has manifold normal functions, such as respiration, intermediate metabolism, storage, and mechanical support and in its widespread distribution throughout the body consists of the blood and lymph, cartilage, bone, the reticular (blood-forming) tissue of the myeloid and lymphatic organs, and loose and dense connective (includ- ing adipose) tissues associated with the skin, omentum, liver, lung, and so forth. The cells of this tissue arise embryonically from the mesen- chyme and may be either fixed or free. Those of the blood and lymph and of the reticular and loose connective tissues are chiefly concerned in defense. The terminology of the connective tissue cells is complicated by the frequent use of several names for the same cell. This condition has arisen (1) because connective tissue is so widespread and involves so many organs that it has been studied by histologists, hematologists, pathologists, and so forth, some of whom have not correlated the knowledge in fields other than their own; and (2) because investigators have disagreed as to the nature and developmental potencies of various cells. In the following brief review we have defined only those cells of the connective tissue which are known to be involved in defense against 832 IMMUNOLOGY the infections to be described herein and have followed in the main Maximow’s views with regard to the origin and potencies of the various cells. We have simplified and used uniform terms wherever possible. The role of the various connective tissue cells in immunity is shown by direct histological studies and by other experimental work. Thus histological studies of defense reactions have demonstrated directly that some cells remove parasites and various types of debris by phagocytosis; that others wall off nonremovable objects and repair damage by filling in cavities, regenerating certain tissues, and so forth; and that still others, such as the eosinophils, show a definite pattern of behavior and seem correlated with certain phases of immunity, although their exact function is still uncertain. On the other hand, removal or impairment by splenec- tomy, blockading procedures, and the like, of an appreciable portion of the connective tissue cells have furnished evidence of the rdle of phagocytes in the immunity of certain infections and of the rdle of the macrophages in the production of antibodies. In the successful carrying out of these studies, certain technical difh- culties have to be recognized and overcome. To study cellular details and especially to see transitional forms, migrating cells, and so forth, early and closely spaced stages in an infection should be studied, fresh material should be used, and this should be adequately fixed and stained by a satisfactory technique. One of these techniques involves fixing in Helly-Maximow’s Zenker formol, preferably embedding in celloidin, staining with dilute Delafield’s hematoxylin, and counterstaining with eosin azure II. In impairing the macrophage system, the time when splenectomy and blockade are performed is important, inasmuch as impairment is partially made good by the host in time. Furthermore, splenectomy is more effective in impairing the macrophage system in certain infections in which the spleen is especially active and in certain laboratory animals having a high spleen weight-body weight ratio. Thus the most conclusive results may be expected when certain blood infec- tions are studied in dogs, rats, and mice splenectomized and blockaded as rapidly and thoroughly as possible; whereas inconclusive or negative results may be expected from inadequate blockade, splenectomy a week or more before infection. In fact, if impairment is slight, the system may even be stimulated to greater activity. A, Predominantly Fixed Connective Tissue cells ——From the strictly IMMUNOLOGY 833 functional aspect of immunity, the predominantly fixed cells of the reticular and loose connective tissues may be divided into two great groups: (1) fixed and free macrophages (including the reticular cells), and (2) the fibroblasts of connective tissue and the endothelial cells lining the ordinary blood vessels. The term macrophage is essentially a physiological designation for almost any large mononuclear connective-tissue cell which is or may become phagocytic. Under macrophages are classified a group of fixed mesenchymal cells, which retain many embryonic characters and a wide range of potencies for development. The concept that the connective tissue of the adult body possesses fixed cells retaining mesenchymal or embryonic potencies for development is largely due to Marchand (1924, review) and Maximow (1927a, review). There are three chief cate- gories: (1) Pericytes (Maximow) which are fixed, undifferentiated, outstretched cells in the adventitia of all of the small blood vessels of loose connective tissue throughout the body; (2) reticular cells, which, together with fibers, form the stroma of all reticular (myeloid and lymphatic) tissues; (3) littoral cells (Siegmund), which line the sinuses or sinusoids of the reticular tissues, the liver, hypophysis, and adrenal (Pls. 1 and 2). Where phagocytic in the liver, they are generally desig- nated Kupffer cells (Pl. 1, Fig. 1; Pl. 2, Fig. 1). The cells lining the sinuses of the reticular tissues are actually reticular cells. The littoral cells are often called endothelial cells or cells of the special endothelium, but this is unfortunate because the littoral cells have wide developmental potencies, whereas the ordinary endothelial cells lining the blood vessels have restricted developmental potencies. There is general agreement that under proper stimuli the cells of these three categories can divide by mitosis, can become phagocytic, can develop into fibroblasts, or can develop into practically any other type of cell of the blood or connective tissue. From the standpoint of the present discussion, it is important that they can become phagocytic either in their fixed position (fixed macrophages) or after rounding up and becoming free (free macrophages). It is not definitely known, however, whether, while engorged, they temporarily or permanently lose their mesenchymal potencies. There may be a difference, for example, between the primitive outstretched reticular cell and the same cell after it has become free and phagocytic. 834 IMMUNOLOGY In addition to the cells which are generally admitted to retain mesen- chymal potencies, free cells occur in the loose connective tissue, which we have called macrophages and which are variously known as histio- cytes, clasmatocytes, rhagiocrine cells, or resting wandering cells. Just as in the case of the phagocytic mesenchymal cells, there is no unani- mity of opinion as to whether these free cells retain all hematopoietic functions, but in any case they can become phagocytic without morpho- logical change, can reproduce by mitotic division, and can transform into fibroblasts. Many other macrophages occur throughout the body, the develop- mental capacities of which have not been adequately studied. Thus the stroma cells of the /amina propria of the intestine probably have develop- mental potencies identical with those of the reticular cells. As would be expected, macrophages in different locations and before and after becoming phagocytic vary somewhat in structure with regard to the amount of their cytoplasm, the size and shape of their nucleus, and the amount and size of the chromatin granules and nucleoli in their nucleus. They generally possess, however, well defined cytoplasm and a large, vesicular, often indented nucleus, in which are found fine chro- matin granules and a few small nucleoli (see reticular cell and macro- phagennPli 3, Fig mir Fibroblasts of loose connective tissue have outstretched, ill-defined cytoplasm and a large, regularly oval, vesicular nucleus containing dust- like chromatin granules and small nucleoli. They can divide by mitosis, are instrumental in repair and in walling off foreign material, but are rarely phagocytic and do not generally develop into other cells (except in bone and cartilage). Endothelial cells line the larger blood vessels and capillaries. (The term as herein used, does not include the littoral cells lining the sinuses and sinusoids of the reticular tissues and elsewhere, which have wide developmental potencies). The endothelial cells can divide by mitosis, can form endothelium of new blood vessels, and can develop into fibro- blasts, but are rarely phagocytic and do not generally develop into other cells. B. Free Blood and Connective Tissue Cells.—In accordance with common usage, cells of the blood and lymph are classified according IMMUNOLOGY 835 to whether they are of myeloid or lymphoid origin. The lymphoid cells of the blood and the cells of the lymph consist of various-sized lymphocytes, which together with monocytes are termed agranulocytes. The myeloid cells of the blood are the various granulocytes (heterophils or polymorphonuclears, eosinophils, and basophils), the erythrocytes, and the platelets. Some authors classify monocytes as lymphoid and others as myeloid cells, but they are classified in this chapter as both, since we believe that they arise from lymphocytes of the lymphatic tissue and from hemocytoblasts (equivalent to lymphocytes) of the bone marrow. The heterophils are functional in immunity by virtue of their obvious phagocytic activities and probably because of their secretion of enzymes. They are end cells, however, which do not reproduce or develop into other cells. Lymphocytes and presumably monocytes, on the other hand, can divide mitotically (Pl. 4, Fig. 3) and both lymphocytes and mono- cytes can develop into macrophages, with all of their developmental potencies. Lymphocytes possess basophil cytoplasm and a relatively large, deeply staining, often indented nucleus, with large acidophil nucleoli Pl. 3, Fig. 1. The monocytes may be the same size as, but in most cases are larger than the medium lymphocytes, their cystoplasm is less basophil and is increased in amount, and their nucleus is more vesicular, more deeply indented with smaller chromatin granules and smaller and more numerous nucleoli (cf. monocytoid lymphocyte in Pl. 3, Fig. 1). As the lymphocytes and monocytes transform into macrophages, they show in- creased amounts of cytoplasm, their nuclei gradually take on macrophage characteristics, and they become phagocytic (polyblasts 1-5, in Pl. 3, Fig. 2). These intermediate forms, together with lymphocytes, mono- cytes, and macrophages, are grouped under the term lymphoid-macro- phage system. There is general agreement that free lymphoid cells, more or less similar to lymphocytes, occur in varying numbers under various physio- logical and pathological conditions in the reticular tissues and the loose connective tissue, and that in such sites they act as “stem” cells of lymphoid and myeloid cells. The nature, classification, and even exact morphology of these different stem cells are subject to such controversy that they are termed by various authors lymphocytes, hemocytoblasts, 836 IMMUNOLOGY lymphoblasts, myeloblasts, monoblasts, and so forth, according to the particular theory of blood formation held by the author (see Bloom, 1938). We have adopted essentially the unitarian viewpoint of Maxi- mow and have called all free mesenchymal stern cells, with wide poten- cies for development, hemocytoblasts in the bone marrow and lympho- cytes in all other locations. Lymphocytes and hemocytoblasts are identical morphologically and probably in their developmental potencies. Under physiological conditions, lymphocytes in lymphatic tissue give rise only to lymphocytes (Pl. 4), and hemocytoblasts in bone marrow give rise only to myeloid cells (erythroblasts, myelocytes, and so forth), but un- der abnormal stimuli they may exhibit their full potencies for develop- ment. In general, these free stem cells are self-perpetuating, but they may arise from the fixed mesenchymal cells of the preceding section. C. So-called Systems of Cells —The foregoing classification of cells should be brought into line with the so-called systems of cells frequently used by various authors. Modern concepts of the cellular basis of im- munity have been largely based on studies of inflammation. Credit should be given to Metschnikoff (1892) for insisting upon the essential role of the mesenchymal cells in inflammation and to Cohnheim, Ziegler, Mar- chand (1924, review), and Maximow (1927a, 1927b, review), among others, for studying the histogenesis of the local inflammatory reactions. Metschnikoff (1892 and 1905, among other studies) laid the whole foundation for the modern concept of the defense function of fixed and mobile cells of the connective tissue by phagocytosis. His concept was essentially physiological. He distinguished (1) microphages, herein designated heterophils; and (2), macrophages, which are identical with macrophages as herein defined, except that he included the phagocytic microglial cells of the brain, which are possibly of mesenchymal origin. The modern understanding of macrophages is based largely upon the studies of vital staining and the storage of colloidal dyes, chiefly by Renaut, Maximow, Goldman, Tschaschin, Kiyono, and Aschoff. The Gefadsswandzellen of the Marchand-Herzog school (see Marchand, 1924) include pericytes and perivascular macrophages (adventitial cells) which are supposed to arise from the endothelium of developing vessels. Aschoff’s (1924, review) reticulo-endothelial system, broadly defined, consists of the macrophages as we have outlined them. It has unfortunately been assumed by most writers that the increase IMMUNOLOGY 837 of macrophages associated with immunity, 1.e., “hyperplasia of the reticulo-endothelial system,” is due to the proliferation of macrophages or cells of the reticulo-endothelial system. This is an admitted source, but detailed studies of a wide variety indicate that most of the new macrophages arise from lymphocytes, with or without the intervention of a monocyte stage (see Pl. 4). In order to include both macrophages and all of their precursors under one term, which would indicate the cytogenesis of macrophages from agranulocytes (lymphocytes and monocytes) as well as from reticulo-endothelial cells, W. H. Taliaferro and Mulligan (1937) proposed the term, lymphoid-macrophage system. This term includes the mononuclear exudate cells, or Maximow’s poly- blasts, which form the cellular exudate in inflammation. ANTIBODIES AND ANTIGENS INVOLVED IN IMMUNITY Infective organisms, derivatives of them, or other foreign, colloidal, protein materials can generally act as antigens. When an antigen is intro- duced parenterally into an animal, it calls forth a substance in the blood of the animal, known as an antibody, which will react with the antigen specifically 72 vivo and generally im vitro and is passively transferable. Such an antibody is often termed an immune antibody, to differentiate it from natural antibodies, which sometimes exist in blood without im- munization. Serum from the blood of an animal containing an antibody is known as antiserum. Some antibodies or antiserums, in addition to reacting with their specific complete antigens, may also react with iso- lated carbohydrate or lipoid parts of the antigen i” vitro. These sub- stances have been differentiated from true antigens by the terms haptenes or partial antigens, since they generally do not stimulate the production of antibodies 72 vivo. Both complete antigens and haptenes have been isolated in high states of purity. Antibodies result from antigenic stimu- lation and are metabolic products of cells. Thus the amount of circulat- ing antibody is often decreased by removing the spleen, which is rich in cells of the lymphoid-macrophage system, or by filling the macro- phages along the blood stream with colloidal or particulate matter. Furthermore, antibodies undoubtedly represent definite substances which are closely associated with the globulin fraction of the serum. It is, how- ever, impossible to say at present whether they are actually globulins and, if they are globulins, whether they are new globulins or the regular 838 IMMUNOLOGY serum globulins slightly modified (see Wells, 1929; Marrack, 1938). Antibodies are variously named according to the effect produced when mixed with antigen. The antibody is a precipitin if it produces a pre- cipitate on mixing with a soluble antigen (precipitinogen). It is an agglutinin if it induces clumping or agglutination of cellular antigens (agglutinogens), such as Bacteria, Protozoa or blood cells. It is an opsonin if it sensitizes the antigen and makes it more readily ingested by phagocytes. It is a lysin if it sensitizes cellular antigens so that, on the addition of a thermolabile component of normal serum known as com- plement or alexin, the cell undergoes death and lysis, during which many of its internal substances diffuse through the cell membrane. In both of the foregoing processes, the antigen is first sensitized by antibody. After such a preparation, it is then lysed by intracellular enzymes (phagocyto- sis) or by extracellular enzymes (lysis) (see Wells, 1929). The antibody is known as an antitoxin if it neutralizes the biological action of a toxic antigen (exotoxin). Definite antitoxins and exotoxins have not been demonstrated in protozoan infections (see W. H. Taliaferro, 1929). An increasing number of immunologists accept the unitarian view- point that the introduction into the body of a single antigen results in the formation of a single antibody, which is an agglutinin, precipitin, and so forth, according to the nature of the antigen or the particular method of testing. This does not mean that a complex cell will not contain many different antigens. Furthermore, a given antibody in a specific infection may act as one type of antibody, and not as another, because of the posi- tion of various antigens on or within the cell (see Topley, 1935). Finally, an antibody-like substance is known as ablastin if it inhibits the reproduction of organisms when mixed én vivo. So far, it has been demonstrated only for certain nonpathogenic trypanosomes. Like other antibodies, it is associated with the globulin fraction of serum and is passively transferable, but differs from them in that it has no im vitro affinity for its specific antigen. In the latter respect, it appears to re- semble certain nonabsorbable antibodies reported in bacterial, virus, and worm infections. The rdle of antibodies is studied by #7 vivo protective (passive trans- fer) and curative tests and by /z vitro studies involving various serologi- IMMUNOLOGY 839 cal tests. Protective and curative tests differ only as regards the time of injecting the serum and organisms. In protective tests the serum is 1n- jected at the same time (or not more than a day before or after) the organisms are injected, whereas, in curative tests the serum is injected some time after the organisms have been injected and generally when they can be found in some particular part of the body. The effect of the latter may obviously be more variable, since the organisms already have a start in the body and may be more difficult to check. THE CELLULAR AND HUMORAL ASPECTS OF IMMUNITY The reader is referred to Maximow (1927b) for a general descrip- tion of the histogenesis of the inflammatory and defense reactions, to Aschoff (1924), Jungeblut (1930), Gay (1931), and Jaffé (1931, and 1938) for a general consideration of the function of cells and, in par- ticular, of macrophages; and to Linton (1929), W. H. Taliaferro (1929 and 1934), and W. H. Taliaferro and Mulligan (1937) for a specific consideration of the role of cells in protozoan immunity. The way in which the cells of the connective tissue, in particular the granulocytes and the cells of the lymphoid-macrophage system, are in- volved in local defense can be seen during the inflammation which fol- lows the introduction of foreign material into connective tissue of the skin. The heterophils migrate early from the blood vessels. Their number and activity depend upon the nature of the inflammatory stimulus and whether it is sterile or septic. They are generally not numerous in proto- zoan infections and soon disappear when the inflammatory material is bacteriologically sterile. Under sepsis, however, they continue to migrate from the blood vessels and to combat the invading organisms in many visible ways—by active phagocytosis and digestion, by the secretion of bactericidal and proteolytic ferments, and the like. They represent an important first line of defense since they are the most easily mobilized cells, but their functions are limited since they generally disintegrate within a few days, ordinarily are recruited only from the blood stream, i.e., do not multiply 7 sétv, and cannot develop into other cells of the area. The cells of the lymphoid-macrophage system are the most important 840 IMMUNOLOGY cells in local defense. The lymphocytes and the monocytes migrate from the blood vessels, as do the heterophils; but unlike the heterophils they are long-lived, may multiply in the tissues, may develop into macro- phages with phagocytic potencies, and from macrophages may progres- sively develop into fibroblasts with reparative functions. As macro- phages, they, together with the macrophages previously present in the area, actively phagocytose and digest certain invading organisms, remove cellular and other debris, and after the acquisition of immunity probably elaborate antibodies which aid in phagocytosis. When large bodies are present, the macrophages may fuse to form foreign body giant cells; when microdrganisms are indigestible, they may form giant cells around them, such as the epithelioid cells of the tubercle; or, when large areas are necrotic, they may surround the area, become transformed into fibro- blasts, and effectively wall it off. The fibroblasts, both those of the local area and those arising from macrophages, react slowly and probably play an active part only in the later stages of local inflammation during regenerative and reparative processes, the formation of scar tissue, and the walling off of foreign bodies. Several other cells may come into play, generally during late stages in the defense reaction. Of these, the eosinophils seem to play a part in the detoxification of foreign proteins and their disintegration products and are particularly prominent after the body has become sensitized to the proteins. Like the heterophils, they do not multiply and cannot de- velop into other cells of the area. Some investigators believe that the plasma cells are also associated with the detoxification of foreign ma- terials. They are not phagocytic, do not seem to have any developmental potencies, and proliferate rarely, if at all. The exact function of the basophils is unknown. Ordinarily, when the stimulant is distributed over a large part of the body, the reaction is designated as a general defense reaction, in contra- distinction to the local defense reaction just described, but as a matter of fact such distribution usually signifies that the stimulant is in the blood stream and is combated by macrophages of organs most closely associated with the blood, such as the spleen, liver, and bone marrow. In some cases, as in malaria, these general reactions can actually be con- sidered local ones in strategically placed organs (see W. H. Taliaferro, IMMUNOLOGY 841 1934). The same types of cells are involved, and the extent to which they are involved depends, as in other sites, upon the nature of the foreign material or infectious agent. The heterophils are often mobilized first, and the lymphoid-macrophage series shows the most pronounced histological changes, with the macrophages seeming to bear the brunt of the activity. Fibroblasts rarely come into contact with foreign material in the blood and are rarely active. Endothelial cells, although they come into contact with hematogenous material, show extremely little histologi- cal change or phagocytic activity. The foregoing account of defense reactions, involving the disposal of foreign material and tissue debris with eventual repair, is characteristic when either antigenic or nonantigenic materials are introduced into a normal animal, but certain quantitative differences are noted when anti- genic materials, including parasites, are introduced into an immune animal. These differences are chiefly due to antibodies and are specific. When antigens are introduced into the immune body, they are generally localized by agglutination if they are cells, or by precipitation if they are in solution, and are made more readily phagocytable by opsonification. Such localization and opsonification are particularly well seen in local reactions in the loose connective tissue. They are often limited to organs such as the spleen, liver, and bone marrow in general reactions, as is well-illustrated in malaria. In trypanosomiasis, these phases have not been completely studied, but ablastin (the reproduction-inhibiting anti- body) at least does not involve either localization or phagocytosis. When antigen and antibody meet in the tissues of an immune animal, not only do localization and opsonification of the antigen occur, but there is generally a much heightened inflammatory reaction (hypersensitivity, sometimes evidenced by a local reaction—the so-called skin test—when suitable amounts of antigen are injected intradermally). Provided this heightened inflammation is not so intense as to overwhelm the body, it represents a speeding up of the whole cellular response in the immune animal. In addition to the specific action of antibodies after the acquisi- tion of immunity, a residual increase in the number of cells of the lymphoid-macrophage system sometimes is seen at strategic sites, which results in a much more rapid mobilization of macrophages during im- munity. This is well illustrated in the spleen in malaria. 842 IMMUNOLOGY ROLE OF IMMUNE PROCESSES IN THE DEVELOPMENT OF PROTOZOAN INFECTIONS GENERAL METHODS The detailed considerations in the succeeding sections deal largely with the rdle of antibodies and cells in modifying the course of infection, together with such allied subjects as recovery, relapse, and immunity to super- and reinfection. The Protozoa offer the advantage of being large enough so that one can ascertain the effect of these processes in a way that is impossible with the smaller bacterial and virus invaders. This analysis has been further facilitated by selecting certain plasmodia and trypanosome infections in which practically all stages in the life cycle of the parasite are accessible for study (1.e., are more or less evenly distributed in the peripheral blood and are not localized in the deeper tissues ) . The course of these infections can be roughly indicated by changes in the number of organisms. Since, however, the number curve is the resultant of the number of parasites produced by reproduction and the number of organisms which die or are actually destroyed, the only deductions that can be drawn from it are that reproduction is going on if the numbers increase, although the rate may be actually decreasing; and that the rate of reproduction is being inhibited, or that the parasites are being killed, or that both activities may be operating, if the numbers remain constant or decrease. The rate of reproduction, if ascertained, however, in conjunction with number counts throughout an infection, will adequately indicate whether the host acquires a defensive mecha- nism, and, if so, whether it is directed toward inhibiting reproduction of the parasites, or destroying the parasites after they are formed, or both. All valid measures of the rate of reproduction so far devised, which are independent of the number of organisms killed, depend upon some measurement of size (since organisms usually grow before they repro- duce) or upon some determination of division forms. A direct measure can be devised for the plasmodia, which divide more or less synchro- nously; but an indirect measure has to be resorted to for the trypanosomes, since they neither divide synchronously nor can their fission rate be ascertained directly as can be done among the free-living forms. The particular criteria used are of course more or less arbitrarily selected IMMUNOLOGY 843 and, to be satisfactory, necessitate a nice adjustment between the validity of the criteria selected and the time required to make measurements. The most convenient measure of the rate of reproduction of the plasmodia so far devised consists in ascertaining the length of the asexual cycle directly (i.e., the time it takes for a young merozoite to become a mature schizont and divide into the next generation of young mero- zoites), in conjunction with the number of merozoites produced. Thus the percentage of segmenters are computed in samples of 50 to 100 parasites from stained blood smears made every 4 to 12 hours whenever parasites can be found. A regularly recurring percentage of segmenters, considered arbitrarily, for example in P. brasilianum, to have 5 or more nuclei by W. H. Taliaferro and L. G. Taliaferro (1934a), indicates a constant rate of reproduction, provided the number of merozoites pro- duced by each segmenter remains approximately constant. The most satisfactory measure of the rate of reproduction among the trypanosomes consists in comparing the percentage of division forms in samples of 50 to 100 forms from stained blood smears made every 6 to 24 hours throughout an infection. Among the pathogenic trypanosomes, in which dividing forms are numerous, division forms may simply be considered as those with some duplication of parts (see Krijgsman, 1933), but among the nonpathogenic Trypanosoma lewisi and T. duttoni, in which actual dividing forms are rare, division forms are considered to be dividing forms plus short young forms 25 1 or less in length (see W. H. Taliaferro and Pavlinova, 1936). The higher the percentage of division forms among the trypanosomes is, the higher the rate of reproduction. Valid measures have also been devised for malaria by L. G. Taliaferro (1925), G. H. Boyd (1929a), Lourie (1934), and Mulligan (1935); and for trypanosomes by Robertson (1912), Krijgsman (1933), and W. H. Taliaferro and L. G. Taliaferro (1922). The last authors’ coefh- cient of variation method depends upon the fact that, within certain limits, the variability in total length increases proportionately as the young and growing forms resulting from reproduction increase. MALARIA This analysis applies only to those malarial species which parasitize erythrocytes and not to such species as Plasmodium elongatum (Huff and Bloom, 1935), which undoubtedly infect other blood and connective 844 IMMUNOLOGY tissue cells. We have omitted from discussion the so-called exo-erythro- cytic and fixed tissue cell stages described in the life cycles of some plasmodia because of the lack of agreement which now exists among malariologists as to their nature (Boyd and Coggeshall, 1938, review). The course of untreated malarial infections has been most thoroughly studied in avian malaria. This work commenced with the careful statis- tical studies of the Sergents (1918) and was extended by Ben Harel 1923), L. G. Taliaferro (1925), G. H. Boyd (1929a), Hartman (1927), Gingrich (1932), Lourie (1934), and others. Treated cases will not be considered in this analysis because treatment itself has been shown greatly to affect the length of the asexual cycle and the number of merozoites produced (G. H. Boyd, 1933; G. H. Boyd and Allen, 1934; Lourie, 1934; and Boyd and Dunn, 1939). Infections with P. cathemerium in canaries are extremely stereotyped and therefore afford an excellent base line for considering the so-called benign infections, which tend to recover and which constitute the ma- jority of malarial infections. When a few parasites are injected into a bird, an incubation period follows during which no parasites can be detected in the peripheral blood. As soon as parasites appear, they increase from day to day at a constant rate (the intersporulation death of parasites will be taken up later), according to a geometrical progres- sion, until sometimes as many as half of the red blood cells are infected (acute rise of infection). At this point, if the bird does not succumb to the infection, recovery is initiated and is manifested by the rapid disap- pearance of many of the parasites from the peripheral blood (crisis). Following the crisis, parasites remain few in number, but may fluctuate to some extent (developed infection). Sooner or later, the number of parasites is reduced to a level at which they can no longer be detected in peripheral blood films; but a few persist, since transfers of large amounts of blood will infect other birds. This latent period may last for several years, but it may be interrupted periodically by spontaneous or induced relapses (much rarer in P. cathemerium than in many other species), which are similar to, though generally quantitatively less than the acute rise of the initial infection, and which are terminated by a crisis. Occasionally, such a relapse may be fatal. By Hegner’s (1926) terminology such an infection is divided into prepatent (incubation), patent (acute rise, crisis, and developed infection), and subpatent IMMUNOLOGY 845 (latent) periods, with second and third patent periods representing first and second relapses and crises. When this infection was analyzed, it was found that the basic rate of reproduction remains comparatively constant, whenever parasites are WINMonkey 119 ——O— No of Parasites = —e— % of Segmeriors C7 ~-*-— Temperature a) fad a> ay S z |= orgs lace 0s 50 a: 54% ve) = be 1034 -<604 2 | = Se E iS 8 ~ = E = = @ — a P10-} 407 -2 80 &. 5 = [: | = oe Ba @ 21 4 @ = = | QT 0- 0 Aa ie . a 4 % ate FIGURE 188. The changes in number of Plasmodium brasilianum and the percentage of segmenters during the acute rise and crisis of the infection in Central American monkey 119. A natural parasiticidal immunity is operative, as evidenced by the inter- and intrasporulation death of parasites; and as acquired immunity is developed at the crisis, further parasiticidal effects are operative, as evidenced by the tremendous death of parasites. The rate of reproduction is temporarily affected, as evidenced by the irregular percentage of segmenters. Had the animal lived, reproduction would have resumed its normal rate, as ascertained from the study of other monkeys similarly infected. (From W. H. Taliaferro, 1932.) demonstrable in the blood, since the schizonts produce between ten and fifteen and a half progeny (merozoites) continuously and produce them every twenty-four hours. (This statement is relatively true, since there is no prolonged inhibition of reproduction. Temporary deviations and fluctuations do occur, however. Thus, Boyd and Allen [1934] and Boyd [1939] found that the number of merozoites produced decreases as the 846 IMMUNOLOGY crisis approaches and then rises thereafter.) Furthermore, since parasites reproduce at a high rate whenever they are found, it seems reasonable to assume that they reproduce at a high rate during latency, when they cannot be found in sufficient numbers to study. This assumption is in accord with the view held by Ross (1910, review), Bignami (1910), James (1913), and others. Essentially the same results were obtained from the studies of P. brasilianum in Panamanian monkeys, except that during the crisis of the initial infection (W. H. Taliaferro and L. G. Taliaferro, 1934a) one asexual cycle may deviate or be retarded for a day or two. This parasite shows a quartan periodicity. Thus in Figure 188 the percentage of segmenters sharply increases and decreases every third day (4/28, 5/1, 5/4) until at the time the number crises is reached (5/7) the percentage of segmenters does not rise as high as before and does not decrease as precipitously. The other so-called benign malarias seem to be similar, as far as the data on the following species go: P. cynomolgi, in both Szlenus rhesus and S. irus; P. knowlesz, in S. irus (Sinton and Mulligan, 1933a, 1933b; Mulligan, 1935); and P. vivax and P. malariae in man. The rapidly fatal infection of P. knowles in S. rhesus is similar to the acute rise of benign infections without a crisis. These statements do not mean that temporary derangements of the cycle may not occur during the crisis, as in P. brasilianum,; or after treatment with quinine, as in P. cathemerium (previously cited); or after changes in host habits, as has been shown to occur in both the latter species (L. G. Taliaferro, 1928; G. H. Boyd, 1929b; W. H. Taliaferro and L. G. Taliaferro, 1934b; and Stauber, 1939). Since there is no prolonged inhibition of reproduction, the number curve can be interpreted chiefly in terms of parasiticidal effects. In other words, the number of parasites after each asexual cycle should increase by the number of progeny in each mature schizont (minus the number of merozoites which develop into sexual forms), provided no death of parasites occurs. The geometrical rate at which P. cathemerium increases does not, however, account for all the progeny produced. They evidently die at all stages of growth and segmentation (L. G. Taliaferro, 1925; Hartman, 1927; Hegner and Eskridge, 1938) and die at a greater rate during the latter part of the acute infection than during the early part (Boyd, 1939). Hegner and Hewitt (1938) and Hewitt (1938) sug- IMMUNOLOGY 847 gested that their death is due to the destruction of multiple infected red cells. It is interesting that in 1888 Golgi noted that malaria would always be progressive until pernicious symptoms were evident, if the parasites arriving at maturity every two days in tertian or every three days in quartan malaria should complete their life cycle. W. H. Taliaferro and L. G. Taliaferro (1934a) found that out of an average of 9 progeny produced by P. brasilianum, never more than 1.5 complete their develop- ment and of the 7.5 which fail to complete their development, about 6 fail to get into new cells and 1.5 die or are killed during intracorpuscular growth. Thus, in Figure 188 on the morning of May 1 there were 60 parasites per 10,000 red blood cells. That evening after sporulation, there were 221, an increase of 3.66 times instead of 9 times. During the subsequent 2.5-day period of growth and division, about two-thirds of these were destroyed, so that on the morning of May 4 there were only 83 per 10,000 red cells. Similar data are furnished by Brug (1934) and W. H. Taliaferro and Mulligan (1937), who worked on infec- tions with P. knowlesi and P. cynomolgi; and by Pijper and Russell (1925, quoted by Sinton ef a/., 1931), by Rudolf and Ramsay (1927), by Sinton et al. (1931), and by Lowe (1934), all of whom worked on one or both of the tertian and quartan malarias of man. Knowles and Das Gupta (1930) believed that the destruction of parasites takes place only during the free merozoite stage. Examination of their tables, how- ever, shows that the infection they studied was made up of several broods of parasites, the sporulation of one of which would obscure a decrease of parasites during the intrasporulation period of another brood. The constant rate of death of large numbers of parasites during the initial part of the infection is a manifestation of natural immunity and represents the suitability of the hosts’ blood to the malarial organism. The crisis, on the other hand, represents the beginning of the immune reaction, when more progeny die than survive and the infection therefore declines. Thus the crisis in Figure 188 takes place after the sporulation of May 4 and between May 5 and May 7, as shown not only by the conspicuous death of parasites during the intersporulation period of May 5 and May 6, but by the relatively slight increase of parasites during the sporulation on May 7. This infection, which was the most acute encountered, caused the death of the monkey on May 9. The temperature 848 IMMUNOLOGY curve is extraneous to the present discussion, but is interesting because of the stiletto-like peaks it shows at each sporulation. Some work on the infection after the crisis has been done on human malaria by Bohm (1918), Knowles and Das Gupta (1930), and Sinton ef al. (1931). Throughout the developed infection, an equilibrium is established between the number of parasites killed and the number produced. Later, the defensive factors are usually successful in suppressing the parasites arising by reproduction, so that latency ensues. Latency may last for years, but may be interrupted by relapses. Although there is no unanimity of opinion on the mechanism of relapse, the best evidence indicates that it represents simply the removal of the defensive factors, so that the parasites, which are continuously reproducing at a constant rate, reaccu- mulate in the blood. The relapse may be fatal, or the defensive factors may again materialize and successfully suppress it. Accordingly, the severity of the relapse depends upon its extent and upon the length of time the defensive factors are removed. These statements are substanti- ated by work on P. cathemerium and P. brasilianum, references for which have already been given. Koch (1899) believed that immunity persisted after complete re- covery, but Wasielewski (1901) suggested, and subsequent workers have supported the conclusion, that parasites remain in small numbers for longer periods than was at first supposed and that this latent infection accounts for the long continued immunity (see Thomson, 1933, review; Chopra and Mukherjee, 1936; Sergent, 1936). More recently, Nauck and Malamos (1935) and Coggeshall (1938) have shown that a certain amount of immunity is retained after complete cure of P. knowlesi. Acquired immunity to malaria can be demonstrated not only by the crisis, recovery from initial infection, and recovery from relapse, but by superinfection. Such immunity is usually species-specific or even strain- specific in human, simian, and avian infections. A series of investigators, beginning with Koch (1899), have worked on this aspect of the subject (for literature and for especial work, see W. H. Taliaferro and L. G. Taliaferro, 1929a, 1934c; Gingrich, 1932; Mulligan and Sinton, 1935; M. F. Boyd ef al., 1936; Manwell, 1938; Redmond, 1939; and Manwell and Goldstein, 1939). The work on human malaria has been recorded chiefly as a result of the use of therapeutic infections in the treatment of paresis (see Mulligan and Sinton, 19332). W. H. Taliaferro and IMMUNOLOGY 849 L. G. Taliaferro (1929a, 1934c) intravenously superinfected birds and monkeys with such large numbers of parasites that they could study the superinfection quantitatively. They found that in immune animals the parasites, after reinfection, begin to be removed at once, with the same effectiveness that they are removed at the time of the crisis in initially infected animals, and that in monkeys the asexual cycle occasionally ex- hibits a transitory delay, accompanied by the production of fewer mero- zoites. In other words, superinfection, since it occurs in an effectively immune animal instead of in an uninfected animal, has no incubation period or acute rise, but begins at once with a crisis and proceeds imme- diately to latency. The problem now arises: what is the mechanism whereby the plas- modia are removed throughout the course of the infection? The death of all of the parasites, whether it takes place before, during, or after the crisis (i.e., during natural or acquired immunity), is associated with phagocytosis by macrophages, chiefly of the spleen, liver, and bone marrow. Other cells play insignificant rdles. Furthermore, the macro- phages phagocytose free parasites, parasitized red blood cells, and resi- dues of parasites such as malarial pigment and uninfected red cells, which are probably injured by the infection. Malarial pigment is the most indigestible part of the parasite red-cell complex, since it is often found in macrophages months after all other vestiges of the parasites have disappeared. Large monocytes containing malarial pigment may be found in the peripheral blood, especially during the crisis and thereafter. A review of the literature covering the considerable amount of work done on this aspect of the subject will be found in W. H. Taliaferro and Mulligan (1937) and involves direct evidence from necropsy findings and indirect evidence from splenomegaly (see Stratman-Thomas, 1935; Coggeshall, 1937; Afridi, 1938) and splenectomy and blockading procedures. Although phagocytosis has been known for years to occur in malarial infections, Cannon and W. H. Taliaferro (1931) and W. H. Taliaferro and Cannon (1936) were the first to study infections and superinfec- tions at closely spaced intervals and to correlate phagocytosis in detail with the course of the infection and with immunity. The rate of phago- cytosis during natural immunity, i.e., before the crisis, increases as the number of parasites increases, but is always comparatively sluggish (PI. 850 IMMUNOLOGY 1, Figs. 1 and 2). When the parasites disappear from the peripheral blood at the time of the crisis, they are not at first phagocytosed, but are filtered out and concentrated in the Billroth cords of the spleen (Pl.#1, Fig. 3). This initial manifestation of acquired immunity probably repre- sents a localization of the parasites due to an antibody, which may be an agglutination, as demonstrated 77 vitro in P. knowlesi by Eaton (1938), or an attachment of parasites to the macrophages, as found in cultures of P. falciparum by McLay (1922). In a few hours or days the con- centrated parasites are ingested in great numbers by the macrophages (Pl. 2, Figs. 1 and 2), owing undoubtedly to an opsonification of the parasites and parasitized red cells by antibody. This antibody probably accounts for the protective property of serum taken from latent infections in avian malaria (Findlay and Brown, 1934), in human malaria (Soti- riadés, 1917; Kauders, 1927; Neumann, 1933; and Lorando and Soti- riadés, 1937), and in simian malaria (Coggeshall and Kumm, 1938; Coggeshall and Eaton, 1938). The fact that protective antibodies have not always been found (W. H. Taliaferro and L. G. Taliaferro, 1929b and 1934b) led W. H. Taliaferro and Cannon (1936) to suggest that antibodies are produced locally in sufficient quantities to be operative in the specific organs 7m stu, but not in sufficient quantities to be easily demonstrated in the peripheral blood. A day or two after concentration in the Billroth cords, the parasites are quickly digested except for the ma- CAPTION FOR PLATE ON FACING PAGE Plate 1. Sluggish phagocytosis of Plasmodium brasilianum by macrophages in control of the American monkeys during the acute rise of the malarial infection (Figs. 1 and 2) and the concentration of P. brasilianum in Billroth cords of the spleen at the initiation of the crisis (Fig. 3). & 1450. (From W. H. Taliaferro and P. R. Cannon, “Cellular Reactions during Primary Infections and Superinfections of Plasmodium Brasilianum and Panamanian Monkeys,” Journal of Infectious Diseases, LIX [ July-August, 1936], 72- 125.) Figure 1. A Kupffer cell in the liver containing a parasite, an unparasitized erythrocyte, and four small masses of malarial pigment. Acute rise of infection. Figure 2. Two macrophages in a Billroth cord in the red pulp of the spleen, containing a small number of residues of parasites, red cells, and malarial pigment. Acute rise of infection. Figure 3. Large numbers of parasitized erythrocytes concentrated in Billroth cords of the spleen. Note that parasites are absent from the venous sinus. Also note that the macrophages in the Billroth cords show the sluggish phagocytosis characteristic of the acute rise and that the littoral cells lining the sinus are not phagocytic. Initiation of crisis of infection. Se ue oS @ Billroth cord Venous Sinus Billroth cord PLATE | Par Mac M Lom 5 tym Mae Pig PLATE il IMMUNOLOGY 851 larial pigment (PI. 2, Figs. 3-5, which are from monkey 119; see also Fig. 188). The pigment is digested within a few months, as may be seen by a study of animals in which relapses or superinfections do not inter- vene. The various tissues gradually return to their normal histological appearance, but as long as immunity lasts they retain their ability to react more quickly than the tissues of nonimmune animals. These data raise the interesting immunological question as to whether the death of the parasites before the crisis and that during or after the crisis are due to the same factors. In other words, is acquired immunity simply an enhancement of the high-grade natural immunity present from the beginning, or is it due to an entirely new set of factors superimposed on the natural condition? Certain facts tend to indicate that the two mechanisms are essentially different. In the first place, during natural immunity, all evidence indicates that phagocytosis is nonspecific, and there is some evidence that only those parasites are phagocytosed which are moribund or otherwise abnormal (Hartman, 1927; Gingrich, 1934; Hegner and Hewitt, 1938; Hewitt, 1938). In the second place, Gingrich found that the injection of large numbers of red cells breaks down CAPTION FOR PLATE ON FACING PAGE Plate 2. Intense phagocytosis of Plasmodium brasilianum by macrophages in Central American monkeys at the height of the crisis of the malarial infection (Figs. 1 and 2) and malarial pigment, the residue of parasites after the intense phagocytosis, in the macrophages shortly thereafter (Figs. 3-5). X 1450. (From W. H. Taliaferro and P. R. Cannon, “Cellular Reactions during Primary Infections and Superinfections of Plasmodium Brasilianum and Panamanian Monkeys,” Journal of Infectious Diseases, LIX [July- August, 1936], 72-125.) Figure 1. A Kupffer cell in the liver containing many recognizable parasitized and unparasitized red cells, and a monocyte (Mon.) containing malarial pigment. Crisis of infection. Figure 2. Two macrophages in the Billroth cord of the spleen containing many recog- nizable parasitized and unparasitized red cells. Crisis of infection. Figure 3. A Kupffer cell in the liver containing malarial pigment. Two days after the crisis. Figure 4. Two macrophages in the bone marrow containing malarial pigment. Two days after the crisis. Figure 5. Four macrophages in the Billroth cord of the spleen containing malarial pigment. Two days after the crisis. Figures 3, 4 and 5 were all taken from monkey 119 (see text Figure 188). Note that the Kupffer cell of the liver contains intermediate amounts of malarial pigment between that found in the macrophages of the spleen and of the bone marrow. 852 IMMUNOLOGY acquired resistance to P. cathemerium, but, even when pushed to the maximum, does not affect natural resistance. This work, however, is open to the possible criticism that the large amounts of pigment in the macro- phages after the crisis, which are not there before the development of immunity, may simply augment the blockading doses to produce the observed effect. In the third place, as pointed out previously, accumulat- ing evidence indicates that the greatly superior mechanism for disposing of parasites associated with acquired immunity is highly specific and is probably associated with an antibody. The limitation of phagocytosis to the macrophages of the spleen, liver, and bone marrow is probably a question of opportunity, as such macro- phages are advantageously placed where they can remove material from the blood stream. The so-called general immunity in malaria, therefore, is actually a local reaction in strategically placed organs (W. H. Talia- ferro, 1934). It should be noted, however, that in overwhelming infec- tions the parasites may be so numerous that secondary complications, such as stasis of the blood, clogging of the capillaries, and hemorrhage may occur in various organs. In such an event the macrophages of the brain, lungs, suprarenals, and kidneys, as weil as those of the bone marrow, liver, and spleen, may actively phagocytose malarial material. CAPTION FOR PLATE ON FACING PAGE Plate 3. Portions of a venous sinus and Billroth cords in the spleen of an uninfected rhesus monkey (Fig. 1) and comparable portions from a rhesus monkey during the late acute rise of an infection with Plasmodium cynomolgi (Fig. 2). X 1400. (From W. H. Taliaferro and H. W. Mulligan, “Histopathology of Malaria with Special Reference to the Function and Origin of the Macrophages in Defence,” Indian Medical Research Memoirs, No. 29 [May, 1937}, pp. 1-138.) Figure 1. The normal constituents of the venous sinus are chiefly the cells of the circulating blood (lymphocytes, granulocytes, and red cells), and those of the Billroth cords are also cells of the circulating blood with a greater proportion of large lymphocytes and, in addition, reticular cells with indeterminate cytoplasm and slightly phagocytic macrophages. Figure 2. The additional constituents of the venous sinus and of the Billroth cords in the spleen of a monkey during the late acute rise of an infection of P. cynomolgi are parasitized red cells and many transitional cells (polyblasts) between lymphocytes and macrophages, containing malarial pigment. The progressive hypertrophy of lymphocytes into macrophages is shown by nuclear changes and by increased amounts of cytoplasm in the lymphocytes (see especially Med Lym 1, which also contains two small granules of malarial pigment) and by further nuclear changes, increased size, and increased phagocytic activity in the polyblasts (see especially polyblasts 1 through 5). Throughout this tran- sitional series, phagocytosis increases approximately with the size of the cell. Monocytoid lymphocyte Sinus Plasma cell Large lymphocyte Sinall lymphocyte Heterophil Macro ye Dew put de 7 BEY oS ®) 5, 24 : Sar Vy So a OA fy 7. 22, , re ; OT hex has ~~ ri Og SP y a ~ @ f oe z has « Sg a I eminent tie Ee - ss Large lymphocyte G Polybl.S Parasite Polybl.L Polybl.2 Dotybl.5 5 Doty. 4 PLATE III Transtttonal Zone Marginal one of Small lymphocytes Secondary nodule Large lymphocyte ~ = £ | Rettcular cell Reticular Mitoses in Macrophage cell A Lymphocytes with tnclusions PLATE IV IMMUNOLOGY 853 The fact has been stressed that the greatly enhanced phagocytic activity of individual macrophages during acquired immunity is specific and is probably related to antibodies. In addition, there is a nonspecific increase of macrophages at strategic points, particularly in the spleen and the bone marrow, due to their cytogenesis from lymphocytes through poly- blast stages and from histogenous macrophages. The latter source is acknowledged by Bruetsch (1927, 1932a, 1932b) and others, and both sources have been demonstrated by W. H. Taliaferro and Cannon (1936) and W. H. Taliaferro and Mulligan (1937). In fact, the chief source of new macrophages is from lymphocytes (Pl. 3). The lympho- cytes themselves arise by mitotic proliferation (Pl. 4). This is a promi- nent part of the so-called lymphoid hyperplasia of the spleen and occa- sionally of other organs, if the malarial infection is long drawn out. In passing it may be mentioned briefly that the parasites destroy red blood cells in large quantities, flood the blood plasma with foreign mat- ter such as corpuscular debris, free malarial parasites and malarial pig- ment, sometimes block the capillaries and damage various tissues, espe- cially the spleen and liver. All these losses and destructions are made good by various nonspecific hyperplastic and reparative activities of the host. The pathological and regenerative changes have been extensively studied, especially in human malaria (see W. H. Taliaferro and Mulli- gan, 1937, for a review of the literature). The foregoing results indicate that acquired immunity against malaria largely involves parasiticidal effects, with no pronounced inhibition of the rate of reproduction for extended lengths of time. The parasiticidal effects can be correlated with phagocytosis. Phagocytosis is sluggish dur- CAPTION FOR PLATE ON FACING PAGE Plate 4. A nodule in the white pulp of the spleen during lymphoid hyperplasia as- sociated with the late acute rise of Plasmodium cynomolgi in a rhesus monkey. (From W. H. Taliaferro and H. W. Mulligan, “Histopathology of Malaria with Special Reference to the Function and Origin of the Macrophages in Defence,” Indian Medical Research Memoirs, No. 29 [May, 1937}, pp. 1-138.) Figure 1. This activated splenic nodule due to malaria is slightly enlarged, shows a pronounced transitional zone and a markedly active secondary nodule, in which occur swollen phagocytic reticular cells and mitoses, among many lymphocytes and a few reticu- lar cells. & 180. Figure 2. A detail of the upper portion of the secondary nodule shown in Figure 1 which consists mainly of medium lymphocytes and swollen, slightly phagocytic reticular cells. Reticular cell A is beginning to mobilize. X 1015. Figure 3. A detail of the lower portion of the secondary nodule shown in Figure 1, which consists mainly of lymphocytes many of which are dividing. x 655. 854 IMMUNOLOGY ing the acute rise of the infection and is greatly enhanced at the time of the crisis, when immunity, which is probably associated with the elabora- tion of antibodies, is developed. Thereafter the infection progressively subsides, unless the immunity is lowered. If the immunity is lowered, the parasites, because their reproduction has not been inhibited, reac- cumulate in the blood until immunity again develops and becomes operative. LEISHMANIASIS The course of kala azar cannot be studied and analyzed as was done in the case of malaria because its causative agent, Lezshmania donovani, is not accessible for study; but it is of interest here because L. donovani lives in the macrophages themselves, as has been shown by Christophers (1904), Meleney (1925), Hu and Cash (1927), and others. It not only lives in the macrophages of the spleen, liver, bone marrow, and intestinal wall, and, in extreme cases, the macrophages of almost all organs and tissues, but proliferation of the macrophages constitutes the chief characteristic of the disease. For a review of the literature, see C. J. Watson, 1928; Linton, 1929. See the former reference also for a seem- ingly similar condition in the little-known histoplasmosis. The parasites, therefore, instead of being digested, find the cytoplasm of the phagocytes a suitable medium in which to grow and multiply. Splenectomy should be particularly illuminating in trying to decide whether the macrophage system is valuable, imperfect as it is, as the only defense the body has; or is deleterious, as being the most suitable location for the parasites. Some work on kala azar has been done (see Laveran, 1917), but further systematic experimental work on animals should prove valuable. The fact that the disease is so often fatal indicates that reproduction of the parasites is continuous, as in malaria. Immunity, nevertheless, is devel- oped in approximately 10 percent of the infections, but it is not apparent whether the suppression of the infection is predominantly due to an increase in the ability of the macrophages to digest the parasites or to an inhibition of reproduction of the parasites. Oriental sore, a cutaneous leishmaniasis caused by L. tropica, on the other hand, usually spontaneously heals and confers an immunity to reinfection. Sections of the skin at the site of the sores often show pro- nounced local accumulations of macrophages. As in the case of the small IMMUNOLOGY 855 percentage of human beings recovering from kala azar, it 1s impossible to decide from the available data whether suppression of the infection is predominantly due to a destruction of the parasites or to an inhibition of their reproduction. The fact that immunity is more or less generalized indicates, however, that some humoral principle is involved. NONLETHAL INFECTION WITH THE Trypanosoma lewist GROUP OF TRYPANOSOMES The trypanosomes differ from the plasmodia in that they live in the blood stream and do not infect the red blood cells. Some are pathogenic. Others are nonpathogenic. Among the latter is a large group of trypano- somes which produce nonlethal infections in rodents, are morphologi- cally identical or similar to T. /ew7si of the rat, and are differentiated almost entirely by their specificity for their rodent hosts. Of these, T. lewisi of the rat and T. duttoni of the mouse have been extensively studied and furnish the basis for the conclusions in the following discus- sion. The number curve of T. /ew7si in the rat when a few parasites are injected, as shown by Steffan (1921), W. H. Taliaferro and L. G. Taliaferro (1922), W. H. Taliaferro (1924), and Coventry (1925), starts, as does a malarial infection, with an incubation period and an acute rise, until the trypanosomes may reach 300,000 or more per cu. mm. Then there is a crisis between the eighth and the fourteenth days, during which most of the parasites are destroyed. Those that remain continue to live in the blood for some time (varying from several weeks to several months), until they are removed either gradually or suddenly. Thereafter they are not found in the blood, and relapses seldom occur, but a few may persist, as ascertained by relapses which sometimes ensue after splenectomy and blockade with India ink, or after other conditions which lower the immunity of the host. Whether a few always persist cannot be determined from the available data. The rat, however, 1s immune to reinfection for long periods, as was first shown by Kanthack et al. (1898). Fatal infections of T. /ewzsi in young rats were first reported by Jiirgens in 1902 (see also W. H. Brown, 1914; Herrick and Cross, 1936; Duca, 1939; Culbertson and Wotton, 1939). They are often complicated by a concomitant occurrence of either or both of the 856 IMMUNOLOGY following: a subnormal condition of the rats or infections, such as Bartonella or paratyphoid. In any case Culbertson and Wotton (1939) have found that fatal infections develop in rats in which the content of ablastin is low. Early investigators (Rabinowitsch and Kempner, 1899; von Wasie- lewski and Senn, 1900; especially Laveran and Mesnil, 1901; MacNeal, 50: Rat 105 S a Coefficient of variation. % of Division Forms Incubation period _- _ _-=-—@=— — — Thousands of trypanosomes per cmm. of blood Day of Infection FIGURE 189. The changes in number of Trypanosoma lewisi and the coefficient of variation and percentage of division forms during the course of infection in rat 105. As acquired immunity develops, the rate of reproduction is inhibited by ablastin, as evidenced by the low coefficient of variation and low percentage of division forms be- ginning at location 1, and the parasites are killed by trypanolysins operative at locations 2 and 3. Whether, in addition, natural immunity operates has not been ascertained. (From W. H. Taliaferro, 1924; division forms added.) 1904; W. H. Brown, 1915) were convinced by their microscopical studies that T. Jewis7 reproduces only during the first few days in the rat, after which the trypanosomes live in the blood as nonreproducing adults. This conclusion has been substantiated by W. H. Taliaferro (1924), Coventry (1925), and Regendanz and Kikuth (1927). Thus in Figure 189 the coefficient of variation of the total length of the trypanosomes and the percentage of division forms, each of which, as will be recalled, measure the rate of reproduction, are high when trypanosomes appear IMMUNOLOGY 857 in the blood on the fourth day after infection and then drop precipitously until on the tenth day of the infection they reach a low level (at location 1, in Figure 189), from which they do not thereafter deviate. Provided such a large number of adult trypanosomes are injected intravenously that they appear in the blood and can be studied immediately, the coeffi- cient is low on the first day and rises precipitously, as may be seen in control rat 980 in Figure 190. The inhibition of reproduction, as will be shown later, is due to the development of an acquired immunity involving an antibody which has been called ablastin (W. H. Taliaferro, 1924, 1932). The rate of reproduction of T. /ew7s7 is similarly retarded and inhibited when grown in an abnormal host, the guinea pig, as ascertained by Coventry (1929). Essentially the same results were found for T. duttoni in the mouse, except that the rate of reproduction is never as high and is not as completely inhibited, according to W. H. Taliaferro and Pavlinova (1936) and W. H. Taliaferro (1938). Hence the trypanosomes are never as numerous during the acute rise and may increase slightly in numbers during the first part of the developed infec- tion. T’. zowensis in the striped ground squirrel, as described by Rouda- bush and Becker (1934), closely parallels the development of T. duttonz. Since T. nabiasi in its natural host, the rabbit, increases in numbers only during the first few days of the infection and thereafter does not show division forms, as reported by Kroé (1936), the rate of reproduction of this trypanosome may also be inhibited. The question arises: Is there a natural immunity during the acute rise of these infections? For it must be realized that in spite of the rise in numbers a constant percentage of the parasites formed may be being killed as was demonstrated in malaria. There are two ways of demon- strating natural immunity. The first applies to the death of the organisms and can be used only in such infections as malaria, in which it can be demonstrated that all of the progeny formed do not survive. This is impossible in the trypanosome infections, in which reproduction is not synchronous and in which no method of ascertaining the total number of progeny produced has so far been devised. The second method applies not only to the death of the organisms, but also to the rate of reproduc- tion, and involves various procedures such as comparisons of the same species in various hosts and splenectomy combined with blockade. Posi- tive experiments of this kind will indicate the existence of a natural im- 858 IMMUNOLOGY munity, but, since natural immunity may be due to many factors, the only conclusion that is warranted from negative experiments is that the experimental method used did not disclose any natural immunity. With these facts in mind, we may conclude the following: Guinea pigs seem to possess a high natural parasiticidal immunity to T. /ew7s7, since T. lewisi hardly increases in numbers at all in spite of the fact that it goes through the same reproductive cycle as it does in the rat. Mice seem to possess a high natural ablastic immunity to T. duttoni, since T. duttoni in splenectomized and blockaded mice reproduce at a higher rate than in normal mice. Rats have not been shown to possess any natural im- munity against T. Jew7s7. The drop in numbers at the crisis (at location 2, in Fig. 189) repre- sents the acquisition of a trypanocidal response on the part of the host, in addition to the ablastic effect, since if only the latter were present the numbers would remain constant. Also, the disappearance of the trypano- somes at the end of the infection (at location 3, in Fig. 189) represents another acquisition of the same type of immune response. The comple- mentary action of the ablastic and trypanocidal effects not only effec- tively suppresses the infection, but also prevents relapses and reinfections for long periods (at least 325 days). Thus a few trypanosomes left from the initial infection or introduced by reinfection may be killed at once, or, if they are not killed at once, their reproduction is inhibited until they are killed. These statements hold for T. duttoni and, as far as they have been tested, for T. nabiasi (previous citations). The three effects of immunity which operate at points 1, 2 and 3 in Figure 189 are all due to humoral antibodies, as tested by passive transfer experiments. They are associated with the globulin fraction of serum, are acquired as a result of specific infection or specific immunization, and are decreased in amount or delayed in time of appearance by splenectomy and blockade. They differ in the following ways: The titer of the three varies independently, as far as can be tested. The trypanocidal effects are due to typical lysins which may, however, act as opsonins 77 vivo. The trypanolysin which terminates the infection kills either adult or dividing trypanosomes taken at any time during the course of infection, whereas that which causes the first number crisis kills only those try- panosomes which have just appeared in a rat’s blood. The parasites that survive the first number crisis are either basically nonsusceptible to this IMMUNOLOGY 859 antibody or acquire a resistance to it. So far, the reproduction-inhibiting properties of ablastin have not been demonstrated 7 vitro, but suitable amounts of serum, containing ablastin together with adult trypanosomes, when tested in vivo in a rat allow the trypanosomes to live, but prevent them from reproducing. (Adult trypanosomes have to be used for this Control Rat 980 650 5 = 600 4 | | } 1! \ 550 | 4 4 Us slit 30 7 5 4 sl J 450 | x | él 1 sl s $ Fae 4 | 4 A = 2 ~390{ | | Ey S ! 5300; 304 Seed Rat 972 | | Pr. ef “a I E I 525 251 4 Experimental Rat 977 4 I 3 lg 2 I 2004.8 204 2] | ! = is z 3 = = = 150) 2 15 3 | i 2 |5 = 7 | 2 = i <= Ss = = = = 104 10) = 12 Js u 2 = 2 / S 2 507 pS | 3 n | Caottiaent Variation | \ S erin | a O24 6° 8S ) OR 2 eB ti ON Pesce aCe Meni ma4 Days after Injection FIGURE 190. The demonstration of ablastin against Trypanosoma lewisi by passive transfer. The rate of reproduction of the trypanosomes, as shown by the constant coeffi- cient of variation, is completely inhibited in experimental rat 977, which received 2 cc. of serum and a large number of adult trypanosomes from seed rat 972; whereas the rate of reproduction goes through the normal cycle in control rat 977, which received a similar number of adult trypanosomes, but no serum, from seed rat 972. (From W. H. Taliaferro, 1924.) test since a curtailment of the reproductive activity of dividing trypano- somes, which is all that could be expected, is difficult to demonstrate with certainty.) Thus the difference between the coefficient of variation curve in experimental rat 977, which was given ablastic serum plus adult trypanosomes, and in control rat 980, which was given normal serum plus adult trypanosomes, is seen in Figure 190. The number curve re- 860 IMMUNOLOGY mains constant in experimental rat 977 because the trypanosomes are not reproducing. As was indicated above, ablastin is not absorbed by the trypanosomes 7” vitro, whereas the trypanocidal antibodies are. There is thus no lasting union of trypanosomes and ablastin, nor is there a sensiti- zation of trypanosomes by ablastin as there is with the trypanolysins. Moreover, if smaller and smaller doses of trypanocidal antibody are given, a point is reached at which the trypanosomes are not killed, but their reproduction remains unaltered. A group reaction can be demon- strated between T. duttoni and T. Jewisi and their ablastins 72 vivo, and between T’. Jewisi and antiduttoni trypanocidal antibody 7m vivo and in vitro; but whether the reaction of anti/ew7s7 trypanocidal antibody against T. duttoni in vivo and im vitro is a true group reaction of an immune antt- lewis? antibody is not evident because normal rat serum is also trypano- cidal to T. duttoni. These statements are based on a series of investiga- tions involving either or both of the following: zm vitro work for the trypanocidal effects and 7 vivo passive transfer experiments for all three effects by W. H. Taliaferro and coworkers (vide infra), Regendanz and Kikuth (1927), Perla and Marmorston-Gottesman (1930) and co- workers, W. H. Taliaferro, Cannon, and Goodloe (1931), and W. H. Taliaferro (1932). Culbertson (1938) has shown that the immunity to T. lewisi is passed through the mother’s placenta and milk to young rats where it persists for several weeks. Later, Culbertson and Wotton (1939) found that the young rats do not appear to produce ablastin as promptly or as well as older rats. Various procedures designed to lower the macrophage function, such as splenectomy, especially if combined with India-ink blockade or some infection such as Bartonella which affects the macrophage system, de- crease the strength and delay the appearance of ablastin and the terminal trypanolytic antibody (Regendanz and Kikuth, 1927; Perla and Mar- morston-Gottesman, 1930; Regendanz, 1932; and W. H. Taliaferro, Cannon, and Goodloe, 1931). This is an effect on acquired immunity. The next question which arises is whether the macrophages or other phagocytic cells assist in passive immunity or in the action of ablastin or the trypanolysins in the body. The work of W. H. Taliaferro (1938) indicates the following: Splenectomy and blockade have no effect on the passive transfer of ablastin, but the following interesting secondary effect results: The passive transfer of ablastin lasts only for a few days. IMMUNOLOGY 861 Thereafter it is not adequately augmented and supplemented by an active ablastic immunity in splenectomized and blockaded animals, as it is in normal rats, because the active immunity is slow in developing and decreased in amount. Splenectomy and blockade definitely decrease the effectiveness of the trypanolysins. It would seem that such an effect could be adequately explained by a decrease in the amount of comple- ment which would prevent the lysis of sensitized trypanosomes, or by a decrease of macrophages which would prevent the removal of opsonized parasites. The fact that previously sensitized trypanosomes are as readily removed in splenectomized and blockaded animals as in normal rats seems to negate both of these suppositions, unless the sensitized trypano- somes are agglutinated and removed mechanically—a possibility which because of technical difficulties has not yet been ruled out. A more likely explanation is that there is an interference with the union of antigen and antibody. It has already been indicated that a lysin differs from an opsonin only in that the terminal lysis and death of sensitized organisms may be effected by extra- rather than intracellular enzymes. Just as in the indirect studies discussed in the preceding paragraph, however, direct studies on phagocytosis have failed to indicate whether phagocytosis or lysis is more important in acquired immunity. Laveran and Mesnil (1901) considered that the parasites are actively phagocytosed, and Roudsky (1911) and Delanoé (1912), studying the acquired immunity of mice to T. /ewis7, came to the same conclusion. Regendanz and Kikuth (1927) believed that the parasites are phagocytosed in a nonspecific way. MacNeal (1904), Manteufel (1909), W. H. Taliaferro (1924), and Coventry (1929), on the other hand, considered that they are lysed. In studying the tissues for evidence of phagocytosis, we are handicapped by the fact that no easily recognizable vestiges of trypanosomes, such as malarial pigment, remain in macrophages for any appreciable length of time. The fact that pigment by itself may be phagocytosed does not invalidate this statement, since the whole complex, consisting of red cell, parasite, and pigment, is often recognized intact in macrophages. To sum up: acquired immunity against nonpathogenic trypanosomes primarily involves ablastin and trypanolysins, the first of which prevents the trypanosomes from undergoing growth and cell division, and the second of which kill the trypanosomes. They are both humoral anti- 862 IMMUNOLOGY bodies which are associated with the globulin fraction of immune serum, are passively transferable, and are probably a product of the lymphoid- macrophage system; but whereas ablastin possesses no 7m vitro affinity for the trypanosomes, the trypanolysins are typical antibodies (ambo- ceptor) and can sensitize the trypanosomes 7m vitro. Furthermore, the macrophage system does not appear to intervene in the passive transfer of ablastin, but in some way functions in the union of antigen and antt- body during the passive transfer of the trypanolysins. CONTINUOUS FATAL TRYPANOSOMIASIS IN THE MOUSE AND SOMETIMES IN THE RAT Most species of trypanosomes appear to be nonpathogenic, and there is a growing mass of evidence that even many of the pathogenic forms Millions of Trypanosomes per cmm. of dbod m3 w ES —— Fe Coefficient of Variation (ae <— Ineclion ip 0 2 "s cme illons of _—— 9 <— Dead g vonatuy Ja} sheg y pit ) UOIPLID\ J JUdI91JJ005 FIGURE 191. The changes in number of Trypanosoma rhodesiense and the coefficient of variation during the course of infection in a mouse. No acquired immunity is de- veloped in the mouse, since the number of trypanosomes more or less steadily increase and their rate of reproduction, as evidenced by the high coefficient of variation, is not inhibited. (Redrawn from W. H. Taliaferro and L. G. Taliaferro, 1922. in man and domesticated animals may be nonpathogenic in their natural hosts (cf. Duke, 1936). Little is known of the course of their infection in their natural hosts, but they have been extensively studied in labo- ratory animals, in which they are all pathogenic. The well-known patho- genic trypanosomes, which produce disease in man and domestic ani- mals, are T. gambiense, T. rhodesiense, T. brucei, T. congolense, T. vivax, T. evansi, T. equinum, and T. eguiperdum. When injected into mice, the parasites almost invariably appear in the blood after a short incubation period, and increase in number more or less steadily until the death of the host. This type of infection is composed of an incuba- tion period and acute rise, with no crisis or developed infection. It sometimes occurs in the rat. Its continuous nature was clearly pointed IMMUNOLOGY 863 out by Massaglia (1907) and is illustrated in Figure 191 by T. rho- desiense in the mouse. Whether there is a natural immunity against the trypanosomes in the mouse cannot be answered at present, because of insufficient data. On the one hand, W. H. Taliaferro, Johnson and Cannon (unpublished work, see W. H. Taliaferro, 1929) reported no effect of splenectomy on mice infected with T. equinum, and on the other hand Schwetz (1934) and Russeff (1935) found a slight effect of splenectomy in mice infected with T. congolense and T. equiperdum respectively. With regard to acquired immunity, most data are in accord in show- ing that the mouse does not develop any appreciable amount. As may be seen in Figure 191, the rate of reproduction of the parasites remains constant and fairly high (the C.V. varies between 8.9 and 10.5 per- cent) and the parasites progressively increase in number until the death of the host (see also control mouse, infected with T. eguimum, in Figure 193, which is drawn on a semilogarithmic scale). In 1933 Krijgsman showed that the rate of increase of T. evans in the mouse and rat is not uniform during the acute rise, but that two periods of high rates of increase, approximately between eighteen and thirty-two hours and sixty and sixty-six hours, alternate with three pe- riods of lower rates of increase at the beginning, middle, and end of the infection. He believed that the terminal low rate of increase is due to a destruction of parasites, as evidenced by the occurrence of more degenerating forms, but that the earlier low rates are due to a partial inhibition of reproduction. He reached this conclusion because he found no increase in degenerating trypanosomes in the blood and in spite of the fact that he found no diminution in the percentage of division forms. He visualized the mechanism of this partial inhibition of re- production as a uniform retardation of all stages of the cycle of growth and cell division. There is no doubt that theoretically such a uniform lengthening of all stages would give a partial inhibition of reproduction, without affecting either the percentage of division forms or the coefficient of variation. Nevertheless, the existence of such a mechanism is doubtful, in view of the fact that in T. /ew7s7, as well as in cells in general, a retardation of cell division is characterized by an increased length of the resting stage (the so-called adult stage of T. lewis’), and not by a gradual slowing down of the whole process with 864 IMMUNOLOGY uniform increases in the length of each stage. Furthermore, Krijgs- man’s inability to find degenerating stages in the blood early in the infection and his finding them during the terminal phases is not con- clusive evidence that trypanosomes are not dying during the early low rates of increase. It is very likely that the macrophages remove such forms less quickly during the latter part than during the early part of the infection, because they have become partially blockaded. The inter- pretation of the varying rates of increase of T. evansi in the mouse, as found by Krijgsman, will therefore have to await further analysis. From an enormous mass of work on the mouse as a carrier of so-called pas- sage strains, however, it appears that no trypanolysin usually develops in the mouse which kills most of them and toward which the residue become antigenically resistant, as has been demonstrated in the infec- tions to be described in the following section. We may accordingly conclude that the course of the infection in the mouse and sometimes in the rat most closely approximates the simplest type of infection, which increases as a geometrical progression and in which little, if any, immunity is acquired of either an ablastic or trypano- cidal type. INTERMITTENT FATAL TRYPANOSOMIASIS IN VARIOUS LABORATORY ANIMALS When the same pathogenic trypanosomes considered in the preced- ing section are grown in the guinea pig, the infection is typically char- acterized by an incubation period, followed by alternate increases (the first is an acute rise and the succeeding ones are relapses) and decreases (crises) in the parasite population until the animal dies. Besides the guinea pig, this kind of infection is observed in rabbits, dogs, cats, and occasionally in rats infected with these same trypanosomes, in man infected with T. rhodesiense and sometimes in mice infected with T. congolense. Sometimes the initial acute rise and crisis do not develop, as is shown in Figure 192. In other animals, such as sheep, the entire infection is of such low grade that trypanosomes are rarely found and then only in thick film. These statements are based on work by Ross and D. Thompson (1910, 1911), J. G. Thomson (1912), W. H. Taliaferro and L. G. Taliaferro (1922), Knowles and Das Gupta (1928), Davis (1931), Krijgsman (1933), Browning ef al. (1934), IMMUNOLOGY 865 and others. The fact that animals often die when trypanosomes are scarce in their blood has been explained as being due to sugar depletion, asphyxiation, toxins, and so forth (see von Brand, 1938, review). A possibility that has not been adequately discussed in the literature is that death shortly after a crisis may be due to the severity of the im- mune reaction, comparable to that seen in overwhelming hypersensi- tivity (cf. graph of malaria, Figure 188). In analyzing such infections, W. H. Taliaferro and L. G. Taliaferro (1922) found that the basic rate of reproduction remains relatively nm wn : bs 5 5 Zl > fK Ss £# \ = S.¢ et E | Fal ce. 52 ! N S = = 35 len, ! 4 Fol Bol = oe SN a 3 \~ g ss oc Bt ear EE aI oe = LSS a G. Ss | Ss) Ay | K | & Days after Injection FIGURE 192. The changes in number of Trypanosoma rhodesiense and the coefficient of variation during the course of infection in a guinea pig. As acquired immunity de- velops, the parasites are killed by trypanolysins operative during the long chronic period and at the two crises thereafter, but the rate of reproduction is not inhibited, as evi- denced by the high coefficient of variation. (Redrawn from W. H. Taliaferro and L. G. Taliaferro, 1922.) constant whenever parasites can be found in the blood in sufficient num- bers for study. Thus in Figure 192 the coefficient of variation remains between 8.5 and 12.0 percent from twenty-three through forty-three days after infection. Knowles and Das Gupta (1928) corroborated this finding for T. evansi in the rat, and Davis (1931) for T. rho- desiense in the cat. There is some evidence that these trypanosomes, when originally isolated in Africa, exhibit infections essentially simi- lar, except that superimposed on the reproductive activity seen in Figure 192 are intermittent periods of heightened reproductive activity. Thus Robertson (1912) reported that periods of active reproduction of T. gambiense in the monkey alternate with periods of less active repro- duction. Since reproduction continues at a relatively high rate, even during the periods of less active reproduction, and is never completely 866 IMMUNOLOGY inhibited, these infections may be considered with the constantly repro- ducing experimental infections discussed in this section. Little is known about natural immunity because, as pointed out in the consideration of the T. Jews? group of trypanosomes, the rate of reproduction of the trypanosomes cannot be measured directly and few attempts have been made to raise the rate of reproduction (see T. duttoni in the mouse) or to increase the percentage of surviving trypanosomes. Nieschulz and Bos (1931), however, reported a slightly shorter incuba- tion period in dogs infected with T. evansi as the result of splenectomy. Acquired immunity, which develops later and which is superim- posed on any natural immunity which may be present, is entirely para- siticidal, with no evidence of an inhibition of reproduction. When the infection shows a typical acute rise in numbers, the first manifesta- tion of acquired immunity is a crisis (see guinea pig, in Figure 193). Thereafter, one or several relapses and crises follow. When a pro- longed chronic low-grade infection ensues, with no typical acute rise as in Figure 192, it is probable that parasiticidal effects of acquired immunity, similar to those produced at the two typical crises later, hold the numbers down. Since all the animals die, the acquired immunity is obviously ineffective. In other words, once the parasites are intro- duced into the host, they reproduce during the entire infection, and although at intervals most of those that have accumulated in the blood are destroyed, the few that escape destruction repopulate the blood again and again until the host dies. An extensive series of 72 vivo and in vitro investigations (see Laveran and Mesnil, 1912; W. H. Taliaferro, 1929, for reviews of the pioneer work) indicate (1) that the periodic destructions of the trypanosomes are due to typical trypanolysins which can be demonstrated 7v vivo and in vitro and which are acquired by the host as a result of specific infec- tion or specific immunization; (2) that the trypanosomes reaccumulate during the relapses not because the trypanolysins disappear, but be- cause the parasites have become resistant to them; and (3) that the relapse trypanosomes differ antigenically and that the majority of them are subsequently killed by a new trypanolysin. In all of this work, the strains of trypanosome used and their continuous maintenance are of paramount importance. The experimental infection is started with what is known as the passage strain and is usually maintained in mice by IMMUNOLOGY 867 serial transfer, since it has been found that strains remain immunologi- cally unchanged for long periods in mice. The trypanosomes which re- populate the blood after each trypanolytic crisis are immunologically different from the passage strain and are resistant to the lysin produc- ing the crisis. They are known as relapse strains and also have to be maintained in mice by serial transfer. Thereafter, as many relapse strains as are studied have to be extracted and maintained separately and con- Guinea Dig (experinjental) —_ 00000 Injected ip with 3cc. of Immune Serurn 8 Crists inf hr = oS Ss Number of Trypanosomes per cmm. of Blood = Killed for Immune Serum 100 YO @ A 8 8 [0 fre PV) i a Gr El ip) 1 Ta @) Days after Injection FIGURE 193. The demonstration of a trypanolysin against a passage strain of Trypano- soma equinum by passive transfer. An artificial crisis is produced in the trypanosome infection in the experimental mouse, which received 0.3 cc. of serum taken from the seed guinea pig after its infection had undergone a crisis, whereas the infection in the control mouse which was not given serum proceeded uniformly until the mouse died. (From W.H. Taliaferro and T. L. Johnson, 1926.) tinuously until the investigation is terminated. Reference to Figure 193 will make this clearer. Both the guinea pig and the two mice were in- fected at appropriate intervals with a passage strain. Serum taken from the guinea pig at the time of the naturally occurring crisis produced an artificial crisis when injected into the experimental mouse, but would have been ineffective had the experimental mouse been infected with the relapse strain, which would have repopulated the blood of the guinea pig after the crisis. (The artificial crisis produced by the immune serum in this so-called curative test is very temporary.) A brief résumé of the investigations follows. 868 IMMUNOLOGY Rouget (1896) first found that the serum of rabbits and dogs, which had been infected with T. eqguiperdum and had become cachetic, exerted a protective action, in a dose of 0.3 cc., on mice infected with the pass- age strain as measured by the survival time of the mice. The fact that immune serum is protective led Laveran and Mesnil (1901) to hope that serotherapy might possibly be developed against pathogenic trypano- somiasis, but so far this hope has not materialized. Schilling (1902) was the first to recognize the phenomenon of trypanolysis in vitro. Rodet and Vallet (1906) studied the lysins systematically, and Massag- lia (1907) showed that the trypanosomes which repopulate the bléod after each trypanolytic crisis are immunologically different from the original strain and are resistant to the lysin producing the crisis. Thus serum from an infected guinea pig before a crisis is only slightly lytic, whereas that during and after the crisis is strongly lytic to the original strain of trypanosomes, but has no deleterious effect on the trypano- somes reappearing after the crisis. Levaditi and Mutermilch (1909) reported that the lysis is a complement-amboceptor reaction (i.e., in- volves a heat labile component of serum and the heat stable antibody), and Leger and Ringenbach (1911 and 1912) found a group specificity between trypanolytic immune serums and different species of pathogenic trypanosomes. W.H. Taliaferro and T. L. Johnson (1926), in a study of the pro- duction of artificial crises (Figure 193) by immune serum against T. equinum in mice, found that zones of inhibition may occur. T. L. John- son (1929), in a continuation of this work, found that the production of the artificial crisis, with resulting lengthening of life in the mouse, is dependent not only upon the amount of immune serum, but upon the absolute number of parasites present and upon the strain of para- site used. For example, when a given serum was injected into mice whose blood showed one to five parasites of a particular strain per microscopic field, it caused lysis of the trypanosomes uniformly in all doses greater than the minimal effective dose; when injected into mice the blood of which contained from ten to twenty-eight parasites of the same strain per microscopic field, it caused alternate zones of lysis and non-lysis (zone phenomenon); whereas when injected into mice whose blood showed fifty parasites per microscopic field, no lysis occurred, no matter what dose of serum was given. Moreover, Johnson was able IMMUNOLOGY 869 to subject this strain to immune serum and to secure a relapse strain which showed the zone phenomenon with one to five parasites per field. Such data give a basis for the interpretation of the variable and often contradictory results obtained by investigators doing only a few tests. The relapse strains can be differentiated from the passage strain not only by their resistance to lysins, but by their behavior in other serologi- cal tests, such as the Rieckenberg blood platelet test (Rieckenberg, 1917; see also Brussin and Kalajev, 1931). They also differ antigeni- cally and therefore stimulate different immune mechanisms, as is shown, for example, by cross-immunity tests. The difference in antigenic constitution of various strains was origi- nally studied by Ehrlich and his coworkers in infections in mice in which artificial crises were produced by incomplete cures with drugs. Of the earlier papers, that of Ritz (1914) is particularly interesting. He incompletely cured a mouse twenty times, during which seventeen immunologically different relapse strains were produced, as tested by cross-immunity in mice after cure. Some of these strains were identical with those of another mouse which had been incompletely cured nine- teen times, during which nine immunologically different strains had been produced. The immunological variations may be inherited, but in time may be lost. Ritz (1916) also showed that the strains arising naturally in the rabbit could be differentiated by the same methods. In the suc- ceeding years, more or less similar studies have been made with both antibody and drug-induced relapse strains. Recently, Lourie and O’Con- nor (1937), in an 7” vitro study of relapse strains after drug treatment, obtained twenty-two relapse strains of which thirteen were immuno- logically distinct. In addition, they ascertained that certain strains tended to occur more frequently than others, that a strain may be a combina- tion of two or several strains, and that individual strains may disappear from such compound strains. The acquisition of this antibody resistance by the trypanosomes, with a concomitant antigenic change, is an interesting case of an environ- mentally induced persistent modification which is inherited for many asexual generations, sometimes through 400 mouse passages. It seems to be similar to the acquisition of drug resistance by free-living Proto- zoa. It can be produced not only im vivo, but also in vitro. It is al- 870 IMMUNOLOGY ways associated with the destruction of many organisms, and hence involves a selection, but the selection is effective within a clone, i.e., within the progeny of a single trypanosome. At the present time, how- ever, it 1s impossible to decide whether such persistent modifications are due to changes in gene constitution, or, if they are not, whether they may eventually lead to such changes (see Robertson, 1929; W. H. Talia- ferro and Huff, 1940; and, in part, Dobell, 1912). Nevertheless, they are of extreme importance in allowing the parasite to overcome the defensive processes of the host and are probably largely responsible for the continued survival of the parasite. The lymphoid-macrophage system and particularly the macrophages along the blood stream, appear to be involved in immunity, as indicated by enlargement and histological changes in the spleen (Laveran, 1908; Van den Branden, 1935; and others) and by the decreased length of life of splenectomized animals infected with various trypanosomes (see Davis, 1931, for most of the work prior to 1931; Nieschulz and Wawo- Roentoe, 1930; Nieschulz and Bos, 1931; Russeff, 1935). Negative results, as might be expected from the complexity of the problem as explained previously, have also been reported by some of the earlier workers (see Davis, 1931), and also by Davis (1931) and Browning et al. (1934), whereas increased length of life was noted in partially blockaded rats infected with T. eguiperdum by Kolmer et al. (1933). Data on splenectomized and blockaded animals which were treated are omitted from consideration because the treatment itself may affect the course of infection. | Whether the trypanocidal antibody acts within the body as a trypano- lysin, or as an opsonin with resulting phagocytosis, or both, has been variously answered. Some authors have maintained that one or the other is the sole method of defense; some that they share equal honors; and some that, although lysis is the fundamental mechanism, phagocytosis is responsible for clearing up the debris, and so forth. No one can doubt the occurrence of phagocytosis after its careful description by sO many competent observers (Neporojny and Yakimoff, 1904; Sauer- beck, 1905; Yakimoff, 1908; Mesnil and Brimont, 1909; Levaditi and Mutermilch, 1910). On the other hand, W. H. Taliaferro and T. L. Johnson (1926) reported the finding of disintegrating trypanosomes IMMUNOLOGY 871 in the blood during experimental crises, which they interpreted as stages in lysis. This question can probably be answered as was the simi- lar question with regard to the trypanolysins in T. /ew/s7 infections. The trypanosomes become sensitized by antibody, and the process may be completed by lysis (extracellular enzymes) on one hand, or by diges- tion within phagocytes (intracellular enzymes) on the other hand. Which occurs may depend to a certain extent on the strength of the antibody. Besides the cellular basis for the production of the trypanocidal anti- body and the codperation of phagocytes in removing sensitized para- sites, Kuhn (1938) has shown a peculiar role of the lymphoid-macro- phage system in passive transfer of anti 7. equiperdum protective serum to mice. Thus immune serum, which is effective in protecting normal mice in doses of 0.4 cc. per 20 gm. body weight, gives only partial pro- tection to splenectomized mice, blockaded with India ink, in doses as high as 1.7 cc. per 20 gm. body weight. Suitable experiments indicate (as in previous work with T. /ewzs7) that this finding is due neither to the lowering of complement nor to the removal of phagocytic cells which might be necessary in removing opsonized parasites, but rather to the prevention of antibody uniting with trypanosomes. An interest- ing, but confusing element in these experiments was that unilateral nephrectomy or ureterotomy was accompanied by a slight reduction in the protective titer of the serum. A comparison of the resistance acquired by hosts against pathogenic and against nonpathogenic trypanosomes is very illuminating. In the first case, the host acquires practically no resistance (mouse) or it pe- riodically forms trypanolysins (guinea pig, dog, etc.) which hardly ever effectively rid the animal of infection because a few of the patho- gens generally become resistant and repopulate the blood again and again until the host dies. In the second case, the host first produces an antibody which inhibits cell division of the parasites and then periodi- cally forms trypanolysins which get rid of the nonreproducing parasites. PRACTICAL APPLICATIONS OF IMMUNE REACTIONS By far the most extensive literature on the immunology of the para- sitic Protozoa deals with experiments fundamentally planned in the hope of achieving some practical method of preventing, curing, or diagnosing 872 IMMUNOLOGY infections. This work has yielded many facts of great interest, but ac- tual practical applications have been limited. ARTIFICIAL IMMUNIZATION The earlier literature on this subject has been critically reviewed by W. H. Taliaferro (1929), to which publication the reader is referred for details. Only a few of the more successful examples are cited. The greatest success with artificial immunization has been attained in Babesia infections of cattle and consists of inducing in young healthy animals a low-grade or latent infection which is frequently controlled with drugs. During this latent infection, the animal possesses a solid immunity to superinfection, similar to the condition in malaria. Like malaria, however, the host’s defenses may weaken and permit severe and even fatal relapses. Mention has been made of the fact that one attack of oriental sore in man generally confers a lasting immunity. As the natural sores oc- cur on the face or other exposed portions of the body and leave dis- figuring scars, it has been the practice in many endemic centers for centuries to inoculate children on unexposed portions of the body. In a sense this is the crudest type of immunization, in that the highly viru- lent virus is employed to induce the ordinary disease. The use of at- tenuated organisms has not met with particular success. Several investigators have been able to immunize laboratory animals with dead trypanosome vaccines. So far, however, such vaccines have not been extensively applied in a practical way and the outlook is not favorable. Among other difficulties, the attainment of an ade- quately polyvalent vaccine can hardly be hoped for, owing to the exist- ence of so many immunologically different strains of trypanosomes. IMMUNOLOGICAL REACTIONS USED IN DIAGNOSIS Considerably more success has followed the practical application of immunological reactions in diagnosis than in immunization, but even here the success has been limited. This is due in part to the technical difficulty of perfecting the tests, especially when only weak reactions ensue, and in part to the fact that they have to be as satisfactory as or better than the demonstration of the parasites, which has been rendered remarkably delicate in certain blood infections, notably malaria and IMMUNOLOGY 873 trypanosomiasis, by the use of stained thick-blood films (see Barber, 1936). From the great mass of literature only the tests which have been perfected or show considerable promise will be mentioned. De- tailed protocols and specific methods of procedure can be found in W.H. Taliaferro (1929) or in some of the more recent articles. A. Specific Immunological Reactions—The reactions between antigen (either complete or haptene) and antibody are so specific that, within certain limits, the presence of a suspected antigen can be ascertained with a known antibody, or, vice versa, a suspected antibody can be verified with a known antigen. Both have been used in diagnosis. When the invading organism liberates some antigen, either whole or partial (haptene) into the blood, sputum, urine, and so forth, the antigen may be detected and identified by its reaction with a high titer immune serum, generally prepared in the laboratory. Sometimes, if the organism iso- lated from an infected host cannot be fully identified by morphological criteria, it may be further classified in this way (see section on im- munological methods of classification). Or, if the invading organism during infection stimulates the formation of a specific antibody in the blood, it may be identified by its reaction with a known antigen which is prepared from the organism in the laboratory. In the Protozoa only the last type of reaction has been extensively used. The specific complement fixation test is one of the most highly stand- ardized laboratory tests. It is based on the fact that antibody will combine with antigen, and the resultant sensitized antigen will then combine fur- ther with complement (a heat labile component of serum), but neither antigen nor antibody will combine with complement alone. In practice, serum suspected of containing an antibody is first heated at 56° C. for twenty minutes to inactivate the complement which it also contains and then is added in varying proportions to a known antigen. To such mix- tures, known quantities of complement (generally fresh guinea-pig serum) are subsequently added. The actual fixation of complement gives no visible sign, but is tested by adding to the system at this point a suspension of red blood cells which have been previously sensitized with their specific lysin (sheep cells and antisheep lysin are generally used). Obviously, if the complement was previously fixed, there will not be enough left to lyse the sensitized cells. In terms of the original test, if the red blood cells undergo lysis and their hemoglobin colors the 874 IMMUNOLOGY solution, the suspected antibody was not present (test negative); if the cells remain entire and unlysed and the supernatant clear, the suspected antibody was present in the serum (test positive). From the foregoing brief résumé, it is obvious that this test demands careful preparation and standardization of the component parts for its successful execution. It differs from the nonspecific complement fixation or Wassermann test widely used in syphilis only in that the test antigen is derived from the immunizing organism or antigen. The test antigen for the Wassermann test, on the other hand, involves the use of a lipoid extracted from normal tissue, such as beef heart. The specific complement fixation test has been most satisfactorily standardized in amoebiasis and in dourine of horses. The successful cultivation of Endamoeba histolytica, the causative agent of amoebic dysentery, made amoebae available in sufficient quantities to provide a suitable antigen, and since the work of Craig in 1927 the complement fixation test for amoebiasis has been intensively studied (see Craig, 1937; Meleney and Frye, 1937; Paulson and Andrews, 1938). The con- sensus of opinion seems to be that the test has to be carefully carried out to be dependable and that at best it can be used only as an adjunct to fecal diagnosis. The sum total of published work through 1910 indicated that com- plement-fixing antibodies could be demonstrated in various trypano- somiases under controlled conditions, but there was little to indicate that they could be used for diagnosis. From 1911 onward, however, the test was perfected and used extensively for the diagnosis of dourine caused by Trypanosoma equiperdum in horses and mules. It was stand- ardized mainly through the efforts of Mohler, Eichhorn, and Buck (1913), E. A. Watson (1920), who used an aqueous antigen; and Dahmen (1922), who used both aqueous and alcoholic extracts. Ac- cording to Watson, the test is often positive before symptoms are ap- parent and during latent stages, and in practice no animal should be considered free of the disease unless negative two months after a last exposure. C. M. Johnson and Kelser (1937) concluded that the test is distinctly valuable in revealing active cases of Chagas’s disease. Little success has attended workers using specific complement fixation in malaria and the leishmaniases, especially kala azar, owing perhaps to the low titer of serums from infected persons and the difficulty of IMMUNOLOGY 875 obtaining antigens. Recently Coggeshall and Eaton (1938) have re- ported good results in simian malaria with an aqueous antigen obtained from heavily infected blood or spleen. The red-cell adhesion test grew out of Rieckenberg’s (1917) blood platelet test. As used by Duke and Wallace (1930), it involves the addition of one drop of a citrated trypanosome suspension to one drop of equal parts of blood from the suspected animal and 2-percent sodium citrate. If the blood comes from an infected animal, red blood cells (occasionally also blood platelets) adhere to the trypanosomes within ten to fifteen minutes. In 1931 Wallace and Wormall concluded that complement is necessary, and H. C. Brown and Broom (1938) found that the concentration of trypanosomes should be between 3,000 and 100,000 per cu. mm. and the red cells between 300,000 and 1,250,000. This test compared favorably with specific complement fixation, when untreated horses infected with T. h7ppicum were tested by W. H. and L. G. Taliaferro (1934d). B. Nonspecific Serological Reactions.—Infection often results in defi- nite changes in serum which can be detected by various physical and chemical means and which, although not specific in the immunological sense, are characteristic enough to be useful in diagnosis. Even when the same changes occur in several infections, they may still be used in conjunction with other criteria or if the infections have different geo- graphical distributions. Several miscellaneous tests have been devised for kala azar which are associated with an increase in the euglobulins of the serum. They in- clude the serum-globulin test of Brahmachari, the aldehyde test of Napier, and the urea-stibamine test of Chopra, Gupta, and David. These tests have been modified and combined by these same and other work- ers. In general, upon the addition of distilled water, formaldehyde, or urea-stibamine in proper proportions to serum from a person infected with kala azar, the mixture becomes characteristically opaque, owing to the formation of a precipitate within a comparatively short time. These tests appear to be extremely useful and Menon ef al. (1936) advocate testing a serum by both the aldehyde and the urea-stibamine test (see Menon ef al., 1936, and Chorine, 1937, for the literature on this subject). Some of these tests may also be of value in trypanosomiasis (see 876 IMMUNOLOGY Hope-Gill, 1938), especially in areas in which kala azar is absent. In 1927 Henry described certain serological tests for the diagnosis of malaria, based on the observation that the serums of malarial sub- jects flocculate in solutions of metharsenate of iron (ferroflocculation test) and of melanin pigment (Henry’s test, or the melanoflocculation test). As a reagent for the Henry test, which was shown by later work to be more sensitive than the ferroflocculation test, Henry (1934) used the filtered supernatant from a suspension of finely ground choroid tissue of ox eye in distilled water. This material after formalin had been added and it had been kept on ice for at least several hours, is added in proper proportions to the serum to be tested, and flocculation is looked for after a half hour or more, preferably by means of the photometer of Vernes, Bricq, and Yvonne. Many subsequent papers on this test have been ably reviewed by Greig, Von Rooyen, and Hendry (1934), Trensz (1936), Villain and Dupoux (1936), de Alda Calleja (1936), Vaucel and Hoang-Tich-Try (1936), and Proske and Watson (1939). The upshot of this work seems to indicate that the test may serve as an adjunct to the search for malarial parasites in diagnosing malaria, but that its use is restricted to laboratories equipped with a photometer and to areas in which kala azar, certain types of leprosy, and certain other diseases are not common. Since it has been shown to be due to an increase of the euglobulin fraction, which flocculates upon dilution with distilled water or weak salt solutions, Proske and Watson (1939) have developed the protein-tyrosin reaction, which is a quanti- tative chemical estimation of the euglobins of the serum. IMMUNOLOGICAL REACTIONS IN RELATION TO CLASSIFICATION Various immunological reactions, since they are frequently spectes- specific, have been used to check and extend other biological classifi- cations. In other words, the more closely two species are related, the stronger, in general, is the group reaction between them. This specificity seems to depend on the basic structure of the antigens and haptenes, which react specifically with immune serum iz vitro. It also probably depends upon the quantitative proportions of the various antigens con- tained in a particular organism (see Wells, 1929). Immunological re- actions can therefore be employed to compare chemical structure with IMMUNOLOGY 877 anatomical structure. The reactions have to be studied, however, to see if, on the one hand, they vary too much within what is a generally recog- nized species, or if, on the other hand, they do not differentiate suffi- ciently among large groups. The extreme specificity within a species may be exemplified by the diversification of a single cell strain of trypanosomes, through the mediation of immune serums or drugs, into a large number of strains which will remain immunologically distinct for long periods. In a sense most of the work on the serology of parasites can be used in classification. For example, an investigator, in attempting to dis- cover a serological test for a given infection, generally considers at once the specificity of the reaction by ascertaining to what extent group reactions with other species exist. On the whole, however, the study of the immunological relationships of organisms can best be attained by using antiserums from artificially immunized laboratory animals. By this method animals such as rabbits, in which antibodies are readily produced, can be immunized until high titer antiserums are obtained. Immunological methods have been employed extensively to estab- lish the identity or nonidentity of various proposed species of Lezsh- mania, which are morphologically identical, and their relationship to certain insect and plant herpetomonads which resemble the cultural forms of Leishmania. This work is fairly consistent in showing that the members of the genus Leishmania are a closely related group and are entirely distinct from the genus Herpetomonas (Noguchi, 1926; Wage- ner and Koch, 1926; Zdrodowski, 1931). Since trypanosomes, like the Leishmanias are frequently morphologi- cally indistinguishable from one another, various immunological tests, as well as biological criteria, have been employed to distinguish them. The in vivo cross-immunity test has been most extensively used (Braun and Teichmann, 1912; Laveran, 1917; Kroé, 1925, 1926; Schilling and Neumann, 1932), but 2” vitro tests have also been used, such as com- plement fixation by Robinson (1926), the phenomenon of “‘attachment’”’ by several authors (see Levaditi and Mutermilch, 1911) and 7m vitro trypanolysis by Leger and Ringenbach (1912) and others. In evaluat- ing the results of these methods, it appears that they need to be re- worked, because of the advance in modern technique and because au- 878 IMMUNOLOGY thorities such as Wenyon (1926) believe that many of the species for- merly recognized as distinct should be combined (cf. Becker, Chapter XVII). The work on piroplasms, although lacking in conclusiveness, has at least served to direct the attention of systematists to the problems of classification (Theiler, 1912; Stockman and Wragg, 1914; du Toit, 1919; Brumpt, 1920). In malaria cross-immunity tests have been extensively employed (Manwell, 1938), whereas in amoebiasis complement fixation tests have occasionally been used (Menendez, 1932). LITERATURE CITED Afridi, M. K. 1938. Observations on extra-abdominal spleen in monkeys infected with P. cynomolgi and P. knowlesi. J. Malar. Inst. India., 1: sa) Alda Calleja, M. de. 1936. Estado actual de los estudios sobre sueroflocula- cion en el paludismo. Med. Paises Calidos, 9: 203-36. Aschoff, L. 1924. Das reticulo-endotheliale system. Ergebn. inn. Med. Kin- derheilk., 26: 1-118. Barber, M. A. 1936. The time required for the examination of thick blood films in malaria studies, and the use of polychromatophilia as an index of anemia. Amer. J. Hyg., 24: 25-31. Ben Harel, S. 1923. Studies of bird malaria in relation to the mechanism of re- lapse. Amer. J. Hyg., 3: 652-85. Bignami, A. 1910. Sulla patogenesi delle recidive nelle febbri malariche. Atti Soc. Studi Malar. 11: 731-45. Translated by W. M. James: Sth. med. J. Nashville, 1913 (Feb.). Bloom, W. 1938. Lymphocytes and monocytes: theories of hematopoiesis. Downey: Handbook of Hematology, 1: 373-436. Bohm. 1918. Hamatologische Studien bei Malaria. Arch. Schiffs- u. Tro- penhyg., 22: 49-55. Boyd, G. H. 1929a. Induced variations in the asexual cycle of Plasmodium cathemerium. Amer. J. Hyg., 9: 181-87. —— 1929b. Experimental modification of the reproductive activity of Plas- modium cathemerium. J. exp. Zool., 54: 111-26. —— 1933. Effect of quinine upon reproduction of the avian malaria parasite, Plasmodium cathemerium. J. Parasit., 20: 139-40. —— 1939. A study of the rate of reproduction of the avian malaria para- site, Plasmodium cathemerium. Amer. J. Hyg., 29 (Sect. C): 119-29. Boyd, G. H., and L. H. Allen. 1934. Adult size in relation to reproduction of the avian malaria parasite, Plasmodium cathemerium. Amer. J. Hyg., 20: 73-83. IMMUNOLOGY 879 Boyd, G. H., and M. Dunn. 1939. Effects of quinine and plasmochin admin- istration upon parasite reproduction and destruction in avian malaria. Amer js Hye:.300(SechG@) ee i7- Boyd, M. F., and L. T. Coggeshall. 1938. A résumé of studies on the host- parasite relation in malaria. Trans. Third Inter. Congress Trop. Med. and Malaria, 2:292-311. Boyd, M. F., W. K. Stratman-Thomas, and S. F. Kitchen. 1936, On the duration of acquired homologous immunity to Plasmodium vivax, Amet. Jnttop: Meds 162 31 1215; Brand, T. von. 1938. The metabolism of pathogenic trypanosomes and the carbohydrate metabolism of their hosts. Quart. Rev. Biol., 13:41-50. Braun, N., and E. Teichmann. 1912. Versuche zur Immunisierung gegen Trypanosoma. Jena. Brown, H. C., and J. C. Broom. 1938. Studies in trypanosomiasis. H. Ob- servations on the red cell adhesion test. Trans. R. Soc. trop. Med. Hyg., 32:209-22. Brown, W. H. 1914. A note on the pathogenicity of T. Jew7s7. J. exp. Med., 19: 406-10. —— 1915. Concerning changes in the biological properties of Trypano- soma lewisi produced by experimental means, with especial reference to virulence. J. exp. Med., 21: 345-64. Browning, C. H., D. F. Cappell, and R. Gulbransen. 1934. Experimental infection with Trypanosoma congolense in mice: the effect of splenec- tomy. J. Path. Bact., 39: 65-74. Bruetsch, W. L. 1927. Ein Beitrag zur Wirkungsweise der Impfmalaria auf den histopathologischen Prozess bei progressiver Paralyse. Z. ges. Neurol. Psychiat., 110: 713-28. —— 1932a. The histopathology of therapeutic (tertian) malaria. Amer. J. Psychiat., 12: 19-65. — 1932b. Activation of the mesenchyme with therapeutic malaria. J. nerv. ment., Dis., 78: 209-19. Brug, S. L. 1934. Observations on monkey malaria. Riv. di Malariol., 13: 3- 25. Brumpt, E. 1920. Les Piroplasmes des bovidés et leurs hétes vecteurs. Bull. Soc. Path. exot., 13: 416-60. Brussin, A. M., and A. W. Kalajev. 1931. Die Bedeutung des Komplements and der Blut-plattchen fiir die Feststellung der Thrombozotobarine. Z. ImmunForsch., 70: 497-521. Cannon, P. R., and W. H. Taliaferro. 1931. Acquired immunity in avian malaria. III. Cellular reactions in infection and superinfection. J. prev. Med. Lond., 5: 37-64. Chopra, R. N., and S. N. Mukherjee. 1936. The trend of immunity studies in malaria. Indian med. Gaz., 71: 34-39. 880 IMMUNOLOGY Chorine, V. 1937. Les Réactions sérologiques dues aux euglobulines. Ann. Inst. Pasteur, 58: 78-124. Christophers, S. R. 1904. A preliminary report on a parasite found in persons suffering from enlargement of the spleen in India. Sci. Mem. Offrs. Med. san. Dept. Gov. India, N.S., 8: 1-17. Coggeshall, L. T. 1937. Splenomegaly in experimental monkey malaria. Amer. J. trop. Med., 17: 605-17. —— 1938. The cure of Plasmodium knowlesi malaria in Rhesus monkeys with sulphanilamide and their susceptibility to reinfection. Amer. J. trop. Med., 18: 715-21. Coggeshall, L. T., and M. D. Eaton. 1938. The complement fixation reaction in monkey malaria. J. exp. Med., 67: 871-81. —— 1938. The quantitative relationship between immune serum and infec- tive dose of parasites as demonstrated by the protection test in monkey malaria. J. exp. Med., 68: 29-38. Coggeshall, L. T., and H. K. Kumm. 1938. Effect of repeated superinfection upon the potency of immune serum of monkeys harboring chronic in- fections of Plasmodium knowlesi. J. exp. Med., 68: 17-27. Coventry, F. A. 1925. The reaction product which inhibits reproduction of the trypanosomes in infections with Trypanosoma lew7si, with reference to its change in titer throughout the course of infection. Amer. J. Hyg., 5: 127-44. —— 1929, Experimental infections with Trypanosoma lewisi in the guinea- pig. Amer. J. Hyg., 9: 247-59. Craig, C. F. 1937. Observations upon the practical value of the complement- fixation test in the diagnosis of amebiasis. Amer. J. publ. Hlth., 27: 689-93. Culbertson, J. T. 1938. Natural transmission of immunity against Trypano- soma lewisi from mother rats to their offspring. J. Parasit., 24: 65-82. Culbertson, J. T., and R. M. Wotton. 1939. Production of ablastin in rats of different age groups after infection with Trypanosoma lewisi, Amer. j. Hye730 ‘Geer @)ealoi=i3° Dahmen, H. 1922. Die Serodiagnostik der Beschalseuche. Arch. wiss. prakt. Tierheilk., 47: 319-53. Davis, L. J. 1931. Experimental feline trypanosomiasis with especial reference to the effect of splenectomy. Ann. trop. Med. Parasit., 25: 79-90. Delanoé, P. 1912. L’Importance de la phagocytose dans l’immunité de la souris a l’égard de quelques flagelles. Ann. Inst. Pasteur, 26: 172-203. Dobell, C. 1912. Some recent work on mutation in microorganisms. J. Genet., 2: 201-20. Duca, C. J. 1939. Studies on age resistance against trypanosome infections: II. The resistance of rats of different age groups to Trypanosoma lewisi, and the blood response of rats infected with this parasite. Amer. J. Hyg., 29: \(Sects.G), 25-32. IMMUNOLOGY 881 Duke, H. L. 1936. Recent observations on the biology of the trypanosomes of man in Africa, Trans. R. Soc. trop. Med. Hyg., 30: 275-96. Duke, H. L., and J. M. Wallace. 1930. ‘Red cell adhesion” in trypanosomiasis of man and animals. Parasitology, 22: 414-56. Du Toit, P. J. 1919. Experimentelle Studien tber die Pferdepiroplasmose. Arch. Schiffs- u. Tropenhyg., 23: 121-35. Eaton, M. D. 1938. The agglutination of Plasmodium knowlesi by immune serum. J. exp. Med., 67:857-70. Ehrlich, P., and K. Shiga. 1904, Farbentherapeutische Versuche bei Trypano- somenerkrankung. Berl. klin. Wschr., 41: 329-32, 362-65. Findlay, G. M., and H. C. Brown, 1934. The relation of the electric charge of the red cells to phagocytosis in avian malaria. Brit. J. exp. Path., 15: 148-53. Gay, F. P. 1931. Tissue resistance and immunity. J. Amer. med. Ass., 97: 195-99. Gingrich, W. 1932. Immunity to superinfection and cross-immunity in malarial infections of birds. J. prev. Med. Lond., 6: 197-246. —— 1934. The effect of an increased burden of phagocytosis upon natural and acquired immunity to bird malaria. J. Parasit., 20: 332-33. Golgi, C. 1888. Il fagocitismo nell’infezione malarica. Rif. med., 4. Greig, E. D. W., C. E. van Rooyen, and E. B. Hendry. 1934. Observations on the melano-precipitation serological reaction in malaria. Trans. R. Soc. trop. Med. Hyg., 28: 175-92. Hartman, E. 1927. Certain interrelations between Plasmodium praecox and its host. Amer. J. Hyg., 7: 407-32. Hegner, R. W. 1926. The biology of host-parasite relationships among Pro- tozoa living in man. Quart. Rev. Biol., 1: 393-418. Hegner, R., and L. Eskridge. 1938. Mortality of merozoites in infections with Plasmodium cathemerium in canaries. Amer. J. Hyg., 28: 299-316. Hegner, R., and R. Hewitt. 1938. The influence of young red cells on in- fections of Plasmodium cathemerium in birds. Amer. J. Hyg., 27: 417-36. Henry, X. 1934. Seroflokkulation bei Malaria. Technik und anwendung in der Praxis. Arch. Schiffs- u. Tropenhyg., 38: 93-100. Herrick, C. A., and S. X. Cross. 1936. The development of natural and artificial resistance of young rats to the pathogenic effects of the parasite Trypanosoma lewisi, J. Parasit., 22: 126-29. Hewitt, R. 1938. Multiple-infected red cells in avian malaria. Amer. J. Hyg., 28: 321-44. Hope-Gill, C. W. 1938. A study of the reaction rate of the serum-formalin reaction in Trypanosoma gambiense sleeping sickness. Trans. R. Soc. trop. Med. Hyg., 31: 507-16. 882 IMMUNOLOGY Hu, C. H., and J. R. Cash. 1927. Considerations of the relationship of the reticulo-endothelial system to kala-azar. Proc. Soc. Exp. Biol. N.Y., 24: 469-72. Huff, C. G., and W. Bloom. 1935. A malarial parasite infecting all blood and blood-forming cells of birds. J. infect. Dis., 57: 315-36. Jaffé, R. 1931. The recticulo-endothelial system in immunity. Physiol. Rev., 11°27 7-32ik —— 1938. The reticulo-endothelial system. J77 Downey: Handbook of Hema- tology, 2: 973-1272. James, W. M. 1913. Notes on the etiology of relapse in malarial infections. Jainfect: Dist al29777-325- Johnson, C. M., and R. A. Kelser. 1937. The incidence of Chagas’ disease in Panama as determined by the complement-fixation test. Amer. J. trop. Med., 17: 385-92. Johnson, T. L. 1929. In vivo trypanolysis with especial reference to ‘‘zones of inhibition,” relapse phenomena and immunological specificity. Amer. J. Hyg., 9: 260-82. Jungeblut, C. W. 1930. Die Palanan des retikulo-endothelialen Systems fiir die Infektion und Immunitat. Eiebat Hyg. Bakt., 11: 1-67. Jurgens, R. J. 1902. Beitrag zur Biologie der Rattentrypanosomen. Arch. Hyg. Berl., 42: 265-88. Kanthack, A. A., H. E. Durham, and W. F. H. Blandford. 1898. On nagana, or tsetse-fly disease. Proc. roy. Soc., 64: 100-18. Kauders, O. 1927. Immunititsstudien bei Impfmalaria. Zbl. Bakt., I, Orig., 104: 158-60. Knowles, R., and B. M. Das Gupta. 1928. Laboratory studies in surra. Indian J. med. Res., 15: 997-1058. 1930. Studies in untreated malaria. Indian med. Gaz., 65: 301-10. Koch, R. 1899. Uber die Entwickelung der Malariaparasiten. Z. Hyg. In- fektKr., 32: 1-24. Kolmer, J. A., and J. F. Schamberg, with the assistance of A. Rule and B. Madden. 1933. The influence of reticuloendothelial “blockade” and splenectomy upon experimental trypanosomiasis and syphilis and the chemotherapeutic properties of arsphenamine and neoarsphenamine. Amer. J. Syph., 17: 176-87. Krijgsman, B. S. 1933. Biologische Untersuchungen ber das System: Wirt- stier-Parasit. 1 & 2 Teil: die Entwicklung von Trypanosoma evansi in Maus und Ratte. Z. Parasitenk. 5: 592-678. Kroo, H. 1925. Beitrag zur Immunbiologie der Trypanosomen. Ueber Stam- meinheit und Arteinheit des Trypanosomo brucei. Z. Hyg. InfektKr., 105: 247-53. —— 1926. Weiterer Beitrag zur Immunbiologie der Trypanosomen. Zur Kritik des Kreuzinokulationsverfahrens als immunbiologische Methode der Artabgrenzung. Z. Hyg. InfektKr., 106: 77-82. IMMUNOLOGY 883 —— 1936. Die spontane, apathogene Tryanosomeninfektion der Kaninchen. Z. ImmunForsch., 88: 117-28. Kuhn, L. R. 1938. The effect of splenectomy and blockade on the protective titer of antiserum against Trypanosoma equiperdum. J. infect. Dis., 63: 217-24. Laveran, A. 1908. Sur quelques altérations de la rate chez les cobayes in- fectés de trypanosomes. Bul. Soc. Path. exot., 1: 393-98. 1917. Leishmanioses. Paris. Laveran, A., and F. Mesnil. 1901. Recherches morphologiques et expéri- mentales sur le trypanosome des rats (Tr. Jewss7 Kent.). Ann. Inst. Pasteur, 15: 673-715. ——— 1912. Trypanosomes et trypanosomiases. Paris. Leger, A., and J. Ringenbach. 1911. Sur la spécificité de la propriété try- panolytique des sérums des animaux trypanosomiés. C. R. Soc. Biol. Paris, 70: 343-45. —— 1912. Sur la spécificité de la propriété trypanolytique des sérums des animaux trypanosomiés. C. R. Soc. Biol. Paris, 72: 267-69. Levaditi, C., and S. Mutermilch. 1909. Recherches sur la méthode de Bordet et Gengou appliquée a l'étude des trypanosomiases. Z. ImmunForsch., Onige 225702222. —— 1910. I. Mécanisme de la phagocytose. C. R. Soc. Biol. Paris, 68: 1079- 81. —— 1911. Le Diagnostic de la maladie du sommeil par |’examen des pro- priétés attachantes du sérum. C. R. Acad. Sci. Paris, 153: 166. Linton, R. W. 1929. The reticulo-endothelial system in protozoan infections. Arch. Pathol., 8: 488-501. Lorando, N., and D. Sotiriades. 1937. Treatment of malaria with immune Blood. Trans. R. Soc. trop. Med. Hyg., 31: 227-34. Lourie, E. M. 1934. Studies on chemotherapy in bird malaria. Ann. trop. Med. Parasit.,. 287) 151-69), 255-77 95 13-23. Lourie, E. M., and R. J. O'Connor. 1937. A study of Trypanosoma rhodest- ense relapse strains in vitro. Ann. trop. Med. Parasit., 31: 319-40. Lowe, J. 1934. Studies in untreated malaria. Numerical studies of the para- sites in relation to the fever. Rec. Malar. Surv. India, 4: 223-41. McLay, K. 1922. Malaria in Macedonia, 1915-1919. Part III. Haematological investigations on malaria in Macedonia. Jour. R. Army med. Cps., 38: 93-105. MacNeal, W. J. 1904. The life history of Trypanosoma lewisi and Trypano- soma brucei. J. infect. Dis., 1: 517-43. Manteufel, P. 1909. Studien ttber die Trypanosomiasis der Ratten mit Be- riicksichtigung der Ubertragung unternatiirlichen Verhiltnissen und der Immunitat. Arb. GesundhAmt. Berl., 33: 46-83. Manwell, R. D. 1938. Reciprocal immunity in the avian malarias. Amer. J. Hyg., 27: 196-211. 884 IMMUNOLOGY Manwell, R. D., and F. Goldstein. 1939. Strain immunity in avian malaria. Amer); Hyg 0.sec. Cs 15-22% Marchand, F. 1924. Die Ortlichen reacktiven Vorginge in Krehl und March- and: Handb. d. Allgemein. Pathol., 4: 78-649. Marrack, J. R. 1938. The chemistry of antigens and antibodies. Special Rep. Ser. Med. Res. Counc., No. 230. Massaglia, M. A. 1907. Des causes des crises trypanolytiques et des rechutes qui les suivent. C. R. Acad. Sci, Paris, 145: 687-89. Maximow, A. 1927a. Bindegewebe und blutbildende Gewebe. In Handb. Mikrosk. Anatomie d. Menschen, 2:232-583. Berlin. —— 1927b. Morphology of the mesenchymal reactions, Arch. Pathol. Lab. Med., 4: 557-606. Meleney, H. E. 1925. The histopathology of kala-azar in the hamster, monkey and man. Amer. J. Path., 1: 147-67. Meleney, H. E., and W. W. Frye. 1937. Practical value and significance of the complement-fixation reaction in amebiasis. Amer. J. publ. Hlth., 27: 505-10. Menendez, P. E. 1932. Serological relationships of Extamoeba histolytica. Amer. J. Hyg., 15: 785-808. Menon, T. B., D. R. Annamalai, and T. K. Krishnaswami. 1936. The value of the aldehyde and stiburea tests in the diagnosis of kala-azar. J. trop. Med. Hyg., 36: 92-95. Mesnil, F., and E. Brimont. 1909. Sur les propriétés protectrices du sérum des animaux trypanosomiés: Races résistantes 4 ces sérums. Ann. Inst. Pasteur, 23: 129-54. Metschnikoff, E. 1892. Lecons sur la pathologie comparée de l’inflammation. Paris. —— 1905. Immunity in infective diseases (Trans. of French book of 1901). Cambridge. Mohler, J., A. Eichhorn, and J. Buck. 1913. The diagnosis of dourine by complement fixation. J. agric., Res., 1: 99-107. Mulligan, H. W. 1935. Descriptions of two species of monkey Plasmodium isolated from Szlenus irus. Arch. Protistenk., 84: 285-314. Mulligan, H. W., and S. A. Sinton. 1933. Studies in immunity in malaria. Rec. Malar. Surv. India, 3:529-68; 809-39. Nauck, E. G., and B. Malamos. 1935. Uber Immunitat bei Affenmalaria. Z. ImmunForsch, 84: 337-58. Neporojny, S. D., and W. L. Yakimoff. 1904. Uber einige pathologischana- tomische Verainderungen bei experimentellen Trypanosomosen. Zbl. Bakt., Ref. 35: 467-68. Neumann, H. 1933. Der Nachweis des parasitiziden Antikérpers bet der Ma- laria des Menschen. Riv. Malariol., 12: 319-34. IMMUNOLOGY 885 Nieschulz, O., and A. Bos. 1931. Uber den Einfluss der Milz auf den In- fektionsverlauf von Surra bei Hunden. Dtsch, tierarztl. Wschr. 39:488- 89. Nieschulz, O., and F. K. Wawo-Roentoe. 1930. Uber den Einfluss der Milzexs- tirpation bei Infektionen mit Trypanosoma gambiense und Schizotry- panum cruzt. Z. ImmunForsch., 65: 312-17. Noguchi, H. 1926. Comparative studies of herpetomonads and leishmanias. J. exp. Med., 44: 327-37. Paulson, M., and J. Andrews. 1938. Complement fixation in amebiasis. A comparative evaluation in clinical practice. Arch. intern. Med., 61:562- 78. Perla, D., and J. Marmorston-Gottesman. 1930. Further studies on T. lew7si infection in albino rats. J. exp. Med., 52: 601-16. Proske, H. O., and R. B. Watson. 1939. The protein tyrosin reaction. Publ. Hlth. Rep. Wash., 54: 158-72. Rabinowitsch, L., and W. Kempner. 1899. Beitrag zur Kenntniss der Blut- parasiten, speciell der Rattentrypanosomen. Z. Hyg. InfaktKr., 30: 251- 94, Redmond, W. B. 1939. The cross-immune relationship of various strains of Plasmodium cathemerium and P. relictum. J. infect. Dis. 64: 273-87. Regendanz, P. 1932. Uber die Immunititsvorgange bei der Infektion der Rat- ten mit Trypanosoma lewisi. Z. ImmunForsch., 76: 437-45. Regendanz, P., and W. Kikuth. 1927. Uber die Bedeutung der Milz fiir die Bildung des vermehrungshindernden Reaktionsproduktes (Taliaferro) und dessen Wirkung auf den Infektionsverlauf der Ratten-Trypano- somiasis (Tryp. lewrs7). Zbl. Bakt., Abt. 1. Orig. 103: 271-79. Rieckenberg, P. 1917. Eine neue Immunititsreaktion bei experimenteller Try- panosomen-Infektion: die Blutplattchenprobe. Z. ImmunForsch., 26: 53-64. Ritz, H. 1914. Uber Rezidive bei experimenteller Trypanosomiasis. Dtsch. med. Wschr., 40: 1355-58. —— 1916, Uber Rezidive bei experimenteller Trypanosomiasis. Arch. Schiffs- u. Tropenhyg., 20: 397-420. Robertson, M. 1912. Notes on the polymorphism of Trypanosoma gambiense in the blood and its relation to the exogenous cycle in Glossina palpalis. Rept. Sleep. Sickn. Comm. roy. Soc., 13: 94-110. — 1929. The action of acriflavine upon Bodo caudatus. Parasitology, 21: 375-416. Robinson, E. M. 1926. Serological investigations into some diseases of do- mesticated animals in South Africa caused by trypanosomes, Eleventh and Twelfth Rept. vet. Res. S. Afr. (Sept.), pp. 9-25. Rodet, A., and G. Vallet. 1906. Contribution 4 l'étude des trypanosomiasis. Arch, Méd. exp., 18: 450-94. 886 IMMUNOLOGY Ross, R. 1910. The prevention of malaria. New York. Ross, R., and D. Thomson. 1910. A case of sleeping sickness studied by pre- cise enumerative methods: regular periodical increase of the parasites disclosed» Prog toy. Soc., BY 8237414-15: —— 1911. A case of sleeping-sickness studied by precise enumerative meth- ods. Further observations. Proc. roy. Soc., B, 83: 187-205. Roudabush, R. L., and E. R. Becker. 1934. The development of Trypanosoma iowensis in the blood of the striped ground squirrel, C/tellus tridecem- lineatus. Iowa St. Coll. J. Sct., 8: 533-35. Roudsky, D. 1911. Mécanisme de l'Immunité naturelle de la souris vis-a-vis du Trypanosoma lewisi Kent. C. R. Soc. Biol. Paris, 70: 693-94. Rouget, J. 1896. Contribution a l’étude du trypanosome des mammiferes. Ann. Inst. Pasteur, 10: 716-28. Rudolph, G. de M., and J. C. Ramsay. 1927. Enumeration of parasites in therapeutic malaria. J. trop. Med. Hyg., 30: 1-8. Russeff, C. 1935. Der Einfluss der Milzexstirpation auf den Verlauf der Dourineinfektion bei verschiedenen Versuchstieren. Z. ImmunForsch., 84: 295-99. Sauerbeck, E. 1905. Beitrag zur pathologischen Histologie der experimentellen Trypanosomen-Infection (mit Trypanosoma brucez). Z. Hyg. InfektKr., 52; 3ie8G: Schilling, C. 1902. Bericht uber die Surra-Krankheit der Pferde und Rinder im Schutzgebiete Togo. Zbl. Bakt. I. Orig. 31: 452-59. Schilling, C., and H. Neumann. 1932. Zur Methodik der immunologischen Differenzierungsmethoden von Trypanosomenstémmen. Arch. Schiffs- u. Tropenhyg., 36: 214-29. Schwetz, J. 1934. L'Influence de la splénectomie sur |’evolution des trypano- somes pathogénes (Tr. gambiense et Tr. congolense) chez les rats et les souris. Bull. Soc. Path. exot., 27: 253-60. Sergent, Ed. 1936. Immunité ou prémunition dans les maladies a hémocyto- zoaires (paludismes, piroplasmoses). Arch. Inst. Pasteur Algé., 14: 413- LG. Sergent, Ed., and Et. Sergent. 1918. Sur le paludisme des oiseaux du au Plasmodium relictum (vel Proteosoma). Ann. Inst. Pasteur, 32: 382-88. Sinton, J. A., Harbhagwan, and J. Singh. 1931. The numerical prevalence of parasites in relation to fever in chronic benign tertian malaria. Indian J. med. Res., 18: 871-79. Sinton, J. A., and H. W. Mulligan. 1933a. Mixed infections in the malaria of the lower monkeys. I. Mixed infections as the cause of apparent varia- tions in the morphology and pathogenicity of simian Plasmodia. Rec. Malar. Surv. India, 3:719-67. — 1933b. Mixed infections in the malaria of the lower monkeys. II. The probable occurrence of mixed infections in some of the older records of monkey malaria. Rec. Malar. Surv. India, 3: 769-808. IMMUNOLOGY 887 Sotiriadés, D. 1917. Essais de sérothérapie dans la malaria. Grece méd., 19: 27-28. Stauber, L. A. 1939. Factors influencing the asexual periodicity of avian imalanias. §) Patasit., 25) 95-116: Steffan, P. 1921. Beobachtungen tiber den Verlauf der kiinstlichen Infektion der Ratte mit Trypanosoma lewisi. Arch. Schiffs- u. Tropenhyg., 25: 241- 47. Stockman, S., and W. G. Wragg. 1914. Cross immunisation with Piroplasma bigeminum and Piroplasma divergens. J. comp. Path., 27: 151-55. Stratman-Thomas, W. K. 1935. Studies on benign tertian malaria. Amer. J. Phyo 22 561-63. Taliaferro, L. G. 1925. Infection and resistance in bird malaria, with special reference to periodicity and rate of reproduction of the parasite. Amer. ye lye. 5: 742-89. —— 1928. Return to normal of the asexual cycle in bird malaria after re- tardation by low temperatures in vitro, J. prev. Med. 2: 525-40. Taliaferro, W. H. 1924. A reaction product in infections with Trypanosoma lewisi which inhibits the reproduction of the trypanosomes. J. exp. Med., 393, T7190: —— 1929. The immunology of parasitic infections. New York. — 1932. Trypanocidal and reproduction-inhibiting antibodies to Trypano- soma lewisi in rats and rabbits. Amer. J. Hyg., 16: 32-84. 1934. Some cellular bases for immune reactions in parasitic infections. J. Parasit. 20: 149-61. —— 1938. The effects of splenectomy and blockade on the passive transfer of antibodies against Trypanosoma lewisi. J. infect. Dis., 62: 98-111. Taliaferro, W. H., and P. R. Cannon. 1936. The cellular reactions during primary infections and superinfections of Plasmodium brasilianum in Panamanian monkeys. J. infect. Dis., 59: 72-125. Taliaferro, W. H., P. R. Cannon, and S. Goodloe. 1931. The resistance of rats to infection with Trypanosoma lewisi as affected by splenectomy. mimer. > Hye. 14: 1-37, Taliaferro, W. H., and C. G. Huff. 1939. The genetics of the parasitic pro- tozoa. (In press.) Taliaferro, W. H., and T. L. Johnson. 1926. Zone phenomena in in vivo trypanolysis and the therapeutic value of trypanolytic sera. J. prev. Med. i 85-123. Taliaferro, W. H., and H. W. Mulligan. 1937. The histopathology of ma- laria with special reference to the function and origin of the macrophages in defence. Indian med. Res. Mem., No. 29. Pp. 138. Taliaferro, W. H., and Y. Pavlinova. 1936. The course of infection of Trypan- osoma duttoni in normal and in splenectomized and blockaded mice. J. Parasit., 22: 20-41. 888 IMMUNOLOGY Taliaferro, W. H., and L. G. Taliaferro. 1922. The resistance of different hosts to experimental trypanosome infections, with especial reference to a new method of measuring this resistance. Amer. J. Hyg., 2: 264-319. — 1929a. Acquired immunity in avian malaria. I. Immunity to superinfec- tion. J. prev. Med. 3: 197-208. —— 1929b. Acquired immunity in avian malaria. I]. The absence of protec- tive antibodies in immunity to superinfection. J. prev. Med. 3: 209-23. —— 1934a. Morphology, periodicity and course of infection of Plasmodium brasilianum in Panamanian monkeys. Amer. J. Hyg., 20: 1-49. — 1934b. Alteration in the time of sporulation of Plasmodium brasilianum in monkeys by reversal of light and dark. Amer. J. Hyg., 20: 50-59. —— 1934c. Superinfection and protective experiments with Plasmodium brasilianum in monkeys. Amer. J. Hyg., 20: 60-72. —— 1934d. Complement fixation, precipitin, adhesion, mercuric chloride and Wassermann tests in equine trypanosomiasis of Panama (murrina). J. Immunol., 26: 193-213. Theiler, A. 1912. Weitere Unterschungen tiber die Anaplasmosis der Rinder und deren Schutzimpfung. Z. InfektKr. Haustiere, 11: 193-207. Thomson, J. G. 1912. Enumerative studies on T. bruce7 in rats and guinea- pigs, and a comparison with T. rhodesiense and T. gambiense. Ann. trop. Med. Parasit., 5: 531-36. — 1933. Immunity in malaria. Trans. R. Soc. trop. Med. Hyg., 26: 483- 514. Topley, W. W. C. 1935. An outline of immunity. Baltimore. Trensz, F. 1936. La Valeur pratique de la mélanofloculation de Henry. Arch. Inst. Pasteur Algér., 14: 353-90. Van den Branden, F. 1935. Sur le rapport du poids de la rate ou du foie au poids du corps, chez des rats blancs (variété albinos de Mus decumanus) non infectés, ainsi que chez les animaux de méme espéce, préalablement infectés de Trypanosoma congolense ou de Trypanosoma brucei, puis guéris ou non guéris par traitement. C. R. Soc. Biol. Paris, 119: 529-30. Vaucel, M., and Hoang-Tich-Try. 1936. Reactions de malaria-floculation au Tonkin. Bull. Soc. méd.- chir. Indochine, 14: 1101-14. Villain, G., and R. Dupoux. 1936. Contribution 4 l'étude serologique du paludisme. Utilisation d’une mélanine artificielle: la M. A. floculation. Arch. Inst. Pasteur Afr. N., 25: 469-551. Wagener, E. H., and D. A. Koch. 1926. The biological relationships of Leishmania and certain herpetomonads. Univ. Cal. Publ. Zool., 28: 365-88. Wallace, J. M., and A. Wormall. 1931. Red-cell adhesion in trypanosomiasis of man and other animals. I]. Some experiments on the mechanism of the reaction. Parasitology, 23: 346-59. Wasielewski, T. K. W. N. von. 1901. Uber die Verbreitung und kiinstliche Ubertragung der Vogelmalaria. Arch. Hyg. Berl., 41: 68-84. IMMUNOLOGY 889 Wasielewski, T. K. W. N. von, and G. Senn. 1900. Beitrage zur Kenntniss der Flagellaten des Rattenblutes. Z. Hyg. InfektKr. 33: 44-72. Watson, C. J. 1928. The pathology of histoplasmosis (Darling) with special reference to the origin of the phagocytic cells. Folia haemat., 37: 70-93. Watson, E. A. 1920. Dourine in Canada, 1904-1920: History, research and suppression. Canada, Dept. Agric. Health of Animals Branch. Wells, H. G. 1929. The chemical aspects of immunity. 2d ed., New York. Wenyon, C. M. 1926. Protozoology. 2 vols., New York. Yakimoff, W. L. 1908. Contribution aux altérations du sang des animaux atteints de trypanosomiases expérimentales. Arch. Sci. biol. St. Pétersb., 13: 243-76. Zdrodowski, P. 1931. Sur la sérologie comparée du groupe de Leishmanies d’origine humaine et canine. Bull. Soc. Path. exot., 24: 37-41. CHAPTER XIX RELATIONSHIPS BETWEEN CERTAIN PROTOZOA AND OTHER ANIMALS HAROLD Kirby, JR.* IN THE LITERATURE in which consideration is given to close rela- tionships between organisms of different species the effort is often made to group the associations discussed under definite categories. The cate- gories are defined, and it is shown in what manner and to what extent each separate association can be referred to its proper position. A reader of this literature soon becomes sensible of the lack of agreement in al- most every major particular. Unlike names are given to the categories, definitions are dissimilar, there is difference of opinion or lack of exact information on the nature of the relationship itself, and the impossibility of making unequivocal distinctions is apparent in many instances. In order to make an advance toward harmony of opinion, the bionomics of many groups of living things must be taken into consideration. The author must here confine his discussion to a statement of what he re- gards as a satisfactory denomination of the types of relationship be- tween Protozoa and other organisms, including other Protozoa, with which they are intimately associated. At the outset, it is apparent that a comprehensive term is necessary to designate all types of relationship between Protozoa and their hosts, whether the Protozoa are epibiotic or endobiotic, whether they live at the expense of their hosts or aid them in some way. Such a term would be applicable also in the instances in which it is uncertain in what sub- division an association belongs. The choice should be between two exist- ing terms, parasitism and symbiosis, the latter of which has etymo- logically exactly the meaning desired. Both words have been used in the general sense. The word parasite, however, has by universal agree- ment been used to designate an organism that lives at its host’s expense, * Assistance rendered by personnel of Work Projects Administration, Official Project number 65-1-08-113, Unit C1, is acknowledged. PROTOZOA AND OTHER ANIMALS 891 obtaining nutriment from the living substance of the latter, depriving it of useful substance, or exerting other harmful influence upon it. In the interests of exactitude, a word should, if possible, express a single defi- nite idea; and therefore it seems undesirable to use parasitism also in a general sense if it can be avoided. There is much justification for applying the term symbiosis to the general relationship under consideration, although many authors have given it a restricted applicability to mutually advantageous associations only. The word in this restricted meaning has, in fact, acquired what- ever sanction general usage confers. Most textbooks in biology and zoology, as well as protozodlogy and parasitology, so define it, the oldest one noted being T. J. Parker’s (1893); and Hegner (1926b) restricted the meaning further, stating that in symbiosis life apart is impossible. J. A. Thomson (1934; with Geddes, 1931; also in the Encyclopaedia Britannica fourteenth ed.) contended that symbiosis is a mutually beneficial internal relationship, and that externally mutual- istic relationships are commensalism. To Haupt (1932) symbiosis includes what others consider commensalism, but does not include parasitism; the same sense is implicit in some dictionary definitions. The extended meaning of the word has been found in only two general biology texts (McFarland, 1913; Eikenberry and Waldron, 1930); it has that meaning also in the article on symbiosis in the New Interna- tional Encyclopaedia (1925). Most important, however, is the report of Hertig, Taliaferro, and B. Schwartz (1937). The sense in which the word was employed by its originator, A. de Bary (1879), is of decisive importance. The three members of the Committee on Terminology of the American Society of Parasitologists, as well as W. Schwartz (1935), appear to have been the only ones among recent authors to understand de Bary. It has been widely stated that he meant symbiosis to designate mutually beneficial relationships (by Caullery, 1922; Hegner, 1926b; as well as by the others cited in the Committee’s report). The Committee gave quotations from de Bary showing clearly that he used symbiosis as a collective term, the subdivisions of which include parasitism and mutualism; he recog- nized two main categories, antagonistic and mutualistic symbiosis. The results of the writer's examination of de Bary’s paper are in complete agreement with their interpretation; there is no ambiguity in de Bary’s 892 PROTOZOA AND OTHER ANIMALS usage. Hertwig (1883) had a similar understanding of the meaning of the word; parasitism and mutualism, he stated, are types of symbiosis. As the Committee pointed out, he changed his usage later. In recent literature other opinions that the words should be used in this original sense have appeared. W. Schwartz (1935) took that atti- tude, although he would restrict symbiosis to the relationship in which there is physiological dependence of one partner on another. Cleve- land (1926) remarked that it would be much better to use it in the gen- eral sense, if the change could be made. Van Beneden (1876) referred to certain associated animals as mutual- ists, before the term symbiosis had been coined. The word conveys the idea of reciprocal benefit, although the examples he described indicate a vague concept on his part of the relationships concerned, and none of them would now be regarded as mutualistic. (He discussed among mutualists parasitic copepods, opalinids, endozoic rotifers, and even Vaginicola on Gammarus). He recognized the three types of associa- tion: commensalism, mutuality, and parasitism. If symbiosis is used in the broad sense, reciprocal relationships should be termed mutualism or mutualistic symbiosis. Protozoa that live in natural cavities of the body, such as the mantle cavities of molluscs and the lumen of the alimentary canal, but do not nourish themselves at the expense of the host, have been termed in- quilines (Caullery, 1922; Grassé, 1935). All inquilines are commensals, but not all commensals are inquilines. There are also ectozoic com- mensals, or ectocommensals; endocommensalism is equivalent to in- quilinism. In a sense, inquilines, like ectozoic symbionts, have not in- vaded the body itself. They occupy cavities open to the outside. Endozoic Protozoa which invade the interior of the body proper, living intracellularly, among tissue cells, or in blood or coelomic cavi- ties, are all parasitic. The conditions of their nutrition necessarily involve strict dependence on the host. Ectozoa and inquilines, which usually are commensals, may become parasites when in their nutritive processes they develop one or another means of using the substance of the host, generally by attack upon, or extraction of substance directly from, the epithelial cells. They are also called parasites when they consume enough material that would otherwise be used by the host to make a difference to it; or when in some way not connected with nutrition they injure the PROTOZOA AND OTHER ANIMALS 893 host without invading its substance. There are few instances among Protozoa in which injurious effects of this type have been proved. There are only two ways in which a protozo6n can directly benefit a larger animal. It may contribute its own body to be used in the nutri- tion of the host; but such a relationship would not constitute mutualism unless some essential substance not otherwise obtained is thus supplied. Another way is action upon the food materials such as to make a sub- stance usable that otherwise would not be. The latter is the situation in the only proved instance of significant mutualism between Protozoa and their hosts—that between flagellates and certain termites, as well as Cryptocercus punctulatus. Another, indirect, benefit might be con- ferred by aid in controlling an injurious organism or substance, but no instance of that is known with certainty. In all its ramifications, the problem of the symbiotic relationships between Protozoa and other animals is far too large for concise treat- ment. It has been considered in various textbooks of protozodlogy, par- ticularly in the chapter on “Ecology, Commensalism, and Parasitism’”’ in Calkins (1933) and in Doflein-Reichenow ( (1927-29). Protozoan relationships are discussed, together with associations in other groups of animals, in Caullery (1922) and Grassé (1935). An important gen- eral article on the subject is that by Wenrich (1935); and a general dis- cussion was published by Fantham (1936). Various aspects have been discussed by Hegner (1924, 1926a, 1926b, 1926c, 1928, 1937), by Metcalf (1923, 1929), by Cleveland (1926, 1934), by contributors to Hegner and Andrews (1930), by Becker (1932, 1933), and by Kirby (i937)? In undertaking to make a contribution to the subject, the author recog- nized two possible paths of approach. Either he could attempt to make a comprehensive scrutiny of the entire expanse of pertinent information, or he could explore in as much detail as possible certain chosen fields of inquiry. The former approach would lead to a generalized account, with selected and perhaps original illustrations; and it would in large part reiterate existing, readily accessible, sometimes commonplace con- cepts. The latter course, although less exhaustive, permits selection for more detailed consideration of certain representative topics; that is the course which has in the main been followed here. In the ecology of symbiotic relationships, an introductory chapter is 894 PROTOZOA AND OTHER ANIMALS provided by accidental and facultative parasitism. Among Protozoa this is best exemplified by certain holotrichous ciliates. As the subject de- velops, instances in which certain genera contain both free-living and symbiotic species come under consideration, together with examples of closely related genera in the two types of habitat. The situation may be illustrated by certain euglenid and some polymastigote flagellates, and again by certain holotrichous ciliates. In certain groups of Protozoa, in- structive series in degree of adaptation and types of relationship appear. One of the most fruitful of these series, in which the range is from incidental commensalism to strict parasitism, is found in the Thig- motricha. Host relationships of a variety of types, which bring out also the adaptation of life cycles to the conditions of symbiotic existence, are found among certain holotrichs of the marine Crustacea, Conidio- phrys pilisuctor, and the Apostomea. There is more or less morpho- logical alteration and adaptation in the symbiotic holotrichs considered above, particularly in the Thigmotricha; and this is also well brought out in the development of attachment structures in Ptychostomidae, Astomata, peritrichs, and certain flagellates of termites. Animals of certain specific groups are characterized by protozoan faunules of particular types. That does not express only the common- place fact that examination of particular hosts will reveal particular symbionts, but makes the point that related hosts often have faunules of similar composition. In this matter of distribution of symbionts among species of hosts is incorporated the problem of specificity in symbiosis, the quality of a symbiont of being restricted to certain hosts. This quality is known as host-specificity. The incorrectness of the term “host-parasite specificity” has been commented on by Hertig, Talia- ferro, and Schwartz (1937). Faunules of two groups of animals are discussed here in their composition, host-specificity, and distributional characteristics: the ciliates of sea urchins, in which there is in part rela- tionship to free-living species; and the Protozoa of termites and Crypto- cercus, in which the symbiotic relationship has reached a maximum de- velopment. Finally, physiological host relationships are discussed in detail in two groups of Protozoa: Ophryoscolecidae in ruminants, and flagellates in termites and Cryptocercus. The former relationship, though long sus- pected of being mutualism, is probably simple commensalism; there is PROTOZOA AND OTHER ANIMALS 895 general agreement on the mutualistic character of the latter. To com- plete this series, relationships of strict parasitism should be discussed from the standpoint of the physiology of the parasite and the effect on the host. The absence of this is readily compensated for, however, in the abundant literature of parasitology; and examples of parasitism are described in almost all groups considered in this chapter. As is apparent, the material is grouped under certain headings; but in every section data bearing on various topics in symbiotic rela- tionships will be found. To group all facts under specific topics would involve much dislocation in other respects; and it has seemed prefer- able to preserve systematic continuity to a considerable extent. ACCIDENTAL AND FACULTATIVE PARASITISM In connection with the origin of host relationships, it is of interest to consider instances in which organisms can develop in both free- living and symbiotic habitats. Accidental and facultative parasitism, therefore, come up for primary consideration. Facultative parasites, as opposed to obligate parasites, are able to live either associated with hosts or not, but parasitism is a natural occurrence in the bionomics of the species. Accidental parasitism is that of a naturally free-living species, which happens through some accident to become parasitic. Although the two types are not identical, they are obviously closely related and categorical separations are not attempted in the discussion. Mercier and Poisson (1923) pointed out that those forms that are most ubiquitous and are preadapted to varied modes of nutrition have the best chance of surviving in the new medium into which they are intro- duced accidentally. For such forms Giard (1880) used the term “‘in- choate parasitism”; Giard had reference to the incomplete and tempo- rary parasitism illustrated by the occurrence of geophilids in the nasal cavities of man. These conditions are met by the ciliate Glaucoma pyriformis. Under natural conditions G. pyriformis ingests bacteria. Hetherington (1933) stated that it is one of the commonest fresh-water Protozoa, appearing in the early stages of the usual infusions of hay, wheat, or lettuce if they are inoculated with pond water. (At that time he named the ciliate Col pidium cam pylum, but later [1936] reported that it is G. pyriformzs.) 896 PROTOZOA AND OTHER ANIMALS In 1923 Lwoff reported that he had succeeded in growing “Col pidium colpoda’’ in pure culture, with neither living nor dead microdrganisms, in a medium of peptone broth. He later revised the identification to Glaucoma piriformis, and in 1932 considered it to be exceptional and unique in its utilization of dissolved nutrient material only. Since then some other ciliates have been maintained in sterile, non-particulate cul- ture; but the number is limited, and some have turned out to be actually G. pyriformis (Hetherington, 1936). It has been shown that G. pyriformis, when introduced into the hemocoele of certain insects, multiplies rapidly and exhibits marked pathogenic potentialities. Lwoff (1924) inoculated the ciliate from pure culture into about thirty caterpillars of Galleria mellonella, all of which succumbed to the infection in from eight to fifteen days. Shortly before death, the blood contained no more leucocytes, but only great numbers of ciliates invading all parts of the body. The ciliates nourished themselves phagocytically at least in part, and contained many globules of fat from the fat bodies of the caterpillar. Janda and Jirovec (1937) injected bacteria-free cultures of G. pyri- forms into the body cavities of various invertebrates and vertebrates, and also brought them into contact with artificially produced wounds. At- tempts to infect annelids, molluscs, crustacea, fish, and amphibia failed, but many insects were successfully inoculated. The ciliates multiplied so rapidly as almost completely to fill the hemolymph in a few days. The fatty tissues especially were destroyed, the ciliates became larger than normal, and the infected insects usually died in a few days. Infec- tion through wounds was achieved only in the aquatic larvae of Aeschna cyanea. Infection by mouth did not occur. Glaucoma that had been para- sitic for some time when returned to the water survived and multi- plied normally. It appears, then, that insects’ blood is a favorable medium in which G. pyriformis may grow, and that the tissues often provide no protec- tion against the organism once it has entered. One would expect that occasionally so common a ciliate might enter an aquatic insect through an external wound or a damaged gut wall, and multiply in the same way with disastrous consequences to the host. That has, indeed, been found to take place. It is possible, as Wenyon (1926) suggested, that Lambornella PROTOZOA AND OTHER ANIMALS 897 stegomyiae (Fig. 194), found by Lamborn (1921) in mosquito larvae (Stegomyia scutellaris) in an earthenware pot in the Malay States, may actually be this species. All the infected larvae died in a few days; the ciliates escaped while the host was still living or soon after death. Keilin (1921), who described the species from formalin-preserved ma- BO ? iD : OD OTRAS Figure 194. Posterior end of larva of Aédes (Stegomyia) scutellaris parasitized by ciliates, Lambornella stegomyiae Keilin (=Glaucoma pyriformis ?). (After Keilin, 1921.) terial, regarded the ciliate as a true parasite, and others have agreed with him, apparently largely because of the epizoic character of the supposed cysts which Keilin found studding the external surface of one mosquito larva. There is no proof that these were cysts of the ciliate—one may, in fact, be justified in thinking it improbable that they were. If they were not, there seems to be no reason why Codreanu 898 PROTOZOA AND OTHER ANIMALS (1930), Lwoff (1932), and others should except it from the list of acci- dental parasites. The ciliates found by MacArthur (1922) in larvae of Theobaldia annulata and studied also by Wenyon (1926) showed but little difference in habit or appearance from Lambornella; and, con- sidering the inadequacy of Keilin’s material, may well have been the same. Wenyon concluded they were Glaucoma pyriformis. Next came the report by Treillard and Lwoff (1924) of the finding of ciliates corresponding to G. pyriformis in larvae of Chironomus plumosus bought at a market and probably obtained in the vicinity of Paris. Of 300 larvae, 13 were parasitized. The ciliates multipled ac- tively, causing death of the host in about eight days. In the cytoplasm were granules of yellow pigment, probably derived from hemoglobin. In 5 of the hosts conjugation was in progress, with all ciliates in any one larva at about the same stage. From another chironomid, Culicoides peregrinus in India, Ghosh (1925) reported Balantidium knowlesu. The ciliates were numerous in the “coelomic cavity’’; there is no statement as to whether the host was a larva or adult, or how many hosts there were. Though Grassé and Boissezon (1929) proposed the new genus Leptoglena for this very inadequately described ciliate, and it seemed to Lwoff (1932) and Codreanu (1930) to be a Glaucoma, it is impossible to recognize it from the description as any one of a considerable number of ciliates. No doubt it belongs in the list of accidental or facultative parasites. The same is true of Twrchiniella culicis, a new genus and species, described, from sections only, by Grassé and Boissezon (1929). The ciliates occurred in the hemocoele of an adult female Cu/ex. Boissezon (1930) suggested that adults may die on the surface of the water and the ciliates may escape and infect larvae; in the original paper it was considered that the parasites lived in larvae, and occurrence in the adult was an impasse. It must, in fact, be rare in adults if the ciliates are as pathogenic as G. pyriformis in other hosts is known to be, for an in- fected larva would then seldom transform into an adult. Codreanu (1930) and Lwoff (1932) considered this ciliate to be Glaucoma. Glaucoma or Glaucoma-like ciliates have been found also in other endozoic habitats than the hemocoele of aquatic larvae of Nemocera. G. parasiticum was observed by Penard (1922) in the gills of Gam- marus pulex, not only on the surface but also in the interior, where it PROTOZOA AND OTHER ANIMALS 899 consumes the soft parts of the parenchyma and blood cells. Penard con- sidered that it may be a temporary parasite only, and is closely related to G. “pyriforme.” There is one record of a similar ciliate in the tissues of a vertebrate. Epstein (1926) studied an infection of very young fish, Abrams brama L., with Glaucoma, probably G. pyriformzs according to Lwoff (1932). Two to three percent were naturally infected in an aquarium at a lake near Moscow. The infection began with the yolk sac, which the ciliates reached through the gut. They then entered the heart and spread throughout the vascular system. In two or three days the hosts suc- cumbed, with all except the resistant parts consumed. The ciliates oc- curred in great abundance in the canal of the spinal cord. Related ethologically to the invasion of the bodies of aquatic larvae of Nemocera by G. pyriformis or related ciliates is the occurrence of the common marine ciliate, Uronema marinum Duj., in the coelom of a sipunculid. Madsen (1931) mentioned the observation by Mrs. E. Wesenberg-Lund of masses of ciliates in several Halicryptus spinulo- sus that had been kept in Copenhagen for several months without food. After some days the sipunculids died, and the ciliates lived longer in the cadavers. He regarded this invasion as following a bacterial infec- tion, Uronema feeding on the bacteria, but did not suggest how the ciliates may have entered Halicryptus. Accidental parasitism similar to that of G. pyriformzs is the relation- ship of Anophrys sarcophaga to crabs, noted by Cattaneo (1888) and studied exhaustively by Poisson (1929, 1930). This marine ciliate normally lives in decomposing animal matter. Under certain circum- stances it invades the hemocoele of Carcinus maenas, but natural in- fection is rare. Cattaneo found it in one of 300; Poisson in 7 of more than 3,000 at the biological station of Roscoff. The ciliates multiply ac- tively in the blood, consuming the amoebocytes, and when these are exhausted feeding on plasma. When the host dies, the ciliates devour bacteria and fragments of tissue, surviving for some hours until de- composition is advanced, when they encyst or die. Artificial transmission was easily accomplished. Of 25 Carcinus maenas inoculated, 20 died within 7 days, usually with a massive infection. Five crabs survived and soon lost the ciliates. Attempts were made to inocu- late 7 other crabs of the genera Cancer, Portunus, Maia, and Eupagurus. 900 PROTOZOA AND OTHER ANIMALS Of these only Portunus depurator developed a heavy infection and died. Some were naturally immune, the serum agglutinating and destroying the ciliates. In others the serum was not toxic 7m vitro, but the ciliates were arrested in certain lymphatic spaces, killed, and phagocytized. Accidental parasitism of a nymph of the hemipteran Nepa cinerea was reported by Mercier and Poisson (1923). A species of Colpoda had invaded the body, probably through a wound in the integument, and produced a tumor the size of a pinhead on the lateroventral surface of the metathorax. The tumor extended part inside and part outside of the body, and in it ciliates were numerous. Large ones contained nu- merous inclusions, especially phagocytized amoebocytes. There were also very small ones with no inclusions; these were believed to be nour- ishing themselves by absorption of dissolved substances. Though locally destructive, the parasite did not prevent growth of the nymph up to the imaginal molt, when it was killed by the observers. Instances of accidental parasitism by Protozoa have been noted in sea urchins. Lucas (1934), in examining Bermuda sea urchins, en- countered transient Protozoa in the body fluids. She stated that these “were normally free-living forms, which probably gained entrance through the water-vascular system, and gave no evidence of coloniza- tion.’ André (1910) reported Explotes charon in certain abundance in the perivisceral fluid of the sea urchin Echinus esculentis, as well as on the surface of the host. Accidental invasion of the body cavity of these marine echinoderms is apparently not infrequent. Warren (1932) studied at Pietermaritzburg, Natal, a ciliate which possibly, according to his account, was a facultative parasite, in the common garden slug Agriolimax agrestis. He considered it to belong to a new genus and species, Paraglaucoma limacis. Kahl (1926) had already established a genus Paraglaucoma for P. rostrata, found in moss in Germany; later he found the species in moss from California and Wisconsin. Apparently Warren knew nothing of Kahl’s work, but the two ciliates appear similar. The length (60-80 y) as reported by Kahl (1931) is greater than that usual in Warren’s form (40 1 in the free- living form; mean lengths 41-63 in the parasitic form); but in 1926 Kahl had reported the length as 45-55 yp. Warren did not report the posterior bristle which Kahl observed. The species also resembles Glaucoma maupasi Kahl, 1926, the ciliate Maupas (1883) described as G. pyriformis. PROTOZOA AND OTHER ANIMALS 901 Warren found the ciliate swarming in certain fecal deposits, and then determined that they live in the lumen of the liver tubules, some at times passing into the stomach and being discharged in ‘fecal cham- bers of mucus.” The incidence of infection varied from 50 to 87 per- cent at different times of the year, and in one slug 18,000 ciliates were present. What seemed to be the same ciliate was found in the “greenish incrustation of earthy matter underneath bricks and flower pots.” This ciliate seemed to have no injurious effect on the slugs, even when present in large numbers. Reynolds (1936) observed ciliates in freshly passed feces of the same species of slug in Virginia. He determined these as Colpoda stezni, but as he gave no illustration or description, and even made the state- ment that the parasitic stage of this (holotrich) resembles (the hetero- trich) Balantidium more closely than it does its own free-living stage, we may not unreasonably consider the systematic status to be unsettled. He determined in sections that the ciliates may be widely distributed in the tissues of the body, and were most abundant in the respiratory chamber and the anterior and posterior ends of the alimentary tract. In one region more than 94 percent of the slugs were infected, in another 25 percent. Infection occurred by ingestion, presumably, of the free- living ciliates in the soil, where C. ste7n7 was also found. Unlike Warren, Reynolds considered that many slugs are killed by the ciliate, and even suggested that the ciliates may be useful in combating molluscan pests. Warren had also examined sections, but did not find invasion of the tissues other than the liver tubules. It is likely that the extensive invasion noted by Reynolds would be more harmful to the slugs. Probably the ciliate described by van den Berghe (1934) as Glaw- coma paedophthora n. sp. belongs in this group of facultative parasites. At any rate, it seems to be a form that has been directly adapted from a free-living habitat to parasitism in the egg masses of Planorbis and Physopsis. At Elizabethville, Belgian Congo, van den Berghe found the ciliates in certain eggs, generally two or three in an egg mass, number- ing from four or five to a great many. They were not found in the genital organs of the snails, and were abundant in the water of the aquarium. Infection of all eggs in a dish took place quickly if ciliates from an infected egg were introduced into the water. In the egg, mul- tiplication from a few ciliates to an intense infection occurred within twelve hours. The embryo was killed by the parasites and within twenty- 902 PROTOZOA AND OTHER ANIMALS four hours had disappeared, the eggshell bursting and the ciliates escap- ing. Though the author stated decisively that the ciliate belongs to the genus Glaucoma, the description and figure do not prove that system- atic position. Along with the adaptation of free-living Protozoa to a symbiotic environment, there should be considered a number of instances of a secondary type of infection of associates in the same hosts. These have been referred to as facultative parasites, from the standpoint of the secondary hosts. Facultative parasitism of Heterocineta janickii on the oligochaete Chaetogaster limnaez, which occurs with the hypocomid ciliate in the mantle cavity of snails, is described below (p. 940). Theil- er and Farber (1932, 1936) found Trichomonas muris present with considerable frequency in oxyurid nematodes in white mice, and divi- sion took place in the intestine of the worms. They even found tricho- monads in nematodes when the flagellates could not be demonstrated elsewhere in the mice. J. G. Thomson (1925) found Gvardia present in abundance in all of hundreds of nematode worms, V zanella sp., from a specimen of the South American rodent Viscacia viscacia. Although he found no trophozoites or cysts of Gzardia elsewhere in the intestine of the rodent, he observed the flagellate from the nematode to be mor- phologically identical with G. viscaciae Lavier. Graham (1935) found Giardia in nematodes, probably Cooperia oncophora, from a bull; but was unable to find the flagellates in the intestine of the bull. A com- parison with G. bovis Fantham would be of interest. As species of Gzar- dia are otherwise exclusively parasites of vertebrates, it is likely that the nematodes with Gvardia had, like those with Trichomonas, been sec- ondarily infected with the mammalian flagellates. Flagellates can evi- dently survive for long and even multiply in the worms, so that their presence in them without simultaneous occurrence in the lumen of the vertebrate intestine is not significant. SYSTEMATICALLY RELATED FREE-LIVING AND SYMBIOTIC PROTOZOA MASTIGOPHORA In addition to the existence of accidental and facultative parasitism, it is significant in connection with the origin of symbiotic relationships PROTOZOA AND OTHER ANIMALS 903 that certain genera contain both free-living and symbiotic species, or that the two types of habitat are occupied by members of closely related genera. The organisms have become closely adapted to their biotic en- vironment, but have not undergone extensive modification. That does not necessarily imply recent adaptation, since stability of characteristics would equally well explain it; but it does indicate a direct origin from free-living types. There are some epibiotic euglenids, including species of Ascoglena and Colacium, Euglena cyclopicola described by Gicklhorn (1925), and Euglena parasitica described by Sokoloff (1933). The last species ad- hered by the anterior end to all of numerous colonies of Vo/vox in a tank in Mexico City, and was not found free in the water. In the green color, stigma, and other structures, except for lack of a flagellum, this is a typical Euglena. It is not certain, however, whether the relation- ship is more than occasional phoresy. E. cyclo picola is normally epibiotic, occurring on Cyclops strennuus and species of Daphnia. Epibiotic eug- lenids have been observed on plankton Crustacea in reservoirs in the vicinity of Berkeley, California. The euglenids, Exglenamorpha hegneri, E. pellucida, and Hegneria leptodactyli, are obligate inquilines of amphibia. They have never been found free-living. Species of Evglena and Phacus, with normal green color and activity, have, however, been found living in frog tadpoles (Alexeieff, 1912; Hegner, 1923; Wenrich, 1924a). This is merely a survival of free-living forms in the intestine, and Alexeieff may be un- justified in terming it facultative parasitism. A colorless euglenid of the genus Menoidium was found living in the intestine of one specimen of Spirobolus marginatus by Wenrich (1935); and it occurred free-living in damp Sphagnum in the aquarium jar. He reported no observations on how long this flagellate might survive in the host. Evglena gracilis fed to the millipeds could be in part recovered alive in one or two days. Euglenamorpha hegneri Wenrich was observed by Hegner (1922) and described by Wenrich (1923, 1924a) and Hegner (1923) from tadpoles of frogs and toads and from H)/a in the North Atlantic states. The typical form has green chloroplasts, a red stigma, and three flagella. In 0.6 percent salt solution it survived for weeks in a hanging drop, and multiplied at first, but continued cultivation was not achieved. A colorless form, distinguished as the variety pellucida by Wenrich, 1s also 904 PROTOZOA AND OTHER ANIMALS present in tadpoles. This differs from the type in several respects, the most important being the lack of a stigma and the presence of from two to six flagella. Most frequently there are from four to six flagella. Ac- cording to Wenrich, six is the doubled number, three new ones growing out very early in preparation for division. In other numbers above three, there are various stages of outgrowth. Division of a flagellate with four flagella results in daughter flagellates with two. Brumpt and Lavier (1924) considered Wenrich’s colorless variety to be a separate species, and Wenrich (1935) seemed inclined to the same opinion. Brumpt and Lavier described a similar colorless form with no stigma, from tadpoles of Leptodactylus ocellatus at Sao Paulo, Brazil, as Hegneria leptodactyli. That flagellate has seven flagella ordinarily, but may have only six. The authors did not mention the presence of an accompanying green form with fewer flagella, and Wenrich (1935) stated that he found the colorless flagellate in some hosts, unaccompanied by the green one. The six-flagellated forms of Hegneria seem to resemble very Closely the six-flagellated forms of Evglenamorpha hegneri vat. pel- lucida, so that it may be necessary to revise the taxonomy of the flagel- lates. One 1s tempted to find, in this interesting series of forms, as Wenrich has brought out, adaptation to the conditions of an endobiotic habitat in loss of chloroplasts and increase of the number of flagella. Endozoic colorless euglenid flagellates of the Astasia type have often been found, especially in Turbellaria, but also in rotifers, Gastrotricha, fresh-water nematodes, fresh-water oligochaetes, nudibranch eggs, and copepods. They usually are in vigorous metabolic movement, and gen- erally lack a flagellum when in the host. Haswell (1892) found them abundant in parenchymal cells in all specimens examined of a rhabdocoele turbellarian in Sydney. A flagellum was present in many but not in most cases. No stigma is mentioned. In 1907 Haswell described a similar euglenid in many specimens of an- other rhabdocoele, within cells of the digestive epithelium and in the spaces between the gut and the body wall. In the host, it was motion- less or executed slow movements, but was more active when freed. No flagellum was present until two hours or more after the organisms were freed from the host. They were kept alive outside of the host for several days, but no euglenids were found normally free in the water. PROTOZOA AND OTHER ANIMALS 905 Playfair (1921), who made his studies in the vicinity of Sydney also, stated that on one occasion he found half a dozen specimens of Astasza margaritifera Schmarda within the tissues of a turbellarian. This species he also found in the water of ponds, not in a free-swimming form and very often lacking a flagellum. This is the only species of Astasza that he reported in the survey of Australian fresh-water flagellates, and his iden- tification is not convincing proof that the form in Turbellaria is the fresh-water species named. Astasia captiva was described by Beauchamp (1911) from the rhabdo- coele Catenula lemnae in France. In one pond almost all individuals were infected, while in another a mile away the flagellates occurred in a small percentage only. In some there were only one or two to a chain of zodids, whereas in others the flagellates were very abundant. They were in con- tinual movement in the “pseudocoele,”’ between the parietal cells. A flagellum was present sometimes even on flagellates in the tissue, but most of the organisms lacked that structure. A colorless rudiment of a stigma, which was invisible in life, was seen frequently in stained prep- arations. Beauchamp stated that no euglenid was seen in other species of rhabdocoeles, including the common Stenostomum leucops. Howland (1928) identified as Astasia captiva an actively metabolic euglenoid flagellate, without flagellum or stigma, which she observed in Stentor coeruleus and Spirostomum ambiguum. S. R. Hall (1931) found euglenids rarely in the mesenchyme of an- other species of Stenostomum and in S. predatorium in Virginia, where Kepner and Carter (1931) doubted the existence of S. Jewcops. While the flagellate was in the host, the flagellum did not extend beyond the edge of the body; but when it was liberated into water the flagellum soon grew out, metabolic movement ceased, and the organism swam rapidly. The euglenids could be kept alive in spring water for three or four days, but attempts to cultivate them failed. When infected hosts were added to a culture of the rhabdocoeles, practically all became in- fected within a week. In one instance, when an infected worm was de- voured by another, several flagellates were observed to pass through the wall of the enteron into the mesenchyme, where they multiplied. There was no apparent effect on the host except in instances in which two or three hundred were present; then the rhabdocoeles became sluggish and bloated, ruptured with liberation of the flagellates, and died. 906 PROTOZOA AND OTHER ANIMALS Because of the presence of a red stigma and bifurcation of the root of the flagellum, Hall assigned this flagellate to the genus Euglena, al- though it is colorless, naming it E. Jeucops. Nieschulz (1922) examined large numbers of the fresh-water nema- tode Trilobus gracilis, in the hope of finding Herpetomonas (— Lepto- monas) bitschli. This was not found, but he reported Astas7a from some specimens, usually only one or two in a host. There was no stigma and no flagellum. He did not state in what part of the body the parasites occurred. In the rotifer Hydatina senta, Astasia has been reported on three occa- sions. Leydig (1857) observed it in the alimentary tract of almost all of the hundreds of rotifers that were examined. Metabolic movements were very active, a red stigma was present, and no flagellum was mentioned. Hudson and Gosse (1889) wrote: ‘‘H. senta, too, suffers from an in- ternal parasite. It... swims up and down its host’s stomach by jerking the contents of its body constantly backwards and forwards.’ Their fig- ures show no flagella, and one, in color, shows a red stigma. Valkanov (1928), without reference to other observers, named the organism he found parasitic in the intestine of the same species of rotifer, A. /y- datinae. In the intestine of gastrotrichs, Astasia-like inquilines were reported by Voigt (1904). He found them in some specimens of a gastrotrich that he later (1909) named Chaetonotus ploenensis, and was unable, despite careful search, to find free-living examples of Astasza in the material. Remane (1936, p. 231) stated that he found the same species in the intestine of another species of Chaetonotus. Astasia doridis was found by Zerling (1933) to be rather abundant in some eggs of the egg masses of the nudibranch Doris tuberculatus at Wimeraux. When heavily parasitized, larvae were destroyed. The para- sites lacked flagella and stigmas and showed intense euglenoid activity. Freed from the eggs, they lived many days with no change in morphology and behavior. The flagellate was not found in the genital tract of the adult. Zerling believed it probable, nevertheless, that adult molluscs are infected by the parasites liberated into sea water at the hatching of in- fected larvae, and that they transmit the parasites to their egg masses. This is the only published record of a euglenid parasite in a marine host. Codreanu and Codreanu (1928) found a considerable percentage of PROTOZOA AND OTHER ANIMALS 907 the fresh-water oligochaete Chaetogaster diastrophus Gruith in the vicin- ity of Bucharest infected by a euglenid parasite that they named Astasia chaetogastris. The flagellates multiplied rapidly in the coelom, and the infection was always fatal in from eight to thirteen days. When freed into water, metabolic movement lessened and a flagellum developed. Both forms had a stigma, and the free form as well as the parasitic one was capable of division. This euglenid is more pathogenic than any other described. One is reminded of the invasion of the hemocoele of insect larvae by Glaucoma pyriformis. Foulke (1884) wrote concerning the fresh-water sabellid Manayunkia speciosa: “Several individuals of Manayunkia were observed to be preyed upon, while still living, by large monads, embedded in one or more of the segments, which were sometimes excavated to a considerable degree.” It is possible that in this statement there is reference to a situa- tion analogous to that of Astasia chaetogastris. Finally, in copepods, occurs Astasia mobilis, which was the first en- dozoic euglenid to be observed (Rehberg, 1882). Alexeieff (1912) studied it in Cyclops, finding it not only in the lumen of the intestine but also twice in the eggs. It sometimes had a flagellum, and a stigma was described. The metabolic activity and some features of the structure of this organism have suggested to some sporozoan affinities. By Labbé (1899), for example, it was included in the genus Monocystis. Alexeieft discussed the possible euglenid origin of Sporozoa, and Stein (1848) had long before remarked upon the apparent relationship between euglenids and Monocystzs. Jahn and McKibben (1937) studied a colorless, stigma-bearing euglenid flagellate whose habitat is given as putrid leaf infusion. They found the root of the flegellum to be bifurcated, as in Euglenidae; whereas in Astasiidae, according to Hall and Jahn (1929), it is not bifurcated. The new genus Khawkinea was established by Jahn and McKibben for flagellates whose characteristics agree with those of Euglena except that they are permanently colorless; and they assigned to this genus not only their new species, K. /alli, but also the free-living form that had been known as Astasia ocellata Khawkine, A. captiva Beauchamp, A. mobilis Alexeieff, A. chaetogastris Codreanu and Codre- anu, and E. /eucops Hall. In the question of the relationship of free-living and endozoic Pro- 908 PROTOZOA AND OTHER ANIMALS tozoa, the flagellate recently discovered in pond water by Bishop (1935, 1936) in England, and by Lavier (1936c) in France is of much inter- est. Bishop found it on four different occasions in the course of thirteen months, in a small pond with thick, black mud and much decaying or- ganic matter; and Lavier found it in samples from two separate places. Many of its characteristics are those of a trichomonad and, as Lavier pointed out, it seems to be the only free-living member of the Tricho- monadidae. There is a slender axostyle, which often is extended into a pointed, posterior projection of the cytosome, or itself projects from the body. Sometimes the flagellate anchors itself to an object by the end of the axostyle. There are three anterior flagella and a trailing flagellum that usually adheres to the body, forming an undulating membrane, but that sometimes, according to Lavier, is free. The nucleus is trichomonad in position, structure, and division, and there is a well-defined para- desmose. Its manner of progression, which differs from that of other free-living forms (Lavier), impressed Bishop with its similarity to the movement of Trichomonas. Bishop (1935) wrote of it under the name “Thichomonas” Keilini n.sp. The flagellate differs from Trztrichomonas in the absence of a costa. Lavier assigned it to the genus Eutrichomastix, which resembles T17- chomonas in all respects except the lack of the costa and undulating membrane. Although it has been shown that the trailing flagellum of Eutrichomastix may adhere to the body under certain conditions, the usual presence of an undulating membrane in the pond flagellate differ- entiates it from that genus. Neither Bishop nor Lavier made any men- tion of the parabasal body or of an attempt to demonstrate it. If this structure, so characteristic of Monocercomonas (Eutrichomastix) and Trichomonas, is present, it would leave no doubt of the trichomonad affinities of the organism; if absent, the flagellate would not show so close a relationship to endozoic forms. Bishop (1939) proposed the new genus Pseudotrichomonas for the organism. It is not possible to state that in this organism there is evidence of the origin of trichomonads, which are widespread and evidently have been adapted for a great period of time to endozoic existence. It may be a survival of an ancestral type; on the other hand, there is the possibility, which Bishop considered, that it might be a parasite of some cold- blooded host that had survived and multiplied in the water. Rosenberg PROTOZOA AND OTHER ANIMALS 909 (1936) found that Tr7trzchomonas augusta sometimes survived in salt solution, on slides ringed with vaseline, for nearly a year. Cleveland (1928b) was able to cultivate indefinitely T. fecal7s in water with feces or tissue, in hay infusion, and in other ways, at temperatures from —3° C. to 37° C.; and this, although it was supposed to have been derived from a warm-blooded host, man. Cleveland also maintained T. augusta in tap water with feces. He did not report on the ability of T. batra- chorum, which Wenrich (1935) stated is morphologically indistinguish- able from T. fecalis, to grow under the conditions supplied. It would not be surprising if flagellates that have such marked ability to survive and even to multiply outside of the host, under such simple conditions, might find natural circumstances occasionally favorable to outside main- tenance of life. They might, at times, be found by collectors. This does not apply immediately to the studies of the species P. kezlini, however, as no endozoic flagellate just like it is now known, and Bishop (1936) found that it would not live in tadpoles; but it raises a general question. Hollande (1939) described as a free-living trichomonad the new genus and species Coelotrichomastix convexas. The flagellate was found in liquid manure. It has four flagella, one of them trailing and said to border an undulating membrane in a deep groove of the body; but there is no costa. There is a unique axostyle, ribbon-like in its posterior part and located superficially near the groove, anteriorly expanded to a hemispherical cupule covering a considerable part of the large nucleus. All parts of the axostyle are covered by small siderophile granules. A very small bacilliform parabasal body was reported. In considering the characteristics of Coelotrichomastix, Hollande failed to comment on the striking similarity in many respects that exists between it and certain flagellates that have been assigned to the genus Tetramitus. This cannot fail to impress the reader of the accounts by Klebs (1893), Bunting (1926), Bunting and Wenrich (1929), and Kirby (1932a). In those papers, furthermore, especially the second and third, one will find facts that suggest the possibility of a different interpretation of certain unexpected characteristics described by Hollande. In assigning ‘Trzcho- mastix’’ salina, originally described by Entz (1904), to Coelotricho- mastix, Hollande made no comment on the writer’s account of what seemed to be the same flagellate under the name Tetramitus salinus (Entz). 910 PROTOZOA AND OTHER ANIMALS Trepomonas agilis, the only species that has been described in that genus, is a common flagellate associated with decaying organic matter in fresh water, and has been found in a coprozoic habitat in human feces (Wenyon and Broughton-Alcock, 1924). The writer on several occa- sions found Trepomonas in salt-marsh pools, associated with marine flagellates and ciliates. Whether it was T. agzl7s was not determined. Flagellates of the genus have also become adapted to an endobiotic habitat in fish, amphibia, and reptiles. Alexeieff (1910) observed Tre- pomonas in Box salpa; and Lavier (1936b) found T. agzlzs once in that fish, where, he stated, it is doubtless an accidental saprozoite. According to Alexeieff (1909) and Lavier (1935), the endozoic Tre pomonas com- mon in amphibia is probably T. ag7/7s. Lavier found the flagellate rather constantly in tadpoles of Rana temporaria, R. esculenta, and Alytes ob- stetricans, and in one adult Triton. He discussed it as an interesting pos- sibility of parasitism in a flagellate normally living free, and possibly finding the endozoic habitat more favorable than the free-living. Das Gupta (1935) found a species of Tvepomonas, usually in small num- bers, in the caeca of three different species of turtles: Terrapene major, Kinosternon hippocrepis, and Chelydra serpentina. A cytological study of the flagellate has recently been made by Bishop (1937). The genus Hexamita includes both free-living and endozoic species. The former are common in fresh water and infusions with decaying or- ganic matter; they also occur in salt water; and a species resembling H. inflatus, and only 11 y long, has been observed by the writer in a salt- marsh pool with decaying algae and a salinity of fifty parts per thou- sand. The type of habitat of the ‘‘trichomonad” named Pseudotricho- monas keilini by Bishop (1939) and of Trepomonas and Tetranutus 1s similar to that of Hexamita; Lavier reported them all from one sample taken in France. Urophagus and Octomastix are considered by most protozodlogists to be synonymous with Hexamita. This was the opinion of Lavier (1936a), who also rejected Octomitus; but he proposed two new genera, Sprro- nucleus and Syndyomita (the latter of which is of the original Octomitus type) for morphological types of Hexamzta-like flagellates in amphibia. Lavier retained the name Hexamita for the common form in amphibia, the type of which, among those he considers, is most like that of free- living Hexamita. This, H. intestinalis, has undergone little modification. The others differ from the free-living type, according to him; but there PROTOZOA AND OTHER ANIMALS 911 seems to be a similarity in type of Spzronucleus to H. rostrata (?), as figured by Wenrich (1935) from the outside of a dead fresh-water snail. At least until a systematic review of the whole group of Hexamzta-like forms is made, it appears to be necessary, for the sake of clarity, to use only the one genus name. The endozoic species of Hexamita are many and are found in a wide variety of hosts. Though given species are restricted to single or related hosts, the tendency to give different names to those in different hosts, without adequate comparison with other described species, has been manifest. Certes (1882) found Hexamita frequently in the stomach of oysters from certain localities on the coast of France. Though he considered this to be H. snflata, he regarded it as a normal, reproducing inhabitant of the stomach; and the identification is in no way positive. In other invertebrates, Hexamita has been recorded from the reproductive organs of the trematode Deropristis inflata in marine eels, but not in the in- testine of the eel (Hunninen and Wichterman, 1936); from the cock- roaches Blatta orientalis (Bishop, 1933), Periplaneta americana, and Cryptocercus punctulatus (Cleveland, 1934); from the horse-leech Haemo pis sanguisugae (Bishop, 1932, 1933); from the milliped Spzro- bolus marginatus (Wenrich, 1935); from the larvae of T7pula (Mac- kinnon, 1912; Geiman, 1932); and from Twbifex (Ryckeghem, 1928). These all occur in the gut, except for the trematode form, as noted, and the one in Twbifex. The latter, furthermore, is the only one in in- vertebrates to which probable pathogenicity has been ascribed. Hexamzta tubifici was encountered at intervals in the course of fifteen years in the body cavity of Twbifex kept in culture in the laboratory in Louvain. Worms that lost their power of activity, appeared whitish, and died were found to have a more or less intense infection with Hexamzita. Ryckeghem considered the question as to whether the flagellate is a parasite or a free-living form invading decomposing tissue. He con- cluded that the former relationship exists, for he found it in living worms in apparently healthy tissue, decomposing chironomid larvae were not invaded by the flagellate, and it was encountered in different collections at long intervals. Each time it was a source of trouble. Hexamita species occur in vertebrates of all classes. Fry and young fingerlings of trout and salmon in hatcheries in the United States were found by Moore (1922, 1923a, 1923b, 1924) and Davis (1923, 1926) O12 PROTOZOA AND OTHER ANIMALS to be extensively infected, especially in the anterior part of the intestine; and they believed the flagellate to be severely pathogenic and to constitute a serious menace to the success of trout culture. The flagellates have been found also in European trout (Moroff, 1903; Schmidt, 1920) and in the fan-tailed darter (Etheostoma flabellare) (Davis, 1926). Davis and Moore did not prove that the flagellates were not secondary in diseased fish, as Schmidt believed. Lavier (1936b) examined 33 species of marine fish and found six species of Hexamita, five of them new, in seven of these. He remarked that the morphology of Hexamzta is much varied if one does not think in general terms, and that an attentive study enables one to recognize clear and constant morphological differences. Hexamita is commonly found in the intestine of amphibia, and has been reported from the intestine of turtles and tortoises, as well as from the bladder of Emys orbicularis (Grassé, 1924) and from the stomach, oesophagus, and small intestine of the snake Natr7x t2grina (Matubayasi, 1937). It occasionally invades the blood of amphibia (Lavier and Gal- liard, 1925; and others) and tortoises (Plimmer, 1912) through a dam- aged intestinal wall. Among birds, Hexamita occurs in pigeons (Noller and Buttgereit, 1923), ducks (Anas boschas, Kotlan, 1923), turkeys (Hinshaw, Mc- Neil, and Kofoid, 1938), and various wild birds in Brazil (Cunha and Muniz, 1922, 1927). Of mammals, rodents especially have been found infected with Hexamita. In addition to rats, mice, ground-squirrels, and woodchucks (Crouch, 1934), the South American hystrichoid rodent Myopotamus coipus |—Myocastor coypus (Molina) | contains a species (Artigas and Pacheco, 1932). Hexamita has also been reported in primates, including man (Cunha and Muniz, 1929; Wenrich, 1933; Chatterji, Das, and Mitra, 1928; Perekropoff and Stepanoff, 1931, 1932). Dobell (1935), discussing all these records except the third, believed that diplozoic forms of Entero- monas, which “‘are very frequently found in feces, in intestinal contents, and in cultures,” were misidentified. As regards Wenrich’s record, how- ever, from Macacus rhesus, this is improbable when one considers his extensive knowledge of the genus as well as the exactness of his de- scription and figures. PROTOZOA AND OTHER ANIMALS 913 From this survey of the distribution of members of the genus Hex- amita, it is apparent that the flagellates are as widespread in animals as are members of the strictly endozoic genus Trichomonas. In their case, how- ever, flagellates equally or more closely related than most of the endozoic forms to the ancestral type are common free-living forms. The endozoic forins, nevertheless, are for the most part as strictly adapted to their habitat as trichomonads. There is no evidence, except possibly in certain species in Amphibia and invertebrates, that the obligate symbionts have been recently adapted from facultatively endozoic forms; any more than that Trichomonas can be supposed to have recently so originated. There is little evidence of parallelism in phylogenetic development in members of these two genera and their hosts (Wenrich, 1935). In most instances Hexamita has been regarded as a commensal in its hosts. A possible exception in an invertebrate host is Hexamita tubifici. In the body cavity of the aquatic annelid the flagellates may be fatal to the host, in a manner comparable to the effect of Glaucoma in dipteran larvae and Astasia in Chaetogaster. Hinshaw, McNeil, and Kofoid (1938b), on the basis of experimental data which they obtained, sug- gested a possible relationship between a condition of enteritis in young turkeys and a heavy infection of Hexamita that occurred in the affected part of the small intestine. They also reviewed reports of possible rela- tionship in other vertebrates between pathological conditions and the occurrence of Hexamita. HOLOTRICHA Among holotrichous ciliates, all types of biotic relationship exist, so that the group is especially favorable for study of the development of symbiosis and host-specificity. In this section will be considered holotrich groups in which free-living and symbiotic species are closely related. In some instances it seems that there has been no more than survival of ordinarily free-living forms in or on a host, where certain conditions of nutrition or protection favored the occurrence of the associate. Per- haps the occurrence of Coleps hirtus on the rhabdocoele Vortex sexden- tatus aS a common epizoon, as recorded by Graff (1882), is a relation- ship of this type. The relationship of Enchelys difflugiarum Penard to Difflugia acumt- 914 PROTOZOA AND OTHER ANIMALS nata (Penard, 1922), and that of E. nebulosa Entz to Cothurnia is ap- parently obligatory predatism; this relationship, of course, is comparable to parasitism, as, if the host were a metazoan and were only partially destroyed by the attacks of the ciliate, we would doubtless consider the latter a true parasite. Haematophagus megapterae Woodcock and Lodge and Metacystis megapterae Kahl are commensals on the bristles of the whale Megaptera nodosa (Kahl, 1930). A number of pleurostomatous and hypostomatous gymnostomes have become associated with animal hosts. In the former group there is Amphileptus claparédei Stein, ‘parasitic on the stalks of colonial Vor- ticellidae” (Kahl, 1933); and A. carchesii Stein in a similar situation. Edmondson (1906) reported that after feeding upon a zodid the latter species (discussed by him as A. me/eagris Ehr.) attaches itself to a stalk. He found that many were present on Carchesium polypinum, clasping the stalks by a deeply cleft posterior end. In addition to these more or less predatory species, there is Lionotus branchiarum (Wenrich) Kahl, described by Wenrich (1924b) as A. branchiarum. It is a true parasite on the gills of the tadpoles of several species of Rana, where it lives in a capsule under the cuticular membrane and occasionally detaches and en- gulfs gill cells. Wenrich (1935) discussed the possibility that A. branchiarum is transitional between a predatory and parasitic status. There is a predaceous, free-swimming phase on the surface of the gills by which other Protozoa may be devoured. Three species of Lionotus, L. impatiens Penard, L. aselli Kahl, and L. hirundi Penard are com- mensal among the gills of Ase//ws; and one, L. agilis Penard, occurs on the ventral surface, among the legs, and on the egg masses of Cyclops. The pleurostome genus Branchioecetes Kahl is very closely related to Loxophyllum, in which Svec (1897) and Penard (1922) put the spe- cies. The two species B. aselli (Svec) and B. gammari (Penard) are commensals on their hosts, to which they adhere by thigmotactic cilia. Commensal hypostomes belong to the genera Trochilia and especially Chilodonella. Trochilia (Dysteropsis) minuta (Roux) has been found free-living as well as commensal with Cyclops, Gammarus, and Asellus (Penard, 1922). Commensalism is widespread in Chilodonella, several species of which are apparently obligatory commensals on fish, others on certain rotifers, amphipods, and isopods. The species on the gills of fish PROTOZOA AND OTHER ANIMALS 915 have often been thought to exert direct or indirect pathogenic action, but proof that they are more than ectocommensals is lacking. Kidder and Summers (1935) distinguished several species on the carapaces of three species of Orchestiidae from beaches in the region of Woods Hole; they noted that no similar ciliates were found free in the sand or sea- weeds, and the commensals lived only a short time when separated from their hosts. C. capucinus Penard, 1922, and C. granulata Penard, 1922, are commensal on Asellas and Gammarus, and C. porcellionis occurs in the gill cavities of the terrestrial isopod Porcellio sp. (Dogiel and Furs- senko, 1921). In aquatic hosts, transmission would take place through the water; in C. porcellionis it must be through survival, at least for a short period, in moist soil. A number of hymenostomes of the new genus Allosphaerium were also described by Kidder and Summers (1935) from the one species of Talorchestia and two of Orchestia that they examined at Woods Hole, Massachusetts. They remarked, concerning the ectocommensal holotrichs of amphipods and isopods, that the external characteristics are singularly well adapted to the environment. They are all small flat forms and possess ventrally placed thigmotactic cilia (Chilodonella, Trochilia, Allosphaerium). When one considers the forces, mainly in the form of water currents, to which they must be subjected and which would tend to effect their removal from the carapace of their various hosts, it is seen that the flatness of their bodies and the adhesive powers of their ventral cilia are of absolute necessity. Existing under the same conditions, it is perhaps not surprising that representatives of two orders of ciliates ex- hibit convergence to such a degree as to render them practically indistinguish- able one from the other except under extreme magnifications. Genera with free-living Trichostomata and Hymenostomata include only a few commensal and parasitic species, but there are numerous genera all members of which are associated with animal hosts. Frontonza branchiostomae was found in abundance at Banyuls-sur-Mer by Cod- reanu (1928) in the atria of most specimens of Branchiostoma lanceo- latum exceeding 3 cm. in length. The genus Glaucoma has been dis- cussed at length under facultative parasitism. Uronema rabaudi was be- lieved by Cépéde (1910) to be a coelomic parasite of Acartia clausi and Clausia elongata, in the empty carapaces of which it was observed. With- out free-living congeners, but similar enough to Uronema to have been put in that genus by Biitschli (1889) and Cuenot (1891), is PAilaster 916 PROTOZOA AND OTHER ANIMALS digitiformis, which was described by Fabre-Domergue (1885) in mucus on the body of Asterzas glacialis, multiplying abundantly on damaged and disintegrating starfish, but disappearing with death of the host. The genus O phryoglena comprises large holotrichous ciliates of which some species are free-living and others endozoic. Kahl (1931) listed eleven of the former and five of the latter. Within the genus there is a range from free-living habits, often with predatism, through commensal- ism to strict parasitism in close relationship to the developmental cycle of the host. The free-living species O. flava, according to Penard (1922), is vo- racious and usually preys upon animals larger than itself, including rotifers, small worms, and small Crustacea, especially Cyclops. It passes under the carapace of Cyclops and consumes the living animal, the soft parts of which are converted into food balls in the cytoplasm. Ophryoglena maligna, described by Penard (1922), preys upon O. flava as a parasite. It invades the cytoplasm, in which the number is one to four or more, and devours the host little by little until it is empty. The ciliates were also found free in the water, but Penard believed that before long they would attach themselves to O. flava. The three species that have been found in the intestine of Turbellaria appear to be commensals. These are O. parasitica, reported by André (1909) from 11 of 234 Dendrocoelum lacteum; O. pyriformis found infrequently by Rossolimo (1926) in Sorocoelis maculosa and Planaria nigrofasciata at Lake Baikal; and O. intestinalis from a large turbellarian of the genus Dicotylus at Lake Baikal. It was shown that the last two species cannot survive long in the water. Truly parasitic, however, are the species reported by Lichtenstein (1921) and Codreanu (1930, 1934) from May-fly naiads. The former found the parasites in the schizocoele and gonads of Baetis sp. near Montpellier; the latter in five Ephemerida from the Alps and the Car- pathians. Codreanu believed that parasitism by these ciliates may occur widely in Ephemeroptera. In young Réthrogena the ciliates occur as cysts, division taking place within the cyst, but in Baetis they are not encysted at any time. When the reproductive organs develop in the females, most or all of the ciliates invade the ovaries, the contents of which they ulti- mately destroy. The May flies nevertheless become adults and in the act of what would normally be egg-laying, ciliates are deposited in the water PROTOZOA AND OTHER ANIMALS 917, instead of eggs. Only the female hosts are able to propagate the infec- tion. Codreanu (1934) remarked that this is the only sufficiently de- fined case of true parasitism of the schizocoele of insects by ciliates. The species found in Baetzs by Lichtenstein was named by him O. collini; that studied in Baetzs by Codreanu (1930) was, he stated, probably the same. Haas (1933) noted the similarity between the oral apparatus of the swarmers of Ichthyophthirius multifiliis Fouq. and that of O phryoglena; Kahl (1935), in consequence, placed that important parasite of fish in the family Ophryoglenidae. Commensalism and parasitism being so well developed in O phryoglena, although along with free-living habits, there are clear ethological relationships between it and Ichthyophthirius. Pleuronema anodontae, the only commensal species of that genus, was reported by Kahl (1926) in small crushed mussels. He stated later (1931) that it is infrequent in Anodonta, but occurs regularly in Sphae- rium species. (Perhaps one should investigate the possibility that this may be one of the Ancistrumidae, not Plezronema.) Very close to Pleuronema is Pleurocoptes hydractiniae Wallengren, an ectocommensal on the hydromedusan Hydractinia echinata. DISTRIBUTIONAL Host RELATIONSHIPS AND HosT-SPECIFICITY IN REPRESENTATIVE SYMBIOTIC FAUNULES GENERAL CONSIDERATIONS There are some generic groups of Protozoa that have a rather wide distribution among animals; these groups are represented by species in hosts widely separated systematically. That is true, for instance, of Hex- amita and Trichomonas among polymastigotes, of Endolimax among endamoebae, of Nosema and Eimeria among sporozoa, of Nyctotherus, Balantidium, and urceolarids among ciliates. These examples have been discussed by Wenrich (1935). The genus Trypanosoma is represented in a very large number of vertebrates of all classes, but is limited to them, as is also Grardia. (The occurrence of Giardia in nematodes is faculta- tive; see p. 902.) A more or less closely restricted host distribution 1s, however, characteristic of many generic, familial, and even higher groups of Protozoa. Entodiniomorphina occur only in certain herbivorous mam- mals, chiefly ruminants and Equidae; opalinids are most likely to have anurous amphibian hosts, although a few have been found in Urodeles, 918 PROTOZOA AND OTHER ANIMALS fish, and reptiles; hypocomids (except the small genus Hypocoma) are parasites of certain groups of molluscs; Astomata are mainly inhabitants of annelids, to which most genera are limited; hypermastigote flagellates occur only in termites and roaches; and certain groups of polymastigotes are restricted to certain groups of termites (p. 923). The problem of host-specificity is ordinarily approached from the standpoint of the individual species; that is, the degree in which it 1s limited to a particular host species. In strict host-specificity, the host ts rigorously determined; there is only one host for a species of symbiont. As has been pointed out by Grassé (1935) and Wenrich (1935), strict host-specificity is not a general phenomenon. Surveys of lists of species and their hosts often bring out many instances in which there 1s only one host for a species, for example, in the genera Giardia, Babesia, Plasmodium, Haemo proteus, Leucocytozoén, and Eimeria. But such data cannot be taken at face value, because the apparent strict host-specificity may be based on insufficient search for the organism in other hosts, or on a tendency of taxonomists to differentiate species on insufficient grounds. More intense study in certain groups, as Trypanosoma and Devescovininae in termites, has shown less rigorous limitation than at first seemed to exist. More commonly, host-specificity is relative. The lim- itation is to more or less related animals; and it depends, as Becker (1933) and Wenrich (1935) have pointed out, on the characteristics of the symbiotic environment, the opportunities for transmission, and the evolutionary tendencies of the Protozoa. The phenomenon is of the same nature as that of the geographical distribution of free-living or- ganisms, though of course it is more complex. It is a commonplace that given animals have characteristic protozoan faunules; this phenomenon is of particular interest when there are faunules of particular types peculiar to major groups. In instances of the highest development of this tendency, it can be predicted what types of Protozoa will be found in unexamined hosts. One may be reasonably certain of finding Opalinidae in anuran species, Ophryoscolecidae in ruminants, and certain types of polymastigotes and hypermastigotes in all termites other than Termitidae . Questions of distributional host relationships and host-specificity will now be considered in greater detail in certain representative symbiotic faunules. Two faunules have been selected for this purpose: ciliates of PROTOZOA AND OTHER ANIMALS O19 sea urchins, and flagellates in termites and roaches. The former is inter- esting also from the standpoint of the relationship of inquilines and free-living forms; the sea-urchin intestine is one of the least specialized environments of its type. The flagellates, however, occupy one of the most specialized of symbiotic habitats. Not only are the circumstances under which they live and are transmitted exceptional, but the hind-gut of the host has actually undergone structural modification to accom- modate them. CILIATES OF SEA URCHINS Faunules of ciliates occur in the greater number of sea urchins that have been examined, but there are some without any. Uyemura (1934), giving positive reports from eight species of sea urchins of Japan, found none in Brissus agassizi, At Amoy, Nie (1934) found none in Tem- nopleurus toreumaticus. Of the species at Yaku Island, Japan, Yagiu (1935) found two uninfected: Colobocentrotus mertensiu and Cidaris (Goniocidaris) biserialis. Powers (1935) found no faunule in Ezcidaris tribuloides at Tortugas, nor were ciliates present in members of the genus Arbacia at Beaufort, Woods Hole, and Naples (1933a), in spite of association with infected species. Why a few species possess no faunules, while so many have ciliates in abundance, is an interesting ques- tion. The intestinal faunules of sea urchins consist mostly of ciliates, which, in whatever part of the world the host occurs, are members of a number of characteristic genera. Most of them are holotrichs; outside of this group are a few species of Metopus and one of Strombilidium, hetero- trichs with many free-living congeners. There are sometimes as many as twelve distinct species in eight genera in Strongylocentrotus pur- puratus (Lynch, 1929); and Yagiu (1933, 1934) found twelve species in Anthocidaris crassispina. On the other hand, from S. franciscanus in Japan, Yagiu (1935) reported only Conchophthirus striatus. Four or five species is perhaps the average infection. The occurrence of amoebae, Chilomastix echinorum (Powers, 1935), nematodes, and rhabdocoeles (Syndesmis) is much less prominent than that of ciliates. In given hosts there is variability in the occurrence of different species of ciliates. Some occur in abundance almost or quite universally; others have a lower incidence, some being of rare occurrence. Plagiopyla minuta 920 PROTOZOA AND OTHER ANIMALS Powers (1933a) occurred in only about 10 percent of Strongylocentrotus drobachiensis, and then there were not more than twelve in a host; whereas some ciliates have been found in all sea urchins of the species. Most often the incidence is not 100 percent. Powers (1933a) pointed out the existence of two groups of ciliates in sea urchins. One group contains diverse species with many free-living congeners, which he regarded as chance or vagrant forms that were en- gulfed with food and survived; the other consists of obligatorily endozoic species. The members of the first group are ‘apparently free-living and only occasionally or accidentally associated with their host.’’ There is, however, no evidence in the literature that many, if any, of the intestinal ciliates in sea urchins are accidentally introduced free-living forms. Though there are species belonging to genera of which most members are free-living, that in itself is no indication that they are not obligatory inquilines. Colpidium echini Russo, found also by Powers (1933a) in all spect- mens of Strongylocentrotus lividus examined at Naples, probably, ac- cording to Kahl (1934), is not a Colpidium. Uronema socialis, described by Powers (1933a) from S. drébachiensis, was later renamed by him (1935) Cyclidium stercoris. Kahl (1934) doubted the generic assign- ment of Colpoda fragilis, described by Powers (1933a) from Tox- opneustes variegatus of Beaufort, North Carolina. These forms, which Powers mentioned in the occasional associate group, together with Plagiopyla, may be obligatory commensals. The Ezplotes sp. found by Powers (1933a) in the gut and outside of S. drébachiensis is possibly an accidental invader. He also reported Trichodina from the sea urchin and in seaweed. Cyclidium stercoris, which occurs in great abundance in S. drébachien- 575, will live and reproduce in sea water (Powers, 1933a); but it is not known that it does so under natural conditions. ““Colpoda” fragilis, on the other hand, is very sensitive to changes in its environment. Many of the ciliates can survive for more or less prolonged periods outside of the host. Entodiscus borealis, one of the strictly endozoic forms, was kept in sea water from fifteen to twenty-three days (Powers, 1933b). Species of Cyclidium occur also in sea urchins of China (Nie, 1934) and Japan (Yagiu, 1933, 1934). Several species of Anophrys have been reported from various echinoids. There is only one free-living species PROTOZOA AND OTHER ANIMALS 921 of Anophrys, A. sarcophaga Cohn, which has been discussed above as a facultative parasite of crabs. Kahl (1934) suggested a relationship of certain of these ciliates to Philaster digitiformis, which occurs, as men- tioned above, on the body of starfish. He was doubtful about the cor- rectness of their position in the genus Anophyrs. There is confusion about the taxonomy of many sea-urchin ciliates. Genera that are restricted to sea urchins, and may be supposed to have evolved in the shelter of these hosts, are Lechriopyla Lynch, Entorhipi- dium Lynch, Entodiscus Madsen, Bigggaria Kahl, Madsenia Kahl. Cryp- tochilidium Schouteden, in part included in Biggaria, has a species in the annelid Phascolosoma vulgaris. Lechriopyla mystax Lynch, commensal in the Pacific Coast sea urchins Strongylocentrotus franciscanus and S. purpuratus, is closely related to Plagiopyla. It is markedly thigmotactic: “Although almost continuously in movement [it] adheres almost constantly to surfaces. The large peristomal groove seems to act as a sucker” (Lynch, 1930). Lechriopyla apparently has diverged from Plagopyla in relation to its obligatory endocommensalism, but there are no profound alterations. Four species of Extorhipidium were distinguished by Lynch (1929) in Strongylocentrotus pur puratus in California. None of these flattened, fan-shaped trichostomes was present in S. franciscanus from the same localities, so there seemed to be marked host-specificity. Uyemura (1934), however, found one of the same species in another sea urchin of Japan, and described a new species, E. fwkuii, which occurs in five hosts of four genera. Related to Entorhipidium is Entodiscus, represented by E. borealis (Hentschel) from several different hosts of the North Atlantic and Japan; and E. sabulonis Powers found in all individuals examined of two species of C/ypeaster at Tortugas. E. borealis is present in great abundance and, with its greatly flattened form, probably in appearance and occurrence suggests Opalina in Amphibia. According to Powers (1933b), besides swimming about in the lumen of the intestine, it ad- heres by the ventral side to the intestinal mucosa. The food vacuoles, he stated, contain rods, probably bacteria, and objects resembling nuclei of epithelial cells. At that time Powers thought that the ciliate might attack the intestinal mucosa, secreting cytolytic enzymes, thus being definitely parasitic; but later (1935) he did not stress this ill-founded conclusion. 922 PROTOZOA AND OTHER ANIMALS Powers (1933a) discussed the possibility that Cryptochilidium echini (Maupas), abundant and universal in Strongylocentrotus lividus at Naples, is a true parasite. In several instances the body was found partly embedded in the intestinal mucosa. As he probably recognized later, this observation does not constitute adequate proof for his conclusion. The genus Cryptochilidium, together with Biggaria, Kahl’s genus for some of the forms described as Cryptochilidium, is well represented in sea urchins of all regions. Metopus histophagus Powers, as the species name indicates, contains in its food vacuoles epithelial cells from the intestine of its host (Powers, 1935); but it was not observed to cause lesions, and probably simply ingests cellular debris, as does M. circumlabens (Lucas, 1934). The species occurs only in C/ypeaster subdepressus of Tortugas. The species M. circumlabens Biggar occurs in a number of hosts at Bermuda, Tor- tugas, Amoy, and Japan, but several other species seem to have a limited host-specificity. Questions of host-specificity and geographical distribution of the ciliates have been discussed by Powers (1935, 1937). He remarked that there 1s little evidence of rigid host-specificity. Species differ in that re- spect. Yagiu (1935) found Cryptochilidium echini and Anophrys elongata in all but one of the host species, examined by him at Yaku Island, which contained any ciliates; and Powers (1935) found Crypto- chilidium bermudense (—Biggaria bermudense) and Anophrys elongata in all sea urchins at Tortugas that were infected with ciliates. Nor are those ciliates limited to those regions; they have been found in various localities. There are some ciliates that have been found in only one or a few hosts, these being sometimes in one region only but also some- times in widely separated localities. There is nothing, however, which leads us to expect that, with the accumulation of more data, most or all of them will not be known to be in various hosts in various parts of the world. There is no limitation to certain genera or other taxonomic groups of sea urchins, as would occur in evolutionary development of assocta- tions with strict specificity. Though no experimental work has been done, it seems likely that cross infection would ordinarily be easy to accomplish; nevertheless it is noteworthy that given species have char- acteristic faunules, and there are a few sea urchins with no faunules, facts that call for experimental investigation of the host relationships. PROTOZOA AND OTHER ANIMALS 923 Another problem that calls for further investigation is the type of faunule in the same host species in different localities, data on which are meager. Strongylocentrotus franciscanus in California harbors Lechriopyla mystax, as well as ciliates of four other genera (Lynch, 1929, 1930); from S. franciscanus at Yaku Island, Japan, Yagiu (1935) re- ported only Conchophthirus striatus; and S. franciscanus examined by Powers (1936, 1937) at Acapulco, Mexico, was found to harbor “en- tirely different ciliates” from those on the coast of California. As regards similarity of faunules, there is the presence of Entodiscus borealis and Madsenia indomita in Strongylocentrotus drobachiensis from both Swe- den and the Bay of Fundy. PROTOZOA OF TERMITES AND THE ROACH Cry ptocercus Flagellates have undergone no more spectacular development than is exemplified in the faunules now existing in certain termites and in Cryptocercus. Elsewhere in that class of Protozoa, in fact, there is noth- ing that is comparable to it. Many groups of the Polymastigida and all but two species of the Hypermastigida have been found only in those insects. There are also a few Protozoa of more ordinary types. Such are among flagellates Trichomonas and related forms, Retortamonas, Mono- cercomonas, Monocercomonoides, Hexamita, and Chilomastix; flagel- lates of these types occur only rather sparingly in higher termites and, except for Trichomonas, in most roaches. There are also Nyctotherus, Balantidium, amoebae, gregarines, and coccidia. But, in insects ancestral to modern termites and roaches, flagellates originating in the Monocer- comonas, Monocercomonoides, and Trichomonas type have undergone a remarkable evolution, giving us the main polymastigote components of the faunules that today exist in lower termites and Cryptocercus. Hy- permastigotes doubtless developed from polymastigotes, but their origin has not been traced. A table of the classification of termites, giving the approximate num- ber of species and the number examined, is given by Kirby (1937). About a quarter of the 1,600 termites are in the four lower families: Mastotermitidae, Hodotermitidae, Kalotermitidae, and Rhinotermitidae; three-quarters are in Termitidae. Flagellate infections in Termitidae are sparse, and the species are small and of common types. In certain Ter- mitidae, faunules have developed consisting mainly of amoebae, which 924 PROTOZOA AND OTHER ANIMALS are almost completely lacking in lower termites. In lower termites there have been recognized, in examinations of less than a third of the known species, 30 genera with 133 species of polymastigotes, and 18 genera with 63 species of hypermastigotes; and certainly thorough study will reveal many more genera and species even in that third. In Cryptocercus punctulatus alone, Cleveland et al. (1934) found 9 genera of hyper- mastigotes with 20 species (only one genus and no species of which occur in termites); and 5 polymastigotes in 3 genera, including Hexamita and Monocercomonoides. Every termite species in the lower families, so far as has been learned, has a flagellate faunule; individual termites lack the Protozoa only in certain phases of the life history, as when they are very young, imme- diately preceding and following a molt, and in certain functional repro- ductive stages. For the most part, any termite of a species, wherever obtained, will be found to have the same group of flagellate species. Sometimes one or more flagellates are absent, but uniformity in com- position of the faunules is the rule. This fact is an aid in termite sys- tematics. Identical faunules do occur in different termite species of cer- tain groups; the fact that the faunules are identical does not necessarily indicate that the hosts belong to the same species. There are often more or less well-marked differences, and this is a strong indication for specific differentiation of the hosts. The flagellates often provide a ready means of distinguishing nymphs in regions where both the termites and their faunules are known. Individual faunules of flagellates in termites may comprise from two to ten or, occasionally, more species. Often a genus is represented in a host by more than one species. In Zootermopsis angusticollis and Z. nevadensis there are three species of Trichonympha (Kirby, 1932b). Nine of sixty-seven hosts of Devescovina contain two species. The genus Foaina is represented by two species in thirty-four, and by three species in three of eighty-three hosts. In Cryptocercus punctulatus Cleveland et al. (1934) differentiated seven species of Trichonympha, four of Bar- bulanympha, three of Leptospironympha, and three of Saccinobaculus. Koidzumi (1921) distinguished six species of Dinenympha in Reticu- litermes 5 peratus. The degree of host-specificity varies in different genera and species. Many species are known from one host only, but as more flagellate PROTOZOA AND OTHER ANIMALS 925 faunules become known the tendency probably will be relatively to re- duce this number. Many species are known from several or many hosts. Trichonym pha agilis probably occurs in all species of Retzculitermes, but has not been found in other termites. Stawrojoenina is widespread in Kalotermes sensu lato, and there are few if any differences between species of different hosts. Of twenty species of the genus Devescovina, only nine have been found in but one host each. On the other hand, there are species with many hosts widely separated geographically. D. glabra has been identified in eighteen termites from Africa, Madagascar, Java, and Sumatra; D. lemniscata has seventeen hosts in Central and South America, the West Indies, Australia, the Pacific Islands, Africa, Madagascar, Java, and India. A unique, elaborately organized deves- covinid, when first found in a Ceylon termite, was thought to be a strictly host-specific form; but it has since been found also in a termite from Australia. The small, simply organized Tricercomitus, which occurs in most if not all species of Kalotermes sensu lato, appar- ently is one species, T. divergens, in all those in which it has been studied. Another species exists in Zootermo psis. Many species of termite flagellates in all groups have a present host distribution which indicates greater stability in characteristics than ex- isted in the same period of time in the insects. Speciation has occurred in the hosts without having taken place in certain of the symbionts. That there are other termite flagellates which have evolved into different spe- cies in single hosts is probable; but we cannot designate any one as cer- tainly rigidly host-specific. Although there are many one-host forms, the situation is such that finding any one of them in a termite, even in another part of the world, would not be astonishing. Even although there is only a single extant host, it would in no instance be unlikely that formerly existing species not directly ancestral served as hosts of the flagellate. But whether a flagellate species occurs in one or in several host species of termites is far from being the question of greatest interest in host distribution. It has, in fact, little significance for general considerations. More important is the fact that there is limitation of certain flagellate types to certain groups of termites. That is true mainly among poly- mastigotes. There are also some very widely distributed flagellate types, but that only adds to the significance of the instances of strict limitation. 926 PROTOZOA AND OTHER ANIMALS Trichomonas, as is not unexpected, is one of the most widely dis- tributed forms, occurring in termites of all families, including Ter- mitidae. Trichonympha, although absent from Mastotermes and Termitidae, not only has a wide distribution among other termites but occurs also in Cryptocercus punctulatus. The genus has been found rep- resented in forty-five termites of ten genera or subgenera in three fam- ilies; and among those that have been studied for detailed characteristics fourteen species have been distinguished. Various Holomastigotidae in termites are related to hypermastigotes of this family in Cryptocercus, although no genus is the same. Several genera are distributed widely in termites, the situation being comparable to that of Trichonym pha. In the distribution of the polymastigote family Pyrsonymphidae, there is a high degree of correlation with the systematic relationships of the hosts. Cleveland ef a/. (1934) extended this family (as Dinenymphidae ) to include other forms than Pyrsonympha and Dinenympha, on the basis of the type of division figure and structural similarities. There are three subfamilies: Saccinobaculinae, in which the flagella are free and there is no attachment organelle; Oxymonadinae, in which the flagella are free and there is an attachment organelle, the rostellum, developed to a high degree; and the Pyrsonymphinae, in which there is a slightly developed attachment organelle and the flagella are adherent to the sur- face of the body for most of its length. Saccinobaculinae have been found only in Cryptocercus punctulatus; Oxymonadinae are known only from Kalotermes sensu lato, in which group they occur in most species; Pyrsonymphinae seem to be restricted to the genus Reticulitermes. It seems possible that evolutionary development of the groups has taken place within the confines of the host groups concerned; although it is unsafe to state that the distribution of the flagellates may not be wider than we now know it to be. The polymastigote subfamily Devescovininae, of which Monocer- comonas (Eutrichomastix) appears to be an ancestral type, is represented in all but five or six of ninety-seven species of Kalotermes sensu lato that have been examined. There has been a most elaborate evolutionary de- velopment in the group; but devescovinids also occur in Mastotermitidae and Hodotermitidae. They appear to be absent, however, from Rhino- termitidae and Termitidae. The polymastigote family Calonymphidae is of particular interest to PROTOZOA AND OTHER ANIMALS 927 the evolutionist, and it appears to have affinities in common with the Devescovininae (Kirby, 1939). It is restricted, except for one enigmatic form that may not belong in the group, to the genus Kalotermes sensu lato. Amoebae rarely occur in lower termites, but among the Termitidae they are not infrequent. Small amoebae were present in almost all species of Amitermes from the United States, Africa, and Madagascar that were examined by the writer; and many larger amoebae, some with un- usual nuclear characteristics, were found consistently in Central Ameri- can and African species of Mzrotermes and in African termites of the Cubitermes group (Kirby, 1927; Henderson, MS). It is likely that fur- ther study of these amoebae will yield results significant for problems of host-specificity. Figure 195. One-day-old nymph of Kalotermes flavicollis, receiving proctodaeal food from the female termite, showing the manner in which infection with flagellates takes place. (After Goetsch, 1936.) Transmission of the flagellates of termites takes place in the active state (see Andrews, 1930). There is no evidence for true encystation, though observations by Trager (1934) and Duboscq and Grassé (1934) indicate a possibility of this in some small polymastigotes. Flagellates of most species disappear prior to each molt except the last. Infection, then, must take place not only at the beginning, but following each molt in the growth period. Refaunation takes place when termites, either nat- urally or experimentally defaunated, are left in contact with normally faunated individuals. Experimentally, termites can be infected by placing flagellate-containing material on the mouth parts. Under natural condi- tions, except for cannibalism, flagellate-containing material can or- dinarily be obtained only directly from the anal opening of another termite, as the Protozoa do not survive long after deposition. Proctodaeal feeding is a common habit among termites. Goetsch (1936) has de- 928 PROTOZOA AND OTHER ANIMALS scribed the early infection of young nymphs of Kalotermes flavicollis by direct application of the mouth parts, accompanied by sucking, to the end of the abdomen of the dedlates (Fig. 195). Certain small polymastigotes are often retained through the molting period (Kirby, 1930; Child, 1934). In Zootermo psis this is true of the minute forms Tricercomitus and Hexamastix. At the last molt the situa- tion differs from that in the preceding molts. Child (MS) reported that in the last molt of Zootermopsis, although the number of flagellates is greatly reduced, all species are carried through from the seventh instar nymph to the winged imago. Cross (MS) and May (MS) have confirmed this fact in Kalotermes minor and Zootermopsis; the shed intima of the nymph, still containing Protozoa, is retained within the gut; and the Protozoa later escape into the lumen of the imago’s intestine. In Cryptocercus the Protozoa are not lost at the time of molting; but then, and only then, most of them form either well-defined cysts (in Trichonympha and Macros pironympha) or resistant stages (Cleveland, et al., 1934). Flagellates are present, often in great numbers, in pellets passed in the first few days after ecdysis. Cleveland found that some pellets, passed immediately after molting, consisted mostly of Protozoa in encysted or resistant form. Reinfection of defaunated roaches took place when they were placed with molting roaches; but not, except occa- sionally with smaller polymastigotes, by association with other infected roaches. Proctodaeal feeding, then, does not have the same role in trans- mission in Cryptocercus as in termites. Cleveland could not find out the exact manner in which infection is first acquired, but thought it probable that it is by association with molting individuals. Once acquired, the faunule persists until the death of the roach. It is probable that cross infection has not been a significant factor in determining the present distribution of flagellates in termites, below the Termitidae at least. Furthermore, the unique characteristics of almost all the flagellates, which have no close relatives except in roaches, indicate that the faunules do not to any great extent include acquisitions from other arthropods or other animals. The present distribution of the flagel- lates, the absence of resistant stages, and the isolated habits of termites support this opinion. If it is sustained by further studies the flagellates of termites will be shown to be easily the leading group of animals for correlative studies in phylogeny of symbionts and their hosts. PROTOZOA AND OTHER ANIMALS 929 If it could be shown that there is resistance to cross infection, such that flagellates introduced experimentally from a natural host species into another one would not survive, this thesis would of course be sup- ported. The writer (1937), however, stated that there seems to be no resistance to cross infection, basing this opinion on experiments by Light and Sanford (1927, 1928) and Cleveland et al. (1934). No expert- ments yet reported, however, have been continued long enough to war- rant any definite conclusion. Unpublished experiments by Dropkin, furthermore, showed that Protozoa of Reticulitermes flavipes, Kalo- termes schwartzi, and K. jouteli could not establish a physiological rela- tionship with Zootermopsis sufficient to permit survival of the termite for more than fifty days in the absence of the normal faunule. Although there has been evolutionary development of the flagellates within termites of groups that exist today, many of the types doubtless go back to ancestral insects. The genus Trichonympha, being found in both termites and Cryptocercus, may be supposed to have passed into both these insects from ancestral protoblattids (Kirby, 1937). The distribu- tion of Trichonympha in termites alone would indicate its antiquity and stability (Kirby, 1932b). The existence of representatives of other hyper- mastigote groups in Cryptocercus indicates the very ancient differentia- tion of those flagellate types. By loss of members of the faunules here and there, together with continued but less drastic evolutionary changes, the present composition of the faunules may have originated. The flagel- lates were probably present in ancestors of Termitidae, but were, in the course of differentiation of those insects, dropped out. The origin of the amoebae needs to be explained; possibly they were acquired later. ADAPTIVE Host RELATIONSHIPS IN MORPHOLOGY AND LIFE HISTORY GENERAL CONSIDERATIONS Structural modifications in animals that live in association with hosts take two general forms. There are morphological changes in direct adaptation to the requirements of the habitat; and there are changes un- related directly to that habitat, but made possible by various factors in it. In the former group, among Protozoa, is the development of or- ganelles of fixation, though this development is not restricted to Protozoa that live in close relationship with other animals. Special adaptations 930 PROTOZOA AND OTHER ANIMALS may appear for nutrition. Probably also in that category is the increase of the number of flagella and the development of undulating membranes and axostyles in certain groups of flagellates. In the latter category are the reduction or loss of cilia, the reduction or loss of mouth structure, the elaborate development of the parabasal apparatus and other or- ganelles in certain polymastigote flagellates, the complex characteristics pai? \ Aare Figure 196. Streblomastix strix attached to the lining of the hind-gut of Zootermopsis angusticollis. (After Kofoid and Swezy, 1919.) of many hypermastigotes, the elaborate morphological specialization of Ophryoscolecidae. Organelles of fixation appear among flagellates in epibiotic dinoflagel- lates (Chatton, 1920; Steuer, 1928); in Streblomastix strix, which often is attached (Fig. 196) to the wall of the hind-gut of its termite host, by a holdfast (Kofoid and Swezy, 1919; Kidder, 1929); in Pyrsonympha and Dinenym pha, which occur free in the gut lumen of Retzcwlitermes or attached by a small, simple, anterior knob (Koidzumi, 1921); and in Oxymonadinae. In the last group the holdfast, which is applied to the intima of the termite gut, is at the end of a rostellum, which may reach a relatively great length and often contains many fibrils (Kirby, 1928; PROTOZOA AND OTHER ANIMALS 931 (Ge immer eee 3 aN mea “yy ia: a= Sim eet. a. See IN. own 1 Motes em ii aes & Rte e £5 Kenn AIS APTN SSSR Wes NS ON NS Z, ZB Zz de mh iy aM eral piri Figure 197. Fixation mechanisms in peritrichs. A, Ellobiophrya donacis, with ring formed by two posterior limbs applied at the ends; B, Ellobiophrya suspended from the bridges uniting the gill filaments of Donax vittatus; C, section of Trichodina pediculus on the ectoderm of Hydra; D, Cyclochaeta (Urceolaria) korschelti from Chiton mar- ginatus. (A, B, after Chatton and Lwoff, 1929; C, D, after Zick, 1928.) Cleveland, 1935). Giardia adheres by a sucking disc to the wall of the small intestine. In some ectoparasitic dinoflagellates, the organelle of fixation 1s pro- longed by rhizoids into the tissues of the host, and apparently nutriment is absorbed by this mechanism. In the polymastigotes in termites, fixation is only to the gut intima; there is no relationship to the epithelial cells. 932 PROTOZOA AND OTHER ANIMALS In gregarines, the epimerite, which often is elaborately developed with hooks or other appendages and inserted into the cell, may serve also for absorption (see Watson, 1915). As regards ciliates, development of fixation habits and structures in the holotrichous groups of Thigmotricha, Ptychostomidae, and Astomata is discussed below. There is among heterotrichs a well-developed fixa- tion apparatus in Licnophora (Stevens, 1901; Balamuth, MS). Urceola- tids have an elaborately organized scopula, a cup-like apparatus Bo 5 - D3 eH Abas: tt ee iii Figure 198. Fixation apparatus of Cyclochaeta (Urceolaria) korschelti, A, seen from above, showing radially arranged ribs bent downward in hook-like points and ring com- posed of overlapping, sickle-shaped individual pieces; B, cross section. (After Zick, 1928.) supported by radially arranged ribs and a ring of denticles (Fig. 197D; Fig. 198) (see for description and illustrations, Zick, 1928, on Cyclo- chaeta (Urceolaria) korschelti; Fulton, 1923, on Trichodina pedt- culus). As has been described in T. pediculus on Hydra and Tricho- dinopsis paradoxa in Cyclostoma elegans, the epithelial cells of the host may be elevated into this sucker (Fig. 197C). A unique attachment mechanism is that of Ellobiophrya donacis, an inquiline of the gill cavity of a lamellibranch. The posterior end of the body of this peritrich PROTOZOA AND OTHER ANIMALS 2 Be, is prolonged into two limbs hollowed into cups at the ends, the homo- logues of halves of the scopula (Fig. 197A). The two cups are sealed firmly together, so that the limbs form a closed ring by which the ciliate is suspended (Fig. 197B) from the framework of the gills (Chat- ton and Lwoff, 1923b, 1929). In Protozoa that live in association with other animals as hosts, the developmental cycle must be adjusted to the requirements of the habitat. This is so arranged as to insure transmission of the organism from one host to another; production of a sufficient number of infective forms so that the likelihood of some reaching a place where development can continue is not too small; protection of the organism, if necessary, in the period when it is out of its host; and often correlation with the life cycle and habits of the host, so that escape from one host and infection of an- other can take place. The situation is most complex in heteroxenous forms, in which the life cycle is shared between two different species of animals, and is correlated with the bionomics of each of them. Cyclic adaptation has perhaps achieved its most perfect development when there is a regular and direct transmission to the next generation through infection of the eggs or embryos. There are not many instances in Pro- tozoa of this last method, which is so perfectly exemplified among the cyclic endosymbionts of insects (Buchner, 1930; W. Schwartz, 1935). Nosema bombycis invades the eggs of silkworms, but this may be in- cidental and is not the only method of transmission. Other instances occur among heteroxenous Sporozoa in the invertebrate host in Babesia and Karyolys7s (see also Lavier, 1925). The developmental cycle and methods of transmission have been con- sidered widely in textbooks and in a general way by many authors, in- cluding Hegner (1924) and Grassé (1935). The situation in certain epibiotic Protozoa is also of considerable interest; and in that connection accounts are given below of the holotrichs Conidiophrys pilisuctor and apostomatous ciliates. THIGMOTRICHA Chatton and Lwoff (1922c) proposed the name Thigmotricha for a group (suborder) of holotrichs including the families Ancistridae, Hy- pocomidae, and Sphenophryidae. Most of these ciliates are inquilines, commensals, or parasites on the gills or palps of molluscs, though some 954 PROTOZOA AND OTHER ANIMALS occur on Protozoa or other invertebrates. They are provided with thigmo- tactic cilia; and they show a series in the evolution of thigmotacticism, in the course of which there is developed a penetrative and absorptive organelle, and in the regression of body ciliature. Chatton and Lwoff (1923a) described Thigmophyra, which was later (1926) placed in a fourth family, Thigmophryidae. Thigmophrya, it was stated, closely resembles Conchophthirus; but, unlike Conchophthirus, it possesses a well-defined thigmotactic area identical with that of other Thigmotricha. Kahl (1934) summarized the characteristics of the suborder Thigmo- tricha and included in it the Conchophthiridae, which he had formerly (1931) treated in the suborder Trichostomata, and which Chatton and Lwoff (see 1937) evidently did not intend to include in their group. Though Calkins (1933) considered adaptations to parasitism in the thigmotrichs as a group, he included most of the genera in the Tricho- stomata, including the Ancistrumidae which Kahl (1931) had put in the Hymenostomata. Calkins, on the other hand, separated Hemzspeira from other Ancistrumidae, putting it in the Hymenostomata. Whether the Thigmotricha constitute a homogeneous group may be questionable (Fauré-Fremiet, 1924, p. 7); but for consideration of the ethological relationships and adaptations to symbiotic existence, the object of interest in this account, it is convenient to treat them together. Most Conchophthiridae occur in the mantle cavity of Pelecypoda, both marine and fresh-water species. Andreula antedonis (André) Kahl (Concho phthirus antedonis André) occurs abundantly in the alimen- tary canal of a crinoid echinoderm; and Uyemura (1934) described as C. striatus a ciliate in the intestine of several sea urchins of Japan. Myxo- phyllum steenstrupii (Stein) lives in the slime covering the body of a variety of land pulmonates. The species of Morgania Kahl and all ex- cept the one species of Conchophthirus Stein (the original spelling by Stein, 1861, not “Conchophthirius” as given by Strand, 1928) men- tioned above are restricted to bivalves. The most detailed studies of the genus Concho phthirus are contained _ in several articles published in 1933-34 by Kidder and by Raabe. Uye- mura (1935) found three species in great abundance in a fresh-water mussel of Japan, Anodonta lauta. There is no doubt that in all parts of the world certain lamellibranchs will be found abundantly infected with these commensals. Only a beginning has been made in their study, as PROTOZOA AND OTHER ANIMALS 935 in that of all the Thigmotricha, insofar as a knowledge of geographical distribution and host-specificity is concerned. In the mantle cavity of the hosts, some species are not localized, whereas others are. Kidder (1934a) found C. curtus and C. magna on all exposed surfaces and also swimming freely in the mantle fluids; C. anodontae, on the other hand, he found to be invariably localized on the nonciliated surface of the palps. The cilia of the flat left side (left if, with Kahl and Raabe, we consider the flattening to be lateral; according to De Morgan and Kidder, it is dorsoventral, and the attach- ment is by cilia of the ventral surface) are thigmotactic. The thigmotactic area usually covers the whole broad side, but in C. dsscophorus there 1s more specialized adhesive apparatus, a circular, sharply outlined area, which occupies only part of the left side, is markedly concave, and 1s provided with differentiated cilia (Raabe, 1934b). C. discophorus swims slowly, and often fastens itself firmly by the thigmotactic region. C. mytili (Fig. 199A) also swims about or clings firmly to surfaces (Kid- der, 1933a). C. anodontae on Elliptio complanatus seems to be most markedly thigmotactic (Kidder, 1934a), remaining quiet, attached to the surface of the palp. Kidder (1933a) found the food vacuoles of C. mytili (Morganta mytili, according to Kahl, 1934) to contain plankton organisms, includ- ing algae, and sperm cells of the host. C. caryoclada (Morgania caryo- clada, according to Kahl) contained mostly algae (Kidder, 1933d). Other species contained algae, bacteria, and sloughed-off epithelial cells. The relationship appears to be simple commensalism, but Kidder (1934a), finding only well-preserved epithelial cells in the food vacuoles of C. magna, was “‘a little in doubt as to its purely commensal rdle.”’ Kidder (1934a) remarked that there is a fair degree of host- specificity. In nature certain species are characteristic of certain molluscs; and the faunules may differ, even though in nature the hosts are very closely associated. Rarely there are as many as three species in one host. A number of species have been found in only one or a few related hosts, but this may be a consequence of the relatively few examinations. A cosmopolitan distribution is characteristic of such species as C. curtus, reported from various fresh-water clams in Europe, the eastern United States, and Japan. Morgania mytili is a commensal of Mytilus edulis in various localities on both sides of the North Atlantic. 936 PROTOZOA AND OTHER ANIMALS In the family Thigmophryidae, Thigmophrya bivalviorum, which occurs on the gills of the marine pelecypods Mactra solida and Tapes pullastra, has a thigmotactic region reduced to an elliptical area in the anterior fifth of the body (Chatton and Lwoff, 1923a). The movements of the cilia of this area are not synchronous with those of the rest of the body. The ciliate swims in the mantle cavity or fixes itself to the gills. The family Ancistrumidae is large and diverse. In general, the ciliates are more sedentary than those previously considered in the order Thigmo- tricha and the thigmotactic area is still more restricted. Although the most frequent habitat is the mantle cavity of Pelecypoda, other molluscs as well as members of other phyla of invertebrates serve as hosts for species of the family. Probably, however, the original hosts were Pelecy- poda. The two principal genera are Ancistruma Strand, 1928 (given incor- rectly as 1926 by Kahl and Kidder) (Fig. 199B, C) and Boverza Stevens, 1901, but there are many others: Eupoterion MacLennan and Connell, Ancistrina Cheissin, Ancistrella Cheissin, Plagiospira Issel (Fig. 199D), Ancistrospira Chatton and Lwoff, Proboveria Chatton and Lwoff, Tzarella Cheissin, Hemispeira Fabre-Domergue (Fig. 200C), Hemis peiro psis Konig (Fig. 200A, B). Kahl (1934) put into the fam- ily, though doubtfully, two ciliates parasitic in Littorina, Protophrya ovicola Kofoid and Isselina intermedia Cépéde. The Ancistrumidae possess more or less conspicuous peristomal cilia; often these rows constitute a prominent fringe. In typical forms the organisms adhere to the surfaces on which they live by the thigmotactic cilia in a tuft at the anterior end. Ancistrella choanomphthali, however, adheres to the gills by its entire concave, ventral surface (Cheissin, 1931). In the Ancistrumidae an evolutionary series is apparent in the shifting posteriorly of the mouth and of the peristome, which becomes spiraled. Chatton and Lwoff (1936b) suggested that the Ancistrumidae constitute so extraordinarily homogeneous a family that we may consider that there is only one genus, subdivided into subgenera, an opinion that expresses the homogeneity, though possibly the conclusion that there should be only one genus is not sound taxonomically. These authors remarked that the characteristics separating the genera or subgenera are purely quanti- tative, consisting of more and more accentuated retrogradation of the mouth and prostomal ciliary lines from the anterior half of the body to the posterior end (Fig. 200K-N). PROTOZOA AND OTHER ANIMALS D5 Other habitats than the mantle cavity of Pelecypoda have been adopted by various species of Ancistruma and Boveria, as well as by members of other genera. Thus Issel (1903) found A. cyclidioides on certain chitons and gasteropods (Natica heraea) as well as on Pelecypoda; and he de- sctibed A. barbatum solely from gasteropods of the genera Fusus and Murex. Adaptation to these hosts is, as was stated above, probably second- ary. In a similar manner, one species of Boveria, the type species B. sub- cylindrica Stevens, is attached to the membrane of the respiratory tree of the holothurian Stichopus californicus (Stevens, 1901). So similar to this, however, that it has been classified as a variety of the same species, B. s. var. concharum Issel, is a Boveria that occurs on the gills of ten of fourteen Pelecypoda that harbor Ancistrumidae at Naples (Issel, 1903). B. labialis lives in the respiratory trees of holothurians as well as on the gills of a clam (Ikeda and Ozaki, 1918). Expoterion pernix, which has many characteristics of a species of Ancistruma, inhabits the intestine of the limpet Acmaea persona (Mac- Lennan and Connell, 1931). The aberrant Hemispeira asteriasi Fabre- Domergue (1888) and Hemispeiropsis antedonis (Cuenot, 1891) occur on echinoderms, the former on the dermal branchiae of a starfish, and the latter on the pinnules of a crinoid (Cuenot, 1894; Konig, 1894). Protophrya ovicola Kofoid occurs upon the surface of the egg capsules in the brood sac of the gasteropod Littorina rudis; and Isselina intermedia is found in the mantle cavity of Littorina obstusata. The two latter species, at least Protophrya, are more truly parasitic than other Ancis- trumidae, and they have undergone some retrogressive changes. For the most part, Ancistrumidae feed on bacteria, diatoms, and other material extracted from the currents of water. Issel (1903) noted that two bivalves constantly rich in the ciliates, Capsa fragilis and Tellina exigua, live under conditions most suitable for offering their inquilines copious food. They occur in calm, muddy water, rich in organic sub- stances. The diet of plankton organisms may be supplemented by sloughed-off epithelial cells, as noted by Stevens (1901) in Boveria subcylindrica and Pickard (1927) in B. teredinidi. The account by Ikeda and Ozaki (1918) of tissue invasion by B. Jabialis is not acceptable without corroboration. The changes said to be undergone by the encysted ciliate within the tissue are bizarre. Protophrya ovicola in the brood sac of Littorina has a destructive chemical action upon the eggs (Kofoid, 1903) and a teratogenic action 938 PROTOZOA AND OTHER ANIMALS on the embryo (Cépéde, 1910). The parasites do not act directly on the embryos, but on the medium, which exerts an injurious effect on embryos in the early stages. Abnormal embryos result, in which the shell is more or less unrolled; not only may the shell be misshapen, but the cells that secrete the shell may fail to function normally. The Hypocomidae (Fig. 199E-1) are true parasites, and occur mostly in marine and fresh-water bivalves and snails. There is no mouth, but the anterior end is provided with a short retractile tentacle. Normally the ciliates are attached to the gills or skin of the mollusc, the tentacle being embedded in an epithelial cell. The parasites obtain nutriment by ex- tracting the contents of the cells to which they are attached, the tentacle combining suctorial functions with those of attachment. The tentacle continues in a tubular structure, extending more or less deeply into the cytoplasm. In many Hypocomidae a fine inner canal has been observed extending from the apex of the tentacle into the deeper cytoplasm. Ac- cording to Chatton and Lwoff (1922c), this adherent organelle is de- rived from structures of Ancistrumidae, where it is indicated in Ancis- truma mytili and is well developed in A. cyclidioides. In relation to the attached parasitic condition of hypocomid ciliates are the regression of the mouth and peristomal ciliature and the reduc- tion of the general ciliature. The former structures have for the most part already disappeared. There is no mouth, but Chatton and Lwoff (1924) stated that in some genera there are residual segments of the adoral ciliary zone. In reduction of the general ciliature there is in Hypocomidae a well- integrated series. The body of Hypocomagalma dreissenae Jarocki and Raabe, 1932, is covered with cilia except for a small ventroterminal area. Ancistrocoma pelseneeri Chatton and Lwoff, as figured by Raabe (1934a), has a larger cilia-free area, occupying a large part of one side of the body, and an anterolateral peristomal fringe. Perhaps the less firm fixation of these forms is also a phylogenetically primitive character. Raabe stated that Ancistrocoma adheres rather weakly to the gills of its host and separates readily. Hypocomagalma swims more rapidly than some other ciliates of the group. Reduction of the ciliature continues through Hypocomides Chatton and Lwoff and Hypocoma Gruber, in which it occupies the inner area of the ventral surface of the body. In Heterocineta (—Hypocomatomorpha) unionidarum Jatocki and Raabe ie eee ine ee Sapte 285 Ua AER; . ¥ = ‘ Ue 3 s , i ii +3, i Ctr << wumuatlunnduuyy, ¢ m,. > ne! Figure 199. Thigmotricha. A, Morgania (Conchophthirus) mytili from Mytilus edulis ; B, C, Ancistruma mytili from Mytilus edulis, B, dorsal view, C, lateral view, tuft of straight tactile cilia near anterior end; D, Plagiospira crinita from Loripes lacteus; E, F, Hypocomina carinata from Mytilus edulis; G-1, Hypocoma parasitica: G, lateral view, tentacle and tubular structure below, H, ventral view, I, two hypocomids attached to Zoothamnium. (A, after Kidder, 1933a; B, C, after Kidder, 1933c D, after Issel, 1903; E, F, after Raabe, 1934a; G-I, after Plate, 1888.) 940 PROTOZOA AND OTHER ANIMALS and Hypocomatidium 5 phaerii Jarocki and Raabe, from the gills of fresh- water mussels, reduction is far advanced to a thigmotactic zone restricted almost entirely to the anterior half of the ventral side. Cilia are not lost altogether in known Hypocomidae. Syringo pharynx pterotracheae, which lives on the gills of the heteropod Pterotrachea coronata either swimming free or fixed to epithelial cells by the rostrum (Collin, 1914), was included by Kahl (1934) among the parasitic gymnostomes. Probably, however, this is a hypocomid ciliate, one with a general body ciliature like Anczstrocoma and Hypo- comagalma. Parachaenia myae, described from Mya arenaria by Kofoid and Bush (1936), may also be a hypocomid, although attachment to the cells of the gills of its host was not described. Chatton and Lwoff’s statement (1926, p. 351) about the prolongation of the anterior indi- vidual in a spur covering the dorsal anterior region of the posterior individual in binary fission of Ancistrocoma pelseneeri is in exact agree- ment with the division process of Parachaenia myae. Another point of agreement with Ancrstrocoma is the type of conjugation. In the shape of the ciliates, the unique attachment of the conjugants by the posterior ends, and the shape and arrangement of the nuclei of P. myae, there 1s almost complete agreement with A. pelseneeri as figured by Raabe (1934a). Among the many genera of Hypocomidae, not all of which can even be named here, the number of which we may expect will be markedly reduced with further study, only the species of Hypocoma (Fig. 199G-1) do not occur on bivalves or snails. They are parasites of Protozoa, and are discussed elsewhere in this book (p. 1083). Hypocomidae are host-specific in marked degree. They are obligatory parasites on certain individual or closely related molluscs, and do not readily infect other molluscs. Jarocki (1935) found that Heterocineta janicki, placed free-swimming into an aquarium with various molluscs, would attach only for periods of from fifteen to eighteen hours in the absence of its natural host Physa fontimalis. When various molluscs, 1n- cluding Bithynia tentaculata, were put together in an aquarium, Hetero- cineta krzysiki, though abundant on the body of Brthynia, did not infect any other species. Sometimes two hypocomids are present on the same host, and Raabe (1934a) noted that there seems to be a tendency to inhibition by one parasite of the development of the other. NY Figure 200. Thigmotricha. A, B, Hemispeiropsis comatulae from Antedon (Comatula) mediterranea; C, Hemispeira asteriasi from Asterias glacialis ; D-H, Sphenophrya dosiniae from Dosinia exoleta: D, adult, E, budding individual, F, young individual, G, longi- tudinal section of Sphenophrya on branchial filament, H, transverse section showing ciliate in furrow between branchial filaments; I, J, Gargarius gargarius (Rhynchophrya cristal- lina) from Mytilus edulis ; K-N, series of diagrams showing retrogradation of peristome in Ancistrumidae: K, Ancistruma, L, Proboveria, M, Boveria, N, Hemispeira. (A, B, after Konig, 1894; C, after Wallengren, 1895; D-H, after Chatton and Lwoff, 1921; I, J, after Raabe, 1935; K-N, after Chatton, 1936.) 942 PROTOZOA AND OTHER ANIMALS In connection with host-specificity among hypocomids, Jarocki’s ob- servation (1934, 1935) that Heferocineta janicki is also a facultative parasite on the oligochaete Chaetogaster limnaei is of considerable in- terest. The oligochaete is usually present as an inquiline in the mantle cavity of Physa fontinalis and other snails. The hypocomids are almost always present on the snails; and they also infect almost all the oligo- chaetes, attaching to various parts of the body and inserting the suctorial tentacles into the hypodermal cells. Parasite-free worms quickly became infected if brought into contact with ciliates either in or out of the mantle cavity. Parasite-free Physa became infected if parasitized olli- gochaetes were introduced into the mantle cavity. The worms pass freely from one host to another, and thus facilitate the spread of the infection. Chaetogaster limnae/ in other snails became facultatively para- sitized by their specific hypocomids; but Heferocineta species could not be introduced into unnatural hosts on the oligochaetes. To two other species of Chaetogaster, Heterocineta janickii became attached tempo- rarily, but soon dropped off. The Sphenophryidae (Fig. 200D-H) ) all occur on the gills of marine lamellibranchs. They are sedentary, immobile, and nonciliated in the adult phase. They are not true parasites; the relationship as defined by Chatton and Lwoff (1921) is “inquilinism complicated by phoresy.” The ciliates are rather large, mostly flattened laterally, and adhere to the surface by a long ventral edge (Fig. 200G). Sometimes they adhere in a very precise and constant position, as Sphenophrya dosiniae in the furrows separating adjacent branchial filaments of Dosinia exoleta (Fig. 200H). There is no mouth opening, and the ciliates apparently feed osmotically; but Mjassnikowa (1930a) found evidence that S. sphaeri ingests cells of the gill epithelium. She may, however, have misinter- preted the nature of certain cytoplasmic spherules. Reproduction is by development of motile buds. Cilia develop from the infraciliature, which consists of a few rows of granules that are present in the vegetative indi- vidual. An unusual sphenophryid is Gargarius gargarius, described by Chat- ton and Lwoff (1934a) from Mytilus edulis at Roscoft. Rhynchophrya cristallina from M. edulis in the Baltic Sea, of which a more complete account was later given by Raabe (1935), is evidently the same ciliate (Fig. 2001, J). Along one surface are two longitudinal, parallel, comb- PROTOZOA AND OTHER ANIMALS 943 like structures, producing a plaited surface suggesting Aspidogaster to Raabe. At the anterior end is a beak-like process, which is embedded in the cells of a filament, whereas the plaited surface adheres to another filament. Nutrition is osmotic. PTYCHOSTOMIDAE The holotrichous ciliates of the family Ptychostomidae are considered by some to be related to the Thigmotricha, if indeed they do not belong in that group. Beers (1938b), following Jarocki (1934), gave prefer- ence to Hysterocinetidae Diesing, 1866, as having priority over Ptycho- stomidae Cheissin, 1932. There is, however, no general recognition of priority in family names, but these are based on the name of the type genus. In this instance it seems that Ptychostomum Stein, 1860, the first described, best known, and largest genus, should not be supplanted as the type by Hysterocineta Diesing, 1866. Rossolimo (1925) suggested that the Thigmotricha and Ptychostomum represent two parallel evolu- tionary series, derived from the same group of free-living organisms, but adapted in somewhat different ways to attachment and the requirements of sedentary life. The family Ptychostomidae now includes some eighteen species, of which nearly half are from Lake Baikal. The ciliates occur in the in- testine of fresh-water oligochaetes, except for Hysterocineta ezseniae de- scribed by Beers (1938b) from a terrestrial oligochaete, and three species from the intestine of gasteropods. In oligochaetes, Ptychostomidae are associated with astomatous ciliates. Cheissin (1932) remarked that there is a tendency for Astomata to be located more anteriorly in the intestine, whereas Ptychostomidae occur in the posterior part. Beers found that 90 percent of H. eiseniae are localized in the third quarter of the gut of Eisenia loénnbergi; and an astomatous ciliate occurs more anteriorly. Cheissin (1928) stated that “Lado psis” (—Hysterocineta) benedictiae is found in the mantle cavity (?) of Benedictia baikalensis; later (1932) he wrote that that ciliate occurs mostly in the intestine and enters the mantle cavity seemingly only accidentally. It may be expected that study of fresh-water oligochaetes in various parts of the world will greatly increase the size of the group. There may then be a tendency to greater subdivision, but at present there are only two genera, Ptychostomum and Hysterocineta, Lada Vejdovsky being a 944 PROTOZOA AND OTHER ANIMALS synonym of the former and Ladopsis Cheissin of the latter. According to Jarocki (1934), the points of distinction are the shape of the macro- nucleus, the size and position of the micronucleus, and the position of the contractile vacuole. Whether such points are sufficient for distinction of genera is questionable. The power of attachment, achieved by a sucker-like organelle, is marked (Fig. 201D). Miyashita (1927) found that Ptychostomum tanishi, when observed in the dissected-out gut, was in part attached to the inner surface of the intestine and in part swam freely in the fluid. Figure 201. Ptychostomidae. A, Ptychostomum rossolimoi from Limnodrilus newaensis ; B, skeletal fibrils of the sucker area of that species; C, Ptychostomum chattoni from Lumbriculus variegatus ; D, Pt. chattoni adherent by sucker to the intestinal wall of the annelid. (A, B, after Studitsky, 1930; C, D, after Rossolimo, 1925.) When put into water, the ciliates eventually attached themselves to sur- faces. Heidenreich (1935) described strong attachment by the sucker in P. rhynchelmis, folds of the intestinal wall being drawn into it. Beers, however, remarked that in H. e/seniae the sucker appears to be only weakly functional, and most specimens were swimming freely in the lumen of the intestine. In relation to the sedentary position, there is more or less marked dorsoventral flattening of the elongated body. According to Studitsky (1930), the first step in the development of the fixation apparatus is represented by the horseshoe-shaped, non- ciliated area in the anterior part of the ventral surface of Ptychostomum Saenuridis, in which no skeletal structures have been described. The next PROTOZOA AND OTHER ANIMALS 945 step is the strengthening of this area by skeletal fibrils, consisting, first, of a set of longitudinal fibrils, and second, of a set of fibrils crossing these. In further development there is differentiation and strengthening of the two systems. The skeletal fibrils form an irregular network in the floor of the sucker. The sucker itself is a simple concavity in P. rossolimoz (Fig. 201A, B) and some other species; sometimes there are a few rows of cilia on an elevated area in its floor. In other species, probably more advanced in the evolutionary series, the border of the sucker is a lip- like elevation. In some forms the sucker is circular (P. tanishi), but often it is pointed posteriorly (P. chatton7, Fig. 201C) or has an opening (P. wrzesniewskii) or indentation (P. elongata). In H. eiseniae it is V- shaped. Cheissin (1932) described myonemes in addition to the skeletal fibrils. Heidenreich (1935) stated that the fibrils of the sucker, as de- scribed by authors, are contractile, thus being not skeletal structures but myonemes. Beers, however, found no myonemes in the species he studied, and concluded that all the fibrils have a supporting function. P. rhyn- chelmis has, Heidenreich stated, unlike other species, a skeleton in the sucker. The sucker is bordered by two sickle-formed skeletal bows, form- ing a ring open posteriorly, and each sickle is prolonged posteriorly in a handle. The two handles form a canal, the neck of which is surrounded by three or four myoneme bands. In Ptychostomidae the oral apparatus is situated at what has been regarded as the posterior end. Beers described a shallow, transverse peris- tomal groove, bordered by lips bearing cilia, and a small cytostome leading into a short, tubular cytopharynx. He found no food vacuoles and no ingestion of ink particles, and consequently concluded that the mouth is non-functional. In this group of ciliates the feeding apparatus is in process of reduction. It functions in some species, possibly together with saprozoic nutrition; in others it has, though still present, little rdle to play in nutrition. ASTOMATA The suborder Astomata is systematically heterogeneous, lacking phylo- genetic unity, as Kahl (1934) remarked. Cépéde (1910) himself noted that fact. The group includes many forms that lack complete descrip- tions. There is no systematic unity to be obtained by bringing together 946 PROTOZOA AND OTHER ANIMALS forms according to the negative characteristic of absence of a cytostome, as ciliates of quite divergent relationships may have suffered regression of that structure. Since a cytostome is not absent in free-living ciliates, even of the most primitive type, it seems most likely that there has been regression, rather than that lack of it is a primitive condition. Further- more, we have to take account of the fact that oral structures may oc- casionally have been overlooked. Cépéde (1910) removed the opalinids from the group, and since then several other genera have been excluded. The Ptychostomidae have gone; in them the mouth structures are not absent, as at first supposed. Protophrya appears to have affinities with the Thigmotricha. Chatton and Lwoff (1935) stated that Metaphrya sagittae from the coelomic cavity of Sagitta sp. is an apostome; and Kofoidella eleutheriae may also be a foettingeriid. The description of Kofosdella, from the gastrovascular canals of a medusa, is too inadequate for systematic purposes; but so far as it goes relationship to Pericaryon, from the gastrovascular canals of Cestus veneris, is not excluded. The macronucleus is described as com- pact and central, and quite variable in size. The macronucleus of Peri- caryon cesticola 1s reticular and peripheral; but it is not impossible that Cépéde, who stated that the supposed macronucleus of Kofo/della could be demonstrated (by Maupas) only after treatment with acetic acid, was referring instead to the trophic mass. The greater part of the Astomata inhabit the intestine of Oligochaeta. In the table of distribution given by Cheissin (1930), records are given of 69 species in the intestine of Oligochaeta, and of 41 elsewhere. Among the latter, omitting Kofoidella and Chromidina, the affinities of which are doubtful, there are 36 species, and 12 of them occur in polychaetes and in the coelom and gonads of oligochaetes. Heidenreich (1935) added 11 species in the intestine of oligochaetes, and Beers (1938a) added one. With about 75 percent of the known species in the intestine of oligochaetes and polychaetes, and the affinities of many of those found elsewhere doubtful, we may correctly consider the Astomata to have a close ecological relationship to that group of animal hosts. Cheissin (1930), examining invertebrates of Lake Baikal for Asto- mata, found none in many Turbellaria, molluscs, and polychaetes, and only a few amphipods had Ano plophrya in the body cavity. Of 24 species of oligochaetes examined, all the Lumbriculidae and most of the others PROTOZOA AND OTHER ANIMALS 947 had one or more species of Astomata; three-quarters of 2,062 individ- uals were infected. Some hosts have several species of the ciliates; in one there are as many as 7, but all may not be present at the same time. Heidenreich (1935) examined worms, mostly oligochaetes and turbel- larians, collected in the vicinity of Breslau. He noted that very few ciliates are found in worms from flowing water, presumably because of the fact that cysts are carried away. There is a certain amount of host-specificity in the group. Hoplo- phryidae and Intoschellinidae appear to be restricted to annelids. Many species have been described from one host only, but some occur in many hosts. Cheissin (1930) found Radiophrya hoplites Rossolimo and Mes- nilella rostrata Rossolimo in most of the Lumbriculidae; the former oc- curred only in worms of that family, the latter was found also in an enchytraeid. There has been a high degree of differentiation of species and genera in the Astomata, although there are comparatively few char- acteristics in which that differentiation can be exhibited. There are two large groups of the Astomata from annelids, those with- out and those with skeletal structures. The former constitute the family Anoplophryidae; the latter were put by Heidenreich (1935) into the two families Hoplitophryidae and Intoshellinidae. The skeletal struc- tures are differentiations of the ectoplasm or endoplasm, or of both, in the form of resistant, refractile, and stainable rods, hooks, rays, or fibrils. They are completely renewed at division. According to the scheme of de- velopment outlined by Heidenreich, the simplest form is a small, ecto- plasmic, skeletal plate with a short tooth that scarcely extends from the pellicle (Evmonodontophrya kijenskiji, Fig. 202K). The plate elon- gates to the rod-like spicule characteristic of Hoplitophrya, in some species of which there is a point projecting from the anterior end of the body. In H. fissispiculata (Ch.) (==Protoradio phrya fissis piculata Cheis- sin) the spicule is divided in a narrow-angled cleft in its posteriar part. This is an approach toward the V-shaped ectoplasmic skeletal element of Radiophrya (Fig. 202 C). The latter is usually provided with an apical point, which projects from the body surface (Fig. 202, J). Some- times a second element, a hook or tooth attached to the central, basal part of the arrowhead and projecting free of the body, is present. This pointed tooth typically projects backward at an angle. In R. hoplites, Cheissin (1930) observed that the tooth is capable of movement. There ot lo, 7h \ aaa 4 | 4 C 4 yg rome Waar LS ATG | Figure 202. Skeletal structures and attachment organelles in Astomata. A, B, Metara- diophrya asymmetrica from the oligochaete Essenia lonnbergi; C, Radiophrya tubificis from Tubifex tubifex; D, Mrazekiella costata (anterior end) from Rhynacodrilus coccin- eus; E, Buchneriella criodrili from Griodrilus lacuum (the sphere around the projecting spine is normally formed in the tissue cell to which the ciliate attaches) ; F, Maupasella nova from Essenia foetida and species of Lumbricus; G, Mesnilella fastigata from Enchy- traeus mobbii; H, Mesnilella maritui from oligochaetes; 1, Intoschellina poljanskyi from Limnodrilus arenarius; J, Radiophrya lumbriculi from Styloscolex sp. and Lamprodrilus sp., attachment apparatus; K, Eum monodontophrya kijenskiji from Tubifex inflatus. (A, B, after Beers, 1938a; C, after Rossolimo and Perzewa, 1929; D, E, F, after Heiden- reich, 1935; G, after Cépéde, 1910; H-K, after Cheissin, 1930.) PROTOZOA AND OTHER ANIMALS 949 is a third skeletal element in Radsophrya; more or less numerous ecto- plasmic skeletal strands attached along the entire inside of the V and extending posteriorly on the ventral region of the body. These spread laterally and, although usually restricted to the anterior part, may reach almost to the posterior end. Skeletal elements of similar type are present in Mrazekiella (Fig. 202D) and Metaradio phrya. The attachment organelle of Metaradiophrya asymmetrica, described by Beers (1938a) from the terrestrial oligochaete Ezsenza lonnbergi in North Carolina, consists of a shaft embedded in the ectoplasm of the anterior part of the body and a stout projecting hook, which Beers found to be immovable (Fig. 202A, B). The left half of the V-shaped element, such as is present in Radio phrya, is lacking. From the attachment organ- elle, skeletal fibrils radiate in the ventral ectoplasm, very close to the surface, the principal group originating near the base of the hook, fol- lowed by an area of the shaft devoid of fibers, then a group of a few short fibers at the posterior end. The asymmetrical arrangement of the fibrils, which is contrasted with the bilaterally symmetrical systems of M. falcifera and M. lumbrici, is the source of the specific name. In other forms the skeleton is completely or partly endoplasmic. In Mau pasella (Fig. 202F) the side arms of the V-shaped element have become reduced and the point has developed into a prominent, project- ing, pointed organelle, that serves for fixation. The longitudinal rays have become endoplasmic. Related to Mamupasella is Buchneriella crio- dvili Heidenreich, which has a particularly well-developed movable spine (Fig. 202E). This penetrates into cells of the intestinal epithelium, anchoring the ciliate firmly. In many ciliates torn from attachment, the end of the spine was surrounded by a globule of differentiated host tissue. In Mesnilella the V-formed element is lacking, and the longitudinal rays are endoplasmic and often reduced in number. A series may be ar- ranged from a many-rayed condition (Fig. 202H) to that in which there is only one spicule, reaching almost the full length of the body (Fig. 202G). Intoschellina has a different type of skeletal apparatus (Fig. 2021). It is an open ring in the ectoplasm surrounding the apex of the body. From this ring three short spines project above the body surface an- teriorly, and three extend in the ectoplasm posteriorly. Two of the posterior spines are short; one, located at one end of the ring, is rela- tively long. 950 PROTOZOA AND OTHER ANIMALS A more or less marked concavity is present on one side near the anterior end of many species of the Astomata mentioned above. This concavity, often supported by skeletal fibrils, may fit easily on the convex surface of the intestinal folds. It is not differentiated as a true sucker, however. The projecting spines and hooks of the skeletal apparatus of many forms serve definitely for attachment. These are adaptations to the requirements of the habitat, but it is a question whether the skeletal apparatus as a whole can be considered to be strictly a fixation apparatus. In the Astomata of the family Haptophryidae, there is a true sucker. If there is a systematic unity in the family, the wide separation of the two groups of hosts, Turbellaria and Amphibia, is noteworthy. The species that have spicules, Lachmannella without and Steinella with an anterior acetabulum-like concavity, occur only in various Turbellaria, and since there are no complete and modern descriptions, comparison with other Haptophryidae is difficult to make. The several species of Haptophrya are better known, especially H. michiganensis Woodhead, 1928, as described by Bush (1933, 1934). H. gigantea has been found in certain European and Algerian frogs and toads; and H. michiganensis in several American salamanders and one frog. Rankin (1937) reported the latter species from 5 of 19 species of North Carolina salamanders, in incidence of 6.3-21.4 percent; and Hazard (1937) found it once in Plethodon cinereus in Ohio, which species Rankin had reported nega- tive. Hazard also found the ciliate in 20 percent of Rana sylvatica in Ohio. There may be some difference in infection in the same host species in different geographic regions. Cépéde (1910) noted that R. esculenta harbors H. gigantea in Algeria, but lacks it in Northern France. Rankin found what he considered to be H. gigantea, together with H. michi- ganensis, in a few of the many Plethodon glutinosus studied. Meyer (1938) reported H. virginiensis, a new species, in R. palustris. The occurrence in a turbellarian of a species often put into the same genus, Haptophrya, is of interest from the standpoint of host-specificity. The species planariarum occurs in various marine and fresh-water Tur- bellaria (Cépéde, 1910), principally in Planaria torva. Bishop (1926) found it in 70 percent of that triclad at Cambridge. Finding certain differences from the forms in vertebrates, she kept it in the genus Sze- boldiellina; but Cheissin (1930), followed by Bush (1934), did not recognize any generic differences. Speculation on the origin of this di- versity of hosts would, with our present information, be vain. 5 j \ Oa an >, Figure 203. Anoplophrya (Collinia) circulans in Asellus aquaticus, A, B, large indi- viduals showing nuclei and pulsating vacuoles; C, terminal portion of antenna broken at end, ciliates enclosed in blood vessel and escaping into water, on contact with which some disintegrate; D, thoracic leg containing ciliates; E, segment of the basal part of an antenna, ciliates carried in opposite directions in the currents of blood. (After Balbiani, 1885.) 952 PROTOZOA AND OTHER ANIMALS Astomatous ciliates that occur in other hosts than annelids, except the Haptophryidae and Chromidinidae, were placed by Cheissin (1930) in the family Anoplophryidae. Heidenreich (1935) separated many of these from that family, without giving them other systematic assignment. So separated by him were the species of the genus Collinia Cépéde, which occur in the hemocoele of amphipods and isopods. According to Cheis- sin (1930) and Summers and Kidder (1936), Collinia is a synonym of Anoplophrya; so that members of that genus occupy very diverse situa- tions. There are several species of the ciliates which are evidently not uncommon in asellids and gammarids. Summers and Kidder believed that there is a relatively strong host-specificity. When Balbiani (1885) described Anoplophrya circulans (Fig. 203), he stated that it was the first example of a ciliate living in the blood of its host (Asellas aquaticus) and circulating with the corpuscles. When the ciliates become too crowded to pass through orifices they consti- tute an obstruction that impedes the circulation. Here and there they pass out through orifices perforating the walls of the arteries, and return with the current to the heart. Only a few continue to the ends of the arteries. As the oxygen is used up in a dead isopod, the ciliates slow down and die; and they ordinarily perish quickly in fresh water. Some, however, survive and encyst on plants or on the legs and antennae of Asellus, later escaping from the cyst and becoming active for a time in the water. The species of Dogzelella are tissue parasites which occur in the pa- renchyma of the mollusc Sphaerium corneum and the rhabdocoeles Stenostomum leucops and Castrada sp. in Russia (Poljanskij, 1925). Poljanskij did not refer to Fuhrmann’s statement (1894, p. 223) that numerous holotrichs occurred in the parenchyma of two individuals of S. leucops near Basel; but he believed that ““Holophrya virginia’ de- scribed by Kepner and Carroll (1923) from the same rhabdocoele in Virginia is Dogzelella. The ciliates seem to have no unfavorable effect upon Sphaerium corneum, even in a moderately heavy infection, but with excessive multiplication the host-parasite balance is disturbed and the molluscs perish from mechanical injury. Rarely, the ciliates may infect the developing embryos in the brood chamber. The forms in rhabdo- coeles are apparently harmless to the host. . Cé pédella hepatica occurs in the hepatic caecum of Sphaerium corneum PROTOZOA AND OTHER ANIMALS 953 in France. An organelle of fixation, a slightly concave plate to which a cone of myonemes is related, is developed at the anterior extremity. The ciliate may penetrate into the hepatic cells. The parasitized cell undergoes degenerative vacuolization, which extends to neighboring cells (Cépéde and Poyarkoff, 1909). Cysts have been found in the liver (Poyarkoff, 1909); these may persist in the outer medium and in- fect a new molluscan host. Another tissue parasite is Orchitophrya stellarum Cépéde, a rare ciliate which was found in 3 of more than 6,000 Asteracanthion rubens (Cépéde, 1910). The infected sea stars were all males, and the ciliates occurred in the gonads, among the reproductive cells. Cépéde found that the parasites were well adapted to life in the sea water, underwent no pathological changes, and survived for a long time. In a putrefying genital gland, removed from the starfish, the ciliates lived well after a day and multiplied. In the host, the parasites bring about what Cépéde termed partial castration. The ciliate absorbs material in the gonad and transforms the contents by so doing and by adding its waste products; and it also brings about mechanically detachment and degeneration of certain sexual cells. Is Orchitophrya an obligate parasite, or is it an acci- dentally invading free-living type, in which Cépéde overlooked the mouth structures? Consideration of instances of accidental parasitism among holotrichs (Glaucoma, Anophrys), as well as of the great in- frequency of the occurrence of Orchitophrya and its ready adaptation to sea water, suggest that the latter may be true. Conidio phrys One of the most complete accounts of the life history and host rela- tionships of an epibiotic ciliate, which is probably a trichostomatous holotrich, is that of Conidiophrys pilisuctor Chatton and Lwoff, 1934 (Fig. 204). In its profound modification in relation to its mode of life, it is approached by no other member of its suborder, and, in fact, by few other ciliates. C. prlisuctor occurs on the secretory hairs, frequently on the thoracic appendages, of a number of freshwater amphipods, espe- cially Corophinm acherusicum, in France. A second species, C. guttipotor Chatton and Lwoff, 1936, is attached to the hairs of Sphaeroma serratum. These ciliates were placed in a new family of Trichostomata, named Pili- suctoridae, by Chatton and Lwoff (1934b), though the International 954 PROTOZOA AND OTHER ANIMALS Rules of Zodlogical Nomenclature demand Conidiophryidae. A complete account of Conidiophrys was given by these authors in their second article (1936a). In a manner suggesting the case of Sacculina, the determination of the systematic position of Conidiophrys is possible only through study of its early development. The form attached to the hairs (Fig. 204A) is immobile, nonciliated (though an infraciliature is present), and is enclosed in a shell-like pellicle which has no opening and beyond the body proper closely en- cases the hair (Fig. 204B). The cucurbitoid trophont undergoes several transverse divisions within the capsule, toward its distal end, producing normally two or three (Fig. 204C), or sometimes as many as six tomites. One specimen was observed with eleven tomites and a twelfth forming, but the distal seven were degenerate (Fig. 204D). When completely formed, the tomite, the longitudinal axis of which is transverse to the longitudinal axis of the trophont, is provided with cilia, with a cytostome Opening on the ventral surface, and with a relatively long, incurved, ciliated cytopharynx (Fig. 204F). Tomites are liberated periodically and have a very short period of free-swimming existence. When the cytostome comes in contact with the end of a secretory hair, this is drawn in and the tomite becomes impaled obliquely on it (Fig. 204F). The form rapidly changes to that of a tear drop and the cilia are lost (Fig. 204G). Growth to the typical trophont proceeds. Chatton and Lwoff maintained that Conidiophrys is not nourished by diffusion from the surrounding water, but depends on the fluid secretion that enters it through the pores at the end of the secretory hairs. Dependence of the trophic form (trophont) upon the host is thus absolute. In discussing the multiplicative polarity of Conidiophrys, fission being localized at the distal pole, Chatton and Lwoff (1936a) speculated con- cerning a possible trophic or humoral influence emanating from the host. Instances of inhibition of division, complete or partial, under the influence of parasitic nutrition are given among parasitic dinoflagellates, apostomatous ciliates, and other Protozoa. (The authors did not com- ment, however, on the absence of any indication of such inhibition in a great number of endozoic forms, a fact which is an impediment to the acceptance of their theoretical explanation.) In Conidiophrys inhibition is exhibited in the removal of the zone of multiplication to a distance eo 2° LEY Peay. *” econ LLG ES Sate os a a ee 4 ee YS Figure 204. Conidiophrys pilisuctor on Corophium acherusicum. A, trophonts on appendage of the amphipod host, attached to hairs; B, trophont at beginning of re- productive period; C, trophont that has formed two tomites, and third forming; D, large trophont with eleven tomites, and a twelfth forming, the distal seven degenerated; E, unattached tomite; F, tomite impaled on a hair by its cytostome; G, young trophont, cilia lost. (After Chatton and Lwoff, 1936a.) 956 PROTOZOA AND OTHER ANIMALS from the pole of communication with the host. When this influence is reduced on the commencement of molting, supernumerary tomites may be produced. There are, Chatton and Lwoff stated, many examples of parasites certain phases of the development of which are conditioned by the molt or sexual maturity of their host. The influence may be chemi- cal, absorbed substances preventing a denaturation of proteins, which may be the essence of cell division. The existence, in trophoepibiotic ciliates, of a trophohumoral gradient of inhibition, susceptible to analysis and analogous to other types of biological gradients, is suggested. APOSTOMEA Though certain ciliates that are now included in the suborder Apostomea have been known for a long time, it is only recently that the group has become well known. Chatton and Lwoff (1935) published an outstanding memoir on the Apostomea, which, they stated, is only the first of three parts. This first part is a monographic study of the genera and species. In the suborder, according to this account, there are two families, by far the more important of which is the Foettingeriidae, with thirteen genera and twenty-six species. In the Opalinopsidae there are only two genera. Chatton and Lwoff expressed doubt that one of these, Opalinopsis, really is an apostome; and the other genus, Chrom- dina, was included by Cheissin (1930) in the Astomata. Kudo (1939) listed the Opalinopsidae in the Astomata. The active, growing, vegetative phase of a foettingeriid ciliate 1s the trophont. The ciliature is in dextral spirals. In the process of growth the basal granules are spaced without multiplying. At the end of the period of growth the organism may encyst, the cilia are lost, and the infracilia- ture undergoes detorsion, the lines becoming meridional. This phase 1s called the protomont. It passes into the multiplicative phase, or tomont, which produces by transverse fission a variable number of tomites. The tomite is a small free-swimming ciliate. The ciliary rows are more or less meridional, with a tendency to turn in a spiral. There is a thigmo- tactic ciliary field, consisting of the parabuccal ciliature. Chatton and Lwoff maintained that the tomite represents the free-living, ancestral type. In twenty-two of the twenty-six species, and possibly in the others also, the tomite becomes fixed to the body surface of a crustacean, and transforms into an encysted phase, the phoront. In the phoront there PROTOZOA AND OTHER ANIMALS oy, is renewed multiplication of the basal granules and torsion of the ciliary lines, leading to the characteristics of the trophont. In the active phases of most species there is a more or less rudimentary, ventrally placed mouth, which is surrounded by a characteristic rosette; sometimes the mouth and rosette are lacking. Almost all the apostomes occur on or in marine animals. Chatton and Lwoff (1935) assigned to the genus Gymnodinioides three species from fresh-water Crustacea, two of which were described by Penard (1922) as Larvulina, commensals on Gammarus, the third by Miyashita (1933) as Hyalospira, from Japanese shrimps. Among the apostomes are the only ciliates with heteroxenous cycles, cycles that alternate as regularly as those in many Sporozoa, though there is no obligatory sexuality. In one group of apostomes, the phoront occurs on copepods, fixed to the integument; and excystation with subsequent development occurs, normally when the host is wounded or is ingested by a predator. The ciliates, however, do not remain long enough in the predator for it to be regarded as a second host. The predators involved are mostly co- elenterates. The hydroid Cladonema radiatum appears to be a very special site for the trophont of Spirophrya sub parasitica, the phoront of which is fixed to the integument of the benthonic copepod Idya furcata. When the copepod is ingested, Spzrophrya excysts and grows rapidly in its remains, accumulating fluid or tissue material in a central vacuole. The trophont does not encyst within the predator, but is expelled with the residues of digestion. Encystation takes place on the carcass of the cope- pod, in the environment, or on the stalk of Cladonema, producing a tomont. This divides into a number of tomites, which may live free for a few days, and eventually degenerate or become fixed to Idya. The phoronts of apostomes of this group will excyst when the copepod mols, but subsequent development is not normal (see Kudo, 1939, Fig. 257). In a second group of apostomes there are encysted phoronts on Crus- tacea, excystation occurs at the molt, and the trophonts develop in the exuvial fluid. Species are associated with a great variety of Crustacea, including Entomostraca, balanids, copepods, and many Malacostraca. The widely distributed genus Gymnodinioides belongs in this group. Polyspira is another genus. P. delagei is phoretic on the gill leaves of pagurids (Eupagurus bernhardus). Excystation occurs at the molt, and Phoront : ae. Em ply pho retic_ cyst \ Youn sanguicolous : trophont or Bee hypertrophont for hypertorndnt) n Tomont T > A intomie =. eee petpal Grown exuvicolous oy PL AVS akg) Rents HOP nor ee. ee ae le sont ee ee Figure 205. Synophrya hypertrophica. A, diagram of cycle of development as a para- site of Portunus or Carcinus; B-D, parasitized branchial lamellae of Portunus holsatus, showing different types of reactional cysts. (After Chatton and Lwoff, 1935.) PROTOZOA AND OTHER ANIMALS 59 the young trophonts grow in the fluid contained in the discarded exo- skeleton. The proteins accumulate in a violet trophic mass, giving the color (which in other genera may be orange, red, and so forth) so characteristic of ciliates of the family Foettingeriidae. Unlike many: apostomes, the trophont does not become encysted. Linear palintomy occurs in the motile stage, producing from eight to sixty-four daughter tomonts, which metamorphose into tomites. In addition to the natural host, the tomites will become fixed on the gills of Portunus holsatus, on which development proceeds normally. In a third group of apostomes, in which there 1s only the genus Synophrya, the trophont is at one stage parasitic in the tissues of the crustacean to which the phoront is attached. Synophrya hypertrophica (Fig. 205) is phoretic on Portunus depuratus, and also on other species of Portunus and Carcinus maenas. The sanguicolous trophonts are in- ternal parasites in the branchial sinus of Portunus or the subcutaneous sinus of Carcinus. They are large, mouthless, immobile, irregularly lobed masses under the integument, enclosed in a double envelope. The re- sulting lesions of Carcinus appears as brown or black spots 1-4 mm. in diameter, found chiefly on the dorsal surface of the carapace. They oc- cur in a high percentage of crabs less than two centimeters in diameter, but not on large crabs. At ecdysis, tomites are produced which disperse in the molted exoskeleton and develop into exuvicolous trophonts. When growth is completed, these encyst as tomonts, each of which produces a number of tomites. The tomites fix themselves and become phoretic cysts on the integument of the gills or branchial cavity of the crab. The parasites then migrate from the cyst into the underlying tissue. A fourth type of cycle is that of Foettingeria actiniarum, which is heteroxenous. It was first known as an inquiline in the gastrovascular cavity of sea anemones, some species of which are almost always in- fected. It has been found in various sea anemones on the coast of France, but it was not found in three species at Woods Hole. Chatton and Lwoff (1935, p. 313) listed ten host species. The ciliates are chymo- trophic. They enter the digestive mass when the coelenterate feeds and there find their sustenance. The ciliates eventually leave the host and encyst, the tomont undergoes palintomy, and the tomites become fixed to a crustacean. The host-specificity of the phoronts is almost nil; the 960 PROTOZOA AND OTHER ANIMALS list of hosts given by Chatton and Lwoff (1935, p. 371) includes cope- pods, ostracods, amphipods, caprellids, the isopod Sphaeroma, and the decapod Carcinus. When the crustacean is ingested by a sea anemone, the phoronts excyst and become young trophonts. Apostomes of the genus Phtorophrya are hyperparasites on other apostomes. The phoront is fixed on the phoront of the host species, and the parasite introduces itself into the body of the other ciliate. It grows rapidly and soon comes to occupy a cyst otherwise empty. Tomites are eventually produced; these leave the empty cyst of the host and swim actively in search of another host phoront. Rose (1933, 1934) reported two unnamed ciliates, considered by him to be Foettingeriidae, parasitic in the oil drop in the oleocyst of the siphonophore Galeolaria quadrivalvis. He thought it probable that the cysts are attached to pelagic copepods. Apostomatous ciliates have been found in the digestive cavity of certain ophiurans and the ctenophore Cestws veneris. Pericaryon cesticola is unusual among Foettingeriidae in adhering firmly to the walls of the gastrovascular cavity of its host. It has an apical stylet, which seems to be an organelle of fixation. Sexual processes have been described in a number of Foettingeriidae. Conjugation is contingent, as in other ciliates, and is of a common type throughout the family. The trophonts conjugate and remain associated during the formation of tomites. At the end of the series of fissions, meiosis Occurs, pronuclei migrate, and the tomites separate. While phoresy on Crustacea is known or presumed to occur in all the Foettingertidae, except in Phtorophrya, the host phoront of which occurs on Crustacea, it is unknown in the Opalinopsidae. The vermiform, elongated (up to 1,200 1), vegetative forms of Chromidina elgans are fixed to the renal cells of cephalopods by an apparently retractile apical papilla. There is no mouth. Multiplication is by simultaneous or succes- sive fissions, producing chains of daughter individuals. The tomite has a buccal ciliature and a buccal orifice, but no rosette. It is believed that a crab may be involved in the cycle. O palinopsis occurs in the liver and intestine of cephalopods, and one species has been found in the liver of the pelagic gasteropod Carinaria mediterranea. PROTOZOA AND OTHER ANIMALS 961 PHYSIOLOGICAL. Host RELATIONSHIPS ILLUSTRATIVE OF MUTUALISM AND COMMENSALISM FLAGELLATES OF TERMITES AND Cryptocercus Before discussing the relationship between the xylophagous flagel- lates and their wood-eating termite or roach hosts, it is desirable to give consideration to the problem of nutrition in some other invertebrates that ingest material consisting largely of cellulose. The most abundant single constituent of wood ts cellulose, which aver- ages in general between about 54 and 64 percent (Pringsheim, 1932 after Schorger). Among other important carbohydrates are hemicellu- loses, which Pringsheim stated is a poorly defined collective name for polysaccharides. A small amount of starch may be present in wood, about 3 to 4 percent, or less; and a certain amount of sugar (Schorger). Lignin is a noncarbohydrate incrustation substance in wood and makes up from about 23 to 28 percent of its bulk (Pringsheim). There are also in wood ash, less than one percent; proteins, a little under one percent, according to Pringsheim, in fir, pine, oak, and beech; fats; waxes; resins; and other substances. Straw and hay have about 30 to 35 percent cellu- lose, about 20 to 30 percent lignin, 3 to 10 percent protein, and 20 to 30 percent starch. The animals that ingest these materials may use one or more of the constituents, and that is not necessarily cellulose. The larva of the goat moth Cossus cossus, though ingesting wood, does not affect the cellu- lose (Ripper, 1930). It has no cellulase and contains no symbiotic micro- Organisms. Ripper found that the carbohydrate used is supplied at least in part by soluble sugars, perhaps also by hemicelluloses. Mansour and Mansour-Bek (1934a) concluded that larvae of the cerambycid Xystro- cerca globosa, with no cellulase and no microdrganisms, derive their sustenance from the relatively high content of sugars and starch in the wood attacked (10.4 percent). Data bearing on the fact that some wood- eating insects seem to make no use of cellulose, but depend on the starch and sugars in the wood, being limited therefore to certain kinds of wood rich in these substances, were discussed by Mansour and Mansour-Bek (1934b). Ullmann (1932) stated that the carbohydrate requirements of invertebrates are met chiefly by sugars and hemicellu- loses. 962 PROTOZOA AND OTHER ANIMALS On the other hand, it has been found that certain termites can sur- vive indefinitely on cotton cellulose or a cellulose-lignin complex (Cleve- land, 1925b); and larvae of the rose beetle Potosia cuprea lived for more than six months on filter paper (Werner, 1926). According to Dore and Miller (1923), the wood that is ingested by Teredo navalis loses in the alimentary tract 80 percent of its cellulose, as well as from 15 to 56 percent of the hemicellulose, but the amount of lignin is not reduced. Digestion of cellulose undoubtedly occurs in the alimentary tract of many beetles, as, for example, the anobiid Xestobium rufovil- losum (Campbell, 1929; Ripper, 1930) and the cerambycids Hylo- trupes bajulus (Falck, 1930), Stromacium fulvum, and Macrotoma pal- mata (Mansour and Mansour-Bek, 1933, 1934a). The wood eaten by this last species was found to have very little soluble sugar and starch (0.47 percent). There are many other instances of cellulose digestion among vertebrates and invertebrates. Yonge (1925) published a review of cellulose digestion in invertebrates, but his statement that no cellu- lase has been found in Insecta is not true today. Xystrocerca globosa is reported to have a strong amylase, as well as maltase and saccharase, enabling it to make use of the starches and sugars in wood (Mansour and Mansour-Bek, 1934a). Tissue-produced cellulase has been demonstrated in a number of gasteropods and insects. Among xylophagous insects, cellulase appears to be produced by the digestive epithelium of certain cerambycid and anobiid larvae. Most cellulose decomposition in nature is brought about by bacteria, filamentous fungi, and certain Protozoa. In many animals that make ultt- mate use of cellulose in nutrition, the material is first acted on by micro- organisms living in the alimentary tract. This is the only method of cellulose breakdown in vertebrates, and it is true also of the process in many invertebrates. Herbivorous mammals harbor bacteria capable of acting on cellulose. Bacillus cellulosam fermentens was isolated by Werner (1926) from larvae of Potosia cuprea, which feed mainly on spruce and pine needles. Bacteria in the intestine of the lamellicorn beetles Oryctus nasicornis and Osmoderma eremita are able to break down cellulose (Wiedemann, 1930). Cleveland et al. (1934) found evidence that symbiotic bacteria are the agents of cellulose decomposi- tion in the xylophagous roach Panesthia javanica. In the above-mentioned animals, bacteria dwell in the lumen of the PROTOZOA AND OTHER ANIMALS 963 gut. Very widespread in wood-eating insects, but by no means restricted to hosts of that group, are the intracellular symbionts studied intensively by Buchner and his associates, as well as by many others (see Buchner, 1930). These bacteria or yeast-like fungi live with their hosts in “cyclic endosymbiosis,” being regularly transmitted to the next generation. Cyclic endosymbionts exist in the termite Mastotermes (Jucci, 1932; Koch, 1938a, 1938b). Buchner believed that these symbionts might play a rdle in the digestive processes of the host, but this opinion, lacking experimental proof, has not been generally adopted (Mansour and Mansour-Bek, 1934b; Schwartz, 1935). Protozoa are present in many of these insects, and sometimes them- selves derive nutriment from cellulose-rich materials. Beetle larvae fre- quently harbor a moderate number of small flagellates (Polymastix, Monocercomonoides) which feed on bacteria (Wiedemann, 1930). As stated above (p. 916), a limited number of small, non-xylophagous flagellates (mainly Trichomonas) and occasionally Nyctotherus are pres- ent in many termites of the family Termitidae (Kirby, 1932b, 1937). Some of these higher termites, especially species of Mérotermes and Cubitermes, harbor large amoebae which ingest wood or other cellulose- rich material on which the termite feeds (Kirby, 1927; Henderson, MS). The wood-feeding roach Panesthia javanica contains two small flagellates, Monocercomonoides and Hexamita; large xylophagous amoebae; smaller amoebae; and a number of ciliates (Kidder, 1937). Mutualistic symbiosis, however, finds its best illustration, so far as Protozoa are concerned, in the abundant and diverse xylophagous flagel- lates of termites other than Termitidae and of Cryptocercus punctulatus. According to some investigators, Protozoa and other organisms of the gut may serve the host as a supplementary food source. Wiedemann’s observation of cellulose-decomposing bacteria in lamellicorn larvae was mentioned above. He believed that the breakdown products are en- tirely used in the metabolism of other bacteria. The bacteria multiply rapidly in the large intestine, where they live in association with the small flagellates. The mid-gut, he found, secretes protease which is in- active in the alkaline medium there, but in the hind-gut, where bac- terial acids accumulate, it digests the bacteria and flagellates. Mansour and Mansour-Bek (1934a, 1934b) and Mansour (1936) have dis- cussed the possibility that the flagellates in termites do not benefit the 964 PROTOZOA AND OTHER ANIMALS hosts in nutritive processes except that, multiplying and being digested continually, they are a direct and supplementary food source for the insects. This seems unlikely, however. The flagellates in termites multiply rapidly for a few days after a molt following which there has been a new infection; then there is little division, and they are destroyed, usually, on the approach of the next molt. What use the host might make of the disintegration products at that time is entirely unknown, but certainly there is no evidence that the Protozoa could be available at any other period as a supplementary food source accounting for gen- eral nutrition (see p. 968). Cyclic endosymbionts seem to be necessary for normal development of the host in some instances. Aschner and Ries (1933) and Aschner (1932, 1934) succeeded in freeing Pediculus of the symbionts that normally inhabit the mycetome, and found that without them larvae died sooner or later. The harmful effects of the absence of symbionts in Pediculus were reduced by rectal injection of yeast extract. Koch (1933a, 1933b) obtained symbiont-free larvae of the anobiid S/todrepa panicea and found that they would not develop normally unless yeast was added to the food. (Koch, however, also reported freeing the saw-tooth grain beetle Orzyaephilus surinamensis of symbionts in the mycetomes by in- cubation [1933b, 1936]; and absence of the microérganisms seemed to be without detrimental effect.) It has been suggested that the symbionts are sources of vitamins or growth factors. It is possible, in the light of these facts, that certain symbiotic Protozoa may be necessary to the life of the host, without participating in the digestive processes or serving as a food source important in bulk. We now come to a consideration of the demonstration—one of the outstanding advances in modern protozodlogy, though not yet complete —that wood-eating flagellates in termites and Cryptocercus are neces- sary for the survival of their hosts in making the products of decomposi- tion of cellulose available for the nutrition of the insects. This has justly received very wide attention, so it is unnecessary to recount all details of the demonstration here (see Cleveland 1924, 1926, 1928a, 1934). Termites feed primarily upon wood. This is especially true of the members of the families Mastotermitidae, Kalotermitidae, and Rhino- termitidae. Many Termitidae attack wood also; others bring into their nests dried grass, ingest soil and extract from it the nutrient materials PROTOZOA AND OTHER ANIMALS 965 it contains, or devour leaves; in fact, almost all types of vegetable mat- ter are utilized by certain members of the group. Hodotermitidae forage for grass and herbs, even eat straw from unbaked bricks; some on the Karroo collect twigs. Kalotermitidae and Rhinotermitidae can live on paper; even, as stated above, cotton cellulose and a lignin-cellulose com- plex (see Cleveland, 1924, 1925b). The wood-boring roach Cry ptocercus punctulatus eats the wood of fallen timber, well-decayed or sound. Cleveland (1923) pointed out that, in the groups of termites that use a uniform diet of wood, all species examined had rich faunas of Protozoa; and this has been confirmed by studies by the writer of more than a hundred additional species. In Termitidae, with varied food habits, such faunas are absent, though there are some Protozoa in many species (Kirby, 1937). Cleveland ef al. (1934) remarked that the correlation of wood feeding and intestinal flagellates is not so close as he at first supposed, since there are some Termitidae that eat wood and have no (xylophagous) flagellates. We know very little of the actual nutrient substances among the varied materials taken in by termites as a group. Matter that has passed through the digestive tract is used extensively by higher termites in building mounds, fungus gardens, and carton nests. Cohen (1933) and Holdaway (1933) analyzed mound material of Ewtermes exitiosus, which contains no Protozoa and feeds on wood. They found cellulose to be much reduced, though some passes out undigested, whereas lig- nin is unaffected. These results agree with those reported by Oshima (1919) after analyses of wood and nest material of Coptotermes formo- sanus, which does contain xylophagous flagellates. Oshima concluded that the principal food of that termite is cellulose and that there is no decrease of lignin. In termites of still another group, Zootermo psis, Hungate (1936, 1938) found essentially the same thing by analyses of uneaten wood and pellets. Tissue-produced cellulase is absent from Cryptocercus punctulatus (Trager, 1932), Kalotermes flavicollis (Montalenti, 1932), and Zoo- termopsis angusticollis (Hungate, 1938). Probably none is present in any Kalotermitidae or Rhinotermitidae, though, in the light of the situation with wood-boring beetles, one should not generalize from limited data. Termitidae have not been investigated for cellulase. Man- sour and Mansour-Bek (1934a) suggested that some termites may be 966 PROTOZOA AND OTHER ANIMALS found to have cellulase, and it is in that higher group that one might be most likely to find the enzyme. The literature on this subject, so far as the writer has determined, contains no discussion of the hemicellulase lichenase, which Ullmann (1932) reported to occur in all the insects, including roaches, and the snails that he tested; and which Oppenheimer (1925) stated is wide- spread in invertebrates. Montalenti (1932) wrote that in the fore-gut of K. flavicollis he found a trace of amylase, which was probably pres- ent in the salivary secretion; in the mid-gut, amylase and invertase, as well as a protease that acted only in acid, though the mid-gut is basic; and in the hind-gut, amylase and invertase probably derived from the mid-gut. Hungate (1938) found amylase in an extract of the fore-gut, and protease in the mid-gut of Zootermopsis angusticollis. On the basis of these findings, it should be possible for the termites to hydrolyze starch; to invert sucrose; to digest the small amount of protein in wood and possibly also some of their own microdrganisms, when the resistance of the latter to the enzyme has been overcome. As remarked above, bacteria in many cellulose-utilizing animals are necessary for the preliminary breakdown of cellulose. Cleveland et al. (1934) suggested the possibility that some Termitidae may profit from the presence of bacteria in the same way. But in those termites that have been examined for cellulose-decomposing bacteria, it appears that the latter cannot account for cellulose digestion. A few positive results have been obtained. Dickman (1931) found them in one of six nitrate- cellulose tubes inoculated with gut contents of Reticulitermes flavipes, and Tetrault and Weis (1937) obtained some from the same termite; but Cleveland (1924) failed in many and varied attempts to isolate cellulose-decomposing bacteria or other fungi from R. flavipes. Beck- with and Rose (1929), using termites of six genera, including one of the Termitidae, obtained cellulose-digesting bacteria in some instances, but not at all in two species. Their results, however, are subject to criticism (Dickman, 1931; Hungate, 1936). Hungate (1936) was un- successful in efforts to show cellulose decomposition by bacteria from the gut of Z. nevadensis, and concluded that bacteria in the alimentary tract are of no importance in the digestion of cellulose. A possible ex- planation of the occasional positive tests is found in Cleveland’s dis- covery that in Cryptocercus punctulatus, feeding on its normal diet of PROTOZOA AND OTHER ANIMALS 967 wood, it is usually possible to obtain cellulose-digesting bacteria in cul- ture from all regions of the alimentary canal, especially the fore-gut. These disappear in time when roaches are fed on paper, and he believed that they are forms living in the wood and accidentally ingested by the insects. Numerous fungi were isolated by Hendee (1933) from wood in- habited by Zootermopsis angusticollis, Reticulitermes hesperus, and Kalotermes minor. Dickman (1931) obtained cellulose-digesting or- ganisms, both bacteria and molds, from material attacked by termites, probably R. flavipes and Zootermopsis sp. Cellulose-decomposing molds were found by Hungate (1936) in burrows and pellets of Zootermop- sis. He concluded, after analyses of sawdust acted on by external or- ganisms and material that had passed through the termites (possibly several times), that cellulose decomposition by bacteria and molds in the wood of the colony is negligible in comparison with that digested in the termites. That fungous action can render cellulose usable by termites is shown, however, by an observation of Cleveland’s (1924). Termites deprived of Protozoa died soon on a cellulose diet, but lived indefinitely when a cellulose-decomposing fungus accidentally developed in certain vials. The flora of spirochetes and other bacteria in the gut of termites, and this applies also to Termitidae, is considerable. They live free in the lumen, attached to certain Protozoa, or attached to the lining of the walls. Spirochetes do not grow on the usual laboratory media (Dick- man, 1931). The possibility that they may participate in digestion of cellulose and hemicellulose in termites was admitted by Cleveland (1928a). In Cryptocercus, however, Cleveland killed the Protozoa, leaving the spirochetes, by contrifuging; and cellulase disappeared in twenty-four hours. The enzyme was not found after defaunated roaches were reinfected with bacteria and spirochetes. Excepting certain castes and brief phases of development, all termites except Termitidae have great numbers of flagellates in the hind-gut. The vestibule, large intestine, and caecum become voluminous organs to accommodate these symbionts. Hungate (1939) estimated that the gut contents containing the Protozoa amount to from a seventh to a fourth of the total weight of Zootermopsis angusticollis. Katzin and Kirby (1939) found the gut contents to be about a third of the weight of 968 PROTOZOA AND OTHER ANIMALS nymphs of Z. angusticollis and Z. nevadensis, and about a sixth of the weight of soldiers. In this fluid gut contents the Protozoa are about as thick as they could possibly be. Hungate, by centrifuging, showed that about half consists of fluid, half of organisms. The organisms are in mass mostly Protozoa, but there are also a great many bacteria and spirochetes. Lund (1930) estimated the number of Trichonympha, Streblomastix, and Trichomonas in Zootermopsis as 54,000; but obvi- ously this would vary greatly with the size of the termite. In Cryptocercus punctulates the colon is enlarged to a relatively greater degree than in termites, becoming ‘‘an immense thin-walled bag completely filled with Protozoa” (Cleveland ef al., 1934). There are probably millions of flagellates in a single full-grown roach. Most species of these flagellates ingest particles of wood. None of them possesses cytostomes. Ingestion is through the surface of the body. In Trichonympha wood ingestion has been described by Swezy (1923), Cleveland (1925a), and Emik (MS). Ordinarily most of the wood in the faunated portions of the hind-gut is enclosed in the cyto- plasm of the flagellates. Cleveland (1924) stated that in Reticulitermes flavipes nearly all the particles of wood are taken into the Protozoa, whereas in Zootermopsis he found many particles free in the lumen of the gut. Bacteria and other flagellates are sometimes ingested by Tricho- nympha collaris (Kirby, 1932b) and other flagellates of termites. This predatory habit is more frequent in some species than in others; and ingestion of other organisms occurs more frequently under the con- ditions of filter-paper feeding. Yamasaki (1937b) observed Dine- nympha in many T. agilis after oxygenation. Wood is, however, the chief and usually the only material taken into holozoic forms. Lund (1930) noted that when Zoofermopsis was fed on cornstarch, many Trichonympha and Trichomonas ingested starch grains. Trichomonas and Hexamastix in Zootermopsis ate, according to Cleveland, able to use starch. Grains of rice starch were taken in by three of the hyper- mastigotes in Cryptocercus, and had some food value for them; and Monocercomonoides in the roach could make full use of starch (Cleve- land et al. 1934). Some flagellates in termites are saprozoic and do not take in solid particles. That is true of Streblomastix in Zootermopsis, of Hoplo- PROTOZOA AND OTHER ANIMALS 969 nym pha in Kalotermes hubbardi, and probably of some forms of Dzne- nympha in Reticulitermes. It is also true of certain very small flagellates. That the flagellates possess an enzyme capable of acting on the cellu- lose of the ingested wood has been clearly demonstrated by a number of investigators. Trager (1932, 1934) proved that Trchomonas termop- sidis produces cellulase. He maintained the flagellate in culture for sev- eral years in the presence of only one species of bacteria, which was not capable of fermenting cellulose or cellobiose. The addition of finely di- vided cellulose to the medium was necessary, and Trichomonas did not live when that was replaced by other polysaccharides. An extract of the ground bodies of the flagellates, concentrated from cultures, acted on cellulose. Emik (MS) obtained fairly pure concentrations of Trichonym pha from Zootermopsis by gravity filtration. Extracts of these concentrates were able to digest certain preparations of cellulose as shown by osazone tests, demonstrating crystals of glucosazone and cellobiosazone. Emik concluded that two enzymes were present, derived from Trichonympha: cellulase, hydrolyzing cellulose to cellobiose; and cellobiase, hydrolyzing cellobiose to glucose. It is not difficult to show the action of cellulase in the contents of the hind-gut, and, in view of the absence of tissue-produced cellulase and the virtual absence of cellulose-digesting bacteria or fungi, the Protozoa must be its source. Both cellulase and cellobiase were found there by Trager (1932). Cleveland et al. (1934) and Hungate (1938) so identified cellulase in flagellates of Cryptocercus punctulatus and Zoo- termo psis angusticollis. Substance stained brown or reddish brown by iodine dissolved in potassium iodide, and assumed, as is customary, to be glycogen, has been found in many of these xylophagous flagellates. The earliest demon- stration, which was discussed critically by Cleveland (1924), was made in Trichonympha agilis by Buscalioni and Comes (1910). Kirby (1932b) mentioned iodine-staining granules in T. campanula. Yama- saki (1937a, 1937b) described abundant glycogen deposits in the species of Trichonym pha, Teratonym pha, Holomastigotes, Pyrsonym pha, Dinenympha, Pseudotrichonympha, Holomastigotoides, and Spiro- trichonym pha in Japanese termites, preparing the material by staining in Ehrlich’s hematoxylin and Best’s carmine after fixation in 90-percent alcohol. Diminution of the glycogen in T. agi/is under conditions of 970 PROTOZOA AND OTHER ANIMALS incubation, starvation, and oxygenation was studied by Yamasaki (1937b). Kirby (1931) stained the axostyle of Trichomonas termop- sidis brown in Lugol’s solution; and stated that this may be taken, as Alexeieff pointed out in the case of Tritrichomonas augusta, to be indic- ative of the possible presence of glycogen in the axostyle. In the light of these results, it seems likely that carbohydrate is stored as glycogen in these Protozoa. (Cleveland ef al. [1934], however, considered it possible that the substance colored by iodine may not be glycogen, but a breakdown product of cellulose which gives the same reaction as glyco- gen. See also page 981.) Since only in the bodies of the flagellates can cellulose be digested, and termites live and develop normally when only cellulose is eaten, the rdle of the symbionts is evident. According to Hungate (1938), about one-third of the total material removed from wood, adding that acted on in the fore- and mid-gut to the soluble materials present, can be obtained without the aid of the Protozoa. It is possible that materials adequate for nutrition of the insect may be obtained in the diet with- out the Protozoa, as Cleveland (1924) found by feeding humus and fungus-decomposed cellulose. Presumably sufficiently rotted wood would also be adequate; Cleveland (1930) stated that defaunated Cryptocer- cus, which dies in two or three weeks on partially decayed wood or cellulose, will live two or three months on completely decayed wood. Cleveland has conclusively demonstrated that continued survival of de- faunated termites and Cryptocercus is impossible on a natural diet of wood. Hungate’s third, therefore, could not provide all necessary sub- stances, it appears; as, if it did, the amount could be multiplied merely by the ingestion of more wood, or further use of that which ordinarily passes to the hind-gut for use of the Protozoa in faunated individuals. Experiments in feeding various cellulose-free carbohydrates to termites have been made by Montalenti (1927) and Lund (1930); and to Cryptocercus by Cleveland (1930, 1934). Montalenti kept Kalo- termes flavicollis alive for several months on soluble starch, alone or mixed with glucose, though the hypermastigotes soon disappeared and the polymastigotes greatly diminished in number. He concluded that the termite could live a long time, if not indefinitely, on soluble carbo- hydrates without Protozoa, but no other worker has confirmed this. Lund’s studies were made to determine the effect of various diets on the PROTOZOA AND OTHER ANIMALS 971 Protozoa of Zootermopsis, not the maximum period of survival of the termites. Cornstarch caused death of the Protozoa and the termites after twenty-three days; the starch was apparently in granular form. (In com- parison with Montalenti’s results, light may be thrown on the discrepancy by the statement of Ullmann (1932) that invertebrates are unable to use the starch of the plant food, but that soluble cooked starch is very well digested by all animals.) Lund used a variety of carbohydrates, on most of which the maximum survival of both Protozoa and termites did not greatly exceed the effects of starvation; and on some they died more quickly. On inulin, dextrin, and lactose Trichomonas and Streblomastix were living in the last termites reported at forty-eight, forty-four, and sixty-five days. Cleveland (1925c) found that Trichomonas (accom- panied by Streblomastix) can keep the host alive from forty to fifty days longer than when no Protozoa are present, but “very few if any [termites ] were able to live indefinitely.’” The hypermastigotes are most important in the mutualistic symbiosis in Zootermopsis. Cleveland et al. (1934) studied the effects of various diets on Cry pio- cercus punctulatus and its Protozoa, using various cellulose-free carbo- hydrates, peptone, gelatin, and glycogen. On no substance did any ex- cept the smaller polymastigotes survive very long; nor did the roaches live more than a few days longer than when water alone was given. These authors found that dextrose is of more food value than the other substances, and considered it likely that a diet including dextrose might be found upon which the insects could live for a long time, if not in- definitely, without Protozoa. Dextrose prolonged the survival period also of defaunated Reticw- litermes flavipes (Cleveland, 1924). Trager (1932) demonstrated dex- trose in the presence of extract from the hind-gut contents of Cry pto- cercus. Cleveland et al. (1934) suggested that dextrose, produced from cellulose by the action of cellulase and cellobiase in the cytoplasm of the flagellates, insofar as it is not used in their metabolism or stored as glycogen, diffuses from their bodies. Hungate (1939), however, identi- fied acetic acid, carbon dioxide, and hydrogen as metabolic products of the Protozoa; and thought it likely that most of the sugar resulting from their digestive processes undergoes anaérobic dissimilation by the Proto- zoa. According to this view, the termites would make use of the acetic acid. OFZ PROTOZOA AND OTHER ANIMALS A further problem arises in the absorption of the substances released from the flagellates by the termite or roach tissues. Either absorption must take place through the chitinous layer of the hind-gut, or fluid must be passed forward into the mid-gut. The problem has been dis- cussed by Buchner (1930) and Cleveland ef a/. (1934). There are dif- ferences of opinion as to whether absorption in the hind-gut is possible, and some authors are inclined to the view that it is. Buchner is one of those. Abbott (1926) found that the hind-gut of Periplaneta aus- tralasiae is permeable to dextrose. Cleveland ef a/. took the opposite view, as a result of osmotic experiments on the colon of Cryptocercus, which showed it to be impermeable to dextrose and water. The peri- trophic membrane also seemed to be largely impermeable to dextrose. The iliac valve controls the passage of materials between the mid-gut and the hind-gut, and when the mid-gut is severed it permits no ma- terial to flow out from the hind-gut. Cleveland concluded that fluid con- taining dextrose passes forward at times through the iliac valve into the space between the peritrophic membrane and the wall of the mid-gut. A problem in the metabolism of xylophagous animals is the source of nitrogen. The small amount of protein present in wood, and the larger amount in straw and hay (important in the case of Hodotermes and some higher termites), may account for the nitrogen metabolism of the Protozoa; but in the absence of action on cellulose outside of the Protozoa it might not be directly available to the termite. Bacteria and molds ingested with the wood might account for some, but that would probably be very little. Pierantoni (1937) hypothesized a fixation of nitrogen by bacteria in the gut, and Green and Breazeale (1937) re- ported the isolation of nitrogen-fixing bacteria from an unidentified species of Kalotermes. Wiedemann (1930) stated that bacteria in certain lamellicorn larvae can use inorganic nitrogen, and the host satis- fies its nitrogen need by digesting these microérganisms. Use by termites and Cryptocercus of some dead Protozoa or pieces of cytoplasm from their bodies (Cleveland ef a/., 1934), while it could not account for any important part of general nutrition (p. 961), might be significant in providing nitrogen. We have seen in the foregoing discussion that many animals ingest substances of which cellulose is an important constituent. In the diges- tive tract of some of them cellulose is broken down, whereas certain PROTOZOA AND OTHER ANIMALS Die others make use only of other constituents of the ingested material. These two nutritional variants may be found in members of the same group, as in Cerambycidae. The decomposition of cellulose may, in some invertebrates, be accomplished by means of a tissue-produced cellulase; in other, even related, forms it may require the action of symbiotic bac- teria. It is possible, though the truth of the hypothesis remains to be shown, that endobiotic bacteria and Protozoa may, in some instances, benefit their hosts as a supplementary food source. There 1s a hypo- thesis also that certain symbiotic microérganisms are a source of vitamins or growth factors, or play a rdle in nitrogen economy. In all termites below Termitidae, except in certain functioning repro- ductive castes, and in the roach Cryptocercus punctulatus, xylophagous flagellates are exceedingly abundant in a specially enlarged part of the hind-gut. These flagellates possess cellulase and cellobiase, and reduce cellulose taken into their cytoplasm to dextrose. The insects possess no tissue-produced cellulase, and few if any cellulose-decomposing bac- teria or other fungi are present. These insects cannot live for long on their usual diet or on cellulose without the flagellates, which presumably release part of the dextrose or its dissimilation products for the use of the host. This may be passed forward into the mid-gut to be absorbed, or perhaps may be absorbed in the hind-gut. To what extent the nitrogen needs of termites may be provided for by occasional digestion of Proto- zoa or fragments of the cytoplasm, or by symbiotic nitrogen-fixing bac- teria, remains to be determined. CILIATES OF RUMINANTS Among the most notable endozoic faunules of Protozoa are the ciliates in ruminants and certain other herbivorous mammals. There are some holotrichs, sparsely represented among the species of ciliates in rumi- nants, but constituting an important and diversified part of the faunules of the caecum and colon of the horse (Hsiung, 1930). There are also a few flagellates and amoebae, but the most characteristic forms belong to the Entodiniomorphina. Of the two families of this suborder of highly organized spirotrichs, Ophryoscolecidae occur chiefly in rumi- nants (see Dogiel, 1927; Kofoid and MacLennan, 1930, 1932, 1933; Wertheim, 1935); and Cycloposthiidae are best known in the horse (see Hsiung, 1930). The latter family is represented also in a number 974 PROTOZOA AND OTHER ANIMALS of other mammals, including the tapir, rhinoceros, chimpanzee, gorilla (Reichenow, 1920), and elephant (Kofoid, 1935). Ciliates in ruminants, except for certain species less constant in oc- currence (as Buxtonella sulcata Jameson, in the caecum of cattle) are localized in the rumen and reticulum. Their relative abundance in the rumen and reticulum is approximately equal (Dogiel and Fedorowa, 1929; Wertheim, 1934a); and they are distributed throughout the con- tents. Dogiel and Fedorowa found that the ciliates are somewhat more abundant in the central part than at the periphery, but that the difference is not very great. Distribution is sufficiently uniform so that counts of a small sample from the rumen have been used for an estimate of the total population. Poor nutrition of the ruminant can cause a rapid reduction in the number of ciliates, and this may be responsible for low counts, in slaugh- terhouse animals, of under 100,000 per cc. (Dogiel and Fedorowa, 1929), under 200,000 per cc. (Wertheim, 1934a), and under 400,000 per cc. (Winogradowa-Fedorowa and Winogradoff, 1929). Under conditions of normal nutrition, many counts above 500,000 per cc. have been obtained. Mangold (1929, 1933) stated that in sheep and goats the normal number remains with much constancy at about 1,000,000 per cc.; and Mowry and Becker (1930) agreed with this as regards goats. Under certain conditions, the population may be much denser than this. By experimental feeding Mowry and Becker obtained a count of nearly 7,600,000 of Entodinium and Diplodinium alone. Ferber (1928) esti- mated that at 900,000 per cc. a gram of rumen contents would contain about one-twentieth of a gram of ciliates. The total number of ciliates in an individual ruminant is enormous. Calculating from a volume of material in the rumen and reticulum of goats of from 2.8 to 5.2 liters, and a ciliate count of from 121,000 to 391,000 per cc., Winogradowa-Fedorowa and Winogradoff (1929) estt- mated a population of from 471,000,000 to 1,548,000,000; for the normal condition these figures should probably be multiplied by about three. In an ox with from 56 to 87 liters and from 70,000 to 117,000 ciliates per cc., there would be nearly 10,000,000,000; and probably the population may be at least five times as dense as that. The ciliates are consistently absent from suckling animals, but, as soon as a diet of plant food begins, the faunule of the rumen and reticu- lum develops. PROTOZOA AND OTHER ANIMALS a7 The kind of food taken has a striking influence on the ciliate popula- tion. Green plant material was regarded as of fundamental importance by Trier (1926), with emphasis on the chlorophyll content. Weineck (1934) expressed agreement with this view, but Westphal (1934b) denied that there is proof of chlorophyll need. In experiments by Mowry and Becker (1930), green fodder alone maintained a low popu- lation. Hay and water alone also maintained a low population, which was more than doubled when cornstarch was added. A much greater increase occurred when a grain mixture, consisting of ground corn, ground oats, wheat bran, and linseed oil meal, was given with hay. There is a limit to this increase, however. Although the densest popula- tion of all was developed on grain alone, there was soon a very great decrease in the number of ciliates. As was pointed out by Mangold (1929), some coarse food is essential. Hay with cornstarch and either plant or animal protein, instead of with grain, also maintained a high level of population density. Apparently in the grain both the starch and the protein constituents are stimulating factors, although the Mangold school has maintained that the protein alone is determinative. Other factors influencing the ciliate population of the rumen that have been discussed are density of the contents and pH. Mowry and Becker (1930) could not corroborate the findings of Dogiel and Fedorowa (1929) and Ferber (1929b) that thick rumen contents con- tain relatively more ciliates than thin fluid contents. Mowry and Becker found the average pH in the rumen of goats to be 7.7, with two-thirds from 7.6 to 7.8 and extremes of from 6.7 to 8.2. Within these limits, there seemed to be no notable changes in the ciliate population that could be attributed to the pH itself. Mangold and Usuelli (1930) found the pH of fresh rumen contents of sheep to be from 7.5 to 7.8. When the ciliates pass into the omasum, abomasum, and intestine, they are destroyed. As nutriment passes posteriorly from the reticulum, a vast number of ciliates must go with it; it is difficult to conceive of any mechanism by which they could be kept back. The population could be maintained only by an adequate rate of multiplication, going on con- tinuously. The rapid disappearance of the ciliates on starvation of the host probably could not be explained simply by starvation, followed by death of the ciliates in the rumen and reticulum; it is more likely that the rate of reproduction declines, and the passage of ciliates into the third stomach rapidly reduces the population. 976 PROTOZOA AND OTHER ANIMALS Several attempts have been made, with varying results, to estimate the reproductive rate by counting dividing ciliates. No adequate deter- mination has been made of the rate of reproduction in a day, a calcu- lation which cannot be based only on the amount of fission seen on one occasion. Rate of reproduction in culture, furthermore, at least in the absence of completely satisfactory culture methods, is not necessarily the same as that in the rumen. Mowry and Becker (1930) found in goats usually less than 0.5 percent of dividing forms, and never as many as one percent. Ferber and Winogradowa-Fedorowa (1929) found in a ram on different occasions from 0.9 to 15 percent in division, the aver- age being 7 percent. Examinations were made twice a day, but they failed to note, as Mowry and Becker pointed out, that from observation of 7 percent dividing forms in two samples per day, it does not follow that 7 percent of the ciliates are dividing in a day. The rate of multipli- cation would probably be much higher than that. Westphal (1934a) found that in culture of certain forms each ciliate divided an average of once in fourteen hours, and the population became 3-fold in a day. It reached in more dilute medium a rate of 5.8-fold in twenty-four hours. Dogiel and Winogradowa-Fedorowa (1930) published a report that from 50 to 90 percent of the ciliates were observed in division in goats under normal conditions of nutrition, and from 12 to 50 percent in slaughterhouse oxen. Westphal (1934a) calculated that there must be daily at least a quadrupling of the number of ciliates. Rumen ciliates live in a chemically complex and delicately balanced environment, and 7 vitro culture has been a difficult problem. Becker and Talbott (1927) and earlier workers failed to obtain more than limited survival. Knoth (1928) obtained longer survival in a medium of rumen fluid, with controlled pH, and with partly anaérobic condi- tions provided by a mixture of carbon dioxide and methane, the maxt- mum being the life of Extodinium, with daily change to fresh solution, for five days. Margolin (1930), in media of hay infusion with rice starch and filter paper acted on by cellulose-decomposing bacteria, the pH being kept at 6.8, reported maintenance of cultures for twenty-four days; but others have been unable to use his methods successfully (Becker, 1932; Westphal, 1934a). Westphal (1934a, 1934b) reported real success with a medium of rumen fluid kept under anaérobic con- ditions, with urea and starch added. There was active multiplication in PROTOZOA AND OTHER ANIMALS O77, the cultures, which, with daily renewal of medium, were kept several weeks, and, he stated, might be continued indefinitely. Entodinium lived particularly well. The rumen ciliates, so far as is known, do not form cysts. Tropho- zoites have been found in the mouth fluids (Becker and Hsiung, 1929), and ruminants have been infected by giving this material with the food (Mangold and Radeff, 1930). Natural transmission is by contact, in common feeding, in which there is a certain period when the tropho- zoites ate exposed to the external environment (Becker and Hsiung, 1929; Mangold and Radeff, 1930; Strelkow, Poljansky, and Issakowa- Keo, 1933). Their ability to withstand external conditions is therefore of crucial significance. The holotrichs and Extodimium are more re- sistant than the larger Ophryoscolecidae. Strelkow ef al. reported that after six hours at room temperature all ciliates were still active, and many survived longer. At 0° C. all continued normal activity for an hour. On dilution of the rumen fluid, they survived for various periods of from one to thirty-two hours; and most were alive after six hours in material two-thirds evaporated. They are thus clearly able to live long enough on feed or in water to infect other animals using the same con- tainers. The interesting report of Fantham (1922) that “species of Entodinium and Diplodinium may be found on wet grass and in aque- ous washings of fresh grass and even of dried grass (fodder) from sheep runs and pasturage’”’ leaves the reader desirous of details con- cerning his observations. It has been found to be a simple matter to bring about elimination of the ciliates from the rumen and the reticulum. Modern investigators have used three defaunation treatments. Mangold and his coworkers have found starvation alone to be satisfactory. The ciliates may ap- parently be absent after only three or four days, but Dogiel and Wino- gradowa-Fedorowa (1930) found six to seven days without water necessary for complete defaunation. They found preferable, however, partial feeding with dry food and water and a liter of milk daily. Milk feeding was used by Falaschini (1935). On the basis of studies made on material kept in the thermostat, Mangold and Usuelli (1930) con- cluded that the increased acidity induced in the rumen contents 1s re- sponsible for the incompatibility of milk and ciliates. The best method of defaunation, according to Strelkow, Poljansky, and Issakowa-Keo ss Si a ees ore | a SASS) " x x [es | TERE Ot ee Fes( | ay SS | = Bs SS Figure 206. Ingestion of plant material by Ophryoscolecidae, A, a piece of grass with three fiber bundles, which are beset with Diplodinium gracile: B, long cellulose fiber rolled up in D, gracile; C, large fragment of grass in Elytroplastron bubalidis ; D, a piece of grass with two D. gracile and four Opisthotrichum janus. The former have ingested the ends of fibers, the latter ingests fine detritus from the surface; E, E. bubalidis, which has partly ingested a large piece of grass. (After Dogiel, 1925.) PROTOZOA AND OTHER ANIMALS Se) (1933), who compared it with milk feeding, is the one discovered by Becker (1929). Becker starved his animals for three days, then gave two doses, twenty-four hours apart, of fifty cc. of 2-percent copper sul- phate, passed through a rubber tube into the rumen. Strelkow ef al. shortened the starvation period to one day and gave three doses of copper sulphate, thus shortening the defaunation period to three days. The ciliates take up plant fragments in the rumen, and Trier (1926) stated that they are apparently exclusively plant feeders. Bacteria, flagel- lates, amoebae, and ciliates may also be ingested, however. According to Dogiel (1925), no Ophryoscolecidae are entirely predatory, as plant debris is always to be found in the plasma. Kofoid and MacLennan (1930, 1932, 1933) and Kofoid and Christenson (1934) recorded the food contents of most of the species they studied, and showed clearly that food habits differ. Some appear to feed only on bacteria and other Protozoa, especially small flagellates. Others use various combinations of plant and animal material, some only plant material, and bacteria with plant debris are ingested by a large proportion. Some species are more in- clined than others to be predatory on ciliates. Entodinium vorax, accord- ing to Dogiel (1925), almost always contains the remains of one or more smaller Entodinium. The plant material is often in relatively small particles, but some ophryoscolecids take in large pieces that may distort the body. Dogiel (1925) described how Diplodinium gracile may seem actually to tear away fibers (Fig. 206A, B); Opisthotrichum janus may bite from the surface of a plant piece the remains of ruptured tissue (Fig. 206D); and D. bubalidis, D. medium, and D. maggii may devour large irregular or flat grass pieces (Fig. 206C), but not fibers. Ostracodinium sp. can take in and roll up large cellulose fibers (Weineck, 1934). Green plant fragments are taken in preference to non-green ones, according to Usuelli (1930b), who offered a choice by feeding hay and barley. A third to a half of the green fragments were in about half of the ciliates; whereas less than 10 percent of them took in non-green fragments, all but a few of which remained free in the lumen. For this selectivity, Usuelli contended, the softer, smoother characteristics of the green plant pieces are responsible. When available, starch grains are ingested avidly by the ciliates, both in the rumen and in the thermostat. Four hours after giving a sheep 980 PROTOZOA AND OTHER ANIMALS fifty grams of cornstarch, 76 percent of the ciliates had taken in grains; and in from four to six hours 87 percent took in rice starch (Usuelli, 1930a). If the amount of starch is not excessive, most of it is eventually taken into the Protozoa. The size of the grains is a factor in ingestion. Figure 207. Ingestion and digestion of starch in Eudiplodinium medium. A, before feeding in culture; B, 2.5 minutes after feeding, starch grains in cytoplasm; C, 12 minutes after feeding, cytoplasm filled with starch; D, 2.5 hours after feeding, abundant deposits of glycogen; E, 16 hours after feeding, some residues of starch, glycogen deposits in certain areas. (After Westphal, 1934a.) For example, fewer ciliates take in potato starch, the diameter of many of the grains of which exceeds 100 1. In material kept in the thermostat, it has been found that fat droplets in milk will be ingested (Ferber, 1928); but ingestion of accessible ma- terial is not indiscriminate (Westphal, 1934a). PROTOZOA AND OTHER ANIMALS 981 That there is digestion of the starch has been well established (Trier, 1926; Westphal, 1934a, 1934b). Figures from Westphal (1934a) are reproduced here (Fig. 207) showing successive stages in the rapid in- gestion by Exdiplodinium medium in culture of rice starch and the dis- solution of this. Although there seems to be no good reason for denying starch-splitting ability to the ciliates themselves, Ullmann (1932) con- sidered it possible that starch-digesting bacteria, taken in with the food and continuing their action in the digestive vacuoles, are responsible. As starch is digested, glycogen (paraglycogen) accumulates in the cytoplasm in granular form. The reserve material is stored in the ecto- plasm, in the region of the gullet and the rectal tube, and sometimes also in the endoplasm. It has been asserted by many that, in addition to the other storage areas, the skeletal plates contain deposits of glycogen (Schulze, 1924; Trier, 1926; Weineck, 1934; Westphal, 1934a, 1934b; MacLennan, 1934). Dogiel disagreed with this concept; and Brown (MS), by a series of chemical tests, solubility tests with sub- stances that would have been expected to extract glycogen, and enzy- matic reactions, found no evidence that the contents of the skeletal prisms is glycogen. Brown pointed out that the results of iodine reac- tions are insufficient in themselves to identify glycogen, as other sub- stances stain in the same way. Glycogen may be built up from simpler carbohydrates, appearing after feeding with dextrose (Trier, 1926; Weineck, 1934) and lactose (Trier). When, after deposits of glycogen have accumulated, the ciliates re- main without food, the reserve is used up in cell metabolism. Trier found that within forty-eight hours after ingestion of the starch most of the accumulated glycogen had disappeared. There is disagreement as to whether the ciliates can use cellulose, though certainly a quantity is ingested. Much quoted has been the state- ment by Dogiel (1925) that, in the endoplasm of the Ophryoscolecidae, cellulose pieces undergo no morphological change and leave by the anus, still with sharp margins and no wrinkling or swelling. The state- ment was evidently based on observations on Diplodinium maggii and D. medium, which ingest large particles (Dogiel and Fedorowa, 1925). Westphal (1934b) reported that he had confirmed this account of ejection of large particles, but Weineck (1934) wrote that it has very 982 PROTOZOA AND OTHER ANIMALS seldom been seen, and that ejected pieces are those with excessively heavy membranes. Reichenow (in Doflein, 1927-29) did not agree that it provides evidence against the use of cellulose, stating that Pro- tozoa, especially when in unfavorable circumstances, may give up nutti- ment useful to them. Usuelli (1930b) saw no microscopic indications of corrosion of ingested fibers, and commented that in any case the green plant fibers, that are the ones chiefly used, contain relatively little cellu- lose. Westphal (1934a, 1934b) denied cellulose digestion in ophryo- scolecids, and showed that, in spite of the presence of cellulose and chlorophyll, cultures died out in the absence of starch. According to Mangold, the colorless pieces persist for a long time, even for four days, and during that time digestible substances are extracted. Earlier opinions that there is digestion of cellulose have been sum- marized by Becker, J. A. Schulz, and Emmerson (1930). P. Schulze (1924, 1927) and Trier (1926) believed that cellulose particles are reduced chemically and structurally. Recently, Weineck (1934) ob- tained positive evidence, including observations on corrosive changes and loss of the original double reactivity in polarized light. No cellulose-splitting enzymes have been isolated from the bodies of ophryoscolecids; that would be difficult in the presence of so many cellulose-decomposing bacteria. It was the suggestion of Trier (1926), and the opinion of Mangold (1929, 1933) and Westphal (1934a), that bacteria taken in with plant fragments are responsible for what cellulose decomposition has been observed within the ciliates. It is, of course, well known that the main rdle, at least, in cellulose-splitting in ruminants is performed by bacteria (Schieblich, 1929, 1932). If the ciliate had a cellulase, according to Mangold (1929), more intensive cellulose decomposition would be observed than has been possible. Doflein (Reichenow ed., 1927-1929) stated that there is no instance of fat digestion in Protozoa. Ferber (1928) observed, in successive rumen samples, that milk-fat droplets ingested iz vivo underwent de- formation and eventually disappeared, but he recognized the probability that bacteria, ingested also, were responsible for this breakdown of fat. Weineck, in experimental feeding with lipoids, found that no fat was ingested, but he observed a small amount in the ciliates that probably had been taken up from plant materials. Inasmuch as the ciliate population can be increased, up to a limit, PROTOZOA AND OTHER ANIMALS 983 by the addition of protein to the diet (Ferber, 1928; Mowry and Becker, 1930), and declines when protein is deficient, it is evident that, as Mangold (1929) stated, the ciliates have an important protein need. They obtain the protein ordinarily from the plant food. Mangold (1929, 1933) thought it unlikely that ingested bacteria or other Protozoa could sufficiently provide for this need. It is normally supplied by the addition of grain. Whether or not the cellulose of the plant food ts fully utilized, the starch and protein of the plant cell plasma supply energy and ma- terials for the activity and growth of the Protozoa. Many investigators who have concerned themselves with the ciliates of ruminants have sought an answer to the question of their possible value to their hosts. The literature was reviewed by Becker, Schulz, and Emmerson (1930); and the subject has been discussed by Becker (1932) and Mangold (1933). Of the various opinions advanced, Zurn’s belief that the ciliates could cause injury has been found entirely untenable. They are present in every ruminant in good condition; and Ferber (1929b) pointed out that the number of ciliates may serve as a guide to the host’s well-being. Favorable conditions of nutrition and optimum physiological activity of the host at the same time favor a large ciliate population; and under the best conditions there may be approximately a doubling of the average density, to 2,000,000 per cc. Unfavorable conditions are rapidly reflected in a decline of that popula- tion. Some, beginning with the first observers of the ruminant faunule (Gruby and Delafond, 1843), have supposed that the relationship is one of mutualistic symbiosis, the ciliates being in one way or another beneficial to their hosts. It is not disputed that great numbers of the ciliates are digested, but mutualism does not follow from that, unless some special contribution is made to the economy of the host. There is still no general agreement as to whether or not the ciliates can break down cellulose, but opinions have been widely published that they aid their hosts in cellulose decomposition. Reichenow (1920) observed morphological changes in ingested cellulose fragments in Troglodytella; and suggested that the significance of this ciliate to its primate hosts, as well as that of Ophryoscolecidae in ruminants, lies in the use of cellulose in constructing their own easily digestible bodies, which serve as animal nutriment for the mammals. In Doflein (Reiche- 984 PROTOZOA AND OTHER ANIMALS now ed., 1927-1929) he expressed the same opinion, and stated that the physiological rdle of these ciliates is evidently similar to that of termite flagellates, admitting, however, that they are not so important to the life of the host as these, because ruminants have other aids in cellulose decomposition. Usuelli (1930b) remarked that the softer green plant parts, which are those chiefly ingested, contain relatively little cellulose; so that even if intracellular digestion of this did take place, it would have little quantitative significance in the decomposition of cellulose in the rumen. Mangold (1929, 1933) and his coworkers have emphasized the rdéle of the ciliates in protein economy, reasoning that their bodies contain a significant part of the nitrogen available to the host, and that they derive this from plant protein, and presupposing that the ruminants can make better use of this animal protein than they can directly of plant protein. The last fact is certainly fundamental to their thesis, but Becker (1932) remarked that there is no proof of it. Becker, Schulz, and Emmerson (1930) found that goats digested slightly more protein when the ciliate population was present; but the difference was so small as to have little significance without further studies. According to analyses by C. Schwarz (1925) of the rumen contents of slaughterhouse cattle, 20 percent of the nitrogen is in the ciliates and 11.7 percent in bacteria. Ferber (1928) found the ciliate nitrogen in sheep and goats, with a population of from 837 to 2079 ciliates per cubic millimeter, to be from 10.27 to 20.33 percent of the total, averaging about 15 percent. Ferber and Winogradowa-Fedorowa (1929) calculated that with a population of 900,000 ciliates per gram and with the total nitrogen 0.166 percent, there would be, in a 3-kilogram rumen content, 150 grams of ciliates. These would contain about 4.7 grams of protein, and the estimate would be nearly doubled by use of the figures of Mangold and Schmitt-Krahmer (1927) for the total nitrogen. There is ciliate protein also in the reticulum, but this amounts to only a fraction of that in the rumen. There are no exact estimates of the amount of ciliate protoplasm di- gested in a day. Ferber and Winogradowa-Fedorowa (1929), on the basis of a fallacious estimate of a 7-percent daily division (see p. 973), calculated that 2 percent of a sheep’s daily protein requirement of thirty grams might be met by the ciliates. In this estimate the higher figures PROTOZOA AND OTHER ANIMALS 985 for total nitrogen were used. Mangold (1933) accepted the estimate that the ciliates provide about one percent of the protein used daily and, under certain circumstances, from 2 to 3 percent. C. Schwarz (1925), however, thought it probable that the greater part of the protein requirements are met by the microdrganisms; and Dogiel and Wino- gradowa-Fedorowa (1930), as well as Westphal (1934a), considered the daily reproductive rate to be much higher than 7 percent. The conclusion that the ciliates are mutualists, participating in the protein economy of the host, was deduced by Ferber (1928, 1929a) from the observed fact that the population density increases at the time of growth, reproduction, and lactation. He himself, like Mowry and Becker (1930), attributed the increase to the increased nutrition and especially to larger protein supply; but, as the latter authors pointed out, the facts do not warrant the deduction. Ferber pursuing the ideas advanced by C. Schwarz (1925), sug- gested that the rdle of the ciliates may be transformation of the protein in the plant food into easily digestible animal protein, and that in times of increased protein need by the host, this activity is enhanced by additions to the ciliate population. The result, of course, would be an increased ratio of ciliate nitrogen to total nitrogen. Becker, Schulz, and Emmerson (1930) remarked that although it is an observed fact that such protein transformations take place, and ciliates are eventually digested, it is doubtful if this substantially benefits the host. Mangold (1933) recognized usefulness to the host, in the mechant- cal rdle of the ciliates in mixture and trituration of the rumen contents, a role which had been suggested by Bundle (1895), Scheunert (1909), and Dogiel (1925). Conclusive evidence that there is any essential aid to the digestive processes in the mechanical activities of the ciliate 1s, however, lacking. It has been found that cattle will develop and reproduce normally on a diet that will produce symptoms of vitamin B (B,) deficiency in other animals. It has therefore been believed that vitamins of the B complex are synthesized within the alimentary tract of ruminants, and the micro- organisms have been investigated in this connection. Bechdel, Honey- well, Dutcher, and Knutsen (1928) found evidence of synthesis of the B complex by bacteria, as others have also reported. Manusardi (1933) investigated the possibility of synthesis of the vitamin (antiberiberic) 986 PROTOZOA AND OTHER ANIMALS by ciliates. Separating by filtration the ciliate, bacterial, and food frac- tions, he fed each to pigeons, together with polished rice. He concluded that it 1s extremely improbable that the ciliate fauna has the capacity of synthesizing vitamin B. The bacterial fraction proved to be the most antiberiberic. Becker was the first to carry on the obvious experiments which should be made in investigating the significance of the ciliates to their hosts. That is defaunation, which led to such dramatic results in Cleveland’s work with termites. If the ciliates perform any necessary function, the effect should become apparent in animals deprived of them. Becker, Schulz, and Emmerson (1930) defaunated four goats, and for periods of two and three weeks made detailed analyses of their use of nutrients. The goats were then reinfected, and after ten days analyses were con- tinued for the same period as before. The presence or absence of ciliates was accompanied by little difference in the coefficients of digestibility. There were no differences of practical significance in the digestion of cellulose, and the goats with ciliates used only slightly more protein than those without. Winogradow, Winogradowa-Fedorowa, and Wer- eninow (1930) had found that raw fiber was 12.8 percent better digested in a normally faunated than in a ciliate-free ram. Becker and Everett (1930) compared during nineteen weeks the growth of seven lambs with ciliates and seven without, the Protozoa having been removed by giving some lambs copper sulphate with milk. They found that the defaunated lambs actually grew a little more rapidly than the others. Poljansky and Strelkow (1935) made observations on growth of four pairs of twin goats for ten months, beginning at the age of from one to two and a half months. The goats were isolated from the parents so early that they did not become naturally infected. One member of each pair was given a ciliate population. In this experiment, also, the ciliate- free goats of three pairs grew a little faster than the others; in one pair the goat with ciliates gained more. Falaschini (1935) compared for a period of fourteen months the growth of four lambs. Two were defaunated by a milk diet after six months, then in five weeks reinfected with ciliates. The other two were defaunated after eight months, then continued on a normal diet with- out ciliates. The growth curves of the four lambs corresponded. PROTOZOA AND OTHER ANIMALS 987 These experiments demonstrate conclusively that, at least in a period of a year or so, the host suffers no apparent detriment from lack of ciliates. The Protozoa perform no necessary role in nutrition, nor are their services necessary to aid the host mechanically or in keeping down the bacterial flora. When present, they may be a source of certain incidental benefits, but apparently the relationship can still best be de- fined in the words of Doflein (1916). He regarded it as ordinary commensalism, any value that the ciliates might have to the host being minimal and incidental. LITERATURE CITED Abbott, R. L. 1926. Contributions to the physiology of digestion in the Australian roach, Periplaneta australastae Fab. J. exp. Zool., 44: 219-54. Alexeieff, A. 1909. Les Flagellés parasites de l’intestin des batraciens in- digénes. C. R. Soc. Biol. Paris, 67: 199-201. — 1910. Sur les Flagellés intestinaux des poissons marins. (Note preé- liminaire.) Arch. zool. exp. gén., (5) 6: N. et R., 5-20. — 1912. Le Parasitisme des Eugléniens et la phylogénie des Sporozoaires sensu stricto, Arch. zool. exp. gén., (5) 10: N. et R., 73-88. André, E. 1909. Sur un nouvel Infusoire parasite des Dendrocoeles (Ophryoglena parasitica n. sp.). Rev. suisse Zool., 17: 273-80. — 1910. Sur quelques Infusoires marins parasites et commensaux. Rev. suisse Zool., 18: 173-87. Andrews, B. J. 1930. Method and rate of protozoan refaunation in the termite Termopsis angusticollis Hagen. Univ. Cal. Publ. Zool., 33: 449-70. Artigas, P. de T., and S. Pacheco. 1932. Sobre um flagellado parasito do Myopotamus coipus. Octomitus myopotami n. sp. Ann. Fac. Med. S. Paulo, 8: 79-81. Aschner, M. 1932. Experimentelle Untersuchungen tiber die Symbiose der Kleiderlaus. Naturwissenschaften, 27: 501-5. — 1934. Studies on the symbiosis of the body louse. I. Elimination of the symbionts by centrifugalisation of the eggs. Parasitology, 26: 309-14. Aschner, M., and E. Ries. 1933. Das Verhalten der Kleiderlaus bei Aus- schaltung ihrer Symbionten. Eine experimentelle Symbiosestudie. Z. Morph. Okol. Tiere, 26: 529-90. Balamuth, W. 1939. Studies on regeneration in Protozoa. I. Cytology and regeneration of Licnophora macfarlandi. MS, Univ. Cal. Library. Balbiani, E. G. 1885. Sur un Infusoire cilié parasite du sang de I’Aselle aquatique (Anoplophrya circulans). Rec. zool. suisse., 2: 277-303. Bary, A. de. 1879. Die Erscheinung der Symbiose. Strassburg. Beauchamp. P. de. 1911. Astasia captiva n. sp. Euglénien parasite de Catenula lemnae Ant. Dug. Arch. zool. exp. gén. (5) 6: N. et R., 52-58. 988 PROTOZOA AND OTHER ANIMALS Bechdel, S. I., H. E. Soneywell, R A. Dutcher, and M. H. Knutsen. 1928. Synthesis of vitamin B in the rumen of the cow. J. biol. Chem., 80: 231-38. Becker, E. R. 1929. Methods of rendering the rumen and reticulum of ruminants free from their normal infusorian fauna. Proc. nat. Acad. Sci. Wash., 15: 435-38. —— 1932. The present status of problems relating to the ciliates of ruminants and Equidae. Quart. Rev. Biol., 7: 282-97. — 1933. Host-specificity and specificity of animal parasites. Amer. J. trop. Med., 13: 505-23. Becker, E. R., and R. C. Everett. 1930. Comparative growths of normal and Infusoria-free lambs. Amer. J. Hyg. 11: 362-70. Becker, E. R., and T. S. Hsiung. 1929. The method by which ruminants acquire their fauna of Infusoria, and remarks concerning experiments on the host-specificity of these Protozoa. Proc. nat. Acad. Sci. Wash., 15: 684-90. Becker, E. R., J. A. Schulz, and M. A. Emmerson. 1930. Experiments on the physiological relationships between the stomach Infusoria of ruminants and their hosts, with a bibliography. Iowa St. Coll. J. Sci., 4: 215-51. Becker, E. R., and M. Talbott. 1927. The protozoan fauna of the rumen and reticulum of American cattle. Iowa St. Coll. J. Sci., 1: 345-71. Beckwith, T. D., and E. J. Rose. 1929. Cellulose digestion by organisms from the termite gut. Proc. Soc. exp. Biol. N.Y., 27: 4-6. Beers, C. D. 1938a. Structure and division in the astomatous ciliate Meta- radiophrya asymmetrica n. sp. J. Elisha Mitchell sci. Soc., 54: 111-25. —— 1938b. Hysterocineta eiseniae n. sp., an endoparasitic ciliate from the earthworm Evsenia lénnbergi. Arch. Protistenk., 91: 516-25. Beneden, P. J. van. 1876. Animal parasites and messmates. New York. Also German and French editions. Berghe, L. van den. 1934. Sur un ciliate parasite de pontes de mollusques d’eau douce Glaucoma paedophthora, n. sp. C. R. Soc. Biol. Paris, 115: 1423-26. Bishop, A. 1926. Notes upon Sieboldiellina planariarum (Siebold), a ciliate parasite of Planaria torva. Parasitology, 18: 187-94. —— 1932. Entamoeba aulastomi Néller. Cultivation, morphology, and method of division; and cultivation of Hexamita sp. Parasitology, 24: 225-322 —— 1933. The morphology and division of Hexamita gigas n. sp. (Flagel- lata). Parasitology, 25: 163-70. —— 1935. Observations upon a “Trichomonas” from pond water. Para- sitology, 27: 246-56. —— 1936. Further observations upon a “Trichomonas” from pond water. Parasitology, 28: 443-45. PROTOZOA AND OTHER ANIMALS 989 — 1937. The method of division of Trepomonas agilis in culture. Para- sitology, 29: 413-18. —— 1939. A note upon the systematic position of “Trichomonas” keilini (Bishop, 1935). Parasitology, 31: 469-72. Boissezon, P. de. 1930. Contribution a l’étude de la biologie et de I’histo- physiologie de Culex pipiens L. Arch. zool. exp. gén., 70: 281-431. Brown, W. H. 1937. Nature and function of the skeletal platelets in the Ophryoscolecidae. MS, Univ. Cal. Library. Brumpt, E., and G. Lavier. 1924. Un Nouvel Euglénien Polyflagellé parasite du tetard de Leptodactylus ocellatus au Brésil. Ann. Parasit. hum. comp.,) 2: 248-52: Buchner, P. 1930. Tier und Pflanze in Symbiose. Berlin. Bunting, M. 1926. Studiees on the life cycle of Tetramitus rostratus Perty. J. Morph., 42: 23-81. Bunting, M., and D. H. Wenrich. 1929. Binary fission in the amoeboid and flagellate phases of Tetramitus rostratus. J. Morph., 47: 37-87. Bundle, A. 1895. Ciliate Infusorien im Coécum des Pferdes. Z. wiss. Zool., 60: 284-350. Buscalioni, L., and S. Comes. 1910. La digestione delle membrane vegetali per opera dei Flagellati contenuti nell’ intestino dei Termitidi e il problema della simbiosi. Atti Accad. gioenia, (5) 3: mem. 17, 1-16. Bush, M. 1933. The morphology of the ciliate Haptophrya michiganensis Woodhead and its relation to the other members of the Astomatea. Trans. Amer, mict. SoG, 5225223-52: —— 1934. The morphology of Haptophrya michiganensis Woodhead, an astomatous ciliate from the intestinal tract of Hemidactylium scutatum (Schlegel). Univ. Cal. Publ. Zool., 39: 251-76. Butschli, O. 1889. Protozoa. III. Abt. Infusoria und System der Radiolaria in Bronn: Klassen und Ordnungen des Thier-Reichs. Leipzig. Calkins, G. N. 1933. The Biology of the Protozoa. 2d ed. Philadelphia. Campbell, W. G. 1929. The chemical aspect of the destruction of oak wood by powder-post and death watch beetles-Lyctws spp. and Xestobium sp. Bio-chem. J., 23: 1290-93. Cattaneo, G. 1888. Su di un Infusorio ciliato, parassito del sangue del Carcinus maenas, Zool. Anz., 11: 456-59. Caullery, M. 1922. Le Parasitisme et la symbiose. Paris. Cépéde, C. 1910. Récherches sur les Infusoires astomes. Anatomie, biologie, éthologie, parasitaire, systématique. Arch. zool. exp. gén (5) 3: 341-609. Cépéde, C., and E. Poyarkoff. 1909. Sur un Infusoire astome Cepedella hepatica Poyarkoff parasite du foie des Cyclas (S. corneum L.). Bull. sci. Fr. Belg., 43: 463-75. Certes, A. 1882. Sur les parasites intestinaux de I’huitre. C. R. Acad. Sci. Paris, 95: 463-65. 990 PROTOZOA AND OTHER ANIMALS Chatterji, G. C., K. N. Das, and A. N. Mitra. 1928. On an Octomitus n. sp. found in the intestinal contents of Hylobates hoolock. J. Dep. Sci. Calcutta Univ., 9: 21-24. Chatton, E. 1920. Les Péridiniens parasites. Morphologie, reproduction, éthologie. Arch. zool. exp. gén., 59: 1-475. — 1936. Les Migrateurs horizontalement polarises de certains Péritriches. De leur signification. Mém. Mus. Hist. nat. Belg., 3: 913-40. Chatton, E., and A. Lwoff. 1921. Sur une famille nouvelle d’Acinétiens, les Sphenophryidae adaptés aux branchies des mollusques acéphales. C. R. Acad. Sci. Paris, 173: 1495-97. —— 1922a. Sur |’évolution des Infusoires des Lamellibranches. Relations des Hypocomidés avec les Ancistridés. Le genre Hypocomides n. gen. CR. Acad. VSciePatis, 175: 787-90. — 1922b. Sur l’évolution des Infusoires des Lamellibranches. Le Genre Pelecyophrya, intermédiaire entre les Hypocomidés et les Sphénophryides. Bourgeonnement et Conjugasion. C. R. Acad. Sci. Paris, 175: 915-17. — 1922c. Sur l’évolution des Infusoires des Lamellibranches. Relations des Sphénophryidés avec les Hypocomidés. C. R. Acad. Sci. Paris, 175: 1444-47, — 1923a. Sur l’évolution des Infusoires des Lamellibranches. Les formes primitives du phylum des Thigmotriches; le genre Thigmophrya. C. R. Acad. Sci. Paris, 177: 81-83. — 1923b. Un Cas remarquable d’adaptation: Ellobiophrya donacis n. g., n. sp., Péritriche inquilin des branches de Donax vittatus (Lamelli- branche). C. R. Soc. Biol. Paris, 88: 749-52. 1924. Sur |’évolution des Infusoires des Lamellibranches: Morphologie comparée des Hypocomidés. Les nouveaux genres Hypocomina et Hypo- comella. C. R. Acad. Sci. Paris, 178: 1928-30. — 1926. Diagnoses de Ciliés thigmotriches nouveaux. Bull. Soc. zool. Fr., D3 45-528 — 1929. Contribution a l'étude de l’adaptation. Ellobiophrya donacis Ch. et Lw. Péritriche vivant sur les branchies de |’Acéphale Donax vittatus da Costa. Bull. biol., 63: 321-49. —— 1934a. Sur un cilié thigmotriche nouveau: Gargarius gargarius n. gen., n. sp., de Mytilus edulis. Bull. Soc. zool. Fr., 59: 375-76. — 1934b. Sur un Infusoire parasite des poils sécréteurs des Crustacés Edriophthalmes et la famille nouvelle des Pilisuctoridae. C. R. Acad. Sci. Paris, 199: 696-99. —— 1935. Les Ciliés apostomes. Morphologie, cytologie, éthologie, évolu- tion, systématique. Premiére partie. Apercu historique et général. Etude monographique des genres et des espéces. Arch. zool. exp. gén., 77: 1-453. —— 1936a. Les Pilisuctoridae Ch. et Lw. Ciliés parasites des poils sécréteurs des Crustacés Edriophthalmes. Polarité, orientation, et desmodexie chez les Infusoires. Bull. biol., 70: 86-144. PROTOZOA AND OTHER ANIMALS 991 1936b. Les Remaniements et la continuité des cinétome au cours de la scission chez les Thigmotriches Ancistrumidés. Arch. zool. exp. gén., 78: N. et R., 84-91. — 1937. Sur l’enkystement d’un Thigmotriche Ancistrumidé: Proboveria loripedis Chatton et Lwoff, et les phénomeénes qui l’accompagnent. C. R. Soc. Biol. Paris, 124: 807-10. Cheissin, E. [E. Chejsin}. 1928. Vorlaufige Mitteilung tiber einige parasitische Infusorien des Bajkal-Sees. C. R. Acad. Sci. U.R.S.S., 1928 (A) : 295-99. — 1930. Morphologische und systematische Studien uber Astomata aus dem Baikalsee. Arch. Protistenk., 70: 531-618. —— 1931. Infusorien Ancistridae und Boveridae aus dem Baikalsee. Arch. Protistenk., 73: 280-304. — 1932. Sur la morphologie et la classification des Infusoires parasitiques de la famille des Ptychostomidae. Trav. Sta. limnol. Lac Bajkal, 2: 29-59. Child, H. J. 1933. The anatomy and histology of the digestive tract and associated organs in Zootermopsis nevadensis (Hagen), with some obser- vations on morphogenesis, behavior during ecdysis, and the protozoan fauna. MS, Univ. Cal. Library. —— 1934. The internal anatomy of termites and the histology of the diges- tive tract 7 Kofoid et al., Termites and termite control. Berkeley, Cal. Cleveland, L. R. 1923. Correlation between the food and morphology of termites and the presence of intestinal Protozoa. Amer. J. Hyg., 3: 444-61. —— 1924, The physiological and symbiotic relationships between the in- testinal Protozoa of termites and their host, with special reference to Reticulitermes flavipes Kollar. Biol. Bull., 46: 178-227. —— 1925a. The method by which Trichonympha campanula, a protozoén in the intestine of termites, ingests solid particles of wood for food. Biol. Bull., 48: 282-88. —— 1925b. The ability of termites to live perhaps indefinitely on a diet of pure cellulose. Biol. Bull., 48: 289-93. —— 1925c. The effects of oxygenation and starvation on the symbiosis be- tween the termite, Termopsis, and its intestinal flagellates. Biol. Bull., 48 :309-26. ——1926. Symbiosis among animals with special reference to termites and their intestinal flagellates. Quart. Rev. Biol., 1: 51-60. — 1928a. Further observations and experiments on the symbiosis between termites and their intestinal Protozoa. Biol. Bull. 54: 231-37. —— 1928b. Tritrichomonas fecalis nov. sp. of man; its ability to grow and multiply indefinitely in faeces diluted with tap water and in frogs and tadpoles. Amer. J. Hyg., 8: 232-55. — 1930. The symbiosis between the wood-feeding roach, Cryptocercus punctulatus Scudder, and its intestinal flagellates. Anat. Rec., 47: 293-94. O92 PROTOZOA AND OTHER ANIMALS —— 1935. The intranuclear achromatic figure of Oxymonas grandis sp. nov. Biol. Bull., 69: 54-63. Cleveland, L. R., S. R. Hall, E. P. Sanders, and J. Collier. 1934. The wood- feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: i-x, 185-342. Codreanu, M., and R. Codreanu. 1928. Un Nouvel Euglénien (Astastia Chaetogastris n. sp.) parasite coelomique d’un Oligochete (Chaetogaster diastrophus Gruith). C. R. Soc. Biol. Paris, 9: 1368-70. Codreanu, R. 1928. Un Infusoire nouveau (Frontonia branchiostomae n. sp.) commensal de l’Amphioxus (Branchiostoma lanceolatum Pall). C. R. Soc. Biol., Paris, 98: 1078-80. — 1930. Sur la phase interne du cycle évolutif de deux formes d’Ophryoglena, Infusoires endoparasites des larves d’Ephéméres. C. R. Acad. Sci. Paris, 190: 1154-57. — 1934, La Présence d’Ophryoglena, Ciliés endoparasites chez les nymphes de l’Ephémére Oligoneuria rhenana Imhoff en France. Ann. Protist., 4: 181-83. Cohen, W. E. 1933. An analysis of termite (Ewtermes exitiosus) mound ma- terial. Jour. Counc. sci. industr. Res. Aust., 6: 166-69. Collin, B. 1914. Notes protistologiques. Arch. zool. exp. gén., 54; N. et R., 85-97. Cross, J. B. A study of Oxymonas minor Zeliff from the termite Kalotermes minor Hagen. Univ. Cal. Publ. Zool. (J press.) Crouch, H. B. 1934. Observations on Hexamita mormotae n. sp., a protozoan flagellate from the woodchuck Marmota monax (Linn). Iowa St. Coll. Je Sei. 82 13-107. Cuénot, L. 1891. Protozoaries commensaux et parasites des Echinodermes. Rev. biol. nord Fr., 3: 285-300. —— 1894. Uber Hemispeiropsis antedonis Cuén., ein an den Comatulen lebendes Infusorium. Zool. Anz., 17: 316. Cunha, A. M. da, and J. Muniz. 1922. Octomitus avium n. sp. Brazil-med., 36: 386-88. — 1927. Estudo sobre os Flagellados intestinaes das Aves do Brazil. Mem. Inst. Osw. Cruz, 20 19-337 — 1929. Nota sobre os parasitas intestinaes do Macacus rhesus com a descripcao de uma nova especie de Octomitus. (Suppl.) Mem. Inst. Osw. Cruz, 53) 34-37. Das Gupta, B. M. 1935. The occurrence of a Trepomonas sp. in the caecum of turtles. J. Parasit., 21: 125-26. Davis, H. S. 1923. Observations on an intestinal flagellate of the trout. J. Parasit., 9: 153-60. —— 1926. Octomitus salmonis, a parasitic flagellate of trout. Bull. U. S. Bur. Fish., 42: 9-26. Dickman, A. 1931. Studies on the intestinal flora of termites with reference to their ability to digest cellulose. Biol, Bull., 61: 85-92. PROTOZOA AND OTHER ANIMALS 993 Diesing, K. M. 1866. Revision der Prothelminthen. Abtheilung: Amastigen. I. Amastigen ohne Peristom. S. B. Akad. Wiss. Wien., Math.-naturw. Cl 22505-79. Dobell, C. 1935. Researches on the intestinal Protozoa of monkeys and man. VII. On the Exteromonas of macaques and Embadomonas intestinalis. Parasitology, 27: 564-92. Doflein, F. 1916. Lehrbuch der Protozoenkunde 4th ed., Jena. Doflein, F., and E. Reichenow. 1927-29. Lehrbuch der Protozoenkunde. Sth ed., Dofleins, Lehrbuch, Jean. Dogiel, V. A. 1925. Uber die Art der Nahrung und der Nahrungsaufnahme bei den im Darme der Huftiere parasitierenden Infusorien. Trav. Soc. Nat. St-Pétersb. (Leningr.) Sect. Zool. et Physiol., 54: 69-93. —— 1927. Monographie der Familie Ophryoscolecidae. Teil I. Arch. Protistenk., 59: 1-288. Dogiel, V. A., and T. Fedorowa. 1925. Uber den Bau und die Funktion des inneren Skeletts der Ophyroscoleciden. Zool. Anz., 62: 97-107. —— 1929. Uber die Zahl der Infusorien im Wiederkauermagen. Zbl. Bakt., (ip) (12: 135-42. Dogiel, V. A., and A. V. Furssenko. 1921. Neue ektoparasitische Infusorien von Landisopoden. Trav. Soc. Nat. St-Pétersb. (Leningr.), 51: 147-58, 199-202. Dogiel, V. A., and T. Winogradowa-Fedorowa. 1930. Experimentelle unter- suchungen zur Biologie der Infusorien des Wiederkauermagens. Wiss. Arch. Landwirtsch. (B)3: 172-88. Dore, W. H., and R. C. Miller. 1923. The digestion of wood by Teredo navalis. Univ. Cal. Publ. Zool., 22: 383-400 Duboscq, O., and P. Grassé. 1934. Notes sur les protistes parasites des termites de France. IX. L’Enkystement des flagellés de Calotermes flavicollis. Arch. zool. exp. gén., 76: N. et R., 66-72. Edmondson, C. H. 1906. The Protozoa of Iowa. Proc. Davenport Acad. Sci., ft 124. Eikenberry, W. L., and R. A. Waldron. 1930. Educational biology. Boston. Emik, L. O. MS,. Studies on the nutrition of Trichonympha. (Univ. Cal. Library, 1937.) MS,. Ingestion of food by Trichonympha. Trans. Amer. micr. Soc. (I7 press). Entz, G. 1904. Die Fauna der kontinentalen Kochsalzwasser. Math. naturw. Ber. Ung., 19: 89-124. Epstein, H. 1926. Infektion des Nervensystems von Fischen durch Infusorien. Arch, russ. protist., 5: 169-80. Fabre-Domergue, P. 1885. Note sur les Infusoires ciliés de la Baie de Concarneau. J. Anat. Paris., 21: 554-68. —— 1888. Etude sur lorganisation des Urcéolaires et sur quelques genres d'Infusoires voisins de cette famille. J. anat. Paris, 24: 214-60. Falaschini, A. 1935. Gli infusori dell’apparato digerente degli erbivori sono 994 PROTOZOA AND OTHER ANIMALS indispensabili per la vita dei loro ospiti? Boll. Lab. Zool. agr. Bachic. Milano, 4 (II): 151-60. Falck, R. 1930. Die Scheindestruktion des Koniferenholzes durch die Larven des Hausbockes (Hytotrupes bajulus L.). Cellulose-chem., 11: 89-91. Fantham, H. B. 1922. Some parasitic Protozoa found in South Africa. -V S. Afr. J. Sel.9 1925332239: — 1936. The evolution of parasitism among the Protozoa. Scientia, Bologna, 59: 316-24. Fauré-Fremiet, E. 1924. Contribution a la connaissance des Infusoires plank- toniques. Bull. biol., (Suppl.), 6: 1-171. Ferber, K. E. 1928. Die Zahl und Masse der Infusorien im Pansen und ihre Bedeutung fiir den Eiweissaufbau beim Wiederkauer. Z. Tierz. Ziicht- Biol., 12: 31-63. — 1929a. Die Verinderungen der Infusorienzahl im Pansen der Wieder- kduer im Zusammenhang mit den Verinderungen des Eiweissumsatzes. Z. Tietz. ZuchtBiol., 15: 375-90. — 1929b. Uber die optimale Beschaffenheit des Panseninhalts der Wieder- kaur. Wiss. Arch. Landw., 1: 597-600. Ferber, K. E., and T. Winogradowa-Fedorowa. 1929. Zahlung und Teilungs- quote der Infusorien im Pansen der Wiederkauer. Biol. Zbl., 49: 321-28. Foulke, S. G. 1884. Some notes on Manayunkia speciosa. Proc. Acad. nat. Sci. Philad., 1884: 48-9. Fuhrmann, O. 1894. Die Turbellarien der Umgebung von Basel. Rev. suisse Zool., 2: 215-90. Fulton, J. F. 1923. Trichodina pediculus and a new closely related species. Proc. Boston Soc: mat, Hist:, 37: 1-29. Geiman, Q. M. 1932. The intestinal Protozoa of the larvae of the crane fly, Tipula abdominalis, J. Parasit., 19: 173. Ghosh, E. 1925. On a new ciliate, Balantidium knowlesi sp. nov., a coelomic parasite in Culicoides peregrinus, Parasitology, 17: 189. Giard, A. 1880. Note sur l’existence temporaire de Myriapodes dans les fosses nasales de l’homme, suivie de quelques reflexions sur le parasitisme inchoatif. Bull. Sci. Fr. Belg., 12: 1-11. Gicklhorn, J. 1925. Notiz tiber Evglena cyclopicola nov. sp. Arch. Protistenk., 51: 542-48. Goetsch, W. 1936. Beitrage zur Biologie des Termitenstaates. Z. Morphol. Okol. Tiere, 31: 490-560. Graff, L. von. 1882. Monographie der Turbellarien. I. Rhabdocoelida. Leipzig. Graham, G. L. 1935. Géardia infections in a nematode from cattle. J. Parasit., 20.1 27-28. Grassé, P. P. 1924. Octomastix parvus Alex., diplozoaire parasite de la Cistude d’Europe. C. R. Soc. Biol. Paris, 91: 439-42. —— 1935. Parasites et parasitisme. Paris. Grassé, P. P., and P. de Boissezon. 1929. Turchiniella culicis n.g., n. sp. PROTOZOA AND OTHER ANIMALS 995 Infusoire parasite de I’hemocoele d’un Cw/ex adulte. Bull. Soc. zool. Fr., 54: 187-91. Green, R. A., and E. L. Breazeale. 1937. Bacteria and the nitrogen metab- olism of termites. J. Bac., 33: 95-96. Gruby, and O. Delafond. 1843. Récherches sur des animalcules se développant en grand nombre dans |’estomac et dans les intestins, pendant la diges- tion des animaux herbivores et carnivores. C. R. Acad. Sci. Paris, 17: 1304-08. Haas, G. 1933. Beitrige zur Kenntnis der Cytologie von Ichthyophthirius multifiliis Foug. Arch, Protistenk., 81: 88-137. Hall, R. P., and T. L. Jahn. 1929. On the comparative cytology of certain euglenoid flagellates and the systematic position of the families Euglenidae Stein and Astasiidae Bitschli. Trans. Amer. micr. Soc., 48: 388-405. Hall, S. R. 1931. Observations on Euglena leucops, sp. nov., a parasite of Stenostomum, with special reference to nuclear division. Biol. Bull., 60: 327-44. Haswell, W. A. 1892. Note on the occurrence of a flagellate infusorian as an intracellular parasite. Proc. Linn. Soc. N. S. W., (2) 7: 197-99. 1907. Parasitic Euglenae. Zool. Anz., 31: 296-97. Haupt, A. W. 1932. Fundamentals of biology, 2d. ed., New York. Hazard, F. O. 1937. Two new host records for the protozoan Haptophrya michiganensis Woodhead. J. Parasit., 23: 315-16. Hegner, R. W. 1922. Frog and toad tadpoles as sources of intestinal Pro- tozoa for teaching purposes. Science, 56: 439-41. —— 1923. Observations and experiments on Euglenoidina in the digestive tract of frog and toad tadpoles. Biol. Bull., 45: 162-80. —— 1924. Parasitism among the Protozoa. Sci. Mon., N.Y., 19: 140-45. —— 1926a. Host-parasite relationships among human Protozoa. Proc. R. Soc. Med., 19: 41-44. —— 1926b. The biology of host-parasite relationships among Protozoa living in man. Quart. Rev. Biol., 1: 393-418. —— 1926c. Homologies and analogies between free-living and parasitic Protozoa. Amer. Nat., 60: 516-25. —— 1928. The evolutionary significance of the protozoan parasites of monkeys and man. Quart. Rev. Biol., 3: 225-44. 1937. Parasite reactions to host modifications. J. Parasit., 23: 1-12. Hegner, R., and J. Andrews (ed.). 1930. Problems and methods of research in protozoology. New York. Heidenreich, E. 1935. Untersuchungen an parasitischen Ciliaten aus Anne- liden. Teil I: Systematik. Arch. Protistenk., 84: 315-92. Hendee, E. C. 1933. The association of the termites, Kalotermes minor, Reti- culitermes hesperus, and Zootermopsis angusticollis with fungi. Univ. Cal. Publ. Zool., 39: 111-34. 996 PROTOZOA AND OTHER ANIMALS Henderson, J. C. Studies of some amoebae from a termite of the genus Cubitermes. Univ. Cal. Publ. Zool. (In press.) Hertig, M., W. H. Taliaferro, and B. Schwartz. 1937. Report of the Com- mittee on Terminology. J. Parasit., 23: 325-29. Hertwig, O. 1883. Die Symbiose oder das Genossenschaftsleben im Thier- reich. Jena. Hetherington, A. 1933. The culture of some holotrichous ciliates. Arch. Protistenk., 80: 255-80. —— 1936. The precise control of growth in a pure culture of a ciliate, Glaucoma pyriformis. Biol. Bull., 70: 426-40. Hinshaw, W. R., E. McNeil, and C. A. Kofoid. 1938a. The presence and dis- tribution of Hexamita sp. in turkeys in California. J. Amer. med. Ass., 93: 160. —— 1938b. The relationship of Hexamita sp. to an enteritis of turkey poults. The Cornell Veterinarian, 28: 281-93. Holdaway, F. G. 1933. The composition of different regions of mounds of Eutermes exitiosus Hill. J. Counc. sci. industr. Res. Aust., 6: 160-65. Hollande, A. 1939. Sur un genre nouveau de Trichomonadide libre: Coelo- trichomastix convexas nov. gen. nov. sp. Bull. Soc. zool. Fr., 64: 114-19. Howland, R. B. 1928. A note on Astasia captive Beauch. Science, 68: 37. Hsiung, T. S. 1930. A monograph on the Protozoa of the large intestine of the horse. Iowa, St. Coll. J. Sci., 4: 359-423. Hudson, C. T., and P. H. Gosse. 1889. The Rotifera: or wheel-animalcules, both British and foreign. London. Hungate, R. E. 1936. Studies on the nutrition of Zootermopsis. I. The rdle of bacteria and molds in cellulose decomposition. Zbl. Bakt., (II), 94: 240-49. — 1938. Studies on the nutrition of Zootermopsis. I. The relative impor- tance of the termite and the Protozoa in wood digestion. Ecology, 19: 1-25: — 1939. Experiments on the nutrition of Zootermopsis. UI. The anaerobic carbohydrate dissimilation by the intestinal Protozoa. Ecology, 20: 230- 45, Hunninen, A. V., and R. Wichterman. 1936. Hyperparasitism: A species of Hexamita (Protozoa, Flagellata) found in the reproductive systems of Dero pristis inflata (Trematoda) from marine eels. J. Parasit., 22: 540. Ikeda, I., and Y. Ozaki. 1918. Notes on a new Boveria species, Boveria labialis n. sp. J. Coll. Sci. Tokyo, 40: art. 6, 1-25. Issel, R. 1903. Ancistridi del Golfo di Napoli. Studio monografico sopra una nuova famiglia di cigliati commensali di molluschi marini. Mitt. Zool. Sta. Neapel, 16: 63-108. Jahn, T. L., and W. R. McKibben. 1937. A colorless euglenoid flagellate, Khawkinea halli n. gen., n. sp. Trans. Amer. micr. Soc., 56: 48-54. Janda, V., and O. Jirovec. 1937. Uber kiinstlich hervorgerufenen Parasitismus PROTOZOA AND OTHER ANIMALS O97 eines freilebenden ciliaten Glaucoma piriformis und Infekionsversuche mit Exglena gracilis und Spirochaeta biflexa. Mém. Soc. Zool. Tchéco- slovaque de Prague, 5: 34-57. Jarocki, J. 1934. Two new hypocomid ciliates, Heterocineta janickii sp. n. and H. /lwoffi sp. n., ectoparasites of Physa fontinalis (L.) and Viviparus fasciatus Miller. Mém. Cl. Sci. Acad. polon. math. nat. (B) 1934: 167-87. — 1935. Studies on ciliates from fresh-water molluscs. I. General remarks on protozoan parasites of Pulmonata. Transfer experiments with species of Heterocineta and Chaetogaster limnae?, their additional host. Some new hypocomid ciliates. Bull. int. Acad. Cracovie, Cl. Sci. math. nat. (B:II) 1935: 201-30. Jarocki, J., and Z. Raabe. 1932. Uber drei neue Infusorien-Genera der Familie Hypocomidae (Ciliata Thigmotricha), Parasiten in Susswasser- muscheln. Bull. int. Acad. Cracovie, Cl. Sci. math. nat. (B:II) 1932: 29-45. Jucci, C. 1932. Sulla presenza di batteriociti nel tessuto adiposo dei Termitidi. Arch. zool. (ital.) Torino, 16: 1422-29. Kahl, A. 1926. Neue und wenig bekannte Formen der holotrichen und heterotrichen Ciliaten. Arch. Protistenk., 55: 197-438. — 1930. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) . I. Allgemeiner Teil und Prostomata, 77 Dahl: Die Tierwelt Deutschlands, 18 Teil, Jena. — 1931. Ibid., Il. Holotricha, 21 Teil. —— Ibid., Il. Spirotricha, 25 Teil. —— 1933. Ciliata libera et ectocommensalia, 72 Grimpe, A. and E. Wagler: Die Tierwelt der Nord- und Ostsee. Lief. 23, Teil II]. C3, 29-146. Leipzig. —— 1934. Ciliata ectocommensalia et parasitica, 72 Grimpe, G. and E. Wagler: Die Tierwelt der Nord- und Ostsee, Lief. 26, Teil II C4, 147-83. Leipsig. 1935. Ibid., IV. Peritricha und Chonotricha, 30 Teil. Katzin, L. I., and H. Kirby, Jr. 1939. The relative weights of termites and their Protozoa. J. Parasit., 25: 444-45. Keilin, D, 1921. On a new ciliate: Lambornella stegomyiae n. g., n. sp., parasitic in the body-cavity of the larvae of Stegomyza scutellaris Walker (Diptera, Nematocera, Culicidae). Parasitology, 13: 216-24. Kepner, W. A., and R. P. Carroll. 1923. A ciliate endoparasitic in Stenostoma leucops. J. Parasit., 10: 99-100. Kepner, W. A., and J. S. Carter. 1931. Ten well-defined new species of Stenostomum. Zool. Anz., 93: 108-23. Kidder, G. W. 1929. Streblomastix strix, morphology and mitosis. Univ. Cal. Publ. Zool., 33: 109-24. —— 1933a. Studies on Conchophthirius mytili De Morgan. I. Morphology and division. Arch. Protistenk., 79: 1-24. 998 PROTOZOA AND OTHER ANIMALS —— 1933b. Studies on Conchophthirius mytili De Morgan. II. Conjugation and nuclear reorganization. Arch. Protistenk., 79: 25-49. —— 1933c. On the genus Ancistruma Strand (Ancistrum Maupas). 1. The structure and division of A. mytilz Quenn. and A. isseli Kahl. Biol. Bull., 64: 1-20. —— 1933d. Conchophthirius caryoclada sp. nov. (Protozoa, Ciliata). Biol. Bull., 65: 175-78. 1933e. On the genus Ancistruma Strand (Ancistrum Maupas). II. The conjugation and nuclear reorganization of A. isseli Kahl. Arch. Protis- tenk., 81: 1-18. —— 1934a. Studies on the ciliates from fresh water mussels. I. The structure and neuromotor system of Conchophthirius anodontae Stein, C. curtus Engl., and C. magna sp. nov. Biol. Bull., 66: 69-90. —— 1934b. Studies on the ciliates from fresh water mussels. II. The nuclei of Conchophthirius anodontae Stein, C. curtus Engl., and C. magna Kidder, during binary fission. Biol. Bull., 66: 286-303. —— 1937. The intestinal Protozoa of the wood-feeding roach Panesthia. Parasitology, 29: 163-205. Kidder, G. W., and F. M. Summers. 1935. Taxonomic and cytological studies on the ciliates associated with the amphipod family Orchestiidae from the Woods Hole district. I. The stomatous holotrichous ectocommensals. Biol. Bull., 68: 51-68. Kirby, H., Jr. 1927. Studies on some amoebae from the termite Mzrotermes, with notes on some other Protozoa from the Termitidae. Quart. Jour. mick. Sci. 7 189-222. — 1928. A species of Proboscidiella ftom Kalotermes (Cryptotermes) dudleyi Banks, a termite of Central America, with remarks on the oxy- monad flagellates. Quart. Jour. micr. Sci., 72: 355-86. — 1930. Trichomonad flagellates from termites. I. Tricercomitus gen. nov., and Hexamastix Alexeieff. Univ. Cal. Publ. Zool., 33: 393-444. — 1931. Trichomonad flagellates from termites. II. Evtrichomastix and the subfamily Trichomonadinae. Univ. Cal. Publ. Zool., 36: 171-262. — 1932a. Two Protozoa from brine. Trans. Amer. micr. Soc., 51: 8-15. — 1932b. Flagellates of the genus Trichonympha in termites. Univ. Cal. Publ. Zool., 37: 349-476. —— 1932c. Protozoa in termites of the genus Amzitermes. Parasitology, 24: 289-304. — 1937. Host-parasite relations in the distribution of Protozoa in termites. Univ. Cal. Publ. Zool., 41: 189-212. — 1939, The Templeton Crocker Expedition of the California Academy of Sciences, 1932, No. 39. Two new flagellates from termites in the genera Coronympha Kirby, and Metacoronympha Kirby, new genus. Proc. Cal. Acad. Sci. (4) 22: 207-20. PROTOZOA AND OTHER ANIMALS 99 Klebs, G. 1893. Flagellatenstudien. Theil I. Z. wiss. Zool., 55: 265-351 (p. 328). Knoth, M. 1928. Neue Versuche sur Ziichtung der im Pansen von Wieder- kauern lebenden Ophryoscoleciden (Ciliata). Z. Parasitenk., 1: 262-82. Koch, A. 1933a. Uber das Verhalten symbiontenfreier Sitodrepalarven. Biol. Zbl., 53: 199-203. — 1933b. Uber kiinstlich symbiontenfrei gemachte Insekten. Zool. Ans., (Supplb.) 6: 143-50. —— 1936, Symbiosestudien. I]. Experimentelle Untersuchungen an Oryzae- philus surinamensis L. (Cucujidae, Coleopt.) Z. Morph. Okol. Tiere, 52-0 137-60. — 1938a. Die Bakteriensymbiose der Termiten. Zool. Anz. (Supplb.) 11: 81-90. — 1938b. Symbiosestudien. III. Die intrazellulare Bakteriensymbiose von Mastotermes darwiniensis Froggatt (Isoptera). Z. Morph. Okol. Tiere, 34: 534-609. Kofoid, C. A. 1903. On the structure of Protophrya ovicola, a ciliate infu- sorian from the brood-sac of Littorina rudis Don. Mark Anniversary Volume, Art. 5, 111-20. New York. — 1935. On two remarkable ciliate Protozoa from the caecum of the In- dian elephant. Proc. nat. Acad. Sci. Wash., 21: 501-6. Kofoid, C. A., and M. Bush. 1936. The life cycle of Parachaenia myae gen. nov., sp. nov., a ciliate parasitic in Mya arenaria Linn. from San Francisco Bay, California. Bull. Mus. Hist. nat. Belg., 12 (22) : 1-15. Kofoid, C. A., and J. F. Christenson. 1934. Ciliates from Bos gaurus H. Smith. Univ. Cal. Pub. Zool., 39: 341-92. Kofoid, C. A., and R. F. MacLennan. 1930. Ciliates from Bos indicus Linn. I. The genus Entodinium Stein. Univ. Cal. Publ. Zool., 33: 471-544. —— 1932. Ciliates from Bos indicus Linn. II. A revision of Diplodininm Schuberg. Univ. Cal. Publ. Zool., 37: 53-152. —— 1933. Ciliates from Bos indicus Linn. III. Epidinium Crawley, Epiplas- tron gen. nov., and Ophryoscolex Stein. Univ. Cal. Publ. Zool., 39: 1-34. Kofoid, C. A., and O. Swezy 1919. Studies on the parasites of the termites. On Streblomastix strix, a polymastigote flagellate with a linear plasmodial phase. Univ. Cal. Publ. Zool., 20: 1-20. Kofoid, C. A., ef al. (ed.) 1934. Termites and termite control. Berkeley, Cal. Koidzumi, M. 1921. Studies on the intestinal Protozoa found in the termites of Japan. Parasitology, 13: 255-309. Konig, A. 1894. Hemispeiropsis comatulae, eine neue Gattung der Urceolari- den. S.B. Akad. Wiss. Wien, 103: 55-60. Kotlan, A. 1923. Zur Kenntnis der Darmflagellaten aus der Hausente und anderen Wasservégeln. Zbl. Bakt., (1) Orig. 90: 24-28. 1000 PROTOZOA AND OTHER ANIMALS Kudo, R. R. 1939. Protozoology. Springfield. Labbé, A. 1899. Sporozoa im Schulze, F. E.: Das Tierreich, Lief. 5, 1-180. Berlin. Lamborn, W. A. 1921. A protozoon pathogenic to mosquito larvae. Parasitol- ogy, 43: 213-15. Lavier, G. 1925. Infections héréditaires par les parasites animaux. Ann. Parasit. hum. comp., 3: 306-21. —— 1935. Sur le parasitisme dans I’intestin d’amphibiens, de Flagellés du genre Trepomonas Duj. C. R. Soc. Biol. Paris, 118: 991-92. —1936a. Sur la structure des Flagellés du genre Hexamita Duj. C. R. Soc. Biol, Parise 120%: 1177-80. —— 1936b. Sur quelques Flagellés intestinaux de poissons marins. Ann. Parasit. hum. comp., 14: 278-89. —— 1936c. Sur un Trichomonadideé libre des eaux stagnantes. Ann. Parasit. hum. comp., 14: 359-68. Lavier, G., and H. Galliard. 1925. Parasitisme sanguin d’un Hexamitus chez un crapaud Bufo calamita. Ann. Parasit. hum. comp., 3: 113-15. Leydig, F. 1857. Uber Hydatina senta. Arch, Anat. Physiol. wiss. Med., 24: 404-16, Lichtenstein, J. L. 1921. Ophryoglena collini n. sp. parasite coelomique des larves d’Ephéméres. C. R. Soc. Biol. Paris, 85: 794-96. Light, S. F., and M. F. Sanford. 1927. Are the protozoan faunae of termites specific ? Proc. Soc. exp. Biol. N.Y., 25: 95-96. — 1928. Experimental transfaunation of termites. Univ. Cal. Publ. Zool., 31: 269-74. Lucas, M. S. 1934. Ciliates from Bermuda sea urchins. I. Metopws. J. R. micr. Soc., 54: 79-93. Lund, E. E. 1930. The effect of diet upon the intestinal fauna of Termopsis. Univ. Cal. Publ. Zool., 36: 81-96. Lwoff, A. 1923. Sur la nutrition des Infusoires. C. R. Acad. Sci. Paris, 176: 928-30. —— 1924, Infection expérimentale 4 Glaucoma piriformis (Infusoire) chez Galleria mellonella (Lépidoptére). C. R. Acad. Sci. Paris, 178: 1106-08. — 1929. Milieux de culture et d’entretien pour Glaucoma piriformis (Cilié) C. R. Soe. Biol. Paris, 100: 635: —— 1932. Réchérches biochemiques sur la nutrition des Protozoaires, le pouvoir de synthése. Monogr. Inst. Pasteur. Lynch, J. E. 1929. Studies on the ciliates from the intestine of Strongy- locentrotus. I. Entorhipidium gen. nov. Univ. Cal. Publ. Zool., 33: 27-56. —— 1930. Studies on the ciliates from the intestine of Strongylocentrotus. Il. Lechriopyla mystax gen. nov., sp. nov. Univ. Cal. Publ. Zool., 33: 307-50. MacArthur, W. P. 1922. A holotrichous ciliate pathogenic to Theobaldia annulata Schrank. J. R. Army med. Cps., 38: 83-92. PROTOZOA AND OTHER ANIMALS 1001 Mackinnon, D. L. 1912. Protists parasitic in the larva of the crane fly, T7pula sp. Parasitology, 5: 175-89. MacLennan, R. F. 1934. The morphology of the glycogen reserves in Poly- plastron. Arch, Protistenk., 81: 412-19. MacLennan, R. F., and F. H. Connell. 1931. The morphology of Expoterion pernix gen. nov., sp. nov., a holotrichous ciliate from the intestine of Acmaea persona Eschscholtz. Univ. Cal. Publ. Zool., 36: 141-56. Madsen, H. 1931. Bemerkungen iiber einige entozoische und freilebenden marine Infusorien der Gattungen Uronema, Cyclidium, Cristigera, As pi- disca und Entodiscus gen. nov. Zool. Anz., 96: 99-112. Mangold, E. 1929. Die Verdauung der Wiederkauer 7 Handbuch der Ernah- rung und des Stoffwechsels der landwirtschaftlichen Nutztiere, als Grund- lagen der Fitterungslehre, 2: 107-237 (Berlin, Springer). — 1933. Die Infusorien des Pansens und ihre Bedeutung fiir die Ernah- rung der Wiederkauer. Biederm. Zbl., (A), n.f. 3: 161-87. Mangold, E., and T. Radeff. 1930. Die Quelle fiir die Infektion des Wieder- kaiiermagens mit Infusorien. Wiss. Arch. Landw. (B), 4: 173-99. Mangold, E., and C. Schmitt-Krahmer. 1927. Die Stickstoffverteilung im Pan- sen der Wiederkauer bei Fiitterung und Hunger und ihre Beziehung zu den Pansen-Infusorien. Biochem. Z., 191: 411-22. Mangold, E., and F. Usuelli. 1930. Die schadliche Wirkung der Milch und der Verinderung der H.- Ionkonzentration auf die Infusorien des Wie- derkiuermagens. Wiss. Arch. Landw. (B), 3: 189-201. Mansour, K. 1936. The problem of the nutrition of wood eating insects. C. R. XIle Int. Cong. Zool., 1: 233-41. Mansour, K., and J. J. Mansour-Bek. 1933. Zur Frage der Holzverdauung durch Insektenlarven. Proc. K. Akad. Wetensch., 36: 795-99. —— 1934a. On the digestion of wood by insects. Jour. exp. Biol., 11: 243-56. —— 1934b. The digestion of wood by insects and the supposed réle of microorganisms. Biol. Rev., 9: 363-82. Manusardi, L. 1933. Gli Infusori ciliati del rumine sintetizzano la vitamina B? Boll. Lab. Zool. agr. Bachic. Milano, 4(1) : 140-48. Margolin, S. 1930. Methods for the cultivation of cattle ciliates. Biol. Bull., Se ellen Matubayasi, H. 1937. Studies on parasitic Protozoa in Japan. I. On flagel- lates parasitic in snakes. Annot. zool. jap., 16: 245-52. Maupas, E. 1883. Contribution a l'étude morphologique et anatomique des Infusoires ciliés. Arch. zool. exp. gén. (2) 1: 427-664. May, E. 1939. The behavior of the intestinal Protozoa of termites at the time of the last ecdysis. MS, Univ. Cal. Library. McFarland, J. 1913. Biology general and medical. 2d ed., Philadelphia. Mercier, L., and R. Poisson. 1923. Un Cas de parasitisme accidentel d’une Neépe par un Infusoire. C. R. Acad. Sci. Paris, 176; 1838-41. 1002 PROTOZOA AND OTHER ANIMALS Metcalf, M. M. 1923. The opalinid ciliate infusorians. Bull. U. S. nat. Mus., 120: 1-484. —— 1929. Parasites and the aid they give in problems of taxonomy, geo- graphical distribution, and paleogeography. Smithson. misc. Coll., 81(8): 1-36. Meyer, S. L. 1938. Haptophrya virginiensis nov. sp., a protozoan parasite of the pickerel frog, Rana palustris Le Conte. Anat. Rec., 72, Suppl.: 54-55. Miyashita, Y. 1927. On a new parasitic ciliate, Lada tanishi n. sp. with pre- liminary notes on its heterogamic conjugation. Jap. J. Zool., 1: 205-18. —— 1933. Studies on a freshwater foettingeriid ciliate Hyalospira cardinae n. g.n. sp. Jap. J. Zool., 4: 439-60. Mjassnikowa, M. 1930a. Sphenophrya sphaerii, ein neues Infusorium aus Sphaerium corneum L. Arch. Protistenk., 71: 255-94. —— 1930b. Uber einen neuen Vertreter der Familie Sphenophryidae aus Myatruncata L. Arch. Protistenk., 72: 377-89. Montalenti, G. 1927. Sull’ allevamento dei termiti senza 1 protozoi dell’ ampolla cecale. R.C. Accad. Lincei, (6) 6: 529-32. — 1932. Gli enzimi digerenti e l’assorbimento delle sostanze solubili nell’ intestino delle termiti. Arch. zool. (ital.) Tonino, 16: 859-64. — 1934. Un interessante caso di simbiosi: i flagellati dell’ intestino delle termiti. Rasseg. faunist. Roma, 1: 25-35. Moore, E. 1922. Octomitus salmonis, a new species of intestinal parasite in trout. Trans. Amer. Fish, Soc., 52: 74-97. — 1923a. Diseases of fish in state hatcheries. 12th Ann. Rep. N.Y. St. Conserv. Comm., 66-79. — 1923b. A report of progress on the study of trout diseases. Trans. Amer. Fish, Soc., 53: 74-94. — 1924. The transmission of Octomitus salmonis in the egg of trout. Trans. Amer. Fish. Soc., 54: 54-56. Moroff, T. 1903. Beitrag zur Kenntnis einiger Flagellaten. Arch. Protistenk., 3: 69-106. Mowry, H. A., and E. R. Becker. 1930. Experiments on the biology of In- fusoria inhabiting the rumen of goats. Iowa St. Coll. J. Sci., 5: 35-60. Nie, Dashu. 1934. Studies of the intestinal ciliates of sea urchin from Amoy. Rep. Mar. Biol. Ass. China, 3: 81-90. Nieschulz, O. 1922. Uber eine Astasia—Art aus dem Sitisswassernematoden Trilobus gracilis Bst. Zool. Anz., 54: 136-38. Noller, W., and F. Buttgereit. 1923. Uber ein neues parasitisches Protozoén der Haustaube (Octomutus columbae nov. spec.) Zbl. Bakt., Refer (1), 75. 239-40. Oppenheimer, C. 1925. Die Fermente und thre Wirkungen. Leipzig. Oshima, M. 1919. Formosan termites and methods of preventing their dam- age: Philipp. J: Sa., 15°319-83. PROTOZOA AND OTHER ANIMALS 1003 Parker, T. J. 1893. Lessons in elementary biology. 2d, ed., London. Penard, E. 1922. Etudes sur les Infusoires d’eau douce. Généve. Perekropoff, G. I., and P. I. Stepanoff. 1931. On an intestinal disturbance in man caused by Octomitus. (In Russian.) J. Mikrobiol., 13: 191-96. — 1932. Zur Frage der Darmerkrankungen des Menschen, die durch Octo- mitus bedingt sind. Zbl. Bakt., Orig. (1) 123: 324-30. Pickard, E. A. 1927. The neuromotor apparatus of Boveria teredinidi Nelson, a ciliate from the gills of Teredo navalis. Univ. Cal. Publ. Zool., 29: 405-28. Pierantoni, O. 1934. La digestione della cellulosa e del legno negli animali e la simbiosi delle termiti. Riv. Fis. Mat. Sci. nat., 9: 57-64. — 1936. La simbiosi fisiologica nei termitidi xilofagi e nei loro flagellati intestinali. Arch. zool. (ital.) Torino, 22: 135-71. —— 1937. Osservazioni sulla simbiosi nei termitidi xilofagi e nei loro flagel- lati intestinali—II. Defaunazione per digiuno. Arch. zool. (ital.) Torino, 25-207. Playfair, G. I. 1921. Australian freshwater flagellates. Proc. Linn. Soc. N.S.W., BOm9o- 146. Plimmer, H. G. 1912. On the blood-parasites found in animals in the Zo- ological Gardens during the four years 1908-1911. Proc. zool. Soc. Lond., 1912: 406-19. Poisson, R. 1929. Apropos de I’ Anophrys maggii Cattaneo, Infusoire parasite du sang du Carcinus maenas L. (Crustacé Décapode) ; Sur son identité avec l’Anophrys sarcophaga Cohn. C. R. Soc. Biol. Paris, 102: 637-39. — 1930. Observations sur Anophrys sarcophaga Cohn (=A. maggii Cat- taneo) Infusoire holotriche marin et sur son parasitisme possible chez certains Crustacés. Bull. biol., 64: 288-331. Poljanskij, J. I. 1925. Drei neue parasitische Infusorien aus dem Parenchym einiger Mollusken und Turbellarien. Arch. Protistenk., 52: 381-93. Poljansky, G., and A. Strelkow. 1935. Uber die Wirkung der Panseninfu- sorien auf das Wachtum der Wiederkauer. Trav. Inst. Biol. Peterhof., 13-14: 68-87. Powers, P. B. A. 1933a. Studies on the ciliates from sea urchins. I. General Taxonomy. Biol. Bull., 65: 106-21. —— 1933b. Studies on the ciliates from sea urchins, II. Entodiscus borealis (Hentschel), (Protozoa, Ciliata), behavior and morphology. Biol. Bull., 65: 122-36. —— 1933c. Studies on the ciliates from Tortugas echinoids. Yearb. Carneg. Instn., 32: 276-80. —— 1935. Studies on the ciliates of sea urchins. A general survey of the infestations occurring in Tortugas echinoids. Publ. Carneg. Instn., 452: 293-326. —— 1936, Ciliates infesting Acapulco sea urchins. J. Parasit., 22: 541. 1004 PROTOZOA AND OTHER ANIMALS —— 1937. Studies of the ciliates of sea urchins. Ann. Rep. Tortugas Lab., 1936-19372 O13: Poyarkoff, E. 1909. Cepedella hepatica, Cilié astome neuveau, parasite du foie des Cyclas. C. R. Soc. Biol. Paris, 66: 96-97. Pringsheim, H. 1932. The chemistry of the monosaccharides and of the poly- saccharides. New York. Raabe, Z. 1933. Untersuchungen an einigen Arten des Genus Conchophthirus Stein. Bull. int. Acad. Cracovie, Cl. Sci. math. nat., (B: II) 1932 (8- 10) : 295-310. — 1934a. Uber einige an den Kiemen von Mytilus edulis L. und Macoma balthica (L.) parasitierende Ciliaten-Arten. Ann. Mus. zool. polon., 10; 289-303. — 1934b. Weitere Untersuchungen an einigen Arten des Genus Conchoph- thirus Stein, Mém. Cl. Sci. Acad. polon., math. nat., (B) 6: 221-35. — 1935. Rhynchophrya cristallina g. n., sp. n. nouvelle forme d’Infusoire de la famille des Sphaenophryidae Chatton et Lwoff. Bull. Inst. océanogr. Monaco, 676: 1-6. Rankin, J. S. 1937. An ecological study of pasasites of some North Caro- lina salamanders. Ecol. Monogr. 7: 171-269. Rehberg, H. 1882. Eine neue Gregarine. Lagenella mobilis n. g. et n. sp. Abh. naturw. Ver. Bremen, 7: 68-71. Reichenow, E. 1920. Den Wiederkauer-Infusorien verwandte Formen aus Gorilla und Schimpanse. Arch. Protistenk., 41: 1-33. Remane, A. 1936. Gastrotricha und Kinorhyncha, Bronn’s Klassen, Bd. 4, Abt. 2, Buch 1, Teil 2, Lief 2, Leipzig. Reynolds, B. D. 1936. Colpoda steini, a facultative parasite of the land slug, Agriolimax agrestis. J. Parasit., 22: 48-53. Ripper, W. 1930. Zur Frage des Celluloseabbaus bei der Holzverdauung xylophager Insektenlarven. Z. vergl. Physiol., 13: 314-33. Rose, M. 1933. Sur un Infusoire Foettingéridé parasite des Siphonophores C. R. Acad. Sci. Paris, 197: 868-69. — 1934. Pluralité des espéces de Foettingeridae (Infusoires Apostomes) parasites des Siphonophores de la baie d’Alger. Bull. Soc. Hist. nat. Afr. INI 257 149-518 Rosenberg, L. E. 1936, On the viability of Tritrichomonas augusta. Trans. Amer, micr. Soc., 55: 313-14. Rossolimo, L. 1925. Infusoires parasites du tube digestif des Oligochétes- genre Ptychostomum St. Arch. russ. protist., 4: 217-33. —— 1926. Parasitische Infusorien aus dem Baikalsee. Arch. Protistenk., 54: 468-509. Rossolimo, L. L., and T. A. Perzewa. 1929. Zur Kenntnis einiger astomen Infusorien: Studien an Skelettbildung. Arch. Protistenk., 67: 237-52. Ryckeghem, J. van 1928. Hexamitus tubifici nov. sp. Ann. Soc. Sci. Brux. (B), 48, pt. 2, Mémoires: 139-43. PROTOZOA AND OTHER ANIMALS 1005 Scheunert, A. 1909. Verdauung. IV. Besondezheiten der Verdauung bei Tieren mit mehrhéligen Magen (Wiederkaduer), Kaltbliitern und Végeln, in Oppenheimer, C., Handbuch der Biochem. des Menschen und der Tiere, 3: 2: 152-70. Jena. (Also, 1924, in 2d ed., vol. 5.) Schieblich, M. 1929. Die Mitwirkung der Bacterien bei der Verdauung im E. Mangold: Handbuch der Ernahrung und des Stoffwechsels der land- wirtschaftliche Nutztiere als Grundlagen der Fiitterungslehre, 2: 310-48. Berlin. —— 1932. Die Bedeutung der normalen Magendarmflora fiir den Wirtsor- ganismus. Biederm. Zbl. (A) n. f. 2: 483-501. Schmidt, W. 1920. Untersuchungen tiber Octomitus intestinalis truttae. Arch. Protistenk., 40: 253-89. Schulze, P. 1924. Der Nachweis und die Verbreitung des Chitins mit einem Anhang tiber das komplizierte Verdauungsystem der Ophryoscoleciden. Z. Morph. Okol. Tiere, 2: 643-66. — 1927. Noch einmal die ‘‘Skelettplatten’”” der Ophryoscoleciden. Z. Morph. Okol. Tiere, 7: 678-89. Schwartz, W. 1935. Untersuchungen iiber die Symbiose von Tieren mit Pilzen und Bakterien. IV. Der Stand unserer Kenntnisse von den physio- logischen Grundlagen der Symbiosen von Tieren mit Pilzen und Bak- terien. Arch. Mikrobiol., 6: 369-460. Schwarz, C. 1925. Die ernahrungsphysiologische Bedeutung der Mikroor- ganismen in den Vormagen der Wiederkauer. Biochem. Z., 156: 130-37. Sokoloff, D. 1933. Algunas nuevas formas de flagelados del Valle de Mexico. An. Inst. Biol. Univ. Mex., 4: 197-206. Stein, F. (Ritter) von. 1848. Uber die Natur der Gregarinen. Arch. Anat. Physiol. wiss. Med., 1848: 182-223. 1860. Uber die Eintheilung der holotrichen Infusionsthiere und Stellte einige neue Gattungen und Arten aus dieser Ordnung. $.B. béhm. Ges. Wiss., Nat.-Math., 1860: 57-63. —— 1861. Uber ein neues parasitisches Infusionsthier (Ptychostomum Paln- dinarum) aus dem Darmkanal von Paludinen und uber die mit demsel- ben zunachst verwandten Infusorienformen. S.B. boéhm. Ges. Wiss., 1861: 85-90. Steuer, A. 1928. Uber Ellobiopsis chattoni Caullery 1910, einen ektoparasitis- chen Flagellaten mariner Copepoden. Arch. Protistenk., 60: 501-10. Stevens, N. M. 1901. Studies on ciliate Infusoria. Proc. Calif. Acad. Sci. (3), 3: 1-42. Strand, E. 1928. Miscellanea nomenclatorica zoologica et paleontologica. Arch. Naturgesch., 92: 30-75. Strelkow, A., G. Poljansky, and M. Issakowa-Keo. 1933. Uber die Infek- tionswege der im Pansen und in der Haube der Wiederkduer befindlichen Infusorien. Arch. Tierernahr. Tierz., 9: 679-97. Studitsky, A. 1930. Eine neue Art der Gattung Prychostomum Stein (Lada 1006 PROTOZOA AND OTHER ANIMALS Vejdovsky) Pt. rossolimoi n. sp. Eine systematische Studie. Zool. Anz., 87: 247-56. Summers, F. M., and G. W. Kidder. 1936. Taxonomic and cytological studies on the ciliates associated with the amphipod family Orchestiidae from the Woods Hole district. II. The coelozoic astomatous parasites. Arch. Protistenk., 86: 379-403. Svec, F. 1897. Beitrage zur Kenntnis der Infusorien Bohmens. I. Die ciliaten Infusorien des unterpocernitzer Teiches. Bull. int. Acad. Prag, Sci. Math. Nat., 4(2): 29-47. Swezy, O. 1923. The pseudopodial method of feeding by trichonymphid flagellates parasitic in wood-eating termites. Univ. Cal. Publ. Zool., 20:391-400. Tetrault, P. A., and W. L. Weis. 1937. Cellulose decomposition by a bac- terial culture from the intestinal tract of termites. J. Bact., 33: 95. Theiler, H., and S. M. Farber. 1932. Trichomonas muris, parasitic in oxyu- rids of the white mouse. J. Parasit., 19: 169. — 1936. Trichomonas muris, parasitic in the oxyurid nematodes, Aspicu- laris tetraptera and Syphacia obvelata, from white mice. Parasitology, 28: 149-60. Thomson, J. A. 1934. Biology for Everyman. London. Thomson, J. A., and P. Geddes. 1931. Life: Outlines of general biology. London. Thomson, J. G. 1925. A Gvardia parasitic in a bursate nematode living in the Viscacha. Protozoology (Suppl., J. Helminth.), No. 1, 1-6. Trager, W. 1932. A cellulase from the symbiotic intestinal flagellates of ter- mites and of the roach, Cryptocercus punctulatus. Bio-chem. J. 26: 1762-71. — 1934. The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite Protozoa. Biol. Bull., 66: 182- 90. Treillard, M., and A. Lwoff. 1924. Sur un Infusoire parasite de la cavité générale des larves de chironomes. Sa sexualité. C. R. Acad. Sci. Paris, 178: 1761-64. Trier, H. J. 1926. Der Kohlehydratstoffwechsel der Panseninfusorien und die Bedeutung der griinen Pflanzenteile fiir diese Organismen. Z. vergl. Physiol., 4: 305-30. Ullmann, T. 1932. Uber die Einwitkung der Fermente einiger Wirbellosen auf polymere Kohlenhydrate. Z. vergl. Physiol., 17: 520-36. Usuelli, F. 1930a. Starkeaufnahme und Glycogenbildung der Panseninfuso- rien. Wiss. Arch. Landw., 3: 4-19. — 1930b. Das Verhalten der Panseninfusorien gegeniiber cellulose und grinen Pflanzenteilen. Wiss. Arch. Landw., 3: 368-82. PROTOZOA AND OTHER ANIMALS 1007 —— 1930c. Gli infusori Ciliati che vivono nell’apparato digerente degli er- bivori. Clin. vet. Milano. Uyemura, M. 1934. Uber einige neue Ciliaten aus dem Darmkanal von japan- ischen Echinoideen (1). Sci. Rep. Tokyo Bunrika Daig. (B)1: 181-91. — 1935. Uber drei in der Sisswasser-Muschel (Anodonta lauta v. Mattens) lebende Ciliaten (Conchophthirus). Sci. Rep. Tokyo Bunrika Daig. (B) 2: 89-100. Valkanov, A. 1928. Protistenstudien. II. Notizen tiber die Flagellaten Bul- gariens. Arch. Protistenk., 63: 419-50. Voigt, M. 1904. Die Rotatorien und Gastrotrichen der Umgebung von Plon. Foschber. biol. Sta. Pl6n, 11: 1-180. 1909. Nachtrag zur Gastrotrichen-Fauna Plons. Zool. Anz., 34: 717-22. Wallengren, H. 1895. Studier 6fver Ciliata infusorier. Arsskr., Lunds Univ. Sa 7 pp: Warren, E. 1932. On a ciliate protozoan inhabiting the liver of a slug. Ann. Natal. Mus., 7: 1-53. Watson, M. E. 1915. Studies on gregarines. Illinois biol. Monogr., 2: 215- 468. Weineck, E. 1934. Die Celluloseverdauung bei den Ciliaten des Wieder- kduermagens. Arch. Protistenk., 82: 169-202. Wenrich, D. H. 1923. Variations in Evglenamorpha hegneri n. g., n. sp., from the intestine of tadpoles. Anat. Rec., 24: 370-71. —— 1924a. Studies on Euglenamorpha hegneri n. g., n. sp., a euglenoid flagellate found in tadpoles. Biol. Bull., 47: 149-74. —— 1924b. A new protozoan parasite, Amphileptus branchiarum, n. sp., on the gills of tadpoles. Trans. Amer. micr. Soc., 43: 191-99. —— 1933. A species of Hexamita (Protozoa, Flagellata) from the intestine of a monkey (Macacus rhesus). J. Parasit., 19: 225-28. —— 1935. Host-parasite relations between parasitic Protozoa and their hosts. Proc. Amer. phil. Soc., 75: 605-50. Wenyon, C. M. 1926, Protozoology, a manual for medical men, veterinarians and zoologists. New York. Wenyon, C. M., and W. Broughton Alcock. 1924. A Trepomonas coprozoic in human faeces. Trans. R. Soc. trop. Med. Hyg., 18: 9. Werner, E. 1926. Der Erreger der Zelluloseverdauung bei der Rosenkaferlarve (Potosta cuprea Fbr.) Bacillus cellulosam fermentans n. sp. Zbl. Bakt., 67 (Il): 297-330. Wertheim, P. 1934a. Uber die Infusorienfauna im Magen von Bos taurus L. Ann. Mus. zool. polon., 10: 251-66. —— 1934b. Warmeversuche mit Wiederkauermageninfusorien. Biol. Zbl., 4990-402. —— 1935. Infusorien aus dem Wiederkauermagen vom Gebiete Jugosla- wiens nebst einer Ubersicht dieser Tierchen vom Balkanhalbinsel-Bereich 1008 PROTOZOA AND OTHER ANIMALS und ein kurzer Bericht tiber die Pferdedarminfusorien, zugleich Revision der Familie Ophryoscolecidae. (Croat, German summary). Arhiva vet., 5: 388-526. Westphal, A. 1934a. Studien wber Ophryoscoleciden in der Kultur. Z. Parasi- temkew 7.37 lata — 1934b. Ein Ziichtungsverfahren fiir die Wiederkauerinfusorien und dessen Ergebnisse fiir die Frage nach der Bedeutung der Infusorien fiir den Wirt. Zool. Anz., (Suppl.) 7: 207-10. Wiedemann, J. F. 1930. Die Zelluloseverdauung bei Lamellicornierlarven. Z. Morph. Okol. Tiere, 19: 228-58. Winogradow, M., T. Winogradowa-Fedorowa, and A. Wereninow. 1930. Zur Frage nach der Einwirkung der Panseininfusorien auf die Verdauug der Wiederkauer. Zbl. Bakt., II, 81: 230-44. Winogradowa-Fedorowa, T., and M. P. Winogradoff. 1929. Zahlungsmethode der Gesamtzahl der im Wiederkauermagen lebenden Infusorien. Zbl. Bakt., 78 (II) : 246-54. Woodhead, A. E. 1928. Haptophrya michiganensts sp. nov., a protozoan parasite of the four-toed salamander. J. Parasit., 14: 177-82. Woodhead, A. E., and F. Kruidenier. 1936. The probable method of infec- tion of the four-toed salamander with the protozoén, Haptoyhrya michi- ganensis. J. Parasit., 22: 107-8. Yagiu, R. 1933. Studies on the ciliates from the intestine of Amthocidaris crassispina (A. Agassiz). I. Cyclidium ozakii sp. nov. & Strombilidium rapulum sp. nov. J. Sci. Hiroshima Univ., (B:I) 2: 211-22. —— 1934. Studies on the ciliates from the intestine of Anthocidaris crassis- pina (A. Agassiz). II. Cryptochilidium sigmoides sp. nov. and Crypto- chilidium minor sp. n. J. Sci. Hiroshima Univ., (B:1) 3: 25-31. —— 1935. Studies on the ciliates from sea urchins of Yaku Island, with a description of a new species, Cryptochilidium ozakii sp. nov. J. Sc. Hiroshima Univ. (B:1)3: 139-47. Yamasaki, M. 1937a. Studies on the intestinal Protozoa of termites. III. The distribution of glycogen in the bodies of intestinal flagellates of termites, Leucotermes (Reticulitermes) speratus and Coptotermes formosanus. Mem. Coll. Sci. Kyoto, (B)12: 211-24. —— 1937b. Studies on the intestinal Protozoa of termites IV. Glycogen in the body of Trichonympha agilis var. japonica under experimental con- ditions. Mem. Coll. Sci. Kyoto, (B)12: 225-35. Yonge, C. M. 1925. The digestion of cellulose by invertebrates. Sci. Prog., 20: 242-48. Zerling, Mlle. 1933. Une Astasza sans flagelle, Astasia doridis n. sp., parasite des pontes de nudibranches. C. R. Soc. Biol. Paris, 112: 643-44. Zick, Karl. 1928. Urceolaria korschelti n. sp., eine neue marine Urceolarine, nebst einem Uberblick tiber die Urceolarinen. Z. wiss. Zool., 132: 355- 403. CHAPTER XX ORGANISMS LIVING ON AND IN PROTOZOA HAROLD Kirpy, JR.’ PROTOZOA as a group may be hosts of a great variety of other organisms. Some of these are epibiotic, and in them the relationship ranges from occasional phoresy to obligatory and constant association. True ectopara- sitism exists in some epibionts, though often the distinction from preda- tism is disputable. Protozoa are not so constituted as to be capable of harboring inquilines; all endobionts are intracellular. Unless autotrophic, therefore, endobiotic forms are parasites in the sense that they are de- pendent on their hosts in nutrition. In many instances, however, the protozoan suffers no apparent detriment from the relationship; and sometimes the association of host and symbiont is constant. (Symbiosis is used as a collective term, including commensalism, mutualism, and parasitism.) It may be, even, that there is mutual advantage; but only autotrophic forms are in a position to confer the commonest type of benefit in mutualism, a nutritive one. It has been suggested, although not demonstrated, that certain intracellular microdrganisms may produce enzymes that function in the nutritional metabolism of the host. Many endobionts are more or less destructive parasites, which cause injury or death; that is true, so far as is known, of all that invade the nucleus. When the association has an obligatory and constant character, as in the occurrence of bacteria on the body surface of certain Protozoa, or of bacteria present in certain areas of the cytoplasm of all or almost all specimens, the error has often been made of interpreting the sym- bionts as structures of the host. When they are present only occasionally, they have sometimes been mistakenly regarded as representing occasional phases in the life history of the host, that is, reproductive phases. In- creasing knowledge of the symbionts of the Protozoa has corrected most * Assistance rendered by personnel of Work Projects Administration, Official Project number 65-1-08-113, Unit C1, is acknowledged. 1010 PARASITES OF PROTOZOA of these errors, but there are sometimes greater difficulties in the way of correct interpretation than might be supposed. A large number of organisms symbiotic with Protozoa are Schizo- mycetes or Phycomycetes. In the latter group, Chytridiales are especially widespread, as parasites of both the cytoplasm and the nucleus. There are in all the major groups of Protozoa species that are symbiotic with other Protozoa. Some groups of these, such as the Metchnikovellidae and endozoic Suctoria, are known only from hosts of this phylum. Only a few Metazoa occur as parasites in Protozoa. Although the relative size relationships are sometimes such as to make it possible, parasitization by these higher forms is not less prevalent than might be expected, in the light of the infrequency, in general, of intracellular parasitism by Metazoa. Cyanellae, chlorellae, and xanthellae have not been included in this account of symbionts of Protozoa. The last two types, at least, are widespread in members of many free-living groups, the former in fresh- water forms, the latter in marine species. Although inhabiting the cytoplasm, the nutritive processes of these organisms are autotrophic; and they are not necessarily dependent on the host. Their relationship to their hosts is often cited as mutualistic, in one way or another. The problem is in part the same as that of the relationship of similar endo- symbionts to many invertebrate animals—a problem that has been rfe- viewed by Buchner (1930). Pascher (1929) discussed the endosym- biosis of blue-green algae in Pawlinella chromatophora and some other Protozoa, as well as in algae. Lackey (1936) described ‘blue chromato- phores” in Paulimella and a number of flagellates, although he failed to recognize them as resembling Pascher’s cyanellae. Goetsch and Scheur- ing (1926) discussed the relationship of the alga Chlorella to proto- zoan and some metazoan hosts. The work of Pringsheim (1928) and others has sustained the thesis of mutualistic symbiosis between Chlorella and Protozoa. Most of the xanthellae have been placed in the crypto- monad genus Chrysidella. Hovasse (1923a) maintained on the basis of nuclear characteristics that xanthellae are dinoflagellates. EPIBIOTIC SCHIZOMYCETES SCHIZOMYCETES ON MASTIGOPHORA Bacteria attached to the surface, either by one end or applied full length, occur on many flagellates. They have been known for some PARASITES OF PROTOZOA 1011 time on Mastigamoeba aspera, small rods besetting the surface having been described by Schulze (1875) in the original description of this type species of the genus. He stated that these rods could best be com- pared with certain bacteria, such as Bacterzum termo. Most of them are applied full length to the body surface. Of other observers, some recog- nized the similarity of the rods to bacteria and others opposed this in- terpretation, but Penard (1905c, 1909) definitely established their bac- terial nature. The bacteria, he found, vary in number, but there are few individuals of the species without them. Lauterborn (1916) stated that a sapropelic flagellate, possibly belonging to the genus Mastv- gamoeba, possessed a yellow-green mantle of radially adherent chloro- bacteria, which he named Chlorobacterium symbioticum. These are the only free-living flagellates, to the writer’s knowledge, on which bacteria have been reported. Many endozoic flagellates bear Schizomycetes. Grassé (1926a, 1926b, and elsewhere) has done much to increase our understanding of those microdrganisms, which many earlier observers had mistaken for pellicular differentiations, cilia, or flagella. Duboscq and Grassé (1926, 1927) found short rods adherent by full length on many specimens of “Devescovina’’ hilli, showed that these are bacteria, and named them Fusiformis hilli; and they also found spirochetes, named Treponema hilli, adherent to all parts of the body sutface. The former report of the rods first established the true nature of the “‘striations,’ which Foa (1905), Janicki (1915), and Kirby (1926b) had described on Devescovina. In many instances the presence of certain microdrganisms is character- istic of the species, and may indeed be considered, together with its morphological characteristics, as an aid in taxonomy. Simpler phoretic relationships, in which the presence of the adherent forms is only occasional, do, however, exist between microdrganisms and some flagel- lates. Examples are the rod-like bacteria occasionally adherent, full length, to Hexamastix claviger (Kirby, 1930); the bacteria sometimes present on the larger forms of Tricercomitus termopsidis (Kirby, 1930); the occasional spirals and rods on Emtrichomastix trichopterae, E. colubro- rum, and Octomitus intestinalis (Grassé, 1926b) ; and the lumen-dwelling types of bacteria and spirochetes often adherent sporadically to various termite flagellates. Boeck (1917) stated that rod-shaped bacteria at times, in certain preparations, covered the body and adhered to the flagella of Giardia microti. 1012 PARASITES OF PROTOZOA Fusiformis-like Rods Adherent Full Length—The genus Fusiformis has the type species F. termitidis Hoelling, 1910. This organism is gen- erally free in the lumen of the hind-gut of termites. It was originally reported in Coptotermes sp. of Brazil, has been observed by the writer Figure 208. Fusiformis-like rods adherent to the surface of flagellates. A, B, Fusiformis grandis and F. melolonthae on Polymastix melolonthae; C, F. melolonthae. on P. melolonthae; D, F. legeri on P. legeri; E, F. lophomonadis on Lophomonas striata; F-J, rod-shaped microédrganisms on Devescovina sp. from Neotermes dalbergiae. (A-E, after Grassé, one 926b; F-J, original.) in C. niger of Panama, and was found by Duboscq and Grassé (1926, 1927) in Glyptotermes ividipennis, where it occasionally adheres to “Devescovina’ hill1, Chromatic granules number from one to sixteen, according to Hoelling’s account; Hoelling identified these with nuclei. In the form studied by the writer in C. m7ger, the chromatic granules are well defined, usually extend the full width of the cell, and in most PARASITES OF PROTOZOA 1013 cases number from one to four. Fusiformis hilli, although much smaller, is similar in shape and contains one or two chromatic granules that occupy the full width. The bacteria were also observed in transverse fission, the occurrence of which, together with the structure, readily distinguishes them from pellicular striations. Ecologically, Fasiformis hilli is closer to F. termitidis than to the “pellicular striations” on species of true Devescovina. It is abundant in the lumen of the intestine, as well as on the surface of Crucinympha hilli, which is not true of the “‘striations.” On the surface it is arranged in a manner scarcely suggesting striations; it sometimes adheres by one end, and it is inconstant in occurrence. Kirby (1938b) noted that of 100 Crucinym pha, 78 had no adherent rods, or very few. Later in the same year that Fuséformis hilli was reported, Grassé (1926a) described F. grandis and F. melolonthae on Polymastix melo- lonthae (Fig. 208A, B, C,); F. legeri on Polymastix legeri (Fig. 208D); and F. lophomonadis on Lophomonas striata (Fig. 208E). F. grandis and F. legeri adhere by one extremity. This article, and the more extended account by Grassé (1926b), first established the true nature of the microdrganisms that adhere by full length to Polymastix and Lophomonas, which had been regarded as pellicular structures. The writer has observed Fus/formis-like microdrganisms on many polymastigote flagellates of termites. They are present on all the twenty species of Devescovina, so constantly that no individual lacks them (Fig. 208A; Fig. 209F-J). On all except D. elongata, the rods appear almost identical with F. lophomonadis of Lophomonas striata, both in their morphology and their morphological relationship to the host flagellate. They are evenly spaced, generally occur over the whole body except the papilla, and are usually situated closer together than Grassé (1926b, Pl. 15) indicated for F. /ophomonadis. They always adhere firmly by the full length, and are not subject to detachment in the ordinary course of technical manipulations. On smears in which the cytosome of the flagellate has been much disturbed, they may have been partly or com- pletely detached, and can then be conveniently studied. Specimens sub- jected to such treatment may sometimes be bent considerably, as was noted by Grassé (1926b) in F. melolonthae. Surface microérganisms were absent from many specimens of Devescovina lemniscata of Neo- termes insularis that had been fed on filter paper soaked previously in 1014 PARASITES OF PROTOZOA 10-percent acid fuchsin, but were never absent from normal material. Presumably the treatment had brought about detachment of many of the bacteria. Generally these slender microdrganisms appear homogene- ous, but chromatic granules can be demonstrated by suitable methods. Similar microorganisms have been found by the writer on three of the five species of Caduceia. On C. nova and C. theobromae (Fig. 209C), they are short, slender, and are confined to a limited, sharply bounded area at the posterior end of the body (Kirby, 1936, 1938a; x ww A Bh gi ILI Many Wiig UL ETT vs (1 YM iy i (CLL MLL sua LM WOU MM yyy i UL l His) iis ; i, Wag nh TINTS x iY ¢ Miah WW G14 Figure 209. Adherent microérganisms on flagellates of termites. A, Fus/formis-like rods on Devescovina sp. from Glyptotermes niger; B, regularly arranged rods on Caduceia sp. from Neotermes greeni; C, investment of spirochaetes and posteriorly localized rods on Caduceia theobromae. (A, B, original; C, after Grassé, 1938.) Grassé, 1937, 1938). On a species of Caduceia from Neotermes greent, they are abundant on the entire surface, except the papilla; and they show a tendency to arrangement in transverse bands, between which, in many specimens, there is regularity in spacing (Fig. 209B). On other genera of Devescovininae from termites, the striation-like bacteria are altogether absent. None have been found in Foaina, on most species of Metadevescovina, on Pseudodevescovina, or on Macro- trichomonas, except for an occasional, irregular occurrence on the sur- face, in a manner comparable to the situation in Cracinympha hill. The absence is the more striking because of their universality on Devescovina and certain species of Caduceia. PARASITES OF PROTOZOA 1015 Rods adhere also to certain hypermastigote flagellates. They were noted by Kirby (1926a) on Staurojoenina assimilis, but were wrongly regarded as pellicular striations; and Cleveland ef al. (1934) found them on Barbulanym pha, Rhynchonympha, and Urinympha of Crypto- cercus punctulatus. Cleveland considered them to be cuticular striations, but noted their resemblance to the adherent bacteria described by Duboscq and Grassé (1926, 1927). Grassé (1938) identified them as bacteria. Spirochetes and Rods Adherent by One End.—Spirochetes occur in great abundance in termites, mostly free in the lumen or attached to the lining of the hind-gut, but also adherent by one extremity to certain flagellates. It is not known whether this phoresy is obligatory or occasional from the standpoint of the spirochete, but the former condition is probable, at least in many instances. The presence of ad- herent spirochetes is especially characteristic of certain Pyrsonymphinae, Oxymonadinae, Devescovininae, and Calonymphidae among polymas- tigotes. Spirochetes are less frequent on hypermastigotes, but do occur on some genera (Holomastigotoides, Koidzumi, 1921; Spzrotricho- nym pha, Sutherland, 1933; Dogiel, 1922a; Spirotrichonym phella, Suther- land, 1933; Rostronympha, Duboscq, Grassé, and Rose, 1937). Cleve- land et al. (1934) did not report them on either polymastigotes or hyper- mastigotes of Cryptocercus. On many of these flagellates, spirochetes are invariably present, either distributed widely on the surface or localized on very definite areas of the body. Localization is illustrated by the distribution of spirochetes on Foaina nucleo flexa and Oxymonas grandis. On the former flagellate, spirochetes are always present on the anterior and posterior parts of the body; many of those on the anterior part are arranged in a row along the surface just over the parabasal filament (Fig. 210C). Grassé (1938) noted in another species of Foaina (mistakenly named by him Parajoenia decipiens) that an anterior tuft of spirochetes obeyed a fixed rule in its distribution. Localization is even more marked in Oxymonas grandis, which bears a dense group of spirochetes on an elongated, limited area at the base of the rostellum (Fig. 210A, B). The rest of the body surface of this flagellate is covered with minute epibiotic bacilli (Fig. 210A). Many observers have mistaken spirochetes for flagella or cilia in a 1016 PARASITES OF PROTOZOA wide range of flagellates of termites; and sometimes the same observer interpreted them correctly on one flagellate and wrongly on another, or even reached different conclusions concerning the filaments on differ- ent parts of the body of the same protozoan. The spirochetes have even Figure 210. Adherent microorganisms on flagellates of termites. A, B, small rods on surface and localized spirochaetes at base of rostellum of Oxymonas grandis; C, spiro- chaetes on the posterior part and on a localized area of the anterior part of Foaina sp. from Cryptotermes merwei. (Original.) been responsible for the erroneous classification of some polymastt- gote flagellates as hypermastigotes. Historical data on the interpretation of adherent spirochetes has been reviewed by Kirby (1924, 1926a); Duboscq and Grassé (1927); and Grassé (1938). Cleveland (1928) discussed their occurrence on flagellates. The spirochetes range in length from only 2 uy, the minimum for PARASITES OF PROTOZOA 1017 Treponema hilli of Crucinympha hilli, to 10 y, which is about the average length of those on species of Devescovina; and up to 20 un, as in the forms on Metadevescovina cus pidis of Kalotermes minor, or even 40 y in the longer species on Caduceia theobromae (Grassé, 1938). On Ste phanonym pha sp. from Neotermes insularis, long spirochetes of from 40 to 60 , are adherent (Fig. 211). Those of an investment or group are often comparatively uniform in length. On Psewdodevescovina uni- Figure 211. Spirochetes adherent to Stephanonympha sp. from Neotermes insularts. (Original. ) flagellata, which is completely covered with spirochetes, simulating a dense coat of cilia, the majority have a length of from 8 to 10 u. Caduceia theobromae is similarly covered by spirochetes from 4 to 6 y1 long, except for the area occupied by the above-mentioned Fusiformzs- like rods (Fig. 209C). The spiral of short spirochetes has only one or two turns. Two turns were counted in those of average length (9 to 13 1.) on Metadevescovina cus pidis, while in the longer ones, up to 20 1, there were three or four. 1018 PARASITES OF PROTOZOA The very long ones on the above-mentioned Stephanonympha had a much larger number of turns, but none like those have been seen on Devescovininae. Normally the spirochetes are in continual, very active flexuous move- ment. They are not rigid, like Spirillaceae, although some observers have compared them with spirilla. Their activity has been described by Koidzumi (1921) on Holomastigotoides hartmanni, Light (1926) on Metadevescovina debilis, Duboscg and Grassé (1927) on “Devescovina’’ hilli, Kirby (1936) on Pseudodevescovina uniflagellata, and by others. They can be studied best in living material by dark-field illumination. Their movements are not synchronized, and are uncodrdinated either in direction or activity. The difference between this movement and that of cilia or flagella has impressed all students who have observed it. As noted by Kirby (1936), they may move at an equally active rate under the same environmental conditions, on moving flagellates, quiet flagellates, dead flagellates, and detached balls of cytoplasm. This activ- ity, together with their form, readily distinguishes them from flagella; but distinction is less easy in fixed material, in which the form is often less evident. The spirochetes do not, in the writer’s experience, detach readily in preparation of smears. Grassé (1938) stated that certain flagellates lacking spirochetes may have lost them in consequence of fixation, but offered no proof that this occurs. Spirochetes of P. wniflagellata were ob- served to be rubbed off by movement of the large flagellate in close contact with the cover glass, and severe manipulation might cause their loss; but that treatment is more drastic than would ordinarily occur in the preparation of specimens. The spirochetes can be removed, however, by relatively simple meth- ods. Light found that treatment with iodine in 70-percent alcohol freed the bodies of most Metadevescovina debilis of spirochetes. Cleveland (1928) discovered that all the spirochetes could be removed, both from the surface of the Protozoa and the lumen of the gut, by feeding the termites on cellulose thoroughly moistened with 5-percent acid fuch- sin. That method was used by Sutherland (1933) to remove spirochetes from Spirotrichonymphella and Stephanonympha; and it has been used in the study of many Devescovininae by the writer. Feeding Kalotermes hubbard: for twelve days on filter paper moistened in 5-percent aqueous PARASITES OF PROTOZOA 1019 acid fuchsin removed the attached spirochetes, and examination by the dark-field method showed that the tertiary flagella described by Light (1926) were absent, proving that they also were spirochetes. The spirochetes of Psewdodevescovina of Neotermes insularis were not re- moved by this method, however, showing that it cannot be depended upon as always effective. Rods of types other than the longitudinally adherent bacteria are less frequent than spirochetes. They occur occasionally on devescovinids, adherent by one end; have been found abundant on Proboscidiella kofoid: (Kirby, 1928), Joenia annectens (Franca, 1918), Oxymonas dimorpha (Connell, 1930), and Microrhopalodina enflata (Duboscq and Grassé, 1934). Occasionally there are also long filamentous organisms, which occur, for example, among the spirochetes on Metadevescovina debilis. Microérganisms adherent by one end to flagellates of termites some- times seem to be actually embedded in the ectoplasm, or to be associated with cytoplasmic differentiations. This was described by the writer (1936) in Pseadodevescovina uniflagellata, and it was noted that the apparent embedded part may stain more deeply than the rest and appear thicker (Fig. 212C). In P. ramosa (Kirby, 1938a) and P. punctata (Grassé, 1938), there are bacteria in the ectoplasm not directly associated with the spirochetes, but in P. wniflagellata the apparent granules are not of the same nature. Grassé (1938) made similar observations on adherent spirochetes of Caduceia theobromae, and interpreted the thick- ening not as part of the spirochete but as a modification of the cytoplasm in reaction to the microdrganism. Rounded corpuscles associated with the point of attachment of spirochetes on Parajoenia grassii wete de- scribed by Janicki (1915) and by Kirby (1937). These bodies seem to be cytoplasmic structures, neither part of the spirochetes nor parasites. A notable instance of a cytoplasmic differentiation, associated with the point of adherence of a microdrganism, was seen by the writer in a species of Macrotrichomonas in Procrytotermes sp. from Madagascar. Rods 2.5-7 4 > Y4-Y% py adhere in large numbers to the posterior part of the body of almost all specimens. Where each of the rods meets the body is a deep-staining, cup-shaped structure (Fig. 212D, E). Rods frequently become detached from these cytoplasmic structures, not being so firmly adherent as are spirochetes. It has been observed in several devescovinid flagellates with complete 1020 PARASITES OF PROTOZOA investment of short spirochetes, that pockets may be formed inward from the surface, enclosing some spirochetes. This was noted by Kirby (1936) in Pseudodevescovina uniflagellata, and by Grassé (1938) in Caduceia theobromae and P. punctata. Grassé noted further that the pockets in the former species may become closed, so that spirochetes are enclosed SSSA oer Tg A RENNIN ay ad ‘= ne ed = Ly 4 Ly Figure 212. A, spirochetes adherent to Trichodinopsis paradoxa; B, microérganisms on the capitulum of the axostyle of Macrotrichomonas pulchra from Glyptotermes dubius ; C, adherent spirochetes on Pseudodevescovina uniflagellata, with enlargement in ecto- plasm at point of adherence of each; D, E, rods adherent to Macrotrichomonas sp. from Procryptotermes sp., showing the cup-shaped structure in the ectoplasm at the end of each rod. (A, after Cépéde and Willem, 1912; B-E, original.) in vacuoles in the cytoplasm. He believed also that the external, fusiform bacilli may at times enter the cytoplasm and be digested; but this opinion may have been based on the presence of an intracytoplasmic symbiont, which actually is quite different, as was noted in Caduceia nova and C. theobromae by the writer (1936, 1938a). The possible physiological relationship between the adherent spiro- chetes and their flagellate hosts has been considered by Cleveland (1928) and Grassé (1938). Cleveland thought at first that they might live in some sort of mutualistic relationship; but he found that when the spiro- PARASITES OF PROTOZOA 1021 chetes were removed, no apparent detrimental effect on the Protozoa developed within three months. Sutherland (1933) found that Stephano- nympha died after detachment of the spirochetes, while Spirotrichonym- phella showed no impairment; but other factors may have been respon- sible for the death of the polymastigote. The writer found a marked reduction in size of Metadevescovina debilis after spirochete detachment, but the relationship was not proved. Grassé (1938) concluded that the relationship with the host is at least not simple phoresy. He discussed the possibility that diffusing substances attract and nourish the spiro- chetes, and that localization in certain regions may be related to certain areas of greatest diffusion, or to the chief phagocytic and absorptive areas. Schizomycetes on Sarcodina.— Lauterborn (1916) found Amoeba chlorochlamys, a sapropelic /imax type of rhizopod, to be characterized by a yellow-green mantle of close-set, radially adherent chlorobacteria. These bacteria, which he named Chlorobacterium symbioticum, were rods about 2 ,, long. When the amoeba was inactive, the mantle com- pletely surrounded it; in activity it opened more or less before advanc- ing pseudopodia. As stated above, Lauterborn found the same micro- organism on a colorless, sapropelic flagellate. It is probable that a similar mantle of bacteria is present on D/- namoeba mirabilis. Leidy (1879) described the surface of the body as “bristling with minute spicules or motionless cils.’’ In the majority of specimens he found a thick investment of hyaline jelly, at the surface of which were abundant, minute, perpendicular rods, termed by Leidy “bacteria-like cils.’’ The rods covering the body surface were some- times absent; and Leidy recorded instances of their disappearance from individuals that possessed them when first observed. Schizomycetes on Ciliophora.—Certain schizomycetes occur in speci- fically phoretic relationship to a number of ciliates and suctorians. The instance earliest known was the adherence of spirochetes to Trichodi- nopsis paradoxa, a peritrich in the intestine of Cyclostoma elegans (Fig. 212A). Earlier authors described this as possessing, unlike other peri- trichs, a general investment of vibratile cilia (Issel, 1906). Issel found basal granules for the supposed cilia; according to Pellissier (1936), these are mitochondria disposed under the pellicle. Observations on microorganisms adherent to Devescovininae (p. 1013) suggest another explanation. Fauré-Fremiet (1909) noted that the filaments have an ¥O22 PARASITES OF PROTOZOA undulatory movement unlike that of cilia, have an uneven distribution, and may quit the host entirely. They differ in staining from true cilia, and their movement remains unmodified, even though the ciliate be crushed. Fauré-Fremiet considered them to be spirilla, but later ob- servers (Cépéde and Willem, 1912; Bach and Quast, 1923; Pellissier, 1936) recognized them as spirochetes. Bach and Quast reported spiro- chetes also in the gut lumen, but found them present only when Tricho- dino psis was also present. Collin (1912) recorded a number of instances of the presence of adherent mircodrganisms on Suctoria. Short, rod-shaped bacteria, many of them in division, were shown adherent full length, in a close-set investment, on Discophrya lyngbyei (Collin, 1912, his Fig. 17). Schizo- phytes adherent by one end, often obviously simple phoretic micro- organisms with no closer relationships, were found on the lorica of Acineta tuberosa and on the tentacles of Choanophrya infundibulifera. Bacteria were adherent in a gelatinous investment on the surface of several species of Paracineta. More recently, especially through the work of Kahl (1928, 1932), the presence of characteristic types of rod-shaped bacteria on the sur- face of certain marine ciliates—chiefly sapropelic—has become known. The rods adhere either by one end, as on Parablepharisma pellitum (Fig. 213G), P. collare (Fig. 213H), Metopus contortus var. pellitus (Fig. 213F), and the stalk of Epzstylis barbata (Fig. 213E); or flat, as on Cristigera vestita (Fig. 213C, D), C. cirrifera (Fig. 213B), Blepha- visma vestitum, Parablepharisma chlamydophorum (Fig. 2131), and species of Sonderia. The presence of adherent bacteria is characteristic of all members of the genus Sonderia. Many of these ciliates are covered by a gelatinous layer, and it is to this that the bacteria adhere (Fig. 213A). Yagiu (1933) and Powers (1933, 1935) found bacilli con- stantly adherent longitudinally to the surface of three species of Cyel7- dium from sea urchins, a different type on each species (Fig. 214). Kirby (1934) believed the protuberances of Metopus verrucosus (Fig. 213]), which are irregular in distribution, to consist of groups of vertically ad- herent bacteria. Kahl questioned this statement, but in view of what we now know of bacteria on flagellates and ciliates it is not improbable. Kahl (1933) maintained that the adherent bacteria are advantageous symbionts, contributing somehow to the nutrition of their hosts; but that = =< |S Figure 213. Bacteria adherent to ciliates, A, bacteria in the gelatinous investment of Sonderia pharyngea Kitby (= S. schizostoma Kahl ?); B, surface bacteria on Cristrgera cirrifera Kahl; C, D, bacteria on Cristigera vestita Kahl; E, bacteria on stalk of Epistylss barbata Gourret and Roeser; F, vertically adherent bacteria on Metopus contortus vat. pellitus Kahl; K, on Parablepharisma pellitum Kahl; H, on Parabl. collare Kahl; I, Parabl, chlamydophorum Kahl, with Ja, longitudinally adherent bacteria; J, vertical rodlets, possibly adherent bacteria, on Metopus verrucosus (da Cunha) (Spirorhynchus ver- rucosus da Cunha). (A, J, after Kirby, 1934; B-D, after Kahl, 1928; E, after Kahl, 1933 from Gourret and Roeser; F-I, after Kahl, 1932.) 1024 PARASITES OF PROTOZOA is only speculation and is, in fact, improbable. Kahl stated that it seemed to him inconceivable that this symbiosis is without advantage to the host. Dogiel (1929) observed Sarcina-like bacterial epiphytes on Didesmis ovalis. These formed a group of regularly quadrangular form, in definite number and arrangement, and were located in a preferred place on the body surface. Other epiphytes, regarded as being probably bacteria, were t 1400 4a eA) & w WS =" G—= x4 | am y, Figure 214. Characteristic bacteria adherent to the pellicle of Cyclidium from the intestine of sea urchins. A, C. rhabdotectum Powers; B, C. ozakii Yagiu; C, C. stercoris Powers. (After Powers, 1935.) found on two species of Diplodinium. These had the form of an elongate oval body, attached by a stalk to the pellicle. They were found also free, ophryoscolecids being only an accidental substrate. ENDOBIOTIC SCHIZOMYCETES The relationship between Protozoa and bacteria that live in the cyto- plasm or, less frequently, the nucleus, is closer than that of the surface forms. The bacteria must obtain all their nutriment from the host, in the body of which they multiply. The association is sometimes a con- stant one, the host seldom, or never, being found without the customary mictodrganisms. These are then probably not detrimental to the host, PARASITES OF PROTOZOA 1025 but have come to occupy a normal place in the metabolism of the combination. Other bacteria occur as occasional endobionts, present in a variable percentage of hosts; often there are certain types that are more or less likely to occur. Some of these are not noticeably detri- mental to the maintenance by the host of normal activity. At the other extreme are some that cause fatal diseases; this is true especially of the nuclear parasites. ASSOCIATIONS OF A CONSTANT CHARACTER Instances in which endobiotic bacteria are always or at least usually present are known chiefly in flagellates of termites and amoebae of the genus Pelomyxa. Doubtless there are many other such associations among Protozoa. Miyashita (1933) found the occurrence of abundant rod-like bacteria in the cytoplasm of Ptychostomum (Hysterocineta) bacteriophilum to be characteristic of the ciliate; and similar rods were seen by Studitsky (1932) in the endoplasm of P. chattoni. Flexuous rods from 8 to 20 y long were observed by Chatton and Lwoff (1929) in all specimens of Ellobiophrya donacis, but not in the mantle cavity of the lamellibranch host of the peritrich. Furthermore, the rods often showed division and were never corroded; so that these authors con- cluded that the microdrganism is a specific symbiont. Schizomycetes in Pelomyxa.—So characteristic are bacteria in Pelomyxa that Penard (1902) designated the genus as always provided with symbiotic bacteria. Greeff (1874), who observed them in Pelomyxa palustris, considered them to be crystals, and he had at first held them to be seminal threads. F. E. Schulze (1875), though noting their similarity to bacteria, agreed with Greeff that the rods are peculiar structures of the Pelomyxa body. Leidy (1879) observed rods in his Pelomyxa villosa (which species, according to Penard [1902], repre- sents nothing in reality, the name having been applied to an aggregate of several species of Pelomyxa), and noticed, as had Greeff, that many appeared to be transversely striated. Bourne (1891) identified the rods as bacteria, and Penard (1893) expressed the same opinion as to their nature. Penard (1902) described the bacteria as having a length of from 10 to 15 y, or sometimes 20 1; and in one individual there were rods of from 40 to 50 . Leiner (1924) found the rods varying in length 1026 PARASITES OF PROTOZOA from 1.5 to 22 y (Fig. 215B). Penard found that all the rods were divided by equidistant transverse partitions, usually into two or three sec- tions. Gould (1894) (Fig. 215A) noted their division into from two to sometimes as many as nine sections; and later (1905), under the name of Veley, she observed transverse fission of the rods. Veley stated that when the rods are set free from the cytoplasm, they are capable of in- dependent movement, of a kind associated with the presence of flagella, Figure 215. Bacteria (Cladothrix pelomyxae Veley, and a small species) in Pelomyxa palustris Greeff. (A, after Gould, 1894; B, after Leiner, 1924.) though none were demonstrated. This movement was at first rapid, and could readily be distinguished from Brownian movement. Leiner ob- served no independent movement. Attempts to cultivate the rods have given inconclusive results. Though Veley (1905) cultured in sheep’s serum, inoculated from washed Pelomyxa, rods which she considered to be identical with those in the cytoplasm, Leiner (1924) concluded that the amoebae cannot be cer- tainly freed of foreign bacteria, and that the sources of error are too great. Veley (1905) named the organism Cladothrix pelomyxae, noting its resemblance to the two existing species of Sphaerotilus, of which genus, according to Buchanan (1925), Cladothrix may probably be regarded as a synonym. The rods are generally aggregated in proximity to the nuclei and refractile bodies (Fig. 215A). Penard (1902) noted that in certain PARASITES OF PROTOZOA 1027 individuals of Pelomyxa vivipara, all the nuclei are enveloped by close- set bacteria applied to the surface. Leiner stated that in P. palustris bac- teria may thickly invest the nuclei, especially in animals in which the refractile bodies are small. Veley observed jointed rods attached to the refractile bodies, and believed that these afford them a point of attach- ment without which the cycle would not be completed. She thought it probable that the refractile bodies, which she considered to be protein in nature, serve the bacteria as a food supply. Leiner found evidence that the long rods extract glycogen from the refractile bodies. Fortner (1934) studied the occurrence of bacteria in different forms of P. palustris. The large, club-shaped, gray-greenish forms were free of, or poor in bacteria. The yellow ones contained very numerous small refractile bodies, with large numbers of bacteria in proximity to these. The small, spherical or pyriform, milky-white type contained no refractile bodies, in place of which were the characteristic bacteria in vacuoles. The white forms he believed to be degenerate, and thought it conceivable that the whole metamorphosis of Pelomyxa might be conditioned by the bacterial infection. Leiner (1924) also noted the variability in the number of the rods and their abundance in yellow animals. He distinguished a second species of parasite, smaller and less numerous than the other, distributed in the cytoplasm (Fig. 215B). Leiner found reason to believe that when the bacteria are excessively abundant, they become definitely injurious to the host. They may cause hypertrophy, structural alteration, and eventual dissolution of the nuclei; and the trophic functions of the cell appear to be disturbed. There is decreased storage of glycogen. When Pelomyxa dies, the bacteria multi- ply extraordinarily. Schizomycetes in Flagellates of Termites ——There are numerous in- stances of a constant association with intracytoplasmic bacteria among flagellates of termites, and often the microdrganisms are restricted to specific areas of the cell. There is not positive proof in all instances discussed below that these are bacteria; but reaction to certain fixatives, staining properties, comparison with known cytoplasmic inclusions, and, frequently, observation of fission stages make it extremely probable. There is no evidence that the bacteria are harmful to the flagellates, though it is possible that as regards certain ones some such evidence may eventually be adduced. They may be referred to as intracellular 1028 PARASITES OF PROTOZOA symbionts with as much justification as may the microdrganisms in the bacteriocytes and mycetocytes of certain insects. Pierantoni (1936) pro- posed a hypothesis concerning the function of bacteria in flagellates of termites. According to this hypothesis, with which Grassé (1938) ex- pressed agreement, the bacteria function in the xylophagous nutrition of the flagellates. The flagellates, which are unique among Protozoa in their xylophagous habits, are, like so many wood-ingesting animals, incapable of digesting cellulose, but depend upon symbiotic bacteria. The bacteria are sometimes localized in “symbiotic organelles,” some- times diffusely distributed in the cytoplasm. The hypothesis rests on grounds similar to that of the supposed réle of the intracellular symbionts in insects—the constant association and frequent localization suggest the likelihood of a fundamental significance in the relationship. The weakness of both hypotheses is patent; there is no physiological evi- dence in their support. Furthermore, the existence of “organelles” of the type mentioned is exceptional in xylophagous flagellates. It is probable that the so-called chromidial zone of Joenia annectens is a bacterial aggregate. It occurs constantly in that hypermastigote, and has been shown or described by all students of the flagellate (Grassi and Foa, 1904, 1911; Franga, 1918; Duboscq and Grassé, 1928, 1933, 1934; Cleveland et a/., 1934; Pierantoni, 1936). According to Duboscq and Grassé, the bodies in this group are rods. The group surrounds the axostyle posterior to the nucleus, often forming a broad ring. Duboscq and Grassé (1934) figured an instance in which they form a spherical group, not encircling the axostyle, in a nondividing flagellate; and ap- parently in division stages they disperse. In the figures presented by these authors there are forms of the small rods that might be interpreted as division stages. Grassi and Foa (1911) concluded that the area they earlier called the chromidial zone acts as a phagocytic organ, since in experimental feeding they found granules of carmine included in it. Duboscq and Grassé regarded the rods as mitochondria, but their reaction to fixatives and stains rather suggests bacteria. They are resistant to fixatives after which true mitochondria are not demonstrable. Pierantoni considered them to be bacteria, and showed differential staining of them and mito- chondria in Altmann-Kull. The writer has observed a comparable aggregate of granules or rods PARASITES OF PROTOZOA 1029 around the axostyle of Devescovina glabra a short distance posterior to the parabasal body. It is conspicuous in the species in several hosts, in some of which it has been found in all specimens from several colonies. Usually the limits of the group are well defined, and it has the form of a thick ring around the trunk of the axostyle. The bodies are generally short rods or granules; in some specimens from one host the group consisted of long rods. Their properties of fixation and staining exclude the possibility that they are mitochondria. In division stages the bacteria are dispersed. Most species of Devescovina have no such bacterial aggregate. Pierantoni found no “‘chromidial zone” in Mesojoenia decipiens, but a similar area was occupied by filamentous bacteria. Kirby (1932a) described as a “proximo-nuclear parasite’ a group of bacteria, usually rod-formed or filamentous and often bent or curved (Fig. 219A), located iN a mass surrounding, or in proximity to the nucleus of all specimens of Trichonympha campanula and T. collaris. The suggestion was made that the organism may depend for its nutrition upon immediate proximity to the source of the nuclear influences upon metabolism. The peripheral granules located in the outer zone of the ectoplasm of the flagella-bearing region of T. collaris and T. turkestanica (Kirby, 1932a), which stain with the Feulgen reaction and show stages of appar- ent division, are probably localized bacterial symbionts. They have not been found in other species of Trichonympha. Ectoplasmic granules, considered by the authors to be bacteria and constant in occurrence and distribution, were described by Kirby (1938a) as immediately under the surface layer of Psewdodevescovina ramosa, and by Grassé (1938) as in a similar position in P. punctata. Similar peripheral bacteria occur in Bullanympha silvestrii (Kirby, 1938b). Grassé found minute bac- teria more deeply situated under the surface of P. brevirostris. Kirby (1932a) discussed the similarity between the peripheral granules of Trichonympha and those described in certain ciliates. A still more remarkable localization is that of certain bacteria which occur on the capitulum of the axostyle. Not all granules that occur in that location are held to be bacteria, but some seem definitely to be. Rods on the capitulum of Pseudodevescovina uniflagellata were described by Kirby (1936) as probably bacteria. These were present in a large percentage of specimens, but not in all; and in only a very 1030 PARASITES OF PROTOZOA small percentage were they present also in the general cytoplasm, par- ticularly the ectoplasm. Rod-shaped microérganisms were found on the capitulum of Macrotrichomonas pulchra from Kalotermes contracti- cornis (Kirby, 1938a) and have since been observed in that species from other hosts (Fig. 212B). Their variability in number and the fact that they do not occur on all specimens, or on any specimens in certain hosts, are in agreement with the view that they are symbionts rather than structures of the flagellate. The greatly expanded capitulum of a large devescovinid flagellate from Neotermes insularis is constantly encrusted with short, stout rod- lets which show evidence of fission. In this remarkable flagellate there are similar rodlets in the peripheral cytoplasm, usually separated by a narrow or a broad bacteria-free band from the capitular group. Bacteria distributed generally in the endoplasm of termite flagellates are frequent and of many types. Some of them are practically constant in occurrence, as are the slender granule-containing rods in the endo- plasm of Caduceia nova and C. theobromae (Kirby, 1936; 1938a). Devescovinids often contain many deep-staining cytoplasmic granules which may be bacteria. Jirovec (1931b) found two kinds of bacteria vety abundant in the cytoplasm of every specimen of Trichonympha serbica he studied. They were not present in T. agzlis, of which T. serbica is probably a synonym, from termites in Spain; so probably they are either present or absent in flagellates of different termite colonies. Para- sites similar to one type (paired cocci) were found by Georgevitch (1929, 1932) in T. serbica. Pierantoni (1936) found minute bacteria present in large numbers in the cytoplasm of Trichonym pha minor and T. agilis, and he reported their occurrence to be constant. ASSOCIATIONS OF AN OCCASIONAL CHARACTER Schizomycetes in Mastigophora—Bacteria in the cytoplasm of flagel- lates of termites have been reported by Kirby (1924) in Dinenympha fimbriata, by Duboscq and Grassé (1925) in Pyrsonympha vertens, by Connell (1930) in Oxymonas dimorpha, by Connell (1932) in Gz- gantomonas lighti, by Powell (1928) in Pyrsonympha major, and by Kirby (1932a) in Trichonympha. Forms like the crescentic organism described by Connell have been seen by the writer in occasional speci- mens of a number of devescovinids. They are not stages of Sphaerita, PARASITES OF PROTOZOA 1031 as Connell suggested that they might be. Organisms with a peg form, differentiated sharply into a clear section at the broader end and a deep-staining section, are frequent in Stephanonympha (Fig. 216A). These have a general resemblance to the peg-formed parasite (Fig. 219D, E) in Trichonym pha cam panula (Kirby, 1932a), but are larger. A unique type is the spindle-shaped organism (Fig. 216B), up to 20 uy in length, with a deeply stainable area at one end, which occurs usually Figure 216. A, microérganisms in Stephanonympha sp. from Kalotermes jeannelanus ; B, spindle-shaped organisms in Caduceia sp. from Neotermes greeni, (Original.) in groups in a species of Caduceia from Neotermes greeni. In most termites the infection of Caduceia was only from 1 to 2 percent, but occasionally about half the flagellates were parasitized. With the larger forms were shorter, slender ones. The smallest forms were usually gathered into stout spindle-shaped groups, suggesting origin by splitting of a short, stout spindle. Many other bacterial parasites in termite flagel- lates have been studied by the writer. Spirochetes frequently invade the cytoplasm of flagellates of termites, 1032 PARASITES OF PROTOZOA especially when the insects are on a filter-paper diet, and have been observed in active movement. Some of these are in vacuoles, but often they lie directly in the cytoplasm. Their condition and activity suggest that many of them are not merely ingested as food, but invade the body as facultative parasites. Motile intracytoplasmic organisms were reported by Kirby (1932a) in Trichonympha from Zootermopsis. They were, indeed, recognized only in consequence of their relatively rapid movements, which left clear tracks in the granular prenuclear endoplasm. One was seen in a nucleus. Nothing is known of the detailed structure or the affinities of these parasites, which vary greatly in size. Abundant, vigorously vibrat- ing bodies were present in many specimens of Psewdodevescovina uni- flagellata, among the particles of wood. Sometimes these were sufficiently numerous to give an appearance of great activity in the cytoplasm. Like the parasite of Trichonympha, they have not been found in preserved material. Few reports have been published of bacteria in flagellates other than these of termites. Yakimoff (1930) named Micrococcus batrachorum (s7¢) a coccus-like form which occurred, grouped in masses of irregular form or isolated, in a small percentage of Trichomonas batrachorum. Dangeard (1902) named Caryococcus hypertrophicus a bacterial parasite of the nucleus of Ezglena deses, which caused a disease developing in great intensity. This was manifested by considerable hypertrophy of the nucleus, discoloration of plastids, and loss of the power of division. No figure of Caryococcus exists. As Dangeard had previously described Nacleophaga, it is probably distinct from that chytrid. Boeck (1917) found rod-shaped bacteria in the cytoplasm of Giardia microti in certain preparations. He considered these to be the same as those which oc- casionally adhered externally, and referred to the relationship as parasit- ism, leading to deleterious results. Schizomycetes in Sarcodina—A number of records exist of cocci and other bacteria in amoebae. Nagler (1910) found a heavy parasitic infection of amoebae, similar to Amoeba albida Nagler, cultivated on an agar plate. The cytoplasm was crowded with small, rounded, deep- staining granules of variable size, considered to be micrococci; and rod-formed and fusiform bacilli, likewise parasites, also occurred. Even- tually all the amoebae were destroyed, with swelling and degeneration PARASITES OF PROTOZOA 1033 of the nuclei. The author regarded the micrococcus as a facultative parasite, which multiplied in the body after ingestion. Mackinnon (1914) reported, in Entamoeba minchini (Léschia hartmann?) in tipulid larvae, a similar organism, which occurred in the nucleus as well as in the cytoplasm; and she stated that Polymastix melolonthae and Mono- cercomonas melolonthae are also infected. Micrococcus was reported by Dangeard (1896) in Sappinia and by Wenyon (1907) in Entamoeba muris. Granules filling the cytoplasm of Mastigina hylae, resembling in size, shape, and distribution the bodies called micrococci by others, were found by Sassuchin (1928b). He mentioned their resemblance to Chytridiales, but no sporangia were shown. Cytoplasmic granules like micrococci were described by Alexeieff (1912) in an amoeba, probably Lecythium sp., and in Tetramitus ros- tratus. He considered these, however, to be produced by multiplication of elementary corpuscles, existing as a corona of small granules around an initial corpuscle. The initial corpuscle was a small spherule with a central nucleus. He thought this to be a chlamydozoén, and named it Chlamydozoin biitschlii, Later Alexeieff (1929) described the same parasite in Monas vulgaris. The evidence, however, that these bodies in amoebae and flagellates are filtrable viruses and his speculations on the relationship of Chlamydozoa and Chytridiales are no more convinc- ing to the reader than his argument that the parasite of cancer is a chlamydozo6n, which he named (1912) Chlamydozodn perniciosum. The reports of Alexeieff are considered here because of the possibility that the granules described are bacterial parasites. The “initial cor- puscles’’ are inclusions of another nature. Mercier (1910) found bacterial filaments made up of short rods in Endamoeba blattae. Eventually they invaded the whole cytoplasm of a parasitized amoeba, which disintegrated. He also observed granular bodies, believed to be parasites, in the cytoplasm of cysts. Bacilli in masses in the cytoplasm of an amoeba, probably Vahl- kam p fia sp., grown on an agar plate, were considered by Epstein (1935) to be parasites. There were no indications of digestion of the bacteria, and the masses increased in volume. In consequence of the infection, division was supposed to be delayed; the nuclei continuing to multiply, several nuclei occurred in the hypertrophied bodies. This article does not carry conviction; the possibility is not excluded that the large 1034 PARASITES OF PROTOZOA multinucleate bodies occur in consequence of abundant nutrition, and it is not proved that the bacteria are parasites. Schizomycetes in Ciliophora and S porozoa.—Bacterial parasites of the nucleus and the cytoplasm have been observed repeatedly in free-living ciliates, and some of these have been studied more intensely than any other bacteria in Protozoa. They are of considerable interest, too, in the history of protozodlogy, having frequently come to the attention of stu- dents, in the latter half of the nineteenth century, and having been variously interpreted. J. Muller (1856) considered the question of spermatozoa in In- fusoria, on the basis of observations by himself and his students, Lieber- kuhn, Claparéde, and Lachmann. They observed undulatory filaments in the cytoplasm of Stentor (which were parasites, as discussed below), and found fine curved threads in hypertrophied nuclei (macronuclei) of Paramecium “aurelia’ (—caudatum) . Miller commented on the presence of these threads in what Ehrenberg had regarded as a seminal gland, but did not commit himself as to their real nature. Claparéde and Lach- mann (1857; 1860-61, p. 259) reported the observation of immobile rods in the nucleus of Chilodon cucullulus and in Paramecium. Believing that the nucleus plays an important réle in “embryo formation” (Sphaerophrya), they advanced the hypothesis that it may at certain times play the rdle in some individuals of a testis, in others of an ovary (1857). Stein (1859) observed hypertrophied macronuclei of Para- mecium “aurelia’ (—caudatum) containing fine, straight rods. He stated that he was at first inclined to regard individuals with such nuclei as males, the nucleus functioning as a testis and producing spermatozoa, while in others it had the rdle of an ovary. Later, he concluded that the spermatozoa developed in the nucleolus (micronucleus), then penetrated the nucleus, and he pointed out the analogy with fertilization in Volvox. Balbiani (1861) observed the rods in nuclei of the same species of ciliate, and noted, as had the others, that they were immobile. He con- cluded that they are parasites, which develop in the interior of the female reproductive organ, and pointed out fundamental differences between them and the ‘‘spermatic filaments’ enclosed in the “nucleolus” of this animal. It was then Balbiani’s contention, developed in 1858 (1858a, 1858b), that Infusoria are hermaphroditic sexual animals, the nucleus (macronucleus) an ovary, and the nucleolus (micronucleus) PARASITES OF PROTOZOA 1035 a testis (the chromosome filaments having been mistaken for sperma- tozoa). Engelmann (1862), finding these parasites in nuclei of Para- mecium caudatum and Blepharisma lateritia, nevertheless continued to regard them as spermatozoa; but Engelmann (1876) wrote of bacteria parasitic in nuclei of Stylonychia mytilus. Butschli (1876), severely criticizing the Balbiani-Stein hypothesis of sexual reproduction, reported rods in the nucleus (macronucleus) of P. aurelia, and agreed with Balbiani that they are parasites. He remarked that similar parasites are sometimes found in the nucleolus (micronucleus) of many Infusoria. A review of the earlier observations is given by Biitschli (1889, p. 1828). Balbiani (1893), giving a figure and a brief discussion of parasites in the macronucleus of Stentor polymorphus, remarked on the fact that the parasitized animal appeared to be perfectly normal and continued its ordinary functions. Zodchlorellae were neither more nor less abundant than usual. The alteration of the nucleus might seem to be equivalent to its artificial extirpation, as the nuclear substance had completely or almost completely disappeared. He discussed these facts in connection with his experimental results in the removal of the macronucleus, and concluded that they were not in disagreement. A minimal amount of the nuclear substance might still have been present and sufficed; or the animal observed might have been in the limited period during which normal life continues. Studies of the nuclear parasites of Paramecium and their effects on the animal were made by Hafkine (1890), Metschnikoff (1892), and Fiveiskaja (1929); and certain observations on the consequences of parasitism were reported by Bozler (1924). A similar parasite, studied in detail by Petschenko (1911), was believed to be cytoplasmic. This author stated that the possibility of confusing microérganisms which live in the micronucleus with those that live in the cytoplasm cannot be denied. Hafkine distinguished three species of Holospora. H. undulata in- vades the micronucleus and is spiraled. At the beginning the organism is a small, fusiform corpuscle. The second species, H. elegans, occurs also in the micronucleus, but is never associated with the first. The vegetative stage is fusiform and more elongated and slender than the others. The third species, H. obtusa, invades the macronucleus. It 1s not spiraled and the two ends are rounded, instead of both or one 1036 PARASITES OF PROTOZOA being pointed. According to Hafkine, multiplication of Holospora takes place in two ways. During development, for a time there is a rapid transverse division. The spiral form of H. wndulata develops after di- vision ceases; the other two species remain straight. The dimensions in- crease in this phase, which was supposed by Hafkine to represent a transformation into resistant spores. H. undulata in this phase loses its pale, transparent aspect, and becomes more refractive at one of its extremities. This modification extends until the whole organism is re- fractive; sometimes it is divided into three or four parts of different refractivity. Of the other reproductive process, there are only traces in H. undulata; but it is more frequent in the other two species. A bud forms at one of the extremities, and grows into a cell like that from which it originated. This type of reproduction, according to Hafkine, makes Holospora transitional between yeasts and Schizomycetes. He maintained, as did Metschnikoff and Fiveiskaja, that there are grounds for not placing the parasite among the typical bacteria. Fiveiskaja studied only H. obtusa in the macronucleus, and did not find any parasitized micronuclei. She found the parasites to be elongated, straight, or slightly curved rods from 12 to 30 1 in length by 0.6 to 0.8 y in width, often showing differential refractivity or stainability. Part, up to half, or all, of a rod might be dark and the other part or other entire rods clear— a characteristic that appears in many of the early drawings (Butschli, 1876; Balbiani, 1893). Petschenko (1911), who studied an organism considered to be a cytoplasmic parasite, named it Drepanospira miilleri and assigned it to the Spirillaceae. He remarked that the external aspect of this organism is the same as that of Holospora undulata and H. elegans. The parasites develop from a group of curved rods in the cytoplasm to a large ellipsoi- dal mass, almost filling the body. In the vegetative period, the micro- organism is a spiral with a nuclear portion near the anterior end, and it shows helicoidal movement. The karyoplasm separates into granules and bands, and endospores are said to be developed. There is a resting period in which the rod is small, curved only once, and the nuclear substance occupies from half to almost all of the cell. There is said to be no cell division, reproduction being only by the endospores. Petschenko stated that the essential difference from Holospora lies in the fact that Hafkine did not establish in his parasites the presence of nuclear ele- PARASITES OF PROTOZOA 1037 ments; but it seems possible that the differences in refractivity noted by Hafkine may have indicated the same structure upon which Pet- schenko based his interpretation of nuclear organization. The latter author found micronuclei to be absent in ciliates with Drepanospira; so that, allowing for differences in detail of observation and interpretation, it seems not impossible that H. wndulata and D. miilleri are actually the same. Hafkine spread the infection by introducing infected paramecia into cultures. In early infection he noted no anomalies in the ciliates, but development of the parasites proceeds rapidly and by the next day all the contents of the infected nucleus are used up. When the parasite fills up a large part of the ciliate, development of the latter is arrested, and the same phenomena appear as in insufficient nutrition. Bozler (1924) and Fiveiskaja (1929) noted the vacuolization of animals in which the macronucleus was parasitized, the accumulation of fat drops and excre- tion granules, and the reduction and disarrangement of the trichocysts. Food-taking, digestion, and defecation continue for a long time, accord- ing to Fiveiskaja, but the number of food vacuoles formed becomes pro- gressively fewer. In late stages food currents are absent, there are no food vacuoles, and the mouth, gullet, and cytopyge may disappear. There is partial atrophy of the ciliary coat. At first there is no change in the activity of the pulsating vacuole; later the shape of the canals changes and the pulsations become slower. Eventually, with large masses of parasites, the pulsations stop and the ciliate dies. Petschenko found that the cytoplasmic (or micronuclear) parasite Dre panos pira miilleri causes the cytoplasm to take on an alveolar char- acter, and that the macronucleus undergoes degenerative changes and may fragment and dissolve. He noted that a chemical action on the cell is indicated, waste products and secretions of the parasite entering the cytoplasm of the cell. There is intoxicatiog of the cell, acting directly on the cytoplasm, indirectly on the nucleus. Bozler stated that the possi- bility that degenerative changes are to be traced back to the influence of a bacterial toxin is not excluded; and Fiveiskaja thought that the changes external to the nucleus were influenced by a substance excreted from the macronuclear parasites, probably a toxin. Destruction of chro- matin, with consequent disturbance of the normal macronuclear control of metabolism, together with the mechanical influence of large masses 1038 PARASITES OF PROTOZOA of parasites, may be sufficient, however, to account for the effect of Holos pora obtusa. Calkins (1904) recorded an infection of the macronuclei of 80 per- cent of Paramecium caudatum, in preparations from one culture, with a parasite that he named Caryoryctes cytoryctoides. The organisms appeared as scattered bodies in parts of each nucleus, and have no resemblance to Holos pora or Nucleophaga. Bacteria are especially prevalent in the cytoplasm of many ciliates that live in decaying matter, and Kahl (1930) stated that often these may be regarded as symbionts (mutualists), rather than parasites. He showed, for example, in Epalxis antiqguorum, \arge symbiotic bacilli which Penard (1922) had described as rods. Recently the occurrence and significance of bacteria in sapropelic ciliates has been the subject of studies by Lieb- mann (1936a, 1936b, 1937). Liebmann (1936a) found in Colpidium colpoda chlorobacteria that appeared to live as facultative symbionts. They were enclosed in vacuoles occurring more or less abundantly in the cytoplasm under conditions of anaérobiosis and H,S content of the water, but not in the presence of oxygen. Their appearance was definitely correlated with the amount of H,S. Similar chlorobacteria were present in the hay infusion from which the Col pidium came, and in normal oxygenated water these were ingested and digested. Under anaérobic conditions with H,S, they remained alive in the vacuoles; and Liebmann believed that through their assimilating activity in the presence of light, H,S is reduced in amount, and energy is contributed to the ciliate. After reserve glycogen is used up, the bac- terial vacuoles may be attacked by digestive processes, and the ciliates die soon thereafter. In many other sapropelic ciliates, Liebmann (1936b, 1937) found, together with dead bacteria, large numbers of living bacteria. These were either packed together in parallel arrangement in bundles (Mefopus) or distributed in the cytoplasm (Chaenia). After a time these symbionts may be digested, and the loss is made up by taking in new saprophytes. When this is prevented, and all symbionts are used in nutrition, the ciliates perish, in spite of filled food vacuoles. Certain living bacteria are therefore necessary for the ciliates’ continued life, under existing anaérobic conditions with hydrogen sulphide. In this connection Lieb- mann suggested that the bacteria split off oxygen, which the ciliates use. PARASITES OF PROTOZOA 1039 Hetherington (1932) mentioned an extensive cytoplasmic invasion of Stentor coeruleus by bacilli, the ciliates losing their bright blue-green color and some of their capacity for motor response. Pale stentors from mass cultures are, he stated, often infected with a great number of bacilli. Numerous instances of physiological regeneration occurred in the recovery of these animals. The report did not indicate whether the bacilli were isolated or grouped. In the cytoplasm of Spirostomum ambiguum, a motile spirillum was found in large numbers by Takagi (1938). He studied nine ciliates and found all infected, and considered it probable that all in the culture were parasitized. One hundred and six were present in one ciliate; in another, 10 of the 67 were undergoing binary fission. A flagellum was detected at one end of the parasite, which swam about actively in the cytoplasm. Takagi stressed the fact that his is the first report of a cyto- plasmic parasite with active motility in a protozoan. He did not com- ment, however, on the observations by Miiller, Claparéde and Lachmann, and Stein, nor on that by Kirby. Miller (1856) mentioned observations by himself, Lieberkithn, Claparéde and Lachmann of motile threads in Stentor; and the isolation of these by the last-named observers, when their motility soon disappeared in the water. Butschli (1889, p. 1831) discussed these observations in his account of parasites of ciliates. The threads occurred in the vacuoles in bundles, and displayed active movement. Claparéde and Lachmann (1857) thought their parasitic nature not improbable, noting their great similarity to certain vibrios. Butschli, while admitting that the threads might be ingested food, be- lieved it more likely that they were parasites. These forms differed from Takagi’s in being in bundles instead of isolated. Mangenot (1934) found rhodobacteria sufficiently abundant in a ciliate identified as Spirostomum teres to impart to it a rose color. They were distributed mostly in the peripheral cytoplasm. He regarded the relationship between them and their host as parasitic or symbiotic, and compared the “rhodelle’”’ association to that with chlorellae, xanthellae, and cyanellae. Irregular aggregations of minute granules (Fig. 217D) were found in the cytoplasm of many individuals of Nyctotherus ovalis by Sassuchin (1928a, 1934). He made various microchemical tests, excluding the possibility that these were glycogen or glycoprotein granules, chondrio- 1040 PARASITES OF PROTOZOA somes, or volutin; and he concluded that they were bacterial parasites, which do not occur in all ciliates. Similar groups of granules were re- ported by Kirby (1932b) in Nyctotherus silvestrianus. Bacteria were found by Hesse (1909) in monocystid gregarines from the seminal vesicles of oligochetes. Each of the species Monocystis lum briculi, M. agilis, M. striata, Rhynchocystis pilosa, and Stomatophora coronata had its own peculiar parasite which was unlike those of the others. Their forms varied, in different species, from ovoid to filamen- tous. Hesse remarked that the bacterial parasites were uncommon, but when present attacked most individuals of a species, and often led to the destruction of the invaded gregarines. SPHAERITA AND NUCLEOPHAGA HISTORICAL ACCOUNT AND DISTRIBUTION In Free-living Protozoa.—Most of the fungi of the order Chytridiales are parasitic in plants or animals (Fitzpatrick, 1930; Minden, 1915). In the lower plants they occur mainly on or in algae; and a considerable num- ber have been found in Phytomastigophora. Though most abundant in this group of Protozoa, they attack also other free-living forms, especially Sarcodina and cysts of ciliates (Biitschli, 1889; see also p. 1059), and many have been encountered in parasitic Protozoa. The chytrids that are known to be hyperparasitic in Protozoa all belong to the genera S phaerita and Nucleophaga. These are the chytrids, also, that have most often been found in free-living species, except for euglenid flagellates. Carter (1856) described “irregular, botryoidal masses, dividing up into spherical cells” in “Astasza’”’ (—Peranema). It is likely that he was observing Sphaerita, and that the enlarged granular nuclei described in Amoeba radiosa (?) were parasitized by Nuacleophaga. The specimens of A. verrucosa, “partly filled with spherical ovules in the granuliferous stage of development,” were probably heavily parasitized by chytrids. Wallich (1863a) found that a large subspherical, granular mass ap- peared in each of the specimens of A. villosa in a saucer; and later from five to a dozen of these masses developed in individual specimens. He observed extrusion and rupture of these, which he regarded as of the nature of nuclei. He evidently was describing an increasingly heavy infestation of a culture of amoebae by Sphaerita. The granulation of the nucleus described by Carter (1863) in A. princips, accompanied by PARASITES OF PROTOZOA 1041 the enlargement of the nucleus to between three and four times its normal diameter, indicates the presence of Nucleophaga. Greeff (1866) mistook the early plasmodial stages of Nucleophaga for young amoebae entering the nucleus of A. ferricola. Stein, who had mistaken parasitic bacteria for reproductive elements of ciliates (p. 1034), observed Sphaerita-like Chytridiales in a number of flagellates (1878, 1883). His plates include figures of what are probably such fungi in Monas guttula, Chlamydomonas alboviridis, Euglena viridis, Trachelomonas volvocina, T. hispida, Phacus pleuronec- tes, Tropidocyphus octocostatus, Anisonema grande (A. acinus), Gleno- dinium pulvisculus, Heterocapsa triquetra, and Dinopyxis laevis. In many of these he represented the escape of minute, flagellated organisms. He likewise concluded that these are reproductive elements, the nucleus undergoing growth and fragmentation, and giving rise thus to endoge- nous germs reproducing the flagellate. This theory of flagellate reproduc- tion was accepted by Kent (1880-82), who confirmed the observations of Stein on Ezglena and other euglenid flagellates. Ryder (1893) com- pared an “‘endoblast”’ figured in E. viridis, from which flagellate “‘germs”’ were said to escape and become amoeboid forms developing into adult euglenas, with Stein’s reproductive stage. Discussion of the early errors of interpretation is given in many of the publications on chytrid para- sites of Protozoa, particularly those of Dangeard (1886b, 1895), Penard (1905b), Chatton and Brodsky (1909), and Mattes (1924). The evidence for Stein’s notion of reproduction did not satisfy Klebs (1883), who pointed out that the ‘““Keimkugel” was a sporangium of “Chytridium spec.,” which, he stated, is one of the most frequent para- sites of Evglena. The problem was studied independently by Dangeard, and he arrived at the same conclusion (1886a, 1886b). The name Sphae- rita endogena was given (1886a) to cytoplasmic chytrids in the rhizopods Nuclearia simplex and a species of Heterophrys, which was later (1886b) named H. dispersa. The illustrated account of the parasite (1886b) in- cluded a report of its occurrence in Evglena viridis. Dangeard (1889a), recorded S. endogena in Phacus pyrum, Trachelomonas volvocina, and T. hispida; later (1889b) he described it in Euglena sanguinea and P. alata; and in 1895 he gave a fairly complete and well-illustrated descrip- tion of the life history of the parasite in Evglena (viridis?). Serbinow (1907) studied the chytrid in E. viridis and E. sanguinea. 1042 PARASITES OF PROTOZOA In more recent work, Sphaerita-like Chytridiales in free-living rhizo- pods and flagellates have been differentiated into several species, but a comparative account of the differential characteristics is lacking. Chat- ton and Brodsky (1909) proposed to give the parasite of euglenids, if separated from S. endogena, the name S. dangeardi; Skvortzow (1927) briefly designated as S. trachelomonadis a parasite of Trachelomonas teres var. glabra and T. swirenkoi in Manchuria; Jahn (1933) differentiated S. phaci from Phacus pleuronectes and P. longicauda, and Gojdics (1939) reported the same species from Exglena sanguinea. Puymaly (1927) failed to recognize S. dangeard/, describing the life history of a chytrid of E. viridis under the name S. endogena. Dangeard (1895), with no great positiveness, proposed the name Psewdosphaerita euglenae for a parasite of E. viridis in which, in the formation of the sporangium, there is fragmentation into islets, and the contour of the sporangium often becomes irregular and cord-like. Mitchell (1928) suggested assign- ment to Psewdos phaerita of a parasite, found in species of Evglena, which showed neither of these characteristics; Jahn (1933) considered at least those Mitchell described in E. viridis to be S. dangeardi. The parasites found by Nagler (1911b) in Evglena sanguinea ate Sphaerita-like; but the form in the cyst, with prominent protuberances, does not resemble Sphaerita. Mainx (1928) found Sphaerita often in E. sanguinea and E. viridis; Giinther (1928) reported it in E. geniculata. Further records of Sphaerita, by Alexeieff (1929), are from Monas vulgaris and Dimastigamoeba gruberi. Since Dangeard’s accounts (1886a, 1886b), Sphaerita in free-living rhizopods has been studied by Chatton and Brodsky (1909) in Amoeba limax, by Penard (1912) in A. alba, and by Mattes (1924) in A. 5 phaeronucleolus. The last observer described two new species, 5. amoebae and S$. plasmophaga. The confused and improbable cycle of Allogromia sp. (Cryptodifflugia sp., according to Doflein, 1909, 1911), outlined by Prandtl (1907), probably was based on a free-living testacean, certain small free-living flagellates, ingested Testacea, and an infection of A. proteus with Sphaerita. Prandtl discussed the observations on supposed reproduction by Carter (1863), Wallich (1863a), Greeff (1866), and even those of Stein (1878), which as stated above were based on para- sitization by chytrids; and he considered that they were really made on “gamete formation” by Allogromia or other parasitic rhizopods. A para- PARASITES OF PROTOZOA 1043 site described by Penard (1912) in A. ferricola appears to be a chytrid, but it is not like typical Sphaerzta. There seems to be only one record of Sphaerita in a free-living ciliate —the brief account of Cejp (1935) of the parasite in Paramecium, up to eleven sporangia occurring in a cell. Bodies like the sporangia of Sphaerita, but with exit tubes, were shown by Collin (1912) in Acéneta tuberosa. Chytrids in other Suctoria, found by Claperéde and Lachmann and by Stein, are mentioned below (p. 1064). Dangeard (1895) established the genus Nacleophaga for a parasite, N. amoebae (not amoebaea as Penard, 1905b, and Doflein, 1907, have it), which he studied in the nucleus of Amoeba verrucosa (A. proteus according to Penard, 1905b). Gruber (1904) found Nuwcleophaga in A. viridis, and supposed it to be different from Dangeard’s species, but according to Penard (1905b) it is probably the same. Penard described N. amoebae in A. terricola and A. sphaeronucleolus; and Doflein re- corded the parasite in A. vespertilio Penard. Mattes also found, in the nuclei of A. terricola and A. sphaeronucleolus, parasites which he named Sphaerita nucleo phaga. He believed that the forms of Penard and Doflein belonged to this same species, those of Dangeard and Gruber each being a different species. Although he did not comment on the relationship of the genera Sphaerita and Nucleophaga, his treatment of the chytrids seems to indicate that he regarded the latter as synonymous with the former. Indeed, no difference exists between the two except the habitat, and the basis for their separation seems scarcely valid. In Endozoic Protozoa—Because most of the studies of Chytridiales in endozoic Protozoa are comparatively recent, there have been few errors of interpretation. At about the time when Stein was describing germ balls in euglenid and other flagellates, Leidy (1881) observed what is clearly Sphaerita in Trichonympha agilis. He “suspected that they are masses of ova-like bodies or spores,” but discussed them as inclusions in the endosarc, not as reproductive elements. Casagrandi and Barba- gallo (1897) described nuclei in E. co// containing small, round bodies, equal in size, and sometimes so numerous as to fill the entire nucleus; and they figured several of them in certain vegetative amoebae (Pl. 2, Fig. 13). These, as suggested by Cragg (1919), were doubtless para- sites, probably Sphaerita, and not nuclear parasites; Cragg suggested also that the account by Craig (1911) of vegetative schizogony in this 1044 PARASITES OF PROTOZOA amoeba was based on parasites. The statement by Craig that the nuclei were visible in life as “brightly refractile masses of granules’ is in keeping with the probability that the supposed nuclei were Sphaerita. Dogiel (1916), finding sporangia of Nucleophaga in Myxomonas poly- mor pha, thought he was observing chromosomes. The same investigator noted what are probably Sphaerita and Nucleophaga in Joenia intermedia and recognized the former as one of the lower fungi, but he hesitated to interpret the latter as parasitic. Early students of Trichomonas (Wen- yon, 1907; Kofoid and Swezy, 1915; Kuczynski, 1918; Mayer, 1920; Wenrich, 1921) showed chytrids without interpreting them correctly. Sphaerita has been found in many parasitic flagellates, especially those in termites. Several species have been differentiated. Cunha and Muniz (1923) gave the name Sphaerita minor to a parasite of Trichomonas muris and T. gallinorum,; chytrids in Trichomonas vitali from Bufo marinus (Pinto and Fonseca, 1926), Trichomonas muris, T. caviae, and Eutrichomastix lacertae (Grassé, 1926b) have been assigned to the same species. Grassé also stated that parasites, probably Sphaerita, invade the plasma of Extrichomastix colubrorum. Sphaerita trichomonadis was described by Crouch (1933) from Trichomonas wenrichi of Marmota monax, and S$, chilomasticis by Cunha and Muniz (1934) from Chilo- mastix intestinalis. Sassuchin (1931) found Sphaerita-like parasites in Chilomastix magna of ground squirrels. In Mastigina hylae, Sassuchin (1928b) noted parasites that are Sphaerita-like in some respects, but are not shown grouped in sporangia; perhaps these are cocci. Among flagellates of termites, Sphaerita, which in no instance has been given a specific name, has been reported or figured in Joenia inter- media (Dogiel, 1917), Staurojoenina assimilis (Kirby, 1926a), Meta- devescovina debilis (Light, 1926), Trichonympha chattoni (Duboscq and Grassé, 1927), Stephanonympha dogieli (Bernstein, 1928), Coro- nympha clevelandi (Kirby, 1929), Oxymonas minor (Zeliff, 1930), Pyrsonympha and Dinenympha (Jirovec, 1931b), Pyrsonym pha elongata (Georgevitch, 1932), Gigantomonas lighti (Connell, 1932), and sev- eral species of Trichonympha (Kirby, 1932a). In undescribed poly- mastigote and some hypermastigote flagellates in the writer's collection, S phaevita has been found to be extremely prevalent. The presence of Sphaerita has been indicated in all species of intestinal amoebae of man: Entamoeba histolytica (Noller, 1921; Lwoff, 1925; PARASITES OF PROTOZOA 1045 Greenway, 1926; Bacigalupo, 1927, 1928), E. coli (Cragg, 1919; Nol- ler, 1921; Epstein, 1922; Lwoff, 1925; Bacigalupo, 1927, 1928), Endo- limax nana (Dobell, 1919; Noller, 1921; possibly Epstein, 1922; Green- way, 1926; Wenyon, 1926; Bacigalupo, 1927, 1928), lodamoeba biitschlii (Noller, 1921; Wenrich, 1937), and Dientamoeba fragilis (Noller, 1921; Wenrich, 1940). Lwoff (1925) thought that the parasite of Entamoeba dysenteriae (histolytica), E. coli, and Endolimax nana 1s identical with Sphaerita endogena; but he provided the name S. normeti for use if it is proved to be a new species. The chytrid is equally prevalent in other endozoic amoebae. Leger and Duboscg (1904) stated that an amoeba in Box boops (E. salpae) is often ravaged by microspheres which lead to its destruction. Wenyon (1907) noted the parasites in Entamoeba muris as ‘vacuoles containing cocci’; Kessel (1924) recorded Sphaerita from the same amoeba. Becker (1926) described S$. endamoebae from Entamoeba citelli, and the same chytrid was found by Sassuchin, Popoff, Kudrjewzew, and Bogenko (1930) in this amoeba of ground squirrels in Russia, though without reference to Becker’s account. A parasite of Hyalolimax cerco pitheci was named S, parvula by Brumpt and Lavier (1935b). Other records are from Endamoeba simulans of termites (Kirby, 1927), Entamoeba bobaci of Marmota bobaci (Yuan-Po, 1928), E. pitheci from Macacus rhesus (Sassuchin et al., 1930), and Entamoeba sp. of cattle (Jirovec, 1933). Third-degree parasitism is that of Sphaerita in entamoebae in Ze/lerzella, reported by Stabler and Chen (1936). In almost all instances, in intes- tinal amoebae, the parasites have been encountered only in the tropho- zoites. Among endozoic ciliates, aside from Chen and Stabler’s (1936) state- ment that Sphaerita has been found in Zelleriella as well as in its entamoeba parasites, the chytrid has been reported only in Nyctotherus and Ophryoscolecidae. Sassuchin (1928a, 1934) found it to be common in N. ovalis from Periplaneta (Fig. 217C, D). In ciliates of antelopes, Dogiel (1929) described Sphaerita diplodiniorum in Diplodinium costa- tum and Ostracodinum gracile. Jitovec (1933) gave the name S. ento- dinii minor to a chytrid in Entodinium simplex (?), and S. entodinu major to one in Entodinium longinucleatum. He observed also a Sphae- vita-like parasite in an undetermined species of Entodinium, and stated that in other Ophryoscolecidae none of these chytrids were observed. 1046 PARASITES: OF PROTOZOR Winogradowa (1936) reported Sphaerita, as well as larger, distributed, probably bacterial, parasites in Entodinium (Fig. 217B). Discussing Joenia annectens and Mesojoenia decipiens, Grassi and Foa (1911) mentioned an enormous enlargement of the nucleus by the presence of a parasite, and reported also a parasite in the cytoplasm. This probably is the first record of Nucleophaga in a flagellate. Its pres- ence in Joenia intermedia and Myxomonas polymorpha (== Giganto- monas herculea), noted by Dogiel (1917, 1916), has been mentioned above. In Hexamastix termitis, Kirby (1930) showed some parasitized nuclei, as Duboscq and Grassé (1933, p. 392) pointed out, but failed to interpret them correctly. The large nuclei with numerous small, unt- form-sized granules contained Nucleophaga, and the parasite has been found, on reéxamination of the material. The parasite has been observed by the writer in many Devescovininae, but not in the smaller species of Foaina. There seems to be a lower limit in the size of nuclei in which it can develop. Nuclear parasites of Trichonympha are considered below (p. 1059). Psewdospora volvocis, a parasite of Volvox with apparent affinities to the Bistadiidae, has been found infected with intranuclear chytrids by Roskin (1927) and by Robertson (1905), the latter of whom misinterpreted the parasite as representing gamete formation by Pseudos pora. Nucleophaga has been found in many endozoic Amoebidae, and several species have been named. Lavier (1935b) reviewed most of the accounts, with the exception of those of Kirby (1927, 1932b) and Sassuchin (1931). The earliest observations were made in Endamoeba blattae (Mercier, 1907, 1910; Janicki, 1909). Tyzzer (1920) found a nuclear parasite in Pygolimax gregariniformis of chickens and turkeys. Two amoebae of man are known to be parasitized: Endolimax nana and lodamoeba biitschli, in which Nucleophaga was first recorded by Noller (1921). Epstein (1922) named Nuacleophaga hypertrophica a nuclear parasite of Endolimax nana; in 1935 he stated that he had studied then (1922) a nuclear infection of both E. nana and I. bitschli. Brug (1926) independently named a nuclear parasite of the latter amoeba N. /ntest- nalis; according to Brumpt and Lavier (1935a), that parasite is the same as the one (Fig. 218F-J) which they also studied in E. nana, and Brug’s name is a synonym for N. Aypertrophica. Kirby (1927) described an unnamed Nuacleophaga (Fig. 218A-E) in Endamoeba dis parata of Miro- PARASITES OF PROTOZOA 1047 termes hispaniolae, and reported it also in E. majestas, E. simulans, and Endolimax termitis; and Sassuchin (1931) found a chytrid in the nucleus of Entamoeba citelli. In Entamoeba ranarum, Lavier (1935a, 1935b) found a parasite described as Nucleophaga ranarum. Although not a protozoan parasite, and one that is of doubtful affinities, the organism named Erythrocytonucleophaga ranae by Ivani¢é (1934), which invades the nuclei of the red blood cells of Rana esculenta, is interesting to con- sider in this connection. LIFE HISTORY AND STRUCTURE Sphaevita—Chytrids of the family Olpidiaceae, to which Sphaerita and Nucleophaga belong, have a one-celled, intramatrical thallus, enclosed from an early period by a delicate membrane, amoeboid in nature, which at maturity changes into a single sporangium or resting sporangium. The sporangium of Sphaerita lacks elongate discharge tubes, the spores escaping through an opening or papilla at one or both ends. The zodspores of chytrids of this family are uniflagellate, according to Minden (1915), Fitzpatrick (1930), and Gwynne- Vaughan and Barnes (1937); but in the spores of many forms in Protozoa, either two flagella or no flagella have been observed. Sphaerita has often been encountered in only a small percentage of the host species, but some records report a high incidence. Noller (1921) found, in certain material, the majority of Endolimax nana and a vety high percentage of Entamoeba coli infected; and Dobell (1919) saw several E. nana infections in which a considerable proportion of the amoebae were parasitized. Sphaerita was present in 80 percent of E. colz and E, histolytica studied by Lwoff (1925). Both Becker (1924) and Sassuchin ef al. (1930) found E. cte//7 in certain ground squirrels very heavily parasitized, and Yuan-Po (1928) reported about 60 percent infection of Entamoeba bobaci. Almost all Chilomastix in a guinea pig contained S. chilomasticis (Cunha and Muniz, 1934). In flagellates of termites, infection varies from light to heavy. In a few instances almost every individual of certain devescovinids on some slides has been para- sitized; on the other hand, the parasite may be infrequent or absent in other host faunules of the same species. Distributional factors would facilitate the presence of the chytrids in higher incidence in endozoic than in free-living Protozoa under natural conditions, but an infection k 1048 PARASITES OF PROTOZOA in free-living amoebae in cultures may develop a high incidence in a short time. Dangeard (1886b) found S. endogena in great abundance in cultures of its two rhizopod hosts; and Ivanié (1925) stated that cultures often perish from severe infection. A host may be parasitized by two or more species (Mattes, 1924; Yuan-Po, 1928), but usually only one species has been distinguished. Brumpt and Lavier (1935b) described two different sphaeritas in two amoebae on the same smears: S. parvula from Hyalolimax cerco- pitheci, and one with larger spores in an Entamoeba of the minuta type. This indicates host-specificity; but various amoebae of man, it appears, contain a common species (p. 1042), and Lwoff (1925) stated that the chytrids do not seem to manifest a narrow host-specificity. The earliest stage in the cytoplasm of the host is a small, amoeboid, uninucleate thallus. Dangeard (1895) described the parasite in Euglena as at first smaller than the flagellate’s nucleus, with a dense, homoge- neous cytoplasm and a vesicular nucleus with a large nucleolus. Mitchell (1928) found the earliest stages to be from 2.5 to 3.5 1, in diameter, with a vesicular nucleus from 1.3 to 1.5 p in diameter. Early stages of the parasites in amoebae have been found as small as 1.5 y (Chatton and Brodsky, 1909) and 2 1 (Mattes, 1924); the latter observer failed to find a distinct nucleus. Sphaerita endamoebae, according to Becker (1926), is from 1.9 to 2.5 1 in its early intracytoplasmic stage, with a fine cell membrane and a relatively large, solid nucleus. Most accounts describe increase in the size of the cytosome and the nucleus before the nucleus begins to divide. In Euglena viridis (Dan- geard, 1895; Mitchell, 1928) the uninucleate thallus may become larger than the host nucleus, and its nucleus becomes correspondingly large. Its shape is spheroidal, ellipsoidal, or elongated. The shape in this phase, together with its size and the presence of vacuoles, is re- garded as of taxonomic significance by Jahn (1933), who distinguished E. phaci on such grounds. Other sphaeritas appear to attain no such size before nuclear multiplication sets in. Sphaerita in Vahlkampfia limax attains only about triple its diameter before nuclear divisions begin (Chatton and Brodsky, 1909). In S. endamoebae, according to Becker (1926), nuclear multiplication keeps pace with growth, and there are binucleate stages no larger than uninucleate ones. Similar development has been noted by the writer in Sphaerita (Fig. 217A) in several PARASITES OF PROTOZOA 1049 species of Devescovininae. There are very few observations on actual nuclear division; Dangeard (1895) interpreted as this some figures he observed, but did not see nuclear division in the larger nuclei of the early stages. Nagler (1911b) reported dumb-bell-shaped figures, as well as granular fragmentation stages, in the parasite of Evglena sanguinea. The outcome, in any event, is in typical Sphaerita a multinucleate thallus, which is converted entire into the sporangium. Parasites, which in some phases are much like Sphaerita but lack a 088. eo soo Figure 217. A, various stages in development of Sphaerita in Devescovina sp. from Neotermes tectonae; B, Sphaerita and other microorganisms in Entodinium sp. and Exudiplodinium sp. from ruminants; C, mature sporangium of Sphaerita from Nyctotherus ovalis ; D, developmental stages of Sphaerita and aggregations of bacteria in Nyctotherus ovalis. (A, original; B, after Winogradowa, 1936; C, D, after Sassuchin, 1928a.) multinucleate structure, have been described, however. Mitchell (1928) reported a parasite in Euglena caudata, which, after growing to a relatively large uninucleate body, underwent repeated division of both nucleus and cytoplasm to form spores. Ivani¢ (1925), describing in free-living amoebae parasites which appear to be Sphaerita-like, stated that the uninucleate forms grow and multiply by binary fission before the plasmodial period begins. When, as is often true in preparations, the cytoplasm of the parasite is not apparent, division of the nuclei may be mistaken for division of individuals within a vacuole. Individual 1050 PARASITES OF PROTOZOA parasites multiplying in this way alone would probably be cocci. That may account for possible errors, not made in the above examples, but probably involved in certain accounts of nuclear parasites, as discussed below. When the parasite has reached a certain size, growth stops and sporulation sets in. In Sphaerita amoebae the size when spores are formed is very variable; sporangia are larger when only a few are pres- ent in an amoeba (Mattes, 1924). The number of spores produced is also variable in this species, ranging from less than a hundred to several hundred. In sporulation the protoplasm simultaneously organizes into membrane-confined bodies around each nucleus, and the spores appear as spheroidal or ellipsoidal structures. The membrane of the sporangium may remain very thin, so as to be scarcely recognizable, as in Sphaerita in Amoeba alba, where groups of spores showed no trace of an envelope (Penard, 1912). Sometimes it becomes more distinct at sporulation; and in the unique case of Sphaerita from Nyctotherus ovalis, according to Sassuchin (1928a), it becomes 1 1 or more thick (Fig 2i7@): The account by Sassuchin ef a/. (1930) and Sassuchin (1934) of the parasite of Entamoeba citelli is not easy to understand. The parasites are said to occur either in groups, varying considerably in size, which resemble sporangia, but around which a membrane was never observed; or arranged singly in the protoplasm. Though these parasites show a spore-like character, the authors did not call them spores, nor did they discuss multiplication. The description by Becker (1926) of Sphae- rita endamoebae from Entamoeba citelli, to which Sassuchin did not refer even in his later article, is in essential agreement with the usual con- cept of the life cycle of Sphaerita. The parasite of Exglena caudata (probably Sphaerita dangeardt) may form as many as 500 spores (Mitchell, 1928). Sporangia of S. endogena contain 100 or more (Dangeard, 1886a). Dogiel (1929) found only from 30 to 40 spores in the “‘spore balls’’ of S. dzplodiniorum. In Sphaerita of Monas vulgaris, nuclear divisions preceding spore forma- tion proceed to stages 16, 32, or sometimes 64 (Alexeieff, 1929). Pinto and Fonseca (1926) mentioned sporangia of only from 7 to 9 “individ- uals” in S. minor of Trichomonas vitali; Canha and Muniz (1934) found from 20 to 30 spores in S. chilomasticis. PARASITES OF PROTOZOA 1051 There is variability in the number of spores and the size of sporangia within a species. That sporulation can occur at different stages of growth was noted by Chatton and Brodsky (1909), in Sphaerita of Vahlkamp fia limax; sporangia ranged from 20 y in diameter down to small ones, with few spores, of 4 yy. Mattes (1924) stated that the size at which sporulation starts in S. amoebae is very variable; and Lwoff (1925), in Sphaerita of entamoebae of man, found that sporulated parasites are of different sizes. The size of the sporangium and the number of spores must not be used indiscriminately for definition of species. The spores are spherical, ovoidal, or ellipsoidal in shape—most fre- quently the first. They range in size, in different species, from a diameter of 0.25 to 0.30 yp (S. parvula, Brumpt and Lavier, 1935b) to elongated forms of from 2.5 to 3 p (Sphaerita from Euglena; Mitchell, 1928; Puy- maly, 1927). Yuan-Po (1928) reported spores of from 2.5 to 4 y ina parasite of Entamoeba bobaci; this is exceptional in Sphaerita of endozoic Protozoa. In the parasite of Nyctotherus ovalis, the spores measure from 1.5 to 2 y (Sassuchin, 1928a). Dangeard (1886a, 1886b) stated that the spores of S. endogena ftom amoebae have a size of 1.5 y. There ap- pears in general to be only a limited variability in the size of mature spores; but Becker (1926) found that in S. endamoebae, the spores of which usually were from 1.0 to 1.6 u, some were as small as 0.5 p. The size and shape of spores is, used discretely, a valuable taxonomic guide. The spore of S. endogena in the rhizopods Nuaclearia simplex and Heterophrys dispersa has, according to Dangeard (1886a, 1886b), a long flagellum (“‘cil’”) placed anteriorly and strongly recurved. Its movements are very active and jerky, and sometimes there is simple rotation in one position. When he studied the zodspores of Sphaerita of Euglena sanguinea (1889b), Dangeard found, in addition to the posteriorly directed flagellum, a very short one directed anteriorly. Serbinow (1907) found only one flagellum on the zodspore of Sphae- vita of Euglena, and thought it possible that Dangeard’s biflagellate zodpores belonged to some other organism, possibly to the parasite of Sphaerita, Olpidium sphaeritae Dang. Serbinow described their jerky, irregular movement. In Sphaerita of E. viridis, Puymaly (1927) also reported biflagellate zodspores, the larger flagellum directed posteriorly, as Dangeard noted again in 1895; and he described the movement as rotation around an axis and rapid, oscillatory swimming. These observa- 1052 PARASITES OF PROTOZOA tions agree with those of Stein (1878, 1883) on the escape of minute, flagellated organisms from the so-called germ balls. Cejp (1935) ob- served two flagella on the zodspores of Sphaerita of Paramecium. Ivani¢ (1925) stated that he repeatedly observed release of the flagellated swarm sports of the parasite (Sphaerita?) of Amoeba jollosi. Mattes (1924), however, though he found flagellated zodspores of Olpidium amoebae of Amoeba sphaeronucleolus, failed to see any flagella or motility in the spores of two Sphaerita species of the same amoeba. The same is true of the observations of all other investigators of the parasite in Protozoa. It appears that Sphaerita in endozoic Protozoa lack flagellated zoéspores, and that most of those of free-living amoebae also do. A central or eccentrically placed nucleus in the spore was reported by Dangeard (1895) in Sphaerita of Euglena, and by Penard (1912) in Sphaerita of Amoeba alba; and it was shown by Cejp (1935) in the parasite of Paramecium. Mitchell (1928) noted a nucleus in the spores of the chytrid of Exglena sanguinea, and he alone described any detail in the nuclear structure. In sphaeritas of endozoic Protozoa, the nucleus has not been found, and there appears to be a thicker spore membrane. The membrane appears in optical section as a well-defined ring, espe- cially in spherical spores. In elongated spores there is frequently a stain- able area at one end, appearing often as a crescentic thickening, as in Sphaerita (Fig. 217C) in Nyctotherus ovalis (Sassuchin, 1928a, 1934). This structure was noted also by Becker (1926) in Sphaerita endamoebae, by Yuan-Po (1928) in the larger species in Entamoeba bobaci, and by Connell (1932) in Sphaerita of Gigantomonas lighti. It has been ob- served by the writer in the parasites in a number of flagellates in ter- mites. Rupture of the sporangium takes place in the cytoplasm of the host, then the body of the host may rupture and the spores be released into the water. In most instances no previously apparent pore or papilla has been shown. Dangeard (1889b) stated that the zodspores of Sphaerita in Euglena sanguinea escape by a papilla at one end. Serbinow’s ac- count (1907) of an elongated or fusiform sporangium in Sphaerita in E. viridis and E. sanguinea, with a short exit papilla at one or both ends, does not apply to most forms that have been placed in the genus. Sphaerita cannot, then, be diagnosed on the basis of this account, as was done by Minden (1915), without excluding many forms. PARASITES OF PROTOZOA 1053 There is slight evidence of fusion of spores in Sphaerita, and none of this carries the conviction of cytological demonstration. Dangeard (1889b) and Puymaly (1927) reported that in sphaeritas of Euglena zodspores may touch or adhere, simulating conjugation of gametes, but that they end by separating. Mattes (1924) found no fusion of spores of Sphaerita amoebae, Chatton and Brodsky (1909) thought copula- tion of spores probable, but did not see it. In Sphaerita of Amoeba alba, Penard (1912) stated that he sometimes encountered the spores in con- jugation; and fusion was reported in Sphaerita-like parasites of Amoeba jollosi by Ivanié (1925), as well as in the so-called gametes of Allo- gromia by Prandtl (1907), which possibly were also Sphaerita. Dangeard (1889b) reported fixation of zodspores to the wall of Euglena sanguinea, and penetration into the cytoplasm. Puymaly (1927) described adherence, loss of flagella, and development of a fine surround- ing membrane; following which the spore probably emits a fine papilla, which perforates the flagellate, and empties abruptly into the cytoplasm. In rhizopods, spores are ingested (Dangeard, 1886a; Chatton and Brodsky, 1909; Mattes, 1924; Lwoff, 1925); this probably is the general method of infection of holozoic Protozoa by the non-flagellated spores. The thallus of Olpidiaceae may develop also into a resting sporangium, which is ordinarily thicker-walled, and may sometimes bear spines, but otherwise in structure and development corresponds to the ordinary sporangium. Spinous cysts or resting sporangia were described by Dan- geard (1889b) in Sphaerita of Euglena sanguinea; by Serbinow (1907) in Sphaerita of E. viridis and E. sanguinea; by Skvortzow (1927) in S. trachelomonadis; and by Mattes (1924) in S. plasmophaga of Amoe- ba sphaeronucleolus. Nucleophaga—Nucleophaga has been found by the writer in low incidence in almost all devescovinid flagellates in termites. Sometimes the infection is greater. In some material of Endamoeba disparata tt was from 6 to 12 percent (Kirby, 1927); Brumpt and Lavier (1935a) found it in 78 percent of trophozoites of E. mana; on one occasion 90 percent of a group of Amoeba sphaeronucleolus were parasitized (Mattes, 1924); and Gruber (1904) lost an entire culture of A. viridis, which had been kept ten years, on account of the fungus. The parasite apparently occurs exclusively in the nucleus. Brumpt and Lavier found it only twice, among thousands of specimens, in the cyto- 1054 PARASITES OF PROTOZOA plasm of E. nana; and in those two instances, as they stated, it prob- ably was not developing there. A given species of Nucleophaga invades certain hosts, and not others. Brumpt and Lavier failed to find it in E. dispar, which was associated with heavily parasitized E. nana. Many related species of devescovinids, however, contain what is probably the same species; although there is more than one species of Nucleophaga in that group of flagellates. In the recent review of studies on Nacleophaga by Lavier (1935b), it was noted that life-history accounts indicate two modes of develop- ment. One is in agreement with the life cycle of Sphaerita, as outlined above. A thallus enlarges and its nuclei multiply, it is converted into a sporangium, and a spore forms around each nucleus (Dangeard, 1895; Penard, 1905b; Mercier, 1907, 1910; Mattes, 1924; Lavier, 1935b). Nucleophaga of Endamoeba disparata (Fig. 218B-E) 1s con- sidered to have this type of life history (Kirby, 1927). In the second type there is no multinucleate structure, and no sporangial membrane, the individual invading parasite (Fig. 218G-J) multiplying repeatedly within the nucleus by division (Epstein, 1922; Brug, 1926; Brumpt and Lavier, 1935a). This is the type of reproduction, evidently, in the nu- clear parasite of Entamoeba citelli, according to Sassuchin (1931); and probably such a parasite would not seem to differ essentially from Caryococcus (Dangeard, 1902). It is not conceivable that there should be such fundamentally different types of development in members of the same genus. Either the latter type is nothing but a misinterpretation of the ordinary chytrid life history, because of failure to see the cytoplasm of the parasite, or the parasite is not Nucleophaga. The figures of Nucleophaga supposed to show this second type of reproduction do not differ essentially from the other accounts—it is probably a matter of differing interpretations of what is actually one form of development. The similarity to Sphaerita appears to be too great to separate Nuwcleo- phaga distantly from that genus; but the relationship of Caryococcus to this needs further investigation. In its early phases, in any event, Nacleophaga appears as a group of granules in the interior of the nucleus, occupying a limited area, whereas the nuclear structure elsewhere is essentially unchanged. This group of granules presumably, though it cannot always be ascertained with certainty, represents the nuclei of a thallus. Nucleophaga amoebae, Figure 218. Nucleophaga. A-E, Nucleophaga in Endamoeba disparata: A, normal nucleus of E. disparata Kirby; B, developmental stage of Nucleophaga; C, D, later stages of Nucleophaga, with residue of chromatin in center; E, surface view of same stage. F-J, Endolimax nana parasitized by Nucleophaga hypertrophica: F, normal amoeba; G, beginning of nuclear parasitism; H, I, multiplication of spherules; J, mature spores, nuclear membrane appears on the point of rupturing. K-O, Nucleophaga in Caduceia theobromae: K, surface view of parasitized nucleus bulged out at one side; L, mature sporangium, with nucleus similarly formed; M, detail of spore; N, parasitized, greatly hypertrophied nucleus; O, normal nucleus, drawn to same scale as K, L, N. (A-E, after Kirby, 1927; F-J, after Brumpt and Lavier, 1935a; K-O, original.) 1056 PARASITES OF PROTOZOA described by Dangeard (1895) in a host he considered to be Amoeba verrucosa, invades the nucleolus, in which it appears at first as a vacuole enclosing a granule—the former its cytoplasm, the latter its nucleus. Growth of the parasite is rapid (Mattes, 1924), and as it proceeds the chromatin is used up. The stainable nuclear material becomes re- stricted to the periphery in a reticulated structure (as noted by the writer in Nucleo phaga of devescovinid flagellates), or to the central zone (Fig. 218C, D), as in hyperparasitized Endamoeba dis parata (Kirby, 1927). Eventually the nuclear material disappears, and the interior is entirely occupied by the parasite. The parasitized nucleus hypertrophies considerably (cf. Fig. 218F and J; O and N), up to several times its original diameter. The para- site must obtain material for its continued growth by diffusion from the cytoplasm through the nuclear membrane. Though Mercier (1910) men- tioned considerable hypertrophy of the nucleus of E. blattae, he showed spores in nuclei in which there seems to have been little enlarge- ment. This is unlike the usual situation. Perhaps the very thick membrane of the nucleus of E. blattae has an influence in restraining the growth of the parasite. Lavier (1935b) noted precocious spore formation in Nucleophaga of Entamoeba ranarum, but stated that it generally occurs when the parasite has attained a large size. In Nucleophaga of devesco- vinid flagellates, notably that in Caduceia theobromae, expansion of the nucleus cannot occur equally in all directions, because of its relation- ship to the axostyle. Instead, it is pushed out on one side, and often has a bilobed figure (Fig. 218K, L). This figure sometimes is retained in the mature sporangium; often it fills out. As has been noted also of Sphaerita, the size of the mature sporangium, as well as the number of spores, is subject to considerable variation in the same species of Nw- cleo phaga. It has been stated that spore formation is in certain forms continuous, and that there may be present in an individual at a given time mature spores and granules corresponding to spores not yet formed (Lavier, 1935b). It is more general, however, for the spores to be formed simultaneously, the entire thallus being converted into the group. Other granules, which have been seen by the writer among the spores, prob- ably represent residual or discarded material; there is no evidence for maturation of later spores. PARASITES OF PROTOZOA 1057 The spores are very much like those of Sphaerita. Their shape is spheroidal or ellipsoidal, and the wall stains more intensely than the contents. Some show a thickening at one side, in crescentic form (Ep- stein, 1922; Brumpt and Lavier, 1935a; Lavier, 1935b). In the interior, often no structure is discernible, or one or two granules are seen, or a central nucleus may be observed. Dangeard (1895) and Epstein (1922) reported a vesicular nucleus; Brumpt and Lavier, however, failed to observe a definite nucleus in the parasite of Endolimax nana, studied by Epstein. In Nucleophaga of Caduceia theobromae, a spheroidal gran- ule of relatively large size was observed toward one end of the spore (Fig. 218M). This may be a nucleus. In size, the spores range from one to 2 py, some being reported as only about one y (Brumpt and Lavier, 1935a), some 2 y (Lavier, 1935b; Penard, 1905b), others as having a variability from about one y to 2 y (Mercier, 1910; Mattes, 1924). No flagella have been observed on spores of Nucleophaga, with the possible exception of those mentioned in the account by Robertson (1905). She described what she supposed to be gametogenesis of Pseudospora volvocis, in a rather complete account of what is probably the development of Nucleophaga. The ‘‘gametes,” as figured and de- scribed, are each provided with one flagellum; and they are reported to fuse, producing a biflagellate zygote. EFFECT ON HOST Minden (1915) wrote (translation) : In the lower plants, mainly algae, which in the widest variety are sought by parasitic Chytridiales, the injuries are usually so striking that these fungi ate designated as dangerous parasites of algae. In a short time large cultures of diatoms, flagellates, and other unicellular organisms may be completely destroyed; but also filamentous algae die cell by cell. The first indication of injury is in the discoloration and disorganization of the cell contents . . finally there remain only granular vestiges. Infection with Sphaerita may be observed in many Protozoa that appear entirely normal, but it often ends fatally. The host may some- times rid itself of the parasite and continue normal life (Dangeard, 1895; Penard, 1912); on the other hand, many observers report death of the host at the time of sporulation. 1058 PARASITES OF PROTOZOA Parasitized euglenid flagellates lose their green color, chlorophyll first being affected and chromatophores degenerating (Dangeard, 1889b, 1895; Puymaly, 1927; Mitchell, 1928; Jahn, 1933). Puymaly found a decrease in flagellar activity, whereas euglenoid movement con- tinued to the last moment and became even more energetic. There is alteration of the nucleus, according to some, though Puymaly stated that there is none; and the cytoplasm becomes vacuolated. Finally, in many cases, the flagellate ruptures and zodspores are liberated. Chatton and Brodsky (1909) found that parasitized amoebae tend to assume a spherical form with radial pseudopodia, instead of progressing; and Sassuchin (1928a) noted a progressive slowing of the ciliary action in Nyctotherus. The pulsating vacuole in these heavily infected hosts slows or loses its rhythm. Degenerative changes were observed in the nucleus of parasitized lodamoeba biitschlii by Wenrich (1937); in that of Entamoeba citelli by Becker (1926); and in the macronucleus of Nyctotherus by Sassuchin (1938a). When the sporangium ruptures, or shortly before, the host may perish (Chatton and Brodsky, 1909; Mattes, 1924; Yuan-Po, 1928; Sassuchin, 1928a), especially if the in- fection is heavy. The fact that in amoebae of man the parasites have been found only in trophozoites has been taken to indicate either that they hinder the amoebae from encysting or that infected cysts degenerate rapidly (Lwoff, 1925). Lwoff therefore pointed out a possible use of Sphaerita as a means of biological control, following Noller’s suggestion that it might be worth while to devote more attention to these natural enemies of amoebae. If one could transmit the infection to carriers of cysts, Lwoff stated, there would be a means of diminishing the number of cysts, this in addition to the inhibition of multiplication. The practicability of this, however, is doubtful. The protozoan whose nucleus is parasitized by Nucleophaga con- tinues activity until the end. Usually there is no apparent change in protoplasmic activity or in structure, aside from the nucleus, even though all stainable chromatin material has disappeared. Lavier (1935a) ob- served increased size and activity in parasitized Entamoeba ranarum, and remarked that the hyperactivity may be provoked by irritation, and may constitute a defense reaction on the part of the amoeba. By the time the parasite reaches the stage of sporulation, however, some changes may PARASITES OF PROTOZOA 1059 have occurred in the cytoplasm (Dangeard, 1895; Sassuchin, 1931), and there may have been some hypertrophy of the host’s body (Epstein, 1922). Epstein, indeed, stated that giant amoebae reaching from 10 to 30 times normal size, may result; but that 1s very much more than 1s usually observed. When the membrane breaks and the spores escape, the host perishes. For that reason, even in heavily parasitized groups of Protozoa, individ- uals with spores dispersed in the cytoplasm are seldom observed. PARASITES OF THE NUCLEUS OF TRICHONYMPHA Except for mention (Kirby, 1940) of the parasites described below, the only report of parasitization of the nucleus of Trichonympha is the description (Kirby, 1932a) of a form in T. saepiculae (Fig. 219B). Numerous spheroidal bodies, each apparently subdivided into compart- ments, filled several nuclei, in which the vestiges of chromatin were con- fined to the central part. Few specimens of this organism were found, and its affinities were not discussed, except for the remark that it 1s unlike Nacleophaga. An unusually interesting parasitization of the nucleus has been studied by the writer in Trichonympha in certain termites of Madagascar and in one from Java. In several series of preparations from Procryptotermes sp. of Madagascar, a large proportion of the hypermastigotes had parasit- ized nuclei. Apparently, in the flagellate from this host two different parasites are involved. One of these has a life history like that of Nuacleophaga: growth of a multinucleate parasite, using up the chro- matin, which is restricted to a peripheral reticulum and finally dis- appears; and formation of numerous spores. The size and structural detail of the spores distinguish them from those of any described Nuacleo- phaga, and suggest a possible affinity to the Haplosporidia. The normal, interkinetic nuclei of these species of Trichonympha have the chromatin in the form of stout, varicose strands which extend throughout the intranuclear area. They may extend entirely to the pe- riphery, but often in the preparations there is a clear outer zone of variable width. In this zone are minute granules. In some nuclei, which possibly show the beginning of kinetic changes, the strands tend to be peripheral, and the central part of the nucleus is occupied by a granular and reticulo-fibrillar matrix. 1060 PARASITES: OF PROTOZOA Figure 219. Micro6rganisms in Trichonympha. A, constantly present organism that forms an aggregate surrounding or near to the nucleus of T. campanula; B, nuclear parasite of T. saepiculae; C, Sphaerita in T. sphaerica; D, E, peg-formed organisms in T. campanula,; F, G, fusiform organisms in T. campanula; H, group of parasites (micro- sporidia?) in T. magna; I, developmental stages of organism shown in H. (After Kirby, 1932a.) In early stages of invasion by the Nucleophaga-like parasite, a body, apparently amoeboid, is observed in the process of penetration into the chromatin mass. In the earliest stages so far found, it is already multi- nucleate. This becomes located in the interior of the chromatin mass, and as it grows its nuclei multiply and the chromatin of the Trzchonym- pha nucleus becomes restricted to a peripheral reticulum (Fig. 220E). PARASI@ES OF PROTOZOA 1061 By the time the parasite reaches its full size, the nucleus of its host has become greatly hypertrophied and has left its normal position. A variable number of spores are produced; in one instance there were only 17, but usually there are from 150 to 200 or more, located within an ellipsoidal membrane 25 to 44 22 to 36 y. The individual spore is ellipsoidal and has a size of 2.5 to 4 y X 2 to 3 y. The spores are larger in size when their number is smaller. The structure of the spores (Fig. 220C-E) is the characteristic of greatest interest in this organism, as nothing like it is known in any other nuclear parasite, or indeed in any known parasite of Protozoa. The nucleus is located at one end, and is usually relatively very large, having a diameter almost equal to the width of the spore. When heavily stained, or when not well fixed, it appears homogeneous, but in good preparations it is resolved into closely packed granules. In the cytoplasm of the spore are a variable number of granules which are relatively large for cytoplasmic granules. These stain intensely with hematoxylin, and possibly are volutin, though no tests could be made to support that view. The cytoplasmic granules are often arranged in an equatorial ring (Fig. 220D), which appears solid in some preparations. Some- times there are no granules outside of the nucleus except in this ring; at the other extreme, the ring constitutes the margin of a solid hemi- spherical mass of granules, which occupies all the area at its end of the spore (Fig. 220E). Between these extremes are conditions in which, in addition to the ring, there are only a few granules at the periphery of the hemispherical area, or more abundant granules in a peripheral, semicircular row at right angles to the ring (Fig. 220C). At the periphery of the mass of mature spores of the hyperparasite from Procryptotermes sp., there is constantly present a single, apparently crystalloid body (Fig. 220B). In different parasites this body is rela- tively uniform in size and shape; and it is generally so located as to cause a protrusion of the membrane. It has the form of a conventional diamond, and stains deeply with Heidenhain’s iron-hematoxylin but not with Delafield’s hematoxylin. It is unlikely that it is to be regarded as residual chromatin. What seems to be a second parasite (Fig. 220G, H) of the nucleus of Trichonympha occurred in from 70 to almost 100 percent of the hypermastigote in certain preparations from the Madagascar Procrypto- Figure 220. Nuclear parasites of Trichonympha sp. from Procryptotermes sp. of Mada- gascar. A-F, Nucleophaga-like parasite, nucleus hypertrophied; G, H, coccoid parasite, nucleus not hypertrophied. A, developmental stage, probably a multinucleate plasmodium, chromatin of nucleus restricted to periphery; B, mature sporangium, diamond-shaped crystalloidal body at periphery of nucleus; C-E, details in structure of spores; F, nucleus ruptured, with some spores in cytoplasm; G, chromatin masses present with parasites, many of latter show crescentic stainable area at one side; H, chromatin peripheral, some dividing forms of parasite. (Original. ) PARASITES OF PROTOZOA 1063 termes. Generally the parasite appears as a mass of spherical bodies, each about one 1 or less in diameter and often with a stainable crescent at one side, located in the central part of the nucleus. The spherical bodies are a good deal smaller than are the nuclei in a plasmodium of comparable size in the other parasite. The chromatin is usually re- stricted to the periphery of the host nucleus; but, except for this re- moval of the central part of the mass, it is little altered; and there is no marked hypertrophy of the nucleus. In the nuclei of some hosts, the proportion being greater on certain slides, rounded bodies with a similar crescentic stainable area are located peripherally, the chromatin mass being concentrated in the center. The distribution of these periph- eral bodies is often such that a common embedding cytoplasm appears unlikely. Further investigation is necessary to establish the nature of this parasite. It may be a bacterial, coccoid parasite of the nucleus, com- parable in certain ways, possibly, to Caryococcus, described by Dangeard (1902). In several instances multinucleate trichonymphas, with all nuclei parasitized, have been found. These are the only multinucleate flagel- lates of this genus that have ever been seen. Cytotomy generally ac- companies division of the single nucleus, but binucleates occasionally occur. PHYCOMYCETES OTHER THAN SPHAERITA AND NUCLEOPHAGA Chytridiales of a number of genera other than Sphaerita and Nucleo- phaga have been found parasitic on Protozoa, especially autotrophic flagellates. Those described up to 1915, in the genera Olpidium, Pseu- dolpidium, Rhizophidium, Phlyctochytrium, Rhizidiomyces, Saccomyces, Rhizophlyctis, and Polyphagus, were discussed by Minden. They occur in or on Exglena, Cryptomonas, Chloromonas, Chroococcus, Gleno- dinium, Haematococcus, Chlamydomonas, Pandorina, and Volvox. OI- pidium arcellae is considered doubtful. Fungus parasites, which are probably Chytridiales, have been found in cysts of a number of ciliates. Stein (1854) found many cysts of V orticella microstoma with up to three or four protuberances perforating the wall and extending a short distance free. Each protuberance was an extension of a rounded body (Muvtterblase) within the cyst. From the terminal opening a thin, gelatinous, clear fluid was reported to escape, 1064 PARASITES OF PROTOZOA forming a globule enclosing about thirty “young,” resembling cer- tain Monas forms, and, when dispersed, having movements like them. Similar structures were shown in a cyst of Vorticella nebulifera. At that time Stein considered this to be a mode of reproduction of Vortzcella. Cienkowsky (1855b) recorded similar bodies in cysts of Nassula am- bigua ("N. viridis”), describing the appearance of clear vacuoles in the cyst contents and the development of “spores,” from many of which a short process broke through the wall of the cyst and permitted the es- cape of the swarm spores. Lachmann (1856) mentioned these observa- tions as showing another kind of reproduction in ciliates. Cohn (1857), however, remarked on the resemblance of these ‘“‘microgonidia,” with their flask-formed “‘mother cells,” to the chytrids of many plants. In their text Claparéde and Lachmann (1860-61) discussed the phenomena as forms of reproduction by embryos, adding observations of their own on similar structures in Urnula epistylidis; but in their footnotes they stated that these were Chytridium. Stein (1859) regarded them as parasites, comparing them with Saprolegniales and in particular with Pythiwm entophytum Pringsheim; but their characteristics are suggestive of Ol pid- zum. Stein recorded similar bodies from cysts of Stylonychia pustulata, Holosticha (Oxytricha) mystacea, dead Toko phrya (Acineta) lemnarum, and Metacineta (Acineta) mystacina (observations of 1854). On the motile bodies escaping from a Vorticella cyst he saw a single flagellum. Species of Olpidium, which differ from Sphaerita in the elongation of the exit tube, occur in certain rhizopods and in Suctoria, as well as in Euglena. O. amoebae was described by Mattes (1924) from Amoeba Sphaeronucleolus; it is said to parasitize a rotifer also. Gdnnert (1935) named O. acinetarum a chytrid which destroyed a culture of Lernaeo- phrya capitata and Podophrya maupasi within a few days. The spores are relatively large, from 2.5 to 3 1 in diameter, equaling the larger ones of Sphaerita; and Mattes found a relatively long posterior flagellum. Rhizophidium and Polyphagus belong to the Rhizidiaceae, in which there is a restricted mycelium. Rhizophidium beauchampi has recently been described by Hovasse (1936) in Exdorina illinoisensis. A heavy infection, exceeding 90 percent, occurred homogeneously in these phyto- monads in a large lake. The zodspore, which has a single long flagellum, becomes fixed to the coenobial surface and germinates by the emission of a tube which penetrates a cell and functions as a sucker. The part PARASITES OF PROTOZOA 1065 of the tube that remains external to the cell swells and becomes a sporangium, in which by simultaneous partitioning from 20 to 100 zo0- spores are produced. Most parasitized colonies had not more than 25 to 28 normal cells, and heavily parasitized colonies may be destroyed. Poly phagus euglenae, whose structure and life history have been de- scribed by Nowakowski (1876), Dangeard (1900b), and Wager (1913), appeared at various times in cultures of Ewglena, which were destroyed in a few days. Serbinow (1907) at Petersburg, and Skvortzow (1927) in East Mongolia found it parasitic on Chlamydomonas. The parasite germinates free in the water, and a single cell, by branched haustoria, may attack many flagellates. A haustorium perforates the cell wall, branches, and the cell contents rapidly disintegrate. A sporangium develops as an outgrowth from the protoplast, and produces a variable, usually very large, number of uniflagellate zodspores. Polyphagus is one of the few Chytridiales in which sexual reproduction has been satis- factorily demonstrated. A zygote is formed by the fusion of two vegeta- tive cells, and becomes a resting spore, with smooth or spinous mem- brane. Skvortzow (1927) reported two other Chytridiales from Eadorina elegans in Manchuria: Phlyctidium eudorinae n. sp. and Dangeardia | mamillata. The latter was originally described by Schréder from Pan- dorina. Phlyctidium is epibiotic, with a haustorium penetrating a cell. The sporangia of Dangeardia are located in the gelatinous sheath of the volvocid. Lagenidium trichophryarum, which belongs near the Chytridiales in the Ancylistales, was described by Goénnert (1935) in Trichophrya epistylidis. The parasite, which appeared once in abundance, was fatal to the suctorian. Lagenidium is rare in Protozoa. Cook’s revision of the genus (1935), which is in the same number of the Archiv fir Protisten- kunde as Gonnert’s article, reports no species from them; the habitat ts filaments of green algae, diatoms, pollen grains, and rhizoids of mosses. Filamentous appendages on the posterior end of certain large fresh- water amoebae (Fig. 221) have long been known. Leidy (1879) ob- served them, and was uncertain as to their nature, regarding them at first as a bundle of mycelial threads dragged behind Amoeba proteus, but finally concluding that they were structural elements of the amoebae. He made the presence of these appendages diagnostic of the new genus 1066 PARASITES OF PROTOZOA Ouramoeba. Korotneff (1880), encountering an amoeba with similar posterior prolongations, created for it a new genus, Longicauda. Penard (1902), as others had already suggested, considered filaments on Amoeba nobilis to be parasites, and reported observation of appendages of different types on A. proteus and A. vespertilio. He noted long, fine filaments also on Pelomyxa tertia. He recounted these observations again later (1905c), and stated that the fungi probably belong very close to the Figure 221. Filamentous fungi (Amoebophilus) parasitic on Amoeba proteus (Oura- moeba vorax Leidy). (After Leidy, 1879.) Entomophthorales or Saprolegniales, resembling in the former group Em pusa, in the latter Leptomitus lacteus. Dangeard (1910) studied filaments on Pelomyxa vorax, and named them Amoebophilus penardi. He gave the name Amoebophilus caudatus to the parasite described by Penard on Amoeba nobilis; and A. korotne ffi to that of “Longicauda amoebina.”’ He thought it possible that they might belong to the Asco- mycetes. Geitler (1937) studied in A. proteus what is apparently the same as Penard’s parasite (Amoebophilus caudatus Dangeard), but he made no reference to Dangeard’s account. Geitler stated that the fungus probably belongs in the Cladochytriaceae of the Chytridiales. PARASITES OF PROTOZOA 1067 Geitler found the fungi on a narrowly defined area of the body, the protruding filaments vertical to the surface of the protoplasm and from 100 to 200 y long. The filaments of a plant are non-septate and arise from a deep-staining, irregularly lobed vesicle, the haustorium, lo- cated in the endoplasm of the amoeba at the limits of the ectoplasm. From the vesicle, which was also noted by Penard, delicate hyphal threads extend through the ectoplasm to the surface of the body, where they broaden and continue as extracellular threads. Basal branching 1s common, and there may also be secondary branching. Infected amoebae show a polar organization, with the fungi at the posterior end; this polarity, Geitler concluded, is probably not called forth by the infec- tion, but was present before. Filaments seen by Penard (1905c) on Amoeba proteus, the same as those shown by Leidy on ‘'Ouramoeba botulicauda,’’ were, when of some length, divided by constrictions into two or more equal parts. The figure of a filament on A. vespertilio shows constrictions marking short subdivisions. Dangeard (1910), who observed nuclei in the fila- ments on Pelomyxa vorax (Amoebophilus penard7), also figured con- strictions demarcating long sections, which he considered to represent budding. The incidence of these parasitic fungi on amoebae is sometimes high. At one period Geitler found 95 percent of A. proteus infected; later the incidence declined. Penard (1902) found the fungi on three out of five A. nobilis, and Dangeard (1910) ona rather large number of Pelomyxa. A filamentous, cylindrical fungus, 0.75 1 in diameter, was found by the writer fairly frequently in certain material of Devescovina hawatr- ensis from Neotermes connexus. A large part or all of the filament was embedded in the cytoplasm, but characteristically a part projected beyond the surface. The surface was penetrated at any point. Fungi which develop in the cytoplasm and then extend projections beyond the surface were described by Penard (1912) in Amoeba terri- cola and A. alba. These parasites, which he found usually fatal to the amoebae, belonged, he thought, in or near the Saprolegniaceae. A fungus assigned to the Saprolegniales was found by Sand (1899) infesting more than half the specimens of Acineta tuberosa collected from the sea at Roscoff. Developing within the cytoplasm, the parasite soon destroyed the cell and formed isolated spheres in the empty lorica. These developed into long tubes, wound in the lorica or projecting free. 1068 PARASITES. OF PROTOZOA One of the tubes terminated in a large, spherical sporangium. Leger and Duboscq (1909c) reported parasitic fungi which developed a mycelium in cysts of the gregarine Nina gracilis. Galleries excavated in the non-protoplasmic parts of calcareous tests of Foraminifera are the work of an organism behaving somewhat in the manner of the mycelium of certain fungi, according to Douvillé (1930). The relationship of this organism to calcareous shells suggests the habitat of Didymella conchae, an ascomycete which Bonar (1936) described from the shells of marine gasteropods and barnacles. PROTOZOA PHYTOMASTIGOPHORA An unusual phoretic relationship described by Penard (1904) existed between a heliozoan and an undetermined species of Chlamydomonas. He found this organism fixed to the surface of Actinosphaerium etch- hornii by its two flagella, which were applied by their full length. Often it was so abundant that the surface of the host was spotted with close-set organisms, and the heliozoan appeared covered with a green envelope. When the chlamydomonads were scattered mechanically, they later re- assembled at the surface of Actinosphaerium. Sokoloff (1933) found a euglenid flagellate, named Euglena parasitica, adherent in abundance to the surface of Volvox coenobia in a tank. There was a conical pro- longation anteriorly by which this adherence was effected; no flagella were mentioned or figured. Sokoloff did not observe the flagellate in the free state. Endozoic, colorless flagellates that probably belong to the genus Khawkinea have often been found, especially in Turbellaria, but also in rotifers, Gastrotricha, fresh-water nematodes, fresh-water oligochaetes, nudibranch eggs, and copepods. In different hosts they occur in the alimentary canal, in tissues, or in the coelom. Howland (1928) identi- fied as Astasia captiva, which Beauchamp had described from a rhabdo- coele (p. 905), an actively metabolic euglenoid flagellate, without flagellum or stigma, found in the cortical ectoplasm of Stentor coeruleus and Spirostomum ambiguum. Jahn and McKibben (1927) assigned this species to their new genus Khawkinea (see p. 907). Parasitic dinoflagellates occur in Tintinnoinea, in Radiolaria, and in other dinoflagellates. In the first two groups, as in so many Protozoa, PARASITES OF PROTOZOA 1069 development of the parasites has been mistaken for a phase of the cyclical development of the host. In the tintinnids these errors were first pointed out by Duboscq and Collin (1910); in the Radiolaria by Chatton (1920b). Chatton (1920b) established the genus Duboscquella for the para- site of tintinnids, and recorded D. tintinnicola as occurring in Codonella galea, Tintinnopsis campanula, and Favella (as Cyttarocylis) ehren- bergii. In the last ciliate, Duboscq and Collin (1910) observed the parasite in abundance at Cette. It is a subspherical body which grows to a large size (100 1.) without apparent inconvenience to the host. Re- peated division gives rise to a dense mass of gametocytes, each of which, after ejection from the host, undergoes two divisions inside or outside of the host, to produce biflagellate gametes. Hofker (1931) found Duboscquella tintinnicola in Favella ehrenbergii and F. helgolandica. Although the enigmatic organism described by Campbell (1926) as Karyoclastis tintinni is apparently not a dinoflagellate, it may be men- tioned here because of its occurrence in this same group of ciliates. Campbell found it to be primarily an intranuclear parasite of Tzntin- nopsis nucula, but, unlike most other described nuclear parasites, it has a cytoplasmic phase. In the macronucleus the parasites occur as numerous small bodies, each with a gray-staining mantle, a clear central area, and within a central granule which undergoes division. The parasites mul- tiply within the nucleus, then the membrane partially disintegrates, and the parasites emerge and form a cloud-like mass in the cytoplasm. Campbell noted that the parasites are distinct in structure from Nzcleo- phaga and Sphaerita. Further investigation is necessary to elucidate the complete life cycle and establish the systematic relationships of Karyo- clastis. Hofker (1931) found a resemblance to Karyoclastis in round bodies associated in the test, in some instances, with Tintinnopsis fum- briata; but he recognized the possibility that their occurrence was the result of a fragmentation phenomenon. Chatton (1920a, 1920b) pointed out that the so-called anisospores, or gametes, in Thalassicolla, Sphaerozoum, and Collozoum (Brandt), the origin of which in the first genus from intracapsular plasmodial masses was described by Hovasse (1923a), belong not to the radiolarians but to the parasitic dinoflagellates similar to Syndinium of the pelagic copepods. Chatton (1923) proposed the genus Merodinium for these 1070 PARASITES OF PROTOZOA organisms, establishing five species for cytoplasmic parasites of Col- lozoum, Sphaerozoum, and Myxosphaera, and a sixth species, in the subgenus Solenodinium, for the intranuclear parasite of Thalassicolla spumida. The dinoflagellate affinities of these organisms are shown by the nuclear structure, the mode of mitosis, and the morphological char- acteristics of the spores. The dinospores are reniform, constricted at the equator, and have two unequal flagella in typical dinoflagellate arrange- ment. Species of Peridinium and related dinoflagellates may be parasitized by Coccidinium, which, according to Chatton and Biecheler (1934), resembles coccidia in the vegetative and multiplicative stages, whereas the spores are typical of dinoflagellates. Chatton and Biecheler (1936) reported having observed copulation and total fusion of spores of two types in Coccrdinium mesnili, and considered this to be the first obser- vation of an indisputable sexual process in an authentic dinoflagellate. Keppen (1899) described from marine dinoflagellates (Ceratium tripos, Ceratium fusus, and Ceratocorys horrida) the parasite Hyalosac- cus cerali1, which he considered to be a parasitic rhizopod. It is impos- sible to obtain a complete understanding of the structure, life history, and relationships of the organism from Keppen’s account and illustra- tions; but certain similarities to Coccidinium are apparent in the structure and nuclear multiplication of the intracytoplasmic stages. Keppen did not describe spores. As did the French authors in Coccidinium, Keppen pointed out a resemblance of Hyalosaccus to coccidia. He considered this to be the same parasite as that observed by Biitschli (1885) in Ceratzum fusus. ZOOMASTIGOPHORA Chlamydomonads may be attacked by Colpodella pugnax, which 1s more of a predator than a parasite. Cienkowsky (1865), who first de- scribed it, found it on Chlamydomonas pulvisculus. Dangeard (1900a) studied it mainly on C, d7/l7, but remarked that it would attack more or less all species of the genus. He never, however, observed it on other Protozoa. The free-swimming Co/podella is colorless, crescentic, about 12 y in length, with a terminal flagellum. It becomes fixed to Chlamy- domonas, perforates its membrane, and within a few minutes the cyto- plasm begins to flow into Colpodella. The envelope of Chlamydomonas PARASITES OF PROTOZOA 1071 is soon emptied, and Co/podella takes on a stouter form and a green color. The substance of its prey collects in a large digestive vacuole and is absorbed. In multiplication, the organism rounds up and undergoes thrice-repeated binary fission within a membrane, from which crescentic flagellates escape. Thick-walled cysts were described by Dangeard. Hollande (1938) gave the name Colpodella raymondi to a parasite found by Raymond (1901) on Chlamydomonas. The parasite, reported Figure 222. A, B, Bodo perforans Hollande, ectoparasitic on Chilomonas paramaecium ; C, ectoparasite of Colpoda cucullus. (A, B, after Hollande, 1938; C, after Gonder, 1910.) Raymond, occurs in one to several spheroidal masses on the surface of Chlamydomonas. Exceptionally there are more than a hundred; the usual size appears to be very much less than that of C. pugnax. According to Raymond, the host appears not to suffer from its presence, at least unless the infection is very heavy. An interesting ectozoic organism on Chilomonas paramecium was named Bodo perforans by Hollande (1938). This flagellate possesses a long, slender rostrum by which it is fixed in a constant position near the anterior end of Chilomonas, at the base of the flagella (Fig. 222A, B). It has two unequal flagella, inserted at the base of the rostrum. Rarely two or three parasites are attached to one host. The rostrum, according to Hollande, penetrates the cytoplasm shallowly; and he found 1072 PARASITES: OF PROVOZOA evidence that material may be extracted from the host. Many parasitized chilomonads had lost their flagella, but otherwise they were apparently not injured. Bodo perforans was rately seen free from attachment. Gregarella fabrearum, studied by Chatton and Brachon (1936) and Chatton and Villeneuve (1937), shows few characteristics that can be used for taxonomic purposes; yet it was considered by them to be a much regressed flagellate, representative of a new group of zo6flagellates, the Apomastigina. It is reported to undergo a cycle of development as follows: ingestion by Fabrea, fixation to the wall of a vacuole, with growth and transverse division, discharge from the body and fixation to the ectoplasm around the cytopyge, where feeding and slower multi- plication take place. The fixed parasites are claviform with the large end adherent, are capable of slow changes of shape, contain large refractile inclusions, and have no flagella or other permanent differentia- tions. The parasite on Colpoda cucullus (Fig. 222C), observed in a hay infusion by Gonder (1910), recalls in some ways certain of these ectozoic flagellates. The organism has a round or oval figure, with the narrowed end extended through the pellicle. The nucleus is single, vesicular, with a large endosome—not an ordinary ciliate type. No fla- gella or cilia were seen. Gonder was vague about its relationships. Small mastigamoebae were recorded by Doflein (Lehrbuch, 1909, and later editions) as not infrequent parasites of Stentor coeruleus; and he figured an instance of heavy infection. Infected ciliates were faded and somewhat contracted, and eventually often burst. Flagellates were found in Craspedophrya rotunda and other Suctoria by Rieder (1936). The incidence was high in a culture of the first species and light in four other species. At first only a few Cras pedophrya were parasitized, but in a few days many were infected. The organisms were colorless, actively metabolic, from 6 to 11 microns in length, and had two flagella 1.5 times the body length, of which one was anterior and the other trailed. They entered the suctorian through the envelope, sometimes at the thinnest place, as over the brood chamber from which a swarmer had escaped, but also elsewhere. They were observed swim- ming about actively within the pellicle, taking up suctorian plasma, and undergoing binary fission. Eventually the flagellates often rounded up and lost the flagella; some left through the pellicle, probably in the way PARASITES OF PROTOZOA 1073 they entered. The host may die and disintegrate, according to Rieder, even when only one or two parasites are present. He considered the organism to be a strict endoparasite of Craspedophrya. A number of enigmatic forms, which at least seem to show certain flagellate relationships, may be considered here. Dangeard (1908) gave the name Lecythodytes paradoxus to a parasite of cysts of Chromulina, which decimated cultures within a few days. Within the cyst the organism is amoeboid, and grows until it occupies the whole interior. Division, he stated, results in eight, or less often four or sixteen zodspores, which escape from the cyst and may infect another host. The zodspores are elongated and narrowed at the ex- tremities, each of which, according to Dangeard, terminates in a long flagellum. Uncertain is the proper systematic position of Sporomonas infusorium, which Chatton and Lwoff (1924a) encountered in the marine ciliates Folliculina elegans, Vorticella sp., and once in Lacrymaria lagenula. Potts found it in Folliculina ampulla at Woods Hole, Massachusetts. In the cytoplasm the parasite occurs as a reniform body, provided with a long flagellum, in active rotation. It increases greatly in size, up to 70 u, and the flagellum is lost. The parasite is then expelled, and multiplication takes place only outside. There is rapidly repeated nuclear division and binary fission without growth (palintomy), resulting in small, virgulate bodies, each provided with a lateral flagellum. Chatton and Lwoff con- sidered this organism to be a flagellate, but discussed its resemblance to Chytridiales. They stated that it differs from that group in multiplying by palintomy, with rapidly repeated mitosis after growth, instead of by syntomy following nuclear multiplication accompanying growth. Mitch- ell (1928), however, described multiplication of the same type as that in Sporomonas in a chytrid of Euglena caudata; and on other grounds also it appears that the distinction is not of crucial significance for classification. The chief differences from chytrid parasites of Protozoa are the expulsion of the organism from the host before multiplication occurs and the active motility, by means of a flagellum, of the early intracytoplasmic growth stages. Georgevitch (1936a, 1936b) assigned to the genus Lezshmania, as L. esocis n. sp., a hyperparasite of Myxidium lieberkihni in the urinary bladder of pike. The intracellular phase is pyriform, with one nucleus 1074 PARASITES: OF) PROTOZOA and a rod-formed structure called by Georgevitch a blepharoplast. Growth, nuclear division, and plasmotomy result in rosettes, usually of eight individuals and a residual body. Longitudinal division also occurs. Flagellated stages were rarely found. The evidence that the parasite is Leishmania is unconvincing, and is certainly inadequate as a basis for extending the distribution of that genus to such an unusual location. SARCODINA The genus Psewdospora, which comprises parasites of true algae and of Volvocidae, has been placed in the Proteomyxa; but there appears to be good reason for accepting the suggestion of Roskin (1927) that it belongs rather in the Bistadiidae of the Amoebida. P. volvocis, first described by Cienkowski (1865), was later reported from Volvox by Robertson (1905) and Roskin (1927). It seems possible that the amoebae found by Molisch (1903) and Zacharias (1909) in Volvox minor (V. aureus) belonged to this genus, though the authors do not refer to earlier observations on parasites of Volvox, and their work is not cited in later accounts of Pseudospora. Roskin (1927) described P. eudorini from Exdorina, in which flagellate Robertson (1905) had te- ported P. volvocis. In its free-living state both these species are small, heliozoa-shaped forms, with immobile or slow-moving pseudopodia. The heliozoan form becomes amoeboid, with lobose pseudopodia, on contact with the host, which it enters. Within the coenobium, the amoeboid form engulfs the cells and undergoes repeated division. After a period, the parasite comes to the surface of the colony, and there is rapid transition to a form with two relatively long flagella. The organism may lose the flagella and become amoeboid, may form cysts and the free heliozoan form, and the heliozoan type may transform into a flagellate, as well as into an amoeboid form. It is generally considered that confusion with parasites is the basis of the accounts of complicated life cycles in Arcella. The earlier ob- servations on this testacean were discussed by Dangeard (1910), who offered convincing evidence that the small amoeboid bodies, supposed to be produced in numbers by repeated exogenous or simultaneous en- dogenous budding (Awerinzew, 1906; Elpatiewsky, 1907; Swarczewsky, 1908), and variously interpreted by authors as reproductive phases of PARASITES OF PROTOZOA 1075 the life cycle, are really parasites. The amoebulae were reported to take on a heliozoa-like form on becoming free; Dangeard noted a similarity to Nuclearia. An amoeboid parasite of Arcel/a was reported by Gruber (1892), and evidently it is quite common, so many have been the ac- counts of it. Doflein (Reichenow ed., 1927-29) and Deflandre (1928) were inclined to accept the parasite interpretation of these supposed reproductive bodies; but Cavallini (1926a, 1926b), without reference to Dangeard’s paper, reported in Arcella vulgaris and Centropyxis aculeata division of the protoplasmic body into many small amoebae, which leave the shell and develop into the mature testaceans. Never- theless, it is probable that binary fission is, as Deflandre (1928) stated, the only mode of reproduction that has been satisfactorily demonstrated in Arcella. Penard (1912) found what he considered to be small parasitic amoebae in Amoeba terricola and other species. They were often ob- served moving actively within the pellicle of dead amoebae, from which they eventually emerged and moved about freely, feeding on bacteria. Penard found indication that these are parasites, the development of which begins in the body of the large amoebae, but proof of this is lacking. There is, to the writer’s knowledge, only one record of amoebae parasitic in a free-living ciliate. Chatton (1910) observed a very small species living as a true parasite in Trichodina labrorum from the rock- fish. Opalinid ciliates are, however, not uncommonly parasitized. The hyperparasites of Opalinidae (Fig. 223A) resemble E. ranarum (Carini and Reichenow, 1935; Brumpt and Lavier, 1936; Stabler and Chen, 1936). Those in different opalinids have not been found to show any taxonomic distinctions; and the systematic name to be used, for some forms at least, is Entamoeba paulista (Carini); to be used, that is, if this amoeba is truly an independent species. Carini and Reichenow were of the opinion that the hyperparasite is either identical with En- tamoeba ranarum ot is a race or species derived from this. Stabler and Chen considered the question of the amoeba’s synonymy with E. ranarum to be still open. Brumpt and Lavier, though recognizing a probable dis- tinction from E. ranarum, discussed the relationship as paranéoxénie, in which an intestinal parasite of the amphibian attacks another parasite that accompanies it, which then becomes a subhost. From the standpoint 1076 PARASITES OF PROTOZOA of general host-parasite relationships, it seems to the writer unlikely that the same amoeba both parasitizes opalinids and lives in the in- testinal lumen; certainly, to date, there is no proof of either possibility. Published reports seem to indicate a greater prevalence of entamoebae in Zelleriella than in other Opalinidae, but they have been found also in Protoopalina, Cepedea, and Opalina (Chen and Stabler, 1936). The geographical distribution of the amphibian hosts of parasitized Opa- linidae has been found to be very wide, but Chen and Stabler stated that “only certain species of anurans and a certain percentage of individuals of a given species harbor the parasitized opalinids.” In a given host, the percentage of ciliates containing hyperparasites varies greatly from none to quite all, as noted by Carini (1933) and Brumpt and Lavier (1936) in Zelleriella of Paludicola signifera, and by Chen and Stabler (1936) in Zelleriella of Bufo marinus. The number of amoebae in a ciliate ranges from one or very few up to a condition in which the cytoplasm of the host is almost completely filled, more than a hundred being present. They have been found mainly in the trophozoites, but may occur also in precystic and encysted ciliates, according to Chen and Stabler (1936), though Brumpt and Lavier (1936) stated that they had never found them in the cysts. Chen and Stabler pointed out the im- portance of the parasite in cysts, in establishing infections in a new generation of the hosts. In opalinids, entamoebae occur either as vegetative forms or as cysts. Stabler and Chen (1936) stated that they apparently feed on the endo- spherules, and they observed instances of mitotic division. It has been noted (Stabler, 1933; Carini and Reichenow, 1935; Stabler and Chen, 1936) that the cysts in the ciliates are all uninucleate; the same observers reported binucleate and quadrinucleate cysts outside of the hosts. It seems, in general, that all the parasites in one ciliate are in the same stage of development; but Brumpt and Lavier (1936) gave an uncon- vincing figure of a Zelleriella said to contain a uninucleate and a bi- nucleate vegetative form, a precystic form, and a uninucleate cyst. Furthermore, in their statement that certain cysts contain two or four nuclei, though most are uninucleate, it is not clear whether or not they found multinucleate cysts in the opalinid host. It has been noted that the parasites seem to have no injurious effect on the host. The fact that parasitized ciliates may yet undergo normal divi- PARASITES ‘OF PROTOZOA 1077 sion (Brumpt and Lavier, 1936; Stabler and Chen, 1936) is the most important evidence for this benignity. No strictly parasitic relationship has been established in any member of the group Heliozoa proper, but Wetzel (1925) discussed as tempo- rary parasitism the association between Raphidiocystis infestans and cili- ates (Fig. 223B). This organism is normally predatory on flagellates and small Infusoria, but when it attacks larger ciliates, such as Para- mecium, Colpidium, Glaucoma, Nassula, and Trachelius, it passes, Dy INNA 4 mn Figure 223. A, trophozoites of Entamoeba sp. in Zelleriella (after Stabler and Chen, 1936); B, Raphidiocystis infestans Wetzel on Paramecium (After Wetzel, 1925.) according to Wetzel, to the parasitic manner of life. The heliozoans become attached by means of pseudopodia to various parts of the body, those pseudopodia gradually shorten, and eventually they lie very flat on the surface. The body of Paramecium may be completely enclosed by many fused Raphidiocystis, which extract dissolved nutriment until only a remnant of the ciliate is left—a process which requires from twenty or thirty minutes to two days—after which the heliozoa disperse. Wetzel discussed the significance of this type of relationship in the phylogenetic origin of parasitism as transitional between strict preda- tism and pure parasitism. 1078 PARASITES OF PROTOZOA Ivanic (1936) stated that the parasite of Entamoeba histolytica, which he described as Entamoebophaga hominis, shows affinities to the Myce- tozoa, but this is hardly apparent from his unconvincing account of the structure and life history. The earliest stages are reported to occur within the host cyst, and growth leads to an amoeboid body. At first uninucleate, this becomes multinucleate. When the cytoplasm of the host cyst has been largely consumed, the parasite breaks out and carries on for a time an active, free-living existence in the intestinal lumen, as an amoeboid organism. In this phase there is nuclear multiplication, binary fission, growth to a giant, multinucleate plasmodium, and endogenous budding. Ivani¢ found evidence that the organism was originally a commensal of the human intestine before it became a parasite of E. histolytica. This bizarre account probably contains a good deal of mis- interpretation and confusion of distinct organisms. SPOROZOA The sporozoan parasites reported in Protozoa belong for the most part to the Microsporidia and Haplosporidia. Dogiel (1906) assigned to the coccidia a parasite, named Hyalosphaera gregarinicola, of a gre- garine from a holothurian. Caullery and Mesnil (1919) considered this systematic determination doubtful, but were certain that the parasite 1s not a Metchnikovella. Dogiel described macrogametes, microgametocytes, and sporulation, but did not observe schizogony. Four microsporidian parasites of Protozoa were listed by Kudo (1924). Three of these are species of Nosema: N. marionis (Thélohan, 1895) Stempell, 1919, in the myxosporidian Ceratomyxa (Le ptotheca) coris from the gall bladder of Coris julis and C. giofredi; N. balantidu Lutz and Splendore, 1908, in Balantidium sp. from Bufo marinus; and N. frenzelinae Leger and Duboscq, 1909, in the gregarine Frenzelina conformis from Pachygrapsus marmoratus. The last species shows a certain amount of correlation with the life cycle of the host, in that sporulation occurs at the moment of encystation of the gregarine. The gregarines develop normally up to a certain point; then the formation of gametes does not take place (Leger and Duboscq, 1909a, 1909c). The fourth species, Perezia lankesteriae, also parasitizes a gregarine, Lankes- teria ascidiae from Ciona intestinalis (Leger and Duboscq, 1909b). Microsporidia are probably much more widespread as parasites of PARASITES OF PROTOZOA 1079 Protozoa than the published accounts indicate. Duboscq and Grassé (1927) showed certain parasites in “Devescovina’ hill: which they considered to be possibly Microsporidia. The organism found by Kirby (1932a) in Trichonympha magna gives certain indications of micro- sporidian affinities (Fig. 219H, 1). An organism with resemblances to Nosema, though enigmatic in relationship, has been observed by the writer in Gigantomonas herculea ftom the termite Hodotermes mos- sambicus. All the above-mentioned Microsporidia are hyperparasites, but there is probably at least one recorded instance of their occurrence in a free-living ciliate. A number of authors have reported “nemato- cysts” in the large vorticellid Epistylis (Campanella) umbellaria; these were discovered by Claparéde and Lachmann (1858). They are arranged in pairs in the ciliate, but are not always present. Fauré-Fremiet (1913), although having often observed Campanella, found ‘‘nematocysts” only once. Chatton (1914) suggested that the structures belonged not to the vorticellid but to a cnidosporidian parasite, and in a recent note Kruger (1933) supported this view. Kriiger observed in the cytoplasm of the ciliate granules that he thought might be nuclei of developmental stages of the parasite. From the standpoint of host-specificity, the Metchnikovellidae are of particular interest, for all members of this family, and there are many, occur in gregarines. The first metchnikovellid was seen by Clapareéde, but he failed to interpret it correctly, mistaking the cysts for spores of the gregarine (Caullery and Mesnil, 1919, p. 232.) This group of Haplosporidia has been studied chiefly by Caullery and Mesnil (1897, 1914, 1919), but contributions have been made also by Awerinzew (1908), Dogiel (1922b), and Schereschewsky (1924). An account of the life cycles of two species of Amphiacantha has been prepared by Stubblefield (MS). MacKinnon and Ray (1931) reported some ob- servations on species of Mefchnikovella from two species of Polyrhab- dina at Plymouth; and Ganapati and Atyar (1937) noted the occurrence of Metchnikovella in Lecudina brasili from a species of Lumbrinereis at Adyar. In the absence of any description or figure, it is not certain that this is not a species of Amphiacantha, as found in related gregarines in Lumbriconerezs elsewhere. Stubblefield (MS) listed twenty species, including the two new spe- cies of Amphiacantha recognized by him, in four genera. The largest 1080 PARASITES OF PROTOZOA genus is Metchnikovella Caullery and Mesnil, with thirteen species; there are three species of Amphiamblys C. and M., three of Am phia- cantha C. and M., and one of Caulleryella Dogiel. All the grega- tines that have been found to contain these hyperparasites occur in annelids, and all but one in marine polychaetes. This one, Metchnikovella hesse? Mesnil, 1908, is found in a monocystid gregarine of the terrestrial oligochaete Fridericia polycheta. The parasitized gregarines belong to various groups, and, according to Caullery and Mesnil (1919), there is no parallelism between the structure of the Metchnikovellidae and that of the gregarine hosts. The host-specificity is apparently on an ethological rather than a phylogenetic basis. The species of Am phiacantha, however, have been found in gregarines of the genus Ophiodina (Lecudina) ot related forms in Lumbriconereis in France and California. Caullery and Mesnil (1919) stated that when there is an infection, the greater part of the gregarines of a host are invaded. Stubblefield (MS) found a high frequency of Amphiacantha in about 20 percent of the worms collected, almost all of which contained gregarines. Published literature gives little information about details of the life cycles of Metchnikovellidae. Caullery and Mesnil (1919) regarded the individualized, nucleated bodies enclosed by the cyst membrane as spores (““germes sporaux”’). When the cysts are ingested by an annelid, these are released in the digestive tract, and penetrate into the cytoplasm of the gregarines. Growth and nuclear division lead in some instances to multinucleate plasmodia. In other instances there are numerous indi- vidual, uninucleate bodies, isolated or arranged in series. Caullery and Mesnil supposed that cysts develop by the formation of a membrane around groups of these cells or the plasmodium. The cyst contents 1s thus either multinucleate or in individualized uninucleate bodies from the beginning. Such a manner of cyst formation is difficult to understand. Stubblefield (MS) prepared an account of the life cycle of Amphia- cantha, which is in closer agreement with that of Haplosporidium. He found evidence for the penetration of the gregarine by an active sporozoite; the growth of the sporozoite, followed by schizogony, to produce trophozoites; the development of the trophozoite into a cyst, which is at first uninculate; nuclear and cytoplasmic division, to pro- duce bodies in the cyst (Fig. 224), which he considered to be gametocytes; the release of these by the rupture of the cyst within the PARASITES OF PROTOZOA 1081 gregarine, after which they undergo reduction, producing gametes which fuse; and finally the development of sporozoites from the zygote. Stubble- field’s observation that cysts rupture within the gregarine host ts in Figure 224. Cysts of two species of Amphiacantha, metchnikovellids parasitic in the gregarine Ophiodina elongata from Lumbricoinereis. (After Stubblefield, MS.) agreement with the statement of Mackinnon and Ray (1931) that the “spores” of Metchnikovella caulleryi have been seen escaping from the cysts into the endoplasm of the gregarines. Caullery and Mesnil stated that Metchnikovellidae seem to have little pathogenic action on the host, particularly in the vegetative stages. What injury there is, is mechanical, when infection is heavy. Ganapati and Aiyar (1937) noted that the entire cytoplasm may be packed with cysts, and 1082 PARASITES; OF) PROTOZOA the body become much misshapen, the gregarine nucleus seemingly degenerating. Stubblefield, however, found that more than six cysts rarely occur in a gregarine. Caullery and Mesnil believed that heavily parasitized gregarines are incapable of completing their sexual develop- ment; and Ganapati and Aiyar remarked that parasitized gregarines were not observed to associate. The affinities of the Metchnikovellidae are uncertain. They have been related to fungi (Chatton, 1913, yeasts), to Microsporidia (Schere- schewsky, 1924), and to Haplosporidia (Awerinzew, 1908). Caullery and Mesnil (1919), while remarking on a certain similarity in nuclear structure to Myxomycetes and Chytridiales, concluded that they are isolated among the lower Protista. Doflein (Reichenow ed., 1927-29) accepted their allocation to the Haplosporidia, and this position was sup- ported by Stubblefield (MS). Caullery and Mesnil (1919) provisionally designated as Bertramza selenidicola a parasite of a species of Selenidium from certain poly- chaetes, and reported a related parasite in Selentdium virgula. Other species of Bertramia are parasites in the body cavities of worms and rotifers. Another parasite with apparent haplosporidian affinities, but unlike Metchnikovella, was observed in a species of Polyrhabdina. It existed as isolated granules and multinucleate masses, the schizonts and sporonts; and as separate ovoid bodies, not enclosed in a cyst, which were evidently spores. Elmassian (1909) found a hyperparasite, Zoomyxa legerz, in Exmeria rouxi, which causes fatal coccidiosis in tench. He considered this to be a haplosporidian, but also discussed its similarities to lower Mycetozoa. It is likely that his account is at least in part incorrect. The parasite 1s said to occur both on the surface of the epithelium and in the cells of Eimeria, the intracoccidian parasitism being accidental. There are said to be several types of schizogony, within coccidia or not; and in this supposed haplosporidian the author described a sexual cycle with coc- cidian-like development of microgametes and macrogametes, and the formation of resistant cysts containing from six to twelve sporozoites. The parasite has pathogenic effects on the nucleus and cytoplasm of Eimeria, causing hypertrophy and eventual dissolution of the cell. Elmassian thought that the effects are brought about by toxic secretions, which act not only on the coccidia but also on the neighboring fish cells. PARASITES OF PROTOZOA 1083 CILIOPHORA Euciliata.—There are few reports of ciliates parasitizing other Proto- zoa, except for Phtorophrya and Hypocoma. Penard (1904) found a ciliate, which seemed to resemble Blepharisma, in a large percentage of the heliozoan Raphidiophrys viridis. The intracytoplasmic forms showed different degrees of development and lived many days in isolated Heliozoa. An immobile organism, with a large contractile vacuole but no cilia or flagella, was found parasitic in three-fourths of a large number of Pseudodifflugia horrida by Penard (1905a). This, he stated, suggested the larger ciliate in the heliozoan, but its affinities are uncertain. Hertwig (1876) reported that a hypotrich bored into the body of Podophrya gemmipara, in the region in which the body is joined by the stalk, and destroyed the acinetid. A number of apostomatous ciliates are parasitic in other Foettin- geriidae. The most completely known of these is Phtorophrya insidiosa Chatton, A. Lwoff and M. Lwoff, 1930, which is parasitic on Gymno- dinioides corophii. The phoront of Phtorophrya is attached to the phoront of Gymnodinioides, which occurs on Corophium acutum. The body of the parasite leaves the phoretic cyst and introduces itself into the body of its host, becoming a parasitic trophont. It grows rapidly, ingesting the cytoplasm of its host, and soon comes to occupy entirely the otherwise empty cyst of Gymnodinioides. By division, four to eight small ciliates, the tomites, are produced. These escape from the host cyst and swim actively in search of another phoront of Gymnodinioides. Chatton and Lwoff (1930, 1935) described also the following in- completely known species of this genus of ciliate parasites: Phtorophrya mendax in the phoronts of Gymnodinioides inkystans; P. fallax in this same host species; P. steweri in Vampyrophrya (2) steuert; P. bathy pela- gica in Vampyrophrya bathy pelagica. The Hypocomidae, like most other Thigmotricha, occur on bivalves or snails, except for species of the genus Hypocoma, which are parasitic on other Protozoa. Hypocoma parasitica (Fig. 199G-1) was found by Gruber (1884) and Plate (1888) on marine vorticellids, especially Zoothamnium, on the coast of Italy. Plate recognized a second species, H. (“‘Acinetoides”) zoothamni. The ciliates occur firmly fixed to the host, and suck out the contents of the zodids. Hypocoma acinetarum Collin is ectoparasitic on Suctoria. Collin (1907) found it on various 1084 PARASITES OF PROTOZOA occasions on Ephelota gemmipara and Acineta papillifera; and Chatton and A. Lwoff (1924b) encountered it on Trichophrya salparum., In Ephelota it attacks chiefly the region in which the stalk is attached to the body. It sucks out plasma, and its presence leads to fragmentation of the nucleus and degeneration of the whole cytoplasmic mass. The parasite then detaches and swims to another suctorian. Hypocoma ascidiarum Collin was found on a tunicate, but probably actually is a parasite of Trichophrya salparum, and may not be different specifically from H. acinetarum (Chatton and A. Lwoff, 1924b). Ectozoic Suctoria—In connection with the relationship between ex- ternally attached Suctoria and their ciliophoran hosts, we must keep in mind the fact that phoresy is widespread among Suctoria. Many forms occur attached to other organisms, and often a species has been found only on a particular host species. The host is not directly concerned in the nutritive processes of the suctorian. Ciliophora may, like many Meta- zoa, serve as hosts for these ectocommensals. Examples of such phoretic forms are Ophryocephalus capitatum Wailes on species of Ephelota; Urnula epistylidis Claparéde and Lachmann on Epistylis and other Suc- toria (see Gonnert, 1935); Tokophrya quadripartita (Cl. and L.) on Epistylis; Trichophrya epistylidis (Cl. and L.); Metacineta mystacina (Ehrbg.) on Carchesium,; and Tokophrya carchesu (Cl. and L.) on Carchesium. Ectocommensalism in such attached forms may be obligatory or facultative. Pseudogemma Collin is more closely adapted to an ectoparasitic man- ner of life on other Suctoria. Reproduction is by internal embryos, and fixation to the host is by a short, stout peduncle embedded in the cyto- plasm. Tentacles are absent, and Collin (1912) considered it possible that the fixation organelle has an absorptive function. Collin listed three species: P. fraiponti Collin, 1909, on Acineta dirisa; P. pachystyla Collin, 1912, on Acineta tuberosa; and P. keppeni Collin, 1912, on Acineta papillifera. The last species is said to have a rounded form and apparently no pedicle, and its location in its host is sometimes external and sometimes almost entirely internal. Collin (1912) believed that it furnishes a natural transition from Psexdogemma to Endosphaera. The species of Allantosoma occur in the intestine of the horse, an endozoic habitat which is unique for Suctoria. According to Hsiung (1928), the species A. dicorniger is strictly a lumen-dweller, and is PARASITES OF PROTOZOA 1085 not attached to any other organism. The other two species have certain relationships to ciliates, but apparently that is only occasional. Hsiung wrote of A. zntestinalis that some were attached to Cycloposthium bipal- matum and Blepharocorys curvigula; Gassovsky (1919) recorded the species as occurring in the colon, rarely the caecum, of horses, without mention of any attachment to ciliates. A. brevicorniger, Hsiung states, is ‘‘often found attached to the body of the ciliate Paraisotricha colpoidea by one tentacle.” Apparently these Suctoria prey upon the ciliates, but are not constantly attached, as obligatory ectoparasites would be. The only account of a suctorian clearly ectoparasitic on Euciliata is Chatton and A. Lwoff’s (1927) description of Pottsia infusoriorum. The chief host is Follzculina ampulla, but it has been found also on Cothurnia socialis. The parasites may occur in numbers on the body of Folliculina, within the lorica. There are four tentacles at one end, on the surface in contact with the host, prolonged rather deeply into the body of the host. Embryos develop endogenously, swim actively, and become fixed to the body of Folliculma. On different occasions, from none to 75 percent of the heterotrichs have been found parasitized with as many as twenty- two parasites. When the number of Pofts/a is large, the host undergoes degeneration. The parasites may survive for a time among the remains, but eventually themselves disintegrate. Tachyblaston ephelotensis, as described by Martin (1909), has a curious life cycle, involving both an external phoretic existence with multiplication, and an intracytoplasmic parasitism, also with multiplica- tion. It seems not impossible that reinvestigation will show that two organisms have been confused in this cycle, since it is so unlike the life histories of other Suctoria. The intracellular phases occur as rounded bodies in Ephelota gemmipara. There is equal division, fol- lowed by the formation of buds. Ciliated “‘spores’ escape from the host and after a brief period of existence become attached to the stalk of Ephelota, developing a stalked lorica. The fixed form undergoes rapid budding. Each bud is provided with a single tentacle, with the aid of which, together with “‘euglenoid changes of shape,” the bud travels up the stalk to penetrate into the body of Ephelota. The internal parasitic phase destroys the cytoplasm of the host. Endozoi Suctoria—The Suctoria that occur as internal parasites of other Ciliophora, and have a wide variety of hosts, belong to the genera 1086 PARASITES OF PROTOZOA Sphaerophyra and Endosphaera. In connection with them, it is interesting to consider the important rdle they have played in the development of protozodlogy. The Acineta theory and the embryo theory of ciliate devel- opment held an important place in the thinking of protozodlogists in the third quarter of the nineteenth century. Stein’s Acineta theory was in the first instance not related to parasitic Suctoria. He came (1849, 1854) to the conclusion that free-living acinetids are the result of metamorphosis of vorticellids, and that they give rise to embryos from which the vorticella form is again produced. This embryo production, of course, is the result of the internal budding process characteristic of acinetids. This theory was successfully attacked by Cienkowski (1855a), Lachmann (1856), and Claparéde and Lach- mann (1860-61). Stein later (1859) modified the Acineta theory as it was originally stated, but still did not admit that acinetids are inde- pendent organisms. The embryos of various Infusoria, he said, have all the characteristics of acinetids; and he believed that various higher Infusoria in their development pass through Acineta-like phases; for example, that podophryids were developmental phases of Paramecium. Authors credit Focke (1844) with the first observation of the so-called motile embryos. He discovered them in Paramecium bursaria, in which they were soon found by many other observers. They were found also in a variety of other Euciliata. As late as 1867, Stein could state that “today no one can doubt that those Infusoria whose reproductive organ- ization consists of nucleolus and nucleus are in fact hermaphrodites, the nucleus playing the rdle of a female, the nucleolus of a male sex organ”; and could maintain that the embryonal spheres were produced from the nucleus. Stein’s thesis, however, had already been discredited. Claparéde and Lachmann (1858-59) had described Sphaerophrya pusilla in water, associated with numerous oxytrichids; yet they were not firm in their opinion that Sphaerophrya might not be an embryo of Oxytricha. It was the view of Balbiani (1860) that the so-called embryos of ciliates were parasites belonging to the genus Sphaerophrya; and in support of this he adduced his observations on entry into ciliates, and on the spread of an infection among Paramecium by the introduction into a sound culture of a few infected ciliates. Metschnikoff (1864) observed the cycle, from separation from one Paramecium host through entry into another, and PARASITES OF PROTOZOA 1087 considered the parasite nature of the so-called embryos to be completely proved. Sphaerophrya shows suctorian characteristics in the presence of tenta- cles. The so-called embryos of certain vorticellids, however, do not have this characteristic; they are simple spherical or ovoidal bodies with equatorial bands of cilia. Stein (1867), in his efforts to combat Balbiant, had only weak arguments against the parasitic nature of the Sphaero- phyra-type ‘embryos’; but he was firm in his conviction that the “em- bryos” of vorticellids (Epistylzs plicatilzs) could not be parasitic Infu- soria. Such a concept, he stated, would be ludicrous. Engelmann (1876), reviewing the whole question in support of the parasite theory (which he had vigorously opposed in 1862), reported having observed the entry of the supposed non-tentaculated embryos of Vortscella microstoma into that host. Thus he proved the parasitic nature of that organism also, a view also stated by Biitschli (1876), and gave it the name Endos phaera. The endozoic forms of Sphaerophrya are but little modified in conse- quence of parasitism, and the majority of species of the genus are entirely free-living. S. stentor7s Maupas is free-living or parasitic in species of Stentor; recently Kalmus (1928) reported it in S. roeselz, The parasites of other ciliates have all been placed in a second species, which also is either free-living or endozoic. Biitschli (1889) and Sand (1899) identified this second species with Claparéde and Lachmann’s free-living S. pusilla. Collin (1912), however, considered it to be S. so/ Metchnikoff which also was originally described as a strictly free-living species. Sand regarded S. so/ as a synonym of S. pusilla. Species of the genus Sphaerophrya differ from those of Podophrya in the absence of a stalk. The body is spheroidal or ellipsoidal, and tentacles radiate from the entire surface. Reproduction is by equal or unequal fission or by external budding, except in S. stentoris, which 1s reported to show a transition to internal budding. The free-swimming forms produced by budding are provided with cilia that are localized at one extremity, in a girdle, or generally distributed; and they possess tentacles. This form, in parasitic phases, penetrates the surface of a ciliate and takes up a position in the cytoplasm, losing cilia and tentacles. There reproduction by division and budding takes place. Endosphaera (Fig. 225) has become more closely adapted to para- sitism. It does not occur as a free-living organism, except briefly in the 1088 PARASITES OF PROTOZOA motile phase that passes from one host to another; and it has no tentacles at any time. Endosphaera has been found in vorticellids of the genera Vorticella, Zoothamnium, Epistylis, Carchesium, Trichodina, and O pisthonecta. All these have been assigned to the species E. engelmanni Entz, the most adequate study of which was published by Lynch and Noble (1931). Génnert (1935) described E. multifilis, reporting tt from the Suctoria Lernaeophrya capitata Perez, Trichophrya epistylidis Cl. and L., Tokophryidae, and Dendrosoma,; and from vorticellids. Figure 225. Opisthonecta henneguyi, parasitized by Endosphaera engelmanni. End. emb., Endosphaera containing an embryo; dis. emb., embryo being discharged through birth pore. (After Lynch and Noble, 1931.) Lynch and Noble found a high incidence of infection in O pssthonecta henneguyi, with as many as twelve parasites, most of which contained one or occasionally two or three internal embryos. They found each parasite to be attached to the pellicle of the host by a short stalk, per- forated by a canal terminating in a birth bore. The spherical embryo, provided with three equatorial bands of cilia, was discharged through this pore. Embryos were observed to attach themselves to the host, and PARASITES OF PROTOZOA 1089 successive stages of penetration were studied in preparations. The authors found no evidence that the parasite pushed an extensible pellicle before it, forming an invaginated chamber in which it dwelt, such as was described by Balbiani (1860) and Biitschli (1876) in Sphaerophrya. Endos phaera was observed in cysts of O pisthonecta, which could account for the survival of the parasite under unfavorable conditions. The embryos of E. mu/tifiliis GOnnert have five bands of cilia. Génnert (1935) observed penetration into Lernaeophrya, preceded by the re- sorption of cilia and the development of a long, mobile, penetrating protoplasmic process. He observed no canal connecting the internal parasite to the surface of the host. Exdosphaera lives, he stated, four or five days, and an embryo may be produced every half hour. Sphaerophrya and Endosphaera appear to be relatively benign para- sites, except when present in large numbers. The effect is then evidently mechanical. Balbiani (1860) remarked that oxytrichids with more than fifty parasites were greatly swollen and deformed, but that ordinarily the host seemed to be not at all inconvenienced. Génnert found that Endo- Sphaera, when present singly, had slight effect on the host, but that the host often perished from multiple infection. THE GENUS AMOEBOPHRYA KOEPPEN Amoebophrya is even more of a zodlogical enigma than is Sticho- lonche, one of its hosts, which Korotneff (1891) wrote of as a “‘zodlogi- cal paradox.”” A modern study of the structure and development of the organism, which would throw light on its affinities, is much to be desired. The evidence that it is a suctorian, accepted by Koeppen (1894), Bor- gett (1897), Sand (1899), and Hartog (1909, Cambridge Natural History), is not convincing. Its assignment to the Mesozoa, made by Korotneff (1891) and Neresheimer (1904, 1908, and later) and agreed to by Collin (1912), does little more than emphasize its enigmatic qualities. Hertwig (1879) described what he regarded as a very peculiar nuclear form within the central capsule of three species of acanthometrid Radio- laria. (Fig. 226D). He stated that he found this body twice in Acantho- Staurus purpurascens and once each in Acanthometra serrata and A. claparédei; and he showed what is doubtless the same thing in Am phi- lonche belonoides. He described this as a large vesicle containing a very 1090 PARASITES OF PROTOZOA large nucleolus, around the sharpened end of which was a conical struc- ture, the membrane of which was marked by circular striations. Fol (1883) found structures, which he described as analogous to those seen by Hertwig, in the ectoplasm of Sticholonche zanclea. Some of the Radiolaria contained spherical bodies, of rather complex structure, which increased in size as the host became older, and contained an ill- defined “‘spiral body.”’ At maturity, these bodies left the host and were capable of rapid movement, comparable to that of very active Infusoria. The free organism had a spiral groove turning from left to right and was completely covered with short, fine cilia. Other specimens of the radiolarian contained a mass of globules, which increased in size and number, finally becoming in volume equal to the rest of the body. Fol advanced two hypotheses in interpretation of these structures: one, that the globules are female reproductive elements, while the spiral body is a sort of spermatophore; the other, which he regarded as also reasonable, that the structures represent parasites. On the last supposition, he stated, it would be difficult to explain the fact that the two kinds of inclusions occur only in different individuals in approximately equal numbers. Korotneff (1891), who studied the “spiral body” in Stzcholonche obtained at Villafranca, concluded that it is a parasite and made the first suggestion as to its affinities. Believing himself to have demon- strated an endoderm of a few cellular elements and a cellular ectoderm, he considered the parasite to be closely related to the orthonectids and possibly a stage in their development. The parasites in both Stscholonche and acanthometrids were studied by Koeppen (1894), who gave them the names Amoebophrya sticho- lonchae and A. acanthometrae, and who was convinced that the organisms are acinetids. He stated that he had studied all phases of development in the same specimen. He based his taxonomic conclusion on supposed development, in the parasitic phase, of an embryo, the spiral body, by internal budding; and on the existence of tentacles for a short period after this embryo became free and lost its cilia. The so-called tentacles soon disappeared, and the body commenced to vary in form, showing slow amoeboid movements. There is no proof that the protoplasmic processes were actually tentacles; evidence is lacking that the behavior was observed repeatedly under normal conditions; and there are no support- ing illustrations. PARASITES OF PROTOZOA 1091 Borgert (1897) found parasitized Sticholonche and acanthometrids in the Gulf of Naples and prepared the most complete existing account of the organisms. Although he disagreed with many of Koeppen’s inter- pretations and found a large number of nuclei in the outer layer, he nevertheless agreed with him that Amoebophrya is a parasitic suctorian. Amoebophrya sticholonchae (Fig. 226A-C) is a common parasite of Sticholonche zanclea in the Mediterranean. Borgert found parasites / fore | (2 Bae Figure 226. Amoebophrya in Radiolaria. A, Sticholonche zanclea containing A. sticholonchae; B, longitudinal section of Amoebophrya in Sticholonche, and, in lower half of figure, section of host and its central capsule; C, A. sticholonchae emerged from its host; D, A. acanthometrae in Acanthometra serrata. (A-C, after Borgert, 1897; D, after Hertwig, 1879.) only in the latter part of March, 1895, though the radiolarian was abun- dant also before and after that period. In its host (Fig. 226A) it 1s an approximately spherical body, located on the concave side of the capsule, transparent, and pale yellowish in color. Within the sphere is a conical body, the point of which is directed toward the body surface of the host (Fig. 226B). The outer surface of the cone is marked by furrows in a close-set, left-wound spiral. The outer surface of the cone is continuous at its base with the inner surface of the sphere, and the spiral furrow continues on the latter. The form of the parasite in this stage has been compared to that of a half-invaginated glove finger. Bor- gert described a large number of very small nuclei arranged in rows between the furrows. In younger parasites there were fewer nuclei, and in an appendix he reported having found a few individuals with single 1092 PARASITES OF “PROTOZOA large nuclei. No cell boundaries were seen, and the nuclei varied in size. The outer layer of the body does not have an epithelial structure; and perhaps the evidence for the nuclear nature of the inclusions is inconclusive. The parasites can easily be induced to leave the host. Borgert found it sufficient to put Stzcholonche in a small amount of water on a slide, when escape was apparently stimulated by the increase in salinity and possibly in temperature. At the beginning of the transformation to the free stage, the tip of the conical part breaks through the surrounding sphere, and cilia appear and become active. The entire body, having become everted, emerges and swims actively in the water. Its form 1s elongated and more or less cylindrical, and it possesses a spiral furrow in which arise abundant small cilia (Fig. 226C). In the interior is a cavity, larger in younger specimens, reduced to a tubular form in older ones, which sometimes is open at the posterior end of the body. Amoebophrya acanthometrae was found in four acanthometrids by Hertwig, in two others by Haeckel, and in a seventh species by Borgert. Borgert stated the probability that the parasite will be found to occur in all acanthometrids the skeletal structure of which permits. He observed it only in uninucleate phases of the host. In 1895 at Naples, after Amoebophrya disappeared from Sticholonche, parasites were found re- peatedly in acanthometrids. Unlike the other species, A. acanthometrae occurs within the central capsule. According to Borgert, it encloses the nucleus of the radiolarian; but this fact does not discommode the latter. Nuclei are extraordinarily small (up to 1 to 2 y), and were not observed at all in some, especially young, specimens. Emergence of the free phase, which is so easy to observe in A. sticholonchae, happens only occasionally. Apparently the nucleus of the host is removed in this process. The free form has a plumper figure than that from Sticholonche, and the cilia are better developed. There remain to be considered the groups of small spherules which occur usually in specimens of Sticholonche without Amoebophrya, though sometimes, contrary to the opinion of Fol (1883), the two are found in a single host. Younger stages, according to Borgert, consist of a spherical protoplasmic mass with a few spherical nuclei. A great number of small nuclei result from division of these. Eventually these nuclei become the center of vesicles, which become free in the host PARASITES OF PROTOZOA 1093 cytoplasm by dissolution of the earlier common plasma mass. Borgert regarded these bodies as parasites of the radiolaria, unrelated to Amoe- bophrya. Though probably it is only an analogy, certain features in their development suggest the life history of Sphaerita. It appears from statements by Neresheimer (1904, 1908) that Amoe- bophrya is not restricted entirely to Radiolaria. Doflein, he wrote, showed him preparations of Noctiluca miliaris in which the parasite was present. METAZOA A number of rotifers live attached to other animals as ectocommensals or ectoparasites. Ehrenberg (1838) found Proales petromyzon (Ehrbg.) and its eggs attached to the branched vorticellids Epzstylis digitalis, Car- chesium polypinum, and Zoothamnium geniculatum, and stated that it devours the vorticellid. Wesenberg-Lund (1929) showed it and its eggs on Zoothamnium. It is a predator rather than a parasite, but differs from ordinary predators in its attachment. Hudson and Gosse (1889), however, found it always free, though often in close association with Epistylis and Carchesium. Approaching closer to parasitism are certain species of Proales, which live in certain algae and Protozoa. P. werneckui (Ehrbg.) occurs rather commonly in galls on Vaucheria; P. parasita (Ehrbg.) is parasitic in Volvox; and P. latrunculus Penard invades the heliozoan Acanthocystis turfacea. Proales parasita was found by Ehrenberg (1838) and Cohn (1858) in Volvox. Plate (1886) described Hertwigia volvocicola from Volvox globator, considering this to be a different species from the preceding. It is listed as a synonym of P. parasita by Hudson and Gosse (1889), but Wesenberg-Lund (1929) considered it again as a separate species. Whether different or not, the habits of the forms are the same. They swim about within Volvox coenobia and feed on the cells. The males live only a day or two, remaining entirely within the host in which they ate hatched. The females may be found within or outside the coenobium. Eggs are laid in the host, where they hatch and, according to Hudson and Gosse, the young rotifer either enters a daughter colony and 1s expelled with it or emerges to swim free. Hudson and Gosse stated that “Volvox appears to suffer little from the depredations of its ungrateful guest.” 1094 PARASITES OF PROTOZOA Penard (1904, 1908-9) has given the most complete account of Proales latrunculus, certain observations on which had been made by Archer (1869), Leidy (1879), and Stokes (1884). Penard studied an epidemic outbreak of the parasite, which eventually carried off most of a group of the Heliozoa. He stated, however, that it is rare, in the sense that many Acanthocystis turfacea in various localities may be examined without encountering it. It is widespread geographically, as indicated by the records from Switzerland, England, and the United States. After being introduced into the body of Acanthocystis, probably, according to Penard, by being ingulfed as prey, the rotifer moves about actively in the cytoplasm. It devours the zodchlorellae and the substance of the heliozoan. In two or three hours an egg may be laid, after which the rotifer may continue to feed and lay a second, smaller egg. The heliozoan occasionally frees itself of the invader, but usually it perishes before the end of the first day. After laying its eggs, the rotifer escapes by an orifice in the then empty envelope of spicules—empty, that is, except for the few small eggs. The young rotifers develop rapidly, hatching in two or three days, when they leave by the orifice through which the parent escaped. As Penard (1908-9) remarked, these rotifers are not true parasites, as they are not adapted to continuous existence in their host. They behave rather as predaceous forms which consume the host from within. One notes a marked specificity to certain hosts or related hosts in the rotifers ectozoic on colonial vorticellids and those endozoic in V aucheria, Volvox, and Acanthocystis. Ehrenberg (1888) on one occasion found the usually ectozoic P. petromyzon within Volvox globator,; and Wesenberg-Lund (1929) stated that Volvox contained also species of Diglena, rotifers that are naturally free-living. Living nematode worms have occasionally been encountered in Pro- tozoa. It is not known whether this ever represents obligatory parasitism, or is only an invasion by accidentally or facultatively parasitic forms. Wesenberg-Lund (1929) stated that free-living nematodes have been found in Volvox, and Schubotz (1908) wrote that Hartmann informed him of having seen nematodes in that flagellate. Schubotz found as many as three nematodes in approximately a tenth of Pycnothrix monocystoides from Procavia capensis. He stated that for entry into this large ciliate, PARASITES; OF PROTOZOA 1095 the worms use openings in the ectosarc or, in undamaged animals, the excretion pore. They are then found wholly or partly in the canal system, whose walls they at times break through. Myers (1938) found nematode worms in the foraminiferan Rofalza turbinata in an incidence, in the colder months of the year, of 5 percent. LITERATURE CITED Alexeieff, A. 1912. Sur un Chlamydozoaire parasite des Protozoaires. Sur le Chlamydozoaire du cancer. Arch. zool. exp. gén., (5) 10: N. et R., 101- KO: —— 1929. Matériaux pour servir a l'étude des protistes coprozoites. Arch. zool. exp. gén., 68: 609-98. Archer, W. 1869. On some freshwater Rhizopoda, new or little known. Quarts). muicts Sa., n:'s,, 9:°250-7.1. Awerinzew, S. 1906. Die Siisswasser-Rhizopoden. Trav. Soc. Nat. St.-Pétersb. (Leningr.), 36: 1-351. (Russian, with German résumé.) —— 1908. Studien uber parasitische Protozoen. Trav. Soc. Nat. St.-Pétersb. (Leningr.), 38: v-xii, 1-139. Bach, F. W., and P. Quest. 1923. Uber Spirochaten im Darme von Cyclostoma elegans Drap. und thre Beziehungen zu Trichodinopsis paradoxa Clap. Zbl. Bakt., 90: 457-60. Bacigalupo, J. 1927. Entameba coli parasitada con Sphaerita. Rev. Soc. argent. Biol., 3: 694-98. —— 1928. Entamoeba coli parasitée par une Sphaerita. C. R. Soc. Biol., Paris, 98:-170-71. Balbiani, E. G. 1858a. Note relativ a l’existence d’une génération sexuelle chez les Infusoires. J. Physiol. Path. Gén., 1: 347-52. —— 1858b. Recherches sur les organes générateurs et la reproduction des In- fusoires. C. R. Acad. Sci. Paris, 47: 383-87. Translation in Ann. Mag. Nat. Hist. (3) 2: 439-43. —— 1860. Note sur un cas de parasitisme improprement pris pour un mode de reproduction des Infusoires ciliés. C. R. Acad. Sci. Paris, 51: 319-22. —— 1861. Recherches sur les phénomeénes sexuels des Infusoires. Arch. physiol. norm. path., 4: 102-30; 194-220; 465-520. —— 1893. Nouvelles Recherches expérimentales sur la mérotomie des In- fusoires ciliés. Ann. micrographie, Paris, 5: 1-25; 49-84; 113-37. Becker, E. 1926. Endamoeba citelli sp. nov. from the striped ground squirrel Citellus tridecemlineatus, and the life history of its parasite Sphaerita endamoebae sp. nov. Biol. Bull., 50: 444-54. Bernstein, T. 1928. Untersuchungen an Flagellaten aus dem Darmkanal der Termiten aus Turkestan. Arch. Protistenk., 61: 9-37, 1096 PARASULES OF PROTOZOA Boeck, W. C. 1917. Mitosis in Giardia microti, Univ. Cal. Publ. Zool., 18: 1-26. Bonar, L. 1936. An unsual Ascomycete in the shells of marine animals. Univ. Cal. Publ. Bot. 19: 187-94. Borgert, A. 1897. Beitrage zur Kenntniss der in Sticholonche zanclea und Acanthometridenarten vorkommenden Parasiten. Z. wiss. Zool., 63: 141- 86. Bourne, A. G. 1891. On Pelomyxa viridis, sp. n., and on the vesicular nature of protoplasm. Quart. Jour. micr. Sci., 32: 357-74. Bozler, E. 1924. Uber die Morphologie der Ernahrungsorganelle und die Physiologie der Nahrungsaufnahme bei Paramecium caudatum Ehrbg. Arch. Protistenk., 49: 163-215. Brug, S. L. 1926. Nucleophage intestinalis n. sp., patasiet der Kern van Endolimax williamsi (Prow.) = Endolimax biitschlii (Prow.) Dutch East Indies Volksgesundheid, 1926: 466-68. Brumpt, E., and G, Lavier. 1935a. Sur une Nacleophaga parasite d’Endolimax nana. Ann, Parasit. hum. comp., 13: 439-44. —— 1935b. Sur un genre nouveau d’amibe parasite Hyalolimax n. g. Ann. Parasit. hum. comp., 13: 551-58. —— 1936. Sur l’hyperparasitisme d’Opalines par des amibes. Ann. Parasit. hum. comp., 14: 349-58. Buchanan, R. E. 1925. General systematic bacteriology; history, nomenclature, groups of bacteria. Baltimore. Buchner, P. 1930. Tier und Pflanze in Symbiose. Berlin. Biitschli, O. 1876. Studien iiber die ersten Entwicklungsvorginge der Eizelle, die Zelltheilung und die Conjugation der Infusorien. Abh. senckenb. naturf. Ges., 10: 213-464. — 1885. Einige Bemerkungen tiber gewisse Organisationsverhialtnisse der sog. Cilioflagellaten und der Noctiluca. Morph. Jb., 10: 529-77. —— 1889. Protozoa. III Abt. Infusoria und System der Radiolaria 7m Bronn: Klassen und Ordnungen des Thier-Reichs, 1. Leipzig. Calkins, G. N. 1904. The life-history of Cytoryctes variolae Guarniere. Stud. Path. Etiol. Variola (Office J. med. Res.) : 136-72. Campbell, A. S. 1926. The cytology of Tintinnopsis nucula (Fol) Laachmann, with an account of its neuromotor apparatus, division, and a new intra- nuclear parasite. Univ. Cal. Publ. Zool., 29: 179-236. Carini, A. 1933. Parasitisme des Zellerielles par des Microorganismes nouveau (Brumptina n. g.) Ann, Parasit. hum. comp., 11: 297-300. Carini, A., and E. Reichenow. 1935. Uber Amébeninfektion in Zelleriellen. Arch. Protistenk., 84: 175-85. Carter, H. J. 1856. Notes on the freshwater infusoria of the island of Bombay. No. 1. Organization. Ann. Mag. Nat. Hist., (2) 18: 115-32; 221-49. —— 1857. Additional notes on the freshwater infusoria in the island of Bombay. Ann. Mag. Nat. Hist., (2) 20: 34-41. PARASITES OF PROTOZOA 1097 — 1863. On Amoeba principes and its reproductive cells, etc. Ann. Mag. Nat. Hist., (3) 12: 30-54. Casagrandi, O., and P. Barbagallo. 1897. Entamoeba hominis s. Amoeba coli (Lésch). Studio biologicoe clinico. Ann. Igiene (sper.), 7: 1-64. Caullery, M., and F. Mesnil. 1897. Sun un type nouveau (Metchnikovella n. g.) dorganismes parasites des grégarines. C. R. Soc. Biol. Paris, 49: 960-62. —— 1905. Recherches sur les Haplosporidies. Arch. zool. exp. gén., (4) 4: 101-81. — 1914. Sur les Metchnikovellidae et autres Protistes parasites des grégarines d’Annélides. C. R. Soc. Biol. Paris, 77: 527-32. — 1919. Metchnikovellidae et autres parasites des Grégarines d’ Annélides. Ann. Inst. Pasteur, 33: 209-40. Cavallini, F. 1926a. The asexual cycle in Centropyxis aculeata and its vari- ability in relation to heredity and environment. J. exp. Zool., 43: 225-43. — 1926b. The asexual cycle of development in Arcella vulgaris. J. exp. Zool., 43: 245-55. Cejp, K. 1935. Sphaerita, parasit Paramecii. Piispévek k poznani houbovych parasiti Protozoi. Spisy Piirodovédeckou Fakultou Karlovy University, Praha (Publ. Fac. Sci. Univ. Charles), 141: 3-7. Cépéde, C., and V. Willem. 1912. Observations sur Trichodinopsis paradoxa. Bull. Sci. Fr. Belg., 45: 239-48. Chatton, E. 1910. Protozoaires parasites des branchies des Labres: Amoeba mucicola Chatton, Trichodina labrorum n. sp. Appendice: parasite des Trichodines. Arch. zool. exp. gén., (5)5: 239-66. —— 1913. Coccidiascus n. g., n. sp., levure ascosporée parasite des cellules intestinales de Drosophila funebris Fabr. C. R. Soc. Biol. Paris, 75: 117-20. —— 1914. Les Cnidocystes du Péridinien Polykrikos schwartzi Butschli. Arch. zool. exp. gén., 54: 157-94. —— 1920a. Les Péridiniens parasites. Morphologie, reproduction, ethologie. Arch. zool. exp. gén., 59 1-475. —— 1920b. Existence chez les Radiolaires de Péridiniens parasites con- siderés comme formes de reproduction de leurs hdtes. C. R. Acad. Sci. Paris, 170: 413-15. —— 1923. Les Péridiniens parasites des Radiolaires. C. R. Acad. Sci. Paris, 177: 1246-49, —— 1934. L’Origine péridinienne des Radiolaires et l’interprétation para- sitaire de l’anisosporogénése. C. R. Acad. Sci. Paris, 198: 309-12. Chatton, E., and B. Biecheler. 1934. Les Coccidinidae, Dinoflagelles coccidio- morphes parasite de Dinoflagelles, et le phylum des Phytodinozoa. C. R. Acad. Sci. Patis, 199: 252-55. —— 1936. Documents nouveau relatifs aux Coccidinides (Dinoflagelles 1098 PARASITES ‘OF PROTOZOA parasites). La sexualite du Coccidinium mesnili n. sp. C. R. Acad. Sci. Paris, 203: 573-76. Chatton, E., and S. Brachon. 1936. Sur un Protiste parasite du Cilié Fabrea salina Henneguy: Gregarella fabrearum n. gen., n. sp., et son evolution. CR. Acad: Scam Panis= 203) 525-272 Chatton, E., and A. Brodsky. 1909. Le Parasitisme d’une Chytridinée du gente Sphaerita Dangeard chez Amoeba limax Dujard. Etude compatra- tive. Arch. Protistenk., 17: 1-18. Chatton, E., and A. Lwoff. 1924a. Sur un flagellé hypertrophique et palintomi- que parasite des Infusoires marins: Sporomonas infusorium (n. g., Nn. sp.) C. R. Soc. Biol. Paris, 91: 186-90. —— 1924b. Sur l’évolution des Infusoires des Lamellibranches: Morphologie comparée des Hypocomidés. Les nouveaux genres Hypocomina et Hypo- comella. C. R. Acad. Sci. Paris, 178: 1928-30. —— 1927. Pottsia infusoriorum n. g., n. sp., Acinétien parasite des Folli- culines et des Cothurnies. Bull. Inst. océanogr. Monaco, 489: 1-12. — 1929, Contribution a l'étude de l’adaptation. Ellobiophrya donacis Ch. et Lw. Péritriche vivant sur les branchies de I’Acephale Donax vittatus da Costa. Bull. biol., 63: 321-49. —— 1935. Les Ciliés apostomes. Morphologie, cytologie, éthologie, évolu- tion, systématique. Premiere partie: Apercu historique et général. Etude monographique des genres et des espéces. Arch. zool. exp. gén., 77: 1-453. Chatton, E., A. Lwoff, and M. Lwoff. 1930. Les Phtorophrya n. g., Ciliés Foettingertidae, hyperparasites des Gymnodinioides, Foettingertidae parasites des Crustaces. C. R. Acad. Sci. Paris, 190: 1152-54. Chatton, E., and S. Villeneuve. 1937. Gregarella fabrearum Chatton et Brachon Protiste parasite du Cilié Fabrea salina Henneguy. La Notion de dépolarisation chez les Flagellés et la conception des Apomastigines. Arch, zool. exp. gén., 78: N. et R., 216-37. Chen, T. T., and R. M. Stabler. 1935. Further studies on the amoebae parasitic in opalinid ciliate protozoans. (Abstract). J. Parasit., 21: 428-29. —— 1936. Further studies on the endamoeba parasitizing opalinid ciliates. Biol. Bull., 70: 72-77. Cienkowsky, L. 1855a. Bemerkungen iiber Stein’s Acinetenlehre. Bull. Acad. Imp. Sci. St.-Pétersb., phys-math., 13: 297-304. Also Quart. J. micr. Sci., 5: 96-103 (1857). —— 1855b. Uber Cystenbildung bei Infusorien. Z. wiss. Zool., 6: 301-6. —— 1865. Beitrage zur Kenntnis der Monaden. Arch. mikr. Anat. 1: 205-52. Claparéde, E., and J. Lachmann. 1857. Note sur la reproduction des Infusoires. Ann, Sci. nat., Zool., 4(8): 221-44. —— 1858-59. Etudes sur les Infusoires et les Rhizopodes. vol. 1, Geneve. Mém. Inst. nat. genev., 5, 6. PARASITES OF PROTOZOA 1099 —— 1860-61. Etudes sur les Infusoires et les Rhizopodes. vol. 2, Geneve. Mem. Inst. nat. genev., 7: 1-291. Cleveland, L. R. 1928. Further observations and experiments on the symbiosis between termites and their intestinal Protozoa. Biol. Bull., 54: 231-37. Cleveland, L. R., S. R. Hall, E. P. Sanders, and J. Collier. 1934. The wood- feeding roach Cryptocercus, its Protozoa, and the symbiosis between Protozoa and roach. Mem. Amer. Acad. Arts Sci., 17: i-x, 185-342. Cohn, F. 1851. Beitrage zur Entwickelungsgeschichte der Infusorien. Z. wiss. Zool., 3: 257-79. — 1857. Uber Fortpflanzung von Nassula elegans Ehr. Z. wiss. Zool., 9: 143-46. 1858. Bemerkungen uber Raderthiere. Z. wiss. Zool., 9: 284-94. Collin, B. 1907. Note préliminaire sur quelques Acinétiens. Arch. zool. exp. gén. (4)7: N. et R., 93-103. — 1912. Etude monographique sur les Acinétiens. II. Morphologie, Physiologie, Systématique. Arch. zool. exp. gén., 51: 1-457. Connell, F. H. 1930. The morphology and life cycle of Oxymonas dimorpha sp. nov., from Neotermes simplicicornis Banks. Univ. Cal. Publ. Zool., 36: 51-66. ——1932. Gigantomonas lighti sp. nov., a trichomonad flagellate from Kalotermes (Paraneotermes ) simplicicornis Banks. Univ. Cal. Publ. Zool., Sie 155-88. Cook, W. R. I. 1935. The genus Lagenidium Schenk, with special reference to L. Rabenhorstii Zopf and L. entophytum Zopf. Arch. Protistenk., 86: 58-89. Cragg, F. W. 1919. A contribution to our knowledge of Entamoeba coll. Indian J. med. Res., 6: 462-84. Craig, C. F. 1911. The parasitic amoebae of man. Philadelphia. Crouch, H. B. 1933. Four new species of Trichomonas from the woodchuck (Marmota monax Linn.). J. Parasit., 19: 293-301. Cunha, A. M. da, and J. Muniz. 1923. Parasitismo de ‘Trichomonas’ pro “Chyttidacae” do genero “Sphaerita’ Dangeard. Brazil-med., 37: 19-20. —— 1934. Observations sur un parasite des Flagellés du genre Chilomastix. @_ RR: SecBiol, Paris, 117: 208-10, Dangeard, P. A. 1886a. Sur un nouveau genre de Chytridinés parasites des Rhizopodes et des Flagellates. Bull. Soc. bot. Fr., 33: 240-42. —— 1886b. Recherches sur les organismes inférieurs. Ann. Sci. nat., (7) Bot., 4: 241-341. —— 1889a. Recherches sur les Cryptomonadinae et les Euglenae Botaniste, fe l33. —— 1889b. Mémoire sur les Chytridinées. Botaniste, 1: 39-74. —— 1895. Mémoire sur les parasites du noyau et du protoplasma. Botaniste, A> 199-248, — 1896. Contribution a l'étude des Acrasiées. Le Botaniste, 5: 1-20. 1100 PARASITES OF PROTOZOA —— 1900a. L’Organisation el le développement du Colpodella pugnax. Botaniste, 7: 5-31. — 1900b. Recherches sur la structure du Polyphagus euglenae Nowak. et sa reproduction sexuelle. Botaniste, 7: 213-61 — 1902. Sur le caryophyséme des Eugléniens. C. R. Acad. Sci. Paris, 134: 1365-66. —— 1908. Sur un nouveau genre, parasite de Chrysomonadinées, le Lecythodytes paradoxus. C. R. Acad. Sci. Paris, 146: 1159-60. —— 1910. Etudes sur le développement et la structure des organismes in- férieurs. Botaniste, 11: 1-311. Deflandre, G. 1928. Le Genre Arcella Ehrenberg. Morphologie- Biologie. Essai phylogénétique et systématique. Arch. Protistenk., 64: 152-287. Dobell, C. 1919, The amoebae living in man. London. Doflein, F. 1907. Studien zur Naturgeschichte der Protozoen. V. Amdben- studien, Erster Teil. Arch. Protistenk., Suppl. 1: 250-93. 1909. Lehrbuch der Protozoenkunde, 2d ed., Jena. Also 3d ed., 1911; 4th ed., 1916; 5th ed. (F. Doflein, and E. Reichenow), 1927-29. Dogiel, V. 1906. Beitrage zur Kenntnis der Gregarinen. I. Cystobia chiridotae nov. sp. II. Hyalosphaera gregarinicola nov. gen. nova spec. Arch. Protistenk., 7: 106-30. —— 1916. Researches on the parasitic Protozoa from the intestine of termites. I. Tetramitidae. Russk. zool. Zh., 1: 1-54. —— 1917. Researches on the parasitic protozoa from the intestine of termites. II. Lophomonadidae. Sci. Res. Zool. Exped. Brit. E. Africa made by V. Dogiel and I. Sokolow in 1914, no. 10. —— 1922a. Untersuchungen an parasitischen Protozoan aus dem Darmkanal der Termiten. III. Trichonymphidae. Arch. russ. Protist., 1: 172-234. —— 1922b. Sur un nouveau genre de Metchnikovellidae. Ann. Inst. Pasteur, 36: 574-77. — 1929. Biologische Notizen tiber Darminfusorien der Huftiere. Arch. russ. protist., 8: 153-62. Douvillé, N. 1930. Parasitisme ou commensalisme chez les Foraminiféres. Les canaux chez les Nummulitides, Soc. geol. France, Livre Jubilaire 1830- 1930, 1: 257-62. Duboscq, O., and B. Collin. 1910. Sur la reproduction sexuée d’un Protiste parasite des Tintinnides. C. R. Acad. Sci. Paris, 151: 340-41. Duboscq, O., and Grassé, P. 1925. Notes sur les Protistes parasites des Termites de France. IV. Appareil de Golgi, mitochondries et vesicules sous-flagellaites de Pyrsonympha vertens Leidy. C. R. Soc. Biol. Paris, 93: 345-48. —— 1926. Les Schizophytes de Devescovina hilli n. sp. C. R. Soc. Biol. Paris, 94: 33-34, —— 1927. Flagellés et Schizophytes de Calotermes (Glytotermes) iridipen- nis Frogg. Arch. Zool. exp. gén., 66: 452-96. PARASITES OF PROTOZOA 1101 —— 1928. Note sur les Protistes parasites des termites de France. L’appareil parabasal de Joenia annectens Grasst. C. R. Soc. Biol., 99: 1118-20. — 1933. L’appareil parabasal des Flagellés. Arch zool. exp. gén., 73: 381-621. —— 1934. Notes sur les Protistes parasites des Termites de France. VII. Sur les Trimitus de Calotermes flavicollis Rossi. VII. Sur Microrhopalodina inflata (Grassi). Arch. zool. exp. gén., 75: 615-37. —— 1937. Les Flagellés de l’Acanthotermes ochraceus Sjost. du Sud- Algérien. C. R. Acad. Sci. Paris, 205: 574-76. Duboscq, O., P.-P. Grassé, and M. Rose. 1937. Les Flagellés de l’ Anacantho- termes ochraceus Sjést. du Sud-Algérien. C. R. Acad. Sci. Paris, 205: 574-76. Ehrenberg, C. G. 1838. Die Infusionsthierchen als vollkommene Organismen. Leipzig. Elmassian, M. 1909. Une Nouvelle Coccidie et un nouveau parasite de la tanche, Coccidium rouxi nov. spec., Zoomyxa legeri nov. gen., NOV. spec. Arch. zool. exp. gén., (5) 2: 229-70. Elpatiewsky, W. 1907. Zur Fortpflanzung von Arcella vulgaris Ehtbg. Arch. Protistenk., 10: 441-66. Engelmann, T. W. 1862. Zur Naturgeschichte der Infusionsthiere. Z. wiss. Zool., 11: 347-93. — 1876. Uber Entwickelung und Fortpflanzung von Infusorien. Morph. |DS 12573-635: Epstein, H. 1922. Uber parasitische Infektion bei Darmamében. Arch. russ. protist., 1: 46-81. 1935. Bacterial infection in an amoeba. J. R. micr. Soc., 55: 86-94. Fauré-Fremiet, E. 1909. Sur un cas de symbiose presente par un Infusoire Gilie Ree soc, Biolk Pariss:67= 113-14) — 1913. Sur les ‘“‘Nématocystes’” de Polykrikos et de Campanella. C. R. Soc. Biol. Paris, 75: 366-68. Fitzpatrick, H. M. 1930. The lower fungi. Phycomycetes. New York. Fiveiskaja, A. 1929. Einfluss der Kernparasiten der Infusorien auf den Stoff- wechsel. Arch. Protistenk., 65: 275-98. Foa, A. 1905. Due nuovi Flagellati parassiti. R. C. Accad. Lincei, (5) 14: 542-46. Focke, G. W. 1845. Ergebnisse ferneren Untersuchungen dere polygastrischen Infusorien. Amtlicher Bericht tber' die zweiundzwanzigste Versammlung deutscher Naturforscher und Artzte in Bremen im September 1844. Verh. Ges. deutscher Naturf. Artzte, 22: 109-10. Fol, H. 1883. Sur le Sticholonche zanclea et un nouvel ordre de Rhizopodes. Mem. Inst. nat. genev., Nr. 15. Fortner, H. 1934. Untersuchungen an Pelomyxa palustris Greeff. Studien zur Biologie und Physiologie des Tieres. I. Teil. Arch. Protistenk., 83: 381- 464, 1102 PARASITES°OF PROTOZOA Franca, C. 1918. Observations sur les Trichonymphides. An. Fac. Med. Porto, (4) 2: 5-14. Ganapati, P. N., and R. G. Aiyar. 1937. Life-history of a dicystid gregarine, Lecudina brasili n. sp., parasitic in the gut of Lumbriconereis sp. Arch. Protistenk., 89: 113-32. Gassovsky, G. 1919. On the microfauna of the intestine of the horse. Trav. Soc. Nat. St.-Pétersbg. (Leningr.), 49: 20-37; 65-69. Geitler, L. 1937. Uber einen Pilzparasiten auf Amoeba proteus und uber die polare Organization des Amébenkorpers. Biol. Zbl., 57: 166-75. Georgevitch, J. 1929. Sur la faune intestinale des Termites de Yougoslavie. C. R. Soc. Biol. Paris, 103: 325-28. —— 1932. Recherches sur les Flagellés des Termites de Yougoslavie. Arch. zool. exp. gén., 74: 81-109. — 1936a. [Studies on a hyperparasite Le/smania esocis nov. spec.} (In Serbian.) Ghlas Srpska Kralj. Akad. Belgrade, 172: 127-37. —— 1936b. Ein neuer Hyperparasit, Leishmania esocis nov. spec. Arch. Protistenk., 88: 90-92. Goetsch, W., and L. Scheuring. 1926. Parasitismus und Symbiose der AlI- gengattung Chlorella. Z. Morph. Okol. Tiere, 7: 220-53. Gojdics, M. 1939. Some observations on Evglena sanguinea Ehrbg. Trans. Amer. micr. Soc., 58: 241-48. Goldschmidt, R. 1907. Lebensgeschichte der Mastigamdben, Mastigella vitrea n. sp. und Mastigina setosan. sp. Arch. Protistenk., (Suppl.) 1: 83- 168. Gonder, R. 1910. Ein Parasit von Colpoda cucullus. Arch. Protistenk., 18: 27D: Gonnert, R. 1935. Uber Systematik, Morphologie, Entwicklungsgeschichte und Parasiten einiger Dendrosomidae nebst Beschreibung zweier neuer Suktorien. Arch. Protistenk., 86: 113-54. Gould, L. J. 1894. Notes on the minute structure of Pelomyxa palustris (Greef). Quart. J. micr. Sci., 36: 295-306: Grassé, P. P. 1926a. Sur la nature des cétes cuticulaires des Polymastix et Lophomonas striata. C. R. Soc. Biol. Paris, 94: 1014-15. —— 1926b. Contribution a l’étude des Flagellés parasites. Arch. zool. exp. gén., 65: 345-602. 1937. Sur un Flagellé termiticole “Caduceia theobromae” Franga. C. R. xii® Cong. Int. Zool.: 1324-29. — 1938. La Véture schizophytique des Flagellés termiticoles: Parajoenza, Caduceia et Pseudodevescovina. Bull. Soc. zool. Fr., 63: 110-22. Grassi, B., and Foa, A. 1904. Ricerche sulla riproduzione dei Flagellati. I. Processo di divisione delle Joenie e forme affini. Nota preliminare. R. C. Accad. Lincei, (5) 13, 2 sem.: 241-53. — 1911. Intorno ai Protozoi dei Termitidi. Nota preliminare. R. C. Accad. Eincer.(6) e205 Wsem:.:725-4 1c PARASITES OF PROTOZOA 1103 Greeff, R. 1866. Uber einige in der Erde lebende Amdben und andere Rhizo- poden. Arch. mikr. Anat., 2: 299-311. — 1870. Unterschungen tiber den Bau und die Naturgeschichte der Vorticellen. Arch. Naturgesch., 1: 353-84. — 1874. Pelomyxa palustris (Pelobius), ein amdbenartiger Organismus des siissen Wassers. Arch. mikr. Anat., 10: 51-73. Greenway, D. 1926. Endolimax nana. Arch. argent Enferm. Apar. dig., p. 174. Gruber, A. 1884. Die Protozoen des Hafens von Genua. Nova Acta K. Leop. Carol., 46: 475-539. — 1892. Eine Mittheilung tiber Kernvermehrung und Schwarmerbildung bei Siisswasserrhizopoden. Ber. naturf. Ges. Freiburg i. B., 6: 114-18. — 1904. Uber Amoeba viridis Leidy. Zool. Jb., (Suppl.) 7 (Festschr. Weissmann) : 67-76. Gunther, F. 1928. Uber den Bau und die Lebensweise der Euglenen, besonders der Arten E. terricola, geniculata, proxima, sanguinea und lucens nov. spec. Arch. Protistenk., 60: 511-90. Gwynne- Vaughan, H. C. I., and B. Barnes. 1937. The structure and develop- ment of the fungi. 2d ed., Cambridge. Hafkine, M. W. 1890. Maladies infectieuses des Paramecies. Ann. Inst. Pasteur, 4: 148-62. Hertwig, R. 1876. Uber Podophrya gemmipara nebst Bemerkungen zum Bau und zur systematischen Stellung der Acineten. Gegenbaurs Jb., 1: 20-82. 1879. Der Organismus der Radiolarien. Jena. Hesse, E. 1909. Contribution a l'étude des Monocystidées des Oligochétes. ANEGh. ZOO) Expy. gen. (5) 3: 27-30V. Hetherington, A. 1932. On the absence of physiological regeneration in Stentor coeruleus. Arch. Protistenk., 77: 58-63. Hoelling, A. 1910. Die Kernverhiltnisse von Fusiformis termitidis, Arch. Protistenk, 19: 239-45. Hofker, J. 1927. The Foraminifera. Siboga Expedition. Leiden. 1931. Studie uber Tintinnoidea. Arch. Protistenk., 75: 315-402. Hollande, A. 1938. Bodo perforans n, sp. Flagellé nouveau parasite externe du Chilomonas paramaecium Ehrenb. Arch. zool. exp. gén., 79: N. et R., 75-81. Hovasse, R. 1923a. Les Peridiniens intracellulaire — zooxanthelles et Syn- diniums — chez les Radiolaires coloniaux. Remarques sur la reproduction des Radiolaires. Bull. Soc. zool. Fr., 48: 247-54. —— 1923b. Sur les Peridiniens parasites des Radiolaires coloniaux. Bull. Soce Zool Fre 48293377 —— 1936. Rhizophidium Beauchampi sp. nov., Chytridinée parasite de la Volvocinée Exdorina (Pleodorina) illinoisensis (Kofoid). Ann. Protist., De D=Gils. Howland, R. B. 1928. A note on Astasia captive Beauch. Science, 68: 37. 1104 PARASITES OF PROTOZOA Hsiung, Ta-Shih. 1928. Suctoria of the large intestine of the horse: Allan- tosoma intestinalis Gassovsky, A. discorniger sp. nov. and A. brevi- corniger sp. nov. Iowa St. Coll. J. Sci., 3: 101-3. Hudson, C. T., and P. H. Gosse. 1889. The rotifera; or wheel-animalcules, both British and foreign. London. Issel, R. 1906. Intorno alla struttura ed alla biologia dell’infusorio Trichodin- Opsis paradoxa Clap. et Lachm. Ann. Mus. Stor. nat. Genova, (3) 2: 334-57. Ivani¢, M. 1925. Zur Kenntnis der Agamogonieperiode einiger Amoeben- parasiten. Zool. Anz., 63: 250-56. —— 1934, Uber einen Kernparasiten der roten Blutzellen beim griinen Frosche (Rana esculenta L.), Erythrocytonucleophaga ranae gen. nov., spy nov. Zblwbakt. ol “Abt. 1133 a6; —— 1936. Uber einen Protoplasmakérperparasiten von Entamoeba histoly- tica Schaudinn (Entamoebophaga hominis gen. nov. spec. nov.). Zbl. Bakt., I Abt., 138: 48-56. Jahn, T. D 1929. On certain parasites of Phacus and Beglen: (Abstract) . Anat. Rec., 44: 249-50. —— 1933, On certain parasites of Phacus and Euglena; Sphaerita phaci, sp. nov. Arch. Protistenk., 79: 349-55. Janicki, C. 1909. Uber Kern und Kernteilung bei Extamoeba blattae Bitschli. Biol. Zbl., 29: 381-93. —— 1915. Untersuchungen an parasitischen Flagellaten. HI Teil: Die Gat- tungen Devescovina, Parajoenia, Stephanonympha, Calonympha. Ueber den Parabasalapparat. Uber Kernkonstitution und Kernteilung. Z. wiss. Zool., 112: 573-691. Jirovec, O. 1931a. Die Silberlinien bei den Pyrsomymphiden. Arch. Pro- tistenk., 73: 47-55. —— 1931b. Symbiose von Bakterien und Trichonympha serbica. Zbl. Bakt., I Abt., 123: 184-86. —— 1933. Beobachtungen Uber die Fauna des Rinderpansens. Z. Parasitenk., 5: 584-91. Kahl, A. 1928. Die Infusorien (Ciliata) der Oldesloer Salzwasserstellen. Arch. Hydrobiol., 19: 50-123. —— 1930. Urtiere oder Protozoa I: Wimpertiere oder Ciliata (Infusoria) I. Allgemeiner Teil und Prostomata, 7 Dahl: Die Tierwelt Deutsch- lands, 18 Teil, Jena. —— 1931. Ibid., II. Holotricha, 21 Teil. —— 1932. Ibid., Ill. Spirotricha, 25 Teil. — 1933. Ciliata libera et ectocommensalia, 72 Grimpe, G., und E. Wagler: Die Tierwelt der Nord-und Ostsee Lief. 23, Teil II. C3, 29-146, Leipzig. —— 1935. Ibid., IV. Peritricha und Chonotricha, 30 Teil. Kalmus, H. 1928. Uber den Bodenfauna der Moldau im Gebiete von Prag. PARASITES OF PROTOZOA 1105 Ein Jahreszyklus. II. Protozoa, etc. Mit einem Anhang: Okologische Beobachtungen und Versuche. Int. Rev. Hydrobiol., 19: 349-429. Kent, W. S. 1880-82. A manual of the Infusoria. London. Keppen, N. A., see Koeppen, N. Kessel, J. F. 1923. On the genus Coxncilmania, budding intestinal amoebae parasitic in man and rodents. Univ. Cal. Pub. Zool., 20: 431-45. — 1924, The distinguishing characteristics of the parasitic amoebae of culture rats and mice. Univ. Cal. Publ. Zool., 20: 489-544. Kirby, H., Jr. 1924. Morphology and mitosis of Dinenympha fimbriata sp. nov. Univ. Cal. Publ. Zool., 26: 199-220. —— 1926a. On Staurojoenina assimilis sp. nov., an intestinal flagellate from the termite, Kalotermes minor Hagen. Univ. Cal. Publ. Zool., 29: 25-102. —— 1926b. The intestinal flagellates of the termite, Cryptotermes hermsi Kirby. Univ. Cal. Publ. Zool., 29: 103-20. 1927. Studies on some amoebae from the termite Mirotermes, with notes on some other Protozoa from the Termitidae. Quart. J. micr., Sci., FB N89=222° — 1928. A species of Proboscidiella ftom Kalotermes (Cryptotermes) dudleyi Banks, a termite of Central America, with remarks on the oxy- monad flagellates. Quart. J. micr. Sci., 72: 355-86. —— 1929. Snyderella and Coronympha, Ewe new genera of puleianelente flagellates from termites. Univ. Cal. Publ. Zool., 31: 417-32. 1930. Trichomonad flagellates from eenites I. Tricercomitus gen. nov., and Hexamastix Alexeieff. Univ. Cal. Publ. Zool., 33: 393-444. 1932a. Flagellates of the genus Trichonympha in termites. Univ. Cal. Publ. Zool., 37: 349-476. —— 1932b. Protozoa in termites of genus Amitermes. Parasitology, 24: 289-304. — 1934. Some ciliates from salt marshes in California. Arch. Protistenk., 82: 114-33. —— 1936. Two polymastigote flagellates of the genra Pseudodevescovina and Caduceia. Quart. J. micr. Sci., 79: 309-35. -—— 1937. The devescovinid flagellate Parajoenia grassii from a Hawaiian termite. Univ. Cal. Publ. Zool., 41: 213-24. —— 1938a. The devescovinid flagellates Caduceia theobromae Franga, Pseudodevescovina ramosa new species, and Macrotrichomonas pulchra Grassi. Univ. Cal. Publ. Zool., 43: 1-40. 1938b. Polymastigote flagellates of the genus Foaina Janicki, and two new genera Cracinympha and Bulanympha. Quart. J. micr. Sci., 81: 1-25. 1940. Microorganisms associated with the flagellates of termites. Pp. 407-8 in Thitd International Congress for Microbiology: Report of Proceedings. New York. 1106 PARASITES OF PROTOZOA Klebs, G. 1883. Uber die Organisation einiger Flagellaten-Gruppen und ihre Beziehungen zu Algen und Infusorien. Unters. Bot. Inst. Tubingen, 1: 233-62. Koeppen, N. [Keppen, N. A.}. 1894. Amoebophrya Sticholonchae nov. gen. et sp. (“Corps spiral” de Fol) Zool. Anz., 17: 417-24. —— 1889. Hyalosaccus Ceratit, nov. gen. et sp., parazit Dinoflagellat. Kiev. obshch. estest. Zap., 16: 89-135. Kofoid, C. A., and O. Swezy. 1915. Mitosis and multiple fission in tricho- monad flagellates. Proc. Amer. Acad, Arts. Sci., 51: 289-378. Koidzumi, M. 1921. Studies on the intestinal Protozoa found in the termites of Japan. Parasitology, 13: 235-309. Korotneff, A. 1880. Etudes sur les Rhizopodes. Arch. zool. exp. gén., (1), 8: 467-82. — 1891. Zoologische Paradoxen. Z. wiss. Zool., 51: 613-28. Kruger, F. 1933. Epzstylis umbellaria mit ‘Nesselkapseln.” Zool. Anz. (Suppl.) 6. Verh. dtsch. zool. Ges., 35: 262-63. Kuczynski, M. H. 1918. Uber die Teilungsvorgange verscheidener Tricho- monaden und ihre Organization im allgemeinen, Arch. Protistenk., 39: 107-46. Kudo, R. 1924. A biologic and taxonomic study of the microsporidia. III. Biol. Monogr., 9: 3-268. Lachmann, C. F. J. 1856. Uber die Organisation der Infusorien, besonders der Vorticellen. Arch. Anat. Physiol. wiss. Med., 1856: 340-98. Lackey, J. B. 1936. Some fresh water Protozoa with blue chromatophores. Biol. Bull., 71: 492-97. Lauterborn, R. 1916. Die sapropelische Lebewelt. Ein Beitrag zur Biologie des Faulschlammes natiirlicher Gewasser. Verh. naturh.-med. Ver. Heidelberg, n. s., 13: 395-481. Lavier, G. 1935a. Action, sur la biologie d’une Entamibe, due parasitism intranucléaire par une Nucleophaga. C. R. Soc. Biol. Paris, 118: 457-59. —— 1935b. Sur une Nuacleophaga parasite d’Entamoeba ranarum. Ann. Parasit. hum. comp., 13: 351-61. Leger, L., and O. Duboscq. 1904. Notes sur les Infusoires endoparasites. Arch, zool. exp. gén., (4) 2: 337-56. —— 1909a. Sur une Microsporidie parasite d’une Grégarine. C. R. Acad. Sci. Paris, 148: 733-34. —— 1909b. Perezia lankesteriae n. g., n. sp., Microsporidie parasite de Lankesteria ascidiae (Ray-Lank.). Arch. zool. exp. gén., (5)1: N. et R., 89-93. 1909c. Etudes sur la sexualité chez les Grégarines. Arch. Protistenk., 17: 19-134. Leidy, J. 1879. Fresh-water rhizopods of North America. U. S. Geol. Sur. Merry Reps.) 12:m12 324: ——— 1881. The parasites of the termites. J. Acad, nat. Sci. Philad., (2)8: 425-47. PARASITES OF PROTOZOA 1107 Leiner, M. 1924. Das Glycogen in Pelomyxa palustris Greef, mit Beitragen zur Kenntnis des Tieres. Arch. Protistenk., 47: 253-307. Liebmann, H. 1936a. Auftreten, Verhalten und Bedeutung der Protozoen bei der Selbstreinigung stehenden Abwassers. Z. Hyg. InfektKr., 118: 29-63. —— 1936b. Die Ciliatenfauna der Emscherbrunnen. Z. Hyg. InfektKr., 118: SDD =i! a6 — 1937. Bakteriensymbiose bei Faulschlammziliaten. Biol. Zbl., 57: 442- 45. Light, S. F. 1926. On Metadevescovina debilis gen. nov., sp. nov. Univ. Cal. Publ. Zool., 29: 141-57. Lutz, A., and A. Splendore. 1908. Uber Pebrine und verwandte Mikro- sporidien. 2. Mitt. Zbl. Bakt., Abt. I, 46: 311-15. Lwoff, A. 1925. Chytridinées parasites des Amibes de homme. Possibilité de leur utilisation comme moyen biologique de lutte contre la dysenterie amibienne. Bull. Soc. Path. exot., 18: 18-23. Lynch, J. E., and A. E. Noble. 1931. Notes on the genus Evdosphaera Engel- man and on its occasional host Opisthonecta henneguyi Fauré-Fremiet. Univ. Cal. Publ. Zool., 36: 97-114. Mackinnon, D. L. 1914. Observations on amoebae from the intestine of the crane-fly larva, Tzpwla sp. Arch, Protistenk., 32: 267-77. Mackinnon, D. L., and H. N. Ray. 1931. Observations on dicystid gregarines from marine worms. Quart. J. micr. Sci., 74: 439-66. Mainx, F. 1928. Beitrage zur Morphologie und Physiologie der Eugleninen. I. Teil Morphologische Beobachtungen, Methoden und Erfolge der Reinkultur. Arch. Protistenk., 60: 305-54. Mangenot, G. 1934. Sur l’association d’une Rhodobactérie et d’un Infusoire. C. R. Soc. Biol. Paris, 117: 843-47. Martin, C. H. 1909. Some observations on Acinetaria. I. The ‘“Tinctin- kérper”’ of Acinetaria and the conjugation of Acineta papillifera. 1. The life-cycle of Tachyblaston ephelotensis (gen. et spec. nov.), with a possi- ble identification of Acinetopsis rara, Robin. Quart. J. micr. Sci., 53: ZDL-89. Mattes, O. 1924. Uber Chytridineen im Plasma und Kern von Amoeba sphaeronucleolus and Amoeba terricolo. Arch. Protistenk., 47: 413-30. Mayer, M. 1920. Zur Cystenbildung von Trichomonas muris. Arch. Pro- tistenk., 40: 290-93. Mecznikow, E., see Metschnikoff, E. Mercier, L. 1907. Un parasite du noyau d’ Amobea blattae Biitschli. C. R. Soc. Biol. Paris, 62: 1132-34. Also Réun. Biol., Nancy, 1907: 52-54. —— 1910. Contribution a I’étude de l’Amibe de la Blatte (Entamoeba blattae Butschli). Arch. Protistenk., 20: 143-75. Metschnikoff, E [Mecznikow, E.}. 1864. Uber die Gattung Sphaerophrya. Arch, Anat. Physiol. wiss. Med., 1864: 258-61. 1108 PARASITES OF PROTOZOA — 1892. Lecons sur la pathologie comparée de l’inflammation. Faite a l'Institut Pasteur en Avril et Mai, 1891. Paris. Minden, M. von. 1915. Chytridiineae zz Pilze I. Kryptogamenfl. Mark Brendenb., 5: 209-422. Mitchell, J. B., Jr. 1928. Studies on the life history of a parasite of the Englenidae. Trans. Amer. micr. Soc., 47: 29-41. Miyashita, Y. 1933. Drei neue parasitische Infusorien aus dem Darme einer Japanischen Stisswagseroligochate. Annot. zool. jap., 14: 127-31. Molisch, H. 1903. Amoeben als Parasiten in Volvox. Ber. dtsch. bot. Ges., ZU 20223, Miller, J. 1856. Einige Beobachtungen an Infusorien. Monatsber. preuss. Akad. Wissensch., 1856: 389-93. Myers, E. H. 1938. The present state of our knowledge concerning the life cycle of the Foraminifera. Proc. nat. Acad. Sci. Wash., 24: 10-17. Nagler, K. 1910. Fakultativ parasitische Micrococcen in Amo6ben. Arch. Protistenk., 19: 246-54. — 191la. Studien tber Protozoen aus einem Almtimpel. I. Amoeba hart- manni n. sp. Anhang: Zur Centriolfrage. Arch. Protistenk., 22: 56-70. — 1911b. Studien uber Protozoen aus einem Almtiimpel. II. Parasitische Chytridiaceen in Evglena sanguinea. Arch. Protistenk., 23: 262-68. Neresheimer, E. 1904. Uber Lohmannella catenata. Z. wiss. Zool., 76: 137-66. 1908. Die Mesozoen. Zool. Zbl., 15: 257-312. Noller, W. 1921. Uber einige Wenig bekannte Darmprotozoen des Menschen und ihre nachsten Verwandten. Arch. Schiffs u. Tropenhyg., 25: 35-46. — 1922. Die wichtigsten parasitischen Protozoen des Menschen und der Were, Teil, Berlin. Nowakowski, L. 1876. Beitrag zur Kenntnis der Chytridiaceen. II. Polyphagus Euglenae. Beitr. Biol. Pfl., ed. by Ferdinand Cohn, 2: 201-19. Pascher, A. 1929. Studien tiber Symbiosen. I. Uber einige Endosymbiosen von Blaualgen in Einzellern. Jb. wiss. Bot., 71: 386-462. Pellissier, M. 1936. Sur certains constituants cytoplasmiques de |’Infusoire cilié Trichodinopsis paradoxa Clap. et Lach. Arch. zool. exp. gén., 78: INSiet R 32-36: Penard, E. 1893. Pelomyxa palustris et quelques autres organismes inférieurs. Arch. Sci. phys. nat. (3), 29: 165-82. — 1902. Faune rhizopodique du bassin du Léman. Genéve. — 1904. Héliozoaires d’eau douce. Genéve. — 1905a. Sur les Sarcodinés du Loch Ness. Proc. roy. Soc. Edinb., 25: 593-608. —— 1905b. Observations sur les Amibes a pellicule. Arch. Protistenk., 6: 175-206. —— 1905c. Notes sur quelques Sarcodinés. Ire partie. Rev. suisse Zool., 13: 585-616. PARASITES OF PROTOZOA 1109 —— 1908-9. Uber ein bei Acanthocystis turfacea parasitisches Rotatorium. Mikrokosmos, 2: 135-43. —— 1909. Sur quelques Mastigamibes des environs de Genéve. Rev. suisse Zool., 17: 405-39. —— 1912. Nouvelles Recherches sur les Amibes du groupe Terricola. Arch. Protistenk., 28: 78-140. 1922. Etudes sur les Infusoires d’eau douce. Geneva. Petschenko, B. de. 1911. Drepanospira miilleri n. g., n. sp. parasite des Para- meciums; contribution a l’étude de la structure des Bacteries. Arch. Protistenk., 22: 248-98. Pierantoni, O. 1936. La simbiosi fisiologica nei termitidi xilofagi e nei loro flagellati intestinali. Arch. zool. (ital.) Torino, 22: 135-71. Pinto, C., and F. da Fonseca. 1926. Trichomonas vitali nova especie. Parasitis- mo das Trichomonas por Sphaerita minor Cunha et Muniz, 1923, e relacdo das especies de Sphaeritas conhecidas. (In Portuguese) . Bol. biol. Fac. Med. S. Paulo, 1926: 34-37. Plate, L. 1886. Beitrige zur Naturgeschichte der Rotatorien. Jena. Z. Naturw., LOE L120. —— 1887. Uber einige ectoparasitische Rotatorien des Golfes von Neapel. Mitt. Zool. Sta. Neapel, 7: 234-63. 1888. The genus Acinetoides, g. n., an intermediate form between the ciliated Infusoria and the Acinetae. Ann. Mag. Nat. Hist., (6) 2: 201-8. (Translation.) Original in Zool. Jahrb., Abt. Anat. Ont., 3: 135-43 (1888). Powell, W. N. 1928. On the morphology of Pyrsonympha with a description of three new species from Reticulitermes hesperus Banks. Univ. Cal. Publ. Zool., 31: 179-200. Powers, P. B. A. 1933. Studies on the ciliates from sea-urchins. I. General Taxonomy. Biol. Bull., 65: 106-21. —— 1935. Studies on the ciliates of sea-urchins. A general survey of the infestations occurring in Tortugas echinoids. Publ. Carneg. Instn., 452: 293-326. Prandtl, H. 1907. Der Entwicklungskreis von Allogromia sp. Arch. Protis- tenk., 9: 1-21. Pringsheim, E. G. 1928. Physiologische Untersuchungen an Paramecium bursaria. Ein Beitrag zur Symbioseforschung. Arch. Protistenk., 64: 289-418. Puymaly, A. de. 1927. Sur le Sphaerita endogena Dangeard, Chytridiaceé parasite des Euglénes. Bull. Soc. bot. Fr., 74: 472-76. Raymond, G. 1901. Sur un Monadinazoosporeé parasite de Chlamydomonas. Microgr. prép., 9: 128-31. Rieder, J. 1936. Biologische und dkologische Untersuchungen an Siisswasser- Suktorien. Arch. Naturgesch., 5: 137-214. 1110 PARASITES OF PROTOZOA Robertson, M. 1905. Pseudospora volvocis, Cienkowski. Quart. Jour. micr. Sci., 49: 213-30. Roskin, G. 1927. Zur Kenntnis der Gattung Psewdospora Cienkowski. Arch. Protistenk., 59: 350-68. Ryder, J. A. 1893. The growth of Ezgleno viridis when constrained princi- pally to two dimensions of space. Contr. zool. Lab. Univ. Pa., 1: 37-50. Sand, R. 1899. Etude monographique sur le groupe des Infusoires tentaculi- féres. Ann. Soc. belge Micr., 24: 57-189; 25: 7-205. Sassuchin, D. N. 1928a. Zur Frage uber die Parasiten der Protozoen. Parasiten von Nyctotherus ovalis Leidy. Arch. Protistenk., 64: 61-70. —— 1928b. Zur Frage uber die ecto- und entoparasitischen Protozoen der Froschkaulquappen. Arch. Protistenk., 64: 71-92. —— 1931. Zum Studium du Darmprotozoenfauna der Nager im Siid-Osten RSFSR. I. Darmprotozoen des Citellus pygmaeus Pallas. Arch. Protis- tenk., 74: 417-28. —— 1933. Materials on hyperparasitism in Protozoa (in Russian). Rev. Microbiol. Saratov, 12: 219-27. —— 1934. Hyperparasitism in Protozoa. Quart. Rev. Biol., 9: 215-24. Sassuchin, D. N., P. P. Popoff, W. A. Kudrjewzew, and W. P. Bogenko. 1930. Uber parasitische Infektion bei Darmprotozoen. Arch, Protistenk., 71: 229-34. Schereschewsky, Helene. 1924. La Famille Metchnikovellidae (C. & M.) et la plaie qu'elle occupe dans le Systéme des Protistes (Russian with French summary). Arch. russ. Protist., 3 (3-4): 137-45. Scherffel, A. 1925a. Endophytische Phycomyceten-Parasiten der Bacillariaceen und einige neue Monadinen. Ein Beitrag zur Phylogenie der Oomyceten (Schréter). Arch. Protistenk., 52: 1-141. — 1925b. Zur Sexualitat der Chytridineen. (Der Beitrage zur Kenntnis der Chytridineen. Teil I. Arch. Protistenk., 53: 1-58. —— 1926a. Einiges ber neue oder ungeniigend bekannte Chytidineen. (Der Beitrage zur Kenntnis der Chytridineen. Teil I]. Arch. Protistenk., 54: 167-260. — 1926b. Beitrige zur Kenntnis der Chytridineen. Teil HI. Arch. Protis- tenk., 54: 510-28. Schouten, G. B. 1937. Nyctotherus ochoterenae n. sp. y Nyctotherus gamarrat n. sp. Protozoarios parasitos de batracios (Evgystoma ovale bicolor Schn. e Hyla venulosa Laur.). An. Inst. Biol. Univ. Méx., 8: 387-92. Schubotz, H. 1908. Pycnothrix monocystoides nov. gen., nov. sp., ein neues ciliates Infusor aus dem Darm von Procavia (Hyrax) capensis (Pallas). Denkschr. med-naturw. Ges. Jena, 13: 1-18. Schulze, F. E. 1875. Rhizopodienstudien. V. Arch, mikr. Anat., 11: 583-96. Serbinow, J. L. 1907. Beitrage zur Kenntnis der Phycomyceten. Organisation u. Entwickelungsgeschichte einiger Chytridineen-Pilze (Chytridineae Schroter). Scripta bot. Petropol, 24: 1-173. PARASITES OF PROTOZOA 1111 Skvortzow, B. W. 1927. Uber einige Phycomycetes aus China. Arch. Protis- tenk., 57: 204-6. Sokoloff, D. 1933. Algunas nuevas formas de flagelados del Valle de Mexico. An. Inst. Biol. Univ., Mex., 4: 197-206. Stabler, R. M. 1933. On an amoeba parasitic in Ze/leriella (Protozoa, Ciliata). J. Parasit., 20: 122. Stabler, R. M., and T. T. Chen. 1936. Observations on an Endamoeba patra- sitizing opalinid ciliates. Biol. Bull., 70: 56-71. Stein, F. (Ritter) von. 1849. Untersuchungen tber die Entwickelung der Infusorien. Arch. Naturgesch., 15; 1: 92-148. —— 1854. Die Infusionsthiere auf ihre Entwickelungsgeschichte untersucht. Leipzig. — 1859. Der Organismus der Infusionsthiere. I. Abtheilung. Algemeiner Theil und Naturgeschichte der hypotrichen Infusionsthiere. Leipzig. pp. 96-100. — 1867. Der Organismus der Infusionsthiere. II. Abtheilung. (1) Dar- stellung der neuesten Forschungsergebnisse tiber Bau, Fortpflanzung und Entwickelung der Infusionsthiere. (2) Naturgeschichte der heterotrichen Infusorien. Leipzig. — 1878. Der Organismus der Infusionsthiere. III. Abtheilung. Flagella- ten. Leipzig. — 1883. Der Organismus der Infusionsthiere. HI. Abtheilung. II. Hialfte die Naturgeschichte der Arthrodelen Flagellaten. Leipzig. Stempell, W. 1909. Uber Nosema bombycis Nageli. Arch, Protistenk., 16: 281-358. — 1918. Uber Leptotheca coris n. sp. und Nosema marionis. Mitt. zool. Inst. Univ. Minster, 1: 1-6. — 1919, Untersuchungen tiber Leptotheca coris n. sp. und das in dieser schmarotzende Nosema marionis Thel. Arch. Protistenk., 40: 113-57. Stokes, A. C. 1884. A microscopical incident. Microscope, 4: 33-35. Stubblefield, J. W. The morphology and life history of Amphiacantha ovalis and Amphiacantha attenuata, two new haplosporidian parasites of gre- garines. Univ. Cal. Library, MS. 1937. Studitsky, A. N. 1932. Uber die Morphologie, Cytologie und Systematik von Ptychostonum Chattoni Rossolimo. Arch. Protistenk., 76: 188-216. Sutherland, J. L. 1933. Protozoa from Australian termites. Quart. J. micr. SC, 762, 145-73. Swarczewsky, B. 1908. Uber die Fortpflanzungerscheinungen bei Arcella vul- garis Ehrb. Arch. Protistenk., 12: 173-212. Takagi, S. 1938. On a bacterial parasite with active motility inhabiting the the internal protoplasm of Spirostomum ambiguum Ehrenb. Annot. zool. Jap: £7: 170-78. Thélohan, P. 1895. Recherches sur les Myxosporidies. Bull. Sci. Fr. Belg., 26: 100-394 (p. 360). 1112 PARASITES OF PROTOZOA Tyzzer, E. E. 1920. Amoebae of the caeca of the common fowl and of the tutkey.—Entamoeba gallinarum, sp. n. and Pygolimax gregariniformis, gen. et spec. nov. J. med. Res., 41: 199-209. Veley, L. J. 1905. A further contribution to the study of Pelomyxa palustris (Greeff). J. Linn. Soc. (Zool.), 29: 374-95. Wager, H. 1913. The life-history and cytology of Polyphagus Euglenae. Ann. Bot. Lond., 27: 173-202. Walker, E. L. 1909. Sporulation in the parasitic Ciltata. Arch. Protistenk., 17: 297-306. Wallengren, H. 1895. Studier dfver ciliata infusorier. Arsskr. Lunds Univ., Ailey Wallich, G. C. 1863a. Further observations on an undescribed indigenous amoeba, with notices on remarkable forms of Actinophrys and Difflugza. Ann. Mag. Nat. Hist., (3) 11: 365-71. — 1863h. Further observations on Amoeba villosa and other indigenous thizopods. Ann. Mag. Nat. Hist., (3) 11: 434-53. Wenrich, D. H. 1921. The structure and division of Trichomonas muris (Hartmann). J. Morph., 36: 119-55. —— 1932. The relation of the protozoan flagellate, Retortomonas gryllo- talpae (Grassi, 1879) Stiles, 1902 to the species of the genus Embado- monas MacKinnon, 1911. Trans. Amer. micr. Soc., 51: 225-38. — 1937. Studies on lodamoeba biitschlii with special reference to nuclear structure. Proc. Amer. phil. Soc., 77: 183-205. — 1940. Studies on the biology of Dientamoeba fragilis. Pp. 408-9 in Third International Congress for Microbiology: Report of Proceedings. New York. Wenyon, C. M. 1907. Observations on Protozoa in the intestine of mice. Arch. Protistenk., suppl. 1: 169-201. 1926. Protozoology, a manual for medical men, veterinarians and zo- ologists. New York. Wesenberg-Lund, C. 1929. Rotatoria, Rotifera - Radertierchen, 77 Kikenthal, W. and T. Krumbach: Handbuch der Zoologie. Berlin. Wetzel, A. 1925. Zur Morphologie und Biologie von Raphidocystis infestans n. sp. einem temporar auf Ciliaten parasitierenden Heliozoon. Arch. Protistenk., 53: 135-82. Winogradowa, T. 1936. Sphaerita, ein Parasit der Wiederkauerinfusorien. Z. Parasitenk, 8: 356-58. Yagiu, R. 1933. Studies on the ciliates from the intestine of Azthocidaris crassispina (A. Agassiz). I. Cyclidium ozakii sp. nov. & Strobilidium rapulum sp. nov. J. Sci. Hiroshima Univ., (B:1) 2:211-22. Yakimoff, W. L. 1930. Zur Frage tiber Parasiten bei Protozoa. Arch. Protis- tenk., 72: 135-38. eet "eee PARASITES OF PROTOZOA iI Ie — 1931. Bolesni domaschnick schirootnich wisiroaemie prosteischmimi (Protozoa) (in Russian). (Cited by Sassuchin, 1934, without journal.) Yakimoff, W. L., and A. P. Winnik. 1933. Die Ansteckung der Oocysten der Kaninchencoccidien durch Bakterien. Arch. Protistenk., 79: 131-32. Yuan-Po, Li. 1928. Entamoeba bobaci n. sp. des tarabagans (Marmota bobac). Ann. Parasit. hum. comp., 6: 339-42. Zacharias, O. 1909. Parasitische AmGben in Volvox minor, Arch. Hydrobiol., 5: 69-70. Zeliff, C. C. 1930. A cytological study of Oxymonas, a flagellate, including the descriptions of new species. Amer. J. Hyg., 11: 714-39. INDEX Abbott, 972 Ablastin, 838; involved in immunity against trypanosomes, 856-62 passim Accessory vacuoles, see Vacuoles, accessory Accidental parasitism, defined, 895; see also Parasitism Acclimatization, to experimental condi- tions, 507; and immunity, inherited, 717-21 Acids, effects on consistency, 55, 61; motor responses to, 333, 342 Actinophrys sol, 600 Actinopoda, sexual reproduction, 598 ff. Active contraction vs. elastic shortening, NO, Dil Activities influencing longevity, 16 Adhesiveness (or stickiness), 71-77; rela- tion to phagocytosis, 74; in animals that live in association with hosts, 930 ff., 944, 948, 949 Adolph, 65, 362, 363, 364, 424, 525 Aérobes, why die in absence of oxygen? 394 Aérobic respiration, see Respiration Afridi, 849 Age changes; sexual immaturity and ma- turity, 714f., 761 Aggregation, in light, 281, 297; in acid region, 342 Agranulocytes, 835 Albuminoid reserves, 160, 162 Alda Calleja, M. de, 876 Alexander, 58 Alexeieff, 160, 903, 907, 910, 970, 1033, 1042, 1050 Alkalies, effects on consistency, 55; motor responses to, 333, 342 Allelocatalytic growth, 533 ff. Allison, 79 Alsup, 314 Alternating current, responses to, 310-14 Altmann’s bioblast theory, 174, 175 Alverdes, 793 Amberson, 356, 363, 369 American Society of Parasitologists, Com- mittee on Terminology, 891 Amoeba, properties of protoplasm as ex- hibited in, 46-50; surface precipitation reaction, 48; consistency, 51-61 passim; surface properties, 63-74 passim; spe- cific gravity, 79, 80, 81; other proper- ties, 82, 84, 90-97 passim; survey of functions having granular basis, 168-75 passim; structure, 272; responses to light, 272-80; to electricity, 305-20, 323; to chemicals, 333-41; dysentery and bacterial complications, 819-22 Amoebae, parasitic in a free-living ciliate, 1075 Amoebophrya Koeppen, 1089-93 Anaérobes, why are they anaérobes, 390- 94 Anaérobic metabolism, measurement of, 385 f. Ana€érobiosis, occurrence of glycolysis and, 386-90 Ancestry, relation to conjugation, 615; necessity of diverse, 696 Ancistridae, 933 Ancistruma, fibrillar system, 228 Ancistrumidae, adaptation, 934-38, 939, 941 André, 900, 916, 934 Andrews, 927 Andrews and von Brand, 365, 368, 369, 485 Anentera, 12 Anesthetics, effect on O2 consumption, 366 Aneurin, or thiamine (vitamin Bi), 490 Angerer, 48, 58 Animals: relationships between certain Protozoa and other animals, 890-1008 (see also under Relationship) Anisogamy, term, 584 Anoplophrya circulans, 951, 952 Antibodies and antigens involved in im- munity, 837-39, 873 Antiserum, 837 Antitoxin, 838 Anus, presence in Infusoria ascertained, 11 Apostomea, 956; adaptation, 957-60 Archer, 1094 1116 Artigas and Pacheco, 912 Aschner, 964 Aschner and Ries, 964 Aschoff, 836, 839 Asexual reproduction, alternation of sex- ual with, 566 f., 571-73; in alternating binary and multiple fission, 569-71 Aspidisca, reorganization in, 22 f. Association, see Relationships Astomata, 945; adaptation, structure, 946- 53; Conidiophrys, 953-56 Attachment, see Adhesiveness Autocatalytic growth, 533 ff. Autogamy (self-fertilization), 654-59; genetic evidence, 757 Avian malaria, 844 Awerinzew, 1074, 1082 606-11, Bach and Quast, 1022 Bacigalupo, 1045 Bacteria, elimination of, before measure- ment of respiration, 358; methods for control or elimination of, 448 ff.; bac- tericidal agents, 463-67; culture, 467; amoebic dysentery and bacterial compli- cations, 819-22; cellulose decomposition by, 962 ff., 966f.; nitrogen-fixing, 972 Bacteriology, Leeuwenhoek the father of, 11 Baitsell, 528 Balamuth, 789, 932 Balantidium coli and B. suis, fibrillar sys- tem, 244; B. sushilii, 245 Balbiani, E. G., 775, 780, 787, 793, 951, 952, 1034, 1035, 1036, 1086, 1087, 1089; interpretation of the process of conjugation, 11, 13, 14 Ball, 66 Bancroft, 281, 287, 320, 321, 323, 324, 327 Barber, 873 Barcroft differential type, 356, 357 Barratt, 362, 366 Barry, M., 13 Barth, 54 Bary, A. de, originator of term symbiosis, 891 Basal apparatus in Paramecium, 195, 198, 200, 204, 226, 227 Basalfibrille, 201, 204, 219 Basal metabolism, measurement, 360 Basophilic granules, 160, 162, 163-65 Bauer and Granowskaja, 800 INDEX Bayliss, 51, 61, 306, 319 Beams, H. W., and King, R. L., 69, 98; Some Physical Properties of the Proto- plasm of the Protozoa (Chap. II), 43- 110 Beauchamp, 905, 907 Bechdel, Honeywell, Dutcher, and Knut- sen, 985 Becker, Elery R., 85, 801, 818, 878, 893, 918, 979, 983, 984, 1045, 1047, 1048, 1050, 1051, 1052, 1058; Certain As- pects of Pathogenicity of Protozoa (Chap. XVII), 818-29 Becker, Elery R., and Derbyshire, R. C., 826 Becker, Elery R., and Everett, R. C., 986 Becker, Elery R., and Hsiung, T. S., 977 Becker, Elery R., and Talbott, M., 976 Becker, Elery R., and Waters, P. C., 824, 826 Becker, Elery R., Schulz, J. A., and Em- merson, M. A., 982, 983, 984, 985, 986 Beckwith and Rose, 966 Becquerel, 58 Beers, 91, 117, 119, 527, 528, 529, 540, 543, 616, 653, 657, 943, 944, 945, 946, 948, 949 Bélai, 26, 223, 253, 260, 594, 598, 599, 611 Bélehradek, 57 Bélehradek and Paspa, 94 Benda, 177 Benedict, 481 Ben Harel, 844 Bensley, 86, 118, 119, 120, 181 Berghe, van den, 901 Berkson et al, 518 Bernard, 534 Bernstein, 1044 Beta granules, 406, 435 Bethe, 258 Bibliography, see Literature cited Bignami, 846 Bills, CG. E.; 802 Biology of the Protozoa, The (Calkins), 3, 616 Biotypes, 712 Biparental reproduction, see Reproduction, biparental Bishop, A., 908, 909, 910, 911, 950; ciliate fibrillar systems, 250, 251 Blaauw, 289 Bles, 72, 77, 125, 167, 368, 384 INDEX Blockade and splenectomy, 832 Blood cells, classified, 834 ff. Bloom, 836 Bodine, 56, 542 Bodine and Boell, 374, 378 Bodine and Orr, 357 Boeck, 1011, 1032 Bohm, 848 Boissezon, 898 Bojewa—Petruschewskaja, 411 Bonar, 1068 Bond, 501, 519, 550 Borgert, 153, 1089, 1091, 1092, 1093 Bott, 593 Bourne, A. G. 1025 Bourne, G., 121 Bourne, G., and Allen, R., 121 Boveri, 638 Boveria, fibrillar system, 228, 229 Bowen, 138, 140, 180, 442, 543, quoted, 443 Bowling, 32, 33 Boyd, G. H., 843, 844, 845, 846 , G. H., and Allen, L. H., 844, 845 Boyd, G. H., and Dunn, M., 844 Boyd, M. F., and Coggeshall, L. T., 844 Boyd, M. F., and Kitchen, S. F., 823 Boyd, M. F., and Stratman-Thomas, W. K., 822, 825 Boyd, M. F., Stratman-Thomas, W. K., and Kitchen, S. F., 848 Boyd, M. F., Stratman-Thomas, W. K., and Muench, H., 823 Bozler, 802, 1035, 1037 Brahmachari, 875 Brain cells, 836 Brand, von, 151, 157, 159, 162, 361, 385, 387, 388, 389, 390, 485, 865 Brand, von, and Jahn, 361, 387, 392 Brand, von, Regendanz, and Weise, 389 Brandt, 78, 97, 787, 1069 Brauer, 208 Braun and Teichmann, 877 Brehme, 795 Bresslau, 87 Brinley, 51, 53, 55, 59 Broh-Kahn and Mirsky, 374, 381, 388, 391, 392, 394 Brown, D. E. S., and Marsland, D. A., 61 Brown, H. C., and Broom, 875 Brown, M. G., et al., 451, 466 Brown, V. E., 138, 199, 435, 436 Brown, W. H., 855, 856, 981 eh Browne, 81 Browning et al., 864, 870 Brues, 55 Bruetsch, 853 Brug, 847, 1046, 1054 Brumpt, 586, 623, 819, 820, 878 Brumpt and Lavier, 904, 1045, 1046, 1048, 1051, 1053, 1054, 1055, 1057, 1075, 1076, 1077 Brussin and Kalajev, 869 Buchanan, R. E., 1026 Buchanan, R. E., and Fulmer, E. I., 495, 547 Buchner, 504, 933, 963, 972, 1010 Buck, 823 Budding division, 28 Buder, 287 Bitschli, O., and Schewiakoff, 202, 204 Buffon, 9 Bullington, 793 Bundle, 985 Bunting, 909. Bunting and Wenrich, 909 Burch, 796 Burge, 384 Burk, 359, 384, 386 Burnside, 530, 796 Burt, 454 Buscalioni and Comes, 969 Buschkiel, 610 Buschkiel and Nerescheimer, 33 Bush, M., 126, 128, 136, 141, 145, 147, 179, 950; ciliate fibrillar systems, 240, 242 Butschli;) ©:, 12, 157, 413.) 566; .915; 1035, 1036, 1039, 1040, 1070, 1087, 1089; interpretation of the process of conjugation, 13, 14; discovery re carbo- hydrate granules of gregarines, 111, fi- brillar system, 201, 202, 208, 210, 213, 217.210: 224.1202 Butts, 53, 54 Cailleau, 478, 384, 485, 486, 492, 493, 500 Cajal, Ramon, y, Santiago, 138, 144 Caldwell, 660 Calkins, Gary N., 16, 18, 21, 28, 29, 30, 32. 33% 344.375. 38) 4551s 85, 86, 112. 117, 161, 431, 529, 647, 649, 666, 699, 714, 893, 934, 1038; General Con- siderations (Chap. I), 3-42; Biology of the Protozoa, 3, 616; ciliate fibrillar bees INDEX : Calkins, Gary N. (continued) systems, 256, 257; fertilization, 583, SOP, SOB, S845 GOs, Gl, GIG, Gl. 618, 624, 627, 630, 632, 633, 637; morphogenesis, 774, 775, 776, 777, 718; 7195 1805 7895 79715 798 Calkins, G. N., and Bowling, R., 233, 591, 602, 611 Calkins, G. N., and Cull, S. W., 581, 626, 634, 636, 689, 702 Campbell, A. S., 160, 166, 1069; ciliate fibrillar systems, 253, 254 Campbell, W. G., 962 Candolle, de, 282 Cannon, P. R., and Taliaferro, W. H., 849 Canti, 138 Capillary manometer, 357 f. Carbohydrates, reserves, 153-59, 173; granules with definite internal structure, 154; differentiation between glycogen and paraglycogen, 157 Carini, 1075, 1076 Carini and Reichenow, 1075, 1076 Carlgren, 319, 324, 327 Carlson, 518 Carter, 1040, 1042 Cartesian diver ultramicromanometer, 358 Casagrandi and Barbagallo, 1043 Catalase, 373; detection of, 384 Cattaneo, 899 Cattle, rumen ciliates, 894, 973-87 (see also under Ciliates of ruminants) Caullery, 891, 892, 893 Caullery and Mesnil, 1078, 1079, 1080, 1081, 1082 Causin, 795, 797 Cavallini, 1075 Cejp, 1043, 1052 Cell division, longevity influenced by re- organization through, 16; in flagellates, 25 ff.; material for study or cytology of, 45; environmental conditions suit- able for most rapid growth and divi- sion, 45; division rates of Protozoa with constant conditions, 527, table, 528; regeneration and, 775-78, 797-801, division cycle, 775-78 Cell membrane, nature of, 62; permeabil- ity, 64-66, 69; other than those of Protozoa, 64 Cell theory, forerunner of, 9; first applied to the Protozoa, 13 Cells, first use of word, 9; longevity of derived structures, 38; macronucleus, 38; micronucleus transcends other struc- tures in ciliate cell, 39; differentia- tion, 44; comparison between protozoan and metazoan, 44, 179ff; relative specific gravity of inclusions and com- ponents of, 80-82; Golgi bodies in metazoan, 140, 143; importance of réle of Golgi apparatus in life history of, 431, 441; link in kinship between all cells apparently established by Golgi apparatus, 441, 443; structures present in all, 432; connective tissue cells involved in immunity: terminol- ogy and classification, 831-37; of the blood and lymph, classified, 834-36; so- called systems of, used by authors, 836 f.; cellular and humoral aspects of immunity, 839-41 Cellular concept of living things, disclos- ure of, 191, difficulties encountered, 191; protoplasmic differentiation con- trasted with cellular differentiation, 192, 260 Cellulose, constituent of wood, 961; use of, in nutrition, 961-73; decomposition, 962 ff., 966f., 983; experiments in feeding cellulose-free | carbohydrates, 970; use by rumen ciliates, 981 f., 983 Centrifuge method, 52; as a research tool, 81 Cépéde, 915, 936, 938, 945, 946, 948, 950, 952, 953 Cépéde and Poyarkoff, 953 Cépéde and Willem, 1020, 1022 Certes, 911 Chakravarty, 158, 163 Chalkley, 45, 57, 59, 67, 73, 83, 84, 85, 121, 340, 518, 523 Chambers, 46, 48, 49, 51, 52, 58, 62, 63, 65, 66, 67, 72, 85, 88, 181, 339, 395, 396 Chambers and Dawson, 76 Chambers and Hale, 58 Chambers and Howland, 54 Chambers and Kempton, 127 Chambers and Pollack, 127 Chambers and Reznikoff, 52, 53, 315 Chapman, 553 Characteristics, inheritance of, 712 f. Characters, inherited; changes in, in uni- parental reproduction, 713-31 Chase and Glaser, 59, 340 INDEX Chatterji, Das, and Mitra, 912 Chatton, E., 587, 930, 941, 1069, 1075, 1079, 1082 Chatton, E., and Biecheler, B., 588, 1070 Chatton, E., and Brachon, S., 1072 Chatton, E., and Brodsky, A., 593, 1041, 1042, 1048, 1051, 1053, 1058 Ghatton, E, and Grasse, P, P., 113, 115 Chatton, E., and Lwoff, A., 75, 1025, 1073, 1083, 1084, 1085; fibrillar com- plex, 200, 223, 228; relationships, 620, 9351; 933, 934, 936, 938; 940; 941, 942, 946, 953, 954, 955, 956, 957, 958, 959, 960 Chatton, E., and Pénard, C., 621 Chatton, E., and Villeneuve, S., 1072 Chatton, E., Lwoff, A., Lwoff, M., 1083 Chatton, E., Parat, M., and Lwoff, A., 165, 180 Cheissin, 151, 936, 943, 944, 945, 946, 947, 948, 950, 952, 956 Chejfec, 649, 660, 775 Chemicals, responses to, 333-44; rhizo- pods, 333-41; ciliates, 342-44 Chen and Stabler, 1045, 1076 Child, C. M., 86, 773, 802, 808 Child, C. M., and Deviney, E., 86, 803 Child (MS), 928 Chilodonella, 27, 630f., 632, 633, 634, 635, 637, 638 Chlamydodon, fibrillar system, 229, 231 Chlamydomonas, division, 589f., 613; kinds of gametic differences in, 589, 667-71; sexuality, 666-87; nature of physiological differences between gam- etes in, 671-78; interpretation of sex- ual phenomena, 678-87; inheritance, 722, 739, 741, 742, 744, 745 Chlorellae, relationship to host, 1010 Cholesterol, 493 Cholodnyj, 344 Chondriome, 170, 432; see also Mito- chondria Chondriosomes, 432; permanence, see also Mitochondria Chopra and Mukherjee, 848 Chopra, Gupta, and David, 875 Chorine, 875 Christophers, 854 Chromatin, value and importance shown, 39; volutin linked with, 162 Chromatin reaction, in derived structures, 183) 21) tr. 181; PES Chromatoidal bodies, 160 Chromidia, the term and its interpretation, 160; associated with mitochondria, 160, 161; term bound up with disproved theories, 165 Chromidial net a definite morphological entity, 161 Chromidial origin of nuclei doubted, 594 Chromosomes, individuality retained in all cell generations, 87; genetic materials in, 710, 711 Ciacco, 123 Cienkowsky, 1064, 1070, 1074, 1086 Ciliates, reorganization of the macronu- cleus and other derived structures, 21- 31; waning vitality, 28 ff., reorganiza- tion by endomixis, 31-36; intracellular micronuclei forming pronuclei, 33; longevity of protoplasm, 34; reorgani- zation by conjugation, 36-39; impor- tance of micronucleus, 39; adhesiveness, 75 ff., 932, 944, 948, 949; ability to combine into composite organelles, 76; color, 83; polarity, 85; contraction, 92, 93; double refraction, 96; segregation granules, 132 ff., (see entries under Seg- regation); fibrillar systems, 191-270 (see also under Fibrillar); responses to light, 295-97; responses to electricity, 321-27; internal processes involved in responses, 324-27; responses to chemi- cals, 342-44; conditions necessary for conjugation, 614, 615 f.; conjugation, 616-39; sexuality in, 621 f., 666, 687- 706; biparental inheritance in diploid ciliates, 750-58; faunules of, in sea urchins, 894, 919-23; first to live in blood of host and circulate with cor- puscles, 952 Ciliates of ruminants, 894, 973-87; total number in an individual, 974; repro- ductive rate, 976; environment, culture, 976; defaunation treatments for elimina- tion of, 977; food, 979 ff., 985; value to host, 983-87; rdle of, in protein economy, 984 f. Ciliophora, Schizomycetes on, 1021-24; Schizomyectes in, 1034-40; parasitizing other Protozoa, 1083-89 Claff, C. L., migration-dilution apparatus, 461-63 Claparéde, 413, 1034, 1039, 1079 Claparéde and Lachmann, 805, 1034, 1120 Claparéde and Lachmann (continued) 1039, 1043, 1064, 1079, 1084, 1086, 1087 ClarkswACnee4s Clark, N. A., 535 Clark, W. M., 396 Classification, of fresh-water species on an ecological basis, 5; Millers, the earliest successful, of Protozoa, 11; cytoplasmic granules, 177-79; immunological reac- tions in relation to, 876-78 Cleveland, L. R., 45, 90, 154, 157, 391, 392, 460; 4635 89278935, °909; "911, 931, 962, 964, 965, 966, 967, 968, 969, 970, 971, 1015, 1016, 1018, 1020 Cleveland, L. R., and Sanders, E. P., 821 Cleveland, L. R., Hall, S. R., Sanders, E. P., and Collier, Jane, 391, 924, 926, 928, 929, 962, 965, 966, 968, 969, 970, 971, 972, 1015, 1028 Clones, 712; single-type, 756; crosses be- tween single- and double-type, 757 Coagulation, irreversible, 61 Coccidiosis in poultry, 824-26 Codreanu, M., and Codreanu, R., 906, 907 Codreanu, R., 897, 898, 915, 916, 917 Goe; 532 Coehn, A., and Barrat, W., 324 Coggeshall, 848, 849 Coggeshall and Eaton, 850, 875 Coggeshall and Kumm, 850 Cohen, Barnett, 395 Cohen, R. A., and Gerard, R. W., 381 Cohen, W. E., 965 Cohn, 208, 1064, 1093 Cohnheim, Ziegler, Marchand, 836 Colas-Belcour and Lwoff, A., 485, 486 Collin, B., 623, 627, 940, 1022, 1043, 1083, 1084, 1087, 1089 Colloidal nature of protoplasm, 49 f., 51 Colonial organisms, growth, 526f.; re- generation in, 804-11 Colonial organisms’ responses, to light, 297-305; shock reaction, 298; kinetic responses, 299; orientations, 300-303; wave length and response, 303; rever- sal in response, 303-5; to electricity, 327-32; orientation, 328; electric charge, 328; mechanics of response, 329-32 INDEX Colonies, temporary: formation and pur- pose, 74 Color of protoplasm, 82 f. Commensalism, term, 818, 891; inqui- lines, 892; physiological host relation- ships illustrative of mutualism and, 961-87 Complement fixation test, 873 f. Composite motor organelles, separation into their components, 76 Conchophthirus, reorganization, 21 f.; fibrillar system, 230, 232, 234 Condensation membrane, 418 Conductivity, fibrillar, 258 Conidiophrys, 953-56 Conjugant meiosis, 624-39; first divi- sion, stages A, B, 626-31; second, stage C, 631f.; third, stage D, and formation of pronuclei, 632-34; stage E, migration of pronuclei and fertiliza- tion, 634f.; stages F, G, H, The ex- conjugants, 635-39 Conjugation, Balbiani’s interpretation of process of, 11, 13, 14; Miller’s ob- servations, 11, 13; tests, 31; reorgani- zation by, 36-39; ciliate, 36 ff., 616- 39; difference between endomixis and, 36; conditions necessary for, in ciliates, 614; defined, 617-23; macronucleus during, 623f.; sexual differences be- tween individuals, 688-99; role of en- vironmental conditions in determining, 700 f.; differences between gamete nu- clei during, 701 f.; significance of di- versities between conjugants and be- tween gamete nuclei, 703-6; regenera- tive ability, 778 f. Conklin, 84, 796 Connell, 163, 1019, 1030, 1031, 1044, 1052 Consistency of protoplasm, 50-61; effect of mechanical agitation, 47, 58; of water, 53; of salts, 53-55, 61; of acids and alkalies, 55, 61; of temperature 55-58; of hydrogen-ion concentration, 58; of narcotics, 59; of radiation, 60; of heavy water, 60; of electric current, 61; hydrostatic pressure, 61; irrevers- ible coagulation, 61 Contractile vacuoles, see Vacuoles, con- tractile Contractility, 90-94; active vs. elastic, 90, 91; Vorticella, 208 ff. 216 ff., 229, 258 INDEX Cook, 367, 1065 Copeland, 343 Copromonas subtilis, 584, 585 f. Coprozoic Protozoa, 6 Copulating cells, see Gametes Copulation, gametic meiosis and fertiliza- tion, 584-601; Sporozoa, 601-6 Corpse, 28 Councilmania lafleuri (Endamoeba colt), life cycle, 569, 572 Coventry, 855, 856, 857, 861 Cowdry, 118 Cowdry and Scott, 121, 135, 138 Cragg, 1043, 1045 Craig, 819, 874, 1043, 1044 Crawley, 606 Gross) J: B) 928 Cross-fertilization, differences self-fertilization and, 606 f. Crossing over and linkage, 737-40, 743- 45 Crouch, 912, 1044 Crozier, 531 Crozier and Harris, 531 Cryptocercus, Protozoa of termites and, 894, 923-29; relationship between flag- ellates of termites and, 894, 961-73 Crystals, specific gravity, 80; defined, 179 Cuenot, 915, 937 Culbertson, 860 Culbertson and Wotton, 855, 856, 860 Cultures, use of in study of Protozoa, 14f.; mediums, 15; technique and significance of control in protozoan, 448-74; ‘“‘pure-mixed”” methods, 448; problem of sterilization, 449-67; im- portance of adequate sterility tests, 467 f.; Establishment of sterilized Proto- zoa in, 468-73; literature cited 473 f., 507-16; food requirements and other factors influencing growth of Protozoa in pure cultures, 475-516; growth in pure, as a population problem, 495- 99; pedigree isolation culture and life cycles, 527-31; methods for growing bacteria-free, pure cultures, 538-44 Cunha, A. M., da, 1023 Cunha, A. M., da, and Muniz, J., 912, 1044, 1047, 1050 Cutler, 154 Cutler and Crump, 523, 524, 535 Cyanellae, relationship to host, 1010 Cyanide, respiratory sensitivity, 376 ff. between 1121 Cyclic adaptation of Protozoa to hosts, 953 Cyclical variations, and regeneration, 775- 80 Cycloposthiidae in mammals, 973 Cyst, vitality of protozo6n within, 4; specific gravity, 79 Cytochrome, 373 Cytochrome-cytochrome oxidase system of hydrogen acceptors, 376-80 Cytokinetic mechanism, 44 Cytology, cytoplasmic granules a special- ized branch of, 111 Cytology of cell division, material for study of, 45 Cytomicrosomes, 432; see also mitochon- dria Cytoplasm, codrdination between nuclear and cytoplasmic division processes, 85 f.; effect of, and its relation to nu- clear constitution, 762-69 Cytoplasmic fission, attachment an im- portant factor in, 73 Cytoplasmic inclusions, 111-90; mito- chondria, 112-26; vacuome hypothesis, 126-29; digestive granules, 129-32; seg- regation granules, 132-38; Golgi bodies, 138-44; excretory granules, 144-50; lipoid reserves, 150-53; carbohydrate reserves, 153-59; protein reserve bodies, 160-66; external secretion, 166-68; granular complex, 168-74; continuity of cytoplasmic granules, 174-77; classifica- tion of cytoplasmic granules, 177-79; comparison with cells of the Metazoa, 179-81; literature cited, 182-90; see also entries under Granules Czermak, 208, 217 Dahmen, 874 Dallasia, see Glaucoma frontata Dallinger, 718 Dangéard, P., 128, 168, 589, 613, 1032, 1033, 1041, 1042, 1043, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1056, 1057, 1058, 1059, 1063, 1065, 1066, 1067, 1070, 1073, 1074, 1075 Daniel, 369 Daniel and Chalkley, 56, 539 Daniels, M. L., cytoplasmic inclusions, 81, 117, 122, 126, 141, 142, 154, 160, 161, 162, 169, Darby, 527, 528, 529, 536, 541 1122 Darkness, adaptation to, 291; growth in relation to light and, 506 f. Das Gupta, 910 Dauermodifikationen, 720, 730 Daugherty, 59 Davenport, 56, 279 IDEN. Isls Shy Sill, OlZ Davis, L. J., 864, 865, 870 Dawson, 529, 650, 791 Dawson and Belkin, 74 Day, H. C., contractile vacuole, 147, 407, 410, 418, 420, 422, 424, 428 Death, cause of, 4; in isolation cultures, 39 Debaisieux, 608 Defaunation methods, 977 Deflandre, 1075 De Garis, C. F., 697, 753; inheritance in Paramecium, 762-69 Degen, 413, 426 Degeneration, inherited: resulting from unfavorable conditions, 716 Dehorne, 626 Dehydrogenases, 372; inhibition of the system, 381 f. Delafield’s hematoxylin, 832 Delage and Herouard, 202 De Lamater, 659 Delanoé, 861 Dellinger, 72, 73, 89, 91 Dembowska, morphogenesis, 774, 775, 777, 778, 784, 786, 790, 797, 798 Density (specific gravity), whole organ- isms, 77-79; relative, of cell inclusions and components, 80-82 Derived and fundamental organization, 4 Development, studies of regeneration and, 772 Developmental cycle of parasite and host, 933 Dewey, V. C., 469 Dextrose, value to wood-eating animals, 971 Diagnosis, immunological reactions used in, 872-76 Dickman, 966, 967 Dierks, K., fibrillar system, 93, 201-4 passim, 218, 219, 220 Diesing, 943 Differentiation, protoplasmic, 44 Difflugia corona, inheritance in, 724-29 Difflugia pyriformis reincorporation, 794 INDEX Diffraction, X-ray- and ultra-centrifuga- tion, 97 f. Digestive function of mitochondria, 123, 124 Digestive granules, 129-32; universality of, rejected, 172; defined, 178 Dileptus gigas, fibrillar system, 235 Diller; Ws F533; G10, 611, "6385 704; 756, 757; endomixis, 647, 649, 650, 651, 654, 655, 656,657, "658," G59; 661; quoted, 654 Dilution, sterilization by, 451-55; com- bined with migration, 460-63 Dimitrowa, A., 407, 413, 424, 499, 535 Dioecious races, 741 Diplodinium, fibrillar system, 251-53 Diploid parthenogenesis, 649 Diploids, biparental inheritance in, 750- 58 Direct current, responses to, 306-10 Division, see Cell division; Fission Diwald, 588, 613 Dixons 356; 3577, 369 Dixon and Bennet-Clark, 317 Dobell, 44, 158, 179, 191, 260, 576, 584, 585, 586, 612, 715, 870, 912, 1045, 1047 Dobell and Jameson, 612 Dobell and Jepps, 819 Doflein, 426, 594, 636, 689, 982, 983, 987, 1042, 1043, 1072, 1075, 1082, 1093 Doflein-Reichenow, 112, 583, 636, 893 Dogiel, 33, 619, 629, 621, 622, 634, 688, 702, 703, 973, 978, 979, 981, 985, 1015, 1024, 1044, 1045, 1046, 1050, 1078, 1079, 1080 Dogiel and Fedorowa, 157, 974, 975, 981 Dogiel and Furssenko, 915 Dogiel and Issakowa-Keo, 70 Dogiel and Winogradowa-Fedorowa, 976, MT SKS) Dore and Miller, 962 Double refraction, 95-97 Douvillé, 1068 Doyle, 116, 124, 174 Doyle and Harding, 425 Dropkin, 929 Dubos, 395 Duboscq and Collin, 1069 Duboscq and Grassé, 129f., 138, 154, 155, 158, 927, 1011, 1012, 1015, 1016, INDEX 1018, 1019, 1028, 1030, 1044, 1046, 1079 Duboscq, Grassé and Rose, 1015 Duca, 855 Dujardin, Felix, 208, 217, 413; quoted, 10, 43; studies by, 11; conclusions on Protozoa, 12, 43; controversy with Ehrenberg re Infusoria, 192, 260 Duke, 862 Duke and Wallace, 875 Dunihue, 130, 137, 436, 439 Duryée, 354, 356, 357 Dusi, H., food, etc., influencing growth, 477, 478, 479, 480, 481, 487, 488, 489, 490, 491, 502, 506 Du Toit, 878 Dyestuffs, use in experiments, 66 Dysentery, amoebic: and bacterial com- plications, 819-22 Eaton, 850 Ecological considerations, 4-8 Ectoparasites, 7 Ectoplasm (plasmagel), 47; elasticity, 87; contractility, 90, 91 Eddy, 533, 542 Edmondson, 914 Edwards:535 55559) 3i5n355 Efimoff, Nekrassow, and Efimoff, 393 Ehrenberg, C. G., 1093, 1094; studies by, 11, 43, 413; conclusions on Protozoa, 12, 13; controversy with Dujardin re Infusoria, 192, 260 fibrillar system, 200, 208, 209, 210, 216, 217, 219 Ehrlich and his coworkers, 869 Eikenberry and Waldron, 891 Eimeria schubergi, life cycle, 573-78 Eisenberg, E., 427, 428 Eisenberg-Hamburg, 340 Eksemplarskaja, 151 - Elasticity, of protoplasm, 87-90; of ir- reversible structures, 89; shortening vs active contraction, 90, 91; Vorticella, ZO9\fF., 217 ff... 258 Electric current, effects of, 61 Electricity, responses to, rhizopods, 305- 20; flagellates, 320; ciliates, 321-27; colonial organisms, 327-32 Elliott, A. M., 360, 468, 518, 537, 540, 542, 543; food, etc., influencing growth, LL25 484, 486, 487, 490, 492, 493, 501, 502, 503, 507 Elliott, A. M., and D. F. Johnson, 501 Ellis J. M., 145, 178; fibrillar system, 246 Elmassian, 1082 Elpatiewsky, 594, 595, 1074 Emerson, 362, 369, 371, 378, 388 Emik, 968, 969 Encyclopaedia Britannica, 891 Encystment, regenerative capacity, 779 Encystment test, 31 Endamoeba coli (Councilmania lafleuri), life cycle, 569, 572 Endomixis, reorganization by, 31-36; dif- ference between conjugation and, 36; macronuclear reorganization, 646-48; endomictic phenomena, 648-54; autog- amy, 654-59; coinage of name, 657; periodicity, 659 f.; genetical studies on, 660-62; literature cited, 662-65; segre- gation of mating types at, 756 Endoparasites, 7 Endoplasm (plasmasol), 47 Endothelial cells, 833, 834 Endozoic Protozoa, defined, 892 Energy, respiration a means of studying, 353 f.; investigations which concern the source of, 368-72 Engelmann, T. W., 29, 30, 34, 37, 96, 97, 1035, 1087; on conjugation, 14; fibrillar system, 198, 201, 208-15 passim, 217, 221; motor responses, 273, 281, 282, 289, 305 Enriques, 27, 622, 630, 633, 689, 703 Entamoeba, pathogenicity of, 819-22 Enterodela, 12 Entodiniomorphina, 973 Entodiscus borealis, fibrillar system, 236, 237 Entorhipidium echini, fibrillar system, 236, 239 Entz Get 25 Entz, Geza, fibrillar systems, 208-18 pas- sim, 258, 259, 909, 914, 1088 Environment, effect upon division and growth, 45; rdle in determining con- jugation, 700 f.; inherited degenerative changes resulting from unfavorable conditions, 716f.; inherited modifica- tions in form and structure, 712-23; variation .. . without obvious action of 1124 Environment (continued) diverse environments, 723-29; external, and regenerative behavior, 774 f. Enzymes, in mitochondria, 120, synthesis of respiratory, 382-84 Ephrussi, 64 Ephrussi and Neukomm, 340 Ephrussi and Rapkin, 54 “Epibionts,” 7 Epidinium caudatum, fibrillar system, 253 Epistylis, fibrils, 214 Epstein, 899, 1033, 1045, 1046, 1054, 1057, 1069 Erdmann, 154, 523, 524, 526, 530, 649, 659, 660, 758 Erdmann and Woodruff, 649, 657 Erythrocytes, 835 Estabrook, 521, 522, 523, 526 Euciliata, parasitic, 1083 f. Euglena, responses to light, 280-90 Euglenids, free-living and symbiotic, 903 ff. Euplotes, reorganization, 23, 24; fibrillar systems (structural analysis, 204-8, inter- pretation, 221-24, conclusions, 257 ff.) ; sexuality, and fertilization, 618, 619, 620, 621, 622, 626, 627, 628, 629, 632, 633, 634, 636, 637, 666, 696, 698, 700, 703 Eupoterion pernix, fibrillar system, 238, 240 Evans, Te G., 357 Everts, 208 Ewles and Speakman, 89 Excretory function of contractile vacuole, 422 ff., 442 Excretory granules, 144-50, 405, 440; mitochondria associated with, 124; uni- versality of, rejected, 173; defined, 178 Existence, struggle for, 553 f. Exogamy, differences between autogamy and, 606 f. Extension and retraction, see Contractility External secretion, 166-68 255 Fabrea salina, fibrillar system, 246 Fabre-Domergue, 916, 936, 937 Facultative parasitism, defined, 895; see also Parasitism Falaschini, 977, 986 Falck, 962 Fantham, 893, 977 Farr, 340 INDEX Fats, see Lipoids Faunules, distributional host relationships and _ host-specificity in representative symbiotic, 894, 917-29; of sea urchins, 894, 919-23; of termites and Crypto- cercus, 894, 923-29 Fauré-Fremiet, 43, 64, 74, 75, 76, 84, 90, 112, 115, 116, 118 120, 122, 146, 148, 160, 166, 167, 175, 180, 410, 419, 437, 526, 527, 534, 649, 805, 807, 934, 1021, 1022, 1079 Fauré-Fremiet, Léon, Mayer, and Plantefol, 394 Fauré-Fremiet, Mayer, Schaeffer, 127 Favella, fibrillar system, 253 Bennie354.05 57 Fenyvessy, von, and Reiner, respiration, 359) 3625-1366; 37s) 37185 388559. 390 Ferber, 974, 975, 980, 982, 985 Ferber and Winogradowa-Fedorowa, 976 984 Ferment theory of the vacuome, 127 Fermor, 610, 654 Fertilization in Metazoa, 583 f. Fertilization in Protozoa, 583-645; copu- lation, 584-606; autogamy, 606-11; zygotic meiosis, 611-14; significance, 614-17; conjugation, 617-23, 37; mac- ronucleus during conjugation, 623 f.; conjugant meiosis, 624-39; literature cited, 639-45 iat, Sul, Se, 7S) Feulgen hydrolysis, 18, 21 Fibers, elasticity, 90, contraction, 90, 92 Fibrillar systems in ciliates, 191-270; ex- amples, 193-228; structural analysis, 193-215, 228-57; Paramecium, 193- 200, 224-28; Holotricha, 193 ff., 228- 44: Heterotricha, 200 ff., 244-51; Sten- tor, 200-204, 218-21; Hypotricha, 204 ff., 255 ff., Euplotes, 204-8, 221-24; Vorticella, 208-15, 216-18; functions interpretation, 215-28, 228-57; Oligo- tricha, 251-54; conclusions, 257 ff.; need of future study, 261; literature cited, 262-70 Fibroblasts, defined, 834 Findlay and Brown, 850 Fine, 532, 542 Finley, 128, 135, 136, 698 983, 984, 137, 168, 438, INDEX Fisher, 47 Fission, asexual reproduction in alternat- ing binary and multiple, 569-71 Fission, cytoplasmic: attachment an im- portant factor in, 73 Fitzpatrick, 1040, 1047 Fiveiskaja, 153, 1035, 1036, 1037 Fixation mechanisms and habits, 71-77, 930 ff., 944, 948, 949 Fixation of complement test, 873 ff. Fixed material, nature of, 47 Flabellula, survey of functions having granular basis, 168-74 passim Flagella, used as organs of attachment, 74; contraction, 93 f. Flagellate responses to electricity, 320 Flagellate responses to light, 280-95; shock reaction and aggregation, 281; orientation, 282-87; wave length and stimulating efficiency, 287-90, 295; kin- etic responses, 290; adaptation to dark, 291; to light, 292-95 Flagellates, reorganization, 24 ff.; division apparently adequate for, 34; adhesive- ness, 74, 930 ff.; color, 83; copulation, 586 ff.; sexuality, 666-87; biparental in- heritance in haploids, 732-40; develop- ment in faunules of termites and Cryp- tocercus, 894, 923-29; relationship be- tween them, 894, 961-73; only free- living, on which bacteria have been reported, 1011; of termites, Schizo- mycetes in, 1013, 1014, 1015, 1027-32 Flather, 423 Flotation, devices aiding, 77 Foa, 1011 Focke, 1086 Foettingeriidae, 956, 959 Fol, 1090, 1092 Folger, H. T., responses to light, 274 ff. Food, effect on rumen ciliates, 974, 975, 985 Food and feeding habits, use of cultures, 14 Food requirements and other factors in- fluencing growth of Protozoa in pure cultures, 475-516; of Protozoa, 476-78; phototrophic nutrition, 477, 478-82; heterotrophic nutrition, 477, 482-87; trophic specialization, 487-89; specific growth factors, or vitamins, 489-93; growth stimulants, 493-95; growth in cultures as a population problem, 495- 1225 97; initial population, 497-99; growth in relation to waste products, 499 f.; growth in relation to food concentration, 500 f.; growth in relation to pH of the medium, 501-3; oxygen relationships, 503 f.; the redox potential, 504f.; growth in relation to temperature, 505 f.; growth in relation to light and darkness, 506f.; acclimatization, 507; literature cited, 507-16; see also Nuttri- tion Food vacuole, permeability of, 69-71 Foraminifera, copulation, 596 ff. Form, inherited environmental modifica- tions in structure and, 721-23 Forrest, 208 Fortner, 93, 420, 428, 791, 1027 Fosse, 423 Foulke, 907 Fragments, behavior of: grafting and re- incorporation, 793-97 Franca, 1019, 1028 Frederikse, 59, 84 Free-living and symbiotic Protozoa, sys- tematically related, 902-17 Frei, 375 Fresh-water Protozoa, classification on an ecological basis, 5 Frey-Wyssling, 97 Friedemann, 385 Frisch, 150, 168, 428 Frontonia, fibrils, 193, 225 Frosch, 463 Frye and Meleney, 822 Fuhrmann, 952 Fulton, 932 Fundamental and derived organization, 4 Fungi, cellulose decomposition, 962 f., 967 Furgason, 458 Furssenko, 809, 8i0 Fusiformis-like rods adherent full length, 1012-15 Fusion of cells, 35 Galadjieff, 660 Galileo, 9 Galvanic current, responses to, 306-10 Gamete broods, 33; differentiation, 35 f. Gamete nuclei, sex differences between, 701f.; significance of diversities be- tween, and between conjugants, 703-6 1126 Gametes, kinds of differences observed in Chlamydomonas, 667-71; morphological differences, 667; functional differences, 667; physiological differences, 668-71; nature of physiological differences be- tween, in Chlamydomonas, 671-78 Gametic meiosis and fertilization, 601 Gamogamy, term, 584 Ganapati and Aiyar, 1079, 1081, 1082 Garnjobst, 780, 789 Garrod) Ea) P3535 Gas analysis, 354, 356 Gas bubbles, 77 Gases, evolution of, other than CO:, 367 f. Gassovsky, 1085 Gastriolar reaction, term, 132 Gastriole, term, 129; digestion associated with, 172, 178 Gatenby, 138, 139, 145 Gause, G. F., 476; growth, 528, 547, 548, 549) 5505 Salk 5555 4 Gaw, 60 Gay, 839 Geiger, Kligler, and Comaroff, 389 Geiman, 911 Geise and Taylor, 476 Geitler, 705, 1066, 1067 Gel, compared with solid, 50 Gelei, Gabor von, fibrillar system, 198 Gelet Jey vons 169) 1455 146, 47, “409; 419, 423, 439, 649; fibrillar system, 193-99, 202, 204, 206, 219, 220, 224- 28, 258 Generation, spontaneous: history and solu- tion of problem, 8 Genetic constitution, defined, 710 Genetic materials, essential properties, 710 Genetics of the Pretozoa (Jennings), 710, TO, FG. Tig. 72152 Georgevitch, 1030, 1044, 1073, 1074 Gerard, 378 Gerard and Hartline, 354, 358 Gerard and Hyman, 376 Gerstein, 528, 529 Ghosh, 898 Giard, 895 Gicklhorn, 903 Giersberg, 53 Giese, 45, 542, 690, 696, 698 Giese and Arkoosh, 690, 696 Gilman, 690, 695, 698, 700 Gingrich, 844, 848, 851 584- INDEX Gitter, diagram, 197 Gladstone et al., 539 Glaser, R. W. and Coria, N. A., cultures, 458-60, 463, 472, 478, 481, 484, 485, 486 Glaucoma (Dallasia) frontata, divisions, 31, 32, 35; fibrillar system, 233 Glaucoma pyriformis, parasitism, 895 ff. Glocker and Reuss, 60 Glutathione, 121, 125, 373 Glycogen, formation of, 155; differenti- ated from paraglycogen, 157; decrease and storage, 159; deposits by rumen ciliates, 981 Glycolysis, measurement of, 385 f.; oc- currence of anaérobiosis and, 396-90 Génnert, 1064, 1065, 1084, 1088, 1089 Goetsch, 927 Goetsch and Scheuring, 1010 Gojdics, 1042 Goldman, 836 Goldschmidt, 586 Golgi, 847 Golgi apparatus, and contractile vacuole, 431-41, 442; general nature of, 431 ff.; presence in all cells, 432; usual form, 441, 442; link in kinship between all cells apparently established, 443; simi- larity in reaction of protozoan and meta- zoan, 443 Golgi bodies, 138-44, 145, 147; vacuome and, different aspects of same thing, 126; structures included, 138; objec- tive criteria used in identification of, 138 ff.; not universally self-perpetuat- ing and permanent, 142; only truly objective criteria, 143; universality of, rejected, 170, 173, 175; compared with lipoid bodies, 170; defined, 177; perm- anence, 181 Gonder, 1071, 1072 Gonium, responses to light, 288, 303 Gordon, 520 Goroschankin, 589 Gould, 1026 Gourret and Roeser, 1023 Gradients, physiological, 802-4 Graff, 913 Grafting and reincorporation: behavior of fragments, 793-97 Graham, 902 Granules, cytoplasmic, 111-90 (see also under Cytoplasmic inclusions ) ; morpho- INDEX wy logical and functional studies of, often separated, 112; segregation, 126, 132- 38, 168, 171, 174, 178; segregation of neutral red by, 127; digestive, 129-32, 172, 178; excretory, 124, 144-50, 173, 178, 405, 440; carbohydrate, 154; baso- philic, 160, 162, 163-65; metachro- matic granules, 160, 162, 165; survey of functions having granular basis, in group of five Protozoa, 168-74; secre- tion, 168, 178; number of types, 169; failure to find general uniformity, 169, 174; comparison on basis of composi- tion, 171; as permanent organelles and as temporary components, 174-77; ac- tive and passive, 175, 177; classifica- tion, 177-79; comparison with cells of the Metazoa, 179-81; unknown, 179; permanence and self-perpetuation, in Protozoa and Metazoa, 181; basal, in Paramecium, 195, 198, 200, 204, 226, 227; relationship between contractile vacuole and, 405 ff., 424, 435, 436, 438 f., 440; beta, 406, 435; osmiophilic, 438 f., 440 Granulocytes, 835 Grassé, 892, 893, 912, 918, 933, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1028, 1029, 1044 Grassé and Boissezon, 898 Grassi and Foa, 1028, 1046 Grave and Schmitt, 260, 343 Gray, 46, 548 Greeff, 201, 208, 213, 1025, 1026, 1041, 1042 Greeley, 54, 57, 306, 326 Green and Breazeale, 972 Greenleaf, W. E., 535 Greenway, 1045 Gregarines, gamete brood, 35; Biitschli’s discovery re carbohydrate granules of, 111; Golgi bodies, 140 Gregory, 541, 543, 628, 629, 631, 796 Grieg, Von Rooyen, and Hendry, 876 Griffin, 23, 205, 221, 647 Griffiths, 422, 424 Gross, 589 Grosse-Allermann, 91 Growth, environmental conditions suitable for most rapid division and, 45; oxida- tion-reduction potential vs. respiration and, 394-96; food requirements and other factors influencing growth of Protozoa in pure cultures, 475-516; specific factors, or vitamins, 489-93; stimulants, 493-95; in cultures as a population problem, 495-99; in relation to waste products, 499 f.; in relation to food concentration, 500 f.; in relation to pH of the medium, 501-3; oxygen relationships, 503f.; in relation to temperature, 505 f.; in relation to light and darkness, 506 f.; methods for meas- urement of, 517-20; individual Protozoa, 520-26; colonial Protozoa, 526f.; pedigree isolation culture and_ life cycles, 527-31; protozoan successions: nonlaboratory, 531 f.; laboratory, 532 f.; autocatalysis and allelocatalysis, 533-37; nutrition and, 537-44; population, 544- 52: struggle for existence, 553 f.; litera- ture cited, 554-64; studies of regenera- tion and, 772 Growth factor, term, 489 Gruber, A., 78, 82, 787, 788, 789, 797, 1043, 1053, 1075, 1083 Gruber, K., 47 Gruby and Delafond, 983 Ginther, 1042 Guyer, 384 Gwynne-Vaughan and Barnes, 1047 Gymnostomes, association with host, 914 Haas, 917 Habenicht, 315 Hackett, 822,823, 825 Haeckel, 201, 208, 1092 Hammerling, 720 Hafkine, 1035, 1036, 1037 Hahnert, 61, 310, 324 Haldane, 356, 553 Haldane-Henderson, methods. of gas an- alysis, 356 Hall) RY P. -135,. 139.9143, 6359).9360, 361, 362, 371, 407, 408, 435, 436, 437, 476, 478, 479, 481, 487, 489, 490, 494, 501, 502, 504, 507, 537, 542, 543; Food Requirements and Other Factors Influencing Growth of Protozoa in Pure Cultures (Chap. TX), 475-516 Hall, R. P., and Dunihue, F. W., 130, 437 Hall, R. P., and Elliot, A. M., 484, 492, 493 Hall, R. P., and Jahn, T. L., 907 1128 HallRo PR: andeloefer: JB 31283166, 167, 482, 483, 500, 535 Hall, R. P., and Nigrelli, R. F., 115, 130, 131, 140 Hall, R. P., and Schoenborn, H. W., 477, 478, 479, 482, 490, 498, 499, 543 Hall, R. P., and his associates, 126, 136, 518 Hall, S. R., 905, 906, 907 Halsey, 530 Hamm, Ludwig, spermatozoa discovered by, 11 Hammett, 543 Hammond, E. C., 537, 548 Hammond, J. C., 227 Hance, 426 Haploids, biparental inheritance in, 732- 40 Haptophrya, accessory bodies from, 179; fibrillar system, 240, 242 Hardy, 47, 518 Hargitt, G. T., and Fray, W. W., cultures, 452'£., 454, 533, 537 Harrington and Leaming, 277 Hartman, E., 844, 846, 851 Hartmann, M., 587, 594, 746, 785, 798, 799, 1094; sexuality, 666-86 passim, 704, 705 Hartmann, M., and Chagas, C., 25 Hartmann, M., and Nagler, K., 593, 595, 596, 613 Hartog, 426, 1089 Harvey, E. B., 58, 71, 78, 426, 430 Harvey, E. N., 58, 60, 64, 80, 363 Harvey, E. N., and Danielli, J. F., 64 Harvey, E. N., and Marsland, D. A., 63, 80, 95 Haswell, 904 Haupt, 891 Haye, contractile vacuole, 148, 406, 407, 409, 410, 418, 419, 420 Hayes, 113, 119 Hay infusion, 15 Hazard, 950 Heat as a bactericidal agent, 464 Heavy water, effect of, 60 Hegerty, 530 Hegner, 796, 844, 891, 393, 903, 933 Hegner and Andrews, 893 Hegner and Eskridge, 846 Hegner and Hewitt, 846, 851 Heidenhain’s iron-alum-hematoxylin, 419 Heidenreich, 944, 945, 946, 947, 948, 949, 952 INDEX Heilbrunn, L. V., 309, 344; protoplasm, tg att Sly SV, De D5 DG, SH, SS), Ga 71, 79, 80, 152; quoted, 111 Heilbrunn, L. V., and Daugherty, K., 53, 54, 60, 153, 319 Heilbrunn, L. V., and Mazia, D., 60 Helly-Maximow’s Zenker formol, 832 Hematochrome, 83 Hemicellulose, ophryoscolecin as, 157 Hemixis and autogamy, 654 ff. Hemocytoblasts, 835, 836 Hendee, 967 Henderson, J. C., 927, 963 Henderson, V. E., 59 Henry, Dora P., 569 Henry, X., 876 Hentschel, 921 Herbivorous mammals, ciliates in, 973 f. Herfs, A., 427, 430 Herrick and Cross, 855 Hertig, M., Taliaferro, Schwartz, B., 891, 894 Hertwig, O., 892 Hertwig, R., 160, 566, 598, 626, 649, 701, 795, 1083, 1089, 1090, 1091, 1092 Hesse, 1040 Heteroautotrophic nutrition, 477, 482 f. Heterogamy, term, 584 Heteromesotrophic nutrition, 477, 483 Heterometatrophic nutrition, 447, 483-87 Heterophils, 835 Heterotricha, fibrillar systems, 244-51; see also Stentor Heterotrophic nutrition, 477, 482-87 Hetherington, 450, 461, 467, 528, 540, 541, 774, 775, 895, 896, 1039 Hewitt, J. H., 792 Hewitt, L. F., 391, 392, 396 Hewitt, R., 846, 851 Heyningen, van, 376 Higgins, H. T., 249 Hill, John, 11 Hindle, 163 Hinshaw, 569 Hinshaw, McNeil, and Kofoid, 912, 913 H-ion concentration, relation between rate of locomotion and, 334, 335, 336 ff., 341 Hirschler, J., 411, 437, 440; cytoplasmic inclusions, 114, 118, 138, 139, 140, 142, 143, 145 Historical facts, protozoa, 8-14 Hoelling, 1012 identified W.) H., “and INDEX Hofer, 787 Hofker, 1069 Holdaway, 965 Hollande, 909, 1071 Holmes, E., 121, 300 Holmes) S: Ji, 372, 781 Hologamy, term, 584 Holotricha, fibrillar systems, 228-44; free- living and symbiotic, 913-17; adapta- tion, 933 ff.; see also Paramecium Holter, 384 Holter and Doyle, 118, 120, 124, 168, 384 Holter and Kopac, 118, 120, 124, 125, 168 Hooke, Robert, 9 Hope-Gill, 876 Hopkins, D. L., 71, 116, 124, 125, 127, 129, 139, 168, 180, 273, 334, 336, 340, 429 Hopkins, D. L., and Mast, 124 lsloysarnres I. S51 TS}, aUil7, ili), ae, 175, 176 Horning, E. S., and Scott, D. H., 137 Horton, 793 Horvath, 199, 206 Hosoi, 802 Host, variability in strains and in host response, 823-26; study of defense mechanisms of, 830 ff.; relationships be- tween certain Protozoa and, 890-1008 (see also under Relationships) Host-specificity, term, 894; holotrich groups, 913 ff.; and distributional host relationships in representative symbiotic faunules, 917-29; approach from stand- point of individual species, 918 Hovasse, 1010, 1064, 1069 Howland, 49, 51, 52, 62, 67, 68, 70, 89, 92, 406, 407, 418, 422, 425, 794, 795, 905, 1068 Howland and Bernstein, 354, 355, 357, 362 Howland and Pollack, 52, 62, 65, 68, 425 Hsiung, 973, 1084, 1085 Hu and Cash, 854 Hudson and Gosse, 906, 1093 Huff and Bloom, 843 Hulpieu, 341 Humoral and cellular aspects of immunity, 839-41 Hungate, 965, 966, 967, 969, 970, 971 Hunninen and Wichterman, 911 Huntington and Winslow, 539 Hutner, 482, 489 1129 Huxley, 525 Huxley and Teissier, 525 Hyaline layer, 49 Hyaloplasm, 50 Hydrogen acceptors, experiments which concern the cytochrome-cytochrome oxi- dase system of, 376-80; which concern other systems, 380 f. Hydrogen-ion concentration, 58 f.; effect on growth, 542, table, 540 f. Hydrostatic pressure effect on consistency, 61; within cell, regulation of, 422, 426 ff., 442 Hyman, 49, 53, 72, 84, 89, 91, 94, 429, 802, 803 Hypocomidae, 933; adaptation, 938-42 Hypotricha, characteristics, 205; fibrillar systems, 255-57; see also Euplotes Hypotrichida, reorganization in, 24 Ichthyophthirius, survey of functions hav- ing granular basis, 168-76 passim; fibril- lar system, 241 Ikeda and Ozaki, 638, 937 Illumination, responses to, see Light Imhof tank sewage, 6 Immaturity and maturity, sexual, 714 f. Immaturity and partial maturity, 761 Immunity and acclimatization, inherited, 717-21 Immunology of the parasitic Protozoa, 830-89; physical bases of immunity, 830-42; cells involved in immunity, 831- 37; antibodies and antigens involved, 837-39, 873; cellular and humoral as- pects, 839-41; role of immune processes in the development of protozoan infec- tions, 842-64; malaria, 843-54; kala azar, 854 f.; infection with . . . trypano- somes, 855-62; continuous fatal trypano- somiasis, 862-64; intermittent fatal try- panosomiasis, 864-71; practical applica- tions of immune reactions, 871-76; arti- ficial immunization, 872; reactions used in diagnosis, 872-76; reactions in rela- tions to classification, 876-78; literature cited, 878-89 Impregnation, Golgi bodies’ relationship to, 143 Indirekt verbindung System, 194, 195, 199, 226 Infections, from coprozoic forms, 6; rdle of immune processes in development of protozoan, 842-64 1130 Inflammation, 836; defense reactions seen during, 839 Infraciliature, 200, 228, 257 Infusionsthierchen als vollkommene Or- ganismen, Die (Ehrenberg), 12 Infusoria, term; Miiller’s studies, 11; Eh- renberg-Dujardin controversy reorgani- zation of, 192, 260 Inheritance, 710-71; types of reproduction and, 171; in uniparental reproduction, 711-13; in biparental reproduction, 711, 732-50; - of © characteristics, 712'f.; changes in inherited characters in uni- parental reproduction, 713-31; age changes, 714f.; degeneration changes resulting from unfavorable conditions, 716f.; acclimatization and immunity, 717-21; environmental modifications in form and structure, 721-23; variation and, occurring without obvious action of diverse environments, 723-29; bi- parental, in haploids: flagellata, 732-40; sex and sex-linked, 740-50; biparental, in diploids: ciliata, 750-58; of mating type in Paramecium aurelia, 753-58; in Paramecium bursaria, 758-61; effect of the cytoplasm and its relation to nuclear constitution, 762-69; literature cited, 769-71 Injury, degree of reorganization and, 781- 84 Inman, Bovie, and Barr, 277 Inquilines, defined, 892; Protozoa not cap- able of harboring, 1009 Intermediate lipoid body, term, 151, 178 Internal parasites, see Parasites International Rules of Zoélogical Nomen- clature, 953 Irreversible coagulation, 61 Isogamy, term, 584 Issel, 936, 937, 939, 1021 Ivani¢, 635, 649, 1047, 1048, 1049, 1052, 1053, 1078 Jacobs, 55, 56 Jacobson, Irene, fibrillar system, 198, 223, 225, 258, 259 Jaffé, 839 Jahn, Theodore Louis, 364, 365, 369, 387, 393, 395, 1042, 1048, 1058; Respira- tory Metabolism (Chap. VI), 352-403; food, etc., influencing growth, 481, 484, INDEX 487, 498, 502, 503, 504, 505, 506, 507; growth, 528, 534, 536, 538, 539, 540, 547, 548, 549, 552 Jahn, T. L., and McKibben, W. R., 907, 1068 James, 846 Jameson, 576, 586, 604, 612 Janda and Jirovec, 896 Janicki, 26, 27, 1011, 1019, 1046 Jarocki, 940, 942, 943, 944 Jarocki and Raabe, 938, 940 Jay, 362, 370, 377 Jenninesy Ee 'S3 74225) 5852052 Ih 22. 525, 588, 606, 615, 617, 636, 714, 724, V3, VIS W295 Ws Wa, Wes 1933 physical properties of protoplasm, 72, 75, 76, 83, 88, 91, 92; motor responses, Dil, 282) 297, 321, 322) 3246 3265542- sexuality, 689, 690, 691, 692, 694, 695, 696, 698, 700, 701, 704; Inheritance in Protozoa (Chap. XV), 710-71; Genetics of the Protozoa, 710, 712, 716, Flgee72l, 152 Jennings, H. S., and Jamieson, C., 793 Jennings, H. S., and Lashley, K. S., 689 Jenseny Wie s793 Jirovec, 154, 156, 1030, 1044, 1045 Jochims, 95 Johns Hopkins University stock R_ of Paramecium, 615 Johnson, C. M., and Kelser, R. A., 874 Johnson, D. F., 468, 476, 484, 486, 501, 502, 507, 538 Johnson, H. P., fibrillar systems, 201-4 passim, 219, 258 Johnson, L. P., 81, 83 Johnson, P. L., 592 Johnson, T. L., 868 Johnson, W. H., 471, 476, 498, 528, 536, 540, 541, 542, 548, 551 Johnson, W. H., and Hardin, 499, 536 Jollos, V., 660, 698; inheritance, 718, 719, 720, 721, 723, 730, 768 Jones, E: P., 541,548, 552 Joness Pay Mie 92 Joschida, 79 Joyet-Lavergne, P., cytoplasmic inclusions, 114, 117, 121, 122, 1125, 140, 4ommise 155, 156, 161, 162, 166, 169, 172, 175, 180 Jucci, 963 Jurgens, 855 Jungeblut, 839 INDEX Kahl, 75, 900, 914, 916, 917, 920, 921, 922, 934, 935, 936, 940, 945, 1022, 1023 1024, 1038 Kala azar, 854 f., 874, 875 Kalmus, 97, 355, 357, 362, 366, 1087 Kamada, 323, 324 Kanda, 79 Kanthack, e¢ al., 855 Katharobic type, 5 Katzin and Kirby, 967 Kauders, 850 Kavanagh and Richards, 534 Kazancev, 163 Kedrowsky, B., cytoplasmic inclusions, 1229126, 127, 128, 129, 1325 133.104, 135, 136, 137, 139, 143, 151, 153, 168, 169, 171, 173, 174, 175, 180 Keilin, 897, 898 Keilin and Hartree, 379 Kempner, 361 Kent, 422, 1041 Kepner, W. A., and Carroll, R. P., 952 Kepner, W. A., and Carter, J. S., 905 Kepner, W. A., and Edwards, J. G., 91 Kepner, W. A., and Reynolds, B. D., 794 Kepner, W. A., and Taliaferro, W. H., 70 Kepner, W. A., and Whitlock, C., 91 Keppen, N. A., see Koeppen, N. Kessel, 1045 Khainsky, 413 Khawkine, 907 Kidder, George W., 21, 22, 33, 536, 620, 627, 628, 635, 636, 637, 638, 647, 649, 650, 653, 658, 930, 934, 935, 939, 963; fibrillar system 228, 230, 232, 234; Technique and Significance of Control in Protozoan Culture (Chap. VIII), 448-74 Kidder, George W., and Claff, C. L., 647 Kidder, George W., and Dewey, V. C., 469 Kidder, George W., and Diller, W. F., 647 Kidder, George W., and Stuart, C. A., 450, 454, 469, 470, 533, 537 Kidder, George W., and Summers, F. M., 915 Kidder, George W., Lilly, D. M., and Claff, C. L., 453, 469 Kimball, R. F., 660, 756, 767; sexuality, 670, 690, 696, 697, 698, 700, 701 Kinetic responses to light, 277-79, 290, 2955, 299 Ltt King, R. L., contractile vacuole, 68, 69, 408, 409, 410, 419, 420, 438, 439 King, R. L., and Beams, H. W., 67, 68, 70, 71, 81, 88, 98; Some Physical Prop- erties of the Protoplasm of the Proto- zoa (Chap. II), 43-110 Kinosita, 323 Kirby, Harold, Jr., 116, 126, 136, 154, 157, 248, 893, 909, 923, 924, 927, 928, 929, 930, 963, 965, 968, 969, 970, HOM, WOU, WO, WOW “ilolss aWOiles. 1017, 1018, 1019, 1020, 1022, 1023, 1029, 1030, 1031, 1032, 1039, 1040, 1044, 1045, 1046, 1048, 1053, 1054, 1055, 1056, 1959, 1060, 1062, 1079; Relationships between Certain Protozoa and Other Animals (Chap. XIX), 890- 1008; Organisms Living on and in Pro- tozoa (Chap. XX), 1009-1113 Kirkman and Severinghaus, 126, 138, 180, 181 Kitching, J. A., 68, 69, 429 Kite, 51, 52, 62, 67 Kiyono, 836 Klebs, 909, 1041 Klee, 649 Klein, B. M., fibrillar system, 194-200, 206, 224-28 Kligler, Geiger, and Comaroff, 389 Kluyver, 396 Kniep, 684, 705 Knoth, 976 Knowles and Das Gupta, 847, 848, 864, 865 Kober and Graves, 519 Koch, A., 963, 964 Koch, R., 848 Koehler, 324, 325, 326 Koehring, 127, 130, 134 K6lliker, 201, 208 Konig, 936, 937, 941 Koeppen, N. [Keppen, N. A.], 1070, 1089, 1090, 1091 Kofoid, Charles A., 198, 206, 594, 936, 937, 974; Life Cycle of the Protozoa (Chap. XI), 565-82 Kofoid, Charles A., and Bush, M., 126, 619, 636, 940 Kofoid, Charles A., and Christenson, J. F., 79 Kofoid, Charles A., and Johnstone, H. G., 463 1132 Kofoid, Charles A., and MacLennan, R. F., 973, 979; fiibrillar system, 252 Kofoid, Charles A., and Swezy, O., 25, 572, 930, 1044 Koidzumi, 924, 930, 1015, 1018 Kolatchev method, 435 Kolkwitz, classification of fresh-water species, 5 Kolmer ef al., 870 Koltzoff, 93, 258, 259 Korotneff, 1066, 1089, 1090 Korr, 381, 395 Korschelt, 203 Koser and Saunders, 492, 494, 537 Kostitzin, 553 Kotlan, 912 Krascheninnikow, 145 Krijgsman, 151, 159, 162, 163, 166, 843, 863, 864 Kroé, 857, 877 Kriger, 1079 Kuczynski, 1044 Kudo, 608, 956, 957, 1078 Kiihne, 208, 217, 305, 315, 323, 332 Kuhn, 871 Kupffer cells, 833, 850”, 851” Labbé, 907 Lachmann, 208, 213, 413, 1064, 1086 Lackey, 6, 83, 387, 532, 1010 Lamborn, 897 Landis, 636, 702, 791 Langmuir, 64 Lankester, 413 Laurens and Hooker, 303 Lauterborn, 5, 1011, 1021 Laveran, 854, 870, 877 Laveran and Mesnil, 856, 861, 866, 868 Lavier, 908, 910, 912, 933, 1046, 1047, 1054, 1056, 1057, 1058 Lavier and Galliard, 912 Lawrie, 484, 503 Lebedew, 591, 593 Le Breton, 127 Le Calvez, 594, 597, 598 Lechriopyla mastax, fibrillar system, 243 Ledermiiller, Infusoria termed by, 11 Leeuwenhoek, Anton von, 8, 9, 11, 12, 208; contributions to microscopic an- atomy and to physiology, 9; description of a protozoén, 10 f.; regarded as the “Father of Protozodlogy and Bacteriol- 1034, 1039, INDEX ogy,” 11; his “little animals” essential nature remained long obscure, 191 Léger, 603, 604 Leger, A., and Ringenbach, J., 868, 877 Léger, L., and Duboscq, O., 604, 1045, 1068, 1078 Leichsenring, 355, 365, 366, 367 Leidy, 1021, 1025, 1043, 1065, 1067, 1094 Leiner, 155, 158, 1025, 1026, 1027 Leishmaniasis, 854 f., 874, 877 Leontjew, 79 Levaditi and Mutermilch, 868, 870, 877 Lewin, 778, 789, 791, 792 Lewis, 90 Leydig, 210, 906 Lichtenstein, 916, 917 Lieberkiihn, 201, 208, 219, 1034, 1039 Liebmann, 1038 Liesche, 592 Life and vitality, 3 f., 34 Life cycle of the Metazoa, 565-67, 581 Life cycle of the Protozoa, 565-82; con- ception of, as a characteristic of every species, 14; pedigree isolation culture and, 527-31; asexual reproduction in alternating binary and multiple fission, 569-71; alternation of asexual and sex- ual reproduction, 571-73; Eimeria schu- bergi, 573-78; Plasmodium vivax, 577, 578; Paramecium caudatum, 578-81; literature cited, 581 f.; mating types in relation to the Maupasian Theory, 699 f.; malarial parasites, 847; adap- tive host relationships in morphology and, 929-60 Light, responses to, 272-305; rhizopods, 272-80; flagellates, 280-95; ciliates, 295-97; colonial organisms, 297-305; growth in relation to darkness and, 506 f. Light, S. F., 1018, 1019, 1044 Lillie, F. R., 785, 786, 787 Lillie, R. S., 324 Lilly, D. M., control of cultures, 469, ATlite Linderstrom-Lang, K., 358 Linear aggregates in protoplasm, 87 Linkage and crossing over, 737-40, 743- 45 Linton, 839, 854 Lipoids, defined, 118”; in mitochondria, 118, 123; interpretation of vacuolar 1066, INDEX system as due to concentration of, 148; reserves, 150-53, 173; boundary be- tween reserve and active, 151; inter- mediate lipoid bodies, 151, 178; visible, 152; compared with Golgi bodies, 170 Lison, 118”, 119, 139 Lister, 9, 596 Literature cited, 39-42, 98-110, 182-90, 262-70, 344-51, 397-403, 443-47, 473 f., 507-16, 554-64, 581 f., 639-45, 662-65, 706-9, 769-71, 811-17, 827-29, 878-89, 987-1008, 1095-1113 “Little animals” of Leeuwenhoek, 10, 191 Littoral cells, 833 Liver cells, plasmosin from, 87 Lloyd, 67, 422 Lloyd and Scarth, 411 Locomotion, relation between rate of, and H-ion concentration, 334, 335, 336 ff., 341 Loeb, J., muscle-tonus, or tropism, theory, 282 Loeb, J., and Budgett, S. P., 51, 324, 332 Loeb, J., and Maxwell, S. S., 289 koeb, 1.74 Hoefer, J.B... 525, 538, 540, 5425 543); food, etc., influencing growth, 476, 477, 478, 480, 481, 482, 484, 485, 486, 487, 495, 501, 502, 507 Loefer, J. B., and Hall, R. P., 481 Longevity, factors influencing, 16-18, 38 f.; division processes inadequate to account for, 28; of ciliate’s protoplasm, 34 Loofbourow and Dyer, 519 Loomis, 58 Looper, 74, 788, 796 Lophomonas, reorganization in, 26, 27 Lorando and Sotiriades, 850 Lotka, 553 Lourie, 834, 844 Lourie and O’Connor, 869 Lowe, 847 Lucas, K., 324; fibrillar system, 247 Lucas, M. S., 900, 922 Luce, 61, 306 Luck and Sheets, 464 Ludford, 138 Ludloff, 324, 325, 326, 331 Ludwig, W., 77, 78, 425, 793 Lund, E. E., 968, 970, 971; fibrillar sys- tem, 198 f., 224, 255 Lund, E. J., 84, 86, 774, 775, 782, 792, LY33 801; respiratory metabolism, 355, 362, 363, 365, 376 Lund, E. J., and Logan, G. A., 71, 78, 332 Luntz, 305, 800 Lutz and Splendore, 1078 Luyet and Gehenio, 58, 67 Lwoff, A., 74, 476, 482, 504, 896, 898, 899, 1044, 1045, 1047, 1048, 1051, 1053, 1058; respiratory metabolism, 360, 362, 366, 367, 371, 377, 378, 379, 382, 383, 384, 388, 390; food, etc., in- fluencing growth, 477, 479, 480, 483, 484, 485, 486, 491, 493, 503 Lwoff, A., and Dusi, H., food, etc., in- fluencing growth, 477, 482, 483, 484, 487, 490, 491, 543 Lwoff, A., and Lederer, E., 482, 493 Lwoff, A., and Lwoff, M., 492 Lwoff, A., and Provasoli, L., 483, 485, Lwoff, A., and Roukhelman, Nadia, 425, 484, 485, 499 Lwoff, M., respiratory metabolism 362, 377, 378, 379, 380, 381, 383, 388; food, etc., influencing growth, 477, 478, 484, 485, 491, 492, 493 Lwoff, M., and Lwoff, A., 478, 484, 487 Lymph, cells, 834 f. Lymphocytes, 835, 836 Lymphoid cells, 835 f. Lymphoid-macrophage system, 835, 837 Lynch, J. E., 113, 114, 119, 126, 136, 145, 752, 788, 919, 921, 923; fibrillar system, 236, 239, 243 Lynch, J. E., and Noble, A. E., 1088 Lyon; 79; 332 Lysin and opsonin, 858, 861 Mac Arthur, 898 McCay, 544 McClendon, 80, 309, 332 McClung, 384 McCoy, 361 McDonald, fibrillar system, 244 MacDougall, M. S., 27, 542, 622, 623, 630, 631, 632, 633, 634, 635, 637, 638, 647; fibrillar system, 229, 231 McFarland, 891 Mackinnon, D. L., 96, 911, 1033 Mackinnon, D. L., and Vleés, F., 84, 96, 97 Mackinnon, D. L., and Ray, H. N., 1079, 1081 McLay, 850 1134 MacLennan, Ronald F., 113, 114, 115, LEZ WO, 122 125, 126aeOmIo e132. 135, 136, 137, 139, 140, 141, 142, 143, 145, 146, 147, 148, 149, 150, 151, 154, 155, 156, 157, 163, 164, 167, 169, 174, 175, 180, 525, 981; Cytoplasmic Inclu- sions (Chap. III), 111-90; fibrillar sys- tem, 241; contractile vacuole, 409, 411, 421, 424, 436, 439, 443 MacLennan, Ronald F., and Connell, F. H., 936, 937; fibrillar system, 238, 240 MacLennan Ronald F., and Murer, H. K., 1198 1205013 1a 749590 Mac Neal, 856, 861 Mc Pherson, Smith, and Banta, 537 Macronucleus, a derived organ, 16; me- rotomy experiment, 16 ff.; changes with metabolism, 18 ff.; during reorganiza- tion, 21 ff., 31 ff., 646-48; during con- jugation, 623 f. Macrophages, 835, 836; defined, 833; cells classified under, 833 f.; structure, 834; phagocytosis by, during malaria, with plates, 849 ff.; system valuable or dele- terious, 854; Le/shmania in, 854 Madsen, 899, 921 Magenthiere (Polygastrica), 12 Maier, 201, 202, 219, 221 Mainx, 481, 684, 705, 1042 Makarov, 59 Malaria, pathogenicity, 822 f.; immunol- ogy, 843-54; phagocytosis, 849 ff.; serological tests, 874, 876, 878 Mangenot, 1039 Mangold, 974, 975, 977, 982, 983, 984, 985 Mangold and Radeff, 977 Mangold and Schmitt-Krahmer, 984 Mangold and Usuelli, 975, 977 Mann-Kopsch material, 435 Manometer, standard methods, 354, 356 f.; micromanometer, 355, 358; capillary, 357 £. Mansour, 963 Mansour and Mansour-Bek, 961, 962, 963, 965 Manteufel, 861 Manusardi, 985 Manwell, 627, 649, 848, 878 Manwell and Goldstein, 823, 848 Marchand, 833, 836 Margolin, 976 Marrack, 838 INDEX Marsh, 363 Marsland, 59, 61, 74 Marsland and Brown, 273 Marston, 127 Martin, 1085 Massaglia, 863, 868 Massart, 78 MastauSs Ose 1525 5406n 4185529 Ole physical properties of protoplasm, 48, AD. 51, 61. 68, 69, 70.72, 13, 070n184s 85, 90, 91; Motor Response in Unicellu- lar Animals (Chap. V), 271-351; ort- entation in Euglena, 283 ff.; conclusions on responses of Volvox, 297 ff., 327 ff. Mast, S. O., and Doyle, W. L., 36, 47, 65, 71, 80, 92, 406, 407, 436; cytoplasmic inclusions, 113-80 passim Mast, S. O., and Fowler, C., 65 Mast, S. O., and Gover, Mary, 287, 290 Mast, S. O., and Hahnert, W. F., 71 Mast, S. O., and Hawk, Brainard, 291, 292 Mast, S. D., and Hulpieu, H. R., 277 Mast, S. O., and Johnson, P. L., 287, 288, 302 Mast, S. O., and Nadler, J. E., reversal in ciliary action, 322, 326, 342 ff. Mast, S. O., and Pace, D. M., 367, 479, 482, 483, 488, 498, 499, 500, 528, 535, 540, 543 Mast, S. O., and Prosser, C. L.,.277, 336, 339, 340 Mast, S. O., and Root, F. M., 91, 788 Mast, S. O., and Stahler, N., 278, 279 Mast, S. O., Pace, D. M., and Mast, L. Re HO, BAO, BIOS a7! Mastigina, 85 Mastigophora, free-living and symbiotic, 902-13; Schizomycetes on, 1010-24: Schizomycetes in, 1030-32 Mattes, 1041, 1042, 1043, 1048, 1050, 1051, 1052, 1053, 1054, 1056, 1057, 1058, 1064 Mattick et al., 520 Matubayasi, 912 Maturity and immaturity, sexual, 714 f.; partial, 761 Maupas, E., 28, 29, 413, 528, 537, 714, 900, 922, 946, 1087; fibrillar system, 192, 198, 205, 208, 216, 221; fer- tilization, 605, 614, 618, 621, 624, 626, 634, 636; sexuality, 690, 696, 699, 700, 701 INDEX Maupasian life cycle, mating types in rela- tion to, 699 f. Maximow, 832, 833, 836, 837, 839 May, 928 Mayer, 1044 Mayer’s hemalum, 419 Measurement of growth, methods, 517-20 Mechanical agitation, effect on consistency, 47, 58 Mechanical support, fibrils, 194 ff., 258 Meiosis, gametic, 584-601; zygotic, 611- 14; conjugant, 624-39 (see also under Conjugant meiosis ) Meldrum, 372 Meleney, 854 Meleney and Frye, pathogenicity, 819, 820, 822, 825, 874 Membranes, cell, 62, 64-66, 69; nuclear, 66 f.; presence or absence of, surround- ing contractile vacuoles, 67, 413-21, 441; division into two types, 414; physiological, 414 ff.; morphological, or permanent, 414, 418, 419, 421 Menendez, 878 Menon ef al., 875 Mercier, 1033, 1046, 1054, 1056, 1057 Mercier and Poisson, 895, 900 Merton, 88, 219, 342 Mesenchymal cells, 833; rdle in inflam- mation, 836 Mesnil, 1080 Mesnil and Brimont, 870 Mesosaprobic type, 5 Messiatzev, 631 Mestre, 519 Metabolic influence, fibrillar complex, 258 Metabolic waste products, 422 Metabolism, activity and changes with, 18- 21, 35; see also Anaérobic, Basal, and Respiratory, metabolism Metachromatic granules, 160, 162, 165 Metallic impregnation methods, in iden- tification of Golgi bodies, 138 Metalnikov, 653 Metazoa, comparison between metazoan and protozoan cells, 44, 179-81; Golgi bodies in cells, 140, 143; differences between protozoan and metazoan or- ganization, 191, 260; life cycles of Pro- tozoa and, 565 ff., 581; analogies in sex phenomena between Protozoa and, 583 f., 600; as parasites in Protozoa, 1010, 1093-95 135 Metcalf, M. M., 33, 69, 115, 405, 406, 409, 435, 591, 893 Metschnikoff, E., 208, 836, 1035, 1036, 1086 Metopus 247 Meyen, 12, 413 Meyer, A., 219 Meyer, S. L., 950 Meyerhof, quotient, 386, 389 Meyers, E., 160, 161 Meyers Ea Gen 35 Microdissection apparatus in structure, 52 “Micrographica’”” (Hooke), 9 Micromanometer, 355; Cartesian diver ultramicromanometer, 358 Micronucleus during reorganization, 28, 29535) fk Microphages, 836 Microscope, discovery and development of, 5) Migration, sterilization by, 455-60; com- bined with dilution, 460-63 Miller, 118, 705 Mills, 540 Milojevic, 604 Milovidov, 161 Minchin, 583, 787 Minden, 1040, 1047, 1052, 1063; quoted, 1057 Minkiewicz, 620 Minnesota, University of, study of Ameba proteus, 592 Mitchell, 539, 1042, 1048, 1049, 1050, HOST O52 LOS Sal ONS Mitochondria, 112-26; identification of, 112, 116; shape, 113; distribution, 115; supposed universality and permanence of, 116, 125, 432; composition, 118; cellular respiration, 121, 122; functions ascribed to, 122 ff.; not a homogeneous group, 126; morphological relationship between paraglycogen and, 155; chro- midia associated with, 160, 161; uni- versality of, rejected, 170, 173, 174; function of carbohydrate storage accom- plished by, 173; defined, 177; perma- nence, 181; terms for, 432; relationship to Golgi apparatus, 432, 437 Mitotic mechanism, 44 Miyashita, Y., 620, 621, 944, 957, 1025 Mizuno, F., 521, 522, 526 circumlabens, fibrillar system, study of 1136 Mjassnikowa, 942 Moewus, F., sexuality, 666-83, 705; cri- tique of works of, on Chlamydomonas, 684-87; inheritance, 710, 722, 723, 732, 733, 734, 736, 738, 739, 740, 742, 743, 744, 745, 746, 747, 748, 749, 750 Mohler, Eichhorn, and Buck, 874 Molina, 912 Molisch, 1074 Mond, J., 538, 544, 551 Monkeys, malarial infection and immu- nity, with plates, 845, 846-54 Monocystis, 601 f. Monocytes, 835 Monod, 803 Monoecious races, 742 Montalenti, 965, 966, 970, 971 Moody, 791, 796 Moore, A. R., 95, 332 Moore, E. L., 649, 777, 779, 780, 782, 785, 788, 790, 911, 912; quoted, 787 Moore, Imogene, 410, 420, 438 Morea, 540, 541 Morgan, de, 935 Morgan, T. H., 704, 785, 786, 797 Morita and Chambers, 52, 67, 69 Moroff, 912 Morphogenesis, Protozoa in connection with problems of, 772-817; physiologi- cal regeneration, 773 f.; some factors in regeneration, 774-93; behavior of fragments: grafting and reincorporation, 793-97; regeneration and division, 797- 801; polarity changes and protoplasmic streaming, 801 f.; physiological gradi- ents, 802-4; regeneration in colonial forms, 804-11; literature cited, 811-17 Morphological membranes, 414, 418, 419, 421 Morphology, adaptive host relationships in life history and, 929-60 Motor organelles, composite: into their components, 76 Motor response in unicellular animals, 271-351; to light, 272-305; to elec- tricity, 305-32; to chemicals, 333-44; literature cited, 344-51 Mottram, 543 Molds, cellulose decomposition, 967 Mowry and Becker, 974, 975, 976, 983, 985 Mueller, 494 Miller, J., 1034, 1039 separation INDEX Miller, O. F., studies by, 11, 43; classi- fication, 11; observation of conjugation, oboe} Miller, R. H., 519 Mulligan, 843, 846 Mulligan and Sinton, 848 Mulsow, 601, 602, 611, 689 Munich school of protozodlogists, 566 Muscle, stalk, 208, 216 Muscle-tonus theory, Loeb’s, 282 Mutualism, inclusion in term symbiosis, 891; defined, 892; physiological host relationships illustrative of commensal- ism and, 961-87 Myeloid cells, 835 f. Myers, 594, 597, 1095 Myonemes, 94; of Stentor, 201 ff., 218 ff., 258, 261 Nadler, 62, 83, 88, 775 Nagler, 27, 1032, 1042, 1049 Nahm, 181 Napier, 875 Narcotics, effects of, 59; responses of Stentor to, 220 Nassonov, D., contractile vacuole and Golgi apparatus, 69, 138, 142, 144, 145, 146, 147, 148, 410, 411, 419, 420, 431, 433-35, 438, 439, 443 Nassonov-Bowen theory re Golgi bodies and secretion, 144 Mauck and Malamos, 848 Naville, fertilization, 576, 602, 607, 608, 609, 611 Necheles, 362 Needham, Joseph, 9, 90, 372, 390, 525 Needham, Joseph, and Boell, E. J., 354, 358 Needham, Joseph, and Needham, D. M., 51, 52, 62, 396 Nelson, 586, 621, 635 Nematode worms, parasitism, 1094 Neporojny and Yakimoff, 870 Neresheimer, E. R., 33; fibrillar system, 201-4 passim, 218, 219, 220, 225, 591, 1089, 1093 Nernst, 324 Neumann, 850 Neuschloss, 720 Neutral red, segregation of, by cytoplasmic granules, 127; granules, not identical with Golgi bodies, 141, 142; in Amoeba, 170; granule, defined, 178 INDEX New International Encyclopedia, 891 Nie, 919, 920 Nielsen, 520 Nieschulz, 906 Nieschulz and Bos, 866, 870 Nieschulz and Wawo-Roentoe, 870 Nigrelli, 116, 134, 135, 136 Nigrelli and Hall, 435 Nirenstein, 428 Nitrogen, source of, 972; ciliate, 984 Noble, 576 Noller, 1044, 1045, 1046, 1047 Noller and Buttgereit, 912 Noguchi, 877 Noland, 531, 591, 621, 623, 629, 637, 688 Novy, 371 Novy, Roehm, and Soule, 356 Nowakowski, 1065 Nowikoff, 423 Nuclear and cytoplasmic division proc- esses, coGrdination between, 85 f. Nuclear membrane, permeability, 66 f. Nuclear purification, 21 ff. Nuclear reorganization processes, 21 ff., 35 ff. Nuclei, chromidial origin doubted, 594; two kinds in ciliates, 687; gamete: dif- ferentiation, 701, 703; effect of cyto- plasm and its relation to, 762-69; in regeneration, 787-93; of Trichonympha, parasitization, 1059-63 Nucleophaga, and Spaerita, 1040-59; his- torical account and distribution, 1040- 47; in free-living Protozoa, 1040, 1043; in endozoic Protozoa, 1046 f.; life his- tory and structure, 1053-57; effect on host, 1058 f. Nussbaum, 787 Nutrition, and growth, 537-44 (see also Food) ; effect on resistance, 826 f.; of wood-eating animals, 961-73; effect upon rumen ciliates, 974, 975, 985 Nyctotherus, fibrillar system, 248-50 Obligate parasites, 895 Oehler, 458, 463, 465, 469, 475 Ogata, 455 Okada, 795 Oligochaeta, host of Astomata, 946 Oligosaprobic type, 5 Oligotricha, fibrillar systems, 251-54 Oligotrichida, sexuality, 688 Oliphant, K., 54, 57, 322, 326, 344 ELS7 Odgamy, term, 584 Opalina, segregation bodies, 132-35; sur- vey of functions having granular basis, 168-75 passim Opalinopsidae, 956, 960 Ophryocystis mesnili, 603, 604 Ophryoscolecidae in ruminants, 973, 977- 82 passim Ophryoscolecin, 157 Oppenheimer, 966 Opsonin and lysin, 858, 861 Optical properties of protoplasm, 82-84 Organic structure, disclosure of cellularity dependent upon analysis of, 191, 260 Organisms living on and in Protozoa, 1009-1113 (see entries under Parasites of Protozoa) Organization, derived and fundamental, 4 Orientation, in light, 279, 282-87, 295, 300-303; electricity, 328 Oshima, 965 Osmic acid methods, in identification of Golgi bodies, 139, 140 Osmiophilic structures, 144-50, 438 f., 440 Osmotic pressure within cell, regulation of, 426 ff. Osterud, 478, 479, 480, 483 Ostwald, 314 Owen, 13 Owens and Bensley, 139 Oxidase, 373; detection of, 384 Oxidation-reduction potential vs. respira- tion and growth, 394-96 Oxygen, effect on anaérobes, 390-94; on a€robes, 394; relationships, 503 f.; ef- fect upon growth, 503, 538; Oxytricha, fibrillar system, 255 Pacinotti, 388 Packard, 60 lexvotatioh, (Gs 184 Jay, SA, Sh, 5G, SOs OWS amoeboid response, 273, 315, 341 Paraglycogen, formation of, 155; morpho- logical relationship between mitochon- dria and, 155; differentiated from glycogen, 157; decrease and _ storage, 159 Paramecium, physical properties of proto- plasm, 51, 55, 57, 60-82 passim, 86, 88, 91, 97; fiibrillar systems, 193-200, 224- 28, 257 ff.; responses to electricity, 321- 27 passim; to chemicals, 342-44; life 1138 Paramecium (continued) cycle, 580f.; fertilization, 588, 610, 615, 616, 617, 626, 636; sexuality in other ciliates and, 687-706; heritable effects of conjugation, 762-69; regen- eration, 774 ff., 793, 798, 799, 802 Paramecium aurelia, Yale 33 year old cul- ture, 615, 653, 654, 659, 660, 661; Johns Hopkins stock R, 615; autogamy, 654 ff.; inheritance of mating type in, 753-58; see also Paramecium Paramecium bursaria, sex reactions, 615; inheritance of mating type in, 758-61 Paramecium caudatum, life cycle, 578-81; see also Paramecium Parasites, parasitic and nonparasitic Pro- tozoa, 6, 892; ectoparasites, 7; endo- parasites, 7; pathogenic aspects, 818-29; three functional categories, 818; ma- larial species, 822f., with plates, 843 ff.; literature cited, 827-29, 878-89; immunology, 830-89; physical bases of immunity, 830-42; rdle of immune processes in the development of proto- zoan infections, 843-64; agent of kala azar, 854f.; trypanosome group, 855- 71; intermittent fatal trypanosomiasis in various laboratory animals, 864-71; practical applications of immune reac- tions, 871-76; immunological reactions in relation to classification, 876-78; parasitism defined, 890; inclusion in term symbiosis, 891, 892; accidental and facultative parasitism, 894, 895-902 Parasites of Protozoa, 1009-1113; epi- biotic Schizomycetes, 1010-24; endo- biotic Schizomycetes, 1024-40; Sphaerita and Nucleophaga, 1040-59; parasites of the nucleus of Trichonympha, 1059-63; Phycomycetes other than Sphaerita and Nucleophaga, 1063-68; Protozoa, 1068- 89; the genus Amoebophrya Koeppen, 1089-93; Metazoa, 1093-95; literature cited, 1095-1113 Parat, 138, 148, 180 Park, 547, 548 Parker, G. H., fibrillar system, 258 Parker, R. C., 660 Parker, I. J., 891 Parnas, 423 Parpart, A. K., sterility technique, 453 f. Parsons, 72, 74 Parthenogenesis, diploid, 649 INDEX Pascher, 93, 590, 613, 671, 679, 1010 Pasteur, Louis, 8, 537 Pathogenicity, certain aspects of, 818-29; problems of virulence and, 818-23; va- riability in strains and in host response, 823-26; nutrition and resistance, 826 f.; literature cited, 827-29; see also Para- sites Patten, M., 791 Patten, R., 119 Patten, R., and Beams, H. W., 81 Paulson and Andrews, 874 Pearl, R., 320, 324, 530, 544, 547, 548 Pedigreed series, nuclear behavior in, 654 Pedigree isolation culture and life cycles, 527-31 Peebles, F., 51, 67, 86 Peebles, F., morphogenesis, 774, 775, 777, Wks WHIDS TO, ses WL, how Pekarek, 51, 52 Pellicle, elasticity, 87 Pellissier, 120, 1021, 1022 Pelomyxa. Schizomycetes in, 1025-27 Penard, E., 73, 76, 91, 92, 93, 97; Proto- zoa and other animals, 898, 899, 914, 915, 916, 957; organisms on and in Protozoa, 1011, 1025, 1026, 1038, 1041, 1042, 1043, 1050, 1052, 1053, 1054, 1057, 1066, 1067, 1068, 1075, 1083, 1094 Peranema tricophorum, responses to light, 290-95 Perekropoff and Stepanoff, 912 Pericytes, cells, 833, 836 Peritrichida, sexuality, Vorticella Perla and Marmorstom-Gottesman, 860 Permeability, surface properties, 64-71; membranes, cells, 64-66, 69; nuclear, 66 f.; vacuoles, contractile, 67-69; food, 69-71; other types, 71 Peroxidase, detection of, 384 Peroxidases, 373 Perty, 413 Peschowsky, 223 Peshpowskaya, 120 Reskett.\Ga ls 53> Peters, A. W., 529, 542, 543 Peters, J. P., and van Slyke, D. D., 356, 385 Peters, R. A., 362, 377 Petersen, W. A., 498, 535 688; see also INDEX Petri-dish method of sterilization by mi- gration, 457 Petschenko, 1035, 1036, 1037 Pfeiffer, 71, 79, 84, 95 Phagocytosis, relation of adhesion to, 74; in malarial infections, wth plates, 849 ff.; in infection with trypanosomes, 860 Phelps, A., 495, 497, 498, 501, 504, 528, 529, 536, 541, 542, 548, 550 Phelps, L. A., 785, 788, 795, 798 Philip and Haldane, 687, 750 Phillips, Ro L.; 537 Philpott, 476 Photoautotrophic nutrition, 477, 478-80 Photomesotropic nutrition, 477, 480 f. Photometatrophic nutrition, 477, 481 f. Phototrophic nutrition, 477, 478-82 Phycomycetes other than Sphaerita and Nucleophaga, 1063-68 Physical properties of the protoplasm, 43- 110 (see entries under Protoplasm) Physiological gradients, 802-4 Physiological membranes, 414 ff. Physiological regeneration, 773 f. Phytomastigophora parasites in Protozoa, 1068-70 Pickard, E. A., 937; fibrillar system, 228, 229, 258 Pierantoni, 972, 1028, 1029, 1030 Pigment granules defined, 179 Pijper and Russell, 847 Pinching, ascribed to centripetal pressure, oT Pinto and Fonseca, 1044, 1050 IHRE, I, 1 SOA, S/T, KKO Pitts, Ro F., and Mast: ‘S: ©) 53, 54> re- sponses to electricity, 334-41 Plant and ciliate conjugation compared, 617 Plasmagel, 47; see also Ectoplasm Plasmalemma, 47; physical characteristics, 49 Plasmasol, recent name for endoplasm, 47 Plasmodia, infectious, 842; rate of repro- duction, 842 f. Plasmodium, malarial parasite, 822 f., with plates, 843 ff. Plasmodium vivax, life cycle, 577, 578; pathogenicity, 822 ff. Plasmosin, 87 Plate, 939, 1083, 1093 Platelets, 835 PSD Plating vs. turbidity test, 467 Platt, 79 Playfair, 905 Plimmer, 912 Poisons, effect on O2 consumption, 366 Poisson, 899 Polarity, of protoplasm, 84; physical- chemical factors involving change in, 85; changes and protoplasmic stream- ing, 801 f. Poljanskij, J. L., 952 Poljansky, G., 114, 115, 154, 631, 636, eS) Poljansky, G., and Strelkow, A., 986 Pollack, 59 Polygastrica (or Magenthiere), 12 Polysaprobic type, 5 Polytoma, inheritance, 743 f., 746, 747 Popoti Me SS. 52s 52255235 52405255 787 Population, growth in cultures as a prob- lem of, 495-97; the initial, 497-99; growth, 544-52 Postgastriole, 129 Poultry, coccidiosis in, 824-26 Powell, 1030 Powers, P. B. A., 919, 920, 921, 922, 923, 1022, 1024; fibrillar system, 236, 237 Poyarkoff, 953 Prandtl, 619, 631, 634, 702, 1042, 1053 Pratje, 588 Pringsheim, E., 590, 1010, 1064 Pringsheim, E. G., food, etc., influencing growth, 475, 476, 477, 478, 480, 482, 483, 488, 493, 494 Pringsheim, E. G. and Ondracek, K., 671, 679, 687 Pringsheim, H., 961 Pronuclei, formation, 632-34; migration, 634 f. Proske and Watson, 876 Protein molecules, results of multipolar character, 86 Proteins, in mitochondria, 118; reserves, 160-66, 171, 174; nomenclature com- plicated, 160, 165; terms that should be dropped or restricted, 165; provision of, by rumen ciliates, 983, 984 f. Protoplasm, conditions under which ani- mation maintained, 34; physical proper- ties, 43-110; Dujardin’s description of, 43; physical properties as exhibited in Amoeba, 46-50; colloidal nature, 49 f., 1140 Protoplasm (continued) 51; consistency, 50-61; surface proper- ties, 61-77; specific gravity or density, 77-82; optical properties, 82-84; struc- tural properties, 84-98; literature cited, 98-110 Protoporphyrin, 382 Protozoa, discovery of, and other histori- cal facts, 8-14; importance in solving question of spontaneous generation and other problems, 8; Leeuwenhoek’s de- scription, 10f.; classification, nomen- clature, 11; differences between proto- zoan and metazoan organization, 191, 260; relationships between certain Protozoa and other animals, 890-1008 (see also under Relationships) ; para- sitic and nonparasitic, 892; ways of benefiting larger animals, 893; free- living and symbiotic, systematically re- lated, 902-17; organisms living on and in, 1009-1113 (see entries under Para- sites of Protozoa); parasitizing other Protozoa, 1068-89; literature cited, see Literature Protozoélogy, indebtedness to microscope, 9; Leeuwenhoek the father of, 11 Provasoli, 481, 484, 486, 495 Prowazek, S., 636, 787; fibrillar system, 205: 221-222 Pruthi, 540, 541, 542 Pseudopodia, contractile, 91, 92 Ptychostomidae, family, species, genera, 943; adaptation, 944 f. Ptychostomum chattoni, fibrillar system, 244 Pitter, 388, 394 Purdy and Butterfield, 458 Puymaly, 1042, 1051, 1053, 1058 Quastel, J. H., 396 Quastel, J. H., and Stephenson, M., 392 Raabe, 934, 935, 938, 939, 940, 941, 942, 943 Rabinowitsch and Kempner, 856 Racial differences, and regeneration, 780 f. Radiation, effects on consistency, 60; used for sterilization, 466 Raffel, 752 Rammelmeyer, 154 Rankin, 950 Rapkine, 64 INDEX Ray, Harendranath, fibrillar system, 245 Ray, J., 282 “Ray-direction theory,” Sachs’s, 282 Raymond, 1071 Reconstruction bands, 23 Red, see Neutral red Redi, 8; quoted 262 Redmond, 848 Redox potential, 504 f. Rees, C. W., 343; fibrillar system, 225.1226. 2272521258 Refraction, double, 95-97 Refractive bodies, 170 Refractive index, 83 Regaud, 177 Regendanz, P., 860 Regendanz, P., and Kikuth, W., 856, 860, 861 Regeneration, of macronucleus, 16 ff.; physiological, 773 f.; factors in, 774-93; and division, 797-801; in colonial forms, 804-11; see also Morphogenesis ; Reproduction Rehberg, 907 Reich, 484, 486, 498, 500, 501, 535, 538, 549 Reichenow, 161, 162, 163, 164, 165, 179, 253, 638, 974, 982, 983, 1075, 1082 Reidmuller 365, 379, 380, 384 Reincorporation and grafting: behavior of fragments, 793-97 Reiner, 385 Reiner and Smythe, 389 Reiner, Smythe and Pedlow, 389 Relationships between certain Protozoa and other animals, 890-1008; symbiosis the comprehensive term for general rela- tionship, 891; externally mutualistic, termed commensalism, 891; protozoan benefits to larger animals, 893; principal discussions and articles on, 893; acci- dental and facultative parasitism, 894, 895-902; systematically related free- living and symbiotic Protozoa, 902-17; distributional host relationships and host-specificity in representative sym- biotic faunules, 917-29; adaptive host relationships in morphology and _ life history, 929-60; physiological host rela- tionships illustrative of mutualism and commensalism, 961-87; literature cited, 987-1008 Remane, 906 198, INDEX Renaut, 836 Reorganization, through cell division, an influence in longevity, 16; by endo- mixis, 31-36; of the macronucleus and other derived structures in Ciliata, 21- 31; by conjugation, 36-39; degree of injury and, 781-84 Reorganization bands, 23 Reproduction, asexual and sexual, 566 f., 571-73; metazoan life cycle, 566; sexual, in all animals and plants? 568; asexual, in alternating binary and mul- tiple fission, 569-71; types of inherit- ance and, 711; measurement of rate, 842; rate, of rumen ciliates, 976; see also Regeneration Reproduction, biparental defined, 711; in- heritance in, 732-50 Reproduction, uniparental: defined, 711; inheritance in, 711-13; changes in in- heritance characters in, 713-31 Reserve bodies, secretion of, 122; defined, 178 Resistance, effect of nutrition on, 826 f. Respiration, concern of mitochondria with, 121, 122, 125; purposes of studying, 353 f.; measurements, table, 362; in- vestigations which concern mechanism of, 372-84 Respiration, aérobic, 354-68; methods of measuring, 354-58; normal rate, 358- 61; effect on Oz consumption, of O2 tension, 361-64; of COz tension, 364 f.; of the physiological state, 365; of tem- perature, 366; of anesthetics and poi- sons, 366; of nutritive substances and other materials, 367; evolution of gases other than COn, 367 f. Respiratory metabolism, 352-403; pur- poses of studying respiration, 353 f.; methods of measuring aérobic respira- tion, 354-58; aérobic respiration, 358- 68; investigations which concern the source of energy, 368-72; investigations which concern the mechanism of respi- ration, 372-84; measurement of an- aérobic metabolism and __ glycolysis, 385 f.; occurrence of anaérobiosis and glycolysis, 386-90; why are anaérobes anaérobes, and aérobes aérobes?, 390- 94; oxidation-reduction potential vs. respiration and growth, 394-96; litera- ture cited, 397-403 1141 Respirometers, 355; sensitivity, tables, 354 Reticular cells, 833 Reticulo-endothelial system, 836 Rey, 125 Reynolds, B. D., 721, 901 Reynolds, M. E., morphogenesis, 777, 778, 780, 784, 791, 792, 797, 798 Reznikoff, 52 Reznikoff and Chambers, 53, 55 Rhizopods, responses to light, 272-80 (shock-reactions, 273-77, kinetic re- sponses, 277-79, orientation, 279); re- sponses to electricity, 305-20 (direct current, 306-10, alternating current, 310- 14, mechanics of response, 314-20) ; responses to chemicals, 333-41 (rate of locomotion and H-ion concentration, 334, 335, 336 ff., 341, mechanics of re- sponse, 338-41) Rhumbler, 72, 413 Richards, Oscar W., 519, 525, 529, 530, 532, 535, 536, 542, 547, 552; Growth of the Protozoa (Chap. X), 517-64 Richards, Oscar W., and Dawson, J. A., D285 929 Richards, Oscar W., and Jahn, T. L., 519 Richards, Oscar W., and Kavanagh, A. K., 547 Riddle and Torrey, 423 Rieckenberg, 869, 875 Rieckenberg blood platelet test, 869, 875 Rieder, 1072, 1073 Rigidity, 87; see also Elasticity Ripper, 961, 962 Ritz, 869 Roach Cryptocercus, Protozoa of termites and, 894, 923-29; relationship between flagellates of termites and, 894, 961-73 Robbie, Boell, and Bodine, 376 Robbins, 340, 341 Robertson, M., 530, 843, 865, 870, 1046, 1057, 1074 Robertson, T. B., 497, 499, 533, 534, 535, 536, 548 Robinson, 877 Rodet and Vallet, 868 Rosel, 787 Root, W. S., 356, 364, 370 Ropiness (or thread formation), 94 f. Roscoff, 899 Rose, 960 Rosenberg, L. E., 167, 908; fibrillar sys- tem, 248, 249 1142 Rosenhof, Résel von, 43 Roskin, G., 92, 93, 96, 1046, 1074; fibril- lar system, 202, 204 Roskin, G., and Levinsohn, L., 384 Ross, 846 Ross and Lotka, 554 Ross and Thompson, 864 Rossolimo, L., 916, 943, 944, 947 Rossolimo, L., and Jakimowitch, K., 21 f. Rossolimo, L., L., and Perzewa, T. A., 948 Rotifers, parasitism, 1093 f. Rottier, 501, 504, 528, 538 Roudabush and Becker, 857 Roudsky, 861 Rouget, C., 208 Rouget, J., 868 Roux, 531, 914 Rudolf and Ramsey, 847 Ruminants, ciliates of, 894, 973-87 (see entries under Ciliates of ruminants) Rumjantzew, 154, 158, 161 Rumjantzew and Suntzowa, 96 Rumjantzew and Wermel, 158, 165 Runyan and Torrey, 804 Russeff, 963, 870 Russell, 519 Ryckeghen, 911 Ryder, 1041 Sachs, “‘ray-direction theory,’ 282 Salts, effect of, on consistency, 53-55, 61; ' motor responses to, 333 ff., 342 ff.; rate of Amoeba's locomotion in sodium and calcium salt solutions, 337, table, 338 Sand, 1067, 1087, 1089 Sandon, 6 Sanford, 929 Sappinia (Amoeba) diploidea, 593, 595, 596 “Sapropelic fauna,” 5 “Sarcode,’ 12, 43, 192 Sarcodina, division processes, 28; Schizo- mycetes on, 1021; Schizomycetes in, 1032-34; parasites in Protozoa, 1074-78 Sassuchin, 153, 1033, 1039, 1044, 1045, 1046, 1047, 1049, 1050, 1051, 1052, 1054, 1058, 1059 Sassuchin, Popoff, Kudrjewzew, and Bogenko, 1045, 1047, 1050 Satina and Blakeslee, 705 Sauerbeck, 870 Saunders, 75, 541 INDEX Scarth and Lloyd, 412 Schaeffer, 49, 66, 70, 71, 72, 73, 82, 83, 86, 90, 91, 279 Schaudinn, Fritz, 14, 566, 574, 575, 577, 596, 598, 604 Schellack, 604, 605 Schereschewsky, 1079, 1082 Scheunert, 985 Schewiakoff, 78, 94, 208 Schieblich, 982 Schilling, 868 Schilling and Neumann, 877 Schirch, 163 Schizomycetes, endobiotic, 1024-40; asso- ciations of a constant character, 1025- 30; in Pelomyxa, 1025-27; in flagellates of termites, 1027-30; associations of an occasional character, 1030-40; in Mastigophora, 1030-32; in Sarcodina, 1032-34; in Ciliophora and Sporozoa, 1034-40 Schizomycetes, epibiotic, 1010-24; on Mastigophora, 1010-24; on Sarcodina, 1021; on Ciliophora, 1021-24 Schlayer, 362 Schleiden, 9 Schmalhausen 522, 526 Schmidt, 90, 92, 96, 97, 413, 912 Schmitt, 64, 354, 357 Schmitt, Bear, and Clark, 97 Schneider, 623 Schoenborn, 477, 482, 483, 490, 502 Schorger, 961 Schouteden, 921 Schréder, O., 7, 608, 1065; fibrillar sys- tem, 201, 202, 203, 204, 219, 220 Schuberg, A., fibrillar system, 193, 194, 195, 198, 201-4 passim 219, 224 Schubotz, 1094 Schultz, 73, 89, 92, 94, 96 SchulzewE Es ole 1025 Schulze, P., 157, 981, 982 Schwammerdam, 10 Schwann, 9 Schwartz, V., 789, 791, 796 Schwartz, W., 891, 892, 933, 963 Schwarz, C., 984, 985 Schwetz, 863 Scott, 586 Scott and Horning, 119 Sea urchins, ciliates of, 894, 919-23 and Synagajewska, 521, INDEX Secretion, of reserve bodies, 122; Golgi bodies’ relationship to, 143; external, 166-68 Secretion granules, formation comparable to segregation granules, 168; defined, 178 Segregation granules, 132-38; term va- cuome substituted for, 126; morphologi- cal variations, 133; composition, 134; formation comparable to secretion granules, 168; universality of, rejected, 171, 174; defined, 178 Seton 4O, Sil, Ss rh Sik, SO) OS Selection, results of long-continued, 726- 29 Self-fertilization 654-59 Serbinow, 1041, 1051, 1052, 1053, 1065 Sergent, Ed., 848 Sergent, Ed., and Sergent, Et., 844 Serological reactions, specific, 873 ff.; non- specific, 875 f. Serotherapy against trypanosomes, 867 f. Severtzoff, 463 Sex, an inherent characteristic of organ- isms? 568; analogies in metazoan and protozoan phenomena, 583 f.; sex, and sex-linked, inheritance, 740-50 Sexuality in unicellular organisms, 666- 709; Chlamydomonas, 666-87; Para- mecium and other ciliates, 687-706; lit- erature cited, 706-9 Sexual reproduction, in all animals and (autogamy), 606-11, plants? 568; alternation of asexual with, 566 f., 571-73 Seyd, 803 Shapiro, 519, 520 Sharp, R. G., fibrillar system, 206, 251, 2525258 Sharp, R. H., 585 Shettles, L. B., responses to light, 292 ff. Shock reaction to light, 273-77, 281, 295, 298 Shoup and Boykin, 376 Shumway, 423 Siebold, Th. von, 413; established the phylum Protozoa, 11; asserted unicel- lularity of Protozoa, 13, 191 Siegmund, 833 Silverline system, 194, 227 Simic, 820 Simpson, 520 1143 Singh, 81 Sinton et al., 847, 848 Sinton and Mulligan, 846 Size, factor in regeneration, 784-87 Skvortzow, 1042, 1053, 1065 Slater, 387 Smith, 528, 539 Snell, 534 Soil-dwelling Protozoa, ecological consid- erations, 6 Soil extracts, 493 Sokoloff, B., 774, 775, 782, 785, 786 Sokoloff, D., 903, 1068 Sonneborn, T. M., 588, 611, 615, 616, 654, 660, 661, 677, 690, 693, 696, 699, 700, 701, 704, 714, 752, 753, 754, 755, 756, 757, 758; Sexuality in Unicellular Organisms (Chap. XIV), 666-709 Sonneborn, T. M., and Cohen, B. M., 615 Sonneborn, T. M., and Lynch, R. S., 697, 766 Sotiriades, 850 Soules 3565 371 Spallanzani, Lazaro, 8, 404 Spasmoneme, 94 Specht, 360, 362, 363, 369, 370, 425 Specific gravity (or density), whole or- ganisms, 77-79; relative, of cell inclu- sions and components, 80-82 Specificity in symbiosis, 894 Spek, 54, 82 Spencer, 653 Spermatozoa, discovery, classification, 11 Sphaerita, and Nucleophaga, 1040-59; historical account and_ distribution, 1040-47; in free-living Protozoa, 1040- 43; in endozoic Protozoa, 1043-46; life history and structure, 1047-53; effect on host, 1057 f. Sphaeromyxa sabrazesi, life cycle, 608 f. Sphenophryidae, 933; adaptation, 941, 942 f. Spindle fibers, 90 Spirochetes and rods adherent by one end, 1015-21 Spirostomum ambiguum, fibrillar system, 250, 251 Splenectomy and blockade, 832 Spontaneous generation, history and solu- tion of problem, 8 Sporozoa, division processes, 28; survey of functions having granular basis, 168- 1144 Sporozoa (continued) 74 passim; fertilization, 601-6; Schizo- mycetes in, 1034-40; parasites in Protozoa, 1078-82 Spurr, 530 Stabler, 1076 Stabler and Chen, 1045, 1075, 1076, 1077 Staining reactions, difficulties with respect to, in both Protozoa and Metazoa, 180 Stalk, vorticellid, 208 ff., 216 ff., 257, 258, 259 Starch, in wood, 961; digestion by wood- eating animals, 971; digestion by rumi- nants, 979 ff. Statkewitsch, 322, 323, 326, 331 Stauber, 846 Steffan, 855 Stein and Schmidt, 422 Stein, F. von, 213, 218, 413, 637, 646, 907, 914, 934, 943, 1034, 1035, 1039, 1041, 1042, 1043, 1052, 1063, 1064, 1086, 1087; view of conjugation, 13 Steinhaus and Birkeland, 552 Stelluti, Francesco, 9 Stem cells, 835 Stempell, 413, 1078 Stentor, responses to light, 295-97 Stentor fibrillar systems, structural anal- ysis, 200-204; interpretation, 218-21; conclusions, 257 ff. Stephenson, 375 Sterilization, problem of, 449-67; general material, 449f.; general methods, 450 f.; special methods and manipula- tions, 451-67; importance of adequate tests, 467; establishment of sterilized Protozoa in culture, 468-73 Stern, 384 Steuer, 930 Stevens, 223, 789, 932, 936, 937 Stickiness, see Adhesiveness Stier, Newton, and Sprince, 519 Stimulants, growth, 493-95 Stimulating efficiency of light, 287-90, PASN5), PMS 10) Stockman and Wragg, 878 Stokes, 32, 33, 422, 1094 Stolé, 788 Stone, W. S., and Reynolds, F. H. K., 455 ff. Stout, 704 Strains and host response, variability in, 823-26 INDEX Strand, 934 StranghGner, 649, 650, 652 Strasburger, 289, 412 Stratman-Thomas, 849 Streaming, protoplasmic: changes, 801 f. Strelkow, 154, 156, 157, 411 Strelkow, Poljansky, and Issakowa-Keo, O77 529719. Structural properties, 84-98; colloids, 49 f.; origin of surface properties, struc- ture and, 62-64; polarity, 84; elasticity, 87-90; contractility, 90-94; ropiness or thread formation, 94 f.; double refrac- tion, 95-97; X-ray diffraction and ultra- centrifugation, 97 f.; analysis of fibrillar systems, 193-215; inherited environ- mental modifications in form and, 721-23 modifications of, in animals that live in association with hosts, 929 ff. Struggle for existence, 553 f. Stuart, Kidder, and Griffin, 467 Stubblefield, 1079, 1080, 1081, 1082 Studitsky, A. N., 270, 944, 1025, 1111; fibrillar system, 244 Stiitzgitter System, 194, 196, 199, 258 Stump, 73 Subdioecious races, 741 f. Subramaniam and Ganapati, 142, 143, 145, 175 Subramaniam and Gopala-Aiyar, 143 Suctoria, ectozoic, 1084 f.; endozoic, 1085- 89 Sudanophil material, 1187; as material, 151; see also Lipoids Sugars, in wood, 961 Sulfhydryl group, 121 Summers, Francis M., 22, 23, 646, 647, 805, 806, 808, 810; Protozoa in connec- tion with Morphogenetic Problems (Chap. XVI), 772-817 Summers, Francis M., and Kidder, G. W., 623, 624, 952 Surface membrane, 62 ff. Surface precipitation reaction, 48 Surface properties of protoplasm, 61-77; structure and origin, 62-64; permeabil- ity, 64-71; adhesiveness (or stickiness), 71-77, 930 ff., 944, 948, 949 Survival of the fittest, 553 f. Sutherland, 1015, 1018, 1021 Svec, 914 Swarczewsky, 594, 1074 and__ polarity 13m Ane reserve INDEX Sweet, 528, 536 Swellengrebel, 162 Swezy, 968 Sydney, 905 Symbiosis, 818, 1009; terms of designat- ing relationship between Prctozoa and their hosts, 890 (see also under Rela- tionships) ; defined, 891 f.; main cate- gories, 891; host-specificity, 894 Symbiotic and free-living Protozoa, sys- tematically related, 902-17 Synophrya, 958, 959 Szent-Gyorgyi and Banga, 381 Takagi, 1039 Taliaferro, L. G., 843, 844, 846 Taliaferro, William H., 717, 838, 839, 840, 845, 852, 855, 856, 857, 859, 860, 861, 863, 866, 872, 873; Immunology of the Parasitic Protozoa (Chap. XVIII), 830-89 Taliaferro, William H., and Cannon, P. R., 849, 850, 851”, 853 Taliaferro, William H., and Huff, C. G., 870 Taliaferro, William H., T. L., 867, 868, 870 Taliaferro, William H., and Mulligan, H. W., 837, 839, 847, 849, 852n, 853 Taliaferro, William H., and Pavlinova, Y., 843, 857 Taliaferro, William H., and Taliaferro, L. G., 75, 843, 846, 847, 848, 850, 855, 862, 864, 865, 875 Taliaferro, William H., Cannon, P. R., and Goodloe, S., 860 Taliaferro, William H., Johnson, T. L., and Cannon, P. R., 863 Tang, 361 Tannreuther, 627, 632 Tartar, V., 781; quoted, 777 Tartar, V., and Chen, T. T.,-715 Taylor, C. V., 147, 206, 319, 343, 783, 784; physical properties of protoplasm, ile 2; (625 167, 68, ‘87, 905" Fibrillar Systems in Ciliates (Chap. IV), 191- 270; contractile vacuole, 408, 409, 413, 416, 420, 438 Taylor, C. V., and Farber, W. P., 783, 790, 795 Taylor, C. V., and Strickland, A. S. R., lez Taylor, H. S., Swingle, W. W., Eyring, H., and Frost, A. A., 60 and Johnson, 1145 Tchakhotine, 60 Teissier, 525, 534 Temperature, resistance of Protozoa to high, 55; effect on consistency, 55-58; on O: consumption, 366; on growth, 505 f., 539 Tennent, Gardiner, and Smith, 139, 180, 181 Tension, effect on O, consumption, 361- 65 Termites, Protozoa of roach Cryptocercus and, 894, 923-29; relationship between flagellates of Cryptocercus and, 894, 961-73; classification, 923; Schizomy- cetes in, 1012 ff., 1027-32 Terry, 327 Testacea, budding division, 28; adhesive- ness, 73 Tetrault and Weis, 966 Theiler, 878 Theiler and Farber, 902 Thélohan, 153, 1078 Thiamine, or aneurin (vitamin B), 490 Thiel, van, 162 Thigmotricha, relation to family Ptychos- tomidae, 943; adaptation, 933-43 Thomson, J. A., 891 Thomson, J. A., and Geddes, P., 891 Thomson, J. G., 848, 864, 902 Thon, 791 Thornton, 53, 56 Thread formation (ropiness), 94 f. Thunberg, 356 Thunberg-Winterstein principle, 357 Tintinnopsis nucula, fibrillar system, 254 Tippett, 518 Tissue, connective: cells involved in im- munity, 831-37 Titration methods, 354, 355 f. Tittler, 649, 784, 791 Topley, 838 Trager, 927, 965, 969, 971 Transparency of protoplasm, 82 Transport concept, 174 Treillard and Lwoff, 898 Trensz, 876 Trichomonas augusta, life cycle, 569, 570 Trichonympha, parasites of the nucleus of 1059-63 Trier, 975, 979, 981, 982 Trophic specializations, 487-89 “Tropism theory,” Loeb’s, 282 Trypanolysins, involved in against trypanosomes, 861 >? immunity 1146 Trypanosoma, segregation granules, 134, 136 Trypanosomes, glycolysis rate, 388 ff.; rate of reproduction, 842; nonlethal in- fection with the Trypanosoma lewisi group of, 855-62; nonpathogenicity, 862; best-known pathogenic, 862; im- munological reactions, 874, 877 Trypanosomiasis, tests, 75; continuous fatal in mouse and sometimes in rat, 862-64; intermittent fatal, in various animals, 864-71 Tschaschin, 836 Turner, John P., 24, 471, 619, 620, 621, 626, 628, 633, 634, 637, 647; fibrillar system, 206-8, 222, 223; Fertilization in Protozoa (Chap. XII), 583-645 Tuzet, 141 Tyzzer, 1046 Uhlenhuth, 180 Ullmann, 961, 966, 971, 981 Ultracentrifugation and X-ray diffraction, 97 f. Ultracentrifuge, research with, 81 Ultra-violet radiation, 60 Unger, 533 Uniparental reproduction, see Reproduc- tion, uniparental Unknown granules, defined, 179 Uric acid, in contractile vacuole, 422 ff. Uroleptus halseyi, preparation for divi- sion, 18, 21; fibrillar system, 256, 257; division, 637 Uroleptus mobilis, preparation for divi- sion, 19, 20; vitality, 29-31; conjuga- tion, 31, 32, 34-38; fertilization, 615, 616, 630, 632, 633 Uronychia, merotomy and NG fie 75 th 783 he Usuelli, 979, 980, 982, 984 Uylmura, 919, 921, 934 regeneration, Vacuolar reaction, term, 132, 176 Vacuolation upon transfer to fresh water, 78 Vacuole, term gastriole substituted for, 129 Vacuoles, accessory (vesicles): relation to contractile vacuole, 412, 416, 424, 439, 441; contribution of osmiophilic gran- ules to formation of, 439 Vacuoles, contractile, 404-47; effects of INDEX fresh and salt water, 66; permeability, 67-69; membrane surrounding? 67; color, 83; osmiophilic structures, 144- 50; function, 173, 421-31, 442; origin, 405-13, 441, 442f.; whether perma- nent or temporary, 410, 419, 441; rela- tion of accessory vacuoles to, 412, 416, 424, 439, 441; structure, 413-21, 441; regulation of hydrostatic pressure, 422, 426 ff., 442; and Golgi apparatus, 431- 41, 442; generalizations re processes associated with, 441-43; outstanding features, 443; literature cited, 443-47 Vacuoles, food: permeability of, 69-71 Vacuoles, other types: in the cytoplasm, Wil Vacuome, hypothesis, 126-29; term sub- stituted for segregation granule, 126; ferment theory, 127; digestive granules, 131; a universal cell constituent, 170; universality of, rejected, 175; present in all cells? 432; identical with Golgi apparatus? 432, 436 Valentin, 96 Valkanov, 602, 613, 906 Van Beneden, 892 Van den Branden, 870 Variation and its inheritance occurring without obvious action of diverse en- vironments, 723-29 Vaucel and Hoang-Tich-Try, 876 Veley, 1026, 1027 Vernes, Bricq, and Yvonne, 876 Verworn, M., 74, 86, 93, 787, 788, 793; motor responses, 282, 305, 320, 324, 326, 332 Vesicles, see Vacuoles, accessory Villian and Dupoux, 876 Virulence, problems of pathogenicity and, 818-23 Viscosity, studies of changes in, 51 ff. Visscher, J. P., 343, 620; 621, 624; fibrillar system, 235 Vitality, and life, 3f., 34; waning, in ciliates, 28 ff.; underlying cause of waning vitality and death of proto- plasm in isolation cultures, 39; effect of conjugation upon, 616 Vitamins, A in the mitochondria, 121, 125; protoporphyrin, 383; specific growth factors, 489-93; B:, 490 ff., Bz, 492: C, 492; ciliate capacity of syn- thesizing B, 985 INDEX Voegtlin and Chalkley, 54 Voigt, 906 Volkonsky, 113, 115, 125, 126, 127, 129, 130, 131, 132, 134, 135, 148, 175, 176, 179 Volterra, 553 Volutin, 160, 161, 162f.; a term with no standard usage, 163; term should be dropped or restricted, 165 Volvox, structure, 297, 298; responses to light, 288, 297-305; responses to elec- tricity, 327-32 Vorticella, body divisions, 212, 218; fibril- lar systems, structural analysis, 208-15; interpretation, 216-18; conclusions, 257 f Wachendorff, 362, 365, 366, 369 Wagener and Koch, 877 Wager, 285, 1065 Walker, E. L., 463, 464 Walker, E. L., and Sellards, A. W., 824 Walker, H. H., 539 Wallace and Wormall, 875 Wallengren, 24, 941 Wallich, 1040, 1042 Wallin, 118 Wang, 531 Warburg, O., respiratory metabolism, 353, 354, 356, 373, 374, 375, 390 Warburg-Keilin system, 380 Warren, 900, 901 Wasielewski, T. K. W. N. von, 848 Wasielewski, T. K. W. N. von, and Senn, G, 856 Wassermann test, 874 Waste products, and growth, 499 f. Water, taking up of, by colloids, 50; effects on consistency, 53; rate of ex- change, 65; contractile vacuole regula- tion of pressure within cell, 426 ff. Water, heavy, 60 Water fleas, 10 Watson, C. J., 854 Watson, E. A., 874 Watson, M. E., 932 Wave length and stimulating efficiency of light, 287-90, 295, 297, 303 Weatherby, J, EH. 148,150, 152, 423, 425; Contzactile Vacuole (Chap. VII), 404-47 Weber, 344 Weber and Weber, 319 1147 Weier, 126 Weineck, E., 975, 979, 981, 982 Weineck, I., 157 Weismann, 28 Wells, 838, 876 Wenrich, 93, 410, 419, 893, 903, 904, SWE, Oilil, Oil, Siler, Gilét ily, Gili. 1044, 1045, 1058 Wenyon, 820, 878, 896, 898, 1033, 1044, 1045 Wenyon and Broughton-Alcock, 910 Wermel, 119, 123, 155, 156 Werner, 962 Wertheim, 973, 974 Weschenfelder, 604, 613, 614 Wesenberg-Lund, C., 1093, 1094 Wesenberg-Lund, Mrs. E., 899 Westphal, 975, 976, 980, 981, 982, 985 Wetzel, 544, 1077 Whole organisms, specific gravity, 77-79 Wipfchterman, 621, 635, 637 iedemann, 962, 963, 972 Willis, 788 Wilson, 160, 162, 825 Wilson and Pollister, 181 Winkler method, 355, 363 Winogradowa, T., 1046, 1049 Winogradow, Winogradowa - Fedorowa, and Wereninow, 986 Winogradowa-Fedorowa doff, 974 Winterstein, 59, 356 Wood, constituents, 961 Woodcock and Lodge, 914, Wood-eating animals, 961-73 Woodhead, 950 Woodruff, Lorande Loss, 34, 476, 498, A958; 5255 52955230955.) 50, 9505 611, 616, 617, 653, 659, 791, 792, 796; on early microscopes, 9; endomixis (Chap. XIII), 646-65 Woodruff, Lorande Loss, and Baitsell, G. Lin SAI S75 S293 Seo Woodruff, Lorande Loss, and Erdmann, R., endomixis, 31, 648, 649, 654, 657, 658, 659, 660 Woodruff, Lorande Loss, and Moore, E. Ik, GBS} Woodruff, Lorande Loss, and Spencer, H., 616, 649, 659 Work Projects 890xz, 1009” Worley, 257 and Winogra- Administration, 5652, 1148 Wright, 519 Wrisberg, 208 Wrzesniowski, 208, 210, 217, 413 Wurmser, 125, 396 Xanthellae, relationship to host, 1010 X-ray diffraction and ultra-centrifugation, 97 f. Yagiu, 919, 920, 922, 923, 1022 Yakimoff, 870, 1032 Yale University, race of Paramecium aurelia, 615, 653, 654, 659, 660, 661 Yamasaki, 154, 156, 157, 159, 968, 969, 970 Yarwood, 576 Yeast, use in cultures, 449, 543 Yellow respiratory pigments, or enzyme, 579 Yocom, H. B., 23, 343, 535, 538; fibril- lar system, 206, 207, 258 Yonge, 962 Yorke, Adams, and Murgatroyd, 388, 389 Youngs] Di Bes yiiaiiS; 1S, Young, Dixie, 647, 653, 797 Young, R. A., 419, 420 Young, R. T., 660 INDEX Yuan-Po, 1045, 1047, 1048, 1051, 1052, 1058 Zacharias, 1074 Zdrodowski, 877 Zederbauer, 588 Zeliff, 1044 Zenker, 413 Zerling, 906 Zhalkovskii, 542 Zhinkin, 153, 154, 157, 159, 388 Zick, 931, 932 Ziegler and Halvorson, 520 Zinger, 123,151) 152; 165 Zodlogical Nomenclature, Rules of, 954 Zo6mastigophora parasites in Protozoa, 1070-74 Zoothamnium, regeneration, 805 ff. Zuelzer, M., 426, 595 Zumstein, 463 Zurn, 983 Zweibaum, 153, 362, 365, 689 Zwischenstreifen, 201, 202 Zygote, start of metazoan life cycle with, 566 Zygotic meiosis, 611-14 International 4 pe